Menopause: Biology and Pathobiology

Contributors Irina D. Burd (383) Department of Obstetrics, Gynecology, and Reproductive Sciences, Robert Wood Johnson ...

Author: Rogerio A. Lobo | Jennifer Kelsey | Robert Marcus

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Contributors

Irina D. Burd (383) Department of Obstetrics, Gynecology, and Reproductive Sciences, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, New Brunswick, New Jersey 08901

Numbers in parentheses indicate the pages on which the authors' contributions begin.

Sanjay K. Agarwal (591) Division of Reproductive Medicine, Department of Obstetrics and Gynecology, Cedars-Sinai Burns and Allen Research Institute, Cedars-Sinai Medical Center, Los Angeles, California 90048; and Department of Obstetrics and Gynecology, University of California, Los Angeles, School of Medicine, Los Angeles, California 90095 Deborah J. Anderson (353) Fearing Laboratory, Department of Obstetrics, Gynecology, and Reproductive Biology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115 Nancy E. Avis (339) Institute for Women's Research, New England Research Institutes, Watertown, Massachusetts 02472 Gloria A. Bachmann (383) Department of Obstetrics, Gynecology, and Reproductive Sciences, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, New Brunswick, New Jersey 08901 John A. Baron (583) Section of Biostatistics and Epidemiology, Dartmouth Medical School, Hanover, New Hampshire 03755 Steven Birken (61) Department of Medicine, Columbia University College of Physicians and Surgeons, New York, New York 10032 Julia E. Bradsher (203) Abt Associates, Inc., Cambridge, Massachusetts 02138 M. Brincat (261) Department of Obstetrics and Gynecology, St. Luke's Hospital Medical School, Gwardamangia MSD 07, Malta

Henry G. Burger (147) Prince Henry's Institute of Medical Research, Clayton, Victoria 3168, Australia John E. Buster (625) Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston, Texas 77030 Peter R. Casson* (625) Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston, Texas 77030

Sybil L. Crawford (159, 175) New England Research Institutes, Watertown, Massachusetts 02472 Susan R. Davis (445) The Jean Hailes Foundation, Clayton, Victoria 3168, Australia; and Department of Epidemiology and Preventive Medicine, Monash University, Melbourne, Victoria 3004, Australia Carol A. Derby (229) New England Research Institutes, Watertown, Massachusetts 02472 Christine Draper (287) Department of Medicine, University of Western Australia, and Department of Endocrinology and Diabetes, Sir Charles Gairdner Hospital, Nedlands, Western Australia 6009, Australia Gary A. Ebert (383) Department of Obstetrics, Gynecology, and Reproductive Sciences, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, New Brunswick, New Jersey 08901

* Current address: Department of Obstetrics and Gynecology, Ottawa Hospital, Ottawa, Canada

xiii

xiv

Gregory F. Erickson (13) Department of Obstetrics and Gynecology, University of California, San Diego, La Jolla, California 92093 Denis Evans (175) Rush Institute on Aging, Chicago, Illinois 60612 Patricia D. Finn (33) Ligand Pharmaceuticals, Inc., San Diego, California 92121 Jeanne Franck (309) Department of Dermatology, Cornell Medical College and New York Presbyterian Hospital, New York, New York, 10021 Robert R. Freedman (215) Departments of Psychiatry and Behavioral Neurosciences and Obstetrics and Gynecology, Wayne State University, Detroit, Michigan 48201 R. Galea (261) Department of Obstetrics and Gynecology, St. Luke's Hospital Medical School, Gwardamangia MSD 07, Malta Ellen B. Gold (175, 189) Department of Epidemiology and Preventive Medicine, School of Medicine, University of California, Davis, Davis, California 95616 Joseph W. Goldzieher (397) Department of Obstetrics and Gynecology, Texas Tech University Health Sciences Center, Amarillo, Texas 79106 Gail A. Greendale (175, 639) Departments of Medicine and Obstetrics and Gynecology, University of California, Los Angeles, School of Medicine, Los Angeles, California 90024 Francine Grodstein (543) Channing Laboratory, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115 Robert E Heaney (481) Creighton University, Omaha, Nebraska 68131 Victor W. Henderson (315) Department of Neurology, University of Southern California, Los Angeles, California 90089 Victoria Hendrick ( l l l ) Department of Psychiatry, University of California, Los Angeles, School of Medicine, Los Angeles, California 90095 Howard L. Judd (591) Department of Obstetrics and Gynecology, Olive View/ UCLA Medical Center, Sylmar, California 91342; and Department of Obstetrics and Gynecology, University of California, Los Angeles, School of Medicine, Los Angeles, California 90095 Margaret R. Karagas (359) Section of Biostatistics and Epidemiology, Dartmouth Medical School, Lebanon, New Hampshire 03756

CONTRIBUTORS

F. S. J. Keating (509) Endocrinology and Metabolic Medicine, Imperial College School of Medicine, St. Mary's Hospital Medical School, London W2 1PG, United Kingdom Jennifer Kelsey (175, 359, 405) Division of Epidemiology, Department of Health Research & Policy, Stanford University School of Medicine, Stanford, California 94305 Stanley G. Korenman (111) Department of Medicine, University of California, Los Angeles, School of Medicine, Los Angeles, California 90095 Galina Kovalevskaya (61) Irving Center for Clinical Research, Columbia University, College of Physicians and Surgeons, New York, New York 10032 Mark A. Lawson (33) Ligand Pharmaceuticals, Inc., San Diego, California 92121 Annie Lo (175) Westat, Inc., Rockville, Maryland 20850 Leslie Lobel (61) Department of Obstetrics and Gynecology, Columbia University, College of Physicians and Surgeons, New York, New York 10032 Rogerio A. Lobo (429) Department of Obstetrics and Gynecology, Columbia University, College of Physicians and Surgeons, New York, New York 10032 Francisco Jos~ L6pez (33) Ligand Pharmaceuticals, Inc., San Diego, California 92121 Cecilia Magnusson (583) Department of Medical Epidemiology, Karolinska Institutet, S- 171 77 Stockholm, Sweden

N. Manassiev (509) Endocrinology and Metabolic Medicine, Imperial College School of Medicine, St. Mary's Hospital Medical School, London W2 1PG, United Kingdom Robert Marcus (405, 495) Department of Medicine, Stanford University School of Medicine, Geriatrics Research, Education & Clinical Center, Veterans Affairs Medical Center, Palo Alto, California 94304 Karen Matthews (175) Department of Psychiatry, University of Pittsburgh, Pittsburgh, Pennsylvania 15261 Donald P. McDonnell (3) Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 277 l0

CONTRIBUTORS

Valerie McGuire (359) Division of Epidemiology, Department of Health Research & Policy, Stanford University School of Medicine, Stanford, California 94305 Sonja M. McKinlay (203) New England Research Institutes, Watertown, Massachusetts 02172 Arshag D. Mooradian (111) Department of Internal Medicine, Saint Louis University School of Medicine, Saint Louis, Missouri 63104 David Morganstein (175) Westat, Inc., Rockville, Maryland 20850 Robert Neer (175) Division of Endocrinology, Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts 02114 AndrOs Negro-Vilar (33) Ligand Pharmaceuticals, Inc., San Diego, California 92121 John O'Connor (61) Department of Pathology and Irving Center for Clinical Research, Columbia University, College of Physicians and Surgeons, New York, New York 10032 Richard L. Prince (287) Department of Medicine, University of Western Australia, and Department of Endocrinology and Diabetes, Sir Charles Gairdner Hospital, Nedlands, Western Australia 6009, Australia Janet H. Prystowsky (309) Department of Surgery, Columbia Presbyterian Medical Center, Columbia University and New York Presbyterian Hospital, New York, New York 10032 Russalind H. Ramos (459) Center for Menopause, Hormonal Disorders, and Women's Health, Sloane Hospital for Women, Columbia Presbyterian Medical Center, New York, New York, 10032 Nancy E. Reame (95) Center for Nursing Research and Reproductive Sciences Program, The University of Michigan, Ann Arbor, Michigan 48109 Robert W. Rebar (135) Department of Obstetrics and Gynecology, University of Cincinnati Medical Center, Cincinnati, Ohio 45267; and the American Society for Reproductive Medicine, Birmingham, Alabama 35216 Clifford J. Rosen (271) St. Joseph Hospital, Maine Center for Osteoporosis Research and Education, Bangor, Maine 04401 Giiran Samsioe (327) Department of Obstetrics and Gynecology, Lund University Hospital, S-221 85 Lund, Sweden Sherry Sherman (175) NIH/NIA, Bethesda, Maryland 20892

XV

Barbara B. Sherwin (617) Department of Psychology and Department of Obstetrics and Gynecology, McGill University, Montreal, H3A 1B 1 Canada Joe Leigh Simpson (77) Departments of Obstetrics and Gynecology and Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030 MaryFran Sowers (175, 245, 535) Department of Epidemiology, School of Public Health, University of Michigan, Ann Arbor, Michigan 48109 Leon Speroff (553) Department of Obstetrics and Gynecology, Oregon Health Sciences University, Portland, Oregon 97201 Margaret G. Spinelli (563) Department of Clinical Psychiatry, Columbia University, College of Physicians and Surgeons; and The New York State Psychiatric Institute, New York, New York 10032 Meir J. Stampfer (543) Departments of Nutrition and Epidemiology, Harvard School of Public Health; and Channing Laboratory, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115 Frank Z. Stanezyk (421) Department of Obstetrics and Gynecology, University of Southern California School of Medicine, Los Angeles, California 90033 Barbara Sternfeld (175, 495) Department of Epidemiology and Biostatistics, Division of Research, Kaiser Permanente, Oakland, California 94611 J. C. Stevenson (509) Endocrinology and Metabolic Medicine, Imperial College School of Medicine, St. Mary's Hospital Medical School, London W2 1PG, United Kingdom Jennifer Tiseh (245) Department of Epidemiology, School of Public Health, University of Michigan, Ann Arbor, Michigan 48109 Anna N. A. Tosteson (649) Clinical Research Section, Department of Medicine and the Center for the Evaluative Clinical Sciences, Department of Community and Family Medicine, Dartmouth Medical School, Hanover, New Hampshire 03755 Michelle P. Warren (459) Department of Obstetrics and Gynecology and Medicine, Columbia University, College of Physicians and Surgeons, New York, New York 10032; and Center for Menopause, Hormonal Disorders, and Women's Health, Sloane Hospital for Women, Columbia Presbyterian Medical Center, New York, New York, 10032

xvi Gerson Weiss (175) Department of Obstetrics and Gynecology, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103 Carolyn Westhoff (607) Columbia University, College of Physicians and Surgeons, New York, New York 10032

CONTRIBUTORS

Preface

lished in recent years, this work represents what we feel will be the first compilation of the entire subject, from basic biology to medical issues and therapeutic considerations. The text has been divided into several areas: basic biology, epidemiology, pathophysiology, and interventions. The chapters in each section are authored by outstanding investigators in the field who are recognized for their expertise. Accordingly, this text will be useful to all who are interested in the field, including basic and clinical investigators, students, residents, fellows, and clinicians. We are indebted to our friends, the contributing authors, and the tireless work of Jenny Wrenn and Jasna Markovac of Academic Press, who have shepherded this project from its inception. Without their help and persistence, this volume would not have been completed so efficiently and professionally, and in a timely manner.

Menopause is defined as the cessation of menstrual flow. Because the age of menopause is largely genetically determined, the average age at which it occurs, approximately 51 years, has not changed over many centuries. However, life expectancy has increased substantially, and the current life expectancy for women is 80 years. If a woman reaches age 54, she can expect to reach the age of 84.3 years. Thus, the years after menopause may account for as much as 40% of a woman's life. Currently in the United States, there are approximately 31 million women over age 55, with estimates of 38 million in 2010 and 46 million in 2020. Similar trends will occur in many parts of the world. Thus, there is a large and ever-increasing population of women in this very important time of life. Many women look forward to this time, even viewing this signal of a change in their reproductive lives as an opportunity for change and for instituting preventive health care. Nevertheless, for many years menopause has not been well understood. Although numerous books on the clinical aspects and the management of menopause have been pub-

Rogerio A. Lobo Jennifer Kelsey Robert Marcus

xvii

2HAPTER

Molecular Pharmacology of Estrogen and

Progesterone Receptors DONALD P.

MCDONNELL

Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710

I. Introduction II. Estrogen and Progesterone Receptors III. Established Models of Estrogen and Progesterone Action IV. Estrogen and Progesterone Receptor Isoforms and Subtypes V. Regulation of Estrogen and Progesterone Receptor Function by Ligands

I. I N T R O D U C T I O N The steroid hormones estrogen and progesterone are small molecular weight lipophilic hormones that, through their action as modulators of distinct signal transduction pathways, are involved in the regulation of reproductive function [1, 2]. These hormones have also been shown to be important regulators in bone, the cardiovascular system, and the central nervous system [3-5]. Despite their different roles in these systems, however, it has become apparent that estrogens and progestins are mechanistically similar [6]. Insights gleaned from the study of each hormone, therefore, have advanced our understanding of this class of molecules as a whole. This review highlights some of the recent mechanistic discoveries that have occurred in the field, and e x -

MENOPAUSE: B I O L O G Y AND PATHOBIOLOGY

VI. Estrogen and Progesterone Receptor Associated Proteins VII. An Updated Model of Estrogen and Progesterone Receptor Action References

plores the subsequent changes in our understanding of the pharmacology of this class of steroid hormones.

II. E S T R O G E N AND PROGESTERONE RECEPTORS The estrogen receptor (ER) and progesterone receptor (PR) cDNAs have been cloned and used to develop specific ligand-responsive transcription systems in heterologous cells, permitting the use of reverse genetic approaches to define the functional domains within each of the receptors [6]. A schematic that outlines the organization of the major functional domains within these two steroid receptors (SR) proteins is shown in Fig. 1. The largest domain (~--300 amino acids) that

Copyright 9 2000 by Academic Press. All rights of reproduction in any form reserved.

4

DONALD P. MCDONNELL An additional activation function (AF-1) is located within the amino terminus of each receptor [ 11 ]. The DNA-binding domain (DBD) is a short region (--~70 amino acid residues) located in the center of the receptor protein [12]. Thispermits the receptor to bind as a dimer to target genes. Within the DBD there are nine cysteine residues, eight of which can chelate two zinc atoms, thereby forming two fingerlike structures that allow the receptor to interact with DNA [13]. All of the information required to permit target gene identification by ligand-activated ERs and PRs is contained within this region.

III. ESTABLISHED OF ESTROGEN PROGESTERONE

FIGURE 1 Establishedmodels of estrogen and progesteroneaction. The classic models of estrogen and progesterone action suggested that, in the absence of ligand, the steroid receptor (SR) exists in the nuclei of target cells in an inactive form. On binding an agonist, the receptorwould undergo an activating transformation event that displaces inhibitory heat-shock proteins (HSP) and facilitates the interaction of the receptorwith specific DNA steroid response elements (SRE) within target gene promoters. The activated receptor dimer could then interact with the general transcription machinery and positively or negatively regulate target gene transcription. In this model the role of the agonist is that of a "switch" that merely converts the ER or PR from an inactive to an active form. Thus, when corrected for affinity, all agonists would be qualitatively the same and evoke the same phenotypic response. By inference,antagonists,compounds that oppose the actions of agonists, would competitivelybind to their cognate receptors and freeze them in an inactive form. As with agonists, this model predicted that all antagonists are qualitatively the same. Within the confines of this classic model it was difficult to explain the molecularpharmacologyof the known ER and PR agonists and antagonists. GTA, General transcriptional activity.

is responsible for ligand binding is located at the carboxyl terminus of each receptor. Crystallographic analysis of the agonist-bound forms of ERs and PRs has indicated that this domain consists of 12 short a-helical structures that fold to provide a complex ligand-binding pocket [7, 8]. The ligandbinding domain also contains sequences that facilitate receptor homodimerization and permit the interaction of apo receptors with inhibitory heat-shock proteins. An activation function (AF-2) required for receptor transcriptional activity is also contained within the ligand-binding domain [9, 10].

MODELS

AND ACTION

The steroid hormones estrogen and progesterone are representative members of a larger family of steroid hormones, all of which appear to share a common mechanism of action. It is generally believed that steroid hormones enter cells from the bloodstream by simple passive diffusion, exhibiting activity only in cells in which they encounter a specific high-affinity receptor protein [14]. These receptor proteins are transcriptionally inactive in the absence of ligand, sequestered in a large oligomeric heat-shock protein complex within target cells [15]. On binding ligand, however, the receptors undergo an activating conformational change that promotes the dissociation of inhibitory proteins [16]. This event permits the formation of receptor homodimers that are capable of interacting with specific high-affinity DNAresponse elements located within the regulatory regions of target genes (Fig. 2) [17]. The DNA-bound receptor can then exert a positive or negative influence on target g e n e transcription. In the classic models of steroid hormone action, it was proposed that progestins and estrogens function merely as switches that, on binding to their cognate receptor, permit

FIGURE 2 The domain structures of the estrogen and progesterone receptors are similar.

CHAPTER 1 Estrogen and Progesterone Receptors conversion of the receptor in an all or nothing manner from an inactive to an active state [ 18]. This implied that ER and PR pharmacology was very simple, and that when corrected for affinity all progestins and estrogens were qualitatively the same. Furthermore, it suggested that antihormones (antagonists) function simply as competitive inhibitors of agonist binding, freezing the target receptor in an inactive state within the cell. Under most experimental conditions this simple model was sufficient to explain the observed biology of known PR and ER agonists and antagonists. However, systems were discovered that did not fit this simple model, indicating that the pharmacology of these receptor systems is more complex than originally believed. Studies probing the complex pharmacology of the antiestrogen tamoxifen have been very informative with respect to understanding the inadequacies of the classic model. Tamoxifen is widely used as a breast cancer chemotherapeutic and has been approved for use as a breast cancer chemopreventive in high-risk patients [ 19, 20]. In ER-positive breast cancers, tamoxifen opposes the mitogenic action of estrogen(s) by binding to the receptor and competitively blocking agonist access. However, it has become clear in recent years that tamoxifen is not a pure antagonist, because in some target organs it can exhibit estrogen-like activity. This is most apparent in both the skeletal system, where tamoxifen, like estrogen, increases lumbar spine bone mineral density, and the cardiovascular system, where both tamoxifen and estradiol have been shown to decrease low-density liproprotein (LDL) cholesterol [21, 22]. These in vivo properties of tamoxifen led to its being reclassified as a selective estrogen receptor modulator (SERM) rather than an antagonist. The observation that different ligand-receptor complexes were not recognized in the same manner in all cells was at odds with the established models of ER action. From a clinical perspective this was an important finding, because it suggested for the first time the possibility of developing compounds that, acting through their cognate receptor, could manifest different activities in different cells. From a molecular point of view, however, the observed pharmacology of tamoxifen begged a reevaluation of the classic model of ER action, and initiated the search for the cellular systems that enable ER-ligand complexes to manifest different biologies in different cells. These ongoing investigations have also provided significant insight into PR action, and have demonstrated that, as in the case of ERs, it will be possible to develop compounds that manifest PRagonist activity in a tissue-selective manner.

IV. ESTROGEN AND PROGESTERONE RECEPTOR ISOFORMS AND SUBTYPES One mechanism to explain the cell-selective action of steroid receptor ligands is the likelihood that they may activate different receptor isoforms (derived from the same gene) or

5

Estrogen Receptor Subtypes 1

595

NH21

hERc~

1

530

NH21

hER[3

I

Progesterone Receptor Subtypes 1

933

1

hPR-A

NH~I

769

I~1

~

I

FIGURE 3 At least two distinct forms of the estrogen and progesterone receptors exist in target cells. DBD, DNA-binding domain; LBD, ligandbinding domain.

subtypes (derived from similar genes). This concept has been well established for the ce- and fi-adrenergic systems, where it has been shown that different receptor subtypes have distinct ligand preferences, and that selectivity can be explained by differences in the expression of these subtypes. Until recently, the parallel between this system and that of the nuclear receptors was not obvious. However, the identification and characterization of ER and PR isoforms and subtypes has shed new light on this issue (Fig. 3).

A. ProgesteroneReceptor Isoforms The progesterone receptor was the first receptor for which bona fide isoforms were shown to exist. Human PRs can exist within target cells in either of two distinct forms, hPR-A (94 kDa) or hPR-B (114 kDa) [23]. These proteins, differing only in that the hPR-B isoform contains an additional 164-amino acid extension at its amino terminus, are produced from distinct mRNAs that are derived from different promoters within the same gene [9]. In most progesterone-responsive tissues these two receptor isoforms are expressed in equimolar amounts. This apparent 1:1 relationship is so widespread that until about 10 years ago the hPR-A isoform was thought to be merely an artifact derived from hPR-B by proteolysis during biochemical fractionation. It has now been established that these two proteins are produced in a deliberate manner by the cell, and that they are not functionally equivalent [23-25]. The first evidence in support of this hypothesis came following the cloning and subsequent functional analysis of the chicken progesterone receptor (cPR) cDNA [26]. Specifically, on expression in

6

DONALD P. MCDONNELL

heterologous cells, it was found that although the A and B forms of cPRs display identical ligand binding preferences, they activate different target genes [26]. It was subsequently shown that the amino-terminal sequences, which distinguish cPR-B from cPR-A, are important in determining target gene selectivity. This concept was reaffirmed when the cloned hPR-B and hPR-A were analyzed in a similar manner [24]. In the systems examined thus far, with few exceptions, it has been observed that hPR-B alone functions as a transcriptional activator in response to progesterone, whereas hPR-A displays minimal or no activity. Further analysis has revealed that hPR-A functions primarily as a ligand-dependent transdominant modulator of the transcriptional activity of hPR-B, the ability of hPR-B to activate target gene transcription being influenced by the cellular concentration of hPR-A [24, 27]. Surprisingly, it was also determined that ligandactivated hPR-A can inhibit the transcriptional activity of agonist-activated ERs, androgen receptors (ARs), and mineralocorticoid receptors (MRs) [24]. Thus, by virtue of having two functionally different receptor isoforms, a single hormone such as progesterone can have completely different functions in target cells.

B. E s t r o g e n R e c e p t o r S u b t y p e s The identification of functionally distinct PR isoforms introduced a new dimension to progesterone action, although it was not until a second estrogen receptor was cloned in 1995 that the general significance of isoforms (or subtypes) in steroid receptor signaling was established [28]. Unlike the case of PRs, ERa and ERfl are encoded by different genes, and although they share significant amino acid homology in their ligand-binding domains, they are not pharmacologically equivalent. Both receptors bind the endogenous estrogen, 17fl-estradiol, with equivalent affinity [29]. However, when binding analysis was extended to additional compounds, significant differences in ligand preferences were noted. The biological and pharmacological consequences of these differences remain to be determined. Although the discovery of ERfl has occurred relatively recently, significant progress has been made in elucidating its role in estrogen signaling. It has been determined that the expression pattern of ERfl does not mirror that of ERce [30, 31]. Expression of both isoforms is found in some tissues, whereas ER/3 alone occurs in others, such as the lung, the urogenital tract, and the colon [29]. The distinct roles of these two receptors in the endocrinology of estrogen have been confirmed by the generation of mice whose ER/3 has been genetically disrupted [32]. The phenotype of these mice is different from that of ERa knockout mice [32]. The specific role for ER/3 in tissues in which it is the only estrogen receptor expressed has not yet been identified. The high degree of amino acid homology between ERa and

ERfl within the DNA-binding domain suggests, but does not prove, that these receptors may regulate the same genes. It is also possible that ER/3 may interact with target genes in a manner that does not require direct contact with DNA regulatory elements within target genes. The identification of estrogen and progesterone receptor isoforms and subtypes and the definition of specific functions that they modulate have introduced a new dimension in steroid hormone action. Understanding the regulatory mechanisms that control the expression levels of the individual forms of each receptor is likely to provide novel targets for pharmaceutical intervention.

V. R E G U L A T I O N

OF ESTROGEN

AND PROGESTERONE RECEPTOR FUNCTION

BY LIGANDS

The finding that ERs and PRs could exist in multiple forms within target cells suggests that some of the tissueselective actions of their cognate agonists and antagonists can be explained by their ability to regulate differentially the action of one specific receptor isoform or subtype. Although a specific example of a receptor subtype-selective steroid receptor ligand has not yet emerged, the fact that such ligands for the retinoic acid receptor(s) have been generated makes the discovery of the ER and PR subtype-selective ligands more likely. Regardless, however, it has become apparent from the study of antiestrogens that the identical ligand operating through the same receptor can manifest different biological activities in different target cells [33]. In breast tissue, for instance, where ERa predominates, all of the known antiestrogens oppose the mitogenic actions of estrogen [34]. In the endometrium, however, where ERa also predominates, it has been found that tamoxifen functions as a partial estrogen mimetic [35, 36], whereas compounds such as raloxifene, GW5638, and ICI182,780 function as pure antiestrogens. Thus, the same compounds, acting through ERa, manifest different biological activities in the breast and the endometrium. This finding is not in agreement with the classic models of ER action that indicate that ligands basically fall into two classes, agonists and antagonists. This paradox has been the subject of much investigation leading to the observation that different compounds can induce different alterations in ER structure and that not all structures are functionally identical. It is implied, therefore (discussed in more detail below) that the cell possesses the cellular machinery to distinguish between these dissimilar complexes and that the identification and characterization of the specific components of these systems are the keys to the development of the next generation of tissue-selective ER and PR modulators. Much of what we know about the effect of ligands on ste-

CHAPTER 1 Estrogen and Progesterone Receptors roid receptor structure has come from studies of different ER-ligand complexes. Initially, using differential sensitivity to proteases, it was demonstrated that the hormone-binding domain within the ER adopts different shapes on binding estradiol and tamoxifen, and that these structures are dissimilar to that of the apo receptor [33, 37]. Thus, receptor conformation is affected by the nature of the bound ligand. This relationship between structure and function was later confirmed by the observation that agonists and antagonists induce different alterations in PR structure [38, 39]. Further analysis has revealed that the majority of the structural changes that occur in the PR are located at the extreme carboxyl tail of the receptor, and that removal of the carboxylterminal 42 amino acids of hPR-B permit the antagonist RU486 to function as an agonist [38]. Interestingly, a similarly positioned domain enables the ER to discriminate between different compounds and, not surprisingly, removal of 35 amino acids from the C-terminal tail of the ER abolishes its ability to distinguish between agonists and antagonists [40]. The recent determination of the crystalline structures of the ER-estradiol and ER-tamoxifen complexes confirmed the important role of the carboxyl tail in determining the pharmacology of steroid receptors [7, 41, 42]. This new structural information has also revealed that agonist activation of the ER permits the formation of a unique surface (or pocket) on the receptor that allows it to interact with the general transcription machinery through the mediation of adaptor or coactivator proteins. In the presence of the antagonist tamoxifen, however, the carboxyl tail of the ER is positioned in such a manner that it occludes this coactivator binding pocket, preventing a productive association with the cellular transcription apparatus. In addition to tamoxifen there are several additional SERMs that manifest distinct activities in vivo. One of these compounds, raloxifene, has been approved as a SERM for the treatment of osteoporosis [43]. This compound distinguishes itself from tamoxifen in that it does not exhibit estrogenic action in the postmenopausal endometrium [44, 45]. Although clearly different biologically, the crystal structures of the ER-tamoxifen and ER-raloxifene complexes were shown to be virtually indistinguishable. Although these results appear to be at odds with the hypothesis that links receptor structure to function, some data from our group have reconciled these potential discrepancies. We have used phage display technology to identify small peptides, the ability of which to bind ERs is affected differentially by the nature of the ligand bound to the receptor [46-48]. The rationale behind this approach is that because of the vast complexity of the peptides available in these libraries, it may be possible to find peptides that have the ability to distinguish between two very similar receptorligand complexes. This approach has led to the identification of a series of high-affinity peptide probes that, in addition to

7 being able to distinguish between ER-estradiol and E R tamoxifen complexes, are also able to distinguish among several different E R - S E R M complexes (Fig. 4). This approach has been extended to the study of the PR and it was similarly observed that various PR ligands manifest different

FIGURE 4 Fingerprinting the surfaces of different ER-ligand complexes using conformation-sensitive peptide probes. (A) Random peptide libraries were constructed in an M13 bacteriophage; each of the resulting bacteriophages expressed a unique random peptide on its surface pilus. Screens were subsequently performed to identify specific peptides (bacteriophage) whose interaction with the ER was influenced by the nature of the bound ligand. The bacteriophages identified in this manner were used to develop an enzyme-linked immunoassay to monitor changes that occur in the ER on its interaction with different ligands. Specifically, a biotinylated estrogen response element (ERE) was used to immobilize recombinant ERs on streptavidin-coated plates. After incubation of this complex with the ligand to be tested, to each well was added an aliquot of a different class of ERinteracting bacteriophage. Binding of the bacteriophage was assessed enzymatically using an anti-M 13 antibody coupled to horseradish peroxidase (HRP). (B) Fingerprint analysis of ER conformation in the presence of different ER ligands. Immobilized ER was incubated in the presence of saturating concentrations of the indicated ligands, and the resulting complexes were incubated with aliquots of bacteriophage expressing eight different peptides. Tam, Tamoxifen; DES, diethylstilbestrol; Prog, progesterone. This figure has been published previously in a similar form [47] and is reproduced and presented here with permission (Copyright 1999 National Academy of Sciences, U.S.A.).

8

DONALD P. MCDONNELL

biologies in different cells, allowing the identification of peptide probes whose interaction with the receptor is influenced by the nature of the ligand bound to the PR. All these findings establish a firm relationship between the structure of a receptor-ligand complex and biological activity, and suggest that novel ER and PR ligands with unique pharmaceutical properties may be developed by exploiting this observation.

VI. ESTROGEN AND PROGESTERONE RECEPTOR

ASSOCIATED

PROTEINS

The estrogen and progesterone receptors are liganddependent transcription factors that, on activation by ligands, associate with specific DNA response elements located within the regulatory regions of target genes [ 14]. The DNA-bound receptor can then positively or negatively influence gene transcription by altering RNA polymerase II activity. However, because RNA polymerase does not appear to interact directly with the steroid receptors, there must be additional factors that allow these two proteins to communicate [ 14]. In the past few years it has become clear that there are at least two functional classes of proteins that are involved in recognizing the activated receptor. One class includes components of the basic transcription machinery, the general transcription factors, whose expression levels are generally invariant from cell to cell. The second class of proteins, "cofactors," is not a part of the general transcription machinery, and can exert either a positive or a negative influence on SR transcriptional activity [49]. Those cofactors that interact with agonist-activated SRs have been called coactivators, whereas those that interact with apo receptors or antagonist-activated receptors have been called corepressors. Interestingly, it has become apparent that differences in the relative expression levels of coactivators and corepressors can have a profound effect on the pharmacology of estrogen and progesterone receptor ligands [25, 50, 51].

have revealed that (1) the expression levels of these coactivators vary from cell to cell, (2) coactivators demonstrate specific receptor preferences, (3) a given receptor can interact with more than one type of coactivator, and (4) the conformation of the receptor adopted in the presence of a specific ligand can determine which coactivators are engaged. These findings strongly support the hypothesis that differential cofactor expression is the most important determinant of estrogen and progesterone receptor pharmacology. With the discovery of the nuclear receptor coactivators and the characterization of their biochemical properties has come a new understanding of the mechanism by which differently conformed receptor-ligand complexes are recognized in the cell. The studies that have been performed with ERs are the most informative. As described previously, it has been shown that ERs in the presence of estradiol undergo a conformational change that allows the presentation of surfaces on the receptors, permitting them to interact with coactivators. Because estradiol induces the same conformational change within ERs in all cells, the phenotypic consequence of the exposure of a cell to estradiol will depend on the properties of the coactivators expressed in that cell (Fig. 5). The

A. C o a c t i v a t o r P r o t e i n s One of the most well-characterized coactivator proteins, steroid receptor coactivator 1 (SRC-1), was identified in a yeast two-hybrid screen as a protein that interacted with agonist-activated PR [52]. Subsequently, this protein has been shown to also interact with the estrogen, glucocorticoid, and androgen receptors. It appears that SRC-1 increases target gene transcription by linking the hormone-activated receptor with the general transcription machinery, stabilizing the transcription preinitiation complex, and nucleating a large complex of proteins that together have the ability to acetylate histones and facilitate chromatin decondensation [53, 54]. In addition to SRC-1, over 30 additional coactivators have been identified and characterized [49]. Cumulatively, these studies

FIGURE 5 A molecular explanation for the tissue-selective agonist/antagonist activity of the SERM tamoxifen. The estrogen receptor undergoes different conformational changes on binding the full agonistestrogen or the SERM tamoxifen. The estradiol-induced conformation allowsthe ER to interact with any coactivator protein expressed in target cells, and thus it can activate transcription. The tamoxifen-induced conformational change, on the other hand, is more restrictive and allows the interaction of the ER with only a subset of available coactivators. In those cells in which the tamoxifen-ER complex can engage a coactivator, this compound can manifest agonist activity.In other contexts tamoxifen functions as an antagonist.

CHAPTER1 Estrogen and Progesterone Receptors situation gets more complicated, however, when considering the role of coactivators in mediating the cell-selective action of SERMs such as tamoxifen. It has been shown that the tamoxifen-induced conformational change within the ER does not allow the coactivator binding pocket to form properly, preventing or hindering the interaction of those coactivators that require the coactivator binding pocket in order to interact with the E R [46]. In cells in which this type of coactivator is important, therefore, tamoxifen can function as an antagonist. It is becoming clear, however, that not all coactivators rely on the coactivator binding pocket to the same degree. Thus, the relative agonist-antagonist activity of tamoxifen depends on the ability of the t a m o x i f e n - E R complex to engage a compatible coactivator in target cells [33]. As the repertoire of cofactors increases, we are likely to find that targeting specific c o f a c t o r - r e c e p t o r complexes will yield pharmaceuticals that manifest their activities in a cell- or tissuerestricted manner.

B.

Corepressor

Proteins

Two nuclear corepressor proteins that appear to be important in ER and PR action have thus far been identified. These proteins, N C o R and SMRT, initially found as proteins that interact with D N A - b o u n d thyroid hormone or retinoid X receptors, repress basal transcription in the absence of hormone [55, 56]. However, it has now been shown that these proteins can interact with either PR or ER, either in the absence of h o r m o n e or in the presence of antagonists [50, 51]. Under these conditions, the corepressors nucleate a large protein complex, which represses target gene transcription by deacetylating histones and facilitating chromatin condensation. The physiological importance of corepressors in ER pharmacology was suggested by the studies of Lavinsky and co-workers, who found that passage of breast tumors in mice from a state of tamoxifen sensitivity to an insensitive state was accompanied by a decrease in the expression level of the corepressor [57]. A similar process, if occurring in humans, could explain how cells become resistant to the antiestrogenic actions of tamoxifen.

AN UPDATED M O D E L O F E S T R O G E N AND P R O G E S T E R O N E RECEPTOR ACTION VII.

On ligand binding, the activated receptor (ER or PR) can interact as a dimer with specific D N A response elements within target genes. It is now apparent that the conformation of the resulting receptor is influenced by the nature of the bound ligand and that the shape of the resulting receptor-ligand complex is a critical determinant of whether it can activate transcription. In the presence of a full agonist, the conformation adopted by the receptor facilitates the displacement

9 of corepressor proteins and recruitment of coactivator proteins, permitting the assembly of a histone-acetylating complex on D N A with a concomitant increase in target gene transcription. Pure antagonists, on the other hand, drive their cognate receptor into a conformation that favors corepressor interaction. The activity of mixed agonists/antagonists appears to relate to their ability to alter receptor conformation differentially, and the ability of corepressor and coactivator proteins within a given target cell to recognize these complexes. Clearly, the classic models of estrogen and progesterone action need to be updated to accommodate the insights that have emerged from the study of the genetics and molecular pharmacology of these two receptors.

References 1. Clark, J. H., and Markaverich, B. M. (1988). Actions of ovarian steroid hormones. In "The Physiology of Reproduction," E. Knobil and J. Neill, eds., pp. 675-724. Raven Press, New York. 2. Clarke, C. L., and Sutherland, R. L. (1990). Progestin regulation of cellular proliferation. Endocr. Rev 11, 266-301. 3. Colditz, G. A., Hankinson, S. E., Hunter, D. J., Willett, W. C., Manson, J. E., Stampfer, M. J., Hennekens, C., Rosner, B., and Speizer, E E. (1995). The use of estrogens and progestins and the risk of breast cancer in postmenopausal women. N. Engl. J. Med. 332, 1589-1593. 4. Barzel, U. S. (1988). Estrogens in the prevention and treatment of postmenopausal osteoporosis. Am. J. Med. 85, 847-850. 5. Grodstein, F., Stampfer, M. J., Manson, J. E., Colditz, G. A., Willett, W. C., Rosner, B., Speizer, E E., and Hennekens, C. H. (1996). Postmenopausal estrogen and progestin use and the risk of cardiovascular disease. N. Engl. J. Med. 335, 453-461. 6. McDonnell, D. P., Vegeto, E., and Gleeson, M. A. G. (1993). Nuclear hormone receptors as targets for new drug discovery. Bio/Technology 11, 1256-1260. 7. Brzozowski, A. M., Pike, A. C., Dauter, Z., Hubbard, R. E., Bonn, T., Engstr6m, O., Ohman, L., Greene, G. L., Gustafsson, J. A., and Carlquist, M. (1997). Molecular basis of agonism and antagonism in the oestrogen receptor. Nature (London) 389, 753-758. 8. Williams, S. P., and Sigler, P. B. (1998). Atomic structure of progesterone complexed with its receptor. Nature (London) 393, 392-396. 9. Giangrande, P. H., and McDonnell, D. P. (1999). The A and B isoforms of the human progesterone receptor: Two functionally different transcription factors encoded by a single gene. Recent Prog. Horm. Res. 54, 291-314. 10. Tzukerman, M. T., Esty, A., Santiso-Mere, D., Danielian, P., Parker, M. G., Stein, R. B., Pike, J. W., and McDonnell, D. P. (1994). Human estrogen receptor transactivational capacity is determined by both cellular and promoter context and mediated by two functionally distinct intramolecular regions. Mol. Endocrinol. 8, 21-30. 11. Gronemeyer, H. (1992). Control of transcription activation by steroid hormone. FASEB J. 6, 2524-2529. 12. Kumar, V., Green, S., Stack, G., Berry, M., Jin, J.-R., and Chambon, P. (1987). Functional domains of the human estrogen receptor. Cell (Cambridge, Mass.) 51, 941-951. 13. Schwabe, J. W. R., Chapman, L., Finch, J. T., and Rhodes, D. (1993). The crystal structure of the estrogen receptor DNA-binding domain bound to DNA: How receptors discriminate between their response elements. Cell (Cambridge, Mass.) 75, 567-578. 14. O'Malley, B. W., Tsai, S. Y., Bagchi, M., Weigel, N. L., Schrader, W. T., and Tsai, M.-J. (1991). Molecular mechanism of action of a steroid hormone receptor. Recent Prog. Horm. Res. 47, 1-26.

10 15. Bagchi, M. K., Tsai, S. Y., Tsai, M.-J., and O'Malley, B. W. (1990). Identification of a functional intermediate in receptor activation in progesterone-dependent cell-free transcription. Nature (London) 345, 547-550. 16. Allan, G. E, Tsai, S. Y., Tsai, M.-J., and O'Malley, B. W. (1992). Ligand-dependent conformational changes in the progesterone receptor are necessary for events that follow DNA binding. Proc. Natl. Acad. Sci. US.A. 89, 11750-11754. 17. Bagchi, M. K., Elliston, J. F., Tsai, S. Y., Edwards, D. P., Tsai, M.-J., and O' Malley, B. W. (1988). Steroid hormone-dependent interaction of human progesterone receptor with its target enhancer element. Mol. Endocrinol. 2, 1221-1229. 18. Clark, J. H., and Peck, E. J. (1979). "Female Sex Steroids: Receptors and Function," 1st ed., Vol. 14. Springer-Verlag, New York. 19. Jordan, V. C., ed. (1996). "Tamoxifen: A Guide for Clinicians and Patients." PRR, Huntington, N.Y. 20. Fisher, B., Costantino, J. P., Wickerham, D. L., Redmond, C. K., Kavanah, M., Cronin, W. M., Vogel, V., Robidoux, A., Dimitrov, N., Atkins, J., Daly, M., Wieand, S., Tan-Chiu, E., Ford, I., and Wolmark, N. (1998). Tamoxifen for prevention of breast cancer: Report of the National Surgical Adjuvant Breast and Bowel Project P-1 Study. J. Natl. Cancer Inst. 90, 1371-1388. 21. Love, R. R., Mazess, R. B., Barden, H. S., Epstein, S., Newcomb, P. A., Jordan, V. C., Carbone, P. P., and DeMets, D. L. (1992). Effects of tamoxifen on bone mineral density in postmenopausal women with breast cancer. N. Engl. J. Med. 326, 852-856. 22. Love, R. R., Wiebe, D. A., Newcombe, P. A., Cameron, L., Levanthal, H., Jordan, V. C., Feyzi, J., and DeMets, D. L. (1991). Effects of tamoxifen on cardiovascular risk factors in postmenopausal women. Ann. Intern. Med. 115, 860-864. 23. Lessey, B. A., Alexander, P. S., and Horwitz, K. B. (1983). The subunit characterization of human breast cancer progesterone receptors: Characterization by chromatography and photoaffinity labelling. Endocrinology (Baltimore) 112, 1267-1274. 24. Vegeto, E., Shahbaz, M. M., Wen, D. X., Goldman, M. E., O'Malley, B. W., and McDonnell, D. P. (1993). Human progesterone receptor A form is a cell- and promoter-specific repressor of human progesterone receptor B function. Mol. Endocrinol. 7, ! 244-1255. 25. Jackson, T. A., Richer, J. K., Bain, D. L., Takimoto, G. S., Tung, L., and Horwitz, K. B. (1997). The partial agonist activity of antagonistoccupied steroid receptors is controlled by a novel hinge domainbinding coactivator L7/SPA and the corepressors NCoR or SMRT. Mol. Endocrinol. 11, 693-705. 26. Conneely, O. M., Dobson, A. D. W., Huckaby, C., Carson, M. A., Maxwell, B. L., Toft, D. O., Tsai, M.-J., Schrader, W. T., and O' Malley, B. W. (1988). Structure-function relationships of the chicken progesterone receptor. In "Steroid Hormone Action," G. Ringold, ed., pp. 227-236. Alan R. Liss, New York. 27. Wen, D. X., Xu, Y.-E, Mais, D. E., Goldman, M. E., and McDonnell, D. P. (1994). The A and B isoforms of the human progesterone receptor operate through distinct signaling pathways within target cells. Mol. Cell. Biol. 14, 8356-8364. 28. Kuiper, G. G. J. M., Enmark, E., Pelto-Huikko, M., Nilsson, S., and Gustafsson, J.-A. (1996). Cloning of a novel estrogen receptor expressed in rat prostate and ovary. Proc. Natl. Acad. Sci. U.S.A. 93, 5925 -5930. 29. Kuiper, G. G. J. M., Carlsson, B., Grandien, K., Enmark, E., H~iggblad, J., Nilsson, S., and Gustafsson, J.-A. (1997). Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors ce and ft. Endocrinology (Baltimore) 138, 863-870. 30. Shughrue, P. J., Komm, B., and Merchenthaler, I. (1996). The distribution of estrogen receptor-fl mRNA in the rat hypothalamus. Steroids 61,678-681. 31. Shughrue, P. J., Lane, M. V., and Merchenthaler, I. (1997). Comparative

DONALD P. MCDONNELL

32. 33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

distribution of estrogen receptor-ce and fi mRNA in the rat central nervous system. J. Comp. Neurol. 388, 507-525. Couse, J. F., and Korach, K. S. (1999). Estrogen receptor null mice: What have we learned and where will they lead us? Endocr. Rev. 20, 358-417. McDonnell, D. P., Clemm, D. L., Hermann, T., Goldman, M. E., and Pike, J. W. (1995). Analysis of estrogen receptor function in vitro reveals three distinct classes of antiestrogens. Mol. Endocrinol. 9, 659-668. Jordan, V. C. (1992). The strategic use of antiestrogen to control the development and growth of breast cancer. Cancer (Philadelphia) 70, 977-982. Kilackey, M. A., Hakes, T. B., and Pierce, V. K. (1985). Endometrial adenocarcinoma in breast cancer patients receiving antiestrogens. Cancer Treat. Rep. 69, 237-238. Gottardis, M. M., Robinson, S. P., Satyaswaroop, P. G., and Jordan, V. C. (1988). Contrasting actions of tamoxifen on endometrial and breast tumor growth in the athymic mouse. Cancer Res. 48, 812-815. Allan, G. F., Leng, X., Tsai, S. Y., Weigel, N. L., Edwards, D. P., Tsai, M.-J., and O'Malley, B. W. (1992). Hormone and antihormone induce distinct conformational changes which are central to steroid receptor activation. J. Biol. Chem. 267, 19513-19520. Vegeto, E., Allan, G. F., Schrader, W. T., Tsai, M.-J., McDonnell, D. P., and O'Malley, B. W. (1992). The mechanism of RU486 antagonism is dependent on the conformation of the carboxy-terminal tail of the human progesterone receptor. Cell (Cambridge, Mass.) 69, 703-713. Wagner, B. L., Pollio, G., Leonhardt, S., Wani, M. C., Lee, D. Y.-W., Imhof, M. O., Edwards, D. P., Cook, C. E., and McDonnell, D. P. (1996). 16a-Substituted anologs of the antiprogestin RU486 induce a unique conformation in the human progesterone receptor resulting in mixed agonist activity. Proc. Natl. Acad. Sci. U.S.A. 93, 87398744. Mahfoudi, A., Roulet, E., Dauvois, S., Parker, M. G., and Wahli, W. (1995). Specific mutations in the estrogen receptor change the properties of antiestrogens to full agonists. Proc. Natl. Acad. Sci. U.S.A. 92, 4206-4210. Tanenbaum, D. M., Wang, Y., Williams, S. P., and Sigler, E B. (1998). Crystallographic comparison of the estrogen and progesterone receptor's ligand binding domains. Proc. Natl. Acad. Sci. U.S.A. 95, 59986003. Shiau, A. K., Barstad, D., Loria, P. M., Cheng, L., Kushner, E J., Agard, D. A., and Greene, G. L. (1998). The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen. Cell (Cambridge, Mass.) 95, 927-937. Delmas, P. D., Bjarnason, N. H., Mitlak, B. H., Ravoux, A. C., Shah, A. S., Huster, W. J., Draper, M., and Christiansen, C. (1997). Effects of raloxifene on bone mineral density, serum cholesterol concentrations, and uterine endometrium in postmenopausal women. N. Engl. J. Med. 337, 1641 - 1647. Sato, M., Rippy, M. K., and Bryant, H. U. (1996). Raloxifene, tamoxifen, nafoxidine, or estrogen effects on reproductive and nonreproductive tissues in ovariectomized rats. FASEB J. 10, 905-912. Ashby, J., Odum, and Foster, J. R. (1997). Activity of raloxifene in immature and ovariectomized rat uterotrophic assays. Regul. Toxicol. Pharmacol. 25, 226-231. Norris, J. D., Paige, L. A., Christensen, D. J., Chang, C.-Y., Huacani, M. R., Fan, D., Hamilton, P. T., Fowlkes, D. M., and McDonnell, D. E (1999). Peptide antagonists of the human estrogen receptor. Science 285, 744-746. Paige, L. A., Christensen, D. J., Gron, H., Norris, J. D., Gottlin, E. B., Padilla, K. M., Chang, C.-Y., Ballas, L. M., Hamilton, E T., and McDonnell, D. E (1999). Estrogen receptor (ER) modulators each induce distinct conformational changes in ERa and ERfl. Proc. Natl. Acad. Sci. U.S.A. 96, 3999-4004.

11

CHAPTER 1 Estrogen and Progesterone Receptors 48. Wijayaratne, A. L., Nagel, S. C., Paige, L. A., Christensen, D. J., Norris, J. D., Fowlkes, D. M., and McDonnell, D. P. (1999). Comparative analyses of the mechanistic differences among antiestrogens. Endocrinology 140, 5828-5840. 49. Horwitz, K. B., Jackson, T. A., Bain, D. L., Richer, J. K., Takimoto, G. S., and Tung, L. (1996). Nuclear receptor coactivators and corepressors. Mol. Endocrinol. 10, 1167-1177. 50. Smith, C. L., Nawaz, Z., and O'Malley, B. W. (1997). Coactivator and corepressor regulation of the agonist/antagonist activity of the mixed antiestrogen, 4-hydroxytamoxifen. Mol. Endocrinol. 11, 657- 666. 51. Wagner, B. L., Norris, J. D., Knotts, T. A., Weigel, N. L., and McDonnell, D. P. (1998). The nuclear corepressors NCoR and SMRT are key regulators of both ligand- and 8-bromo-cyclic AMP-dependent transcriptional activity of the human progesterone receptor. Mol. Cell. Biol. 18, 1369-1378. 52. Onate, S. A., Tsai, S., Tsai, M.-J., and O'Malley, B. W. (1995). Sequence and characterization of a coactivator for the steroid hormone receptor superfamily. Science 270, 1354-1357. 53. Spencer, T. E., Jenster, G., Burcin, M. M., Allis, C. D., Zhou, J., Mizzen, C. A., McKenna, N. J., Onate, S. A., Tsai, S. Y., Tsai, M.-J., and

54.

55.

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57.

O'Malley, B. W. (1997). Steroid receptor coactivator- 1 is a histone acetyltransferase. Nature (London) 389, 194-198. Shibata, H., Spencer, T. E., Onate, S. A., Jenster, G., Tsai, S. Y., Tsai, M.-J., and O'Malley, B. W. (1997). Role of co-activators and corepressors in the mechanism of steroid/thyroid receptor action. Recent Prog. Horm. Res. 52, 141-165. H6rlein, A. J., N~iar, A. M., Heinzel, T., Torchia, J., Gloss, B., Kurokawa, R., Ryan, A., Kamei, Y., S6derstr6m, M., Glass, C. K., and Rosenfeld, M. G. (1995). Ligand-independent repression by the thyroid hormone receptor mediated by a nuclear receptor co-repressor. Nature (London) 377, 397-403. Chen, J. D., and Evans, R. M. (1995). A transcriptional co-repressor that interacts with nuclear hormone receptors. Nature (London) 377, 454-457. Lavinsky, R. M., Jepsen, K., Heinzel, T., Torchia, J., Mullen, T.-M., Schiff, R., Del-Rio, A. L., Ricote, M., Ngo, S., Gemsch, J., Hilsenbeck, S. G., Osborne, C. K., Glass, C. K., Rosenfeld, M. G., and Rose, D. W. (1998). Diverse signaling pathways modulate nuclear receptor recruitment of N-CoR and SMRT complexes. Proc. Natl. Acad. Sci. U.S.A. 95, 2920-2925.

~HAPTER

Ovarian Anatomy and Physiology GREGORY F.

ERICKSON

Department of Obstetrics and Gynecology, University of California, San Diego, La Jolla, California 92093

VI. Accelerated Loss in OR: Activin Hypothesis VII. New Data on the Effects of Activin VIII. Statement of Conclusion References

I. Introduction II. Statement of the P r o b l e m

III. The Primordial Follicle IV. The Adult Ovary V. Ovary Reserve

I. INTRODUCTION

II. STATEMENT OF THE P R O B L E M

A characteristic feature of reproductive capacity in women is its cyclical nature, a feature that is strikingly reflected by the growth and development of oocytes and follicles within the ovaries [1]. Typically, the tissues of the adult ovaries undergo dramatic cyclical changes, which in turn are reflected in cyclical changes in the production of the steroid hormones, estradiol (E 2) and progesterone (p4), as well as key regulatory proteins, such as inhibin. The regulated expression of these ligands is critically important in the mechanisms that underlie fertility in women, including the ovulation of an oocyte with the potential for producing a normal embryo. The process begins at puberty with the first cycle and ends at the menopause with the permanent cessation of menses. The causative event in the menopause is the disappearance of primordial follicles in the ovaries, i.e., the loss of ovary reserve (OR). Consequently, the size of a woman's OR (number of primordial follicles) has great impact on when the menopause begins. Therefore, to understand the menopause from either a physiological or a pathophysiological perspective, we must understand the interactions between OR, folliculogenesis, and the menstrual cycle during aging. This chapter reviews what is currently known about these relationships. MENOPAUSE: BIOLOGY AND PATHOBIOLOGY

Today, the menopause occurs in most women at --~51 years of age. Demographic studies have demonstrated that the mean life expectancy of women in developed countries [1] has increased from - 4 5 years in 1850 to - 8 2 years in 1998 (Fig. 1) [ l a]. This is an important observation because it indicates that most women today will live one-third to onehalf of their lives postmenopausally, i.e., they will live - 30 years after the menopause. Clinicians can therefore expect to extend care increasingly to large numbers of women of advanced reproductive age in whom ovarian dysfunction will be a major cause of infertility and morbidity. If one considers that the vast majority of fertility and gynecologic problems in the aging woman are a direct consequence of the age-related decrease in ovary reserve, it becomes apparent that the disappearance of primordial follicles is one of the critical events in the life of all women. One female function most adversely affected by the agerelated decrease in OR is decreased fecundity. The basis for this age-related change is the failure of dominant follicles to release eggs that can undergo normal embryonic development [2-5]. Results from in vitro fertilization (IVF) [6,7] have demonstrated that this decrease becomes particularly evident in patients after 36 years of age, when pregnancy 13

Copyright 9 2000 by Academic Press. All rights of reproduction in any form reserved.

1

4

G

R

E

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FIGURE 1 Changesin the life expectancy and age of the menopause in women over the past 150 years. From Nachtigall [la], with permission.

rates with self-produced oocytes decline sharply by --~65%, i.e., from ---25% per transfer in women ~<30 years of age to --~9% per transfer in women after the age of 36 years [7]. A similar age-related decrease in female fecundity has been found using artificial insemination with donor semen (AID) [8] and gamete intrafallopian transfer (GIFT) [9]. The low fecundity rate continues through --~44 years, after which viable pregnancies almost never occur [9-11 ]. These facts, together with the delay in childbearing by women in developed countries, have set the stage for an increase in reproductive problems and disorders attributable to female aging, in particular infertility. An important point is that women between 36 and 44 years of age can exhibit regular menstrual cycles [12]. This is important because it argues that the decrease in fecundity in these older women is not the result of the failure of the aged ovaries to produce dominant follicles, presumably the cyclical activity reflects the ability of these dominant follicles to undergo the physiological changes that typically occur during selection, ovulation, luteinization, and luteolysis. One of the main lines of evidence in support of this theory is that nearly normal quantities of androgen [ 13], estrogen, and progesterone [14,15] appear to be secreted from the aged dominant follicles during the menstrual cycle. This evidence supports the idea that the dominant follicles that develop in these older women are fully capable of expressing a nearly normal pattern of steroidogenic activity. By contrast, studies of oocytes from aged dominant follicles have demonstrated the existence of alterations that contribute negatively to pregnancy. For example, investigators have found that aneuploidy increases significantly in embryos that develop from oocytes isolated from the mature follicles of women >35 years of age [16]. Thus, one is led to the conclusion that (1) the endocrine and gametogenic function of dominant follicles can become dissociated in women after --~36 years of age and (2) the aberrant expression of cellular responses in the egg would appear to be the basis for the age-related decrease in fecundity. Understanding how the developmental potential of the

O

R

Y

F. ERICKSON

aged oocyte is altered independently of changes in the granulosa and theca cells is a fundamental question in ovary research. Although relatively little is known about this problem, an interesting role for ovary reserve (the number of primordial follicles) has been suggested from analysis of folliclestimulating hormone (FSH) and inhibin levels and pregnancy rates. An important point to emerge from the clinical studies is that the number of follicles within the ovaries, not oocyte age, is the main determinant predicting pregnancy in older women [9]. That is, the incidence of pregnancy with self-produced oocytes is highest in older women with adequate OR, i.e., those with no significant elevation in plasma FSH levels and whose ovaries contain a comparatively large number of mature Graafian follicles [6,13,17]. Given this relationship, it is not unreasonable to propose that the selective deteriorative changes that occur in aging oocytes are either correlated with or causally connected to a significant decrease in OR, rather than aging itself. There is a fundamental question: How does this occur?

IIl. THE PRIMORDIAL

FOLLICLE

Before addressing this question, we must understand some basic biology of the primordial follicles or OR. The primordial follicles represent a pool of nongrowing follicles from which all dominant preovulatory follicles are selected [1]. Thus, primordial follicles are, in a real sense, the fundamental reproductive units of the ovary. Morphologically, each primordial follicle is composed of an outer single layer of squamous epithelial cells that are termed granulosa or follicle cells, and a small (approximately 15/xm in diameter) immature oocyte arrested in the dictyotene stage of meiosis; both cell types are enveloped by a thin, delicate membrane called the basal lamina (Fig. 2). By virtue of the basal lamina, the granulosa and the oocyte exist in a microenvironment in which direct contact with other cells does not occur. Although small capillaries are occasionally observed in proximity to primordial follicles, these follicles do not have an independent blood supply [1]. The mean diameter of a nongrowing primordial follicle is 29/xm [18]. All the primordial follicles present in a woman's ovaries are formed before birth. Developmentally, the primordial follicles are formed in the cortical cords of the fetal ovaries between the sixth and ninth months of gestation [1]. During this period, all the germ cells are stimulated to initiate meiosis. Because the oocytes in the primordial follicles have entered meiosis, all oocytes that are capable of participating in reproduction during a woman's life are formed at birth, i.e., the human ovaries acquire a lifetime quota of eggs before birth. Soon after primordial follicle formation, some are recruited (activated) to initiate growth. As successive recruitment proceeds over time, the size of the pool of primordial follicles becomes progressively smaller (Fig. 3) [ 18a]. Between the times of birth and menarche the number

CHAPTER2 Ovarian Anatomy and Physiology

15 of primordial follicles (and thus oocytes) decreases from several million to several hundred thousand (Fig. 3). As a woman ages, the number of primordial follicles (OR) continues to decline, until at menopause they are difficult to find (Fig. 4) [ 18b].

IV. THE ADULT OVARY

FIGURE 2 Electron micrograph of a human primordial follicle showing oocyte nucleus (N), Balbiani body (.), and granulosa or follicle cells (arrowheads). From [ 1], Erickson, G. F. (1995). The Ovary: Basic Principles and Concepts. In "Endocrinology and Metabolism" (Felig, E, Baxter, J. D., Broadus, A. E., Frohman, L. A., Eds.), 3rd ed., pp. 973-1015. McGrawHill, with permission.

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FIGURE 3 Changes in the total number of oocytes in human ovaries during aging. In the embryo at early to midgestation, the number of oocytes increases to almost 7 million. Shortly thereafter, the number falls sharply to about 2 million at birth. The enormous loss (--~70%) of oocytes in the embryo between 6 and 9 months is caused by apoptosis. The store of eggs continues to diminish with age until no oocytes are detected in the ovaries at about 50 years of age. From [18a], Baker, T. G. (1971). Radiosensitivity of mammalian oocytes with particular reference to the human female. Am. J. Obstet. Gynecol. 110, 746-761, with permission.

In this section, we will deal with the anatomy and physiology of folliculogenesis as it occurs in normal women during the reproductive years. We will focus our attention on the manner in which the developmental program is expressed in a recruited primordial follicle as it develops to the ovulatory stage or dies by atresia. An underlying principle of the human ovaries is that of the 2 million primordial follicles, only 400 of so will complete their development and undergo ovulation and corpus luteum formation; all others (99.9%) will die by atresia after recruitment (Fig. 5) [ 18c]. Therefore, the very essence of folliculogenesis is selection.

A. Anatomy The adult human ovary is a mass of follicles, luteal tissue, blood vessels, nerves, and connective tissue elements, all of which form a relatively heterogeneous assemblance of histological units. It is the continuous and progressive change in the follicles and corpora lutea that gives rise to the cyclical changes in the menstrual cycle. During the reproductive years, the normal human ovaries are oval-shaped bodies that each measure 2.5-5.0 cm in length, 1.5-3 cm in width, and 0.6-1.5 cm in thickness [1]. The medial edge of the ovary is attached by the mesovarium to the broad ligament, which extends from the uterus laterally to the wall of the pelvic cavity. The surface of the ovary is covered by an epithelial layer of cuboidal cells resting on a basement membrane. This layer, termed the germinal or serous epithelium, is continuous with the peritoneum. Underlying the serous epithelium is a layer of dense connective tissue termed the tunica albuginea. The ovary is organized into two principal parts: a central zone, the medulla, which is surrounded by a particularly prominent peripheral zone, the cortex (Fig. 6). Embedded in the connective tissue of the cortex are the follicles containing the female gametes, oocytes. The number and size of the follicles vary depending on the age and reproductive state of the female. The existence of follicles of different sizes (primordial, primary, secondary, tertiary, Graafian, and atretic)reflects specific changes associated with their growth, development, and fate. At the end of the follicular phase of the menstrual cycle, the Graafian follicle that reaches maturity secretes its ovum into the peritoneal cavity (Fig. 6). After ovulation, the wall of the ovulating follicle develops into an endocrine structure, the corpus luteum. If implantation does

16

GREGORY F. ERICKSON

FIGURE 4 Photomicrographs of the cortex of human ovaries from birth to 50 years of age. Arrowheads indicate small, nongrowing primordial follicles with a single layer of squamous granulosa cells. From Erickson [ 18b], with permission.

not occur, the corpus l u t e u m deteriorates and eventually bec o m e s a n o d u l e of dense connective tissue t e r m e d the c o r p u s a l b i c a n s . O t h e r cells in the cortex are the s t e r o i d o g e n i c cells t e r m e d s e c o n d a r y interstitial cells. T h e s e cells, which are derived f r o m the theca c o m p a r t m e n t of atretic follicles, are found in nests or cords t h r o u g h o u t the life of the female. At the m e d i a l b o r d e r of the cortex is a mass of loose connective tissue, the medulla. This tissue contains a n e t w o r k of

c o n v o l u t e d b l o o d vessels and associated nerves that pass through the connective tissue toward the cortex (Fig. 6). A distinct group of t e s t o s t e r o n e - p r o d u c i n g cells, the hilus cells, lie in the m e d u l l a at the hilum of the ovary [19]. The arterial supply to the ovary originates from two prin-

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FIGURE 5 Folliculogenesis is a highly selective process. Of the 2 million primordial follicles at birth, only 400 or so are brought to ovulation and luteinization by FSH and LH. From [18c], Soules, M. R., and Bremmer, W. J. (1982). The menopause and climacteric: Endocrinologic basis and associated symptomatology. J. Am. Geriatr. Soc. 30, 547-561, with permission.

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FIGURE 6 Diagram summarizing the anatomy and histology of the human ovary during the reproductive years. Development of the follicles and

corpus luteum occurs within the cortex, whereas the spiral arteries, hilus cells, and autonomic nerves are located in the medulla. From [1], Erickson, G. F. (1995). The Ovary: Basic Principles and Concepts. In "Endocrinology and Metabolism" (Felig, P., Baxter, J. D., Broadus, A. E., Frohman, L. A., Eds.), 3rd ed., pp. 973-1015. McGraw-Hill, with permission.

CHAPTER2 Ovarian Anatomy and Physiology cipal sources: one, the ovarian artery, arises from the abdominal aorta; the other is derived from the uterine artery [ 1]. These two vessels, which enter the mesovarium from opposite directions, form an anastomotic trunk and become a common vessel called the r a m u s o v a r i c u s artery. At frequent intervals this artery gives rise to a series of primary branches, which enter the hilum like teeth on a rake. In the hilum, numerous secondary and tertiary branches are given off to supply the medulla (Fig. 6).

17 THECA INTERNA

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B. P h y s i o l o g y Typically, the human ovaries produce a single dominant follicle that secretes into the oviduct a mature egg that is ready to be fertilized at the end of the follicular phase of the menstrual cycle. Each dominant follicle begins with the recruitment of a primordial follicle into the pool of growing follicles. It is not known exactly how recruitment occurs, but it appears to be controlled by one or more local ovarian regulatory factors by autocrine/paracrine mechanisms. In a broad sense, all growing follicles can be divided into two groups, healthy and atretic, according to whether apoptosis (programmed cell death) is present in granulosa cells [20,21 ]. As a consequence of successive recruitments, the ovaries appear to always contain a pool of small Graafian follicles from which a prospective dominant follicle can be selected. Once selected, a dominant follicle typically grows and develops to the preovulatory state. Those follicles that are not selected become committed to die by atresia. 1. ENDOCRINOLOGY OF FOLLICULOGENESIS

Regardless of age, follicle growth and development are brought about by the combined action of FSH and luteinizing hormone (LH) on the follicle cells. FSH and LH bind to specific high-affinity receptors on the membranes of the granulosa and theca interstitial cells, respectively. The interaction of these ligands with their receptors activates signal transduction pathways that stimulate mitosis and differentiation responses in the granulosa and theca cells [22,23]. Physiologically, these signaling pathways act in parallel to regulate the expression of specific genes in a precise quantitative and temporal fashion. There are two major endocrine responses associated with folliculogenesis. The first is that the actions of FSH and LH stimulate the production of large amounts of estradiol by the dominant follicle. This important gonadotropin-dependent response is called the twogonadotropin/two-cell concept Fig. 7) [23a]. Because the estradiol response appears to be specific to a dominant follicle, the levels of plasma estradiol can be used as a marker for the status of the dominant follicle. The second endocrine response is the marked increase in the production of inhibin by FSH [24]. With respect to aging, observations support the possibility that localized changes in inhibin production may

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play a role in the accelerated loss of OR. We will return to this issue later. 2. CHRONOLOGY

In women, folliculogenesis is a very long process [22]. In each menstrual cycle, the dominant follicle that is selected to ovulate originates from a primordial follicle that was recruited to grow about 1 year earlier. Whatever the course of development or the final destiny (atresia or ovulation), all follicles undergo various progressive changes (Fig. 8). The very early stages of folliculogenesis (class 1, primary and secondary; class 2, early tertiary) proceed very slowly. Consequently, it requires -->300 days for a recruited primordial follicle to complete the preantral or hormone-independent period. The basis for the slow growth is the very long doubling time (---250 hr) of the granulosa cells. When follicular fluid begins to accumulate at the class 2 stage, the Graafian follicle begins to expand relatively rapidly (Fig. 8). As the antral (hormone-dependent) period proceeds, the Graafian follicle passes through the small, (classes 3, 4, and 5), medium (classes 6 and 7), and large (class 8) stages. A dominant follicle that survives to the ovulatory stage requires about 4 0 - 5 0 days to complete the whole antral period. Selection of the dominant follicle is one of the last steps in

18

GREGORYF. ERICKSON

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FIGURE 8 The chronology of folliculogenesis in the human ovary. Folliculogenesis is divided into two major periods, preantral (gonadotropin independent) and antral (FSH dependent). In the preantral period, a recruited primordial follicle develops to the primary/secondary (class 1) and early tertiary (class 2) stages, at which time cavitation or antrum formation begins. The antral period includes the small Graafian (0.9-5 mm, classes 4 and 5), medium Graafian 6 - 1 0 mm, class 6), large Graafian (10-15 mm, class 7), and preovulatory (16-20 mm, class 8) follicles. Time required for completion of preantral and antral periods is ---300 and ---40 days, respectively. Number of granulosa cells (gc), follicle diameter (mm), and atresia (%) are indicated. From Gougeon [24a], with permission.

the long process of folliculogenesis. The dominant follicle, which is selected from a cohort of class 5 follicles, requires - 2 0 days to develop to the stage wherein it undergoes ovulation. Those follicles that are not selected become atretic. Atresia can occur at each stage of graafian follicle development, but atresia appears most prominent in follicles at the class 5 stage (Fig. 8) [24a]. 3. SELECTION The dominant follicle that will ovulate its egg in the next cycle is selected from a cohort of healthy, small Graafian follicles (4.7 + 0.7 mm in diameter) at the end of the luteal

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phase of the menstrual cycle [22]. Morphologically, each cohort follicle contains a fully grown egg, about 1 million granulosa cells, a theca interna containing several layers of theca interstitial cells, and a band of smooth muscle cells in the theca externa (Fig. 9) [24b]. Selection is a quintessential aspect of ovary physiology. It is characterized by a high sustained rate of granulosa mitosis. Shortly after the midluteal phase, the granulosa cells in all the cohort class 4 and class 5 follicles show a sharp increase (---twofold) in the rate of granulosa mitosis [22]. The first indication that the prospective dominant follicle has been selected is that the granulosa cells of the chosen follicle continue dividing at a fast rate while proliferation slows in the nondominant cohort follicles. Because this distinguishing event is seen at the late luteal phase, it has been concluded that selection occurs at this point in the cycle. As mitosis and follicular fluid accumulation continue (Fig. 8), the dominant follicle grows rapidly during the follicular phase, reaching 6.9 + 0.5 mm at days 1-5, 13.7 + 1.2 mm at days 6-10, and 18.8 +_ 0.5 mm at days 11-14. In nondominant follicles, growth and expansion proceed more slowly, and with time, atresia becomes increasingly more evident (Fig. 8). Rarely does an atretic follicle reach ->9 mm in diameter, regardless of the stage in the cycle. FSH is obligatory for follicle selection, and no other ligand by itself can serve in this regulatory capacity. Physiologically, the mechanism of selection is causally connected to the secondary rise in plasma FSH (the primary FSH rise being the midcycle preovulatory surge of FSH and LH). The secondary rise in plasma FSH begins a few days before the progesterone and estradiol concentrations reach baseline values at the end of luteal phase, and it continues through the first week of the follicular phase (Fig. 10) [24c]. The importance of the secondary rise in FSH is demonstrated by the fact that the dominant follicle will undergo atresia if the FSH levels are decreased. Consequently, the secondary rise in FSH is obligatory for the selection of a dominant follicle that will ovulate in the next cycle. One of the major conse-

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FIGURE 9 Diagrammatic representation of the histologic architecture of a Graafian follicle. Reprinted from [24b], Mol. Cell. Endocrinol. 29, G. F. Erickson. Primary cultures of ovarian cells in serum-free medium as models of hormone-dependent differentiation, 2149. Copyright 1983 with permission from Elsevier Science.

CHAPTER2 Ovarian Anatomy and Physiology

19

quences of the secondary FSH rise is that a critical threshold level of FSH accumulates in the follicular fluid of the chosen follicle. In normal class 5 to class 8 follicles, the mean concentration of follicular fluid FSH increases from --~1.3 mIU/ ml (--~58 ng/ml) to --~3.2 mIU/ml (--~143 ng/ml) through the follicular phase [25]. In contrast, FSH concentrations are low or undetectable in the microenvironment of the nondominant cohort follicles. Thus, the selection and the continued growth of a dominant follicle involve a progressive increase in the concentration of FSH within its microenvironment. Once activated, the dominant follicle becomes dependent on FSH for its survival. The regulation of FSH levels in follicular fluid is totally obscure. FSH triggers a marked activation of mitosis and differentiation of the granulosa cells, which in turn is reflected in a progressive increase in estradiol and inhibin synthesis and follicular fluid accumulation (Fig. 10). One of the effects of the increased estradiol and inhibin production is that the secondary rise in FSH is suppressed (Fig. 10). When this oc-

curs, the concentration of FSH falls below threshold levels and the development of the nondominant follicles stops. It is noteworthy that mitosis in these atretic follicles can be markedly stimulated by treatment with human menopausal gonadotropin (hMG) during the early follicular phase. Thus, if FSH levels within the microenvironment are increased, the nondominant follicle could perhaps be rescued from atresia.

C. The Role of FSH The major FSH-dependent changes that occur during the development of the dominant follicle are summarized in Fig. 11 25a]. The granulosa cells are the only cell types known to express FSH receptors. It follows, therefore, that FSH-mediated effects in the dominant follicle are at the levels of the granulosa cells. In dominant follicles, the FSHinduced differentiation of the granulosa cells involves three major responses, increased steroidogenic potential, mitosis, and LH receptors. 1. S T E R O I D O G E N I C P O T E N T I A L

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20

GREGORY F. ERICKSON

bind to upstream DNA regulatory elements called AMP response elements (CREs), whereby they regulate gene transcription. In this regard the FSH signal mechanisms stimulate the expression of specific genes that control the level of estradiol production by the granulosa cells [26]. The major steroidogenic genes induced by FSH include those for P450 aromatase (P450arom) and 17fl-hydroxysteroid dehydrogenase (17fl-HSD) (Fig. 11). The temporal pattern of expression of these genes has an important role in generating the normal pattern of estradiol production by the dominant follicle during the follicular phase of the cycle (Fig. 10). FSH also acts on the granulosa cells to increase their potential for luteinization as reflected by in vitro experiments with cultured granulosa cells from human follicles at different stages of development [27]. The mechanisms by which this progesterone potential remains suppressed during folliculogenesis in vivo remains unknown, but a putative FSH-dependent luteinizing inhibitor has been proposed [27].

3. INDUCTION OF L H RECEPTOR

The ability of LH and human chorionic gonadotropin (hCG) to activate the ovulatory cascade in the dominant follicle is dependent on the expression of a large number of LH receptors on the granulosa cells [1]. Studies have clearly demonstrated an obligatory role of FSH in the induction of LH receptor (Fig. 11). A key feature of LH receptor expression in the granulosa layer is that it is suppressed throughout most of folliculogenesis. That is, the number of LH receptors remains low in granulosa cells during the early and intermediate stages of dominant follicle growth, but then increases sharply to very high levels at the preovulatory stage. The acquisition of LH receptors implies that when the LH ligand enters the microenvironment of the dominant follicle in the late follicular phase, it can act on the granulosa cells to regulate their function, perhaps even replacing FSH as the principal regulator of granulosa cytodifferentiation.

2. MITOSIS

The granulosa cells in the dominant follicle have the ability to divide at a relatively rapid rate throughout the follicular phase of the cycle, increasing from about 1 • 106 cells at selection to over 50 • 106 cells at ovulation [22,23]. Despite its overall importance to ovarian physiology, it remains unclear how granulosa proliferation is controlled. There is evidence in humans that FSH stimulates the rate of granulosa cell division in vivo and in vitro (Fig. 11), but the mechanism by which FSH stimulates mitosis is not understood.

D. T h e R o l e o f L H There are two hormones, LH and insulin, that regulate steroidogenesis in the interstitial tissue, and both function as stimulators of androgen production [28]. Each hormone interacts with a transmembrane receptor and the binding event is transduced into an intracellular signal that stimulates transcription and translation of specific steroidal genes (Fig. 12). Throughout the life of a woman, LH acts as a critical positive regulator of ovary androgen biosynthesis [28]. The L H -

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FIGURE 12 Diagramof the LH and insulin signal transduction pathways in ovarian interstitial cells leading to increased androstenedione production. Redrawn from Erickson [28], with permission.

CHAPTER2 Ovarian Anatomy and Physiology receptor interactions in the interstitial cells are critically important in estradiol production by virtue of their ability to promote the production of androstenedione, the P450arom substrate (Fig. 7). The action of the LH-receptor signaling pathway in the ovary interstitial cells results in the expression of a battery of genes leading to increased androgen synthesis (Fig. 12). The role of LH in stimulating androgen production has been intensively studied in women because of its involvement in infertility and hyperandrogenism, such as in polycystic ovary syndrome [29,30]. There are four families of interstitial cells in the human ovaries (Fig. 6), the theca interstitial cells (TICs), secondary interstitial cells (SICs), theca lutein cells (TLCs), and hilus cells (HCs). The TICs, SICs, and TLCs are related to each other by a developmental sequence occurring during folliculogenesis and luteogenesis, a process called thecogenesis [31]. The formation of the TICs, SICs, and TLCs involves a developmental; process that encompasses both proliferation and differentiation [28,32]. Because thecogenesis is accompanied by mitosis, it contributes to total interstitial mass and therefore total androgen potential. LH promotes androgen synthesis through activation of the LH/hCG receptor/AMP-dependent protein kinase A signal transduction pathway (Fig. 12). The heterotrimeric G proteins act as transducers that couple LH/hCG-bound receptors to adenylate cyclase, which forms the second messenger, cAME cAMP activates PKA, which in turn phosphorylates specific serine and threonine residues on substrate proteins. The phosphorylated proteins generate cytoplasmic and nuclear responses that can lead to increased steroidogenesis. Androstenedione is the principal steroid produced by TICs, and treatment with LH increases its production in a time- and dose-dependent manner [28]. This concept explains in part the regulated production of androstenedione in normal women and its overexpression in women with chronically elevated levels of plasma LH. At the molecular level, activation of the LH signaling cascade leads to the stimulation of gene transcription, most notably genes for P450c22 and P450cl 7 [33]. The fact that the level of transcription and translation of these genes increases during folliculogenesis argues that LH-induced differential gene expression plays a physiological role in androstenedione production by human TICs during the menstrual cycle. It has been known for many years that the rate-limiting step in steroidogenesis involves the translocation of cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane, where it is metabolized to pregnenolone by P450c22. This protein, called steroidogenic acute regulatory (STAR) protein, has been isolated and cloned [34]. An important concept is that StAR is obligatory for LH-induced steroidogenesis. In the human ovary StAR is expressed in the interstitial cells, including the TICs [35]. It is noteworthy that there is convincing evidence that insulin signaling plays a role in regulating interstitial cell function in women [28]. Insulin receptors with protein tyro-

21 sine kinase (PTK) activity have been demonstrated in human ovaries. In situ hybridization and immunohistochemical studies have revealed that insulin receptors are expressed in TICs of Graafian follicles (both dominant and cohort) and in SICs [36]. Insulin stimulates androgen production by isolated TICs and SICs, and the stimulation is believed to be mediated by the insulin receptor. Activation of the insulin receptor signaling pathway can function alone to increase TIC/SIC androgen production and, importantly, the pathway can interact with the LH receptor pathway to further enhance the signals evoked by each receptor (Fig. 12). The cross-talk between the insulin and LH receptor pathways could be clinically relevant because of the development of hyperandrogenism in women with hyperinsulinemia.

E. I n t r a o v a r i a n C o n t r o l As discussed, the development of the dominant follicle is directed by the endocrine hormones FSH and LH. These ligands bind to receptors that are coupled to the AMP/protein kinase A signal transduction pathways, which in turn are coupled to differential gene activity in a quantitative and temporal fashion. An important concept to emerge in the past decade is that growth factors, which are themselves products of the ovary, modulate (either amplify or attenuate) FSH and LH action. All growth factors are ligands that can act in an autocrine/paracrine manner to regulate the timing and degree of hormone-dependent folliculogenesis. This is the autocrine/paracrine or growth factor concept (Fig. 13) [36a]. There are five different classes of growth factors: insulin-like growth factor (IGF), transforming growth factor/3 (TGF-/3), transforming growth factor-a (TGF-c0, fibroblast growth factor (FGF), and cytokines; all five classes have been described within follicles of human ovaries [37]. The principle that arises from all the evidence is that growth factors act by autocrine and paracrine mechanisms to cause plus and minus changes that determine whether a follicle lives or dies. The current challenges are to understand how specific growth factor families exert control of ovary functions and how these modulations are integrated into the overall pattern of physiology and pathophysiology during the life of the female.

V. OVARY R E S E R V E As discussed earlier, the number of ovarian primordial follicles decreases with age from birth through the menopause (Fig. 3). Importantly, studies [38,39] of human ovaries have established the concept that the rate of loss of OR (primordial follicles) is not constant during aging, with a significant accelerated decrease in OR occurring at about 37 years in most women (Fig. 14).

22

GREGORY F. ERICKSON

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FIGURE 13 Comparison of the autocrine-paracrine and endocrine concepts. H, Hormone. From [36a], Erickson, G. F. (1994). Non-gonadotropic regulation of ovarian function: Growth hormone and IGFs. In "Ovulation Induction: Basic Science and Clinical Advances (Excerpta Medica Int'l Congress Series)," (Filicori, M.,and Flanigni, C., Eds.), pp. 73-84. With permission from Elsevier Science.

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Major events during a woman's life that impact on fertility and fecundity.

A. Regulation Morphometric analysis of normal ovaries has demonstrated that the rate of recruitment (initiation of primordial follicle growth) accelerates sharply in women at --~38 years of age. Consequently, there is a biexponential decrease in OR in women [38,39]. It can be seen (Fig. 15) that the number of primordial follicles decreases steadily for more than three decades, but when the pool of primordial follicles reaches a critical number o f - 2 5 , 0 0 0 at 37.5 + 1.2 years, the rate of loss of primordial follicles accelerates ---twofold. Consequently, the OR is reduced to 1000 primordial follicles at --~51 years of age [38,39], which corresponds to the median age at natural menopause [40]. If the earlier rate of decrease in primordial follicles persisted, menopause would not be expected until the female reached 71 years of age. An important point in this natural process is that the number of primordial follicles within the ovaries of any given woman who reaches 38 years of age is variable, i.e., important individual differences in OR exist. As seen in Fig. 15, some women reach the critical threshold of 25,000 primordial follicles in their late twenties, whereas others do not reach this threshold until their forties. It seems therefore that age alone has limited predictive value for accurately determining a woman's OR. The significance of this variability is demon-

strated by the fact that women who continue to menstruate regularly after the age of 45 have 10 times more primordial follicles than do those with irregular menses [41 ]. Further, a higher level of primordial follicles is functionally coupled with a higher pregnancy rate in older women [6,13,17]. It can be argued, therefore, that the OR determines the number of maturing Graafian follicles, which in turn determines menstrual activity, which in turn determines fecundity. In a real sense, OR may be of greater importance than a woman's chronological age in predicting fertility. If we accept this argument, then OR, not age, would be the fundamental factor in determining the decrease in fecundity at - 3 8 years. Hence the question: What is the underlying mechanism for the accelerated recruitment at --~38 years of age? Although the answer to this question is not known, it is reasonable to assume that regulatory molecules are involved. In this regard, there are two possibilities: the decrease of one or more necessary inhibitors and/or the increase in one or more stimulators. Despite its physiological importance, very little is known about the mechanisms of recruitment in any species. Evidence from animal studies indicates that the rate of recruitment of primordial follicles can be influenced by several regulatory factors (Table I). One particularly interesting result with aging rats is that the rise in plasma FSH follow-

CHAPTER 2 Ovarian Anatomy and Physiology

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FIGURE 15 The age-related decrease in the total number of primordial follicles within both human ovaries, from birth to the menopause. As a result of recruitment (initiation of primordial follicle growth), the number of primordial follicles decreases progressively from --~1,000,000 at birth to 25,000 at 37 years. At age 37, the rate of recruitment increases sharply, and the number of primary follicles declines to 1000 at the menopause, i.e., at about 51 years. From Faddy et al. [38], with permission.

ing unilateral ovariectomy is associated with a significant reduction in the number of primordial follicles within the ovaries; the most striking feature is that the effect was observed only in old rats [42]. These studies support the potentially important concept that increased levels of FSH might function to accelerate the rate of recruitment during aging. Hence the question: To what extent is the accelerated loss of OR in aging women a consequence of increased circulating FSH? This is an important question because we know that a significant elevation of plasma FSH is observed in women when the loss of OR is accelerated at --~38 years [11,17], and the increased plasma FSH corresponds to the time that fecundity drops. Nonetheless, the question of whether the age-related increase in FSH in

TABLE I Known Modulators of Primordial Follicle Number in Laboratory Animals Regulator Follicle-stimulating hormone Thymus removal Starvation Growth hormone/prolactin Morphine sulfate Epidermal growth factor

Effect on ovary reserve

Ref.

Decrease Decrease Increase Increase Increase Increase

42 43 44 44 46 47

women is causally connected to the stimulation of recruitment remains unanswered. Experiments in mice show that the rate of recruitment can be modulated by several factors, including the thymus, restricted food intake, prolactin (PRL) and/or growth hormone (GH), opiates, and epidermal growth factor (EGF) (Table I). Experiments with neonatal mice indicate that thymectomy leads to a dramatic loss of primordial follicles by apoptosis [43]. Because apoptotic primordial follicles are rarely seen in aging women [38,39], it seems unlikely that a thymus factor plays an important role in the accelerated loss of OR at 38 years. In another study, a 50% reduction in food intake was found to increase the number of primordial follicles, suggesting starvation may increase the OR [44]. This could be potentially important in humans, but the question of whether starvation elicits a similar effect in women needs to be carefully examined. Studies using the sterile Snell dwarf mouse indicate that their ovaries contain significantly more primordial follicles than do those of the wild type. Precisely how this occurs is uncertain, but it has been theorized that the endocrine state resulting from chronically low GH and/ or PRL might be involved [45]. Finally, experiments done in mature mice have shown that the administration of either morphine sulfate [46] or epidermal growth factor [47] leads to a sustained reduction in the rate of primordial follicle recruitment, followed by an increase in OR. These studies, albeit limited, support the concept that the

24

GREGORY F. ERICKSON

rate of recruitment can be modulated, either increased or decreased, by regulatory elements. Although the clinical significance of these animal data is unknown, they raise the intriguing idea that it may be possible to slow down the rate of recruitment. If true, these data could have important implications for increasing the OR, which in turn could have important implications for fecundity in older women.

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At the present time, there is considerable interest in the hypothesis that increased plasma FSH concentrations consequent to decreased ovary inhibin A and B production may be involved in the mechanisms underlying accelerated recruitment and decreased fecundity in women after 36 years of age (see also Chapter 9). Therefore, to understand the physiological mechanism underlying the age-related increase in FSH, one needs to understand the structure of inhibin and agerelated changes in inhibin production in women. Inhibin is a member of the TGF-fl superfamily [48]. Inhibin molecules are composed of two heterodimeric proteins, a common ce subunit and one of two distinct/3 subunits termed flA or fiB (Fig. 16). The two subunits (a and flA or fiB) are held together by disulfide bonds, producing two different inhibins termed inhibin A and inhibin B. By contrast, the activins are built of two types of the/3 subunits, generating dimeric proteins called activin A, AB, or B. It should be mentioned here that the differential regulation of ce subunit expression might be expected to have profound effects on the levels of inhibin and activin produced by the ovary; that is, a high and low level of a subunit expression would be expected to result in relatively high and low levels of inhibin and activin production, respectively (Fig. 16). It is now clear that a monotropic rise in FSH occurs in women during aging [12]. The rise in FSH, which precedes that of LH by almost 10 years, becomes detectable after 36 years of age [14]. A detailed examination of FSH and LH concentrations in young and old women during the cycle [49] revealed that serum FSH, but not LH, is significantly elevated in older women throughout the menstrual cycle (Fig. 17). It is certainly of interest that the increase in plasma

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FIGURE 17 The daily serum FSH and LH levels throughout the menstrual cycle of 11 women in each group (mean +_ SE). Reprinted by permission from [49], The gonadotropin secretion pattern in normal women of advanced reproductive age in relation to the monotropic FSH rise, by Klein, N. A., Battaglia, D. E. Clifton, D. K., Bremner, W. J., and Soules, M. R., Journal of the Society for Gynecologic Investigation, 3, 27-32. Copyright 1996 by the Society for Gynecologic Investigation.

FSH coincides with the accelerated loss in OR. Presumably, some alteration has occurred in the negative feedback mechanism of FSH production in aging women, which is reflected in an increase in plasma FSH levels. The most likely explanation for this observation is that aging in women leads to a significant decrease in inhibin production. Direct evidence that the changing FSH profiles in aging women are accompanied by a concomitant decrease in plasma inhibin during the follicular phase of the cycle has been reported [50]. The strong evidence that inhibin exerts a negative feedback effect on pituitary FSH secretion in animals [51,52] supports the theory that decreased ovary inhibin production might be responsible for the increased FSH levels in women after 36 years, which in turn might be responsible for the decreased fecundity that can occur at this time. Direct evidence to support this theory has come from studies [ 15] showing that women aged 35 years or more produce less inhibin in response to exogenous gonadotropin than do women less than 35 years (Fig. 18). By contrast, no significant differences in plasma estradiol (and progesterone) are detectable in these women (Fig. 18). Studies in normal

CHAPTER 2 Ovarian Anatomy and Physiology

25

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FIGURE 18 The selective decrease in the inhibin response during ovarian hyperstimulation with human menopause gonadotropin (hMG) in aging women. Inhibin levels, but not estradiol levels, are significantly lower in women 35 years or older, hMG, Human menopausal gonadotropin; CC, clomiphene citrate; hCG, human chorionic gonadotropin. From [15], Hughes, E. G., Robertson, D. M., Handelsman, D. J., Hayward, S., Healy, D. L., DeKretser, D. M. (1990). Inhibin and estradiol responses to ovarian hyperstimulation: Effects of age and predictive value for in vitro fertilization outcome. J. Clin. Endocrinol. Metab. 70, 358-364. 9 The Endocrine Society.

cycling women have revealed a selective decrease in plasma inhibin levels during the follicular phase of the menstrual cycle, beginning at ---36 years of age [15,50]. It should be noted that the fully processed dimeric inhibin A has been shown to be the predominant circulating form in women before and after treatment with menopause gonadotropin [53]. Thus, a functional link between aging in women and decreased expression of ovary inhibin A is suggested (see Fig. 10). However, an important point to emerge from inhibin studies [24c] in normal women during the cycle indicate that important changes in inhibin B can be detected during the luteal-follicular transition (Fig. 19). Indeed, Klein et al. [54] have presented evidence for a role of decreased inhibin B in the monotropic FSH rise in aging women. From all these data, it seems reasonable to propose that a decreased ability of the ovaries to produce inhibin B (and perhaps inhibin A as well) may be the underlying cause of the monotropic rise in FSH in women after ---35 years of age. The question of whether these changes in inhibin and FSH negatively affect the egg has not been resolved. However, the data fit with a prediction that the decrease in inhibin, which begins around the time of the accelerated recruitment

at 37 years of age, may be involved, directly or indirectly, in the mechanisms that cause poor oocyte quality in aging women. What cells in the ovary are responsible for inhibin production? Studies using in situ hybridization and immunohistochemistry have documented the tissue-specific expression of the ce,/3A, and/3B subunits of inhibin in normal human ovaries during the menstrual cycle [55,56]. Yamoto et al. [55] found that the three inhibin subunit proteins are selectively expressed in the granulosa cells of growing follicles; however, there are important differences between the preantral and Graafian follicle stages. Here, the most striking difference is that granulosa cells in preantral follicles express relatively high levels of the/3A and/3B proteins, but the ce subunit proteins appears undetectable [55]. By contrast, the granulosa cells in the healthy antral follicles express all three subunit proteins, and the levels of ce,/3A, and/3B proteins become very high in the dominant follicle, particularly at the preovulatory stage [55]. By virtue of the different pattern of ce subunit expression during folliculogenesis, it would appear that granulosa cells in preantral follicles (primary, secondary, and early tertiary) produce activin, whereas those in healthy Graafian (antral) follicles produce inhibin. The question of whether the dominant follicle produces activin remains to be answered. An in vivo study on inhibin secretion by human ovaries presents a compelling case that the entire pool of healthy

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FIGURE 19 Plasma concentrations of inhibin A and B, and estradiol and FSH during the luteal-follicular transition in normal cycling women. Data were aligned with respect to the day of the intercycle FSH peak (mean _ SE). From [24c], Groome, N. P., Illingworth, P. J., O'Brien, M., Pai, R., Rodger, E E., Mather, J. P., and McNeilly, A. S. (1996). Measurement of dimeric inhibin B throughout the human menstrual cycle. J. Clin. Endocrinol. Metab. 81, 1401-1405. 9 The Endocrine Society.

26

GREGORY F. ERICKSON

Graafian follicles synthesize and secrete inhibin [57]. First, the concentration of inhibin is higher in the ovarian veins than in the peripheral circulation during the normal menstrual cycle. This finding indicates that plasma inhibin comes from the ovaries. Second, the amount of inhibin secreted during the late follicular phase is similar in the veins of both ovaries [57]. Therefore, it seems likely that the Graafian follicles in both ovaries (dominant and nondominant) secrete inhibin during the follicular phase. These data implicate all follicles (healthy and atretic) in the production of inhibin during the follicular phase of the menstrual cycle, i.e., peripheral inhibin levels reflect the total number of developing Graafian follicles. It is possible that this concept could have relevance to the lower levels of inhibin seen in older women. It has been shown that women exhibit an age-related decrease in the total number of Graafian follicles in the ovaries. Collectively, these observations offer a plausible explanation for the reduced levels of circulating inhibin after 36 years of age, i.e., fewer Graafian follicles in turn results in lower plasma inhibin levels, leading in turn to increased FSH levels. Another possible explanation is that a decrease in the expression of the a and/or the/3 subunits in the granulosa cells plays a role in the lower level of circulating inhibin in women after 36 years of age. This possibility implies that an age-related defect or alteration occurs in the granulosa cells, which leads to the underexpression of inhibin (but not estradiol) after --~35 years. Indeed, there is evidence from studies with cultured granulosa lutein cells to support this idea [58,59].

VI. ACCELERATED ACTIVIN

L O S S I N OR:

HYPOTHESIS

It is now of interest to discuss the potential causal connection between the monotropic rise in FSH and the mechanisms underlying the accelerated loss of OR at --~38 years of age. The existence of/3A and/3B proteins in the granulosa cells of secondary and early tertiary follicles argues that these proteins serve some function in preantral folliculogenesis in humans. The fact that the a subunit proteins appear undetectable in these follicles suggests that they may dimerize to form activin [55]. Therefore, one could hypothesize that activin may be an autocrine/paracrine regulator of preantral folliculogenesis. This is of interest because activin appears to be a potent inducer of FSH receptors in granulosa cells [60]. Furthermore, it has been shown that activin accelerates folliculogenesis [61,62]. These observations support the possibility that activin may play a part in the accelerated loss of OR through increasing granulosa FSH sensitivity, which could in turn may play a role in the pathogenesis of the egg in an old dominant follicle by causing a premature overexpression of its development. How might this occur? Three different isoforms of activin (Fig. 16), isolated

from porcine follicular fluid [63-65], have been shown to be disulfide-linked homodimers of the inhibin/3A subunit (activin A; M r 24,000) or the /3B subunit (activin B; M r 22,000), or a heterodimer composed of a/3A and/3B subunit protein (activin AB; M r 23,000). The isoforms are present in equimolar concentrations in follicular fluid pooled from all antral follicles [65]. So far, there is no evidence for activin in follicular fluid of dominant follicles. Originally, activin was found to be a stimulator of FSH secretion in vitro [63,64] and in vivo [65-69]; however, subsequent studies demonstrated that activin exerts a wide range of positive and negative effects in many different target cells [70]. Activin achieves these effects by binding to a novel family of transmembrane receptors with protein serine/threonine kinase activity [71]. In women, plasma levels of free activin are low and do not change substantially during the cycle [72]. In women, plasma levels of free activin are low and do not change substantially during the cycle [72]. Thus, it seems likely that activin regulates follicular function physiologically by autocrine/paracrine mechanisms. It has been shown that developing follicles indeed produce and respond to activin. As discussed earlier, the/3A and /3B subunits are selectively expressed in human granulosa cells of healthy follicles between the secondary and preovulatory stages [55,56]. It seems likely that in the absence of the a subunit, activin may function in initiating or maintaining the growth and development of preantral follicles during the gonadotropin-independent stages of folliculogenesis. Studies in the rat have shown that FSH can stimulate activin expression in granulosa cells in vivo [73,74], and convincing evidence that rat granulosa cells from preantral follicles actually secrete dimeric activin has been reported [75]. Further, the mRNAs for activin receptor subtype II (Act RII and Act RIIB) have been identified in rat follicles [76,77], being present in the oocytes and granulosa cells [78]. Moreover, specific binding of radiolabeled activin to these cells has been demonstrated [79-81 ]. Collectively, these results support the hypothesis that human granulosa cells in preantral follicles may produce and respond to activin, and importantly, this process may be amplified by FSH. Much of our understanding of the biological effects of activin in the ovary has come from studies in laboratory animals. There is evidence suggesting that the autosecretion of activin may play a role in regulating follicle growth and development. Most striking is the observation that activin is a potent stimulator of FSH receptor expression in rat granulosa cells [77,82]. Thus far, activin is the only ligand known to induce FSH receptors. This may have relevance to the acquisition of FSH receptors in the granulosa cells, which occurs early in preantral follicle development, e.g., at the primary and secondary stages [83,84]. Another important effect of activin is that it can prevent FSH-induced receptor down-regulation [85]. Therefore, the concept emerging is that the activin produced by granulosa cells might play an

27

CHAPTER 2 Ovarian Anatomy and Physiology

important physiological role in the induction and maintenance of FSH receptors in the granulosa cells during folliculogenesis (Fig. 20) [86]. How might this situation impact the OR and fecundity in aging women? Because FSH stimulates activin production and the FSH levels are elevated in women after 36 years of age, one could postulate that these two elements might act synergistically to accelerate the rate of granulosa cytodifferentiation and folliculogenesis in aging ovaries with respect to the OR, and one could propose the following cascade process. The granulosa cells in preantral follicles synthesize, secrete, and respond to intrinsic activin. One major response to the autosecretion of activin is the expression and maintenance of FSH receptors. The relatively high level of FSH after 36 years has a stimulatory effect on the autocrine activin mechanism. This results in a synergistic interaction between the two signal transduction pathways, which leads to accelerated growth and differentiation responses in the granulosa cells. In this hypothesis, the relatively high amounts of activin could have a strong stimulatory effect on oocyte development in the presence of high FSH. These potent stimulatory effects are then theorized to produce an "overripe" egg lacking a normal meiotic spindle in the aged dominant follicle. Clearly, further work is needed to test the validity of this new hypothesis. Nonetheless, this idea is consistent with the data of Gougeon et al. [39] showing an accelerated loss of developing preantral follicles in women after 37 years. Further, both the fact that activin and FSH interact to in-

crease markedly LH receptor [87] and estradiol production [88] in rat granulosa cells, and the fact that activin can accelerate meiotic maturation [89] and promote antrumlike formation [62], e.g. accelerate FSH-dependent granulosa cytodifferentiation, are consistent with this hypothesis. There is evidence that activin can influence physiological responses in vivo. Doi et al. [90] found that FSH action in vivo can be amplified by exogenous activin. That is, injected activin enhances follicle growth, FSH receptor, number, and estradiol production in intact and hypophysectomized immature rats. These observations are important because they demonstrate that the positive effects of activin on granulosa and other follicle processes observed in vitro will also occur in vivo. Therefore, these results further support the contention that interactions between the autocrine growth factor, activin, and the elevated FSH levels might have a strong stimulatory effect on granulosa cells, which leads to the acceleration of follicle growth and development after 36 years. Interestingly, the fact that the length of the follicular phase of the menstrual cycle is significantly shorter in older women [17] fits with this prediction. It should be mentioned that negative effects of activin on folliculogenesis have also been reported. Foremost is the study by Woodruff et al. [69], who showed that activin injection into the ovary bursa of immature rats caused oocyte degeneration, granulosa pyknosis, and decreased mitosis. Therefore, it is also possible that high levels of activin might induce atresia and trigger oocyte demise in the rat.

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FIGURE 20 The activin autocrine hypothesis for accelerated granulosa cytodifferentiation and folliculogenesis. From Erickson [86], with permission.

28

GREGORY F. ERICKSON VII.

NEW

DATA

ON THE

EFFECTS

OF ACTIVIN The results of our study of activin action in adult cycling rats are relevant to this new hypothesis [61 ]. We found that the administration of recombinant human activin A to rats produced dramatic structure/function changes in folliculogenesis. The most striking results are as follows. First, activin stimulated a twofold increase in the n u m b e r of large Graafian follicles during the follicular phase of the cycle. The data suggested that activin increased the size of the pool of early tertiary preantral follicles, and their growth and development to the preovulatory stage [61]. Interestingly, nearly all of these large follicles contained apoptotic granulosa cells and therefore they were classified as atretic (Fig. 5). Based on these results, we conclude that activin provides a multifunctional stimulus in v i v o that includes both the stimulation and the inhibition of follicle cell activities. Second, these large atretic follicles ovulated prematurely, i.e., --~24 hr earlier than normal. Histologically, the ovulatory changes evoked by activin paralleled those described for normal physiological o v u l a t i o n m t h e c a l swellings, the initiation of germinal vesicle breakdown, cumulus expansion, stigma formation, release of egg cumulus complexes, and morphological luteinization of the follicle wall [61 ]. These observations provide the first evidence that a ligand, namely activin, can significantly shorten (by 25%) the length of the follicular phase of the normal estrous cycle. This necessarily implies that dominant follicle development and ovulation were accelerated in response to activin administration. Third, we found that the activin-exposed eggs in the oviducts and in the large ovulating follicles were arrested in metaphase I and appeared degenerate (Fig. 6). This finding confirms and extends other studies showing that activin acts in the rat ovary to affect oocyte quality negatively. There is evidence that A C T RII receptors are strongly expressed in the rat oocytes [78] and that activin can accelerate meiotic maturation in isolated rat oocytes [89]. Therefore, this negative action of activin might be mediated by the activin signaling pathway present in the rat egg. The mechanisms and the physiological/pathophysiological implications for the multifunctional actions of activin remain to be elucidated. Nevertheless, our observations support the proposition that the autosecretion of activin may contribute to an acceleration of follicle development that could result in the premature ovulation of overripe eggs in cycling w o m e n by autocrine/paracrine mechanisms.

VIII.

STATEMENT

OF CONCLUSION

F r o m the preceding discussion, it is clear that the primary problem in the dominant follicle that leads to reduced fe-

cundity in older w o m e n is the susceptibility of the egg to meiotic nondisjunction and aneuploidy. A potentially important theory to explain the problem was developed in this discussion. Evidence indicating that an age-related decrease in the production of ovary inhibin leads to a monotropic rise in FSH, which in turn is reflected in the acceleration of the loss of OR by virtue of accelerating the rate of recruitment, was discussed. Further, it was suggested that specific interactions between granulosa-derived activin and increased FSH receptor and ligand may act synergistically to further accelerate the rate of granulosa and oocyte cytodifferentiation: this functional response might then lead to accelerated development of the dominant follicle, which in turn is reflected in the age-related shortening of the follicular phase. At the level of the oocyte, these changes are reflected in an increased potential for aneuploidy.

References 1. Erickson, G. E (1995). The ovary: Basic principles and concepts. In "Endocrinology and Metabolism" (P. Felig, J. D. Baxter, A. E. Broadus and L. A. Frohman, eds.), 3rd ed., pp. 973-1015. McGraw-Hill, New York. l a. Nachtigall, L. E. (1995). The aging woman. In "Gynecology and Obstetrics" (J. J. Sciarra, ed.), Vol. 1, Chap. 28. Lippincott, Philadelphia. 2. Sauer, M. V., Paulson, R. J., and Lobo, R. A. (1990). A preliminary report on oocyte donation extending reproductive potential to women over 40. N. Engl. J. Med. 323, 1157-1160. 3. Navot, D., Bergh, P. A., Williams, M. A., Garrisi, G. J., Guzman, I., Sandier, B., and Grunfeld, L. (1991). Poor oocyte quality rather than implantation failure as a cause of age-related decline in female fertility. Lancet 337, 1375-1377. 4. Sauer, M. V., Paulson, R. J., and Lobo, R. A. (1993). Pregnancy after age 50: Application of oocyte donation to women after natural menopause. Lancet 341, 321-323. 5. Sauer, M. V., Miles, R. A., Dahmoush, L., Paulson, R. J., Press, M., and Moyer, D. (1993). Evaluating the effect of age on endometrial responsiveness to hormone replacement therapy: A histologic, ultrasonographic, and tissue receptor analysis. J. Assist. Reprod. Genet. 10, 47-52. 6. Padilla, S. L., and Garcia, J. E. (1989). Effect of maternal age and number of in vitro fertilization procedures on pregnancy outcome. Fertil. Steril. 52, 270-273. 7. Piette, C., de Mouzon, J., Bachelot, A., and Spira, A. (1990). In-vitro fertilization: Influence of women's age on pregnancy rates. Hum. Reprod. 5, 56-59. 8. Schwartz, D., and Mayaux, M. J. (1982). Female fecundity as a function of age. N. Engl. J. Med. 306, 404-406. 9. Qasim, S. M., Karacan, M., Corsan, G. H., Shelden, R., and Kemmann, E. (1995). High-order oocyte transfer in gamete intrafallopian transfer patients 40 or more years of age. Fertil. Steril. 64, 107-110. 10. Penzias, A. S., Thompson, I. E., Alper, M. M., Oskowitz, S. P., and Berger, M. J. (1991). Successful use of gamete intrafallopian transfer does not reverse the decline in fertility in women over 40 years of age. Obstet. Gynecol. 77, 37-39. 11. Wood, C., Calderon, I., and Crombie, A. (1992). Age and fertility: Results of assisted reproductive technology in women over 40 years. J. Assist. Reprod. Genet. 9, 482-484. 12. Sherman, B. M., and Korenman, S. G. (1975). Hormonal characteris-

29

CHAPTER 2 Ovarian A n a t o m y and P h y s i o l o g y

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25.

25a.

tics of the human menstrual cycle throughout reproductive life. J. Clin. Invest. 55, 699-7067. Navot, D., Rosenwaks, Z., and Margolioth, E. J. (1987). Prognostic assessment of female fecundity. Lancet 2, 645-647. Lee, S. J., Lenton, E. A., Sexton, L., and Cooke, I. D. (1988). The effect of age on the cyclical patterns of plasma LH, FSH, estradiol and progesterone in women with regular menstrual cycles. Hum. Reprod. 3, 851-855. Hughes, E. G., Robertson, D. M., Handelsman, D. J., Hayward, S., Healy, D. L., and DeKretser, D. M. (1990). Inhibin and estradiol responses to ovarian hyperstimulation: Effects of age and predictive value for in vitro fertilization outcome. J. Clin. Endocrinol. Metab. 70, 358-364. Munne, S., Alikani, M., Tomkin, G., Grifo, J., and Cohen, J. (1995). Embryo morphology, developmental rates, and maternal age are correlated with chromosome abnormalities. Fertil. Steril. 64, 382-391. Scott, R. T., Toner, J. P., Muasher, S. J., Oehninger, S., Robinson, S., and Rosenwaks, Z. (1989). Follicle-stimulating hormone levels on cycle day 3 are predictive of in vitro fertilization outcome. Fertil. Steril. 51, 651-654. Gougeon, A., and Chainy, G. B. N. (1987). Morphometric studies of small follicles in ovaries of women at different ages. J. Reprod. Fertil. 81, 433-442. Baker, T. G. (1971). Radio sensitivity of mammalian oocytes with particular reference to the human female. Am. J. Obstet. Gynecol. 110, 746-761. Erickson, G. E (1986). An analysis of follicle development and ovum maturation. Semin. Reprod. Endocrinol. 4, 233-254. Soules, M. R., and Bremner, W. J. (1982). The menopause and climacteric: Endocrinologic basis and associated symptomatology. J. Am. Geriatr. Soc. 30, 547-561. Erickson, G. F., Magoffin, D. A., Dyer, C. A., and Hofeditz, C. (1985). The ovarian androgen producing cells: A review of structure/function relationships. Endocr. Rev. 6, 371-399. Hsueh, A. J., Billig, H., and Tsafriri, A. (1994). Ovarian follicle atresia: A hormonally controlled apoptotic process. Endocr. Rev. 15, 707-724. Erickson, G. F. (1997). Defining apoptosis: Players and systems. J. Soc. Gynecol. Invest. 4, 219-228. Gougeon, A. (1996). Regulation of ovarian follicular development in primates: Facts and hypotheses. Endocr. Rev. 17, 121-155. McNatty, K. P., Moore-Smith, D., Osathanondh, R., and Ryan, K. J. (1979). The human antral follicle: Functional correlates of growth and atresia. Ann. Biol. Anim. Biochim. Biophys. 19, 1547-1558. Erickson, G. F. (1978). Normal ovarian function. Clin. Obstet. Gynecol. 21, 31-52. Groome, N. P., Illingworth, P. J., O'Brien, M., Cooke, I., Ganesan, T. S., Baird, D. T., and McNeilly, A. S. (1994). Detection of dimeric inhibin throughout the human menstrual cycle by two-site enzyme immunoassay. Clin. Endocrinol. (Oxford) 40, 717-723. Gougeon, A. (1986). Dynamics of follicular growth in the human: A model from preliminary results. Hum. Reprod. 1, 81-87. Erickson, G. E (1983). Primary cultures of ovarian cells in serum-free medium as models of hormone-dependent differentiation. Mol. Cell. Endocrinol. 29, 21-49. Groome, N. P., Illingworth, P. J., O'Brien, M., Pai, R., Rodger, E E., Mather, J. P., and McNeilly, A. S. (1996). Measurement of dimeric inhibin B throughout the human menstrual cycle. J. Clin. Endocrinol. Metab. 81, 1401-1405. McNatty, K. P., Hunter, W. M., McNeilly, A. S., and Sawers, R. S. (1975). Changes in the concentration of pituitary and steroid hormones in the follicular fluid of human graafian follicles throughout the menstrual cycle. J. Endocrinol. 64, 555-571. Erickson, G. F. (1994). Polycystic ovary syndrome: Normal and abnor-

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63. Vale, W., Rivier, J., Vaughan, J., McClintock, R., Corrigan, A., Woo, W., Karr, D., and Spiess, J. (1986). Purification and characterization of an FSH releasing protein from porcine ovarian follicular fluid. Nature (London) 321,776-779. 64. Ling, N., Ying, S.-Y., Ueno, N., Shimasaki, S., Esch, F., Hotta, M., and Guillemin, R. (1986). Pituitary FSH is released by a heterodimer of the/3-subunits from the two forms of inhibin. Nature (London) 321, 779 -782. 65. Nakamura, T., Asashima, M., Eto, Y., Takio, K., Uchiyama, H., Moriya, N., Ariizumi, T., Yashiro, T., Sugino, K., Titani, K., and Sugino, H. (1992). Isolation and characterization of native Activin B. J. Biol. Chem. 267, 16385-16389. 66. Schwall, R., Schmelzer, C. H., Matsuyama, E., and Mason, A. J. (1989). Multiple actions of recombinant activin-A in vivo. Endocrinology (Baltimore) 125, 1420-1423. 67. Rivier, C., and Vale, W. (1991). Effect of recombinant activin-A on gonadotropin secretion in the female rat. Endocrinology (Baltimore) 129, 2463-2465. 68. Carroll, R. S., Kowash, E M., Lofgren, J. A., Schwall, R. H., and Chin, W. W. (1991). In vivo regulation of FSH synthesis by inhibin and activin. Endocrinology (Baltimore) 129, 3299-3304. 69. Woodruff, T. K., Krummen, L. A., Lyon, R. J., Stocks, D. L., and Mather, J. E (1993). Recombinant human inhibin A and recombinant human activin A regulate pituitary and ovarian function in the adult female rat. Endocrinology (Baltimore) 132, 2332-2341. 70. DePaolo, L. V., Bicsak, T. A., Erickson, G. E, Shimasaki, S., and Ling, N. (1991). Follistatin and activin: A potential intrinsic regulatory system within diverse tissues. Proc. Soc. Exp. Biol. Med. 198, 500-512. 71. Mathews, L. S. (1994). Activin receptors and cellular signaling by the receptor serine kinase family. Endocr. Rev. 15, 310-325. 72. Demura, R., Suzuki, T., Tajima, S., Mitsuhashi, S., Odagiri, E., Demura, H., and Ling, N. (1993). Human plasma free activin and inhibin levels during the menstrual cycle. J. Clin. Endocrinol. Metab. 76, 1080-1082. 73. Meunier, H., Cajander, S. B., Roberts, V. J., Rivier, C., Sawchenko, P. E., Hsueh, A. J. W., and Vale, W. (1988). Rapid changes in the expression of inhibin ce-,/3A-, and/3B-subunits in ovarian cell types during the rat estrous cycle. Mol. Endocrinol. 2, 1352-1363. 74. Meunier, H., Roberts, V. J., Sawchenko, R E., Cajander, S. B., Hsueh, A. J. W., and Vale, W. (1989). Periovulatory changes in the expression of inhibin ce-,/3A-, and j3B-subunits in hormonally induced immature female rats. Mol. Endocrinol. 3, 2062-2069. 75. Miyanaga, K., Erickson, G. F., DePaolo, L. V., Ling, N., and Shimasaki, S. (1993). Differential control of activin, inhibin, and follistatin proteins in cultured rat granulosa cells. Biochem. Biophys. Res. Commun. 194, 253-258. 76. Feng, Z. M., Madigan, M. G., and Chen, C. L. C. (1993). Expression of type II activin receptor genes in the male and female reproductive tissues of the rat. Endocrinology (Baltimore) 132, 2593-2600. 77. Nakamura, M., Minegishi, T., Hasegawa, Y., Nakamura, K., Igarashi, S., Ito, I., Shinozaki, H., Miyamoto, K., Eto, Y., and Ibuki, Y. (1993). Effect of an activin A on follicle-stimulating hormone (FSH) receptor messenger ribonucleic acid levels and FSH receptor expressions in cultured rat granulosa cells. Endocrinology (Baltimore) 133, 538544. 78. Cameron, V. A., Nishimura, E., Mathews, L. S., Lewis, K. A., Sawchenko, R E., and Vale, W. W. (1994). Hybridization histochemical localization of activin receptor subtypes in rat brain, pituitary, ovary, and testis. Endocrinology (Baltimore) 134, 799-808. 79. LaPolt, E S., Soto, D., Su, J. G., Campen, C. A., Vaughan, J., Vale, W., and Hsueh, A. J. (1989). Activin stimulation of inhibin secretion and messenger RNA levels in cultured granulosa cells. Mol. Endocrinol. 3, 1666-1673. 80. Xiao, S., and Findlay, J. K. (1991). Interaction between activin and follicle-stimulating hormone-suppressing protein and their mecha-

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CHAPTER 2 Ovarian A n a t o m y and Physiology

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nisms of action on cultured rat granulosa cells. Mol. Cell. Endocrinol. 79, 99-107. Woodruff, T. K., Krummen, L., McCray, G., and Mather, J. E (1993). In situ ligand binding of recombinant human [125I]activin-A and recombinant human [125I]inhibin-A to the adult rat ovary. Endocrinology (Baltimore) 133, 2998-3006. Xiao, S., Robertson, D. M., and Findlay, J. K. (1992). Effects of activin and follicle-stimulating hormone (FSH)-suppressing protein/follistatin on FSH receptors and differentiation of cultured rat granulosa cells. Endocrinology (Baltimore) 131, 1009-1016. Presl, J., Pospisil, J., Figarov~i, V., and Krabec, Z. (1974). Stagedependent changes in binding of iodinated FSH during ovarian follicle maturation in rats. Endocrinol. Exp. 8, 291-298. Zeleznik, A. J., Schuler, H. M., and Reichert, L. E. (1981). Gonadotropin-binding sites in the Rhesus monkey ovary; Role of the vasculature in the selective distribution of human chorionic gonadotropin to the preovulatory follicle. Endocrinology (Baltimore) 109, 356-362. Nimrod, A., and Lamprecht, S. A. (1980). Hormone-induced desen-

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sitization of cultured rat granulosa cells to FSH. Biochem. Biophys. Res. Commun. 92, 905-911. Erickson, G. F. (1997). Dissociation of endocrine and gametogenic ovarian function. In "Perimenopause" (R. Lobo, ed.), Serono Symp., pp. 101-118. Springer-Verlag, Berlin. Nakamura, K., Nakamura, M., Igarashi, I. S., Miyamoto, K., Eto, Y., Ibuki, Y., and Minegishi, T. (1994). Effect of activin on luteinizing hormone-human chorionic gonadotropin receptor messenger ribonucleic acid in granulosa cells. Endocrinology (Baltimore) 134, 2329-2335. Mir6, F., Smyth, C. D., and Hillier, S. G. (1991). Development-related effects of recombinant activin on steroid synthesis in rat granulosa cells. Endocrinology (Baltimore) 129, 3388-3394. Itoh, M., Igarashi, M., Yamada, K., Hasegawa, Y., Seiki, M., Eto, Y., and Shibai, H. (1990). Activin A stimulates meiotic maturation of the rat oocyte in vitro. Biochem. Biophys. Res. Commun. 166, 1479-1484. Doi, M., Igarashi, M., Hasegawa, Y. U., Eto, Y., Shibai, H., Miura, T., and Ibuki, Y. (1992). In vivo action of activin-A on pituitary-gonadal system. Endocrinology (Baltimore) 130, 139-144.

~HAPTER

Regulation of the Hyp oth al ami c -- Pituitary Gonadal Axis: Role of Gonadal Steroids " and lmpl"~cat~ons for the Menopause FRANCISCO

Josr L6PEZ, P A T R I C I A

D. FINN, MARK A. LAWSON,

AND ANDRI~S NEGRO-VILAR Ligand Pharmaceuticals, Inc., San Diego, California 92121

I. Introduction II. Functional Organization of the Hypothalamic-Pituitary- Ovarian Axis III. Anatomical and Biochemical Correlates of Gonadal Steroid Hormone Action in the Central Nervous System

IV. Ovarian Steroid Action in the Central Nervous SystemnControl of Reproduction V. The Menopause: Consequences of Steroid Hormone Loss on the Central Nervous System References

I. I N T R O D U C T I O N

monthly chance of becoming pregnant. This regular ovulatory pattern continues for the life span of females in most species except humans, for whom reproduction ceases in midlife with the onset of menopause. Because of an increasing life expectancy, women spend an increased portion

The window of opportunity for conception to occur in most mammalian species is cyclical. Women produce a viable egg roughly every month, providing, therefore, a

MENOPAUSE: BIOLOGY AND PATHOBIOLOGY

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Copyright9 2000by AcademicPress. All rightsof reproductionin any formreserved.

34

L 6 P E Z ET AL.

of their life in the postmenopausal state. In fact, 35 million American w o m e n are currently postmenopausal and this n u m b e r increases at a rate of over a million every year [1]. M e n o p a u s e is characterized by a marked decrease in estrogen production from the ovaries. The estrogen decrease that occurs not only results in a loss of reproductive function, but is also associated with a n u m b e r of conditions that reduce the quality of life of this population of women. It has b e c o m e clear over the past 1 0 - 1 5 years that the ubiquitous presence of the estrogen receptor (ER) in body tissues and organ systems provides the substrate for estrogenic regulation or modulation of multiple body functions. That knowledge has run in parallel with ever increasing evidence of the beneficial effects of estrogen replacement therapy after the menopause. What started originally as an approach to treat vasomotor symptoms and vaginal dryness has evolved into a comprehensive therapeutic approach that can span several decades after the m e n o p a u s e and is intended to prevent and/ or treat osteoporosis, cardiovascular disease, cognitive dysfunction, and possibly other neurodegenerative disorders such as Alzheimer disease. Central to this expansive approach to h o r m o n e replacement therapy is not only a more refined understanding of the mechanisms that lead to estrogen's beneficial effects on many tissues and functions, but also to an understanding of the undesired side effects that result from estrogenic activity at sites that are not the intended therapeutic targets (i.e., uterus and breast). The concept of tissue selectivity in estrogen action has e m e r g e d in recent years and provides the cellular and molecular basis to understand the diversity of estrogen activity and the distinct m o d e of action of full versus partial estrogen agonists [ 2 4]. It is hoped that a better understanding of these issues will lead to a better h o r m o n e replacement therapy in postmenopausal women.

II. FUNCTIONAL ORGANIZATION OF THE H Y P O T H A L A M I C PITUITARY-OVARIAN AXIS A. Functional Organization Multiple physiological factors influence reproductive fitness, as illustrated by the alteration of normal menses by factors such as stress and metabolic state. However, the principal components of the reproductive endocrine axis, the luteinizing hormone-releasing h o r m o n e (LHRH) neurons of the forebrain, the pituitary gonadotroph, and the gonads, each have principal and distinct roles in modulating reproductive function. H o r m o n a l feedback regulation appears to occur at all levels of this axis (Fig. 1). Therefore, a complex regulatory system is implicated in the mechanisms that lead to the release of the egg. In spite of the complexity, it is interesting that a small n u m b e r of neurons in the forebrain, par-

FIGURE 1 Schematic representation of the hypothalamic-pituitarygonadal axis in females. Neurons located in the forebrain synthesize the decapeptide luteinizing hormone-releasing hormone (LHRH), also known as gonadotropin-releasing hormone. The activity of LHRH-producing neurons is controlled by multiple afferent terminals, which either stimulate (positive sign on top of the diagram) or inhibit (negative sign on top of the diagram) the LHRH neuronal network. LHRH is secreted at the level of the median eminence into the hypophyseal portal system in a pulsatile manner. Via the portal circulation, LHRH reaches the anterior pituitary (AP), where it stimulates (positive sign close to arrows beneath LHRH) the production of the pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating (FSH) hormone. Gonadotropins, in turn, are secreted in a pulsatile manner into the bloodstream to regulate (positive sign near the gonad) gonadal homeostasis in terms of gametogenesis and production of gonadal hormones such as estradiol (E2), progesterone (P), and inhibin. Gonadal hormones exhibit trophic effects on sex accessory tissues and contribute to the regulation of the hypothalamic-pituitary axis by the establishment of the classical long-loop negative feedback (LL-). Under some conditions estradiol is able to stimulate the activity of the LHRH neuronal network, resulting in the ovulatory surge of gonadotropins. This type of control mechanism is known as positive feedback (LL+). Two other feedback loops have been demonstrated in the hypothalamic-pituitary-gonadal axis. These involve the gonadotropin-dependent negative regulation of LHRH production (negative short-loop feedback; SL-) and the LHRHdependent inhibition of LHRH production constituting the ultrashort-loop negative feedback (USL-). NL, Neurointermediate lobe of the pituitary gland.

ticularly the hypothalamus, are responsible for all aspects of reproduction. These neurons, which n u m b e r 2 0 0 0 - 3 0 0 0 in humans, synthesize the decapeptide L H R H , also known as gonadotropin-releasing hormone, which in essence represents the conductor of reproductive function. The absence of L H R H results in an absolute impairment of reproduction as

CHAPTER 3 Role of Gonadal Steroids in Menopause evidenced by the phenotype of the hypogonadal mouse in which there is a mutation on the LHRH gene leading to the synthesis of a nonfunctional prohormone [5]. A similar reproductive impairment is observed in humans with Kallmann's syndrome, in which there is an absence of LHRH neurons in the forebrain [6]. In these abnormal conditions, reproductive function can be recovered by introduction of either a normal gene, in the case of the mice [7], or by exogenous LHRH treatment, in the case of humans with Kallmann's syndrome [8]. Neurons of the LHRH system originate outside of the brain in the olfactory placode and migrate into the ventral medial forebrain [9]. Rather than residing in a discrete anatomical location, LHRH-containing neurons form a loose continuum of scattered cells that span areas such as the diagonal band of Broca, dorsal septum, bed nucleus of the stria terminalis, lateral and medial preoptic areas, and mediobasal hypothalamus [10-16]. In many rodent species, including mice and rats, the majority of LHRH neurons are found in the rostral medial preoptic area, near the organum vasculosum of the lamina terminalis, and very few neurons are found caudal to the medial preoptic area [10,12]. In primates, including humans, many LHRH neurons are also found in the medial preoptic area. However, because LHRH neurons migrate more caudally, many are also located in the mediobasal hypothalamus [ 11,13-15]. Although LHRH neuronal perikarya are scattered throughout many areas of the forebrain, rather than concentrated in specific nuclei, the majority of LHRH axon terminals converge in the median eminence through the preoptico-infundibular tract [17]. At the level of the median eminence, LHRH terminals establish contacts with fenestrated capillaries, forming the hypophyseal portal system. Via this specialized vascular system, secreted LHRH is transported to the anterior pituitary, where it interacts with specific membrane receptors in gonadotrophs to stimulate the production and secretion of the gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). The interactions between the different regulatory sites of the hypothalamic-pituitary-gonadal axis are schematically represented in Fig. 1. The pituitary gonadotropins, LH and FSH as well as thyroid-stimulating hormone, belong to the family of heterodimeric pituitary glycoprotein hormones [18]. All of these glycoproteins contain a common a subunit that is linked noncovalently with a hormone-specific/3 subunit (Fig. 2). Despite their similar structures and sequence homologies, these hormones exhibit only marginal cross-reactivity between their respective receptors. A common structural feature of these proteins is the presence of multiple glycosylation sites on both subunits. For example, FSH contains four such glycosylation sites, two on each of the ce and/3 subunits [19]. Luteinizing hormone and FSH control gonadal function via interactions with specific membrane receptors present in various cell types within the gonad. The recep-

35

FIGURE 2 General structural features of the pituitary glycoprotein hormone family. Luteinizing hormone (LH), follicle-stimulating hormone (FSH), and thyroid-stimulatinghormone (TSH) belong to the family of pituitary heterodimeric glycoprotein hormones. The three proteins share a common ce subunit and have hormone-specific/3 subunits. Chorionic gonadotropin (CG) producedby the placenta is highlyhomologousto pituitary LH and interacts with LH receptors; it is thus included in the diagram for the sake of completeness.

tors transducing gonadotropin signals belong to the seventransmembrane-spanning domains, G protein-coupled receptors. In general, gonadotropin receptors are coupled to the cellular response via activation of adenylate cyclase and the subsequent production of cyclic adenosine monophosphate (cAMP) [20]. Gonadotropin actions on the gonad encompass production of steroid and peptide hormones as well as gametogenesis. Through negative feedback mechanisms gonadal ster o i d s - e s t r o g e n , progesterone, and testosterone--close the regulatory loop in the hypothalamic-pituitary-gonadal axis by inhibiting the release of LHRH, and the gonadotropins do so by acting at the hypothalamus and pituitary (Fig. 1; longloop feedback). Estrogen is the only gonadal steroid that has the ability to also exert positive feedback action on the hypothalamus and pituitary. The positive feedback effects of estrogen play a fundamental role in initiating the series of events that lead to the preovulatory surge of gonadotropins in spontaneous ovulators, including humans (reviewed in Section III,A,2). In spite of extensive work on the central actions of sex steroids in the control of reproductive functions, the molecular mechanisms involved in these actions remain elusive. It is clear, however, that gonadal steroid effects are mediated by discrete subpopulations of neurons that are strategically located to control reproductive functions. Direct effects of sex steroids on LHRH neurons have not yet been conclusively documented in vivo due to the failure to detect ERs or estrogen binding in LHRH neurons [21-24]. However, ER immunoreactivity in LHRH neurons in the medial preoptic area of rats has been demonstrated [25]. More recently, Skynner et al. (1999) have shown the presence of ERce and ERfl mRNA in single preoptic area neurons by nested RT-PCR. Although it is intriguing to speculate that estrogen can act directly on LHRH neurons, the identification of ERs in

36

L6PEZ ET AL.

LHRH neurons needs to be explored further. Whereas the direct action of estrogen on LHRH neurons remains controversial, it is clear that estrogen acts on neurons that lie in close proximity to LHRH neurons, such as GAB A-containing neurons, as well as on other sets of neurons that innervate LHRH neurons, such as fl-endorphin-containing neurons, whose cell bodies reside in the hypothalamus, and norepinephrine-containing neurons, whose cell bodies are located in the hindbrain. Furthermore, there is ample evidence that these neuronal populations mediate at least some of estrogen's action on the LHRH neuronal network [ 16,26]. Additional findings indicate that sex steroids may also affect LHRH neuronal function by nongenomic mechanisms or by actions that do not require the presence of classical nuclear steroid receptors (for a review, see Ref. 27). Glycoprotein hormones such as activin and follistatin may also exert negative feedback actions on the hypothalamic-pituitary-gonadal axis. Release of these hormones from the pituitary may constitute a short-loop feedback to the hypothalarrius. Evidence for this arises from the observation of LH/chorionic gonadotropin receptors on LHRH neurons [28,29]. Follistatin transcription and release are increased by LHRH in primary pituitary cell cultures under conditions that inhibit FSH synthesis [30], and the presence of follistatin receptors on neurons in the hypothalamus closely associated with LHRH neurons suggesting a possible central nervous system (CNS) site of action [31 ]. Additional data suggest the possibility that follistatin may indirectly influence LHRH neuron activity through antagonism of activin action [32].

B. D i s t r i b u t i o n o f L H R H N e u r o n s in the F o r e b r a i n It is generally thought that LHRH neurons are sparsely distributed in the medial forebrain of primates and are most abundant in the medial preoptic area and mediobasal hypothalamus. However, this view has been challenged. In humans, the distribution of LHRH mRNA-expressing neurons has been mapped using a cDNA probe complementary to mammalian LHRH. Many LHRH mRNA-expressing neurons were found both inside and outside the medial forebrain in humans [33]. In fact, more than 12,000 LHRH mRNA-expressing neurons per subject were counted, a number nearly 10-fold more than had been reported for other primate species by other investigators using in situ hybridization or immunocytochemistry [ 11,14,15]. These authors divided LHRH neurons into subtypes, types I through III, based on morphological characteristics and mRNA expression levels. Type I and type II LHRH neurons are small in size, whereas type III is large (Table I)[33a]. In addition, type I neurons display the classical oval-fusiform morphology typical for LHRH neurons in other species [17]. In contrast, type II neurons are small, oval, and have more rounded somata (Table I). Type III neurons exceed 500/xm 2 in profile area, are round to oval in morphology, and are characterized by prominent Nissl staining located in the periphery of the cells. Additionally, type I LHRH neurons express very high levels of LHRH, whereas type II neurons express the least amounts of mRNA for LHRH. Levels of LHRH mRNA in type III neurons are intermediate between types I and II (Table I).

TABLE I Subpopulations of LHRH Neurons in Humans: Differential Regulation by the Postmenopausal Reduction of Estrogen Levels LHRH neuron subtype Parameter

Type I

Type II

Type III

Size a

Small

Small

Large

Level of mRNA expression in LHRH neurons`"

Heavy

Light

Intermediate

Shape"

Oval to fusiform

Round to oval

Round to oval

Major location of the cells`"

Mediobasal hypothalalamus and the medial preoptic area

Scattered in the dorsal preoptic area, the septal area, the amygdala and the substantia innominata

Putamen and the magnocellular basal forebrain complex

50% increase

No change

Not evaluated

LHRH mRNA levels: changes in postmenopausal women b

a Characteristics of the different subsets of LHRH neurons were taken from the paper by Rance et al. [33].

b LHRH mRNA levels were determined by in situ hybridization and reported by Rance's laboratory in 1996 [33a].

CHAPTER3 Role of Gonadal Steroids in Menopause The distribution patterns of the three subtypes of LHRH neurons are also somewhat distinct. Type I cells are located in areas that have been previously shown to contain LHRH neurons in other primate species [11,13-15]: i.e., the medial preoptic area and mediobasal hypothalamus. Moreover, these cells are similar in number to that reported for LHRH neurons found in other primate species [11,14,15,34]. In contrast, type II LHRH neurons were found to be numerous in the medial forebrain, the medial septum, medial preoptic area, and the lateral forebrain. Small numbers of cells are also found in the bed nucleus of the stria terminalis and in striatal areas such as the globus pallidus and ventral pallidum. Type III LHRH neurons were found in high numbers in lateral forebrain areas such as the putamen, sublenticular substantia innominata, and nucleus basalis of Meynert. Table I summarizes the characteristics and distribution of the different types of LHRH neurons. Since the original report on the distribution of LHRH mRNA-expressing neurons in human brain [33], there have been other reports verifying the presence of previously undetected populations of LHRH neurons in the lateral forebrain of both fetal and adult rhesus monkeys [35,36]. Like the traditional well-described LHRH neurons implicated in gonadotropin secretion, these previously undetected populations of LHRH neurons that reside in the lateral forebrain arise from the olfactory placode and begin their migration into forebrain areas slightly earlier than the LHRH neurons destined to reside in the medial forebrain [35]. The distribution of LHRH-immunoreactive neurons in the lateral forebrain of fetal rhesus macaques is similar to the distribution of LHRH mRNA-expressing neurons described in human lateral forebrain [33,35]. Interestingly, these lateral forebrain LHRH neurons are detectable with only one of five antisera made to mammalian LHRH, suggesting that these LHRH neurons may be distinct from those that reside in the medial forebrain [35]. Further analysis of these lateral forebrain LHRH neurons revealed that they also express the enzyme EP24.15, which is capable of cleaving LHRH between the fifth and sixth amino acids [37]. Taken together these data suggest that lateral forebrain LHRH neurons contain an amino-terminal five-amino acid cleavage product (LHRH 15) of the LHRH decapeptide. The physiological significance of LHRH-expressing neurons in the lateral forebrain is unknown. Few, if any, lateral forebrain neurons project to the median eminence, the site where the vast majority of medial forebrain LHRH neurons establish contacts with fenestrated capillaries to release LHRH into the hypophyseal circulation to elicit gonadotropin release from the pituitary [ 16]. Several of the lateral forebrain areas, in which LHRH neurons are found in humans, appear to be sites for estrogen's action in nonhuman primates [38,39], yet few of these areas are directly implicated in reproductive function. Instead, several of these areas undergo changes with aging that are associated with the development

37 of Alzheimer disease [40]. What role, if any, the LHRH gene may play in these areas is still unknown.

III. ANATOMICAL BIOCHEMICAL GONADAL

AND

CORRELATES

STEROID

IN THE CENTRAL

OF

HORMONE

NERVOUS

ACTION

SYSTEM

Steroid hormones exhibit their regulatory functions by modulating the activity of ligand-dependent transcription factors (for reviews on the mechanism of action of steroid hormones, see Refs. 41 and 42; see also Chapter 1). In addition, a growing body of evidence supports the notion that steroid hormones also exert effects that are independent of those mediated via nuclear receptors [27]. This dual genomic and nongenomic action of steroid hormones as well as the identification of multiple nuclear receptors complicate our understanding of the biology of steroid hormones and their regulatory actions on LHRH production and secretion. With menopause, there is a cessation of ovarian function, resulting in the decreased production of sex steroids, progesterone, and, in particular, estrogens. It is necessary to understand the mechanisms by which, and the sites where, ovarian steroids act in the CNS to appreciate the consequences of estrogen withdrawal and to develop strategies for safe and effective estrogen replacement therapies. The following discussion summarizes general concepts of estrogen signal transduction mechanisms as well as the distribution of ERs in the brain.

A. E s t r o g e n R e c e p t o r S i g n a l Transduction Mechanisms Interactions of the ER with transcription factors and subsequent binding to hormone response elements (HREs) provide a mechanistic framework by which the selectivity of estrogen action can be explained. The multiple transcription factors involved in the model of nuclear receptor activation of genes has been integrated into a tripartite pharmacological model [3]. As illustrated in Fig. 3, three modes of action combined with three ligand-dependent conformations of the receptor make up this model. In the first mode of action, initial binding of the hormone in the receptor induces a conformational change to the receptor that results in binding of the receptor/ligand complex to a hormone response element (HRE). In addition, ligand-receptor interaction leads to dimerization and recruitment of the general transcription factor complex. A second mode of action involves an adaptor protein driving final activation of transcription and involving the interaction of the receptor complex with specific HREs in the promoter region of particular genes. Third, recruitment of other transcription factors acting as coactivators with the receptor eliminates the need for specific HREs in

38

FIGURE 3 Schematic depiction of the tripartite receptor pharmacological model to explain pleiotropic actions of steroid hormones. Steroid receptors interact with various ligands (L1 through L3), resulting in a change in the spatial conformation of the ligand receptor complex (represented on the left side of the figure by the diverse shapes of the receptor dimers). As a consequence of the different ligand-dependent conformations, various responses can be achieved in the tissues of interest. The specific responses are conformation dependent and probably use different mechanisms to modulate gene expression. In the first mechanism represented, the receptor uses a hormone response element (HRE) present on the gene of interest. After interacting with the HRE, the receptor recruits general transcription factors (GTFs) to form a complex that activates transcription of the gene. In the second mechanism, interaction with ligand 2 leads to a different conformation of the receptor that uses the HRE, but needs an adaptor protein to interact with the general transcription machinery. The third mechanism is HRE independent, but requires an interaction with other transcription factors to elicit activation of gene transcription. From [3] Katzenellenbogen, J. A., O'Malley, B. W., and Katzenellengoben, B. S. (1996). Tripartite steroid hormone receptor pharmacology: Interaction with multiple effector sites as a basis for the cell- and promoter-specific action of these hormones. Mol. Endocrinol. 10, 119-131. 9 The Endocrine Society. the target gene. Diverse conformations of the receptor may be generated by interaction with different ligands (Fig. 3, L 1 through L3), providing a molecular mechanism for the pleiotropic actions of the steroid receptor complex in response to pharmacological or physiological agents interacting with the receptor. The identification of a second ER confers an additional level of complexity by adding a level of selectivity, which depends on the discrete distribution of the various receptor subtypes. The classical, human ER was cloned and identified in 1985 as a ligand-dependent transcription factor that binds estrogens and antiestrogens [43]. Ten years later, Kuiper et al. [44], using a rat prostate cDNA library, isolated another ER, which has been designated fl (the classical ER has been defined as a). Various functional domains were defined within the ERa coding sequence [45], two of which are involved in DNA- and ligand-binding interactions (Fig. 4). In addition, two other functional domains, AF-1 and AF-2, were defined; the AF-1 domain is involved in hormone-

L 6 P E Z ET AL.

independent activation of the receptor, whereas the AF-2 domain participates in hormone-dependent activation [45]. A comparison of the human ERc~ and ERfl cDNAs illustrates that the DNA-binding domain is almost 100% conserved in both receptors (Fig. 4), suggesting that both receptors interact with similar HREs on DNA. In contrast, the similarity of the ligand-binding domains is poor (59%; Fig. 4). This difference may be exploited by the identification of novel ligands that display differential receptor specificity, allowing the possibility to modulate estrogen action selectively via ERa, ERfl, or both. A variety of molecular entities corresponding to the ERfl sequence have been cloned. Shortly after the initial description of the receptor, a human homolog was cloned from the testis [46], which encodes a protein that is eight amino acids shorter than the rat ERfl. Later, the human sequence was extended to the first rat methionine codon, adding the eight amino acids [47]. At the same time, the mouse homolog was cloned [48], showing a similar eight-amino acid extension. Further efforts have resulted in the identification of extended forms of ERfl. A clone that encodes 45 additional amino acids was recently isolated in humans [49]. In addition, another clone for the rat sequence, which contains an additional 64 amino acids, has been described (GenBank accession number AJ002602). Figure 4 depicts the homology of the short and long forms of the rat ERfl compared to the ERa. These longer forms of the receptor have also been identified in the mouse and are accessible in GenBank. The existence of these molecular entities raises questions about the validity of our current cloning strategies [50], because it is difficult to assess if any or all of the isolated clones represent physiologically relevant ERfl. In addition to extended N terminal receptor subtypes, various reports [50a,b,c] identify

AF-I i

ERo~

DNAHORMONE BINDING BINDING/AF-II 180 263302

A/B

I

C

IDI

E

553 595

I FI

HINGE i

ERI3 Short

96 18%

i

ERI3 Long

166 211

A/B I C I D I 97% 30%

149 A/B

30%

449 477

E

IF I

59%

18%

219 264

I c ]DI 97% 30%

502 530

E

IF I

59%

~8%

FIGURE 4 Functional estrogen receptor (ER) domains (A though F) and identity between the human ERa and ERfl short and long forms. Activation function I (AF-I) resides in the N terminus of the estrogen receptor, whereas activation function II (AF-II) resides in the C terminus. Notice that the DNA-binding domain of the ER is highly similar (97% homologous) among the ce and/3 forms of the receptor. In contrast, the identity in the hormone-binding domain is low (59% homologous), suggesting the potential for subtype-selective ligands. The ERa, ER/3 short form, and ER/3 long form protein sequences were obtained from GenBank accession numbers NM000125, X99101, and AB006590, respectively. They were aligned using the Clustal W algorithm [45a] and Omiga 1.1.3 software (Oxford Molecular Limited).

CHAPTER3 Role of Gonadal Steroids in Menopause isoforms of ERfl with amino acid insertions in the ligand binding domain, and this has been observed in humans as well as rats. It is apparent (Fig. 3) that interaction of estrogen with its receptor causes a receptor-ligand complex dimerization that associates with DNA and subsequently modulates gene transcription. Studies looking at colocalization of ERs have been able to identify cell types that express both subtypes of the ER, particularly in the CNS (see Section II,B). Colocalization of both receptor subtypes in the same neuron would imply that E R a and ERfl could conceivably form heterodimers to control gene transcription (Fig. 5). Pettersson et al. demonstrated, using a mammalian two-hybrid system, that both subtypes of the ER could form functionally active heterodimers [51 ]. These observations have been confirmed using immunoprecipitation [49]. The possibility that ERce and ER/3 form functionally active heterodimers raises the question of whether heterodimers would interact with classical estrogen response elements (EREs) or other DNA response elements (Fig. 5). Heterodimer formation, therefore, could contribute to the pleomorphic action of estrogens in different tissues and in response to different ligands. ERce and ERfl bind estradiol with similar affinity [52], and both act at EREs to regulate gene expression [53]. However, they differ in their ligand-dependent activation of AP-l-regulated gene expression. In the presence of 17/3estradiol, ERce, but not ERfl, activated gene transcription via AP-1, whereas in the presence of antiestrogens both receptors activated gene transcription via AP-1 [53]. This pro-

FIGURE 5 The existence of two estrogen receptor subtypes, ERa and ERfl, that coexist in somepopulations of cells suggestthat ER heterodimers could modulate gene transcription. Estrogen receptor c~and fl homodimers have been shown to modulate transcription via interactions with classical estrogen response elements (EREs). However,ERfl homodimers could also use nonconsensus ERE (other REs) to modulateactivityof additional genes. Likewise, ERcdERfl heterodimers modulate transcription via interaction with classical EREs and possibly other nonconsensus EREs in ERa/ERfl responsive genes. Estradiol is represented in the figure by a gray triangle. Notice the change in receptor conformation after estradiol is bound to the receptor.

39 vides an additional biochemical substrate by which liganddependent conformations of the receptor could induce differential gene activation utilizing diverse signal transduction mechanisms.

B. Distribution of ERc~ and ERfl in the Central Nervous System The gonadal steroid, estradiol, has widespread effects on the brain. The sites of estradiol's action in the CNS have been explored by examining binding sites for estradiol using autoradiography as well as expression of ER mRNA and protein using in situ hybridization and immunocytochemistry, respectively. Since the discovery that there are two subtypes of ER, a and fl (see Section II,A), studies have been conducted in several species to compare the distribution of these different receptor subtypes and gain some insight on the possible role of ERfl based on its specific anatomical location compared to that of ERa. The distribution of estrogen binding sites found in the brain of females of different species exhibits many commonalties [54]. In all vertebrates examined to date, estrogenbinding cells are found in the medial preoptic area, the mediobasal hypothalamus, the limbic forebrain including the septum, bed nucleus of the stria terminalis, and corticomedial amygdala, as well as the midbrain tegmentum. Furthermore, in rodents there is evidence that the cell groups that bind estrogen in the brain participate in hormonally controlled functions and behaviors, such as gonadotropin secretion and mating behavior [54,55]. Estrogen receptor fl mRNA and protein distribution in the brain has been described only in rats, monkeys, and E R a knockout mice (ERaKO) [38,56-59]. Although there are some discrepancies among the reported distribution of both ER subtypes in rats, the data so far show that both E R a and ERfl are abundantly expressed in limbic forebrain areas such as the bed nucleus of the stria terminalis, the medial amygdala, the medial preoptic area, and the periventricular nucleus of the hypothalamus (see Table II) [57,58]. Estrogen receptor c~ mRNA is expressed at much higher levels than is ERfl mRNA in two mediobasal hypothalamic areas, the ventromedial and arcuate nuclei, whereas ERfl mRNA is more highly expressed (and possibly exclusively) in the supraoptic and paraventricular hypothalamic nuclei, the cerebral cortex, and the cerebellum. Less clear is the differential expression of E R a and ERfl mRNA in some other areas of rat brain. Laflamme and colleagues reported higher expression of ERc~ mRNA, compared to ERfl mRNA, in the CA3 area of hippocampus and exclusive expression of ERa in the locus coeruleus [58], one of the major sites of noradrenergic neurons innervating the forebrain. In contrast, Shughrue et al. [57] reported the opposite pattern than that observed by Laflamme et al. [58] in the hippocampus and equal expression

40

L6PEZ

TABLE II

ET AL.

Distribution of ERa and ERfl in the Central Nervous System of Female Rats a H y b r i d i z a t i o n signal r e p r e s e n t i n g m R N A b D e n s i t y of labeled cell bodies r

Relative intensity of labeling d

B r a i n area

ERa

ERfl

ERa

ERfl

Isocortex

-/+

+/+ + +

Hippocampus (CA1-CA3)

-/+

+ + +

Basal n u c l e u s of M e y n e r t Medial amygdala

+ + + + +

+ + + + +

N.L. + + + e

N.L. + +/+ + +

-/+

+ +

-/+

Lateral s e p t u m

+ +

+

+ +/+ + +

+

Medial septum

+

+ +

+ +

-

+ + + + +

+ + + + + +

+ + + + +f

+ + +f

N u c l e u s of the diagonal b a n d B e d n u c l e u s of the stria terminalis (posterior) Substantia innominata M e d i a l preoptic area Periventricular hypothalamic nucleus S u p r a o p t i c nucleus

N.L.

N.L.

-

-

+ + + +

+ + + +

+ + +

+ +/+ + +

+ + +

+

+ +

+

-

+ + + +

-

+ + +/+ + + +

-

+ +/+ + + +

P a r a v e n t r i c u l a r h y p o t h a l a m i c nucleus V e n t r o m e d i a l h y p o t h a l a m i c nucleus

+ .qt_.qt_ e

_ g

.qt_ --I- + + h

A r c u a t e nucleus

+ + +

+

+ + +

+

+ +

+

+ + +

-/+

Dorsal r a p h e Locus coeruleus

+

+ +

N.L. + + +

N.L. -

Cerebellum

-

+ + /+ + +

-

+ /+ +

P e r i a q u e d u c t a l gray

-/+

+

+ +/+ + + + ..]_[..1_ + h

a D a t a a c c o r d i n g to S h u g h r u e et al. [57] and L a f l a m m e et al. [58]. bN.L., Not listed; - , undetectable signal; + , low but positive signal; + + , moderate signal; + + + , strong signal; + + + + , very strong signal. CFrom S h u g h r u e et al. [57]. d F r o m L a f l a m m e et al. [58]. e

Posterior part.

f Principal part. g Ventrolateral part. h C a u d a l part.

of ERa and ERfl mRNA in the locus coeruleus. Table II summarizes the localization of the two receptor subtypes in a variety of brain areas according to both research groups. Shughrue and colleagues [56] found ERfl mRNA to be expressed in ERaKO mice in many of the same areas in which it is expressed in normal rats, including the bed nucleus of the stria terminalis, medial amygdala, medial preoptic area, and paraventricular nucleus of the hypothalamus. Of particular interest was the low level of ERfl mRNA expression in the supraoptic nucleus in ERceKO mice, an area in rats that shows a high degree of both ERfl mRNA and protein expression [57-59]. However, ERceKO mice are exposed to very high levels of estrogen as a consequence of the lack of negative feedback effects of estrogen mediated by ERa. Therefore, these changes in ERfl expression may be the result of the different endocrine milieu to which these animals are exposed. Pau et al. used a reverse transcriptase polymerase chain reaction (RT-PCR) and in situ hybridization to determine the differential expression of ERa and ERfl mRNA in rhesus macaque brain [38]. Because the full-length cDNAs for the

two subtypes of ER have not yet been cloned in the monkey, the authors refer to their findings as putative sites of ER expression. In most areas examined in female monkey brain, both ERce and ERfl mRNA were expressed. These areas include the septum, amygdala, medial preoptic area, medial basal hypothalamus, and paraventricular hypothalamic nucleus. Estrogen receptor ce mRNA alone was found in areas such as the frontal cortex and locus coeruleus. As the question of differential expression of the ER subtypes is investigated in more species and as additional specific antibodies that discriminate ERa and ERfl become available, a clearer picture of their pattern of expression in the brain should emerge and a better understanding of their physiological role should arise. Colocalization of ERa and ERfl mRNAs in the same cells in brain has been studied. In female rats, many cells express ERfl mRNA and are immunoreactive for ERce in the preoptic part of the periventricular nucleus, the posterior part of the bed nucleus of the stria terminalis, and the medial nucleus of the amygdala [60]. A smaller number of double-labeled cells are found in the medial preoptic area, and only a few double-

CHAPTER3 Role of Gonadal Steroids in Menopause labeled cells are found in the ventromedial and arcuate nuclei of the hypothalamus. The consequences of this coexpression of the two ER subtypes are presently unknown; however, it is likely that cells that express one or both of the ER subtypes respond to estrogen differently. In addition, cells in which ERa and ERfl are expressed may express an array of genes that could be activated by the heterodimer a/fl (see Section II,A). Studies in female rats suggest that specific areas of the brain may become less sensitive to estrogen with aging. Comparisons of nuclear ER concentrations in the medial preoptic area and hypothalamus show a decline with aging under several endocrine conditions [61-63]. Under one of these paradigms, Wise and Parsons [64] evaluated female sexual behavior, and the deficit in sexual behavior paralleled the decline in ER concentrations, suggesting that the deficit in behavior resulted from aging-dependent estrogen insensitivity. Whether similar mechanisms are operative in humans is yet to be determined.

C. D i s t r i b u t i o n o f P r o g e s t e r o n e R e c e p t o r in the C e n t r a l N e r v o u s S y s t e m In all vertebrate species studied to date, progesterone receptor (PR) is found in the medial preoptic area and mediobasal hypothalamus [65]. In most species, PR is also found in other regions of the brain, including the limbic forebrain, midbrain tegmentum, cortex, and cerebellum [65,66]. Similar to ER binding, PR binding may decrease with aging in rats. Wise and colleagues showed a decline in the concentration of PR binding in the medial preoptic area and mediobasal hypothalamus of middle-aged rats compared to young rats after 2, but not 4, days of estradiol treatment [64]. Furthermore, this decline in PR in middle-aged rats correlated with deficits in all aspects of female reproductive behavior tested [64]. In contrast, Brown and colleagues did not detect a decrease in progesterone binding in various microdissected regions, including the medial preoptic area and mediobasal hypothalamus in young compared to older rats [63]. However, unlike the paradigm used by Wise and Parsons [64], Brown et al. [63] exposed the animals to estrogens for 3 days and attained higher circulating levels of estrogens, thus it may be that under certain hormonal conditions there is a decline in PR binding with aging. From a strictly functional point of view, PRs can be divided into two distinct anatomical classes; one class is induced by estradiol and the other is not [66]. A variety of studies examining several species, and using either immunocytochemistry for PR binding of radiolabeled progesterone or in situ hybridization for PR mRNA, show that estradiol induces PRs in the medial preoptic area and mediobasal hypothalamus [67-72]. Furthermore, estrogen and progesterone receptors are localized in the same cells in these areas

41 of the guinea pig brain [73]. In rats, the time course of PR induction by estrogen and the subsequent decline of PRs is similar to that observed in the onset and decline of reproductive behavior and the LH surge, demonstrating an association between these events [74-77]. Additionally, studies in ERaKO mice have provided some mechanistic insights into the estrogen-dependent regulation of PRs. In animals lacking ERce, it has been shown that estradiol induces both PR mRNA and protein in the medial preoptic area and mediobasal hypothalamus (albeit at lower levels than in wildtype animals) [78,79]. These observations suggest that both ERce and ERfl are involved in estrogen-dependent induction of PR expression in these areas. In other areas of the brain, including the medial amygdala, thalamus, and cortex, estrogen does not induce PR expression [68,69], even though ERs are present in these areas. Whether both receptors are located in the same cells in these areas remains a question and the absence of such colocalization could explain the lack of effect of estradiol on PRs in these specific areas of the brain.

IV. O V A ~ A N

STEROID

IN T H E C E N T R A L CONTROL

ACTION

NERVOUS

SYSTEM--

OF REPRODUCTION

Although it is clear that ovarian steroids act directly on the CNS to control reproductive function, the mechanisms by which this is accomplished are yet to be elucidated. The role of LHRH as a primary regulator of reproductive function suggests that feedback regulation of LHRH synthesis and release by ovarian steroids is the most direct route for modulating reproductive function. Yet, there is little evidence that ovarian steroids act directly on LHRH neurons [21,23-25]. In contrast, much evidence suggests that other neurotransmitter and neuropeptide systems mediate the effects of ovarian steroids on LHRH neurons [77,80]. A selective description of possible mechanisms by which ovarian steroids act on the brain to control reproductive function follows.

A. Effects o f E s t r o g e n s o n the L H R H N e u r o n a l N e t w o r k 1. NEGATIVE FEEDBACK EFFECTS

A classical action of ovarian steroids, particularly estrogen, on LHRH secretion is the suppression of LHRH release via negative feedback (Fig. 1). Although there is much evidence for this action of estradiol on the hypothalamicpituitary axis, the actual mechanisms underlying this effect are not clear. The paucity of LHRH neurons and their lack of distinct anatomical organization have hampered the analysis of the regulation of LHRH gene expression and secretion in

42 mammalian model systems. Therefore, studies of mRNA synthesis and LHRH secretion have proved difficult, and data are highly dependent on the experimental model used. However, due to the development of in vitro cell models and increased sensitivity of techniques for analysis of mRNA levels, our knowledge of LHRH gene expression and release has increased dramatically. Measurement of changes in LHRH mRNA levels in response to estrogen in rodents by in situ hybridization has provided conflicting results, and these have been reviewed extensively [81-83]. Overall, in studies that find a change in LHRH gene expression with ovariectomy, the change found is small, making it unlikely that the large increase in LHRH secretion observed with estrogen withdrawal is solely subserved by a change in mRNA synthesis. Instead, ovarian steroids may be acting at the level of mRNA translation and/or LHRH secretion to elicit their effects. In 1990, Mellon and colleagues [84] developed an immortalized mouse LHRH neuronal cell line GT1, which provides a model to address mechanistic problems on the regulation of LHRH neurons in vitro. Like LHRH neurons in vivo, GT1 cells secrete LHRH in a pulsatile manner [8587]. Furthermore, several groups have shown that GT1 cells express functional ERs. Poletti et al. [88] determined that ERs are present in a subclone of the GT1 cells, based on whole cell binding experiments. Estrogen receptors in immortalized LHRH neurons are low in abundance, but have high affinity for estradiol [88,89]. The low binding capacity of ERs in GT1 cells (6.2 fmol/mg cytosolic protein) [88,89] compared with MCF-7 cells, a human breast carcinomaderived cell line (60 fmol/mg cytosolic protein) [90,91], may help to explain why the majority of in vivo studies failed to detect ERs in LHRH neurons [21,23-25]. The presence of ERa mRNA transcripts in GT1-7 cells has been demonstrated by RT-PCR followed by Southern hybridization [89]. Identification of ERa mRNA- and estradiol-binding sites in immortalized LHRH neurons does not preclude the existence of ERs that are incapable of mediating transcriptional activation. However, transient transfection studies have shown that endogenous ERs can be activated sufficiently by estradiol to drive transcription of a luciferase reporter gene via an estrogen response element (ERE)-dependent mechanism [89]. Although the construct used in these studies contains a consensus vitellogenin ERE, the fact that estrogen action occurred at physiological concentrations of the steroid indicates that LHRH neurons may respond to estrogens under in vivo conditions by activating or repressing particular genes. The pure antiestrogen, ICI182-780, and other antiestrogens specifically block this response [89], suggesting that this effect is ER dependent. Further evidence for functional ERs in GT1 neurons comes from demonstrations of estradiol-dependent modulation of androgen receptors in the GTI-1 subclone [88] and galanin gene expression in the GT1-7 subclone [89].

L6PEZ ET AL.

Examination of the effects of estrogen on transcription of the LHRH gene in cultured LHRH-expressing neuronal cell lines has not revealed direct regulation by ERs through binding to regulatory sequences. Moreover, evaluation of cloned rat and mouse LHRH promoter sequences indicates that they do not contain recognizable EREs. However, the human LHRH promoter does contain an ERE that binds purified ER from calf uterus in DNase footprinting studies [92]. These observations imply that if LHRH neurons do indeed produce functional ERs as was shown by immunocytochemistry [25] and via nested RT-PCR [92a], the necessary cisacting elements are present in the human LHRH gene for ER-dependent regulation. From a molecular perspective, it is extremely difficult to delineate the mechanism(s) by which estradiol elicits ER-dependent gene expression in vivo in LHRH neurons. However, the functional relevance of this site in LHRH-expressing neurons has yet to be demonstrated in LHRH neurons or cell lines. 2. P O S I T I V E FEEDBACK EFFECTS In spontaneous ovulators, including humans, the rising tide of estradiol during the first phase of the reproductive cycle exerts positive feedback effects both on the brain to induce the hypersecretion of LHRH and on the pituitary to increase its sensitivity to LHRH. These events result in the generation of a preovulatory gonadotropin surge and culminate in ovulation [55,93]. The molecular mechanisms governing the hypersecretion of LHRH and the resulting preovulatory surge of LH are still not well understood, however, it is clear that these physiological processes are dependent on the action of the ovarian steroids, estradiol and progesterone, on the CNS [55,93]. Plasma levels of LHRH in the portal circulation increase at, or very near, the time of the LH surge [94-97]. It remains a subject of some controversy whether this increase in LHRH secretion and the steroid-induced LH surge are associated with alterations in the cellular content of LHRH mRNA. In rats and ferrets, the results of studies examining levels of LHRH mRNA relative to a spontaneous or an ovarian steroid-induced LH surge are contradictory [98108]. Even when the general findings among a set of these studies agree, the reports are in discord with one another on the time course of the rise and fall in LHRH gene expression relative to the LH surge. Several laboratories generally have found either little difference in LHRH mRNA over the estrous cycle or at the time of an LH surge [100102,104,107,108], or have found that LHRH mRNA gene expression increases prior to, or at the time of, a preovulatory LH surge in at least a subset of LHRH neurons [98100,103-106]. The sources of the discrepancies among the many studies that have examined LHRH mRNA relative to the LH surge remain largely unknown. (For a thorough examination of these issues, see Refs. 81 and 82.) Those studies that reported a change in LHRH gene expression prior to

CHAPTER 3 Role of Gonadal Steroids in Menopause or at the time of the LH surge show that the increase is relatively small. This scarce change is unlikely to account for the large increase in LHRH secretion at this time, making it likely that other mechanisms, such as translation, processing, and release, contribute to hypersecretion.

AT

3. ACTIVATION OF L H R H NEURONS THE TIME OF THE L H SURGE

Expression of c-Fos or other immediate early gene products by individual neurons can be used as a marker of cell activation and has been used to gain a better understanding of the molecular mechanisms underlying LHRH secretion. Work in several laboratories has shown increased expression of c-Cos mRNA and protein in LHRH neurons near the time of the LH surge in rats [ 107,109-116], mice [ 117], hamsters [118,119], and sheep [120]. This phenomenon is restricted to the time of the LH surge and depends on synaptic transmission [ 110,114]. This suggests that increased transcription of one or more genes in LHRH neurons accompanies the release of LHRH and may be responsible for initiating events that are crucial for the LH surge mechanism. The time course of c-Cos mRNA and protein expression in LHRH neurons has been examined in the rat. Lee and colleagues found that the number of LHRH neurons expressing c-Fos increases during the ascending phase of the LH surge [113]. In a more detailed study, Finn and colleagues found that levels of c-Cos mRNA in LHRH neurons are significantly elevated only after serum LH levels begin to rise, 2 hours before the peak of the LH surge [107]. Concomitantly with the occurrence of the LH surge, LHRH neurons also express Jun, another immediate early gene product [ 112]. Fos and Jun form a heterodimer, AP-1, which is known to regulate expression of a variety of genes [ 121]. Among the possible target genes for AP-1 regulation in LHRH neurons are the genes for LHRH and galanin (GAL) (this is discussed in Section III,A,4). The pattern of c-Fos expression in rat LHRH neurons changes during aging [ 122-124]. Specifically, fewer LHRH/ c-Fos double-labeled neurons were found in middle-aged compared to young rats at the time of either a preovulatory or a sex steroid-induced LH surge. Furthermore, c-Fos induction was delayed and abbreviated in LHRH neurons of middle-aged rats compared to younger animals. These data suggest differences in the molecular physiology of LHRH neurons during the aging process. Witkin and colleagues evaluated c-Fos in LHRH neurons at the time of the surge in female rhesus macaques and observed little expression of the immediate early genes [ 125]. There were no differences in the number of double-labeled cells in intact and ovariectomized females and among ovariectomized females treated with oil, estradiol, or estradiol and progesterone [ 125]. Luteinizing hormone levels were verified and the animals were sacrificed at the time of unchanging or ascending LH levels. The findings in this study can be interpreted either as showing that LHRH neurons in primates are

43 not activated at the time of the LH surge, that LHRH neurons may be activated, but express immediate early gene products other than c-Fos, or that the animals were sacrificed before c-Fos levels in LHRH neurons increased. In summary, it is clear that in at least some species LHRH neurons increase expression of c-Fos at the time of the LH surge. Although it is known that c-Fos and other immediate early genes increase gene expression, the identity of the genes induced in LHRH neurons by immediate early genes and the consequences of their induction remain unknown. 4. GALANIN AS A MARKER OF SEXUALLY DIMORPHIC EFFECTS OF ESTROGEN

Galanin is widely distributed throughout the CNS [126] and is expressed in rats in a subset of LHRH neurons in the diagonal band of Broca and rostral medial preoptic area [ 127,128]. This observation represented the first example of the existence of another neuropeptide in the LHRH neuronal network, and further studies have corroborated the presence of GAL mRNA [129]. Given the participation of endogenous GAL in the control of gonadotropin secretion in female rats during proestrus (reviewed in Section III,B,1), and the responsiveness of GAL to estrogen in other tissues [130], including brain [131,132], it was posited that GAL within LHRH neurons is sensitive to gonadal steroids and may contribute to the molecular mechanisms underlying the hypersecretion of LHRH that induces the preovulatory surge of LH from the pituitary. The expression of GAL mRNA and peptide in rat LHRH neurons is sexually dimorphic and extremely sensitive to estrogen. The sexual dimorphism favors females, in which the majority of LHRH neurons, about 6 0 - 8 0 % , express GAL mRNA/peptide [ 129,131 ]. In male rats, only 10-15% of LHRH neurons express GAL. This same pattern exists at the level of LHRH axon terminals at the median eminence, where GAL and LHRH are copackaged in the same secretory vesicles [133]. The copackaging of these two peptides provides the anatomical and biochemical bases for our observation of cosecretion of LHRH/GAL into hypophyseal portal blood [ 134]. The sexual dimorphic expression of GAL in LHRH neurons is due to the action of sex steroids acting both in adulthood (activating effects of sex steroids) and during early perinatal development (organizing effects of sex steroids). The number of LHRH neurons coexpressing GAL does not change in adult male rats with orchidectomy and subsequent testosterone or estradiol replacement therapy, whereas in female rats ovariectomy drastically reduces the number of LHRH neurons expressing GAL (from 80% to approximately 15%) [135]. Estradiol (or testosterone) therapy restores the number of double-labeled neurons to control levels [ 135]. However, if male rats are neonatally orchidectomized, their LHRH neuronal system is responsive to estrogen in adulthood, demonstrating that the system is sexually differ-

44 entiated [135]. Thus, colocalization of GAL in a subset of LHRH neurons represents a situation in which a primary phenotype (LHRH expression), which is not sexually dimorphic, is expressed in all neurons and another phenotype (GAL expression), which is sexually dimorphic, is expressed in a subset of neurons. Functionally, the observations imply that at least three subsets of LHRH neurons exist in the forebrain of male and female rats: (1) a subset that does not express GAL under any steroidal condition; (2) a subset that expresses GAL independent of steroidal input; and (3) a subset that expresses GAL only under the appropriate steroidal input (Fig. 6). The neurons of this third subset, although present in the brains of both males and females, are much more numerous in the female brain (Fig. 6). Gene expression studies in rats demonstrate a close association between the induction of GAL mRNA in LHRH neurons and the production of an LH surge. Galanin mRNA levels in LHRH neurons increase nearly two-fold between the morning and afternoon of a preovulatory or steroid-induced LH surge [102,107,136]. A thorough examination of GAL gene expression in LHRH neurons relative to a steroid-induced LH surge reveals that the actual increase in GAL mRNA levels in LHRH neurons occurs after LH levels rise and, once elevated, remain high well after the completion of the surge [107]. This rise in GAL mRNA levels in LHRH neurons does not occur if the LH surge is blocked by disrupting synaptic transmission by any number of treatments [136,137]. These data taken together show that the induction of GAL gene expression in LHRH neurons is closely associated with an LH surge and are consistent with a role

L6PEZ ET AL.

for GAL release from LHRH neurons in the LH surge mechanism. The rise in GAL mRNA levels in LHRH neurons at the time of an LH surge is sex steroid dependent and sexually differentiated. Treatment of ovariectomized rats with estradiol at levels sufficient to elicit a small LH surge results in an increase in GAL mRNA levels in LHRH neurons [138]. When estradiol treatment is followed by progesterone administration, a larger release of LH is elicited and higher levels of GAL mRNA in LHRH neurons are attained. Administration of progesterone alone, however, has no effect on serum LH levels or on GAL mRNA concentrations in LHRH neurons [138]. As perinatal exposure to testosterone precludes the ability of rats primed with ovarian steroids to undergo an LH surge, it also precludes the ability of sex steroid priming to elicit GAL gene expression in LHRH neurons [139]. Thus, the same mechanisms responsible for the sexual differentiation of the LH surge mechanism appear to be responsible for sexual differentiation of the induction of GAL gene expression in LHRH neurons in response to ovarian hormone stimulation. As reviewed in Section III,A,3, LHRH neurons express c-Fos mRNA and protein at the time of the LH surge and there is evidence providing strong support for the hypothesis that GAL gene expression is activated by c-Fos. First, there are elements in the 5' flanking region of the rat GAL gene that are able to bind members of the Fos/Jun family of transcription factors [ 140]. Second, both c-Fos and GAL mRNA are induced in LHRH neurons coincident with a preovulatory or steroid-induced LH surge [102,107,109-116,136139]; the induction of both is dependent on synaptic transmission [110,114,136,137]. Third, GAL mRNA is expressed in the majority of the LHRH neurons that colocalize c-Fos protein at the time of a steroid-induced LH surge [115]. Fourth, in estrogen-primed rats, progesterone increases both the number of LHRH neurons that express c-Fos and the level of GAL gene expression in LHRH neurons [111,138]. Finally, the first detectable rise in GAL mRNA levels in LHRH neurons occurs 2 hr after c-fos mRNA is induced in these neurons [ 107], providing sufficient time for translation of c-fos mRNA into c-Fos protein and the initiation of GAL gene expression by an AP-1-dependent mechanism.

B. N e u r o t r a n s m i t t e r a n d N e u r o p e p t i d e S y s t e m s I m p l i c a t e d in L H R H / L H FIGURE 6 Functional subpopulations of luteinizing hormone-releasing hormone (LHRH) neurons. Expression of galanin in luteinizing hormonereleasing hormone neurons is sexually dimorphic and neonatally determined. In this respect, LHRH neurons can be classified as those that never express galanin, those that express galanin under basal conditions and galanin levels are not regulated by estradiol, and those that express galanin only after estrogen treatment. The latter cell subpopulation occurs primarily in female rats and in neonatally orchidectomized males.

Regulation

1. GALANIN Galanin [141] has emerged as an important modulator of reproductive events (see Ref. 126 for a recent review). Galanin stimulates LH secretion in rats, and these effects may be mediated, at least in part, by increasing LHRH secretion. This conclusion is based on the following observations:

CHAPTER 3 Role of Gonadal Steroids in Menopause (1) GAL stimulates LHRH release from arcuate nucleusmedian eminence fragments obtained from male or female rats [128,142,143]; (2) infusion of GAL into the cerebral ventricles stimulates LH secretion in estradiol-primed, ovariectomized rats [143,144], and this effect is blocked by prior infusion of the GAL receptor antagonist, galantide [143]; and (3) infusion of galantide alone into the brain blunts the LH secretion that occurs with either a steroid-induced or spontaneous LH surge [143], as does passive immunization against GAL [145]. Based on these and other observations of higher GAL expression in LHRH neurons of females compared to males, and the close association between induction of GAL gene expression in LHRH neurons and the LH surge, it has been postulated that the GAL derived from LHRH neurons facilitates LH secretion. In fact, early studies showed direct effects of GAL on basal and LHRHinduced LH secretion from the anterior pituitary gland in vitro [127,134]. 2. NOREPINEPHRINE

Noradrenergic neurons play a role in the release of LHRH and LH, as demonstrated by experiments in rodents, rabbits, and primates [80]. In each of these species, disruption of noradrenergic activity results in a suppression of pulsatile LH secretion. This suppression appears to be mediated by ce-adrenergic receptors [80]. In monkeys, the release of norepinephrine from the stalkmedian eminence is pulsatile and the pulses of norepinephrine occur synchronously with pulses of LHRH [146]. celAdrenergic stimulation at the stalk-median eminence level, either by norepinephrine or methoxamine, increases LHRH secretion, whereas cel-adrenergic blockade by prazosin decreases LHRH release [146-148]. Infusions of other adrenergic compounds that act through ce2- or/3-adrenergic receptors have no effect on LHRH secretion [147,148]. The action of norepinephrine on LHRH secretion appears to be mediated, at least in part by prostaglandin E 2, because norepinephrine-stimulated LHRH secretion at the stalk-median eminence is associated with an increase in extracellular prostaglandin E 2 levels, and infusion of prostaglandin E 2 stimulates LHRH secretion [147,148]. Noradrenergic input also appears to be important for the generation of an LH surge in rats. Noradrenergic turnover (a measure of neuronal activity) increases in the medial preoptic area and median eminence at the time of an estrogeninduced LH surge [ 149]. In addition, the episodic release of norepinephrine increases in the medial preoptic area between the morning and afternoon on the day of the estrogen-induced LH surge [150]. An increase in noradrenergic turnover is also found in these same areas coincident with the LH surge in ovarian steroid-primed rats as well as in regularly cycling rats [149,151]. Additionally, disruption of noradrenergic input by lesions or surgical transection blocks the LH surge in regularly cycling rats [152,153], however

45 these effects are transient. Similarly, inhibition of noradrenergic transmission results in the blockade of the LH surge [ 152-156], and these effects appear to be mediated through a-adrenergic receptors. Noradrenergic input may be of less importance to the generation of an LH surge in primates, because ovulation occurs in monkeys even after complete dennervation of the medial basal hypothalamus [157], which severs ascending noradrenergic inputs from the hindbrain. However, this persistence of function may be more a reflection of redundancy in the neural circuitry controlling ovulation than a demonstration that noradrenergic input does not normally affect LHRH secretion at the time of the LH surge in primates. Noradrenergic neurons are restricted to various cell groups in the hindbrain, of which several accumulate estrogen [ 158]. These same cell groups send noradrenergic projections to the hypothalamus [ 159], but it is unknown whether these inputs are estrogen sensitive. In the rat, neurons containing dopamine fi-hydroxylase (a marker of noradrenergic and adrenergic neurons) synapse near, but not on, LHRH neurons [1601, suggesting an indirect effect of norepinephrine on LHRH secretion. However, the immortalized LHRH-containing cell line GT1 does express fil-adrenergic receptors [161], suggesting that a direct interaction occurs between noradrenergic and LHRH neurons. 3. OPIOIDS

Opioids exert tonic inhibitory effects on LHRH and LH secretion in mammals. These effects appear to be mediated by two opioids, fl-endorphin and dynorphin [77,162]. Estrogen can exert direct effects on fl-endorphin- and dynorphinproducing neurons, because subsets of these neurons bind estrogen [ 163,164]. However, it is currently unknown which ER subtype mediates the effects of estrogens on these subpopulations of neurons. Studies in rats indicate several effects of estrogen on opioid systems. For example, ovariectomy increases and estrogen replacement decreases mRNA levels for the precursor of fl-endorphin, proopiomelanocortin, in the arcuate nucleus [165-167]. On the morning of proestrus, when estrogen levels are high, fl-endorphin levels decrease in the arcuate nucleus, the site where it is synthesized, and fl-endorphin levels increase in the median eminence, the site from which it is released into the hypophyseal portal blood [168,169]. Thus, estrogen also appears to influence the transport and/or release of this peptide. A direct action of fl-endorphin on LHRH secretion is supported by studies showing direct synaptic contacts between LHRH and fl-endorphin-containing neurons at the level of the medial preoptic area in rats [ 1 7 0 172] and the mediobasal hypothalamus in primates [173]. Although a study examining the expression of opioid receptors in rat LHRH neurons reported negative results [174], this may be due to a lack of sensitivity in the technique employed. The immortalized LHRH cell line G T I - 1 does

46

L6PEZ ET AL.

express the 6 opioid receptor and both the G T I - 1 and GT1-7 lines respond to opioids with decreases in LHRH secretion [ 175-177]. Although it is clear that opioids influence LHRH/LH secretion, whether these effects are direct or mediated by other neurons is yet to be elucidated. 4. SUBSTANCE P Tachykinins, or neurokinins, are a class of peptides including tachykininA, tachykininB (also known as substance K), and substance P, which are derived from protachykinin precursors. Tachykinins are widely distributed in the brain and are particularly abundant in the medial preoptic area and mediobasal hypothalamus (see Brownstein et al. [ 178] and Ljungdahl et al. [ 179] for a complete description). Infusion of substance P peripherally or directly into the ventricles of the brain stimulates LH secretion in estrogenprimed ovariectomized rats [ 180], whereas blockade of substance P effects, with intracerebroventricular infusion of antiserum, suppresses LH secretion in ovariectomized rats [ 180]. These effects could be explained by direct effects of substance P on LHRH neurons, because substance P-containing neurons appear to form synaptic contacts with LHRH neurons [181]. Substance P levels are responsive to estrogen and fluctuate in regions of the forebrain during the estrous cycle [ 182,183]. Moreover, estrogen treatment of ovariectomized rats increases substance P levels in the medial preoptic area preceding the daily rise in LH secretion [184] and increases substance P immunoreactivity in the medial amygdala [185]. This rise in substance P levels may be subserved by rises in mRNA levels, because estrogen treatment of ovariectomized rats increases tachykinin mRNA levels in the ventromedial nucleus of the hypothalamus and in the medial amygdala [ 186,187]. The effects of estrogen on a subpopulation of substance P neurons in the mediobasal hypothalamus appear to be direct because those neurons concentrate estrogen [ 188].

rapidly around age 35 [194]. This is illustrated in Fig. 7 [195a,195b], which shows the number of remaining ovarian follicles in relation to age. The rate of follicular loss during the first 30 years progresses steadily. Assuming this decline continues at a constant rate, extrapolation reveals that the age at follicular exhaustion would be 80 years. However, this extrapolation is not correct, because around age 35 years the rate of follicular disappearance increases dramatically [190], and follicular exhaustion actually occurs at a much earlier age. The age-related reduction in the follicular pool and the ensuing infertility are associated with a marked elevation in levels of FSH during the follicular phase of the menstrual cycle [ 196-201 ]. This increase in FSH levels commences at age 35 years [197] and is followed several years later by increases in LH levels [196,197,201]. Elevated levels of gonadotropins are the consequence of a loss in ovarian estradiol secretion [198,199]. In particular, elevated follicular levels of FSH in women over age 45 years are associated with reductions in total inhibin levels [202], contributing, therefore, to a differential elevation of pituitary gonadotropins. Inhibin is a dimeric glycoprotein produced by some cell types of the gonad. It is composed of an ce and one of two/3 subunits,/3A and/3B, resulting in two types of inhibins, A (ce-/3A) and B (ce-/3B) [203,204]. Studies have characterized in detail the levels of both inhibins during female reproductive aging [205]. Inhibin B levels are reduced during the follicular phase of the menstrual cycle in older women (Fig. 8). This decrease in inhibin B concentration is accompanied by an elevation in serum FSH levels during the

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Reproductive aging is characterized by a gradual reduction of the ovarian follicular pool, which underlies the associated decline in fertility and endocrine changes observed as menopause progresses [ 189-192]. The endocrinological aspects of the menopausal transition have been extensively reviewed [193]. This issue is further discussed in Chapter 9. Rather, the most evident changes in menopause as they relate to the action (or lack thereof) of estrogen on the hypothalamic-pituitary-gonadal axis will be highlighted. The decline in fertility begins in a moderate, progressive fashion in the third decade of life [194,195], but begins to accelerate

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CHAPTER

3 Role of Gonadal Steroids in Menopause

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FIGURE 8 Mean inhibin B, follicle-stimulating hormone (FSH), estradiol (E2), inhibin A, and progesterone (P4) levels in cycling women 2 0 - 3 4 years old ( 9 and 3 5 - 4 6 years old (e). Hormone levels are depicted as centered to the day of ovulation (., P < 0.04; **, P < 0.02, when comparing the two age groups). From [205], Welt, C. K., McNicholl, D. L, Taylor, A. E., and Hall, J. E. (1999). Female reproductive aging is marked by decreased secretion of dimeric inhibin. J. Clin. Endocrinol. Metab. 84, 105-111. 9 The Endocrine Society.

early follicular phase, which results in sustained elevated concentrations of estradiol (Fig. 8). Furthermore, reduced levels of inhibin B are observed during ovulation, resulting in slightly higher FSH levels in older women after the ovulatory surge of FSH and during the luteal phase. Inhibin A concentrations show a different pattern of secretion, i.e, inhibin A levels do not change during the follicular phase of the menstrual cycle and are significantly reduced during the luteal phase (Fig. 8). Luteinizing hormone and progesterone levels remain unchanged among the younger and older age groups (Fig. 8) [205]. Therefore, it appears that low inhibin B levels in older cycling women may be the earliest marker of accelerated follicular loss and reproductive aging [205] (see Chapter 9). The different subsets of LHRH neurons described in Section I,B may represent distinct functional pools of LHRH neurons. It has been shown that LHRH mRNA levels are increased only in the type I neurons during the menopause [33a]. These changes indicate that estrogens under physiological conditions somehow reduce production of LHRH mRNA. Consequently, the increase in LHRH mRNA would be related to an increase in production of the peptide, which would drive an increase in gonadotropins as observed in the menopausal period. The data described so far present an in, teresting view with respect to the action of estrogens in the gonadal axis. The consequences of reduced ovarian steroid action in the CNS are poorly understood, especially in terms

of regulation of the LHRH neuronal network function. However, it is clear that LHRH levels increase with menopause due to the diminished negative feedback of ovarian steroid hormones. Menopause is not a consequence of inadequate production of LHRH. Estradiol exhibits activational effects that are translated to elevations in the expression of various genes; for example, estrogens stimulate the expression of the GAL gene in LHRH neurons [206]. Conversely, estrogens also exhibit potent repression of various gene products, such as/3-endorphin [165-167], some of which are intimately related to the reproductive aging process. Similarly, LHRH mRNA elevation in a subset of LHRH neurons during the menopause [33a] would represent such a repression by estradiol. Thus, determining the physiological consequences of differential gene regulation by estradiol should increase our understanding of the pathophysiology of the menopausal period. Sex steroid secretion is eventually compromised in postmenopausal women [207], consistent with the follicular depletion. However, the consequence of estrogen withdrawal on brain function is poorly understood. Early studies described neuronal hypertrophy in postmenopausal women in the subventricular nucleus (a subdivision of the arcuate nucleus), suggesting a relationship to steroid depletion. More detailed studies have confirmed the original observations by clearly demonstrating that neurons in the arcuate nucleus of postmenopausal women are 30% larger than those of their

48

L 6 P E Z ET AL.

premenopausal counterparts [208,209]. Evidence for the involvement of estradiol depletion as the hypertrophic driver was provided by the observation of ER transcripts in the arcuate nucleus of pre- and postmenopausal women [208]. This anatomical association (ER mRNA presence and hypertrophy) strongly suggests that estrogen negative feedback occurs by a direct effect on a subpopulation of ER mRNAcontaining neurons in the arcuate nucleus [208]. In addition, this hypertrophic response has also been observed in older men [209], even though this effect is smaller in males (22%) than in postmenopausal females (30%) [208,209], reflecting the diverse endocrine status of males and females during reproductive aging. With respect to the identity of the hypertrophied neurons, Rance and Young III [210] demonstrated that the hypertrophied neurons located in the arcuate nucleus contain the tachykinins, substance P and neurokinin B. In addition, the expression of both of these neuropeptides was elevated in postmenopausal compared to premenopausal women [210]. This increase appears to be due to estrogen withdrawal and not to hypothalamic aging. This is based on evidence in monkey studies that show an increase in tachykinin mRNA levels after ovariectomy and abolition of the effect after estrogen replacement therapy [211 ]. These data, together with the evidence in rodents indicating that substance P has a stimulatory role in the control of gonadotropin secretion [180,212-215], suggest that substance P and tachykinins, in general, may be involved in the increased levels of gonadotropins observed during menopause. There are several possible mechanisms by which estrogen withdrawal at menopause results in increased levels of substance P. One possibility is that estrogen acts directly on tachykinin-producing neurons of the arcuate nucleus to inhibit substance P production, and this suppression is released with estrogen withdrawal at menopause. An alternate possibility is that estrogen is stimulatory to a system that inhibits substance P production. Estrogen is stimulatory (not inhibitory) to substance P production in rats, therefore these models are of little help in elucidating molecular mechanisms operating in humans. Such a task may require a nonhuman primate model whose reproductive physiology is more similar to humans. Thus, resolution of the mechanisms by which substance P levels are increased and the consequences of this increase in postmenopausal women await further investigation.

A. H o t F l u s h One of the most common and troubling physiological manifestations of menopause is the hot flush (hot flash). Hot flushes occur in about 85% of menopausal women whether the menopause occurs naturally or surgically [216,217]. They often first occur within months of the last menses and can persist for years after ovarian quiescence. Hot flushes

have been characterized by a variety of investigators [218222]. They begin by an ascending flush of the upper body, starting at the thorax caused by cutaneous vasodilatation. This results in a feeling of warmth. Blood pressure is stable, but changes in heart rate often occur. The vasodilatation causes a decrease in body core temperature, resulting in a sensation of cold that often elicits shivering. Hot flushes often occur at night (night sweats) and disrupt normal sleep patterns, leading to complaints such as irritability, fatigue, and forgetfulness. Estrogen replacement virtually eliminates hot flushes, demonstrating their origin in the estrogen-withdrawal that occurs with ovarian follicular depletion at menopause [222-224]. Hot flushes appear to result from a dysfunction of thermoregulatory centers in the hypothalamus and are correlated with pulses of circulating estrogen and gonadotropin secretion in menopausal women (see Fig. 9; see also Chapter 13) [225-228]. In studies employing frequent blood sampling, hot flushes have been temporally correlated with pulses of LH (Fig. 9) [229,230], yet it is unlikely that LH release initiates hot flushes. Hot flushes occur in women with disrupted pituitary function [231,232] or who have been treated with an LHRH agonist to suppress LH pulses [233,234]. Moreover, the rise in LH following discontinuation of the latter treatment does not induce hot flushes [235], further evidence against an LH-initiated mechanism for hot flushes. Because LH levels reflect LHRH release from the hypothalamus, hot flushes are also closely associated with LHRH secretion, but like LH, LHRH release appears to be correlated with, not causative of, hot flushes. This conclusion is supported by studies showing that women with abnormal LHRH

FIGURE 9 Schematicrepresentation of the consequences of the ovarian insufficiency occurring during menopausal transition. During menopause, the exhaustion of ovarian follicles results in a decrease in circulating levels of estradiol and inhibin. Under physiological conditions estrogenexhibits a tonic inhibitory influence on both the LHRH neuronal system and the thermoregulatory center in the hypothalamus. The lack of estrogen-dependent inhibition results in increased secretion of gonadotropins and the advent of hot flush episodes that are coincident with secretory episodes of LH release. This coincidence can be dissociated by treatment with LHRH analogs, suggesting an association between these phenomenarather than a cause-effect relationship.

CHAPTER3 Role of Gonadal Steroids in Menopause secretion and associated reproductive impairments experience hot flushes in response to estrogen withdrawal [236]. Thus, hot flushes occur in the absence of LHRH secretion. However, in women in which suppression of LHRH secretion appears to result from perturbations in neurotransmitter/ neuropeptide systems that control LHRH release [237,238], estrogen withdrawal did not result in hot flushes. It is currently thought that estrogen withdrawal influences neuropeptide/transmitter systems that act at the medial preoptic area/ hypothalamus to cause an overall lowering of the thermoregulatory set point. The association of hot flushes with increased LH secretion is due to the associated stimulation of LHRH neurons that also reside in these areas of the brain. In summary, during menopause, the exhaustion of ovarian follicles results in a decrease in circulating levels of estradiol and inhibin. Under physiological conditions estrogen exhib, its a tonic inhibitory influence of both the LHRH neuronal system and the thermoregulatory center in the hypothalamus (Fig. 9). The lack of estrogen-dependent inhibition results in increased secretion of gonadotropins and in the appearance of hot flush episodes that are coincident with secretory episodes of LH release. Noradrenergic neurons are one of several neurotransmitter and neuropeptide systems that have been implicated in the hot flush mechanism. Noradrenergic neurons are responsive to estrogen [239], and a subset of noradrenergic neurons projects to the medial preoptic area [240]. Furthermore, norepinephrine modulates LHRH secretion (reviewed in Section II,B,2) and thermoregulation [241 ]. The strongest evidence for hot flushes being mediated by disruption in noradrenergic systems comes from studies showing that clonidine, an c~-adrenergic agonist, reduces the frequency of hot flushes in postmenopausal women [242-245]. The exact mechanism by which clonidine reduces hot flush frequency is unknown, but possible mechanisms include centrally mediated stabilization of thermoregulatory centers in the medial preoptic area/hypothalamus and/or peripherally mediated blockade of vasomotor symptoms. The latter mechanism is supported by the observation that clonidine treatment in humans reduced the increase in forearm blood flow produced by epinephrine, norepinephrine, and angiotensin [246]. In the CNS, there is evidence suggesting that clonidine's action is dependent on stimulation of a2-adrenergic receptors. In addition, there are data indicating that clonidine may be effective by stimulating imidazoline-preferring binding sites, a potential new family of noradrenergic receptors [247]. The mechanisms by which estrogen alters noradrenergic systems are unclear. Some studies show that estrogen can increase central noradrenergic activity by increasing the activity of the rate-limiting enzyme in its synthesis [248], decreasing levels of an enzyme responsible for its catabolism [249], and inhibiting its reuptake [250]. Other studies show that estrogen lowers noradrenergic activity. In rats, ovariectomy (estrogen withdrawal) increases [251-253] and estro-

49 gen replacement decreases [254] hypothalamic noradrenergic activity. Thus, if hot flushes are caused by changes in noradrenergic input due to estrogen withdrawal, it is unclear whether this is the result in an overall increase or decrease in activity. Norepinephrine applied directly to the medial preoptic area of nonhuman primates stimulates many of the same physiological changes associated with hot flushes, such as peripheral vasodilatation, heat loss, and a decrease in core body temperature [255,256]. This supports the idea that an increase in noradrenergic activity may cause hot flushes. However, unlike women experiencing hot flushes, the animals in these studies also showed bradycardia and hypotension; therefore, this model does not completely mimic the physiological changes occurring in women during a hot flush. This may be due to restricted application of the norepinephrine to the medial preoptic area, or perhaps an increase in noradrenergic activity is not fully responsible for hot flushes. Estrogen withdrawal may result in changes in noradrenergic activity, and such changes could account for disruption in thermoregulatory centers producing hot flushes; however, other systems such as the opioid system are also implicated in the etiology of hot flushes. Opioid-containing neurons richly innervate the medial preoptic area [257] and subsets of opioid-containing neurons bind estrogen [258], similar to noradrenergic neurons. Also, like norepinephrine, opioids influence both reproduction (reviewed in Section III,B,3) and thermoregulation [259,260]. Several observations support the idea that opioids are involved in the etiology of hot flushes. First, placebo treatment in women significantly decreases hot flush frequency [261 ]. This effect could be mediated by an increase in opioid transmission since placebo effects can be blocked by blockade of opioid neural transmission [262]. Second, opiate and estrogen withdrawal share many of the same physiological symptoms, including hot flushes and sleep disturbances; however, opiate withdrawal is associated with additional symptoms that do not occur with estrogen withdrawal. Possible explanations for this dissociation are that opioids interact with other neural systems to induce the common symptomatology. Alternatively, a subset of opioid-producing neurons, those responsive to estrogen, may account for the symptoms present with both opiate and estrogen withdrawal, whereas the remaining opioid-producing neurons, those not sensitive to estrogen, are responsible for the additional symptoms seen with opiate withdrawal. Intriguingly, opiatewithdrawal symptoms can be alleviated in rats by estrogen treatment [263] lending support to the latter hypothesis. It has been shown in women that proopiomelanocortin (POMC; one of the precursor of opioids) mRNA levels in the hypothalamus decrease after menopause [264]. However, using a primate model (cynomolgus monkeys), the same group has shown that POMC mRNA levels are similar in untreated, estrogen-treated, and estrogen/progesteronetreated ovariectomized animals [211], suggesting that the

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decrease in POMC gene expression observed in postmenopausal women is due to hypothalamic aging, not to ovarian steroid withdrawal [211 ]. Presently, nothing is known about the physiological relevance of increased substance P levels in the arcuate nucleus of postmenopausal women (see Section III,B,4 for a more detailed description). However, like several of the neurotransmitters mentioned above, tachykinins have also been implicated in the etiology of some types of flushing, and might therefore play a role in hot flushes in menopausal women. In healthy men, substance P infusion induces both vasodilatation [265,266] and flushing. Furthermore, in individuals with carcinoid tumors, pentagastrin infusion, food, or alcohol can induce flushing, and this flushing is usually associated with increases in tachykinin levels [267-272]. Concurrent treatment with a somatostatin analog will decrease both hot flushing and tachykinin levels [268-272]. Although there is a strong association between increased tachykinin levels and flushing, the association is not absolute, suggesting that tachykinins are not the only cause of flushing in these individuals. Whether tachykinins, especially of central origin, play a role in the postmenopausal flush is yet to be determined.

B. C o g n i t i v e F u n c t i o n s a n d M o o d Many investigators have examined the effect of estrogen on cognition in postmenopausal women using a variety of tests that assess cognition. Although there are discrepancies in the results of these studies, in general, women receiving estrogen replacement after natural or surgical menopause score higher on a very limited number of memory tests. Thus, estrogen does not appear to enhance cognition globally, but rather appears to enhance only specific aspects of cognition (see Chapter 20). Some early studies of estrogen replacement in postmenopausal women found that estrogen improved memory as assessed by self-report and by objectively administered memory tests [224,273], whereas other studies did not [274,275]. Sherwin and colleagues undertook a series of studies examining the effects of estrogen replacement in women who had undergone surgical menopause. Memory tests were administered to a group of women prior to surgery. Postsurgery, the women were treated with estrogen, androgen, or estrogen and androgen for 3 months, treated with placebo for the fourth month, and then randomly crossed over to a different hormonal treatment group and treated for an additional 3 months. In the Paragraph-Recall Test of short-term verbal memory, scores did not differ significantly pre- and postoperatively in women who received sex steroids, whereas scores were lower post-operatively in women who received placebo [276]. Similarly, women who underwent surgical menopause were in another study in which patients were

treated with estrogens and no differences were observed on a retention test of new material (Paired-Associate Test) preversus postsurgery, whereas there was a decline in scores postsurgery in women treated with placebo [277]. In addition, scores on the Paragraph-Recall Test increased in the estrogen-treated women after surgery, but were merely maintained in the placebo-treated group [277]. Finally, there were no significant group differences pre- and postsurgery for two other memory tests (immediate or delayed visual recall) [277]. In a third study involving a larger number of women who underwent surgical menopause, Sherwin and colleagues found that women treated with estrogen scored higher postsurgery on specific cognitive tests (immediate and delayed recall of the Paired-Associate Test), whereas women treated with placebo had lower scores postsurgery [278]. Less has been done to examine estrogen's effects on cognition in women undergoing natural menopause. In one study, women between 45 and 60 years did not show any differences on two subtests of the Weschler Adult Intelligence Scale when treated with estrogen or with placebo, either when scores were compared within groups pre- versus postsurgically or when compared between groups postsurgically [279]. Other studies have found that postmenopausal women who had or were currently using estrogen scored higher on some cognitive tests involving verbal memory (Category Fluency Test, Mini-Mental State Examination, or immediate and delayed Paragraph Recall), but not on other verbal, visual, or spatial memory tests [280,281 ]. Taken together then, these studies examining the effect of estrogen replacement on cognitive functioning in women undergoing estrogen withdrawal show that estrogen appears to have positive effects on some specific aspects of verbal memory. However, this conclusion should be taken with caution, because most studies finding positive effects of estrogen on cognitive measures did not control for the effect of hot flushes that accompany estrogen withdrawal in a majority of women. This is important because hot flushes occur frequently at night, disrupting sleep, and this effect alone could be responsible for the poorer performance of women after estrogen withdrawal than before. Resolution of this confounding factor awaits the results of current studies examining the effect of estrogen replacement on cognition in postmenopausal women. At the present time, little is known about the neural mechanisms underlying estrogen's effects on memory; however, a likely site of action is the hippocampus. The hippocampus is intimately involved in learning and memory, and ERce and ERfl are both found in the hippocampus [58,282]. Studies in rats demonstrate that estrogen withdrawal after ovariectomy results in a decrease in dendritic spines in specific regions of the hippocampus and that this decrease can be reversed with estrogen treatment [283]. Moreover, the density of dendritic spines on a subtype of hippocampal neurons varies over the estrous cycle, with the highest number found at proestrus, coincident with peak estrogen levels and

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CHAPTER 3 Role of Gonadal Steroids in Menopause the lowest number at estrus, coincident with basal estrogen levels [284]. Changes in dendritic spines are thought to be one of the mechanisms involved in learning and m e m o r y [285]. Thus the dynamic changes estrogen exerts on dendritic morphology could be a factor explaining at least some of the effects of estrogen on cognition. In addition to cognition, estrogen may influence m o o d in women. Estrogen replacement therapy compared to placebo has been shown to decrease depression scores in postmenopausal w o m e n [279]. In a study of w o m e n over age 50 years, depression scores were found to increase with age in w o m e n that did not receive estrogen replacement, but did not increase in w o m e n who received estrogen replacement [286]. This positive association between m o o d and estrogen is also seen in w o m e n undergoing surgical menopause. Sherwin and Gelfand found, in a prospective, cross-over study of w o m e n who underwent surgical menopause, that depression scores were higher in w o m e n treated with placebo than in w o m e n who were treated with sex steroid hormones (estrogen and/or androgen) [287]. The scores of these w o m e n were also higher than those of w o m e n who underwent a hysterectomy only [287]. Similar results were obtained in another group of w o m e n who, after undergoing a surgical hysterectomy and ovariectomy 4 years earlier, reported more positive moods after 2 years of treatment with either estrogen or estrogen plus testosterone, compared to untreated w o m e n [276]. In all of these studies, estrogen was given in doses used for estrogen replacement therapy in postmenopausal women, and the w o m e n treated were not clinically depressed. A study of depressed w o m e n given estrogen at doses used for estrogen replacement showed a decrease in depression scores in a majority of w o m e n classified as mildly depressed but not in a majority of w o m e n classified as clinically depressed [288]. However, in a study of w o m e n with severe, refractory depression, estrogen enhanced m o o d when given at pharmacological doses [289]. Thus, estrogen treatment at doses used for replacement therapy appears to have a favorable effect on m o o d in women, but does not appear to alleviate symptoms of clinically depressed women. Estrogen may mediate its effects on m o o d through the neurotransmitter, serotonin. A deficit in serotoninergic brain activity is implicated in the etiology of depression, and increasing serotoninergic activity with serotonin reuptake inhibitors presently serves as one of the most effective treatments for depression [290]. Estrogen increases serotoninergic activity in a variety of ways, including decreasing serotonin catabolism [249], decreasing the m R N A for the transporter responsible for its reuptake [291 ], increasing the m R N A levels of the rate-limiting enzyme in its production [292], and reducing serotonin 1A autoreceptors [293]. Thus, with the advent of menopause, it is likely that serotoninergic activity decreases, which could result in an increase in negative moods in at least a population of women. Currently, the ER has not been localized to serotoninergic cells of rats

[294], but estradiol does induce the progestin receptor in serotoninergic neurons in primates [295] suggesting that these neurons also express the ER. The presence of both estrogen and progestin receptors in these neurons would provide a mechanism by which sex steroids could directly modulate serotoninergic activity, thereby influencing mood.

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7HAPTER /

Gonadotropins and Menopause: New Markers S T E V E N B I R K E N 1 Department of Medicine, Columbia University College of Physicians and Surgeons, New York, New York 10032

JOHN O' CONNOR

Department of Pathology and Irving Center for Clinical Research, Columbia University College of Physicians and Surgeons, New York, New York 10032

G A L I N A KOVALEVSKAYA Irving Center for Clinical Research, Columbia University College of Physicians and Surgeons, New York, New York 10032 LESLIE LOBEL

I. II. III. IV.

Department of Obstetrics and Gynecology, Columbia University College of Physicians and Surgeons, New York, New York 10032

V. Changes in Gonadotropin Patterns in Menopause VI. Gonadotropin Fragments as Urinary Analytes References

Introduction Gonadotropin Parameters of the Normal Menstrual Cycle Structures of Gonadotropins and Their Fragments Assay of Gonadotropins

I. I N T R O D U C T I O N

from the corpus luteum; and chorionic gonadotropin (hCG), the hormone of pregnancy, which rescues the corpus luteum and continues steroid support for the developing fetus. The fourth glycoprotein hormone, thyroid-stimulating hormone (hTSH), stimulates production of thyroxine to control bodily metabolism [1]. All of the glycoprotein hormones are produced by the pituitary except for hCG, which is primarily a product of trophoblast cells in the placenta. A slight quantity of hCG appears to be of pituitary origin and is released along with hLH in nonpregnant individuals [2-6]. All four human glycoprotein hormones are thought to have evolved from a single ancestral gene [7,8]. Although the a subunits are identical among all four glycoproteins in terms of pri-

The gonadotropins are heterodimeric glycoprotein hormones composed of a common ce subunit and hormone-specific/3 subunits. There are four members of this family of hormones in humans (h), three of which are directly involved in reproduction: follicle stimulating hormone (hFSH), which promotes maturation of the follicle and ovulation; luteinizing hormone (hLH), which provides the final stimulus for ovulation and stimulates steroid secretion

1To whom correspondence should be addressed.

MENOPAUSE: BIOLOGY AND PATHOBIOLOGY

61

Copyright9 2000 by AcademicPress. All rights of reproductionin any form reserved.

62 mary structure, there are some differences in glycosylation patterns, especially in the addition of sulfate to hLH in place of some of the terminal sialic acid sugar residues [9,10]. hCG and hLH have nearly the same fi subunit structures and are the most closely related of the glycoprotein hormones in both structure and function [1,8,11]. hCG differs from hLH mainly in its extension at its carbohydrate-rich fi COOH-terminal region which endows the hormone with a much longer circulating half-life than that of hLH, namely, 24 versus 1 hr [11,12].

BIRKEN ET AL.

Ovulation Panel 1

LH

-20

Panel 2

mIU/mgCr

-40

FSH

II. G O N A D O T R O P I N P A R A M E T E R S OF THE N O R M A L M E N S T R U A L C Y C L E The menstrual cycle is characterized by a distinctive pattern of secretion of two gonadotropins, hLH and hFSH. Both hormones are secreted in a pulsatile manner under control of gonadotropin-releasing hormone (GnRH) [ 13-16]. The pulsatile pattern of circulating gonadotropins is essential for proper gonadotropin activity at its target receptors, i.e., the ovary in females and the testis in males. Approximately hourly gonadotropin pulsatility is found both in young normal cycling women and in women of later reproductive age, although the rate of pulsation may alter somewhat during aging [13-19]. As illustrated by the classical Fig. 1 of the menstrual cycle, the gonadotropins regulate the concentrations of estradiol and progesterone, which are the effector molecules of the reproductive system. An intricate feedback system of the circulating steroids and peptides controls gonadotropin secretion [20]. hFSH concentrations are most critically controlled because their precise regulation ensures development of only one dominant follicle [22,22]. hFSH appears to be controlled by several negative and positive feedback systems in addition to circulating steroids, including inhibins and activins [23,24]. Higher gonadotropin concentrations characteristic of reproductive aging in women are discussed later in this chapter. The temporal profile of the pituitary gonadotropins undergoes a substantial change as a woman proceeds through her reproductive years; with increasing ovarian senescence, a consequent diminution of sex steroid production disrupts the monthly cyclicity and the menopausal period ensues. The changes in gonadotropin secretion concomitant with menopause are best understood under terms of the gradual alterations in the normal menstrual cycle as a woman approaches the menopausal transition. The normal menstrual cycle is defined to commence with the first day of menstrual bleeding and in the human is of 2 4 - 3 2 days duration. Several cytokines, including epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), transforming growth factor-fi (TGF-fi), activin, and inhibin are involved in follicular growth and maturation [25]. Control of the circulating gonadotropins hLH and hFSH is primarily under the regulation of the hypothalamic GnRH

-40

Follicular ~ Luteal i[~ Phase ._l _. Phase ._

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Panel 3

mIU/mgCr

-100

Estrone Conjugates

ng/mgCr -50

-25

Panel 4

PDG

~g/mgCr

I

-15 -10

I

-5

0

5

10

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Days FIGURE 1 Urinary hormone profiles throughout the normal menstrual cycle. Panel 1: In the follicular phase, the levels of (preovulatory) human luteinizing hormone (hLH) remain relatively constant, with sharply higher hLH values occurring around midcycle LH surge (ovulation defined as day 0). During the luteal phase, hLH decreases to low levels until the late luteal phase, when it again begins to rise and a new cycle begins. Panel 2: Human follicle-stimulating hormone (hFSH) levels are elevated in the follicular phase, reflecting the primary role this hormone plays in the maturation of the dominant follicle, hFSH induces its own receptors and by doing so enables the cellular machinery to produce all of the protein factors necessary for cellular growth and function. At ovulation, there is a secondary spike of hFSH, coincident with hLH; afterward, hFSH levels decline until late in the luteal phase; when the diminishing levels of estradiol and progesterone derepress hFSH production and levels again rise at the beginning of a new cycle. Panel 3: This panel profiles estrone glucuronide and sulfate, the principal excretory metabolites ofestradiol and estrone. In the circulation, there is a rise ofestradiol coincident with the periovulatory gonadotropin surge and a secondary rise in the luteal phase under the influence of low levels of gonadotropins. Panel 4: The profile of pregnandiol-3-glucuronide (PDG), the major urinary metabolite of circulating progesterone, the hormone synthesized by the corpus luteum and peaking in the luteal phase. Progesterone exerts local effects within the corpus luteum and promotes the maturation of the endometrium. The corpus luteum is "rescued" by hCG in a conceptive cycle; otherwise it collapses, forming the corpus albicans prior to initiation of a new cycle.

and under feedback control from the inhibins and steroids produced by the dominant follicle [25]. GnRH modulates the release of both hLH and hFSH from the pituitary [26]. The menstrual cycle is segmented into three phases (see Fig. 1): the follicular phase, during which a follicle (the dominant follicle) destined to ovulate forms; the ovulatory phase, during which the dominant follicle ruptures and re-

63

CHAPTER4 Gonadotropins and Menopause: New Markers leases a mature ovum; and the luteal phase, whereby the ruptured follicle undergoes a process known as luteinization, in which ovarian thecal and granulosa cells enlarge and produce increased quantities of progesterone. During the preovulatory or follicular phase of the menstrual cycle (10-16 days), under the influence of increased hFSH from the pituitary, ovarian tissue surrounding the ovum destined to ovulate (i.e., the dominant follicle) undergoes proliferation and a cavity is formed within the follicle. The events involved in the mechanism of recruitment of one follicle (the dominant follicle) for ovulation are complex, involving the action of hLH and other growth factors. Estradiol production within the follicle is increased. Coincidentally, the tissues of the uterus, under the influence of increased estradiol, undergo proliferation and thickening. Ovulation commences on days 12-15 of the cycle. The egg, with its surrounding zona pellucida, breaks through the wall of the ovary and escapes into the abdominal cavity near the oviduct [27]. The ovulatory event is preceded by an increased release of hLH from the pituitary, which drives the granulosa cells of the dominant follicle to produce progesterone in addition to estradiol. These cells increase in size and lipid content, produce a yellow pigment (lutein), and form the corpus luteum subsequent to the expulsion of the ovum. The corpus luteum is vascularized in response to increased production of angiogenic factors elaborated by the granulosa and theca cells of the ovary. Immediately following ovulation, the uterine endometrial cells undergo a transformation in preparation for the implantation of the fertilized ovum (blastocyst). The luteal phase of the cycle (13-14 days) is characterized by increased production of progesterone, which in turn results in changes in uterine endometrial cell morphology. The endometrial glandular cells form secretory vacuoles, which contain glycogen. By days 2 0 - 2 1 of the cycle, secretion from these cells peaks and the uterine environment is optimized for implantation [27]. Progesterone production by the corpus luteum peaks coincident with maximum vascularization. Unless the corpus luteum is maintained by the hCG produced in a conceptive cycle, the corpus luteum undergoes structural and functional degeneration (luteolysis). Although both estrogen and prostaglandins have been invoked in the mechanism of luteolysis, the exact process remains unresolved. The pattern of urinary hormone metabolite secretion by the corpus luteum is illustrated in Fig. 1. Estrogen concentrations fall subsequent to ovulation, with a secondary rise occurring during the middle of the luteal phase, and again falling toward the end of the cycle. Progesterone and hydroxyprogesterone concentrations rise in parallel with estrogen, whereas inhibin A, which remains relatively depressed and static during the follicular phase, exhibits a surge in the luteal phase with a concomitant decrease in circulating hFSH. As the concentrations of estrogen and progesterone decrease in the late luteal phase, hFSH again begins to increase, initiating a new cycle.

III. STRUCTURES OF GONADOTROPINS AND THEIR FRAGMENTS Although the primary structures of the gonadotropins were determined between 1970 and 1975, more than 20 years elapsed until any three-dimensional structure became known, due to problems with crystallization of hormones with high carbohydrate content. The primary structures of the ce and fl subunits of hCG, hLH, and hFSH are shown in Fig. 2B. The ce subunit (Fig. 2A) is common to all of the glycoprotein hormones, whereas the fl subunit differs in each. The fl subunits of hLH and hCG are very similar. The structure of the hLH/3 subunit in Fig. 2B depicts its form

1

10

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50

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50

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Thr-Phe-Lys-Glu-Leu-Val-Tyr-Glu-Thr-Val-Arg-Val-Pro-Gly-Cys-Ala-His70

80

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100

Lys-Cys-Asp-Ser-Asp- Ser-Thr-Asp-Cys-Thr-Val-Arg-Gly-Leu-Gly-Pro-SerII0 Tyr-Cys-Ser-Phe-Gly-Glu-Met-Lys-Glu

FIGURE 2 (A) Primary structure of the ce subunit, common to all of the glycoprotein hormones. (B) Primary structure of the fi subunit of hLH as encoded by its mRNA. The lightly shaded area indicates the amino acids present in the hLHficf; the darkly shaded area indicates additional amino acids present in some of the core molecules. The brackets, both solid and dotted, indicate amino acids encoded by hLHfl mRNAsthat are usually not found in circulating or excreted hLH, but only within the pituitary tissue. (C) The fi subunit of hFSH. N-linked carbohydrate is present on residues 7 and 24 in FSH fi and residue 30 in hLH fl subunits. CHO indicates glycanmoiety.

64

BIRKEN ET AL.

in the circulation as well as its original biosynthetic form within the pituitary. Even when hLH is extracted from pituitary tissue, most of its very hydrophobic, leucine-rich seven-residue fl COOH-terminal region is missing (as indicated in brackets in Fig. 2B). This hydrophobic structure is involved in internal transport of hLH during its synthesis within the pituitary and is thought to be cleaved off prior to release into the circulation and certainly does not exist at all in urinary hLH [28]. A partially deglycosylated form of hCG was finally crystallized and its structure solved in 1994 [29-31]. The structures of both subunits strongly resembled each other (mainly fl sheet with a small a-helical region in ce; see Fig.3) [29,31 ], a finding that could not be deduced from the diverse primary amino acid sequences of each subunit. Both subunits consisted of a large loop and two smaller loops, with the fl subunit having an unusual seatbelt capture portion that held alpha in its embrace (Fig. 3). The two subunits are held together by noncovalent forces, chiefly hydrogen bonding and hydrophobic interactions; further stabilization is achieved by the region of the fl subunit starting at about residue 92, which folds around the ce loop 2 region. The seatbelt loop is held fast by an intradisulfide bridge between residues 26 and 110 [29,31]. This unexpected disulfide bridge surprisingly does not inhibit dissociation of subunits in acid buffers or chaotropic reagents. The manner in which dissociation takes place in relation to the seatbelt interaction is not yet known, but it is assumed that there is either enough structural alteration to widen the seatbelt to allow a to slip out, or that the

FIGURE 3

26-110 disulfide bridge exists in an equilibrium between closed (oxidized) and open (reduced) states. Solution of the gonadotropin structure also pointed out the homology of the gonadotropins to disulfide knot growth factors such as TGF-fl. Recombinant studies support the evolution of heterodimeric gonadotropins from an ancient homodimeric molecule, based on a recombinant construction made from a combination of segments from ce and fl subunits that resulted in a homodimeric bioactive molecule [7]. Although the three-dimensional structures of hLH, hFSH, and hTSH have not been individually solved, it is likely that they all resemble hCG structurally, and each has been modeled based on the hCG structure [29,31 ]. Carbohydrate plays an important role in the bioactivity of glycoprotein hormones. Some carbohydrate groups are important for signal transduction at the receptor [32] whereas other carbohydrate groups are important for long-term survival in the circulation [ 12]. Baenziger and colleagues made important observations concerning sulfation of some carbohydrate groups in hLH. They found that sulfated glycoproteins bind to a sulfate/sugar receptor in the liver and contribute to the pulsatility of hLH, which is necessary for its biological activity [9,33-37]. The pulses of hLH are accentuated not only by pulse release from the pituitary but also by rapid withdrawal of a portion of the circulating hLH by the liver receptor, which increases the downslope of the pulse. Human FSH is only slightly sulfated unlike the other FSH isoforms of the hormone in other mammals [35-37]. Figure 4 illustrates the nature of the carbohydrate moi-

Three-dimensional structures of hLH isoforms. The noncovalently bound heterodimer structure shown for hLH (a) is based on the solved structure for hCG, which is presumed to be very similar. The "seatbelt" region of the fl subunit, which wraps around a is indicated. The a subunit appears as a string in this representation for convenience, although the structures of both a and fl are mostly fl sheet except for a small a-helical structure in the a subunit, which is indicated on the illustration. In vivo, it is presumed that hLH dissociates into free hLHfl (b) and then is degraded into the hLHfl core fragment (c) in a body compartment such as the kidney. The hLHfl core fragment is a stable molecule that is rapidly cleared and excreted into the urine. The structures of hLHfl and hLHfl core fragment are likely different from their structures within the hLH molecule, but for convenience are shown here as the same.

CHAPTER4 Gonadotropins and Menopause: New Markers

FIGURE 4 Carbohydrate chains attached to asparagines of hLH and hFSH represented schematicallybased on the studies of Baenziger [35]. Carbohydrate formsexhibitmicroheterogeneityas indicated. A, Sialicacid; e, galactose; m, N-acetylgalactosamine; O, sulfate; 5, N-acetylglucosamine; O, mannose.

eties in hLH and hFSH, as well as some of the microheterogeneity that results in the very wide range of isoelectric points in the glycoprotein hormones [35-37]. Human LH is partially sulfated whereas hFSH is much less so (Fig. 4). Sialic acid is the most common terminal sugar for the gonadotropins in general and is responsible for their acid isoelectric points. The sulfate groups that replace sialic acid groups on half of the hLH molecules maintain the same acid nature of hLH, giving it a negative charge at physiological pH. Figure 4 has been greatly simplified to indicate the major N-linked carbohydrate moieties present on gonadotropins. There are literally dozens of variations, including molecules lacking both sialic acid and sulfate circulating in the bloodstream [9,35-37]. The various structures and their relative proportions in humans and other mammals have been described in detail by Baenziger [35-37].

IV. A S S A Y O F G O N A D O T R O P I N S A. B i o a s s a y This chapter focuses on measurement of gonadotropins and gonadotropin-derived polypeptides. In this section we provide the background information necessary to understand the complexity involved in measuring these diverse molecules.

65 The measurement of gonadotropins has proceeded from intact animal bioassays, in which administration of the hormone produces a measurable change in an organ weight or an increase in production of a hormone by that organ. A primary example of this technique is the mouse uterine weight assay for hLH (or hCG), because both of these hormones bind to the same receptor. The end point of the assay is an increase in mouse uterine weight after administration of either gonadotropin [38]. In general, intact animal bioassays are not usually performed due to the high cost, substantial interanimal variability, and lack of assurance that the animal model response will be identical to the human response. Despite these shortcomings, intact animal bioassays do have the advantage of taking into consideration both the intrinsic activity of the hormone at the receptor and, additionally, the circulating half-life of the hormone. This latter a component of biological activity is not evaluated in either cell or tissue preparations derived from the test animal or, more recently, in stable cell lines that have been genetically engineered to express the human gonadotropin receptor [39-41 ]. Most bioassays in current use rely on cell preparations from intact animals. Biologically active hFSH is measured by employing the in vitro immature rat Sertoli cell aromatase bioassay, i.e., measuring the amount of estrogen produced under cellular stimulation by hFSH [42]. Similarly, the biological activity hCG or hLH is routinely determined using a cultured rat Leydig cell preparation, the end point being an increase in cellular testosterone production under the influence of hLH [43]. In some instances, the measurement of the increased production of the initial intracellular product of receptor activation, cyclic AMP, rather than the hormonal endproduct, is employed in bioassays. A recent development in bioassay technology is development of cell lines that have been genetically engineered to express the functional human gonadotropin receptor, i.e., either the LH/CG receptor or the FSH receptor (44). This procedure allows for a more precise estimation of biological activity of human hormones than does use of the rodent receptor.

B. R a d i o r e c e p t o r A s s a y An alternative to the bioassay is the radioreceptor assay, in which intact cells or solubilized cell membranes from hormone target tissue containing the appropriate gonadotropin receptor are employed as the binding protein [45,46]. It should be noted that hormonal binding to receptor does not per se signify biological activity. It is well recognized that there exist isoforms of the gonadotropins, presumably with altered carbohydrate structure, with diminished or no biological activity despite their sometimes high affinity for the receptor [47].

66

BIRKEN ET AL.

Unfortunately, technical difficulty in performing these assays, variation in response of different systems/preparations, and difficulty in obtaining a pure reference preparation have limited use of these assays to research, with only modest clinical applications.

C. Immunoassay Attempts to simplify and improve both precision and accuracy of hormone measurements have resulted in the development of hormone-specific binding assays. Early applications of this technique utilized a naturally occurring hormone-binding protein, e.g., thyroxine binding globulin for T4 assay [48]. However, the majority of binding assays utilize specific antibodies developed against the gonadotropins. The first immunologically based assays employed polyclonal antibodies raised in animals. These immunoassays, which generally recognized multiple epitopes on the gonadotropin molecule, had the ability to detect many of the circulating isoforms, subunits, and fragments of the gonadotropin indiscriminately. A further assay refinement came with the development of monoclonal antibody technology. Monoclonal antibodies afford a continuous supply of a binding reagent of defined epitope specificity. This has led to the development of the immunometric assay, the most common formulation of which involves two monoclonal antibodies, each directed to a different epitope on the gonadotropin molecule. One of these antibodies (the capture antibody) is immobilized on a solid support and serves to extract the analyte from the matrix (usually blood or urine). The second antibody (the detection antibody) is labeled, e.g., with ~25I or an enzyme, and binds to a second epitope on the analyte molecule. The assay response is directly proportional to the quantity of analyte present in the specimen. The practical consequences of this development in the gonadotropin field have been an increase in the sensitivity of the measurements, but more importantly, the ability to specifically and independently measure intact hormone isoforms, free subunits, and metabolic fragments, especially in the urine. It has been demonstrated for hCG that urinary measurements can provide more complete and, in many instances, more clinically useful information than can serum determinations. [For example, hCG fl core fragment (hCGflcf), found in measurable quantities only in urine, is a much more effective marker of nontrophoblastic malignancy and some pregnancy disorders than is serum hCG] [49-52]. The core fragments are discussed in detail in Sections IV,D and VI. It should be noted that highly specific assays sometimes confer an important disadvantage compared to the less specific polyclonal-based older assay systems. Monoclonal antibody-based systems are directed to one epitope for each an-

tibody in the system and some such assays tend to detect poorly, or not at all, some of the isoforms of the gonadotropins. Thus, studies have documented cases in which the biological/immunological activity ratio changes throughout pregnancy [43,53] and with pregnancy disorders (hCG) [54], and throughout the human menstrual cycle (hLH or hFSH) [55] and with reproductive aging [56]. In extreme cases, investigators have documented the presence of either only biologically active or immunologically active gonadotropin in a subject. As will be described later, the way to avoid this difficulty may be to focus assays on gonadotropin fragments, which have much less epitope diversity than the holohormone or its subunits and which can be measured more reliably. The substantial variation that exists in the results obtained from both bioassays and immunoassays at different research centers highlights the need to identify stable gonadotropin markers of well-defined structure. The urinary fl core fragments of the parent gonadotropin molecules have the potential to fulfill this need.

D. fl Core Fragments of Gonadotropins as Stable Urinary Analytes Although the structures of the gonadotropins are very important to their biological functions, their structures are equally important to their immunochemical measurement (see Sections IV, A-IV, C). Such measurements can be performed in both blood and urine, although gonadotropinderived additional molecular species not detectable in blood appear in the urine. These molecules are derived from proteolysis of the fl subunits of gonadotropins. Currently, only fl core fragments of hLH and hCG have been reported to appear along with their parent heterodimeric and whole subunit parents in urine [57-60]. Although hFSH fragments should prove of greatest interest for menopausal studies, the fl core fragments of hCG and hLH are first described, because they have been isolated and characterized, and measurement systems for them have been developed; the information they provide should also prove relevant to hFSH fragments. The fl core fragments of hCG and hLH are very similar in structure. The fl core structures consist of approximately half of the fl subunit, comprising two disulfide-bridged chains. Figure 5 shows the subtle structural differences in the hCGflcf and hLHflcf (see Fig. 3 for the likely proteolytic origin of such fragments), hCGflcf is found mainly in urine, from which it has been isolated [57,59,61,62]. It is composed of fl residues 6 - 4 0 covalently linked to residues 55-92 by disulfide bridges [57]. hLHflcf has, thus far, been isolated only from pituitary extracts. Based on immunochemical studies using antibodies developed to pituitary hLHflcf, an

CHAPTER4 Gonadotropins and Menopause: New Markers 1

67

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Arg-Cys-Gly-Pro-Cy A r g A r g e,,.. -rk..+ e,,.. , , p Cys C!y C!y P r e Lys Asp His P r e L e u T ~ r t44 Cy3 A s p H i s P r e G ! n Le'-'- Ser Cly~Leu Let: P~e L e u F I G U R E 5 Comparison of the primary structures of the fl core fragments of hCG (top) and h L H (bottom). Areas absent from the cores are indicated by the cross-out lines. Peptide bond cleavages are indicated by vertical arrows. N-linked carbohydrate moieties are present on residues 13 and 30 of h C G / 3 core (top) and on residue 30 of h L H fl core (bottom).

analogous core form exists in urine [59,60,63]. Although hLHflcf (pituitary form) is very similar in primary structure to hCGficf, the second peptide component was found to be heterogeneous, being either residue 4 9 - 9 3 or 5 5 - 9 3 [58,59,63]. Note that hLHficf includes the Cys at residue 93, giving the core an even number of Cys residues, whereas hCGflcf ends at residue 92 and has an odd number of Cys residues (Fig. 5). Another point must be considered in making these comparisons, hCGficf was isolated from urine and hLHflcf was isolated from a pituitary extract. We already have evidence that the urinary form of hLHflcf is different in structure from the pituitary form on the basis of high-performance liquid chromatography (HPLC) properties [59,60,63]. However, the immunochemical system we have developed recognizes both pituitary and urinary forms of hLHflcf very well, and so both forms can be quantified. We have immunochemical evidence of the existence of a urinary hFSH/3 core fragment (hFSHflcf) (but not a pituitary hFSHflcf), although the structure of hFSHflcf has not yet been determined. All of these cores arise from a proteolytic process within a body tissue compartment and then are released into the urine (Fig. 3). What is remarkable about hCGficf is that it is homogeneous in urine in terms of its two peptide constituents. Frequently, proteolytic processes result in considerable peptide heterogeneity due to incomplete proteolytic bond cleavages. In the case of hCGficf, this proteolytic process proceeds to completion within a body compart-

ment, likely the kidney, and has not been duplicated by protease mixtures in vitro despite much effort [64,65]. The two fl core fragments have very desirable properties in terms of urinary analytes: (1) both molecules are present in urine in substantial concentrations relative to the parent hormone, faciliting their measurement; (2) both represent the end point of a proteolytic process within a tissue compartment, usually the kidney but sometimes the pituitary (hLHficf) or placenta (hCGficf), and are highly stable molecules; and (3) the fl core fragments are potent immunogens and are easily measured by most fl-directed antibodies as well as by specific antibodies generated against the cores [66-68] because they display a unique determinant. In contrast, as described earlier, hLH and hFSH are excreted in a variety of isoforms and as such present a diversity of epitopes, which complicates their measurement. For example, hLH may appear as isoforms undetectable by some immunochemical commercial and research assay systems [63,69,70], although they are still a part of the bioactive pool of hLH. These "invisible" hLH isoforms can complicate hLH measurements in some patients [63,71]. Figure 6 depicts the results of one such analysis of first morning void urine samples, which show an absent hLH periovulatory surge [63]. The heterodimeric gonadotropins are not very stable in urine because they tend to dissociate slowly into free subunits during multiple freeze-thaw cycles or simply during

68

BIRKEN ET AL.

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FIGURE 6 Profiles of urinary hLH molecular forms in a normally ovulating subject who did not express measurable intact hLH in either of two hLH assays (A). Note that both hLHfl and hLHflcf surges are clearly apparent. (B) The corresponding urinary steroid metabolite patterns for the cycle. It can be inferred from the steroid profiles that the subjects experienced normal ovulatory cycles, even in the absence of detectable intact hLH. Concentrations were normalized to creatinine. Day 1 is the first day of menses. Reproduced from [63]; O'Connor, J. E, Kovalevskaya, G., Birken, S., Schlatterer, J. P., Schechter, D., McMahon, D. J., and Canfield, R. E. (1998). The expression of the urinary forms of human luteinizing hormone beta fragment in various populations as assessed by a specific immunoradiometric assay. Hum. Reprod. 13, 826-835, with permission of the authors and

Human Reproduction.

prolonged storage. Core molecules are extremely stable for weeks at room temperature in the presence of a bacteriostat [59,60,63,72]. We have found that hLHflcf is stable for 40 or more freeze-thaw cycles [63]. These core molecules are, therefore, candidates for use as new, stable urinary markers of menopause.

V. CHANGES IN GONADOTROPIN PATTERNS IN MENOPAUSE Both hLH and hFSH concentrations rise significantly in menopausal women, having escaped the negative feedback inhibition of the steroid hormones usually produced by the ovary, hFSH pituitary secretion also rises due to the absence of inhibin B in menopausal women. Klein et al., Welt et al., as well as Santoro and colleagues showed a parallel decline of inhibins, especially inhibin B, with the rise of FSH during the follicular phase of perimenopausal women [24,73,74] (see also Chapter 9). Burger also showed that in early perimenopause, serum hFSH rose rapidly as inhibin B declined, with no significant fall in inhibin A [75]. Later in perimenopause inhibin A fell markedly. The escape of hFSH control due to inadequate luteal phase inhibin regulation may lead to

perimenopausal ovarian hyperstimulation with excessive development of a number of follicles [20] (see Fig. 7B). Inhibin B levels may fall precipitously low before the number of hFSH-responsive follicles decline. This situation may be responsible in part for menopausal symptoms such as unpredictable mood swings, headaches, and fluid retention. Inhibin control of hFSH may be more critical than the negative feedback of high estrogen levels at this stage of the menopausal transition. Burger proposed this scenario for the high FSH levels as women approach menopause: "With increasing age, the gradual decline in follicle numbers in the ovary leads to a fall in inhibin B levels, and a consequent rise in FSH sufficient to maintain ovulatory function and continued secretion of E 2 and inhibin A, mainly by the dominant follicle" [75]. Burger goes on to hypothesize that as follicle numbers decline further, leading to menstrual irregularity, inhibin B falls much further leading to a jump again in hFSH concentration in the circulation and continued dominant follicle development. When no more follicles are available at the time of cessation of menses, ovulatory function stops and inhibin A and E 2 levels fall with the final additional rise in hFSH concentration. Figure 7 summarizes these changes in pictorial format. In a review of reproductive aging in the rodent, Wise et al. hypothesized that the origin of the perimenopausal changes in gonadotropins, both their lowered rate of pulsation and their rise in concentration, is due to changes in the GnRH patterns, such as lower frequency of GnRH pulsation favoring increased release of hFSH over hLH [ 15,16]. Wise et al. attribute the change in GnRH pulsatility to deterioration of pacemakers within the brain, chiefly the suprachiasmatic nuclei that send branches to GnRH-generating neurons. A deterioration of this key neural pacemaker may initiate the gradual disintegration of neurotransmitter rhythms that are critical for precise and regular gonadotropin secretion [15,16]. Elderly postmenopausal women, compared to prematurely menopausal women, do appear to have increased asynchrony of GnRH secretion, although circadian rythmicity appears to be preserved [ 19]. Other studies in support of the neuroendocrine hypothesis include those of Matt et al., which indicated alterations in both interpulse intervals and pulse width of hLH in older cycling women as compared to younger women [76]. Studies of Reame et al. likewise found faster midluteal pulses in perimenopausal women as compared to midreproductive age women [56]. Soules, Battaglia, and Klein recently described their evaluation of the two competing hypotheses regarding reproductive aging [77,78]. Both hypotheses consider a rise in hFSH as contributing to exhaustion of the remaining follicles. The neuroendocrine hypothesis, however, considers slowing of the GnRH pulse generator as the cause of hFSH elevation whereas the ovarian hypothesis holds that it is the lower inhibin B levels that prompt the hypothalamus and pituitary to increase hFSH, which accelerates loss of the remaining follicles.

69

CHAPTER 4 G o n a d o t r o p i n s and M e n o p a u s e : N e w Markers

A

B

Premenopausal W o m e n Reproductive A g e < 35 years

~GnRH

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FIGURE 7 Summary illustration of hormonal changes during reproductive aging of women. Key: e, Dominant follicle; 1", level elevated in proportion to length of arrow (bold indicates significant elevation); $, level reduced in proportion to length of arrow (bold indicates significant reduction); arrows that are N-shaped indicate that a level is not always elevated; zig-zag lines indicate oscillating levels and pulsations (width and height symbolize frequency and amplitude, respectively); slanted lines crossed by two parallel bars indicate a disruption of an effect. (A) Premenopause: hFSH under control of GnRH (+ effect, bold arrow signifies strong effect), inhibins A and B from developing follicles ( - effect, dashed bold arrow signifies strong negative effect), and estrogen ( - effect). (B) Early perimenopause: hFSH begins to be variable while inhibin declines (although the decline in inhibin is somewhat questionable). Estrogen concentrations rise and hFSH is variably higher resulting in more follicle development and higher estrogen levels. (C) Late perimenopause: hFSH levels high, inhibin levels lower, and HLH variably higher. A dominant follicle still may develop. (D) Postmenopause: hFSH levels high with pulsatile pattern similar to that in a younger woman but with greater amplitude (note: pulsating hormone levels shown only for hFSH and hLH in postmenopausal women); same for hLH. GnRH patterns variable, inhibin absent, and estrogen very low due to absence of dominant follicle; no follicle development. (E) Postmenopausal elderly: hFSH levels high but pulsation rate slower; same for hLH. GnRH low and quite variable, no inhibin, and very low estrogen. (F) Postmenopause, premature ovarian failure (POF): high hFSH levels but pulsation pattern similar to that of a younger woman, as is hLH. No inhibin. GnRH levels and patterns similar to those of younger women. A portion of this illustration is based on Figure 7 from [20], Prior, J. (1998). Perimenopause: the complex endocrinology of the menopausal transition. Endocrine Reviews 19, 397-428. 9 The Endocrine Society.

Wise et al. support the neuroendocrine hypothesis [15,16]. Soules and colleagues have made a number of observations that are not consistent with either the ovarian hypothesis, in terms of inhibin levels in aging women, or the neuroendocrine hypothesis, in terms of similar GnRH pulsation patterns during aging. Nonetheless, they reach the overall conclusion that the ovarian hypothesis can better predict the changes in the gonadotropin levels and the number of follicles that are a precursor to menopause. Burger and others [79,80] have presented interpretations for the different stages during the transition to menopause. Prior has presented a synthesis of these various proposals

[20]. We have attempted to simplify this summary in Fig. 7. In this illustration, we present six panels illustrating the changing gonadotropin patterns from young premenopausal women to early and late perimenopausal, to postmenopausal, and to elderly postmenopausal women. Also included are young women experiencing premature ovarian failure. In many perimenopausal patients hLH and hFSH ovarian receptors were very low and such receptors were absent in postmenopausal patients [81 ]. This led Vihko to suggest that high serum gonadotropin levels act in concert with low or absent ovarian receptors for these hormones. Santoro demonstrated that there is also an age-related

70

BIRKEN ET AL.

alteration in hypothalamic or pituitary function that acts to decrease what would be even higher hLH and hFSH levels. In studies of women with premature ovarian failure compared to normal age menopausal women, it was apparent that hLH secretion was greater in the younger women along with greater pulse amplitudes, although pulse frequency was similar in both groups [19]. Therefore, the changes in gonadotropin secretion as women age demonstrate a decline in capabilities of the pituitary to produce gonadotropins in response to GnRH and/or a decline in GnRH during aging. These capacity differences in gonadotropin secretion in women with premature ovarian failure as compared to normal age menopausal women are also apparent in the gonadotropin fragment analysis patterns, which will be reviewed later. Gonadotropin bioactivity also increases in menopause along with the total immunoreactive concentration ofgonadotropins [82]. Changes in sialylation (increased sialic acid content) with decreasing circulating steroids contribute to the increase in bioactivity of the gonadotropins after menopause. Studies of biological to immunological gonadotropin ratios show that postmenopausal women display nearly twice the ratio value than do normal young cycling women or older perimenopausal women [82]. Although the high concentrations of hFSH and hLH after menopause are apparent, these vary greatly during the transition to menopause, the perimenopause. Santoro provides an excellent illustration of the changes in gonadotropin and steroid levels among 11 midreproductive age women and 11 perimenopausal women during the menstrual cycle [18] (Fig. 8). The rise in overall hFSH concentrations during the

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FIGURE 8 Daily hLH, hFSH, estrone conjugates (El), and PDG excretions (mean _ SEM) corrected for creatinine and standardized to the day of presumed ovulation (day 0) in 11 regularly menstruating perimenopausal women (open circles) compared with 11 younger women (closed circles). E] was higher in the perimenopausal women (P = 0.023) and integrated PDG was lower (P = 0.015). Reproduced from [18], Santoro, N., Brown, J. R., Adel, T., and Skurnick, J. H. (1996). Characterization of reproductive hormonal dynamics in the perimenopause. J. Clin. Endocrinol. Metab. 81, 1495-1501. 9 The Endocrine Society.

early follicular phase and an overall rise in estrogen in perimenopausal women are apparent in this illustration. Although hFSH increases prior to a rise in hLH levels during perimenopause, a great range of possible values makes such gradual increases of little diagnostic value in determining the perimenopausal stage [20,79,80]. Likewise, estrogen values are quite variable during perimenopause, being even higher in some perimenopausal women than in young, normal cycling women. Despite the use of day-3 serum FSH and/or circulating estrogen concentrations as indicative of the perimenopausal state, these single point measurements are far from conclusive; further exploration should provide new and improved markers that can indicate proximity to menopause.

VI. G O N A D O T R O P I N AS URINARY

FRAGMENTS

ANALYTES

The gonadotropins, being heterodimeric hormones whose subunits are noncovalently bound together, are unstable in urine. Although it is possible to decrease their instability by addition of additives such as glycerol and by minimizing freeze-thaw cycles, intact gonadotropins are not ideal molecules for accurate quantification in urine [83-86]. The gonadotropins undergo proteolytic cleavages during transit through the kidney and their stability in urine is likely to be further reduced by such damage [57,59,64,68,87]. Most commercial gonadotropin assays are certified as quantitatively accurate in blood but only qualitatively accurate in urine due to such problems [88,89]. It is easier to quantify glycoprotein hormones in blood, but each sampling requires a technician or physician or even extremes, such as an indwelling catheter for multiple samplings. Large-scale studies using blood specimens cannot be conducted for this reason, but such studies can readily be performed in urine if an appropriate, highly stable analyte can be identified. The/3 core fragments of the gonadotropins are ideal molecules for urinary measurement. The two gonadotropins of significance to menopause are hFSH and hLH, with the former being the most relevant. Although we have evidence for the presence of a urinary hFSHflcf, only/3 core fragments of hCG and hLH have been isolated, characterized, and measured thus far. The hCGflcf has been used extensively in urinary measurement systems for the past decade. It is extremely stable, as attested to by a variety of groups [72], and is generally present in higher concentrations than heterodimeric hCG in urine. Measurement applications include certain cancers and various problem pregnancies, including Down syndrome. The utility of hCGflcf in the cancer marker field (used only informally for this purpose) is limited to monitoring the recurrence of hCGsecreting tumors after therapy. The higher molar concentration of this fragment as compared to hCG in urine leads to greater sensitivity of detection. The hLHflcf epitope is similarly stable in urine. Current assays for the hLHflcf are based

CHAPTER4 Gonadotropins and Menopause" New Markers on the isoform isolated from pituitary tissue, the urinary form of this molecule has not yet been purified [59,60,63]. hLH is more difficult to measure accurately in urine than is hCG because it is less stable than hCG and frequently displays isoforms not recognized by many monoclonal antibody-based immunochemical measuring systems [63,71,9093] (see Fig. 6). In addition, hLH may appear to be absent from urine after having completely dissociated into subunits or after having been completely metabolized to smaller fragments. FSH also presents measuring problems due to dissociation of subunits in urine. This problem has occurred in analyses of FSH in nonhuman primates, which are used as models for studies of human reproduction. One lab has recently proposed boiling all monkey urine samples to cause complete dissociation of FSH into subunits and then measuring the released fl subunit [94]. However, if any heterodimeric hormone is proteolytically cleaved, such boiling may cause irreversible loss of some epitopes and total fl concentration may not represent total FSH originally present in the specimen. In all of these situations,/3 core fragment measurement provides the most stable and consistent reflection of gonadotropins in circulation. These fragments have reached an end point in proteolysis and are not further degraded in urine as long as microbial growth is inhibited. While acknowledging that FSHflcf would most likely provide the best menopausal marker, because we have already developed an immunochemical system for measurement of hLHflcf, we have applied these measurements first to assess their utility in studies of women in menopausal transition. As previously described, we demonstrated that hLHflcf appeared in high concentrations in women during the menstrual cycle starting on the day of the LH surge, peaking 1-2 days after the surge [63]. Figure 9 shows the hormone profiles of 15 normal cycling premenopausal women and the temporal relationship in the appearance of hLH, hLHfl, hCG, and hLHflc. The X axis is normalized to day 0, this being the zenith of the hLH surge appearance in urine. This delayed appearance of hLHflcf after the hLH surge suggests that circulating hLH is sequestered in a body compartment (likely kidney tissue) and is excreted after 2 4 - 4 8 hr of proteolytic processing, hLHflcf can be easily measured in urine but not in serum. This is shown in Figure 10 and implies that the kidney creates the fragment by absorbing hLH from the blood and excreting it later into the urine. It is also possible that some of the hLHcf is directly secreted by the pituitary into the bloodstream but is cleared so rapidly that its circulating concentration remains very low. Our original hypothesis was that postmenopausal women, who do not experience the large midcycle surge of hLH but instead maintain a continuous pulsatile high concentration of hLH, would tend to display a high plateau level of hLHflcf. Essentially, this would result from an integration of the multitude of hLH pulses in the time delay during the proteolytic processing steps. We tested this hypothesis by examining first morning void urines for 10 consecutive days in a series

71

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FIGURE 9 Hormone profiles in the urine of normally cycling women (n = 15). Concentrations were presented as mean _+ SE, femtomoles/milligram creatinine (fmol/mg C). hLH concentration has been measured using two different immunoradiometric assays (n = 8 for the hLH-2 assay). Steroid hormone ratios are calculated using estrone-3-glucuronide (E1-3-G) and pregnandiol-3-glucuronide (Pd-3-G) (• 103). Day 0 is the day of hLH surge. Reproduced from [63], O'Connor, J. F., Kovalevskaya, G., Birken, S., Schlatterer, J. E, Schechter, D., McMahon, D. J., and Canfield, R. E. (1998). The expression of the urinary forms of human luteinizing hormone beta fragment in various populations as assessed by a specific immunoradiometric assay. Hum. Reprod. 13, 826-835, with permission of the authors and Human Reproduction.

of postmenopausal women. The resultant data did not agree with our hypothesis. Instead, very large-amplitude pulses of hLHflcf, which could not readily be correlated with hLH surges, appeared in the urine of these women. One such pattern of hLHflcf excretion in a postmenopausal woman is illustrated in Fig. 11. Note the very large fluctuations of hLHflcf (between 0 and 600 fmol/mg creatinine), which do not correlate with urinary hLH, even considering the 24to 48-hr time delay in the appearance of the core after an hLH surge (Fig. 9). Analysis of 10 consecutive first morning void urine samples from cycling women of reproductive age (< 35 yr) during the follicular phase (day 1 was the first day of menses) indicated that much shallower fluctuations occurred in these women, prior to the hLH surge at the time of ovulation. Indeed, by integrating the area under the peaks of graphs of hLHflcf in femtomoles/milligram of creatinine versus day of collection, it was possible to differentiate young cycling women from postmenopausal women, even with the occasional high spikes in some young women

72

BIRKEN ET AL.

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~ "r" _1 '-

40 20 o 200

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FIGURE 10 hLH and hLHflcf in serum and urine of the same patient, o, hLH- 1 ; r~, hLH2; A, hLHfl; e, hLHflcf. The serum levels of intact hLH (o) and hLHflcf (e) indicate that there is an insignificant amount of hLHflcf detected in the blood. The lower panel illustrates the urinary values for hLH and hLHflcf for the same days of collection. The surge of hLH (day 0) and the surge of hLHflcf (1-2 days later) are detected in urine, but the peak of hLHflcf lags that of the intact hLH by 1-2 days, suggesting that urinary hLHflcf is a consequence of the peripheral or renal metabolic processing of intact hLH. Reproduced from [63], O'Connor, J. E, Kovalevskaya, G., Birken, S., Schlatterer, J. P., Schechter, D., McMahon, D. J., and Canfield, R. E. (1998). The expression of the urinary forms of human luteinizing hormone beta fragment in various populations as assessed by a specific immunoradiometric assay. Hum. Reprod. 13, 826-835, with permission of the authors and Human Reproduction.

during the follicular phase. We selected 10-day intervals as a convenient research set, and specimens could be easily collected by volunteer subjects and stored frozen until samples were brought to the laboratory. In a future marker assay, the collection protocol is unlikely to consist of such a large sample set. When samples are analyzed from women still experiencing regular menstrual cycles, the 10-day interval collection provides a convenient starting point within the cycle and encompasses the follicular phase that most closely corresponds to the postmenopausal state of relatively low circulating steroids. For regularly cycling women, the mean of the areas under the peaks of 10 subjects was 278 with a median area of 169. The mean of the areas among postmenopausal subjects ranged between approximately 1000-4000 with medians in the ranges of 900-3000. The postmenopausal subjects differed significantly from the population of normal cycling women by the amplitude and area under the peaks of the daily fluctuations of this fragment. We com-

pared the areas under the peak of the lowest hLHflcf levels of postmenopausal women with the highest core levels of premenopausal women and could statistically differentiate the two groups even in this worst-scenario sampling situation. Perimenopausal women fall in between, with some clearly in the postmenopausal pattern and some in a premenopausal pattern. Figures 12 and 13 illustrate analysis of two perimenopausal women. The patterns of hLHflcf shown in Fig. 12 are typical of those seen in normal cycling women of midreproductive age (see Fig. 9) [63]. A peak hLHflcf appears 1-2 days after the hLH surge, after the presumed metabolic breakdown of circulating hLH into hLHflcf within a tissue compartment. Figure 13 depicts a perimenopausal woman with a typical postmenopausal pattern (see Fig. 11), with many very large hLHflcf peaks, not coordinated to particular hLH peaks in the urine. However, we must keep in mind the earlier discussion of the problems of accurately measuring hLH, in urine and the phenomenon of "invisible" urinary hLH, which we have encountered several times in our own laboratories [63,71 ]. We are in the process of developing assays for a similar hFSHflcf. With the combination of the two fragment assays,

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FIGURE 11 The 60-day patterns of hLHflcf and hLH in first morning void urine collections from a postmenopausal woman. In the upper panel the hLHflcf is normalized to creatinine, whereas in the lower panel the hLH is measured by the DELFIA assay on glycerol-preserved urines.

73

CHAPTER 4 Gonadotropins and Menopause: New Markers

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Day of Urine Collection FIGURE 12 The 60-day patterns of hLH/3cf and hLH in first morning void urine collections from a perimenopausal woman. In the upper panel the hLH/3cf is normalized to creatinine, whereas in the lower panel the hLH is measured by the DELFIA assay on glycerol-preserved urines. The pattern of hLH/3cf resembles that of midreproductive age women (see Fig. 9).

hLH/3cf and hFSH/3cf, it may be possible to develop urinary assays that will define the stage of m e n o p a u s a l transition. Because changes in h F S H concentrations usually precede those of hLH, it is envisioned that the hFSH/3cf assay, when developed, m a y provide a stronger discriminant function than the hLH/3cf assay. In summary, the gonadotropin patterns in b l o o d and urine undergo significant alterations during the m e n o p a u s a l transition. The d e v e l o p m e n t of sensitive assays for stable proteolytically derived fragments of h L H and h F S H in urine will provide new markers to determine the phases of the menopausal transition. The standardization of these assays with stable h o r m o n a l metabolites should obviate the p r o b l e m s often encountered in c o m p a r i s o n and analysis of data collected from different laboratories and thereby ease collection of data in large-scale epidemiological studies. Furthermore, implementation of assays based on stable h o r m o n a l metabolites will heighten awareness in the general clinical c h e m i s t r y c o m m u n i t y o f the utility of metabolic by-products as markers for other physiologically relevant proteins.

0

10

20

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40

50

60

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Day of Urine.Collection FIGURE 13 The 60-day patterns of hLHflcf and hLH in first morning void urine collections from a perimenopausal woman. In the upper panel the hLI-I/3cfis normalized to creatinine, whereas in the lower panel the hLH is measured by the DELFIA assay on glycerol-preserved urines. The pattern of hLHflcf resembles that of a postmenopausal woman. Part of this figure is reproduced from Birken, S., Santoro, I. V., Maydelman, Y., Kovalevskaya, G., Lobo, R., Freeman, E. W., Warren, M., McMahon, D., and O'Connor, J. (1999). Differences in urinary excretion patterns of the hLH beta core fragment in premenopausal, perimenopausal, and postmenopausal women. Menopause 6(4), with permission of the authors and Lippincott Williams & Wilkins.

Acknowledgments This work was supported by NIH grants RO1-AG 13783 and ROlES07589. We wish to express appreciation to Nanette Santoro for thoughtful advice.

References 1. Pierce, J. G., and Parsons, T. E (1981). Glycoprotein hormones: Structure and function. Annu. Rev. Biochem. 50, 465-495. 2. Birken, S., Maydelman, Y., Gawinowicz, M. A., Pound, A., Liu, Y., and Hartree, A. S. (1996). Isolation and characterization of human pituitary chorionic gonadotropin. Endocrinology (Baltimore) 137, 14021411.

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CHAPTER 4 Gonadotropins and Menopause: New Markers

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76 74. Welt, C. K., McNicholl, D. J., Taylor, A. E., and Hall, J. E. (1999). Female reproductive aging is marked by decreased secretion of dimeric inhibin. J. Clin. Endocrinol. Metab. 84, 105 - 111. 75. Burger, H. G., Cahir, N., Robertson, D. M., Groome, N. P., Dudley, E., Green, A., and Dennerstein, L. (1998). Serum inhibins A and B fall differentially as FSH rises in perimenopausal women. Clin. Endocrinol. (Oxford) 48, 809-813; published erratum: Ibid., 49(4), 550. 76. Matt, D. W., Kauma, S. W., Pincus, S. M., Veldhuis, J. D., and Evans, W. S. (1998). Characteristics of luteinizing hormone secretion in younger versus older premenopausal women. Am. J. Obstet. Gynecol. 178, 504-510. 77. Klein, N. A., and Soules, M. R. (1998). Endocrine changes of the perimenopause. Clin. Obstet. Gynecol. 41,912-920. 78. Soules, M. R., Battaglia, D. E., and Klein, N. A. (1998). Inhibin and reproductive aging in women. Maturitas 30, 193-204. 79. Burger, H. G. (1996). The endocrinology of the menopause. Maturitas 23, 129-136. 80. Burger, H. G. (1996). The menopausal transition. Bailliere's Clin. Obstet. Gynecol. 10, 347-359. 81. Vihko, K. K. (1996). Gonadotropins and ovarian gonadotropin receptors during the perimenopausal transition period. Maturitas 23 (Suppl.), S19-$22. 82. Schmidt, P. J., Gindoff, P. R., Baron, D. A., and Rubinow, D. R. (1996). Basal and stimulated gonadotropin levels in the perimenopause. Am. J. Obstet. Gynecol. 175, 643-650. 83. Saketos, M., Sharma, N., Adel, T., Raghuwanshi, M., and Santoro, N. (1994). Evalution of time-resolved immunofluorometric assay and specimen storage conditions for measuring gonadotropins. Clin. Chem. (Winston-Salem, N.C.) 40, 749-753. 84. Kesner, J. S., Knecht, E. A., and Krieg, E. F., Jr. (1995). Stability of urinary female reproductive hormones stored under various conditions. Reprod. Toxicol. 9, 239-244. 85. Livesey, J. H., Hodgkinson, S. C., Roud, H. R., and Donald, R. A. (1980). Effect of time, temperature and freezing on the stability of im-

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~HAPTER

Genetic Programming In Ovarian

Development

and Oogenesis JOE LEIGH SIMPSON

Departments of Obstetrics and Gynecology and Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030

I. II. III. IV.

Ovarian Differentiation Requires Only One X (Constitutive) Polygenic and Stochastic Control over Oocyte Number Monosomy X X Chromosomal Mosaicism: 45,X/46,XX and 45,X/47,XXX V. Pitfalls in Localizing Ovarian Maintenance Genes to Specific Regions of the X

VI. VII. VIII. IX. X. XI.

I. O V A R I A N

Failure of germ cell development is associated with complete ovarian failure, resulting in lack of secondary sexual pubertal development (primary amenorrhea). A decreased number but not a total absence of germ cells is more likely associated with premature ovarian failure, presenting with infertility or secondary amenorrhea (see Chapter 8). Yet complete and premature ovarian failure may be different manifestations of the same underlying pathogenic and etiologic processes. Many different genetic mechanisms are pertinent to the processes m chromosomal abnormalities, Mendelian mutations of autosomal or X-linked genes, and polygenic/ multifactorial factors. In this contribution, we enumerate clinical disorders associated with germ cell abnormalities, deducing etiologic factors responsible for ovarian differentiation and oogenesis in normal females.

MENOPAUSE:

BIOLOGY AND PATHOBIOLOGY

Genes on the X Short Arm Genes on the X Long Arm Nature of X Ovarian Maintenance Determinants Autosomal Chromosomal Abnormalities Autosomal Genes (Mendelian) To What Extent Is Premature Ovarian Failure Genetic? References

REQUIRES

DIFFERENTIATION

ONLY ONE X

(CONSTITUTIVE) In the absence of the Y chromosome, the indifferent embryonic gonad always develops into an ovary. Germ cells exist in 45,X human fetuses [ 1]. Oocyte development initially exists even in 46,XY phenotypic females, such as in infants with XY gonadal dysgenesis [2] or the genito-palatocardiac syndrome [3]. Oocyte development in the presence of a Y chromosome is also well documented in mice [4]. Thus, the pathogenesis of germ cell failure in humans can be deduced to be increased germ cell attrition. If two intact X chromosomes are not present, ovarian follicles in 45,X individuals usually degenerate by birth. Genes on the second

77

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78

JOE LEIGH SIMPSON

X chromosome are thus responsible for ovarian maintenance, rather than for ovarian differentiation.

II. P O L Y G E N I C CONTROL

OVER

AND STOCHASTIC OOCYTE

NUMBER

It is to be expected that oocyte number (reservoir) will be low in some women simply on statistical (stochastic) grounds. Normal distribution exists for all common anatomic traits (e.g., height), and this principle should apply to oocyte number and reservoir at birth. That a normal distribution of germ cell number exists in ostensibly normal females is well established in animals but difficult to prove in humans. Different rodent strains show characteristic breeding duration, implying genetic control over either the rate of oocyte depletion or the number of oocytes initially present. It follows that some ostensibly normal (menstruating) women may have decreased oocyte reservoir or increased oocyte attrition on a genetic basis, analogous to animal models. In humans a genetic basis for the above can be presumed by analogy to the heritability of age at human menopause, a characteristic that clearly shows familiar tendencies. Assessing heritability of age at menopause is complicated because iatrogenic behavior (e.g., hysterectomy) and other confounding factors (e.g., leiomyomata or uterine cancer) must be taken into account. However, several studies within the past decade have directly addressed the issue. Cramer et al. [5] performed a case control study on 10,606 United States women who were between 45 and 54 years of age. Women with an early menopause ( 4 0 - 4 5 years) were age-matched with controls who were either still menstruating or had experienced menopause after age 45 years. Of 129 early menopause cases (<46 years), 37.5% had a family history of a similarly affected mother, sister, aunt, or grandmother. Only 9% of controls had such a relative (odds ratio after adjustment, 6.1; 95% C.I., 3.9 to 9.4). As predicted on the basis of polygenic expectations, the odds ratio was greatest (9.1) for sisters and for when menopause occurred prior to 40 years. The frequency of galactose1-phosphate uridylyltransferase (GALT) variants (N314D or Q188R) did not deviate from expected in early menopause cases, in contrast to previous studies by the same authors [6]. Torgerson and colleagues [7] reported that if women underwent menopause during the 5-year centile aged 4 5 - 4 9 years, the likelihood was increased that menopause would occur in a similar 5-year centile in their daughter. Twin studies have been used to estimate heritability of age at menopause. Two studies have shown similar results [8]. Snieder et al. [9] studied 27 monozygotic (MZ) and 353 dizygotic (DZ) twin pairs in the United States. For age at menopause correlation (r) was 0.58 in MZ twins and 0.39 in DZ twins (h 2 = 63%). Treolar et al. [8,10] performed a simi-

lar study in 1177 MZ and 711 DZ Australian twin pairs. For age at menopause, correlations (r) were 0.49-0.57 for MZ and 0.31-0.33 for DZ. Heritability (h 2) was 31-53% [8]. Differences between MZ and DZ groups held when iatrogenic menopause (hysterectomy for leiomyomata or endometriosis) was taken into account.

III. M O N O S O M Y X The complement most frequently associated with ovarian dysgenesis is 45,X. The proportion of 45,X individuals in a given sample will depend on the method of ascertainment. Fewer 45,X individuals will be detected if primary amenorrhea is the presenting complaint than if short stature or various somatic anomalies are the presenting complaints. Primary amenorrhea is more likely to be the presenting complaint in women examined by gynecologists, whereas short stature is likely to be evaluated in children examined by pediatricians. Overall, about 50% of all patients with gonadal dysgenesis have a 45,X complement; 25% have sex chromosomal mosaicism with a structural abnormality (e.g., 45,X/46,XX). Far fewer have a structurally abnormal X or Y chromosome or no detectable chromosomal abnormality [11,12]. In 80% of cases the paternally derived X has been lost [13]. With one possible exception to be noted, the phenotypes of 45,X m and 45,XP cases do not differ. (Xm,X of maternal origin; XP,X of paternal origin). That is, no evidence exists in general for imprinting [14,15]. In structurally abnormal X chromosomes, it is also the paternal X that is lost [16,17]. The above suggests that X m and XP chromosomes are lost at random [ 18]. Because 45,Y is lethal, the theoretical percentage of 45,X m cases would be 67%, not greatly different from the 80% actually observed.

A. G o n a d s In most 45,X adults with gonadal dysgenesis, the normal gonad is replaced by a white fibrous streak, 2 to 3 cm long and about 0.5 cm wide, located in the position ordinarily occupied by the ovary. A streak gonad is characterized histologically by interlacing waves of dense fibrous stroma, indistinguishable from normal ovarian stroma (Fig. 1). That germ cells are usually completely absent in adults but present in 45,X embryos is the basis for the belief that the pathogenesis of germ cell failure is increased atresia, not failure of germ cell formation. Speed [19,20] has shown that in monosomy X oogenesis ceases in meiosis I at or before the pachytene meiotic stage. Degenerating pachytene oocytes are observed. A variety of pairing abnormalities and disruptions are seen at later stages of meiosis I, but oocytes in dictyotene are rare.

CHAPTER5 Genetic Programming in Ovarian Development

Ogata and Matsuo [ 18] argue that the ovarian failure found in monosomy X is caused by generalized (nonspecific) meiotic pairing errors, the extent of ovarian failure correlating with extent of pairing failure. Ovarian rete tubules, which probably originate from either mesonephric tubules or medullary sex cords, are present in the median portion of most streak gonads. Hilar cells are usually detected in streak gonads of patients past the age of expected puberty. That 45,X humans manifest streak gonads is not so obvious as one might expect. Relatively normal ovarian development occurs in many other monosomy X mammals (e.g., mice). These observations are, incidentally, at odds with the hypothesis [19,20] that ovarian failure merely reflects meiotic pairing errors. If the hypothesis were true, the monosomy X mouse should not differ from the monosomy X human. The more likely explanation is that in humans not all loci on the normal heterochromatic (inactive) X are inactivated. In addition, X inactivation never exists in oocytes, with X reactivation of germ cells occurring before entry in meiotic oogenesis [21 ]. X inactivation could also occur only after some crucial time of differentiation, beyond which only a single euchromatic (active) X is necessary for continued oogenesis.

79

B. S e c o n d a r y S e x u a l D e v e l o p m e n t Although streak gonads are usually present in 45,X individuals, about 3% of adult cases menstruate spontaneously and 5% show breast development (Table I). Occasionally, the interval between menstrual periods appears normal in 45,X patients, and fertile patients have been reported. Although an undetected 46,XX cell line should always be suspected in menstruating 45,X patients, it is plausible that a few 45,X individuals could be fertile, inasmuch as germ cells are present in 45,X embryos. The rare offspring of 45,X women are probably not at greatly increased risk for chromosomal abnormalities [22,23], although theoretically they should be. Some authors disagree with this statement [24], and work with X-specific fluorescence in situ hybridization (FISH) probes suggests that low-grade 45,X/46,XX mosaicism may be increased in women experiencing repeated abortion [25]. Irrespective, menstruation and fertility occur so rarely that 45,X patients should be counseled to anticipate primary amenorrhea and sterility. After hormone therapy is initiated in such women, uterine size becomes normal. This permits 45,X women to carry pregnancies in their own uterus after receipt of donor embryos or donor oocytes mixed with their husband's sperm.

80

JOE LEIGH SIMPSON TABLE I

Somatic Features Associated with 45,X C h r o m o s o m a l C o m p l e m e n t G r o w t h a

Body size Decreased birth weight Decreased adult height (141-146 cm) Intellectual function Verbal IQ > performance IQ Cognitive deficits (space-form blindness) Craniofacial Premature fusion of sphenooccipital and other sutures, producing brachycephaly Abnormal pinnae Retruded mandible Epicanthal folds (25%) High-arched palate (36%) Abnormal dentition Visual anomalies, usually strabismus (22%) Auditory deficits; sensorineural or secondary to middle ear infections Neck Pterygium coli (46%) Short, broad neck (74%) Low nuchal hair (71%) Chest Rectangular contour (shield chest) (53%) Apparent, widely spaced nipples Tapered lateral ends of clavicle

Cardiovascular Coarctation of aorta or ventricular septal defect (10-16%) Renal (38%) Horseshoe kidneys Unilateral renal aplasia Duplication of ureters Gastrointestinal Telangiectasias Skin and lymphatics Pigmented nevi (63%) Lymphedema (38%) due to hypoplasia of superficial vessels Nails Hypoplasia and malformation (66%) Skeletal Cubitus valgus (54%) Radial tilt of articular surface of trochlear Clinodactyly V Short metacarpals, usually IV (48%) Decreased carpal arch (mean angle 117 ~ Deformities of medcal tibial condyle Dermatoglyphics Increased total digital ridge count Increased distance between palmar triradii a and b Distal axial triradius in position t'

aModified from Simpson [11].

IV. X CHROMOSOMAL MOSAICISM: 45,X/46,XX AND 45,X/47,XXX If nondisjunction or anaphase lag occurs in the zygote and embryo, two or more cell lines may result (mosaicism) (Fig. 2). The final c o m p l e m e n t will depend on the stage at which abnormal cell division occurs and on the types of daughter cells that survive following nondisjunction or anaphase lag. Detection of mosaicism depends on the n u m b e r of

NORMAL MITOSIS

cells analyzed per tissue and on the n u m b e r of tissues analyzed [11,12]. The most c o m m o n form of mosaicism associated with gonadal dysgenesis is 45,X/46,XX. Individuals with a 45,X/ 46,XX c o m p l e m e n t predictably show fewer anomalies than do 45,X individuals. Simpson [11] tabulated that 12% of 45,X/46,XX individuals menstruate c o m p a r e d with only 3% of 45,X individuals. A m o n g 45,X/46,XX individuals, 18% undergo breast development c o m p a r e d with 5% of 45,X in-

MITOTIC NONDISJUNCTION

FIGURE 2 Diagrammatic representation of the products of normal mitosis and mitosis characterized by nondisjunction of a Y chromosome. If all daughter cells survived, the complement would be 45,X/46,XY/47,XYY [ 12].

CHAPTER5 Genetic Programming in Ovarian Development dividuals. Mean adult height is greater with a 45,X/46,XX complement than with 45,X; more mosaic (25%) than nonmosaic (5%) patients reach adult heights greater than 152 cm [11]. Somatic anomalies are less likely to occur in 45,X/ 46,XX than in 45,X individuals. 45,X/47,XXX occurs less often but is phenotypically similar to 45,X/46,XX. Individuals with 45,X/46,XY may also show bilateral streak gonads; however, more often they show a unilateral streak gonad and a contralateral dysgenetic testis (mixed gonadal dysgenesis).

V. P I T F A L L S I N L O C A L I Z I N G O V A R I A N MAINTENANCE GENES TO SPECIFIC REGIONS OF THE X Delineating the region (genes) on the X responsible for ovarian maintenance is the first step in understanding normal ovarian differentiation and in producing gene products of therapeutic benefit. Until the past decade, phenotypickaryotypic correlations to deduce location of gonadal and somatic determinants relied solely on metaphase analysis. Prometaphase karyotypes allow 1200 band analysis (traditional GTG banding 400-500), but each band still contains considerable DNA. More refined analysis is now possible using polymorphic DNA markers that allow precise resolution far beyond the capacity of light microscopy. Progress has, nonetheless, been slow compared to that achieved in delineating the regions of the Y necessary for testicular differentiation (SRY) or spermatogenesis (DAZ). Several impediments have resulted in a relative lack of progress. One is that the incidence of X deletions is very low. Analyzing only cases ascertained by population-based methods is impractical because no individuals with X deletions were recovered among 50,000 consecutively born neonates [26]. Most del(Xp) or del(Xq) individuals have been identified only because they manifested clinical abnormalities, exceptions being familial cases or cases detected in fetuses at the time of prenatal genetic diagnosis undertaken because of their mother's advanced maternal age. Doubtless many less severely affected individuals escape detection. Mode of ascertainment should ideally be considered in phenotypickaryotypic analysis, but in reality this is impractical because sample sizes are too small. Inevitably biases of selection arise. Another pitfall impeding molecular analysis of X ovarian maintenance genes is that analysis is not always derived from individuals who are well-studied cytogenetically. Mosaicism in nonhematogenous tissues has not always been excluded to the extent reasonably possible. Individuals with unstable aberrations (rings, dicentrics) should probably be excluded from phenotypic-karyotypic deductions because monosomy X and other cell lines may arise secondarily, sometimes in

81 tissues (e.g., gonads) relatively inaccessible to study. Utilizing X/autosome translocations for analysis may also be hazardous because of vicissitudes of X inactivation, and because autosomal regions are not devoid of significance for gonadal differentiation.

VI. GENES ON THE X SHORT

ARM

A. 46,X, d e l ( X p ) or 4 5 , X / 4 6 , X , d e l ( X p ) D e l e t i o n s Deletions of the short arm of the X chromosome show variable phenotype, depending on the amount of Xp persisting. The most common breakpoint for terminal deletions is Xpl 1 (Fig. 3). In 46,X, del(X)(pl 1), only proximal Xp remains; the del(Xp) chromosome thus appears acrocentric or telocentric. Chromosomes characterized by progressively more distal breakpoints have been reported: Xp21, 22.1, and 22.3. X/autosomal translocations leading to Xp interstitial deletions have been reported, and are analytically useful albeit subject to caveats noted in the previous section. Availability of polymorphic DNA markers now allow precise determinations of breakpoints in terminal deletions, but still

FIGURE 3 A normalX chromosomeand deletions of the X chromosome derived fromthree differentpersons [32].

82

JOE LEIGH SIMPSON

relatively few cases have been subjected to refined molecular analysis. Approximately half of 46,X, del(Xp)(pll) individuals show primary amenorrhea and gonadal dysgenesis. The others menstruate and usually show breast development. In one tabulation by the author of 27 reported del(X)(p 11.2 11.4) individuals, 12 menstruated spontaneously; however, menstruation was rarely normal [27]. Additional compilations have not materially altered these conclusions [28-30]. Ogata and Matsuo [ 18] estimate that 50% of del(X)p 11 cases show primary amenorrhea, with 45% showing secondary amenorrhea. Ovarian function is thus observed more often in individuals with a del(Xpl 1) chromosome than in 45,X individuals. Women with more distal deletions [del(X)(p21.1 to p22.1.22)] menstruate more often, but many are still infertile or even have secondary amenorrhea (Fig. 4). Thus, Xp [X(pter --->p21)] retains a role in ovarian development [2830]. The distal region of importance must involve Xp21,

22.3 22.2 22.1 21 11.4 11.3 11.2 11.1 11 12

13

B. Isochromosomes for Xq m

~

9

"

II ~

"iii

US

amain m

9

m m

m|m

9

mm9 21

mm

22 23 24

ano

mm

A

9

9

25 26

22.1, or 22.2 because del(X)(p22.3) cases do not show primary amenorrhea. Most women with deletions of Xp are short in stature. Thus, statural determinant(s), i.e., regions with genes, must exist on Xp. Because del(Xp) women may menstruate but still be short, regions on Xp responsible for ovarian and statural determinants must be distinct [28-32]. Clinically it is important to realize that del(Xp) women may be short despite manifesting normal ovarian function. Both mother and daughter may show the same Xp deletions, not only in association with X/autosome translocation but also in association with terminal deletions. In 1977 Fraccaro et al. [31] first emphasized familial distal Xp deletions. Among 10 del(Xp) cases subsequently studied by James et al. [16] were two mother-daughter pairs; only 6 of their 10 cases arose de novo. Familial cases involved deletion at Xp 11 as well as at Xp22-12 are also reported. Xp interstitial deletions involving Xp 11-22 and Xp 11.422.3 [33,34] have been reported.

mm

27

28 FIGURE 4 Ovarian function associated with simple deletions of the X chromosome. All cases are characterized by banding studies and reasonable exclusion of mosaicism [27]. n, 1o amenorrhea; A, 2 ~ amenorrhea oligomenorrhea; o, fertility or regular menses.

Division of the centromere in the transverse rather than in the longitudinal plane results in an isochromosome, a metacentric chromosome consisting of isologous arms. Both arms are structurally identical and contain the same genes. An isochromosome for the X long arm [i(Xq)] differs from a terminal deletion of Xp in that not just the terminal portion but all of the Xp is deleted. Many isochromosomes for Xq are in reality isodicentrics, the clinical significance of which is that a minute portion of Xp is duplicated and retained in addition to duplication of the entire Xq. An isochromosome for the X long arm is the most common X structural abnormality, but coexisting 45,X cell lines (mosaicism) are typical. Nonmosaic cases are relatively uncommon. 46,X,i(Xq) individuals almost always have streak gonads and primary amenorrhea. Occasionally menstruation is observed, but surveys continue to agree with early reports published by the author [ 11 ] in showing rarity of menstruation [18]. The near complete lack of gonadal development in 46,X,i(Xq) contrasts to that in 46,X, del(X)(p 11) individuals, about half of who menstruate or develop breasts. The contrast is in greater with more distal Xp deletions. Phenotypic differences could be explained if gonadal determinants were present at several different locations on Xp, one locus being deficient in i(Xq) yet retained in del(X)(p 11). Alternatively, 46,XX cells may be associated with del(Xp) more often than generally appreciated. Irrespective, duplication of Xq, that is, i(Xq), fails to compensate for deficiency of Xp. One explanation is that gonadal determinants on Xq and Xp have different functions. Another is that all loci on i(Xq) chromosomes are completely inactivated. It seems unlikely that

CHAPTER5 Genetic Programming in Ovarian Development TABLE II

83

Ovarian Functiona Ovarian failure (%) in X deletions Complete (primary amenorrhea or streak gonads)

Partial (secondary amenorrhea or abnormal menses)

Monosomy X (45,X)

88

12

0

Short arm deficiency del(X)(p 11) del(X) (p21 - 22.2 ) del(X)(p22.3) i(Xq) idic(Xq)

50 13 0 91 80

45 25 0 9 20

5 62 100 0 0

Long arm deficiency del(X)(ql3-21) del(X)(q22-25) del(X)(q26-28) idic(Xp)

69 31 8 73

31 56 67 27

0 13 25 0

Deficiency

No failure (presumed normal)

a As tabulated on the basis of cases reviewed in 1995 by Ogato and Matsuo [ 18]. Ogato and Matsuo provided data in the first two columns, with the assumption being that the remainder of cases have normal ovarian function (e.g., 5% in del(X)(pll). Publications surveyed overlap in large part those used for analysis by Simpson [27] (see Fig. 4).

duplication of Xq per se produces abnormalities but is unknown, given that 47,XXX often appears clinically normal. Almost all reported 46,X,i(Xq) patients are short. Their mean height seems to be less than that of 45,X patients (Table II). The mean height of nonmosaic 46,X,i(Xq) patients is 136 cm [11], and many somatic features of the Turner stigmata are observed [ 11 ]. Somatic anomalies occur as frequently in 46,X,i(Xq) individuals as in 45,X individuals, and the spectrum of anomalies associated with the two complements is in general similar. James et al. [16] attempted an extensive molecular analysis of i(Xq), confirming short statue and finding relative deficiency of pterygium coli (webbing of the neck.) This observation suggests a protective effect for Xq with respect to pterygium coli.

VII. GENES ON THE X L O N G A R M

A. 46,X, del(Xq) and 45,X/46,X, del(Xq) Deletions Deletions of the X long arm are well known [27-30] and vary in composition. If the breakpoint leading to a terminal deletion originates at band Xql 3, the derivative chromosome resembles No. 17 or No. 18; a breakpoint at band Xq21 produces a chromosome resembling No. 16 (see Fig. 3).

Almost all deletions originating at Xql3 are associated with primary amenorrhea, lack of breast development, and complete ovarian failure [30]. Xq 13 thus seems to be an important region for ovarian maintenance. Key loci could lie in proximal Xq21, but not more distally, given that del(X) (q21) to (q24) individuals menstruate far more often (Fig. 4). Menstruating del(X)(q21) women might have retained a region that contained an ovarian maintenance gene, whereas del(X)(ql 3 or q21) women with primary amenorrhea might have lost such a locus [30]. Molecular attempts at mapping the region of Xq most integral for ovarian development have been reported. Sala et al. [35] studied seven X/autosome translocations involving Xq21-22. Four cases showed primary amenorrhea; the other three were described as follows: (1) one case with secondary amenorrhea, elevated gonadotropins, and small ovaries; (2) one case with secondary amenorrhea; and (3) one case with amenorrhea, absent breast development, and small ovaries. A region of Xq spanning 15 mb encompassed breakpoints in all seven cases. Breakpoints in four other X/autosome translocations studied by Philippe et al. [36] were also localized to the same region. The YAC contig encompassing these breakpoints spanned most of the Xq21 region and extended between DXS233 and DXS 1171 [37]. An X q - Y p homologous region is contained within this contig [38]. That breakpoints associated with ovarian failure spanned the entire Xq21 region makes it unlikely that a single gene causes ovarian failure, unless in these balanced X/autosome translocations ovarian failure is the result not of disruption of a gene per se, but rather is reflective of generalized cytologic perturbation. To this end several other observations are of note. First, a normal female has been observed having an X/autosome breakpoint in Xq21 (case 5513 of Philippe et al. [36]). If not explained in other ways (e.g., a 46,XX cell line), one must conclude that not all of Xq21 is obligatory for ovarian development. This concept would be at odds both with existence of a critical region between Xql 3 and Xq25 [39-42] as well as with the hypotheses that ovarian failure reflects generalized meiotic breakdown caused by any type of rearrangement [20]. Second, observation of a normal female with a breakpoint-involved Xq21 [36] is consistent with existence of many distinct ovarian genes in the same region. Third, in two but not the other five cases of Sala et al. [35] ovarian failure was accompanied by choroiderma. Again, this favors existence of multiple genes (contiguous gene syndrome). Sala et al. [35] concluded that eight different genes are responsible for X maintenance on Xq 21. In more distal Xq deletions, the more common phenotype is not primary amenorrhea but premature ovarian failure [28,29,43,44]. Although distal Xq seems less important than proximal Xq for ovarian maintenance, the former must still have regions important for ovarian maintenance. Informative cases have included terminal deletions originating at

84

JOE LEIGH SIMPSON

various sites and two interstitial deletions [43,45]. These interstitial deletions point out hazards of interpretation without molecular-based studies. Although there is no clear demarcation into discrete regions, it is heuristically useful to stratify terminal deletions into those occurring in regions Xq 13 --9 21, Xq22-25, and Xq 26-28. Table II shows the extent of ovarian function tabulated by Ogato and Matsuo [18] using such stratification. Figure 4 shows the author's tabulation in different format. Both estimates are based on pooled cases, and both are generally consistent. Distal Xq deletions are not infrequently familial. Some familial Xq deletions are derivative of Xq autosome translocations, but familial terminal or interstitial deletions also exist [45]. Familial Xp terminal or interstitial deletions have been characterized by various breakpoints between Xq25 and Xq28. Breakpoints near or in Xq27 seem most common. Some families have been ascertained for reasons other than premature ovarian failure, a case reported by our group having been ascertained following amniotic fluid analysis in a fetus [46]. This suggests that additional families would be detected if prometaphase analysis or polymorphic molecular studies were more routinely performed in premature ovarian failure. Distal Xq deletions seem to have a less severe effect on stature than do proximal deletions. Somatic anomalies of the Turner stigmata are uncommon and perhaps no more common than in the general population.

VIII. NATURE MAINTENANCE

OF X OVARIAN DETERMINANTS

Clearly the X chromosome is necessary for ovarian maintenance, preventing premature germ cell attribution and permitting progression beyond meiotic pachytene. Moreover, we have deduced that several different regions seem to contain crucial genes. At the least, key regions exist on proximal Xp and proximal Xq; an unknown number of determinants exist on distal Xp and distal Xq. As noted, Sala et al. [35] believed that eight different genes exist Xq21 alone. Ultimately both the number of individual genes and their gene product(s) will be determined. This would have both prognostic and potential therapeutic value, given that recombinant technology allows synthesis of a protein gene product(s) once the DNA sequence is known. At present little is known about the nature of ovarian maintenance gene products. Jones et al. [46] proposed that a key gene product is DFFRX; its locus is located on Xp 11.4 and homologous to a locus on Yql 1.2. Both genes escaped inactivation in two de novo (X)(pl 1.2) deletions. James et al. [16] considered DFFRX an unlikely candidate after observing ovarian function despite haploinsufficiency; however, neither of the two cases of James et al. [16] were completely normal clinically,

for which reason a role for DFFRX in gonadal development is not categorically excluded. An attractive candidate gene for a role in ovarian maintenance is the human homolog of the Drosophila melanogaster gene diaphanous (dia). This gene causes sterility in male and female Drosophila [47]. Sequence comparisons between dia and the relevant human expressed sequence tag (EST) DRE25 show significant homology. DRE25 in turn maps to human Xq22 [47]. As we have already noted, Xq22 is a key region for ovarian maintenance. The product of Drosophila dia is a member of a family of proteins that help establish cell polarity, govern cytokinesis, and reorganize the actin cytoskeleton. Studying familial premature ovarian failure, an Xq21/autosome translocation alluded to earlier [48] was found to be associated with disruption of DRE25 [49]. Perturbation involved the last intron, producing a human DIA characterized by truncated transcripts; the transcript was unstable and could not be translated. This mechanism seems relevant to the phenomenon of mRNAs in oocytes and embryos remaining untranslated for long periods of time, presumably until their proteins become necessary later in differentiation. On the other hand, human DIA is expressed not only in developing ovaries, but also in testis and other tissues. This suggests that in humans the role dia plays in oogenesis is neither primary nor specific. Four Xq 21 ~ Xqter deletions associated with premature ovarian failure recently by the same group [50] also indicated that dia is not necessarily involved in del(Xq) deletions. In one of the four cases, loss of exons at the 3' end of dia was detected; however, in the other three no perturbations of dia were evident. A broader biologic question can be posed concerning X ovarian maintenance determinants. Do the various regions contain gene(s) coding for different gene products? If so, the presumption would be that all these genes are either essential or at least contribute to normal ovarian differentiation. If different genes exist, the prospect of alternative therapeutic options is raised given that various gene products will eventually all be synthesized. However, it would seem hazardous evolutionarily if perpetuation of the species were to depend on transcription and translation of an entire cascade of ovarian differentiation genes, perturbation of any of which would be deleterious if not lethal (genetically). Moreover, ovarian disturbance associated with many X deletions is rarely complete. We have noted that terminal deletions involving either proximal Xp or proximal Xq may be associated with complete ovarian failure, but more distal deletions of Xp or Xq are far more likely to be associated with premature ovarian failure or normal ovarian function. Teleologically, it might be more attractive if all X ovarian maintenance determinants were to produce the same gene product or perhaps products capable of interaction (dimerization, for example). This would seem to be more conservative evolutionarily because mutation or deletion of a single locus would not be singularly catastrophic. If such a scenario

CHAPTER5 Genetic Programming in Ovarian Development were true, an ineluctable corollary would be that the X ovarian genes act in threshold fashion, thus exerting their primary effect through an autosomal gene. One mode of action might involve transcriptional or translational regulation of DNA-binding proteins.

85 ments is important because their offspring are at increased risk for gametes showing unbalanced segregation.

X. A U T O S O M A L

GENES

(MENDELIAN)

A. X X G o n a d a l D y s g e n e s i s IX. A U T O S O M A L ABNORMALITIES

CHROMOSOMAL

A. T r i s o m y Autosomal trisomy has long been known to affect adversely ovarian development. The question has always been whether this effect is mediated by nonspecific meiotic perturbation or by chromosome-specific genes, perhaps acting in double dose. Trisomies 13 and 18 are frequently associated with ovarian failure, as indicated by necropsy observations in stillborns or deceased neonates. Few longitudinal data are available for trisomy 13 or 18 because few affected females survive until menarche. In trisomy 21, however, ovarian function may be normal. There seems to be no objective information on age at menopause. Pregnancies occur in trisomy 21 females [51]. About onethird of offspring are aneuploid (fewer than the theoretically expected 50%). Ostensibly normal ovarian function in trisomy 21 suggests that specific ovarian genes exist on chromosomes 13 and 18. If nonspecific meiotic breakdown is merely secondary to an uneven number of chromosomes, the effect should be the same effect with chromosome 21 as with chromosomes 18 and 13.

B. T r a n s l o c a t i o n s Chromosomal rearrangements, specifically balanced autosomal reciprocal translocations, are not infrequently observed in otherwise normal women with complete or partial ovarian failure. As with autosomal trisomy it is unclear whether this association reflects disruption of autosomal loci integral for ovarian preservation and oogenesis. That no chromosome(s) is consistently involved suggests nonspecific meiotic perturbation. In fact, men who are azoospermic or oligospermic but otherwise normal clinically show balanced autosomal translocations far more often than expected: about 1% of men requiring intracytoplasmic sperm injection (ICSI) show a balanced autosomal rearrangement, typically a balanced translocation [52]. A problem of comparable magnitude probably exists in women, but relative accessibility of ovaries makes cytogenetic studies more difficult. In both sexes the pathogenesis presumably leading to meiotic breakdown involves malalignment or failure of synapis. Recognizing individuals with autosomal rearrange-

Gonadal dysgenesis histologically similar to that occurring in individuals with an abnormal sex chromosomal complement may be present in 46,XX individuals, as shown by the author over 25 years ago [53]. Mosaicism has been reasonably excluded in affected individuals, although mosaicism restricted to the embryo can never be excluded. The general term XX gonadal dysgenesis can be applied to those individuals. 1. PHENOTYPE

Many different forms of 46,XX gonadal dysgenesis exist, but the prototypic form of XX gonadal dysgenesis not associated with somatic anomalies is clearly inherited in autosomal recessive fashion. Affected individuals are normal in stature (mean height, 165cm) [54], and Turner stigmata are usually absent. Frequent reports of consanguinity have long made it clear that autosomal recessive genes are responsible. More recent segregation analysis by the author and colleagues revealed the segregation ratio to be 0.16 for female sibs. Thus, two-thirds of gonadal dysgenesis cases in 46,XX individuals are genetic [55]. The one-third of cases that are nongenetic (phenocopies) could be due to infection, infarction, infiltrative, or autoimmune phenomena. Of considerable clinical interest is the variable expressivity. In some families one sib has streak gonads, whereas another affected individual had primary amenorrhea and extreme ovarian hypoplasia (presence of a few oocytes) [5359]. If the mutant gene responsible for XX gonadal dysgenesis is capable of variable expression, the gene may be responsible for some sporadic cases of premature ovarian failure. 2. MECHANISM OF GENE ACTION

The mechanism underlying failure of germ cell persistence in most forms of XX gonadal dysgenesis is unknown, but several hypotheses seem reasonable. One is perturbation of meiosis, a general mechanism already invoked to explained germ cell breakdown in both monosmy X and balanced chromosomal translocations. In plants and lower mammals meiosis is known to be under genetic control. Surely this is true in humans as well, for which reason one would predict existence of mutations that would be manifested as ovarian failure and infertility in otherwise normal women. Other possibilities include interference with germ cell migration, abnormal connective tissue milieu, or gonadotropin receptor perturbation (see later). Table III lists

86 TABLE III

JOE LEIGH SIMPSON

Mouse Genes Affecting Germ Cell Number or Gametogenes

Gene

Description/function

gcd

Germ cell deficiency

dhh

Desert hedge hog/protein signaling

BMP 8B

Bone morphogenetic protein 8B/TGF-/3 family

Dazla

RNA-binding protein

Igf-I

Insulin-like growth factor-I

Nblb2

Basic helix-loop transcription factor

Ztx

Zinc-finger protein

Hsp 70-2, Hsc 7 O t p

Heat-shock protein

Mhl, Pms2

Mismatch repair

ATM

Cell check point

Dazla

RNA binding (premeiotic)

as 50 to 100 families should identify chromosomal region(s) worthy of sequencing. This method was, in fact, applied successfully in Finland to elucidate the form of XX gonadal dysgenesis due to follide-stimulating hormone (FSH) receptor mutation (see later.)

B. P e r r a u l t S y n d r o m e ~ X X

Gonadal Dysgenesis

with Neurosensory Deafness A distinct variant of XX gonadal dysgenesis is that associated with neurosensory deafness. This condition is called Perraut syndrome. Like XX gonadal dysgenesis without deafness, Perrault syndrome is autosomal recessive [54-64].

C. F S H R e c e p t o r M u t a t i o n some genes in mice and other species that deleteriously affect germ cell development or gametogenesis. Attractiveness of the human homologs of these genes as explanations for XX gonadal dysgenesis is underscored by the phenotype of various murine "knockout" models. Often the only abnormality is ovarian or testicular failure or abnormalities restricted to gametogenesis. This is true even though these murine and Drosophila genes would have been predicted to act in ways seemingly disparate from those expected of genes affecting germ cells or gametogenesis. These genes may affect cell cycle checkpoints, DNA repair genes, or heat-shock proteins (which as chaperone proteins, protect unoccupied steroid receptors). Usually, meiotic breakdown occurs at or before the pachytene stage. That germ cells are affected is not a surprise, but that abnormalities seem restricted to the reproductive system is. The model for the murine gene germ cell deficiency (gcd) [60] is especially attractive. This murine autosomal recessive gene produces decreased numbers of germ cells in both ovary and testes. Its human homolog could be responsible for some cases of XX gonadal dysgenesis. Even more plausibly, gcd could be responsible for the syndrome of germ cell deficiency in both sexes (see later). In the absence of candidate genes such as those cited above, identifying autosomal genes responsible for the various forms of XX gonadal dysgenesis is more difficult. An investigator might await the fortuitous family in which an autosomal translocation cosegregates with XX gonadal dysgenesis. Sporadic cases of gonadal dysgenesis have long been associated with reciprocal autosomal translocations, but for years there seemed to be little consistency in the chromosome involved. The alternate approach is a "brute force" genome-wide search for relevant gene(s), utilizing sib-pair analysis, with the polymorphic DNA markers readily available throughout the genome. Using sib-pair analysis, as few

In Finland Aittomaki et al. [58,59] searched hospitals and cytogenetic labs to identify 75 patients country-wide having XX gonadal dysgenesis, defined in 46,XX women as primary or secondary amenorrhea and serum FSH >- 40 mlU/ ml. These 75 included 57 sporadic cases and 18 cases having affected relatives (seven different families). Most cases were found in north central Finland, a sparsely populated part of the country. The overall frequency of the disorder in Finland was 1 per 8300 liveborn females, a relatively high incidence attributed to a founder effect. Segregation ratio of 0.23 for female sibs was consistent with autosomal recessive inheritance, as was the consanguinity rate of 12%. Sib-pair analysis using polymorphic DNA markers were next used to localize the gene to a specific region. Chromosome 2p, a region that had previously been known to contain genes for both the FSH receptor (FSHR) and the luteinizing hormone (LH) receptor (LHR). One specific mutation (C566T:alanine to valine) in exon 7 was observed in six multiplex families [59,65]. That C566T was not found in all Finnish XX gonadal dysgenesis cases indicates genetic heterogeneity. The C566Tnegative cases could represent the same disorder discussed previously (XX gonadal dysgenesis with no somatic anomalies). Consistent with this is that the C566T mutation is rarely detected in samples from women with 46,XX ovarian failure who reside outside Finland [66]. Layman et al. [66] found no mutations in the FSHR gene in 35 46,XX women having hypergonadotopic hypogonadism. Of the 35, 15 had primary amenorrhea and 20 had secondary amenorrhea. Three of the 35 had one or more affected sisters. Liu et al. [67] found no sequence abnormalities in one multigeneration primary ovarian failure (POF) family, 4 sporadic POF cases and 2 cases of hypergonadotopic hypogonadism cases. Irrespective of apparently being found mostly in Finland,

CHAPTER5 Genetic Programming in Ovarian Development TABLE IV Phenotypes of Finnish XX Gonadal Dysgenesis, with and without the C566T Mutation a Measure

C566T

Wild-type566

Primary amenorrhea Height (cm) Delayed puberty FSH (IU/liter) Follicles (ultrasound)

22/22 160.5 6/16 61 6/12

22/30 165.8 10/20 84 1/11

aData of Aittomakiet al. [65].

it was a surprise to many that at least one form of XX gonadal dysgenesis was caused by mutation involving FSHR. It had been expected that ovaries associated with gonadotropin receptor mutations would be characterized by numerous but undeveloped primordial follicles, not streak gonads. This is the phenotype said to exist in the so-called Savage syndrome, considered to be due to a gonadotropin-resistance syndrome. Aittomaki et al. [65] later contrasted the phenotype of C566T XX gonadal dysgenesis with non-C566T XX gonadal dysgenesis. Subjects with the former, examined by ultrasound, were more likely to have ovarian follicles (Table IV). C566T XX gonadal dysgenesis thus showed some features expected for gonadotropin resistance; however, FSH was clearly elevated and the phenotype was far more reminiscent of the bilateral streak gonads and prototypal XX gonadal dysgenesis.

D. Inactivating L H R e c e p t o r M u t a t i o n s Another trophic hormone receptor gene in which a mutation causes gonadal dysgenesis is the LH receptor. Sultan and Lumbroso [68] tabulated the 46,XY cases, nine having female phenotype and two having micropenis. All 46,XX cases occurred in sibships in which an affected 46,XY male had Leydig cell hypoplasia. Latronico et al. [69] reported primary amenorrhea in a 22-year-old woman. In that family, three males and one female had a homozygous nonsense (stop) mutation at codon 554 (C554X). The newly produced stop codon resulted in a truncated protein having five rather seven transmembrane domains. The affected female had breast development but only a single episode of menstrual bleeding at age 20 years; LH was 37 mIU/ml; FSH, 9 mIU/ml. The mutation reduced signal transduction activity of the LH receptor gene. In a 46,XX case reported by Toledo e t al. [70], secondary amenorrhea occurred; LH and FSH were 10 and 9 mIU/ml, respectively. The mutations was Ala593Pro. Activating LH receptor mutations seem to have little effect in females, although in males precocious puberty occurs

87 [68]. No females with activating LHR have shown precocious puberty.

E. X X G o n a d a l D y s g e n e s i s a n d M u l t i p l e Malformation Syndromes Mutant genes that act on multiple organ systems are called pleiotropic. The pleiotropic combination of XX gonadal dysgenesis and neurosensory deafness, termed Perrault syndrome, has already received comment. Several other syndrome are recognized: XX gonadal dysgenesis and cerebellar ataxia [71]; XX gonadal dysgenesis, microcephaly, and arachnodactyly [72]; XX gonadal dysgenesis and epibulbar dermoid [73]; and XX gonadal dysgenesis, short stature and metabolic acidosis [74]. These four disorders are presumably all autosomal recessive in nature, based on multiple affected sibs. Contiguous gene syndrome (chromosomal deletions) or other non-Mendelian mechanisms are not excluded. No molecular progress has been made toward elucidating genes responsible for the syndromes described above, but the gene responsible for one autosomal dominant syndrome has been localized. The blepharophimosis-ptosis syndrome has long been recognized as associated with ovarian failure [75,76]. Using several large kindreds, sib-pair analysis using polymorphic DNA variants [77] localized the gene for blepharophimosis-ptosis to chromosome 3 (3q21-24). This region contains no obvious candidate genes. Puzzling with respect to the blepharophimosis-ptosis syndrome is the report of Fraser e t al. [78] showing that ovaries in blepharophimoisis-ptosis were unresponsive to gonadatropins. This is reminiscent of the phenotype associated with the Finnish XX gonadal dysgenesis C566T FSHR mutation [65]. In each of the multiple malformation syndromes associated with ovarian failure, the underlying biologic question must be posed. Does the seemingly pleiotropic gene cause both the somatic anomalies and the ovarian failure? Do the somatic and nonsomatic phenotypes involve only closely linked genes, i.e., contiguous gene syndrome? Could an unrecognized parental chromosomal rearrangements exist? In turn, do any of these genes play pivotal roles in normal ovarian differentiation and maintenance? Or, is perturbation of ovarian development merely secondary, perhaps occurring through generalized disturbance of connective tissue?

E F r a g i l e X and E x p a n s i o n o f the T r i p l e t Nucleotide Repeat CGG A special category of pleiotropic genes affecting ovarian development is those involving expansion of triplet nucleotide repeats. The prototype is the fragile X syndrome, caused

88

JOE LEIGH SIMPSON

by mutation of the F M R 1 gene on Xq27. "Fragile" refers to the tendency toward chromosomal breakage when affected Cells are grown in vitro in folic acid-deficient media. Several fragile sites exist, FRAXA and F R A X E in particular. In FRAXA, affected males show mental retardation, characteristic facial features, and large testes. The molecular basis involves presence of 230 or more (CGG) n repeats; the normal number of repeats in males is only 6-50. When heterozygous females show 50-200 repeats, a permutation is said to be present. During female (but not male) meiosis the number of triplet repeats may increase (expand). A woman with a FRAXA premutation may thus have an affected son if her X were to expand during meiosis to more than 230 repeats and if that son were to inherit that expanded rather than the normal X. Females may also be affected, but show a less severe phenotype than males. Females with the FRAXA premutation may show premature ovarian failure. Schwartz et al. [79] reported that fragile X carrier females more often showed oligomenorrhea than do noncarrier female relatives (38 vs. 6%). Premature ovarian failure was increased with FRAXA premutation (25 vs. 8%). Murray et al. [80] reported an extensive analysis of 1268 controls, 50 familial POF, and 244 sporadic POF cases. Table V shows the frequency of FRAXA in each. Of familial cases, 16% of showed FRAXA premutation; among sporadic cases, 1.6% showed POE In the same sample POF was not increased in F R A X E . Consistent with the above data are observations that FRAXA carrier women respond poorly to ovulation-inducing agents, thus producing fewer oocytes and fewer embryos in ART [81 ]. Although the consensus is that FRAXA is indeed associated with POF, Kenneson et al. [82] do not agree. They believe that the phenomenon is explained best by a contiguous gene syndrome m two separate but closely linked loci that may or may not be detected in a given individual or family. That Xq27-28 contains both F M R 1 ( F R A X A ) and an ovarian maintenance gene is consistent with, but does not prove, the hypothesis.

TABLE V Frequency of FRAXA Alleles on the X Chromosomes of 25 Women with Familial and 122 with Sporadic Premature Ovarian Failure a Phenotype

Allele

Number of CGG repeats

Familial POF

Sporadic POF

Normal

Minimal Common Intermediate Premutation Full mutation

0-10 11-40 41-60 61-200 >200

0 44 2 4 0

0 236 6 2 0

1 1237 30 0 0

50

244

1268

Total aData of Murray et al. [80].

The manner by which FRAXA premutation could produce POF remains obscure. That ovulation also seems difficult to induce, at least one other triplet nucleotide expansion disorder--myotonic dystrophymraises the possibility that nucleotide expansion per se is not salutary for ovarian development. However, no such correlation exists in Huntington disease, another triplet nucleotide expansion disorder. More importantly, women with complete FRAXA expansion do not always show POF. G. M y o t o n i c D y s t r o p h y a n d C T G Nucleotides Expansion Myotonic dystrophy is another nucleotide expansion disorder. It is an autosomal dominant disorder characterized by muscle wasting (head, neck, and extremities), frontal balding, cataracts, and male hypogonadism (80%) caused by testicular atrophy. Female hypogonadism is much less common and not well documented; however, it is usually assumed that age of menopause does not seem to be decreased. Pathogenesis involves nucleotide expansion of CTG, with (CTG),, occurring in the 3' untranslated region. Normally there are 5-27 CTG repeats. Heterozygotes usually have at least 50 repeats, and severely affected individuals have 600 or more. As in FRAXA, poor response to ovulation induction is observed. Sermon et al. [83] report fewer embryos per cycle than in standard assisted reproductive technologies (ART) and few pregnancies in preimplantation genetic diagnosis. Neither fragile X nor myotonic dystrophy need ordinarily be considered in the differential diagnosis of primary ovarian failure. However, these disorders occasionally are the explanation for POE H. G a l a c t o s e m i a In galactosemia enzyme deficiencies prevent synthesis of glucose from galactose. Several different enzymes and their mutant alleles, usually autosomal recessive in nature, may be responsible. Especially of interest is the form of galactosemia caused by deficiency of galactose-1-phosphate uridylyltransferase, controlled by a gene on 9p. In addition to renal, hepatic, and ocular damage, ovarian failure may be associated. Initially Kaufman et al. [84] reported 12 of 18 female cases had premature ovarian failure (POF). Waggoner et al. [85] later reported that 8 of 47 (17%) females with galactosemia had ovarian failure. Pathogenesis presumptively involves galactose toxicity after birth, given that maternal enzymes should be protective in utero. Consistent with this idea is that a neonate with galactosemia showed normal ovarian histology [86]. Given the clinical severity of galactosemia and necessity for childhood dietary treatment, it seems highly unlikely that undiagnosed galactosemia would prove to be the cause of

CHAPTER5 Genetic Programming in Ovarian Development ovarian failure in women presenting solely with primary amenorrhea or premature ovarian failure. Of greater general interest, therefore, was the report in 1989 by Cramer et al. [6] that GALT heterozygotes were at increased risk for POF. However, the same author later failed to observe GALT abnormalities in another sample of women with early menopause [5]; Kaufman et al. [87] likewise failed to confirm. Moreover, not all homozygotes for human galactosemia are abnormal, nor are even transgenic mice in which GALT is inactivated [88]. In summary, homozygous but probably not heterozygous galactose-l-phosphate uridylyltransferase deficiency is associated with ovarian failure.

I. D e f i c i e n c y o f 1 7 c e - H y d r o x y l a s e / 17,20-Desmolase Sex steroid synthesis requires intact adrenal and gonadal biosynthetic pathways. Various genes and their products (enzymes) are necessary to convert cholesterol to testosterone or androstenedione and, hence, to estrogens. An enzyme block may have varying consequences, depending on its site of action. The most common adrenal biosynthetic problem is female pseudohermaphroditism due to deficiency of 21- or 1 lfl-hydroxylase. However, ovarian development is normal in both these adrenal biosynthetic defects. If the enzyme 17ce-hydroxylase is not present (Fig. 5), neither androgens nor estrogens are synthesized. Thus, primary amenorrhea occurs in 46,XX cases. In 46,XY cases lack of androgens results in lack of virilization. However, the situation is complex because 17ce-hydroxylase and 17,20desmolase (lyase) are governed by a single gene on 10q. The gene C Y P 1 7 codes for a cytochrome P450 enzyme and is

I BIOSYNTHETIC

Acetate

89 located on 10q. About 150 cases have been reported, and most are male (46,XY) pseudohermaphrodites. Mutations have usually been point mutations rather than deletions or gene conversions. C Y P 1 7 mutations have been described that affect only 17,20-desmolase function [89,90]. In females, deficiency of 17ce-hydroxylase/17,20-desmolase (CYP17) should be considered a rare cause of hypergonadotropic hypogonadism. Ovaries are hypoplastic and sometimes streaklike in appearance. Oocytes appear incapable of reaching diameters greater than 2.5 mm [91 ]. However, stimulation with exogenous gonadotropins can mature follides to produce oocytes capable of fertilization in vitro [92]. Hypertension caused by hypervolemia occurs because of mineralocorticoid excess, an important diagnostic clue. If hypertension is not present, clinical presentation is similar to XX gonadal dysgenesis without somatic anomalies. Diagnosis is based on elevated progesterone, deoxycorticosterone, and corticosterone coupled with decreased testosterone and estrogen.

J. A r o m a t a s e M u t a t i o n s Conversion of androgens (A4-androstenedione) to estrogens (estrone) requires cytochrome P450 aromatase, an enzyme that is the gene product of a single 40-kb gene located on chromosome 15q21.1 [93]. The gene consists of 10 exons. Ito et al. [94] reported a mutation in this C Y P 1 9 (P450arom) gene in a 18-year-old 46,XX woman with primary amenorrhea and cystic ovaries. The patient was a compound heterozygote for two different point mutations in exon 10. The mutant protein had no activity. The above cases notwithstanding, deficiency of the aromatase enzyme is more often associated with genital

PATHWAYS ]

Cholesterol

Pregnenolone Progesterone

c

C

D 17 ~ - O H pregnenolone --~ Dehydroep~androsterone B JrB D E 17 c~ - OH progesterone ---~Androstenedione -l~Testosterone

II - deoxycorticosterone

II _ deoxycortisol

Corticosterone

Cortlsol

Estrone ~ E s t r a d l o l

Aldosterone

FIGURE 5 Importantadrenal and gonadal biosynthetic pathways. Letters designate enzymes required for the appropriate conversions. (A) 20a-Hydroxylase,22a-hydroxylase, and 20,22-desmolase; (B) 38fl-ol-dehydrogenase; (C) 17ce-hydroxylase; (D) 17,20-desmolase;(E) 17-ketosteroidreductase; (F) 21-hydroxylase;(G) 11-hydroxylase.(From Simpson [12].

90

JOE LEIGH SIMPSON

ambiguity. Shozu et al. [95] detected placental aromatase deficiency manifesting as maternal virilization during the third trimester. The 46,XX infant was born with genital ambiguity (female pseudohermaphroditism). Adrenal enzyme defects were not evident. The molecular basis of the mutation was an 87-bp insert in exon 6 of the aromatase gene, altering the splice junction site to produce a novel protein with 29 additional amino acids. Aromatase mutation in 46,XX female infants has been associated with genital ambiguity [94] or clitoromegaly [96]. In latter cases clitoral enlargement occurred at puberty, but breast development did not. Multiple ovarian follicular cysts were evident. FSH was elevated, estrone and estrodial were low. Estrogen and progesterone therapy resulted in a growth spurt, decreased FSH, decreased androstenedione and testosterone, breast development, menarche, and fewer follicular cysts. Molecular studies demonstrated compound heterozygosity for CYP19 point mutations.

K. P o l y g l a n d u l a r A u t o i m m u n e S y n d r o m e and O v a r i a n A n t i b o d i e s Type I polyglandular autoimmune syndrome is an autosomal recessive disorder especially common in the Finnish and Iranian Jewish populations [97]. Affected individuals show moniliasis due to immune deficiencies, and deficiencies of the parathyroids, adrenals, and gonads exist. The frequency of hypergonadotropic ovarian failure is 60%. The gene is localized to 2q22.3 [98]. The murine homolog is the autoimmune regulation gene (AIRE). Type I polyglandular autoimmune syndrome ovarian failure is uncommon outside the Finnish and Iranian Jewish populations. Type II polyglandular autoimmune syndrome, also called Schmidt syndrome, is heterogenesis. Inheritance is autosomal dominant. Gonadal failure occurs, as well as adrenal, thyroid, and pancreatic hypofunction. Systemic defects involve the hematological, gastrointestrial, and ocular systems and the tegument (hair). Immunologic dysfunction is often pronounced and moniliasis common. HLA-B8 is associated, as are, to a lesser extent, DR3 and DR4 [99]. A related issue is the considerable literature concerning a potential relationship between POF and antiovarian antibodies. These antibodies may be either of generalized nature or specific against a single cellular component (e.g., gonadotropin receptor, stromal cells, zona pellucida). Antiovarian antibodies as a cause of ovarian failure were reviewed by Anasti [100]. To this author, a casual relationship seems less likely than a secondary effect (epiphenomenon), arising only after damage has occurred for unrelated reasons. Even if causative, POF and not primary amenorrhea would be expected. Similar reasoning applies as well to oophoritis and ovarian failure.

L. G e r m Cell Failure in B o t h Sexes In several sibships, male and female sibs have each shown germ cell failure. Affected females show streak gonads, whereas males show germ cell aplasia (Sertoli cell-~6nly syndrome, or del Castillo phenotype). In two families, parents were consanguineous, and in each no somatic anomalies coexisted [ 101,102]. In three other families, coexisting patterns of somatic anomalies suggested distinct entities. Hamet et al. [103] reported germ cell failure, hypertension, and deafness; A1-Awadi et al. [104] reported germ cell failure and alopecia; Mikati et al. [105] reported germ cell failure, microcephaly, and short stature. These families demonstrate that the same autosomal gene is capable of deleteriously affecting germ cell development in each sex, presumably acting at a site common to early germ cell development. Elucidating such genes could have profound implications for understanding normal germ cell development and gametogenesis. To this end the noteworthy murine model for gcd (germ cell deficiency) is worth recalling. This autosomal recessive disorder is characterized by germ cell deficiency in both male and female mice.

M. 4 6 , X X A g o n a d i a In agonadia, the gonads are absent, the external genitalia are abnormal, and all but rudimentary Mtillerian or Wolffian derivatives are absent. Almost all affected individuals are 46,XY. External genitalia usually consist of a phallus about the size of a clitoris, underdeveloped labia majora, and nearly complete fusion of labioscrotal folds. A persistent urogenital sinus is often present. By definition, gonads cannot be detected, and neither normal Mtillerian derivatives nor normal Wolffian derivatives are ordinarily present. Rudimentary structures may be present along the lateral pelvic wall. In about one-half the cases, somatic anomalies coexist, i.e., craniofacial and vertebral anomalies and mental retardation [ 106]. Because almost all cases are 46,XY, pathogenic explanations have always focused on loss of testes early in embryogenesis. Any pathogenic explanation for agonadia in 46,XY individuals must take into account not only absence of gonads (usually testes), but also abnormal external genitalia and lack of internal genital ducts. Two general explanations have been invoked: (1) Fetal testes functioned long enough to complete male differentiation, but then disappeared, hence the synonymous designation "testicular regression syndrome." (2) Gonadal, ductal, and genital systems concomitantly developed abnormally as result of either defective anlagen, defective connective tissue, action of a teratogen, or other mechanisms. Given existence of both heritable tendencies [107] and frequent coexistence of somatic anomalies, defective connective tissue has seemed plausible

CHAPTER5 Genetic Programming in Ovarian Development in certain cases. Alternatively, cases with and without somatic anomalies could be etiologically distinct (genetic heterogeneity). Of interest in the present context of causes of primary amenorrhea, are there even rarer reports of 46,XX agonadia? Sporadic cases were reported by Duck et al. [108] and Levinson et al. [ 109]. Mendonca et al. [ 110] reported agonadia without somatic anomalies in phenotypic sibs of unlike chromosomal complements (46,XY and 46,XX). Kennerknecht et al. [111] reported agonadism, hypoplasia of the pulmonary artery and lung, and dextrocardia in XX and XY sibs.

91 pressivity. Thus, the autosomal recessive mutations responsible for certain forms of XX gonadal dysgenesis may be manifested as less severe ovarian pathology. In Finnish cases ascertained by Aittomaki [58,59], POF also not infrequently coexisted in the same kindred as complete ovarian failure (COF). This applied to both the FSH receptor mutation cases (C566T) as well as the non-C566T cases. XX gonadal dysgenesis genes may therefore be responsible for familial premature ovarian failure.

C. A u t o s o m a l D o m i n a n t P O F

XI. TO WHAT EXTENT IS PREMATURE OVARIAN FAILURE G E N E T I C ? As already discussed, premature ovarian failure can result from several different genetic mechanisms. These include (1) X chromosomal abnormalities, (2) autosomal recessive genes causing the various types of XX gonadal dysgenesis, and (3) autosomal dominant genes whose action is restricted to POF. The first two mechanisms have already been considered in detail, so we shall focus here only the third. However, prior to doing so it is useful to recall the role that the former two etiologies play in POE

A. X C h r o m o s o m a l A b n o r m a l i t i e s Premature rather than complete ovarian failure is not rare in the X abnormalities. At least 10 to 15% of 45,X/46,XX individuals menstruate, compared to fewer than 5% of 45,X individuals [ 11]. The former percentage represents a minimum because many mosaic individuals are so mildly affected that they are never detected clinically. Spontaneous menstruation occurs in about half of all 46,X, del(X)(pl 1) and 46,X, del(X)(p21 or 22) individuals, who often present with secondary amenorrhea and premature ovarian failure. Deletions or X/autosomal translocations involving regions Xp22 and Xq26 are more likely to be associated with premature ovarian failure than with complete ovarian failure. Recall also that women with the FRAXA premutation ( F M R 1 ) show an increased frequency of premature ovarian failure, a phenomenon that may or may not be the result of perturbations of the terminal Xq ovarian maintenance genes.

B. A u t o s o m a l R e c e s s i v e P O F In some families we have noted that the propositus may have 46,XX gonadal dysgenesis and streak gonads, with a sib having only ovarian hypoplasia with some oocytes. These sibships suggest that the mutant gene responsible for XX gonadal dysgenesis is capable of exerting variable ex-

An entity different from the above is ostensibly idiopathic premature ovarian failure transmitted in more than one generation [ 112,113]. This suggests autosomal dominant inheritance, although in some of these families subtle X deletions or FRAXA premutations could have existed. This mechanism has already been noted to exist in the blepharophimosisptosis syndrome. Mattison et al. [114] studied five families. That these families were probably ascertained from a very large population base raises the concern that the familial aggregates could have been observed by chance or on the basis of polygenic factors. In none were ovarian antibodies present. Useful information on idiopathic autosomal dominant POF is coming from Italian investigators. Defining POF as cessation of menses for 6 months or longer, Vegetti et al. [115] studied 81 women under age 40 years. After excluding 10 cases of presumptively known etiology (5 abnormal karyotypes, 3 previous ovarian surgery, 1 prior chemotherapy, 1 galactosemia), pedigree analysis was performed. Of the remaining 71 cases, 23 (31%) had an affected female relative. The median age of subjects with a positive family history was older (37.5 yr) than those without such a history (31 yr). Family members were affected in a pattern consistent with autosomal dominant inheritance. Transmission occurred through both male and female members. Neither blepharophimosis-ptosis syndrome nor fragile X were observed. In the Italian study several different genes could be involved, but the familial nature of POF seems firmly established.

References 1. Jir~isek,J. (1976). Disorders of sexual differentiation. In "Principles of Reproductive Embryology" (J. L. Simpson, ed.), pp. 51-111. Academic Press, New York. 2. Cussen, L. K., and McMahon, R. (1979). Germ cells and ova in dysgenetic gonads of a 46,XY female dizygote twin. Arch. Dis. Child. 133, 373-375. 3. Greenberg,F., Greesick, M. V., Carpenter, R. J., Law, S. W., Hoffman, L. E, and Ledbetter, D. H. (1987). The Gardner-Silengo-Wachtel syndrome: Male pseudohermaphroditism with micrognathia, cleftpalate, and conotruncal cardiac defect. Am. J. Hum. Genet. 26, 59-64.

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4. Evans, E. E, Ford, C. E., and Lyon, M. F. (1977). Direct evidence of the capacity of the XY germ cell in the mouse to become an oocyte. Nature (London) 267, 430-431. 5. Cramer, D. W., Xu, H., and Harlow, B. L. (1995). Family history as a predictor of early menopause. Fertil. Steril. 64, 740-745. 6. Cramer, D. W., Harlow, B. L., Barbieri, R. L., and Ng, W. G. (1989). Galactose-l-phosphate uridyl transferase activity associated with age at menopause and reproducutive history. Fertil. Steril. 51, 609-615. 7. Torgerson, D. J., Thomas, R. E., and Reid, D. M. (1997). Mothers' and daughters' menopausal ages: Is there a link? Eur. J. Obstet. Gynecol. Reprod. Biol. 74, 63-66. 8. Treloar, S. A., Do, K. A., and Martin, N. G. (1998). Genetic influences on the age at menopause. Lancet 352, 1084-1085. 9. Snieder, H., MacGregor, A. J., and Spector, T. D. (1998). Genes control the cessation of a woman's reproductive life: A twin study of hysterectomy and age at menopause. J. Clin. Endocrinol. Metab. 83,

26.

27.

28.

29.

1875-1880.

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CHAPTER 5 Genetic Programming in Ovarian Development

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JOE LEIGH SIMPSON association among blepharophimosis, resistant ovary syndrome, and true premature menopause. Fertil. Steril. 50, 747-751. Schwartz, C. E., Howard-Peebles, P. N., Bugge, M., Mikkelsen, M., Tommerup, N., Hull, C., Hagerman, R., Holden, J. J., and Stevenson, R. E. (1994). Obstetricial and gynecological complications in fragile X carriers: Multicenter study. Am. J. Med. Genet. 51, 400-402. Murray, A., Webb, J., Grimley, S., Conway, G., and Jacobs, P. (1998). Studies of FRAXA and FRAXE in women with premature ovarian failure. J. Med. Genet. 35, 637-640. Black, S. H., Levinson, G., Harton, G. L. Palmer, F. T., Sisson, M. E., Schoener, C., Nance, C., Fugger, E. F., and Fields, R. A. (1995). Preimplantation genetic testing (PGT) for fragile X (fraX). Am. J. Hum. Genet. 57, A31. Kenneson, A., Cramer, D. W., and Warren, S. T. (1997). Fragile X premutations are not a major cause of early menopause. Am. J. Hum. Genet. 61, 1362-1369. Sermon, K., De Vos, A., Van de Velde, H., Seneca, S., Lissens, W., Joris, H., Vandervorst, M., Van Steirteghem, A., and Liebaers, I. (1998). Fluorescent PCR and automated fragment analysis for the clinical application of preimplantation genetic diagnosis of myotonic dystrophy. Mol. Hum. Reprod. 4, 791-796. Kaufman, F. R., Kogut, M. D., Donnell, G. N., Goebelsmann, U., March, C., and Koch, R. (1981). Hypergonadotropic hypogonadism in female patients with galactosemia. N. Engl. J. Med. 304, 994-998. Waggoner, D. D., Buist, N. R., and Donnell, G. N. (1990). Long-term prognosis in galactosaemia: Results of a survey of 350 cases. J. Inherit. Metab. Dis. 13, 802-818. Levy, H. L., Driscoll, S. G., Porensky, R. S., and Wender D. F. (1984). Ovarian failure in galactosemia N. Engl. J. Med. 310, 50. Kaufman, F. R., Devgan, S., and Donnell, G. N. (1993). Results of a survey of carrier women for the galactosemia gene. Fertil. Steril. 60, 727-728. Leslie, N.D., Yager, K., and Bai, S. (1995). A mouse model for transferase deficiency galactosemia. Am. J. Hum. Genet. 57, 191, A38. Geller, D. H., Auchus, R. J., Mendonca, B. B., and Miller, W. L. (1997). The genetic and functional basis of isolated 17,20-1yase deficiency. Nat .Genet. 17, 201-205. Biason-Lauber, A., Leiberman, E., and Zachmann, M. (1997). A single amino acid substitution in the putative redox partner-binding site of P450c17 as cause of isolated 17,20-1yase deficiency. J. Clin. Endocrinol. Metab. 82, 3807-3812. Araki, S., Chikazawa, K., Sekiguchi, I., Yamauchi, H., Motoyama, M., and Tamada, T. (1987). Arrest of follicular development in a patient with 17 alpha-hydroxylase deficiency: Folliculogenesis in association with a lack of estrogen synthesis in the ovaries. Fertil. Steril. 47, 169172. Rabinovici, J., Blankstein, J., Goldman, B., Rudak, E., Dor, Y., Pariente, C., Geier, A., Lunenfeld, B., and Mashiach, S. (1989). In vitro fertilization and primary embryonic cleavage are possible in 17ahydroxylase deficiency despite extremely low intrafollicular 17/3estradiol. J. Clin. Endocrinol. Metab. 68, 693-697. Simpson, E. R., Michael, M. D., Agarwal, V. R., Hinshelwood, M. M., Bulun, S. E., and Zhao, Y. (1997). Cytochromes P450 11: Expression of the CYPI9 (aromatase) gene: An unusual case of alternative promoter usage. FASEBJ. 11, 29-36. Ito, Y., Fisher, C. R., Conte, E A., Grumbach, M. M., and Simpson, E. R. (1993). Molecular basis of aromatase deficiency in an adult female with sexual infantilism and polycystic ovaries. Proc. Natl. Acad. Sci. U.S.A. 90, 11673-11677. Shozu, M., Akasofu, K., Harada, T., and Kubota, Y. (1991). A new cause of female pseudohermaphroditism: Placental aromatase deficiency. J. Clin. Endocrinol. Metab. 72, 560-566. Mullis, P. E., Yoshimura, N., Kuhlmann, B., Lippuner, K., Jaeger, P., and Harada, H. J. (1997). Aromatase deficiency in a female who is compound heterozygote for two new point mutations in the P450

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SHAPTER (

Neuroendocrlne " Reg ulat "on o f the Perimenopause Transition NANCY E.

I. II. III. IV.

REAME

Center for Nursing Research and Reproductive Sciences Program, The University of Michigan, Ann Arbor, Michigan 48109

V. The Neuroreproductive Axis in Postmenopause VI. Brain Aging and Reproductive Senescence VII. Future Studies References

Definition of Perimenopause Ovarian Determinants of Reproductive Aging Dynamic Gonadotropin Secretion in Young Women Gonadotropin Changes during Perimenopause

I. D E F I N I T I O N

year 2025, the number of postmenopausal women in the United States is expected to double [5]. Given the increasing life span of women in the United States, the number of years spent in the postmenopausal state is significant. Thus, the cessation of menses and the resulting hypoestrogenism may have important health consequences for the quality of life of a large and growing proportion of the population. Urinary continence, bone metabolism, cardiovascular function, memory and cognition, the synchrony of daily biorhythms, and the aging process itself have all been shown to be influenced by estrogen. Moreover, compared to other organ systems, the female reproductive system is unique in that it undergoes spontaneous failure at a relatively young age, thus making it an excellent model for the study of the aging process free of chronic disease. The concept of the perimenopause was first introduced by Treolar and colleagues in 1967 [6] when they conducted a cross-sectional analysis of several hundred menstrual cycle calendars obtained from women across the reproductive life span. In that study, the critical marker of aging was menstrual irregularity, defined as a change in genital bleeding to

OF PERIMENOPAUSE Reproductive aging is a continuum that begins with a steep decline in fertility by age 35 years, long before the final menstruation (menopause) occurs at age 51 years on average [ 1]. The climacteric, which is that period of time when reproductive function declines, is associated with a discordant rise in follicle-stimulating hormone (FSH); this rise in FSH is viewed as monotropic, because luteinizing hormone (LH) levels remain normal. Progressive loss of regular menstrual cyclicity and depletion of responsive follicles from the ovary are also associated with the climacteric [2]. According to the definition of the World Health Organization [3], the perimenopause is the period immediately prior to menopause when endocrinological, biological, and clinical features of approaching menopause commence, continuing for at least the first year after menopause. As the year 2000 approaches, there will be some 35 million perimenopausal women in the United States, with half a million women added annually to the midlife population [4]. By the

MENOPAUSE: BIOLOGY AND PATHOBIOLOGY

95

Copyright9 2000by AcademicPress. Allrightsof reproductionin anyformreserved.

96

NANCY E. REAME

either longer or shorter flow intervals. However, menstrual cycle irregularity is now known to be a late feature of the perimenopause transition, brought about by neuroendocrine events that occur well before cyclic ovulation is disrupted. Premature menopause (premature ovarian failure) is a condition causing amenorrhea, hypoestrogenism, and elevated gonadotropins in women less than age 40 years [7]. In a survey of 2000 women [8], the age-specific incidence was 1 in 100 by age 40 years, and 1 in 1000 by age 30 years. Although most cases have an autoimmune etiology, some cases are idiopathic. In addition to the premature loss of fertility, health consequences for these young women appear to be similar to consequences for older hypoestrogenic women, and include higher cardiac risk and bone loss acceleration. The clinical picture of this disordered folliculogenesis has been likened to the natural perimenopausal stage of diminished ovarian reserve when transient fluctuations of follicular development and atresia occur prior to complete cessation of ovarian function. However, compared to the perimenopause, women with premature menopause show much higher FSH concentrations, perhaps due to the younger reproductive axis or, alternatively, due to less restraint from differences in inhibin [9]. Despite the inevitability of menopause for all women who live long enough, the events leading up to and following the last menses are highly variable in duration and magnitude from woman to woman, thus contributing to the wide range in age at menopause (ages 45-55 years). In a study of ovarian function in 17 women in this age range, the number of follicles present in the ovary varied from more than 1000 in those still cycling to a few hundred in perimenopause, and to none in the postmenopausal state [ 10]. Moreover, although the mean duration of the perimenopausal transition is approximately 3.5 years, nearly 10% of women will have no perimenopause transition whatsoever, but rather the persistence of regular cycles until an abrupt cessation of menstruation [ 11 ]. Such variability in the onset and regulation of the menopause experience suggests that its underlying etiology is multifactoral and dependent on a variety of external and internal influences. This chapter describes the evidence to date that aging effects at all three levels of the hypothalamic -pituitary- ovarian (HPO) axis may contribute to the pronounced hypersecretion of pituitary gonadotropins that occurs at the close of the reproductive years.

II. O V A R I A N

DETERMINANTS

OF REPRODUCTIVE

AGING

A. R i s i n g F S H Studies in the past decade have clarified that the hallmark sign of reproductive aging is the disproportionate increase in circulating FSH vs. LH, especially prominent in the early

follicular phase. This discordant increase in FSH occurs gradually across the midreproductive years, becoming pronounced in women over 40 years old [12,13]. The etiology of this rise of one gonadotropin but not the other has been the topic of investigation. It is now generally accepted that gonadotropin-releasing hormone (GnRH) is the stimulating factor for both LH and FSH and any divergence in their secretion can be explained by (1) differential sensitivities of LH and FSH to variations in the dose or frequency of pulsatile GnRH secretion, (2) the gonadal hormonal milieu, including sex steroid and nonsteroidal factors, and/or (3) alterations in the pituitary tone of activin and/or follistatin. In keeping with these assumptions, the prevailing view is that the monotropic increase in FSH results from a greater sensitivity to declining ovarian feedback on the hypothalamicpituitary-ovarian axis compared to LH. Because the increase in basal FSH occurs in conjunction with normal or even elevated levels of estradiol, it is presumed that diminished input from nonsteroidal factors, notably inhibin from the declining pool of follicles, is responsible for the dampened inhibitory tone of ovarian feedback [14,15].

B. A g e C h a n g e s in L H Evidence for a clear age-related increase in LH secretion in regular-cycling women has been more equivocal than for FSH. In a study of 94 subjects ages 24 to 50 years, there was a significant increase in mean FSH secretion detected by age 35 years, with no aging effects observed in LH secretion until age 45 years [ 12]. In another study by the same authors of 500 regularly cycling, infertile women, the rise in FSH levels was observed to begin as early as age 28 years [13]. In addition, a statistically significant increase in mean LH levels during the follicular phase could be detected by age 35 years, followed by a further increase in women older than age 40 years. Unlike prior investigations, the authors concluded that an increase in both FSH and LH concentrations occurred in women with regular ovulatory cycles quite early in reproductive life and could be used as the earliest endocrine markers of reproductive aging. The previously undetected rise in LH prior to age 40 years may have been uncovered in this cohort due to the very large sample size. The clinical relevance and mechanisms for such a subtle age-related change in LH concentrations await further study. The current view is that the regulation of LH secretion is relatively resistant to the incipient decline in ovarian reserve, with perhaps only a subtle rise in concentrations until just prior to menopause, when the ovarian pool of responsive follicles is dramatically reduced along with a steep decline in estradiol and gonadal inhibin [ 10]. The fact that ovariectomy of premenopausal women is associated with a dramatic increase (four- to sixfold) in LH levels within 1 - 4 weeks of surgery [ 16] has served as evidence that ovarian inhibition is

CHAPTER6 Perimenopause Neuroendocrine Regulation the major input to the GnRH-mediated regulation of LH secretion. It should be noted that in the absence of negative feedback signals (estradiol and inhibin) after ovariectomy, the discordance in gonadotropin secretion is particularly evident, with FSH levels rising earlier and remaining persistently higher than LH, suggesting that the unrestrained, endogenous GnRH pulse generator may directly or indirectly favor FSH secretion.

97

II []

Young Older

9

9

6

_J D 6 ~SIS

._1 -~

"r _1

3

3

0

~

0

C. G o n a d a l P e p t i d e s

ao ~

--~'l::~300 t

Reports now suggest that an age-related decline in ovarian inhibin-B is the primary trigger for the selective FSH rise in the menstrual cycles of older women [ 17] (see Chapter 2). However, to presume that a singular deficiency in inhibin activity could account for age-related elevations in FSH fails to consider the other component gonadal peptides known to mediate FSH regulation. The inhibin a, fiA, and fiB protein subunits are members of the transforming growth factor-fi family of peptides. Produced in the ovary and pituitary, they are encoded by distinct genes and dimerize to give rise to inhibin A (ce,fiA), inhibin B (ce,fiB), activin A (flA, fiA), activin AB (flA, fiB), and activin B (fiB, fiB). Inhibins and activins have opposing effects on FSH secretion: inhibins suppress FSH, and activins stimulate FSH production [ 18]. Follistatins, monomeric proteins distinct from both inhibins and activins, have functional overlap with inhibins in suppressing FSH release [19], through their action as binding proteins for both activins and inhibins [20]. Inhibin A (from the mature follicle and corpus luteum) and inhibin B (from small antral follicles) are secreted in reciprocal, mirror-image patterns of each other across the ovulatory menstrual cycle, acting in tandem to regulate the cyclic profile of FSH secretion [21 ]. Studies have now documented the negative influence of estradiol, inhibin, and follistatin, and the positive roles of activin and GnRH in regulating FSH release. With the development of highly specific assays for most of these gonadal peptides, investigations of the combined steroidal and nonsteroidal ovarian milieu during reproductive aging are now possible. Noteworthy is the finding that in postmenopausal women, circulating levels of both follistatin [22] and activin [23] are elevated. We and others have begun to test the hypothesis that changes in the overall tone of ovarian feedback may contribute to the age-related rise in FSH. When measured simultaneously, the secretory profiles of the inhibins and activin A have been demonstrated to be altered across the menstrual cycle of women over age 40 years. Compared to youngeraged controls, activin A demonstrates higher concentrations that do not change across the cycle, whereasthe overall inhibin tone is reduced [24,25]. The pool of developing follicles may be more sensitive to the age-related effects on in-

12

e"

.--_o200 I-

20

o~ tU 100

10

ID t2 ffl

0

0 G.

0

Follicular

Luteal

Follicular

Luteal

FIGURE 1 Mean concentrations of gonadotropins and sex steroids in young (mean age = 27.9 + 2 years) and older (mean age = 43.7 + 1 years) cycling women.., Group differences; LH, p < 0.05; FSH,p < 0.001. From Ref. 24, N. E. Reame, T. L. Wyman,D. J. Phillips, D. M. de Kretser, and V. Padmanabhan (1998). Net increase in stimulatory input resulting from a decrease in inhibin B and an increase in activin A may contribute in part to the rise in follicular phase follicle-stimulating hormone of aging cyclic women. J. Clin. Endocrinol. Metab. 83, 3302-3307. 9 The Endocrine Society.

hibin production than the corpus luteum, because the decline in inhibin B is already apparent in women as young as age 35 years, prior to a decline in FSH or luteal phase inhibin A [26]. Both total and and free follistatin concentrations in the periphery do not appear to be influenced by age or cycle phase [24] (Figs. 1 and 2). This altered proportion of dimers in older women has been explained by a reduction in inhibin a subunit available from the depleted ovarian pool to combine with the/3 subunits to form inhibins, thus favoring activin formation and, in turn, enhanced FSH secretion [25]. In a group of perimenopausal women with irregular cycles, the suppression of FSH during hormone replacement therapy was associated with a suppression of activin A in the face of unchanged inhibin B and follistatin [27,28]. Because activins appear capable of direct pituitary stimulation of FSH secretion and are produced in the pituitary [29], this finding is compatible with the view that estradiol's negative feedback action on FSH release may involve indirect, paracrine as well as direct, endocrine mechanisms. However, this hypothesis must await further investigation until more sensitive assays are developed for activin B and activin AB, as well as for measurement of the free (biologically active) form of activins. Moreover, it is not known whether the low levels at which these FSH regulators circulate in the peripheral blood are of sufficient magnitude to play an endocrine role.

98

NANCY E. REAME

InhibinA

Total inhibin a

Inhibin B

m YoungI ['~ Older___l

400 ._1

E O. v t'-

25 200 t-t'n

Free Follistatin

Total Follistatin ~-, 600 E <

.J

6~

U~3 c"

.c_

400

4

..~

co

._ ,,.., 0

<

O tt.

200

2

Follicular

Luteal

Follicular

Luteal

Follicular

Luteal

FIGURE 2

Mean concentrations of gonadal proteins from the same subjects as in Fig. 1. Total inhibin is a derived number from the sum of inhibin A and inhibin B . . , Group differences; p < 0.05. From Ref. 24, N. E. Reame, T. L. Wyman, D. J. Phillips, D. M. de Kretser, and V. Padmanabhan (1998). Net increase in stimulatory input resulting from a decrease in inhibin B and an increase in activin A may contribute in part to the rise in follicular phase follicle-stimulating hormone of aging cyclic women. J. Clin. Endocrinol. Metab. 83, 3302-3307. 9 The Endocrine Society.

III. D Y N A M I C G O N A D O T R O P I N S E C R E T I O N IN Y O U N G W O M E N The classic studies of Knobil and colleagues conducted in the late 1970s clearly demonstrated that a pulsatile pattern of gonadotropin-releasing hormone was essential for physiological gonadotrope function. Since then, much has been learned about the precise nature of the pulsatile rhythms in both reproductive health and illness through the assessment of pulsatile luteinizing hormone as a surrogate marker of GnRH pulse generator activity. LH episodes originate from periodic activation of the pituitary gonadotroph by intermittent hypothalamic GnRH stimulation. The release magnitude of episodic gonadotropin secretion is defined, among other determinants, by the pituitary responsiveness and the capacity of GnRH to prime the gonadotroph. Serial measurement of FSH concentrations is a less accurate estimate of GnRH secretion due to a reduced and delayed release by the pituitary as well as a slower metabolic clearance rate when compared to LH. A. T h e M e n s t r u a l C y c l e Numerous cross-sectional [30-32] as well as longitudinal studies [33] of pulsatile LH characteristics during the ovulatory menstrual cycle have demonstrated that LH pulse frequency increases during the follicular phase from approxi-

mately one pulse every 90-100 min to one pulse per hour at the time of the LH surge. LH pulse frequency is maintained during the LH surge, but slows during the luteal phase under the influence of the corpus luteal steroids (and central endogenous opioids), with one pulse every 2 - 6 hr varying in amplitude. Thus, the ability to change GnRH amplitude and slow frequency appears to be a critical requirement for the maintenance of cyclic ovulatory function. The dynamic gonadotropin secretory patterns in relation to HPO function have now been well documented for many of the reproductive endocrine disorders and shown to differ from that of the

TABLE I Dynamic Gonadotropin Activity" Hormonal component Central opioid tone FSH (mlU/mil) LH (mlU/ml) LH pulse interval (min) LH pulse amplitude Estradiol Androgens

Postmenopause

Cycle day 6

HA h

PCO c

Absent >30 > 30

Absent <- 10 <--10

Present < 10 < 10

Absent --<10 > 10

60-100 High Very low Low

60-100 Low Low Very low

> 100 Variable Very low Low

60 High Elevated Elevated

a Modified from Ref. 33a, with permission from Springer-Verlag. b HA, Hypothalamic amenorrhea. c p c o , Polycystic ovarian syndrome.

CHAPTER6 Perimenopause Neuroendocrine Regulation ovulatory menstrual cycle (Table 1) [33a]. This body of knowledge provides important context for examining dynamic changes in the perimenopause years.

B. G o n a d o t r o p i n C h r o n o b i o l o g y and Sleep Effects Superimposed on the cyclic changes in pulsatile gonadotropin secretion that occur as a function of the menstrual cycle are the effects of circadian rhythms and sleep. As reviewed by Van Cauter [34], the accumulated evidence suggests that all hormones of the hypothalamo-pituitary axis are influenced by both sleep (irrespective of the time of day) and circadian rhythmicity (regardless of the sleep-wake status). In terms of GnRH activity, circadian effects appear to influence pulse amplitude, whereas sleep affects pulse frequency. LH exhibits a nocturnal, sleep-dependent decline in basal concentrations that is restricted to the early follicular phase [35]. Using a 20-min sampling frequency, Soules et al. [36] demonstrated that this nocturnal inhibition was due to a slowing of LH pulse frequency during the early morning between 1 a.m. and 5 a.m., with a corresponding increase in LH pulse amplitude. The sleep-related decline in LH secretion that is unique to the early follicular phase is believed to be mediated by hypothalamic opioid activity, because pulse frequency can be enhanced after naloxone administration [37], whereas dopaminergic blockade has no effect [38]. Rossmanith and colleagues [39] showed that during the early follicular phase, LH and FSH responses to 25 ~g of GnRH are markedly blunted when assessed in awake subjects at night; this blunting could be completely prevented for LH but not FSH when GnRH was administered during sleep. They concluded that the increased LH pulse amplitude observed during sleep in the early follicular phase was not due to increased pituitary responsiveness, given that there was no circadian effect on the priming effect of GnRH dosing. The physiologic relevance of sleep-related changes in LH in the early follicular phase is not known, but it has been suggested to be important for the maintenance of adequate cyclicity and normal folliculogenesis [40].

C. S y n c h r o n i c i t y of O t h e r H o r m o n e s w i t h Nighttime Gonadotropin Secretion Circadian and sleep-entrained variabilities in the release of other pituitary tropic hormones, such as TSH [41] and prolactin [42], have also been reported. In addition, estradiol concentrations have been observed to exhibit sleep and nighttime enhancement [39]. Changes in reproductive hormone release coincident with sleep may represent mani-

99 festations of entrained links between CNS regulation and endocrine function. In rodents, the suprachiasmatic nucleus (SCN) has been identified as the pacemaker for circadian rhythmicity [43]. Several important physiological events presumed to be controlled by the SCN pacemaker occur during the night and include secretion of melatonin, suppression of vasopressin secretion, increased sensitivity to the phaseshifting effects of light pulses, and high electrical activity and glucose utilization in the SCN. How these various phenomena interface with diurnal features of the reproductive axis requires further definition. It has been demonstrated that the ultradian fluctuations in leptin, the obesity gene product, show pattern synchrony with those of both LH and estradiol in young women during the follicular phase, a time when nocturnal slowing of LH pulses was evident [44]. The investigators postulated that in addition to its trophic effects, leptin may contribute to reproductive axis organization by regulating the minute-to-minute oscillations in the levels of LH and estradiol in the critical period before ovulation. Thus, at night, as leptin levels rise, the pulsatility profile of LH changes from high frequencylow amplitude to low frequency-high amplitude, becoming synchronous with leptin pulsatility. Such a view would help explain the disruption of HPO function that is characteristic of states of low leptin synthesis, such as anorexia nervosa. Although melatonin controls seasonal reproductive cyclicity in some mammalian species, its role as a pacemaker of the human reproductive axis is controversial. Cagnacci et al. [45,46] speculated that melatonin may play a role in timing diurnal LH modifications, because melatonin administration enhanced basal daytime LH secretion, LH pulse amplitude, and responses to a low-dose GnRH challenge during the follicular phase, but not in the luteal phase. However, others [47] have not found a consistent melatonin effect on follicular-phase LH levels. In addition, the circadian rhythm of melatonin secretion does not change significantly across the menstrual cycle and supraphysiologic melatonin concentrations did not decrease the midcycle LH surge response [48-50].

D. S e a s o n a l V a r i a t i o n Seasonal variability in pulsatile LH secretion was suggested by the findings Of Martikainen et al. [51 ], who studied normal volunteers in Finland for 6 daytime hours of the midfollicular phase during peak differences in seasonal daylight (December vs. May). Although mean concentrations, pulse frequency, and amplitude of both LH and FSH did not differ by season, the area under the curve was significantly higher during the winter. A seasonal effect has also been observed for the timing of the LH surge, with ovulation more likely to occur in the morning during the spring and in the evening during autumn and winter [52]. Levels of nighttime LH have

100

NANCY E. REAME

been shown to be higher in the summer at midcycle, at a time when the nocturnal rise in melatonin was reduced [53].

E. FSH Chronobiology Whether FSH secretion changes over the 24-hr period remains controversial. When measured every 15 min, a robust circadian rhythm has been described in young women but is diminished after menopause [54]. In that study, a cosine rhythm with a nighttime decline in transverse mean FSH was observed in the early and late follicular phase despite no evidence of circadian rhythmicity in LH or estradiol. Although diminished compared to the early follicular phase, the comparable timing of the FSH acrophase in the late follicular and midluteal phases, and its presence in the postmenopause group, prompted the investigators to propose a central, rather than peripheral, feedback mechanism for the circadian rhythmicity. The authors concluded that their findings provided further evidence for a dissociation in the hypothalamic regulation of pituitary LH and FSH secretion in women. Other studies have failed to confirm a circadian rhythm in FSH [55]. Using an analytical technique to define discrete secretory bursts measured at 10-min intervals over 24 hr, FSH secretion was maximal during the late follicular phase (high estradiol) and in postmenopausal women unrestrained by estrogen [56]. Although FSH and LH secretory bursts demonstrated a significant concordance rate of 25%, the relatively high rate of nonconcordance prompted the investigators to propose that distinct mechanisms other than a single releasing hormone probably operate to regulate differentially the secretion of each gonadotropin. Diurnal variations in LH and FSH were not described.

IV. GONADOTROPIN CHANGES DURING PERIMENOPAUSE

A. Older Cyling Women The fact that FSH is elevated in normal cycling women over age 40 without concomitant decreases in ovarian steroids would also support the possibility of an aging change in the GnRH signal. Attention has turned from the mechanisms that give rise to the early increase in FSH to the causes of the more subtle alterations in pulsatile LH secretion that lead to the magnified secretory profile of the postreproductive years. Collectively, the data are conflicting. Our studies [57] of daytime pulsatile LH secretion where blood was sampled every 10 min across three phases of the same menstrual cycle showed a gradual rise in pulse frequency with advancing age (Fig. 3). In contrast, in other studies, LH pulsatile secretion in older cycling women was observed to be similar [58] or reduced [59] compared to younger controls. The use of less frequent sampling (every 20 min) in one

FIGURE 3 Effectsof age on pulsatile LH secretory characteristics. Age groups are in years. All subjects were studied across the same menstrual cycle. FOLL, Early follicular phase; ML, midluteal phase; LL, late luteal phase.., p < 0.05; ***,p < 0.001. From Ref. 55, N. E. Reame,R. P. Kelch, I. Z. Beitins, M. Y. Yu, C. Zawacki, and V. Padmanabhan (1996). Age effects on FSH and pulsatile LH secretion across the menstrual cycle of premenopausal women. J. Clin. Endocrinol. Metab. 81, 1512-1518. 9 The Endocrine Society. study and the preliminary nature of the other report may in part account for the differences in findings. In our studies, the enhancement of both LH pulse frequency and amplitude occurred prior to any overt reductions in cyclic estradiol or progesterone concentrations and was phase dependent. Using an intensive sampling protocol (every 10 min for 8 daytime hr), subjects of ages 4 0 - 5 0 years demonstrated shorter cycles, higher mean FSH across all 3 study days, and higher mean LH in the follicular and late luteal phase compared to the youngest age group ( 2 0 - 3 4 years). In keeping with earlier findings of a gradual rise in basal LH levels with age [13], we observed a strong

CHAPTER6 Perimenopause Neuroendocrine Regulation 41.3

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FIGURE 4 Variationsin dynamicgonadotropinsecretionon cycle day 6 in three ovulatorysubjects over age 40 years with similarestradiolconcentrations (pg/ml). ,, LH pulse. See text for discussion.

correlation between increasing age and higher transverse mean LH in the follicular phase (r = 0.42, p = 0.008) and late luteal studies (r = 0.60, p = 0.0001). Unlike previous studies, our ability to document this age-related increase in a sample of only 32 women is probably related to the large number of data points per study (n = 49 over 8 hr) used to calculate mean concentrations. Additionally, the age effects we observed were most evident in the late luteal phase, a time that is typically less represented in daily sampling studies due to the high variability of cycle length in older women. In the women over age 40 years, individual secretory patterns of LH and FSH across the menstrual cycle were highly variable. Figure 4 depicts examples of individual secretory patterns for LH and FSH (open circles) from three older subjects to highlight the variability present on cycle day 6 of a presumed ovulatory cycle. (Ovulation was presumed based on the presence of a midcycle urinary LH surge, and progesterone values ranging between 5 and 10 ng/ml when measured every 30 min during the midluteal phase study.) In five subjects, elevated FSH secretion persisted across the cycle in the presence of normal changes in pulsatile LH secretion; five others exhibited a failure to slow LH pulse frequency and increase amplitude in the luteal phase with or without enhanced FSH secretion. The remaining six subjects exhibited gonadotropin profiles similar to those observed in the youngest age group. In addition to the cross-sectional cohort, we had the rare opportunity to restudy an individual at multiple time points across the climacteric. Figure 5 presents gonadotropin patterns across two ovulatory menstrual cycles of the same subject studied a decade apart. In this particular woman, despite a strikingly similar gonadotropin profile in the follicular phase at both time points, there is a shorter luteal phase by age 45 years as evidenced by an earlier decline in progesterone concentrations. The markedly lower basal estradiol values in the later study may account for the failure of FSH to suppress in the luteal phase. The absence of large-amplitude LH pulses in the midluteal phase by age 45 years was a common finding in the cross-sectional study.

Taken together, these data suggest that (1) the age-related increase in FSH concentrations in ovulatory women, although more pronounced, is associated with phase-dependent enhancement of pulsatile LH secretion; (2) the higher LH concentrations are brought about by changes in both pulse frequency and amplitude; and (3) these age effects preempt overt reductions in cyclic estradiol or progesterone concentrations.

B.

Age-RelatedOligomenorrhea

We have begun a series of studies in perimenopausal women to assess the effect of intermittent follicular activity on the HPO axis. Eligibility criteria include a change in menstrual cycle regularity in the past year, 45 years of age or older, the onset of hot flashes or other estrogen-deficiency symptoms, and a basal serum FSH value of 20 mIU/ml or greater. Figure 6 presents data from three 8-hr studies of dynamic LH secretion conducted in the same subject on cycle day 6 (left panel), day 26 (middle panel), and day 33 (right panel) of a 43-day menstrual cycle. The typical gonadotropin profile of a postmenopausal woman observed in the first study is dramatically altered during an episode of abnormal follicular development, as evidenced by the ultrasound detection of an ovarian cyst. Under the influence of the 20-fold increase in estradiol concentrations, there is a marked suppression of FSH to nearnormal concentrations. The reversal of the LH:FSH ratio in the presence of reduced LH pulse amplitude and frequency is reminiscent of the normal luteal phase despite the absence of significant progesterone exposure. These data serve to highlight the range of HPO secretory activity present during the perimenopause years and the need for caution when attempting to document menopausal status from a single hormone determination at any given point in time. A crosssectional, epidemiologic study of perimenopausal women in Australia revealed much overlap and high variability in single FSH and estradiol determinations for age and body mass index-matched women with regular cycles versus those with irregular menstrual function [60].

102

NANCY E. REAME

0.7 80

0.3 70

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FIGURE 5 Longitudinalassessment of gonadotropin secretory patterns in a regularly cycling woman studied at a 10year interval. The x axis is clock hours. Day refers to menstrual cycle day (day 1 = first day of menses). From Ref. 33a, with permission from Springer-Verlag.

C. GnRH Stimulation Test as a Probe of Pituitary Aging Conflicting evidence exists about age effects on pituitary reserve in perimenopausal women when assessed with a GnRH challenge test. An early study demonstrated that the gonadotropin responses to high-dose (100 ~g) and low-dose (10 ~g) GnRH are similar in hypogonadal women and in women during the early follicular phase, but are maximally different during the late follicular phase [61 ]. This difference is presumed to reflect the effect of rising estradiol levels in the young women, and in turn, increasing pituitary reserve relative to pituitary sensitivity to GnRH [62]. To determine whether menstrual cycle irregularity during the perimenopause may be related to increased gonadotropin bioactivity, Schmidt et al. [63] performed GnRH challenge tests using a 100-~g dose in the early to midfollicular phase. The perimenopausal group was compared to young, regularcycling women, a group of older, cycling women, and a postmenopausal group. Although the perimenopause was associated with magnified gonadotropin levels, similar to those of the postmenopausal group at both base line and after stimulation, only postmenopausal women demonstrated increased LH bioactivity. Because estradiol in the earlymidfollicular phase in the perimenopausal group was not lower than in young cycling women and there were no group

differences in androgens, the authors concluded that steroidal feedback differences did not explain the enhanced LH bio/immuno ratio in the postmenopausal years. Gonadal peptides were not assessed. To what extent the 5- to 10-year age difference between the peri/postmenopause groups and the older cycling women may have contributed to the magnified GnRH response and bioactive secretion was not explored. Using a GnRH dose of 25 /zg as a measure of nearmaximal pituitary release capacity (sensitivity), Fujimoto et al. [64] compared gonadotropin responses in young and older cycling women in the early follicular phase (Fig. 7). They showed that the percentage change in both FSH and LH was higher in the young versus older group despite similar levels of estradiol and inhibin, suggesting a diminished pituitary responsiveness in the older cohort.

V. THE N E U R O R E P R O D U C T I V E AXIS IN P O S T M E N O P A U S E After menopause, disproportionately higher levels of FSH versus LH are the norm, with only gradual declines in both gonadotropins occurring after the seventh decade [65]. The enhanced LH secretion in postmenopausal women is associated with a pattern of relatively high pulse amplitude and

CHAPTER 6

Perimenopause Neuroendocrine Regulation

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A. Changes in Dynamic Gonadotropin Secretion

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older and younger women during the early follicular phase of a normal menstrual cycle. From Ref. 64. Reprinted with permission from the American Society for Reproductive Medicine (Fertility and Sterility, 1996, Vol. 65, pp. 539-544).

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a regular frequency similar to that of the follicular phase [66]. Although the hypothalamic content of GnRH decreases in postmenopausal women [67], GnRH gene expression in the medial basal hypothalamus is increased after menopause without any reductions in area or neuron number compared to that of younger controls [68]. Such data suggest that the

Pulsatile LH secretion of postmenopausal women has been compared with that of young women with premature ovarian failure as a way to distinguish age effects on the neuroreproductive axis. Although LH pulse frequency is similar in the two age groups, young hypogonadal women exhibit higher basal concentrations and greater pulse amplitude compared to women with age-appropriate menopause [66]. Moreover, there is less suppression of LH and FSH by estradiol replacement in the younger group [69]. The lower concentrations of gonadotropins secreted by the older group have been cited as evidence for an age-related effect on hypothalamic-pituitary function. To what extent possible differences in gonadal peptide activity may influence the differential gonadotropin secretion was not explored. Because its half-life has been estimated to be two- to fourfold shorter, the glycoprotein free ce subunit (FAS), although tightly correlated with LH secretion, has been proposed as a more sensitive marker of GnRH secretion at fast pulse frequencies, such as in postmenopausal women [70]. FAS may also be more resistant than LH to down-regulation. GnRH agonist administration to women after menopause results in persistent elevation of FAS despite suppression of LH levels [71]. More importantly, perhaps, Hall and colleagues [72] have proposed that the quality of the LH architecture may change as a function of menopause. Disappearance of endogenous LH after GnRH receptor blockade is prolonged in postmenopausal women, compared to young, cycling controis. The disappearance of FAS was not altered, suggesting that age differences in LH relate to LH microheterogeneity rather than to renal clearance factors [72]. The bulk of neuroendocrine studies of postmenopausal

104

NANCY E. REAME

women have been undertaken not for the purpose of assessing aging mechanisms per se, but rather to simulate aspects of the underlying LH pulse signal free of the influence of endogenous ovarian hormones, i.e., a "castrate" model. Despite the widely held view that LH and FSH secretions after menopause change to a uniform picture of high-amplitude, high-frequency secretion (approximately hourly), estimates of pulsatile LH secretion have varied markedly from study to study, with reported mean pulse frequencies ranging from 60 to 120 min [30,66,73-76] and mean LH concentrations ranging from a low of 19 mlU/ml [39] to values exceeding 75 mlU/ml in the two subjects studied by Yen and colleagues [30]. Within the same study, individual variability of pulsatility patterns is high. In the nine subjects studied by Couzinet [76], pulse frequency ranged from 4 pulses/8 hr to 8 pulses/8 hr, with the range spread equally across the sample. Mean LH concentrations averaged 28.8 IU/liter. High inter-group variability in LH pulse patterns was also noted by Rossmanith et al. [66], who reported a mean pulse frequency of 4 pulses/8 hr, a frequency similar to the luteal phase, for their group of seven subjects. Thus, these data when examined closely, demonstrate a level of variability in LH pulsatile secretion similar to that reported across the highly variable menstrual cycle of young women. A number of differences in methodologies and sample selection may contribute to the variable findings in LH pulse profiles after menopause. Previous studies have used assay methods with varying sensitivities, different criteria to define LH pulses, and heterogeneous study groups. To ensure an adequately depleted ovarian milieu, studies have required subjects to be at least 2 years postmenopausal and without a current history of estrogen replacement therapy (ERT). However, the minimum selection criteria have been interpreted broadly by different investigators: studies have included heterogeneous samples of women ranging in gynecologic age from 2 to 20 years beyond menopause and histories of past use of ERT as recent as 6 - 8 weeks before study. In addition, the type of menopause (surgical vs. spontaneous) has not been controlled for, so that samples ranging in size from 5 to 15 subjects have included both oophorectomized, premenopausal women in their early 40s and spontaneously menopausal women in their 60s, adding further to the heterogeneity of populations. Although it has been assumed that these differences in subject traits and characteristics are benign with respect to the study paradigm, the applicability of the findings to the understanding of the normal climacteric may be limited.

B. Gonadotropin Chronobiology after Menopause Rossmanith and Lauritzen [40] studied 24-hr pulsatile patterns of LH in postmenopausal women compared to

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women at three different menstrual cycle phases (Fig. 8). In addition to the sleep-entrained rise in LH in the early follicular phase, when secretory profiles were fitted to cosinor functions, diurnal excursions in LH secretion were observed in 14 of the 16 early follicular-phase women, 11 of the 14 late follicular-phase women, in 12 of the 15 midluteal-phase women, and in 7 of the 8 postmenopausal women. However, the postmenopausal women demonstrated the smallest deviation from mesor levels (the value around which the os-

CHAPTER6 Perimenopause Neuroendocrine Regulation

105

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cillation occurs) and a marked shift was observed in the acrophase time from early afternoon in the cycle to early morning hours after menopause. This dampened rhythmicity in pulsatile LH secretion after menopause was attributed to the loss of sex steroid sensitivity of opioid action [40]. FSH secretion was not assessed. Another study using similar clinical protocol and analytical methods uncovered a blunted circadian rhythm in FSH secretion in postmenopausal women compared to young, cycling controls [54]. In that study, a cosine rhythm with a nighttime decline in transverse mean FSH was observed in the early and late follicular phase and was markedly attenuated after menopause (Fig. 9). In contrast to the findings of Rossmanith and Lauritzen [40], these investigators observed no evidence of circadian rhythmicity in LH or estradiol under similar conditions. Such disparate findings suggest additional studies are warranted to clarify the exact nature of the underlying gonadotropin rhythmicities.

C. Pulsatile Testosterone Secretion in Hypogonadal Women Thanks to the elegant studies of Judd and colleagues [77], who measured ovarian vein and peripheral plasma hormone concentrations before and after ovariectomy, the follicular-

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depleted ovary has been shown to be the major source of testosterone after menopause. These studies as well as others showing dramatic testosterone suppression after GnRH agonist treatment led Adashi [78] to conclude that the postmenopausal ovary, rather than being a defunct endocrine gland in "end-organ failure," is gonadotropin dependent and responsive to LH. With the advent of the more sensitive immunofluorometric assays (IFMAs), the low concentrations of testosterone normally present in perimenopausal women are now within the range of assay sensitivity, thus making it feasible for the first time to characterize reliably its dynamic secretion. As a further test of the hypothesis that the climacteric ovary is a gonadotropin-responsive, androgenproducing gland, we assessed the relationship between pulsatile LH secretion and episodic release of testosterone (T) in comparison to cortisol secretion in hypogonadal females [79]. Figure 10 presents plasma concentrations of LH, testosterone, and cortisol sampled over 8 daytime hours from a 50-year-old postmenopausal woman with an FSH value > 5 0 mIU/ml and an estradiol level of 5 pg/ml. In this individual, testosterone secretion was episodic and the concordance between T and LH pulses was 71% (p < 0.01), but no significant cross-correlation of secretory patterns was observed. In contrast, there was no significant relationship between pulses of T and cortisol, although a strong positive cross-correlation was observed at a 0 time lag (r = 0.58; p < 0.01). These preliminary data suggest that although the adrenal gland may

106 serve as the rhythm generator for basal testosterone secretion, pituitary LH contributes to the pulsatile release of T in postmenopausal women.

VI. BRAIN AGING AND REPRODUCTIVE SENESCENCE There is currently renewed debate over the relative contribution of the ovaries and the hypothalamic-pituitary unit to the initiation of the human menopause [80]. As reviewed by Wise and colleagues [81,82], heterochronic ovarian transplant studies clearly implicate the hypothalamic-pituitary axis as a primary mediator of both the monotropic FSH rise and the reproductive aging in the rat [83]. Ovulation can be restored in senescent ovaries when transplanted to the kidney capsule of young females [84], and CNS-acting agents can reinitiate estrous and ovulation in aged animals [85]. Conversely, although ovarian transplants from young donors to old recipients in the mouse will double the number of cyclic ovulations, they fail to prevent cycle lengthening [86]. Taken together, such data support a clear influence of the hypothalamic-pituitary system in the onset of reproductive senescence in rodents. Drawing on data from their work, Wise and colleagues [80] have proposed a competing hypothesis on the trigger for menopause. It is their view that preemptive aging changes in the brain lead to the alterations in folliculogenesis, gonadal peptide activity, and gonadotropin augmentation. Several lines of evidence from her laboratory suggest that changes in a variety of neurotransmitter systems that regulate GnRH secretion and possibly circadian, diurnal, and ultradian oscillation are altered with age and may contribute to reproductive senescence. The observation that changes in pulsatile LH release can be detected in middle-aged rats that showed no deterioration in the regularity of their estrous cycles suggested that aging of the hypothalamic pulse generator occurs early during the transition to acyclicity and may play a causative role in age-related transition. The change in LH pulsatility has been shown to correlate with changes in the diurnal pattern of activity or gene expression of norepinephrine, serotonin, and fl-endorphin. Based on these findings, Wise et al. propose that multiple pacemakers in the brain are likely to govern the orchestration of complex neurochemical events that give rise to reproductive cyclicity [80]. Moreover, they hypothesize that the accelerated loss of follicles in women after age 35 years may reflect an age-related desynchronization in the rhythmicity of pulsatile GnRH secretion [82]. Specifically, they postulate that a progressive deterioration of the 24-hr rhythmicity of the biological clock located in the suprachiasmatic nucleus of the hypothalamus leads to an uncoupling of the coordinated neurosecretory inputs that govern the activity of the GnRH pulse generator. Such insults, they propose, would

NANCY E. REAME

lead to a dampening of the GnRH pulse frequency, and in turn the preferential increase in the release of FSH over LH. Some additional lines of evidence would support such a central cause for gonadotropin disturbances. In the rhesus monkey, the frequency of GnRH pulses has been shown to influence the LH:FSH ratio: slow pulses produce mainly FSH, fast pulses produce LH [87]. Moreover, disturbances in diurnal LH secretion are key features of the HPO axis in patients with reproductive endocrine disorders [88]. Although contrary to our findings of enhanced (rather than diminished) LH pulsatility in older ovulatory women, the idea of derangements in the circadian controls of GnRH secretion clearly merits further examination given the growing body of evidence for age-related declines in other neuroendocrine systems mediated by hypothalamic function. For example, studies have demonstrated diminished function of the somatotropic axis of premenopausal women [89,90], and menopause-related effects on the nighttime suppression of cortisol [91]. Prolactin is pulsatile and magnified with sleep in postmenopausal women but is dampened overall due to a lower pulse frequency compared to normal cycling, younger women [92]. These lines of evidence would suggest that subtle aging deficits in hypothalamic function may exist much earlier than previously believed, but to what extent these alterations are relevant to the initiation of menopause remains to be determined. Studies underway in our laboratory are addressing whether the perimenopausal changes in gonadotropin secretion occur first at night, as in puberty, and if menopause is heralded by a suppression of the sleepinduced changes in LH pulsatility. In summary, there is heightened interest in the role of central aging deficits in the etiology of the menopause. A fundamental question is whether GnRH secretion increases at the time of menopause, and if so, whether this is mediated by declines in the integrity of central circadian pacemakers. This hypothesis provides important new directions for studies of the HPO axis at menopause. An understanding of the factors that interact and initiate the process of hypoestrogenism in aging women is needed to develop strategies for alleviating the negative aspects of the menopause and better comprehend the process of biologic aging.

VII. FUTURE

STUDIES

With the advent of specific assays for the bioactive forms of the gonadal peptides and their subunits, it will be possible in the near future to assess systematically the component roles of the inhibins, activins, and follistatin as local and central mediators of aging changes in gonadotropin secretion (Fig. 11). Such information should greatly add to our understanding of the ovulatory process and the abnormalities associated with premature menopause and infertility. Cross-sectional studies of hormonal profiles have for the

CHAPTER 6 Perimenopause Neuroendocrine Regulation

107

FIGURE 11 A model for the changes in gonadotropin and estrogen secretion observed in older, cycling women: proposed aging effects on the HPO axis during the early follicular phase. Age effects at both the level of central pacemakers and the ovary may act together to promote the rise in FSH, enhanced LH pulsatility, and hyperestrogenism.

most part relied on daily or weekly blood measures of LH, FSH, and sex steroids collected from healthy volunteers or wives of infertility patients. Although only women with regular menstrual cycles were studied, few additional clinical data, such as smoking history, parity, body weight characteristics, or diet, were reported. Such factors have been linked to ovarian function [2] and may help explain the controversy about whether estradiol levels increase, decrease, or remain unchanged with age in women who continue to experience regular menstrual cycles. Although seldom reported, it is presumed that the majority of neuroendocrine studies of the menopause transition have been limited to predominantly white, middle-class samples of patients or volunteers. How race, socioeconomic status, and lifestyle factors (e.g., smoking history, diet, body fat

characteristics, exercise) may collectively or independently govern the nature and timing of perimenopause events is unknown. Thus, studies are needed of the influence of extragonadal factors on the aging of the HPO axis. In 1992, the National Institute on Aging launched a multisite, longitudinal study of the hormonal and systemic effects of the natural menopause transition in African-American, Hispanic, Asian-American, and white women as a way to address the limited information available about determinants of the menopause experience, especially for women of color. Early findings suggest that there are significant differences across racial and ethnic groups in menopause symptomatology, such as hot flash severity, which are not accounted for by body mass index, smoking, or socioeconomic factors [93].

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Acknowledgments This work was supported by NIH grants RO1 NR01373, 5M01RR00042, U54 HD29184, and RO 1 AG15083. The author is indebted to her colleague Vasantha Padmanabhan, Ph.D., for her thoughtful review of an earlier version of this manuscript.

18.

19.

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sociated with the monotropic FSH rise in older, ovulatory women: A study of serum and follicular fluid levels of dimeric inhibin A and B in spontaneous menstrual cycles. J. Clin. Endocrinol. Metab. 81, 2742 -2745. Ying, S. Y. (1988). Inhibins, activins and follistatins: Gonadal proteins modulating the secretion of follicle-stimulating hormone. Endocr. Rev. 9, 267-293. Shimasaki, S., Koga, M., Esch, F., Mercado, M., Cooksey, K., Koba, A., and Ling, N. (1988). Porcine follistatin gene structure supports two forms of mature follistatin produced by alternate splicing. Biochem. Biophys. Res. Commun. 152, 717-723. Nakamura, T., Takio, K., Eto, Y., Shibai, H., Titani, K., and Sugino, H. (1990). Activin-binding protein from rat ovary is follistatin. Science 247, 836-838. DePaulo, L. V., Bicsak, T. A., Erickson, G. E, Shimasaki, S., and Ling, N. (1991). Follistatin and activin: A potential intrinsic regulatory system within diverse tissues. Proc. Soc. Exp. Biol. Med. 198, 500-512. Wakatsuki, M., Shintani, Y., Abe, M., Liu, Z.-H., Shitsukawa, K., and Saito, S. (1996). Immunoradiometric assay for follistatin: Serum immunoreactive follistatin levels in normal adults and pregnant women. J. Clin. Endocrinol. Metab. 81,630-634. Harada, K., Shintani, Y., Sakkamoto, Y., Wakatsuki, M., Shitsukawa, K., and Saito, S. (1996). Serum immunoreactive activin A levels in normal subjects and patients with various diseases. J. Clin. Endocrinol. Metab. 81, 2125-2130. Reame, N. E., Wyman, T. L., Phillips, D. J., de Kretser, D. M., and Padmanabhan, V. (1998). Net increase in stimulatory input resulting from a decrease in inhibin B and an increase in activin A may contribute in part to the rise in follicular phase follicle-stimulating hormone of aging cyclic women. J. Clin. Endocrinol. Metab. 83, 3302-3307. Santoro, N., Adel, T., and Skurnick, J. H. (1999). Decreased inhibin tone and increased activin A secretion characterize reproductive aging in women. Fertil. Steril. 71,658-662. Welt, C. K., McNicholl, D. J., Taylor, A. E., and Hall, J. E. (1999). Female reproductive aging is marked by decreased secretion of dimeric inhibin. J. Clin. Endocrinol. Metab. 84, 105-111. Reame, N. E., Zuliani, G. C., Lukacs, J., Lukacs, N., Rolfes-Curl, A., and Padmanabhan, V. (1998). Circulating levels of activin decrease in response to hormone replacement therapy. Menopause 5(4), 267 (Abstr. No. P45). Reame, N., Lukacs, J., Olton, P., and Padmanabhan, V. (1999). Differential effects of ovulation and hormone replacement therapy on circulating levels of inhibin A, inhibin B, activin A and follistatin in perimenopausal women. 81st Annu. Meet. Endocr. Soc., Abstract P2-55, p. 292. San Diego. Mather, J. P., Woodruff, T. K., and Krummen, L. A. (1992). Paracrine regulation of reproductive function by inhibin and activin. Proc. Soc. Exp. Biol. Med. 201, 1-15. Yen, S. S. C., Tsai, C. C., Naftolin, E, Vandenberg, G., and Ajabor, L. (1972). Pulsatile patterns of gonadotropin release in subjects with and without ovarian function. J. Clin. Endocrinol. Metab. 34, 671-675. Santen, R. J., Bardin, C. W. (1973). Episodic luteinizing hormone secretion in man. J. Clin. Invest. 52, 2617-2628. Backstrom, C. T., McNeilly, A. S., Leask, R. M., and Baird, D. T. (1982). Pulsatile secretion of LH, FSH, prolactin, oestradiol and progesterone during the human menstrual cycle. Clin. Endocrinol. (Oxford) 17, 29-42. Reame, N. E., Sauder, S. E., Kelch, R. P., and Marshall, J. C. (1984). Pulsatile gonadotropin secretion during the human menstrual cycle: Evidence for altered frequency of gonadotropin-releasing hormone secretion. J. Clin. Endocrinol. Metab. 59, 328-337. Reame, N. E. (1997). Gonadotropin changes in the perimenopause. In "Perimenopause" (R. Lobo, ed.), p. 161. Springer-Verlag, New York.

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follicular luteinizing hormone secretion during the dark season. Fertil. Steril. 65, 7188-7220.

52. Testart, J., Frydman, R., and Roger, M. (1982). Seasonal influence of diurnal rhythms in the onset of the plasma luteinizing hormone surge in women. J. Clin. Endocrinol. Metab. 55, 374-377. 53. Kivela, A., Kauppila, A., Ylostalo, P., Vakkuri, O., and Leppaluoto, J. (1988). Seasonal, menstrual and circadian secretions of melatonin, gonadotropins and prolactin in women. Acta physiol. Scand. 132, 321327. 54. Mortola, J. E, Laughlin, G. A., and Yen, S. S. C. (1992). A circadian rhythm of serum follicle stimulating hormone in women. J. Clin. Endocrinol. Metab. 75, 861-864. 55. Veldhuis, J. D., Iranmanesh, A., Johnson, M. L., and Lizarralde, G. (1990). Twenty-four hour rhythms in plasma concentrations of adrenohypophyseal hormones are generated by distinct amplitude and/or frequency modulation of underlying pituitary secretory bursts. J. Clin. Endocrinol. Metab. 71, 1616-1623. 56. Booth, R. A., Weltman, J. Y., Yankov, V. I., Murray, J., Davison, T. S., Rogol, A. D., Asplin, C. M., Johnson, M. L., Veldhuis, J. D., and Evans, W. S. (1996). Mode of pulsatile FSH secretion in gonadal hormone-sufficient and deficient women. J. Clin. Endocrinol. Metab. 81, 3208-3214. 57. Reame, N. E., Kelch, R. P., Beitins, I. Z., Yu, M. Y., Zawacki, C., and Padmanabhan, V. (1996). Age effects on FSH and pulsatile LH secretion across the menstrual cycle of premenopausal women. J. Clin. Endocrinol. Metab. 81, 1512-1518. 58. Klein, N. A., Battaglia, D. E., Clifton, D. K., Bremner, W. J., and Soules, M. R. (1996). The gonadotropin secretion pattern in normal women of advanced reproductive age in relation to the monotropic FSH rise. J. Soc. Gynecol. Invest. 3, 27-32. 59. Matt, D. W., Weltman, J. Y., Evans, W. S., Kauma, S. W., Veldhuis, J. D., James, C. A., and Board, J. A. (1994). Follicular phase LH pulse frequency is decreased in middle-aged regularly cyclic women. 76th Annu. Meet. Endocr. Soc., Anaheim, CA, 1994, Abstr. No. 710. 60. Burger, H. G., Dudley, E. C., Hopper, J. L., Shelley, J. M., Green, A., Smith, A., Dennerstein, L., and Morse, C. (1995). The endocrinology of the menopausal transition: A cross-sectional study of a populationbased sample. J. Clin. Endocrinol. Metab. 80, 3537-3545. 61. Wang, C. F., Lasley, B. L., Lein, A., and Yen, S. S. C. (1976). The functional changes of the pituitary gonadotrophs during the menstrual cycle. J. Clin. Endocrinol. Metab. 42, 718-728. 62. Hoff, J. D., Lasley, B. L., Wang, C. F., and Yen, S. S. C. (1977). The two pools of pituitary gonadotropin: Regulation during the menstrual cycle. J. Clin. Endocrinol. Metab. 44, 302-312. 63. Schmidt, P. H., Gindoff, P. R., Baron, D. A., and Rubinow, D. R. (1996). Basal and stimulated gonadotropin levels in the perimenopause. Am. J. Obstet. Gynecol. 175, 643-650. 64. Fujimoto, V. Y., Klein, N. A., Battaglia, D. E., Bremner, W. J., and Soules, M. R. (1996). The anterior pituitary response to a gonadotropinreleasing hormone challenge test in normal older reproductive-age women. Fertil. Steril. 65, 539-544. 65. Rossmanith, W. G., Liu, C. H., Laughlin, G. A., Mortola, J. E, Suh, B. Y., and Yen, S. S. C. (1990). Relative changes in LH pulsatility during the menstrual cycle: Using data from hypogonadal women as a reference point. Clin. Endocrinol. (Oxford) 32, 647-660. 66. Rossmanith, W. G., Handke-Vesely, A., Wirth, U., and Scherbaum, W. A. (1994). Does the gonadotropin pulsatility of postmenopausal women represent the unrestrained hypothalamic-pituitary activity? Eur. J. Endocrinol. 130, 485-493. 67. Parker, C. R., and Porter, J. C. (1984). Luteinizing hormone-releasing hormone and thyrotropin-releasing hormone in the hypothalamus of women: Effects of age and reproductive status. J. Clin. Endocrinol. Metab. 58, 488-491. 68. Rance, N. E., and Uswandi, S. V. (1996). Gonadotropin-releasing hormone gene expression is increased in the medial basal hypothalamus

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80. Wise, R M., Krajnak, K. M., and Kashon, M. L. (1996). Menopause: The aging of multiple pacemakers. Science 273, 67-70. 81. Wise, E M., Scarbrough, K., Lloyd, J., Cai, A., Harney, J., Chiu, S., Hinkle, D., and McShane, T. (1994). Neuroendocrine concomitants of reproductive aging. Exp. Gerontol. 29, 275-283. 82. Wise, E M. (1998). Menopause and the brain. Sci. Am. (Special Issue Women's Health: A Lifelong Guide) 9 (Summer), 79-81. 83. Sopelak, V. M., and Butcher, R. L. (1982). Contribution of the ovary versus hypothalamus-pituitary to termination of estrous cycles in aging rats using ovarian transplants. Biol. Reprod. 27, 29-37. 84. Ascheim, E (1983). Relation of neuroendocrine system to reproductive decline in female rats. In "Neuroendocrinology of Aging" (J. Meittes, ed.), pp. 73-101, Plenum, New York. 85. Quadri, S. K., Kledzik, G. S. and Meites, J. (1973). Reinitiation of estrous cycles in old constant-estrous rats by central-acting drugs. Neuroendocrinology 11,248-255. 86. Nelson, J. E, and Felicio, L. S. (1990). Hormonal influences on reproductive aging in mice. Ann. N.Y. Acad. Sci. 592, 8-12. 87. Wildt, L., Hausler, A., Marshall, G., Hutchison, J. S., Plant, T. M., Belchetz, E E., and Knobil, E. (1981). Frequency and amplitude of gonadotropin-releasing hormone stimulation and gonadotropin secretion in the rhesus monkey. Endocrinology (Baltimore) 109, 376-385. 88. Khoury, S., Reame, N. E., Kelch, R. E, and Marshall, J. C. (1991). Diurnal patterns of pulsatile LH secretion in hypothalamic amenorrhea: Reproducibility and response to opiate blockade and an alpha-2 andrenergic agonist. J. Clin. Endocrinol. Metab. 64, 755-762. 89. Wilshire, G. B., Loughlin, J. S., Brown, J. S., Adel, T. E., and Santoro, N. (1995). Diminished function of the somatotropic axis in older reproductive-aged women. J. Clin. Endocrinol. Metab. 80, 608- 613. 90. Cano, A., Catelo-Branco, C., and Tarin, J. (1999). Effect of menopause and different combined estradiol-progestin regimens on basal and growth hormone-releasing hormone-stimulated serum growth hormone, insulin-like growth factor-l, insulin-like growth factor binding protein (IGFBP)-I, and IGFBP-3 levels. Fertil. Steril. 71, 261-267. 91. Van Coevorden, A., Mockel, J., and Laurent, E. (1991). Neuroendocrine rhythms and sleep in aging men.Am. J. Physiol. 260, E851-E861. 92. Katznelson, L., Riskind, R N., Saxe, V. C., and Klibanski, A. (1998). Prolactin pulsatile characteristics in postmenopausal women. J. Clin. Endocrinol. Metab. 83, 761-764. 93. Gold, E. B., Sternfeld, B., Kelsey, J. L., Brown, C., Mouton, C., Reame, N., Salamone, L., and Stellato, R. (2000). The relation of demographic and lifestyle factors to symptoms in a multi-racial/ethnic population of women 40-55 years of age. Am. J. Epidemiol., in press.

_~HAPTER

Changes in Men as They Age: The Manopause STANLEY G. ARSHAG D.

KORENMAN

MOORADIAN

VICTORIA HENDRICK

I. II. III. IV. V. VI.

Department of Medicine, University of California, Los Angeles, School of Medicine, Los Angeles, California 90095 Department of Internal Medicine, Saint Louis University School of Medicine, Saint Louis, Missouri 63104 Department of Psychiatry, University of California, Los Angeles, School of Medicine, Los Angeles, California 90095

Introduction Age-Related Changes in the Male Reproductive System Age-Related Changes in Other Hormonal Systems Other Changes with Aging Related to Hormonal Factors Androgen Effects on Sexual and Reproductive Function Nonerectile Sexual Dysfunctions

VII. VIII. IX. X.

I. I N T R O D U C T I O N

a "viropause" remains controversial. A syndrome is usually defined as a set of symptoms, signs, and diagnostic features that are all due to a single etiology, e.g., Cushing's syndrome is due to the overproduction of cortisol or administration of excess glucocorticoids. By that definition the manopause is not a syndrome, but neither are some features of the menopause. Perhaps whether a syndrome even exists is not an interesting question. If the manopause is defined to include concomitantly appearing changes that may influence e a c h other and that become apparent in middle age, then it may indeed exist. Clinical features associated with aging in men include sexual dysfunction, hypogonadism, and psychological changes. In Fig. 1 the pie chart shows a separation of these three clinical features although they are really connected and

Although the menopause may be defined by the final occurrence of menses at a defined time in each woman, many biological changes occur in women prior to that last ovulation. Among these are loss of bone mineral density, lipoprotein alterations, increased body weight and altered composition, development of hot flashes, and alterations in mood and perceptions of well-being, in addition to the endocrine system changes. In men, although there is no comparable signal event associated with aging, analogous changes in bone density, lipoproteins, and psychological factors as well as alterations in endocrine status take place. The question of whether these and other changes represent a syndrome, a "manopause," or

MENOPAUSE:

BIOLOGY AND PATHOBIOLOGY

Erectile Dysfunction Manopause and Mental Health Psychological State and Sexual Function Conclusions References

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Copyright 9 2000 by Academic Press. All rights of reproduction in any form reserved.

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KORENMAN ET AL.

A. A n a t o m i c C h a n g e s o f H y p o t h a l a m i c Pituitary Testicular System

FIGURE 1 Problems of the aging man. The parallel lines between the three pie sections are to indicate that the components interact with one another even though they may be independent. do influence each other. Hypogonadism affects mood, depression influences sexual function, anxiety affects androgen production, and erectile dysfunction affects mood and selfesteem. These will be discussed later in more detail. There are other changes that occur in men as they age, and to cover them fully would require an entire volume, rather than a chapter. Among the issues of concern are benign prostatic hyperplasia, prostate carcinoma, atherosclerosis and its sequelae, lipid alterations, changes in body mass and composition, altered glucose sensitivity, changes in muscle strength and composition, declining bone density, and the changes in endocrine systems other than the reproductive system. To cover all of these thoroughly is beyond the scope of this chapter. Therefore, we will deal primarily with androgens, sexual function, and psychological findings and introduce other elements more in passing. As the reader will see, the information available is inadequate in many areas. Connections between findings are hard to establish. Another difficulty is the emphasis of studies on men over the age of 60 years. However, in our analysis, we will emphasize investigations that include men from ages 40 to 60 years, the age range of interest. One obvious conclusion from this analysis is the need for much more research into aging of the male.

II. AGE-RELATED MALE

REPRODUCTIVE

CHANGES

I N THE

SYSTEM

Aging is often associated with significant histological and functional changes of the male reproductive system [1-3]. These changes are aggravated by a variety of chronic organ system diseases that are commonly found in elderly men [4]. Thus, the distinction between an age-related change and a disease state is sometimes blurred. Nevertheless, the preponderance of currently available data suggest that aging, independent of disease, is associated with altered reproductive function in men.

Striking structural changes occur in the testis with age [5]. These include thickening of the basement membrane of seminiferous tubules, peritubular fibrosis, thinning of the germinal epithelium, germ cell arrest, and narrowing and collapse of tubules with reduction in the number of Sertoli cells. However, areas of spermatogenesis are usually preserved until late in life. The number of Leydig cells in the testes may be reduced with age, although some studies have failed to find an age-related decrease in Leydig cell number [3,5]. The presence of inflammatory cells near degenerating seminiferous tubules suggests that autoimmune damage of testes occurs with age. The finding of increased serum sperm agglutinating antibodies with age is of questionable significance [3,5]. These antibodies may well be another nonspecific manifestation of age-related dysregulation of the immune system. The pituitary gland also undergoes histological changes with age. The incidence of pituitary adenomas and empty sellae increases with age [6], and numbers of eosinophilic cells decrease and numbers of basophilic and chromophobic cells proportionately increase [7,8]. Aging, particularly during the transition from youth to middle age, is associated with a significant decline in the number and size of cells producing growth hormone, with hypertrophy and relative hyperplasia of thyrotrophs [7,8]. The number of prolactinsecreting cells is not altered in aging men. Because of the large reserve capacity of the endocrine glands, the correlation between histological changes and changes in endocrine function is generally poor. However, some of the structural changes, such as loss of Leydig or Sertoli cells, undoubtedly contribute to the age-related alterations in hypophyseal-gonadal function.

B. N o r m a l P i t u i t a r y - T e s t i c u l a r P h y s i o l o g y The hypothalamic-pituitary testicular axis is outlined in Fig. 2. Testosterone, the principal circulating androgen, is secreted by testicular Leydig cells under luteinizing hormone (LH) stimulation. Sperm production and inhibin [a Sertoli cell hormone that inhibits follicle-stimulating hormone (FSH) secretion] are stimulated by FSH. The inhibitory effect of testosterone on LH secretion and of inhibin on FSH secretion establishes the negative feedback loop of the axis. Testosterone mostly slows the hypothalamic gonadotropin-releasing hormone (GnRH) pulse generator whereas estrogens derived from testosterone seem primarily to affect gonadotropin pulse height at the pituitary level [9]. The gonadotropin-secreting cells and Leydig cells are adapted to

CHAPTER 7 Changes in Aging Men

113 The biological effects of T are due to its interaction with androgen receptors or the interaction of its metabolites, such as DHT or estradiol (E2), with the androgen or estrogen receptor. In addition, DHT may act as antiestrogen by competing for the estrogen receptor [15]. These receptors then mediate the effects of those hormones on specific genes involved in the phenotypic expression of various tissue functions. Androgens can also act on various enzymes, especially in the liver through receptor-independent mechanisms. The biological effects of androgens and their active metabolites are manifest in essentially every organ system [ 16]. These biological effects, as well as the androgen economy, are altered with age.

FIGURE 2 The hypothalamic-pituitary-testicular axis and relation to peripheral responses. A simplified version of the interactions related to testosterone secretion and physiological actions. SHBH, Sex hormone binding globulin; FSH, follicle-stimulating hormone; GNRH, gonadotropin-releasing hormone; LH, luteinizing hormone.

the pulsatile release of GnRH. Thus, continuous LH or human chorionic gonadotropin stimulation of the testis results in down-regulation of LH receptors and loss of steroidogenic response [10]. Testosterone (T) secretion shows diurnal and seasonal variation [ 11 ]. The peak plasma concentration of T occurs at 6 : 0 0 - 8 : 0 0 a.m., falls slowly by about 35% during the day, and begins to rise again at about midnight. The plasma concentrations of T also tend to be higher in spring and summer months. In plasma, up to 54% of T is bound to sex h o r m o n e binding globulin (SHBG) and 45% is weakly bound to albumin. For most tissues, the available T is the sum of the albumin-bound fraction and the dialyzable fraction This sum of T fractions is referred to as the "bioavailable" testosterone (BT) [12]. SHBG also binds other 17fi-hydroxylated steroids, including estradiol, and its secretion by the liver is enhanced by estrogen and suppressed by androgens. Secreted T is metabolized to active or inactive compounds in various target tissues. In the liver, T is metabolized to inactive reduced metabolites, which are then conjugated to yield glucuronides or sulfates for excretion into the urine [13]. In adults, reproductive tissue T is converted to 5cedihydrotestosterone (DHT) via the enzyme 5a-reductase. In adipose tissue, in Leydig and Sertoli cells, and in certain brain nuclei, T is converted to estradiol by the aromatase enzyme, whereas muscle responds directly to T as the active hormone. In reproductive tissue, DHT is further metabolized to 3ce,17fi-androstanediol and to 3/3 metabolites. These metabolites undergo glucuronide conjugation in the prostate and may play a pivotal role in androgen removal from that organ [13,14]. Thus, specific metabolism of T in various tissues allows generation and clearance of different active compounds.

C. A g e - R e l a t e d

C h a n g e s in t h e H y p o t h a l a m i c -

Pituitary-Gonadal

Axis

In men, age-related changes in the reproductive system occur at multiple levels (Table I). Circulating testosterone declines longitudinally with age at the rate of 100 ng/dl/ decade of life [17-19]. This is accompanied by an increase in SHBG such that free and bioavailable testosterone concentrations drop to an even greater extent. The increase in SHBG may be due in part to the decreased mean concentration of T and maintenance of the E 2 level commonly seen with age. The age-related fall in unbound (free) T and bioavailable T (free and albumin bound fraction) with age

TABLE I Age-Related Changes in the Male Reproductive System Reproductive tissue structural changes

Testes: thickening of basement membrane of seminiferous tubules; peritubular fibrosis; germ cell arrest Prostate: hyperplasia, cancer Epididymus: regression of secretory epithelium Seminal vesticles: modest changes in epithelial components Hormonal changes

Decreased serum total testosterone level Decreased bioavailable testosterone Decreased clearance of testosterone Decreased accumulation of 5a-reduced steroids in reproductive tissues Increased plasma binding of testosterone Increased mean LH and FSH levels Decreased LH pulse frequency Increased incidence of both hypergonadotrophic and hypogonadotrophic hypogonadism Changes in sexuality

Decreased libido Increased latency Reduced frequency and rigidity of erections Decreased ejaculatory volume Reduced orgasmic contractions

1

1

4

K

O

R

E

[ 18,20] has been documented, although not in every study. As a result of declining T production and increased binding, mean serum gonadotropin concentrations rise slowly with age, although inappropriately low serum gonadotropin values are reported, suggesting that pituitary hypothalamic dysfunction is also common in elderly subjects [20-22]. The normal diurnal fluctuation in plasma T is attenuated in the elderly so that the morning peak is much lower [ 11 ]. Compared to young men, healthy older men have a reduced LH pulse frequency but the pulse amplitude is maintained. The age at which this begins is unknown. The reduced diurnal fluctuations of T with age are possibly due to the decreased frequency of LH pulses [23]. However, some studies have failed to find an age-related change in LH pulse frequency [24]. In addition to changes in LH pulse frequency, there is also an age-related decay in the 24-hr rhythm of LH and FSH, and the circannual rhythm of FSH is lost while the circannual rhythm of LH seems to be preserved [25,26]. The gonadotropin-suppressing activity of T or DHT is enhanced in healthy elderly men [27]. This may partly account for reduced FSH/LH response to GnRH in aging men [28]. It is noteworthy that Muta et al. [29] found that LH suppressibilty by T or estradiol is reduced in older men. This study, unlike that of Winters et al. [28], included subjects with relatively more severe primary testicular failure. In healthy aging men, treatment with clomiphene citrate stimulates LH and FSH secretion similarly to that in young men [30]. However, older men showed a smaller rise in serum testosterone, bioavailable T, and estradiol, suggesting a reduced testicular response to LH. Primary alterations in testicular function are supported by the observed histological changes in Leydig cell or Sertoli cell number and reduced spermatogenesis. Sertoli cell inhibin production declines significantly by the fifth decade [31 ]. This accounts in part for the increase in mean FSH levels found in aging men. Recent studies have indicated that inhibin B, which is inducible by exogenous FSH, is the only inhibin detectable in adult men and appears to be the physiologically relevant inhibin [32]. The Leydig cell response to stimulation with exogenous human chorionic gonadotropin (hCG) or endogenous LH is also reduced with age [33,34], at least partially due to a decrease in pregnenolone production, which limits the availability of substrate for conversion to testosterone. Aging is often associated with alterations in androgen action and metabolism. The metabolic clearance rate of both testosterone and DHT is reduced [35]. T reduction to 5/3 metabolites is increased. There is also an increase in testosterone conversion to estradiol [36]. This, together with the reduction in E 2 clearance with age, accounts for the increased Ez/T ratio in aging men [36]. It is believed that the enzyme responsible for converting T to DHT, namely 5ce-reductase, is inducible by androgens [37]. This concept is also sup-

N

M

A

N

ET AL.

ported by the studies of scrotal testosterone patches showing that increased conversion of T to DHT locally was possibly due to induction of 5ce-reductase in scrotal skin [38]. This observation is relevant to changes found in aging in which the concentration of 5ce-reduced androgens in reproductive tissues is diminished [39]. Thus, it is possible that the lower circulating androgen levels in older men permits diminished 5a-reductase activity and therefore reduced responsiveness of the reproductive tissues to androgens. However, the capacity of DHT-degrading enzymes in prostatic epithelium, such as 3ce- and 3fl-hydroxysteroid reductase, is also reduced with age [40]. The effect of those changes on overall androgen metabolism is small. It is noteworthy that inherent genetic differences determine sensitivity to T effects. The number of CAG trinucleotide repeats in the androgen receptor gene correlates with age of onset of prostate cancer [41]. Although many factors, including coexisting diseases and nutritional changes, have been linked to the age-related decline in T production, careful studies suggest that chronic illness plays a minor role and that age is the more powerful risk factor. However, there is a wide individual variation in the rate of decline in reproductive function and some men can maintain both normal T production and spermatogenesis well into advanced age [ 17,18].

D. A n d r o g e n E f f e c t s With age there is a loss of lean body mass, primarily muscle mass. Androgens can cause muscle hypertrophy without altering muscle cell number [16]. They also potentiate muscular growth produced by GH [16]. Thus, it is possible that some of the age-related loss of muscle mass is related to T and GH deficiency. Certain muscle groups, such as cardiac muscle or diaphragm, that are not highly sensitive to androgens or are independent of lifestyle changes do not show a significant loss of mass with age. This suggests that regular exercise would prevent some of the age-related losses of muscle mass. However, other muscle groups, notably temporalis, levator pubii, ischiocavernosus, and bulbospongiosus muscle, are androgen dependent. These muscle groups atrophy as androgen availability to tissues decreases with aging.

E. O t h e r E f f e c t s o f A n d r o g e n s Androgens affect the hematopoietic system, particularly erythropoiesis, and may have some antiinflammatory effects. Androgens stimulate erythropoietin production and also have direct stimulatory effects on the stem cells of the bone marrow [42]. Thus, in the elderly, reduced T may contribute to reduced hematopoiesis. Although androgens alter

CHAPTER7 Changes in Aging Men

115

the expression or activity of various hepatic and renal enzymes, these effects do not seem to be of any relevance to the changes seen in these organs with age. Similarly, the relevance of the effect of T on feeding behavior or body fat distribution with aging is not clear. In general, low doses of T increase adipose tissue, especially in the upper body area, whereas high physiological doses of T suppress adipose tissue mass and lipoprotein lipase as a result of aromatization to estrogens [43-45]. It is possible that the changes in adipose tissue distribution with age are related to altered T secretion. Testosterone therapy may also reduce adiposity and improve muscular strength [46,47]. The role of T or DHEA in preventing osteoporosis in men is modest compared to the known role of estrogens in women. Nevertheless, in aging men there is a direct relationship between bone mass and plasma T levels [48,49]. Mild to moderate androgen insufficiency occurs frequently in older men. Considering the diversity of the biological actions of androgens, it is likely that some of the common physiological changes with age are related to androgen deficiency. Therefore, it is possible that some older men with androgen deficiency would benefit from androgen replacement therapy. Multicenter, placebo-controlled trials are needed to establish the long-term safety and efficacy of T treatment in men.

III. A G E - R E L A T E D IN OTHER

HORMONAL

CHANGES SYSTEMS

The biological consequences of altered androgen status with age are often modulated by the other hormonal changes commonly found in aging men (Table II). These additional hormonal systems will be discussed briefly.

A. G r o w t h H o r m o n e The changes of growth hormone (GH) physiology with age and the known biological correlates of these changes constitute the rationale for the preliminary attempts at "rejuvenating" older subjects with GH therapy. Although pituitary GH content may not change with age, sleep-induced GH secretion, the peak amplitude, and the 24-hr plasma levels of GH are reduced in older subjects [50-53]. The response to a variety of GH stimulators is also altered with age in some but not all studies. The response to GH releasing hormone (GHRH) [54,55] or insulin-induced hypoglycemia [56] may be reduced. The reduction in GH production could be partly attributed to reduced growth hormone releasing hormone receptors [57] and more importantly to increased pituitary sensitivity to inhibition by somatostatin [58].

TABLE II Hormonal Changes Commonly Found in Aging Men System/activity

Change

Hypothalamic-pituitary unit Growth hormone Basal Nocturnal peak IGF-I IGFBP- 1 IGFBP-3 ACTH Basal Response to CRH TSH Basal Response to TRH LH/FSH

N or decreased Decreased in males Decreased or N

Thyroid

See Table III

Adrenal Cortisol basal concentrations Sensitivity to dexamethasone supression DHEA + DHEA sulfate concentrations Aldosterone concentrations

N Decreased Decreased Decreased

Testis

See Table I

Calcium/bone PTH 1,25-(OH)2D

Decreased Decreased

Water metabolism Arginine vasopressin in response to Osmotic stimuli Baroreceptor stimuli Atrial natriuretic factor

Decreased N Decreased

Carbohydrate metabolism Insulin Glucagon

Increased Increased

N in males a Decreased Decreased Decreased Decreased N N

aN, No change.

In addition to the changes in GH secretion, there are changes in insulin-like growth factor-I (IGF-I) and its binding proteins. Thus, with age, IGF-I is decreased and IGF binding protein-3 (IGFBP-3) is also decreased [59]. In contrast, IGF binding protein- 1 (IGFB P- 1), an inhibitor of IGF-I bioactivity, is increased with age [60]. The changes in GH secretion, along with reduction in bioactivity of IGF-I, may contribute to the age-related increase in bone loss and muscle wasting. However, so far GH replacement has failed to demonstrate clear anabolic effects in elderly subjects. Of note is that the age-related changes in GH secretion vary greatly among populations. Nutritional factors and coexisting diseases often influence GH secretion. There appears to be significant gender-related differences; in women, basal GH levels decline with advancing age [61] whereas in men they remain unchanged [62]. The more marked fall in basal GH that occurs in older women may be related to estrogen

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KORENMAN ET AL.

deficiency. Although testosterone, through its conversion to estrogen, has a positive modulating influence on GH secretion, the age-related reduction in T is not sufficient to cause a decrease in GH levels; perhaps, because of an increased conversion rate, estrogen levels do not fall in men as they age and may not be reduced in the GH cells.

B. T h y r o i d H o r m o n e E c o n o m y Aging is commonly associated with an altered thyroid hormone (TH) economy (Table III) [62a], although the feedback control system maintains the same plasma concentrations of total or free T 4 and T 3. The production and clearance rates of thyroid hormones are decreased in parallel [63-65]. The TH binding to serum proteins is usually not altered [63]. The pituitary thyroid-stimulating hormone (TSH) response to thyrotropin-releasing hormone (TRH) may be reduced in aging men but is normal in elderly women [60,63]. Thyroid gland response to endogenous or exogenous TSH may also be reduced. The incidence of primary thyroidal failure, as evidenced by increased plasma TSH levels, increases with age, especially in women. It has been estimated that 4.4% of the population over the age of 60 years has primary thyroidal failure [66]. In addition to the changes in thyroid hormone economy, the tissue responsiveness to thyroid hormones is reduced

T A B L E II I

Age-Related Changes in Thyroid Hormone Economy a

Parameter h Radioactive iodine uptake

Changes Decreased

T 4 production

Decreased or unaltered

T 3 production

Decreased or unaltered

T 4 degradation

Decreased or unaltered

T 3 degradation

Decreased or unaltered

Serum T 4 concentration (total or free)

Unaltered

Serum T 3 concentration (total or free)

Unaltered or decreased

Serum thyroid hormone binding capacity (T 3 resin uptake)

Unaltered

Serum TSH

Unaltered or increased

Circadian TSH variation

Decreased

TSH response to TRH

Unaltered or decreased, especially in men

TSH rise in response to thyroid hormone deficiency

Decreased

Thyroid response to TSH

Decreased or unaltered

a Reprinted from Mooradian and Wong [62a], with permission of the publisher. b T4, Thyroxine; T 3, triiodothyronine; TSH, thyroid-stimulating hormone, TRH, thyrotropin-releasing hormone.

with age [65]. This is partly related to reduced TH transport across the cellular plasma membrane [67,68] and the altered biology of transcription factors involved in the expression of genes responsive to TH [69]. The TH receptor affinity or capacity is not significantly altered with age [68]. The clinical correlates of the age-related changes in tissue responsiveness to TH are the finding of classical features of hypothyroidism in biochemically euthyroid elderly individuals. In addition, postprandial thermogenesis is also blunted partly because of loss of fl-adrenergic receptor activity in brown adipose tissue [70]. It is also possible that the age-related reduction in thyroid hormone responsiveness contributes to reduced metabolic rate and blunted postprandial thermogenesis. These changes may partially account for the cold intolerance and increased risk of hypothermia in the elderly.

C. A d r e n a l P h y s i o l o g y l.

GLUCOCORTICOIDS

In humans, the hypothalamic-pituitary adrenal axis remains intact with age. The plasma cortisol level remains stable [71], and the pituitary adrenocorticotropic hormone (ACTH) response to exogenous corticotropin releasing hormone (CRH) or to intravenous metyrapone does not change [72,73]. Cortisol production and clearance rates are both decreased proportionately with aging. The circadian rhythm of cortisol secretion remains intact, although in men over 40 years, a phase advance in cortisol secretion is observed [74]. In this group, the peak and nadir of cortisol secretion occur approximately 3 hr earlier than in younger controls [74]. The cortisol response to ACTH or to insulin-stimulated hypoglycemia is usually unaltered in healthy elderly subjects [56]. An increased cortisol response to CRH, despite the normal ACTH response, suggests a diminished sensitivity of ACTH to negative feedback by glucocorticoids in older men. The early phase of ACTH inhibition by hydrocortisone is blunted in the elderly (>age 65 years), although the late phase remains intact. This could be related to changes in blood-brain transport of cortisol [72]. The elderly have a higher serum cortisol response to perioperative stress or during depression and the dexamethasone suppression test often fails to yield the expected drop in plasma cortisol levels [75]. It is tempting to speculate that this relative increase in cortisol level with age may contribute to some age-related changes in body composition, notably osteopenia. 2.

MINERALOCORTICOIDS

Aldosterone secretion decreases with age, probably because of reduced plasma renin activity [76]. This reduction is evident both at basal conditions and during stimulation with salt restriction, upright posture, or ACTH [76]. An

CHAPTER7 Changes in Aging Men age-related reduction in aldosterone clearance partially offsets the decreased aldosterone production rate. The reduced plasma renin activity and aldosterone production may contribute to the orthostatic hypotension commonly found in the elderly and may expose them to a higher risk of developing hyperkalemia following administration of angiotensinconverting enzyme inhibitors. 3. ADRENAL ANDROGENS

One of the most dramatic age-related changes in the hormonal system is adrenal androgen production. In men, dehydroepiandrosterone (DHEA) secretion declines progressively between 20 and 96 years of age [77]. The serum level of DHEA in older men is approximately 5 - 3 0 % of that seen in young men [77]. This "adrenopause" is probably the result of reduced 17,20-desmolase activity with age. Weight loss in overweight men but not women is associated with a 125% rise in serum DHEA sulfate, which suggests that agerelated increase in adiposity or insulin resistance may contribute to reduced DHEA levels in aging men [78]. The biological implications of the decline of DHEA in aging humans are still not clear. Experiments in animals who do not secret DHEA suggest that DHEA may be implicated in longevity and have protective effects in tumorigenesis, atherosclerosis, and age-related memory disturbances [79]. In one epidemiological study, death from cardiovascular disease in men over the age of 50 years was inversely related to DHEA sulfate levels [80]. DHEA administration reduces plasma plasminogen activator inhibitor type 1 and tissue plasminogen activator concentrations in men [81]. DHEA also inhibits platelet activity [82]. These effects may help prevent heart disease in men. More interventional studies are needed to establish the clinical relevance of DHEA in the biology of aging. 4. ADRENAL MEDULLA

Elevated plasma levels of epinephrine and norepinephrine (NE) have been found in healthy octogenarians compared to younger subjects [83,84]. Plasma dopamine levels do not change with age. The increased NE levels are due to an increased production and decreased clearance rate. This is accompanied by a decrease in platelet az-adrenergic receptors [85] and cardiac fl-adrenergic transmission [86]. The NE response to upright posture, during the cold pressor test, following glucose ingestion, and during insulin tolerance testing is increased in the elderly, whereas the NE and epinephrine response to exercise may be reduced in healthy elderly men [84]. The clinical consequences of these changes are not apparent but they may contribute to orthostatic or postprandial hypotension [87]. They could also be related to increased vascular resistance and therefore contribute to hypertension and the need for after-load reduction, especially in those with congestive heart failure.

117 D. C a l c i u m a n d B o n e M e t a b o l i s m Whereas age-related bone loss is a common phenomenon in both men and women, the process is accelerated by coexisting hormonal deficiencies, notably estrogen deficiency during the menopause and androgen deficiency in men. In healthy men, radial bone mineral content decreases by 1% per year whereas vertebral bone mineral content decreases by 2.3% per year [88]. Parathyroid hormone (PTH) secretion increases with age, as production of 1,25-dihydroxycholecalciferol (calcitriol) and intestinal calcium absorption are reduced [89]. Nutritional deficiency vitamin D and limited exposure to sunlight, coupled with reduced conversion of 25a-hydroxycholecalciferol to calcitriol, contribute to the reduced calcitriol levels seen in elderly men [89,90] Age-related osteopenia is the result of multiple factors, including altered dynamics of bone cell populations inherent to aging p e r s e aggravated by multiple nutritional and hormonal changes including deficiencies of sex steroids, GH, IGF-I, and calcitriol.

E. C a r b o h y d r a t e M e t a b o l i s m One of the major consequences of the age-related hormonal changes is the emergence of type 2 diabetes. This is the result of both altered insulin secretion and action with age [91]. These changes may be partly due to decreased physical activity and altered body composition favoring accumulation of central adiposity. The incidence of type 2 diabetes increases progressively with age starting at about age 40. Approximately 20% of the population in the United States over the age of 65 years has type 2 diabetes mellitus and at least 40% have glucose intolerance [91,92]. A decline in insulin secretory capacity with age along with reduced insulin sensitivity is common [91]. The plasma levels of glucagon and its clearance rate remain unaltered. Clinical diabetes, especially when poorly managed, causes a variety of complications that are associated with a poor quality of life. Increased glycation of various proteins and enhanced lipid peroxidation accelerate the age-related deterioration of various organ systems. In particular, body composition and vigor are adversely affected. Older subjects with diabetes are at increased risk of dehydration and malnutrition. Optimization of blood glucose control reverses most of the short-term and possibly long-term complications of diabetes.

E Water Metabolism The age-related changes in water and electrolyte homeostasis are summarized in Table IV. The total body water and

118

KORENMAN

TABLE IV

Biological Changes with Age

Body composition Increased: body weight until the sixth decade, thereafter declines; central adiposity Normal:extracellular fluid volume Decreased: lean body mass, bone mass, muscle mass; intracellular and total body water. Cardiovascular system Increased: stroke volume, end-diastolic volume, systolic blood pressure Normal: cardiac output, myocardial contractility Decreased: heart rate, ejection fraction Pulmonary function Increased: residual volume, closing volume Normal: total lung capacity Decreased: vital capacity, arterial PaO2, elastic recoil, maximum expiratory flow rate, maximum voluntary ventilation Digestive system Increased: frequency of teritary contractions in esophagus Normal: motility of stomach and intestine Decreased: salivation, taste, peristalsis of esophagus, acid/pepsin production; colonic motility Hormonal system See Tables I-III Central nervous system Increased: incidence of neurodegenerative diseases and depression, difficulty in learning new tasks Normal: overall intelligence Decreased: speed of cognitive processing, memory I-Iematopoietic/immune system Increased: incidence of anemia, certain hematological malignancies Normal: complete blood count, B cells, macrophages Decreased: progenitor cells, certain components of complement, T cells, intracellular bactericidal activity

intracellular fluid volume are decreased with age while extracellular blood volume is maintained. Elderly men have reduced thirst perception [93]. Basal arginine vasopressin (AVP) secretion may increase with age [94]. The osmolar threshold (the level of plasma osmolarity that will initiate AVP secretion) is lower in the elderly. However, AVP responses to volume and pressure changes are reduced, and the renal response to AVP is also blunted with age [94]. This results in a reduced capacity to conserve water that predisposes the elderly to dehydration, especially when water access is limited or during excess water losses as a result of intercurrent illness. A reduced capacity to generate angiotensin, a potent stimulator of AVP and thirst, also limits the ability of older subjects to maintain water homeostasis. The ability to maintain salt and water balance is further compromised by changes in atrial natriuretic peptide (ANP) secretion [95]. Baseline ANP is increased in elderly subjects and the expected reduction in ANP following dehydration is blunted. The natriuretic effect of ANP is probably preserved, although hemodynamic responses to ANP may be reduced

ET AL.

[95]. The increased ANP secretion with age may contribute to the suppression of plasma renin activity and aldosterone secretion.

IV. O T H E R RELATED

CHANGES

WITH

TO HORMONAL

AGING

FACTORS

A variety of other physiological and structural changes occur with age [96] (Table IV). Some of these are probably related to processes inherent to aging per se, whereas others are secondary to lifestyle changes or nutritional and hormonal alterations. Consequent to the loss of muscle mass, the basal metabolic rate is reduced with age [96]. The reduced skeletal muscle mass with age, along with changes in cardiovascular and pulmonary physiology, results in reduced exercise capacity and low maximum oxygen consumption (VO 2 max). It appears that the changes in pulmonary function are more important than the changes in the cardiovascular system in limiting exercise capacity in the elderly [97]. The partial pressure of arterial oxygen (PaO2) declines steadily with age while P a C O 2 is not significantly altered [97]. Weak respiratory muscles, decreased lung compliance, and increased chest wall stiffness account for most of the age-related changes in pulmonary functions, some of which may be related to cigarette smoking. The other components of age-related loss of lean body mass are bone loss and altered body water content. Hormonal factors, such as loss of androgens, GH, and IGF-I and nutritional factors such as calcium and vitamin D deficiency, along with genetic factors, account for the bone loss (Table V) [98].

TABLE V

Epidemiological Correlates with Erectile Dysfunction a

Positively associated with ED Age Cigarette smoking Depression, inward looking or with expressed anger Diabetes, treated with medications (more severe) Cardiovascular disease, treated Use of vasodilators (however they were defined) Not grossly associated with ED Hypertension Alcohol intake over a wide range Allergies Serum cortisol or DHT level Inversely associated with ED Dominance DHEA level in blood HDL cholesterol level in blood a Adapted from Feldman et al. [98].

CHAPTER7 Changes in Aging Men V. A N D R O G E N

EFFECTS

AND REPRODUCTIVE

119 ON SEXUAL

FUNCTION

The biological actions of androgens are far reaching, and reduced androgen availability in aging men contributes to a host of biological changes. Testosterone acts on most body tissues. However, the biological effects in aging tissue do not always correlate with plasma concentrations, because local tissue factors such as conversion to DHT or E 2, or metabolism to glucuronides, modulate its activity. Thus, although T concentrations may decrease with age, some T-sensitive organs, especially the prostate, commonly undergo hyperplasia. Although androgens have a permissive role for prostatic tissue growth and development, their precise role in benign prostatic hyperplasia (BPH) is not clear. This may be related to altered T metabolism in aging prostate such that in B PH the ratio of DHT to 3ce-androstanediol is increased compared to normal prostate [16]. The androgen dependency of the other accessory sex organs is also well established. Secretory epithelium of epididymus regresses following castration. Exogenous androgen treatment restores some but not complete secretory function [16]. The seminal vesicles, in particular the epithelial component, are androgen dependent and so is spermatogenesis. However, these organs change modestly with age, suggesting that the age-related reduction in T availability is not sufficient to result in a clinically relevant change in these organs. Androgens do not appear to alter erections in response to erotic films [99,100]. To what extent do androgens play a role in erectile function in the adult male and what are the effects of declining androgen availability with aging on sexual function? Of course androgens are necessary both in utero and after birth for the proper development of the male external and internal genitalia. Growth of the penis and testes during puberty is also totally dependent on adequate androgen availability. It has already been noted that adequate T concentrations are necessary for normal libido [ 101 ]. Pharmacological reduction of circulating T in young men reduced sex drive and responses although the erectile response to erotic stimuli was unchanged [ 102]. It has been reported that severe hypogonadism is responsible for less than 7% of cases of erectile dysfunction (ED) [103,104]. An important role for T is suggested by studies demonstrating in other species that castration reduces erectile capacity, which can be preserved with dihydrotestosterone (DHT) [105]. In rats, T facilitates centrally mediated erections and yawning (a sexual response) [106]. Androgens stimulate the sexually dimorphic brain nuclei and increase the size and dendritic spread of the spinal cord motor neurons innervating the bulbospongiosus and ischiocavernosus muscles [107]. They may also affect the penile vascular re-

sponse mechanism [ 108]. In men, androgens are responsible for normal seminal fluid and prostatic secretions and the frequency of nonerotic or nocturnal erections [99]. The frequency of nocturnal penile erections correlates with circulating testosterone [109]. The reduction in testosterone status after age 45 is generally paralleled by a gradual decline in sexual desire, arousal, and activity [110]. The magnitude of this decline varies widely [ 111] and is frequently the result not of aging but of other factors, including medications, depressed mood, alcohol use, obesity, and chronic illnesses (e.g., diabetes, vascular disease). Despite reduced libido, sexual enjoyment and satisfaction do not decline with age [112]. An important predictive factor for sexual enjoyment in aging men, as in younger men, is the quality of the marital relationship [ 112]. As distinguished from libido, the role of a decline in T in ED in men as they age has not been well characterized, but in one study there was no relationship between the mild hypogonadism of aging and ED. Both conditions were common, but independently segregated [21 ]. Many men seek androgen replacement to improve erectile function, but the use of testosterone for this purpose in eugonadal men is usually unsuccessful [20,113]. Testosterone supplementation does, however, increase sexual interest [ 114].

VI. NONERECTILE SEXUAL DYSFUNCTIONS Sexual dysfunction in men consists of a small group of problems including early or premature ejaculation, retarded or lack of ejaculation, loss of libido, and erectile d y s function.

A. E j a c u l a t o r y D y s f u n c t i o n The mechanism of ejaculation encompasses seminal emission, ejaculation, and bladder neck closure. Afferent stimuli include activation of higher centers of sexual response to reach a threshold. Sympathetic nerves then cause smooth muscle contraction in the epididymis, vas, seminal vesicle, and prostate to produce filling of the prostatic urethra with the seminal emission. Finally, contraction of the bulbospongiosus and ischiocavernosus muscles and contraction of the bladder neck lead to propulsion of the semen out of the penis while the sensation of orgasm is experienced. 1. PREMATURE EJACULATION Early or premature ejaculation is perhaps the most common disorder of sexual function in men, affecting at least a third. Ejaculation usually occurs very close to the time of

120

K O R E N M A N ET AL.

vaginal penetration, in the most severe cases before vaginal penetration. Milder cases are associated with ejaculation after a few seconds of thrusting. This condition improves with sexual experience and age but persists in a substantial number of men well into the fourth and fifth decade. It is thought to be due to anxiety associated with sexual activity. For men with premature ejaculation, sex therapy behavioral techniques are beneficial but usually do not suffice [115]. Pharmacological interventions targeted at augmentation of serotonin function have been reported in numerous studies to be highly effective for this condition. In particular, the use of standard doses of the serotoninergic agents fluoxetine, paroxetine, sertraline, and clomipramine have been found to prolong significantly latency to ejaculation [116-121 ], with improvement noted as early as 1 week following initiation of medication [120]. One study found that clomipramine produced the greatest increase in latency time, although it was associated with more side effects than the serotonin reuptake inhibitors (fluoxetine, paroxetine, and sertraline). Following discontinuation of the serotonin reuptake inhibitors, premature ejaculation has been observed to recur in 90% of treated men [ 117]. 2. RETARDED EJACULATION Retarded ejaculation, which is unusual, is sometimes found in association with the use of antipsychotic drugs and with certain antidepressives [ 122,123]. Sometimes removal or a change of medication will reverse the condition. Retrograde ejaculation is one of the consequences of prostate surgery. Most commonly this is due to surgical damage to the vesical-urethral sphincters, making it easier to pass the ejaculate into the bladder than through the urethra. This may also occur with diabetic autonomic neuropathy, in which the same sphincters become dysfunctional. Retrograde ejaculation is usually treated with reassurance but many men complain that their sensation of orgasm and release is substantially reduced in the absence of an ejaculate.

VII. ERECTILE

DYSFUNCTION

Erectile dysfunction, on the other hand, is progressively common with age and has many etiologies and risk factors. There have been extensive publications and reviews of the field [ 124-127] and we will not try to recapitulate here the history of research in the area. In the past year the problem of ED has mutated from an underdiagnosed disorder managed by a few physicians to a public phenomenon characterized by media frenzy, numerous jokes, and an intense debate featuring patients, health care providers, and government, regarding whether treatment of ED with sildenafil or other oral agents should remain covered by insurance. What kind of problem is ED and how often does it occur?

A. E p i d e m i o l o g y At the 1993 National Institutes of Health Consensus Development conference on ED [ 128], erectile dysfunction was formally defined as "an inability of the male to achieve an erect penis as part of the overall multifaceted process of male sexual function." Although the adopted definition seemed refreshingly simple and useful at the time, in practice it has become too vague. For example, how erect? Does a nonrigid but usable erection count as ED or normal erectile function? How is a full erection for masturbation and on awakening, but no erection in the presence of a partner, to count? What should we call variable erectile response? These questions pertain not only to research determining the prevalence of the condition but also to difficult questions as to support of the treatment of ED by health insurers. Feldman and colleagues in the Massachusetts Male Aging Study (MMAS) [98] developed a nine-point questionnaire and divided ED into three levels of dysfunction, with the most severe being a complete absence of sexual response and "mild" being an occasional failure of certain aspects of response. By these criteria, in a community-based group of men from ages 40 to 70 years, 9.6% had complete ED, 25.2% had moderate ED, 17.2% had minimal Ed, and 48% had no ED. Although this approach engendered a degree of criticism, until the advent of sildenafil this partition was effective in the selection of patients for treatment, because men usually wished to be treated only if they were seriously affected by the problem. With the advent of sildenafil, the target population could conceivably become 52% of men ages 4 0 - 7 0 years and much higher percentages of older men [129]. If, of the 110 million American males over age 40 years, 50 million had ED, and they wanted to have sex once weekly, that would require 52 • 50 million pills at $8.00/pill or nearly $21 billion/year for this single indication. Obviously, a more precise, medically determined objective diagnosis of ED is required. Table VI lists factors found in the MMAS [98] to be associated with an increase of ED and factors decreasing ED. Factors inhibiting sexual function include coronary artery disease and diabetes, especially if treated (more severe) and if associated with smoking cigarettes. Depression and anger, whether internalized or expressed, were highly associated with ED. This is of particular importance because depression is very strongly associated with loss of libido (see above) and is greatly underdiagnosed in men, especially in middleaged men (see below). Studies of populations in the medical system, however, although biased because of the patterns of referral and the expertise of the practitioner, demonstrate a high prevalence of hypertension, coronary heart disease, and diabetes, as well as treatment for each, associated with ED [103,104]. Gener-

CHAPTER 7 Changes in Aging Men

121

TABLE VI Condition

Criteria for Depressive C o n d i t i o n s Symptoms

Duration

Major depressive episode

Five or more of the following symptoms present for the same 2-week period (must include symptom 1 or 2): 1. Depressed mood most of the day nearly every day 2. Markedly diminished interest or pleasure in almost all activities 3. Significant weight loss when not dieting, or weight gain 4. Insomnia or hypersomnia 5. Psychomotor agitation or retardation 6. Fatigue or loss of energy 7. Feelings of worthlessness or excessive/inappropriate guilt 8. Diminished ability to think/concentrate, or indecisiveness 9. Recurrent thoughts of death

2 weeks

Dysthymic disorder

Depressed mood most of the day more days than not for at least 2 years (two or more of the following symptoms while depressed): 1. Poor appetite or overeating 2. Insomnia or hypersomnia 3. Low energy or fatigue 4. Low self-esteem 5. Poor concentration/indecisiveness 6. Feelings of hopelessness

At least 2 years

Adjustment disorder with depressed mooda

Development of emotional or behavioral symptoms in response to an identifiable stressor occurring within 3 months of the onset of the stressor; predominant manifestations are depressed mood, tearfulness, or feelings of hopelessness (these symptoms are clinically significant as evidenced by either of the following criteria): 1. Marked distress in excess of what would be expected from exposure to the stressor 2. Significant impairment in social/occupational functioning

Subclinical depression

Depressive symptoms that do not meet criteria for major depression, dysthymia, or adjustment disorder with depressed mood

a

Occurs within 3 months of stressor and does not persist beyond 6 months after stressor terminates

a During the 2-year disturbance, the person has never had a major depressive episode and has never been without the above symptoms for more than 2 months. Symptoms must cause significant distress or impairment in functioning and are not due to the effects of a substance or general medical condition. b The disturbance should not meet criteria for another psychiatric disorder and does not represent bereavement. Once the stressor has terminated, the symptoms do not persist more than 6 months.

ally, in those studies, the definition was limited to those with a c o m p l e t e inability to c o m p l e t e sexual i n t e r c o u r s e for at least 3 m o n t h s . Other clinical associations with E D i n c l u d e d pelvic surgical p r o c e d u r e s and m a j o r p e r i p h e r a l vascular disease as well as n e u r o l o g i c a l disorders s u c h as m u l t i p l e sclerosis. T h e drugs associated with E D i n c l u d e d p r i m a r i l y vasodilators, t r e a t m e n t s for d e p r e s s i o n and psychosis, and h o r m o n e s or drugs affecting the reproductive e n d o c r i n e syst e m [98,122,123].

B. Erectile Mechanism W h e n at rest, the penis m a i n t a i n s a state of flaccidity t h r o u g h a - a d r e n e r g i c a l l y m e d i a t e d c o n t r a c t i o n of c a v e r n o s a l and vascular s m o o t h m u s c l e , inhibiting b l o o d flow into the organ [130,131 ]. As the result of an erotic stimulus, received t h r o u g h one or m o r e of the five senses or via m e m o r y (fan-

tasy), inhibition of the s y m p a t h e t i c d i s c h a r g e takes place and a p a r a s y m p a t h e t i c d i s c h a r g e is initiated, with p r e s y n a p t i c t e r m i n a l s in the pelvic p l e x u s [ 1 3 2 - 1 3 4 ] . Postsynaptically, the signals travel by n o n a d r e n e r g i c , n o n c h o l i n e r g i c ( N A N C ) nitric oxide (NO) nerves to t e r m i n a t e in the s m o o t h m u s c l e of the c a v e r n o s a l arteries and t r a b e c u l a r sinusoids [135]. (Fig. 3). T h e s e m u s c l e s relax w h e n N O stimulates g u a n y l y l cyclase to c o n v e r t G T P to cyclic GMP. In s m o o t h m u s c l e , cyclic G M P inhibits Ca entry and facilitates Ca loss [136, 137]. In the a b s e n c e of sufficient C a 2+ s m o o t h m u s c l e relaxes, allowing the heart to p u m p m u c h m o r e b l o o d into the corpora, i n d u c i n g penile swelling. O t h e r n e u r o t r a n s m i t t e r s that have b e e n related to erectile f u n c t i o n include prostaglandin E 1 (PGE1) and other stimulators of a d e n y l y l cyclase, vasoactive intestinal p e p t i d e (VIP), endothelin, calcitoninrelated peptide, and histamine. T h e y p r o b a b l y play a m i n o r role in the h u m a n u n d e r p h y s i o l o g i c a l conditions. I n c r e a s e d inflow of b l o o d alone will not result in an

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FIGURE 3 Neurogenic mediation of penile vasodilatation via smooth muscle relaxation. On the left the postganglionic autonomic nerve is seen stimulating a smooth muscle cell, on the right. The group of cells in the left lower section represent endothelial cells. ARG, Arginine; CIT, citrulline, GC, guanylyl cyclase; DHT, 5a-dihydrotestosterone EFS, electric field stimulation (nerve discharge); ACH, acetylcholine; PGE~, prostaglandin E I.

erection; it requires nearly complete inhibition of venous return. That is accomplished passively by the unique anatomy of the penis, in which the expanding corporal sinusoids compress the subtunical plexus of veins draining the corpora cavernosa against the unyielding tunica albuginea [138]. The subtunical plexus, in turn, is drained through veins penetrating the tunica albuginea that are also compressed during stimulation, so that at maximum erection, penile blood flow is nearly zero [ 139,131 ]. Penile blood pressure may exceed systemic pressure at that time as a result of contraction of the ischiocavernosus muscle, which acts as a constriction ring at the base of the penis [ 140]. Ejaculation is neurally mediated in response to the filling of the prostatic urethra with semen and achievement of the "orgasmic plateau" of sexual stimulation, via contraction of the bulbospongiosus muscle [ 140].

C. Inadequate Erectile Function What happens to cause ED? First, there can be inhibition of the central nervous system centers mediating the response to erotic stimuli. Both testosterone deficiency (see above) and depression (see below) reduce libido substantially and together they are responsible for the majority of cases of reduced sexual interests in adult men. Second, the integrity of the neural pathways mediating an erection can be interrupted by spinal cord injury, pelvic surgery (usually due to resection of a prostate or colon cancer),

or autonomic neuropathy such as in Type I diabetes mellitus, or by primary neurological diseases such as multiple sclerosis [ 141 ]. A number of medications and recreational drugs affect the neural response at the periphery so as to contribute to ED [98,122,123,142,143]. Third, there may be a failure of response to the neural signals as a result of diminished NO synthesis, which has some relation to intact neural pathways, adequate androgen availability, and cavernosal smooth muscle integrity. This is commonly found in association with diabetic ED [105,144,145]. There is also evidence of enhanced contractility due to increased ce-adrenergic sensitivity in ED [146]. Fourth, ED is very commonly associated with abnormalities of the intrinsic tissues of the corpora cavernosa, including disrupted muscle fibers, an increase of dense connective tissue in the perisinusoidal area, and a reduction of tunical elastic fibers, which probably prevent adequate compression of the subtunical venous plexus [144,147-149]. This is commonly associated with atherosclerotic disease and diabetes and is thought to be due in part to ischemia. Fifth, the blood supply to the penis may be compromised by arterial atheromatous disease, a very low cardiac output, or arteriolar disease [ 150]. These conditions, once thought to be common and irreversible, probably account for a small proportion (less than 20%) of cases of ED. Failure of venous occlusion is very common in ED [ 151, 152], and although once it was considered to be a common etiologic factor, it is now believed to be largely a conse-

CHAPXER7 Changes in Aging Men quence of an inadequate filling rate and a degree of scarring in the perisinusoidal tissues except in cases of penile trauma, in which damage to large vessels is not uncommon, and in Peyronie's disease, in which peripheral fibrous plaque formation often inhibits venous compression [ 148]. Penile vein ablative surgery has been generally unsuccessful in the management of ED. How conditions associated with a high incidence of ED [98,124] produce the effects listed above is not fully understood, and until a minimally invasive acceptable method of biopsying the corpora cavernosa is developed we have no good way to correlate penile structure and ultrastructure with function and disease.

D. D i a g n o s t i c A s s e s s m e n t With the advent of sildenafil and other oral medications to come, the role of the health system in the diagnosis and treatment of ED has changed. No longer must physicians carefully elicit information about sexual function from reluctant patients. Rather, they often have to ensure that the patient requesting treatment indeed has an erectile problem as opposed to other sexual disorders or a desire for a somewhat enhanced lifestyle. Prior to initiating therapy, physicians must take a detailed sexual history that includes the nature of the dysfunction-weak or absent erection, erection of short duration, curved or distorted erection; the duration and progression of the condition; prior level of sexual activity, including repertoire and frequency as well as partner's interest, availability, and satisfaction. A careful review of the presence of nocturnal and especially morning erections gives considerable information about the erectile potential of the individual with simple therapies [153], although the long-standing attempt to differentiate psychogenic from organic ED by these means seems irrelevant. However, psychological elements, which should be elucidated, are present in virtually every case of ED. In this context, a simple questionnaire designed to evaluate male sexual dysfunction, e.g., the International Index of Erectile Function, can be used by clinicians [154]. Honest answers to this line of inquiry will provide an understanding of the degree of ED, relationship issues, and, in concert with the remainder of the medical assessment, medical and life style risk factors that contribute to the problem. Associated factors including the patient's endocrine status also need to be assessed during the history and physical examination. For laboratory testing, in patients who are regularly followed, we simply measure a TSH and bioavailable T, and if both are normal, we proceed. A low bioavailable T will precipitate measurement of prolactin and LH and a very low bioavailable T will precipitate an MRI of the pituitary gland, especially in younger men [155]. In the vast majority of men over the age of 40, and more particularly over the age

123 of 50, ED will be multifactorial in origin and susceptible to simple therapies (see below). For those with unusual problems, such as penile trauma or severe Peyronie's disease, evaluation by a skilled urologist is required.

E. T r e a t m e n t For men with bone fide ED, it is difficult to withhold initial therapy when a simple and effective agent such as sildenafil is available. The drug, a pill, is taken 1-2 hr prior to anticipated sexual activity [156]. It acts as an inhibitor of phosphodiesterase V. Phosphodiesterase V is responsible for degrading cyclic GMP to GMP, eliminating its biological effect (Fig. 3). As noted previously, cyclic GMP is the second messenger stimulated by NO in the corpus cavernosum. It is responsible for inhibiting Ca e+ intake and increasing Ca e+ egress from the smooth muscle of the corpora cavernosa, relaxing the arteries and sinusoids. Inhibition of phosphodiesterase V (PDE V) maintains the level of cyclic GMP for a much longer time, facilitating the erection. To be effective, the drug requires a substantial innervation of the penis. The drug does not affect libido. The side effects of sildenafil are attributed to its lack of perfect specificity. Patients may experience headaches, flushes, gastrointestinal distress, visual blurring, or a bluish haze during the 5 hr or so that the drug is active but they willingly accept the side effects if the primary effect is delivered (Fig. 4). In this study, note that with increased dosage, which produced increased efficacy, the discontinuation rate declined despite increased side effects. There are no reports of long-term consequences of sildenafil, which is taken only when intercourse is anticipated. It has been quite successful in restoring erectile function in over two-thirds of men with moderate ED [ 156]. The most significant issues are an absolute contraindication of sildenafil use

ADVERSE EVENTS Dose Response Study

20

Discontinuation Rate

10

7 o

7 c:~

0 4030-

Percent

20-

m~ 0

P

25

50

100

Dose of Sildenafil

H=headache

F=flushing

D=dyspepsia

V=visual disturbance

FIGURE 4 Adverse effects and discontinuation of sildenafil therapy. Derived from the data of Goldstein et al. [ 156].

124 in anyone taking nitrates in any form and an absolute contraindication of the use of nitrates in anyone who has recently used sildenafil. How recently? No one knows for sure. The drugs, in combination, can and have produced vascular collapse. A few deaths have been reported in association with the use of sildenafil in older men, by and large either using nitrates or with preexisting heart disease. It is likely that the death rate is not in excess of what is expected for this population. The next line of therapy in ED is based on the introduction of PGE 1 and or other agents, mainly papaverine and phentolamine, into the corpora cavernosa, by direct injection. Prostaglandin E l (alprostadil) is available in an injectable form (Caverject, Upjohn; EDEX, Schwarz Pharma) [157], and in a form that allows introduction through the urethra [158]. PGE 1 stimulates adenylyl cyclase to produce cyclic AMP. This second messenger inhibits Ca 2§ entry into smooth muscle cells, causing their relaxation. These agents affect the penis directly. They require neither an erotic stimulus nor an intact neural system. Thus, they are useful in spinal cord injury or after radical prostatectomy or colectomy. Their main problems are the necessity to introduce them directly into the penis and their propensity to produce hypotension in about 1% of patients, especially those with severe cardiovascular disease. This is particularly significant with the medicated urethral system for erection (MUSE), which requires up to 1 mg of PGE 1, whereas the injection therapies require only up to 20/zg to be introduced. Intracavernosal injection produces a satisfactory result for about two-thirds of men tested, whereas MUSE is effective for only about one-third. Vacuum tumescence devices can produce an erection in about 90% of the men who attempt them [ 159]. They consist of a plastic cylinder in which the pendulous penis is placed. The cylinder is connected to a vacuum pump, and on evacuation, blood is drawn into the penis. To keep the blood there, an obstructing band is slipped over the base of the penis when a full erection is achieved. These devices are efficient and inexpensive over time. They produce a somewhat abnormal erection in that all of the tissues, not only the corpora, are engorged, and obstruction of blood flow sometimes leads to cooling of the penis. This procedure does not interfere with a patient's medications, nor does it produce hypotension. However, there is a loss of spontaneity with sex, the erection is sometimes "on a hinge," sometimes ejaculation through the obstructing band is a problem, and, in some instances, application of excessive negative pressure produces petechiae. Also a degree of manual dexterity and skill is required to get the device to work. Penile prostheses were once the primary therapy for ED. The recent advent of much less costly and invasive approaches had made them more of a last resort. They produce a satisfactory erection in about 80% of the men who have them. Unfortunately, the rigid rod versions have a tendency

KORENMAN ET AL.

to explant and the versions including a pump mechanism have a tendency to undergo mechanical failure [ 160]. Vascular surgery is employed in specialized urology centers to establish appropriate blood flow to the penis. It is indicated only for specialized conditions such as penile or pelvic trauma to a young person. Considering that sildenafil is a small molecule, it is highly likely that other oral agents with the appropriate specificity will become available for therapy. With the cloning of penile inducible nitric oxide synthase [ 161 ], it may be possible to develop gene therapy or very specific small molecules that can enhance or preserve NO synthesis or concentrations. Numerous other components of this now well-understood system are susceptible to pharmacological attack as well.

VIII. MANOPAUSE AND MENTAL HEALTH A. D e p r e s s i o n in M i d d l e - A g e d and Elderly M e n ~ E p i d e m i o l o g y and R i s k Factors Although a number of epidemiological studies have assessed the relationship between mood and aging in women [ 162-164], the psychological changes accompanying aging in men have received little attention. Studies of mood in middle-aged men are particularly scarce. However, several authors have reported high rates of depressed mood, insomnia, mood swings, irritability, impotence, decreased libido, weakness, and lethargy in this population [165,166]. Because these symptoms may not meet criteria for major depression, as defined by the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) (see Table VII), they are likely to be missed in epidemiological studies of psychiatric disorders. Among the elderly (older than 65 years), epidemiological surveys report lower rates of major depression as compared to younger populations [ 167,168]. However, studies exploring the prevalence of subsyndromal depression, i.e., depressive symptoms not meeting criteria for major depression, have consistently found a high rate among elderly persons [169-171]. Despite the high prevalence of depressive symptomatology among older persons, the symptoms are seldom recognized or treated [ 169,172]. This undertreatment may reflect clinicians' attribution of the symptoms to physical illnesses or to understandable responses to adversity [ 173,174]. Also, the greater tendency among elderly patients to express psychological distress through somatic symptoms contributes to oversights in the diagnosis of depressive disorders [ 174]. Factors associated with depressive symptoms in older men include limited economic resources, poor health, Caucasian race, and impaired sexual functioning [165-177].

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CHAPTER 7 C h a n g e s in A g i n g M e n

TABLE VII

Criteria for Anxiety and Panic Syndromes

Condition Panic disorder without agoraphobia

Symptoms Recurrent 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

unexpected panic attacks consisting of the following symptoms a: Palpitations, pounding heart, or accelerated heart rate Sweating, trembling, or shaking Sensations of shortness of breath or smothering Feeling of choking, chest pain, or discomfort Nausea or abdominal stress Feeling dizzy, unsteady, lightheaded, or faint Derealization (feelings of unreality) or depersonalization (being detached from oneself) Fear of losing control or going crazy Fear of dying Numbness or tingling sensations Chills or hot flashes

At least one of the attacks has been followed by the following symptoms: 1. Persistent concern about having additional attacks 2. Worry about the implications of the attack or its consequences (e.g., losing control, having a heart attack, or going crazy) 3. A significant change in behavior related to the attacks Panic disorder with agoraphobia

Same as above, but with anxiety about being in places or situations from which escape might be difficult or embarrassing or in which help may be unavailable b

Generalized anxiety disorder

Excessive anxiety and worry occurring more days than not, about a number of events and activities. Difficulty in controlling the worry, and the anxiety and worry are associated with three or more of the following symptoms (with at least some symptoms present for more days than not for the past 6 months)c: 1. Restlessness or feeling keyed up or on edge 2. Being easily fatigued 3. Difficulty concentrating, mind going blank 4. Irritability 5. Muscle tension 6. Sleep disturbance (difficulty falling or staying asleep, or restless, unsatisfying sleep)

aThe panic attacks are not due to the direct physiological effects of a substance (e.g., a drug of abuse or a medication) or a general medical condition (e.g., hyperthyroidism). bThe situations are avoided or endured with distress or anxiety about having a panic attack or paniclike symptoms, or require the presence of a companion. CThe anxiety, worry, or physical symptoms cause clinically significant distress or impairment in social, occupational, or other important areas of functioning, and are not due to the direct physiological effects of a substance (e.g., a drug of abuse or a medication) or a general medical condition (e.g., hyperthyroidism), and do not occur exclusively during a mood disorder, a psychotic disorder, or a pervasive developmental disorder.

Poor physical function is also a risk factor for depressive symptoms and, conversely, depressive symptoms are associated with subsequent physical decline in elderly persons [169,178]. Being widowed, divorced, or separated are additional risk factors for depressive symptoms in this population [176,177]. Negative stereotypes of aging also are likely to impact a man's mood and self-image [179]. For many men, retirement produces a sense of letdown and can contribute to depressive symptoms [ 180]. Retirement frequently represents a loss of prestige, income, status, purpose, and workrelated friendships [ 181]. Depressive symptoms related to retirement occur most frequently in men whose lives and self-identity centered around their work or in men who have had to retire because of poor health or inability to maintain

their jobs [ 181 ]. Men who are healthy, active, and have adequate financial resources and extended social networks are least likely to experience difficulty with retirement [181].

B. E v a l u a t i o n and T r e a t m e n t o f D e p r e s s e d M o o d in A g i n g M e n Depressive symptoms in aging individuals are often missed by hospital physicians [ 182]. A careful assessment of mood is important in this population, particularly because somatic complaints may mask symptoms of depressed mood [183]. Any patient presenting with fatigue, changes in

126 appetite or sleep, and reduced libido should be evaluated for depressed mood. Depression rating scales such as the Geriatric Depression Scale [184] help screen for major depression. Unusual thought content should also be explored, because approximately 50% of depressed men over age 60 years experience delusional depression. Common presentations include delusions of being ill (somatic delusions) and of being followed or spied on (persecutory delusions) [183]. Criteria for depressive illnesses and anxiety syndromes are presented in Tables VI and VII. Patients who endorse depressive symptoms but do not meet full criteria for major depression or dysthymia may still benefit from treatment. The relationship between the depressive symptoms and psychosocial stressors (e.g., death of a family member, loss of a job, onset of an illness, or relocation) should be explored. Knowledge of factors that may have triggered the depressive mood changes is important in the choice of intervention. Interpersonal psychotherapy is particularly helpful for men who have undergone recent life transitions, because it focuses on strategies to cope with role changes or grief or to modify unrealistic expectations about relatives and other people in one's life. Cognitive-behavior therapy is an alternative approach based on training people to identify and challenge self-defeating thoughts, such as "I'm no longer working, therefore people will find me boring." Certain medications (e.g., antihypertensive agents) have been linked with depressive mood changes, thus a complete determination of the patient's medication usage should be obtained. Laboratory studies should include thyroid function testing and a bioavailable T level to rule out hypothyroidism and hypogonadism as contributing factors to the depressive symptoms. Antidepressant medications can be very helpful in promoting the recovery from major depression. Currently the most commonly used antidepressant medications are the serotonin reuptake inhibitors (fluoxetine, sertraline, and paroxetine), and they are generally well tolerated and are relatively safe in overdose. Typical side effects from these medications include gastrointestinal symptoms and impairment of sexual function, mostly libido. The tricyclic antidepressants (e.g., nortriptyline, desipramine, doxepin, and amitriptyline) have less effect on sexual function but can produce sedation, orthostatic hypotension, blurry vision, constipation, and EKG changes. Other available antidepressant medications include venlafaxine, nefazodone, bupropion, and the monoamine oxidase inhibitors (tranylcypromine and phenelzine). The antidepressants that are least likely to affect sexual function are bupropion and nefazodone. Monoamine oxidase inhibitors have the disadvantage of requiring very close attention to dietary guidelines and drug-drug interactions, to avoid the possibility of a "tyramine reaction," which can produce an abrupt and dangerous rise in blood pressure. When using antidepressant medications with men aged 65 years or older, the starting dose should be approximately half

K O R E N M A N ET AL.

that used for younger populations. Patients should be reminded that beneficial effects may not become fully apparent until after 4 - 6 weeks of treatment. Once a patient has experienced a positive response, he should be maintained on the same dose for a minimum of an additional 6 months. Long-term follow-up should continue after resolution of the depression, because the likelihood that a depressive episode will recur exceeds 50% for individuals aged 60 years or older [ 185]. Ideally, medications should be used in combination with psychotherapy and life style changes. Depressive symptoms may discourage men from maintaining healthy habits, such as exercising and not smoking, and following healthy diets. Depressed mood has also been significantly associated with a lower likelihood of engaging in walking, gardening, and exercise [186]. Unhealthy aspects of the patient's life style should therefore be explored, such as being sedentary, using alcohol and nicotine, and tendencies toward isolation, and the patient should be encouraged to exercise regularly and to be involved in stimulating activities. For men with limited social support networks, referrals to group therapy is beneficial. Screening and treatment of depressed mood in older people is a cost-effective intervention in terms of health and well being per dollar spent [ 187]. Appropriate treatment also appears to increase the number of years during which older persons are free of disability [ 169]. More research is needed, however, on specific screening and treatment interventions for middle-aged and elderly men, particularly because these populations are growing with the aging of the baby-boom generation.

C. A n d r o g e n s and the Central N e r v o u s S y s t e m Declines in bioavailable T may well account for the reduced libido with age. The latter is believed to be a central nervous system (CNS)-related effect of T that may be modulated through E 2 produced locally [18]. Androgens are necessary but not sufficient for maintaining normal libido. In older men, unlike young men, higher plasma T levels are associated with greater sexual activity [97,99] Also, latency to erection stimulated by erotic material correlates with T levels. In hypogonadal men, T replacement restores sexual interest and improves the latency, frequency, and magnitude of the nocturnal penile tumescence and the frequency of early morning erections [ 100,115]. The effect of T on the CNS extends beyond sexual behavior. T has been shown to alter mood, memory, ability to concentrate, and the overall sense of vigor and well being [ 117119]. A number of studies have examined the relationship between mood and levels of testosterone in men. However, most have included wide age ranges rather than focusing on middle-aged or elderly men. Some of these studies have

CHAPTER7 Changes in Aging Men

127

found testosterone levels in men with major depression to be lower [88], whereas others have found no significant difference from controls [189,190]. Methodological problems may explain the discrepant findings, including a lack of control for time of day of blood-drawing, total T versus free or bioavailable T, age distribution, body mass index (BMI; high B MI is associated with decreased T binding and thus lower total T values), cortisol levels (which may affect the hypothalamic-pituitary-gonadal axis), and medication use. One study that did control for medical illness, age, alcohol use, weight, and use of medications found no significant differences in free or total testosterone among 12 patients with major depression compared with 12 controls. It did identify a trend for lower testosterone levels (10% lower total testosterone and 20% lower free testosterone) in the depressed group [190]. Replication of this study in a population of middle-aged men and with a larger sample size would be of immense interest. In the only study of T levels in middleaged men, a high level of psychosocial stress was inversely related to free T levels in a sample of 439 men aged 51 years [191]. The authors concluded that psychosocial stress may be associated with premature aging in middle-aged men.

IX. P S Y C H O L O G I C A L

STATE

AND SEXUAL FUNCTION A. P s y c h o l o g i c a l C a u s e s o f E D Negative expectations of changes of sexual functioning with age may contribute to erectile difficulties [192]. Other potential causes include marital conflict, employment-related problems, family illnesses, boredom, poor communication of sexual needs, and lack of interest from one's spouse. An important and underrecognized etiology for sexual dysfunction is depressed mood. In a study of 1709 men, moderate to complete ED was found 1.82 times more in those men with depressive symptoms (as assessed by Center for Epidemiological Studies--Depression Scale) compared to those without symptoms, after controlling for age, health, medication use, demographic factors, and hormone levels [ 193]. Depressed mood has also been associated with reduced penile rigidity and nocturnal penile tumescence (NPT) time [194,195]. In the Massachusetts Male Aging Study [98], measures of depressed mood and anger were strongly correlated with ED, and were postulated to result from elevations in blood catecholamines, producing vasoconstriction and thereby inhibiting the physiological events necessary for normal sexual function. Because ED can dramatically affect mood and self-confidence [165], a vicious cycle may develop in which depressed mood and anxiety concerning sexual performance exacerbates erectile difficulties.

B. P s y c h o l o g i c a l E v a l u a t i o n a n d T r e a t m e n t of Sexual Dysfunction Premature ejaculation is the most common male sexual dysfunction, occurring in approximately 36-38% of men [ 196]. Psychological factors, including high levels of anxiety [197] and lack of intimacy with one's partner [198], are linked with this condition. Although psychogenic factors are also common in young men with ED, in men over age 50 years, most have organic etiologies for the sexual dysfunction [ 199] and often psychological issues exacerbated by ED [200]. An evaluation of sexual dysfunction, therefore, should include a psychological assessment. A review of situational factors associated with the dysfunction is essential in evaluating the extent to which the problem may have a psychogenic origin. For example, a man's sexual problems may arise only when he feels criticized or rejected, or only when he is under pressure at work. When present, alcohol and substance abuse will impair sexual function. Partners should be present during the evaluation, because they can provide useful observations and the relationship between the two can be explored. A tense or conflictual relationship is a major impediment to successful restoration of sexual function, and couples' therapy should be recommended as part of the treatment strategy. A woman's reaction to her partner's sexual difficulties should also be assessed, because she may view the man's sexual problems as a reflection on herself and feel hurt or angry. An open discussion, in which common reactions are described and normalized, can help bolster the couple's mutual trust and support. Even if an organic etiology for the sexual dysfunction is identified, psychological evaluation is still beneficial because the couple's emotional reactions may exacerbate the problem. Marital therapy may be necessary in cases in which either partner experiences persistent frustration or hostility toward the other. Sex therapy techniques can also be of great benefit, and include structured sexual exercises, psychodynamic exploration of emotional conflicts, and cognitivebehavioral strategies [ 199].

X. C O N C L U S I O N S In the 1960s, the claim was that we all began to go downhill after the age of 30, and we should never trust anyone over 30. Well, that crowd is all in its 50s now. What does happen to men as they age and what can we learn to make that inevitable process healthier and more enjoyable? Must the acquisition of wisdom invariably be associated with "settling" of the body? We really do not know and the information presented here provides only an antipasto to what should be a rich scientific repast. Only by much more intensive investigation of men as

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t h e y p a s s t h r o u g h t h e i r 4 0 s a n d 5 0 s w i l l w e b e a b l e to r e c o m mend soundly behaviors and evaluations that will not only i m p r o v e h e a l t h a n d w e l l b e i n g , b u t , in t h e l o n g r u n , p e r h a p s

19.

r e d u c e h e a l t h c a r e c o s t s as w e l l .

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2 H A P T E R {~

Premature Ovarian Failure ROBERT W. REBAR

I. II. III. IV.

Department of Obstetrics and Gynecology, University of Cincinnati Medical Center, Cincinnati, Ohio 45267; and the American Society for Reproductive Medicine, Birmingham, Alabama 35216

V. Evaluation of Patients with Hypergonadotropic Amenorrhea VI. Therapy References

Early Reports of Premature Ovarian Failure Clinical Features of Premature Ovarian Failure Prevalence of Premature Ovarian Failure Etiology of Premature Ovarian Failure

genes are important in controlling the number of oocytes ovulated and hence presumably the timing of the cessation of reproductive function [3]. Although these data are difficult to extrapolate to humans, given what is known about the control of ovarian function by the X chromosome [4], it is not difficult to believe that inherited tendencies are important. Any role for ovarian inhibin and its feedback action on pituitary FSH secretion also remains to be explored. Also potentially important in the regulation of the onset of menopause is the hypothalamic-pituitary axis. Although oocyte depletion may provide the major reason for the occurrence of menopause in humans, numerous animal studies document changes in neurotransmitter and in central nervous system (CNS) feedback responses to estrogen with aging. Of particular note is the observation that aging ovaries transplanted to young rodents cycle normally whereas young ovaries transplanted to aged animals do not function well [5]. Once more, however, extrapolation of such data to humans is most difficult. The concept that young women under the age of 40 with "hypergonadotropic" amenorrhea by definition should have depletion of their oocytes and premature ovarian failure was supported by the findings of Goldenberg and colleagues [6]. They reported in 1973 that women who had basal FSH concentrations greater than 40 mIU/ml [Second International Reference Preparationmhuman menopausal gonadotropin, (2nd IRP-hMG] without exception had no viable oocytes on ovarian biopsy.

I. EARLY REPORTS OF PREMATURE OVARIAN FAILURE Menopause, defined strictly as the last episode of menstrual bleeding, typically occurs around age 51 and is generally considered premature if it occurs before the age of 40 years. In fact, de Moraes and Jones [1] first defined premature menopause, or premature ovarian failure, as consisting of the triad of amenorrhea, hypergonadotropinism, and hypoestrogenism in women under the age of 40 years. Why the cessation of reproductive life should occur prematurely has been of great interest to clinicians and remains enigmatic in the majority of cases. How little is known about premature menopause is less surprising in view of how little is known about normal menopause. The events that signal menopause are unclear. Depletion of oocytes is obviously an important factor, and it has been documented that follicle depletion accelerates just prior to menopause [2]. Although a few follicles may be present at menopause, they do not respond to folliclestimulating hormone (FSH) and luteinizing hormone (LH). In an unsuccessful effort tastimulate follicular development and estradiol secretion, the hypothalamus signals the pituitary gland to secrete still more FSH and LH. Thus, an increase in serum FSH concentrations is an early sign heralding the cessation of ovarian function. Preliminary studies in strains of mice indicate that specific MENOPAUSE:

BIOLOGY AND PATHOBIOLOGY

135

Copyright 9 2000 by Academic Press. All rights of reproduction in any form reserved.

136

ROBERT W. REBAR

The belief that the ovarian "failure" observed in such young women was permanent was first questioned by a number of isolated case reports documenting the initiation or resumption of cyclic menses and/or pregnancy in affected women. Several large series have now confirmed these case reports [7]. In one of those reports, we documented pubertal progression in two young girls with elevated circulating FSH and multiple endocrine deficiencies (i.e., hypoparathyroidism and hypoadrenalism) and suggested that waxing and waning autoimmune dysfunction might account for the transient nature of the ovarian failure [8]. O'Herlihy and coworkers [9] reported that up to one-fourth of younger women with FSH values in the menopausal range will resume ovulation spontaneously and a few will even conceive. In 1982 we reported that 9 of 18 young women presumed to have ovarian failure had circulating estradiol typical of women with functioning ovarian follicles and that 4 of the 9 women who had ovarian biopsies had viable oocytes [10]. In addition, circulating concentrations of serum progesterone typical of ovulation were noted in 5 women, and a spontaneous pregnancy occurred in one. Aiman and Smentek [11] reported that 18% of 157 women reported in the literature who had ovarian biopsies had specimens containing apparently viable oocytes. They also noted that 14 of the women had conceived after the ovarian failure had been diagnosed. A number of recent series have confirmed ovarian follicular activity in many women with ovarian failure. Hague et al. [12] reported evidence of ovarian follicular activity in 12 (17.1%) of 93 women with amenorrhea and elevated FSH concentrations. By pelvic ultrasound Conway and colleagues [ 13] identified follicular activity in 65 of 109 women (60%) with "idiopathic" premature ovarian failure. Bone mineral density was lower in women in whom ovaries were not identified on ultrasound (n -- 26) than in those in whom TABLE I

follicles > 4 m m were identified (n = 57). Similarly, Nelson and colleagues [14] documented ovarian follicular activity by serum estradiol levels greater than 50 pg/ml in nearly half of 65 women with karyotypically normal spontaneous premature ovarian failure and imaged an antral follicle in over 40% [ 15] of the women. These observations led us to suggest that this disorder involved more than just the premature cessation of ovarian function and might more appropriately be termed "hypergonadotropic amenorrhea" [ 13 ] - - at least until such time as it was apparent that the premature loss of ovarian function was permanent.

II. CLINICAL

FEATURES

OF PREMATURE

OVARIAN

FAILURE

To define the clinical spectrum of women with hypergonadotropic amenorrhea, Rebar and Connolly [ 16] compiled data from 115 affected women seen sequentially between 1978 and 1988. Initial inclusion criteria were (1) amenorrhea of 3 or more months' duration, (2) age under 40 years at the onset of the amenorrhea, and (3) circulating FSH of more than 40 mlU/ml on at least two occasions. A number of interesting differences and similarities existed between those with primary and those with secondary amenorrhea and are summarized in Table I. In over 75% of the patients, symptoms of estrogen deficiency, most commonly hot flushes and/or dyspareunia, were evident, but these symptoms were far more common in those with secondary amenorrhea. Chromosomal abnormalities and failure to develop mature secondary sex characteristics were far more common in those with primary amenorrhea. Chromosomal abnormalities were present in over half

Features of Women with Primary and Secondary Amenorrheaa

Feature

Primary amenorrhea

Number of patients Symptoms of estrogen deficiency Incomplete sexual development Karyotypic abnormalities Immune abnormalities Spinal bone density <90% of controls Progestin-induced withdrawal bleeding Pregnancies before diagnosis Evidence of ovulation after diagnosis Pregnancies after diagnosis

18 (15.7) 4 (22.2) 16 (88.9) 10 (55.6) 4 (22.2) 3/4 (75) 2/9 (22.2) 0 0 0

Secondary amenorrhea 97 (84.3) 83 (85.6) 8 (8.2) 6/45 tested (13.3) 16 (16.5) 13/22 (59.1) 36/70 (51.4) 33 (34.0) 23 (23.7) 8(8.2)

Significant differenceb p < 0.001 p < 0.001 p < 0.001 p < 0.01 NS NS NS p < 0.025 P < 0.05 NS

aAdapted from Rebar and Connolly [16] with permission from the American Society for Reproductive Medicine (Fertility and Sterility, 1990, Vol. 53, pp. 804-810). Values in parentheses are percentages of women diagnosed with a type of amenorrheaand associated features. bResults using X2 test. NS, not significant.

CHAPTER 8 Premature Ovarian Failure the women with primary amenorrhea, who tended to have deletions of all or a part of one X chromosome, whereas those with secondary amenorrhea more commonly had an additional X chromosome. Easily detected immune disturbances were present in approximately 20% of the patients. Thyroid abnormalities were most common, with five women having Hashimoto's thyroiditis, two developing primary hypothyroidism, one developing subacute thyroiditis, and one having Graves' disease. Three asymptomatic patients had high titers of antimicrosomal antibodies. One of the women had vitiligo and hypoparathyroidism, one had Addison's disease, and one additional woman had insulin-dependent diabetes mellitus. A relatively small number of the women in this series, all with secondary amenorrhea, had received chemotherapy with alkylating agents and in some cases radiation therapy as well before developing hypergonadotropic amenorrhea. The effects of alkylating agents and radiation therapy on ovarian function have been recognized for several years [ 17]. As more young women with childhood malignancies, especially the various leukemias and lymphomas, are treated and cured, the incidence of patients with induced amenorrhea will no doubt increase. Four of the patients with secondary amenorrhea and normal karyotypes had a family history of early menopause prior to age 40. Four others reported a temporal relationship between the onset of amenorrhea and various infections, including chicken pox, shigellosis, malaria, and an undefined viral syndrome. Spinal bone density, as evaluated by dual-photon absorptiometry, was less than 90% (range 62-105%; mean 85.7%) of the mean value observed in age-matched controls in 16 of the 26 women who underwent such testing. Progestin-induced withdrawal bleeding, presumably an indication of endogenous estrogen activity, occurred in just under 50% of the women tested. Withdrawal bleeding even occurred in 2 of the 9 individuals with primary amenorrhea who were challenged. There was, however, no correlation between the response to exogenous progestin and subsequent ovulation. None of the women with primary amenorrhea ever ovulated or conceived with her own oocytes. In contrast, over one-third of the women with secondary amenorrhea were pregnant at least once before developing hypergonadotropic amenorrhea and almost one-quarter had evidence of ovulation after the diagnosis was established. Yet only 8% of those with secondary amenorrhea later conceived. Twenty-five of the patients with secondary amenorrhea were treated with clomiphene citrate to induce ovulation, but only four (16%) ovulated as determined by serial ultrasound and serum progesterone levels. Because each of the four who ovulated had evidence of spontaneous episodic ovulation before therapy, it is unclear if the clomiphene actually induced ovulation or if ovulation occurred in association with clo-

137 miphene on the basis of chance alone. Fourteen women were suppressed for 1 to 3 months with large doses of exogenous estrogen and then were administered human menopausal gonadotropins (from 50 to 100 ampules, with each ampule containing 75 IU of FSH and 75 IU of LH). We subsequently administered menotropins to five additional women suppressed previously for 1 to 3 months with a gonadotropinreleasing hormone agonist. Two of the patients suppressed with the agonist had evidence of significant follicular activity and ovulation, and one conceived. Thus, ovulation induction is unlikely to be successful in these women. Twelve women with secondary amenorrhea had ovarian biopsies, with apparently viable oocytes noted in seven of the specimens. Yet two of the eight subsequent pregnancies occurred in women with no follicles observed on biopsy. Fully seven of these eight pregnancies occurred while the patients were taking exogenous estrogen; the remaining pregnancy in this series occurred in response to clomiphene. Five of the eight pregnancies resulted in live term births, two ended in spontaneous abortion, and one ended in elective abortion. Only three patients with primary amenorrhea underwent gonadal biopsy: the two with 46,XY karyotypes had dysgerminomas. The one additional patient had fibrous streaks. These observations lead to the obvious conclusion that hypergonadotropic amenorrhea is a heterogeneous disorder. No doubt many of these young women have premature ovarian failure, but clearly others do not, as documented by subsequent ovulations and pregnancies. It would also seem logical to conclude that premature ovarian failure might be the end result of several varied disorders. Ovarian biopsy cannot be recommended in view of documented pregnancies in women who had no follicles found on biopsy. Because of the low incidence of ovulation and pregnancy among women undergoing ovulation induction, it is likewise difficult to recommend such efforts. Moreover, these clinical observations stress the importance for subsequent management of measuring basal FSH concentrations in all amenorrheic women. It is clear that progestin-induced withdrawal cannot be used to distinguish women with chronic anovulation from those with impending ovarian failure.

III. PREVALENCE OF PREMATURE OVARIAN FAILURE Estimation of the prevalence of premature ovarian failure in the general population is difficult, de Moraes-Ruehsen and Jones [ 1] found that 7% of 300 consecutive women presenting with amenorrhea had premature ovarian failure. Aiman and Smentek [ 11 ] combined the observations of several investigators to estimate the frequency of the disorder among American women. Based on the assumptions that 43 million

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American women were of reproductive age in 1985 and that the incidence of amenorrhea was 3%, they concluded that the frequency of premature ovarian failure is approximately 0.3%, with 129,000 American women being affected in that year. Alper and colleagues [18] estimated that 5 to 10% of women with secondary amenorrhea have this disorder. Coulam and co-workers [19] examined the medical records of 1858 women living in Rochester, Minnesota, in 1950 and calculated that the risk of experiencing menopause prior to age 40 was 0.9%.

IV. ETIOLOGY OF PREMATURE OVARIAN FAILURE de Moraes-Ruehsen and Jones [1] suggested three possible explanations for the early completion of atresia that they believed to exist in women with hypergonadotropic amenorrhea and premature ovarian failure: (1) decreased germ cell endowment, (2) accelerated atresia, and (3) postnatal germ cell destruction. Because these possibilities cannot apply to individuals in whom many follicles still remain, some block to gonadotropin action in ovarian follicles must exist. In view of data that even postmenopausal women may have a few remaining follicles [20,21] and previously cited information that follicle number decreases rapidly in the last several months before menopause [2], it is possible that a few women who are truly "perimenopausal" will ovulate and even conceive. Among the various causes of hypergonadotropic amenorrhea that can be identified, it is clear that some are present only in those who have no oocytes whereas others may have the potential for ovulation and spontaneous pregnancy. Possible causes of hypergonadotropic amenorrhea and premature ovarian failure are listed in Table II.

A. Genetic and Cytogenetic Causes 1. ~ F A M I L I A L ~ P R E M A T U R E OVARIAN F A I L U R E Several reports have described individual families with vertical transmission of premature ovarian failure, implying autosomal dominant, sex-linked inheritance [22-24]. In such families the etiology might well be due to one of the three reasons postulated by de Moraes-Ruehsen and Jones [ 1]. It certainly is well recognized that the number of oocytes differs widely among various strains of mice [25]. Moreover, both individual mice [3,25] and humans [15,26,27] have markedly different rates of follicular atresia. It is possible that the etiology of premature ovarian failure that occurs in some individuals with the neurological disorder myotonia dystrophica also is due to a decreased endowment in germ cell number or to accelerated atresia [28].

TABLE II Causes of Hypergonadotropic Amenorrhea Genetic and cytogenetic etiologies "Familial" premature ovarian failure FSH receptor mutations Fragile X premutations Structural alterations or absence of an X chromosome Trisomy X with or without mosaicism In association with myotonia dystrophica Enzymatic defects 17ce-Hydroxylase deficiency Galactosemia Physical insults Ionizing radiation Chemotherapeutic agents Viral infection Cigarette smoking Surgical extirpation Immune disturbances In association with other autoimmune disorders Isolated Congenital thymic aplasia Defects in gonadotropin structure or actions (genetic?) Secretion of biologically inactive gonadotropin ce or fl subunit defects Gonadotropin receptor or postreceptor defects Circulating FSH-binding inhibitors Idiopathic

Molecular biology has provided explanations for some familial cases of hypergonadotropic ovarian failure. Aittom~iki and colleagues [29] demonstrated a mutation in exon 7 of the FSH receptor gene located on chromosome 2p in which an Ala to Val substitution at residue 189 is present in six Finnish families with multiple affected women. The histological appearance of the ovaries of women with this mutation showed hypoplasia with few primordial follicles [30]. None had the appearance of complete ovarian dysgenesis with streak ovaries. That such FSH receptor abnormalities are rare causes of ovarian failure is suggested by a study in Great Britain failing to identify any such mutations in 30 women with sporadic premature ovarian failure and in 18 women with familial premature ovarian failure [31 ]. It is now clear that women carrying one X chromosome with a fragile X premutation have an increased prevalence of premature menopause [32]. Fragile X syndrome is the most common inherited form of male retardation and is caused by an expansion of a trinucleotide repeat sequence in the first exon of the FMR1 gene (Xq27.3). The full fragile X mutation occurs when the number of trinucleotide repeats exceeds 200, when gene transcription fails and the FMR1 protein is not expressed [33]. In normal individuals there are less than 50 trinuleotide repeats at this fragile site and a fragile X premutation is said to occur when between 50 and 200 tri-

CHAPTER8 Premature Ovarian Failure nucleotide repeats are present. At least one other group has reported that fragile X premutations occur at least 10 times more frequently in women with premature ovarian failure than in the general population [34]. However, no causal link has yet been shown. Because the FMR1 gene is expressed both in the brain and in the gonad, the fragile X premutation may not be as "innocent" as presumed [35].

139 moieties on gonadotropin molecules are altered such that they are biologically inactive or their metabolism is changed. Unfortunately, this postulate does not coincide with experimental data suggesting a direct effect of galactose on the oocyte. Pregnant rats fed a 50% galactose diet delivered pups with significantly reduced numbers of oocytes, apparently due to decreased germ cell migration to the genital ridges [48].

2. GONADAL DYSGENESIS

Studies of individuals with gonadal dysgenesis have documented that two intact X chromosomes are needed for normal maintenance of oocytes. It is known that the gonads of 45,X fetuses contain the normal complement of oocytes at 20 to 24 weeks of fetal age, but that those oocytes rapidly undergo atresia so that virtually none remains at birth [36]. Structural abnormalities of the X chromosome also can affect ovarian function and have been found in women with premature ovarian failure [7,11,37]. Even documented submicroscopic deletions of a portion of X chromosome can apparently lead to premature ovarian failure [38]. 3. TRISOMY X WITH OR WITHOUT MOSAICISM

An excess of X chromosomes also may be found in some women who develop premature menopause [39]. Patients identified to date have developed normal secondary sex characteristics and only later presented with secondary hypergonadotropic amenorrhea. Reports of the triple-X syndrome associated with immunoglobulin deficiency [40] and Marfan syndrome [41], together with the observation that control of T cell function may be related to the X chromosome [42], suggest a possible association between immunological abnormalities and triple-X females with premature ovarian failure.

C. P h y s i c a l Insults Destruction of oocytes by any of several environmental insults, including ionizing radiation, various chemotherapeutic agents, certain viral infections, and even cigarette smoking, may occur [49]. 1. IONIZING RADIATION

Approximately 50% of individuals who receive 400 to 500 rads to the ovaries over 4 to 6 weeks, as may occur in treatment for Hodgkin's disease, will develop permanent hypergonadotropic amenorrhea [ 17,50,51 ]. For any given dose of radiation, the older the woman, the greater her likelihood of developing amenorrhea. It appears that a total of 800 rads is sufficient to result in permanent sterility in all women [50,51]. That amenorrhea following radiation therapy is not always permanent was reported by Jacox in 1939 [52]. The transient nature of amenorrhea in some women suggests that some follicles may be damaged but not destroyed by relatively low doses of radiation. Although surgical transposition of the ovaries outside the field of irradiation is now common practice, it is not clear just how fertile such women ultimately are. 2. CHEMOTHERAPEUTIC AGENTS

B. E n z y m a t i c D e f e c t s 1. 17a-HYDROXYLASE DEFICIENCY The rare women with deficiency of the 17a-hydroxylase enzyme are identified easily because of the associated findings of primary amenorrhea, sexual infantilism, hypergonadotropinism, hypertension, hypokalemic alkalosis, and increased circulating levels of deoxycorticosterone and progesterone [43-45]. Ovarian biopsies have revealed numerous large cysts and follicular cysts, with complete failure of follicular maturation [45]. 2. GALACTOSEMIA

Women with galactosemia develop amenorrhea with elevated gonadotropin levels even when treatment with a galactose-restricted diet begins at an early age [46,47]. Although the etiology of premature ovarian failure in galactosemia is unknown, it is tempting to speculate that the carbohydrate

As more and more young women treated for childhood malignancies, especially leukemias and lymphomas, survive long-term, it has become obvious that chemotherapeutic agents may produce temporary or permanent ovarian failure [17,53-57]. Alkylating agents, particularly cyclophosphamide, are most likely to affect reproductive function. In general, the younger the woman at the time of therapy, the more likely it is that ovarian function will not be compromised by chemotherapy. It may well be that it is the number of oocytes present at the time of therapy that determines if ovarian function will be compromised: the greater the number of oocytes, the more likely it is that normal ovarian function will persist. The frequency of congenital anomalies does not appear to be increased in the children of women previously treated with chemotherapy [58]. It has been suggested, however, that one agent, dactinomycin, may be associated with an increased risk of congenital heart disease, and further studies in this area are clearly needed.

1

4

0

R

O

B

E

3. VIRAL AND OTHER AGENTS

Although several viruses are believed to have the potential to cause ovarian destruction, confirming that such is the case in humans is difficult. Morrison and colleagues [59] reported three presumptive cases in which "mumps oophoritis" preceded premature ovarian failure, including a mother and daughter pair, in which the mother experienced mumps parotitis and abdominal pain during pregnancy just prior to delivery of the daughter. Although there is no evidence that cigarette smoking will lead to premature menopause, data do exist documenting that cigarette smokers experience menopause several months earlier than do nonsmokers [60].

D. I m m u n e D i s t u r b a n c e s Any role for immune disturbances in the etiology of hypergonadotropic amenorrhea remains controversial. It is clear that several autoimmune abnormalities may occur in association with hypergonadotropic amenorrhea (Table III). However, the prevalence of autoimmune abnormalities in normal women is unknown, and it may be that it is not increased in ovarian failure. Moreover, it is not clear if autoimmune disturbances play any role in the development of hypergonadotropic amenorrhea. As is characteristic for other autoimmune disturbances, the ovarian "failure" in affected women may wax and wane, and pregnancies may occur, at least early in the disease process. In a literature review tabulating 380 cases of premature ovarian failure, LaBarbera and colleagues [61] noted that 17.5% had a definite associated autoimmune disorder. Additional evidence that hypergonadotropic amenorrhea may have an autoimmune etiology in at least some cases has been provided by sporadic case reports documenting return of ovarian function following either immunosuppressive therapy or recovery from an autoimmune disease [62-64].

TABLE III

T

Adapted from Rebar et

al.

[7] and LaBarbera et

Juvenile rheumatoidarthritis Keratoconjunctivitisand Sj6gren's syndrome Malabsorption syndrome Myasthenia gravis Polyendocrinopathies(type I, type II, and unspecified) Primary biliary cirrhosis Quantitative immunoglobulinabnormalities Rheumatoid arthritis Systemic lupus erythematosus Thyroid disorders, including Graves' disease and thyroiditis Vitiligo al.

W. REBAR

In a few cases lymphocytic infiltrates suggesting autoimmune dysfunction have been observed in ovarian biopsy specimens [65]. Still other immune abnormalities have been identified in some patients with premature ovarian failure. Enhanced release of leukocyte migration inhibition factor (MIF) by peripheral lymphocytes has been observed following exposure of the lymphocytes to crude ovarian proteins [66,67]. A significant association of early ovarian failure with HLA-DR3 has been noted [68], perhaps suggesting a genetic susceptibility to autoimmunity in some individuals. Several years ago McNatty and colleagues [69] reported complementdependent cytotoxic effects on cultured granulosa cells, as documented by inhibition of progesterone production and cell lysis, in sera from 9 of 23 patients with hypergonadotropic amenorrhea and Addison's disease. Cellular immune abnormalities involving numbers and/or function of peripheral monocytes and of subsets of T cells and B cells have also been noted in women with premature ovarian failure [70]. Indirect immunofluorescence of ovarian biopsy specimens has revealed antibodies reacting with various ovarian components in some patients [71]. Circulating immunoglobulins to ovarian proteins have been detected by immunocytochemical techniques by several investigators [61]. Utilizing a solid-phase, enzyme-linked immunosorbent assay, we have detected antibodies to ovarian tissue in 22% of karyotypically normal women with premature ovarian failure [72,73]. The most thoroughly documented study to the present time remains that of Chiauzzi and colleagues [74], who documented that two patients with ovarian failure and myasthenia gravis had circulating immunoglobulin G that blocked binding of FSH to its receptor. However, it is important to reiterate that ovarian autoantibodies may not be the cause of ovarian failure. Rather, the ovarian failure may result from cell-mediated autoimmunity, and autoantibodies may appear only because of the resultant cell death. However, Anasti and colleagues [75] failed to demonstrate the

Possible Autoimmune Disorders Associated with Premature Ovarian Failure a

Alopecia Anemia, both acquired hemolyticand pernicious Asthma Chronic active hepatitis Crohn's disease Diabetes mellitus Glomerulonephritis Hypoadrenalism (Addison's disease) Hypoparathyroidism Hypophysitis Idiopathic thrombocytopeniapurpura a

R

[61].

CHAPTER8 Premature Ovarian Failure presence of blocking antibodies to LH or FSH receptors in any of 38 premature ovarian failure patients studied. In recent years there has been increasing interest in the relationship between the immune and reproductive systems. Miller and Chatten [76] documented that congenitally athymic girls dying before puberty had ovaries devoid of oocytes on autopsy. Data from our laboratory suggest that the thymus gland may be necessary early in development for normal gonadotropin function. Congenitally athymic mice, well known to develop premature ovarian failure, have lower gonadotropin concentrations prepubertally than do their normal heterozygous littermates [77]. These hormonal alterations, as well as the accelerated loss of oocytes, can be prevented by thymic transplantation at birth [78]. In comparing ovarian development in the rodent to that of the primate, it is essential to recognize that development occurring during the first few weeks of life in the mouse occurs in utero in the human female. Thus, thymic ablation in fetal rhesus monkeys in late gestation is associated with a marked reduction in oocyte number at birth [79]. One possible explanation for the association of thymic aplasia and ovarian failure may be found in our observation that peptides produced by the thymus can stimulate release of gonadotropin-releasing hormone (GnRH) and consequently LH [80]. In recent years it has become evident that organ-specific autoimmunity may be directed against intracellular enzymes, particularly those involved in hormone synthesis [70,81 ]. For example, thyroid peroxidase is a major thyroid autoantigen for autoimmune hypothyroidism, and the 21-hydroxylase enzyme is the foremost autoantigen in Addison's disease. One group has identified 3fi-hydroxysteroid dehydrogenase as an autoantigen in 20% of women with premature ovarian failure [82]. This, too, may merely be an epiphenomenon of ovarian inflammation rather than causal for the development of ovarian failure. From a theoretical viewpoint, identifying patients with an autoimmune etiology for their hypergonadotropic amenorrhea is important because affected patients might be treated effectively early in the disease process before all viable oocytes are destroyed.

141 cases of male pseudohermaphroditism with immunologically active but biologically inactive LH are well documented [84,85]. Altered forms of immunoreactive LH and FSH have been reported in urinary extracts from women with premature ovarian failure compared to those from oophorectomized and postmenopausal women [86], suggesting that metabolism and/or excretion of gonadotropins is altered in some cases of this disorder. However, using two different probes for the fl subunit of FSH (as well as two probes for the FSH receptor gene), one group failed to find any mutations in a small group of patients [87]. These findings do not rule out mutations in other patients or in different portions of the molecule. Interference with F S H action at the ovarian level also might lead to early ovarian failure. Defects in FSH receptor structure (as reported in the Finnish study [29]), FSH receptor antibodies (as noted), competitive inhibitors to FSH binding, or defects in postreceptor systems that mediate hormone action are each theoretically possible. Sluss and Schneyer [88] reported identifying two individuals out of a group of 27 with hypergonadotropic amenorrhea (and intermittent evidence of ovarian function) whose sera had lowmolecular-weight FSH receptor-binding activity that was an antagonist of FSH action. Even when this inhibitor was removed from the serum, however, FSH levels were elevated in both patients. These studies cannot eliminate the possibility that this FSH binding inhibitor is merely produced secondarily to development of ovarian FSH insensitivity. Other possible defects in gonadotropin action remain to be identified. Clearly all of these disorders might well be genetic abnormalities.

E

Idiopathic

The diagnosis of idiopathic causes of premature ovarian failure is one of exclusion, but presently no definitive etiology is identified in most cases of hypergonadotropic amenorrhea. It is likely that additional causes of this entity will be recognized as more is learned about premature ovarian failure.

E. D e f e c t s in G o n a d o t r o p i n S t r u c t u r e or A c t i o n It is possible to envision that abnormal structure, secretion, or metabolism of gonadotropins in some women forms the basis for early ovarian failure. The concept of secretion of altered molecular forms of FSH with reduced or absent biological activity leading to accelerated follicular atresia even as a rare cause for premature ovarian failure is appealing. This is especially true given evidence that normal concentrations of gonadotropins are required early in development: fetal hypophysectomy in rhesus monkeys leads to the newborn having no oocytes in their ovaries [83]. Moreover,

G. R e s i s t a n t O v a r y S y n d r o m e : A Term No Longer Useful As originally defined, the resistant ovary, or "Savage," syndrome was found in young amenorrheic women with (1) elevated peripheral gonadotropin levels, (2) normal but immature follicles in the ovaries, (3) a 46,XX karyotype, (4) mature secondary sex characteristics, and (5) decreased sensitivity to stimulation with exogenous gonadotropin [89]. Individuals fulfilling these criteria might easily have any

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of several different etiologies for their hypergonadotropic amenorrhea. Moreover, regardless of the etiology, these features may be common to all individuals with hypergonadotropic amenorrhea at some time during the disease process prior to final loss of all oocytes. As a consequence, use of the term resistant ovary will become less and less useful as understanding of ovarian failure increases, and the terminology is already of severely restricted value.

V. EVALUATION OF PATIENTS WITH HYPERGONADOTROPIC AMENORRHEA Young women with hypergonadotropic amenorrhea should be evaluated to identify (1) specific, potentially treatable causes and (2) other potentially dangerous associated disorders. A thorough history and physical examination are always warranted. A maturation index and evaluation of the cervical mucus may help determine if any endogenous estrogen is present. Simple laboratory tests should be performed to exclude thyroid disease, hypoparathyroidism, adrenal insufficiency, diabetes mellitus, and other forms of immune dysfunction. The extent of such testing is unclear, but a reasonable set of tests is listed in Table IV. In addition to the clinical evaluation of estrogen status, measurement of circulating LH, FSH, and estradiol concentrations on more than one occasion may help determine if any functional follicles are present. If the estradiol concentration is greater than 50 pg/ml or if the LH level is greater than the FSH (in terms of mIU/ml) in any sample, then a few viable oocytes still must be present. Irregular uterine bleeding, indicative of continuing estrogen production, also suggests the presence of remaining functional oocytes. Identifiable follicles on transvaginal ultrasonography also can be used to identify women with remaining TABLE IV Evaluation of Hypergonadotropic Amenorrhea in Young Women a Complete history and physical examination Maturation index Karyotype (? limited to women with onset before age 30) Complete blood count with differential, sedimentation rate, total serum protein and albumin/globulin ratio, rheumatoid factor, antinuclear antibody Fasting blood glucose, serum calcium and phosphorus, evaluation of adrenal status T 4, thyroid-stimulating hormone, antithyroglobulin, and antimicrosomal antibodies or antithyroid-stimulating immunoglobulins Serum LH, FSH, and estradiol on at least two occasions Evaluation of bone mineral density a Adapted from Rebar et al. [7].

oocytes [90] and are present in a large percentage of affected women [ 12,13,90]. If available, testing of the patient's serum for antibodies to endocrine tissues, including ovary, may be of some value. The difficulty with this recommendation is the fact that there are no readily available tests for antibodies to any specific antigens. In addition, as noted previously, antibodies may develop because of cytotoxicity in the ovary and may not cause ovarian failure. In which patients chromosomal studies should be conducted is also unclear. It would seem prudent to obtain a karyotype in women with the onset of hypergonadotropic amenorrhea prior to age 30 to identify those with various forms of gonadal dysgenesis, individuals with mosaicism, those with trisomy X, and those with a portion of a Y chromosome. If a Y chromosome is present, gonadal extirpation is warranted because of the increased risk of malignancy [91-93]. Chromosomal evaluation also may be warranted to rule out familial transmission in women who develop hypergonadotropic amenorrhea after the birth of daughters. Although controversial, ovarian biopsy does not appear justified in women with hypergonadotropic amenorrhea and a normal karyotype. It is not clear how the results would alter therapy. Aiman and Smentek [ 11 ] reported one of their two patients who eventually conceived had no oocytes present on biopsy. Similarly, Rebar and Connolly [ 16] noted that two of eight subsequent pregnancies among 97 women with secondary hypergonadotropic amenorrhea occurred in women with no follicles present in ovarian tissue obtained by laparotomy. As also noted by Aiman and Smentek [11], if five sections of an ovarian biopsy are examined and each is 6/zm thick, then the presence of follicles is sought from a sample representing less than 0.15% of an ovary measuring 2 • 3 x 4 cm. Thus, the absence of follicles on biopsy may not be representative of the remainder of the ovary. Moreover, affected individuals almost always require estrogen replacement regardless of the results of the biopsy. Evaluation of bone density appears warranted in women with hypergonadotropic amenorrhea because of the high prevalence of osteopenia [13,16,90]. Periodic assessment may be warranted, regardless of therapy, to assess the rapidity of bone loss. Similarly, monitoring patients for the development of autoimmune endocrinopathies may be warranted even if all tests are normal when the patient is first evaluated; development of other disorders after diagnosis of hypergonadotropic amenorrhea does occur [16].

VI. THERAPY It is reasonable to treat all young women with hypergonadotropic amenorrhea with exogenous estrogen regardless of whether they are interested in childbearing. The accelerated bone loss frequently accompanying this disorder can be

CHAPTER 8 Premature Ovarian Failure prevented by administration of exogenous estrogens [90]. So, too, may the increased risk of cardiovascular disease present in women with estrogen deficiency [94]. Although it also appears that women with premature ovarian failure are at reduced risk of breast cancer [95] (and probably venous thrombosis), the hope is that administration of exogenous estrogen merely returns these relative risks to those found in normal premenopausal women. In addition, almost all spontaneous pregnancies in this disorder occur during or following estrogen administration [16,96]. Even with exogenous estrogens, however, the probability of spontaneous pregnancy appears to be less than 10%. The pregnancy rate is low despite the fact that one-fourth or more of women ovulate after the diagnosis of hypergonadotropic amenorrhea is made. Because of the possibility of pregnancy, women taking exogenous estrogens in any form, even as part of oral contraceptive agents, should be advised to contact their physician if they develop any signs or symptoms of pregnancy or do not have withdrawal bleeding. Although it may not be necessary to advise the use of barrier forms of contraception, the possibility of pregnancy must be discussed. Either oral contraceptives or sequential estrogen-progestin therapy may be utilized, but sequential therapy is more physiologic. It is important to remember that these young women may require twice as much estrogen as do menopausal women to alleviate signs and symptoms of hypoestrogenism. Several isolated case reports have suggested that ovarian suppression with estrogen or a GnRH agonist followed by stimulation with human menopausal gonadotropin may be efficacious in inducing ovulation and allowing conception [97-100]. Most of these reports emanate from one group of investigators. Larger studies suggest that the possibility of successful ovulation induction and pregnancy is small indeed and may be no greater than what concurs spontaneously in these patients [ 14,16,101 ]. I n v i t r o fertilization involving oocyte donation clearly provides individuals with hypergonadotropic amenorrhea the greatest likelihood of bearing children. The first successful case of oocyte donation in humans was reported in 1984. A young woman with ovarian failure was given oral estradiol valerate and progesterone pessaries to prepare the endometrium for transfer of a single donated oocyte following fertilization with her husband's sperm [102]. Since then, several programs utilizing oocyte donation have been successful because of (1) improvements in transvaginal ultrasonography, allowing follicular aspiration and oocyte collection without surgery, (2) improvements in success with embryo cryopreservation and subsequent embryo transfer to the donor at a later time, and (3) improved ability to synchronize artificial cycles in the recipient with the hyperstimulation cycles in the donor, generally by use of GnRH agonists [103-105]. A number of replacement protocols have been developed for the donor with hypergonadotropic amenorrhea, including use of oral, transvaginal, and transdermal administration of

143 estradiol and oral, and transvaginal and intramuscular administration of progesterone. If pregnancy develops from the transferred embryo, given the absence of functional gonads in the recipient, exogenous supplementation with estradiol and progesterone must be continued until placental production of progesterone is well established. Success rates generally have exceeded those observed in standard in v i t r o fertilization programs [103-106]. Thus, oocyte donation offers the possibility of pregnancy to all women with premature ovarian failure so long as a normal uterus is present.

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ing in vitro fertilization and embryo donation in a patient with primary ovarian failure. Nature (London) 307, 174-175. Chan, C. L. K., Cameron, I. T., Findlay, J. K., Healy, D., Lecton, J. F., Lutjen, P. J., Renou, P. M., Rogers, P. A., Trounsen, A. O., and Wood, E. C. (1987). Oocyte donation and in vitro fertilization: Clinical state of the art. Obstet. Gynecol. 42, 350-362. Sauer, M. V., and Paulson, R. J. (1989). Oocyte donation for women who have ovarian failure. Contemp. Obstet. Gynecol. November, pp. 125-135. Lydic, M. L., Liu, J. H., Rebar, R. W., Thomas, M. A., and Cedars, M. I. (1996). Success of donor oocyte IVF-ET in recipients with and without premature ovarian failure. Fertil. Steril. 65, 98-102. Rebar, R. W., and Cedars, M. I. (1994). Hypergonadotropic amenorrhea. In "Ovulation Induction: Basic Science and Clinical Advances" (M. Filicori and C. Flamigni, eds.), pp. 115-121. Elsevier, Amsterdam.

~HAPTER

Perimenopausal Changes in FSH, the lnhibins, and the Circulating Steroid Hormone Milieu HENRY G.

BURGER

Prince Henry's Institute of Medical Research, Clayton, Victoria 3168, Australia

I. Introduction and Definitions II. Current Concepts of Ovarian Physiology as a Function of Age III. Major Longitudinal Studies of Steroids in Relation to the Final Menstrual Period

IV. Hormonal Studies during the Menopausal Transition V. Conclusions References

strual cycles and have no symptoms of approaching menopause. Studies of the hormonal changes occurring during the perimenopause have been based on various experimental designs and definitions. In some instances hormonal changes have been recorded as a function of age with little attention paid to menstrual cycle status [4,5]. In the few longitudinal studies reported, the FMP has been used as a reference point with hormonal changes described in terms of time intervals before and after that point [6-9]. Very few studies have reported on hormone changes in relation to changes in menstrual cycle characteristics, such as the first self-reported change in the amount of menstrual flow, in the frequency of menstruation, or in the combination of changes in flow and frequency. This approach has been adopted in the Melbourne Women's Midlife Health Project [10,11 ], for which data will be provided below. The changes reported, particularly from longitudinal studies, in circulating concentrations of estradiol (E2) and

I. INTRODUCTION

AND DEFINITIONS The World Health Organization has defined the menopause as the permanent cessation of menstruation resulting from loss of ovarian follicular activity [1 ]. The perimenopause is defined as that period which commences when the first features of approaching menopause begin until at least 1 year after the final menstrual period (FMP). The term menopausal transition has been applied to that portion of the perimenopause which ends with the FMP [2]. The menopausal transition as studied in a group of North American women had a duration of approximately 4 years [3]. Thus the overall average duration of the perimenopause is 5 years. It is strongly recommended that the term perimenopause be used in this way, and not applied loosely to women in their forties and early fifties who continue to have regular menM E N O P A U S E : B I O L O G Y AND PATHOBIOLOGY

147

Copyright 9 2000 by Academic Press. All rights of reproduction in any form reserved.

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in relation to the occurrence of the FMP are examined in this chapter. The sparse data available on the circulating inhibins are reviewed and data from a longitudinal community-based study of women in the menopausal transition are presented. Menopausal status has been defined in terms of menstrual cycle status. Changes in progesterone and in androgens are reviewed briefly. estrone (El),

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II. CURRENT CONCEPTS OF OVARIAN PHYSIOLOGY AS A FUNCTION OF AGE Circulating concentrations of follicle-stimulating hormone (FSH), luteinizing hormone (LH), E2, progesterone (P) and the inhibins during the normal menstrual cycle have been described [12,13] (Figs. 1 and 2). The menstrual cycle

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CHAPTER9 Perimenopausal Hormone Changes

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women were compared with a young control group showed increased levels of urinary estrogens and a fall in urinary pregnanediol excretion in the luteal phase of the cycle, despite the continuance of regular cyclicity [14]. The occurrence of unchanged or even elevated E 2 concentrations despite rising FSH is most plausibly explained by a decline in circulating INH concentration with age, as has been demonstrated in one study [15]. It is probable that the decline is particularly in INH-B [16]. Luteal-phase P concentrations showed no change with age in some studies and, as indicated, a decline in others. Follicular-phase LH concentrations rise only in the oldest group of regularly cycling women. Measurement of E 2 and INH in both ovarian veins of regularly cycling women has shown that E 2 concentrations are significantly higher in the vein draining the ovary containing the dominant follicle as compared to the contralateral ovarian vein, whereas INH concentrations are equivalent in both ovarian veins regardless of the side of the dominant follicle [ 17]. Although both E 2 and inhibins have been shown to be products of the ovarian granulosa cell, differential mechanisms appear to govern the secretion of the two inhibins. INH-B appears to be a product of the cohort of developing follicles, whereas INH-A together with E 2 is derived particularly from the dominant follicle [13,18]. It is postulated that the circulating concentration of INH-B may reflect the number of follicles recruited from the primordial pool, a number that decreases with increasing age [19]. The ability of the dominant follicles of older women to produce E 2 and INH-A appears to remain intact while regular cycles continue [ 12,16]. This has also been demonstrated in the E 2 response to ovarian hyperstimulation for the purposes of in vitro fertilization, with the response being age invariant [20]. This contrasts with the situation for production of total INH, which declines with increasing age in response to ovarian hyperstimulation [20]. In vitro studies of granulosa cells obtained at oocyte aspiration for the purposes of in vitro fertilization have also shown a diminished ability of granulosa cells from older women to produce INH-A in comparison with those from younger women [21]. Estradiol plays a central role in female reproductive function and has been described previously as the physiological basis of the fertile period [22]. Teleologically, it could be hypothesized that preservation of E 2 secretion would be desirable for as long as possible in the human female. Consequences of the loss of E 2 include the development of estrogen deficiency symptoms and undesirable health outcomes such as loss of bone and increased susceptibility to atherosclerosis and myocardial infarction. The decreased INH-B production in older women with elevated FSH concentrations in the follicular phase of the cycle can be postulated to increase secretion of FSH, which in turn provides increased drive to maintain E 2 secretion as the overall ovarian follicle number decreases.

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FIGURE 2 Plasma concentrations of (a) inhibin A and inhibin B, (b) estradiol and progesterone, and (c) LH and FSH, during the female menstrual cycle. Data displayed with respect to the day of midcycle LH peak. Mean concentrations are shown ___SE. From [13], Groome, N. P., Illingworth, P. J., O'Brien, M., Rodger, P. A. L., Rodger, F. E., Mather, J. F., and McNeilly, A. S. (1996). Measurementof dimeric inhibin B throughout the human menstrual cycle. J. Clin. Endocrinol. Metab. 81, 1401-1405.

is characterized by relatively stable and low values for E 2 and inhibin A (INH-A) during the first half of the follicular phase, with a subsequent rise to a midcycle peak 24 hr prior to the midcycle ovulatory luteinizing hormone surge. After abrupt falls, there are secondary peaks of E 2 and INH-A secretion, parallel to that of P, during the luteal phase, with a subsequent fall leading to the onset of menses. In contrast, inhibin B (INH-B) concentrations rise and fall closely related to those of FSH during the early follicular phase, show a midcycle peak, and subsequently decline to their lowest points during the luteal phase (Fig. 2). The pattern of E 2 and INH-A is preserved when levels are examined as a function of increasing age in regularly cycling women. Thus in a large cross-sectional study of women ranging from 24 to 50 years of age [ 12], all cycling regularly, early follicular-phase concentrations of E 2 w e r e unchanged and in fact slightly higher in the oldest group of subjects, despite a progressive rise in circulating FSH (Fig.l). Another study in which older

150

HENRY G. BURGER

III. MAJOR LONGITUDINAL STUDIES OF STEROIDS IN RELATION TO THE FINAL MENSTRUAL PERIOD A small number of studies have examined the changes in circulating E 2 and E 1, and in some cases in androgens, in relation to FMP [6-9]. Such changes do not appear to be due to changes in steroid metabolism [23]. Most data relate to follicular-phase steroid hormone concentrations, but several have examined circulatory steroids, including P, during the luteal phase. The data reported by Rannevik et al. [9] are typical of the few published investigations of gonadotropin and estrogen around the FMR The study group consisted of 160 women from the Malm6 Perimenopausal Project. It had a 12-year duration and 152 of the 160 women were included over that time period. The mean age at the onset of menopause was 52.1 years. Estradiol concentrations were measured for 7 years prior to the FMP and remained relatively constant (means of 461-515 pmol/liter) until 6 months prior to the FMR In 154 observations 1 to 6 months prior to the FMP, mean E 2 was still 383 pmol/liter but had fallen to 182 pmol/liter 1 to 6 months after the FME A gradual further decline ensued to 171 pmol/liter in the 7 to 12 months after the FMP, with levels reaching 72 pmol/liter 97 to 108 months after that reference point. Concentrations of E~ behaved similarly, with a very small decrease in the 1 to 6 months before the FMP, a moderate decrease 1 to 6 months afterward (from 299 to 216 pmol/ liter), and a gradual decrease to 133 pmol/liter, 97 to 108 months after the FMP (Fig. 3). No data were provided on geometric mean levels in these subjects nor on the relationship between hormonal and self-reported menstrual cycle

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status other than FMR It was, however, noted that during the premenopausal period there was an increasing frequency of inadequate luteal function, with low P concentrations. Similar data for the period after FMP were reported by Longcope et al. [7]. Data based on samples collected during the luteal phase were also broadly similar [24], with E 2 preserved until a few months before the onset of amenorrhea and then gradually falling thereafter, and with P levels falling progressively from 4 years prior to the onset of amenorrhea. From these data it appears that the FMP occurs during a time of relatively steeply falling E 2 concentrations, though substantial further falls occur for many months after that major selfreported endpoint.

IV. HORMONAL STUDIES DURING THE MENOPAUSAL TRANSITION A. FSH and Estradiol Relatively few studies have focused specifically on the endocrinology of the menopausal transition, when the most noteworthy characteristic is significant hormonal variability. A landmark study was that of Sherman and Korenman [25], who reported on 50 complete menstrual cycles in 37 women. Ten women aged 18 to 30 years, with a history of regular cycles, served as a control group. Six cycles were examined in regularly cycling women aged 46 to 51 years, in which it was noted that the follicular phase of the cycle was shorter than in the younger women and that E 2 concentrations during the early follicular phase were significantly lower than those observed in the younger women, but that serum FSH was strikingly increased throughout the cycle, despite the occurrence of E 2 concentrations that might have been expected to suppress its secretion. Daily hormone concentrations were also measured in 2 women, one aged 49 and one aged 50 who were clearly in the menopausal transition. Two of the cycles studied were anovulatory, but were nevertheless characterized by increasing E 2, and initially postmenopausal values of LH and FSH, which subsequently fell with the rise of E 2. An anovulatory cycle was followed by a cycle that demonstrated evidence of follicular maturation. Metcalf and colleagues [6,26] examined the excretion of FSH, LH, estrogens, and pregnanediol in weekly urine samples collected for 14 to 87 weeks from 31 perimenopausal women aged 36 to 55 years. Their study concentrated particularly on the gonadotropin changes, but wide fluctuations in estrogen excretion were noted. These authors stated "about the only conclusion that can be made with confidence concerning pituitary-ovarian function in individual perimenopausal women is that it is unsafe to generalize." In a more recent paper, Metcalf [27] concluded "in older women, a good menstrual history is probably the single most useful measure of ovarian status." Hee et al. [28] confirmed the

CHAPTER 9 Perimenopausal Hormone Changes

151

variability of perimenopausal E 2 concentrations and added data on INH in a small longitudinal study of three volunteer women who had developed irregular cycles at age 4 5 - 4 6 years. Abrupt decreases in E 2 and INH into the postmenopausal range were followed by values characteristic of reproductive-aged women.

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B. Studies Including Inhibins The Melbourne Women's Midlife Health Project is based on a cross-sectional survey of a randomly selected population sample of 2001 Melbourne women, all Australian born and between 45 and 55 years at the time of their interview in May, 1991 [29]. A longitudinal study of 437 women was undertaken to examine many aspects of the menopausal transition. The data from the first year of this longitudinal study have been subjected to cross-sectional analysis in terms of menstrual cycle history. Of the subjects, 27% reported no change in menstrual frequency or flow, 23% reported a change in flow with no change in frequency, 9% reported a change in frequency without change in flow, 28% reported a change in both frequency and flow, and by the time of blood sampling 13% reported that at least 3 months had elapsed since their last menstrual period. Mean age increased from 48.5 years in the first group to 51.4 years in the last group. The data are shown in Fig. 4. Although unadjusted E 2 values were slightly lower in the groups experiencing a change in frequency or a change in frequency and flow (88 and 82% of those without any change), the only statistically significant decline in E 2 occurred in those who had no menses for at least 3 months, when the geometric m e a n E 2 concentration was 42% of that observed in the first group. When the E 2 data were adjusted for age and body mass index, the only significant change was again in the group with 3 months or more of amenorrhea, with the E 2 geometric mean being 54% of that of the Group 1 women. It must be emphasised that there was a broad spread of E 2 values, with some being > 1500 pmol/liter. Such high levels may reflect hyperstimulation of granulosa cells by elevated FSH levels, and could give rise to symptoms of breast fullness and fluid retention. Immunoreactive inhibin concentrations were significantly lower (71% of those in the first group) in those experiencing a change in frequency and flow and had fallen to 38% in those with 3 months or more of amenorrhea. Following adjustment for age and body mass index, only the change in the final group was significant, with the adjusted geometric mean being 53 % of that in Group 1. These data, particularly when examined without adjustment for age and body mass index, suggested that the decreases in inhibin levels occurred before decreases in E2, consistent with the hypothesis that declining concentration inhibin provides a mechanism for allowing FSH to rise, so as to maintain early follicular-phase

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levels relatively intact. When thie data were analyzed without reference to menstrual cycle status and purely as a function of age, a marked decline in INH was observed, inverse to rising FSH levels, whereas E 2 w a s relatively constant until the age of approximately 51 or 52 years, when it too declined steeply. A further cross-sectional analysis of data was undertaken on 110 subjects aged 4 8 - 5 9 years in the third year of the longitudinal-phase Melbourne Women's Midlife Health Project [ 11 ] (Fig.5). Subjects were divided into those calledpremenopausal, with no reported change in menstrual cycle pattern; early perimenopausal, with a reported change in cycle frequency in the preceding year but a bleed in the preceding 3 months; late perimenopausal, with no menses in the preceding 3-11 months; and postmenopausal, with no menses for more than 12 months. The hormone concentrations in the premenopausal subjects were used as reference points for the other groups. Early perimenopausal subjects had significantly lower levels of INH-B (13.5 ng/liter compared with 48 ng/liter) in the presence of a small, statistically nonsignificant rise in FSH (21.4 compared with 13.5 E2

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fluctuate widely in individual women during the menopausal transition. Grouped data show that mean changes in hormone levels become significant around the FMP, with a decrease in INH-B concentration in early perimenopausal women being the most important and significant initial endocrine event at that time.

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F I G U R E 5 Geometric mean levels (with lower 95% confidence intervals) of FSH, IR-INH, INH-A, INH-B, and E 2 as a function of menopausal status in a group of 110 women from the Melbourne Women's Midlife Health Project. Values with the same superscript (a, b, or c) not statistically different: values with differing superscripts are different, P < 0.05. IR, Immunoreactive. From Burger et al. [ 11 ], with permission of Blackwell Science Ltd.

IU/liter). There was no significant change in E 2 or INH-A. In late perimenopausal subjects, INH-A had fallen (geometric mean 4.2 ng/liter compared with 9.6 ng/liter), whereas INH-B was unchanged and FSH had risen signficantly to 72.2 IU/liter. E 2 also fell significantly to 89 pmol/liter (compared with 306 pmol/liter in the premenopausal group). The postmenopausal subjects showed no further significant changes in the peptide hormones or in FSH but E 2 fell further to 41 pmol/liter. A significant inverse correlation was noted between FSH and E 2, FSH and INH-A, and FSH and INH-B. The data were interpreted as consistent with negative feedback roles for both the dimeric inhibins and E 2 as contributors to the regulation of FSH secretion during the menopausal transition. Overall, circulating E 2 and inhibin concentrations may

Faddy and Gosden [30] have developed a mathematical model to describe the rates of growth and death of follicles in human ovaries in women between 19 and 50 years of age. Their study was based on the number of follicles at three successive stages of development, counted in histological sections of ovaries from 52 regularly cycling normal women. Their model predicts that the number of follicles growing from a stage at which there are at least two layers of granulosa cells surrounding an oocyte that has increased in size will decrease from 51 per day at age 2 4 - 2 5 years to only 1 per day at age 4 9 - 5 0 years. It could be hypothesized that circulating INH-B concentrations may provide an index of the numbers of those follicles progressing from that relatively early stage of development and that INH-B levels decline when there has been a substantial decline in numbers proceeding to maturation, late in reproductive life. In contrast, providing that a competent follicle is able to develop to dominance, or to a size sufficiently large to maintain production of steroids, E 2 and INH-A secretion would be expected to be relatively preserved. It has also been shown that the ability of granulosa cells from the follicles of older women to respond to human chorionic gonadotropin (hCG) stimulation by secretion of INH declines markedly, as does their secretion of P [31 ]. In addition, as noted above, the granulosa cells from older women in fact secrete lower amounts of INH-A into a culture medium than do those from younger women, though whether such secretory properties of granulosa cells obtained after ovarian hyperstimulation reflect the secretory abilities of single dominant follicles in older women could be questioned [21]. Thus it is hypothesized that declining numbers of follicles with declining INH-B levels lead to a reciprocal rise in FSH, which in turn stimulates the rate of follicular development, and maintains the capacity of the ovary to develop a dominant follicle until late in reproductive life. Preservation of that capacity results in preservation of circulating E 2 and INH-A within the normal range. The preserved E 2 is postulated to maintain quality of life and bony and vascular health. Validation of this hypothesis will require measurements of dimeric inhibin as a function of aging during the menopause transition, and comparison of changes in its levels with those of E 2, studies that have been reported [ 11 ] or are in progress. Ultrasound monitoring of the ovary would be necessary to assess follicular growth. Differential measurements of INH-A and INH-B have shed further light on pituitary-ovarian relationships during this phase of waning reproductive function.

CHAPTER 9 Perimenopausal Hormone Changes

153

C. Progesterone

75 e-v

It is well known, from studies in which basal body temperature has been used as a marker of ovulatory function, that anovulatory cycles become more prevalent as a function of increasing a g e m 3 - 7 % of cycles were found to be anovulatory between ages 26 and 40 years, compared to 1215% between 41 and 50 years [32]. A large study of lutealphase P concentrations [24] noted that the frequency of nondetectable P gradually increased as the FMP approached. Interestingly, these authors noted that in 11.5 % of their cases the endometrium was found to be secretory, whereas P concentrations were below 3 nmol. This discordance was seen most frequently in the period from 3 to 1 years before the FMP. The lack of a luteal-phase rise in P is a striking feature of the postmenopause compared with the reproductive period. Rannevik et al. [9] reported that the frequency of cycles with P values indicative of ovulation (concentrations > 10 nmol/liter) decreased from about 60% to less than 10% during the 6 years preceding the FMP. Ovulatory P concentrations were found in 62.2% of women 72 to 61 months premenopausal, and in 4.8% who were 6 to 0 months premenopausal, whereas all serum P measurements were less than 2 nmol/liter postmenopausally. There is some controversy regarding the maintenance of P secretion during the luteal phase in older regularly cycling women. Lee et al. [ 12] showed that P secretion was well preserved in a group of regularly cycling women aged 46 to 50 years, whereas Santoro et al. [14] showed decreased urinary pregnanediol excretion in a group of regularly cycling women aged 43 to 52 years, compared with women aged 19 to 38 years.

D. A n d r o g e n s Variable findings have been reported in regard to the changes in circulating androgens in relation to the FMP. Rannevik et al. [9] reported a small but significant decline in testosterone (T), androstenedione (A), and sex hormone binding globulin (SHBG) during the 2 years around the menopause. Thus T fell from 1.7 nmol 1 to 6 months before the FMP to 1.4 nmol 13 to 24 months afterward and 1.2 nmol 85 to 96 months afterward. SHBG fell from 4.0 mg/liter 1 to 6 months before the FMP to 3.5 mg/liter 85 to 96 months afterward but the ratio T/SHBG was unchanged over that period. The data for A were not specifically listed. Longcope et al. [7] did not see any change in T and A over 80 months from the FMP but noted that the mean concentrations of T in all their subjects, including those still having cyclic menses, were significantly less than those of a group of normal young women sampled on days 5 to 7 of the cycle, and suggested that there is a decrease in the ovarian secretion of T prior to the menopause. It is noteworthy that a recent report

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[33] found that there was a steep decline in total serum T concentration with age, such that the levels in a woman aged 40 were approximately 50% of those in a woman aged 21 (0.61 nmol compared with 1.3 nmol) (Fig. 6). Percentage of free T did not vary significantly with age but free T concentration clearly showed a steep decline. The ratio of dehydroepiandrosterone (DHEA) to T and dehydroepiandrosterone sulfate (DHEAS) to T were age invariant because of the declines of DHEA and DHEAS with age. Other studies have suggested that total T levels decrease by approximately 20% and A decreases by approximately 50% with natural menopause [34]. Vermeulen [35] showed that postmenopausal women aged 51 to 65 years had lower mean levels of T (1.03 nmol), A (3.45 nmol), and dihydrotestosterone (DHT) (0.33 nmol) in comparison with women aged 18 to 25 years, i.e., with T (1.53 nmol), A (5.80 nmol), and DHT (1.04 nmol). The effects of ovariectomy on androgen profiles were reported by Judd et al. [36] and Hughes et al. [37]. Before the menopause, oophorectomy results in a decrease of circulating A and T by about 50%, the decrease in the latter being due in large part to the decrease in A. Postmenopausally, removal of the ovaries results in a 50% decline in T and a much lesser decline in A. The postmenopausal ovary secretes more T but less A than its premenopausal counterpart. [34]. In light of the recent report of Zumoff et al. [33], and the difficulty in demonstrating a significant decline in T around the FMP, it may be that the apparent decline in T at the menopause is related as much to aging as to decreased ovarian function in those women with intact ovaries. In the Melbourne Women's Midlife Health Project, there was no significant change seen in total T or in the T/SHBG ratio as a function of changing menopausal status [ 10].

154

HENRY G. BURGER

V. C O N C L U S I O N S 7.

The perimenopause is a time of markedly fluctuating hormone levels. Attempts to define menopausal status purely on the basis of single measurements of FSH or E 2 are unlikely to yield useful information. Though E 2 concentrations appear to be preserved in regularly cycling women at least until the age of 50, INH-B declines and FSH rises. The establishment of menstrual irregularity is marked by a decrease in the follicular-phase concentrations of INH-B, an increase in FSH, but relative preservation of E 2 and INH-A until the time of the FMP. The frequency of anovulatory cycles increases markedly as the FMP approaches. It is difficult to demonstrate substantial changes in androgen concentrations in the immediate perimenopausal period, though levels postmenopausally appear to be lower than those of young regularly cycling women, perhaps as a function of increasing age rather than menopausal status. Hormonal measurements are of little diagnostic value during the perimenopause other than for the purposes of physiological study. The issue of the appropriate reference points for the study of the perimenopause remains unclear.

8.

9.

10.

11.

12.

13.

14.

Acknowledgments 15. The collaboration of my colleagues in the Melbourne Women's Midlife Health Project (Lorraine Dennerstein, Emma Dudley, John Hopper, Adele Green, John Wark, Peter Ebeling, and Janet Guthrie) is acknowledged. David Robertson and his staff at Prince Henry's Institute of Medical Research provided the INH-A and INH-B assays for which Nigel Groome, Oxford Brookes University, Oxford, UK, provided the reagents. Mr. N. Balazs and his staff in the Department of Chemical Pathology, Monash Medical Centre, provided the FSH and estradiol measurements. The Melbourne Women's Midlife Health Project is supported by grants from the Victorian Health Promotion Foundation and the Public Health Research and Development Committee of the Australian National Health and Medical Research Council. Support for the hormone assays has also been provided by Organon Australia Pty Ltd.

References 1. World Health Organization (1981). "Research on the Menopause. Report of a WHO Scientific Group," Tech. Rep. Ser. 670. WHO, Geneva. 2. World Health Organization (1996). "Research on the Menopause in the 1990's," Tech. Rep. Ser. 866. WHO, Geneva. 3. McKinlay, S. M., Brambilla, D. J., and Posner, J. G. (1992). The normal menopause transition. Maturitas 14, 103-115. 4. Sherman, B. M., West, J. H., and Korenman, S. G. (1976). The menopausal transition: Analysis of LH, FSH, estradiol, and progesterone concentrations during menstrual cycles of older women. J. Clin. Endocrinol. Metab. 42, 629-636. 5. Velasco, E., Malacara, J. M., Cervantes, E, Diaz de Le6n, J., Divalos, G., and Castillo, J. (1990). Gonadotropins and prolactin serum levels during the perimenopausal period: Correlation with diverse factors. Fertil. Steril. 53, 56-60. 6. Metcalf, M. G., Donald, R. A., and Livesey, J. H. (1981). Pituitary-

16.

17.

18.

19.

20.

21.

22.

23.

ovarian function in normal women during the menopause transition. Clin. Endocrinol. 14, 245-255. Longcope, C., Franz, C., Morello, C., Baker, R., and Conrad-Johnston, C., Jr. (1986). Steroid and gonadotropin levels in women during the perimenopausal years. Maturitas 8, 189-196. Rannevik, G., Caristr6m, K., Jeppsson, S., Bjerre, B., and Svanberg, L. (1986). A prospective long-term study in women from premenopause to postmenopause: Changing profiles of gonadotrophins, oestrogens and androgens. Maturitas 8, 297-307. Rannevik, G., Jeppsson, S., Johnell, 0., Bjerre, B., Laurell-Boruli, Y., and Svanberg, L. (1995). A longitudinal study of the perimenopausal transition: Altered profiles of steroid and pituitary hormones, SHBG and bone mineral density. Maturitas 21, 103-113. Burger, H. G., Dudley, E. C., Hopper, J. L., Shelley, J. M., Green, A., Smith, A., Dennerstein, L., and Morse, C. (1995). The endocrinology of the menopausal transition: A cross-sectional study of a populationbased sample. J. Clin. Endocrinol. Metab. 80, 3537-3545. Burger, H. G., Cahir, N., Robertson, D. M., Groome, N. P., Green, A., and Dennerstein, L. (1998). Serum inhibins A and B fall differentially as FSH rises in perimenopausal women. Clin. Endocrinol. 48, 809-813. Lee, S. J., Lenton, E. A., Sexton, L., and Cooke, I. D. (1988). The effect of age on the cyclical patterns of plasma LH, FSH, oestradiol and progesterone in women with regular menstrual cycles. Hum. Reprod. 3, 851-855. Groome, N. P., Illingworth, P. J., O' Brien, M., Rodger, P. A. L., Rodger, E E., Mather, J. E, and McNeilly, A. S. (1996). Measurement of dimeric inhibin B throughout the human menstrual cycle. J. Clin. Endocrinol. Metab. 81, 1401-1405. Santoro, N., Brown, J. R., Adel, T., and Skurnick, J. H. (1996). Characterization of reproductive hormonal dynamics in the perimenopause. J. Clin. Endocrinol. Metab. 81, 1495-1501. MacNaughton, J., Bangah, M., McCloud, P., Hee, J., and Burger, H. (1992). Age related changes in follicle stimulating hormone, luteinizing hormone, oestradiol and immunoreactive inhibin in women of reproductive age. Clin. Endocrinol. 36, 339-345. Klein, N. A., Illingworth, P. J., Groome, N. P., McNeilly, A. S., Battaglia, D. E., and Soules, M. R.(1996). Decreased inhibin B secretion is associated with the monotropic rise of FSH in older, ovulatory women: A study of serum and follicular fluid levels of dimeric inhibin A and B in spontaneous menstrual cycles. J. Clin. Endocrinol. Metab. 81, 27422745. Illingworth, P. J., Reddi, K., Smith, K. B., and Baird, D. T. (1991). The source of inhibin secretion during the human menstrual cycle. J. Clin. Endocrinol. Metab. 73, 667-673. Roberts, V. J., Barth, S., EI-Roeiy, A., and Yen, S. S. C. (1993). Expression of inhibin/activin subunits and follistatin messenger ribonucleic acids and proteins in ovarian follicles and the corpus luteum during the human menstrual cycle. J. Clin. Endocrinol. Metab. 77, 1402-1410. Richardson, S. J., Senikas, V., and Nelson, J. E (1987). Follicular depletion during the menopausal transition: Evidence for accelerated loss and ultimate exhaustion. J. Clin. Endocrinol. Metab. 65, 1231-1237. Hughes, E. G., Robertson, D. M., Handelsman, D. J., Haywood, S., Healy, D. I., and de Kretser, D. M. (1990). lnhibin and estradiol responses to ovarian hyperstimulation: Effects of age and predictive value for in vitro fertilization outcome. J. Clin. Endocrinol. Metab. 70, 358 -364. Seifer, D. B., Gardiner, A. C., Lambert-Messerlian, G., and Schneyer, A. L. (1996). Differential secretion of dimeric inhibin in cultured luteinized granulosa cells as a function of ovarian reserve. J. Clin. Endocrinol. Metab. 81,736-739. Burger, H. G. (1984). The physiological basis of the fertile period. In "Fertility and Sterility," R. F. Harrison and B. W. Thompson, eds., pp. 51-8. MTP Press, Lancaster, England. Longcope, C. (1990). Hormone dynamics at the menopause. Ann. N.Y. Acad. Sci. 592, 21-30.

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CHAPTER 9 P e r i m e n o p a u s a l H o r m o n e Changes 24. Trevoux, R., De Brux, J., Castanier, M., Nahoul, K, Soule, J.-R, and Scholler, R. (1986). Endometrium and plasma hormone profile in the peri-menopause and postmenopause. Maturitas 8, 309-26. 25. Sherman, B. M., and Korenman, S. G. (1975). Hormonal characteristics of the human menstrual cycle throughout reproductive life. J. Clin. Invest. 55, 699-706. 26. Metcalf, M. G., and Donald, R. A. (1979). Fluctuating ovarian function in a perimenopausal woman. N.Z. Med. J. 89, 45-47. 27. Metcalf, M. G. (1988). The approach of menopause: A New Zealand study. N.Z. Med. J. 101, 103-106. 28. Hee, J., MacNaughton, J., Bangah, M., and Burger, H. G. (1993). Perimenopausal patterns of gonadotrophins, immunoreative inhibin, oestradiol and progesterone. Maturitas 18, 9-20. 29. Dennerstein, L., Smith, A.M., Morse, C., Burger, H. G., Green, A., Hopper, J., and Ryan, M. (1993). Menopausal symptoms in Australian women. Med. J. Aust. 259, 232-236. 30. Faddy, M. J., and Gosden, R. G. (1995). A mathematical model of follicle dynamics in the human ovary. Hum. Reprod. 10, 770-775. 31. Pellicer, A., Mari, M., de los Santos, M. J., Sim6n, C., Remohi, J., and Tarin, J. J. (1994). Effects of aging on the human ovary: The secre-

32. 33.

34. 35. 36.

37.

tion of immunoreactive (a-inhibin and progesterone. Fertil. Steril. 61, 663-668. Doring, G. K. (1969). The incidence of anovular cycles in women. J. Reprod. Fertil. 6, 77-81. Zumoff, B., Strain, G. W., Miller, L. K., and Rosner, W. (1995). Twenty-four hour mean plasma testosterone concentration declines with age in normal premenopausal women. J. Clin. Endocrinol. Metab. 80, 1429-30. Judd, H. L. (1976). Hormonal dynamics associated with the menopause. Clin. Obstet. Gynecol. 19, 775-788. Vermeulen, A. (1976). The hormonal activity of the postmenopausal ovary. J. Clin. Endocrinol. Metab. 42, 247-253. Judd, H. L., Lucas, W. E., and Yen, S. S. (1974). Effect of oophorectomy on circulating testosterone and androstenedione levels in patients with endometrial cancer. Am. J. Obstet. Gynecol. 118, 7 9 3 798. Hughes, C. L. Jr., Wall, L. L., and Creasman, W. R. (1991). Reproductive hormone levels in gynaecologic oncology patients undergoing surgical castration after spontaneous menopause. Gynecol. Oncol. 40, 42-45.

7 H A P T E R 1(

Epidemiology." Methodologic Challenges in the Study of Menopause SYBIL L.

I. II. III. IV.

CRAWFORD

New England Research Institutes, Watertown, Massachusetts 02472

Introduction Study Design Data Collection Methods Measurement Issues

V. Analytic Considerations

VI. Conclusion References

I. I N T R O D U C T I O N

care patterns, and a subject's own perceptions of menopause. Statistical data analyses in both cross-sectional and longitudinal studies often require handling issues such as biases of recall and selection and censored and missing data, and complicated data structures such as daily symptoms or reproductive hormone levels. Methodologic challenges in the study of menopause are discussed in this chapter. Topics covered include study design issues for both observational studies and clinical trials of hormone replacement therapy; types of data collection instruments; measurement issues, particularly ascertainment of menopause status; and analytic concerns, including choice of appropriate statistical techniques for different types of data.

The menopause is a complex and multifaceted phenomenon, one that is very challenging to study for a number of reasons. First, study design can be logistically and scientifically demanding. Community-based samples are critical for an examination of menopause in the general population, as opposed to nonrepresentative clinic-based samples, but the former are much more costly to obtain. Moreover, choice of the length of followup and eligibility criteria are complicated by variation within and across women in experiences of menopause transitions. In addition, the menopause involves multiple domains, including physiologic as well as psychologic and lifestyle changes. This has implications for selection of data collection methods, in order to obtain a complete picture. A number of measurement issues also arise in a study of menopause, including how to define menopause status. Many measures, particularly those derived from self-report, are affected by physiologic changes as well as by sociodemographic characteristics, psychologic factors, culture, healthMENOPAUSE: BIOLOGY AND PATHOBIOLOGY

II. STUDY DESIGN A number of issues involved in the design of studies of menopause are summarized in this section. The first topic considered is the choice of a sampling flame and the importance of a population-based sample. Issues relevant to 159

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observational studies include cross-sectional versus longitudinal design, choice of age range, and eligibility criteria for menopause status. For examining the role of hormone replacement therapy, the use of observational studies versus clinical trials is discussed, and eligibility criteria used in several large clinical trials are presented.

A. Choice of Sampling Frame Many early studies of menopause relied on clinic-based samples [1], such as patients at menopause clinics. Such samples are relatively cost-efficient to obtain and response rates are likely to be relatively high. These advantages are greatly offset, however, by the atypical nature of such groups. Women seeking health care, particularly those seeking treatment for menopausal symptoms, differ in a number of ways from other menopausal-aged women, with respect to characteristics likely to be related to menopausal and healthrelated factors under study. For example, they have greater access to health care and are more worried about their health, are more likely to report menopausal symptoms, are more likely to undergo a surgical menopause, are more likely to receive psychiatric treatment, and tend to experience more long-term ill health preceding menopause [2-9]. In short, a sample of patients, particularly those from a menopause clinic, is unlikely to provide adequate representative data on the experience of menopause in the general population. Moreover, the range of variability in characteristics of interest may be restricted, affecting the ability to detect associations with outcomes [10]. Thus, it is critical to sample from a frame including women in the general community. A related consideration is sufficient representation from traditionally understudied subgroups, particularly women of lower socioeconomic status (SES) and races and ethnicities other than non-Hispanic Caucasians [4,11-14]. Compared with non-Hispanic Caucasian women, particularly those with high levels of SES indicators such as education, we know relatively little about the menopausal experience for these women. Sampling frames used in past and current communitybased studies include census lists [11,15], driver's license lists [ 16], health maintenance organization patient lists [ 11 ], utility lists [ 11 ], telephone directories [ 17], and random-digit dialing [ 11 ]. Formal statistical generalization to the population of interest requires use of probability sampling from a sampling frame in which all members of the target population have a known nonzero probability of being sampled [18]. In practice, this may be costly or logistically difficult to achieve, particularly when attempting to sample hard-tofind subgroups less likely to be included on sampling frames. Studies may need to combine information from multiple sampling frames, as was done by several field sites involved in the Study of women's Health Across the Nation (SWAN) [ 11].

Obtaining sufficient numbers of racial/ethnic minorities or low-SES women may be particularly challenging, and may require techniques such as household enumeration, or "snowball" sampling in which potential subjects are referred to the study by already-identified subjects, both of which were employed by SWAN field sites [11]. Note that computation of sampling probabilities under this latter approach can be difficult or impossible, because the probability that a woman is "sampled" as a snowball is typically not known [ 11 ]. In turn, this affects the ability to use the sample to generalize to the population of interest. Thus, there can be trade offs between obtaining a probability sample and sampling sufficient numbers from hard-to-find subgroups such as low-SES subjects.

B. Issues in Observational Studies of Menopause This section considers study design issues relevant for observational studies of menopause, where the natural history of menopausal transitions m including surgical menopausem will be observed. I. CROSS-SECTIONAL VERSUS LONGITUDINAL DESIGN

This issue is particularly important for studies of menopause, due to the relatively lengthy duration of the entire menopausal experience for an individual woman. In a crosssectional study of menopause, one cannot observe withinsubject changes in health occurring concurrently with withinsubject menopause transitions. Thus, inferences regarding the role of menopause are based on between-woman comparisons, e.g., comparing age-matched pre- and postmenopausal women [ 19]. Such analyses assume that cross-sectional estimates of menopause status or reproductive age are equivalent to those estimated from longitudinal observations, an assumption that is not always correct [12,20-25]. In addition, retrospective data are more subject to recall error [26]. Accurate assessment of temporal sequences also is much more difficult with cross-sectional data, e.g., determining whether attitudes prior to the menopause transition predict subsequent menopausal experiences [2,24,27], and again requires assumptions regarding the applicability of between-woman differences. Choice of cross-sectional or longitudinal design, however, often is determined in large part by considerations of cost. The followup time necessary to capture the full menopausal period, from pre- to peri- to postmenopausal (definitions of these terms are presented in Section III), is relatively long. In Caucasians, the median time elapsed between the onset of perimenopause and the final menstrual period (FMP) has been estimated as 3.8 years [27]. Moreover, note that this interval does not include the length of time that a woman is in the study prior to reaching perimenopause. Thus a cross-sectional study can be a cost-efficient way to exam-

CHAPTER 10 Methodologic Challenges in Menopause Studies ine both menopausal transitions, pre- to perimenopause and peri- to postmenopause, in a short time. However, interpretation of results is subject to the caveat noted above regarding use of between-woman comparisons to make inferences regarding within-woman changes. Note that a median length of perimenopause of 3.8 years implies that a large percentage of women will transition from peri- to postmenopause in under 4 years. Length of this transition, however, is not entirely random but is associated with a number of characteristics, including smoking [27], which affects a number of important variables (e.g., cardiovascular disease risk factors), as well as the age at onset of perimenopause [27]. Women with a shorter perimenopause also are less likely to report menopausal symptoms or to be characterized as depressed [2,23]. Thus, care must be used when generalizing from women with a short perimenopause to the full population. An important advantage of following the same individuals through the entire menopausal periodmfrom pre- to peri- to postmenopause--is the enhanced ability to assess the presence of curvilinear (nonlinear) relationships between health outcomes and menopause status or reproductive age, measured as time before/after the final menstrual period [4,22,28]. For example, we can examine whether within-subject bone loss accelerates around the onset of perimenopause, and decelerates after FMP. Such analyses are more complicated and require assumptions when performed on cross-sectional data. 2. CHOICE OF AGE RANGE

A common goal of menopause studies is to distinguish the roles of menopause or reproductive age and chronologic age. Hence, the age range needs to be selected accordingly, with women observed to experience menopause transitions at different chronologic ages in order to reduce confounding between reproductive and chronologic age. The appropriate age range depends in part on whether the study is crosssectional or longitudinal; in general, the age range should be broader for a cross-sectional than for a longitudinal study, since the latter involves following subjects as they age, thereby widening the effective observed age range. For a cross-sectional study of natural menopause, the age range should include the full set of ages at which women typically become menopausal [26], approximately 40 years through 55 years, as was done in SWAN's cross-sectional phase [ 11]. A study of surgical menopause may need to include women younger than 40 years, as many women have a hysterectomy prior to age 40, particularly for diagnoses such as endometriosis [29]. African-American women also tend to undergo hysterectomy at a younger age than do Caucasians [29]. The age range should not be so large, however, as to involve cohort effects, where younger women are different from older women, e.g., with respect to characteristics such as use of oral contraceptives [28]. Another consideration for choice of age range is the

161 prevalence of smoking in the population under study. Smokers tend to experience menopause earlier than do nonsmokers, by 1-2 years on average [10,15,30,31]. Investigators may want to consider a lower age range for smokers than for nonsmokers, in order to capture the pre- to perimenopause transition in both groups. For a longitudinal study of initially premenopausal women, it is important to consider an upper age limit for recruitment. Later menopause has been linked to a number of observed characteristics, most importantly lower rates of smoking, and possibly higher body mass index [30,32,33], both of which are associated with key outcomes such as cardiovascular disease risk and bone density [34,35]. Thus, older women who are still premenopausal are atypical of premenopausal women in general, exhibiting "survivorship" bias [4]. In addition, these women tend to have a shorter perimenopause [27] and as a consequence may experience fewer menopausal symptoms [2,23]. In short, data from older premenopausal women may not be generalizable to the population at large. Note that this group's menopausal experience is important scientifically. A longitudinal study, however, will collect relevant data as women age into this group. For a longitudinal study with followup of subjects of under 4 - 5 years, where the full menopausal period may not be observed, choice of age range is determined in part by the stage of menopause of greatest interest. For example, a study focusing on the transition from pre- to perimenopause should sample primarily younger women, e.g., an age range centered around 4 7 - 4 8 years, the estimated median age for onset of perimenopause in Caucasians [27]. In contrast, a study of postmenopausal women should include primarily older women, e.g., aged 5 0 - 7 9 years, as in the Women's Health Initiative [36]. 3. MENOPAUSE STATUS ELIGIBILITY CRITERIA As with age range, the appropriate choice of eligibility criteria involving menopause status depends in part on whether the study is cross-sectional or longitudinal. For a cross-sectional design, one may want to be more inclusive, sampling women in a variety of menopause stages~including surgical menopause--in order to include data from all phases of menopause in the study. In fact, including only older postmenopausal women can be problematic when the outcome is age at menopause, due to recall bias [4,26]. For longitudinal studies, within-woman changes in menopause status or reproductive aging are observed directly, so that eligibility criteria regarding initial menopause status can be more restrictive than in a cross-sectional study. The aims of a study also affect the choice of status categories or reproductive age. Choice of status eligibility criteria which are appropriate for one set of scientific goals may not be adequate for others. For example, the Postmenopausal Estrogen-Progestin Intervention Study (PEPI) had as its primary goal to compare the impact of different hormone

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replacement therapy regimes on measures of cardiovascular disease risk [37,38]. To this end, the design employed sampling restrictions in terms of chronologic age and years since menopause (natural or surgical). A consequence of this design was an artificial collinearity between these two variables, i.e., a collinearity that does not exist in the general population, where age and years since menopause are not restricted. Thus, analyses attempting to identify predictors of age at menopause (not an original study goal) were hampered by the inability to distinguish covariates of age at menopausemthe outcome of interestmfrom correlates of chronologic age [38]. As this example demonstrates, careful thought should be given to potentially competing study aims when selecting eligibility criteria. Past studies of menopause vary with respect to inclusion or exclusion of surgically menopausal subjects. These women differ in a number of ways from women experiencing natural menopause, including better access to health care, and lower prior levels of health [4,24,39]. Their menopausal experiences and subsequent disease risk also are likely to diverge from those of naturally menopausal women, in part because of different characteristics prior to surgery, but also because their reproductive hormonal profile~e.g., rapidity and timing of changes in hormone levels w differs as well [24,40]. Thus, one cannot generalize results regarding menopause and health outcomes, such as cardiovascular disease risk factors or menopausal symptoms, from surgically menopausal women to naturally menopausal women. Because surgically menopausal women are not a random subsample of all women going through menopause, however, omitting them from analyses implies that the resulting description of the menopausal experience for the general population is in some sense incomplete. A common recommendation is to include these w o m e n ~ i n cross-sectional studies, include women with a prevalent hysterectomy or bilateral oophorectomy, and in longitudinal studies, continue to follow women with an incident surgical menopausembut study them separately from other women. It may be useful to consider surgically menopausal women as a separate stratum, both for sampling and for data analyses [4,22,36,37].

C. Issues in S t u d i e s o f H o r m o n e Replacement Therapy This section compares the use of observational studies and clinical trials in the study of hormone replacement therapy (HRT), and summarizes eligibility criteria used in several recent trials. 1. OBSERVATIONAL STUDIES VERSUS CLINICAL TRIALS

Clinical trials are preferable to observational studies of this topic due to selection bias [4,41-43]. Many studies have

found that prior to initiation of HRT, users are more likely to exhibit characteristics associated with better health, including higher SES (as indicated by income and education), higher use of health care, more exercise, lower body mass index, and a better risk profile for cardiovascular disease [41,44-49]. Thus part of the difference between users and nonusers in outcomes such as cardiovascular disease risk factors in observational studies is likely due to preexisting differences in health and related characteristics [50]. Comparing women prescribed and not prescribed HRT, many past studies took place when physicians were reluctant to prescribe HRT for women at high risk for cardiovascular disease (CVD) [47], reflected in higher observed CVD risk among nonusers. Even considering only ever-users, women who continue to use HRT differ from those prescribed HRT but who discontinue use. The former group is more tolerant of HRT and is less likely to experience adverse effects [4,51]. In fact, short-term users have been found to have greater subsequent cardiovascular disease risk compared to long-term users [52]. Sturgeon and colleagues also noted a "healthy user survivor effect," whereby women who developed an illness discontinued use of HRT [53]. In addition, long-term HRT users are by definition "compliant." In clinical trials, compliers on both study arms have been found to fare better than noncompliers, and the magnitude of the effect of compliance with placebo was similar to that found for HRT in two meta-analyses [54,55]. Thus, part of the difference in health outcomes or disease risk between users and nonusers may be due to a compliance effect. Moreover, it is difficult to control completely for all such biases. For example, it is not straightforward to adjust appropriately for education and SES when modeling health outcomes [4,43,45,46]. Even in a population that was relatively homogeneous with respect to SES, users differed from nonusers regarding behaviors affecting health promotion and disease prevention [41 ]. In addition, some biases involved in prescribing and compliance may not be observed or known [41,43,56]. In summary, estimates of the benefit of HRT obtained from observational studies are likely to be somewhat overstated [43,53,56]. To assess the impact of HRT on outcomes such as cardiovascular disease risk or events, it is important to look to results from clinical trials such as PEPI, the Women's Health Initiative, or the Heart and Estrogen/Progestin Replacement Study (HERS) [36,42,57]. Such trials avoid selection bias by employing random assignment to treatment arm, including a placebo arm [57].

2. ELIGIBILITY CRITERIA IN CLINICAL TRIALS o r H R T In the final stage of sample selection, both PEPI and HERS included only subjects with a high likelihood of complying with treatment arm, by requiring 80+ % compli-

CHAPTER 10 Methodologic Challenges in Menopause Studies ance during a run-in phase [57,58]. Current HRT users recruited to PEPI were required to stop treatment [58]. The trials also excluded women with contraindications for use of HRT [57,58]. Both restrictions are justified on logistical and ethical grounds. They may, however, limit generalizability of results somewhat to compliant subjects who are potentially able to be long-term HRT users. Moreover, short-term (3 months) cessation of HRT may be insufficient to achieve "wash-out" of its effects, e.g., on rate of bone turnover. McKinlay suggests that the most appropriate subjects for a clinical trial of HRT are those with no prior use [4]. Current users are less likely to report adverse effects for the HRT arm (because they have been taking HRT, and hence they can tolerate it), and are more likely to be unblinded on the placebo arm, whereas past u s e r s m w h o may have discontinued use due to adverse effects m may be less likely to be able to tolerate the HRT arm. Residual effects of HRT also may be an issue with current users. The availability of women with no prior HRT use varies by geographic region, however [59,60].

III. DATA COLLECTION

METHODS

As in all studies, there is typically a trade off between retrospective and prospective data collection in menopause studies, with a lower cost but also potentially lower accuracy for retrospective data collection. Moreover, as in studies of other topics, subject burden increases with the level of detail of data collection, and hence more detailed data collection regimes such as: daily hormone measurements tend to be employed with a selected subset (either by design, or by default due to subject nonresponse); consequently, the resulting data are less generalizable [4,24,61 ]. Thus, menopausal studies, which can involve relatively demanding data collection methods such as daily menstrual calendars, need to balance scientific rigor with participant burden. In addition, the menopause is a highly multifaceted phenomenon, involving changes in physiologic, epidemiologic, and psychosocial factors. Thus, investigators should consider collection of data in a number of domains [ 12]. These multidisciplinary data will provide better, more complete information for key study goals and will make efficient use of the large effort needed to recruit the participant sample. As an example, a recent study noted a link between depression and low bone density [62], possibly related to low estrogen concentrations, and another study found an association between bone density levels and breast cancer [63]; such findings are useful in a number of disciplines, including endocrinology, psychology, and oncology. This section presents a summary of data collection methods and types of instruments used in studies of menopause to collect information in various domains, moving from least to most detailed or demanding.

163 A. M a i l e d S u r v e y s This type of instrument has been used in past large epidemiologic cross-sectional surveys, such as the first phase of the Massachusetts Women's Health Study [15]. Such an instrument is fairly unobtrusive for subjects. It is also relatively inexpensive compared with other modes of data collection, although this advantage is offset somewhat by the need to conduct telephone followup of nonrespondents. For example, in the cross-sectional phase of the Massachusetts Women's Health Study, the initial response rate to the mailed survey was 57%; telephone followup of initial nonrespondents raised the combined final response rate to 77%. Moreover, the initial respondents to the mailed survey differed from nonrespondents to the mailed survey who subsequently responded by telephone, with higher levels of education and access to health care among the former [64]. Thus, it may be necessary to employ a mixed mode in order to reduce nonrespondent bias. By necessity, all data collected on a mailed pen-and-paper survey are self-reported. Thus, the investigators cannot verify data such as prescription medications, or anthropometric measures such as height or weight. Separate data coding and data entry also are required, unless scannable forms are used.

B. T e l e p h o n e S u r v e y s This mode has been used in a number of menopause studies, including the cross-sectional survey in the Melbourne Women's Midlife Health Project [ 17], the Healthy Women's Study [16], the longitudinal phase of the Massachusetts Women's Health Study [15] and in SWAN's cross-sectional phase [ 11 ]. Telephone surveys are useful not only for primary data collection, but for initial screening to identify and recruit specific cohorts. SWAN's cross-sectional telephone survey, for example, included questions used to assess cohort eligibility, particularly menopause status--which is not available on sampling frames; many field sites recruited eligible participants into the cohort at the conclusion of the telephone interview [11]. Depending on available technology, computeraided telephone interviewing can be used, which eliminates extra steps in data coding and data entry and leads to more accurate data collection. As with mailed surveys, however, there may be some bias, in this case due to subjects without telephone or with unlisted telephone numbers. Thus, a mixedmode approach may be needed, combining home visits with telephone interviews, as in SWAN [ 11 ].

C. I n - H o m e or I n - C l i n i c Visits Data collection has been carried out in subjects' homes or in local clinics in a number of recent menopause studies,

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including the Healthy Women's Study, the Melbourne Women's Midlife Health Project, the second phase of the Massachusetts Women's Health Study, and SWAN's longitudinal phase. Many anthropometric measurements have been taken in a subject's home, including height, weight, blood pressure, and girth; blood and urine specimens also can be collected [65]. For longitudinal studies, it is important to employ similar collection methods for an individual subject at each visit in order to estimate within-woman changes, because measures such as blood pressure can vary by setting. Other physiologic data collected in clinics in studies of menopause include bone density and carotid ultrasound [ 11 ]. Collection of blood or urine specimens also can be done, as in the Melbourne Women's Midlife Health Project [17], the Massachusetts Women's Health Study [65], and SWAN [ 11 ]. This may be logistically difficult, however, depending on what is being measured. Specimens may need to be taken on a particular day of the menstrual cycle (e.g., days 2 - 5 in the early follicular phase for regularly cycling women), or at a certain time of day, for assessing concentrations of reproductive hormones. Fasting samples may be required for accurate measurements, e.g., of glucose. Studies examining the relationship of reproductive hormones to cardiovascular disease risk factors may impose multiple conditions on the specimen collection protocol. Consequently, the study should consider accommodation of subjects' schedules by allowing them to "drop in" for specimen collection on a different day from the rest of the data collection, as in SWAN [ 11 ]. This type of data collection, particularly when done at a clinic, involves considerably more participant burden than a mailed or telephone survey, and response rates are correspondingly lower. Comparing response rates for the crosssectional and longitudinal phases of SWAN, for example, the latter were substantially lower [ 11 ].

D. D a i l y C a l e n d a r s Daily collection of self-reported data is useful for measuring a series of similar, recurrent events such as menstrual bleeding or premenstrual or menopausal symptoms. Retrospective recall of these events is poor [22,66-69]. Several large menopause studies have employed calendars, including SWAN and the Massachusetts Women's Health Study. Response rates tend to be lower than for mailed or telephone surveys [67], with most of the dropouts occurring at the beginning of data collection. Depending on the length of the data collection period and on the amount of data women are asked to record, there may be fairly substantial data coding and data entry requirements. For a study of menopausal transitions or the perimenopausal period, a longer period of data collection may be required than for studying premenopausal women, e.g., 2 + years, in order to capture perimenopausal changes in bleeding over time.

The calendars are self-administered in a woman's home, which means that no review by study personnel is possible until after the calendar is sent back to the study site. Retrospective data recording is also an issue; data quality is less accurate when data are collected retrospectively rather than prospectively. Johannes and colleagues pilot-tested an electronic calendar for daily collection of menstrual bleeding and symptoms. The device recorded the time and date of data entry by the participant. In the pilot study comprising a month's data collection, all 23 subjects entered at least one day's data late, i.e.,after the day on which bleeding or symptoms occurred [70]. These results indicate that it is critical to take measures to ensure high-data quality, particularly when using traditional paper instruments. Calendar instruments should be very simple to understand, in order to minimize mistakes and respondent burden. Subjects should be asked to return completed calendars frequently, e.g., monthly, so that participation and data quality can be monitored, and to limit the amount of retrospective data recording. Completed calendarsmparticularly the first several calendars--should be examined in order to identify errors. Researchers may want to send a letter to participants noting commonly made errors, or even to make retraining calls to respondents whose cal' endars demonstrate a large number of problems.

E. D a i l y S p e c i m e n C o l l e c t i o n Daily collection of specimens such as serum or urine can be very informative, particularly in the study of perimenopause, during which hormone concentrations fluctuate widely even within an individual woman [71-76]. Thus such measures are highly superior to annual serum or urine samples, with respect to capturing within-woman variability. Such collection is expensive to conduct and requires a great deal of subject cooperation, however; consequently, sample sizes often are relatively small.

IV. M E A S U R E M E N T

ISSUES

A number of measurement issues arise in the study of menopause, particularly the determination of a woman's menopause status. Various researchers also have noted methodological difficulties in the measurement of menopausal symptoms, as well as cultural or ethnic differences in reporting of variables related to menopause.

A. D e f i n i t i o n s o f M e n o p a u s e Status Indicators used to define menopause status have included age, menstrual bleeding, levels of reproductive hormones, and a woman's self-report.

CHAPTER 10 Methodologic Challenges in Menopause Studies 1. CHRONOLOGIC AGE

Early studies of menopause used chronologic age as a proxy for postmenopause [12,24,77]. This is a very poor measure of menopause status, however, because the final menstrual period occurs over a wide age range [27]. A comparison of menses-based and age-based definitions using data from a case-control study of breast cancer [78] indicated that--using a menses-based definition as the "gold standard"msensitivity and specificity for the age-based definitions differed for cases and controls. In particular, there were more premenopausal women classified incorrectly as postmenopausal among cases than among controls, because age at menopause (by the menses-based definition) was later in cases than in controls. Thus studies of breast cancer employing age-based definitions of menopause status may suffer from this differential misclassification, which affects estimation of the association between menopause status and disease. 2. MENSTRUAL BLEEDING Past World Health Organization Working Group meetings [79,80] have recommended use of the following definitions for menopause status categories, based largely on observed menstrual cycle patterns, which are assumed to reflect underlying endocrinological changes or levels [81 ]: a. Natural menopause: the permanent cessation of menstruation, determined retrospectively after 12 consecutive months of amenorrhea without any other pathological or physiological cause. b. Perimenopause: the period just prior to the final menstrual period through the first year after the final menstrual period, beginning at the onset of endocrinologic and menstrual changes. c. Premenopause: the entire reproductive period prior to the FME d. Induced menopause: the cessation of menses due to removal of both ovaries with or without removal of the uterus, or iatrogenic ablation of ovarian function. e. Premature menopause: natural menopause occurring before age 40. Also known as premature ovarian failure. f. Postmenopause: dating from the FMP, including both natural and surgical menopause.

Note that the perimenopausal period as defined above overlaps with both the first 12 months of postmenopause after the FMP, and the premenopause. Metcalf [75] distinguishes premenopause as menstruating at regular intervals, whereas perimenopause begins with the onset of irregular cycling and continues after the FMP until hormone levels stabilize. Other uses of these terms in the literature [82-85] separate pre-, peri-, and postmenopause, with premenopause ending at the onset of endocrinologic or menstrual changes, and perimenopause concluding with the FMP.

165 3. ENDOCRINE MEASURES The decade prior to the FMP is characterized by an increase in variability in reproductive hormone concentrations, even though a woman may continue to have her normal menstrual bleeding. Abrupt changes in these hormones may occur, with values typical of postmenopause followed by those seen in younger reproductive-aged women [71-75,86]. Although hormone concentrations stabilize 1-2 years after the FMP [87-89], no sharp changes occur at the time of the FMP [74]. To categorize women regarding menopausal status, a cutoff of follicle-stimulating hormone (FSH) of 3 5 - 4 0 IU/liter is commonly used in clinical practice and in research studies [90,91 ]. Some studies have employed cutoffs of FSH greater than 10-20 IU/liter to indicate perimenopause [84,92]. The above discussion indicates, however, that endocrine variables cannot be considered reliable indicators of menopausal status [71,72,87], particularly based on a single serum or urine sample, because within-woman values fluctuate widely during perimenopause, and patterns are variable across women [76]. Hormone concentrations also vary by chronological age as well as by time before the FMP [71,76,93-97]. Although average values within and across women demonstrate general trends during this period, no single cutoff value is likely to be accurate as a predictor of status [98]. 4. SELF-DEFINITIONS

Women's perceptions of their own menopause status vary by culture and race/ethnicity [81], and do not agree completely with categorizations based on bleeding patterns [99]. Self-reported menopause status may not be appropriate for epidemiological purposes [81 ], but may be of interest in its own right or in studying women's experiences during the transition through menopause [99]. Assessment of years since the FMP from self-report on a cross-sectional survey can be inaccurate, because recall becomes increasingly unreliable with greater time elapsed since the FMP and there is evidence for digit preference [33,100-102] (see Section IV, C).

B. C h a r a c t e r i z i n g P e r i m e n o p a u s e The definition of natural menopause presented in Section IV,A,2 has become an accepted standard [82], and investigators have turned their attention to better characterizing perimenopause, for which no standard definition exists [5,82]. Treloar [103,104] defined the onset of perimenopause as the start of an increase in the variability in cycle length. Two later studies also identified menstrual irregularity as a perimenopausal indicator, using as a "gold standard" either an FSH level of at least 15 IU/liter [84] or subsequent transition to postmenopause within 3 years [82]. Menopausal symptoms, particularly hot flashes and night

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sweats, also were indicative of perimenopause. In the latter study, changes in menstrual flow were associated with status before controlling for irregularity, but were not independently related to status [82]. Further refinement of the characterization of perimenopause is needed [5,105]. Two studies have suggested a distinction between different stages of perimenopause, based on menstrual bleeding regularity [83,106]. Early perimenopause corresponds to self-reported changes in frequency of menstrual bleeding, whereas late perimenopause is defined by prolonged (more than 6 months) amenorrhea. Differentiation of these two stages appears to be informative in terms of summarizing the sequence of bleeding patterns, and for prediction of subsequent transition to postmenopause [83,106]. In addition, the simultaneous incorporation of multiple sources of data, e.g., symptoms, bleeding patterns, and reproductive hormone profiles, has been suggested as an area of future study [ 107].

C. R e l i a b i l i t y o f S e l f - R e p o r t e d D a t a Many epidemiologic studies of menopause ask a woman to report the date of her last menstrual period or the date of surgical menopause--used to estimate her age at menop a u s e m o r patterns of use of hormone replacement therapy. Several studies have investigated the reliability and reproducibility of such self-reported information, by comparison with medical records or by repeated interviewing of subjects over time. Considering menopause status, reliability and reproducibility tend to be relatively high for the type of menopause, i.e., natural versus surgical [108-110]. Reliability and reproducibility also are better for age at surgical menopause than for age at natural menopause [68,69,108-112]. In one study [108], women tended to underestimate age at menopause. This error has implications for estimating the association between age at menopause and disease. For example, breast cancer has been found to be positively related to later age at menopause, whereas osteoporosis and cardiovascular disease have been linked to an earlier menopause. If selfreported age at menopause is misclassified as compared with true age at menopause, the association of age at menopause with disease will be underestimated for breast cancer [ 108, 112] and overestimated for cardiovascular disease or osteoporosis [ 108]. Digit preference in reporting of age at menopause also was observed in several studies [102,111], particularly for ages ending in "0" and "5." For self-reported age at menopause, reliability tended to decrease as the time since the final menstrual period increased [ 109,111 ]. One study found, however, that reproducibility was higher as time since menopause increased, for women with an earlier menopause; thus it may be that women with a relatively young age at menopause (under 40) recall this event better [108].

Regarding recollections of patterns of HRT use, reliability was highest for ever-use [110,113-115]. For epidemiological studies, a single self-report question may be sufficiently accurate for this piece of information [ 113]. Selfreported details of use, including length of use, dose, and reasons for starting/stopping, however, were less reliable [110,113-114]. Lower reliability was related to subject characteristics such as higher age of the subject [114-115] and longer time elapsed since last use [ 113-115]. In summary, self-report may be adequate for basic data such as type of menopause or ever-use of HRT, but less appropriate for more detailed data such as the age at final menstrual period or length of HRT use. Thus, researchers may need to consider prospective designs or other sources of information, e.g., medical records abstraction, for these data.

D. M a s k i n g o f " N a t u r a l " M e n o p a u s e T r a n s i t i o n s Medical interventions, particularly exogenous reproductive hormone use and removal of the uterus and/or ovaries, affect characteristics commonly used to define menopause status [28]. Depending on the regime, use of HRT or oral contraceptives can alter menstrual bleeding, so that their use essentially masks menopausal status defined in terms of menstrual cycling [26,31,116]. Hormone use also can complicate classification based on endocrinological criteria, because it may affect reproductive hormone levels [116119]. Self-defined menopause status also varies by HRT use [99]. Surgical menopause, either from a hysterectomy or oophorectomy, obviously affects menstrual bleeding patterns. Moreover, women with a simple hysterectomy (ovaries not removed) may experience ovarian failure earlier than other women [ 120]. In short, straightforward determination of natural menopause status and timing of natural transitions that would have occurred in the absence of medical interventions is not possible. Implications for data analyses are discussed in Section V, and potential analytic strategies are presented.

E. M e a s u r e m e n t o f M e n o p a u s a l S y m p t o m s A number of methodological problems in past studies of menopausal symptoms have been identified. First, in order to avoid influencing a subject to associate certain symptoms with menopause, it is important to ask about general health and symptoms, including symptoms suspected of being related to menopause [69]; researchers should not identify symptoms a priori as being menopausal, or ask women what symptoms they experienced during menopause. Questions should include both positive and negative symptoms [3,5]. The reference period should be fairly short, e.g., 1 to 4 weeks, in order to minimize inaccuracies in recall [22,69]. Symptom reporting also is affected by cultural norms, as discussed in

CHAPTER 10 Methodologic Challenges in Menopause Studies the following subsection. In addition, researches should employ a standard scale, so that results across different studies can be compared [5,121 ]. A commonly used scale, the B lattKupperman index, is widely used but has been shown to be inadequate. Problems with this scale include development on a possibly unrepresentative sample of women and arbitrary weighting of items [122].

F. C u l t u r a l D i f f e r e n c e s in R e p o r t i n g Ethnicity and culture have been found to affect experiences and perceptions of menopause; this in turn leads to ethnic or cultural differences in reporting of menopauserelated data. For example, symptom reporting varies by culture and geographic region, with lower rates found in Asian and Central American populations than in the United States and Western Europe [ 123-125]. In Mayan Indians, one study [123] found no self-reported hot flashes, despite the occurrence of endocrine changes that were similar to those seen in women in the United States. Japanese women tend to report headaches, shoulder stiffness, and joint pain, whereas vasomotor symptoms appear to be rare and tend not to be associated with menopause status [126]. Ethnicity and culture also are related to sexual attitudes, values, and behavior [22], as well as to perceptions and reporting of menstrual bleeding [66,67]. These differences need to be accounted for in studies of menopause, by using culturally appropriate instruments [66,67,125].

V. A N A L Y T I C

CONSIDERATIONS

This section summarizes a variety of issues involved in analyses of data collected in studies of menopause.

A. M e t h o d s for E s t i m a t i n g A g e at N a t u r a l M e n o p a u s e Data from either cross-sectional (i.e., prevalence) or longitudinal (incidence) studies may be used for estimation of age at natural menopause. 1. PREVALENCE DATA Distributions of recalled age at menopause from crosssectional data can be analyzed using techniques such as Kaplan-Meier plots and Cox proportional hazards modeling [26]. Typically, prevalent cases of natural menopause are asked when their periods stopped, and data for pre- or perimenopausal women are censored at their current age. Techniques that do not account for censoring, e.g., histograms of age at menopause in the subset of prevalent naturally menopausal women, lead to estimates of age at menopause that are biased downward [26]. As noted in Section III, use of retrospective recall of age

167 at the FMP may be problematic; consequently, self-reporting of 12 + months amenorrhea (yes~no) at the time of the interview may be more accurate. Use of this outcome variable suggests estimation of a binary logistic regression of 12+ months of amenorrhea on chronologic age. Median age at menopause (or other percentiles) then can be estimated from the logistic regression model as a function of the intercept and slope [15,127]. Median age at menopause can be estimated for various subgroups, e.g., smokers and nonsmokers, by stratification on the characteristic of interest [15]. Note that a logistic regression analysis excludes women with a prevalent surgical menopause. For Caucasians, several studies suggest that this exclusion is appropriate, and that a competing risks model is not necessary [128,129]. It is unclear whether this holds for other racial/ethnic groups, however, particularly for African-Americans, who have a much higher rate of hysterectomy [29]. 2. INCIDENCE DATA As noted earlier, data from a prospective design, where information on menopause transitions is collected as they occur, is preferable to a retrospective design in terms of accuracy. Covariates also can be assessed prior to transitions, with less recall bias [ 10]. Longitudinal analyses of incidence data often employ hazards modeling, for example, estimating the probability that an event--such as the F M P - - occurs during the study, given that it has not occurred earlier. If the age at the FMP can be measured precisely, e.g., using menstrual calendars, then one can use techniques such as Cox proportional hazards modeling. If menopause status is ascertained only at intervals, e.g., at an annual interview, one can employ an approach used by Brambilla and McKinlay [ 10], which involves multinomial modeling of conditional probabilities of menopause status categories at each interview, including natural menopause, surgical menopause, and not yet menopausal. Analyses of age at natural menopause may need to take into account the competing risk of surgical menopause, although longitudinal analyses by Brambilla and McKinlay in non-Hispanic Caucasians suggest that this is not necessary for this racial/ethnic group [ 10], consistent with cross-sectional findings. B. A n a l y t i c M e t h o d s for M e n s t r u a l C a l e n d a r D a t a Goals of analyses of data from menstrual calendars typically include characterization of the distribution and patterns of menstrual segment lengths. Because bleeding may or may not correspond to a menstrual cycle, the term "segment" is sometimes used rather than "cycle" [67]. Many studies focus on segment length or bleeding length rather than on heaviness of menstrual flow [66,67,130,131 ]. The latter has been found to be less informative, e.g., in defining menopause status [82,83]. The majority of past studies utilizing menstrual calendars have been done in premenopausal women, with the exception of Treloar [103,104], who

168 followed women from menarche to the FME This subsection summarizes issues involved in the analysis of calendar data from premenopausal--regularly cycling--women, as well as analytic complications arising from the study of perimenopausal, i.e., irregular, bleeding. 1. ISSUES IN ANALYSIS OF PREMENOPAUSAL CALENDARS

Data can be analyzed using either a menstrual segment or an individual woman as the unit of analysis. Both approaches are useful, and the appropriate choice depends on the question of interest [132]. If the goal is inference regarding the distribution of segment length for an individual in the population of interest, then the woman should be used as the unit of analysis. An example is the reference period method, where each woman's bleeding patterns are summarized for a standard unit of time, typically 90 days [67]; this provides a cross-sectional summary for each subject. A related issue is length bias. In general, the observation period is fixed and the number of observations (segments) per woman varies. Consequently, women with shorter-and hence more--segments observed are overrepresented in analyses using the segment as the unit of analysis. Thus, using the segment as the unit of analysis can give estimates of segment length that are biased downward. In contrast, bywoman analyses give each woman the same weight. Because the observation period is usually defined in terms of calendar time rather than in terms of completion of a menstrual segment, the last segment is only partially observed. This censoring can cause bias in estimates, because the probability that its length is unknown is related to the length of the segment, with longer segments more likely to be censored [133]. The resulting bias may be small if the data collection period is relatively long. Belsey and Farley [67] summarize a number of analytic methods proposed to handle this censoring, including omitting the last segment from analyses; including it only in estimation of variability but not mean length; truncation, which affects variability estimates; and methods for handling right-censored survival data [ 133]. A number of statistical techniques have been employed in the analysis of menstrual segment lengths, all of which handle the correlation between multiple segments observed in the same subject. Methods also should account for length bias. Techniques such as growth curve modeling may not be particularly useful, because the number of observations (segments) is inherently part of the data to be observed. Methods that explicitly examine within-woman correlation between segments include estimation of segment-to-segment probabilities [106,130], e.g., whether long segments tend to be followed by shorter segments, as well as estimation of autocorrelation to assess the dependence between segment lengths as a function of the lag between segments [130]. Techniques used to model segment length include randomeffects modeling with a random intercept, or equivalently, a

SYBIL L. CRAWFORD

generalized estimating equation (GEE) approach with exchangeable correlation; this assumes that a woman's segment lengths vary randomly about her own mean [130]. Methods that incorporate covariates for segment length include GEE techniques [134,135], Poisson modeling [136], and autoregressive modeling [133]. Harlow and Zeger also employed a mixture model approach to characterize the distribution of segment lengths, whereby one component consisted of "normal" length segments and the other component included very long segments [130]. 2. ANALYTIC COMPLICATIONS FROM PERIMENOPAUSAL DATA Additional analytic issues arise in the study of perimenopausal menstrual segment lengths, due to increased menstrual irregularity. A key question is what constitutes a menstrual segment. World Health Organization definitions require at least one bleeding ~ not spotting~day followed by at least one bleed-free day [67]. Some analysts of premenopausal data omit spotting episodes from analyses entirely [67,137,138]. Spotting episodes, however, may be quite informative in the study of perimenopausal bleeding patterns. Johannes and colleagues, for example, found spotting episodes to be more common in the early perimenopause, indicating the utility of spotting episodes in distinguishing perimenopausal stages [106]. Thus a "conservative" definition of menstrual episodes or segments, whereby any spotting or bleeding is considered separately, may be in order. As noted in Section IV, perimenopause is characterized by within-woman changes in bleeding patterns, particularly an increase in irregularity. Hence the autocorrelation structure for segment lengths within an individual woman may be very different from the exchangeable correlation observed in studies of regularly cycling premenopausal women. To capture this, we may need a more complicated autocorrelation structure. Moreover, irregularity itself is not a welldefined concept. Bleeding patterns during the perimenopause may vary not only across women, but within women as well, and may depend on proximity to the final menstrual period [82,83,106]. Thus models of perimenopausal segment lengths need to allow autocorrelation structures to vary both within and across women. Finally, the complete interval of perimenopausal menstrual bleeding may not be observed during the period of study. Data may be subject to either left or right censoring, or both, depending on the woman's initial menopause status, the length of her perimenopause, and the length of the calendar data collection. Also, as just noted, bleeding patterns may change for an individual over time, depending on proximity to the final menstrual period [82,83,106]. Thus the menstrual segments observed during the study may not be representative of a woman's entire perimenopausal period, unlike segments observed in regularly cycling women. Analyses of perimenopausal segment length should account for this censoring.

CHAPTER 10 Methodologic Challenges in Menopause Studies C. M e t h o d s for C o m b i n i n g D a t a C o l l e c t e d at D i f f e r e n t F r e q u e n c i e s Menopause studies often involve different types of instruments, collecting data at varying frequencies, e.g., annual clinic visits, monthly symptom reporting collected via calendars, and daily urine specimens. Scientific questions of interest may require combining these data, as in assessment of the relationship between reproductive hormone levels measured annually and symptom patterns observed in monthly menstrual calendars, or in a comparison of daily menstrual calendar data to self-reported data on bleeding patterns from an annual interview. Possible approaches include "collapsing" the data measured at a higher frequency of measurement. For example, Johannes and colleagues summarized each woman's 12 months of bleeding patterns in terms of within-woman mean and variance of segment length [ 106]. The correlations of these summaries with annual reproductive hormone values then was computed. The time scale of one of the measures also can be adapted, as was done in several analyses of predictors of menstrual segment length [131,135]. Time-varying covariates such as weight were measured less frequently than monthly, and the schedule of measurement did not correspond to a woman's menstrual segments. To include these variables as predictors, the investigators defined the value of a covariate corresponding to a particular menstrual segment as the average of that covariate during the first 14 days of that segment or of the preceding segment. Another approach is to analyze only data measured concurrently, e.g., estimating the correlation between reproductive hormone concentrations measured at an annual clinic visit and characteristics of the menstrual segment occurring during that annual clinic visit. This has the disadvantage of ignoring other, possibly relevant, data, however.

D. M a s k i n g o f M e n o p a u s e S t a t u s D u e to U s e o f H R T As noted in Section IV, use of HRT prior to observation of 12 + months of amenorrhea results in an inability to assess a woman's "true" menopause status in the absence of HRT, or to determine her age at "natural" menopause. A variety of analytic techniques have been employed or proposed to handle these women in analyses where natural menopause status or transitions are variables of interest. A common approach is to omit ever-users or concurrent users from analyses [28,139-141]. However, as discussed in Section II, HRT users are not a random sample of all women traversing the menopause. Thus analyses that omit these women completely do not describe experiences in the overall

169 population. In longitudinal analyses in which baseline data are available prior to initiation of HRT, one can include some data from these women by censoring their observations at the time of HRT initiation, or omitting observations concurrent with HRT use [ 19]. Another technique is to analyze menopause status for non-HRT users, then compare users and nonusers, omitting menopause status as a variable [142]. This method has the advantage of including users in analyses. HRT users may be a mixture of "natural" menopause status categories, however, so that putting them in a single category may not be appropriate. For the same reason, treating HRT users as a separate menopause status category may distort the estimated association between menopause status and other variables. Other analyses have combined HRT users with postmenopausal women [85]. Many women, however, begin HRT use prior to permanent cessation of menses [49], and thus are likely to be dissimilar to naturally postmenopausal women. Inclusion of HRT users with this latter group could weaken or bias estimated associations of postmenopausal status with other characteristics. HRT use may be included in analyses as a covariate, and menopause status defined in terms of observed bleeding patterns or estrogen levels regardless of HRT use status [23]. For some outcomes, such as depression or sexual activity, the source of estrogen may be relatively unimportant to the question of interest, so that the outcome can be modeled as a function of a marker of the total estrogen exposure (endogenous and exogenous combined), and an indicator for HRT use (yes/no). For other outcomes, however, the distinction between endogenous and exogenous estrogen may be of greater relevance. For example, endogenous estrogen has little liver exposure compared with oral preparations of exogenous estrogen [40,143], and thus using total estrogen as a predictor may not be applicable for outcomes such as circulating lipids. Perhaps the most appropriate general approach is to consider "natural" menopause status as missing for HRT users, and employ techniques developed to handle missing data. Menopause status is unlikely to be missing completely randomly, so that analyses would require techniques that assume either data missing at random (related only to observed data) or nonignorable missingness (related to the unobserved "true" menopause status) [144,145]. Note that all approaches used in this situation necessarily rely on assumptions that are untestable, because "true" menopause status is not observable.

E. H a n d l i n g S u r g i c a l M e n o p a u s e in A n a l y s e s As with HRT use, surgical menopausemhysterectomy and/or bilateral oophorectomy--effectively masks or censors a woman's natural menopause status or transitions that

170 would have occurred in the absence of medical intervention. Often surgically menopausal women are omitted from data anlayses [28]. Similar to HRT users, however, surgically menopausal women are not a random subsample of all women, nor are they a small subsamplemover one-third of women in the United States undergo a hysterectomy by age 60 [ 146]. Thus results from analyses omitting these women will not be generalizable to the entire population [ 129]. Analyses of age at menopause as a potential risk factor for diseases such as breast cancer sometimes assign a "mean" or "typical" age at the FMP to surgically menopausal women. This imputation process, however, does not reflect the underlying distribution of the age at natural menopause, and thus distorts the associations of age at the FMP with disease outcomes [147]. Hysterectomy or oophorectomy status also can be included as a covariate or stratifying factor in analyses. Type of menopause can be included as a predictor, comparing naturally postmenopausal women to surgically menopausal women, as in PEPI [37]. Given the many differences between these two groups, it may be necessary to stratify data analyses on type of menopause [4,81 ]. Data may also be treated as censored for surgically menopausal women. For example, in survival analyses of age at natural menopause using prevalence data, data from surgically menopausal women may be censored at the time of surgery [31 ]. This approach assumes that a woman's experience in the absence of surgery is similar to that of women observed to have a natural menopause [129]. This may be accurate after controlling for predictors of type of menopause, such as access to medical care; that is, natural status may be missing at random. This assumption is essentially untestable, however. Finally, analyses may employ competing risks modeling. Such techniques can be used either with prevalence data, to estimate models for 12+ months of amenorrhea (yes~no) [128], or self-reported age at the FMP or surgery [129], or longitudinal incidence data [ 10].

E Assessing Associations between Menopause Status or Reproductive Age and Health Outcomes For cross-sectional data, any inferences regarding the role of menopause transition within a woman are done using between-women comparisons. Depending on the available data, analyses may employ categorized menopause status (pre, peri, post, surgical), reproductive age defined as time before/after the FMP, or levels of reproductive hormones. Analyses need to control for important confounders such as age, by including them as model predictors or by age matching [19]. For longitudinal data, one can directly examine withinwoman changes in outcomes concurrent with within-woman

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changes in menopause status. For example, one can model successive differences in the outcome, e.g., change in serum cholesterol or bone density from one annual visit to the next, as a function of corresponding successive changes in menopause status [23,142]. Longitudinal data also permit better assessment of temporal sequences of events, such as whether fluctuations in reproductive hormone levels precede or follow more overt signs of perimenopause such as increased menstrual irregularity. For cross-sectional data, statistical methods include linear and logistic regression, depending on the outcome variable, including concurrent menopause status as a covariate. Longitudinal analytic techniques must account for within-subject correlation of multiple observations. Approaches that consider each observation separatelymdata are not collapsed within a woman m include generalized estimating equation methods, repeated measures modeling, and random-effects modeling. This last method can be used, for example, to identify women with an extreme menopausal trajectory, e.g., "fast" losers of bone density. Other methods collapse data within a woman, e.g., a paired t-test of a subject's mean outcome level prior to the FMP versus the corresponding mean after the FMP [140]. Alternatively, one can apply spline analysis, fitting within-woman slopes before and after the FMP for a piecewise linear model, and compare pre-FMP and post-FMP slopes [ 139-141 ]. Choice of functional forms is key. For example, analysts of bone density data often use log of years since menopause as a predictor of bone density [92]; this functional form implies that bone loss is more rapid for women in early postmenopause. It is important to determine the presence or absence of curvilinear relationships, e.g., whether bone loss accelerates in perimenopause and levels off after the FME Depending on the outcome of interest, it is also critical to distinguish pre- from perimenopause rather than combining all observations prior to the FMP, because acceleration of changes due to menopause may occur well before the FMP [28]. One should also consider the amount of change in reproductive hormone concentrations in addition to absolute concentrations; rapid hormonal changes may be associated with outcomes such as symptoms [5,148-150].

G. Confounding, Effect Modification, and Stratification A key confounding factor related to both status and many health outcomes of interest is smoking. Smokers have an earlier menopause [10,15,30,31], higher levels of risk factors for cardiovascular disease [34], and a higher risk of low bone density and osteoporosis [35]. Without controlling for smoking status, the role of the menopause transition in changes in levels of disease risk is overstated [4,30,150]. Other potential

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confounders to consider include body mass index, possibly parity, and oral contraceptive use [30,31-33]. Relationships between menopause status and outcomes such as cardiovascular disease risk also may differ across subpopulations, e.g., smokers versus nonsmokers. In analyses of blood pressure and lipids, for example, menopause status played a larger role for nonsmokers [4]. Thus, analyses may need to include appropriate interaction terms between menopause status and smoking status, or even to stratify on smoking status. Type of menopause~surgical or naturalmis another potential stratification factor. As noted in Section II, surgically menopausal women have very different experiences before, during, and after the menopausal transitions. Consequently, including the type of menopause as a covariate may not be sufficiently complex to assess the role of surgical versus natural menopause; analyses may need to be stratified on type of menopause [4].

VI. CONCLUSION In closing, it is important to note comments by Lock [ 125]. The emphasis of much of the studies of menopause to datemparticularly in the United States and Europemhas been on its negative health consequences, e.g., experience of menopausal symptoms, loss of bone density, and increase in cardiovascular disease risk. Cross-cultural studies suggest, however, that the menopause is not universally a time of decline in health, and that influences other than biology, such as culture, are involved in women's menopausal transitions. Lock proposes that investigators identify factors that are associated with a positive menopausal experience, and that research take into account variables from a variety of domains including lifestyle and psychosocial as well as physiological.

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SYBIL L. CRAWFORD 134. Harlow, S. D., and Campbell, B. (1996). Ethnic differences in the duration and amount of menstrual bleeding during the postmenarcheal period. Am. J. Epidemiol. 144, 980-989. 135. Harlow, S. D., and Campbell, B. C. (1994). Host factors that influence the duration of menstrual bleeding. Epidemiology 5, 352-355. 136. Collett, D., and Weerasooriya, N. (1993). A modelling approach to the analysis of menstrual diary data. Stat. Med. 12, 955-965. 137. Treloar, A. E., Boynton, R. E., Behn, B. G., and Brown, B. W. (1967). Variation of the human menstrual cycle through reproductive life. Int. J. Fertil. 12, 77-126. 138. Rodriguez, G., Faundes-Latham, A., and Atkinson, L. E. (1976). An approach to the analysis of menstrual patterns in the critical evaluation of contraceptives. Stud. Fam. Plann. 7, 42-51. 139. van Beresteijn, E. C. H., Korevaar, J. C., Huijbregts, P. C. W., Schouten, E. G., Burema, J., and Kok, F. J. (1993). Perimenopausal increase in serum cholesterol: A 10-year longitudinal study. Am. J. Epidemiol. 137, 383-392. 140. Jensen, J., Nilas, L., and Christiansen, C. (1990). Influence of menopause on serum lipids and lipoproteins. Maturitas 12, 321-331. 141. Falch, J. A., and Sandvik, L. (1990). Perimenopausal appendicular bone loss: A 10-year prospective study. Bone 11,425-428. 142. Crawford, S. L., Casey, V. A., Avis, N. E., and McKinlay, S. M. (2000). A longitudinal study of weight and the menopause transition: Results from the Massachusetts Women's Health Study. Menopause, in press. 143. Longcope, C., Herbert, E N., McKinlay, S. M., and Goldfield, S. R. (1990). The relationship of total and free estrogens and sex hormonebinding globulin with lipoproteins in women. J. Clin. Endocrinol. Metab. 71, 76-72. 144. Little, R. J. A., and Rubin, D. B. (1987). "Statistical Analysis with Missing Data." Wiley, New York. 145. Rubin, D. B. (1987). "Multiple Imputation for Nonresponse in Surveys." Wiley, New York. 146. National Center for Health Statistics, Pokras, R., and Hufnagel, V. G. (1987). "Hysterectomies in the United States, 1965-84," Vital Health Stat., Ser. 13, No. 92, DHHS Publ. no. (PHS) 88-1753. U.S. Gov. Printing Office, Washington, DC. 147. Pike, M. C., Ross, R. K., and Spicer, D. V. (1998). Problems involved in including women with simple hysterectomy in epidemiologic studies measuring the effects of hormone replacement therapy on breast cancer risk. Am. J. Epidemiol. 147, 718-721. 148. Schmidt, E J., and Rubinow, D. R. (1991). Menopause-related affective disorders: A justification for further study. Am. J. Psychiatry 148, 844-852. 149. Brincat, M., Magos, A., Studd, J. W., Cardozo, L. D., O'Dowd, T., Wardle, P. J., and Cooper, D. (1984). Subcutaneous hormone implants for the control of climacteric symptoms: A prospective study. Lancet 1, 16-18. 150. Stampfer, M. J., Colditz, G. A., and Willett, W. C. (1990). Menopause and heart disease: A review. Ann. N. Y Acad. Sci. 592, 286-294.

~HAPTER 1

SWAN: A Multi c enter,

Multiethnic, CommunityBased Cohort Study

of Women and the Menopausal Transition MARYFRAN SOWERS,* SYBIL L. CRAWFORD, t BARBARA STERNFELD,; DAVID M O R G A N S T E I N , wE L L E N B . G O L D , G A I L A . G R E E N D A L E , # D E N I S E V A N S , * * R O B E R T N E E R , tt K A R E N M A T T H E W S , ~ S H E R R Y S H E R M A N , w167 ANNIE LO, wGERSON WEISS, A N D J E N N I F E R K E L S E Y ## *Department of Epidemiology, School of Public Health, University of Michigan, Ann Arbor, Michigan 48109; tNew England Research Institutes, Watertown, Massachusetts 02472; *Department of Epidemiology and Biostatistics, Division of Research, Kaiser Permanente, Oakland, California 94611; ~Westat, Inc., Rockville, Maryland 20850; IIDepartmentof Epidemiology and Preventive Medicine, School of Medicine, University of California, Davis, Davis, California 95616; #Departments of Medicine and Obstetrics and Gynecology, University of California, Los Angeles, School of Medicine, Los Angeles, California 90024; **Rush Institute on Aging, Chicago, Illinois 60612; ttDivision of Endocrinology, Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts 02114; **Departmentof Psychiatry, University of Pittsburgh, Pittsburgh, Pennsylvania 15261; ~NIH/NIA, Bethesda, Maryland 20892; IIIIDepartmentof Obstetrics and Gynecology, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103; and ##Division of Epidemiology, Department of Health Research & Policy, Stanford University, School of Medicine, Stanford, California 94305

I. II. III. IV. V. VI.

Appendix A. SWAN Investigators Appendix B. Specific Sampling and Recruiting Strategies by Sites with List-Based Primary Sampling Frames Appendix C. Specific Sampling and Recruiting Strategies by Sites with RDD-Based Primary Sampling Frames References

Introduction Overview of the Study Design Data Collection Sampling and Recruitment Strengths and Limitations of SWAN Summary

I. I N T R O D U C T I O N

completely understood [1,2]. Furthermore, much of what is known is based on data from Caucasian women, from women who are self-referred to menopause clinics, or from convenience samples of women seen in the clinical setting

Menopause is a universal phenomenon of women, yet, as discussed in other chapters in this book, it is inMENOPAUSE:

BIOLOGY AND PATHOBIOLOGY

175

Copyright 9 2000 by Academic Press. All rights of reproduction in any form reserved.

176 for other health problems. In the next two decades, approximately 40 million American women will experience the menopause [3] and by the year 2005, it is estimated that $3-5 billion will be spent annually for hormone replacement therapy (HRT) and the physician monitoring of that HRT use [4]. Additionally, study of the menopause poses special methodological challenges because of its transitional nature, the potential for involving multiple organ systems (i.e., bone, lipids, mental health), and the potential contribution of varied social, behavioral, and cultural factors (see Chapter 10). Thus, study of the menopausal transition is both important and complex. To address many of the knowledge deficits about the menopausal transition identified in chapters of this book, the Study of Women's Health Across the Nation (SWAN), a multisite, longitudinal study of the natural history of menopause, was funded by the National Institute on Aging, the National Institute of Nursing Research, and the Office of Research on Women's Health. The overall goal of SWAN is to describe the chronology of the biological and psychosocial characteristics of the menopausal transition and the effect of this transition on subsequent health and risk factors for agerelated chronic diseases. Because investigation of the menopausal experience in minority women has been neglected, SWAN placed special emphasis on including minority populations. This would allow SWAN to describe the sociocultural, lifestyle, psychological, and biological characteristics of these groups in relation to the menopausal transition [5]. In addition, emphasis was placed on recruiting a sample of women that was community or population based, rather than volunteer or clinically based, so that the sample would be representative of women from the full spectrum of socioeconomic status and cultural experiences. The specific aims of SWAN, shown below, are being addressed in a representative cohort of initially premenopausal women who are socially and culturally diverse. The aims are as follows: 1. To characterize the symptomatology, hormonal, and bleeding pattern characteristics related to the menopausal transition. 2. To investigate the hormonal and menstrual bleeding pattern characteristics related to change in bone mineral density, cardiovascular status markers, measures of carbohydrate metabolism, and body composition during the menopausal transition. 3. To examine the relations of psychosocial factors, personality characteristics, and behaviors, including lifestyle behaviors, as they may relate to age at onset, symptoms, and physiological changes of the menopausal transition. 4. To discern what changes observed over time are related to the menopausal transition as compared to age-related changes, including those changes that appear to accelerate the aging process.

SOWERS ET AL.

5. to describe and quantify cultural and ethnic differences among women with respect to midlife aging and the menopausal transition among the four race/ethnic groups of the cohort, in addition to non-Hispanic Caucasians. This chapter is an overview of the SWAN study design and includes a brief description of SWAN's comprehensive data collection. The data being collected mirrors the specific aims, reflecting the belief that the biologic process of menopause occurs within the context of diverse personality characteristics, psychosocial factors, and behavioral attributes as well as an ethnic and cultural context. Consequently, the methods used to recruit this important sample of multiethnic women are described and the strengths and limitations of those methods are discussed.

II. O V E R V I E W

OF THE STUDY DESIGN

SWAN is organized as a prospective, multicenter, multiethnic, multidisciplinary study of the natural history of the menopausal transition, under the auspices of a cooperative agreement between the National Institutes of Health and seven sites with clinical examination facilities, a data coordinating center, and two laboratories. Appendix 1 shows the locations, investigators, and roles of those investigators. SWAN includes a large and representative sample of African-American, non-Hispanic Caucasian, Chinese, Hispanic, and Japanese women. The study design, developed in a collaborative process, consists of a cross-sectional study and a longitudinal cohort study, both of which employ common protocols across the seven sites with clinical examination facilities. Focus groups were conducted to inform the development of the study design and the protocols and to ensure the relevance and the appropriateness of the protocols to the multiethnic cohort. The SWAN Cross-sectional Study consisted of a 15- to a 20-minute telephone interview (or face-to face interview in those instances in which no telephone number could be associated with the sampled respondent). The interview was administered to 16,065 women aged 40-55 years who were randomly selected from sampling frames established at each site with clinical examination facilities (described more fully in Section IV). The two purposes of the SWAN Cross-sectional Study were to identify women eligible for study longitudinally and assess, cross-sectionally, those factors associated with the age at natural menopause, the prevalence of surgical menopause, symptoms of menopause, health status, and health care use. Additional information about the eligibility criteria, sampling frames, and characteristics of participants are discussed in Section IV. On completion of the interview, eligibility for the longitudinal study was determined and women meeting the eligibility criteria were invited to join that cohort. The annual examinations of the SWAN Longitudinal Study include

CHAPTER 11 SWAN Cohort Study TABLE I

177

The Breadth of Measures and Their Frequency of Ascertainment in the SWAN Longitudinal Study

Type of measurement

Frequency a

Questionnaire

Type of measurement

Frequency

a

Specimen data b

Socioeconomic status Medical history Psychosocial environment Lifestyle behaviors Menstrual status Natural/surgical menopause Symptoms Use of medical services Use of medications Quality of life Sexual activity Food frequency

Annual Annual Annual Annual Annual Annual Annual Annual Annual Annual Annual Base line, F/U-04

Clinic measurements

Blood (serum) E 2, FSH, SHBG, DHEAS, testosterone TSH Glucose and insulin Fibrinogen, factor VII, PAI-1, TPA antigen Lipid profile, HDL subfractions, lipoprotein (a) Biochemical bone turnover markers (at five sites) Serum repository specimens

Annual Base line Annual Base line Base line, F/U-01, 02, 03, 05 Base line, F/U-01, 02, 03 Annual

Urine N-Teleopeptides (at five sites) Urine repository specimens

Annual Annual

Other data collection beyond annual evaluation

Anthropometry Blood pressure Bone density (at five sites)

Annual Annual Annual

Abstract medical records for hysterectomy Menstrual calendars Daily Hormone Study (subsample of 900)

Monthly One cycle, annually

a Note: F/U denotes a follow-up examination; the number denotes which follow-up examination. bAbbreviations: E2, estradiol; FSH, follicle-stimulating hormone; SHBG, sex hormone binding globulin; DHEAS, dehydroepiandrosterone sulfate; TSH, thyroid-stimulating hormone; PAI-1, plasminogen activator inhibitor-1; TPA, tissue plasminogen activator; HDL, high-density lipoprotein.

questionnaires, blood and urine specimen collection, and physical measures (Table I). Because the SWAN Longitudinal Study is focused on the menopausal transition, unique data collection activities are required. For example, the annual examinations are scheduled for days 2 - 5 after bleeding c o m m e n c e s to standardize serum h o r m o n e measures to the early phase of the menstrual cycle. In addition, the cohort is followed with monthly menstrual cycle calendars and a subset of the cohort participates in daily urine collection as well as maintaining a daily symptom diary for one complete menstrual cycle on an annual basis. The following section describes the data collection more fully.

III.

DATA A.

COLLECTION Theoretical

Approaches

As a multidisciplinary study, the SWAN data collection instruments and approaches were developed to address the potential contribution of the multiple theories surrounding the study of the menopausal transition [6]. For example, the biological approach ascribes the experience of the menopause particularly within the framework of alterations in metabolism and endocrine status. The psychological~psychosocial approach maintains the importance of stressors and losses as catalysts for symptoms. The sociocultural/environmental approach indicates that cultural constructs and lifestyle factors define our responses toward the menopause and the presentation of potential symptoms. Finally, the feminist

theory views the menopause as a normal developmental stage with its own unique challenges. The instruments and data collection activities of SWAN have reflected an inclusive approach that acknowledges the need for and value of each of these perspectives, while minimizing the reductionist approach to studying and interpreting the characteristics of the menopause transition.

B. Types of Data The types of data collected from SWAN participants in the annual examinations are shown below in examples that include the type of construct and contributing variables. Construct

Variable

Acculturation

Language used, cultural and religious practices, dietary practices Weight changes associated with each pregnancy, weight cycling Use of contraceptive methodologies Use of hormone preparations, past use of oral contraceptives, and current contraception methodology Smoking history and current passive smoke exposure; current caffeine and alcohol consumption; diet and dietary practices, including use of supplements; amount and frequency of physical activity practices, including planned exercise

Body size history Contraception Hormone use practices

Lifestyle behaviors

178

SOWERS ET AL.

Construct

Variable

Construct

Variable

Menstrual status

Current menstrual bleeding characteristics and their variation according to timing, duration, and intensity; usual premenstrual symptoms, if any

Bone status and its turnover

Psychological status

Depression, hostility, and stress

Bone mineral density of the femoral head, lumbar spine, and total body (from five sites with bone densitometry facilities); biochemical measures of bone formation and resorption

Recent medical care utilization

Frequency of prevention behaviors, including Pap smear, physical breast exam, and physician visit for health problem or routine check-up; use of complementary and alternative health approaches; health insurance

Carbohydrate and energy metabolism

Glucose, insulin and thyroid-stimulating hormone concentrations (the latter at base line)

Clotting factors

Fibrinogen, factor VIIc, plasminogen activator inhibitor-1, tissue plasminogen activator antigen

Relationships

Number, nature, and satisfaction with relationships; life satisfaction

Lipid metabolism

Reproductive history

Age at menarche, gravidity, parity, pregnancy losses, infertility, lactation practices

Total cholesterol, triglycerides, highand low-density lipoprotein cholesterol, high-density lipoprotein cholesterol subfractions, lipoprotein (a)

Reproductive hormones

Self-perception

Quality of life, health status, degree of physical activity

Estradiol, follicle-stimulating hormone, sex hormone binding globulin, progesterone, and testosterone

Sexuality

Types of practices, satisfaction, and attitudes toward sex

Significant life events

Marriage, divorce, death or birth in family, change in or loss of job, illness, social support, occupational stress (autonomy)

Significant medical history

Diagnosis by a physician of hypertension, cardiovascular disease, malignancies, or thyroid disease; fractures, pelvic surgery, urinary incontinence, current medications, family history of health events

Sociodemographic status

Age, birth date, birthplace, marital status, level of education, income of household, occupation and the physicality of one's work, household composition

Social environment

Discrimination, religiosity, and spirituality

The interview data will be linked with other information being collected annually that describes the physical and hormone status of enrollees. The general areas of interest and the variables that contribute to the constructs are shown below.

Construct

Variable

Blood pressure

Resting systolic and diastolic blood pressure, resting heart rate

Body composition and body topology

Weight, height, waist and hip circumference, body composition (the latter from five sites with bone densitometry facilities)

Two additional data collection elements, monthly menstrual cycle calendars and daily specimen/diary collection, are important in more precisely characterizing the transitional process. The monthly menstrual calendars provide a record of the changing characteristics of menstrual bleeding from month to month. These monthly calendars also include a record of the use of oral contraceptives or other hormones, symptoms, and the occurrence of any gynecological surgery or procedures. A more extensive calendar is in use at three clinical sites to ascertain lifestyle factors including dieting, shift work, exercise practice, and smoking behavior, as well as stress. A Daily H o r m o n e Study (DHS) contributes to the SWAN specific aims by providing a more complete understanding of the variation in hormone concentrations throughout menstrual cycles (or equivalent time periods) of the perimenopausal transition and characterizing changes in the nature and frequency of within-cycle events, such as ovulation. Blood and urine specimens are being collected from a subsample of 900 women, with participation at each of the seven sites and from each of the race/ethnic groups as well as the non-Hispanic Caucasian women. Participants collect daily urine specimens for one complete menstrual cycle each year. These urine specimens are assayed for excretion products of the pituitary (the gonadotropins, luteinizing hormone, and follicle-stimulating hormone) and the ovary (estrone conjugate and pregnanediol glucuronide). The goal is to describe the changes in the hormone concentrations at important points during the menopausal transition and prior to the final menstrual period. The DHS also includes a daily diary to characterize symptoms and social dimensions of each day during the cycle of the daily urine collection.

CHAPTER 11 SWAN Cohort Study

179

TABLE II The Site-Specific Recruiting Goals for Race/Ethnicity Percentage in the SWAN Longitudinal Study of the Menopausal Transition in Seven Geographic Locales a Primary race/ethnic self-identification (%) AfricanAmerican

Geographic locale Detroit, Michigan (Ypsilanti/Inkster) Chicago, Illinois (Morgan Park/Beverly) Boston/Cambridge, Massachusetts Pittsburgh, Pennsylvania (Allegheny County) Oakland, California (plus Hayward and Richmond) Newark, New Jersey (Hudson County) Los Angeles, California (South Bay/Sawtelle)

Chinese Hispanic Japanese Caucasian

66 55 45 33

33 45 55 66 45 33 45

55 66 55

a Each site was to recruit at least 450 women to the SWAN Longitudinal Study with the proportion of primary race/ ethnicity among women described in the table.

Collectively, the monthly menstrual calendars and the Daily Hormone Study help to refine the definition of the menopause by providing more frequent and supplemental data during the transitional period. It is anticipated that an outgrowth will be the provision of more comprehensive understanding of the bleeding and hormone markers of the onset of perimenopause and the stages within the transition process.

recruitment strategy that they considered optimal for the Study's scientific questions, the characteristics of the local site (including access to clinical facilities), and the specific minority population to be evaluated. The result was the use of multiple sampling frames and multiple sampling approaches implemented in a coordinated manner. SWAN thus also provides the opportunity to describe and evaluate the various sampling frames, the sampling approaches to recruiting women from those frames, and the relative impact of using the various sampling frames and approaches.

IV. SAMPLING AND RECRUITMENT A. Overview

B.

The SWAN sampling and recruiting was implemented in seven locations in the United States: Boston, Chicago, the Detroit area, Los Angeles, Newark, Pittsburgh, and Oakland, California. The recruitment goal for each of the seven sites was to obtain representative samples of at least 450 women [non-Hispanic Caucasian women and one designated minority group (African-American, Chinese, Hispanic, and Japanese)] in a proportion specific for each site (see Table II). To achieve this goal, each site developed a sampling and

SWAN Recruitment

As indicated previously, recruitment for SWAN was undertaken as a two-step process (Table III). The first recruitment step involved a cross-sectional study to act as a sampiing frame for the SWAN Longitudinal Study. The second recruitment step was the development of a longitudinal study cohort from among the SWAN Cross-sectional Study enrollees. To be eligible for participation in SWAN Cross-sectional Study, women had to meet the following criteria:

TABLE III Summary of Sampling Units Contacted to Determine Eligibility in the Two-Step Process to Identify SWAN Longitudinal Study Enrollees Recruitment step Cross-sectional study recruitment, sampling units contacted Longitudinal study recruitment, units from the cross-sectional study

Sampling units

No. eligible

No. recruited

Response rate (%)

202,985

34,985

16,065

46.6

3,306

50.7

16,065

6,521 a

aThere were 36 Caucasian women included in this table who were "filtered out" (i.e., eligible to enter the cohort, but not recruited because target recruitment had been met).

180

S O W E R S ET AL.

1. Primary residence in designated geographic area 2. Ablility to speak English or designated other language m Spanish, Cantonese, or Japanese 3. Age 4 0 - 5 5 years at time of contact 4. Cognitive ability to provide verbal informed consent 5. Membership in a specific site's targeted ethnic groups To identify women eligible for the cross-sectional study, sites screened the constituent sampling units from the sampiing frames. Depending on the site, the sampling units were the households, telephone numbers, or individual names of women; the sampling frames were the listings of the sampiing units. Study-wide, 202,485 sampling units from sampiing frames were evaluated, leading to the identification of 34,446 eligible women. Of these, 16,065 women completed the SWAN Cross-sectional Study. The eligibility criteria for the SWAN Longitudinal Study wereas follows: 1. Aged 4 2 - 5 2 years 2. No surgical removal of the uterus and/or both ovaries 3. Not currently using exogenous hormone preparations affecting ovarian function 4. At least one menstrual period in the previous 3 months 5. Self-identification with one of each site's designated race/ ethnic group From the SWAN Cross-sectional Study, 6557 women were identified as eligible for longitudinal study. Of these women, 36 Caucasian women were "filtered out," i.e., they were not asked to participate in the longitudinal study because the site had met its target sample size. Of the remaining 6521 women, 3306 were recruited for the SWAN Longitudinal Study (see Table IV). This is the cohort currently being followed.

TABLE IV

Geographic Primary locale frame type" Boston Chicago Detroit

List List List

Frames

To identify the cohort for the longitudinal study, sites had to address successfully three competing requirements. These requirements were to (1) identify populations representative of a defined and diverse community, (2) recruit women from a specified race/ethnic minority group in a proportion significantly greater than the groups' proportion in the general United States population, and (3) implement the recruitment in a defined and circumscribed geographic locale so that relatively intense longitudinal clinical studies could be sustained. To meet these requirements and to be cost-efficient, the seven sites selected study communities that had a relatively higher density of the particular racial/ethnic minority group designated for recruitment. Then, individual sites utilized a variety of sampling frames from which the sample(s) would be drawn. In general, these sampling frames included telephone numbers randomly generated from random digit dialing (RDD)-based and list-based frames (Table IV). The following sections describe both types of frames in the context of the SWAN geographic locations and racial/ethnic group requirements. Appendices 2 and 3 provide specific information about the sampling approach at each clinical site. 1. RANDOM DIGIT DIALING-BASED FRAMES

The sampling unit for RDD frames was a telephone number and the only eligibility information available from an RDD frame consisted of the telephone number itself, i.e., the geographic location associated with the telephone number's exchange. Three sites [Newark area, Pittsburgh area, and Los Angeles (Table IV)] use an RDD-based sampling frame as the major frame. Two of these sites (Newark and Los Angeles) used list-assisted RDD-based sampling, and the Pitts-

The Primary Sampling Frame, Supplemental Frames, and Type of Supplemental Information Provided to the Primary Frame According to Geographic Locales Primary frameb

Oakland List Los Angeles RDD

City census listing Enrollmentlist from earlier study Electricalutility company customer listing for communitycensus HMO enrollment list RDD 3+ approach

Pittsburgh Newark

RDD RDD 3+ approach

RDD RDD

C. T h e S a m p l i n g

Supplemental frames" None None None None VRL, telephone directory list, ethnic organization membershiplists, snowball VRL Snowball

Supplemental information added to frames Telephone numbers, face-to-facecontact None Telephone directory, race from organization lists and VRL, face-to-facecontact None

Telephone directory None

RDD, Random digit dialing. is a variation in random digit dialing that increases the likelihood that telephone numbers are households and not commercialfirms. cVRL, Voter registration list.

a

b 3+

CHAPTER 11 SWAN Cohort Study burgh site used voter registration lists as their important secondary frame. Sites with a primary RDD sampling frame implemented the following steps: 1. Each telephone number was screened to determine if it represented a household. 2. The household was then screened to verify that the household was in the target geographic area and to determine if any woman age-eligible for the cross-sectional study was in residence. 3. Personnel then determined whether the household included at least one age-eligible woman who was Caucasian or was from the site's designated racial/ethnic minority group. All three of the sites that used the RDD sampling frame supplemented the RDD frame with list-based or "snowball" (referral by other participants) sampling frames. 2. LIST-BASED FRAMES At four sites, lists representing households (Detroit area) or individual women (Boston, Chicago, and Oakland areas) comprised the primary sampling frames. The list-based frames were varied and included a state-mandated census in Boston, an electrical utility customer listing in the Detroit area, a census from a previous study in the Chicago area, and a health maintenance organization (HMO) enrollment list in the Oakland, California area. Although each of these sites recruited its entire sample using a list sampling frame, only one site had a single frame that included all the information necessary to determine eligibility a priori (age, address, telephone, geographic area, race/ethnicity, gender) for recruiting to the SWAN Cross-sectional Study. That single list-based frame had been developed in a previous research study.

D.

Sampling Strategies

Specific sampling procedures varied across sites and were a function of the sampling frame(s) used and the level of information available with the frame(s). Sampling procedures included conducting a census, implementing an area probability sampling, and identifying acquaintance networks with snowballing. For example, the Detroit site conducted a census in which every household in the selected geographic area was enumerated and contacted, with the probability of selection being 1. Area probability samples were developed and implemented in the Chicago, Oakland, and Pittsburgh areas, where women were sampled with a known probability of selection that was <1. In addition to using their R D D samples, the sites at Los Angeles and Newark also used snowball sampling. With snowball procedures, selection probabilities could not be determined. To minimize the likelihood of selection bias from factors such as seasonal variation or systematic sampling, sites

181 organized their contacts with the sampling units into "batches." Each batch was a random sample within the overall sample from the particular site. The approximate size of each batch was derived from the number of sampling units that the site projected could be contacted in a 2-month time period. For those households with more than one eligible woman, a single eligible woman was sampled by one of two approaches. Five sites sampled by selecting the woman in the household with the most recent birthday (month and day only), an approach referred to as the "birthday" method. Two sites (Los Angeles and New Jersey) sampled the first woman contacted who was willing to provide information. Only one household member was sampled to avoid clustering of women within households for study variables such as health status and health care use. All sites used computer-assisted telephone interviewing (CATI) with standardized interviews and scripts to facilitate contact in a consistent manner, thereby minimizing the opportunity for information bias. When telephone numbers were unavailable for a sizable proportion of the population, as was the case for the Detroit area census, interviewers directly contacted those households without listed telephone numbers, face to face.

E. C o m p u t a t i o n

of the Response Rates

C o m m o n disposition codes were used to define the status of each sampling unit eligible for contact and to facilitate the computation of the response rates (see Table V). Response rates for the SWAN Cross-sectional Study were calculated as follows:

TABLE V Disposition Codes for Units of Telephone Numbers, Households, or Individual Women in SWAN 1. Unusablesampling unit (i.e., business or fax telephone number, deceased woman) 2. No contact, unknown usability (i.e., busy signals, answering machine, never home, moved and cannot be traced) 3. Contactmade, unknown cross-sectional eligibility (i.e., hang-up, refusal to be screened for the cross-sectional screening interview) 4. Contactmade, ineligible for the cross-sectional interview 5. Contactmade, cross-sectional eligible, unknown cohort eligibility (incomplete cross-sectional screening interview) 6. Completedcross-sectional screening interview; cohort ineligible or cohort eligible and an attempt was made to recruit into the cohort 7. Completedcross-sectional screening interview; cohort eligible but no attempt was made to recruit into the cohort (i.e., "filtered" at the point of cohort recruitment because of adequate recruitment) 8. Cross-sectional eligible but no attempt was made to recruit for crosssectional interview (i.e., "filtered" at point of cross-sectional interview)

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S O W E R S ET AL.

1. The proportion of eligible women was calculated among women with known eligibility (disposition codes 4 through 8):Pe = (5 + 6 + 7 + 8)/(4 + 5 + 6 + 7 + 8). 2. This proportion then was applied to the total number of sampling units with unknown eligibility (disposition codes 2 and 3), to estimate the potential number of eligible women among those with unknown eligibility (assuming it was the same as those with known eligibility): pe (2 + 3) = Eu. 3. The total number of women whom sites attempted to recruit for the Cross-sectional Study was computed as the sum of known eligible women recruited (5 + 6 + 7) and the estimated potential number of women among those with unknown eligibility (Eu): (5 + 6 + 7) + E u = R. 4. The participation rate was estimated as the number of women participating in and completing the interview divided by the estimated total number of eligible women whom the site attempted to recruit: (6 + 7)/R. Women known to be eligible but not asked to participate in the cross-sectional interview [because a sufficient number had been recruited (disposition 8)] are included in the calculation of percentage eligible. They were excluded from the calculation of the cross-sectional participation rate because they were not actually recruited to complete the crosssectional study. Table VI gives the distribution of sampling units for the SWAN Cross-sectional Study disposition codes by site and indicates the greater efficiency from those frames containing more eligibility criteria information. The percentage of unusable units sampled (disposition code 1) was lower for list-based sites ( 0 - 1 4 % ) as compared with the sites using primarily RDD-based sampling ( 1 9 - 3 5 % ) . Sites primarily using a list-based frame also had a lower ( < 11%) percentage of sampled units with no contact due to busy signals, answering machines, never being home or moved and inability to trace (disposition code 2) as compared with the RDD-

TABLE VI

based sampling (11-25%). The two list-based sites whose frames had substantial eligibility and recruiting information (an H M O enrollment list and a list from a previous research study) required the fewest number of sampling units (<3500) to reach their cohort targets. In contrast, those sites whose frames did not include information about the eligibility criteria information sampled 24,283 to 78,914 sampling units to meet their specific cohort targets. The percentage of sampled units with contact made but unknown cross-sectional eligibility (i.e., hang-ups and disposition code 3) varied widely across sites, but was not related to RDD-based versus list-based frame status. The percentage of known ineligible units (disposition code 4) did not differ greatly across sites, with the exception of the Detroit area, where the investigators were conducting a census of all households and were not allowed into three apartment dwellings. The percentage of eligible women who began but did not complete an interview (disposition code 5) was uniformly low across all sites (less than 3%).

E

Response Rates

A total of 202,985 sampling units were screened for potential participation in the SWAN Cross-sectional Study. Of these, 34,985 were defined as eligible and 16,065 completed the interview, for an overall response rate of 46.6%. Of these, 6521 women were cohort-eligible and were asked to participate in the SWAN Longitudinal Study; a total of 3306 women entered the cohort, for an overall response rate of 50.7% (Table III). Response rates did not vary statistically by age, marital status, parity, or menopausal status (see Table VII) whether considering the overall group or just non-Hispanic Caucasian women. However, women with a high school education or less (response rates of 37.3 and 40.8%, respectively) were

The Number and Percentage of Sampling Units Used in the SWAN Cross-Sectional Study by Site and Common Disposition Codes Number (percentage) per site"

Disposition code 1 2 3 4 5 6 7 8 Site total a

Boston (L)

Chicago(L)

Detroit(L)

Los Angeles (R)

Newark(R)

Pittsburgh(R)

Oakland(L)

Totalacross sites

2517 (13.6) 9524 (51.3) 1188 (6.4) 2778 (15.0) 328 (1.8) 2233(12.0) 0 (0.0) 0 (0.0)

26 (1.1) 43 (1.8) 203 (8.3) 742 (30.3) 43 (1.8) 1393(56.8) 0 (0.0) 0 (0.0)

1006 (4.1) 2587 (10.6) 2504 (10.3) 14,937(61.5) 662 (2.7) 2551 (10.5) 36 (0.1) 0 (0.0)

10,804(24.8) 7909 (18.1) 4749 (10.9) 17,360(39.8) 104 (0.2) 2242 (5.1) 0 (0.0) 446 (1.0)

15,504(19.6) 19,636(24.9) 21,249(26.9) 18,774(23.8) 261 (0.3) 3490 (4.4) 0 (0.0) 0 (0.0)

11,239(35.3) 3540 (11.1) 1552 (4.9) 12,027(37.8) 596 (1.9) 2604 (8.2) 0 (0.0) 278 (0.9)

8 (0.2) 308 ( 9 . 3 ) 434 (13.1) 1025 (30.9) 25 (0.8) 1516(45.7) 0 (0.0) 4 (0.1)

41,100(20.2) 43,547(21.4) 31,879(15.7) 67,643(33.3) 2019 (1.0) 16,029 (7.9) 36(0.02) 728 (0.4)

2450

24,283

43,614

78,914

31,836

3320

18,568

L, List based; R, RDD based. Numbers in parentheses are percentages.

202,985

183

CHAPTER 11 SWAN Cohort Study

TABLE V I I

Longitudinal Cohort Percentage Participation among Cohort-Eligible Women a Overall sample participating in cohort (%)

Caucasians participating in cohort (%)

Overall

50.1 (48.9-51.4)

48.0 (46.2-49.7)

Age (years) 42-45 46-49 50-52

49.4 (47.7-51.1) 51.9 (49.9-53.8) 47.4 (43.7-51.1)

47.3 (44.9-49.8) 49.5 (46.7-52.4) 45.1 (39.8-50.4)

Education Less than high school High school degree Some college College degree Postcollege

40.8 37.3 52.4 56.1 62.1

24.7 29.7 51.0 52.5 60.8

Smoking Never smoked Past smoking Current smoking

51.6 (49.9-53.2) 52.2 (49.8-54.8) 42.3 (39.6-45.0)

49.8 (47.3-52.3) 51.7 (48.5-54.9) 38.1 (34.3-41.9)

Difficulty in paying for basics Very hard Somewhat hard Not hard at all

42.5 (38.9-46.2) 47.0 (44.8-49.1) 53.5 (51.9-55.2)

38.6 (32.2-45.0) 44.2 (41.0-47.5) 50.6 (48.4-52.8)

Marital status Never married Married/living as married Separated/widowed/divorced

50.1 (46.8-53.4) 51.0 (49.5-52.5) 47.7 (45.1-50.3)

47.3 (42.3-52.3) 48.7 (46.6-50.8) 45.6 (41.5-49.8)

Parity 1 + children No children

49.8 (48.4-51.1) 52.0 (49.0-55.0)

47.1 (45.2-49.1) 50.6 (46.9-54.2)

Race/ethnicity African-American Caucasian Chinese Hispanic Japanese

54.2 48.0 69.2 34.1 63.1

(51.9-56.6) (46.2-49.7) (64.4-73.9) (30.8-37.5) (58.6-67.6)

Site Boston Chicago Detroit Los Angeles Newark Oakland Pittsburgh

48.9 73.7 58.9 53.1 29.6 67.3 42.8

(45.7-52.2) (70.1-77.2) (55.7-62.1) (49.9-56.3) (27.2-32.1) (63.8-70.8) (39.8-45.7)

54.9 76.5 59.7 44.1 23.4 65.2 41.4

(50.2-59.6) (71.4-81.6) (54.7-64.8) (39.6-48.5) (19.9-26.9) (60.0-70.4) (37.8-45.0)

Self-reported health Excellent Very good Good Fair Poor

52.5 52.9 48.8 44.1 40.5

(49.8-55.2) (50.9-55.0) (46.5-51.0) (40.7-47.5) (32.7-48.3)

50.8 48.5 45.4 43.6 38.1

(47.5-54.1) (45.8-51.2) (41.8-48.9) (36.5-50.7) (23.2-53.0)

Menopause status Premenopausal Early perimenopausal

49.4 (47.7-51.0) 51.1 (49.3-52.9)

Subject characteristic

(36.8-44.9) (34.9-39.7) (50.2-54.6) (53.2-59.0) (59.3-64.9)

(15.9-33.5) (26.4-33.0) (47.7-54.2) (48.6-56.4) (57.4-64.2)

47.4 (45.0-49.8) 48.5 (45.9-51.0)

apercentages are +95% CI. Participation is according to sociodemographic and lifestyle characteristics in the full sample (n = 6445) and restricted to Caucasians only (n = 3170). The 95% confidence intervals allow the comparison of frequency within each sociodemographic or lifestyle variable; statistically significant differences are shown in bold.

less likely to participate than women with some college education (response rate of 52.4%). Women with a postcollege educational experience were the most likely to participate and had a response rate of 62.1%. Likewise, those women who reported that it was somewhat hard or very hard to pay for basics (i.e., food, shelter and health care) were less likely to participate. Their response rates were 47.0 and 42.5%, respectively, as compared to the response rate of 53.5% for women who reported no difficulty in paying for basics. As reported in Table VII, women who currently smoked were significantly less likely to participate than women who had never smoked or who had quit smoking. Participation varied according to self-reported race/ethnicity status, with Chinese (69.2%) and Japanese (63.1%) being the most likely to participate followed by AfricanAmerican (54.2%), non-Hispanic Caucasian (48.0%), and Hispanic women (34.1%). Because Chinese, Japanese, and Hispanic women were recruited at single sites, the degree of participation might be representative of other site characteristics rather than race or ethnicity. The site differences in response rates, ranging from 29.6% at Newark to 73.7% in Chicago, were believed to reflect, in part, differences in site recruitment strategies. Therefore, we evaluated response rates based on whether the site's recruitment was primarily a list-based or an RDD-based approach (see Table VIII). The response rate for sites whose primary frames were list based was 60.7% compared with a 40.3% response rate for RDD sites. Table VIII shows that although sites that relied primarily on the RDD-based approach had lower percentage participation as compared to participation at list-based sites, similar characteristics were likely to be associated with nonparticipation. Irrespective of recruitment strategy, women with less education and women who smoked or had difficulty in paying for basics were less likely to participate. Importantly for this study, response rates according to menopause status did not vary by recruitment strategy.

V. STRENGTHS AND LIMITATIONS OF SWAN SWAN has successfully recruited and enrolled a large community-based sample of women with substantial representation from five racial/ethnic groups. To do so, SWAN incorporated a wide range of sampling frames and diverse sampling approaches, including the extensive use of listbased frames and supplemental frames or supplemental information to the primary frames. Theoretically, it would have been desirable to select participants as a national probability sample so that the study findings could be generalized to the national population of midlife women, similar to the National Health and Nutrition Examination Surveys (NHANES) [7]. However, the demands of efficiently implementing a longitudinal study that requires intensive and ongoing (annual and

184

SOWERS ET AL. TABLE VIII Longitudinal Cohort Percentage Participation According to Sociodemographic and Lifestyle Characteristics a

Subject Characteristics Overall Age (years) 42-45 46-48 50-52 Education Less than high school High school degree Some college College degree Postcollege Smoking Never smoked Past smoking Current smoking Difficulty in paying for basics Very hard Somewhat hard Not hard at all Marital status Never married Married/living as married Separated/widowed/divorced Parity 1+ children No children Race/ethnicity African-American Caucasian Chinese Hispanic Japanese Self-reported health Excellent Very good Good Fair Poor Menopause status Premenopausal Early perimenopausal

At list-based sites

At RDD-based sites

60.7 (59.0-62.4)

40.3 (38.7-42.0)

59.9 (57.4-62.3) 63.4 (60.7-66.1) 55.3 (50.2-60.5)

40.1 (37.8-42.4) 41.0 (38.3-43.7) 39.1 (33.9-44.3)

48.7 (41.8-55.7) 44.8 (41.0-48.6) 62.8 (59.7-65.9) 69.0 (65.1-72.8) 71.6 (68.1-75.0)

36.6 (31.7-41.5) 31.5 (28.4-34.6) 42.7 (39.7-45.8) 43.5 (39.5-47.6) 49.6 (45.2-54.0)

63.3 (60.9-65.7) 63.1 (59.6-66.5) 51.8 (47.9-55.7)

42.2 (39.9-44.4) 41.6 (38.1-45.1) 33.3 (29.7-36.8)

58.5 (52.6-64.4) 56.5 (53.3-59.6) 63.5 (61.3-65.7)

32.4 (27.9-36.8) 38.8 (35.9-41.6) 43.3 (41.0-45.6)

57.2 (53.0-61.4) 63.4 (61.2-65.6) 56.9 (53.3-60.6)

38.5 (33.2-43.7) 41.4 (39.4-43.4) 37.5 (33.8-41.2)

60.4 (58.5-62.3) 62.1 (58.0-66.2)

40.0 (38.3-41.9) 41.5 (37.2-45.8)

56.4 (53.8-59.1) 62.7 (60.2-65.2) 69.2 (64.4-73.9) -m

45.7 (40.4-50.9) 36.4 (34.1-38.6) 34.1 (30.8-37.5) 63.1 (58.6-67.6)

64.7 (61.0-68.4) 65.4 (62.5-68.3) 56.3 (53.1-59.4) 55.6 (50.7-60.5) 46.4 (34.5-58.2)

41.1 (37.4-44.8) 41.9 (39.1-44.7) 41.4 (38.3-44.5) 33.6 (29.1-38.0) 35.7 (25.4-46.0)

61.4 (59.0-63.8) 60.0 (57.5-62.4)

39.3 (37.1-41.5) 41.8 (39.2-44.3)

apercentages are +_95%CI. Data are from cohort-eligible women with sites categorized as using list-based sampling (n = 3099) or using RDDbased sampling (n = 3346). RDD-based sites were Los Angeles, Newark, and Pittsburgh locales; list-based sites were Boston, Chicago, Detroit, and Oakland locales. The 95% confidence intervals allow the comparison of frequency within each sociodemographicor lifestyle variable, with statistically significant differences shown in bold.

monthly) clinical data collection could not be met with a national probability sample. Typically, exclusive reliance on list-based frames has been viewed with caution by survey researchers because of concerns about inadequate coverage (and attendant nonrepresentativeness) or the inadequacy of information related to eligibility (and attendant inefficiency). However, in SWAN, the response rate was higher at those sites using lists as their primary sampling frame and the response characteristics (i.e., education level, menopause status) were similar for both list-based and RDD-based sites. Potentially, this is attributable to the combining of several lists prior to sample selection or the use of multiple lists to provide supplemental information to facilitate recruitment after samples were selected. Nonetheless, the response rates suggest that bias from inadequate coverage by list-based frames was no different than the bias that might have resulted from the RDD-based frames. SWAN identified supplemental approaches to use with random digit dialing sampling in small geographic areas because the technique is less efficient in small areas than in large areas [8]. These supplemental approaches helped overcome the widely recognized disadvantages of telephone sampling frames, i.e., approximately 8% of the population in the United States does not have current telephone service, and this rate varies widely depending on the socioeconomic status [9,10]. The degree to which using list-based RDD ameliorated this issue is unknown; however, future studies may need to add four to six lists and use list-assisted RDD. These SWAN recruiting efforts demonstrate the need to have a carefully considered sampling strategy that incorporates flexible approaches and numerous frames. No methodology is clearly right or wrong; nonetheless, it is obvious that frames with the most information related to eligibility criteria were the most efficient. This study adds to our understanding of the complexities of sampling and the need for multiple methods of recruiting to identify information applicable to a broadly diverse population. The importance of SWAN's recruitment strategies can be most appreciated within the context of ethnicity and culture. Almost no studies have simultaneously and comparatively investigated the prevalence of menopausal symptoms in multiethnic/multicultural populations, although there are reported cultural/ethnic differences in symptoms, age at menopause, and bleeding characteristics [ 11 ]. It is unclear if these differences (if there are true differences) are due to hormonal differences (i.e., lower serum estradiol and testosterone levels) or other physiological differences (i.e., differences in immune responses). The differences could readily be associated with cultural perceptions of the menopause and aging, dietary practices, physical activity, body composition (more fat mass or less lean mass), or reproductive practices reflected in parity or age at first conception. Furthermore, the

CHAPTER 11 SWAN Cohort Study role of culture and acculturation (the process of incorporating the customs, norms, identification, and social and working activities from different societies in shaping health status, health belief, and health behaviors) has not been widely applied to the menopausal transition. SWAN has also incorporated data-gathering approaches consistent with major theories and approaches to the menopause [6]. Thus, the biological theory ascribes the experience of the menopause to alterations in metabolism and endocrine status. Typically, with this theory, there is a pronounced focus on ovarian function and, in some, attendant focus on hormone replacement or alternative hormone sources. The psychological/psychosocial approach considers the importance of stressors and losses, particularly as catalysts for symptoms. The approach then suggests the need for social supports, well-developed relationships, and coping skills for a "successful" transition. The sociocultural/environmental approach suggests that culture frames our behaviors and attitudes toward the menopause. The external environment (i.e., passive smoking, occupational exposures, or workplace demands) modifies our biological assets and, in doing so, frames our responsiveness to the transitional events. Finally, feminist theory, with its views that the menopause is a normal developmental stage, urges an understanding of how women achieve control of the experience and become active participants, addressing the challenges of symptoms. The information-gathering activities of SWAN have reflected an inclusive approach that acknowledges the value of each of these to a complex process.

VI. SUMMARY The of SWAN study employs a prospective design that includes sufficient pre- and postmenopausal observations to ensure the separation of menopause-related and age-related physiological changes. Other attributes include the comprehensive standardized data collection related to biological, behavioral, physiological, social, environmental, and cultural factors; specialized data collection methodologies suitable to address the monthly and yearly variation in behavioral and biological patterns; generalizability to community-dwelling populations recruited from major United States population centers; sufficiently large sample sizes and numbers of data points to ensure reliable estimates of associations and relevant effect sizes; and inclusion of sufficient numbers of racial/ethnic minorities to provide comparative information with the non-Hispanic Caucasian population. Because of these attributes, SWAN can contribute new and substantive knowledge about women's health in general and the menopause transition in particular. SWAN is the first national study to describe women at midlife, an understudied age group. Its multidisciplinary approach provides the opportu-

185 nity to consider the contributions of both culture and biology so that we may better understand health in American women.

APPENDIX A. SWAN INVESTIGATORS Clinical Sites Boston Principal Investigator: Robert Neer, M.D. Coprincipal Investigator: Joel Finkelstein, M.D. Coinvestigators: Josh Alexander, Ph.D.; Andrew Arnold, M.D.; David MacLaughlin, Ph.D.; Richard Pasternak, M.D. Biostatistician: David Schoenfeld, Ph.D. Project Manager: Tracy Thomas, B.A. Chicago Principal Investigator: Lynda Powell, Ph.D. Coprincipal Investigator: Denis Evans, M.D. Biostatistician: Peter Meyer, Ph.D. Project Manager: Diedre Wesley Data Manager: Gerard Kaszubowski Detroit Principal Investigator: MaryFran Sowers, Ph.D. Coprincipal Investigators: Sioban Harlow, Ph.D.; John Randolph, M.D. Coinvestigators: Carolyn Sampselle, Ph.D.; Nancy Reame, Ph.D. Biostatisticians: Roderick Little, Ph.D.; M. Anthony Schork, Ph.D. Project Manager: Vanessa Harris, M.P.H. Data Managers: Ruth Sanchez-Pena, M.S.; Gavin Welch, M.P.H. Los Angeles Principal Investigator: Gail Greendale, M.D. Coprincipal Investigator: Stanley Korneman, M.D. Project Manager: Miriam Schocken, Ph.D.

Newark Principal Investigator: Gerson Weiss, M.D. Coprincipal Investigator: Nanette Santoro, M.D. Biostatistician: Joan Skurnick, Ph.D. Project Manager: Ann Reinert Data Manager: Pat McTerrell Oakland Principal Investigator: Ellen Gold, Ph.D. Coprincipal Investigator: Barbara Sternfeld, Ph.D. Coinvestigators: Barbara Abrams, Ph.D.; Shelley Adler, Ph.D.; Gladys Block, Ph.D.; Maradee Davis, Ph.D.; Bruce Ettinger, M.D., William Lasley, Ph.D.; Marion Lee, Ph.D.; Helen Schauffler, Ph.D.; Barbara Sommer, Ph.D. Biostatistician: Steven Samuels, Ph.D.

1

8

6

S

O

W

Project Manager: Sarah Rowell, M.S. Data Manager: Marianne O'Neill Rasor, M.A.

Pittsburgh Principal Investigator: Karen Matthews, Ph.D. Coprincipal Investigator: Jane Cauley, Ph.D. Coinvestigators: Joyce Bromberger, Ph.D.; Charlotte Brown, Ph.D.; Kim Sutton-Tyrell, Ph.D.; Sidney Wolfson, M.D. Data Manager: Nancy Remaley, M.S.I.S.

Coordinating Center Principal Investigator: Sonja McKinlay, Ph.D. Coprincipal Investigator: Sybil Crawford, Ph.D. Project Directors: Juli Bradsher, Ph.D.; Kay Johannes, Ph.D. Project Manager: Patricia McGaffigan Data Coordinator: Beth Willis

Laboratory~University of Michigan Principal Investigator: Rees Midgley, M.D. Coprincipal Investigator: Daniel McConnell, Ph.D. Coinvestigator: Barry England, Ph.D. Laboratory Manager: Kimberly Gonzalez, M.T. Systems Analyst: Mark Davis, B.S.

Laboratory~Medical Research Laboratory (MRL) Principal Investigator: Evan Stein, M.D. Coinvestigator: Paula Steiner

Steering Committee Chair

E

R

S

E T AL.

phone numbers were identified through individual contact. The sampling plan was implemented initially by New England Research Institutes, Inc. and subsequently by the California Survey Research Services.

B. Chicago Site The Chicago Health and Aging Project (CHAP) database, a census of all residents initiated prior to SWAN, served as the sampling frame for the Morgan Park and Beverly neighborhoods in Chicago. The CHAP frame ultimately included the complete name, address, gender, age, and race of individuals. About 1% of records with missing age, gender, or race data on the CHAP frame was excluded from SWAN sampling. The sampling approach involved stratification by race (African-American and Caucasian). A random number generated from a uniform distribution between 0 and 1 was assigned to each woman in the sampling frame. Subjects were paired with random numbers based on their position in the sampling group. A sampling fraction was computed as the required race-specific sample size divided by the racial/ethnicspecific frame size. Women whose random numbers were less than or equal to the sampling fraction were included in the sample, i.e., simple random sampling was employed within each race stratum.

Jennifer Kelsey, Ph.D.

Project Officers National Institute on Aging (NIA): Sherry Sherman, Ph.D.; Marcia Ory, Ph.D. National Institute of Nursing Research (NINR): Carole Hudgings, Ph.D.

A P P E N D I X B. SPECIFIC S A M P L I N G AND R E C R U I T I N G STRATEGIES BY SITES WITH LIST-BASED P R I M A R Y SAMPLING FRAMES A. Boston Site The list-based frame used by the Boston SWAN site used the Spring, 1995 Massachusetts Census from all 22 wards in Boston. This census is updated annually and contains the name, address, gender, and age of the residents, but not their race/ethnicity or telephone numbers. The listed age, however, was not always accurate because this census arbitrarily assigns an age if the actual age is missing. Telephone numbers were obtained for some women from Survey Sampling, Inc., the white pages, and directory assistance. Selected tele-

C. Detroit Area Site A census was conducted of all households in the 40 target Census Block Groups in the Ypsilanti community and the 46 Census Block Groups in the Inkster community (located in the Detroit area). The sampling frame for the Ypsilanti and Inkster communities was based on a household list from the commercial electric utility company. The list contained every household name and address in the geographical area of interest (100% coverage) but did not include gender, telephone number, age, or ethnicity. The probability of selection for each household was 1. Prior to contacting the household sampling units within a given Block Group, the U.S. Census Block Groups were randomly assigned to sampling batches to minimize selection bias. In order to contact households, telephone numbers were obtained from cross-matching with the local telephone listing, a reverse telephone directory, and a commercial listing. About 45% of households were matched with a telephone number. Interviewers contacted those households (face to face) without a telephone number to determine if there was an eligible woman in residence, and, if appropriate, conducted the cross-sectional interview. There were more Caucasian women than African-American women in the census

CHAPTER 11 SWAN Cohort Study

187

area. Therefore, Caucasian women were subsampled from the Cross-sectional Study for the Longitudinal Study at a rate of 25% using an 8-sided die for the last 8 months of recruitment. The sampling plan was developed and implemented by site investigators and staff.

D. O a k l a n d A r e a Site The membership list of the Kaiser Permanente Medical Care Program (KPMCP), which insures approximately 30% of the population in the San Francisco Bay area, acted as the sampling frame for the Oakland, California area [12]. This frame included name, age, gender, address, and telephone number, but not race/ethnicity. From the membership roles, two lists were assembled, one of female members with Chinese surnames and one of female members with non-Chinese surnames [ 13]. All of the Chinese-surnamed women whose home zip codes mapped to either the Richmond, Oakland, or Hayward Kaiser facilities and who were in the appropriate age range were included (n = 2446). The list was randomly ordered using a random number generator and then divided sequentially into batches of 100 and sampled until the cohort recruitment goal of 250 Chinese women was achieved (1400 sampling units required). A similar approach was used for the Caucasian women. In that instance, the list consisted of 4418 women randomly selected from the approximately 47000 nonChinese-surnamed women members of the appropriate age and residing in the same geographic area. Ultimately, 1650 sampling units were sampled to achieve the cohort recruitment goal of 200 Caucasian women. The sampling plan was developed and implemented by site investigators and staff.

APPENDIX

C. S P E C I F I C

SAMPLING

AND R E C R U I T I N G S T R A T E G I E S BY SITES WITH RDD-BASED PRIMARY SAMPLING

a sampling frame that contains all listed households and a significant portion of the unlisted households. Thus, the sampling frame consists of all phone numbers found in any eight-digit sequence that contains at least one listed telephone number. To reduce the number of unprofitable (nonhousehold) calls, but at the cost of some bias caused by removing some telephone households from the frame, the expanded list was developed in an abbreviated fashion in the following way. For each eight-digit sequence in the master list, there was a count of the number of listed numbers. The sampler eliminated any sequence with fewer than a specified number of listed numbers. Some market research houses have used a 2 + or a 3 + standard in which sequences with at least two or at least three listed telephone numbers are retained. The Los Angeles site used a 3+ selection method that eliminated residential household blocks with zero, one, or two listed telephone numbers before initiating random sampling. The screening of households was conducted by California Survey Research Services. Although the Los Angeles SWAN site recruited from census tracts with a higher density of Japanese persons ( 6 - 2 0 % of the population) to meet its recruiting goal, the RDD-based sample required supplementation with lists that were devoted entirely to the recruitment of Japanese women. Thus, the Los Angeles site identified all women aged 4 0 - 5 5 years with a first, middle, or family Japanese name from voter registration lists and sampled 100% of these women. The Los Angeles site also used a frame containing listed telephone numbers with Japanese surnames and sampled 100% of these listings. Additionally, the University of California, Los Angeles (UCLA) site also used snowball sampling. The snowball sample for UCLA consisted of referral by Japanese participants of up to five women without regard to the eligibility of that participant.

FRAMES A. L o s A n g e l e s Site

The RDD samples from South Bay and Sawtelle in the Los Angeles area were created from the RDD frame maintained by Survey Sampling Institute. The list-assisted random digit dialing method combines a number of available phone lists (including white pages, drivers' licenses, and vehicle registrations) into a Master List of the first eight digits of a phone number (the area code, the exchange, and two more digits). This Master List is expanded by a factor of 100 by adding all possible two-digit sequences (00-99) to each Master List entry. This expanded list can be used directly as

B. N e w a r k Site The RDD samples for the Newark area were created from the RDD frame maintained by the Survey Sampling Institute, and the screening of households was conducted by California Survey Research Services. Hudson County in New Jersey was stratified into five areas: Hoboken City, Union City, West New York Township, Jersey City, and the remainder of Hudson County so that census tracts containing higher than average densities of Hispanic households could be oversampled. Random digit dialing was then applied to telephone households in those census tracts. A 3 + selection method was also applied at this site in the same manner as the methodology used at the Los Angeles site. The New Jersey site also used snowball sampling. In New Jersey, snowball sampling involved asking women who

188

SOWERS ET AL.

completed the cohort base line but were ineligible for the cohort to provide the names of up to five w o m e n who were cohort age-eligible and who lived in the target areas in Hudson County.

and samples from the 22 zip codes in which a substantial number of African-American w o m e n were known to reside.

References C. P i t t s b u r g h S i t e The major sampling approach implemented at the Pittsburgh site was RDD. Samples of random telephone numbers for households were generated with probability proportional to size across all nonbusiness telephone exchanges (central office codes, or COCs) and working blocks according to the density of the listed residential telephone numbers in the exchange. Area c o d e - C O C - w o r k i n g block combinations (including the first eight digits of the area code) and exchange were selected systematically whereas the last two numbers of the 10-digit telephone number were randomly generated. This systematic selection of exchanges (COCs) and working blocks provided a self-weighting, equal-probability sample. The first-stage selection of a telephone number represented the selection of a household. If a household was found to contain more than one age-eligible female, a second-stage randomized selection of a female was made using the birthday method. The Pittsburgh sites supplemented their R D D sampling with voter registration lists (VRLs) to improve their capacity to oversample their designated ethnic groups and/or target the age group of interest. The VRLs for Pittsburgh included information on gender, birth date, and address for all registered voters and ethnic identification for about 85% of registered voters in Allegheny County. Telephone numbers for the V R L sample were obtained from the Cole Directory for Pittsburgh and Allegheny County, the white pages, and directory assistance. Several different strategies were used; systematic samples were drawn from the voter registration list, including samples reflecting all of Allegheny County

1. Diczfalusy, E. (1986). Menopause, developing countries and the 21st century. Acta Obstet. Gynecol. Scand., Suppl. 134, 45. 2. U.S. Congress, Office of Technology Assessment (1986). "The Menopause, Hormone Therapy and Women's Health," OTA- Bp-BA-88. U.S. Govt. Printing Office, Washington, DC. 3. Skolnick, A. A. (1992). At third meeting, menopause experts make the most of insufficient data. JAMA, J. Am. Med. Assoc., 268, 2483-2485. 4. Weinstein, M. C., and Tosteson, A. N. A. (1990). Cost-effectiveness of hormone replacement. Ann. N.Y. Acad. Sci. 592, 162-72. 5. NIH Guide (1993). "Menopause and Health in Aging Women," Vol. 22, No. 32. National Institutes of Health, Washington, DC. 6. Barile, L.A. (1997). Theories of menopause. Brief comparative synopsis. J. Psychosoc. Nurs. 35,(2), 36-39. 7. Montaquila, J. M., Mohadjer, L., and Khare, M. (1998). The enhanced sample design of the future National Health and Nutrition Examination Survey (HANES). Proc. Am. Star. Assoc: Sect. Surv. Res. Methods. 8. Giesbrecht, L.H. (1996). Estimating coverage bias in RDD samples with current population survey data. Proc. Am. Stat. Assoc. Sect. Surv. Res. Methods 1,503-508. 9. Mohadjer, L. (1988). Stratification of prefix areas for sampling rare populations. In "Telephone Survey Methodology" (R. M. Groves, P. P. Biemer, L. E. Lyberg, J. T. Massey, W. L. Nicholls, and J. Waksberg, eds.), pp. 161-173. Wiley, New York. 10. Thornberry, O. T., Jr., and Massey, J. T. (1988). Trends in U.S. telephone coverage across time and subgroups. In "Telephone Survey Methodology" (R. M. Groves, P. P. Biemer, L. E. Lyberg, J. T. Massey, W. L. Nicholls, and J. Waksberg, eds.), pp. 25-49. Wiley, New York. 11. Sowers, M. E, and LaPietra, M. (1995). Menopause: Its epidemiology and potential association with chronic diseases. Epidemiol. Rev. 17, 287-302. 12. Krieger, N. (1992). Overcoming the absence of socioeconomic data in medical records: Validation and application of a census-based methodology. Am. J. Public Health 82, 703-710. 13. Choi, B. C. K., Hanley, A. J. G., Holowaty, E. J., and Dale, D. (1993). Use of surnames to identify individuals of Chinese ancestry. Am. J. Epidemiol. 138, 723-734.

~HAPTER 1

Demogr aphics, Environmental Influences, and Ethnic and International Differences in thc Menopausal Experience ELLEN B.

GOLD

Department of Epidemiology and Preventive Medicine, School of Medicine, University of California, Davis, Davis, California 95616

I. Introduction II. Demographic Characteristics III. Ethnic and International Differences

IV. Environmental Influences V. Conclusions References

I. I N T R O D U C T I O N

sition. Therefore, this chapter begins with a discussion of the methodologic issues, and is followed by a review of factors studied to date that have been suspected or shown to affect the nature of the transition.

Although menopause is a universal phenomenon among women, the timing of the onset and the signs and symptoms of the perimenopause, menopausal transition, and final menstrual period are not [1]. Most of our knowledge and perceptions of the experience of menopause are derived from studies largely of white women, and many have been studies of clinic-based, rather than population-based, samples of women. Thus, until recently, much of the picture of the menopause experience may have been affected by the nature of the samples of women studied. In addition, a number of methodologic issues arise, which must be considered in conducting and comparing sutdies of the menopausal tranMENOPAUSE: BIOLOGY AND PATHOBIOLOGY

A. Methodologic Concerns Most studies of the menopausal transition have been cross-sectional, rather than longitudinal, in design, providing opportunity for distortion of the true picture of the menopausal experience, particularly for understanding risk factors that precede, rather than accompany or follow, the menopause transition. Further, definitions of menopause have 189

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190 varied depending on number of months of amenorrhea, and scales and time frames for assessing symptoms have varied from study to study. Studies have also varied in whether and which factors have been included in controlling simultaneously for the effects of multiple confounding variables. 1. AGE AT MENOPAUSE

The analysis of age at natural menopause in a number of studies has been calculated only as a simple mean, rather than using the less biased survival or multivariable regression analysis approaches, which include more information and observations, because women are included but withdrawn if they have experienced surgical menopause or are still premenopausal [2]. Also, accuracy of reporting of age at menopause can vary by whether menopause was natural and by duration from time of menopause to time of the interview about menopause [3]. Further, in some studies reporting age at menopause, it is unclear if the age at the final menstrual period is being reported, which appears to be more frequent, or if the age at cessation of menses plus one year of amenorrhea, apparently a more rare occurrence, is what is reported [4]. Finally, in future studies, an accurate picture of the true age at menopause may become even more difficult to discern as more women are prescribed and take oral contraceptives or other hormone replacement medication prior to the final menstrual period. 2. PRESENTATION AND SYMPTOMS OF MENOPAUSE A number of methodologic issues may influence reporting and thus the prevalence of menopausal symptoms observed in different studies. First, a lack in consistency of symptoms included, in scales used to assess them, or in the time frame for assessment may lead to differences in ascertainment. Second, a failure to recognize colloquial or culturally specific expressions for certain symptoms, even for hot flashes (which can be considered a Western colloquialism), may account for some of the differences in prevalence between different populations. An extension of this problem is failure to recognize and address cultural sensitivities in reporting symptoms, which may also be affected by who asks the questions, and may result in underreporting of specific or even most symptoms. Third, most studies do not incorporate hormonal measures and may use inadequate questions about menstrual bleeding so that perimenopausal status cannot be well established. Further, a lack of comparability among studies with regard to the age group of women studied may also be reflected in the resulting differences in prevalence rates of symptoms. Finally, in some studies, age is used as a surrogate for menopausal status, so that status is presumed based on age rather than on menstrual function, thus introducing misclassification of menopause-related symptoms and possible lack of comparability with studies that have asked about menstrual function.

ELLEN B. GOLD

In recent years, more information has begun to appear regarding differences in the timing and presentation of menopause experienced by samples of women of different socioeconomic, ethnic, and lifestyle backgrounds, resulting in a fuller and more varied picture and also greater insights (and questions) regarding the physiology underlying the menopausal experience. This field of investigation will benefit and greater understanding will result if this trend continues, along with increased standardization of instruments and methods so that a clearer picture of the menopausal experience will emerge.

B. S u m m a r y o f U n d e r l y i n g P h y s i o l o g y Although the physiologic changes that accompany the menopause are described in detail elsewhere in this volume (Chapters 1-9), a brief summary is given here to provide relevant context to the signs and symptoms of the menopause experience and to the factors that affect them that are discussed in this chapter. The cessation of menstruation that defines menopause is believed to be due to a cessation of ovulation due to a loss of ovarian follicles, which produce estrogen. This loss results in a number of endocrine changes, particularly the decline in ovarian production of estradiol, the most biologically active form of estrogen [5,6], as well as increased circulating concentrations of follicle stimulating hormone (FSH) and decreased concentrations of inhibin, which normally inhibits the release of FSH [5]. Age at menopause is thought to be more sensitive to varying rates of atresia of ovarian follicles [7] than to the absolute number of oocytes depleted [8]. It has been widely held that as circulating estrogen concentrations decline in the perimenopause, variations in the timing of menstrual bleeding and in the nature of bleeding may occur. Menstruation may occur at irregular intervals due to irregular maturation of residual follicles, with diminished responsiveness to gonadotropin stimulation, or to anovulatory uterine bleeding after estrogen withdrawal without evidence of corpus luteum function [9]. As menstrual cycles become increasingly irregular, uterine bleeding may occur after an inadequate luteal phase or without ovulation or evidence of corpus luteum function [9], usually indicated by a short luteal phase, characteristic of women over age 40 [ 10,11 ]. Such cycles may be associated with insufficient FSH in the follicular phase, in turn resulting in lower luteal-phase estrogen and progesterone secretion. The absence of the corpus luteum, resulting in estrogen secretion (even hyperestrogenicity [10,12]) unopposed by progesterone, may lead to profuse blood loss. Conversely, relatively low concentrations of estrogen that may accompany the menopause transition also may lead to intermittent spotting. Thus, the nature and timing of bleeding may vary both within and between

CHAPTER 12 Demographics, Environment, Race, and Nationality women, but little is known about host, environmental, or lifestyle factors that may affect such variation. Extraovarian sources, such as adipose tissue, convert androstenedione to testosterone and estrone postmenopausally [ 13-16], although postmenopausal estrone production is only about one-third that of premenopausal production [7,17] but may be elevated in perimenopausal women in both the follicular and the luteal phases [10]. This peripheral production of estrone postmenopausally has been related to the amount of body fat [14,18] but has not been consistently related to age, height, or years since menopause. Also, in the perimenopause, FSH concentrations increase [6] without a concomitant increase in luteinizing hormone (LH) [19]. Reduced concentrations of estrogen and progesterone and increases in FSH [ 19] affect the central nervous system [2023] and result in vasomotor instability, leading to the characteristic hot flashes or flushes in some women. Two longitudinal studies have shown that vasomotor symptoms in the peri- and postmenopause are related to serum estrogen levels [20,24]. Hot flashes may also be more prevalent in women who experience irregular menses prior to menopause than in women who experience an abrupt cessation of menses [25]. One other longitudinal study has reported that the prevalence of symptoms appears to peak during the perimenopause transition and to decrease after menopause [26]. In the international literature, some have reported that about 4 0 - 6 0 % of perimenopausal and 6 0 - 8 0 % of menopausal white women experience hot flashes [27], and a substantial majority of these report that they are moderate or severe [28,29]. Discrepancies in the prevalence of hot flashes partially reflect inconsistencies in research methods and study populations

[30]. Other symptoms in hormone-receptive tissues may also occur in the perimenopause and postmenopause [22]. Changes in the vagina and vulva may result in atrophy, pruritus, dryness, bleeding, and dyspareunia. Estimates of the prevalence of vaginal dryness range from 12-34%, depending on the age group of women studied [20,28,31-34]. The embryology of the urinary and genital systems is shared, and the urethral epithelium and submucosa are affected by estrogen [35,36]. Although about a quarter of midlife women report some form of incontinence, its frequency does not appear to be related to menopausal status as determined from menstrual changes or serum FSH or estradiol concentrations, even though rates of incontinence increase during the time of menopause and then decline thereafter [37]. Whereas mood changes and sleep disturbances may also occur at this time, the causal time sequence of vasomotor symptoms, mood changes, and sleep disturbances and the factors that influence their occurrence and/or perception have not been clarified. Thus, for example, it is not known if hormonal changes affect mood and sleep independent of their effect on vasomotor instability.

191 A variety of physical symptoms, such as headaches, joint pain, aches in the back of the neck and shoulders, constipation, and dizzy spells have been thought to increase during the peri- and postmenopausal years [38]. However, the empirical data are inconclusive in identifying which, if any, are more prevalent during different stages of the menopause transition. For example, some investigators have found that women report significantly more frequent joint pain and dizzy spells when perimenopausal than they did when premenopausal [24], and others have found a greater proportion of peri- and postmenopausal women reporting aches and pains, but not dizzy spells, compared to premenopausal women [39]. Still others have found no association between any somatic symptoms and menopausal status [40]. Although some factors are known to be associated with early age at menopause and risk of experiencing symptoms, the relation of many have not been examined, most have not been examined in relation to duration of the perimenopause, and the endocrinologic effects of known risk factors in perimenopausal women still remain to be adequately explored.

II. D E M O G R A P H I C CHARACTERISTICS A. A g e at N a t u r a l M e n o p a u s e a n d O n s e t o f the P e r i m e n o p a u s e Age at natural menopause has traditionally been defined as the age at the final menstrual bleeding, which is followed by at least 12 months of amenorrhea. Some researchers have suggested that the age at which natural menopause occurs may be a marker of aging and health [41-43]. Crosssectional data indicate that endocrine changes characteristic of the onset of the perimenopause begin at around 45 years [44]. The median age at menopause in white women from industrialized countries is between 50 and 52 and at perimenopause is 47.5 years [26,45-48], with slight evidence of increasing age over time [48-51 ]; these onsets may vary by race/ethnicity (see Section III,A) and may be affected by lifestyle factors (discussed in Section IV,A). 1. SOCIOECONOMIC STATUS

Lower social class, as measured by a woman's level of education completed or by her own or her husband's occupation, has been associated in more than one study with an earlier age at menopause [46-48,52]. One study found that education was more strongly associated than occupation [47]. Most studies that have examined the relation of marital status have found that single women have menopause at an earlier age and that this association cannot be explained by nulliparity [47,53,54].

192 2. MENSTRUAL AND REPRODUCTIVE HISTORY Age at menopause may be a marker for hormonal status or changes earlier in life [55]. In a landmark longitudinal study of largely white, well-educated women, those whose median menstrual cycle length between the ages of 20 and 35 years was less than 26 days were reported to have menopause 1.4 years earlier than women with cycle lengths between 26 and 32 days, whereas a later natural menopause (mean = 0.8 year later) was observed in women with cycle lengths of 33 days or longer [56]. In addition, variability in cycle length of 9 or more days was also associated with a later age at menopause in this and other studies [47,57], although one early study reported an earlier menopause in women with irregular menses [48]. Increasing parity, particularly in women of higher socioeconomic status (SES), has also been associated with later age at menopause [45-47,50,52,54,55,58], consistent with the theory that menopause occurs after sufficient depletion of oocytes [58]. Although some studies report no familial relationship, one study has reported that age at menopause is positively associated with maternal age at menopause [52], and one study has shown genetic control of age at menopause in a study of twins [59]. Age at menarche has been fairly consistently observed not to be associated with age at menopause, after adjusting for parity and cycle length [47,48,50,53,60-62], as has prior spontaneous abortion, age at first birth, or history of breastfeeding [47,61,62]. Women who have used oral contraceptives (OCs) have also been reported to have a later age at menopause [47,52,62,63], an observation that is also consistent with the theory that OCs delay depletion of oocytes. However, the finding is not wholly consistent across studies, because one study reported that this delay became nonsignificant after a time-dependent adjustment for when OCs were used [47], and another study reported that OC users had a significantly earlier natural menopause than did nonusers, although this association was not consistent across 5-year age groups [45].

B. P r e s e n t a t i o n a n d S y m p t o m s o f M e n o p a u s e 1. SOCIOECONOMIC STATUS Although the majority of menopausal white women report vasomotor symptoms (hot flushes or flashes or night sweats), the prevalence varies greatly by socioeconomic status. Estimates of the incidence of hot flushes in menopausal white women range in population studies in the United States and worldwide from 24 to 93% [27]. Less educated women report more hot flashes and irritability compared to more educated women [39,64-68]. One large cross-sectional study reported increased prevalence of all symptoms associated with difficulty in paying for

ELLEN B. GOLD

basics [49]. One relatively small cross-sectional study found that women who reported mood changes or irritability that they believed were related to the menopausal transition were significantly more likely to report more other everyday complaints [69]. This was in contrast to women who reported hot flashes, sweating, or headaches associated with the menopausal transition who did not report more other everyday complaints. Homemakers have been shown to report hot flashes for a longer period compared to employed women [70], although working women of lower SES report more stress and tension during menopause [70,71 ], and worsening work stress has been associated with increased reporting of vasomotor symptoms, general health symptoms, and sexual difficulties [39]. 2. MENSTRUAL AND REPRODUCTIVE HISTORY The relationship of menstrual characteristics to the probability of experiencing menopausal symptoms largely remains unexplored, and reproductive history has been examined in a few studies but with somewhat inconsistent results. One cross-sectional study reported that women having natural menopause before age 52 years had a significantly greater reporting of hot flashes [66]. In another study, multiparous women had a lower prevalence of hot flashes compared to nulliparous women or women with abrupt cessation of menses [25]. However, another study showed menopausal symptoms to be associated positively with increasing parity [72], and some studies report no differences in symptom reporting frequency by parity [67,73,74]. Two longitudinal studies and one retrospective study have shown that reporting of menopausal vasomotor symptoms was more frequent among women who reported experiencing premenstrual tension before menopause [64,75,76], a finding that may be related to higher FSH levels in menstruating women with hot flushes [77]. One of these studies also reported that women with vasomotor symptoms were significantly more likely to report that their mothers also had vasomotor symptoms than were women without symptoms [75]. Most studies show symptoms to be more prevalent in hysterectomized women [39] and among women who experience an early menopause. In summary, later age at menopause may be a marker of health and longevity [41-43]. Studies have fairly consistently shown that lower socioeconomic status [46-48, 50,52], single marital status [47,53,54], regular menstrual cycles [47,57], nonuse of oral contraceptives [47,52,62,63], and lower parity [45-47,50,51,54,55,58] are associated with earlier menopause. Age at menarche [47,48,50,53,60-62], prior spontaneous abortion, age at first birth, and prior breastfeeding [47,61,62] are not associated with age at menopause. Lower socioeconomic status [39,64-68] and history of premenstrual tension [64,75,76] are associated with greater menopausal symptom reporting. However, the relation of parity to prevalence of symptoms has been inconsistent across studies.

CHAPTER 12 Demographics, Environment, Race, and Nationality III. ETHNIC

AND

INTERNATIONAL

DIFFERENCES

A. A g e at N a t u r a l M e n o p a u s e a n d at O n s e t o f P e r i m e n o p a u s e 1. ETHNIC DIFFERENCES

African-American [57] and Latina [78] women have been observed to have natural menopause about 2 years earlier than white women, despite their increased average body mass relative to white women (see Section IV,A,2). However, one small study in Nigeria reported the average age at menopause to be 52.8 years [79], nearly 2 years higher than that generally reported for white women in industrialized nations. Mayan women, despite their high parity, have been reported to experience menopause at about age 45 years [80]. Further, Mexican-American women may have shorter bleeding periods and follicular phase lengths [81]. In contrast, Asian and Caucasian women tend to be of similar age at menopause [82], although Thai women have been reported to have a lower median age at menopause (49.5 years), despite their high parity (see Section II,A,2) [60], and Filipino Malay women have been reported to have an average age at menopause of 47-48 years [83]. 2. INTERNATIONALDIFFERENCES A number of reports tend to indicate that women living in developing countries (including Indonesia, Singapore, Pakistan, Chile, and Peru) experience menopause several years earlier than do those in developed countries [63,84-87]. Some work has also indicated that women living in urban areas have a later menopause than do women in rural areas [88]. Women living at high altitude in the Himalayas or in the Andes of Peru have been shown to undergo menopause 1-1.5 years earlier than those living at lower altitudes or in less rural areas [63,89-91 ]. It is unclear if these geographic differences in the age at natural menopause reflect socioeconomic, environmental, racial/ethnic, or lifestyle differences, and whether and how these affect physiology.

B. P r e s e n t a t i o n a n d S y m p t o m s o f M e n o p a u s e

193 women. Among Filipino Malay women aged 4 0 - 5 5 years, reporting of vasomotor or circulatory symptoms occurred in 63%, and nervous or psychological symptoms (particularly headache or irritability) were reported by 79%, although only 31% consulted a physician, a rate that was higher among women with a vocational or college education [83]. It is unclear whether these ethnic differences in symptom frequencies are due to differences in cultural perceptions of menopause and reporting symptoms [ 100], diet [ 101 ], physical activity or body mass [102], differences in use of herbs or plant-estrogen-containing products [103], or in the use of acupuncture (which lowers excretion of the vasodilating neuropeptide calcitonin gene-related peptidelike immunoreactivity) [ 104] between Asian and Caucasian women, or to differences in serum estradiol levels (lower in Asian women in relatively nonsystematic studies that did not indicate adequate control of the day of the menstrual cycle on which blood was drawn for estrogen assays) [ 102,105]. Mayan women report no hot flashes [80], despite hormone profiles similar to those of Western women [ 106]. On the other hand, African-American and Hispanic women have been reported to have a higher prevalence of vaginal dryness compared to Caucasian women [20,49,67]. Some researchers believe that differences in the prevalences of symptom reporting reflect negative cultural stereotypes of aging and of the menopause experience [ 107,108] and are related to mental health [32]. However, others believe that because some studies report a frequency of hot flashes, night sweats, and vaginal dryness in countries such as Indonesia and Southeast Asia, for example, similar to that seen in Western countries, the latter view may be too simplistic [109]. Rather, cultural values of menopause as well as climate, dietary habits, and lifestyle may also be related. In summary, ethnicity appears to be related to both age at menopause and symptom reporting. African-American [57] and Latina [78] women have an earlier menopause than do Caucasian or some Asian [83] women, although not all Asian women [60,83]. Women in less developed countries also experience menopause earlier [63,84-87]. Additionally, Mayan [80] and Asian [60] women report fewer hot flashes, whereas African-American and Hispanic women have a higher prevalence of vaginal dryness than do Caucasian women [20,49,67].

1. ETHNIC DIFFERENCES Although the majority of Caucasian women experience menopausal symptoms [27,29], the reported frequency is much lower in most Asian women that have been studied [29,49,92-97], although one retrospective study reported no difference in symptom prevalence between Japanese and Caucasian menopausal women in Hawaii [98]. Further, some estimates of the prevalence of hot flashes have varied in similar Asian populations, e.g., from 23 [60] to 69% [99] in Thai

IV. E N V I R O N M E N T A L

INFLUENCES

A. A g e at N a t u r a l M e n o p a u s e a n d O n s e t o f the P e r i m e n o p a u s e 1. SMOKING

Perhaps the single most consistently shown (micro) environmental effect on menopause is that women who

194 smoke stop menstruating 1 to 2 years earlier than comparable nonsmokers [45,46,50,52,57,110-114] and have a shorter perimenopause [26]. In some studies heavy smokers have been observed to have an earlier menopause than light smokers, suggesting a dose-response effect of smoking on atrophy of ovarian follicles [52,113-117]. However, former smokers have only a slightly earlier age at menopause than never smokers, and increased time since quitting diminishes the difference [ 115,118], suggesting a reversible effect. The polycyclic aromatic hydrocarbons in cigarette smoke are known to be toxic to ovarian follicles [ 119,120] and thus could result in premature loss of ovarian follicles and early menopause in smokers, although the fact that former smokers have only a slightly earlier menopause than nonsmokers is not wholly consistent with this, even though the latter could reflect a duration and thus a dose effect. Because drug metabolism is enhanced in smokers [ 121 ], estrogen also may be more rapidly metabolized in the livers of smokers, possibly leading to an earlier decline in estrogen levels [122]. Smoking has also been observed to have antiestrogenic effects [123]. Greater prevalence of hysterectomy among premenopausal smokers than nonsmokers [115,124] does not appear to account for the earlier menopause in smokers [ 125]. Although one study reported that nonsmoking women whose spouses smoked had an age at menopause resembling that of smokers [ 126], very little is known about the effect of passive smoke exposure on age at menopause. 2. B o o r MASS AND COMPOSITION

A number of studies have examined the relation of body mass to age at menopause, and the findings have been rather inconsistent. Some studies have reported both increased body mass [indicated by weight for height] and upper body fat distribution [indicated by waist-to-hip ratio] to be positively associated with later age at menopause [45,122,124] and increased sex hormone concentrations [127], although other studies report no significant association of body mass with age at menopause [46,47,57,128,129]. Some studies have found a relationship between weight [ 117] or increased upper body fat distribution [ 128] and earlier age at menopause, particularly in smokers. One study reported earlier menopause in women on weight reduction programs or who had gained more than 26 pounds between the ages of 20 and 45 years [57]. Some of these discrepant findings may be explained by differences in study design (cross-sectional or retrospective vs. prospective) and/or analysis (e.g., inadequate or varying control of confounding variables and/or survival analysis vs. comparison of crude means). In general, the better designed and analyzed studies show no relationship. Although body mass and composition may be related to age at menopause and risk of developing symptoms, they are also related inversely to physical activity, alcohol consumption, and education and positively related to infertility and parity [130]. Further research is needed to examine the independent con-

ELLEN B. GOLD

tribution or interactive effect of body mass and composition and these other factors on the age at and course of menopause, using appropriate longitudinal study design and data analysis techniques that control for the effects of multiple confounding variables simultaneously. 3. PHYSICAL ACTIVITY

Exercise results in changes in a number of endocrine parameters [estradiol, progesterone, prolactin, luteinizing hormone, and follicle-stimulating hormone], both during and after intense physical activity [131-133], with concentrations of these hormones tending to be lower at rest [131,132,134]. Athletes experience a later age at menarche and increased incidence of anovulation [135] and amenorrhea [136] and, in those who menstruate, a shortened luteal phase and reduced mean and peak progesterone levels [130,134]. Although exercise is associated with decreased concentrations of reproductive hormones and frequency of ovulation, few studies have examined the effect of exercise on age at menopause, although one study of modest size has reported no relationship [57]. 4. OCCUPATIONAL]ENVIRONMENTAL FACTORS

Almost nothing is known about the effects of occupational or other environmental factors on age at and course of menopause, although occupational exposures and stressors [such as shift, hours worked, and hours spent standing and heavy lifting] have been shown to increase risk of adverse pregnancy outcomes [137-140] and to affect menstrual cycle length and variability and fecundability [141-144]. In addition, a number of environmental exposures, such as to DDT and polychlorinated biphenyls, have been shown to have estrogenic activity and may be associated with an increased risk of breast cancer [145,146], although this association has not been consistently observed [147,148]. Thus, it is reasonable to expect that occupational and environmental exposures may be related to endocrine disruption that is reflected in altered age at menopause. It is estimated that 40 million women in the United States alone, and several hundred million worldwide [149], will experience the menopausal transition in the next two decades, due to the aging of the "baby boomer" generation [150]. Approximately 70% of American women have worked outside the home [151 ]. Thus, this period in reproductive epidemiologic research presents a prime opportunity to learn more about the effects of occupational and environmental exposures on the menopause transition in these women. 5. DIET

A study from Papua New Guinea has suggested that malnourished women have cessation of menses about 4 years earlier compared to well-nourished women [152], consistent with other studies showing that women with greater weight [88,117] and height [53] may have a later age at menopause.

CHAPTER 12 Demographics, Environment, Race, and Nationality Vegetarians have also been observed to have an earlier age at menopause in one report [ 153]. Inclusion of meat in the diet of vegetarians has been observed to increase the episodic releases of LH and FSH and the length of the menstrual cycle [154]. Thus, meat may modify the interaction of hormones along the hypothalamic-pituitary-ovarian axis. At least one study has reported that increased meat or alcohol consumption is significantly associated with later age at menopause after adjusting for age and smoking [52]. Dietary fiber (whose intake tends to be inversely related to meat intake) may interrupt enterohepatic circulation of sex hormones, leading to the lower estrogen concentrations observed in vegetarian women [ 155]. Premenopausal women administered soy have shown increased plasma estradiol concentrations and follicular phase length, delayed menstruation, and/or suppressed midcycle surges of LH and FSH [ 156]. In postmenopausal women fed soy, FSH and LH did not decrease significantly, nor did sex hormone binding globulin (SHBG) increase, and little change occurred in endogenous estradiol or body weight, although a small estrogenic effect on vaginal cytology was observed [157]. The role of dietary phytoestrogens, fat, protein, and other nutrients in affecting age at menopause and/ or risk and severity of menopausal symptoms in perimenopausal and postmenopausal women remains to be studied systematically.

B. P r e s e n t a t i o n a n d S y m p t o m s o f M e n o p a u s e 1. SMOKING

Smokers have been shown to have lower serum estradiol and estrone concentrations among postmenopausal women [158] and lower urinary estrogen among premenopausal women [159]. These hormonal effects may be related to the findings in the few studies undertaken that smokers report more hot flashes and irritability than do nonsmokers [39,65], as well as more change in sexual desire [66]. Current smoking has been associated with increased reporting of symptoms during the menopausal transition in a number of studies [49,66,68,160]. However, one study reported no significant increase in hot flashes in smokers [67] but showed that thin women who smoked premenopausally had the greatest increase in hot flashes. Further, virtually no information is available about the relation of passive smoke exposure or whether smoking affects severity or frequency of symptoms. 2. BODY MASS AND COMPOSITION Much of the early clinical literature suggested that higher body weight might reduce the probability of experiencing symptoms, particularly hot flashes [73,161,162], due to higher circulating estrogen levels in heavier women due to peripheral production of estrone in adipose tissue. However,

195 most subsequent studies have not reported this [49]. One study showed no relation of body mass index (BMI) to reporting of hot flashes in nonsmokers [67]. Another study reported that women with lower body fat reported more hot flashes [163], and one small study reported significantly higher BMI in women reporting hot flashes, pins and needles, backaches, aches/stiffness in joints, shortness of breath, and fluid retention [20]. Additionally, two population-based studies have reported no significant increase in weight at the menopause [164,165], and one large study reported no increase in waist-to-hip ratio with menopause [ 128]. 3. PHYSICAL ACTIVITY

The effects of physical activity on symptoms reported during the menopause transition are covered in detail elsewhere in this volume (Chapter 34) and thus will only be summarized briefly here for completeness. Serum concentrations of estradiol, progesterone, prolactin, LH, and FSH all tend to increase during and after intense exercise [131-133], whereas resting values tend to be lower in athletes [ 132,134]. The findings from various studies regarding the effect of physical activity on reporting of symptoms, particularly vasomotor symptoms, have been inconsistent, perhaps due to differences in techniques in assessing physical activity and in sample sizes. Midlife women who participate in an exercise program have been reported in some studies to experience less frequent and less severe vasomotor symptoms, despite the fact that lower estrogen concentrations are associated with higher levels of physical activity [166,167]. However, this has not been consistent in other crosssectional or case-control studies, some of which have found no association of physical activity with symptoms [66,168171], and others have found a protective effect [49,172]. Because the onset of hot flashes is accompanied by lower circulating concentrations of plasma fl-endorphins [173], and physical activity increases secretion of endogenous opioid peptides, particularly fl-endorphins [ 174], exercise may prevent symptoms. Exercise also appears to have antidepressant effects [ 175,176] and thus may also be associated with wellbeing and with fewer midlife psychological symptoms, including negative mood and change in sexual desire [66]. 4. DIET

A number of dietary factors are considered to play a role in production, metabolism, and excretion of estrogen, in phases of the menstrual cycle, and in severity of menopausal symptoms. Vegetarian women have been shown to have lower plasma estrone and estradiol concentrations, perhaps due to lower saturated fat intake [177]. Further, Asian women, who consume less fat, excrete two to four times as much estrogen and have substantially lower plasma estrone and estradiol concentrations than do Caucasian women [102,105]. The relation of fat, alcohol, protein, or other nutrient

196 [such as antioxidant] intake to risk of experiencing menopausal symptoms has not been well studied. Nonetheless, some reports have indicated that alcohol may be estrogenic and may contain phytoestrogens [178], and that alcohol intake is inversely associated with levels of SHBG [127, 179,180]. Plant sterols have also been under study with regard to their effects on circulating hormones, menstrual cycles, and menopausal symptoms. Phytoestrogen is a term that includes classes of compounds that are nonsteroidal and either of plant origin or derived from metabolism of precursors in plants eaten by humans [181] (see also Chapter 33). The main classes of compounds are isoflavones and lignans. They structurally resemble estradiol and have been shown to have weak estrogenic activity, compete with estradiol for binding to estrogen receptors in tissues [182,183], and when ingested have estrogenic and antiestrogenic effects, depending on the concentrations of circulating endogenous estrogens and estrogen receptors [ 184,185]. In rats, the most potent of these, coumestrol, suppressed estrous cycles but did not behave as a typical antiestrogen [ 186]. Soy products are rich in phytoestrogens, which have been detected in high concentrations in the plasma or urine of individuals who consumed soy or other phytoestrogens [187]. Other less concentrated dietary sources of phytoestrogens include rice, corn, alcohol, cereal bran, whole wheat, and beans [188]. In Japanese women, phytoestrogen excretion is 100 times higher and endogenous estrogen excretion is 100 to 1000 times higher than in American and Finnish women [189]. Differences in phytoestrogen intake may be a (partial) explanation for the differences in frequencies of menopausal symptoms observed in Asian and Caucasian women, although this is not currently known. Urinary excretion of phytoestrogens and the concentration of plasma sex hormone binding globulin have been positively associated with dietary intake of fiber, which has been inversely related to plasma percentage free estradiol [190]. In postmenopausal women supplemented with soy or wheat flour (which contains less potent enterolactones), statistically significant (40 and 25%, respectively) reductions in hot flashes were observed, whereas vaginal cell maturation was unchanged and FSH was decreased [191 ]. In addition, in a small randomized trial of a 12-week phytoestrogen-rich diet, postmenopausal women on the diet showed significantly increased SHBG, significant reduction in hot flashes and vaginal dryness, and significant increases in serum concentrations of phytoestrogens, though no significant change in estradiol [ 181 ]. In summary, environmental factors do influence the menopausal transition. Active smoking has been consistently associated with a 1- to 2-year earlier menopause [45, 46,50,52,57,110-114], in a dose-response relationship, although the role of passive smoke exposure is uncertain. Findings regarding the relations of body weight and body

ELLEN B. GOLD

composition to age at menopause and symptom reporting have been inconsistent. The relations of diet, physical activity, and occupational or other environmental factors to age at menopause have largely not been investigated. Active smoking has also been associated with increased symptom reporting during the menopause transition [39,49,65,66, 68,160]. However, findings that relate physical activity to symptom reporting have been inconsistent. Phytoestrogen intake has been related to reduced hot flashes [ 181,191 ], but the role of other dietary factors is only beginning to be explored.

V. C O N C L U S I O N S Despite important methodologic differences and the limitations in the study designs used and the populations studied in the accumulating literature on the menopausal experience, an interesting and complex picture is emerging. A number of demographic (e.g., education, employment, race/ethnicity), menstrual and reproductive, and lifestyle (e.g., smoking and diet) factors appear to be important determinants of the age at which menopause occurs and to have meaningful relationships to the varied symptom experience of women. AfricanAmerican and Latina ethnicity, smoking, lower parity, vegetarian diet and undernutrition, and lower socioeconomic status have been found fairly consistently to be associated with earlier menopause. Symptom reporting varies by ethnicity, with less reporting of vasomotor symptoms in most Asian populations and increased reporting of vaginal dryness in African-American and Hispanic women. History of premenstrual tension, smoking and lower socioeconomic status have been associated with increased symptom reporting, whereas dietary phytoestrogen intake appears to reduce hot flashes. However, a number of the relationships are inconsistent (e.g., the role of body mass and composition and physical activity), possibly due to varying methodologic approaches and limitations, and others remain largely unexplored (e.g., passive smoke exposure and occupational and other environmental exposures). Thus, much remains to be learned about how these factors affect hormones at the physiologic level and thus determine the onset of the perimenopause, the timing of the final menstrual period, and the occurrence of the constellation of symptoms that are associated with the menopause transition. Furthermore, increased understanding of the underlying physiologic bases of these influences needs to include potential racial/ethnic differences in physiologic responses to lifestyle factors and other environmental exposures, as well as increased understanding of the cultural contexts, cultural differences, and cultural sensitivities that affect the presentation and experience of the menopausal transition. Increasing knowledge about these relationships ultimately offers women and their health care providers

197

CHAPTER 12 Demographics, Environment, Race, and Nationality

choices based on deeper understanding as to the variety of alternatives available to deal with the individual presentations of menopause. Acknowledgments The author is indebted to the following collaborators for their contributions to this study of the natural history of the menopause: Drs. Barbara Abrams, Shelley Adler, Gladys Block, Maradee Davis, Bruce Ettinger, Bill Lasley, Marion Lee, Marianne O'Neill Rasor, Steven Samuels, Helen Schauffler, Barbara Sommer, and Barbara Sternfeld.

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7 H A P T E R 1!

Distinguishing the Effects of Age from Those of Menopause JULIA E. BRADSHER Abt Associates, Inc., Cambridge, Massachusetts 02138 SONJA M. MCKINLAu New England Research Institutes, Watertown, Massachusetts 02172

IV. Conclusions References

I. Introduction II. Statement of the Problem III. Manifestations of Aging versus Menopause

I. I N T R O D U C T I O N

by the final menstrual period and internally by a decline in ovarian function, which results in infertility. Coinciding with this profound change, chronic diseases increase in prevalence. At the same time, aging processes both precede and succeed menopause, some of which are independent of menopause. Some authors have suggested that the age at natural menopause is a biological marker of the general health and aging of a woman [2,3]. In their study of Seventh Day Adventist women, Snowden and colleagues [2] found that early age at menopause was associated with excess mortality. However, their study used self-reported age at menopause as the major predictor, which is demonstrably unreliable [4]. Cooper and Sandler [3], in a more recent study, demonstrated that menopause may serve as a biological marker of aging and health and that hormonal changes associated with the menopause may be a significant risk factor for certain diseases in later life. This approach to menopause as a biological marker, however, suffers from a

In the United States and other western countries, women live an average of 7 5 - 8 0 years and most women can expect to live approximately 30 of these years beyond the menopause [1]. The menopause is a universal phenomenon and this decline in ovarian function is increasingly implicated as a significant risk factor in chronic disease and other conditions observed in the aging female. An overarching issue in the biology and epidemiology of menopause is its relationship to the underlying aging process. This is one of the least understood aspects of menopause and one with methodological and theoretical importance. As part of the physiological changes that occur during the normal aging process, women experience a unique and profound change at about two-thirds of the way along the life span and about half-way through the adult life cycle. This change, the menopause transition, is marked externally MENOPAUSE:

BIOLOGY AND PATHOBIOLOGY

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204 reductionistic viewpoint, which limits the scope of the menopause event to its biological processes. Research efforts in disaggregating the role of aging from that of menopause run the risk of inappropriate medicalization of the menopause [5]. Utian [6] has proposed a model to discriminate between the health conditions associated with aging versus those associated with menopause. In short, Utian postulates that characteristics associated with estrogen deficiency (such as hot flashes) should be considered unique to menopause (as opposed to aging). However, the endocrine-deficiency model of Utian is limited and does not explain all changes that occur around the time of the menopause transition [5,7]. Also lacking in the biomedical/endocrine-deficiency approach is the view that both aging and the menopause are part of a normal life trajectory for all women. This chapter first describes the methodological problems and theoretical concerns with addressing the relationship between aging and menopause (a more detailed review of the statistical and methodological challenges in studying menopause is found in Chapter 10). Next, evidence is presented in five areas of importance in distinguishing aging from menopause: musculoskeletal changes in women, cardiovascular disease, cognition, depression, and decline in sexuality. This chapter concludes with research recommendations based on the issues described herein.

II. STATEMENT OF THE P R O B L E M One of the greatest methodological challenges to menopause research and to addressing the relationship between aging and menopause is the limitation of available data. Many of the menopause studies reported in the medical and epidemiological literature suffer from a host of methodological flaws [8,9; see Chapter 10 of this volume], thereby making their conclusions suspect and potentially biased. Available data from most existing studies suffer from limited sample sizes and are based on a clinical population, that is, women seeking medical care. In addition, many studies have not included a sufficient age range of women, or have been cross-sectional or of a limited duration to allow for separating out the relationship between age and menopause. In fact, in many studies of the menopause, age is used as a proxy indicator for menopause status [9], thereby removing the possibility of addressing this concern at the outset. In order to address adequately the relationship between aging and menopause, prospective cohort studies must be undertaken. Such studies must include a large enough sample size and allow for generalization to the general community. These studies must also include sufficient pre- and postmenopausal data points, thereby establishing an adequate premenopausal baseline and inclusive change data over time. By doing so, one can estimate the changes that occur before

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or after menopause, and therefore independently of, estrogen decline and its associated changes. Without sufficient base line data, for example, the magnitude of phenomena associated with the menopause cannot be compared to a normative standard established prior to the decline in circulating hormone levels [10]. As Rostosky and Travis [9] have stated, in the study of menopausal symptoms, the absence of base line data may cause the "appearance of extensive symptomatology associated with menopause as opposed to other, supposedly more normal or health states." Finally, in order to examine carefully and separate out effects of aging from the menopause, comparable data on parallel processes in men and women should be compared. Most of the research conducted on menopause and reported in the research and medical literature focus on the biological processes and exclude the broader psychosocial context of women's lives [9]. As Rostosky and Travis have noted, "problems of menopause are viewed as embedded in women's biology and seldom viewed in the context of social structure" [9]. By adopting the biomedical model, research on menopause suffers the pitfalls of "gerontology in medicine" [ 11 ]. The interests of medicine direct the nature and scope of inquiry and equate the needs of medicine with those of society. There is also a tendency to view the signs and symptoms associated with the menopause as universal across all women and unique to menopause. However, it has been hypothesized that women's responses to physiological changes at menopause are not unique to menopause, but may be unique to a subset of women who respond similarly to a range of life events that may or may not be hormonally triggered. That is, women's responses are not event driven but are subject driven. Neugarten and Kraines [12] hypothesize that psychological and symptom responses to menarche are very similar to menopause responses in some women. These same women may respond distinctively to pregnancy, postpartum, and premenstrual changes, which are all hormonally based.

III. MANIFESTATIONS

OF AGING

VERSUS MENOPAUSE A. B o n e L o s s , O s t e o p o r o s i s , a n d F r a c t u r e s Peak bone mass in well-developed Caucasian societies is estimated to occur in the third or fourth decade of life [ 1315], after which bone mass declines. During their lifetime women lose about 50% of their trabecular bone and 30% of their cortical bone, about half of which is lost during the 10 years after the menopause [16-18]. Osteoporosis, defined somewhat loosely as the end stage of bone loss, affects over 20 million Americans and is manifest as approximately 1.3 million osteoporotic fractures in the United States each year [19,20]. Of all Caucasian women, 30%

CHAPTER 13 Distinguishing the Effects of Age/Menopause eventually sustain osteoporotic fractures [19,20], two times higher than their male counterparts [21,22] and 50% higher than in African-American women [23]. For the past two decades, it has been hypothesized that the higher hip fracture rates in older women (65 years and over) are a direct consequence of a menopause-related acceleration in bone loss [15,19,24,25]. This hypothesis states that bone loss is due to increased bone resorption and a compensatory, but inadequate, increase in bone formation. Cortical bone loss occurs at a rate of approximately 1-2% per year and continues at this rate for at least the first decade of estrogen decline. Trabecular bone loss is faster, with rates of 2 - 3 % per year in the forearm and allegedly as high as 1-2% per year in the vertebrae during the first few years of estrogen decline. However, only recently have prospective studies begun to document and quantify such an acceleration [17,26-29]. Although bone loss appears to accelerate when menses cease, it is not clear when this menopause-related bone loss begins. Ovarian function declines gradually in the normal menopause transition, over about 4 years [30], and serum estradiol concentrations begin to decrease a year or more before menses cease. Some cross-sectional studies suggest that bone mass of the spine or proximal femur decreases prior to the menopause, though it is difficult to distinguish pre- from perimenopausal loss in these reports [31,32]. Furthermore, historical differences in peak bone mass (a cohort effect) might explain these data. Some cross-sectional studies have failed to detect premenopausal bone loss from the spine [19,25-27] whereas some longitudinal studies of pre- or early perimenopausal women have failed to detect significant decreases in spine, radius, or total body bone mass [33-35]. In contrast, other longitudinal studies have reported significant premenopausal [36,37] or late perimenopausal [33,34] bone loss from the radius and spine. In a longitudinal assessment, women who smoked, who had less prior oral contraceptive use, who were older, and who had menopause without use of estrogen replacement therapy had greater amounts of bone loss. Physical activity, alcohol intake, body mass index, muscle area, prior breast feeding, and calcium intake were not associated with base line bone mineral density or 5-year rate of bone loss [37]. Data from the Study of Osteoporotic Fractures suggest that weight, estrogen exposure, physical activity, dietary calcium, and alcohol intake are associated with higher bone mineral density in postmenopausal women [38]. In their evaluation of the effects of endogenous sex steroids on bone mineral density in elderly white women, Greendale and colleagues [39] found that bioavailable estrogen was the strongest predictor of bone mineral density in these women, although bioavailable testosterone was associated with bone mineral density of the distal radius and serum dehydroepiandrosterone (DHEA) was associated with bone mineral density of the spine, hip, and midradius. Finally, data from the Post-

205 menopausal Estrogen/Progestin Intervention (PEPI) study demonstrated that, during the 36-month duration of the trial, postmenopausal women who were assigned to placebo demonstrated decreased bone mineral density (BMD) at the spine and hip, whereas women assigned to estrogen therapy had increased BMD [40]. Two reports suggest that chronological age, body mass, smoking, exercise, and other life style factors may play a larger role in bone loss than does menopause [17,29]. Data from the Massachusetts Women's Health Study (MWHS), a 4-year longitudinal study of 427 Caucasian women who were 45 to 55 years of age on entry, indicate that bone loss is easily detectable in the late perimenopause and is equally rapid in the 12 months before and after the last menstrual period [29]. However, insufficient premenopausal data on bone mass make these decreases difficult to interpret as definitively menopause related [29]. Another area of concern is hip fracture. Reliable estimates of the impact of bone density and prior rates of bone loss on these fractures have not been produced, primarily because fracture occurs more than 15 years after the final menstrual period and prospective follow-up of an adequate cohort of women is expensive. Moreover, the risk of falling appears to contribute considerable predictive information to subsequent fracture, independent of bone density [41-44]. Many epidemiological studies have examined correlates of bone mineral density and bone loss in pre- and postmenopausal women. Heredity may be an important determinant of premenopausal bone mineral density [17,36,37]. Available information on cultural differences in bone density and risk of hip fracture [45-51] reveals contradictions that underscore, first, the inability of bone density and rate of loss to predict fracture reliably and, second, the multiple risk factors likely to predict fracture. Current knowledge relating bone density to menopause can be summarized as follows: 1. Reliable estimates of accelerated bone loss at different bone sites in different populations in the perimenopause are limited. Reports on Caucasian women indicate that there may be some accelerated loss associated with menopause. Some postmenopausal women lose bone much more rapidly than others [28,52] do. It is not yet fully clear why some postmenopausal women are "fast losers" of bone. 2. Risk factors for accelerated bone loss have not been reliably established from adequate, prospective databases in different ethnic/racial groups, although several have been proposed. Sowers and colleagues [37] reported that smoking, lack of parity, later age of menarche, lower body weight, and lower age at first pregnancy were associated with lower base line bone mineral density in pre- and perimenopausal women. 3. The role of ovarian decline, at menopause, in bone loss has been largely inferred through the demonstrated

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beneficial effect of exogenous hormones (in clinical trials) in slowing or preventing bone loss in women. The relationship with endogenous estrogen decline has not been established directly.

B. C a r d i o v a s c u l a r D i s e a s e Cardiovascular disease (CVD) is the major cause of death in women in the United States. Both in absolute numbers and on a percentage basis, it accounts for more deaths in women (45%) than in men (39%), considering all ages [53]. The misconception that CVD is a male disease arises principally from its earlier onset in men. At ages 4 5 - 4 9 years, the incidence of CVD in men is about four times the rate in women, but by ages 6 5 - 6 9 years this ratio declines to about two, and by 85 years it is only slightly greater than one [53]. The fact that more women than men survive to older ages accounts for the greater proportion of women dying from CVD causes. This fact underscores the importance of understanding those factors that differentiate women most susceptible to developing CVD. It is hypothesized that ovarian hormone decline at menopause has pathophysiological consequences for CVD. Postmenopausal women and those who have undergone bilateral oophorectomy appear to have a higher risk of CVD than do premenopausal women of similar ages, suggesting that menopause might accelerate atherogenesis. The Nurses Health Study suggests that when the effects of age and smoking, two major risk factors for CVD and for the occurrence of menopause, are statistically controlled, increased risk of CVD is apparent only among women who have had a bilateral oophorectomy and are not on postmenopausal hormone replacement therapy (HRT). A comprehensive review of 20 observational studies comparing rates of CVD in women who have ever used HRT versus those who have not used such therapy concludes that HRT is associated with about a 50% reduction in CVD risk in postmenopausal women [54]. These results have been challenged as potentially reflecting a selection bias because women who are in better health or who have better access to health care may preferentially receive HRT [55]. These results may also be biased by inappropriate selection of comparison groups in study design [8]. More recently, the PEPI trial [56] provided support and the Heart and Estrogen/ progestin Replacement Study (HERS) trial [57] provided limited support for the seemingly large protective effect of exogenous hormones. The positive effect on high-density lipoprotein (HDL) cholesterol of exogenous hormones in PEPI, which peaked and regressed in a 3-year period, is almost certainly at least partially due to a first pass through the liver of orally administered hormones. Studies of the natural history of risk factors for CVD as women traverse the perimenopause can aid in understanding

the effects of menopause versus aging in cardiovascular disease. Previous studies, such as the Framingham Heart study, Massachusetts Women's Health Study, Nurses Health Study, and the Pittsburgh Healthy Women Study, have been useful in elucidating the effects of changes in menstrual bleeding on key CVD risk factors. However, their findings are not conclusive because bleeding patterns are a crude marker of ovarian aging; measures were taken relatively infrequently (e.g., biennial); populations under study are Caucasian, relatively healthy educated women; and recent findings in cardiology have pointed to new biological markers that have not been assessed in these studies. Large prospective databases have established, in Caucasian and African-American populations, the primary risk factors for subsequent heart disease. Although the relative contributions in men and women and in different populations may vary, they include primarily smoking, diabetes, high relative body weight, high and/or uncontrolled blood pressure, high total cholesterol, and increases in some lipids or lipid fractions. Clearly, these and other risk factors, such as low level of exercise and high fat diet, are interrelated [58-60]. Menopause has not been as an important risk factor in such databases. Additional data indicate that some of these risk factors are established well before menopause, including diabetes and body weight increases [61,62]. Moreover, there is evidence that cardiovascular disease is manifest, in terms of myocardial infarcts, in the fourth and fifth decades, before menopause [63,64]. Reported rates of cardiovascular mortality in women, from many countries, indicate smooth exponential trends with age, similar to those in men. Longitudinal data from a population-based study suggest that age-related factors (body mass, exercise) are much more strongly related to cardiovascular risk than is menopause status [64a]. Thus, the observational, population-based and clinical trial-based evidence is that cardiovascular disease is age related and not menopause related. It is well established that total, low-density lipoprotein (LDL), and HDL cholesterol are important risk factors for CVD. Some evidence suggests that low levels of HDL cholesterol and high levels of triglycerides are particularly important risk factors in women. This has been reinforced in a recently completed, large primary prevention trial including women [65]. There may be racial/ethnic differences in the effects of the menopause on lipids and lipoproteins. Two studies on minority women, Pima and African-American, show no variability in lipids according to menopausal status [66,67]. However, a report from the Atherosclerosis Risk in Communities (ARIC) study showed cross-sectionally that postmenopausal women, both African-American and Caucasian, had higher concentrations of total cholesterol, LDL cholesterol, apoprotein B, and lipoprotein a [Lp(a)] [68,69].

CHAPTER 13 Distinguishing the Effects of Age/Menopause C. C o g n i t i o n There has been considerable interest in the role that estrogen plays in cognitive functioning in women [70]. However, it is important to distinguish cognitive function changes that are associated with the normal aging process from physiological changes related to the menopause transition. Studies have demonstrated that memory performance declines with increasing age [70,71 ], but that age-related deficits are limited to certain skills, such as short-term memory [70]. On the other hand, memory tasks such as digit-span and long-term memory for remote past events show virtually no change with age [70-73]. An increasing body of knowledge provides laboratory and clinical data supporting the notion that diminishing levels of circulating estrogen levels are linked to decline in various measures of cognitive function. It is now understood, however, that estrogen levels do not significantly decline until less than 1 year prior to the transition into postmenopause, in normally transitioning women. In their small comparative study of peri- and postmenopausal women, Halbreich and colleagues [74] found that the age-related decline in performance of some cognitive functions accelerated following the menopause. They concluded that these changes may be attributed to a combination of biological age, a change in the hormonal milieu, and/or the social aspects of menopause. Research suggests that estrogenic influences on brain functions are specific and affect various brain functions differently. Some research has demonstrated that estrogen's effect on memory and other factors such as mood may be related to the hormone's ability to enhance neurotransmitters such as serotonin and acetylcholine within the central nervous [75]. However, randomized trials that firmly establish cause and effect have not been done [76]. Given the speculation on the effect of estrogen on cognition, it is a small step to consider the role of estrogen replacement therapy in the prevention of Alzheimer's disease and its progression in some individuals. It has been observed that older women who have used estrogen replacement therapy (ERT) at some prior time are 4 0 - 7 0 % less likely to develop Alzheimer's disease, depending on dose strength and duration of hormone exposure [76]. However, like earlier observational studies relating HRT to CVD, these small observational study reports suffer from a range of selection and recall biases that make interpretation difficult [8] (see Chapter 21).

D. D e c l i n e in S e x u a l i t y Menopause is a frequently cited cause of reduced sexual interest and activity in older women. However, several com-

207 munity-based studies of sexuality in midlife and in older women have documented that most women who have partners engage in sexual behavior [77-81]. Although limited by their cross-sectional nature, by narrow definitions of sexual behavior (usually frequency of heterosexual intercourse), and by incomplete control for the multiple determinants of sexuality, existing studies suggest that prevalence of sexual intercourse tends to decline with age and perhaps with menopausal status. However, the actual degree of and determinants of this apparent decrease remain unknown, and whether all sexual behaviors (vs. intercourse only) change over time is also uncertain. In men, a decline in sexual activity appears to continue with age [81,82], despite availability of partners. In women, the decline in sexual activity is increasingly a function of not having a viable partner as well as other factors [80,83]. Moreover, sexual drive and interest among women appear not to be related to estrogen and progestin [77,84-86], although lower estrogen levels have been associated with vaginal dryness [87-89] and hot flashes [90]. Because vaginal dryness and hot flashes have also been associated with reduced frequency of intercourse [91,92], the primary relationship (i.e., causal path) is not clear. There is documentation that sexual activity in women and men begins to decline in the late 30s or early 40s, well before menopause [83].

E. D e p r e s s i o n There is no reliable population-based evidence that menopause accelerates depression. Indeed, what reliable evidence exists indicates that any increase in depressive symptoms associated with menopause is transitory and related more directly to the length of the transition and bothersomeness of accompanying discomforts (including sleep disruption from hot flashes) [5,93]. No direct relationships between depression and decline in reproductive hormones (estradiol, primarily) have been reliably reported. Indeed, Avis and colleagues [93] reported evidence of no direct relationship. Despite the previously presented evidence to the contrary, a long-held notion that continues to persist among women in general [93] as well as among clinicians [94,95] is that menopausal women are more likely than other women to become depressed [96-98]. Much of the research that gives rise to this perception is based on clinic or patient populations of women seeking treatment [99]. Although some crosssectional research has shown more frequent depressed mood among peri- or postmenopausal women than among premenopausal women [81,100-102], most studies have not [5, 96,103]. Longitudinal data from the Massachusetts Women's Health Study suggest that experiencing a long perimenopausal period (at least 27 months) is associated with a slightly increased risk of depression [104]. Both cross-sectional and

208 longitudinal data, however, show that psychosocial factors [5,100,101,105] and premenopausal depression [99,106] account for more of the variation in depressed mood than menopause. In a comparison of women from Massachusetts, Canada, and Japan, the rates of reporting feeling blue and depressed were highest in the United States sample and lowest in the Japanese sample [107]. Within the Japanese samples, the highest rates were found among the premenopausal women, whereas in the United States sample, the highest rates were in the perimenopausal women. In the Canadian sample no differences by menopausal status were observed. These findings argue against a direct link between decline in estrogen and depression. Attempts to associate menopause and depression through cross-cultural comparisons, however, are complicated by variations in the meaning of menopause as well as the reporting of depressive symptoms [108,108a]. Irritability, tension, anxiety, and pounding heart have also been associated with peri- and postmenopause. As with depression, the associations have been made largely by clinical and anecdotal reports. Some epidemiological data suggest a slight increase in irritability during the transition [109,110] or postmenopausally [ 111 ], whereas others do not. Little is known about the relation between mood changes during the menopause and hormone levels. Two studies among premenopausal women found conflicting results [112], and a study of perimenopausal women (ages 4 0 - 5 5 ) found no difference in hormonal profile between those with high-level and those with low-level psychological symptoms [84]. When studying the psychological symptoms associated with menopause, it is important to reiterate the issue raised earlier in the discussion of theoretical issues. It has been hypothesized that women's responses to the physiological changes associated with menopause are not event driven, but are subject driven [4]. Therefore, psychological symptoms such as depressed mood may not be unique to menopause but, rather, may be the effect of a clustering among some women to respond similarly to a range of life events that may or may not be hormonally triggered. Current knowledge relating depression to menopause can be summarized as follows: 1. The majority of menopausal women do not show signs of mood disorders. Community-based studies consistently show that only a minority of menopausal women show signs of depression or other psychological symptoms. The majority of depression at menopause can be attributed to non-menopause-related factors, such as health problems or social circumstances. 2. Subgroups of women may be at increased risk of depression during menopause, e.g., women who experience severe forms of stress, such as surgical menopause.

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IV. C O N C L U S I O N S One of the least understood aspects of menopause is its relationship to the underlying aging process. It has been suggested that age at natural menopause is a biological marker of aging and health. However, there are methodological and theoretical challenges to this biomedical approach that raise the question as to whether aging and menopause are so directly related. This chapter has outlined some of the substantive areas of research in which this relationship may be examined. In general, research is insufficient to separate out fully effects of menopause from effects of aging. Progress has been made, but further research is needed in which rigorous methodology is used and operating paradigms are challenged. The WHO Report on Research on Menopause in the 90s [ 113] underscores the current state of menopause research and the need for further research. In order to more carefully address aging versus menopause, the following recommendations may be drawn from this chapter: 1. Studies are needed that have sufficient sample sizes and that can be generalized to the population. 2. Prospective studies are needed that include pre- and postmenopausal data points, thereby establishing an adequate premenopausal baseline and change over time. 3. Studies are needed that include comparable data on men and women in order to establish clearly the role of estrogen decline and other hormone changes in the underlying physiological process (bone loss, for example). 4. Studies are needed that consider the social context and the social construction of aging and menopause and that consider the cross-cultural differences in the definitions and experiences of aging and the menopause. 5. Studies are needed that will consider the hypothesis that the subjective responses to hormonally based events across the life course may occur differently for some women than others and that these experiences may not be unique to menopause. 6. More randomized clinical trials are needed to establish firmly the role of exogenous estrogens in addressing in later life health conditions that are attributed to the menopause.

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~ H A P T E R 14

Menopausal Hot Flashes ROBERT R.

I. II. III. IV. V.

FREEDMAN

Departments of Psychiatry and Behavioral Neurosciences and Obstetrics and Gynecology, Wayne State University, Detroit, Michigan 48201

Epidemiology of Hot Flashes Descriptive Physiology Objective Measurement of Hot Flashes Endocrinology Thermoregulation and Hot Flashes

VI. VII. VIII. IX.

I. E P I D E M I O L O G Y OF HOT FLASHES

at rates of 1 0 - 2 5 % [9], and Mexican Mayan women not at all [ 10]. Reasons for these findings are not known. Perhaps women from rural and non-Western cultures demonstrate physiologically defined hot flashes as frequently as Western women but are acculturated in some way to not report them. Or, they may actually have fewer physiologically defined flashes. This could be due to factors such as diet, because some foods such as yams and soy products contain substantial amount of phytoestrogens which may help ameliorate hot flashes [ 11 ]. The answers to these questions are unknown and represent important avenues for further research.

Hot flashes are the most common symptom of the menopause and occur in the vast majority of postmenopausal women. Their prevalence among naturally menopausal women has been reported to be 68 [1] to 82% [2] in the United States, 60% in Sweden [3] and 62% in Australia [4], with a median age of onset of approximately 51 years [5]. Among ovariectomized women, the prevalence of hot flashes is approximately 90% [2,6]. In one study, Feldman et al. [2] found that 64% of women experienced hot flashes for 1-5 years and Kronenberg [5] reported the median length of the symptomatic period to be 4 years. Studies of risk factors for menopausal hot flashes have found few strong effects. There is some evidence that thin women who smoke during the premenopausal period are more likely to report hot flashes [7] than are heavier nonsmokers. No significant association has been found between the report of hot flashes and socioeconomic status, age, race, parity, age at menarche, age at menopause, or number of pregnancies [5,7]. Cultural factors do affect the reporting of hot flashes. Compared to Western women, women from Indonesia report hot flashes at rates of only 10-20% [8], Chinese women

MENOPAUSE:

BIOLOGY AND PATHOBIOLOGY

Circadian Rhythms Sleep Treatment of Hot Flashes Summary and Conclusions References

II. DESCRIPTIVE P H Y S I O L O G Y A. S e l f - R e p o r t D a t a Kronenberg [5] conducted an extensive questionnaire study of hot flashes in 506 women ranging in age from 29 to 82 years. Of those reporting current symptoms, 87% had daily hot flashes and one-third of these reported more than 10 per day. Hot flashes generally lasted 1-5 min, with about 6% lasting more than 6 min. About 40% of the women recognized a premonition that a hot flash was about to begin.

215

Copyright 9 2000 by Academic Press. All rights of reproduction in any form reserved.

216

ROBERT R. FREEDMAN

The experience of a hot flash was most often described as sensations of heat, sweating, flushing, chills, clamminess, and anxiety. Sweating was reported most often in the face, head, neck, and chest, but rarely in the lower body.

B. P h y s i o l o g i c a l D a t a 1. SKIN TEMPERATURE AND BLOOD F L O W

Peripheral vasodilation, as evidenced by increased skin temperature, occurs during hot flashes in all body areas that have been measured (Fig. 1) [12]. These areas include the fingers, toes, cheek, forehead, forearm, upper arm, chest, abdomen, back, calf, and thigh [ 12-15]. Finger blood flow [ 14], and hand, calf, and forearm blood flow [ 16] also increase during hot flashes. Thermographic measurements during hot

flashes yielded data similar to those obtained with skin temperature [ 17]. 2. SWEATING AND SKIN CONDUCTANCE Sweating and skin conductance, an electrical measure of sweating, also increase during hot flashes (Fig. 1). Molnar [13] obtained sweat prints with iodized paper during hot flashes and reported profuse sweating on the forehead and nose, moderate sweating on the sternum and adjacent areas, and little or no sweating on the cheek and leg. The total body sweat rate was estimated to be about 1.3 g/min. In our laboratory, we measured sweat rate and skin conductance simultaneously from the sternum [12]. Sweat rate was recorded by capacitance hygrometry using a 3.5-cm-diameter plastic chamber attached over the sternum. Compressed air, regulated at 200 ml/min, was dried over CaCO 2 and passed

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CHAPTER 14 Menopausal Hot Flashes

217

through the chamber. Skin conductance level was also recorded from the sternum using a 0.5-V constant voltage circuit and disposable Ag/AgC1 electrodes. Both measures increased significantly during 29 hot flashes recorded in 14 women (Fig. 2). Measurable sweating occurred during 90% of the flashes and there was a close time correspondence between both measures. 3. CORE BODY TEMPERATURE

Homeotherms regulate core body temperature between upper thresholds, where sweating and peripheral vasodilation occur, and a lower threshold, where shivering occurs. If core body temperature were elevated in women with hot flashes, their symptoms of sweating and peripheral vasodilation could be explained. However, measurements of esophageal [14], rectal [13], and tympanic [15] temperatures were not elevated prior to hot flashes. These studies all found declines of about 0.3~ following hot flashes, probably due to increased heat loss (peripheral vasodilation) and evaporative cooling (sweating). However, esophageal and rectal temperatures have long thermal lag times and might respond too

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Elevations in core body temperature can be caused by increased metabolic rate (heat production) and by peripheral vasoconstriction (decreased heat loss). In the last study [ 12] we sought to determine if either of these factors accounted for the core body temperature elevations preceding hot flashes. Twenty-nine flashes were recorded in 14 postmenopausal women. Significant elevations in metabolic rate (about 15%) occurred but were simultaneous with sweating and peripheral

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slowly to appear along with the rapid peripheral events of the hot flash [ 18]. Additionally, it has been shown that tympanic temperature does not reliably measure core body temperature because it is affected by peripheral vasodilation and sweating [ 19]. We therefore conducted several studies in which we measured core body temperature using an ingested radiotelemetry pill, which has a faster response time than the esophageal and rectal methods (Fig. 3). The pill is swallowed 90 min before an experiment, to allow its egress from the stomach, and the signals are detected by a wire antenna and stored in a small digital recorder. The typical transit time through the gut is about 24 hr, during which the recorder samples the data every 30 sec. Hot flashes are recorded on a separate device, using the sternal skin conductance level as the marker. In the first study, 10 symptomatic women were recorded using ambulatory monitoring for 24 hr [20]. Of 77 hot flashes recorded, 46 (60%) were preceded by small but significant increases in core body temperature. In a second study, conducted during sleep in a temperature-controlled laboratory, 37 hot flashes occurred in 8 postmenopausal women [21]. Significant core temperature elevations preceded 24 of the flashes (65%), whereas rectal temperature had not significantly changed (Fig. 4). These results were replicated during a daytime study in the laboratory [ 12].

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FIGURE 2 Time course of skin conductance and sweating in 29 hot flashes. From Freedman [12]. Reprinted by permission from the American Society for Reproductive Medicine (Fertility and Sterility, 1998, Vol. 70, 1-6).

FIGURE 3 Radiotelemetry pill.

218

ROBERT R. FREEDMAN

5. HEART RATE

Modest increases in heart rate, about 7-15 beats/min [ 13,22,23], occur at approximately the same time as the peripheral vasodilation and sweating.

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Temperature from the dorsum of one finger was proposed as the first physiological marker for menopausal hot flashes [24]. In 7 symptomatic women, 41 skin temperature elevations > 1~ occurred within approximately 1 min of the subjective hot flash. However, the duration of the temperature elevations averaged 31 min, whereas the duration of subjective flushing was 2.3 min. Also, precise definitions of the onset and offset of the temperature elevations were not reported.

Minutes

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1. F I N G E R TEMPERATURE

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FIGURE 4 (A) Core body temperature (means) recorded from ingested telemetry pills during menopausal hot flashes. Time zero is the beginning of the sternal skin conductance response (in C). (B) Rectal temperature (means) during menopausal hot flashes. (C) Sternal skin conductance (means) during menopausal hot flashes. From Freedman and Woodward [21 ]. Reprinted by permission from the American Society for Reproductive Medicine (Fertility and Sterility, 1996, Vol. 65, 1141-1144).

vasodilation and did not precede the core temperature elevations (Fig. 1). Peripheral vasoconstriction did not occur. Thus, increased metabolic rate and peripheral vasoconstriction did not account for the core body temperature elevations in these women.

Subsequently, skin conductance recorded from the sternum was investigated as a hot flash marker. Tataryn et al. [25] found that 98% of 128 subjective flashes in 8 postmenopausal women were accompanied by elevations in sternal skin conductance compared to 82% for finger temperature and 81% for decreased tympanic temperature. All of these changes were significantly reduced by estrogen administration in 4 of the women. However, the precise characteristics of the skin conductance responses were not defined. Our laboratory subsequently sought to determine these characteristics [23]. Sternal skin conductance level, finger temperature, and heart rate were recorded for 4 hr in 11 postmenopausal and 8 premenopausal women. Twenty-nine subjective hot flashes were indicated by pushbutton in the first group. All of these were accompanied by an increase in sternal skin conductance ---2/xmho/30 sec. One skin conductance elevation occurred without a button press. All skin conductance elevations occurred within 66 sec of the button press. No skin conductance elevations occurred in the premenopausal women. Thus, there was a concordance of 95% between the skin conductance criterion and the reports of the subjects. Significant elevations in skin temperature and heart rate occurred during the flashes but were not as sensitive or specific as the skin conductance elevations. We replicated these findings in 18 symptomatic and 8 asymptomatic postmenopausal women [26]. There was a concordance of 80% between the sternal skin conductance criterion (2/xmho/30 sec) and the subjective reports (button press) in 15 flashes recorded in the symptomatic women. No events occurred in the asymptomatic women. Our findings were then independently replicated by an-

CHAPTER 14 Menopausal Hot Flashes other laboratory [27]. In two separate studies of 20 symptomatic women, a concordance of 90% was obtained between the sternal skin conductance criterion and subjective reports. Measurements of finger temperature and blood flow were less predictive and did not improve the concordance rate when added to the skin conductance measure.

B. A m b u l a t o r y M o n i t o r i n g To evaluate treatment studies it would be useful to have a method that could be used outside the laboratory over longer periods of time. We therefore developed methods for recording sternal skin conductance on ambulatory monitors for 24 hr. Using the same basic circuit and electrodes we found a concordance of 86% between the skin conductance criterion and button presses in 43 flashes recorded in 7 symptomatic women [23]. No such changes occurred in the 8 premenopausal women. We replicated these findings in a second study [26]. A concordance of 77% was obtained in 149 flashes recorded in 10 symptomatic women. Twelve skin conductance responses occurred in 8 putative asymptomatic women, representing a false response rate of about 8%. These ambulatory monitoring procedures were then successfully used to demonstrate the efficacy of a behavioral treatment for hot flashes in two subsequent studies [28,29].

C. P r o v o c a t i o n T e c h n i q u e s For laboratory investigations, it would be useful to provoke hot flashes reliably as opposed to waiting for them to occur during extended recording periods. Sturdee [22] observed that peripheral warming provoked objective and subjective hot flashes in 7 of 8 symptomatic women. We therefore sought to define this procedure operationally. Two 40 x 60-cm circulating water pads maintained at 42~ were placed on the torso of 11 supine symptomatic women in a 23~ room [23]. Eight hot flashes occurred within 30 min. A concordance of 73% was obtained between the skin conductance criterion (2/xmho/30 sec) and subjective report (button press). These findings were replicated in a subsequent study in 14 symptomatic women with a concordance of 84%. In this study, 25 hot flashes occurred during a 45-minute heating period. No objective or subjective responses occurred in 8 asymptomatic women.

IV. E N D O C R I N O L O G Y A. E s t r o g e n s Because hot flashes accompany the decline of estrogens in the vast majority of naturally and surgically menopausal

219 women, there is little doubt that estrogens play a role in the genesis of hot flashes. However, estrogens alone do not appear responsible for hot flashes because there is no correlation between the presence of this symptom and plasma [30], urinary [31 ], or vaginal [31 ] concentrations. No differences in unconjugated plasma estrogen concentrations were found in symptomatic versus asymptomatic women [32]. Additionally, clonidine significantly reduces hot flash frequency without altering circulating estrogen values [33]. Prepubertal girls have low estrogen production without hot flashes and hot flashes occur in the last trimester of pregnancy when estrogen production is high. Nevertheless, estrogen administration in hormone replacement therapy virtually eliminates hot flashes [34,35].

B. G o n a d o t r o p i n s Because gonadotropins become elevated at menopause, their possible role in the initiation of hot flashes has been investigated. Although no differences in luteinizing hormone (LH) concentrations were found between women with and without hot flashes [36], a temporal association was found between LH pulses and hot flash occurrence [37,38]. However, subsequent investigation revealed that women with a defect of gonadotropin-releasing hormone (GnRH) secretion (isolated gonadotropin deficiency) had hot flashes but no LH pulses and women with abnormal input to GnRH neurons (hypothalamic amenorrhea) had some LH pulses but no hot flashes [39]. Additionally, hot flashes occur in hypophysectomized women, who have no LH release [40], in women with pituitary insufficiency and hypoestrogenism [41], and in women with LH release suppressed by GnRH analog treatment [42,43]. Thus, LH cannot be the basis for hot flashes.

C. O p i a t e s It was observed that alcohol-induced flushing in subjects taking chlorpropamide, a drug that stimulates insulin release and lowers blood glucose, was related to opiate receptor activation [44]. Lightman et al. [45] subsequently found that naloxone infusion significantly reduced hot flash and LH pulse frequencies in six postmenopausal women. However, DeFazio et al. [46] attempted to replicate this study and found no effects. Tepper et al. [47] found that plasma fl-endorphin concentrations decreased significantly before occurrence of menopausal hot flashes whereas Genazzani et al. [48] found significantly increased values preceding hot flashes. Thus, there is no consistent evidence of the involvement of an opioidergic system in menopausal hot flashes.

220

ROBERTR. FREEDMAN D. C a t e c h o l a m i n e s

There is considerable evidence that norepinephrine plays an important role in thermoregulation mediated, in part, through ce2-adrenergic receptors [49]. Injection of norepinephrine into the preoptic hypothalamus causes peripheral vasodilation, heat loss, and a subsequent decline in core body temperature [49]. Additionally, there is considerable evidence that gonadal steroids modulate central noradrenergic activity [50]. Studies of plasma norepinephrine have not found increased concentrations prior to or during hot flashes [ 14,37]. However, brain norepinephrine content cannot be measured in plasma, due to the large amounts derived from peripheral organs [51 ]. We therefore measured plasma 3-methoxy-4-hydroxyphenylglycol (MHPG), the main metabolite of brain norepinephrine, to determine if central norepinephrine concentrations were elevated during hot flashes [52]. We studied 13 symptomatic and 6 asymptomatic postmenopausal women who were supine with an intravenous line in a 23~ room. Blood samples were drawn at the beginning and end of a 60-min period and during a hot flash, if one occurred. The same procedures were followed during a 45-min heating period. Basal MHPG levels were significantly higher in the symptomatic women (p < 0.0001, Table I) and increased significantly during resting and heat-induced flashes. There were no hot flashes or significant MHPG changes in the asymptomatic women, whose blood drawing times were yoked to those of 6 symptomatic women. However, approximately 50% of the free MHPG that enters the blood is metabolized peripherally to vanillylmandelic acid (VMA), and VMA formation can compete with MHPG production [53]. Thus, fluctuations in peripheral VMA formation could potentially distort measurements of plasma MHPG. Therefore, we measured both compounds simultaneously before and after hot flashes in 14 symptomatic women [12]. Plasma MHPG concentrations increased significantly (p < 0.02) between the preflash (3.7 ___ 1.4 ng/ml) and postflash (5.1 ___ 2.3 ng/ml) blood samples whereas

VMA levels did not significantly change (6.2 ___ 1.8 ng/ml vs. 6.1 ___ 2.5 ng/ml). Thus, there is evidence of increased brain norepinephrine content before hot flashes and these increase significantly when a flash occurs. Clonidine, an ce2-adrenergic agonist, reduces central noradrenergic activation and hot flashes [54-56]. Yohimbine, an ce2-adrenergic antagonist, increases central noradrenergic activation. We sought to determine if clonidine would ameliorate hot flashes and if yohimbine would provoke them in controlled laboratory conditions [57]. Nine symptomatic postmenopausal women, aged 4 3 - 6 3 years, served as subjects. Six asymptomatic women, aged 4 6 - 6 1 years, served as a comparison group. All women were in good health and had been amenorrheic for -- 2 years. In two blind laboratory sessions, subjects received either intravenous clonidine HC1 (1/xg/kg) or placebo followed by a 60-min waiting period and then by 45 min of peripheral heating. In two additional blind sessions, subjects received yohimbine HC1 (0.032-0.128 mg/kg intravenously)or placebo. Clonidine significantly (p = 0.01) increased the length of heating time needed to provoke a hot flash compared to placebo (40.6 ___ 3.0 min vs. 33.6 __+ 3.6 min) and reduced the number of hot flashes that did occur (2 vs. 8) (Fig. 5). In the symptomatic women, six hot flashes occurred during the yohimbine sessions and none during the corresponding placebo sessions, a statistically significant difference (p < 0.015). No hot flashes occurred in the asymptomatic women during either session (Fig. 6). These data support the hypothesis that a2-adrenergic receptors within the central noradrenergic system are involved in the initiation of hot flashes and are consistent with the idea that brain norepinephrine is elevated in this process. Animal studies have shown that yohimbine increases norepinephrine release by blocking inhibitory presynatic a2-adrenergic re-

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FIGURE 5 The occurrence of a hot flash during body heating was delayed after 1/xg/kg clonidine, comparedto placebo. No hot flashesoccurred in an asymptomaticwoman. From Freedman et al. [57]. Reprintedwith permission from the American College of Obstetricians and Gynecologists (Obstetrics and Gynecology, 1990, Vol. 76, 573-578).

221

CHAPTER 14 Menopausal Hot Flashes 10 -

gens modulate adrenergic receptors in many tissues [50]. It is possible, therefore, that hypothalamic ce2-adrenergic receptors are affected by the estrogen withdrawal associated with the menopause. As noted above, a decline in inhibitory presynatic ce2 receptors would lead to increased central norepinephrine concentrations and this is consistent with evi, dence from animal studies.

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FIGURE 6 A hot flash, indicated by a sternal skin conductance response, occurred after intravenous infusion of 0.032 mg/kg yohimbine in a menopausal woman with hot flashes. No responses occurred in the matched placebo session or in an asymptomatic women givenhigher doses. From Freedman et al. [57]. Reprinted with permission from the American College of Obstetricians and Gynecologists (Obstetrics and Gynecology, 1990, Vol. 76,573-578).

ceptors [58]. These autoreceptors mediate the turnover of norepinephrine through a feedback mechanism, and a reduction in their number and/or sensitivity would result in increased norepinephrine release [59]. This mechanism is consistent with human studies showing that yohimbine elevates and clonidine reduces plasma levels of M H P G [60]. Therefore, the yohimbine provocation and the clonidine inhibition of hot flashes in symptomatic women may reflect a deficit in inhibitory ce2-adrenergic receptors not seen in asymptomatic women. Additionally, the injection of clonidine into the hypothalamus reduces body temperature and activates heat conservation mechanisms, effects that are blocked by yohimbine [61]. Thus, ce2-adrenoceptors in the hypothalamus may be responsible for the events of the hot flash that are characteristic of a heat dissipation response. There is considerable evidence demonstrating that estro-

V. THERMOREGULATION AND HOT FLASHES Increased thermosensitivity at menopause has been noted in the literature for many years and is reflected in reports of increased hot flash frequency and duration during warm weather [62,63]. Peripheral heating has been demonstrated to provoke hot flashes in most of our symptomatic subjects [23], and this has been found by others as well [22]. As noted earlier, core body temperature (Tc) in homeotherms is regulated by hypothalamic centers between the thresholds of Tc for sweating and peripheral vasodilation and shivering (Fig. 7). According to this mechanism, the heat dissipation responses of hot flashes (sweating, peripheral vasodilation) would be triggered if body temperature were elevated or the sweating threshold lowered. We previously demonstrated that peripheral heating induced hot flashes in symptomatic but not in asymptomatic postmenopausal women nor in premenopausal women [23, 26]. These data suggested that the sweating threshold was reduced in symptomatic postmenopausal women. Considerable research in humans and animals has shown that conditions that alter the sweating threshold tend to alter the shivering threshold in the same direction [49]. We therefore tested to see if the T~ shivering threshold was reduced in symptomatic women, similar to their reduction in sweating threshold. We found that the shivering threshold was elevated rather than reduced in symptomatic compared to asymptomatic women [64]. This result implies that the thermoneutral zone

T

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Zone

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BRAIN NE CLON

t

MHPG?

t

FIGURE 7 We have shown that the thermoneutral zone is narrowed in symptomatic women. Elevated brain norepinephrine (NE) in animals reduces this zone. Yohimbine (YOH) elevates brain norepinephrine and should reduce this zone. Conversely, clonidine (CLON) should widen it. HF, Symptomatic women; Non-HF, asymptomatic women; MHPG, 3-methoxy-4-hydroxyphenylglycol (the primary brain NE metabolite).

222

ROBERT R. FREEDMAN

is narrowed in postmenopausal women with hot flashes. This hypothesis would explain the ability of small Tc elevations, as we found with the telemetry pill, to trigger the heat loss mechanisms of the hot flash (sweating, cutaneous vasodilation) and would also explain the shivering observed following many of the episodes. We therefore measured the thermoneutral zone in symptomatic and asymptomatic postmenopausal women, hypothesizing a reduction in the former group. We studied 12 symptomatic and 8 asymptomatic postmenopausal women [65]. We measured body temperature using a rectal probe, the ingested telemetry pill, and a weighted average of rectal and skin temperatures and determined the sweating and shivering thresholds for each. In a subsequent session, we raised body temperature to the sweating threshold using exercise. The symptomatic women had significantly smaller interthreshold zones compared to the asymptomatic women on all three measures of body temperature (Table II). Sweat rates were significantly higher in the former group. During exercise, all of the symptomatic and none of the asymptomatic women demonstrated hot flashes. Animal studies have shown that increased brain norepinephrine narrows the width of the interthreshold zone [49]. Conversely, clonidine reduces norepinephrine release, raises the sweating threshold, and lowers the shivering threshold in human studies [66]. Thus, we suggest that elevated brain norepinephrine narrows the thermoregulatory interthreshold zone in symptomatic postmenopausal women. This zone was so small as to be virtually zero using our methods. We propose that small elevations in core body temperature trigger hot flashes when the sweating threshold is crossed. Core

TABLE II Sweating Thresholds, Shivering Thresholds, and Interthreshold Zones for Rectal Temperature, Telemetry Pill Temperature, and Mean Body Temperature a

body temperature falls following hot flashes and patients often report shivering at this time. This likely represents the point where the shivering threshold is crossed, although this has not been directly measured.

VI. CIRCADIAN RHYTHMS The circadian rhythm of Tc is well known, and similar variations in other thermoregulatory parameters, such as heat conductance and sweating, have also been demonstrated. These patterns suggest that the thermoregulatory effector responses of hot flashes might also demonstrate temporal variations. A previous study showed circadian rhythmicity of self-reported hot flashes in some menopausal women, but no physiological data were collected [67]. We recruited and screened 10 symptomatic and 6 asymptomatic postmenopausal women [20]. Each received 24-hr ambulatory monitoring of sternal skin conductance level to detect hot flashes as well as ambient temperature, skin temperature, and Tc. The last measure was recorded using the ingested radiotelemetry pill. Cosinor analysis demonstrated a circadian rhythm (p < 0.02) of hot flashes with a peak around 1825 hr (Fig. 8). This rhythm lagged the circadian rhythm of Tc in symptomatic women by about 3 hr. Tc values of the symptomatic women were lower than those of the asymptomatic women (p < 0.05) from 0000-0400, and at 1500 and 2200 hr. The majority of hot flashes were preceded by elevations in Tc, a statistically significant effect (p < 0.05). Hot flashes began at significantly (p < 0.02) higher levels of Tc (36.82 ___0.04~ compared to all nonflash periods (36.70 ___ 0.005~ These data are consistent with the hypothesis that elevated Tc serves as part of the hot flash triggering mechanism.

VII. SLEEP A. T h e r m o r e g u l a t i o n a n d S l e e p

Temperature measurement (~ Group

Sweating

Shivering

Interthreshold

Symptomatic Asymptomatic P value

37.4 _+ 0.06 37.7 _ 0.05 0.001

Rectal 37.4 ___ 0.06 37.3 _+ 0.16 NS

0.0 _+ 0.06 0.4 ___ 0.18 0.005

Symptomatic Asymptomatic P value

37.2 ___ 0.09 37.5 +__0.14 0.008

T e l e m e t r y pill 37.2 ___ 0.15 37.1 ___ 0.09 NS

0.0 ___ 0.11 0.4 + 0.18 0.005

Symptomatic Asymptomatic P value

37.2 ___ 0.07 37.6 ___ 0.04 0.0003

Mean body 36.4 ___ 0.06 36.1 ___ 0.18 0.02

0.8 ___ 0.09 1.5 ___ 0.20 0.0006

a Values are means __+ S.E. P values for group differences, unpaired T-tests; NS, not significant.

Our observations of Tc elevations preceding hot flashes are supported by several other findings of our current work. Previous research had shown that external body heating applied 2 hr before sleep induced significantly higher amounts of subsequent stage 4 (slow wave) sleep [68]. We then observed [69] that postmenopausal women with hot flashes had significantly more stage 4 sleep than did asymptomatic women and that the number of hot flashes occurring 2 hr before sleep was significantly and positively correlated with the amount of subsequent stage 4 sleep. In our most recent work we used ambient cooling to reduce the frequency of hot flashes in the 2-hr period before sleep, which significantly reduced the amount of subsequent stage 4 sleep. Three consecutive nights of sleep recording were conducted on 11 symptomatic and 7 asymptomatic

CHAPTER 14 Menopausal Hot Flashes

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Hour F I G U R E 8 Hot flash (HF) frequency and core body temperature over 24 hr. Hot flash frequency in 10 symptomatic women shown as bars. Curves: best-fit cosine curve for hot flash frequency ( . . . . . ); 24-hr core temperature data for 10 symptomatic women ( o - - o ) with best-fit cosine curve (--); 24-hr core temperature data in 6 asymptomatic women (D ..... n) with best-fit cosine curve ( ..... ). From R. R. Freedman, D., Norton, S., Woodward, and G. Cornelissen (1995). Core body temperature and circadian rhythm of hot flashes in menopausal women. J. Clin. E n d o c r i n o l . M e t a b . 80(8), 2 3 5 4 - 2 3 5 8 . 9 The Endocrine Society.

menopausal women. Ambient temperature during the 2 hr before sleep was set at either neutral (23~ warm (30~ or cold (18~ Hot flash frequencies were lower in the cold condition (p < 0.02), and in the neutral condition (p < 0.01) relative to the warm condition. The percentage of time spent in stage 4 sleep was greater following both the neutral and warm conditions in the symptomatic group, compared to the asymptomatic group (p < 0.01). Within the symptomatic group, time spent in stage 4 sleep was significantly lower following the cold condition (p < 0.025), compared to the warm condition. These results demonstrate that the association between hot flashes and slow wave sleep can be selectively reversed by suppressing hot flashes with cooling prior to sleep onset.

flash frequency and in nighttime awakenings as well as increased rapid eye movement (REM) sleep. However, the causal relationships, if any, between hot flashes and sleep variables are not known.

VIII. T R E A T M E N T OF H O T F L A S H E S

A. Hormone Replacement Therapy Virtual elimination of hot flashes by hormone replacement therapy has been established [34,74]. Initially, estrogen alone was given, but to provide uterine protection women with a uterus are now given estrogen plus a progestin in combination or cyclically [hormone replacement therapy (HRT)].

B. Sleep Disturbance B. Laboratory sleep studies have demonstrated that symptomatic postmenopausal women wake up more frequently during the night than do asymptomatic women. Erlik et al. [70] first reported that objectively measured hot flashes were accompanied by EEG-defined waking episodes in 45/47 flashes recorded in 8 symptomatic women. This relationship did not occur in asymptomatic women. Three additional studies [71-73] showed that administration of estrogen to symptomatic women resulted in significant declines in hot

c~-AdrenergicAntagonists

As noted above, clonidine decreases central noradrenergic activation as well as thermally induced hot flashes [57]. In a double-blind cross-over study involving a small number of women, Clayden et al. [54] found that oral clonidine reduced hot flash frequency by 44% after 4 weeks and by 56% at 8 weeks, significantly better than the placebo group. In 11 symptomatic women, Schindler et al. [33] found that clonidine significantly reduced hot flash

224

ROBERTR. FREEDMAN

frequency and was well tolerated. Using objective measurements of hot flashes, Laufer et al. [55] found that clonidine significantly reduced hot flash frequency compared with baseline and with placebo. However, 4 of the subjects withdrew due to side effects. Using transdermal clonidine, Nagamani et al. [75] found an 80% decrease in hot flash frequency in the active drug group compared to 36% in the placebo group. Reported side effects were minimal. Thus clonidine, particularly in transdermal form, may be a useful treatment for hot flashes in women for whom HRT is contraindicated.

tance. The paced respiration group showed a significant decline in hot flash frequency (again about 50%) compared to no change in the control group. However, there were no significant changes in any biochemical measure for either group. Thus, the mechanism through which paced respiration reduces hot flash frequency remains to be determined. Two other small studies [78,79] have also found significant amelioration of hot flashes through behavioral relaxation procedures. Thus, behavioral treatments my be useful for women in whom HRT is contraindicated or who choose not to take medications.

C. B e h a v i o r a l T r e a t m e n t s

D. A l t e r n a t i v e T r e a t m e n t s

Although hormone replacement therapy offers effective control of hot flashes, it may be contraindicated for some women. We have presented evidence that central sympathetic activation is increased in women with hot flashes. Behavioral relaxation methods have been shown to reduce sympathetic activity in normal subjects and in some clinical populations [76]. Therefore, we treated seven menopausal women with hot flashes using a combination of progressive muscle relaxation exercises and slow deep breathing [77]. Seven additional women were assigned to receive a control procedure, alpha wave EEG biofeedback. The relaxation procedure significantly reduced objective symptoms recorded in the laboratory and diary-recorded hot flash frequency (by about 50%) compared to the control procedure. This investigation demonstrated that a combination of muscle relaxation exercises and slow deep breathing significantly reduced hot flash frequency in a small group of subjects. However, because two treatment procedures were combined, it was not possible to determine which component was responsible for the therapeutic effect. The physiological data showed that respiration rate was the only recorded variable that was significantly altered during training. Therefore, a second study was performed in which one group of subjects received slow deep breathing alone, another group received muscle relaxation exercises alone, and a third group received alpha EEG biofeedback [28]. Treatment outcome was assessed by ambulatory monitoring of sternal skin conductance responses, described earlier. Only the paced respiration group showed a significant decline in hot flash frequency (about 50%), decreased respiration rate, and increased tidal volume. There were no significant changes shown by the other two groups. We then sought to determine if reduced sympathetic activation was the mechanism by which paced respiration ameliorates hot flashes [29]. We therefore measured plasma MHPG, epinephrine, norepinephrine, and platelet ce2-receptors during paced respiration or alpha EEG biofeedback in 24 symptomatic women. Treatment outcome was again assessed by ambulatory monitoring of sternal skin conduc-

1. ACUPUNCTURE

One controlled study has thus far been published on the effects of acupuncture on hot flashes. Wyon et al. [80] randomized 24 symptomatic women to receive either active or placebo acupuncture for 8 weeks. Both groups showed significant declines in reported hot flash frequency at 4 weeks, 8 weeks, and 3 months posttreatment. 2. PHYTOESTROGENS

Phytoestrogens are plant compounds with estrogen-like biological activity [11 ]. Their possible beneficial effects on hot flashes are inferred from the low reported prevalence of symptoms in countries, such as Indonesia and Japan, with diets rich in these compounds (see discussion on epidemiology, Section I). A controlled study of 145 women in Israel compared a phytoestrogen-rich diet (tofu, soy drink, miso, flax seed) with the usual Israeli diet [81]. Both groups showed significant declines in hot flash scores but the decline in the active treatment group was significantly greater. Similar findings were obtained by Murkies et al. [82], who compared soy flour dietary supplementation with wheat flour supplementation (control group). The most recent investigation compared soy protein supplementation with casein (placebo) in 104 symptomatic women [83]. At week 12, the soy group had a significantly greater reduction in hot flash frequency (45%) compared to placebo (30%). Thus, dietary supplementation with phytoestrogens may be useful for treating hot flashes, although the effects are not dramatic when compared with placebo.

IX. S U M M A R Y

AND CONCLUSIONS

Hot flashes are the most common symptom associated with menopause, although prevalence estimates are lower in some rural and non-Western areas. The symptoms are characteristic of a heat-dissipation response and consist of sweating on the face, neck, and chest, as well as peripheral

225

CHAPTER 14 Menopausal Hot Flashes

vasodilation. Although hot flashes clearly accompany the estrogen withdrawal at menopause, estrogen alone is not responsible because levels do not differ in symptomatic and asymptomatic women. Until recently it was thought that hot flashes were triggered by a sudden, downward resetting of the hypothalamic setpoint, because there was no evidence of increased core body temperature. However, we obtained such evidence, using a rapidly responding ingested telemetry pill. We then found that the thermoneutral zone, within which sweating, peripheral vasodilation, and shivering do not occur, is virtually nonexistent in symptomatic women but is normal (about 0.4~ in asymptomatic women. Thus, we believe that small temperature elevations preceding hot flashes acting within a reduced thermoneutral zone constitute the triggering mechanism. We also demonstrated that central sympathetic activation is elevated in symptomatic women; in animal studies, this reduces the thermoneutral zone. Clonidine reduces central sympathetic activation, widens the thermoneutral zone, and ameliorates hot flashes. Estrogen virtually eliminates hot flashes but its mechanism of action is not known. Behavioral relaxation procedures reduce hot flash frequency to the same extent as clonidine (about 50%) but their mechanism of action is also not understood.

11. 12. 13. 14.

15.

16.

17. 18.

19.

20.

21. 22.

Acknowledgment Research conducted by the author was supported by NIH Merit Award, AG-05233, from NIA.

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226 34. Johnson, S. (1998). Menopause and hormone replacement therapy. Med. Clin. North Am. 82(2), 297-320. 35. Kenemans, E, Barentsen, R., and van de Weijer, E (1996). Practical HRT. Med. Forum Int. 36. Campbell, S. (1976). Intensive steroid and protein hormonal profiles on postmenopausal women experiencing hot flashes and a group of controls. In "Management of the Menopause and Post-Menopausal Years" S. Campbell (ed.). MTP Press, London. 37. Casper, R. F., Yen, S. S. C., and Wilkes, M. M. (1979). Menopausal flushes: A neuroendocrine link with pulsatile luteinizing hormone secretion. Science 205, 823-825. 38. Tataryn, I. V., Meldrum, D. R., Lu, K. H., Frumar, A. M., and Judd, H. L. (1979). LH, FSH, and skin temperature during menopausal hot flush. J. Clin. Endocrinol. Metab. 49, 152-154. 39. Gambone, J., Meldrum, D. R., Laufer, L. Chang, R. J., Lu, J. K. H., and Judd, H. L. (1984). Further delineation of hypothalamic dysfunction responsible for menopausal hot flashes. J. Clin. Endocrinol. Metab. 59(6), 1092-1102. 40. Mulley, G., Mitchell, R. A., and Tattersall, R. B. (1977). Hot flushes after hypophysectomy. Br. Med. J. 2, 1062. 41. Meldrum, D. R., Erlik, Y., Lu, J. K. H., and Judd, H. L. (1981). Objectively recorded hot flushes in patients with pituitary insufficiency. J. Clin. Endocrinol. Metab. 52(4), 684-687. 42. Casper, R. E, and Yen, S. S. C. (1981). Menopausal flushes: Effect of pituitary gonadotropin desensitization by a potent luteinizing hormone releasing factor agonist. J. Clin. Endocrinol. Metab. 53, 1056-1058. 43. DeFazio, J., Meldrum, D. R., Laufer, L. Vale, W., Rivier, J., Lu, J. K., and Judd, H. L. (1983). Induction of hot flashes in premenopausal women treated with a long-acting GnRH agonist. J. Clin. Endocrinol. Metab. 56(3), 445-448. 44. Leslie, R. D. G., Pyke, D. A., and Stubbs, W. A. (1979). Sensitivity to enkephalin as a cause of non-insulin dependent diabetes. Lancet 1, 341-343. 45. Lightman, S. L., Jacobs, H. S., Maguire, A. K., McGarrick, G., and Jeffcoate, S. L. (1981). Climacteric flushing: Clinical and endocrine response to infusion of naloxone. Br. J. Obstet. Gynecol. 88, 919-924. 46. DeFazio, J., Vorheugen, C., Chetkowski, R., Nass, T., Judd, H. L., and Meldrum, D. R. (1984). The effects of naloxone on hot flashes and gonadotropin secretion in postmenopausal women. J. Clin. Endocrinol. Metab. 58(3), 578-581. 47. Tepper, R., Neri, A., Kaufman, H., Schoenfield, A., and Ovadia, J. (1987). Menopausal hot flushes and plasma fl-endorphins. Obstet. Gynecol. 70, 150-152. 48. Genazzani, A. R., Petraglia, F., Facchinetti, F., Facchini, V., Volpe, A., and Alessandrini, G. (1984). Increase of proopiomelanocortin-related peptides during subjective menopausal flushes. Am. J. Obstet. Gynecol. 149, 775-779. 49. Brtick, K., and Zeisberger, E. (1990). Adaptive changes in thermoregulation and their neuropharmacological basis. In "Thermoregulation: Physiology and Biochemistry" (E. Schtinbaum and E Lomax, eds.), pp. 255-307. Pergamon, New York. 50. Insel, E A., and Motulskey, H. J. (1987). Physiologic and pharmacologic regulation of adrenergic receptors. In "Adrenergic Receptors in Man" (E A. Insel, ed.), pp. 201-236. Dekker, New York. 51. Lambert, G. W., Kaye, D. M., Vaz, M., Cox, H. S., Turner, A. G., Jennings, G. L., and Elser, M. D. (1995). Regional origins of 3-methoxy-4-hydroxyphenylglycol in plasma: Effects of chronic sympathetic nervous activation and devervation, and acute reflex sympathetic stimulation. J. Autom. Nerv. Syst. 55, 169-178. 52. Freedman, R. R., and Woodward, S. (1992). Elevated az-adrenergic responsiveness in menopausal hot flushes: Pharmacologic and biochemical studies. In "Thermoregulation: The Pathophysiological Basis of Clinical Disorders" (Lomax and Sch6nbaum, eds.), pp. 6 - 9 . Karger, Basel.

ROBERT R. FREEDMAN 53. Kopin, I. J., Blombery, E, Ebert, M. H., Gordon, E. K., Jimerson, D. C., Markey, S. E, and Polinsky, R. J. (1984). Disposition and metabolism of M H P G - C D 3 in humans: Plasma MHPG as the principal pathway of norepinephrine metabolism and as an important determinant of CSF levels of MHPG. In "Frontiers in Biochemical and Pharmacological Research in Depression" (E. Usdin, et al., eds.), pp. 57-68. Raven Press, New York. 54. Clayden, J. R., Bell, J. W., and Pollard, E (1974). Menopausal flushing: Double blind trial of a non-hormonal medication. Br. Med. J. 1, 4 0 9 412. 55. Laufer, L. R., Erlik, Y., Meldrum, D. R., and Judd, H. L. (1982). Effect of clonidine on hot flushes in postmenopausal women. Obstet. Gynecol. 60, 583-589. 56. Schmitt, H. (1977). The pharmacology of clonidine and related products. Handb. Exp. Pharmacol. 39, 299-396. 57. Freedman, R. R., Woodward, S., and Sabharwal, S. C. (1990). ce2Adrenergic mechanism in menopausal hot flushes. Obstet. Gynecol. 76(4), 573-578. 58. Goldberg, M., and Robertson, D. (1983). Yohimbine: A pharmacological probe for study of the a 2- adrenoceptor. Pharmacol. Rev. 35, 143180. 59. Starke, K., Gothert, M., and Kilbringer, H. (1989). Modulation of neurotransmitter release by presynaptic autoreceptors. Physiol. Rev. 69, 864-989. 60. Charney, D. S., Heninger, G. R., and Sternberg, D. E. (1982). Assessment of a2-adrenergic autoreceptor function in humans: Effects of oral yohimbine. Life Sci. 30, 2033-2041. 61. Zacny, E. (1982). The role of az-adrenoceptors in the hypothermic effect of clonidine in the rat. J. Pharm. Pharmacol. 34, 455-456. 62. Molnar, G. W. (1981). Menopausal hot flashes: Their cycles and relation to air temperature. Obstet. Gynecol. 57(6)(Suppl.), 52-55. 63. Kronenberg, F., and Barnard, R. M. (1992). Modulation of menopausal hot flashes by ambient temperature. J. Therm. Biol. 17(1), 43-49. 64. Freedman, R. R., and Woodward, S. (1995). Altered shivering threshold in postmenopausal women with hot flashes. Menopause 2(3), 163-168. 65. Freedman, R. R., and Krell, W. (1999). Reduced thermoregulatory null zone in postmenopausal women with hot flashes. Am. J. Obstet. Gynecol. 181(1), 66-70. 66. Delaunay, L., Bonnet, E, Liu, N., Beydon, L., Catoire, E, and Sessler, D. I. (1993). Clonidine comparably decreases the thermoregulatory thresholds for vasoconstriction and shivering in humans. Anesthesiology 79, 470-474. 67. Albright, D. L., Voda, A. M., Smolensky, M. H., Hsi, B., and Decker, M. (1989). Circadian rhythms in hot flashes in natural and surgicallyinduced menopause. Chronobiol. Int. 6(3), 279-284. 68. Horne, J. A., and Reid, A. J. (1985). Night-time sleep EEG changes following body heating in a warm bath. Electoencephalogr. Clin. Neurophysiol. 60, 154-157. 69. Woodward, S., and Freedman, R. R. (1994). The thermoregulatory effects of menopausal hot flashes on sleep. Sleep 17(6), 497-501. 70. Erlik, Y., Tataryn, I. V., Meldrum, D. R., Lomax, P., Bajorek, J. G., and Judd, H. L. (1981). Association of waking episodes with menopausal hot flushes. JAMA, J. Am. Med. Assoc. 245(17), 1741-1744. 71. Thomson, J., and Oswald, I. (1977). Effect of oestrogen on the sleep, mood, and anxiety of menopausal women. Br. Med. J. 2, 1317-1319. 72. Schiff, I., Regestein, Q., Tulchinsky, D., and Ryan, K. J. (1979). Effects of estrogens on sleep and psychological state of hypogonadal women. JAMA, J. Am. Med. Assoc. 242(22), 2405-2414. 73. Scharf, M. B., McDannold, M. D., Stover, R., Zaretsky, N., and Berkowitz, D. V. (1997). Effects of estrogen replacement therapy on rates of cyclic alternating patterns and hot-flush events during sleep in postmenopausal women: A pilot study. Clin. Ther. 19(2), 304-311. 74. Ettinger, B. (1998). Overview of estrogen replacement therapy: A historical perspective. Proc. Soc. Exp. Biol. Med. 217 (1), 2-5.

CHAPTER 14 Menopausal Hot Flashes 75. Nagamani, M., Kelver, M. E., and Smith, E. R. (1987). Treatment of menopausal hot flashes with transdermal administration of clonidine. Am. J. Obstet. Gynecol. 156(3), 561-565. 76. Hoffman, J. W., Benson, H., Arns, E A., Stainbrook, G. L., and Langsberg, G. L. (1982). Reduced sympathetic nervous system responsivity associated with the relaxation response. Science 215, 190-192. 77. Germaine, L. M., and Freedman, R. R. (1984). Behavioral treatment of menopausal hot flashes: Evaluation by objective methods. J. Consult. Clin. Psychol. 52(6), 1072-1079. 78. Irvin, J. H., Domar, A. D., Clark, C., Zuttermeister, E C., and Friedman, R. (1996). The effects of relaxation response training on menopausal symptoms. J. Psychosom. Obstet. Gynecol. 17, 202-207. 79. Wijima, K., Melin, A., Nedstrand, E., and Hammar, M. (1997). Treatment of menopausal symptoms with applied relaxation: A pilot study. J. Behav. Ther. Exp. Psychiatry 28(4), 251-261.

227 80. Wyon, Y., Lindren, R., Lundeberg, T., and Hammar, M. (1995). Effects of acupuncture on climacteric vasomotor symptoms, quality of life, and urinary excretion of neuropeptides among postmenopausal women. Menopause 2(1), 3-12. 81. Brezezinski, A., Adlecreutz, H., Shaoul, R., R6sler, A., Shmueli, A., Tanos, V., and Schenker, J. G. (1997). Short-term effects of phytoestrogen-rich diet on postmenopausal women. Menopause 4(2), 8994. 82. Murkies, A. L., Lombard, C., Strauss, B. J., Wilcox, G., Burger, H. G., and Morton, M. S. (1995). Dietary flour supplementation decreases post-menopausal hot flushes: Effect of soy and wheat. Maturitas 21(3), 189-195. 83. Albertazzi, E, Pansini, E, Bonaccorsi, G., Zanotti, L., Forini, E., and De Aloysio, D. (1998). The effect of dietary soy supplementation on hot flushes. Obstet. Gynecol. 91, 6-11.

_~HAPTER | .

Cardiovascular Pathophysiology CAROL A.

DERBY

New England Research Institutes, Watertown, Massachusetts 02472

IV. Studies of Menopause and Cardiovascular Risk Factors V. Conclusions References

I. Introduction II. Sex-Specific Trends in Cardiovascular Risk with Age III. Epidemiologic Studies of Menopause and Cardiovascular Outcomes

Data regarding endogenous estrogen levels and cardiovascular risk are rare. One cross-sectional and one prospective study have examined circulating estrogen concentrations and cardiovascular disease in postmenopausal women, both with negative results [3,10]. Cauley et al. [10] found no cross-sectional association between estrone concentrations and coronary artery occlusion in a series of 87 postmenopausal women evaluated with coronary arteriography. The only prospective study to date found that age-adjusted endogenous estrogen concentrations did not predict cardiovascular death or fatal ischemic heart disease in a cohort of 651 postmenopausal women [3 ]. The remaining evidence for the hypothesis that menopause increases cardiovascular risk is based on comparisons of age-specific disease rates in men and women, observational studies of cardiovascular outcomes in women with menopause at an early age, and studies of risk factor changes around the time of menopause. This chapter reviews these lines of evidence based on vital statistics data, epidemiologic studies of menopause and cardiovascular outcomes, and studies of menopause in relation to blood pressure, lipids, and hemodynamic factors. Direct effects of estrogen on the arterial wall are also briefly summarized.

I. I N T R O D U C T I O N Cardiovascular diseases are the leading cause of death among women in the United States and in most developed countries [1]. In the United States, all cardiovascular diseases combined claim the lives of one-half million women annually, twice the number of deaths attributable to all forms of cancer [ 1,2]. Among women over the age of 50, these diseases account for over 50% of all deaths, and are among the major causes of morbidity and disability in postmenopausal women [2]. It has been hypothesized that the cessation of ovarian function with menopause is associated with increased cardiovascular risk, and that the increase is mediated by estrogen. However, whether menopause is independently associated with increased cardiovascular risk remains a topic of debate. The best evidence in support of this hypothesis is from studies of exogenous estrogen replacement [3-5]. However, these data are based predominantly on observational studies, and there is concern that results may be biased by selection factors that determine who uses hormone replacement therapy [6-9]. The cardiovascular consequences of hormone replacement therapy are discussed in Chapter 37.

MENOPAUSE:

BIOLOGY AND PATHOBIOLOGY

229

Copyright 9 2000 by Academic Press. All rights of reproduction in any form reserved.

230

CAROL A. DERBY

II. SEX-SPECIFIC CARDIOVASCULAR WITH

TRENDS

IN

RISK

AGE

Rates of coronary disease are very low among young women and increase sharply with age, with the largest apparent increase around the age of 50 years. Women have lower age-specific rates of fatal coronary heart disease (CHD) than do men at every age [5]. The sex ratio for fatal CHD is remarkably consistent across populations with varying rates of heart disease and lifestyles, with a 2.5- to 4.5-fold excess risk in men aged 4 5 - 6 9 years [11]. The sex differential in CHD deaths is greatest around age 50 years and diminishes thereafter [5,12-16]. The consistency of an apparent protective trait in women along with the decline in the sex ratio for CHD deaths around the age of menopause have been cited as evidence that natural menopause increases CHD risk [3,12-15,17]. However, closer examination of vital statistics data does not support this hypothesis. If natural menopause were responsible for increased CHD mortality, then the rate of increase in mortality rates with age would be expected to accelerate in the postmenopausal period. That is, when plotted on a log scale, the slope of the curve for age-specific death rates would be steeper in women over the age of 50 years compared with that for younger women. Examinations of vital statistics data for the United States [5,12,16], New York state [14], and England and Wales [13] have all failed to demonstrate an inflection point in the rate of increase in CHD mortality with age. This is in contrast to plots of the mortality rates for breast cancer, which show a clear shift around the age of menopause [5,16]. Similarly, close inspection of sex-specific rates of increase in CHD mortality with age suggest that menopause is not responsible for diminution of the male-to-female mortality ratio around age 50 years. Rather, the narrowing of the gender gap in CHD mortality is due to a slowing of the rate of increase in male death rates after age 50 years, and is not attributable to a postmenopausal shift in the rates of change for women [12,13,16]. Furthermore, although the average age of menopause has been reported to be fairly consistent across countries and over time [18], the age at which the male-to-female ratio of CHD deaths peaks has been shown to vary from 30 to 35 years in the Netherlands, to 55 to 60 years in Japan [16]. In summary, there is a widely held belief that estrogen protects premenopausal women from cardiovascular disease and that this protection is substantially reduced with the menopause [19]. However, analyses of age-specific disease rates in men and women have consistently refuted this assumption. Thus, vital statistics data do not support the hypothesis that menopause is accompanied by an increase in cardiovascular risk.

III. EPIDEMIOLOGIC OF MENOPAUSE

STUDIES

AND

CARDIOVASCULAR

OUTCOMES

Studies of menopause and cardiovascular risk have yielded inconsistent results, particularly studies of natural menopause. In part, this may be attributed to a range of methodologic issues. Many studies have not distinguished between natural and surgical menopause, or between women with hysterectomy alone and those with bilateral oophorectomy [15,20-22]. Definitions of menopause are not consistent across studies and often rely on self-report [15,22-29]. The definition of early menopause has ranged from 35 to 50 years, with age at menopause frequently based on recall. Inaccurate recall of age at menopause may be particularly problematic for studies of natural menopause. In some instances, specific age of menopause is not reported [23,28]. Risk estimates from many studies of coronary disease and early menopause are based on small numbers of cases, particularly in the premenopausal control group [24,29,30]. Several studies have included a large proportion of nonspecific, or "soft," cardiovascular end points such as angina, with very few more definitive cases such as myocardial infarction [21,29-33]. Because the majority of longitudinal studies begin with women at or very close to menopause, there are few longitudinal data on changes during the menopausal transition period preceding the actual cessation of menses. This perimenopausal period is characterized by wide fluctuations in circulating estrogen and may mark the beginning of estrogen-mediated changes in cardiovascular risk factors [18]. In general, prospective studies have been based on follow-up times that are relatively short given the lengthy natural history of cardiovascular disease. Many studies have not controlled for confounding by smoking, a major cardiovascular risk factor that is also associated with younger age at menopause [ 17]. Finally, it is very difficult to discriminate between changes related to aging and those attributable to menopause per se. Precise adjustment for age is crucial yet is often lacking [17]. A detailed discussion of methodologic issues in the study of menopause is included in Chapter 10.

A. S t u d i e s w i t h T y p e o f M e n o p a u s e U n s p e c i f i e d A few published studies relating early menopause to cardiovascular risk have failed to specify whether menopause was natural or surgical. Two of these [21,22] suggested that early menopause is associated with increased risk of cardiovascular disease, whereas a third [20] found no increased risk of fatal coronary heart disease among women with early menopause. Oliver [21] described a case-series of women with clinical coronary heart disease under the age of 45

CHAPTER 15 Cardiovascular Pathophysiology years, and reported that compared with women in the general population, a high proportion had experienced menopause (20% compared with 3 to 5%). Similarly, a population-based case-control study in Gothenberg, Sweden, showed that women with clinical heart disease were significantly more likely than healthy controls to have reached menopause prior to age 50 years [22]. In contrast, a case-control study reported by Mann and Inman [20] found no elevated risk of fatal coronary heart disease among women with menopause before age 50 years. Given that the type of menopause was not specified, and that a control group was either missing entirely [21 ] or was not well defined [20,22], inferences from these studies are limited.

B. S t u d i e s o f N a t u r a l M e n o p a u s e A number of studies have examined the relationship between natural menopause and cardiovascular risk, with inconsistent results. Sznajderman and Oliver [33] described a case-series of women evaluated for premature cessation of menstruation between the ages of 35 and 40 years, and determined the proportion that developed clinical features of ischemic heart disease 15 to 20 years later. When compared with a general population sample, the women with early menopause had a sevenfold increased risk. However, the interpretation of this study is limited by the lack of an appropriate control group and by the small number of cases. In a cross-sectional study, Witteman et al. [24] measured the extent of calcification in the abdominal aorta in a population-based sample of women aged 4 5 - 5 5 years. After adjustment for age and coronary risk factors, women with natural menopause were estimated to have a 3.4-fold elevated risk of calcifications compared with premenopausal women (95% confidence interval, 1.2-9.7). Among women with natural menopause, there was evidence for an increasing trend in risk of calcification with increasing time since menopause. However, estimates were based on small numbers of women with calcifications, and the temporal association between outcome and exposure could not be determined. Case-control studies have suggested either a positive association between early menopause and cardiovascular risk [34], or no association [23,25]. A study in Rochester, Minnesota compared incident cases of coronary heart disease prior to age 60 years and hospital-based controls on the proportion with menopause before age 50 years [34]. The estimated relative risk of myocardial infarction for women with early menopause was 1.3, and was not statistically significant. A hospital-based case-control study by LaVecchia et al. [23] also found no association between natural menopause and risk of myocardial infarction prior to age 55 years. This study did not specify age of menopause in cases and controls.

231 The largest reported case-control study of cardiovascular disease and menopause was based on data from the baseline interview of the Nurses Health Study [25]. Women under 56 years of age, with a history of myocardial infarction (N = 279), were age matched with over 5000 control women who had no history of infarction. The estimated relative risk of myocardial infarction for women with natural menopause compared with premenopausal women was 0.9 with a 95% confidence interval of 0.6-1.3. Multivariate adjustment for cardiovascular risk factors, including smoking, further attenuated this association. Myocardial infarction risk was elevated in women with natural menopause before age 35 years, although the relative risk for this age stratum was based on few cases and was not statistically significant. Prospective studies of natural menopause and cardiovascular risk have also failed to yield consistent results, with some suggesting increased risk in postmenopausal women [27,29,30] and others showing no evidence for elevated risk [26,28]. The Framingham study compared coronary disease event rates for 1934 women with natural menopause during follow-up and age-matched premenopausal women [29,30, 35]. Among women aged 4 5 - 5 4 years, the relative risk for coronary heart disease was 2.7, and was statistically significant. This risk estimate is based on a total of only 10 cases, the majority of which were angina. Risk estimates in other age strata were not significant [29]. An update from this study has reported a relative risk of 4.1 for natural menopause in women aged 5 0 - 5 9 years [35]. None of these analyses were adjusted for chronological age or smoking status. A study by van der Schouw et al. [27] has provided additional data from a large population-based study with 20 years of follow-up on 12,115 women initially aged 5 0 65 years. The age-adjusted hazard ratio for age at menopause was 0.982 per year (95% confidence interval, 0.968-0.996). Thus, for each year of delay in menopause (surgical and natural combined), cardiovascular mortality risk decreased by 2%. Adjustment for cardiovascular risk factors, including smoking, did not alter these results substantially. However, when natural menopause was analyzed separately, the hazard ratio did not reach statistical significance (0.98; 95% confidence interval, 0.97-1.00). These results may be limited by the fact that menopausal status and age of menopause were based on self-report. A population-based prospective study in Gothenburg, Sweden [26] examined 12-year incidence rates for various cardiovascular end points in relation to menopausal age in a cohort of 1462 women. Relative risks for myocardial infarction and angina were slightly elevated regardless of whether early menopause was defined at 40, 45, or 50 years. However, none of these estimates was statistically significant, and no adjustment was made for potential confounding by smoking, and menopausal age was determined by respondent recall.

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The largest prospective study of natural menopause and cardiovascular risk is the Nurses Health Study, which reported on 12 years of follow-up for 121,700 women aged 3 0 55 years at baseline [28]. After adjustment for age in 5-year increments, relative risk of coronary disease women with natural menopause was 1.7 (95% confidence interval, 1.12.8). However, more precise adjustment for age, in 1-year increments, showed no increased cardiovascular risk with natural menopause (relative risk 1.2, 95% confidence limits, 0.8-1.8). This study highlights the importance of adjusting closely for chronological age and smoking status [ 17]. Similarly, another study of women in Italy who were followed for 16 years found that although cardiovascular morbidity and mortality were higher among women with natural menopause as compared with premenopausal women, these differences were no longer apparent after age adjustment [36].

C. S t u d i e s o f S u r g i c a l M e n o p a u s e In contrast to the inconclusive findings for natural menopause, studies of bilateral oophorectomy have been more consistent, suggesting that early bilateral oophorectomy increases risk of cardiovascular disease. Nevertheless, it should be noted that studies of surgical menopause are limited by many of the methodologic issues outlined above. Furthermore, comparisons across studies are complicated by inconsistencies in the reference groups used. Oophorectomized women have been compared with women who had hysterectomy only, with premenopausal women, or with naturally menopausal women. Overall, autopsy studies suggest that bilateral oophorectomy is associated with an increased degree of coronary atherosclerosis. Autopsy studies provide clear definitions of ovarian status as well as anatomic definitions of disease end points. However, the data are cross-sectional, selection factors that determine inclusion in autopsy series are difficult to quantify, and control for confounding factors is limited by lack of information. Parrish et al. [37] studied the autopsy records of 80 women who had bilateral oophorectomy prior to age 50 years, comparing the extent of coronary atherosclerosis with that in age-matched women with intact ovaries. Bilateral oophorectomy was associated with more extensive atherosclerosis only among women with surgery prior to age 40 years. Wuest et al. [38] compared autopsy findings for oophorectomized women, a control series of women, and a series of men. Women with bilateral oophorectomy had more extensive coronary atherosclerosis than did female controls, and oophorectomized women had disease rates that approximated those of men in the same age groups. Similarly, Rivin and Dimitroff [39] compared the autopsy records of women with bilateral oophorectomy before age 50 years and those from a series of 600 control women. The oophorectomized

group had two to three times the rate of severe atherosclerosis compared to controls. The latter two studies are limited in that the composition of the female control group with respect to menopause status and the comparability of the ages for cases and controls are not clear. In contrast to these studies, Novak and Williams [40] found no significant difference in the extent of atherosclerosis in oophorectomized women compared with controls. Analyses by age at menopause also showed no effect, although the sample sizes in the age strata were quite small and there was a wide range of ages at death. A few cross-sectional studies have examined anatomically defined atherosclerosis in samples of women undergoing arteriography or noninvasive radiographic screening tests. Manchester et al. [41 ] compared the prevalence of angiographically documented coronary artery disease for 20 women with history of bilateral oophorectomy at least 5 years prior to angiography, and a control group of 65 women. All women were under the age of 50 years, and had been referred for angiography for the evaluation of angina. The majority of the control group (95%) was premenopausal. The relative odds of significant coronary artery disease (greater than a 50% occlusion) was 0.6 (p = 0.4) for oophorectomized women compared with controls. This estimate was based on a small sample of women, and only five in the oophorectomy group had significant coronary disease. In addition, arteriography studies are subject to a number of selection biases that may limit their generalizbility [42]. A much larger cross-sectional study of anatomically defined disease found that women with bilateral oophorectomy had 5.5 times the risk of calcifications in the abdominal aorta than did premenopausal women of the same age [24]. Women with hysterectomy and intact ovaries had no excess risk. These results support the hypothesis that increased cardiovascular risk in oophorectomized women is due to loss of ovarian function. Oliver and Boyd [43] have also reported results consistent with this hypothesis. Women with either bilateral or unilateral oophorectomy before age 35 were compared with respect to the incidence of clinical coronary heart disease in the 25 years following surgery. Those with bilateral oophorectomy appeared to have an elevated risk of coronary heart disease compared with either the unilateral oophorectomy group (odds ratio approximately 10) or with a general practice sample (odds ratio approximately 8). In contrast, the coronary heart disease rate for women with unilateral oophorectomy was similar to that in the general medical practice sample. In general, case-control studies of myocardial infarction and surgical menopause also suggest that surgical menopause increases risk. However, in several of these studies sample sizes were small and results were not statistically significant. At least one study has suggested that factors leading to gynecologic surgery may be related to increased cardiovascular risk independent of ovarian status [44]. Ritterband et al. [44] reviewed hospital records from 10 years prior

CHAPTER 15 Cardiovascular Pathophysiology to identify women with oophorectomy and a group with hysterectomy only before the age of 35. The women were evaluated for history or physical evidence of coronary heart disease. Coronary disease rates for women with hysterectomy only and those with bilateral oophorectomy were similar even after adjusting for age and estrogen replacement therapy. When compared with a control group of their sisters, both gynecologic surgery groups had twice the rate of heart disease, although the number of cases was small and differences were not statistically significant. Robinson et al. [31] also compared cardiovascular disease prevalence in women with history of oophorectomy or hysterectomy only prior to age 45. In contrast to the previous study, the estimated risk of clinical coronary heart disease was approximately 4.0 times higher for women with oophorectomy, and the difference was statistically significant. Winklestein et al. [15] studied 50 women with myocardial infarction and compared the prevalence of surgical menopause with prevalence in paired neighbor controls and with controls from a random probability population sample. Myocardial infarction patients had a higher probability of having had surgical menopause, although the result was only of borderline statistical significance. This study did not distinguish between surgical menopause with or without bilateral oophorectomy. In another hospital-based case-control study, La Vecchia et al. [23] studied women with myocardial infarction (MI) before age 55 years, and a group of hospital controls. They reported no association between surgical menopause and acute myocardial infarction (estimated relative risk 0.7, 95% confidence interval, 0.39-1.23). It was not clear whether the surgical menopause group excluded women with hysterectomy only and intact ovaries. In a similar study, Beard et al. [34] identified women with coronary heart disease prior to age 60 years, and compared the prevalence of surgical menopause with a group of age-matched controls from the Mayo Clinic. The relative risk for surgical menopause compared with natural menopause was 1.6, and was of borderline statistical significance (95% confidence interval, 1.0-2.5). Johansson et al. [32] identified a group of women who had undergone bilateral oophorectomy between 1904 and 1910, and attempted to obtain follow-up information regarding their cardiovascular experience. This was compared with the cardiovascular status of women with surgery for uterine prolapse. Among women under the age of 65 at follow-up, the estimated odds ratio for coronary heart disease was 3.0 but was not statistically significant. This study is limited in that follow-up information was obtained for less than half of the women with oophorectomy, and risk estimates were based on only 17 cases and 17 controls. The largest case-control study of surgical menopause and cardiovascular risk was from the Nurses Health Study [25]. Bilateral oophorectomy was associated with an overall estimated relative risk of 2.9 (95% confidence interval, 2.1-

233 4.0). Risk associated with oophorectomy increased with decreasing age at surgical menopause. Women with hysterectomy without bilateral oophorectomy had no increased risk of acute myocardial infarction (estimated relative risk 0.9, 95% confidence interval, 06-1.3). This finding is consistent with the cross-sectional results of Witteman et al. [24], and in contrast to the case-control study by Ritterband et al. [44]. Prospective data also suggest that bilateral oophorectomy is associated with increased cardiovascular risk. In the Framingham study, women with surgical menopause tended to have elevated risk of coronary heart disease, although, the association was significant only among women aged 4 0 - 4 4 years [29,30]. In addition, the number of CHD cases was small, and angina constituted the majority of cases. A more recent prospective study estimated the hazards ratio for age at menopause among women with hysterectomy only and for those with bilateral oophorectomy [27]. Age at menopause was not associated with cardiovascular mortality among those with hysterectomy only. Among women with oophorectomy, there was a significant inverse relation between age of menopause and cardiovascular mortality risk, with a 6% reduction in annual risk with each year increase in age of menopause. The Nurses Health Study found that after adjusting for age and smoking, women with hysterectomy and bilateral oophorectomy had a relative risk of 2.2 (95% confidence interval, 1.2-4.2) [28]. This study provides some of the strongest evidence to date that increased cardiovascular risk following surgical menopause is the result of decreased estrogen levels. Although women with surgical menopause had a twofold increased risk of cardiovascular disease, women who used hormone replacement therapy after bilateral oophorectomy had no greater risk than did premenopausal women.

D. S u m m a r y Epidemiological evidence that natural menopause increases cardiovascular risk is lacking. A number of studies ranging from case series to cohort studies have shown inconsistent results. This inconsistency may be the result of a number of methodologic issues, variations in study design, or definitional inconsistencies. Given that the natural history of cardiovascular disease can span decades and that ovarian function declines gradually for a period of 10 to 15 years prior to the cessation of menses, the lack of an abrupt increase in cardiovascular risk following natural menopause is not surprising [5,17,30]. Data regarding surgical menopause have been more consistent suggesting that bilateral oophorectomy prior to age 40 or 45 years is associated with increased cardiovascular risk. The lack of increased risk in women with bilateral oophorectomy who take hormone replacement therapy is the

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best evidence that estrogen is the mediating factor in this relationship. However, factors leading to surgical menopause may be related to cardiovascular risk and it is not possible to separate these factors from the effects of decreased postsurgical estrogen levels [44,45]. Thus, the role of menopause in the development of atherosclerotic disease remains controversial. Larger studies, using standard definitions and with long-term follow-up beginning prior to the perimenopausal period, are needed to clarify the relation of natural menopause to cardiovascular risk. The inferences from studies of surgical menopause would be strengthened by additional data regarding factors leading to surgery. Finally, studies regarding the role of endogenous estrogen levels in relation to cardiovascular outcomes are needed to confirm whether there is a causal link between menopausal declines in estrogen and cardiovascular risk.

IV. STUDIES OF MENOPAUSE AND CARDIOVASCULAR RISK FACTORS An alternative approach to evaluating the influence of menopause on cardiovascular risk is to examine the relationship between menopausal status and cardiovascular risk factors. In this section the epidemiological evidence for associations between menopause status and the traditional risk factors--smoking, blood pressure, and lipids--is discussed. Also discussed is the association between menopause and hemodynamic factors and evidence for direct effects of estrogen on arterial walls. The associations of menopause with carbohydrate metabolism and body composition are described in Chapter 16.

A. Smoking Although menopause status is not a determinant of smoking behavior, smoking is a potentially strong confounder in studies of cardiovascular risk and menopause [17,28,45]. Smoking is well established as a coronary risk factor, both independently [46] and via positive associations with blood pressure and lipids [47-49] and an inverse association with body mass index [50]. In addition, smoking has consistently been associated younger age of menopause. Women who smoke cigarettes experience menopause 1 to 2 years earlier than do nonsmoking women [51-53]. Proposed mechanisms include lower estrogen levels, more rapid estrogen metabolism, accelerated aging of ovarian follicles, and impaired estrogen receptor binding in smokers [54]. An understanding of this association is important to the interpretation of studies regarding menopausal changes in cardiovascular risk.

B. Blood Pressure Blood pressure increases gradually with age in both men and women. However, there is a cross-over in the relative levels of blood pressure after the age of menopause. Although women have lower levels of systolic and diastolic blood pressure prior to age 60 years, men tend to have lower levels at older ages [55]. Similarly, hypertension is less prevalent in women until middle age, when the sex difference disappears or reverses [2]. These patterns have led to the hypothesis that estrogens may protect premenopausal women from hypertension. Studies showing blood pressure fluctuations with the menstrual cycle have also suggested an association with female hormones [56-58]. However, there is no clear evidence linking menopause with adverse changes in blood pressure. Four decades ago, Taylor et al. [59] observed that arterial hypertension was no more common in a series of postmenopausal women than in the general population. The interpretation of this study was limited given that inferences were based on a case series and that the study lacked an adequate control group. Nevertheless, subsequent studies have failed to produce consistent evidence that would refute Taylor's early finding of no association between menopause and blood pressure. Cross-sectional studies of blood pressure in relation to menopause status have yielded discrepant results. These have shown no difference between premenopausal and postmenopausal women [56,60-62], or have found increased systolic [63], decreased systolic [64], or increased diastolic blood pressure [65] in postmenopausal women. Wu et al. [56] found in increased prevalence of both hypotension and hypertension in postmenopausal women, whereas Portaluppi et al. [61] reported no difference in the prevalence of hypertension after adjusting for differences in age and body mass index. In general, prospective studies have failed to show an association between menopause and blood pressure. Data from the Framingham study indicate no relationship between changes in menopausal status and changes in systolic or diastolic blood pressure [66]. These comparisons considered the effects of natural or surgical menopause with and without bilateral oophorectomy. Matthews et al. [67,68] followed a cohort of women through the menopause transition from pre- to peri- to postmenopause, and found no significant changes in either systolic or diastolic blood pressure. A longitudinal population study in Gothenberg, Sweden also observed no increase in blood pressure for women who became postmenopausal during follow-up [69]. In fact, this study reported that systolic blood pressure decreased with increasing time since menopause. Declining blood pressure with increasing time since menopause was also observed in a cohort of 168 perimenopausal women followed for 7 years in Sweden [70].

CHAPTER 15 Cardiovascular Pathophysiology In contrast, two recent prospective studies have shown increases in systolic blood pressure with menopause. Poehlman et al. [71] followed a small sample of 38 women for 6 years, and found that systolic blood pressure increased significantly more among women who became postmenopausal during follow-up. The observed blood pressure changes were not correlated with changes in body fatness. A larger study reported by Staessen et al. [72] followed 315 women for 5 years and showed an increase in systolic blood pressure of 3 - 4 mm Hg for postmenopausal women, but no change among premenopausal women. This difference persisted after adjustment for age, changes in body mass index, and antihypertensive medication use. A higher incidence of hypertension among postmenopausal women was also reported, although this was not statistically significant after adjustment for confounders. Increases in blood pressure around the time of menopause may be the consequence of aging, rather than changing hormonal status. Increasing blood pressure with age has been documented for women with unchanged menstrual status [67-69]. A 30-year follow-up study of over 4000 female atomic bomb survivors in Japan found an increasing trend in systolic blood pressure with age, but no change in the slope of this trend around the time of either natural or surgical menopause [73]. Similarly, van Beresteyn et al. [70] observed increasing systolic blood pressure with chronological aging, but no blood pressure effect of menopause. Weight gain around the time of menopause, rather than menopause per se, may also influence blood pressure levels. Excess body weight and excess abdominal fat are positively correlated with increased blood pressure [47], and an ageassociated weight gain has been observed among women around the time of menopause [67,74]. Both cross-sectional [56,61] and prospective studies [70,74] have shown a positive correlation between weight gain and blood pressure among women of menopausal age, but no independent blood pressure effect of menopause. In contrast, others have reported that adjustment for body mass index did not explain a positive relation between menopause and systolic blood pressure [63,71,72]. In summary, the preponderance of evidence from epidemiological studies does not support a causal association between menopause and adverse changes in blood pressure. Whether menopause exerts an independent effect on blood pressure is difficult to assess given the joint effects of age and blood pressure. If menopause does exert an adverse effect, the lack of consistency across numerous studies suggests that it may be weak, and limited to systolic blood pressure.

C. L i p i d s Perhaps the best evidence for an effect of menopause on CVD risk is that suggesting adverse changes in the lipid pro-

235 file around the time of menopause. This evidence is based on sex differences in age trends for lipoproteins and results from cross-sectional and longitudinal population studies and studies of postmenopausal hormone replacement therapy. Total cholesterol levels increase with age in both sexes. However, among men, the increase begins in the third decade of life, whereas in women the age-related increase is delayed until the fifth decade [75]. Similarly, although low-density lipoprotein (LDL) cholesterol increases with age for men and women, the timing of the increase differs by sex. Women experience a more gradual increase until middle age, when their rate of increase accelerates and that of men levels off [76,77]. In both sexes, increasing LDL with age is thought to be the result of an age-dependent decline in LDL receptors [78]. Animal experiments have shown that estrogens increase LDL receptors [79,80]. These observations have led to the hypothesis that age-related increases in LDL for women are modulated by the presence of estrogen, and that increasing LDL levels around the time of menopause may be the result of a reduction in LDL receptors triggered by declining estrogen levels [77,81 ]. In contrast, sex-specific trends in high-density lipoprotein (HDL) cholesterol with age suggest that differential levels of testosterone rather than estrogen determine sex differences [77]. During adolescence, HDL levels rise slowly in girls, and decline abruptly in boys in conjunction with sexual maturation [76,77,82]. HDL levels remain constant in adults, with levels in men remaining below those in women, even after menopause [54,76]. The majority of observational studies, conducted in various populations with differing definitions and study designs, have supported an association between menopause and increasing serum cholesterol levels. These include studies in the United States [30,65,66,71,83-85], Europe [60,64,69, 86,87], Britain [88], Japan [73,89], and China [56]. Most comparisons have been for women with natural menopause and premenopausal controls, with higher total cholesterol levels in the postmenopausal group. This has been observed in both cross-sectional [56,60,64,87,89] and longitudinal studies [30,66,69,86]. Similar findings have been reported when the postmenopausal group included both naturally and surgically menopausal women [30,62,63,65]. One crosssectional [88] and one prospective study [73] have compared total cholesterol levels in naturally menopausal women with those in men. Razay et al. [88] reported that age predicted increasing total cholesterol levels in women, and that women over 50 years of age had higher levels than did younger women. The lack of change in total cholesterol levels with age in men was interpreted as suggesting that the observations in women were due to menopause. It is important to note that this evidence is indirect, and the study is limited by the fact that postmenopausal status was based solely on age. Akahoshi et al. [73] compared longitudinal changes in lipids for women who became naturally menopausal and

236 age-matched male controls. A sharp increase in total cholesterol levels around the time of menopause was observed only in the women. Increased serum total cholesterol levels in postmenopausal women appear to be independent of age and are not explained by increased body mass index among postmenopausal women [56,60,63,65,66,73,85,87,89], although not in all studies [36,90]. A cross-sectional study by Campos et al. [90] and a prospective analysis reported by Casiglia et al. [36] each reported that differences in total cholesterol levels for pre- and postmenopausal women were no longer statistically significant after adjustment for age [36], or age and body mass index [90]. Total cholesterol levels also appear to be elevated among women with surgical menopause, although fewer studies have addressed this issue. A small, cross-sectional study reported by Notelevitz et al. [83] showed elevated total cholesterol levels in young women with bilateral oophorectomy compared to age-matched women with intact ovaries. Hjortland et al. [66] demonstrated prospectively that relative to premenopausal controls, the increase in total cholesterol levels for women with surgical menopause was similar to the increase observed for women with natural menopause. Similarly, the prospective study of Akahoshi et al. [73] showed that serum cholesterol levels increased abruptly with menopause, whether surgical or natural, and that no such change occurred in an age-matched group of male controls. There are few data regarding the relation of menopause to serum cholesterol levels in nonwhite women. The few studies to have examined this issue suggest that racial differences exist [84,85]. A report from the Evans County Cardiovascular Disease Study [85] showed no significant relationship between menopause status and total cholesterol levels in black women, and a significant relationship among whites that was independent of age, body mass index, and smoking. A more recent cross-sectional analysis of data from the Minnesota Heart Survey found similar results. Demirovic et al. [84] reported higher total cholesterol levels in white postmenopausal women compared with premenopausal controls. In contrast, among blacks, postmenopausal women did not have significantly higher total cholesterol levels than did premenopausal women. The lack of an association between menopause and serum cholesterol levels has also been reported among Pima Indian women in the southwestern United States [91]. More information is required regarding menopausal changes in minority women. Increases in total cholesterol with menopause appear to be attributable to increases in LDL [30,63,67,71,86,90]. This is consistent with the hypothesis that declining LDL receptors in response to decreasing estrogen levels mediate menopausal changes in lipids and lipoproteins. A relationship between menopause and LDL levels has been observed in cross-sectional [60,63,90] and prospective studies [67,

CAROL A. DERBY

71,86]. Studies have not consistently controlled for age, although those that did incorporate age controls have suggested an independent effect [60,63,90]. In contrast, Matthews et al. [68] have studied women as they transition from premenopause through perimenopause, and concluded that elevations in LDL are associated with aging and not with menopausal status. Cross-sectional results from the Framingham Offspring Study [90] have suggested that menopause may be related to changes in the quality as well as the quantity of LDL. Postmenopausal women in this study were shown to have significantly increased numbers of small, dense LDL particles compared to premenopausal women of the same age. These particles, rich in apoprotein-B, have been associated with risk of premature coronary artery disease [92,93]. Several studies have shown a relation between increased triglyceride levels and either natural [56,63,69,71,87] or surgical menopause [83]. However, this has not been shown consistently. Other studies have reported either no association or have shown that adjustment for age attenuated the association between menopause status and triglycerides [36,60,90]. Studies of HDL and menopause have also yielded inconsistent findings. Cross-sectional studies have found menopause to be associated with decreased [83,90] or increased [56] HDL, or have failed to show an association [30,60, 63,84,89,94]. Prospective studies have tended to show decreasing HDL with menopause [67,71,86]. Jensen et al. [86] longitudinally studied premenopausal women as they transitioned through menopause, and reported that HDL declined gradually beginning in the 2 years preceding the cessation of menses. Thus, the apparent discrepancy between crosssectional and prospective studies might be explained by the inability of cross-sectional analyses to detect small, gradual changes in HDL [86]. In contrast, Matthews et al. [68] have suggested that changes in HDL may be the effect of aging rather than menopause status. The failure of observational studies to demonstrate a consistent relation between menopause and HDL seem to conflict with studies of exogenous hormone therapy, which have consistently shown increased HDL with treatment [95-97]. This apparent discrepancy may be due to the direct hepatic effects of pharmacological doses of oral estrogen on lipid metabolism [77]. The HDL effects of hormone replacement therapy appear to be attenuated with transdermal hormone administration, which attains hormone levels more closely resembling physiologic levels and does not stimulate the liver on absorption [98]. Lipoprotein(a) [Lp(a)] has been established as a coronary risk factor [99,100], yet data regarding the relation of menopause to Lp(a) are limited. Jenner et al. [101] reported no differences in Lp(a) levels for postmenopausal and premenopausal women, after adjusting for age. This is in contrast to

CHAPTER 15 Cardiovascular Pathophysiology the cross-sectional study reported by Heinrich et al. [102] that showed significantly higher Lp(a) levels in postmenopausal women. This study also showed that concentrations of Lp(a) levels changed slightly in both men and women prior to age 45 years, whereas after age 45, levels in women increased steadily. Further studies are needed to determine the effects of menopause on Lp(a). The best evidence that estrogens mediate changes in lipoproteins with menopause is derived from studies of women taking exogenous hormone replacement therapy (Chapter 37). Although these findings have been generally consistent, the majority are based on observational data, and concerns have been raised regarding the influences of selection biases on the characteristics of women who take hormone replacement therapy [6,7,9]. However, in the largest randomized placebo controlled trial to date, the Postmenopausal Estrogen/Progestin Interventions (PEPI) Trial, estrogen replacement therapy, either opposed or unopposed, was associated with significant increases in HDL and decreases in LDL [95]. Data directly linking endogenous estrogen levels with lipid and lipoprotein changes are limited and inconsistent. Wu et al. [56] showed that premenopausal women had increased estradiol levels relative to postmenopausal women, and that estradiol levels were inversely associated with total cholesterol. In contrast, a cross-sectional study of women who had been postmenopausal for an average of 9 years found that endogenous estrogens were not related to lipids [103]. Similarly, a prospective study reported by BarrettConnor and Goodman-Gruen [3] found that estrone concentrations were only weakly related to total cholesterol levels. A few studies have examined the time course of lipid and lipoprotein changes in relation to menopause, and have provided indirect evidence that these changes are mediated by declining estrogen levels. Declining estradiol concentrations have been shown in women approaching menopause [ 104,105], and several studies have suggested a temporal relationship between lipid and lipoprotein changes and this perimenopausal decline. Jensen et al. [86] showed that lipid changes were correlated with decreasing estrogen and increasing gonadotrophins during the perimenopausal period. Akahoshi et al. [73] demonstrated prospectively that increased total cholesterol preceded natural menopause by approximately 3 years. Hjortland et al. [66] also found higher premenopausal serum total cholesterol levels in women approaching menopause compared with those who remained premenopausal during follow-up, although the timing of evaluations in relation to menopause may have caused these differences. Lindquist and Bengtsson [64] found that premenopausal increases in total cholesterol were related to the time remaining until menopause. Finally, a cross-sectional study of total and HDL cholesterol levels in premenarchal girls and menstruating, pregnant, and post-

237 menopausal women also suggested that ovarian hormones influence lipid levels [89]. The timing of lipid changes in relation to surgical menopause also suggests a temporal association with the loss of ovarian function. Serum cholesterol levels have been shown to increase abruptly following bilateral oophorectomy [73]. In addition, the Framingham study reported that total cholesterol levels increased significantly in women with bilateral oophorectomy, but not among women with hysterectomy without oophorectomy or with unilateral oophorectomy [66]. In summary, epidemiologic data have consistently shown a relation between menopause and increased serum total cholesterol levels. This increase may be attributable to increased levels of LDL cholesterol, possibly as the result of decreased LDL receptors in response to declining estrogen levels. Increased triglyceride levels may also occur with menopause, although these data have been less consistent. There is no consistent evidence that menopause per se has a deleterious effect on HDL cholesterol levels. Important issues must be considered when weighing the available evidence relating menopause to adverse changes in the lipid profle. Both menopause and lipid levels are strongly related to age, as suggested by the gradual increases observed in both men and women with chronologic age. Thus, the effects of menopause independent of age are difficult to assess. Second, many of the data supporting a causal role of estrogen in lipid changes with menopause are derived from studies of exogenous hormone replacement therapy. Few studies have examined the relation of endogenous hormones to lipid levels, and these have been inconsistent, or have provided only indirect evidence by establishing a temporal association. Additional studies are needed to understand the timing, duration, and extent of lipid changes throughout the menopause transition and to establish whether these changes are determined by changes in endogenous ovarian hormone levels.

D. H e m o s t a t i c C h a n g e s Elevations of plasma fibrinogen [ 106-108] and factor VII [ 106,109,110] have been associated with increased risk of coronary heart disease. There is evidence that these coagulation factors may be influenced by estrogen. Fibrinogen increases with age [ 111 ], and in women this increase begins in the fifth decade [112]. Studies of fibrinogen during pregnancy and throughout the menstrual cycle also suggest an influence of endogenous estrogen [ 113]. The use of hormone replacement therapy, either estrogen alone [95,114] or estrogen plus progestin [95,115], has been associated with reduced concentrations of fibrinogen. Factor VII activity increases with the use of estrogen alone, although these changes normalize with the addition of progestins [115].

238 Population-based data regarding the effect of menopause on hemostatic factors are sparse and are primarily crosssectional. Several cross-sectional studies have reported elevated fibrinogen concentrations in postmenopausal women compared with premenopausal women [ 111,113,116-121 ]. Other cross-sectional comparisons have shown no differences in fibrinogen concentration by menopause status [63, 112,122]. Lee et al. [120,121] reported that although levels were elevated in postmenopausal women, menopause status explained only a small portion of the total variation in fibrinogen. Lindoff et al. [118] compared premenopausal women with women who had been postmenopausal for less than 18 months, and with a second group of women who had been postmenopausal for longer than 18 months. These comparisons suggested that increases in fibrinogen levels were related to the time elapsed since menopause. Meade et al. [117] prospectively observed that circulating fibrinogen increased in women who became postmenopausal during follow-up. Fibrinogen concentrations have been correlated with BMI, lipoproteins, and smoking [ 121,123], which are also related to menopause status. Although some studies have adjusted comparisons for age and these other factors [ 111,113,116, 117,120,121], others have not [63,112,118,119,122]. Thus, the lack of consistency across studies may be due in part to confounding and differential levels of adjustment. Cross-sectional studies have also shown elevated values for factor VII in postmenopausal women compared with those who are premenopausal [111-113,117]. This association has also been observed prospectively [116]. Increased factor VII may be a consequence of increased triglycerides in postmenopausal women [111,124]. Very-low-density lipoprotein cholesterol concentrations may rise after menopause [76], and studies in men have shown that triglycerides may increase factor VII activity [ 125]. Enhanced coagulability due to elevations of fibrinogen and factor VII in postmenopausal women may be offset by a concurrent increase in antithrombin III activity in postmenopausal women [118,126]. Postmenopausal women have been shown to have higher levels of antithrombin III compared with premenopausal women [113,116,118] or men [116]. In summary, most information regarding the effect of menopause on hemostatic factors is derived from studies of hormone replacement therapy. Studies of menopausal changes in hemostatic factors in untreated women are primarily cross-sectional. Some, but not all, of these have shown elevated concentrations of fibrinogen and factor VII in postmenopausal women. Whether these changes are attributable to estrogen or to changes in other factors at menopause has not been clearly demonstrated. Furthermore, because increased antithrombin III levels have also been shown

CAROL A. DERBY

in postmenopausal women, the net impact on risk of thrombosis may be minimal.

E. Direct Effects of Estrogens on the Vascular Wall Estrogen receptors are present in the cardiovascular system [ 127,128], and the direct effects of estrogens on the vascular wall may influence both the development of atherosclerosis and the regulation of arterial blood flow [ 124,129,130]. It has been estimated that the majority of the cardiovascular benefit of hormone replacement is attributable to direct effects of estrogen on the arterial wall, whereas only 2 0 - 3 0 % is attributable to lipid effects [ 126,131,132]. The molecular mechanisms by which estrogen affects arterial wall functions are not fully understood. Proposed mechanisms for the vasodilatory effects of estrogen include effects on calcium channels, potentiation of endothelium-dependent vasodilation, and increased synthesis of prostacyclin [124,126,130,133,134]. Estrogens may inhibit atherosclerosis by acting on vascular connective tissue, impeding vascular smooth muscle cell proliferation, reducing LDL accumulation in the arterial wall, inhibiting platlet aggregation, inhibiting stress-induced endothelial injury, and inhibiting the formation of foam cells [124,126,130,135]. Evidende for these effects is based on in vivo and in vitro animal studies [127,136-142] and studies of the effects of estrogen administration in postmenopausal women [131, 143-145]. Hormone replacement has been shown to enhance vascular tone and to inhibit progression of atherosclerosis [ 146-152]. Human studies of the effects of estrogen on vascular tone and atherogenesis are limited due to the lack of noninvasive techniques. Evidence for menopausal effects in women not treated with hormones is limited to data from studies of agerelated changes in vascular reactivity. In men, flow-mediated vasodilation of the brachial artery declines after the age of 40, whereas in women the decline begins only after age 50 and is steep at the time of menopause [153,154]. Flow-mediated vasodilation has also been shown to vary throughout the menstrual cycle [ 155]. Taddei et al. [ 156] studied the relationship of age to endothelial function in premenopausal women less than 45 years old, postmenopausal women over the age of 45, and men. Among men, there was a constant age-related decline in the vasodilatory response to acetycholine, whereas in women there was only a gradual decline up to age 49, after which vascular response to acetylcholine decreased more quickly than in men. The sex difference in agerelated endothelial dysfunction was interpreted as evidence that menopause influences endothelium-dependent vasodilation. Finally, Gangar et al. [ 144] demonstrated that pulsatility index in the internal carotid arteries of postmenopausal

239

CHAPTER 15 Cardiovascular Pathophysiology w o m e n , an indicator of blood flow impedance, was correlated with length of time since menopause. In summary, estrogens m a y help maintain vascular tone and protect the arterial wall from atherosclerotic processes. These m e c h a n i s m s may potentially explain the major portion of m e n o p a u s a l effects on cardiovascular risk. However, the existing evidence is based on the effects of h o r m o n e rep l a c e m e n t or e x p e r i m e n t a l administration of estrogens. Additional studies are needed to determine the impact of m e n o pausal changes in e n d o g e n o u s h o r m o n e levels on arterial wall function. Elucidation of these processes m a y be key to understanding the influence of m e n o p a u s e on the developm e n t of atherosclerotic disease.

V. C O N C L U S I O N S Cardiovascular disease rates are higher in p o s t m e n o p a u sal than in p r e m e n o p a u s a l w o m e n , and the m a l e - t o - f e m a l e ratio of disease rates diminishes after the age of m e n o p a u s e . W h e t h e r these trends are due to declining ovarian function with m e n o p a u s e remains controversial. Vital statistics data describing sex-specific trends with aging do not support the c o m m o n l y held belief that cardiovascular disease rates in w o m e n accelerate as a result of menopause. E p i d e m i o l o g i c a l studies e x a m i n i n g cardiovascular risk in relation to natural m e n o p a u s e have also failed to demonstrate increased risk with m e n o p a u s e , with results inconsistent across studies. Studies of surgical m e n o p a u s e have been m o r e consistent, suggesting a relationship b e t w e e n bilateral o o p h o r e c t o m y at an early age, and increased cardiovascular risk. Given the lengthy natural history of atherosclerosis, an abrupt increase in risk of clinical cardiovascular disease m i g h t not be expected at the age of m e n o p a u s e . The influence of m e n o p a u s e on risk m i g h t be m o r e gradual, m e d i a t e d t h r o u g h changes in cardiovascular risk factors. E p i d e m i o l o g i c a l studies have not shown a consistent effect of m e n o p a u s e on blood pressure. However, m e n o p a u s e has been associated with adverse changes in lipids and lipoproteins. W h e t h e r m e n o p a u s e per se is associated with adverse changes in hemostatic factors remains unclear. B e c a u s e estrogens appear to have favorable effects directly on arterial wall function, declining estrogen levels with m e n o p a u s e m a y facilitate the developm e n t of atherosclerosis and impaired blood flow. N u m e r o u s m e t h o d o l o g i c a l issues have limited prior studies of m e n o pause and cardiovascular disease. A major limitation of the current body of k n o w l e d g e is the lack of data regarding the relationship of e n d o g e n o u s h o r m o n e s to cardiovascular disease and related risk factors. Also lacking are data regarding racial differences in changes with m e n o p a u s e , and information regarding changes that occur during the p e r i m e n o p a u s a l period. To address these issues and to clarify the influence of m e n o p a u s e on cardiovascular risk, future studies m u s t in-

clude large, multiracial populations, with l o n g - t e r m followup and the ability to correlate biological and clinical markers of cardiovascular disease with e n d o g e n o u s h o r m o n e concentrations.

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ease: Serum lipids and blood pressure effects. Am. J. Obstet. Gynecol. 161, 1847-1853. Tikkanen, M. J. (1996). The menopause and hormone replacement therapy: Lipids, lipoproteins, coagulation and fibrinolytic factors. Maturitas 23, 209-216. Dahlen, G. H., Guyton, J. R., Attar, M., Farmer, J. A., Kautz, A., and Gotto, A. M. (1986). Associations of levels of lipoprotein Lp(a), plasma lipids and other lipoproteins with coronary artery disease documented by angiography. Circulation 74, 758-765. Hoefler, G., Harnoncourt, F., Paschke, E., Mirtl, W., Pfeiffer, K. H., and Kostner, G. M. (1986). Lipoprotein Lp(a)-a risk factor for myocardial infarction. JAMA, J. Am. Med. Assoc. 256, 2540-2545. Jenner, J. L., Ordovas, J. M., Lamon-Fava, S., Schaefer, M. M., Wilson E W. F., Castelli, W. E, and Schaefer, E. J. (1993). Effects of age, sex, and menopausal status on plasma lipoprotein(a) levels: The Framingham Offspring Study. Circulation 87, 1135-1141. Heinrich, J., Sandkamp, M., Kokott, R., Schulte, H., and Assmann, G. (1991). Relationship of lipoprotein(a) to variables of coagulation and fibrinolysis in a healthy population. Clin. Chem. (Winston-Salem, N.C.) 37, 1950 - 1954. Cauley, J. A., Gutai, J. E, Kuller, L. H., and Powell, J. G. (1990). The relation of endogenous sex steroid hormone concentrations to serum lipid and lipoprotein levels in postmenopausal women. Am. J. Epidemiol. 132, 884-894. Adamopoulos, D. A., Loraine, J. A., and Dove, G. A. (1971). Endocrinological studies in women approaching the menopause. J. Obstet. Gynaecol. Br. Commonw. 78, 62-79. Sherman, B. M., West, J. H., and Korenman, S. G. (1976). The menopausal transition: Analysis of LH, FSH, estradiol and progesterone concentration during menstrual cycles of older women. J. Clin. Endocrinol. Metab. 42, 629-636. Meade, T. W., Mellows, S., Brozovic, M., Miller, G. J., Chakrabati, R. R., North, W. R., Haines, A. E, Stifling, Y., Imeson, J. D., and Thompson, S. G. (1986). Haemostatic function and ischemic heart disease: Principal results of the Northwick Park Heart Study. Lancet 2, 533-537. Kannel, W. B., Wolf, P. A., Castelli, W. E, and D'Agostino, R. B. (1987). Fibrinogen and risk of cardiovascular disease: The Framingham Study. JAMA, J. Am. Med. Assoc. 258, 1183-1186. Wilhelmsen, L., Svardsudd, K., Korsan-Bengsten, K., Larsson, B., Welin, L., and Tibblin, G. (1984). Fibrinogen as a risk factor for stroke and myocardial infarction. N. Engl. J. Med. 201, 501-505. Dalaker, K., Hjermann, I., and Prydz, H. (1985). A novel form of Factor VII in plasma from men at risk for cardiovascular disease. Br. J. Haematol. 61, 205-222. Hoffman, C. J., Miller, R. H., Lawson, W. E., and Hultin, M. B. (1988). Elevation of factor VII activity and mass in ischemic heart disease and in young subjects at high risk of ischemic heart diseases. Circulation 78(Suppl. II), 206. Folsom, A. R., Wu, K. K., Davis, C. E., Conlan, M. G., Sorlie, P. D., and Szklo, M. (1991). Population correlates of plasma fibrinogen and factor VII, putative cardiovascular risk factors. Atherosclerosis 91, 191-205. Balleisen, L., Bailey, J., Epping, E H., Schulte, H., and van de Loo, J. (1985). Epidemiological study on factor VII, Factor VIII and fibrinogen in an industrial population: I. Baseline data on the relation to age, gender, body-weight, smoking, alcohol, pill-using, and menopause. Thromb. Haemostasis 54, 4 7 5 - 479. Meilahn, E. N., Kuller, L. H., Matthews, K. A., and Kiss, J. E. (1992). Hemostatic factors according to menopausal status and use of hormone replacement therapy. Ann. Epidemiol. 2, 445-455. Manolio, T. A., Furberg, C. D., Shemanski, L., Psaty, B. M., O'Leary, D. H., Tracy, R. E, and Bush, T. L. (1993). Associations of postmenopausal estrogen use with cardiovascular disease and its risk factors in older women. Circulation 88 (Part 1), 2163 -2171.

115. Nabulsi, A. A., Folsom, A. R., White, A., Patsch, W., Heiss, G., Wu, K. K., and Szklo, M. (1993). Association of hormone replacement therapy with various cardiovascular risk factors in postmenopausal women. N. Engl. J. Med. 328, 1069-1075. 116. Meade, T. W., Dyer, S., Howarth, D. J., Imeson, J. D., and Stifling, Y. (1990). Antithrombin III and procoagulant activity: Sex differences and effects of the menopause. Br. J. Haematol. 74, 77-81. 117. Meade, T. W., Haines, A. E, Imeson, J. D., Stirling, Y., and Thompson, S. G. (1983). Menopausal status and haemostatic variables. Lancet 1, 22-24. 118. Lindoff, C., Petersson, F., Lecander, I., Martinsson, G., and Astedt, B. (1993). Passage of the menopause is followed by haemostatic changes. Maturitas 17, 17-22. 119. Iso, H., Folsom, A. R., Sato, S., Wu, K. K., Shimamoto, T., Koike, K., Iida, M., and Komachi, Y. (1993). Plasma fibrinogen and its correlates in Japanese and US population samples. Arteriosclerosis Thromb. 13, 783 -790. 120. Lee, A. J., Lowe, G. D. O., Smith, W. C. S., and Tunstall-Pedoe, H. (1993). Plasma fibrinogen in women: Relationships with oral contraception, the menopause and hormone replacement therapy. Br. J. Haematol. 83, 616-621. 121. Lee, A. J., Smith, W. C. S., Lowe, G. D. O., and Tunstall-Pedoe, H. (1990). Plasma fibrinogen and coronary risk factors: The Scottish Heart Health Study. J. Clin. Epidemiol. 43, 913-919. 122. Pinto, S., Rostagno, C., Coppo, M., Paniccia, R., Prisco, D., Bruni, V., Rosati, D., and Abbate, R. (1990). No signs of increased thrombin generation in menopause. Thromb. Res. 58, 645-651. 123. Stefanick, M. L., Legault, C., Tracy R. P., Howard, G., Kessler, C. M., Lucas, D. L., and Bush, T. L. (1995). Distribution and correlates of plasma fibrinogen in middle-aged women: Initial findings of the postmenopausal estrogen/progestin interventions (PEPI) study. Arterioscler. Thromb. Vasc. Biol. 15, 2085-2093. 124. Samaan, S. A., and Crawford, M. H. (1995). Estrogen and cardiovascular function after menopause. J. Am. Coll. Cardiol. 26, 1403-1410. 125. Skartlein, A. H., Lyberg-Beckman, S., Holme, I., Hjermann, I., and Prydz, H. (1989). Effect of alteration in triglyceride levels on factor VII-phospholipid complexes in plasma. Arteriosclerosis 9, 798-801. 126. Gupta, S., and Rymer, J. (1996). Hormone replacement therapy and cardiovascular disease. Int. J. Gynecol. Obstet. 52, 119-125. 127. Losordo, D. W., Kearney, M., Kim, E. A., Jekanowski, J., and Isner, J. M. (1994). Variable expression of the estrogen receptor in normal and atherosclerotic coronary arteries of premenopausal women. Circulation 89, 1501 - 1510. 128. Karas, R. N., Patterson, B. L., and Mendelsohn, M. E. (1994). Human vascular smooth muscle cells contain functional estrogen receptors. Circulation 89, 1943-1950. 129. Pines, A., Mijatovic, V., van der Mooren, M., and Kenemans, E (1997). Hormone replacement therapy and cardioprotection: Basic concepts and clinical considerations. Eur. J. Obstet., Gynecol. Reprod. Biol. 71, 193-197. 130. Rosano, G. M. C., Chierchia, S. L., Leonardo, F., Beale, C. M., and Collins, P. (1996). Cardioprotective effects of ovarian hormones. Eur. Heart J. 17(Suppl D), 15-19. 131. Baron, Y. M., Galea, R., and Brincat, M. (1998). Carotid artery wall changes in estrogen-treated and -untreated postmenopausal women. Obstet. Gynecol. 91,982-986. 132. Stevensen, J. (1995). The metabolic and cardiovascular consequences of HRT. Br. J. Clin. Pract. 49, 87-90. 133. Glasser, S. E, Selwyn, A. E, and Ganz, E (1996). Atherosclerosis: Risk factors and the vascular endothelium. Am. Heart J. 131, 379-384. 134. Collins, E, Rosano, G., Jiang, C., Lindsay, D., Sarrel, E M., and Poole-Wilson, E A. (1993). Cardiovascular protection by oestrogen-A calcium antagonist effect? Lancet 341, 1264-1265. 135. Wild, R. A. (1996). Estrogen: Effects on the cardiovascular tree. Obstet. Gynecol. 87, 27s-35s.

CHAPTER 15 Cardiovascular Pathophysiology 136. Williams, J. K., Adams, M. R., and Klopfenstein, H. S. (1990). Estrogen modulates responses of atherosclerotic coronary arteries. Circulation 81, 1680-1687. 137. Williams, J. K., Adams, M. R., Herrington, D. M., and Clarkson, T. B. (1992). Short-term administration of estrogen and vascular responses of atherosclerotic coronary arteries. J. Am. Coll. Cardiol. 20,452-457. 138. Williams, J. K., Shively, C. A., and Clarkson, T. B. (1994). Determinants of coronary artery reactivity in premenopausal female cynomolgus monkeys with diet-induced atherosclerosis. Circulation 90, 983-987. 139. Clarkson, T. B., Anthony, M. S., and Klein, K. P. (1994). Effects of estrogen treatment on arterial wall structure and function. Drugs 47(Suppl. 2), 42-51. 140. Clarkson, T. B., Hughes, C. L., and Klein, K. E (1995). The nonhuman primate model of the relationship between gonadal steroids and coronary heart disease. Prog. Cardiovasc. Dis. 38, 189-198. 141. Gisclard, V., Miller, V. M., and Vanhoutte, P. M. (1988). Effect of 17/3estradiol on endothelium-dependent responses in the rabbit. J. Pharmacol. Exp. Ther. 244, 19-22. 142. Hayashi, T., Fukuto, J. N., Ignarro, L. J., and Chaudhuri, G. (1992). Basal release of nitric oxide from aortic rings is greater in female rabbits than in male rabbits: Implications for atherosclerosis. Proc. Nat. Acad. Sci. U.S.A. 89, 11259-11263. 143. A1-Khalili, F., Eriksson, M., Landgren, B., and Schenck-Gustafsson, K. (1998). Effect of conjugated estrogen on peripheral flow-mediated vasodilation in postmenopausal women. Am. J. Cardiol. 82, 215"218. 144. Gangar, K. E, Vyas, S., Whitehead, M., Crook, D., Meire, H., and Campbell, S. (1991). Pulsatility index in internal carotid artery in relation to transdermal oestradiol and time since menopause. Lancet 338, 839-842. 145. Herrington, D. M., Braden, G. A., Williams, J. K., and Morgan, T. M. (1994). Endothelial-dependent coronary vasomotor responsiveness in postmenopausal women with and without estrogen replacement therapy. Am. J. Cardiol. 73, 951-952. 146. Gruchow, H. W., Anderson, A. J., Barbriak, J. J., and Sobocinski, K. A. (1988). Postmenopausal use of estrogen and occlusion of coronary arteries. Am. Heart J. 115, 954-963.

243 147. Sullivan, J. M., Vander Zwaag, R., Lemp, G. F., Hughes, J. R, Maddock, V., Koetz, F. W., Ramanathan, K. B., and Mirvis, D. M. (1988). Postmenopausal estrogen use and coronary atherosclerosis. Ann. Intern. Med. 108, 358-363. 148. Volterrani, M., Rosano, G. M. C., Coats, A., Beale, C., and Collins, E (1995). Estrogen acutely increases peripheral blood flow in postmenopausal women. Am. J. Med. 99, 119-122. 149. Reis, S. E., Gloth, S. T., Blumenthal, R. S., Resar, J. R., Zacur, H. A., Gerstinblith, G., and Brinker, J. A. (1994). Ethinyl oestradiol acutely attenuates abnormal coronary vasomotor responses to acetylcholine in postmenopausal women. Circulation 89, 52-60. 150. Collins, E, Rosano, G. M. C., Sarrel, E M., Ulrich, L., Adamopoulos, S., Beale, C. M., McNeill, J. G., and Poole-Wilson, E A. (1995). Oestradiol-17-fl attenuates acetylcholine-induced coronary arterial constriction in women but not men with coronary heart disease. Circulation 92, 24-30. 151. Gilligan, D. M., Badar, D. M., Panza, J. A., Quyyumi, A. A., and Cannon, R. O., III (1994). Acute vascular effects of estrogen in postmenopausal women. Circulation 90, 786-791. 152. Gilligan, D. M., Quyyumi, A. A., and Cannon, R. O., III (1994). Effects of physiological levels of oestrogen on coronary vasomotor function in postmenopausal women. Circulation 89, 2545-2551. 153. Celermajer, D. S., Sorensen, K. E., Bull, C., Robinson, J., and Deanfield, J. E. (1994). Endothelium-dependent dilation in the systemic arteries of asymptomatic subjects relates to coronary risk factors and their interaction. J. Am. Coll. Cardiol. 24, 1468-1474. 154. Celermajer, D. S., Sorensen, K. E., Spiegelhalter, D. J., Georgakopoulos, D., Robinson, J., and Deanfield, J. E. (1994). Aging is associated with endothelial dysfunction in healthy men years before age-related decline in women. J. Am. Coll. Cardiol. 24, 471-476. 155. Hashimoto, M., Akishita, M., Eto, M., Ishikawa, M., Kozaki, K., Toba, K., Taketani, Y., Orimo, H., and Ouchi, Y. (1995). Modulation of endothelium-dependent flow-mediated dilation of the brachial artery by sex and menstrual cycle. Circulation 92, 3431-3435. 156. Taddei, S., Virdis, A., Ghiadoni, L., Mattei, E, Sudano, I., Bernini, G., Pinto, S., and Salvetti, A. (1996). Menopause is associated with endothelial dysfunction in women. Hypertension 28, 576-582.

7HAPTER 1(

Insulin Bo dy 9 Resistance, " Weight Obesity, Body Co os t on, and the Menopausal Transition MARYFRAN SOWERS AND JENNIFER TISCH Department of Epidemiology, School of Public Health, University of Michigan, Ann Arbor, Michigan 48109

V. Linking Body Composition and Carbohydrate Metabolism with Hormone Status VI. Summary Appendix References

I. I n t r o d u c t i o n

II. Mechanisms for Sex Hormones, Body Composition/ Topology, and Insulin Regulation III. Ovarian Hormone Status and Body Composition/Body Topology IV. Carbohydrate Metabolism and Change in Ovarian Hormone Status

I. I N T R O D U C T I O N

women. Differences in body composition have been linked to mortality, heart disease, gall bladder disease, certain cancers, osteoporosis, and arthritis in pre- and postmenopausal women [ 1-4]. Additionally, differences in body composition are also associated with a number of undesirable metabolic characteristics, including glucose intolerance, hyperinsulinemia, elevated triglycerides, low high-density lipoprotein (HDL) cholesterol concentrations, and potentially plasma leptin concentrations [5,6]. This suggests that if menopauserelated events influence body composition, the metabolic impact is particularly important for those diseases linked to disordered carbohydrate metabolism, including diabetes and heart disease in women.

Possible changes in body composition associated with events linked to a menopausal transition have not been well characterized, although a limited number of studies, with selected populations, have provided some preliminary information. Furthermore, although there is some evidence of differences in body composition according to race or ethnicity, the preponderance of existing data are limited to studies of Caucasian women or use methodological approaches that preclude comparability between race/ethnic groups. It is increasingly well appreciated that body composition, including topology, is related to disease conditions in

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Copyright 9 2000 by Academic Press. All rights of reproduction in any form reserved.

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This chapter is organized into four topics. First, we review potential mechanisms by which the events of the menopausal transition, including change in sex steroid hormone concentrations, are related to body composition changes or to change in measures of carbohydrate metabolism. Second, studies of body size are related to three states closely associated with the menopausal transition. These states include not only the menopausal transition but also surgical menopause (with a particular focus on oophorectomy) and the use of hormone replacement therapy. Third, we relate studies of the menopausal transition, surgical menopause, and hormone replacement therapy to measures of carbohydrate metabolism. Finally, we relate potential body composition changes in the menopausal transition to changes in carbohydrate metabolism and insulin resistance. The Appendix encompasses a description of methods used to evaluate body sizemincluding weight, obesity indices, body composition assessment, and body distribution (topology)--for those readers unfamiliar with body composition methodology.

FOR SEX BODY COMPOSITION/ AND INSULIN REGULATION

Weight gain and body topology

Menopause

Impaired Carbohydrate Metabolism

FIGURE 1 Model 1: Hormone changes with menopause lead to weight gain and change in body topology and, in turn, this leads to impaired carbohydrate metabolism.

Rarely have investigators explored the multiple pathways through which sex steroids (or their change with the menopausal transition), body composition, and measures of carbohydrate metabolism interact to influence the health of women. This review addresses mechanisms and studies that relate sex steroid concentrations to body composition and to measures of carbohydrate metabolism. We summarize several suggested mechanisms for the interaction between sex steroid hormones and body composition. It should be noted that these proposed mechanisms tend to evoke increasing insulin resistance as an integral part of the process or designate insulin resistance as a result of the process (see Figs. 1 and 2)

II. M E C H A N I S M S HORMONES, TOPOLOGY,

It is hypothesized that sex steroid hormones influence tissue mass, both lean and fat mass, albeit this has limited support in the literature. Because the menopausal transition is associated with marked shifts in gonadotropin and ovarian hormone concentrations, it is further hypothesized that the menopausal transition has a major impact on total weight and body composition, promoting an increase in fat mass and a redistribution of that fat mass to the abdomen. It is further believed that this influence is independent of a weight accumulation with aging. Several mechanisms have been proposed as to how the change in sex steroid hormones associated with the menopausal transition influences body composition and its topology. It is difficult to disaggregate the mechanisms associated with the accumulation of fat tissues (obesity) as compared to the role of fat patterning, suggesting either an immature understanding of these mechanisms or that body composition and its patterning are intrinsically linked and should not be conceptually divorced from each other. It is believed that the change in sex steroid hormones associated with the menopausal transition also affects the activities of insulin. It is hypothesized that there is probably a direct and indirect role. The direct role is probably mediated through hepatic glycogen deposition or sensitivity of the muscle bed to insulin action. An indirect mechanism may be related to an increasing insulin resistance associated with a change in body composition, particularly an increase in fat mass and decrease in muscle mass.

A. E n e r g y B a l a n c e It has been suggested that any increase in weight with the menopausal transition (or with a change in sex steroid concentrations) is a direct function of direct energy intake coupled with resting energy expenditure (REE). Resting energy expenditure decreases with increasing age, a decline that is proposed to begin with the menopausal transition. Furthermore, if there were a decline in physical activity, the combined effect would be to further increase the likelihood of weight gain [7]. The hypothesis indicates the difficulty in separating an age effect from an menopausal transition effect. Recent studies have reported that with age, an increase in adiposity and a decline in physical activity are associated with a decline in the levels of insulin-like growth factor I (IGF-I). IGF-I has anabolic growth-promoting effects of

HormoneChange]

~,, odyTopology

Impaired Carbohydrate Metabolism

Weight Increase

FIGURE 2 Model 2: Hormone change with menopause (estradiol, progesterone, and testosterone) and increasing weightgain leadto modification of body topologyand impaired carbohydratemetabolism.

CHAPTER 16 Body Composition, Insulin, and the Menopause growth hormone and insulin-like activities. Poehlman et al. [8] have shown that decline in IGF-I was related to a decline in physical activity and they have proposed that this decline in IGF-I might be related to the change in body composition with menopause. Studies have inadequately addressed the role of sex steroids in the regulation of IGF-I particularly in relation to body composition.

B. L e p t i n Estrogen may also influence body composition though its interaction with leptin. Leptin may be the hormone produced by body stores to signal adequate energy stores (adipose tissue) for reproduction. In some [9,10], but not all [11] studies leptin concentrations are higher in premenopausal women than postmenopausal women. Furthermore, estrogen regulates leptin production in rats and human subjects in vivo [9].

C. L i p o p r o t e i n L i p a s e and F a t t y A c i d D e p o s i t i o n Another mechanism includes a direct mechanism of estrogen concentrations linked to the patterning of android and gynoid fat. Estrogen is postulated to regulate directly lipoprotein lipase (LPL) activity in the gluteofemoral adipocytes. LPL is a key enzyme in the regulation of fatty acid deposition. It is proposed that during the premenopausal period, lipid deposition in the gluteofemoral adipocytes is facilitated by LPL via estrogen pathways in order to assure adequate energy stores for reproduction. For example, during lactation there is mobilization of energy from the gluteofemoral adipocytes to the breast for milk, a physiological mechanism mediated by suppression of estrogen and controlled by prolactin. Furthermore, there is some evidence of enlargement of femoral subcutaneous adipocytes to support the demands of pregnancy and lactation and this enlargement is reported to disappear with menopause [ 12]. Concurrently, it has been suggested that progesterone competes with glucocorticoid receptors [13,14] and may protect adipocytes from glucocorticoid effects during the late luteal phase of the menstrual cycle, a protection that would be increasingly compromised with the menopausal transition. It is extrapolated that with the events of the menopausal transition, LPL levels are minimally or no longer under the influence of estrogen (and potentially progesterone) and the gluteofemoral adipocytes no longer function as the major source of energy storage. These mechanisms have been suggested as being reflected in the relative location of regional fat deposition. In premenopausal women, the gluteofemoral pool is associated with higher LPL activity and low lipolytic activity. If the abdominal pool is the source of high lipolytic activity, there is rapid turnover of nonesterified fatty acids. These fatty acids

247 drain into the portal system and may compete with glucose as fuel, leading to insulin resistance. In postmenopausal women, the femoral pool is associated with lower lipoprotein lipase activity while the abdomen is associated with increasing visceral fat disposition and with higher lipolytic activity. In women using hormone replacement therapy (HRT), the femoral pool would again reflect stimulated lipoprotein lipase activity while the abdominal pool would have lower lipoprotein lipase activity. Thus, in premenopausal women, there is greater activity in the gluteofemoral region. With the menopausal transition, the abdomen becomes the "default" region, in the absence of estrogen replacement [ 15].

D. Stress and the C R F - A C T H - A d r e n a l

Axis

An extension of these mechanisms is based in a belief that increased glucocorticoid stimulation [ 16] is manifest in the increased size of the abdominal visceral adipose pool. In the states of depression, dysphoria, maladaption, and chronic stress, as well as in smoking behavior and alcohol use, chronic hypothalamic stimulation is reflected as increased activity in the CRF-ACTH-adrenal axis. This stress-based condition is proposed to occur with the menopausal transition [ 1].

E. W e i g h t and A n d r o g e n i s m A modification of these mechanisms suggests that with an increase in weight, there is an increase in insulin resistance (reflected as increased insulin concentrations). This is further associated with an inhibition of sex hormone binding globulin which, in turn, is associated with increased free testosterone concentrations and greater androgenicity, reflected by more centroid obesity [ 1,17-19]. Investigators have suggested a number of mechanisms whereby estrogen status is related to insulin action [17-19]. These mechanisms typically relate to glycogen status in the liver or muscle uptake of glucose for energy. The mechanisms are as follows: 1. Estrogens may have a direct effect on glycogen deposition in the liver. The impact of a menopausally related decline in estradiol concentrations and a relative increase in estrone has not been investigated relative to glycogen deposition. 2. Alternatively, estrogens may enhance the permissive effect of corticosteroids on hepatic glycogen deposition. 3. Estrogen decreases glucagon secretion; however, again the secretion of glucagon relative to shift in the estrogen levels with the menopausal transition has not been investigated. 4. Estrogen may enhance sensitivity of glucose uptake in muscle. The change in sex steroid concentrations may have a dual impact with respect to muscle mass. If the loss of estrogen minimizes glucose uptake in muscle, and if there is a loss

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of muscle mass (in the presence of an increase in fat mass), the menopausal transition could pose a substantial impact both directly and indirectly on carbohydrate metabolism.

diminution of ovarian estrogen and progesterone achieved over a period of years), and hormone replacement therapy (with a rapid reintroduction of estrogen and progestins).

A. W e i g h t as a M e a s u r e o f B o d y C o m p o s i t i o n E

Summary 1. WEIGHT GAIN AND MENOPAUSE

The mechanisms by which changes in sex steroid hormone concentrations may affect body composition, body topology, or insulin activity are incompletely understood. A major hypothesis suggests that changes in body composition and insulin activity with the menopausal transition may be a function of change in energy expenditure and the decline in concentrations of growth factors. These changes would include an increase in fat mass relative to lean and a greater likelihood of the deposition of that fat mass in the abdomen as visceral fat. A second hypothesis links estrogen, and potentially progesterone, with lipoprotein lipase activity, particularly in the gluteofemoral region to optimize energy stores for reproduction, a mechanism that is minimized with the diminution of estradiol and progesterone production during and after the menopausal transition. A third mechanism, with a focus on the stress-related responses of the adrenal cortex, may suggest an approach to increasing the size of the abdominal visceral pool, but is less closely linked with the change in ovarian function. Finally, there is the mechanism that with loss of ovarian function with either natural or surgical menopause, the ratio of estrogen to androgen (testosterone) shifts and that a more androgenic state is more likely to be associated with the deposition of fat viscerally. Changes in sex steroid hormone concentrations may directly influence glycogen deposition and/or secretion, or through glucose uptake in muscle. It is quite possible that each of these mechanisms contribute interactively during and after the menopausal transition in a dynamic process.

III. OVARIAN HORMONE BODY COMPOSITION/BODY

STATUS AND TOPOLOGY

Because most studies have not simultaneously considered the interaction of the menopausal transition (or change in sex steroid concentrations), body composition, and measures of carbohydrate metabolism, we are forced to review these components independently. In this section, we review findings from studies that have reported measures of weight and body mass index (BMI). We will also review the more direct measures of body composition, including dual energy X-ray absorptiometry (DXA), underwater weighing and bioelectrical impedance (BIA), and body topology (waist-to-hip ratio) in three conditions in which there are changes in ovarian hormone status. These three states include oophorectomy (with a rapid and typically complete loss of ovarian estrogen and progesterone), natural menopause (with a more erratic

Weight gain, particularly after the menopausal transition, is thought to be a common occurrence [20]; however, remarkably few studies have simultaneously considered both age and menopausal status in relation to weight gain. For example, Kirchengast [21] reported an increase in weight across three age groups of Viennese women recruited from groups defined in clinical settings. The "older" premenopausal group (aged 32-41 years) weighed 64.3 kg; the perimenopausal group (aged 3 8 - 4 9 years) weighed 67.6 kg and the postmenopausal group (47-64 years) weighed 72.4 kg. The investigators excluded all potential enrollees with a BMI greater than 30 kg/m 2. They reported this weight increase as a function of menopausal status without adjusting for an age contribution. The longitudinal studies of weight suggest no association of change in weight with menopause after adjusting for the contribution of age. For example, in a 3-year follow-up of Pittsburgh women, Wing et al. [20] reported that similar weight gain was seen across time in both pre- and postmenopausal women. Similarly, in a 6-year follow-up of women in G6teborg, Sweden, Lindquist [22,23] concluded that the increase in weight appeared to be age, not menopause, related. These investigators excluded HRT users (7.5% of their population) and women with surgical menopause (9% of their population) from their analyses. The patterns of agerelated weight change may not be simple linear increases in the peri- and postmenopausal periods. In the 1996 report from G6teborg, investigators reported that women who remained premenopausal at two examinations gained weight; women who were pre- and then postmenopausal likewise gained weight, and yet women who were postmenopausal at both examinations lost weight in the interim time between evaluations. Thus, there are reports from a limited number of crosssectional studies of an association between the menopausal state and weight increase. However, rarely do these studies consider whether this association reflects an additional contribution of menopausal status to age or whether the weight gain is really just an age-related phenomenon that happens to coincide with a menopausal transition. The longitudinal studies suggest that when the contribution of age is considered, menopausal status per se is not a statistically important contributor to weight gain. Furthermore, although weight gain reflects an increase in total mass, it does not reflect changes in the constituents of the mass (lean vs. fat tissue) nor does it reflect any changes in the regional distribution or topology of that mass.

CHAPTER 16 Body Composition, Insulin, and the Menopause 2. WEIGHT GAIN WITH OOPHORECTOMY In considering oophorectomy as a model for evaluating the role of weight gain and the hormone change with menopause, it is important to distinguish the difference between oophorectomy and hysterectomy with and without ovarian conservation. Obviously, women who have hysterectomy with ovarian conservation do not reflect the same endogenous hormonal environment as those women without ovaries. These studies are not reporting data on weight alone, but instead use BMI, which takes into consideration weight per unit of height and show no association. 3. WEIGHT GAIN AND HRT USE

Studies of weight in women using hormone replacement therapy do not particularly clarify the nature of the relation. Short-term studies may show an increase in weight. For example, Gambacciani et al. [24] reported that a group of 12 control women not using HRT increased their body weight by 1.9 kg in 12 months whereas those using HRT, on average, had no weight increase. Yet, in a study that spanned 15 years, Kritz-Silverstein and Barrett-Connor [25] reported that there was little difference in the weight change between those women who continuously used estrogen replacement therapy (ERT) as compared to those without such use. Finally, the Postmenopausal Estrogen/Progestin Intervention (PEPI) Trial reported a very modest increase in weight in the placebo group during the 3 years of the trial as compared to the four treatment groups, but none of the differences was statistically different [26]. 4. SUMMARY

249 the average difference is approximately 1 BMI unit (killigrams per meter squared), translatable to a difference of approximately 10 pounds [27-29]. Longitudinal studies of BMI and the menopausal transition suggest a different relation than do the cross-sectional studies. In the longitudinal studies, the increase in BMI during the menopausal transition is not independent of age. This is observed across a number of populations including United States women in Pittsburgh [20] and Framingham [30], Japanese women in Nagasaki [31 ], and Swedish women in G6teborg [22,23]. These reports also span several age cohorts. The Framingham women were evaluated during the 1960s and 1970s whereas the Pittsburgh women were observed during the 1980s and 1990s, suggesting these observations are not limited to unique cohorts of women. The differences in findings of the cross-sectional studies as compared to the longitudinal studies suggest the need to determine whether the cross-sectional studies reflect clinic populations, who will have a different experience related to the age at which they will have their menopausal transition. Are premenopausal women in cross-sectional studies chronologically aged 35, 40, or 45 ? How long will it be before they experience the year of amenorrhea associated with menop a u s a l - 5 , 7, 11, or 13 years? Cross-sectional studies are potentially forced to misclassify women according to their menopausal status based on age and information from a single point of time. Typically, the cross-sectional studies frequently enroll many fewer women than do longitudinal studies. These characteristics may explain the discrepancies in findings between the cross-sectional and longitudinal studies. 2. B M I AND OOPHORECTOMY

The evidence is highly inconsistent as to the role of weight gain accompanying transitions associated with the menopausal period. Weight gain does occur, but there is substantial difficulty in linking this weight gain just to the events of a transition. Most of the studies, which are typically crosssectional in design, do not try to separate the independent effects of age and menopause status. Additionally, readers should exercise caution in generalizing the findings of the studies to external populations, because there are substantial opportunities for selection bias in using or observing clinical populations.

B. B o d y M a s s I n d e x as a M e a s u r e of Body Composition 1. BMI AND THE MENOPAUSAL TRANSITION Measures of weight alone do not consider the amount of body tissue per unit of height; thus, the use of BMI (weight/ height 2) is a better measure of body habitus. There have been a number of cross-sectional studies that have reported a greater BMI among postmenopausal women as compared to premenopausal women, after adjusting for age. Typically,

A Finnish study [32] indicates that women with hysterectomy and preservation of at least one ovary had a greater BMI than did those who had not undergone hysterectomy. 3. BMI ANn HORMONE REPLACEMENT In a number of prospective studies and clinical trials, there have been conflicting findings as to whether estrogen or hormone replacement therapy is associated with an increase in B MI. For example, the results from the Nurses Health Study (wherein BMI was self-reported) indicated that estrogen use was associated with lower BMI [33]. In a study among Pittsburgh women, Matthews et al. [34] reported greater weight gain among estrogen users. Notelovitz [35], Nachtigall [36], and Utian [37] have each reported no influence of estrogen replacement on weight following menopause. The Pittsburgh investigators have argued that estrogen users are a highly selective group and that there are differential factors, including baseline weight, that affect their weight change patterns as compared to women who do not use HRT [38]. These contradictory findings indicate the nature of problems associated with making definitive statements about weight or BMI and the menopause. Prospective studies of

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women transitioning the menopause are uncommon. Rarely do prospective studies report the role of initial or baseline weight in explaining the amount of weight or BMI change. Furthermore, information about factors that influence weight, such as smoking, diet, and exercise habits, are frequently unavailable or not considered in the statistical adjustment. Increasingly, there is an appreciation of the kinds of bias that can lead to an inappropriate interpretation of the true association between menopause, HRT use, and weight. Many studies may not consider the age of menopause and whether the menopause was natural or surgical. The latter could introduce significant bias if there is an important association of socioeconomic status and weight, and those with lower socioeconomic status are more likely to have a surgical menopause. Among those studies of hormone replacement therapy, the sample sizes are frequently small and limit the ability to generalize. Formulations of hormone replacement are mixed. The preparations are strikingly different and include conjugated equine estrogens, alkalyted estrogens, or estrogen/progestin combinations. Doses are wide ranging. The evaluation of hormone replacement therapy also rarely accounts for why women were taking the therapy and how this might influence weight change pattern. Thus, the change in BMI could readily be different for the 40-year-old woman with a hysterectomy/oophorectomy associated with dysfunctional uterine bleeding as compared to the 55-year-old woman with postmenopausal hot flashes. 4. SUMMARY Currently, there is evidence that weight gain (as an absolute measure of weight or BMI) around the menopausal transition is likely to be linked to age, and is not unique to the menopausal transition. This has been most clearly demonstrated in prospective studies whose populations are not specifically selected from clinical patients. This is in direct contradiction to the commonly held perceptions of weight gain during the menopausal transition. Although there is little to support the concept of a major increase in weight with the menopause, supplementary data are needed to address whether there is a shift in the proportion of fat relative to the amount of lean tissue. Current data also do not address the potential shift in the location of large amounts of fat tissue to visceral stores in the abdomen.

C. Direct Measures of Body Compartment (Fat and Lean) Only a few studies have involved more direct measures of the lean and fat compartments in relation to menopause. Kirchengast [21] reported an increase in percentage body fat as measured by DXA across three age groups of Viennese women recruited from clinical populations. The older premenopausal group (aged 32-41 years) was 32.4% fat, the

perimenopausal group (aged 3 8 - 4 9 years) was 36.8%, and the postmenopausal group (47-64 years) was 38.1% fat. Two other studies using DXA [39,40] reported an 8-9% greater fat mass in postmenopausal women than in premenopausal women. Other studies, using more direct measures of body composition, have found little body composition change in relation to menopausal status [41-44]. Thus, approximately half of the studies suggest an increase in percentage body fat whereas the other half do not. Limited evidence from the very few longitudinal studies suggests that the sizes of body compartments (fat and lean mass) change with both ovarian aging and chronological aging. Sowers has shown that, on average, the 0.9 kg total weight gain annually was associated with a 1.4 kg increase in the fat compartment and a 0.5 kg decrease in the lean compartment, after adjusting for age [2]. The population includes more than 400 pre- and perimenopausal women, aged 2 5 45 years, and those women with hysterectomy were more likely to have an increase in the size of the fat compartment. In a widely cited study incorporating data from just 38 women, aged 4 4 - 4 8 years, who were assessed with underwater weighing, Poehlman [8] described a "menopause" effect in size of the fat compartment in a 6-year follow-up. Essentially, the definitive studies have not been undertaken to characterize the change in the size of the fat and lean body compartments with menopausal transition, after accounting for age effects. 1. BODY COMPOSITION AND EXOGENOUS HORMONE USE With estrogen/progestin preparations, Haarbo et al. [45] reported no impact on fat mass in 62 early postmenopausal women, consistent with the work of Hassager and Christiansen [46], who reported that estrogen replacement prevented an increase in fat mass. 2. SUMMARY Although suggestive of an increase in fat mass, the limited number of studies considering the sizes of the fat and lean compartments are insufficient to determine if there are actual shifts in the proportion of fat and lean with the menopausal transition. Furthermore, it is uncertain that these changes might occur apart from that which would be observed with age alone. Likewise, there is insufficient information about the differential impact of exogenous hormone replacements that include progestins as compared to those based on conjugated equine estrogens. Although speculation, conjecture, and hypotheses abound, evidence is minimal.

D. Body Topology (Patterning) and the Menopause There is extensive evidence of the influence of ovarian aging with respect to body composition topology [44]. Usually,

CHAPTER 16 Body Composition, Insulin, and the Menopause this association is described with waist-to-hip ratio (WHR). The abundance of evidence of the influence of ovarian aging on body topology may be a function of the relative ease of measuring WHR in larger populations. It is more costly and more difficult to use computerized tomography to measure body topology. Alternatively, total body DXA scans indicate differences in the estimate of android (upper body segment, trunk) and gynoid (hip and thigh region) distributions; however, this measurement does not differentiate between subcutaneous fat and visceral adipose tissue and whether the visceral fat tissue is saturated adipose tissue. These latter depots are thought to be most deleterious to health. Current data from the dozen or so cross-sectional studies of body fat distributions are contradictory in their findings. Approximately half of the studies suggest a shift to a more negative topological profile with menopause (following adjustment for age and/or BMI). The remaining studies suggest retaining a positive profile, reflecting a gynoid, not android, patterning [8,21,27-29,40,42-44,47-53]. In two longitudinal studies, there was apparently a menopause-based change in body topology. Poehlman [8] reported a menopause-related change in 38 women followed for 6 years. Bjorkelund [54] reported 1237 women from G6teborg, aged 38-60, to have an increase in waist and a decrease in the hip circumference. They suggested the change in body composition was a gradual process that started well before the cessation of menstrual bleeding and the process continued at a slower rate in the postmenopause. In a longitudinal study comparing pre- and perimenopausal women, Sowers reported that perimenopausal women had a greater waist girth relative to hip girth than the younger premenopausal women, after adjusting for age and other important variables such as smoking, physical activity, and parity [2]. 1. BODY TOPOLOGY AND EXOGENOUS HORMONE USE

Following menopause, it appears that there is an increase in visceral fat mass that is preventable, at least in part, by hormonal replacement therapy [24,45,55]. For example, Gambacciani et al. [24] reported that a group of 12 women using HRT, on average, had no weight increase whereas those not using HRT increased their body weight by 1.9 kg in 12 months. Simultaneously, in women not using HRT, the fat was patterned as an increase in total body fat mass to the trunk and arms. In the HRT-treated group (n = 15), there were no pattern modifications to the trunk. Studies by Haarbo [45], Kaye [56], den Tonkelaar [29], and Wing [20] each have reported that postmenopausal hormone use was associated with lower waist-to-hip ratio, albeit den Tonkelaar et al. [47] indicated that the association was no longer present following adjustment for age and obesity in their crosssectional study. In contrast, the PEPI Trial found no difference in the waist-to-hip ratio of those treated for 3 years with four different hormone preparations versus those in the placebo-control group [26].

251 2. SUMMARY

It appears that a redistribution of adipose tissue may occur with menopause based on longitudinal studies of the menopausal transition and the use of exogenous hormones. More studies are needed, however, that incorporate factors infrequently considered when evaluating waist-to-hip ratio, including ethnicity, the baseline weight, the type of preparation or formulation of replacement therapy used, smoking behavior, and socioeconomic status. Furthermore, more viable measures of both visceral adiposity and saturation of adipocytes are needed for implementation in both clinical and epidemiological studies. For example, both published and unpublished studies have shown that waist circumference explained more variation in HDL cholesterol and triglyceride concentrations than did WHR [4].

IV. C A R B O H Y D R A T E AND CHANGE HORMONE

METABOLISM

IN OVARIAN

STATUS

Carbohydrate metabolism and insulin resistance are believed to be important for two reasons. First, as carbohydrate metabolism becomes increasingly disordered and insulin resistance increases, greater numbers of women will have both diagnosed and undiagnosed diabetes. Second, insulin resistance is an important risk factor for coronary heart disease (CHD) in both men and women. Fasting plasma insulin concentrations correlate well with measures of insulin resistance using clamp studies, albeit not as glucose tolerance becomes abnormal [57]. Fasting insulin concentrations are correlated with other risk factors for coronary disease, including increases in blood pressure, circulating cholesterol and triglycerides, and decreases in HDL cholesterol in both Caucasians and African-Americans [58,59]. Some have postulated that changes in insulin and insulin sensitivity at the menopause may play a central role in women's increased risk for coronary heart disease at older ages, but findings have been conflicting. Fasting insulin concentrations are related to body fat distribution and weight gain [60,61 ].

A. A n i m a l S t u d i e s More than 30 years ago, Foglia [62] demonstrated that in castrated, pancreatectomized rats, estrogen replacement was associated with an initial deterioration in glycemic control followed by subsequent protection as pancreatic islet cells were regenerated. More recently, it was shown that castration increased the incidence of diabetes in female rats and this could be reversed with the administration of estrogen or testosterone [63]. In general, the work in animals suggests a beneficial effect for estrogen on insulin secretion in castrated and

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intact rats. Investigators have generally observed an increased weight gain in ovariectomized rats as well as an increased insulin responsiveness in treated animals as compared to untreated animals [ 19]. Animal studies have also suggested other ovarian hormones, apart from estrogen, need to be considered. For example, there is some evidence from animal studies of a direct effect of progesterone and progestins on islet cells. Progesterone enters the islet cells, binds to cytosolic receptors, and enters the nucleus [64,65]. Progesterone and progestins can reduce glucose uptake in skeletal muscle and the uptake of glucose into lipid [19], resulting in a progesterone-induced resistance to insulin action in peripheral tissues.

B. H u m a n S t u d i e s There are relatively few studies of the effects of human menopause, either natural or surgical, on carbohydrate metabolism. Several studies have included both pre- and postmenopausal groups, although it is frequently unclear as to the perimenopausal status of the premenopausal women. 1. NATURAL AND SURGICAL MENOPAUSE

The role for menopause, surgical or natural, in carbohydrate metabolism is ill defined. One cross-sectional study showed a fall in insulin concentrations when comparing 426 pre- versus postmenopausal Italian women [42,43]. In contrast, another cross-sectional study of Turkish women [66] identified no difference in the insulin:glucose ratio among women with premature ovarian failure and natural menopause. Other studies have described increased fasting insulin concentration with menopause and suggested that the loss of ovarian function may explain this increase [58,67,68]. Two longitudinal studies have examined measures of carbohydrate metabolism and menopausal status. Natural menopause was not associated with increased glucose concentrations [30] in the Framingham Study. Likewise, natural menopause was not associated with the response to oral glucose tolerance tests in the participants of the Healthy Women's Study [34]. In the Framingham Study, ovariectomy was not associated with increased glucose levels [30]. These inconsistencies in study design and the measures used to evaluate different aspects of carbohydrate metabolism suggest the importance of determining whether the decline of estrogen and progesterone hormones at the menopause initially reduces both insulin secretion and elimination. It is also important to determine whether increasing insulin resistance at the cellular level induces an increase in circulating insulin concentrations, what causes the insulin resistance, and when the insulin resistance is likely to be sufficiently great as to generate a clinical diagnosis of disease. Godsland [ 19] suggested that postmenopausal women have a reduction in glucose-induced insulin secretion with a compensatory reduc-

tion in insulin elimination [69]. In this situation, detection of a menopausal difference will be difficult because a deficiency in insulin secretion but a compensatory reduction in insulin elimination could result in no net change in glucose tolerance or circulating insulin concentrations.

2. H o r m o n e R e p l a c e m e n t and Carbohydrate Metabolism The literature on hormone replacement and carbohydrate metabolism is much more extensive than the literature associated with natural or surgical menopause without replacement. Although there is an advantage in terms of frequency of a greater number of studies, the findings may require interpretation within the type and amount of the estrogen and/ or progestin used or within the degree to which carbohydrate metabolism was normal at the time treatment was initiated. For example, studies have shown that 2-hr insulin secretion in postmenopausal women did not differ between those who received hormone replacement therapies and those who did not receive therapy [26]. However, in 25 postmenopausal women with non-insulin-dependent diabetes mellitus (NIDDM), treatment with 17fl-estradiol for 3 months resulted in the diminution of hyperandrogenic characteristics and improved glucose homeostasis as well as plasma lipids [70]. In an excellent review, Godsland [19] concluded that estradiol replacement in postmenopausal women is associated with improved insulin sensitivity. When the replacement therapy was conjugated equine estrogens (Premarin) at the 0.625-mg/day dose, there was either an improvement or no change in insulin sensitivity, whereas administration of the 1.25-mg/day dose was associated with deterioration in insulin sensitivity. The deterioration in insulin sensitivity with the higher doses may reflect a secondary effect of increasing glucocorticoid activity. When the therapeutic regimen was alkylated estrogen, such as ethinyl estradiol and mestranol, there was deterioration in glucose tolerance and insulin resistance. Transdermal methods of delivery, in which exposure to the liver is averted, were not associated with changes in carbohydrate metabolism. Formulations incorporating progestins probably did not have the same effect as those based on estrogens [71]. A number of mechanisms have been proposed to explain the potential glucose tolerance and insulin resistance seen in formulations that are estrogen/progestin combinations and these mechanisms parallel those associated with mechanisms described in relation to body composition. These include increased circulating fatty acid levels, impact on growth hormone concentration, increased glucocorticoid activity in the obese via increased synthesis and decreased corticosteroid half-life, and reduced insulin receptor concentrations [ 19].

CHAPTER 16 Body Composition, Insulin, and the Menopause 3. SUMMARY

In animal models, estrogen deficiency is associated with impaired carbohydrate metabolism and increased risk of diabetes. This was reversible with the administration of an estrogen replacement. In humans, there is relatively little information about the process of the menopausal transition and measures of carbohydrate metabolism. There is substantially more information in women generated through studies of both estrogen replacement therapy and hormone replacement therapy, suggesting that the ovarian hormones estrogen and progesterone can have an impact on carbohydrate metabolism. However, the impact of a hormone replacement regimen depends on the dose of the estrogenic material, the formulation (i.e., alkylated vs. conjugated equine estrogens), the delivery system (tablet vs. transdermal), and the presence or absence of progestins. Estrogen replacement could result in improvement of markers of carbohydrate metabolism and insulin status. However, higher estrogen concentrations have resulted in deterioration of glucose tolerance, potentially through increased corticosteroid action and tryptophan metabolism. Understanding these mechanisms may be important linkage of body composition to subsequent risk of heart disease and/ or diabetes. There has been much focus on the role of estrogen status in the link between body composition and glucose tolerance, whereas the importance of progesterone and progestins is probably inadequately appreciated. In the menopause, ultimately, there is not only a diminution of estradiol production, there is also a loss of progesterone activity. Progesterone and some progestagens induce insulin resistance and this element must be considered in future studies that attempt to describe the impact of the menopausal transition on carbohydrate metabolism.

V. LINKING BODY COMPOSITION AND CARBOHYDRATE METABOLISM WITH H O R M O N E STATUS Cross-sectional studies have indicated that visceral adiposity is associated with alterations in glucose/insulin homeostasis. Furthermore, there is an association of age and menopausal status with the visceral adiposity, most frequently expressed as the ratio of size of the waist circumference to the hip circumference. Based on these relationships, it has been speculated that those experiencing the menopausal transition will be more likely to present with disruptions in glucose/insulin homeostasis, even to the point of having greater incidence of diabetes. In one of the few studies of carbohydrate metabolism and measured visceral adipose tissue (AT), Lemieux et al. [72,73] suggested that the group with the greatest gain in visceral AT had the greatest

253 deterioration in glucose/insulin homeostasis. Unfortunately, sample sizes were inadequate to evaluate the role of the menopausal transition of some study enrollees through the menopause. Other studies have linked body composition and carbohydrate metabolism with menopausal status. In the Healthy Women's Study, women transitioning the menopause had increases in fasting glucose and insulin concentrations, but these increases were simultaneously seen in premenopausal women, and appeared to be independent of measures of body composition. This is interpreted as indicating that changes in carbohydrate metabolism markers were reflecting aging [34]. In a study of pre- and postmenopausal women, postmenopausal women had a reduced pancreatic insulin secretion and increased insulin as compared to the premenopausal women [60,69]. The investigators suggest that additionally, menopausally associated changes are then accompanied by increasing insulin resistance with aging.

VI. SUMMARY Some investigators have postulated that changes in insulin and insulin sensitivity at the menopause may play a role in women's increased risk for coronary heart disease at older ages; however, the findings have been conflicting. Fasting insulin concentrations are correlated with other risk factors for coronary disease, including increases in blood pressure, total cholesterol and triglycerides, and decreases in HDL cholesterol [58,59]. In spite of the importance in understanding the role of body composition and the hormone changes of the menopausal transition in relation to carbohydrate metabolism, there is much to be learned of the temporal sequence of the relationship and the magnitude of the contribution. Most current studies are not longitudinal, thus making difficult the establishment of the temporal sequence of events. The use of weight and BMI rather than the more direct markers of body composition does not allow for the accounting of changes in the lean and fat compartments or their topological redistribution. Likewise, markers of body topology or patterning have primarily been based on circumference measures that become less precise as obesity increases. The markers of carbohydrate metabolism are limited in their capacity to not only describe trends, but also supply adequate information to inform potential understandings of mechanisms. In spite of the aforementioned limitations, emerging methodologies for the more direct measure of body composition and the understanding that waist circumferences are probably adequate to approximate visceral adipose tissue will facilitate the development of this area. Furthermore, a number of studies of the menopausal transition, including the Study of Women's Health Across the Nation (SWAN) in the United States, will allow the longitudinal and simultaneous measure

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of ovarian hormone levels, body composition, glucose, and insulin. It is anticipated that the product will be a more complete understanding of the subtle changes that occur across a relatively long time span (estimated to be 8-10 years). This understanding is important because several of the major chronic diseases arising in the last third of the life span are thought to be affected by body composition, obesity, and impaired glucose metabolism.

APPENDIX

Measuring Body Composition Body composition technically is the amount and type of tissue represented in a living organism. Within humans, this has been largely restricted to understanding the size of three compartments: (1) the amount of fat, (2) the amount of lean, and (3) the amount of bone mineral content, which is frequently included as a subcompartment of the lean tissue mass. The size of these compartments is evaluated typically for the sake of describing obesity (the amount of excess fat in relation to the amount of lean mass), starvation (the loss of both fat mass and lean mass), or the loss of bone mineral density as a risk for osteoporotic fracture. More recently, it has come to be understood that not only is the amount of fat and lean mass important, but among the more corpulent, the distribution of that fat mass is also important. Because measurement of body composition and its distribution is technically difficult in humans, particularly in large numbers of humans in epidemiology studies, a number of measures have been developed that act (with more or less success) as surrogates for the measures of body composition and its distribution. In this section, we describe how well weight and body mass index approximate body composition and then describe three techniques that more directly estimate body composition, including underwater weighing, dual X-ray densitometry, and bioelectrical impedance (BIA). To describe distributions of adipose tissue, only one measure will be described (waist-to-hip ratio), because much of the clinical and epidemiological work uses this approach.

Weight Body weight is representative of the sum of protein, fat, water, and bone mineral mass, and does not provide any information on these four chemical components. In normal adults, there is a tendency to have increased fat deposition with age, concurrent with a reduction in muscle protein. Such changes are not evident in body weight measurements and can only be evaluated by determining body fat and/or fat-free mass. Kvist et al. [74] measured whole-body adipose tissue volume by computed tomography (CT), in a group of men and women differing in adiposity. One of the best pre-

dictors was weight. This observation is similar to those by Ross et al. [75] for obese men and women. They also reported that whole-body lean tissue from magnetic resonance imaging (MRI) in obese men and women can be predicted from weight.

Body Mass Index (Quetelet Index) The body mass index was originally described by the French biostatistician, Quetelet, and named after him. It represents weight relative to stature [weight (kg)/height (m2)] and has been associated with mortality and morbidity in a J-shaped curve. The very low values have been associated with increased mortality from digestive and pulmonary diseases, and higher values associated with increases in cardiovascular diseases, diabetes, and other chronic diseases [76]. Values of B MI less than 20 are regarded as indicative of underweight, values greater than 25 are indicative of overweight, and values greater than 30 indicate obesity [77]. A BMI of 20-25 is the "ideal" index range associated with the lowest risk of illness for most people. Measures of height and weight are generally reliable and have small technical errors of measurement. Using BMI for field studies is beneficial because the measure is highly reproducible. It provides important information apart from the independent measures of weight and height in that it controls for differences in weight normally accounted for by age, gender, ethnicity, and height, thereby facilitating the assessment of obesity in different populations. Although weight adjusted for height is highly correlated with the amount of fat mass (correlations of 0.80 to 0.90), this is a less appropriate approach to the measurement of muscle mass, protein status, or lean tissues (correlations of 0.4 to 0.6). The lack of correlation between BMI and lean mass is relevant. It is in the lean compartment that metabolism of glucose takes place and where insulin resistance is manifest. Furthermore, both fat and lean compartments independently predict menstrual cycle length [78], suggesting that the lean mass may be related to sex hormone relationships. For example, the lean compartment has now been shown to be a better predictor of bone mass and its change in white [79] and Japanese women [80].

Anthropometry Anthropometry is the process of measuring various dimensions of the human body. It is the science that deals with measurement of the size, weight, and proportional dimensions. Anthropometric measurements are of two types: growth and body composition measurements.

Skinfold Thickness The use of skinfold thickness to predict body fat is one of the most common field anthropometric techniques in body composition assessment. Skinfold thickness has been used

CHAPTER 16 Body Composition, Insulin, and the Menopause extensively as a means for estimating body density and fatness. Administered correctly, skinfolds can give accurate results. These techniques are widely used in clinical and field studies because they are relatively easy to administer to large groups of individuals and the equipment is inexpensive (skinfold calipers, tape measure, and anthropometer). The main purpose of skinfold measurements is to estimate general fatness and the distribution of subcutaneous adipose tissue. Fat is pinched between a two-pronged caliper on designated body sites such as triceps, biceps, abdomen, iliac crest, just below the scapula, the thigh and the chest. A skinfold caliper is designed specifically for simple accurate measurement of subcutaneous tissue. The skinfold measurements are used in multiple equations to predict percentage body density; this has been reviewed extensively by Brodie et al. [81 ]. The equations make several assumptions about the relationships between skinfold thickness and adiposity. The first assumption is that a fixed relationship exists between subcutaneous and deep adipose tissue. Second, subcutaneous adipose tissue is representative of the total body fat. Third, selected skinfold thicknesses and adiposity change in proportion to each other. Fourth, fat-free mass is relatively constant for a given body size and skinfold thickness. In addition, there are individual differences that can invalidate these established equations. For specific populations, the validity of the equations rests on the assumption that, for each population of interest, the composition of the fat-free mass is similar. For purposes of generalizability, these equations are not appropriate because many of them are specific to a particular population, and are dependent on the age, sex, nutritional status, and genetic background [81]. Therefore, care must be used in choosing appropriate equations for the population of interest. In addition to these limitations in the application of the equations, there are logistical limitations associated with the skinfold measurement process that can result in an inappropriate estimation of the subcutaneous fat thickness and, consequently, total body fat. Some of the problems associated with this method are inability to palpate the fat/muscle interface, difficulty in obtaining interpretable measurements in obese subjects, use of calipers that do not exert constant pressure at the skinfold site, and incorrect site location. A critical disadvantage in the use of skinfolds is that it is limited to the assessment of subcutaneous fat only, and cannot accurately distinguish between abdominal visceral adipose tissue and saturated adipose tissue. A ratio of triceps and subscapular skinfolds has been used to characterize the relative amount of appendicular to axial fat. Skinfold thicknesses have low correlations with fat-free mass (approximately 0.2) but they are highly correlated with percentage body fat (r = 0.7-0.9), and these correlations do not differ significantly among the common measured sites [82-84]. Although there are relatively high correlations between skinfold thicknesses at single sites and percentage

255 body fat, no one skinfold thickness is an accurate predictor of percentage body fat, albeit triceps skinfolds appear to have the best predictability in population studies [83].

Measures of B o d y C o m p o s i t i o n In field-based studies, measures of body composition have historically been limited to simple measures using weight related to height (power indices) as measures of adiposity. The use of more complex measures of body composition have typically been precluded by the logistical demands of implementation, including number of persons to be evaluated, the lack of available facilities, and time required for implementation.

Underwater Weighing Hydrodensitometry, or underwater weighing, is the classic approach to determining body composition. Based on principles promulgated by Archimedes, the technique generates knowledge of two compartments, the fat mass and the fat-free mass. When a body is submerged in water, there is a buoyant counterforce equal to the weight of the water that is displaced. Because bone and muscle have greater density than water, a person with a larger percentage of fat-free mass will weigh more in the water. Conversely, a larger amount of fat mass will make the body lighter in the water. The individual is measured for the amount of water displacement by submerging in water while sustaining a 30-sec forced expiration. This step is required because air trapped in the lungs also contributes to the amount of water displaced by the subject. The underwater weight is recorded at the end of the forced expiration. This is then compared to the subject's weight in air to obtain body density. Estimates of the fat body and the fat-free body densities are used to calculate the size of these two body composition compartments. The fat-free mass is a heterogeneous compartment that could be further subdivided according to its primary constituents: water (73.8%), protein (19.4%), and mineral (7.8%). Although not feasible for implementation in field studies, the hydrodensitometry approach is used as the gold standard for validating other methods [85-87]. This methodology is compromised because densitometry equations were developed from direct analysis of white cadavers [85] and will lead to the systematic underestimation of relative fatness in American Indian women, black women, and Hispanic women. The fat-free body density in these race/ethnic groups exceeds the assumed value of 1.1 g/ ml [88].

Dual X-Ray Densitometry Assessment using dual X-ray densitometry was developed in the late 1980s and early 1990s as an alternative

256 approach to underwater weighing [89-91]. Currently, the measurement of body composition by dual X-ray densitometry is rapidly replacing underwater weighing as the gold standard for body composition and is logistically more viable than is underwater weighing for studies of clinical and epidemiological populations [89,90]. All DXA devices consist of three components. First, there is an energy source capable of emitting low and high energy levels. Then, a series of detectors capable of discerning amounts of energy attenuation is linked to electrical signals, and these signals provide information used in solving simultaneous equations. Finally, a mechanical system is required to allow the integrated movement of energy source and detectors to determine changes in the degree of attenuation throughout different parts of the body. The fundamental premise is that the amount of density of tissue can be estimated by measuring the amount of ionizing energy transferred through material from a source transmitting at higher and lower energy levels. The estimated fat content in bonefree lean tissue is derived by the constant attenuation of pure fat (Rf = 1.18-1.21) and the attenuation of bone-free lean tissue (R l = 1.399), where R is the attenuation coefficient. The ratio of the attenuation at the lower energy relative to the higher energy in soft tissue [for the low- and high-energy Xrays (40 and 70 keV)] is a function of the proportion of the R values for fat and lean in each pixel (area unit). Traditionally, body composition has been expressed as a two-compartment system of fat tissue and lean tissue (that included muscle, bone, and water). Measurement using DXA allows for the description of a three-compartment system composed of adipose, muscle/water, and bone mineral content. The accuracy of body composition by dual X-ray densitometry is highly correlated with that of hydrodensitometry and the precision error with scans repeated a week apart is less than 2%. There are limitations in DXA measures of body composition. DXA cannot be undertaken in the morbidly obese for two reasons. First, the table is only mechanically stable to weights between 260 and 290 pounds (depending upon the manufacturer), but deforms with loads beyond those weights, jeopardizing the entire system. Second, an assumption underlying the use of DXA is that measurements are not affected by the anteroposterior thickness of the body. However, studies have consistently shown that thickness greater than 25 cm does have an impact on evaluating the energy signal and typically overestimates the fat mass [92]. The sensitivity to change in hydration status has the potential to affect the bone-free lean tissue; however, studies indicate that this is a relatively minor source of error [93]. Last, the estimation of body composition is a function of the 40-50% of the pixels that do not contain bone. Thus, measurements of regions of the body (including the thorax and the arm, which may have relatively fewer pixels) without bone are more prone to measurement error.

SOWERS AND TIscI4

Bioelectrical Impedance A third methodology, bioelectrical impedance, is lower in cost than DXA and is logistically easier to implement in field settings. All BIA devices consist of essentially an alternating electrical current source (usually less than 0.25 V), cables, electrodes for inducing the current into the body, and a system for sensing the voltage drop due to impedance from body tissues. This approach operationalizes the assumption that the electronic conduction in biological tissues is mainly ionic, that is, electrical charges are transferred by ionized salts, bases and acids dissolved in body fluids. Thus, simplistically conceived, the body has highly conductive intracellular and extracellular materials that can be measured, separated by insulating layers of materials such as lipids. The measures generated by the technique, which are resistance and reactance, can be used to derive estimates of total body water and, by extension, lean tissue and fat mass [94]. Because fat-free mass is composed of water, proteins, and electrolytes, conductivity is greater in fat-free mass than in fat [95]. Hence, conductivity is greater in lean than in fat tissue. Thus, this technology establishes a twocompartment model of body composition (fat tissue and lean tissue, which includes bone) based on the transmission speed of low-level, alternating current. Furthermore, specific equations have been validated for blacks [96], Hispanics [97], and Asians [88].

M e a s u r e s o f B o d y T o p o l o g y (Patterning) The terms topology and distribution refer to the relative amount of a tissue in different physical regions of the body. The term patterning is also used in discussions of regional body composition. It is ordinarily used to characterize a specific pattern of tissue distribution. Terms associated with body topology include gynoid obesity, android obesity, and central adiposity. Excess fat concentrated mostly in the abdomen is described as android obesity, whereas fat mostly below the waist and particularly in the hips is described as gynoid obesity. Central adiposity is located in the axial skeleton in amounts disproportionately greater than adiposity observed in the limbs or appendicular skeleton. The ultimate goal for many of the measures of topology is to act as a surrogate marker for the amount of visceral adiposity rather than a measure of subcutaneous adiposity.

Waist-to-Hip Ratio The ratio of hip and waist circumferences (WHR) represents the most commonly used measure of body topology. Recent reports have characterized individuals whose fat is concentrated mostly in the abdomen (android obesity) as more likely to develop many of the health risks associated with obesity, compared to those with gynoid obesity. A

257

CHAPTER 16 Body Composition, Insulin, and the Menopause W H R o f 0.8 or greater in w o m e n is indicative of android obesity. Percentiles are available for the W H R from a large French sample [98] (8646 men; 9747 women) 1 7 - 6 0 years of age and a large Danish sample [99] (1527 men; 1467 women) 3 5 - 6 5 years of age. Conceptually, android obesity is associated with excess visceral adipose deposits. However, the accuracy of the W H R in distinguishing abdominal visceral adipose tissue from subcutaneous adipose tissue is not defined. Estimates of abdominal visceral adipose tissue as well as subcutaneous tissue increase with increasingly greater body mass index, although the proportions of subcutaneous and visceral fat may begin to shift in w o m e n at around 60 years of age [ 100]. In obese individuals, changes in visceral adipose tissue after weight loss are not well related to changes in the W H R [ 101 ]. Thus, conceptually, measures of body topology cannot be considered independently of measures of composition. Circumferences contributing to the waist-to-hip ratio are relatively easy to measure with measuring tapes; however, they can be difficult to measure in persons who are markedly overweight. Standardized protocols for m e a s u r e m e n t of waist-to-hip ratio have not been developed. For example, protocols have not been developed to encourage the measurement of the waist on inspiration or expiration or to define how the hip circumference should be taken if there is sizable abdominal girth. The location of the waist moves up and down with changes in weight and muscle tone, so typically long-term reproducibility is problematic. It is more difficult to interpret trunk circumferences than limb circumferences because the trunk includes organs in addition to various tissues. Interpretation of hip circumference is uncertain because the hips include large amounts of adipose tissue and muscle and are affected by pelvic size and shape.

Summary M e a s u r e m e n t of body composition typically characterizes three compartments: (1) the amount of fat mass, (2) the amount of lean mass, and (3) the amount of bone mineral content (which, in some methodologies, is a subcompartment of the lean tissue mass compartment). It is increasingly well appreciated that body composition is related to chronic disease conditions in women, including diabetes. It is now understood that not only is the amount of fat and lean mass important, but among the more corpulent, the distribution of the fat mass is also important. It is also believed that sex h o r m o n e s may have a regulatory role in body composition and topology. Because m e a s u r e m e n t of body composition and component distribution is technically difficult in humans, particularly in epidemiological studies, a n u m b e r of measures have been used (with more or less success) as surrogates for the m e a s u r e m e n t of body composition and c o m p o n e n t distribu-

tion. These include weight and w e i g h t / h e i g h t indices as surrogate measures of body composition and the waist-to-hip circumference ratio as a measure of body topology. Increasingly, sophisticated measures of body composition are being applied in clinical and epidemiologic studies. These include the use of dual X-ray densitometry and bioelectrical impedance. Although these are likely to give more robust measures of both the fat and the lean compartments, a more technical understanding of the underlying assumptions used to generate information is required.

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259 71. Crook, D., Godsland, I. E, Worthington, M., Felton, C. V., Proudler, A. J., and Stevenson, J. C. (1993). A comparative metabolic study of two low-estrogen-dose oral contraceptives containing desogestrel or gestodene progestins. Am. J. Obstet. Gynecol. 169, 1183-1189. 72. Lemieux, S., Prud'homme, D., Tremblay, A., Bouchard, C., and Despres, J. P. (1996). Anthropometric correlates to changes in visceral adipose tissue over 7 years in women. Int. J. Obes. 20, 618-624. 73. Lemieux, S., Prud'homme, D., Nadeau, A., Tremblay, A., Bouchard, C., and Despres, J. P. (1996). Seven-year changes in body fat and visceral adipose tissue in women. Association with indexes of plasma glucose-insulin homeostasis. Diabetes Care 19, 983-991. 74. Kvist, H., Chowdbury, B., Grangard, U., Tylen, U., and Sjostrom, L. (1988). Total and visceral adipose tissue volumes derived from measurements with computed tomography in adult men and women. Am. J. Clin. Nutr. 48, 1351-1361. 75. Ross, R., Shaw, K. D., Rissanen, J., Martel, Y., de Guise, J., and Avruch, L. (1994). Sex differences in lean and adipose tissue distribution by magnetic resonance imaging: Anthropometric relationships. Am. J. Clin. Nutr. 59, 1277-1285. 76. Meisler, J. G., and St. Jeor, S. (1996). Summary and recommendations from the American Health Foundation's Expert Panel on Healthy Weight. Am. J. Clin. Nutr. 63(3, Suppl.), 474S-477S. 77. Health and Welfare Canada (1988). Promoting healthy weights: A discussion paper. Health Services and Promotion Branch, Health and Welfare, Ottawa. In "Principles of Nutritional Assessment" (R. S. Gibson, ed.), pp. 163-186. Oxford University Press, Oxford. 78. Symons, J. P., Sowers, M. E, and Harlow, S. D. (1997). Relationship of body composition measures and menstrual cycle length. Ann. Hum. Biol. 24, 107-116. 79. Sowers, M. E, Kshirsagar, A., Crutchfield, M., and Updike, S. (1992). Joint influence of fat and lean body composition compartments on femoral bone mineral density in premenopausal women. Am. J. Epidemiol. 136, 257-265. 80. Douchi, T., Oki, T., Nakamura, S., Ijuin, H., Yamamoto, S., and Nagata, Y. (1997). The effect of body composition on bone density in pre- and postmenopausal women. Maturitas 27, 55-60. 81. Brodie, D., Moscrip, V., and Hutcheon, R. (1998). Body composition measurement: A review of hydrodensitometry, anthropometry, and impedance methods. Nutrition 14, 296-310. 82. Frerichs, R. R., Harsha, D. W., and Berenson, G. S. (1979). Equations for estimating percentage of body fat in children 10-14 years old. Pediatr. Res. 13, 170-174. 83. Lohman, T. G., Boileau, R. A., and Massey, B. H. (1975) Prediction of lean body weight in young boys from skinfold thickness and body weight. Hum. Biol. 47, 245-262. 84. Boileau, R. A., and Lohman, T. G. (1977). The measurement of human physique and its effect on physical performance. Orthop. Clin. North Am. 8, 563-581. 85. Siri, W. E. (1956). The gross composition of the body. Adv. Biol. Med. Phys. 4, 239-280. 86. Siri, W. E. (1961). Body composition from fluid spaces and density: Analysis of methods. In "Techniques for Measuring Body Composition" (J. Brozek and A. Henschel, eds.), pp. 223-224. Natl. Acad. Sci., Nat. Res. Counc., Washington, DC. 87. Brozek, J., Grande, F., Anderson, J. T., and Keys, A. (1963) Densitometric analysis of body composition: Revision of some quantitative assumptions. Ann. N. Y. Acad. Sci. 110, 113-140. 88. Heyward, V.H. (1996). Evaluation of body composition. Current issues. Sports Med. 22, 146-156. 89. Cullum, I. D., Ell, P. J., and Ryder, J. R. (1989). X-ray dual photon absorptiometry: A new method for the measurement of bone density. Br. J. Radiol. 62, 587-592. 90. Mazess, R. B., Peppier, W. W., Chestnut, C.H., III, Nelp, W. B., Cohn, S. H., and Zanzi, I. (1981). Total body and lean body mass by dual-

260

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92.

93.

94.

95.

96.

SOWERS AND TISCH photon absorptiometry. II: Comparison with total body calcium by neutron activation analysis. Calcif Tissue Int. 33, 361-363. Mazess, R. B., Peppier, W. W., and Gibbons, M. (1984). Total body composition by dual-photon (153Gd) absorptiometry. Am. J. Clin. Nutr. 40, 834-839. Laskey, M. A., Lyttle, K. D., and Barber, R. W. (1992). The influence of tissue depth and composition on the performance of the Lunar dualenergy X-ray absorptiometer whole-body scanning mode. Eur. J. Clin. Nutr. 46, 39-45. Going, S. B., Massett, M. P., Hall, M. C., Bare, L. A., Root, P. A., Williams, D. P., and Lohman, T. G. (1993). Detection of small changes in body composition by dual-energy x-ray absorptiometry. Am. J. Clin. Nutr. 57, 845-850. Boulier, A., Fricker, J., Thomasset, A., and Apfelbaum, M. (1990). Fat-free mass estimation by the two-electrode impedance method. Am. J. Clin. Nutr. 52, 581-585. Lukaski, H. C., and Bolonchuk, W. W. (1988). Estimation of body fluid volumes using tetrapolar bioelectrical impedance measurements. Avia. Space Environ. Med. 59, 1163-1169. Ainsworth, B. E., Stolarczyk, L. M., Heyward, V. H., Berry, C. B.,

97.

98.

99.

100.

101.

Irwin, M. L., and Mussulman, L. M. (1997). Predictive accuracy of bioimpedance in estimating fat-free mass of African-American women. Med. Sci. Sports Exercise 29, 781-787. Stolarczyk, L. M., Heyward, V. H., Goodman, J. A., Grant, D. J., Kessler, K. L., Kocina, E S., and Wilmerding, V. (1995). Predictive accuracy of bioimpedance equations in estimating fat-free mass of Hispanic women. Med. Sci. Sports Exercise 27, 1450-1456. Tichet, J., Vol, S., Balkau, B., LeClesiau, H., and D'Hour, A. (1993), Android fat distribution by age and sex: The waist-hip ratio. Diabetes Metab. 19, 273-276. Heitmann, B. L. (1991). Body fat distribution in the adult Danish population aged 35-65 years: An epidemiological study. Int. J. Obes. 15,535-545. Enzi, G., Gasparo, M., Biondetti, ER., Fiore, D., Semisa, M., and Zurlo, E (1986). Subcutaneous and visceral fat distribution according to sex, age, and overweight, evaluated by computed tomography. Am. J. Clin. Nutr. 44, 739-746. van der Kooy, K., Leenen, R., Seidell, J.C., Deurenberg, E, Droop, A., and Bakker, C. J. (1993). Waist-hip ratio is a poor predictor of changes in visceral fat. Am. J. Clin. Nutr. 57, 327-333.

7HAPTER 1 q

Influence of Estrogen on Collagen R. GALEA AND M. BRINCAT Department of Obstetrics and Gynecology, St. Luke's Hospital Medical School, Gwardamangia MSD 07, Malta

I. II. III. IV.

Introduction Structure of Collagen Age-Related Changes in Collagen Skin

V. Carotid Arteries VI. Urinogenital System VII. Concluding Remarks References

I. I N T R O D U C T I O N

the proteoglycans (long chains of repeating disaccharides attached to specific core proteins).

An intriguing question regarding complex multicelluar organisms concerns what holds their myriad cells together. Certainly one of the primary factors contributing to the solution of this challenge is the group of tough, fibrous, collagenous proteins within connective tissue. Connective tissues of various types (bone, tendon, cartilage, etc.) are sites where the majority of the extracellular matrix resides. The matrix is composed of secreted fibrous proteins embedded in a gellike polysaccharide ground substance. Adhesion proteins that keep cellular components linked to each other are also present. Various types of extracellular matrix occur, such as the basal lamina or basement membrane. Basal laminae form the resting platform of epithelial cells or, alternatively, surround muscle fibers, adipocytes, and peripheral nerves. Differences among the various types of extracellular matrices result from variations on this general repertoire of complex macromolecules. Collagen is the major primary structural protein of the extracellular matrix; elastin and fibrillin are also structural proteins. Other complex macromolecules of extracellular matrix are the specialized proteins (fibronectin, laminin) and MENOPAUSE:

BIOLOGY AND PATHOBIOLOGY

II. S T R U C T U R E OF C O L L A G E N Collagen is the major protein component of most vertebrate connective tissues. In mammals, collagen constitutes about 25-35% of total protein [1]. It is present in virtually every animal tissue and provides an extracellular framework for all metazoan animals. The large family of collagen proteins comprises at least 19 different members in human tissues. These proteins are made up of about 30 distinct polypeptide chains encoded by 30 independent genes. Collagen molecules are characterized by the formation of three lefthanded polypeptide helical chains tightly coiled around each other in a ropelike fashion to form a right-handed supercoil. The collagen helix is more extended than an ce helix because it has three amino acid residues per turn with a pitch of 0.94 mm, giving rise to 0.31 nmol per residue. All collagens contain greater or lesser stretches of this triple helix. (Fig. 1) [2]. Two basic ce chains have been identified in collagen (eel 261

Copyright 9 2000 by Academic Press. All rights of reproduction in any form reserved.

262

FIGURE 1 Collagen fiber and fibril structure, showing putative locations of pores and hole zones. Reprinted with permission from Einhorn [2].

and a2), each consisting of just over 100 amino acids in groups of three, organized in the basic collagen triple helix configuration. The triple helix domains of collagens consist of repeats of the amino acid sequence Gly-X-Y. Hydroxyproline usually occupies position X; position Y is usually occupied by proline. The ring structure of the latter amino acids is responsible for the stabilization of the helical conformations of the polypeptide chains. Hydroxyproline is formed in the endoplasmic reticulum by posttranslational alteration of proline residues by means of the enzyme, prolyl hydoxylase. These proline residues would have already been incorporated into collagen polypeptide chains. The hydroxyl groups of these modified amino acids tend to form hydrogen bonds between adjacent polypeptide chains, providing further stabilization to the triple helix. This very stable complex forms the basic building unit of collagenous structures. Proline and hydroxyproline constitute 20-25% of total amino acids in collagen. Collagen biosynthesis has several unusual features. The first of these is an extensive use of the principle of spontaneous self-assembly seen in the formation of crystals. The three polypeptide chains of the protein fold into a triple helical conformation by a process that begins with the formation of a small nucleus of the triple helix at the carboxyterminal end of the molecule and is followed by propagation of the nucleus in a zipperlike fashion. The self-assembly of collagen monomers into fibers is an entropy-driven process reminiscent of crystallization. Collagens can be divided into two main groups, fibrillar and nonfibrillar. Other small groups also occur, such as the network-forming and anchoring filaments. In the form of fi-

GALEA AND BRINCAT

bers, collagen acts to transmit forces, to dissipate energy, and to prevent mechanical failure in normal tissues. Deformation of collagen fibers involves molecular stretching and slippage, fibrillar slippage, and, ultimately, defibrillation. Type I is the most abundant collagen type, constituting 90% of total body collagen. It predominates in skin and bone. Skin also contains an amount of the very similar type III collagen. Type II collagen is specifc to cartilage. In contrast to glycosaminoglycans, which allow rapid diffusion of water molecules, thereby creating tissue turgor with application of compressive force, collagen fibrils resist tissue stretching. Although the great majority of total body collagen is remarkably stable, a fraction of collagen in all tissues is continuously degraded and replaced throughout life. As these constituents are excreted or metabolized without reincorporation into new collagen, the process of collagen degradation can be tracked by measurement of its breakdown products in urine. For decades, measurement of urinary hydroxyproline was the classical assay for this purpose. However, it was not ideal because only about 10% of the total daily production of hydroxyproline is excreted, the remainder being processed in the liver. Recent interest has centered on nonmetabolized products, such as the pyridinium cross-links, pyridinium (PYD) and deoxypyridinium (D-PYD), which exist primarily in type I collagen of bone. These compounds are formed by the action of the enzyme lysyl oxidase to condense the amino acids lysine and hydroxylysine in adjacent collagen fibers, resulting in the formation of mature nonreducible covalent cross-links PYD and D-PYD. When bone is resorbed, proteolytic degradation of collagen releases free PYD and D-PYD into the circulation for renal clearance and urinary excretion. Measurement of free PYD, D-PYD, and their peptide-bound forms has found recent application in the assessment of patients with bone disorders, for whom the assays may have diagnostic utility and aid in the monitoring of patients on therapy. Another collagen marker that may have utility in management of bone disease is the type I procollagen carboxyterminal extension peptide. This molecule is released as an intact subunit from the intact procollagen molecule during collagen biosynthesis. It circulates in plasma, in which its concentration offers a reflection of bone collagen turnover rate.

III. A G E - R E L A T E D

CHANGES

IN COLLAGEN Age is associated with changes in the quality, type, and amount of collagen. For example, type III collagen is more abundant in the skin of young animals than in that of older animals. This may indicate gene switching comparable to the switch in the bone marrow from fetal hemoglobin to the

CHAPTER 17 Influence of Estrogen on Collagen adult form, hemoglobin A. Growth of connective tissue involves an increased rate of collagen biosynthesis and this is reflected in an increased tissue level of intracellular posttranslation enzyme activities. Both the rates of translation and the levels of these enzymes decrease with age [3]. The menopausal state is certainly characterized by a lack of endogenous estrogen, which is temporally related to reductions in tissue collagen content. Albright [4] speculated that postmenopausal osteoporosis was part of a generalized connective tissue disorder, having observed that the skin of osteoporotic women was thin. Because of reductions in skin collagen and in the density and collagen content of bone, McConkey et al. [5] showed that transparent skin on the back of the hand was most common in women over 60 years of age, and the prevalence of osteoporosis in women with transparent skin was 83% versus 12.5% in women with opaque skin. To investigate the relationship between age, bone mass, and skin thickness, our group evaluated differences in bone density measurements and skin thickness in a group of postmenopausal women. Bone density was assessed using dualenergy X-ray absorptiometry (DXA) of the lumbar spine and proximal femur; skin thickness was measured using high-frequency (22 MHz) ultrasound, a technique that correlates with skin collagen content. Results showed that skin thickness and bone density were much lower in women who had sustained an osteoporotic fracture than in controls (Fig. 2). Women with a fracture had bone mass values that were about 20% below the mean control value. When skin thickness measurements were combined with bone density, the accuracy of predicting the presence of fracture was increased (Fig. 3).

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The skin is one of the largest organs in the body, containing a sizeable proportion of total body collagen. This organ is composed of populations of cells of diverse embryonic origin, which under normal conditions exist side by side in complete harmony, forming a complex mosaic. Anatomically skin is made up of two main layers. The epidermis forms a thin outer layer and is composed of keratinocytes (keratin-producing cells) of ectodermal origin intermingled with melanin-producing cells, the melanocytes, which arise from a specialized embryonic ectodermal tissue, the neural crest. The other deeper layer is the dermis, a stroma that forms the main bulk of the skin and is intimately bound with the overlaying epidermis; fingerlike processes, or dermal papillae, project upward into corresponding recesses in the epidermis. In contrast to the epidermis, the dermis is predominantly fibrous and contains blood vessels. It is of mesodermal origin, as is all connective tissue (including bone). It

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also contains several structures derived from the embryonic ectoderm, e.g., sweat glands and hair follicles. The fibers present in the dermis consist of two main types of fibrous protein, collagen (97.5%) and elastin (2.5%) [6]. Collagen fibers are responsible for the main mass and resilience of the dermis. This collagen is disposed mainly in a parallel arrangement to the skin surface. The elastin fibers, on the other hand, form a subepidermal network and are only thinly

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Scattergrams of (a) lumbar vertebrae 2 to 4 and (b) femur region (Ward's triangle) bone density measurements vs. skin thickness measurements in postmenopausal women who had sustained an osteoporotic fracture and in normal postmenopausal women on no treatment. The sensitivity and accuracy of the methods are also shown.

CHAPTER 17 Influence of Estrogen on Collagen distributed. Collagen is produced by fibroblasts. These cells contain abundant endoplasmic reticulum where secreted proteins such as collagen are synthesized from the main types of procollagen found in connective tissues, types I, II, III, V, and XI. The menopausal decrease in estrogen leads to a decline in both skin thickness and collagen content, giving rise to a characteristic phenotype of dry, wrinkled, flaky, and easily bruised skin. This estrogen decrease can be prevented by estrogen replacement therapy [7-9]. Atrophy of the dermis after the menopause is due to a decrease in the dermal skin collagen content of the dermis. The amounts of hydroxyproline and glycosylated hydroxylysine [10,11] in type I collagen and of immature and reducible cross-links decrease with age [12]. To what extent these changes are fundamental to the aging process is still unknown. These decreases are not only arrested but are reversed by hormone replacement therapy [13]. There is now strong evidence, as seen from the National Health and Nutrition Examination Survey [ 14], that estrogen use prevents dry skin and skin wrinkling, thus extending the potential benefits of postmenopausal estrogen therapy to include protection against selected age- and menopause-associated dermatologic conditions. Estrogens have in fact been shown to enhance the dermal content of water, glucosaminoglycans (GAGs), and collagen. Both estrogen and androgen receptors have been identified on dermal fibroblasts [ 15]. In a study to identify specific estrogen-sensitive structures [ 16], normal human skin was examined for the binding of the estrogen receptor (ER) D5 antibody, which is associated with p29, a 29-kDa protein found in the cytoplasm of normal estrogen-sensitive cells. Strong and specific staining was seen in the epidermis, with a gradient showing the most intense staining in the granular layer. Similar positive staining was seen in the hair follicles and sebaceous glands. Variable staining was seen in the eccrine glands and vessels. These findings demonstrate that p29 is present in these structures, and hence that estrogens may exert a specific effect on these tissues. Estrogen could increase the rate of collagen production [ 17] by altering the degree of polymerization of GAGs. Estrogens increase the hydroscopic qualities [18], probably through enhanced synthesis of dermal hyaluronic acid [ 19]. Collagenous fibrils were found to be less fragmented in the dermis of women treated with estrogens [20]. Studies have been carried out on skin changes in various connective tissues and in endocrine disorders. Black [21,2 l a] and Shuster [22] looked at the relationship between skin thickness and skin collagen in systemic sclerosis, in osteoporotics of mixed etiology, and in hirsute women and found a good correlation between the two. In a small study, Black [21] demonstrated changes in the collagen content and thickness of the skin in osteoporotics (mixed etiology) treated with androgens, when compared to osteoporotics who had not been on this treat-

265 ment. Shuster et al. [22] found an increase in skin collagen in women with hirsutism, but the increase was not statistically significant. In scleroderma, Black [21 ] demonstrated a decrease in total collagen content and skin thickness in affected areas. In clinically normal areas, the skin thickness was decreased but the collagen content was not significantly altered. Arho [23] did not show any difference between skin thickness and collagen content in patients with scleroderma when compared to normal patients. He also found a good correlation between skin thickness and collagen content in a number of patients with a variety of endocrine and collagen disorders. The conditions looked at by Arho, apart from normal skin, were acromegaly, rheumatoid arthritis, lupus erythematosus, scleroderma, prurigo besnier, psoriasis, and Cushing's syndrome. Patients with prurigo besnier and scleroderma had normal skin collagen contents. Acromegaly produced both thicker skin and higher skin collagen content whereas patients with Cushing's syndrome and those who had been treated with costicosteroids had thinner skin and lower skin collagen content than controls. Reports on the skin collagen changes with age are conflicting. Shuster and Bottoms [24,25] showed that the best way of expressing skin collagen content was by measuring the collagen content of a skin biopsy per square millimeter of skin surface. This method takes into account the possibility of changes in the total mass of the dermis. Shuster and Bottoms reported a reduction in total skin collagen with age, but this was not confirmed by Reed and Hall [26]. Shuster and Black found that the amount of collagen present in skin was at all ages higher in males than in females. Hall [27] confirmed that skin collagen was higher in males than in females, but once again could not significantly confirm the decline of skin collagen with age. With increasing age past the menopause, the skin tends to get thinner and this is reversed with adequate estrogen replacement therapy [28]. The dermal skin thickness, composed as it is of connective tissue made up of diverse macromolecules, including collagen, reflects more than just the effect of estrogen on collagen. For example, castrated rats that were given estrogens had a 70% increase in their glycosaminoglycans content in 2 weeks [ 17]. Similar increases in women would lead to skin thickness increases that far outstrip what would be expected from collagen content increases alone. Most studies have measured the thickness of dermis, because this is the layer that is mainly connective tissue. Subcutaneous tissue is an added variable, and studies using Harpenden's callipers, which include subcutaneous fat, have been inconsistent [29]. Ultrasound can also be used to measure skin thickness [30]. Ultrasound examination has been used by dermatologists when assessing dermal malignancies. Good reproducibility has been obtained by using highfrequency (22 MHz) ultrasound to determine the thickness of the skin, excluding the subcutaneous tissues, and studies have shown that skin thickness increases with estrogen

266 replacement therapy. Skin changes and bone changes with estrogen at the menopause are linked [28,31 ]. In animals, estrogens appear to alter the vascularization of the skin [32], and a change in the connective tissue of the dermis occurs, as is reflected by increased mucopolysaccharide incorporation, hydroxyproline turnover, and alterations in ground substance. In addition to increased dermal turnover of hyaluronic acid, the dermal water content is enhanced with estradiol therapy [19]. Rauramo [33] observed that oral estrogen therapy in castrated women caused thickening of the epidermis for 3 months and this persisted for 6 months. Punnonen [34], using two different strengths of estrogens in castrated women, showed a statistically significant thickening of the epidermis after 3 months with both strengths, but this thickness persisted only with the lower dose. A third of patients on the higher dose actually developed epidermal thinning, possibly reflecting a toxic effect of high dosage. The mechanisms explaining this bimodal effect are unclear. Other authors were also able to show beneficial skin changes using both topical [8,35] and implanted estradiol [36]. Topical estradiol gel has also been shown to increase skin collagen content as measured by skin hydroxyproline. Skin blister fluids were assayed and an increase of both procollagen type I carboxy-terminal propeptide (PICP) and procollagen type I N-terminal propeptide (PINP) occurred with the gel. In comparing a group of patients who had been on estradiol and testosterone implants from 2 to 10 years to a group of untreated postmenopausal women it was shown that the treated group had a significantly greater skin collagen content than the untreated group. Optimum skin collagen was obtained after 2 years of an optimum estrogen regimen. Estrogen levels that are too high or too low are associated with lower levels of collagen [37]. The same conclusions were also reported in relation to epidermis [38]. The decline in skin collagen content after the menopause occurs at a much more rapid rate in the initial postmenopausal years than later on. Some 30% of skin collagen is lost in the first 5 years after the menopause [37], with an average decline of 2.1% per postmenopausal year over a period of 20 years. The increase in skin collagen content after 6 months of sex hormone therapy depends on the collagen content at the start of treatment [37]. In women with a low skin collagen content, estrogens are initially of therapeutic and later of prophylactic value, whereas in those with mild loss of collagen content in the early menopausal years estrogen is of prophylactic value only. Thus a deficiency in skin collagen can be corrected but not overcorrected. Skin collagen content has been shown to have a strong correlation with skin dermal thickness measured radiologically [37]. Using 100-mg subcutaneous estradiol implants, significant increases in skin thickness and the metacarpal index occurred over a 1-year period, with most of the increase occurring in the first 6 months.

GALEA AND BRINCAT

Brincat and Castelo-Branco [35,37,39,40] have shown that following the menopause, skin collagen content and skin thickness are increased in women on estrogen replacement therapy compared to age-matched women on no treatment. Prospective studies have shown that skin thickness, skin collagen, and bone mass increase in postmenopausal women who start estrogen replacement. When postmenopausal women were treated with topical estrogens [41], it was found that after treatment for 6 months, elasticity and firmness of the skin had markedly improved and the wrinkle depth and pore sizes had decreased by 61-100%. Furthermore, skin moisture was also increased and the measurement of wrinkles using skin profilometry revealed significant, or even highly significant, decreases in wrinkling. On immunohistochemistry analyses of the same subjects, significant increases of type III collagen labeling were combined with increased numbers of collagen fibers at the end of the treatment period. The mechanical properties of the skin have been shown to be improved with estrogen replacement therapy in other studies [42]. In these studies, the mechanical properties were defined by extensibility and elasticity measurements using a computerized device. Computerized measurements of skin deformability and viscoelasticity revealed differences between women on estrogen therapy, postmenopausal women on no treatment, and nonmenopausal controls. This parallels the changes noted elsewhere with skin collagen. A steep increase in skin extensibility was evidenced during the perimenopause in untreated women. Estrogen replacement therapy appeared to limit the age-related increase in cutaneous extensibility, thereby exerting a preventive effect on skin slackness. No effect of estrogen replacement therapy was found on other parameters of skin viscoelasticity. Estrogen replacement therapy has a beneficial effect on some mechanical properties of skin and thus may slow the progress of intrinsic cutaneous aging. Brincat et al. [36,43,44] found significant correlations between the skin (dermal) collagen, skin thickness (measured radiologically), and the metacarpal index, both in postmenopausal women who had been on estrogen replacement therapy and in untreated postmenopausal women. The common factor is the connective tissue present in all three sites. These findings were independent of the woman's age, number of years since the menopause, original skin thickness, or metacarpal index. Attempts to identify a range of values of skin thickness that could be used as a screening test for menopausal osteoporosis have been underway for some time [45].

V. CAROTID ARTERIES Considerable epidemiological evidence favors an important cardioprotective effect of hormone replacement therapy [46]. This has been attributed to the favorable serum lipopro-

CHAPTER 17 Influence of Estrogen on Collagen

267

tein profile brought about by this treatment on postmenopausal women [47]. However, only 2 0 - 2 5 % reduction in coronary artery plaque formation can be thus accounted for [48]; other mechanisms of cardioprevention are therefore present. One such mechanism could be due to postmenopausal connective tissue changes that mimic those in skin and

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-

Controls vs Oral HRT p = N.S. Contols vs Implants p <0.05 Oral HRT vs Implants p = N.S.

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19 Controls n = 51

b

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bone. Using high-frequency ultrasound, our group looked at the differences in the layers of one of the major blood vessels, the carotid artery. One study therefore investigated differences in the externa and media of the carotid artery in two groups of postmenopausal women on estrogen replacement and an untreated group, the intima was also assessed. The results showed that the media, the layer containing the greatest amount of collagen, was thicker in the estrogen-treated groups when compared to the untreated group. The intima of the untreated group was shown to be significantly thicker than that of the treated group (Fig. 4). This signifies that there was less atherosclerotic plaque in the treated group, compared to the untreated group. However, in women on a higher dose of estrogen, such as those with an estradiol implant, the intima was as thick as in the untreated group. The result was postulated to be due to the collagen portion of the intima next to the media increasing in size at the expense of the decrease in atherosclerotic plaque [49,50]. It is thus postulated that these changes are brought about by estrogen. If these arterial changes induced by hormone replacement therapy also occur in the coronary arteries, and some suggestion of this has been made [51 ], then these may partially explain the cardioprotective effect of this treatment in postmenopausal women.

.

VI. U R I N O G E N I T A L

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FIGURE 4

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Plots of the mean (_ SD) thickness of the different layers of

t h e c a r o t i d a r t e r i e s (a, e x t e r n a ; b, m e d i a ; c, i n t i m a ) w o m e n o n n o t r e a t m e n t (controls)

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The female genital and urinary tracts, which develop in close proximity to each other, both arise from the embryological urogenital sinus and are both estrogen sensitive. The epithelium of the vagina and of the proximal and distal urethra has a high concentration of estrogen receptors. Fluctuations in sex steroids, in particular estrogens, result in symptomatic and urodynamic changes in the lower urinary tract. This has been demonstrated during the menstrual cycle, in pregnancy, and following the menopause. After the menopause the vagina sustains a decrease in vascularity and becomes thin, inflamed, and ulcerated. The cervix atrophies and retracts, and atrophy of the uterine corpus results in a return to the 1:2 corpus:cervix ratio of childhood. Postmenopausal genital atrophy is associated with dyspareunia, apareunia, pruritus vulvae, and incontinence. It has been estimated that about 40% or more of women over 60 years of age complain of insufficient control of micturition. All of these symptoms improve with estrogen replacement therapy [52]. The most common complaints in postmenopausal females regarding the urinogenital system are stress incontinence, urgency, urge incontinence, frequency (diurnal and nocturnal), dysuria, and voiding difficulties. In continent women, the maximum urethral pressure must exceed the intravesical pressure at all times except during micturition. This positive closure pressure is produced by the four functional layers of the urethra, namely, the epithelium, the

268 connective tissue, the vascular tissue, and the muscle. It seems that stress incontinence is not likely to be the result of the menopause, whereas urge incontinence and other irritative bladder symptoms may be related. Connective tissue is an important component of the female urethra and collagen is its most abundant structural protein. An investigation into skin collagen content and urethral pressure profiles [53] showed that for all the parameters studied, a greater collagen content was correlated with improved sphincteric function. The area under the curve of the resting pressure profile of the urethra showed a significant positive correlation, with collagen content at the proximal, but not the distal, part of the urethra. The effect of collagen on the urethral closure pressure, therefore, is greater in the proximal than in the distal part of the urethra. Histological measurements in the urethra show that collagen fibrils occupy twice the volume of muscle [54] and elastin fibers probably play only a minor role, because they are not abundant in the urethra [55]. As in other sites in the body, the collagen in the female urethra is produced by fibroblasts, which have estrogen receptors. Estrogens may improve incontinence by increasing urethral resistance, raising the sensory threshold of the bladder, increasing ce-adrenoreceptor sensitivity in the urethral smooth muscle [56], or possibly by a combination of these factors. Cells in the intermediate and superficial layers of the vagina, urethra, and bladder have also been shown to increase [57]. Biopsies of paraurethral tissue suggest that the decline in estrogen levels at menopause might be altering connective tissue. Collagen content was almost double in premenopausal biopsies compared to values in postmenopausal women. Estrogen therapy results in an increase of mRNA for collagen I and III, which may imply that the changes occurring after menopause are due to an increased turnover [58]. Urethral pressure profilometry has been the main parameter used to assess the efficacy of estrogen therapy. Caine and Raz [59] reported an increased maximum urethral pressure together with symptomatic improvement in 26 out of 40 women with stress incontinence who were treated with oral conjugated estrogens. Favorable results were also obtained by Faber and Heindrich and Hilton and Stanton. The latter [60,61 ] also reported significant subjective improvement in the symptoms of stress incontinence, urgency, and frequency. In a double-blind placebo-controlled study by Wilson e t al. [62], 36 women with urodynamically proved genuine stress incontinence were treated with cyclical oral piperazine estrone sulfate for 3 months. Although there was symptomatic improvement, there was no significant difference in observed responses, urethral pressure profiles, or the quantity of urine lost. In other placebo-controlled studies [53] (using a 50-mg estradiol implant vs. placebo) 46 women were investigated by urethral pressure profilometry and a pad-weighing test after 3 months of treatment. No significant subjective evidence

GALEA AND BRINCAT

of improvement was reported, although there was a significant improvement in bladder base descent as seen on the videocystourethrogram. Control of micturition is a complex process; estrogen deficiency is only one of several factors contributing to a loss of control. Absorption of estrogens is high when the vaginal mucosa is atrophic and graduallydecreases as the vaginal mucosa matures under hormonal influence [63]. Although there is, as yet, no conclusive evidence that estrogen alone cures stress incontinence, its use as an adjunct to other methods of treatmentmsuch as surgery, exercise, and biofeedback, as well as other d r u g s m m a y be beneficial in the management of postmenopausal urinary stress incontinence.

VII.

CONCLUDING

REMARKS

Connective tissue in the body represents a complex mass of cells and an extracellular matrix; these are intimately related to each other and provide a milieu in which all the other cells are able to perform their functions and relate to each other. This connective tissue is acutely sensitive to sex steroids. Modifications in the noncollagenous extracellular matrix are outside the scope of this chapter, but the scanty evidence that exists suggests profound and rapid changes in molecules such as GAG, hyaluronic acid, and other mucopolysaccharides and noncollagenous molecules. The components of collagen, which are vitally important, undergo measurable changes with estrogen deprivation, but these changes are preventable and to a degree the components are replaceable with appropriate estrogen therapy. Early evidence suggests that selective estrogen receptor modulators (SERMS) such as raloxifine have an effect similar to that of estrogen on certain markers of collagen turnover.

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269

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50.

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57.

58.

59. 60.

61.

62.

63.

amine in combination for stress urinary incontinence in postmenopausal women. Urology 32, 273-280. Samsioe, G., Janssen, I., Mellstrom, D., and Svandborg, A. (1985). Occurrence, nature and treatment of urinary incontinence in a 70 year old female population, Maturitas 7, 335-342. Falconer, C., Ekman Ordeberg, G., Ulmsten, U., Westergren Thorsson, G., Barchan, K., and Malmstr6m, A. (1996). Changes in paraurethral connective tissue at menopause are counteracted by estrogen. Maturitas 24(3), 197-204. Caine, M., and Raz, S. (1973). The role of female hormones in stress incontinence. Proc. Congr. Inter. Soc. Urol., 16th, Amsterdam. Hilton, E, Tweddel, A. L., and Mayne, C. (1990). Oral and intravaginal oestrogens alone and in comination with alpha adrenergic stimulation in genuine stress incontinence. Int. Urogynecol. J. 12, 80-86. Hilton, E, and Stanton, S. L. (1978). The use of intravaginal estrogen cream in genuine stress incontinence. A double blind clinical trial. Urol. Int. 33, 136 - 143. Wilson, E D., Paragher, B., Butler, B. et al. (1987). Treatment with oral piperalzine oestrone sulphate for genuine stress incontinence in postmenopausal women. Br. J. Obstet. Gynaecol. 94, 568-574. Samsioe, G. (1998). Urogenital agingmA hidden problem. Am. J. Obstet. Gynecol. 178(5), $245-$249.

2 H A P T E R 1{

The Growth Hormone IGF-I Axis and Menopausc CLIFFORD J.

ROSEN

St. Joseph Hospital, Maine Center for Osteoporosis Research and Education, Bangor, Maine 04401

I. Introduction II. Physiology of the GH/IGFs III. Diseases in Postmenopausal Life and Their Relationship to the GH/IGF-I Axis

IV. Summary References

I. I N T R O D U C T I O N

nance of life quality and musculoskeletal balance. These investigations also raised expectations that manipulation of the GH/IGF system in postmenopausal women could modify the risk of certain diseases. In this chapter, the focus is on the circulatory GH/IGF regulatory system, how it is regulated, and what importance these systems might hold in respect to menopause and its long-term manifestations. In particular this discussion centers on the role estrogen plays in modulating GH secretion, IGF-I production, and somatomedin activity in skeletal and reproductive tissues. Deficiencies in gonadal steroid production during menopause are likely to have a major impact on the GH/IGF-I axis, and are certain to play some role in several diseases that occur after menopause.

Menopause is associated with major changes in several endocrine systems, not the least of which is the growth hormone/insulin-like growth factor-I (GH/IGF-I) axis. This hormonal system not only controls growth during adolescence but also is vitally important for the maintenance of the musculoskeletal system throughout life. Moreover, body composition and fat mass may be important feedback regulators of GH secretion. Over the past decade major advances have taken place in our understanding of growth hormone secretion, its regulation, its effects on target tissues, and the complex circuitry that permits IGF-I to mediate GH activity at the tissue level. Although the role of estrogen in priming GH secretion in young adults during growth has been known for several decades, we are now just beginning to appreciate how sex steroids may be critical for maintaining GH activity in later life. The advent of recombinant peptide technology has pushed clinical trials with rhIGF-I and/or rhGH in several disease states, including diabetes mellitus, obesity, renal failure, and osteoporosis. More importantly, the use of rhGH as replacement therapy for growth hormone deficiency (GHD) syndromes has highlighted the importance of GH in the mainteMENOPAUSE:

BIOLOGY AND PATHOBIOLOGY

II. P H Y S I O L O G Y O F T H E G H / I G F s A. G r o w t h H o r m o n e / G r o w t h - H o r m o n e Releasing Peptides

1. GH SECRETION The regulation of GH secretion from the pituitary is complex and involves elaboration of discrete neurosecretory 271

Copyright 9 2000 by Academic Press. All rights of reproduction in any form reserved.

272 peptides from the hypothalamus. Hypothalamic releasing factors include growth-hormone-releasing hormone (GHRH) and somatostatin [1]. Two distinct GH-releasing peptides (GHRPs) of 40 and 44 amino acids have been isolated form the hypothalamus. In addition, several synthetic growth hormone-releasing peptides have been developed and have been shown to act through a distinct GH releasing receptor. MK0677, a potent spiroindoline derivative, stimulates the GH secretagogue receptor in vivo [2,3]. Similarly, hexarelin, a 2methyl derivative of GHRP-6, can also provoke GH release from the pituitary [3]. Somatostatin, a small 14-amino acid polypeptide produced in several endocrine tissues, inhibits GH synthesis and release [ 1]. Together the GHRPs and somatostatin control GH release, although other neuromodulators can influence GH secretion (e.g., y-amino-n-butyric acid, endogenous opiods, and neuropeptide Y). Both GHRPs and their synthetic analogs have been utilized in clinical trials to raise serum GH and IGF-I in hopes of enhancing metabolic profiles and improving musculoskeletal performance. GH secretion is pulsatile (due to episodic secretion of the GHRPs) and circadian, with the highest pulses occurring between 0200 and 0600 hr [4]. Rising estrogen and androgen concentrations during puberty increase the magnitude of the GH pulse [3]. All these changes result in an increase in the production of IGF-I from the liver and other sites. IGFBP-3, a major IGF binding protein, and acid-labile subunit (ALS) are also induced by GH. Besides somatostatin (SMS), IGF-I can also inhibit GH release and likely is very important as another feedback mechanism on GH [2,4]. In sum, GH secretion is a complex pulsatile process with circadian variation. It is regulated by several hypothalamic factors and by IGF-I. Each of these controlling determinants in turn can be affected by endogenous or exogenous estrogen. 2. SEX STEROID REGULATION OF GH SECRETION Gonadal steroids influence GH secretion at several levels and are defined by striking gender differences. Pituitary secretion of a coherent burst of GH is the result of a brief G H R H somatostatin imbalance in favor of GHRH [4,5]. In rodents and humans, the pattern of GH release over 24 hr is more disorderly in females than in males [4-6]. This effect has been quantified by an entropy statistic [4]. Postmenopausal women treated with estrogens demonstrate increased disorderliness of GH release [2,4]. In addition, the amount of GH secreted per burst frequency is greater in females than in males, although this difference disappears at menopause [4]. In premenopausal women the greatest GH secretion occurs during the late follicular phase, concurrent with increases in estradiol and IGF-I [4,7]. Other factors also contribute to gender differences. For example, women have a greater sensitivity to GH release by GHRH than do men [3,8,9]. Also, serum estradiol is the predominant positive correlate of the mean serum GH concentration and GH pulse amplitude in both men and

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women (both pre- and postmenopausal) [2,4,7,8]. Estradiol may also modulate somatostatin release, thereby enhancing GH secretion in response to arginine [2,4]. Finally, oral administration of estrogen to postmenopausal women can enhance GH secretion, in part by decreasing serum IGF-I levels (see later). Although originally thought to be a function of hepatic action on oral estrogens, a study by Friend et al. demonstrated that at higher doses of percutaneous estradiol, serum IGF-I levels decline and GH secretion increases [ 10]. Gender differences in GH secretion can also be accounted for by body composition. Although several other determinants regulate GH secretion (age, nutritional status, and exercise), gender influences these factors. For example, premenopausal women are relatively protected against the age-associated fall in GH, and are less sensitive than men to the GH-suppressing effect of increased body fat [2,4]. Moreover, oxygen consumption is a more positive determinant of GH secretion in women than in men [4,7]. In respect to body composition, it is clear that in both sexes, increased body fat is associated with reduced GH secretion. For example, GH secretion is inversely related to leptin concentrations [11]. This effect is almost certainly mediated through hypothalamic pathways, although the mechanisms have not been defined. The percentage of body fat may override the actions of sex steroids, although more studies are needed to define the interrelationship between estradiol, body composition, and GH status. In summary, the sex steroids play a major role in modulating GH secretion during adult life. Both qualitatively (i.e., greater disorderliness of GH secretion in women) and quantitatively (i.e., more GH pulses in women than men) estrogen is a major factor in controlling GHRH and GH output [4]. These effects may also be important in the menopausal state, when acute estrogen deficiency results in changes in body and tissue composition. Moreover, as noted below, gonadal steroids also modulate the final common pathway for GH activity, IGF-I.

B. I G F - I a n d I G F - I I S t r u c t u r e and F u n c t i o n 1. I G F s AND I G F B P s

The IGFs are 7-kDa polypeptides that share structural homology with proinsulin [12]. These proteins were initially called 'somatomedins' because of their growth-promoting properties in numerous tissues, and the inability to suppress their activity with antiinsulin antibodies [13]. Both growth factors are found in high concentration in serum and nearly every mammalian cell type can synthesize and export IGF-I and IGF-II (see Fig. 1). The IGF regulatory system in each organ is tissue specific, but all share common components. IGFs circulate in a molar ratio of 2:1 (IGF-II:IGF-I) [6]. However, neither is. free, but rather is bound to a series of high-affinity IGF-specific binding proteins (IGFBPs), of

CHAPTE~ 18 IGF-I Axis and Menopause

273

1

IGFBPs {.4 .5)

[

J

IGFs

FIGURE 1 The circulatory IGF regulatory system is complex and composed of IGFs, IGF binding proteins (IGFBPs), and IGF receptors. The major circulatory IGFBP is IGFBP-3, but all the IGFBPs shuttle IGFs to and from the intravascular space. Growth hormone is the principal regulator of IGF-I synthesis in the liver.

which six have been fully characterized [14]. These binding proteins share about 50% sequence homology and contain highly conserved cysteine residues [14]. In serum, their concentrations range from 100 to 5000 ng/ml, and except for IGFBP-3 are relatively unsaturated [12-15]. A family of IGF-specific, low-affinity IGFBP-related proteins (IGFBrps) have been identified [16]. Their precise physiologic role has not been defined, although they possess the capacity to act on target cells independent of the IGFs. Serum IGFBPs hold IGFs within the circulation and in extracellular tissue as inactive peptides. IGFBP-3 and IGFBP-5 have extracellular matrix binding sites that provide alternative storage sites for both IGFs [15,17,18]. In bone, for example, IGFBP-5 has very strong affinity for the hydroxyapatite crystal and is the principal binding protein holding IGF-I and IGF-II within the matrix [ 17]. All but one of the IGFBPs, IGFBP-3, are small enough to translocate from the circulation into tissue, shuttling IGFs to or from cells. The IGFBPs clearly serve as a reservoir for the IGFs, but also can enhance or inhibit IGF activity, depending on tissue-specific factors. Although both IGFs are mitogens, IGF-II is much more active during prenatal life than is IGF-I. On the other hand, IGF-I is the principal regulator of linear growth. There is a wealth of information about the role of IGF-I in skeletal homeostasis, whereas much less is known about IGF-II despite its relative abundance. Both IGFs act as skeletal mitogens, and each can be activated by a series of cytokines, calcium analogs, or other growth factors. 2. I G F - I R AND IGFBP-PROTEASES

IGF receptors and IGFBP proteases comprise two other essential components of all tissue IGF regulatory systems. There are two extramembrane IGF receptors [19]. The type I IGF receptor (IGF-IR) is expressed ubiquitously and shares significant sequence homology with the insulin receptor [ 19].

It can bind insulin, IGF-I, or IGF-II. The presence of the type I receptor may confer special properties on the cell for several reasons. First, receptor binding to ligand can prevent programmed cell death or apoptosis [20]. Also, the presence of IGF-IR on the surface of some neoplastic cells may signify a more proliferative cell type. Third, interference with the type I IGF-IR can result in tumor cell death [20]. There are three different properties mediated by the IGFIR. These include mitogenicity, transforming activity, and antiapoptotic activity [20,21]. The type II IGF-II receptor (IGF-IIR) is structurally very different from the type I receptor and contains a mannose 6-phosphate binding site [ 19]. It does not bind insulin and preferentially binds IGF-II over IGF-I. Its precise role in cell growth is unclear, although it is a target for disposal of intracellular proteins [19]. Signaling from both IGF receptors occurs after ligand binding; this is followed by autophosphorylation of the receptor [19-21]. Two major substrates of the receptor, IRS-I and IRS-2, are phosphorylated and then interact with a number of src homology 2 (SH2) domain-containing proteins [ 19-22]. These interactions eventually lead to activation of downstream signaling proteins and kinases. The other unique component of the IGF regulatory system is a group of IGFBP-specific proteases. These proteases cleave intact IGFBPs, thereby altering binding of the IGFs to IGFBPs [23]. Some of them act only on certain tissues and are under the control of autocrine, paracrine, and hormonal influences. In particular, IGFBP tissue-specific proteases can act as comitogens by cleaving intact binding proteins into smaller fragments that bind the IGFs less avidly [24]. In addition, the smaller IGFBP fragments may actually act as agonists to enhance IGF bioactivity [25]. Finally, some of these proteases act on extracellular components, thereby permitting cells to penetrate organic matrices [26]. All these activities add to the layer of complexity surrounding a particular IGF regulatory system whether it be in bone or in the circulation. Much progress has been made in defining constituents of the skeletal IGF system and its regulation.

C. T h e S k e l e t a l I G F R e g u l a t o r y S y s t e m 1. IGF-I The IGFs are the most abundant growth factors in bone [ 17]. As in the circulation, IGF-II is present in much higher concentrations than IGF-I. Both decline with age in cortical and trabecular sites, although the relative ratio of IGF-II: IGF-I is preserved. In rodent bone and serum, IGF-I is the more abundant growth factor [27]. Both IGFs are stored in the skeletal matrix bound to IGFBP-5 and hydroxyapatite. Acid hydrolysis during bone resorption may be the mechanism whereby inert growth factors are activated (see Fig. 2). But active growth factors are also synthesized in the skeleton and are regulated by three major hormones: growth

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FIGURE 2 The skeletal IGF regulatory system is composed of ligands (IGF-I and IGF-II), IGF binding proteins (IGFBPs), IGFBP-specificproteases, and IGF receptors. At the onset of remodeling, osteoblasts are activated to secrete IGFs. With bone resorption, IGFs are released from the skeletal matrix and togetherwith osteoblast-synthesizedIGFs recruit new osteoblasts for bone formation.OC, Osteoclast; OB, osteoblast. hormone, parathyroid hormone (PTH), and 1,25-dihydroxyvitamin D [27,28]. Estadiol also can be a potent inhibitor of IGF-I synthesis. Within the IGF-I gene of rat osteoblasts, McCarthy and colleagues identified a cyclic AMP promoter element that is down-regulated by 17/3-estradiol [29]. This may have major significance in terms of gender responsiveness to anabolic factors, which work through IGF-I, and in understanding how estrogen may dampen stimuli that increase bone formation. IGF-II, the predominant IGF produced by human osteoblasts, is regulated by systemic hormones (e.g., progesterone and glucocorticoids) and local factors (e.g., electromechanical stimuli and BMP-7) [30]. IGF-I expression has also been noted in osteoclasts, and both IGFs can recruit premature osteoclasts to bone surfaces [31]. Undifferentiated osteoclasts possess an IGF-I receptor, although it is not clear whether there is an active autocrine IGF loop in differentiated osteoclasts. Based on these studies, there is strong evidence that IGF-I is a major coupling factor that permits bone remodeling to be balanced between resorption and formation. 2.

SKELETAL I G F B P s AND IGFBP PROTEASES

All six IGFBPs are expressed by bone cells. IGFBP-1, -2, -4, and -6 are inhibitory to skeletal cells under most circumstances, whereas IGFBP-3 and -5 are stimulatory [32]. IGFBP-5 is the first binding protein shown to have agonistic

effects on osteoblasts independent of the IGFs [33]. Growth hormone enhances IGFBP-3 and IGFBP-5 activity, whereas 1,25-dihydroxyvitamin D and PTH stimulate IGFBP-4 synthesis [33,34]. Insulin can cause suppression of IGFBP-1 production in bone cells, and glucocorticoids stimulate IGFBP-1 synthesis [35,36]. The IGFs can also regulate their own bioactivity. For example, IGF-I increases IGFBP-3 and IGFBP-5 tissue expression in bone and it simultaneously decreases IGFBP-4 production [27]. Within the skeleton, IGFBP-specific proteases, are produced as well as other proteases such as matrix metalloprotease, cathepsin D, and plasmin [25,36]. These enzymes are capable of breaking down different proteins as well as the IGFBPs. Complete characterization of IGFBP-specific proteases in bone has not been successful to date. In addition to the transport and modulation of IGFs by the IGFBPs, it is now apparent that some IGFBPs can have IGFindependent activity. For example, IGFBP-3 can be activated by the tumor suppressor protein, p53, which in turn can inhibit mitogenesis [37]. IGFBP-3 can also suppress breast cancer growth irrespective of IGF-I [37,38]. Similarly, IGFBPrp-1, a member of the new low-affinity IGFBP family (which may number four), has also been shown to have IGF-independent effects on breast cancer cells [16,39]. These properties suggest that this unique superfamily of IGFBPs exhibit other properties that could be tailored for certain therapeutic par-

275

CHAPTER 18 IGF-I Axis and Menopause adigms. For example, in the skeleton, agents that affect the IGFBPs are already being tested to target and enhance the bone-forming properties of IGF=I.

D. Regulation of Serum IGFs 1. GROWTrI HORMONE There is a dynamic equilibrium between circulating levels of IGF-I and tissue production of this peptide. However, caution must be exercised when interpreting changes in serum IGF-I as alterations in local tissue production. Indeed, there can be tremendous divergence in specific regulatory factors that affect hepatic synthesis versus those that control tissue production (Table I). But certain hormones are common determinants of IGF-I expression in most tissues. Since its discovery as a sulfation factor more than 40 years ago, IGF-I has been considered a mediator of growth hormone activity in bone [40]. In the skeleton, growth hormone stimulates osteoblast and chondrocyte production of IGF-I [41 ]. Osteoblasts also make IGFBP-3 in response to GH, and there is some in vitro evidence that IGFBP-4 production is enhanced by GH [27,32]. Serum concentrations of IGF-I reflect growth hormone secretion to a certain degree and therefore have been

TABLE I

Factors That Affect Circulating IGF-I Concentrations

Major direct determinants of circulating IGF-I levels Growth hormone Protein-calorie intake Catabolic stressors Illnesses Sepsis Trauma Anorexia/bulemia nervosa Thyroxine Insulin Binding affinityof the acid-labile subunit for IGFBP-3/IGF-I Indirect determinants that act through GH Aging Body fat (?leptin) Estrogens Androgens ?Adrenal androgens (e.g., DHEA) ?Inflammatorycytokines Exercise Other determinants that could directly affect IGF-I expression Zinc Parathyroid hormone (PTH) PTH-related peptide Estrogens Androgens Adrenal androgens Platelet-derived growth factor Inflammatory cytokines

used clinically as a surrogate indicator of GH status. Indeed, low serum IGF-I is found in growth hormone deficiency states of children and adults, whereas high levels of IGF-I are found in acromegaly [42]. In these disease states, serum values are bound to reflect skeletal content and activity, although there are no studies in humans with growth hormone deficiency or excess to prove that thesis. In rats and mice, alterations in serum IGF-I are also reflected in cortical bone content of IGF-I. 2. NUTRITION Although GH represents the principal hormonal regulator of circulating IGF-I, other determinants can affect IGF-I concentrations both in serum and at the tissue level. The nutrient status of an individual can profoundly affect serum IGF-I concentrations [43]. For example, protein-calorie malnutrition severely limits IGF-I synthesis in the liver and leads to a 50% reduction in circulating concentrations even among healthy volunteers [44]. Starvation is also associated with reduced bone formation and increased bone resorption. These changes are due to declining skeletal IGF-I concentrations as well as alterations in postreceptor GH action, a decreased number of GH receptors, and alterations in several IGFBPs [45]. Yet these effects occur despite a marked increase in GH production. Thus during protein calorie malnutrition, there is peripheral resistance to GH leading to dissociation between GH and IGF-I. As noted above, not only do serum levels of IGF-I decline dramatically, but the bioactivity of IGF-I is also markedly reduced by malnutrition. In part this may be related to a marked increase in IGFBP-1. Nutrient intake and insulin status both determine serum IGFBP-1 concentrations and the extent of phosphorylation of IGFBP-1, which in turn determines IGF binding affinity [46]. With starvation IGFBP-1 increases and binds IGF-I more avidly. This occurs because of a decline in substrate availability and suppressed insulin concentrations. Because IGFBP-1 is also synthesized by osteoblasts, it is conceivable that this IGFBP might contribute to the marked impairment in bone formation noted with starvation. Similarly, during chronic insulin deficiency, serum IGFBP-1 concentrations are high and this could lead to growth retardation in poorly controlled type I insulin-dependent diabetes [47]. Moreover, osteopenia and poor bone formation have been noted in IDDM, and this might be a function of high skeletal production of IGFBP-1. Another inhibitory IGFBP that is increased in some chronic diseases and could impact bone formation is IGFBP4. This binding protein is principally regulated in bone by PTH [48]. However, in one study, the highest levels of IGFBP-4 were found in elders who sustained a hip fracture and had undergone significant weight loss prior to their injury [49]. This would imply that there may be other regulatory factors associated with poor nutrition (e.g., cytokines) that could trigger local production of inhibitory IGFBPs. A

276 marked change in the bioactivity of IGF-I due to IGFBP perturbations may be the major cause of growth retardation in malnourished children. In addition to IGFBP changes, there is also evidence that zinc deficiency, a common accompaniment of protein-calorie malnutrition, can inhibit IGF-I synthesis in liver and bone [44]. Zinc repletion in experimental animals leads to increased hepatic IGF-I expression, although longitudinal studies in adults have not shown a direct rise in serum IGF-I due to zinc supplementation alone [44]. 3. AGE Other factors regulate circulating concentrations of IGFI, and these can affect the skeleton. Advanced age is associated with a progressive decrease in serum IGF-I as GH secretion declines approximately 14% per decade of life [43,5052]. Thus over a lifetime, GH production is reduced nearly 30-fold. This decrement is due to increased somatostatinergic tone and a generalized reduction in the pulses of GH releasing hormones and GH-releasing peptides [53]. Declining sex steroid production may also negatively impact the GH/IGF-I axis [54]. Alterations in body composition and specifically increases in visceral body fat can feed back negatively on the hypothalamic G H - G H R H axis, possibly via leptin [55]. The sum of altered GH secretion in the elderly includes low circulating values of serum IGF-I and IGFBP-3. Aging also affects circulating IGFBPs in both men and women. Serum IGFBP-4, an inhibitory binding protein, increases with advancing age in both men and women [32, 34,48]. On the other hand, values of IGFBP-3 and IGFBP-5 are much lower in older individuals than in younger ones [56]. There is evidence that serum IGFBP-1 concentrations are higher in the elderly than in younger people [57]. These changes in stimulatory and inhibitory IGFBPs are consistent with in vitro evidence that senescent cells have impaired cellular responsiveness to the IGFs [58]. In particular, a recent study demonstrated that osteoblasts from older patients are resistant to IGF-I stimulation [48]. Although these age-associated changes in IGFs could be lead to osteoporosis, there is still much debate about the role IGFs and IGFBPs play in respect to determining overall bone density and fracture risk. 4. SEX STEROIDS As noted above, gonadal steroids can have a profound effect on growth hormone secretion, thereby indirectly affecting serum IGF-I. However, estrogens and androgens can also regulate IGF-I at the tissue level independently of GH. For example, one consistent finding in regard to circulating IGF-I, whether measured during puberty or after menopause, is a striking gender difference that cannot be related to alterations in hypothalamic releasing peptides or GH secretion. Males exhibit a 10-15% higher serum IGF-I concentration than females across all ages after puberty, even though GH secretion is greater in females than males [59]. The cause for this gender difference is not clear, although it is known

CLIFFORD J. ROSEN

that estrogen can suppress IGF-I production in several tissues, including liver and bone. On the other hand, subacute estrogen deficiency associated with the peri- and menopausal state, is associated with a marked decline in serum IGF-I [59]. Several attempts have been made to link high or low serum concentrations of IGF-I to the pathogenesis of several chronic diseases, including osteoporosis, breast cancer, and Alzheimer's disease. One cause for an age-associated decline in serum IGF-I is reduced sex steroid production [60]. However, the picture is complex in part because both estrogen and testosterone can affect pituitary GH release as well as tissue IGF-I expression. There is strong evidence that total and free testosterone concentrations in serum correlate with GH secretory bursts in pubertal boys [61 ]. Also, administration of testosterone to younger men with hypogonadism, and boys with isolated gonadotropin-releasing hormone (GnRH) deficiency, leads to an increase in serum levels of IGF-I [62]. But the precise mechanism and site of action in the hypothalamus or pituitary are not known, in part because androgens are converted to estrogens via aromatization and this may positively affect GH secretion. Such a mechanism may be extremely important in respect to the aging skeleton, because new cross-sectional and longitudinal data demonstrate that total estradiol concentrations are a better predictor of bone mineral density in the elderly male than is serum testosterone [63]. Furthermore, osteoblasts possess the capacity to aromatize androgens to estrogens, thereby providing a local site for regulation [64]. And, in males with osteoporosis and deficient aromatase activity, exogenous estrogens, not androgens, partially restore bone mass [65,66]. Several lines of evidence suggest that there may be a strong causal relationship between endogenous estrogens and serum IGF-I in postmenopausal women. First, crosssectional studies have demonstrated that serum estradiol levels correlate with IGF-I in both men and women [67]. Second, both cross-sectional and now longitudinal data have demonstrated that serum IGF-I declines during the early menopausal years [68,69]. Third, several preliminary studies suggest that low serum IGF-I levels in elderly women are related more closely to years since menopause than to chronological age [70]. Adrenal androgens may also affect circulating IGF-I. For example, dehydroepiandrosterone sulfate (DHEA-S) levels decline with age and absolute levels in postmenopausal women correlate with serum IGF-I [71]. Similarly, in premenopausal women with adrenal androgen excess and insulin resistance, serum IGF-I levels are relatively high [71]. Also, in a randomized placebo-controlled trial of DHEA, serum IGF-I levels rose in both elderly men and women [72]. Finally, there is some preliminary evidence that in a subset of adolescent women with eating disorders, DHEA-S increases serum IGF-I [M.S. LeBoff, personal communication; 73]. Hence, some evidence supports the thesis that weak adrenal androgens may have a positive impact on serum and

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277

possibly skeletal IGF-I. There are no data on adrenal androgen action on the IGFBPs. Further randomized trials will have to determine if these compounds prevent bone resorption, stimulate bone formation, or both. Gonadal steroids regulate several IGFBPs as well as IGFI. Estrogen stimulates production of IGFBP-4 and inhibits osteoblastic IGFBP-3 synthesis [32,74]. On the other hand, testosterone stimulates IGFBP-3 synthesis and activates a critical IGFBP-3 protease, prostate-specific antigen [75]. Progesterone, PTH, dexamethasone, and 1,25-dihydroxyvitamin D also stimulate IGFBP-4 production in bone cells, although it is unclear precisely how these hormones impact skeletal activity of IGF-I [76]. However, it is certain that there are multiple layers of IGF regulation that could be impacted by alterations in gonadal steroids. 5. GENETIC CONTROL OF SERUM IGF-I

There is tremendous heterogeneity in serum IGF-I concentrations among healthy adults. Normal values range from 100 to 300 ng/ml, and although GH remains the major regulatory factor controlling serum concentrations, it is clear that other determinants are operative [77]. Indeed it is likely that the IGF-I phenotype is a continuous variable representing a complex polygenic trait. Hence, there should be some element of heritability for IGF-I expression that may or may not be controlled at the pituitary level. Commuzie et al. demonstrated among Mexican-Americans strong heritability for serum IGF-I [78]. In a preliminary cross-sectional motherdaughter pair study, serum IGF-I was found to correlate very closely despite a 30-year age difference [79]. Moreover, in recombinant inbred strains of mice, serum IGF-I and bone density correlate very closely and IGF-I cosegregates with bone mineral density (BMD) across two generation, suggesting strong heritability for IGF-I [80]. In a study by Rosen et al. it is reported that a polymorphism in a noncoding region upstream of the transcription start site of the IGF-I gene is associated with significantly lower levels of serum IGF-I in both men and women [81 ]. The reason for these differences with respect to this polymorphism is not clear, but there is preliminary evidence to suggest that estrogen may be important in this regard. For example, estrogen is known to bind to a cyclic AMP-inducible protein, thereby interfering with PTH-mediated IGF-I expression. This effect is not dependent on the estrogen response element (ERE) in the IGF-I gene. This site is very close to the polymorphic dinucleotide repeat associated with variable IGF-I levels. Thus, there may be several mechanisms whereby estrogen could affect IGF-I synthesis. These new lines of evidence also suggest there may be important genetic regulation of serum and tissue IGF-I expression. 6. OTHER REGULATORY FACTORS FOR

IGF-I

The major control over IGF-I synthesis in the liver is growth hormone. Nutritional determinants, possibly including zinc, regulate IGF-I message expression in the liver.

Heritable factors may also be important. Deficient gonadal steroids can affect serum IGF-I at the level of hepatic transcription or at the pituitary/hypothalamic level. Estrogen also enhances IGF-I production in the uterus. But, in addition to those factors, adequate insulin is a prerequisite for IGF-I expression in the liver [82,83]. Thyroxine has recently been shown to up-regulate IGF-I expression in rat femorae and this may explain the mechanism of hyperthyroidism-induced bone turnover [83]. PTH is a major stimulator of skeletal IGF-I expression in rat, mouse, and human bone cells [84]. This effect is most pronounced when it is administered intermittently. Anti-IGF-I antibodies block collagen biosynthesis and other anabolic properties induced by PTH [85]. Whether there is a gender difference in skeletal PTH responsiveness remains to be determined, although several preliminary studies suggest that male inbred strains of mice, as well as male growth hormone-deficient mice, have a more vigorous bone density response to PTH than do females [L. R. Donahue, personal communication]. In humans, there are no side-by-side gender studies with PTH to determine if this effect also occurs. However, it should be noted that the most vigorous skeletal response to PTH has been reported in men [86]. In conclusion, many factors affect serum IGF-I concentrations. These may be very important in defining specific disease states and their manifestations. In particular, gonadal steroids can have a major effect on the GH/IGF-I axis from "north to south," i.e., from the hypothalamic releasing factors all the way through to tissue-specific expression of IGFI. Evidence that estrogen can affect GH secretion and IGF-I expression supports the hypothesis that menopausal-related changes in several tissues could modify the risk of developing several chronic diseases.

III. DISEASES IN P O S T M E N O P A U S A L LIFE AND THEIR R E L A T I O N S H I P TO THE G H / I G F - I AXIS A. O v e r v i e w Three major diseases occur with higher frequency during the postmenopausal years. Breast cancer, osteoporosis, and heart disease are also the three major causes of death in women after age 50 years. Each of these disorders has multiple etiologic factors that contribute to the ultimate presentation, but estrogen plays a key pathogenetic role in all three conditions. For example, chronic estrogen deficiency is linked to low bone mineral density and an accelerated rate of atherogenesis. On the other hand, lifelong exposure to estrogen enhances a woman's risk for breast cancer. In each situation, IGF-I may be an important pathogenic pathway. In this section, a review of the interrelationship between IGF-I, gonadal steroids, and risk of disease is undertaken.

278 B. H e a r t D i s e a s e , I G F s , a n d G o n a d a l S t e r o i d s Numerous cohort and cross-sectional studies have demonstrated that estrogen replacement in the postmenopausal period reduces the risk of atherosclerotic disease [87]. However, there is a paucity of randomized placebo-controlled trials assessing the efficacy of ERT on cardiovascular risk. Still, studies using surrogate markers such as cholesterol, liproprotein a [Lp (a)], and HDL do suggest that estrogen replacement can enhance favorable cholesterol profiles [87, 88]. Similarly, oral HRT lowers serum IGF-I and IGFBP-3 [89], but the precise role of the GH/IGF-I axis in modulating estrogen's action on the vascular tree remains unclear. Independent of estrogen, the effects of reduced growth hormone secretion on the cardiovascular system have provoked considerable interest. It is well established that GH secretion declines by 10-15% per decade after age 50 years [60]. This drop coincides with a progressive increase in cardiovascular risk. Some completed long-term studies of GHD patients have shown there is a much higher risk of atherosclerosis and mortality from heart disease in these patients [90,91 ]. Chronic growth hormone replacement reduces total cholesterol and may decrease overall cardiovascular risk [92]. Therefore, as growth hormone secretion falls with advanced age, it is possible that atherogenesis is accelerated. Further support for that hypothesis relates to the marked changes in body composition that result from the aging process. For example, alterations in GH secretion can be related to intraabdominal fat and the waist-to-hip ratio, both powerful risk factors for subsequent heart disease [2]. Further longterm studies will be needed to define how latent GH deficiency in elderly individuals affects cardiovascular disease and whether rhGH "replacement" can affect risk profiles. Finally, the role of IGF-I in certain vascular events may shed light on the interrelationship of the IGFs to sex steroids. For example, IGF-I is a very potent mitogen for vascular smooth muscle proliferation and migration. Moreover, this peptide is present in very high concentrations within the circulation. Despite those facts, high levels of serum IGF-I have not been associated with a greater overall risk of cardiovascular disease. However, in a longitudinal study of atherosclerotic patients undergoing balloon coronary angioplasty for single-vessel disease, Craig et al. have demonstrated that the highest quartile of serum IGF-I was associated with a nearly twofold greater risk of coronary artery restenosis within 3 months of treatment [93]. Moreover, men tend to have greater rates of coronary restenosis than do women, and have higher serum IGF-I levels [93]. Also, somatostatin analogs have been shown to prevent coronary artery restenosis following angioplasty. Thus, one component of atherosclerotic risk may be related to the local effects of IGF-I. Once again, however, caution should be used in interpreting how systemic levels of IGF-I relate to the peptide's action at tissuespecific sites. Still, there is strong evidence that serum IGF-I

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is down-regulated by estrogen and therefore is likely to have an effect downstream. Further studies are needed to assess how IGF-I and estrogen interrelate in the atherogenic process.

C. O s t e o p o r o s i s Osteoporosis is defined as a decrease in bone mass that leads to an increase in skeletal fragility. In the United States alone, more than 20 million postmenopausal women have low bone mass and are at major risk for fracture. For over 50 years it has been known that estrogen deprivation is associated with hypercalcuria and fractures [94]. Moreover, it is widely accepted that estrogen replacement in menopausal women can prevent bone loss. Estrogen deprivation during menopause leads to a cascade of events in the bone remodeling unit, provoking an increase in bone resorption. This sequence involves several cytokines that are produced by osteoblasts and serve to recruit osteoclasts to bone surfaces. Although the GH/IGF-I axis is not directly implicated in postmenopausal bone loss due to estrogen deprivation, it is likely that gonadal steroids influence both serum and skeletal bioavailability of the IGFs. In order to understand this process more completely, it is essential to define how bone mass is developed. Two processes are involved in the final determination of adult bone mass at any age: (1) peak bone mass acquisition and (2) rate of bone loss. The high prevalence of osteoporosis in women and the evidence that estrogen replacement prevents bone loss suggest that the latter process (i.e., bone loss) is the major feature of low bone mass. However, in recent years, the importance of peak bone mass has taken on new meaning. 1. PEAK BONE MASS AND ITS RELATIONSHIP TO ESTROGEN AND I G F s

Peak bone mass is acquired by the end of the second decade and remains one of the most important factors in determining adult bone density at any point in an individual's life [95]. Longitudinal studies with DXA suggest that the most rapid acquisition phase for bone is between the ages of 12 and 16 years, a time when linear bone growth is just beginning to deaccelerate [96]. Coincident with several hormonal surges (e.g., estrogens and androgens) at that time, GH pulses are more frequent and of greater magnitude [97, 98]. Serum IGF-I levels are also at their highest point in life at this time [99]. Hence, ascertaining the role of IGF-I in acquisition of peak bone mass has become a major thrust for investigators. Several lines of evidence support the importance of IGFI in the process of peak bone mass acquisition. First, growthhormone-deficient (but otherwise healthy) mice (lit~lit) have reduced volumetric bone mineral density throughout life as a result of markedly reduced serum IGF-I concentrations [100]. Similarly, male and female adolescents with acquired

CHAPTER 18 IGF-I Axis and Menopause GH deficiency have lower peak bone mass than do agematched normals [101]. Second, normal inbred strains of mice have differences in bone density that correspond to similar differences in serum and skeletal IGF-I [85]. Inbred strains acquire peak bone mass by 16 weeks of age, a time when large interstrain differences in serum IGF-I are first noted [102]. Also, osteoblasts from high-density mice express more IGF-I than do cells from low-density mice. Third, the acquisition of bone mineral density in adolescence corresponds to a similar rise in serum IGF-I [103]. Fourth, in a longitudinal trial with young girls (ages 11-12), milk supplementation increased bone density more than placebo, and the rise in B MD corresponded to a greater increase in serum IGF-I than in the nontreated group [ 104]. Finally, surges in sex steroids (both androgens and estrogens) promote peak bone mass acquisition and also enhance GH release from the pituitary, thereby increasing serum IGF-I [105]. Although these studies indirectly suggest that IGF-I and peak BMD are closely related, they do not establish cause and effect. Furthermore, the mechanism whereby peak bone mass is enhanced by IGF-I has not been fully elucidated. For example, the highest bone mass in mice is noted in strains with reduced bone resorption [D. J. Baylink, personal communication]. Similarly, African-American males and females have higher bone density than do Caucasians, but this may be a function of slower bone turnover rather than an increase in bone formation. Peak bone mass in adolescence is associated with a marked increase in the size of the skeleton as well as incremental changes in mineralization. There is a strong gender effect on size and this could affect measurement of peak bone mass and future fracture risk. Using volumetric bone density measurements rather than two-dimensional area determinations, investigators have noted that much of the male-female difference in apparent BMD disappears, except in the vertebrae, where males continue to have greater true volumetric density [106]. Because IGF-I promotes periosteal growth, and boys have higher serum IGF-I levels than girls, it is conceivable that IGF-I could affect size, which in turn could affect fracture risk. However, in studies of adult patients with Laron dwarfism (i.e., growth hormone resistance syndrome), area BMD measurements were much lower than age- and sex-matched controls, but volumetric determinations failed to reveal differences between the two groups [R. Rosenfeld, personal communication]. These findings would suggest that IGF-I is not the only factor responsible for peak bone mass acquisition. Clearly, further studies will be needed to define how IGF-I affects peak bone mass. 2. IGF-I, ESTROGENS,ANDBONE L o s s Several cross-sectional studies have demonstrated a correlation between serum IGF-I and bone density across a wide age range in men and women [107-109]. In one study, serum and skeletal IGF-I levels showed a dramatic age-related de-

279 cline that could be superimposed on an age-related drop in bone density [108]. In addition to the IGF changes, there are data to suggest that IGFBP-3 may affect bone mass in healthy and osteoporotic males [110-112]. However, despite their strengths, all these studies suffer from the absence of longitudinal data relating bone density, bone loss, or fracture risk to IGF-I. As noted previously, numerous factors affect serum IGF-I, and some of these are the same determinants of age-related bone loss. For example, declining estrogen production in men and women has been linked to low bone mass in males and females, as well as to a decline in serum IGF-I [63,67]. The nutrient status of individuals can have a profound effect on serum IGF-I, and malnourished elders, who are more likely to fall, to have low serum vitamin D levels, and to have suffered recent weight loss, are independent predictors of fracture risk. Prospective data on fracture risk and the IGFs are even less convincing although a pattern has recently emerged. Three large cohorts have been analyzed for the relationship of IGF-I to bone density. In the Study of Osteoporotic Fractures (SOF), a cohort of more than 9000 women, the lowest quartile of IGF-I has been associated with a significant and independent risk for hip fracture (RR = 1.7/1.12.2) [D. Bauer, personal communication]. This finding might be considered intuitive based on the realization that serum IGF-I integrates several coincidental processes that produce a catabolic state (e.g., acute illness, starvation, immobility, and age) as well as predicting risk for fracture and frailty. Since SOF is a prospective observational study, it adds more strength to the thesis that IGF-I may be related to osteoporosis. In addition to the relationship of IGF-I to bone density, another study demonstrated that the lowest levels of estradiol were associated with the lowest BMD and greatest risk of fracture [113]. Whether this effect is partially mediated through IGF-I is not known. In another longitudinal observational study, the Framingham Heart Study, it was recently noted that the lowest quartile of IGF-I was associated with the lowest BMD at multiple skeletal sites, even when adjusted for covariables such as body weight and protein intake [D. Kiel, personal communication]. However the relationship of IGF-I to bone mass only held for women, not men. In the Rancho Bernardo cohort, serum levels of IGF-I were also higher in men than in women, and bone density was also greater at all sites in males than females [67].. However, as noted previously, these studies cannot prove cause and effect. Men with the syndrome of idiopathic osteoporosis, a unique subset of osteoporotic patients, have undergone careful reexamination. These individuals are middle aged but have very low bone mineral density and suffer from debilitating spinal fractures. A small percentage of these men have hypercalcuria but the majority have no identifiable cause for their disease. In 1992, Johansson et al. reported that these men had low serum IGF-I concentrations [114]. Subsequently, Reed et al. also noted low serum IGF-I and

280 hypercalcuria in males with this syndrome [115]. On bone biopsy, these men were found to have low bone turnover. More recently, Kurland et al. reported on 25 males with idiopathic osteoporosis who had low serum IGF-I levels and reduced bone formation on bone biopsy [116]. New studies in this cohort revealed that growth hormone dynamics were normal [117], Ebeling et al., in a preliminary study of men with a similar phenotype, noted that first-degree male relatives of those subjects had low spine bone mineral density and increased body fat [118]. These findings suggest that IGF-I may play a pathophysiologic role in maintenance of adult bone density or acquisition of peak bone mass. That thesis was reinforced by a study of men with idiopathic osteoporosis, in which a homozygous polymorphism in a noncoding region of the IGF-I gene was twice as common in affected men than in those in the general population [81]. This genotype, labeled 192/192, is also associated with 1 5 20% lower serum IGF-I levels than any other combination of alleles. But even more germane to menopause and this discussion is the increasing realization, from studies in families with mutations of the estrogen receptor or the aromatase gene, that one of the major determinants of bone mass in men is serum estradiol. In some studies, the correlation between estradiol and bone density in men is quite strong [119]. This relationship may be mediated through IGF-I, thus providing another link between the GH/IGF-I axis and estrogen. In sum, there are several lines of evidence, some direct, some indirect, that serum IGF-I may be related to bone mineral density and that it may be regulated by gonadal steroids. Whether serum IGF-I can be utilized as a diagnostic tool in males for osteoporosis remains to be determined. 3. INTERVENTION TRIALS WITH rhGH AND rhlGF-I IN POSTMENOPAUSAL OSTEOPOROSIS

Low bone mineral density as a result of chronic growth hormone deficiency in adulthood can lead to osteoporotic fractures [120]. Recently, the United States Food and Drug Administration approved the use of recombinant human GH (rhGH) for growth hormone deficiency (GHD) in adults. In part, this indication was based on compelling data from the United States and Europe that rhGH treatment for GHD increases BMD at several skeletal sites after 2 years of treatment [ 120,121 ]. These effects are more pronounced in males than in females, although this may be related to hormonal replacement with estrogens, which could dampen both the IGF-I response to GH and skeletal activation of remodeling (see later). No trials have shown that rhGH can increase bone mass in the elderly to the extent noted in GHD, even though both skeletal and serum IGF-I levels are low initially and are increased by exogenous administration [96]. The reasons for the disparate response between young and old skeletons to rhGH or rhlGF-I are not entirely clear, although studies sug-

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gest that low levels of IGF-I may not be the only pathogenic mechanism in age-related osteoporosis. Originally, Rudman et al. reported a 1.6% increase in lumbar BMD following 6 months of rhGH treatment to elderly males with low serum IGF-I [122]. These men were not randomized to treatment or placebo but were selected for very low levels of IGF-I. Subsequent follow-up of that cohort failed to show a significant effect of rhGH on the spine, hip, or total body B MD [ 123]. Other short-term studies have also been unable to show a strong positive effect from rhGH treatment on bone mineral density, even though markers of bone turnover rise [124-127]. Similarly, Holloway et al. could not establish a benefit from rhGH treatment alone that was greater than treatment with the antiresorptive medication, calcitonin [ 127]. Furthermore, MacLean et al. reported that total body B MD decreased after 1 year of low-dose rhGH in elderly men and women who were classified as frail by indices of physical performance [128]. Moreover, there was no correlation between serum IGF-I and measures of physical performance in that same cohort either at baseline or after treatment [D. Kiel, personal communication]. It is even more notable that the results of these studies were negative despite consistent and significant increases of serum IGF-I into the young normal range. Taken together, these data would suggest that IGF-I deficiency was not the pathogenic factor in age-related osteoporosis, or that other factors, including the IGFBPs, limit the bioactivity of IGF-I in the skeleton of older individuals. However, it should be noted that long-term studies with rhGH or rhlGF-I in elderly individuals have not gone beyond 24 months. Based on previous GH trials, it might take several years to see a beneficial effect from growth factor treatment, especially in respect to the aging skeleton [120,121]. In addition, it will be critical to assess the effects of gender on skeletal responsiveness, especially in elderly individuals The absence of an anabolic skeletal effect in the elderly has not deterred investigations with rhlGF-I and IGF-I/ IGFBP-3 as antiosteoporotic treatments. Ebeling et al. investigated several doses of rhlGF-I in younger postmenopausal women and found that bone turnover was stimulated and the lowest dose of rhlGF-I could increase bone formation more than resorption [129]. Few side effects were noted with rhlGF-I at doses of 30 and 60/zg/kg/day [ 129]. Ghiron et al. administered low-dose IGF-I (15/xg/kg/day, twice daily) to elderly women and found a selective increase in bone formation without changes in bone resorption [ 126]. These data suggest that rhlGF-I in low doses may have an effect on bone turnover, and potentially (although not measured in the Ghiron study) on BMD. The findings by Ghiron et al. are also consistent with short-term studies by Grinspoon and colleagues in young fasting women [45]. In those studies, much higher doses of rhlGF-I were well tolerated and produced an increase in bone formation that exceeded bone resorption. These effects were pronounced considering that those

CHAPTER 18 IGF-I Axis and Menopause women exhibited a decline in serum IGF-I and a rise in IGFBP-1, another inhibitory IGF binding protein [45]. Serum levels of IGFBP-3 are reduced in some osteoporotic patients, and because of the concern about hypoglycemia during rhlGF-I treatment, an alternative approach for using rhlGF-I in age-related osteoporosis has emerged. IGF-I complexed to IGFBP-3 and administered daily as a soluble complex subcutaneously has been shown to increase serum IGF-I concentrations markedly in the young and elderly without serious adverse effects. Based on earlier animal studies, the IGF-I/IGFBP-3 complex can strongly enhance bone formation and bone mass [130]. Dose studies using IGF-I/IGFBP-3 complex (0.3-6.0 mg/kg) in young volunteers and healthy elderly adults has shown that this agent is safe and well tolerated [D. Rosen, personal communication]. No episodes of hypoglycemia were noted, even at high doses of complex. Similarly, in a phase I trial, 7 consecutive days of rhlGF-I/IGFBP-3 at doses of 0.5-2.0 mg/kg/day by continuous subcutaneous infusion via minipump produced no serious side effects to healthy elders. Furthermore, procollagen peptide (a marker of bone formation) increased 50% over the 7-day period and remained elevated for an additional 7 days after discontinuation of treatment [D. Rosen, personal communication]. Despite a concomitant rise in deoxypyridinoline with complex administration, this rise did not persist posttreatment. Thus, this form of IGF-I administration could have utility in patients with osteoporosis. In conclusion, the evidence is overwhelming that rhGH treatment, which restores IGF-I levels to young normal ranges in growth hormone-deficient adults, leads to a substantial increase in bone mineral density in those patients. This effect seems even more pronounced in males than in females, although the reason for this is at present not clear. On the other hand, elders treated with rhGH or rhlGF-I do not demonstrate an increase in bone mass after 1 year of therapy. This suggests that although IGF-I may be important in the acquisition of peak bone mass, age-related osteoporosis is not solely a function of reduced GH secretion or deficient serum or skeletal IGF-I.

D. B r e a s t C a n c e r , I G F s , a n d S e x S t e r o i d s Although heart disease claims more lives, breast cancer remains the most feared disorder of postmenopausal life. It is beyond the scope of this chapter to define the multiple factors that contribute to breast cancer risk. However, long-term estrogen replacement has been associated with a 30% greater risk of cancer (see Chapter 40). Moreover, lifetime estrogen exposure may be an independent risk for subsequent mammary tumor. The relationship of the GH/IGF axis to breast cancer growth and metastases has been studied for many years. Indeed, in the late 1960s and 1970s, hypophysectomy was utilized to reduce further metastases in women with ad-

281 vanced disease. IGF-I receptor expression has also been associated with a poor prognosis in patients with breast cancer. More recently, evidence has emerged to suggest that IGF-I may be important as an indicator of disease risk and in the pathogenesis of neoplastic growth. Although it has been known for several decades that acromegalics are at higher risk for colonic neoplasms, the association between serum IGF-I and cancer risk has taken on a new and potentially very important dimension. For the past half-decade, evidence has gradually emerged that IGF-I may be important in epithelial cell growth and transformation. Most of that work focused on breast cancer, although IGF-I has long been known to be central for prostatic growth and proliferation. The publication of a large prospective study that identified a relationship between circulating IGF-I and future breast cancer risk has tremendously heightened interest in the causality between IGF-I and tumor growth. In this study, women in the Nurses Health Study were examined prospectively using serum IGF-I levels to assess risk of subsequent breast cancer. Hankinson et al. noted that among premenopausal women less than age 50 years, there was a 4.5 relative risk of breast cancer in the highest quartile of plasma IGF-I compared to the lowest quartile [131 ]. Moreover, adjustment for IGFBP-3 increased the predictive value of IGF-I in this study [131]. Indeed IGFBP-3 was shown to be inversely related to risk, whereas IGF-I was positively related to risk. Other studies, some of which have utilized antineoplastic drugs, have provided further indirect evidence that the IGF regulatory system is involved in the pathogenesis of mammary neoplasia. For example, it has been shown that fenretinide, a synthetic retinoid with antitumor efficacy, reduced plasma IGF-I levels and increased IGFBP-3 concentrations, especially among premenopausal women [132]. Also, tamoxifen has been shown to down-regulate IGF-I induction of tyrosine phosphorylation of the IGF-I receptor and inhibited IRS-1 signaling in MCF-7 cells [133]. Dunn et al., utilizing a p53-deficient mouse model, demonstrated that diet reduction lowered serum IGF-I by one-fourth, increased apoptosis, and decreased tumor progression [134]. Furthermore, IGF-I administration to these diet-restricted mice increased cell proliferation sixfold. When rhGH, rhGH + IGF-I, or saline was administered for 7 weeks to aged rhesus monkey there was a marked increase in mammary glandular size and epithelial proliferation index, especially in the GH/IGF-I treatment group [135]. There was also a direct correlation in that study between serum levels of IGF-I and overall mitogenic effects. Clarke et al. have noted that 14 days of estradiol treatment to nude athymic mice with human breast xenografts results in a threefold up-regulation of IGFIR mRNA, whereas treatment with progesterone results in a 50% reduction in receptor mRNA [136]. Taken together, these experimental data suggest that the IGF-I system is involved in tumor development and progression. Despite these indirect lines of evidence, several concerns about causality

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must be considered. These include the overall duration of tissue exposure to a specific level of IGF-I, the marked rise in relative risk despite only modest increases in serum IGF-I, and the precise circulatory-to-tissue relationship that would permit extrapolation of risk to a much larger population. These mitigating factors will need to be explored in greater detail before causality is established.

IV. S U M M A R Y There is now very strong evidence that growth hormone and insulin-like growth factor-I are essential for maintaining homeostatic balance in the skeleton and other tissues over the lifetime of an individual. IGFs are one of several coupling factors that link bone resorption to bone formation, thereby maintaining adult bone mass. IGF-I also mediates linear growth and acts in an endocrine manner to modulate growth hormone activity in adolescence and during most stages of adult life. More importantly, emerging data suggest that IGF-I expression is heritable, and that the absolute levels of serum IGF-I can affect the expression of several disorders of the menopausal period. On balance, there can be little debate that the IGFs are essential growth factors for the proper function of many tissues. However, more longitudinal studies will be needed to confirm causal relationships between changes in circulating IGF-I and bone mineral density, breast cancer, and heart disease. The therapeutic role of GH in modulating changes in postmenopausal tissues also remains to be determined. But, there are clearly effects from HRT that are mediated through the IGF regulatory system. Hence, the interaction between gonadal steroids and the growth hormone IGF-I axis will continue to remain an area of intense investigations.

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men improves body composition but not functional activity. Ann. Intern. Med. 124, 708-716. Thompson, J. L., Butterfield, G. E., and Marcus, R. (1995). The effects of recombinant rhlGF-I and GH on body composition in elderly women. J. Clin. Endocrinol. Metab. 80, 1845-1852. Ghiron, L., Thompson, J., Hallloway, L., Hintz, R. L., Butterfield, G., Hoffman, A., and Marcus, R. (1995). Effects of rhlGF-I and GH on bone turnover in ederly women. J. Bone Miner. Res. 10, 1844-1877. Holloway, L., Kohlmeier, L., Kent, K., and Marcus, R. (1997). Skeletal effects of cyclic recombinant human GH and salmon calcitonin in osteopenic postmenopausal women. J. Clin. Endocrinol. 82,1111-1117. MacLean, D., Kiel, D. P., and Rosen, C.J. (1995). Low dose rhGH for frail elders stimulates bone turnover in a dose dependent manner. J. Bone Miner. Res. 10, (Suppl.1), 458. Ebeling, P., Jones, J., O'Fallon, W., Janess, C., and Riggs, B. L. (1993). Short term effects of recombinant IGF-I on bone turnover in normal women. J. Clin. Endocrinol. Metab. 77, 1384-1387. Bagi, C. M., Brommage, R., DeLeon, L., Adams, S., Rosen, D., and Sommer, A. (1994). Benefit of systemically administered rh IGF-I/ IGFBP-3 on cancellous bone in ovariectomized rats. J. Bone Miner Res. 9, 1301-1305. Hankinson, S. E. et al. (1998). Circulating concentrations of IGF-I and the risk of breast cancer. Lancet 351, 1593-1596. Torrisi, R. et al. (1998). Effect of fenretinide on plasma IGF-I and IGFBP-3 in early breast cancer patients. Int. J. Cancer 76, 787-790. Guvakova, M. A., and Surmacz, E. (1997). Tamoxifen interferes with the IGF-I receptor signaling pathway in breast cancer cells. Cancer Res. 57, 2606-2610. Dunn, S. E. et al. (1997). Dietary restriction reduces IGF-I levels which modulates apoptosis, cell proliferation and tumor progression in p53 deficient mice. Cancer Res. 57, 4667-4672. Ng, S. T., Zhou, J., Adesanya, O. O., Wang, J., LeRoith, D., and Bondy, C. A. (1997). Growth hormone treatment induces mammary gland hyperplasia in aging primates. Na. Med. 3,(10), 1141-1144. Clarke, R. B., Howell, A., and Anderson, E. (1997). Type I IGF receptor gene expression in normal human breast tissue treated with estrogen and progesterone. Br. J. Cancer 75, 251-257.

--, ( _ H A P T E R l~

Bone and Calcium RICHARD L. PRINCE AND CHRISTINE DRAPER Department of Medicine, University of Western Australia, and Department of Endocrinology and Diabetes, Sir Charles Gairdner Hospital, Nedlands, Western Australia 6009, Australia

I. Biology of Ovarian Hormone Effects on the Skeleton II. Review of Bone Structure and Function III. Integration of Bone Cell Activity

IV. Estrogen Effects on Epithelial Calcium Transport V. Estrogen and the Physiology of Calcium Homeostasis References

chanical role in locomotion and its physiological role in the extracellular homeostasis of calcium and acid-base balance. Preservation of a functionally strong skeleton before and during pregnancy and lactation is critically important in reproductive success, because fracture in childhood and young adult life may impair reproductive ability. Pregnancy is a high estrogen state not associated with significant bone loss [1]. Lactation, on the other hand, is a low estrogen state associated with a very significant reduction in bone mass, at the end of which bone mass rises markedly, as do estrogen concentrations [2]. Thus it is possible that the important effects that estrogen has on bone and calcium balance have their origins in regulation of the supply of calcium to the fetus and infant. Estrogen also plays a role in the restoration of the bone calcium supply for the next child, and in doing so preserves the mechanical strength of the skeleton. In performing this task estrogen has direct effects on the three principal organs of calcium and phosphate homeostasis--the bone, the gut, and the kidney. Thus estrogen has a spectrum of action similar to that of calcitriol, a well-recognized calciotropic hormone. An obvious difference is that estrogen production is not directly regulated by the extracellular calcium concentration. Rather, by direct effects on tissues regulating calcium transport, estrogen directs the activity of the other calcitropic hormones that secondarily maintain calcium homeostasis. In this way the effects

I. B I O L O G Y OF O V A R I A N H O R M O N E E F F E C T S ON T H E S K E L E T O N Menopause, whether normal or premature, plays an important role in the development of bone disease. The biological basis for the bone loss occurring after estrogen removal is the subject of this chapter. Of the two major ovarian hormones, estrogen and progesterone, estrogen has been considered to have the major role. Why the reproductive hormones have such an important effect on the bone and calcium system is unclear. Two broad groups of action must be distinguished: first, a role in the developing skeleton, and second, a role in the mature skeleton. Estrogen plays a vital role at puberty to develop a mature skeleton. Normal estrogen action includes direct stimulation of chondrocyte activity through a receptor-mediated effect. This is followed by fusion of the growth plate, probably associated with apoptosis. The development of a strong skeletal structure during growth ("peak bone mass") is an important basis for the reduction of fracture propensity in old age. In the adult skeleton, in keeping with their central role in reproduction, the physiological effects of reproductive hormones could be seen in Darwinian terms as aiming to optimize the chances of reproductive success. In skeletal terms this relates to the two principal roles of the skeleton, its me-

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Copyright9 2000by AcademicPress. All rightsof reproductionin any formreserved.

288 of estrogen on bone mineral metabolism may be aimed at skeletal maintenance during reproductive life. For over 50 years it has been considered that there are two major processes contributing to bone loss in women: postmenopausal or estrogen-deficiency-related osteoporosis, usually considered to be important within 10 years of menopause, and age-related osteoporosis. Nordin et al. calculated the theoretical contributions of the two processes to the observed bone loss and concluded that postmenopausal bone loss may account for the loss of 12% of the skeleton, whereas age-related bone loss may account for loss of 20% of the skeleton [3]. It is of course a simplification to separate the etiology of bone loss in women into two categories, because there is no clear cutoff between the two processes. Indeed, in recent years it has become clear that the skeletal effects of age are independent and additive to those of estrogen deficiency. Estrogen replacement is the best recognized treatment for the prevention of bone loss (Fig. 1). Even the low concentrations of endogenous estrogen found in many postmenopausal women play an important role in the prevention of fracture [4]. It is vital to understand the mechanism whereby estrogen and estrogen-like agents prevent bone loss. Two main concepts need to be addressed when considering the cause of the bone loss following estrogen deficiency. The first is the mechanism of the direct effect of estrogen deficiency on bone loss. The principal mechanism involved here is the increase in bone remodeling that occurs on all trabecular and endocortical bone surfaces consequent to estrogen deficiency. If it continues long enough this remodel-

FIGURE 1 The effects over 2 years of estrogen and progesterone (piperazine estrone sulfate, 1.25 mg; medroxyprogesterone acetate 2.5 mg) compared to calcium supplementation (calcium lactate gluconate, 1 g) and placebo tablets at the distal forearm. Hormone therapy was clearly most effective. Calcium supplementation, even in these women averaging 5 years past the menopause, clearly prevented bone loss at this site. This indicates that calcium balance plays an important role in estrogen-related bone loss. From Ref. 94, Prince et al. (1991). Copyright 9 1991 Massachusetts Medical Society. All rights reserved.

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ing always results in bone loss due to a relative increase in osteoclastic bone resorption compared to osteoblastic bone formation. The second concept is the role of estrogen in maintaining calcium balance due to direct effects on the kidney and possibly the intestine. Loss of these actions results in a negative calcium balance that stimulates increased bone turnover via mechanisms that include parathyroid hormone (PTH):mediated bone resorption. The increase in osteoclastic bone remodeling induced by the calcium deficit also resuits in bone loss that is partially correctable by calcium supplementation. It is the triple effect of estrogen deficiency on bone, kidney, and intestine that largely accounts for postmenopausal and age-related osteoporosis [5]. These various mechanisms will be elucidated further in this chapter. In skeletal terms the menopause serves no beneficial biological function; indeed, the protective effect of estrogen on the skeleton is lost, resulting in a pathological degree of bone fragility. Osteoporotic fracture, that is, fracture occurring after minimal trauma, will affect nearly half the postmenopausal female population [6]. Nevertheless, the increase in life expectancy is such that at least in Western societies over one-third of women's lives could be lived in an estrogen-deficient state. This massive demographic change accounts for the increased importance of the skeletal biology of the menopause in the past 30 years.

II. R E V I E W OF B O N E S T R U C T U R E AND FUNCTION Adult bone is a dynamic tissue consisting of a mineralized extracellular matrix and mesenchymal-derived osteogenic and hemopoietic cells. Bone serves a wide range of functions. It provides protection for the brain and spinal cord, it is a site for hematopoiesis and for attachments of muscles that act as levers for movement, and it plays a role in calcium and acid-base homeostasis. The bone matrix has organic (35%) and inorganic (65%) components. The inorganic component consists primarily of calcium and phosphate, deposited as hydroxyapatite. Bone is categorized into two types, trabecular or cancellous bone and cortical bone. Cortical bone is arranged in Haversian systems. These are the product of remodeling events within bone, resulting in concentric deposits of lamellar bone with central nutrient blood vessels. Cortical bone has a higher true volumetric density than does trabecular bone, whereas trabecular bone has a larger surface area per unit volume and a greater rate of metabolic activity. Because of its larger surface area, trabecular bone is more labile. The cells contained in bone derive from two lineages. Osteoblasts and osteocytes are derived from mesenchymal precursors whereas osteoclasts are derived from a hematopoietic cell lineage.

CHAPTER 19 Bone and Calcium A. O s t e o b l a s t s The principal role for cells of the osteoblast lineage is bone formation, achieved through direct matrix synthesis. Another major role for these cells is the modulation of bone resorption through the regulation of osteoclast activity. Preosteoblasts, the precursors of mature osteoblasts, derive from pluripotential stem cells in the marrow stroma that can give rise to a variety of tissues, including muscle, cartilage, adipose tissue, and fibrous tissue [7]. Osteoblasts are cuboidal, lie on the matrix surface, and are connected by gap junctions. They are responsible for the synthesis of the bone matrix, in particular collagen, on which hydroxyapatite is deposited.

B. O s t e o c y t e s During the formation of bone, osteoblasts that are encased in the bone matrix become osteocytes. They lie in and completely fill the lacunae of the bone and are connected by cellular processes and gap junctions to each other and to osteoblasts. The cellular processes are packed with microfilaments. During differentiation from osteoblasts the cell loses many of the osteoblastic organelles. It is thought that osteocytes are involved in ion exchange and in signaling. They may influence the activity of osteoblasts, osteoclasts, and bone lining cells in particular in relation to mechanical stress effects on the skeleton as a result of movement. Osteocytes have been shown to produce transforming growth factor fi (TGF-fi) and possibly other cytokines.

C. B o n e L i n i n g Cells Osteoblasts may also differentiate into bone lining cells. Bone lining cells are flattened cells that have lost their synthetic capacity. They are separated from mineralized bone by a very thin fibrous tissue layer. The lining cells maintain this layer, which prevents osteoclast access to the bone surface. Lining cells may also secrete collagenases that remove the surface layer of fibrous tissue, thus permitting the osteoclasts access to the bone surface in order to commence resorption.

D. M a t r i x P r o t e i n s Many bone proteins are secreted by bone cells. However, not all matrix proteins are synthesized by osteoblasts; for example, albumin and ce2HS-glycoprotein are derived from the blood. The organic component of the bone matrix consists primarily of type I collagen (90%), which gives the bone ten-

289 sile strength. Other important proteins include glycoproteins such as osteonectin and alkaline phosphatase; glycoaminoglycans (decorin, biglycan, and fibromodulin); T-carboxyglutamic acid (gla)-containing proteins osteocalcin and matrix gla protein; and the integrin-binding family of proteins such as vitronectin, fibronectin, osteopontin, and bone sialoprotein [8]. Many of these proteins are attached to collagen fibrils and have a role in structural organization.

E. O s t e o c l a s t s The principal function of the osteoclast is bone resorption. Osteoclasts are derived from the hematopoietic granulocyte-macrophage colony-forming unit (GM-CFU), which is also the stem cell for monocytes and macrophages [9]. They are found on endosteal bone surfaces, in Haversian systems, and occasionally on periosteal bone surfaces. When resorbing bone the osteoclast is highly polarized and contains several different plasma membrane domains. Osteoclasts are large multinucleated cells, containing from two to several hundred nuclei, a large number of mitochondria, electron-dense secretory granules, a well-developed Golgi apparatus, and a ruffled border. Next to the ruffled cell border is a clear zone, which is rich in actin filaments and free from organelles [10].

III. INTEGRATION OF BONE CELL ACTIVITY The structure of the mature adult skeleton is the result of four different cellular mechanisms. These are endochondral and intramembranous ossification together with modeling and remodeling on preformed surfaces. The interplay of these mechanisms determines the form of the adult skeleton. Estrogen plays an important role in these mechanisms.

A. B o n e R e m o d e l i n g Bone remodeling involves osteoclast-mediated bone resorption coupled to and followed by osteoblast mediatedbone formation occurring in the same area [ 11 ], so that the original shape of the trabecula or cortex is reestablished (Fig. 2, see color plate). The process occurs on trabecular surfaces and within cortical bone as a cutting cone. When it occurs on the endosteal surface of cortical bone it may result in the development of trabecular structures in a process known as endocortical trabecularization. The discrete locations at which bone remodeling occurs are referred to as bone remodeling units and consist of an osteoclast-derived

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pit and active osteoblasts that replace the resorbed bone. The rate of turnover is determined by the number of active bone remodeling units at any given time. In the adult skeleton approximately 10% of the skeleton is remodeled each year. Although trabecular bone accounts for only 20% of the total skeleton, it accounts for 50% of bone remodeling. The biological rationale for this apparently rather pointless exercise has been debated. Two possibilities have been suggested: first, it may be necessary to replace bone damaged by mechanical stress so that bone structure remains strong. Second, it is clear that bone remodeling is an important part of the regulation of extracellular homeostasis. Thus bone remodeling is one of the most important biological processes that occur in bone, supporting as it does the two major biological roles of the mature skeleton. Furthermore, it is the principal mechanism by which bone is lost or gained in the adult skeleton. 1. BONE RESORPTION

Bone resorption is initiated by the migration of osteoclasts and osteoclast precursors to a specific site and the retraction of bone lining cells at that site to expose the bone surface. The osteoclasts fuse to the bone surface to form bone-resorbing osteoclasts. This attachment is achieved by interactions between integrins and matrix proteins containing the RGD amino acid sequences (arginine-glycine-asparagine), such as osteopontin. Different RGD peptides and antibodies against the vitronectin receptor inhibit the attachment of osteoclasts and hence bone resorption. Once osteoclast attachment has occurred the osteoclasts transport protons across the membrane by both a Na,+H + antiporter and either a H+-ATPase or a H,+ K + antiporter, reducing the pH in the area to less than three. Production of these hydrogen ions occurs through the hydration of carbon dioxide and is catalyzed by carbonic anhydrase II. Resorption of bone mineral by hydrogen ion occurs by the following reaction: Cal0(PO4)6(OH) 2 +

8H + ---> 6 H P O 4 2 + 10Ca 2+ + 2H20

The released calcium and phosphorous then enter the extracellular space by a process of transcytosis across the osteoclast. Osteoclastic bone resorption also occurs through the secretion of lysosomal enzymes into the lacunae by exocytosis. During resorption osteoclasts also synthesize and deposit osteopontin into the resorption lacunae. Osteopontin is a phosphorylated glycoprotein thought to facilitate the adhesion or detachment of the osteoclast to the bone surface. Bone morphogenetic proteins (BMPs) are also released by osteoclasts during resorption. A "reversal phase" follows, lasting 1 to 2 weeks. During this phase a new group of mononuclear cells that remain uncharacterized smooth the surface of the pit and deposit the cement line ready for osteoblastic bone formation.

2. CIRCULATING M A R K E R OF BONE R E S O R P T I O N Markers of bone resorption include tartrate-resistant acid phosphatase (TRAcP), hydroxyproline, hydroxylysine glycosides, pyridinoline, and deoxypyridinoline. Tartrate-resistant acid phosphatase is a lysosomal enzyme contained in the osteoclast and released during resorption [12]. Unfortunately, TRAcP can originate in blood cells [13], thereby diminishing this marker as a specific index of bone resorption. Hydroxyproline is a posttranslationally modified amino acid that is abundant in all collagens [14]. It is produced by posttranslational hydroxylation of proline residues in procollagen. Hydroxyproline is released during bone resorption and cannot be reutilized in collagen synthesis [13]. The free amino acid form is either excreted in the urine (10-20%) or metabolized by the liver. The proportion of hydroxyproline excreted in the urine remains constant with changes in bone turnover [ 12], and thus hydroxyproline does give a measure of the metabolic state of the bone. Dietary sources of hydroxyproline, such as meat and gelatin, also influence its urinary excretion, though this can be overcome by taking fasting urine samples [12]. However, hydroxyproline lacks sensitivity because it is poorly correlated with bone resorption when assessed by either calcium kinetics or bone histomorphometry [ 13]. Hydroxylysine is another amino acid unique to collagen [15]. It is much less abundant than hydroxyproline [13], but when released by bone resorption is not metabolized and is almost totally excreted in the urine [ 12] and is thus a more sensitive marker [ 13]. Hydroxylysine is also unaffected by diet. There are two main hydroxylysine glucosides; fl-1-galactosyl-O-hydroxylysine is seven times more prevalent in bone than ce-l,2-glucosyl-galactosyl-O-hydroxylysine, which is more prevalent in skin collagen. The major problem with the use of fl-1-galactosyl-O-hydroxylysine as a measure of bone resorption is the difficulty of measurement and an interassay CV of greater than 6% [ 12]. Pyridinoline and deoxypyridinoline are nonreducible cross-links that stabilize the collagen chains and are released from the bone matrix during resorption [ 15]. Although pyridinoline is prevalent in bone, cartilage, and other connective tissues, the ratio of pyridinoline to deoxypyridinoline in urine is similar to that in bone, suggesting that pyridirioline in urine is derived predominantly from bone [16]. Deoxypyridinoline is present in significant amounts only in bone. Urinary pyridinoline and deoxypyridinoline are not metabolized in vivo and are correlated with the rate of bone turnover as measured by iliac crest biopsy, calcium kinetics, and bone histomorphometry [ 13,15]. Pyridinoline and deoxypyridinoline have a diurnal variation, with the highest excretion occurring at night, following a pattern similar to that of osteocalcin [ 15]. At present, although there is no international standard or widespread use of an internal standard [12], pyridinoline and deoxypyridinoline are the most sensitive

CHAPTER 19 Bone and Calcium markers of bone resorption. Measurement techniques include ultraviolet absorbance of metabolites separated on high-performance liquid chromatography (HPLC) or radioimmunoassay using antibodies raised to the cross-links. Other potential markers of bone resorption include crosslinked amino-terminal telopeptide of type I collagen (INTP) and cross-linked carboxyterminal telopeptide of type I collagen (ICTP). The ICTP fragment is derived from the crosslinking the carboxyterminal telopeptides of two a 1 chains of collagen and the helical region of an a 1 or a2 collagen chain and is released during bone resorption [12]. The ICTP concentration in serum has been shown to be well correlated with bone resorption measured by either calcium kinetic studies or bone histomorphometry [14]. It is probably specific for bone because it contains cross-links that do not occur in type I collagen from other tissues. ICTP has a circadian rhythm like that of pyridinoline, deoxypyridinoline, and osteocalcin. INTP is rich in pyridinoline and deoxypyridinoline and after release from the matrix during resorption it is excreted in the urine [ 14]. INTP has a diurnal rhythm similar to that of pyridinoline, deoxypyridinoline, osteocalcin, and ICTP [12]. 3. BONE FORMATION

Bone formation commences with the differentiation of osteoblasts from preosteoblasts. Osteoblasts synthesize the precursors of type I collagen and type I procollagen and secrete them into the extracellular space, where they are cleaved to release collagen. The collagen molecules then assemble in fibrils and are stabilized by intramolecular and intermolecular cross-links. Osteoblasts also synthesize and secrete other extracellular proteins, including alkaline phosphatase and the bone matrix protein osteocalcin. For bone mass to be maintained the described processes of bone resorption and bone formation must be balanced. One possible mechanism to achieve balance is that bone resorption produces factors that then modulate osteoblastic bone formation. These factors may be released from the matrix as it is resorbed or may be released by the osteoclast. IGF-I and TGF-fl have both been hypothesized as possible bone coupling factors [17]. Another hypothesis is that the osteopontin secreted by the osteoclasts may act as a signal for osteoblast activation to commence [ 18]. 4. CIRCULATING MARKER OF BONE FORMATION Serum markers of bone formation include alkaline phosphatase, osteocalcin, and procollagen I extension peptides [13]. In bone, alkaline phosphatase is localized in osteoblast membranes and is released into the circulation [ 15]. Serum alkaline phosphatas e activity is the most commonly used marker for bone formation but lacks sensitivity and specificity because isoenzymes in the liver, kidney, intestine, and placenta also circulate in blood [19]. Rather than total

291 alkaline phosphatase activity, measurement of bone alkaline phosphatase using antibodies specific for the bone isoforms increases the sensitivity of this marker. Osteocalcin (bone gla-protein) is a small noncollagenous protein that is specific for bone and teeth [20]. It contains T-carboxyglutamic acid, a unique calcium-binding amino acid, that is synthesized by a vitamin K- and carbon dioxidedependent posttranscriptional modification of specific glutamic acid residues [21]. Osteocalcin is predominantly synthesized by osteoblasts and incorporated into the extracellular matrix, but a small proportion is released into the circulation, where it can be analyzed by radioimmunoassay [20]. However, osteocalcin has a diurnal variation, so if used as a bone marker the sample must be taken at a consistent time [22]. 5. MINERALIZATION

After osteoblasts secrete the matrix proteins there is a lag time of about 3 weeks prior to mineralization. Although this process of mineralization is not well understood, products of osteoblasts such as alkaline phosphatase are known to have a major role. Osteopontin and osteocalcin are also expressed by the osteoblasts during matrix mineralization. The mineralization process may involve the formation of matrix vesicles and may take as long as 5 months. The osteoblasts active at the site either become entombed in the calcium bone matrix, to become osteocytes, or become bone lining cells. These lining cells are involved in reinitiating osteoclastmediated bone resorption. 6. REGULATORS OF BONE REMODELING Osteoclastic resorption is altered rapidly by both PTH and calcitonin. PTH-stimulated osteoclastic resorption is probably indirect through effects on the osteoblast, because osteoclasts do not contain PTH receptors. Calcitonin appears to inhibit bone resorption directly because osteoclasts possess calcitonin receptors. The most potent bone-resorbing hormone is calcitriol. Calcitriol receptors have been found in osteoblasts but the data in osteoclasts are currently uncertain. Other cytokines and colony-stimulating factors (CSFs) have a role in regulating osteoclastic bone resorption. Interleukins (IL-1, IL-3, and IL-6), tumor necrosis factor-a, tumor necrosis factor-fl (lymphoxotoxin), transforming growth factor-a, BMP7, granulocyte-macrophage colony-stimulating factor, platelet-derived growth factor (PDGF), and macrophage colonystimulating factor all increase osteoclast formation. IGF-I, IGF-II and interferon-y (IFN-T) decrease osteoclast formation [ 10]. There has been much interest in a new regulatory system for osteoclast differentiation involving a membranebound protein, osteoclast differentiation factor (ODF; also TRANCE, RANKL) [23]. Another circulating protein, osteoprotogerin (OPG), binds to ODF and inhibits osteoclastogenesis.

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B. R o l e o f E s t r o g e n in B o n e R e m o d e l i n g Estrogen plays an important role in maintaining bone mass in the female by suppressing remodeling and maintaining a balance between osteoblast and osteoclast activity. At menopause a rise in bone resorption occurs and lasts for the remainder of life [24]. This increase in osteoclastic activity is the principal recognized defect that leads to menopausal bone loss. However, if there were not an associated relative defect in osteoblast bone formation, the increase in turnover would not matter. This defect is relative in that the rate of bone formation is actually increased at menopause but not at a rate sufficient to maintain skeletal mass. Estrogen deficiency results in an increase in osteoclast activity in two ways, increased activation frequency of bone remodeling units and increased resorption depth of the bone lacunae. The latter results in perforation of trabecular plates and endocortical trabecularization. There is also some evidence that osteoblasts have a reduced ability to refill the resultant lacunae, although whether this is an aging effect, a specific disease effect, or a true effect of estrogen deficiency is uncertain [25]. The relative balance between osteoclastmediated bone resorption and osteoblast-mediated bone formation determines whether bone is gained or lost. Shortterm estrogen deficiency earlier in life is not necessarily associated with bone loss. During lactation total body bone mass decreases 2 - 3 % while lumbar spine density decreases 4%. However, postlactation this bone loss is completely regained [2,26] so long as the duration of lactation does not exceed 9 months or so. Similarly, the induction of hypogonadism with long-acting gonadotropin-releasing hormone analogs for the management of endometriosis causes significant bone loss, bone density returns to baseline if the treatment is restricted to 6 months [27]. However, longer term hypogonadism appears to be a problem as judged by studies of bone density in subjects with secondary amenorrhea due to prolactinoma [28]. Thus it is possible that long-term hypogonadism is associated with trabecular perforation and destruction of surfaces for osteoblast formation to occur irrespective of age.

essarily be limited. In reviewing current ideas on the regulation of remodeling it is important to recognize that the activities of the two main cell types, the osteoclast and osteoblast, depend not only on the activity of the individual cell but also on the number of cells recruited to the resorption site. Thus the control of the cell population by control of the rate of differentiation from precursor cells is thought to be an important regulatory step. At the level of the differentiated individual cell, regulation involves control of the activity of the cell and its longevity. 1. ESTROGEN RECEPTORS IN BONE CELLS

a. Osteoblasts Since the first description of estrogen receptors and mRNA for estrogen receptor ce within cultured osteoblast like cells was published [29,30], it has been considered that one major mechanism of the action of estrogen is via this cell type. Estrogen receptor ce mRNA has been shown in human osteoblasts and osteoblast progenitor cells using in situ techniques [31 ]. The discovery of a novel estrogen receptor transcript, estrogen receptor fl [32], raises interesting possibilities for tissue-specific regulation of activity. Estrogen receptor fl has now been reported to be present in human and rat osteoblast-like cells [33,34]. b. Osteoclasts Some workers have reported the presence of estrogen receptors within mature osteoclasts. Although this may be true for avian species [35], in mammals the receptor is located within osteoclast precursor cells [36]. Using a reverse transcriptase polymerase chain reaction (RT PCR), it has been claimed that estrogen receptor ce is found in human osteoclasts derived from giant cell tumors of bone [37]; unfortunately, the material used was contaminated with stromal cells. Using more specific estrogen probes and in situ hybridization techniques, no receptor or mRNA for estrogen receptor a or fl has been found in osteoclasts, although abundant receptor and message was found in the stromal cells [38,39]. Furthermore, in situ hybridization data from mature human bone has not shown any estrogen receptor a mRNA in osteoclasts [31 ]. Thus the balance of the evidence is that estrogen does not have any direct effect on the mature human osteoclast.

C. C e l l B i o l o g y o f E s t r o g e n R e g u l a t i o n of Bone Remodeling As indicated above there is evidence that estrogen plays a critical role in each of the processes that determine skeletal health or disease. At the menopause the principal effect on bone of estrogen deficiency is increased bone remodeling. Because the nature and interactions of all the factors involved in the coordinated activity of the osteoclasts and osteoblasts of the remodeling unit have not been fully investigated, the description of the effects of estrogen must nec-

2. ESTROGEN REGULATION OF OSTEOCLAST ACTION Estrogen suppression of bone resorption in the oophorectomized rat is accompanied by inhibition of carbonic anhydrase II and tartrate-resistant acid phosphatase mRNA and is considered to be an indirect action of estrogen [40]. The current model for regulation of this process involves a variety of cytokines that promote the differentiation of osteoclast precursors from GM CFUs and then stimulate their activity. There are a large number of cytokines potentially involved in this process; perhaps best known are IL-1 and IL-6. The

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FIGURE 3 Estrogen regulation of cytokines in bone. (A) Normal bone remodeling with estrogen (E2) acting as an inhibitor. (B) Estrogen deficiency. Lack of estrogen action on peripheral blood monocytes results in reduced secretion of IL-1, TNF, and GM-CSF; consequently, osteoclast differentiation and activation are increased. Lack of estrogen results in greater secretion of IL-6, which results in increased osteoclast differentiation and secretion and activation of more enzymes that directly activate osteoclasts. PBM, peripheral blood monocyte; OB, osteoblast; ST, stromal cell; OC, osteoclast; PTH, parathyroid hormone; CSF, colony-stimulating factor. Adapted with permission from Ref. 41, Horowitz (1993). Copyright 1993 American Association for the Advancement of Science.

relationship between these factors in estrogen deficiencyinduced bone resorption has been elegantly reviewed [9,41 ] (Fig. 3). A second important way in which stromal cells may regulate osteoclast action relates to the role of transforming growth factor-fi (TGF-fi) in the inhibition of bone resorption [42,43]. An estrogen concentration of 10 -8 M has been demonstrated to increase TGF-fi concentration in the media of human osteoblast-like cell lines [44]. It has been suggested that this TGF-fi is deposited in the bone matrix. At the time of bone resorption, under the influence of the acidic environment, it would be available to inhibit osteoclast activation. This would occur at the appropriate time to stop bone resorption, ready for the reversal phase and subsequent bone formation. Finally, estrogen may regulate the life cycle of the osteoclast by inducing apoptosis [31 ]. 3. E S T R O G E N R E G U L A T I O N OF O S T E O B L A S T ACTION

The first evidence that estrogen has direct in vitro effects on bone cells was obtained by the demonstration of stimulation of alkaline phosphatase activity in the rat osteoblastic osteosarcoma UMR106 cell in a dose-dependent manner [45]. This finding has been confirmed in normal human osteoblast cells [46]. Subsequently, estrogen stimulation in UMR106 cells of aminotransferase, creatinine kinase, aspartate transaminase, lactate dehydrogenase, and transferrin was demonstrated with maximal activity at 10-11M estrogen [47]. These enzymes have been shown to be stimulated by estrogen in a number of in vivo systems [47]. There are also

data that estrogen can directly stimulate IGF-I in rat bone, which can stimulate osteoblast activity [48]. The biological significance of estrogen action on the osteoblast in terms of bone formation remains controversial. Without doubt, during estrogen deficiency a net increase in osteoblastic activity results from an increased activation frequency of bone resorption units. In view of the fact that this increased bone turnover is always associated with overall bone loss, a more subtle question remains. This relates to the relative impairment of bone formation in relation to bone resorption during the increased bone turnover associated with postmenopausal estrogen deficiency. The question arises as to whether this is due to relative decreased bone formation or relative increased bone resorption. Either way there is a relative defect in bone formation in menopausal estrogen deficiency. It should be noted that the osteoblast defect does not extend to impairment of fracture healing, which appears perfectly normal after menopause, perhaps because the cellular mechanisms involve endochondral bone formation rather than bone remodeling as a primary event. The issue is complicated by evidence that other influences maintaining osteoblast activation may be causative in the age-related defect in bone formation associated with osteoblast senescence [25,49]. Possibilities include reduction of osteoblast activation as a result of decreases in physical activity and the stress-strain effects these induce at the bone surface. Certainly in animal experiments it is clear that mechanical effects stimulate periosteal bone formation [50,51]. It is possible that the strain-related increase in bone density in

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postmenopausal women [52] is due to stimulation of osteoblastic activity.

D. Bone Modeling In adult life bone modeling occurs as a result of de novo osteoblast action on periosteal surfaces. This process causes radial enlargement of long bones during growth and continues during old age. The molecular mechanisms controlling periosteal bone apposition are not well understood. It is clear that mechanical forces applied to the skeleton may induce dramatic bone formation on these surfaces, initially in the form of woven bone [53], and estrogen is thought to play a role in this process. Estrogen receptor mRNA is expressed in the periosteum of rat calvariae and has been shown to regulate calvarial cell activity [54,55]. In the adult female human estrogen replacement appears to stimulate periosteal bone formation in the phalanges [56].

E. Endochondral and Intramembranous Ossification Although modeling and remodeling are the mechanisms by which the menopausal estrogen loss affects the skeleton, it is important to note that two other processes, endochondral and intramembranous ossification, are important in the developing skeleton. It is the growth of the skeleton in childhood and adolescence that determines the maximum skeletal dimensions and thus may determine the propensity to fracture in postmenopausal life. Endochondral ossification is the primary process by which most bones in the body grow in length. As such, this process is the primary determinant of frame size. The process involves the development of chondroblasts into chondrocytes, which lay down the extensive extracellular matrix. This calcifies as the primary spongiosa. It is important to realize that estrogen plays a critical role in the growth and fusion of the growth plate, both in males and females. The importance of estrogen is shown by the abundance of estrogen receptors or estrogen receptor mRNA in human chondrocytes [31,57]. Estrogen also plays a critical role in chondrocyte biology. In the absence of the estrogen receptor, or an adequate circulating concentration of estrogen, the mature skeleton is seriously compromised [58-60]. This is due to lack of fusion of the growth plate and impairment of peak bone mass. This may be due to a lack of apoptosis of the mature chondrocyte [61 ]. It should be noted that a role for chondrocytes in bone formation following fracture has been suggested [62]. Thus the effects of estrogen on chondrocyte biology may be relevant to the adult skeleton. Intramembranous ossification is important in the development of platelike non-weight-bearing bones of the skull and clavicle. It differs from endochondral bone formation

in that the osteoblasts develop in a fibroblast matrix rather than in a cartilage matrix on the surface of the bone. During development, intramembranous ossification is associated with woven bone formation, which is later remodeled to lamellar bone.

IV. ESTROGEN EFFECTS ON EPITHELIAL CALCIUM TRANSPORT The second important concept that needs to be highlighted is that estrogen directly regulates calcium and phosphate transport across cell membranes. This, of course, may also involve the regulation of calcium movement into and out of the bone compartment across the bone lining cells by estrogen. Two other critical membranes involved in calcium and phosphorus transport are found in the kidney and intestine. The concept involves acceptance of these organs as estrogen targets. The evidence for this is outlined below. Calcium membrane transport is dependent on transcellular and paracellular calcium transport. Transcellular calcium flux in epithelial cells is the result of the specialized structure of these cells, creating a functional polarity between the apical and basolateral membranes. In the epithelial cell, the apical membrane may be exposed to a relatively high concentration of calcium compared to the low intracellular calcium concentration, so calcium traverses it along a concentration gradient, utilizing specific transport systems. At the basolateral membrane, calcium must move against a calcium gradient, requiring specific transport mechanisms [63,64]. Two distinct transport mechanisms are known to exist in the plasma membranes of the renal and intestinal epithelia that remove calcium from the cell, the plasma membrane calcium pump (PMCP) and the Na,+Ca2+ exchanger (NCE). Evidence also exists that both of these systems exist in bone cells [63,65]. Further, transcellular calcium transport has been shown to be important in dentine mineralization by the odontoblast [66]. Two important calcium-binding proteins also play a role in moving calcium from the apical to basolateral membranesmcalbindin D28K, present in distal tubular cells in the kidney, and calbindin D9K, present in intestinal cells. The calbindins have been shown to increase diffusion rates of physiological concentrations of calcium [67]. The plasma membrane calcium pump The PMCP is a CaZ+Mg 2+ dependent ATPase of molecular weight 135,000 [68]. The pump consists of 10 putative transmembrane helices with a high degree of homology in the catalytic domains of the rat and human sequences. The PMCP is coded by four separate genes in both human and rat DNA and alternate splicing results in potentially more than 30 isoforms [68]. Calmodulin increases the affinity and the Vmax of this system [68] in both kidney and intestine.

CHAPTER 19 Bone and Calcium

295

The sodium calcium exchanger The NCE is a secondary active transport system, using the electrochemical gradient produced by sodium ATPase activity [69]. Three different isoforms have been discovered that are coded from a single gene, with almost identical homology between species. It is a 970-amino acid protein with a primary structure that contains 11 transmembrane-spanning regions and a large cytoplasmic loop between transmembrane segments 6 and 7. The NCE is particularly abundant in cells that handle large fluxes of calcium across their membranes, such as contractile and neuronal cells. The orientation of the NCE is determined by the predominance of two inwardly directed electrochemical gradients generated by plasma membrane sodium and calcium pumps. This electrochemical gradient is determined by the net activity of the PMCP, the sodium pump, cell organelle calcium sequestering, and the membrane potential difference. Depending on these factors it can operate in a calcium influx or calcium efflux mode, or if the gradient is neutral, Na,+Ca2+ exchange ceases [69].

A. Biology of Estrogen Effects on the Kidney

on the extracellular calcium balance. Approximately 70% of calcium reabsorption occurs in the proximal tubule [70] and is largely passive and voltage dependent. It is associated with active reabsorption of sodium, glucose, and other solutes. In the distal tubule sodium and calcium reabsorption can be uncoupled, for instance with thiazide diuretics [71]. It is in this segment that regulation by parathyroid hormone and cAMP occurs [71,72]. This suggests that fine regulation of calcium excretion by hormonal control of the NCE and the PMCP occurs in the distal tubule. The mechanisms involved in calcium transport and their regulation are outlined in Fig. 4. In the kidney the NCE is located only in the distal tubule and has been shown to be the primary mechanism by which PTH modulates renal calcium reabsorption [73]. The PMCP is present in both proximal and distal tubules, with a higher affinity in the distal tubule indicating that its role in the proximal tubule may be the maintenance of intracellular calcium concentrations rather than the translocation of large amounts of calcium [74]. Localization studies in the human kidney using monoclonal antibodies to the human red blood cell calcium pump could detect only the calcium pump in the distal tubule [75], indicating that lower affinity plasma pump activity observed in proximal tubules is of a different type.

1. PHYSIOLOGY OF RENAL CALCIUM HANDLING

In humans the kidneys filter approximately 100 to 200 mmol of calcium per 24 hr, of which about 98% is reabsorbed. Because of the high rate at which calcium cycles across the renal tubular membrane it is possible for subtle variations in the rate of reabsorption to have profound effects

2. R O L E OF CALCIUM-SENSING R E C E P T O R IN REGULATION OF ION TRANSPORT IN THE KIDNEY

In the kidney, paracellular calcium transport is regulated by the extracellular ionized calcium concentration that acts on the recently cloned calcium-sensing receptor [76,77]. The

LUMEN

PERITUBULAR SPACE -77mV

II

.' D 28K

i

Ca2+

/

~

0.5mM ~

ESTROGEN

2K+ " ~-]-~ ..

Ca2+~; .........";i'--"~'1

[~A_~

~

+

/

J

"3Na

~DE~ATE CYCLAs~ADP . ~ . . l. ~l ~ C a

~

e

tl

I

I

lmM

PTH "

C a 2+

I

FIGURE 4 In the kidney, calcium is filtered at the glomerulus and reabsorbed along the tubule. The distal tubule is a critical regulatory segment for calcium homeostasis. The diagram shows that in this segment calcium moves down an electrochemical gradient generated by PTH, which appears to regulate calcium absorption at the apical membrane and also the sodium calcium exchanger at the basolateral membrane. It also shows that estrogen directly stimulates calcium reabsorption by an effect on transcription of calbindin D28K. There is also an effect on the plasma membrane calcium pump (calcium ATPase), perhaps mediated through calbindin action.

296 interaction of calcium with this receptor results in a decrease in hormone-dependent sodium chloride reabsorption. This is the result of the inhibition of hormone-dependent cyclic AMP production and a decrease in the Na+,K + 2C11- cotransporter and K + channel activity as a result of phospholipase A activation; this, in turn, decreases both the lumen positive potential that drives paracellular calcium transport and the countercurrent multiplier that concentrates urine, resulting in increased urine calcium excretion [78]. 3. R O L E OF ESTROGEN IN REGULATION OF ION TRANSPORT IN THE KIDNEY

A direct effect of estrogen on the kidney to regulate calcium reabsorption requires the demonstration of renal estrogen receptors. A number of studies have indeed shown that in the rat kidney functional estrogen receptors are present in both the proximal and distal tubule [79,80]. Immunocytochemical evidence for the presence of a guinea pig renal estrogen receptor has been presented [81]. The receptor appeared to be localized to glomeruli, interstitial cells, and blood vessels, but not tubular cells. If the latter data are correct, any action of estrogen on the tubule would have to be via a paracrine route. Paracrine effects of estrogen on the kidney may involve IGF in view of evidence that estrogen stimulates IGF-binding protein 4 mRNA in the rat kidney and uterus but not the liver [82]. Estrogen also induces calcium-dependent nitric oxide synthase in the guinea pig kidney [83]. Evidence for a direct action of estrogen on kidney cell calcium transport has now been obtained using cell lines derived from the renal epithelium. In these studies physiological concentrations of estrogen have been shown to have a direct biphasic effect on PMCP-mediated plasma membrane transport in Madin-Darby bovine kidney (MDBK) cells. There was an initial inhibition followed by a twofold stimulation. These data suggest a direct receptor-mediated action [84]. The concept of direct effects of estrogen on renal ion transport is supported by evidence that phosphorus transport is directly regulated by estrogen ovariectomized rats [85,86]. In vivo studies of estrogen effects on calcium handling in perfused rat kidneys have shown a paradoxical response to an increased renal calcium load. There was no detectable effect on calcium excretion under basal conditions but an increase in the excretion of a calcium load in the presence of estrogen [87]. This effect was PTH dependent in that parathyroidectomy abolished the estrogen effect and it was only present during mild volume expansion. This result suggests a possible proximal tubule site of action for this effect of estrogen and indicates the complexities of estrogen action on the kidney. 4. ESTROGEN EFFECTS ON RENAL CALBINDIN

A major factor in transcellular calcium transport in the kidney is calbindin D28K. This calcium-binding protein is

PRINCE AND DRAPER

colocated with the PMCP in the distal nephron in both human [88] and rat [64] kidney cells. Of particular interest is the evidence for a calbindin D28K stimulatory effect on basolateral membrane PMCP activity [89], suggesting a coordinated role of calbindin D28K and the PMCP in regulating transcellular calcium transport. This would be expected to facilitate transcellular calcium transport by increasing calcium transport across the apical membrane [90]. This is particularly relevant in view of the comparable action of the classic calciotropic hormone calcitriol to increase calbindin D28k in distal tubule [91], and to increase calcium transport across both the apical and basolateral membranes of the distal, but not the proximal, tubule [92]. Estrogen increases renal calbindin D28k mRNA concentrations in the oophorectomized rat after 8 hr of exposure [93]. Estrogen effects in a distal tubule kidney cell from the MDBK line have been studied and have shown direct stimulation of calbindin D28k mRNA. 5. CLINICAL STUDIES OF ESTROGEN ACTION ON THE KIDNEY

The factors that increase renal calcium reabsorption are numerous and may include volume depletion, PTH, 1,25dihydroxyvitamin D, and estrogen. At the menopause a significant increase in renal calcium excretion occurs and persists indefinitely [24]. This suggests that estrogen deficiency is a major cause of increased renal calcium excretion, which is likely to be due to a primary effect on the kidney [94]. Clinical studies have indicated that estrogen status regulates the amount of filtered calcium that is reabsorbed [95,96] such that in the estrogen-deficient state more calcium is lost in the urine. A study by McKane et al. has confirmed an estrogen effect on the stimulation of renal calcium reabsorption [97]. Indeed, the effect of estrogen deficiency may be a prominent cause of the rise in PTH with aging [98].

B. B i o l o g y o f E s t r o g e n Effects on the G u t The absorption of calcium occurs by transcellular and paracellular mechanisms (Fig. 5). In general, the paracellular route is considered to be unregulated, although there is some evidence that vitamin D can stimulate the nonsaturable phase of calcium transport [99]. The driving force behind the paracellular route is thought to be concentration gradient that is driven by solvent drag. Paracellular movement of calcium takes place throughout the length of the intestine and may account for two-thirds of calcium flux in the rat intestine. In humans, passive paracellular absorption appears to have an absorption efficiency of about 15%. Thus at high dietary intakes it would be possible to supply the calcium requirement to maintain extracellular homeostasis from this source. Paracellular movement favors absorption only in the duodenum, with paracellular calcium secretion occurring in the jejunum

CHAPTER 19 Bone and Calcium

297

C a 2+

~

1.OmM-~-'---~

( ~

.._._---~ ~

Ca 2+

LUMEN

ATP - ~ 2K+ ~ C__J__.~ Na+ BLOOD

g

3Na+ ~

~-~Ca2+

ATP

~ Ca2+

1.5mM

i RE E 0R

CALCITRIOL ESTROGEN -55mV FIGURE 5 Calcium transport across the intestine. Calcium regulation across the bowel wall is most active in the duodenum. In this area calcium absorption is regulated by calcitriol and also to some extent by estrogen, both of which stimulate the activity of the plasma membrane calcium pump, perhaps by effects on calbindin D9K.

and ileum, indicating that net calcium absorption is determined by transcellular mechanisms as well the net difference between paracellular absorption and secretion [ 100]. There are two mechanisms of transcellular transport, active transport and a transcellular vesicular mechanism termed transcaltachia. Transcellular calcium transport in the enterocyte is summarized in Fig. 5. NCE activity has been shown in the intestine in both rats [101] and humans [102] and it appears to have approximately 20% of the calcium translocating activity of the PMCP in basolateral membrane preparations [101]. This suggests that PMCP is the more important mechanism for translocation of calcium in the intestine. In support of this is the observation that the activity of the PMCP in the rat declines with age [103] and this activity and mRNA expression appear to be stimulated by calcitriol [ 103]. The intestinal PMCP has a calbindin D9K binding domain, but the interaction of calbindin D9K does not appear to result in an increase in PMCP activity [ 104]. 1. E S T R O G E N E F F E C T S ON G U T C A L C I U M A B S O R P T I O N

Studies of estrogen effects on calcium absorption in the rat intestine have in general supported the concept of a reduction in calcium absorption in the absence of estrogen. A variety of techniques have been used, including studies in intact rats and studies on everted gut sacs. As in the kidney, a direct estrogen effect in this organ requires the presence of an estrogen receptor that has been identified in the intestinal crypt cell IEC-6 cell line [ 105] and in rat duodenum [ 106]. In estrogen-deficient rats, estrogen replacement increases intestinal calcium absorption, but this effect is decreased with increasing age, indicating an age-dependent effect of estrogen on intestinal calcium transport [107]. Estrogen has been shown to increase calcium uptake by duodenal cells from estrogen-deficient rats

in a dose-dependent manner. This effect was negated by inhibitors of DNA transcription and protein translation, indicating direct estrogen effects on transcription and translation to increase calcium transport [ 106]. 2. C L I N I C A L S T U D I E S OF E S T R O G E N A C T I O N ON G U T C A L C I U M A B S O R P T I O N

In view of evidence for a reduction in calcium absorption at the menopause [ 108] it has been suggested that there may be a direct effect of estrogen on gut calcium absorption. Other data have suggested that estrogen may potentiate the effect of 1,25-dihydroxyvitamin D on the stimulation of calcium absorption [ 109]. However direct evidence for an effect of estrogen on calcium absorption in aging humans remains controversial [ 110]. In addition to the direct effects of estrogen on the gut, there are also indirect estrogen effects via inhibition of bone resorption, with a consequent increase in the calcium requirement to recalcify skeletal resorption sites. This may result in a temporary rise in calcitriol concentrations for up to 2 years with the associated stimulation of gut calcium transport by the classical vitamin D-mediated pathway.

V. E S T R O G E N OF CALCIUM

AND THE PHYSIOLOGY

HOMEOSTASIS

In classical physiology the major role of the calcitropic hormones PTH and calcitriol (1,25-dihydroxy vitamin D) is considered to be to maintain constant extracellular calcium concentrations. Under conditions of reduced extracellular fluid calcium concentrations there is an increase in PTH that has three main effects: stimulation of renal tubular calcium reabsorption, an increase in bone resorption to liberate calcium into the extracellular space, and stimulation of

298

PRINCE AND DRAPER

CALCIUM ~ %

BONE RESORPTION

t

~

~

RENAL CALCIUM

f

1 L

FIGURE 6 PTH and calcitriol regulation of extracellular calcium homeostasis. Negative feedback between calcitriol and PTH ensure that a deficiency of calcitriol-mediated gut calcium absorption is compensated for by a rise in parathyroid hormone.

calcitriol formation in the kidney (Fig. 6). The raised calcitriol concentrations stimulate active transport of calcium from the intestinal lumen into the extracellular space. Gut calcium absorption consists of two mechanisms, an active, high-affinity, low-capacity, calcitriol-regulated system [111] that is especially important under circumstances of a low calcium intake, and a passive, paracellular, high-capacity system that is driven by the concentration gradient of calcium between the lumen of the gut and the extracellular fluid. Increasing luminal calcium by dietary supplement will increase absorption by both pathways. Thus PTH alters calcium fluxes at the bone, the kidney, and, indirectly through its renal effects, the gut to restore extracellular calcium homeostasis. PTH concentrations then fall under the influence of the raised calcium, sensed by the calcium receptor in the parathyroid gland [ 112], and by a feedback action of calcitriol to inhibit PTH production [ 113]. Evidence points to the presence of a calcium receptor within the kidney that regulates extracellular calcium concentration independently of PTH [77,114]. The effect of these PTH and calcitriol homeostatic responses has been demonstrated in pre- and postmenopausal women subject to low calcium intake [ 115,116]. In general they are not modified by estrogen deficiency per se except insofar as estrogen influences the flux of calcium into and out of the bone, intestine, and kidney.

A. Effects of Ovarian Hormone Deficiency on Extracellular Calcium Homeostasis There is no doubt that a negative calcium balance develops at the menopause. Menopausal loss of bone must result

in a negative calcium balance because the vast majority of calcium in the body resides in the hydroxyapatite of the skeleton. Estrogen deficiency directs increased bone resorption, directly impairs gut calcium absorption, and directly increases renal calcium excretion. These effects may result in an increase, no change, or a reduction in extracellular calcium balance, depending on the relative activity of the calcium transport mechanisms in the three organs. At menopause renal calcium excretion rises and gut calcium absorption falls [94,108], with a possible rise in gut calcium excretion [ 117]. During this time there is evidence for a reduction in calcium transport across the intestine [108,118,119]. The fasting urine calcium excretion also rises at the menopause and persists indefinitely [24]. The reason for these multiorgan effects relates to the two previously mentioned principal effects of estrogen deficiency on bone and calcium biology: increased bone remodeling and loss due to the removal of the direct protective effect of estrogen on bone cells, and the increased loss of calcium from the body due to loss of the stimulatory effects of estrogen on calcium reabsorption in the kidney and absorption in the gut. In acute estrogen deficiency after GnRH analog administration there is a sufficient increase in the calcium flux out of bone to measurably suppress PTH and calcitriol [120]. This homeostatic response results in a reduction in gut calcium absorption as calcitriol falls and an increase in renal calcium excretion as PTH falls (Fig. 7). At lower levels of bone resorption such as occur with natural menopause, it is not possible to detect a reduction in PTH or calcitriol concentrations. This is because the relative balance between the bone, gut, and renal effects is such that the flux of calcium out of the bone is matched by an identical flux of calcium out of the kidney and intestine. Indeed there

CHAPTER 19 Bone and Calcium

LOW DIETARY CALCIUM

LOW GUT CALCIUM ABSORPTION

299

ESTROGEN DEFICIENCY

HIGH BONE RESORPTION AND BONE LOSS

HIGH URINE SODIUM

HIGH UR/NE

CALCIUM

LOW CALCITRIOL ] EXTRA CELLULAR CALCIUM BALANCE INCREASED

.PTH SUPPRESSED [ FIGURE 7 Roleof estrogen in early postmenopausalbone loss: the lack of estrogen-mediatedsuppression of bone resorption ensures a major movement of calcium from the bone compartment to the extracellular compartment. This is excreted via the kidney and by the bowel. This excretion is facilitated by the relative suppression of PTH and calcitriol.

is no detectable change in PTH or calcitriol for the first 10 years after natural menopause [5,24]. The reintroduction of estrogen in estrogen-deficient postmenopausal women results in the reversal of the mechanisms described with a temporary fall in plasma calcium as the skeletal mass rises. This occurs as a result of refilling of resorption sites with newly formed hydroxyapatite. The resulting temporary stimulation of PTH and calcitriol may last up to 2 years [5]. Stimulation of calcitriol results in an acute increase in gut calcium absorption [121 ] and the stimulation of PTH results in increased renal calcium and reabsorption [97]. Thus there are some acute changes associated with early estrogen replacement that result in increases in PTH and calcitriol, which are directed to correction of total body calcium balance. In this regard PTH and calcitriol are acting to supplement estrogen action on the intestine and kidney. Because of the variable and confusing relation between the effects of estrogen on the three organs of calcium homeostasis, it has been suggested by some authors that there is a direct effect of estrogen on the production of PTH and calcitriol. To date no direct evidence for these effects has been produced [5].

B. Age-Related Osteoporosis~The Importance of Defects in Estrogen and Vitamin D Action An outline of the physiological interactions important in the development of a negative calcium balance in aging is

shown in Fig. 8. The regulation of calcium homeostasis by calcitropic hormones is relevant to our understanding of the negative calcium balance that occurs in elderly individuals. With increasing age there is a rise in circulating PTH concentrations [24,122,123]. This is due to a negative calcium balance as a result of a primary increase in renal calcium loss and a fall in gut calcium absorption as a result, in part, of estrogen deficiency. Thus estrogen deficiency plays a significant causative role in age-related bone loss. The elevation in PTH occurs as a result of the negative calcium balance. This stimulates bone resorption that then induces bone loss. In these circumstances the effect of estrogen deficiency on the kidney and intestine is a more important cause of bone loss than the direct effect on bone. The effectiveness of calcium supplementation in treating age-related bone loss is due to its ability to correct the estrogen-deficiency-induced negative calcium balance. The importance of the negative calcium balance has been shown by the reversal of the bone loss of estrogen deficiency and aging by correction of this negative balance. Many randomized trials show that dietary calcium supplementation is partially effective in preventing bone loss in estrogen-deficient women [24,124-126]. Furthermore, fracture rates have been reduced by calcium supplementation in preliminary randomized controlled trials [127-129]. It is important to realize that calcium supplementation, in addition to reducing bone loss, also reduces measures of bone resorption and secondarily bone formation [94,129]. It is highly likely that this is due, at least in part, to the suppression of circulating PTH concentrations noted following calcium supplementation [94,129].

300

PRINCE AND DRAPER

LOW DIETARY CALCIUM AND VITAMIN D

LOW GUT CALCIUM ABSORPTION

ESTROGEN

DEFICIENCY

HIGH URINE SODIUM

HIGH BONE RESORPTION AND BONE LOSS

HIGH URINE CALCIUM

/

I PTHr C ASED I EXTRA CELLULAR CALCIUM BALANCE DECREASED FIGURE 8 Role of estrogen in late postmenopausal bone loss: once the high rate of bone resorption due to loss of the direct inhibitory effect of estrogen on the bone has occurred, the indirect effects of estrogen deficiency on the kidney and bowel result in a negative extracellular calcium balance, which results in high bone resorption, in part by stimulation of parathyroid hormone. This negative extracellular calcium balance is exacerbated in old age by low dietary calcium intake, and vitamin D deficiency is exacerbated by early renal failure preventing the formation of calcitriol. In addition, high salt intakes exacerbate urine calcium

loss.

In view of the evidence for defects in the vitamin D pathway in age-related bone loss it would be simplistic to attribute the whole of bone loss after the menopause to defects in estrogen regulation of calcium metabolism. A second abnormality of calcitropic hormones that occurs with aging is a relative reduction in the concentrations of calcitriol [118,130,131] and free calcitriol [24]. There has been some suggestion that there is a rise in total calcitriol with aging [132] but this may be due to a rise in vitamin D-binding protein [24]. Furthermore, it only continues until the middle of the 60th year of age, after which even the total calcitriol concentration falls [ 133]. The fall in calcitriol with aging is associated temporally with the reduction of gut calcium absorption [118,134]. From what has been said above, a fall in calcitriol would be most unexpected in the face of a negative calcium balance. Therefore it seems very likely that the fall in calcitriol is a pathophysiological abnormality that is implicated in the rise in PTH and excess loss of bone calcium with aging. There are several reasons why calcitriol concentrations fall with age. First, because calcitriol is manufactured in the kidney, its fall is associated with age-related declines in renal function [24]. Second, in subjects with low sunlight exposure; not compensated for by dietary vitamin D, there is a fall in skin vitamin D production with resulting fall in 25-hydroxyvitamin D, the substrate for calcitriol formation in the kidney [135]. Because of difficulties with the calcitriol assay it has

not always been possible to show a fall in this hormone in the presence of vitamin D deficiency. Vitamin D deficiency is, however, associated with a raised PTH and is thereby associated with bone loss [ 136,137]. Finally, there is evidence for a specific defect in circulating calcitriol in osteoporotic fracture patients compared with age-matched controls [ 138].

C. Skeletally Active Estrogen Receptor Agonists Other Than Classical Estrogens The concept of estrogen antagonists and partial agonists has been understood at a conceptual level for many years. Evidence that classical estrogen antagonists such as tamoxifen could be partial agonists on bone tissue has also been known for some years [139]. This concept has now been extended to a tissue-specific role for estrogen-like compounds and has been named the selective estrogen receptor modulator (SERM) concept. The mechanisms that specify bone activity of estrogen while excluding activity on other organs have not yet been elucidated. Nevertheless, a large number of compounds that have been synthesized demonstrate tissue specificity. The potential for targeting therapy to specific tissues without encountering deleterious effects on other tissues is an attractive concept. There are also many naturally occurring plant estrogen-like compounds that also demonstrate tissue-specific actions.

CHAPTER 19 Bone and Calcium 1. PHYTOESTROGENS Phytoestrogens are plant-derived molecules that either inherently or after conversion by intestinal flora exert estrogenic and/or antiestrogenic effects [140]. There are several categories of phytoestrogens, including isoflavonoids, coumestans, and resorcyclic acid lactones (lignins). Structurally, they are related to estrogen (Fig. 9). The foods that contain different phytoestrogens vary widely and thus many foods containing one phytoestrogen have no detectable amount of another phytoestrogen. Phytoestrogens were initially studied because of their agricultural significance. Isoflavones were found to be responsible for infertility in sheep. Based on epidemiologic human data they are thought to have anticarcinogenic, antiviral, and fungicidal effects [ 141 ]. Phytoestrogens inhibit enzymes involved in the growth and proliferation of cells and consequently the growth and proliferation of many types of cancers, including those of the breast, prostate, and colon [ 142]. They also exhibit other properties, including antioxidant, radical scavenging, hypolipidemic, and serum cholesterol lowering. a. Phytoestrogens as Estrogen Analogs The estrogenic properties of phytoestrogens are attributed to their affinity to bind estrogen receptors. Phytoestrogens stimulate the transcription of the human estrogen receptor and competitively bind to it [143]. Binding has been demonstrated in the uterus of various species [ 144], in cancer cell lines [ 145], and in the ovine pituitary gland and hypothalamus [146]. Phytoestrogens exert an estrogenic or antiestrogenic effect, depending on the phytoestrogen, the dose and the preexisting hormonal milieu. Also, phytoestrogens and synthetic estrogen analogs that act as estrogen antagonists in classical tissues, such as the uterus and breast, may act as agonists in nonclassical tissues, such as the bone. Phytoestrogens have a lower binding affinity for the estrogen receptor than do steroidal estrogens. Coumestans have about 1/20th and isoflavones 1/200th the affinity of estradiol to bind uterine estrogen receptors. Besides the lower binding affinity, the phytoestrogen-receptor complex formed is less stable and is less able to be transformed to bind the DNA, which results in a shorter duration of binding to the DNA [144]. The description of phytoestrogen binding to a second estrogen receptor (ER)/3 in addition to ER c~ is of interest [ 147]. Coumestrol increases estrogen and progesterone cytosol receptor concentrations [ 148] and nuclear estrogen-receptor binding [149], and stimulates prostaglandin H synthase (PHS) in vitro [149a]. Thus the transduction pathway for coumestrol largely mimics that of estradiol. When injected with estradiol, coumestrol can reduce the physiological effect of estradiol [138]. Thus, coumestrol can act as both an estrogen agonist and an estrogen antagonist. Although the effect of coumestrol and genistein mimic

301 most of the effects of estradiol in the rat uterus (water inhibition, vascular permeability, etc.), not all the effects of estradiol are either mimicked or inhibited [149,150]. This may be due to different types of estrogen receptors or that the phytoestrogen receptor complex formed may bind only to some estrogen response elements. Phytoestrogens also exhibit some effects that are not mediated by the estrogen receptor. b. Phytoestrogen Effects on Bone and Calcium Because phytoestrogens, by definition, have estrogenic or antiestrogenic effects, their effects on bone are of interest. Coumestrol derivatives KCA 098 have been shown to improve bone strength by increasing bone calcium and phosphorous content in oophorectomized rats, with inhibition of PTH, prostaglandin E2, 1,25-dihydroxyvitamin D 3 and interleukin-lfl [151]. The radiologic density of rat femurs suggested a higher bone density in rats treated with KCA 098 than in their oophorectomized controls [ 151 ]. In cell cultures derived from 9-day chick or rat embryonic femurs, coumestrol has been shown to reduce bone resorption and increase bone formation [152]. Coumestrol acted in the same way as estrogen, inhibiting PTH, calcitriol, and prostaglandin E2-dependent bone resorption. Coumestrol acted in a dose-dependent manner, but was less potent than estradiol. Dietary soybean protein has been shown to prevent bone loss in the femur and lumbar vertebra of oophorectomized rats [153]. One possible explanation for this is the phytoestrogen content of soybeans. Soybeans contain the isoflavones daidzein and genistein [154,155], which are located primarily in the protein component [156]. Genistein, the primary isoflavone in soy, has been shown to increase calcium content, alkaline phosphatase activity, and DNA content in bone cultures [157]. Further, 10 -7 M genistein completely inhibited the decrease in bone calcium content induced by bone-resorbing factors, but did not further enhance the inhibitory effect of oestrogen (10 -9 M) on PTHstimulated bone resorption [158]. Genistein has also been shown to inhibit bone loss in lactating rats [ 159]. The clinical relevance of these observations needs further evaluation. Nevertheless, the importance of estrogen replacement in the treatment of age-related bone loss has been recognized for many years [160]. Further epidemiological data point to the critical clinical value of the presence of low circulating concentrations of estrogen in the development of fracture in postmenopausal women [4]. Thus it is possible that the dose-response relationships between daily doses of phytoestrogen, especially coumestrol, and fracture prevention is such that these compounds could play a major role in a public health program of fracture prevention. 2. E f f E c T s o r SYNTHETIC S E R M s ON BONE

A variety of synthetic SERMs are under active investigation as therapeutic agents for the prevention and treatment

302

PRINCE AND DRAPER

coH

OH

HO

0 Coumestrol

Estradiol

0

CH HO

OH

HO Diethylstilbestrol

Diadzein

,OH

OCH3 OH

O

O

Genistein

Formononetin

O H ~ C H 2 O H OCH3

OH" ~

CH2OH

~0 /

OH Enterodiol

Biochanin A

OHio

HO

O

CH3 O

HO

H "OH

OH Enterolactone

Zearalanol

FIGURE 9 There are many phytoestrogens. Shown here are their structure and relationship to 17fl-estradiol; daidzein and genistein and their precursors, formononetin, and biochanin A are all found in relatively high concentrations in soy. Enterolactone and enterodiol are the principal metabolites of the lignan group of phytoestrogens. Coumestrol is a potent flavinoid found in young legumes such as alfalfa, clover, soy, or split bean sprouts. Diethylstilbestrol, a synthetic estrogen, is shown for comparison. The structure of zearalanol (a fungal estrogen) is also shown.

303

CHAPTER 19 Bone and Calcium

o f b o n e loss. T h e s e i n c l u d e r a l o x i f e n e [ 1 6 1 - 1 6 5 ] , i d o x i f e n e , a n d d r o l o x i f e n e . T h e s e c o m p o u n d s all d e m o n s t r a t e affinity for the e s t r o g e n r e c e p t o r a n d h a v e activity that is r e l a t i v e l y b o n e specific. T h e m o l e c u l a r b i o l o g y o f t h e s e specific effects

17. 18.

has n o t y e t b e e n s a t i s f a c t o r i l y r e s o l v e d . T h e c l i n i c a l effects o f t h e s e n e w c o m p o u n d s p o i n t to the e x t r a o r d i n a r y flexib i l i t y o f e s t r o g e n effects on d i f f e r e n t t i s s u e s and give rise to m u c h o p t i m i s m that a d e t a i l e d u n d e r s t a n d i n g o f e s t r o g e n ac-

19.

t i o n s h o u l d l e a d to specific t a r g e t e d t h e r a p i e s . 20.

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PRINCE AND DRAPER women. N. Engl. J. Med. 323, 878-883. 126. Cumming, R. G., and Nevitt, M. C. (1997). Calcium for the prevention of osteoporotic fractures in postmenopausal women. J. Bone Miner Res. 12, 1321-1329. 127. Chevalley, T., Rizzoli, R., Nydegger, V., Slosman, D., Rapin, C.-H., Michel, J.-P. et al. (1994). Effects of calcium supplements on femoral bone mineral density and vertebral fracture rate in vitamin-D-replete elderly patients. Osteoporosis Int. 4, 245-252. 128. Recker, R. R., Kimmel, D. B., Hinders, S., and Davies, K. M. (1994). Anti-fracture efficacy of calcium in elderly women. J. Bone Miner. Res. 9(1), S 154. 129. Reid, I. R., Ames, R. W., Evans, M. C., Gamble, G. D., and Sharpe, S. J. (1995). Long-term effects of calcium supplementation on bone loss and fractures in postmenopausal women: A randomized controlled trial. Am. J. Med. 98, 331- 335. 130. Tsai, K. S., Heath, H., III, Kumar, R., and Riggs, B. L. (1984). Impaired vitamin D metabolism with aging in women. J. Clin. Invest. 73, 1668-1672. 131. Davis, J. W., Ross, P. D., Johnson, N. E., and Wasnich, R. D. (1995). Estrogen and calcium supplement use among Japanese-American women: Effects upon bone loss when used singly and in combination. Bone 17, 369-373. 132. Epstein, S., Bryce, G., Hinman, J. W., Miller, O. N., Riggs, B. L., Hui, S. L. et al. (1986). The influence of age on bone mineral regulating hormones. Bone 7, 421- 425. 133. Eastell, R., Yergey, A. L., Vieira, N. E., Cedel, S. L., Kumar, R., and Riggs, B. L. (1991). Interrelationship among vitamin D metabolism, true calcium absorption, parathyroid function, and age in women: Evidence of an age-related intestinal resistance to 1,25-dihydroxyvitamin D action. J. Bone Miner Res. 6, 125-132. 134. Devine, A., Prince, R. L., Kerr, D. A., Dick, I. M., Kent, G. N., Price, R. I. et al. (1993). Determinants of intestinal calcium absorption in women ten years past the menopause. Calcif Tissue Int. 52, 358360. 135. Krall, E. A., Sahyoun, N., Tannenbaum, S., Dallal, G. E., and DawsonHughes, B. (1989). Effect of vitamin D intake on seasonal variations in parathyroid hormone secretion in postmenopausal women. N. Engl. J. Med. 321, 1777-1783. 136. Dawson-Hughes, B., Dallal, G. E., Krall, E. A., Harris, S., Sokoll, L. J., and Falconer, G. (1991). Effect of vitamin D supplementation on wintertime and overall bone loss in healthy postmenopausal women. Ann. Intern. Med. 115, 505-512. 137. Khaw, K.-T., Sneyd, M.-J., and Compston, J. (1982). Bone density parathyroid hormone and 25-hydroxyvitamin D concentrations in middle aged women. Br. Med. J. 305, 273-276. 138. Prince, R. L., Dick, I. M., Lemmon, J., and Randell, D. (1997). The pathogenesis of age-related osteoporotic fracture: Effects of dietary calcium deprivation. J. Clin. Endocrinol. Metab. 82, 260-264. 139. Love, R. R., Mazess, R. B., Barden, H. S., Epstein, S., Neewcomb, P. A., Jordan, V. C. et al. (1992). Effects of tamoxifen on bone mineral density in postmenopausal women with breast cancer. N. Engl. J. Med. 326, 852-856. 140. Dwyer, J. T., Goldin, B. R., Saul, N., Gualtieri, L., Barakat, S., and Adlercreutz, H. (1994). Tofu and soy drinks contain phytoestrogens. J. Am. Diet. Assoc. 94(7), 739-743. 141. Adlercreutz, H., Fotsis, T., Bannwart, C., Wahala, K., Makela, T., Brunow, G. et al. (1986). Determination of urinary lignans and phytoestrogen metabolites, potential antiestrogens and anticarcinogens, in urine of women on various habitual diets. J. Steroid Biochem. Mol. Biol. 25, 791-797. 142. Adlercreutz, H. (1995). Phytoestrogens: Epidemiology and a possible role in cancer protection. Envioron. Health Perspec. 103(Suppl. 7), 103-112. 143. Miksikek, R. J. (1994). Interaction of naturally occurring nonsteroidal

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CHAPTER 19 FIGURE 2 Bone remodeling: following activation of osteoclastic bone resorption by bone lining cells, bone is resorbed, forming a Howship's lacuna. At reversal, a cement line is laid down and bone formation commences by active plump osteoblasts. The newly formed osteoid takes some weeks to calcify. From Lindsay and Kelly, 1996 [ 11].

CHAPTER 20 FIGURE 1 (a) Skin tear due to fragility of forearm skin in an 87-year-old female without hormone replacement therapy. (b) Lesion taped closed to increase rate of healing by approximating the epidermal edges of the full-thickness wound. (c) Wound healed, with linear scar.

CHAPTER 20 FIGURE 2 (a) Transdermal patch on skin to deliver hormone replacement therapy. (b) Skin irritation from the transdermal patch. Irritant dermatitis, secondary to the adhesive used to attach the patch to the skin.

2 H A P T E R 2(

Menopause and the Skin JANET H. PRYSTOWSKY Department of Surgery, Columbia Presbyterian Medical Center, Columbia University and New York Presbyterian Hospital, New York, New York 10032

JEANNE FRANCK Department of Dermatology, Cornell Medical College and New York Presbyterian Hospital, New York, New York 10021

I. II. III. IV. V.

VI. Topical Hormone Replacement Therapy VII. Psychological Effects of Menopausal Changes Related to Skin Appearance-- Self-Image VIII. Summary References

Introduction Dryness Sweating Atrophy Slowed Wound Healing

I. I N T R O D U C T I O N

the skin of menopausal women compared to premenopausal women and/or postmenopausal women on hormone replacement therapy. Fortunately, the changes that occur are largely preventable or reversible when hormone replacement therapy is instituted at the onset of menopause or shortly thereafter. Thus, many of the intrinsic aging changes that occur in postmenopausal women are attributed to estrogen deficiency, and the greater impact of endogenous androgens, e.g., masculinization of facial hair, may be a reflection of a deficiency of progesterone [2]. During premenopause, at approximately age 4 0 - 5 0 years, ovulation becomes less frequent and progesterone is deficient, whereas menopause occurs typically between 50 and 55 years and is associated with the disappearance of all follicles and estrogen deficiency. Thus, some advocate progestins during perimenopause, followed by estrogens in combination with progestins once menopause is confirmed [3].

Numerous changes occur in the skin of menopausal women. These include dryness, increased sweating, wrinkling, general sagging of the skin (including breast ptosis), altered barrier function, thinning of the skin, and decreased wound healing. Skin appendages are also influenced and excess facial hair and thinning scalp hair are signs of the relative hyperandrogenism that accompanies menopause. Although other changes may also be present in menopausal skin, e.g., pigmentation or skin cancer growths, which are secondary to a lifetime of sun exposure or other extrinsic etiologies, they are not directly related to menopause and therefore will not be discussed. The menopausal perturbations of the skin alter the appearance and functional capacity of the skin in several ways that make the skin weaker and unattractive [1 ]. Although the functional impairment of the skin, as in decreased wound healing, may be medically most significant, it is unwise to discount the aesthetic problems induced by menopause because they may cause significant psychological distress and self-image disturbances. Over the past decade, numerous studies have been conducted to measure the content and functional changes of MENOPAUSE: BIOLOGY AND PATHOBIOLOGY

II. D R Y N E S S Menopausal patients often complain of dry, itchy skin. Dry skin is caused by the loss of an effective water barrier to 309

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310 prevent evaporation of water from the epidermis. Sebum provides an effective film to decrease insensible water loss. However, sebum production diminishes in menopause and hyposeborrhea is a contributing factor to the complaints of rough, dry skin in menopausal women [4]. Hormone replacement therapy was observed to alleviate symptoms from hyposeborrhea. Further evidence of the adverse influence of menopause on dry skin was obtained from a study that evaluated skin water-holding capacity in postmenopausal women to determine whether hormone replacement therapy had an effect [5]. Skin capacitance and transepidermal water losses were measured on normal-looking skin and at the site of plastic occlusion. Investigators demonstrated that the water-holding capacity of the stratum corneum was significantly increased at the plastic occlusion stress test site in women receiving transdermal estrogen compared to menopausal women not receiving transdermal estrogen [5]. Improved water-holding capacity is associated with improved barrier function of the skin. The lack of an effective barrier may also result from stripping of the skin surface sebum and other natural moisturizers by harsh soaps, astringents, or other cleansers. Frequently, patients will claim that they have used the same soap for decades and have difficulty accepting advice to change their bathing habits. They typically will have fewer dry skin symptoms if they use a milder cleanser and less frequent bathing to compensate for the menopausal decrease in natural moisturizing of the skin. Treatment with lotions containing aor fl-hydroxy acids or urea helps to plump the epidermis, through unknown mechanisms, which leads to a more hydrated and resilient skin surface. Notably, these products make skin cracking and hyperkeratosis less evident. Other moisturizers are helpful because they leave a thin film of oil on the surface of the skin, which decreases evaporation of water from the underlying epidermis. In contrast to the weaker barrier function described above in postmenopausal women, some studies using the detergent sodium lauryl sulfate have surprisingly shown that premenopausal skin was more sensitive than postmenopausal skin. When forearm skin was treated with a low concentration of sodium lauryl sulfate, premenopausal patients developed irritant dermatitis more intensely than did postmenopausal patients. Increased transepidermal water loss and skin capacitance as well as visual scoring were utilized to measure the development of irritant dermatitis [6]. Sodium lauryl sulfate-induced irritant contact dermatitis was also compared on forearm and vulvar skin; it was determined that labia majora skin is not more reactive to sodium lauryl sulfate compared to forearm skin. In addition, capacitance, a reflection of skin hydration, was found to be more sensitive than transepidermal water loss in monitoring vulvar irritant dermatitis. Age-related differences in irritant reaction were seen in the forearm, but not the vulva when premenopausal and postmenopausal women were studied [7].

PRYSTOWSKY AND FRANCK

These findings suggest that postmenopausal skin is more resistant to the detergent action of sodium lauryl sulfate but are not consistent with clinical experience and may reflect unique properties of sodium lauryl sulfate or the population studied. Differences observed between pre- and postmenopausal forearm skin could be confounded, for example, by the degree of photoaging present at the time of the studies.

III. SWEATING In addition to the physical unpleasantness of hot flushes, patients experiencing hot flushes suffer disturbing ramifications from these unpredictable drenching sweats. Typically, they will be embarrassed by the impact of copious quantities of sweat--soiled clothing, mined hairdo, and flushed appearance (suggesting nervousness, which confers a psychosocial disadvantage in many situations). Hot flushes occur in about 70% of women undergoing menopause; they are associated with estrogen withdrawal and, fortunately, disappear with estrogen-based hormone replacement therapy [8]. The exact mechanism of hot flushes has not been elucidated but is likely to be related to heat-loss mechanisms. Flushes represent a disturbance in thermoregulation and occur in parallel to changes in skin temperature, blood flow, pulse rate, and pulses of luteinizing hormone. A study of hot flushes has revealed that an increase in core body temperature precedes a majority of menopausal hot flushes and is likely to be a trigger of this heat-loss phenomenon [9] (see Chapter 14). Topical antisweating remedies may be of benefit for small regions of the body; they are generally considered impractical for hot flushes because of the extent of the sweating reaction.

IV. ATROPHY Wrinkles that occur due to sagging of skin over underlying muscles are a hallmark sign of facial aging. The sagging occurs for many reasons, including intrinsic aging and extrinsic aging of the skin and subcutaneous tissue. Intrinsic aging occurs due to the chronological aging process alone and includes the effects from the hormonal changes in menopause. Extrinsic aging of the skin occurs in addition to intrinsic aging and is represented by changes due to sun exposure, chemicals, and other environmental insults on the skin. A representative example of intrinsically aged skin is the buttocks or other area of the body that has received minimal sun exposure. The skin develops atrophy clinically evident as the epidermis takes on a "cigarette paper" crinkling. In contrast, a notable example of extrinsic aging (photoaging) would be the face and forearms of a career golfer or tennis player with fair skin. Patients with lighter skin (types I and II) are more vulnerable to sun damage and demonstrate more significant extrinsic aging changes than do patients with darker skin

CHAPTER20 Menopause and the Skin types. The hormonal changes, which occur as a result of menopause, contribute to intrinsic aging of the skin. Both intrinsic and extrinsic aging of the skin cause notable weaknesses in the structural proteins elastin and collagen, which enhance the sagging or wrinkling attributes of the skin. This discussion focuses on the effects related to intrinsic aging only. It has been demonstrated that collagen levels in the skin taken from a non-sun-exposed area (lower abdomen) decline in women over 50 years old and after menopause [10]. Hormone replacement therapy induced increases in skin collagen content during 12 months of therapy. Collagen loss in the skin was prevented in postmenopausal women treated with hormone replacement therapy [10]. Thus, treatment of patients with estrogen can both prevent and/or restore collagen levels in postmenopausal women [11]. In women with high levels of collagen, these levels are maintained, and in women with low levels of collagen they are increased compared to levels demonstrated at the start of hormone replacement therapy. These beneficial effects contribute toward increased skin resilience and thickness. These data suggest that hypoestrogenism in postmenopausal years has a significant effect on skin and collagen content. Changes in skin collagen levels are closely correlated with decreases in bone mass [12]. In support of these findings, a group of investigators found that skinfold thickness of the back of the hand correlated with the presence of osteoporosis. Thus, the lower the skin fold thickness, the greater the probability of underlying osteoporosis [13]. The anecdotal clinical observation of "thin skin and weak bones" is held up when evaluated scientifically. A similar positive correlation between skin thickness and bone density was obtained when skin thickness was measured using ultrasound and compared to bone densitometry [14] (see Chapter 17). Type III collagen levels in the skin were found to correlate positively with hormone replacement therapy in postmenopausal women [15]. Patients receiving subcutaneous estradiol and testosterone for a median of 8.0 years had significantly greater levels of type III collagen in skin biopsies from the lateral thigh than did those not on replacement therapy. When patients previously not on hormone replacement therapy were treated for 6 months, the proportion of type III collagen increased significantly. Of note, type III collagen predominates in the gastrointestinal and vascular connective tissues and also represents approximately 10% of the total collagen in the adult human dermis (type I collagen predominates in bone and tendon and forms the bulk of skin collagen). Most studies of collagen content in skin have concentrated on type III collagen for quantitative measurements because of the ease of the assay compared to other structural proteins. Skin collagen content has also been found to correlate with urethral pressure [16]. Thus, the beneficial effect of estrogens on urethral function may be mediated by collagen.

311 Evidence for a role of menopause in elastin fiber changes was found by studying elastic fibers from sun-protected buttock skin of menopausal women by light and electron microscopy. In three women (ages 3 0 - 3 7 years) with histories of premature menopause, the elastic fibers had degenerative changes, which included coalescence of cystic spaces into lacunae, peripheral fragmentation, granular degeneration, and splitting of the fibers into strands. Comparable changes in elastin fibers due to intrinsic aging would typically be seen in patients 20 years older than these patients, suggesting that premature estrogen deprivation may have caused these findings [ 17]. This limited study suggests that the hormonal changes that occur in menopause could contribute to weakening the elastic fibers in the skin, making it easier to tear when stretched (see Section V). The effects of race and hormone replacement therapy on wrinkling, dryness, and atrophy were ascertained in close to 4000 postmenopausal patients aged 40 years and greater [18]. The odds of wrinkling were substantially lower in estrogen users; estrogen use was not associated with skin atrophy. When age, body mass index, and sunlight exposure were adjusted for, estrogen use was associated with a statistically significant decrease in the likelihood of senile dry skin. The prevalence of atrophy, dry skin, and wrinkled skin was lower in African-American women than in white women. This is consistent with the well-known protective effects of melanin for protection of photoaging (extrinsic). Thus, estrogen use appears helpful in decreasing dry skin and wrinkles to correct intrinsic aging due to menopausal hormone changes [4]. In summary, skin thickness has been demonstrated to increase in thickness by 10-20% in women treated with hormone replacement therapy compared to women who were not treated [4]. These benefits are likely to be helpful in preventing the hormonal aging changes contributing to skin atrophy and fragility in the elderly. However, hormonal therapy is not expected to counteract the actinically induced changes in the skin, which contribute to atrophy and skin cancer.

V. SLOWED W O U N D HEALING Atrophic skin tears very easily (Fig. l a), requiring repair with paper strips (Fig. lb) because the wounds are full thickness and sutures frequently tear the skin further. When these wounds heal, they form linear scars on the forearms and legs of women. (Figure l c). In the vulva, atrophic skin causes decreased sexual endurance and dyspareunia. To measure the resilience of the skin, age-related changes in viscous deformation and biological elasticity in forearm and vulvar skin have been determined [19]. The mechanical properties of the skin were compared in nonmenopausal women, menopausal women with hormone replacement therapy, and

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menopausal women without hormone replacement therapy [9]. Looking at the volar forearm using a computerized suction device, a steep increase in skin extensibility was seen in perimenopausal women who were untreated. Hormone replacement therapy appeared to limit the age-related increase in skin elasticity. These findings suggest that hormone replacement therapy might help prevent skin slackness and formation of skin tears. An adverse effect of estrogen deficiency on wound healing has been observed in a study of wound contraction in rats that were oophorectomized and a significant reduction in plasma estradiol concentrations was measured [20]. When full-thickness wounds were made in the flank skin, slower wound contraction was observed in rats wounded 4 months after oophorectomy. Earlier time points showed no difference in wound contraction, demonstrating that there is a delayed effect on wound healing [ 16]. In humans, an impairment of granulation tissue formation was observed in postand perimenopausal patients compared to premenopausal patients [21]. The delayed wound healing in menopausal women is an added concern because this occurs in addition to the increase in skin fragility from atrophy. Thus, not only is there an increase in tendency toward forming wounds, but those that form will be likely to heal more slowly in the absence of hormone replacement therapy.

VI. TOPICAL

HORMONE

REPLACEMENT

THERAPY

Hormone replacement therapy (HRT) consisting of estrogen with or without a progestin has been advocated to help treat or prevent changes that occur related to menopause. These include hot flushes, vaginal and urinary tract atrophy, sexual problems, aging skin, and affective symptoms. In addition, HRT it is seen as a preventive measure for osteoporosis and cardiovascular disease. However, increased risk for development of malignancy, usually either uterine or breast, is cited as a potential concern that contributes to patient fear of hormone replacement therapy and limits how widely it is utilized [ 19]. The mode of delivery of hormone replacement therapy, i.e., topical versus oral, has different considerations with respect to the skin and the direct effects of topical estrogen treatment on the skin are discussed below. Topical estrogen can be delivered to the skin as a way to provide low doses of estrogen directly to a target tissue of concern, e.g., the aging face or vulva, without delivering a systemic dose. Alternatively, the skin may be used as the topical route of delivery of a systemic dose of estrogen to provide full hormone replacement therapy. This approach avoids the need for oral ingestion of a pill. Topical treatment of the aging face with estrogen has been demonstrated to decrease various aging skin symptoms in

perimenopausal women [22]. Investigators have used 0.3% estriol cream or 0.01% estradiol cream for 6 months. The estriol group showed slightly superior results compared to the estradiol group. To minimize the risk of systemic hormonal side effects, the concentration of the hormone and size of the application field should be limited if this approach is to be taken. For systemic hormone replacement therapy, transdermal estradiol patches are affixed to the skin and provide a percutaneous delivery of estradiol into the systemic circulation at a constant rate for up to 4 days. Because this method avoids first-pass hepatic metabolism, premenopausal levels of estradiol can be maintained in postmenopausal women using very low dosages, thus helping to prevent bone mineral density loss and other changes, such as vaginal cytology, which occur during menopause. This form of estradiol delivery is tolerated well, with the chief side effect being local irritation at the application site [23] (Fig. 2). To minimize side effects from transdermal delivery, the delivery system needs to accommodate differences in climate. A hot and humid climate decreases the tolerance to estradiol patches. A study in Thailand demonstrated that patients treated with an estradiol-containing topical gel that dried within 3 min had fewer side effects on the skin than did patients treated with an estradiol patch. Patients treated with the patch developed itching, vesicular rash, and residual pigmentation in 58% of patients whereas no skin side effects were seen with the gel. Both the patch and the gel were equivalent pharmacokinetically [24]. Short-term (50 days) local effects of topically applied estrogens do not appear to have any notable effects on epidermal hydration (electrical capacitance and conductance) and mechanical properties [25]. In another investigation, estriol treatment for 3 weeks to the abdominal skin of 14 postmenopausal women resulted in thickening of elastic fibers in the papillary dermis, with better orientation, and slight increase in number when compared to control patients that received a similar ointment without estriol. Epidermal thickness was increased slightly in a few of the patients and no changes were observed in epidermal cell size, mitotic activity, dermal vascularization, or inflammatory infiltrate in the specimens taken before and after the treatment or between the treatment groups. An infrequent but untoward effect of topical sensitization to a transdermally administered drug is allergy induction that will preclude systemic administration of the drug by a different (e.g., oral) route. A case has been described in which contact eczema developed with use of a transdermal estrogen system [26]. Generalized eczema later developed after oral administration of the estrogen derivative. Patch testing confirmed that the patient was allergic to the active drug 17fl-estradiol. Whether the patient would have developed an allergy to the drug administered orally alone cannot be determined. It is conceivable that challenging the patient

CHAPTER 20 Menopause and the Skin topically with the e s t r o g e n drug lead to a h e i g h t e n e d risk of allergic sensitization; this is a possible side effect of topical e s t r o g e n administration.

VII. P S Y C H O L O G I C A L E F F E C T S OF MENOPAUSAL CHANGES RELATED TO SKIN APPEARANCE--SELF-IMAGE Patients with skin c h a n g e s related to aging and m e n o pause frequently express c o n c e r n b e c a u s e these c h a n g e s are a visible and a frequent r e m i n d e r that they are getting older every time they look at t h e m s e l v e s in the mirror. W o m e n w h o have had a m a s t e c t o m y or other disfiguring surgery m a y be m o r e sensitive to the visible c h a n g e s in their facial appearance. Referral to a d e r m a t o l o g i s t or c o s m e t i c s u r g e o n can assist patients with the p r o b l e m s associated with the c h a n g e s in their skin and appearance and at the s a m e t i m e screen for p r e c a n c e r o u s and cancerous lesions. Topical retinoids, ce- and f l - h y d r o x y acids, c h e m i c a l peels, d e r m a b r a sion, and laser resurfacing are a m o n g the m a n y a p p r o a c h e s to r e j u v e n a t i n g the skin for w o m e n in the m e n o p a u s a l p h a s e of their life. T h e s e agents and p r o c e d u r e s principally help a m e l i o r a t e the extrinsic aging c h a n g e s that a c c o m p a n y the intrinsic aging c h a n g e s that occur in m e n o p a u s e . H o r m o n e r e p l a c e m e n t therapy is an i m p o r t a n t c o m p o n e n t of a facial r e j u v e n a t i o n strategy.

VIII. S U M M A R Y H o r m o n e r e p l a c e m e n t therapy has b e e n d e m o n s t r a t e d to e n h a n c e skin thickness and this is likely to be s e c o n d a r y to its effects on the structural proteins, collagen, and elastin. H o r m o n e r e p l a c e m e n t t h e r a p y has also b e e n correlated with i m p r o v e d w o u n d h e a l i n g and tissue resilience. T h e s e positive c h a n g e s support the c o n c e p t that in the a b s e n c e of contraindicating factors, h o r m o n e r e p l a c e m e n t t h e r a p y s h o u l d be strongly c o n s i d e r e d to help m a i n t a i n skin integrity in m e n o p a u s a l w o m e n [27]. A l t h o u g h local reactions to topical a d m i n i s t r a t i o n have occurred, these have g e n e r a l l y b e e n mild; topical h o r m o n e r e p l a c e m e n t t h e r a p y appears to be a c o n v e n i e n t and useful way to provide h o r m o n e t h e r a p y to m e n o p a u s a l w o m e n in a safe and effective manner. Finally, it should be r e m e m b e r e d that h o r m o n e r e p l a c e m e n t t h e r a p y is an i m p o r t a n t c o m p o n e n t of aesthetic r e j u v e n a t i o n of the aging face.

References

1. Bologna,J. L. (1993).Dermatologic and cosmetic concerns of the older woman. Clin. Geriatr. Med. 9, 209-229.

3 13 2. DeLignieres, B. (1991). Ovarian hormones and cutaneous aging. Rev. Fr. Gynecol. Obstet. 86, 451-454. 3. Mechain, C., and Kuttenn, F. (1993). Natural history of menopause. Rev. Prat. 43, 2597-2601. 4. Vaillant, L., and Callens, A. (1996). Traitement hormonal substitutif et vieillissement cutane. Therapie 51, 67-70. 5. Pierard-Franchimont, C., Letawe, C., Goffin, V., and Pierard, G. E. (1995). Skin water-holding capacity and transdermal estrogen therapy for menopause: A pilot study. Maturitas 22, 151-154. 6. Eisner, P., Wilhelm, D., and Maibach, H. I. (1991). Effect of lowconcentration sodium lauryl sulfate on human vulvar and forearm skin. Age-related differences. J. Reprod. Med. 36, 77-81. 7. Eisner, P., Wilhelm, D., and Maibach, H. I. (1990). Sodium lauryl sulfate-induced irritant contact dermatitis in vulvar and forearm skin of premenopausal and postmenopausal women. J. Am. Acad. Dermatol. 23, 648-652. 8. Backstrom, T. (1995). Symptoms related to the menopause and sex steroid treatments Ciba Found. Symp. 191,171-180; discussion: pp. 180186. 9. Woodward, S. (1996). Core body temperature during menopausal hot flushes. Fertil. Steril. 65, 1141-1144. 10. Castelo-Branco, C., Duran, M., and Gonzalez-Merlo, J. (1992). Skin collagen changes related to age and hormone replacement therapy. Maturitas 15, 113-119. 11. Moniz, C. F., Magos, A., de Trafford, J., and Studd, J. W. (1987). Skin collagen changes in postmenopausal women receiving different regimens of estrogen therapy. Obstet. Gynecol. 70, 123-127. 12. Castelo-Branco, C., Pons, F., Gratacos, E., Fortuny, A., Vanrell, J. A., and Gonzalez-Merlo, J. (1994). Relationship between skin collagen and bone changes during aging. Maturitas 18, 199-206. 13. Orme, S.M. (1994). Is a low skinfold thickness an indicator of osteoporosis? Clin. Endocrinol. (Oxford) 41, 283-287. 14. Gruber, D. (1995). Measuring skin thickness in perimenopausal women. Correlation with bone density and hormone parameters. UItraschall Med. 16, 22-24. 15. Laurent, G., Watson, N., and Studd, J. (1993). Type III collagen content in the skin of postmenopausal women receiving oestradiol and testosterone implants. Br. J. Obstet. Gynaecol. 100, 154-156. 16. Punnonen, R., Vaajalahti, P., and Teisala, K. (1987). Local oestriol treatment improves the structure of elastic fibers in the skin of postmenopausal women. Ann. Chir. Gynaecol., Suppl. 202, 39-41. 17. Bolognia, J. L., Braverman, I. M., Rousseau, M. E., and Sarrel, P. M. (1989). Skin changes in menopause. Maturitas 11, 295-304. 18. Dunn, L. B., Damesyn, M., Moore, A. A., Reuben, D. B., and Greendale, G. A. (1997). Does estrogen prevent skin aging? Results from the First National Health and Nutrition Examination Survey (NHANES I). Arch. Dermatol. 133, 339-342. 19. Eisner, P., Wilhelm, D., and Maibach, H. I. (1990). Mechanical properties of the human forearm and vulvar skin. Br. J. Dermatol. 122, 607614. 20. Calvin, M., Dyson, M., Rymer, J., and Young, S. R. (1998). The effects of ovarian hormone deficiency on wound contraction in a rat model. Br. J. Obstet. Gynaecol. 105, 223-227. 21. Gniadecki, R., Wyrwas, B., Kabala, A., and Matecka, J. (1996). Impairment of granulation tissue formation after menopause. J. Endocrinol. Invest. 19, 215-218. 22. Schmidt, J.B., Binder, M., Macheiner, W., Kainz, C., Gitsch, G., and Bieglmayer, C. (1994). Treatment of skin ageing symptoms in perimenopausal females with estrogen compounds. A pilot study. Maturitas 20, 25-30. 23. Balfour, J. A., and McTavish, D. (1992). Transdermal estradiol. A review of its pharmacological profile, and therapeutic potential in the prevention of postmenopausal osteoporosis. Drugs Aging 2, 487-507. 24. Sentrakul, P., Chompootaweep, S., Sintupak, S., Tasanapradit, P., Tunsaringkarn, K., and Dusitsin, N. (1991). Adverse skin reactions to trans-

314 dermal oestradiol in tropical climate. A comparative study of skin tolerance after using oestradiol patch and gel in Thai postmenopausal women. Maturitas 13, 151-154. 25. Jemec, G. B., and Serup, J. (1989). Short-term effects of topical 17 beta-oestradiol on human post-menopausal skin. Maturitas 11, 2 2 9 234.

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26. E1 Sayed, F., Bayle-Lebey, P., Marguery, M. C., and Bazex, J. (1996). Systemic sensitization to 17-beta estradiol induced by transcutaneous administration. Ann. Dermatol. Venereol. 123, 26-28. 27. Pierard, G. E., Letawe, C., Dowlati, A., and Pierard-Franchimont, C. (1995). Effect of hormone replacement therapy for menopause on the mechanical properties of skin. J. Am. Geriatr. Soc. 43, 662-665.

2HAPTER 2

Estrogen Therapy and the Brain VICTOR W. HENDERSON

I. II. III. IV. V.

Department of Neurology, University of Southern California, Los Angeles, California 90089

Chapter Overview Estrogen and the Brain Estrogen Therapy and Cognitive Skills Estrogen Therapy, Affective Disorders, and Mood Estrogen Therapy and Alzheimer's Disease

VI. Estrogen Therapy and Stroke VII. Estrogen Therapy and Parkinson's Disease VIII. Concluding Perspective on Estrogen and the Brain References

I. C H A P T E R O V E R V I E W

II. E S T R O G E N A N D T H E B R A I N The brain is a key target organ for gonadal steroids, including estrogen. Some actions of estrogen on the central nervous system--particularly organizational effects that occur in early stages of prenatal and postnatal development-are conceptualized as permanent. Other estrogen actions, both short- and long-term, are viewed as more malleable and are thus of more particular therapeutic interest [1]. Within a few years of a woman's last menstrual cycle, the ovarian production of estrogen has essentially ceased. Only small amounts of estrogen are produced from the peripheral conversion of androgenic precursors [2], and circulating concentrations of estrogens plummet [3]. A woman will spend about 40% of her adult life in a state of relative estrogen deprivation unless she uses replacement hormones after the menopause. The CNS consequences of menopausal estrogen loss are only partially understood, but they may be germane to both normal and pathological brain function. A number of estrogen actions could affect brain processes important in health and disease [ 1] (Table I). These include

Estrogen acts on the brain through receptor- and nonreceptor-mediated mechanisms. After the menopause, a marked decline in circulating concentrations of estrogen has the potential to influence central nervous system (CNS) functions important in health and disease. Estrogen appears to influence cognitive abilities and mood, but it is unsettled whether the behavioral effects of hormone therapy are clinically germane to most healthy women. Estrogen modulates processes that could have an impact on several neurological disorders prevalent in an aging population. Data on disease prevention, supported by strong biological plausibility, are well developed with respect to Alzheimer's disease, for which most epidemiological studies report significant associations between the use of estrogen therapy and reduced Alzheimer risk. Preliminary studies do not suggest an important link between estrogen and Parkinson's disease. There is a plausible rationale, but as yet no persuasive evidence from human studies, to consider a protective effect of estrogen against ischemic cerebrovascular disease.

MENOPAUSE: BIOLOGY AND PATHOBIOLOGY

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Copyright9 2000by AcademicPress. Allrightsof reproductionin anyformreserved.

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TABLE I

Effects of Estrogen on Brain Functiona

Organizational actions Effects on neuronal number, morphology, and connections occurring during critical stages of development Neurotrophic actions Neuronal differentiation Neurite extension Synapse formation Interactions with neurotrophins Neuroprotective actions Protection against apoptosis Antioxidant properties Antiinflammatory properties Augmentation of cerebral blood flow Enhancement of glucose transport into the brain Blunting of corticosteroid response to behavioral stress Interactions with neurotrophins Effects on neurotransmitters Acetylcholine Noradrenaline Serotonin Dopamine Glutamate Gamma aminobutyric acid Neuropeptides Effects on glial cells Effects on proteins involved in Alzheimer's disease Amyloid precursor protein Tau protein Apolipoprotein E a Modified from Henderson [ 1]. Different actions of estrogen are not mutually exclusive.

effects mediated by specific intranuclear receptors [4] that regulate the synthesis of specific gene products, as well as more rapid effects that occur independently of genomic activation and implicate putative receptors on the plasma membrane [5,6]. Within the CNS, the identification of two different classes of intranuclear estrogen receptors (a and fl) with distinct topographical distributions [7] is but one indication that estrogen can modulate functions of specific neuronal subsets. Interestingly, estrogen has an impact on glial cells [8] as well as on neurons. Estrogen is both neurotrophic and neuroprotective (Table I). Estrogen may modulate neuronal growth and development through interactions with a class of proteins know as neurotrophins [9]. In vivo [10,1 l] and in laboratory animals [12], estrogen promotes the sprouting of nerve processes of responsive neurons, as well as the formation of synaptic connections [ 13,14]. Estrogen may protect neurons from a variety of exogenous insults, including oxidative stress, excitatory neurotoxicity, and ischemia [15-19], and it may help prevent programmed cell death (apoptosis) [20]. Effects on the immune system [21 ], the behavioral stress response [22], cerebral blood flow [23], and glucose transport across the

blood-brain barrier [24] may be important in some disease states. This chapter critiques observational and experimental findings on estrogen that are clinically relevant to normal brain functioning in the postmenopausal period. The effect of estrogen therapy in two neurodegenerative disorders, Alzheimer's disease and Parkinson's disease, is also considered, as well as the possible impact of estrogen on stroke. Conceptual and factual gaps impede completion of a scientific edifice on which to. base interventional decisions, but for some clinical issues at least the intimation of a coherent structure has taken form. Potential effects of estrogen on neuropsychiatric disorders whose greatest clinical impact occurs before the menopause (for example, migraine headache, multiple sclerosis, epilepsy, and schizophrenia) are not addressed in this chapter.

III. ESTROGEN THERAPY AND COGNITIVE SKILLS Experimental studies of memory using adult ovariectomized rats suggest that estrogen replacement improves certain learning and memory behaviors. For example, estrogen treatment after ovariectomy is reported to boost performances on water escape [25], passive avoidance [26], and spatial memory (radial maze) [27] tasks. Human studies have also examined estrogen effects on cognition. A common paradigm has been to consider psychometric performance at different phases of the menstrual cycle. Findings are controversial and not fully coherent [2833]. Verbal fluency, manual speed, articulatory agility, or creativity may be better during the late follicular or midluteal phase of the menstrual cycle, when plasma levels of estrogen are elevated, than during the menses, when estrogen levels are low. Conversely, performance on spatial tasks may be enhanced during the menses. Both verbal fluency [34] and verbal learning [35] are reported to improve when male-tofemale transsexuals are treated with estrogen. Given claims that, on average, some tasks are more easily performed by women (e.g., certain verbal skills, fine motor skills) and others by men (e.g., certain visuospatial skills) [36-38], one broad interpretation of the literature is that estrogen maintains or enhances skills at which women tend to excel vis-avis men but has no effect on, or even impedes, skills viewed as more easily undertaken by men. A population-based study of postmenopausal women reported that women who used estrogen performed better than nonusers on several psychometric measures [39]. Observational studies of healthy community-dwelling older women also suggest that women who use (or who previously used) hormone replacement have better verbal memory or other cognitive skills [40-44]. In contrast, studies in two large co-

CHAPTER21 Estrogen Therapy and the Brain horts failed to verify appreciable differences between postmenopausal women who did and did not use estrogens [45,46], and relatively high serum concentrations of estrogen do not appear to protect postmenopausal women from ageassociated cognitive decline [47] Cognitive effects of estrogen given after the menopause have been analyzed only occasionally in randomized controlled trials. Several investigations show an advantage for treated women on verbal memory or other tasks [48,49]. In one study of verbal memory, the ability to recall information from a paragraph-length story declined in women whose ovarian function had been suppressed with a gonadotropinreleasing hormone agonist; memory scores returned to baseline after estrogen was introduced [50]. In this study, estrogen loss did not affect other cognitive measures. Some authors, in fact, report no effect of estrogen replacement therapy on cognition [51 ].

IV. ESTROGEN THERAPY, AFFECTIVE DISORDERS, AND MOOD Women are more prone to suffer from depression than men [52]. Low or falling levels of estrogen may adversely influence mood. Low mood is more often reported during the premenstrual phase of the menstrual cycle and the postpartum period. Conversely, during the follicular phase of the menstrual cycle, a woman may enjoy a greater sense of wellbeing [30]. Whether low mood is also more prevalent following the climacteric, however, remains controversial [53]. The pharmacological therapy of depression commonly involves agents that act on the monoamines noradrenaline and serotonin, and speculatively, estrogen effects on mood could be mediated through these neurotransmitters [54-58]. Severe depression has been the focus of two randomized treatment trials of estrogen. In one study of 40 psychiatric inpatients with unipolar depression, symptoms abated significantly after 3 months of treatment with very high doses of oral estrogens [59]. Subjects included both premenopausal and postmenopausal women. Another study of 61 women with major depression beginning within 3 months of childbirth found transdermal estrogen to be effective, with improvement in postnatal depression evident within a month of initiating therapy [60]. Of broader clinical relevance, most studies of estrogen and mood have involved women without diagnosed depression. In the late postmenopausal period, women who receive estrogen replacement report fewer depressive symptoms than do other women [61 ]. Estrogen replacement after the menopause is reported to reduce anxiety, improve mood, and enhance subjective well-being [62-68]. Of some concern, apparent benefits on mood may be reduced when estrogen is administered in combination with a progestogen [69].

317

V. ESTROGEN THERAPY AND ALZHEIMER'S DISEASE The most common cause of dementia is Alzheimer's disease, accounting for one-half to three-fourths of all cases. In Alzheimer's disease, memory deficits and other signs of cognitive dissolution begin insidiously and progress gradually over a period of a decade or longer. The clinical and pathological phenotype is similar regardless of underlying etiology, but different genetic and nongenetic factors can culminate as the clinical entity of Alzheimer's disease. Highly penetrant autosomal dominant forms of the disord e r - w i t h dementia symptoms first appearing in the fourth, fifth, or sixth decades of life--are often caused by point mutations in genes encoding the presenilin proteins (chromosomes 14 and 1) and the amyloid precursor protein (chromosome 21) [70]. More commonly, Alzheimer's disease emerges as "lateonset" dementia after about age 60 years. In late-onset disease, so-called susceptibility genes can influence risk, but they do not lead to dementia in an all-or-none manner. The first such identified gene is the chromosome 19 gene that encodes for apolipoprotein E, a lipid transport protein important for neuronal repair [71 ]. Increased risk results from possession of the e4 allele of apolipoprotein E [72], which is associated with a reduction in neuronal plasticity [73]. Gender also plays a role, and women who possess a copy of the e4 allele appear at greater risk than do men of the same genotype [74]. Other susceptibility genes are suspected but remain to be confirmed. Other factors also are postulated to increase Alzheimer risk, including advanced age, reduced educational attainment, female gender, head injury, and depression [75]. Pharmacological exposures of interest include nonsteroidal antiinflammatory drugs [76-78] and estrogen (see below), both associated with decreased risk. Neurotrophic and neuroprotective effects of estrogen are of obvious import to Alzheimer's disease (Table I). Estrogen effects on various neurotransmitter systems are another means by which estrogen might have an impact on Alzheimer's disease. For example, the pharmacological management of Alzheimer patients has largely focused on acetylcholine, a key transmitter involved in attention and memory [79]. Cholinergic neurons of the basal forebrain region possess receptors for estrogen [80], and these nerve cells are clearly affected in Alzheimer's disease [81 ]. Experimentally, estrogen treatment elevates cholinergic markers in the basal forebrain and in projection target areas of basal forebrain neurons [82,83]. In ovariectomized rats, estrogen prevents learning deficits induced by the pharmacological blockade of cholinergic (muscarinic) receptors [84]. Female Alzheimer patients treated with an agent that prevents the degradation of acetylcholine appear to respond better if they use

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TABLE II

Study

Interventional Studies of Estrogen for Women with Alzheimer's Disease a Year of publication

Total number of subjects b

Treatment duration

Randomized, double-blind trial?

Improvement claimed? c

Fillit et al. [93]

1986

7

6 weeks

No

Yes

Honjo et al. [94]

1989

14

6 weeks

No

Yes

Honjo et al. [95]

1993

14

3 weeks

Yes

Yes

Fillit [96]

1994

8

3 months

Yes

No

Ohkura et al. [97]

1994

6 weeks 6 weeks

No No

No Yes

Ohkura et al. [98]

1994

30

6 weeks

No

Yes

Ohkura et al. [99]

1994

20

5 months

No

Yes

Ohkura et al. [ 100]

1995

7

variable ( 5 - 4 5 months)

No

Yes

Asthana et al. [ 101 ]

1997

10

8 weeks

Yes

Yes

Birge [ 102]

1997

20

9 months

Yes

Yes

5 inpatients 6 outpatients d

a Modified from Henderson [ 103] with permission of the publisher. b Subjects included Alzheimer patients treated with oral or transdermal estrogen and, in some studies, Alzheimer controls who were either untreated or who were given placebo. c Criteria for improvement varied among different studies, and improvement when present did not necessarily indicate improvement on all outcome measures. dlnpatients in this study were more severely demented than were outpatients.

concomitant estrogen [85]. In addition to cholinergic effects, estrogen influences noradrenaline and serotonin [58,86], other neurotransmitter systems disrupted by Alzheimer's disease. Estrogen affects fl-amyloid and tau, key biochemical features of Alzheimer pathology, fl-Amyloid is deposited in neuritic plaques of Alzheimer brain, and a phosphorylated form of tau protein is the major constituent of neurofibrillary tangles. Estrogen modifies metabolism of the amyloid precursor protein, reducing formation of the fl-amyloid fragment [87]. Estrogen may also contribute to microtubule stability by facilitating the induction of tau, a microtubuleassociated protein [88]. There is also a link between estrogen and apolipoprotein E. Estrogen-dependent neuronal sprouting does not occur in the absence of apolipoprotein E [89]. Apolipoprotein E levels are reduced in Alzheimer brain [90], and estrogen increases the expression of central nervous system apolipoprotein E [91 ]. A. E s t r o g e n and A l z h e i m e r ' s D i s e a s e S y m p t o m s It is observed that women with Alzheimer's disease who take estrogen perform substantially better in several cognitive domains than women do with Alzheimer's disease (equivalent in age, education, and duration of dementia symptoms) who do not take estrogen [92]. Several small formal interventional trials have attempted to treat Alz-

heimer symptoms with estrogen [93-102] (Table II). Results have been encouraging, with positive findings in virtually all studies (reviewed by Henderson [103]). However, only a few have been conducted as randomized controlled trials [95,101,102], and complete details are available for only one of these [95]. Meaningful conclusions await results of larger randomized double-blind, placebo-controlled trials.

B. E s t r o g e n a n d A l z h e i m e r ' s Disease: Primary Prevention If estrogen therapy protects against Alzheimer's disease, then it might be expected that relatively fewer women with this diagnosis would use estrogen. Studies of Alzheimer patients support this prediction (Fig. 1). In 1994, Birge [104] in St. Louis reported that not a single person among 158 women in his Alzheimer clinic was taking estrogen. That same year, Henderson et al. [105] in Los Angeles compared 143 women with Alzheimer's disease and 92 older women without dementia, who were enrolled in a longitudinal study of aging and dementia. Fewer than half as many cases as controls used estrogen (7% versus 18%). Similar results were subsequently described by Mortel and Meyer [106] in Houston, who compared 93 clinic cases (12% estrogen use) to a convenience sample of 148 controls (20% use). Note that these analyses were based on current estrogen use. If

CHAPTER21 Estrogen Therapy and the Brain

319

FIGURE 1 Postmenopausal estrogen therapy and the risk of Alzheimer's disease. Data published in the 1990s are presented for case-control and cohort studies that evaluated the effects of estrogen therapy on Alzheimer's disease risk. A protective effect of therapy is suggested by relative risk estimates (odds ratios) less than 1. For each study, the upper limit of the 95% confidence interval is also given, when available. Statistically significant studies (indicated by asterisks) are those in which the upper confidence limit was also less than 1. Studies in which information on estrogen exposure was collected prospectively (i.e., before the onset of dementia symptoms) are represented by solid black bars. Data are from Broe et al. [108] (Australia), Graves et al. [109] (Seattle), Birge [104] (St. Louis), Henderson et al. [105] (Los Angeles), Brenner et al. [ 115] (Puget Sound), Mortel and Meyer [ 106] (Houston), Paganini-Hill and Henderson [ 114] (Leisure World), Tang et al. [ 116] (New York), van Duijn et al. [ 110] (Rotterdam), Lerner et al. [ 111 ] (Cleveland), Kawas et al. [ 117] (Baltimore), Waring et al. [ 118] (Rochester, Minnesota), and Baldereschi et al. [112] (Italy). Leisure World data of Paganini-Hill and Henderson [113] from 1994 are not shown, because Alzheimer cases in this study were included as a subset of cases reported in 1996 [114], and estimates of relative risk were similar in the two studies.

demented women are more likely to be prescribed estrogen in an attempt to palliate symptoms, the resulting bias would underestimate the magnitude of any protective effect. More importantly, if there was a tendency for women who develop cognitive deficits not to initiate estrogen therapy or to discontinue estrogen, then a protective effect could be erroneously inferred. Indeed, estrogen users tend to be healthier than nonusers [ 107], and in contrast to these results, earlier case-control studies from Australia and Seattle [108,109] had found no significant association between estrogen therapy and the risk of developing Alzheimer's disease. More recent epidemiological studies, however, have generally supported inferences initially based on analyses of current use (Fig. 1). In a Dutch population-based study of early-onset disease, van Duijn et al. [ 110] found that women with Alzheimer's disease were significantly less likely to have used estrogen (but see below), as did Lerner et al. [ 111 ] in Cleveland (25% of 88 cases versus 45% of 176 controls), and Baldereschi et al. [112] in a large population-based Italian study that included 92 Alzheimer cases and 1476 women without dementia (3% versus 12% estrogen use). Several cohort and nested case-control analyses have had access to information on estrogen exposure that was col-

lected prospectively, before the onset of dementia symptoms. The largest such study [113,114] was based in Leisure World, a predominantly White upper middle class retirement community in southern California. The Leisure World cohort was established by postal survey in the early 1980s, with information on estrogen exposure collected from each woman at the time of her enrollment. Death certificate records were subsequently obtained for deceased participants. For female cohort members who died before 1996, PaganiniHill and Henderson [ 114] identified 248 Alzheimer's disease cases and 1198 controls from death certificate records. Postmenopausal estrogen therapy had been reported by 39% of women later identified as Alzheimer cases and 48% of women classified as controls. Different routes of estrogen administration (oral, oral plus injection or cream, injection or cream) were each associated with significant risk reductions [114]. In Leisure World, diagnoses were not based on in-person assessments, and death records almost certainly missed a number of Alzheimer cases; however, this misclassification was most likely nondifferential with respect to whether the subject had ever used estrogen, and findings may therefore have substantially underestimated the true association between estrogen therapy and Alzheimer risk reduction.

320 Findings in Leisure World contrast with those obtained from older women enrolled in a large health maintenance organization in the Puget Sound area of Washington state. Brenner et al. [ 115] used computerized pharmacy records to compare estrogen use of 107 women with Alzheimer's disease and 120 nondemented control subjects. Alzheimer cases had been carefully ascertained, and virtually identical proportions of cases (49%) and controls (48%) filled at least one prescription for estrogen. For oral estrogens, the corresponding frequencies were 23% and 28% respectively (this difference favoring estrogen users was not statistically significant). Positive results, however, are reported from New York, Baltimore, and Rochester, Minnesota, where subjects had also been examined in person to determine the presence of Alzheimer's disease. In a community-based cohort from New York City, 167 new cases of Alzheimer's disease were identified by Tang et al. [116]. Only nine (6%) Alzheimer cases, but 15% of women who did not develop dementia, had used oral estrogens at some point after the menopause. Among women with Alzheimer's disease, the nine women who had received estrogen were older than those who had never used estrogen, implying that estrogen therapy may have delayed the onset of dementia symptoms among this small group of estrogen users. Estrogen appeared protective for women who possessed the e4 allele of apolipoprotein E as well as for women who did not have this particular allele. This finding differs from that of van Duijn et al. [110], who found that protective effects of estrogen were apparent only among those early-onset cases of familial Alzheimer's disease with the e4 allele. In the Baltimore Longitudinal Study of Aging, Kawas et al. [117] considered the effects of oral and transdermal estrogen. Nine (26%) of 34 women who developed Alzheimer's disease had reported estrogen use, compared to 47% of 472 older women who remained free of dementia. In a population-based study from Rochester, Minnesota, preliminary analyses by Waring et al. [118] found medical record documentation that 5% of 222 women with Alzheimer's disease had used estrogen for at least 6 months after the menopause, compared to 12% of matched controls. Findings from several of these studies are somewhat strengthened by a positive dose-response relationship. Thus, in Leisure World, the apparent protective effect of estrogen was significantly associated with larger doses of estrogen (within the range of dosages commonly prescribed for postmenopausal use) [114]. A significant association with longer durations of therapy and the degree of risk reduction was reported from Leisure World [114], New York [ 116], and Rochester [ 118], but not from Baltimore [ 117]. Post hoc analyses in the generally negative study from Puget Sound indicated that women who filled the largest number of estrogen prescriptions experienced the lowest Alzheimer risk [115].

VICTOR W. HENDERSON

VI. ESTROGEN

THERAPY

AND S T R O K E Stroke represents cerebral damage or dysfunction caused by vascular disease intrinsic or extrinsic to the central nervous system. Stroke is the third leading cause of death, exceeded only by heart disease and cancer. The incidence of stroke climbs dramatically with age. Rates of stroke are lower in women than men, but sex-specific rates are more concordant in years after the menopause. There are many causes of stroke. An abrupt reduction in cerebral perfusion due, for example, to vascular occlusion typically leads to ischemic and hypoxic damage; perfusion can also be disrupted directly or indirectly (e.g., mass effect or secondary vasospasm) by vascular rupture into the brain substance or the subarachnoid space surrounding the brain. In the older woman, a strong body of coherent evidence suggests lower rates of coronary artery disease in women who use estrogen therapy after the menopause [ 119]. Many risk factors for cerebrovascular disease, including increasing age, hypertension, smoking, diabetes, and hyperlipidemia [120], also predispose to cardiovascular disease. Atherosclerosis within large extracranial vessels supplying blood to the brain (carotid and vertebral arteries) predisposes to cerebral infarction. In the heart, angiographic studies of the coronary arteries clearly link estrogen use to significant reductions in atherosclerosis [ 121 ], attributable in large part to favorable effects on lipoproteins, lipid peroxidation, fibrinolysis, and vascular endothelial function. In light of these observations, it is not surprising that estrogen also appears to prevent or delay atherosclerotic narrowing of the carotid arteries [122]. Estrogen-induced augmentation of cerebral blood flow [23,123] might also benefit cerebral function in the presence of cerebrovascular disease. However, despite similarities between ischemic cardiovascular disease and ischemic cerebrovascular disease, effects of estrogen on stroke are inconsistent across studies; indeed most studies show no significant benefit of estrogen on the overall incidence of stroke [124]. By contrast, a favorable effect of estrogen on stroke mortality is often observed [124], an observation consistent with experimental models of stroke [19]. The discrepancy between apparent cardioprotective effects of estrogen and less clear-cut neuroprotective effects could have several explanations. First, the efficacy of estrogen even for heart disease was challenged by the overall absence of positive findings in a recent randomized trial of estrogen plus progestogen for the secondary prevention of heart attack in women with preexisting coronary artery disease [125]. Perhaps more importantly, in some studies discrepancies may reflect a failure or inability to distinguish among different forms of stroke. For example, risk factors for hemorrhagic and ischemic strokes would be ex-

CHAPTER21 Estrogen Therapy and the Brain pected to differ substantially. Even for ischemic disease, mechanisms and risk factors are not identical for heart and brain [126], and it is noteworthy that analyses limited only to ischemic cerebrovascular disease have not found the degree of risk reduction anticipated on the basis of studies of heart disease [ 127,128]. At present, therefore, effects of estrogen on stroke risk remain uncertain, and a better understanding must await additional data. Similar uncertainties surround the use of estrogen to diminish a woman's risk of dementia caused by cerebrovascular disease, often referred to as multiinfarct dementia. One study compared a group of 65 women with a clinical diagnosis of vascular dementia to 148 women without dementia [106]. Demented women were less likely to be current users of estrogen therapy than controls, but differences were not statistically significant.

VII. ESTROGEN

THERAPY

AND PARKINSON'S DISEASE Parkinson's disease is a common progressive neurodegenerative disorder of the basal ganglia. Key symptoms are tremor, rigidity, and bradykinesia. Most parkinsonian symptoms are attributed to the prominent loss of dopaminecontaining neurons in the substantia nigra, and drugs that increase brain levels of dopamine or activate dopamine receptors lead to clinical improvement. In contrast, hyperkinetic movement disorders such as chorea typically benefit from treatment with dopamine antagonists. Both pregnancy [129] and oral contraceptive use [130] are associated with reversible chorea, suggesting an association between sex hormones and movement disorders. Although the substantia nigra and other portions of the extrapyramidal motor system do not appear to contain large numbers of estrogen receptors [131], estrogen influences dopaminergic activity and motor behaviors mediated by dopamine [132-135]. Nevertheless, evidence is tenuous that estrogen therapy modifies hypokinetic or hyperkinetic symptoms of patients with movement disorders. Younger women with Parkinson's disease occasionally report that disease symptoms worsen at about the time of menstruation [136]. Case reports suggest that estrogen may precipitate parkinsonian symptoms in women receiving neuroleptic therapy [137] and aggravate symptoms in women with Parkinson's disease [ 138,139]. Estrogen may prevent the loss of dopaminergic neurons experimentally induced by neurotoxins [140]. However, in one population-based community survey, estrogen use after the menopause did not appear to affect the risk of developing uncomplicated Parkirlson's disease, although estrogen may have reduced the risk of dementia sometimes associated with this neurodegenerative disorder [ 141 ].

321 VIII. CONCLUDING

PERSPECTIVE

ON ESTROGEN AND THE BRAIN Estrogen exerts both discrete and pervasive effects on brain function. Available evidence implies that estrogen therapy has the potential to benefit mood and certain cognitive skills, but the magnitude of these effects and their clinical import for healthy older women remain controversial. Estrogen actions on physiological and biochemical processes relevant to neurological disease suggest also that this sex steroid could be an important modulator of various pathological states. Progesterone, like estrogen, influences brain function, and initial observations in several clinical conditions raise the possibility that some apparent estrogen benefits could be offset by the administration of a progestogen [69,85,100]. Because women with a uterus cannot receive prolonged unopposed estrogen therapy, future investigations should be directed toward understanding the specificity and magnitude of progestogen effects. The spectra of both receptor- and nonreceptor-mediated actions differ for different estrogens, and the selective estrogen receptor modulators (SERMs) are active only in certain tissues. To the extent that a given estrogen is associated with a clinically important response, future studies will be needed to define more clearly mechanisms of action with respect to the particular response. Such knowledge will permit more rational drug selection and facilitate the rational development of new, more potent, and specific compounds. The possibility that some estrogenic compounds, including the phytoestrogens, may antagonize as well as promote certain estrogenic activities must also be considered. The exciting possibility that estrogen replacement may reduce a woman's risk of Alzheimer's disease is supported by more recent epidemiological data, but the surety of estrogen for primary prevention will be achieved only after results of large-scale randomized controlled treatment trials become available. One of these, an ancillary study of the Women's Health Initiative in the United States, involves approximately 7500 older nondemented women randomly assigned to receive oral conjugated estrogens (with or without a progestogen, depending on the presence or absence of a uterus) or placebo [142]. Results of this ongoing clinical trial are not expected, however, until several years into the third millennium. Initial findings with regard to estrogen and symptomatic treatment (tertiary prevention) in Alzheimer patients are encouraging but not compelling. As noted above, anticipated results of randomized double-blind, placebo-controlled trials may provide more certain guidance. Considerably less is known about possible estrogen effects on Parkinson's disease than on Alzheimer's disease, and current limited findings do not justify large-scale randomized controlled trials in this extrapyramidal disorder. Clearly,

322

VICTOR W. HENDERSON

additional basic and clinical data are welcome. With respect to cerebrovascular disease broadly considered, one cannot conclude from the epidemiological data that estrogen has a primary preventive role. However, the favorable impact of estrogen on serum lipids and vascular reactivity implies that estrogen might still have a role in the primary or secondary prevention of certain kinds of ischemic stroke. Moreover, neuroprotective effects of estrogen also imply a potential role in limiting neurological damage in the setting of acute vascular occlusion.

18.

19.

20.

21.

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~ H A P T E R 2"~

The Gynourinary System GORAN SAMSIOE

Department of Obstetrics and Gynecology, Lund University Hospital, S-221 85 Lund, Sweden

I. Introduction II. Anatomy and Physiology III. Innervation

IV. Mechanisms of Continence V. Treatment Modalities for Female Urinary Incontinence References

I. I N T R O D U C T I O N

50 years [1-8]. Results are variable due mainly to different definitions of incontinence and sometimes to the selection of the women who were studied. From observational studies it can be inferred that urinary incontinence is three times more prevalent in women than in men and that prevalence increases with age. If data from various population-based studies are pooled, incontinence in women seems to increase by 3 - 5 % at age 20 years, 8 - 9 % at age 30 years, and 12-15% at age 50 years. We have completed [9] a study with a detailed questionnaire on urogenital problems concomitant with an interview of each subject. Among the 3000 women studied we can confirm the overall prevalence of poor control of micturition in about one-third of the cases. However, it should be pointed out that some women complain of this only in specific situations, e.g., concomitantly with bronchitis. Incontinence was associated with parity, overweight, and hysterectomy, confirming previous data, but the condition was also associated with a family history of diabetes. As can be expected, there is a variation between prevalence and incidence of various symptoms related to urogenital aging depending on the setup of a given study (see Table III). In comparison with osteoporosis and cardiovascular disease, much less attention has been paid to problems incurred by urogenital aging. Given the present composition of the population in modern Western countries, 8% of the total population experience urogenital problems [8]. In the United States alone, 20 million women suffer from these socially disabling symptoms. The prevalence figures obtained by questionnaire surveys are often higher than the clinical

Advancing age is associated with a decline in a large number of physiological processes. The urogenital system is no exception. Symptoms and signs from urologic structures and the female sex organs often parallel each other, because they originate from a common fetal structure (i.e., the urogenital sinus). Symptoms and signs of lost urogenital integrity increase with age (Table I), but can be modulated by hormonal status as well as lifestyle and environmental factors. By introducing measures that have an impact on lifestyle, environment, and the hormonal situation, it is possible to influence the urogenital aging process and thereby reduce disabling symptoms and signs in some elderly women.

Demography The aging process as well as hormonal insufficiency will ultimately occur in all women. Lifestyle changes often accompany these two processes. There is a huge spectrum of interindividual and intraindividual degree of sensitivity to these changes; symptoms and signs of urogenital aging are therefore highly variable within an individual as well as between individuals (Table II). Several questionnaire-based surveys have been carried out to outline and delineate the nature and occurrence of urogenital problems in women, especially after the age of MENOPAUSE: BIOLOGY AND PATHOBIOLOGY

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TABLE I Common Symptoms of Urogenital Aging Urological symptoms

Vaginal symptoms

Frequent micturation Recurrent cystitis Urethritis Dysuria Urge incontinence Stress incontinence Mixed type of incontinence

Vaginal dryness Loss of lubrication Dyspareunia Vaginitis Vaginal discharge Vulvar itching and burning

II. A N A T O M Y AND P H Y S I O L O G Y A. Bladder The inner surface of the human bladder is lined with a transitional epithelium, which is folded when the bladder is empty and distended when the bladder is full. Below the epithelium, the lamina propria forms a layer of fibroblastic TABLE II Scandinavian Guidelines for Assessment of Female Incontinence a

Patient history

Pelvic examination

Urine test Frequency/volume chart Stress test Pad test a Adapted from Ref. 1.

Country All Denmark France Germany Italy Netherlands United Kingdom

Severity of Urogenital Tract Symptoms a Number affected (% of population)

Severe (%)

Moderate (%)

Mild (%)

869 (29.3) 107 (22.8) 139 (28.4) 129 (26.2) 202 (39.5) 121 (23.4) 171 (35.0)

11.2 13.1 16.5 12.5 9.4 5.8 10.5

40.8 46.7 38.9 34.9 43.6 28.9 48.6

48.0 40.2 44.6 52.7 47.0 65.3 40.9

aAdapted from Ref. 8, Maturitas, Vol. 27, Barlow et al., Prevalenceof urinary problems in European countries, pp. 239-248, Copyright 1997, with permission from Elsevier Science.

experience suggests. Many women feel embarrassed and do not readily address problems related to lost urogenital integrity unless specifically asked. The dollar costs for incontinence have been estimated to be $ 8 - 1 0 billion per annum, corresponding to 2 - 3 % of the total costs of the health care system. [ 10,11 ]. There can be little doubt that incontinence is the major urogenital problem encountered by women. Consequently the focus here is on pathophysiology, diagnosis, and treatment of this disabling disorder. Other problems will be mentioned in brief.

Factor

TABLE III

Assessment Type and severity of incontinence Effect of quality of life Other diseases/drug affecting the lower urinary tract Drinking and voiding habits Pelvic mass Mucosal atrophy Genital descensus Blood, glucose, protein Infection Clarifies patient history Basis for behavioral advice Objective incontinence Quantification of incontinence

connective tissue that contains numerous blood vessels. In the upper part of the bladder, i.e., the detrusor, the smooth muscle cells lack a specific organization. Muscle fibers can travel between each of the layers and branch into a longitudinal or circular direction. Close to the bladder neck, the muscle bundles are arranged in three layers m inner longitudinal, middle circular, and outer longitudinal. In addition, the large-diameter muscle fascicles of the bladder detrusor are replaced by much finer muscle fibers. The triangle between the two urethral orifices and the internal urethral meatus is referred to as the trigone of the bladder. The trigone is also composed of three muscle layers, a superficial layer, a deep layer, and a detrusor layer [12].

B. Urethra The human female urethra is 3 - 4 cm long. It runs inside the adventitial coat of the anterior wall of the vagina and penetrates and passes through the pelvic floor musculature [13]. Its lining changes gradually from transitional to nonkeratinized stratified squamous epithelium. The muscle arrangement in the female differs from that in the male. A thin layer of circular smooth muscle envelops the longitudinal fibers throughout the length of the female urethra [12]. The longitudinal smooth muscle layer of the urethra does not contribute to any "sphincteric" function. It contracts concomitantly with the detrusor muscle layers during micturition to shorten and widen the urethra. The lamina propria is composed of connective tissue, longitudinally oriented smooth muscle cells, and a dense network of blood vessels [14]. The lamina propria contains estrogen receptors and consequently atrophies at menopause, often resulting in clinical symptomatology such as incontinence [ 12]. The striated muscle component of the female urethra, also known as the rhabdosphincter, is thickest around the middle third, and thinner in the proximal and distal thirds. The adjacent periurethral striated muscle is derived from the muscles of the pelvic floor [13]. The anatomical

CHAPTER22 The Gynourinary System features of the lower urinary tract differ in various species. The description herein refers to the bladder and urethra of humans. Hence, results obtained in the experimental animal should be interpreted with care, especially when discussing a perceived clinical relevance in humans.

329 content may further slow down protein turnover, with possible deleterious consequences for the aging organism, e.g., a decrease in elasticity. 1. PARAURETHRAL COLLAGEN AND THE MENOPAUSE

The bladder receives blood supply from the vesicle branches of the internal iliac artery. The veins form the vesicle plexus and drain into the internal iliac veins, whereas lymphatic drainage passes to the external iliac lymph nodes.

Menopause seems to induce a paraurethral connective tissue enriched in collagen concentration and with different mechanical properties due to higher cross-linking [15]. A concomitant decrease in the proteoglycan/collagen ratio suggests a tissue with some increase in load-bearing ability, but a decrease in elasticity [ 16]. The changes are not accompanied by any change in mRNA levels, indicating a decrease in degradation.

D. C o l l a g e n

2. PARAURETHRAL COLLAGEN AND STRESS URINARY INCONTINENT WOMEN OF FERTILE AGE

C. B l o o d S u p p l y

The major structural component of the bladder wall is collagen. This structural protein belongs to a family of proteins that make up approximately 30% of the dry weight of human tissues. Currently, there are 20 different collagen types with approximately 30 unique genes, each of which codes for a unique collagen polypeptide or ce chain. Collagen fibers have the tensile strength of fine steel wire and it is this remarkable strength that provides tissues with their characteristic mechanical properties. Although collagens are major structural proteins, they also have other functions, including those related to cell adhesion, the binding of growth factors, and filtration of proteins. The most abundant collagen is type I, whose molecular structure is the prototype for all collagen fibers. The basic building block of collagen fibers is the collagen molecule, which is composed of three polypeptide chains called a chains. Collagen fibers are composed of molecular units that are synthesized initially as a "pro" form called procollagen. After procollagen molecules are secreted, extracellular enzymatic processing converts the pro forms into collagens. Intracellularly, each of the three polypeptide chains forms left-handed helical coils. The strength and diversity of these coils are related to the chemical composition of different collagen types. Collagen molecules can exist as either homopolymers, composed of identical a chains, or as heteropolymers, composed of dissimilar chains. For example, type III collagen, which is found along with type I in the bladder, is a homopolymer, whereas type I collagen is a heteropolymer composed of two ce1 and a2 chains. Factors such as age, mechanical stress, hormones, enzymes and their inhibitors, growth factors, and cytokines are involved in the regulation of connective tissue metabolism. Collagen turnover becomes slower with increasing age. The collagen concentration is unchanged during fertile age but after menopause increases in different organs [15]. The simultaneous progressive formation of stable collagen molecule networks through the continuous increase in stable cross-link

A higher collagen concentration in combination with higher cross-linking has been demonstrated in stress urinary incontinent (SUI) women of fertile age compared to continent controls [16]. These biochemical changes are accompanied by ultrastructural differences in terms of significantly larger collagen fibril diameter. These alterations focus on a possible disturbance in the fibrillization process [ 17]. The urethra is a fiat tube with a slitlike orifice that is held closed by atmospheric pressure and requires muscular force to open as well as to close. This might be the reason why a stiffer urethra does not close properly, resulting in poor control of micturition and the typical after-drip in men. It is possible that the pathophysiology of stress urinary incontinence in women is different in women of fertile age [17] and in women after menopause [ 18]. In women of fertile age with advanced stress incontinence, significant changes in the extracellular matrix of the paraurethral connective tissue have been shown [ 17]. These changes result in a stiffer, less supporting paraurethral tissue, but no major changes in biochemistry or ultrastructural degradation of collagen.

III. INNERVATION A. P a r a s y m p a t h e t i c P a t h w a y s Parasympathetic supply to the bladder derives from the sacral parasympathetic nucleus located at spinal level $ 2 - $ 4 , which in turn receives input from the pontine micturition center in the central nervous system (CNS). Preganglionic axons travel in the pelvic nerve to ganglia located within the pelvic plexus, to vesical ganglia (located on the serosal surface ofthe bladder), or to intramural ganglia of the bladder wall. Postganglionic axons leave the ganglia to supply the smooth muscle of the lower urinary tract. Activation of parasympathetic postganglionic nerves contracts the smooth muscle of the detrusor during micturition. The role of the parasympathetic nervous system in urethral function is less clear [ 19,20].

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B. S y m p a t h e t i c P a t h w a y s Preganglionic sympathetic axons are derived from spinal level T10-T12 and leave the spinal cord via the splanchnic nerves. The fibers are conveyed to the inferior mesenteric ganglion, where they either synapse with postganglionic cell bodies or pass straight through without synaptic contact. They pass via the pelvic plexus or directly to the bladder and urethra via the hypogastric nerve. Some preganglionic axons pass down the sympathetic chain to the lumbosacral level of the spinal cord, where they either synapse with cell bodies of the sympathetic chain ganglia or pass directly into the pelvic nerve [ 19]. A substantial part of urethral tone, an important factor for maintenance of intraurethral pressure, is mediated through stimulation of ce-adrenoceptors by released noradrenaline [20].

C. S e n s o r y A f f e r e n t I n n e r v a t i o n The hypogastric pathway is implicated in sensations associated with bladder filling and bladder pain; the pudendal pathway is implicated in a sensation that micturition is imminent and the pelvis pathway is implicated in both sensations. Sensory afferents travel in the pelvic nerve through the dorsal root ganglia to the dorsal horn of the sacral spinal cord [21 ].

D. I n t r a m u r a l G a n g l i a Intramural ganglia lie along nerve trunks, are surrounded by a capsule, and are concentrated to the trigone region. Immunoreactivity to vasoactive intestinal peptide, somatostatin, substance P, neuropeptide Y, galanin, tyrosine hydroxylase, or nitric oxide synthase [22-24], for example, has been demonstrated in nerve cell bodies of intramural ganglia. It has been suggested that the presence of these transmitters does not imply regulation of smooth muscle activity, but that the ganglia rather act by relaying and integrating the extrinsic nervous input to the bladder [ 19]. The possibility that nitric oxide (NO) is involved in inhibitory transmission in the outlet region of the urinary tract has been investigated, and it was demonstrated that NO is responsible for nonadrenergic, noncholinergic (NANC) relaxation of this region in several species, including humans [25-27]. NO was demonstrated to relax urethral tissue and to be involved in nerve-evoked relaxations.

E. C a r b o n M o n o x i d e as a B i o l o g i c a l M e s s e n g e r Carbon monoxide (CO) has been suggested to be involved in neuromuscular transmission in the autonomic nervous system. CO is formed by the cleavage of heme into Fe

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and biliverdin. It has been proposed that CO causes smooth muscle relaxation by raising intracellular levels of cyclic guanosine monophosphpate [28,29]. Such observations are of interest also for the ostensible biochemical differences between smokers and nonsmokers.

IV. M E C H A N I S M S

OF CONTINENCE

A. U r i n e S t o r a g e a n d M i c t u r i t i o n The function of the bladder and urethra is to store urine and, when appropriate, to evacuate it. The bladder forms the reservoir by accommodating increasing volumes of urine, and the outlet region increases its resistance. Several factors contribute to the ability of the outlet region to maintain continence. The proximal portion of the urethra lies within the abdominal cavity, and this position allows the pressure to rise along with any rise in intraabdominal pressure by passive transmission. The periurethral striated muscle seems to play an important role in maintaining continence during, for example, coughing and sneezing [30], but the rhabdosphincter and the properties of the smooth muscle of the urethra also contribute to maintaining continence. The lamina propria is also believed to "seal" the female urethra, thereby contributing to urethral closure [31]. The previously described meshwork of detrusor muscle is ideally suited for emptying the spherical bladder. The urethral opening is not only a consequence of the bladder contraction, but also the drop in urethral pressure before contraction of the bladder [32]. The factors contributing to this drop are still largely unknown. The mechanism of urinary continence is very complex and only partly understood. Basically, continence depends on two factors: normal urogenital tract support and normal sphincteric function [32]. The supportive mechanisms of the vesical neck and the urethra consist of the levator ani muscles, the arcus tendineus fasciae pelvis, and the endopelvic fascia around the vagina and urethra. In particular, the ligament connections between the pubic bone and the vagina as well as the interrelationship between the urethra and the anterior vaginal wall are both of great importance for the continence mechanism [31,32]. All pelvic floor muscles connected to the urogenital system are intimately involved in the mechanisms of urinary continence. Disturbance of any part of this delicate system may contribute to the development of incontinence. However, when one element is abnormal, other mechanisms may be able to compensate and maintain continence. Stress urinary incontinence is caused by a deficiency of the closure pressure in the urethra concomitant with an increase in intraabdominal pressure in the absence of detrusor activity [33]. There are probably several types of stress incontinence. Each type of stress incontinence reflects the malfunction of one or a battery of anatomic components of the

CHAPTER22 The Gynourinary System supportive and/or the sphincteric system. Until recently the predominating theory behind the occurrence of stress incontinence has been the so-called pressure transmission theory. In stress incontinent women this theory suggests that the intraabdominal pressure increase (caused by coughs, jumps, etc.) will not be properly transmitted from the intraabdominal pressure sphere, where the bladder is located, down to the urethra, and therefore the bladder pressure will exceed the urethra pressure during a short period of time, allowing urine to leak. Another pathophysiological view, when it comes to surgical treatment of stress incontinence, is to look to tentative anatomical dysfunctions behind the disorder. This is an old concept for which there has been renewed interest [32,34]. The integral theory suggests that continence is maintained by a complicated interplay between specific urogenital structures, aiming to support a kink off the midurethra at stress situations. If this is correct, the surgical treatment of the patient's symptoms should aim at creating functional pubourethral ligaments supporting the midurethra and reinforcing the suburethral vaginal wall. Simultaneously the connective tissue "gluing" these structures and the pelvic floor muscles together should be reinforced. Another theory emphasizes a strong anatomical support of the urethra from below, implying that the urethra may be closed by forces acting from above, for instance, an abdominal pressure transmitted at stress situations [33]. Accumulating evidence indicates that a more integrated, comprehensive view regarding the different structures, both inside and outside the urethra, is needed to explain the mechanisms of continence [32,34,35]. According to these theories, SUI is caused by defects in the supporting tissues that both actively and passively stabilize the urethra in its correct anatomical position. To a certain degree, a dysfunction in the intrinsic urethral mechanism created by mucosal and submucosal compression also contributes to incontinence. Only quite small defects in the closing mechanisms may well induce a risk of incontinence. The margin between continence and incontinence may be a tension resulting in only a few millimeters of displacement in the structures described. The micturition cycle can be separated into three phases: bladder filling, activation of the micturition reflex, and bladder emptying. During filling (stretch) passive bladder wall properties are activated and build up intramural tension, which in turn triggers bladder afferent nerves, leading to a desire to void. Activation of the micturition reflex, in the meaning of initiation of voiding, is a condition of nervous control. Emptying (contractility) is a neuromuscular activity. When the micturition reflex has been activated, pressure falls in the urethra and the pelvic floor becomes silent as evidenced by the electromyelogram (EMG) pattern. The detrusor muscle contracts, the bladder neck opens, and urine is passed. The bladder emptying is related to the various morphological and functional aspects of detrusor contractility, including also intramural changes.

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V. TREATMENT MODALITIES FOR FEMALE URINARY INCONTINENCE From a clinical standpoint it is often wise to differentiate between stress and urge incontinence, because treatments may be quite different. However, a huge overlap between the two conditions occurs and therefore it is sometimes difficult to treat them separately. Some types of treatments are common to both stress and urge incontinence, such as pelvic floor exercise, estrogen, or electrostimulation. Based on our present knowledge, pelvic floor muscle exercise should be the first choice of treatment for stress urinary incontinence.

A. S u r g e r y Stress incontinence is often treated by a surgical procedure such as laparoscopic colposuspension, a procedure using tension-free vaginal tape, needle bladder neck suspension, or endoscopic transuretheral injections. One of the major problems is to find and interpret the diagnostic tests and from there choose the best approach. Some 200 surgical procedures have been described for this condition. Conceivably the great number of procedures reflects disappointing results. It may also reflect a poor and insufficient understanding of the pathophysiology behind the condition [36,37]. Most of the methods for stress incontinence aim at supporting the pelvic floor and/or lifting at least part of the urethra upward, thereby producing continence. The most commonly used procedure is the so-called Marshal-Marchetti fixation of the paraurethral tissues. This procedure was modified by Burch, and the Burch colposuspension has now more or less become the routine operative method of choice because it can also be performed by a laparoscopic technique [37a]. A pivotal problem with all surgical procedures is the longterm result. It goes without saying that the underlying pathophysiology cannot be cured by surgery. Hence recurrence rates of the disease are fairly high irrespective of surgical procedure and it would seem pointless to perform radical surgery. Invasive procedures are, however, often accompanied by bleeding and subsequent formation of scar tissue, which in turn may help to support the pelvic floor. Nonetheless, it may be best to use less extensive surgical procedures, such as the tension-free vaginal tape (TVT) procedure as described by Ulmsten and co-workers [38]. Even if long-term results of such a procedure are largely unknown, it is conceivably neither worse nor better than any other surgical procedure. However, TVT involves minimal surgical trauma, thus it can possibly be reiterated if needed. Electrical stimulation to treat stress and urge incontinence is an additional option. The results of randomized control clinical trials evaluating the effect of this procedure are conflicting [39-43]. There is a need for more randomized

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trials with a sufficient sample size and use of sensitive, reproducible, and valid outcome measures [44]. Functional electrical stimulation has many theoretical advantages. One of the major problems is patient selection, because there is no protocol to select the patients who are most likely to benefit from electrical stimulation. In clinical practice, favorable results have been presented repeatedly. Long-term data have been presented and after approximately 10 years most of the treated women had symptoms of urge incontinence [45]. This was a minor problem among a third of them. A majority of the women who answered the treatment follow-up questionnaire were satisfied with maximal electric stimulation as a treatment modality [46,47]. In a review on the actual practice of surgical management of urinary incontinence in Norway and Finland, it was found that 90% of the facilities performed preoperative urodynamic evaluation. Burch colposuspension was used by more than 90% of the facilities in the two countries. Generally, the number of procedures per center and per department was low: 29 and 12%, respectively, did not perform any followup after surgery. Hence, long-term results are difficult to evaluate. Operative skill, when using the Burch colposuspension, is clearly related to the long-term results.

B. B l a d d e r T r a i n i n g a n d R e t r a i n i n g This type of program requires the participant to resist or inhibit the sensation of urgency, postponing voiding and urinating according to a timetable. Urge incontinence is often referred to as an overactive bladder syndrome. Overactivity leads to a frequent desire to void and the bladder gradually becomes smaller and smaller. The bladder training and retraining program aims at suppressing the desire to void, with the goal to gradually increase bladder volume. About 80% of the studies showed marked primary improvement, but results are somewhat difficult to evaluate because different inclusion criteria were used [48-52].

C. P e l v i c M u s c l e E x e r c i s e s Contractions of the pubococcygeous muscles improve urethral resistance. The use of weighted vaginal cones, for example, may serve as an adjunct to pelvic muscle strengthening [53,54].

D. E s t r o g e n Loss of estrogen influence leads to atrophy of the urethral mucosa, which in turn results in a widening of the urethra and thereby a decrease in resistance to flow. Control of micturition calls for increased muscular activity that is hard to

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achieve, because estrogen deficiency reduces blood flow through the urogenital tissues, thereby impeding muscular function. With a decline in blood flow, important immune system functions may also be affected, facilitating, in combination with the wide urethra, microbial migration into the urinary bladder. This, together with the vaginal reservoir of urinary pathogenic microorganisms, makes it understandable that there is a high frequency of symptomatic and asymptotic bacteriuria, as well as recurrent cystitis [55]. In stress incontinence anatomical changes are the main factor and consequently a surgical approach remains the first line treatment of this condition, even if estrogens improve the condition somewhat. Albeit limited, data support the notion that estrogen effects in stress incontinence may be mediated via influence on collagen and neuromuscular metabolism, which are of importance for the supportive function of the pelvic floor. This may well include an effect on intraurethral pressure. 1. QUALITY OF LIFE In elderly women, incontinence is a major reason for permanent admission to nursing homes. The use of estrogen replacement therapy, especially low-dose topical applications, may substantially reduce both incontinence and recurrent urinary tract infections, thereby making it possible to live outside an institution. This is a major aspect, affecting both quality of life and the costs of caring for those with incontinence. Women with incontinence often suffer socially, because they are afraid of situations in which they cannot reach a toilet almost immediately. Many elderly women avoid long travel trips, visits to the theater or other public places, and cultural events, and often remain confined to their own homes, which has a serious impact on their quality of life. For women with an active sex life, the loss of lubrication and glandular functions severely impairs sexual desire. Treatment of this condition improves quality of life, not only for the woman but also for her partner. 2. ESTROGEN THERAPY

As mentioned previously, atrophy does not begin until endogenously produced estrogens are markedly lower than those required for endometrial proliferation. This observation opens a therapeutic window. It is possible to target urogenital atrophy while minimizing the risk of endometrial proliferation. Estrogen aimed at controlling urogenital symptoms therefore could and should be given at much lower doses than is common when treating other climacteric complaints. Vaginal estrogens can also be given without a progestogen. For estradiol, daily doses of 50-100/xg are recommended in systemic patches to ensure relief of vasomotor symptoms, whereas 8-10/xg of vaginally delivered estradiol seems sufficient to combat urogenital symptoms and signs. In other words, only 10-15% of the dose required to alleviate hot flushes is needed to achieve clinical improvement of urogeni-

CHAPTER22 The Gynourinary System

333

tal estrogen deficiency symptoms. Instead of using ultralowdose estradiol, it is possible to administer estriol, which is a short-acting estrogen. Estriol is available as tablets, cream, and vaginal pessaries. Vaginal administration is preferred, because the therapeutic window with tablets is fairly narrow and may lead to endometrial proliferation and possibly an increased risk of cancer [56] (see Table IV) [56a]. The clinical efficacy of estriol and of ultralow doses of estradiol have been shown in a number of clinical studies of lower urinary tract infections [5,57], various forms of incontinence [3, 6,58], and vaginal problems [59,60]. Marked changes may occur within the lower urogenital tract with little or no influence on endocrine parameters such as sex steroid hormone levels, sex hormone binding globulin, or gonadotropin levels. Even lipid metabolic parameters, such as triglycerides, are not influenced [61 ]. Lower urinary tract infections are markedly decreased and the recurrence of lactobacilli is probably of great importance. However, several unresolved issues remain in this respect [62]. Systemic absorption via the vaginal route is negligible after 3 - 4 weeks of treatment. Hence, low-dose estrogen treatment offers no protection for osteoporosis or cardiovascular disease, but also causes no increased risk for breast cancer or any other side effect. Consequently, neither absolute nor relative contraindications exist for low-dose estrogen therapy via the vaginal route. However, in the elderly, long-standing low-dose estrogen therapy may produce some systemic effects [56,63]. In 1991, well-established vaginal delivery systems of estrogens aimed at the treatment of urogenital estrogen deficiency symptoms were made available as over-the-counter (OTC) preparations in Sweden. Experience with OTC preparations of vaginal estrogens has been positive; no negative effects have been encountered. With low-dose OTC vaginal estrogens, the costs of treatment and prevention of estrogen deficiency symptoms of the urogenital tract can be limited to the cost of the medication. There is no need for compulsory follow-up programs. Trained nurses may counsel elderly patients and answer questions about low-dose estrogen treatment by vaginal administration.

TABLE IV

In the vagina, estrogen stimulates maturation of the urethral mucosa, as shown by an increase in superficial and intermediate squamous cells and a decrease in parabasal or transitional cells [64]. Interestingly, several studies have shown an estrogen-induced maturation of urethral cytology to correlate with clinically improved urinary stress incontinence following administration of estrogen [65,66]. Further evidence explaining these cytological changes and suggesting a hormonal effect on the urogenital tract and therefore possibly incontinence was the discovery of estrogen and progesterone receptors within the urethra and bladder trigone [67,68]. Additionally, estrogen and progesterone receptors have been demonstrated in the levator ani and round ligament [69] and most recently in the uterosacral ligament [69]. Although the significance of these hormonal receptors is uncertain, several possible contributions to continence at the level of these supporting tissues exist. These include increased collagen content and strength, which has been suggested to be positively influenced by estrogen [64,68], and the probable effect of estrogen on increasing periurethral blood flow, which has been shown to occur in rabbits [70] and is well established in vaginal tissue, a site of similar receptors [64]. Another physiologic explanation for an estrogen effect on incontinence is the demonstration, at least in animal models, of an increase in the concentration of ce-adrenergic receptors in the lower urinary tract in response to estrogen [71 ] and evidence from human studies of an estrogen-mediated enhancement of urethral response to ce-adrenergic agonists [72,73]. Improvement in urethral pressure profiles and symptomatic improvement of incontinence following estrogen administration have been described [74], but lack of a control population limits the interpretation. Rud [75] studied stress continent and incontinent women treated with oral estradiol or estriol for 3 weeks and noted a significant increase in maximal urethral pressure and urethral length at rest. Bhatia e t al. [65] also showed an increase in urethral closure pressure in 54% of 11 women with urodynamically proved stress incontinence treated with estrogen vaginal cream for

Vaginal Ultrasound of the Endometrium in Postmenopausal Women a Tibolone

Parameter Women (n) Endometrial thickness (mm, _+SD) Uterine volume (ml) Mean ovarian volume (ml)

Low-dose estrogens

Base line

6 months

Base line

6 months

36 3.2 _+0.3 42 4.2

36 3.2 _+0.7 42 3.9

36 3.0 +_0.1 45 3.9

36 2.9 + 0.8 46 3.9

aFrom Ref. 56a, Maturitas, Vol. 26, Botsis et al., Vaginal ultrasound of the endometriumin postmenopausal women with symptoms of urogenital atrophy on low-dose estrogen or tibolone treatment: A comparison, pp. 57-62, Copyright 1997, with permission from Elsevier Science. The women had symptoms of urogenital atrophy; low-dose estrogens were vaginally administered.

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6 weeks. In 1996, Fantl et al. [76] randomized 83 postmenopausal incontinent women (urodynamically proved to have either genuine stress or detrusor instability) to either cyclic hormone replacement or placebo and found no significant change in the number of incontinent episodes per week, volume of urine lost, or frequency or nocturia after 3 months of therapy. In 1993, Elia and Bergman published a comprehensive review of clinical studies of estrogen and urinary stress incontinence [77]. They found in their review of randomized, placebo-controlled studies symptomatic improvement in the majority of studies, but no objective urodynamic improvement. Fantl et al. [86] published the first report of the Hormones and Urogenital Therapy Committee, a meta-analysis of estrogen therapy in the management of urinary incontinence in postmenopausal women, looking at peer-reviewed original articles with an estrogen-treated group, confirmed diagnosis, and both subjective and objective outcomes data. For controlled studies they found an overall symptomatic improvement rate with estrogen of 64-75%; however, improvement with placebo was 10-56%. Five of the seven studies reported significant improvement with estrogen, and the overall improvement rate was highly significant (p < 0.01). When restricted to stress incontinence only, the improvement rate was still significant, but at a lower rate (26 vs. 46% for all patients). For uncontrolled studies, symptomatic improvement rates with estrogen ranged from 8 to 89% (overall, 64%), and when confined to genuine stress incontinence from 34 to 73% (overall, 53%). Only three of the controlled studies recorded urodynamic data. Of these, only Henalla et al. [78] showed a highly significant increase in maximal urethral closure pressure in 34 women treated with vaginal conjugated estrogens for 3 months. The Hormone and Urogenital Committee concluded that estrogen subjectively improves urinary incontinence in postmenopausal women but found no strong evidence of an effect of estrogen on urodynamic or other objective outcomes.

E. O t h e r P h a r m a c o l o g i c a l M o d a l i t i e s Pharmacological treatments of incontinence due to detrusor overactivity (urge incontinence) include anticholinergic agents such as tertiary amines. Tricyclic agents such as immipramin have been shown to be effective in the decrease of incontinence frequency [79]. Strategies in pharmacological treatment of incontinence due to urethral sphincter insufficiency are designed to increase bladder output resistance. Drugs include those with direct c~-adrenergic agonist activity, which may be enhanced by concomitant estrogen treatment, fi-Adrenergic blocking drugs, which might allow unopposed stimulation of a-receptor-mediated contracted muscle responses, are also of theoretical interest.

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lb URGE INCONTINENCE

Antimuscarinic drugs have a long-standing tradition in the treatment of urge incontinence [80-82]. Atropine and related substances are tertiary amines easily absorbed from the gastrointestinal tract. However, these substances also readily penetrate into the CNS and central nervous side effects are therefore a limiting factor in clinical usage. The quaternary amines such as propantelin, emepronium, and tolterodin display less of these effects. However, oral bioavailability is variable and fairly low (5-10%). Mouth dryness, being the most common of these side effects, limits the use of these drugs. Tolterodin and metabolites seems to have some selectivity for the bladder compared to the salivary glands [83]. Calcium channel blockers such as nifedipin are theoretically interesting. However, peripheral vasodilatation is often pronounced and effects on bladder are still debatable. Hence these drugs are at present no alternative for the patient and clinician [84,85]. Oxybutynin displays mainly antimuscarinic effects, although a direct relaxing effect on muscular tissues has been demonstrated. Again, a low oral bioavailability and fairly high percentage of typically antimuscarinic side effects such as mouth dryness, constipation, and visual disturbances limit its use. Side effects are dose dependent and lower doses than recommended for full therapeutic effect are often attractive, because side effects are then negligible [86,87]. fl-Receptor agonists have also been used because such agents can relax human bladder muscle cells and increase bladder capacity. Therapeutic effects in patients have been obtained by terbutalin, but often at the expense of tachycardia. It can be concluded that the therapeutic effect is less distinct than with anticholinergics and the specific indications, if any, of this family of drugs need to be established. a-Adrenenergic receptor antagonists may also act on bladder function. But the adrenergic innervation of the bladder is sparse and of doubtful clinical significance in humans. Nonetheless, prazosin given to patients with uninhibited neurogenic bladder contractions seems to have a limited effect. Prostaglandin synthethase inhibitors and some antidepressive agents have also been used with various success. Usage has often been hampered by side effects and much remains to be clarified until such agents can be used clinically on a larger scale basis. 2. PHARMACOLOGICAL TREATMENT OF STRESS INCONTINENCE The primary cause of genuine stress incontinence is the anatomical change brought about by a weakness of the supporting tissues of the pelvic floor. This in turn is associated with vaginal delivery, abdominal surgery, or other injuries. In urodynamic investigations it has been found that the intra-

CHAPTER 22 The Gynourinary System urethral pressure is low in stress incontinent women [88,89]. Data suggest that at least two a-adrenergic receptor agonists found to be effective in clinical trials, norefedrin and midodrin, increase intraurethral pressure. It should be pointed out that the lowered intraurethral pressure is only a minor contributor to the overall clinical picture, which limits the use of any pharmacological agent only to milder cases [90,91 ]. Due to an insufficient knowledge of the specific pharmacological mechanism of drugs for treating incontinence, it is difficult to design an effective pharmacological protocol for urge and especially stress incontinence. 3. VAGINALSYMPTOMS

Typically the first vaginal symptom is a feeling of dryness, due to atrophy of the mucus-producing glands of the vaginal wall. In the vagina, atrophy yields a thin mucosa susceptible not only to infections but also to stress during intercourse. Petechial bleedings are not uncommon and will lead to vaginal obliteration most pronounced in the fundal region of the vagina. Itching and a feeling of burning are well known symptoms in the vulva and vagina as a result of atrophic vaginitis. Also, atrophy of the minor lips can lead to burning sensations and itching in the vulva, but it should be remembered that several dermatological conditions (such as lichen ruber, psoriasis, eczema, and lichen sclerosus) yield similar vulvar symptoms and differential diagnosis must always be considered. Infections such as candidiasis may be superimposed and should also be considered in this context. Vaginal symptoms are especially overt in women who have a male partner capable of and interested in sexual activity. There is a general belief that sexual activity in elderly women is low, but in the study by Barlow et al. [8] of women aged between 55 and 74 years, about half reported to be sexually active. Among these, frequent intercourse, e.g., once per week or more, was reported by 23% of women in general and by 14% of women between 70 and 74 years. There was a difference between countries, which may reflect cultural differences: intercourse frequency was relatively lower in the United Kingdom and Germany and higher in Italy and Denmark. Of those women reporting sexually activity in the past year, 22% reported a loss of interest. However, the most common cause for reduced sexual activity (66%) was loss of partner. Male factors, inclusive of impotence, were reported by 10% of women. In this report by the women there was also a striking difference between lost male capacity, with 13% of Danish women reporting this problem and only 6% of women in Italy and the United Kingdom reporting this. 4. LIBIDO

Sexual activity is dependent not only on vaginal function but also on libido per se. Loss of sexual desire is often a symptom of depression and may accompany several chronic

335 diseases, both psychiatric and somatic. There can be little doubt that sex steroids contribute to brain dimorphism and to neuroplasticity as well as psychoplasticity. Estrogen is a prudent activator of sexual attraction; the hormone causes and maintains secondary sex characteristics and is included in nocturnal activation of the neurovegetative pathways that lead to vaginal and clitoral congestion during REM phases of sleep [92,93]. Estrogens have been shown to activate pheromone secretion in sweat and sebaceous glands. These pheromones contribute to the scent of women and are of great importance in sexual attraction [95,98]. Human sexuality is rooted in three main dimensions: biological, motivational-affective, and cognitive. Human sexuality is mainly relational. Couple satisfaction and premenopausal quality of sexuality are strong predictors for the quality (and quantity?) of postmenopausal sex life [95-98]. Hormonal influence is of importance for the level of sexual arousal and the quality of physical and physiologic response. However, hormones cannot influence the direction of sexual arousal. Optimal estrogenic influence could be considered a very important factor for female sexuality, but it is the androgenic influence that enhances vital energy, libido, and sexual activity both in men and women [97,98]. It should be remembered in this context that peak concentrations of androgens during the menstrual cycle coincide with the estradiol peak at ovulation. Administration of combined oral contraceptives and HRT may increase prolactin. Prolactin may inhibit libido via the dopaminergic system in the central nervous system. Progesterone and most progestogens seem to have a mild inhibiting effect. Oxytocin is also of importance. It peaks in plasma at orgasm and in animals it is responsible for sexual satiety after extended mating. The relation of estrogen treatment to libido is complex. Vasointestinal peptide is a neurotransmitter that is closely linked to variations in sex steroids, and estrogens are a potent stimulator. VIP in turn translates sexual desire into a vascular response that leads to liquid transudation in the vagina [92]. By increasing the number of glands as well as glandular function, estrogen contributes further to the lubrication of the vagina, thereby rendering dyspareunia less common. On the other hand, systemic estrogen treatment may increase the level of sex hormone binding globulin and thereby decrease the free androgen fraction, which may have a negative impact on libido. But estrogen also has a positive effect on the sense of general well being and on mood swings and sleep disturbances. By doing so, a general interest in sex is conceivably increased, but this should be regarded as an indirect effect of estrogen. Local estrogen treatment in the form of a vaginal ring, a vaginal tablet, a pessary, or cream improves local function only. Hence, there are no effects on general well being or mood. The vaginal lamina propria contains a plexus of large

3

3

6

G

O

but thin veins. One of the important physiologic response factors for sexual arousal is vasocongestion of the vaginal wall. N o r m a l function of this plexus could well be of importance for vaginal p h y s i o l o g y [92]. It is also well k n o w n that sexually active w o m e n have fewer vaginal p r o b l e m s than do those w h o do not have a partner [101]. L o c a l s t i m u l a t o r y factors, directly or indirectly, c o n t r i b u t e to vaginal integrity. Sexual activity is dep e n d e n t on the p s y c h o l o g i c a l and p h y s i o l o g i c a l integrity of both the m a l e and the f e m a l e counterpart. I m p o t e n c e in m e n gradually increases with age and erectile d y s f u n c t i o n is a c o m m o n p r o b l e m . Unfortunately, m e n are less p r o n e than w o m e n to c o n s u l t doctors for sexual dysfunction. A chronic prostatitis could often be treated and other m e a s u r e s to help or a m e l i o r a t e erectile d y s f u n c t i o n are available today. In order to have a g o o d sex life, a c o u p l e m u s t be able to c o m m u n i c a t e their p r o b l e m s to one another. U n d e r s t a n d i n g the p r o b l e m s of the partner is of great i m p o r t a n c e to the success of other modalities of t r e a t m e n t of sexual d y s f u n c t i o n both in m e n and w o m e n . U s i n g the M c C o y Sex Scale, N a t h o r s t - B 6 6 s and H a m m a r recently r e p o r t e d [ 102] that a c o n t i n u o u s c o m b i n e d r e g i m e n c o n t a i n i n g 2 m g of estradiol and 1 m g of n o r e t h i s t e r o n e acetate significantly i m p r o v e d several p a r a m e t e r s of i m p o r t a n c e for sexual function. T h e same was true for the preparation tibolone, w h i c h is a steroid with w e a k estrogenic, w e a k progestogenic, and s o m e a n d r o g e n i c properties. T h e difference in effect of the two preparations was small. T h e result of this study suggests that a n d r o g e n t r e a t m e n t could be used for imp r o v i n g sexual function in w o m e n . Similar results have been obtained also by Sherwin et al. using c o m b i n a t i o n s of estrogens and a n d r o g e n s [97]. T i b o l o n e m a y be used to treat w o m e n with e n d o g e n o u s l y deprived a n d r o g e n influence. This is not u n c o m m o n in o o p h o r e c t o m i z e d w o m e n ; the p o s t m e n o p a u s a l ovary is responsible for a substantial part of female a n d r o g e n production t h r o u g h o u t life. E m b a r r a s s i n g increase of libido is a side effect s o m e t i m e s e n c o u n t e r e d in H R T e m p l o y i n g a n d r o g e n i c p r o g e s t o g e n s , especially in patients of a d v a n c i n g age. O f interest is the fact that the s t i m u l a t o r y effect of oxytocin on libido, w h i c h has been well d o c u m e n t e d in animals, seems also to be true for h u m a n s . Indeed, H R T influences oxytocin; e s t r o g e n s stimulate and p r o g e s t o g e n s attenuate estrogen effects [ 103].

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82. Naglo, A. S. et al. (1981). Influence of atropine and isoprenaline on detrusor hyperactivity in children with neurogenic bladder. Scand. J. Urol. Nephrol. 15, 97-102. 83. Stahl, M. M. S., Ekstr6m, B., Sparf, B. et al. (1995). Urodynamic and other effects of tolterodine: A novel antimuscarinic drug, for treatment of detrusor overactivity. Neurourol. Urodyn. 14, 647-655. 84. Forman, A. et al. (1978). Effects of nifedipine on the smooth muscle of the human urinary tract in vitro and in vivo. Acta Pharmacol. Toxicol. 43, 11-118. 85. Andersson, K. E., and Forman, A. (1978). Effects of calcium channel blockers on urinary tract smooth muscle. Acta Pharmacol. Toxicol., Suppl. 2, 90-95. 86. Yarker, Y. E., Goa, K. L., and Fitton, A. (1995). Oxybutynin: A review of its pharmacodynamic and pharmacokinetic properties, and its therapeutic use in detrusor instability. Drug Aging 6, 243-262. 87. Baigrie, R. J. et al. (1988). Oxybutynin: Is it safe? Br. J. Urol. 319, 22. 88. Lindholm, P., and Lose, G. (1986). Terbutaline (Bricanyl | in the treatment of female urge incontinence. Urol. Int. 41, 158-160. 89. Castleden, C. M., and Morgan, B. (1980). The effect of/3-adrenoceptor agonists on urinary incontinence in the elderly. Br. J. Clin. Pharmacol. 10, 619-620. 90. Caine, M. (1986). The present role of alpha-adrenergic blockers in the treatment of benign prostatic hyperplasia. J. Urol. 136, 1-4. 91. Petersen, T. et al. (1989). Prazosin treatment of neurological patients with detrusor hyperreflexia and bladder emptying disability. Scand. J. Urol. Nephrol. 23, 189-194. 92. Graziottin, A. (1966). Libido. In "The Year Book of the Royal College of Obstetricians and Gynecologists" (J. W. W. Studd, ed.), pp. 235244. RCOG Press, London. 93. Levin, R. J. (1992). The mechanisms of human female sexual arousal. Annu. Rev. Sex. Res. 3, 1-48. 94. Arimondi, C., Vannelli, G. B., and Balboni, G. C. (1993). Importance of olfaction in sexual life: Morphofunctional and psychological studies in man. Biomed. Res. (India) 4, 43-52. 95. MacPhee, D. C., Johnson, S. M., and Van der Veer, M. M. (1995). Low sexual desire in women: The effect of marital therapy. J. Sex. Marital Ther. 21, 159-182. 96. McCoy, N. L. (1995). Menopause and sexuality. In "Optimizing Hormone Replacement Therapy" (M. K. Beard, ed.), pp. 32-36. McGraw-Hill, Minneapolis, MN. 97. Sherwin, B.B., Gelfand, M. M., and Brender, W. (1985). Androgen enhances sexual motivation in females: A prospective, crossover study of sex steroid administration in the surgical menopause. Psychosom. Med. 47, 339-351. 98. Frock, J., and Money, J. (1992). Sexuality and menopause. Psychother. Psychosom. 57, 29-33. 99. Sands, R., and Studd, J. (1995). Exogenous androgens in postmenopausal women. Am. J. Med. 98, 76-69. 100. Casson, P. R., and Casson, S. A. (1996). Androgen replaement therapy in women: Myths and realities. Int. J. Fertil. Menopausal Stud. 41, 412-422. 101. Bachmann, G. A. (1990). Impact of vaginal health on sexual function. J. Clin. Pract. Sex., (Spec. Issue) pp. 18-21. 102. Nathorst-B66s, J., and Hammar, H. (1997). Effect on sexual life: A comparison between tibolone and a continuous estradiolnorethisterone acetate regimen. Maturitas 26, 15-20. 103. Bossnar, T., Forsling, M., and Akerlund, M. (1995). Circulating oxytocin and vasopressin is influenced by ovarian steroid replacement in women. Acta Obstet. Gynecol. Scand. 74, 544-548.

~ H A P T ER 2:~

Is There a Relationship between Menopause and Mood? NANCY E. AvIs

I. II. III. IV.

Institute for Women's Research, New England Research Institutes, Watertown, Massachusetts 02472

V. Empirical Research: Hormones and Mood VI. Conclusions References

Introduction Theories of Menopause and Mood Methodological Issues Empirical Research: Menopause Status and Mood

In 1896 the German psychiatrist Kraepelin introduced the concept of involutional melancholia as an agitated depression with hypochondriac or nihilistic delusions that was more common in women than in men and first appeared during middle age [2]. Although Kraepelin later conceded that involutional melancholia was not a separate clinical entity, but rather a form of manic depression, other psychiatrists continued to argue that it was a distinct clinical entity. In 1966 Robert Wilson wrote in his best-selling book, "Feminine Forever," that "the menopausal woman is not normal; she suffers from a deficiency disease with serious sequelae and needs treatment" [3]. Women experiencing this normal end of reproduction are thought to experience regret, to evidence signs of clinical depression (involutional melancholia), to present with a broad range of accompanying symptoms, to be high utilizers of physicians' services, and generally to consume a disproportionate share of medical resources [3,4]. In 1980, the American Psychiatric Association concluded that there was not a unique form of depression occurring at middle age and subsumed involutional melancholia under the broader category of major depression in DSM-III [5].

I. I N T R O D U C T I O N The belief that menopause leads to mood disturbances such as increased irritability, nervousness, and depression has a long history. Some of the earliest writings on menopause refer to psychological or mood characteristics of women at this time. In 1777 John Leake stated in his book, "Chronic or Slow Diseases Peculiar to women," that the cessation of menses led to "pain and giddiness of the head, hysteric disorders, colic pain and a mid-life female weakness." He further stated that at this time women are sometimes affected with low spirits or melancholy. In 1882, Tilt referred to menopause as an event bearing "evil effects." Menopause was viewed as a physiological and psychological crisis. In 1887 Farnham summarized the relation between menopause and psychiatric disorder as "the ovaries, after long years of s e r v i c e . . , become irritated, transmit their irritation to the abdominal ganglia, which in turn transmit the irritation to the brain, producing disturbances in the cerebral tissue exhibiting themselves in extreme nervousness or in an outburst of actual insanity" [ 1]. MENOPAUSE: BIOLOGY AND PATHOBIOLOGY

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Copyright 9 2000 by Academic Press. All rights of reproduction in any form reserved.

340 This notion that women become depressed, irritable, and suffer from other mood disturbances during menopause continues. This belief prevails among women in general [6] as well as among clinicians [7,8]. Such a characterization has received reinforcement both from pharmaceutical advertisements in professional medical journals [9,10] and from patient images used and developed during the course of medical education [ 11-13]. Estrogen replacement therapy (ERT) is touted as a mental tonic [14,15] and in general surveys, high percentages of women believe that women become depressed, irritable, or suffer other negative moods during the menopause [ 16 - 19]. How did these beliefs come about and are they supported by the scientific literature? In this chapter, theories proposed to explain a relation between menopause and mood disturbances are presented. Some of the methodological issues involved in the study of menopause and mood are then reviewed. These methodological issues help explain how these beliefs developed as well as point to challenges in drawing conclusions from the research. The scientific literature on menopause and mood is then reviewed. Finally, the theories in light of the evidence are reviewed and the conclusions that can be drawn are summarized.

II. THEORIES OF MENOPAUSE AND M O O D Four theories or hypotheses are generally offered to explain possible associations between menopause and mood disturbances. The first two hypotheses are related to declining estrogen levels but differ in the directness of the effect. The first theory is the symptom or domino hypothesis, which posits that depressed mood is caused by vasomotor symptoms associated with changing estrogen levels [20,21]. This has been referred to as the domino hypothesis in that vasomotor instability leads to chronic sleep deprivation, which in turn leads to irritability and mood changes [22]. Second is the biochemical hypothesis, which associates a decline in estrogen directly with biochemical changes in the brain that lead to mood changes. As early as 1953, Malleson wrote that emotional symptoms at menopause are due to estrogen deficiency [23]. This hypothesis derives support from the similarity between the pathophysiology of depression and the neurobiologic effects of estrogen. The pathophysiology of depression is thought to involve the dysregulation of several neurotransmitter and neuromodulatory systems: serotonergic, noradrenergic, cholinergic, dopaminergic, and y-aminobutyric acid (GABA) systems [24]. The neurobiologic effects of estrogen are thought to include decreased monoamine oxidase activity (which increases the bioavailability of catecholamines), the enhancement of serotonin neurotransmission, the enhancement of cholinergic transmission, antidopaminergic effects in certain brain areas, the modulation of GAB A receptors, a decrease of fl-endorphin

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function, the modulation of progesterone receptors, and the modification of sleep and circadian rhythms [25]. The effects of estrogen that are of greatest interest to depression researchers involve the cholinergic and serotonergic system. Estrogen increases acetycholine synthesis by increasing choline acetyltransferese [25]. Estrogen is thought to enhance serotonergic transmission by decreasing monoamine oxidase activity, increasing free tryptophan (a precursor of serotonin) availability to the brain, and enhancing the transport of serotonin [26]. The biochemical hypothesis differs from the symptom hypothesis in that it posits a direct association between estrogen decline and depression. According to the symptom hypothesis, vasomotor symptoms mediate the association between decreased hormonal levels and depression. The biochemical hypothesis also posits that these neurobiologic changes may occur at other times of hormonal fluctuations, such as puberty, during the premenstrual phase of the menstrual cycle, and postpartum [27-29]. The third hypothesis is the psychoanalytic view, which posits that onset of menopause is a critical event in the life of a middle-aged woman and is a threat to her adjustment and self-concept [30]. In the 1940s, psychoanalytic writers such as Helen Deutsch and Ruth Benedek viewed menopause in terms of reproductive loss. Deutsch even went so far as to say that the inability to reproduce was a psychological death for all women. Benedek [31 ], on the other hand, also believed that menopause provided a release of construc, tive energymthat menopause desexualizes emotional needs, thus freeing energies. This notion of increased energy following menopause is also seen in Margaret Mead's writings about postmenopausal zest. Fourth, the social circumstances perspective states that menopause per se is not associated with depression or other mood disturbances, but rather it is various life events and circumstances coincidental with menopause that are related to depression [32,33]. This perspective views midlife as a time of numerous changes in women's lives (e.g., children leaving or returning home, changes in health, and increased health problems and death of aging parents) that may increase a woman's risk for depression and other psychological disturbances.

III. M E T H O D O L O G I C A L ISSUES Prior to reviewing the literature in this area, several important methodological issues are important to consider. These issues highlight both how these beliefs have come about, as well as the difficulties in drawing conclusions across studies.

A. C l i n i c v e r s u s P o p u l a t i o n - B a s e d S a m p l e s Much of the research that gives rise to the perceived relationship between menopause and mood changes is de-

CHAPTER23 Menopause and Mood rived from clinic or patient samples of women who have sought treatment for menopause-related problems. These patient samples present a biased view of the natural menopause transition and have resulted in a clinical stereotype o f the "typical" menopausal women who experiences a broad range of often diffuse symptoms. Such clinical stereotypes appear to have resulted in misdiagnosis and treatment of clinically diagnosable conditions, unrelated to menopause, when presenting symptomatology was labeled as "menopausal." This was noted as early as 1944 [34] and is clearly evident in recent population-based studies of the impact of estrogen replacement therapy on subsequent mortality [35,36]. These two studies (British and Swedish, respectively) reported elevated risks of suicide among ERT users, which may reflect clinical depression inappropriately treated with estrogens. Data from patient-based studies represent only women who seek medical care. This is particularly problematic for samples of women seeking treatment for acute menopausal symptoms. Studies have shown that fewer than half of menopausal women seek menopause-related treatment [37-42], and those who do seek treatment tend to report more life stress and suffer more from clinical depression, anxiety, and psychological symptoms than do nonpatient samples [38,40]. Some studies are based on general clinic samples of women who have not specifically sought treatment for menopause symptoms. However, general clinic samples are biased as well. Population-based research demonstrates that not all women have annual checkups [43]. Patient-based samples are biased in terms of education, socioeconomic status, other health problems, and incidence of general depression [37, 43-45]. Ballinger has reported that women aged 4 0 - 5 5 from the general population who were identified as psychiatric cases had more frequent contact with general practitioners than did matched normal controls [44], and that a higher proportion of women identified as psychiatric cases were referred to a gynecology outpatient clinic compared to those from a general population [45]. Avis and McKinlay [43] found that women who were health care utilizers tended to report worse health and more physical a n d psychological symptoms, stress, and have higher rates of depression compared to nonutilizers. In a similar analysis among Australian women, Morse et al. [40] found that utilizers reported worse health, and more psychosomatic symptoms. Thus, although patient-based samples provide data on how some women experience menopause, they clearly represent a biased sample and do not provide information on the proportion of women not interacting regularly with the medical care system. Further, such studiesare likely to overestimate the prevalence of mood disorders among peri- or postmenopausal women. As data from population-based, as well as communitybased, samples have become available, it is apparent that this research presents a very different picture than does patientbased research and challenges many of our long held beliefs about menopause.

341 B. D e f i n i n g M e n o p a u s a l Status The inconsistency with which researchers have defined menopause status presents a major challenge in comparing studies in this area [46]. In relating mood effects to menopause status, it is important to use consistent definitions of pre-, peri-, and postmenopause. Some of the earlier studies [47] used age as a surrogate for menopause status. However, this is an imprecise measure of status, because women between 45 and 55, for instance, can be pre-, peri-, or postmenopausal. Pansini et al. [48] have shown the considerable age overlap among menopause status groups. The standard epidemiological definition of natural menopause is 12 consecutive months of amenorrhea in the absence of surgery or other pathological or physiological cause (e.g., pregnancy, lactation) that would terminate menstruation [49]. Although perimenopause has been less consistently defined, the currently accepted epidemiological definition is as having menses in the past 12 months with changes in regularity, or no menstrual cycle in the past 3-11 months [50, 51 ]. However, some studies have classified women as postmenopausal following only 6 months of amenorrhea. These women would be classified as perimenopausal by other investigators. Some studies combine pre- and perimenopausal or peri- and postmenopausal women for analysis. In reviewing the literature, I have tried to take into account how the authors defined menopause status, using the World Health Organization (WHO) classification as the standard. An additional consideration is the distinction between surgical and natural menopause. Surgical menopause is defined as occurring when a surgical procedure stops menstruation. Women who have had a bilateral oophorectomy or a~hysterectomy with or without removal of the ovaries are generally included in this category. Women who have a surgical menopause tend to be younger, of poorer health, and use health care more frequently [52-54]. Their menopause experience is also quite different, in that they experience more sudden hormonal changes, as well as a surgical procedure. A number of studies have found that women who have had a surgical menopause report more distress than do women who experience a natural menopause [55-58]. Therefore, women experiencing a surgical menopause must be considered separately from those experiencing a natural menopause. Further, the effects of a bilateral oophorectomy are also different from those of a hysterectomy.

C. M e a s u r e m e n t o f M o o d Another challenge in this area is the wide variation in the measurement of mood. Several studies have used standard measures of depressive symptoms or depression such as the Center for Epidemiologic Studies Depression Scale, known as the CES-D [56,59-62], the Beck Depression Inventory [63], or the General Health Questionnaire [64]. These

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measures are all indicative of depressive disorder. Other studies have used a set of symptoms derived from factor analyses of symptom lists that are labeled depressed mood or dysphoria [65,66] or psychological symptoms [55,61]. Factor analysis is a frequently used statistical technique to determine how a set of items "go together." Still other studies have used a single symptom from a symptom list, such as feeling blue or depressed [67] or irritable [68,69]. These different measurement approaches raise two issues. First, it is critical to distinguish between significant affective disorders of clinical significance and minor symptoms of mood disturbances [70]. This distinction is important not only from a research perspective, but clearly has implications for treatment. Second, one needs to be careful in comparing studies because of different outcomes being measured. Most of the studies using a factor labeled depressed or negative mood combine both feelings of sadness or depression with feelings of nervousness, anxiety, or irritability [55,60,65,66,71,72]. Further, some negative mood lists include insomnia or trouble sleeping (which may be related independently to menopause and/or cause mood disturbances) [60,65,66,73], whereas other studies have sleep problems as a separate factor [71,72]. For those studies that use an index or scale consisting of several different mood states, it is not possible to examine the impact of menopause on specific moods. Use of factor scales with different symptoms or moods does not allow one to make distinctions among moods. Thus, it is difficult to tease out specific mood changes associated with menopause. Further, it should also be pointed out that depression or feeling depressed is clearly the mood that has been the most studied. Other specific mood states often studied are feeling nervous (or tense) and anxiety. Rarely has general moodiness or mood lability been studied.

D. S y m p t o m R e p o r t i n g Additional problems arise from the way symptom lists are sometimes used. Problems occur with the use of menopausal checklists and with unreliable time frames for recall. Many early studies [69,74], as well as studies today, have made use of menopausal checklists that ask women to check off symptoms they have experienced due to menopause. Such a method of symptom reporting has two problems. One problem is that women will use their own definition of menopause. Not only does this lead to inconsistencies across women, but some women may define "being menopausal" by their symptoms. A second problem with this approach is that it requires women to determine which symptoms are due to menopause. This second problem perpetuates stereotypes of menopausal symptoms. Women will attribute symptoms to menopause that are part of their stereotyped belief of what women experience during this time. Parlee [75] ar-

gues with respect to menstrual symptom reporting that recollected symptom data are more vulnerable to the influence of stereotypical beliefs about the psychological concomitants of menstruation. The time lapse leads women to substitute the symptoms they "believe" to be associated with a particular phase for the symptoms they actually had, but do not recall with accuracy. Thus, assumptions regarding menopausal symptoms, or stereotypical views of menopause, become perpetuated with the use of menopausal checklists that ask women to report symptoms they experienced during menopause. Studies also differ in terms of the time frame used for symptom reporting. Some studies will ask women to report symptoms they experienced in the past 2 weeks, whereas others ask about longer periods of time, even up to the past year. It is generally accepted that the longer the time period of recall, the less accurate the recollection. Excellent discussions of these methodological problems related to symptom lists are provided by Kaufert and Syrutuik [76] and Kaufert, Gilbert, and Hassard [60].

IV. EMPIRICAL RESEARCH: MENOPAUSE STATUS AND MOOD This next section reviews the empirical research on menopause status and mood. This research includes factor analyses, cross-sectional studies, longitudinal studies, and studies that have looked at other variables related to mood.

A. F a c t o r A n a l y s e s One line of research that addresses the relation between menopause and mood is the examination of how symptoms cluster or group together. It can be argued that if psychological symptoms or mood changes are hormonally based, they should group with vasomotor symptoms, if the same hormones are responsible for both. A number of researchers have used the techniques of cluster analysis or factor analysis to determine how symptoms group together. One of the earliest such studies was conducted by McKinlay and Jefferys [77]; they conducted cluster analyses of eight symptoms (including sleeplessness and depression), menopause status, and various sociodemographic factors among a sample of 638 women aged 4 5 - 5 4 years. An examination of the clustering of these variables shows that, except for hot flashes, none of the symptoms clustered with menopause status. Factor analysis, as previously mentioned, is a statistical technique to determine how a set of items are related. The first reported factor analysis of symptoms was conducted by Greene [78], who factor analyzed a list of 30 symptoms from a study of 50 women, aged 4 0 - 5 5 years, who were patients at a hormone replacement clinic in London. Greene

CHAPTER23 Menopause and Mood found three factors, or clusters of symptoms, which he labeled vasomotor, somatic, and psychological. Since Greene's small clinic-based study, numerous large, population-based and clinic-based studies have conducted factor analyses of symptom lists. In a study of 682 London women aged 45-65 years from a general clinic, Hunter, Battersby, and Whitehead [71] found the following seven factors from a list of 36 symptoms: somatic symptoms, depressed mood, cognitive difficulties, anxiety/fears, sexual functioning, vasomotor symptoms, and sleep problems. Holte and Mikkelsen [73] report results of a factor analysis of 21 symptoms from 1566 Norwegian women aged 45-55 years who were not taking HRT that resulted in five factors, which they labeled vasomotor complaints, mood lability, nervousness, vague somatic complaints, and urogenital complaints. Dennerstein et al. [66] conducted a factor analysis of 22 symptoms among a population-based sample of 1897 women aged 45-55 years in Melbourne, Australia and found seven first-order factors: dysphoria, vasomotor, cardiopulmonary, skeletal, digestive, respiratory, and general somatic. When they conducted a secondorder analysis, they found three factors: vasomotor, general respiratory, and psychosomatic. Collins and Landgren [65] report results of a study of 1324 Swedish women, all aged 48 years. From a 20-item symptom list, they found the following four factors: negative mood, vasomotor symptoms, decreased sexual desire, and positive well being. This was the only study that included aspects of positive mood. In a small community-based study of 301 Seattle women aged 35-55 years and not taking HRT, Mitchell and Woods [72] found five factors among 28 symptoms: dysphoric mood, vasomotor symptoms, somatic symptoms, neuromuscular, and insomnia. In a population-based study of 1498 British women aged 47 years, Kuh, Wadsworth, and Hardy [55] found that 20 symptoms produced five factors: psychological, general somatic, vasomotor, sexual difficulties, and markers of aging. One study reported by Avis et al. [67] compared the factor structure of symptoms in three distinct populations (Canada, U.S., and Japan). All three studies were based on similar sample characteristics (i.e., community-based; aged 45-55 years; and pre-, peri-, and naturally postmenopausal women). Although Avis et al. [67] found different factor structures for the three countries, in all three samples vasomotor symptoms were distinct from psychological symptoms. These studies differ in terms of the specific symptoms studied, number of symptoms included in the list (ranging from 20 to 36), and time frame for symptom reporting (from the past 2 weeks up to 1 year). Studies also differ in sample characteristics such as age of sample (two studies included only women aged 47 or 48 years, whereas others included an age range), composition of sample (some exclude women taking estrogen), and whether the sample was clinic based or community based. Studies derive from a range of countries,

343 including the United States [59,67,72], Canada [60,67], Australia [66], Great Britain [55,71], Sweden [67], Norway [73], and Japan [67]. Despite these differences, the results are overwhelmingly consistent; in every study, vasomotor symptoms load on a separate factor from psychological symptoms. Although some analyses result in more than one mood, none of these psychological or mood symptoms load on the same factor as the vasomotor symptoms.

B. C r o s s - S e c t i o n a l Studies R e l a t i n g M e n o p a u s a l Status and M o o d Numerous cross-sectional studies have examined the relationship between menopause status and mood. Most of these studies do not find a relation between menopause status and depression or dysphoric or negative mood. These include studies using measures such as the CES-D [56,6062], a psychiatric interview [79,80], dysphoric mood [66], negative affect [81], nervousness and mood lability [82], anxiety/fears [71 ], psychological symptoms [55,61 ], and individual symptoms of irritability, anxiety, and/or depression [44,83]. Several studies, however, have found a relation between menopause status and mood, mostly in the perimenopause [64,65,67,71]. Avis et al. [67] conducted an analysis of responses to feeling blue or depressed by menopause status among women in the United States, Japan, and Canada. They found that for women in the United States sample, periand surgical menopausal women reported higher rates of "feeling blue or depressed" in the past 2 weeks than did preor postmenopausal women. There was no relation between status and symptom reporting for the Canadian or Japanese samples. Ballinger [64] reports a higher number of psychiatric cases (as defined by the General Health Questionnaire) among perimenopausal women in a sample of 539 women aged 40-55 years recruited from patient lists of six general practitioners. However, she also found that this association was due more to age than to menopausal status. Hunter, Battersby, and Whitehead [71] found higher rates of depressed mood (including loss of interest in things, lack of enjoyment, feeling miserable and sad, no feelings of well-being, life not worth living, irritability, and loss of appetite) among peri- and postmenopausal women in a sample of 682 women aged 4 5 - 6 5 years recruited from an ovarian screening clinic. They did not find a relation between status and symptoms labeled anxiety/fears or sleep problems. Although their sample was not recruited from a menopause clinic, it is a clinic sample, rather than a population-based sample. In a large Swedish sample of 1324 women all aged 48 years, Collins and Landgren [65] found higher rates of negative mood (including tension, depression, spells of crying, early wakening, insomnia, and difficulty concentrating)

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among postmenopausal women. Collins and Landgren defined postmenopausal as 6 months of amenorrhea and therefore this category of women probably included some women who would be classified as perimenopausal, using the WHO criteria. In multivariate analysis, only vasomotor symptoms were significantly related to status. They did not analyze the group of perimenopausal women, which was quite small. In a survey of 3000 women aged 4 0 - 6 0 years, Jaszmann, van Lith, and Zaat [69] also found higher rates of complaints of irritability among perimenopausal women, but did not conduct statistical tests. Huerta et al. [84] found higher scores of depression on a modified Hamilton depression and Bech-Rafaelson scale among women 1-year postmenopause in a sample of 222 women aged 3 6 - 6 1 years old recruited from a Social Security Hospital in Mexico. They did not find any differences in anxiety. In summary, although the majority of cross-sectional studies do not find a significant relation between menopause status and various measures of mood disturbances, several studies have reported slight increases among perimenopausal women. These studies, however, generally have not controlled for vasomotor symptoms. The one study that did control for symptoms found that the relation disappeared with vasomotor symptoms included in the model [65].

C. P r o s p e c t i v e a n d L o n g i t u d i n a l S t u d i e s The majority of studies in this area have been crosssectional. Such cross-sectional studies limit researchers to inferring causality from apparent associations [85]. Crosssectional studies can neither control for premenopausal characteristics of women (unless asked retrospectively) nor separate effects of aging from those of menopause. Longitudinal cohort designs facilitate identification of those associations that are most likely to reflect a cause-effect relationship through observation of temporal sequences in events or rate changes. Longitudinal designs can also separate effects of aging from effects of menopause Several longitudinal or prospective studies have been conducted [55,57,59,63,80,86-88]. Each of these studies analyzes their longitudinal data somewhat differently. In their 3year longitudinal study of Manitoba women, Kaufert et al. [57] did not find that the onset of menopause was related to increased depression using the CES-D scale. Also using the CES-D scale, Woods and Mitchell [88] did not find that onset of menopause was related to increased depression in a population-based sample of 347 midlife women in Seattle. The Pittsburgh Healthy Women study compared psychological characteristics and symptoms between pre- and postmenopausal women after 3 years of follow-up of a sample of 541 initially premenopausal women. Controlling for age and baseline levels of characteristics, they found that natural menopause did not adversely affect anxiety or depression [63]. Hallstr6m and Samuelsson [80] conducted a psychiat-

ric interview on 899 women from the general population in Sweden, on two occasions, 6 years apart. They found no increase in onset of mental disorder at menopause. Holte [86] reports a longitudinal follow-up of 59 women from his cross-sectional study who were premenopausal at the beginning of the study and postmenopausal at its end, 4 years later, and not taking HRT. Holte did not find an increase in anxiety or depression (as measured by Goldberg's General Health Questionnaire). Kuh et al. [55] followed a nationally representative sample of British women born in 1946. Their paper reports results of surveys completed by over 1200 women when they were 36 years of age and again when they were 47 years. Psychological symptoms at age 47 were unrelated to natural menopause status, except for a slight rise in irritability among perimenopausal women. However, women who had had a hysterectomy or were on HRT reported significantly more psychological symptoms. They further found that psychological symptoms at age 47 were strongly related to current family life and work stress, anxiety, depression, and health problems at age 36. Two exceptions to these findings are reported by Avis et al. [59] and Hunter [87]. Using data from the Massachusetts Women's Health Study (MWHS), Avis et al. [59] addressed the effect of change in menopause status on depression as measured by the CES-D scale, while controlling for prior depression. To study change in menopause status, a menopause transition variable was created that took into account a woman's menopausal status at the two time points (27 months apart) at which depression was measured (referred to as T~ and T2). Women were classified into five categories: premenopausal at both T~ and T2 (pre-pre), premenopausal at T~ and perimenopausal at T2 (pre-peri), perimenopausal at both T I and T2 (peri-peri), pre-or perimenopausal at T~ and postmenopausal at T2 (pre/peri-post), and postmenopausal at both T~ and T2 (post-post). Across all menopause statuses, those women who were classified as depressed at T~ had higher rates of depression at T2 (see Fig. 1). For women who were not depressed at T~, the rate of depression at T2 increased slightly as women moved from pre-pre to pre-peri, and was highest for women who remained perimenopausal for at least 27 months. The rate of depression began to decrease as women moved from peri- to postmenopause, and was lowest for those women who were postmenopausal for at least 27 months. Controlling for premenopausal depression, there was still a slight increase in depression among the peri-peri women, that is, women who were classified as perimenopausal at the beginning and end of the 27month period. In further analyses of the MWHS data, Avis et al. examined whether this increased rate of depression among the periperi women could be attributed to symptoms associated with menopause (i.e., hot flashes, night sweats, and menstrual problems). When menopausal symptoms were added to the regression model, it became a significant predictor of T2 de-

CHAPTER23 Menopause and Mood

345

FIGURE 1 Percentageof women classified as depressed at T2 by depression status at T1and menopausal transition. From Ref. [59]. Reprinted from Annals of Epidemiology, 4, Avis, N. E., Brambilla, D., McKinlay, S. M., and Vass, K. A longitudinal analysis of the association between menopause and depression, pp. 214-220. Copyright 1994, with permission from Elsevier Science.

pression, and menopausal transition was no longer statistically significant. Over all menopause transition categories, those women who reported experiencing hot flashes, night sweats, and/or menstrual problems consistently showed higher rates of depression. Figure 2 shows the relation between depression and symptoms. Hunter [87] describes follow-up data of women from their original Southeast England Cohort. She reports data on 36 women who were initially premenopausal and became peri- or postmenopausal 3 years later. She found that the women reported significantly more depressed mood at follow-up. However, she did not have a comparison group to control for age and she did not control for vasomotor symptoms in analysis. There was no change in anxiety. She found no evidence of an increase in clinical depression as defined by the General Health Questionnaire. In summary, longitudinal studies provide no evidence that onset of perimenopause leads to increased depression. One study that examined length of the perimenopause [59] did find a small increase in depression associated with a long perimenopause, which was not significant when vasomotor symptoms were included in the model.

D. P s y c h o s o c i a l a n d H e a l t h F a c t o r s Cross-sectional studies of depression and menopause have shown that psychosocial factors account for more of the variation in depressed mood among women at the time of meno-

pause than does menopause itself [55-57,62,64,82,89,90]. Greene and Cooke [90] conducted a detailed cross-sectional study of postmenopausal women in Glasgow and found that stressful life events involving exits from a woman's social network were associated with reports of psychological symptoms. These events accounted for a greater proportion of the variation in reports of psychological symptoms than did menopause status. Bart [91] analyzed mental hospital records of 533 women aged 4 0 - 5 9 years and found that the lack of meaningful roles and the consequent loss of selfesteem, rather than hormonal changes, seemed to account for menopausal depression. In a survey of women aged 4 0 - 5 5 years from general practitioner lists, Ballinger [64] found that the death of a parent and changing patterns of relations with children were related to psychiatric morbidity. In a cross-sectional analysis of 2500 women from the Massachusetts Women's Health Study, McKinlay et al. [56] looked at the relative contributions of health and social circumstances and menopause to depression. They found that the variables most associated with depression were lower education, marital status (widowed, divorced, and separated women have higher rates), physical health, and stress from worry about others. Other studies have also shown that socioeconomic status [71,82], stress [66], negative attitudes toward menopause and aging [55,66], and negative expectations of menopause [82] are related to more negative mood during menopause. In longitudinal research, Hunter [87] found that negative stereotyped beliefs about the menopause and being under

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FIGURE 2 Percentageof women classified as depressed at T2 by menopausal transition and menopausal symptomsat T2. From Ref. [59]. Reprinted from Annals of Epidemiology, 4, Avis, N. E., Brambilla, D., McKinlay, S. M., and Vass, K. A longitudinal analysis of the association between menopause and depression, pp. 214-220. Copyright 1994, with permission from Elsevier Science.

stress before the menopause, together with not working outside the home and being working class, were related to depression. Together, these factors accounted for 51% of the variation in depressed mood when these women became peri- or postmenopausal. Hallstr6m and Samuelsson [80] found that the weighted sum of life events, but not menopause, was associated with onset risk of mental disorder. Kaufert, Gilbert, and Tate [57] found that the likelihood of becoming depressed was increased for women with areas of current stress in their livesmparticularly if it was related to husbands or children. Other studies have found that ill health is significantly related to depression [56,57,81,88].

E. O t h e r V a r i a b l e s R e l a t e d to M o o d Longitudinal data suggest that prior depression is the primary factor related to depression during menopause. In addition to the previously reported findings of Avis et al. [59], Hunter [87] also found that depressed mood during the menopause was most strongly predicted by premenopausal depression. In their prospective study, Kuh et al. [55] found depression at age 36 years was highly related to depression at age 47. In their cross-sectional study, Porter et al. [61 ] found that depression was higher among those women who reported a past history of depression or anxiety. Studies have also found that women who report greater mood disturbances at menopause also report problems re-

lated to menstrual cycle or reproduction. These include previous or current premenstrual symptoms or complaints [29,65,66,88], dysmenorrhea [65], and postpartum depression [29]. Stewart and Boydell [29] divided women attending a menopausal clinic into groups experiencing high or low levels of psychological distress. They found that those reporting high levels of psychological distress were significantly more likely to report past psychiatric history, dysphoric premenstrual syndrome (PMS), and postpartum depression. Woods and Mitchell [88] found that both a history of PMS and postpartum depression differentiated women with consistently depressed mood (measured at two points in time, 1 year apart) from those recovering from depressed mood or not depressed. Holte and Mikkelson [82] found that women who reported experiencing greater depression during their menstrual periods earlier in life were more likely to report nervous complaints and mood lability at age 4 5 - 5 5 years. Menopause status was unrelated to either nervous complaints or mood lability. It is not clear from these findings, however, whether this pattern of symptom reporting related to reproductive events is hormonally based or is a result of greater symptom sensitivity or greater symptom reporting in general.

E

Cultural Differences

It is well recognized that menopause is experienced differently across cultures [ 18,92-94]. For example, hot flashes are uncommon in Mayan women [92]. Japanese and Indonesian

CHAPTER23 Menopause and Mood women report far fewer hot flashes than do women in Western societies [ 18,67,95]. South Asian women do not explicitly associate any physical or psychosocial difficulties with the physiological process of menopause [96]. Most menopause studies include women of only one culture. Cross-cultural comparisons are thus hampered by differences in study methodology. A few studies, however, have used similar methodology across cultures. Cross-cultural studies show that there is a cultural component to the relation between menopause and mood [67,68]. Avis et al. [67] compared Massachusetts, Canadian, and Japanese women aged 4 5 - 5 5 years in their rates of reporting feeling blue and depressed in the past 2 weeks. The highest rates in general were reported by the North American samples. In the Japanese and Canadian samples, the highest rates of feeling blue or depressed were found among the premenopausal women, whereas in the United States sample the highest rates were in the perimenopausal women. Also, there were essentially no differences by menopause status in the Canadian sample. In a study of the climacteric in seven Southeast Asian countries, Boulet et al. [68] found an inconsistent pattern of psychological complaints and menopause status across countries. Anxiety, irritability, and depression were each reported at higher rates by peri- or postmenopausal women. However, the pattern of symptom reporting varied by status across countries. These different patterns across countries clearly argue against a direct link between decline in estrogen and mood. Attempts to associate menopause and mood (particularly depression) through cross-cultural comparisons, however, are complicated by variations in the meaning of menopause as well as in the presentation of psychological symptoms [97].

347 pausal women aged 4 0 - 5 5 years [99]. Over half of their sample consisted of women attending a gynecology outpatient clinic. In general, they found no differences in hormone profiles of women with high or low levels of psychological symptoms. However, they did find that among 18 women in the early postmenopause group (last menstrual period 1 to 4 years ago), those with higher symptom scores had significantly higher concentrations of estradiol compared to women with lower scores. The 48 women who were recruited from the gynecology clinic were interviewed for depression using a clinical interview. There were no significant differences in hormone profiles between the depressed and nondepressed groups. Cawood and Bancroft [83] conducted a study in which they concurrently measured mood (according to the multiple affect adjective checklist) and hormones among 145 women aged 4 0 - 6 0 years recruited from the community. Women completed an initial interview and four subsequent interviews 1 week apart. They found no relation between circulating estrone (El) or estradiol (E2) and depression or positive affect plus sensation seeking (the only moods analyzed). However, they did find that feelings of tiredness were significantly related to depression and positive affect. Tiredness was also positively correlated with hot flushes. In the Massachusetts Women's Health Study, we are examining the relation between hormones (specifically E2) , symptoms, and depression among women traversing the menopause. Preliminary evidence does not suggest a relation between hormones and depression. However, experiencing hot flashes or night sweats or having trouble sleeping do appear related to depression.

B. HRT and Mood V. EMPIRICAL RESEARCH: HORMONES AND MOOD

A. Studies Examining the Relation between Endogenous Hormones and Mood The most direct evidence of a relation between hormones and mood would come from studies concurrently measuring both hormones (specifically estrogen) and mood among menopausal women. Unfortunately, very few such studies exist. Chakravarti et al. [98] studied 82 premenopausal women seen at a menopause clinic. They found that although mean plasma estradiol concentrations were associated with vasomotor symptoms, there was no clear association between hormone levels and symptoms (including depression and irritability). In 1987, Ballinger, Browning, and Smith studied hormone profiles and psychological symptoms in 85 perimeno-

It has been hypothesized that if estrogen is related to mood, then HRT users should show less mood disturbance than nonusers. Reviews in this area, however, have concluded that there is not enough convincing evidence at this point that estrogen treatment improves psychological symptoms or mood [46,87,100]. Most studies of HRT and mood are observational and thus subject to selection bias. It has been well established that estrogen users tend to be better educated and healthier than nonestrogen users. These variables are also negatively related to depression. Although the extensive literature on selection factors to estrogen use has generally not included psychological factors of women choosing to take estrogen, undoubtedly there are psychological selection factors as well. Thus, any study comparing users and nonusers must adequately control for these selection factors [100]. Further, some observational studies have actually found greater mood disturbances among current or previous HRT users [55,58,61,65,100-102]. Matthews et al. [63] followed women aged 4 2 - 5 0 years who were initially premenopausal for 3 years. They found that women who

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became postmenopausal during this time and had taken HRT within the previous 2 months increased significantly in depression scores from baseline to follow-up examinations, whereas women who became postmenopausal and had not taken HRT in the previous year did not. Reviews of well-controlled clinical trials find inconsistent results [46,103]. Some studies report evidence of positive effects of estrogen replacement therapy significantly greater than for placebo, whereas other studies do not. Many clinical trials of HRT do not adequately control for a placebo effect and/or are conducted on selected cohorts [103]. Because several studies have clearly demonstrated a significant placebo effect with HRT [104-107], it is thus critical that any well-designed study has a control group and be double blind. Clinical trials conducted on small numbers of depressed women have provided inconsistent results [ 100,103]. Other studies have been conducted on women undergoing a surgical menopause. Although these studies more consistently show positive effects of estrogen on mood, we cannot necessarily generalize these results to the general population [ 100]. In both observational studies and clinical trials, any positive effect of HRT on mood must also take into account the effect of HRT on vasomotor symptom reduction. As reviewed by Holte [46], almost all of the clinical trials concluding that HRT influences mood have lacked adequate control for the effects of HRT in relieving vasomotor complaints. A recent meta-analysis on the effect of HRT on depressed mood did not even mention vasomotor symptoms [108]. Finding that estrogen users report more positive mood alone does not tell us whether this is due to a direct effect of estrogen or to symptom reduction. A study by Alder [109], controlling for vasomotor symptoms and nonmenopausal social factors, found that minor stresses contributed more to psychological symptoms than did hormone levels or time since insertion of estrogen implants.

C. N e u r o b i o l o g y o f E s t r o g e n Most of the research on the neurobiology of estrogen has been conducted on animals. However, a few small studies provide some support for the effects of estrogen on aspects of the serotonin system in menopausal women. Klaiber et al. [ 110] report that postmenopausal women are more likely to have increased monoamine oxidase activity in comparison with premenopausal women. Gonzales and Carrillo [111] found that postmenopausal women have lower blood serotonin concentrations than do premenopausal women and that an increase in serotonin follows estrogen replacement therapy in postmenopausal women. However, decreases in gonadal hormones are not related in a simple or direct way to psychological distress during meno-

pause. Sex steroid concentrations per se have not distinguished symptomatic from asymptomatic women [22,112].

VI.

CONCLUSIONS

Menopause is thought to alter central nervous system function as manifested by hot flashes, mood changes, and sleep disturbances. However, only vasomotor symptoms such as hot flashes and night sweats have been clearly and consistently associated with menopause. Epidemiologic studies of menopause and mood do not show consistent evidence of a relation between menopause and depression or other negative moods in the general population. This conclusion is supported by reviews of the literature on menopause and depression [25,113]. Factor analyses of symptom lists have quite consistently shown that vasomotor symptoms load on a factor separate from psychological symptoms. Community-based or population-based studies consistently show that in the general population of women, only a minority of peri- or postmenopausal women evidence signs of depression or other mood disturbance. Although some studies have found more negative mood among perimenopausal women, it appears that the majority of mood disturbance seen at the time of menopause can be attributed to prior depression, vasomotor symptoms, or non-menopause-related factors such as health problems or social circumstances. The domino theory that vasomotor symptoms lead to mood disturbances has some support. A number of studies have found a strong association between vasomotor complaints and depressed mood both cross-sectionally [65,82,100,114,115] and longitudinally [59,86,114]. These findings suggest that studies need to control for vasomotor symptoms in analyses of any direct effect of menopause on mood. They also provide some support for the domino theory that vasomotor symptoms have an impact on mood. However, many studies do not find a relation between menopause and mood, even when vasomotor symptoms have not been controlled for. Further, other studies suggest that not all women who have vasomotor symptoms experience mood disturbances. Further research should examine more closely whether asymptomatic women show fluctuations in mood. Research provides consistent evidence that social circumstances can account for much of the mood effects during the menopause transition. Studies that have included measures of stress or life changes have typically found that social factors are highly related to mood, and often more so than menopause status. Thus, there is strong support for the social circumstances perspective that menopause per se is less responsible for mood effects than are life events of middleaged women. The psychoanalytic view posits that onset of menopause has a psychological impact on women. According to this

CHAPTER 23 Menopause and Mood view, the greatest psychological impact of menopause should be found among women as they transition from pre- to perimenopause. Longitudinal studies have not found any evidence for increased mood disorders at the time of this transition. Thus, the research does not provide support for this hypothesis. Although onset of menopause may have a psychological impact on some women, they clearly appear to be in the minority. Biochemical research on the neurobiology of estrogen and mood is an active area of investigation, but there is insufficient evidence at this time to support a biochemical hypothesis for menopause and mood disturbances. Researchers have shown that estrogen can alter brain neurotransmitter activity in several ways, although the majority of this research has been conducted in animals and the extent to which neurotransmitter activity interacts with menopause-related mood changes has yet to be determined [22]. There is no clear understanding of the mechanisms whereby hormonal changes mediate change in mood [ 116]. A review by Blehar and Oren [117] on the psychobiology of depression concluded that biological factors (compared to genetic, environmental, and psychological factors) are the weakest in terms of what research can tell us about depression. Menopausal symptoms are related to depression, but correlational studies of mood and hormone status have failed to show a relation between depressed mood and estrogen concentrations. Evidence suggests that the rate of hormonal changes may be the important regulatory variable, rather than absolute hormonal concentrations [22], which may explain why associations between hormones and mood disorders are stronger for women undergoing a bilateral oophorectomy than for naturally menopausal women. Further, even though all women experience a decline in estrogen following menopause, not all women report mood disturbances. These results suggest that other factors mediate the relation between hormonal level and depressed mood. There is no evidence that menopause is associated with increased mood disturbance on a population level, but an unanswered question is whether some women may be more vulnerable to mood effects of hormonal changes. Several researchers have proposed that whereas menopause does not cause mood disturbances in all women, there may be a subgroup of women at higher risk for depression or other mood changes [28,112,116]. It has been suggested that women with a history of PMS have an increased sensitivity to hormone changes [118] and that women with previous affective disorders that are cyclic or associated with reproductive events are at higher risk [25]. Although the postpartum period is not always associated with depression, women with previous depression, a history of premenstrual dysphoric disorder, or a history of bipolar disorder are at increased risk for postpartum depression [ 117]. However, it is premature to conclude that this pattern is related to a hormonal imbalance.

349 Other factors that may be related are coping style or a greater sensitivity to symptoms. Further, because most studies involve retrospective reporting of reproductive and psychiatric history, there is the inherent problem of selective recall among women experiencing problems at the time of the study. Further research needs to focus on developing a better understanding of women who may be at risk for mood effects of the menopause transition and whether there is a pattern in response to periods of hormonal fluctuations. Are such effects due to abnormal biochemical changes resulting from hormonal fluctuations, a woman's general sensitivity to symptoms, or a coping style? Prospective research needs to follow women during their reproductive years to examine continuity with respect to responses to times of hormonal change. It is also possible that some women show improved mood as a result of menopause. If there is a subgroup of women who show improved mood and a subgroup of women who show mood disturbance, these two groups of women may offset each other and be obscured when looking at population data as a whole. This possibility should be examined in future studies. Clearly, depression is the most frequently studied mood in relation to menopause. It has been studied as a psychiatric disorder as well as a mild dysphoric mood. In fact, depression is the only affective disorder to receive significant research attention. Studies have also looked at groupings of 9symptoms often labeled psychological symptoms, negative affect, or dysphoria. However, individual symptoms such as nervousness, irritability, or mood lability have rarely been studied. Further, little research has focused on mood fluctuations (moodiness) and minor mood changes. Further research needs to focus more on these two symptoms.

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36. Hunt, R., Vessey, M., McPherson, K., and Coleman, M. (1987). Longterm surveillance of mortality and cancer incidence in women receiving hormone replacement therapy. Br. J. Obstet. Gynaecol. 94, 620-635. 37. Avis, N. E., Crawford, S. L., and McKinlay, S. M. (1997). Psychosocial, behavioral, and health factors related to menopause symptomatology. Women's Health: Res. Gender, Behav. Policy 3, 103-120. 38. Ballinger, S. E. (1985). Psychosocial stress and symptoms of menopause: A comparative study of menopause clinic patients and nonpatients. Maturitas 7, 315-327. 39. Brown, J. R. W. C., and Brown, M. E. A. (1976). Psychiatric disorders associated with the menopause. In "The Menopause: A Guide to Current Research and Practice" (R. J. Beard, ed.). University Park Press, Baltimore, MD. 40. Morse, C., Smith, A., Dennerstein, L., Green, A., Hopper, J., and Burger, H. (1994). The treatment seeking women at menopause. Maturitas 18, 3. 41. Rose, L. (1977). What is menopause? In "The Menopause Book" (L. Rose, ed.), pp. 201-245. Hawthorne Books, New York. 42. Weideger, P. (1977). "Menstruation and Menopause." Delta, New York. 43. Avis, N. E., and McKinlay, S. M. (1990). Health-care utilization among mid-aged women. Ann. N. Y Acad. Sci. 592, 228-238. 44. Ballinger, C. B. (1976). Psychiatric morbidity and the menopause: Clinical features. Bri. Med. J. 1, 1183-1185. 45. Ballinger, C. B. (1977). Psychiatric morbidity and the menopause: Survey of a gynaecological out-patient clinic. Br. J. Psychiatry 131, 83-89. 46. Holte, A. (1998). Menopause, mood and hormone replacement therapy: Methodological issues. Maturitas 29, 5-18. 47. Bungay, G. T., Vessey, M. P., and McPherson, C. K. (1980). Study of symptoms in middle life with special reference to the menopause. Br. Med. J. 281, 181-183. 48. Pansini, F., Albertazzi, P., Bonaccorsi, G., Calisesi, M., Campobasso, C., Zanotti, L., Bagni, B., and Mollica, G. (1994). The menopausal transition: A dynamic approach to the pathogenesis of neurovegetative complaints. Eur. J. Obstet. Gynecol. Reprod. Biol. 57, 103109. 49. World Health Organization Scientific Group (1996). Research on the menopause in the 1990's. WHO Tech. Rep. 886, 12-14. 50. Brambilla, D. J., McKinlay, S. M., and Johannes, C. B. (1994). Defining the perimenopause for application in epidemiologic investigations. Am. J. Epidemiol. 140, 1091-1095. 51. Cooper, G. S., and Baird, D. D. (1995). The use of questionnaire data to classify peri- and premenopausal status. Epidemiology 6, 625-628. 52. Brett, K. M., Marsh, J. V. R., and Madans, J. H. (1997). Epidemiology of hysterectomy in the U.S.: Demographic and reproductive factors in a nationally representative sample. J. Women's Health 6, 309-316. 53. Kjerluff, K., Langenberg, P., and Guzinski, G. (1993). The socioeconomic correlates of hysterectomies in the United States. Am. J. Public Health 83, 106 - 108. 54. Roos, N. P. (1984). Hysterectomies in one Canadian province: A new look at risks and benefits. Am. J. Public Health 74, 39-46. 55. Kuh, D. L., Wadsworth, M., and Hardy, R. (1997). Women's health in midlife: The influence of the menopause, social factors and health in earlier life. Br. J. Obstet. Gynaecol. 104, 923-933. 56. McKinlay, J. B., McKinlay, S. M., and Brambilla, D. (1987). The relative contributions of endocrine changes and social circumstances to depression in mid-aged women. J. Health Soc. Behav. 28, 345-363. 57. Kaufert, P. A., Gilbert, P., and Tate, R. (1992). The Manitoba Project: A reexamination of the link between menopause and depression. Maturitas 14, 143-155. 58. O'Connor, V. M., DelMar, C. B., Sheehan, M., Siskind, V., FoxYoung, S., and Cragg, C. (1995). Do psycho-social factors contribute more to symptom reporting by middle-aged women than hormonal status? Maturitas 20, 6 3 - 69. 59. Avis, N. E., Brambilla, D., McKinlay, S., and Voss, K. (1994). A lon-

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CHAPTER 23 Menopause and Mood

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2 H A P T E R 2~

Immunologic Aspects of Menopausc D E B O R A H J. A N D E R S O N

Fearing Laboratory, Department of Obstetrics, Gynecology, and Reproductive Biology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115

IV. Hormone Replacement Therapy--Effects on Autoimmunity and Immune Function V. Conclusions References

I. Effects of Steroid Hormones on the Immune System II. Somatic Aging of the Immune System III. Effects of the Immune System and Its Decline on Other Events Associated with Menopause

I. E F F E C T S

eases is severalfold higher in women than in men (Table I) [5a], and many autoimmune diseases in women first appear or peak during periods of major hormonal changes (puberty, menopause). High estrogen states exacerbate systemic lupus erythematosus (SLE), but often decrease the severity of autoimmune arthritis [6]. All three of the principal groups of sex hormones (estrogens, progestins, and androgens) have been shown to affect various immune functions. High-affinity estrogen receptors are expressed on CD8 + T cells, thymocytes, macrophages, and endothelial cells [6]. Estradiol stimulates antigenspecific primary antibody responses, probably due to inhibition of CD8 + T cells that normally regulate B cell function. The estrogenic influence on macrophages and other antigen-presenting cells is weak, but inhibition of macrophage interleukin-1 (IL-1) production and an increase in phagocytic activity have been reported [6]. Although administration of estradiol to ovariectomized mice results in thymic atrophy [6], little is known about effects of estrogens on thymic function or T cell development. Effects of progesterone on immune functions may be primarily mediated through glucocorticoid receptors. Lymphocytes apparently do not express classical progesterone receptors, but both macrophages and lymphocytes express

O F STEROID

HORMONES ON THE IMMUNE SYSTEM A. S y s t e m i c I m m u n i t y Menopause in women results from a sharp decline in ovarian function that normally occurs during the fourth or fifth decade of life. Postmenopausal women experience markedly decreased ovarian production of estradiol, progesterone, testosterone and inhibin, and increased pituitary production of follicle stimulating hormone (FSH) and luteinizing hormone (LH) [1]. There is also evidence that many postmenopausal women experience reduced adrenal endocrine function, evidenced by lower circulating concentrations of cortisol and androstenedione [1,2]. Sex and adrenal steroid hormones influence the immune system in a variety of ways [3-5]. It has been known for several years that sexually mature female animals usually produce higher titers of antibodies than do age-matched males following immunization, and several inbred animal models of autoimmune diseases show more severe symptoms and/or earlier onset of disease among female animals. The prevalence of many serious clinical autoimmune disMENOPAUSE: BIOLOGY AND PATHOBIOLOGY

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TABLE I Increased Prevalence of Autoimmune Diseases in Women a Prevalence/disease Common (> 1 in 1000) Rheumatoid arthritis Pernicious anemia Graves' disease Hashimoto's thyroiditis Type 1 diabetes mellitus Multiple sclerosis Moderately common (<1 in 1000) Systemic lupus erythematosus Sj6gren's syndrome Chronic active hepatitis Celiac disease

Rare (<1 in 10,000) Scleroderma Polymyositis Goodpasture's syndrome Primary biliary cirrhosis Autoimmune hemolytic anemia Immune thrombocytopenic purpura Pemphigus vulgaris Bullous pemphigoid Dermatitis herpetiformis Myasthenia gravis

Age at onset (years)

Female: male ratio

35-50 60-70 20-40 40-60 12-20 30

3:1 1.5:1 6:1 9:1 1:1 1.5:1

20-40 50 10-25, 5 0 - 6 0 0.5-2.5 45-65 45-65 none >35 >60 20-40 40-60 >60 20-40 20-30

9:1 9: 1 3:1 1: 1 3:1 2:1 1: 1 9:1 1.5:1 4:1 1:1 1: 1 1:1 3:1

a Adapted from Ref. 5a, Oilier and Symmons (1992). Autoimmunity. In "Medical Perspective Series" (A.P. Read and T. Brown, eds.), p. 74. BIOS Scientific Publishers, Oxford.

glucocorticoid receptors [7]. Glucocorticoids have potent T cell immunosuppressive and antiinflammatory effects; they exert these effects directly through inhibition of many monocyte and T cell cytokine genes, especially IL-1, tumor necrosis factor (TNF), and IL-2 [8]. Certain progestins having a pregnane-type structure bind to and activate the glucocorticoid receptor, and have glucocorticoid-like immunosuppressive effects, including (1) inhibition of T cell activation, (2) increased survival time of homografts, (3) increased ease of tumor induction, and (4) induction of lymphocytopenia [5]. It is of interest that synthetic progestins of the 19nortestosterone type do not bind to the glucocorticoid receptor, and appear to have no effects on immune function [5]. Glucocorticoids also can promote antibody responses through T suppressor cell inhibition, and a recent study demonstrated that progesterone treatment of human cells in vitro suppressed cellular immunity but promoted the humoral arm of the immune system by inducing T helper (Th) cell secretion of Th-2-type cytokines (IL-4 and IL-5) that promote differentiation of B cells and antibody synthesis [9,10]. Progesterone also affects various mechanisms of inflammation, including (1) modulation of IL-1 and TNF-ce production by monocytes and (2) inhibition of inducible nitric oxide (NO) synthase gene expression and NO production in murine macrophages [ 11 ]. Furthermore, there is extensive evidence for an interaction between ovarian and adrenal endocrine

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functions. Female animals have greater basal concentrations of circulating corticosterone and greater glucocorticoid responses to stress [4]. Ovariectomy results in depressed basal and stimulated concentrations of circulating glucocorticoids to values similar to those of intact males. Replacement of estradiol in ovariectomized females induces an increase in base line and stimulated concentrations of corticosterone, whereas androgens have the opposite effect [12]. Furthermore, cortisol concentrations in postmenopausal women were increased following hormone replacement therapy (HRT) [2]. Therefore, many of the systemic effects attributed to ovarian hormones may be mediated by glucocorticoids, which are the most potent steroid regulators of immune and inflammatory responses. Androgens likewise generally have predominantly immunosuppressive effects. Testosterone does not interact with glucocorticoid receptors, but it is conceivable that other androgenic steroids could activate glucocorticoid receptors due to a high degree of homology between various steroids and also their receptors [4]. Furthermore, postreceptor mechanisms could also be involved. Glucocorticoids and androgens share the same hormone response elements in sensitive genes [ 13], suggesting that these hormones may have synergizing effects on gene expression. Androgen receptors have been described in human thymocytes, but not in peripheral lymphocytes. All of the sex steroids can cause thymic atrophy, and several lines of evidence suggest that effects of sex hormones on this gland may contribute to their actions on immune responses [14].

B. M u c o s a l I m m u n i t y Immune defense of mucosal surfaces in the genital tract appears to be particularly affected by ovarian hormones. In rodents, estradiol up-regulates antigen presentation and immunoglobulin A (IgA) levels in the uterus, and progesterone partially inhibits this effect [15]. Estrogens have also been shown to elevate IgM production, and increase the expression of the polymeric Ig receptor that mediates transport of IgA and IgM across the mucosal epithelium [16]. Changes in IgA concentrations have been documented in genital tract secretions of women during the menstrual cycle, with lowest levels recorded at midcycle [17]. In rodents and humans, estrogens increase the thickness of the vaginal epithelium and promote the secretion of mucins and other important protective factors, such as complement component C3 by reproductive tract epithelial cells [ 18]. Thus, estrogens appear significantly to promote mucosal immunity and protection of the genital tract against infections. Other mucosal immune functions appear to be influenced by progestins. Macrophages play an important role in mucosal immunity, and are particularly sensitive to effects of progesterone [19]. The secretion of several cytokines, including leukemia inhibition factor (LIF), granulocyte/

CHAPTER24 Immunologic Aspects of Menopause macrophage colony-stimulating factor (GM-CSF), IL-1, and transforming growth factor /3 (TGF-fl), which have immunoregulatory functions, are up-regulated in the human female reproductive tract during the luteal (progesteronedominated) phase of the menstrual cycle [ 18]. In addition, a population of natural killer (NK)-like cells is recruited to the uterine stroma during the luteal phase of the menstrual cycle; because these cells do not express progesterone receptors [20], this effect may be an indirect effect of progesterone. It has been demonstrated that progesterone inhibits the induction of chemokine receptor expression by T cells, and inhibits the production of chemokines by activated CD8 + T cells [21 ]. Because chemokines and chemokine receptors are important regulators of T cell migration through tissues, these data suggest that progesterone may have an inhibitory effect on T cell migration. It has been known for some time that progesterone enhances susceptibility to sexually transmitted infections in animal models, perhaps due to epithelial thinning or to changes in the local immune defense [22,23]. In contrast, it has been reported that women are more susceptible to chlamydial salpingitis during the menstrual and proliferative (estrogen-dominated) phases of the menstrual cycle [24]. More research is needed on the subject of sex steroid hormone effects on mucosal immune defense and genital tract infections in women. Sex steroids have been implicated as cofactors in the sexual transmission of the human immunodeficiency virus type 1 (HIV-1). Steroid hormones can bind to regulatory sequences of HIV-1 and modulate viral expression [25,26]. Epidemiological studies indicate that women taking oral contraceptives may be more susceptible to HIV-1 infection [27]. No data are available on effects of hormone replacement therapy on susceptibility to sexually transmitted infections in postmenopausal women.

II. S O M A T I C A G I N G OF THE IMMUNE SYSTEM The immune system changes with age: in particular, immune function of the T cell compartment of the immune system peaks at puberty and gradually declines thereafter with age. During puberty, sex and adrenal steroids trigger thymic involution by inducing extensive apoptotic death of thymocytes [28]. The endocrine function of the thymus, which is essential for T cell maturation, also declines with age, giving rise to the concept of "thymic menopause" [29]. The intrathymic environment is composed of a complex network of paracrine, autocrine, and endocrine signals involving interleukins and thymic peptides, which operate synergistically to enable the maturation of T lymphocytes. With aging there is a decline in the production of naive lymphocytes by the central lymphoid organs, which leads to reduced diversity in antigen specificities recognized by the immune

355 system. Thus, there is a progressive decline in the ability of the immune system to react with foreign antigens, and increased reactivity to autoantigens. The age-related thymic involution is ascribed to both intrinsic and extrinsic factors. Steroid hormones and circulating levels of IL-lfl and IL-2 are extrinsic factors that affect thymic function [29]. There is also evidence that peripheral immune functions are affected by aging. Although reports in this area are often conflicting, there is a general consensus that T cell proliferation is impaired and cytokine profiles are altered in aged men and women. Reported alterations in cytokine profiles include decreased production of IL-2 (T cell growth hormone) and increased production of certain proinflammatory cytokines (IL-lfl, IL-6, TNF-ce) and cytokines that promote humoral immunity (IL-4, IL-5). These changes in T cell activity and cytokine profiles could contribute to some of the immunopathologic states associated with aging, including osteoporosis, atherosclerosis, and autoimmune disease [30].

III. EFFECTS OF THE IMMUNE SYSTEM AND ITS DECLINE ON

OTHER EVENTS ASSOCIATED WITH MENOPAUSE A. C y t o k i n e s and O v a r i a n F u n c t i o n The immune system influences ovarian function through cytokines. Certain T cell cytokines, such as interferon-y, which is produced during cell-mediated immune responses, down-regulate the production of estradiol and progesterone by granulosa cells in vitro [31 ]. Other cytokines may actually have a fundamental role in normal ovarian physiology. The normal ovary contains many macrophages, and two of their products, IL-1 (a and/3) and TNF-a, have been shown to promote steroid synthesis by luteinized ovarian granulosa cells, affecting their ability to proliferate [32]. If these cytokines play a critical role in the normal physiology of the ovary, then the changes in cytokine production that occur with aging could have an impact on ovarian function.

B. A u t o i m m u n i t y and P r e m a t u r e O v a r i a n F a i l u r e Several lines of evidence suggest that autoimmunity plays a role in premature ovarian failure (POF). There is a known association between autoimmune disease and POF: approximately 10% of women with POF will also have Hashimoto's autoimmune thyroiditis, and 1-2% will have adrenal insufficiency [33]. Furthermore, POF is often associated with high titers of circulating antiovarian antibodies and/or lymphocytic infiltration of the ovary [34]. Little is known about the etiology or natural history of autoimmune ovarian failure in women. Studies in animals have shown that ovarian antibodies develop in certain strains of rodents following disrup-

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tion of T cell tolerance (i.e., by neonatal thymectomy) [35], and also that humoral antibodies, cellular immunity, and subsequent ovarian failure can be elicited by immunization with ovarian antigens [36]. A study of the immunopathogenesis of ovaries from women with POF revealed that the granulosa cells in POF but not in normal ovaries express high levels of Class II histocompatibility antigens, which are involved in antigen presentation and have been implicated in the induction of tissue-specific autoimmune disorders [37]. This provides further evidence that many cases of clinical POF have an autoimmune component. Because the incidence and titers of autoantibodies increase with age, it is also possible that autoimmunity plays a role in the decline of ovarian function associated with the menopause.

C. B o n e R e s o r p t i o n Several studies have shown that the cytokines IL-1, IL-6, and TNF-ce play a role in bone remodeling. In particular, IL-1 has been implicated in the pathogenesis of postmenopausal osteoporosis because of its potent stimulatory effects on bone resorption in vitro and in vivo [38]. Ovariectomy in rats causes a rapid increase in bone turnover and a marked decrease in bone density, which is abrogated by administration of estradiol. In this model, ovariectomy is also associated with increased IL-1 production by bone marrow cells, and the ovariectomy-induced bone loss was significantly decreased by IL-1 receptor antagonist treatment (which blocks the action of IL- 1) [39]. Studies in menopausal women have also shown that circulating IL-1 levels and production of IL-1 by macrophages are increased after the menopause, and that IL-1 and other proinflammatory cytokines also play a role in bone resorption in humans [38,40]. These studies indicate that IL-1 and possibly other proinflammatory cytokines play an important causal role in the mechanism by which menopause induces bone loss in women. Furthermore, receptor antagonist (RA) therapy, such as with IL-IRA, which decreases IL-1 production by bone marrow cells, may provide an important therapeutic intervention for osteoporosis.

IV. H O R M O N E

REPLACEMENT

THERAPY~EFFECTS AUTOIMMUNITY IMMUNE

ON

AND

FUNCTION

Aging women experience a progressive decline in T cell immunity and increased production of autoreactive lymphocytes and antibodies. These effects are primarily attributable to the somatic aging and decline of the thymus, which is the gatekeeper of T cell development, programming, and function. A decline in T cell specificity and function directly affects antibody synthesis, because B cell differentiation and

antibody secretion are directly regulated by cytokines produced by CD8 + (suppressor) and CD4 § (helper) T cells. Circulating levels of proinflammatory cytokines such as IL-1 are also increased in postmenopausal women, which could contribute to the immunopathogenesis of osteoporosis and arthritis. Hormone replacement therapy (HRT) has been reported to increase circulating concentrations of cortisol in postmenopausal women, which could cause mild T cell immunosuppression [2]. Furthermore, patients with premature ovarian failure were found to exhibit a reduction in the ratio of CD4 § to CD8 + T cells in peripheral blood, but this condition was reversed by estradiol replacement therapy [41]. Estrogen replacement therapy also reduced levels of circulating IL-1 in postmenopausal women to premenopausal values, indicating that the beneficial effect of estrogen replacement therapy on osteoporosis may be mediated through its suppressive effect on proinflammatory cytokine production, because these factors have been shown to promote bone resorption [40]. Estrogen replacement therapy is recommended for postmenopausal women with rheumatoid arthritis [42], but must be used with extreme caution in women with SLE [43].

V. C O N C L U S I O N S In postmenopausal women, natural immunoregulatory effects of endogenous ovarian and adrenal steroids may be reduced, whereas exogenous estrogen and progestins administered in HRT may restore, suppress, or amplify some of these effects. More clinical studies are needed on the effects of menopause, with or without HRT, on general immunological functions and clinical disorders arising from or exacerbated by chronic immunosuppression or shifts in immunoregulation, including autoimmunity, neoplasia, and infectious diseases. Special attention should be given to neoplastic and infectious diseases of the genital tract, because immune defense functions at this site are clearly affected by ovarian steroid hormones.

References 1. Burger, H. G. (1999). The endocrinologyof the menopause.J. Steroid Biochem. Mol. Biol. 69, 31-35. 2. Helgason, S., Carlstrom, K., Damber, J. E., Selstam, G., and von Schoultz, B. (1981). Effects of various estrogens on circulating androgens and cortisol during replacement therapy in post-menopausal women. Maturitas 3, 301-308. 3. Grossman,C. J. (1985). Interactions between the gonadal steroids and immune system. Science 227, 257-261. 4. DaSilva, J. A. (1995). Sex hormones, glucocorticoids and autoimmunity: Facts and hypotheses.Ann. Rheum. Dis. 54, 6-16. 5. Paavonen,T. (1994). Hormonalregulation of immuneresponses. Ann. Med. 26, 255-258. 5a. Oilier,W., and Symmons,D. P. M. (1992). Autoimmunity.In "Medical

CHAPTER 24 Immunologic Aspects of Menopause

6.

7.

8.

9.

10.

11. 12.

13.

14.

15.

16.

17. 18. 19. 20.

21.

22.

23. 24.

Perspective Series" (A. E Read and T. Brown, eds.), p. 74. BIOS Scientific Publishers, Oxford. Cutolo, M., Sulli, A., Seriolo, B., Accardo, S., and Masi, A. T. (1995). Estrogens, the immune response and autoimmunity. Clin. Exp. Rheumatol. 13, 217-226. Shust, D., Anderson, D. J., and Hill, J. A. (1996). Progesterone-induced immunosuppression is not mediated through the progesterone receptor. Hum. Reprod. 11,980-985. Sternberg, E. M., and Wilder, R. L. (1993). Coricosteroids. In "Arthritis and Allied Conditions. A Textbook of Rheumatology" (D. J. McCarty and W. J. Koopman, eds.), 12th ed., pp. 665-682. Lea & Febiger, Philadephia. Piccinni, M.-E, Guidizi, M.-G., Biagiotti, R., Beloni, L., Giannarini, L., Sampognaro, S., Parronchi, E, Manetti, R., Annunziato, E, Livi, C., Romagnani, S., and Maggi, E. (1995). Progesterone favors the development of human T helper cells producing Th2-type cytokines and promotes both I1-4 production and membrane CD30 expression in established Thl cell clones. J. Immunol. 155, 128-133. Wang, Y., Campbell, H. D., and Young, I. G. (1993). Sex hormones and dexamethasone modulate interleukin-5 gene expression in T lymphocytes. J. Steroid Biochem. Mol. Biol. 44, 203. Chao, T. (1993). The effects of female sex hormones on macrophage function. Diss. Abstr. Int. 54. Lesniewska, B., Miskowa, B., Nowak, M., and Malendowicz, L. K. (1990). Sex differences in adrenocortical structure and function. The effect of ether stress on ACTH and corticosterone in intact gonadectomized and testosterone or estradiol-replaced rats. Res. Exp. Med. 190, 95 -103. Beato, M., Chalepakis, G., Schauer, M., and Slater, E. E (1989). DNA regulatory elements for steroid hormones. J. Steroid Biochem. 32, 737-747. Sobhon, E, and Jirasattham, C. (1974). Effects of sex hormones on the thymus and lymphoid tissue of ovariectomized rats. Acta Anat. 89, 211-225. Wira, C. R., and Rossoll, R. M. (1995). Antigen-presenting cells in the female reproductive tract: Influence of sex hormones on antigen presentation in the vagina. Immunology 84, 505-508. Wira, C. R., Kavshic, C., and Richardson, J. (1999). Role of sex hormones and cytokines in regulating the mucosal immune system in the female reproductive tract. In "Mucosal Immunology" (E L. Ogra, J. Mestecky, M. E. Lamm, W. Streber, J. Bienenstock, and I. D. McGhee, eds.), pp. 1449-1461. Academic Press, San Diego, CA. Kutteh, W. H., and Mestecky, J. (1994). Secretory immunity in the female reproductive tract. Am. J. Reprod. Immunol. 31, 40-46. Anderson, D. J. (1996). The importance of mucosal immunology to problems in human reproduction. J. Reprod. Immunol. 31, 3-19. Miller, L., and Hunt, J. S. (1996). Sex steroid hormones and macrophage function. Life Sci. 59, 1-14. King, A., Gardner, L., and Loke, Y. W. (1996). Evaluation of oestrogen and progesterone receptor expression in uterine mucosal lymphocytes. Hum. Reprod. 11, 1079-1082. Vassiliadou, N., Tucker, L., and Anderson, D. J. (1999). Progesteroneinduced ihibition of chemokine receptor expression on peripheral blood mononuclear cells correlates with reduced HIV-1 infectability in vitro. J. Immunol. 162, 7510-7518. Kaushic, C., Murdin, A. D., Underdown, B. J., Wira, C. R. (1998). Chlamydia trachomatis infection in the female reproductive tract of the rat: Influence of progesterone on infectivity and immune response. Infect. Immun. 66, 893-898. Parr, M. B., and Parr, E. L. (1997). Protective immunity against HSV-2 in the mouse vagina. J. Reprod. Immunol. 36, 77-92. Sweet, R. L., Blankfort-Doyle, M., Robbie, M. O., and Schacter, J. (1986). The occurrence of chlamydial and gonococcal salpingitis during the menstrual cycle. J. Am. Med. Assoc. 225, 2062-2064.

357 25. Furth, E A., Westphal, H., and Hennighausen, L. (1990). Expression from the HIV-LTR is stimulated by glucocorticoids and pregnancy. AIDS Res. Hum. Retroviruses 6, 553-560. 26. Kolesnitchenko, V., and Snart, R. S. (1992). Regulatory elements in the human immunodeficiency virus type 1 long terminal repeat (LTR) responsive to steroid hormone stimulation. AIDS Res. Hum. Retroviruses 8, 1977-1980. 27. Wang, C. C., Kreiss, J. K., and Reilly, M. (1999). Risk of HIV infection in oral contraceptive pill users: A meta-analysis. J. AIDS 21, 51-58. 28. Goya, R. G. (1992). Hormones, genetic program and immunosenescence. Exp. Clin. Immunogenet. 9, 188-194. 29. Hadden, J. W., Malec, E H., Coto, J., and Hadden, E. M. (1992). Thymic involution in aging. Prospects for correction. Ann. N.Y. Acad. Sci. 673, 231-239. 30. Rose, N. R. (1994). Thymus function, aging and autoimmunity. Immunol. Lett. 40, 225-230. 31. Best, C. L., Griffin, E M., and Hill, J. A. (1995). Interferon gamma inhibits luteinized human granulosa cell steroid production in vitro. Am. J. Obstet. Gynecol. 172, 1505-1510. 32. Mori, T., Takakura, K., Fujiwara, H., and Hayashi, K. (1996). Immunology of ovarian function. In "Reproductive Immunology" (R. Bronson, N. Alexander, D. Anderson, W. Branch, and W. Kutteh, eds.), pp. 240-274. Blackwell, Cambridge, MA. 33. Rebar, R. W., and Bronson, R. A. (1996). Premature ovarian failure. In "Reproductive Immunology" (R. Bronson, N. Alexander, D. Anderson, W. Branch, and W. Kutteh, eds.), pp. 309-321. Blackwell, Cambridge, MA. 34. La Barbera, A. R., Miller, M. M., Ober, C., and Rebar, R. W. (1988). Autoimmune etiology in premature ovarian failure. Am. J. Reprod. Immunol. Microbiol. 16, 115-122. 35. Taguchi, O., Nishizuka, Y., Sakakura, T., and Kojima, A. (1980). Autoimmune oophorithis in thymectomized mice: Detection of circulating antibodies against oocytes. Clin. Exp. Immunol. 40, 540-553. 36. Rhim, S. H., Millar, S. E., Robey, E, Luo, A. M., Lou, Y. H., Allen, E, Dean, J., and Tung, K. S. (1992). Autoimmune disease of the ovary induced by an 8 amino acid zona pellucida peptide. J. Clin. Invest. 89, 28-35. 37. Hill, J. A., Welch, W. R., Faris, H. M., and Anderson, D. J. (1990). Induction of Class II major histocompatibility complex antigen expression in human granulosa cells by interferon gamma: A potential mechanism contributing to autoimmune ovarian failure. Am. J. Obstet. Gynecol. 162, 534-540. 38. Cohen-Solal, M. E., Graulet, A. M., Denne, M. A., Gueris, J., Baylink, D., and deVernejoul, M. C. (1993). Peripheral monocyte culture supernatants of menopausal women can induce bone resorption: Involvement of cytokines. J. Clin. Endocrinol. Metab. 77, 16481653. 39. Kimble, R. B., Vannice, J. L., Bloedow, D. C., Thompson, R. C., Hopter, W., Kung, V. T., Brownfield, C., and Pacifici, R. (1994). Interleukin-1 receptor antagonist decreases bone loss and bone resorption in ovariectomized rats. J. Clin. Invest. 93, 1959-1967. 40. Cantatore, F. E, Loverro, G., Ingrosso, A. M., Lacanna, R., Sassanelli, E., Selvaggi, L., and Carrozzo, M. (1995). Effect of oestrogen replacement on bone metabolism and cytokines in surgical menopause. Clin. Rheumatol. 14, 157-160. 41. Ho, E C., Tang, G. W., and Lawton, J. W. (1993). Lymphocyte subsets and serum immunoglobulins in patients with premature ovarian failure before and after oestrogen replacement. Hum. Reprod. 8, 714-716. 42. VanVollenhoven, R. E, and McGuire, J. L. (1994). Estrogen, progesterone and testosterone: Can they be used to treat autoimmune diseases? Cleveland Clin. J. Med. 61, 276-284. 43. Sanchez-Guerrero, J., Liang, M. H., Karlson, E. U., Hunter, D. J., and Colditz, G. A. (1995). Postmenopausal estrogen therapy and the risk for developing systemic Lupus erythematosus. Ann. Intern. Med. 122, 430-433.

~HAPTER 2.

Cancers of the Female Reproductive System MARGARET R.

I. II. III. IV.

KARAGAS

Section of Biostatistics and Epidemiology, Dartmouth Medical School, Lebanon, New Hampshire 03756

JENNIFER KELSEY

Division of Epidemiology, Department of Health Research & Policy, Stanford University School of Medicine, Stanford, California 94305

VALERIE McGUIRE

Division of Epidemiology, Department of Health Research & Policy, Stanford University School of Medicine, Stanford, California 94305

V. Ovarian Cancer VI. Summary and Conclusions References

Introduction Breast Cancer Endometrial Cancer Cancer of the Cervix

I.

INTRODUCTION

Whereas in premenopausal women the main source of estrogen is the ovaries, in postmenopausal women direct ovarian estrogen production is minimal. In postmenopausal women most estrogen derives from the conversion of adrenal androgens, particularly androstenedione, mainly in adipose tissue. Progesterone, produced largely by the corpus luteum of the ovary during the luteal phase of the menstrual cycle in premenopausal women, appears only at extremely low concentrations in postmenopausal women, and is of adrenal origin. Prolactin, which is secreted by the anterior pituitary and which promotes lactation, declines markedly at menopause. In women, as in men, the most potent androgen is testosterone. In premenopausal women, testosterone is derived from the ovary and adrenal glands and from the peripheral conversion of androstenedione. Androstenedione also comes from the ovaries and adrenal glands. In postmenopausal women, the secretion of androstenedione from the ovaries decreases considerably, so that the main source of androstenedione is the adrenal glands. The stromal and hilar cells of the ovary continue to secrete testosterone in postmeno-

Breast, uterine, and ovarian cancers comprise 45% of the reported malignancies in women in the United States [1]. Sex hormones are strongly suspected to be involved in the pathogenesis of breast cancer and ovarian cancer and their etiologic role is well established for cancer of the endometrium. The role of sex hormones, if any, in the etiology of cancer of the cervix is not well understood. Menopause has a profound impact on the production of sex hormones, and thus affects the pathophysiology of cancers of the breast and uterus. However, the effect of menopause on the pathophysiology of ovarian cancer has yet to be established. Bernstein and Ross [2] and Adashi [3] have reviewed the changes in sex hormones that occur at the time of menopause. Estrogen concentrations substantially decrease due to the continuous loss of ovarian follicles and, unlike the situation in premenopausal women, a higher proportion of the estrogen in postmenopausal women is estrone than estradiol. MENOPAUSE: BIOLOGY AND PATHOBIOLOGY

359

Copyright9 2000by AcademicPress. All rightsof reproductionin any formreserved.

360 pausal women. Data are inconsistent as to whether testosterone concentrations slightly decrease, remain the same, or slightly increase in postmenopausal women compared to premenopausal women. If the concentration of testosterone does decrease, it does so not nearly so much as estrogen. Dehydroepiandrosterone (DHEA) and its sulfate (DHEAS), weak androgens that can be metabolized to androstenedione and testosterone, are produced by the adrenal glands. Concentrations of DHEA and DHEAS decrease with age. Following cessation of follicular development, a decrease in 17fl-estradiol and inhibin secretion occurs [3a]. These two factors are responsible for negative feedback to the hypothalamus and pituitary. The absence of negative feedback allows gonadotropin concentrations to rise. Folliclestimulating hormone (FSH) rises earlier and to a greater extent than luteinizing hormone (LH). FSH concentrations reach maximal values in the perimenopausal and immediate postmenopausal years, but remain high during the menopausal years. The rate of cell proliferation in the breasts declines with age starting at around 3 5 - 4 5 years. This results in a decrease in glandular epithelium and some involution of acinar and lobular tissue. Eventually the ducts degenerate, leaving small areas of atrophic parenchymal tissue surrounded by connective tissue and fat. Although glandular tissue may constitute about one-third of the breast in premenopausal women, only about 5% of the breast in postmenopausal women is glandular. The proportion of fat increases with age, and represents the largest component of the breast in postmenopausal women. Postmenopausal breast involution is not uniform, however, and one part of the breast may lose all its lobules while another has a pattern similar to that of a premenopausal woman. It is possible that the presence of such well formed lobules is a risk factor for breast cancer. Declining concentrations of estrogens appear to play a central role in breast involution, but other factors such as gonadotrophins, glucocorticoids, growth factors, and their tissue receptors may also be involved. Although very high doses of estrogen may partially delay or reverse the breast involution in postmenopausal women, they do not restore the breast to its premenopausal state. The doses of estrogen used in hormone replacement therapy do not seem to have much effect on breast involution [4]. Cellular proliferation and differentiation of the endometrium depends on the presence of estrogen and progesterone. In the premenopausal years, estradiol produced by ovarian follicles stimulates the proliferation of the glandular, stromal, and vascular endothelial cells that comprise the uterine mucosa. Progesterone secreted by the corpus luteum induces endothelial cell differentiation into a secretory state. In the luteal phase, there is a decline in estrogen stimulation as concentrations of cytoplasmic estradiol receptors diminish and there is an increase in 17fl-hydroxysteroid dehydrogenase conversion of estradiol to estrone [5]. During the perimenopausal period, the endometrium may undergo dis-

KARAGAS ET AL.

ordered proliferation as a result of anovulatory cycles. As estrogen concentrations decline, the endometrium becomes progressively inactive [6], resembling the early proliferative phase of the menstrual cycle with a small glandular epithelium, but devoid of mitoses. Postmenopausally, the endometrium is thin, atrophic, and amitotic with a compact stroma [5]. In postmenopausal women, there is a potential for abnormal proliferation of the endometrium as a result of peripheral production of estrogens or estrogen supplementation, whereas the endometrium becomes inactive with prolonged progestin treatment [6]. This progestin effect is also seen in women who use progestin-containing oral contraceptives [6]. The cervix is responsive to estrogens and progestins, and shares embryological origins with the endometrium [7]. The endocervix contains mucus-secreting columnar cells that extend into the cervical os at birth, whereas the ectocervix is lined with stratified squamous epithelium. At puberty, the endocervical cells at the junction of the ectocervix undergo squamous metaplasia, forming the transformation zone. It is these cells that are susceptible to neoplastic transformation [8]. During pregnancy, the columnar epithelium expands into the ectocervix, where areas of metaplasia can occur, a phenomenon sometimes observed in women taking oral contraceptives [7]. During the menstrual cycle, the stratified squamous epithelium of the ectocervix proliferates and undergoes maturation under the influence of estrogen. Progesterone causes exfoliation of the outermost layer, an effect also observed with estrogen deficiency or androgen administration. Postmenopausally, the stratified epithelial tissue is no longer stimulated and becomes atrophic, thin, and unstratified [7]. The transformation zone frequently retracts in postmenopausal women, but the degree to which this occurs is highly variable [7].

II. B R E A S T C A N C E R Breast cancer is the most common cancer among women in the United States, accounting for about 30% of new cases of cancer [1 ]. As a cause of death among cancers in women, it ranks second only to lung cancer. Figure 1 shows that until the late 1980s, age-specific incidence rates of breast cancer had increased for a period of 15-20 years, particularly in postmenopausal women. Since the late 1980s, however, incidence rates have been relatively constant. It is believed that at least some of the previous increase was attributable to better detection because of the more widespread use of mammography. With mammography, not only are some tumors being detected at an earlier stage than before, but lesions that were not previously considered cancer are now being called cancer. In particular, most cases of ductal carcinoma in situ are asymptomatic and detectable only by mammography. From 1983 to 1992 there was a 200% increase in the diagnosis of ductal carcinoma in situ in the United States [9].

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Some of these tumors may never progress and become clinically apparent. Thus, it is unclear how much of the apparent increase in breast cancer incidence rates is real and how much is attributable to changing methods of detection and diagnosis. Figure 2 presents incidence rates in the United States by race in the 5 years following the establishment of the cancer

Surveillance, Epidemiology, and End Results (SEER) program in the United States and the most recent years for which data are available [ 10]. It can be seen that in the most recent years, incidence rates are highest in blacks before about 45 years of age, but after this age rates are higher in whites than in blacks and lowest in the "other" category, most of whom are Asian. This figure also shows that incidence rates have

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362

KARAGAS ET AL.

been increasing particularly rapidly in the f o r m e r l y low-risk groups, such that the recent rates in blacks are close to the earlier rates in whites, and the recent rates in the " o t h e r " group are approaching those of the earlier rates in blacks. For m a n y years incidence and mortality rates have been highest in North A m e r i c a and northern Europe, intermediate in southern E u r o p e and South A m e r i c a , and lowest in Asia and Africa. However, one of the m o s t striking characteristics of breast cancer e p i d e m i o l o g y in recent years has been the rapid increase in incidence rates in several Asian and eastern E u r o p e a n countries as they have b e c o m e more westernized [11]. Table I [ 12,13] shows that risk factors for breast cancer associated with a relative risk of greater than 4.0 are increasing age, birthplace in North A m e r i c a or northern Europe, having a m o t h e r and a sister with a history of breast cancer especially if at a young age, having a history of biopsy-confirmed benign proliferative breast disease with atypia, and TABLE I

having certain breast cancer susceptibility genes. Factors associated with a relative risk of around 2 . 1 - 4 . 0 are nodular densities on m a m m o g r a m , a history of cancer in one breast, having a m o t h e r or a sister with a history of breast cancer, having biopsy-confirmed benign proliferative breast disease without atypia, having hyperplastic epithelial cells without atypia in nipple aspirate fluid, and being exposed to radiation to the chest in m o d e r a t e to high doses. In addition to certain d e m o g r a p h i c characteristics, factors associated with a relative risk of around 1 . 1 - 2 . 0 are removal of the ovaries before age 40, nulliparity and small n u m b e r s of full-term pregnancies for breast cancer occurring after age 40, multiparity for breast cancer occurring before about age 40, late age at first full-term pregnancy, early age at menarche, late age at menopause, a history of p r i m a r y cancer in the e n d o m e t r i u m or ovary, obesity for breast cancer occurring after age 50, thinness for breast cancer diagnosed before age 50 years, and tallness [14]. There is also strong evidence, although it is

Established Risk Factors for Breast Cancer in W o m e n a

Factor

High-risk group

Low-risk group

Relative risk greater than 4.0

Age group Country of birth Mother and sister with history of breast cancer, especially if diagnosed at an early age Biopsy-confirmed benign proliferative breast disease with atypia Atypical epithelial cells in nipple aspirate fluid Mutations in BRCA 1 or BRCA2, breast cancer at early age

Old North America, northern Europe

Young Asia, Africa

Yes Yes Yes Yes

No

No No fluid produced No

Relative risk of 2.1-4.0

Nodular densities on mammogram History of cancer in one breast Mother or sister with history of breast cancer Biopsy-confirmed benign proliferative breast disease without atypia Hyperplastic epithelial cells without atypia in nipple aspirate fluid Radiation to chest in moderate to high doses

Densities occupying >75% of breast volume Yes Yes Yes Yes Yes

Parenchyma composed entirely of fat No No No No fluid produced No

Relative risk of 1.1-2.0

Socioeconomic status Marital status Place of residence Place of residence (within United States) Race/ethnicity, breast cancer at ->45 years of age Race/ethnicity, breast cancer at <40 years of age Religion Removal of ovaries before age 40 Parity, breast cancer at ->40 years of age Parity, breast cancer at <40 years of age Age at first full-term pregnancy Age at menarche Age at menopause History of primary cancer in endometrium, ovary Weight, breast cancer at ->50 years of age Weight, breast cancer at <50 years of age Height

High Never married Urban Northern White Black Jewish No Nulliparous Parous ->30 years -< 11 years ->55 years Yes Obese Thin Tall

Low Ever married Rural Southern Hispanic, Asian Hispanic, Asian Seventh-day Adventist, Mormon Yes ->4 Nulliparous <20 years _>15 years <45 years No Thin Obese Short

aAdapted from Kelsey [13] (J. L. Kelsey, Breast cancer epidemiology: Summary and future directions. Epidemiol. Rev. 15, 256-263, 1993), with permission from Epidemiological Reviews, and from Kelsey and Bernstein [12], with permission, from the Annual Review of Public Health, Volume 17, 9 1996, by Annual Reviews http://www.Annual Reviews.org.

363

CHAPTER 25 Cancers perhaps not definitive, that alcohol consumption [ 15], recent use of oral contraceptives, especially if it has been for several years [ 16], long-term and recent use of hormone replacement therapy [ 17], lack of physical activity [ 18], and lack of breast feeding [ 19] increase the risk for breast cancer. Although it has beenproposed that a diet high in fat increases the risk for breast cancer, results from prospective epidemiologic studies generally do not support this hypothesis [20]. Epidemiologic studies are currently underway to examine hypothesized protective effects of foods rich in antioxidant vitamins and in phytoestrogens. Several of these risk factors indicate that sex hormones are important in the etiology of breast cancer. It is believed that the manner in which these hormones affect cancer risk is by controlling the rate of cell division, the differentiation of cells, and the number of susceptible cells [21 ]. Thus, when sex hormone concentrations change substantially around the time of menopause and the breast undergoes involution, it would be expected that the epidemiology of breast cancer would to some extent change too. One indication that menopause affects breast cancer risk is the shape of the age-specific incidence curve (Fig. 1). Although the rate of increase of incidence rates of most non-hormone-dependent adult cancers is constant with age, the incidence rates for breast cancer rise steeply until about age 50 years, then increase less rapidly. The rate of increase after about 50 years of age varies by locale [ 11 ]. In high-risk countries, such as most of those in North America and northern Europe, the incidence continues to increase throughout the life span, but at a less rapid rate after age 50 years. In intermediaterisk countries, such as those in southern Europe and Latin America, the rate of increase is less rapid, and in low-risk countries, such as many countries in Asia and Africa, there is little increase in incidence rates after menopause. Incidence rates at one point in time in low-risk countries may actually show a decrease in incidence rates after menopause, but there are no examples of cohort-specific incidence rates decreasing after menopause, at least until old age. Pike e t al. [22] point out that this strongly suggests that whatever factors cause the incidence rates to increase before menopause for the most part bring about irreversible changes. In other words, if it were not for menopause, the lifetime risk of breast cancer would be much greater than it is. Additional pieces of evidence that menopause affects breast cancer risk are that oophorectomy at an early age reduces the risk [23], and that the earlier the age at natural menopause the lower the risk (Table II) [17,24]. For both natural and artificial menopause, the relative risk increases by about 2.8% for each year older at menopause [ 17]. Also, in female dogs who are oophorectomized before their first estrous cycle, the risk of breast cancer is close to zero [25], suggesting that functioning ovaries are almost essential for the development of breast cancer. At a given age, it has been found that women who are still menstruating are at higher risk than those who are postmenopausal [27,26] (Table III).

TABLE II Relative Risks of Breast Cancer by Age at Natural Menopause and Age at Bilateral Oophorectomy, Nonusers of Hormone Replacement Therapy, Combined Results from 51 Epidemiological Studies a,b Menopausal status and age at menopause (years) Postmenopausal <35 35-39 40-44 45-49 50-54 >-55 Perimenopausal Premenopausal

Natural menopause

Bilateraloophorectomy beforenatural menopause

0.46 0.51 0.62 0.70 0.81 0.85

0.48 0.65 0.65 0.72 }

0.90

0.77 1.00

a Reference group is premenopausal women. Estimates are adjusted for study, age at diagnosis, parity, and age at birth of first child. bSource: Collaborative Group on Hormonal Factors in Breast Cancer [17].

That sex hormones are involved in the etiology of breast cancer is further suggested by the increased breast cancer risk among women whose menarche occurs at an early age, nulliparous women, women who have small numbers of children, women who give birth to their first child at a relatively late age, and possibly women who do not breast feed their children [27]. Key [21] proposes that breast cancer risk increases with early menarche and late menopause because of increased exposure of the breasts to estrogen, and possibly progesterone. He also suggests that first pregnancy and multiparity probably reduce breast cancer risk through the hormonally induced differentiation of breast cells and the corresponding reduction in the number of susceptible cells. In addition, women with high bone mass are at elevated risk for breast cancer, a relationship possibly attributable to the TABLE III Relative Risks of Breast Cancer by Menopausal Status and Years since Natural Menopause and Years since Bilateral Oophorectomy, Combined Results from 51 Epidemiological Studies a,b Menopausal status and time since menopause (years) Postmenopausal >-15 10-14 5-9 1-4 Perimenopausal Premenopausal

Natural Bilateraloophorectomy menopause beforenatural menopause 0.50 0.59 0.66 0.70

0.48 0.52 0.70 0.86 0.77 1.00

a Reference group is premenopausalwomen. Estimates are adjusted for study, age at diagnosis, parity, and age at birth of first child. bSource: Collaborative Group on Hormonal Factors in Breast Cancer [17].

364 association between both high bone mass and breast cancer risk with estrogen [28,29]. It has also been hypothesized [30,31] that exposure to maternal estrogens while in utero may increase risk. A variety of studies, both of the case-control design and of subgroups of women at particularly high or low risk for breast cancer, have indicated that higher concentrations of estrogen in postmenopausal women are associated with an increase in breast cancer risk. However, until recently, there has been little evidence that such a relationship exists for premenopausal women, possibly because of difficulties in measurement of estrogen in premenopausal women [2]. Several prospective studies [32-35], in which sera were collected and stored before breast cancer developed, have confirmed that higher serum estrogen concentrations in fact predict a higher risk for subsequent breast cancer. Two of the reports [32,33] were based on postmenopausal women only, whereas two others [34,35] included both premenopausal and postmenopausal women. Another prospective study [36] did not find an association between estrogen and breast cancer. Nevertheless, taken as a whole, these prospective studies provide direct evidence that estrogen is involved in the etiology of breast cancer. Results regarding progesterone have been less compelling, and until recently, results of studies concerned with androgen concentrations had been inconsistent. However, two case-control studies [37,38] and one prospective study [33] indicate that high concentrations of testosterone in postmenopausal women are associated with an increased risk for breast cancer, thus increasing the likelihood that testosterone is etiologically involved. Like estrogen, testosterone increases rates of cell division. Also, at higher concentrations, more testosterone is converted in adipose tissue to estrogen. Testosterone has a greater affinity for sex-hormone binding globulin than does estrogen, so it may indirectly enhance breast cancer risk by increasing the amount of free estradiol that is not bound to sex-hormone binding globulin. A recent study [39] that initially found testosterone concentration to be positively associated with breast cancer risk reported that this association was substantially reduced when estradiol bound to sex-hormone binding globulin and total estradiol were taken into account statistically. This result is consistent with the hypothesis that testosterone has an indirect effect on breast cancer risk, that is, by influencing the amount of bioavailable estrogen or by affecting the concentrations of estrogen precursors [39]. It is possible that testosterone assumes greater relative importance in postmenopausal women because testosterone concentrations, at most, decline only slightly after menopause, whereas estrogen concentrations decrease substantially [40]. After menopause, some risk factors emerge that are not apparent in premenopausal women. One is hormone replacement therapy (see Chapter 40). Although data have been somewhat inconsistent, a combined analysis [ 17] suggests an increasing risk for breast cancer with increasing length of

KARAGAS ET AL.

use, such that the relative risk is 1.35 for women who have used replacement estrogen for 5 years or longer. The elevation in risk is comparable to that associated with later age at menopause. The risk is mainly seen in current users, and may be slightly greater in those using a combined estrogen/ progestin regimen than in those using estrogen alone. Another risk factor that emerges in postmenopausal women is obesity. Whereas in premenopausal women thinness appears to confer a greater risk [ 14], postmenopausal obese women and/or women who have gained weight during adulthood [41] have an elevated risk. It is believed that the increased risk in obese postmenopausal women occurs mainly because of the greater conversion of androstenedione to estrogens in obese women [42] and because of the lower concentrations of sex-hormone binding globulin in obese women, thus increasing the amount of estrogen available to target tissues [43]. In premenopausal women, peripheral conversion from androstenedione is a relatively minor source of estrogen compared to what is secreted from the ovaries. There have been reports that other established or probable breast cancer risk factors are more or less strong in premenopausal or postmenopausal women, including lack of breast feeding, alcohol consumption, and low levels of physical activity, but evidence for a differential effect by menopausal status is not at all consistent. Although parity is associated with an increased risk for breast cancer in young women and decreased risk in women over about 4 0 - 4 5 years of age, this cross-over in risk is probably attributable to the time since the last pregnancy rather than to menopause [27]. Breast cancer susceptibility genes such as BRCA1 and B R C A 2 affect mainly younger women. It may be that the hormonal milieu of a premenopausal woman is required to progress to breast cancer or that in premenopausal women a sufficient induction period has already elapsed for mutations present at birth to be manifested. Preliminary data [44] suggest that a relatively high proportion of breast cancers developing in carriers of BRCA1 or B R C A 2 have a hormoneresistant phenotype at the time they are diagnosed. Mammographic patterns change with menopause. The proportion of women in which the breast parenchyma is occupied by diffuse or nodular densities (the DY pattern), a pattern associated with an elevated risk for breast cancer, decreases with increasing age, particularly near menopause. The proportion of breasts in which the parenchyma is composed entirely of fat (the N 1 pattern), and in which the parenchyma is mostly fat but in which there is some ductal prominence (P1), correspondingly increases. These patterns are associated with a lower risk for breast cancer. Evidence suggests that these changes are attributable more to menopausal status than to age [45]. The efficacy of screening for breast cancer by mammography is established for women of ages 5 0 - 6 9 years on the basis of randomized controlled trials and cost-effectiveness analyses. However, whether women of ages 4 0 - 4 9 years should undergo screening mammography has been contro-

CHAPTER25 Cancers

365

versial [46,47]. One reason for the lower effectiveness of mammograms in premenopausal women is probably that their radiographically denser breasts may obscure breast tumors and make mammography less sensitive. Another possible explanation is that some of the tumors may be more aggressive in younger women and that by the time these aggressive tumors are detected it is already too late for treatment to be effective [48]. In addition, the lower incidence of breast cancer in younger women contributes to the lower cost-effectiveness of mammography in this group [47]. Tamoxifen and raloxifene are said to be antiestrogens in the breast. The Breast Cancer Prevention Trial in North America reported a 49% reduction in invasive breast cancer incidence among women randomly assigned to take tamoxifen (55% in women age 60 and older, 51% in women ages 5 0 - 5 9 , and 44% in women aged 49 or younger). However, in postmenopausal women this beneficial effect is almost balanced by the increased risk for adverse events such as endometrial cancer, pulmonary embolism, and deep venous thrombosis [49]. Thus, on the basis of this trial, tamoxifen would not seem useful as a preventive agent except in very high risk women. Other smaller randomized trials in England [50] and Italy [51] have found either no protection or at most a very slight reduction in breast cancer incidence among women assigned to take tamoxifen. Possible reasons for the discrepant results between the North American and European trials are suboptimal compliance in the Italian study, younger groups of women in both of the European trials, and the inclusion in the English trial of more women with a family history of breast cancer at an early age than in the North American trial [52]. Longer follow-up and more

data on mortality, in addition to breast cancer incidence, are clearly needed. Raloxifene is being touted as an alternative to hormone replacement therapy in postmenopausal women who wish to reduce their risk of osteoporosis and of breast cancer without increasing their risk for endometrial cancer. A randomized trial has in fact reported a 76% reduction in risk of breast cancer during 3 years of raloxifene treatment, including a 90% reduction in risk for estrogen receptor positive breast cancer and a 12% nonsignificant reduction in risk for estrogen receptor negative breast cancer [53]. For a further discussion of trials involving tamoxifen and raloxifene, see Chapter 28.

III. E N D O M E T R I A L CANCER Endometrial cancer is the most common gynecologic malignancy in the United States and the fourth leading cancer in women. It accounts for 6% of all reported cancers [1 ] and ranks ninth as a cause of cancer deaths in women in the United States [ 1]. Endometrioid adenocarcinomas comprise the vast majority of endometrial cancers. Adenocarcinomas with squamous metaplasia or differentiation, papillary serous carcinomas, and clear cell carcinomas are relatively uncommon, but are associated with a worse prognosis [54,55]. Incidence rates of endometrial cancer rose somewhat during the 1960s in the United States, then increased dramatically in the early 1970s, particularly among women ages 45 to 64 years, a trend that mirrored the prevalence of unopposed estrogen use [56]. Rates dropped steeply after 1975 (Fig. 3)

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366

KARAGAS ET AL.

the lower prevalence of hormone replacement therapy among blacks and other women, compared to Caucasians [59]. The major risk factors for endometrial cancer are summarized in Table IV [60]. In addition to age and country of residence, factors related to a greater than fourfold relative risk of endometrial cancer include unopposed estrogen therapy for 5 or more years [61,62] and polycystic ovary syndrome [63,64]. Obesity, late age at menopause, nulliparity or fewer pregnancies, and, most recently, tamoxifen use are associated with a relative risk from 2.1 to 4. White race, pelvic irradiation, a family history of endometrial cancer, and a prior history of breast cancer are associated with relative risks of 1.3 to 2.0 [60,65-68]. A reduced incidence of endometrial cancer has been observed in women who have used combined oral contraceptives or depo-medroxyprogesterone acetate [60] and who smoked cigarettes [69]. Women who have endometrial hyperplasia or estrogen-producing theca-granulosa cell tumors appear to be at high risk for this disease, but data are limited, making precise risk estimation difficult [60]. In addition to the risk factors listed in Table IV, diets high in fat and total calories and with fewer fruits, vegetables, and soy products, tall stature, and physical inactivity have been suggested as being related to a greater endometrial cancer risk, but the evidence is inconclusive [65,70,71 ]. Hypertension and diabetes were associated with endometrial cancer risk in some studies but not in others [70,72]. Use of an intrauterine device as a contraceptive was related to a reduced risk of endometrial cancer in a number of studies [73], but further data are needed. The results of studies examining

when an association between estrogen use and endometrial cancer risk was reported (Chapter 41). It should be noted that in many cancer registries, including the SEER registries (until 1992), endometrial cancers were coded as cancers of the corpus uteri and, occasionally, uterus not otherwise specified. Because other types of corpus uterus cancers are rare, rates of cancer of the corpus uteri are a reasonable approximation of rates of endometrial cancer. Also, the data shown from the SEER registries [10] in Figs. 3 and 4 represent rates of endometrial cancer in all women, including those who have had their uterus removed. Although the inclusion in the denominator of women who have had a hysterectomy markedly underestimates incidence rates for women at risk for endometrial cancer, the overall time trends are not affected [57]. Reported incidence rates of endometrial cancer are highest in North America and northern Europe and among the Maori in New Zealand [58]. In Asia, such as in Shanghai and Tianjin, China, and in Japan, rates are relatively low [58]. Japanese and Chinese in Hawaii have only slightly lower rates than whites in Hawaii, suggesting that environmental factors, in part, explain the variability in the occurrence of these malignancies between Asian and Caucasian women [58]. As shown in Fig. 4, after age 45 years, rates of endometrial cancer are higher in white women than in women classified as black or "other," with the latter category including Asians. Also shown in Fig. 4 is that the temporal decline in endometrial cancer rates is not observed in blacks as it is in whites and in other women. Although the reasons for racial/ethnic differences are unclear, they may be related to

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Incidence rates of invasive cancer of the corpus uteri by age, race, and time period.

367

CI-IAPTER25 Cancers TABLE IV

Established Risk Factors for Endometrial Cancer a

Factor

High-risk group

Low-risk group

Relative Risk > 4 . 0

Age Geographic area Use of estrogen replacement therapy Polycystic ovary syndrome

70-75 years North America, northern Europe >5 years Yes

<40 years Asia, Africa None No

Relative risk of 2 . 1 - 4 . 0

Weight Parity Age at menopause Use of combination oral contraceptives Use of DMPAb Use of Tamoxifen

Obese Nulliparous ->55 years No No Yes

Thin -->4 <45 years ->4 years Yes No

Relative risk of 1 . 3 - 2 . 0

White race Family history of endometrial cancer History of primary cancer in breast Cigarette smoking Pelvic irradiation

Yes Yes Yes No Yes

No No No Yes No

Associations that appear strong r

Endometrial hyperplasia with atypia Estrogen-producing ovarian tumors

Yes Yes

No

No

a Adapted from Kelsey and Whittemore [60], Annals of Epidemiology, Vol. 4, J. L. Kelsey and A. S. Whittemore, Epidemiology and primary prevention of cancers of the breast, endometrium, and ovary. A brief overview,pp. 89-95. Copyright 1994, with permission from Elsevier Science. bDMPA, Depo-medroxyprogesteroneacetate. CBased on clinical observations, but difficult to quantify exactly.

effects of several other reproductive factors, such as age at first and last pregnancy, age at menarche, spontaneous and induced abortion, and breast feeding, have been inconsistent [65,70,74]. There is some evidence that endometrial carcinogenesis may differ by histological subtype. In a multinstitutional case-control study, endometrioid adenocarcinomas were strongly related to body mass index and menopausal estrogen use, but these associations were not observed in the smaller group of serous carcinomas [75]. Serous tumors of the endometrium are more common among black than white women [55]. Unlike endometrioid carcinomas, they often occur in the presence of endometrial intraepithelial carcinoma and in the absence of hyperplasia [76]. In several case series, overexpression of p53 was present in a high percentage of serous and intraepithelial tumors and only a small fraction of endometrioid carcinomas [76]. These findings have led to speculation that serous carcinomas may arise from an estrogen-independent pathway. The pathogenesis of endometrioid carcinomas undoubtedly involves endogenous or exogenous estrogens and progestins, and possibly other sex steroids. The underlying hypothesis is that estrogens stimulate the proliferation of endothelial cells forming the uterine mucosa, and that these cells have the potential for neoplastic transformation unless

they undergo differentiation into secretory cells induced by progesterone during the normal menstrual cycle or through progestin supplementation. As shown in Fig. 3, endometrial cancer is primarily a disease of postmenopausal women. In the United States, incidence rates are relatively low before age 40 years and increase sharply until the sixth or seventh decade of life. Postmenopausal therapy may be responsible for some of this rise, along with residual endogenous estrogens and the virtual absence of progesterone in the postmenopausal period. Prior to menopause, women may be relatively protected by progesterone. The age-specific incidence curve for endometrial cancer has shifted to the right since the late 1970s. Women over 70 years of age from 1993 to 1995 and women over 65 years of age from 1978 to 1982 had higher endometrial cancer rates than women of comparable ages from 1973 to 1977. The reasons for this shift are unknown, but could represent the effects of the growing use of hormone replacement therapy among older women in the United States [77] and the lower proportion of older women who use progestins [78]. Women who undergo a later menopause are at higher risk of endometrial cancer than those who undergo menopause at an early age (Table V) [79-82]. Table V excludes studies that did not present relative risk estimates [83], including one

368

KARAGAS ET AL.

TABLE V

Relative Risk of Endometrial Cancer by Age at Menopause: Case-Control and Cohort Studies

First author/year

Total no. of cases/ controls or cohort size

Case-control studies Wynder/1966 [79] a

112/200

Elwood/1977 [86]

212/1198

Weiss/1980 [85] r

322/289

Kelsey/1982 [70] La Vecchia/1984 [ 102]

167/903 283/566

Shu/1991 [64]

268/268

Austin/1991 [71]

168/334

Kalandidi/1996 [80] de Waard/1996 [81 ]

145/298 147/900

Cohort studies Kvale/1991 [82]

422/62,079

McPherson /1996 [74]

167/24,848

Baanders-Van Halewyn/1996 [93]

111/25,000

Age at menopause (years)

Odds ratio or relative risk

--<45 45-49 50-54 ->55 >49 49-51 ->52 -<48 ->52 Per 10 years <50 50-52 ->53 ->45 46-48 49-50 ->51 <-45 46-49 50 - 5 4 ->55 Per year <50 50-51 52-53 ->54

1.0 b 1.3 1.7 5.0 1.0 ~ 1.2 1.7 1.0 b 2.6 1.9 1.0 b 1.5 2.6 1.0 b 1.0 2.3 1.9 1.0 h 0.8 1.6 2.0 1.05 1.0b 1.0 1.4 2.6

--<45 -->54 < 45 45-49 50 - 5 4 ->55 <50 50-51 52-53 ->54

1.0/' 2.54 1.Oh 0.62 1.04 1.87 1.0 h 1.2 1.5 2.6

a Crude odds ratios computed from data provided in the manuscript. b Reference category. c Restricted to estrogen nonusers.

study that reported no relation with age at menopause [84]. Some of the variability across studies may relate to inaccurate reporting, especially among women who experience perimenopausal bleeding as an early symptom of endometrial cancer [84]. Also, use of postmenopausal hormones may obscure the time when natural menopause occurs, particularly among women who have menstrual-like bleeding from cyclic estrogens and progestins. In an analysis restricted to nonusers of estrogen, the relative risk for endometrial cancer was 2.6 among women who underwent menopause at age 52 years or older compared to those with menopause at age 48 years or younger [85]. One explanation for an increased risk may be that women who undergo a late

menopause are exposed to a longer perimenopausal period of anovulatory, progestin-deficient cycles of ovarian estrogen secretion [86]. If total duration of a woman's menstrual life were related to endometrial cancer risk, then one might expect that an earlier age at menarche also would increase risk, and although there is some evidence for this, results are inconsistent [65,87]. Further evidence for the etiologic involvement of endogenous hormones is that certain aspects of reproductive history affect risk. Most notably, nulliparous and nulligravid women have a high incidence of endometrial cancer, and risk decreases with greater number of pregnancies or births (Table IV). The magnitude of the associations varies to some

CHAPTER 25 Cancers

extent across studies; however, most find that women who have had more than four pregnancies have less than half the risk of endometrial cancer compared to nulligravid women [65,74,83]. The protective effect of pregnancy could stem from the prolonged periods in which progesterone is the dominant sex hormone, and during which endometrial cell division is thereby suppressed. The effect of nulligravidity could partly be a consequence of ovulatory disturbances leading to infertility. Indeed, in many, but not all, epidemiologic studies, an elevated risk of endometrial cancer is observed in relation to infertility [87,88]. In one study [63], the association was most pronounced among women with infertility apparently due to ovarian factors, with a relative risk of 4.2 (95% CI = 1.7-10.4) for ovarian sources of infertility and 1.7 overall (95% CI = 1.1-2.6) [63]. A large medical-record-based study found an excess risk of endometrial cancer specifically among women who had progesterone deficiency but adequate estrogen concentrations [standardized incidence ratio (SIR) = 9.4; 95% CI = 5.0-16.0] [88]. These findings are also supported by clinical studies of anovulatory women [89]. Some epidemiologic studies have found that women in the general population with menstrual irregularity [90] or periods of low flow [84] are at an elevated risk of endometrial cancer, but those findings need further confirmation. Breast feeding also suppresses ovulation, but reduces estrogens to a greater extent than progesterone. Although prolonged lactation was related to a reduced risk of endometrial cancer in an international study [91 ], studies in the United States have not found a relation [84]. The larger studies that have measured endogenous hormone concentrations have found higher values of estrogen in endometrial cancer cases than in controls [71,92]. In a casecontrol study reported by Potischman and colleagues [92], postrnenopausal endometrial cancer cases had higher serum concentrations of several estrogens, particularly estrone. In the smaller group of premenopausal women, higher concentrations of progesterone were found in controls than in endometrial cancer cases, but circulating estrogen concentrations were similar. High concentrations of sex-hormone binding globulin were associated with a diminished risk among both pre- and postmenopausal women. These findings support the hypothesis that unopposed estrogens are a major risk factor for endometrial cancer in postmenopausal women, and that progesterone deficiency may play a more important role in premenopausal women. A possible role of androgens in the pathogenesis of endometrial cancer has been raised in recent case-control studies, although the mechanisms for any association are unknown. Higher serum concentrations of androstenedione were found in endometrial cancer cases than in controls in two studies [71,92], and in both premenopausal and postmenopausal women [92], although no difference was seen in urinary excretion of this hormone in another study [93]. The observed association could be an incidental finding because andro-

369 stenedione increases the potential for aromatization to estrone, but the relation held even after adjustment for estrogen [92]. Testosterone has not been measured, but hirsutism, a symptom of hyperandrogenism, was associated with endometrial cancer risk in at least two studies [84,94]. Use of estrogen replacement therapy without progestins is one of the most important determinants of risk in postmenopausal women (see Chapter 41). Use of unopposed estrogens for more than 10 years is associated with almost a 10-fold increase in endometrial cancer risk [61 ]. The relative risk is higher among women who use 1.25 mg/day of conjugated estrogens rather than 0.3 or 0.625 mg/day, although the latter doses were associated with a relative risk of 3.5 to 4.0. Women whose use of estrogens has occurred recently, i.e., within the past 4 years, are at greater risk than previous users, although there is some evidence that those who stopped for 5 or more years still have about a twofold elevated incidence compared to nonusers [61 ]. The few epidemiologic studies evaluating use of estrogen plus progestin replacement therapy have found no evidence of an overall increase in risk in women who used progestin for 10 days a month or longer [95-97]. However, compared to women who used estrogen alone, risk is only modestly reduced among women who add a progestin for fewer than 10 days per month [95-97]. The data regarding long-term use of estrogen/progestin therapy are inconsistent. In one study, 5 or more years of use of estrogen with a progestin for at least 10 days a month was associated with a 2.5-fold elevation in risk for endometrial cancer [96], whereas in another study, there was essentially no increase [97]. The epidemiologic data regarding oral contraceptives also indicate that pills dominated by estrogen are carcinogenic to the endometrium whereas progestins can be protective. For instance, sequential oral contraceptives (21 days of an estrogen followed by 7 days of a progestin) were associated with an increased risk of endometrial cancer [65]. In contrast, women who used combined oral contraceptives containing both estrogen and progestin or depo-medroxyprogesterone acetate are at a reduced risk of endometrial cancer for many years even following cessation of use [60]. Presumably, other progestin-only oral contraceptives are protective as well, but this question has not been specifically addressed. In nearly all epidemiologic studies, a lower incidence of endometrial cancer has been observed in cigarette smokers, and in nearly all studies that have evaluated premenopausal and postmenopausal women separately, a stronger effect is found among postmenopausal women [85]. This reduction in risk is greater for current than for former smokers in most studies, and also appears to be more pronounced among heavier smokers. Among postmenopausal women, the effect has been noted in both non-users and users of estrogen replacement therapy, although there is some indication of an enhanced protective effect among hormone users. Although smoking does not influence endogenous estrogen concentrations, smoking is associated with an earlier menopause [98]

370 and may reduce the bioavailability of estrogens through alterations in adrenal steroid production, metabolic inactivation of estrogens, or inhibition of aromatase [99,100]. Another important risk factor among postmenopausal women is obesity. In several case-control studies, an association was found among both premenopausal and postmenopausal women [70,90,101,102]. Body fat would be expected to increase risk of endometrial cancer in postmenopausal women because of increased conversion of androstenedione to estrogen by aromatase in adipose tissue and, in turn, the reduction in sex-hormone binding globulin, leading to higher exposure to "free" estrogens. The elevated risk observed in premenopausal women could be a result of ovulatory disturbances and progesterone deficiency observed in young obese women [89]. The impact of obesity on the overall incidence rate of endometrial cancer is far greater in postmenopausal women because their underlying rates of disease are markedly higher than in younger women. Family history of endometrial cancer likely plays a role in the pathogenesis of this cancer. As with other cancers, this relationship may be stronger in younger women [66]. In a study conducted by Kelsey et al. [70], women with a mother or sister with endometrial cancer or ovarian cancer were at a two- to threefold elevated risk for endometrial cancer. Similar findings were reported by Gruber and Thompson [66], and in his study, a family history of colorectal cancer was also modestly related to endometrial cancer risk [66]. Hereditary nonpolyposis colorectal cancer (HNPCC) includes multiple types of adenocarcinomas, including colon, endometrial, and ovarian cancers, and the genes associated with this familial syndrome code for enzymes involved in DNA mismatch repair. The possible role of mismatch repair defects in the etiology of sporadic endometrial cancers requires further exploration. Endometrial hyperplasia is often a precursor to endometrioid carcinoma, and the occurrence of malignant transformation appears to be largely, but not solely, confined to hyperplasia with cellular atypia [103]. In the hyperplastic endometrium, the ratio of glandular to stromal tissue is increased compared with the normal proliferative endometrium, and morphologic abnormalities can be seen in the glandular structures [ 103]. The development and progression of these lesions appear to be sensitive to the effects of estrogens and progestins [65,104]. In a nested case-control study [93], urinary excretion of estrogens was higher in women who later developed endometrial hyperplasia, compared to controls. Use of unopposed estrogens increases the occurrence of endometrial hyperplasia, and addition of progestin reduces this risk. Indeed, progestins are used clinically to treat the milder forms of hyperplasia. Obesity is a risk factor for endometrial hyperplasia, but this is observed only in postmenopausal women [93,105]. The relation between reproductive and menstrual characteristics and endometrial hyperplasia is less clear [93,105]. Screening asymptomatic women for endometrial cancer

KARAGAS ET AL.

is not generally recommended in light of the relatively low incidence of the disease [ 106]. Presently, the American Cancer Society recommends endometrial biopsy at the time of menopause for high-risk women, but even this is controversial [ 106]. The Pap smear is not a sensitive or specific test for endometrial cancer [106]. A progesterone "challenge test" may identify some cases of endometrial hyperplasia, but this test is rarely performed [107]. In clinical practice, endometrial biopsies are largely reserved for symptomatic women such as those with abnormal bleeding or suspicious findings on clinical examination [107]. The newer regimens of postmenopausal therapy have potentially important implications for a woman's risk of developing endometrial cancer. The incidence of endometrial cancer is about 2.6 times higher among breast cancer patients treated in randomized trials of adjuvant tamoxifen, compared to tamoxifen-untreated women, a result consistent with most observational studies [108]. In the Breast Cancer Prevention Trial of women at high risk for breast cancer, an increased incidence of endometrial cancer also was observed in tamoxifen-treated women compared to placebo controls [ 109]. Based on very few women, an English prevention trial found the same trend [50]. Although studies are limited, there is some evidence that the adverse effects of tamoxifen on endometrial cancer may be limited to postmenopausal women [ 110] and that tamoxifen may lead to more advanced stage disease [111 ]. Raloxifene, a new selective estrogen receptor modulator (SERM), is believed to be an estrogen agonist in the endometrium on the basis of animal studies [112]. In limited human data, raloxifene was not associated with endometrial proliferation in postmenopausal women treated for 2 months [112]. Preliminary results of the Multiple Outcomes of Raloxifene Evaluation (MORE) trial, a placebo-controlled clinical trial of raloxifene, indicated no differences in the incidence rates of endometrial cancer in the two treatment arms of the study, but there was limited power to detect an effect [53]. Thus, the effects of raloxifene on endometrial carcinogenesis are as yet unknown.

IV. C A N C E R

OF THE CERVIX

Invasive carcinoma of the cervix is a relatively uncommon malignancy in the United States, with 13,700 new cases and 6300 deaths estimated among women in 1998 [ 1]. Globally, cancer of the cervix remains an important health issue, ranking second as a cause of cancer among women worldwide [58]. In the United States, incidence rates are higher in blacks and other races compared with whites (Fig. 6). The rates of invasive cervical cancer have markedly declined in the United States (Fig. 5), especially among blacks (Fig. 6). This decline is thought to be attributed in part to Pap smear screening and effective treatment of early and preneoplastic disease [114]. The pathogenesis of cervical

CHAPTER 2 5

371

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cancer is dominated by human papilloma virus (HPV) infection, and several high-risk forms of the virus have been identified [ 115,116]. Presently, there is no evidence that menopause influences risk of cervical cancer. Data regarding exogenous estrogens and progestins and reproductive history are either sparse or inconclusive [ 116]. Use of oral contraceptives, depo-medroxy-

progesterone acetate, and higher parity was associated with an elevated risk of cervical cancer in several studies [ 115], yet it is uncertain whether these factors act independently of HPV infection [ 115]. A number of studies have found that oral contraceptives are more strongly related to adenocarcinoma of the cervix, the histologic type of about 10% of cervical cancers, than to squamous cell carcinoma [ 117]. This association

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may explain the rising incidence rates of adenocarcinoma of the cervix among young women in the United States [115,117]. In a cohort study, a twofold higher incidence of dysplasia and carcinoma in situ of the cervix was found among women who had in utero exposure to diethylstilbestrol (DES) [118]. However, a complicating factor is whether women who were exposed to DES were more rigorously screened compared to unexposed women [118]. Smoking is also a risk factor for invasive carcinoma of the cervix [115,116]. Tobacco carcinogens are found in cervical mucosa, and the effects are thought to involve direct DNA damage or local immune system suppression rather than a mechanism relating to endogenous hormones. Some studies have raised the possibility that endogenous or exogenous factors might act synergistically with HPV infection [87,115]. The viral DNA contains hormone response elements, and malignant transformation in vivo is enhanced by sex hormones. The possibility that postmenopausal therapy affects risk of cervical cancer is largely unexplored, including the effects of tamoxifen or other selective estrogen receptor modulators. Recommendations for Pap smear screening do not change after menopause. According to American Cancer Society guidelines, women who have three normal annual Pap smears and pelvic exams may be screened less frequently based on the advice of their physicians [114]. Atrophic changes in the postmenopausal cervix lead to an increased susceptibility to infection, which can result in false positive results [7]. The chance of a false negative smear potentially increases as well, due to the retraction of the transformation zone following menopause [7].

V. O V A R I A N

CANCER

Ovarian cancer is the fifth most common cancer among women in the United States, accounting for about 5% of new cases of cancer, and is the leading cause of death from gynecologic malignancies [ 1]. Women aged 50 years and older comprise 73% of all cases [119]. In addition, 83% of the older women will be diagnosed at advanced stages of disease compared with 54% in women less than age 50 years [119]. Epithelial ovarian tumors, sex-cord stromal tumors, and germ cell tumors are the major types of ovarian tumors. Epithelial ovarian cancer is the most common histopathologic type, accounting for 90% of malignant ovarian tumors, and originates from cells of the surface germinal epithelium of the ovary [120]. The most common histopathologic subgroup of epithelial ovarian cancer is serous adenocarcinoma, followed by mucinous adenocarcinoma and endometrioid adenocarcinoma. Of the epithelial ovarian tumors, approximately 15% are classified as tumors of low malignant potential, which are characterized by cellular stratification with variable nuclear atypia but without evidence of stromal invasion [120]. These borderline tumors are most common in women under age 40 years. The risk factors associated with invasive and borderline epithelial tumors appear to be similar [ 121 ]. Figure 7 shows that incidence rates of invasive ovarian cancer have remained nearly constant over the past several decades. Ovarian cancer is uncommon in women younger than age 40 years, after which the incidence rates increase, peaking between ages 7 0 - 7 9 years, then decreasing slightly. The incidence rates are highest in whites, intermediate in

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blacks, and lowest in "other" races, most of whom are Asian (Fig. 8). In women under age 40 years, incidence rates are similar in all racial groups. Although the ovarian cancer incidence rates are higher for women over 55 years of age compared with younger women, the rates increase less rapidly with age after 55 years than before [122]. One possible explanation is that the postmenopausal ovary may be less exposed or less susceptible than the premenopausal ovary to endogenous or exogenous carcinogens. Globally, incidence rates of ovarian cancer are highest in Europe and North America, with particularly high rates in Scandinavia. Incidence rates are lowest in Japan, parts of Africa, and China [58]. When Asian women move from countries with a low incidence of ovarian cancer to higher risk areas, their descendants' risk for ovarian cancer will approach that of native-born women within a few generations [ 123]. The major established risk factors for epithelial ovarian cancer are summarized in Table VI. Age and geographic area are associated with a fourfold differential in risk. Factors associated with a relative risk of 2.1-4.0 are family history of ovarian cancer, nulliparity or a small number of pregnancies, and lack of oral contraceptive use. Evidence from a collaborative analysis of 12 case-control studies suggests that the protective effect of oral contraceptives may be stronger in postmenopausal women than in premenopausal women [124]. Although the majority of epithelial ovarian cancers are sporadic, 5 - 1 0 % are estimated to be inherited [125]. The inherited breast-ovarian cancer syndrome has been linked to germline mutations in the BRCA1 tumor suppressor gene [126]. Estimated risks of BRCA1 carriers developing an

ovarian malignancy by age 70 years range from 21% [127] to 40% [ 126], risks that are considerably higher than the corresponding 1% in the general population [127]. A relative risk of 1.2-2.0 is associated with a lack of breast-feeding and having a history of a primary cancer in the breast or endometrium. Possible risk factors, for which the evidence is not as strong, include late age at menopause and early age at menarche [ 128], perineal talc use [ 129], fertility drugs [ 130], galactose from lactose in milk [ 131 ], caffeine consumption [132], a history of mumps [133], and hormone replacement therapy [ 134]. Tubal ligation and hysterectomy may be protective [ 130]. The causes of ovarian cancer are poorly understood. Epidemiologic studies suggest that reproductive hormones are involved in its etiology [ 123,128,135,136]. For several years, two main models have been proposed for the etiology of ovarian cancer [137-139], and a third hypothesis has now been put forth [135]. Fathalla [137] proposed that incessant ovulation, without pregnancy-induced rest periods, contributes to ovarian carcinogenesis. The ovarian surface epithelium undergoes rapid proliferation 24 hr after ovulation, and the formation of clefts and inclusion cysts within the ovarian stroma are most pronounced after ovulation. As the traumatized epithelium of ruptured follicles is recurrently repaired, it is exposed to estrogen-rich follicular fluid that may act as a mitogen to facilitate proliferation and lesion repair. Pike [140] quantified Fathalla's hypothesis by proposing that ovarian cancer incidence rates were proportional to ovarian epithelial "tissue age" measured in units of cellular mitosis. Ovulation induces a transient increase in mitotic activity in the epithelium and each ovulation increases tissue age. Thus,

374

KARAGAS ET AL.

TABLE VI

Established Risk Factors for Ovarian Cancer a

Factor

Age Geographic area Family history of ovarian cancer Parity Use of combination oral contraceptives

High-risk group Relative risk > 4.0 70-75 years North America, northern Europe Relative risk of 2.1-4.0 Yes Nulliparous No

Relative risk of 1.3-2.0 No Breast-feeding Yes History of primary cancer in breast or endometrium

Low-risk group

< 40 years Asia, Africa No -->4 ->6 years > 1 year No

a Adapted from Kelsey and Whittemore [60], Annals of Epidemiology, Vol. 4, J. L. Kelsey and A. S. Whittemore, Epidemiology and primary prevention of cancers of the breast, endometrium, and ovary. A brief overview, pp. 89-95. Copyright 1994, with permission from Elsevier Science.

pregnancy and use of oral contraceptives may protect against ovarian cancer by inhibiting ovulation. Early age at menopause, if it is in fact a protective factor, would have its effect by decreasing the total number of lifetime ovulatory cycles. The second theory, the gonadotropin hypothesis, suggests that high concentrations of pituitary gonadotropins increase cancer risk by stimulating the ovarian surface epithelium [139]. According to this theory, excessive gonadotropin secretion results in increased estrogenic stimulation of the epithelial cells, thereby increasing the risk for ovarian cancer. This would be especially true of the early postmenopausal years when both gonadotropins and the age-specific ovarian cancer rates are high. Risch [ 135] proposed a third model. He hypothesized that ovarian cancer risk is increased by androgenic stimulation of the ovarian epithelium and is reduced by progesterone. Premenopausally, the ovaries secret androgens at a higher rate than estrogen. Epithelial cells, especially those within inclusion cysts, have exposure to paracrine ovarian androgens as well as circulating androgens. Postmenopausally, the ovary continues to secrete androstenedione, dehydrotestosterone, testosterone, and, to a lesser extent, DHEA, with concentrations lower than in premenopausal women but higher than in oophorectomized women [ 141,142]. Several epidemiologic studies suggest a possible association between androgens and risk of epithelial ovarian cancer. In a prospective cohort study, androstenedione and DHEA concentrations were 50% higher in both pre- and postmenopausal women who developed epithelial ovarian cancer compared with controls [ 143]. In this same study, serum gonadotropin concentrations were lower in women at increased risk for epithelial ovarian cancer, a finding inconsistent with the gonadotropin theory. In a case-control study, women with epithelial ovarian cancer were more likely than control subjects to have had a physician diagnosis of polycystic ovarian syndrome [144], a condition characterized by increased serum LH, normal or low FSH, and increased androstenedione and testosterone con-

centrations [145]. Several studies have reported that oral contraceptives suppress ovarian testosterone production 3 5 70% [135]. Progesterone concentrations are elevated during pregnancy, and pregnancy is associated with a reduced risk for ovarian cancer. Ovarian cancer risk is reduced among users of oral contraceptives containing progestin only and those containing estrogen and progestin [128,135]. Another study reported increased apoptosis of ovarian epithelial cells in monkeys treated with combined or progestin-only oral contraceptives [146], suggesting that progesterone may increase death in cells capable of becoming malignant. Reduced expression of progesterone receptor was reported in cultured ovarian carcinomas compared to cultured normal epithelial cells [ 137]. Thus, decreased progesterone stimulation can be associated with ovarian tumorigenesis. Each model is consistent with some epidemiologic findings but not others. The "incessant ovulation" theory would suggest that late age at menopause and early age at menarche should increase the risk for ovarian cancer [128]. Eight casecontrol studies reported an association between age at menopause and ovarian cancer risk,with odds ratios of 1.4 to 4.6 for women who reached menopause at age 50 years or older [ 128]; however, only three of these studies reported a statistically significant result [ 148-150]. Seven case-control studies reported no association, as did two cohort studies [128]. Likewise, conflicting results were noted for an association between early age at menarche and ovarian cancer risk [ 128]. These exposures may be subject to recall bias. Imprecise recall of age at first or last menses could obscure an association with age at menopause [122]. In some women with ovarian cancer, early menopause may be a preclinical symptom of an ovarian malignancy [ 122]. The protective effect of pregnancy supports all three theories, although the role of incomplete pregnancies, either due to spontaneous or induced abortions, on the risk of epithelial ovarian cancer is inconclusive [123,128]. Pregnancy suppresses ovulation and causes pituitary gonadotropin levels to

CHAPTER25 C

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decrease sharply. Pregnancy also greatly increases estrogen, androstenedione, and progesterone concentrations. In a combined analysis of 12 case-control studies, risk for ovarian cancer was reduced 40% for the first full-term pregnancy, and, a further 14% reduction in risk with pregnancy was noted with each subsequent birth [130]. In this same analysis, the investigators estimated the percent reduction in risk for epithelial ovarian cancer per year of anovulation brought about by term pregnancies. They reported a decrease in risk that was greater for women aged less than 55 years than for women 55 years or older [124]. Lactation suppresses the pituitary gonadotropins and leads to anovulation in some women. The risk of ovarian cancer is decreased in women with a history of lactation compared with women who do not breast-feed [123,128]. In a collaborative analysis of 12 case-control studies, the risk of ovarian cancer decreased almost 1% for each month of lactation, with the protective effect strongest in the early months following delivery [130]. This is the period when ovulation is more likely to be suppressed. The reduction in risk for ovarian cancer was similar for both pre- and postmenopausal women [122]. It is well established that nulliparous women are at increased risk for ovarian cancer [130]. Whether infertile women are at elevated risk for ovarian cancer is controversial [123,128,151]. In a pooled analysis of three case-control studies, ovarian cancer risk was increased among women who were infertile compared with women without infertility, with the greatest increase in risk for nulliparous women who had used infertility drugs [130]. In a cohort study, infertile women who had taken the infertility drug clomiphene for 12 cycles or more were at increased risk for ovarian cancer [152]. Other studies have also found such associations [151]. Fertility drugs increase a woman's endogenous concentrations of gonadotropins, estrogen, and progesterone. Whether fertility drugs, if they do increase risk, act as a direct carcinogen, or induce or promote malignant cell transformation through increased cell division, is not known [ 151 ]. Combined oral contraceptives contain both estrogen and progestin. The oral contraceptives suppress the midcycle gonadotropin surge and inhibit ovulation. The evidence is very convincing that oral contraceptives reduce the risk of ovarian cancer [123,128]. A meta-analysis estimated a relative risk of 0.64 (95% confidence intervals, 0.57-0.73) for ever use of oral contraceptives [ 153]. Longer duration of oral contraceptives increases the protection against epithelial ovarian cancer. It has been estimated that there is a 50% reduction in risk after 5 years on oral contraceptives [153,154], with each year of oral contraceptive use contributing a 10-12% reduction in risk [ 153]. The protective effect of oral contraceptives against ovarian cancer is apparent at all ages at diagnosis [128], and for all histologic types, with the exception of mucinous tumors [155]. In the collaborative analysis of 12 case-control studies, the investigators estimated the percent

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reduction in risk for epithelial ovarian cancer per year of anovulation brought about by oral contraceptive use [124]. They reported a greater decrease in risk for women 55 years or older compared with women less than 55 years of age. One possible explanation is that the use of the early higher potency formulations by the older women may confer greater protection than the low-potency formulations used by the younger women [ 156]. The difference in risk conflicts with the ovulation hypothesis, but is more consistent with the gonadotropin theory, because the low-potency oral contraceptive may be less effective at inhibiting pituitary secretion of gonadotropin [157]. Only two studies assessed the risk associated with the new lower dose estrogen and progestin biphasic or multiphasic oral contraceptives, and both reported a protective effect [158,159]. Neither of these studies was able to compare pre- and postmenopausal women. Two case-control studies have assessed the effect of total number of ovulatory cycles in women on the risk of ovarian cancer. In the collaborative analysis, the investigators reported a statistically significant increasing trend in risk with increasing years of ovulation for women less than 55 years of age, but not the older women [124]. Another study suggests that premenopausal women with more lifetime ovulatory cycles are at increased risk of having p53-positive tumors if they develop ovarian cancer [ 160]. p53 is a tumorsuppressor gene whose protein regulates cell proliferation by binding to specific regions of DNA and initiates cell death if the damage is irreparable [ 161 ]. Because ovulation increases cell proliferation, the Fathalla-Pike hypothesis predicts that ovulation may induce p53 transitions [ 162]. The majority of epidemiologic studies have reported a decreased risk for ovarian cancer after tubal ligation and/or hysterectomy [123,128]. It has been hypothesized that surgery prevents exogenous agents, such as growth factors or other toxins, from entering the peritoneal cavity [ 163]. Studies have shown that women who have already undergone a tubal ligation have no additional protection from ovarian cancer following a hysterectomy [ 123]. The protective effect of these surgical procedures does not diminish until 20 to 25 years after the surgery [ 164]. Published data on the role of hormone replacement therapy and risk of ovarian cancer are conflicting [ 128]. Hormone replacement therapy decreases the secretion of pituitary gonadotropins, although not to premenopausal concentrations [165]. If the gonadotropin hypothesis is true, hormone replacement therapy should decrease the risk for ovarian cancer. Most epidemiologic studies have shown no association [128], or a modest increase in risk of 40 to 70% [ 166,167]. In a meta-analysis, the use of hormone replacement therapy for more than 10 years was associated with a 20% increase in risk for epithelial ovarian cancer [134]. These studies included women who had used unopposed estrogen. Whether hormone replacement therapy that includes estrogen and progestin affects risk for ovarian cancer

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requires further study. Two case-control studies suggest an increased risk for epithelial ovarian cancer and hormone replacement therapy in women diagnosed with endometrioid tumors [155,168]. Ovarian cysts are reported to be a common side effect of tamoxifen treatment in both pre- and postmenopausal women with breast cancer, although an increase in ovarian cancer risk has not been found [169]. One study reported an increased incidence of ovarian cysts in premenopausal women with breast cancer treated with tamoxifen compared with premenopausal women with breast cancer who were not treated with tamoxifen [ 170]. These cysts disappeared when tamoxifen treatment was discontinued. To date, there have been no published studies on the effects of raloxifene on the human ovary. Screening for ovarian cancer among healthy women is not advised at this time [171]. CA125 is the most widely used marker for the detection and management of ovarian cancer [ 172]. Unfortunately, this marker is not highly sensitive and lacks specificity. This test is not consistently elevated in women diagnosed with early-stage tumors. The addition of ultrasound as a second screening test increases the specificity, but detects only about half of the early-stage tumors [173].

Vl.

SUMMARY

AND CONCLUSIONS

In summary, menopause clearly affects the incidence of breast and endometrial cancers. Indeed, as Pike [22] has pointed out, if it were not for menopause, the incidence of breast cancer might increase with age in a linear fashion throughout the life span, so that even more women would develop breast cancer than already do. Evidence points to a critical role of endogenous sex hormones in the pathogenesis of both breast and endometrial cancers, but in somewhat different ways. Factors that increase concentrations of estrogens almost certainly increase risk of breast cancer, and, although the evidence is by no means conclusive, progestins may enhance the effect of estrogen alone. Estrogens are known to increase the risk for endometrial cancer, but progestins protect against the potential carcinogenic effect of estrogens on the endometrium. Estrogen replacement therapy is an important risk factor for endometrial cancer, and probably increases the risk for breast cancer as well, but to a lesser extent. Progestin added to estrogen replacement therapy for at least 10 days during the month appears to lower endometrial cancer risk. The effect on breast cancer risk of adding progestin to estrogen is not certain, but it either increases or has no effect on the risk associated with estrogen alone. It does not decrease the risk. After menopause, women not using hormone replacement therapy have low concentrations of circulating estrogens, and those of progesterone are close to zero. Obesity, which

results in greater estrogen production, increases the risk for both breast and endometrial cancers in postmenopausal women. Later age at menopause, which is associated with longer exposure to estrogen and possibly to estrogen unopposed by progestin, also increases the risk for both of these cancers. Other breast cancer risk factors, such as early age at menarche, low parity, and late age at first birth, are thought to affect breast cancer risk in part through hormonal pathways. The changes brought about by these risk factors have already occurred several years before menopause occurs, but their effect is still apparent well after menopause. For endometrial cancer, potentially protective factors such as high parity and oral contraceptive use likely operate through hormonal mechanisms, particularly through exposure to progestins. Again, these exposures have taken place long before menopause occurs, but they have effects lasting well after menopause. Thus, at the present state of knowledge, during the postmenopausal years body weight and use of hormone replacement therapy are the two major characteristics that a woman can still change in order to affect her subsequent risk for both of these cancers. Menopause appears to have little or no effect on risks of cervical or ovarian cancers. The role of hormone replacement therapy on risks of these cancers requires further study, especially the risk for ovarian cancer among long-term users and users of combined estrogen and progestin formulations. Newer selective estrogen receptor modulators are under development to provide safer options than hormone replacement therapy, but such safety is far from established at this time. The rapid advances in molecular genetics should help to clarify the interplay between genetic factors and hormonal influences on the development of these neoplasms. There are also intriguing findings that androgens may be involved in breast and possibly endometrial carcinogenesis. Further research is needed in this area. In conclusion, although much progress has been made in the understanding of the pathogenesis of breast, uterine, and ovarian cancers, new approaches to the prevention of these malignancies remains a priority.

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CHAPTER 25 Cancers

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45. 46.

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51.

52. 53.

54. 55.

56.

57. 58.

59.

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Thun, M. J., and Heath, C. W., Jr. (1995). Estrogen replacement therapy and fatal ovarian cancer. Am. J. Epidemiol. 141, 828-835. Negri, E., Tzonou, T., Beral, V., Lagiou, P., Trichopoulos, D., Parazzini, F., Francheschi, S., Booth, M., and La Vecchia, C. L. (1999). Hormonal therapy for menopause and ovarian cancer in a collaborative re-analysis of European studies. Int. J. Cancer 80, 848- 851. Weiss, N. S., Lyon, J. L., Krishnamurthy, S., Dietert, S. E., Lift, J. M., and Daling, J. R. (1982). Noncontraceptive estrogen use and the occurrence of ovarian cancer. JNCI, J. Natl. Cancer Inst. 68, 95-98. Shushan, A., Peretz, T., Uziely, B., Lewin, A., and Mor-Yosef, S. (1996). Ovarian cysts in premenopausal and postmenopausal women with breast cancer. Am. J. Obstet. Gynecol. 174, 141-144. Cohen, I., Figer, A., Tepper, R., Shapira, J., Altaras, M. M., Yigael, D., and Beyth, Y. (1999). Ovarian overstimulation and cystic formation in premenopausal tamoxifen exposure: Comparison between tamoxifen-treated and nontreated breast cancer patients. Gynecol. Oncol. 72, 202-207. Westhoff, C. L. (1996). Ovarian cancer. Annu. Rev. Public Health 17, 85-96. Rustin, G. J. S., van der Burg, M. E. L., and Berek, J. S. (1993). Tumor markers. Ann. Oncol. 4, $71-$77. Woolas, R. P., Xu, F. J., Jacobs, I. J., Yu, Y. H., Daly, L., Berchuck, A., Soper, J. T., Clarke-Pearson, D. L., Oram, D. H., and Bast, R. C., Jr. (1993). Elevation of multiple serum markers in patients with Stage I ovarian cancer. J. Natl. Cancer Inst. 85, 1748-1751.

2 H A P T E R 2(

Menopausal Sexuality GLORIA A. B ACHMANN, IRINA D. BURD, AND GARY A. EBERT Department of Obstetrics, Gynecology, and Reproductive Sciences, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, New Brunswick, New Jersey 08901

V. Other Factors VI. Partner, Relationship, and Menopausal Sexuality VII. Interventions for Sexual Dysfunction References

I. Introduction

II. Menopausal Sexuality III. Role of Estrogen IV. Androgens and Postmenopausal Sexual Function

logic changes there are a variety of nonhormonal changes that usually occur during this transition; these temporal changes in personal life are an adjunct to the factors that Aging and menopause have often been misperhave determined a woman's sexual drive throughout her lifeceived as the end to the sexual vitality and enjoyment of time. Therefore, even though menopause is a universal event, one's intimate relationships. Existence of cultural stereoit would be difficult to devise a model that would serve as types about menopause being the end to fertility and therefore the loss of sexuality have served as a self-fulfilling - a stereotype for the relationship between menopause and sexual function for every woman. Instead, it is vital to review prophecy. However, these notions of sexual retirement coall of the variables that play a role in overall sexual function, inciding with menopause are being disproved, not only in including the sex response cycle, the pathophysiology of the the realm of psychosocial research but also at the anatomic menopause, the age at the menopause, the impact that aging and physiologic levels. Through the pioneering work of exand menopause may produce, whether the menopause is perts such as Freud, Kinsey, Wolpe, and Masters and Johnnatural or surgical, endocrine factors, cultural and social son, it became clear that sexual dysfunction is in the realm influences, and various concurrent illnesses, as well as the of medical pathology and that a global approach to both availability and sexual vitality of an intimate partner. This sexual function and sexual dysfunction, especially in the chapter presents the foundation for addressing the intricate menopausal years, is imperative. The belief that sexual funcchanges occuring at menopause that may impact on sexual tion is not a mere dimension of our personality and solely performance and the pharmocologic and counseling intera product of our psychosocial environment but as much of a ventions that are efficacious and can be offered to the aging natural endocrinologic and physiologic process as respirasubset of women. tory, cardiac, or digestive systems was revolutionized by Masters and Johnson [1]. This concept of sexuality, being of clinical importance and greatly impacted by the overall physical, emotional, and psychological health of the person, II. M E N O P A U S A L SEXUALITY has promoted health care for menopausal women through the development of algorithms that address sexual health issues. As a woman enters the menopausal years, a variety of When evaluating sexual function in the perimenopausal changes inevitably occur in her sexual performance, and for and menopausal woman, in addition to marked endocrinothe most part these changes adversely affect sexual health. I. I N T R O D U C T I O N

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384 Sexual alterations are not only influenced by lifestyle, culture, religion, partner, and environment but also by the rate and intensity of the changes in the endocrinologic milieu unique to each woman. As a result of numerous variables influencing an older female's lifestyle and surrounding environment, menopausal women may choose to build their own "schema" of sexual function and expression and select the factors that represent their perception of menopause and sexuality and what changes to expect in the transition process [2]. However, negative sexual schemas often serve as self-fulfilling prophecies and perpetuate negative attitudes toward sexual health. When psychologic dependence on a particular negative schema--whether correct or incorrect--of sexual function occurs, women may attribute depression and decreased sexual arousal solely to menopause, especially when other disruptive symptoms, such as vasomotor flushes and sleep disruption, also are present. Furthermore, some women may use entry into menopause to validate depression or lack of sexual desire for their partner. Other women may feel that menopause can be identified as the major reason for their release from obligatory sexual exchange with their partner, which during the reproductive years was felt as a burden and duty in a relationship without sexual attraction. Understanding the interaction between a couple, being aware of the woman's schemas, and providing effective counseling and education about menopause may foster improved communication, an increased sense of well-being, and improved sexual functioning in the menopausal and postmenopausal years. Additionally, because menopause is associated with aging, menopausal changes and aging changes are often superimposed. Historically, the image of elderly women has not been associated with sexual vitality, sexual allure, sexual attraction, or sexual excitement. Because fecundity has been evolutionarily associated with sexual activity, sexual exchange is often misconceived as being synonymous to fertility, and therefore it has followed that that the loss of reproductive capabilities with menopause often has been translated into "asexuality." Moreover, in institutions that care for the aging population, such as nursing homes, the view of the elderly as "functional" children often permeates and with this labeling the elderly are not considered sexually attractive or capable of exhibiting sexual excitement [3]. These social views often adversely affect the aging woman's self-perception of herself as a sexual being and dampen her sexual vigor. Other factors, relating more to aging than menopause, may also influence a woman's perception of herself and her sexuality. Wrinkles, gray hair, and loss of skin elasticity usually have a negative impact on the woman's self-perception of being sexually attractive. These alterations make aging women feel uncomfortable about their body image and may decrease their sexual desire, especially if they think that

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these changes reflect loss of femininity and loss of attractiveness to their sexual partner [4]. In addition to the negative impact of these cultural views, older women experience an increased prevalence of sexual dysfunctions during this transition because of effects of hormonal changes on the breasts, urogenital system, and skin. These changes are often interpreted as the harbingers to the end of sexual life. Many studies, including the one by Rosen et al. (which surveyed 329 women, ages 18-72 years), have shown that frequency of sexual dysfunctions increases across the life span, with a rather dramatic increase in menopausal women [5]. The majority of sexual problems that surface in perimenopausal and menopausal women can be linked to the changes in gonadal steroid status. A decline in serum concentrations of estrogen, progesterone, and androgens is associated with physical, physiologic, anatomic, and behavioral changes that adversely affect sexual function. Perimenopausally, ovarian function is unstable and declines rapidly as the number of follicles decrease (atresia) to low levels, and the remaining follicles are poorly responsive to gonadotropic stimulation. Changes in hypothalamus/pituitary responsiveness may also play a key role in the endocrinologic fluctuations that occur at this time [6]. These marked changes in endocrine function are not only reflected in vasomotor symptoms but also are frequently mirrored in a dysfunctional uterine response as noted by the high incidence of dysfunctional uterine bleeding at this time, which in turn often adds to sexual problems [7]. Women unable or unwilling to have coital activity with vaginal bleeding or spotting will be obligated to endure long periods of abstinence during the menopausal transition. With advanced age, there is an increased prevalence of mitochondrial DNA mutations in the ovary, a change that has been shown to influence menstrual status and later is strongly associated with amenorrhea [8]. Through ultrasound techniques, a marked decrease in blood flow to the ovaries has been demonstrated to be responsible for hypoperfusion of the ovary, probably leading to chromosomal DNA mutations [9]. It has been hypothesized that follicular atresia is brought about by programmed cell death (apoptosis) [10] and these changes in DNA and meiotic instabilities could serve as contributing signals toward cell death. With declining ovarian function and loss of functional follicles, the major producer of estrogen, blood levels of estrogen decline. Increasing follicle-stimulating hormone (FSH), declining inhibin (INH), and fluctuating estradiol levels characterize the beginning of the menopause and ultimately lead to a chronic hypoestrogenic state [7,11]. Urogenital atrophy in older females chronically deprived of estrogen is one of the major reasons for an increase in sex dysfuntion at this time. Symptoms associated with atrophic urogenital changes include vaginal dryness, vulvovaginal pruritis, coital pain, postcoital bleeding, urinary urgency and urinary frequency, and recurrent urinary tract infections [ 12]. Because of these

CHAPTER26 Menopausal Sexuality changes in urogenital integrity, coitus is often an unpleasant experience for the menopausal woman because it is marked by pain and it often follows that there is reduced sexual interest and sexual activity. Menopausal women also describe urinary frequency, burning, and pressure postcoitally, which also detracts from their interest in sexual exchange. In additional to coital pain and bleeding, urinary incontinence reflects changes in the urogenital tract with menopause that may represent another element contributing to menopausal sexual decline. In a study of 902 postmenopausal Swedish women [13], 20% had complaints of urinary incontinence and 70% related the commencement of this complaint to the onset of the menopause. This disorder, which is embarrassing to women, also may lessen libido because of the concern that incontinence will occur during sexual activity. In a study done by Sutherst, about 46% of women reported that urinary incontinence adversely effected their sexual function [ 14]. The major role of androgen is in the arena of sex drive motivational activities such as libido, arousal, and sexual fantasy [ 15]. Ovarian androgen concentrations decrease over several years, correlating with natural ovarian atrophy occurring during menopause; ovarian androgen concentrations decrease abruptly with surgical menopause. In the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV), Hypoactive Sexual Desire Disorder (HSDD) is characterized by absence of sexual desire and lack of sexual fantasies, causing marked distress in interpersonal life. There are several subtypes in regard to context (generalized versus situation), to etiological factors (psychological endocrinological or combined factors), and to onset (lifelong versus acquired). Declining concentrations of gonadal steroids (androgens) in menopause and perimenopausally are associated with acquired HSSD. Premenopausal women who undergo oophorectomy with or without hysterectomy experience sudden endocrinologic shifts and often suffer significantly from loss of sex desire. In a study done by Utian [16], subjects who had undergone hysterectomy, irrespective of whether the ovaries were preserved or removed, experienced decreased libido. In women who have ovarian preservation with hysterectomy, this decline in drive may be related to changes in vascular perfusion to the ovaries [ 16]. In a study done by Parker et al. [17] it was reported that postradical hysterectomy subjects with ovarian retention experienced early loss of hormone function similar to those who had their ovaries removed. Physiologic studies conducted by Masters and Johnson on the sexual response of an aging woman pioneered the research on this topic, and their data have been supported by other investigators (Fig. 1) [1,18]. Menopausal and postmenopausal sexual activity is characterized by a decrease in vaginal vascularity and diminished vasocongestion, a limited quantity of vaginal secretions often leading to inadequate lubrication, loss of sensitivity in genital and breast tissue,

385

FIGURE 1 Sexualresponse cycle in menopausal female.

and a decrease in contractility of the uterus and orgasmic platform (lower third of the vagina), even though orgasmic ability does not change [ 1]. Data from other investigations complement Masters and Johnson's research by reporting that there is a definitive decline in vaginal lubrication, reduced sexual frequency, and less frequent orgasms with age [ 19]. It is clear that most of the sexual response changes are directly impacted by changes in the endocrine system, because the greatest percentage of menopausal sexual dysfunctions are directly or indirectly related to changes in circulating estrogen and androgen levels [20]. Other limitations in sexual function, from nonendocrine factors, will be exacerbated by these endocrinologic changes [19]. For example, decreased libido from gonadal hormone loss may be exacerbated by coexistent psychosocial factors (depression, alcohol, or drug abuse), chronic medical illnesses (diabetes, hypertension, or cancer), changes in partner's appearance, and problems in the relationship. Therefore, a decrease in sex arousal may be caused by estrogen/androgen decline but may also be contributed to by a relationship conflict, a past sexual trauma, chemotherapy, or medications such as anticholinergics.

III. ROLE OF ESTROGEN Estrogen plays important roles in the sex response cycle and in the maintenance of urogenital, breast, and uterine integrity. With menopause, this hypoestrogenic environment impacts on urogenital health by interfering with muscular, neurologic, and vascular integrity. Estrogen receptors are located in the endothelium of the vasculature and play a role in local production of vasodilatory prostaglandin, which influences local and systemic blood flow [21]. Estrogen loss also is related to changes in central and peripheral nervous

386 function and the capacity to develop muscle tension [ 11 ]. An end result of these changes is that vaginal function is compromised and vaginal secretions are decreased and the composition of vaginal secretions is changed [22]. In a subset of 52 postmenopausal women, Semmens et al. reported that there was a decrease in vaginal blood flow, a loss of vaginal secretions, and an increase in the pH of vaginal secretions from acidic levels of less than 4.5 to more alkaline levels by an average of 1 pH unit [22]. Additionally, these investigations reported a decrease in transvaginal potential difference indicating that there was a deficiency of the active transporting mechanism in the vaginal epithelium. On administration of estrogen replacement therapy to these subjects, a return to premenopausal levels of vaginal circulation, a significant increase in vaginal secretions, a decrease in pH and an increase in transvaginal potential (normalization of the active transport) were noted. These results proved the pivotal role of estrogen on vaginal circulation, secretion, and electropotential for transudation and that the changes that occur during menopause are reversible. However, complete reversibility of vaginal atrophy may be dependent on the duration of estrogen deprivation. Vaginal dryness due to estrogen loss not only causes painful intercourse but over time may lead to decreases in sexual desire [23]. Reversing urogenital atrophy is often helpful in eliminating dyspareunia and thereby increasing sexual desire. Moreover, these data point to the fact that vaginal circulation, one of the main factors in the sex response cycle, may be increased to reproductive-age levels with estrogen replacement therapy, therefore eliminating the deficits in congestion of the labia minora, the lower one-third of vagina, and the clitoris, which are contributors to sexual arousal. Because vaginal tissue in the reproductive-age female is stimulated by estrogen through nuclear receptors, clinical signs of estrogen deprivation include loss of vaginal elasticity, with the vaginal vault becoming thinner and paler in color and the vaginal barrel becoming shorter. Additionally, the vaginal surface loses most of its rugae, becomes friable, and with minimal trauma may become covered with petechiae and/or may bleed. Often, adhesions form during the healing phase after vaginal vault trauma, which increases vaginal distortion, contributing to dyspareunia [ 12,24]. Another sequella in an estrogen-deprived vagina is the loss of vaginal tone, which may interfere with the menopausal woman's ability to experience full satisfaction of coitus. Not only does vaginal relaxation create an uncomfortable pressurelike feeling, but the condition also decreases sexual sensation and response and predisposes the older woman to urinary and bowel dysfunction [25]. Urogenital atrophy not only is the most common etiology of sexual problems in the menopausal women but also translates to nonsexual complaints such as recurrent vaginitis or frequent urinary tract infections [12]. Both sexual and nonsexual symptoms make the sexual experience less enjoyable and may lead to a subsequent decrease in sexual interest.

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Estrogen loss also has an impact on vulvar health. In a study done by Oriba and Maibach [26], it was shown that the estrogen-deprived vulva loses most of its collagen, adipose, and water-retaining ability. Without appropriate lubrication, unprotected atrophic vulvar tissue becomes irritated with minimal manual stimulation or penile friction during sexual intercourse. With decreased levels of gonadal steroids, the uterus becomes atrophic and fibrous, which adds to sexual dysfunction [24]. With uterine change, older women may experience painful spasms of uterine musculature during orgasm and the regular, recurrent contractions of uterine musculature during orgasm may turn into painful, spastic ones. In older women, painful uterine spasms may be ameliorated with estrogen supplementation [ 1]. Estrogen also plays a role in supporting both an overall sense of well-being and sexual well-being by eliminating vasomotor symptoms and sleep disturbances. This dependence on estrogen of sleep and temperature regulation and other central nervous system (CNS) effects has been termed the "mental tonic effect," because estrogen was found to have a monoamine oxidase inhibitory effect leading to increased noradrenaline synthesis, increased CNS activation, and eventually catecholamine depletion [27-29]. This relationship does not necessarily suggest that estrogen could be used for symptoms of depression, because improvement in an overall sense of well being is not equivalent to being on an antidepressant [27]. Rather, improvements in selfperception may show positive effects on sexual physiology and self-assessment of being sexually attractive, which may improve sexual functioning. Overall, estrogens have several positive effects on sexuality (Table I) in addition to their global effects on vasomotor symptoms and on reducing the risk of cardiovascular disease and osteoporosis, and possibly a positive effect on patients with Alzheimer's disease or a positive role in preventing its presentation. Unopposed estrogen use is associated with an increased incidence of endometrial cancer [30]. For example, in a study done by Notelovitz et al. [31 ], endometrial histology was evaluated in 238 postmenopausal women taking different estrogen doses (0.3, 0.625, or 1.25 mg) and compared with the histology of women given placebo in a prospective, randomized, double-blind 2-year clinical trial. The incidence

TABLE I Impact of Hormones on Sexual Function: Estrogen Urogenital integrity Vaginal dryness Vaginal infections Vasomotor symptoms Insomnia General sense of well being

CHAPTER26 Menopausal Sexuality of histological changes in the placebo group was comparable to that in the group taking 0.3 mg of unopposed estrogen, whereas the other doses of estrogen led to an increased prevalence of endometrial hyperplasia; it was suggested that 0.3 mg is a minimal dose to use unopposed for longer periods of time without progestogens. Estrogen/progestogen combinations are usually used in order to reduce the incidence of endometrial hyperplasia. Progestogens may have a negative dose-dependent effect on the central nervous system, in regard to psychological and sexual functions. These negative effects on mood and sexuality seem to be attenuated once the estrogen/progestin dose ratio is increased [32].

IV. ANDROGENS AND POSTMENOPAUSAL

SEXUAL FUNCTION

Androgens used in conjunction with estrogen have been used as another intervention for improving sexual health in the menopausal female (Table II). Androgen concentrations begin to decline perimenopausally, usually from decreases in adrenal production. In the premenopausal years, both the adrenals and the ovaries produce androgens (androstenedione, testosterone and dehydroepiandrosterone), although only adrenals produce dehydroepiandrosterone sulfate (DHEAS). A further decline in androgen production is associated with ovarian failure, either with natural or surgical menopause [33]. Data have shown that this decline in androgen decreases libido and sexual arousal and has a negative effect on the number of sexual fantasies [19]. Positive effects of androgen use were noted as early as the 1940s, a time when androgens were also used in the treatment of estrogendependent breast cancer [34]. When the loss of libido is not caused by other variables, such as prescribed medication, various endocrine and vascular disorders, psychiatric illnesses, etc., estrogen/androgen may be beneficial in improving sex drive motivational activities. Hence, the appropriate evaluation of the patient is imperative to rule out other causes of loss of sexual interest [15]. Androgens used for replacement therapy also may have an impact on improving a general feeling of well being, through their effects on psychological and affective functioning. In a study done by Sherwin and Gelfand [35], 53 surgically menopausal females were separated into four groups:

TABLE II Impact of Hormones on Sexual Function: Estrogen/Androgen Libido Psychological and affectivefunction Vasomotor symptoms Vaginal dryness Mastalgia

387 estrogen, estrogen/androgen, androgen, and placebo. It was noted that estrogen/androgen and androgen groups showed better appetite, energy, and sense of self-being compared to placebo and estrogen groups. Later studies by other investigators showed that androgens decrease irritability and nervousness as well as decrease insomnia in some instances [36]. These effects of androgens are especially beneficial in menopausal women with sexual complaints, because the improvement in overall quality of life will positively impact sexual health as well. Androgen/estrogen replacement may also enhance sexual health in the treatment of mastalgia, a condition that often detracts from a woman's desire to engage in sexual activity. It has been proposed by Pye et al. [37] that estrogen decline during the transitional years may be responsible for this phenomenon. Decline in estrogen exposure perimenopausally and in the early menopause causes the up-regulation of estrogen receptors in breast tissue and its sensitization to estrogen. With increased estrogen, either through the woman's own surges or estrogen replacement therapy, breast tissue becomes very sensitive and patients report severe pain in the breast. However, the addition of androgen to estrogen may help to alleviate this problem. Some data suggest that androgens act synergetically with estrogen for the relief of vaginal dryness as well as the relief of vasomotor symptoms [15] and the treatment of osteoporosis [38]. Androgen replacement alone may adversely affect lipid profiles, particularly high-density lipoprotein (HDL) cholesterol. For example, it was found that testosterone administration to menopausal subjects may prevent the increase in circulating HDL seen in women taking only estrogen [39]. However, when used with estrogen, androgen does not show a markedly negative impact on lipid profile and may actually prove beneficial in some patients through a decrease in circulating triglyceride.

V. OTHER FACTORS Other important determinants of the way a menopausal woman evaluates how she feels sexually include her lifestyle, the society that she lives in, her libido throughout her lifetime, concurrent physical illnesses, surgeries, medications that she is taking, and cultural and psychological influences, as well as the presence of a sexually active partner. In a study done by Koster and Garde [40], sexual desire was investigated through interviews and questionnaires and was found to be correlated with health status, former sexual activity, partner availability, and social status. An extremely strong determinant is past sexual experience. Health status is another key determinant of sense of well being, life satisfaction, and sexuality. Because older people are usually at higher risk for various malignancies and chronic illnesses compared to younger individuals, they may be more susceptible to become depressed and anxious.

388 The physical changes associated with chronic illness as well as the emotional issues may influence sexual activity and dampen sexual vigor. For example, women undergoing surgeries or procedures (e.g., radiation therapy) for malignancies often experience changes that are brought about by interactions of interpersonal, psychological, anatomical, and physiological factors, which may influence sexual function as well. Furthermore, many women hold the erroneous belief that without a uterus they will be unable to enjoy sexual activity or achieve orgasms [2]. In fact, any kind of surgery that threatens "femininity," such as mastectomy or other kinds of gynecologic surgeries, can have negative sexual ramifications. To avert sexual problems requires counseling prior to and after the surgery [3 ]. On the other hand, those who undergo radical surgery or radiation therapy for a pelvic malignancy may discover that this procedure has a direct effect on their sexual function. Even though radical hysterectomy may bring about relief from pain and bleeding, it often results in nerve severance and may create some anatomical disfigurement. Common consequences of the radiation therapy for cervical cancer include loss of vaginal lubrication, vaginal friability, and pelvic fibrosis. These effects, combined with feelings about malignancy, often result in loss of self-esteem with consequent loss of sexual interest and arousal [41 ]. Chronic illnesses also have an effect on self-esteem and sexual function. Psychiatric disorders, diabetes, arthritis, hypertension, and cardiovascular illnesses are the most vivid examples of such a relationship. Despite the controversy that exists in regard to the effect of gonadal steroids on depression it has been shown that more than 65% of women develop some signs of depression concurrent at the time of the menopause [42]. This could be attributed to the effects of androgen decline, the replacement of which in menopause often results in increased psychological function and heightened perception of well being [15]. Because mood states are correlated with sexual function [43], better mood is related to greater sexual satisfaction, whereas depressed mood is associated with decreased sexual function. Specifically, it has been reported that depression correlates with lower libido, a decrease in lubrication, and a decline in sex frequency [44]. It can be inferred that decreased psychological function might be associated with lower sexual function and/or satisfaction. Diabetes, a chronic illness associated with older age, may impair sexual functioning in both males and females. Although the impact of diabetes on sexual function in women is not as extensively studied as its impact on erectile function in men, some data exist to support the relationship. In a physiologic study done by Wincze et al., [45], arousal in diabetic patients was compared to that of controls by examining capillary engorgement through vaginal photoplethsmography and by recording sexual difficulties that were reported

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through questionnaires. Even though the groups did not differ in the rates of reported sexual difficulties, it was found that physiological arousal of diabetic patients was markedly decreased [45]. Moreover, in another study, a decline in libido and anotgasmia was found to be slightly more prevalent in women with type II diabetes [46]. Women undergoing hemodialysis or peritoneal dialysis in the later stages of diabetes may report anorgasmia and decreased libido [47]. Fatigue associated with the procedure, the underlying disorder, or depression associated with chronic disease may all contribute to this increased level of sexual dysfunction. Hypertension is also a frequently encountered disorder affecting sexual function. In females, the most common associated side effects are vaginal dryness and a decline in sexual interest. The loss of optimal sexual function may be related either to central or peripheral effects of the antihypertensive medication or to the diagnosis of hypertension. These adverse effects are superimposed on the compromised vaginal integrity of menopausal woman with declining gonadal steroids and may cause an exacerbation of urogenital symptoms. Because these side effects were found to be associated mostly with antihypertensive medication, careful monitoring of the patient with dose reduction and medication changes when needed may prove beneficial [3]. As with hypertension, arthritis and chronic pain are commonly associated with older age. These conditions can produce pain during intercourse, especially if sexual positions are not modified, and, consequently, decrease sexual desire. In a study done by Yoshino and Uchida, it was found that half of the married women with rheumatoid arthritis reported decreased libido and frequency of intercourse [48]. Therefore, finding a mutually comfortable position may enhance sexual pleasure and alleviate possible sexual problems associated with long-term avoidance of a sexual relationship with a spouse. Breaking a sex avoidance cycle is key in the treatment algorithms. Aging as well as chronic illnesses are associated with an increased number of prescribed and consumed medications. Medications often have idiosyncratic effects and may not have the same sexual side effects in every individual. Furthermore, it takes time for the full sexual side effects of a new medication to be reported. In addition to antihypertensive medication, as discussed earlier, four other major groups of medications may affect sexual function through their side effects. These include psychotropic drugs, minor tranquilizers, antiparkinsonian agents, and chemotherapeutic agents such as tamoxifen (Table III.) As with antihypertensive medication, other medication may exacerbate sexual dysfunction, thus the physician should inquire about changes in sexual function during regular patient visits. For example, drugs such as tamoxifen, which has antiestrogenic effects, have been shown to have a negative effect on sexual behavior by influencing libido, vaginal lubrication, mood state, and vasomotor symptoms [49].

CHAPTER26 Menopausal Sexuality TABLE III

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Geriatric Pharmacology Affecting Sexuality Drug

Antihypertensives Psychotropics Monamine oxidase inhibitors Tricyclic antidepressants Neuroleptics Selective serotonin reuptake inhibitors Antiparkinsonian agents Minor tranquillizers Chemotherapeutic agents (tamoxifen)

Effect

abuse, performance anxiety, anger and relationship issues have to be taken into account, because they can be sources of or important contributors to the disorder.

Reduced lubrication, depressive symptoms Reduced arousal Increased orgasmic difficulty Reduced genital sensation Loss of libido, anorgasmia Anticholinergric effects Decline in sexual enjoyment Decreased libido, mood changes

Currently, much attention has been given to selective serotonin reuptake inhibitors (SSRIs), which belong to a class of antidepressants that have been used not only for depression but for panic and obsessive compulsive disorders (OCDs). Even though SSRIs (e.g., fluoxetine, sertaline, and paroxetene) are considered to be extremely safe, they may be implicated in a loss of libido, a decline in sex frequency, and anorgasmia. Sexual dysfunction was reported in 2 0 - 4 0 % of the patients taking an SSRI [44]. Interestingly, in contrast to men, decreased psychological arousal and decreased libido in depressed women tend to remit with continued SSRI use. However, if the side effects persist, switching to a different class of antidepressant medication, such as buproprion, venlafaxine, etc., becomes inevitable [50,51]. Unfortunately, these alternatives to SSRIs are not recommended for panic disorder or OCD and symptoms of these disorders may be exacerbated by such a medication change. Similarly, drug and alcohol abuse must be considered in the differential diagnosis for women who present with sexual dysfunction. Alcohol adversely affects sexual function, and even premenopausal women experience sexual dysfunction with its use. In one study, 95 % of alcoholic women had lower estradiol concentrations and experienced both dyspareunia and vaginal dryness [52]. These detrimental effects may exacerbate the presentation of menopausal changes and make sexual intercourse an unpleasant experience, with further dampening of sexual vigor and avoidance of coitus. Another psychiatric problem diagnosed by the Diagnostic and Statistical Manual (DSM-IV) criteria is psychogenic sexual dysfunction, categorized in regard to desire, arousal, orgasm, and pain. As reported by Schover and Jensen [53], psychogenic dysfunction in desire can be either loss of interest or aversion to sex; disorders of the arousal phase can be a reduction in genital lubrication, decreased response to visual or erotic stimuli, or reduced intensity of experience; and psychogenic disorders of orgasms can lead to a reduced frequency of reaching orgasms or painful orgasms. When dealing with any sexual dysfunction, organic disorders should be ruled out first. However, previous sexual trauma, sexual

VI. PARTNER, RELATIONSHIP, AND MENOPAUSAL SEXUALITY In a study by Greendale et al. [43], sexual function in 874 postmenopausal women of ages 45 to 64 years was assessed. Sexual activity was reported by 64% of the women, with the leading reasons for sexual inactivity being absence of a partner (64%) and or a partner's physical problem (20%). These results confirm other data generated by a study of 46- to 54year-old female members of a health insurance plan [54] and a large population-based Swedish study [55]. There are several reasons for older women being without a sex partner. First, changes in social structure brought about by the feminist movement and increasing mobility of the workforce changed the way women perceive their relationship with their environment and their family and, hence, increased the number of single, divorced, and widowed older women. Second, in regard to heterosexual relationships, there are less single older men available, and those who are interested and physically fit can pair with younger women as condoned by societal morals, whereas older women pairing with younger men is usually not accepted by most of society. Third, those who do find an intimate partner are often discouraged by their families to continue the relationship because of the fear that the woman may be at risk of being exploited and the family might be deprived of their inheritance. Finally, a widowed woman may perceive a new relationship as a betrayal to her late husband and feel guilty about forming another sexual relationship. As result, many women often perceive being alone as the end to their sexual lives and stop engaging in any sexual activities, including masturbation. These women often need to be counseled, because the absence of a sexual partner is not the end to their sexual life and satisfaction of their sexual needs. Women who are not in a relationship may be tactfully advised to experiment with vibrators and deal with their sexual feelings and needs through self-stimulation and fantasy. By addressing issues of guilt associated with masturbation, the clinician can alleviate the anxiety that might be present. There are videos addressing the topic of sexual health; these may be supplemental in assisting older women without partners to explore alternative ways of sexual activity. Although scant data exist on homosexual relationships, as we enter the next millennium there are increasing number of menopausal women presenting with issues relating to alternative lifestyles. Unfortunately, there are limited research data on the topics of lesbian relationships and relationships involving several partners; these need to be addressed in the future [56].

390 A healthy relationship with mutual attraction, respect, and free communication between partners is an important component of sexual love and intimacy. It has been found that when healthy couples age and sex activity has been gratifying and important to them in the past it is likely to continue satisfactorily into old age. In a study done by Hawton et al., enjoyment of sexual activity depended on factors such as marital adjustment and duration of the relationship [57]. Another important factor influencing sexual expression in a heterosexual relationship is the health of the aging male. Aging males experience changes in sexual desire as well as erectile function. Changes in spontaneous erection and the need for greater manual stimulation become important; the erection becomes less firm, the refractory period lengthens, and ejaculation becomes less forceful. These changes may be misinterpreted by a woman and may be perceived by her as a lessened interest in her aging body. This could lead to avoidance behavior and reduction in seductive behavior, which are important for enhancement of the sexual response in the male. Without open communication or counseling, these inevitable changes that occur with aging may bring anger and resentment into the relationship, which may destroy sexual love and intimacy. Therefore, clarification of the nature of the male's "sexual dysfunction" during counseling or couples therapy may prove to be of benefit. Additionally, the aging male may also experience changes in sex response cycle [1], and the decision to be sexually active is also dependent on many variables. The most prominent factors for sustained sexual activity in older men were found by Martin [58] to be interest and frequency of sexual activity between the ages of 20 and 40 years. Decreased sexual drive and sexual frequency patterns in 20s and 30s predicted sexual inactivity earlier in their life cycle. Overall, male sexual function declines with age and the rates of impotence increase. It was reported that rates change dramatically from 5% at the age of 40 years to about 25% at the age of 65 years, even though the rates for sexual satisfaction do not decrease [59]. Many couples may perceive male erectile dysfunction as the end of their sexual relationship, because coitus is considered to be the most important part of the sexual relationship. Therefore, it is important to assure both sexual partners that with decreased or absent erectile function there not only are other ways of sexual intimacy and exploration of sexuality (such as kissing, cuddling, and oral and manual stimulation), but also various available treatments.

VII. INTERVENTIONS FOR SEXUAL DYSFUNCTION Sexuality and sexual dysfunction may not be easy topics for a menopausal patient to discuss with her physician. A1-

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though it is often difficult to commence sexual history with a patient whose shame, lack of comfort, guilt, or religious beliefs about the procreational purpose of sex prohibit communication on this topic, because of a sexual problem it often proves to be beneficial for the overall health picture of older females to inquire about sexual needs and individualized sexual outlets. Given the overview of factors that play a role in determining sexuality in menopausal years, physician intervention should be individualized and tailored in regard to the most likely etiology. Because the average age for menopause is 51 years, initiating the conversation on the topic of menopause should be started early during the perimenopausal transition (around the mid-40s). Education and counseling at this time may alter preexistent self-made negative schemas about menopause, enhance an overall sense of well being, and aid the older woman's perception of herself as a sexual person. Before the onset of menopausal symptoms, it is important to counsel perimenopausal women about expected changes in the hormonal milieu, and the impact of these changes on sexual desire and urogenital function, and the benefits of hormone replacement therapy and other interventions in order to make the transition a smoother experience. Although the benefits of counseling are well known, time constraints limit the amount of intervention a clinician can accomplish during an office visit. One effective and direct counseling approach as proposed by Dunn [3] utilizes the 4Rs for resolving sexual problems. The components of this technique are reframe, reeducate, refocus, and refresh. With reframing the clinician assists the patient in forming links between emotions and sexual functioning. For example, in serious life stresses, such as loss of a parent or friend, mental tensions, marital discords, or serious illnesses, a patient may lose sight of how the stress negatively impacts overall health and sexuality. Sudden loss of libido in the older female may be puzzling and worrisome. Reframing the situation and explaining how the surrounding milieu may have an impact on sexual function may bring reassurance for further sexual encounters. Reeducation, another component of this therapeutic approach, refers to the central role of the practitioner in providing the necessary sex information in order to allay stress and anxiety. For example, if a menopausal woman complains of vulvovaginal irritation, including dyspareunia and painful contractions during orgasm, education about changes in the sex response cycle, hormonal alterations, and the availability of hormonal treatment is important. Reeducation on the topic of vulvar hygiene also may prove to be helpful, such as keeping vulvovaginal area dry after exercise and swimming and/or using loose-fitting, absorbent undergarments. Refocusing involves redirecting the attention of the patient away from the problem. For example, if an aging woman is concerned that her male partner is not performing in the usual manner and she is afraid that he has lost interest

CHAPTER 26 Menopausal Sexuality in her aging body, refocusing would encourage her to spend more time in emphasizing and exploring sensual and pleasurable experiences rather than focusing on sexual scenarios that are no longer effective. Spending time in a non-goaloriented sexual mode may improve both male and female performance. Refreshing is the fourth component and counsels the woman to revive a relationship in which lovemaking has become status quo. As the partners age, the "old script" for lovemaking may not be as effective as it was when the couple was younger. For example, in chronic illnesses such as arthritis, the routine coital old position may not be comfortable or may even be painful. Refreshing the couple's sexual script with both sexual and nonsexual suggestions, such as holding hands, dancing, kissing, and viewing erotic videos, may be beneficial for the revival of the relationship and sexuality in aging partners. In addition to education and counseling, a menopausal woman should be offered hormone replacement therapy. Because the majority of sexual problems that commence in the menopausal woman stem from estrogen and androgen deficiency, hormone replacement therapy will be helpful in decreasing vulvovaginal complaints and sex drive motivational activities and consequently will improve the total sense of general well being in older females. Because steroids are lipid-based compounds, administration of hormone replacement treatment may be either systemic or local. Although hormones alone will not correct all sexual difficulties in the menopausal woman, especially if the problems are due to issues that are not related to the hormonal milieu, they will often improve the situation even when the sexual complaints are not directly related to estrogen and androgen. After adequate counseling, patients who complain of vaginal irritation, pressure, burning, pain with intercourse, or postcoital bleeding should be offered estrogen replacement therapy. If there are no indications for estrogen replacement therapy other than urogenital changes, local estrogen can be used either in the form of a cream or in the form of a estradiol-releasing vaginal ring. However, if the patient has other reasons for estrogen use, such as osteoporosis, cardiovascular disease, or Alzheimer's disease risk reduction or vasomotor symptoms, systemic therapy should be prescribed. It is important to remember that many times adequate systemic concentrations of estrogen that are delivered by either the oral or the transdermal route may not be adequate to reverse the urogenital aging changes, and, therefore, supplemental local estrogen may be necessary. Patients who are given estrogen replacement therapy may also note an improvement in sexual motivational activities, because many of the accompanying menopausal complaints may be ameliorated or eliminated with estrogen, such as vasomotor symptoms, sleep disturbances, and emotional lability. However, women with loss of sex desire or a decline in sexual arousal will respond more positively to estrogen/androgen replace-

391 ment therapy. Therefore, first-line intervention for patients who complain of sexual problems as they relate to urogenital atrophy should be estrogen replacement therapy. However, patients who are on adequate estrogen and do not have total reversal of sexual problems may benefit from the addition of androgen. In a study by Berger [60], a high percentage of women who were on estrogen alone continued to complain of poor quality of life as well as sexual desire symptoms. When they were crossed over to estrogen/androgen therapy, a marked improvement in both sexual problems and in quality of life was noted. The same study design was employed by Sarrell and colleagues [61 ], who found that the high percentage of sexual complaints that emerged in women who previously had been adequately controlled with estrogen replacement therapy was reduced with the addition of androgens. In this study, after a washout period, half of the group was put on esterified estrogens and the other half on esterifled estrogens plus methyltestosterone. After 4 weeks, there was a marked improvement in both sexual desire and sexual sensation in the estrogen/androgen group, whereas in the estrogen-alone group, the women did not show a significant improvement in their sexual dysfunction. Therefore, in patients who complain of loss of sex drive, the addition of androgen to estrogen therapy should be considered. Estrogen/ androgen therapy can be prescribed in the same manner as estrogen-alone replacement therapy, in that patient response, rather than serum hormone concentrations, can be monitored. Measurement of hormone concentrations is necessary only in patients who are not responding to estrogen or estrogen/androgen, and interpersonal issues have been ruled out. "Free" testosterone is the most beneficial hormone to assess. Women who are placed on estrogen/androgen may also note an improvement in vaginal blood flow as compared to the improvement noted with estrogen alone. When other medications may be affecting sex health, either changing medications, such as the antihypertensives or antidepressants, may ameliorate the problems. Sexual problems are not specifically a "female" or "partner" issue, but rather affect couples, so that it is important to address each partner's physical, emotional, and sexual health. For instance, many times in the heterosexual couple, a male's difficulty with erectile function is directly related to the female's vaginal dryness, and once the dryness problem is ameliorated either with hormone replacement therapy or an over-the-counter lubricant, the male's sexual dysfunction also improves. Reinforcing the idea of changing sexual scripts and encouraging activities of sexual gratification that do not depend only on intercourse are also helpful. Last, when any kind of sexual problem surfaces, whether at or around the menopause, or if it has been a long-standing problem noted even before the menopause, if it cannot be corrected in the medical office, a recommendation for counseling with a sex therapist should be considered regardless of the patient's age. Menopausal patients often need the same interventions used by younger patients,

3

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s u c h as u s e o f v i b r a t o r s , c h a n g e s in coital p o s i t i o n , or changes

in

sexual

stimulus

(oral/genital

stimulation

or

manual genital stimulation), and these suggestions should be p a r t o f the s e x u a l i n t e r v e n t i o n o f f e r e d to m e n o p a u s a l w o m e n a n d their p a r t n e r s .

References 1. Masters, W. H., and Johnson, V. E. (1981). Sex and aging process. J. Am. Geriatr. Soc. 29(9), 385-390. 2. Kingsberg, S. A. (1996). Maintaining and evaluating quality of life after menopause. Female Patient (Suppl.), May, pp. 19-24. 3. Dunn, M. E., Umlauf, R.L., and Mermis, B. J. (1992). The rehabilitation situations inventory: Staff perception of difficult behavioral situations in rehabilitation. Arch. Phys. Med. Rehabil. 73(4), 316-319. 4. Leiblum, S. R. (1991). Sex: Midlife and beyond. Amer. Fertil. Soc. Postgrad. Course: Sex. Dysfunction: Patient Concerns Prac. Strategies, Orlando, FL, 1991. 5. Rosen, R. C., Taylor, J. E, Leiblum, S. R., and Bachmann, G. A. (1993). Prevalence of sexual dysfunction in women: Results of a survey study of 329 women in outpatient gynecological clinic. J. Sex. Marital Ther. 19, 171. 6. Sherman, B., and Korenman, S. (1975). Hormonal characteristics of the human menstrual cycle throughout reproductive life. J. Clin. Invest. 55, 699-706. 7. Klein, N. A., and Soules, M. (1998). Endocrine changes of the perimenopause. C/in. Obstet. Gynecol. 41(4), 912-920. 8. Suganuma, N., Kitagawa, T., Nawa, A., and Tomoda, Y. (1993). Human ovarian aging and mitochondrial DNA deletion. Horm. Res. 39, 16-21. 9. Van Blerkom, J. (1996). The influence of intrinsic and extrinsic factors on the developmental potential and chromosomal normality of the human oocyte. J. Soc. Gynecol. Invest. 3, 3-11. 10. Hsueh, A., Billig, H., and Tsafriri, A. (1994). Ovarian follicle atresia: A hormonally controlled apoptotic process. Endocr. Rev. 15, 707-724. 11. Warren, M. E, and Kulak, J. (1998). Is estrogen replacement indicated in perimenopausal women? Clin. Obstet. Gynecol. 41(4), 976-987. 12. Bachmann, G. A. (1994). Vulvovaginal complaints. In "Treatment of the Postmenopausal Woman," (R. A. Lobo, ed.), Raven Press, New York. 13. Iosef, C. S., and Bekassy, Z. (1984). Prevalence of genito-urinary symptoms in the late menopause. Acta Obstet. Gynecol. Scand. 63, 257-260. 14. Sutherst, J. R. (1979). Sexual dysfunction and urinary incontinence. Br. J. Obstet. Gynecol. 86, 388-398. 15. Young, R. L. (1996). Androgen use in menopausal therapy. Female Patient (Suppl.), May, pp. 1-5. 16. Utian, W. H. (1975). Effects of hysterectomy, oophorectomy and estrogen therapy in libido. Int. J. Gynecol. Obstet. 13, 97-100. 17. Parker, M., Bosscher, J., Barnhill, D., and Park, R. (1993). Ovarian management during radical hysterectomy in the premenopausal patient. Obstet. Gynecol. 82(2), 187-190. 18. Roughan, E A., Kaiser, E E., and Morley, J. E. (1993). Sexuality and the older woman. Clin. Geriatr. Med. 9(1), 87-106. 19. Pariser, S. E, and Neidermier, J. A. (1998). Sex and mature woman. J. Women's Health 7(7), 849-859. 20. Bachmann, G. A. (1993). Sexual function in the perimenopause. Obstet. Gynecol. Clin. North Am. 20, 379-389. 21. Hickok, L. R., Toomey, C., and Speroff, L. (1993). A comparison of esterified estrogens with or without methyltestosterone: Effects on endometrial histology and serum lipoproteins in postmenopausal women. Obstet. Gynecol. 82(6), 919-924.

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22. Semmens, J. E, and Wagner, G. (1982). Estrogen deprivation and vaginal function in postmenopausal women. JAMA, J. Am. Med. Assoc. 248(4), 445-448. 23. Bachmann, G. A., Leiblum, S. R., Bernard, S., et al. (1985). Corrolates of sexual desire in post-menopausal women. Maturitas 7, 211-216. 24. Semmens, J. E, and Semmens, E. C. (1984). Sexual function and the menopause. Clin. Obstet. Gynecol. 27(3), 717-723. 25. Bachmann, G. A., Leiblum, S., and Hymans, H. N. (1985). Sexual expression during climacteric years. Sex. Med. Today, February, pp. 61-65. 26. Oriba, H. A., and Maibach, H. I. (1989). Vulvar transepidermal water loss (TEWL) decay curves. Effect of occlusion, delipidation and age. Acta Derm.-Venereol. 69(6), 461-465. 27. Utian, W. H. (1972). The mental tonic effect of estrogens administered to oophorectomized females. S. Afr. Med. J. 46, 1079. 28. Klaiber, E. L., Broverman, D. M., and Vogel, W. (1972). The effects of oestrogen therapy on plasma MAO activity ad EEG driving responses of depressed women. Am. J. Psychiatry 128, 1429-1498. 29. Klaiber, E. L., Broverman, D. M., Vogel, W., and Korbayasti, V. (1979). Oestrogen therapy for severe persistent depression in women. Arch. Gen. Psychiatry 36, 550-554. 30. Speroff, L. (1994). The menopause: A signal for the future. Jn "Treatment of the Postmenopausal Woman" (R. A. Lobo, ed.), Raven Press, New York. 31. Notelovitz, M., Varner, R. E., Rebar, R. W., et al. (1997). Minimal endometrial proliferation over a two-year period in postmenopausal women taking 0.3 mg of unopposed esterified estrogens. Menopause 4(2), 80-88. 32. Sherwin, B. B. (1991). The impact of different doses of estrogen and progestin on mood and sexual behavior in postmenopausal women. J. Clin. Endocrinol. Metab. 72(2), 336-343. 33. Davis, S. R., and Burger, H. G. (1996). Androgens and the postmenopausal woman. J. Clin. Endocrinol. Metab. 81(8), 2759-2763. 34. Geist, S. H. (1941). Androgen therapy in the human female. Ther. Symp. 1, 154-161. 35. Sherwin, B. B., and Gelfand, M. M. (1985). Differential symptom response to parenteral estrogen and/or androgen administration in the surgical menopause. Am. J. Obstet. Gynecol. 151, 153-160. 36. Notelovitz, M., Watts, N., Timmons, C., et al. (1992). Effects of estrogen plus low dose androgen vs. estrogen alone on menopausal symptoms in oophorectomized/hysterectomized women. Abstr. North Am. Menopause Soc., p. 101. 37. Pye, J. K., Mansel, R. E., and Hughes, L. E. (1985). Clinical experience of drug treatments for mastalgia. Lancet 2, 373-377. 38. Raisz, L. G. (1996). The role of androgens in the pathogenesis and treatment of the postmenopausal osteoporosis. Supportive Female Patient, May, pp. 11-18. 39. Whitehead, M. (1994). Progestins and androgens. Fertil. Steril. 62, 1615-1675. 40. Koster, A., and Garde, K. (1993). Sexual desire and menopausal development. A prospective study of Danish women born in 1936. Maturitas 16, 49-60. 41. Huang, E., and Bachmann, G. (1999). Analysis of sexual function posthysterectomy. J. Am. Assoc. Gynecol. Laporoscopy (in press). 42. Anderson, E., Hamburger, S., Liu, J. H., and Rebar, R. W. (1987). Characteristics of menopausal women seeking assistance. Am. J. Obstet. Gynecol. 156, 428-433. 43. Greendale, G. A., Hogan, P., and Shumaker, S. (1996). Sexual functioning in postmenopausal women. J. Women's Health 5, 445-458. 44. Modell, J. G., Katholi, C. R., Model, J. D., and DePalma, R. L. (1997). Comparative sexual side effects of buprion, fluoxetine, paroxetine and sertraline. Clin. Pharmocol. Ther. 61,476. 45. Wincze, J. E, Albert, A., and Bansal, S. (1993). Sexual arousal in diabetic females: Physiologic and self-report measures. Arch. Sex. Behav. 22, 587.

CHAPTER 26 Menopausal Sexuality 46. Jensen, S. B. (1985). Sexual relationships in couples with diabetic partner. J. Sex. Marital Ther. 11,259-270. 47. Levy, N. (1978). Sexual factor and rehabilitation. Dial Transplant. 7, 591-594. 48. Yoshino, S., and Uchida, S. (1981). Sexual problems in women with rheumatoid arthritis. Arch. Phys. Med. Rehabil. 62, 122-123. 49. Cuzick, J., and Baum, M. (1985). Tamoxifen and contralateral breast cancer. Lancet 1,282. 50. Piazza, L. A., Markowitz, J. C., Kocsis, J. H., et al. (1997). Sexual functioning in chronically depressed patients treated with SSRI antidepressants: A pilot study. Am. J. Psychiatry 154, 1757. 51. Kavoussi, R. J., Segraves, R. T., Hughes, A. R., et al. (1997). Doubleblind comparison of bupropion sustained release and sertraline in depressed outpatients. J. Clin. Psychiatry 58, 532. 52. Seki, M., Yoshida, K., and Kashimura, M. (1997). [A study on sexual dysfunction in female patients with alcoholics.] Nippon Rinsho 55, 3035. 53. Schover, L. R., and Jensen, S. B., eds. (1988). "Sexuality and Chronic Illness. A Compehensive Approach." Guilford Press, New York.

393 54. Pfeiffer, E., and Davis, G. C. (1972). Determinants of sexual behavior in middle and old age. J. Am. Geriatr. Soc. 20, 151-158. 55. Lindgren, R., Berg, G., Hammar, M., and Zuccon, E. (1993). Hormonal replacement therapy and sexuality in a population of Swedish postmenopausal women. Acta Obstet. Gynecol. Scand. 72, 292-297. 56. Glick, P. C. (1979). The future marital status and living arrangements of the elderly. Gerontologist 19, 301-309. 57. Hawton, K., Gath, D., and Day, A. (1994). Sexual function in a community sample of middle-aged women with partners: Effects of age, marital, socioeconomic, psychiatric, gynecological, and menopausal factors. Arch. Sex. Behav. 23, 375. 58. Martin, C. E. (1981). Factors affecting sexual functioning in 60-79 year old married males. Arch. Sex. Behav. 5, 10. 59. Meston, C. M. (1997). Aging and sexuality. West. J. Med. 60. Berger, H. G., Hailes, J., et al. (1984). The management of persistent menopausal symptoms with estradiol-testoterone implants. Maturitas 6, 351-358. 61. Sarrell, P. M. et al. (1998). J. Reprod. Med. 43, 847-857.

7 H A P T E R 2q

Historical Perspectives JOSEPH W. GOLDZIEHER

Department of Obstetrics and Gynecology, Texas Tech University Health Sciences Center, Amarillo, Texas 79106

IV. Sociocultural Perspectives References

I. Introduction II. Biological Considerations III. Psychologic Aspects

not many women arrived at the age of menopause. In the Middle Ages life was "solitary, nasty, brutish and short" [Thomas Hobbes], with average life expectancy around 35 yearsmalso not conducive to a population of menopausal women. At the beginning of the nineteenth century life expectancy of a girl child at birth was 36.5 years; if she survived to age 20, she could expect to live to age 56. By the end of that century, American women's life expectancy was up to 45 years. Now, near the end of the millennium, it is nearly 80 years. This implies a rapid growth, numerically and percentage-wise, of the menopausal population, as shown by successive demographic pyramids of the United States (Figs. 1-3). By the year 2050 it is estimated that there will be more than 70 million women over the age of 50 years in the United States. Paradoxically, the population explosion in the second half of the twentieth century, as exemplified by Mexico (Figs. 4 and 5), has decreased the proportion, but not the number of menopausal women. In any event, it is clear that the number of women reaching the age of adult-onset ovarian failure (climacteric/menopause) will become very large and will require an increasing social- and health-oriented commitment. Three perspectives must be considered in any overview of therapeutic intervention in menopausal women: the biological, the psychological, and the sociocultural.

I. I N T R O D U C T I O N In the wild, mammals are fertile until they die. However, when nonhuman primates are allowed to live in a protected environment to great age, they eventually show signs of progressive ovarian failure. This is most easily studied in the baboon, in which the female sex skin is a reliable indicator of ovarian hormone status. After about 16 years of age, female fertility decreases, cycles become irregular, evidence of inadequate progesterone production can be seen, and eventually an unstimulated sex skin supervenes. Similar cycle decay occurs in rhesus and cynomolgus monkeys in their 20s and 30s. Chimpanzees will cycle regularly until they are well into their 30s; after age 40 their cycles become increasingly sporadic and about a fourth have no ovarian activity by this time. The age of onset of ovarian failure is difficult to determine in the large apes (orangs and gorillas) for technical reasons. Fertility is a central issue in all human populations, as documented by fertility rites of one kind or another in every known culture. Declining fertility with age was undoubtedly recognized in hunter-gatherer groups if enough of the females survived to age 40 years and beyond. Aristotle (384322 BC) noted that menstruation ceased by age 40inconsiderably earlier than in present times. Thus, if life expectancy at birth was around 2 0 - 2 5 years in Greek and Roman times,

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Copyright 9 2000 by Academic Press. All rights of reproduction in any form reserved.

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JOSEPH W. GOLDZIEHER

FIGURE 1

II. B I O L O G I C A L

Population pyramid for the United States, 1980. From the U.S. Census Bureau, International Data Base [1].

CONSIDERATIONS

The permanent cessation of menses at the end of a period of declining fertility was always an important milestone in an aging woman's life. By inevitable recourse to the post hoc fallacy, every illness, malaise, and symptom that had no obvious explanation was attributed to this phenomenon. The herbal medicines that were used in olden times by midwives and other purveyors of traditional remedies are interestingly described by Greer [lb], although it is somewhat difficult to penetrate the fog of feminist rhetoric (some of today's alternative medicines are direct descendants of herbal folklore remedies ~). In later times, when "physicians" of one kind or another intervened, the outcomes were likely to be harmful if not fatal. The usual treatments were bleeding, purging and pukingmand the prescribing of mixtures that might include, among other things, salts of lead, arsenic, or mercury, herbs containing potent alkaloids, or various revolting biological products. The rationale was based on the

1Germany's Commission E reviewed data on safety and efficacy of more than 1400 herbal agents; only 8 were judged possibly effective for one or more menopausal complaints: balm (Melissa officinalis), black cohosh (Cimicifuga racemosa), chaste tree (Vitex agnus-castus), gingko (Gingko biloba), ginseng (Panax quinquefolius), passion flower (Passiflora incarnata), saint-john's-wort (Hypericum perforatum), and valerian. Agents that did not pass include agrimony, angelica, catnip, chamomile, damiana, dandelion, dong quai, fenugreek, gotu kola, hops, licorice root, life root, sage, and sarsaparilla (see Israel and Youngkin [lc]). There are many phytoestrogens, e.g., lignans, isoflavones, coumestans, and resorcylic acid lactones. Some have antiestrogenic effects as well. The soy estrogens, especially genistein, are under intensive investigation as partial estrogens ("selective estrogen receptor modulators").

idea that the menopausal woman no longer discharged excrementitious humors from the womb and that some form of removal, even the induction of "vicarious menstruation" (cupping, leeches, or venesection) was desirable. John Freind (1729) [2], a mathematician, historian, and physician, tried to squeeze menopausal symptomatology and management into a mathematical paradigm. In the end, he felt that women, "inasmuch as they heap up a great quantity of Humours by living continually at home and not being used to hard Labour or exposed to the Sun, should receive a discharge of this Fulness, as a remedy given by nature" (i.e., menstruation). However, he was optimistic about the ultimate outcome, for he felt that "no very bad symptoms happen in elderly women although the menses should be wanting." Menville de Ponsan [3] in 1840 believed that the symptoms of menopause that women experienced were the death throes of the womb; however, he held out the hope that "when the vital forces cease to conspire [sic] over the uterus, they will increase those of the spirit and the rest of the body. The critical age passed, women have the hope of a longer life than men, [and] their spirit acquires more precision, understanding and vivacity." Others, however, stressed menopausal women's vulnerability to nervous and mental, as well as physical, illness; the 1888 Surgeon-General's "Index Catalogue," at the subject of menopause, noted: "See also: insanity in women, and uterus (cancer of)." So much for the prescientific era. The biological aspects of menopause could not be addressed rationally until the hormonal nature and events of the reproductive cycle became known. Even then, there remained the problemmstill

CHAPTER 27 Historical Perspectives

399

FIGURE 2 Populationpyramid for the United States, 1990. From the U.S. Census Bureau, International Data Base [1].

not entirely s o l v e d m o f which perceived changes were dependent on the hormonal milieu and which were due to concomitant events in the process of "aging," whatever that ambiguous term might mean. The interaction of these two f o r c e s m h o r m o n a l and a g i n g - - c a n be extremely complex, even at the biological level. Postmenopausal osteoporosis responds to estrogens up to a p o i n t - - b u t in the very elderly, there appears to be an "osteoporosis of aging." It turns out that these elderly have a diminished ability to convert vitamin D precursors to calcitriol as well as increased resistance to the action of this vitamin/hormone. In the absence of enough calcitriol and adequate calcium absorption, relative

hyperparathyroidism ensues, leading to further osteoporosis. Thus, age-associated events interact intimately with hormonal events. Before World War I, research in reproductive physiology remained largely observational, concentrated on cyclical and seasonal changes in laboratory and domestic animals. However, as early as 1912, Europeans had prepared ovarian tissue extracts that had progestational and, eventually, estrogenic activity. In the remarkable decade of 1926 to 1936, all the steroid sex hormones were extracted from tissue or urine, their structures identified, and pure products made available, experimentally and clinically, for study of their functions. At the

FIGURE 3 Populationpyramid for the United States, 2025. From the U.S. Census Bureau, International Data Base [1].

400

JOSEPH W. GOLDZIEHER

FIGURE 4 Populationpyramid for Mexico, 1970. From the United Nations, 1996 Revision [la].

same time placental, pituitary, and urinary tropic hormones were identified and their functions demonstrated. Interestingly, these early biologists who prepared gonadal extracts with hormonal activity were acutely aware of their vast clinical potential, even to the extent of predicting their application to hormonal fertility controlmthis in 1921! [4] m w h i c h would not become a reality for another four decades [5]. In the ensuing years, details of the hypothalamic-pituitary-gonadal axis were explicated; reproductive physiology acquired a rational basis. Clinical application of these find-

ings was not far behind, but was limited by the scarcity and cost of hormonal materials. Estradiol and its derivatives were available only as injectables until the late 1930s, when the Germans synthesized orally active ethinyl estradiol and Dodds in England prepared a large series of stilbenes, of which the most active was diethylstilbestrol, given by him to science and medicine without a patent. Progestins presented a similar problem, because progesterone isolated from ox bile was a short-acting, unpleasant injectable, prohibitively expensive at $1000/g. In 1942 the former Parke-

FIGURE 5 Populationpyramid for Mexico, 1980. From the U.S. Census Bureau, International Data Base [1].

CHAPTER27 Historical Perspectives Davis chemist, Russell Marker, working independently in a tin-roofed garage in Mexico City, synthesized 2 kg of progesterone from a precursor he found in a readily available Mexican yam, approached a company called Syntex, and broke the Schering monopoly. Progesterone eventually sold for $80/g. However, therapeutic use of progestins did not become a reality until the chemists synthesized orally active anhydrohydroxyprogesterone (Pranone) and, later, 17acetoxyprogestins and progestational 19-nortestosterone derivatives. Clinicians were well aware, long before the sex hormones were identified, of the symptomatic consequences of ovariectomy in women of reproductive age, and of the similar but slower prodromal development of symptoms during the "climacteric." When replacement therapy became a reality, the situation was considered by some to be analogous to another deficiency s t a t e E h y p o t h y r o i d i s m ~ w h i c h could be managed quite satisfactorily by permanent thyroid administration. However, with estrogen replacement therapy (ERT) the general attitude was different. In a 1937 book entitled "Practical Endocrinology" [6], ERT was not even mentioned. In 1940 Shorr, on the other hand [7], felt that replacement therapy was appropriate but that the body would eventually have to adjust to the hypoestrogenic state and ERT could be discontinued. (This notion, undocumented except for vasomotor symptoms, is still to be found, more than a halfcentury later, in the Food and Drug Administration (FDA)mandated package literature.) Many physicians at this time still felt that menopause was characterized chiefly by disturbances of the nervous system and that, as these subsided, replacement therapy would no longer be needed. Most women, in turn, regarded menopause as a natural, possibly difficult stage to be got through, and were not in tune with the notion of a permanent hormonal deficiency state that needed lifelong replacement. Moreover, although they were very aware of the troublesome signs and symptoms of menopause, they were not then aware of its long-term dangers, and of the preventive-medicine aspects of ERT. Early on, a few gynecologists emphasized concomitant problems of menopause such as accelerated coronary disease, hypertension, and urogenital problems in addition to the well-recognized disorders of the autonomic nervous system. They also explored the potential of sex hormones in the treatment of a variety of gynecological disorders. Nevertheless, establishment medicine followed the precept-standard in other therapeutic s i t u a t i o n s ~ o f using as little medication as needed to treat symptoms and for as short a time as possible. This was the attitude of the American Medical Association's Council on Pharmacy well into the second half of the twentieth century until it was dissolved, and is still the formal position of the FDA. The concept of a deficiency state that needs replacement was not and still is not in vogue with the medical establishment. No one in the Council (or the FDA) apparently considered that there might

401 not be premonitory symptoms in the case of a potentially lethal hip fracture or first heart attack. Fuller Albright, the great Boston endocrinologist, had demonstrated as far back as 1941 [8] that hypogonad women decreased in height unless estrogens were given, in which case their stature remained stable. Despite this clear demonstration of the effect of estrogen deficiency on bone and the prevention of osteoporosis by ERT, it was to be another halfcentury before the profession began to address this disease seriously. A few gynecologists with extensive clinical experience bucked the hands-off trend. In 1966 a New York City specialist, Robert Wilson, published a popular book entitled "Feminine Forever" [9], in which he recommended lifelong replacement therapy for nearly all women, by analogy with hypothyroidism and other deficiency states. For these opinions, and for the temerity to pander his wares to the laity by way of a best-seller, he was summarily expelled from his local specialty society and vilified for years thereafter by his erstwhile colleagues. Nova ex veteris was not about to occur on their watch, if they could prevent it. The apathy of the medical profession is not difficult to understand. Endocrinologists, who knew the most about reproductive hormones and their physiology, had surrendered this field to the gynecologists. (Fuller Albright once remarked that the specialty of endocrinology specialized in handing off its discoveries to other specialties.) Few clinic i a n s - e v e n gynecologistsEwere familiar with the field of sex hormones, having had no formal training in this domain. Many of the symptoms they were asked to treat were subjective, and results were often difficult to distinguish from placebo effects. Prescribing and managing ERT or hormone replacement therapy (HRT) was time-consuming, unremunerative, and presumably endless. Even if only a small proportion of menopausal women were symptomatic and therefore required intervention, the numbers were still awesome. Clinicians' conservative philosophy also stemmed from concerns about estrogen-induced malignancy. In 1932 Lacassagne [10] in France reported that selected, susceptible strains of male mice (which incidentally happened to be infected with mouse mammary tumor virus) developed breast tumors on estrogen treatment. The fact that the virus (not present in humans) was essential, that other mouse strains were resistant to the tumorigenic effects of estrogen, and that removal of endogenous prolactin (by hypophysectomy) prevented tumor development altogether did not deter extrapolation to human females. In the light of additional animal research and the fact that epidemiological studies of human breast cancer showed statistical associations with various reproductive events, the estrogens-cause-cancer belief grew, fostered by media scare coverage and to a certain extent also by the vested interests of the cancer research/treatment community. This albatross hangs around the neck of hormone therapy to this day, even in the face of mounting evidence

402 that the incidence of breast cancer in the case of ERT, or in users of estrogen-containing contraceptive formulations and even after pregnancy in a woman who has had breast cancer, is little c h a n g e d m i f at a l l - - a n d that the benefits significantly outweigh the worst-case risk estimate of breast cancer increment. A similar concern, from time of the earliest availability of estrogen therapy, was the issue of endometrial cancer, again on the basis of animal experiments. Clinical studies were consistently negative until 1975, when Ziel and Finkle [11] and others reported an increased relative risk in long-term users of "unopposed" estrogens. This has been amply confirmed, as has the observation that risk can be completely avoided by the timely use of progestins. Despite the clearcut resolution of this issue, it continues to be a red herring repeatedly dragged out by the media, by regulatory agencies, and by others, with the inevitable effect of frightening away potential candidates for HRT. In the decades following Wilson's "Feminine Forever," experimental and epidemiological knowledge about estrogen deficiency and replacement therapy has increased vastly, especially with respect to bone and cardiovascular disease, breast and colon cancer, and autonomic and cognitive effects. What has been the consequence of this surfeit of information on physicians' prescribing habits and patients' compliance? Not very much. In 1990 Grisso et al. [ 12] studied the proportion of physicians who prescribed ERT for the majority of their postmenopausal patients. Regardless of venue (academic, health maintenance organization, or private practice), less than 6% of cardiologists did so, and the percentage among general internists, who often suggested calcium and exercise, was as low or lower; even among gynecologists, 43% or fewer were consistent prescribers. Women who specifically seek treatment for menopause (about 20% of American women) tend to get short shrift [ 13]. As late as 1994, a survey in England [14] showed that 56% of gynecologists and 65% of general practitioners considered ischemic heart disease to be a relative or absolute contraindication to ERT. A 1993 Finnish survey [15] also had found that 24% of nonspecialist physicians believed that HRT increased the risk of cardiovascular disease. With such prescribing practices in the Western world, it is not surprising that the 1981 - 1982 Massachusetts Women's Health Study [ 16] found, in a general population sample of women aged 45-55 years, that only 8.1% reported the use of hormones. Ravnikar in 1987 [17] reported that 2 0 - 3 0 % of patients never filled their prescription and 20% discontinued within 8 months. Brett and Madans [18] reported that only 20% of initial users continue for 5 years or more. Clearly, prescriber apathy still prevails and women certainly are not persuaded by their physicians of the efficacy, safety, and preventive-medicine aspects of HRT. In the deathless words of the comic-strip character Pogo, "We have seen the enemy and it is us."

JOSEPH W. GOLDZIEHER

III. PSYCHOLOGIC

ASPECTS

According to the 1990 Harvard/World Health Organization/World Bank study [ 19], of the major causes of disability in women of developed regions, four of the first five diagnoses are psychiatric: unipolar depression, dementia, schizophrenia, and bipolar disorder. In 1973 [20] a large longitudinal study found a notable deterioration in mental health in Swedish women nearing the menopause. A postal survey conducted in Oxford, England in 1980 yielded similar findings [21], as did a 1993 survey in the United States [22]. However, other studies have reported no increase in moderate or severe depressive symptoms with menopause [23]. Such statistical associations yield no information with regard to causality, and any effects directly attributable to hormone deficiencies might well be confounded by physiologic changes that accompany aging, poor nutrition, or lack of exercise. However, vasomotor symptoms may disrupt sleep, leading to chronic sleep deficiency, which is associated with irritable m o o d m a clear-cut chain of hormone-dependent events with psychologic consequences. The psychoanalytic school has addressed menopause and its treatment in various ways, starting with Deutsch [24], who in 1924 wrote, "Woman's last traumatic experience as a sexual being, the menopause, is under the aegis of an incurable (sic) narcissistic wound." Harris [25] points out that such traditional misogynistic Freudian views have been modified by ego psychology and object relations theory in later years. The psychoanalytic form of psychotherapy for menopause-associated problems has been largely replaced by novel psychotropic drugs and the neurochemistry of depression is rapidly being elucidated [26]. By 1987, a study by Ballinger [27] of menopausal Australian women concluded that, "physical vulnerability, brought on by hormonal changes, interacts with psychological vulnerability to life stress so that those with good coping skills are better able to override the effects of hormonal changes and so suffer fewer and less severe psychological symptoms." Investigations of the effects of hormonal intervention on psychological symptomatology have always been beset by technical problems, but studies, especially on cognitive function and mood, have expanded greatly since the 1970s [28,29] and promising publications on the effect of ERT on Alzheimer's disease are appearing.

IV. S O C I O C U L T U R A L

PERSPECTIVES

Any event as ubiquitous as menopause is necessarily embedded in a sociocultural context. Patriarchal societies determined the status of women, and menopause was dismissed as a natural event, although surrounded by a halo of traditional mythology. In the post-Renaissance West, menstrual

403

CHAPTER 27 Historical Perspectives p h e n o m e n a i n c l u d i n g m e n o p a u s e were simply not discussed in polite society, and w o m e n were often c h a r a c t e r i z e d as illness-prone, frail creatures (at least in the m i d d l e and u p p e r classes). W h e n their s y m p t o m a t o l o g y required intervention, this was likely to do m o r e h a r m than good, as m e n t i o n e d earlier. M e n o p a u s a l s y m p t o m a t o l o g y is by no m e a n s pervasive and h o m o g e n e o u s , but specific to distinct cultural or population groups, as n o t e d as early as 1857 [30] and 1897 [31] and also m o r e r e c e n t l y [32]. N e u g a r t e n et al. [33], G r e e n e [34], and M c Q u a i d e [35] have described the i m p a c t on midlife and m e n o p a u s a l p e r c e p t i o n s of sociocultural factors in various c o n t e m p o r a r y populations. Clearly, individual attitudes and e x p e c t a t i o n s are f o r m e d within a cultural milieu, and t h e s e m a s well as the p e r c e p t i o n s and prejudices of the potential t h e r a p i s t s m d e t e r m i n e the nature and level of intervention, if any. A f t e r W o r l d W a r II, and especially after the 1960s, the f e m i n i s t m o v e m e n t attained increasing p r o m i n e n c e in parts of W e s t e r n society. Paradoxically, it vigorously o p p o s e d h o r m o n a l i n t e r v e n t i o n of any kind, even opposing h o r m o n a l contraceptives [36], w h i c h in fact gave w o m e n m o r e control over their reproductive f u n c t i o n than any previous event in history. F e m i n i s t s were e q u a l l y opp o s e d to the " m e d i c a l i z a t i o n " of m e n o p a u s e , seeing it as an e n c r o a c h m e n t on w o m e n ' s a u t o n o m y by the m e d i c a l establ i s h m e n t (read e x p e n s i v e m a l e g y n e c o l o g i s t s and g r e e d y d r u g c o m p a n i e s ) . O v e r the past decade or so, the rhetoric has subsided s o m e w h a t , and it r e m a i n s to be seen w h e t h e r this special interest g r o u p has had any significant i m p a c t on public acceptance of the idea that the p h e n o m e n o n of m e n o pause implies i m p o r t a n t health and quality-of-life c o n c e r n s , f o u n d e d on b i o l o g i c a l as well as sociocultural factors. In the decade of the 1990s the ubiquity and cost of postm e n o p a u s a l o s t e o p o r o s i s b e c a m e r e c o g n i z e d , especially as n e w n o n h o r m o n a l c o m p o u n d s to prevent or treat this disorder were developed. This caused the p h a r m a c e u t i c a l i n d u s t r y to take an aggressive interest in p r o m o t i n g k n o w l e d g e of and n e w attitudes t o w a r d the c o n s e q u e n c e s of m e n o p a u s e . O f course, the bias of p h a r m a c e u t i c a l c o m p a n i e s is to i n f o r m the public about the p r o b l e m s to w h i c h their particular product is relevant, but in the process we are seeing, for the first time, a h i g h - i m p a c t activity that will create a public d e m a n d for the m e d i c a l c o m m u n i t y to p e r f o r m services that it has long neglected.

References 1. The United States Census Bureau, International Data Base. l a. The United Nations, 1996 Revision. lb. Greer, G. (1992). "The Change. Women, Aging and the Menopause," pp. 160-234. Knopf, New York. 1c. Israel, D., and Youngkin, E. Q. (1997). Herbal Therapies for perimenopausal and menopausal complaints. Pharmacotherapy 17, 970-984. 2. Freind, J. (1729). "Emmenologia" (T. Dale, transl.). Cox, London.

3. Menville de Ponsan, C. F. (1840). "The Critical Age of Women, the Maladies they can Undergo at This Stage of Their Life, and the Means to Combat or Prevent Them," p. 47. BailliOre, Paris. 4. Haberlandt, L. (1921). Uber hormonale sterilisierung des weiblichen Tierk6rpers. Muench. Med. Wochenschr. 68, 1577-1579. 5. Perone, N. (1994). The progestins. In "Pharmacology of the Contraceptive Steroids" (J. Goldzieher and K. Fotherby, eds.), pp. 5-20. Raven Press, New York. 6. Goldzieher, M. A. (1937). "Practical Endocrinology," 2nd ed. Appleton Century, New York. 7. Shorr, E. (1940). The menopause. Bull. N.Y. Acad. Med. [2] 16, 453474. 8. Albright, F., Smith, P. H., and Richardson, A. M. (1941). Postmenopausal osteoporosis: Its clinical features. J. Am. Med. Assoc. 116, 2465-2474. 9. Wilson, R. A. (1966). "Feminine Forever." M. Evans, New York. 10. Lacassagne, A. L. (1932). Apparition des cancers de la mammelle chez la souris m~le soumis a des injections de folliculine. C. R. Seances Soc. Biol. Ses. Fil. 195, 632-638. 11. Ziel, H. K., and Finkle, W. D. (1975). Increased risk to endometrial cancer among users of conjugated estrogens. N. Engl. J. Med. 293, 1167-1170.

12. Grisso, J. A., Baum, C. R., and Turner, B. J. (1990). What do physicians in practice do to prevent osteoporosis? J. Bone Miner. Res. 5, 213219. 13. Morse, C. A., Smith, A., Dennerstein, L., Green, A., Hopper, J., and Burger, H. (1994). The treatment-seeking woman at menopause. Maturitas 18, 161-173. 14. Norman, S. G., and Studd, J. W. W. (1994). A survey of views on hormone replacement therapy. Br. J. Obstet. Gynaecol. 101, 879887. 15. Hemminki, E., Topo, E, Malin, M., and Kangas, I. (1993). Physicians' views on hormone therapy around and after the menopause. Maturitas 16, 163-173. 16. Avis, N. E., Kaufert, E A., Lock, M., McKinlay, S. M., and Vass, K. (1993). The evolution of menopausal symptoms. Baillibre's Clin. Endocrinol. Metab. 7, 17-32. 17. Ravnikar, V. A. (1987). Compliance with hormone therapy. Am. J. Obstet. Gynecol. 156, 1332-1334. 18. Brett, K. M., and Madans, J. H. (1997). Use of postmenopausal hormone replacement therapy: Estimates from a nationally representative cohort study. Am. J. Epidemiol. 145, 536-545. 19. Anonymous (1996). "The Global Burden of Disease-- 1996," p. 236. Harvard, Cambridge University Press/WHO/World Bank, Cambridge, MA and Geneva. 20. Hallstrom, T. (1973). "Mental Disorder and Sexuality in the Climacteric: A Study in Psychiatric Epidemiology." Scandinavian University Books, Goteborg, Sweden. 21. Bungay, G. T., Vessey, M. E, and McPherson, C. K. (1980). Study of symptoms in middle life with special reference to the menopause. Br. Med. J. 281, 181-183. 22. Kessler, R. C., McGonagle, K. A., Swartz, M., Blazer, D. G., and Nelson, C. B. (1993). Sex and depression in the National Comorbidity Survey I: Lifetime prevalence, chronicity and recurrence. J. Affective Disord. 29, 85-96. 23. Pearlstein, T., Rosen, K., and Stone, A. B. (1997). Mood disorders and menopause. Endocrinol. Metab. Clin. North Am. 26, 279-294. 24. Deutsch, H. (1924). The menopause. Int. J. Psychoanal. 65, 55-62. 25. Harris, H. (1990). A critical view of three psychoanalytic positions on menopause. In "The Meanings of Menopause" (R. Formanek, ed.), pp. 65-78. Analytic Press, Hillsdale, NJ. 26. Owens, M. J., and Nemeroff, C. B. (1994). The role of serotonin in the pathophysiology of depression. Clin. Chem. (Winston-Salem, N.C.) 40, 288-295. 27. Ballinger, S. (1995). Psychosocial stress and symptoms of menopause:

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30. 31.

A comparative study of menopause clinic patients and non-patients. Maturitas 7, 315-327. Henderson, V. W., Watt, L., and Buckwalter, J. G. (1996). Cognitive skills associated with estrogen replacement in women with Alzheimer's disease. Psychoneuroendocrinology 21, 421- 430. Haskell, S. G., Richardson, E. D., and Horwitz, R. I. (1997). The effect of estrogen replacement therapy on cognitive function in women: A critical review of the literature. J. Clin. Epidemiol. 50, 1249-1264. Tilt, E. J. (1857). "The Change of Life in Health and Disease." Churchill, London. Currier, A. F. (1897). "The Menopause." Appleton, New York.

JOSEPH W. GOLDZIEHER 32. Greendale, G. A., and Sowers, M. (1997). The menopause transition. Endocrinol. Metab. Clin. North Am. 26, 264-277. 33. Neugarten, B., Wood, V., Krainer, R., and Loomis, B. (1968). Women's attitudes toward the menopause. In "Middle Age and Aging," (B. Neugarten, ed.), pp. 193-200. University of Chicago Press, Chicago. 34. Greene, J. G. (1990). Psychosocial influences and life events at the time of menopause. In "The Meanings of Menopause" (R. Formanek, ed.), pp. 79-115. Analytic Press, Hillsdale, NJ. 35. McQuaid, S. (1998). Women at midlife. Soc. Work 43, 21-31. 36. Seaman, B. (1969). "The Doctors' Case Against the Pill." P. H. Wyden, New York.

~ H A P T E R 2l

Prevention Trials in

Perimenopausal and Postmenopausal Women JENNIFER KELSEY Division of Epidemiology, Department of Health Research & Policy, Stanford University School of Medicine, Stanford, California 94305

ROBERT MARCUS Department of Medicine, Stanford University School of Medicine, Geriatrics Research, Education & Clinical Center, Veterans Affairs Medical Center, Palo Alto, California 94304

I. II. III. IV. V. VI. VII.

Introduction Hormone Replacement Therapy: Background The Postmenopausal Estrogen/Progestin Trial The Women's Health Initiative Heart Estrogen/Progestin Replacement Study The Women's Health Study The Breast Cancer Prevention Trial

VIII. Recent Trials of Agents That Reduce Bone Loss and Fracture Incidence IX. Study of Tamoxifen and Raloxifene X. Conclusion References

I. I N T R O D U C T I O N

recurrence of coronary heart disease; and the Women's Health Study (WHS), a study of aspirin, fl-carotene, and vitamin E as protective agents against coronary heart disease and cancer. Although the Breast Cancer Prevention Trial (BCPT) study of tamoxifen to reduce the incidence of breast cancer was undertaken in women over a relatively wide age range (35-74 years), we also discuss this trial because it has relevance to perimenopausal and postmenopausal women. Finally, we describe trials of recently developed agents designed to prevent or treat osteoporosis, including alendronate, which is a bisphosphonate, and raloxifene, the first selective estrogen receptor modulator (SERM) to be approved by the United States Food and Drug Administration (FDA) for osteoporosis prevention. We begin with a discussion of what has previously been learned about risks and benefits of hormone replacement therapy and why it was felt that randomized trials were needed to determine its long-term risks and

In recent years several large intervention trials have been undertaken to evaluate ways of reducing disease occurrence among perimenopausal and postmenopausal women. This chapter describes and critically reviews several of the major randomized prevention trials that are currently being undertaken or were recently completed in the United States. Included in this discussion are the Postmenopausal Estrogen/Progestin Interventions (PEPI) trial, a study of the effects of several hormone replacement regimens on predictors of coronary heart disease; the Women's Health Initiative (WHI), a study of the long-term effects of a low-fat dietary pattern, hormone replacement therapy, and calcium/ vitamin D supplementation on the incidence of several diseases; the Heart Estrogen/progestin Replacement Study (HERS), a study of hormone replacement therapy to prevent MENOPAUSE:

BIOLOGY AND PATHOBIOLOGY

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Copyright 9 2000 by Academic Press. All rights of reproduction in any form reserved.

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KELSEY AND MARCUS

benefits. This chapter is in part adapted from other material on intervention trials in women [ 1,2].

II. H O R M O N E THERAPY:

REPLACEMENT

BACKGROUND

Conjugated estrogens, consisting of a mixture of active estrogens from the urine of pregnant horses, were first approved for marketing in the United States in 1942, under the provisions of the Food, Drug, and Cosmetics Act of 1938. They were first approved because they fulfilled the requirement of being safe for their intended use in the treatment of menopausal symptoms, vaginitis, and amenorrhea. Starting in 1962, it was required that efficacy as well as safety be demonstrated. The FDA found a group of estrogen products to be "probably effective" for selected cases of osteoporosis. In 1986, after the publication of two randomized trials [3,4], the FDA upgraded the status of estrogen to "effective" for the treatment of postmenopausal osteoporosis. Although some of the trials described in this chapter emphasize potential protective effects of hormone replacement against cardiovascular disease, the FDA has not approved estrogen use for this purpose. Manufacturers of hormone replacement therapy may not discuss or distribute promotional materials regarding protective effects against cardiovascular disease. The FDA has taken the position that evidence supporting cardioprotective effects is not sufficient for approval. Hormone replacement therapy began to be widely used in the United States during the 1950s. In addition to its beneficial effect on menopausal symptoms, it was believed by some that estrogen would permit women to be forever young. Most women who received estrogen were prescribed conjugated estrogens (Premarin). Until 1992, Premarin was the only oral estrogen approved by the FDA as a hormone replacement agent. Use of oral estrogen increased greatly from the early 1960s to the mid-1970s [5]. It was generally used for relatively short periods of time to counteract symptoms of vasomotor instability. In the mid-1970s, after evidence strongly indicated that continuous administration of estrogen was associated with an increased risk for endometrial hyperplasia and endometrial cancer [6,7], prescriptions for estrogen declined sharply for about 5 years. Then, use increased again [5,8,9] as strategies were introduced to protect the uterus while providing sufficient estrogen. To protect the uterus against hyperplasia and cancer, progestin, most often medroxyprogesterone acetate, was added to the estrogen, either as part of an intermittent cyclic regimen (e.g., 5 - 1 0 mg/day for 12 days each month) or as a continuous regimen (e.g., 2.5 mg/day without interruption). Prescriptions for medroxyprogesterone acetate increased about fivefold between 1982 and 1992 [9].

During recent years research on long-term effects of estrogen replacement has focused on three areas: use of estrogen to prevent or retard loss of bone mass and thereby to reduce the risk for osteoporotic fractures, the possible adverse effect of estrogen on breast cancer risk, and the possible protective effect of estrogen against coronary heart disease. The protective effect of estrogen replacement on loss of bone mass is well established, and the addition of progestin does not reduce the beneficial effect of estrogen alone on bone mass [ 10,11]. The effect of estrogen on breast cancer risk has been less firmly established, although a recent metaanalysis suggests an elevation in risk with increasing length of use [12]. A relative risk of 1.35 for breast cancer was estimated for women who have used estrogen for 5 years or more. The risk in women who used progestin along with estrogen was just slightly greater than the risk in those using estrogen alone, but the number of women using the combination therapy was not large enough to be sure. Observational epidemiologic studies have indicated a substantial protective effect of estrogen replacement on the risk for coronary heart disease morbidity and mortality [13]. Estrogen appears both to produce beneficial changes in circulating lipoproteins known to affect coronary heart disease [14-16] and to decrease fibrinogen concentrations [17,18]. In regard to serum lipids, estrogen replacement increases the concentrations of high-density lipoprotein (HDL) cholesterol, which is an important predictor of coronary heart disease in women, and lowers those of low-density lipoprotein (LDL) cholesterol, which is a predictor of some importance in women. The changes in HDL cholesterol may account for half of the overall effect of estrogen in reducing cardiovascular disease mortality [16]. Some evidence suggested that progestins suppress the beneficial changes of estrogen alone on HDL cholesterol [15,19,20]. Therefore, when it became part of clinical practice to add progestins to the estrogen for protection of the uterus, there was concern that this modification might negate the beneficial effects of estrogen alone. This concern was important to the development of the PEPI trial.

III. THE POSTMENOPAUSAL ESTROGEN/PROGESTIN

TRIAL

The PEPI trial was designed to examine the effects of unopposed estrogen and three estrogen/progestin regimens on various coronary heart disease risk factors in women of ages 4 5 - 6 4 years. The risk factors included HDL cholesterol, fibrinogen, insulin, and blood pressure [21]. When the idea of such a trial was first proposed in the mid-1980s, a trial with coronary events or mortality as endpoints was considered too expensive. Therefore, it was decided to use predictors of coronary heart disease, rather than coronary heart disease it-

CHAPTER28 Prevention Trials

407

self, as endpoints. The selected endpoints were generally accepted as being independent predictors of coronary heart disease, and the relationships between their levels and risk of coronary heart disease were well established. The trial was initiated by the National Institutes of Health. The coordinating center was at the Bowman Gray School of Medicine. Seven clinical centers randomized 875 women, stratified by clinical center and hysterectomy status, from December 1989 to February 1991, to one of the following five groups: (1) placebo, (2) conjugated equine estrogen, 0.625 mg/day, (3) conjugated equine estrogen, 0.625 mg/day plus medroxyprogesterone acetate, 10 mg/day for the first 12 days of the 28-day cycle, (4) conjugated equine estrogen, 0.625 mg/day plus medroxyprogesterone acetate, 2.5 mg/day, and (5) conjugated equine estrogen, 0.625 rag/day plus micronized progesterone, 200 mg/day for the first 12 days of each 28-day cycle. Women were to be seen at 3-, 6-, and 12-month intervals after randomization during the first year, and every 6 months for the next 2 years, for a total of 3 years. Each year, physical examination, mammography, and endometrial biopsy were performed. The results were presented by the intention-to-treat principle, whereby women were considered to be in the group to which they had been randomly assigned, regardless of their compliance with that regimen. Results for adherent women only were quite similar, a finding to be expected in view of the high degree of adherence among women in the PEPI trial [22]. Overall, about 80% of hysterectomized women and 75% of women with an intact uterus had pill counts exceeding 80% compliance at their 36-month visit. Table I shows that during the 3 years of the trial, HDL cholesterol decreased slightly in the placebo group (decrease of 1.2 mg/dl), increased in the two groups assigned to take estrogen plus medroxyprogesterone acetate (increases of 1.2

TABLE I

to 1.6 mg/dl), and increased more in the groups assigned to take conjugated estrogen plus cyclic micronized progesterone (increase of 4.1 mg/dl) and conjugated estrogen alone (increase of 5.6 mg/dl). Compared with placebo, all the treatment groups on average experienced decreases in mean LDL cholesterol and increases in triglyceride. Mean fibrinogen concentration increased to a greater extent in the placebo group than in any of the active treatment groups, but the magnitude of the changes did not differ significantly among the active treatment groups. Although not included in the table, systolic blood pressure increased and postchallenge insulin concentrations decreased during the 3 years in all groups; the extent of change did not differ by group. Compared with all other groups, unopposed estrogen was associated with a greatly increased frequency of development of adenomatous or atypical hyperplasia (34 versus 1%) and of hysterectomy (6 versus 1%). Results regarding HDL cholesterol are of particular interest, because some evidence suggests that HDL cholesterol is an especially important predictor of coronary heart disease in women. Because women randomized to estrogen alone or to estrogen in combination with micronized progesterone had the largest increases in HDL cholesterol, these compounds would be expected to have the most favorable effect on incidence of coronary heart disease. However, the high proportion of women developing endometrial hyperplasia among those using estrogen alone dampens enthusiasm for this regimen. Thus, in this trial conjugated estrogen with cyclic micronized progesterone had the most favorable effect on HDL cholesterol among the regimens that were not associated with an increased risk for endometrial hyperplasia. However, the long-term effects of this agent have not been well studied. The PEPI trial is also providing information on other end-

PEPI Trial: Mean Changes for Selected Outcome Measures by Treatment Group a Treatment group

Outcome measure

Placebo

HDL cholesterol (mg/dl) - 1.2 (-2.2,-0.2) b LDL cholesterol (mg/dl) -4.1 (-6.5, -1.8) Triglycerides (mg/dl) -3.2 (-7.2, 0.7) Fibinogen (g/liter) 0.10 (0.04, 0.16)

CEE only

CEE MPA (cyclic)

CEE MPA (continuous)

CEE MP (cyclic)

5.6(4.5,6.7)

1.6(0.5,2.7)

1.2(0.1,2.2)

4.1 (3.1,5.1)

-14.5 (-16.8, -12.1)

-17.7 (-20.1, -15.4)

-16.5 (-18.8, -14.2)

-14.8 (-17.0, -12.5)

13.7 (9.3, 18.0)

12.7 (8.5, 16.8)

11.4 (7.0, 15.9)

13.4 (9.1, 17.7)

-0.02 (-0.08, 0.04)

0.06 (0.00, 0.12)

0.01 (-0.04, 0.07)

0.01 (-0.04, 0.07)

a Mean changes expressed as differencebetween average of all follow-up data and average of all baseline data; table adapted from the Writing Group for the PEPI trial [21]. /'Values in parentheses are 95% confidence intervals; CEE, conjugated equine estrogen; MPA, medroxyprogesterone acetate; MR micronized progesterone.

408 points. It clearly showed that, compared to placebo, all the active treatment regimens had a favorable effect on bone mineral density in the lumbar spine and hip over the 3-year period of the trial [11]. Women in all treatment groups gained weight, but gain in mean weight, and also in waist and hip girth, was greatest in women assigned to placebo and least in those taking unopposed estrogen. The magnitude of the difference, however, was only about 1 kg over the 3-year period [23]. Future reports from PEPI will focus on physical, cognitive, and affective symptoms, on mammographic parenchymal density, carbohydrate metabolism, and other outcomes of interest. Much useful information has been gained from the PEPI trial. Because women were randomized to the various treatment groups, this study adds substantially to the body of evidence from observational studies indicating that hormone replacement therapy reduces the risk of coronary heart disease and that the association does not occur solely because healthier people tend to use hormone replacement. The magnitudes of the changes in HDL cholesterol levels associated with use of estrogen alone or estrogen with micronized progesterone suggest that the risk for coronary heart disease would be reduced by 2 0 - 2 5 % among women using these compounds. The favorable effect on fibrinogen concentrations with or without the addition of progestin to estrogen has been confirmed in a more recent study [24], and would be expected to be associated with a further substantial decrease in risk for coronary heart disease. The increase in HDL cholesterol and decrease in circulating fibrinogen compared to placebo suggest that estrogen has both a long-term beneficial effect and an immediate effect, in turn indicating that the greatest degree of protection will be achieved in long-term current users. However, the HERS trial, described later in this chapter, raises the possibility of a short-term increase in risk for a coronary event on initiation of an estrogen/progestin regimen, followed by a longer term protective effect. Thus, some uncertainty remains. Moreover, it should be noted that the finding of a 10% per year incidence of adenomatous or atypical endometrial hyperplasia among women in the estrogen-alone group provides strong evidence that women taking this regimen should be under annual endometrial surveillance. Limitations of the PEPI trial include its use of predictors of coronary heart disease as endpoints, rather than coronary heart disease itself, and its relatively short period of followup. Accordingly, this trial allows only rough estimates of the expected magnitude of the decreased risk for coronary heart disease incidence or mortality among women using the various regimens. Premarin was the only type of estrogen used in this trial; the effects of other types of estrogens or of estrogens administered transdermally cannot be evaluated from this study. In addition, the trial had small numbers of racial and ethnic minorities, so that the results cannot necessarily be generalized to them. Thus, although the PEPI trial was

KELSEY AND MARCUS

considerably less expensive than larger trials (described below) and provided much useful information, it leaves unanswered some key questions that can only be properly addressed in larger trials of longer duration.

IV. T H E W O M E N ' S HEALTH

INITIATIVE A. I n t r o d u c t i o n

The overall goals of the WHI are to test methods of reducing the risk for cardiovascular disease, breast cancer, colorectal cancer, and osteoporotic fractures in women. Many other health outcomes will be monitored as well. It is the largest and most expensive research study ever funded by the National Institutes of Health, with a budget of $628,000,000 over the 15-year period 1992-2007. It includes a randomized trial of about 64,500 women and an observational study of about 100,000 women in the age range 5 0 - 7 9 years, of diverse race/ethnicity and socioeconomic status [25]. Only the randomized trial component of the Women's Health Initiative is considered here. The three branches of the randomized trial are designed to test hypotheses concerning the effects on disease incidence of (a) dietary modification, (b) hormone replacement, and (c) calcium/Vitamin D (see Table II). Women may participate in one, two, or three of the branches of the trial. The large number of enrollees is needed primarily to test the hypothesis that dietary modification can reduce the incidence of breast cancer. The WHI was originally proposed in 1991 by the then Director-designate of the National Institutes of Health, Bernadette Heeley. The concept of such a trial received a great deal of support from the Congressional Caucus on Women's Issues, and the study is funded by the United States Congress as a separate line item in the budget of the National Institutes of Health. The coordinating center is at the University of Washington. Recruitment began in 16 Vanguard Clinical Centers if the fall of 1993. Twenty-four additional clinical centers were added in early 1995 [25-27]. Recruitment was completed in the spring of 1998.

B. D i e t a r y M o d i f i c a t i o n B r a n c h The dietary modification branch of the WHI has the primary aim of testing the hypothesis that a low-fat eating pattern reduces the risks of breast cancer and colon cancer. A secondary hypothesis is that a low-fat eating pattern reduces the risk of coronary heart disease. Evidence that a diet high in fat increases the risk for breast cancer is derived mostly from comparisons of breast cancer incidence rates in countries with various levels of per

CHAPTER 28 Prevention Trials TABLE II

409

Women's Health Initiative: Expected Number of Women Randomized to Various Components a Hormone replacement therapy branch b

Total

PERT

Control

ERT

Control

Not in hormone Replacement branch

19,200 28,800

1210 1815

1210 1815

990 1485

990 1485

14,800 22,200

16,500

4538

4538

3712

3712

64,500

7563

7563

6187

6187

Intact uterus Dietary modification branch Intervention Control Not in dietary modification branch Total

48,000

No uterus

37,000

27300 aln each cell it is expected that 70% of women will participate in the calcium/vitamin D branch, for an estimated total of about 45,000 women. Table adapted from the Women's Health Initiative Study Group [25]. b Abbreviations: PERT, progestin/estrogen replacement therapy; ERT, estrogen replacement therapy.

capita fat consumption, studies of migrants from one country to another, and animal studies. Epidemiologic case-control studies at most suggest a weak association between fat consumption and risk of breast cancer, and most cohort studies show no association. In fact, a recent meta-analysis of cohort studies of the relationship between dietary fat intake during adulthood and breast cancer found no association [28]. Also, it has been hypothesized that if a high-fat diet is involved in the etiology of breast cancer, it may be fat intake early in life rather than in adulthood that is important [29]. On the other hand, evidence is strong that people who have diets high in meat, protein, and fat have an elevated risk of colon cancer, and that persons with diets high in vegetables and fibers are at reduced risk [30]. It is uncertain which of these foods are actually of etiologic importance, because people who eat large amounts of meat, protein, and fat tend to eat relatively small quantities of vegetables and fibers. Nonetheless, most epidemiologists agree that this general dietary pattern is related to colon cancer risk. It is generally accepted that a diet low in fat, saturated fat, and cholesterol lowers total cholesterol and LDL cholesterol, both of which are risk factors for coronary heart disease. A diet low in fat may also reduce HDL cholesterol [31]. Because, as mentioned above, a high HDL cholesterol level protects against coronary heart disease, especially in women [32,33], the net effect of the low-fat dietary pattern on coronary heart disease risk in women is not entirely clear. Some 48,000 women are enrolled in the dietary modification branch of the trial. Of these, 40% are in the intervention group and 60% are in the control group. It is intended that women in the intervention group attain a diet with (1) total fat intake of no more than 20% of daily calories, (2) saturated fat intake less than 7% of daily calories, (3) at least five daily servings of fruits and vegetables, and (4) at least six daily servings of grain products. In addition, women in both the intervention and control groups are being given a stan-

dard packet of health promotion materials, including information on a healthy diet. Group meetings with a nutritionist, self-monitoring tools, and one individual dietary counseling session are also being used to try to help the women in the intervention group to change to and maintain a low-fat eating pattern. The average intervention period is expected to be 9 years.

C. H o r m o n e R e p l a c e m e n t B r a n c h When the WHI began, the primary aim of the hormone replacement branch was to test the hypotheses that estrogen replacement therapy with progestin, and estrogen replacement therapy alone, reduce the risk of coronary heart disease. A secondary hypothesis was that these same regimens reduce the risk of osteoporotic fractures. The incidence of breast cancer and endometrial cancer are being monitored during and after the trial. It was mentioned previously that almost all observational epidemiologic studies find that estrogen replacement therapy reduces the risk of coronary heart disease. However, there is concern that a tendency for healthier women to use hormone replacement therapy [34,35] may make it appear that the beneficial effect is greater than it really is. When the WHI began, results from the PEPI trial had not been published, but small randomized trials had shown that estrogen replacement therapy affects HDL cholesterol and LDL cholesterol levels such that a reduction in risk for coronary heart disease would be expected [ 14,15]. The effects of progestin and estrogen replacement together are less certain, because this combination of hormones has not been used for as long a period of time. However, it seems clear that progestin and estrogen therapy together decrease loss of bone mass at least to the same extent as estrogen alone [11]. The beneficial effects of a combined progestin and estrogen regimen on

410 coronary heart disease may be less than those of estrogen alone [19,20]. As mentioned above, results of studies concerned with the effect of progestin and estrogen replacement therapy of risk of breast cancer have been inconclusive. Some 27,500 women are enrolled in the hormone replacement branch. Women without a uterus were randomized to either (1) conjugated equine estrogen alone (0.625 mg/day) or (2) placebo. At the beginning of enrollment, women with a uterus were randomized to one of three groups: (1) conjugated equine estrogen alone (0.625 mg/day), (2) conjugated equine estrogen (0.625 mg/day) plus continuous low-dose progestin (2.5 mg/day), and (3) placebo. However, when results of the PEPI trial were published showing that a relatively high proportion of the women taking estrogen alone had to be taken off it because of endometrial bleeding, the women in this group were switched to the estrogen plus progestin group. WHI investigators have been working closely with the external Data and Safety Monitoring Board to develop algorithms that take into account risks and benefits and to consider earlystopping criteria and trial-reporting procedures. The results of the hormone replacement branch of the WHI are likely to be of some use. Obtaining information on long-term risks and benefits of estrogen plus continuous low-dose progestin among women of various ages and racial/ethnic groups will be important for women considering using this regimen. Valuable data on adverse side effects such as uterine bleeding in women of various ages and on possible beneficial effects such as on memory will also be important. Nevertheless, several questions about its design have arisen. One major question is why this trial was started before the results from the PEPI trial were available, because the findings from the PEPI trial brought about a major change in the hormone replacement branch shortly after it began. Second, it is unfortunate that only one regimen, conjugated estrogen with continuous low-dose progestin, is being tested in the women with intact uteri, and another single regimen, conjugated estrogen, in the women without a uterus. Even at the time the trial started there were many questions about the optimal dose of progestin and about other forms of estrogen. Now, just a few years after the WHI began, there are many more options available to women regarding doses, modes of administration, and forms of estrogen and progestin. In addition, the first selective estrogen receptor modulator, raloxifene, has now been approved by the FDA, and more are expected to be approved over the next few years. The hormone replacement branch will provide no information about these other replacement regimens, and this trial will provide little information to assist women in deciding which of many possible regimens are most likely to be optimal for them. A third problem is that if the primary aim is indeed to determine whether hormone replacement therapy affects the incidence of coronary heart disease, it would seem much more cost-effective if women at low risk for coronary

KELSEY AND MARCUS

heart disease had not been included in this branch. Finally, there has been concern that as new information about risks and benefits of replacement therapy is reported from other on-going studies, women who have been randomized may wish to switch from their assigned treatment. To date, this has not proved to be a major problem, but as time goes on and women become aware of the large number of options available to them, this could become a problem.

D. C a l c i u m / V i t a m i n

D Supplementation Branch

The primary aim of the calcium/vitamin D supplementation branch is to test the hypothesis that supplemental calcium and vitamin D reduce the risk of hip fracture. Secondary aims are to test the hypothesis that this supplementation reduces the risk of other fractures and of colorectal cancer. At three clinical centers, changes in bone mass in the hip and spine will also be monitored. Most randomized trials indicate that use of calcium supplementation somewhat retards loss of bone mass [36]. Bone mass is a moderately strong predictor of hip fracture. Results from observational studies are inconsistent as to whether calcium intake affects hip fracture risk. However, a randomized trial in frail older women in France [37] showed a substantial reduction in fracture incidence among elderly women taking supplemental calcium with vitamin D. Also, Recker et al. [38] found that calcium supplementation reduced the incidence of vertebral fractures in older women who already had at least one vertebral fracture. Available evidence does not permit a firm conclusion to be drawn as to whether the addition of vitamin D to supplemental calcium increases the protective effect of calcium alone against bone loss. Evidence is inconsistent as to whether supplemental calcium and/or vitamin D protects against colorectal cancer [30]. Participants in the dietary modification and hormone replacement branches are invited at their 1-year anniversary to enroll in the calcium/vitamin D branch as well. It is expected that 45,000 women will participate. Half of the participants are randomized to a regimen of calcium carbonate plus vitamin D and half are randomized to placebo. This branch of the trial is the least costly and probably the least controversial. It is unlikely that these supplements will have adverse effects, and this component of the trial may be able to increase knowledge about the long-term effects of this supplementation on fracture risk of women of different ages. Also, it may be able to address the question of whether simultaneous hormone replacement therapy enhances any effect of calcium/vitamin D. Because there is some interest in the hypothesis that vitamin D protects against certain cancers, including breast cancer, this component of the WHI should be able to shed some light on that issue as well. It is unfortunate, however, that this trial was not designed to com-

CHAPTER28 Prevention Trials

41 1

pare the effect on fractures of calcium supplementation alone compared to calcium supplementation plus vitamin D. This is likely to remain an important unanswered question.

V. HEART ESTROGEN/PROGESTIN REPLACEMENT STUDY The primary objective of HERS was to determine whether hormone replacement therapy is associated with a reduced risk of fatal coronary heart disease and nonfatal and fatal myocardial infarction among women with an intact uterus who have already been diagnosed with coronary heart disease [39]. Secondary objectives were to examine the effects of hormone replacement therapy on other outcomes among women with diagnosed coronary heart disease. Among the other outcomes of interest were other cardiovascular changes such as venous thromboembolic events, and cancer, osteoporotic fractures, uterine bleeding, and symptomatic side effects. The coordinating center was at the University of California, San Francisco. The study was funded by W y e t h Ayerst pharmaceutical company. HERS was a double-blind randomized trial that enrolled 2763 women younger than 80 years of age during the 18month period from February, 1993 to September, 1994. The women were recruited form coronary care units, catherization laboratory data bases, generalized mailing to women in targeted age groups, and advertisements. The women were randomized in equal numbers either to a daily pill containing 0.625 mg conjugated estrogen plus 2.5 mg medroxyprogesterone acetate or to placebo. Eighteen centers in the United States participated. Annual follow-up, which lasted for 5 years, included cardiovascular and gynecologic examinations, electrocardiogram, mammography, and a questionnaire concerned with various symptoms [40]. The trial ended in 1998. The study found that during the average follow-up period

TABLE I I I

HERS: Relative Risk of a Primary Coronary Heart Disease Event a

Year since randomization

Relative risk

95% confidence interval

1

1.52

1.01-2.29

2

1.00

0.67-1.49

3

0.87

0.55-1.37

4 and 5

0.67

0.43-1.04

Total

0.99

0.80-1.22

a Includes nonfatal myocardial infarction and coronary heart disease death. Relative risk is the risk in those assigned to estrogen/progestin replacement therapy relative to those assigned to placebo. Adapted from Hulley et al., [41 ].

of 4.1 years, this estrogen/progestin replacement therapy did not affect the likelihood of coronary heart disease events (relative risk of 0.99) [41 ] (Table III). During the first year of the trial, the rate of coronary events was higher in the group taking the hormone replacement therapy, whereas in later years of the trial the rate was lower in the women taking replacement therapy. Thus, the results of this trial are somewhat different from those of observational studies, almost all of which have shown protection against coronary heart disease from hormone replacement therapy. One reason for the discrepant results could be selection bias, that is, the decreased risk for coronary heart disease found in the observational studies could be attributable to the tendency of healthier women who are at lower risk for coronary heart disease to be more likely to use hormone replacement compared to women at higher risk. However, there are other plausible explanations. HERS did indicate protection after 2 or more years of use, and in fact the relative risk decreased with time in a linear fashion after the first few months of the trial. The HERS investigators hypothesize that there might be an immediate prothrombotic, proarrhythmic, or proischemic effect of the hormone regimen followed by a long-term beneficial effect, possibly as a result of beneficial effects on lipoproteins. Most observational studies have not considered the question of whether there is an immediate short-term increase in risk on initiation of use. Also, most observational studies have included younger, healthy women, and it is uncertain that the results of this study pertain to such women. PEPI indicated that the addition of medroxyprogesterone acetate to estrogen reduces the beneficial effect of estrogen alone, so that the results of HERS may pertain only to the particular regimen used in this trial. The lipoprotein changes achieved in HERS (8% rise in HDL cholesterol, 14% decrease in LDL cholesterol), however, were similar to those seen with estrogen alone in PEPI. At this time, it would seem prudent for women with coronary heart disease not to initiate hormone replacement therapy until additional data are available from other randomized trials. Only sketchy data on fracture incidence are available from HERS, but no significant difference in fracture incidence was observed between the estrogen/progestin and placebo arms. It must be noted that routine spine radiographs were not taken in this trial, so no information is available concerning vertebral compression fracture. In addition, the study participants were not preselected to be at a particularly high risk for fracture, so the study may have had low power to detect a difference. Nevertheless, the failure to observe even a trend toward skeletal protection after several years of hormone replacement therapy is surprising and disappointing. Finally, the women on hormone replacement therapy had increased risks for thromboembolic events and gallbladder disease. Observational studies have reported increased risks

412

KELSEY AND MARCUS

for idiopathic thromboembolism with both estrogen replacement therapy [42-45] and estrogen plus progestin replacement therapy [43,45]. The association with gallbladder disease is plausible because estrogen has been shown to increase biliary cholesterol content [46]. The excess incidence of thromboembolic events in HERS was 4.1 per 1000 womanyears, which is higher than the rate indicated by recent observational epidemiologic studies. The authors attributed this higher rate to the older age and greater number of risk factors for thrombotic events in the HERS participants. These probable risks for thromboembolism and gallbladder disease are additional considerations when a woman is trying to decide whether to use hormone replacement therapy.

VI. THE WOMEN'S

HEALTH

STUDY

The WHS was designed to be a randomized trial of the benefits and risks of low-dose aspirin, fl-carotene, and vitamin E on cardiovascular disease and cancer [47,48]. It was initiated by investigators at Harvard University and is funded by the National Institutes of Health. Considerable evidence suggests that low-dose-aspirin reduces the risk of coronary heart disease [48]. Low-dose aspirin reduces the tendency of platelets to aggregate, thus decreasing the likelihood that clots or thrombi will form. Randomized trials in both men and women have shown that aspirin reduces the risk for myocardial infarction, stroke, and vascular death among patients with cardiovascular disease. In 1982 the Physicians' Health Study, a large randomized double-blind placebo-controlled trial of the primary prevention of cardiovascular disease in apparently healthy male physicians, was initiated to test the effects of aspirin and/3carotene on risk for cancer and cardiovascular disease. In 1988 the aspirin component of this trial was stopped prematurely because a substantially reduced risk of a first myocardial infarction had been noted among those assigned to take aspirin [49]. However, the mortality rate from all cardiovascular causes was very similar among those assigned aspirin and those not assigned aspirin, and the rate of moderate to severe or fatal hemorrhagic stroke was higher among the aspirin users, although this latter finding was based on small numbers. Thus, decisions about aspirin use even among men are not entirely clear-cut. There were several reasons for initiating a study of aspirin among women [48]. Because the Physicians' Health Study included only men, any recommendations regarding aspirin use in women had to be extrapolated from men. Results from observational epidemiologic studies of the risk for cardiovascular disease among female aspirin users have been inconsistent. In addition, although women have lower rates of myocardial infarction than do men, the rates of stroke are

similar. Thus, the net balance of risks and benefits could be different in women and men. Various epidemiologic and laboratory studies have suggested that antioxidants such as fl-carotene and vitamin E may reduce the incidence of cardiovascular disease and cancer [50,51]. It has been hypothesized that antioxidant vitamins scavenge free radicals and excited oxygen molecules, thus preventing damage to DNA that can lead to cancer. Regarding cardiovascular disease, it has been hypothesized that antioxidant vitamins may inhibit the oxidation of LDL cholesterol into an especially atherogenic form and that these vitamins also help preserve endothelial function [52]. Foods such as fruits and vegetables that are high in fl-carotene and vitamin E also have relatively high levels of other substances that might inhibit cancer. Thus, whether supplementation with fl-carotene and vitamin E (as opposed to other constituents of fruits and vegetables either singly or in combination) will reduce the risk for cancer and cardiovascular disease is not known. Because any reduction in risk associated with antioxidants is expected to be on the order of only 2 0 - 3 0 % [52], and because people with diets high in antioxidants are likely to have healthier than average lifestyles in other respects [53], randomized trials are needed to provide conclusive evidence. With these considerations in mind, the WHS was started in 1991 [48]. A total of 39,876 apparently healthy women ofage 45 years and older were recruited from among nurses, physicians, dentists, and other health professionals. A two-by-twoby-two factorial design was employed in which women were randomized to one of eight combinations of placebo, aspirin, fl-carotene, and vitamin E (Fig. 1). By recruiting health professionals for the study, various efficiencies are gained. In other studies it has been found that health professionals can provide quite accurate and complete information over long periods of time. Studies of health professionals can also be conducted entirely by mail at a considerable savings of money compared to trials in which participants must be seen at a central clinic. In January, 1996, the fl-carotene arm of the trial was stopped because results from the Physicians' Health Study in men found no overall evidence of benefit or risk for coronary heart disease or cancer after more than 10 years of use. In addition, two other trials [54,55] reported an increased risk for lung cancer and cardiovascular disease mortality among heavy smokers, most of whom were men, taking/3carotene supplements. The aspirin and vitamin E components of the trial continue. The WHS is a cost-effective way of testing hypotheses regarding aspirin and certain nutritional supplements in women. Although some might question the representativeness of the study population on which it is based, it is hard to see why findings from women health professionals, who come from a range of social classes and backgrounds, would not apply to other women as well.

CHAPTER28 Prevention Trials

413

Approximately 40,000 Postmenopausal Health Professionals

20,000 Aspirin

20,000 Aspirin placebo

10,000 Beta, Beta-carotene pl 9lacebo

5,000 Vitamin E

5,000 Vitamin E

5,000 Vitamin E

placebo

5,000 Vitamin E

placebo

10,000 Beta-carotene

placebo

5,000 Vitamin E

5,000 Vitamin E placebo

5,000 Vitamin E

5,000 Vitamin E placebo

FIGURE 1 Design of the Women's Health Study. The fl-carotene arm was stopped in January 1996. Adapted from Buring and Hennekens [47].

VII. THE BREAST CANCER PREVENTION TRIAL The BCPT, initiated by the National Institutes of Health and with a coordinating center at the University of Pittsburgh, was a randomized double-blind placebo-controlled trial to test the efficacy of tamoxifen (Nolvadex) in the prevention of breast cancer. It included women who were at least 35 years of age and whose risk of breast cancer was at least as high as that of an average 60-year-old woman in the United States. Tamoxifen, when used as a chemotherapeutic agent in women with breast cancer, has been shown to reduce the incidence of contralateral breast cancer and to delay breast cancer recurrence [56]. Compared to other chemotherapeutic agents, serious side effects and adverse reactions have been reported to be rare [57-59]. In view of these desirable properties, the BCPT was started to determine if tamoxifen can reduce the incidence of breast cancer in healthy women. A secondary aim was to determine whether tamoxifen reduces the incidence of cardiovascular diseases, as suggested by some previous trials. Osteoporotic fractures were also monitored, since a few studies had indicated some protection against loss of bone mass among postmenopausal women. The trial was begun in 1992 and included 13,388 women assigned in equal numbers to tamoxifen or placebo. The trial was halted early in 1998, 14 months earlier than planned, because it showed a 49% reduction in invasive breast cancer incidence among the women who took tamoxifen compared

to the placebo group [60]. No difference was found in the number of heart attacks in the two groups. Women in the tamoxifen group had 19% fewer fractures of the hip, lower forearm, and spine than did those in the placebo group. Tamoxifen use was associated with an increased incidence of three uncommon but serious conditions, including endometrial cancer, pulmonary embolism, and deep vein thrombosis. In fact, in postmenopausal women, the risks for these adverse outcomes almost outweighed the reduction in risk for breast cancer [60]. The encouraging early results of the BCPT need to be interpreted with caution. First, as shown in Table IV, two smaller randomized trials in England [61 ] and Italy [62] have found either no reduction in risk or at most a very slight reductions in risk for breast cancer among women assigned to take tamoxifen. Possible reasons for the discrepant results between the North American and Italian trials are suboptimal compliance in the Italian study, younger groups of women in both of the European trials, and the inclusion in the English trials of more women with a family history of breast cancer at an early age compared to those in the North American trial [63]. Longer follow-up and more data on mortality as well as breast cancer incidence are clearly needed. Second, in addition to the potentially life-threatening side effects mentioned above, women taking tamoxifen often experience vasomotor symptoms such as hot flashes and various gynecologic problems [61,64]. These are troublesome enough that compliance is sometimes a problem in breast cancer patients. Healthy women may be even less likely to take a

414

KELSEY AND MARCUS

TABLE I V

Incidence of Breast Cancer in Three Randomized Trials of Tamoxifen to Prevent Breast Cancer by Treatment Assignment a

Trial Breast Cancer Prevention Trial, North America [60]

Number in trial at baseline

Mean months of follow-up

Incidence rates b Tamoxifen

Placebo

Relative risk

13,388

48

3.4

6.8

0.51

Royal Marsden Hospital Chemoprevention Trial, England [61 ]

2471

60

4.7

5.0

0.94

Italian Tamoxifen Prevention Study, Italy [62]

5408

46

2.1

2.3

0.91

a Adapted from Pritchard [63] and Fisher et al. [60]. ORates per 1000 woman years.

medication that makes them feel uncomfortable. In the BCPT, noncompliance (defined as discontinuation of protocol therapy for other than a protocol-specific reason) was 33% among women in the trial for three years [L. Ford, personal communication, 1995]. In the Italian study [62] 28% of women dropped out over the follow-up period, which had a median of 46 months. Third, one study [65] reported an increased risk of hip fracture among breast cancer patients randomized to tamoxifen. This finding needs to be evaluated in other studies. Fourth, there is concern that tamoxifen may stimulate breast tumor growth in some patients [66]. In both premenopausal and postmenopausal breast cancer patients who initially responded to tamoxifen but who later developed resistance, the breast tumors in some instances appear dependent on tamoxifen for growth. Finally, there have been reports of new primary breast cancers years after tamoxifen has been discontinued. In particular, a higher-than-expected incidence of estrogen-receptor negative contralateral tumors following tamoxifen treatment has been noted [66,67]. In addition, in late 1995, the National Surgical Adjuvant Breast and Bowel Project stopped its study of long-term use of tamoxifen as adjuvant therapy for early-stage breast cancer when routine review of data found no additional benefit for women taking tamoxifen for more than 5 years [68]. Thus, many questions remain about the use of tamoxifen to reduce the incidence of breast cancer. Long-term follow-up is needed.

VIII. RECENT TRIALS OF AGENTS THAT REDUCE BONE LOSS AND FRACTURE

INCIDENCE

Although estrogen has been administered for skeletal protection for several decades, trials with adequate statistical power to assess the effect of osteoporosis regimens on fracture have been undertaken only in recent years. The FDA has only recently required that new osteoporosis drugs, other

than estrogens and their analogs, be shown to decrease the incidence of fracture. This provision takes recognition of the experience from two clinical trials of sodium fluoride showing that increased bone mineral density is not necessarily synonymous with improvement in skeletal integrity [69,70]. Since 1994, industry has operated according to a comprehensive FDA Guidance [71 ] that prescribes the nature of preclinical (animal) studies that need to be carried out and the type of bone quality testing that needs to support evidence of changes in bone mass or bone mineral density. Consequently, during the 1990s several large, well-designed industry-sponsored multicenter clinical trials were undertaken to test the skeletal benefits of new osteoporosis drugs in postmenopausal women. In this section we discuss completed trials involving the potent bisphosphonate, alendronate, and the selective estrogen receptor modulator, raloxifene, both of which have received FDA approval for osteoporosis prevention.

A. A l e n d r o n a t e ( F o s a m a x )

Bisphosphonates is a term given to a group of drugs related to the naturally occurring molecule, pyrophosphate, in which the oxygen bridge is replaced by carbon, thus rendering the compound completely inaccessible to cleavage by alkaline phosphatases. The first-generation bisphosphonate, etidronate (Didronel), has been used in the treatment of Paget's disease of bone since the 1960s. In the early 1990s a strategy for applying this antiresorptive agent to conservation of bone mass and treatment of osteoporosis was reported in two independent studies [72,73]. In both studies, the drug was administered cyclically, 2 weeks every 3 months, for several years. Cyclic administration was required because, unique among drugs of this class, etidronate at effective antiresorptive doses also inhibits the deposition of mineral on bone matrix, thereby increasing the risk for osteomalacia if use is continuous. Both studies clearly showed a beneficial

CHAPTER28 Prevention Trials effect of cyclic etidronate on bone mineral density. However, although several hundred women were enrolled, neither study actually achieved adequate statistical power to establish antifracture efficacy; consequently, the FDA did not approve this drug for an osteoporosis indication.

B. F r a c t u r e I n t e r v e n t i o n T r i a l Subsequent generations of bisphosphonates show considerable improvement in the ratios of antiresorptive effects to mineralization inhibition, permitting their continuous use. One potent bisphosphonate, alendronate (Fosamax), was used in the largest osteoporosis clinical trial to date. Sponsored by the manufacturer, Merck Research Labs, the Fracture Intervention Trial (FIT) enrolled about 6000 women at multiple centers across the United States. FIT was a composite of two related but distinct studies. The first was an evaluation of antifracture efficacy of alendronate among about 2000 women who had evidence of vertebral compression fracture on entry to the trial (Vertebral Fracture Arm). The second arm enrolled about 4000 women who fulfilled bone mineral density criteria for osteoporosis but who had not yet sustained a compression deformity (Clinical Fracture Arm). Participants were randomized to receive placebo or alendronate, 5 mg/day, with an intent to stay on the assigned regimen for 3 years. Unfortunately for the integrity of the study design, it was recognized from results of smaller phase II studies that came to light during the course of FIT that a 10-mg dose offered greater skeletal response without adding significantly to the incidence of adverse experiences. Therefore, the treatment dose was increased to 10 mg for the last year of the trial. Moreover, during year 3, the FIT Data Safety Monitoring Board determined that drug efficacy was established for the Vertebral Fracture Arm and that it was not ethically justified to continue half of the women on placebo. Accordingly, the Vertebral Fracture Arm was brought to an early conclusion. Thus, although the FDA approved alendronate at a 10-mg daily dose for treatment of osteoporosis, much of the benefit documented by FIT represents the effect of the 5-mg dose. Despite the deviations from the original study protocol described above, the results from the FIT Vertebral Fracture Arm were impressive [74]. Women assigned to alendronate experienced a 50% reduction in new vertebral fracture, a 90% reduction in the incidence of multiple vertebral fractures, and a 50% reduction in all nonvertebral fractures. In addition, 22 hip fractures were observed for women assigned to placebo, and only 11 in the alendronate group. Women assigned to alendronate sustained less height loss than those on placebo. The substantial decrease in fracture incidence associated with alendronate exceeds that which could have been predicted from the observed rises in bone mineral density and published relationships between bone mineral density deficit

415 and fracture incidence. This result confirms the wisdom of FDA in requiring fracture incidence to be the primary endpoint of osteoporosis trials. The explanation for this discrepancy is not immediately clear, but it seems likely that a high rate of bone remodeling is itself conducive to fracture, and that effective suppression of remodeling by alendronate had a salutary effect independent of bone mineral density. It was also found in FIT that approximately one out of three vertebral fractures is symptomatic to the point that patients seek medical attention for fracture-related symptoms. Although it had been widely understood that many compression fractures were asymptomatic, FIT represents the first prospective confirmation and quantification of this view. The FIT Clinical Fracture Arm has now been completed and published [75]. With respect to "clinical" fractures, that is, fracture events that resulted in a patient seeking medical attention, alendronate reduced the incidence by 14%, although this reduction was not statistically significant. However, when the analysis was restricted to the subset of women whose baseline bone mineral density values were more than 2.5 standard deviations below the normal young adult mean for women, a 36% reduction in clinical fractures did achieve significance, and the "Number Needed to Treat" for preventing a fracture was 15. With respect to vertebral deformities, alendronate reduced the overall incidence in the entire group by 44%. Thus, alendronate did offer significant antifracture protection. Within this highly jeopardized group of women who met bone mineral density criteria for osteoporosis, those in the lowest tertile of bone mineral density experienced greater fracture protection than did women in the higher tertiles. At present, FIT participants are being offered an opportunity to remain under observation for several additional years. In FIT, as in all formal trials, alendronate has proven to be well tolerated. There had been concern about the propensity for this drug (as for most drugs of this class) to cause esophageal irritation and symptoms of heartburn. In FIT, the overall incidence of adverse experiences was very low, and the incidence of esophageal symptoms associated with alendronate did not differ from that observed with placebo. Nevertheless, esophageal symptoms do remain the major adverse experience with alendronate, particularly among patients who neglect to take the medication exactly as instructed to reduce the likelihood of esophageal irritation (with a full glass of water, no chewing or sucking of the tablet, and remaining in upright position for at least 30 min after taking the pill).

C. E a r l y P o s t m e n o p a u s a l I n t e r v e n t i o n C o h o r t In 1997, the FDA extended approval of alendronate for the prevention of bone loss in recently menopausal women who choose not to take hormone replacement therapy.

416

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FIGURE 2 Meanpercent change from baseline in bone mineral density of the hip in postmenopausal women given estrogen/progestin (• 5 mg alendronate (A), 2.5 mg alendronate (m), or placebo (e). From Hosking et al. (1998) [76]. Copyright 1998 Massachusetts Medical Society. All rights reserved. Evidence to support this indication included interim results from another large clinical trial [76]. In the Early Postmenopausal Intervention Cohort (EPIC), 6000 healthy women who were about 5 years postmenopausal were randomly assigned to receive placebo, alendronate at either 2.5 or 5 mg/ day, or hormone replacement therapy. The hormone replacement therapy regimen differed by study center. Participants in the United States received 0.625 mg of conjugated estrogens and continuous medroxyprogesterone acetate, 5 mg, on a daily basis. Women in European centers received estradiol plus norethindrone, an androgenic progestin. At the 2-year interim analysis, women in all active treatment groups had conserved and actually gained bone mineral density compared to the placebo group. However, women receiving hormone replacement therapy showed significantly greater gains in bone mineral density than did women assigned to alendronate (see Fig. 2 for changes in hip bone mineral density), and women in the European centers showed the greatest increases, presumably reflecting the additive skeletal effects of norethindrone. Thus, alendronate, 5 mg/day, does constitute an effective alternative to hormone replacement therapy for skeletal protection of early menopausal women. However, the interim EPIC results show that alendronate is by no means superior to hormone replacement therapy for this purpose, and that there is presently no basis for recommending that women who are doing well on hormone replacement therapy be switched to alendronate.

teristic changes in the 3-dimensional structure of the estrogen receptor that are induced by binding of the drugs. Different SERMs may have different tissue-specific effects. For example, the SERM tamoxifen is a triphenylethylene that behaves as a partial agonist in the uterus; another SERM, raloxifene, a benzothiophene, behaves as a complete antagonist in the uterus. Thus, raloxifene was developed to have beneficial effects on bone and on lipid metabolism, while antagonizing estrogen in both the uterus and breast. In fact, in the Multiple Outcomes of Raloxifene Evaluation (MORE)randomized trial among postmenopausal women with osteoporosis, a 76% reduction in breast cancer risk was found after 3 years among women treated with raloxifene [78]. Accordingly, SERMs such as raloxifene have been proposed as an alternative to hormone replacement therapy in postmenopausal women for the prevention and treatment of osteoporosis and possibly the prevention of breast cancer, while not adversely affecting the uterus. Raloxifene is the first SERM to be approved by the FDA for postmenopausal therapy. Raloxifene (Evista, Eli Lilly Co.) received FDA approval for osteoporosis prevention in early 1998. At its approved dose of 60 mg/day, raloxifene increases bone mineral density at the spine and hip. Raloxifene's skeletal protective effect has been shown by several relatively small placebo-controlled clinical trials carried out in North America and Europe, and by one somewhat larger study, in which raloxifene treatment for 2 years increased bone mineral density at the spine and hip by about 2% compared to placebo [79] (see Fig. 3 for data on change in bone mineral density of the hip). This increase was slightly less than what had been observed in various studies with hormone replacement therapy or alendronate therapy. Raloxifene was shown to be well tolerated, although its antiestrogenic actions on the hypothalamus resulted in an increase in hot flashes in some women. In the MORE randomized trial among postmenopausal women

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SERMs are agents that produce estrogenlike effects on some tissues and antagonize estrogen in others. SERMs under development are those with actions mimicking the beneficial effects of estrogen on bone and lipid metabolism while antagonizing estrogen in reproductive tissue [77]. The precise mechanisms by which SERMs produce these tissue-selective actions are not fully understood, but appear to reflect charac-

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FIGURE 3 Meanpercent change from baseline in bone mineral density of the hip in postmenopausal women given 150 mg raloxifene (ram), 60 mg raloxifene ( ~ ) , 30 mg raloxifene (---), or placebo (.... ). From Delmas et al. (1997) [79]. Copyright 1997 Massachusetts Medical Society. All rights reserved.

CHAPTER 28 Prevention Trials

4 17

with osteoporosis [80], after 3 years a 30% reduction in risk for vertebral fracture among women using 60 mg/day of raloxifene and a 50% reduction in risk among those using 120 mg/day were noted. The protection was seen both among those with and without prevalent vertebral fractures at baseline. Little protection, however, was seen against nonvertebral fractures, including hip fracture.

IX.

STUDY

OF TAMOXIFEN

AND

RALOXIFENE In 1999, the Study of Tamoxifen and Raloxifene (STAR) was started. The primary objective of STAR is to compare the effectiveness of tamoxifen and raloxifene in reducing the risk for developing breast cancer and also to compare the frequency of side effects. About 22,000 postmenopausal women age 35 years or older who do not have breast cancer but who are at increased risk for breast cancer will be ramdomized to receive either 20 mg of tamoxifen daily or 60 mg of raloxifene daily for 5 years. About 400 clinical sites in the United States and Canada will recruit women. The trial is funded by the National Cancer Institute, with a coordinating center at the University of Pittsburgh.

of a short-term increase in risk for coronary heart disease events among women with diagnosed coronary heart disease who were taking a estrogen/progestin replacement regimen is troublesome and needs to be evaluated in other studies. Trials of the newer agents, such as alendronate, raloxifene, and tamoxifen, have not been of long enough duration to evaluate their many potential important risks and benefits. Thus, long-term monitoring through various types of studies, both observational and randomized, will be highly important. The appearance of SERMs makes even more complex a woman's decision about replacement therapies. In any individual case, reliance on the results of large randomized trials may provide assistance but not necessarily an ultimate determination of the correct choice. It will remain important for each woman and her physician to have a reasonable sense of the extent of her menopausal symptoms, her risks for skeletal, cardiovascular, breast, and perhaps other diseases, her attitude toward taking medications, her concern about side effects, and her likely quality of life with and without replacement therapy. These considerations will help a woman reach a decision with potentially far-ranging consequences.

References X. CONCLUSION Many options are now available to perimenopausal and postmenopausal women to reduce their menopausal symptoms and to decrease their risk for certain major diseases (although possibly at the expense of increasing their risk for other diseases). Still more choices will be available over the next several years. However, the l o n g - t e r m risks and benefits of many of these agents are unknown. The on-going intervention trials should be able to provide some information to help women make choices, but there will still be large gaps in knowledge. If compliance and retention are adequate, the WHI can provide some information about the long-term effects of what is currently considered to be a healthy diet and it should also be able to quantify the long-term effects of supplementation with calcium and vitamin D. The WHS will be useful in determining whether aspirin or vitamin E supplementation reduces the incidence of cardiovascular disease and cancer. These results may be particularly useful to healthy women who are averse to taking potent medications. However, the major trials of hormone replacement therapy, such as the WHI and HERS, include only single hormone replacement regimens compared to placebo. Now that a variety of doses and modes of administration of hormone replacement and new agents such as SERMs are available, WHI and HERS will provide information on only a small piece of what needs to be known. Also, the finding in HERS

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moxifen: Preliminary findings from the Italian randomised trial among hysterectomised women. Lancet 352, 93-97. Pritchard, K. I. (1998). Is tamoxifen effective in prevention of breast cancer? Lancet 352, 80-81. Bush, T. L., and Helzlsouer, K. J. (1993). Tamoxifen for the primary prevention of breast cancer: A review and critique of the concept and trial. Epidemiol. Rev. 15, 233-243. Kristensen, B., Ejlertsen, B., Mouridsen, H. T., Andersen, K. W., and Lauritzen, J. B. (1996). Femoral fractures in postmenopausal breast cancer patients treated with adjuvant tamoxifen. Breast Cancer Res. Treat. 39, 321-326. DeGregorio, M. W., Maenpaa, J. U., and Wiebe, V. J. (1995). Tamoxifen for the prevention of breast cancer: No. 12. Important Adv. Oncol., pp. 175-185. Rutqvist, L. E., Cedermark, B., Glas, U., Mattsson, A., Skoog, L., Somell, A., Theve, T., Wilking, N., Askergren, J., Hjalmar, M.-L., Rotstein, S., Perbeck L., and Ringborg, U. (1991). Contralateral primary tumors in breast cancer patients in a randomized trial of adjuvant tamoxifen therapy. J. Natl. Cancer Inst. 83, 1299-1306. Anonymous (1995). NSABP halts B-14 trial: No benefit seen beyond 5 years of tamoxifen use. J. Natl. Cancer Inst. 87, 1829. Riggs, B. L., Hodgson, S. F., O'Fallon, W. M., Chao, E. Y., Wahner, H. W., Muhs, J. M., Cedel, S. L., and Melton, L. J. 3rd (1990). Effect of fluoride treatment on the fracture rate in postmenopausal women with osteoporosis. N. Engl. J. Med. 322, 802-809. Kleerekoper, M., Peterson, E. L., Nelson, D. A., Phillips, E., Schork, M. A., Tilley, B. C., and Parfitt, A. M. (1991). A randomized trial of sodium fluoride as a treatment for postmenopausal osteoporosis. Osteoporosis Int. 1, 155-161. Food and Drug Administration (1994). "Guidelines for Preclinical and Clinical Evaluation of Agents Used in the Prevention and Treatment of Postmenopausal Osteoporosis." FDA, Rockville, MD. Storm, T., Thamsborg, G., Steiniche, T., Genant, H. K., and Sorenson, O. H. (1990). Effect of cyclical etidronate therapy on bone mass and fracture rate in women with postmenopausal osteoporosis. N. Engl. J. Med. 322, 1265-1271. Watts, N. B., Harris, S. T., Genant, H. K., Wasnich, R. D., Miller, P. D., Jackson, R. D., Licata, A. A., Ross, P., Woodson, G. C., 3rd, Yanover, M. J., Mysiw, W. J., Kohse, L., Rao, M. B., Steiger, P., Richmond, B., and Chestnut, C. H., III (1990). Intermittent cyclical etidronate treatment of postmenopausal osteoporosis. N. Engl. J. Med. 323, 73-79. Black, D. M., Cummings, S. R., Karpf, D. B., Cauley, J. A., Thompson, D. E., Nevitt, M. C., Bauer, D. C., Genant, H. K., Haskell, W. L., Marcus, R., Ott, S. M., Torner, J. C., Quandt, S. A., Reiss, T. F., Ensrud, K. E, for the Fracture Intervention Trial Research Group (1996). Randomised trial of effect of alendronate on risk of fracture in women with existing vertebral fractures. Lancet 348, 1535-1541. Cummings, S. R., Black, D. M., Thompson, D. E., Applegate, W. B., Barrett-Connor, E., Musliner, T. A., Palermo, L., Prineas, R., Rubin, M., Scott, J. C., Vogt, T., Wallace, R., Yates, A. J., La Croix, A. Z., for the Fracture Intervention Trial Research Group (1998). Effects of alendronate on risk of fracture in women with low bone density but without vertebral fractures: Results from the Fracture Intervention Trial. JAMA, J. Am. Med. Assoc. 280, 2077-2082. Hosking, D., Chilvers, C. E. D., Christiansen, C., Ravn, P., Wasnich, R., Ross, P., McClung, M., Balske, A., Thompson, D., Daley, M., and Yates, A. J. (1998). Prevention ofbone loss with alendronate in postmenopausal women under 60 years of age. N. Engl. J. Med. 338, 4 8 5 - 492. Bryant, H. U., and Dere, W. H. (1998). Selective estrogen receptor modulators: An alternative to hormone replacement therapy. Proc. Soc. Exp. Biol. Med. 217, 45-52. Cummings, S. R., Eckert, S., Krueger, K. A., Grady, D., Powles, T. J.., Conley, J. A., Norton, L., Nickelsen, T., Bjarnason, N. H., Morrow, M., Lippan, M. E., Black, D., Glusman, J. E., Costa, A., and Jordan, U. C.

420 (1999). The effect of raloxifene on risk of breast cancer in postmenopausal women. Results from the MORE randomized trial. J. Am. Med. Assoc. 281, 2189-2197. 79. Delmas, E D., Bjarnason, N. H., Mitlak, B. H., Ravoux, A. C., Shah, A. S., Huster, W. J., Draper, M., and Christiansen, C. (1997). Effects of raloxifene on bone mineral density, serum cholesterol concentrations, and uterine endometrium in postmenopausal women. N. Engl. J. Med. 337, 1641-1647.

KELSEY AND MARCUS

80. Ettinger, B., Black, D. M., Mitlak, B. H., Knickerbocker, R. K., Nickelsen, T., Genant, H. K., Christiansen, C., Delmas, E D., Zanchetta, J. R., Stakkestad, J., GlUer, C. C., Krueger, K., Cohen, F. J., Eckert, S., Ensrud, K. E., Avioli, L. V., Lips, E, and Cummings, S. R., from the Multiple Outcomes of Raloxifene Evaluation (MORE) Investigators (1999). Reduction of vertebral fracture risk in postmenopausal women with osteoporosis treated with raloxifene. Results from a 3-year randomized clinical trial. J. Am. Med. Assoc. 282, 637-645.

7 H A P T E R 2~

Estrogens: Different Types and Properties FRANK Z. STANCZYK

Department of Obstetrics and Gynecology, University of Southern California School of Medicine, Los Angeles, California 90033

III. Potency of Estrogens References

I. Steroidal Estrogens II. Nonsteroidal Estrogens

has two hydroxyl groups, is the most biologically active estrogen produced in the body. Oxidation of the hydroxyl group at carbon 17 of estradiol gives rise to estrone. Addition of a hydroxyl group at carbon 16 of estradiol yields estriol. Transformation of the hydroxyl group of estradiol at carbon 17 from fl orientation to ce orientation gives rise to 17ce-estradiol, which generally is considered to have no estrogenic activity. Endogenous concentrations of this estrogen have not been detected. However, some data suggest that 17a-estradiol may relieve postmenopausal symptoms [3].

Estrogens can be categorized according to their chemical structure into two groups: steroidal and nonsteroidal. Each group can be subdivided further into natural and synthetic compounds.

I. S T E R O I D A L

ESTROGENS

All steroidal estrogens have the same basic structural characteristics [ 1,2]. They are related to the parent structure, estrane, which has 18 carbons, and contain an aromatic A ring as well as oxygenated functional groups at carbons 3 and 17. The presence of a hydroxyl group on the aromatic ring constitutes a phenolic ring.

2. ESTROGEN METABOLISM Estradiol is readily converted to estrone and both are metabolized, primarily in the liver, to a variety of hydroxylated products [4]. Hydroxylation of the two substrates occurs at carbon 1, 2, 4, 6, 7, 11, 14, 15, 16, or 18, with the hydroxyl groups in c~ or/3 orientation (Fig. 2). However, there are two major pathways of estrogen metabolism: 2- and 16cehydroxylation. 16a-Hydroxylation of estrone and its subsequent reduction at carbon 17 give rise to estriol, mentioned earlier. Of the various hydroxylated estradiol and estrone metabolites, estriol appears to be the only metabolite that has some estrogenic activity [5]. Estriol is also quantitatively one of the most important conjugated estrogen metabolites found

A. N a t u r a l S t e r o i d a l E s t r o g e n s

1. CLASSIC ESTROGENS There are three principal namely estrone, estradiol, and gens are often referred to as they were the first estrogens to

natural steroidal estrogens, estriol (Fig. 1). These estro"classic" estrogens because be isolated. Estradiol, which

MENOPAUSE: BIOLOGY AND PATHOBIOLOGY

421

Copyright 9 2000 by Academic Press. All rights of reproduction in any form reserved.

422

FRANK Z. STANCZYK

0

OH

OH ...

HO

HO Estrone (E1)

OF

HO Estradiol (52)

Estriol (53)

FIGURE 1 Chemical structure of estrone, estradiol, and estriol. Adapted from Ref. 1.

in urine. In addition to the 16ce-hydroxylated pathway, the 2-hydroxylated pathway yields quantitatively important urinary metabolites; these include conjugated 2-hydroxyestrone and its methoxy derivative, i.e., 2-methoxyestrone. 4-Hydroxylation of estrone is also an important pathway of estrogen metabolism, however, it appears to be quantitatively of lesser magnitude than 16-hydroxylation. Both 4hydroxyestrone and 16a-hydroxyestrone can react directly with DNA or can be metabolized further into reactive oxygenated compounds, and both have been implicated in increased risk of cancer [6]. The presence of a hydroxyl group at carbon 2 or 4 of a phenolic ring, which is characteristic of estrogens, gives rise to a catechol group (Fig. 3). Estrogens that contain this group are referred to as catechol estrogens. There is evidence that catechol estrogens may play an important role in the regulation of catecholamines by competing for the enzyme, catechol-Omethyltransferase, which catalyzes methylation of the catecholamines dopamine and norepinephrine. It has been proposed that catechol estrogens may control hormonal actions of the catecholamines by inhibiting their methylation, thereby preventing their inactivation. 15ce-Hydroxylated estrogens are found in relatively high concentrations in late pregnancy. The human fetal liver appears to be a highly active site of 15ce-hydroxylase activity. In the late stages of human pregnancy, urinary 15ce-

18

OH

OH 3

2

;

10

7

15

hydroxyestriol, also called estetrol, is excreted in amounts that exceed those of all other estrogens except estriol and 16c~-hydroxyestrone. Estrone and estradiol, as well as the hydroxylated metabolites of these estrogens can undergo conjugation, forming sulfated and glucuronidated metabolites. Quantitatively the most important circulating estrogen in women is estrone sulfate. The large pool of estrone sulfate may be conceptualized as a slowly metabolized estrogen reservoir. Circulating concentrations of estrone sulfate are three- to fivefold higher than those of estrone glucuronide. In contrast to the conjugated metabolites of estrone and estradiol, circulating concentrations of unconjugated hydroxylated metabolites of these precursors are very low. For example, during the menstrual cycle, circulating levels of 2-hydroxyestrone and estriol are only about 10 pg/ml, in contrast to concentrations of estrone sulfate and estrone glucuronide, which are at least one to two orders of magnitude higher. Studies have been performed to test individual urinary estrogen conjugates for predicting ovulation and determining the start and end of the fertile period [7]. Data show that urinary levels of estrone glucuronide, estradiol-3-glucuronide, estradiol- 17-glucuronide, and estriol- 16-glucuronide rise early and steeply prior to ovulation. Subsequent studies chose estrone glucuronide and estriol-16-glucuronide for development of a noninvasive immunochemical test (e.g., urinary dipstick assay) to determine the fertile period. During pregnancy, circulating estrogen conjugates are present in very high concentrations in both the fetus and mother. The placenta secretes estrone, estradiol, and estriol in varying proportions into the maternal and fetal circula-

0

16 ~=

HO

T ;

HO

HO

FIGURE 2 Sites (arrows) of hydroxylation of estradiol. Adapted from Ref. 4, Zhu and Conney (1998). Carcinogenesis (London) 19, 1-27, by

catechot

permission of Oxford University Press.

FIGURE 3 Structure of catechol. From Ref. 2.

2-hydroxyestrone

CHAPTER29 Estrogens: Different Types and Properties tions; these estrogens can be conjugated or undergo hydroxylation at various carbons and then be conjugated. In the fetus, the predominant form of conjugation is sulfurylation. In the mother, however, both sulfated and glucuronidated estrogen metabolites are found. Four major estrogens in the maternal circulation include estriol-3-sulfate, estriol3-glucuronide, estriol- 16-glucuronide, and estriol-3-sulfate16-glucuronide [8]. Although most of the conjugated estrogens are excreted in urine, part of the circulating conjugated estrogens will undergo enterohepatic circulation, primarily forming glucuronides of the estrogens at carbon 3, e.g., estriol-3-glucuronide. 3. EQUINE ESTROGENS Ring B unsaturated estrogens are present in high concentrations in pregnant mare urine and are important constituents of a conjugated equine estrogen preparation (Premarin, Wyeth-Ayerst Laboratories) used for estrogen replacement treatment in postmenopausal women. Approximately 45 % of this preparation is composed of several different sulfated ring B unsaturated compounds, which include the sulfated forms of equilin and 17a-dihydroequilin and constitute 25 and 15% of this preparation, respectively. Equilin has the same chemical structure as estrone, except that it has a double bond between carbons 7 and 8. Minor sulfated equine estrogens present in the preparation include equilenin (which has double bonds between carbons 6 and 7 as well as between 8 and 9), the 17c~- and 17fi-reduced forms of equilenin, and 17fi-dihydroequilin. Although the latter compound is a minor constituent (1-2%) of the conjugated equine preparation, it has been shown to be about eight times more potent than estradiol as a uterotropic agent [9]. Minor sulfated ring B saturated estrogens are also found in the equine preparation and include 17/3estradiol, 17c~-estradiol, and AS,9-estrone.

B. S y n t h e t i c Steroidal E s t r o g e n s A number of potent steroidal estrogens have been synthesized and are used orally or parenterally. Esterification of estradiol at carbon 3 or carbon 17 with fatty acids yields long-acting, oil-soluble estrogenic preparations that are used therapeutically [5]. As a general rule, the greater the molecular weight of the esterified estrogen, the more pronounced is the biologic activity of the compound. These products are usually administered intramuscularly. When they are given orally, they are usually not as potent by this route. For example, estradiol benzoate, which is synthesized by esterification of estradiol with benzoic acid, is only about half as potent when administered orally in comparison to its administration intramuscularly. Structural modification of the estradiol molecule by insertion of an ethinyl group at carbon 17 yields ethinylestradiol, which has estrogenic activity and high oral activity (Fig. 4).

423

OH --C=

CH~O

OH ~C--~--CH

H Mestranol

Ethinyleslradiol

FIGURE 4 Chemical structures of mestranol and ethinylestradiol. From Ref. 2, from J. Cell Sci. Suppl., by permission of the Company of Biologists Ltd.

This compound plays an important role as an estrogenic component of oral contraceptives. Modification of ethinylestradiol by formation of a methyl ether at carbon 3 gives rise to mestranol (Fig. 4). This estrogen has been widely used as a component of oral contraceptive formulations, but is now used less frequently. The estrogenic effect of mestranol is due to its rapid demethylation in the liver, forming ethinylestradiol. The metabolism of ethinylestradiol is similar to that of the natural estrogens [ 10]. It undergoes extensive hydroxylation at carbons 2 and 16. In addition, the 2- and 3-methyl ethers of ethinylestradiol have also been identified as major metabolites. Both ethinylestradiol and its hydroxylated metabolites undergo extensive conjugation. In the circulation, the principal form of ethinylestradiol is ethinylestradiol sulfate. A series of novel synthetic steroidal estrogens with potent antioxidant properties have been synthesized chemically and are referred to as "scavestrogens" [11,12]. It is well established that estradiol is a naturally occurring antioxidant, which may explain the cardioprotective effect of postmenopausal estrogen replacement therapy. 17ce-Dihydroequilin sulfate and 17ce-dihydroequilenin sulfate, which are constituents of the conjugated equine estrogen preparation described earlier, appear to have more potent antioxidant properties than does estradiol [13]. However, in vitro studies show that formation of A 8,9 derivatives of estradiol and 17ceestradiol gives rise to scavestrogens, which not only have antioxidative activities similar to those of the parent molecules, but also have iron-chelating and superoxide anion radical inhibitory properties [11]. Synthetic estrogens with even greater radical-scavenging activities have been prepared by addition of methylated phenolic derivatives at carbon 17 in conjunction with formation of a A9,11 double bond in ring C of estradiol [ 12].

II. N O N S T E R O I D A L E S T R O G E N S Numerous nonsteroidal estrogens have been isolated from natural sources or have been synthesized. These compounds differ in chemical structure from steroidal estrogens, and can be subdivided into natural and synthetic types, similar to the steroidal estrogens. Their affinities for the estrogen receptor vary from very weak to highly potent.

424

FRANK Z. STANCZYK

A. N a t u r a l N o n s t e r o i d a l E s t r o g e n s Phytoestrogens are nonsteroidal diphenolic compounds that bind weakly (relative to estradiol) to the estrogen receptor. They have also been reported to be antiestrogenic and inhibitors of estrogen binding. Two classes of phytoestrogens have been identified in biological specimens (blood, urine, and feces) from humans, namely, lignans and isoflavonoids [ 14]. There are differences in chemical structure between the two groups (Figs. 5 and 6). Lignans can be subdivided into plant and animal (or mammalian) types on the basis of their origin. Plant lignans can be structurally modified by intestinal bacteria to form animal lignans. The main plant lignans are matairesinol and secoisolariciresinol. They are found in various seeds, such as sesame seed (matairesinol) and linseed (secoisolariciresinol), various grains (mainly matairesinol), and whole soybeans. In animals, matairesinol and secoisolariciresinol are metabolized to enterodiol, which can be transformed to interolactone. The main isoflavonoid phytoestrogens are daidzein and genistein. They are found in soybeans and various soy products (tofu, soy milk, miso) and, to a lesser extent, other legumes. Daidzein is converted to equol and, to a lesser extent, desmethylangolensin in animals. Green tea contains daidzein and genistein (isoflavonoids), as well as secoisolariciresinol (lignan). In natural foods, phytoestrogens occur as glycones (also called glycosides), i.e., they are bound to monosaccharides,

disaccharides, and polysaccharides by glycoside linkages. This linkage is formed when an alcohol group reacts with an aldehyde group. Thus, the hydroxyl group of a phytoestrogen can react with the aldehyde group of a sugar, forming a glycone. In the gut, bacteria are able to hydrolyze glycones. When a glycone is hydrolyzed, it forms a sugar moiety and a nonsugar moiety. The latter is called an aglycone and represents the phytoestrogen moiety without the carbohydrate portion. It is known that phytoestrogens undergo extensive metabolism in the body, similar to that of steroids, i.e., they are reduced, hydroxylated, and/or conjugated [14]. However, little is known about the nature of phytoestrogen metabolites. A number of studies have investigated the estrogenic activity of phytoestrogens and show that they are weak estrogens. Studies using the rat uterus model to assess estrogenicity show that the affinities of phytoestrogens for the estradiol receptor vary widely; however, phytoestrogens generally have affinities which are at least 1000 times lower than that of estradiol ( k d = 1 • 10 - 9 1 • 10-10).

B. S y n t h e t i c N o n s t e r o i d a l E s t r o g e n s The most important nonsteroidal estrogens are the stilbestrois, which are derivatives of stilbene (1,2-diphenylethylene). One of the best known estrogenic stilbene derivatives

eo COH O

HO

HO

O OMe

[~OMe OMe Arctigenin

[~OMe OH Matairesinol

HoC o

OH Secoisolariciresinol

.OC

O" O.

o ~OH Enterolactone

OH

OH Enterodiol

FIGURE 5 Structuresof plant and mammalianlignans. From Ref. 14.

CHAPTER 29 Estrogens: Different Types and Properties

HO

425

HO

o.

o

Ho~O'~o

// "OMe

~ Formononetin

BiochaninA HO

"OMe Coumestrol

HO

OH

O

L[..~

OH

H o ~ O ' ~

oH

OH

Genistein

MeO" .,...,..~ O , ~

Daidzein

HO

Glycitein

HO

/1

o

]~.~/C)H

C

"OMe

)H

O-Desmethylangolensin

Equol

FIGURE 6 Structuresof isoflavonoids. From Ref. 14.

is diethylstilbestrol (Fig. 7) because it was widely used therapeutically in the 1940s and 1950s. It is synthesized from estrone and is a highly potent orally active estrogen. In comparison, estrone is orally inactive and not as estrogenic as diethylstilbestrol. However, published reports about potential oncogenic effects of diethylstilbestrol have dramatically limited its use as a therapeutic agent. Modification of the chemical structure of the diethylstilbestrol molecule has resulted in products with greatly reduced estrogenicity. Many of the structural elements observed in more modern nonsteroidal estrogens include spatial arrangement of three aromatic rings with a basic side chain attached to one of them. Triphenylethylene derivatives that were developed into clinically useful agents in the 1960s and 1970s include clomiphene and tamoxifen (Fig. 8). Clomiphene is an effective antifertility agent in labora-

/CH2

HO

---

tory animals, but paradoxically induces ovulation in infertile women [ 15]. It is marketed as an impure mixture of isomers with opposing biologic activities. One isomer is an estrogen, whereas the other is an antiestrogen. Although clomiphene showed some promise in the treatment of breast cancer, the drug was developed only for induction of ovulation. Substitution of an ethyl group for the chloral group of the clomiphene molecule gives rise to the antiestrogen tamoxifen [ 15]. Although this compound, like clomiphene, is also an antifertility agent and induces ovulation in infertile women, preliminary studies showed its potential efficacy in the treatment of advanced breast cancer. Tamoxifen had several advantages

~ N o

\

O

OH

C/ H3

FIGURE 7 Structureof diethylstilbestrol. Adapted from Ref. 2.

I) c~ CLOMIPHENE

TAMOXIFEN

FIGURE 8 Structuresof clomiphene and tamoxifen. From Ref. 16.

426

FRANK Z. STANCZYK

/

/

o ~ N \

o ~ N \

minor route

HO Tamoxifen

4-Hydroxytamoxifen

major route f

0 ~NH

N-desmethyltamoxifen FIGURE 9 Importantmetabolitesof tamoxifen. Adaptedfrom Ref. 16.

over other antiestrogenic drugs; these include (1) higher antitumor potency, (2) low side effects, and (3) potential for longterm treatment. These advantages led to its clinical success in treatment of all stages of breast cancer during the past 25 years and, more recently, to its proposed use for the prevention of breast cancer. The two major routes of tamoxifen metabolism include 4hydroxylation and progressive degradation of the dimethylaminoethane side chain yield (Fig. 9) [ 16]. The former reaction yields 4-hydroxytamoxifen, which is quantitatively a minor metabolite in serum, but has a high binding affinity for the estrogen receptor. Progressive demethylation of the tamoxifen side chain begins with formation of N-desmethyltamoxifen, which is quantitatively the principal serum metabolite. Further demethylation gives rise to didesmethyltamoxifen. Removal of the dimethylaminoethane side chain does not affect the biological actions of the altered tamoxifen molecule. In addition to the three metabolites just described, other circulating metabolites of tamoxifen include ce-hydroxytamoxifen, tamoxifen-N-oxide, ce-hydroxy-N-desmethyltamoxifen, and 4-hydroxy-N-desmethyltamoxifen. The glucuronides of 4-hydroxytamoxifen, dihydroxytamoxifen, and a monohydroxylated N-desmethyltamoxifen have been identified in urine.

In contrast to tamoxifen, the novel antiestrogen, raloxifene (Fig. 10) [15], is less estrogenic in the rat and mouse uterus, and is a potent inhibitor of estrogen-stimulated effects in vitro. Although raloxifene exhibits antitumor properties in animal models, it is not superior to tamoxifen in this

/ o

o

01HO Raloxifene

FIGURE 10 Structureof raloxifene. AdaptedfromRef. 16.

CHAPTER 29 Estrogens: Different Types and Properties respect. Raloxifene has been shown to maintain bone density in postmenopausal women.

III. POTENCY OF ESTROGENS Potency of natural estrogens has been determined in various test systems. The tests can be divided into three types: (1) bioassays, (2) clinical assays, and (3) in v i t r o receptorbinding studies. However, problems encountered in estimating estrogen potency are well recognized. A major source of the difficulties can be found in variables associated with each test. These include (1) type of animal species, (2) type of tissue or target organ, (3) specific response of tissue or target organ, (4) route and dose of estrogen administration, (5) temporal considerations, (6) vehicle for drug administration, and (7) extrapolation of data from animals to humans. On the basis of the above tests, generalizations have been made about potencies of the three classic estrogens. Estradiol is considered to be the most potent estrogen, and estrone has been reported to be 5 0 - 7 0 % less active than estradiol [5]. Estriol is generally considered to be the weakest of the three classic estrogens, with a potency that is one-tenth of that of estradiol. Transformation of the 17fl-hydroxyl to the 17ce-orientation or hydroxylation and/or conjugation of any of the three classic estrogens gives rise to products that are generally considered to be inactive. In recent years, potency of estrogens has been compared on the basis of metabolic consequences of the hepatic firstpass effect of estrogens. The liver responds to the high concentrations of estrogens emerging in the hepatic portal vein blood by increasing the synthesis of "estrogen-sensitive" proteins such as sex hormone-binding globulin (SHBG), corticosteroid-binding globulin (CBG), thyroxine-binding globulin (TBG), renin substrate, various coagulation and fibrinolytic factors, high-density lipoprotein (HDL), and apolipoproteins. In addition, potency of estrogens has been compared on the basis of their effects on the pituitary, specifically on follicle-stimulating hormone (FSH) and luteinizing hormone (LH) secretion. Biologic effects of various doses of conjugated estrogens and ethinylestradiol in postmenopausal women have been demonstrated [17,18]. The smallest doses of conjugated equine estrogens to elicit significant reduction of FSH and LH in either premenopausal or postmenopausal women were 0.3 and 0.625 mg, respectively. With increasing doses (0, 0,15, 0.30, 0.625, and 1.25 mg) of these estrogens, there is a stepwise increase in "estrogen-sensitive" proteins, specifically SHBG, CBG, TBG, and renin substrate. The lowest doses that gave significant elevations above premenopausal levels were 0.15 mg for SHBG, 0.625 mg for CBG, and 0.3 mg for TBG and renin substrate. No significant differences were found between premenopausal and postmenopausal women with respect to the four parameters at base line.

427 In contrast to conjugated equine estrogens, much smaller doses, specifically 10 and 20/xg, of ethinylestradiol are required to reduce significantly base line postmenopausal serum FSH and LH, respectively. It has also been shown that 50 # g of ethinylestradiol lowers postmenopausal serum FSH and LH levels by 75 and 60%, respectively. As shown with conjugated equine estrogens, no significant differences were found between premenopausal and postmenopausal women with respect to the estrogen-sensitive proteins at base line. There was a stepwise increase in the four parameters with increasing doses of ethinylestradiol (0, 5, 10, 20, and 50/zg). With the 5-/xg dose, SHBG, TBG, and renin substrate increased significantly above premenopausal values, whereas the 10-/xg dose was required to increase CBG significantly. With the 50-/zg dose, SHBG and renin substrate levels were three to four times higher than premenopausal values.

References 1. Stanczyk, F. Z. (1998). Structure-function relationships and metabolism of estrogens and progestogens.In "Estrogens and Progestogensin Clinical Practice" (I. S. Fraser, R. E S. Jansen, R. A. Lobo, and M. I. Whitehead, eds.), pp. 27-39. Churchill-Livingstone,London. 2. Stanczyk,E Z. (1998). Steroidhormones.In "Mishell's Textbookof Infertility, Contraception and ReproductiveEndocrinology" (R. A. Lobo, D. R. Mishell,Jr., R. J. Paulson, and D. Shoupe, eds.), 4th ed., pp. 46-66. Blackwell Science,Malden, MA. 3. Oettel, M., Heller, R., Grabner, R., Losche, W., Krause, S., and Romer, W. (1996). The local vascular effects of 17a-estradiolvs. 17fl-estradiol. l l th Congr. Euro. Assoc. Gynecol. Obstetr., Budapest, Hungary, 1996, p. 71. 4. Zhu, B. T., and Conney, A. H. (1998). Functional role of estrogen metabolism in target cells: Review of perspectives. Carcinogenesis (London) 19, 1-27. 5. Henzl, M. R. (1986). Contraceptivehormones and their clinical use. In "Reproductive Endocrinology,Physiology,Pathophysiologyand Clinical Management" (S. S. Yen and R. E. Jaffe, eds.), pp. 643-682. Saunders, Philadelphia. 6. Service, R. E (1998). New role for estrogen in cancer? Science 279, 1631-1633. 7. Stanczyk, F. Z. Miyakawa, I., and Goebelsmann, U. (1980). Direct radioimmunoassay of urinary estrogen and pregnanediol glucuronide during the menstrual cycle. Am. J. Obstet. Gynecol. 137, 443-450. 8. Goebelsmann,U., and Jaffe, R. B. (1971). Oestriol metabolismin pregnant women.Acta Endocr. 66, 679-693. 9. Bhavnani, B. E, and Woolover, C. A. (1991). Interaction of ring B unsaturated estrogens with estrogenreceptors of human endometriumand rat uterus. Steroids 56, 201-209. 10. Roy, S., Bernstein L., and Stanczyk, E Z. (1988). Analysis of oral contraceptive risks. In "Female Contraception" (B. Rumenbaum, T. Rabe, and L. Kiesel, eds.), pp. 21-55. Springer-Verlag,Berlin. 11. Romer, W., Oettel, M., Droescher, E, and Schwarz, S. (1977). Novel "scavestrogens" and their radical scavenging effects, iron-chelating, and total antioxidative activities: AS,9-Dehydroderivatives of 17ce-estradiol and 17/3-estradiol.Steroids 62, 304-310. 12. Romer, W., Oettel, M., Mezenbach, B. Droescher, E, and Schwarz S.

428 (1997). Novel estrogens and their radical scavenging effects, ironchelating, and total antioxidative activities: 17ce-Substituted analogs of A9~ll)-dehydro-17fl-estradiol. Steroids 62, 688-694. 13. Subbiah, M. T. R., Kessel, B., Agrawal, M., Rojan, R., Abplanalp, W., and Rymaszewski, Z. (1993). Antioxidant potential of specific estrogens on lipid peroxidation. J. Clin. Endocrinol. Metab. 77, 1095-1097. 14. Adlercreutz, H., and Mazur, W. (1997). Phyto-oestrogens and Western diseases. Ann. Med. 29, 95-120. 15. Jordan, V. C. (1997). The origin of antiestrogens. In "Estrogens and Antiestrogens" (R. Lindsay, D. W. Dempster, and V. C. Jordan, eds.), pp. 9-20. Lippincott-Raven, Philadelphia.

FRANK Z. STANCZYK 16. Jordan, V. C., Piette, M., and Cisneros, A. Metabolism of antiestrogens. In "Estrogens and Antiestrogens" (R. Lindsay, D. W. Dempster, and V. C. Jordan, eds.), pp. 29-41. Lippincott-Raven, Philadelphia. 17. Geola, F. L., Frumar, A. M., Tataryn, V., Lee, K. H., Hershman, J. M., Eggena, P., Sambhi, M. P., and Judd, H. L. (1980). Biological effects of various doses of conjugated equine estrogens in postmenopausal women. J. Clin. Endocrinol. Metab. 51,620-625. 18. Mandel, F. P., Geola, F. L., Lu, J. K. H., Eggena, P., Sambhi, M. P., Hershman, J. M., and Judd, H. L. (1982). Biologic effects of various doses of ethinyl estradiol in postmenopausal women. Obstet. Gynecol. 59, 673-679.

~HAPTER 3(

Progestogens ROGERIO A.

LOBO

Department of Obstetrics and Gynecology, Columbia University, College of Physicians and Surgeons, New York, New York 10032

I. Introduction II. Rationale for Use of Progestogens III. Progestogen Biologic Aspects

I.

IV. Conclusions References

INTRODUCTION

with the risk in women who do not use estrogen, the relative risk of endometrial cancer is 2.1 after 2 to 5 years of estrogen use and increases to 3.5 after 6 years of use [5]. Overall, the summary relative risk is 2.8 (2.6-3.0) [4]. However, because vaginal bleeding is an early symptom of this disease, there is usually an early diagnosis and endometrial cancers attributable to unopposed estrogen use are usually welldifferentiated lesions with a good prognosis [6]. Epidemiologic and histologic studies have clearly demonstrated that the addition of progestogens to estrogen replacement therapy not only decreases the risk of endometrial cancer [7-9] but can also reverse established endometrial hyperplasia to normal endometrium [10]. The duration of progestogen exposure is important. For more than 10 days of use, the relative risk is 1.1 (0.9-1.3), and with continuous combined regimens the risk is estimated to be 1.3 [ 11 ]. A consensus view is that because progestogens are protective against endometrial disease, some regimen should be employed in women with a uterus [1,2]. The 3-year Postmenopausal Estrogen/Progestin Interventions (PEPI) trial proved once again that unopposed estrogen is hazardous in women with a uterus. Over 60% of women developed various degrees of endometrial hyperplasia with 34% developing adenomatous or atypical hyperplasia [12]. Because of these and other data, no longer is unopposed estrogen treatment considered appropriate under normal circumstances, and this has influenced the design of the ongoing National Institutes of Health-sponsored Women's Health Initiative

For more than a decade, the most confusing and controversial issue in hormonal replacement therapy has been the use of progestogens. In an attempt to clarify some of the key issues, an international consensus meeting was convened in 1989, chaired by the author and Malcolm Whitehead [ 1]. Although many studies have been published since then, clinical management has not changed appreciably. This was one of the conclusions of a subsequent progestogen consensus meeting, cochaired by the author and Ian Fraser in the fall of 1998 [2]. This chapter focuses on our knowledge of progestogens, their indication(s), potential side effects, and the various regimens that are used by postmenopausal women.

II. RATIONALE FOR USE OF PROGESTOGENS It has been known for some time that postmenopausal women who receive unopposed estrogen therapy have an increased incidence of hyperplasia, which may progress to endometrial adenocarcinoma under the continued influence of estrogen (see Chapter 25) [3]. This association of unopposed estrogen and endometrial cancer has been demonstrated in 30 case-control studies and 7 cohort studies [4]. Compared MENOPAUSE: BIOLOGY AND PATHOBIOLOGY

429

Copyright9 2000by AcademicPress. Allrightsof reproductionin any formreserved.

430

ROGERIO A. LOBO

(WHI) study as well as the decision of institutional review boards to approve new studies involving estrogen and progestogens. Nevertheless, in practice, there are women who cannot or choose not to take progestogens because of side effects and personal reasons. In this scenario, close monitoring is mandatory and the dose of estrogen should be reduced.

A. E f f e c t s o n the B r e a s t In women without a uterus, the consensus view is that progestogens are unnecessary [1,2] and may decrease some of the benefits of estrogen, which will be discussed below. Nevertheless, there are a minority of investigators who believe that the use of progestogen should be beneficial in women without a uterus because it protects the breast [13]. There are others, however, who conclude that progestogens may further increase the risk of breast cancer, whether or not estrogen imparts this risk [14]. Over 10 years ago data from Ferguson and Anderson first suggested that mitotic activity of the breast increased in the luteal phase [ 15,16]. These data have been confirmed by others [17-19]. Thus, although progestogen decreases the proliferation of the endometrial epithelium, it increases the proliferation of the mammary epithelium, thus suggesting a risk rather than protection. However, the situation is quite complex. Proliferation of breast tissue in the normal luteal phase is a coordinated event and is followed by apoptosis or cell death. Nevertheless, Spicer and Pike have argued that this cycle of events may increase genetic risk and accordingly the propensity to develop breast cancer [20]. Experimental data are at odds with this theory. Some ex vivo studies in mice, using human tissue, have supported the view that progestogens do not stimulate growth [21] and other in vitro tissue culture studies have shown an inhibitory effect of progesterone [22]. Thus, the data regarding breast tissue are quite inconsistent, and are clearly different from the data showing a protective effect of progestogens on the endometrium. It follows, therefore, that progestogens should not be routinely used in hysterectomized women specifically for a breastprotective effect. In terms of treatment efficacy, the only other organ system whereby progestogens may have some benefit is in bone remodeling. Systems such as the central nervous system and cardiovascular system do not appear to be benefited by progestogens; whether there is a negative influence of progestogens will be discussed below.

B. E f f e c t s o n B o n e Progesterone during the menstrual cycle has been thought to lead to an increase in bone mass, with anovulatory women having lower bone mass [22a,22b]. However, this observa-

tion has not been confirmed by others [23,24]. Although high doses of progestogens alone have been shown to have a positive influence on bone mass [25,26] as well as to exert a proliferative effect on bone cells in in vitro studies [27], most clinical studies do not show an additional beneficial effect when progestogens are added to estrogen replacement. In the PEPI trial [28], although the intention-to-treat analysis suggested a benefit of the addition of medroxyprogesterone acetate [MPA], this could not be confirmed in the analysis of adherent subjects. There may, however, be a difference in bone mass between the various progestogens. Although the evidence for a beneficial or additive effect of progesterone on the 17acetoxy progestogens is weak, some evidence exists for the beneficial use of 19-nor progestogens such as norethindrone acetate. Data from Christiansen and Speroff [29,30] have suggested an additive effect of norethindrone acetate on bone mass, resulting in an increase in bone formation. It is clear that routine doses of progestogens do not inhibit bone resorption.

III. P R O G E S T O G E N BIOLOGIC ASPECTS A. P h a r m a c o l o g y For practical purposes, native progesterone is the only natural compound with significant biologic function. Significant reduction in potency occurs with 17a-hydroxylation. However, in an esterified form, long-acting 17-OH progestogens are in clinical use as parenteral progestogens (17cehydroxyprogesterone valerate and caproate). Absorption of oral progesterone is inefficient. Rapid hepatic hydroxylation and conjugation (first-pass effects)occur, resulting in increases in pregnanediol-3-glucuronide, the major urinary metabolite. Nevertheless, oral crystalline progesterone, which has been used with some efficacy in postmenopausal women [31 ], results in progesterone concentrations of 5 to 10 ng/ml when 300-mg doses are used. A more efficient means of ingesting progesterone is with a micronized product, Utrogestin or Prometrium, now available in the United States, which may be administered orally in doses of 100 to 200 mg. When administered in divided doses (100 mg at 9 a.m. and 200 mg at 9 p.m.), peak serum progesterone concentrations of more than 10 ng/ml are encountered (Fig. 1) [32]. These findings are associated with significant endometrial progestational activity. The PEPI trial demonstrated that 200 mg daily for 12 days affords beneficial endometrial protection [12]. Oral administration of progesterone is subject to first-pass effects. Characteristic of these changes is the rapid conversion of progesterone to desoxycorticosterone, an effect that

431

CHAPTER 30 Progestogens

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FIGURE 1 Meanconcentrations of progesterone, pregnanediol 3c~-glucuronide, 17-hydroxyprogesterone, and 20c~-dihydroprogesterone in the peripheral plasma of postmenopausal women before, during, and after administration of oral progesterone. Pretreatment, days 1 and 2; after 5 days of treatment with 100 mg of progesterone at 9 hr and 200 mg at 21 hr, day 7; and posttreatment, days 9 to 11. Adapted from Ref. 32, by permission from the American Society for Reproductive Medicine (Fertility and Sterility, 1986, Vol. 46, p. 402). does not occur readily with the systemic route [33,34]. When equal doses of progesterone are administered orally and intramuscularly, despite a threefold increase in serum levels with the intramuscular route, the ratio of desoxycorticosterone/progesterone was much higher than with the oral route (Fig. 2). These data, which confirm hepatic effects characteristic of first'pass metabolism, are interesting but await clinical relevance. Native progesterone is well absorbed vaginally and rectally [35,36] as well as nasally [37], but does not compare with the high concentrations achieved by the intramuscular route. These routes of administration may result in higher circulating progesterone compared to the oral route, because the progesterone is not subject to first-pass changes.

B. Synthetic Progestogens Of the two classes of synthetic progestogens, the 17acetoxy group (medroxyprogesterone acetate) has properties closest to native progesterone. Apart from the 17-acetoxy function of this class, the manipulations at C-6 (CH 3 for medroxyprogesterone acetate) produce increased progestational effects and oral efficacy. The 19-norprogestogens, by virtue of an ethinyl group at C-17 and the removal of the nineteenth carbon from C-10, have increased progestational activity, oral efficacy, and reduced androgenic activity, even though this class is derived from testosterone. However, despite the C-19 ethinyl group, which tends to nullify androgen action, characteristics of some members of this group

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(particularly norethindrone and levonorgestrel) are their androgenic side effects. Oral absorption of these synthetic progestogens is variable. For this and other reasons, it has been difficult to ascribe potency ratios for these various progestogens. The most commonly used progestogen is medroxyprogesterone acetate, which constitutes the majority of prescriptions for postmenopausal patients in the United States. Absorption of medroxyprogesterone acetate after a 10-mg oral dose is fairly rapid, reaching concentrations of 3 to 4 ng/ml within 1 to 4 hr, and declining to 0.3 to 0.6 ng/ml by 24 hr. However, these characteristics are variable, and major differences have been noted in medroxyprogesterone acetate pharmacokinetics in postmenopausal women (Fig. 3) [38]. Similar characteristics occur with administration of norethindrone. After ingestion of 1 mg, peak concentrations occurred within 1 hr in 16% of women, within 2 hr in 51%, and after 2 hr in 33% [39]. Although these data pertain to oral contraceptive users, at least this degree of variability would be expected in postmenopausal women. In the case of 1 mg of norethindrone, peak values of 5 ng/ml occur, which then decline to less than 1 ng/ml within 24 hr. However, these concentrations, although higher than those achieved with 10 mg of medroxyprogesterone acetate, are still only 60% of those achieved by parenteral administration.

FIGURE 3 Plasmalevels of medroxyprogesterone acetate after oral administration. Measurement of medroxyprogesterone acetate by radioimmunoassay. Adapted from Ref. 38, J. C. Cornette, K. T. Kirton, and G. W. Duncan; Measurement of medroxyprogesterone acetate by radioimmunoassay. J. Clin. Endocrinol. Metab. 33, 459. 9 The Endocrine Society.

This is not the case with levonorgestrel, in which oral and parenteral administration lead to similar levels [40]. Thus, we see significant first pass effects and portal-hepatic inactivation with these three progestogens. Medroxyprogesterone acetate is most affected, followed by norethindrone. Levonorgestrel is least affected. These data also correlate with the known differences in potency of these progestogens (levonorgestrel being the most potent) and with the variability observed in postmenopausal endometria (medroxyprogesterone acetate having the greatest variability), as reviewed elsewhere. The newer 19-nor progestogens related to norgestrel (norgestimate, desogestrel, and gestodene) are not widely used in postmenopausal women. The profiles and characteristics of these compounds, however, are similar to those of levonorgestrel.

C. Progestogen Metabolism Progesterone is metabolized primarily along the pathway that produces pregnanedione, pregnenolone and pregnanediol [41,42]. Table I summarizes the most important progesterone metabolites. Overall, about 50% of endogenous progesterone follows the 5ce pathway [43,44]. Another 35% follows 3/3 metabolism, and 9% is processed through the 20ce pathway. In vivo, only 3% is transformed into desoxycortisone, mostly in the kidney, where it has mineralocorticoid activity. Very little progesterone is 17-hydroxylated through peripheral metabolism, although the ovary (corpus

CHAPTER30 Progestogens

433 TABLE I

Important Progesterone Metabolitesa Route of serum metabolite concentration

Chemical Progesterone 5a-dihydroprogesterone [5a-(allo)pregnan-3,20-dione] 5/3-Dihydroprogesterone 3/3-Dihydroprogesterone 20a-Dihydroprogesterone (20a-hydroxypregn-4-en-3-one) 20/3-Dihydroprogesterone (20/3-hydroxypregn-4-en-3-one) 21 a-Desoxycorticosterone (21a-deoxycortisone) 17-Hydroxyprogesterone

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100

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50

33

50

35 10

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Vaginal

Corpus luteum/liver Liver + Liver +

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luteum) is an important source of 17-hydroxyprogesterone during the menstrual cycle [45]. In spite of major differences in all other aspects of progesterone metabolism between species, all species studied appear to share a high metabolic clearance rate for progesterone [46]. About 75% of progesterone metabolism takes place in the liver and spleen. Much of the rest (for example, 21-hydroxylation) occurs in the kidney and this metabolism is probably potentiated by estrogen [47,48]. 21Hydroxylase activity is also found in the aorta, thymus, and spleen [48,49]. The role of the brain in progesterone metabolism is complex. The brain has been shown to account for 10% of the extrasplanchnic clearance of progesterone from the blood of rhesus monkeys [46], but there is considerable variation between species and between different parts of the brain in the ability to metabolize progesterone. Higher concentrations are found in the midbrain and hindbrain, suggesting that these, in addition to the limbic and hypothalamic areas, are sites of sex behavior and gonadotropin control in addition to the limbic and hypothalamic areas. Oral administration of progesterone results in high concentrations of 20a-dihydroprogesterone, as well as other metabolites, namely, 5a- and 3ce-dihydroprogesterone, and 21 adesoxycortisone, which are the result of poor absorption of progesterone. Micronization of progesterone increases bioavailability, as demonstrated by the pattern of metabolites in Fig. 1 [32]. Changing the route of progesterone administration dramatically changes the level of production and distribution of metabolites. For instance, vaginal progesterone produces the expected high amounts of 20a-dihydroprogesterone and low amounts of 17-hydroxyprogesterone, but it produces virtually no 5ce- or 5fl-dihydroprogesterone [43], which result from first-pass hepatic metabolism. These metabolites are

significant products after oral administration (Fig. 4), [50]. Among the metabolites, the structure of the A ring is critical. Without the A-4-3 ketone feature, the steroid is not a progestogen. Furthermore, the 5ce position is a specific binding site for gamma-aminobutyric acid (GABA) activity, resulting in central nervous system (CNS) effects [51 ]. The 19-nor derivatives alter this A ring to develop enhanced binding activity [52]. Structural changes in progesterone, such as alterations in the C-3 or C-17 position, produce progestogens that have increased oral bioavailability. 1. BINDING

Although roughly 80% of circulating progestogens are bound to serum proteins, such as albumin, the affinity is weak, with little physiologic relevance. The dissociation constant is on the order of 10-4 kDa. About 18% of progesterone is bound by cortisone-binding globulin (CBG), and this increases to 40% during pregnancy as CBG levels increase [53]. A small amount is also bound to sex hormone binding globulin. Tissue binding also occurs. Uteroglobulin, a small secretory protein expressed in the uterus and several other tissues, is strongly induced by progestins and binds progesterone with high specificity [54]. Breast cyst fluid contains large amounts of apolipoprotein D, which also binds progesterone. Because cystic breast disease increases the risk of breast cancer by a factor of 2 - 4 , further investigation into this chemical and its relationship to progesterone and carcinogenesis is warranted. 2. ELIMINATION

The 5a (more than the 5fl) pathway is the primary elimination route for progesterone. Next in importance are the 3a, 3fi, and 20ce pathways, followed by the conjugates, glucuronides, and sulfates. Hydroxylated metabolites are

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eliminated principally in the urine, accounting for 50% of a labeled injection of progesterone, whereas ketonic compounds, 30% in the same study [55], are excreted chiefly in the bile. The principal urinary metabolite is 3c~-pregnanediol glucuronide.

D. Individual Variability Another important observation is the large variation in these hormones between individuals. An example of this variation is found in Cornette's study [38] of serum concentrations of medroxyprogesterone acetate after oral administration (Fig. 3). Although the levels of MPA in this study reflect an older assay for MPA, the approximate twofold variation in peak values is typical of reduced and variable bioavailability with various progestogens. Progestogens with less bioavailability (e.g., progesterone and MPA) versus those with greater bioavailability (e.g., 19-norprogestogens) are subject to greater variability. Variation is great not only between individuals but also within the same individual during different physiologic states. Various stages of pregnancy, different phases of the menstrual cycle, and menopausal changes all cause dramatic changes in progesterone metabolism [45]. On a theoretical basis, therefore, substantial differences in estrogen concentrations in postmenopausal women would be expected to influence progesterone metabolism.

E. Progestogen Action on the Endometrium Simplistically, progestogens prevent endometrial proliferation because of an antiestrogenic effect. Progestogens inhibit estrogen action by inhibiting estrogen receptor functions but also through a variety of other biochemical mechanisms. Three important concepts are key to the understanding of the

progestogen protective effects: (1) protective biochemical changes with progestogens occur prior to morphological changes; (2) biochemical and morphological changes may be dissociated, and it is the former changes that are more important; and (3) progestogen length of treatment is more important than dose. Progestogens inhibit nuclear estradiol receptors and therefore inhibit mitotic activity and thymidine incorporation as well as induce other biochemical changes [56] (Fig. 5). Progestogens promote isocitric and estradiol dehydrogenous activity, which enhances the conversion of extracellular estradiol to the weaker estrone, and progestogens enhance aryl sulfatase activity, which promotes the formation of estrone sulfate for elimination. These key enzymatic steps are time dependent but are significantly present within 6 - 7 days after progestogen exposure [56]. Low doses are sufficient to inhibit these enzymatic changes. Thus 0.3 mg of norethindrone is as effective as 5 mg for inhibiting nuclear estradiol receptors (Fig. 6). Morphological changes, however, lag behind biochemical changes and full secretory changes may not occur at all with lower doses of progestogens. Thus doses as low as 2.5 mg of medroxyprogesterone acetate can inhibit biochemical changes induced by estrogen (and therefore should be protective) yet may not result in secretory formulation of the endometrium. We demonstrated several years ago that the proliferative effects of 0.625 mg of conjugated equine estrogen (CEE) could be inhibited equally well by 2.5, 5, or 10 mg of MPA, although morphology was vastly different (Fig. 7) [57]. Proof that biochemical changes are more important than morphology may be found in a study by Moyer [58]. With cyclic doses of micronized progesterone (200 mg), mitotic activity was inhibited although the endometrium did not exhibit full secretory changes and some were weakly proliferative. Yet with this regimen, which included moderate doses of estrogen for 5 years, there were no cases of hyperplasia. Morphological changes more significantly influence the

CHAPTER 30 Progestogens

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FIGURE 6 Effects of progestins on estrogen-primed, postmenopausal endometrium. REN, Nuclear estradiol receptor; epithelial labeling index; [3H]thymidine labeling of glandular epithelium. The mean secretory level was given a value of 1 (dashed horizontal line), and each of the postmenopausal values was calculated as a proportion of the mean secretory level. The results are given as mean _+ SEM of at least six analyses. Adapted from Ref. 59, by permission from the American Society for Reproductive Medicine (Fertility and Sterility, 1986, Vol. 46, pp. 1062-1066).

436

ROGERIOA. LOBO E

Progestogen Potency

Although it has been difficult to ascribe differences in potency of progestogen used to prevent hyperplasia, a summary has been provided by King and Whitehead [59], (Table II) using a sequential progestogen regimen. As pointed out earlier, whereas 2.5 mg of MPA provides adequate protection against the proliferative effects of 0.625 mg of CEE, 10-mg doses are required to achieve full secretory changes. However, as little as 0.3 mg of norethindrone (NET) suffices to afford significant biochemical and morphological changes. A "consensus" score weighs in the effects on biochemistry and morphology. Equivalent doses are suggested to be 200 mg of micronized progesterone, 5 mg of MPA, and 0.35 mg of NET.

G. C l i n i c a l T r i a l s o n P r o g e s t o g e n s U s e d to P r e v e n t H y p e r p l a s i a

FIGURE 7 Concentrationsof cytosolic estrogen receptors in the three groups of postmenopausal women receiving no therapy (B), conjugated equine estrogens (CEE), and estrogen plus various dosages of medroxyprogesterone acetate (MPA). *, p < 0.001; **, p < 0.025. Adapted from Ref. 57.

pattern of withdrawal bleeding. With sequential regimens, the more fully secretory the endometrium is, the more bleeding and cramps will occur, which are characteristics of cyclic menstruation. On the other hand, low doses of progestogens, which may protect the endometrium biochemically, may lead to more irregular bleeding because of unpredictable morphological changes. This in turn is more marked if there is lower bioavailability, in the case of certain progestogens, and due to individual variability in responses between women. TABLE II

In prospective randomized clinical trials, the effects of progestogen on estrogen-induced hyperplasia have been assessed. Estrogen at a dose equivalent of 0.625 mg of conjugated equine estrogens results in a hyperplasia rate of approximately 20% per 1 year [60]. Although this is principally cystic hyperplasia, longer duration of unopposed estrogen has been shown to result in more severe lesions [12]. Higher doses, such as 1.25 mg of CEE, result in a hyperplasia rate of 57%. MPA either in continuous (2.5 mg) or sequential regimens (either 5 or 10 mg), for 10-14 days, leads to a rate of hyperplasia that is similar to placebo [60]. A sequential regimen of micronized progesterone, 200 mg for 12 days, was similarly effective [12]. The finding that sequential regimens of MPA at 5 or 10 mg give a similar protective effect is in keeping with the notion that the length of exposure is an extremely important feature of progestogen treatment. The placebo effect of approximately 1% hyperplasia risk

Oral Doses of Progestins Required to Elicit Premenopausal Secretory Changes" Dose (mg)

Progestin Norethindrone Levonorgestrel Medroxyprogesteroneacetate Progesterone

Nuclear estradiol Estradiol Morphologic receptor dehydrogenase value <0.35 0.15 < 2.5 200

<0.35 <0.075 < 2.5 250

0.35 0.075 > 10 >300

Consensus b 0.35 0.075 5 200

a Adapted from Ref. 59, by permission from the American Society for Reproductive Medicine (Fertility and Sterility, 1986, Vol. 46, pp. 1062-1066). b Dose tested (mg) that best fits all the parameters analyzed.

CHAPTER 30 Progestogens

437

at 1 year has become a standard against which all newer regimens are expected to achieve for Food and Drug Administration (FDA) approval. Additional data have also suggested that lower doses of esterified estrogens, such as 0.3 mg (slightly less potent than CEE), result in a rate of hyperplasia that is similar to placebo [61]. Higher doses were shown to increase the risk in a fashion similar to that of CEE (Fig. 8). At least three other studies have confirmed the original observation by Ettinger [62], that progestogen exposure for 14 days every 3 months is sufficient to reduce the risk of hyperplasia, with the equivalent of CEE, 0.625 mg, at 1 year. Although this is a reasonable option for some women, there are no published long-term studies to date, and the withdrawal bleeding that occurs is usually heavy and prolonged. Progestogen therapy is protective to the endometrium but it can only prevent the excess risk associated with unopposed estrogen use. A base line rate of endometrial disease, including cancer, can still occur. This speaks to the need for continuous surveillance. The use of ultrasound and other modalities has been reviewed extensively elsewhere [63]. It is important to realize that endometrial cancers, which develop with unopposed estrogen exposure, are of the "endometrioid" type. These lesions are well differentiated, are positive for estrogen and progesterone receptors, and generally do not result in excess mortality [6]. Endometrial cancers, which can develop de novo, with or without hormonal replacement, are usually of the "serous" type. These lesions are poorly differentiated and are usually devoid of or have low abundance of estrogen and progesterone receptors. These cancers can develop in the presence of an atrophic endometrium and may arise even with an "idealized" hormonal regimen. Such lesions have been described in women after many years of continuous combined therapy [64]. Thus any new unexpected finding needs to be evaluated thoroughly in postmenopausal women, regardless of the treatment regimen. PROGESTOGEN REGIMENS AND BLEEDING PATTERNS

Various regimens are now used for women with a uterus (Table III). It is commonly accepted to use estrogen, whether

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TABLE III Various Options for Endometrial Protection with Progestogens Sequential for 12-14 days each month Continuous combined mdaily dose Sequential combinedmestrogen with progestogen daily for 25 days Cyclophasic or "pulsed" Intermittent--every 3 months Intrauterine progestogen

it is oral or nonoral, on a daily or continuous basis. Daily and continuous regimens are the most popular and have been in use for the longest time. They also have been the focus of numerous studies, as discussed earlier. The pattern of bleeding with sequential regimens (12-14 days of progesterone exposure) is usually predictable, with approximately 80% of women experiencing withdrawal bleeding [65]. However, it is important to note that with sequential regimens, if the dose of progestogen is low and there is no secretory transformation of the endometrium, with time bleeding decreases and a high rate of amenorrhea (80-90%) can be achieved after 5 years [58]. Whereas larger doses of progestogen result in cyclic predictable withdrawal bleeding such as with normal menstruation, lower doses may cause more irregular bleeding to occur before day 12 or 14 of progestogen administration. Bleeding prior to day 9 of progestogen administration usually signifies a nonsecretory endometrium [66]. The continuous combined regimen causes more irregular bleeding in the first few months, because atrophy is being established and is more likely to be prolonged in younger postmenopausal women. By the end of 1 year, over 80% of women achieve amenorrhea [64,65]. For many women, the constant irregular bleeding early on is a deterrent to continuing this regimen; however, other women, particularly those who achieve amenorrhea early, readily accept this option. Larger doses of progestogen used continuously may lead to amenorrhea earlier [65]. A sequential combined regimen constitutes the use of the routine continuous regimen, but only for 25 days each m o n t h - - f o r example, CEE (0.625 mg) and MPA (2.5 mg) daily for only 25 days. This regimen has been advocated for those women with continuous spotting on the daily continuous regimen and has been reported to result in a greater percentage of amenorrhea [67,68]. The hormone-free days (5 per month) are thought to be beneficial in preventing abnormal vascular patterns in the endometrium caused by continuous progestogen exposure [69]. A new "pulsed" or cyclophasic regimen has been introduced. This regimen employs continuous estrogen but with an intermittent (pulsed) progestogen exposure, 3 days on and 3 days off [70]. This approach has been advocated (on a theoretical basis) to minimize progestogen exposure and to allow more effective progestogen action by not achieving

438

ROGERIO A. LOBO

continuous down-regulation of receptors [70]. Early studies have suggested an acceptable bleeding profile [71] and a metabolic profile [72] that may be somewhat more beneficial than with continuous progestogen exposure. More long-term studies are needed. The intermittent (every 3 months) regimen, discussed previously [62], is an option for women who do not tolerate more constant progestogen use. However, patients on this regimen often dread the progestogen dosing every 3 months, because it is followed by heavy bleeding. Also, there has been some concern that on a long-term basis, this regimen may not be as protective as other regimens. Because the uterus is the target of progestogen use, more direct approaches have been advocated. Vaginal administration of progesterone achieves higher endometrial, as compared to systemic, concentrations [73-75] (Fig. 9). Vaginal tablets, capsules, or gels have been used with success to limit systemic effects, particularly in women who are relatively intolerant to progestogen use because of side effects. The most direct approach has been to use an intrauterine device (IUD). The progesterone-impregnated IUD (Progestasert) has been shown to be acceptable for 1 year [76] as a means to prevent hyperplasia and bleeding. A newer levonorgestrel IUD that releases levonorgestrel for 3 - 4 years is being studied and shows great promise in causing amenorrhea [77]. The only concern is that this regimen may not be possible or acceptable to all women because of difficulties in the insertion of an IUD in a postmenopausal uterus. Progesterone creams, which have been sold for topical use on the skin, result in minimal blood concentrations of progesterone. When large doses are used, blood concentrations increase but are unpredictable [77a,77b]. These methods are inefficient from an absorption perspective and require large amounts on a constant basis. They are a systemic form of therapy, in contrast to providing progesterone locally as with the "uterine" methods described above.

a 20-

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2

3

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H. Potential Concerns with Progestogen Administration Progestogen effects on the breast and bone were discussed in Section II. Advocates for using progestogens in a woman without a uterus point to a beneficial effect on bone mass. However, if there are beneficial effects on bone, these are likely to be relatively minor. At the same time, there remains some concern that progestogens may increase breast tissue proliferation and thus the risk of breast cancer [14]. An analysis by Greendale using the PEPI data set showed that all progestogen regimens lead to greater breast density in mammographic studies [78]. Additional potential concerns of progestogen exposure, which may lead to limitations of their use, are potential effects on cardiovascular function and CNS effects.

intravaginally administered micronized P (o; n = 15) and after intramuscularly administered P (o; n = 5); *, p < 0.05 compared with intravaginal. (b) Endometrial P concentrations (mean _+ SE) in group I (intravaginal micronized P; n = 15), group II (intramuscular P; n = 5), and control group (normal ovulating women; n = 4); *, p < 0.05 compared with controls. Adapted from Ref. 73, by permission from the American Society for Reproductive Medicine (Fertility and Sterility, 1994, Vol. 62, pp. 485-490).

1. C A R D I O V A S C U L A R E F F E C T S OF P R O G E S T O G E N S

Cardiovascular effects can be divided into those that are lipid/lipoprotein related and other effects. As reviewed previously [79,80], the major effect of added progestogen is an attenuation of the high-density lipoprotein (HDL)cholesterol rise that occurs with oral estrogen. This effect is influenced by the dose and type of progestogen and the length of exposure [79,81,82]. Micronized progesterone (MP) appears to have the least effect, as demonstrated in the PEPI

CHAPTER 30 Progestogens

439

trial [83] (Fig. 10). The study by Lobo et al. [80], which compared several regimens of MPA plus CEE with CEE alone showed that all MPA regimens decreased the HDL cholesterol rise by 50%. Although there is a slight attenuation of the rise in total triglycerides with progestogens, there is no beneficial or deleterious effect on total or lowdensity lipoprotein (LDL) cholesterol or in lipoprotein (a) [Lp(a)] [80,83]. Because most of the progestogen-mediated lipoprotein changes (HDL cholesterol, triglycerides) are related to oral first-pass effects, there is less of an effect with transdermal progestogen and other alternate delivery systems. Indeed, with transdermal estrogen there is also a minimal effect on lipoproteins. Perhaps the nonlipid effects are of less concern in that up to 70% of the benefit of estrogen has been considered to be nonlipid/lipoprotein related [84]. Estrogen-mediated blood flow (in all vascular levels) is attenuated by progestogen [85-88]. Progestogens attenuate the benefit of estrogen on coronary atherosclerosis and have a negative effect on endothelium-based vasodilation and vasomotion [89,90] (Figs. 11

and 12) [90a,90b]. These effects are influenced by the type and dose of progestogen. There are now several studies, along with those mentioned above, that suggest an attenuating effect of MPA specifically on estrogen-induced vasomotion and atherosclerosis [89-93]. In some cases, the effect of estrogen is eliminated. Progesterone appears to have the most inert effect (Fig.11) [89,91]. Two observational cohort studies, however, have suggested that the addition of progestogens to estrogen does not attenuate the cardiovascular benefit [92,93]. Nevertheless, we do not know about pill compliance with these regimens or the characteristics of the women in these cohorts who took progestogen. The Heart and Estrogen/progestin Replacement Study (HERS) trial [94] showed no secondary prevention benefit of a continuous combined regimen compared to placebo in older women with advanced coronary disease. Although these data are unexpected and there may be several reasons for these findings, they do suggest that continuous progestogen exposure should be used with caution in women with known coronary disease, particularly those who have

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FIGURE 10 Mean percent change from base line by treatment arm for high-density lipoprotein cholesterol (top left), low-density lipoprotein cholesterol (top right), triglycerides (bottom left), and total cholesterol (bottom right). CEE, Conjugated equine estrogens; MPA, medroxyprogesterone acetate; ME micronized progesterone. Adapted from Ref. 83, J. Am. Med. Assoc. 273, 199-208. Copyright 1995 American Medical Association.

440

FIGURE 11 Estrogenreplacement therapy vs. hormone replacement therapy: primary prevention of coronary artery atherosclerosis. The data from these two studies demonstrate that coronary artery atherosclerosis was reduced by about one-half when treated (TX) with estrogen (E2) replacement or estrogen plus progesterone (E2+) replacement. The data also indicate that medroxyprogesterone acetate (MPA) administered continuously attenuates the atheroprotective effect of unopposed conjugated equine estrogens (CEE). (A) Adapted from Ref. 89. (B) Adapted from Ref. 90.

ROGERIO A. LOBO diovascular function include effects on carbohydrate tolerance and coagulation. Progestogens in large doses are known to induce hyperinsulinemia and insulin resistance, and may attenuate the increase in insulin sensitivity observed with moderate doses of estrogen (Fig. 13) [97-98a]. These effects are magnified by the dose of progestogen and the route of administration, with the oral route having the most significant effect. There are no apparent adverse effects of added progestogens on coagulation risk factors [80,83]. Procoagulant changes induced by estrogen are not different with added progestogen and there is evidence for minor beneficial changes, such as higher levels of plasminogen [80].

2. PSYCHOLOGICAL EFFECTS OF MOOD DISTURBANCES Whereas estrogen has been shown to improve mood and psychological well being in postmenopausal women, progestogens have been shown to have an adverse effect, at least in some women [99-101]. Again, the type, dose, and duration of progestogen use are important variables that contribute to these effects [ 102]. Progestogen "intolerance" in terms ofdepressive mood and other psychological symptoms (irritability, anxiety) is a major reason for noncompliance with any hormonal regimen. Although MPA has been most implicated in causing m o o d disturbances, a recent double-blind cross-over study has shown that norethindrone may cause adverse effects on m o o d when compared to the effects of estrogen alone (Fig. 14) [ 101 ]. In addition, there may be "somatic" side effects of adding progestogens, including the feelings of breast tenderness, bloating, and menstrual cramps.

FIGURE 12 Treatment(TX) employing estrogen (E2) replacement therapy (ERT) vs. hormone replacement therapy (HRT): coronary artery reactivity. Findings demonstrating that coronary vasdodilatory response to acetylcholine is dependent on estrogens and, furthermore, are reversed when medroxyprogesterone acetate (MPA) is added, either cyclically or continuously. CEE, Conjugated equine estrogens. (A) Adapted from Ref. 90a. (B) Adapted from Ref. 90b. recently had a myocardial infarction. MPA in particular, according to studies in monkeys, may have an adverse vasoconstrictive effect on coronary vessels [91]. Whether this effect is relevant only to monkeys and not to women is not clear at present. The cohort studies showing no attenuating effect of progestogens on coronary disease [92,93] employed older sequential regimens of progestogen administration rather than a continuous regimen. The clinical studies showing adverse effects of progestogens on coronary architecture and vasodilation have been carried out in animals; human studies are now in progress. To date only the more indirect measures have been assessed, such as effects on blood flow, circulating levels of nitrites/ nitrates (endothelium-derived relaxing factor), and prostacyclin metabolites [95,96]. Other potentially adverse aspects of progestogen on car-

FIGURE 13 Percentagechanges from base line after conjugated equine estrogen (CEE) and CEE with medroxyprogesteroneacetate (MPA) in postmenopausal women, with values as indicated. *, significant change from base line, p < 0.05. Adapted from Ref. 98a, by permission of Elsevier Science from Comparison of estimates of insulin sensitivity in pre and post menopausal women using the insulin tolerance test and the frequently sampled intravenous glucose tolerance test, by S. R. Lindheim, T. A. Buchanan, D. M. Duffy, M. A. Vijod, T. Kojima, F. Stanczyk, and R. A. Lobo, Journal of the Society for Gynecologic Investigation, Vol. 1, pp. 150-154. Copyright 1994 by the Society for Gynecologic Investigation.

CHAPTER 30 Progestogens

441

Trend F Tests Linear

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FIGURE 14 Trend analysis, profile of mood states tension-anxiety (POMS T-A) factor, long-menopausalduration attribute group (increased scores reflect improved mood); n = 17. PLCB, placebo; E, estrogen alone; E + P, estrogen plus progestin. Adapted from Ref. 101, Psychoneuroendocrinology, Vol. 21; E. L. Klaiber, D. M. Broverman, W. Vogel, L. G. Peterson, and M. B. Snyder; Individual differences in changes in mood and platelet monoamine oxidase (MAO) activity during hormonal replacement therapy in menopausal women, pp. 575-592. Copyright 1996, with permission from Elsevier Science. The very severe adverse progestogen effects occur in 1 0 20% of w o m e n on h o r m o n e r e p l a c e m e n t therapy. In the PEPI trial, however, no significant adverse effects of p r o g e s t o g e n s could be demonstrated [103], although the test instruments m a y not have been sufficiently sensitive to detect changes. Progestogens may also attenuate the beneficial effects of estrogen on stress responses [ 104]. P o s t m e n o p a u s a l w o m e n have enhanced biophysical (blood pressure, heart rate) and n e u r o h o r m o n a l (epinephrine) responses to stress and these are improved by estrogen; yet this i m p r o v e m e n t is blunted by progestogen. These findings may be most relevant for cardiovascular health, but the data also suggest that progestogens may alter psychological aspects of dealing with stress. Beneficial cognitive effects of estrogen may be attenuated by progestogens, although there are few c o m p l e t e d studies on this point. In one clinical study on cognition [105], progestogens did adversely affect certain cognitive domains. The mechanisms for this possible effect are likely to be multiple and may involve reduction in cerebral blood flow as well as interference with serotonin and GAB A pathways, as alluded to previously. In addition, progestogens m a y interfere with the protrusion of dendritic spines from neuronal dendrites [ 106]. IV.

CONCLUSIONS

Progestogens should be considered as essential for protecting the e n d o m e t r i u m from developing endometrial disease with u n o p p o s e d estrogen. Even with " i d e a l " progestogen therapy certain undifferentiated endometrial cancers can develop, which confirms the necessity for continued surveillance of all w o m e n for all types of cancer. B e c a u s e some potential adverse effects can occur with progestogens, there

should be flexibility with prescribing patterns. In addition, progestogens should be used with caution in w o m e n with severe coronary disease. In this setting, options include the use of u n o p p o s e d low-dose estrogen, micronized progesterone, and nonsystemic forms such as transvaginal or intrauterine therapy. These options also remain for w o m e n who have side effects and are intolerant to progestogens.

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26.

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32.

33.

34.

35.

36.

37.

38.

39. 40.

41.

42.

D. R. (1982). Effects of progestins on bone metabolism in postmenopausal women. J. Reprod. Med. 27(Suppl. 8), 511-514. Gallagher, J. C., Kable, W. T., and Goldgar, D. (1991). Effect of progestin therapy on cortical and trabecular bone: Comparison with estrogen. Am. J. Med. 90, 171-178. Lau, K.-H. W., Wang, S. P., Linkhart, T. A., Demarest, K. T., and Baylink, D. J. (1994). Picomolar norethindrone in vitro stimulates the cell proliferation and activity of a human osteosarcoma cell line and increases bone collagen synthesis without an effect on bone resorption. J. Bone Miner. Res. 9, 695-703. Writing Group for the PEPI Trial (1996). Effects of hormone therapy on bone mineral density: Results from the Post-menopausal Estrogen/Progestin Interventions (PEPI) trial. JAMA, J. Am. Med. Assoc. 276, 1389-1396. Christiansen, C., and Riis, B. J. (1990). Five years with continuous combined oestrogen/progestogen therapy: Effects on calcium metabolism, lipoproteins, and bleeding pattern. Br. J. Obstet. Gynaecol. 97, 1087-1092. Speroff, L., Rowan, J., Symons, J., Genant, H., and Wilborn, W. (1996). The comparative effect on bone density, endometrium, and lipids of continuous hormones as replacement therapy (CHART Study): A randomized controlled trial. JAMA, J. Am. Med. Assoc. 276, 1397-1403. Whitehead, M. I., Townsend, P. T., Gill, D. K., Collins, W. P., and Campbell, S. (1980). Absorption and metabolism of oral progesterone. Br. Med. J. 280, 825-827. Padwick, M., Endacott, J., Matson, C., and Whitehead, M. I. (1986). Absorption and metabolism of oral progesterone when administered twice daily. Fertil. Steril. 46, 402. Ottosson, U. B., Carlstrom, K., Damber, J. E., and von Schoultz, B. (1984). Serum levels of progesterone and some of its metabolites including deoxycorticosterone after oral and parenteral administration. Br. J. Obstet. Gynaecol. 91, 1111-1119. Ottosson, U. B., Carlstrom, K., Damber, J. E., and von Schoultz, B. (1984). Conversion of oral progesterone into deoxycorticosterone during postmenopausal replacement therapy. Acta Obstet. Gynecol. Scand. 63, 577. Nillius, S. J., and Johansson, E. D. B. (1971). Plasma levels of progesterone after vaginal, rectal or intramuscular administration of progesterone. Am. J. Obstet. Gynecol. 110, 470-477. Whitehead, M. I., Siddle, N. C., and Townsend, P. T. (1982). The use of progestins and progesterone in the treatment of climacteric and postmenopausal symptoms. In "Progesterone and Progestin" (C. W. Bardin, E. Milgrom, and P. Mauvis-Jarvis, eds.). Raven Press, New York. Steege, J. E, Rupp, S. L., Stout, A. L., and Bernhisel, M. (1986). Bioavailability of nasally administered progesterone. Fertil. Steril. 46, 727-729. Cornette, J. C., Kirton, K. T., and Duncan, G. W. (1971). Measurement of medroxyprogesterone acetate by radioimmunoassay. J. Clin. Endocrinol. Metab. 33, 459. Fotherby, K. (1983). Variability of pharmacokinetic parameters for contraceptive steroids. J. Steroid Biochem. 19, 817- 820. Humpel, M., Wendt, H., Pommerenke, G., Weiss, C., and Speck, U. (1978). Investigations of pharmacokinetics of levonorgestrel to specific consideration of a possible first-pass effect in women. Contraception 17, 207-220. Mahesh, V. B., Brann, D. W., and Hendry, L. B. (1996). Diverse modes of action of progesterone and its metabolites. J. Steroid Biochem. Mol. Biol. 56, 209-219. Acedo, A. R., Landgren, B. M., Cekan, Z., and Diczfalusy, E. (1976). Studies on the pattern of circulating steroids in the normal menstrual cycle. 2. Levels of 20-alpha-dihydroprogesterone, 17-hydroxyprogesterone and 17-hydroxypregnenolone and the assessment of their value for ovulation prediction. Acta Endocrinol. (Copenhagen) 82, 600-616.

CHAPTER 30 Progestogens 43. Backstrom, T., Andersson, A., Baird, D. T., and Selstam, G. (1986). The human corpus luteum secretes 5ce-pregnane-3,20-dione. Acta Endocrinol. (Copenhagen) 111, 116-121. 44. Broom, T. J., Johnson, D. W., Phillipou, G., and Seamark, R. F. (1983). Applications of heavy isotope tracers to clinical studies: Progesterone urinary production rate determination in the menstrual cycle and pregnancy. J. Clin. Endocrinol. Metab. 56, 346-351. 45. Florensa, E., Harrison, R., Johnson, M., and Youssefnejadian, E. (1977). Plasma 20ce-dihydroprogesterone, progesterone and 17hydroxyprogesterone in normal human pregnancy. Acta Endocrinol. (Copenhagen) 86, 634-640. 46. Little, B., Billiar, R. B., Rahman, S. S., Johnson, W. A., Takaoka, Y., and White, R. J. (1975). In vivo aspects of progesterone distribution and metabolism. Am. J. Obstet. Gynecol. 123, 527-534. 47. Casey, M. L., MacDonald, E C., and Winkel, C. A. (1985). Deoxycorticosterone biosynthesis in kidney tissue of experimental animals: Characterization of steroid 21-hydroxylase activity in guinea pig kidney. Ann. N.Y. Acad. Sci. 458, 232-237. 48. Winkel, C. A., Simpson, E. R., Milewich, L., and MacDonald, E C. (1980). Desoxycorticosterone biosynthesis in human kidney: Potential for the formation of a potent mineralocorticosteroid in its site of action. Proc. Natl. Acad. Sci. U.S.A. 77, 7069-7073. 49. Winkel, C. A., Parker, C. R., Jr., Simpson, E. R., and MacDonald, E C. (1980). Production rate of deoxycorticosterone in women during the follicular and luteal phases of the ovarian cycle: The role of extraadrenal 21-hydroxylation of circulating progesterone in deoxycorticosterone production. J. Clin. Endocrinol. Metab. 51, 13541358. 50. de Lignieres, B., Dennerstein, L., and Backstrom, T. (1995). Influence of route of administration on progesterone metabolism. Maturitas 21, 251-257. 51. Lan, N. C., and Gee, K. W. (1994). Neuroactive steroid actions at the GABA A receptor. Horm. Behav. 28, 537-544. 52. Baulieu, E. E. (1997). Neurosteroids: Of the nervous system, by the nervous system, for the nervous system. Recent Prog. Horm. Res. 52, 1-32.

53. Dunn, J. E, Nisula, B. C., and Rodbard, D. (1981). Transport of steroid hormones: Binding of 21-endogenous steroids to both testosterone-binding globulin and corticosteroid-binding globulin in human plasma. J. Clin. Endocrinol. Metab. 53, 58-68. 54. Peter, W., Beato, M., and Suske, G. (1989). Recombinant rabbit uteroglobin expressed at high levels in E. coli forms stable dimers and binds progesterone. Protein Eng. 3, 61-66. 55. Sandberg, A. A., and Slaunwhite, W. R., Jr. (1958). The metabolic fate of Ca~4-progesterone in human subjects. J. Clin. Endocrinol. Metab. 18, 253. 56. Whitehead, M. I., Townsend, E T., Pryse-Davies, J., Ryder, T. A., and King, R. J. (1981). Effects of estrogens and progestins on the biochemistry and morphology of the postmenopausal endometrium. N. Engl. J. Med. 305, 1599-1605. 57. Gibbons, W. E., Moyer, D. L., Lobo, R. A., Roy, S., and Mishell, D. R., Jr. (1986).Biochemical and histologic effects of sequential estrogen/progestin therapy on the endometrium of postmenopausal women. Am. J. Obstet. Gynecol. 154, 456-461. 58. Moyer, D. L., de Lignieres, B., Driguez, E, and Pez, J. E (1993). Prevention of endometrial hyperplasia by progesterone during longterm estradiol replacement: Influence of bleeding pattern and secretory changes. Fertil. Steril. 59, 992-997. 59. King, R. J., and Whitehead, M. I. (1986). Assessment of the potency of orally administered progestins in women. Fertil. Steril. 46, 10621066. 60. Woodruff, J. D., and Pickar, J. H. (1994). Incidence of endometrial hyperplasia in postmenopausal women taking conjugated estrogens (Premarin) with medroxyprogesterone acetate or conjugated estrogens alone: The Menopause Study Group. Am. J. Obstet. Gynecol. 170, 1213-1223.

443 61. Genant, H. K., Lucas, J., Weiss, S., Akin, M., Emkey, R., McNancyFlint, H., Downs, R., Mortola, J., Watts, N., Yang, H. M., Banav, N., Brennan, J. J., and Nolan, J. C. (1997). Low-dose esterified estrogen therapy: Effects on bone, plasma estradiol concentrations, endometrium, and lipid levels. Estratab/Osteoporosis Study Group. Arch. Intern. Med. 157, 2609-2615. 62. Ettinger, B., Selby, J., Citron, J. T., Vangessel, A., Ettinger, V. M., Hendrickson, M. R. (1994). Cyclic hormone replacement therapy using quarterly progestin. Obstet. Gynecol. 83, 693-700. 63. Parsons, A. K., and Londono, J. L. (1999). Detection and surveillance of endometrial hyperplasia and carcinoma. In "Treatment of the Postmenopausal Woman: Basic and Clinical Aspects" (R. A. Lobo, ed.), 2nd ed., Chapter 47, pp. 513-538. Lippincott-Williams & Wilkins, New York. 64. Leather, A. T., Savvas, M., and Studd, J. W. (1991). Endometrial histology and bleeding patterns after 8 years of continuous combined estrogen and progestogen therapy in postmenopausal women. Obstet. Gynecol. 78, 1008-1010. 65. Archer, D. E, Pickar, J. J., Bottiglioni, E, for the Menopause Study Group (1994). Bleeding patterns in postmenopausal women taking continuous combined or sequential regimens of conjugated estrogens with medroxyprogesterone acetate. Obstet. Gynecol. 83, 686-692. 66. Padwick, M. L., Pryse-Davies, J., and Whitehead, M. I. (1986). A simple method for determining the optimal dosage of progestin in postmenopausal women receiving estrogens. N. Engl. J. Med. 315, 930-934. 67. Marengo, M., Rodriguez, V. D., and Gil, D. E. (1996). Hormonal replacement therapy (HRT): Evaluation of a novel 25 days regimen with percutaneous estradiol (PE2) and micronized oral progesterone (MP). Proc. Inter. Congr. Menopause, 8th, Sydney, 1996, Abstract. 68. Gillet, J. Y., Andre, G., Faguer, B., Erny, R., Buvat-Herbaut, M., Domin, M. A., Kuhn, J. M., Hedon, B., Drapier-Faure, E., and Barrat, J. (1994). Induction of amenorrhea during hormone replacement therapy: Optimal micronized progesterone dose: A multicenter study. Maturitas 19, 103-115 69. de Lignieres, B., and Droguez, E C. (1998). Long-term experience with oral micronized progesterone and effects on the endometrium. Contemp. Obstet./Gynecol., Suppl. 43, 10-17. 70. Casper, R. F., and Chapdelaine, A. (1993). Estrogen and interrupted progestin: A new concept for menopausal hormone replacement therapy. Am. J. Obstet. Gynecol. 168, 1188-1194. 71. Gaubel, E, Lim, E C., and Creasy, G. (1999). Effect of 17fi-estradiol/ norgestimate cyclophasic hormone replacement therapy on endometrial histology. Obstet. Gynecol. 93(Suppl. 4) 81S, (abstr.). 72. Lobo, R. A., Zacur, H. Z., Caubel, P., and Lane, R. (2000). A novel pulsed regimen of norgestimate preserves the beneficial effects of 17fi-estradiol on lipids and lipoprotein profiles. Am. J. Obstet. Gynecol. 182(1), in press. 73. Miles, R. A., Paulson, R. J., Lobo, R. A., Press, M. E, Dahmoush, L., and Sauer, M. V. (1994). Pharmacokinetics and endometrial tissue levels of progesterone after administration by intramuscular and vaginal routes: A comparative study. Fertil. Steril. 62, 485-490. 74. Casanas-Roux, E, Nisolle, M., Marbaix, E., Smets, M., Bassil, S., and Donnez, J. (1996). Morphometric, immunohistological and threedimensional evaluation of the endometrium of menopausal women treated by estrogen and Crinone, a new slow-release vaginal progesterone. Hum. Reprod. 11, 357-363. 75. Fanchin, R., De Ziegler, D., Bergeron, C., Righini, C., Torrisi, C., and Frydman, R. (1997). Transvaginal administration of progesterone. Obstet. Gynecol. 90, 396-401. 76. Shoupe, D., Meme, D., Mezrow, G., and Lobo, R. A. (1991). Prevention of endometrial hyperplasia in postmenopausal women with intrauterine progesterone. N. Engl. J. Med. 325, 1811-1812. 77. Wollter-Svensson, L. O., Stadberg, E., Andersson, K., Mattsson, L. A., Odlind, V., and Persson, I. (1997). Intrauterine administration of levonorgestrel 5 and 10 ~g/24h in perimenopausal hormone replace-

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ROGERIO A. LOBO ment therapy. A randomized clinical trial during one year. Acta Obstet. Gynecol. Scand. 76, 449-454. Cooper, A. J., Spencer, C., Whitehead, M. I., Ross, D., Barnard, G. J., and Collins, W. E (1998). Systemic absorption of progesterone from progest cream in postmenopausal women. Lancet 351, 1255-12561 Burry, K. A., Patton, E E., and Hermsmeyer, K. (1999). Percutaneous absorption of progesterone in postmenopausal women treated with transdermal estrogen. Am. J. Obstet. Gynecol. 180, 1504-1511. Greendale, G. A., Raboussin, B. A., Sie, A., Singh, R., Olson, L. K., Gatewood, O., Bassett, L. W., Wasilauskas, C., Bush, T., and BarrettConnor, E. (1999). Effects of estrogen and estrogen-progestin on mammographic parenchymal density. Postmenopausal Estrogen /Progestin Interventions (PEPI) Investigators. Ann. Intern. Med. 130, 262-269. Lobo, R. A. (1991). Effects of hormonal replacement on lipids and lipoproteins in postmenopausal women. J. Clin. Endocrinol. Metab. 73, 925-930. Lobo, R. A., Pickar, J. H., Wild, R. A., Walsh, B., Hirvonen, E., and the Menopause Study Group (1994). Metabolic impact of adding medroxyprogesterone acetate to conjugated estrogen therapy in postmenopausal women. Obstet. Gynecol. 84, 987-995. Jensen, J., Nilas, L., and Christiansen, C. (1990). Influence of menopause on serum lipids and lipoproteins. Maturitas 12, 321-331. Jensen, J., Nilas, L., and Christiansen, C. (1986). Cyclic changes in serum cholesterol and lipoproteins following different doses of combined postmenopausal hormone replacement therapy. Br. J. Obstet. Gynaecol. 93, 613-618. Writing Group for the PEPI Trial (1995). Effects of estrogen or estrogen/progestin regimens on heart disease risk factors in postmenopausal women, The Postmenopausal Estrogen/Progestin Interventions (PEPI) Trial. JAMA, J. Am. Med. Assoc. 273, 199-208. Collins, E (1999). Estrogen-blood flow and vasomotion. In "Treatment of the Postmenopausal Woman: Basic and Clinical Aspects" (R. A. Lobo ed.), 2nd ed., Chapter 34, pp. 385-390. LippincottWilliams & Wilkins, Philadelphia. Killam, A. E, Rosenfeld, C. R., Battaglia, E C., Makowski, E. L., and Meschia, G. (1973). Effect of estrogens on the uterine blood flow of ovariectomized ewes. Am. J. Obstet. Gynecol. 115, 1045-1052. Resnik, R., Brink, G. W., and Plumer, M. H. (1977). The effect of progesterone on estrogen-induced uterine blood flow. Am. J. Obstet. Gynecol. 128, 251-254. Hsueh, A. J. W., Peck, E. J., and Clark, J. H. (1975). Progesterone antagonism of the oestrogen-induced uterine growth. Nature (London) 254, 337-339. Whitehead, M. I., Fraser, D., Schenkel, L., Crook, D., and Stevenson, J. C. (1990). Transdermal administration of oestrogen/progestogen hormone replacement therapy. Lancet 335, 310-312. Adams, M. R., Kaplan, J. R., Manuck, S. B., Koritnik, D. R., Parks, J. S., Wolfe, M. S., and Clarkson, T. B. (1990). Inhibition of coronary artery atherosclerosis by 17-beta estradiol in ovariectomized monkeys: Lack of an effect of added progesterone. Arteriosclerosis 10, 1051-1057. Adams, M. R., Register, T. C., Golden, D. L., Wagner, J. D., and Williams, J. K. (1997). Medroxyprogesterone acetate antagonizes inhibitory effects of conjugated equine estrogens on coronary artery atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 17, 217-221. Williams, J. K., Adams, M. R., and Klopfenstein, H. S. (1990). Estrogen modulates responses of atherosclerotic coronary arteries. Circulation 81, 1680-1687. Williams, J. K., Honore, E. K., Washburn, S. A., and Clarkson, T. B. (1994). Effects of hormone replacement therapy on reactivity of atherosclerotic coronary arteries in cynomolgus monkeys. J. Am. Coll. Cardiol. 24, 1757-1761. Miyagawa, K., Rosch, J., Stanzcyk, E, and Hermsmeyer, K. (1997). Medroxyprogesterone interferes with ovarian steroid protection against coronary vasospasm. Nat. Med. 3, 324-327.

92. Levine, R. L., Chen, S.-J., Durand, J., Chen, Y. E, and Oparil, S. (1996). Medroxyprogesterone attenuates estrogen-mediated inhibition of neointima formation after balloon injury of the rat carotid artery. Circulation 94, 2221-2227. 93. Register, T. C., Adams, M. R., Golden, D. L., and Clarkson, T. B. (1998). Conjugated equine estrogens alone, but not in combination with medroxyprogesterone acetate, inhibit aortic connective tissue remodeling after plasma lipid lowering in female monkeys. Arterioscler. Thromb. Vasc. Biol. 18, 1164-1171. 94. Hulley, S., Grady, D., Bush, T., Furberg, C., Herrington, D., Riggs, B., and Vittinghof, E. (1998). Randomized trial of estrogen plus progestin for secondary prevention of coronary heart disease in postmenopausal women. Heart and Estrogen/progestin Replacement Study (HERS) Research Group. JAMA, J. Am. Med. Assoc. 280, 605- 613. 95. Pines, A., Fisman, E. Z., Levo, Y., Averbuch, M., Lidor, A., Droy, Y., Finkelstein, A., Hetman-Peri, M., Moshkowitz, M., and Ben-Ari, E. (1991). The effects of hormone replacement therapy in normal postmenopausal women: Measurements of Doppler-derived parameters of aortic flow. Am. J. Obstet. Gynecol. 164, 806-812. 96. Ganger, K. E, Vyas, S., Whitehead, M., Crook, D., Meire, H. and Campbell, S. (1991). Pulsatility index in internal carotid artery in relation to transdermal oestradiol and time since menopause. Lancet 338, 839-842. 97. Wagner, J. D., Martino, M. A., Jayo, M. J., Anthony, M. S., Clarkson, T. B., and Cefalu, W. T. (1996). The effects of hormone replacement therapy on carbohydrate metabolism and cardiovascular risk factors in surgically postmenopausal cynomolgus monkeys. Metab. Clin. Exp. 45, 1254-1262. 98. Lindheim, S. R., Presser, S. C., Ditkoff, E. C., Vijod, M. A., Stanczyk, E Z., and Lobo, R. A. (1993). A possible bimodal effect of estrogen on insulin sensitivity in postmenopausal women and the attenuating effect of added progestin. Fertil. Steril. 60, 664-667. 98a. Lindheim, S. R., Buchanan, T. A., Duffy, D. M., Vijod, M. A., Kojima, T., Stanczyk, F., and Lobo, R. A. (1994). Comparison of estimates of insulin sensitivity in pre and post menopausal women using the insulin tolerance test and the frequently sampled intravenous glucose tolerance test. J. Soc. Gynecol. Invest. 1, 150-154. 99. Sherwin, B. B. (1991 ). The impact of different doses of estrogen and progestin on mood and sexual behavior in postmenopausal women. J. Clin. Endocrinol. Metab. 72, 336-343. 100. Hoist, J., Backstrom, T., Hammarback, S., and von Schoultz, B. (1989). Progestogen addition during oestrogen replacement therapy: Effects on vasomotor symptoms and mood. Maturitas 11, 13-20. 101. Klaiber, E. L., Broverman, D. M., Vogel, W., Peterson, L. G., and Snyder, M. B. (1996). Individual differences in changes in mood and platelet monoamine oxidase (MAO) activity during hormonal replacement therapy in menopausal women. Psychoneuroendocrinology 21,575-592. 102. Smith, R. N., Holland, E. E, and Studd, J. W. W. (1994). The symptomatology of progestin intolerance. Maturitas 18, 87-91. 103. Greendale, G. A., Reboussin, B. A., Hogan, P., et al. (1998). Symptom relief and side effects of postmenopausal hormones: Results from the Postmenopausal Estrogen/Progestin Interventions Trial. Obstet. Gynecol. 92, 982-988. 104. Lindheim, S. R., Legro, R. S., Morris, R. S., Wong, I. L., Tran, D. Q., Vijod, M. A., Stanczyk, E Z., and Lobo, R. A. (1994). The effect of progestins on behavioral stress responses in postmenopausal women. J. Soc. Gynecol. Invest. 1, 79-83. 105. Rice, M. M., Graves, A. B., McCurry, S. M., Bowen, J, McCormick, W., and Larson, E. B. (1999). Does progestin modify the beneficial effects of estrogen on cognition? Findings from the Karne Project. Personal communication. 106. Wooley, C. S., and McEwen, B. S. (1993). Roles of estradiol and progesterone in regulation of hippocampal dendritic spine density during the estrous cycle in the rat. J. Comp. Neurol. 336, 293-306.

7HAPTER 3

Androgens SUSAN R.

DAVIS The Jean Hailes Foundation, Clayton, Victoria 3168, Australia; and Department of Epidemiology and Preventive Medicine, Monash University, Melbourne, Victoria 3004, Australia

I. Introduction II. Androgen Physiology in the Reproductive Years III. Physiological and Nonphysiological Causes of Testosterone Deficiency in Women IV. Clinical Consequences of Androgen Insufficiency and Evidence for Benefits of Testosterone Replacement in Women

I.

V. Potential Risks of Testosterone Replacement in Women VI. Which Women Are Most Likely to Benefit from Testosterone Replacement? VII. How Should Testosterone Replacement Be Prescribed? VIII. Conclusions References

INTRODUCTION

closely parallels increasing age. Hence many women experience symptoms of androgen deficiency in their late reproductive years. The symptoms usually develop insidiously and most women are not aware that such symptoms have a biological basis. Therefore it is important to question women directly about the presence of such symptoms when hormone replacement therapy is being considered, and again at subsequent consultations. Otherwise many women for whom androgen replacement therapy may be appropriate and beneficial may remain unidentified and untreated. The use of androgen therapy in women is no longer restricted to those who have undergone either a spontaneous menopause in their middle years or who have had surgical menopause, but is increasingly being used in younger women who have experienced premature ovarian failure. Other possible novel indications that are currently being evaluated include management of the premenstrual syndrome, adjunctive therapy for systemic lupus erythematosis and rheumatoid arthritis, and alleviation of the wasting syndrome of human immunodeficiency virus (HIV) infection (see Table I).

Androgens have important physiological effects in women. Not only are they the precursor hormones for estrogen biosynthesis in the ovaries and extragonadal tissues, but androgens also appear to act directly, via androgen receptors, throughout the body. Androgen levels decline with increasing age in women and it is now accepted that many postmenopausal women experience a variety of physical symptoms secondary to androgen depletion, as well as physiological changes that affect their quality of life. Affected women complain of persistent fatigue, lack of well being, and loss of libido, symptoms easily attributable to psychosocial and environmental factors. However, when these symptoms are clearly linked to low circulating bioavailable testosterone levels, androgen replacement will result in significant improvement in symptomatology. In contrast to the acute fall in circulating estrogen at the time of menopause, the decline in circulating testosterone and the adrenal preandrogens commences in the decade preceding the average age of natural menopause and most

MENOPAUSE: BIOLOGY AND PATHOBIOLOGY

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SUSAN R. DAVIS

TABLE I

Clinical Indications for Testosterone Replacement in Women

Current indications

Symptomatic testosterone deficiency following natural menopause Symptomatic testosterone deficiency due to surgical menopause, chemotherapy, or irradiation Premature ovarian failure Premenopausal loss of libido with diminished serum free testosterone Potential indications

Management of premenstrual syndrome Glucocorticosteroid-induced bone loss Premenopausal bone loss Management of wasting syndromes secondary to HIV and malignancy Premenopausal iatrogenic androgen deficiency states, including GNRH-analog treatment of endometriosis Adjunctive therapy for rheumatoid arthritis or systemic lupus erythematosus

II. A N D R O G E N P H Y S I O L O G Y IN THE R E P R O D U C T I V E YEARS Androstenedione (A), and dehydroepiandrosterone (DHEA) are produced by both the ovaries and the adrenals, with the adrenals also being the main site of production of DHEA sulfate (DHEAS). Approximately half of the circulating testosterone is produced by peripheral conversion of these preandrogens to testosterone, with A being the main precursor [ 1]. The ovaries are a primary site of testosterone synthesis and circulating DHEAS is an important precursor for ovarian intrafollicullar production of testosterone and dihydrotestosterone (DHT) [1 ]. Whether there is direct secretion of testosterone by the adrenals is controversial. Testosterone is further metabolized to the potent androgen DHT in various peripheral sites. There is significant cyclicity in plasma levels of A and testosterone in regularly ovulating women, with increases in the mean circulating levels of both of these hormones in the middle third of the menstrual cycle [2,3]. This is followed by a second rise in A production by the corpus luteum during the late luteal phase. Ovarian androgens are produced by the thecal cells under the control of luteinizing hormone (LH). The blood level of DHEA in adulthood is higher than any other circulating steroid except cholesterol. DHEA secretion is acutely stimulated by adrenocorticotropic hormone (ACTH) [4,5]; however DHEAS, which has a long plasma half-life, does not acutely increase following ACTH administration [1] Adrenal androgen and cortisol production are not always linked. Circulating adrenal androgen levels have been observed to be normal or suppressed in acute stress [6], severe systemic illness [7], anorexia nervosa [8], and Cushing's syndrome [9], which are otherwise characterized by elevated cortisol levels. Increased adrenal androgen production may also be seen in association with hyperprolactinemia

[5], although the majority of women with this disorder have normal androgen levels. DHEA and DHEAS are converted peripherally into A and then into the potent androgens testosterone and DHT as well as to estrogens. Under normal physiological conditions only 1-2% of total circulating testosterone is free or biologically available. The rest is bound by sex hormone binding globulin (SHBG) and albumin, with SHBG binding 66% of total circulating testosterone [ 10]. The binding affinity for steroids bound by SHBG is DHT > testosterone > androstenediol > estradiol > estrone [11]. SHBG also weekly binds DHEA, but not DHEAS [11]. Therefore variations in the plasma levels of SHBG impact significantly on the amount of free, or bioavailable, testosterone [ 11 ]. Elevations in estradiol and thyroxine increase SHBG, whereas increases in testosterone, glucocorticosteroids, growth hormone, and insulin suppress SHBG production. In women androgens may act directly via the androgen receptor, or indirectly after conversion to estrogen. Androgens are the precursor hormones for estrogen production not only in the ovaries but also in extragonadal tissues, including bone, adipose, and brain. Therefore maintenance of physiological circulating androgen levels in women ensures adequate supply of substrate for estrogen biosynthesis in extragonadal sites, such as bone, in which high tissue estrogen concentrations may be required physiologically (for example, maintenance of bone mineralization and prevention of bone loss). That the primary source of estrogen in extragonadal sites is that produced locally from androgens also explains the apparent threshold dose of estrogen replacement postmenopausally below which bone loss continues, and the observation that whereas standard estrogen replacement therapy has little effect on libido [12-15], most parameters of sexuality improve when extremely high doses of estrogen are administered [16]. Furthermore, this hypothesis may also explain the gender discrepancy between the development of osteoporosis and dementia, which occur much later in life in men than in women, because men maintain adequate circulating testosterone levels for the extragonadal production of estrogen in bone and brain well into their latter years.

III. PHYSIOLOGICAL AND N O N P H Y S I O L O G I C A L CAUSES OF T E S T O S T E R O N E DEFICIENCY IN W O M E N The mean circulating levels of testosterone decline continuously with increasing age from the early reproductive years, such that the levels of women in their 40s are approximately half of those of women in their 20s [17]. Although the percentage of free testosterone does not vary with age,

CHAPTER31 Androgens an absolute decline in free testosterone with age has been reported [17]. DHEAS levels also fall linearly with age, and this contributes to the decline in the level of their main metabolite, testosterone [18,19]. In the late reproductive years there is failure of the midcycle rise in free testosterone that characterizes the menstrual cycle in young ovulating women [3]. This occurs despite preservation of normal free testosterone levels at other phases of the cycle. The mean plasma concentrations of testosterone in women transiting the menopause are also significantly lower than in younger ovulating women sampled in the early follicular phase [20]. Acutely, across the perimenopausal period, neither A, DHT, nor the ratio of total testosterone to SHBG (the free androgen index, or FAI) appears to change [ 10,20]. Following menopause, direct ovarian secretion appears to account for up to 50% of testosterone production, with the adrenal gland being a less important source [21]. Ovarian stromal hypertrophy and hyperplasia may persist or develop after menopause, probably secondary to elevated LH levels and individual sensitivity, resulting in increased testosterone production [22]. Alternatively, the ovaries may become fibrotic and a poor source of sex steroids, and in such women the adrenal gland becomes the main androgen source following postmenopause [22]. After menopause peripheral conversion of A remains a major source of circulating testosterone [21]. A sudden decline in androgens is a feature of ovariectomy, with both testosterone and A decreasing acutely by about 50% [23]. Other iatrogenic causes of testosterone deficiency include chemical ovariectomy, for example, the use of gonadotropin-releasing hormone (GnRH) antagonists for the treatment of fibroids or endometriosis, postchemotherapy or radiotherapy, and the administration of exogenous estrogens or glucocorticosteroids. In general, circulating free testosterone is suppressed in women using either the combined oral contraceptive pill or oral estrogen replacement therapy [24,25]. This is a result of an increase in SHBG combined with suppression of LH production by the pituitary and hence lessened stimulus for the ovarian stromal production of testosterone. These effects are amplified in older women whose overall androgen production is declining [24,25]. Treatment with oral glucocorticosteroids results in ACTH suppression and hence reduced adrenal androgen production [26]. In the premenopausal years other pathophysiological states such as hypothalamic amenorrhea and hyperprolactinemia are characterized by low circulating testosterone levels and bone loss. Similarly women with premature ovarian failure experience significant loss of bone that not uncommonly persists despite adequate standard estrogen/progestin therapy [27], and it is likely that young women with either ongoing hypothalamic amenorrhoea or premature ovarian failure require testosterone replacement in order to prevent progressive bone reabsorption.

447

IV. CLINICAL CONSEQUENCES OF ANDROGEN INSUFFICIENCY AND EVIDENCE FOR BENEFITS OF TESTOSTERONE REPLACEMENT IN WOMEN A. S e x u a l D y s f u n c t i o n Multiple interacting factors influence libido and frequency and enjoyment of sexual activity in women. These include general health status, the physical and social environment, education, past experiences, current expectations, and the cultural milieu. Prevailing myths are that sexuality in women declines with increasing age, and that older women mostly continue to be sexually active in order to please their partner. Indeed one study of women in nursing homes reported that 25% of women over the age of 70 masturbate [28 ]. Most women who have a natural menopause do not report loss of sexual desire, erotic pleasure, or orgasm. However, there is an age-related reduction in sexual frequency among women and lessening of coital frequency associated with the menopausal transition independent of age [29]. Androgens appear to be important in female sexuality, with reduced androgen levels in the late reproductive years and beyond contributing to the decline in sexual interest experienced by a proportion of women. Compared with premenopausal women, women in their postmenopausal years report fewer sexual thoughts or fantasies, have less vaginal lubrication during coitus, and are less satisfied with their partners as lovers [29]. A low testosterone level is most closely correlated with reduced coital frequency [30]. rn the Melbourne Women's Midlife Health Study, 62% of women aged 45 to 55 years reported no change in sexuality in the preceding year, although 31% reported a decrease [31]. In the latter group, the decline, was significantly and adversely associated with menopause rather than with age. In a study of sexagenarian women, the only hormone positively correlated with sexual desire was circulating free testosterone [32]. Estradiol and testosterone are both present in the human female brain. The highest concentrations of estradiol have been reported in the hypothalamus and the preoptic area; the highest concentrations of testosterone have been reported in the substantia nigra, the hypothalamus, and the preoptic area [33]. The concentration of testosterone is severalfold higher than estradiol in each of these regions, with the highest ratio of testosterone to estradiol demonstrated in the hypothalamus and preoptic area. This distribution corresponds with high aromatase activity found in these regions in animals [34]. It is most probable that androgen effects within the central nervous system are mediated directly via androgen receptors and as a consequence of local aromatization of androgen precursors to estrogen. Support for specific direct

448 androgen actions within the brain comes from cross-sex hormone therapy studies in transsexuals [35]. In one such study, the administration of androgens to female-to-male transsexuals led to increases in sexual motivation and arousability, where as the combination of antiandrogens and high-dose estrogen given to male-to-female transsexuals had the opposite effect [34]. Androgen treatment of female-to-male transsexuals also resulted in improved visuospatial ability, lessened verbal fluency, and increased anger readiness, whereas the reverse, including an increased tendency to indirect angry behavior, was seen in the male-to-female subjects [35]. Research addressing the effect of women anticipating an adverse influence of menopause on their sexuality has shown that there is a weak correlation between anticipated changes in sexuality and what actually occurs [29]. The greatest predictor of postmenopausal sexual satisfaction is the quality of the sexual aspects of a woman's life in her premenopausal years, although having a satisfying sexual relationship prior to menopause does not preclude a woman from experiencing androgen-responsive sexual dysfunction subsequently. Undoubtedly, the availability of a sexual partner and the effects of age on the health and sexual interest of that person contribute to the apparent decline in sexuality among some women with increasing age. Estrogen replacement improves vasomotor symptoms, vaginal dryness, and, possibly, general well-being, but has little effect on libido [12,13]. Oral estrogen replacement therapy improves sexual satisfaction in women with atrophic vaginitis causing their dyspareunia, but women lacking this symptom benefit little or not at all [ 15,36]. In some cases, the commencement of estrogen replacement may suppress free testosterone levels and cause relative testosterone deficiency in the postmenopausal years, as described above, and hence precipitate a decline in sexual desire [24,37]. We, and others, have demonstrated improvements in a several aspects of sexuality in postmenopausal women treated with exogenous testosterone over and above the effects achieved with estrogen alone (Fig. 1). Sustained improvements in intensity of sexual drive, arousal, frequency of sexual fantasies, satisfaction, pleasure, and relevancy were observed in a cohort of postmenopausal women. In contrast to the consistently observed increases in sexual motivation, improvements in coital frequency and orgasm vary considerably between studies. The failure for testosterone supplementation to improve coital frequency may be attributed to the participants in these studies being women with very long-term relationships, with established sexual patterns, such that change is less likely. Frequency of sexual activity as a measure of efficacy of androgen therapy in women can also be misleading, because often this is more a measure of a male partner's sexual appetite rather than the woman's. The only study in which the addition of testosterone replacement did not show any benefit over estrogen

SUSANR. DAVIS Sexuality Score Summary Statistics 5

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FIGURE 1 Summary graph of the effects on sexuality of either three monthly 50-mg estradiol implants alone (o) or 50-mg estradiol plus 50-mg testosterone (m) in postmenopausal women. The grand mean (i.e., means of 6, 12, 18, and 24 months) for each sexuality parameter is adjusted for baseline as a covariate. Error bars represent standard error of the differences between means. If the error bars do not overlap for a single parameter the difference is significant with a P value < 0.05 [ 16]. Reprinted from Maturitas 21; S. R. Davis, P. I. McCloud, B. J. G. Strauss, and H. G. Burger; Testosterone enhances estradiol's effects on postmenopausal bone density and sexuality, pp. 227-236. Copyright 1995, with permission from Elsevier Science.

alone was of women being treated for generalized menopausal symptoms rather than low libido [38]. Testosterone replacement should also be considered as part of the management of young women with premature menopause, particularly Turner's syndrome. Women who are sexually active when they develop premature ovarian failure are often very disturbed by their diminished libido. Alternatively, young women who have not become sexually active, who have either primary or secondary premature ovarian failure, should be fully informed about the option of androgen replacement or perhaps in some instances should be offered low-dose androgen replacement as a component of their hormone replacement regimen. Whether premenopausal women who complain of loss of libido and who have low bioavailable testosterone levels should be offered androgen replacement is controversial. Certainly such women do appear to be at greater risk of premenopausal bone loss, and it is the authors' experience that some symptomatic premenopausal women with low measurable testosterone levels clinically appear to benefit from androgen therapy. At present this is a subgroup of

CHAPTER31 Androgens women for whom clear recommendations cannot be made. However, their symptoms should not be dismissed or too readily attributed to other psychosocial factors, because it is possible that relative testosterone deficiency is a significant contributing factor. Currently, management of such women needs to be very open minded and therapy individualized, but clinical studies are needed before specific therapeutic guidelines for the premenopausal woman with low libido and low testosterone levels can be made. Testosterone replacement is unlikely to benefit women in whom other factors play a dominant role in their sexual dysfunction. Clinically discerning this may be quite straightforward, for example, in the case of postovariectomy, but extremely difficult in some women, and therefore an insightful psychosocial and sexual history is essential when evaluating the appropriateness of testosterone therapy in a woman.

B. A n d r o g e n s a n d B o n e F u n c t i o n Androgenic steroids have an important physiologic role in the development and maintenance of bone mineralization in women and men, although, the mechanisms of androgen action on bone are still a matter of debate. The skeletal effects of androgens appear to be mediated in part via the estrogen receptor after local aromatization of androgens to estrogen, and mutations in either the estrogen receptor gene or the aromatase gene are associated with osteoporosis [39,40]. Abundant aromatase activity has been reported in fetal osteoblasts and cell lines of osteoblastic origin [41]. There is also evidence that androgens act directly on bone. Androgen receptors have been demonstrated in human osteoblast-like cell lines, and androgens have been shown to stimulate directly bone cell proliferation and differentiation [42,43]. DHT has been shown to increase alkaline phosphatase activity, type I procollagen synthesis, and insulin-like growth factor II messenger RNA in the SAOS2 cell line [44]. Androgen receptor mRNA is expressed in human osteoblasts, osteocytes, hypertrophic chondrocytes, marrow mononuclear cells, and vascular endothelial cells within bone, but not in osteoclasts [45]. The pattern and number of cells expressing the androgen receptor are similar in female and male tissues [45]. In addition, androgen receptors are up-regulated in osteoblastic cells by both testosterone and DHT in vitro [46]. Total and bioavailable testosterone and DHEAS, not estradiol, are the greatest predictors of bone mineral density (BMD) and bone loss in premenopausal women [47-50]. Women who experience bone loss confined to the hip prior to menopause, a not uncommon occurrence, have lower total and free testosterone concentrations by 14 and 22%, respectively, than do those who do not significantly lose bone [49]. Consistent with these findings, hyperandrogenic women have higher BMD, after correction for body mass index, than

449 do their normal female counterparts [51]. In the premenopausal years BMD is also strongly positively correlated with body weight [48]. In obesity SHBG is suppressed with a resultant increase in free testosterone [52], and this may partially explain the relationships between obesity, free testosterone, and increased B MD, with the greater endogenous levels of biologically active free testosterone in more corpulent women directly enhancing bone mass. Androgen insufficiency may also be a factor underlying bone loss in young women with premature ovarian failure. Despite adequate standard estrogenprogestin therapy, twothirds of such women have significantly reduced BMD to levels associated with increased hip fracture risk. Of these, 47% have reductions in BMD within 18 months of their diagnosis [27]. In postmenopausal women low circulating free testosterone is predictive of subsequent height loss (a surrogate measure of vertebral compression fractures) and hip fracture [11,53,54]. Circulating DHEA and DHEAS are positively correlated with BMD in aging women [55-57] and the progressive decline in DHEA with increasing age is believed to contribute to senile osteoporosis. It is unlikely that these adrenal preandrogens directly influence bone metabolism but that their effects are mediated indirectly following conversion to estradiol, A, or testosterone. Suppression of adrenal production of DHEA and DHEAS with chronic glucocorticosteroid therapy may also contribute to the pathogenesis of osteoporosis and osteopenia, which are known complications of this therapy in women and men. DHEA or testosterone administration may be effective in preventing and or treating this common and serious side effect of glucocorticosteroid therapy. Women treated with oral DHEA have restoration of circulating A, DHT, and testosterone to premenopausal levels, as well as increases in DHEA and DHEAS, with no changes in circulating levels of estrone or estradiol from baseline [58]. Similarly, the daily percutaneous administration of 10 ml of a 20% DHEA solution results in an increase in circulating total testosterone of approximately 50%, with no consistent effect on estradiol or estrone [50]. Circulating DHEAS, but not estradiol, in postmenopausal women is positively correlated with BMD [55], and daily application of a 10% DHEA cream has been reported to increase hip BMD in older women [59]. Thus DHEA therapy may prove, in time, to be an alternative to administering testosterone replacement to androgen-deficient women. Studies of both oral and parenteral estrogen and estrogenplus-testosterone therapy in postmenopausal women have shown beneficial effects of testosterone replacement on BMD [16,53,54]. Oral esterified estrogen-methyltestosterone treatment has been shown to increase spinal BMD over a 2-year period, in contrast to estrogen-only therapy, which prevented bone loss [60]. The estrogen-methyltestosterone combination not only suppresses biochemical markers of

450

SUSAN R. DAVIS

bone reabsorption (as seen with estrogen alone), but is also associated with increases in markers of bone formation [61 ]. Treatment of postmenopausal women with nandrolone decanoate has been shown to increase vertebral B MD and has been used successfully for many years to treat osteoporosis [62]. Combined estradiol and testosterone replacement with subcutaneous implant pellets increases bone mass in postmenopausal women [63,64], with the effects in the hip and spine being greater than with estradiol implants alone [16] (see Figs. 2 and 3). Thus it appears that estradiol alone has an antireabsorptive effect on bone in postmenopausal women, whereas the addition of testosterone, either orally or parentally, enhances bone formation. This is further supported by the above-mentioned studies of men, either with a mutation of the gene encoding the aromatase enzyme [40] or a mutation of the estrogen receptor [39]. Increasing BMD is clinically important only if it is associated with enhanced mechanical strength and a reduced fracture rate. As yet, no studies have addressed the impact of androgens on fracture incidence. However, the effects of androgens on the mechanical properties of bone have been studied in feral female cynomolgus monkeys [65]. In this pri-

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FIGURE 3

The effects of hormonal implants on bone mineral density (g/ cm 2) in the femoral trochanter (Troc); estradiol, E (o); estradiol plus testosterone, E + Te (m). Error bars represent standard error of the differences between means (S.E.D.s). Inner error bars are used to compare means between times for the same treatment. The comparison between the treatment groups is made with the outer error bars. If error bars do not overlap, that is differ by more than 2 S.E.D.s, the means are significantly different by a P value of at least 0.05 [16]. Reprinted from Maturitas 21; S. R. Davis, P. I. McCloud, B. J. G. Strauss, and H. G. Burger; Testosterone enhances estradiol's effects on postmenopausal bone density and sexuality, pp. 227-236. Copyright 1995, with permission from Elsevier Science.

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The effects of hormonal implants on bone mineral density (g/ cm 2) in the lumbar spine (L1-L4); estradiol, E (o); estradiol plus testosterone, E + Te (m). Error bars represent standard error of the differences between means (S.E.D.s). Inner error bars are used to compare means between times for the same treatment. The comparison between the treatment groups is made with the outer error bars. If error bars do not overlap, that is, differ by more than 2 S.E.D.s, the means are significantly different by a P value of at least 0.05 [16]. Reprinted from Maturitas 21; S. R. Davis, P. I. McCloud, B. J. G. Strauss, and H. G. Burger; Testosterone enhances estradiol's effects on postmenopausal bone density and sexuality, pp. 227-236. Copyright 1995, with permission from Elsevier Science.

mate model, increases in intrinsic bone strength and resistance to mechanical stress were associated with increased BMD following testosterone therapy. Treatment also resulted in increased bone torsional rigidity and bending stiffness. In summary, current data indicate androgen replacement, in the form of testosterone, and possibly DHEA, is potentially an effective alternative to the prevention of bone loss and the treatment of osteopenia and osteoporosis. Because prospective data confirming a reduction in fracture rate with such therapy are lacking, specific guidelines cannot be given for this sole indication. However, androgen replacement as nandrolone decanoate has been successfully used to treat osteoporosis in elderly women, and testosterone therapy may be appropriate for this indication in naturally and surgically postmenopausal women. The addition of androgen to standard hormone replacement regimens may be necessary to prevent bone loss in young hypogonadal women in whom androgen insufficiency is aggrevated by exogenous estrogen. The use of testosterone to prevent bone loss in individuals on long-term glucocorticosteroids has not been studied but warrants further research.

CHAPTER31 Androgens C. T e s t o s t e r o n e a n d the P r e m e n s t r u a l S y n d r o m e Studies indicate that premenstrual syndrome (PMS) represents individual vulnerability to the effects of circulating steroids. Variations in testosterone levels during the menstrual cycle may influence behavioral changes such as those seen in PMS, and significantly lower levels of testosterone throughout the menstrual cycle have been reported in women who suffer PMS compared with controls [66,67]. Although there are no published randomized studies to support the use of testosterone supplementation for this condition, testosterone replacement is used in the management of PMS in some clinical centers in the United Kingdom and Australia. Randomized trials evaluating such therapy are nearing completion, and positive outcomes from these are necessary before testosterone therapy for PMS can be recommended for widespread use.

451 treated with concurrent estrogentestosterone therapy [16]. Gain in fat-free mass probably reflects increased muscle mass. Because aging is associated with loss of muscle mass, this is a beneficial effect of testosterone therapy in the older woman, and may contribute to preservation of muscle strength and skeletal stability. However, the ethics of treating an older woman who participates in competitive (Master's) sport with testosterone replacement for medical indications has become a significant issue that needs to be resolved. Testosterone levels are frequently lower in HIV-positive premenopausal women, and testosterone replacement is associated with an increase in fat-free mass and body cell mass in HIV-positive men [76,77]. Augmentation of testosterone levels in HIV-positive premenopausal women using a transdermal patch is associated with overall increased mean body weight and body mass index as well as improved quality of life [78]. In the future, there may be a therapeutic role for androgens in the alleviation of the wasting syndrome of HIV and perhaps even cachexia of other chronic diseases.

D. T e s t o s t e r o n e a n d A u t o i m m u n e D i s e a s e Although immunogenetic factors are the major determinants of the development of immune-mediated diseases, gender and age also play a role. Androgens appear to suppress both cell-mediated and humoral immune responses and it has been proposed that higher testosterone levels in men may be protective against autoimmune disease [68-70]. The direct administration of testosterone replacement has resulted in symptomatic improvement in postmenopausal women with rheumatoid arthritis [71] and both pre- and postmenopausal women have been observed to have reductions in disease activity with DHEA therapy associated with increases in levels of circulating DHEA, DHEAS, and testosterone [72]. Further evaluation of potential beneficial effects of testosterone administration in women with rheumatoid arthritis and systemic lupus erythematosis is required.

E. B e n e f i c i a l Effects on B o d y C o m p o s i t i o n In postmenopausal women, neither measured nor estimated free testosterone is associated with waist-to-hip ratio measurements and there does not appear to be a direct relationship between androgens and visceral adiposity in this population [73]. Contrary to popular belief, neither oral nor parenteral hormone replacement therapy significantly increases body weight [16,74,75]. There is also no evidence that the addition of testosterone replacement causes weight gain [16]. With increasing age women tend to lose muscle mass and replace this with increased fat mass coupled with an overall tendency for weight gain in most Western societies. We have reported an increase in fat-free mass and a reduced fat mass: fat-free mass ratio in postmenopausal women

V. POTENTIAL RISKS OF TESTOSTERONE REPLACEMENT IN W O M E N A. C a r d i o v a s c u l a r D i s e a s e R i s k There are concerns regarding potential adverse metabolic effects of androgen replacement in women, particularly with respect to possible detrimental effects on lipid and lipoprotein metabolism and vascular function. However, available clinical data are extremely reassuring in that androgen replacement therapy in women does not appear to be associated with the undesirable metabolic consequences seen in women with androgen excess or with an increase in cardiovascular risk. 1. TESTOSTERONE REPLACEMENT AND LIPIDS LIPOPROTEINS

AND

Menopause, both natural and surgically induced, is associated with the development of a more adverse lipoprotein profile, which is unrelated to endogenous testosterone levels [79]. Postmenopausal estrogen replacement therapy lowers total and low-density lipoprotein (LDL) cholesterol, and these favorable effects are not diminished with either oral or parenteral testosterone replacement [ 16,60,61 ]. Parenteral testosterone replacement does not affect high-density lipoprotein (HDL) cholesterol [ 16]; however, HDL cholesterol and apolipoprotein A1 decrease significantly when oral methyltestosterone is administered with oral estrogen [80,81]. Whereas oral estrogen replacement is associated with increased triglyceride levels, concomitant administration of oral methyltestosterone results in a reduction in triglycerides [61].

452 Measurement of circulating lipids is a clinical surrogate for lipoprotein-lipid metabolism. More direct measurement of lipid metabolism is probably a better indicator of the effects of exogenous steroid therapy on cardiovascular risk. Combined oral esterified estrogen and methyltestosterone therapy reduces arterial LDL degradation and cholesterol ester content in cynomolgus monkeys, which does not differ from the effects observed when estrogen is given alone [82]. Combined estrogen and methyltestosterone therapy is also associated with reduced plasma concentrations of apoplipoprotein B, reduced LDL particle size, and increased total body LDL catabolism [82]. Small LDL particles are more susceptible to oxidation and are hence considered to be more atherogenic. However, because estrogens appear to increase oxidative modification of LDL in the arterial wall [82], the reduction in LDL particle size observed with both oral estrogen and combined therapy may not be deleterious but may merely reflect selective removal of large LDL particles from the circulation. 2. EFFECTS OF ANDROGENS ON THE MYOCARDIUM AND VASCULAR FUNCTION The incidence of coronary heart disease in postmenopausal women is not associated with levels of circulating testosterone or DHEAS [83,84]. Intracoronary testosterone administration to anaesthetized male and female dogs induces increases in coronary artery cross-sectional area peak flow velocity and calculated volumetric blood flow, which is blocked by pretreatment with an inhibitor of nitric oxide synthesis [85]. The beneficial effects of estrogen replacement therapy on coronary artery reactivity in cynomolgus monkeys is not lost with the addition of oral methyltestosterone [86]. Cardiac myocytes and fibroblasts contain functional ce and/3 estrogen receptors, and cardiac myocytes express cytochrome P450 aromatase [87]. Incubation of cardiac myocytes with A or testosterone results in transactivation of an estrogen receptor-specific reporter, with A resulting in a significantly higher induction than testosterone [87]. Furthermore, both A and testosterone up-regulate inducible nitric oxide synthase in cardiac myocytes [87]. The capacity for cardiac myoctes to synthesize estrogen from androgenic precursors and activate downstream target genes is greater in cells from female than from male animals [87]. Thus, evaluating the data to hand, the administration of testosterone to women does not appear to affect the key events in coronary artery lipid metabolism or coronary artery function. Parenteral testosterone replacement does not negate the favorable effects exerted by exogenous estrogen on lipid and lipoprotein levels, whereas oral methyltestosterone use not only opposes the HDL cholesterol-elevating effects of oral estrogen, but also reduces HDL cholesterol levels below baseline values. It is not known whether this effect of oral methyltestosterone in the setting of concomitant lowering of total cholesterol, LDL cholesterol, and triglyceride

StJSAN R. DAVIS levels is detrimental. Certainly a high cardiac risk profile, which includes a low HDL cholesterol level, should be considered a relative contraindication to oral testosterone replacement, but should not influence the use of parenteral testosterone replacement therapy.

B. Androgens and Breast Cancer It is not known whether there are any relationships between endogenous androgen levels and breast cancer, because epidemiological studies have shown both positive and negative associations. Secreto and others reported positive associations between endogenous androgen levels and breast cancer in pre- and postmenopausal women [80,88]. In contrast, Bulbrook et al. observed that low urinary androgen levels were associated with early onset of breast cancer and a higher relapse rate [89]. In postmenopausal women with breast cancer, age-adjusted mean values of total and free testosterone have found to be higher than in controls [90] and intratumor concentrations of 5a-DHT and estradiol have been reported to be greater than circulating levels of these hormones [91 ]. Androgen receptors are found in over 50% of breast tumors [91 ], and are associated with longer survival in women with operable breast cancer and a favorable response to hormone treatment in advanced disease [92]. There is also evidence that the mechanism by which highdose medroxyprogesterone acetate exerts a negative effect on breast cancer growth is mediated via the androgen receptor [93]. It is not known whether there is any relationship between exogenous androgen therapy and the incidence of breast cancer.

C. Testosterone Replacement and Clinical Side Effects The potential masculinizing effects of androgen therapy include development of acne, hirsutism, deepening of the voice, and excessive libido. These cosmetic side effects are rare if supraphysiological hormone levels are avoided [14, 15,36,63,64,94,95]. Other adverse metabolic effects are also rare with judicious therapy. Fluid retention is uncommon and appears to be more idiosyncratic than dose related. Hirsutism, androgenic alopecia, and/or acne are relatively strong contradictions to androgen replacement. Enhancement of libido is currently the most common indication for testosterone therapy, however, circumstances in which this would be an undesirable effect is a relative contraindication to therapy. Absolute contraindications include pregnancy and lactation, as well as known or suspected androgen-dependent neoplasia (see Table II). Hepatocellular damage has been reported with high-dose oral 17ce-alkyl androgens, but not in currently used doses or other oral formulations [96].

CHAPTER31 A TABLE II

n

d

r

o

g

Contraindications to Testosterone Therapy

Relative

Severe acne Moderate - severe hirsutism Androgenic alopecia Circumstances in which enhanced libido would be undesirable Absolute

Pregnancy or lactation Known or suspected androgen-dependentneoplasia

D. Summary of the Potential Risks of Testosterone Replacement Syndromes of endogenous androgen excess are clearly associated with increased cardiovascular risk, perturbations in lipid and carbohydrate metabolism, a more android weight distribution, and virilization. In contrast, clinical data to date do not indicate testosterone maintained close to, or within, the normal female reproductive range has any adverse metabolic consequences (see Table III). With respect to b r e a s t cancer risk, whether women who have endogenously high testosterone levels are at increased risk is controversial, and there is no evidence that there is an increase in risk with socalled "physiological" exogenous androgen replacement therapy after menopause. Physicians should always be cognizant of the anxiety most women have about breast cancer when prescribing any hormone therapy.

VI. WHICH WOMEN ARE MOST LIKELY TO BENEFIT FROM TESTOSTERONE REPLACEMENT? The indication for androgen replacement therapy for a woman is most often low libido and diminished well being, and hence is based on clinical assessment, with evaluation of the outcome of treatment based on the subjective self-

TABLE III Potential Dose Related Side Effects of Androgen Therapy in Women Masculinization: hirsutism, acne, temporal balding, voice deepening, clitoromegaly Fluid retention (more often idiosyncratic) Lipids: oral testosterone may adversely affect serum levels of HDL cholesterol and apolipoprotein A1. Drug interactions: C-17-substituted derivatives of oral testosterone may decrease anticoagulantrequirements. Androgens may elevate serum levels of oxyphenbutazone and in diabetic patients may rarely affectinsulin requirements. Hepatocellular damage has been associated with high-dose 17-a-alkylandrogens given orally.

e

n

s

4

5

3

assessment of response and reporting by the patient. Biochemical measurements are of limited value, although the free androgen index (FAI) may be a guide. The clinical settings in which the administration of testosterone is most likely to enhance a woman's health and well being are listed in Table I. Future indications may also include wasting states, such as in HIV-infected individuals and malignancy-related cachexia, the prevention or treatment of bone loss (particularly iatrogenic bone loss from glucocorticosteriod therapy or premenopausal bone loss), and PMS. Some women present seeking clinical assistance for low libido and/or inadequate restoration of well being despite apparently sufficient estrogen/progestogen replacement. Others may volunteer such problems while attending their physician for another reason; however, many women suffer silently, unaware of any available therapeutic options. A significant proportion of women will not raise the issue of diminished libido because they find the topic awkward. Sadly, many young women who suffer low/absent libido following treatment of a malignancy have difficulty discussing the issue with their physician because they feel their problem will be perceived as trivial relative to their disease recovery or remission. Following treatment with chemo- or radiotherapy, the symptoms of the iatrogenic menopause and androgen deficiency, including fatigue, loss of well being, depression, and reduced libido, can be difficult to distinguish from the overall physical toll of cancer treatment, and frequently premature menopause and associated androgen insufficiency go undiagnosed and untreated. Therefore all "at risk" women should be directly questioned about the symptoms of androgen deficiency and made aware of the therapeutic possibilities available. Testosterone replacement therapy is becoming an accepted component of hormone replacement therapy for the restoration of sexual and general well being in women who have undergone a surgical menopause. Testosterone replacement in women who have undergone natural menopause and especially women who have premature ovarian failure continues to be a neglected component of hormone replacement therapy. Clearly androgen replacement is a far more sensitive issue for women than is estrogen replacement in general. It is essential that physicians offering this therapy are sensitive to the enormous variations in women's knowledge, expectations, sexual practices, and needs and always tailor treatment to the needs of the individual. The management of young women with premature menopause, particularly those with primary amenorrhea, for example, Turner's syndrome, who have never been sexually active, is more difficult. Testosterone replacement should be considered for such women experiencing persistent fatigue, lack of well being, and low libido despite adequate estrogen and progestogen replacement. However, it is obviously difficult for a woman to identify herself as having inadequate libido when she has never

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experienced what may be "normal." Alternatively, young women who have not been sexually active should have the option of testosterone replacement explained to them. Whether young women with premature ovarian failure should be recommended to use testosterone replacement to prevent bone loss, specifically from the neck of the femur, is yet to be established.

VII. HOW SHOULD TESTOSTERONE REPLACEMENT BE PRESCRIBED? Currently no form of testosterone replacement is officially available in the United States for the treatment of loss of libido in women; however, oral methyltestosterone can be prescribed for menopausal symptoms unresponsive to estrogen replacement alone, and testosterone implants have been approved for replacement therapy for women in the United Kingdom. The current international therapeutic options in terms of androgen replacement therapy are listed in Table IV. To achieve a good therapeutic response in terms of enhanced libido with testosterone replacement, testosterone levels often need to be restored to at least the upper end of the normal physiologic range for young, ovulating women. The doses of testosterone required to achieve such an effect usually result in an initial postadministration peak testosterone level that is supraphysiologic regardless of the mode of administration. The oral estrogen/androgen preparation available in the United States is in two strengths, esterified estrogen at 0.625 mg plus methyltestosterone at 1.25 mg, or esterified

TABLE IV Androgen Replacement Therapy Formulations Used for Women Formulation

Dose range

Frequency

Route

Methyltestosterone a (in combination with esterified estrogen)

1.25-2.5 mg

Daily

Oral

Testosterone undecanoate

4 0 - 8 0 mg

Daily or alternate days

Oral

Nandrolone decanoate

25-50 mg

6 - 1 2 times weekly

Intramuscular

Mixed testosterone esters

50-100 mg

4 - 6 times weekly

Intramuscular

Testosterone implants

50 mg

3 - 6 times monthly

Subcutaneous

Transdermal testosterone patch b

150/zg

Every 3.5 days

Topical

a Currently available in the United States. b Undergoing clinical trial.

estrogen at 1.25 mg plus methyltestosterone at 2.5 mg. Methyltestosterone is not available in most other countries because liver damage has been reported with long-term highdose therapy [96]. More recent data do not support any short-term (12 months) detrimental effects of the available doses of methyltestosterone combined with estrogen on hepatic enzymes or blood pressure [96]. It has been observed that women who use esterified estrogens combined with methyltestosterone report a lower instance of nausea compared with women receiving conjugated equine estrogen alone [96]. Testosterone undecanoate is an oral androgen prescribed for replacement therapy in hypogonadal men. Its clinical use in women has been little studied, although in some countries its prescription for women is quite widespread. It is believed to be absorbed via the lymphatics, hence, for optimal absorption, it is recommended testosterone undecanoate be ingested with fat, for example, with a glass of milk. However, supraphysiologic peaks of total testosterone have been reported with a dose as low as 20 mg [97]. The clinical role for testosterone undecanoate as a replacement therapy for women at present remains unclear and further research into the safety and efficacy of this agent is required before its use can be recommended. There has been considerable clinical experience with the administration of testosterone implants in postmenopausal women, particularly in the Commonwealth countries, and these are approved for use in women in the United Kingdom. These implants are fused crystalline implants, 4 - 5 mm in diameter, containing testosterone BP (British Pharmacopoeia) as the active ingredient. A dose of 50 mg is extremely effective and does not cause virilizing side effects [16]. This dose is obtained by bisecting a 100-mg implant under sterile conditions. The implant is inserted, under local anaesthesia, subcutaneously, usually into the lower anterior abdominal wall using a trocar and cannula. This therapy provides a slow release of testosterone with an approximate duration of effect, for a 50-mg implant, of between 3 and 6 months. There is marked individual variation in this period, therefore testosterone levels must be carefully monitored in that a testosterone level should be measured prior to the administration of each subsequent implant. The author recommends that additional testosterone implants should not be inserted unless total testosterone is within the normal range for young women. Rarely is a 100-mg testosterone implant necessary to achieve an adequate therapeutic effect. Mean circulating testosterone levels approximately three times the upper limit of normal have been reported 4 weeks following the administration of 100-mg testosterone pellets [97]. In contrast, 6 weeks after the insertion of a 50-mg testosterone implant, the mean circulating testosterone levels in postmenopausal women are just above the upper limit of normal for young ovulating women [95]. Mixed testosterone esters (50-100 mg) are occasionally

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administered in doses four to six weeks apart as an intramuscular injection to women with androgen deficiency symptoms. Clinically this therapy results in a more rapid onset of effects, such that women report enhanced libido within 2 to 3 days of treatment. In contrast, women generally report a delay in restoration of libido of 10 to 14 days after insertion of a testosterone implant. The pharmacokinetics of mixed testosterone esters administered intramuscularly to women have not been studied, although many women report increased acne and occasional clitoromegaly with this therapy. The development of a transdermal testosterone matrix patch, specifically for use in women, may provide a new option for women requiring testosterone replacement. The patch, which is currently undergoing early clinical trial, delivers 150/xg of testosterone per day over a 3- to 4-day period (twice weekly application). This should increase circulating testosterone levels, on average, by approximately 25 ng/dl, (about 1 nmol/liter). There are some obvious advantages of patch technology over both oral and implant therapy; however, as with other hormone patches, some women may experience skin irritation or simply prefer a less conspicuous form of treatment. Nandrolone deconoate, a weakly aromatizable androgen, is approved in some countries for the treatment of postmenopausal osteoporosis. The dose, administered intramuscularly, should not exceed 50 mg and the frequency of treatment is best titrated against the patient's gross build. It is prudent that treatment is not given less than every 6 weeks and, because these women are usually elderly and not on estrogen therapy, they should be very carefully monitored for masculinization. In most women, treatment with nandronone decanoate resuits in cessation of bone loss over time and in some women an increase in BMD. Alternatives, which are not currently or generally available, are transdermal testosterone as a cream or gel, or parenteral testosterone via a vaginal ring. Although the transdermal preparations may be regionally available on specific prescription from compounding pharmacists, there are no pharmacokinetic data available or published clinical experience pertaining to their use. The contraindications to and side effects of testosterone replacement are listed in Tables II and III. Again it is emphasized that with judicious dosing and careful patient monitoring, side effects of testosterone replacement are rare. All postmenopausal women treated with testosterone replacement should be using concurrent estrogen replacement therapy. There are no clinical data available regarding the use of testosterone replacement in postmenopausal women who are not on estrogen; however, one would predict that such use would result in adverse metabolic and cosmetic side effects. The only clinical situation in which androgen therapy has been used in postmenopausal women without concurrent estrogen is the administration of the anabolic steroid, nandrolone decanoate. Women treated with this steroid must be

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very carefully monitored for virilization, which may occur when this compound is given unopposed by estrogen.

VIII.

CONCLUSIONS

Testosterone levels in women fall substantially during the reproductive years, with little further change at the time of spontaneous menopause. A major fall in circulating testosterone occurs following bilateral oophorectomy. The biological availability of the hormone is determined by the levels of SHBG, with factors that raise SHBG levels, leading to a fall in bioavailable testosterone. It is increasingly being accepted that androgen deficiency in women underpins a variety of symptoms and pathophysiological conditions and that in certain subsets of women androgen replacement therapy is of clinical benefit. A substantial body of evidence indicates that testosterone is an important determinant of female sexuality, and that androgen replacement enhances certain aspects of sexual function, particularly in women who have undergone oophorectomy. Many women are ill at ease discussing their loss of sexual desire, particularly those who have undergone chemotherapy, and often comment that they feel the issue will be viewed by their physician as trivial relative to their recovery or remission. Also, the symptoms of iatrogenic menopause can easily be attributed to side effects of chemotherapy or to other psychosocial factors in premenopausal women, or those who have undergone a premature menopause. Therefore it is the treating physician's responsibility to facilitate discussion of sexuality in all "at risk" women and the possibility of low circulating androgen levels as an underlying cause for those with positive symptomatology should be evaluated. Physiologic-range androgen replacement may also have a role in the maintenance or restoration of bone mass, both following menopause and after pharmacologic glucocorticoid therapy. Further research into the biological actions of androgens in bone and clinical studies of testosterone replacement and fracture are needed to define the appropriate clinical application of androgens in the prevention and treatment of bone loss in women. Although more controversial, premenopausal women with either spontaneous or iatrogenic androgen deficiency also warrant consideration for androgen replacement, as do women experiencing glucocorticosteroid-induced bone loss and possibly premenopausal bone loss. The role of androgen therapy in the premenstrual syndrome should be further elucidated by results from randomized placebo-controlled studies that are currently underway. The role of testosterone as an anabolic agent in women with wasting diseases remains to be established. At levels in or just above the physiologic range, testosterone in women does not appear to have major adverse effects

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on cardiovascular risk factors or on vascular function. Its actions, if any, in breast cancer risk remain uncertain. The inclusion of testosterone therapy in postmenopausal hormone therapy regimens is increasing but is still limited by the lack of availability of preparations and formulations designed specifically for use in women. Advances, particularly involving transdermal technologies, are occurring in the effort to optimize the methods of androgen replacement in women, as well as in men.

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dehydroepiandrosterone: 50 patients treated up to 12 months J. Rheumatol. 25, 285-289. Goodman-Gruen, D., and Barrett-Connor, E. (1995). Total but not bioavailable testosterone is a predictor of central adiposity in postmenopausal women. Int. J. Obes. 19, 293-298. Darling, G. M., Johns, J. A., McCloud, P. I., and Davis, S. R. (1997). Estrogen and progestin compared with simvastatin for hypercholesterolemia postmenopausal women. N. Engl. J. Med. 337, 595-601. Writing Group for the PEPI Trial (1995). Effects of estrogen or estrogen/progestin regimens on heart disease risk factors in postmenopausal women. JAMA, J. Am. Med. Assoc. 273, 199-208. Engelson, E. S., Goggin, K. J., Rabkin, J. G., and Kotler, D. P. (1996). Nutrition and testosterone status of HIV positive women. Proc. Int. Conf. AIDS, 11th, Vancouver, p. 332. Engelson, E. S., Rabkin, J. G., Rabkin, R., and Kotler, D. P. (1996). Effects of testosterone upon body composition. J. Acquired Immune Defic. Syndr. Hum. Retrovirol. 11, 510-511. Miller, K., Corcoran, C., Armstrong, C., Caramelli, K., Anderson, E., Cotton, D., Basgoz, N., Hirschhorn, L., Tuomala, R., Schoenfeld, D., Daugherty, C., Mazer, N., and Grinspoon, S. (1998). Transdermal testosterone administration in women with acquired immunodeficiency syndrome wasting: A pilot study. J. Clin. Endocrinol. Metab. 83, 27172725. Wakatsuki, A., and Sagara, Y. (1995). Lipoprotein metabolism in postmenopausal and oophorestomized women. Obstet. Gynecol. 85, 523-528. Secreto, G., Toniolo, P., Pisani, P., Recchione, C., Cavalleri, A., Fariselli, G., Totis, A., DiPetro, S., and Berrino, F. (1989). Androgens and breast cancer in premenopausal women. Cancer Res. 49, 471476. Hickok, L. R., Toomey, C., and Speroff, L. (1993). A comparison of esterified estrogens with and without methyltestosterone: Effects on endometrial histology and serum lipoproteins in postmenopausal women Obstet. Gynecol. 82, 919-924. Wagner, J. D., Zhang, L., Williams, J. K., Register, T. C., Ackerman, D. M., Wiita, B., Clarkson, T. B., and Adams, M. R. (1996). Esterified estrogens with and without methyltesterone decrease arterial LDL metabolism in Cynomolgus monekys. Arterioscler. Thromb. Vasc. Biol. 16, 1473-1479. Barrett-Connor, E., and Goodman-Gruen, D. (1995). Prospective study of endogenous sex hormones and fatal cardiovascular disease in postmenopausal women. Br. Med. J. 311, 1193-1196. Barrett-Connor, E., and Goodman-Gruen, D. (1995). Dehydroepiandrosterone sulfate does not predict cardiovascular death in postmenopausal women. The Rancho Bernardo Study. Circulation 91, 17571760.

85. Chou, T. M., Sudhir, K., Hutchison, S. J., Ko, E., Amidon, T. M., Collins, P., and Chatterjee, K. (1996). Testosterone induces dilation of canine coronary conductance and resitstance arteries in vivo. Circulation 94, 2614 - 2 6 1 9 . 86. Honore, E. K., Williams, J. K., Adams, M. R., Ackerman, D. M., and Wagner, J. D. (1996). Methyltestosterone does not diminish the beneficial effects of estrogen replacement therapy on coronary arter reactivity in cynomolgus monkeys. Menopause: J. North Am. Menopause Soc. 3, 20-26. 87. Grohe, C., Kahlert, S., Lobbert, K., and Vetter, H. (1998). Expression of oestrogen receptor alpha and beta in rat heart: Role of local oestrogen synthesis J. Endocrinol. 156, R1-R7. 88. Secreto, G., Toniolo, P., Berrino, E., Recchione, C., Cavalleri, A., Pisani, P., Totis, A., Fariselli, G., and DiPetro, S. (1991). Serum and urinary androgens and risk of breast cancer in postmenopausal women. Cancer Res. 51, 2572-2576. 89. Bulbrook, R. D., and Thomas, B. S. (1989). Hormones are ambiguous risk factors for breast cancer. Acta. Oncol. 28(6), 841-847. 90. Berrino, F., Muti, P., Michelli, A., Bolelli, G., Krogh, V., Sciajno, R., Pisani, P., Panico, S., and Secreto, G. (1996). Serum sex hormone levels after menopause and subsequent breast cancer. J. Natl. Cancer Inst. 88, 291-296. 91. Recchione, C., Venturelli, E., Manzari, A., Cavalteri, A., Martinetti, A., and Secreto, G. (1995). Testosterone, dihydrotestosterone and oestradiol levels in postmenopausal breast cancer tissues. J. Steroid Biochem. Mol. Biol. 52, 541-546. 92. Bryan, R. M., Mercer, R. J., Rennie, G. C., Lie, T. H., and Morgan, F. J. (1984). Androgen receptors in breast cancer. Cancer (Philadelphia) 54, 2436 -2440. 93. Birrell, S. N., Roder, D. M., Horsfall, D. J., Bentel, J. M., and Tilley, W. D. (1995). Medroxyprogesterone acetate therapy in advanced breast cancer: The predictive value of androgen receptor expression. J. Clin. Oncol. 13, 1572-1577. 94. Burger, H. G., Hailes, J., and Menelaus, M. (1984). The management of persistent symptoms with estradiol-testosterone implants: Clinical, lipid and hormonal results. Maturitas 6, 351-358. 95. Burger, H. G., Hailes, J., Nelson, J., and Menelaus, M. (1987). Effect of combined implants of estradiol and testosterone on libido in postmenopausal women. Br. Med. J. 294, 936-937. 96. Barrett-Connor, E., Timmons, M. C., Young, R., Wiita, B., and Estratest Working Group (1996). Interim safety analysis of a two-year study comparing oral estrogen-androgen and congugated estrogens in surgically menopausal women. J. Women's Health 5, 593-602. 97. Buckler, H. M., Robertson, W. R., and Wu, F. C. W. (1998). Which androgen replacement therapy for women? J. Clin. Endocrinol. Metab. 83, 3920-3924.

~ H A P T E R 3~

Alt ernatlve " Th er ap"les to Hormone Replacement Therapy MICHELLE P.

WARREN

RUSSALIND H.

RAMOS

Department of Obstetrics and Gynecology and Medicine, Columbia University, College of Physicians and Surgeons, New York, New York 10032; and Center for Menopause, Hormonal Disorders, and Women's Health, Sloane Hospital for Women, Columbia Presbyterian Medical Center, New York, New York 10032 Center for Menopause, Hormonal Disorders, and Women's Health, Sloane Hospital for Women, Columbia Presbyterian Medical Center, New York, New York 10032

I. Introduction II. Phytoestrogens III. Herbs

I.

IV. Selective Estrogen Receptor Modulator: Raloxifene V. Recommendations References

INTRODUCTION

Menopause is an integral part of the process of aging, specifically denoting the cessation of menses. It is a state of hormonal dysfunction secondary to ovarian failure, and, as such, it is responsive to hormone replacement therapy (HRT). However, not all women can, or prefer to, take HRT. The issue of who should be treated raises complicated questions. Despite the potential health benefits of mammalian est r o g e n s - c o m m o n l y conjugated equine estrogens (CEEs) in the United States and 17/3-estradiol in Europe--for b o n e s , coronary arteries, lipoprotein metabolism, the genitourinary system, the brain, and the eyes, estrogens have the disadvantage of being tissue agonists for breasts and endometrial tissue. In addition, for women with an intact uterus, addMENOPAUSE: BIOLOGY AND PATHOBIOLOGY

459

ing progestin to estrogen poses unwanted side effects, i.e., continuing menstrual periods, and other symptoms, such as bloating and depression. Conventional HRT may cause other potential side effects in some women, such as gallbladder disease, breast tenderness, and mood changes, among others. Thus, alternative therapies that include natural products such as phytoestrogens and herbs, as well as raloxifene, which is a selective estrogen receptor modulator (SERM), have attracted increasing attention from the general public. These alternative therapies have been suggested to protect against breast and endometrial cancer, to obviate the need for progestin, and to have less side effects, yet still provide health benefits. This chapter will review the classification, dietary sources and metabolism, biological effects, and potential clinical Copyright9 2000by AcademicPress. All rightsof reproductionin any formreserved.

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effects of natural products, including phytoestrogen and herbs, and the medical alternative, raloxifene.

isoflavones, whereas the soy sprout is a potent source of coumestral, the major coumestan [4]. Phytoestrogens in the diet may have a role in modulating hormone-related diseases based on their structural similarity to 17/3-estradiol and diethylstilbestrol, as seen in Fig. 2, with a similar distance between the t w o - O H groups on equol, enterolactone, and enterodiol and that on 17/3-estradiol, which is a factor essential for strong binding to the estrogen receptor. The structure of phytoestrogens is also similar to the weak estrogen/antiestrogen tamoxifen, which is used for the treatment and prevention of breast cancer. Also, the presence and position of t h e - O H groups on the phytoestrogen compounds, estradiol and diethylstilbestrol, are considered one of the prerequisites for estrogenic activity [5]. Phytoestrogens are attenuated estrogens [6]. In vitro studies performed with human cell cultures have examined the relative estrogenic effects of different phytoestrogens [7]. Estimates of the relative potencies of these compared to estradiol (E2) at (100%) are coumestrol (0.202), genistein (0.084), equol (0.061), and daidzein (0.013). Importantly, however, the same levels of bioactivity were produced by the isoflavones and by E 2 when tested at concentrations sufficiently high to elicit maximal responses. This indicates that the estrogen receptor complexes formed by E 2 and isoflavonoids are functionally equivalent. The comparative dissociation constant of genistein for the estrogen receptor determined in competitive binding experiments is 100 to 10,000 times higher than that of estradiol and diethylstilbestrol [8]. It has been accepted that phytoestrogens are "weak estrogens," having less binding affinity for the estrogen receptor as compared to estradiol. It has been discovered that there are at least two forms of the estrogen receptor (ER): the ce form, which is the "classic" estrogen receptor, and the/3 form. It is interesting to note that genistein and daidzein bind to both forms of estrogen receptor and have about seven times greater affinity for ERce than for ER/3 [9,10]. Genistein and daidzein

II. P H Y T O E S T R O G E N S Phytoestrogens, estrogen-like compounds found in plant products, have been shown to have both estrogenic and antiestrogenic properties. Following the publication of the AllenDoisy bioassay for estrogens in 1923 [ 1], plant extracts were first reported to exhibit estrogenic activity in 1926 [2]. By 1975, several hundred plants had been found to exhibit estrogenic activity on bioassay or to contain estrogenically active compounds [3]. Phytoestrogens have been identified in bile, urine, semen, blood, and feces in humans and animals. The rapidly growing body of literature on the geographic differences in the incidence and prevalence of many diseases--for example, coronary heart disease and breast, endometrial, and ovarian cancers, as well as menopausal symptoms, especially hot flushesmhas implicated several etiologic factors, including racial characteristics, diet, and lifestyle. This has provoked interest in diet, in particular the idea that certain foods may contain different biologically active compounds. Epidemiologic studies have suggested that consumption of a diet rich in phytoestrogen, commonly seen in Asian populations, especially Japan, has reduced the risk for the so-called Western diseases, i.e., cardiovascular disease and breast and endometrial cancers, as well as menopausal symptoms, especially hot fushes. There are three main classes of phytoestrogens, isoflavones, lignans, and coumestans, which occur in either plants or their seeds. Resorcyclic acid lactones exhibit estrogenic activity and are produced by molds that commonly contaminate cereal crops and hence are better termed mycoestrogens (Fig. 1) [3a]. A single plant often contains more than one class of phytoestrogen. For example, the soy bean is rich in

Dietary estrogens

I

I

Naturally occurring

Synthetic contaminants

I Ovarian steroids

Phytoestrogens

I

I

I

Mycoestrogens

Growth promoters

(diethylstilbestrol)

I Xenoestrogens (DDT, PCB)

I

I,so,,avono, sll ,0nans II o,hers I I

I

I

I,so,,avonesI Icoumes,ansI FIGURE 1 Sources and classification of dietary estrogens. From Ref. [3a], A. Murkies, G. Wilcox, and S. Davis. Phytoestrogens. The Journal of Clinical Endocrinology and Metabolism 83(2), 297-303, 1998. 9 The Endocrine Society.

HO

O

HO

-

O ~ / O H

O 1a. Formononetin

OH o ~. r.~~~Q_~5 HO

OH [ ~

HO

~

HO

O 1b. Daidzein

OCH1

'

~

CH10

O 1d. Equol*

"/ O H

O 1c. Genistein

OCH1

OGluc OGluc

HO

OH

2a. Secoisolariciresional diglucoside

2b. Enterodiol*

O HO~ ~ ~ ~ 2c. Enterolactone*

OH

HO 3. 1713-Estradiol

[

yoH

O ~ N

~

HO 4a. Diethylstilbestrol

4b. Tamoxifen

4c. Ipriflavone

FIGURE 2 A comparison of the chemical structures of isoflavones (formononetin, daidzein, genistein, and equol), lignans (secoisolariciresinol diglucoside, enterodiol and enterolactone), endogenous estrogen (17/3-estradiol), and synthetic molecules (the potent synthetic estrogen diethylstilbestrol, the potent synthetic antiestrogen tamoxifen, and the potent isoflavone derivative ipriflavone). Tissues and biological fluids contain mammalian phytoestrogens that are derived from plant phytoestrogens [these compounds are indicated by asterisks (*)]. From Ref. [11], D. Tham, C. Gardner, and W. Haskell. Potential health benefits of dietary phytoestrogens: A review of the clinical, epidemiological, and mechanistic evidence. The Journal of Clinical Endocrinology and Metabolism 83(7), 2223-2235, 1998. 9 The Endocrine Society.

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have about 37% of the binding affinity for ERfl and about 5% for ERa as compared to 17fl-estradiol [10]. In addition, because phytoestrogens exhibit a weaker binding affinity to both estrogen receptors than do endogenous estrogens, they may have much higher concentrations in the body [ 11,12].

A. I s o f l a v o n e s 1. DIETARY SOURCES AND METABOLISM

Isoflavones are the most common forms of phytoestrogens. They have a common diphenolic structure resembling the structure of the potent synthetic estrogens diethylstilbestrol and hexestrol. Genistein and daidzein are two of the major isoflavones found in humans. Genistein and daidzein are parent compounds, which are metabolized from their plant precursors, biochanin A and formononetin, respectively. In plants, isoflavones are inactive when present in the bound form as glycosides, but when the sugar residue is removed, these compounds become activated. These plant compounds undergo fermentation by intestinal microflora, with both metabolites and unfermented parent (aglycone) compounds being liable to absorption. In the body, the parent compounds are reconjugated to glucuronides, but others do not undergo any further metabolism in the body and are excreted in the urine [13]. In the colonic microflora, daidzein may be metabolized to equol or to O-demethylangolensin (O-Dma) and genistein may be metabolized to p-ethyl phenol. Daidzein, genistein, equol, and O-Dma are the major phytoestrogens detected in the blood and urine of humans and animals [11 ]. Isoflavones are found in a variety of plants, including fruits and vegetables, but they are predominantly found in leguminous plants and are especially abundant in soy [ 11 ] (Table I). The isoflavone contents of different varieties, crops, and harvest years of soy beans vary substantially. In addition, different soy products (e.g., tofu and soy bean concentrates) have variable isoflavone content, which can be attributed to various processing steps. For example, processed soy products, such as hot dogs and tofu yogurt, may contain only one-tenth of the isoflavone content of whole soy beans (p.2-0.3 vs. 2 4 mg isoflavone/g) [14]. Several factors influence the bioavailability of isoflavones. Fermentation, as in the case of tempeh, a fermented soy bean product, enhances urinary isoflavone recovery [15]. Several investigators have reported that individual variability in colonic microflora plays an important role in determining the preferred pathways of isoflavone metabolism and the bioavailability of isoflavones [11 ]. 2. BIOLOGIC EFFECTS Genistein, the most extensively studied isoflavone, is an inhibitor of tyrosine protein kinases [ 16], DNA topoisomerases I and II [17] and ribosomal $6 kinase [18]. Other properties include inhibition of angiogenesis [19] and differentiation of cancer cell lines [20]. Genistein is reported to inhibit tumor promoter-induced hydrogen peroxide formation, and scav-

enge exogenously added hydrogen peroxide in human cell culture [21]. Other isoflavones, such as daidzein, apigenin, and prunectin, have also been shown to be potent hydrogen peroxide scavengers and antioxidants [22]. The antioxidant potencies of isoflavones are structurally related and closely associated with the presence of hydroxyl groups at positions 4' and 5' and with the position of the aromatic ring [ 11 ]. Tyrosine kinase inhibition is important because these receptor enzymes are involved in control of mitogenesis, cell cycle regulation, cell survival, and cellular transformation via growth factor binding. Growth control factors modulated by tyrosine kinases include epidermal growth factor, transforming growth factor a, platelet-derived growth factor, and insulin and insulin-like growth factors, and all have been implicated in tumor growth [23]. DNA topoisomerase II and ribosomal $6 kinase inhibition may lead to protein-linked DNA strand breaks, arrest of tumor cell growth, and differentiation induction of several malignant cell lines [24]. Tyrosine kinase mediation of mammary tumor cells to milk-producing, growth-arrested cells has been reported [25]. Possible mechanisms for the antiproliferative properties of genistein include prevention of cell mutations by stabilization of cell DNA and reduction of cell oxidants, reduction in capacity of malignant cells to metastasize by inhibiting angiogenesis and subsequent tumor growth, as well as inducing cell differentiation [26]. In addition, isoflavones also possess antihypertensive and antiinflammatory properties [23]. B. L i g n a n s 1. DIETARY SOURCES AND METABOLISM

Lignans are compounds possessing a 2,3-dibenzylbutane structure and exist as minor constituents of many plants, where they form the building blocks for the formation of lignin (as distinguished from lignan) found in the plant cell wall. They are constituents of higher plants (gymnosperms and angiosperms), such as whole grains, legumes, vegetables, and seeds, with exceptionally high concentrations of lignans found in flax seed [11] (Table II). The chemical structure of plant lignans differs somewhat from that of mammalian lignans (Fig. 2). Most of the structural changes occur in the colon, liver, and small intestine during enterohepatic circulation. Mammalian lignans differ in structure from plant lignans in that they have phenolic hydroxyl groups in the meta position only in their aromatic rings. Once in the colon, they are absorbed and then are conjugated with glucuronic acid or sulfate in the liver, reexcreted through the bile duct, deconjugated by the bacteria, and reabsorbed. Some reach the kidney and are excreted in the urine [15]. Lignans are excreted in the urine as conjugated glucuronides and in feces in the unconjugated form [ 11]. The major mammalian lignans are known by the common names enterolactone and enterodiol, which are the products of colonic bacterial metabolism of the plant lignans matairesinol

463

CHAPTER 32 Alternatives to HRT

TABLE I

Study food Wang and Murphy [14,14a] b Soy bean 1 Soy bean 2 Roasted soy beans Soy flour Soy granule Textured vegetable protein 1 Textured vegetable protein 2 Protein isolate 1 Protein isolate 2 Protein concentrate (alcohol extraction) Tofu Tempeh Miso 1 Miso 2 Soy hot dog Soy bacon Tempeh burger Tofu yogurt Soy parmesan Cheddar cheese 1 Cheddar cheese 2 Mozarella cheese Flat noodle Dwyer et al.[13a] c Tofu, brand 1 Tofu, brand 2 Tofu, brand 3 Tofu, brand 4 Soy drink Soy-based specialty formula 1 Soy-based specialty formula 2

F o o d Sources of Isoflavones a Total isoflavones

Daidzein

Genistein

Glycetin

1176 4215 2661 2014 2404 2295 2261 621 987

365 1355 941 412 917 799 831 89 191

640 2676 1426 1453 1225 1175 1185 373 640

171 184 294 149 262 321 245 159 156

73 532 865 647 389 236 144 386 282 88 43 197 123 127

0 238 405 272 107 55 26 95 103 26 0 83 24 15

19 245 422 281 227 129 83 255 162 6 4 62 56 56

54 49 38 94 55 52 35 36 17 56 39 52 43 56

289 260 313 292 28 3 5

76 73 97 86 7 0 1

213 187 216 206 21 3 4

a Values are in micrograms per gram of food source. b Glucoside, malonyl, acetyl, and aglycone forms of isoflavones combined. c Values shown are averages of two lots, one purchased in June and the other in December of 1992. Biochanin A, coumestrol, and formononetin were assayed, but found in only negligible amounts. From Ref. [11], D. Tham, C. Gardner, and W. Haskell. Potential health benefits of dietary phytoestrogens: A review of the clinical, epidemiological, and mechanistic evidence. The Journal of Clinical Endocrinology and Metabolism 83(7), 2223-2235, 1998. 9 The Endocrine Society. Adapted with permission from Refs. [14,14a]. Copyright 1994 American Chemical Society.

and secoisolariciresinol, respectively [27]. The h i g h e r the dietary intake of precursors, the higher the m a m m a l i a n lignan p r o d u c t i o n in the colon and the higher the e x c r e t i o n rate in the urine. Clinical trials p e r f o r m e d by K i r k m a n et al. [28] d e m o n s t r a t e d that the e x c r e t i o n of the lignans, enterodiol and e n t e r o l a c t o n e , was h i g h e r during a c a r o t e n o i d (carrot and spinach) and cruciferous (broccoli and cauliflower) v e g e t a b l e diet than during a v e g e t a b l e - f r e e diet, s u g g e s t i n g that these v e g e t a b l e s m a y p r o v i d e a source of m a m m a l i a n lignan precursors. B e c a u s e dietary m e t a b o l i s m of lignans as well as isoflavones is d e t e r m i n e d p r e d o m i n a n t l y by the gastrointestinal flora, antibiotic use or b o w e l disease and g e n d e r will m o d i f y m e t a b o l i s m [11 ].

2. BIOLOGIC EFFECTS M a n y plant lignans have b e e n s h o w n to have anticarcinogenic, antiviral, bactericidal, and fungistatic activities [ 2 9 31 ]. E n t e r o l a c t o n e , the m o s t a b u n d a n t m a m m a l i a n lignan, is a m o d e r a t e inhibitor of p l a c e n t a l a r o m a t a s e and c o m p e t e s with the natural substrate a n d r o s t e n e d i o n e for the e n z y m e . O t h e r e x p e r i m e n t s with a c h o r i o c a r c i n o m a cell line (JEG-3) s h o w e d that e n t e r o l a c t o n e is very readily transferred f r o m cell culture m e d i a into the cells [32]. M o s t of the lignans, as well as flavonoids, are only w e a k inhibitors. Ho w e v e r , a diet rich in v e g e t a b l e s may, due to the a b u n d a n c e of these c o m p o u n d s in the diet, lead to sufficient c o n c e n t r a t i o n s (e.g.,

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WARREN AND RAMOS

TABLE II Study food Thompson et al. [26a] Flax seed meal Flax seed flour Cereals Triticale Wheat Oats Brown rice Corn Rye Barley Cereal brans Oat bran Wheat bran Rice bran Oilseeds Soy bean Sunflower seeds Peanuts Legumes, dried whole Lentil Kidney Navy bean Pinto bean Vegetables Garlic Asparagus Carrot Sweet potato Broccoli Mushroom Celery Cucumber Tomato Fruits Pear Plum Strawberry Banana Orange Canteloupe Apple

Food Sources of Lignans a

C. P o t e n t i a l C l i n i c a l E f f e c t s o f P h y t o e s t r o g e n s

Total lignans

Enterodiol

Enterolactone

675.4 526.8

85.2 118.2

590.2 408.6

9.2 4.9 3.4 3.0 2.3 1.6 1.1

5.2 4.1 2.5 1.7 2.0 0.7 0.4

4.0 0.8 0.9 1.3 0.3 0.9 0.7

6.5 5.7 1.8

2.6 2.7 1.3

3.9 3.0 0.5

8.6 4.0 1.6

6.9 2.0 1.0

1.7 2.0 0.6

17.9 5.6 4.6 2.0

7.9 3.3 3.5 1.5

10.0 2.3 1.1 0.5

4.1 3.7 3.5 3.0 2.3 0.6 0.3 0.3 0.2

0.8 ! .4 2.8 2.4 1.6 0.4 0.2 0.2 0. I

3.3 2.4 0.6 0.6 0.7 0.1 0.1 0.1 0. I

1.8 1.5 0.8 0.7 0.4 0.4 0.3

!. ! 0.5 0.4 0.6 0.3 0.2 0.3

0.7 ! .0 0.4 0. I 0.1 0.2 0.0

aValues are expressed as mammalian lignan production by fecal flora (micrograms) from foods (per gram). From Ref. [11], D. Tham, C. Gardner, and W. Haskell. Potential health benefits of dietary phytoestrogens: A review of the clinical, epidemiological, and mechanistic evidence. The Journal of Clinical Endocrinology and Metabolism 83(7), 2223-2235, 1998. 9 The Endocrine Society.

in fat cells) to reduce conversion of androstenedione to estrone, lowering risk for estrogen-dependent cancer [33]. Lignans may also affect cholesterol homeostasis, in that they have been shown to inhibit the activity of cholesterol-7ahydroxylase, the rate-limiting enzyme in the formation of primary bile acids from cholesterol [34].

Phytoestrogens are thought to decrease the risk for cardiovascular disease, breast and endometrial cancers, and osteoporosis, to provide relief for menopausal symptoms, notably hot flushes, and possibly to improve memory, mood, and sleep patterns. These potential clinical effects may be attributable to a variety of potential mechanisms that could either be estrogen receptor dependent, owing to their structural similarity to the estrogens 17fl-estradiol and diethylstilbestrol, or independent, owing to their influence on enzymes, protein synthesis, cell proliferation, angiogenesis, calcium transport, Na+,K+-adenosine triphosphatase, growth factor action, vascular smooth muscle cells, lipid oxidation, and cell differentiation [26]. Based on current epidemiologic studies, clinical trials, and mechanistic data on this field, phytoestrogens have been regarded as beneficial. This section discusses the relationship of phytoestrogens to diseases and conditions, with consideration to the possible mechanisms.

I. CARDIOVASCULARDISEASE Estrogen decreases the risk of cardiovascular disease, as illustrated by the lower incidence among premenopausal women as compared to men. However, incidence rises as women reach menopause, approaching that of males. Hormone replacement therapy decreases this risk. The protective effects of estrogen may be manifested through lipid changes, with decreases in low-density lipoprotein (LDL)cholesterol and increases in high-density lipoprotein (HDL)cholesterol, and vascular effects, in both vasomotor tone and vessel wall compliance [26]. In postmenopausal women, phytoestrogens act as an estrogen agonist and may produce effects similar to those of estrogen. Several lines of evidence, including epidemiologic, clinical trial data, and basic science, suggest the plausibility of a causal, inverse relationship between phytoestrogens and cardiovascular disease. The well-established low rates of cardiovascular diseases and the high intakes of dietary phytoestrogens in Asian populations relative to those in other industrialized countries are consistent with a potential protective effect of phytoestrogens [35]. Although there are confounding variables such as differences in dietary fat and cholesterol, the consumption of soy protein has been shown to alter lipid levels [36]. A number of human clinical trials have reported variable effects of phytoestrogens on serum lipid. A review of the effects of soy consumption on lipid levels produced results with a range in cholesterol concentrations from + 1 to - 12% in normocholesterolemic human subjects [37]. Hypercholesterolemic subjects were found to have large decreases (1234 %) in 11 studies, moderate to minimal decreases ( 1- 10%) in four studies, no change in one study, and small increases (1 and 6%) in two others [23]. A 9% reduction in total cholesterol was observed in a small study of normolipemic premenopausal women given a 60-g soy protein supple-

CHAPTER32 Alternatives to HRT ment [38]. In another study, moderately hypercholesterolemic (mean, 6.0 mmol/liter) postmenopausal women were given 45 g/day of either soy flour or wheat flour for 12 weeks [39]. Follow-up done after 12 weeks revealed an insignificant decrease in circulating cholesterol for both treatment groups, with no significant difference between the soy and the wheat flour groups in cholesterol levels, despite significantly higher urinary concentrations of phytoestrogens for the women taking the soy flour. Consumption of 25 g of soy protein-enriched bread resulted in a decreased total serum cholesterol and increased HDL cholesterol in hypercholesterolemic men [40]. Gooderham et al. [41] reported no significant cholesterol effects in a study of 20 healthy men who were given 60 g/day of either soy protein or casein for 28 days, despite the 100- to 150-fold increase in plasma isoflavone concentrations of genistein and daidzein during the soy protein diet. A soy bean protein diet in subjects with Type II hyperlipoproteinemia may lower cholesterol on average by 20% [42]. A meta-analysis of 38 published controlled clinical trials of soy protein consumption that averaged 47 g/day and serum lipid and lipoprotein concentrations found that consumption of soy protein was associated significantly with mean reductions in total cholesterol (9.3% decrease), LDL cholesterol (12.9% decrease), and triglycerides (10.5% decrease) [43]. Other data have suggested that the soy protein is needed for reduction of plasma cholesterol, and that the purified isoflavone without the protein moiety will not exert this beneficial effect [44]. A randomized, double-blind cross-over trial [45] was conducted among 51 nonhypercholesterolemic and normotensive perimenopausal women, to investigate the effect of soy protein supplementation with known levels of phytoestrogens on cardiovascular risk factors and menopausal symptoms. Women were randomly assigned for 6-week periods to one of the three diets: (1) 20 g of complex carbohydrate (comparison diet), (2) 20 g of soy protein containing 34 mg of phytoestrogens, either given in a single dose, or (3) split into two doses, and then subsequently randomized to the remaining two interventions. Compared with the carbohydrate placebo diet, significant declines in total cholesterol (6% lower) and low-density lipoprotein cholesterol (7% lower) were observed on both soy diets. No significant differences were noted in HDL cholesterol or triglyceride. In contrast to estrogen replacement therapy [46], soy protein does not increase, and may actually reduce triglyceride concentrations, whereas traditional HRT has been shown to increase HDL cholesterol. Figure 3 compares the lipid and lipoprotein values among the treatment diets and illustrates the beneficial effect of the soy protein supplement on lipids and lipoproteins. A significant decline in the diastolic blood pressure (5 mmHg lower) was noted in the twice-daily soy diet, compared with the placebo diet. The twice-daily group showed significant improvement in the severity of vasomotor symptoms and in hypoestrogenic symptoms. Adherence was excellent in both groups, signifying high tolerability. It is inter-

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FIGURE 3 Differences in lipids and lipoproteins between treatment groups adjusted. FromRef. [47a], S. Washburn,G. Burke, T. Morgan,and M. Anthony. Effectof soy protein supplementationon serumlipoproteins,blood pressure, and menopausalsymptomsin perimenopausalwomen.Menopause 6(1), 7-13. esting to note that based on mortality data from the lipid research clinic study, a 6% decrease in total cholesterol levels would be expected to reduce coronary heart disease risk by approximately 12% [47], suggesting a significant effect of dietary soy supplementation on the primary prevention of coronary heart disease. Two proposed mechanisms for the hypocholesterolemic effect of phytoestrogens are the up-regulation of the LDL receptors and/or the inhibition of the endogenous cholesterol synthesis. Phytoestrogens in soy protein may stimulate the clearance of cholesterol, probably by up-regulating LDL receptors, and thereby increasing LDL receptor activity [48]. Lipoprotein a [Lp(a)] is a cholesterol-carrying particle in the blood that is structurally similar to LDL, with the addition of the apoprotein (a) [apo(a)] moiety. Although cholesterol levels are used as predictors of ischemic heart disease, Lp(a) is now acknowledged as a strong, independent predictor of heart disease, stroke, and failure of therapeutic intervention [49]. The only compounds found to alter circulating Lp(a) are estrogens and other sex steroids, which may decrease Lp(a) concentrations by 35% [50]. Phytoestrogens, having a structural similarity with endogenous estrogens and estrogen receptor-binding capabilities, may suggest a potential means of lowering Lp(a), warranting further studies to test this. An important cardiovascular risk factor, arterial compliance, diminishes with menopause. This may lead to systolic hypertension and increase the left ventricular work. A clinical trial [44] has been done to assess the effects of increased isoflavone intake on arterial compliance and plasma lipids in

466 postmenopausal women. The trial included a 3- to 4-week run-in period, a 5-week placebo period, a 5-week 40-mg period, and a 5-week 80-mg period. Arterial compliance was assessed using ultrasound as a pressure (carotid artery) and volume (outflow into aorta) relationship. Arterial compliance increased significantly by 23% in the 80-mg period and slightly less in the 40-mg period as compared to the placebo period. These findings of improved arterial compliance with increased isoflavone intake may represent a potential new therapeutic approach in improving cardiovascular function after menopause. Independent of the possible role of soy protein in the reduction of plasma cholesterol concentrations, studies of cultured vascular cells have demonstrated that increased concentrations of isoflavonoids alter cellular processes associated with lesion development [51 ]. In low-density cultures of proliferating endothelial cells, genistein induced marked morphologic changes. When concentrations were increased up to 25 /xmol/liter, genistein induced a highly spread morphology compatible with growth arrest. In contrast, confluent quiescent endothelial cells did not exhibit toxicity signs even at genistein concentrations up to 200/xmol/liter [52], suggesting that only proliferating cells are targeted by genistein, leaving quiescent, nondividing cells unaffected. Proteolytic degradation of the extracellular matrix by endothelial cells is controlled by angiogenic factors, such as basic fibroblast growth factors (bFGF), that induce the production of urokinase-type plasminogen activator and its physiological inhibitor, plasminogen activator inhibitor- 1. Experiments have demonstrated that genistein markedly reduced both bFGF-stimulated and basal levels of both plasminogen activator and plasminogen activator inhibitor-1 activities in bovine microvascular endothelial cells. Moreover, genistein inhibited the bFGF-induced migration of endothelial cells in wounded confluent monolayers of endothelial cells [52]. These data may represent a more complex interference of genistein with early events of angiogenesis. The role of genistein as a protein tyrosine kinase inhibitor may be responsible for an antithrombolytic effect [53]. An increase in tyrosine phosphorylation at tyrosine residues of platelet proteins is associated with the platelet's activation, and protein tyrosine phosphorylation is subsequently increased after thrombin stimulation [54]. Genistein, therefore, may reduce tyrosine phosphorylation, decreasing platelet activation, leading to a reduction in the deposition and aggregation of platelets and a decrease in the progression of atherosclerosis. Cardiovascular disease being a major health concern, this collection of data on phytoestrogens reflects a promising role in its prevention and treatment. Preventing chronic diseases such as cardiovascular disease by means of dietary intervention is an attractive and cost-effective health benefit. 2. CANCER

It has been well accepted that the incidence and prevalence of cancer differ among the various populations of the

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world. Hormone-related cancers of the breast, ovary, endometrium, and prostate have been reported to vary by as much as 5- to 20-fold among populations, and migrant studies indicate that the differential is largely attributable to environmental factors rather than to genetics [55,56]. Japan has consistently been reported to have the lowest risk of hormonedependent cancers [55]. Migrants to Western countries from Asia who maintain their traditional diet do not increase their risk of these diseases [57], whereas an increased risk for these diseases accompanies a change toward a westernized diet [58]. The highest rates of these cancers are typically observed in populations with Western lifestyles that include relatively high-fat, meat-based, low-fiber diets, whereas the lowest rates are typically observed in Asian populations with Eastern lifestyles that include plant-based diets with a high content of phytoestrogens [55,59]. Isoflavones and lignans have been reported to reduce the proliferation of cells. Research studies indicate that these effects are largely dependent on specific conditions, such as the concentration of phytoestrogen, the presence or absence of other endogenous estrogens, and the particular cell line (e.g., estrogen receptor dependent or independent). Enterolactone, enterodiol, and synthetic mammalian lignan derivatives inhibit growth in human breast cancer cell lines by 18-20% [60]. Lignans seem to be cytostatic in that 95% of treated cells remain viable. The mechanism of effect is mediated by inhibition of plasma membrane ATPase (other than the NA+,K+-dependent ones). The effects of synthetic and naturally occurring flavonoids were tested on the same breast cancer cell line [61]. Inhibition of cell proliferation has been noted among all compounds tested, daidzein concentration requiring 15.3 mg/ml to inhibit exponential cell growth by 5 0 % . Endogenous and exogenous sex hormones have been associated with various cancers. High concentrations of biologically active estrogens and androgens are associated with increased risk of breast and ovarian cancer in women and prostate cancer in men. Diets that lower these estrogen or androgen levels are associated with low risk of breast, ovarian, and prostate cancers [62]. For breast, ovarian, endometrial, and colon cancers, there is a consistent relationship between reproduction, exogenous hormone use, and risk factors. Despite the positive associations of endogenous and exogenous estrogens with these cancers, plant estrogens are inversely associated with cancer. Some phytoestrogens have been shown to inhibit enzymes that are involved in the production of estrone from the androgens (e.g., aromatase), thus denying the tumor a source of endogenous estrogen [63]. Lignans and isoflavones seem to stimulate sex hormone binding globulin (SHBG) synthesis in the liver, most likely reducing the biologic effects of sex hormones [13,64]. An increase in SHBG leads to a decrease in the percentages of free testosterone and free estradiol and in the reduction of both the albuminbound and the free fraction of the sex hormones. This results in a reduction in the metabolic clearance rate of the steroids, thereby reducing their biologic activity. The urinary ex-

CHAPTER 32 Alternatives to HRT cretion of isoflavones and lignans in both premenopausal and postmenopausal Finnish women correlates positively with plasma SHBG and negatively with plasma percentage free estradiol and percentage free testosterone [ 13]. In vitro studies using HepG2 liver cancer cells showed that enterolactone [65], genistein [66], and daidzein [M. Carson et al., unpublished data] stimulate SHBG synthesis. The anticarcinogenic effects of genistein may be partly attributed to its antioxidant properties. Studies have reported that genistein strongly inhibits tumor-promoter-induced H202 formation both in vivo and in vitro. The fact that genistein potentially inhibits oxidant formation and protooncogene expression suggests that the antioxidant properties and antiproliferative effects of genistein may at least in part be responsible for the anticarcinogenic mechanisms [22]. a. B r e a s t Cancer. A dietary component has long been implicated in the etiology of breast cancer. Subjects with breast cancer or at high risk of breast cancer excrete low amounts of lignans and isoflavonoids [64,67], but subjects living in areas with low risk of hormone-dependent cancers have higher levels [68]. Japanese women, in their homeland, who have breast cancer have a higher number of in situ tumors with fewer nodal metastases, and those with metastases have less nodal spread, compared with women with breast cancer in the United States or Britain [23]. The rates of breast and endometrial cancers are lower in Japan, where soy is a staple of the diet, compared to the rates in the United States, where very little soy is eaten. Hirayama 69] reported an inverse relationship between the consumption of miso (soy bean paste soup) and breast cancer risk among Japanese women. In a case-control study, Ingram et al. [70] reported a substantial reduction in breast cancer risk among women with a high intake (as measured by excretion) of phytoestrogens--particularly the isoflavonic phytoestrogen equol and

467 the lignan enterolactone. These results may be important in the prevention of breast cancer. In a study of Asian-Americans of Chinese, Japanese, and Filipino heritage, it was reported that tofu consumption was significantly and inversely associated with breast cancer [71 ]. In Fig. 4 [7 l a], note that breast and endometrial cancer rates are lower in Japan, where soy consumption is high, as compared to the United States, where soy consumption is low. b. E n d o m e t r i a l Cancer. There is a 30-fold difference in the incidence of endometrial cancer between various cancer registration areas [72]. Rates range from 25 per 100,000 in parts of the United States to 2 per 100,000 in Singapore and Japan [55]. Large differences in rates are also reported in this study between white Hawaiians (23/100,000) and Japanese Hawaiians (15/100,000). Endometrial cancer rates do not seem to be increased by dietary phytoestrogen as compared to conjugated estrogens. In a multiethnic casecontrol study [73] comparing various dietary factors in 332 endometrial cancer patients in Hawaii and 511 age- and ethnicity-matched controls, including Japanese, Caucasian, Chinese, Filipino, and Native Hawaiian populations, high consumption of soy and other legumes has been reported to be associated with half the risk of developing endometrial cancer. High-fiber and low-fat diets were correlated with reduced risk. There are no sufficient data regarding the effect of high levels of dietary phytoestrogens on endometrial growth, but no increased incidence in endometrial cancer has been observed from areas with high dietary levels of phytoestrogens. c. Colon Cancer. Phytoestrogens may be protective with regard to colon cancer. Some epidemiologic evidence in Japan [74] has demonstrated lower incidence of colon cancer in areas with high tofu consumption. Three case-control

FIGURE 4 (A) Age-adjustedbreast cancer rates for women35-74 years of age in 1985.Modifiedfrom Ursin et al. (B) Age-specific endometrial cancer rates for the years 1983-1987. Modifiedfrom Parkin et al. [159], from Archer [71a].

468 studies concerning colon cancer incidence and soy product consumption suggest a trend toward a lower risk of colon cancer with increasing consumption, although neither trial reached statistical significance [75]. In vitro studies have been performed on well-differentiated adenocarcinoma-type colorectal cell lines using biochanin A, genistein, and diadzein, among others [76]. Biochanin A and genistein inhibited cell growth via activation of a signal transduction pathway for apoptosis. Although other mechanisms of cell death were considered, such as inhibition of tyrosine kinase, topoisomerases I and II, and ribosomal $6 kinase, or induction of cytochromes P450, this study suggests a process for isoflavone-induced apoptosis not requiring protein synthesis. Estradiol, progesterone, and dihyrotestosterone receptors have been identified in primary colon cancer tumors [77] and it has been suggested [78] that these receptors may play a role in the pathogenesis of colon cancer. A review of the epidemiologic, clinical trial, animal model, and cell culture data suggests that phytoestrogens are strong candidates for a role as natural cancer-protective compounds. This hypothesis is strongly supported by the high dietary levels of these compounds in countries or areas with low cancer incidence. However, the current evidence is not yet sufficient and further research is needed to clearly establish the association between phytoestrogens and cancers. 3. OSTEOPOROSIS

In the elderly, continual loss of bone is a natural process of aging. In addition, osteoporosis is also related to gender and hormone deficiency. Women have a higher incidence of osteoporotic fractures as compared to men due to lower peak bone mass, and among postmenopausal women, the decrease in estrogen production accelerates bone loss. Hormone replacement therapy has been documented to be effective in the reduction of postmenopausal osteoporosis [79]. Research studies on the relationship between phytoestrogen intake and osteoporosis have been very limited, but there is a reason to believe that there may be some effect on bone. Epidemiologically, bone density is lowest in Asian women and highest in African-American women, with white women in the middle [80]. However, it is interesting to note that the incidence of hip fracture is lower in Asians (and also African-Americans), as compared to whites [81 ], despite the fact that Asians have thinner bones. The reasons for the differences are unknown, although several factors may be contributory, such as body habitus, exercise, and diet. In a study done by Ho et al. [82], the rates of hip fracture in Hong Kong and the United States were compared; for men and women 85 years old and above, the rates in Hong Kong were roughly one-third of the rates in the United States. In a double-blind clinical trial done by Potter et al. [83], the effect of soy protein and phytoestrogens on bone mineral density among hypercholesterolemic postmenopausal women was examined. Subjects were randomly assigned to 40 g of

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protein/day from either soy protein with a high concentration of isoflavones, soy protein with a moderate concentration of isoflavones, or a casein nonfat dry-milk group. No significant differences were found among the three groups in bone density studies of the hip or total body. Significant increases were found in lumbar bone mineral density and bone mineral content (2%) among those who took the soy protein with a high isoflavone content compared to the casein nonfat dry-milk group. The group receiving the moderate concentration of isoflavone had intermediate and nonsignificant changes in the lumbar spine. This is consistent with an estrogenic effect; conjugated estrogens have a more pronounced effect on trabecular than on cortical bone, and therefore affect bone density more in the vertebrae than in femoral bone. A number of studies done on ipriflavone (7-isopropoxyisoflavone), an isoflavone derivative, have rendered indirect evidence on the potential benefits of phytoestrogens regarding bone metabolism. Ipriflavone, at a dose of 600 mg/day, maintained bone density when given to premenopausal women taking gonadotropin-releasing hormone (GnRH) agonists [84], as well as maintained or increased bone density in postmenopausal women [85]. One of the main metabolites in humans of ipriflavone is daidzein [86], which forms around 10% of the breakdown products or approximately 60 mg of daidzein daily. There were some animal studies done to support the hypothesis of the potential beneficial effect of phytoestrogens on bone and these effects may involve nonestrogenic mechanisms. Soy bean intake and genistein, which is the predominant isoflavone in the soy beans, have been tested on bone density in the oophorectomized rat model, In a study by Fanti et al. [87], it has been reported that genistein protects against loss of trabecular bone volume and bone mineral density and appears to increase osteoblastic activity without affecting osteoclasts. Because estrogen appears to suppress osteoclastic activity (bone resorption) rather than osteoblastic activity (bone formation), the effect of genistein on bone may not be an estrogen-like mechanism. Another study in which rats were injected subcutaneously with genistein (5 /zg/g body weight) has reported that in the genistein-treated group, there was less bone loss and a higher rate of bone formation [88]. An abstract done by Anderson and colleagues [89] has reported that genistein in low doses was equivalent to conjugated equine estrogens in maintaining bone mass in ovariectomized rats. Genistein appears to have a biphasic effect. In a model of extreme bone loss, it is more effective at low rather than high doses. In another study, four groups of rats were compared: oophorectomized rats, sham-operated rats, oophorectomized rats fed with a milk protein diet with 17/3estradiol, and oophorectomized rats fed with soy bean protein isolate [90]. Oophorectomized rats showed significant decreases in bone density; both 17fi-estradiol and soy bean protein isolate have been shown to protect against this bone

CHAPTER 32 Alternatives to HRT loss. A primate study has reported that soy protein did not prevent increased cortical bone turnover in ovariectomized macaques [91], which is different from what has been reported in the rat studies. Although the data supporting the specific role of phytoestrogens on bone are limited, there is, nevertheless, some evidence that isoflavones may benefit bone. There have been inconsistencies in the results from animal studies with regard to bone density conservation, but one consistent finding is that, unlike estrogen, soy does not reduce oophorectomy-induced bone turnover. Soy appears to affect bone by stimulating osteoblastic activity rather than reducing osteoclastic activity. 4. OTHER MENOPAUSAL SYMPTOMS

The most common symptom of menopause is vasomotor instability (manifested as hot flushes and profuse sweating). It has been postulated that the consumption of foods containing phytoestrogens contributes to the lower rate of hot flushes among Asian women. The incidence of hot flushes varies in frequency from 70 to 80% of menopausal women in Europe [92], to 57% in Malaysia [93], and 18 and 14% in China [94] and Singapore [95], respectively. Significant differences in the consumption of soy products exist among the populations in these geographic areas. It is possible that the estrogenic effects of phytoestrogens may be responsible for modifying the frequency and severity of vasomotor symptoms among these populations. Hot flushes were reported to be lower among women in Japan than those in Canada, which may be attributed to the high phytoestrogen intake in Japan [96]. In a study done by Adlercreutz [97], urinary isoflavone and endogenous estrogen levels were analyzed among women from Japan, America, and Finland. The urinary isoflavone in the Japanese women was excreted at 100-1000 times the amount of the endogenous estrogens in omnivorous women. A double-blind, randomized, placebocontrolled study [98] was done in Italy among 104 postmenopausal women comparing the effects of daily intake of 60 g of isolated soy protein with 60 g of the milk protein casein for 12 weeks. Women receiving soy experienced a 45% reduction in hot flushes, whereas the placebo group experienced a 30% reduction (signifying the placebo effect). Another clinical trial done among 145 Israeli postmenopausal women found that the 78 women assigned to a phytoestrogen-rich diet had a significant decrease in hot flushes and less vaginal dryness. In yet another study among 58 women comparing the effects of daily intake of 45 g of soy flour with 45 g of wheat flour for 12 weeks [39], it was found that the soy group had significantly fewer hot flushes. Four studies have been done to test possible estrogenic effects of dietary phytoestrogens on vaginal epithelial cells. Significant improvements in the vaginal maturation index was found among 25 women who were supplemented with 4 g of soy flour daily [99]. Another study found improve-

469 ments in vaginal cytology [ 100]. The other studies had negative results. The Murkies study did not find significant changes in vaginal cytology, and the remaining study [101 ] showed an increase in the percentage of vaginal superficial cells but did not achieve statistical significance. A synthetic phytoestrogen based on zearalenone, a resorcyclic acid lactone, has estrogenic activity similar to that of conjugated estrogens with regard to the effects on flushes, dyspareunia, and vaginitis [102]. In this double-blind controlled study, conjugated estrogens and a zearalenone analog were equally effective in the treatment of menopausal symptoms and both were superior to placebo. Increased vaginal maturation was also seen in both treated groups compared with placebo. Based on these data, phytoestrogens appear to have efficacy and tolerability. Thoughtful consideration of the consumption of a diet rich in phytoestrogen to alleviate menopausal symptoms seems reasonable.

III. HERBS Although the conventional HRT remains the cornerstone for the treatment of menopausal symptoms, most notably hot flushes, herbal medicines (also known as phytopharmaceuticals) are also recognized for their efficacy and safety. Considerable interest in this area has been a current trend and a significant number of women acknowledge and prefer to use herbal therapy in alleviating their symptoms. This may be attributed to the misconception that herbal therapies are all "natural," and therefore safe. Commission E of the German Federal Health Authorities is the only agency that regulates the safety and efficacy of herbs and phytomedicinals. Medicinal herbs are defined as crude drugs of vegetable origin utilized for the treatment of disease states, often of a chronic nature, or to attain or maintain a condition of improved health [103]. This includes tinctures, extracts, and similar preparations that, as such, are called phytomedicinals (plant medicines). Herbs are known to have medicinal effects, and as such must be considered drugs, though not requiring prescription, and are purchased over the counter (OTC) and therefore are not Food and Drug Administration (FDA) approved. Safety has been an issue, and health care providers thus have an obligation to be well informed and updated, and to elicit as much information as possible, as to what herbal medicines patients are using, how much, and how often, and to offer the best advice for safety.

A. Menopausal Symptoms and Selected Herbal Therapies The postmenopausal period gives rise to a constellation of symptoms that cause discomfort and distress, at times severe enough to affect the quality of life of postmenopausal

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women. These symptoms, caused by a decrease in circulating estrogen during menopause, include vasomotor instability (manifested as hot flushes and profuse sweating), genitourinary changes, decreased libido, sleep disturbances, mood changes, and memory and concentration difficulties, among others. For these menopausal symptoms, the traditional management is HRT, but selected herbal therapies for specific complaints have also been shown to be efficacious. 1. VASOMOTOR INSTABILITY

A hot flush is the most common symptom among postmenopausal women. It is a sudden, transient sensation of heat or burning, starting from the head down through the neck, and may progress in a wavelike fashion over the entire body. In most cases, the hot flush is accompanied by profuse perspiration, particularly involving the head, neck, chest, and back, and is often followed by chills. Many women claim a prodromal feeling described as anxiety, pressure in the head, or a tingling sensation for a few minutes, signaling an impending flush. Several factors may trigger a hot flush. These would include a warm environment, certain foods (spicy foods, alcohol, caffeine, and hot drinks), stress, and many others. Hot flushes can be mild, moderate, or severe. They can last from a few seconds to a few minutes and may occur infrequently or very often in a day. Although the underlying dynamics of the hot flush are not clear, it appears that central temperature-regulating control is lost secondary to decreases in estrogen, with subsequent loss of autonomic control of the peripheral vasculature [ 104]. The flush is not a release of heat that has accumulated, but is an inappropriate, sudden excitement of the mechanisms that control heat release [105]. Recommendations found in the lay press have included black cohosh, dong quai, ginseng, gotu kola, licorice root, sage, and sarsaparilla in relieving hot flushes. Commission E has approved chaste tree, also called chasteberry (Vitex agnus castus), for menopausal symptoms, including hot flushes. 2. GENITOURINARY CHANGES

Decreasing estrogen levels generally cause atrophy of the genital organs. The mucosa becomes pale, thin, dry, and inelastic. The major problem is atrophy of the vaginal mucosa, which may construct the vaginal introitus and cause dyspareunia, which affects sexual functioning and may cause psychological stress. Atrophy of the genital organs also increases susceptibility to trauma and infection. Some degree of urinary incontinence is common among postmenopausal women. In most cases, this is not caused by gross anatomic changes, but is due to changes in the supporting structures of the bladder neck. Maximal urethral closure pressure decreases with aging [ 106]. Lubricants are frequently advised, including commercial water-soluble lubricants, vegetable oils, and unscented oils such as mineral oil. Some authors suggest a combination of

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herbs for these conditions as a tea, douche, or ointment [107-109]. Cultural factors can influence the use of herbs. Two studies evaluated the use of herbs to enhance sexual experience in Zimbabwen women [ 110,111 ]. Most women in both studies have claimed during interviews that they routinely inserted herbal preparations into the vagina to achieve satisfactory sexual experience. The goal was to facilitate "dry sex," believed to be an essential part of a successful marital relations. Herbs recommended by the lay press for various vaginal complaints include agrimony, chaste tree, dong quai, and witch hazel. 3. M o o d CHANGES AND SLEEP, MEMORY, AND CONCENTRATION DIFFICULTIES The physiologic changes associated with menopause are often accompanied by psychologic and social changes, at times making it difficult to separate their clinical effects. Though most of the physiologic changes can be explained by decreasing estrogen concentrations, understanding the psychologic phenomena is more complicated. A significant number of women report insomnia, mood changes, memory and concentration difficulties, and depression, among others. Most of these symptoms can be attributed to various factors from the past lifestyle and to the life changes that occur during menopause. Although somatic symptoms are usually relieved by HRT, psychologic symptoms are not always alleviated, and to many women, herbal therapies have helped, coupled with modifications of habits and lifestyle, including cessation of smoking, decreasing alcohol intake to the minimum, getting sufficient exercise, eating a balanced and healthy diet, and reducing of stress. Herbs recommended by the lay press for these problems include balm, chamomile, damiana, ginkgo, gotu kola, hops, passion flower, scullcap, and valerian. Commission E has approved balm (Melissa officinalis), black cohosh (Cimicifuga racemosa), and passion flower (Passiflora incarnata) for nervous disturbances; ginseng (Panax quinquefolius) for loss of concentration; ginkgo (Ginkgo biloba) for mood swings; St. John's wort (Hypericum perforatum) for depression; and valerian for insomnia. 4. SEXUALITY

The issue of sexuality is as important as the other issues of menopause. Problems dealing with sexuality in the menopausal period have been regarded as very distressing and have brought feelings of inadequacy, frustration, and a loss of wellbeing, among others, to many women. Most women experience some changes in sexual functioning during menopause, and these include decrease or loss of sexual desire and diminished sexual activity and responsiveness. Several factors influence sexual function. These include changes in the physiologic, psychologic, sociocultural, and interpersonal factors. Physiologic changes occurring during menopause and related to sexual function include decrease in muscle

CHAPTER32 Alternatives to HRT tension, lack of increase in breast size during sexual stimulation, delay in reaction time of the clitoris, delay and decrease in vaginal lubrication, decrease in vaginal expansion, and many others. Most of these changes are probably caused by alterations in sensory stimulation and blood flow secondary to a decrease in estrogen levels [106]. Aging and change in body image can also affect sexual function. Herbal therapies recommended by the lay press sources include chaste tree, damiana, ginseng, gotu kola, and sarsaparilla.

B. R e v i e w o f S e l e c t e d H e r b a l T h e r a p i e s This section is limited to the eight herbal therapies judged by Germany's Commission E to be possibly safe and efficacious in the treatment of menopausal symptoms. The symptoms, however, are varied and are not limited to hot flushes, vaginal dryness, and insomnia. The following discussions represent a comprehensive review of herbal therapies used in the menopausal woman. 1. BALM (Melissa officinalis)

Preparations of the leaves of Melissa are commonly known as lemon balm (Melissa officinalis L.), a member of the mint family (Lamiaceae, formerly Labiatae) [112]. Melissa contains about 0.1-0.2% volatile oil, composed mainly of oxygenated compounds such as citral (isomers a and b), geraniol, caryophyllene oxide, and polyphenols (caffeic acid, protocatechuic acid, etc.); a tannin composed of chlorogenic, caffeic, and rosmarinic acids; flavonoids; and other compounds [ 113]. Historically, the leaves (primarily in tea form) have been used for their calmative, spasmolytic, and carminative activity. In Germany, it was allowed to use lemon balm preparations to alleviate difficulty in falling asleep due to nervous conditions and to treat functional gastrointestinal symptoms [ 113]. Balm has been suggested by the lay press for the relief of tension, anxiety, stress reactions, and depression, owing to its vasodilatation effect on the circulatory system [ 105]. In addition, it has been said to be effective in menopause for "increased tension" [ 114]. Scientifically, balm has been regarded to have sedative, antispasmodic, antihormonal, and antiviral actions in various animal models [ 115], thought to be due to the presence of volatile oils. More recently, antibacterial and antiviral activities have been confirmed. Oxidative products of caffeic acid and derivatives were found to have antiviral activity [103]. A European ointment corresponding to 0.7 g of leaf per gram of ointment has become available in the American market. Commission E approved balm for the treatment of nervous disturbances. Balm is recommended to be used with caution. A tincture of 2 - 6 ml three times/day is suggested, or 2 - 3 teaspoons of dried herb steeped in 1 cup of boiling water twice/day.

471 2. BLACK COHOSH (Cimicifuga racemosa)

Black cohosh consists of the dried rhizome and roots of

Cimicifuga racemosa (L.) Nutt., a member of the buttercup family (Ranunculaceae), which is indigenous to the rich woods of the eastern deciduous forest of North America [112]. The naturally occurring triterpene constituents of Cimicifuga include acetin, cimi-cifugoside, acetylacteol, 27deoxyactein, cimi-genol, and deoxyacetylacetol. The flavonoid constituents include formonetin, and the aromatic constituents include isoferulic and salicylic acids [ 116]. This herb was introduced to American medicine by Native Americans, who called it "squaw root" because it was predominantly used to treat uterine disorders and female complaints by promoting or restoring healthy menstrual activity; soothing irritation and congestion of the uterus, cervix, and vagina; relieving pregnancy-related pain or distress; and promoting a quick and uncomplicated delivery and uterine involution and recovery [ 117]. Its ability to lower blood pressure has been confirmed in both human and animal studies [117] and it has been used for centuries in America, Europe, and China for treating hypertension. Black cohosh has been used since the early 1940s in Germany as a natural hormonal agent for treating disturbances of the hypophysis, and authorities certified a favorable benefit:risk ratio for its use for "premenstrual and dysmenorrheic as well as climacteric neurovegetative complaints" [ 118]. Several clinical trials have been conducted to prove the safety and efficacy of black cohosh. In an essay on the clinical effectiveness of Cimicifuga published in a German medical journal [ 119], the author concluded that Cimicifuga has a hormone-like and slightly euphoric effect. In a study among 629 patients with menopausal complaints who were given the Cimicifuga extract Remifemin at a dose of 40 drops, twice/ day, for 6 - 8 weeks, improvement in menopausal symptoms in approximately 80% of the patients was observed, and complete disappearance of individual symptoms have been reported by some patients [ 120]. Two open studies were conducted wherein patients with menopausal complaints who either had contraindications to hormone replacement therapy (HRT) or refused to take HRT were given Remifemin at 40 drops, twice/day, for 3 months. The authors concluded that the herbal extract is a promising therapeutic regime in cases of refusal of hormonal therapy, possesses high therapeutic efficacy, shows outstanding tolerance, leads to very good patient compliance, and shows positive results without the use of hormones [ 121,122]. In a randomized, double-blind study, the effects of Remifemin given four tablets daily were compared to effects of receiving 0.625 mg of conjugated estrogens daily plus control effects of three placebo tablets and control effects of four placebo tablets. The group receiving the herbal extract had a notable increase in the degree of proliferation of the vaginal epithelium, as well as a significant improvement of the somatic parameters in comparison to the

472 estrogen and placebo group. The conjugated estrogens showed a slight influence on the vaginal epithelium, and the control group showed no clear improvement of the menopausal ailments [ 124]. In a group of 60 hysterectomized patients under 40 years old, who had at least one intact ovary and still complained of climacteric symptoms, the effects of estrogens were compared with Remifemin. Remifemin was shown to be just as effective in improving postoperative ovarian functional deficits after hysterectomy in young women compared with estriol, conjugated estrogens, and an estrogen-gestagen combination [123]. In an open, controlled comparative study with 110 patients, Remifemin was compared to placebo on gonadotropin secretion in menopausal women with typical menopausal ailments. The Remifemin group (2 tablets, twice/day) had a significant luteinizing hormone (LH) suppression compared to the placebo group (2 tablets, twice/day). Neither group showed a significant effect on the follicle-stimulating hormone (FSH) serum concentration. The herbal extract showed an estrogen-like mode of action with selective LH suppression in menopausal women and no effect on FSH, unlike the effects of estrogen therapy [ 157]. Commission E has approved the use of black cohosh for the treatment of dysmenorrhea, premenstrual syndrome (PMS), and nervous conditions associated with menopause. There are limited data on black cohosh toxicity. Ingestion of leaves and roots may cause nausea and vomiting. Additive hypotensive effects may occur in patients taking both black cohosh and antihypertensive agents [125]. Commission E recommends daily doses of 4 0 - 2 0 0 mg of black cohosh, with use not to exceed 6 months [112]. The herb is usually taken as a decoction or ethanol tincture in amounts corresponding to the stated dosage.

3. CHASTE TREE, CHASTEnERRV (Vitex

agnus castus) This herb consists of the fruits of Vitex agnus castus L., a shrub or small tree native to West Asia and southwestern Europe that has become naturalized in much of the southeastern United States. It is a member of the verbena family (Verbenaceae) [ 112]. The fruits contain flavonoids, which are considered to be the primary active components. These include the major flavonoid casticin (quercetagetin-3,6,7,4'-tetramethyl ether), as well as orientin (the 3,6,7,4'-tetramethyl ether of 6-hydroxykaempferol), and quercetagetin [126]. Chaste tree (chasteberry) is very popular in Europe in treating PMS and was considered to be the best herb in maintaining emotional balance before and during menses, as well as in menopause [107-109,127,128]. Some consider it as a "natural replacement alternative for estrogen replacement therapy" [127]. The lay press has suggested its use for menopause symptoms such as hot flushes, and it is said to stimulate pituitary function, alter LH and FSH secretions, and increase natural levels of progesterone. It is said to decrease excessive bleeding, due to its properties as a progesterone precursor [ 107-109,128].

WARREN AND RAMOS

It is reputed to have both aphrodisiac and anaphrodisiac properties [ 108]. It was used in ancient times to decrease libido in temple priestesses [ 109], and is still taken in Europe to treat "excessive sexual desire" [ 127]. In a clinical survey conducted by German gynecologists [ 129], the effect of a chaste tree preparation (Agnolyt) at a dose of 40 drops daily for 166 days was evaluated on 1542 women diagnosed with premenstrual syndrome, of which 90% reported complete relief of symptoms after an average treatment of 25.3 days. This herb may inhibit secretion of prolactin by the pituitary gland and thus may have a role in correcting some types of amenorrhea and hyperprolactinemia, and in increasing milk production in lactating women [ 130]. The German Commission E allows use of chasteberry preparations for PMS, mastalgia, menopausal symptoms, and inadequate lactation. Adverse effects include itching, rashes, and gastrointestinal discomfort. Animal experiments indicate a possible interference with dopamine receptor antagonists. Preparations include alcoholic extracts (tinctures) of the pulverized fruits, formulated to provide an average daily dose equivalent to 20 mg of the crude fruit, or 3 0 40 mg of the fruits in decoction [112].

4. GINKGO (Ginkgo biloba) Ginkgo consists of the dried leaves of Ginkgo biloba (maidenhair tree), the only surviving member of the ginkgo family (Ginkgoaceae), and appropriate formulations thereof. Active ingredients include flavones; bioflavonoids such as the glycosides kaempferol, quercetin, and isorhamnetin; and organic acids. Novel diterpene lactones unique to ginkgo include ginkgolides (A, B, C, and M) and bilobalide, a sesquiterpene. Of the diterpenes, ginkgolide B is considered the most active [112]. Extracts are made from dried leaves through a complex extraction process; during a multistep procedure, active ingredients are enriched and unwanted substances are eliminated. Finally, the liquid extract is dried to give 1 part extract from 50 parts raw drug (leaves) [131]. The effects of ginkgo may be caused by single active ingredients or by the combined action of the many agents found in the extracts. The main indications for ginkgo are peripheral vascular disease such as intermittent claudication, and, more important, cerebral insufficiency, which can be manifested by symptoms such as difficulties of concentration and memory and absent mindedness, confusion, lack of energy, tiredness, decreased physical performance, depressive mood, anxiety, dizziness, tinnitus, and headache [131]; these symptoms are not uncommon during the menopause. It is suggested that ginkgo may slow the aging process [109]. Several pharmacologic and clinical studies of ginkgo leaf extract have shown a positive effect on vasoregulating activity of arteries, capillaries, and veins (increased blood flow) in various circulatory disorders such as cerebral insufficiency, postthrombotic syndrome, varicose conditions, short-term memory loss, cognitive disorders secondary to

CHAPTER32 Alternatives to HRT depression, dementia, tinnitus, vertigo, and obliterative arterial disease of the lower limbs [112]. In vitro studies have shown that G. biloba extract has free-radical scavenging properties [160], probably owing to the flavonoid component. This could mean that ginkgo can stimulate relaxation of contracted blood vessels. Ginkgo is licensed in Germany for the treatment of cerebral dysfunction with the following symptoms: difficulties of memory, dizziness, tinnitus, headaches, and emotional instability with anxiety. It is also licensed as a supportive treatment for hearing loss during cervical syndrome and for peripheral arterial circulatory disturbances with intact circulatory reserve (intermittent claudication) [131]. No serious side effects have been noted for the ginkgo extract. In very rare cases, mild gastrointestinal disturbances, headache, and allergic skin reactions have been claimed. The recommended daily dose is 120-160 mg of standardized ginkgo leaf extract [ 132]. 5. GINSENG (Panax ginseng~Oriental; Panax quinquefolius--American)

This herb consists of the dried root of Asian ginseng (Panax ginseng C. A. Meyer) and American ginseng (Panax quinquefolius L.), both members of the ginseng family (A. Raliaceae) [ 112]. Asian ginseng contains as its primary active components at least 18 triterpenoid saponins, including ginsenosides. Other constituents include a trace of volatile oil, starch, polysaccharides (panaxans A - U in a concentration of 7-9%), pectin, free sugar, polyacetylenes, and the like [ 113 ]. American ginseng is regarded to be a better temperature regulator and provides more cooling properties [ 127]. Ginseng has been used for thousands of years and has been regarded as an aphrodisiac and a tonic. Several pharmacologic and clinical studies have been done to test its properties, which include radioprotective, antitumor, antiviral and metabolic properties; central nervous system, reproductive performance, and lipid metabolism enhancement, and antioxidant, cholesterol-lowering, and endocrinologic activities [ 133,134]. Some evidence exists showing that ginseng can facilitate resistance to stress. In Europe, several clinical studies have made use of extracts standardized to contain between 4 and 7% ginsenosides. The results were positive and reports include shortened reaction time to visual and auditory stimuli, elevated respiratory quotient, increased alertness, improved power of concentration, enhanced grasp of abstract concepts, and better visual and motor coordination [ 112]. The German Commission E monograph allows the use of ginseng as a tonic to treat fatigue, diminished work capacity, and loss of concentration, in addition to its use as a general aid during convalescence, but has recommended its use for only up to 3 months [135]. Ginseng is generally considered to be safe, although excessive use for a significant period of time can produce some adverse effects such as nervousness and hypertension [ 103]. Data are insufficient to recommend dosing.

473 6. PASSION FLOWWR (Passiflora

incarnata)

Commission E has approved the use of passion flower for "nervous unrest" conditions. It is marketed mainly in Europe, incorporated in some sedative-hypnotic mixtures [ 130]. The lay press has claimed that it relieves anxiety, insomnia, restlessness, muscle tension, and headaches due to nervous tension, and that it induces sleep without a hangover effect, making it the herb of choice for temporary insomnia [108,127,128]. Although it has been regarded as a "safe and natural tranquilizer" by the lay press [107], there is insufficient evidence concerning its efficacy and safety. There has been an isolated report of an elderly man developing a hypersensitivity reaction that was manifested as an erythematous rash, blister, and purpura over the chest after taking a preparation that included passion flower [112]. This was inconclusive though, because the preparation contained other ingredients. Currently, little information is available to make recommendations. 7. ST. JohN'S WORT (Hypericum perforatum) This drug consists of the dried flowering tops of Hypericum perforatum L., a member of the St. John's wort family (Hypericaceae). It is indigenous in Europe and naturalized in Asia, Africa, North America,and Australia. The chemical constituents of this herb are 0.05-0.3 % of naphthodianthrone pigments, especially hypericin and pseudohypericin; flavonoids, including hyperoside (hyperin), quercetin, isoquercetin, rutin, and nadkaempferol; various flavonols; and small amounts of volatile oils, among other components

[113]. Based on four clinical trials published since 1987, St. John's wort is best classified as an antidepressant, although sedative, anxiolytic, and antiinflammatory properties have been claimed to be found in this herb [ 112]. In a randomized, placebo-controlled, double-blind study [ 136], the effects of 300 mg of St. John's wort extract (standardized to a hypericin content of 0.9 mg), and a placebo preparation, on 105 patients diagnosed with mild to moderate depression were compared. The results showed a 67% response in the treatment group as compared to a 28% response in the placebo group. The end points used were depressive mood indicators such as feelings of sadness, hopelessness, helplessness, and uselessness, as well as emotional fear, and difficult or disturbed sleep. The researcher group, composed of a psychiatrist, an internist, and a general practitioner, concluded that St. John's wort can be recommended for the treatment of mild to moderate depression, and that compared with the synthetic antidepressants it produced side effects of minor significance. The xanthone and flavonoid fractions of the plant are believed to have monoamine oxidase (MAO)-inhibiting properties [ 103]. St. John's wort may cause photodermatitis, and lightskinned individuals are advised to avoid skin exposure to

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direct sunlight after taking this herbal preparation [113]. Until additional information is available, patients should be aware of possible food and drug interaction warnings similar to those for traditional MAO inhibitors. The recommended dosage is 2 - 4 g of the herb (calculated to contain 0.2-1.0 mg hypericin) in capsule form, to be taken once or twice daily, for mild antidepressant action or mild disturbances [ 137]. 8. VALERIAN(Valeriana

officinalis)

This drug consists of preparations of the dried or fresh root of Valeriana officinalis L., a member of the valerian family (Valerianaceae). Its natural range includes eastern, southeastern, and east-central Europe, extending to south Sweden and the Southern Alps. Locally naturalized westward in Europe, the plant is likewise naturalized in eastern North America [ 112]. The active constituents of Valerian have not been specifically identified, although numerous chemical components have been identified from the root and its essential oil. Valepotriates, a group of unstable iridoids, have been shown to possess sedative activity, as have some valepotriate degradation products. Other biologically active components may include valerenic acid and valerenone and/or an interaction of these constituents. Valerenic acid and the esters of eugenyl and isoeugenyl are spasmolytic [112]. Based on the review of published data, two types of valerian effects can be postulated: in low dosages, the drug acts thymoleptic-sedatively; in higher dosages, anticonvulsivespasmolytic effects will be additionally observed [138], and these different effects can probably be attributed to the different active components found in valerian. Commission E has approved its use as a "sedative and sleep-inducing preparation to mediate states of excitation and difficulty falling asleep due to nervousness" [ 161 ]. Valerian has been considered to be safe and to have low toxicity, but has been linked to four cases of severe liver damage [139]. In controlled clinical trials, there have been reports of headaches excitability, uneasiness, and cardiac changes [125]. The recommendation for this drug is 1-2 capsules at night or 2 - 3 g once or twice daily, as an antianxiety agent [ 112].

IV. S E L E C T I V E MODULATOR:

ESTROGEN

RECEPTOR

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E

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AND RAMOS

postmenopausal woman, the ideal estrogen would be one that promotes all of the positive effects, without stimulation of the uterus or breast. Thus an "ideal" estrogen would be one that promotes estrogenic effects at the desirable target tissues. Alternatives to estrogens are now available that achieve estrogenic effects selectively by activating estrogen receptors only in certain sites. This class of compound, now known as selective estrogen receptor modulators, or SERMs [140], also have estrogen antagonist effects on certain organs. In fact, it is as estrogen antagonists that most of these compounds were initially developed. These compounds now represent an important potential therapeutic alternative for women in the menopause. Two early SERMs include clomiphene citrate [141], a drug now used to induce ovulation, and tamoxifen, a drug used to treat breast cancer [ 142,143]. Tamoxifen was found to protect bone [ 144,145] and increase HDL cholesterol similar to estrogen [ 142,146]; subsequent studies have shown a cardiovascular benefit [ 147-149], but tamoxifen was also found to stimulate the endometrium and in some cases promote endometrial cancer [150-152]. Another drug, raloxifene, has shown more estrogen agonistic effects while behaving as an estrogen antagonist at the breast and the uterus [140]. Thus two important reproductive organs are not stimulated by this drug, but beneficial effects have been noted at the level of the bone and the lipid profile [153,154]. This combination of effects makes raloxifene unique. Some of the negative thromboembolic effects appear to occur, however, with an incidence of thromboembolism similar to estrogen use. Raloxifene also differs from estrogen in its lipid effects, in that it decreases LDL and triglycerides but is neutral on HDL. Long-term effects on cardiovascular events are not yet known. Other side effects such as leg cramps and leg swelling may occur, and there is no beneficial effect on hot flashes; in some cases hot flashes may increase. The most impressive effect of raloxifene to date has been its ability to reduce the development of breast cancer. Estrogen receptor-positive cancers are significantly reduced in patients on raloxifene. Thus, in high-risk patients this may be a very useful alternative to estrogen therapy. The development of new SERMs reflects the increasing specificity and complexity of alternative treatments for women postmenopause [ 155].

V. R E C O M M E N D A T I O N S

RALOXIFENE

Estrogens are known to have multiple effects on the body that appear to delay the effects of aging; these include protective effects on the heart, bones, brain, retina, colon, and skin. Unfortunately, this hormone also stimulates reproductive organs, in particular, the breasts and the uterus. In the

Alternative treatments with herbal remedies and phytoestrogens are receiving increasing attention because of interest in these compounds and their easy access in health food stores, without prescription. Many of these compounds have pronounced physiologic effects as well as undesirable side effects. They also may encourage patients to believe that they

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CHAPTER 32 Alternatives to H R T

TABLE I I I Herbal therapy Balm

(Melissa officinalis)

Black cohosh

(Cimicifuga racemosa)

Chaste tree, chasteberry (Vitex

agnus castus)

Ginkgo

(Ginkgo biloba)

Ginseng

(Panax quinquefolius)

Passion flower

(Passiflora incarnata) St. John's wort

(Hypericum perforatum)

Valerian

Herbs Judged Possibly Effective for Perimenopausal Lay press claims

Evidence

Symptoms by Germany's Adverse effects

Commission

Ea

Recommendations

Relief of tension, anxiety, stress reactions and depression

Has sedative, antispasmodic, antihormonal, and antiviral actions in various animal models; approved by Commission E for treatment of nervous disturbances

No information available, but should be used with caution

Use with caution; a tincture of 2 - 6 ml, 3 • or 2 - 3 tsp dried herb steeped in 1 cup boiling water, 2 X/day

Said to be a source of estrogen and progesterone

Approved by Commission E for treatment of dysmenorrhea, PMS, and nervous conditions associated with the menopause

Data on toxicity limited; ingestion of leaves and roots may cause nausea and vomiting; additive hypotensive effects may occur in patients taking black cohosh along with antihypertensive agents

Commission E recommends doses of 4 0 - 2 0 0 mg/day, not to exceed 6 months; usually taken as a decoction or ethanol tincture in amounts corresponding to stated dosage

Popular in Europe for treating Approved by Commission E for symptoms of PMS and the PMS, mastalgia, menopausal menopause; reputed to symptoms, and poor lactation; have both aphrodisiac and may inhibit secretion of anaphrodisiac properties prolactin

May cause an itchy rash and gastrointestinal discomfort; may interact with dopaminereceptor antagonists

Prepared as a tincture; extract provides an average daily dose equal to 20 mg of the crude fruit or 3 0 - 4 0 mg fruit in a decoction

Suggested that herb improves memory and relieves senility

Effective in the treatment of circulatory disorders, vertigo, tinnitus, dementia, mood swings; postulated to act as a free radical scavenger

Gastrointestinal disturbances, headache, allergic reactions; larger doses may produce restlessness; may reduce clotting time, so should be used with caution in patients receiving anticoagulants; severe poison ivy-like dermatitis may occur after contact with fruit pulp

120-160 rag/day of standardized ginkgo leaf extract

Considered a "wonder herb" by the Chinese, who have used it for >5000 years

Controversy exists in the scientific literature regarding its merits; no evidence of efficacy in humans; approved by Commission E to treat fatigue, diminished work capacity, and loss of concentration, but use not to exceed 3 months

Various adverse effects reported; women should be cautioned not to take with hormone therapy in light of case reports of uterine bleeding

No well-designed studies to support efficacy or dosing

Said to relieve anxiety, insomnia, restlessness, and muscle tension

Insufficient evidence of efficacy or safety; approved by Commission E for "nervous unrest" conditions

Little information available

Insufficient information available

Touted as a mild tranquilizer, an antidepressant, and an aid for menstrual cramps

Four published clinical trials since 1987 show efficacy in depression compared with placebo

May cause photodermatitis; until further information is available, patients should be aware of possible food and drug interaction warning similar to those that apply to traditional MAO inhibitors

Usual dosage is 2 - 4 g hypericin in capsule form, 12•

Said to have sedative and antianxiety properties; widely used in France

Approved by Commission E as a "sedative and sleep-inducing preparation to mediate states of excitation and difficulty falling asleep due to nervousness"; studies in humans support a tranquilizing, sedative, and hypnotic effect

Generally thought to be safe, but has been linked to 4 cases of severe liver damage; headaches, excitability, uneasiness, and cardiac changes reported in controlled clinical trials; not all active ingredients have been identified

1-2 commercial capsules HS; 2 - 3 g once or twice a day as an antianxiety regimen

a Bachman [ 156].

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are undergoing appropriate treatment for a medical condition when in fact the therapeutic efficacy for a medical condition has not been determined and an opportunity for intervention is lost. Physicians should realize that m a n y of these products are in fact drugs, although they make no claims of preventing or treating disease [130]. This is because they are sold as food products and are thus protected from Federal Food and Drug Administration regulation. Another p r o b l e m is that they are not subjected to F D A standards and thus their composition is highly variable. Physicians prescribing these drugs need to be knowledgeable about their effects, but sources for standardized information are lacking. The F D A keeps a list of herbs recognized as safe, but this is based on food use. The only regulating agency for herbs and phytomedicinals is G e r m a n y ' s Commission E, established in 1978 to review the safety and use of more than 1400 preparations. C o m m i s s i o n E publishes m o n o g r a p h s on the safety and efficacy of herbs and phytomedicinals, and these are a useful resource [130]. Only eight of the many herbs reviewed by C o m m i s s i o n E have been j u d g e d to have some efficacy for treatment of m e n o p a u s a l symptoms. These include balm, black cohosh, chasteberry, ginkgo, ginseng, passion flower, St. John's wort and valerian (Table III), [156]. Recent studies on soy products and other plant products containing soy suggest that isoflavones are effective in reducing menopausal symptoms, and r e c o m m e n d a t i o n s will be based on studies that have demonstrated effectiveness. C o m m i s s i o n E did not approve other herbs, which include licorice root, agrimony, angelica, catnip, chamomile, damania, dandelion, dong quoi, hops, sage, fenugreek, gotukola, and sarsaparilla [130]. Soy products appear to have promise for the relief of hot flushes [26,97]. The few studies done in a double-blind placebo protocol show an effect beginning at 4 to 6 weeks of treatment. Effects include a decrease in the number and/or the severity of hot flushes and improvement in sleep patterns [39,45,97,100]. The dose of isoflavone is 60 mg daily and can be taken in the form of dietary supplementation (including soy products and flaxseed) [39,100]. Diet can include tofu, tempeh, chick peas, soy beans, and soybean powder or milk. The milk can have a high caloric content, and patients should be forewarned. Isoflavone tablets are available both from soy and from red clover sources. Most of the published work has been done on the soy sources. These tablets are helpful in patients who are suffering acutely from hot flushes and find it difficult to change their diets [26,45,97,98]. In order to obtain full cardiovascular benefit, however, it should be noted that the full protein fiber moiety is necessary for the cholesterol benefit, and extracts may have the protein fiber removed. The safety of isoflavones in breast cancer patients has not been determined, but indirect evidence indicates that they may be beneficial and would be particularly useful in this group. M o r e studies are necessary before specific reco m m e n d a t i o n s can be made.

R

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AND RAMOS

References 1. Allen, E., and Doisy, E. A. (1923). An ovarian hormone. Preliminary report on its localization, extraction and purification, and action in test animals. JAMA, J. Am. Med. Assoc. 181, 819-821. 2. Loewe, S., Lange, F., and Spohr, E. (1927). Uber weiliche sexual hormone (Thelytropine). Biochem. Z. 180, 1-26. 3. Farnsworth, N. R., Bingel, A. S., Cordell, G. A., Crane, F. A., and Fong, H. H. S. (1975). Potential value of plants as sources of new antifertility agents. II. J. Pharm. Sci. 64, 717-754. 3a. Murkies, A., Wilcox, G., and Davis, S. (1998). Phytoestrogens. J. Clin. Endocrinol. Metab. 83(2), 297-303. 4. Price, K. R., and Fenwick, G. R. (1985). Naturally occurring estrogens in foods--A review. Food Addit. Contam. 2, 73-106. 5. Martucci, C. P., and Fishman, J. (1993). Enzymes of estrogen metabolism. Pharmacol. Ther. 57, 237-257. 6. Bannwart, C., Adlercreutz, H., Wahala, K., Brunow, G., and Hase, T. (1987). Isoflavonic phytoestrogens in humans, identification and metabolism. Eur. J. Cancer Clin. Oncol. 23, 1732. 7. Markiewiez, L., Garey, J., Adlercreutz, H., and Gurpide, E. (1993). In vitro bioassays of nonsteroidal phytoestrogens. J. Steroid Biochem. Mol. Biol. 45, 399-405. 8. Martin, P., Horwitz, K., Ryan, D., and McGuire, W. (1978). Phytoestrogen interaction with estrogen receptors in human breast cancer cells. Endocrinology (Baltimore) 103, 1860-1867. 9. Wang,T., Sathyamoorthy, N., and Phang, J. (1996). Molecular effects of genistein on receptor mediated pathways. Carcinogenesis (London) 17, 271-275. 10. Kuiper, G., Carlsson, B., Grandien, K., Enmark, E., Haggblad; J., Nilsson, S., and Gustaffson, J. (1997). Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors alpha and beta. Endocrinology (Baltimore) 138, 863870. !!. Tham, D., Gardner, C., and Haskeli, W. (1998). Potential health benefits of dietary phytoestrogens: A review of the clinical, epidemiological, and mechanistic evidence. J. Clin. Endocrin. Metab. 83, 2223-2235. 12. Adlercreutz, H., and Mazur, W. (1997). Phyto-oestrogens and Western diseases. Ann. Med. 29, 95-120. 13. Adlercreutz, H., Hockerstedt, K., Bannwart, C., Bloigu, S., Hamalainen, E., Fotsis, T., and Ollus, A. ( !987). Effect of dietary components including lignans and phytoestrogens on enterohepatic circulation and liver metabolism of estrogens and on sex hormone binding globulin (SHBG). J. Steroid Biochem. 27, ! 135-1144. 13a. Dwyer, J. T., Goldin, B. R., Saul, N., Gualtieiri, L., Barakat, S., and Adlercreutz, H. (1994). Tofu and soy drinks contain phytoestrogens. J. Am. Diet Assoc. 94, 739-743. 14. Wang, H., and Murphy, P. A. (1994). lsoflavone composition of American and Japanese soybeans in Iowa: Effects of variety, crop year, and location. J. Agric. Food Chem. 42, 1674-1677. 14a. Wang, H., and Murphy, P. A. (1994). lsoflavone content in commercial soybean foods. J. Agric. Food Chem. 42, 1666-1673. 15. Hutchins, A. M., Lampe, J. W., Martini, M. C., Campbell, D. R., and Slavin, J. L. (1995). Vegetables, fruits, and legumes: Effect on urinary isoflavonoid phytoestrogen and lignan excretion. J. Am. Diet. Assoc. 95, 769-774. 16. Akiyama, T., Ishida, J., Nakagawa, S., Ogawara, H., Watanabe, S., Itoh, N., Shibuya, M., and Fukami, Y. (1987). A specific inhibitor of tyrosine-specific protein kinase. J. Biol. Chem. 262, 5592-5595. 17. Okura, A., Arakawa, H., Oka, H., Yoshinari, T., and Monden, Y. (1988). Effect of genistein on topoisomerase activity and on the growth of [val 12] Ha-ras-transformed NIH 3T3 cells. Biochem. Biophys. Res. Commun. 157, 183-189. 18. Linassier, C., Pierre, M., Le Pecq, J. B., and Pierre, J. (1990). Mecha-

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19.

20.

21.

22.

23. 24.

25.

26. 26a.

27.

28.

29.

30. 31.

32.

33.

34.

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152. Dijkhuizen, F. R, Brolmann, H. A., Oddens, B. J., Roumen, R. M., Coebergh, J. W., and Heintz, A. E (1996). Transvaginal ultrasonography and endometrial changes in postmenopausal breast cancer patients receiving tamoxifen. Maturitas 25, 45-50. 153. Walsh, B. W., Kuller, L. H., Wild, R. A., Paul, S., Farmer, M., Lawrence, J. B., Shah, A. S., and Anderson, E W. (1998). Effects of raloxifene on serum lipids and coagulation factors in healthy postmenopausal women. JAMA, J. Am. Med. Assoc. 279, 1445-1451. 154. Delmas, E D., Bjarnason, N. H., Mitlak, B. H., Ravoux, A. C., Shah, A. S., Huster, W. J., Draper, M., and Christiansen, C. (1997). Effects of raloxifene on bone mineral density, serum cholesterol concentrations, and uterine endometrium in postmenopausal women. N. Engl. J. Med. 337, 1641-1647. 155. Cummings, S. R., Eckert, S., Krueger, K. A., Grady, D., Powles, T. J., Cauley, J. A., Nerton, L., Nickelsen, T., Bjarnason, N. H., Morrow, M., Lippman, M. E., Black, D., Glusman, J. E., Costa, A., and Jordan, V. C. (1999). The effect of raloxifene on risk of breast cancer in postmenopausal women. JAMA, J. Am. Med. Assoc. 281, 2189-2197. 156. Bachman, C. (1998). "Recognition and Management of the Perimenopausal Patient in Clinical Practice," Monogr. Embryon. Inc., Somerville, NJ. 157. Duker, E. M., Kopanski, H. J., and Wuttke, N. (1991). Effects of extracts from Cimicifuga racemosa on gonadotropin release in menopausal women and ovariectomized rats. Planta Med. 57, 420. 158. Ursin, G., Bernstein, L., and Pike, M. C. (1994). Breast cancer. In "Trends in Cancer Incidence and Mortality" (R. Doll, J. F. Fraumeni, and C. S. Muir, eds.), Vol. 19/20, pp. 241-264. Cold Spring Harbor Laboratory Press, Plainview, NY. 159. Parkin, D. M., Muir, C. S., Whelan, S. L., Gao, Y. T., Ferlay, J., and Powell, J., eds. (1992). Cancer incidence in five continents. Lyon: International Agency for Research on Cancer Scientific Publications No. 120. 6, 301-431,486-509. 160. Pincemail, J., Dupuis, M., Nasr, C., Hans, P., Haag-Berrurier, M., Anton, R., and Deby, C. (1989). Superoxide anion scavenging effect and superoxide dismutase activity of Ginkgo biloba extract. Experientia. 45, 708-712. 161. Monographie (1985). Valerianae radix (Baldrianwurzel). Bundesanzelger, Bonn.

...HA T E R

3:

Calcium Nutriture: A Model System for Understanding Menopause--Nutrient Interactions ROBERT P.

HEANEY

Creighton University, Omaha, Nebraska 68131

I. Introduction II. Calcium

III. Vitamin D References

I. I N T R O D U C T I O N

agrees that good nutrition is a sound plan, most of us seem to tolerate substantial departures from optimal eating without apparent consequences. Moreover, we all know individuals who do all the wrong t h i n g s - - e a t a high-fat diet, drink too much, smoke, exercise too littlemyet who charge into their declining years with seeming vigor and sometimes great zest. So there is a certain cognitive dissonance between what we know from theory and what we seem to know from experience. There are, I think, several explanations. First, the effects of bad nutrition are cumulative and, for the most part, are slow to develop. Thus we do not perceive them in a way that allows us to connect cause and effect. Second, many of the consequences of bad nutrition are isolated, discrete events--heart attacks, strokes, osteoporotic fractures--probabilistic in character and remote in time from the lifestyle indiscretions that may have caused them. Finally, the exceptional cases are just that--exceptions,

Good nutrition is important throughout life. It follows that nutrition is important at menopause as well. However, the events occurring at menopause--the loss of gonadal hormones and their effects on various body tissues and organs--are not primarily nutritional in nature, and hence even the best nutrition cannot prevent or reverse them. In that sense, nutrition cannot properly be considered an "intervention" at menopause in the way that hormone replacement therapy can be. The importance of nutrition at menopause is more subtle. It is expressed in several ways: (1) reduced estrogen levels decrease the ability to get by on marginal intakes of certain nutrients, and (2) disorders that are due in part to pathological responses to chronic adaptation to low calcium intakes begin to manifest. It is a commonplace experience that, although everyone

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Copyright 9 2000 by Academic Press. All rights of reproduction in any form reserved.

482 individuals with above-average intrinsic reserves and hence able to sustain physiological assaults for longer than average periods of time. Because our lives are never controlled experiments, individually we have no way of knowing what might have happened had we chosen differently. If, by simple chance, any given woman is likely to sustain one fracture from age 50 until the time she dies, and if she actually sustains two or three fractures over that period, how is she to interpret that experience? As due to her failure to drink milk as a teen-age girl? Or as bad luck? How does she even know that it is bad luck, i.e., how does she know what to expect? Ailsa Goulding, in her study of childhood fractures [ 1], reported that parents of children who sustained as many as six to eight fractures during adolescence considered their offspring to be "accident-prone," rather than wondering whether they had flimsy bones. ~ The connection between real causes and their effects, especially if remote in time, is generally perceptible only at a public health level or in randomized controlled trials, and is often extremely difficult to perceive in individual cases. In fact, as in all probabilistic matters, we can form judgments only by observing large numbers of people. In the last analysis we know that good nutrition is important for individuals because, when those individuals are aggregated, the group will experience fewer heart attacks, strokes, or osteoporotic fractures than would a comparable group of individuals whose nutrition was poor in respect to the nutrients important for those outcomes. But there is another consequence of inadequate diets that goes beyond direct nutrient deficiency effects. Inadequte diets cause the body to react, that is, to attempt to compensate for the vagaries of intake. Such compensation is an evolutionary adaptation necessary to tide animal species over temporary periods of food scarcity. The mechanisms by which the body compensates for an inadequate calcium intake will be described in more detail in what follows. But the fact of adaptation is important to grasp at the outset because menopause, in addition to its other effects, impairs a woman's ability to adapt to a low-calcium diet. This change requires heightened adaptive responses and, because many women cannot respond sufficiently, it is the basis for the increase in the calcium requirement at menopause. What this increase really means is that a woman can no longer get by on the marginal intake she may have had through much of her adult life, that is, she can no longer adapt as well as she could before. For all these reasons it is both appropriate and important to discuss nutrition in the context of menopause. Most of the general principles discussed here apply to

1Goulding et al. [1] showed, in fact, that children who fractured had significantly less bone mass compared to nonfracture control children.

ROBERT P. HEANEY

many nutrients. The focus is primarily on calcium and vitamin D, in part because the changes in bone mass that occur at menopause focus a woman's attention on precisely these two nutrients, and in part because these two serve as a useful model system, showing how the issues get complex very quickly. It is now well established that calcium and vitamin D play a key role in maintaining bone health and protecting against late-life osteoporotic fractures. But their nutritional role is not exhausted in these effects. Calcium, particularly, is now recognized to be important in the regulation of blood pressure, in reducing risk of colon cancer and in mitigating the symptoms of premenstrual syndrome (PMS); and vitamin D inadequacy, in addition to its bony effects, may be implicated in certain cancers as well. Each of these aspects of calcium and vitamin D nutrition is briefly described in what follows. (Other nutritional topics, such as diets high in phytoestrogens, are covered elsewhere in this volume.)

II. C A L C I U M Calcium is an essential cofactor for countless intracellular processes, from muscle contraction to signal transduction. Calcium's functionality is so broad, in fact, that essentially all cells have found it necessary to restrict calcium concentration in the cell sap to something like three to four orders of magnitude below that of the extracellular fluid (ECF) surrounding the cell. These low cytosolic concentrations permit cells to use calcium as a nearly universal second messenger, admitting controlled quantities into critical cellular compartments when specific functions are to be activated, and promptly pumping it out, either into the extracellular space, or into intracellular vesicles, when the cell action is to be shut off. Intracellular storage of calcium is the rule, rather than the exception. Examples include the sarcoplasmic reticulum of muscle and calcium phosphate crystals in mitochondria. One may speculate that, given the critical character of calcium to intracellular function, most cells have developed means to maintain their own supply and may, thus, have only limited dependence on extracellular calcium. However, multicellular life requires integrative functions and, therefore, the maintenance of critical concentrations of many factors in the ECF of complex organisms. Here a constant high calcium concentration (in the range of 1.25 mM) is essential for a variety of functions, ranging from synaptic transmission to blood clotting. The means whereby ECF [Ca 2+] is stabilized vary across the higher vertebrates. Fish and amphibia have access to the calcium in the surrounding water, and buffer the concentration of calcium in their extracellular fluids by controlling calcium fluxes across the gill membranes. But terrestrial vertebrates, dependent on periodic food ingestion for their calcium, need both an internal

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source and an internal sink of calcium for homeostasis. Bone serves that homeostatic purpose.

from the bony reserves (by increasing bone resorption and then scavenging the calcium released in the process). The aggregate effect of them all, as Fig. 1 indicates, is to prevent or reverse a fall in ECF (Ca2+).

A. R e g u l a t i o n of C a l c i u m H o m e o s t a s i s The concentration of calcium in extracellular fluid is tightly controlled by adjusting both the renal calcium threshold and flows of calcium into and out of the ECF. These processes, in turn, are regulated by parathyroid hormone (PTH), 1,25-dihydroxyvitamin D (1,25(OH)zD), and calcitonin. PTH acts to correct a fall in calcium concentration by a complex set of interacting effects, summarized in Fig. 1 [2]. These actions include (in the probable order of the appearance of their effects): (1) decreased renal tubular reabsorption of serum inorganic phosphate (Pi), (2) increased resorptive efficiency of osteoclasts already working on bone surfaces, (3) increased renal lce-hydroxylation of circulating 25(OH)-vitamin D to produce the chemically most active form of vitamin D, (4) increased renal tubular reabsorption of calcium, and (5) activation of new bone remodeling loci. These effects interact and reinforce one another in important ways, indicated by the connections between the loops of Fig. 1. For example, the reduced ECF Pi caused by the immediate fall in tubular reabsorption of phosphate is a potent stimulus to the synthesis of 1,25(OH)zD, and it also increases the resorptive efficiency of osteoclasts already in place and working in bone. 1,25(OH)zD directly increases intestinal absorption of both ingested calcium and the endogenous calcium contained in the digestive secretions and it also is necessary for the full expression of PTH effects in bone, particularly the maturation of cells in the myelomonocytic line that produce new osteoclasts. Together, these effects reduce losses through the kidneys, improve utilization of dietary calcium, and remove calcium

Bone Resorption

PT.

~ Abs ~1,;

9 Renal Excr. Ca

FIGURE 1 Schematic depiction of the three-arm control loop regulating extracellular fluid [Ca2+], showing specifically the response to a drop in [Ca2+]; PTH, parathyroid hormone. Adapted from Arnaud [2]. Copyright Robert P. Heaney, 1981. Reproduced with permission.

B. A d a p t a t i o n to C a l c i u m I n s u f f i c i e n c y Under the conditions in which human physiology evolved, calcium intake would have averaged --~0.8 mmol (32 mg)/ kg/day [3], and, when adequate food was available, the gut would have served as a nearly continuous source of calcium. The mechanisms for dealing with a fall in ECF (Ca 2+), just described, would have been called into play only infrequently, i.e., at times of food shortage or for long intervals between meals. Thus these are basically emergency measures. By contrast, under contemporary conditions, adult diets often contain less than 0.2 mmol (8 mg) Ca/kg/day. The reason for the fall in the calcium content of the human diet from paleolithic to historic times is that cereal grains and legumes, which constitute more than half the total food intake of agriculture-based populations, are typically poor sources of calcium. Neither food was prominent in preagricultural diets. The much lower calcium intake of contemporary diets requires adaptive mechanisms to be invoked frequently, and in some individuals, constantly. The ability to adapt to reduced intake or increased loss varies, and depends on genetics, lifestage, and environmental factors. Calcium is a good illustration of these three kinds of factors. Women vary considerably in their calcium absorption efficiency. A part of this variability is certainly hereditary (although the precise mechanistic basis for this genetic effect remains unclear). Similarly, gifts at puberty are able to absorb calcium much more efficiently than they will be able to do at menopause. And finally, other factors such as hormonal status and coingested nutrients (such as salt and protein) influence the adaptive ability to reduce calcium losses in the face of inadequate intakes [3]. Most young individuals seem able to adapt to low intakes reasonably well, that is they can largely protect what skeletal mass they have so long as they are not subjected to unusual stresses. However, even in the young, adaptation may never be fully optimal, i.e., although they can build a skeleton during growth, its mass probably does not reach its full genetic potential on low intakes. Moreover, this ability to get by on low calcium intakes declines with age. Various policymaking bodies have recognized this decline by recom, mending higher calcium intakes for the elderly [4,5]. But there is a sense in which the requirement in the elderly uncovers the true requirement for all ages, that is, it reveals the intake that protects the skeleton without requiring constant adaptation.

484

ROBERT P. HEANEY

It is at the point of regulating ECF (Ca2+), i.e., adapting to low calcium intake, that the system influences both bone health and blood pressure regulation (and possibly the manifestations of PMS, as well). Adaptation, although obviously a necessary ability, may exert undesirable effects when constantly invoked. Medical science is familiar with this phenomenon in the case of stressmwith its high adrenergic hormone levels and high secretion of adrenal glucocorticoids. Less well understood, but gradually becoming clearer, are the counterpart effects following from constant high secretion of PTH with its cascade of mechanisms. First is an increase in parathyroid cell mass [6]. Associated with this phenomenon is an age-specific increase in incidence of hyperparathyroidism among postmenopausal women [7]. Whether the two phenomena are causally related is not settled, but it is true for other organ systems that constant stimulation promotes neoplasia, and it would be surprising if this same relationship were not true for the parathyroid glands as well. Other likely consequences of constant adaptation, described in more detail below, are preeclampsia, hypertension, and colon cancer. Optimal calcium intake means not just adequacy for the skeleton, but reduction in risk of such other disorders as well.

C. O p t i m a l C a l c i u m I n t a k e In approaching an estimation of the optimal calcium intake, it is useful to bear two points in mind: calcium was a surfeit nutrient in the environment in which human physiology evolved, and calcium exhibits what is termed "threshold" behavior. Calcium is a unique nutrient in at least two senses. It is about the only nutrient with a reserve that has acquired a major function in its own right, i.e., bone provides structural support to resist gravity and make locomotion and mechanical work possible. In brief, we walk on our calcium reserve. Calcium is unique in a second respect. It is about the only nutrient that will not accumulate in the body as a result of ingesting more than we need. This is a little appreciated but extremely important feature of calcium nutrition. It is a reflection first of the environmental abundance of calcium during human evolution and second of the fact that calcium is not stored as such, but as bone tissue. Bone mass is driven by osteoblast and osteoclast cell function, not by dietary intake, and the balance between osteoblast and osteoclast activity is regulated by a classical negative feedback loop designed to optimize the bending of bone under mechanical usage. A shortage of mineral will limit bone mass acquisition, but a surplus will not increase bone mass beyond genetically determined limits, as modified by current mechanical loading. The first point and its consequences are examined in the following discussion; the second point will be discussed in Section II,H.

The Paleolithic Calcium Intake and the Modern Diet

Calcium is the fifth most abundant element in the environment in which life evolved, and is present in high concentrations in the foods consumed by evolving hominids and hunter-gatherer humans [3]. Accordingly, human physiology is optimized to prevent calcium intoxication, not to deal with chronic shortages. This is expressed as low intestinal absorption efficiency, weak renal conservation, and completely unregulated dermal losses. (Contrast this behavior with that of sodium, a scarce mineral in the primitive environment. Sodium is absorbed at nearly 100% efficiency and, in trained individuals, both urinary and sweat losses can be reduced nearly to zero.) Gross intestinal calcium absorption in adults, at prevailing intakes, averages about 30%, and because substantial quantities of calcium enter the digestive tract with gastrointestinal secretions, net absorption is generally --~10% [8,9]. Additionally, many dietary components interfere with renal calcium conservation, notably sodium [3], net acid production [10,11], and sulfur-containing amino acids [12]. These agencies create a floor below which urinary calcium cannot be reduced, despite the effect of PTH on increasing renal tubular calcium reabsorption. Finally, resting dermal losses (i.e., without sweating) may be in the range of 1.5 mmol (60 mg)/day [13], and sweating can cause losses five to ten times that large [14]. The net result of all these excretory forces is that total obligatory calcium losses in sedentary adults on typical diets are generally in the range of 4 6 mmol (160-240 mg)/day. With a diet like that of our paleolithic ancestors [i.e., a calcium density of 1.75-2.0 mmol (---70-80 mg)/100 kCal], low absorption efficiency would still be quite sufficient to offset regular obligatory losses and to adapt to day-to-day variability in intake and excretion (see below). Having said this, it is useful to bear in mind that there is a huge disproportion between the body's needs for calcium and the calcium content of even a poor diet. Even at the peak of the adolescent growth spurt, bony growth could be accommodated nicely by the calcium contained in little more than a single serving of milk, if that calcium could be absorbed efficiently and fully conserved once absorbed. So, in theory at least, even calcium-poor diets could be adequate. This fact may explain why nutritional scientists had been slow to accept the life-long importance of a high calcium intake for contemporary humans. If the calcium was in the diet and the body was not fully accessing it, thenmit was arguedmthe body did not really need it. That conclusion was recognized as wrong after publication of several randomized, controlled trials, 2 showing that

2See the review by Heaney [15] for a summary of these trials, as well as the more recent compilationof studies by the Food and Nutrition Board of the National Academyof Science [5].

CHAPTER33 Menopause-Nutrient Interactions the body would indeed use extra calcium and improve calcium balance if the mineral were provided by a high enough intake. Failure to retain calcium at low intakes reflected not absence of need but the fact that human absorption and conservation efficiencies for calcium are simply not up to the challenge of a low intake, particularly when the bony reserves are so accessible. (As already noted, there was no evolutionary need for hominids to develop the type of absorptive and excretory conservation that today's diets demand, because, in the course of evolution, available foods provided a surplus of calcium. Although our diets are modern, our physiologies are paleolithic.)

D. C a l c i u m H o m e o s t a s i s a n d B o n e R e m o d e l i n g Despite high levels of PTH secretion and up-regulation of intestinal calcium absorption efficiency on low intakes, full adaptation to low intakes may not be so easily accomplished on contemporary diets. A more detailed look at the control system will make clear why that is so. Two distinct, but interconnected, feedback loops are involved. The first, the loop maintaining ECF (Ca2+), has already been summarized. The other is the loop regulating bone remodeling. Briefly, bone is constantly being remodeled, a process that generally begins with activation at a local site, followed by osteoclastic erosion into the bony surface, and later by osteoblastic in-filling of the resulting cavity. The loop regulating these processes seeks to optimize bone stiffness. Local sensors in bone, probably located in the osteocytes and their processes, detect the amount of local bending during usage. If that bending is greater than optimalmin other words, if the bone is bending too m u c h m t h e apparatus sends out signals to activate bone remodeling and strengthen the region experiencing excessive loads. The balance between the amount of bone resorbed and the amount deposited is thus regulated by a system intrinsic to bone. Two features of this remodeling scheme are important in our context. First, although PTH is secreted in response to demands of the ECF (Ca2+), not in response to bone bending, this hormone is nevertheless the principal determinant of the bone remodeling activation threshold, i.e., the intensity of signal needed to get the remodeling process going. Hence it determines the amount of remodeling in the total skeleton. Second, most bone remodeling is asynchronous (resorption first, formation later). This means that, under steady-state conditions, calcium released by resorption at one site will be matched by calcium required by formation at another. However, this asynchrony also means that a change in the rate at which new loci of remodeling are initiated increases or decreases osteoclastic resorption before there is a corresponding change in mineralization of forming bone at sites activated previously. Thus, an increase in PTH makes

485 surplus calcium available immediately, because acutely more bone is being resorbed than is currently being formed. Later, when newly activated sites reach their formation phase, there will be a demand to repay the loan of the previous "surplus" calcium. This phase lag between resorption and formation, which is normally of several weeks' duration, is admirably suited to mammalian calcium homeostasis in an environment only transiently depleted of calcium. Generally the diet, rich in calcium, supplies the animal's needs, but in periods of fasting or famine, when food becomes less available, the internal calcium reserves are tapped. Later, when food sources are plentiful again, the loan is automatically repaid from the diet. The second loop, already discussed, regulates calcium homeostasis. A unique feature of the ECF [Ca 2+] control loop, important in this context, is that PTH operates through three independent effector mechanisms (gut, kidney, and bone). Although PTH acts on the kidney to increase renal tubular calcium reabsorption and thereby decrease renal excretory losses, this would not be enough by itself. Reducing losses does not compensate for a deficiency; it just slows its progression. Thus it is the gut and the bone that must provide the calcium needed to compensate for increased losses or reduced intake. But the three effector mechanisms are linked only by the common level of circulating PTH that bathes and stimulates them all. Variation in the relative responsiveness of these effectors explains such phenomena as ethnic differences in requirement and shifts in requirement with age and at menopause.

E. Q u a n t i t a t i v e O p e r a t i o n o f the S y s t e m Although the operation of the calcium regulatory system, or any feedback loop, must first be sketched out qualitatively, in the final analysis it is the quantitative operation of the system that will determine what ultimately happens. In this case that will be what happens to the size of the calcium reserve, i.e., the mass of the skeleton. This quantitative working of the system for adjusting inputs and losses in response to dietary and other perturbations is often ignored. For example, it is commonly, if erroneously, assumed that, because intestinal calcium absorption efficiency is normally low, up-regulation will fully compensate for declines in intake or increases in excretory loss. But quantitative analysis of the system shows the fallacy of that assumption. In the face of increased demand, ECF [Ca 2+] tends to fall, because shortfalls are not fully offset from food calcium absorbed at the prior rate. The result is an increase in PTH secretion, which activates more bone resorption, increases renal conservation, and increases calcium absorption efficiency. The net effect with respect to total bone mass depends both on the relationship between the responsiveness of the three effector organs and on their capacity to provide the needed calcium [16].

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Sensitivity of the effectors is genetically and hormonally determined, whereas capacity to respond is largely determined by unregulated factors outside the control loop, such as the calcium content of the diet and factors that influence obligatory loss. If for some reason the response of one or the other of these effectors is blunted, PTH rises further, forcing more response from the other two effectors. Conversely, if one effector (such as bone) is highly responsive to PTH, the hormone level rises less because the needed calcium is readily supplied from the nearly limitless skeletal reserves. As a result, less improvement in external calcium utilization ensues. Similarly, if the gut is unresponsive or the diet is so low in calcium that its capacity to yield the needed amount is exceeded, then PTH secretion rises further and bone is driven to meet the needs of the ECF [Ca2+]. The two key insights here are (1) it is ECF [Ca 2+] that is being regulated, not bone mass, and (2) the dose-response curves for the three effector organs are independent of one another. Examples of different patterns of effector responsiveness abound. Thus, American blacks (and probably African blacks as well) have a bony resorptive apparatus relatively resistant to PTH [ 17,18]. As a result, they develop and maintain a higher bone mass than do Caucasians and Orientals, despite an often poor diet. As predicted from the foregoing, they exhibit higher PTH and calcitriol levels [18], but lower bone remodeling indices. In this way they utilize and conserve diet calcium more efficiently than Caucasians. The opposite situation occurs at normal menopause. Because estrogen appears to decrease bony responsiveness to PTH, estrogen loss at menopause increases the skeletal response to PTH. This is a part of the explanation for the increase in recommended calcium intake after menopause [4,5]. Obese individuals also increase their bone mass as they gain weight [19], and they lose less bone at menopause [20]. Like blacks, they have high circulating PTH levels and (presumably) a relatively resistant bone remodeling apparatus.

E Age-Related Changes in Operation of the Control System Important changes occur in the inputs and settings of this system with age. Calcium intake among women in the United States falls from early adolescence to the end of life. In NHANES-II, median calcium intake was 793 mg in early adolescence, 550 mg in the 20s, and 474 mg at menopause [21]. At the same time, absorption efficiency also falls with age. 3 Peripubertal girls absorb calcium with about 45%

3A part of this absorptive decline is due to estrogen deficiency, which both decreases renal la-hydroxylation of 25(OH)D and appears to have a small effect on the intestinal mucosa. A further part may be due to decrease in mucosal mass, which, in animals, is known to vary with food intake.

greater efficiency for the same intake than do perimenopausal women [22]. In general, after age 40 years, absorption efficiency drops by about 0.2 absorption percentage points per year, with an added 2.0 percentage point drop across menopause [8]. In concrete terms, if a 40-year-old woman absorbed a standard load at an efficiency of 30%, the same woman, at age 65 and deprived of estrogen, would absorb at an efficiency of 22.8%, or almost a 25% worsening in absorptive performance. To complicate the situation further, renal calcium clearance rises at menopause [22a], an effect seen most clearly with low calcium intakes, when urinary calcium will typically be as much as 36% higher than at premenopause [22b]. Vitamin D status declines with age as well [23,24], although this is also a function of solar exposure, dermal vitamin D synthetic efficiency, and milk consumption. In Europe, where solar vitamin D synthesis is low for reasons of latitude and climate, and milk is generally not fortified, serum 25(OH)D concentration drops from over 100 nmol/liter (40 ng/ml) in young adults to under 40 nmol/1 (16 ng/ml) in individuals over age 70 years [23,24]. Not surprisingly, serum PTH rises with age as a consequence of this aggregate of age-related changes. A 24-hr integrated PTH is 70% higher in healthy 65-year-old United States women consuming diets containing 800 mg Ca per day than in third-decade women on the same diets [6]. That this difference is due to insufficient absorptive input is shown by the fact that the difference can be completely obliterated by increasing calcium intake in the older women [6].

G. Two Examples of System Operation As implied in the foregoing, it is a quantity that is being optimized (i.e., ECF [Ca2+]); this is accomplished by the algebraic sum of various quantitative inputs and outputs. Two examples will serve to illustrate the importance of this quantitative approach. One highlights the contrast between calcium handling at menarche and menopause just described, and the second describes the response of the system at any given age to a fixed increase in obligatory loss. 1. M E N A R C H E AND MENOPAUSE True trabecular bone density increases by about 15% across menarche [25], and about the same quantum of bone is lost across menopause [26]. Curiously, administration of estrogen to women more than 3 years postmenopausal has generally failed to reproduce the pubertal increase in B MD, and it has been customary to say, in recent years, that apart from whatever remodeling transient estrogen replacement therapy (ERT) may produce in postmenopausal women [27], the principal effect of ERT on bone is to stabilize bone mass, rather than to cause restoration of what had been lost. But this conclusion was drawn without attending to the quanti-

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TABLE I Net Calcium Absorption at Menarche and Menopause

Ca intakea Ca absorption efficiencyb Endogenous fecal Cac Net Ca absorption

Menarche

Menopause

793 rag/day 35.2% 67 mg/day 212 mg/day

474 mg/day 30.5% 102 mg/day 42 mg/day

aNHANES-II median values [21]. bHeaney et al. [8]; O'Brien et al. [22]; values adjusted to intake. CHeaney et al. [28].

tative aspects of the age-related changes in the calcium economy, summarized in the foregoing. Table I [28] assembles published data for median calcium intake and mean data for absorption efficiency and endogenous fecal calcium loss, and shows very clearly how quantitative changes occurring in the 40 years from menarche to menopause account for the rather different performance of the two age groups. In brief (and despite an intake less than recommended), a peripubertal girl is able to achieve net absorption of over 200 mg Ca from the median diet of her age cohort, whereas an early menopausal woman extracts less than one-fifth as much from hers. The drop in intake amounts to about 40%, but the drop in net absorption is 80%. As Table I shows, this is the resultant of lower intake, lower absorption efficiency, and higher digestive juice calcium losses. Given the level of total body obligatory losses at midlife, this absorbed quantity is simply not sufficient to support an estrogen-stimulated increase in BMD. As would be predicted from this understanding, higher calcium intakes permit estrogen to produce in postmenopausal women the same sort of bony increases seen at puberty (see Section II,G,3). 2. RESPONSE TO AUGMENTED LOSSES

As already noted, it is commonly (and uncritically) considered that the absorptive apparatus is able to compensate both for a change in intake and for a change in excretory loss. However, quantitative considerations make it clear that this depends entirely on the level of calcium in the diet. Thus, an individual increasing his/her sodium intake by an amount equivalent to a single daily serving of a fast-food, fried chicken meal experiences an increase in urinary calcium of about 1 mmol (40 mg)/day [29]. Without compensating adjustments in input to the ECE [Ca 2+] drops. PTH, of course, rises, and with it, synthesis of 1,25(OH)2D, resulting ultimately in better extraction of calcium from the diet. Published data allow rough estimation that a calcium drain of this magnitude produces an increase in 1,25(OH)2D of about 6 - 7 pmol/liter [30], and dose-response data for 1,25(OH)2D indicate that this stimulus would increase calcium absorption efficiency by about 2 - 3 absorption percentage points [31]. A 2 - 3 % increase in extraction from a

50-mmol (2000-mg) diet yields 1-1.5 mmol ( 4 0 - 6 0 mg) of extra calcium, more than enough to offset the increased urinary loss, whereas from a 5-mmol (200 rag) diet, the same absorptive increase yields less than 0.1 mmol (4 mg). 4 Thus, on a high-calcium diet, the body easily compensates for varying drains: both bone and ECF (Ca 2+) are protected. But on a low-calcium diet, although the ECF (Ca 2+) is protected, bone is not. Why does serum 1,25(OH)zD not rise more on a low-calcium diet? Simply because the lce-hydroxylation step is responding to PTH. Bone calcium meets much (or most) of the ECF need, and PTH secretion is regulated by ECF (Ca2+), not by bone mass. In brief, as the body adjusts to varying demands, the portion of the demand met by bone will be determined both by factors influencing bony responsiveness and by the level of diet calcium, the one critical component of the system that is not regulated. However, it must also be stressed that, although an adequate calcium intake is a necessary condition for bone building and for adaptation to varying calcium demands, it is not by itself sufficient. Calcium alone will not stop estrogen-deficiency bone loss nor disuse loss (because neither is due to calcium deficiency). But by the same token, recovery from immobilization or restoration of bone lost because of hormone deficiency will not be possible without an adequate supply of the raw materials needed to build bone substance.

H. T h e C a l c i u m R e q u i r e m e n t 1. THRESHOLD BEHAVIOR

As noted briefly earlier, calcium is a threshold, or plateau, nutrient, a concept illustrated in Fig. 2 [32]. What is meant by this term is that, when one graphs bony response against calcium intake, starting from deficient levels, response rises as a function of intake up to some point (the threshold), above which response plateaus. This behavior is most straightforwardly brought out during growth (as in Fig. 2B), when the amount of bone that can be amassed is strictly limited by intake. But when calcium intake is high enough to supply the demands of the genetic program, as modified by current mechanical loading, these latter factors become controlling and calcium intake no longer exerts an effect. Additional calcium will make no more bone and is simply excreted. But the same basic relationship holds for all life stages, even when bone may be undergoing some degree of involution. The threshold concept is generalized to all life situations in Fig. 3A, which shows schematically what the intake/ retention curves look like during growth, maturity, and

4This is because extraction efficiency is already relatively high on low intakes, and there is less calcium still unabsorbed on which the mucosacan work to extract additional calcium.

488

ROBERT P. HEANEY

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FIGURE 2 Threshold behavior of calcium intake. (A) Theoretical relationship of bone accumulation to intake. Below a certain value--the threshold--bone accumulation is a linear function of intake (the ascending line); in other words, the amount of bone that can be accumulated is limited by the amount of calcium ingested. Above the threshold (the horizontal line), bone accumulation is limited by other factors and is no longer related to calcium intake. (B) Actual data from two experiments in growing rats, showing how bone accumulation does, in fact, exhibit a threshold pattern. The arrows and asterisks denote the minimum requirement. Redrawn from data in Forbes, et al. [32]. Copyright Robert P. Heaney, 1992. Reproduced with permission.

cium nutrition in this life stage is to move the older adult to point A and thereby to make certain that insufficient calcium intake is not aggravating any underlying bone loss. This difference is well illustrated by a study of calcium supplementation by Reid et al. in postmenopausal women [33]. Initially, BMD in the calcium-supplemented group rose above the starting value, i.e., there was apparent bone gain; but this reflected the expected remodeling transient [27]. Data for the second and subsequent years of the study showed continuing bone loss, despite the high calcium intake. However, the calcium-supplemented group was losing significantly less rapidly than the placebo-treated controls [34]. The study was not dose-ranging, but, for the sake of illustration, if one assumes that the intake achieved in the calcium-supplemented subjects was above the threshold of Fig. 3B, then the continuing bone loss reflects nonnutritional factors. Thus the calcium-supplemented individuals in the Reid study can be said to have been at point A in Fig. 3B, and the placebo-treated individuals, at point B. 2. F U N C T I O N A L I N D I C A T O R OF

involution. In brief, the plateau occurs at a positive value during growth, at zero retention in the mature individual, and sometimes at a negative value in the elderly. (Available evidence suggests that the plateau during involution is negative in the first 3 - 5 years after menopause, rises to or toward zero for the next 10-15 years, and then becomes increasingly negative with age in the old elderly.) In Fig. 3B, which shows only the involutional curve, there are two points located along the curve, one below (B) and one above (A) the threshold. At A, calcium retention is negative for reasons intrinsic to the skeleton, whereas at B, involutional effects are compounded by inadequate intake, which makes the balance more negative than it needs to be. Point B (or below) is probably where most older adults in the industrialized nations would be situated today. The goal of cal-

NUTRITIONAL ADEQUACY

FIGURE 3 (A) Schematic intake and retention curves for calcium for

The functional indicator of nutritional adequacy for such a threshold nutrient is termed "maximal retention" and can be located in Figs. 2A and 3A at the points on the horizontal axes corresponding to the asterisks above the curves. This intake represents the minimum requirement for skeletal maintenance. Calcium retention in this sense is "maximal" only in that further intake of calcium will produce no further retention. 5 This approach was used by the Food and Nutrition Board of the National Academy of Sciences for the first time in its development of intake recommendations for calcium in 1997 [5]. Most other nutrients, when ingested above the optimal intake level, continue to increase their body reserves, often to the point of producing toxicity (e.g., energy and the fatsoluble vitamins). But because the calcium reserve is in the form of bone tissue, and bone tissue mass is regulated by cellular processes regulated by mechanical controls, any calcium intake in excess of skeletal need is simply excreted. The fact that the body cannot store bone above the level of current skeletal need is what creates the plateau. When the requirement is defined in terms of the concepts set forth in Figs. 2 and 3, then, for a population, the average of the individual minimum requirements for bone health is given by the mean of the intakes at which the individuals reach the plateau or threshold value. Current best estimates for this average minimum requirement are in the range of 2 5 - 3 0 mmol (1000-1200 mg) Ca/day for mature adults, rising with age to 30-37.5 mmol (1200-1500 mg) Ca/day by

three life stages. Retention is greater than zero during growth, zero at maturity, and may be negative during involution. (B) The involution curve only. Point B designates an intake below the maximal calcium retention threshold, and point A, an intake above the threshold. Copyright Robert E Heaney, 1998. Reproduced with permission.

5This is in contrast to treatment with hormones or drugs, which, under appropriate conditions, can clearly lead to further bone augmentation.

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CI-IAeTER33 Menopause-Nutrient Interactions age 75 [4,5]. These are a v e r a g e requirements. Assuming a population coefficient of variation for the distribution of requirements around the mean on the order of 10%, the corresponding best estimate of a recommended daily allowance (RDA) for calcium would be 30-35 mmol (1200-1400 mg) Ca/day for mature adults, rising to 35-45 mmol (14001800 mg) Ca/day for the elderly. During growth, intakes below an individual's minimum lead to bone mass less than the genetically programmed amount, and in the aged, low intakes exaggerate or account for much of age-related bone loss. The result in both instances is bony weakness and increased fracturability, i.e., osteoporosis. The importance of calcium in protecting against these outcomes is underscored by more than 30 investigatorcontrolled studies of calcium supplementation, essentially all showing that high calcium intakes enhance bone acquisition during growth and reduce age-related bone loss and/or fractures (see Footnote 2, page 484). 3. C A L C I U M AS A NECESSARY COMPONENT OF

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FIGURE 4 Mean skeletal response to estrogen replacement therapy at three measurement sites, with (white) and without (black) supplemental calcium, from 31 published randomized, controlled trials of estrogen in postmenopausal women. Calcium intake in the unsupplemented group averaged 589 mg/day and in the supplemented studies, 1189 mg/day. Redrawn from Nieves et al. [37]. Copyright Robert R Heaney, 1998. Reproduced with permission.

P R EVENTI VE R E G I M E N S

The need for supplemental calcium has become increasingly clear as awareness of the essential role of calcium has grown in recent years, and increasing numbers of studies of therapy of osteoporosis have combined calcium with other agents. Probably the earliest report of such combination therapy, a retrospective compilation of Mayo Clinic osteoporosis treatment data [35], showed a synergistic effect on reducing vertebral fracture rate in osteoporotic women by adding calcium and vitamin D to regimens of ERT and fluoride, with the greatest reduction being found with the combination of all four agents. More recently, Davis et al. [36] showed a substantial enhancement by calcium of the bone protective effect of ERT in postmenopausal women. And, most convincing of all, Nieves et al. [37], in a meta-analysis of 31 published randomized, controlled trials using ERT, segregated trials according to whether calcium was added to the ERT regimen. There was a strikingly greater, and more than additive, effect when calcium was incorporated into the regimen. Figure 4 displays the data from this meta-analysis.

quired for skeletal maintenance. The figure indicates diagrammatically the self-evident fact that, if intake is less than optimal, adaptation will be required. As described earlier, this adaptation takes the form of increased production of PTH and 1,25(OH)2D, which, other things being equal, improve intestinal calcium absorption efficiency and renal calcium conservation. An individual's capacity to adapt at any given intake determines whether skeletal mass will be protected. This capacity clearly declines with age, just as do cardiopulmonary, renal, and other organ system reserves. By age 7 5 - 8 0 years, the ability to adapt to suboptimal calcium intakes is virtually zero. This is the underlying factual basis

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I. C a l c i u m and N o n s k e l e t a l Illnesses As noted earlier, low calcium intake has been implicated not only in osteoporosis, but also in hypertensive disorders and colon carcinogenesis. How can a single mineral have tissue effects so diverse--ranging from bone mass to arteriolar smooth muscle--particularly because blood calcium is usually maintained at normal levels irrespective of calcium intake and/or deficiency? The answer is, in part, suggested by Fig. 5, which indicates the paleolithic intake, the probable optimum intake, and the age-varying minimum intake re-

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490 behind the rise with age of calcium intake recommendations (which are pegged to skeletal effects, not to total health). However, although bone mass can often be maintained at daily intakes of 15-20 mmol Ca (600-800 mg), as is the case for African-Americans, it does not follow that such intakes are thereby optimal. Even intakes above the minimum for skeletal maintenance may exert a health toll, to the extent that the required adaptation may produce negative effects. Intakes falling within the shaded zone of Fig. 5 require a continuous adaptive response. Augmented sensitivity to chronically high levels of PTH and 1,25(OH)zD is believed to be behind a portion of the cases of essential hypertension, preeclampsia, pregnancy-associated hypertension, and PMS. Clearly these are all multifactorial disorders (just as is osteoporosis), and low calcium intake is only one of the many factors involved. Nevertheless, high calcium intakes have been shown to reduce the risks and/or the severity of all of them [38-43]. Even with respect to bone mass, there is considerable individual and ethnic variability in the capacity to adapt sufficiently to suboptimal intakes, i.e., in the location of the dashed line in Fig. 5. African-Americans, for example, despite lower calcium intakes than United States whites, have bones about 10-15% more massive. They manage this by more efficient intestinal calcium absorption and better renal calcium conservation [ 18]. But to produce these adaptations they carry substantially higher circulating levels of PTH and 1,25(OH)zD than do Caucasians on similar intakes. Moreover, while their bones are protected, they suffer an exceptionally high prevalence of hypertension and cerebrovascular accidents. PTH and/or 1,25(OH)zD raise cytosolic (Ca 2+) slightly [44] and may thereby provide the basis for increased arteriolar tone. Some portion of the hypertensive disease burden in the African-American population can be traced to their low calcium intakes, because elevated calcium intake has been shown to lower blood pressure and this effect is greater in individuals who are already hypertensive [41 ]. Although the evidence is virtually conclusive with respect to the efficacy of high calcium intake in osteoporosis, the hypertensive disorders, and PMS, the strongest data to date with respect to colon cancer are found largely in animal studies. Human evidence is confined to precancerous colon lesions, which have been shown to be reversed by high calcium intakes [43]. The probable mechanism is as follows. On high calcium intakes, net calcium absorption in the intestine averages about 10%, leaving the vast bulk of dietary calcium in the food residue, where it complexes in the colon with unabsorbed free fatty acids and bile acids. Both substances are mucosal irritants and function as promoters for colon cancer. Calcium, by forming soaps with these acids, neutralizes their irritant effects; thus calcium functions as an antipromoter. This is an important, if not strictly nutritional, function of calcium, particularly for contemporary high-fat diets, because levels of both free fatty acids and bile acids

ROBERT P. HEANEY

rise in the food residue as fat intake rises. At low calcium intakes this protection by calcium is ineffective, both because there is much less calcium to start with, and because absorptive extraction is higher, even less calcium reaches the colon. Current best estimates are that calcium intakes of about 4 0 - 4 5 mmol (1600-1800 mg)/day will reduce the risk of preeclampsia by about 60% [38], the symptoms of premenstrual syndrome by about 50% [40], and the incidence of hip fracture by about 30-50% [45]. Similarly, a high-calcium intake, as part of a diet high in fruits and vegetables, has been estimated to reduce the risk of stroke and myocardial infarction by nearly 30% [41 ]. In brief, these diverse conditions are all seen to result in part from the decline in human calcium intake over the past roughly 4000 years (from paleolithic to modern times).

J. C a l c i u m S o u r c e s Optimally the calcium needed for full realization of these nutritional benefits will be supplied by foods incorporated into a balanced diet. For menopausal women this will often best be accomplished through increased intakes of low-fat dairy products; however, fortified foods and calcium supplements can help meet a target figure of 4 0 - 4 5 mmol/day (1600-1800 mg). The principal calcium supplement form on the United States market today is calcium carbonate, the active ingredient in Caltrate, Os-Cal, Tums, and the generic oyster shell preparations. Calcium supplements should be taken with meals, because this is the normal way all nutrients enter our bodies. Utilization is better both when calcium sources are taken with other foods and when the daily requirement is spread out over several doses [46,47]. Gastric acid is definitely not needed to absorb calcium taken as part of a meal, even for the less soluble phosphate and carbonate salts. Despite advertising claims to the contrary, absorbability is very nearly the same for most widely used calcium supplements [48]. Citrate absorption is not better, for example, than carbonate absorption [49]; and the slight superiority of calcium in the form of calcium citrate malate or one of the amino acid chelates can be economically offset by taking an occasional extra dose of one of the cheaper carbonate preparations. However, calcium citrate, calcium citrate malate, calcium phosphate, and the other calcium sources available provide useful alternatives for women who, for one reason or another, do not like the cheaper carbonate forms. Magnesium supplementation is not needed for the body to utilize the calcium in these sources [50]. However, magnesium deficiency, such as may occur with malabsorption syndromes or intestinal fistulas, leads both to dysregulation of the calcium economy and to reduced bone mass. Magnesium supplementation is necessary to correct these defects.

CHAPTER33 Menopause-Nutrient Interactions Sprue syndromes can sometimes be silent, thus it may be that there are a few cases of osteoporosis that would benefit from both calcium and magnesium supplementation. However, for the majority, magnesium has not been shown to confer any additional benefit over calcium alone.

III. VITAMIN D

491 reinforced by the fact that it could be cured by consuming certain fish liver oils (arguably foods). At one time nutritionists argued that a healthy individual could obtain all the nutrients he or she needed simply by consuming a balanced diet. With vitamin D, not a true nutrient, that has never been even remotely true. There is a pressing need, at high latitudes, to supplement dermal vitamin D production. Doing so is analogous to providing clothing and shelter to help maintain body temperature.

A. Background Vitamin D is a fat-soluble secosterol normally produced in the skin in response to ultraviolet B radiation (UV-B), which causes a photolytic conversion of 7-dehydrocholesterol to previtamin D. In equatorial East Africa, where Homo sp. evolved, daily dermal production of vitamin D would have exceeded 25/xg (1000 IU) [51]. Skin pigmentation slows, but does not prevent, this dermal synthesis, and may have functioned to protect against vitamin D intoxication. As modern humans migrated to higher latitudes, they lost their skin pigmentation. This change was necessary to increase the efficiency of dermal synthesis and thereby to compensate for the decline in UV-B exposure (caused by the increased cloudiness of, for example, northern Europe, the annual decrease in UV-B in winter, and the latitude-dependent attenuation of UV-B by the atmosphere, even in summer). Natural selective forces would have weeded out individuals unable to make this change, because the consequence of inadequate vitamin D production would have been rickets, with severe consequences for reproductive success. Vitamin D is stored in body fat depots, circulates complexed to a D-binding protein in plasma, and is successively hydroxylated at the 25-position in the liver and then at the lee position in the kidney, to produce 1,25(OH)zD, the hormonally most active form of the vitamin. The 25-hydroxylation step is only weakly regulated and is a function mainly of circulating levels of vitamin D. By contrast, the renal lee hydroxylation step producing 1,25(OH)zD is tightly controlled both by PTH and by serum Pi. (la-Hydroxylation rises with a rise in PTH and with a fall in Pi-) 1,25(OH)2D binds to a specific vitamin D receptor found in many tissues, inducing the synthesis, in at least some of them, of a calcium-transporter protein, one of a family of proteins called calbindins. Vitamin D is, strictly speaking, an accidental nutrient. It is not present in most foods in any important quantity and would not have been a component of the primitive diet. Scientific recognition of the morbidity caused by its absence happened to occur at the same time that the true nutritional deficiency diseases were being elucidated. Until that time the prevailing paradigm had been that all disease was caused by a harmful presence (bacteria or toxins), not by an absence. Hence vitamin D deficiency got lumped together with the true nutritional deficiency diseases. This categorization was

B. Functions of Vitamin D 1. INTESTINAL CALCIUM ABSORPTION

The best attested effect of vitamin D is the induction of the calcium transport carrier protein in intestinal mucosal cells, necessary for active calcium absorption from the intestinal contents into the blood. The efficiency of calcium absorption from standard meals varies directly with the logarithm of 1,25(OH)zD production [31 ]. The importance of vitamin Dmediated active calcium absorption is widely recognized, and can be illustrated by comparison with passive absorption of calcium. Gross passive absorption amounts to roughly 1 0 15% of the ingested intake. At intakes of 20-25 mmol (800-1000 mg)/day, that means 2.0-3.75 mmol (80-150 rag). This is usually not sufficient to offset the calcium contained even in the digestive secretions [3.5 _+ 0.75 mmol (140 _+ 30 mg/day)], let alone extraintestinal routes of loss; hence net intestinal absorption of calcium is zero or even negative in the absence of vitamin D, particularly on contemporary calcium intakes, and total body calcium balance will, of course, be even more negative. Even at intakes as high as 2000 mg/day, passive absorption does not produce enough calcium to support normal growth during adolescence. In any event, failure to absorb sufficient calcium to offset renal and dermal losses in the adult, or to support growth in young persons, leads to elevated secretion of PTH and a fall in serum Pi. When vitamin D is deficient these changes are not able by themselves to increase intestinal absorption. PTH levels continue to rise, producing very low Pi levels that lead to osteoblast/chondroblast dysfunction and accumulation of unmineralized osteoid in bone, manifested as osteomalacia and rickets. 2. EXTRAINTESTINALEFFECTS

Vitamin D receptors are widespread in tissues throughout the body and 1,25(OH)zD exhibits several effects less well understood than the facilitation of dietary calcium absorption. Perhaps the clearest example is the fact that 1,25(OH)2D is necessary for PTH to mobilize bone calcium, a fact that partially explains why hypocalcemia sometimes occurs in vitamin D deficiency syndromes, despite the presence of still large skeletal calcium reserves. Vitamin D also exerts important if incompletely characterized effects on the skin.

492

ROBERT P. HEANEY

These are manifested both in the alopecia found with genetic defects in the vitamin D receptor [52] and in the often dramatic response of psoriasis to topical 1,25(OH)2D (or closely related compounds) [53]. Other functions are hinted at in the observation that 1,25(OH)2D reverses the glucose intolerance, insulin resistance, and lipid abnormalities of end-stage renal disease [54]. Additionally vitamin D functions in a way that can perhaps best be characterized as promoting cell differentiation. In vitro this behavior is seen, for example, in the differentiation of mouse myeloid leukemia cells in culture [55], a finding paralleled in the clinic in the quite dramatic reversal by 1,25(OH)2D of certain myelodysplasias, including myelomonocytic leukemia [56]. Although hormonally the kidney is the only significant source of 1,25(OH)2D, the lcehydroxylase is expressed in many tissues and it is likely that local conversion from 25(OH)D circulating in the blood allows these tissues to synthesize 1,25(OH)2D and use it in an autocrine or paracrine fashion. Instances of response to exogenous 1,25(OH)2D, such as psoriasis or myelodysplasia, may represent local mechanisms that are somehow defective in these disorders. (The fact that these defects can be overridden by administered 1,25(OH)2D simply hints at an underlying autocrine mechanism, rather than indicating that high blood levels of 1,25(OH)2D are in themselves necessary or beneficial.) This interpretation is consistent with the fact that under primitive conditions, with abundant dermal synthesis of vitamin D and high dietary calcium intakes, blood 25(OH)D levels would normally have been high but 1,25(OH)2D levels, low.

C. Optimal Vitamin D Intake Until very recently, estimates of the vitamin D requirement were related to the oral dose required to prevent rickets and osteomalacia, which may be as low as ---2/zg/day ( 8 0 100 IU). The inadequacy of this approach has been recently recognized and now the accepted functional indicator of vitamin D status is the serum level of 25(OH)D [5]. But there is still no general agreement as to precisely what value of that indicator is optimal and where the lower limit of acceptable normal is located. Parfitt has done the field an important service in codifying the effects of vitamin D insufficiency on the calcium economy, thereby defining what he has termed hypovitaminosis D osteopathy [57]. In this scheme he recognizes three grades of osteopathy, as set forth in Table II. Note that calcium malabsorption and the expected responses in PTH and bone remodeling are found in all three grades. Where the grades differ is in their effect on bone mass and bone histology. Clinical osteomalacia is present only in HVOiii, while HVOii exhibits osteomalacia only on biopsy. Thus clinical rickets and osteomalacia reflect only the most severe degrees of vitamin D insufficiency. At less extreme

TABLE II

Hypovitaminosis D Osteopathy a

Manifestation

HVOi

HVOii

HVOiii

Ca malabsorption Increased PTH Elevated alkaline phosphatase Serum Ca Serum P Bone remodeling Histologic osteomalacia Bone apposition Bone loss

~ u,~ u," Normal Normal High No Yes Yes

t,," ~ ~ Normal Low Variable Yes Yes Variable

t,," !,," t,," Normalto low Low Low Yes No No

aModified after Parfitt [57]. HVOi, HVOii, and HVOiii represent the three grades of hypovitaminosis D osteopathy, in order of increasing severity.

degrees, the principal disturbance is a reduction in calcium absorption, with resulting bone loss and hormonal evidences of the body's attempt to compensate. In other words, mild and moderate degrees of vitamin D deficiency produce not clinical osteomalacia, but osteoporosis. This approach to the problem assists in defining the lower limit of normal for circulating 25(OH)D. That limit would be the value below which PTH secretion begins to rise. Various studies indicate that this limit is in the range of 80 nmol 25(OH)D/liter (32 ng/ml) [24,57-59], and some studies suggest that the true value may even be as high as 120 nmol/ liter (48 ng/ml) [60,61 ]. Young adults and outdoor workers usually have 25(OH)D values at least this high, but older individuals, especially those with limited sun exposure, regularly exhibit lower levels, and laboratory reference ranges commonly extend down to 15-20 ng/ml (37.5-50 nmol/liter). These "normal" limits are almost certainly too low, and in the management of postmenopausal women it will be important to ensure a vitamin D intake sufficient to maintain young adult 25(OH)D levels. That will be at least 15/zg (600 IU/day). Failure to meet this target will not cause osteomalacia in most cases, but will limit the individual's ability to utilize dietary calcium, and hence will either contribute to postmenopausal bone loss or will limit the efficacy of hormonal preventive regimens (or both). As will be evident from the foregoing, this approach to the intake requirement is related solely to the calcium economy and to protection of bone. If serum 25(OH)D is important as a precursor compound for cell level autocrine regulation in a variety of tissues, the level needed for optimal support of such function is unknown.

References 1. Goulding, A., Cannan, R., Williams, S. M., Gold, E. J., Taylor,R. W., and Lewis-Barned, N. J. (1998). Bone mineral density in girls with forearm fractures. J. Bone Miner Res. 13, 143-148.

493

CHAPTER 33 M e n o p a u s e - N u t r i e n t Interactions 2. Arnaud, C. D. (1978). Calcium homeostasis: Regulatory elements and their integration. Fed. Proc. 37(12), 2557-2560. 3. Eaton, B., and Nelson, D. A. (1991). Calcium in evolutionary perspective. Am. J. Clin. Nutr. 54, 281 S-287S. 4. NIH Consensus Conference (1994). Optimal calcium intake. JAMA, J. Am. Med. Assoc. 272, 1942-1948. 5. Food and Nutrition Board, Institute of Medicine (1997). "Dietary Reference Intakes for Calcium, Magnesium, Phosphorus, Vitamin D, and Fluoride." National Academy Press, Washington, DC. 6. McKane, W. R., Khosla, S., Egan, K. S., Robins, S. E, Burritt, M. E, and Riggs, B. L. (1996). Role of calcium intake in modulating agerelated increases in parathyroid function and bone resorption. J. Clin. Endocrinol. Metab. 81, 1699-1703. 7. Heath, H., III, Hodgson, S. F., and Kennedy, M. A. (1980). Primary hyperparathyroidism. Incidence, morbidity, and potential economic impact in a community. N. Engl. J. Med. 302, 189-193. 8. Heaney, R. P., Recker, R. R., Stegman, M. R., and Moy, A. J. (1989). Calcium absorption in women: Relationships to calcium intake, estrogen status, ~ind age. J. Bone Miner. Res. 4, 469-475. 9. Nordin, B. E. C., Polley, K. J., Need, A. G., Morris, H. A., and Marshall, D. (1987). The problem of calcium requirement. Am. J. Clin. Nutr. 45, 1295-1304. 10. Berkelhammer, C. H., Wood, R. J., and Sitrin, M. D. (1988). Acetate and hypercalciuria during total parenteral nutrition. Am. J. Clin. Nutr. 48, 1482-1489. 11. Sebastian, A., Harris, S. T., Ottaway, J. H., Todd, K. M., and Morris, R. C., Jr. (1994). Improved mineral balance and skeletal metabolism in postmenopausal women treated with potassium carbonate. N. Engl. J. Med. 330, 1776-1781. 12. Schuette, S. A., Hegsted, M., Zemel, M. B., and Linkswiler, H. M. (1981). Renal acid, urinary cyclic AMP and hydroxyproline excretion as affected by level of protein, sulfur amino acid and phosphorus intake. J. Nutr. 111, 2106-2116. 13. Charles, E (1989). Metabolic bone disease evaluated by a combined calcium balance and tracer kinetic study. Dan. Med. Bull. 36, 4 6 3 479. 14. Klesges, R. C., Ward, K. D., Shelton, M. L., Applegate, W. B., Cantler, E. D., Palmieri, G. M. A., Harmon, K., and Davis, J. (1996). Changes in bone mineral content in male athletes. JAMA, J. Am. Med. Assoc. 276, 226-230. 15. Heaney, R. E (1993). Nutritional factors in osteoporosis. Annu. Rev. Nutr. 13, 287-316. 16. Heaney. R. E (1965). A unified concept of osteoporosis. Am. J. Med. 39, 877-880. 17. Bell, N.H., Greene, A., Epstein, S., Oexmann, M. J., Shaw, S., and Shary, J. (1985). Evidence for alteration of the vitamin D-endocrine system in blacks. J. Clin. Invest. 76, 470-473. 18. Aloia, J. F., Mikhail, M., Pagan, C. D., Arunachalam, A., Yeh, J. K., and Flaster, E. (1998). Biochemical and hormonal variables in black and white women matched for age and weight. J. Lab. Clin. Med. 132, 383-389. 19. Matkovic, V., Jelic, T., Wardlaw, G. M., Ilich, J. Z., Goel, E K., Wright, J. K., Andon, M.B., Smith, K. T., and Heaney, R. P. (1994). Timing of peak bone mass in Caucasian females and its implication for the prevention of osteoporosis. J. Clin. Invest. 93, 799-808. 20. Ribot, C., Tremollibres, E, Pouilles, J.-M., Bonneu, M., Germain, E, and Louvet, J.-E (1988). Obesity and postmenopausal bone loss: The influence of obesity on vertebral density and bone turnover in postmenopausal women. Bone 8, 327-331. 21. Carroll, M. D., Abraham, S., and Dresser, C. M. (1983). "Dietary Intake Source Data: United States, 1976-80," Vital Health Stat. Ser. 11, No. 231, DHHS Publ. No.(PHS) 83-1681. National Center for Health Statistics, Public Health Service, U.S. Govt. Printing Ofrice, Washington, DC. 22. O'Brien, K. O., Abrams, S. A., Liang, L. K., Ellis, K. J., and Gagel,

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R. E (1996). Increased efficiency of calcium absorption during short periods of inadequate calcium intake in girls. Am. J. Clin. Nutr. 63, 579-583. Nordin, B. E. C., Need, A. G., Morris, H. A., and Horowitz, M. (1999). Biochemical variables in pre- and postmenopausal women: Reconciling the calcium and estrogen hypotheses. Osteoporos. Int. 9, 351-357. Heaney, R. E, Recker, R. R., and Ryan, R. A. (1999). Urinary calcium in perimenopausal women: Normative values. Osteoporos. Int. 9, 13-18. McKenna, M. J., Freaney, R., Meade, A., and Muldowney, F. E (1985). Hypovitaminosis D and elevated serum alkaline phosphatase in elderly Irish people. Am. J. Clin. Nutr. 41, 101-109. Francis, R. M., Peacock, M., Storer, J. H., Davies, A. E. J., Brown, W. B., and Nordin, B. E. C. (1983). Calcium malaborption in the elderly: The effect of treatment with oral 25-hydroxyvitamin D 3. Eur. J. Clin. Invest. 13, 391-396. Gilsanz, V., Gibbens, D. T., Roe, T. F., Carlson, M., Senac, M. O., Boechat, M. I., Huang, H. K., Schulz, E E., Libanati, C. R., and Cann, C. (1988). Vertebral bone density in children: Effect of puberty. Radiology 166, 847-850. Genant, H. K., Cann, C. E, Ettinger, B., Gordan, O. S., Kolb, F. O., Reiser, U., and Arnaud, C. D. (1984). Quantitative computed tomography for spinal mineral assessment. In "Osteoporosis" (C. Christiansen et al. eds.), pp. 65-72. Glostrup Hospital, Department of Chemistry, Copenhagen, Denmark. Heaney, R. E (1994). The bone remodeling transient: Implications for the interpretation of clinical studies of bone mass change. J. Bone Miner. Res. 9, 1515-1523. Heaney, R. E, and Recker, R. R. (1994). Determinants of endogenous fecal calcium in healthy women. J. Bone Miner. Res. 9, 16211627. Itoh, R., and Suyama, Y. (1996). Sodium excretion in relation to calcium and hydroxyproline excretion in a healthy Japanese population. Am. J. Clin. Nutr. 63, 735-740. Dawson-Hughes, B., Stem, D. T., Shipp, C. C., and Rasmussen, H. M. (1988). Effect of lowering dietary calcium intake on fractional whole body calcium retention. J. Clin. Endocrinol. Metab. 67, 62-68. Heaney, R. E, Barger-Lux, M. J., Dowell, M. S., Chen, T. C., and Holick, M. E (1997). Calcium absorptive effects of vitamin D and its major metabolites. J. Clin.Endocrinol. Metab. 82, 4111-4116. Forbes, R. M., Weingartner, K. F., Parker, H. M., Bell, R. R., and Erdman, J. W., Jr. (1979). Bioavailability to rats of zinc, magnesium and calcium in casein-, egg- and soy protein-containing diets. J. Nutr. 109, 1652-1660. Reid, I. R., Ames, R. W., Evans, M. C., Gamble, O. D., and Sharpe, S. J. (1993). Effect of calcium supplementation on bone loss in late postmenopausal women. N. Engl. J. Med. 328, 460-464. Reid, I. R., Ames, R. W., Evans, M. C., Gamble, O. D., and Sharpe, S. J. (1995). Long-term effects of calcium supplementation on bone loss and fractures in postmenopausal women: A randomized controlled trial. Am. J. Med. 98, 331-335. Riggs, B. L., Seeman, F., Hodgson, S. F., Taves, D. R., and O'Fallon, W. M. (1982). Effect of the fluoride/calcium regimen on vertebral fracture occurrence in postmenopausal osteoporosis. N. Engl. J. Med. 306, 446-450. Davis, J. W., Ross, E D., Johnson, N. E., and Wasnich, R. D. (1995). Estrogen and calcium supplement use among Japanese-American women: Effects upon bone loss when used singly and in combination. Bone 17, 369-373. Nieves, J. W., Komar, L., Cosman, E, and Lindsay, R. (1998). Calcium potentiates the effect of estrogen and calcitonin on bone mass: Review and analysis. Am. J. Clin. Nutr. 67, 18-24. Bucher, H. C., Guyatt, G. H., Cook, R. J., Hatala, R., Cook, D. J., Lang, J. D., and Hunt, D. (1996). Effect of calcium supplementation

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on pregnancy-induced hypertension and preeclampsia. JAMA, J. Am. Med. Assoc. 275, 1113-1117. Lipkin, M., and Newmark, H. (1995). Calcium and the prevention of colon cancer. J. Cell Biochem., Suppl. 22, 65-73. Thys-Jacobs, S., Starkey, E, Bernstein, D., and Tian, J. (1998). Calcium carbonate and the premenstrual syndrome: Effects on premenstrual and menstrual symptomatology. Am. J. Obstet. Gynecol. 179, 444-452. Appel, L. J., Moore, T. J., Obarzanek, E., Vollmer, W. M., Svetkey, L. E, Sacks, F. M., Bray, G. A., Vogt, T. M., Cutler, J. A., Windhauser, M. M., Lin, E-H., and Karanja, N. (1997). A clinical trial of the effects of dietary patterns on blood pressure. N. Engl. J. Med. 336, 11171124. Hyman, J., Baron, J. A., Dain, B. J., Sandler, R. S., Haile, R. W., Mandel, J. S., Mott, L. A., and Greenberg, E. R. (1998). Dietary and supplemental calcium and the recurrence of colorectal adenomas. Cancer Epidemiol., Biomarkers Prev. 7, 291-295. Holt, E R., Atillasoy, E. O., Gilman, J., Guss, J., Moss, S. E, Newmark, H., Fan, K., Yang, K., and Lipkin, M. (1998). Modulation of abnormal colonic epithelial cell proliferation and differentiation by low-fat dairy foods. JAMA, J. Am. Med. Assoc. 280, 1074-1079. Hruska, K. A., Goligorsky, M., Scoble, J., Tsutsumi, M., Westbrook, S., and Moskowitz, D. (1986). Effects of parathyroid hormone on cytosolic calcium in renal proximal tubular primary cultures. Am. J. Physiol. 251(2, Pt 2), 188-198. Chapuy, M. C., Arlot, M. E., Duboeuf, F., Brun, J., Crouzet, B., Arnaud, S., Delmas, E D., and Meunier, E J. (1992). Vitamin D 3 and calcium to prevent hip fractures in elderly women. N. Engl. J. Med. 327, 1637-1642. Heaney, R. E, Smith, K. T., Recker, R. R., and Hinders, S. M. (1988). Meal effects on calcium absorption. Am. J. Clin. Nutr. 47, 707-709. Heaney, R. E, Weaver, C. M., and Fitzsimmons, M. L. (1990). The influence of calcium load on absorption fraction. J. Bone Miner. Res. 11, 1135-1138. Heaney, R. P., Recker, R. R., and Weaver, C. M. (I 990). Absorbability of calcium sources: The limited role of solubility. Calcif Tissue Int. 46, 300-304. Heaney, R. P., Dowell, M. S., and Barger-Lux, M. J. (1999) Absorption of calcium as the carbonate and citrate salts, with some observations on method. Osteoporosis Int. 9, 19-23.

50. Spencer, H., Fuller, H., Norris, C., and Williams, D. (1994). Effect of magnesium on the intestinal absorption of calcium in man. J. Am. Coll. Nutr. 13, 485-492. 51. Vieth, R. (1999). Vitamin D supplementation, 25-hydroxyvitamin D levels, and safety. Am. J. Clin. Nutr. 69, 842-856. 52. Malloy, E J., Pike, J. W., and Feldman, D. (1997). Hereditary 1,25dihydroxyvitamin D resistant rickets. In "Vitamin D" (D. Feldman, F. H. Glorieux, and J. W. Pike, eds.), pp. 765-787. Academic Press, San Diego, CA. 53. Smith, E. L., Pincus, S. H., Donovan, L., and Holick, M. F. (1988). A novel approach for the evaluation and treatment of psoriasis. J. Am. Acad. Dermatol. 19, 516-528. 54. Mak, R. H. K. (1997). 1,25-Dihydroxyvitamin D 3 corrects insulin and lipid abnormalities in uremia. Kidney Int. 53, 1353-1357. 55. Van Leeuwen, J. E T. M., and Pols, H. A. P. (1997). Vitamin D: Anticancer and differentiation. In "Vitamin D" (D. Feldman, F. H. Glorieux, and J. W. Pike, eds.), pp. 1089-1105. Academic Press, San Diego, CA. 56. Mellibovsky, L., Dfez, A., P6rez-Vila, E., Serrano, S., Nacher, M., Aubfa, J., Supervfa, A., and Recker, R. R. (1998). Vitamin D treatment in myelodysplastic syndromes. Br. J. Haematol. 100, 516-520. 57. Parfitt, A. M. (1990). Osteomalacia and related disorders. In "Metabolic Bone Disease and Clinically Related Disorders" (L. V. Avioli, and S. M. Krane, eds.), 2nd ed., pp. 329-396. Saunders, Philadelphia. 58. Chapuy, M.-C., Preziosi, E, Maamer, M., Arnaud, S., Galan, E, Hercberg, S., and Meunier, E J. (1997). Prevalence of vitamin D insufficiency in an adult normal population. Osteoporosis Int. 7, 4 3 9 443. 59. Thomas, M. K., Lloyd-Jones, D. M., Thadhani, R. I., Shaw, A. C., Deraska, D. J., Kitch, B. T., Vamvakas, E. C., Dick, I. M., Prince, R. L., and Finkelstein, J. S. (1998). Hypovitaminosis D in medical inpatients. N. Engl. J. Med. 338, 777-783. 60. Dawson-Hughes, B., Harris, S. S., Krall, E. A., and Dalla, G. E. (1997). Effect of calcium and vitamin D supplementation on bone density in men and women 65 years of age or older. N. Engl. J. Med. 337, 670-676. 61. Kinyamu, H. K., Gallagher, J. C., Rafferty, K. A., and Balhorn, K. E. (1998). Dietary calcium and vitamin D intake in elderly women: Effect on serum parathyroid hormone and vitamin D metabolites. Am. J. Clin. Nutr. 67, 342-348.

2 H A P T E R 3~

Exercise BARBARA STERNFELD Department of Epidemiology and Biostatistics, Division of Research, Kaiser Permanente, Oakland, California 94611 ROBERT MARCUS Department of Medicine, Stanford University, School of Medicine, Geriatrics Research, Education & Clinical Center, Veterans Affairs Medical Center, Palo Alto, California 94304

I. II. III. IV.

V. Physical Activity and Change in Disease Risk Factor Status VI. Conclusions References

Introduction Physical Activity and Age at Menopause Physical Activity and Vasomotor Symptoms Physical Activity and Psychological and Somatic Symptoms

menopausal transition and poses questions and directions for future research and public health policy in this area.

I. I N T R O D U C T I O N Regular physical activity confers numerous health benefits, ranging from increased longevity and decreased risk of coronary heart disease, non-insulin-dependent diabetes, hypertension, colon cancer, and hip fracture to an improved sense of well-being and functional status [ 1]. These benefits are the long-term consequence of physiological adaptations of the musculoskeletal, cardiovascular, metabolic, and hormonal systems to varying modes and intensity of activity. Although these adaptations occur at whatever stage in the life cycle regular physical training occurs (childhood, adolescence, young adulthood, middle age, old age) and persist as long as training continues, they may either complement, conflict with, or be obscured by physiological changes and challenges that accompany different developmental stages. To date, understanding the effects of physical activity on the course of the menopausal transition and the implications of those effects on the current and future health status of midlife women have been of only minimal interest to most exercise scientists. As a result, little knowledge exists regarding the relationships between physical activity and age at menopause, symptoms of menopause, and changes in disease risk factor status that occur during the menopausal transition. This chapter reviews the current state of understanding of the effects of regular physical activity on the MENOPAUSE:

BIOLOGY AND PATHOBIOLOGY

II. P H Y S I C A L

ACTIVITY

AND

AGE AT MENOPAUSE A substantial body of literature exists associating vigorous exercise training, particularly in activities such as gymnastics, figure skating, and ballet dancing, with a late age at menarche [2-6]. Retrospective studies consistently find a later age at menarche among athletes who trained prior to menarche compared with those who started training after menarche [3,6]. However, because the population distributions of age at initiation of training and age at menarche overlap, a retrospective study design, in which the comparison groups are defined on the basis of one of the variables of interest (age at initiation of training), results in a biased (i.e., nonrandom) sample. This creates a correlation between age at initiation of training and age at menarche in the sample, which may not exist in the population and seriously compromises the credibility of these findings [7]. Furthermore, both biological factors, particularly genetic predisposition [8,9], and social factors, including nutritional status, family size, and social norms [4,9], may confound the association between exercise and sexual maturation. Nevertheless, in two 495

Copyright 9 2000 by Academic Press. All rights of reproduction in any form reserved.

496

STERNFELD AND MARCUS

prospective, epidemiological studies that controlled for confounding factors, greater premenarche participation in sports activity in general [10] or in selected sports (gymnastics, dance, figure skating, synchronized swimming, and diving) [11] was related to delayed menarche. In contrast, little is known about the relationship between physical activity and age at menopause. In seven studies that have examined predictors of age at menopause [12-18], only one reported on the effect of physical activity [18]. In that study, women were followed prospectively, and physical activity at baseline was assessed by self-report using the Alumni Study questionnaire [19], which asks about participation in sports and exercise and number of stairs climbed, and city blocks walked. Age at menopause did not differ by activity level, either between women above and below the median energy expenditure or between women above and below the 20th percentile of energy expenditure [2000 kcal per week). Hypothetically, physical activity might be associated with later age at menopause if activity results in later menarche and later menarche results in later menopause. However, age at menarche is not consistently associated with age at menopause [13,15], nor is there a proved causal relationship between activity and age at menarche. On the other hand, physical activity has been associated with later onset of and fewer ovulatory cycles [20,21] as well as longer or more variable cycles [22], and the opposite factors, early onset of regular menstrual cycles [13] and shorter cycle length [15], have been correlated with earlier age at menopause. In summary, no data exist to support the hypothesis of a relation between activity and late menopause, and the null findings of Bromberger et al. [18] suggest that activity may not independently impact age at menopause at all. This remains an empirical question deserving further investigation.

III. PHYSICAL VASOMOTOR

ACTIVITY

AND

SYMPTOMS

As discussed earlier in this volume, of all the physical and psychological symptoms commonly reported by midlife women, only vasomotor symptoms (hot flashes and night sweats) clearly vary by menopausal status, having a relatively low prevalence in premenopausal women, then rising during peri- and early postmenopause, and declining with increasing years postmenopause [23,24]. Although the physiology of the hot flash is not fully understood, various neuroendocrine substances such as catecholamines, cortisol, and endogenous opioids may be implicated in this process [25]. Evidence for the role of fl-endorphin in the pathogenesis of the hot flash comes primarily from experiments with naloxone, an opiate antagonist. Administration of naloxone in

the morphine-dependent rat causes symptoms similar to those of the hot flash, including a sudden increase in peripheral tail temperature, decreased core temperature, and a luteinizing hormone (LH) surge [26,27]. Indeed, many of the symptoms of opiate withdrawal in humans, such as hot flushes and perspiration, increased heart rate, insomnia, irritability, and joint and muscle aches, mirror those experienced by peri- and postmenopausal women [25]. Physical activity, which causes a range of neuroendocrine responses, may increase production and metabolism of endogenous opioids both acutely and chronically. A single bout of vigorous exercise raises serum concentration of all endogenous opioids, particularly fl-endorphin, one to five times above basal levels [28,29], and is accompanied by increases in adrenocorticotropic hormone (ACTH) and cortisol [29]. Exercise training may augment this acute response [30, 31 ]. In a comparison of female eumenorrheic and amenorrheic athletes and sedentary controls, resting levels of plasma endorphins were higher in the athletes throughout the menstrual cycle, regardless of menstrual status [28]. Although peripheral concentration of endogenous opioids may or may not reflect or influence concentrations in the brain [32], the exercise-associated increase in peripheral levels suggests a biological basis for hypothesizing that physical activity may reduce either the frequency or perceived intensity of menopausal vasomotor symptoms. On the other hand, higher levels of body fat appear to decrease vasomotor symptoms because adipose cells are the primary source of estradiol once ovarian production and secretion decline [33]. Active women tend to be leaner than sedentary women, thus greater physical activity might, in reality, increase the risk of hot flashes. Furthermore, if both body fat and endorphin levels reduce the likelihood of vasomotor symptoms, the effects may not be observable if the two factors tend to vary inversely with each other. Existing investigations of physical activity and vasomotor symptoms in midlife women have generally failed to establish any relationship [34-39]. As ~ o w n in Table I, six out of nine studies found no difference in the prevalence of vasomotor symptoms between active and sedentary women. Five of these six were cross-sectional study designs that included women of varying menopausal status. Sample sizes ranged from a few hundred [37] to more than 1000 [36], and the methods used to assess activity ranged from a simple global question about participation in regular exercise [36] to a detailed quantitative activity history [34,35,38]. Despite differences in study population and activity methodology, none found any difference in prevalence of vasomotor symptoms according to activity level once menopausal status was taken into account. The sixth null study used a popoulation-based casecontrol design in which cases (n = 82] were defined by the frequency of vasomotor symptoms in the 3 months following

CHAPTER 34 Exercise the last menstrual period (LMP), and activity was assessed by recall for the 12-month period prior to the LMP [39]. Controls (n -- 89] were women who were also 3-12 months since the LMP but who reported no or only infrequent hot flashes or night sweats during the same reference period. Having frequent vasomotor symptoms showed no association with participation in physical activity in the year before menopause. This lack of relationship was observed consistently, regardless of the domain of activity under consideration (recreational, housework, childcare, or occupational) and regardless of variables such as body mass index and other symptoms. In contrast, three studies have observed fewer vasomotor symptoms in active women compared with sedentary women [40,41,41 a]. The earliest study to find a protective effect was a cross-sectional survey of Swedish women in which 142 regular participants in an exercise program were compared with 1246 controls randomly selected from the population [40]. Among naturally menopausal women (at least 6 months since LMP, not on hormone replacement therapy), the prevalence of moderate to severe hot flashes was 21.5% in the exercise group and 43.8% in the control group. Unfortunately, the activity level of the controls was not assessed, nor was adjustment made for any age difference or differences in length of time since LMP between the two groups. These methodological shortcomings could have caused the difference in the observed prevalence of vasomotor symptoms in the two groups to be much greater than it truly was. On the other hand, a more recent study from this same group [41] also observed lower prevalence of hot flashes among highly active post-menopausal women compared to postmenopausal women who participated in little or no exercise (5% vs. 14-16%, p < 0.05). In that survey, 35% of the sample was on hormone replacement therapy (HRT), and women on HRT were more likely to be active than those who were not. Although women were asked about hot flashes since experiencing menopause, which presumably preceded initiation of HRT, the possibility of confounding by HRT cannot be dismissed. The third report of an inverse relation between physical activity and risk of vasomotor symptoms comes from the Study of Women's Health Across the Nation (SWAN), a multicenter, prospective, population-based study of the menopause transition in women of diverse ethnicities in the United States. In 12,000 findings from a cross-sectional survey of over 16,000 women between the ages of 40 and 55 years of varying menopausal status, the proportion reporting hot flashes and night sweats in the previous 2 weeks decreased from 52.5% in those who rated themselves much less physically active than other women of their age to 32.6% in those rating themselves as much more active [41 a]. With adjustment for menopausal status, age, race/ethnicity, education, body mass index, smoking, and other confounders, the

497 odds ratios for hot flashes associated with activity level rated "much less than others" and "less than others" were 1.71 [95% confidence interval (CI) = 1.42 - 2.07] and 1.33 (95% CI = - 1.16-1.54), respectively, relative to ratings of "much more active." In summary, the literature generally suggests either no relationship between physical activity and experience of vasomotor symptoms or perhaps a weak protective association. Differences in findings are not easily explained by methodology or quality of the study. Although none of the studies relied on clinical samples, all, except for the case-control study [39], which is limited by the accuracy of recall, are cross-sectional studies and, therefore, are limited in the ability to establish appropriate temporal sequence. Because no prospective data yet exist to indicate whether women who are more physically active prior to menopause experience fewer vasomotor symptoms during or after menopause, any conclusion about the effect or lack of effect of activity on vasomotor symptoms must remain tentative. Given the transient nature of hot flashes, any protective effect of physical activity may be more of an acute than a chronic response. The decrease in reported vasomotor symptoms found immediately after an exercise class compared with immediately prior to the class [37] supports this hypothesis and is consistent with an acute rise in plasma/3endorphins following vigorous activity [29,32]. This could explain the negative findings of the case-control study that assessed habitual activity during a time prior to the period of time for which vasomotor symptoms were assessed and the negative findings of most of the cross-sectional surveys [34,36,38] that assessed exercise and hot flashes during the same period of time but when respondents were in a resting state. To date, no prospective studies or randomized clinical trials have addressed this issue, either in terms of an adaptive effect of regular physical activity or of an acute effect. Such studies could provide new insights into whether physical activity helps to prevent or mitigate the frequency or intensity of vasomotor symptoms.

IV. P H Y S I C A L PSYCHOLOGICAL SOMATIC

ACTIVITY

AND

AND

SYMPTOMS

Despite the generally null findings with regard to vasomotor symptoms, most studies of midlife women have found physical activity to have a direct relationship with positive mood, vigor, and general well-being [34-36,42] and an inverse relationship with negative symptoms, such as depression, anxiety, problems with memory and concentration, difficulty sleeping, and decreased sexual desire [36,37,42]. Studies have also generally found that active midlife women

498 TABLE I Study design

STERNFELD AND MARCUS

Summary of Studies Investigating Relationship between Physical Activity and Symptoms in Midlife Women Sample

Activity measure

Findings: vasomotor symptoms

Findings: other symptoms

Ref.

Crosssectional

Participants in exercise program, ages 50-58, in Sweden, compared with randomly selected population controls, ages 5 2 54; varying menopausal status (n = 142 vs. 1246)

Participation in exercise program

21.5% of active women vs. 43% of controls reported moderate or severe hot flashes (postmenopausal only)

Not reported

[40]

Crosssectional

Australian women ages 34-62, volunteering for bone density study; varying menopausal status (n = 386)

Energy expenditure in recreational, occupational, and housework activity

No relation

Inverse relation between recreational activity and dizziness, rapid heart beat, pins and needles, joint pain, and other general health symptoms; direct relation between occupational activity and same outcomes

[35]

Crosssectional

Australian women in bone density study returning for follow-up exam; varying menopausal status (n = 279)

Energy expenditure in recreational, occupational, and housework activity in past 12 months, based on intensity of activities, duration, and frequency

No relation

Crosssectional

Population-based sample of Australian women, ages 45-55; varying menopausal status (n = 2001) Subset of cohort in Dennerstein study; varying menopausal status (n = 728)

Exercise for fitness or recreation at least once a week

Not reported

Inverse relation with negative affect; direct relation with positive affect and overall well being

[42]

Energy expenditure in recreational activity in past 2 months, based on intensity of activities, duration, and frequency

No relation

Direct relation with self-rated health

[38]

Crosssectional

Population-based sample of Swedish women, 48 years old; varying menopausal status (n = 1,324)

Participation in regular exercise (yes/no)

No relation

Inverse relation with negative mood and reduced sex drive; direct relation with wellbeing

[36]

Crosssectional (Study I)

Australian volunteers; varying menopausal status (n = 220)

Aerobic activity at least twice a a week for 30 min a time for past 3 months

No relation

Inverse relation with depressed mood, anxiety, fears, fatigue, tension, problems with memory and concentration, sexual dysfunction, sleep problems; direct relation with vigor and perceived attractiveness

[37]

Experiment (Study II)

Australian volunteers who were regular exercisers; varying menopausal status (n = 47)

Aerobic activity at least twice a a week for 30 min a time for past 3 months

Fewer symptoms reported following exercise class, independent of change in mood

Fewer somatic symptoms reported following exercise class, independent of change in mood

[37]

Crosssectional

Population-based sample of Swedish women, ages 55-56, naturally menopausal (n = 793)

Physical activity score based on intensity of activity and time per week

5% of highly active reported moderate or severe hot flashes vs. 14-16% of less active

Not reported

[41]

Crosssectional

[34]

(continues)

499

CHAPTER 34 Exercise

TABLE I Study design

Sample

Case-control Casesdefined as women 48-52 years old, 3-12 months since LMP with frequent vasomotor symptoms (n = 82); controls same chronological and biological age without vasomotor symptoms (n = 89) CrossCommunity-based samples sectional of African-Americans, Caucasians, Chinese, Hispanics, and Japanese women, ages 40-55, from seven areas of the United States; varying menopausal status (n = 12, 899)

(continued) Findings: vasomotor symptoms

Findings: other symptoms

Ref.

Activity score based on No relation intensity of activity and frequency; separate scores for recreational, occupational, and household activity

Activity attenuated relation between psychological and vasomotor symptoms

[39]

Rating of activity level relative to other women of same age

Increased risk of forgetfulness, stiffness/sorenessin joints, heart pounding, difficulty sleeping, vaginal dryness, and urine leakage associated with less activity, after adjustment for age, menopausal status, ethnicity, and other factors

[41a]

Activity measure

Increased risk associated with less activity, after adjustment for age, menopausal status, ethnicity, and other factors

report fewer somatic symptoms, including stiffness and soreness in the joints, headaches, and heart pounding [35,37]. For example, in the data from the SWAN cross-sectional survey, the odds ratios associated with activity less than one's peers, after adjustment for ethnicity, age, menopausal status and other confounders, was 1.65 (95% CI = 1.33 - 2.05) forheart pounding and 2.33 (95% CI = 1.92-2.82) for stiffness or soreness in the joints [41 a]. These patterns of association between physical activity and somatic and psychological symptoms are independent of menopausal status and are similar in magnitude in pre-, peri-, and postmenopausal women [35,36,42]. A substantial body of literature indicates that regular physical activity improves various dimensions of mental health, increasing sense of general wellbeing, positive mood, and self-esteem, and decreasing anxiety and depression [43]. These associations are independent of socioeconomic status and physical health, but may depend on whether the activity is volitional (enjoyable) rather than obligatory; housework, for example, is not associated with the same elevation in mood as is recreational activity [43]. Although various mechanisms have been proposed to explain the effects of activity on mental health, including increased release of endorphins, increased levels of neurotransmitters (specifically, dopamine and seritonin), therapeutic effects of elevated body temperature, and distraction from stressful stimuli [44], all of these hypotheses remain speculative. Nevertheless, they lend credibility to the cross-sectional findings discussed above that show lower prevalence of psychological symptoms in active, midlife women. On the other hand, the lower prevalence of somatic symptoms reported by active mid-

life women could be more a marker for better health status than a real effect of physical activity itself on the specific symptom.

V. PHYSICAL ACTIVITY AND CHANGE IN DISEASE RISK FACTOR STATUS A. Cardiovascular Disease Increased rates of cardiovascular disease (CVD), particularly coronary heart disease (CHD), in women around the time of the menopause and following oophorectomy suggest that the decrease in estradiol that accompanies the menopause may, itself, be a CVD risk factor [45,46]. Studies comparing the effects of estrogen replacement therapy (ERT) and/or hormone replacement therapy on lipids and lipoproteins, clotting factors, body composition, and other CVD risk factors also provide evidence that menopause raises the risk of CVD morbidity and mortality [46]. However, studies of ERT/HRT may suffer from serious selection bias that results from the fact that women who use ERT/HRT are different from those who do not [47], and many of the changes in CVD rates and risk factor status may be more age related than hormonerelated. Nevertheless, midlife women do experience increased risk of CVD compared to younger women, and, as with men, physically active women of all ages have lower risk than sedentary women. For more than a decade, physical inactivity has been counted as one of the major risk factors for CHD. In a 1987 review by Powell et al., the results of 43 studies of physical

500

activity and coronary heart disease were evaluated in terms of adequacy of the activity assessment, the ascertainment of the outcome, and the epidemiological methods [48]. The conclusion was that "better" studies consistently found about an 80% increase in risk of heart disease associated with inactivity, an increase similar in magnitude to that associated with hypertension, hypercholesterolemia, and smoking. Furthermore, the risk associated with inactivity could not be explained by preexisting illness because follow-up for coronary events in the prospective studies generally did not begin until a few years after the baseline assessment of activity. The authors concluded that, given criteria for inference of causality, the inverse relation between activity and heart disease could reasonably be considered a causal one. A meta-analysis by Berlin and Colditz confirmed that conclusion [49]. Unfortunately, only five of the studies reviewed by Powell et al. included women at all or reported on women separately from men [50-54]. More reports of the relation between activity and CHD incidence or mortality in women have begun to appear [55-58]. Although some of the findings are not as definitive as they are in men, owing, in part, to smaller numbers of events [57,59], the overall direction of findings is similar. For example, in a case-control study of women with nonfatal myocardial infarction conducted in a large health maintenance organization, the adjusted odds ratio for the highest quartile of energy expenditure relative to the lowest was 0.40 (95% CI = 0. 25-0.63) with an apparent dose response from lowest to highest quartile [58]. A similar protective relationship was observed for energy expenditure in nonstrenuous activity and in brisk walking. As with men, there is a protective relation with physical fitness as well as with activity; in a study of 7080 women followed for an average of 8 years, higher levels of aerobic fitness, as determined by a graded treadmill exercise test, were significantly associated with lower risk of both all-cause and CVD mortality [60]. The protective effect of activity on CVD incidence and mortality may be mediated in part by a beneficial effect of physical activity on blood pressure, lipids and lipoproteins, glucose intolerance and insulin resistance, and clotting factors, all of which are implicated in the pathogenesis of CVD. Numerous cross-sectional studies indicate that physically active women of both pre- and postmenopausal status have a more optimal cardiovascular risk factor profile compared with their sedentary peers. For example, with regard to blood pressure, a cross-sectional analysis of 4576 Dutch women, ages 4 9 - 7 0 years, found that both mean systolic and diastolic blood pressures were significantly lower in those in the highest tertile of sports activity compared with those in the lowest [61 ]. Similarly, baseline data for 533 participants in the Healthy Women's Study, a prospective study of midlife, initially premenopausal women, showed a significant, inverse linear trend between blood pressure and self-reported physi-

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cal activity [62]. The baseline data from the Healthy Women's Study also showed that levels of high-density lipoprotein cholesterol (HDL-C) and a HDL2-C subfraction increased as activity increased and levels of low-density lipoprotein cholesterol (LDL-C) decreased [62]. Similarly, an analysis of 283 nonsmoking, postmenopausal women showed that physical fitness was directly related to HDL-C and inversely related to total cholesterol (TC), the TC/HDL-C ratio, and triglycerides [63]. Many other studies support these findings of an association between physical activity and a more favorable atherogenic lipid profile [57,64-71], although the extent to which body weight or body fat either confounds or mediates the relationships is not entirely clear [59,72,73]. Despite some inconsistency [74], higher levels of physical activity in women are also generally associated with lower levels of fasting plasma glucose and insulin and improved glucose tolerance [62,75] as well as with reduced risk of developing non-insulin-dependent diabetes [76], a negative health outcome in its own right and also a risk factor for subsequent CHD. Finally, although data in women are limited regarding the relationship between activity and hemostatic factors, an inverse association between activity and plasma fibrinogen has been reported in both young adult [77] and postmenopausal women [78-80]. As with lipids and lipoproteins, the association with fibrinogen may be partly explained by body mass [78-80]. Other clotting factors, specifically tissue plasminogen activator (tPA) and plasminogen activator inhibitor (PAI-1), also showed a favorable relation with physical activity in a population-based sample of 774 Swedish women, ranging in age from 25 to 64 [81] and in a small sample of highly active and sedentary postmenopausal women [75]. Evidence from prospective studies, including both exercise training interventions and observational studies of change in CVD risk factors over time, is somewhat more equivocal, particularly in terms of lipids and lipoproteins, for which the effects depend on a number of intervening variables, including changes in body weight, amount, duration, type of exercise, dietary change [59,71,82-84], and, perhaps, menopausal status [85,86]. Few studies have examined the effectiveness of exercise training on reducing blood pressure in hypertensive women, and limited observational data suggest only minimal association between change in activity and change in blood pressure on the population level [8789]. Few, if any, prospective studies have examined change in activity and change in hemostatic factors in either pre- or postmenopausal women. Studies of physical activity and CVD risk factors in women have begun to address specifically the question of whether the effects of activity modify effects of aging and hormone use. Two cross-sectional comparisons of active and sedentary pre- and postmenopausal women suggest that, although mean arterial and systolic blood pressure were higher in the postmenopausal women, regardless of activity level

CHAPTER34 E

x

e

r

c

i

[90,91], decreased cardiac output, stroke volume, and oxygen consumption [90] and increased systemic vascular pressure [90] and central arterial stiffness [91 ] were not observed in the active older women, regardless of HRT use but were observed in older sedentary women [90]. Also, in a small randomized trial in postmenopausal women that was designed to compare the effects on lipids and lipoproteins of moderate exercise alone, estrogen replacement therapy alone, and exercise and ERT combined, significant decreases in systolic blood pressure relative to a control group, as well as significant decreases in total cholesterol, triglycerides, and LDL-C and increases in HDL-C, were apparent in all three experimental groups. Although the magnitude of the changes was similar in the estrogen and estrogen plus exercise arms and greater than those in the exercise alone arm, only the exercise and estrogen plus exercise groups demonstrated improvement in physical fitness [92]. A similar study conducted in somewhat older postmenopausal women that used combination HRT rather than ERT found independent and complementary effects for exercise and drug therapy [93]. These findings indicate that exercise may attenuate some of the deterioration in cardiovascular risk factors that occurs with age and/or change in menopausal status, but the magnitude of attenuation may perhaps not be as great as or may be different in nature from that achieved with hormone replacement. A small number of prospective studies designed to follow midlife, initially premenopausal women through the menopausal transition also suggest that physical activity contributes to the maintenance of a more optimal CVD risk factor profile over time. In the Healthy Women's Study, in which women were followed over a 3-year period, neither baseline physical activity nor change in activity was related to changes in diastolic blood pressure, LDL-C, or triglycerides [89]. However, decreased activity was related to decreased HDL-C (although not significantly after adjustment for change in weight and menopausal status), whereas increased activity was associated with maintenance of HDL-C. Also, decreased activity was related to decreased HDL2-C, independently of change in weight and menopausal status [89]. Additional evidence comes from the Women's Healthy Lifestyle Project, a randomized dietary and behavioral clinical trial designed to prevent elevations in cardiovascular risk factors in initially premenopausal midlife women. After 6 months, the experimental group (n = 253] had a significant increase in physical activity, compared to no change in the control group. The experimental group also had significantly greater decreases in both systolic and diastolic blood pressure, as well as more favorable mean changes in total choles' terol, LDL-C, triglycerides, and the HDL-C/TC ratio, compared with controls [94]. Unfortunately, this study could not determine the degree to which the changes in risk factors were attributable to change in activity specifically or to change in diet or weight.

s

e

5

0

1

To date, no studies have actually followed women from pre- to postmenopause with large and varied enough sample sizes to be able to untangle the effects of aging from the effects of change in menstrual status from the effects of physical activity and other lifestyle behaviors on CVD risk factors. Until such studies are conducted, the importance of promoting physical activity during the menopausal transition as a way to prevent immediate worsening of CVD risk factors will remain largely unproved.

B. W e i g h t G a i n / C h a n g e in F a t D i s t r i b u t i o n Increases in weight [95], increases in body fat [96], and redistribution of body fat from the periphery to the center [96] typically occur in women as they age. What, if any, aspect of this process of change in body composition and topology may be attributable specifically to the change in hormonal status that defines the menopausal transition is not yet clear. Although the midlife women in the Healthy Women's Study gained an average of 2.25 kg over a 3-year follow-up period, the weight gain of those who remained premenopausal was the same as those who became postmenopausal [97], suggesting an aging effect but not a hormonal one. Similarly, a large cross-sectional survey Of factors associated with fat distribution in obese women found that the waist-to-hip ratio, a measure of truncal fat, varied by age and weight but not menopausal status [98]. In contrast, treatment with hormone replacement therapy appears to preserve a more peripheral fat distribution [99] and is associated with lower body mass index, waist-to-hip ratio, and percentage of body fat, independently of socioeconomic and life style factors [ 100]. This suggests that loss of estradiol during the menopausal transition, and not just aging, may contribute to changes in body composition and fat distribution. A similar suggestion emerges from a longitudinal study in which an acceleration of loss of muscle mass, as determined by rate of loss of total body potassium, along with accelerated loss of skeletal mass, was noted in the first 3 years postmenopause compared with the pre- and perimenopausal period [101]. Decreased lipolytic response in abdominal adipocytes in postmenopausal women compared with premenopausal women may account for increased central fat disposition but could be mediated either hormonally and/or by age [ 102]. Despite the widely held belief that greater physical activity reduces the risk of weight gain and subsequent obesity, data to support that belief are surprisingly limited [ 1]. Although exercise training intervention studies show that physical activity promotes fat loss while preserving lean muscle tissue [95], the effect of physical activity on weight change over time in the general population is less established. Cross-sectional studies in large, population-based samples that include young, middle-aged, and/or elderly

502 women generally report small but significant inverse associations between activity and body mass index (BMI) or other measures of adiposity [61,62,65,103,104] and waist-to-hip ratio or other measures of fat distribution [61,105-107]. In two longitudinal studies, baseline recreational activity was independently and inversely associated in a dose-response way with weight gain during the follow-up period [89,108], but in another, baseline activity was not related to weight change between baseline and follow-up, although activity reported at the follow-up was [ 109]. In the PEPI trial, a large randomized controlled trial of the effects of postmenopausal estrogen and progestin, higher baseline activity was significantly associated with lower weight gain and less change in waist circumference, independently of treatment group [ 110]. Change in activity has also been associated with change in weight, with increased activity resulting in an attenuation of weight gain [ 104,111 ], and maintenance of a low activity level over time increasing risk of a large weight gain, relative to maintenance of a high activity level [109]. However, inconsistent and somewhat paradoxical study findings, such as increased risk of weight gain in women who increased their activity level compared with those who maintained a high activity level [ 109], suggest that weight change may be as much a cause of activity change as a consequence of it [95]. The effect of habitual physical activity or change in physical activity on body fat and fat distribution during the transition from pre- to postmenopausal status has not received much attention. In a cross-sectional study designed to examine the relationship of exercise and age to resting metabolic rate (RMR) [112], postmenopausal sedentary women had an RMR, adjusted for fat mass and fat-free mass, that was about 10% lower than that of premenopausal sedentary women. In contrast, no difference in RMR was observed between postmenopausal and premenopausal active women. This suggests that the decline in RMR that accompanies aging or change in hormonal status may be attenuated or avoided with physical activity. On the other hand, in a small longitudinal study in which RMR, recreational physical activity, fat mass, and waist-to-hip ratio were measured 6 years apart in 35 initially premenopausal women [113], RMR and activity decreased and fat mass and waist-to-hip ratio increased in those who became postmenopausal (n = 18) relative to those who did not. Unfortunately, because this study only reported between-group differences, the impact of activity or change in activity on the apparently menopausally mediated changes in RMR, body composition, and fat distribution is not clear. Yet another perspective comes from the Healthy Women's Study. Over a 3-year follow-up period, during which time some of the cohort transitioned from preto postmenopausal status, both baseline activity and change in activity were inversely associated with change in weight, independently of baseline weight, menopausal status, or hormone use [89]. In this cohort, menopausal status was not associated with weight change, thus these findings imply that

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the influence of activity on weight gain during the menopausal transition is simply the same as it is during any other period of adult aging. In conclusion, at this point, the extent to which physical activity modifies either the rate or magnitude of changes in body composition and fat distribution that may accompany the change in menopausal status is largely unaddressed and poorly understood.

C. B o n e L o s s The well-established transient increase in the rate of bone loss that takes place as a consequence of menopausal estrogen deprivation is discussed in detail in Chapter 35 of this volume. Although timely and long-term administration of estrogen conserves bone and is associated with substantial reductions in fracture risk, many women prefer not to embark on HRT and seek to achieve skeletal protection by consuming more calcium and performing more exercise. However, the evidence that such lifestyle modification is effective at preventing menopause-related bone loss is largely absent. In fact, although increased calcium intake may somewhat ameliorate menopausal bone loss, it does not obliterate it [ 114]. And, in contrast to the substantial body of literature exploring the skeletal effects of exercise in premenopausal women and women 10 years or more beyond menopause, the effects in peri- or early menopausal women have received only minimal attention. This may reflect the desire of investigators to focus specifically on the effects of exercise alone, without having to contend with complexities introduced by studying women who are also experiencing transient increases in bone loss due to estrogen deficiency. Physical activity affects the bone status of peri- and early menopausal women in two ways: by having affected peak bone mass and by influencing rate of age-related adult bone loss. Of the two, variation in peak bone mass at skeletal maturity greatly exceeds variation in the rate of adult bone loss. Therefore, bone mass at the time women begin the menopausal transition is a powerful influence on bone mass 10 years later. Although about 75% of peak bone mass is determined by genetic endowment, among the modifiable environmental influences on bone acquisition, habitual physical activity accounts for much of the nongenetic component. Observational studies and well-controlled exercise intervention trials in young women have shown that exercise training enhances bone density in a site-specific manner. Both resistance and endurance exercise training reliably induce small, but significant, increases in lumbar spine bone mineral density (BMD) [115-117]. Although these studies have not generally demonstrated increased BMD at the proximal femur, such improvement has been observed in two reports, a 2-year program of combined aerobic and weight training [ 117] and a high-impact (jumping) study in women aged 3 5 - 4 5 years [ 118]. In one of the earliest attempts to explore the relationship

CHAPTER 34 Exercise of habitual activity to age-related bone loss, Talmage and colleagues [ 119] observed, cross-sectionally, a lower rate of bone loss with age in athletic versus nonathletic women. In addition, the athletic group appeared to have had a delayed onset of the acceleration in bone loss that occurred after age 50 years compared with nonathletic women. However, the biases inherent in the cross-sectional design necessitate confirmation of this observation by a prospective study. Whether exercise ameliorates menopause-related bone loss remains unclear. Pruitt and colleagues [120] conducted the single published study of the skeletal effect of exercise in "early" postmenopausal women. Participants were randomly assigned to an exercise group or to control status for a 9-month intervention. The exercise mode was a moderately intense program of supervised progressive resistance using weight-lifting machines and involving all major muscle groups of the arms, legs, and trunk. On completion of the study, the exercise group had maintained BMD at the lumbar spine whereas the control group had sustained a 3.6% loss. At the other measured sites, proximal femur and forearm, no significant protective effect of exercise was observed. This report has been interpreted to indicate that exercise may offer skeletal protection during the menopause at the spine but not at other locations. However, several issues limit validity of this interpretation. Although the trial participants were substantially younger than those in other studies of postmenopausal women, they still averaged 8 years from last menses. Therefore, this is not a study of women who are actually progressing through the menopausal transition or who have recently entered the phase of most rapid bone loss. Furthermore, even in younger women, studies using exercise machines have failed to show an effect of exercise on the proximal femur. Among the components of exercise that initiate skeletal activity, impact (the rate at which strain is introduced to the bone) appears to play an important role. Because most exercise machines are designed, for safety purposes, to minimize impact to bones and joints, they may not administer a training stimulus to the hip or forearm adequate for inducing a skeletal response. Also, because the average healthy middle-aged woman may spend 4 - 8 hr each day in upright activity, it is possible that the loads induced by the training regimen did not constitute a significant increment to daily loading exposure. Thus, to resolve whether exercise can minimize menopause-related bone loss will require additional study with women of a more appropriate menopausal age and employing a training program of sufficient intensity and duration to provide the skeleton a novel and incremental loading environment. At present, predicting what such a study might show is difficult. Although exercise attenuates bone loss in oophorectomized animals [ 121-123], it is not clear that this would occur in estrogen-deprived humans. Indeed, a normal skeletal response to exercise may require the presence of estrogen. Young women with disruption of their estrogenic milieu, such as those with exercise-associated amenorrhea or who

503 have received gonadotropin-releasing hormone (GnRH) therapy for endometriosis, experience bone loss due to increased osteoclastic bone resorption even in the presence of prodigious exercise. In surgically postmenopausal women taking estrogen, 1 year of resistance exercise significantly increased spine, total body, and radial midshaft BMD compared with estrogen-replaced, nonexercising controls who merely maintained BMD [124], whereas a similar protocol in estrogendeplete women had no effect on BMD [M. Notelovitz, personal communication]. The skeletal effect of exercise results from two types of loading. The first comes from direct impacts on the bone; the second comes from tension generated by muscular contraction that is transmitted to insertion points on bone. Such muscle-generated loads actually constitute a large proportion of total skeletal load environment under normal circumstances [125,126]. Consequently, muscle strength is directly related to bone mass [ 127,128]. Loss of muscle strength precedes the loss of bone and the recovery of muscle strength precedes recovery of bone mass, albeit with a substantial delay reflecting different rates of adaptation for these two tissues [ 129]. As with the age-related loss of bone, progressive decreases in muscle mass and strength with normal aging have been recognized for many years, and older women, in particular, frequently exhibit deficits in muscle strength that are sufficiently profound as to interfere with accomplishing the activities of daily life. It is relevant, therefore, to ask whether some component of this process, referred to as sarcopenia, may be related to the menopausal transition. At present, it is impossible to rule out such a connection, but there is no substantial evidence that it exists. For instance, muscle strength may actually begin decreasing shortly after age 30, during the height of reproductive life [ 130], and muscle strength of older women who have not taken HRT may be no different from that of long-term hormone replacement users [ 131 ]. Even if no obvious relationship between estrogen and muscle strength exists, and even though the effects of exercise on bone in menopausal women are modest, encouraging all women to participate in regular and frequent exercise as a means to promote and maintain leg strength throughout life is strongly warranted. The primary goal that one hopes to achieve through exercise is a reduction in fractures, not conservation of BMD. Hip fractures are the fractures that most devastate quality of life, carry the greatest repercussions for survival, and confer the largest societal cost. Most often, hip fractures are the immediate and direct consequence of a fall. Falls underlie an important proportion of vertebral fractures as well. Because muscle weakness is a consistently important antecedent factor for falls and an independent risk factor for hip fracture [ 132], a major goal accomplished with exercise, in terms of skeletal benefit, is a reduction in falls. Therefore, the recommendation to incorporate leg-strengthening exercises into an overall program of regular physical activity is an essential part of a sound public health policy.

504

VI. CONCLUSIONS As discussed throughout this chapter, numerous questions remain unanswered regarding the role of habitual physical activity in maintaining the quality of life and health status of w o m e n as they move through the menopausal transition. The following questions are the most pressing among these: 9 Does participation in regular physical activity decrease the frequency and/or intensity of vasomotor symptoms? If so, what is the " d o s e " of activity (frequency, duration, intensity) necessary to achieve this effect, and is it a result of exercise training or an acute effect? 9 Does participation in regular physical activity prevent or attenuate the adverse changes in blood pressure, lipids and lipoproteins, and hemostatic and metabolic factors that accompany midlife, either because of age and/or change in hormonal status? If so, how does that attenuation compare with the effects of hormone replacement therapy? 9 Does participation in regular physical activity diminish the magnitude of age- and/or hormone-related increases in weight, body fat, and central fat deposition that occur in midlife? If so, what dose of activity is necessary and what is the impact of regular physical activity in the period preceding the perimenopause? 9 Does participation in regular physical activity reduce the accelerated loss of bone density that accompanies the decline in ovarian function that characterizes the menopause or prevent age and/or hormone-related loss in muscle strength and lean muscle mass? If so, what types of physical activities produce these effects and to what extent does the effect depend on prior exercise training? Answers to these questions require large-scale longitudinal studies that follow women from pre- to postmenopause and in which the full ranges of physical activity patterns and changes in patterns commonly observed in the general population are represented. These studies also need to include substantial numbers of nonwhite women in order to ensure the generalizability and relevance of the findings. In addition, carefully designed, randomized exercise trials in perimenopausal and early postmenopausal women are called for in order to determine the effects of different doses of activity on outcomes such as bone and muscle loss, fat gain, and blood pressure change and to evaluate the effects in comparison with other interventions. Even without definitive answers to the questions posed above, the public health policy recommendation regarding physical activity for women (and men) of all ages and stages of life is quite clear. Participation in regular physical activity, at or above the level r e c o m m e n d e d in the 1996 Surgeon General's Report [1] (moderate intensity activity for a total of 30 min a day on most days of the week), confers significant health benefits. Currently 20% or fewer of the population of w o m e n in the United States meets this recommendation [ 1].

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Promoting regular physical activity among midlife women, therefore, has great potential for reducing the societal disease burden of sedentary living as well as for improving the quality of life and sense of well being for individual women.

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507 109. Williamson, D. E, Madans, J., Anda, R. E, Kleinman, J. C. Kahn, H. S., and Byers, T. (1993). Recreational physical activity and 10-year weight change in a US national cohort. Int. J. Obes. 17, 279-286. 110. Espeland, M. A., Stefanick, M. L., Kritz-Silverstein, D., Fineburg, S. E., Waclawiw, M. A., James, M. K., and Greendale, G. A. (1997). Effect of postmenopausal hormone therapy on body weight and waist and hip girths. Postmenopausal Estrogen-Progestin Interventions Study Investigators. J. Clin. Endocrinol Metab. 82, 1549-1556. 111. Klesges, R. C., Klesges, L. M., Haddock, G. K., and Eck, L. H. (1992). A longitudinal analysis of the impact of dietary intake and physical activity on weight change in adults. Am. J. Clin. Nutr. 55, 818-822. 112. Van Pelt, R. E., Jones, E P., Davy, K. E, DeSouza, C. A., Tanaka, H., Davy, B. M., and Seals, D. R. (1997). Regular exercise and the agerelated decline in resting metabolic rate in women. J. Clin. Endocrinol. Metab 82, 3208-3212. 113. Poehlman, E. T., Toth, M. J., and Gardner, A. W. (1995). Changes in energy balance and body composition at menopause: A controlled longitudinal study. Ann. Intern. Med. 123, 673-675. 114. Riis, B., Thomsen, K., and Christiansen, C. (1987). Does calcium supplementation prevent postmenopausal bone loss? A double-blind, controlled clinical study. N. Engl. J. Med. 316, 173-177. 115. Snow-Harter, C., Bouxsein, M. L., Lewis, B. T., Carter, D. R., and Marcus, R. (1992). Effects of resistance and endurance exercise on bone mineral status of young women: A randomized exercise intervention trial. J. Bone Miner. Res. 7, 761-769. 116. Lohman, T., Going, S., Pamenter, R., Hall, M., Boyden, T., Houtkooper, L., Ritenbaugh, C., Bare, L., Hill, A., and Aickin, M. (1995). Effects of resistance training on regional and total bone mineral density in premenopausal women: A randomized prospetive study. J. Bone Miner. Res. 10, 1015-1024 117. Friedlander, A. L., Genant, H. K., Sadowsky, S., Byl, N. N., and Gluer, C. C. (1995). A two-year program of aerobics and weight-training enhances BMD of young women. J. Bone Miner. Res. 10, 574-580. 118. Heinonen, A., Kamus, E, Sievanen, H., Oja, E, Pasanen, M., Rinne M., Uusi-Rasi, K., and Vuori, I. (1996). Randomized controlled trial of effect of high-impact exercise on selected risk factors for osteoporotic fractures. Lancet 348, 1343-1347. 119. Talmage, R., Stinnett, S. S., Landwehr, J. T., Vincent, L. M., and McCartney, W. H. (1986). Age related loss of bone mineral density in non-athletic and athletic women. Bone Miner. 1, 115-125. 120. Pruitt, L. A., Jackson, R. D., Bartells, R. L., and Lehnhard, H. J. (1992). Weight-training effects on bone mineral density in early postmenopausal women. J. Bone Miner. Res. 7, 179-195. 121. Tuukkanen, J., Peng, Z., and Vaananen, H. K. (1991). Effect of exercise on osteoporosis induced by ovariectomy in rats. Calcif Tissue Int. 49, Suppl., $80. 122. Barengolts, E. B., Curry, D. J., Bapna, M. S., and Kukreja, S. C. (1991). Effects of exercise on bone loss in sham-operated or oophorectomized rats. J. Bone Miner. Res. 6, S 105. 123. Barengolts, E. B., Curry, D. J., Bapna, M. S., and Kukreja, S. C. (1993). Effects of two non-endurance exercise protocols on established bone loss in ovarietomized adult rats. Calcif Tissue Int. 52, 239-243. 124. Notelovitz, M., Martin, D., and Tesar, R. (1991). Estrogen therapy and variable-resistance weight training increase bone mineral in surgically menopausal women. J. Bone Miner. Res. 6, 583-590. 125. Pauwels, R. (1965). "Gesammelte Abhandlungen zur Funktionellen Anatomie des Bewegungsapparates." Springer, Berlin. 126. Lu, T. W., Taylor, S. J., O'Connor, J. J., and Walker, E S. (1997). Influence of muscle activity on the forces in the femur: An in vivo study. J. Biomech. 30, 1101-1106. 127. Snow-Harter, C., Bouxsein, M., Lewis, B., Charette, S., Weinstein, E,

508 and Marcus, R. (1990). Muscle strength as a predictor of bone mineral density in young women. J. Bone Miner. Res. 5, 589-595. 128. Villa, M. L., Marcus, R., Delay, R. R., and Kelsey, J. L. (1995). Factors contributing to skeletal health of postmenopausal MexicanAmerican women. J. Bone Miner. Res. 10, 1233-1242. 129. Sievanen, H., Heinonen, A., and Kamus, E (1996). Adaptation of bone to altered loading environment: A biomechanical approach using x-ray absorptiometric data from the patella of a young woman. Bone 1,55-59.

STERNFELD AND MARCUS 130. Kallman, D. A., Plato, C. C., and Tobin, J. D. (1990). The role of muscle loss in the age-related decline of grip strength: Cross-sectional and longitudinal perspectives. J. Gerontol. 45, M82-M88. 131. Taaffe, D. R., Villa, M. L., Delay, R., and Marcus, R. (1995). Maximal muscle strength of elderly women is not influenced by oestrogen status. Age Ageing 24, 329-333. 132. Whipple, R. H., Wolfson, L. I., and Amerman, E M. (1987). The relationship of knee and ankle weakness to falls in nursin ghome residents: An isokinetic study. J. Am. Geriatr. Soc. 35, 13-20.

~ H A P T E R 3.

Estrogens and Osteoporosis F. S . J. K E A T I N G , N . M A N A S S I E V , A N D J. C . S T E V E N S O N Endocrinology and Metabolic Medicine, Imperial College School of Medicine, St. Mary's Hospital Medical School, London W2 1PG, United Kingdom

I. Magnitude of the Disease II. Definition of Osteoporosis III. Bone Mineral Density

IV. Bone Remodeling V. Treatment of Postmenopausal Osteoporosis References

I. M A G N I T U D E OF T H E D I S E A S E

able cost of osteoporosis in the United States may be as high as $10 billion annually [3], and this figure does not include costs of long term domiciliary care. Indeed, the growth in the elderly population of the United States is already exceeding the figures predicted, and if this trend were to continue, a tripling of the incidence of hip fracture may occur by 2040 [4,5]. A similar aging of the population is predicted in underdeveloped countries as infant and child mortality rates improve, and the consequent rise in osteoporosis incidence that will eventually ensue may impose an overwhelming financial burden on the health care systems of these countries. In this chapter we define osteoporosis, discuss the critical process of bone remodeling in the pathogenesis of this condition, and describe what is currently understood about preventive and treatment strategies. In addition to describing the effects of hormone replacement therapy, we also consider the effects of other hormones and hormonelike agents such as raloxifene, tibolone, and calcitonin, as well as other pharmacologic approaches.

Although previously underestimated as a major public health concern, osteoporosis is now recognized as one of the most important diseases facing women (and to some lesser extent men) of advancing age, eventually affecting about 50% of the elderly female population of the western world. Postmenopausal osteoporosis accounts for the majority of incidences of the disease, but, although it is beyond the scope of this chapter, it should be remembered that secondary causes of the disease account for a significant number of new cases of fracture each year. The extent of osteoporosis is highlighted by statistics (extrapolated from epidemiological studies) showing approximately 1.5 million fractures oc, curring each year in white women as a consequence of the disease in the United States [1]. Of these fractures, around 700,000 are vertebral fractures, 250,000 are hip fractures, the same number are wrist fractures, and 300,000 involve other sites. A 50-year-old white woman has a 15.6% lifetime risk of symptomatic vertebral body fracture, and the corresponding values for hip and wrist fractures are 17.5 and 16%, respectively, with a 39.7% lifetime risk of fractures at any of these three sites [2]. If the values for asymptomatic vertebral fractures, and fragility fractures at other sites are added, the size of the problem becomes apparent. Moreover, the situation is only likely to worsen as the population of the developed world continues to age. Already, the directly attributMENOPAUSE: BIOLOGY AND PATHOBIOLOGY

II. D E F I N I T I O N OF O S T E O P O R O S I S Osteoporosis has been defined as "a disease characterized by low bone mass and microarchitectural deterioration of bone tissue, leading to enhanced bone fragility and a consequent increase in fracture risk" [6]. Osteoporosis (porous 509

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bone) implies a histological diagnosis of reduced bone tissue and deranged architecture, but normal mineralization; however, the clinical diagnosis of the disease has been made traditionally on the occurrence of fragility fractures, principally at the femoral neck, the spine, and the distal forearm. The main obstacle to such a fracture-based diagnosis was that a patient previously described as normal became immediately osteoporotic on the occurrence of a fragility fracture, and this was inconsistent with our knowledge of the natural history of the disease. Such an approach also led inevitably to a delay in diagnosis of disease until "end stage" was reached, and opportunity was wasted to prevent the occurrence of the fracture in the first place. To overcome this dilemma in diagnosis, attempts have been made to define osteoporosis as a disease diagnosed at a certain low level of bone density, in the same way that hypertension as a disease is defined beyond a set level of blood pressure. It follows that for such a diagnosis to be viable, accurate, noninvasive, and reproducible methods of measuring bone density must be available. The advent of dualenergy X-ray absorptiometry fulfilled these criteria at minimal radiation exposure, and is currently the most prevalent method of bone densitometry in the United Kingdom. Although much debate continues as to the correct level of bone mineral density (BMD) below which to assign a diagnosis of osteoporosis, the World Health Organization (WHO) proposed a stratified classification of osteoporosis in 1994 to encompass both systems of diagnosis, and this is summarized in Table I [7]. Thus an individual would be termed osteoporotic if their B MD were less than 2.5 standard deviations below the young adult range, regardless of whether a fracture had occurred. A further classification including values of BMD between 1 and 2.5 SD below the young adult mean was also included, and such individuals were termed osteopenic (low bone mass) and said to be at higher risk of future osteoporosis than the normal population.

TABLE I World Health Organization Classification of Osteoporosis a Classification

Description

Normal

Bone mineral density not more than 1 standard deviation below the young adult mean

Osteopenia

Bone mineral density between 1 and 2.5 standard deviations below the young adult mean

Osteoporosis

Bone mineral density more than 2.5 standard deviations below the young adult mean

Established (severe) osteoporosis

Bone mineral density more than 2.5 standard deviations below the young adult mean in the presence of one or more fragility fractures

a From W H O data, 1994 [7].

III. BONE MINERAL DENSITY A. L o w B o n e D e n s i t y a n d F r a g i l i t y F r a c t u r e s Prospective studies have shown that with declining BMD, the risk of fragility fractures increases progressively and continuously [8,9], and fracture risk increases by up to threefold for every standard deviation decrease in BMD [10]. At 2.5 SD below the young adult mean, 30% of postmenopausal women will be identified as having osteoporosis using BMD measurements from the spine, hip, or forearm, and this is roughly the equivalent lifetime risk for fractures at these sites [ 10]. Decreased bone mass is associated with increased fracture risk across all skeletal sites, but is most significant in terms of mortality and morbidity at the hip. There is an almost exponential rise in hip fracture incidence after age 50 years until at least the ninth decade, and incidence rates reach approximately 3% per year among white women over the age of 85 years in northern Europe and the United States [11]. The associated mortality with hip fracture is around 20% at 1 year postfracture, and this often follows an extended period of hospitalization [5]. Morbidity is also high; 10% of women sustaining hip fracture become dependent on others to carry out activities of daily living, and almost 50% depend on long-term institutionalized nursing care [ 12]. Although mortality rates are lower for vertebral and wrist fractures, morbidity is significant. Even asymptomatic vertebral fractures may lead to a reduction in activities of daily living through progressive skeletal deformities such as kyphoscoliosis, and persistent pain may impinge on the patient's life both physically and psychologically. Painful, symptomatic vertebral fractures often necessitate periods of bed rest up to 6 months. Incidence rates for vertebral fractures are more difficult to report due to the large number of subclinical fractures and variation in the methodology employed to assess vertebral deformity, but the same pattern of age-related exponentially increased incidence rate is also seen with vertebral fracture as with hip fracture. One study of a population in the United States showed an annual incidence of 0.5% at age 50 years, rising to 4% at age 85 years [ 1]. However, more data are available concerning the prevalence of vertebral fracture, and one study reported rising age-related prevalence rates reaching a maximum of 20% at age 70 years in a population of 2063 women in Puerto Rico and Michigan [13]. More recent data showed a prevalence of wedge fracture of around 60% in women over 70 years and a prevalence of crush fracture of 10% [14]. Distal forearm fractures also show a steep rise in incidence in women with increasing age, but the rise tends to occur at an earlier age, and by around 60 years of age, the incidence rate begins to level off to around 0.5% of women

CHAPTER 35 Estrogens and Osteoporosis per year [ 15]. It is thought that the classic fall onto the outstretched hand, producing the distal radius fracture, becomes less common with advancing age as decreased proprioceptive and reflex responses diminish the speed of this defensive mechanism. Consequently, more falls occur directly onto the lower limb, which may in part contribute to the increase in hip fracture observed with increasing age. Distal radius fracture accounts for 50,000 hospital admissions per year in the United States and over 6 million restricted activity days in people over 45 years of age [16]. Persistent morbidity is also frequent, with chronic pain, loss of function, neuropathies, and posttraumatic arthritis, and many patients report poor functional outcome at 6 months postfracture [3].

B. Genetic and Environmental Influences on Bone Mass

511 TABLE III Some Candidate Genes for Studying the Genetic Contribution to Osteoporosis a Gene studied Vitamin D receptor

Estrogen receptor

Transforming growth factor/3 Interleukin-6

Collagen type I genes Collagenase

Peak bone mass is reached soon after the end of linear growth, and is largely genetically determined, although environmental factors can influence the eventual final peak bone mass achieved (Table II). Twin studies and family studies of bone mineral density have shown that genetic factors are probably the single most important influence on bone mass and osteoporotic fracture risk. Studies have revealed that up to 7 0 - 8 5 % of the interindividual variation in bone mass is genetically determined [ 17]. Several genes are thought to contribute to the determination of bone mass rather than a single gene or pair of genes contributing major effects. However, at present the exact number and precise function of these genes remain unclear. Candidate gene studies have identified potential contributors to osteoporotic risk, and these are shown in Table III. Much work is still outstanding as to the relative contributions, if at all, of these and other genes, and further elucidation of the human genome will in-

TABLE II

Factors Affecting Bone Mass

Genetic Racial Geographical Environmental In attaining peak bone mass Hormonal Physical activity Diet After peak mass is achieved Smoking Alcohol Physical activity Hormonal Diet

Rationale for study Vitamin D regulates bone cell differentiation, bone turnover, and calcium homeostasisby interaction with the vitamin D receptor Estrogen regulates bone turnover and skeletal growth by interaction with the estrogen receptor TGF-/3 is present in bone matrix and is thought to couple bone resorption and bone formation IL-6 regulates osteoclastdifferentiation and may mediate some actions of sex hormones in bone Type I collagen is the most abundant protein in bone This is a degradativeenzyme involved in resorption of bone matrix

a Data from Ralston [17].

evitably produce further candidate genes that may be shown to contribute to bone density. Other evidence from identical twin studies has shown that variation in bone mass increases with aging, suggesting that bone loss may be more independent of genetic factors [18]. Studies of incidence rates of osteoporotic fractures show that racial and geographical factors are important determinants of fracture risk, with European and American women having generally higher fracture rates than women from developing nations [19]. Within developed nations, racial variations in fracture rates are also observed; for example, black women in the United States display a hip fracture rate approximately one-third that of white American women [20]. Hip fracture rates among black African women are lower still than American black women [19], and this is despite the evidence from the limited number of studies available demonstrating that black African women have similar or slightly less bone density than American whites, and considerably less bone mass than American blacks [21,22]. The above evidence would suggest that within populations, bone density variation is the most influential variable of fracture rates, but that other variables play an important role in the observed differences of fracture rates between countries. Peak bone mass declines from around middle age, although earlier in the proximal femur [23], and both men and women lose about 0 . 3 - 0 . 5 % of their bone mass every year. Again, adverse environmental factors can decrease these losses still further. Women experience an additional accelerated period of bone loss following their menopause, which

512 may be up to 10-fold higher than the premenopausal rate of bone loss, and for the first 5 to 10 years of postmenopausal life, this loss can be even higher still. Consequently, bone mass may fall to about half of its peak value by around 80 years of age. The accelerated rate of bone loss in postmenopausal women results from increased bone turnover due to the effect of estrogen deficiency on the bone remodeling process, as described below.

IV. BONE REMODELING As with almost all adult bone diseases, osteoporosis develops from a disorder of the bone remodeling process. Adult skeletal bone consists of cortical (compact) and trabecular (cancellous) bone, and is continuously repaired and reformed by a process known as remodeling. It appears that bone remodeling is an essential process in land-based vertebrates to maintain the integrity of the skeleton. Cortical bone is dense and forms some 85% of the total body bone, predominantly in the long shafts of the appendicular skeleton. It is laid down concentrically around central canals, Haversian systems, containing blood, lymphatics, nerves, and connective tissue. Trabecular bone, although forming only 15% of the adult bone, is relatively prominent at the ends of long bones and in the inner parts of flat bones. It consists of interconnecting trabeculae interspersed by the bone marrow. In the lumbar spine, as much as 65% of the bone is of the trabecular type. Bone remodeling occurs at discrete sites, known as bone remodeling units, on the bone surface of both trabecular and cortical bone. The process basically involves the removal of mineralized bone and its replacement with newly formed and mineralized osteoid. Central to the process are the actions of osteoclasts and osteoblasts. Osteoclasts, derived from the hematopoietic precursors of the monocyte-macrophage lineage, perform the resorption of mineralized bone by acidification and proteolytic digestion; osteoblasts, descended from bone marrow pluripotent stromal stem cells, are responsible for the formation and subsequent mineralization of bone matrix. Remodeling always follows in a sequential, closely coupled pattern of resorption followed by formation, and the process takes about 3 - 4 months to complete, with resorption occupying the first 10 days or so. Remodeling occurs in discrete packages on both cortical (in the haversian systems) and trabecular bone (on the trabecular surfaces), and at any one time multiple bone remodeling units are at different stages of the bone remodeling process. In the development of osteoporosis, an imbalance exists between resorption and formation, with net bone loss. The most important influences on this are the effects of aging and the loss of gonadal function, as will be discussed below.

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A. B o n e R e s o r p t i o n Bone resorption first involves activation of the quiescent bone surface, with retraction of the lining cells (specialized osteoblasts) and proteolytic digestion of the endosteal membrane, resulting in exposure of the mineralized bone ready for osteoclastic resorption. The underlying mechanisms governing activation are not well understood; however, it seems that matrix metalloproteinases (released from osteoblasts) are involved in the removal of the endosteal membrane. It is also possible that activation is prompted by mechanical stresses acting on specific sites, transmitted via the osteocytic-canalicular network of the bone [24]. Activation is followed by recruitment of the osteoclast precursors to the exposed bone and differentiation of these precursors into the mature osteoclast. A series of steps achieves this, commencing with fusion of the precursors into a multinucleated cell. It has been demonstrated that osteoclastic bone resorption is more effective the higher the number of nuclei within the cell [25]. Mbalaviele and co-workers have shown that osteoclastic fusion may occur in a fashion similar to trophoblastic cell fusion in the placenta utilizing E-cadherin, a hemophilic calcium-dependent cell attachment molecule [26,27]. Following fusion of the precursors, attachment to the bone surfaces occurs via integrins on the osteoclast cell membrane, which bind to bone matrix molecules through specific RGD (ArgGly-Asp) sequences [28]. The osteoclast then undergoes polarization with realignment of the intracellular organelles, and the development of a ruffled border, a specialized area of the cell membrane in proximity to the bone surface, associated with the expression of the vacuolar ATPase proton pump, the SRC protooncogene, and proteolytic enzymes required in the resorptive process. Following resorption, osteoclastic action is terminated by cell apoptosis, characterized by the morphological changes of nuclear chromatin condensation, and separation of the osteoclast from the bone surface. This apoptosis may be the common end pathway in the action of both estrogens and bisphosphonates on bone, and Parfitt has linked the excessive osteoclastic activity associated with estrogen deficiency to the absence of an apoptosis stimulus [29].

B. B o n e F o r m a t i o n Bone formation commences with chemotaxis of osteoblasts and their precursors to the site of the resorptive defect, and it is thought that this occurs as a result of the action of local factors released by the resorbing bone. In vitro studies by Mundy and co-workers demonstrated that cells with osteoblastic properties were chemotactically attracted by local factors produced by the resorbing bone [30,31 ]. Transforming growth factor/3 (TGF-/3) has been demonstrated to be

CHAPTER35 Estrogens and Osteoporosis chemotactic for bone cells [32] and is released from resorbing bone. Platelet-derived growth factors (PDGFs) are also possible mediators in this process, and have been demonstrated to be chemotactic for certain mesenchymal cells, monocytes, neutrophils, and smooth muscle cells [25]. It is also possible that structural proteins such as collagen and bone gla protein act as chemotactic stimulants to osteoblastic congregation at resorptive sites [30,31 ]. Following chemotaxis, the osteoblasts undergo proliferation, and it is likely that growth factors such as the TGF-fl superfamily, PDGF, insulin growth factors I and II (IGF-I and -II), and fibroblast growth factors (FGFs) are mediators in this process. The precursors then undergo differentiation into mature osteoblasts. Bone-derived growth factors such as IGF-I and bone morphogenetic protein 2 (BMP-2) have been suggested as mediators in this stage, and interestingly, TGF/3 may actually inhibit osteoblast differentiation, suggesting that it acts as a trigger early in the process of osteoblast recruitment and proliferation, but must be removed or inactivated prior to differentiation of the osteoblasts. Differentiated osteoblasts subsequently lay down the new osteoid in the resorption cavity, which is then mineralized. Cessation of osteoblast activity is the final stage of bone remodeling, and again local factors, possibly TGF-fl, are central to this process.

513 TABLE IV Systemic Hormones and Local Factors Involved in the Control of Bone Remodeling Systemic hormones

Parathyroid hormone Triiodothyronine Growth hormone Glucocorticoids 1,25-DihydroxyvitaminD Sex steroids Cytokines and growth factors Bone formationstimulators Transforming growth factor/3 Insulin-like growth factors Platelet-derivedgrowth factors Fibroblast growth factors Bone morphogeneticproteins Bone resorption stimulators Interleukins 1, 6, 8, and 11 Platelet-derivedgrowth factors Macrophage colony-stimulatingfactor Granulocyte/macrophagecolony-stimulatingfactor Epidermal growth factor Tumour necrosis factor Fibroblast growth factors Leukemiainhibitory factor Bone resorption inhibitors Interferon-y Interleukin-4

C. Control of Remodeling A complex interrelationship between systemic hormones, mechanical stresses, and locally derived cytokines, prostaglandins, and growth factors is responsible for the control of bone remodeling; the major compounds involved in this control are shown in Table IV. Adults show annual resorption and reformation of approximately 25% of their trabecular bone, but only around 3% of their cortical bone. Trabecular bone has a higher surface-to-volume ratio, and up to 85% of the surface of trabecular bone is in contact with the bone marrow. It thus follows that control of remodeling is primarily under local control [33]. Local control of remodeling is primarily effected by a large number of cytokines and growth factors, some of which can exert influence on both osteoblasts and osteoclasts, and many of which act interdependently. For example, the precursor of mature osteoblasts, the stromal-osteoblastic precursor cell, is stimulated both by local factors, such as interleukin (IL)-1 and tumour necrosis factor (TNF), and by systemic hormones, such as parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D3, to produce in turn cytokines and growth factors, in particular IL-6 and IL-11, and these cytokines regulate the differentiation pathway of preosteoclasts. Indeed, the list of cytokines and colony-stimulating factors involved in the development of osteoclasts, and therefore stimulation of their action, is

large and includes IL-1,-3,-6, and- 11, TNF, granulocytemacrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), leukemia inhibitory factor, and stem cell factor [33]. It seems that IL-6 may have a pivotal role in the osteoclastogenic process, in that it has been implicated in the stimulation of differentiation of the granulocyte-macrophage colony-forming units (GMCFUs) into osteoclast precursors [34], also stimulates osteoclast formation, and in combination with IL-1, stimulates bone resorption [33]. It is also possible that IL-6 can stimulate mature osteoclasts after receptors for IL-6 were demonstrated on human osteoclastoma cells, which increased the resorptive activity of these cells [35]. As observed previously, it has been shown that normal remodeling involves close coupling of the resorptive and formative processes. Here again, it is thought that local factors are primarily involved in the regulation of this coupling. In particular, the TGF-fl superfamily may be especially important. Bone resorption leads to the release of TGF-fl as already discussed, and this stimulates osteoblast precursor proliferation. The exposure, however, is transient, and subsequently the proliferating cells undergo differentiation into mature osteoblasts and express bone morphogenetic proteins (BMPs), which are autostimulatory on osteoblasts, propagating the formation of mineralized bone.

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V. T R E A T M E N T POSTMENOPAUSAL

OF OSTEOPOROSIS

A. E s t r o g e n s In his classic observations of the link between osteoporosis and estrogen deficiency, Albright noted that 40 of 42 women with osteoporotic fractures were postmenopausal [36]. It was first proposed that estrogen deficiency led to a decrease in osteoblast activity and a subsequent decrease in bone mass [37], but this explanation proved to be inadequate when a consistent decline in osteoblast activity was not shown in postmenopausal women [38]. Various studies have showed that the cessation of ovarian function is associated with an increase in bone turnover and a consequent decline in bone mass [39]. The most usual cause of estrogen decline is the onset of menopause, either natural or surgical, and studies across all cultures have demonstrated bone loss. In addition, estrogen decline can result from ovarian failure secondary to anorexia, hyperprolactinemia, pituitary failure, and excessive exercise. Declining estrogen levels lead to an increase in the frequency of activation of remodeling cycles, with a subsequent increased rate of resorption and formation, as shown by the increased levels of markers for bone turnover in the serum and urine (serum alkaline phosphatase and osteocalcin; excretory products of type I collagen, e.g., hydroxyproline or pyridinoline cross-links of collagen) [40]. An increase in the number of osteoclasts in trabecular bone is observed following the loss of ovarian activity. It has also been suggested that osteoclast aggression is increased in the estrogen-deficient state, possibly due to the previously mentioned delay in osteoclast apoptosis at the termination of resorption, resulting in larger resorption cavities that can only be partially filled by osteoblast activity. Furthermore, with increased frequency of remodeling, the likelihood of simultaneous remodeling on opposite sides of trabeculae is increased, with a greater chance of complete transection of trabeculae and a consequent loss of template on which further bone formation can occur, leading to the observed loss of bone mass and architecture characteristic of the disease (see also Chapter 19). Fundamental differences exist between the patterns of bone loss seen with aging and loss of ovarian function. Postmenopausal bone loss is characteristically associated with excessive osteoclastic activity, as discussed above, whereas bone loss associated with aging has been seen to be more related to a progressive decline in the number of osteoblasts available compared to the demand [41]. The type of this bone loss also differs, with trabecular bone more affected in postmenopausal bone loss and cortical bone primarily affected by age-associated bone loss. Although the effects of estrogen deficiency on bone are

well established, it has been difficult to describe the exact role of estrogens in modulating bone remodeling. It is known that pharmacological doses of estrogens suppress the increased bone remodeling rates in postmenopausal women [42-44], and estrogen loss at menopause is associated with an apparent partial release from inhibition of skeletal resorption [45]. Estrogen seems to act both directly and indirectly on bone. Eriksen et al. demonstrated evidence of estrogen acting through the classical estrogen receptor-mediated mechanism on cultured human osteoblast-like cells [46]. Komm et al. showed that estrogen could act, via a direct receptormediated action, on osteoblast-like cells to enhance levels of type I procollagen and transforming growth factor/3 mRNA levels [47], and argued that estrogen thus governed the transcriptional activity of the TGF-fl gene in osteoblasts, which in turn could positively control the transcriptional activity of the type I collagen gene, thus providing a possible explanation for the observed effects of estrogen on bone remodeling. The indirect actions of estrogen on bone are mediated via cytokines and local growth factors. It has been proposed that TNF-a and IL-1 both affect the initial step in estrogen deficiency bone loss. Both are released from the peripheral blood monocytes in excessive amounts in ovariectomized women and act as stimulators of bone resorption. This effect is antagonized by estrogen replacement. Further work on the TNF-ce cytokine has shown that permanent suppression of its activity can lead to protection against the increased bone turnover and bone loss associated with oophorectomy in mice [48]. Further work on cytokines has shown that IL-6 production from bone marrow stromal and osteoblastic cell lines is inhibited by estrogen [49], and that this is mediated by an estrogen receptor-mediated inhibitory effect on the transcription gene for IL-6. Studies on ovariectomized mice showed that administration of 17fl-estradiol prevented the IL-6-mediated increases in osteoclast populations in trabecular bone. It can therefore be proposed that estrogen deficiency leads to in increase in expression of IL-6 and a consequent increased rate of osteoclastogenesis and therefore bone resorption. Furthermore, IL-6-deficient ovariectomized mice do not seem to undergo this bone loss associated with increased osteoclast numbers [33].

B. H o r m o n e R e p l a c e m e n t T h e r a p y Epidemiological and clinical data have consistently shown that hormone replacement therapy (HRT) results in a cessation of bone loss, and an improvement in bone density with a subsequent reduction in risk of osteoporotic fracture [50-63]. Earlier studies tended to use bone density measurements from the distal radius and the metacarpals, and therefore potential difficulty existed in attempting to extrapolate the results of these studies to the hip and spine, areas where

CHAPTER35 Estrogens and Osteoporosis trabecular bone is more prevalent. However, cross-sectional follow-up data from these earlier studies did show that similar effects were noted in the spine and hip [64]. Hormone replacement can now be administered via a number of routes, and these are summarized in Table V. There are several types of estrogens in common use in HRT preparations, and the most commonly used are conjugated equine estrogens, estradiol valerate, 17/3-estradiol, and estrone sulfate. Studies have elucidated the optimum dosages of these estrogens for bone conservation and the results are summarized in Table VI [65-71]. A higher dosage may be required for women who have undergone a premature menopause either due to surgery or for medical reasons, and more elderly women embarking on HRT may conserve bone on lower doses than listed, with the added benefit of minimizing the estrogenic side effects common in this group of patients. Unopposed estrogen use is advocated for women having undergone total hysterectomy, but additional progestogens are usually prescribed for women with an intact uterus, to avoid the potential for endometrial hyperplasia and carcinoma. The addition of progestogen, either sequentially for 12 days each month, or as "bleed-free" continuous combined preparations, has been shown to abolish any increased risk of endometrial carcinoma over nonusers of HRT [72]. Both methods of combined HRT have been shown to produce bone-conserving effects, and there is some evidence to suggest that continuous combined HRT has a greater effect than sequential HRT [73,74]. Progestogens derived from 19norethisterone have been shown to be effective in reducing bone loss [73,75], possibly through their androgenic properties. Less androgenic progestogens, such as medroxyprogesterone acetate, have no effect on bone density. Although there is near unanimity among researchers on the beneficial effects of estrogen on bone density, far more debate exists regarding the timing and duration of treatment. HRT is not well tolerated in a substantial number of women, and much attention has been drawn to the potential adverse effects of therapy, namely, the possible increased risk of breast cancer and venous thromboembolism. Hormone replacement is often commenced in the perimenopausal period, when symptomatic relief is often the most pressing

TABLE V

Estrogen Replacement

Route

Preparation

Oral

Tablets

Transdermal

Patches, gels

Vaginal

Creams/gels, rings, pessaries

Subcutaneous

Implants

Intramuscular Intranasal

515 TABLE VI Commonly Used Estrogens and Standard Dosages Required to Preserve Bone Density Estrogen

Bone-conserving dose

Ref.

Conjugated oral estrogens

0.625 mg daily

65

Oral estradiol valerate

1-2 mg daily

66

Oral 17/3-estradiol

1-2 mg daily

67

Transdermal 17/3-estradiol

50/xg daily

58

Percutaneous 17/3-estradiol

1.5 mg daily

68

Subcutaneous 17/3-estradiol

25-50 mg every 6 months

69-71

indication for treatment, at least in the perception of the women affected. The duration of therapy has often been short term, although most clinicians now advocate use for several years following the natural cessation of ovarian function, and longer in the case of a surgically induced menopause. Although many clinicians believe that a reduction in fracture risk has been shown in epidemiological studies of estrogen use in these younger women, a study from the Framingham cohort suggested that the initial benefit of HRT on bone density had waned in women over the age of 75 who had used HRT in the immediate postmenopausal period, and that estrogen therapy in the first decade following the menopause could not be reliably expected to protect against osteoporotic fractures many years later [50]. However, there are major concerns regarding these conclusions. High bone density is associated with longevity, and thus the nonuser group of these elderly women would represent a biased group in terms of bone density. Because estrogen is associated with a reduction in all-cause mortality, the users could include those with low bone density who would survive into old age because of their treatment. Two further studies showed that current users of estrogen had a lower risk of osteoporotic hip fracture than past users of estrogen when both groups were compared with "never" users [51,53]. The above evidence raises questions regarding the optimum timing and duration of treatment with HRT. One effective regimen would appear to be lifelong treatment commencing at the time of menopause, which would be expected to reduce the risk of fracture in women of 80 years of age by about two-thirds over never-users of HRT [76]. This, however, may be an entirely unacceptable approach to women due to adverse effects of treatment, and a possible increasing risk of breast cancer development with duration of therapy. Other proposed strategies have included commencing estrogen treatment following osteoporotic fracture, or starting many years following the menopause, when women are at high risk of fracture. Both strategies can be justified in terms of published evidence, but carry obvious drawbacks with respect to the acceptability of commencing HRT at a late age and the increased incidence of estrogenic side effects, such as breast tenderness, in this age group. Using very low doses

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of estrogen, initially and gradually increasing the dose if necessary, may circumvent this latter problem. In all probability, the optimum decision regarding duration and timing of treatment with HRT will continue to be driven by the patients' symptomatology and the desire to prevent osteoporosis and heart disease, and will be tempered by the acceptability and associated risks of long-term treatment.

C. S e l e c t i v e E s t r o g e n R e c e p t o r M o d u l a t o r s A search for safer and more acceptable alternatives to HRT led to the evaluation of several compounds, grouped together as "antiestrogens." Initially, these compounds were used in the treatment of hormonally responsive cancers, such as breast cancer. However, antiestrogens have a wide spectrum of activity, from being pure estrogen antagonists to mixed agonists/antagonists. The most familiar are the triphenylethylenes, such as clomiphene, tamoxifen, 3-hydrotamoxifen (toremifene), 4-iodopyrrolidintamoxifen (droloxifene), and TAT-59. Clomiphene and tamoxifen are in widespread clinical use, and toremifene (Fareston) has been approved for the treatment of advanced breast cancer. Other agents with mixed agonist/antagonist properties include benzothiophenes, such as raloxifene (formerly keoxofene), and structurally distinct molecule such as ormeloxifene (centchroman). ICI 182 780 has been found to be a pure estrogen antagonist and is now under clinical investigation for the treatment of tamoxifen-resistant breast cancer. The term "selective estrogen receptor modulator" (SERM) was proposed to define more precisely those compounds that can bind to, and activate, estrogen receptors (ERs) but that have effects on target tissues that may vary from those of estradiol [77]. As currently used, SERM classification includes all compounds previously described, excluding pure antiestrogens, such as ICI 182 780. Research activity increased markedly following the discovery that tamoxifen-treated postmenopausal women with breast cancer had lower cholesterol and more preserved bone mass compared with untreated control patients. In order to understand the mechanism of action of SERMs, a brief review of the action of steroid hormones at the cellular level is necessary. Free steroids are thought to diffuse passively to all cells because there is no evidence as yet of an active transport mechanism. Steroids are preferentially retained in target cells as stable complexes with intracellular receptor proteins (i.e., estrogen receptors) that are steroid and tissue specific. The receptor is thought to be a ligand-activated transcription factor, the steroid being the ligand. The ER has six structural domains of overlapping functions, labeled A through F. Binding of the receptor by the steroid results in activation of the receptor molecules, which leads to conformational changes in the receptorligand complex. This activation process allows the receptor-

ligand complex to bind to specific sites on DNA, termed nuclear acceptor sites. Once bound to the nuclear acceptor sites, the activated steroid-receptor complex acts as a transcription factor. This leads to specific mRNA and protein synthesis. The newly synthesized protein leaves the cell and exhibits its effects on other cells and on the metabolism in general. Transfer of the steroid in the cell and nuclear binding of the steroid-receptor complex are rapid, occurring within minutes. Following nuclear binding, mRNA synthesis occurs within several hours, and finally protein synthesis and turnover are seen within 12-24 hr. The major physiological effects of steroids in cells are seen within 12-36 hr. During puberty and reproductive life estrogen exerts its effect via the mechanism just described on all cells and tissues susceptible to its action, including the reproductive system, breasts, cardiovascular system, central nervous system, skeleton, skin, and connective tissues. After the menopause, estrogen deficiency leads to involutional changes at these sites. Estrogen replacement, as HRT, reverses these changes, but acts indiscriminately on all estrogen-sensitive tissue. In postmenopausal women, not all of the effects of estrogen are desirable or acceptable. The concept of selective estrogen receptor modulation is that estrogen action on certain receptors is retained (cardiovascular system, the skeleton, central nervous system, skin, connective tissue, and lower reproductive and urinary tracts), leaving receptors in the breasts and the uterus inactive. Intensive research is currently aimed at understanding the mechanism of the selectivity of estrogen agonist and estrogen antagonist behavior by SERMs. The estrogen receptor lies at the center of this mechanism. Emerging evidence from several groups indicates the presence of multiple transcription pathways for ligand-bound estrogen receptors and the existence of multiple estrogen receptor subtypes. The estrogen receptor contains multiple transcription-activating functions (e.g., AF-1 and AF-2) that account for some of the tissue-selective effects of SERMs. At the cellular and molecular level, antiestrogens can act in several ways. Some antiestrogen compounds bind the estrogen receptor but fail to activate it; some activate the estrogen receptor, and the complex binds to the acceptor site but fails to regulate fully gene transcription or expression. There may also be alternative pathways different from the classical one through which drugs may initiate cellular response. It appears that at least some SERMs may act by binding to the cell DNA without the DNA-binding domain of the estrogen-receptor complex [78]. EFFECTS OF S E R M s ON POSTMENOPAUSAL

OSTEOPOROSIS Tamoxifen is a nonsteroidal estrogen receptor antagonist/ agonist widely used for palliative and adjuvant treatment of breast cancer. Preliminary clinical studies demonstrated the efficacy of tamoxifen in the treatment of advanced breast cancer [79,80]. It was originally felt that the antiestrogenic

CHAPTER 35 Estrogens and Osteoporosis effect of tamoxifen would lead to bone loss but subsequent work demonstrated that tamoxifen did not appear to have an antiestrogenic effect on bone [81]. The effect of tamoxifen on bone depends on the menopausal status of the woman. In postmenopausal women 2 0 - 4 0 mg of tamoxifen administered daily led to bone preservation or increased bone density of between 0.61 and 1.71% at 2 years [82,83]. In premenopausal women slight bone loss was shown in one study [84]. The bone-preserving effects of long-term tamoxifen treatment (20 mg daily) are likely to be at best only modest. At the end of one 5-year study, tamoxifen-treated patients had a bone density 2% above base line, whereas untreated patients had a 2.7% loss compared to base line [85]. Another study found no increase in bone mineral density in postmenopausal women treated with 40 mg of tamoxifen for 7 years, in comparison to the base line [86]. In spite of being a bonepreserving agent in one observational study, it was found that tamoxifen does not appear to offer protection against fracture, and may even increase the risk [87]. Unfortunately, in addition to general side effects such as flushing, tamoxifen's usefulness as a postmenopausal treatment for preserving bone density would be severely limited by its estrogen a g o nist effect on the endometrium, where increased incidences of hyperplasia and carcinoma have been observed. Another SERM, raloxifene, was also investigated as a potential therapy for breast cancer. In vitro studies demonstrated the ability of raloxifene to prevent estrogen binding to the ER; it exhibited antitumor activity in carcinogeninduced rodents [88], and also in estradiol-dependent proliferation of MCF-7 breast cancer cells [89]. However, it demonstrated a lack of antitumor effect in a small trial in postmenopausal women with breast cancer resistant to tamoxifen [90]. Through a number of animal studies, it became evident that raloxifene has a SERM profile distinct from that of tamoxifen. Most animal studies were performed on a rat model. Raloxifene preserved both bone tissue in ovariectomized rats and the biomechanical properties of the bone (load-to-fracture ratio in the vertebra and shear-tofailure ratio in the femoral neck) [91 ]. Raloxifene is primarily an antiresorptive drug. Like estrogen, it reduces bone remodeling in estrogen-deficient early postmenopausal women and induces a positive shift in calcium balance. In one study 60 mg/day of raloxifene was compared with 0.625 mg/day of conjugated equine estrogen (CEE) and 5 mg/day of medroxyprogesterone acetate (MPA) from day 1 to day 14. Raloxifene resulted in improved intestinal calcium absorption, a fall in urinary calcium excretion, and reduced bone resorption [92]. Results from the largest clinical study to date were reported by Delmas et al. [93]. This prospective, randomized study involved 601 postmenopausal women. The study subjects were given 60 mg of raloxifene for 24 months. At this dose raloxifene led to an increase of 2.0-2.4% of bone mineral density in the spine and hip, with no effect on endometrial thickness. Raloxifene was

517 well tolerated with no significant difference in the adverse events between groups. However, the withdrawal rate in the study was high at 25%, and when measured as absolute gain in B MD rather than gain over placebo, the increase in B MD was less dramatic. However, it may be that reduction in bone turnover, rather than simple increase in density, is important for fracture prevention by reducing the likelihood of trabecular perforation. Preliminary data have shown that raloxifene reduces the incidence of new vertebral fractures, particularly in women with preexisting established osteoporosis [94]. Raloxifene appears to be well tolerated. Compared to placebo, women taking raloxifene experience more hot flushes and muscle cramps. The incidence of venous thromboembolism is increased two- to threefold, similar to HRT-treated patients, but importantly, raloxifene has no effect on the endometrium, acting as an antiestrogen on this tissue. The development of raloxifene and other SERMs has been a major recent advance in the prevention of postmenopausal osteoporosis and holds much promise for the future.

D. P h y t o e s t r o g e n s a n d I s o f l a v o n e s Isoflavones are naturally occurring plant chemicals that are currently receiving attention as potential alternative therapies for a range of hormone-dependent conditions, including cancer, coronary heart disease, osteoporosis, and the menopause. Isoflavones are present in legumes, but red clover and soya products contain the highest concentrations among foods so far analyzed. There are plausible mechanisms to explain the potential health benefits of isoflavones. Because dietary isoflavones are structurally similar to estradiol but have 100-1000 times weaker estrogen activity, they have the capacity to bind to the estrogen receptor and exert partial estrogen agonist/antagonist effects, which may be tissue specific. At certain concentrations, which may depend on many factors, including receptor numbers, extent of protein mimics, and ability to initiate estrogen-like actions, isoflavones may antagonize and inhibit estrogen action. This tissue-selective estrogen action is currently driving pharmacologists to develop selective estrogen receptor modulators (SERMs). If dietary isoflavones can be shown to exert tissueselective effects, these compounds would therefore have the potential to act as estrogen agonists, which may prove beneficial in postmenopausal women with respect to risk factors for heart disease, menopausal symptoms, and osteoporosis. However, under some conditions, these compounds may act as antiestrogens, which may assist in preventing the development of breast cancer. The main isoflavone compounds are biochanin, formononetin, daidzein, genistein, and equol. When ingested, these compounds are metabolized, absorbed, and, like estrogens, undergo first-pass metabolism in the enterohepatic circulation.

518 Phytoestrogens are effective in decreasing the severity of hot flushes in postmenopausal women. Dietary supplementation with 45 g of soya or wheat flower for 12 weeks significantly reduced the number and the severity of hot flushes, the reduction being more pronounced in the soy group [95]. A larger randomized controlled trial investigated the effect of 60 g of isolated soy protein or 60 g of placebo (casein) daily in 104 postmenopausal women [96]. The 60 g of isolated soy protein contained 40 g of proteins and 76 mg of isoflavones. The 60 g of casein contained 40 g of protein but no isoflavones. At the end of the 12-week study period there was a 45% reduction in the number of hot flushes in the soy group and a 30% reduction in the placebo group, which reached statistical significance. Unfortunately, there was a high withdrawal rate, largely due to gastrointestinal side effects such as constipation, bloating, nausea, and vomiting. Soy flour supplementation (45 g daily) or linseed (25 g daily) but not red clover sprout (10 g dry seed daily) given for 6 weeks to postmenopausal women led to vaginal cell maturation with an increase in the maturation index [97]. These studies do suggest that phytoestrogens may be beneficial to the health of postmenopausal women. A synthetic isoflavone called ipriflavone has been developed. Its effective dose is 600 mg daily in three divided doses. Inhibition of bone resorption has been demonstrated in several models both in vitro and in vivo. It is possible that the effect of ipriflavone on the bone may be due to the inhibition of recruitment and/or differentiation of preosteoclasts [98]. Clinical randomized controlled studies evaluating the effect of ipriflavone on bone have been reported. Ipriflavone given to early postmenopausal women aged 4 0 - 4 9 years led to a 1.1% improvement in the bone density of the lumbar spine after 12 months of treatment and a 1.2% improvement after 24 months. There was a 1.8% bone loss in the placebo group after 12 months and a 3.7% loss after 24 months [99]. No changes in hot flush frequency, vaginal cytology, or osteocalcin level were observed and there was a reduction in urinary hydroxyproline/creatinine ratio in the group receiving ipriflavone. A larger study involving 193 postmenopausal women aged between 50 and 65 years of age with lumbar BMD less than 1 SD below the mean for aged-matched healthy young women (Z score) was conducted and women were randomized to 600 mg ipriflavone/day or placebo and followed for 24 months. Lumbar BMD, serum skeletal alkaline phosphatase, serum osteocalcin, urinary excretion of calcium/creatinine, and hydroxyproline/creatinine ratio were measured every 6 months. BMD was measured with dual-energy X-ray absorptiometry (DXA) scanning with a coefficient of variation of 0.5% using a calibration phantom and < 1 % in vivo. There was a 1% increase in the lumbar BMD in the ipriflavone group and 0.7% decrease in the placebo group. The differences were statistically significant but should be interpreted with caution, bearing in mind the coefficient of variation of the equipment [100]. Ipriflavone

KEATING ET AL.

has been found to be effective in treatment of established postmenopausal osteoporosis. Given in the usual dosage of 600 mg a day for 2 years it led to improvement in the radial B MD by 4% [101]. In all the studies ipriflavone was well tolerated. The commonest side effects were gastrointestinal, followed by skin reactions and CNS symptoms such as headache, depression, and drowsiness. There is currently much research interest in phytoestrogens and ipriflavone, probably prompted by consumer interest in more "natural" alternatives to conventional menopausal treatments.

E. C a l c i t o n i n Calcitonin is a peptide hormone that acts as a physiological antagonist to parathyroid hormone. Calcitonin is produced by the parafollicular (C cells) in the thyroid gland. Naturally occurring calcitonin is a 32-amino acid peptide, but there is considerable variability in sequence among species. Calcitonin from salmon is 10-100 times more potent than mammalian forms in lowering serum calcium in animals. The secretion of calcitonin, which has a circulating halflife of 2-15 min, is influenced directly by blood calcium levels, with an increase in calcium causing an increase of calcitonin secretion, leading to a hypocalcemic effect in situations of high bone turnover. Calcitonin acts by inhibiting osteoclast-mediated bone resorption and also by stimulating renal calcium clearance. It also reduces the number of active osteoclasts and the rate at which new osteoclasts are formed. Receptors for calcitonin are found on osteoclasts [102-103] and renal tubular cells. Other calcitonin-binding receptors are thought to be present in the brain, gastrointestinal tract, and immune system, and the presence of these may help to explain the analgesic effects exerted by the hormone directly on cells in the hypothalamus and related structures. Human calcitonin is not in general clinical use at present. Salmon, eel, and pork calcitonin are used instead. Salmon calcitonin (salcatonin) differs from human calcitonin in 16 amino acid residues. It is 4 0 - 5 0 times more potent and has a longer circulating half-life than human calcitonin. Salcatonin is available in injectable or intranasal formulations [104]. Following intranasal administration, peak plasma concentrations are seen 3 0 - 4 0 min later, compared to 1525 min after intravenous or subcutaneous administration. Intranasal administration provides approximately 25-50% of the biological activity of the same dose administered through injection. The elimination half-life of salcatonin is around 4 5 - 9 0 min. The administration of salcatonin may cause nausea, vomiting, facial flushing, tingling of the hands, and an unpleasant taste. Rarely, serious allergic reactions may occur, including bronchospasm, angiodema, or even anaphylactic shock. Nasal irritation may also occur. Approximately 4 0 - 7 0 % of patients receiving intranasal or parenteral salca-

CHAPTER35 Estrogens and Osteoporosis tonin therapy for more than 6 months will develop specific antibodies. However, these antibodies do not appear to decrease the effect of salcatonin on bone [ 105]. The precise role of calcitonin in bone loss is not entirely clear. Levels are elevated during growth, pregnancy, and lactation, suggesting that a physiological role of calcitonin is prevention of unwanted bone resorption [106]. The role of endogenous secretion of calcitonin in postmenopausal osteoporosis is also not fully established. Plasma calcitonin is lower in women than in men [ 107], and lower in women with osteoporosis [108,109], although not all studies have found this [ 110]. However, pharmacological doses of calcitonin directly inhibit osteoclast activity. THERAPEUTIC USE OF CALCITONIN IN POSTMENOPAUSAL WOMEN

Calcitonin given for 1 to 2 years to early postmenopausal women, within 5 years of their final menstrual period, resulted in an increase in BMD of the lumbar spine. Doses of 50 IU daily given by subcutaneous injection led to an increase in vertebral BMD [ 111 ]. Intranasal calcitonin appears to require a minimum dose of 200 IU daily [ 112,113]. The effect on vertebral BMD is greater in older women, and little effect is seen on the proximal femur [113]. Older women with established osteoporosis showed similar results, and decreased vertebral fracture rates, which varied between 3 5 - 6 0 % after 24 months of treatment [114,115]. A recent meta-analysis reviewed 18 clinical trials involving calcitonin [ 116]. The pooled positive change was found to be 1.97% in vertebral BMD and 0.32% in proximal femur BMD, with an aggregated number of vertebral fractures prevented by the treatment in the order of 59.2 per 1000 patient-years. However, calcitonin was not shown to be effective in the prevention of bone loss in one study of 40-year-old or older perimenopausal women, with irregular menstrual cycles [117].

E Tibolone Tibolone (Livial, Org OD 14) is a synthetic molecule that has been used to treat vasomotor symptoms of the menopause. It is structurally related to norethisterone and possesses a combination of estrogenic, progestogenic, and androgenic activities. In animal studies it was shown to have 1-10% of the estrogenic activity of ethinyl estradiol, 12% of the progestogenic activity of norethisterone, and 2% of the androgenic activity of methyl testosterone [ 118]. Tibolone is effective in treating vasomotor symptoms [ 118]. It has been shown that tibolone is effective in preventing the accelerated bone loss in early postmenopausal women [ 119] and oophorectomized women [120,121]. Tibolone is effective in the treatment of established osteoporosis. A 2-year study of tibolone treatment in a group of women with osteoporosis and a mean age of 69 years led to an 8% increase in lumbar bone

519 density [122]. Similarly positive results were seen in a subsequent recent study, which also revealed that a lower dose of 1.25 mg/day was as effective as the standard dose of 2.5 mg/day in the preservation of bone density [123]. Tibolone is safe and well tolerated [123], and its sideeffect profile is similar to HRT, with the most frequently reported symptoms being vaginal bleeding and discharge, pruritis vulvae, breast tenderness, acne, and hirsutism. Other side effects include weight changes, ankle edema, headache, and visual disturbances. Endometrial atrophy occurs in 90% of users [ 124], but should breakthrough bleeding occur after 3 months of treatment, this would merit investigation [ 125]. The metabolic effects of tibolone have also been studied, and administration led to a decrease in total cholesterol, highand low-density lipoproteins, triglycerides, apolipoproteins A1 and B, and lipoprotein (a) [126,127]. Coagulation and fibrinolysis were also favorably affected, with decreased fibrinogen and PAI-1 and increased plasminogen levels [128]. Its long-term effects on cardiovascular disease development are unknown, as are any effects on other tissues, such as brain and breast.

G. Bisphosphonates 1. PHYSIOLOGY

Bisphosphonates are stable analogs of pyrophosphate and share the same properties. Like pyrophosphate, they inhibit the precipitation of calcium phosphate from solutions, inhibit crystal aggregation, and inhibit the dissolution of crystals of hydroxyapatite. Bisphosphonates display a high affinity for hydroxyapatite and bone mineral, and this essentially limits their effect to bone. In a study using autoradiography with tritium-labeled bisphosphonate, the highest concentration of the drug was found on the surface of the bone [ 129]. B isphosphonates were first recognized as inhibitors of bone resorption about 30 years ago [130,131]. They are effective in Paget's disease, hypercalcemia of malignancy, osteolytic bone metastasis, and osteoporosis. Gastrointestinal absorption of bisphosphonates is very low and if taken with, or soon after, even light meals, none will be absorbed. Approximately 20% of the circulating drug is retained by the skeleton [ 132], the rest being rapidly excreted unmodified by the kidney. Bisphosphonates bind strongly with hydroxyapatite crystals in the bone. Their skeletal half-life is long, perhaps up to 10 years or longer with certain bisphosphonates. The duration of biological activity is very long in Paget's disease (months to years) but is shorter in patients with hypercalcemia of malignancy, and even shorter in healthy postmenopausal women or in patients with other metabolic bone disease [133]. Little has been published on toxicity of bisphoshonates. Older studies examining etidronate have revealed little toxicity and no carcinogenicity, mitogenicity, or

5

2

0

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E

A

teratogenicity [134,135]. Reproductive and developmental toxicity studies with alendronic acid and pamidronic acid have been conducted with rats and rabbits [136,137]. No selective developmental toxicity was observed. However, at very high daily doses of pamidronate (10 times higher than the recommended human doses), a low incidence of shortened fetal long bones was noticed, associated with markedly low body weights. These effects are related to the mechanism of action of bisphosphonates. The drug prevents bone resorption in the pregnant animal, thus denying a source of calcium for the growing fetus. In addition, it crosses the placenta, preventing fetal bone modeling. These findings suggest that bisphosphonates should be avoided in pregnancy unless absolutely indicated. 2. M O D E OF A C T I O N

The mode of action of bisphosphonates has been studied intensively and appears to involve several possible mechanisms. At lower doses bisphosphonates bind preferentially in the resorption pits under the osteoclasts, whereas at higher doses they may affect the mineralization associated with osteoblasts [ 138]. The formation (adsorption) of the calcium-bisphosphonate phase on the surface of the bone or enamel changes the bone physicochemical characteristics and can significantly reduce the rate of dissolution of the organic and inorganic substance. This leads to a shallowing of the resorption pits and to a reduced rate of osteoclast migration and formation of new pits. At the cellular level, it appears that bisphosphonates are toxic to osteoclasts, leading to cellular death [139,140]. As osteoclasts digest the bone surface of a bisphosphonate-treated subject, bisphosphonate is released and internalized by the osteoclast [ 141 ]. Once internalized, bisphosphonate may interfere with the cytoskeleton, lead to loss of the ruffled border, or may be directly cytotoxic [ 142]. The microinjection of bisphosphonates into isolated osteoclasts disrupts the cytoskeletal ring of action in polarized, resorbing osteoclasts [143]. Bisphosphonates may also affect enzymes and then signal transduction cascades known to be responsible for osteoclast formation and activity [144]. It is also possible that bisphosphonates affect bone resorption through releasing a resorption inhibition factor of low molecular mass ( < 10,000 Da) from osteoblasts [ 145,146]. 3. ETIDRONATE

Etidronate administered continuously over a period of 6 - 1 2 months decreases bone resorption by 50%. However, new bone formation (bone mineralization) is also reduced. To overcome this problem, etidronate is given cyclically in doses of 400 mg/day for 14 days in every 3 months. Given early after the menopause etidronate preserved bone mineral density in the spine and femoral neck over a 2-year period compared with placebo [147,148]. Serum al-

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kaline phosphotase and osteocalcin were reduced by 12 and 14%, respectively, and the urinary N-telopeptide/creatinine ratio was reduced by 23% in etidronate-treated patients. In another study [149], etidronate increased spinal BMD by 1.8-3.8%. The difference between the treatment and the placebo group was between 3.14 and 5.0% at 2 years. The women were stratified in three groups according to their menopausal status: 1-3, 4 - 5 , or 7-10 years postmenopause. In the femoral neck, the difference between the groups was 1.8% after 2 years, which was statistically significant. Serum osteocalcin decreased by 36 and 25% after 1 and 2 years, respectively, in the treatment group, compared to the base line. Urinary deoxypiridinoline/creatinine was 26.3-22.5% lower at 1 and 2 years, respectively, compared to the base line. A 2-year study performed by Meunier et al. [150] in early postmenopausal women showed preservation of spinal BMD in the study group (+0.6% over the base line), whereas there was 2.3 % loss in the placebo group, the difference being statistically significant. There was no change in the femoral neck in the etidronate group over the base line, but 2% loss in the placebo group, the difference being significant. The results of the studies are consistent with the mechanism of action of antiresorptive agents. The biochemical results are in agreement with this, showing significant suppression of markers of bone remodeling, indicating reductions in bone resorption and bone formation. Cyclical etidronate treatment significantly increased the BMD of the spine in patients with established osteoporosis, and decreased the rate of vertebral fractures, compared with placebo over a 6-year period [ 151 ]. This study confirmed earlier results reporting the effectiveness of etidronate in the treatment of postmenopausal osteoporosis [152-154]. Harris et al. also showed a significant increase in the hip BMD after 3 years of treatment [154], although this may not necessarily translate to lower fracture rates at this site. A more recent case-control study examined the use of etidronate and the risk of nonvertebral fracture in patients with an established diagnosis of osteoporosis and found that women taking cyclical etidronate had a significantly reduced risk of hip fracture (by 34%), compared to controls (relative risk, 0.66) [155]. The incidence of wrist fracture was also lower in the treatment group, but the difference was not statistically significant. Between 5 and 15% of patients do not respond well to cyclical etidronate therapy [156]. However, before categorizing a patient as a nonresponder, the clinician should take into account that it may take 1 year for a response to be observed in the spine and up to 3 years in the hip. Also, due to the limitations of the bone densitometry equipment, at 95% confidence intervals a change in BMD of at least 2.8 times the precision error of the skeletal site measured must occur before one can conclude that a real change in the BMD has occurred.

CHAPTER 35 Estrogens and Osteoporosis Clinical data and experience administering cyclical etidronate have been accumulating for many years. There have been a few reports of osteomalacia (one report per 3.6 million cycles). Long-term treatment does not tend to decrease the bone quality because the incidence and rate of vertebral fractures appears to be lowest in patients with the longest exposure [ 157]. Etidronate administered orally is very well tolerated, with gastrointestinal disturbances in subjects treated no higher than in the placebo group. There have been isolated reports of pseudogout, severe hypocalcaemia, and erythema multiforme. The oral absorption of etidronate is very poor and the medication should be taken on an empty stomach with fasting continued for a further 2 hr. 4. ALENDRONATE Alendronate is 100 times more potent than etidronate. It is poorly absorbed after oral administration, with bioavailability of 0.75%, even when taken after an overnight fast and 2 hr before breakfast. Oral absorption and disposition were linear over a dose range of 5 - 8 0 mg, but there is wide variability both within subjects and between subjects in the absorption. Reducing the interval before food from 2 hr to 30 min reduces the absorption by 40%, but giving the medication even 2 hr after a meal makes absorption negligible [158]. Alendronate is effective in increasing the bone mineral density in the spine and in reducing the incidence of fractures in women with postmenopausal osteoporosis [ 159]. At 3 years, the mean differences in B MD between women receiving 10 mg of alendronate daily and those receiving placebo were 8.8% in the spine and 5.9% in the femoral neck. The increase seen in the spinal BMD with alendronate is similar to that seen with etidronate: 8.2% after 3 years of intermittent cyclical etidronate treatment. Alendronate is effective in reducing the number of vertebral and other clinical fractures in postmenopausal women with low BMD and at least one existing vertebral fracture. The relative risk of sustaining a new vertebral fracture after 3 years of treatment with alendronate compared to those treated with placebo was 0.46. The relative risk of any clinical fracture was 0.72 and for hip fracture and wrist fracture 0.49 and 0.52, respectively [ 160]. The efficacy of alendronate in preventing nonvertebral fractures was confirmed in a meta-analysis, with women over 65 years of age experiencing the greatest benefit [161]. The risk reduction for hip and wrist fractures was 54 and 61%, respectively, but the risk reduction seen in the incidence of hip fractures was not statistically significant. Alendronate in half the normal dosage (5 mg/day instead of 10 mg/day) was effective in preventing bone loss in early postmenopausal women. Two years of treatment resulted in a 3.5% gain in lumbar spine BMD. In this smaller dosage, aledronate was well tolerated, with a side-effect profile similar to that of placebo [ 162].

521 Oral aledronate is less well tolerated than etidronate. One major side effect is upper gastrointestinal disturbances and especially chemical esophagitis with erosions or ulcerations and exudative inflammation accompanied by thickening of the esophageal wall. The incidence of any adverse effect on the esophagus is estimated at 2:4750, with the incidence of serious or severe reactions at approximately 1:9700 patients treated [ 163]. The incidence of gastroesophageal side effects can be partially reduced by careful instruction of the patient; in particular, the medication should be taken with a large glass of water and an upright position should be maintained for at least 30 min after administration, and preferably until after eating. 5. OTHER BISPHOSPHONATES

A number of other bisphosphonates are now in clinical use or development. These include clodronate, tiludronate, pamidronate, risedronate, and ibandronate. Although some of them have been used in postmenopausal women to prevent bone loss or to improve BMD, none of them is currently licensed for this purpose in most countries. Tiludronate (100 mg/day) over 6 months led to preservation of spinal bone density in postmenopausal women [164]. In two studies postmenopausal women with breast cancer were randomized to receive 1600 mg of clodronate or placebo in addition to adjuvant tamoxifen or toremifene therapy. Two years of treatment with clodronate led to a gain of 2.9% in the spinal BMD and 3.7% in the hip [165]. Lower doses of clodronate (400 mg/day), given either continuously or cyclically (for 1 month out of every 3 months), also appeared to be effective. Twelve months of cyclical clodronate treatment led to a 3.3% increase in spinal bone density and maintained the bone density of the hip [166]. It has been reported that clodronate may decrease tumor metastases and improve survival when given to patients with metastatic breast cancer [ 167,168]. Pamidronate is another amino-bisphosphonate that has been shown to be active both intravenously and orally. It is currently used intravenously for the treatment of malignant hypercalcaemia, but the oral form has been shown to prevent and reverse postmenopausal bone loss [ 169] and corticosteroid-induced osteoporosis [170]. Gastrointestinal side effects from oral administration have prevented the oral form from being generally used for osteoporosis. Risedronate (5mg/day), when given to early postmenopausal women, leads to significant increase in lumbar spine and femoral neck B MD and is well tolerated [ 171 ]. Ibandronate, a newer bisphosphonate, is under evaluation, and preliminary studies suggest that it may be useful in the treatment of osteoporosis [ 172]. One current drawback is that it needs to be administered intravenously, although patients who experience difficulty with oral bisphosphonates may find this a palatable alternative.

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H. C a l c i u m C A L C I U M PHYSIOLOGY AND M E T A B O L I S M

There is about 0.7-1.5 kg of calcium in the average adult body, over 98% of which is in the skeleton. Only a minor proportion of the total calcium (about 0.5%) is exchangeable. The average daily dietary intake of calcium adults in the United States is 600-800 mg, less than half of which is absorbed. Calcium absorption increases in pregnancy, lactation, and during rapid growth in children, and decreases with advancing age. Most calcium is absorbed in the proximal small intestine. Maintenance of calcium balance is dependent on the efficiency of intestinal hbsorption. Vitamin D stimulates calcium absorption from the gut. Parathyroid hormone (PTH) decreases the urinary loss of calcium by stimulating calcium reabsorption from the proximal tubule in the kidney. Intestinal disease or dietary calcium deprivation leads to decreased calcium bioavailability from the gut. The role of calcium in the prevention and treatment of osteoporosis has been controversial, with arguments presented both for and against the concept of calcium supplementation for all postmenopausal women [173,174]. There is now evidence from an 18-month randomized controlled trial that increased dietary calcium (increased milk intake of 300 ml over the usual daily consumption of 150 ml) in adolescent girls aged 12.2 years old led to increased bone mineral density [175]. It would appear therefore that a higher than recommended calcium intake leads to significant gain in bone mineral density. However, the effect of calcium on the bone of adolescents may be to cause acceleration in the accrual of peak bone mass rather than an overall increase. In adults following estrogen withdrawal, increased bone resorption occurs; with increased plasma calcium levels, and consequently increased urinary calcium excretion, together with reduced parathyroid hormone levels and calcitriol production. Estrogen administration lowers urinary calcium and hydroxyproline values and plasma alkaline phosphatase activity [176]. These effects are largely mediated through the direct effect of estrogen on the bone via estrogen receptors on the bone cells [46] and possibly via the direct effect of estrogen on tubular reabsorption of calcium via estrogen receptors in the kidneys [177] (see also Chapter 19). To counteract these losses in estrogen-depleted postmenopausal women, calcium supplementation may be considered. Calcium absorption decreases with age, and it has been estimated that 1000 mg/day of extra calcium is needed to counteract the decreased calcium absorption and increased calcium excretion [174]. Cross-sectional case-controlled studies have examined the link between calcium intake and hip fracture rates in men and women over the age of 50 years [ 178-180]. Women with lower calcium intake were at higher risk of hip fracture (adjusted relative risk, 1.9) compared to controls.

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Some double-blind, randomized, placebo-controlled studies [181-183], but not all [184], have generally confirmed the findings of case-controlled studies, showing that calcium supplementation between 500 and 1000 mg/day slows the rate of postmenopausal bone loss in women of at least 3 years postmenopause. The ages of the women participating in these studies were between 40 and 70 years. The serum parathyroid hormone concentrations, serum alkaline phosphatase concentration, and urinary hydroxyproline excretion tended to be lower in the calcium group. Inevitably, it follows that optimal intake of calcium should be maintained throughout life. The amount varies according to sex, age, and ethnicity. Factors affecting calcium absorption, such as vitamin D, are important. Other factors, such as certain medications or food components, can negatively influence absorption. Optimal calcium intake may be achieved through diet, calcium-fortified foods, calcium supplements, or various combinations of these. Calcium is provided in tablets or sachets. In normal individuals, the absorption of calcium citrate is better than that of calcium carbonate [185]. A dose-range study found that the calcium absorption curve is initially steep (between 0 and 500 mg) and then plateaus. It appears that the amount of calcium absorbed from 500 mg of calcium citrate is greater than is absorbed from 2000 mg of calcium carbonate [186]. In addition, calcium citrate leads to greater inhibition of PTH, compared to calcium carbonate [ 187]. These data might be important for patients with decreased stomach acidity. The secretion of gastric acid decreases with age and as many as 50% of apparently healthy people over 50 years old may have decreased secretion. The absorption of calcium citrate is independent of the presence of gastric acid in the stomach, whereas in persons with low gastric acid the absorption of calcium carbonate given alone is insignificant [188,189]. However, when calcium carbonate is taken with food, it is absorbed normally even by patients with achlorhydria. Also, the amount of calcium per pill is higher with carbonate preparations, so that fewer pills are required to satisfy the same desired intake. High levels of calcium intake have several potential adverse effects. In dosages greater than 5000 mg/day, calcium toxicity may occur, with high serum calcium levels, severe renal damage, and ectopic calcium deposition (milk alkali syndrome). This may also occur when calcium carbonate is overused as an antacid. Adverse effects have not usually been observed with doses less than 1500 mg/day. Gastrointestinal side effects such as constipation are rare at dosages less than 1500 mg/day.

I. V i t a m i n D Vitamin D is not a vitamin but a hormone precursor. When the skin is exposed to sunlight or certain artificial

CHAPTER 35 Estrogens and Osteoporosis lights, ultraviolet light penetrates the epidermis and causes transformation of 7-dehydrocholesterol (provitamin D3) to vitamin D 3. At body temperature, it takes about 24 hours for the formation of vitamin D 3 in the skin. Aging decreases the capacity of the skin to produce vitamin D3, and a greater than fourfold reduction occurs after the age of 70 years. Topical sunscreens, latitude, time of the day, and area of exposure also affect vitamin D 3 production. Latitude is especially important. In the winter months at >42 ~, ultraviolet light falls at a sharper angle than in the summer and is completely absorbed by the ozone layer. As a consequence, little or no vitamin D 3 c a n be synthesized in these geographical areas. When there is insufficient production of vitamin D 3 in the skin, oral vitamin D intake becomes important. Vitamin D 3 undergoes two hydroxylation steps before attaining full physiological activity. The first step occurs in the liver, where 25-hydroxyvitamin D, 25(OH)D, is produced. The second step occurs in the kidney and depends on the body's requirements for calcium. If there is a tendency toward hypocalcemia, parathyroid hormone stimulates the production of 1,25-dihydroxycholecalciferol [calcitriol; 1,25(OH)zD]. In the presence of normo- or hypercalcemia, metabolism of 25(OH)D is directed by a 24-hydroxylase enzyme toward the formation of 24,25 dihydroxycholecalciferol, which is metabolically inactive. Calcitriol, or 1,25(OH)2D, is the physiologically important metabolite, acting on its target tissues via steroid receptors. Calcitriol binds to the vitamin D receptor (VDR) and forms a complex. This complex interacts with a receptor accessory factor to form a heterodimer, which in turn interacts with nuclear vitamin D response elements. In bone, this interaction leads to expression of mRNA and increased production of osteocalcin and osteopontin. In the intestine, calcium-binding protein is synthesized, leading to increased absorption of calcium and phosphate into the circulation [190,191]. Although it is clear that vitamin D has an important role in normal bone homeostasis and calcium metabolism, its role in postmenopausal osteoporosis is less evident. As mentioned earlier, peak bone mass is dependent on genetic and environmental factors. The genetic component is currently thought to be responsible for around 80% of the peak bone mass [ 17]. Morrison et al. reported that the vitamin D genotype was responsible for up to 75% of the total genetic component of bone density in healthy individuals. When a particular genotype was used as a predictor of low bone density (more than 2 SD below mean young normal density), it was found that women with the BB genotype would reach this level 18.4 years after the menopause, compared with 29 years for the bb genotype and 22 years for the heterozygote genotype, Bb [192]. It appears that premenopausal individuals with the bb genotype not only have higher bone density but also have different response to vitamin D administration than those with the BB genotype. When given vitamin D,

523 those with the bb genotype respond with higher levels of osteocalcin and lower levels of parathyroid hormone [ 193]. Although the above findings may expand our knowledge of bone metabolism, they have by no means been consistent, because other studies have found no correlation between low bone density and genotype [194] or between osteoporosis and genotype [195]. The elderly are more prone to vitamin D deficiency. The absorption of vitamin D decreases with age and formation in the skin may also decrease with less exposure to sunlight, especially in those with poor mobility. In addition, older people may have less oral vitamin D intake, in part due to a lower intake of fortified milk. In one study, it was found that less than one in three brands of milk contained 80-120% percent of the amount of vitamin D stated on the label, and some of them contained none at all [ 196]. Unfortunately, there are few clinical studies of vitamin D as a sole agent in the prevention or treatment of osteoporosis. Most clinical studies examining vitamin D in different clinical settings (i.e., postmenopause, liver disease, corticosteroid osteoporosis, chronic renal failure, or rheumatoid arthritis) have also used calcium in the treatment. Calcium (500 mg) and 700 IU vitamin D supplement appeared to increase the bone mineral density in men and women older than 65 years [197]. The incidence of nonvertebral fractures was lower in the treatment group. A larger study using different regimen of 1200 mg Ca and 800 IU vitamin D daily in healthy, ambulatory elderly women (mean age, 84 years) reported on rates of fractures, bone density change, and parathyroid hormone and vitamin D blood levels [198]. It found a 43 and 32% reduction in the number of hip fractures and in the total number of nonvertebral fractures, respectively. There was a significant decrease in the level of parathyroid hormone in the treatment group. Bone density in the proximal femur increased 2.7% in the treatment group and decreased 4.6% in the placebo group over 18 months. In a randomized study [199] comparing the effect of 0.25 mg of vitamin D or 1000 mg of calcium daily in women with osteoporotic vertebral fractures (mean age, 6 3 - 6 7 years), it was found that after 3 years of treatment the vitamin D group suffered significantly less new vertebral fractures--9.9 per 100 patientyears versus 31.5 per 100 patient-years. This effect was mainly restricted to women with less than five osteoporotic fractures from the outset, indicating that vitamin D supplementation should be started sooner rather than later. There was a significant reduction of nonvertebral fractures in the vitamin D group. In studies of the elderly, the effect of vitamin D on bone density may be largely due to correcting subclinical vitamin D deficiency rather than affecting osteoporosis. There is legitimate concern about the ability o f vitamin D to induce hypercalcemia, nephrocalcinosis, or renal stones. In the studies reviewed, vitamin D was found to be safe and well tolerated, with no significant adverse reactions.

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J. Exercise ass and muscle mass are positively correlated [200]. Bone gain is also positively correlated with physical activity [201 ]. Static loads are inefficient, whereas dynamic loads are efficient in increasing the B MD. Changes in the strain, rather than the absolute magnitude of the load, may be most important; higher rates and frequencies of changes in strain produce higher bone density. Studies in animals have demonstrated that increasing strain magnitude initiates a dose-dependent elevation of bone mass [202]. During locomotion, the magnitude and rate of limb bone strains are higher with heel strike when running or landing from a jump. Gymnastic exercise is thus more effective in increasing the bone mass compared with cycling or swimming [203]. Young gymnasts (mean age, 21 years) gained about 2% in the lumbar spine and 1.6% in the femoral neck in a study lasting 8 months, indicating that bone mineral density of the clinically relevant sites of the lumbar spine and femoral neck can respond dramatically to mechanical loading characteristic of gymnastic-type exercises in college-aged women [204]. In professional tennis players, the BMD of the playing arm was found to be up to 30% greater that of than the nonplaying arm [205]. Former elite athletes have demonstrated 12.1% higher BMD in the hip and 8.7% higher in the spine compared with controls many years after stopping competitive training [206]. In the same study active controls (defined as those vigorously exercising for more than 1 hr per week) had higher bone density compared to inactive ones. The mechanism of action of exercise on the bone is not clear. The theory of mechano-transabduction, which concerns the flow of fluid through interstices of bone, provides a possible explanation, suggesting that loading causes fluid flow that either affects the cell directly by local deformation, or by some electrical effect related to streaming potentials. Low rate strains would cause relatively sluggish fluid movements, whereas high rate strains could cause rapid local fluid movements with potentially greater effectiveness in initiating cellular events [207]. Evidence that exercise is important in preserving bone density has also derived from observation of paralyzed individuals. Studies involving paraplegics and tetraplegics have showed conclusively that there is a significant demineralization of the skeleton over time [208]. Paraplegic patients able to stand in leg braces showed higher BMD in the proximal femur than those unable to do so, demonstrating the beneficial effect of mechanical loading on the bone in osteoporosis secondary to paraplegic conditions [209]. Confirmation that loading is important in maintaining bone density has also derived from studies on astronauts. After only 3 weeks in space, experimental dogs and rats began to lose bone mass. Similar findings were noted in astronauts after 24 days of flight, with losses of between 14 and 18% in the calcaneus [210].

The effect of exercise seems to be independent of estrogen status in women. In premenopausal women aged between 35 and 45 years, high-impact exercise (including aerobic and step exercises and high-impact jumping exercises), performed 3 times a week over a period of 18 months, led to a significant increase in bone mineral density at the femoral neck compared with controls [211 ]. Exercise also improved muscle performance and dynamic control in these subjects. However, female athletes who become estrogen deficient lose bone significantly, and thus exercise alone is not sufficient to prevent estrogen-deficiency bone loss. EFFECT OF EXERCISE ON THE BONE DENSITY OF MENOPAUSAL WOMEN

It seems reasonable to extend the above findings and physiological considerations to the prevention of osteoporosis and fractures in postmenopausal women, because prevention of fractures involves not only maximizing bone density but also addressing the indirect factors that contribute to falls, i.e., coordination, muscle strength, and balance. High-intensity strength training over 1 year led to improvements in muscle mass, strength, and balance, and to bone density gains in estrogen-depleted women aged between 50 and 70 years. The gain in the femoral neck and the lumbar spine was 0.9 and 1%, respectively, in the exercise group, compared to losses of 2.5 and 0.019%, respectively, in the control group [212]. The Leisure World Study examined the risk of hip fracture in a cohort of men and women with a mean age of 73 years. The study was prospective and 8600 women were included in the analysis. Women engaging in active exercise for more than 1 hr a day had a significantly decreased risk of hip fracture compared with those exercising for less than 0.5 hr or not at all (relative risk, 0.6) [213]. A further group of men and women over 70 years of age, based in the community, were assigned to either an intervention group (which included exercise) or a control group. Those in the intervention group suffered fewer falls and fewer fractures compared with the control group [214]. It seems, therefore, that exercise is effective through all age groups. A population-based approach encouraging moderate exercise from childhood to old age may contribute toward the preservation of bone mass, as well as improving muscle strength, agility, and balance. The consequent decreased risk of falls [215] together with improvement in bone density should logically lead to fewer fractures and thus less morbidity and mortality.

K. Anabolic Therapies, Parathyroid Hormone, Fluoride, and Androgens 1. PARATHYROID HORMONE Parathyroid hormone is an 84-amino acid polypeptide chain produced in the cells of the parathyroid glands. Its

CHAPTER35 Estrogens and Osteoporosis function is to maintain the extracellular fuid (ECF) calcium concentration. However, the complete polypeptide chain is not necessary for the biological action of the hormone. Synthetic fragments containing the amino-terminal sequence residues 1-34 exert the known biological actions of the hormone. Any fall in plasma calcium concentration outside the physiological range leads to increased PTH production and secretion. PTH acts directly on the bone and kidney, and indirectly on the intestine through its effects on the synthesis of 1,25(OH)zD, to increase serum calcium concentration. PTH secretion leads to increased serum calcium via an increased rate of dissolution of bone mineral, a reduction in the renal clearance of calcium, and an increase in the efficiency of calcium absorption from the intestine. PTH exerts its effect through the mineral ion transport of the kidney and the bone, and by stimulating the renal 25-hydroxyvitamin D 1-a-hydroxylase, PTH increases intestinal calcium absorption. Once serum calcium increases, it suppresses further secretion of PTH via a direct feedback control mechanism. PTH has two main actions on bone, altering calcium availability and bone remodeling. The effect on calcium availability is rapid, within minutes, and is crucial for short-term calcium homeostasis. The effect on bone remodeling becomes apparent after some hours, with increased numbers of osteoclasts and a subsequent increase in the remodeling of bone. However, the exact effect of PTH on bone remodeling is not completely understood. Receptors for PTH are found on osteoblasts but not on osteoclasts. Thus, the action of PTH on osteoclasts is thought to be indirect, through cytokines released from osteoblasts to stimulate osteoclast differentiation from their precursors. PTH action on the bone depends on the mode of secretion. Chronic and greatly increased PTH secretion, as in severe hyperparathyroidism, leads to significant bone resorption and the well-defined bone disease osteitis fibrosa cystica. Chronic intermittent lower dose hormone administration leads to an anabolic action on the skeleton, with increased bone density. Animal data suggest a pronounced anabolic effect of PTH on the skeleton, with an increase in bone mass and bone strength [216,217]. 2. P T H IN OSTEOI'OROSlS An early study of PTH in the treatment of postmenopausal osteoporosis was performed by Reeve et al. [218]. The primary end point was prevention of new osteoporotic fractures. The mean age of the women enrolled in the study was 63 years, and all had at least one osteoporotic vertebral fracture. No new fractures were recorded during the study. However, the study was not randomized and there were no measurements of bone mineral density. In a later 6-month randomized controlled trial, subcutaneous PTH administration was associated with preserved bone mineral density in the lumbar spine, as compared to a 2.8% loss of BMD with placebo, when measured with DXA scanning [219]. How-

525 ever, no change in BMD was observed in the distal radius, and a slight decrease in femoral BMD was observed in the subjects, who were all under current treatment with a gonadotropin-releasing hormone analog for endometriosis and fibroids. In a 3-year randomized controlled trial [220], subcutaneous PTH was given to patients between 59 and 64 years of age with established osteoporosis, already receiving estrogen treatment. Compared with the control group, the B MD of patients taking both HRT and PTH showed an increase of 13% at the vertebrae and 2.7% at the hip, with significantly fewer vertebral fractures in the PTH-treated group. These clinical studies have suggested a possible beneficial effect of PTH in osteoporosis secondary to estrogen deficiency. However, PTH is relatively expensive and inconvenient to administer. Long-term and larger studies are needed to define an appropriate role for PTH in the treatment of osteoporosis. 3. FLUORIDE In the late 1930s, it was noticed that workers exposed to industrial fluoride had bones with abnormally high density. This observation led to the first clinical experiment with sodium fluoride, which was given to patients with Paget's disease of bone or to patients with osteoporosis. It resulted in positive calcium balance [221]. Fluoride is present in the food chain from a variety of sources, the commonest of which are fluorinated water, toothpaste, and mouthwash. Absorption occurs rapidly in the stomach, mainly as hydrofluoric acid. Absorption decreases with age, and the presence of cations, such as calcium, magnesium, and aluminum, can decrease absorption by 2 0 - 3 0 % . Fluoride given in large dosages (25-100 rag/day) often leads to a chemical gastritis, but this is uncommon when slow-release tablets are used. Fluoride acts both on bone cells and on bone mineral, increasing the recruitment of osteoblasts and decreasing the activity of osteoclasts. It displaces the hydroxyl group of hydroxyapatite to form fluoroapatite. These crystals of fluoroxyapatite are more densely packed and are less susceptible to the action of osteoclasts [222], but the fluoroxyapatite structure may be more brittle. 4. EFFECT OF FLUORIDE TREATMENT ON OSTEOPOROSIS

Riggs et al. performed a 4-year randomized trial using 75 mg/day of fluoride in postmenopausal women with established osteoporosis [223]. This led to a striking improvement in bone density, increasing by 35% at the spine, 12% at the femoral neck, and 10% at the femoral trochanter, although a 4% decrease in the B MD at the radial shaft was noted. Fewer vertebral fractures were noted in the fluoride-treated patients, but this effect did not reach clinical significance. However, fewer nonvertebral fractures were seen in the placebo group, the effect of which was clinically significant. was concluded that the newly formed bone had greater

526 stallinity but decreased elasticity and thus was of inadequate strength. The authors were particularly troubled by the higher rate of long bone fracture in the humerus, femur, and tibia of fluoride-treated patients, as well as the high rate of side effects such as nausea, vomiting, epigastric pain, and leg pains. They thus felt that the fluoride-calcium combination was not suitable in the treatment of osteoporosis. In a later randomized controlled study, lasting almost 5 years, Pak et al. [224] administered 50 mg/day of slowrelease sodium fluoride and 400 mg/day of calcium to women with postmenopausal osteoporosis. They reported a yearly increase in bone density of 4 - 5 % at the lumbar spine and 2.38% at the femoral neck, but no change at the radial shaft. The treatment group experienced significantly fewer new vertebral fractures, and the incidence of side effects, including gastrointestinal upset and microfractures, was not different between the groups. It was thought that the intermittent dosing of slow-release fluoride, of lower dosage than previously used, contributed to the limitation of side effects seen with this study. Two editorials were published as a response to the interim and full analyses of this study, concluding that slow-release sodium fluoride, 50 mg/day, was a safe and effective method of increasing bone density to prevent first osteoporotic fractures [225,226]. The use of fluoride has been endorsed by the United Kingdom Consensus Group [227]. 5. ANDROGENS

Androgen production in the female is greater than commonly appreciated. The role of androgens in the female includes acting as precursors for estrogen production, anabolic effects, stimulation of axillary and pubic hair growth, sebum production, stimulation of bone formation, and stimulation of erythropoetin production in the kidneys. Androgens are produced in the ovaries and adrenal glands, and from peripheral conversion in adipose tissue. During reproductive life, the relative contribution from these sources varies. The ovaries and adrenals produce androstenedione, testosterone, and dehydroepiandrosterone (DHEA), and the adrenals also produce DHEA sulfate (DHEAS). Androstenedione, DHEA, and DHEAS are converted peripherally to testosterone, dihydrotestosterone (DHT), and estrogen. Only 1-2% of the total circulating testosterone is free or biologically active, the rest being bound to sex hormone binding globulin (SHBG) and albumin. In women, alterations in the level of SHBG have a dramatic effect on the free levels of testosterone in plasma, because SHBG binds 66% of total circulating testosterone. Increased levels of estradiol and thyroxine increase SHBG, whereas testosterone, glucocorticoids, excessive growth hormone, high insulin levels, and obesity suppress SHBG. The daily androstenedione and testosterone production in premenopausal women is thought to be about 3.2 and 0.26 mg, respectively [228]. In premenopausal women, 25 % of testosterone is produced by the

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ovaries, 25% by the adrenals, and 50% by peripheral conversion. In postmenopausal women, 50% of testosterone is produced by the ovaries, 10% by the adrenals and 40% by peripheral conversion, and the overall androgen production decreases with age. Age-related decrease in androgen production starts premenopausally and androgen levels fall by half between the ages of 20 and 40 years [229]. After the menopause, the process continues. The age-related decline is particularly noticeable for DHEA and DHEAS. Following natural menopause, the levels of DHEA and DHEAS are 40 and 30% less than premenopausal values, respectively, and those of androstenedione and testosterone are 50 and 70% less than their premenopausal values, respectively [230]. After oophorectomy, the levels of testosterone and androstenedione fall by 50% in previously premenopausal women and by 50 and 21%, respectively, in previously postmenopausal women [231 ]. Androgen receptors have been found in osteoblastic cells [232]. Androgens directly stimulate human bone cell proliferation and differentiation [233]. Testosterone is a precursor of estrogen, and one study of testosterone implants revealed that 3 months following implant insertion, estrogen levels had increased from 59.8 to 120 pmol/liter [234]. In a further study of postmenopausal women with adrenocortical failure (Addison's disease), they had 3-10 times lower levels of androstenedione, DHEA, DHEAS, and testosterone compared to controls and significantly lower radial bone density, both distally and at the midshaft [235]. A number of studies have been performed using parenteral testosterone (as subcutaneous implants or injections) and oral methyl testosterone. Two years of treatment with estrogen and methyl testosterone led to a 3.4% increase in spine bone mineral density compared to the control group receiving estrogen only. The treatment was well tolerated, with acne and increased hair growth the most common side effects [236]. Methyl testosterone at daily doses of 1.25-2.5 mg has been found to be safe and effective, and avoids liver dysfunction. However, total cholesterol, high-density lipoprotein, triglycerides, and apolipoprotein A 1 decreased in estrogen/methyl testosterone groups compared to base line [237]. A further metabolic problem with androgens is that they induce insulin resistance. Androgens have a positive skeletal effect. In one study, concurrent 100-mg testosterone implants and 75-mg estradiol implants led to a 5.7% increase in bone density at the spine and a 5.2% increase at the femoral neck compared to controls on conjugated equine estrogens and progestogens [238], although the design and control of the study was less than ideal. Testosterone administration in the form of 50-mg subcutaneous implants every 6 months is well tolerated, with signs of androgen excess being exceedingly rare provided treatment is properly monitored. General experience shows that if implants are withheld when serum testosterone is above the normal range, side effects can be avoided [239]. In further studies, treatment of postmenopausal women with

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nandrolone decanoate injections led to increased vertebral BMD [240,241] and increased BMD at the distal radius [242,243] and resulted in reduced fracture rates [241]. Women administered testosterone have also reported increased well being and libido [244,245]. Nonoral delivery of androgens and the avoidance of potent testosterone esters would appear to be prudent.

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and fracture incidence among postmenopausal women on oestrogen with osteoporosis. Lancet 350, 550-555. Rich, C., and Enskinck, J. (1961). Effect of sodium fluoride on calcium metabolism of human beings. Nature (London) 191, 184-185. Kleerekoper, M. (1998). The role of fluoride in the prevention of osteoporosis. Endocrinol. Metab. Clin. North Am. 27(2), 441-452. Riggs, B. L., Hodgson, S. F., O'Fallon, W. M., Chao, E. Y., Wahner, H. W., Muhs, J. M., Cedel, S. L., and Melton, L. J., 3rd (1990). Effect of fluoride treatment on the fracture rate in postmenopausal women with osteoporosis. N. Engl. J. Med. 322(12), 802-809. Pak, C. Y. C., Sakhaee, K., Adams-Huet, B., Piziak, V., Peterson, R. D., and Poindexter, J. R. (1995). Treatment of postmenopausal osteoporosis with slow-release sodium fluoride. Final report of a randomized controlled trial. Ann. Intern. Med. 123(6), 401-408. Heaney, R. P. (1994). Fluoride and osteoporosis. Ann. Intern. Med. 120, 689-690. Kleerekoper, M. (1995). Osteoporosis and the primary care physician: Time to bone-up. Ann. Intern. Med. 123, 4 6 6 - 467. Eastell, R., Boyle, I. T., Compston, J., Cooper, C., Fogelman, I., Francis, R. M. Hosking, D. J., Purdie, D. W., Ralston, S., Reeve, J., Reid, D. M., Russell, R. G., and Stevenson, J. C. (1998). Management of male osteoporosis: Report of the UK Consensus Group. Q. J. Med. 91(2), 71-92. Carr, B. R. (1993). The ovary. In "Textbook of Reproductive Medicine" (B. R. Carr and R. E. Blackwell, eds.), p. 199. Appleton & Lange, Norwalk, CT. Zumoff, B., Strain, G. W., Miller, L. K., and Rosner, W. (1995). Twenty-four-hour mean plasma testosterone concentration declines with age in normal premenopausal women. J. Clin. Endocrinol. Metab. 80, 1429-1430. Meldrum, D. R., Davidson, B. J., Tataryn, I. V., and Judd, H. L. (1981). Changes in circulating steroids with aging in postmenopausal women. Obstet. Gynecol. 57, 624-628. Judd, H. L., Lucas, W. E., and Yen, S. S. C. (1974). Effect of oophorectomy on circulating testosterone and androstenedione levels in patients with endometrial cancer. Am. J. Obstet. Gynecol. 118, 793-798. Colvard, D. S., Eriksen, E. F., Keeting, P. E., Wilson, E. M., Lubahn, D. B., French, E S., Riggs, B. L., and Spelsberg, T. C. (1989). Identification of androgen receptors in normal human osteoblastic-like cells. Proc. Natl. Acad. Sci. U.S.A. 86(3), 854-857. Kasperk, C. H., Wergedal, J. E., Farley, J. R., Linkhart, T. A., Turner, R. T., and Baylink, D. J. (1989). Androgens directly stimulate proliferation of bone cells in vitro. Endocrinology (Copenhagen) 124(3), 1576-1578.

533 234. Thom, M. H., and Studd, J. W. W. (1981). Hormonal profile in postmenopausal women after therapy with subcutaneous implant. Br. J. Obstet. Gynaecol. 88, 426-433. 235. Devogelaer, J. P., Crabbe, J., and De Deuxchaisnes, C. N. (1987). Bone mineral density in Addison's disease: Evidence for an effect of adrenal androgens on bone mass. Br. Med. J. 294, 798-800. 236. Watts, N. B., Notelovitz, M., Timmons, M. C., Addison, W. A., Wiita, B., and Downey, L. J. (1995). Comparison of oral estrogens and estrogens plus androgen on bone mineral density, menopausal symptoms, and lipid-lipoprotein profiles in surgical menopause. Obstet. Gynecol. 85(4), 529-537; erratum: Ibid. 85(5, Pt. 1), 668 (1995). 237. Hickok, L. R., Toomey, C., and Speroff, L. (1993). A comparison of oesterified estrogens with and without methyltestosterone: Effects on endometrial histology and serum lipoproteins in postmenopausal women. Obstet. Gynaecol. 82, 919-924. 238. Savvas, M., Studd, J. W., Norman, S., Leather, A. T., Garnett, T. J., and Fogelman, I. (1992). Increase in bone mass after one year of percutaneous oestradiol and testosterone implants in postmenopausal women who have previously received long-term oral oestrogens. Br. J. Obstet. Gynaecol. 99(9), 757-760. 239. Davis, S. R., and Burger, H. G. (1996). Androgens and the postmenopausal women. J. Clin. Endocrinol. Metab. 81, 2759-2763. 240. Need, A. G., Horowitz, M., Bridges, A., Morris, H. A., and Nordin, B. E. (1989). Effects of nandrolone decanoate and antiresorptive therapy on vertebral density in osteoporotic postmenopausal women. Arch. Intern. Med. 149(1), 57-60. 241. Geusens, P., and Dequekere, J. (1986). Long-term effects of nandrolone decanoate, 1 alpha-hydroxyvitamin D 3 or intermittent calcium infusion therapy on bone mineral content, bone remodeling and fracture rate in symptomatic osteoporosis: A double-blind controlled study. Bone Miner. 1, 347-357. 242. Passeri, M., Pedrazzoni, M., Pioli, G., Butturini, L., Ruys, A. H., and Cortenraad, M. G. (1993). Effects of nandrolone decanoate on bone mass in established osteoporosis. Maturitas 17(3), 211- 219. 243. Sherwin, B. B., and Gelfand, M. M. (1985). Differential symptom response to parenteral estrogen and/or androgen administration in the surgical menopause. Am. J. Obstet. Gynecol. 151, 153-160. 244. Burger, H., Hailes, J., Nelson, J., and Menelaus, M. (1987). Effects of combined implants on oestradiol and testosterone and libido in postmenopausal women. Br. Med. J. 294, 936-937. 245. Myers, L. S., Dixen, J., Morrissette, D., Carmichael, M., and Davidson, J. M. (1990). Effects of estrogen, androgen, and progestin on sexual psychophysiology and behavior in postmenopausal women. J. Clin. Endocrinol. Metab. 70(4), 1124-1131.

SHAPTER 3f

Osteoarthritis and Menopause MARYFRAN

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Department of Epidemiology, School of Public Health, University of Michigan, Ann Arbor, Michigan 48109

III. Observations Related to Clinical or Epidemiological Studies IV. Summary References

I. I n t r o d u c t i o n

II. Pathogenesis: Potential Mechanisms for the Role of Estrogen and Osteoarthritis

acteristics such as repetitive trauma and congenital anomalies. However, there is an ongoing debate as to whether estrogen levels contribute to the pathogenesis of osteoarthritis [ 1]. This debate has largely focused on the role of estradiol levels (the major estrogen in premenopausal women), with some investigators suggesting a protective effect for higher levels and other investigators identifying a negative role. This unresolved debate was initially motivated by four observational studies from clinical and free-living populations. As early as 1925, Cecil and Archer consolidated the observations from a case series of women and suggested that the menopause appeared to be a risk factor for the development of osteoarthritis [4]. In 1949, Stecher et al. [5] reported that Heberden's nodes (the enlargement of the terminal interphalangeal joints of the fingers) are more frequently observed in women and that their occurrence could be related to the climacteric (perimenopause). In 1958, observations by Kellgren and Lawrence in a British population suggested that the prevalence of osteoarthritis increased markedly after age 55 years, particularly in women [6]. These were complemented by studies in the United States in a Michigan middle-aged population that indicated a steeper rise in the prevalence of OA among women than among men [7]. Nevertheless, the actual role of circulating estrogen levels remains unresolved. Furthermore, understanding the role of estrogen in the onset, progression, and severity of osteo-

I. I N T R O D U C T I O N Osteoarthritis (OA) is a degenerative process involving all of the major joint tissues, including articular cartilage, synovium, and subchondral bone. OA is characterized by the progressive erosion of articular cartilage, including the loss of one of its major constituents, sulfated proteoglycans. Changes in cells and matrix lead to a softening, fibrillation, ulceration and loss of articular cartilage, sclerosis, and eburnation of the subchondral bone, osteophytes, and subchondral cysts [ 1]. Osteoarthritis (OA) is a highly prevalent condition that will ultimately affect 8 out of 10 elderly individuals, both males and females, at some joint location [ 1]. It is one of the most common reasons for a patient to visit a general practitioner [1]. OA involves both non-weight-bearing joints (principally of the hand) and weight-bearing joints of the hip or knee. Pain, joint tenderness, deformity, and limitation of motion are primary presentations. It is estimated that there are more that 17 million cases of osteoarthritis in the United States, making OA a major cause of morbidity and disability [2]. OA is estimated to result annually in 4 million hospitalizations, nearly 70,000 total knee replacements, and more than 30,000 total hip replacements [3]. Major risk factors for osteoarthritis include aging, obesity, and previous injury [ 1] along with other important char-

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arthritis is now made both more complex and more critical with the introduction of the selective estrogen receptor modulators (SERMs), such as tamoxifen [8]. We will address this debate in the following manner. First, we will introduce mechanisms whereby estrogen might influence the pathogenesis of osteoarthritis. These mechanisms can provide the backdrop against which investigations using animal models, clinical studies, and epidemiological studies can be evaluated. These studies will be organized into the following general areas: age- and gender-related studies, studies addressing the menopausal transition, studies evaluating measured hormone concentrations, and studies of hormone replacement therapy.

II. PATHOGENESIS: POTENTIAL MECHANISMS FOR THE ROLE OF ESTROGEN AND OSTEOARTHRITIS Osteoarthritis is believed to be the result of biochemical changes occurring in cartilage that affect two matrix components, proteoglycans and type II collagen. There is a progressive depletion of cartilage proteoglycan over time. Additionally, there is an alteration in the degree of type II collagen hydration. Accompanying this are changes in ultrastructure properties of the collagen, changes that are marked by infiltration of type I collagen, the collagen associated with bone. These alterations in the proteoglycan content of cartilage matrix, accompanied by structural damage, make the cartilage less resistant to compression or mechanical stress. Repair mechanisms are needed to respond to these underlying changes in cartilage. In contrast to rheumatoid arthritis, in which the primary source of degradative enzymes is the synovium, the primary source of enzymatic activity in OA is probably in the chondrocytes. The enzyme families associated with the chondrodytes include the metalloproteases, the serine proteases, and the thiol proteases. There have been multiple mechanisms proposed to relate estrogens to the pathogenetic properties associated with OA (see Table I). These mechanisms will each be briefly discussed.

TABLE I Potential Mechanisms for Estrogen in the Pathogenesis of Osteoarthritis An indirect role of estrogens via the maintenanceof bone stiffness A direct role of estrogens in collagen synthesis A direct role of estrogen receptors in collagen synthesis A direct role of estrogens for the suppression of collagen synthesis An indirect role for estrogen in modulating the excess expression of cytokines

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A. I n d i r e c t R o l e o f E s t r o g e n via I n f l u e n c e on M a i n t e n a n c e o f B o n e Stiffness Early observational studies reported that osteoporosis and osteoarthritis rarely coexisted with each other, suggesting that people with low bone density did not have OA. Based on that concept, Radin [9] hypothesized that the presence of stiffer subchondral bone was more likely to damage the overlying cartilage under conditions of mechanical stress or load-bearing, resulting in osteoarthritis. A corollary to this hypothesis was related to hormone replacement therapy (HRT); if a woman with osteoporotic bone healed while using estrogens, then the healed bone may be stiffer and subsequently the woman is more likely to experience damage to overlying cartilage, i.e., develop osteoarthritis. Current research continues to support the observations that bone mineralization processes are different in osteoporosis and osteoarthritis, albeit the relationship is more complex than a simple failure to coexist together. As discussed later, none of the current work addresses the role of estrogen in these processes.

B. D i r e c t R o l e o f E s t r o g e n Collagen Synthesis In tissue culture systems, estrogen is reported to stimulate collagen synthesis in the rat uterus and femur [ 10]. In bovine aortic smooth muscle cells, estrogen was associated with a shift of type I to type III procollagen fractions [11]. In partially meniscectomized rabbits, the administration of estrogen increased the frequency of OA and accentuated the severity of the OA [ 12]. Thus, the in vivo response to estrogen administration may differ from that seen in vitro. If estrogen were to have a direct role in osteoarthritis, then the demonstration of estrogen receptors in cartilage-related cells would be important. Work by Sheridan [ 13] in baboons suggests the presence of estrogen receptors in the chondroblasts and chondrocytes of laryngeal elastic and hyaline cartilage and in the articular cartilage of the mandibular condyle. Likewise Tsai and Liu [14] have reported the presence of estrogen receptors in the cartilage of rabbit femoral condyle. It has been suggested that the up-regulation of estrogen receptors represents the effort to repair arthritic changes in cartilage [ 14]. However, the presence of estrogen receptors in critical cell types has not been universally reported. Sheridan [13] has reported that cells important to arthritic processes may have estrogen receptors [15,16] in some cells from dogs, rabbits, and baboons, but not all studies demonstrate this [17]. There appears to be substantial interspecies variation in the response of cell culture systems to estrogen. As an extension, the potential implication

CHAPTER36 Osteoarthritis and Menopause for human response to estrogen may be difficult to predict from cell culture systems, given this substantial interspecies variation. The role definition, and the attendant ambiguity, is not only a function of the species or system in which the experiment is being conducted. Natsazky summarizes that in chondrocytes, 17fl-estradiol inhibits cell proliferation [18-22], stimulates RNA synthesis [23], and stimulates 35804 incorporation [23-25] and collagen production [22,23], depending upon the 17fl-estradiol concentration as well as the model used. Further, Nasatzky [26] reports that the sexspecific effects of 17fl-estradiol in cultured chondrocytes appear to be a function of receptor number. Thus, not only is it important to anticipate whether there are estrogen receptors on the critical cell types, but also to understand factors leading to the up-regulation of those estrogen receptors and how the responses will vary according to type and amount of estrogen administration.

C. P o s s i b l e I n d i r e c t R o l e o f E s t r o g e n in M o d u l a t i n g E x c e s s E x p r e s s i o n o f C y t o k i n e s Associated with Osteoarthritis Estrogens may have an indirect role in OA expression through their influence on cytokine expression, when controlled cytokine action plays a role in normal tissue turnover and repair. Cytokines that have been extensively studied in relation to OA include the interleukins (IL-1, IL-6) and transforming growth factor-fl, and tumor necrosis factor-a. It is recognized that uncontrolled or excessive cytokine action, apparently particularly that of IL-1, will result in excessive cartilage degradation, pathologic change in joints, and an inflammatory response in the synovium [27]. Estradiol was associated with an increase in the synthesis and release of IL-1 in macrophages [28]. Estradiol (as well as testosterone) has also been shown to modulate the IL-6 release induced by IL-1. In contrast, hydrocortisone has been shown to decrease the IL-6 activity released by IL-1-induced chondrocytes [29]. In summary, there are at least three major mechanisms through which estrogen may play a role in the processes associated with the development and/or progression of osteoarthritis. There may be direct roles either through proteoglycan or collagen synthesis, or conversely through the suppression of collagen synthesis. Estrogen action may also affect cartilage indirectly through the maintenance of bone stiffness or by modulating the excess expression of cytokines. In complex human systems, each of these actions may act in synchrony or in opposition to each other. The complexity and the multiplicity of potential mechanisms provide ample opportunity to observe conflicting outcomes. The end

537 result is that at any one time in a particular process, estrogen may appear to have both a beneficial and a protective effect.

III. OBSERVATIONS RELATED TO CLINICAL OR EPIDEMIOLOGICAL STUDIES A. A g e - a n d G e n d e r - R e l a t e d T r e n d s In various geographic populations, osteoarthritis presents with an increasing in prevalence in women, beginning at the midlife. There is also a difference in joint presentation between males and females. For example, as early as the 1950s, observations by Kellgren and Lawrence in a British population suggested that the prevalence of osteoarthritis increased markedly after age 55, particularly in women [6]. These studies were complemented by studies in other geographic locales. In the United States, the Tecumseh Community Health Study reported a steeper rise in the prevalence of OA among women than among men during their middle ages [7]. Likewise, in a Dutch population, there was a rise in OA among women and men at midlife, though the prevalence of OA in specific joints was different in men and women [30]. The aforementioned gender differences and the pattern of increasing prevalence in persons between 50 and 60 years of age suggested that estrogen levels in premenopausal women might have a role in protecting women from OA. A second perspective was that the relative absence of estrogen as a result of the menopause might precipitate the presentation with OA. These possibilities led investigators to consider those conditions in which estrogen status had been modified, including menopause, hysterectomy/oophorectomy, and the provision of estrogen through hormone replacement therapy.

B. M e n o p a u s e - R e l a t e d S t u d i e s Stecher et al. [5] were among the first to report that age at menopause was correlated with the age of onset of Heberden's nodes (r = 0.46), a manifestation of osteoarthritis of the hand. Stecher based his report on a case series of 99 women ranging in age from 33 to 65 years. Simultaneously in that report, Stecher cited historical treatises by early clinicians from the early 1800s, including Haygarth, Duckworth, and Monroe, who also related the presence of Heberden's nodes with the onset of the menopausal period [5]. Stecher concluded that Heberden's nodes occur in genotypically susceptible women, with the menopause a contributory factor. In the 1950s, Kellgren and Moore [31 ] defined a subset of osteoarthritis referred to as primary generalized osteoarthri-

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tis (GOA). Their study group was composed of women with arthritic involvement of joints in the hands, spine, and knees, who were believed to have arthritis that might respond to estrogen supplementation. However, in this small clinical trial, no benefit was demonstrated in this subset [31]. Ehrlich, in 1975, described another subtype of hand OA that appears to be erosive and of inflammatory nature [32] and about which there has been speculation as to its estrogen sensitivity. Whereas Stecher, Kellgren, and Moore, as well as Ehrlich, focused on the presentation of OA in the hands, the arthritis of the menopause defined by Cecil and Archer in their 1925 manuscript [4] focused on knee OA. Approximately 25% of their patients with arthritis of the knee simultaneously reported that they were becoming menopausal. These investigators also noted that these menopausal patients with knee OA tended to be overweight women and without evidence of an infectious source as an explanation of their knee OA. In light of these observations, the investigators administered an "ovarian extract" in an uncontrolled clinical study, but they were unable to demonstrate a benefit [4]. More contemporary studies have failed to link the menopausal transition with the presentation of OA. For example, Cicuttini [33] reported less OA among premenopausal women as compared to postmenopausal women after adjusting for age, weight, previous injury, and estrogen replacement therapy (ERT) use. However, the 95% confidence intervals around the measure of association included the null value of no association. Samanta et al. reported no association between age at menopause among women with osteoarthritis as compared to age-matched controls [34]. It is noteworthy that, in humans, menopause has been associated with an increased production of IL-1 by peripheral blood mononuclear cells, a condition that is apparently reversed by estrogen replacement [35]. It is logical that the greater estrogen levels in premenopausal women would be associated with suppression of IL-1 and less frequent presentation of OA. However, if IL-1 production is part of the cytokine response in OA and there are lower circulating estrogen levels in the postmenopause, a biological mechanism for a greater incidence of OA in the postmenopause can be considered. Studies involving the menopausal transition involve several complexities that make it difficult to study in relation to OA. First, although there is ultimately diminution in estradiol levels, estradiol levels fluctuate substantially during the perimenopausal period. Thus, to better consider a menopausal effect, investigators should define when the menopausal transition began and whether any effect in relation to OA or its attendant biological processes was related to changes in estradiol levels or to diminution of estradiol levels. Then, investigators would have to consider the amount of time that would be required before menopausal change

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could be temporally associated with either the development or progression of the OA.

C. H y s t e r e c t o m y / O o p h o r e c t o m y S t u d i e s Another strategy to examine the role of estrogen in OA would be to evaluate the status of women who have experienced a hysterectomy or oophorectomy. Schouten et al. [36] used this approach to examine the role of estrogen and radiographically defined osteoarthritis (ROA) by characterizing the 690 women of the Zoetermeer community between the ages of 45 and 54 years. They used four menopausal designations, including premenopausal, postmenopausal, hysterectomy, and oophorectomy. The investigators found no association of ROA, bony swelling, or Heberden's nodes with these menopause state designations after accounting for age. The lack of association reported by Schouten et al. is inconsistent with reports by Spector et al. in British women [37]. In a series reported in 1989, Spector, Brown, and Silman [37] reported that there was approximately two times greater frequency of hysterectomy and gynecological surgery in women with OA. The data, presented in a letter to the editor, were not adjusted for age, body size, or smoking. The investigators suggested the following pathway between hysterectomy and osteoarthritis. Both dysfunctional uterine bleeding and fibroids are conditions attributable to estrogen excess and both are major conditions associated with hysterectomy. By extension, the investigators then suggested that OA is also associated with high estrogen levels (because it occurs with two times greater frequency in women with hysterectomy and gynecological surgery). This suggests an adverse role for estrogens relative to OA. In a second series, Spector [38] reported that women with hysterectomy were more likely to experience OA of the knee and carpometacarpal joint of the hand compared to controls, following adjustment for age, obesity, parity, and smoking status. It should be noted that the hysterectomized group apparently included women whose ovaries were conserved as well as those without ovarian conservation. A more clear demarcation of the status of the ovaries in the study group would allow for a greater appreciation of the role of ovarian estrogen in these findings. These aforementioned studies demonstrate well the contradictory results from studies of the estrogen and OA relationship. There are difficulties in comparing information from human gynecologic surgery to that from animal models using ovarian oblation, because of interspecies differences and differences in why different women will have gynecologic surgery. There are inconsistencies in studies of different human population groups. It is critical that the studies define whether the gynecologic surgery included removal of both ovaries. It is also important to determine the reason for

CHAPTER36 Osteoarthritis and Menopause the surgery. Women who have surgery for reasons not related to estrogen status (such as dysfunctional uterine bleeding vs. contraception) would tend to undermine the ability to extrapolate assumptions from hysterectomy to a causal mechanism relevant to OA.

D. H o r m o n e R e p l a c e m e n t T h e r a p y S t u d i e s in H u m a n s The literature about hormone replacement and OA, like that of hysterectomy and oophorectomy, is contradictory, with studies reporting increased risk of OA, protective associations for OA, and no association. Felson reported that among members of the Framingham Study (mean age of 77 years at radiographic examination), there was no association of estrogen use with radiographically defined OA of the knee [39]. A subsequent report by Hannan et al. [40] in the same study population provided additional detail but confirmed the earlier report by Felson, identifying that among women who used estrogen replacement therapy, there was evidence of no association. Samanta et al. also reported no association between HRT use among women with osteoarthritis as compared to age-matched controls [34]. This is consistent with the work of Oliveria et al. [41], who summarized a series of studies about estrogen replacement therapy and OA. They reported a negative association between prevalent osteoarthritis and estrogen use [41], and a similar trend was also observed among long-term users. Other studies have reported important associations of OA and ERT use. Two of the four groups in the Study of Osteoporotic Fractures (a study of fractures in white women who were at least 65 years old) included radiographs of the hip in one of their examinations. They found that current users of ERT and HRT had 46% lower risk of moderate to severe radiographic OA findings compared with women who had never used replacement therapy. The risk of osteoarthritis of the hip was similar in past ERT users and never users of ERT [42]. Similarly, in the Chingford Study population of approximately 1000 London area women, the prevalence odds ratio indicated a lower frequency of radiographically defined knee OA among women currently using HRT (a protective association). Likewise, there was a protective association between current HRT use and hand OA; however, the 95 % confidence intervals included the value for the null hypothesis of no association. There appeared to be no protective effect for ever users of HRT [43]. In contrast, Holbrook et al. [44] reported that those women of the Rancho Bernardo Study (in southern California) with self-reported OA were more likely to also be taking estrogen replacement therapy. This cooccurrence of selfreported OA and ERT, however, suffers from a lack of tem-

539 porality and does not indicate that the provision of estrogen (via ERT) gives rise to OA. These studies include a number of complexities. For example, these users of estrogen replacement used ERT for an extended time. Thus, study enrollees were typically well beyond the age of the transition and the studies did not adequately address the number of years since menopause. Furthermore, these studies do not identify the reasons for initiating replacement. Potentially, enrollees would include women with both surgical and natural menopause. There are also issues around the definition of OA. It is well recognized that there is only minimal overlap among women who self-report with OA as compared with the radiographically defined OA. More women are likely to selfreport OA than would be described using OA and common classification scales such as the Kellgren-Lawrence criteria. It also appears that hand and knee OA (even when both are radiographically defined) may have different factors associated with their development.

E. H o r m o n e A d m i n i s t r a t i o n in A n i m a l M o d e l s In animal studies, Rosner [45] reported that estradiol valerate did not prevent experimentally induced osteoarthritis in animals. Even though there was evidence of suppression of proteoglycan synthesis by estradiol, the administration of estradiol did not alter severity of the osteoarthritis. Tsai and Liu [14] have reported that higher estradiol levels are associated with the development of knee OA in rabbits, but that the manifestation of the pathology was duration and dose dependent. Studies using the selective estrogen receptor modulator tamoxifen have also been evaluated in relation to experimentally induced osteoarthritis in rabbits. Rosner et al. [8] reported that whereas the administration of estradiol was associated with a worsening of the arthritic pathology, the administration of tamoxifen was not associated with a worsening of the pathology. Likewise, Tsai and Liu have reported that tamoxifen injected concurrently with estradiol benzoate in the early stages of knee OA in rabbits minimized the chondrodestructive effects of estradiol [46]. In animal models, Silberberg et al. [47,48] paradoxically demonstrated attenuation of joint disease in adult female mice who were oophorectomized. In their administration of estrogen therapy, the oophorectomized mice showed an apparent increased cartilage matrix condensation and fibrillation and reduction of chondrocyte proliferation. 1. ANIMAL STUDIES USING ~/[EASuRED HORMONE CONCENTRATIONS There is a small body of literature developed to describe the role of estrogen concentrations in cartilage and its me-

540 tabolism. Bellino surmised that neither the articular cartilage nor chondrocytes from rabbits could synthesize estrogen; however, these cells would be capable of responding to estrogen in metabolic processes [49].

2. HUMAN STUDIES USING MEASURED HORMONE CONCENTRATIONS Endogenous estrone and estradiol concentrations appear to be similar in women with varying degrees of prevalent OA [50]. However, the concentrations of sex hormone binding globulin are reported to be lower in women with generalized OA than in controls, suggesting the opportunity for higher "free" circulating estrogen concentrations [51 ]. Spector et al. contrasted the sex steroid concentrations in controls and women with generalized osteoarthritis (defined by clinical evidence of Heberden's nodes or the presence of clinical and radiological evidence of OA in the hands and knees). After stratifying on age, women with generalized OA had a significantly higher average testosterone concentration compared to controls in the age group 4 1 - 5 2 years. There were lower levels of sex hormone binding globulin in women with generalized OA (as compared to controls) in the age group 5 3 - 6 1 years. There was no association with circulating estradiol; however, the investigators do not define estradiol in relation to phase within the menstrual cycle of the premenopausal women. Thus, it is difficult to determine if there might have been an estradiol association, if the investigators had accounted for this major source of variation. There is less variation in testosterone levels associated with phase of a menstrual cycle as compared to the variation in estradiol levels during the menstrual cycle of premenopausal women [51 ].

IV. S U M M A R Y The hypothesized relationships between osteoarthritis and ovarian hormone levels usually involve estrogen, but the hypotheses that have emerged are bidirectional. A protective effect for estrogen was first suggested by observations of increased risk of OA during the menopausal transition. The investigators attributed the increased risk of OA to the decline in circulating estrogen levels. Studies, both in v i v o and in vitro, suggest that estrogen may be chondrodestructive, potentially though the up-regulation of estrogen receptors. The role for estrogen was further explored in studies of the menopause and in studies of surgical menopause, under the assumption that oophorectomy was a more definable time point at which there was loss of circulating estrogen. Likewise, hormone replacement therapy has been evaluated with the understanding that a factor that is causal of disease can be identified when that factor is replaced in a system in which it is absent. The results of studies that address these issues are frequently contradictory.

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There are several reasons that studies to date may be so contradictory. First, consistently defining OA has been difficult. It is unclear as to how much of any relationship is related to the initiation, progression, or severity of the osteoarthritis. It is equally unclear which of the subtypes of OA are being addressed in these studies. Because OA is a heterogeneous condition, it is quite possible that the estrogen relationship may be more important in hand OA, or in generalized osteoarthritis, which includes the hand, but is less applicable to the hip or knee. Estrogen may be related to OA only of specified etiologiesmthat is, whether there is a substantial inflammatory component or immune response as compared to a more mechanical modality. The use of animal models is not particularly informative in sorting out the heterogeneity of OA responses characterizing the human disease. It is well recognized that there are important substantial interspecies differences affecting the interpretation of studies in animal models. Studies in humans frequently have insufficient sample sizes to sort out whether the response is related to the initiation, progression, or severity of the disease(s). There are also difficulties in characterizing the exposures to hormones. In studies of hormone replacement therapies, responses have varied according to the dose and type of formulation, as well as the timing in the disease course. Studies have also tended to address issues related to estrogen, but failed to consider other important hormones such as androgen levels. In light of these limitations, well-defined and wellexecuted studies in this area are critical. Osteoarthritis is a major cause of functional limitation and results in substantial expenditure of dollars for health care. The female population is significantly affected by osteoarthritis and, on reaching age 60, women experience a major shift in their estrogen production and a shift in their estrogen:androgen ratio. A more complete understanding can have an important impact in the population and could provide an opportunity for needed therapeutic interventions.

References 1. March, L. M. (1997). MJA practice essentials: Osteoarthritis. M.J.A. 166(2), 98-103. 2. Arthritis National Research Foundation (1996). Available at http:// www. curearthritis, org

3. Praemer, A., Furner, S., and Rice, D. P. (1992). "Musculoskeletal Conditions in the United States." American Academy of Orthopedic Surgeons, Park Ridge, IL. 4. Cecil, R. L., and Archer, B. H. (1925). Arthritis of the menopause. JAMA, J. Am. Med. Assoc. 84, 75- 84. 5. Stecher, R. M., Beard, E. E., and Hersh, A. H. (1949). Heberden's nodes: The relationship of the menopause to degenerativejoint disease of the fingers. J. Lab. Clin. Med. 34(2), 1193-1202. 6. Kellgren, J. H., and Lawrence, J. S. (1958). Osteoarthrosis and disk degeneration in an urban population. Ann. Rheum. Dis. 17, 388-397.

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CHAPTER 36 Osteoarthritis and M e n o p a u s e 7. Mikkelsen, W. M., and Duff, I. F. (1970). Age-specific prevalence of radiographic abnormalities of the joints of the hands, wrists and cervical spine of adult residents of the Tecumseh, Michigan, Community Health Study area, 1962-1965. J. Chronic Dis. 23, 151-159. 8. Rosner, I. A., Malemud, C. J., Goldberg, V. M., Papay, R. S., Getzy, L., and Moskowitz, R. W. (1982). Pathologic and metabolic response of experimental osteoarthritis to estradiol and an estradiol antagonist. Clin. Orthop. Relat. Res. 171, 280-286. 9. Radin, E. L., and Rose, R. M. (1986). Role of subchondral bone in the initiation and progression of cartilage damage. Clin. Orthop. 213, 34 -40. 10. Smith, Q. T., and Allison, D. J. (1966). Changes of collagen content in skin, femur and uterus of 17fl-estradiol benzoate-treated rats. Endocrinology (Baltimore) 79, 486-492. 11. Beldekas, J. C., Smith, B., Gerstenfeld, L. C., Sonenshein, G. E., and Franzblau, C. (1981). Effects of 17fl-estradiol on the biosynthesis of collagen in cultured bovine aortic smooth muscle cells. Biochemistry 20, 2162-2167. 12. Tsai, C., and Liu, T. (1993). Estradiol-induced knee osteoarthritis in ovariectomized rabbits. Clin. Orthop. Relat. Res. 291, 295-302. 13. Sheridan, P. J., Aufdemorte, T. B., Holt, G. R., and Gates, G. A. (1985). Cartilage of the baboon contains estrogen receptors. Rheumatol. Int. 5, 279-281. 14. Tsai, C. L., and Liu, T. K. (1992). Osteoarthritis in women: Its relationship to estrogen and current trends. Life Sci. 50, 1737-1744. 15. Young, P. C.M., and Stack, M. T. (1982). Estrogen and glucocorticoid receptors in adult canine articular cartilage. Arthritis Rheum. 25, 568-573. 16. Rosner, I. A., Manni, A., Malemud, C. J., Boja, B., and Moskowitz, K. W. (1982). Estrogen receptors in articular chondrocytes. Biochem. Biophys. Res. Commun. 10, 1378-1382. 17. Kan, K. W., Cruess, R. L., Posner, B. I., Guyda, H. J., and Solomon, S. (1984). Hormone receptors in the epiphysial cartilage. J. Endocrinol. 103, 125-131. 18. Herbai, G. (1971). Studies on the site and mechanism of action of the growth inhibiting effects of estrogen. Acta Physiol. Scand. 90, 262-271. 19. Corvol, M. T., Malemud, C. J., and Sokoloff, L. (1972). A pituitary growth-promoting factor for articular chondrocyte in monolayer culture. Endocrinology (Baltimore) 120, 1422-1429. 20. Scranton, J. P. E., McMaster, J. H., and Diamond, P. E. (1975). Hormone suppression of DNA synthesis in culture chondrocytes and osteosarcoma cell lines. Clin. Orthop. Relat. Res. 112, 340-348. 21. Takahashi, M. M., and Noumura, T. (1987). Sexually dimorphic and laterally asymmetric development of the embryonic duck syrinx: Effect of estrogen on in vitro cell proliferation and chondrogenesis. Dev. Biol. 121,417-422. 22. Nasatzky, E., Schwartz, Z., Boyan, B. D., Soskolne, W. A., and Ornoy, A. (1993). Sex-dependent effects of 17fl-estradiol on chondrocyte differentiation in culture. J. Cell. Physiol. 154, 359-367. 23. Nasatzky, E., Schwartz, Z., Soskolne, W. A., Boyan, B. D., and Ornoy, A. (1992). Sex-related effects of sex hormone on the production of matrix by chondrocyte in vitro. J. Bone Miner. Res. 7, 51-158. 24. Corvol, M. T., Carrascosa, A., Tsagris, L., Blanchard, O., and Rappaport, R. (1987). Evidence for a direct in vitro action of sex steroids on rabbit cartilage cells during skeletal growth: Influence of age and sex. Endocrinology (Baltimore) 120, 1422-1429. 25. Blanchard, O., Tsagris, L., Rappaport, R., Duval-Beaupere, B. G., and Corvol, M. (1991). Age-dependent responsiveness of rabbit and human cartilage to sex steroids in vitro. J. Steroid Biochem. Mol. Biol. 40, 711-716. 26. Nasatzky, E., Schwartz, Z., Soskolne, W. A., Brooks, B. P., Dean, D. D., Boyan, B. D., and Ornoy, A. (1994). Evidence for receptors specific for 17-estradiol and testosterone in chondrocyte cultures. Connect. Tissue Res. 30, 277-294. 27. Pelletier, J. P., DiBattista, J. A., Roughley, P., McCollum, R., and

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Martel-Pelletier, J. (1993). Cytokines and inflammation in cartilage degradation. Rheum. Dis. Clin. North Am. 19, 545-568. Hu, S. K., Mitcho, Y. L., and Rath, N. C. (1988). Effect of estradiol on interleukin 1 synthesis by macrophages. Int. J. Immunopharmacol. 10, 247-252. Guerne, P. A., Carson, D. A., and Lotz, M. (1990). IL-6 production by human articular chondrocytes. Modulation of its synthesis by cytokines, growth factors, and hormones in vitro. J. Immunol. 144, 4 9 9 505. Van Saase, J. L . C . M . , Van Romunde, L. K.J., Cats, A., Vandenbroucke, J. P., and Valkenburg, H. A. (1989). Epidemiology of osteoarthritis: Zoetermeer survey. Comparison of radiological osteoarthritis in a Dutch population with that in 10 other populations. Ann. Rhuem. Dis. 48, 271-280. Kellgren, J. H., and Moore, R. (1952). Generalized oseoarthritis and Heberden's nodes. Br. Med. J. 1, 181-184. Ehrlich, G. E. (1975). Osteoarthritis beginning with inflammation. JAMA, J. Am. Med. Assoc. 232, 157-159. Cicuttini, E M., Spector, T., and Baker, J. (1997). Risk factors for osteoarthritis in the tibiofemoral and patellofemoral joints of the knee. J. Rheumatol. 24, 1164-1167. Samanta, A., Jones, A., Regan, M., Wilson, S., and Doherty, M. (1993). Is osteoarthritis in woman affected by hormonal change or smoking? Br. J. Rheumatol. 32, 366-370. Pacifici, R., Rifas, L., McCracken, R., Vared, I., McMurtry, C., Avioli, L. V., Peck, L. V., and Peck, W. A. (1989). Ovarian steroid treatment blocks a postmenopausal increase in blood monocyte interleukin-1 release. Proc. Natl. Acad. Sci. USA 86, 2398-2402. Schouten, J. S.A.G., van den Ouweland, E A., and Valkenburg, H. A. (1992). Natural menopause, oophorectomy, hysterectomy and the risk of osteoarthritis of the dip joints. Scand. J. Rheumatol. 21, 196-200. Spector, T. D., Brown, G. C., and Silman, A. J. (1989). Increased rates of previous hysterectomy and gynaecological operations in women with osteoarthritis. Br. Med. J. 297, 899-900. Spector, T. D., Hart, D. J., Brown, P., Almeyda, J., Dacre, J. E., Doyle, D. V., and Silman, A. J. (1991). Frequency of osteoarthritis in hysterectomized women. J. Rheumatol. 18, 1877-1883. Felson, D. T. (1990). The epidemiology of knee osteoathritis: Results from the Framingham Osteoarthritis Study. Semin. Arthritis Rheum. 20, 42-50. Hannan, M. T., Felson, D. T., Anderson, J. J., Naimark, A., and Kannel, W. B. (1990). Estrogen use and radiographic osteoarthritis of the knee in women: The Framingham Osteoarthritis Study. Arthritis Rheum. 33, 525-532. Oliveria, S. A., Felson, D. T., Klein, R. A., Reed, J. I., and Walker, A. M. (1996). Estrogen replacement therapy and the development of osteoarthritis. Epidemiology 7, 415-419. Nevitt, M. C., Cummings, S. R., Lane, N. E., Genant, H. K., and Pressman, A. R. (1994). Current use of oral estrogen is associated with a decreased prevalence of radiographic hip OA in elderly white women. Arthritis Rheum. 37(Suppl.)315, $212 (abstr.). Spector, T. D., Nandra, D., Hart, D. J., and Doyle, D. V. (1997). Is hormone replacement therapy protective for hand and knee osteoarthritis in women?: The Chingford Study. Ann. Rheum. Dis. 56, 4 3 2 434. Holbrook, T. L., Wingard, D. L., and Barrett-Connor, E. (1990). Selfreported arthritis among men and women in an adult community. J. Commun. Health 15, 195-208. Rosner, I. A.,Goldberg, V. M., Getzy, L., and Moskowitz, R. W. (1979). Effects of estrogen on cartilage and experimentally induced osteoarthritis. Arthritis Rheum. 22, 52-58. Tsai, C., and Liu, T. (1992). Inhibition of estradiol-induced early osteoarthritic changes by tamoxifen. Life Sci. 50, 1943-1951. Silberberg, M., and Silberberg, R. H. (1963). Modifying action of estrogen on the evoluation of osteoarthrosis in mice of different ages. J. Endocrinol. 72, 4 4 9 - 451.

542 48. Silberberg, R., Goto, G., and Silberberg, M. (1958). Degenerative joint disease in castrate mice. I. Affects of oophorectomy at various ages. Arch. Pathol. 65, 438-441. 49. Bellino, F. L. (1992). Estrogen metabolism, not biosynthesis, in rabbit articular cartilage and isolated chondrocytes: A preliminary study. Steroids 57, 507-510.

MARYFRAN SOWERS 50. Cauley, J. A., Kwoh, C. K., Egeland, G., Nevitt, M. C., Cooperstein, L., Rohay, J., Towers, A., and Gutai, J. E (1993). Serum sex hormones and severity of osteoarthritis of the hand. J. Rheumatol. 20, 1170-1175. 51. Spector, T. D., Perry, L. A., and Jubb, R. W. (1991). Endogenous sex steroid levels in women with generalised osteoarthritis. Clin. Rheumatol. 10, 316-319.

~ H A P T E R 3",

The Epidemiology of Cardiovascular Disease and Postmenopausal Hormone Therapy FRANCINE GRODSTEIN

M E I R J. S T A M P F E R

Channing Laboratory, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115 Departments of Nutrition and Epidemiology, Harvard School of Public Health; and Channing Laboratory, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115

I. Introduction II. Primary Prevention of Cardiovascular Disease and Stroke III. Secondary Prevention of Cardiovascular Disease

IV. Association of Hormones with Lower Risk of CHD: Cause and Effect or Selection? V. Summary References

women, and that hormone replacement therapy (HRT) after menopause might decrease the risk. Attention has also been given to the possibility of protecting menopausal women against stroke through the use of HRT. CHD and stroke share many risk factors; furthermore, some evidence suggests that, as with CHD, surgical removal of the ovaries increases the frequency of cerebrovascular disease [3 ]. This chapter summarizes the epidemiological investigations regarding the association between postmenopausal hormone use and cardiovascular diseases, including both primary and secondary prevention of coronary heart disease

I. I N T R O D U C T I O N Cardiovascular diseases remain the leading cause of death in women. In particular, rates of coronary heart disease (CHD), although relatively low among premenopausal women, rise sharply with age. Moreover, the ratio of rates for men and for women grows smaller with increasing age [ 1]. This observation led to speculation that functioning ovaries in premenopausal women were protective. The increased risk of CHD among young women with bilateral oophorectomy [2] further supports the view that hormones play an important role in reducing the risk of CHD in premenopausal

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and stroke. The majority of epidemiologic studies address the use of estrogen alone, because this was standard practice at the time those studies were conducted; progestin was added to the estrogen routine to reduce the increased risk of uterine cancer due to unopposed estrogen use, but this is a relatively recent practice. Thus, much of the currently available evidence can be most directly applied to women taking estrogen replacement; however, we do include a section in this chapter specifically examining the data on combined hormone therapy and cardiovascular diseases.

II. PRIMARY P R E V E N T I O N OF CARDIOVASCULAR DISEASE AND STROKE A. C o r o n a r y H e a r t D i s e a s e Several study designs have been used to examine the association between hormone use and heart disease: hospitaland community-based case-control studies, cross-sectional studies, and prospective studies. In the hospital-based casecontrol design, patients with a history of prior hormone use among patients hospitalized for heart disease are compared to patients with the same history hospitalized for other reasons. It is essential to choose controls from among patients diagnosed with diseases unrelated to estrogen. This can be difficult because many diseases are associated in some way with estrogens. For example, in several investigations, many of the controls were subjects admitted for treatment of fracture. These studies were conducted before it was widely appreciated that hormone therapy reduces the incidence of osteoporosis and fracture. Such controls would be less likely to have taken hormones compared to similarly aged women in the population. Their inclusion in a study would tend to reduce the magnitude of the apparent inverse association between hormone therapy and risk of heart disease. Even in patients whose illness is not related to estrogen, physicians may be reluctant to prescribe hormones to avoid the possibility of interactions with other medications or simply to reduce the patient's burden of medications. Generally, one might expect hospital-based case-control studies to underestimate the protective effect of hormones on heart disease, and in fact only the hospital-based case-control studies do not find consistent protection against heart disease for hormone users. Six hospital-based case-control studies [4-9] have examined the relation between estrogen use and subsequent risk of heart disease. The relative risks observed in these studies range from 4.2 to 0.5, with most showing no association. Of the two investigations that found strong positive associations, one [5] was based on only 14 cases of coronary disease, had very low participation (10% of those initially

eligible), and was restricted to women under the age of 46, and the other [9] used controls in whom 40% had orthopedic disorders. In contrast, relative risks from the population-based casecontrol studies of myocardial infarction (MI) and hormone use, in which the subjects are all chosen from the general population, range from 0.3 to 1.2 [10-20]. Only one study reported a relative risk that was not below 1 [17], although one study, that combined stroke and MI cases [ 16] found essentially null results and a subsequent study also reported little relation between current hormone use and MI [20]. Heckbert et al. [ 19] closely examined the effect of duration of hormone use on risk of heart disease in a study using 850 cases and 1974 controls from the Group Health Cooperative in Seattle, Washington; they found increasing protection with increasing duration of current use, with a relative risk of 0.91 for less than 1.8 years of use and 0.55 for 8.2 years or more of use. In the cross-sectional studies, the degree of coronary artery occlusion is assessed among users and nonusers of postmenopausal hormones in women presenting for coronary arteriography [21-25]. It is likely that this design could overestimate the benefit of hormone use on the risk of CHD, because hormone users have greater contact with the health care system and may be more likely to have angiography than nonusers with the same equivocal symptoms. Hence, it is possible that the estrogen-using group in these investigations is artificially enriched with women who have nonsignificant disease. Nonetheless, the magnitude of such an effect could explain only a small fraction of the apparent benefit of hormone therapy observed by these studies. And, when one study examined symptoms in users and nonusers in order to address this issue, no difference was found between the two groups, suggesting the absence of any important bias. The results of the four cross-sectional studies [21-23,25] examining women with severe occlusion (defined as 70% or more stenosis in two studies, and as an average of or greater than 50% in the other two) compared to those with no stenosis were nearly identical; each observed a statistically significant decrease of about 60% for the risk of severe coronary disease among women using hormones compared with nonusers. In a fifth study, the outcome was defined as coronary artery disease or greater than 24% stenosis [24], and Hong et al. also found a significant decrease of disease in hormone users, with an 87% reduction in prevalence. Controlling for total cholesterol and triglyceride levels in the Gruchow et al. study [22] did not change the results at all. However, when high-density lipoprotein (HDL) cholesterol was included in the regression model, the observed association was substantially reduced. This effect is consistent with the view that elevations in HDLs [and a decrease in low-density lipoproteins (LDLs)] most likely mediate at

CHAPTER37 Cardiovascular Disease and Hormone Therapy least part of the apparent benefit of estrogen. Usually, it is inappropriate to adjust for these variables because they are in the causal pathway and controlling for them is, effectively, controlling for the end point. When one does include HDL levels in the model, it is equivalent to asking what the result of estrogen use is on coronary risk above and beyond its effect on HDL levels. Prospective studies have important advantages over casecontrol studies, chiefly in avoiding the problems of recall bias and control selection. Most of the prospective studies followed women with and without hormone exposure, and thus had an internal control group; this design is preferable because the exposed and unexposed individuals are generally comparable. However, in a few studies, all subjects were taking hormone therapy. At the end of the follow-up period, their mortality experience is compared with national statistics. In most instances, patients given estrogen tend to be healthier than the general population, in part by virtue of their socioeconomic status and connection with the medical care system. As a result, studies that compare hormone users to a general population probably overestimate the benefit of hormone therapy. One potential limitation of most prospective studies is that hormone use is often assessed only at baseline. With long follow-up in such studies, there can be substantial misclassification of hormone use because many women will stop or start taking hormones after the baseline assessment; this would lead to an underestimate of the benefit. All the prospective studies [26-52] have observed a protective effect of hormone therapy on coronary heart disease incidence and mortality, although the results from the Framingham Study [34,35] are equivocal. As expected, those using national statistics for comparison [27,28,43,44] found some of the strongest apparent benefits, with relative risks ranging from approximately 0.3 to 0.4. The Nurses' Health Study [33] is the largest prospective cohort to investigate hormone use and heart disease. The study was established in 1976 when 121,700 married female registered nurses, aged 30 to 55 years, completed a mailed questionnaire. Information on coronary risk factors and hormone usage was updated by means of follow-up questionnaires sent every 2 years. In the analysis of hormones and heart disease, a total of 59,337 postmenopausal women without prior coronary heart disease were followed for up to 16 years; 584 nonfatal myocardial infarctions and 186 confirmed coronary deaths were documented. Most of the benefit was observed for current hormone users, who had a 40% lower risk of heart disease compared to nonusers [relative risk (RR = 0.60, with 95% confidence interval (CI), 0.43-0.83] after adjustment for a wide array of CHD risk factors. There was little effect of duration of current use; the protection was similar for short-term (RR = 0.53 for less than 2 years) and long-term (0.70 for 10 or more

545 years) users. However, the relation was attenuated among past hormone users (RR = 0.85; 95% CI, 0.71-1.01); in particular, the inverse association appeared to diminish 3 or more years after the cessation of estrogen use (RR = 0.69 for women who had stopped estrogen use less than 3 years in the past and RR = 0.81 after 3 to 5 years). The multivariate-adjusted relative risk of coronary disease for current users of 0.625 mg of conjugated estrogen was 0.53 (95% CI, 0.36-0.78) compared to never users, and only modest protection (not statistically significant) was observed with the 1.25-mg dose (RR = 0.82; 95% CI, 0.51-1.33). The inverse association was observed regardless of age; for women under age 50, the relative risk for coronary disease among current estrogen users was 0.18 (95% CI, 0.05-0.60); for those 5 0 - 5 9 years, 0.71 (95% CI, 0.52-0.96); and for women 6 0 71 years, 0.66 (95% CI, 0.44-1.01). The Nurses' Health Study cohort includes predominantly young postmenopausal women, and data for women of older ages are sparse. Ettinger et al. [55] studied 454 women with an average age of 77 years at the end of follow-up, and reported a relative risk of 0.40 (95% CI, 0.16-1.02) for coronary death among long-term hormone users. In the Leisure World cohort [39,40], hormone status and other cardiovascular risk factors were ascertained in 8807 women, aged 4 0 101 years, living in a retirement community in 1981 (median age = 73 years); 203 deaths due to MI were identified over 7 89 years of follow-up. The rate of fatal myocardial infarction was substantially reduced among hormone users (RR = 0.60, p < 0.001). Current hormone users, defined by the single baseline questionnaire, were afforded the greatest protection (RR = 0.51). Results from all the studies of coronary disease were combined in a meta-analysis [53] (Fig. 1). The relative risks for all studies of heart disease ranged from 0.17 to 4.2, with a summary relative risk of 0.65 (95% CI, 0.61-0.69); again, users of a variety of hormone regimens are included in these studies, but the majority are of oral conjugated estrogen alone. Evidence from many of these studies indicates that current hormone users enjoy greater protection against heart disease than do past users. Thus, combining investigations of current, past, and ever use in a summary estimate such as this is misleading, because the results will be directly affected by the proportion of past and current use in the studies included. Summary estimates based on analyses of current use, where such data were provided, were recalculated; as expected, the estimates were lower than those derived by combining studies of any hormone use. For the populationbased case-control studies, the pooled relative risk for current hormone use was 0.69 (95% CI, 0.50-0.95); for the cross-sectional studies, it was 0.39 (95% CI, 0.31-0.48); and for the internally controlled prospective studies, the summary estimate was 0.60 (95% CI, 0.50-0.72). The pooled relative risk for current hormone use, combining all three

546

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FIGURE 1 Heart disease and postmenopausal hormones: meta-analysis of ever and current users. From Grodstein and Stampfer [5 3].

study designs, was 0.53 (95% CI, 0.47-0.60); we believe that this figure represents the best summary of the impact of hormone therapy on CHD risk from observational data.

B. C e r e b r o v a s c u l a r D i s e a s e Fewer investigations of stroke than of heart disease have been conducted. Three population-based case-control studies of stroke and hormone use find relative risks ranging from 0.15 to 1.22 [54-57]. One of these studies comes from a retirement community [56], and compares 216 women with fatal or nonfatal first stroke identified by hospital discharge diagnostic indices and medical center death records to 1056 age-matched controls residing in the community during the same time period. The relative risk overall was 0.97, although there was some variation in risks when the data were examined by type of stroke (from 2.79 for transient ischemic attack to 0.49 for embolic infarction). However, the numbers in each of these groups tended to be small and none of the relative risks attained statistical significance. Stroke was not associated with the duration of hormone use or with past versus current use, but long-term and current users were a minority of the population. In another study, based on 1422 cases from the Danish National Patient Register and 3171 controls [57], the association between nonfatal stroke and hormone use was examined by the type of stroke. Analyses were done separately for different types of hormone regiments. Current unopposed estrogen use was not related to thromboembolic infarction

(RR = 1.16; 95% CI, 0.86-1.58), but there were decreased risks of subarachnoid and intracerebral hemorrhages (RR = 0.52; 95% CI, 0.23-1.22 and RR = 0.15; 95% CI, 0.021.09, respectively); however, the numbers of cases of hemorrhagic stroke were relatively small and the relative risk estimates were not statistically significant. For current use of combined estrogen and progestin, no association was found for thromboembolic infarction (RR = 1.17), subarachnoid hemorrhage (RR = 1.22), or intracerebral hemorrhage (RR = 1.17). Ten prospective studies have examined the influence of hormone use on risk of stroke [32-34,37,40,44,50,52,5861]. Five of these studies with internal controls [37,40,52, 58,59,61] found lower risk among hormone users, with relative risks ranging from 0.23 to 0.79; two of these were statistically significant [40,61]. Three large investigations [33,50,60] found no effect of hormones on the risk of cerebrovascular disease incidence, whereas the Framingham Study [34] observed significantly elevated rates of stroke among hormone users, in particular, atherothrombotic brain infarctions, though the number of cases was small. One of the largest investigations examining stroke risk was the Nurses' Health Study [33], with 552 cases of fatal and nonfatal first stroke. Hormone therapy was not associated with the incidence of stroke, with a relative risk of 1.03 among current hormone users and 0.99 for past users. Similarly, no association was observed specifically for subarachnoid hemorrhage (RR = 0.90), although there appeared to be a slight increase in the risk of ischemic stroke (RR 1.40). In addition, there was a trend of increasing risk of

CHAPTER37 Cardiovascular Disease and Hormone Therapy stroke with increasing dose of estrogen used (RR = 0.64 for 0.3 mg, RR = 1.24 for 0.625 mg, and RR = 1.44 for 1.25 mg). Interestingly, in an analysis of mortality [62], however, there was an apparent overall decrease in stroke death for current hormone users (RR = 0.68; 95% CI, 0.39-1.16, based on 28 cases who were current hormone users), consistent with the results of two other studies that specifically focus of deaths due to stroke [40,52,59]. In the Leisure World Study [40], the end point was stroke mortality. Of 8807 women followed, 92 died from stroke. The relative risk of stroke death was 0.63 for hormone users compared to nonusers, although this was not statistically significant. The greatest apparent protection was found among current users and those who used hormones 1 year in the past, with a relative risk of 0.3 (p < 0.05) for mortality due to stroke, compared to nonusers. In summary, it appears that hormone use is not related to stroke incidence, but may be associated with a better prognosis after stroke.

C. E s t r o g e n C o m b i n e d w i t h P r o g e s t i n a n d Primary Prevention of Cardiovascular Diseases Progestin use was quite uncommon during the period that most of the epidemiological studies were conducted. Hence, most of the data are related directly to use of estrogens alone. Currently, progestins are prescribed along with estrogens in women with a uterus to reduce or eliminate the excess risk of endometrial cancer due to unopposed estrogen. However, progestin alone tends to raise LDL and lower HDL, and may thus detract from the beneficial effect of estrogens on the lipid profile; in clinical trials, significant decreases in LDL and increases in HDL were found for women assigned to estrogen combined with progestin, but for HDL, the elevation among users of estrogen with medroxyprogesterone acetate was less than that for users of estrogen alone. Furthermore, although estrogen therapy improves blood flow, limited studies suggest that this benefit may be diminished with the addition of progestin [68,69]. Nonetheless, the observational epidemiologic studies of primary prevention of heart disease strongly suggest that there is a similar decrease in the risk for users of estrogen alone and combined with a progestin. In a case-control study with 502 cases of myocardial infarction and 1193 controls, Psaty et al. [ 18] reported relative risks of 0.68 (95% CI, 0.381.22) for current users of conjugated estrogen with medroxyprogesterone acetate (MPA) and 0.69 (95% CI, 0.47-1.02) for users of conjugated estrogen alone. In a follow-up study of the population of Uppsala, Sweden, Falkeborn et al. [49] also found that MI was reduced by 47% (RR = 0.53; 95% CI, 0.30-0.87) in women taking estrogen (estradiol valerate or conjugated estrogen) with a progestin (levonorgestrel,

547 MPA, and norethisterone acetate) and 31% (RR - 0.69; 95% CI, 0.54-0.86) in those taking estrogen alone. In the Nurses' Health Study [33], the risk of CHD was diminished for women using estrogen with progestin (primarily MPA; RR - 0.39; 95% CI, 0.19-0.78) or estrogen alone (RR = 0.60; 95% CI, 0.43-0.83), after adjusting for an array of coronary risk factors. In one case-control study of women in Massachusetts [ 17], the relative risk of MI for ever use of combined therapy was 1.2 (95% CI, 0.6-2.4), although the confidence interval is quite wide, and the investigators also found no protection against MI for women using estrogen alone (RR = 0.9; 95% CI, 0.7-1.2). In a meta-analysis of all the studies of estrogen and progestin, the summary relative risk was 0.61 (95% CI, 0.45-0.82). Only three studies [33,57,61] have examined the relation of stroke with combined hormone therapy. Pedersen et al. [57] found no association between current use of combined therapy and subarachnoid hemorrhage (RR = 1.22), intracerebral hemorrhage (RR - 1.17), thromboembolic infarction (RR = 1.17), or transient ischemic attack (RR - 1.25). In the Nurses' Health Study [33], there was also no relation between current use of estrogen and progestin and stroke risk (RR = 1.09; 95% CI, 0.66-1.80). However, in the Swedish cohort, Falkeborn et al. [61] did report a reduced risk of stroke for users of estrogen and progestin (RR = 0.61; 95% CI, 0.40-0.88).

III. SECONDARY PREVENTION OF CARDIOVASCULAR D I S E A S E Espeland et al. [63] studied 186 postmenopausal women with evidence of early or subclinical carotid artery atherosclerosis who participated in a trial of lipid-lowering medication. During 4 years of follow-up, intimal media thickness progressed (mean = 0.015 mm/year) in women taking placebo who never used hormone therapy but regressed in those taking hormones (mean = 0.012 mm/year); hormone use had little additional benefit for the group of women assigned lovastatin. In addition to the longer term effects on atherosclerosis, short-term benefits may also be important. In a clinical trial of 11 postmenopausal women with coronary artery disease (>-70% stenosis of one or more coronary arteries), Rosano et al. [64] reported that estrogen administration (1 mg Estrace) 40 min before a treadmill test increased total exercise time and time to ST depression, and reduced symptoms on exertion in the women on estrogen compared to those given placebo. In the Leisure World Study [40], a prospective study, hormone users with a history of angina or myocardial infarction at baseline had approximately a 35% decreased risk of mortality compared to those not taking hormones (mean duration of hormone use at baseline was 8 years). Simi-

548 larly, in the Lipid Research Clinics cohort [36], among women with prevalent cardiovascular disease, those taking hormones (n = 74) had an 80% lower cardiovascular death rate than nonusers (n = 162) (death rates = 13.8/10,000 and 66.3/10,000, respectively; relative risk = 0.21; approximate 95% confidence interval, 0.03-1.6). Kim et al. [65] followed 293 women after coronary angioplasty, and compared the 100 subjects who used hormones both before angioplasty and during follow-up to 193 subjects who had never used hormones. Survival at 4 and 7 years was 98 and 95% in the estrogen group versus 90 and 78% in nonusers. In addition, survival or freedom from infarction at 7 years was 89% for women taking hormones and 66% for nonusers. In a similar study of 1091 women after coronary artery bypass surgery, Sullivan et al. [66] reported that hormone users had improved survival at 5 and 10 years (98.8 vs. 80.7% and 69.3 vs. 46.3%, respectively). After adjusting for confounding factors such as age and number of diseased vessels, hormone use was still inversely related with survival (relative risk = 0.34; p = 0.001). Finally, Sullivan et al. [48] conducted a study of 2268 women presenting for angiography: 446 with no detectable coronary artery disease, 644 with mild to moderate disease, and 1178 with severe disease. Among those with no disease, the 5-year survival of hormone users was the same as nonusers. However, among those with mild to moderate disease, estrogen users had better 5-year survival (98 vs. 91%). The difference was even more marked for those with severe disease (97 vs. 81%). Thus, the most substantial benefit was for women with the worst disease at baseline. Results from the Heart and Estrogen/progestin Replacement Study (HERS) [67], the first large-scale randomized trial of hormone therapy and cardiovascular disease, have been reported. A total of 2763 women with coronary disease were randomized to 0.625 mg of oral conjugated estrogen combined with 2.5 mg of continuous medroxyprogesterone acetate (n = 1380) or placebo (n = 1383). Overall, among these women with coronary disease, there was no overall protection against second cardiovascular events for women assigned to treatment compared to those given placebo (RR = 0.99; 95% CI, 0.80-1.22). However, there was a strong relation between duration of treatment and risk of major coronary events; in particular, there was a decreasing risk of heart disease with increasing duration of hormone use (p-trend = 0.009). In the first year of the trial, the risk of major coronary disease increased 52% among treated women, with most of this risk concentrated in the first 8 months of treatment; in the second year, there was no relation between treatment and disease (RR = 1.00), and in the third year the relative risk was 0.87. By the fourth to fifth years of the trial, rates of coronary events were 33% lower in women assigned to hormone therapy; the average duration of treatment was 4.1 years and 25% of women assigned to treatment had discontinued hormone use at the end

GRODSTEIN AND STAMPFER

of 3 years, thus the decreased risk observed during years 4 5 is likely a substantial underestimate of the benefits of hormone therapy. Although this decreased risk of coronary disease with long-term hormone therapy is consistent with the large body of observational evidence on primary prevention, the increased risk of second coronary events in the shortterm was unexpected; indeed, there are no other available data equivalent to those from HERS showing the short-term effects of hormone use on clinical disease. Perhaps a susceptible group of women experience adverse effects (i.e., thrombotic complications) of hormone therapy in the short-term. An increase in venous thrombosis is consistently supported by both observational studies and the HERS trial. Finally, for secondary prevention of stroke, in a trial of aspirin and stroke in patients who had experienced transient ischemic attacks [58], hormone users had a relative risk of 0.16 (p = 0.01) for stroke or death from any cause and a relative risk of 0.23 (p = 0.06) for stroke, compared to nonusers.

IV. A S S O C I A T I O N

OF HORMONES

WITH LOWER RISK OF CHD: CAUSE AND EFFECT

OR SELECTION?

The findings from the observational studies that hormone users are at generally lower risk from cardiovascular disease do not necessarily imply cause and effect. Women and their physicians decide on hormone therapy. Often the health status of women will have an important influence on this decision and on the results of studies that examine them. Thus, some have argued that hormone use is merely a marker rather than a cause of good health. Most of the observational studies reviewed here have provided some information bearing on this critical point. One way to judge the evidence for this position is to examine results of studies in which all the women were judged eligible by their physicians to receive hormone therapy. Only two small studies of primary prevention of CHD meet that criterion [30,31 ]; the summary relative risk from those two studies was 0.22 (95% CI, 0.06-0.88). These findings do not support the hypothesis that selection of healthy women for hormone use can explain the lower rate of CHD among users. With a similar intent, the Nurses' Health Study [33] tried to evaluate whether increased medical care of women using postmenopausal hormones might be responsible for the benefit observed. In an analysis limited to women who reported regular physician visits (50% of the cohort), results were similar to those found in the larger population of all subjects: the relative risk for major coronary heart disease was 0.52 (95% CI, 0.37-0.74) for current hormone use. Another approach is to examine the risk profile of hormone users and nonusers to determine if there is a consistent

549

CHAPTER 37 Cardiovascular Disease and Hormone Therapy

pattern of higher risk among the nonusers, and to assess whether the differences, if any, are sufficient to explain the large decrease in risk among hormone users. Barrett-Connor [70] observed that, in a cohort of postmenopausal women, those taking hormones reported more intensive health care behavior, including frequent screening tests such as blood cholesterol measurement and mammograms. An examination of determinants of hormone therapy in 9704 women participating in a large, multicenter study of osteoporotic fractures [71 ] found that hormone users tended to be better educated, less obese, and drank alcohol and participated in sports more often than nonusers. Similarly, in a prospective study of randomly selected premenopausal women, Matthews et al. [72] observed a better cardiovascular risk factor profile prior to hormone use among the women who subsequently took hormones at menopause than among women who did not. However, many of the large studies reviewed here are based in homogeneous groups, chosen because of their common profession or community. In the Nurses' Health Study, all women are registered nurses with access to health care and knowledge, and the distribution of established coronary risk factors was similar among current and never users of estrogens [33]. The same findings were observed in the Lipid Research Clinics Program Follow-up Study [36] (Table I). In both of these investigations, multivariate control for risk fac-

TABLE I

Risk Factor Profiles of Estrogen Users and Nonusers

Study of osteoporotic fractures a % Risk factor

Current

Past

Never

15.8

18.2

27.0

43.2 75.5

43.5 73.5

76.1

BMI --> 27.3 (kg/m2) d

Waist/hip ratio > 0.84

tors had only modest impact on the relative risk estimates. In the Leisure World Study [40] of women in a retirement community, the age-adjusted relative risk of all-cause mortality was 0.80 (95% CI, 0 . 7 0 - 0 . 8 7 ) for hormone users compared to nonusers; after further adjustment for high blood pressure, history of angina, MI or stroke, alcohol use, smoking, body mass index, and age at menopause, the relative risk was virtually the same (RR = 0.79; 95% CI, 0.71-0.88), implying an equivalent risk status for users and nonusers. In addition, to further examine this issue, the Nurses' Health Study conducted an analysis limited to a subgroup of low-risk women (i.e., those with no diagnosis of hypertension, diabetes, or high serum cholesterol who were nonsmokers and had a Quetelet's index below 32 kg/m2). Even with such restrictions, the relative risk for coronary disease was almost 40% lower for current hormone users. In summary, to explain the benefit as a result of confounding by health status, one would have to presume unknown risk factors that are extremely strong predictors of CHD and very closely associated with hormone use. Compliance with use of hormones has also been considered as a marker for low risk of heart disease [70], suggesting to some that the behavioral characteristics of hormone users are more important to their decreased risk of CHD than the hormone that they are taking. This argument is based on findings from clinical trials, in which subjects who were

Risk factor

Current

Past

Never

Current smoker

11.2

14.7

14.5

37.0 63.0

Hypertension Diabetes

23.2 2.7

25.0 3.8

21.8 3.5

74.8

66.4

9.9

11.2

28.6

36.2

41.1

10.6

28.1

29.8

33.5

High serum cholesterol Parental MI e before age 60 years Vigorous physical activity >-- once per week BMI --> 29 (kg/m 2)

aCauley et al. [71]. bStampfer e t al. [32]. CBush e t al. [36]. dBMI, Body mass index. eMI, Myocardial infarct.

Lipid Research Clinics Program c %

Nurses' Health Study b % Risk factor

Current/past Never

16

25

33 12

31 10

7.6

Alcohol

82

79

10.0

9.3

24.7

25.7

48.2

43.1

42.4

Mean BMI (kg/m 2) Age

53.8

52.6

9.8

13.3

15.0

129.0

127.7

79.9

79.5

234.8

235.2

Systolic blood pressure Diastolic blood pressure Cholesterol

550

compliant placebo takers had a better o u t c o m e than n o n c o m pliant subjects on placebo [73]. However, it is unclear to what extent these findings from drug therapy trials can be extended to the interpretation of observational studies, in which the subjects t h e m s e l v e s have chosen to use medication. It is possible, for example, that clinical trial participants with s y m p t o m s of preclinical disease m a y selectively stop taking their r a n d o m l y assigned regimen, perhaps explaining part of the apparent benefit of compliance. F u r t h e r m o r e , alt h o u g h few studies have specifically e x a m i n e d this issue, there does not consistently appear to be a greater protection against heart disease in l o n g - t e r m estrogen users c o m p a r e d to short-term users, indicating that the characteristic of longterm c o m p l i a n c e cannot explain the apparent benefit of horm o n e therapy. A l t h o u g h the overall null results of the H E R S trial have been suggested to d e m o n s t r a t e that " e x p e r i m e n t a t i o n trumps observation" [74], in fact no observational data are currently available to e x a m i n e h o r m o n e therapy in a way c o m p a r a b l e to the H E R S trial; there are no studies of c o m b i n e d horm o n e therapy for secondary prevention, and few observational studies have investigated the short-term effects of horm o n e use on cardiovascular disease. The data from H E R S on longer term h o r m o n e use (33% decrease with 4 - 5 years of treatment) are c o m p l e t e l y consistent with (and included in the 95% confidence interval for) the 4 0 % lower risk of myocardial infarction in the l o n g - t e r m prospective observational studies of p r i m a r y prevention.

V. SUMMARY The p r e p o n d e r a n c e of evidence from the epidemiologic studies strongly supports the view that p o s t m e n o p a u s a l horm o n e therapy can substantially reduce the risk for coronary heart disease in healthy w o m e n , although there appears to be an increased risk of venous t h r o m b o e m b o l i s m , and the data on stroke are inconclusive. The only large-scale clinical trial of h o r m o n e use indicates that c o m b i n e d therapy in w o m e n with established coronary disease leads to a short-term increase in the risk of second events, with a longer term decrease that is consistent with the observational data. Although the results from general population studies may be influenced in part by inherent characteristics of h o r m o n e users and nonusers, m u c h of the epidemiologic data are derived from studies in more h o m o g e n e o u s populations in which health status and health-seeking behavior are not largely related to estrogen use. Clinical trials of h o r m o n e therapy for p r i m a r y prevention are currently underway, but it will be at least a decade before their o u t c o m e is known. Findings from observational studies, in conjunction with the abundant biologic evidence described elsewhere in this book, lend substantial foundation for a causal relation between h o r m o n e therapy and r e d u c e d risk of heart disease.

GRODSTEIN AND STAMPFER

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552 57. Pedersen, A. T., Lidegaard, O., Kreiner, S., and Ottesen, B. (1997). Hormone replacement therapy and risk of non-fatal stroke. Lancet 350, 1277-1283. 58. American-Canadian Co-operative Study Group (1986). Persantine aspirin trial in cerebral ischemia. Part III: Risk factors for stroke. Stroke 17, 12-18. 59. Paganini-Hill, A., Ross, R. K., and Henderson, B. E. (1988). Postmenopausal oestrogen treatment and stroke: A prospective study. Br. Med. J. 297, 519-522. 60. Boysen, G., Nyobe, J., Appleyard, M., Sorensen, E S., Boas, J., Somnier, F., Jensen, G., and Schnohr, E (1988). Stroke incidence and risk factors for stroke in Copenhagen, Denmark. Stroke 19, 1345-1353. 61. Falkeborn, M., Persson, I., Terent, A., Adami, H. O., Lithell, H., and Bergstrom, R. (1993). Hormone replacement therapy and the risk of stroke. Arch. Intern. Med. 153, 1201-1209. 62. Grodstein, F., Stampfer, M. J., Colditz, G. A., Willett, W. C., Manson, J. E., Joffe, M., Rosner, B., Fuchs, C., Hankinson, S. E., Hunter, D. J., Hennekens, C. H., and Speizer, F. E. (1997). Postmenopausal hormone therapy and mortality. N. Engl. J. Med. 336, 1769-1775. 63. Espeland, M. A., Applegate, W., Furberg, C. D., Lefkowitz, D., Rice, L., and Huninnghake, D. (1995). Estrogen replacement therapy and progression of intimal-medial thickness in the carotid arteries of postmenopausal women. Am. J. Epidemiol. 142, 1011-1019. 64. Rosano, G. M. C., Sarrel, P. M., Poole-Wilson, P. A., and Collins, P. (1993). Beneficial effect of estrogen on exercise-induced myocardial ischaemia in women with coronary artery disease. Lancet 342, 133-36. 65. Kim, S. C., O'Keefe, J. H., Ligon, R. W., Giorgi, L. V., Cavero, P. G., and Hartzler, G. O. (1995). Estrogen improves long-term outcome after coronary angioplasty. Circulation 92 (suppl. 1), 674 (abstr.).

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66. Sullivan, J. M., E1-Zeky, F., Vander Zwaag, R., and Ramanathan, K. B. (1994). Estrogen replacement therapy after coronary artery bypass surgery: Effect on survival. J. Am. Coll. Cardiol. 23, 49A. 67. Hulley, S., Grady, D., Bush, T., Furberg, C., Herrington, D., Riggs, B., and Vittinghoff, E. (1998). Randomized trial of estrogen plus progestin for secondary prevention of coronary heart disease in postmenopausal women. JAMA, J. Am. Med. Assoc. 280, 605-613. 68. Sarrel, E M., Lindsay, D., Rosano, G. M. C., and Poole-Wilson, E A. (1992). Angina and normal coronary arteries in women: Gynecologic findings. Am. J. Obstet. Gynecol. 167, 467-472. 69. Sullivan, J. M., Shala, B. A., Miller, L. A., Lerner, J. L., and McBrayer, J. D. (1995). Progestin enhances vasoconstrictor responses in postmenopausal women receiving estrogen replacement therapy. Menopause 2, 193-199. 70. Barrett-Connor, E. (1991). Postmenopausal estrogen and prevention bias. Ann. Intern. Med. 115,455-456. 71. Cauley, J. A., Cummings, S. R., Black, D. M., Mascioli, S. R., and Seeley, D. G. (1990). Prevalence and determinants of estrogen replacement therapy in elderly women. Am. J. Obstet. Gynecol. 163, 14381444. 72. Matthews, K. A., Kuller, L. H., Wing, R. R., Meilahn, E. N., and Plantinga, P. (1996). Prior to estrogen replacement therapy, are users healthier than nonusers? Am. J. Epidemiol. 143, 971-978. 73. Coronary Drug Project Research Group (1980). Influence of adherence to treatment and response of cholesterol on mortality in the coronary drug project. N. Engl. J. Med. 303, 1038-1041. 74. Petitti, D. B. (1998). Hormone replacement therapy and heart disease prevention: Experimentation trumps observation. JAMA, J. Am. Med. Assoc. 280, 650-652.

~ H A P T E R 3t

Interventions for the Control of Symptoms LEON SPEROFF

Department of Obstetrics and Gynecology, Oregon Health Sciences University, Portland, Oregon 97201

I. Introduction II. Disturbances in Menstrual Patterns III. Symptoms Due to a Decrease in Estrogen

IV. Symptoms Associated with Progestin Treatment V. Conclusion References

I. I N T R O D U C T I O N

to associate the middle years of life with negative experiences. The events that come to mind are impressive: onset of a major illness or disability [and even death] in a spouse, relative, or friend; retirement from employment; financial insecurity; the need to provide care for very old parents and relatives; and separation from children. And thus, it is not surprising that a middle-age event, the menopause, shares in this negative outlook. The scientific study of all aspects of menstruation has been hampered by the overpowering influence of social and cultural beliefs and traditions. Problems arising from life events have often been erroneously attributed to the menopause. But data [especially more reliable community-based longitudinal data] now establish that the increase in most symptoms and problems in middle-aged women reflects social and personal circumstances, not the endocrine events of the menopause [1-7]. The Massachusetts Women's Health Study, a large and comprehensive prospective, longitudinal study of middleaged women, provides a powerful argument that the menopause is not and should not be viewed as a negative experience by the vast majority of women [4,8]. The menopausal cessation of menses was perceived by these women (as by the women in other longitudinal studies) as having almost

Changes in menstrual function are not symbols of some ominous "change." There are good physiologic reasons for changing menstrual function, and understanding the physiology will do much to reinforce a healthy, normal attitude. Attitude and expectations about the menopause are very important. Women who have been frequent users of health services and who expect to have difficulty at the menopause do experience more symptoms and higher levels of depression [1,2]. The symptoms that women report are related to many variables within their lives, and the hormonal change at menopause cannot be held responsible for the common psychosocial and life style problems we all experience. It is time to stress the normalcy of this physiologic event. Menopausal women do not suffer from a disease [specifically a hormone deficiency disease], and postmenopausal hormone therapy should be viewed as specific treatment for symptoms in the short term and preventive pharmacology in the long term. The belief that behavioral disturbances are related to manifestations of the female reproductive system is an ancient one that has persisted to contemporary times. This belief regarding the menopause is not totally illogical; there is reason

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BIOLOGY AND PATHOBIOLOGY

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no impact on subsequent physical and mental health. This was reflected by women expressing either positive or neutral feelings about menopause. An exception was the group of women who experienced surgical menopause, but here ~ there is good reason to believe that the reasons for the surgical procedure were more important than the cessation of menses. There are two overall objectives, one longterm and one shortterm, for medical intervention at the perimenopausal time of life. A long-term goal is to provide and reinforce a program of preventive health care. The issues of preventive health care for women are familiar ones. They include family planning, cessation of smoking, control of body weight and alcohol consumption, prevention of cardiovascular disease and osteoporosis, maintenance of mental wellbeing (including sexuality), cancer screening, and treatment of urologic problems. The short-term objective is to help patients alleviate disruptive symptoms. The symptoms frequently seen and related to decreasing ovarian follicular competence and then estrogen loss during the perimenopausal and early postmenopausal years are as follows: 1. Disturbances in menstrual pattern, including anovulation and reduced fertility, decreased flow or hypermenorrhea, irregular frequency of menses, and, then ultimately, amenorrhea. 2. Vasomotor instability (hot flushes and sweats). 3. Atrophic conditions: atrophy of vaginal epithelium; formation of urethral caruncles; dyspareunia and pruritus due to vulvar, introital, and vaginal atrophy; general skin atrophy; urinary difficulties such as urgency and abacterial urethritis and cystitis.

II. D I S T U R B A N C E S IN MENSTRUAL

PATTERNS

Throughout the perimenopausal period, there is a significant incidence of dysfunctional uterine bleeding. Although the greatest concern provoked by this symptom is endometrial neoplasia, the usual finding is nonneoplastic tissue displaying estrogen effects unopposed by progesterone. This results from anovulation in premenopausal women and from extragonadal endogenous estrogen production or estrogen administration in postmenopausal women. In all women, whether premenopausal or postmenopausal, whether on or off hormone therapy, specific organic causes (neoplasia, complications of unexpected pregnancy, or bleeding from extrauterine sites) must be ruled out. In addition to careful history and physical examination, dysfunctional uterine bleeding requires evaluation. Transvaginal ultrasonographic measurement of endometrial thickness can be utilized in postmenopausal women to avoid unnecessary biop-

sies [9]. A total endometrial thickness greater than 4 mm requires biopsy. If the uterus is normal on examination, for reasons of both accuracy and cost-effectiveness, the method of biopsy should be an office aspiration curettage. Less than 10% of postmenopausal women cannot be adequately evaluated by office biopsy. Most commonly, the reason is the inability to enter the uterine cavity. In such instances, an in-hospital dilatation and curettage [D and C] is in order. Furthermore, if the uterus is not normal on pelvic examination, the office endometrial biopsy must yield to an in-hospital D and C with hysteroscopy in order to achieve accuracy of diagnosis. If vulva, vagina, and cervix appear normal on inspection, perimenopausal bleeding can be assumed to be intrauterine in origin. Confirmation requires the absence of abnormal cytology on the Pap smear. The principal symptom of endometrial cancer is abnormal vaginal bleeding, but carcinoma will be encountered in patients with bleeding in only about 1-2% of postmenopausal endometrial biopsies [10,11]. Normal endometrium is found over half the time, polyps in approximately 3%, endometrial hyperplasia about 15% of the time, and atrophic endometrium in the rest of patients with postmenopausal bleeding. Postmenopausal bleeding should always be taken seriously. Approximately 10% of patients who have benign findings at the initial evaluation will subsequently develop significant pathology within 2 years [ 11]. The persistence of abnormal bleeding demands repeated evaluation. Additional procedures include the following: 1. Colposcopy and cervical biopsy for abnormal cytology or obvious lesions. 2. Endocervical assessment by curettage for abnormal cytology (the endocervix must always be kept in mind as a source for abnormal cytology). 3. Hysterogram, hysteroscopy, or ultrasonography with the uterine instillation of saline if bleeding persists to determine the presence of endometrial polyps or submucosal fibroids. The pathologic reading, "tissue insufficient for diagnosis," when a patient is on estrogen/progestin treatment, often represents atrophic, decidualized endometrium that yields little to the biopsy instrument. The clinician must be confident in his or her technique, knowing that a full investigation of the intrauterine cavity has been accomplished, then as long as the patient does not persist in bleeding, this reading can be interpreted as comforting and benign, the absence of pathology. In the absence of organic disease, appropriate management of uterine bleeding is dependent on the age of the woman and endometrial tissue findings. In the perimenopausal woman with dysfunctional uterine bleeding associated with proliferative or hyperplastic endometrium (uncomplicated by atypia or dysplastic constituents), periodic oral

CHAPTER38 Interventions for the Control of Symptoms progestin therapy is mandatory, such as 5 - 1 0 mg medroxyprogesterone acetate (or an equivalent amount of other progestins) given daily for at least the first 10 days of each month. If hyperplasia is present, follow-up aspiration curettage after 3 - 4 months is required, and if progestin treatment is ineffective and histological regression is not observed, formal curettage is an essential preliminary to alternate therapeutic surgical choices. Because hyperplasia with atypia carries with it a risk of cancer [even invasive], hysterectomy is the treatment of choice. When monthly progestin therapy reverses hyperplastic changes [which it does in 9 5 - 9 8 % of cases] and controls irregular bleeding, treatment should be continued until withdrawal bleeding ceases. This is a reliable sign (in effect, a bioassay) indicating the onset of estrogen deprivation and the need for the addition of estrogen. If vasomotor disturbances begin before the cessation of menstrual bleeding, the combined estrogen/progestin postmenopausal program can be initiated as needed to control the flushes.

5 55 portant to change because even with the lowest dose oral estrogen contraceptive available, the estrogen dose is fourfold greater than the standard postmenopausal dose, and, with increasing age, the dose-related risks with estrogen become significant. One approach to establish the onset of the postmenopausal years is to measure the concentration of folliclestimulating hormone (FSH) annually, beginning at age 50, being careful to obtain the blood sample on day 6 or 7 of the pill-free week (when steroid levels have declined sufficiently to allow FSH to rise). Friday afternoon works well for patients who start new packages on Sunday. When FSH is greater than 20 IU/liter, it is time to change to a postmenopausal hormone program. Because of the variability in FSH levels experienced by women around the menopause, this method is not always accurate [ 14]. But there is no harm in retesting after another year or two on low-dose oral contraceptives. Some clinicians are comfortable allowing patients to enter their mid-50s on low-dose oral contraception, and then empirically switching to a postmenopausal hormone regimen.

A. Oral Contraceptives for Older Women If contraception is required, the healthy, nonsmoking perimenopausal older woman should seriously consider the use of oral contraception. The anovulatory woman cannot be guaranteed that spontaneous ovulation and pregnancy will not occur. The use of a low-dose oral contraceptive will at the same time provide contraception and prophylaxis against irregular, heavy anovulatory bleeding and the risk of endometrial hyperplasia and neoplasia. A traditional postmenopausal hormone regimen is often utilized to treat a woman with the kind of irregular cycles usually experienced in the perimenopausal years. This addition of exogenous estrogen without a contraceptive dose of progestin when a woman is not amenorrheic or experiencing menopausal symptoms is inappropriate and even risky (exposing the endometrium to excessively high levels of estrogen). And most importantly, a postmenopausal hormonal regimen does not inhibit ovulation and provide contraception [12]. The appropriate response is to regulate anovulatory cycles with monthly progestational treatment along with an appropriate contraceptive method or to utilize low-dose oral contraception. An oral contraceptive that contains 20/zg estrogen (the lowest available dose) provides effective contraception, improves menstrual cycle regularity, diminishes bleeding, and relieves menopausal symptoms [ 13].

III. S Y M P T O M S D U E TO A D E C R E A S E IN E S T R O G E N The menopause should serve to remind patients and clinicians that this is a time for education. Certainly preventive health care education is important throughout life, but at the time of the menopause, a review of the major health issues can be especially rewarding. Besides the general issues of good health, attention is now being focused (partly because of their relationship to postmenopausal hormone therapy) on cardiovascular disease and osteoporosis. During the menopausal years, some women will experience severe multiple symptoms, whereas others will show no reactions or minimal reactions that can go unnoticed until careful medical evaluation. The differences in menopausal reactions and symptoms across different cultures are poorly documented, and indeed, they are difficult to ascertain. Individual reporting is so conditioned by sociocultural factors that it is hard to determine what is due to biological versus cultural variability [ 15,16]. For example, there is no word to describe a hot flush in Japanese, Chinese, and Mayan [ 17]. Nevertheless, there is reason to believe that the nature and prevalence of menopausal symptoms are common to most women, and that variations among cultures and within cultures reflect not physiology, but differences in attitudes, societies, and individual perceptions [ 18-21 ].

B. Changing from Oral Contraceptives to Postmenopausal Hormone Therapy

A. Vasomotor Instability ~ The Hot Flush

A common clinical dilemma is when to change from oral contraception to postmenopausal hormone therapy. It is im-

The vasomotor flush is viewed as the hallmark of the female climacteric, experienced to some degree by most

556 postmenopausal women (see Chapter 14). The term hotflush is descriptive of a sudden onset of reddening of the skin over the head, neck, and chest, accompanied by a feeling of intense body heat and concluded by sometimes profuse perspiration. The duration varies from a few seconds to several minutes and, rarely, for an hour. The frequency may be rare to recurrent every few minutes. Flushes are more frequent and severe at night (when a woman is often awakened from sleep) or during times of stress. In a cool environment, hot flushes are fewer, less intense, and shorter in duration compared with a warm environment [22]. In one longitudinal follow-up of a large number of women, fully 10% of the women experienced hot flushes before menopause, whereas in other studies as many as 1525% of premenopausal women reported hot flushes [2,2325]. The frequency has been reported to be even higher in premenopausal women diagnosed with premenstrual syndrome [26]. In the Massachusetts Women's Health Study, the incidence of hot flushes increased from 10% during the premenopausal period to about 50% just after cessation of menses [23]. By approximately 4 years after menopause, the rate of hot flushes declined to 20%. In a community-based Australian survey, 6% of premenopausal women, 26% of perimenopausal women, and 59% of postmenopausal women reported hot flushing [27]. Although the flush can occur in the premenopause, it is a major feature of postmenopause, lasting in most women for 1-2 years, but in some [as many as 25%] for longer than 5 years. In cross-sectional surveys, up to 40% of premenopausal women and 85% of menopausal women report vasomotor complaints [25]. In the United States, there is no difference in the prevalence of vasomotor complaints in surveys of black and white women [28,29]. The exact estimates on prevalence are hampered by inconsistencies and differences in methodologies, cultures, and definitions [30]. The physiology of the hot flush is still not understood, but it apparently originates in the hypothalamus and is brought about by a decline in estrogen. However, not all hot flushes are due to estrogen deficiency. Unfortunately, the hot flush is a relatively common psychosomatic symptom, and women often are unnecessarily treated with estrogen. When the clinical situation is not clear and obvious, estrogen deficiency as the cause of hot flushes should be documented by elevated levels of FSH. The correlation between the onset of flushes and estrogen reduction is clinically supported by the effectiveness of estrogen therapy and the absence of flushes in hypoestrogen states, such as gonadal dysgenesis. Only after estrogen is administered and withdrawn do hypogonadal women experience the hot flush. Although the clinical impression that premenopausal surgical castrates suffer more severe vasomotor reactions is widely held, this is not borne out in objective study [31 ].

LEON SPEROFF

Although the hot flush is the most common problem of the postmenopause, it presents no inherent health hazard. The flush is accompanied by a discrete and reliable pattern of physiologic changes [32]. The flush coincides with a surge of luteinizing hormone (LH), not FSH, and is preceded by a subjective prodromal awareness that a flush is beginning. This aura is followed by measurable increased heat over the entire body surface. The body surface experiences an increase in temperature, accompanied by changes in skin conductance, and followed by a fall in core temperaturem all of which can be objectively measured. In short, the flush is not a release of accumulated body heat but is a sudden inappropriate excitation of heat release mechanisms. Its relationship to the LH surge and temperature change within the brain is not understood. The observation that flushes occur after hypophysectomy indicates that the mechanism is not dependent on or due directly to LH release. In other words, the same hypothalamic event that causes flushes also stimulates gonadotropin-releasing hormone [GnRH] secretion and elevates LH. This is probably secondary to hypothalamic changes in neurotransmitters that increase neuronal and autonomic activity [33]. Premenopausal women experiencing hot flushes should be screened for thyroid disease and other illnesses. Flushes and sweating can be secondary to diseases, including pheochromocytoma, carcinoid, leukemias, pancreatic tumors, and thyroid abnormalities [34,35]. Clinicians should be sensitive to the possibility of an underlying emotional problem. Looking beyond the presenting symptoms into the patient's life will be an important service to the patient and her family that eventually will be appreciated. This is far more difficult than simply prescribing estrogen, but confronting problems is the only way of reaching some resolution. Prescribing estrogen inappropriately [in the presence of normal levels of gonadotropins] only temporarily postpones by a placebo response dealing with the underlying issues. A striking and consistent finding in most studies dealing with menopause and hormonal therapy is a marked placebo response in a variety of symptoms, including flushing. In an English randomized, placebo-controlled study of women being treated with estrogen implants and requesting repeat implants, there was no difference in outcome in terms of psychological and physical symptoms comparing the women who received an active implant to those receiving a placebo [36]. A common clinical problem encountered is described in the following scenario: a woman will occasionally undergo an apparent beneficial response to estrogen, only to have the response wear off in several months. This leads to a sequence of periodic visits to the clinician and ever-increasing doses of estrogen. When a patient reaches a point of requiring large doses of estrogen, a careful inquiry must be undertaken to search for a basic psychoneurotic or psychosocial problem. To help persuade a patient that her symptoms are not due to

CHAPTER38 Interventions for the Control of Symptoms low levels of estrogen, it is very helpful and convincing to measure the patient's blood level of estradiol and share the result with her. A summary of the data on the hot flush is given below: Premenopausal hot flush: Postmenopausal hot flush: No flushes: Daily flushing: Duration:

10-25% of women 15-25% of women 15-20% of women 1-2 years average; 5 or more years: 25%

Other causes: Psychosomatic Stress Thyroid disease Pheochromocytoma Carcinoid Leukemia Cancer OTHER TREATMENT OPTIONS FOR HOT FLUSHES

When patients with hot flushing cannot take estrogen, transdermal clonidine, applied with the 100/xg dose once weekly, can be effective [37,38]. Side effects are minimal and a modest impact can be expected. Clonidine, bromocriptine, and naloxone given orally are only partially effective for the relief of hot flushes and require high doses with a high rate of side effects. Bellergal (a combination of belladonna alkaloids, ergotamine tartrate, and phenobarbital) treatment is slightly better than a placebo, but it is also a potent sedative [39]. Veralipride, a dopamine antagonist that is active in the hypothalamus, is relatively effective in inhibiting flushing at a dose of 100 mg daily [40,41 ]. Mastodynia and galactorrhea are the major side effects. Medroxyprogesterone acetate (10-20 mg daily) and megestrol acetate (20 mg twice daily) are also effective, but concerns regarding exogenous steroids (especially in patients who have had breast cancer) would apply to progestins as well [42,43]. Methyldopa, in doses of 500-1000 mg/day, is approximately twice as effective as a placebo, suggesting a role for adrenoreceptors in the hot flush mechanism [44]. Venlafaxine hydrochloride is an antidepressant that inhibits serotonin reuptake; it effectively reduced hot flush frequency in a dose of 25 mg daily [45]. Vitamin E, 800 IU daily, is only slightly more effective than placebo [46]. Tibolone Tibolone is a steroid, related to the 19-nortestosterone family, that is effective for preventing bone loss and treating hot flushes in a dose of 2.5 mg/day [47,48]. Tibolone is metabolized into three steroid isomers with varying estrogenic, progestogenic, and androgenic properties. The metabolites differ in their activities and dominance accord-

557 ing to the target tissue. Thus, tibolone provides estrogenic effects on bone and hot flushing, but it induces atrophy of the endometrium [49]. Its beneficial impact on bone (2.5-mg dose) is comparable to standard hormonal therapy [50]. A lower dose of 1.25 mg daily also provides bone protection, but it is less effective and there is more vaginal bleeding. In the endometrium, tibolone is converted locally (by endometrial 3fl-hydroxysteroid dehydrogenase/isomerase) to its A4-progestational isomer; hence, tibolone exerts a progestational effect on the endometrium [51]. Tibolone has an estrogenic effect on the vagina, and women report improvements in the symptoms of vaginal dryness and dyspareunia and an increase in sexual enjoyment and libido [52]. Although a short-term reduction in high-density lipoprotein (HDL) cholesterol is an undesirable consequence with tibolone treatment, the long-term impact on the risk of cardiovascular disease is unknown [53]. In a 2-year study, the unfavorable effect on lipoproteins was accompanied by beneficial changes in coagulation factors consistent with enhanced fibrinolysis and unchanged coagulation [54]. Overall, it is possible that some favorable activity on the cardiovascular system is maintained. A major advantage of tibolone (2.5 mg daily) is its low (10-20%) incidence of causing bleeding. Because tibolone inhibits breast cell proliferation in vitro, it is possible that future studies will indicate that tibolone offers some protection against breast cancer. Tibolone also has a beneficial impact in short-term studies on insulin resistance in normal women and in women with non-insulin-dependent diabetes mellitus [55,56].

B. A t r o p h i c C o n d i t i o n s With extremely low estrogen production, atrophy of vaginal mucosal surfaces takes place, accompanied by vaginitis, pruritus, dyspareunia, and stenosis. Genitourinary atrophy leads to a variety of symptoms that affect the ease and quality of living. Urethritis with dysuria, urgency incontinence, and urinary frequency are further results of mucosal thinning, in this instance, of the urethra and bladder. Recurrent urinary tract infections are effectively prevented by postmenopausal intravaginal estrogen treatment [57]. Vaginal relaxation with cystocele, rectocele, and uterine prolapse, and vulvar dystrophies are not a consequence of estrogen deprivation. Although it is argued that genuine stress incontinence will not be affected by treatment with estrogen, others contend that estrogen treatment improves or cures stress incontinence in over 50% of patients due to a direct effect on the urethral mucosa [58,59]. Most cases of urinary incontinence in elderly women are a mixed problem with a significant component of urge incontinence that definitely can be improved by estrogen therapy. Dyspareunia seldom brings older women to our offices. A

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basic reluctance to discuss sexual behavior still permeates our society, especially among older patients and physicians. Gentle questioning may lead to estrogen treatment of atrophy and enhancement of sexual enjoyment. Objective measurements have demonstrated that vaginal factors that influence the enjoyment of sexual intercourse can be maintained by appropriate doses of estrogen [60]. Both patient and clinician should be aware that a significant response can be expected by 1 month, but it takes a long time to restore the genitourinary tract fully (6-12 months), and clinicians and patients should not be discouraged by an apparent lack of immediate response. Furthermore, sexual activity by itself supports the circulatory response of the vaginal tissues and enhances the therapeutic effects of estrogen. Therefore sexually active older women will have less atrophy of the vagina even without estrogen. A decline in skin collagen content and skin thickness that occurs with aging can be considerably avoided by postmenopausal estrogen therapy [61-64]. The effect of estrogen on collagen is evident in both bone and skin; bone mass and collagen decline in parallel after menopause, and estrogen treatment reduces collagen turnover and improves collagen quality [65,66]. Although it is uncertain whether estrogen treatment can affect physical appearance, at least one study demonstrated not only an increase in facial skin thickness, but an improvement in wrinkles with topical estrogen [67]. More impressively, data from the United States First National Health and Nutrition Examination Survey indicated that estrogen use was associated with a lower prevalence of skin wrinkling and dry skin [68]. However, smoking is a major risk factor for facial skin wrinkling, and hormone therapy cannot diminish this impact of smoking [69]. One of the features of aging in men and women is a steady reduction in muscular strength. Many factors affect this decline, including height, weight, and level of physical activity. However, women currently using estrogen have been reported to demonstrate a lesser decline in muscular strength, although at least one study could detect no impact of estrogen [70-72]. This is an important issue because of the potential protective consequences against fractures, as well as a benefit due to the ability to maintain vigorous physical exercise.

C. M e n o p a u s e a n d M e n t a l H e a l t h The view that menopause has a deleterious effect on mental health is not supported in the psychiatric literature or in surveys of the general population (see Chapter 39) [24,25, 73,74]. The concept of a specific psychiatric disorder (involutional melancholia) has been abandoned. Indeed, depression is less common, not more common, among middle-aged women, and the menopause cannot be linked to psychological distress [1-7,75]. The longitudinal study of premeno-

pausal women indicates that hysterectomy with or without oophorectomy is not associated with a negative psychological impact among middle-aged women [76]. And longitudinal data from the Massachusetts Women's Health Study document that menopause is not associated with an increased risk of depression [77]. Although women are more likely to experience depression than men, this sex difference begins in early adolescence, not at menopause [78]. The United States National Health Examination Followup Study includes both longitudinal and cross-sectional assessments of a nationally representative sample of women. This study has found no evidence linking either natural or surgical menopause to psychologic distress [79]. Indeed, the only longitudinal change was a slight decline in the prevalence of depression as women aged through the menopausal transition. Results in this study were the same in estrogen users and nonusers. A negative view of mental health at the time of the menopause is not justified; many of the problems reported at the menopause are due to the vicissitudes of life [80,81]. Thus, there are problems encountered in the early postmenopause that are seen frequently, but their causal relation with estrogen is unlikely. These problems include fatigue, nervousness, headaches, insomnia, depression, irritability, joint and muscle pain, dizziness, and palpitations. Indeed, men and women at this stage of life both express a multitude of complaints, but these do not reveal a gender difference that could be explained by a hormonal cause [82]. Attempts to study the effects of estrogen on these problems have been hampered by the subjectivity of the complaints (high placebo responses) and the "domino" effect of what reduction of hot flushes does to the frequency of the symptoms. Using a double-blind cross-over prospective study format, Campbell and Whitehead concluded many years ago that many symptomatic "improvements" ascribed to estrogen therapy result from relief of hot flushesma domino effect [83]. A study of 2001 Australian women aged 45-55 years focused on the utilization of the health care system by women in the perimenopausal period of life [84]. Users of the health care system in this age group were frequent previous users of health care, less healthy, and had more psychosomatic symptoms and vasomotor reactions. These women were more likely to have had a significant previous adverse health history, including a past history of premenstrual complaints. This study emphasized that perimenopausal women who seek health care help are different from those who do not seek help, and they often embrace hormone therapy in the hope it will solve their problems. Similar findings have been reported in a cohort of British women [85]. It is this population that is seen most often by clinicians, producing biased opinions regarding the menopause among physicians. We must be careful not to generalize to the entire female population the behavior experienced by this relatively small group

CHAPTER38 Interventions for the Control of Symptoms of women. Most importantly, perimenopausal women who present to clinicians often end up being treated with estrogen inappropriately and unnecessarily. Nevertheless, it is wellestablished that a woman's quality of life is disrupted by vasomotor symptoms, and estrogen therapy provides impressive improvement [86,87]. Patients are grateful to be the recipients of this "domino" effect. Emotional stability during the perimenopausal period can be disrupted by poor sleep patterns. Hot flushing does have an adverse impact on the quality of sleep [88]. Estrogen therapy improves the quality of sleep, decreasing the time to onset of sleep and increasing the rapid eye movement (REM) sleep time [86,89,90]. Perhaps flushing may be insufficient to awaken a woman but sufficient to affect the quality of sleep, thereby diminishing the ability to handle the next day's problems and stresses. An improvement in insomnia with estrogen treatment can even be documented in postmenopausal women who are reportedly asymptomatic [90]. Thus, the overall "quality of life" reported by women can be improved by better sleep and alleviation of hot flushing. However, it is still uncertain whether estrogen treatment has an additional direct pharmacologic antidepressant effect or whether the mood response is an indirect benefit of relief from physical symptoms and, consequently, improved sleep. Utilizing various assessment tools for measuring depression, improvements with estrogen treatment have been recorded in oophorectomized women [91,92]. In the large prospective cohort study of the Rancho Bernardo retirement community, no benefit could be detected in measures of depression in current users of postmenopausal estrogen compared with untreated women [93]. Indeed, treated women had higher depressive symptom scores, presumably reflecting treatment selection bias; symptomatic and depressed women seek hormone therapy. Nevertheless, estrogen therapy is reported to have a more powerful impact on women's wellbeing beyond the relief of symptoms such as hot flushes [94]. In elderly depressed women, improvements in response to fluoxetine were enhanced by the addition of estrogen therapy [95].

IV. S Y M P T O M S

ASSOCIATED

WITH

PROGESTIN TREATMENT Many women do not tolerate treatment with progestational hormones. Typical side effects include breast tenderness, bloating, and depression. These reactions are significant detrimental factors with continuance of treatment. However, appropriately designed, placebo-controlled studies fail to document adverse physical or psychological effects with short-term treatment utilizing medroxyprogesterone acetate [96,97]. This suggests that the side effects of medroxyprogesterone acetate are related to duration of treatment or that only studies with large numbers of subjects will detect the

559 small percentage of women who have problems (and both explanations are probably true). Can the progestational agent be administered less frequently? We are secure in our position, supported by clinical data, that a monthly estrogen/progestin sequential or a daily combination program effectively prevents endometrial hyperplasia. Experience with other regimens is very limited. The administration of medroxyprogesterone acetate every 3 months was associated in one study with longer, heavier menses and unscheduled bleeding, and a 1.5% incidence of hyperplasia at 1 year, whereas in another study, overall bleeding was less, but the incidence of hyperplasia was approximately 4% [98,99]. In yet another study, there was no endometrial hyperplasia encountered by 143 women who completed 2 years of treatment; however, the progestin administered every 3 months was of high dosage, 20 mg medroxyprogesterone acetate daily for 14 days [ 100]. Most impressively, the Scandinavian Long Cycle Study, a clinical trial scheduled to last 5 years, was canceled after 3 years because of a 12.5% incidence of endometrial hyperplasia and one case of endometrial cancer [101]. Therefore, if a patient chooses this regimen, more intensive endometrial monitoring is required; an annual endometrial biopsy is strongly recommended. Indeed, any program that differs from the standard regimen is untested by clinical studies of sufficient length and patient numbers and, therefore, requires periodic surveillance of the endometrium. Some patients are very sensitive to medroxyprogesterone acetate. These patients are often relieved of their symptoms by switching to norethindrone. In a sequential regimen, the dose of norethindrone is 0.7 mg [available in the progestinonly, minipill oral contraceptive; each pill contains 0.35 mg norethindrone]. In the continuous, combined regimen, the dose of norethindrone is 0.35 mg daily. Progesterone can be administered in a vaginal gel that allows the delivery of very low doses that can effectively protect the endometrium with low systemic levels because of a first-pass effect on the uterus [102]. The administration of 90 mg every 2 days produces secretory changes in the endometrium [ 103]. An application of the 4% commercial preparation twice weekly protects the endometrium and is associated with amenorrhea in most patients. In a sequential regimen, the one applicator of the 4% preparation should be applied daily for at least 10 days each month. No long-term studies are available documenting endometrial safety and metabolic effects.

V. C O N C L U S I O N The menopause is a physiologic event that brings clinicians and patients together, providing the opportunity to enroll patients in health maintenance. The failure to respond

560

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appropriately (by either clinician or patient) easily leads to a loss of the patient from a practice, but equally, if not more, important is the probability that the loss of a patient from a practice means that another woman has lost her involvement in a p r e v e n t i v e h e a l t h c a r e p r o g r a m . opinion, the menopause

Contrary

to p o p u l a r

is n o t a s i g n a l o f i m p e n d i n g d e c l i n e ,

but, r a t h e r , a w o n d e r f u l p h e n o m e n o n

that can signal the start

of something positive, a good health program.

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92. Sherwin, B. B. (1988). Affective changes with estrogen and androgen replacement therapy in surgically menopausal women. J. Affective Disord. 14, 177-187. 93. Palinkas, L. A., and Barrett-Connor, E. (1992). Estrogen use and depressive symptoms in postmenopausal women. Obstet. Gynecol. 80, 30-36. 94. Limouzin-Lamothe, M.-A., Mairon, N., Joyce, C. R. B., and Le Gal, M. (1994). Quality of life after the menopause: Influence of hormonal replacement therapy. Am. J. Obstet. Gynecol. 170, 618-624. 95. Schneider, L. S., Small, G. W., Hamilton, S. H., Bystritsky, A., Nemeroff, C. B., Meyers, B. S., and the Fluoxetine Collaborative Study Group (1997). Estrogen replacement and response to fluoxetine in a multicenter geriatric depression trial. Am. J. Geriatr. Psychiatry 5, 97-106. 96. Kirkham, C., Hahn, E M., Van Vugtl, D. A., Carmichael, J. A., and Reid, R. L. (1991). A randomized, double-blind, placebo-controlled, cross-over trial to assess the side effects of medroxyprogesterone acetate in hormone replacement therapy. Obstet. Gynecol. 78, 93-97. 97. Prior, J. C., Alojado, N., McKay, D. W., and Vigna, Y. M. (1994). No adverse effects of medroxyprogesterone treatment without estrogen in postmenopausal women: Double-blind, placebo-controlled, crossover trial. Obstet. Gynecol. 83, 24-28. 98. Ettinger, B., Selby, J., Citron, J. T., Vangessel, A., Ettinger, V., and Hendrickson, M. R. (1994). Cyclic hormone replacement therapy using quarterly progestin. Obstet. Gynecol. 83, 693-700. 99. Williams, D. B., Voigt, B. J., Fu, Y. S., Schoenfeld, M. J., and Judd, H. L. (1994). Assessment of less than monthly progestin therapy in postmenopausal women given estrogen replacement. Obstet. Gynecol. 84, 787-793. 100. Hirvonen, E., Salmi, T., Puolakka, J., Heikkinen, J., Granfors, E., Hulkko, S., M~ik~ir~iinen, L., Nummi, S., Pekonen, F., Rautio, A.-M., Sundstr6m, H., Telimaa, S., Wilen-Rosenqvist, G., Virkkunen, A., and Wahlstr6m, T. (1995). Can progestin be limited to every third month only in postmenopausal women taking estrogen? Maturitas 21, 39-44. 101. Bjarnason, K., Cerin, ~,., Lindgren, R., Weber, T., and The Scandinavian Long Cycle Study Group (1999). Adverse endometrial effects during long cycle hormone replacement therapy. Maturitas 32, 151-170. 102. Miles, R. A., Press, M. F., Paulson, R. J., Dahmoush, L., Lobo, R. A., and Sauer, M. V. (1994). Pharmacokinetics and endometrial tissue levels of progesterone after administration by intramuscular and vaginal routes: A comparative study. Fertil. Steril. 62, 485-490. 103. Ross, D., Cooper, A. J., Pryse-Davies, J., Bergeron, C., Collins, W. E, and Whitehead, M. I. (1997). Randomized, double-blind, dose-ranging study of the endometrial effects of a vaginal progesterone gel in estrogen-treated postmenopausal women. Am. J. Obstet. Gynecol. 177, 937-941.

~ H A P T E R 3~

Effects of Steroids on Mood/Depression MARGARET G.

I. II. III. IV.

SPINELLI

Department of Clinical Psychiatry, Columbia University, College of Physicians and Surgeons; and The New York State Psychiatric Institute, New York, New York 10032

V. The Hypothalamic-Pituitary Axes VI. Steroid Hormones: Mechanism and Modulation of Neuronal Effects VII. Conclusion References

Introduction Mood Disorders across the Female Life Cycle Brain Neurotransmitters and Mood Catecholestrogens: The Estrogen-Mediated System in the Brain

I. I N T R O D U C T I O N

Depression is a state change characterized by a persistently depressed or irritable mood [4]. Mood symptoms are associated with neurovegetative signs of increased or decreased appetite, weight, or sleep. Perceptual changes such as poor self-esteem and feelings of impoverishment may be prominent. The cognitive changes of major depression such as loss of memory and concentration may mimic early dementia and confront the physician with a difficult differential. Depression is twice as prevalent in women than in men. The Epidemiological Catchment Area study [1 ] found that the lifetime prevalence of depression for men is 2.3-4.4% and 4.9-8.7% for women. The preponderance of depression for women is during the childbearing years, with a prevalence of 7.5-10.4% from ages 23 to 44 years. Although clinical studies have been unable to demonstrate direct associations between hormones and mood [2], modern research has focused on the activity of the hypothalamic-pituitary axis and its interaction with the central nervous system. The neurohormonal basis of mood disorders in women is understood as steroid hormone changes that

Gender differences in mood disorders are well recognized and have been replicated in large clinical epidemiological studies [1]. Several hypotheses have been proposed for the etiology of this gender predominance. Once thought to be due to psychosocial and/or genetic factors, these differences are now proposed to have neurohormonal etiologies [2]. Both Western and cross-cultural studies agree that menopause is a normal physiological state characterized by elevated serum concentrations of leuteinizing hormones and follicle-stimulating hormone and reduced values of circulating estrogen and progesterone. It was believed that menopausal mood changes were a result of increasing chronological age or manifestations of vasomotor symptoms that contribute to depression. We now understand a biological hypothesis centered around the relationship between decreasing levels of gonadal hormones and depressed mood. Some women experience an abnormal vulnerability to this hormonal flux [3].

MENOPAUSE:

BIOLOGY AND PATHOBIOLOGY

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Copyright 9 2000 by Academic Press. All rights of reproduction in any form reserved.

564 trigger alterations in brain neurochemicals. In turn, reciprocal feedback from the brain monoaminergic system modulates the hypothalamic-pituitary axis. The link between gender and mood is initially recognized at adolescence. An equal prevalence of affective disorders exist in boys and girls until the age of puberty [5], when the onslaught of reproductive hormones is associated with an increased incidence of depression in girls as compared to boys. These affective states have a temporal relationship to hormonal status, a trend that continues as shifts in reproductive hormones persist throughout the female life cycle. Reproductive hormone production and altered mood states are demonstrated in the puerperium, the premenstruum, and perimenopause [6-8]. The view of menopause as a time of depression and melancholia for women has been challenged [9]. The term involutional melancholia was introduced by Kraeplin in 1896. Although the United Kingdom was skeptical of this diagnostic category as far back as the 1930s, it took longer to discard this concept in America. Weissman [10] discovered that mood disorders were not more prevalent in menopausal women than women at any other time of their life cycle. She concluded that there is no evidence for a category of involutional melancholia, a term no longer included in the World Health Organization's International Classification of Diseases [3,9,11 ]. By contrast, perimenopause appears to be a time of particular vulnerability for women. It has been suggested that the hormonal fluctuations during the immediate premenopoausal years are responsible for this affective instability [12]. Similarly, surgical menopause induces mood changes because of the rapid hormonal loss at this time.

MARGARET G. SPINELLI

ity. With successful antidepressant treatment [14], the HPA system normalizes. As the mood normalizes, the HPA axis is more susceptible to normal cortical feedback inhibition by cortisol (Fig. 1). Cortisol is the major glucocorticoid secreted from the adrenal cortex [15]. Under stress, both serotonin (5-hydroxytryptamine; 5 HTHT) and norepinephrine (NE) regulate release of cortisol releasing factor, which, in turn, activates the secretion of ACTH from the anterior pituitary. Sporadic bursts of ACTH increase plasma cortisol. The effects of corticosteroids on the electrical properties of brain cells have been described [16]. Temporary fluctuations in corticosteroid levels after acute stress modulate neurotransmitter responses in the hippocampus, a brain structure involved in mood and cognition. This may explain mood disturbances observed in association with stress-related disorders. Cortisol changes correlate with sleep electroencephalogram (EEG) activity. EEG patterns in depressed patients show short rapid eye movement (REM)-stage latency, decreased slow-wave sleep, and sleep continuity disturbance. When Jarrett et al. [ 16] measured cortisol with sleep EEG function, the short cortisol latency in the sleep measure correlated with the short REM latency in depressed subjects. Gender differences in brain physiology are reflected in the electrical activity of the brain and are related to the hormonal status of the reproductive cycle. Altered EEG alpha wave activity reflects changes in gross electrical activity that parallel altered hormone levels [ 17]. The brain monoaminergic pathways involved in steroid feedback appear to be the underlying mechanism.

B. F e m a l e G o n a d a l Steroids A. D e p r e s s i o n a n d N e u r o e n d o c r i n e R e g u l a t i o n The neuroendocrine system is complex and well integrated. It involves the release of anterior pituitary hormones by hypothalamic factors and feedback control by circulating target organ hormones. The entire system is controlled by internal biological rhythms or external events affecting the hypothalamus [ 13]. Patients with depression tend to have neuroendocrine dysregulation, which includes a hyperactive hypothalamicpituitary-adrenal (HPA) axis and nonsuppression of dexamethasone by cortisol [14]. This dysregulation also extends to pregnant and postpartum depressed women [3]. This HPA axis dysregulation may be a result of disturbed physiology of the hypothalamic and limbic system centers that control secretion of corticotropin releasing factor (CRF) and adrenocorticotropic hormone (ACTH). Alternatively, abnormal neurophysiology and central nervous system function may cause the depressed state and HPA axis overactiv-

Estrogen is the most likely component of the hypothalamic pituitary-ovarian axis related to mood. Estrogen has demonstrated antidepressant properties in vivo and in vitro, whereas progesterone has opposite effects [2]. Lipophilic ovarian hormones easily cross the blood-brain barrier. Estrogen receptors are widely disseminated in the brain [18] at sites such as the pituitary, hypothalamus, limbic forebrain, and monoaminergic neurons in the brain stem. Estrogen modulates the synthesis of central nervous system (CNS) enzymes, peptides, neurotransmitters, and receptors, all of which contribute to altered affective states. It also affects monoamine synthesis and turnover, and spontaneous electrical activity in the brain [2]. Both estrogen and progesterone affect neurotransmitter function [ 19] of dopamine (DA), norepinephrine, serotonin, and y-aminobutyric acid (GABA). Progesterone decreases NE concentration in the rat brain, suggesting its etiologic role in depression. Progesterone mediates DA release from

565

CHAPTER 39 Effects of Steroids on Mood/Depression

Antidepressant Hypothalamic paraventricular nucleus

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-

1 +

Hippocampus J ~ . .

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?

,ucocor ico,d/t +

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Adrenal CTH

;

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Antidepressant FIGURE 1 A novel mechanism for antidepressants acting on the HPA system. Schematicrepresentationof the antidepressant-inducedincreases in glucocorticoid receptor (GR) and mineralocorticoid (MR) gene expression, suggesting a novel mechanism of action for these drugs on the hypothalamic-pituitary-adrenocortical system. Stimulatory (+) and inhibitory (-) actions of neural inputs to brain regions involved in HPA system regulation, and the sites of corticosteroid regulation are shown. The sites at which antidepressants have stimulatory actions on GRs or MRs or both, are indicated. It is not yet known if antidepressants also regulate GR concentrations in lymphocytes. Reprinted from [14], Trends in Neuroscience 18; N. Barden, J. M. H. M. Reul, and F. Holsboer; Do antidepressants stabilize mood through actions on the hypothalamicpituitary-adrenocortical system? pp. 6-11. Copyright 1995, with permission from Elsevier Science.

the corpus striatum, whereas estrogen modulates dopamine transmission and release in the nucleus accumbans [20]. Gonadotropin-releasing hormone (GnRH) is released from the hypothalamus to cause secretion of luteinizing and follicle-stimulating hormones (LH/FSH), with subsequent ovarian steroid production [21]. GnRH has demonstrated some antidepressant effects in animal models and humans. Because GnRH neurons are close to fibers containing NE and 5HT, it has complex actions on mental status. Paradoxically, GnRH treatment may facilitate depression [22], yet it also abolishes negative mood symptoms in the premenstruum by impairing ovarian cyclicity [21 ]. This complicated situation extends to neurotransmitter interactions and is further complicated by the likelihood that the h y p o t h a l a m i c pituitary-gonadal (HPG) axis is also interactive with the h y p o t h a l a m i c - p i t u i t a r y - a d r e n a l axis and the h y p o t h a l a m i c -

pituitary-thyroid (HPT) axis, all implicated in affective disorders.

II. MOOD DISORDERS ACROSS THE FEMALE LIFE CYCLE Affective disorders in women are associated with specific times of the reproductive life cycle [23]. Because there appear to be alterations in the steroid hormones with negative mood states, recent thinking is focused on the existence of a particular vulnerability in some women to the normal hormonal changes. In addition, women with affective disorder at one particular phase of the female life cycle often experience recurrence at other times of hormonal flux. In a menopause clinic, Stewart and Boydell [23] screened

566 44 women who scored high on a psychological distress scale and compared them to 42 women in a low-distress group. Women with high distress were more likely than the lowdistress women to report a past psychiatric diagnosis, depressive symptoms associated with oral contraceptive use, premenstrual depression, postpartum blues, or postpartum depression. Association between female affective disorders, steroid hormones, and brain neurotransmitter function supports a neurohormonal etiology that is demonstrated along the life cycle continuum. In order to understand this relationship of steroid-related mood states to menopause, the biological underpinnings of other female life cycle phases will be reviewed. In particular, a review of each phase and the association with substrate changes along the HPG axis are considered. Findings on the relationship of mood and the neuroendocrine system have challenged the field of neuroendocrinology. Studies reveal underlying mechanisms for the interaction of gender and mood. Gender differences in the central nervous system begin in the perinatal period on exposure to sex steroids. This process is responsible for the development of dimorphic brain morphology and function [24]. Differences in neuromorphology, neurochemistry, and physiology may contribute to the prevalence of neuropsychiatric disorders in women, and may account for the contrast in processing emotions between the sexes. The limbic, hippocampal, and hypothalamic (HT) systems are sexually dimorphic. The hypothalamus demonstrates gender differences in distribution of neurotransmitters and shape of synapses. Position emission tomography (PET) scans reveal 15% greater cerebral cortical blood flow and glucose metabolism in women compared to men. Because both male and female reproductive hormones are derived from cholesterol, there is considerable overlap in circulating estradiol in normal men and women. Therefore estrogen is not found exclusively in women, nor androgen in men. Hamilton et al. [25] reviewed the variability of gonadal hormones between sexes. Estrogen receptors are found in male and female rodents. An example of a hormone found predominantly in one sex but having similar effects in both is androgen which increases libido in women and in hypogonadal men. Progesterone elevates basal body temperature in both. Steroids may have gender-specific effects in some brain regions. Interconversion among hormones can occur in the brain so that estrogen, for example, may mediate testosterone's effects on some monoamine systems, such as DA. The diurnal variation of cortisol and testosterone is greater in men than women. Although both hormones vary by season in men, women demonstrate more seasonal behavior changes. Therefore it does not appear that the amplitude of hormonal changes correlate with changes in behavioral symptoms.

MARGARET G. SPINELLI

A. P r e m e n s t r u u m A reported 75% of women complain of premenstrual somatic and behavioral symptoms during the 10- to 14-day luteal phase of the menstrual cycle [26]. However, only 3 - 8 % fulfill diagnostic criteria described in the fourth edition of the "Diagnostic and Statistical Manual of Mental Disorders" (DSM-IV) [4]. These criteria for premenstrual dysphoric disorder (PMDD) include depression, irritability, and sleep and appetite changes that are serious enough to interfere with social, occupational, or family functioning [27]. PMDD is described under the general category of mood disorders. The international classification of diseases [ 11] considers this disorder under the category of medical genitourinary disorders. DSM-IV criteria for this diagnosis include severe incapacitating behavioral changes that are 30% worse in the luteal phase than in the follicular phase of the cycle. Physical symptoms of premenstrual syndrome (PMS) are not necessary to fulfill criteria for this disorder. The fact that women with diagnosed PMDD are more likely to suffer major depressive episodes in later life supports a common vulnerability [28] that is likely represented in the activity of the hypothalamic-pituitary-gonadal axis. In addition, the likelihood of suicide and psychiatric admissions in women increases at this time, and women with existing psychiatric illness experience worsening symptoms [28]. Because symptoms intensify with the rise of progesterone after ovulation and with the rapid fall in both estrogen and progesterone, the relationship of mood with altered gonadal steroids has been a primary area of study. Data on the relationship of steroid hormones, mood and the menstrual cycle are discussed in Section III. A review by Roca [7] suggests that an identifiable pattern of estrogen and progesterone secretion with premenstrual symptoms has not been established. The dynamic nature of the HPG system and the complicated interaction with CNS substrates create a difficult area for neuroendocrine research. Although basal hormone studies reveal no conclusive evidence of correlation to premenstrual symptoms [7], dynamic endocrine studies of the hypothalamic pituitary axis have revealed only inconsistent results. Halbreich et al. [29] found temporal relationships between hormones and premenstrual symptoms associated with the higher levels of progesterone, its decline over time, and the ratio of progesterone to estrogen. A time lag of 4 - 7 days between progesterone changes and the onset of clinical symptoms was demonstrated. Walker and Bancroft [30] examined the possible hormonal relationship to mood during the normal menstrual cycle compared to a cycle on oral contraceptives (OCs). OCs block, but provide a constant exogenous source of, steroid hormones, while the dynamics of a normal menstrual cycle continues. The study evaluated effects of ovulation blockade with

CHAPTER39 Effects of Steroids on Mood/Depression and without cyclical changes in progesterone. Patterns of menstrual cycle changes were compared in three groups of women. There were 35 women in the "monophasic group" on low-dose "combined pills," which provide stable levels of estrogen and progestagen; 30 women in the "triphasic group" on low-dose pills received increasing doses of progestagen, mimicking the luteal rise in progesterone. Another 57 women in a "nonpill" control group used nonsteroidal contraceptives. A visual analog scale rated mood, energy, tension, irritability, and physiological changes. The "monophasic group" had less breast tenderness and a tendency toward menstrual rather than premenstrual changes. The only group effect was slightly lower mood scores throughout the cycle for the combined group, which received stable doses of exogenous estrogen and progestin. Although none of the groups demonstrated cyclical variability in symptoms, the authors stress the study limitation, which was the absence of a group with severe premenstrual symptomatology. Although hormone intervention studies with estrogen or progesterone have not demonstrated therapeutic efficacy in premenstrual dysphoric disorder, several studies have shown that GnRH agonists that suppress ovulation are effective [21 ]. Contrary to some beliefs, oral contraceptives show no benefit in treatment of premenstrual disorders.

B. Puerperium Postpartum blues, depression, and psychosis were once thought to be three separate entities. Research now suggests a spectrum of severity in each of these states, notable for the rapid shift in female hormones. 1. POSTPARTUM BLUES

The postpartum period begins with maternity "blues" in 39-85% of new mothers [31]. This syndrome of mood lability is self-limiting and subsides without treatment within 2 weeks. In an effort to investigate prospectively the etiology of postpartum blues, O'Hara et al. [31] followed 182 women from the second trimester of pregnancy through 9 weeks postpartum. Women who reported a history of depression, premenstrual dysphoria, or increased antepartum depressive symptoms were more likely to experience postpartum blues. In this series, O'Hara found no differences in demographic measures, socioeconomic status, or obstetrical complications. The relationship between hormonal variations and mood symptoms was also studied. The authors found that women experiencing the blues had significantly higher free estriol concentrations at week 38 of gestation, with a greater decrease in estriol from mean prepartum values to those at day 1 postpartum. Postpartum blues did not correlate with other hormone measures.

567 2. POSTPARTUM DEPRESSION One in ten women begins motherhood with a depressive episode [6], and 1 in 500 or 1000 suffers postpartum psychosis [32-34]. It is a well-supported fact that psychiatric hospital admissions increase seven times in the first three postpartum months compared to prepregnancy [34]. Of these mothers, 80% receive a diagnosis of affective disorder, most commonly major depression. Symptoms of depression tend to fit the diagnostic pattern of DSM-IV major depression [4]. Clinical markers of postpartum depression include profound anxiety, sleeplessness, and egodystonic obsessional thoughts of harming the infant. There is a gradual rise in estrogen and progesterone during gestation. With the loss of placenta at delivery, hormone levels plummet within 24 to 48 hr. This precipitous drop from the extremely high levels of gestation is a first step in the sequence of biological events that may trigger psychiatric symptoms in the vulnerable woman [2]. Because alterations in serotonergic and noradrenergic transmission are well correlated with affective disorders, and gonadal hormones affect monoamine transmission, this chemical cascade profoundly alters brain chemistry. 3. POSTPARTUM PSYCHOSIS When mood symptoms are associated with abnormal perceptual experiences and loss of contact with reality, a postpartum psychosis has occurred [32,33]. Postnatal psychosis begins abruptly within the first week of delivery. It may also occur up to 6 weeks after childbirth. The onset begins with insomnia, restlessness, anxiety, and hyperactivity associated with depressed or elated mood. Often thought disordered, the woman may have delusional beliefs about herself or her infant. Wisner et al. [33] found that women with postpartum psychosis compared to those with nonpostpartum psychosis displayed prominent cognitive disorganization, hallucinations, bizarre behavior, impaired insight and sensorium, and disorientation. These symptoms indicate organic etiology as suggested by cognitive examinations. The precipitous onset and the unusual psychotic symptoms, such as tactile, olfactory, and visual hallucinations, are generally recognized as those representative of physiologic influences such as toxic substance or hormonal etiology [4].

C. Menopause Modern literature has dispelled the myths associated with menopause and mood [12]. Mood variations are more common in the perimenopause, a phase associated with fluctuating gonadal hormones. Although menopause is not a pathological state, many women experience somatic symptoms such as hot flashes and insomnia [3]. The biologically sensitive woman may demonstrate neurohormonal vulnerability

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MARGARET G. SPINELLI

associated with affective states, but other elements such as psychosocial stress, aging, and environmental factors also play a role in the etiology of depression. Because depression during menopause does not manifest differently than during another time of life [ 10], it is not categorized as a psychiatric diagnosis. Evidence for benefit of hormone replacement therapy (HRT) for psychological symptoms of menopause are inconclusive. In women with surgical menopause, the evidence for beneficial response to HRT is more convincing, particularly if androgen is also administered [8,35]. Several studies have addressed methodological problems in menopausal research. In a prospective study of menopausal women, Hunter [36] found significant but small increases in depressive symptoms in peri/postmenopausal women compared to premenopausal women. Although 6 of 36 subjects became depressed, the author suggests that past depression as well as cognitive and social factors account for 51% of the variance in the depressed women with a history of depression. Patterns of affective change differ in those with natural menopause compared to perimenopause and surgical menopause [12]. Of 95 subjects in an Edinburgh Menopause Clinic, 78 women had natural menopause and 17 had total abdominal hysterectomy (TAH) with or without bilateral salpingo-oophorectomy (BSO). Of the 78 who experienced a natural menopause, 35 were depressed and 43 had no mood changes. However 83% of the depressed subjects had previous depression, a well-established precipitant for recurrence. Fourteen of the 43 (33%) without mood symptoms had a previous occurrence. A clear peak of illness was found in the first-time onset in the perimenopausal period (4 years on either side of last menstrual period). In fact, 35% of women experienced their first episode of depression at this time. Overall there was a high percentage of subjects with recurrent depression at menopause and a high percentage experiencing their first episode of depression in the perimenopausal period. In addition, the percentage of depressed women is within the range of those reported for other types of outpatient clinics. An issue of note is the self-referred nature of this samplem 50% came with a chief complaint of mood disorder and therefore represent a selective population. When the time distribution of first-episode depression was clustered by chronological age, no clustering was noted. However, when the episodes were distributed by age at menopause, they found a cluster of 17 (35%) in the perimenopausal period.

D. E x o g e n o u s S t e r o i d A d m i n i s t r a t i o n Exogenous administration of steroid hormones may also contribute to negative affective states. This relationship has been demonstrated with oral contraceptives [30]. Some women complain of depressed mood, which appears to be

associated with the progesterone component of treatment. Although this has improved with low-dose pills, some cannot tolerate the affective response with treatment. GnRH agonists, synthetic derivatives of the native decapeptide produced by the hypothalamus, reverse the suppression of LH and FSH by the anterior pituitary gland. Agents such as leuprolide and goserelin acetate cause ovarian suppression with vasomotor instability, flushes, and emotional lability. Although the agonists have been implicated in depression and psychosis [22], they paradoxically relieve symptoms of premenstrual disorder [21]. The manipulation of the HPG axis with GnRH analogs induces an abrupt hormonal change that, in turn, induces negative affective states. These hormonal interventions have been used for endometriosis and assisted reproduction [22]. The difficulties that face the infertile couple often cause interpersonal difficulties and mood symptoms. In addition, biological interventions that induce affective symptoms in some women further complicate this difficult time. Ovulation suppression with GnRH agonists induces a reversible medical menopause that results in remitted PMS symptoms [21 ] Treatment with these agents is limited by the loss of bone density. In an effort to maintain the therapeutic response without loss of bone density, estrogen/progesterone add-back therapy is used. The authors tested the effectiveness and safety of longterm administration of GnRH plus hormone replacement for women with moderate to severe PMS. Over a 12-month period, this study evaluated physiological and psychological variables in 10 women [20] with regular menstrual cycles who complained of a 25% increase in PMS during the luteal phase of the cycle. Four-week cycles of intramuscular injections of placebo or leuprolide acetate were tested with all patients, followed by 12 cycles of GnRH (7.5 mg). Conjugated equine estrogen (0.625 mg/day) was started for 6 consecutive days within the first cycle and was increased as needed. Medroxyprogesterone acetate (10 mg/day) was taken orally for 10 days after 4, 8, and 10 days of GnRH therapy. A significant decrease in all symptoms was demonstrated. There were no changes in lipids, no evidence of uterine hyperplasia, and no statistically significant loss of bone density. The authors concluded that GnRH hormone agonist therapy, with hormonal add-back, is effective in treating and improving PMS symptoms over a 12-month period. Female gonadal steroids influence platelet imipramine binding. A likely mechanism for premenstrual relief of symptoms associated with these hormones is the 5HT system. The GnRh agonist o-Trp6-LHRH (Decapeptyl) was given to women undergoing assisted reproduction to determine the effect of platelet serotonin transporter density in these women compared to those without pretreatment with Decapeptyl [37]. In this open-label trial of 19 women, 10 received active

CHAPTER39 Effects of Steroids on Mood/Depression drug and 9 were treated with human menopausal gonadotropin (Pergonal). Hamilton rating scales [38] were compared and platelet plasma samples were collected for estrogen, progesterone, FSH, and LH. The GnRH analog induced ovarian suppression reflected by low plasma estradiol levels, whereas Pergonal induced ovarian stimulation. Elevated depression and anxiety scores were observed in the Decapeptyl group and were associated with a significant decrease in density (flmax) of platelet imipramine binding sites. No change in/~max w a s observed in the Pergonal-treated group. The authors concluded that ovarian suppression is associated with depressed and anxious mood and decreased serotonin transporter density. Clinical correlates of mood lability with GnRH agonists were further described by Warnock and Bundren [22]. Four premenopausal women with no prior psychiatric history developed severe anxiety, mood disorder, and menopausal symptoms following GnRH agonist therapy for endometriosis. The first case received a leuprolide injection of 3.75 mg monthly for endometriosis. At 2 weeks after the first injections, she experienced depression, panic attacks, and suicidal ideation. All symptoms were alleviated by sertraline, and leuprolide continued without adverse mood effects. The second case experienced depression with psychotic features 1 month after the second injection of 3.75 mg (intramuscular) for polycystic ovarian disease. Irritability and auditory hallucinations responded to sertraline (50 mg/day) while she continued the next 4 months of GnRH agonist treatment. A third case received leuprolide for infertility, but rejected treatment for depression. The final case of endometriosis experienced paranoia, volatile physical outbursts, and stalking behavior after 2 weeks of agonist treatment. Sertraline reversed symptoms. This data prompted Warnock et al. to perform a retrospective pilot study [39] of 42 subjects with laparoscopydiagnosed endometriosis treated with 24 weeks of GnRH agonist therapy. Their mood was assessed with the Hamilton Depression Rating Scale [38]. The 22 patients receiving sertraline had fewer depressive symptoms but did not differ significantly in physical symptoms from 20 control women who received a GnRH agonist alone. Patients requiring a GnRH agonist (Lupron) may benefit from concomitant sertraline therapy, particularly if they have a personal or family history of depression. Another hormonal intervention to cause altered mood states is the FSH and LH analog clomiphene, which induced at least one case of mania identified after ovulation induction with gonadotropins [40]. The patient was a 34-year-old woman with no previous personal family history of psychiatric disorders. Several months after receiving clomiphene without result, she began ovulation induction with human chorionic gonadatropin and urofol litropin (FSH induction) during days 8-12 of her cycle, then human chorionic gonadotropin (LH induction) on days 13 and 14. Midway into

569 a course of ovulation induction she had a 1-month manic episode associated with racing thoughts, heightened mood, bizarre activity, insomnia, and hypersexuality. This manic state was followed by severe depression with sucidality, successfully treated with sertraline (50 mg). This information demonstrates the association of mood changes with the precipitous decline of serum estradiol similar to that in women who experienced surgical menopause after TAH/BSO. In addition, further support is offered for precipitous hormonal changes as triggers for CNS changes via the HPG axis.

III. BRAIN NEUROTRANSMITTERS AND M O O D Changes in monoamines have been implicated in the etiologies of affective, anxiety, and psychotic disorders [13]. Monoamine neurotransmitters in the brain are called biogenic amines and include the catecholamines (norepinephfine, epinephrine, and dopamine) and an indoleamine (serotonin) [13]. The catecholamine (CA) hypothesis suggests that depression is a result of deficient CAs at important central adrenergic receptors, with the deficiency playing a role in stress and emotion. Hormones that vary during the menstrual cycle, menopause, childbirth, and oral contraceptive therapy perturb these central neurotransmitter and neuromodulator systems [41]. The potential effects of estrogen and progesterone include alterations of monoamine oxidase, dopamine, norepinephrine and serotonin turnover, and modulation of ce-adrenergic receptor density in the brain. Each of these CNS substances has been implicated in mood disorders (Fig. 2). Neurotransmitters are stored within nerve terminals and released into the synaptic cleft; they transfer messages via actions on pre- or postsynaptic receptors. The monoamine system is regulated by enzymes within the nerve terminals. Specifically, neurotransmitters are inactivated by the

Hormone Estradiol

Progesterone

Brain function Monoamine oxidase Catechol-o-methyltransferase Dopamine turnover hypothalamus o~-Adrenergic receptor density in hypothalamus o~-Adrenergic receptor density in cortex Seizure threshold Monoamine oxidase Catechol-o-methyltransferase Serotonin turnover in limbic area Seizure threshold

FIGURE 2 Effects of gonadal hormones on brain function. From Pajer [2].

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MARGARET G. SPINELLI

enzymes monoamine oxidase (MAO) and catechol-omethyltransferase (COMT) [ 13]. Several receptors have been identified for each neurotransmitter. Both classes of adrenergic receptors (ce and/3) are sensitive to norepinephrine, and/3 receptors are sensitive to both epinephrine and norepinephrine [ 13].

to changes in serotonergic function. 5HT uptake inhibitors [46] such as fluoxetine (Prozac) [47] and sertraline (Zoloft) [48] have demonstrated efficacy in controlled clinical treatment trials. Rapid relief of symptoms occurs with subclinical doses of the SSRIs and supports these neurohormonal underpinnings [46].

A. S e r o t o n i n ( 5 H T )

B. N o r e p i n e p h r i n e

The monoamine serotonin (5-hydroxytryptophan) is synthesized from the essential amino acid tryptophan, with tryptophan hydroxylase as the rate-limiting step in synthesis. Several subtypes of 5HT receptors have been identified. The fact that changes in brain serotonergic activity underlie mood disorders is well replicated in the literature and has been reviewed by Mann et al. [42]. Tryptophan, the precursor of 5HT, is reduced in the cerebrospinal fluid of depressed patients. The brains of suicide victims compared to victims of homicide show decreased 5HT levels, an acute reduction in 5HT receptor activity, and an increase in 5HT receptor numbers [41]. Thus, 5HT binding is increased in the brains of suicide victims, in brains of depressed individuals postmortem, and in platelets of depressed individuals obtained postmortem. Serotonin, found in neurons, may also be found in other cells, such as the platelet [43]. The platelet is the peripheral model for the brain neuron because of its similar morphology and 5HT uptake sites. Levels of platelet 5HT correlate with those in the brain, making the platelet an accessible model for investigation of central monoamine function. 5HT is also involved in the activity of the HPA axis. Specifically, 5HT is implicated in the regulation of gonadotropins because 5HT neurons stimulate gonadatropin release through its interaction with 5HT receptors via projections to the median eminence [41 ]. 5HT axons also terminate on luteinizing hormone, releasing neurons in the preoptic area. Altered hormone states and 5HT levels have been implicated in premenstrual disorders [44]. Altered 5HT sensitivity is suggested by several mechanisms. Imipramine receptor binding in platelets, a reflection of 5HT activity, is decreased in women with premenstrual depression. Cortisol response to tryptophan is blunted during the luteal phase of the cycle compared to the midfollicular phase. Women with premenstrual disorder have decreased platelet 5HT uptake and diminished whole blood 5HT during the luteal phase. Premenstrual symptoms may be disabling enough [45] to require pharmacotherapy. For these women, irritability, tension, and depressed mood disrupt family, occupational, and social functioning. Other than ovulation suppression, the serotonin-specific reuptake inhibitors (SSRIs) appear to be the most effective treatment for premenstrual dysphoric disorder. Although this disorder occurs during normal hormonal shifts, the mechanism of symptoms appears to be due

Norepinephrine and epinephrine also belong to the class of neurotransmitters called catecholamines. CAs are made from tyrosine and are released from the adrenergic nerve fibers or the adrenal medulla [ 15], They bind to receptor molecules on the plasma membrane; these receptor molecules transduce CA interactions into a physiological response. NE is found in highest concentrations in the hypothalamus. The monoamine hypothesis postulates that noradrenergic deficiency in the brain also plays a role in the pathogenesis of some depression [49]. Knowledge of abnormalities in mood disorders results, in part, from the fact that antidepressants decrease the number of central nervous system fladrenergic receptors. Diminished fl receptor number is a genetically determined risk factor for mood disorders. With reference to the HPA axis and HPG axis feedback mechanisms, disorders of the monoamine systems are predictive of disorders in hormone secretion of the anterior pituitary [49]. Inversely, disorders of the anterior pituitary can be indicative of disorders in the monoaminergic transmission in the hypothalamus. Release and concentration of hormones into the blood can therefore be regarded as a general indicator of neuronal activity in some monoamine systems. One other mechanism that underlies the neuroendocrine hypothesis of depression is decreased luteinizing hormone in menopause, which is understood as deficient noradrenergic activity in the hypothalamus. This action supports the hypothesis that the anterior pituitary lobe is under monoamine control. Clinical correlates of circulating catecholamines are demonstrated in the postpartum period. Specifically, women with 1 day of postpartum blues have significantly lower levels of noradrenaline and adrenaline on that day compared to surrounding days [50].

C. D o p a m i n e Dopamine, a catecholamine modulated by estrogen transmission, is implicated in psychotic disorders. Dopamine originates from the amino acid precursor tyrosine [43]. The biochemical explanation for schizophrenia is derived from the observations that the only consistent feature among the antipsychotic drugs used to treat the disease is their ability to antagonize DA receptors. It is postulated that

CHAPTER39 Effects of Steroids on Mood/Depression schizophrenia may be related to a relative excess of central dopaminergic neuronal activity [43]. Estrogen modulates the activity of DA, the neurotransmitter implicated in the onset of psychosis. Growth Hormone (GH) is a measure of hypothalamic dopamine D 2 receptor function in the brain and is peripherally measured by apomorphine agonist activity. Wieck [20] studied this relationship in women with a history of psychosis. Fifteen women with a history of psychosis and 15 control psychiatrically healthy women were injected with apomorphine. Seven of the 15 at-risk women had a recurrence of psychosis after delivery and 8 at-risk women remained well. The 7 women with recurrence of psychosis had an increase in GH response to apomorphine, suggesting an increase in DA receptor activity in the hypothalamus, whereas control women and those without recurrence had no such response. In a similar study, McIvor et al. [51 ] evaluated 14 women at 36 weeks gestation and 3 months postpartum. All had a history of depression. Five women relapsed in the postpartum period and demonstrated an increased sensitivity of DA receptor function. Both authors concluded that this increased DA activity in the postpartum period predicts depression and anxiety disorders and is likely due to estrogen's effects on DA transmission.

D. y - A m i n o b u t y r i c A c i d GAB A is an inhibitory amino acid neurotransmitter. It is at the GAB A receptor that benzodiazepines exert anxiolytic, sedating, and anticonvulsant effects [52,53). The GABA-A receptor complex in the brain is the most prevalent of the two known GABA receptors in the mammalian CNS [43]. GABA-A has both agonist and antagonistic properties. Natural steroids interact with GABA, with physiological and pathological consequences [52,53]. GAB A levels are low in the CSF of depressed subjects, and GABA agonists relieve depression [52]. In fact, antidepressants inhibit GABA uptake and stimulate its release. Steroid regulation of GABA activity is further evidence of their role in affective disorder. Further implications for affective change at various phases in the female life cycle are explained by these neuroendocrine relationships in the HPA and HPG axes.

IV. CATECHOLESTROGENS: THE ESTROGEN-MEDIATED SYSTEM IN THE BRAIN Neuroendocrine interaction is facilitated by catecholestrogens (CEs), which are part of a major route of estrogen metabolism in the brain. Because they have the potential for

571 interaction with both CA- and estrogen-mediated systems, CEs provide the mechanism by which estrogen affects mood. Catecholestrogens are 2- and 4-hydroxylated metabolites of estrogen and estradiol [54]. Three catecholestrogens that interact with the catecholamine system are thought to be mediators of estrogen metabolism. The interactions between steroids and brain monoamines is understood as the activity of these biological substrates along the HPA axis. CEs are mainly formed in the hypothalamus and the limbic system, where they inhibit the synthesis, inactivation, and degradation of catecholamines. This competitive inhibition occurs in a dose-dependent manner at pharmacological doses because physiological doses of CE are unable to compete with CA.

V. T H E H Y P O T H A L A M I C PITUITARY

AXES

Because many endocrinopathies present with mood symptoms, the hypothalamic-pituitary axes have became an important area of research. Brain neurotransmitters linked to endocrine disorders control hypothalamic peptides that regulate the release of pituitary hormones. These neurobiological mechanisms have encouraged the emerging area of study called psychoneuroendocrinology [ 13]. The function of the neuroendocrine system and mood is a complicated biological process. The cascade of events that begin in the brain and end in the target organ may be impaired at any level of the brain, hypothalamus, or pituitary. The effects of any positive or negative feedback loop may be differentiated by this biological process [2].

A. T h e H y p o t h a l a m i c - P i t u i t a r y - A d r e n a l

Axis

Early neuroendocrine studies in depressed subjects were focused on the HPA axis. These disturbances are demonstrated as consistently elevated plasma cortisol levels and the inability of dexamethasone to suppress cortisol activity [55]. This abnormal cortisol reaction is understood as a failure in normal negative feedback regulation of the HPA axis and results in fragmented secretory activity and higher plasma cortisol concentration. Decreased monoamine (NE and DA) concentrations are correlated with high cortisol levels in depression. A significant negative correlation was found between the cerebrospinal fluid norepinephrine levels and predexamethasone cortisol level in depressed patients with a positive dexamethasone suppression test (DST) response. Pretreatment data also showed cortisol had a signficant negative correlation with dopamine in the cerebrospinal fluid. Positive DST responders exhibit a negative response with treatment and recovery from illness. The nocturnal pattern of

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MARGARET G. SPINELLI

cortisol secretion also returns to that of normal age- and sexmatched controls.

B. T h e H y p o t h a l a m i c - P i t u i t a r y - G o n a d a l A x i s Although the HPG axis is less well studied, an increasing awareness of the relationship of mood with altered sex steroids has encouraged further investigation. The gonadal hormones estrogen and progesterone influence a myriad of CNS processes. This complex system is vulnerable to numerous and diverse insults by stimuli that may influence specific steps in HPG functioning or the balance between hormone levels. The etiology of affective disorders related to the reproductive life cycle is likely represented in the biological cascade of events that begins in the cortex and influences feedback mechanisms [2] Such activity includes stimulation of GnRH release in the hypothalamus. The pulsatile activity of GnRH influences FSH and LH secretion from the anterior pituitary, which in turn causes ovarian secretion of estrogen, progesterone, androgens, and inhibin. The feedback loops from these peripheral hormones influence higher level functions such as neurotransmitter activity, GnRH, and gonadotropins. During the reproductive years, GnRH is secreted in a pulsatile fashion every 6 0 - 9 0 min, resulting in normal concentrations of FSH and LH. This mechanism causes downregulation of GnRH receptor numbers and subsequent desensitization of pituitary gonadotrophs. The loss of endogenous ovarian gonadotropin stimulation causes a severe hypoestrogen state [22]. In addition to PMS, GnRH analogs are used in gonadal hormonedependent conditions such as endometriosis, polycystic ovary syndrome, neoplastic diseases [37], and as adjuvant for in vitro fertilization. Clinical correlates of GnRH agonists previously described further attest to neuroendocrine regulation of mood and psychosis.

VI. S T E R O I D MECHANISM OF NEURONAL

HORMONES: AND MODULATION EFFECTS

Steroid hormones provide feedback control over the HPA axis and the HPG axis by regulating the synthesis of respective trophic hormones and the induction and regulation of the synthesis of proteins, transmitters, hormones, and receptors [56]. The adrenal cortical hormones are steroid hormones and are produced by the adrenal cortex, the gonads, and other reproductive structures such as the placenta. The hypothalamus [15] receives signals from the CNS and higher cortical systems via neurotransmitters. The hypothalamus then exer-

cises control over hormone release by the pituitary, a structure that lies directly below the hypothalamus. Fluctuations in the HPG axis cause perturbations in neurochemistry, which in turn may induce behavioral changes. Because steroid hormones are lipid soluble, they easily cross cell membranes, where they may regulate gene transcription for neuropeptides and for enzymes associated with neurotransmission and structure and breakdown of synapses [56]. The mechanisms of steroid actions on the brain have both fast and slow functions. Fast steroid actions are nongenomic activities that occur over minutes to seconds. Hormones bind to membrane receptors, causing changes in calcium channels, modulation of GAB A receptors, and changes in excitable membrane properties. The slow reaction occurs inside the cell via receptormediated gene expression in the nucleus of enzymes and neurotransmitter receptors [41,52,53]. Inside the cell, hormones bind to the receptors, which in turn become DNA transcription factors. Each transcription factor contains three main regions, a DNA-binding site, a hormone-binding region, and a region for transcriptional regulation. The binding hormone induces a change in the receptor molecule, which causes DNA binding and transcription of the gene. Over minutes to hours, steroids regulate the gene transcription of neuropeptides, enzymes for neuronal transmission, and factors for synapse regulation. An example of steroid effects on neuronal excitability is the GAB A-A receptor, which regulates chloride conductance in neurons. This activity is enhanced by GAB A agonists such as anxiolytics, hypnotics, anticonvulsants, and anesthetics but is reduced by convulsants [52,53]. Bidirectional regulation of various steroids on GAB A receptor function results in altered neuronal excitability. This mechanism provides important communication between body and brain, integrating responses to external stimuli or internal signals. This neuronal excitability was first described with the anesthetic action of intravenously injected cholesterol [53] and the rapid hypnotic effect of progesterone and deoxycorticosterone. In addition, cortisol provokes epileptogenic seizures and dramatic alterations in electroencephalograms [ 16]. Neuronal effects are also demonstrated by estrogen [57]. The effects of transdermal estrogen replacement on EEG mapping were monitored in a double-blind placebocontrolled trial of 69 depressed menopausal women. The women ranged in age from 45 to 60 years and had no previous hormone replacement therapy. Each woman was randomly assigned to a 3-month treatment with transdermal estradiol (Estraderm TTS; ETTS); 50 txg twice weekly, or placebo. Menopause was defined as 6 months up to 5 years without menses, estrogen under 55 pg/ml, and FSH > 19 mu/ml. Surgically menopausal women were included if surgery had occurred between 2 months to 5 years previously. Estradiol increased and FSH decreased in the treatment group, whereas those measures remained stable in the pla-

CHAPTER 39 Effects of Steroids on Mood/Depression

573

cebo group. Overall, depressed mood improved and was associated with significant interdrug differences in brain function, particularly over the left temporal region. The ETTS patients demonstrated changes in brain wave activity, however no changes occurred in the placebo-treated patients, suggesting further evidence for estrogen's action on fluctuating CNS chemistry.

Beck Depression Inventory [60] compared to controls, findings that were not dose related. Although this trial does not support estrogen as treatment for depression, its antidepressant properties are demonstrated in this sample of postmenopausal women. In animal studies, ovarian steroids demonstrate a complex effect on the serotonergic system. The acute effect of estrogen exposure causes an immediate reduction of 5HT receptor density throughout the brain. However, a delayed effect of 4 8 - 7 2 hr caused a selective increase in brain regions that contain estrogen receptors [ 18]. Early work of Biegon and McEwen [18] demonstrated the aforementioned bimodal mechanism of estrogen on 5HT function in the female rat brain. Estrogen's rapid effects on brain membranes occur by modifying 5HT receptor availability. Slower change on the same receptors occurs via interaction with intracellular estrogen receptors and DNA transcription. The effects of estradiol on serotonin receptors in brains of ovariectomized female rats in vivo and in vitro were investigated. Initially, ovariectomized female rats were injected with estradiol and control animals were injected with a control substance. Both were sacrificed after 1 hr. In a second experiment, the animals were sacrificed after 48 and 72 hr. Steroid hormones were added directly to the serotonin assay tubes and tissue from the whole forebrain or cortex was preincubated in steroid containing buffer for 2 hr. The study demonstrated estradiol's biphasic effect on serotonin receptors, i.e., estradiol's ability to decrease the concentration of 5HT1 receptors within 1-2 hr of injection (Figs. 3 and 4) followed by delayed (72-hr) effects in 5HT2 receptors. Estrogen exposure acutely affected all areas of the brain but the delayed effects occurred only in estrogencontaining areas such as the amygdala, the mediobasal hypothalamus, and the preoptic area. In sum, this animal model of estrogen modulation effects

A. E s t r o g e n Estrogen is produced by the follicle and corpus luteum of the ovary and by the placenta in the second and third trimester of pregnancy [3]. The ovary secretes estradiol ( E 2 ) and estrone (El) whereas the placenta produces E 2 and E 3 plus estriol (E3) [15]. Kendall [58] demonstrated the relationship of ovarian hormone secretion to the serotonin uptake system. Estrogen reduces 5HT2 receptor binding during prolonged imipramine (IMI) treatment in rat brain cerebral cortex, a mechanism that depends on the presence of sex steroids. This effect is blocked in ovariectomized rats and may be restored with estrogen administration. Estrogen's effect on platelet IMI binding sites, its displacement of tryptophan from binding sites on plasma albumin, and degradation of monoamine oxidase (MAO) all support estrogen's ability to increase brain neurotransmitters, particularly at the site of the synapse. Although these substrate effects maintain estrogen's bioavailability for central nervous system function, clinical studies show conflicting effects of estrogen on psychological function. For example, in a placebo-controlled clinical treatment trial Ditkoff et al. [59] administered conjugated equine estrogen (0.65 and 1.25 mg) for 3 months to postmenopausal women. No subject had major depressed mood, but the estrogen-treated group showed significant improvement on the

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Estrogenic modulation of brain serotonin receptors, showing the acute effect of an estradiol injection on serotonin binding to the forebrains of ovariectomized female rats. (A) Specific binding curves obtained in a single experiment. (B) Scratchard Analysis of the binding data. Control: K d = 4.0, B m a x - - 190 fmol/mg of protein. Injection of 2/zg: K d = 2.0, Bmax = 90 fmol/mg of protein. Injection of 25/zg: K d -- 2.0, Bmax -- 52 fmol/mg of protein. B/F, Bound/free. From Biegon and McEwen [18].

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MARGARET G. SPINELLI

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on the 5HT system demonstrates support for the affective changes in women who are susceptible to rapidly changing hormone levels. 1. ESTROGEN, MOOD, AND REPRODUCTION

The clinical application of Biegon and McEwen's work to postnatal depression is demonstrated by Hamilton and Sichel [61 ]. For some women, the postpartum period may be a time of acute sensitivity to the rapid decline in estrogen. However, postpartum depression is treated with the standard psychopharmacological interventions used for nonpuerperal conditions. If the immediate postpartum hormonal decline is a risk factor, a logical preventive intervention might be to halt the neurohormonal cascade by interfering with steps in this event. Hamilton administered estrogen prophylactically to postpartum women at risk for recurrence. In 50 women with past histories of moderate to severe postpartum mood disorders, a single injection of a longacting estrogen was given immediately after delivery, followed by a taper of oral conjugated estrogen over 14 days. Statistically the risk of postpartum recurrence is 30-50%, however, there were no postpartum recurrences in these atrisk patients. Although there were no instances of thromboembolic events, this adverse effect at high doses of estrogen must be considered. In a subsequent study, Sichel et al. [62] administered high-dose oral estrogen as the first dose at delivery, supplemented by subcutaneous heparin and followed by transdermal estrogen patches. Seven women with a history of postpartum psychosis and four with a history of postpartum depression were treated with high dose oral estrogen (10 mg) administered daily in decreasing doses over 4 weeks. Heparin (5000 U subcutaneously) was administered twice daily

for the first week. Only one patient had a postpartum recurrence. All others required no psychotropic medications for the first year after delivery, an effect that is likely due to replacing the rapid loss of hormone at a critical time in the puerperium. This preliminary result demonstrates the antidepressant properties of estrogen in postpartum depressed women. In view of the risk of thromboembolism and other adverse events, prophylactic use of estrogen for recurrent postpartum affective disorder remains experimental. Data are insufficient and further research is warranted [62]. One mechanism of action that explains the antidepressant effects is estrogen's interference with the enzymatic degradation of norepinephrine accomplished by the catecholestrogen metabolites of estradiol. When catecholestrogens competitively inhibit catechol-o-methyltransferase, the action of NE is potentiated. In addition, estrogen's inhibition of monoamine oxidase makes NE more available for synaptic action in adrenergic neurons. 2. ESTROGEN, MOOD, AND MENOPAUSE

Klaiber et al. [63] administered estrogen to pre- and postmenopausal women to determine its effect on depression and central adrenergic function. A group of 23 subjects who had failed to respond to 2 years of antidepressant treatment were given large doses of conjugated estrogen in double-blind fashion. In this group, 10 women had psychotic symptoms associated with the affective disorder. Placebo was administered to 17 women with comparable illness. The treatment cycle for premenopausal patients was three menstrual cycles and 12 weeks for noncycling women. After a 2- to 3-week placebo washout, the treatment group received oral conjugated estrogen begun at 5 mg/day, titrated

CHAPTER39 Effects of Steroids on Mood/Depression up to 25 rag/day as tolerated. Weekly Hamilton Depression Rating Scales [38] were administered and twice weekly blood samples were drawn for MAO activity. On day 21 of treatment, premenopausal women were given 2.5 mg of methoxyprogesterone for 5 days, then estrogen and progestin were discontinued to allow menstruation. After 3 months the treatment group demonstrated a significant decline in the Hamilton scores compared to the control group, indicating clinical improvement. However, overall scores remained fairly high. As predicted, estrogen decreased plasma MAO activity in the depressed subjects; the plasma MAO activity of the placebo group increased for unclear reasons. Therefore, this study supports the antidepressant effects of estrogen, and clinically demonstrates the underlying biological mechanism for these effects. Although the risks of large doses for a long duration must be considered, estrogen may be a good adjunctive treatment for partial response to antidepressant medication. Such adverse events include thromboembolic events and endometrial hyperplasia and therefore warrant caution. Halbreich et al. [64] investigated the mechanism of estrogen replacement on serotonergic activity. The serotonin agonist 1- (meta-chlorophenyl) piperazine (m-CPP) was administered to postmenopausal women. Cortisol and prolactin responses to m-CPP are indicative of serotonergic activity. A blunted response indicates diminished serotonergic activity. The study included 18 normal postmenopausal women, 11 of whom were also treated with estrogen transdermal patches (Estraderm, 0.1 mg); these women were compared to 15 normal women of reproductive status. Menopause was determined by the absence of menses for 2 years, absence of menopausal symptoms for 1 year, and no detection of progesterone. Without estrogen, the prolactin and cortisol responses to m-CPP were blunted in postmenopausal women compared to control reproductive women. Estrogen replacement increased the hormonal responses. The diminished 5HT response in postmenopausal women likely contributes to affective vulnerability, and may explain the mood-enhancing effect of estrogen. This finding further suggests that damaged receptors may be an important factor in the inhibition of steroid effects. The unpredictable nature of this finding is suggested by variable mood states induced by estrogen replacement therapy (ERT). Oppenheim [65] described the mood-altering effects of estrogen in a case of rapid cycling mood. Although rapid cycling is a direct complication of antidepressant medication, estrogen induced the state in a 72-year-old woman who had a partial response to antidepressant treatment. Oral conjugated estrogen (Premarin) was added at 0.625 rag/day and increased to 4.375 mg/day over 30 days. By day 5 of treatment, her moods cycled from elation and hypomania to depression every 1 to 3 days. Hypomania was demonstrated as elation, aggression, and overactivity associated with im-

575 pulsivity and shopping sprees. Other cases of induced hypomania have been reported [66]. An interesting caveat was Chouinard's [67] clinical case report of the mood stabilization effects of combined estrogen and progesterone in two women with bipolar disorder resistant to lithium and/or tryptophan. Schneider and colleagues [68] combined estrogen with antidepressant medication in a randomized double-blind trial of elderly depressed women on fluoxetine (20 mg/day). The 72 patients who received estrogen replacement therapy were compared to 286 who did not. Patients on ERT and fluoxetine had a 40.1% greater mean Hamilton improvement than 17% of patients on ERT and placebo. This combination provides a reasonable alternative for elderly depressed outpatients who fail to respond to monotherapy. The particular response in women on ERT may best be explained by an inadequate response to SSRIs because of a chronic hypoestrogenic state. Because 5HT2 receptor binding appears to be estrogen dependent, some elderly depressed women may fail SSRIs because of inadequate estrogen modulation of receptor activity. In sum, estrogen appears to improve mood in nondepressed postmenopausal women, but appears to be ineffective in women with major depression.

B. P r o g e s t e r o n e The female sex hormones accumulate and are metabolized in the brain, where CNS activity is increased by estrogen and decreased by progesterone. Because progesterone opposes effects of estrogen on monoamine function, it has opposite effects on mood. Depressogenic effects of progesterone are attributed to its action on MAO [2]. In contrast to estrogen, progesterone decreases the degradation of monoamine oxidase. The subsequent increase in the enzymatic breakdown of monoamines makes these neurotransmitter substrates less available at the site of the synapse. As previously described, negative mood is sometimes induced by progesterone and attenuated by a higher estrogen:progesterone dose ratio. Progesterone and deoxycorticosterone are major hormones released by the adrenals and ovaries, which contain enzymes capable of forming barbiturate-like substrates from inactive hormones. These metabolites of progesterone (3a, 5ce-tetrahydroprogesterone; THP) and deoxycorticosterone (3ce,5ce-tetrahydrodeoxycorticosterone; THDOC) are allosteric agonists of the GABA-A receptor [24,52,53]. THP which is increased during the luteal phase and in pregnancy acts as a sedative, anxiolytic, anticonvulsant, and proanesthetic. However, pregnenolone sulfate (PS) and dehydroepiandrosterone sulfate (DHEAS) are antagonists with antidepressant properties. The substantial amounts of steroids in the CNS, their fast

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turnover, and alterations during physiological states once again imply their active role in CNS functions 1. PROGESTERONE, MOOD,AND REPRODUCTION Although progesterone appears to be depressogenic, it has been studied as a potential prophylactic agent for postpartum depression. Dalton [69] administered intermuscular progesterone at delivery to 27 women with prior postpartum depressions then daily for 7 days. This was followed by progesterone suppositories two times per day for 60 days. By 6 months after delivery, none of the women had developed postpartum depression. Because of major methodological problems such as lack of double-blind conditions, these data remain inconclusive. Other trials of progesterone prophylaxis [70] dispute Dalton's results. Further support was provided when Harris et al. [71 ] queried the relationship of puerperal mood, progesterone, and cortisol. In a prospective study of 120 primiparous women, saliva was collected twice daily from 2 weeks antepartum to 35 days postpartum to determine cortisol and progesterone levels. Seven women developed major depression. Decreased evening cortisol levels in the immediate peripartum period were associated with postnatal depression; however, no relationship of mood to progesterone was detected. Similar to other reports, this provides no support for a treatment strategy of progesterone augmentation after delivery. 2. PROGESTERONE,

MOOD,AND MENOPAUSE

Progesterone-induced depressive states cause considerable noncompliance in clinical populations of postmenopausal women on hormone replacement therapy. It is not well known that a relationship between mood symptoms is reported with progestins. Consequently, the patient may be categorized as "postmenopausal onset of depression." Specifically, concerns over noncompliance include the risk of cardiovascular disease, osteoporosis, and gastrointestinal cancer [9]. Magos et al. [72] reproduced this state in a placebocontrolled prospective study in 58 postmenopausal hysterectomized women who were treated with subcutaneous estradiol (150 mg) and testosterone (100 mg) implants (Orgenon). Norethisterone (19-nor steroid progestagen), 2.5 and 5 mg/ day, was given for 7 days and placebo was given for two periods of 7 days. Psychological, physiological, and behavioral variables were assessed. The group included 70 women with an average age of 48.3 years who had hysterectomies for nonmalignant conditions within the previous 22 years. Asymptomatic women were judged to be postmenopausal either by a history of bilateral salpingo-oophorectomy or by a plasma FSH measurement of >20 IU/liter before HRT with estradiol and testosterone. There was a five-period design, each period representing 7 days. The first and last periods were used as

baseline and were tablet flee. Norethisterone or an identical placebo was allocated during the middle three periods. A total of 58 women completed the study; 39 were treated with 5 mg and 19 with 2.5 mg. A significant increase in psychological symptoms such as depression, anxiety, irritability, and several variables such as pain, concentration, and water retention showed worsening with 5 mg/day of the progestagen. Although there was a trend in some variables at low doses, there were no statistical differences between the drug and placebo. The dose relationship suggests that it may be advantageous to prescribe minimum dosages of progestational agents to achieve effects. The author addresses the similarity of norethisterone-induced behavioral change to the complaints of premenstrual syndrome and suggests that PMS is a model for this condition. In order to determine if a relationship exists between a history of PMS and adverse response to progesterone, oral medroxyprogesterone acetate was given in conjunction with transdermal estrogen [73] to two groups of women with TAH/BSO. The study involved 24 women with a history of PMS and 24 with no history; they all received estrogen (100 /zg) on days 1 through 25 and oral medroxyprogesterone acetate (10 mg daily) or placebo for 12-25 days in a random double-blind cross-over design. A history of PMS did not predict a difference in mood or physiological symptoms between groups. The authors suggest that using a single cycle may be a limiting factor, and further investigation should include several cycles of therapy. Alternatively, Siddle et al. [74] found no mood changes associated with progesterone. Equine estrogen (1.25 mg/ day) was administered continuously to two groups of subjects who were subsequently randomized to dydroegesterone (20 mg/day) for 12 days each month for 3 months, then 10 mg in the same fashion for the subsequent 3 months. Group 1 followed this protocol while group 2 received the hormone regimen in reverse order. There were no significant differences in mood or anxiety symptoms when either of the dydroegesterone doses were administered. The authors refer to previous unpublished findings in which they found 10-20% of women experienced a PMS syndrome induced by progestins, suggesting that this syndrome is therefore limited to a subsection of women. Klaiber et al. [75] addressed the varied outcome of positive mood states with estrogen, and reversal of these effects with the addition of progestin. The author posited that the opposite steroid effects may relate to their opposite action on the adrenergic and serotonergic function. In menopause, these opposite effects do not occur in all women, but may be caused by other variables. This receptor modulation was clinically demonstrated [76] by comparing the role of estrogen therapy in women with a short duration of menopause (12.9 + 6.1 months) to women with a long duration of menopause (76.6 + 52.3 months). ERT is associated with improved mood in post-

CHAPTER39 Effects of Steroids on Mood/Depression menopausal women, but may not always exert the expected physiological and behavioral effects. Because the duration of menopause determines estradiol levels, lower levels are found in women with a longer duration of menopause. Klaiber et al. [76] followed 38 psychiatrically healthy menopausal women between ages 45 and 65 years with no menses for 6 months and FSH >50 mlU/ml. In a doubleblind cross-over design, the women were studied over five 28-day cycles using two randomly assigned groups (A and B). After a 28-day placebo entry (phase I), group A received estropipate (1.5 mg/day) for two 28-day cycles (phase II), then crossed over to placebo treatment for phase III. Norethindrone (1 mg/day) was administered for 10 days of each cycle to both groups during the ERT cycle. Group B received placebo for phases I and II, then ERT for phase III. Hormones were assayed and mood was monitored by the Hamilton Depression Rating Scale [38]. Pretreatment variables included the duration of menopause, age, and serum levels of estradiol, testosterone, and FSH. The adrenergic/serotonergic variable was platelet M A t activity. The short-duration group had higher mean pretreatment estradiol levels than the long-duration group. However, the short-duration group had lower mean estradiol levels during treatment. Both groups showed a significant improvement in mood when estrogen was administered alone. Differences in mood response occurred when estrogen and progestin were administered. The short-duration group had no significant decline in mood symptoms compared to controls when progestin was administered. The long-duration group tended to lose mood improvement gained with estrogen when progestin was added. The long-duration group had higher estrogen levels on hormone replacement therapy and more dysphoric mood on progestin. Women characterized by short menopausal duration, high pretreatment serum estradiol and testosterone levels, and low pretreatment serum FSH levels were less adversely affected by the estrogen/progesterone combination than were women with long menopausal duration, low pretreatment serum estradiol and testosterone levels, and high pretreatment serum FSH levels. In contrast to women in menopause for a long duration, women in menopause for a short duration had higher circulating pretreatment estradiol levels, unimpaired receptors, and a higher density of estradiol receptors. Therefore, addition of progestin and reduction in estradiol receptor density may not be sufficient to adversely affect mood states. Higher serum estradiol levels were associated with decreased M A t activity during HRT. As expected, high estradiol levels in the long-duration group did not protect against dysphoric mood during progestin treatment, yet the shortduration group had no mood changes on progestin despite lower mean estradiol. It appears that higher pretreatment estradiol levels in the short-duration group were protective against depressive mood changes induced by a progestin.

577 In euthymic patients, decreased M A t is a marker of adrenergic and serotonergic function and correlates positively with DA and 5HT metabolites in the CSF. Women with adequate adrenergic and serotonergic function have a less negative mood response to progestins. A reasonable conclusion for estrogen's inability to sustain positive mood changes in the presence of high estradiol levels in the long-duration group is likely due to impaired receptors. Platelet M A t activity in long-duration women negatively correlate with serum estradiol levels during HRT, suggesting that menopausal women with a higher level of serum estradiol during HRT have poor adrenergic and serotonergic function. Klaiber concluded that these effects may involve the adrenergic and serotonergic neural system by the following mechanism. Because estradiol increases the density of adrenergic and serotonergic receptors, estrogen plus progesterone reportedly decreased the concentration of adrenergic and CNS estradiol receptors induced by estrogen deprivation. Progestin may adversely affect mood states by reducing the effects of estradiol on CNS adrenergic and serotonergic function. Because M A t activity is a genetic marker of adrenergic and serotonergic activity, Klaiber inquired about enzyme activity. Findings illustrated that estrogen's mood-elevating effects in women with low pretreatment platelet M A t activity and impaired pretreatment mood states occurred on estrogen alone, but addition of progesterone impaired mood. Women with higher platelet M A t activity were resistant to the negative effects of progestin on mood. Women whose platelet M A t activity increased during HRT had less adverse mood response on progestin than did those with decreased M A t . In summary, impaired CNS adrenergic and serotonergic function makes nondepressed menopausal women vulnerable to the antiadrenergic and antiserotonergic effect of the estrogen/progesterone combination, whereas optimal adrenergic and serotonin function may protect against the adverse response of progesterone/estrogen. These biological mechanisms support the thesis that steroid hormones have profound effects on female mood states.

C. Androgens Androgens are produced in the Leydig cells of the testes in men and in adrenals in both sexes. The main secretory product from the testes is testosterone. Results of attempts to determine a relationship between testosterone and mood are varied. Testosterone has been implicated in PMS [77] and synthetic derivatives of testosterone have been implicated in depression. Erikson et al. [77] measured serum levels of free testosterone and found them significantly higher throughout the menstrual cycle in subjects with PMS. However, Danazol, a

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synthetic derivative of 17 ce-ethinyl testosterone, suppresses ovarian activity and has been implicated in the treatment of PMS [78]. In a randomized double-blind cross-over controlled trial, 16 women who were treated with Danazol experienced relief of severe PMS symptoms when compared to the 12 women on placebo. Seven (43%) of the Danazol group had responses in the asymptomatic range on 200-mg (twice daily) doses compared to 8.3% on placebo. Another relationship between sex steroid hormones and mood was illustrated with a precursor of testosterone and estrogen, dehydroepiandrosterone. Because levels of DHEA and its sulfate (DHEAS) decrease with aging, Wolkowitz [79] recruited six middle-aged and elderly patients with major depression and low basal plasma hormone levels and administered DHEA for 4 weeks. When dosing was adjusted for levels in the normal range for younger healthy patients, depression and memory improved significantly and directly correlated with plasma levels of the steroid. The author suggests two possible mechanisms for improved mood: the return of youthful DHEA levels as well as the role of DHEA as a precursor for testosterone and estrogen, which appear to have mood-elevating effects. Seidman and Rabkin [80] demonstrated an open case series of testosterone replacement in men with refractory depression and low normal serum testosterone levels. After 2 months of ineffective treatment with SSRI, each of five men received a 400-mg injection of testosterone enanthate every 2 weeks for 8 weeks. There was a significant improvement in Hamilton Depression Rating Scales from entry week through week 8. The authors suggest that testosterone should be administered with caution in men with abnormal prostate examination or elevated prostate-specific antigen levels. Although the study is limited by the number of subjects and the absence of a control group, it provides support for further study.

1. ANDROGENS, MOOD, AND REPRODUCTION The relationship of testosterone to menopause has been a recent focus of study. The profound decrease of estrogen during natural menopause is a consequence of ovarian follicle depletion. Testosterone levels increase with LH secretion which causes ovarian hyperplasia, therefore testosterone levels may vary. Estrogen and testosterone are depleted after TAH/BSO, whereas women with natural menopause produce testosterone until the fifth menopausal year [35]. 2. ANDROGENS, MOOD, AND MENOPAUSE In an attempt to clarify effects of HRT, Sherwin [8] administered estrogen and androgen in a prospective, doubleblind cross-over design to healthy, surgically menopausal women. All subjects anticipated a total abdominal hysterectomy and bilateral salpingo-oophorectomy for benign disease. During the preoperative period, 12 patients were randomly assigned to an estrogen/androgen (EA) group, 11 to an estrogen (E) group, 10 to an androgen (A) group, and 10

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to a placebo (PBO) group. A fifth group was composed of hysterectomy controls without oophorectomy (CON). Intramuscular hormones were administered monthly for 3 months of the postoperative year. Although the study found correlations between hormone levels and improved mood in depressed women, Sherwin cautions against generalizing to the clinical populations because of the induction of physiological hormone levels and the absence of objective criteria to measure mood. Sherwin and Gelfand [35] investigated the administration of HRT with androgens in order to determine if the affective status of surgically menopausal women treated with estrogen alone would differ from those treated with an estrogen/androgen combination. Subjects were healthy women without psychiatric history, matched for age and socioeconomic status. All subjects were at least 2 years postoperative TAH/BSO. The study population was separated into three groups. Group 1 received EA intramuscularly every month for the entire postoperative period. Group 2 was composed of women who were given a monthly injection of estrogen (E) alone, and a control group (CON) received no hormonal intervention. All subjects had a hormone washout 8 weeks prior to baseline. The EA group received 1 ml of estrogen/androgen combined drug (Climactron); the E group received Delstrogen. Objective mood scales and hormone samples were evaluated at intervals in 44 subjects, including 22 women in EA, 11 women in E, and 11 women in CON. There was no difference in basal hormone levels between the groups. Women receiving either hormone preparation had higher estrogen levels, associated with improved mood, and felt more elated and energetic than the CON group. Mood covaried with the physiological range of estrogen and supraphysiological levels of testosterone. The testosterone group had the most improved mood scores, most likely due to the very high levels. All symptoms improved when estrogen and testosterone levels were high. The hormone-treated groups had lower depression scores than the placebo group, which corresponded to their higher levels of circulating estrogen and testosterone. Heightened hostility was detected in the androgen-treated group. In conclusion, because of slow testosterone metabolism, testosterone remained at supraphysiological levels. Therefore, conclusions apply only to high testosterone levels, but cannot predict mood at physiological levels. The mechanism by which estrogen maintains higher serotonin levels is by decreasing MAO activity in the amygdala and hypothalamus. The mechanism of androgen action is not well understood, but it may be aromatized to estrogen to provide added antidepressant effect. Because individual responses to HRT (particularly testosterone) are difficult to predict, the same authors [82] measured levels of bound and unbound steroid. Both steroids bind to sex hormone binding globulin (SHBG). In plasma,

CHAPTER39 Effects of Steroids on Mood/Depression estrogen increases and testosterone decreases SHBG. Only the free unbound portions of circulating testosterone are presumed biologically active. The study aimed to determine the SHBG profile induced by chronic administration of an estrogen/androgen preparation. Ten healthy women in the second postoperative year after TAH/BSO received 1 ml estrogen/androgen drug intramuscularly every 28 days for 2 years [82]. After an 8-week washout period, baseline blood samples were drawn. A combined hormone injection was followed by psychometric tests and steroid hormone levels. High circulating hormone levels corresponded with improved mood in women with normal affective states. Testosterone levels remained fairly high and stable. Estrogen levels decreased after 2 days. However, one subject had abnormally increased SHBG levels associated with dysphoric mood and one had abnormally low levels that were associated with improved depression score. Sherwin suggests that total plasma hormone levels are altered by SHBG, and provide insufficient information. Levels of SHBG may be important to understanding mood and clinical response in some women, thereby providing additional information when dosing.

VII. CONCLUSION Thus far, several researchers report mood-enhancing effects of HRT, but others fail to demonstrate this pattern. Generalizations from data on HRT, such as biological outcomes and psychological variables, are difficult to make. Modern studies have addressed the inconsistent methodology in menopause research. Evaluation of outcome must consider qualitative measures and pre-existing variables such as natural versus surgical menopause, history of depressed mood, and duration of menopause. In order to aggregate data, Zwiefel et al. [82] performed a meta-analysis to examine the effectiveness of HRT on menopausal depressed mood. Methodologies and existing variables were systematically evaluated, addressing variables such as sample size, level of depression, reliability of depression rating scales, type and dose of HRT, and length of treatment. Studies were obtained from literature searches (1974 to 1995) and from correspondence with major contributors to research. Inclusion criteria for the 26 studies were the use of valid measures of depression and administration of hormone therapy. The average number of subjects per study was 47.15 (SD = 28.94). Study variables included type of design, assignment to groups, application of double- or single-blind conditions, recruitment procedures, and demographics. Most studies did not provide demographic information, such as level of education, socioeconomic status, income, urban or rural location, marital status, and race. Although most studies included postmenopausal women,

579 few used perimenopausal women or both. In addition, menopause was determined by various measures, such as TAH/ BSO, FSH levels, amenorrhea for 12 months, estrogen levels, and presence of menopause symptoms. Mechanisms of menopause were classified as TAH/BSO, natural menopause, or TAH without BSO; some studies did not specify. Hormones varied by dose, route of administration and type of steroids. Some studies utilized progesterone or testosterone. Most studies included subjects who were either not depressed or were experiencing only mild levels of depression. Most studies included in the meta-analyses used adequate sample sizes, controlled research designs, random assignment, and valid, reliable outcome measures for depression. Overall results of the meta-analyses demonstrated that depressed mood scores decreased from pretreatment to posttreatment for estrogen versus estrogen plus progesterone comparison treatment. Estrogen was a more effective treatment. Significant effect scores were associated with HRT. The average treatment subject had lower levels of depressed mood compared to control subjects. Estrogen plus progesterone reduced the effect of ERT on depressed mood. The androgen alone and androgen plus estrogen treatment yielded a large effect. The results of this meta-analysis suggest that estrogen reduces depressive symptoms in nonclinically depressed women, whereas progesterone alone and in combination with estrogen is associated with smaller reductions in depressed mood. Androgen alone and in combination with estrogen was associated with improved mood. In summary, there were a variety of methodological approaches in the use of study design, depression measures, and length of treatment, all associated with variation in effect size. Further issues that must be addressed include optimal combination of hormones, dose-response relationships, effectiveness of HRT across demographic variables such as race and socioeconomic economic status, and treatment outcomes as a function of menopausal status, type of menopause, and severity of symptoms. Several suggestions were made to improve further studies, such as improved reporting of demographic information and reporting effective doses of hormones. In this author's experience, the onset of mood and anxiety symptoms associated with HRT remains underdiagnosed and undertreated. Because noncompliance with HRT may have serious implications for the postmenopausal woman, the prescribing physician should inquire about mood in women, who may hesitate to report depresssive symptoms and who may therefore discontinue HRT in an attempt to self-treat. In addition, particular attention should be given to women with existing mood disorders such as chronic depression or bipolar mood disorders, or with a previous history of puerperal psychiatric illness. The developing field of perinatal and reproductive psychiatry encourages liaison between the fields of mental health

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a n d r e p r o d u c t i v e events. I m p r o v e d c o m m u n i c a t i o n b e t w e e n the p r o f e s s i o n a l s w o r k i n g in p s y c h i a t r y , o b s t e t r i c s , a q d gynecology may increase awareness of mood disorders induced b y e n d o g e n o u s or e x o g e n o u s steroids. S t a n d a r d p s y c h o p h a r macology intervention easily provides treatment for those w o m e n in n e e d o f i n t e r v e n t i o n .

Acknowledgment The author is the recipient of the Research Scientist's Development Award for Clinicians from the National Institute of Mental Health, NIMH Grant No. 1 K20 MH01276-01.

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CHAPXER 39 Effects of Steroids on Mood/Depression

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582 79. Wolkowitz, O. M., Reus, V. I., Roberts, E., Manfredi, F., Chan, T., Raum, W. J., Ormiston, S., Johnson, R., Canick, J., Brizendine, L., and Weingartner, H. (1997). Dehydroepiandrosterone (DHEA) treatment of depression. Biol. Psychiatry 41, 311-318. 80. Seidman, S. N., and Rabkin, J. G. (1998). Testosterone replacement therapy for hypgonadal men with SSRI-refractory depression. J. Affective Disord. 48, 157-161.

MARGARET G. SPINELLI 81. Sherwin, B. B., and Gelfand, M. M. (1987). Individual differences in mood with menopausal replacement therapy: Possible role of sex hormone-binding globulin. J. Psychosom. Obstet. Gynaecol. 6, 121131. 82. Zweifel, J. E., and O'Brien, W. H. (1997). A meta-analysis of the effect of hormone replacement therapy upon depressed mood. Psychoneuroendocrinology 22, 189-212.

~HAPTER 4(

Hormone Replacement Therapy and Breast Cancer CECILIA MAGNUSSON

JOHN A. BARON

I. II. III. IV.

Department of Medical Epidemiology, Karolinska Institutet, S-171 77 Stockholm, Sweden Section of Biostatistics and Epidemiology, Dartmouth Medical School, Hanover, New Hampshire 03755

Background Steroid Hormones and Breast Cancer Patterns of HRT Use Epidemiological Studies of the HRT/Breast Cancer Association

V. Discussion References

II. STEROID H O R M O N E S AND BREAST CANCER

I. B A C K G R O U N D Hormone replacement therapy (HRT)--i.e., prescription of estrogens with or without progestins to counter the decline and loss of ovarian function at m e n o p a u s e - - i s increasingly used in developed countries. The obvious rationale for the growing popularity of HRT is the substantial beneficial effect on menopausal symptoms [1] as well as a probable preventive effect on osteoporosis [2] and possibly also on ischemic heart disease [3]. Yet, evidence that ovarian hormones play a fundamental role in breast cancer etiology [4] raises the serious concern of an increased risk of this cancer following HRT. Because breast cancer incidence is relatively high and hormone replacement is common, an association between the two would have important public health implications.

MENOPAUSE: BIOLOGY AND PATHOBIOLOGY

There is compelling evidence that ovarian hormones, e.g., estrogen and possibly progesterone, have a principal role in the development of breast cancer (see Chapter 25). There is a 100-fold difference in incidence between women and men, and premenopausal oophorectomy is clearly protective. Further, the changes in incidence with age differ from the steady increase seen for non-hormone-dependent cancers. Breast cancer incidence increases rapidly with age up to age 50 years, which is roughly the mean age at natural menopause and consequently the time of cessation of ovarian estrogen and progesterone production [4]. After this age there is a marked decline in the rate of increase of incidence with age (Fig. 1). Finally, many established breast cancer risk factors

583

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3.00

2.50

O O O O O

2.00

(1) x...

o C

1.50

0

Jo

1.00

0.50

0.00 2024-

2529-

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I

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I

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I

I

I

3034

3539

4044

4549

5054

5559

6064

6569

7074

7579

8084

I 85+

Age (years)

FIGURE 1 Age-specific breast cancer incidence in Sweden in 1995.

(i.e., age at menarche, parity, age at giving birth, age at menopause, and postmenopausal obesity) are associated with exposure to ovarian steroids [5]. Estrogens are known to stimulate proliferation of the breast epithelium [6,7], and a quantitative review of prospective data concluded that risk of breast cancer was significantly increased among women with higher serum levels of estradiol [8]. Progesterone was formerly thought to antagonize the proliferatory effect of estrogen and thereby reduce breast cancer risk. Results from in vitro studiesmin which cells are separated from the complex hormonal milieu of breast tissue in v i v o m d o suggest that progestins can antagonize estrogen-stimulated growth of human breast cells [9,10]. Yet, this assumption must now be questioned [4]. In vivo studies have repeatedly shown that the mitotic activity of breast epithelium peaks during the luteal phase of the menstrual cycle, when progesterone levels are at their highest [ 11-14]. Further, Cline et al. found that conjugated estrogens with continuous addition of progesterone-like progestins induced greater proliferation of breast epithelial cells in macaques than did conjugated estrogens alone [15]. Finally, there is evidence that mammographic density, a risk factor for breast cancer [ 16], is higher in women taking combined estrogen-progestin replacement therapy than in those taking estrogen without progestins [17]. In addition to estrogens and progesterone, breast cancer risk is likely influenced by several other endogenous factors [18-20]. These endocrine

factors were thought to exert their carcinogenic effect only through stimulation of epithelial proliferation [21]. However, new research hints that estrogen metabolites can bind to DNA and trigger damage directly [22].

III. PATTERNS OF HRT USE The use of HRT has increased dramatically since the introduction of these drugs in the 1960s [23,24]. In 1992, an estimated 31.7 million prescriptions were dispensed in the Untied States for oral menopausal estrogens and an additional 4.7 million for transdermal estrogen; approximately one in six to one in four postmenopausal women were taking menopausal hormones. Ten years previously, the number of dispensed prescriptions for oral estrogen was 13.6 million, and transdermal preparations had not yet been marketed. Recent data from Sweden indicate that the prevalence of replacement hormone use ranges from 20 to 35% of women in their 50s [25,26], and sales statistics indicate a threefold increase in use during the past two decades (studies by Apoteket AB, Stockholm). HRT comprises a heterogeneous group of hormonal drugs (Table I) whose clinical use has changed over time. Earlier, oral estrogens alone were usually prescribed: in the United States these were mainly conjugated estrogens from equine urine (0.325, 0.625, or 1.25 mg) and in Europe estra-

CHAPTER40 Hormone Replacement and Breast Cancer Hormonal Compounds Used for Hormone Replacement Therapy

TABLE I

Compound

Comment

585 scientific effort. Reports on the relation between HRT and breast cancer risk started to appear in the 1970s and were soon followed by numerous other publications [28].

Estrogens Estradiol

Conjugated estrogens

Estrone sulfate Methallenstril Ethinyl estradiol Estriol Dienestrol

Medium potency, most common HRT estrogen in Europe; administered orally, transdermally, or intramuscularly Medium potency, most common HRT estrogen the United States; administered orally or transdermally Medium potency Medium potency High potency, mainly used in oral contraceptives Low potency, used in Europe orally and topically for relief of urogenital symptoms Low potency, used topically

Progestins Medroxyprogesterone acetate Dydrogesterone Norethisterone acetate Levonorgestrel Lynestrenol

17-Hydroxyprogesterone derivative, most common HRT progestin in the United States 17-Hydroxyprogesterone derivative 19-Nortestosterone derivative, most common HRT progestin in Europe 19-Nortestosterone derivative 19-Nortestosterone derivative

diol valerate (1 or 2 mg). Because of repeated reports of an increased risk for endometrial cancer in users of estrogen only [2], combined estrogen-progestin regimens--presumably with smaller effects on endometrial cancer risk [2,27]-are now prescribed to women with an intact uterus. Progestins can be added cyclically (most often 10 days per cycle) or continuously (every day), and they may be both testosterone-like (19-nortestosterone derivatives, i.e., norethisterone acetate, levonorgestrel, or lynestrenol--mainly used in Europe) or progesterone-like (17-hydroxyprogesterone derivatives, i.e., medroxyprogesterone acetate--mainly used in the United States). Estrogen and progestin are also administered through different routes, earlier mainly orally, and to a less extent, intramuscularly, but lately also transdermally. Oral estriol and topical treatment (assumed to have negligible systemic effects) are also used in some countries for alleviation of urogenital atrophy.

IV. E P I D E M I O L O G I C A L

STUDIES

OF THE HRT/BREAST CANCER

ASSOCIATION

The evident plausibility of a causal influence of HRT on breast cancer development and the important public health implications of such an association have motivated extensive

A. E s t r o g e n R e p l a c e m e n t T h e r a p y ~

The

Collaborative Study In 1997, findings from a collaborative project were published summarizing the effects of HRT on breast cancer risk [28]. This endeavor involved the gathering and reanalysis of 90% of the available epidemiological data on the topic. The main analyses were based on 17,949 women with breast cancer and 35,916 women without, all of whom were postmenopausal and in whom age at menopause and HRT use could be established. The median year of birth in these women was 1925. The estimates of breast cancer risk after HRT were combined using the Mantel-Haenzel technique, after stratification for study, age, time since menopause, body mass index (BMI), parity, and age at first birth. Cases included in the study were diagnosed with their breast cancer, on average, in 1985, when most HRT use consisted of estrogens without addition of progestin. Nonetheless, explicit information on the types of preparations used was available for only 39% of the eligible women. In these women, 80% had used primarily estrogen alone (75% of whom reported use of conjugated estrogens), and 12% had used primarily combined estrogen-progestin treatment. Thus the overall results from this study mainly refer to risk of breast cancer after use of estrogen without addition of progestins. This report concluded that use of hormone replacement therapy within the past 5 years was associated with breast cancer risk in a duration-dependent way (relative risk per year of use, 1.023; 95% confidence interval, 1.011-1.036) (Table II). The magnitude of this effect was comparable to the influence of delaying menopause (in these data, 2.8% increase in relative risk per year of delay). There was no indication of an excess breast cancer risk 5 or more years after cessation of use (Table II), and the association between recent HRT use and breast cancer risk was largely confined to localized tumors. The findings also supported an interaction between BMI and HRT: the adverse effect of treatment was more pronounced among relatively lean women (BMI < 25.0 kg/m 2) than among those who were overweight or obese (BMI -> 25.0 kg/m2). The relative risks after 5 or more years of use within 5 years of diagnosis was 1.52 (95% confidence interval, 1.29-1.79) for women with a BMI <25.0 kg/m 2 and 1.02 (95% confidence interval, 0.82-1.26) for women with a BMI _> 25.0 kg/m 2. No evidence of interactions between HRT and any other factors on breast cancer risk was found. The collaborative study also reinforced the importance of methodological issues, in particular the necessity for careful

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MAGNUSSON AND BARON

TABLE II

Breast Cancer Studies in Relation to Timing of Use of Hormone Replacement Therapy a Cases/controls

RR b

95% CI c

12,467/23,568

1.00

Reference

Last use < 5 years before diagnosis <1 1-4 5-9 10-14 -->15

368/860 891/2037 588/1279 304/633 294/514

0.99 1.08 1.31 1.24 1.56

0.84-1.17 0.96-1.21 1.12-1.53 1.00-1.53 1.21-2.00

Last use -- before diagnosis <1 1-4 5-9 - 10

437/890 566/1256 151/374 93/233

1.12 1.12 0.90 0.95

0.96-1.31 0.98-1.28 0.72-1.13 0.71-1.26

Duration of use (years) Never-use

a Mainly use of estrogen replacement therapy. Pooled estimates from a collaborative study, including 90% of the available epidemiologic data (Collaborative Group on Hormonal Factors in Breast Cancer, 1997). Adjusted for study, age at diagnosis, time since menopause, body mass index, parity, and age at first birth. bRR, Relative risk. c CI, Confidence interval.

adjustment for age at menopause, for any estimate of breast cancer risk after HRT use [29]. It was shown that failure to control for age at menopause led to a substantial underestimation of relative risks among recent users of HRT. This is easily understood because duration of HRT use in women of a given age will correlate with age at menopause. The earlier the menopause (early menopause being protective against breast cancer), the longer the duration of HRT use, rendering age at menopause an important negative confounder of the association between HRT and breast cancer risk. The authors therefore suggested that women with unknown age at menopause should be excluded from analyses of the association between HRT use and breast cancer risk. Unfortunately, only a partial understanding of the effect of HRT on breast cancer risk can be obtained from the collaborative project. The estimates of risk after treatment pertain mainly to use of estrogens without addition of progestins, and combined regimens are most commonly used today. Furthermore, no data on the effect of different estrogenic compounds, routes of administration, or doses were presented. Finally, only about 2% of the cases included had used long-term HRT in the past, and the absence of an effect of past use on HRT could be due to chance. We have published data from a population-based case-control study of breast cancer, including 2563 cases and 2845 controls, giving some support to the influence of long-term use of HRT in the distant past [30]. In this study, women who had used HRT for more than 10 years and had discontinued treatment more than 10 years previously had a more than twofold increase in breast cancer risk as compared to women who never used

HRT (a statistically significant estimate that, however, was based on few subjects).

B. Combined Estrogen-Progestin Treatment Although most investigators have examined the association between use of estrogen alone and breast cancer risk, there are some data that pertain to combined regimens (Tables III and IV). If the hypothesis that the mitogenic effect of estrogen is augmented by progesterone is true [4], combined estrogen-progestin treatment might confer a higher risk of breast cancer than estrogen taken alone. In support of this possibility, Bergkvist et al. [31 ] found a fourfold relative risk ofbreast cancer in Swedish long-term users of combined treatment in comparison to never-users (an estimate based on 10 cases) (Table III). An excess risk in ever-users of sequential estrogen-progestin replacement was also demonstrated in a Danish study [32], and an American study reported a similar (but nonsignificant) relative risk for combined treatment [32a] (Table IV). During 1995, results from three population-based studies in the United States were presented, all examining the influence of combined treatment [33-35] (Tables III and IV). Only one of these [33] demonstrated an effect of combined treatment, reporting a relative risk for recent use of 1.41 (95% confidence interval, 1.15-1.74); lack of power precluded analyses of long-term use. It should be noted that different estrogenic compounds were used for HRT in the American studies (mainly conjugated estrogens) and the European studies (mainly estradiol). When reviewing the literature on combined estrogenprogestin therapy and breast cancer risk, the most striking impression is the critical lack of data. Only Newcomb et al. [35], who conducted the largest case-control study to date on breast cancer, had a practical ability to examine the association in any detail. This study showed remarkably null associations with use of any type of HRT, either long-term or recent. In the collaborative analysis, women who had recently used combined treatment for at least 5 years had a relative risk of 1.53 (95% confidence interval, 0.80-2.92) as compared to neverusers [28]. Thus, epidemiological data on the different types of combined treatment (e.g., progesterone- or testosteronederived progestins, cyclic or continuous addition) are scarce. However, different combined regimens have been used for a relatively long time in Sweden, which is why we attempted to assess the influence of these drugs on breast cancer risk in our case-control study [30]. Use for more than 10 years of any combined regimen conferred a statistically significant threefold increase in breast cancer risk as compared to never-use, an association similar to that with long-term use of estrogen only. Interestingly, we found apparent evidence of a more pronounced adverse effect of long-term use of continuously combined regimens than of long-term cyclical treatment (with testosterone-derived progestins).

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CHAPTER 40 H o r m o n e R e p l a c e m e n t and B r e a s t C a n c e r

TABLE I I I

B r e a s t C a n c e r R i s k in R e l a t i o n to T y p e o f H R T a Estrogen-progestin, RR

Estrogen only, RR

Ref.

Person-years of follow-up

No. of cases

Ever

Ever Current long term Current long term

31

133,375

253

--

1.8 b

40

313,902

1185

1.0

1.2

1.3 b

1.4b

33

725,550

1935

--

~

1.3 b

~

54

297,977

634

0.9

--

--

Ever

--

Ever long term Current

Sweden, prescription-based; measure of longterm use, ->9 and ->6 years, respectively. Estimates were adjusted for age and period

4.4

1.2b

1.4

Comments

1.2

United States; measures of long-term use, -> 20 years and ->4 years, respectively. Estimates were adjusted for age and education; associations were more pronounced for breast cancer in situ

1.4 b

United States; estimates were adjusted for age; ages at menarche, first birth, and menopause; type of menopause; parity; family history; and benign breast disease. Associations were more pronounced among older postmenopausal women Sweden, prescription-based; estimates were adjusted for age and period. These results are derived from further follow-up of the cohort in Ref. [31].

1.3 b

a From cohort studies reporting on combined therapy; RR, relative risk. b Lower bound of confidence interval does not include unity.

TABLE I V

B r e a s t C a n c e r R i s k in R e l a t i o n to T y p e o f H R T a Estrogen-progestin, RR

Estrogen only, RR

Ref.

No. of cases/ controls

Ever

32

1486/1336

1.0

32a

1686/2077

1.1

0.9

1.2

1.7

32b

699/685

1.0

1.6 b

1.4 b

1.2

34

537/492

0.9

1.0

0.9

0.9

0.4

0.9

35

3130/3698

1.0

1.0

0.9

1.0

1.0

0.9

55

435/1740

1.3

--

30

2563/2845

2.7 b

--

1.9 b

Ever Current long term Current long term

Ever

Ever long term Current

1.4 b

1.0

2.4

1.6 b

3.0 b

a From case-control studies reporting on combined therapy; RR, relative risk. b Lower bound of confidence interval does not include unity.

Comments Denmark; estimates were adjusted for age and place of residence United States; hospital-based; measure of long-term use, -> 15 years. Estimates were adjusted for age and type of menopause United States; measure of long-term use, -->10 years. Estimates adjusted for age and type of menopause; response rates were below 75 % United States; measures of long-term use, ->20 and ->8 years, respectively. Estimates were adjusted for age, age at first birth, and family history of breast cancer; the response rate among controls was below 75% United States; measure of long-term use, -> 15 and ->5 years overall and recently, respectively. Estimates were adjusted for age, state, type of menopause, time since, body mass index, family history, benign breast disease, alcohol consumption, and education Sweden; measure of long-term use, -> 11 years. Estimates were adjusted for age, age at menarche, age at first birth, parity, body mass index, family history, and use of oral contraceptives. Study nested within cohort of participants of mammography screening program Sweden; measure of long-term use, -> 11 years. Estimates were adjusted for age, age at menarche, age at first birth, parity, age at menopause, and type of menopause

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MAGNUSSON AND BARON

C. I n f l u e n c e o f H R T o n N a t u r a l H i s t o r y o f Breast Cancers When assessing the risks and benefits of HRT, not only breast cancer incidence but also survival must be considered. Most studies, excluding that of Colditz et al. [33], have not found an excess breast cancer mortality among women who used HRT, in spite of a possibly increased incidence [3638]. Brinton found an association between HRT and breast cancer that was more pronounced for in situ and small tumors [39], a finding that was confirmed by other studies [40] and the collaborative study [28]. Furthermore, case fatality has been reported to be lower [41] and prognostic tumor characteristics less disadvantageous [42] in patients with HRT prescriptions than in those without. These findings are not readily interpreted. Possibly, women with HRT are more intensely followed, and therefore have breast cancer detected at an earlier stage than other women. If so, the reported association between HRT and breast cancer incidence may be spurious and due to detection bias (see below). Because mammography sensitivity and specificity might be lowered in women with HRT [43], the potential influence of surveillance bias on the HRT/breast cancer association is, however, not easily discerned. Alternatively, exogenous and endogenous hormones might have a biologic influence on the natural history of breast cancer, perhaps through interference with the metastatic process [44,45].

D. L i m i t a t i o n s o f O b s e r v a t i o n a l S t u d i e s For several reasons, investigation of the association between breast cancer risk and HRT poses substantial difficulties. The various categories of HRT present several problems. In the past 50 years, numerous HRT regimens have been employed, including various estrogen compounds in various doses, with (or without) various progestational agents added in various patterns. The inevitable result is that some subjects will misreport their HRT history, and that investigators will struggle to devise reasonable groupings of the regimens used. This misclassification of exposure history will often tend to obscure an association between HRT and breast cancer. However, it is also possible that inaccurate reporting of HRT history could exaggerate an HRT association. For example, in research designs in which cases of breast cancer are compared with controls without breast cancer, the cases may tend to overreport hormone use, while the controls may tend to overlook use that actually occurred. There has been some investigation into whether this pattern of distorted reporting actually occurs; fortunately, it appears that this is not a substantial problem [28,46]. Another serious problem is that women who use HRT are

a selected group. Until recently, HRT was used mainly for relief and prevention of menopausal symptoms, or for hormone replacement after oophorectomy/hysterectomy. Women taking the drug for these indications are relatively estrogen deficient [20] and are therefore at lower than average risk of hormone-related diseases such as breast cancer. Further selection pressures are added by the physician prescribing HRT, because many clinicians will ensure that patients do not have endometrial or breast cancer before prescribing hormone replacement. The negative work-up (e.g., mammogram and endometrial aspirate) will remove from the treated groups women with occult cancer (and probably women with precancerous lesions such as certain benign breast diseases and endometrial hyperplasia). This selection also tends to place the treated women in a group with a below average risk for being diagnosed for breast (and endometrial) cancer. If the women who take HRT are inherently at lower than average risk of breast (and endometrial) cancer, then even the observation that these women have an average risk would imply that HRT is actually increasing breast cancer risk above the pretreatment level. Moreover, because women who become ill may stop HRT, only relatively healthy women will continue HRT for prolonged periods of time, adding an element of a continuous bias toward healthy women taking these drugs [47,48]. On the other hand, there are factors that tend to exaggerate the association between HRT and breast cancer risk. In most countries, women who use HRT must obtain a prescription for the drugs, and the current standard of care is for the prescribing physicians to monitor these patients for problems such as breast cancer. The enhanced medical surveillance of these women in comparison to women not given HRT would tend to increase the risk of detection of breast (or endometrial) cancer. This extra opportunity for diagnosis among HRT users would create a tendency for HRT to be associated with the diagnosis of breast cancer even if there is no underlying tendency for HRT to increase the risk of the tumors. Other factors may tend to lead to an underestimation or an overestimation of the true association between HRT and breast cancer. In general, women who take HRT are better educated and have higher incomes compared to women who do not take HRT [49-51 ]. Women using hormone replacement therapy are also more likely to smoke cigarettes, drink alcohol, and to be relatively lean [49-51 ]. These traits may well affect the risk of breast cancer (and other diseases), and so will have to be taken into account in analyses.

V. D I S C U S S I O N In summary, a causal association between HRT and breast cancer is biologically plausible because estrogen is a known breast epithelial mitogen and possibly also a mutagen. There

589

CHAPTER 40 H o r m o n e R e p l a c e m e n t and Breast C a n c e r

is evidence of a moderate effect of long-term recent use of HRT on breast cancer risk. However, important questions remain--particularly the impact of past long-term use, and the effects of different preparations (e.g., type and dose of estrogen, type and regimen of combined estrogen-progestin treatment). Moreover, it cannot be ruled out that methodological obstacles have distorted the reported association between HRT and breast cancer. There are some indications that breast cancer after HRT has less malignant tumor features and improved prognosis as compared to other tumors. Yet, it is not known if these indications reflect a true biologic effect of HRT on the natural history of breast tumors or if they are due to biases (for example, detection bias because of increased health awareness or medical surveillance of women on HRT). The risk-benefit assessment of HRT is complicated, because such treatment likely entails important advantageous health effects, including improved quality of life and reduced risk of osteoporotic fractures. In addition, reduced risks of cardiovascular disease (see Chapter 37), and also possibly colon cancer [52] and Alzheimer's disease [53] (see Chapter 21), have been discussed as possible beneficial effects of HRT. Furthermore, it is important to view risks and benefits in their proper time window. Breast cancer is relatively common in women 5 0 - 6 0 years of age (when HRT use is common), whereas cardiovascular disease and especially osteoporosis are diseases of elderly women. Therefore, it is crucial to establish not only the magnitude of the association between different HRT regimens and risk of disease, but also how long the association persists after cessation of use.

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~HAPTER 4

HRT and Risk of Endometrial Cancer SANJAY

K.

AGARWAL

HOWARD L.

JUDD

Division of Reproductive Medicine, Department of Obstetrics and Gynecology, Cedars-Sinai Burns and Allen Research Institute, Cedars-Sinai Medical Center, Los Angeles, California 90048; and Department of Obstetrics and Gynecology, University of California, Los Angeles, School of Medicine, Los Angeles, California 90095 Department of Obstetrics and Gynecology, Olive View/UCLA Medical Center, Sylmar, California 91342; and Department of Obstetrics and Gynecology, University of California, Los Angeles, School of Medicine, Los Angeles, California 90095

IV. Physiology of Endometrial Shedding V. Summary References

I. Endometrial Cancer II. Estrogen and Progestin Therapy III. Endometrial Surveillance

I. E N D O M E T R I A L C A N C E R

bibliographies, and consultations with experts. Studies were excluded from the meta-analysis if an inappropriate comparison group was used, if relative risk (RR) estimates or confidence intervals (CIs) were not published, and the authors could not retrieve the information necessary to calculate these values, or if there were fewer than five endometrial cancer cases included in a specific study. Summary estimates were calculated of the risk of endometrial cancer using metaanalytic statistical methods described by Greenland [39], which are based on the assumption of fixed effects. In Table 1 is shown the summary RR estimates for endometrial cancer and estrogen usage [38]. Among ever-users of unopposed estrogen compared to never-users, the RR was 2.3 (95% CI, 2.1-2.5). The summary RR estimate for casecontrol studies was slightly higher than for cohort studies. Among case-control studies, those with gynecologic controis had a higher summary risk estimate than those with hospital-based or community controls. The summary RR

The major complication of unopposed estrogen replacement is the development of endometrial carcinoma. Endometrial cancer ranks third among the most common cancers in women in the United States, and it is the most common gynecologic malignancy. The American Cancer Society estimated the occurrence of approximately 36,100 new cases and 6300 deaths due to endometrial cancer in the United States in 1998 [ 1]. In 1975, two case-control studies reported an association between estrogen replacement therapy and an increased risk of endometrial carcinoma [2,3]. Since then, more than 30 epidemiologic studies have been published linking estrogen use with the development of this tumor [4-37]. A metaanalysis of these articles has been published [38]. It was based on a literature search of English-language studies published between 1970 and 1994 using MEDLINE, a review of

MENOPAUSE:

BIOLOGY AND PATHOBIOLOGY

591

Copyright 9 2000 by Academic Press. All rights of reproduction in any form reserved.

592

AGARWAL AND JUDD

TABLE I Relative Risk from Meta-Analysis: Postmenopausal Estrogen Therapy and Endometrial Cancer a RR b

95% CI r

No. of studies

2.3 d 1.7 d 2.4 a 2.2 c 3.3 e 2.4 d

2.1-2.5 1.3--2.1 2 . 2 - 2.6 2 . 0 - 2.5 2.7-4.0 2.0-- 2.9

29 4 25 10 6 10

Conjugated estrogen dose (milligrams) 0.3 3.9 0.625 3.4 _>1.25 5.8

1.6-9.5 2.0-5.6 4.5-7.5

3 4 9

Duration of use (years) <1 1-5 5-10 >10

1.4 2.8 5.9 9.5 e

1.0-1.8 2.3-3.5 4.7-7.5 7.4-12.3

9 12 10 10

Regimen Intermittent and cyclic Continuous

3.0 ~ 2.9 ~

2 . 4 - 3.8 2.2-3.8

8 8

Type of estrogen Conjugated Synthetic h

2.5 g 1.3 ~

2.1 - 2.9 I. 1-- 1.6

9 7

Time since last use (years) --<1 1-4 -->5

4.1 ~ 3.7 2.3

2.9-5.7 2.5-5.5 1.8-3.1

3 3 5

Stage/invasiveness Stages 0 - 1 Stages 2 - 4 Noninvasive Invasive

4.2 1.4 6.2 3.8 e

3.1-5.7 0.8-2.4 4.5 - 8 . 4 2.9-5.1

3 3 4 6

Death from endometrial cancer

2.7

0 . 9 - 8.0

3

Ever-users of estrogens All eligible studies Cohort studies Case-control studies Hospital controls Gynecologic controls Community controlsf

aFrom Grady et al. [38]. Reprinted with permission from the American College of Obstetricians and Gynecologists (Obstetrics and Gynecology, 1995, Vol. 85, pp. 304-313.) b Pooled relative risk from meta-analysis. cpooled 95% CI from meta-analysis. dp homogeneity, <0.0001. ep homeogenity, <0.01. fCommunity controls include residential, neighborhood, and popula'tion-based controls. gP homogeneity, <0.05. h Synthetic estrogens include primarily ethinyl estradiol, estradioi valerate, estriol, and uspecified other estrogens; diethyistilibesterol and estrogen combined with androgen excluded, except in cases where such use was lumped with all synthetic estrogens.

estimate for endometrial cancer was higher among users of conjugated estrogens than among users of synthetic estrogens. There was also a dose-response relationship of conjugated estrogens and endometrial cancer ranging from 3.9 for 0.3 mg to 5.8 for -> 1.25 mg of medication. The summary RR was appreciably higher with prolonged duration of estro-

gen use with the RR for less than 1 year of use being 1.4 and the RR for more than 10 years of use being 9.5. There was no significant difference in the RR estimates between interrupted and daily use of estrogen. With regard to stage, grade, and invasiveness, the RRs were higher for early stage and grade and for noninvasiveness. The summary RR for death due to endometrial cancer was also elevated at 2.7 (95% CI, 0.9-8.0). Last, the summary RR was still elevated 5 or more years after discontinuation of unopposed estrogen therapy (RR 2.3; 95% CI, 1.8-3.1). The main limitation of this meta-analysis is that all of the data, with one exception [40], were from observational studies, which are subject to confounding. Misclassification attributable to inclusion of women with endometrial hyperplasia among endometrial cancer cases could spuriously increase risk [41], but studies based on careful review of pathologic findings still show an increased risk of endometrial cancer in women who have taken unopposed estrogens [7,41,42]. Increased risk of endometrial cancer in women who have taken unopposed estrogens might also be attributable to biased selection of controls, or recall bias among women using estrogen [7,41 ]. However, the consistent report of increased risk despite differing control groups and adjustment for confounding variables, and with increasing dose and duration of use, make it unlikely that the association is due to bias. It is not surprising that estrogens have been incriminated in the development of endometrial cancer. Unopposed estrogen stimulates endometrial growth [43]. However, the mechanisms by which estrogens promote growth of the endometrium have only been partially worked out. Estrogen increases (DNA) synthesis, as measured by tritiated thymidine labeling, and the content of both nuclear estradiol and progesterone receptors [44,45]. Uterine cells are extremely sensitive to estrogen in vivo, but in vitro they fail to respond or only respond to pharmacologic quantities of estrogen [46]. This lack of response in vitro has given rise to the proposal that estrogen-induced proliferation in vivo may be mediated by polypeptide growth factors. Growth factors stimulate cell proliferation through binding to specific receptors. Both epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) have been implicated in growth of reproductive tissues [46,47]. Exposure of cells to EGF usually activates a biochemical cascade resulting in DNA synthesis and mitosis. IGF-I circulates in plasma and binds to a specific receptor that resembles the insulin receptor. Receptors for both polypeptides are found in the endometrium [48]. Estrogen has been shown to up-regulate the gene for EGF as well as EGF receptors in the uterus of rats and mice. Estradiol also increases both IGF-I and the transcription of DNA for the message for IGF-I in the uterus of rats. Similar findings have been forthcoming with human endometrium. An antibody specific for EGF has been shown to inhibit uterine and

CHAPTER41 HRT and Risk of Endometrial Cancer vaginal growth, and in vivo exposure to EGF alone mimics the induction of uterine and vaginal growth and differentiation similar to those induced by estrogen in mice [49]. These results suggest that EGF acts as an estrogen-inducible physiologic mediator of mouse reproductive tract growth in vivo. Similar findings are awaited in human endometrium. There is a cascade of changes of endometrial histology associated with estrogen administration. In the absence of estrogen, the endometrium is atrophic. With exposure to estrogen, the endometrium begins to grow (proliferate). If estrogen exposure is continued, it can overgrow (hyperplasia) and ultimately become cancerous. Figure 1 shows histologic views of complex (adenomatous) hyperplasia with and without atypia and well-differentiated carcinoma of the endometrium. Proof that estrogens stimulate endometrial hyperplasia will be presented below. Proof that estrogens promote the transformation of hyperplasia to cancer is not available because no ethical physician would attempt, or Internal Review Board would allow, such a prospective study to be conducted. Thus, some other type of study must be substituted for a randomized trial to establish that endometrial hyperplasia is a precursor lesion for endometrial cancer. The most compelling studies are those that have identified women with endometrial hyperplasia. For some reason, these women did not undergo medical therapy or a hysterectomy. In subsequent years, some of these subjects developed endometrial cancer. Of the nearly 600 women followed in this manner, approximately 20% developed endometrial cancer during the next 10 months to 30 years [50,51]. This percentage is much greater than would be expected by chance. Two studies broke down the type of hyperplasia. The first followed 216 women and found that 22.1% with adenomatous hyperplasia, 57.1% with atypical hyperplasia, and 58.5% with carcinoma in situ developed endometrial cancer with a mean interval of 18 years from the diagnosis of hyperplasia to cancer [52]. The second study reported that 1, 3, and 23% developed endometrial cancer with a precursor lesion of simple, complex, or atypical complex hyperplasia,

593 respectively [53]. The mean follow-up of this latter report was 13.4 years. It should be noted that the vast majority of the women in both studies were not on estrogen replacement. The following discussion describes the proofs that estrogen promotes the development of endometrial hyperplasia: In the 1970s, several British reports determined retrospectively and prospectively the percentages of women on estrogen replacement who developed hyperplasia. For example, cystic or adenomatous hyperplasia developed in 10 and 2% of 98 women treated with various estrogen preparations during a study that lasted for a mean of 9.7 months [54]. In a second report, cyclic high- and low-dose estrogen preparations (conjugated equine estrogens, estrone sulfate, and estradiol valerate) were administered to 102 subjects for an average of 15.1 months [55]. Endometrial biopsy specimens showed that 23 and 17% developed cystic or adenomatous hyperplasia, respectively, from the high-dose group. For the low-dose group, the percentages were 12 and 12% for the same endometrial lesions, respectively. A review of 745 women was conducted and showed that the incidence of hyperplasia with high-dose estrogen (1.25 mg of conjugated estrogens or its equivalent) was 14.8%, and with low-dose estrogen (0.625 mg of conjugated estrogens or its equivalent) it was 7.0% [56]. The mean follow-up was 21.6 months. Three large, randomized, double-blind, prospective studies have examined the effect of hormone replacement on the endometrium [57-59]. Two of these studies were also placebo controlled [58-59]. These studies have unequivocally shown that continuous unopposed estrogen replacement at doses that prevent bone loss increases the occurrence of endometrial hyperplasia over that of placebo, and estrogen/ progestin replacement reduces the occurrence of hyperplasia back to that seen with use of placebo. The first trial to be reviewed was a National Institute of Health (NIH)-sponsored study called the Postmenopausal Estrogen/Progestin Intervention (PEPI) trial [58]. This study cohort consisted of 875 healthy postmenopausal volunteers of all races between the ages of 45 and 64 years at entry who

FIGURE 1 Representative histologic views of (A) adenomatous hyperplasia, (B) adenomatous hyperplasia with atypia, and (C) welldifferentiated carcinomaof the endometrium.

594 gave written, informed consent to participate in the study. The cohort involved in the endometrial study comprised 596 women with a uterus. The women were recruited at seven study clinics in the United States. The eligibility and exclusion criteria for this trial, reviewed elsewhere [60], included cessation of menses for at least 1 year but not more than 10 years prior to enrollment, a follicle-stimulating hormone (FSH) level of at least 40 mIU/ml, and a normal or atrophic endometrial biopsy result at baseline. Women were excluded if they had breast or endometrial cancer, or any other cancer, except nonmelanomatous skin cancer, diagnosed less than 5 years before baseline, serious medical illness, or severe menopausal symptoms. Participants discontinued hormone replacement therapy 2 months prior to the first screening visit. Treatment group assignment was stratified by clinical center and uterine status and was assigned using a computergenerated randomization schedule developed and installed by the PEPI Coordinating Center. Women were randomized to one of the following treatments in 28-day cycles: placebo, 0.625 mg/day of conjugated equine estrogens (CEEs), 0.625 mg/day of CEEs plus 10 mg/day of medroxyprogesterone acetate (MPA) for the first 12 days of each cycle, 0.625 mg/day of CEEs plus 2.5 mg/day of MPA, or 0.625 mg/day of CEEs plus 200 mg/day of micronized progesterone (MP) for the first 12 days of each cycle. Active drugs and placebo were prepared in identical forms. The 2.5- and 10-mg doses of MPA were specially prepared for identical appearance. All women took two pills (one of CEEs or matching placebo and one of MPA or matching placebo) daily and two capsules (each with 100 mg of MP or matching placebo) for the first 12 days of each cycle. Study medications and matching placebos were supplied for the PEPI trial as follows: CEEs, Wyeth-Ayerst Laboratories, Philadelphia, Pennsylvania (Premarin); MPA, The Upjohn Company, Kalamazoo, Michigan (Provera); and ME Schering-Plough Research Institute, Kenilworth, New Jersey (micronized progesterone). Scheduled visits occurred at 3, 6, and 12 months during the first year of the study and at 6-month intervals for the remainder of the 3-year study. Included among the data collection and procedures at annual visits were pelvic examination and endometrial biopsy. Unscheduled visits were conducted as required to respond to problems noted by the participant or the local clinician. Endometrial tissue was obtained using standard biopsy techniques, without regard to the day of the woman's hormonal cycle. Most biopsies were performed with a Pipelle cannula and the remainder with vacuum or suction aspiration or a Novak-type curette. Biopsy results for women in whom the operator was certain of entry into the uterus but was unable to obtain tissue (due to presumed atrophy) were classified as normal. The 18 women in whom entry into the uterus was not possible at baseline were not assigned to a study

AGARWAL AND JUDD

group. If this occurred at follow-up visits, the woman discontinued study drugs (n = 14). Unscheduled biopsies were performed at any time to evaluate abnormal or problematic vaginal bleeding, or as a follow-up to an earlier diagnosis of hyperplasia. Biopsy slides were reviewed by a local pathologist and then were reviewed by independent central readers. Slides with a discrepancy between the local and the central reading were reviewed by a third pathologist. In most cases the final diagnosis was based on agreement between two of the three pathologists. When there was disagreement among the three pathologists, the PEPI gynecologist, who had reviewed the participant's clinical course, selected the final diagnosis. The following criteria and terminology were used for the diagnosis of the endometrial biopsies as reviewed in Hendrickson's textbook entitled "Major Problems in Pathology" [61 ]. In simple (cystic) hyperplasia, there was an increase in both the stromata and the glandular elements. The glands were cystically dilated and lined by cuboidal proliferative cells without cytologic atypia. The glands were separated by a dense cellular stroma composed of ovoid cells with prominent nuclei. Small blood vessels were present. In complex (adenomatous) hyperplasia, there was a marked increase in the number of glands that appeared to be close together with little intervening stroma between them. The glands were lined by a stratified epithelium in which mitotic figures were occasionally present. The stromata were also very cellular and had mitotic figures. Atypical hyperplasia was characterized by the presence of cytologic atypia in the epithelium of the glands. The nucleus was larger and rounded and could have prominent nucleoli, and there were irregularities of the nuclear membrane. Occasional mitosis could be seen. In adenocarcinoma, there was marked cytologic atypia with large, prominent nuclei and a complex cribriform pattern. Necrosis, mitosis, and nuclear pleomorphism were present. Some women underwent a dilatation and curettage (D and C) or a hysterectomy as part of the follow-up. In seven women, the result from a D and C or hysterectomy was more serious than the result from the previous biopsy. In these cases the diagnosis reported herein is based on the findings of these procedures and not on the results of an endometrial biopsy. Conditions requiring premature unmasking were limited to serious issues related to participant safety. The study protocol required cessation of study medications and unmasking for women with biopsy results classified as complex (adenomatous) hyperplasia, atypia, or cancer. Women with simple (cystic) hyperplasia were continued on study medications and were not unmasked. Because vaginal bleeding may be a symptom requiring an intervention for participant safety, a mechanism for an unmasked review of bleeding data that maintained the masking of participants and clinical staff was required. A consult-

CHAPTER 41 HRT and Risk of Endometrial Cancer

595

ing gynecologist, not otherwise involved in the PEPI study, was notified the first time each woman experienced vaginal bleeding. This gynecologist reviewed the data about bleeding and then obtained from the PEPI Coordinating Center partial information on drug assignment indicating that the participant was receiving placebo, estrogen plus a sequential progestational agent, estrogen plus a continuous progestational agent, or estrogen only. After reviewing this information, the gynecologist gave a recommendation on whether an unscheduled biopsy should be performed. All the analyses in this study were by intention to treat. Treatment effects were assessed on either mean changes from baseline of the primary end points (e.g., high-density lipoprotein cholesterol, fibrinogen, and insulin) or rates of change on linear models (blood pressure) [62]. PEPI was designed to provide statistical power exceeding 80% for detecting prespecified differences in each of the four. primary outcomes measured with an overall type 1 error controlled at 0.05. Secondary outcome measures, such as endometrial histology, used nominal P values. Differences among treatment regimens for baseline characteristics were assessed with analysis of variance and the Fisher exact test. Frequencies and percentages describe the rates of events, and differences in rates among treatment regimens were assessed with the Fisher exact test. Log rank tests were used to compare the distributions of the time to diagnoses of hyperplasia among treatment regimens. The 596 women with a uterus who were randomly assigned to the five treatment regimens had similar sociodemographic, lifestyle, and menopause-related characteristics [63]. Records of interruptions in administration of the study drug were filed for participants who stopped taking their medications for more than a week. A total of 74.5% (444) of

TABLE II

the women continued to take the study drug for at least 80% of the follow-up period. However, fewer of the participants assigned to CEEs only (43.7% [52]) took the study drug for at least 90% of the follow-up period compared with 80 to 85% (96 to 102) of the participants in the other four groups (P < 0.001). A summary of the number and types of procedures used to obtain samples of the endometrium during the course of the study is presented in Table II. Approximately 120 endometrial biopsies were performed at baseline for each of the study groups. At the end of the 3-year trial, a total of 527 PEPI participants (88%) underwent biopsies. Reductions in the number of annual biopsies for all groups were due to study dropouts or participants' refusal to have another biopsy. A total of 174 unscheduled endometrial biopsies were performed. Ten (8.4%) of 119 women taking placebo had 11 unscheduled biopsies, and 79 (66.4%) of 119 women taking estrogen only had at least one unscheduled biopsy (P < 0.001). These 79 women had 66.1% (115/174) of all unscheduled biopsies. One woman receiving placebo had a D and C, whereas 21 participants receiving estrogen-only therapy had a total of 24 D and Cs (P < 0.001). There was also a significant difference (P = 0.04) in the number of hysterectomies across all the treatment regimens. Two women taking placebo had hysterectomies during the trial, one for adenocarcinoma of the endometrium and one for an ovarian cystadenoma. Seven women taking CEEs alone had hysterectomies, six for atypical hyperplasia and one for complex (adenomatous) hyperplasia. Tables I I I - V summarize the endometrial histology results for the course of the study. Of the 2418 biopsies performed during the trial, 164 (6.7%) required the opinion of the arbiter

Endometrial Procedures Performed during the PEPI Trial a Treatment regimenb

Procedure

Placebo

CEE only

CEE+MPA (cyclic)

Annual endometrialbiopsy baseline Follow-up visit, 12 months Follow-up visit, 24 months Follow-up visit, 36 months Unscheduled biopsyc,d Dilation/curettage Hysterectomy

119 115 112 102 11/10 1 2

119 110 104 98 115/79 24/21 7

118 117 112 108 20/16 2/2 3

CEE+MPA (continuous) CEE + MP 120 115 111 109 11/9 1 0

120 115 110 110 17/14 0 2

Total 596 (100%) 572 (96%) 549 (92%) 527 (88%) 174/126 28/25 14

aFrom The Writing Group for the PEPI Trial [58]. b CEE, Conjugated equine estrogens; MPA, medroxyprogesteroneacetate; ME micronized progesterone. CTotal number of procedures/numberof women. d p < 0.001 for placebo compared with CEE only; P = 0.38 for placebo compared with CEE + MPA (cyc), CEE + MPA (con), and CEE + MP. e p < 0.001 for placebo compared with CEE only; P = 0.43 for placebo compared with CEE + MPA (cyc), CEE + MPA (con), and CEE + MP. f P = 0.04 for overall comparison among groups.

596

AGARWAL AND JUDD

TABLE III Induction of Endometrial Hyperplasia or Cancer with Placebo in Large Clinical Trials a Follow-up (months)

Study

Baseline number

6

12

24

36

PEPI [58] CHART [59]

119 137

0/93

0/115 0/83

0/112 0/67

3/1026 1/59

a The incidence of endometrial hyperplasia is expressed as the number of cases per number of evaluable subjects at each interval time point. b One patient had adenocarcinoma.

pathologist. In 30 cases (1.2%), three different opinions were reported and the diagnoses were assigned by the clinic gynecologist. The data presented in PEPI represent the most abnormal endometrial histology result during follow-up for each participant. A total of 506 women (85%) had normal results for all follow-up biopsies. Endometrial hyperplasia or adenocarcinoma was reported for 90 women (15%). Among the 119 women assigned to placebo, one case each of simple (cystic) hyperplasia, complex (adenomatous) hyperplasia, and adenocarcinoma of the endometrium occurred (Table III). In women given CEEs alone, 74 of 119 (62.2%) developed some type of endometrial hyperplasia and 41 of 119 (34.4%) had complex hyperplasia or atypia (Table IV). These women were more likely to develop simple, complex, or atypical hyperplasia as their most abnormal diagnosis compared to women given placebo (27.7 vs. 0.8%, 22.7 vs. 0.8%, and 11.8 vs. 0%, respectively; P < 0.001). Figure 2 shows the estimated survival functions of the time to the diagnosis of simple (cystic), complex (adenomatous), and atypical hyperplasia as the most abnormal biopsy result. The curves indicate the estimated proportion of women remaining free of a specific type of hyperplasia during a given length of time for placebo, estrogen alone, and the combined estrogen/progestin regimens. Women in the placebo group had longer intervals free of all three types

TABLE IV

of hyperplasia compared to women in the CEE only group. The data below each graph show the number of women who developed hyperplasia during each year of follow-up. For the CEE only group, 25 (21%), 29 (24.4%), and 20 (16.8%) women developed some type of hyperplasia as their most abnormal result during the first, second, and third years of the study, respectively. Of these women, 15 (12.5%), 14 (11.8%), and 12 (10.1%) had the more serious diagnosis of complex (adenomatous) or atypical hyperplasia for the same respective years. Among the 55 women with simple (cystic) hyperplasia as their most abnormal diagnosis, 5 persisted with this diagnosis at subsequent biopsies; a total of 38 reverted to normal (18 spontaneously and 20 with intervention) and one had incomplete follow-up data. An additional 11 women progressed to a more serious form of hyperplasia. If hormone replacement is continued in women with simple (cystic) hyperplasia, it should be anticipated that a minority will develop a more serious lesion in the next 1 to 2 years. Thus, caution should be exercised in following this approach. Based on the timing of the occurrence of the most abnormal biopsy result in each participant (Fig. 2), the development of all three types of hyperplasia remained relatively constant across the 3 years of treatment. If the yearly occurrence of hyperplasia persisted in subsequent years, it would be anticipated that a majority of women taking CEEs alone would have the more serious types of complex (adenomatous) or atypical hyperplasia after about 5 years of therapy. The second prospective trial that used a placebo group was the Continuous Hormones As Replacement Therapy (CHART) study [59]. This was a placebo-controlled, doubleblind, parallel-group, randomized clinical trial conducted at 65 study centers that enrolled a total of 1265 subjects to be treated for no more than 2 years. A sample size of 110 evaluable subjects per treatment group was needed at the end of month 24 to detect, at a 0.05 significance level and power of 0.95, a difference in hyperplasia rate, assuming a 12% rate in the unopposed ethinyl estradiol (EE 2) groups and 1% rate in the combination groups.

Induction of Endometrial Hyperplasia with Estrogen Alone in Large Clinical Trials a

Study

Regimen

Baseline number

Menopause Study Group [57] PEPI [58] CHART [59]

CEE, 0.625 mg CEE, 0.625 mg EE2, 1/~g EE2, 2.5/zg EE2, 5.0/zg EE2, 10.0/.~g

345 119 141 137 141 143

Follow-up (months) 6

12

18

24

36

21/298

57/283 25/110 0/75 0/90 1/94 9/61

29/104 1/64 1/67 2/90 10/18

20/98

1/67 0/84 2/92 10/31

0/87 0/104 0/107 5/99

aThe incidence of hyperplasia is expressed as the number of cases per number of evaluable subjects at each evaluation time point.

597

CHAPTER 41 HRT and Risk of Endometrial Cancer TABLE V

Postmenopausal Estrogen/Progestin Use and Risk of Endometrial Cancer a

1.00

\

t~

Study group

Type of study

RR

95% CI

E

0.75 -

c-

Nachtigall et al. [ 4 0 ] Hammond et al. [29] Gambrell et al. [30] Persson et al. [34] Voigt et al. [25]

Jick et al. [26] Brinton and Hoover [28]

Randomized trial Cohort Cohort Cohort Case control Progestin use < 10 days/month 10 days/month Case control Case control

b b 0.2 c 0.9 1.6

b b 0.1-0.6 d 0.4-2.0 0.6-3.90

cO 1:::

o {3. 0.50

s

0.25 -,

J

0 2.0 0.9 1.9 1.8

0.7-5.3 0.3-2.4 0.9-3.8 0.6-4.9

12 24 36 Duration of time, mo

Placebo CEE only CEE + Progestin

0 10 2

0 15 5

48

1 8 3

Simple (cystic) hyperplasia

a From Grady et al. [38]. Reprinted with permission from the American College of Obstetricians and Gynecologists (Obstetrics a n d G y n e c o l o g y , 1995, Vol. 85, pp. 304-313). bNo endometrial cancer observed in the estrogen/progestin group. cRelative risk (RR) calculated by the authors from published crude data. dThe 95% confidence interval (CI) calculated by the authors from the published crude data. All subjects were healthy volunteers who met inclusion and exclusion criteria that (1) confirmed their postmenopausal status [FSH >-- 40 mIU/ml; estradiol, 20 pg/ml, as measured at a central laboratory (SciCor Inc., Indianapolis, Indiana)] using commercially available radioimmunoassay methods and (2) confirmed their good health status [e.g., low-density lipoprotein cholesterol (190 mg/dl); spinal trabecular bone density between 90 and 150 m g / c m 3, an atrophic endometrium, and no major illnesses]. Subjects were excluded if there was a recent history of vaginal bleeding, baseline m a m m o g r a p h y suggestive of malignant disease, chronic use of medications that affect bone calcium metabolism, or significant vasomotor symptoms that required medical treatment. Subjects were required to have not taken oral or transdermal estrogen replacement for 6 months prior to randomization. The study was approved by each center's Institutional Review Board, and all subjects provided written informed consent prior to enrollment. Each participant was randomized according to a code prepared by Parke-Davis Biometrics Department using computer-generated random numbers. The randomization code was prepared in blocks of 9. There were four norethindrone acetate (1 m g ) - e t h i n y l estradiol ( N A - E E 2 ) dosecombination treatment groups and four unopposed EE 2 treatment groups plus a placebo group (Tables III-V). The dosing regimen was one tablet daily for the duration of the study. At each clinic visit during the study (end of 1, 3, 6, 9, 12, 18, and 24 months), subjects returned unused medication and it was examined. Subjects were asked if they had missed more than 7 consecutive days of medication (noncompliance). If the subject was considered noncompliant, she was withdrawn from the study.

1.00

E

0.75 -

ct.O _ x_ O

0.50 -

0 O..

0.25 -, 0

I

i

i

12 24 36 Duration of time, mo 0 12 1

Placebo CEE only CEE+ Progestin

0 8 0

48

1 7 1

Complex (adenomatous) hyperplasia

1.00

E

h_ L 0.75 -

ctO 1::7

o 0.50 0 13_

0.25 -, 0 Placebo GEE only CEE+ Progestin

.....

i

i

i

12 24 36 Duration of time, mo 0 3 0

0 6 0

!

48

0 5 1

Atypia

FIGURE 2 Kaplan-Meier estimates of time to a diagnosis. The data below each graph show the number of annual cases of the same types of hyperplasia during follow-up according to the women's most abnormal endometrial biopsy result. The dotted line indicates placebo; solid line, conjugated equine estrogens (CEE) only; broken line, CEE + progestin. From the Writing Group for the PEPI Trial [58].

598

AGARWAL

Endometrial biopsy samples were obtained from each subject at baseline and at the end of months 6, 12, 18, and 24 of treatment using vacuum aspiration curettage (Vabra). A blinded safety protocol, which required that any treatment group with a rate of hyperplasia that either exceeded twice the rate in the placebo group or 6% of the sample size for that group was to be terminated, was run concurrent with the clinical protocol. Histology on the blinded biopsy samples was conducted by a central pathologist. The degree of proliferation was also graded (mildly, moderately, or markedly) based on a priori criteria that included several quantitative parameters. Severity scores were assigned to each biopsy category for statistical analysis as follows: atrophic, 1; mildly proliferative, 2; moderately proliferative, 3; markedly proliferative, 4; and hyperplastic, 5. Unfortunately, the method used to grade the endometrium in this study was not referenced. Data on endometrial effects were analyzed using categorical data methods for percentage of subjects with hyperplasia. Primary comparisons were individual or combined N A - E E 2 treatment groups versus the corresponding EE 2 treatment group and dose response of EE 2 treatment groups including the placebo group. Comparisons of N A - E E 2 to EE 2 were tested using the Fisher exact test for 2 • 2 tables. Linear dose-response testing used the Cochran-MantelHaenszel correlation test, based on the rank of each estrogen dose. In addition, analysis of endometrial severity scores was conducted comparing unopposed EE 2 groups with the corresponding N A - E E 2 group. Estimates of the cumulative probability of hyperplasia were commuted using the life table method. The cumulative probabilities of hyperplasia over time were compared between the 10-/xg EE 2 and the

1-mg NA/10-/zg EE 2 treatment groups only. If a subject developed hyperplasia, she was terminated from the study. A total of 1265 postmenopausal women were enrolled at 65 study sites. The treatment groups did not differ significantly for age, months since last menstrual period, height, weight, or baseline bone mineral density. All enrolled women had an atrophic endometrial biopsy at baseline. Based on a priori stopping rules, subjects in the 10-1zg EEz-only group were terminated from the study early owing to a high rate of hyperplasia for that treatment group. All remaining treatment groups had similar rates of withdrawal that ranged from 22 to 30%, which was slightly greater than the estimated rate prior to the start of the study. Excluding the 10-1zg EE 2 group, over 73% of the subjects completed the study. Other compliance issues were not provided in the paper. Table III shows the number of biopsies that showed hyperplasia in the placebo group. During the first 18 months, none of the biopsied subjects had hyperplasia. Only one biopsy of 59 taken at 24 months showed hyperplasia. Figure 3 shows the mean endometrial severity score at 24 months in the placebo group. It was 1.5, with 1 being atrophic and 2 being mildly proliferative. Table IV shows the number of biopsies that were positive in women receiving EE 2 only for 24 months. There was a dose-response effect of EE 2, with 0 to 1 biopsies showing hyperplasia at each time interval with the 1- and 2.5-/.~g/day dosages, and 0 to 2 biopsies showing hyperplasia at each time point with the 5-/zg dosage. As mentioned, with the 10-/xg/day dosage of EE 2, the treatment group was terminated early (per protocol) owing to an unacceptably high rate

n

5 Ckl C" .,..., C O

4

B

3

D

.~ 2

m

E O o

>, L m > O0

E

0 "0 e-

c"

N

Placebo

0.2-1

0.5-2.5

1-5

AND JUDD

1-10

Norethindrone acetate,mgEthinyl estradiol, l.tg

1

2.5

5

10

Ethinyl estradiol, #g

FIGURE 3 Meanendometrial severity score _ SE after 2 years of treatment. The asterisks indicate statistically significant (P -< 0.045) differencesamong unopposed ethinyl estradiol groups and ethinyl estradiol dose-matched

CHAPTER41 HRT and Risk of Endometrial Cancer of endometrial hyperplasia. The mean endometrial severity score shown in Fig. 3 rose with increasing doses of EE 2, to a mean of nearly 4, which corresponded to markedly proliferative endometrium with the 10-~g/day dosage. It is now clear that in direct comparisons to placebo, two of these preparations (CEE, 0.625 mg; EE 2, 10/xg), which are the lowest doses that prevent bone loss without calcium supplementation, also induce statistically significant increases of endometrial hyperplasia in women given these medications for 1 to 3 years. The third trial was conducted by the Menopause Study Group and will be discussed later in this chapter. The trial also assessed 0.625 mg CEEs. Thus, in the two reports that studied CEEs, the total percentages of women who developed hyperplasia at 1 year of therapy were similar [Menopause Study Group, 57 of 283 total biopsies (20.1%) [57], vs. PEPI, 25 of 115 biopsies (21.7%)] [58]; however, the percentages who developed the more serious diagnoses of complex or atypical hyperplasia were different [Menopause Study Group, 2 adenomatous and 0 atypical of 283 biopsies (0.7%) [57], vs. PEPI, 12 complex (adenomatous) and 3 atypical of 115 biopsies (13.4%)] [58]. There are various potential explanations for this difference. In PEPI, the women were sampled without regard to the day of the hormonal cycle, whereas the Menopause Study group sampled women between days 22 and 28 of the hormonal cycles. Second, in PEPI, seven subjects had worse hyperplasia on either a D and C or hysterectomy specimen and these diagnoses were substituted for the biopsy results. In the Menopause Study Group trial, only biopsy samples were reported. Third, PEPI was sponsored by NIH and the resuits were calculated in the absence of corporate sponsors. The Menopause Study Group was sponsored by WyethAyerst Laboratories. Fourth, the PEPI study included several women whom the arbiter pathologist had diagnosed with simple hyperplasia with atypia in the CEE-only group but who had not been identified by the central or clinic pathologists. The Menopause Study Group reported no one with atypical hyperplasia. Last, this difference between the two studies may not be statistically significant and may have occurred only by chance. Several conclusions can be drawn from the two placebocontrolled trials. First, estrogen only therapy at dosages of 0.625 mg/day of CEEs and 10/zg/day of EE 2 unequivocally and significantly increase the occurrence of endometrial hyperplasia over that seen with the use of placebos. This conclusion was reinforced by the inclusion only of women with normal (PEPI) or atrophic (CHART) endometrium at baseline. This conclusion is also supported by the many observational studies and small clinical trials that have shown an exaggerated occurrence of hyperplasia with estrogen-only therapy. Second, if women on estrogen-only therapy develop simple (cystic) hyperplasia and are continued on the medication, a minority (20%) will develop a more serious form of hyperplasia in the next 1-2 years. Third, the occurrence

599 of hyperplasia was steady across the 3 years of the PEPI trial. If the same rate of occurrence that was seen during the first 3 years continued, it would be anticipated that more than 50% of women would have complex or atypical hyperplasia after 5 years of therapy. Fourth, there was a dose-response relationship with estrogen (EE2) and the occurrence of endometrial hyperplasia in the CHART study with the highest dose (10 ~g/day) necessitating early termination of this group. Fifth, in PEPI, the numbers of unscheduled biopsies and D and Cs necessary to follow the women on CEEs alone were significantly greater than those required to follow the women on placebo. Sixth, the lowest doses of CEEs (0.625 mg/day) and EE 2 (10/xg/day) that prevent bone loss from the spine and hip (PEPI) [64,65] and the spine (CHART) [59] would be anticipated to result in sufficient occurrence of hyperplasia to necessitate discontinuation after a few years of estrogen-only therapy in a majority of women if routine endometrial biopsies were performed on a yearly basis.

II. E S T R O G E N AND PROGESTIN T H E R A P Y Several studies published in the 1980s suggested that the combined administration of estrogen with a progestin would reduce the occurrence of endometrial cancer. Of particular note was a study from the Wilford Hall Air Force Hospital, which reported that the incidence of endometrial carcinoma was 390.06 per 100,000 women-years for those who used unopposed estrogens compared with 245.5 per 100,000 women-years for nonhormone users [66]. In women who took an estrogen and a progestin, the incidence was 49 cases per 100,000 women-years. These data were interpreted to indicate that the addition of a progestin not only avoids the enhanced risk of those who take estrogen but reduces the risk to levels substantially lower than those seen in nonhormone users. Although this study has been quoted frequently, it suffered from several methodologic problems. Of particular concern was the likelihood that the groups were not equivalent for the risk of developing endometrial cancer when they were assigned therapy. The risk of cancer in the untreated women was high (245.5 per 100,000 women-years) compared with the national figures (approximately 100 per 100,000 women-years). This indicated that women with some risk factors of this cancer were not given estrogen replacement. Results of the seven studies that provided data on risk for endometrial cancer among users of estrogen plus progestin are presented in Table V. In two of these studies [29,40], three or fewer endometrial cancers occurred; one included only hypoestrogenic women [29] and another did not adjust

600 the results for the confounding effects of age [30]. Although the overall summary RR for endometrial cancer among women who took estrogen plus progestin was 0.8 (95% CI, 0.6-1.2), the direction of the effect was different in cohort versus case-control studies: 0.4 (95% CI, 0.2-0.6) and 1.8 (95% CI, 1.1-3.1), respectively. Two studies provided information on the effect of the number of days per month that the progestin was used with estrogen. In one, women who took progestin for fewer than 10 days per month had an RR for endometrial cancer of 2.0, compared to an RR of 0.9 for those who took progestin for at least 10 days per month [25]. In the other, the RR for endometrial cancer was 1.8 and did not vary by the number of days per month that the progestin was used [28]. In terms of the so-called protective effect of progestin on the endometrium, the type, potency, dosage, monthly duration, and frequency of administration all appear to be important variables; however, limited data are available about these issues. For type, the first is progesterone. This can be administered as a micronized formulation for oral use or as a vaginal suppository. Intramuscular injections are available but are rarely used for replacement therapy. The second is a C-21 progestin. These are derivatives of progesterone that have been modified chemically so that they can pass through the gastrointestinal tract and be active when given orally. These include MPA and megestrol acetate. The third is a 19nortestosterone derivative. These are derivatives of testosterone in which carbon at position 19 has been removed and an ethinyl group has been placed at position 17. These substances have both progestin and androgen effects, with the progestin actions predominating. In the United States, these include norethindrone, norethindrone acetate, and levonorgestrel. The potency of a number of progestins to protect the endometrium has been studied [67]. The end point was the ability to elicit an endometrial response comparable with that of a secretory phase endometrium. The relative potencies were shown to be levonorgestrel (8.000), norethindrone (1.000), MPA (0.090), and progesterone (0.002). Some questions have been raised about these results because of bioavailability problems with the MPA in this study. Two groups of investigators have compared the biochemical and histologic changes elicited by various progestins in regard to dose [68,69]. Both groups treated women with 0.625 or 1.25 mg of CEEs. The women were then given different doses of progestin, and their endometrium was sampled by biopsy at a specific interval after the progestin was begun. The British group performed the biopsies on the subjects on day 6 of progestin [68]. These investigators showed that the 1-mg dose of norethindrone was equally effective as the 5-mg dose in reducing DNA replication and estradiol receptor formation, but that it was not as efficient in forming a secretory endometrium. The 0.15-mg dose of levonorgestrel was equally effective as the 0.50-mg dose at

AGARWAL AND JUDD

altering the biochemical and histologic changes of progestin. For MPA, there was a dose-dependent suppression of DNA synthesis between 2.5 and 10.0 mg [70]. The levels of estradiol receptor were reduced by the 10.0-mg dose into the range seen in the secretory phase of the cycle but were not statistically different from values observed in younger women during the proliferative phase of their cycles. For histologic changes, the 10.0-mg dose elicited suboptimal responses. American investigators performed biopsies of subjects on day 11 of MPA [69]. They reported that the 5- and 10-mg doses suppressed estradiol receptor equally but that only the 10-mg dose resulted in a homogeneous secretory pattern within the endometrium. The duration of progestin use each month is another important variable. At a cellular level the action of progestin is rapid. Norethindrone (5 mg) has been shown to reduce thymidine labeling and nuclear estradiol receptors while raising estradiol dehydrogenase and isocitric dehydrogenase activity of the endometrium after 3 days of administration [45]. Maximal effects were seen at 6 days. Clinically, one observational study showed that the occurrence of hyperplasia was 12% with estrogen alone, 2% with estrogen plus 7 to 10 days of a progestin, and 0% with estrogen plus 13 days of a progestin each month [54]. This was after a mean treatment time of 9.7 months. Another observational study reported 32 and 18% hyperplasia with high-dose and low-dose estrogen therapy, 18% with lowdose estrogen therapy, and 3 - 4 % with sequential estrogen and progestin replacement, with the progestin being given for 6 to 10 days per month [55]. The mean treatment time was 15.1 months. Several small drug trials evaluating the endometrial response to single or multiple regimens of estrogen and progestin or comparing the impact of estrogen alone with that of an estrogen and a progestin have been published [71-74]. For the most part, these studies have been prospective, randomized, and double blind. Each has shown that the addition of a progestin to estrogen replacement has reduced the occurrence of hyperplasia. There are now three large clinical trials that have compared the occurrence of hyperplasia in women given estrogen or estrogen and a progestin [57-59]. The methodology of the PEPI and CHART trials have already been reviewed. The third trial, which was by the Menopause Study Group, was a prospective double-blind, parallel study conducted with healthy postmenopausal women at 99 sites in the United States and Europe [57]. Eligibility criteria were women between 45 and 65 years of age with an intact uterus, who were at least 12 months since their last menstrual period, had a serum FSH level higher than the lower limit found in postmenopausal women for a given laboratory (most were between 25 and 35 mIU/ml), and had not used estrogen or progestin medications for at least 2 weeks. At all sites approval of Institutional Review Boards was obtained and the

CHAPTER41 HRT and Risk of Endometrial Cancer participants gave informed consent before enrollment in the study. The participants were randomly assigned to one of five treatment groups for 13 cycles (1 year). All women were given CEEs at a dosage of 0.625 mg/day and MPA or a matching MPA placebo daily. Two of the groups received MPA (2.5 or 5.0 mg) daily. Two other groups received 5.0 or 10.0 mg for the last 14 days of each 28-day hormonal cycle. The last group was given CEEs plus placebo for MPA daily. The study was not controlled with a double-placebo group. All medications were supplied by Wyeth-Ayerst Laboratories (Philadelphia, Pennsylvania; CEEs, Premarin; MPA, Cycrin). Endometrial biopsies were performed at baseline and between days 22 and 28 of cycles 6 and 13. Other biopsies were taken at any time if medically indicated. If hyperplasia developed, the participant was withdrawn from the study. Approximately 75% of biopsies were obtained with the Pipelle suction curette, the Novak curette, or the Vabra aspirator. The remaining 25% were obtained with 14 other curettes. With a hypothesized incidence of endometrial hyperplasia of 7.5% in the CEE-alone group and 2% in the CEE/ MPA groups, a sample size of 215 patients per treatment group was estimated to provide 80% power to detect at least one statistically significant difference at the 0.0125 level (Bonferroni adjustment for four multiple comparisons). To allow for a 20% discontinuation rate, an enrollment of about 270 patients per group was planned. To ensure uniformity of interpretation, all endometrial biopsy specimens were evaluated by a single pathologist at The Johns Hopkins Hospital (Baltimore, Maryland). The terminology used to report endometrial hyperplasia in this study (cystic or adenomatous hyperplasia without atypia; cystic or adenomatous hyperplasia with atypia) corresponds to the alternative classifications of simple hyperplasia, complex hyperplasia; simple atypical or complex atypical hyperplasia, respectively. The criteria and terminology used have been described in the literature and were similar to the criteria and terminology used in the PEPI trial [75,76]. If a subject developed hyperplasia, she was terminated from the study. A total of 1724 postmenopausal women were enrolled. The population that completed this 1-year study with endometrial biopsy data valid for analysis was composed of 1385 subjects. Four women were 44 years old, but their data were included in this report. The prestudy characteristics of the subjects were not different statistically (p > 0.05) among treatment groups for any demographic characteristic. Among women taking the continuous combined regimens, approximately 20% of the biopsy specimens had either no tissue or no endometrial tissue identified. Those who took the sequential regimens had either no tissue or no endometrial tissue in only 10% of biopsy specimens, and those in the conjugated estrogens-alone

601 group had either no tissue or no endometrial tissue in approximately 15% of biopsy specimens. Endometrial hyperplasia developed in 23 (2%) of 1469 patients included in the evaluation of the 6-month data (Tables IV and V). The incidence with each of the CEE/ MPA regimens was significantly lower than with CEE alone. There were no statistically significant differences between the lower dose and higher dose MPA regimens or between the continuous combined and the sequential regimens. The evaluation of the 12-month data (including 6-month data) showed that endometrial hyperplasia had developed in 62 (4%) of the 1385 patients included in this analysis. It should be mentioned that all subjects were counted just once. The incidence of endometrial hyperplasia was significantly lower with each of the CEE/MPA regimens than with CEE alone. There were a total of five cases of endometrial hyperplasia in the two lower dose MPA groups and no cases of hyperplasia in the two higher dose groups. This difference approached statistical significance (P = 0.06). During the course of this study, two women developed endometrial cancer during the thirteenth cycle. One of them received CEE and sequential MPA (10 mg), and the other was given CEE alone. Both women underwent hysterectomy. The second large clinical trial that compared estrogen alone with estrogen/progestin (E/P) was the PEPI trial [58]. The methodology and placebo and estrogen-only results were reviewed above. In PEPI, among women taking one of the three E/P regimens, 16 (13.6%) taking cyclic MPA, 9 (7.5%) taking continuous MPA, and 14 (11.7%) taking cyclic MP had unscheduled biopsy rates that were similar to those of women receiving placebo (P = 0.338), but significantly lower than women receiving estrogen only. Women receiving the E/P regimens underwent zero to two D and Cs per regimen, similar to the rate of women receiving placebo (P = 0.43), but lower than those receiving CEEs alone (Table II). Five women receiving the E/P regimens had hysterectomies, one for atypical hyperplasia, one for persistent vaginal bleeding, two for uterine leiomyomas, and one for an ovarian cystadenoma. Table VI summarizes the endometrial histology results of the women taking one of the E/P regimens. Ten cases of simple (cystic) hyperplasia, two of complex (adenomatous) hyperplasia, and one of atypical hyperplasia were distributed among the three E/P groups. There was no difference in the occurrence of abnormal biopsy specimens between the women who received placebo and those who received one of the three E/P regimens (P = 0.16). However, the incidence of hyperplasia was significantly less in the women taking E/P than in women receiving CEEs alone. The last large trial was the CHART study [59]. Again, the methodology and the placebo and EE2-only results were reviewed earlier. A significantly greater percentage of subjects developed endometrial hyperplasia while receiving 10/zg of EE e compared with the 1 mg NA/10/zg ofEE e combination

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TABLE V I

Temporal Induction of Endometrial Hyperplasia with Estrogen/Progestin in Large Clinical Trials a Follow-up (months)

Study

Regimen

Baseline number

Menopause Study Group [57]

CEE, 0.625 mg + MPA, 10 mg • 14 days MPA, 5 mg • 14 days MPA, 5 mg four times daily MPA, 2.5 mg four times daily

345 345 345 345

CEE, 0.625 mg + MPA, 10 mg • 12 days MPA, 2.5 mg four times daily MPA, 200 mg • 12 days

118 120 120

NA mg/EE 2/xg 0.2/1 0.5/2.5 1/5 1/10

139 136 146 145

PEPI [581

CHART [59]

6

12

0/292 1/293 0/291 1/295

0/272 3/277 0/272 2/279

18

1/117 0/115 2/115 0/84 0/75 0/92 0/92

0/80 0/69 9/65 0/71

0/67 0.50 0/70 0.63

24

36

1/112 0/111 2/110

4/108 1/109 2/110

1/69 0/57 0/65 0/65

aThe incidence of endometrial hyperplasia is expressed as the number of cases per number of evaluable subjects at each evaluation time point.

(Tables IV and V). As the dose of unopposed EE 2 increased, there were increased percentages of subjects with hyperplasia. This contrasted to the N A - E E 2 combination groups, which had no significant dose trend at any time point with or without the placebo group included. The protective effect of the addition of NA to EE 2 on the endometrium was demonstrated by the life table analysis, which yielded a probability of developing hyperplasia of 0.24 in the 10-/xg EE 2 treatment group by 19 months in contrast to 0 probability for the 1-mg NA/10-p~g EE 2 group at the end of the study. The probability of hyperplasia over time was statistically significantly greater for the 10-/xg EE 2 treatment group compared with the 1-mg NA/10-/xg EE 2 treatment group (P < 0.001).

III. ENDOMETRIAL

SURVEILLANCE

The traditional and definitive procedure for diagnosis of endometrial hyperplasia or cancer has been the fractional dilatation and curettage. Under general anesthesia, this has been shown to produce adequate material for histological evaluation in up to 94% of cases [77]. Major problems with this diagnostic procedure include cost, possible medical complications, and inconvenience, and it is for these reasons that alternative screening procedures have been sought. Less expensive and invasive office endometrial sampling procedures such as those employing the Pipelle device, Novak curette, and Vabra aspirator are replacing dilatation and curettage. The real test of these devices is their sensitivity at detecting endometrial cancer. In a study of 40 patients al-

ready known to have endometrial carcinoma as diagnosed by other procedures, 39 (97.5%) were confirmed as having endometrial carcinoma by Pipelle biopsy specimens [78]. The Vabra aspirator, Novak curette, and dilation and curettage have also been shown to have similar diagnostic capabilities [79]. In general, the Pipelle device is best tolerated by patients. Although the sensitivitities for these office procedures are similar to that reported for dilatation and curettage, isolated reports of women with endometrial cancer missed by these instruments continue to trouble gynecologists. There are two situations when ultrasound-based evaluation of abnormal postmenopausal bleeding is particularly valuable: (1) to screen for discreet abnormalities of the endometrium such as polyps, which may not be sampled adequately by office biopsy and may require hysteroscopic excision, and (2) in the presence of cervical stenosis, rendering office endometrial biopsy impossible, in which case a reassuring sonographic evaluation may render a surgical diagnostic procedure unnecessary. A number of studies have been published using transvaginal sonography to differentiate women at risk for the presence of endometrial hyperplasia or cancer from those with a normal or atrophic endometrium [80-83]. Most have focused on the measurement of endometrial thickness and have demonstrated that an endometrial thickness of greater than 5 mm is a valid cutoff. Women with an endometrial thickness of greater than 5 mm should undergo endometrial sampling to exclude cancer or precancerous lesions [80,81]. Reports have indicated that the use of color doppler with transvaginal ultrasonography improves specificity without compromising sensitivity. One study attempted to character-

CHAPTER41 HRT and Risk of Endometrial Cancer

603

FIGURE 4 Ultrasoundimages during saline infusion sonography. (A) During early saline infusion and (B) after maximal uterine distention, demonstrating the presence of a subserosal fibroid. Both pictures are from the same woman and illustrate how saline infusion sonography can help improve measurementof endometrial thickness and detection of focal intrauterine lesions.

ize uterine tumors by their color flow patterns [84]. They found the mean intratumoral resistance index value for endometrial carcinomas to be 0.34 and for leiomyomata 0.58, and suggested that a value of less than 0.4 should be regarded as one of malignancy. In the absence of other confirmatory reports, these data remain of interest, but are considered research tools at the present time. Not surprisingly, the use of saline infusion sonography (SIS) in the evaluation of this common clinical situation has been the focus of a number of reports. Figure 4 shows how SIS can assist with the ultrasonic measurement of endometrial thickness and with the detection of focal intrauterine lesions. A direct comparison of SIS to hysteroscopy in 47 asymptomatic postmenopausal women with the sonographic finding of a thickened endometrium ( > 15 mm) was conducted by Wolman et al. [85]. A prospective, doubleblind design was used in which all subjects underwent SIS and then hysteroscopy a week later by an examiner unaware of the SIS findings. An additional three patients failed SIS due to cervical stenosis. In summary, the authors detected a sensitivity of 86% and a specificity of 100% for SIS in this population. Similar results have been obtained by other investigators, and further refinements such asthe concomitant use of color doppler with pulsatility and resistance indices are currently under evaluation.

IV. P H Y S I O L O G Y

OF

ENDOMETRIAL SHEDDING An exciting and rapidly developing area of research involves the role of matrix metalloproteinases (MMPs) in endometrial shedding. This family of enzymes, which is found in endometrium and other tissues, is involved in the degradation of most components of the extracellular matrix as seen with menstruation. The balance between MMP produc-

tion and that of specific tissue inhibitors of MMP action regulates their impact. It has been shown that endometrial MMPs are expressed in menstrual cycle-specific patterns, consistent with regulation by steroid hormones. Although long-term hormone replacement is advocated for the protection of bone and the heart, a majority of women commencing with HRT stop within the first 2 years. In those with a uterus, vaginal bleeding is commonly cited as a reason for discontinuing this potentially beneficial therapy. It is possible, therefore, that therapies may be developed that directly modulate MMP activity so as to decrease vaginal bleeding seen with E/P, thereby increasing compliance.

V. SUMMARY Based on the available literature, the lowest doses of estrogen that prevent bone loss from the spine and hip have been partially established. These include CEE (0.625 mg) [63,64], piperazine estrone sulfate (1.25 mg) [86], and transdermal estradiol (0.05 mg) [87]. The lowest doses of two other estrogens that prevent bone loss from the spine have been partially established. These include EE 2 (10/zg) [59] and micronized estradiol (0.5 mg) [88]. From the review just completed, it is clear that estrogenonly therapy at doses that will prevent postmenopausal bone loss will stimulate endometrial hyperplasia. Because longterm therapy with estrogens is necessary to prevent osteoporotic fractures, it is expected that many older women will use estrogen for several to many years. It is also clear that the addition of a progestin, whether it be MPA, MP, or NA, given at the doses studied, with either continuous or sequential regimens, will reduce the occurrence of hyperplasia seen with estrogen-only therapy to that occurring with the administration of placebo. It is possible, even likely, that the same or different progestins could be

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administered using the same or different doses, the same or different schedules, or the same or different regimens, which could also reduce the occurrence of estrogen-stimulated endometrial hyperplasia. One possible example may be the use of a levonorgestrel-releasing intrauterine device. If this is the case, then future studies of the nature of those reviewed here will be needed to establish this claim.

18.

19.

20.

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65. 66. 67. 68.

69.

70.

71.

72.

73.

74.

75.

76. 77. 78.

one acetate or conjugated estrogens alone. Am. J. Obstet. Gynecol. 170, 1213-1223. Writing Group for the PEPI Trial (1996). Effects of hormone replacement therapy on endometrial histology in postmenopausal women. JAMA, J. Am. Med. Assoc. 275, 370-375. Speroff, L., Rowan, J., Symons, J., Genant, H., and Wilborn, W., for the CHART Study Group (1996). The comparative effect on bone density, endometrium, and lipids of continuous hormones as replacement therapy. (CHART Study). JAMA, J. Am. Med. Assoc. 276 (17), 13971403. Writing Group for the PEPI Trial (1995). The postmenopausal estrogen/progestin interventions (PEPI) trial: Rationale, design and conduct (I). J. Controlled Clin. Trials 16 (Suppl.), 3S-19S. Hendrickson, M., and Kempson, R. (1980). "Major Problems in Pathology," Vol. 12, pp. 285-318. Saunders, Philadelphia. Writing Group for the PEPI Trial (1995). The postmenopausal estrogen/progestin interventions (PEPI) trial: Baseline characteristics of participants (IV). J. Controlled Clin. Trials 16 (Suppl.), 54S-72S. Writing Group for the PEPI Trial (1995). Effects of estrogen or estrogen/progestin regimens on heart disease risk factors in postmenopausal women. JAMA, J. Am. Med. Assoc. 273, 199-208. Lindsay, R., Hart, D. M., and Clark, D. M. (1984). The minimum effective dose of estrogen for prevention of postmenopausal bone loss. Obstet. Gynecol. 63, 759-763. Writing Group for the PEPI Trial (1996). Effects of hormone therapy on bone mineral density. JAMA, J. Am. Med. Assoc. 276, 1389-1396. Gambrell, R. D. (1989). Prevention of endometrial cancer with progestogens. Maturitas 8, 1-21. King, R. J. B., and Whitehead, M. I. (1986). Assessment of potency of orally administered progestins in women. Fertil. Steril. 46, 1062-1066. Fraser, D., Whitehead, M. I., Endacott, J., Morton, J., Ryder, T. A., and Pryse-Davies, J. (1989). Are fixed-dose oestrogen/progestogen combinations ideal for all HRT users? Br. J. Obstet. Gynaecol. 96, 776 -782. Gibbons, W. E., Moyer, D. L., Lobo, R. A., Roy, S., and Mishell, D. R. (1986). Biochemical and histologic effects of sequential estrogen/progestin therapy on the endometrium of postmenopausal women. Am. J. Obstet. Gynecol. 154, 456-461. Lane, G., Siddle, N. C., Ryder, T. A., Pryse-Davies, J., King, R. J. B., and Whitehead, M. I. (1986). Is Provera the ideal progestogen for addition to postmenopasual estrogen therapy? Fertil. Steril. 45, 345-352. Gelfand, M. M., and Ferenczy, A. (1989) A prospective 1-year study of estrogen and progestin in postmenopausal women: Effects of endometrium. Obstet. Gynecol. 4, 398-402. Clisham, P. R., Cedars, M. I., Greendale, G., Fu, Y. S., Gambone, J., and Judd, H. L. (1992). Long-term transdermal estradiol therapy: Effects of endometrial histology and bleeding patterns. Obstet. Gynecol. 79, 196-201. Williams, D. B., Voigt, B. J., Fu, Y. S., Schoenfeld, M. J., and Judd, H. L. (1994). Assessment of less than monthly progestin therapy in postmenopausal women given estrogen replacement. Obstet. Gynecol. 84 (5), 787-793. Ettinger, B., Selby, J., Citron, J., Vangessel, A., Ettinger, M., and Hendrickson, M. (1994). Cyclic hormone replacement therapy using quarterly progestin. Obstet. Gynecol. 83, 693-700. Novak, E. R., and Woodruff, J. D. (1979). "Novak's Gynecologic and Obstetric Pathology with Clinical and Endocrine Relations," 8th ed., pp. 171-237. Saunders, Philadelphia. Kurman, R. J., ed. (1987). "Blaustein's Pathology of the Female Genital Tract," 3rd ed., pp. 257-372. Springer-Verlag, New York. Grimes, D. A. (1982). Diagnostic dilation and curettage: A reappraisal. Am. J. Obstet. Gynecol. 142, 1-6. Stovall, T. G., Photopulos, G. J., Poston, W. M., Ling, F. W., and Sandles, L. G. (1991). Pipelle endometrial sampling in patients with known endometrial carcinoma. Obstet. Gynecol. 77, 954-956.

606 79. Stovall, T. G., Solomon, S. K., and Ling, E W. (1989). Endometrial sampling prior to hysterectomy. Obstet. Gynecol. 73, 405-409. 80. Fleischer, A. C., Kalemeris, G. C., Machin, J. E., Entman, S. S., James, A. E., Jr. (1986). Sonographic depiction of normal and abnormal endometrium with histopathologic correlation. J. Ultrasound Med. 5, 445 -452. 81. Granberg, S., Wikland, M., Karlsson, B., Norstr6m, A., and Friberg, L. G. (1991). Endometrial thickness as measured by endovaginal ultrasonography for identifying endometrial abnormality. Am. J. Obstet. Gynecol. 164, 47-52. 82. Lin, M. C., Gosink, B. B., Wolf, S. I., Feldesman, M. R., Stuenkel, C. A., Braly, E S., and Pretorius, D. H. (1991). Endometrial thickness after menopause: Effect of hormone replacement. Radiology 180, 427432. 83. Nasri, M. N., and Coast, G. J. (1989). Correlation of ultrasound findings and endometrial histopathology in postmenopausal women. Br. J. Obstet. Gynecol. 96, 1333-1338.

AGARWAL AND JUDD 84. Kurjak, A., and Zalud, I. (1991). The characterization of uterine tumors by transvaginal color doppler. Ultrasound Obstet. Gynecol. 1, 50-52. 85. Wolman, I., Jaffa, A. J., Hartoov, J., Bar-Am, A., and David, M. E (1996). Sensitivity and specificity of sonohysterography for the evaluation of the uterine cavity in perimenopausal patients. J. Ultrasound Med. 15, 285-288. 86. Harris, S. T., Genant, H. K., Baylink, D. J., Gallagher, J. C., Karp, S. K., McConnell, M. A., Green, E. M. and Stoll, R. W. (1991). The effect of estrone (Ogen) on spinal bone density of postmenopausal women. Arch. Intern. Med. 151, 1980-1984. 87. Field, C. S., Ory, S. J., Wahner, H. W., Herrmannn, R. R., Judd, H. L., and Riggs, B. L. (1993). Preventive effects of transdermal 17/3estradiol on osteoporotic changes after surgical menopause: A 2-year placebo-controlled trial. Am. J. Obstet. Gynecol. 168, 114-121. 88. Ettinger, B., Genant, H., Steiger, E, and Madvig, E (1992). Lowdosage micronized 17/3-estradiol prevents bone loss in postmenopausal women. Am. J. Obstet. Gynecol. 166, 479-488.

2 H A P T E R 4~

Risk of Pulmonary Embolism/Venous Thrombosis CAROLYN WESTHOFF

Columbia University, College of Physicians and Surgeons, New York, New York 10032

IV. Incidence and Prognosis V. Clinical Recommendations References

I. Clinical Entities II. Diagnosis and T r e a t m e n t III. P a t h o p h y s i o l o g y

I. C L I N I C A L

in the lower extremity only, and the remaining one-third presents as a pulmonary embolus with or without a symptomatic clot in the leg. Clots in the leg may be silent, recognized only after a pulmonary embolism has occurred, if then, or they may come to clinical attention due to swelling and pain in the extremity. In clinically recognized cases of deep vein thrombosis of the leg the main symptoms are swelling (88%), pain (56%), and tenderness (55%). Symptoms such as redness, palpable venous cord, or a positive Homan's sign are present in only a minority of confirmed cases [2]. Pulmonary emboli present with dyspnea (80%), pleuritic chest pain (60%), and cough (41%) [3]. On ausculation, 60% of angiogram-confirmed cases have audible crackles. There is no important difference between men and women in the frequency of these presenting complaints. Because of difficulties in diagnosing these conditions, it is likely that many milder cases may be unrecognized. The sequelae of venous thrombosis are immediate death due to pulmonary embolism, recurrent symptomatic or asymptomatic thrombosis with repeated risk of embolism, and the development of postphlebitic syndrome. The

ENTITIES

Venous thrombosis mainly presents in the deep veins of the leg or in the lung, but can also occur elsewhere, including the brain, retina, liver, mesentery, and upper extremities. Clots originating in the upper extremity are relatively rare, and are usually sequelae of medical procedures such as catheter placement [ 1]. Thrombophlebitis is a clinical syndrome of pain and swelling in the leg that originates as a valve pocket thrombus, most often in the soleal vein or posterior tibial or popliteal veins, during some period of stasis. Such a thrombus may propagate upward to the femoral and iliac veins where pieces then break off into the venous circulation to become trapped in the lung as a pulmonary embolus. It is thought that distal leg clots do not embolize until after upward propagation to the thigh; however, clots may originate in the thigh or pelvis rather than in the leg. Further propagation in the lung or showers of emboli can be fatal. In most cases of pulmonary embolism it is possible to identify an apparent source clot in the lower extremity. About two-thirds of clinically recognized cases of venous thromboembolism (VTE) present with a venous thrombosis MENOPAUSE: BIOLOGY AND PATHOBIOLOGY

607

Copyright 9 2000 by Academic Press. All rights of reproduction in any form reserved.

608

CAROLYN WESTHOFF

postphlebitic syndrome encompasses chronic swelling, skin changes including ulceration, and chronic pain interfering with ambulation [4]. These chronic symptoms can also be associated with superficial phlebitis. The main goal of treatment of deep-vein thrombophlebitis (DVT) is to prevent pulmonary embolism (PE), prevent recurrence of thrombosis, and prevent the postphlebitic syndrome.

II. D I A G N O S I S

AND TREATMENT

The method of diagnosis of clots depends primarily on the location of the suspected clot. Proximal thrombi (that is, above the knee) are diagnosed by real-time B-mode compression ultrasound with high sensitivity and high positive predictive value. This test is safe, noninvasive, and widely available. If the patient has had clots previously, compressability may be abnormal, and thus sonography will be less helpful for diagnosis of recurrent clots; it is also less useful for distal clots. Contrast venography is still the standard for diagnosis of thrombi, and is more accurate than sonogram when distal thrombi are suspected. Because venography is invasive and more difficult to perform, its use is limited to cases in which sonography is not helpful. Magnetic resonance imaging is proving to be useful for diagnosis, but has not replaced other modalities, except perhaps in pregnant women [5]. A suspected pulmonary embolus is first evaluated by a chest radiograph plus perfusion scan or by a ventilationperfusion (V/Q) scan in which anatomical mismatches between air flow and blood flow in the lung may indicate the location of an embolus. The advantage of doing the perfusion scan as the first diagnostic test is that it is noninvasive and does not require highly specialized skills to carry out. A multicenter study, Prospective Investigation of Pulmonary Embolism Diagnosis (PIOPED), was carried out to assess the performance characteristics of V/Q scans used to assess 931 adults suspected of PE [3]. A high-probability perfusion scan in women has a sensitivity of 41% and a positive predictive value for a PE of 86%. An intermediate- or high-probability V/Q scan has a sensitivity of 84% and a positive predictive value of 44%. Thus, scans that are intermediate probability or nondiagnostic usually require proceeding to a pulmonary angiogram for a definitive diagnosis in order to avoid unnecessary treatment for those patients who will prove, by angiogram, to be negative for PE. Conventional treatment of DVT and PE has been intravenous unfractionated heparin during hospitalization followed by 3 to 6 months of oral anticoagulant therapy (OAT). Despite the ongoing risk of DVT recurrence, OAT is typically not continued past 6 months due to the risk of hemorrhage. Initial thrombolytic therapy in the acute phase is not widely used due to the risks of hemorrhage. Use of thrombectomy or inferior vena cava filters is considered in selected patients with large thrombi or when adequate anticoagula-

tion has not prevented recurrences. Also, treatment has been initiated with subcutaneous low-molecular-weight heparin (LMWH) used at home without laboratory dose adjustment. This appears to be at least similar to unfractionated heparin in the prevention of thrombus extension, death, hemorrhage, and recurrence of VTE events [6]. An advantage of LMWH is that it can be used at home without laboratory monitoring; a disadvantage is its greater expense. The duration of subsequent therapy with OAT is extended, possibly for life, in patients who have a continuing risk for VTE, such as those with cancer and those with an inherited predisposition.

III. PATHOPHYSIOLOGY The underlying causes of venous thrombosis have not been better described than by Virchow, who proposed in the nineteenth century that hypercoagulability, injury to the vessel wall, and circulatory stasis cause intravascular thrombosis. The disease was not described in antiquity, and Dexter proposes that the emergence of venous thrombosis as a major medical problem is due to the modern increase in chairsitting [7]. Multiple factors are required for a venous clot to occur, because each factor alonemthat is, stasis or injury or hypercoagulabilitymis only rarely associated with a clinically evident thrombus. Most of the recognized risk factors for VTE have a an obvious connection to stasis, injury, or hypercoagulability. The coagulation system is a dynamic balance between procoagulant and anticoagulant factors. The coagulation system factors related to arterial thrombosis appear to be distinct from those related to venous thrombosis. The risk of venous clots has been linked to deficiencies of several anticoagulant factors. In particular, inherited deficiencies of protein C, protein S, and antithrombin III confer an increased risk of clinically apparent venous clots. Acquired or inherited resistance to activated protein C (APC resistance) also leads to increased risk of thromboses. APC resistance can be measured genotypically or phenotypically. Genetic assays aim to identify in factor V an autosomal recessive point mutation called the Leiden mutation [8], which causes APC resistance. Phenotypic assays measure the resistance to cleavage of the factor V complex in the presence of activated protein C; this phenotype favors coagulation. The APC-resistent phenotype is present in individuals with the Leiden factor V mutation [9], and also, in a brief report from the World Health Organization Monitored Investigation of Cardiac Disease (MONICA) survey, the APC-resistent phenotype has been associated with oral contraceptive users, hormone replacement therapy users, and those with a body mass index greater than 30 kg/m 2 [10]. Screening of asymptomatic individuals for these inherited predispositions is not indicated on any routine basis. A genetically determined abnormality in prothrombin (a procoagulant) has been identified in thrombophilic families

CHAPTER 42 Pulmonary Embolism/Venous Thrombosis [ 11 ]. This and other specific inherited or acquired abnormalities of the coagulation system are suspected to increase the risk of venous thrombosis. Additional genetic studies will continue to pinpoint specific mutations in clotting factors that increase susceptibility to venous thrombosis, but these are likely to be rare and are currently not well established.

IV. INCIDENCE

AND PROGNOSIS

Several cohort studies have quantified the incidence of venous thrombosis and pulmonary embolism by identification of all new cases arising in a defined population. The first of these was the Tecumseh Community Health Study, whereby 9000 individuals of all ages in a city in Michigan were questioned and examined repeatedly in several cycles from 1957 to 1969 [12]. Most of the 169 first thrombotic events and the 63 recurrent events were identified by report of the participants without further validation. The incidence of these events increased with age, and men and women over age 40 experienced similar rates of DVT and PE. Because there were only 45 cases reported by women over age 40, the incidence rates are imprecise, but nonetheless they are similar to those obtained subsequently in larger studies with more uniform and complete surveillance of the population. These investigators estimated there would be about 250,000 clinically recognized cases annually in the United States, of which nearly half would occur in women over age 40 years. The Worcester DVT study calculated incidence rates for VTE based on discharge diagnoses from all 16 hospitals covering the Worcester Standard Metropolitan Statistical Area (1985 population, 379,953) from July, 1985 through December, 1986. All medical records with an eligible discharge diagnosis were reviewed to identify cases living in the catchment area and to confirm the diagnoses [2]. In total there were 615 cases included in the a n a l y s i s - - 4 0 5 initial episodes and 210 recurrent episodes. When extrapolated to the United States population, this study predicts about 170,000 initial and 99,000 recurrent cases annually, with nearly half of these in women over 40 years old. This study also identified an increase in risk with age, and similar incidence rates for men and women. Age-specific incidence rates for first events in women over 40 are given in Table I; the number of subjects used to calculate these rates was estimated from information in the paper. Overall, 12% of patients with a first episode died in the hospital, 5% after DVT, and 23% after PE. Case fatality was similar for men and women and increased with age. Immediate case fatality was only 2% below age 40 years, but was 10% from ages 40 to 59 years and 11% from ages 60 to 79 years. All patients in the Worcester cohort who were discharged alive were followed for 2 to 3.5 years, and 30% of the patients died during the follow-up interval. Long-term survival depended on age, but not on sex or whether the initial event was a PE or a DVT.

609 TABLE I Incidence of Venous Thrombosis and Pulmonary Embolism in Women > 4 0 Years Old a Venous thrombosis

Pulmonary embolism

Age (years)

Worcester

Malm6

Worcester

40 -49 50-59 60-69 70-79 Cases (N)

1.0 4.2 9.9 21.1 129b

9.7 10.3 21.7 42.9 189

0 1.1 6.2 6.9 540

NHS 1.3 1.8 3.2 344

a Cases per 10,000 women per year. Rates from Worcesterinclude only first events; rates from Malm6 and the NHS may include patients with recurrent thromboses or emboli. bThe numbers of cases from Worcester were calculated from data in the paper.

A similar study evaluated DVT in Malm6, Sweden, a city of 230,000. Subjects included all patients referred for phlebography (venogram) for suspected DVT in 1987 [13]. There was only one source of diagnostic testing for this population, and phlebography was the test of choice at the time of the study. There were 366 tests positive for DVT of 1009 referrals, including 189 women aged 40 years or older. Some of these cases may have been diagnosed and treated solely as outpatients, in contrast to most other studies that have considered only inpatient cases. Patients presenting with a primary pulmonary embolus were not included in this study, but PE was clinically suspected in 5% of the cases with DVT. As in the previous cohorts, the incidence rates increased with age and were similar in men and women. The incidence rates in Malm6 may appear higher than those in Worcester because of the possible inclusion of outpatients, the inclusion of a few patients with PE in the DVT group, and because new (76%) and recurrent (24%) cases were not separated. Fatality rates were not presented. Participants in the Nurses' Health Study (NHS) were evaluated for the occurrence of PE between 1976 and 1992 [ 14]. Medical records were examined for all of the 280 PEs that were reported by questionnaire or by death certificate. All but 36 of these cases occurred in women 40 years or older. Age-specific incidence rates are shown in Table I; a rate for women older than 70 years is not calculated because very few members of the cohort had yet reached that age. Mortality rates were not presented. Based on the Tecumseh, Worcester, Malm6 and NHS cohorts, about one-quarter to one-third of all cases of PE and VTE among women over 40 years old are recurrences. The risk of a recurrence, even after 6 months of anticoagulant treatment, is much greater than the risk of a first event. In an 8-year study of 355 consecutive, new VTE cases in Padua, Italy, where the cases were diagnosed from 1986 to 1987,

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CAROLYN WESTHOFF

78 patients experienced a recurrent venous thrombotic event during follow-up, including 9 fatal pulmonary emboli [4]. The recurrences accrued gradually with a cumulative recurrence rate of 30% at 8 years. Those cases whose primary VTE was secondary to surgery or trauma had a decreased risk of recurrence. Whether sex or age influenced recurrence risk was not presented. Mortality at 8 years was 30% and was mainly related to cancer. After 8 years of follow-up, 29% of subjects developed post-thrombotic syndrome. In a similar study from Cleveland [15], 124 cases with venogramconfirmed VTE diagnosed in 1984 and 1985 were followed until recurrence or death; follow-up ended in 1992. During follow-up, 42% of the cases died; the main predictors of death were cancer, stroke, or age greater than 75 years at the initial diagnosis. Chronic symptoms of pain, swelling, or discoloration in the affected leg were reported by 42%, and a recurrence was diagnosed in 15% of patients. These followup studies indicate that chronic symptoms and recurrence are common after a first VTE even in patients who receive a full 6-month course of anticoagulation. Little else is known about the distribution of VTE in the general population. A study of all 23,000 VTE hospital discharges in California from 1991 through 1994 found that about 75 % of cases are idiopathic and 25% are secondary to cancer, surgery, trauma, or another medical event [ 16]. This study also revealed that the risk among African-Americans was higher than among whites for idiopathic events [relative risk (RR), 1.3; 95% confidence interval (CI), 1.1-1.5]. The risk for Hispanics for idiopathic events was lower (RR, 0.6; 95% CI, 0.5-0.7) as was the risk for Asians (RR, 0.26; 95% CI, 0.2-0.3). The risk for secondary VTE was similar for whites, African-Americans, and Hispanics, but was lower for Asians.

prevalence is about 3%, and is even higher among patients with recurrent VTE. The relative risk of VTE from family studies is about 8 and from a population-based study is 6.5, which shows good agreement [19-21]. DVT of the leg is the most common manifestation, and one-half of predisposed individuals will not experience a first clinical event until after age 40 years. The prevalence of protein S deficiency has not been described in the general population. Among patients with a first VTE episode, the prevalence is about 1-2%, and seems to be higher among patients with recurrent thrombosis [21]. There may be phenotypic variations in protein S deficiency, and the relative risks for VTE for individuals with this class of clotting abnormality have not been precisely quantified. Antithrombin III deficiencies are rare, occurring in only 0.02% of unselected blood donors [ 18], in whom the relative risk for VTE may be as high as 50 [21,22], and the first VTE event will often occur before age 25 years, which makes this the most serious of the inherited predispositions. The most recently described and most common of the inherited predispositions is APC resistance due to a point mutation in factor V [23]. The population prevalence is between 3 and 6% and is greater among whites than in other racial groups [24]. The relative risk of VTE in individuals with this mutation is about 8 compared to the unaffected population, and the risk may be substantially higher for homozygotes [25]. A first event in an affected individual may occur at any age. Altogether, about 30% of individuals with a first VTE event occurring in the absence of a predisposing clinical situation will have one of these underlying genetically determined abnormalities of the coagulation system. The APC resistance due to the Leiden mutation is the most common of these underlying abnormalities [8].

B. R i s k s o f V T E w i t h C l i n i c a l l y A. R i s k s o f V T E w i t h C o n g e n i t a l

Identified Risk Factors

or A c q u i r e d C a u s e s o f H y p e r c o a g u l a b i l i t y Familial clustering of VTE has allowed the investigation and recognition of several inherited disorders of coagulation that are associated with a dramatically increased risk of VTE. In population-based studies, however, the risk of thrombosis in the presence of specific genetically determined abnormalities is sometimes lower than the risk as estimated in studies of affected families; this suggests that for certain abnormalities, members of strongly affected families may have additional, unrecognized abnormalities that generate higher risks than seen in otherwise unselected individuals with the same defect [17]. Bearing in mind these discrepancies, the frequency of each of the major genetic predispositions and estimates of the associated risk will be presented. The prevalence of protein C deficiency has been estimated in large studies of blood donors to be about 0.2-0.4% [18]. Among patients experiencing a first VTE episode, the

The strongest risk factors for VTE have been readily identified clinically without the use of laboratory or epidemiological studies to establish risk. Immobilization with or without injury is the hallmark of these risk factors. VTE associated with any of these antecedent factors is generally referred to as secondary VTE because the clot is presumed to have arisen as a result of a specific precipitating event. In most series of consecutive cases, from 40 to 80% of all subjects are considered to have a VTE that is secondary to a clinically recognized precipitating event. In studies to assess more subtle causes of VTE, those VTE patients with any of these major risk factors are excluded. Due to this approach, it is essentially unknown how cofactors might increase risk in the large proportion of VTE patients who have a precipitating factor. Studies of clinical risk factors have not presented separate analyses regarding menopausal women, but there is no a p r i o r i reason to suspect important differences in risk factors by age or sex. Overall, there is an increase in

CHAPTER42 Pulmonary Embolism/Venous Thrombosis TABLE II

Risk

Risk Factors for VTE in Hospitalized Women Aged > 4 0 Years Old a Reason for hospitalization

<10%

Minor surgery (<30 min) Minor trauma or medical illness

10-40%

Minor trauma, surgery, or illness with past VTE history Surgery (> 30 min), including urologic, gynecologic, general, cardiothoracic, or neorological surgery Cancer, heart, or lung disease Major trauma or burns

>40%

Major surgery, illness, or trauma with past history of VTE Fracture or orthopedic surgery of pelvis, hip, or leg Pelvic or abdominal surgery for cancer Lower limb paralysis (e.g., stroke) or amputation

a

Adapted from THRIFT Consensus Group [26].

VTE risk after age 40 years, but much of this increase is probably due to the increased prevalence of the underlying medical risk factors with increasing age. Many VTE events occur after hospitalization for another problem, and are not themselves the primary reason for the hospitalization. Risk of VTE occurring in patients who are already hospitalized has been well characterized. A risk classification for women over age 40 modified from the Thromboembolic Risk Factors (THRIFT) Consensus Group [26] is presented in Table II. Women who fall into the low-risk group do not warrant prophylactic anticoagulation, but for those in the moderate- or high-risk groups routine prophylaxis is advisable. A meta-analysis of the incidence of DVT following general surgery indicates that effective prophylactic measures include low-dose heparin, graduated elastic compression stockings, and intermittent pneumatic compression [27]. In the PIOPED study several risk factors were present among women who were referred for evaluation for possible PE [3]; these included surgery within 3 months before the onset of symptoms (38%), immobilization (35%), malignancy (28%), a history of previous phlebitis (18%), stroke (7%), and trauma (7%). After evaluation, these factors were associated with a 50% or greater increased risk of a positive angiogram among the women in the study population. An increased risk of either deep vein thrombosis or pulmonary embolus has been identified in such patients in numerous studies [28].

C. Other Risk Factors for Primary or Idiopathic VTE Few studies have evaluated causes of VTE after excluding cases with strong precipitating factors. The main exposures of interest have been weight, smoking, chronic medical conditions, and use of exogenous hormones. Use of hor-

611 mones will be discussed separately below. Some studies have identified modestly increased risks for VTE in women with chronic medical conditions such as hypertension, diabetes mellitus, and gall bladder disease [29,30]. Because evaluation of hormone use has often been a primary goal of the analyses, women with these conditions have often been excluded in order to control for confounding. Overall, evaluation of risk associated with the common chronic medical conditions has not been illuminating. Obesity, when defined as a body mass index, has generally been found to be a risk factor for VTE. Obesity had no effect on VTE risk only in the Walnut Creek Contraceptive Drug Study, in which obesity was defined as weight 15% above the cohort mean [31 ]; this study included 38 cases, of which 23 were 40 years or older. In the Nurses' Health Study there were 125 women who reported an idiopathic pulmonary embolism. Among these women, obesity (defined as a BMI of 29 kg/m 2) was associated with a relative risk of 2.9 (95% confidence interval, 1.5-5.4). Most of these cases were older than 40 years, and the increased risk associated with obesity held for all ages [ 14]. In a United Kingdom cohort that included 292 female VTE cases aged 5 0 - 7 9 years, a BMI of 26 kg/m 2 or greater had a relative risk for VTE of 2.0 (95% confidence interval, 1.4-2.9) compared to women with a BMI of 25 kg/m 2 or less [30]. Large studies of oral contraceptives and thrombosis included younger study populations, but all have identified obesity as a risk factor for VTE [32-34]. The evidence supports an increased risk of VTE in obese women in the menopausal age group as well as in younger women. There are no data concerning VTE and obesity in men. The data for smoking are less consistent. Studies in younger women find little or no increased risk of VTE in current smokers [32-34]. The large United Kingdom cohort of women aged 5 0 - 7 9 years identified a relative risk of 1.2 (95% confidence interval, 0.9-1.7) for VTE among current smokers compared to never smokers [30]. Among the 125 idiopathic PE cases from the NHS, there was a RR of 1.9 (95% confidence interval, 0.9-3.7) for women who smoked 2 5 - 3 4 cigarettes daily and a RR of 3.3 (95% confidence interval, 1.7-6.5) for women who smoked 35 or more cigarettes daily compared to nonsmokers [ 14]. In sum, cigarette smoking is not an important explanatory variable for VTE in women, regardless of age.

D. Hormonal Risk Factors for Venous Thromboembolism Case reports followed very soon by epidemiologic studies showed that use of the old, high-dose oral contraceptives (OCs) were associated with an increased risk of venous thrombosis, including pulmonary embolus [35]. As the dose of estrogen in the OC decreased there was a concomitant decrease in VTE risk [36]. This led to the conclusion that the

612

CAROLYN WESTHOFF

TABLE III Current HRT Use and VTE Risk The Early "Negative" Studies Study

Years

Number of cases

Adjustedrelative risk (95% CI)

BCDSP [39] Nachtigall [40] Petitti [ 2 9 ] Devor [ 4 1 ]

1972 1969-1978 1969-1976 1980-1987

18 30 17 121

2.3 (0.6-8.0) a 0.85 (ns; CI not provided) 0.7 (0.2-2.5) b 0.6 (0.2-1.8)

a Relative risk and CI not provided in original publication, but subsequently estimatedby Douketis [54]. NS, not significant. b90% CI.

risk of VTE in OC users was linked to the estrogen dose, a concept that was little challenged over the past two decades [37]. Because the dose of estrogen in hormone replacement therapy (HRT) is so much lower than the dose in even the lowest dose OCs, it seemed unlikely that HRT would be unlikely to cause any problem with venous clots [38]. The original studies (Table III) that attempted to assess risk of VTE in HRT users supported this line of thought. The first report came from the Boston Collaborative Drug Surveillance Program [39] and reported on 18 cases of VTE. Of the cases, 14% were HRT users, and of the controls, 8% were users; if the study were larger these percentages would have indicated an increased risk. The authors correctly concluded "significant associations were not present"; however, the small number of cases and rare use of HRT meant that the study did not have statistical power to detect an association. The next report came from the Walnut Creek Contraceptive Drug Study [29,31], and included 17 cases, of whom 12% used HRT; 15% of the controls used HRT. This does not indicate any hint of increased risk, but again, due to the small number of cases and limited use of HRT the study lacked statistical power to detect an association. Both of these studies excluded the majority of cases to focus on idiopathic VTE only. In contrast, Nachtigall [40] excluded none of the cases; in this randomized controlled trial of chronically ill, permanently hospitalized women, 84 women were randomized to an oral HRT regimen for 10 years, and 84 controls received placebo. These 168 women experienced 30 VTE e v e n t s - - a number that is at least 10 times greater than the largest expected number calculated from incidence rates in Table I. The high rate of VTE in this population may have been due to limited mobility of the subjects during their chronic hospitalization. There are also cases of superficial thrombophlebitis included, and no requirement for confirmatory diagnostic tests. There was no indication of increased VTE risk among the women randomized to HRT; however, it is difficult to interpret this finding in this unusual population. Devor [41 ] also included all cases of VTE in a hospitalbased case-control analysis. Based on 121 cases, there was no evidence of excess risk in the HRT users. The overall

analysis showed no increase in risk associated with HRT, but when subsets of cases were excluded (such as women with a previous thrombosis) the relative risk increased. As is true for other consecutive, unselected series of VTE cases, most of the cases in this study were secondary to a known predisposing factor. These four early evaluations of the H R T - V T E association did not indicate an increased risk, but the first two studies were too small for a precise or sophisticated analysis and the randomized trial included an unusual population. Finally, the larger case-control study included so many women with secondary VTE and other serious diseases that it is difficult to assess whether HRT would have been given to these women by the physicians who were caring for their medical illnesses. With the clarity of hindsight, it appears that none of these early studies had a design or number of cases appropriate to address the question.

E. S t u d i e s o f t h e H R T - V T E

Association

Starting in 1996, a series of new large studies began to be published (Table IV), suggesting an increased risk of both deep vein thrombosis and pulmonary embolus among current users of hormone replacement therapy. These new studies include data from two large prospective cohort studies, the Nurses' Health Study [42] and the Oxford-Family Planning Association cohort [43], from an historical cohort study of the Group Health Incorporated (GHI) medical plan database [44], from an historical cohort study using record linkage with the United Kingdom General Practice Research Database [30], from a record linkage study using medical databases in Italy [45], from a hospital-based case-control study in the United Kingdom [46], and, finally, from a randomized controlled trial of women with heart disease, the Heart and Estrogen/progestin Replacement Study (HERS) trial, in the United States [47]. All of these studies in Table IV consider just those cases with a first episode of VTE. All of the studies excluded cases and controls with risk factors for VTE (e.g.,

TABLE IV Risk of Primary VTE among Current HRT U s e r s m S u b s e q u e n t Studies Adjusted relativerisk (95% CI) Number of cases Currentuse First-yearuse

Study

Years

Grodstein [42] Daly (letter) [43] Daly [ 4 3 ] Jick [44] Gutthann [30] Varas [ 4 5 ] Hulley [ 4 7 ]

1976-1992 1982-1993 1993-1994 1980-1994 1991-1994 1991-1995 1993-1997

a Pulmonary embolus only.

123a 18 103 42 292 171 46

2.1 (1.2-3.8) 2.2 (0.6-7.9) 3.5 (1.8-7.0) 3.6 (1.6-7.8) 2.1 (1.4-3.2) 2.3 (1.0-5.3) 2.9 (1.5-5.6)

Not calculated Not calculated 6.7 (2.1-21.3) 6.7 (1.5-30.8) 4.6 (2.5-8.4) 2.9 (1.2-6.9) 3.3 (1.1-10.1)

CHAPTER 42 Pulmonary Embolism/Venous Thrombosis cancer, fracture, and stroke), as well as excluding cases with superficial phlebitis. All of the studies described specific diagnostic criteria that were required for inclusion as a case. Although the methodology differed and the stringency of the diagnostic criteria varied somewhat between studies, the relative risks shown in Table IV are nonetheless extremely similar, and indicate a risk among current HRT users that is two to three times the risk among nonusers. Unlike previous smaller studies, these studies were able to assess risks in some subgroups of HRT users. The most consistent finding was that the increased risk of VTE was greatest in or limited to the first year of HRT use. Until recently, the usual approach to analysis was to look for increased risk as the time of exposure increased, e.g., the risk of lung cancer increases with increasing years of cigarette smoking. The notion of looking for an early, transient risk is more recent and an understanding of these consistent results is not yet established. Other studies indicate that the increased risk of VTE in OC users may also be early and transient [34,48]. Other subgroup analyses have attempted to assess conventional markers of risk such as dose. In these analyses, there was an indication of an increased risk of VTE with higher estrogen doses in one study [32], but no d o s e - r e s p o n s e effect in the others that were able to assess dose [42,46,49]. The studies did generally agree on little or no excess risk in past users [42,45,46,49]. The populations that were studied often included only a few HRT users or a limited range of HRT regimens; therefore, the investigators had a limited ability to compare risks between regimens. In addition to the studies listed above, the Postmenopausal Estrogen/Progestin Intervention (PEPI) trial also evaluated phlebitis in women randomly assigned to five different HRT regimens; although there was a suggestion of an increased risk of VTE among the subjects receiving active treatment, this outcome was too rare to allow comparisons between the groups [50]. Taken together, these studies have provided little evidence to distinguish the risks of opposed versus unopposed estrogen, of oral versus other routes of administration, or between different formulations. Overall, there appears to be a small, perhaps transient, increase in risk that is not limited to or avoided by any particular HRT regimen. The majority of VTE cases in women in the menopausal age group are secondary to other identifiable risk factors. The incidence of cases that might be attributed to HRT use can be calculated from the cohort studies. In the GHI cohort there were about two extra VTE cases per 10,000 HRT users per year [44]. An estimate based on United Kingdom incidence data similarly suggested about two additional cases per 10,000 HRT users per year [46]. In the HERS study the excess risk was about 7 cases per 1000 in year 1 and about 2 cases per 1000 in years 4 and 5 [47]; however, women were selected for that randomized trial based on diagnosed cardiovascular disease, and they appear to have a much higher baseline risk for VTE than do women in

613 the general population. The HERS results do suggest that women with an increased baseline risk of VTE may experience many more cases of VTE if using HRT than is seen among low-risk women. All of these studies have looked at traditional estrogenbased hormone replacment regimens. Some data are accumulating about the risk of VTE in women using the other, newer selective estrogen receptor modulators. Tamoxifen has the longest and widest use of these and has been associated with increased risk of VTE at a magnitude similar to the studies of estrogen [51]. Tibolone, a nonestrogen treatment for hot flashes, was evaluated in some of the European studies with relative risks for VTE slightly lower than those seen for estrogen; however, the number of users of tibolone in these studies was small and therefore the relative risk estimate thus far must be considered imprecise [46]. Raloxifene use in placebo-controlled clinical trials has been associated with a relative risk of VTE of about 3.0 [52], and this excess is noted in the product labeling [53].

V. C L I N I C A L

RECOMMENDATIONS

Venous thromboembolism is a common problem among women in the menopausal age group, and is most likely to occur among women with predisposing medical problems. In large part VTE increases with age because the predisposing problems become more common with advancing age. Among healthy, low-risk women, even after age 40, the risk of VTE is probably about 1 - 2 new cases per 10,000 women per year. In this population, the risk of VTE for women using HRT may increase two- to threefold from this low level, yielding somewhere between 1 and 6 additional cases per 10,000 HRT users. This is comparable both in relative and absolute terms to the increased risk seen among younger women who use oral contraceptives. As with younger women and OCs, this risk of VTE in HRT users needs to be communicated to potential HRT users so that they can weigh this along with all other risks and benefits in making a decision to use HRT. Among menopausal women with chronic medical problems or women who will be having surgery or hospitalization for some other reasons, the baseline risk of a VTE is substantially higher. The results from the HERS trial [47] indicate that the women in that trial with known heart disease had a baseline risk of VTE of at least 20/10,000 women per year and that HRT use in these women increased that risk threefold. The proportional increase was the same as that seen in low-risk women, but because their baseline risk of VTE was higher, this increase in risk may translate into 2 0 40 additional cases per 10,000 users per year. Because most of the studies agree that the excess risk is concentrated in the first 1-2 years of HRT use, this information is most important for women who are considering whether to initiate HRT.

614

CAROLYN WESTHOFF

Although there are no specific relevant data regarding the risk of VTE among hospitalized HRT users, it appears prudent to consider discontinuing oral HRT in women who are going to undergo major surgery or who develop a medical problem that is associated with immobilization or other disease that is associated with a high risk of VTE. Among women who are currently being treated with anticoagulants, there are no data to indicate whether HRT use would modify their risk of a recurrent event.

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16. White, R., Zhou, H., and Romano, E (1998). Incidence of idiopathic deep venous thrombosis and secondary thromboembolism among ethnic groups in California. Ann. Intern. Med. 128, 737-740. 17. Rosendaal, E (1997). Risk factors for venous thrombosis: Prevalence, risk, and interaction. Semin. Hematol. 34, 171-187. 18. Tait, R. C., Walker, I. D., Reitsma, E H., Islam, S. I., McCall, F., Poort, S. R., Cankie, J. A., and Bertina, R. M. (1995). Prevalence of protein C deficiency in the healthy population. Thromb. Haemostasis 73, 87-93. 19. Allaart, C., and Bri~t, E. (1994). Familial venous thrombophilia. In "Hemostasis and thrombosis" (A. Bloom, C. Forbes, D. Thomas, et al., eds.), pp. 1349-1360. Churchill-Livingstone, New York. 20. Bovill, E. G., Bauer, K. A., Dickermann J. D., Callas, E, and West, B. (1989). The clinical spectrum of heterozygous protein C deficiency in a large New England kindred. Blood 73, 712-717. 21. Koster, T., Rosendaal, F. R., Briet, E., van der Meer, E J., Colly, L. E, Trienekens, E H., Poort, S. R., Reitsma, E H., and Vandenbroucke, J. E (1995). Protein C deficiency in a controlled series of unselected outpatients: An infrequent but clear risk factor for venous thrombosis (Leiden thrombophilia study). Blood 85, 2756-2761. 22. Hiejboer, H., Brandjes, D. P., Buller, H. R., Sturk, A., and Ten Cate, J. W. (1990). Deficiencies of coagulation-inhibiting and fibrinolytic proteins in outpatients with deep-vein thrombosis. N. Engl. J. Med. 323, 1512-1516. 23. Dahlb~ick, B., Carlsson, M., and Svensson, E (1993). Familial thrombophilia due to a previously unrecognised mechanism characterized by poor anticoagulant response to activated protein C: Prediction of a cocafactor to activated protein C. Proc. Natl. Acad. Sci. U.S.A. 90, 1004-1008. 24. Ridker, E M., Hennekens, C. H., Lindpainter, K., Stampfer, M. J., Eisenberg, P. R., and Miletich, J. E (1995). Mutation in the gene coding for coagulation factor V and the risk of myocardial infarction, stroke, and venous thrombosis in apparently healthy men. N. Engl. J. Med. 332, 912-917. 25. Vandenbroucke, J., and Helmerhorst, E (1996). Risk of venous thrombosis with hormone-replacement therapy. Lancet 348, 972. 26. THRIFT Consensus Group (1992). Risk of and prophylaxis for venous thromboembolism in hospital patients. Br. Med. J. 305, 567-574. 27. Clagett, G., and Reisch, J. (1988). Prevention of venous thromboembolism in general surgical patients. Results of meta-analysis. Ann. Surg. 208, 227-240. 28. Salzman, E., and Hirsch, J. (1994). The epidemiology, pathogenesis, and natural history of venous thrombosis. In "Hemostasis and Thrombosis: Basic Principles and Clinical Practice" (R. Coleman, J. Hirsch, V. Marder, and E. Salzman, eds.), pp. 1275-1296. Lippincott, Philadelphia. 29. Petitti, D., Wingerd, J., Pellegrin, E, and Ramcharan, S. (1979). Risk of vascular disease in women: Smoking, oral contraceptives, noncontraceptive estrogens, and other factors. JAMA, J. Am. Med. Assoc. 242, 1150-1154.

30. Gutthann, S. P., Garcia Rodriguez, L. A., Castellsague, J., and Oliart, A. D. (1997). Hormone replacement therapy and risk of venous thromboembolism: Population based case-control study. Br. Med. J. 314, 796-800. 31. Petitti, D., Wingerd, J., Pellegrin, E, and Ramcharan, S. (1978). Oral contraceptives, smoking, and other factors in relation to risk of venous thromboembolic disease. Am. J. Epidemiol. 108, 480-485. 32. Jick, H., Jick, S., Gurewich, V., Myers, M. W., and Vasilakis, C. (1995). Risk of idiopathic cardiovascular death and nonfatal venous thromboembolism in women using oral contraceptives with differing progestagen components. Lancet 346, 1589-1592. 33. World Health Organization Collaborative Study of Cardiovascular Disease and Steroid Hormone Contraception (1995). Effect of different progestagens in low oestrogen oral contraceptives on venous thromboembolic disease. Lancet 346, 1582-1588.

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CHAPTER 42 P u l m o n a r y E m b o l i s m / V e n o u s Thrombosis 34. Spitzer, W. O., Lewis, M. A., Heinemann, L. A., Thorogood, M., and MacRae, K. D. (1996). Third generation oral contraceptives and risk of venous thromboembolic disorders: And international case-control study. Br. Med. J. 312, 83-88. 35. Inman, W. H., Vessey, M. E, Westerholm, B., and Engelund, A. (1970). Thromboembolism disease and the steroidal content of oral contraceptives: A report to the Committee on Safety of Drugs. Br. Med. J. 2, 203209. 36. Gerstman, B. B., Piper, J. M., Tomita, D. K., Fergus, W. J., Stadel, B. V., and Lundin, F. E. (1991). Oral contraceptive estrogen dose and the risk of deep venous thromboembolic disease. Am. J. Epidemiol. 133, 32-37. 37. Realini, J., and Goldzieher, J. (1985). Oral contraceptives and cardiovascular disease: A critique of the epidemiological studies. Am. J. Obstet. Gynecol. 152, 729-798. 38. Lobo, R. (1992). Estrogen and the risk of coagulopathy. Am. J. Med. 92, 283-285. 39. Boston Collaborative Drug Surveillance Program (1974). Surgically confirmed gallbladder disease, venous thromboembolism, and breast tumors in relation to postmenopausal estrogen therapy. N. Engl. J. Med. 290, 15-19. 40. Nachtigall, L., Nachtigall, R., Nachtigall, R., and Beckman, E. (1979). Estrogen replacement therapy II: A prospective study in the relationship to carcinoma and cardiovascular and metabolic problems. Obstet. Gynecol. 54, 74-79. 41. Devor, M., Barrett-Connor, E., Renvall, M., Feigal, D., Jr., and Ramsdell, J. (1992). Estrogen replacement therapy and the risk of venous thrombosis. Am. J. Med. 92, 275-282. 42. Grodstein, F., Stampfer, M., Manson, J., Colditz, G., Willett, W., Rosner, B., Speizer, E, and Hennekens, C. (1996). Postmenopausal estrogen and progestin use and the risk of cardiovascular disease. N. Engl. J. Med. 335, 453-461. 43. Daly, E., Vessey, M. P., Painter, R., and Hawkins, M. (1996). Casecontrol study of venous thromboembolism risk in users of hormone replacement therapy. Lancet 348, 1027. 44. Jick, H., Derby, L., Myers, M., Vasilakis, C., and Newton, K. (1996). Risk of hospital admission for idiopathic venous thromboem-

45.

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53. 54.

bolism among users of postmenopausal oestrogens. Lancet 348, 981-983. Varas-Lorenzo, C., Garcia-Rodriguez, L., Cattaruzzi, C., Troncon, M., Agostinis, L., and Perez-Gutthann, S. (1998). Hormone replacement therapy and the risk of hospitalization for venous thromboembolism: A population-based study in Southern Europe. Am. J. Epidemiol. 147, 387-390. Daly, E., Vessey, M. P., Hawkins, M. M., Carson, J. L., Gough, P., and Marsh, S. (1996). Risk of venous thromboembolism in users of hormone replacement therapy. Lancet 348, 977- 980. Hulley, S., Grady, D., Bush, T., Furberg, C., Herrington, S., Riggs, B., and Vittinghoff, E. (1998). Randomized trial of estrogen plus progestin for secondary prevention of coronary heart disease in postmenopausal women. JAMA, J. Am. Med. Assoc. 280, 605-613. Lidegaard, O. (1998). Thrombotic diseases in young women and the influence of oral contraceptives.Am. J. Obstet. Gynecol. 179, $62-$67. Grodstein, F., Stampfer, M., Goldhaber, S., Manson, J., Colditz, G., Speizer, F., Willett, W., and Hennekens, C. (1996). Prospective study of exogenous hormones and risk of pulmonary embolism in women. Lancet 348, 983-987. Writing group for the PEPI Trial (1995). Effects of estrogen or estrogen/progestin regimens on heart disease risk factors in postmenopausal women. JAMA, J. Am. Med. Assoc. 273, 199-208. Fisher, B., Costantino, J., Redmond, C., Poisson, R., Bowman, D., Couture, J., Dimitrov, N. V., Wolmark, N., Wickerham, D. L., Fisher, E. R. et al. (1989). A randomized clinical trial evaluating tamoxifen in the treatment of patients with node-negative breast cancer who have estrogen-receptor-positive tumors. N. Engl. J. Med. 320, 479-484. Delmas, P., Mitlak, B., and Christiaansen, C. (1998). Effects of raloxifene in post menopausal women (letter). N. Engl. J. Med. 338, 13131314. Eli Lilly and Company (1998). "Evista| Product Information." Eli Lilly and Company. Douketis, J. D., Ginsberg, J. S., Holbrook, A., Crowther, M., Duku, E. K., and Burrows, R. E (1997). A reevaluation of the risk for venous thromboembolism with the use of oral contraceptives and hormone replacement therapy. Arch. Intern. Med. 157(14), 1522-1530.

~HAPTER

4":

Combined Estrogen/ Androgen Replacement Therapy: Benefits and Risks BARBARA B.

SHERWlN

Department of Psychology and Department of Obstetrics and Gynecology,

McGill University, Montreal, H3A 1B 1 Canada

IV. Risks of E/A Replacement Therapy V. Future Directions References

I. Introduction

II. Historical Background III. Benefits of E/A Replacement Therapy

II. H I S T O R I C A L B A C K G R O U N D

I. I N T R O D U C T I O N

Although combined estrogen/androgen replacement therapy for postmenopausal women is still not a common treatment, the concept of combined therapy actually developed over 60 years ago. Soon after testosterone propionate had been synthesized in the mid-1930s, it was used to treat a variety of gynecological disturbances such as menorrhagia, mastitis, dysmenorrhea, and menopausal symptoms in oophorectomized women [1]. In one of the earliest studies, menopausal women received estrogen and 25 to 50 mg of testosterone propionate daily [2]. The therapy resulted in the serendipitous finding that sexual appetite and response were significantly greater than that experienced with estrogen alone. Following this observation, many studies originally undertaken to assess the efficacy of various therapeutic agents for the management of menopausal symptoms almost universally reported increased libido as an effect of exogenous androgen [3-5]. Several investigators reported, however, that the effects of androgen administration on libido

Controlled studies have prodided a voluminous amount of information on the benefits and risks of estrogen replacement therapy in menopausal women, yet there is still a paucity of data bearing on the efficacy of combined estrogen/androgen (E/A) replacement therapy. Although it is not clear why this is true, it is likely that outdated notions of female sexuality, the historical attribution of menopausal symptoms to neurotic illness, and the clinical sequelae of androgen-excess endocrinopathies in women may all have played a role in the apparent reluctance of medical scientists and practitioners to investigate and prescribe combined E/A preparations as replacement therapy to selected postmenopausal women. Historically, there has also been a reluctance, on behalf of the medical community, to acknowledge that there was any physiological role for androgens in women. This chapter reviews the benefits and risks of combined E/A therapy in the postmenopause and then underlines methodological issues that bear on research in this area.

M E N O P A U S E : B I O L O G Y AND PATHOBIOLOGY

617

Copyright 9 2000 by Academic Press. All rights of reproduction in any form reserved.

618 were dependant on previous sexual functioning; women who described "very little or no libido" in the past reported no change in sexual desire following testosterone pellet implants, whereas those who "had once had libido but lost it" reported a marked increase after treatment [6,7]. Greenblatt et al. assessed the relative efficacy of hormones administered singly or in combination to menopausal women [8]. Satisfactory relief of all symptoms was reported by 96.9% of patients who received estrogen alone, and by 89.6% who received an estrogen/androgen combination (diethylstilbestrol 0.25 mg/day and methyltestosterone, 5 mg/day). Only 23.5 % of those who received androgen alone reported symptom relief whereas 83.3% of the subjects in the placebo group reported no improvement of symptoms. Most noteworthy, 66.6% of the patients stated a preference for the estrogen/androgen combination because of the increased well-being and libido they experienced while on this regimen. These findings were confirmed in a study by Caldwell and Watson, who found that postmenopausal women who received a combined estrogen/androgen preparation showed improved physical capacity, increase in weight, and improvement in memory and in ability to learn new material [9]. Despite the consistency in the findings of these early studies, they lacked control groups, used unsystematic methods of data collection, and the majority were merely anecdotal in nature. A second (though somewhat weak) source of support for the libido-enhancing effects of androgen comes from the clinical reports of its administration in the treatment of estrogen-dependant breast cancers [10,11]. This treatment was based on the belief that large doses of androgen would oppose the effects of estrogen and thus halt spread of the disease. These women spontaneously reported increased libido as a result of the hormone therapy. However, the massive doses of testosterone propionate used in those cases (1200 mg/week), the confounds inherent in this gravely ill subject sample, and the uncontrolled nature of the reports detract from the meaningfulness of these findings. A third series of studies undertaken in the late 1950s and early 1960s similarly concerned women who underwent several surgical procedures in an effort to halt the continuing spread of metastatic breast cancer. Following mastectomy and oophorectomy, no changes in sexual desire were obvious [12]. However, following adrenalectomy, carried out because of progression of the malignant disease, 14 out of 17 patients reported the sudden absence of all sexual desire. In a later study of seven patients who had had oophorectomy 1 to 7 years before the adrenals were removed, all reported almost total loss of sexual feelings and responsivity after adrenalectomy [ 13]. The authors concluded that the radical decrease in libido in these women following their adrenalectomies was due largely to their near total loss of any source of endogenous androgen production. Despite the fact that these patients were gravely ill and the data were uncontrolled and

BARBARA B. SHERWIN

anecdotal, for over 20 years these studies were frequently cited as evidence of androgen's libido-enhancing properties in women.

III. BENEFITS OF E/A REPLACEMENT

THERAPY A. E n e r g y L e v e l

It has been known for a long time that androgens have anabolic properties. In hypogonadal men treated with testosterone, lean body mass increased by 11% and muscle mass in the upper arm and the thigh increased by 21% and 12%, respectively [14]. There is little question that androgenic steroid administration to prepubertal boys, to hypogonadal men, and to women results in increased nitrogen retention and increased muscle mass. The observation that androgens have energizing properties may be a result of the general anabolic effects of this steroid hormone on the central nervous system. There is a dearth of information on whether exogenous androgen administered along with estrogen to postmenopausal women influences their energy level and sense of wellbeing. The sole study that measured these parameters systematically reported an increase in ratings of energy level and wellbeing in premenopausal women who had had their uterus and ovaries removed and randomly received a combined E/A preparation or androgen alone postoperatively [15]. This did not occur in women who were given estrogen alone or placebo.

B. S e x u a l F u n c t i o n i n g Effects of testosterone on various components of mating behavior have been studied intensively in female nonhuman primates. On the whole, these studies show that the administration of estrogen alone to ovariectomized rhesus monkeys was associated with a decrease in proceptive behavior (i.e., attempts to solicit mounts from the male, a behavioral homology of sexual desire in women). Implantation of minute amounts of testosterone, via a cannula, into the anterior hypothalamus of estrogen-treated ovariectomized and adrenalectomized unreceptive female rhesus monkeys resulted in restoration of their proceptivity without affecting other aspects of sexual behavior, such as attractivity [ 16]. These studies on testosterone and sexual behavior in female nonhuman primates serve to underline two points. One is that there is a specificity of action of testosterone on compoments of sexual behavior such that it enhances proceptivity (the animal's motivation to engage in sexual behavior) but has no effect on its attractivity or its receptivity to males.

CHAPTER43 Estrogen/Androgen Replacement Therapy Second, the fact that a very small dose of testosterone implanted in the hypothalamus was effective in restoring sexual desire in rhesus monkeys [ 16] suggests that testosterone exerts an effect on sexual desire in female rhesus directly on the brain and not via an influence on peripheral tissues. Several correlational studies have tested the association between circulating levels of this sex steroid and aspects of sexual behavior in postmenopausal women. Leiblum and colleagues [ 17] reported that neither estradiol nor testosterone discriminated between sexually active and inactive untreated postmenopausal women, but sexually active women had less vaginal atrophy compared to the inactive women. In a longitudinal study of perimenopausal women, plasma testosterone levels were positively associated with coital frequency [18]. Moreover, a positive correlation occurred between testosterone levels and sexual desire and sexual arousal in premenopausal women over the age of 40 years [19]. Other epidemiological studies that have investigated changes in sexual functioning in peri- and postmenopausal women failed to measure circulating levels of hormones [20,21]. One recent population-based study in middle-aged women failed to find an association between testosterone levels and any aspect of sexual functioning [22]. Another and perhaps more powerful paradigm for investigating the role of testosterone in women involves administering hormone replacement therapy to women who have just undergone total abdominal hysterectomy (TAH) and bilateral salpingo-oophorectomy (BSO). When both ovaries are removed from premenopausal women, circulating testosterone levels decrease significantly within the first 2 4 - 4 8 hr postoperatively [23]. The fact that these women are deprived of ovarian androgen production following this surgical procedure has provided a rationale for administering both estrogen and androgen as replacement therapy. In Britain and Australia, subcutaneous implantation of pellets containing estradiol and testosterone has been used as a treatment for menopausal symptoms for several decades. This route of sex-steroid administration results in a slow constant release of the sex hormones over a period of at least 6 months. Women complaining of decreased libido despite treatment with estrogen received subcutaneous implants of 40 mg estradiol and 100 mg testosterone [24]. Patients reported a significant increase in libido by the third postimplantation month. These findings gained support from a double-blind study of women complaining of loss of libido despite treatment with oral estrogens [25]. They randomly received a subcutaneous implant containing either estrogen alone or estrogen plus testosterone. After 6 weeks, the loss of libido in the estrogen-alone implant group remained, whereas the combined estrogen/testosterone group showed significant symptomatic relief. In a recent prospective, 2-year, single-blind randomized trial, 34 postmenopausal women received either estradiol 50-mg implants or estradiol 50-mg plus testosterone 50-mg

619 implants administered every 3 months [26]. Women who received the combined implant had a significantly greater improvement compared with those receiving estrogen alone, in sexual activity, satisfaction, pleasure, and orgasm. We undertook a series of studies to investigate effects of E/A replacement therapy on aspects of sexual functioning. In one study, premenopausal women who needed to undergo total abdominal hysterectomy and bilateral salpingooophorectomy for benign disease were tested preoperatively [27]. Postoperatively, women randomly received either a combined E/A preparation, estrogen alone, androgen alone, or placebo. These drugs were all depot preparations and were administered intramuscularly once a month. Those who received one of the androgen-containing preparations (E/A or androgen alone) had higher scores on measures of sexual desire, sexual arousal, and number of sexual fantasies compared to women who received estrogen alone or placebo. This occurred despite the fact that estrogen treated women had supraphysiological levels of estrogen [15], had no hot flashes [ 15], and their mood was equally as positive as those who received the combined E/A drug [28]. These findings suggested that testosterone enhanced the cognitive, motivational aspects of sexuality in women such as sexual desire and interest and that it did so directly and not secondary to its enhancement of other aspects of physical and/or psychological functioning. These results were confirmed in a longitudinal study of women who had undergone TAH and BSO 4 years earlier and had been maintained for at least 2 years on either the combined E/A depot preparation or on the same dose of estrogen alone administered intramuscularly [29]. A third group had undergone TAH and BSO and remained untreated. Levels of sexual desire and interest were greatest in the women treated with E/A preparation and covaried with their plasma testosterone level throughout the treatment month as the intramuscular drug was being metabolized. The androgenic enhancement of sexual motivation in women treated with the combined intramuscular drug has been shown to persist with long-term chronic administration of monthly injections that cause an initial surge in estradiol and testosterone levels and metabolize slowly over a period of several weeks [30]. Twenty women who were dissatisfied with their estrogen or estrogen/progestin therapy received a placebo for 2 weeks and then were randomized to either 1.25 mg esterified estrogens combined with 2.5 mg methyltestosterone or to 1.25 mg esterified estrogen alone for 8 weeks [31]. Women who received the combined E/A drug reported improved sexual sensation and sexual desire compared both to their previous estrogen therapy and to the postplacebo baseline assessments, whereas no changes in sexual functioning occured in the women treated with estrogen alone. Taken together, findings from the subcutaneous implant pellet studies and from the prospective studies on surgically menopausal women given an intramuscular E/A preparation

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provide compelling evidence that E/A replacement therapy acts to increase overall energy level over and above estrogen alone and to enhance sexual desire and sexual arousal in postmenopausal women. Frequency of sexual activity was less affected by combined E/A therapy perhaps because its determinants are more complex and involve couple issues. Rather, these findings strongly suggest that, in women, just as in men [32] testosterone has its major impact on the cognitive motivational, or libidinal, aspects of sexual behavior such as desire and fantasies. Moreover, studies on nonhuman primates suggest the likelihood that testosterone exerts this effect on sexual desire via mechanisms that impact directly on the brain rather than by an effect on peripheral tissues [ 16]. Finally, the results of these studies also allow the observation that although estrogen is important for the integrity of vaginal tissues, it does not maintain sexual desire and interest in postmenopausal women.

C. B o n e M e t a b o l i s m Androgen-specific receptors have been demonstrated in osteoblastic cells in women [33] and androgens directly stimulate bone cell proliferation [34]. In a cross-sectional study of hormone levels and bone mineral density (BMD) in premenopausal women, a significant positive correlation was evident between the percentage of free testosterone and BMD, but not between BMD and free estradiol after controlling for body wieght [35]. There is some evidence that testosterone has anabolic effects on protein metabolism. When women receiving 0.625 mg esterified estrogen/day were compared to those receiving the same dose of estrogen plus 1.25 mg/day methyltestosterone (E/A), urinary excretion markers of bone resorption decreased equally in both groups [35]. Whereas the women treated with estrogen alone had a reduction in serum markers of bone formation, those who received the combined E/A drug had an increase in their serum markers of bone formation. There are very few prospective randomized trials of effects of E/A preparations on B MD. In one study, women either received a subcutaneous implant of 50 mg of estradiol or an implant containing the same amount of estradiol plus 100 mg of testosterone [36]. After 3 years, there was no change in BMD in the estrogen-alone group but a 2.5 % increase in B MD occurred in the combined E/A implant group. Unfortunately, in this study, only the metacarpal bone density, which is not at risk for osteoporotic fracture, was measured. In a prospective, randomized study, women received either 1.25 mg esterified estrogen/day or the same amount of estrogen plus 2.5 mg methyltestosterone/day for 2 years [37]. Both treatment regimens prevented bone loss at the spine and hip. However, only the combined E/A drug was associated with a significant increase in spinal bone mineral

density compared with pretreatment baseline. In summary, these findings indicate that whereas exogenous estrogen prevents bone loss in postmenopausal women, the addition of testosterone to an estrogen replacement regimen actually increases bone density.

IV. RISKS OF E/A REPLACEMENT

THERAPY

A. Effects o n L i p o p r o t e i n L i p i d s A meta-analysis of observational studies demonstrated a 50% reduction of heart disease risk in postmenopausal women taking estrogen [38]. It has been estimated that approximately 50% of the cardiovascular risk reduction provided by exogenous estrogen is mediated by lipoprotein changes [39]. Although the coadministration of a progestin, either continuously or cyclicly, attenuates the beneficial effect of estrogen on high-density lipoprotein (HDL) cholesterol, the lipid profile in hormone-treated women is preferable to that in placebo-treated women [40]. The addition of testosterone to an estrogen replacement therapy regimen may partially offset the beneficial effects of estrogen on risk factors for cardiovascular disease. Oral estrogen/androgen replacement reduced both total cholesterol and low-density lipoproteins (LDLs) more than oral estrogen alone [37,41]. However, the addition of testosterone to the regimen also decreased HDL levels relative to estrogen alone, thereby resulting in a detrimental increase in the ratio of HDL/total cholesterol [37,41 ]. However, combined preparations also lower triglyceride levels compared to those with estrogen alone, which is thought to be beneficial. In a prospective, controlled study, 291 surgically menopausal women received either 0.625 or 1.25 mg conjugated equine estrogen (CEE) or 0.625 or 1.25 mg esterified estrogen (EE) combined with 1.25 or 2.5 mg methyltestosterone, respectively [42]. At 6 and 12 months, decreases in triglycerides and HDL cholesterol were observed for both doses of estrogen/androgen, whereas increases were observed in women who were given CEE. Total cholesterol and LDL decreased in all treatment groups by 6 months. Furthermore, no adverse effects on serum creatinine or liver function tests were observed in any treatment group. Studies of nonoral routes of administration of estrogen/ androgen drugs tell a somewhat different story. Subcutaneous implantation of estradiol (40 mg) and testosterone (100 mg) did not cause any changes in cholesterol, triglycerides, or HDL cholesterol from pretreatment levels [43]. Farish and colleagues [44] likewise found that subcutaneous pellets of estradiol (50 mg) and testosterone (100 mg) had no effect on HDL fractions, but testosterone appeared to enhance slightly the LDL cholesterol-lowering effect of estradiol.

CHAPTER43 Estrogen/Androgen Replacement Therapy Following 2 years of treatment with an intramuscular combined estrogen/androgen depot preparation, Sherwin and associates [45] reported that combined replacement therapy did not adversely affect the lipoprotein cholesterol profile in these women compared with patients treated with parenteral estrogen alone and surgically menopausal women who were untreated. Other evidence suggests that the route of administration modulates the response to hormone therapy. For example, percutaneous and vaginal administration of estradiol do not cause the increases in triglycerides and very lowdensity lipoprotein (VLDL) observed during oral therapy [46]. Thus, it seems likely that parental routes of administration of combined estrogen/androgen drugs in the postmenopause may not cause the detrimental effect on HDL seen with oral preparations, as parenteral routes of administration bypass the so-called hepatic first-pass effect. Based on the evidence available to date, it would seem that, compared to exogenous estrogen administered alone, oral estrogen/androgen preparations cause a decrease in HDL cholesterol levels and in triglyceride levels. On the other hand, parenteral routes of estrogen/androgen administration seem not to change lipid and lipoprotein levels compared to effects of parenterally administered estrogen only. However, it must also be acknowledged that estrogens administered via nonoral routes have a lesser beneficial effect on lipid metabolism compared to oral preparations. It also now seems clear that non-lipoprotein-mediated effects of estrogen provide cardioprotection. Examples of such mechanisms are reduced atherosclerotic plaque formation unrelated to HDL cholesterol levels [47], and estrogen-induced vasodilatation [48]. In this regard, it is both interesting and potentially important that treatment of ovariectomized cynomolgus monkeys with oral estrogens improved endothelium-mediated vasodilatation of their atherosclerotic coronary arteries, and the addition of oral methyltestosterone did not alter this response [49]. Moreover, administration of an oral combined estrogen/androgen preparation caused an increase in vaginal blood flow measured with laser doppler velocimetry equal to that which occurred with estrogen alone [50]. This suggests that the addition of androgen to an estrogen replacement regimen does not compromise peripheral blood flow. Moreover, prospective clinical trials that measured blood pressure found no changes in either systolic or diastolic blood pressure in women treated with a combined estrogen/androgen drug compared to pretreatment baseline and compared to women given estrogen only [37,42,44,48-50].

B.

Symptoms of Virilization

There is scanty empirical data from controlled studies on the incidence of hirsutism in postmenopausal women treated with combined estrogen/androgen preparations. A single ex-

621 ception is a recent study that assessed hair growth on the upper lip, chin, and sideburn area using a modified Ferriman-Gallwey scale [42]. The incidence of facial hirsutism was 3% among CEE recipients and 6% for women who had received estrogen and androgen. Interestingly, in this study, which had four treatment groups of different doses of CEE (0.625 and 1.25 mg) and of a combined drug (0.625 and 1.25 mg EE plus methyltestosterone (MT) 1.25 and 2.5 mg, respectively), all the cases of hirsutism (mild or moderate severity) occurred in women taking the high dose of either CEE or of the combined E/A drug. In that study, acne was also reported by 3 % of the women taking the combined E/A drug. Indeed, safety surveillance data on Estratest and Estratest HS (Solvay Pharmaceuticals, Marietta, Georgia)indicate that, of all adverse events reported between 1989 and 1996, the most commonly reported were alopecia (11.1%), acne (5.8%), and hirsutism (4.5%) [51]. Surprisingly, the percentage of adverse events characterized as virilization was similar for both the higher dose (1.25 mg EE + 2.5 mg MT) and the lower dose (0.625 mg EE + 1.25 mg MT) combined drugs. In contrast, our own clinical experience with an intramuscular combined E/A drug suggests that approximately 20% of women who receive 150 mg testosterone enanthate intramuscularly every 4 weeks along with estrogen will develop mild hirsutism manifested by an increased growth of hair on the chin and/or upper lip. When the dose is reduced to 75 mg testosterone enanthate per month, less than 5% of women have increased hair growth. Moreover, hair growth decreases or usually stops entirely when the patient is switched to treatment with estrogen alone. There is good reason to believe that, in women, hirsutism is a dosedependent side effect of exogenous testosterone. Its development would depend also on the amount of estrogen given in combination, because both sex steroids influence the production of sex hormone-binding globulin (SHBG), which, in turn, determines the concentration of free, or biologically available, testosterone. It is also noteworthy that voice alteration constituted only 0.5% of all adverse events reported with Estratest. Neither has deepening of the voice occurred with the doses of the injectible E/A preparation used in our own studies.

C. Effects on E n d o m e t r i a l H i s t o l o g y Of course, the postmenopausal uterus must be protected from excessive estrogenic stimulation during estrogen replacement therapy. The relevant question, therefore, is whether the addition of androgen to an estrogen replacement regimen should be coadministered with more or less progestin than would be used to prevent endometrial hyperplasia with estrogen alone. In a study that compared the effect of oral estrogen alone with an oral combined estrogen/androgen preparation on endometrial histology, similar

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changes in estrogen-stimulated proliferative growth occurred in both groups after 6 months of treatment [35]. The majority of women developed a moderately proliferative endometrial histology pattern and no woman displayed hyperplastic changes. Others have found endometrial hyperplasia both in women treated with estrogen alone and in those given a combined preparation [52]. It would seem, therefore, that the addition of testosterone neither facilitates nor antagonizes the stimulatory effect of estrogens on the endometrium. This suggests that a progestational agent should be added to the hormone regimen when a combined estrogen/ androgen preparation is used.

V. F U T U R E

DIRECTIONS

Although controlled studies on the clinical efficacy of combined estrogen/androgen preparations in postmenopausal women are relatively few in number, the consistency of their findings makes them compelling. All of them demonstrate that the addition of testosterone to an estrogen replacement regimen increases the cognitive aspects of sexuality such as desire and interest, and also heightens energy level and well-being over and above treatment with estrogen alone. These findings support the notion that in women, just as in men, testosterone is important for the maintenance of libido, or sexual desire. Because human sexuality is complex and multidetermined, this statement assumes that physiological levels of testosterone are one determinant of sexual desire in premenopausal women and deficits in behavior may occur sometime after the menopause when the production of testosterone decreases. This is predicated on the findings that exogenous testosterone increased circulating levels of the hormone and restored sexual desire in several studies in both naturally and surgically menopausal women. At the present time, there are no data available to address whether the administration of testosterone to premenopausal women complaining of decreased sexual desire and who have normal ovarian and adrenal function would be efficacious. Although such studies have never been done, it is likely that the sexual dysfunction in healthy premenopausal women would not be hormonally related and testosterone treatment geared toward inducing supraphysiological levels would be unwise. Rather, the available literature supports the opinion that the treatment of postmenopausal women with combined estrogen/ androgen drugs should strive to reinstate physiological levels of both sex hormones when clinically indicated. If it is true that testosterone maintains sexual desire in women, as research findings indicate, then it would be expected that postmenopausal women might experience an even greater loss of libido when treated with estrogen alone. This is because estrogens increase SHBG whereas androgens reduce it, with the consequence being a reduction in bioavailable androgens. When normally postmenopausal women

were given 2 mg/day of oral micronized estradiol, testosterone levels decreased by 42%, dehydroepiandrosterone sulfate (DHEAS) levels fell by 23%, and dehydroepiandrosterone (DHEA) levels decreased by 11% [53]. Free testosterone was reduced even more profoundly because SHBG levels increased 160% following treatment with micronized estradiol. Therefore, the decrease in testosterone production by the ovarian stoma during perimenopause causes a deficiency in testosterone that is compounded in women given exogenous estrogen, which reduces the bioavailability of the already diminished pool of androgens. Unfortunately, most of the studies on combined estrogen/androgen preparations failed to measure plasma levels of hormones or of SHBG, and so it is not known what the optimal dose of each, given in combination, should be in order to maintain SHBG levels within the physiological range. Route of administration of combined estrogen/androgen preparations is also an important consideration in view of the lack of effect of parenteral preparations on the lipid profile compared to oral combined preparations. Ideally, of course, one would want to maintain the beneficial effects of estrogen on HDL while attenuating any HDL-lowering effects of the testosterone. Virilizing side effects of combined estrogen/androgen therapy in the postmenopause occur in some proportion of women. Unfortunately, the occurrence of hirsutism, acne, or alopecia with combined preparations has rarely been studied systematically. Therefore, it is not known what absolute doses of estrogen and testosterone or, perhaps more importantly, what dose ratio of these two hormones would have the least probability of inducing undesirable androgenic effects. Intuitively, a dose of testosterone that induces physiological (and not supraphysiological) levels of circulating testosterone in postmenopausal women would likely minimize the risk of virilizing side effects. Maintaining SHBG levels within the normal female range by balancing the dose of each hormone might also be important in this regard. Finally, it has been suggested that ethnic differences might play a role in determining response to combined estrogen/ androgen therapies. Conventional wisdom holds that women of Mediterranean origin who are dark-haired and darkskinned may be more likely to experience hirsutism compared to light-haired, light-skinned women of Northern European origin following treatment with estrogen/androgen drugs. Once again, this possibility has not been studied systematically and remains within the realm of clinical report. Clinical experience with combined estrogen/androgen drugs has provided some guidelines for identifying the subset of postmenopausal women who might benefit from this therapy. As has already been mentioned, sexual function is multidetermined and it is quite likely that sexual desire and interest may be diminished during the perimenopause secondary to other symptoms that frequently occur at that time such as hot flashes, sleep disturbance, and atrophic vaginitis.

CHAPTER 43 Estrogen/Androgen Replacement Therapy

B e c a u s e e s t r o g e n controls hot flashes and restores the integrity o f the vaginal tissues, e s t r o g e n r e p l a c e m e n t t h e r a p y s h o u l d be first-line t r e a t m e n t for w o m e n c o m p l a i n i n g o f sexual d y s f u n c t i o n s at the time o f m e n o p a u s e . S h o u l d the c o m p l a i n t persist after other s y m p t o m s h a v e abated f o l l o w ing t r e a t m e n t with e s t r o g e n alone, then t r e a t m e n t with a c o m b i n e d p r e p a r a t i o n s h o u l d be attempted. A w o m a n ' s prem e n o p a u s a l history is also relevant in treatment. W o m e n w h o report that a d e c r e a s e in sexual desire c o i n c i d e d in t i m e with the h o r m o n a l c h a n g e s that c h a r a c t e r i z e the m e n o p a u s e are m o r e likely to r e s p o n d s u c c e s s f u l l y to a c o m b i n e d drug. In contrast, w o m e n w h o r e p o r t a l o w level o f sexual desire or interest that is lifelong are less likely to r e s p o n d c o m p l e t e l y to a h o r m o n a l i n t e r v e n t i o n alone and m a y require marital or c o u p l e t h e r a p y in addition to h o r m o n e treatm e n t to r e s o l v e the p r o b l e m . Finally, s o m e p o s t m e n o p a u s a l w o m e n w h o are b e i n g treated with e s t r o g e n c o m p l a i n o f severe fatigue that i m p e d e s t h e m f r o m c a r r y i n g out their norm a l activities. T h e s e w o m e n m a y also benefit f r o m c o m b i n e d e s t r o g e n / a n d r o g e n t h e r a p y a l t h o u g h they n e e d to be i n f o r m e d that the t r e a t m e n t m a y also stimulate sexual feelings that m a y not be w e l c o m e d by some. T h e r e is g r o w i n g a c c e p t a n c e o f the fact that c o m b i n e d e s t r o g e n / a n d r o g e n drugs are a useful addition to the a r m a m e n t a r i u m o f t r e a t m e n t options for p o s t m e n o p a u s a l w o m e n . C o m b i n e d p r e p a r a t i o n s h a v e a s u p e r i o r efficacy c o m p a r e d to e s t r o g e n alone on i n c r e a s i n g b o n e density, on the alleviation o f fatigue, and in r e s t o r i n g sexual desire and interest in postm e n o p a u s a l w o m e n . H o w e v e r , m u c h w o r k r e m a i n s to be d o n e to d e t e r m i n e the safest and m o s t effective doses and r o u t e o f a d m i n i s t r a t i o n o f such p r e p a r a t i o n s as well as their p o s s i b l e l o n g - t e r m effects.

References 1. Greenblatt, R. B. (1942). Androgenic therapy in women. J. Clin. Endocrinol. 2, 665-666. 2. Shorr, E., Papanicolaou, G. N., and Stimmel, B. F. (1938). Androgens in postmenopausal women. Proc. Soc. Exp. Biol. Med. 38, 759-768. 3. Carter, A. C., Cohen, E. J., and Shorr, E. (1947). The use of androgens in women. Vitam. Horm. (N. Y.) 5, 317-391. 4. Groome, J. R. (1939). Androgens in women. Lancet 2, 722-724. 5. Silberman, D., Radman, H. M., and Abarnel, A. R. (1940). Hormone therapy for menopause. Am. J. Obstet. Gynecol. 39, 332-338. 6. Greenblatt, R. B, Mortara, F., and Torpin, R. (1942). Sexual libido in the female. Am. J. Obstet. Gynecol. 44, 658-663. 7. Kupperman, H. S, and Studdiford, W. E. (1953). Endocrine therapy in gynecologic disorders. Postgrad. Med. 14, 410- 425. 8. Greenblatt, R. B, Barfield, W. E, Garner, J. F, Calk, G. L, and Harrod, J. P. (1950). Evaluation of an estrogen, androgen, estrogen-androgen combination and a placebo in the treatment of the menopause. J. Clin. Endocrinol. 10, 1547-1558. 9. Caldwell, B. M, and Watson, R. (1952). An evaluation of psychologic effects of sex hormone administration in aged women. J. Gerontol. 7, 228-244. 10. Foss, G. L. (1951). The influence of androgens on sexuality in women. Lancet 1, 667-669.

623 11. Kennedy, B. J. (1973). Effects of massive doses of sex hormones on libido. Med. Aspects Hum. Sex 7, 67-75. 12. Waxenberg, S. E, Drellich, M. G, and Sutherland, A. M. (1959). Changes in female sexuality after adrenalectomy. J. Clin. Endocrinol. 19, 193-202. 13. Drellich, M. G, and Waxenberg, S. E. (1966). Erotic and affectional components of female sexuality. In "Science of Psychoanalysis" (J. Masserman, ed.), pp. 45-55. Grune & Stratton, New York. 14. Bhasin, S., Storer, T., Strakova, J., Phillips, J., Phillips, C., Berman, N., Bunnell, T., and Casaburi, R. (1994). Testosterone increases lean body mass, muscle size and strength in hypogonadal men. Clin. Res. 42, 74A. 15. Sherwin, B. B, and Gelfand, M. M. (1985). Differential symptom response to parenteral estrogen and/or androgen administration in the surgical menopause. Am. J. Obstet. Gynecol. 151, 153-160. 16. Everitt, B. J., and Herbert, J. (1975). The effects of implanting testosterone propionate in the central nervous system on the sexual behavior of the female rhesus monkey. Brain Res. 86, 109-120. 17. Leiblum, S., Bachmann, G., Kemmann, E., and Colburn, D. (1983). Vaginal atrophy in the postmenopausal woman: The importance of sexual activity and hormones. JAMA, J. Am. Med. Assoc. 249, 21952198. 18. McCoy, N., and Davidson, J. (1985). A longitudinal study of the effects of menopause on sexuality. Maturitas 7, 203-210. 19. F16ter, A., Nathorst-B66s, J., Carlstr6m, K., and von Schoultz, B. (1997). Androgen status and sexual life in perimenopausal women. Menopause 4, 95- 100. 20. Dennerstein, L., Smith, A. M. S., and Morse, C. A. (1994). Sexuality and the menopause. J. Psychosom. Obstet. Gynaecol. 15, 59-66. 21. Hallstrom, T. (1977). Sexuality in the climacteric. Clin. Obstet. Gynecol. 4, 227-239. 22. Dennerstein, L., Dudley, E. C., Hopper, J. L., and Burger, H. (1997). Sexuality, hormones and the menopausal transition. Maturitas 26, 83-93. 23. Longcope, C. (1981). Metabolic clearance and blood production rates of estrogen in postmenopausal women. Am. J. Obstet. Gynecol. 111, 779-785. 24. Burger, H. G, Hailes, J., Manelaus, M., Nelson, J., Hudson, B., and Balazs, N. (1984). The management of persistent menopausal symptoms with oestradiol testosterone implants: Clinical, lipid and hormonal results. Maturitas 6, 351-358. 25. Burger, H. G., Hailles, J., Nelson, J., and Menelaus, M. (1987). Effects of combined implants of estradiol and testosterone on libido in postmenopausal women. Lancet 294, 936-937. 26. Davis, S. R., McClaud, P., Strauss, B. J. G., and Burger, H. (1995). Testosterone enhances estradiol's effects on postmenopausal bone density and sexuality. Maturitas 21, 227-236. 27. Sherwin, B. B., Gelfand, M. M., and Brender, W. (1985). Androgen enhances sexual motivation in females: A prospective cross-over study of sex steroid administration in the surgical menopause. Psychosom. Med. 7, 339-351. 28. Sherwin, B. B. (1988). Affective changes with estrogen and androgen replacement therapy in surgically menopausal women. J. Affective Disord. 14, 177-187. 29. Sherwin, B. B., and Gelfand, M. M. (1987). The role of androgen in the maintenance of sexual functioning in oophorectomized women. Psychosom. Med. 49, 397-409. 30. Sherwin, B. B. (1985). Changes in sexual behavior as a function of plasma sex steroid levels in postmenopausal women. Maturitas 7, 225-233. 31. Sarrel, P., Dobay, B., and Wiita, B. (1998). Estrogen and estrogenandrogen replacement in postmenopausal women dissatisfied with estrogen-only therapy. J. Reprod. Med. 43, 129-134. 32. Bancroft, J., and Wu, F. C. W. (1983). Changes in erectile responsiveness during androgen replacement therapy. Arch. Sex. Behav. 12, 59-66.

624 33. Colvard, D. S., and Eriksen, E. F., Keeting, E E. Wilson, E. M., Lubahn, D. B., French, F. S., Riggs, B. L., and Spelsberg, T. C. (1989). Identification of androgen receptors in normal human osteoblast-like cells. Proc. Natl. Acad. Sci. U.S.A. 86, 854-857. 34. Kasperk, C. H., Wergedal, J. E., Farley, J. R., Linkhart, T. A., Turner, R. T., and Baylink, D. G. (1989). androgens directly stimulate proliferation of bone cells in vitro. Endocrinology (Baltimore) 124, 15761578. 35. Raisz, L. G., Wiita, B., and Arctic, A. (1996). Comparison of estrogen alone and estrogen plus androgen on biochemical markers of bone formation and resorption in postmenopausal women. J. Clin. Endocrinol. Metab. 81, 37-43. 36. Barlow, D. H., Abdalla, H. I., Roberts AD. (1986). Long-term hormone therapy: Hormonal and clinical effects. Obstet. Gynecol. 67, 321325. 37. Watts, N. B., Notelovitz, M., Timmons, M. C., Addison, W. A., Wiita, B., and Downey, L. J. (1995). Comparison of oral estrogens and estrogens plus androgen on bone mineral density, menopausal symptoms, and lipid-lipoprotein profiles in surgical menopausal women. Obstet. Gynecol. 85, 529-537. 38. Grady, D., Rubin, S. M., Petitti, D. B., Fox, C. S., Black, D., Ettinger, B., Ernster, V. L., and Cummings, S. R. (1992). Hormone therapy to prevent disease and prolong life in postmenopausal women. Ann. Intern. Med. 117, 1016-1037. 39. Bush, T. L., Barrett-Connor, E., Cowan, L. D., Criqui, H. H., Wallace, R. B., Suchindran, C. M., Tyroler, H. A., and Rifkind, B. M. (1987). Cardiovascular mortality and noncontraceptive use of estrogen in women: Results from the Lipid Research Clinics Program Follow-up Study. Circulation 75, 1102-1109. 40. Writing Group for the PEPI Trial (1995). Effects of estrogen or estrogen/progestin regimens on heart disease risk factors in postmenopausal women. JAMA, J. Am. Med. Assoc. 273, 199-208. 41. Hickok, L. R., Toomey, C., eroff, L. (1993). A comparison of esterified estrogens with and without methyltestosterone: Effects on endometrial histology and serum lipoproteins in postmenopausal women. Obstet. Gynecol. 82, 919-924. 42. Barrett-Connor, E., Timmons, C., Young, R., and Wiita, B. (1996). Interim safety analysis of a two-year study comparing oral estrogen-

BARBARA B. SHERWIN

43.

44.

45.

46.

47.

48. 49.

50.

51.

52.

53.

androgen and conjugated estrogens in surgically menopausal women. J. Women's Health 5, 593-602. Teran, A. Z., and Gambrell, R. D., Jr. (1988). Androgens in clinical practices. In "Androgens in the Menopause" (L. Speroff, ed.), pp. 1422. McGraw-Hill, New York. Farish, E., Fletcher, C. D., Hart, D. M. Azzawi, E. A., Abdalla, H. I., and Gray, C. E. (1984) The effect of hormone implants on serum lipoproteins and steroid hormones in bilaterally oophorectomized women. Acta Endocrinol. (Copenhagen) 106,116-123. Sherwin, B. B., Gelfand, M. M., Schucher, R., and Grabor, J. (1987). Postmenopausal estrogen and androgen replacement and lipoprotein lipid concentrations. Am. J. Obstet. Gynecol. 156, 414-419. Mischell, D. R., Jr., Moore, R. E., Roy, S., Brenner, C. F., and Cage, M. A. (1978). Clinical performance and endocrine profiles with contraceptive vaginal rings containing a combination of estradiol and dnorgestrel. Am. J. Obstet. Gynecol. 130,155-161. Adams, M. R., Clarkson, T. B., Koritnik, D. R., and Nash, H. A. (1987). Contraceptive steroids and coronary artery atherosclerosis of cynomolgus macaques. Fertil. Steril. 47, 1010-1018. Sarrel, P. (1990). Ovarian hormones and the circulation. Maturitas 590, 287-298. Honor6, E. H., Williams, J. K., Adams, M. R., Ackerman, D. M., and Wagner, J. D. (1996). Methyltestosterone does not diminish the beneficial effects of estrogen replacement therapy on coronary artery reactivity in cynomolgus monkeys. Menopause 3, 20-36. Sarrel, P. (1998). Ovarian hormones and vaginal blood flow: Using laser Doppler velocimetry to measure effects in a clinical trial of postmenopausal women. Int. J. Impotence Res. 10, $91-$93. Phillips, E., and Bauman, C., (1997). Safety surveillance of esterified estrogens - methyltestosterone (Estratest-Estratest HS ) replacement therapy in the United States. Clin. Ther. 19, 1070-1084. Gelfand, M. M., Ferenczy, A., and Bergeron, C. (1989). Endometrial response to estrogen-androgen stimulation. In "Menopause Evaluation Treatment and Health Concerns" (C. B. Hammond, F. B. Haseltine, and I. Seniff, eds.), pp. 29-40. Alan R. Liss, New York. Casson, P. R., Elkind-Hirsh, K. E., Buster, J. E., Hornsby, E J., Carson, S. A., and Snables, M. C, (1997). Effect of postmenopausal estrogen replacement on circulating androgens. Obstet. Gynecol. 90, 995-998.

~HAPTER 4 ~

DHEA: Biology and Use Therapeutic Intervention JOHN E.

BUSTER AND P E T E R R. CASSON 9

IV. DHEA Replacement V. Conclusions References

I. Introduction

II. Androgens: Origin and Control III. DHEA, DHEAS, and Androgen Metabolites: Increase during Childhood and Decline with Age

I. I N T R O D U C T I O N

elderly individuals could retard maladies of age, including cardiovascular disease, neoplasia, diabetes, immunosenescence, osteoporosis, muscular wasting, decreased libidinal interest, and depression [6-8]. Despite burgeoning interest in this field, it is not clear when or how to administer DHEA to menopausal women. Oral DHEA is nontheless widely consumed in the United States as a food supplement. This chapter reviews work describing the origin and regulation of DHEA and DHEAS, the fate of their downstream metabolites, and the impact of declining androgen production with age. It further examines conditions that accelerate age-related decline of DHEA and DHEA sulfate production and examines the case for replacing DHEA in elderly women.

Dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS) are 19-carbon steroids originating principally from the zona reticularis of the human adrenal cortex. They circulate in abundance and have production rates far higher than any other circulating steroid. Ubiquitous tissue sulfatases rapidly interconvert DHEAS to DHEA, which has a higher clearance rate and shorter halflife than DHEAS. DHEA has virtually no androgenic activity of its own but is converted intracellularly to bioactive androgens and estrogens. Measurement of circulating DHEAS, a stable clinical marker of adrenal androgen secretion, provides a rough index of the circulating pool of available prohormone [1-3]. Circulating DHEAS concentrations and production decline with advancing age. In the elderly, DHEAS concentrations and production are about 10% of reproductive age peaks. This decline, sometimes called "adrenopause," occurs concurrently with involution of the zona reticularis of the adrenal cortex [1-5]. Adrenopause represents a senescent endocrine deficiency similar to menopause. If it is truly a deficiency state, restitution of adrenal androgen levels in

MENOPAUSE: BIOLOGY AND PATHOBIOLOGY

Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston, Texas 77030

II. ANDROGENS: ORIGIN AND C O N T R O L The literature traditionally identifies five androgens (Table I) as clinically important: DHEAS, DHEA, androstenedione ( A 4 A ) , testosterone (T), and dihydrotestosterone (DHT). In women, these androgens have widely differing

625

Copyright 9 2000 by Academic Press. All rights of reproduction in any form reserved.

626

BUSTER AND CASSON

Anter,oPitu,taryr 1Lt

TABLE I Androgen Concentrations and Relative Biological Potency in Reproductive-Age Women Using Testosterone as a Standard of 1.0 a

Androgen

Relative potency bioas say

Dihydrotestosterone Testostrone Androstenedione DHEA DHEAS

5 1 0.1 0.01 0.001

DHEA-S

>90~

/ 7.r A

+ACTH+c~176~ AnteriOrPituitary ~ 9 DHEA-S

, Adrenal Gland

serum concentrations, production rates, potencies, and origins (Table I). Although the basic 19-carbon (C~9) androstane nuclear structure is held in common (Fig. 1), effects on target tissues differ. As examples, DHEAS is associated with immunomodulation, enhancement of insulin effect, and osteoporosis protection, whereas libidinal drive, sex hair development, seborrhea, and acne are more associated with T [6-10]. Interconversions between DHEAS, T, and other androgens blur these distinctions [9-12]. The sources, regulation, and interconversion of these five androgens, as influenced by aging and menopausal status, are depicted in Figs. 2 and 3. They are described in detail in the following discussions.

A. Dehydroepiandrosterone and Dehydroepiandrosterone Sulfate DHEA and DHEAS, collectively designated DHEA(S), are prohormones without known receptors or specific target tissues. DHEAS and DHEA are secreted daily in milligram amounts by the zona reticularis (ZR) of the human adrenal cortex [11]. The ZR, the sole secretory source of DHEAS, is unique to humans and higher primates [4,13]. At 8 to

18 16 14

2 4

15

6

FIGURE 1 Androstane structural nucleus (19 carbons) common to the five clinically important androgens.

~ LH~

)-Estradi

a Adapted from Casson and Carson [6].

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Ovary

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Representative concentration ng/dl ng/dl ng/dl ng/dl ng/dl

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CDIHYDROTESTOSTERONE)

FIGURE 2 Androgen dynamics in (A) premenopausal and (B) postmenopausal women. Menopausal levels of luteinizing hormone (LH) drive ovarian stroma to produce increased testosterone, compensating in part for the age-related loss of adrenal androgens and androgen precursors.

16 mg/day (much less after menopause), DHEAS production exceeds that of all other steroids [11,12]. With circulating concentrations of 100- to 1000-fold greater than any other androgen, DHEAS circulates in a pool with very slow turnover. Unconjugated DHEA, also originating from the ZR, and in addition secreted by ovarian stroma (DHEAS is not), is converted peripherally to DHEAS. Unconjugated DHEA is a metabolic intermediate to and from DHEAS to A4A, T, and DHT (Fig. 3). Its production rate is 6 - 8 mg/day in reproductive years, but because it has a considerably higher metabolic clearance rate than DHEAS, its pool turns more quickly in circulating concentrations that are much lower than those of DHEAS [ 11,12]. From circulating DHEAS, bioactive androgens and estrogens are synthesized within the cells of their site of a c t i o n ~ the target tissues [9,14-16]. DHEAS is extracted from the circulation and transferred into target cells as DHEAS (Fig. 4). Once in the cell, DHEAS is converted to DHEA by steroid sulfatase, to A4A by intracellular 3fl-hydroxysteroid dehydrogenase (3/3 HSD), and then to T by 17fl-hydroxysteroid dehydrogenase (17/3 HSD) (Figs. 3 and 4). Testosterone thus formed is acted on by 5ce-reductase to produce DHT, or alternatively by aromatase to produce 17fl-estradiol (E 2) m depending on local biological requirements and steroidogenic architecture (Figs. 3 and 4) [9,14-16]. It thus appears in both women and men (more in women) that much of the biologically significant androgen production originates from circulating DHEAS with interconversion and then action occurring in target tissues. In breast tissue, for

CHAPTER 44 DHEA: Biology and Therapeutic Use

627

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o

Androstenedlone

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Dlhydrotestosterone

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FIGURE 3 Interconversionsof five clinically important androgens from circulating dehydroepiandrosteronesulfate: androstenedione, testosterone, and dihydrotestosteroneare formed within target tissues from circulating DHEAS. Estrone and estradiol and are also formed within target tissues from their corresponding A4 androgens, androstenedione and testosterone.

example, approximately 75% of estrogen in premenopausal women originates from circulating DHEAS. After menopause, virtually 100% of breast tissue estrogen comes from this source [14-16]. In men, as another example, over half of prostatic T arises from circulating DHEA [14,17,18] (Fig. 4). Male castration, which drops circulating T precipiADRENAL CORTEX HEA~ DS-ST <- D DHEA-S ~_~SULFATASE -> BLOOD

END ORGAN

t

DHEA-S

-

DHEA

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FIGURE 4 Intracrine activity of human target tissues. These are the known biosynthetic steps involved in the formation of androgens (A) from circulating DHEAS and estrogensin human target tissue. 17OH-HSD, 17/3hydroxysteroid dehydrogenase;3/3-HD, dehydrogenase/A4,5-isomerase(R) steroid receptor. Modified from Labrie [14], Mol. Cell. Endocrinol., Vol. 78, F. Labrie, Intracrinology, pp. Cl13-Cl19. Copyright 1991, with permission from Elsevier Science.

tously, reduces prostatic DHT only by 5 0 - 6 0 % . In men, therefore, DHEAS, originating exclusively from the ZR, continues to circulate in abundance even after castration and therefore continues to exert a significant androgen effect within the prostate [ 14,17,18]. Even steroid-secreting organs utilize circulating DHEAS as a prohormone. The ovary, for example, extracts and converts circulating DHEAS in granulosa cells of maturing follicles [ 19,20]. Extracted DHEAS is converted to intrafollicular T, which modulates oocyte maturation [ 19,20]. DHEAS secretion, as mentioned previously, originates exclusively from the ZR [4,13]. DHEAS secretion is regulated by adrenocorticotropic hormone (ACTH) and several coregulatory factors that include prolactin, insulin-like growth factor (IGF-I), estrogen, and the ZR cell mass itself [21-27]. The ZR is one of three adult zones of the adrenal: the outer zona glomerulosa (ZG), the middle zona fasciculata (ZF), and the inner zona reticularis (ZR) (Fig. 5) [4,27]. The ZR becomes histologically distinct during childhood and assumes an increasing percentage of adrenal mass during the years surrounding adrenarche [28,29] (Fig. 6). Steroidogenic architecture within zones is determined by gene expression unique to the cells of that zone (Fig. 7). Thus, ZR cells in

628

BUSTER AND CASSON

izing hormone (LH), continues to secrete DHEA in significant amounts [5,11,12,30-32]. In aging women, DHEAS production and its circulating concentrations decline dramatically [1-4]. This decline is associated with fragmentation of the ZR in a cellular process closely resembling apoptosis [33]. This apoptotic process and the ZR atrophy that results are believed to be the principal mechanism for age-related decline in adrenal androgens (Fig. 8). The corpus luteum-like ZR may thus be seen in nature as a "temporary tissue" [4].

B. Androstenedione: The m4 Analog of DHEA

FIGURE 5 Histologic section of the adult adrenal cortex. The adrenal cortex is divided into three zones: the outer zona glomerulosa (ZG), the middle zona fasciculata (ZF), and the innermost zona reticularis (ZR). Steroidogenic enzyme architecture unique to each zone directs the profile of steroid products secreted by each. Adapted from Bargmann, [28].

culture have very low expression of 313 HSD. Correspondingly, 3/3 HSD is easily detected in ZF cells, which produce m4 steroids in abundance: A n A and cortisol, not DHEA. Thus, the ZR embodies a morphologically distinctive cell type that is genetically programmed with steroidogenic machinery devoted solely to production of A5 androgens: DHEAS and DHEA [4,27] (Figs. 5-7). DHEA, not DHEAS, is also secreted by the premenopausal ovary in modest amounts [5,11,12]. The postmenopausal ovarian stroma, under the influence of high levels of undulating pituitary lutein-

C. Testosterone: A Bioactive Metabolite of DHEA

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Androstenedione ( A 4 A ) , the A4 analog of DHEA, is also a circulating prohormone [9,11,14]. It has no specific receptors or target tissue activity. In normal women, most A 4 A found in androgen-responsive tissues has been converted to A4A from circulating DHEAS through DHEA [9,11,14]. This conversion is facilitated by 3/3 HSD in target tissue cells [9,14]. Circulating A 4 A is secreted both by ovarian stroma and by the adrenal ZF (the zone that secretes cortisol) [4,13,27]. It is not produced by adrenal ZR cells, which lack 3/3 HSD activity and therefore produce DHEA [13,27]. During premenopausal years, approximately 50% of secreted A n A originates from the ZF, whereas the other 50% originates from ovarian stroma [11,12]. Variations in circulating A a A reflect its dual origins: its adrenal origin by a circadian variation and its ovarian origin by a periovulatory surge during midcycle [12,34] (Fig. 9). In aging women, circulating A a A decreases slightly [1,12,35]. This is because even though adrenal secretion decreases, ovarian secretion continues. Presumably this represents the response of the ovarian stroma to high menopausal LH, allowing the ovary to compensate in part for a diminishing adrenal contribution [30-32,36,37].

Mass of adrenal cortex as a function of age and zona reticularis detected histologically as a percentage of harvested glands. Total adrenal mass increases steadily during childhood. Zona reticularis is detectable histologically as early as age 6 and is present in virtually all glands by the age of 14 years. No reticular zone, open areas; focal reticular zone, striped areas; continuous reticular zone, black areas. Adapted from Dhom [29].

Testosterone is a biologically potent androgen with specific receptors and target tissues. It is secreted into the circulation by both the adrenal ZF (cortisol and A n A ) and the ovaries [13,27,30-32,36,37]. As mentioned previously, it is also formed within target tissue from circulating DHEAS [9]. During reproductive years, approximately 25% of circulating T originates from the ovary, about 25% from the ZF, and about 50% from peripheral A 4 A conversion [11]. During reproductive years, T levels show a periovulatory rise at midcycle in association with the LH surge [34] (Fig. 9). Circulating T has no circadian change [11,12]. In aging women, circulating T remains stable well into the 80s [12,36,37]. Although ovarian volume decreases

CHAPTER44 DHEA: Biology and Therapeutic Use

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FIGURE 7 Steroidogenic architecture of the zona reticularis. DHEA and DHEAS are secreted by a discrete layer of cells in the adult human adrenal cortex, the zona reicularis. The key molecular feature of the zona reticularis that results in the production of DHEA(S) is its low expression of the enzyme 3fl-hydroxysteroid. The zona fasciculata has a high level of 3fi-hydroxysteroid, resulting in the synthesis of the glucocorticoid, cortisol. Other key enzymatic differences between the zones that result in the production of different steroids by the zones are shown. Adapted from Hornsby [12]. some 30%, the ovarian stroma, driven by high undulating, menopausal LH, secretes T in increasing abundance (Figs. 10-12) [38-41]. Circulating T thus decreases little after menopause even though adrenal androgen production of DHEA(S) and A4A declines. A substantial ovarian contribution of T after menopause is well documented by a considerable ovarian veinperipheral vein step-down gradient that is even greater in postmenopausal women (Fig. 9) [40,41 ]. Removal of postmenopausal ovaries produces a 40 to 50% decrement in circulating T (Fig. 12) [5]. Given that considerable target tissue T is transformed from circulating DHEAS and

FEMALE

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Days in Relation to LH Peak FIGURE 8 In a process resembling apoptosis, there is a decrease in the number of functional zona reticularis cells with age. These 12 adrenal glands (from patients 18 to 84 years old) are from one of a very small number of studies of the changes in the reticularis with aging. It shows increasing zona reticularis irregularity rather than simple involution of the zone. Solid black: zona reticularis; white: zona fasciculata and zona glomerulosa; striped, medulla; stipple, central vein. Adapted from Kreiner [33].

FIGURE 9

Midcycle rise in ovarian A4A (A) and T (B) in a group of women before and after 1 month of dexamethasone suppression of adrenal androgen production. Both A4A and T have a midcycle increase in concentration associated with periovulatary period. Adapted from Abraham [34], G. E. Abraham (1974). Ovarian and adrenal contribution to peripheral androgens during the menstrual cycle. J. Clin. Endocrinol. Metab. 39, 340. 9 The Endocrine Society.

630

BUSTER ANDCASSON Growing

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200

1000

100

500

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30

30

20

20

10

10

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Estradiol

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FIGURE 10 Schematic representation of the most salient differences between the reproductive-age (top) and postmenopausal (bottom) ovary. The stromal compartment comprises virtually all of the postmenopausal ovary. The postmenopausal ovarian stroma synthesizes and secretes considerable DHEA, A4A, and T. Adapted from Nicosia [39].

given that menopausal DHEAS concentrations are very low, the impact of menopausal oophorectomy from T withdrawal at target tissues is probably clinically significant.

D. Dihydrotestosterone: The Ultimate Androgen Dihydrotestosterone, believed to be the most potent of androgens, is present primarily in target tissues. Its circulating

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FIGURE | 2 Serum androgen and estrogen levels in 16 postmenopausal women before (E3) and after (i) ovariectomy. Ovariectomy produces a dramatic decrease of T and some decrease of A4A 9 estradiol and estrone are minimally effected. Adapted from Judd [5].

concentrations are very low in women [34]. It is secreted by the adrenal ZF (cortisol) in small amounts [4,13]. DHT is produced from 5a reduction of the 4 - 5 double bond in the A ring of T. In target tissues, where the receptor DHT complex has far greater affinity for genome receptor sites than does the receptor for T, DHT is a potent androgen (Figs. 3 and 4) [14]. Target tissue conversion of T to DHT is believed to act as an androgen amplifier mechanism that sequesters androgens in a nonaromatizable and very potent configuration.

III. DHEA, DHEAS, AND ANDROGEN METABOLITES: INCREASE DURING CHILDHOOD AND DECLINE WITH AGE

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function of the postmenopausalovary: Concentrationof androgens and estrogens in ovarian and peripheral vein blood. J. Clin. Endocrinol. Metab. 39, 1020. 9 The Endocrine Society.

DHEA(S) production begins to increase at ages 7 to 8 years in girls (Fig. 13). At this time, the ZR first appears as a structure identifiably separate from other adrenal zones (Figs. 5 and 6) [28,29,42]. This increase is associated with clinical benchmarks of adrenarche: pubic and axillary hair, emerging libidinal interest, muscle mass, and strength, increased bone mass, maturation of the immune system, and increasing stature (Table II; Fig. 14) [42]. DHEA(S) concentrations reach their lifetime zenith during the decades of the 20s and 30s and then begin a gradual decline that continues into the 80s and 90s (Fig. 13) [2,3]. Although circulating T, A 4 A , and DHT fall only modestly with advancing age, it is likely that tissue production and target tissue impact fall more dramatically in concert with declining DHEAS production, because DHEAS is the principal prohormone to these steroids (Fig. 3) [ 1]. During this decline, identified clinically as postmenopausal se-

CHAPTER44 DHEA: Biology and Therapeutic Use

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nescence, events much the opposite of adrenarche occur: loss of pubic and axillary hair, decreased libidinal interest, loss of muscle mass and strength, loss of bone mass, immunosenescence, and decline of adult stature (Table II) [43-46]. Because DHEAS is an important prohormone, it is reasonable to speculate this decline is linked to events of aging. It follows that restoration of youthful circulating DHEAS may attenuate some aspects of aging. A. C o n d i t i o n s A c c e l e r a t i n g the D H E A and A n d r o g e n D e c l i n e Declining androgen production is accelerated by misadventures that often occur in advancing years: 1. Oophorectomy: Postmenopausal oophorectomy decreases circulating T by 40-50%. Perimenopausal and

TABLE II Postmenopausal Senescence as a Model of Reverse Adrenarche a Adrenarche

Menopausal senescence

Increasing sex hair Increasing libido Increasing bone density Increasing stature Increasing muscle mass Immune maturation

Loss of sex hair Loss of libido Loss of bone density Loss of stature Loss of muscle mass Immunosenescence

a Events after menopause may be considered analogous to adrenarche except that they are associated with declining DHEAS production.

postmenopausal oophorectomies are associated with decreased libidinal interest and depression, and may have other long-term liabilities, including loss of bone mineral density, accelerated immunosenescence, and increased insulin resistance [5]. 2. Pituitary/adrenal insufficiency: Both Sheehan's syndrome and Addison's disease are models of androgen depletion. Both are associated with muscle wasting, loss of pubic and axillary hair, decreased libidinal interest, osteoporosis, and immunosenescence. 3. Chronic illness: Anorexia nervosa, advanced neoplasia, and burn trauma are all associated with low androgen concentrations and clinical manifestations of androgen depletion [46-48]. 4. Estrogen replacement: Estrogen replacement to menopausal women suppresses circulating DHEAS, A 4 A , and T. LH levels are also suppressed, and levels of sex hormone binding globulin are increased (Fig. 15) [49]. 5. Corticosteroid therapy: Corticosteroids suppress ACTH and therefore ZR secretion of DHEA(S) [26]. The clinical picture of Cushing's syndrome includes androgen depletion, loss of pubic and axillary hair, osteoporosis, muscle wasting, and immunosuppression. Plausibly, the serious side effects of corticosteroid suppression could be attenuated by coadministration of DHEA [50]. B. Clinical E x p r e s s i o n o f D H E A and D H E A M e t a b o l i t e D e f i c i e n c y There are no agreed on diagnostic criteria for androgen deficiency. In women, androgen deficiency is subtle in pre-

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sentation and slow to evolve. Though not immediately lethal, there is evidence that it may accelerate aging and increase mortality. As mentioned above, events of adrenarche, a clinical model of increasing androgen effect in women (Table II), are reversed with advanced age. These events have traditionally been accepted with resolve as inevitable and unpreventable ravages of aging. New strategies to replace androgens in women are under active investigation. These are discussed in the following section.

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IV. D H E A R E P L A C E M E N T A. R a t i o n a l e for D H E A R e p l a c e m e n t in the E l d e r l y The rationale for replacing DHEA is supported by animal studies, epidemiological evidence, and clinical trials. Specific reasons for which DHEA replacement may be beneficial closely parallel the rationale for estrogen replacement: cardioprotection, obesity control, insulin sensitivity, lipid effects, bone turnover, growth hormone (GH) axis impact, immune function, and cognitive effects.

CHAPTER 44 DHEA: Biology and Therapeutic Use

633

1. D H E A AND CARDIOPROTECTION Animal evidence implies a cardioprotective effect from exogenously administered DHEA. In rabbit models of accelerated atherogenesis (heterotopic heart transplantation or aortic intimal balloon injury), DHEA administration significantly retards atherogenesis [7,8]. Epidemiologic literature on cardioprotective effects of adrenal androgens is more contentious. The initial evaluation of the Rancho Bernardo cohort indicated that an increase in serum DHEAS of 100/zg/dl was associated with a 36% reduction in overall cardiovascular disease and a 48% reduction in cardiovascular mortality, even after adjustment for multiple risk factors [51 ]. This effect, however, was seen only in men, although another study of this cohort did demonstrate a significant association between high DHEAS levels and elevated high-density lipoprotein (HDL) in women. The evaluation of this cohort published in 1995 demonstrated a mild cardioprotective effect of higher elevated DHEAS levels, in both men and women [52]. Other epidemiologic data are similarly equivocal. Some reports demonstrate an inverse relationship between DHEAS levels and premature myocardial infarction (MI) in young men. DHEAS is also lower in men with angiographically demonstrated coronary artery disease, and in heart transplant patients with accelerated posttransplant atherosclerosis. However, other studies have not demonstrated a reproducible cardioprotective effect of DHEA. Unrecognized confounding factors may affect DHEAS levels and may explain these equivocal results. Factors that increase adrenal androgen levels include cigarette smoking, alcohol consumption, and obesity. Conversely, DHEAS levels are attenuated by estrogen replacement (Fig. 16), chronic illness, and hyperinsulinemia [53]. There is also a strong heritable and racial component to individual DHEAS levels. Long-term clinical trials of DHEA replacement to deter-

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mine whether any cardioprotective effects occur do not exist. Short-term clinical trials to date examine only the effect of DHEA on surrogate cardiovascular end points such as lipoprotein profiles. In a prospective, double-blind, randomized trial of 10 healthy young men, Nestler and colleagues administered DHEA (1600 mg/day) or placebo over 28 days, and they observed a significant decline in low-density lipoprotein in the DHEA group [54]. However, subsequent clinical trials of DHEA replacement in various populations have demonstrated either no effect on lipids, or overall androgenization [55]. We have shown that 6 months of 25 mg of oral micronized DHEA given to in postmenopausal women results in a 12% decline in HDL, with a concurrent decline in apolipoprotein A1, both indicative of increased atherogenic risk [56]. In perimenopausal women, Barnhardt and colleagues observed that oral DHEA (50 mg/day) given over 3 months also induced a progressive decline in total cholesterol and HDL [57]. It may be, therefore, that oral administration of DHEA, even in low doses, may adversely effect the lipoprotein profile of women. The putative cardioprotective effect of DHEA may not be lipoprotein mediated. Other possible cardioprotective mechanisms postulated on the basis of human trials include a fibroblast antiproliferative effect (secondary to glucose-6phosphate dehydrogenase inhibition), decreased platelet aggregation, or increased fibrinolysis. DHEA also may have an indirect inotropic effect on the aged heart by augmenting serum insulin-like growth hormone levels (IGF-I) [56]. It also may have secondary beneficial cardiac effects via its antiobesity and insulin-sensitizing actions. 2. D H E A AND OBESITY

In both rodents and dogs, DHEA supplementation has antiobesity effects. Again, the clinical trials remain more equivocal. Nestler and colleagues gave young men 1600 mg/ day of DHEA for over 1 month and demonstrated a decline in body weight and an increase in lean body mass [54]. However, two further investigations in the same population or in obese young men did not confirm these results. In elderly subjects, Yen and colleagues performed a double-blind, parallel, randomized controlled trial of 100 mg/day of oral DHEA administration. They found increased lean body and decreased fat mass in men, but not in women [58]. We have done a 1-month study of 25 mg/day of oral micronized DHEA administered to older men and also noted decreased weight with increased lean body mass [59], but we have not seen any reproducible effects on body morphology in postmenopausal women. Thus, if there is an effect of DHEA replacement on obesity in humans, the effect appears mild and also may be gender limited. 3. D H E A AND INSULIN SENSITIVITY

In rats, DHEA administration reduces the onset and ameliorates the severity of genetic and drug-induced diabetes. A

634 case report treatment of severe Type II diabetes with DHEA and subsequent improvement has been published [60]. A 17hr infusion of intravenous DHEA in women with polycystic ovarian disease can increase postreceptor pyruvate dehydrogenase (PDH) activity, thought to be a postreceptor marker of insulin effect [61 ]. These studies prompted us to examine whether DHEA replacement may augment insulin sensitivity in older men and women. In a placebo-controlled, double-blind crossover trial in postmenopausal women (with 3-week treatment periods), we showed that 50 mg/day of oral micronized DHEA augmented T lymphocyte insulin binding and degradation [62], a marker of clinical insulin sensitivity (Fig. 16). This enhanced insulin effect was associated with a trend toward decreased areas under the curve (AUC) for glucose and insulin after an oral glucose load. Subsequently, we performed a parallel, blinded, randomized, controlled trial giving 50 mg/day of oral micronized DHEA to postreproductive women [63]. We measured insulin sensitivity with an intravenous glucose tolerance tests, with data analysis by the minimal modeling technique, and found DHEA replacement had an ameliorating effect on observed study-induced declines in insulin sensitivity. Despite these data, other studies have not shown an insulin-sensitizing effect of DHEA. However, Diamond and colleagues recently demonstrated that application of 10% DHEA cream for 12 months in older women resulted in significant declines in basal glucose and insulin levels [64]. In an early study of 1600 mg/day of DHEA replacement in obese young men, hemoglobin A 1c did decline [65]. In summary, it appears that if insulin-sensitizing effects of DHEA exist, they may well not be dramatic and may be gender limited to women. 4. D H E A AND BONE TURNOVER

Although data on DHEA and bone metabolism are extremely limited, the fact that the pattern of bone gain and loss in human life closely parallels adrenal androgen secretion raises the possibility of an association between the two phenomena. Some epidemiologic investigations have correlated bone loss with DHEAS levels, particularly in the very elderly. Other rationales for a role of DHEA in bone turnover include the existence of frequent and disastrous bone loss with long- term corticosteroid administration, a situation in which adrenal androgen secretion is greatly reduced. Interleukin-6 (IL-6), a mediator of bone reabsorption in osteoporosis, decreases with DHEA supplementation [66]. In vitro studies in this area show promise. In human osteoblast cell cultures, DHEA has a mitogenic effect, mediated through transforming growth factor-fl and the androgen receptor [67]. This effect is not blocked by 5a-reductase or 3fl-hydroxysteroid dehydrogenase inhibition, indicating a direct DHEA/androgen receptor effect, or an effect mediated by A 5 metabolites of DHEA.

BUSTER AND CASSON

Despite these hints that DHEA may play a role in bone turnover, the clinical data remain limited. In our 3-week and 6-month studies in postmenopausal women, we have measured urinary hydroxyproline, hydroxylysine, and collagen cross-links, and have not seen reproducible declines [56,62]. In similar populations, others have not seen bone mineral density (BMD) changes with DEXA-scan, albeit with short durations of treatment. However, a study by Labrie and colleagues, using 12 months of 10% DHEA cream applied topically, demonstrated increases in bone mineral density at the hip in conjunction with declines in plasma bone alkaline phosphatase and urinary hydroxyproline [68]. Serum osteocalcin, a marker of bone formation, was increased twofold over control values. Whether DHEA replacement has bonesparing effects remains an area for further investigation. 5. D H E A AND GROWTH HORMONE

Growth hormone (GH) declines markedly with age and has been under scrutiny as a possible hormonal replacement therapy to prevent some of the sequelae of aging. While our study with recombinant GH demonstrates some promise in increasing muscle mass, strength, and well-being in elderly individuals, others do not confirm it. Also, this therapy is expensive and requires daily injection. The effects of GH are in large part mediated by either hepatic or end-organ production of IGF-I, previously known as somatomedin. In several clinical trials of DHEA replacement in aged individuals, serum IGF-I levels, both total and free, are increased in concert with decreasing IGF-I binding protein-3 levels. This was first demonstrated by Morales and colleagues in a placebo-controlled trial in men and women [69], and subsequently confirmed by us [56] and by Yen [58] in 6-month and year-long trials. Diamond and colleagues have also demonstrated an augmentation of serum IGF-I levels and action with 12 months of 10% DHEA cream in 15 postmenopausal women [64]. Thus, it appears clear that in both men and women, physiologic DHEA replacement augments serum IGF-I levels by about 50%. This effect may be due to augmentation of hepatic and end-organ IGF-I secretory response to circulating GH. That this augmentation of IGF-I secretion may have clinical benefits is demonstrated by the finding of increased muscle strength in men with DHEA replacement [58]. 6. D H E A AND IMMUNE FUNCTION In aging individuals, a decline in cellular-mediated immune competence is thought to occur, a phenomenon termed immunosenescence. This decline in immune function seems to be mediated by decreased interleukin-2 (IL-2) or IL-2 receptor levels, and is also associated with increased IL-6 levels. The concept that the age-related decline in DHEA levels may somehow be linked to immunosenescence was first demonstrated by Schwartz and colleagues, who showed that DHEA supplementation prevents spontaneous or mutagen-

CHAPTER44 DHEA: Biology and Therapeutic Use

635

induced carcinogenesis in rats [70]. Although the DHEA doses used were extremely high, subsequent studies in mice have demonstrated that both in vivo and in vitro DHEA augments lymphocyte IL-2 production. In humans, one in vitro study indicates that DHEA augments lymphocyte IL-2 production [71 ]. There is now also increasing clinical data demonstrating the immunoaugmentory effects of physiologic DHEA replacement in the elderly. DHEAS, 50 mg, given twice a day at the time of vaccination, increases influenza (but not tetanus) titer response over that seen with placebo [72]. In another randomized, blinded trial, 7.5 mg of subcutaneous DHEAS given at the time of influenza vaccination increased hemagglutination inhibition (HI) antibody response, particularly in subjects with lower initial titers, and lower serum endogenous DHEAS levels [73]. In 1993, we demonstrated that DHEA significantly augments natural killer cell cytotoxicity (Fig. 17) and number in postreproductive women and decreases stimulated lymphocyte IL-2 response [66]. Yen's group subsequently confirmed these data in a 6-month study of 100 mg of oral DHEA given to elderly men and women [58], and also demonstrated increases in serum IL-2 levels and lymphocyte expression of surface IL-2 receptor. It appears now that one of the more clearly delineated effects of DHEA replacement in humans is functional enhancement of the aging immune system. Whether this finding results in clinically significant beneficial effects is an issue that remains to be addressed.

7. COGNITIVE EFFECTS DHEA has demonstrated neurotropic actions at the yaminobutyric acid (GABA) receptor. This steroid enhances the maze performance of mice and has a beneficial effect on

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memory in these animals. It also promotes the growth of mouse brain explant in vitro. Human studies are limited. In a single-dose, double blind, randomized, controlled trial, DHEA significantly augmented rapid eye movement sleep over placebo [74]. Given the benefits of rapid eye movement sleep to overall sleep quality, this finding may contribute to the postulated enhancement in well-being seen in subjects given DHEA, shown by Morales et al., where a libido-independent increase in a sense of wellbeing (as measured by objective scales) was seen with DHEA (50 mg/day) [69].

B. D H E A Preparations and Their B ioavailability The bioavailability of orally administered adrenal androgens was first reported in 1982 in the form of a case report describing the administration of 25 mg/day of DHEAS for 1 year to a 19-year old male with hypogonadal hypogonadism [75]. Administration of this compound did not result in puberty, but serum DHEAS rose to peripubertal levels (200 to 250/xg/dl), with development of high testosterone levels (150 ng/dl). Thus, even at this early stage, significant bioconversion of orally administered adrenal androgen to more potent androgens was demonstrated. Later, Nestler and colleagues gave 1600 mg/day of DHEA to five healthy young men for 28 days [54]. Surprisingly, at this dose their serum DHEAS levels increased only 2.5- to 3.5-fold. Androstenedione increased 2-fold, and estrone, estradiol, sex hormonebinding globulin, and total and free testosterone all remained unaltered. The effect of this report was to perpetuate administration of the high doses reported in the literature, although Mortola and colleagues later demonstrated that administration of a similar dose to postmenopausal women greatly elevated all downstream androgens to supraphysiologic levels, androgenizing both glucose tolerance and lipid profiles [55]. The use of physiologic replacement doses then were considered by investigators working in the area on the basis of the fact that the combined production rates of DHEA and DHEAS are in the range of 50 mg/day. We performed an initial dose-ranging study to ascertain what levels of our oral micronized DHEA preparation (Belmar Pharmacy, Lakewood, Colorado) were needed to reproduce the premenopausal adrenal androgen milieu without adverse androgenization. On the basis of these single-dose studies, we postulated that the optimal oral replacement dose of this preparation of DHEA was 50 mg/day [76]. Concurrently, we addressed the issue of nonoral administration by performing a randomized, placebo-controlled, blinded, single-dose trial of oral compared to vaginal micronized DHEA administration [77]. After DHEA administration we sampled blood over a 12-hr period and calcualted areas under the curve for DHEA, DHEAS, and T. Comparison of these AUCs showed that with oral administration there is significant bioconversion to

636

BUSTER AND CASSON

DHEAS and T; with vaginal administration, this bioconversion is dramatically attenuated, with most of the DHEA appearing in the circulation as the native steroid. In our 3-week trial of 50 mg/day of oral micronized DHEA in postmenopausal women, we demonstrated supraphysiologic elevations of 23-hr postdose DHEAS and T, indicating that oral bioavailability of this preparation was more efficient than initially postulated and that 25 mg/day may be a more appropriate dose [62]. However, in a subsequent study, 25 mg/day of oral micronized DHEA, administered over 6 months, was subject to significant dose attenuation [56]. At the end of the trial, serum DHEA and DHEAS levels (again 23-hr postdose) were not significantly different from placebo values. We believe that any further trials of DHEA supplementation would require dose titration on the basis of serum DHEAS and T values to overcome this dose-attenuation effect. Given the adverse lipid effects seen with oral administration, the possibility of nonoral (vaginal, sublingual, or transcutaneous) administration becomes germane to avoid hepatic first-pass effects. Indeed, DHEA 20% cream has been effectively administered transcutaneously with physiologic elevations of DHEA, DHEAS, and downstream metabolites. Importantly, in these studies there were also changes in the growth hormone axis and in bone turnover, indicating that even with nonoral administration beneficial salutary effects exist, and, thus, may not be medicated by hepatic first-pass effect [64,68]. Much more investigation needs to be done on doses of DHEA and routes of administration used in human trials. The future of DHEA replacement therapy may very well lie with nonoral administration.

C. S i d e E f f e c t s Although reports of the possibility of adverse effects of this potent oral steroid exist in the literature, to date there is only one noted [76]. This occurred in one of our single-dose studies, in which a woman who was given 150 mg developed transient jaundice and hepatic dysfunction a week later. Her baseline blood serum, assayed retrospectively, demonstrated false-positive hepatitis C titers and positive antimitochondrial antibodies. Whether her hepatic dysfunction was a direct result of DHEA administration is not known, but the previously documented adverse hepatic effects of oral steroids, particularly androgens, give pause for concern. Although no other side effects have been noted, the lipid changes seen in women, both in our 6-month study and in Barnhardt and colleagues' investigation, raise some concern about chronic oral administration of this compound [56,57]. Additionally, the theoretical potential for side effects such as hirsutism are of concern because of the rapid biotransformation of this compound to the more potent androgens when administered orally. In animal models of oral DHEA administration, increases in liver size and induction hepatic carci-

noma are seen. Finally, the possible effects of chronic DHEA administration on subclinical prostate cancer or benign prostatic hyperplasia must also be considered carefully.

V. C O N C L U S I O N S It appears that physiologic DHEA administration to aging humans may have multiple beneficial effects. In men, but possibly not in women, it may have an antiobesity effect. Conversely, in women, but possibly not in men, DHEA may promote insulin sensitivity. DHEA may beneficially affect bone turnover and clearly augments the growth hormone/ IGF-I axis. Additionally, DHEA is likely an immunoaugmentory substance, reducing some of the declines in immune competence seen with aging. It is still puzzling how DHEA achieves these effects. Several investigators have noted either a membrane-bound or cytosolic DHEA receptor, but despite intensive effort, this work remains to be replicated. Labrie and colleagues postulate that DHEA, by virtue of end-organ bioconversion to a mix of estrogenic and androgenic metabolites that interact with androgen and estrogen receptors, creates a set of tissueunique physiologic effects [78]. This potential mechanism of action of DHEA is termed intracrinology and remains the most plausible theory of DHEA action at present. DHEA also may act in humans to exert multiple beneficial effects by augmenting end-organ and hepatic production of IGF-I in response to GH. It now has been well demonstrated that IGF-I is an immunoaugmentory substance in its own right and that its receptors and effects are noted in bone, muscle, and fat. Such an augmentation of IGF-I action would also result in an overall decline in serum growth hormone levels through hypothalamic-pituitary negative feedback, resulting in enhanced insulin sensitivity. Indeed, IGF-I therapy has been studied in clinical trials as a treatment for diabetes. Clearly the idea of DHEA replacement as an antiaging therapy is rapidly evolving. Future studies of DHEA replacement in humans are needed, with dose titration, possibly nonoral administration, and with and without concurrent estrogen replacement. Even more important are development of basic science investigations into the area, looking at the mechanism of adrenarche and adrenopause, and at the secretory control of DHEA and DHEAS. Finally, elucidating a mechanism of action that explains the multiple beneficial effects of these compounds would lend tremendous credence to this field. Unfortunately, at this point, popularization of this compound has far outpaced credible scientific investigation. We do not recommend using DHEA clinically at this time because of the current inability to assess the cost-benefit relationship of the practice. The future of DHEA replacement has some potential for attenuating certain aspects of aging, and the subject is certainly worthy of further investigation.

CHAPTER 44 D H E A : Biology and Therapeutic Use

References 1. Labrie, F., B61anger, A., Cusan, L., Gomez, J. L., and Candas, B. (1997). Marked decline in serum concentrations of adrenal C19 sex steroid precursors and conjugated androgen metabolites during aging. J. Clin. Endocrinol. Metab. 82, 2396-2402. 2. Ravaglia, G., Forti, P., Maioli, F., Boschi, F., Bernardi, M., Pratelli, L., Pizzoferrato, A., and Gasbarrini, G. (1996). The relationship of dehydroepiandrosterone sulfate (DHEAS) to endocrine-metabolic parameters and functional status in the oldest-old. Results from an Italian study on healthy free-living over-ninety-years-olds. J. Clin. Endocrinol. Metab. 81, 1173-1178. 3. Orentreich, N., Brind, J. L., Rizer, R. L., and Vogelman, J. H. (1984). Age changes and sex differences in serum dehydroepiandrosterone sulfate concentrations throughout adulthood. J. Clin. Endocrino. Metab. 59,551-555. 4. Hornsby, P. J. (1995). Biosynthesis of DHEAS by the human adrenal cortex and its age-related decline. Ann. N.Y. Acad. Sci. 774, 29-46. 5. Judd, H. L., Lucas, W. E., and Yen, S. C. S. (1974). Effect of oophorectomy on circulating testosterone and androstenedione levels in patients with endometrial cancer. Am. J. Obstet. Gynecol. 118, 793-798. 6. Casson, P. R., and Carson, S. A. (1996). Androgen replacement therapy in women: Myths and realities. Int. J. Fertil. 41, 412-422. 7. Casson, P. R., Hornsby, P. J., Ghusn, H. F., and Buster, J. E. (1997). Dehydroepiandrosterone (DHEA) replacement in postmenopausal women: Present status and future promise. Menopause 4, 225-231. 8. Casson, P. R., and Buster, J. E. (1997). DHEA replacement after menopause: HRT 2000 or nostrum of the 90's? Contemp. Obstet. Gynecol. 42, 119-133. 9. Labrie, E, B61anger, A., Simard, J., Lu-Th6, V., and Labrie, C. (1995). DHEA and peripheral androgen and estrogen formation: Intracrinology. Ann. N.Y. Acad. Sci. 774, 16-28. 10. Labrie, F., B61anger, A., Cusan, L., and Candas, B. (1997). Physiological changes in dehydroepiandrosterone are not reflected by serum levels of active androgens and estrogens but of their metabolites: Intracrinology. J. Clin. Endocrinol. Metab. 82, 2403-2409. 11. Longcope, C. (1986). Adrenal and gonadal androgen secretion in normal females. Clin. EndocrinoL Metab. 15, 213 -227. 12. Longcope, C. (1990). Hormone dynamics at the menopause. Multidiscip. Perspect. Menopause 592, 21-30. 13. Endoh, A., Kristiansen, S. B., Casson, P. R., Buster, J. E., and Hornsby, P. J. (1996). The zona reticularis is the site of biosynthesis of dehydroepiandrosterone and dehydroepiandrosterone sulfate in the adult human adrenal cortex, resulting from its low expression of 3fl-hydroxysteroid dehydrogenase. J. Clin. Endocrino. Metab. 81, 3558-3565. 14. Labrie, E (1991). Intracrinology. Mol. Cell. Endocrinol. 78, C l 1 3 Cl18. 15. Martel, C., Melner, M. H., Gagne, D., Simard, J., and Labrie, F. (1994). Widespread tissue distribution of steroid sulfatase, 3fl-hydroxysteroid dehydrogenase/A5-A4 isomerase (3fl-HSD), 17fl-HSD, 5a-reductase and aromatase activities in the rhesus monkey. Mol. Cell. Endocrinol.

104, 103-111. 16. Simpson, E. R., and Zhao, Y. (1996). Estrogen biosynthesis in adipose tissue. Significance in breast cancer development. Ann. N.Y. Acad. Sci. 184, 18-26. 17. B61anger, B., B61anger, A., Labrie, F., Dupont, A., Cusan, L., and Monfette, G. (1989). Comparison of residual C-19 steroids in plasma and prostatic tissue of human, rat and guinea pig after castration: Unique importance of extratesticular androgens in men. J. Steroid Biochem. 32, 695-698. 18. Roy, A. K. (1992). Regulation of steroid hormone action in target cells by specific hormone-inactivating enzymes. Proc. Soc. Exp. Biol. Med. 199, 265-272. 19. Haning, R. V., Jr, Flood, C. A., Hackett, R. J., Loughlin, J. S., and McClure, R. J. ( 1991). Metabolic clearance rate of dehydroepiandrosterone

637 sulfate, its metabolism to testosterone, and its intrafollicular metabolism to dehydroepiandrosterone, androstenedione, testosterone, and dihydrotestosterone in vivo. J. Clin. Endocrinol. Metab. 72, 1088-1095. 20. Haning, R. V., Jr, Hackett, R. J., Flood, C. A., Loughlin, J. S., and Zhao, Q. Y. (1993). Plasma dehydroepiandrosterone sulfate serves as a prehormone for 48% of follicular fluid testosterone during treatment with menotropins. J. Clin. Endocrinol. Metab. 76, 1301-1307. 21. Feher, T., Szalay, K. S., and Szilagyi, G. (1985). Effect of ACTH and prolactin on dehydroepiandrosterone, its sulfate ester and cortisol reduction by normal and tumorous human adrenocortical cells. J. Steroid Biochem. 23, 153-157. 22. Klein, N. A., Andersen, R. N., Casson, P. R., Buster, J. E., and Kramer, R. E. (1992). Mechanisms of insulin inhibition of ACTH-stimulated steroid secretion by cultured bovine adrenocortical cells. J. Steroid Biochem. Mol. Biol. 41, 11-20. 23. Polderman, K. H., Gooren, L. J. G., and van der Veen, E. A. (1994). Testosterone administration increases adrenal response to adrenocorticotropin. Clin. Endocrinol. (Oxford) 40, 595-601. 24. Poderman, K. H., Gooren, L. J. G., and van der Veen, E. A. (1995). Effects of gonadal androgens and estrogens on adrenal androgen levels. Clin. Endocrinol. (Oxford) 43, 415-421. 25. Parker, J. R., Stankovic, A. K., Falany, C. N., and Grizzle, W. E.(1995). Effect of TGF-fl on dehydroepiandrosterone sulfotransferase in cultured human fetal adrenal cells. Ann. N.Y. Acad. Sci. 774, 326-328. 26. Parker, L. N., Sack, J., Fisher, D. A., and Odell, W. D. (1978). The adrenarche: Prolactin, gonadotropins, adrenal androgens, and cortisol. J. Clin. Endocrinol. Metab. 46, 386-401. 27. Hornsby, P. J. (1997). DHEA: A biologist's perspective. J. Am. Geriatr. Soc. 45, 1395-1401. 28. Bargmann, W. (1951). "Histologic and mikroskopische Anatomie des Menshen," Vol. 2. Thieme, Stuttgart. 29. Dhom, G. (1973). The prepuberal and puberal growth of the adrenal (adrenarche). Beitr. Pathol. 150, 357-377. 30. Lucisano, A., Russo, N., Acampora, M. G., Fabiano, A.' Fattibene, M., Parlati, E., Maniccia, E., and Dell'Acqua, S. (1986). Ovarian and peripheral androgen and oestrogen levels in post-menopausal women: Correlations with ovarian histology. Maturitas 8, 57-65. 31. Nagamani, M., Hannigan, E. V., Dillard, E. A., Jr., and Dinh, T. V. (1986). Ovarian steroid secretion in postmenopausal women with and without endometrial cancer. J. Clin. Endocrinol. Metab. 62, 508-512. 32. Ushiroyama, T., and Sugimoto, O. (1995). Endocrine function of the peri- and postmenopausal ovary. Horm. Res. 44, 64-68. 33. Kreiner, E., and Dohm, G.(1979). Altersveranderungen de menschilichen Nebenniere. Zentralbl. Allg. Pathol. Anat. 123, 351-356. 34. Abraham, G. E. (1974). Ovarian and adrenal contribution to peripheral androgens during the menstrual cycle. J. Clin. Endocrinol. Metab. 39, 340. 35. Burger, H. G., Dudley, E. C., Hopper, J. L., Shelley, J. M., Green, A., Smith, A., Dennerstein, L., and Morse, C. (1995). The endocrinology of the menopausal transition: a cross-sectional study of a populationbased sample. J. Clin. Endocrinol. Metab. 80, 3537-3545. 36. Longcope, C., Hunter, R., and Franz, C. (1980). Steroid secretion by the postmenopausal ovary. Am. J. Obstet. Gynecol. 138, 564-568. 37. Vermeulen, A. (1976). The hormonal activity of the postmenopausal ovary. J. Clin. Endocrinol. Metab. 42, 247-253. 38. Burger, H. G. (1996). The endocrinology of the menopause. Maturitas 23, 129-136. 39. Nicosia, S. V. (1986). Ovarian changes during the climacteric. In "The Climacteric" (M. Notelovitz and EA. van Keep, eds.), pp. 179-199. Plenum, New York. 40. Judd, H. L., Judd, G. E., Lucas, W. E., and Yen, S. C. C. (1974). Endocrine function of the postmenopausal ovary: Concentration of androgens and estrogens in ovarian and peripheral vein blood. J. Clin. Endocrinol. Metab. 39, 1020. 41. Chang, R. J., and Judd, H. L. (1981). The ovary after menopause. Clin. Obstet. Gynecol. 24, 181-191.

638 42. Babalola, A. A., and Ellis, G. (1988). Serum dehydroepiandrosterone sulfate in a normal pediatric population. Clin. Biochem. 18, 182-189. 43. Vermeulen, A. (1996). Dehydroepiandrosterone sulfate and aging. Ann. N.Y. Acad. Sci. 774, 121-127. 44. Casson, E R., Anderson, R. N., Herrod, H. G., Stentz, E B., Straughn, A. B., Abraham, G. E., and Buster, J. E. (1993). Oral dehydroepiandrosterone in physiologic doses modulates immune function in postmenopausal women. Am. J. Obstet. Gynecol. 169, 1536-1539. 45. Garg, M., and Bondada, S. (1993). Reversal of age-associated decline in immune response to pnu-imune vaccine by supplementation with the steroid hormone dehydroepiandrosterone. Infect. Immun. 61, 22382241. 46. Wild, R. A., Buchanan, J. R., Myers, C., and Demers, L. M. (1987). Declining adrenal androgens: An association with bone loss in aging women. Proc. Soc. Exp. Biol. Med. 186, 355-360. 47. Parker, C. R., and Banter, C. R. (1985). Divergence in adrenal steroid secretory pattern after thermal injury in adult patients. J. Trauma 25, 508-510. 48. Findling, J. W., Buggy, B. E, Gilson, I. H., Brummitt, C. E, Bernstein, B. M., and Raft, H. (1994). Longitudinal evaluation of adrenocortical function in patients infected with the human immunodeficiency virus. J. Clin. Endrocrinol. Metab. 79, 1091-1096. 49. Casson, E R., Elkind-Hirsch, K. E., Buster, J. E., Hornsby, E J., Carson, S. A., and Snabes, M. C. (1997). Effect of postmenopausal estrogen replacement on circulating androgens. Obstet. Gynecol. 90, 995-998. 50. Kalimi, M., Shafogoj, Y., Loria, R., Padgett, D., and Regelson, W. (1994). Anti-glucocorticoid effects of dehydroepiandrosterone (DHEA). Mol. Cell. Biochem. 131, 99-104. 51. Barrett-Connor, E., and Goodman-Gruen, D. (1987). Absence of inverse relation of dehydroepiandrosterone sulfate with cardiovascular mortality in postmenopausal women. N. Engl. J. Med. 137, 711. 52. Barrett-Connor, E., and Goodman-Gruen, D. (1995)The epidemiology of DHEAS and cardiovascular disease. Ann. N.Y. Acad. Sci. 774, 259 -70. 53. Casson, E R., Elking-Hirsch, K. E., Carson, S. A., Hornsby, P. J., Buster, J. E., and Snabes, M. C. (1997). Postmenopausal estrogen replacement suppresses circulating androgens. Obstet. Gynecol. 90, 995-998. 54. Nestler, J. E., Barlascini, C. O., Clore, J. N., and Blackard, W. G. (1988). Dehydroepiandrosterone reduces serum low density lipoprotein levels and body fat but does not alter insulin sensitivity in normal men. J. Clin. Endocrinol. Metab. 66, 57-61. 55. Mortola, J., and Yen, S. S. C. (1990). The effects of dehydroepiandrosterone on endocrine-metabolic parameters in postmenopausal women. J. Clin. Endocrinol. Metab. 71,696-704. 56. Casson, E R., Santoro, N., Elkind-Hirsch, K. E., Carson, S. A., Hornsby, E J., and Buster, J. E. (1998). Postmenopausal dehydroepiandrosterone (DHEA) adminstration increases insulin-like growth factorI (IGF-I) and decreases high density lipoprotein (HDL): A six month trial. Fertil. Steril. 70, 107-110. 57. Barnhardt, K. T., Rader, D., Freeman, E., Kapoor, S. K., Smith, E, and Nestler, J. E. (1997). The effect of DHEA replacement on the endocrine and lipid profiles of perimenopausal women. Fertil. Steril. Abstr. 0-081 (presented at the 53rd Annual Meeting of the American Society for Reproductive Medicine, Cincinnati, Ohio, November 1997). 58. Yen, S. C. C., Morales, A. J., and Khorram, O. (1995). Replacement of DHEA in aging men and women: Potential remedial effects. Ann. N.Y. Acad. Sci. 774, pp. 128-142. 59. Ghusn, H. E, Taffet, G., Jaweed, M., LeBlanc, A., Casson, P. R., Rodriguez, G. E, and Oregno, C. (1996). DHEA improves lean body mass of older men. 49th Annu. Sci. Meet. Am. Gerontol. Soc., Washington, DC, Oral presentation. 60. Buffington, C. K., Pourmotabbed, G., and Kitabchi, A. E. (1993). Case report: Amelioration of insulin resistance in diabetes with dehydroepiandrosterone. Am. J. Med. Sci. 306, 320-324.

BUSTER ANt) CASSO~ 61. Schriock, E. D., Buffington, C. K., Givens, J. R., and Buster, J. E. (1994). Enhanced post-receptor insulin effects on women following dehydroepiandrosterone infusion. J. Soc. Gynecol. Invest. 1, 74-78. 62. Casson, E R., Faquin, L. C., Stentz, F. B., Straughn, A. B., Andersen, R. N., and Abraham, G. E. (1995). Replacement of dehydroepiandrosterone (DHEA) enhances T-lymphocyte insulin binding in postmenopausal women. Fertil. Steril. 3, 1027-1031. 63. Bates, G. W., Egeman, R. S., Umstot, E. S., Buster, J. E., and Casson, E R. (1995). Dehydroepiandrosterone attenuates study-induced declines in insulin sensitivity in postmenopausal women. Ann. N.Y. Acad. Sci. 774, 291-293. 64. Diamond, E, Cusan, L., Gomez, J. L., B61anger, A., and Labrie, E (1996). Metabolic effects of 12 month percutaneous dehydroepiandrosterone replacement therapy in postmenopausal women. J. Endocrinol. 150, $43-$50. 65. Usiskin, K. S., Butterworth, S., Clore, J. N., Yadon, A., Ginsberg, H. N., Blackard, W. G., and Nestler, J. E. (1990). Lack of effect of dehydroepiandrosterone in obese men. Int. J. Obes. 14, 457-463. 66. Casson, E R., Anderson, R. N., Herrod, H. G., Stenz, R. B., Straughn, A. B., and Abraham, G. E. (1993). Oral dehydroepiandrosterone in physiologic doses modulates immune function in postmenopausal women. Am. J. Obstet. Gynecol. 169, 1536-1539. 67. Kasperk, C. H., Wakley, G. K., Hierl, T., and Ziegler, R. (1997). Gonadal and adrenal androgens are potent regulators of human bone cell metabolism in vitro. J. Bone. Miner. Res. 12, 464-471. 68. Labrie, E, Diamond, E, Cusan, L., Gomez, J. L., B61anger, A., and Cadas, B. (1997). Effect of 12 month dehydroepiandrosterone replacement therapy on bone, vagina, and endometrium in postmenopausal women. J. Clin. Endocrinol. Metab. 82, 3498-3505. 69. Morales, A. J., Nolan, J. J., Nelson, J. C., and Yen, S. S. C. (1994). Effects of replacement dose of dehydroepiandrosterone in men and women of advancing age. J. Clin. Endocrinol. Metab. 78, 13601367. 70. Schwartz, A. G., and Pashko, L. L. (1993). Cancer chemoprevention with the adrenocortical steroid dehydroepiandrosterone and structural analogs. J. Cell. Biochem. 176, 73-79. 71. Suzuki, T., Suzuki, N., Daynes, R. A., and Engleman, E. G. (1991). Dehydroepiandrosterone enhances IL-2 production and cytotoxic effector function of human T cells. Clin. Immunol. Immunopathol. 61, 202-211. 72. Araneo, B. A., Dowell, T., Woods, M. A., Daynesra, R. A., Judd, M., and Evans, T. (1995). DHEAS as an effective vaccine adjuvant in elderly humans. Ann. N. Y. Acad. Sci. 774, 232-248. 73. Degelau, J., Guay, D., and Hallgren, H. (1997). The effect of DHEAS on influenza vaccination in aging adults. J. Am. Geriatr. Soc. 45, 747-751. 74. Freiss, E., Trachsel, L., Guldner, J., Schier, T., Steiger, A., and Holboer, F. (1995). DHEA administration increases rapid eye movement sleep and EEG power in the sigma frequency range. Am. J. Physiol. 268, E107-E113. 75. Cohen, H. N., Hay, I. D., Beastall, G. H., and Thompson, J. A. (1982). Failure of adrenal androgen to induce puberty in familial cytomegalic adrenocortical hypoplasia. Lancet 12, 1471 - 1472. 76. Buster, J. E., Casson, E R., Straughn, A. B., Dale, D., Umstot, E. S., and Chiamori, N. (1992). Postmenopausal steroid replacement with micronized dehydroepiandrosterone: Preliminary oral bioavailability and dose proportionality studies. Am. J. Obstet. Gynecol. 166, 11631170. 77. Casson, P. R., Straughn, A. B., and Milem, C. A. (1996). Delivery of dehydroepiandrosterone (DHEA) in premenopausal women: Effects of micronization and non-oral administration. Am. J. Obstet. Gynecol. 174, 649-653. 78. Labrie, E, B61anger, A., Simard, J., Lun-Th6, V., and Labrie, C. (1995). DHEA and peripheral androgen and estrogen formation: Intracrinology. Ann. N.Y. Acad. Sci. 774, 16-28.

-m

z

~HAPTER 4.

Menopause Issues for Older Women GAlL A. GREENDALE

Departments of Medicine and Obstetrics and Gynecology, University of California, Los Angeles, School of Medicine, Los Angeles, California 90024

IV. Conclusions References

I. Introduction II. Selected Syndromes and Late Menopausal Women III. Therapeutic Considerations in Late Menopausal Women

I. I N T R O D U C T I O N

rise [1]. Other conditions that may be sex steroid related, such as tooth loss, cataracts, urinary tract infection, and urinary incontinence are also more prevalent compared to younger reproductive age women. Older women may also suffer from cognitive or physical limitations, which require appropriate tailoring of intervention strategies. Therefore, the manner in which we conceive of menopause-related health care of the late postmenopausal women must differ somewhat from that for women in early postmenopause.

A. O v e r v i e w This chapter focuses on some aspects of menopause that are of special relevance to the care of late menopausal women. For the purpose of this chapter, women who are at least 10 years postmenopause are considered to be late menopausal. Because there is a wide range of age at menopause, it is important to distinguish between "menopausal age," which refers to years postmenopause, and chronological age. For example, a woman who is 60 years old and whose menopause was at age 45 is in the late postmenopausal period, whereas a 60-year-old woman whose menopause was at age 56 is in the early postmenopause. This chapter also considers features of menopause-related health care that pertain to women of older chronological age (approximately 70 years and older). Virtually all women will experience natural menopause by age 60; therefore, women greater than 70 years are in the late postmenopause. During the late postmenopausal period, the incidence of clinical cases of many of the chronic conditions (e.g., fractures, heart disease) associated with menopause begins to

M E N O P A U S E : B I O L O G Y AND PATHOBIOLOGY

B. E m e r g i n g M e n o p a u s a l S y n d r o m e s The first section of this chapter discusses four conditions that have only recently been associated with exogenous or endogenous estrogens: periodontal disease, tooth loss, cataracts, and urinary tract infection. These areas of menopauserelated research are relatively early in development. However, the health, quality of life, and functional implications of each make them important considerations in older women. The association between menopause, postmenopausal hormone use, and cognition, which is also a new area of menopauserelated research, is covered in Chapter 21.

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Copyright 9 2000 by Academic Press. All rights of reproduction in any form reserved.

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GAIL A. GREENDALE

C. Primary versus Secondary Prevention Especially relevant to the care of older women is the question of whether therapies begun in the late postmenopause (when chronic conditions may have become established) are as effective as those started earlier (when the goal is primary prevention). For most menopause-associated chronic diseases, possible differences in the success of primary versus secondary disease prevention have only begun to be studied. This chapter explores the question of primary versus secondary prevention in the context of osteoporosis. Similarly, an important concern is whether postmenopausal hormone treatment is beneficial in the setting of established heart disease (discussed in Chapter 37).

D. Therapeutic Considerations Unfortunately, there are few data regarding the acceptance and side effect profiles of postmenopausal hormones in older women. However, clinical experience suggests that there are some age-related differences in both of these areas. This chapter therefore presents an approach to systemic postmenopausal hormone therapy that may be acceptable specifically to older women. Vaginal estrogen therapy and pessary use will also be highlighted, because they relate to the postmenopausal care of older women.

II. SELECTED SYNDROMES AND LATE MENOPAUSAL WOMEN

A. Menopause, Hormone Replacement Therapy, and Dental Health Tooth retention is associated with maintenance of good health and nutritional status in older persons. It is also a determinant of quality of life and positive self-esteem [2]. Tooth loss is highly prevalent in the United States. A national survey conducted in the 1980s found that 41% of those aged greater than 65 years were edentulous [3]. Factors leading to tooth loss are numerous and probably interrelated. These include oral health status (e.g., advanced caries, periodontal disease), access to dental care, dental practice patterns, economic factors, and systemic conditions (e.g., diabetes and, possibly, osteoporosis) [3,4]. The contribution of menopause-related physiological changes to the causes of alveolar bone loss, tooth loss, gingivitis, and periodontitis and the potential role for estrogen therapy as a means to prevent these conditions are research areas receiving increasing investigation [5]. Periodontitis is a disease characterized by inflammation surrounding the gingival unit (gingiva and alveolar mucosa)

extending to the periodontal ligament, alveolar bone, and cementum [6]. Periodonitis is defined as the loss of attachment of both the periodontal ligament and the bony support of the tooth (generally referred to as "attachment loss") [7] Thus, loss of attachment and alveolar bone resorption are features that distinguish gingivitis (inflammation of the gingival tissue) from periodontitis (loss of attachment) [6,8]. The adult form of periodontitis occurs in persons greater than 35 years of age. Several studies estimate that the prevalence of periodontitis is higher in men than women, after adjustment for the higher rate of poor oral hygiene associated with the male gender [7]. The observed sex difference in periodontal disease occurrence raises the possibility that sex steroid hormones play a role in disease prevention. There is limited but implicative evidence of an association between attachment loss and both years since menopause and systemic bone loss. A cross-sectional study of postmenopausal women with early periodontal disease who were not taking estrogen or other bone-active drugs found that the degree of attachment loss was related to years since menopause [9]. Other cross-sectional studies correlate less favorable attachment with low systemic bone mineral density (BMD) and clinical osteoporotic fracture [10,11]. These findings do not establish an etiological link between menopause and attachment loss. It is possible that menopause and periodontal disease are not directly associated, but that periodontitis-mediated inflammatory bone resorption becomes more readily apparent as attachment loss when bone mass is already low due to postmenopausal osteoporosis. On the other hand, one may postulate a direct relation between estrogen concentrations (and therefore menopause) and inflammatory-mediated gingivitis and bacterial plaque formation. For example, gingival prostaglandin synthesis is lowered in the presence of estrogen [12]; bacterial peroxidases, which combat plaque formation, are enhanced by estrogen [13]; and gingival fluid concentrations of interleukins, particularly IL-1/3 and IL-6 (proposed mediators of bone resorption), are lower in women with periodontitis who are taking estrogen compared to those who are not [ 14]. One small nonrandomized longitudinal study offers intriguing evidence that women with established periodontitis may benefit from estrogen therapy [15]. Blinded computerassisted densitometric image analysis (CADIA)of alveolar bone was performed over a 12-month period in 24 patients who were undergoing supportive periodontal therapy. Those who were taking estrogen showed a statistically significant increase in alveolar bone density, whereas those who were not taking estrogen experienced a significant loss. Tooth loss is easier to study than attachment loss, because women can self-report the number of their remaining teeth or nondental personnel can easily and quickly count the number of teeth present. In cross-sectional analyses, a higher number of remaining teeth has been positively associated

CHAPTER45 Menopause Issues for Older Women with higher BMD at multiple skeletal sites [16,17]. These findings have been substantiated in a longitudinal study in which incident tooth loss during 7 years of follow-up was highly related to the rate of bone loss over that time period [ 18]. Several cross-sectional reports associating higher tooth number with postmenopausal hormone use are emerging from large community-based cohort studies [ 19-21 ]. In the Leisure World Study [ 19], the multiply adjusted risk ofhaving fewer than 25 teeth was 0.76 in women who had ever used estrogen. (Adjustment factors included age, smoking, sociodemographic characteristics, dental access, and hygiene.) Current and past estrogen users were equally protected and there was a significant relation between duration of estrogen use and presence of more than 25 teeth. Women who had everused estrogen were also less likely to be edentulous (relative risk, 0.64); a significant effect of duration of estrogen use was also present for edentia. Results from the Framingham cohort are similar [20]. In women aged 7 2 - 9 5 years at the time of the study visit, controlled for age, smoking status, and education, estrogen users had an average of 12.5 teeth whereas nonusers had an average of 10.7 teeth. Each 4.2-year increment in duration of estrogen use was associated with one more tooth being retained. Findings from the Nurses' Health Study are somewhat different than those from the former two cohorts [21 ]. A protective effect of estrogen for tooth retention was substantially stronger in the current users (relative risk, 0.73) than in the past users (relative risk, 0.95). Tooth retention was unrelated to duration of estrogen use, however. A detailed study of dental health behavior was conducted in a subgroup, and the dental behavior adjusted results in that subgroup were not different from those of the entire cohort. None of these cohort studies directly address the question of how soon after menopause estrogen use should be initiated to protect against tooth loss. The mechanism for tooth preservation associated with estrogen use remains speculative. As noted above, better underlying bone strata could offer more "resistance" to loss of attachment, and estrogen's antiinflammatory properties might also directly prevent gingivitis and periodontal disease. Estrogen users are generally healthier, more educated, and have more access to care compared to nonusers (referred to as a "healthy user effect"); to the extent possible, the cohort studies summarized here adjusted for these potential confounders. The observed relation between duration of estrogen use and degree of tooth retention does not entirely mitigate concerns about a healthy user effect, but does add further support to the findings. In summary, evidence for a direct effect of estrogen in preventing gingivitis and periodontal disease is promising but sparse; evidence for an overall tooth maintenance effect, however, is mounting. It may be reasonable to add tooth retention to the discussion of possible benefits of hormone use. Current data do not allow us to assess whether women must

641 institute estrogen soon after the menopause in order to obtain the potential tooth retention benefit.

B. M e n o p a u s e , H o r m o n e R e p l a c e m e n t T h e r a p y , and Lens Opacities Age-related lens opacities and cataracts (lens opacities resuiting in visual impairment) are common. In the Framingham eye study, the prevalence of lens opacities increased from 41.7 to 91.1% as age ranged from 52 to 85 years [22,23]. In the National Health and Nutrition Examination Survey, lens opacities were found in 12.2% of persons aged 4 5 - 5 4 years and increased to 57.6% of those aged 6 5 - 7 4 years. In the latter study, the prevalence of cataract causing visual loss in the oldest age group was 28.5% [24]. The economic and quality-of-life effects of cataracts are large. Cataracts are the leading cause of blindness worldwide [25]. In the United States, cataract extraction and intraocular lens implantation is the most common surgical procedure performed in Medicare enrollees, estimated to cost 3.4 billion dollars in 1991 [26]. Cataracts lead to limitations in vision-related daily activities, such as driving and reading [27]. There are sex differences in cataract occurrence. The Beaver Dam Eye Study found that two cataract types (nuclear sclerosis and cortical) occur more commonly in women and that the incidence of nuclear cataract increased significantly with age only in women [28,29]. Similarly, a recent crosssectional study conducted in London reported that the ageadjusted cataract prevalence ratio was 1.2 in females compared to males [30]. The observed sex difference in cataract rates raises the possibility of an association of cataracts and sex steroids, either endogenous or exogenous. A few preliminary attempts have been made to address these hypotheses, but results are inconclusive. With respect to endogenous s e x steroids, investigators from the Blue Mountains Eye Study (Australia) noted a higher prevalence of all three types of cataracts among women between later menarche (a marker for shorter duration of endogenous estrogen exposure). There was no association, however, between age at menopause and cataracts [31]. In the Beaver Dam Eye Study, younger age at menarche was protective for nuclear sclerosis, and the odds of cortical opacities were lower in women with older age at menopause [29]. Data regarding postmenopausal hormone use and cataract occurrence are limited and contradictory. Current users of hormones who were aged greater than 65 years had a lower prevalence of cortical cataract (odds ratio, 0.4) in the Australian study [31 ]. This study did not directly measure duration of use, but it is plausible that current, older users of postmenopausal hormones were those with longer duration of exposure. Conversely, women with nonsurgical menopause

642 were at higher risk for posterior subcapsular cataract (odds ratio, 2.1), suggesting that estrogens and or progestogens may enhance this form of cataract development. A small case-control study [32] reported that the prevalence of lens autofluorescence, a measure of nuclear cataract formation, was lower among women who were current estrogen users. Finally, a newly derived rat model of cataract development suggests that there may be a role for endogenous estrogen and cataract prevention [33]. In this model, transforming growth factor-fl is used to induce lens damage to rat lenses in culture. Lenses from castrated female rats are more susceptible to damage induced by this agent than are lenses of noncastrated rats or castrated rats that have been treated with exogenous estrogens. In summary, the linkage between cataracts, menopause, and hormone therapy is uncertain. The fundamental explanation for the observed differences in the relations between cataract types and reproductive hormone use remains obscure. Basic mechanistic studies of this potential relation are needed, as is more epidemiological exploration.

C. Menopause, Hormone Replacement Therapy, and the Lower Urinary Tract Chapter 22 reviews in detail the pathophysiology of genitourinary system changes in the menopause. Here we highlight some aspects of this topic as they relate to older women. Urinary incontinence (UI) is common in older age, with approximately 5% of ambulatory women aged 6 5 - 7 4 years and 15% of those aged greater than 75 years experiencing clinically significant incontinence. This figure soars to 50% among female nursing home residents [34]. Urinary tract infections (UTIs) are also very common in older women, leading to substantial morbidity. For example, the genitourinary tract is the leading cause of bloodstream infection in older persons [35]. In women, older age (and presumably concomitant lower estrogen concentrations) is associated with several genitourinary (GU) changes, such as atrophy of the bladder trigone, diminished sensitivity of the ce-adrenergic receptors of the bladder neck and urethral sphincter, and thinning of the urethral mucosa [36]. Based on these observed alterations in the GU tract, a relation between menopause and urinary incontinence is biologically plausible. However, some studies report an increase in the prevalence of urinary incontinence with menopause, whereas others do not [37,38]. Similarly, intervention studies using estrogen for stress and urge UI have had mixed results [39-42]. In the setting of these inconclusive and conflicting data, many clinicians employ a treatment trial of estrogen for stress and urge UI. It is important to recognize, however, that the causes of UI in older women, especially those residing in long-term care settings, are likely to be multifactorial. In addition to the role played by menopause-related anatomical and physiological changes,

GAIL A. GREENDALE

comorbidity (such as congestive heart failure), medication use (such as diuretics), diminished ambulatory capability (limiting the capability to toilet), and cognitive impairment may each contribute to the loss of continence [34]. Ouslander [43] has reviewed behavioral and medical interventions targeted at each of these factors. A causal relation between menopause and UTI has not been established, but physiological changes that occur in older women suggest that the two may be linked. The higher vaginal pH and atrophy of the vaginal epithelium that occur after menopause are thought to lead to increased susceptibility to UTI by allowing gram-negative colonization of the perineum and urethra [44,45]. It is postulated that estrogen supplementation, by improving these local host defenses against gram-negative vaginal colonization, may be an effective means of lowering the rate of recurrent UTI. Uncontrolled, unmasked studies offered initial support for the hypothesis that estrogen prevents recurrent UTIs. One series reported complete resolution of recurrent UTIs in five women who had had at least three infections during the 3 months prior to instituting vaginal estrogen therapy [45]. Corresponding reduction of vaginal pH with return of gramnegative vaginal flora to normal flora was noted in this study. Similarly, Privette and colleagues found that estrogen treatment (either oral or vaginal) led to almost total irradication of recurrent UTIs in a group of 12 patients with a history of frequent UTIs [44]. Randomized trials have been few, but their results have been striking. The first of these randomized women to oral estriol or placebo, and found that 75% of those on active treatment had a reduced rate of UTI after 12 weeks, whereas only 40% of those assigned to placebo improved [46]. Even more remarkable are results from one 8-month randomized controlled trial of vaginal estriol for women with recurrent UTIs [47]. In this trial, the treated group experienced 0.5 UTI episodes per patient-year, whereas the corresponding rate in the control group was 5.9 UTI episodes. Corresponding improvements in pH and bacterial flora were noted in the treated groups in both of these trials. In summary, although data on estrogen and UTI prevention are limited, the relative safety of estrogen (especially vaginally) in contrast to the morbidity and mortality of recurrent UTIs makes a vaginal estrogen trial worth considering in this circumstance. (For further discussion, see Section III,B.)

D. Osteoporosis: Patterns of Bone Loss with Aging and Treatment Issues in Older Women Chapter 19 presents detailed information regarding menopause and osteoporosis. This section concentrates on aspects of the natural history of osteoporosis and its treatment that are germane to older women. Until recently, based on the idea that bone loss slowed or

CHAPTER45 Menopause Issues for Older Women halted in older women, initiating treatment in women in the seventh or eighth decade of life was often felt to be unnecessary and nonhelpful. The realization that the rate of bone loss not only does not slow, but actually increases with advancing age [48-52] necessitates reconsideration of this position. Although we now know that bone loss continues to be a problem well past menopause, specific data on the effectiveness of initiation of treatment in later life are limited. Intervention trials initiated in women who were 10 or even 20 years postmenopausal, some of which included women with established osteoporosis by fracture criteria [53-57], generally reported that the response of B MD to estrogen was not less in older women or in women with prior fragility fracture. Results from the Rancho Bernardo Study, a large observational study of chronic diseases, address the timing of initiation and the need to continue postmenopausal hormone therapy for osteoporosis prevention [58]. The investigators considered five categories of women: never-users, past early-users (began prior to age 60 but no longer using), past late-users (began after age 60 and no longer using), current late-users (began after age 60 and continued to use), and current continuous-users (started prior to age 60 and continue to use). Among current hormone users, there was no substantial or statistically significant difference in BMD between those who initiated treatment prior to or after age 60 years. B MDs of past users, irrespective of whether hormones were initiated soon after the menopause, or continued for up to 10 years, were similar to those who had never used estrogen. These studies suggest that late initiation of estrogen remains efficacious with respect to B MD preservation and that estrogen use must be continued indefinitely, because even long-duration former use does not confer BMD benefit. The observation that BMD can be stabilized by hormone treatment in older women does not assure, however, that fracture risk will be lowered. One concern is that if substantial microarchitectural connectivity has already been lost, increasing BMD might not be tantamount to gaining structural integrity. (For example, trabeculae that are not communicating may become wider as a result of hormone therapy, but this might not translate into improved bone strength.) An analysis from the Study of Osteoporotic Fractures (SOF), a large community-based observational study, examined the question of actual fracture prevention in relation to timing of hormone use [59]. Analogous to the BMD results previously reviewed, compared to never-users of estrogen, current users were protected against hip fracture (relative risk, 0.60), wrist fracture (relative risk, 0.39), and all nonspinal fractures (relative risk, 0.66). Compared to neverusers, former users, regardless of duration of use, were not at lower risk of fractures. Importantly, when current users were further subdivided into those who began hormones within 5 years of menopause versus those who began later, only current users who started early were at reduced fracture

643 risk (relative risks for hip, wrist, and all nonspinal fractures were 0.29, 0.29, and 0.50, respectively). Thus, the SOF results concur with other observational data that hormone replacement therapy (HRT) use must be substantial to be effective, but differ in that fractures were not prevented unless HRT was instituted within 5 years of menopause. Some insight into this conundrum may be gained from fracture intervention studies. There are scant data in the case of estrogen, but in one small clinical trial, transdermal 17fiestradiol appeared to have decreased the incidence of vertebral fracture in women with preexisting osteoporotic fracture [57]. More compelling evidence that starting treatment late is effective comes from large randomized controlled trials of alendronate for primary and secondary vertebral fracture prevention [60,61 ]. Stratified analyses by age greater than or less than 75 years demonstrate equivalent fracture prevention in the younger and older age groups [60]. Additionally, alendronate prevents further compression of partially compressed vertebral bodies as well as preventing new vertebral fractures in women with prior vertebral fractures. In summary, recent observational studies reporting continued and even increasing bone loss with greater age underscore the necessity for considering preventive or remedial strategies in older postmenopausal women. Current data support long-term continuation of estrogen therapy to prevent osteoporosis in older women. Appropriate recommendations for women who have not been previously treated are more difficult to determine at present. Several small intervention studies show a BMD-preserving effect of estrogen begun at later ages. The cohort study that examined fracture outcomes in relation to the onset of estrogen use after the menopause found that an early start was required for fracture prevention. Alendronate treatment, however, works well when instituted in late postmenopausal and older aged women and in those with established vertebral fracture.

III. THERAPEUTIC IN LATE MENOPAUSAL

CONSIDERATIONS WOMEN

A. S y s t e m i c E s t r o g e n a n d P r o g e s t o g e n T r e a t m e n t No formal data describe the attitudes of older women toward hormone treatment or characterize the side effects HRT in older compared to younger women. However, clinical experience suggests that one major concern of older women and their families is the potential return of vaginal bleeding due to hormone treatment. Bleeding patterns accompanying hormone treatment vary by regimen. Most of the detailed information regarding treatment-related bleeding patterns, however, comes from early postmenopausal women [62-64]. When estrogen is given with a cyclic monthly progestogen, approximately 70% of women have predictable cyclic bleeding that continues indefinitely. Continuous combined estrogen and progestin

644

GAIL A. GREENDALE

produce unpredictable, light to moderate spotting and bleeding; however, the bleeding ceases in approximately 90% of women within 12 months of treatment [62-64]. Bleeding terminates with continuous combined treatment because the treatment induces atrophy of the endometrial lining. One might postulate that older women taking continuous combined hormone therapy would experience less bleeding and earlier cessation of bleeding compared to younger women, because older women already have a more atrophic endometrium at the beginning of treatment. When continuous combined hormone replacement with conjugated equine estrogen and medroxyprogesterone acetate was used, women with longer duration of menopause (e.g., greater than 2 years) at the start of treatment experienced less bleeding and had more amenorrhea than those with more recent menopause [64]. When continuous combined 17fl-estradiol and norethisterone was administered to women with a mean age of 65 years, only 30% of the 20 women on active treatment had vaginal bleeding, which was light and ceased in all instances by 6 months of treatment [54]. Overall, it is reasonable to recommend continuous combined hormone treatment as the preferred postmenopausal hormone regimen in those older women who wish to minimize resumption of vaginal bleeding. Some older women may wish to avoid systemic doses of estrogens and progestogens, but may have conditions that would benefit from local estrogen treatment (for example, vaginal atrophy, recurrent UTI, and UI). It is appropriate to consider vaginally applied estrogens in these instances.

B. V a g i n a l E s t r o g e n s Is vaginal estrogen local therapy? This question has been approached by evaluating the effects of vaginally applied estrogens on estrogen-responsive tissues (e.g., uterus, liver), by

TABLE I Generic name

Trade name

Estradiol tablet

Vagifem

Estradiol ring

Estring

Estriol cream

Ovestin, Synopause

measuring proteins and hormones that change in response to estrogen administration (e.g., sex hormone-binding globulin or follicle-stimulating hormone), and by measuring systemic concentrations of estrogens during treatment with vaginal preparations. Serum estrogen values must be interpreted with caution, especially when the prescribed agent is conjugated equine estrogens, which are composed of multiple estrogenic compounds that are not detected in the serum assays for estradiol or estrone. The absence of change in these variables is interpreted as an indicator that the vaginally administered estrogen acts only locally. Table I summarizes the results of some topical vaginal estrogen studies. A 52-week unmasked trial of low-dose (25 /xg) 17fl-estradiol vaginal tablets reported no biopsyassessed endometrial hyperplasia at the conclusion of treatment in 43 women [65]. Three women did develop weakly proliferative endometrial histology, indicating some estrogen effect on endometrial cells. This study also measured estradiol, follicle-stimulating hormone, and luteinizing hormone, none of which changed as a result of treatment. A shorter (22-week) study of two doses of vaginal estradiol in 20 postmenopausal women similarly found no hyperlasia in the group given 25-/xg vaginal tablets [66]. Pooled data from 12 studies that tested low doses of estriol vaginal cream (0.5 mg daily for 2 - 3 weeks and then 0.5 mg twice per week) suggest that endometrial stimulation does not occur with that regimen [67]. No endometrial proliferation was evident in 61 biopsies taken after 6 months of use and 58 biopsies performed after 12 months of treatment [67]. Serum estriol concentrations do rise, however, as a result of estriol vaginal cream use [68]. The endometrial stimulatory effect of a vaginal ring that releases 17fl-estradiol, 7.5/xg per 24 hr, has been tested in the United States and Australia [69]. These unmasked, nonplacebo controlled studies used a progestin challenge test (PCT) at 13 weeks to evaluate endometrial safety (bleeding after administration of progestin indicates

Vaginal Estrogen Preparations

Description 25/xg of 17fl-estradioI per tablet Silicon elastomerring with core containing 2 mg of estradiol (releasing 7.5/xg/24 hr Contains 1 mg/g

Evidence for local effect

Frequency One tablet twice weekly Replace every 90 days

0.5 mg of estriol twice weekly

Low serum Ej, E 2 levels; no endometrial hyperplasiaa Low serum E lb, E2 levels; no elevation of SHBG; progestin challenge test negative Low serum E ~,E2 levelsb; elevation in E 3 level; no elevation of SHBG; no endometrial hyperplasiaa

a Endometrial hyperplasia assessed by biopsy. Rarely, proliferative endometrium occurs.

bEstrone levels increased to 950 pmol/hr (within "postmenopausalrange" but greater than baseline value).

CHAPTER45 Menopause Issues for Older Women endometrial stimulation). In the Australian study, 5 of 122 participants had positive PCTs and in the United States trial 2 of 58 challenges were positive. Four of the seven women with bleeding following PCT underwent biopsy that was negative for hyperplasia. Although the 17/3-estradiol ring has not caused hyperplasia by the PCT criterion, two other studies raise some concerns about a possible systemic effect. In one study, estrone levels increased to 950 pmol/liter after 48 weeks of ring use (within the "postmenopausal range" but higher than baseline) [70]. Surprisingly, one investigation found that the vaginal estradiol ring preserved bone density [71]. Two older vaginal preparations, conjugated equine estrogens cream and 17fl-estradiol vaginal cream, are not discussed here because there are no comprehensive data available for low-dose vaginal application of these products. In summary, the endometrial safety of low-dose estriol vaginal cream, low-dose 17fl-estradiol vaginal tablet, and the vaginal estrogen ring appears to be reasonably well established. At most, these medications produce weakly proliferative endometrial tissue. However, concerns remain about potential effects on other tissues. Therefore, in women in whom no systemic exogenous estrogen exposure is desired, these agents should be used with caution.

C. N o n e s t r o g e n V a g i n a l T h e r a p y Vaginal atrophy can be managed to some degree with vaginal moisturizers. Examples include Astroglide, GyneMoistrin, Moist Again, and Replens. Replens decreases vaginal pH, thus may offer some benefit in UTI prevention (although it has not been specifically tested for this purpose) [72].

D. P e s s a r i e s Prolapse of the uterus and bladder results from several factors, such as multiparity, birth trauma, increased intraabdominal pressure, and atrophy of the vaginal epithelium related to low estrogen levels. Pelvic prolapse may lead to uncomfortable symptoms such as a feeling of pelvic pressure or heaviness. Vaginal bleeding may occur if the prolapse is substantial enough to lead to erosions (this generally happens in the case of second- or third-degree prolapse, with extension of the cervix to the level of or outside the introitus). In some instances, urinary incontinence may be caused or exacerbated by uterine or bladder prolapse [34]. In older women, particularly those whose comorbidity places them at unacceptably high surgical risk, pessaries represent an important alternative to surgical management of prolapse. As in younger women, pessaries may also be useful as a temporizing measure until a later definitive prolapse repair is executed, or they may be used as a preoperative diag,

645 nostic aid to assess how anatomic reconstruction will affect lower urinary tract function. There are many types of pessaries. The choice of the pessary type depends on several factors: the degree of prolapse, the anatomic support desired, the anatomy of the patient, and the ability of the patient to self-manage the pessary. The major types of pessaries are illustrated in Fig. 1. Pessaries that require complex fitting or uterine manipulation will not be discussed, but are reviewed by Zeitlin and Brubaker [73,74]. A ring pessary is simple for a generalist physician to prescribe and for the patient to use. It looks like the outer ring of a diaphragm, and is fit (between the symphysis pubis and vaginal apex) in a similar fashion. The flexible ring facilitates insertion, which may be a significant issue in women with a narrowed vaginal canal. Drainage of vaginal secretions is not substantially blocked, which is relevant to patients with limited capability to remove and clean the pessary ~themselves. The ring's major shortcoming is that it offers limited pelvic support, and therefore is not adequate in cases of substantial prolapse. The cube pessary, which looks like a cube with concave sides, adheres to the vaginal walls via the suction created by its concavities. It is even easier to fit and place than the ring, because it is simply inserted in the vagina and does not require further manipulation. The cube offers more structural support than does the ring pessary and can be used with fairly advanced prolapse. Drainage around the cube is limited, therefore self-management skills are more important. Pessary care includes assessment of stability, managing vaginal secretions, and surveillance for vaginal erosion. Asking the patient to walk around in the privacy of the examining room assesses stability. Asking her to attempt toileting after the pessary has been placed is also suggested. Managing secretions is easy if the patient is capable of removing, washing, and reinserting the pessary every few days (in the case of the cube) or weekly (in the case of the ring). However, many older women cannot or do not wish to withdraw and replace the pessary. In a clinic that manages pessaries in women in their 70s and 80s, we have found that we can manage pessary cleansing and assessment with short visits every 2 to 4 weeks, depending on the pessary type. To prevent erosion of thinned vaginal epithelium by the pessary, some authors recommend prophylactic use of vaginal estrogen (in women who are not taking systemic postmenopausal hormones) [75,76]. However, other practitioners manage this on a case-by-case basis, rather than using vaginal estrogens in all postmenopausal patients with pessaries.

IV. C O N C L U S I O N S Research in menopause and its implications for health and quality of life has burgeoned in the last decade. As the field continues to mature, increased attention to specific

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GAIL A. GREENDALE

FIGURE 1 Pessaries: (A) ring (silicone, folding), (B) ring (with support, silicone, folding), (C) Shaatz (silicone, folding), (D) Gellhorn (silicone, 95% rigid), (E) Gellhorn (acrylic, rigid), (F) Gellhorn (silicone, flexible), (G) Risser (silicone, folding), (H) Smith (silicone, folding), (I) tandem cube (silicone, flexible), (J) cube (silicone, flexible), (K) Hodge with knob (silicone, folding), (L) Hodge (silicone, folding), (M) Hodge (with support, silicone, folding), (N) Gehrung (silicone, folding), (O) donut (silicone), (P) incontinence dish (with support, silicone, folding), (Q) incontinence dish (silicone, folding), (R) ring incontinence (silicone, folding), (S) Inflatoball (latex). Photograph courtesy of Milex Products, Inc., Chicago, Illinois.

issues for late menopausal and older women is appropriate. Important priorities include a more complete understanding of the natural history of menopause-related syndromes in older women; increased attention to the relative effectiveness and side effects of interventions in late menopausal and older versus younger women; and a more thorough investigation of newly emerging areas, such as the relation between menopause and cataracts, that are of particular relevance in maintaining health and functional independence in old age.

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Management of Functional Impairment," Clin. Prac. Guideline, No. 4, AHCPR Publ. No. 93-0542. U.S. Dept. of Health and Human Services, Rockville, MD. Klein, B. E. K., Klein, R., and Linton, K. L. P. (1992). Prevalence of age-related lens opacities in a population. Ophthalmology 99, 5 4 6 552. Klein, B. E. K., Klein, R., and Ritter, L. L. (1994). Is there evidence of an estrogen effect on age-related lens opacities? Arch. Ophthalmol. 112, 85-91. Reidy, A., Minassian, D. C., Vafidis, G., Joseph, J., Farrow, S., Wu, J., Desai, P., and Connolly, A. (1998). Prevalence of serious eye disease and visual impairment in a north London population: Population based, cross sectional study. Br. Med. J. 316, 1643-1646. Cumming, R. G., and Mitchell, P. (1997). Hormone replacement therapy, reproductive factors, and cataract. Am. J. Epidemiol. 145, 242-249. Benitez del Castillo, J. M., del Rio, T., and Garcia-Sanchez, J. (1997). Effects of estrogen use on lens transmittance in postmenopausal women. Ophthalmology 104, 970-973. Hales, A. M., Chamberlain, C. G., Murphy, C. R., and McAvoy, J. W. (1997). Estrogen protects lenses against cataract induced by transforming growth factor-fi (TGFfi). J. Exp. Med. 185, 273-280. Ouslander, J. G. (1992). Geriatric urinary incontinence. Disease-AMonth 2, 65-149. Reuben, D. B., Yoshikawa, T. T., and Bedsine, R. W., eds. (1996). "Geriatrics Review Syllabus: A Core Curriculum in Geriatric Medicine," 3rd ed. Kendall/Hunt Publishing Company for the American Geriatrics Society, Dubuque, IA. Griebling, T. L., and Nygaard, I. E. (1997). The role of estrogen replacement therapy in the management of urinary tract infection in postmenopausal women. Endocrinol. Metab. Clin. North Am. 26, 347-360. Milsom, I., Ekelund, P., Molander, U., Arvidsson, L., and Areskoug, B. (1993). The influence of age, parity, oral contraception, hysterectomy, and menopause on the prevalence of urinary incontinence in women. J. Urol. 149, 1459-1462. Rekers, H., Drogendijk, A. C., Valkenburg, H. A., and Riphagen, F. (1992). The menopause, urinary incontinence and other symptoms of the genito-urinary tract. Maturitas 15, 101-111. Hinton, P., Tweddell, A. L., and Mayne, C. (1990). Oral intravaginal estrogens alone and in combination with alpha adrenergic stimulation in genuine stress incontinence. Int. Urogynecol. J. 1, 80-86. Walter, S., Wolf, H., and Barlebo, H. (1978). Urinary incontinence in postmenopausal women treated with estrogens. Urol. Int. 33, 135-143. Fantl, J. A., Cardozo, L., and McClish, D. K. (1994). Estrogen therapy in the management of urinary incontinence in postmenopausal women: A meta-analysis. Obstet. Gynecol. 83, 12-18. Fantl, J. A., Bump, R. C., Robinson, D., McClish, D. K., and Wyman, J. F. (1996). Efficacy of estrogen supplementation in the treatment of urinary incontinence. Obstet. Gynecol. 88, 745-749. Ouslander, J., Leach, G., Staskin, D., Abelson, S., Blaustein, J., Morishita, L., and Raz, S. (1989). Prospective evaluation of an assessment strategy for geriatric urinary incontinence. J. Am. Geriatr. Soc. 37, 715-724. Privette, M., Cade, R., Peterson, J., and Mars, D. (1988). Prevention of recurrent urinary tract infections in postmenopausal women. Nephron 50, 24-27. Parsons, C. L., and Schmidt, J. D. (1982). Control of recurrent lower urinary tract infection in the postmenopausal woman. J. Urol. 128, 1224 -1226. Kirkengen, A. L., Andersen, P., Gjersoe, E., Johannessen, G. R., Johnsen, N., and Bodd, E. (1992). Oestriol in the prophylactic treatment of recurrent urinary tract infections in postmenopausal women. Scand. J. Primary Health Care 10, 139-142. Raz, R., and Stamm, W. E. (1993). A controlled trial of intravaginal estriol in postmenopausal women with recurrent urinary tract infections. N. Engl. J. Med. 329, 753-760.

648 48. Jones, G., Nguyen, T., Sambrook, E, Kelly, E J., and Eisman, J. A. (1994). Progressive loss of bone in the femoral neck in elderly people: Longitudinal findings from the Dubbo osteoporosis epidemiology study. Br. Med. J. 309, 691-695. 49. Burger, H., de Laet, C. E. D. H., van Daele, E L. A., Weel, A. E. A. M., Witteman, J. C. M., Hofman, A., and Pols, H. A. E (1998). Risk factors for increased bone loss in an elderly population. Am. J. Epidemiol. 147, 871-879. 50. Ensrud, K. E., Palermo, L., Black, D. M., Cauley, J., Jergas, M., Orwoll, E. S., Nevitt, M. C., Fox, K. M., and Cummings, S. R. (1995). Hip and calcaneal bone loss increase with advancing age: Longitudinal results from the study of osteoporotic fractures. J. Bone Miner. Res. 10, 17781787. 51. Greenspan, S. L., Maitland, L. A., Myers, E. R., Crasnow, M. B., and Kido, T. H. (1994). Femoral bone loss progresses with age: A longitudinal study in women over age 65. J. Bone Miner. Res. 9, 1959-1965. 52. Hannan, M. T., Felson, D. T., and Anderson, J. J. (1992). Bone mineral density in elderly men and women: Results from the Framingham Osteoporosis Study. J. Bone Miner. Res. 7, 547-553. 53. Writing Group for the PEPI Trials (1996). Effects of hormone therapy on bone mineral density: Results from the Postmenopausal Estrogen/ progestin Interventions (PEPI) trial. JAMA, J. Am. Med. Assoc. 276, 1389-1396. 54. Christiansen, C., and Riis, B. J. (1990). 17fl-Estradiol and continuous norethisterone: A unique treatment for established osteoporosis in elderly women. J. C/in. Endocrinol. Metab. 71, 836-841. 55. Quigley, M. E. T., Martin, P. L., Burnier, A. M., and Brooks, E (1986). Estrogen therapy arrests bone loss in elderly women. Am. J. Obstet. Gynecol. 156, 1516-1523. 56. Lindsay, R., and Tohme, J. F. (1990). Estrogen treatment of patients with established postmenopausal osteoporosis. Obstet. Gynecol. 76, 290-295. 57. Lufkin, E. G., Wahner, H. W., O'Fallon, W. M., Hodgson, S. G., Kotowicz, M. A., Lane, A. W., Judd, H. L., Caplan, R. H., and Riggs, B. L. (1992). Treatment of postmenopausal osteoporosis with transdermal estrogen. Ann. Intern. Med. 117, 1-9. 58. Schneider, D. L., Barrett-Connor, E., Morton, and D. J. (1997). Timing of postmenopausal estrogen for optimal bone mineral density. JAMA, J. Am. Med. Assoc. 277, 543-547. 59. Cauley, J. A., Seeley, D. G., Ensrud, K., Ettinger, B., Black, D., and Cummings, S. R. (1995). Estrogen replacement therapy and fractures in older women. Ann. Intern. Med. 122, 9-16. 60. Ensrud, K. E., Black, D. M., Palermo, L., Bauer, D. C., Barrett-Connor, E. Quandt, S. A., Thompson, D. E., and Karpf, D. B. (1997). Treatment with alendronate prevents fractures in women at highest risk. Arch. Intern. Med. 157, 2617-2624. 61. Black, D. M., Cummings, S. R., Karpf, D. B., Cauley, J. A., Thompson, D. E., Nevitt, M. C., Bauer, D. C., Genant, H. K., Haskeil, W. L., Mar-

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cus, R., Ott, S. M., Torner, J. C., Quandt, S. A., Reiss, T. E, Ensrud, K. E., for the Fracture Intervention Trial Research Group (1996). Randomized trial of effect of alendronate on risk of fracture in women with existing vertebral fractures. Lancet 348, 1535-1541. Archer, D. E, Pickar, J. H., and Bottiglioni, E (1994). Bleeding patterns in postmenopausal women taking continuous combined or sequential regimens of conjugated estrogens with medroxyprogesterone acetate. Obstet. Gynecol. 83, 686-692. Magos, A. L., Brincat, M., Studd, J. W. W., Wardle, E, Schlesinger, E, and O'Dowd, T. (1985). Amenorrhea and endometrial atrophy with continuous oral estrogen and progestogen therapy in postmenopausal women. Obstet. Gynecol. 65, 496-499. Woodruff, J.D., and Pickar, J. H. (1994). Incidence of endometrial hyperplasia in postmenopausal women taking conjugated estrogens (Premarin) with medroxyprogesterone acetate or conjugated estrogens alone. Am. J. Obstet. Gynecol. 170, 1213-1223. Mettler, L., and Olsen, E G. (1991). Long-term treatment of atrophic vaginitis with low-dose oestradiol vaginal tablets. Maturitas 14, 1231 Mattson, L. A., Cullberg, G., Eriksson, O., and Knutsson, E (1989). Vaginal administration of low-dose oestradiol--Effects on the endometrium and vaginal cytology. Maturitas 11,217-222. Vooijs, G. E, and Geurts, T. B. E (1995). Review of the endometrial safety during intravaginal treatment with estriol. Eur. J. Obstet. Gynecol. Reprod. Biol. 62, 101 - 106. Kicovic, E M., Cortes-Prieto, J., Milojevic, S., Haspels, A. A., and A1jinovic, A. (1992). The treatment of postmenopausal vaginal atrophy with ovestin vaginal cream or suppositories: Clinical, endocrinological and safety aspects. Maturitas 2, 275-282. Nachtigall, L. E. (1995). Clinical trial of the estradiol vaginal ring in the U.S. Maturitas 22, $43-$47. Smith, E, Heimer, G., Lindskog, M., and Ulmsten, U. (1995). Oestradiol-releasing vaginal ring for treatment of postmenopausal urogenital atrophy. Maturitas 16, 145-154. Naessen, T., Bergund, L., and Ulmsten, U. (1997). Bone loss in elderly women prevented by ultralow doses of perenteral 17fl-estradiol. Am. J. Obstet. Gynecol. 177, 115-119. Nachtigall, L. E. (1994). Replens versus local estrogen in menopausal women. Fertil. Steril. 61, 178-180. Zeitlin, M. P. and Lebherz, T. B. (1992). Pessaries in the geriatric patient. J. Am. Geriatr. Soc. 40, 635-639. Brubaker, L. (1994). The pessary: An important gynecological option. Menopausal Med. 2, 1-4. Nichols, D. H., and Julian, P. J. (1985). The vaginal pessary. In "Ambulatory Gynecology" (D.H. Nichols and J.R. Evard, eds.), pp. 193199. Harper & Row, Philadelphia. Greenhill, J. P. (1972). The nonsurgical management of vaginal relaxation. Clin. Obstet. Gynecol. 15, 1083-1097.

HAPTER 4(

Deci "s on Analy AppliedsIs "

to Postmenopausal Hormone Replacement Therapy ANNA N. A. TOSTESON

I. II. III. IV. V.

Clinical Research Section, Department of Medicine and the Center for the Evaluative Clinical Sciences, Department of Community and Family Medicine, Dartmouth Medical School, Hanover, New Hampshire 03755

Introduction Decision Analysis Review of Studies Treatment Regimens Treatment Risks and Benefits

VI. Estimated Changes in Life Expectancy VII. Limitations VIII. Summary References

I. I N T R O D U C T I O N

II. DECISION

Accounting for the multifaceted effects of hormone replacement therapy (HRT) on health is a challenging task. Although the potential benefits include menopausal symptom relief and reduced incidence of osteoporotic fractures and heartdisease, the potential harms include increased incidence of endometrial and breast cancers. Quantitative syntheses of clinical and epidemiological data using decision analysis have provided valuable insights into the net health effects of HRT [ 1,2] and have played a role in clinical guideline development [3]. In this chapter, decision analysis is introduced, applications of decision analysis to postmenopausal hormone, replacement therapy are reviewed, and updated estimates of the effect of hormone replacement therapy on life expectancy are reported.

MENOPAUSE" BIOLOGY AND PATHOBIOLOGY

ANALYSIS

Decision analysis is a quantitative approach to decision making under conditions of uncertainty [4]. This technique has been promoted for assessing the value of health interventions, due in part to the uncertainty that is inherent in the practice of medicine [5-7]. Decision analysis facilitates identification of the clinical management strategy that, on average, provides the best health outcome. Use of decision analysis is particularly appealing for interventions, such as hormone replacement therapy, that have the potential to affect many diseases. When both costs and health effects are analyzed simultaneously, decision analysis has also provided a framework for assessing the cost effectiveness of many health interventions including HRT [8-16]. More importantly, by systematically varying assumptions in extensive analyses, decision-

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Copyright9 2000by AcademicPress. All rightsof reproductionin any formreserved.

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ANNA N. A. TOSTESON o

analytic techniques are used to highlight key assumptions that dictate which clinical management strategy is "best." When substantial uncertainty surrounds critical assumptions, decision-analytic techniques can be used to quantify explicitly the expected effect of a health intervention under multiple, clearly defined scenarios. For example, decision analysis can be used to estimate the range of likely health effects for combined estrogen-progestin therapy as the assumed influence of treatment on breast cancer is varied even though the epidemiological evidence is scant (see Chapter 40). The steps involved in decision analysis include problem definition, model development, parameter estimation for probability and outcome values, and analysis. To assess the effects of long-term use of hormone replacement therapy on life expectancy, a previously described decision-analytic Markov state transition model was modified to reflect up-todate assumptions regarding the risks and benefits of treatment with hormone replacement therapy [11]. The model follows hypothetical cohorts of women who are initially well, until death or age 99 years, and tracks the annual occurrence of breast cancer, heart disease, and hip fracture. Annual age-specific incidences of these diseases are also updated. To estimate the average effect of hormone replacement therapy on life expectancy, a cohort of untreated women was first simulated. Next, a cohort of treated women was simulated. The difference in estimated life expectancy between the treated and untreated cohorts forms the basis for the projected health benefit of long-term hormone replacement therapy. By systematically evaluating the effect of treatment for cohorts of women at various risks for osteoporosis, breast cancer, and heart disease, estimates are provided that can help clinicians when counseling individual women. Customized information on the risks and benefits of treatment should enhance informed decision making [ 17]. A review of selected studies that have used decision-analytic approaches to estimate the health effects of hormone replacement therapy is provided in the next section.

III. REVIEW

OF STUDIES

Over the past 20 years, a number of studies have used decision-analytic techniques to estimate the average change in life-expectancy associated with long-term use of hormone replacement therapy [ 1,2,8-16,18]. In addition to estimating the health impact of HRT, most studies also estimated the net increase in cost associated with long-term HRT use and have suggested that HRT provides good value for the resources expended (i.e., it is cost-effective). Estimated changes in life expectancy for 50-year-old women initiating treatment with hormone replacement therapy have ranged from losses of 6 months to gains of over 3 years, depending on a woman's base line risk for breast cancer, heart disease, and osteopo-

rosis [1,2,8-16,18]. These wide-ranging estimates reflect both evolving scientific knowledge regarding the effect of HRT on disease incidence and the differing treatment regimens and durations evaluated. In the first analysis to estimate life expectancy changes for 50-year-old women treated with estrogen combined with progestin relative to an untreated cohort, Weinstein and Schiff [9] in 1982 reported modest gains of less than 1 month (7 to 26 days) for women treated for 10 or 15 years, respectively. This analysis was completed before sufficient epidemiological evidence was available to estimate the effect of hormone replacement therapy on coronary heart disease. More recently, studies have evaluated changes in coronary heart disease incidence, and projected gains in life expectancy have increased [1,2,12-15,18]. Although Tosteson and colleagues estimated net increases in life expectancy that ranged from 0.7 to 2.9 months, the treatment durations considered did not exceed 15 years [ 12]. With lifelong estrogenprogestin treatment, Grady and colleagues projected gains in life expectancy of 1.2 to 12 months for average risk women [ 1]. The health benefits of long-term hormone replacement therapy are closely linked to the absolute risks of breast cancer, heart disease, and osteoporosis in the population of interest. For example, Grady and colleagues projected larger gains for women at high risk for coronary heart disease, ranging from 7.2 to 19.2 months, and potential losses for women at high risk for breast cancer [1 ]. When Col and colleagues considered women's risk for breast cancer and heart disease simultaneously, the population subgroup that would benefit most from long-term treatment was women at high risk for heart disease and low risk for breast cancer [2]. More recently, Col and colleagues also integrated varying osteoporosis risks [ 18]. For long-term use of combined estrogenprogestin therapy, a loss of 5 months of life was projected for women at low risk of osteoporosis and heart disease, but at high risk for breast cancer. In contrast, women at high risk for osteoporosis and heart disease and low risk for breast cancer may gain 28.8 months (2.4 years). In addition to the absolute risks of each disease, the estimated health effects also depend critically on the relative risk reduction (or increase) that is conferred with long-term use of hormone replacement therapy. Until clinical trial results become available, there will be ongoing uncertainty and debate over these potential benefits (or harms). Decision analysis is particularly useful in the context of uncertain treatment effects, because several alternative scenarios for the influence of HRT on breast cancer, heart disease, and osteoporosis can be posed and evaluated quantitatively. By varying the assumed relative risks of long-term treatment across an interval considered reasonable (i.e., conducting "sensitivity analyses"), a range of estimated health effects is defined. To provide up-to-date estimates of the potential health effects of hormone replacement therapy we evaluate

CHAPTER46 Decision Analysis in Postmenopausal Therapy several treatment regimens and make explicit assumptions about treatment risks and benefits, as described in the next sections.

IV. T R E A T M E N T

REGIMENS

The long-term use of two HRT regimens is considered. First, for women with a uterus, a 15-year regimen of combined continuous HRT (e.g., 0.625 mg conjugated estrogens/ 2.5 mg medroxyprogesterone acetate) is evaluated. Second, for women who have had a hysterectomy, a 15-year course of unopposed estrogen (e.g., 0.625 mg conjugated equine estrogen) taken daily is evaluated.

V. TREATMENT RISKS AND BENEFITS A. O s t e o p o r o s i s and H i p F r a c t u r e Osteoporosis is associated with the increased incidence of skeletal fracture [19]. The most common "osteoporotic" fractures are those of the hip, spine, and wrist. Here, the decision-analytic model tracks hip fracture incidence, because of the excess mortality that is associated with these fractures. Using Olmsted County, Minnesota, data, one-half of the mortality observed in the year following hip fracture is attributed to the fracture [11]. Hip fracture incidence is assumed to be reduced by 75% for women treated with either hormone replacement therapy regimen [1]. Because some epidemiological studies have reported more favorable risk reductions for long-term current HRT users [20], a re-

TABLE I

651 duction of 50% was considered under the most favorable assumption set for both unopposed estrogen and combined estrogen-progestin therapy (Table I). Under all assumption scenarios, the hip fracture treatment benefit was assumed to remain for only 5 years following the termination of therapy [21,22].

B. B r e a s t C a n c e r The influence of hormone replacement therapy on breast cancer incidence remains uncertain and has been the focus of several meta- and pooled analyses [ 1,23]. Unopposed estrogen use is associated with an increased risk of breast cancer, which increases with duration of use [23]. The base line scenario for unopposed estrogen used a relative risk of 1.35 from 5 years of treatment until 2 years beyond termination of treatment (Table I). The effect of combined estrogen-progestin therapy on breast cancer incidence and mortality is more uncertain [1,23] (see Chapter 40) and was addressed under three scenarios. In the base line scenario, the increased risk of breast cancer for combined therapy was assumed equivalent to the effect of unopposed estrogen. Under the most favorable scenario, breast cancer risk was not affected by combined therapy. Finally, under the least favorable scenario, the risk of breast cancer was assumed to increase by 50%.

C. H e a r t D i s e a s e Epidemiological studies have consistently found that unopposed estrogen use is associated with lower risk of

Summary of Relative Risks for Women Treated with Hormone Replacement Therapy Relative to Untreated Women Incidence of

Treatment regimen and assumption scenario

Breast cancer

Heart disease

Hip fracture

Endometrial cancer

Unopposed estrogen therapy Base line a Most favorable Least favorable

1.35 b 1.35 b 1.35 b

0.65 0.65 0.85

0.75 0.50 0.75

n/a n/a

Combined estrogen-progestin therapy Base line a Most favorable Least favorable

1.35 b 1.00 1.50

0.85 0.65 1.00

0.75 0.50 0.75

1.0 1.0 1.0

n/a c

a The term "base line" refers to the current best-guess assumption scenario regarding the effect of treatment on disease incidence. blncreases to 1.35 after 5 years of use, and increased risk remains throughout duration of treatment. Elevated risk returns to normal 2 years after termination of treatment. Cn/a, Not applicable.

6

5

2

A

N

N

coronary heart disease. A meta-analysis of the effect of unopposed estrogen on coronary heart disease mortality [ 1] reported a relative risk of 0.63 (95% CI, 0 . 5 5 - 0 . 7 2 ) for women ever using unopposed estrogen relative to nonusers. In this analysis, a 35% risk reduction in coronary heart disease incidence is assumed and the benefit of treatment continues for only 2 years beyond termination of therapy. Because reductions in coronary heart disease incidence may have been overestimated in observational studies, the risk reduction for unopposed estrogen is limited to 15% under the unfavorable scenario. Although data on the effects of combined estrogenprogestin therapy on coronary heart disease risk remain limited, it has generally been believed that some protective benefit exists. Indeed, studies of the mechanism of disease support this hypothesis [24] as does the scant but growing epidemiological evidence on combined therapy [25,26] (see Chapters 28 and 37). However, recent randomized clinical trial results regarding hormone replacement therapy among women with existing coronary heart disease challenge this notion [27]. Because of this uncertainty, we evaluate three scenarios for combined estrogen-progestin therapy. Under the base line scenario, the incidence of coronary heart disease is reduced by a conservative 15%. Under the most favorable scenario, combined therapy is projected to reduce the coronary heart disease incidence the same as unopposed estrogen (reduction of 35%). Under the least favorable scenario, combined therapy is projected to have no effect on coronary heart disease incidence (Table I). For all treatment regimens, coronary heart disease benefits are only effective until 2 years beyond treatment termination.

A

N. A.

D.

Other Diseases

The only diseases considered separately with respect to their impact on life expectancy were heart disease, breast cancer, and hip fracture. Although hormone replacement therapy may also influence the incidence of endometrial cancer, deep vein thrombosis [28], colon cancer [29], stroke [30,31], and Alzheimer's disease [32], these factors are not explicitly considered in the analysis. Age-specific rates of death from other causes were based on United States life tables [33].

VI. LIFE

ESTIMATED

CHANGES

On average, a 15-year course of treatment with unopposed estrogen was projected to extend life by 5.5 months and treatment with estrogen combined with progestin was projected to extend life by 2.9 months (Table II). As expected, long-term use of hormone replacement therapy resulted in larger gains in women at higher risk (twofold greater than average risk) for heart disease and smaller gains in women at higher risk for breast cancer. Overall, women at higher risk for heart disease and hip fracture and lower risk for breast cancer were projected to have the largest life expectancy gains. Women at lower risk for heart disease and hip fracture and higher risk for breast cancer would see minimal gains, if any. Indeed, under base line assumptions, when treated with combined therapy, such women are expected to lose approximately 15 days of life.

Change (months) with 15-yeartreatment"

Average risk Higher risk for heart disease Higher risk for breast cancer Higher risk for heart disease and fracture and lower risk for breast cancer Lower risk for heart disease and fracture and higher risk for breast cancer

IN

EXPECTANCY

TABLE II Estimated Changes in Life Expectancy among Different Population Subgroups

Population subgrouph

TOSTESON

Unopposed estrogen in Estrogen-progestin in women with hysterectomy womenwith a uterus + 5.5 (2.9-7.7) +8.1 (3.9-10.0) +4.1 (1.6-6.1)

+ 2.9 (0.4-8.9) +3.9 (0.5-10.9) + 1.6 ( - 1.1-8.4)

+9.4 (5.6-13.0)

+5.6 (2.7-13.5)

+ !.0 (-0.5-2.1)

-0.5 (-2.7-5.0)

a Numbers in parentheses give the range of estimates obtained when the most favorable and unfavorable assumption sets (Table I) were evaluated. bHigher risk: women with risks twofold greater than the average risk for women; lower risk: women with risks 50% lower than the average risk for women; unless otherwise indicated women are considered at average risk for all diseases.

CHAPTER46 Decision Analysis in Postmenopausal Therapy To put the projected benefits of hormone replacement therapy into perspective, it is instructive to compare our findings with those reported by Tsevat and colleagues [34] for modification of coronary heart disease risk factors among populations of 35-year-old women. For example, gains in life expectancy of less than 1 year (8.4 months) were projected if smoking were eliminated among all 35-year-old women in the United States (gains were 1.8 to 3.3 years among smokers). In comparison to these estimates, the potential health benefits of long-term hormone replacement therapy are noteworthy. The application of decision analysis to hormone replacement therapy provides useful information for health care providers who must customize treatment recommendations to individual patients with known risk factors. Col and colleagues have developed algorithms linking individual patient risk factors with the incidence of coronary heart disease and breast cancer for use in counseling women regarding use of hormone replacement therapy [2,18]. These analyses provide systematic and detailed estimates of the health effects of hormone replacement therapy based on state-of-the-art knowledge regarding the risks and benefits of postmenopausal therapy. Such estimates are useful for informing women of the potential effects of hormone replacement therapy [17,35,36], but are subject to limitations as described in the next section.

V I I . LIMITATIONS The formal quantitative method of decision analysis was used to evaluate the health benefits of treatment with postmenopausal hormone replacement therapy. Health benefits were characterized based on changes in life expectancy rather than on changes in morbidity (i.e., health-related quality of life). This limitation is significant, because many women initiate postmenopausal treatment to alleviate symptoms of menopause. However, the majority of women initiating treatment do not continue with treatment long term [37]. Any benefits of menopausal symptom relief or overall enhanced well-being were not accounted for in this analysis [38]. Likewise, changes in morbidity associated with the increased or decreased incidence of disease affected by hormone replacement therapy were also not considered. This limitation may be of most relevance for osteoporosis-related fractures, because only reductions in hip fracture incidence were counted as a benefit of long-term treatment. The majority of osteoporotic fractures do not affect mortality, but have a measurable impact on morbidity that should be considered [39]. Estimated health benefits for hormone replacement therapy were limited to initiation of a 15-year treatment regimen at age 50 years. Longer treatment durations will provide

653 more favorable outcomes regarding fracture and heart disease incidence, but will be less favorable with regard to breast cancer incidence. On balance, however, others who have estimated the net health effects of longer term therapy have projected larger net gains in life expectancy [1,2,181. Initiation of treatment at older ages (e.g., 65 years) may also be worthy of consideration, because many women do not initiate long-term hormone replacement therapy at the time of menopause [40]. To the extent that older women consider treatment initiation for prevention of osteoporotic fractures, there are new pharmaceutical agents such as alendronate [41-43], a bisphosphonate, and raloxifene [44-46], a selective estrogen receptor modulator, to consider. In a recent study that considered all three interventions in 50-yearold women, life expectancy gains for raloxifene, which mimics the action of estrogen on bone and lipids but causes no stimulatory effect on breast or uterine tissues, were larger than for hormone replacement therapy among those at high risk for breast cancer and moderate to low risk for heart disease [ 18]. However, hormone replacement therapy had larger gains projected among women at moderate to high risk for coronary heart disease.

VIII. SUMMARY Projected life expectancy changes for 50-year-old women undergoing a 15-year course of treatment were updated based on current estimates of the effect of hormone replacement therapy on the risk of breast cancer, heart disease, and hip fracture. Unopposed estrogen was evaluated for women with a prior hysterectomy and combined continuous estrogenprogestin therapy was considered for women with a uterus. Gains in life expectancy for 15 years of treatment ranging from 0.4 to 8.9 months were projected for women at average risk for heart disease, breast cancer, and hip fracture. Gains were largest (2.7 to 13.5 months) among women with higher than average risk for heart disease and hip fracture and lower than average risk for breast cancer. In contrast, smaller gains and potential losses (-2.7 to 5.0 months) were noted for women at lower than average risk for heart disease and hip fracture and higher than average risk for breast cancer. This analysis did not account for potentially important healthrelated quality-of-life factors, which may be critical for individual patient decision making. Nonetheless, estimates of the average health effect for long-term hormone replacement therapy among women with various risk profiles should provide useful information for health care providers when counseling patients regarding postmenopausal hormone replacement therapy decisions. Individual decisions regarding long-term use of postmenopausal therapy remain a personal matter worthy of careful deliberation.

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ANNA N. A. TOSTESON

Acknowledgment Supported by Grant AG12262 from the National Institute on Aging, U.S. Public Health Service.

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19. National Osteoporosis Foundation (1998). Osteoporosis: Review of the evidence for prevention, diagnosis and treament and cost-effectiveness analysis: Status report. Osteoporosis Int. 8(Suppl 4), S 1-$88. 20. Weiss, N., Ure, C., Ballard, J., Williams, A., and Daling, J. (1980). Decreased risk of fractures of the hip and lower forearm with post-menopausal use of estrogen. N. Engl. J. Med. 303, 1195-1198. 21. Felson, D., Zhang, Y., Hannan, M., Kiel, D., Wilson, P., and Anderson, J. (1993). The effect of postmenopausal estrogen therapy on bonedensity in elderly women. N. Engl. J. Med. 329(16), 1141-1146. 22. Cauley, J., Seeley, D., Ensrud, K., Ettinger, B., Black, D., and Cummings, S. (1995). Estrogen replacement therapy and fractures in older women. Ann. Intern. Med. 122(1), 9-16. 23. Collaborative Group on Hormonal Factors in Breast Cancer (1997). Breast cancer and hormone replacement therapy: Collaborative reanalysis of data from 51 epidemiological studies of 52,705 women with breast cancer and 108,411 women without breast cancer. Lancet 350, 1047-1059. 24. Mendelsohn, M., and Karas, R. (1999). The protective effects of estrogen on the cardiovascular system. N. Engl. J. Med. 340, 1801-1811. 25. Grodstein, F., Stampfer, M., Manson, J., Colditz, G., Willett, W., Rosner, B., Speizer, E, Hennekens, C. (1996). Postmenopausal estrogen and progestin use and the risk of cardiovascular disease. N. Engl. J. Med. 335(7), 453-461. 26. Barrett-Connor, E., and Grady, D. (1998). Hormone replacement therapy, heart disease, and other considerations. Annu. Rev. Public Health 19,55-72. 27. Hulley, S., Grady, D., Bush, T., Furberg, C., Herrington, D., Riggs, B., and Vittinghoff, E. (1998). Randomized trial of estrogen plus progestin for secondary prevention of coronary heart disease in postmenopausal women. J. Am. Med. Assoc. 280, 605-613. 28. Daly, E., Vessey, M., Hawkins, M., Carson, J., Gough, P., and Marsh, S. (1996). Risk of venous thromboembolism in users of hormone replacement therapy. Lancet 348, 977-980. 29. Hebert-Croteau, N. (1998). A meta-analysis of hormone replacement therapy and colon cancer in women. Cancer Epidemiol. Biomarkers Prev. 7, 653-659. 30. Nanda, K., Bastian, L., Hasselblad, V., and Simel, D. (1999). Hormone replacement therapy and the risk of colorectal cancer: A meta-analysis. Obstet. Gynecol. 93(Suppl. S, Part 2), 880-888. 31. Fung, M., Barrett-Connor, E., and Bettencourt, R. (1999). Hormone replacement therapy and stroke risk in older women. J. Women's Health 8, 359-364. 32. Paganini-Hill, A., and Henderson, V. (1994). Estrogen deficiency and risk of Alzheimer's disease in women. Am. J. Epidemiol. 140(3), 256-261. 33. National Center for Health Statistics (1996). "Vital Statistics of the United States, 1992: Life Table." NCHS, Hyattsville, MD. 34. Tsevat, J., Weinstein, M., Williams, L., Tosteson, A., and Goldman, L. (1991). Expected gains in life expectancy from various coronary heart disease risk factor modifications. Circulation 83(4), 1194-1201. 35. O'Connor, A., Tugwell, P., Wells, G., Elmslie, T., Jolly, E., Hollingworth, G., McPherson, R., Drake, E., Hopman, W., and Mackenzie, T. (1998). Randomized trial of a portable self-administered decision aid for postmenopausal women considering long-term preventive hormone therapy. Med. Decis. Making 18, 295-303. 36. Newton, K., Lacroix, A., Leveille, S., Rutter, C., Keenan, N., and Anderson, L. (1998). The physician's role in women's decision making about hormone replacement therapy. Obstet. Gynecol. 92, 580-584. 37. Ettinger, B., Li, D., and Klein, R. (1996). Continuation of postmenopausal hormone replacement therapy: Comparison of cyclic versus continuous combined schedules. Menopause 3(4), 185-189. 38. Tosteson, A., Gabriel, S., Kneeland, T., Moncur, M., Manganiello, P., Schiff, I., Ettinger, B., and Melton, L. J. (2000). Has the impact of hormone replacement therapy on health-related quality of life been undervalued? J. Women's Health, in press.

CHAPTER 46 Decision Analysis in Postmenopausal Therapy 39. Tosteson, A. (1997). Quality of life in the economic evaluation of osteoporosis prevention and treatment. Spine 22(24S), 58S. 40. Cauley, J., Cummings, S., Black, D., Mascioli, S., and Seeley, D. (1990). Prevalence and determinants of estrogen replacement therapy in elderly women. Am. J. Obstet. Gynecol. 163, 1438-1444. 41. Liberman, U., Weiss, S., Broil, J., Minne, H., Quan, H., Bell, N., Rodrigues-Portales, J., Downs, J., Dequeker, J., Favus, M., Seeman, E., Recker, R., Capizzi, T., Santora, A., Lombardi, A., Shah, R., Hirsch, L., Karpf, D., for the Alendronate Phase III Osteoporosis Treatment Study Group (1995). Effect of oral alendronate on bone mineral density and the incidence of fractures in postmenopausal osteoporosis. N. Engl. J. Med. 333(22), 1437-1443. 42. Black, D., Cummings, S., Karpf, D., Cauley, J., Thompson, D., Nevitt, M., Bauer, D., Genant, H., Haskell, W., Marcus, R., Ott, S., Torner, J., Quandt, S., Reiss, T., Ensrud, K., Group for the Fracture Intervention Trial Group (1996). Randomized trial of effect of alendronate on risk of fracture in women with existing vertebral fractures. Lancet 348, 1535-1541.

655 43. Meunier, E (1997). Oral alendronate increases bone mineral density and reduces vertebral fracture incidence in postmenopausal osteoporosis. Br. J. Rheumatol. 36(Suppl. 1), 15-19. 44. Delmas, E, Bjarnason, N., Mitlak, B., Ravoux, A., Shah, A., Huster, W., Draper, M., and Christiansen, C. (1997). Effects of raloxifene on bone mineral density, serum cholesterol concentrations, and uterine endometrium in postmenopausal women. N. Engl. J. Med. 337(23), 16411647. 45. Walsh, B., Kuller, L., Wild, R., Paul, S., Farmer, M., Lawrence, J., Shah, A., Anderson, E (1998). Effects of raloxifene on serum lipids and coagulation factors in healthy postmenopausal women. J. Am. Med. Assoc. 279, 1445-1451. 46. Cummings, S., Eckert, S., Krueger, K., Grady, D., Powles, T., Cauley, J., Norton, L., Nickelsen, T., Bjarnason, N., Morrow, M., Lippman, M., Black, D., Glusman, J., Costa, A., and Jordan, V. (1999). The effect of raloxifene on risk of breast cancer in postmenopausal women: Results from the MORE randomized trial. J. Am. Med. Assoc. 281, 21892197.

Index

A

y-Aminobutyric acid (GABA), mood regulation and steroid effects, 571-572 Androgens, s e e Androstenedione; Dehydroepiandrosterone; Dehydroepiandrosterone sulfate; Dihydroxytestosterone; Testosterone Androstenedione levels in aging, 359, 628 menopausal transition levels, 153 ovarian synthesis, 21,628 reproductive physiology in women, 446 ANP, s e e Atrial natriuretic peptide Anthropometry, body composition measurement, 254 Arginine vasopressin (AVP), age-related changes in males, 118 Aromatase, mutations, 89- 90 Arthritis, s e e Osteoarthritis Aspirin, Women's Health Study, 412 Atrial natriuretic peptide (ANP), age-related changes in males, 118 Autoimmune disease premature ovarian failure incidence, 140, 355 ovary autoantibodies, 90, 140-141,355-356 polyglandular autoimmune syndrome, 90 testosterone role, 451 AVE s e e Arginine vasopressin

Activin cells expressing in ovary, 25 follicle-stimulating hormone induction and ovary reserve reduction, 26-28, 97 isoforms, 26, 97 overview of actions, 26-28 Acupuncture, hot flash treatment, 224 Age of onset, menopause endometrial cancer risk factor, 367-368 estimation from epidemiology studies incidence data, 167 prevalence data, 167 factors affecting body mass, 194 diet, 194-195 ethnicity, 193 exercise, 194, 496 menstrual and reproductive history, 192 occupational/environmental factors, 194 smoking, 193-194 socioeconomic status, 191 heritability, 78 international differences, 193 mean, 13, 96, 190 Alcoholism, sexual activity impact in menopause, 389 Alendronate early postmenopausal intervention, 415-416 Fracture Intervention Trial, 415 mode of action, 520 osteoporosis treatment, 521 Alzheimer's disease genetics, 317 hormone replacement therapy effects biological rationale, 317-318 primary prevention, 318-320 symptom response, 318 risk factors, 317

B Bioelectrical impedance, body composition measurement, 256 Biofeedback, hot flash treatment, 224 Bisphosphonates, s e e Alendronate; Etidronate; Osteoporosis Black cohosh, herbal therapy in menopause, 471-472 Blood pressure, cardiovascular disease risk in menopause, 234235 BMD, s e e Bone mineral density BMI, s e e Body mass index Body composition compartments, 254, 257 direct measures of body compartment, 250 exercise effects in menopause, 501-502

657

658 Body composition ( c o n t i n u e d ) measures anthropometry, 254 bioelectrical impedance, 256 body mass index, 254 dual energy X-ray absorptiometry, 255-256 skinfold thickness, 254-255 underwater weighing, 255 weight, 254 menopause status linkage studies, 253 steroid hormone effects energy balance, 246-247 insulin action and weight, 247-248 leptin, 247 lipoprotein lipase and fatty acid deposition, 247 stress and glucocorticoid stimulation, 247 testosterone effects in women, 451 Body mass, see also Obesity effects on menopause onset age, 194 presentation and symptoms, 195 exercise effects in menopause, 501-502 hormone replacement therapy effects, 249 menopause status linkage studies, 253 oophorectomy and weight gain, 249 weight gain in menopause, 248 Body mass index (BMI) hormone replacement therapy effects, 249-250 measurement, 254 menopause effects, 249 oophorectomy effects, 249 Body temperature, see Core body temperature Body topology measures overview, 256 waist-to-hip ratio, 256-257 menopause, 250-251 Bone, see a l s o Calcium; Fracture; Osteoporosis aging effects, distinguishing from menopause effects, 204-206 androgen effects, 449 bone lining cells, 289 calcium supplementation effects, 288 composition, 288 dehydroepiandrosterone replacement therapy effects, 634 endochondral ossification, 294 estrogen role in peak bone mass acquisition, 287-288 exercise effects in menopause, 502-503,524 hormone replacement therapy effects, 288 intramembranous ossification, 294 lactation, bone loss, 287 males, age-related changes, 117 matrix proteins, 289 osteoblasts, 289 osteoclasts, 289 osteocytes, 289 progesterone effects, 430, 438 remodeling calcium homeostasis, 485 cytokine effects, 356

INDEX estrogen role osteoblast receptors and regulation, 292-294 osteoclast receptors and regulation, 292-293 overview, 292 formation and markers, 291, 512-513 mineralization, 291 overview, 289-290, 512 regulators, 291, 512-513 resorption and markers, 290-291, 512 types and structure, 288 Bone mineral density (BMD), treatment effects in osteoporosis prevention and treatment bisphosphonates alendronate, 521 etidronate, 520-521 ibandronate, 521 mode of action, 520 pamidronate, 521 pharmacology, 519 residronate, 521 tiludronate, 521 toxicity, 519-520 calcitonin, 518-519 calcium, 522 estrogen/androgen replacement therapy effects, 620 exercise effects, 524 fluoride, 525-526 hormone replacement therapy, 514-516 parathyroid hormone, 524-525 phytoestrogens, 517-518 selective estrogen receptor modulators estrogen receptor binding, 516 raloxifene, 517 tamoxifen, 516-517 testosterone, 526-527 tibolone, 519 vitamin D, 522-523 Breast, compositional changes in aging, 360 Breast cancer Breast Cancer Prevention Trial with tamoxifen, 405, 413-414 cultural differences, 360-363 epidemiology, 360-363,583-584 genetics, 364 growth hormone/insulin-like growth factor-I axis role, 281-282 hormone replacement therapy studies decision analysis, 651 estrogen-progestin replacement therapy, 363-364, 586 estrogen replacement therapy, 585-586 limitations of observational studies, 588 natural history effects, 588-589 testosterone replacement effects in women, 452 menopause risk, 363 phytoestrogen effects, 467 prevention raloxifene, 365 tamoxifen, 365 risk factors, 362-364 screening, 364-365 sex hormone role

INDEX

659

estrogen, 363-364, 583-584 progesterone, 364 testosterone, 364

C Calcitonin osteoporosis treatment, 518-519 regulation of secretion, 518 Calcitriol bone remodeling regulation, 291 calcium homeostasis role, 298-299 Calcium adaptation to insufficiency, 483-484, 487,489-490 epithelial transport estrogen effects, 294-295 overview, 294 plasma membrane calcium pump, 294 sodium/calcium exchanger, 295 fluxes, 482-483 functions, overview, 482-483 gut absorption aging effects, 486-487 estrogen effects, 297-298 mechanisms, 296-297 vitamin D role, 491 homeostasis aging effects, 299-300 bone remodeling, 485 estrogen deficiency effects, 298-300 feedback loops, 485-486 overview of physiology, 298 parathyroid hormone role, 483,485-486 phytoestrogen effects, 301 selective estrogen receptor modulator effects, 300-301,303 vitamin D role, 300, 483 intake functional indicator of adequacy, 488-489 non-skeletal illness prevention, 489-490 optimal levels, 484-485,488-490 threshold behavior, 487-488 kidney handling aging effects, 486 calcium-sensing receptor, 295-296 estrogen effects calbindin induction, 296 clinical studies, 296 ion transport regulation, 296 physiology, 295 osteoporosis treatment, 522 supplementation bone effects, 288 combination therapy effectiveness, 489 fracture reduction, 299 sources, 490-491 Women's Health Initiative study, 410-411 Cancer, s e e Breast cancer; Cervical cancer; Colon cancer; Endometrial cancer; Ovarian cancer Carbohydrate metabolism, s e e Insulin

Cardiovascular disease (CVD) aging effects, distinguishing from menopause effects, 206 aspirin trials, 412 dehydroepiandrosterone replacement therapy effects, 633 epidemiologic studies of menopause and outcomes natural menopause studies, 231-232 overview, 230, 233-234 risk factors and menopause status blood pressure, 234-235 hemostatic changes, 237-238 lipid profiles, 235-237 smoking, 234 surgical menopause studies, 232-233 unspecified menopause studies, 230-231 epidemiology, 543 estrogen role circulating levels, 229 vascular wall effects, 238-239 exercise and risk reduction, 499-502 growth hormone/insulin-like growth factor-I axis role, 278 hormone replacement therapy studies, s e e a l s o Heart Estrogen/ Progestin Replacement Study; Postmenopausal Estrogen/ Progestin Interventions cause and effect versus selection, 548-550 decision analysis, 651-652 primary prevention of coronary heart disease, 544-547 secondary prevention, 547-548 phytoestrogens and risk reduction, 464-466 postmenopausal mortality, 229 progesterone effects, 438-440, 547 sex-specific trends in aging, 230 testosterone replacement effects in women lipid profile effects, 451-452 myocardium and vascular function effects, 452 fi-Carotene, Women's Health Study, 412 Carotid artery, hormone replacement therapy effects, 266-267 Cataract hormone replacement therapy effects, 641-642 sex differences in occurrence, 641 Catecholestrogens, mood effects, 571 CBG, s e e Corticosteroid-binding globulin Cerebrovascular disease, s e e Stroke Cervical cancer cervix changes in aging, 360 epidemiology, 371-372 screening, 372 Chaste tree, herbal therapy in menopause, 472 Cholesterol, menopause levels and cardiovascular disease, 236 Chromosomal translocation, ovarian function, 85 Clomiphene applications, 425 effects in mood disorders, 569 Clonidine, hot flash treatment, 220-221,223-225,557 Cognition aging effects, distinguishing from menopause effects, 207 dehydroepiandrosterone replacement therapy effects, 635 estrogen effects in menopause, 50-51 herbal therapies, 470 hormone replacement therapy effects, 316-317

660

INDEX

Collagen age-related changes, 262-263 biosynthesis, 262 carotid artery, hormone replacement therapy effects, 266-267 estrogen regulation of synthesis and arthritis, 537 markers, 262 skin menopause effects, 265-266, 311 structure, 263,265 structure, 261-262 types, 262 urogenital system, menopause effects, 267-268, 329 Colon cancer, phytoestrogen effects, 467-468 Core body temperature, hot flashes, 217,222 Corticosteroid, androgen suppression, 631 Corticosteroid-binding globulin (CBG), estrogen response, 427 Cortisol, dysregulation in depression, 564, 571-572 Cortisone-binding globulin (CBG), progesterone binding, 433 CTG nucleotide expansion, ovarian function in disorders, 87-88 Cultural differences age of onset, menopause, 193 breast cancer, 360-363 endometrial cancer, 366 menopause data reporting, 167 menopause status, ethnicity effects on presentation and symptoms, 193 mood and menopause, 346-347 ovarian cancer, 372-373 perimenopause study design considerations, 106-107 CVD, s e e Cardiovascular disease

D Data collection, menopause studies combining data collected at different frequencies, 169 cultural differences in reporting, 167 daily calendars, 164 daily specimen collection, 164 home or clinic visits, 163-164 mailed surveys, 163 measurement of menopausal symptoms, 166-167 overview, 163 reliability of self-reported data, 166 telephone surveys, 163 Decision analysis, hormone replacement therapy life expectancy impact, 650-653 limitations, 653 overview, 649-650, 653 review of studies, 650-651 risk/benefit analysis breast cancer, 651 heart disease, 651-652 hip fracture, 651 osteoporosis, 651 steps, 650 treatment regimens, 651 Deep vein thrombosis, s e e Venous thrombosis Dehydroepiandrosterone (DHEA) expression in aging, 359 age-related changes in males, 117 biosynthesis, 625-628

bone effects, 449 deficiency, clinical definition, 631-632 replacement therapy preparations and bioavailability, 635-636 rationale bone protection, 634 cardioprotection, 633 cognition, 635 diabetes protection, 633-634 immune function, 634-635 insulin-like growth factor I augmentation, 634 obesity reduction, 633 overview, 632, 636 side effects, 636 reproductive physiology in women, 446 Dehydroepiandrosterone sulfate (DHEAS) aging effects on levels, 625,628, 630-631 biosynthesis, 625-628 bone effects, 449 conditions accelerating age-related decline, 631 insulin-like growth factor-I, effects on levels, 276-277 metabolism, 626-627 regulation of secretion, 627 reproductive physiology in women, 446 Demographics historical perspective, 397 Mexico population pyramids, 397, 399-400 United States population pyramids, 397-399 Depression, s e e a l s o Mood aging effects, distinguishing from menopause effects, 207-208 exercise effects, 499 gender differences, 563-564 hormone replacement therapy effects, 317 hypothalamic-pituitary-adrenal axis dysregulation, 564, 571572 males epidemiology, 124 erectile dysfunction association, 120-121, 127 evaluation and treatment, 125-126 risk factors, 124-125 testosterone effects, 126-127 postpartum depression, 567, 574, 576 progesterone effects, 440-441 sexual activity impact in menopause, 388 sleep alterations, 564 17,20-Desmolase, deficiency, 89 DHEA, s e e Dehydroepiandrosterone DHEAS, s e e Dehydroepiandrosterone sulfate Diabetes, type 2 dehydroepiandrosterone replacement therapy effects, 633634 males in aging, 117 sexual activity impact in menopause, 388 Diet, s e e a l s o Phytoestrogens consequences of bad nutrition, 481-482 effects on menopause onset age, 194-195 presentation and symptoms, 195- 96 Women's Health Initiative study, 408-409 insulin-like growth factor-I, diet effects on levels, 275-276

661

INDEX Diethylstilbestrol, structure and uses, 425 Dihydroxytestosterone biosynthesis, 630 potency, 630 Dopamine, mood regulation and steroid effects, 570-571 Dual energy X-ray absorptiometry (DXA), menopausal studies, 250-251,255-256 DXA, s e e Dual energy X-ray absorptiometry Dynorphin, luteinizing hormone and luteinizing hormonereleasing hormone, regulation of secretion, 45-46

E ED, s e e Erectile dysfunction EGF, s e e Epidermal growth factor Ejaculatory dysfunction premature ejaculation, 119-120 retarded ejaculation, 120 Endometrial cancer age at menopause as risk factor, 367-368 cultural differences, 366 effects raloxifene, 371 tamoxifen, 371 endometrial changes in aging, 360 epidemiology, 365-367, 591 growth factors, 592-593 histology, 367 hormone replacement therapy effects Continuous Hormones as Replacement Therapy study, 596600 estrogen/androgen replacement therapy effects, 621-622 meta analysis of relative risk, 591-592 overview, 369-370, 386-387, 402 Postmenopausal Estrogen/Progestin Interventions study, 593 -596 progestin protection in combination therapy, 599-604 hyperplasia association, 593 pathogenesis, 370 phytoestrogen effects, 467 risk factors, 366-367, 369-370 sex hormone role, 367-369, 592-593 smoking, protective effect, 370 surveillance and screening, 370-371,602-603 Endometrium estrogen effects, 367-369, 592-593 progesterone effects administration routes, 438 bleeding patterns and progestogen regimens, 437-438 estradiol receptor inhibition, 434 hyperplasia prevention trials, 436-438, 599-604 protection, 429, 436, 441 withdrawal bleeding, 436 shedding, matrix metalloproteinase role, 603 /3-Endorphin exercise effects, 496 hot flash role, 219 luteinizing hormone and luteinizing hormone-releasing hormone, regulation of secretion, 45-46 Energy balance, estrogen effects, 246-247

Epidemiology, s e e a l s o Cardiovascular disease; Data collection, menopause studies; Mood; Study design, epidemiology; Study of Women's Health Across the Nation age at menopause, estimation incidence data, 167 prevalence data, 167 menstrual calendar data analysis overview, 167-168 perimenopausal data, 168 premenopausal calendar analysis, 168 Epidermal growth factor (EGF), primordial follicle number regulation, 23 ER, s e e Estrogen receptor Erectile dysfunction (ED) androgen deficiency role, 119 depression association, 120-121, 127 diagnostic assessment, 123 epidemiology, 118, 120-121 erectile mechanism, 121-122 etiologies, 122-123 psychological causes, evaluation, and treatment, 127 treatment alprostadil, 124 prosthetics, 124 sildenafil, 123-124 vacuum tumescence, 124 vascular surgery, 124 Estradiol, s e e Estrogen Estrogen, s e e a l s o Hormone replacement therapy body composition effects energy balance, 246-247 insulin action and weight, 247-248 leptin, 247 lipoprotein lipase and fatty acid deposition, 247 stress and glucocorticoid stimulation, 247 bone remodeling role osteoblast receptors and regulation, 292-294 osteoclast receptors and regulation, 292-293 overview, 292 breast cancer effects, 363-364, 583-586 calcium effects epithelial transport, 294-295 gut absorption, 297-298 homeostasis, 298-300 renal handling calbindin induction, 296 clinical studies, 296 ion transport regulation, 296 carbohydrate metabolism effects, 251-253 cardiovascular disease circulating levels, 229 vascular wall effects, 238-239 central nervous system effects, overview, 315-316 classic estrogens equine estrogens, 423 metabolism of estrone, estradiol, and estriol, 421-423 types, 421 cognition effects in menopause, 50-51 collagen effects in skin, 265-266 domains, 4

662 Estrogen (continued) effects on bone, 514 ethinylestradiol, structure and metabolism, 423 expression in aging, 47-48, 190-191,359 final menstrual period levels, 150 growth hormone regulation, 272 history of use, 400 hormone replacement therapy, 34 hot flush role, 48-49, 219 immune system effects, 353 insulin-like growth factor-I, effects on levels, 276-277 luteinizing hormone-releasing hormone neurons, effects on network c-Fos expression during luteinizing hormone surge, 43 galanin marker of sexually dimorphic effects, 43-44 negative feedback, 41-42 positive feedback, 42-43 menopausal transition levels, 150-152, 154 menstrual cycle levels, 62-63, 148-149 mood effects in menopause, 51,347-348 nonsteroidal estrogens, see also specific compounds natural, 424 synthetic, 424-427 osteoarthritis role animal studies, 539-540 bone stiffness maintenance, 536 collagen synthesis, 536-537 cytokine modulation, 537 levels in disease, 540 overview, 535-536 overview of action, 4-5 peak bone mass acquisition role, 287-288 potency assays, 427 scavestrogens, 423 secretion from aged dominant follicles, 14 structure, 421 tissue selectivity in action, 34 Estrogen receptor (ER) carboxyl tail in pharmacology, 7 coactivator proteins, 8-9 corepressor proteins, 9 domains, 3-4, 38 expression in aging, 48 ligand effects on function, 6 - 8 modeling of ligand binding, 9 phage display peptide binding, 7 raloxifene binding, 7 subtypes activation of AP-1-regulated gene expression, 39 central nervous system distribution, 39-41 heterodimerization, 39 sequence homology between subtypes, 38 types, 5-6, 38 tamoxifen binding, 5, 7-9, 516 Ethinylestradiol, structure and metabolism, 423 Ethnicity, see Cultural differences Etidronate mode of action, 520 osteoporosis treatment, 520-521 trials in menopause, 414-415

INDEX

Exercise benefits, overview, 495, 504 effects on menopause body weight and composition, 501-502 bone, 502-503 cardiovascular disease risk reduction, 499-502 onset age, 194, 496 presentation and symptoms hot flash, 496, 498-499 mental health, 499 overview, 195 prospects for study, 504 menarche age of onset in athletes, 495-496 osteoporosis treatment, 524

F Fecundity, age-related decrease, 13-14 Feminism, impact on menopause interventions, 403 Fluoride, osteoporosis treatment, 525-526 Follicle-stimulating hormone (FSH) activin induction and ovary reserve reduction, 26-28 assays bioassay, 65 immunoassay, 66 radioreceptor assay, 65-66 urinary fl core fragments, 66-68, 70-73 circadian rhythm postmenopause, 104-105 premenopause, 100 dynamic secretion following menopause, 103-104 estrogen response, 427 expression in aging, 24, 46, 68-70, 95-96, 102-103, 190191 folliculogenesis luteinizing hormone receptor induction, 20 mitosis stimulation, 20 overview, 17 selection role, 18-19 steroidogenic potential effects, 19-20 glycosylation, 35, 64-65 indications for hormone replacement therapy, 555 inhibin expression inhibition, 24-25 menopause transition levels, 150 - 152, 154 menstrual cycle levels, 62-63, 148-149 perimenopause levels age-related oligomenorrhea, 101 gonadotropin-releasing hormone stimulation test as pituitary aging probe, 102 older cycling women, 100-101 primordial follicle number regulation, 22-24 receptor mutations, 86-87, 138, 141 signal transduction, 19-20 structure, 35, 62-64 urinary marker of menopause, 70-73 Fracture aging effects, distinguishing from menopause effects, 204206 bisphosphonate prevention, see Alendronate; Etidronate

INDEX

663

epidemiology, 510-511 hormone replacement therapy, decision analysis in hip fracture, 651 lifetime risk, 509 morbidity and mortality, 510-511 Fragile X syndrome, ovarian function, 87-88, 138-139 FSH, s e e Follicle-stimulating hormone

G GAB A, s e e y-Aminobutyric acid Galactosemia, ovarian function, 88-89, 139 Galanin luteinizing hormone and luteinizing hormone-releasing hormone, regulation of secretion, 44-45 marker of estrogen sexually dimorphic effects, 43-44 Genitourinary system, s e e Urogenital system Germ cell failure, overview, 90 GH, s e e Growth hormone GHRP, s e e Growth hormone-releasing peptide Ginkgo, herbal therapy in menopause, 472-473 Ginseng, herbal therapy in menopause, 473 Glucocorticoid lymphocyte receptors, 353-354 males, age-related changes, 116 stimulation in menopause, 247 Growth hormone (GH) insulin-like growth factor-I, effects on levels, 275 males, age-related changes, 115-116 postmenopausal disease role breast cancer, 281-282 cardiovascular disease, 278 osteoporosis bone loss, 279-280 clinical trials with recombinant hormones, 280-281 peak bone mass acquisition role, 278-279 overview, 277 primordial follicle number regulation, 23 secretion, regulation, 271-272 Growth hormone-releasing peptide (GHRP), types, 272 Gynourinary system, s e e Urogenital system

H hCG, s e e Human chorionic gonadotropin HDL, s e e High-density lipoprotein Heart Estrogen/Progestin Replacement Study (HERS) cardiovascular disease outcomes, 411-412, 548, 550 objectives, 411 overview, 405, 411, 417 venous thromboembolism outcomes, 612-613 Herbs history of menopause treatment, 398-399 menopausal therapies Commission E recommendations, 475-476 genitourinary changes, 470 hot flush, 470 sexuality, 470-471 sleep, mood, and cognition, 470 regulation of use, 469, 476

types and indications in menopause balm, 471 black cohosh, 471-472 chaste tree, 472 ginkgo, 472-473 ginseng, 473 passion flower, 473 St. John's wort, 473-474 HERS, s e e Heart Estrogen/Progestin Replacement Study Hexarelin, growth hormone, regulation of secretion, 272 High-density lipoprotein (HDL) estrogen/androgen replacement therapy effects, 620-621 menopause levels and cardiovascular disease, 235-237 testosterone effects in women, 451 Hormone replacement therapy (HRT) Alzheimer's disease risk reduction, 207 androgen/estrogen replacement therapy, s e e Testosterone attitudes towards prescription, 401-402 body mass effects, 249-250 body topology effects, 251 bone effects, 288 breast cancer studies estrogen-progestin replacement therapy, 363-364, 586 estrogen replacement therapy, 585-586 limitations of observational studies, 588 natural history effects, 588-589 carbohydrate metabolism effects, 252-253 cardiovascular disease risk reduction, 206 carotid artery effects, 266-267 central nervous system effects Alzheimer's disease biological rationale, 317-318 primary prevention, 318-320 symptom response, 318 cognition, 316-317 depression, 317 mood, 51,317, 347-348, 558-559, 568, 579-580 overview, 315-316, 321-322 Parkinson's disease, 321 stroke, 320-321 costs, 176 decision analysis, s e e Decision analysis, hormone replacement therapy dehydroepiandrosterone replacement therapy, s e e Dehydroepiandrosterone disadvantages, 459 endometrial cancer effects Continuous Hormones as Replacement Therapy study, 596-600 meta analysis of relative risk, 591-592 overview, 369-370, 386-387,402 Postmenopausal Estrogen/Progestin Interventions study, 593 -596 progestin protection in combination therapy, 599-604 formulations, 584-585 history, 401-402, 406 hot flash treatment, 223 immune function effects, 356 incontinence management administration, 332-333

664 Hormone replacement therapy (HRT) ( c o n t i n u e d ) efficacy, 333-334 mechanism of action, 333 quality of life improvement, 332 late menopause effects cataract, 641- 642 dental health, 640-641 osteoporosis, 642-643 urinary incontinence, 642 urinary tract infection, 642 masking of natural menopause transitions data analysis in studies, 169 menopause study design effects, 166 osteoarthritis effects, 539 osteoporosis treatment, 514-516 ovarian cancer effects, 375-376 safety, overview, 406 sexual dysfunction intervention, 391 studies, s e e Heart Estrogen/Progestin Replacement Study; Postmenopausal Estrogen/Progestin Interventions; Women's Health Initiative study design eligibility criteria in clinical trials, 162-163 observational studies versus clinical trials, 162 topical therapy, 312- 313 trends in use, 584 venous thromboembolism risks risks and clinical recommendations, 612-614 Hot flash circadian rhythm, 222 core body temperature, 217,222 dietary effects, 196 epidemiology in menopause, 48, 215,556-557 exercise effects, 496, 498-499 heart rate, 218 herbal therapies, 470 hormonal etiology /3-endorphin, 219 estrogens, 219 luteinizing hormone, 219 norepinephrine, 220-221 overview, 48-49, 556 measurement ambulatory monitoring, 219 finger temperature, 218 provocation techniques, 219 skin conductance, 218-219 metabolic rate, 217-218 non-menopausal causes, 556-557 noradrenergic system disruption, 49 opioid role, 49-50 self-reported descriptions, 215-216 skin temperature and blood flow, 216 sleep hot flash relationship, 223 thermoregulation, 222-223 smoking effects, 215 substance P role, 50 sweating and skin conductance, 216-217 thermoregulation, 221-222

INDEX

treatment acupuncture, 224 biofeedback, 224 clonidine, 220-221,223-225,557 hormone replacement therapy, 223,556 phytoestrogens, 224, 460, 469 tibolone, 557 HRT, s e e Hormone replacement therapy Human chorionic gonadotropin (hCG) assays bioassay, 65 immunoassay, 66 radioreceptor assay, 65-66 urinary fl core fragments, 66-68 sites of synthesis, 61 structure, 62-64 Hydroepiandrosterone, s e e Dehydroepiandrosterone 17c~-Hydroxylase, deficiency, 89, 139 Hypergonadotropic amenorrhea, s e e Premature ovarian failure Hypothalamic- pituitary- ovarian axis brain aging and reproductive senescence, 105 - 106 feedback loops, 34-36 functional organization, 34-36 gonadotropin-releasing hormone stimulation test as pituitary aging probe, 102 luteinizing hormone-releasing hormone neurons anatomy, 35 distribution in forebrain, 36-37 estrogen effects on network c-Fos expression during luteinizing hormone surge, 43 galanin marker of sexually dimorphic effects, 43-44 negative feedback, 41-42 positive feedback, 42-43 neurotransmitters and neuropeptides in secretion dynorphin, 45-46 fl-endorphin, 45-46 galanin, 44-45 norepinephrine, 45 substance P, 46 overview of reproductive function, 34-35 steroid receptors, 35-36 subtypes, 36-37 mood regulation, 572 Hypothalamic-pituitary- testicular system anatomic changes in aging, 112 overview of normal function, 112-113

I Ibandronate, osteoporosis treatment, 521 IGF-BP, s e e Insulin-like growth factor binding protein IGF-I, s e e Insulin-like growth factor-I IGF-II, s e e Insulin-like growth factor-II IL-I, s e e Interleukin- 1 Incontinence assessment, 327-328 hormone replacement therapy administration, 332-333 efficacy, 333-334

INOEX mechanism of action, 333 quality of life improvement, 332 sexual activity impact, 385 stress urinary incontinence and paraurethral collagen, 329 treatment bladder training and retraining, 332 pelvic muscle exercises, 332 stress incontinence, 334-335 surgery, 331-332 urge incontinence, 334 urine storage and micturition, 330-331 Inhibin expression cells expressing in ovary, 25 Graafian follicles, 26 inhibition by follicle-stimulating hormone, 24-25 menstrual cycle, 46-47 follicle-stimulating hormone regulation, 97 menopausal transition levels, 151-152, 154 production in aging, 17, 24 structure, 24, 46 types, 24, 46, 97 Insulin androgen production role, 21 carbohydrate metabolism and change in ovarian hormone status animal studies, 251-252 human studies, 252-253 overview, 251 estrogen effects on action, 247-248 hormone replacement therapy effects on carbohydrate metabolism, 252 resistance, s e e Diabetes type 2 Insulin-like growth factor-I (IGF-I) dehydroepiandrosterone replacement therapy effects, 634 growth factor mediation, overview, 271 postmenopausal disease role breast cancer, 281-282 cardiovascular disease, 278 osteoporosis bone loss, 279-280 clinical trials with recombinant hormones, 280-281 peak bone mass acquisition role, 278-279 overview, 277 receptors, 273 serum level regulators age, 276 genetic control, 277 growth hormone, 275 nutrition, 275-276 parathyroid hormone, 277 sex steroids, 276-277 skeletal system, 273-274 structure and function, 272-273 Insulin-like growth factor-II (IGF-II) receptors, 273 skeletal system, 273-274 structure and function, 272-273 Insulin-like growth factor binding protein (IGFBP) effects on levels

665 aging, 276 nutrition, 275-276 sex steroids, 277 proteases, 273-274 skeletal system, 274-275 types, 272-273 Interleukin- 1 (IL- 1) aging effects on expression, 356 bone resorption stimulation, 356 effects on ovarian function, 355 Ipriflavone, osteoporosis treatment, 518

L Late menopause cataracts hormone replacement therapy effects, 641-642 sex differences in occurrence, 641 definition, 639 dental health hormone replacement therapy effects, 640-641 periodontitis rates, 640 genitourinary tract, hormone replacement therapy effects urinary incontinence, 642 urinary tract infection, 642 osteoporosis, hormone replacement therapy effects, 642-643 primary versus secondary prevention measures, 640 sexual activity, 447 therapeutic considerations overview, 640 pessaries, 645 systemic hormone treatment, 643-644 vaginal estrogens and moisturizers, 644-645 LDL, s e e Low-density lipoprotein Leeching, history of menopause treatment, 398-399 Levonorgestrel, pharmacology, 432 LH, s e e Luteinizing hormone LHRH, s e e Luteinizing hormone-releasing hormone Libido, s e e Sexual activity Life expectancy, hormone replacement therapy impact, 650-653 Lipoprotein(a), menopause levels and cardiovascular disease, 236-237 Low-density lipoprotein (LDL) estrogen/androgen replacement therapy effects, 620-621 menopause levels and cardiovascular disease, 235-237 testosterone effects in women, 451-452 Luteinizing hormone (LH) androgen biosynthesis regulation, 20-21 assays bioassay, 65 immunoassay, 66 radioreceptor assay, 65-66 urinary fl core fragments, 66-68, 70-73 circadian rhythm postmenopause, 104-105 premenopause, 99 dynamic secretion following menopause, 103-104 estrogen response, 427 expression in aging, 46, 68-70, 96-97, 102-103 folliculogenesis, 17

666

INDEX

Luteinizing hormone (LH) ( c o n t i n u e d ) glycosylation, 64-65 hot flash role, 219 menopausal transition levels, 150-151 menstrual cycle levels, 62-63, 98-99, 148-149 perimenopause levels age-related oligomenorrhea, 101 gonadotropin-releasing hormone stimulation test as pituitary aging probe, 102 older cycling women, 100-101 receptor induction by follicle-stimulating hormone, 20 mutations, 87 signal transduction, 21 seasonal variability, 99-100 structure, 35, 62-64 urinary marker of menopause, 70-73 Luteinizing hormone-releasing hormone (LHRH) agonist effects in mood disorders, 568-569 circadian rhythm, 99 depression effects, 565 expression in aging, 47 hormone response element binding, 37-38 neurons anatomy, 35 distribution in forebrain, 36-37 estrogen effects on network c-Fos expression during luteinizing hormone surge, 43 galanin marker of sexually dimorphic effects, 43-44 negative feedback, 41-42 positive feedback, 42-43 neurotransmitters and neuropeptides in secretion dynorphin, 45- 46 fl-endorphin, 4 5 - 4 6 galanin, 44-45 norepinephrine, 45 substance P, 46 overview of reproductive function, 34-35 steroid receptors, 35-36 subtypes, 36-37 pulsatile secretion in brain aging, 106

M Magnesium, supplementation, 490-491 Male menopause, s e e Manopause Manopause adrenal physiology glucocorticoids, 116 hydroepiandrosterone, 117 mineralocorticoids, 116-117 neurotransmitters, 117 androgen replacement therapy, 115 bone loss, 117 clinical features, 111-112 definition, 111 depression epidemiology, 124 erectile dysfunction association, 120-121,127

evaluation and treatment, 125-126 risk factors, 124-125 testosterone effects, 126-127 diabetes, 117 ejaculatory dysfunction premature ejaculation, 119-120 retarded ejaculation, 120 erectile dysfunction androgen deficiency role, 119 depression association, 120-121, 127 diagnostic assessment, 123 epidemiology, 118, 120-121 erectile mechanism, 121-122 etiologies, 122-123 psychological causes, evaluation, and treatment, 127 treatment alprostadil, 124 prosthetics, 124 sildenafil, 123-124 vacuum tumescence, 124 vascular surgery, 124 growth hormone, age-related changes, 115-116 hypothalamic-pituitary-testicular system anatomic changes in aging, 112 overview of normal function, 112-113 impact on female sexuality, 390 pulmonary function, 118 thyroid hormone, age-related changes, 116 water metabolism, 117-118 Medroxyprogesterone, pharmacology, 431-432 Memory, s e e Cognition Menopause status assessing associations with health outcomes, 170-171 ethnicity effects on presentation and symptoms, 193 measures age, 165 endocrine measures, 165 menstrual bleeding, 165 overview, 164, 190 self-definitions, 165 Menstrual cycle age of onset in athletes, 495-496 disturbances evaluation, 554-555 oral contraceptive therapy, 555 gonadotropin levels, 62-63, 98-99, 148-149 phases, 62-63 Mineralocorticoids, age-related changes in males, 116-117 Monosomy X, s e e X chromosome Mood, s e e a l s o Depression catecholestrogens, 571 empirical research of menopause status correlates cross-sectional studies, 343-344 cultural differences, 346-347 factor analysis, 342-343 health factors, 346 prospective and longitudinal studies, 344-345 psychological history, 346 psychosocial factors, 345-346

INDEX

667

estrogen effects menopause, 574-575 overview, 347-348, 564-565, 569, 572-575 postpartum depression, 574 exercise effects, 499 gender differences, 563-566 gonadotropin-releasing hormone agonist effects, 568-569 herbal therapies, 470 historical background menopause studies, 339 views in menopause, 402 hormone replacement therapy effects, 51, 317, 347-348, 558559, 568, 579-580 hypothalamic-pituitary-ovarian axis regulation, 572 methodological issues in study clinic versus population-based samples, 340-341 measurement instruments, 341-342 menopause status definition, 341 symptom reporting, 342 neurotransmitters and steroid effects ),-aminobutyric acid, 571-572 dopamine, 570-571 norepinephrine, 570, 574 overview, 569-570 serotonin, 570, 573-575 postpartum disorders blues, 567 depression, 567 psychosis, 567 premenstrual disorders, 566-567 progesterone effects menopause, 576-577 overview, 440-441,564-565,569, 575-576 postpartum depression, 576 prospects for menopause studies, 349 skin changes and self-image, 313 testosterone effects, 577-579 theories of menopause and mood biochemical hypothesis, 340, 349 domino hypothesis, 340, 348 psychoanalytical view, 340, 348-349 social circumstances perspective, 340, 348 Mucosal immunity, sex steroid modulation, 354-355 Myotonic dystrophy, ovarian function, 88

N Norepinephrine hot flash role, 220-221 luteinizing hormone and luteinizing hormone-releasing hormone, regulation of secretion, 45 mood regulation and steroid effects, 570, 574 Norethindrone, pharmacology, 432 Nutrition, s e e Diet

O Obesity, s e e a l s o Body mass dehydroepiandrosterone replacement therapy effects, 633 venous thromboembolism risks, 611

OR, s e e Ovary reserve Oral contraceptives effects on menopause onset, 192 menstrual disturbances, therapy, 555 transition to hormone replacement therapy, 555 venous thromboembolism risks, 611-612 Osteoarthritis epidemiology age-related trends, 537 gender-related trends, 537 hysterectomy/oophorectomy studies, 538-539 menopause-related studies, 537-538 overview, 535 estrogen role animal studies, 539-540 bone stiffness maintenance, 536 collagen synthesis, 536-537 cytokine modulation, 537 levels in disease, 540 overview, 535-536 hormone replacement therapy effects, 539 risk factors, 535 tamoxifen effects, 539 Osteoporosis aging effects, distinguishing from menopause effects, 204-206 bisphosphonate prevention, s e e Alendronate; Etidronate bone mineral density aging effects, 511-512 fracture risk, 510 genetic and environmental influences, 511 definition and classification, 278, 509-510 economic impact, 509 epidemiology, 509 growth hormone/insulin-like growth factor-I axis role bone loss, 279-280 clinical trials with recombinant hormones, 280-281 peak bone mass acquisition role, 278-279 hormone replacement therapy decision analysis, 651 late menopause effects, 642-643 phytoestrogen effects, 468-469 treatment bisphosphonates alendronate, 521 etidronate, 520-521 ibandronate, 521 mode of action, 520 pamidronate, 521 pharmacology, 519 residronate, 521 tiludronate, 521 toxicity, 519-520 calcitonin, 518-519 calcium, 522 estrogen effects on bone, 514 exercise, 524 fluoride, 525-526 hormone replacement therapy, 514-516 parathyroid hormone, 524-525

668

INDEX

Osteoporosis ( c o n t i n u e d ) phytoestrogens, 517-518 selective estrogen receptor modulators estrogen receptor binding, 516 raloxifene, 416-417, 517 tamoxifen, 516-517 testosterone, 526-527 tibolone, 519 vitamin D, 522-523 Ovarian cancer causal theories, 373-374 cultural differences, 372-373 epidemiology, 372-373 histology, 372 hormone replacement therapy effects, 375-376 protection oral contraceptives, 375 pregnancy, 374-375 hysterectomy or tubal ligation, 375 risk factors, 373 screening, 376 Ovary anatomy, 15-17 autoantibodies and ovarian function, 90, 140-141 folliculogenesis atresia, 17-18 chronology, 17-18 endocrinology, 17 growth factors and intraovarian control, 21 selection, 18-19 interstitial cell types, 21 X chromosome in development, s e e X chromosome Ovary reserve (OR), s e e a l s o Primordial follicle activin hypothesis, 26-28 heritability, 78 loss and menopause onset, 13-14, 21,46 primordial follicle age-related decrease, 14-15 anatomy, 14 development, 14 regulators, 22-24

P Pamidronate, osteoporosis treatment, 521 Parathyroid hormone (PTH) age-related changes, 486 bone remodeling regulation, 291 calcium homeostasis role, 298-300, 483,485-486 insulin-like growth factor-I, effects on levels, 277 osteoporosis treatment, 524-525 Parkinson's disease, hormone replacement therapy effects, 321 Passion flower, herbal therapy in menopause, 473 Patterning, s e e Body topology PEPI, s e e Postmenopausal Estrogen/Progestin Interventions Perimenopause characterization in studies, 165-166 cultural differences in study design, 106-107 definition, 95-96, 147, 165 final menstrual period, steroid levels, 150

gonadotropin levels age-related oligomenorrhea, 101 gonadotropin-releasing hormone stimulation test as pituitary aging probe, 102 older cycling women, 100-101 menopausal transition androgen levels, 153 estradiol levels, 150-152, 154 follicle-stimulating hormone levels, 150-152, 154 inhibin levels, 151-152, 154 luteinizing hormone levels, 150-151 overview, 147 progesterone levels, 153 ovarian determinants of reproductive aging, 96-97 Periodontitis, rates and hormone replacement therapy effects in late menopause, 640-641 Perrault syndrome, XX gonadal dysgenesis, 86 Pessary, late menopause patient management, 645 Physical activity, s e e Exercise Phytoestrogens calcium homeostasis effects, 301 cancer effects breast cancer, 467 colon cancer, 467-468 endometrial cancer, 467 overview, 466-467 cardiovascular disease risk reduction, 464-466 dietary effects on menopause presentation and symptoms, 196 estrogen receptor affinity, 424, 460, 462 glycosides, 424 hot flash treatment, 224, 460, 469 isoflavones biological effects, 462 dietary sources and metabolism, 462 lignans biological effects, 463-464 dietary sources and metabolism, 462-463 osteoporosis effects, 468-469, 517-518 structures, 301-302, 461 supplementation, 476 types, 424, 460 PMS, s e e Premenstrual syndrome POF, s e e Premature ovarian failure Polyglandular autoimmune syndrome, ovarian function, 90 Postmenopausal Estrogen/Progestin Interventions (PEPI) aims of study, 406-407 bone outcomes, 408 endometrial cancer outcomes, 593-596 limitations, 408 lipid profile outcomes, 407 overview, 405 treatment groups, 407-408 Postmenopause, s e e Late menopause Postpartum mood disorders blues, 567 depression, 567 psychosis, 567 PR, s e e Progesterone receptor Premature ovarian failure (POF) clinical features, 136-137

INDEX definition, 135 etiology aromatase mutations, 89-90 autoimmune disease incidence, 140, 355 ovary autoantibodies, 90, 140-141,355-356 polyglandular autoimmune syndrome, 90 chemotherapy, 139 17,20-desmolase deficiency, 89 follicle-stimulating hormone receptor mutations, 86-87, 138, 141 fragile X syndrome, 87-88, 138-139 galactosemia, 88-89, 139 germ cell failure, 90 17ce-hydroxylase deficiency, 89, 139 ionizing radiation, 139 luteinizing hormone receptor mutations, 87 mechanism of gene action, 85-86 multiple malformation syndromes, 87 myotonic dystrophy, 88 overview, 135-136, 138 Perrault syndrome, 86 viruses, 140 X chromosome causes, s e e X chromosome 46,XX agonadia, 90-91 evaluation of hypergonadotropic amenorrhea patients, 142 follicular function, 136 idiopathic disease, 141 incidence, 96 phenotype, 85 prevalence, 137-138 resistant ovary syndrome, definition and utility of term, 141-142 treatment of hypergonadotropic amenorrhea patients, 142-143 Premenstrual syndrome (PMS) mood disorders, 566-567 testosterone role, 451 Primates, uniqueness of menopause, 397 Primordial follicle, s e e a l s o Ovary reserve age-related decrease, 14-15, 22, 46 anatomy, 14 development, 14 regulation of numbers, 22-24 PRL, s e e Prolactin Progesterone, s e e a l s o Hormone replacement therapy bone effects, 430, 438 breast cancer effects, 364 breast effects, 430 cardiovascular effects, 438-440 depression and mood effects, 440-441 domains, 4 endometrium effects administration routes, 438, 559 bleeding patterns and progestogen regimens, 437-438 estradiol receptor inhibition, 434 hyperplasia prevention trials, 436-438 protection, 429, 436, 441 withdrawal bleeding, 436 final menstrual period levels, 150 history of use, 400-401

669 immune system effects, 353-355 menopausal transition levels, 153 menstrual cycle levels, 62-63, 148-149 metabolism administration route dependence, 433 binding proteins, 433 elimination, 433-434 first-pass effects, 430-431 metabolites, 432-433 tissues in metabolism, 433 mood effects menopause, 576-577 overview, 440-441,564-565,569, 575-576 postpartum depression, 576 overview of action, 4-5 pharmacology of progestogens, 430-431 potency, 436 rationale for inclusion in hormone replacement therapy, 429-430 secretion from aged dominant follicles, 14 side effects of therapy, 559 synthetic progestogens, 431-432 variability in serum concentrations, 434 Progesterone receptor (PR) carboxyl tail in pharmacology, 7 central nervous system distribution, 41 coactivator proteins, 8-9 corepressor proteins, 9 domains, 3 - 4 estradiol induction, 41 isoforms, 5 - 6 ligand effects on function, 6 - 8 modeling of ligand binding, 9 phage display peptide binding, 7-8 subtypes, 5 Prolactin (PRL) expression in aging, 359 primordial follicle number regulation, 23 Prolapse, late menopause patient management, 645 PTH, s e e Parathyroid hormone Pulmonary embolism diagnosis, 608 incidence, 609-610 pathophysiology, 608-609 risk factors age, 611,613 hereditary hypercoagulability disorders, 610 hormone replacement therapy risks and clinical recommendations, 612- 614 immobilization and hospitalization, 610-611 obesity, 611 oral contraceptives, 611-612 treatment, 608

R Raloxifene breast cancer effects, 365,474 calcium homeostasis effects, 301,303 collagen turnover effects, 268

670

INDEX

Raloxifene ( c o n t i n u e d ) endometrial cancer effects, 371 estrogen receptor binding, 7 osteoporosis prevention, 416-417 treatment, 517 overview of effects, 474 structure and applications, 426-427 tissue-specific effects, 416 Residronate, osteoporosis treatment, 521 Resistant ovary syndrome, definition and utility of term, 141-142

S St. John's wort, herbal therapy in menopause, 473-474 Selective estrogen receptor modulator (SERM), s e e Raloxifene; Tamoxifen SERM, s e e Selective estrogen receptor modulator Serotonin estrogen effects in menopause, 51 mood regulation and steroid effects, 570, 573-575 Sex hormone binding globulin (SHBG), estrogen response, 427 Sexual activity aging effects, distinguishing from menopause effects, 207, 384 factors affecting function alcoholism, 389 depression, 388 diabetes, 388 health status, 387-388 medications, 388-389 past sexual experience, 387 herbal therapies, 470-471 hormonal control, 335 hypoactive sexual desire disorder, 385 incontinence impact, 385 interventions for dysfunction education and counseling, 390-391 hormone replacement therapy, 391 sex therapy, 391-392 treatment of decreased libido, 335-336 masturbation in older women, 447 overview of menopause sexuality, 383-384 partners and relationships, 389-390 sex hormone roles androgens, 385,387 estrogen, 384-387 sexual response cycle, 385 testosterone effects in women libido, 447 brain activity, 447-448 hormone replacement therapy rationale, 448 premenopausal replacement, 448-449 urogenital atrophy effects, 384-385 uterine and vulvar changes, 386 vaginal blood flow and dryness, 385-386 SHBG, s e e Sex hormone binding globulin Skin anatomy, 263 conductance and hot flashes, 216-219

hormone replacement therapy effects, 558 menopause effects atrophy, 310- 311 collagen, 263,265-266, 311 dryness, 309-310 overview, 309 self-image, 313 sweating, 216-217, 310 wound healing, 311-312 topical hormone replacement therapy, 312- 313 Skinfold thickness, body composition measurement, 254-255 Sleep depression alterations, 564 estrogen effects, 386 herbal therapies, 470 hormone replacement therapy effects, 559 hot flash relationship, 223 thermoregulation, 222-223 Smoking confounding effect in menopause studies, 170-171 menopause effects cardiovascular disease risk, 234 hot flash association, 215 onset age, 193-194 presentation and symptoms, 195 endometrial cancer, protective effect, 370 Socioeconomic status, effects on menopause age of onset, 191 presentation and symptoms, 192 Somatostatin, growth hormone, regulation of secretion, 272 STAR, s e e Steroidogenic acute regulatory protein Steroid receptor coactivator 1, receptor ligand modulation, 8 Steroidogenic acute regulatory protein (STAR), androgen production role, 21 Stroke causes, 320 hormone replacement therapy effects, 320-321 primary prevention studies, 546-547 secondary prevention studies, 548 Study design, epidemiology age range selection, 161 aging effects, distinguishing from menopause effects bone loss, osteoporosis, and fractures, 204-206 cardiovascular disease, 206 cognition, 207 depression, 207-208 overview, 203-204, 208 sexual activity, 207 cross-sectional design, 160-161 longitudinal design, 160-161 menopause status eligibility criteria, 161-162 sampling frame choice, 160 Study of Women's Health Across the Nation (SWAN) aims, 176 clinical sites, 185-186 data collection overview, 164 theoretical approaches, 177 types of data, 177-179

INDEX

671

funding, 176 recruitment, 179-180, 186-187 response rates analysis, 182-183 computation, 181-182 sampling frames list-based frames, 181, 186-187 overview, 160, 180 random digit dialing-based frames, 180-181, 187-188 sampling by site, 186-188 sampling strategies, 181 strengths and limitations, 183-185 study design, overview, 176-177 Substance P expression in aging, 48 hot flush role, 50 luteinizing hormone and luteinizing hormone-releasing hormone, regulation of secretion, 46 Surgical menopause, handling in menopause data analysis, 169-170 SWAN, s e e Study of Women's Health Across the Nation

T Tamoxifen advantages, 425-426 Breast Cancer Prevention Trial, 405, 413-414 breast cancer effects, 365 endometrial actions, 6 endometrial cancer effects, 371,474 estrogen receptor binding, 5, 7-9 metabolism, 426 osteoarthritis effects in animal models, 539 osteoporosis treatment, 516-517 ovarian cyst association, 376 tissue-specific effects, 416 TBG, s e e Thyroxine-binding globulin T cell aging effects, 355 estrogen receptors, 353 Testosterone aging effects on levels in women, 445,455,628-631 autoimmune disease role, 451 binding proteins, 113, 153 biosynthesis, 628 body composition effects, 451 bone effects, 449-450 breast cancer effects, 364 deficiency in women, causes, 446-447 depression prevention in men, 126-127 effects in men adipose tissue, 115 bone mass, 115 hematopoiesis, 114-115 musculature, 114 estrogen/androgen replacement therapy benefits bone metabolism, 620 energy level, 618 sexual function, 387, 618-620

historical background, 617-618 indications, 622-623 risks cardiovascular lipid profile, 620-621 endometrial hyperplasia, 621-622 virilization, 621-622 formulations and doses for replacement therapy in women, 454-456 hypothalamic-pituitary-testicular system, 112-115 immune system effects, 354 indications for replacement therapy in women, 445-446, 453454 menopausal transition levels, 153,622 mood effects, 577-579 osteoporosis treatment, 526-527 premenstrual syndrome role, 451 pulsatile secretion in hypogonadal women, 105 replacement therapy risks in women breast cancer, 452 cardiovascular disease lipid profile effects, 451-452 myocardium and vascular function effects, 452 side effects, 452-453 reproductive physiology in women, 446 sexual activity effects in women brain activity, 447-448 hormone replacement therapy rationale, 448 libido, 447 premenopausal replacement, 448-449, 455 TGF-fl, s e e Transforming growth factor-fl Thyroid hormone, age-related changes in males, 116 Thyroxine-binding globulin (TBG), estrogen response, 427 Tibolone hot flash treatment, 557 osteoporosis treatment, 519 Tiludronate, osteoporosis treatment, 521 TNF-a, s e e Tumor necrosis factor-or Transforming growth factor-fl (TGF-fl), bone remodeling regulation, 512-513 Trisomy, ovarian function, 85, 139 Tumor necrosis factor-or (TNF-ce), effects on ovarian function, 355

U Ultrosonography, endometrial imaging, 602-603 Underwater weighing, body composition measurement, 255 Urogenital system aging symptoms, 327-328 anatomy and physiology bladder, 328 blood supply, 329 urethra, 328-329 atrophy and sexual activity, 384-385 collagen changes in aging, 268, 329 herbal therapies, 470 hormone replacement therapy effects, 557-558 hormone replacement therapy, late menopause effects urinary incontinence, 642 urinary tract infection, 642 vaginal estrogens and moisturizers, 644-645

672

INDEX

Urogenital system ( c o n t i n u e d ) incontinence assessment, 327-328 hormone replacement therapy administration, 332-333 efficacy, 333-334 mechanism of action, 333 quality of life improvement, 332 stress urinary incontinence and paraurethral collagen, 329 treatment bladder training and retraining, 332 pelvic muscle exercises, 332 stress incontinence, 334- 335 surgery, 331-332 urge incontinence, 334 urine storage and micturition, 330-331 innervation carbon monoxide messenger, 330 intramural ganglia, 330 parasympathetic pathways, 329 sensory afferent innervation, 330 sympathetic pathways, 330 menopause effects, 267-268 severity of menopausal symptoms, 327-328 vagina dryness in menopause, 191 symptom management, 335,644-645 Uterine cancer, s e e Endometrial cancer

V Vagina, s e e Sexual activity; Urogenital system Venous thrombosis diagnosis, 607-608 incidence, 609-610 pathophysiology, 608-609 risk factors age, 611,613 hereditary hypercoagulability disorders, 610 hormone replacement therapy risks and clinical recommendations, 612-614 immobilization and hospitalization, 610-611 obesity, 611 oral contraceptives, 611-612 sequelae, 607-608 sites, 607 treatment, 608 Viropause, s e e Manopause Vitamin D biosynthesis, 491 calcium homeostasis effects, 300, 483 functions differentiation factor, 492 intestinal calcium absorption, 491 skin, 491-492 metabolism, 491 optimal intake, 492 osteoporosis treatment, 522-523 supplementation, Women's Health Initiative study, 410-411 Vitamin E, Women's Health Study, 412

W Weight, s e e Body mass WHI, s e e Women's Health Initiative WHS, s e e Women's Health Study Women's Health Initiative (WHI) aims of study, 408 calcium/vitamin D supplementation branch, 410-411 dietary modification branch, 408-409 hormone replacement branch, 409-410 overview, 405 Women's Health Study (WHS) aspirin effects, 412 fl-carotene effects, 412 overview, 405, 412, 417 vitamin E effects, 412

X X chromosome monosomy X mechanism of chromosomal loss, 78 ovarian differentiation in 45,X individuals, 77-78 premature ovarian failure, 139 secondary sexual development, 79 streak gonads, 78-79 mosaicism and phenotypes 45,X/46,XX, 80-81 45,X/47,XXX, 81 ovarian maintenance genes nature of maintenance determinants, 84-85 pitfalls in localization, 81 45,X/46,X,del(Xp), 81-82 45,X/46,X,del(Xq), 83-84 46,X,del(Xp), 81-82 46,X,del(Xq), 83-84 46,X,i(Xq), 82-83 premature ovarian failure, overview of genetic causes autosomal dominant failure, 91 autosomal recessive failure, 91 chromosomal abnormalities, 91 triple X syndromes, 139 XX gonadal dysgenesis aromatase mutations, 89-90 17,20-desmolase deficiency, 89 follicle-stimulating hormone receptor mutations, 8687 fragile X syndrome, 87-88 galactosemia, 88-89 germ cell failure, 90 17cr-hydroxylase deficiency, 89 luteinizing hormone receptor mutations, 87 mechanism of gene action, 85-86 multiple malformation syndromes, 87 myotonic dystrophy, 88 ovary autoantibodies, 90 Perrault syndrome, 86 phenotype, 85 polyglandular autoimmune syndrome, 90 46,XX agonadia, 90- 91