Endometriosis: Advances and Controversies

ENDOMETRIOSIS edited by

Togas Tulandi McGill University Montreal, Quebec, Canada

David Redwine St. Charles Medical Center Bend, Oregon, U.S.A.

M A R C E L

MARCELDEKKER, INC. D E K K E R

NEWYORK BASEL

Although great care has been taken to provide accurate and current information, neither the author(s) nor the publisher, nor anyone else associated with this publication, shall be liable for any loss, damage, or liability directly or indirectly caused or alleged to be caused by this book. The material contained herein is not intended to provide specific advice or recommendations for any specific situation. Trademark notice: Product or corporate names may be trademarks or registered trademarks and are used only for identification and explanation without intent to infringe. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress. ISBN: 0-8247-4777-1 This book is printed on acid-free paper. Headquarters Marcel Dekker, Inc., 270 Madison Avenue, New York, NY 10016, U.S.A. tel: 212-696-9000; fax: 212-685-4540 Distribution and Customer Service Marcel Dekker, Inc., Cimarron Road, Monticello, New York 12701, U.S.A. tel: 800-228-1160; fax: 845-796-1772 Eastern Hemisphere Distribution Marcel Dekker AG, Hutgasse 4, Postfach 812, CH-4001 Basel, Switzerland tel: 41-61-260-6300; fax: 41-61-260-6333 World Wide Web http://www.dekker.com The publisher offers discounts on this book when ordered in bulk quantities. For more information, write to Special Sales/Professional Marketing at the headquarters address above. Copyright n 2004 by Marcel Dekker, Inc. All Rights Reserved. Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher. Current printing (last digit): 10 9 8 7 6 5 4 3 2 1 PRINTED IN THE UNITED STATES OF AMERICA

Foreword

Endometriosis is an enigma. The chapters in this book represent the endeavors of several experts in the field to review what is known about endometriosis in an objective manner. Yet what emerges is how little is known about endometriosis in spite of the innumerable studies conducted and the quantity of information that has accumulated. Opinions about endometriosis abound, and this book is filled with diverse opinions. Those opinions serve to raise questions for further research and to emphasize further how enigmatic this disorder remains. Because of the myriad of symptoms that may be associated with endometriosis, practitioners are dedicated to attempting to alleviate the symptoms associated with the disorder even in the absence of an understanding of its pathophysiology. Endometriosis is common. Every health care provider who interacts with women has dealt with many cases. Every woman knows others with the disorder. Everyone has an opinion and advice. Yet treatment remains far from satisfactory for many. The authors have provided their opinions as to the best approaches to therapy in these patients with complex medical problems. iii

iv

Foreword

Endometriosis engenders emotional responses in patients and health care practitioners alike. To women suffering from endometriosis, the disorder seems ill defined and frequently the cause of considerable pain, infertility, surgical procedures, the need for expensive medications, frustration, and anger. To those who provide care to affected women, endometriosis conjures up images of difficult patients whose treatment is often less than satisfactory. If the purpose of this book is to stimulate discussion and thereby research, it will fulfill that goal admirably. If the purpose is to educate readers, it will succeed at that goal as well. If the purpose is to serve as a resource, it will do so commendably. I would encourage individuals interested in endometriosis to read these chapters carefully, to ponder the ideas contained herein, and to commit themselves to serving their patients and future generations of women with a better understanding of this disorder. Only when basic science and research succeed at providing more complete answers will we be able to conquer this disease. Robert W. Rebar, M.D. Executive Director American Society for Reproductive Medicine Birmingham, Alabama

Preface

The science surrounding endometriosis is rapidly evolving and, in some cases, tenets that are widely accepted one year may not be applicable the next. New inventions and discoveries continue to be added and many controversies surround the management of this condition. This book reflects the latest thinking on endometriosis, thereby addressing new concepts and different treatment modalities. The contributors are physicians and researchers, leaders in the field with many years of experience in their topics. The subjects discussed in the first eight chapters include epidemiology, pathophysiology, distribution of endometriosis, genetics, basic and clinical research aspects, immunology, and possible serum markers for this condition. Chapter 9 deals with the mechanism of endometriosis-related infertility and Chapters 10 through 18 are dedicated to the new developments in medical and surgical management of endometriosis. Aromatase inhibitors are a promising new treatment, and the relationship between endometriosis and the outcome of in vitro fertilization are examples of the many advances and controversies in the management of endometriosis. v

vi

Preface

This is a book for students, residents, fellows, basic and clinical researchers, and practicing gynecologists. Readers will gain a better understanding of endometriosis and its management, as well as insight into designing new investigations. We are grateful to the contributors for their comprehensive and authoritative reviews. We would also like to acknowledge the staff of Marcel Dekker, Inc., for their support and expertise in publishing medical texts. Togas Tulandi David Redwine

Contents

Foreword Robert W. Rebar Preface Contributors 1. Epidemiology of Endometriosis Philippe R. Koninckx 2. Pathogenesis of Endometriosis: Peritoneal Endometriosis, Ovarian Endometriosis, and Rectovaginal Adenomyosis Jacques Donnez and Jean Squifflet

iii v xi 1

19

3. Anatomical Sites of Endometriosis Haya Al-Fozan and Togas Tulandi

45

4. Genetics of Endometriosis Stephen Kennedy

55 vii

viii

Contents

5.

Research Aspects of Endometriosis Dan C. Martin

69

6.

Animal Models for Research on Endometriosis Thomas M. D’Hooghe, Sophie Debrock, Joseph A. Hill, Daniel C. Chai, and Jason M. Mwenda

81

7.

Immunology of Endometriosis and Immunotherapy Emre Seli, Neal G. Mahutte, Murat Berkkanoglu, and Aydin Arici

99

8.

Serum and Peritoneal Markers for Endometriosis Tommaso Falcone and Mohamed A. Bedaiwy

123

9.

How Does Endometriosis Cause Infertility? Ariane Germeyer and Linda C. Giudice

151

10.

Medical Therapy for Endometriosis: An Overview Eric S. Surrey

167

11.

Treatment with Aromatase Inhibitors Serdar E. Bulun, Bilgin Gurates, Zongjuan Fang, Mitsutoshi Tamura, David Langoi, Gonca Imir, Sanober Amin, Santanu Deb, and Sijun Yang

189

12.

Endometriosis: Medical Treatment with Progestagens Robert Lahoud and Robert F. Harrison

203

13.

Gonadotropin Releasing Hormone Agonist and Antagonist for Endometriosis Robert L. Barbieri

219

Management of Endometriosis After Hysterectomy and Bilateral Salpingo-Oophorectomy David Redwine

245

14.

15.

Treatment of Ovarian Endometrioma Haya Al-Fozan and Togas Tulandi

263

16.

Treatment of Endometriosis-Related Pelvic Pain Kevin Jones and Christopher Sutton

273

Contents

17.

18.

ix

In Vitro Fertilization or Superovulation? Evidence, Flaws, and Advice Ian S. Tummon

295

Does Endometriosis Affect the Results of In Vitro Fertilization? Simon M. Kelly, William M. Buckett, and Seang Lin Tan

315

Index

325

Contributors

Haya Al-Fozan Department of Obstetrics and Gynecology, McGill University, Montreal, Quebec, Canada Sanober Amin Department of Obstetrics and Gynecology, University of Illinois at Chicago, Chicago, Illinois, U.S.A. Aydin Arici, M.D. Department of Reproductive Endocrinology, Yale University School of Medicine, New Haven, Connecticut, U.S.A. Robert L. Barbieri, M.D. Department of Obstetrics, Gynecology, and Reproductive Biology, Brigham and Women’s Hospital, Boston, Massachusetts, U.S.A. Mohamed A. Bedaiwy, M.D. Department of Gynecology and Obstetrics, The Cleveland Clinic Foundation, Cleveland, Ohio, U.S.A.

xi

xii

Contributors

Serdar E. Bulun Department of Obstetrics and Gynecology, University of Illinois at Chicago, Chicago, Illinois, U.S.A. Murat Berkkanoglu, M.D. Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, Connecticut, U.S.A. William M. Buckett Department of Obstetrics and Gynecology, McGill University, Montreal, Quebec, Canada Daniel C. Chai U.S.A.

Fertility Center of New England, Reading, Massachusetts,

Santanu Deb Department of Obstetrics and Gynecology, University of Illinois at Chicago, Chicago, Illinois, U.S.A. Sophie Debrock, Ph.D. Department of Gynecology and Obstetrics, Leuven University Fertility Center, Leuven, Belgium Thomas M. D’Hooghe, M.D., Ph.D. Department of Gynecology and Obstetrics, Leuven University Fertility Center, Leuven, Belgium Jacques Donnez, M.D., Ph.D. Department of Gynecology, Catholic University of Louvain, Brussels, Belgium Tommaso Falcone, M.D. Department of Gynecology and Obstetrics, The Cleveland Clinic Foundation, Cleveland, Ohio, U.S.A. Zongjuan Fang Department of Obstetrics and Gynecology, University of Illinois at Chicago, Chicago, Illinois, U.S.A. Ariane Germeyer, M.D. Department of Gynecology and Obstetrics, Stanford University School of Medicine, Stanford, California, U.S.A., and Department of Reproductive Medicine, University of Heidelberg, Heidelberg, Germany Linda C. Giudice, M.D., Ph.D. Department of Gynecology and Obstetrics, Stanford University School of Medicine, Stanford, California, U.S.A. Bilgin Gurates Department of Obstetrics and Gynecology, University of Illinois at Chicago, Chicago, Illinois, U.S.A.

Contributors

xiii

Robert F. Harrison, M.A., M.D., D.Sc., F.R.C.S., F.R.C.O.G., F.R.C.P. Department of Obstetrics and Gynecology, Rotunda Hospital, Dublin, Ireland Joseph A. Hill U.S.A.

Fertility Center of New England, Reading, Massachusetts,

Gonca Imir Department of Obstetrics and Gynecology, University of Illinois at Chicago, Chicago, Illinois, U.S.A. Kevin Jones, M.D., M.B., Ch.B., M.R.C.O.G., M.Sc. Department of Obstetrics and Gynecology, Great Western Hospital, Swindon, Wiltshire, England Stephen Kennedy, M.D., M.A., M.R.C.O.G. Nuffield Department of Obstetrics and Gynecology, University of Oxford, John Radcliffe Hospital, Oxford, England Simon M. Kelly, M.D., F.R.A.N.Z.C.O.G. Department of Obstetrics and Gynecology, McGill University, Montreal, Quebec, Canada Philippe R. Koninckx, M.D., Ph.D. Department of Obstetrics and Gynecology, Catholic University of Leuven, Leuven, Belgium Robert Lahoud, M.B.B.S., M.Med., F.R.A.N.Z.C.O.G. West, Westmead, Australia

IVF Australia

David Langoi Department of Obstetrics and Gynecology, University of Illinois at Chicago, Chicago, Illinois, U.S.A. Neal G. Mahutte, M.D. Department of Obstetrics and Gynecology, Dartmouth College School of Medicine, Lebanon, New Hampshire, U.S.A. Dan C. Martin, M.D. Department of Obstetrics and Gynecology, University of Tennessee, Memphis, Tennessee, U.S.A. Jason M. Mwenda, Ph.D. Department of Reproductive Biology, Institute of Primate Research, Nairobi, Kenya David Redwine, A.B., M.D. Oregon, U.S.A.

Endometriosis Institute of Oregon, Bend,

xiv

Contributors

Emre Seli, M.D. Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, Connecticut, U.S.A. Jean Squifflet, M.D. Department of Gynecology, Catholic University of Louvain, Brussels, Belgium Eric S. Surrey, M.D. Colorado Center for Reproductive Medicine, Englewood, Colorado, U.S.A. Christopher Sutton, M.A., M.B., B.Ch., F.R.C.O.G. Department of Obstetrics and Gynecology, Royal Surrey County Hospital, Guildford, Surrey, England Mitsutoshi Tamura Department of Obstetrics and Gynecology, University of Illinois at Chicago, Chicago, Illinois, U.S.A. Seang Lin Tan, M.B.B.S., F.R.C.O.G., F.R.C.S.(C)., M.MED(O&G) Department of Obstetrics and Gynecology, McGill University, Montreal, Quebec, Canada Togas Tulandi, M.D., F.R.C.S.C., F.A.C.O.G. Department of Obstetrics and Gynecology, McGill University, Montreal, Quebec, Canada Ian S. Tummon, M.D., F.R.C.S.(C). Department of Obstetrics and Gynecology, Mayo Clinic, Rochester, Minnesota, U.S.A. Sijun Yang Department of Obstetrics and Gynecology, University of Illinois at Chicago, Chicago, Illinois, U.S.A.

1 Epidemiology of Endometriosis Philippe R. Koninckx Catholic University Leuven Leuven, Belgium

INTRODUCTION Endometriosis is still a poorly understood condition. This is despite the high and still increasing publication of more than 500 articles per year. For example, there were 455, 426, 448, 504, and 534 articles in the past 5 years, respectively. Endometriosis is considered to be one of the most important causes of pelvic pain and infertility. The exact prevalence is unknown, as a laparoscopy is required to make the diagnosis and the recognition varies with the experience and the interest of the laparoscopist. Moreover, the pathophysiology is poorly understood, which makes it difficult to formulate and test simple hypotheses. The definitions of endometriosis have changed over time, contributing to biases in the literature. In the mid-eighties, the concept of nonpigmented or subtle endometriosis was introduced, and from the nineties onwards the recognition of deep endometriosis has progressively increased. The revised American Fertility Society (rAFS) classification is widely used. Yet, it has never been validated as a classification for pain or infertility. 1

2

Koninckx

Taken together, the absence of an easy, non invasive diagnosis, the changing definitions, and the absence of a clear understanding of the pathophysiology and the absence of a validated classification system are the reasons why there are still many controversies surrounding endometriosis. We therefore consider it a prerequisite to introduce definitions and biases, and the problems of pathophysiology and classification systems, before discussing epidemiology.

DEFINITIONS AND BIASES IN THE LITERATURE Endometriosis is defined as the presence of endometrial glands and stroma outside the uterus. It was described at the turn of the century as severe lesions such as ovarian ‘‘chocolate cysts’’ [1] and as adenomyosis externa [2–7]. In 1899, Russell [8] wrote, ‘‘On the microscopic study of the ovary, we were astonished to find areas which were an exact prototype of the uterine glands and interglandular connective tissue.’’ In the following decades, endometriosis was described as a disorder causing pain and requiring surgery. During that period, other sites [9] were described, and endometriosis was reported as an ‘‘accidental’’ finding during surgery for other gynecological disorders [10–12]. Only after the introduction of endoscopy in the late 1960s, blackpuckered endometriosis lesions were recognized as a frequent observation in women with pain or infertility. Following the description of nonpigmented endometriosis in the mid eighties [13–16], the prevalence of the disease increased [17–25]. In the 90s, the awareness of deep infiltrating endometriosis increased progressively, together with the recognition that this type of endometriosis was not always diagnosed during surgery. Subtle Endometriosis Following the recognition of nonpigmented endometriosis [26] in the mid80s, the race to find smaller implants led to a series of articles describing polypoid lesions [14–16,27,28], white and red vesicles, flamelike lesions, and finally microscopic endometriosis [29–31]. The latter is visible only under the microscope or by scanning electron microscopy [32,33]. This led to the suggestion that microscopic endometriosis could be present in all women, which induced techniques such as peritoneal washings [34] or blood painting [35] to diagnose endometriosis. The interest in nonpigmented endometriosis was further triggered by the observation that these lesions were morphologically very active, leading to the speculation that this activity is due to secretion of ‘‘active’’ substances in peritoneal fluid [36], which could explain infertility and pain. These lesions

Epidemiology

3

can be stimulated to develop by substances in peritoneal fluid and suppressed by medical treatment [37–42]. It has been postulated that the severity of endometriosis is better assessed by its degree of activity, rather than by its extent [43]. We will use the term ‘‘subtle endometriosis’’ throughout the text to refer to these lesions. I prefer to define them as small, superficial, and active lesions without surrounding sclerosis and without the hemosiderin black spots. Subtle lesions contain gland and stroma, and thus fit the definition of endometriosis. However, recognition and definition of these lesions remain controversial. Its recognition increases with the awareness and with experience of the surgeon. Morphologic confirmation of endometriosis rarely exceeds 60 (57%) [44]. This is generally attributed to technical problems of excision of these small lesions and detecting them after processing. Endometriosislike lesions are also well recognized. Their prevalence is unknown. The concept of microscopic endometriosis makes the issue more complicated. According to this concept, all women would have endometriosis. The data to support it have been anecdotal, and a study in baboons showed that the incidence is low [45]. The newest concept is ‘‘nonimplanted endometriosis’’ in peritoneal fluid that has to be distinguished from retrograde menstruation. Typical Endometriosis Typical lesions are described as black puckered lesions surrounded by a sclerotic area and by a typical vascular pattern, suggesting angiogenesis. We may assume that an experienced surgeon can readily recognize these lesions. Yet, at least two problems exist in their detection and reporting. The first is that the exact prevalence of endometriosislike lesions is unknown. The second is endometriosis on the diaphragm. This has been considered rare, but the number of surgeons who systematically inspect the diaphragm in steep Trendelenburg, with a 30-degree scope, is very low. Even for this lesion, the histological confirmation rarely exceeds 80% (76% [44], even 50% [46]). Cystic Ovarian Endometriosis Distinguishing cystic ovarian endometriosis from cystic corpus luteum can be difficult. Women with ultrasound findings of persistent endometriotic cyst of more than 4 months, including those who were treated with GnRHa or OCP, often were found to have only cystic corpus luteum at surgery. These clinical observations do not allow any conclusion about its prevalence, but are consistent with the report that ovarian cysts can develop during ovarian downregulation [47].

4

Koninckx

Imaging, such as ultrasound and CT scanning, has a sensitivity of 70% to 80% and a specificity of 90% to 95% [48–52]. This is a valuable method of preoperative diagnosis. Ovarian blood flow measurement does not seem to improve its specificity or sensitivity [48]. Measurement of CA125 in the endometrioma fluid increased the sensitivity and specificity to nearly 100% [53,54]. Unfortunately, a rapid test to assist an intraoperative diagnosis is not yet available. In general, cystic ovarian endometriosis is associated with adhesions [25], whereas a ‘‘chocolate cyst’’ without adhesions is most likely a cystic corpus luteum. The presence of severe adhesions, especially in the fossa ovarica, should raise the suspicion of an endometriotic cyst. Diagnostic accuracy can be further increased by inspection of the inside of the cyst by ovarioscopy [55] or laparoscopy [56]. ‘‘Those with a flattened appearance and red or red and brown mottled ridges generally were endometriosis and those with a dark uniform base, an intracavitary clot, or a yellowish rim generally were corpus lutea or albicans.’’ [57]. A second problem is that the pathology report often reveals ‘‘compatible with endometriosis,’’ without a positive identification of endometrial glands and stroma. This is not unusual, especially for larger cysts. However, it is rarely addressed in the literature, making it difficult to know how accurate the diagnosisis is. Different treatments give different results and recurrence rates. During the microsurgery era, the procedure was done by excising the cyst wall and suturing the ovarian opening [58]. Today, there are several endoscopic techniques. Aspiration and rinsing of cystic ovarian endometriosis has been attempted, but the recurrence rate is high [59–61]. For cysts of less than 5 cm, the method of stripping the cyst from the ovary is rapid and relatively easy [62]. Closure of the ovary can be achieved by tissucol or sutures. The cyst wall can be vaporized [63] or destroyed with unipolar or bipolar coagulation. Another method is focal treatment [64]. To understand the rationale of focal treatment, the rediscovery of the work of Hughesdon [31, 65,66] is important. By serial sections of ovarian endometrioma, it was postulated that it developed from invagination of the ovarian cortex. The difficulty in diagnosis, and the different techniques used for treatment should be taken into account when interpreting results and prevalence of endometriosis [67–70].

Deep Endometriosis In the 90s it was realized that deep endometriosis was a frequent disease, either recognized during laparoscopic surgery [25,71] or by clinical examination during menstruation [72]. The endoscopic excision of endometriosis has

Epidemiology

5

revealed that endometriosis deeper than 5 to 6 mm is associated with pain and infertility. Three subtypes were described [73]. Type I is characterized by a large area of typical and sometimes subtle endometriotic lesions surrounded by white sclerotic tissue. Only during excision does it become obvious that the endometriosis infiltrates deeper than 5 mm. Typically the endometriotic area becomes progressively smaller and as it grows deeper, the lesion becomes cone shaped. Type II lesions are characterized by retraction of the bowel. Clinically they are recognized as a prominent bowel retraction around a small typical lesion. In some women, however, endometriosis might not be seen by the laparoscope, and the bowel retraction is the only clinical sign. Diagnosis is not difficult, because during the laparoscopy an induration under the bowel can be felt. Otherwise, it is diagnosed only during excision. Type III lesions are spherical endometriotic nodules above or inside the rectovaginal septum. Typically, these lesions are felt as painful nodules. In some women, vaginal examination reveals some dark blue cysts (3–4 mm) in the posterior fornix. Sclerosing endometriosis invading the sigmoid is similar to the rectal endometriosis, but is situated 10 cm above the rectovaginal septum. This is another rare form of deep endometriosis, which we proposed to classify as type IV. The literature on deep endometriosis is confusing and controversial. The diagnosis of deep endometriosis cannot be established by clinical examination. Even during menstruation, high located deep endometriosis cannot be palpated. Large lesions can be diagnosed by contrast enema, transvaginal or transrectal ultrasound, or MRI. The sensitivity of these imaging techniques for detecting smaller lesions is unknown. As my expertise and awareness developed, I realized that a substantial amount of these lesions, especially the smaller rectovaginal lesions or the type IV lesions, have been previously missed at surgery. Biases and Shifts in the Literature In the past 20 years, there has been a gradual evolution in the recognition of the different stages of endometriosis. Subtle endometriosis has been recognized since the mid-80s. This has increased the apparent prevalence of endometriosis. It has also change our perception of ‘‘normal women.’’ Traditionally, women with minimal and mild endometriosis were almost exclusively those with typical lesions, whereas ‘‘normal women’’ have subtle endometriosis only. This evolution alone explains why the association between luteinized unruptured follicle (LUF) syndrome and minimal endometriosis systematically reported before 1985 disappeared from the literature. Luteinized unruptured follicle syndrome has been associated with typical lesions, but not with subtle lesions.

6

Koninckx

The awareness of these changes is important in understanding and interpreting the data reported in the literature. This is essential when discussing prevalence, and when comparing older data with more recent observations. The bias of confusing cystic ovarian endometriosis and cystic corpora lutea will have little effect on the reported prevalence, although in some series cystic corpora lutea could be as high as 30%. As surgeons became more aware of deep endometriosis over the years, its reported prevalence increased. The recognition that the small lesions tend to go unnoticed even during laparoscopy, mainly because of lack of performing diagnostic methods, leads to the conclusion that the prevalence of deep endometriosis is underreported. This is especially important in studies concerning pelvic pain. CLASSIFICATION OF ENDOMETRIOSIS Revised AFS classification is a point scoring system. We found that class I consisted of superficial lesions with a total area of less than 3 cm2; class II, superficial lesions with a total area of more than 3 cm2; classes III and IV comprised mainly cystic ovarian endometriosis. The contribution of adhesions to the rAFS classification and its association with cystic ovarian endometriosis easily explains this [25]. It should be stressed that the rAFS has never been validated as a tool to evaluate infertility or pain objectively. As the clinical importance of subtle lesions is questionable, it might be preferable to regroup women with subtle lesions into a separate class. Deep lesions are found in all four rAFS classes, but mainly in classes I and II. When we regrouped these lesions into a separate class, we found that cystic ovarian endometriosis and deep lesions are those that correlate with pain. Without regrouping, these associations disappear. This is because milder endometriosis groups are variably contaminated with deep endometriosis. PATHOPHYSIOLOGY Sampson and Metaplasia Theory Sampson’s retrograde menstruation, implantation, and the metaplasia theory focuses on the implantation/metaplasia of cells. It refers to subtle and small initial lesions that will subsequently grow and develop to more severe disease. It is an attractive theory because of the abundance of data demonstrating retrograde menstruation as a frequent phenomenon in all women and the presence of viable endometrial cells in the peritoneal fluid, which have the capacity to implant, grow, and infiltrate superficially. According to this hypothesis, the development into advanced condition may be influenced

Epidemiology

7

by a decreased cellular immunity, a lower natural killer (NK) cell activity, peritoneal fluid cytokines and growth factors, or low peritoneal fluid steroid concentrations in the luteal phase. Each step in the pathophysiology has been documented. This theory, however, cannot explain why progression occurs in some women only. It holds that progression of endometriosis, once established, is unavoidable, albeit at a different speed and to a different stage according to modulating factors. This theory considers endometriosis as normal endometrial cells behaving abnormally in an abnormal environment, that is, the peritoneal milieu. However, this theory is not supported by all [74]. The key event in the process is implantation or metaplasia, which has been the subject of many investigations. The early subtle lesions become very important. The Endometriotic Disease Theory The endometriotic disease theory (EDT) [75] considers retrograde menstruation, viable endometrial cells in peritoneal fluid, and occasional implantation of these cells as a normal physiological phenomenon. The non implanted and implanted cells are normally removed by the defense mechanisms of the body, such as macrophages. Attachment and implantation occur when the mesothelial layer is damaged by trauma, infection, or low-grade inflammation, for example, irritation caused by CO2 pneumoperitoneum, or by abundant retrograde menstruation. It seems logical that attachment and implantation must occur more frequently when more viable cells are present in peritoneal fluid. These cells can temporarily grow and develop, depending on the environment. When left alone, they can also disappear spontaneously. This may result in some fibrotic or scar tissue as the remnant of local inflammation. It contains some endometrial cells, shielded from the bloodstream and immunocompetent cells similar to bacteria in an abscess. Endometriosis is caused by cellular modification, such as genetic mutation, as observed in many benign tumors. This cellular modification occurs more frequently in genetically predisposed persons, and it is facilitated by other factors such as total body irradiation, or by chemical pollutants, such as dioxins. The probability that such an event occurs is higher when more cells are present seems logical. The type of cellular modification, together with local factors such as the peritoneal fluid microenvironment or the intraovarian milieu, will determine whether they will develop into typical lesions, deep endometriosis, or cystic ovarian endometriosis, and whether the morphological characteristics will be chocolate cysts, endometrial glands and stroma, or adenomyosis externa. This theory also refers to subtle lesions as a normal physiological condition, occurring intermittently in all women. Typical, cystic, and deep endo-

8

Koninckx

metriosis are considered as benign tumors, originating from a cellular modification transforming endometrial cells into endometriotic cells. Endometriotic disease is the presence of abnormal cells in an abnormal environment. Pathophysiology and Prevalence The theories of pathophysiology of endometriosis are essential in discussions of prevalence. Indeed, according to the EDT, subtle endometriosis is a physiological condition, occurring intermittently in all women, and these lesions should not be considered a disease [59]. According to Sampson/metaplasia theory, subtle lesions are the early stages of endometriosis and extremely important because they are very active. Accordingly, it is logical to examine the pelvis for these early and small endometriosis lesions and to treat them to prevent progression. The epidemiology of the four major presentations of endometriosis will be discussed separately.

EPIDEMIOLOGY Although neither the ideal design nor the ideal case and control groups are likely to be achievable in epidemiologic studies of endometriosis, better subject-selection strategies may improve the validity of studies that are obliged to depart from the ideal [76]. Subtle Endometriosis Following the description of non pigmented endometriosis in the eighties [13– 16], the prevalence of the disease increased from 5% to 20% to 60% to 80% in women with infertility or pelvic pain [17–25,77]. The prevalence clearly increases with the awareness and the experience of the surgeon. In all series, the underlying biases of no confirmation or, at best, limited confirmation by pathology should be recognized. The prevalence of subtle lesions decreases with age for unknown reasons [25,78]. No studies have demonstrated an association with any of the variables considered important, such as early menarche, short cycles, abundant or painful periods, infertility, race, dioxin, or total body radiation. Typical Endometriosis Taking into account only typical endometriosis, the prevalence of asymptomatic endometriosis varies from 4% in women undergoing tubal ligation to 50% in teenagers with intractable dysmenorrhea. The prevalence in women

Epidemiology

9

with pain or infertility ranges between 40% and 70% [25,46]. In general, the incidence is estimated to be 1.3 per 1000 women aged 15 to 44 [79]. In a recent large study in Norway, the lifetime risk for endometriosis was 2.2% [80]. In this study, early menarche, frequent menstruations, pelvic pain, infertility, and nulliparity were associated with endometriosis. In a controlled study of women with infertility and a normal partner, compared with women with azoospermic partner, stage I endometriosis is not more common in infertile women than in control women. However, stage II endometriosis was more frequent (3.3% vs 5.7%) in infertile women [81]. There is a non validated clinical impression that endometriosis could vary with race blacks having lower rates of endometriosis and Asians have higher rates than Caucasians. According to the Sampson’s theory, abundant retrograde menstruation is a predisposing factor for endometriosis. This seems to be clinically and experimentally supported by increased prevalence of endometriosis in women and in primates with obstructed uterine outflow. Indeed, women with endometriosis have more abundant periods, and early menarche. However, a recent review failed to demonstrate this association [82]. Endometriosis is clearly associated with dysmenorrhea, but it is unknown whether this is a cause or a consequence. Dioxin has been suggested to be causally related to endometriosis. This hypothesis was formulated in 1994 [83] based on indirect observations that the incidence and severity of endometriosis increased in primates treated with dioxins [84,85]. In the human, final proof is still lacking [86]. The Seveso accident, with massive pollution, suggests a nonsignificant doubling of prevalence [87]. Also, breast-fed infants, possibly exposed to dioxins in milk, have a lower incidence of endometriosis in adult life [88]. Total body radiation is associated with increased prevalence of endometriosis in primates [89]. Little evidence is available to support this in the human. Endometriosis is a hereditary disease [90–98]. The prevalence among first-degree relatives is seven times higher than in control groups. In monozygotic twins the prevalence is up to 15 times higher. The lower natural killer cell activity in plasma and in peritoneal fluid [71,99–106] has fueled speculation about the role of the immune system in endometriosis [107–110]. To date, however, no association has been found between the prevalence of endometriosis and chronic immunosuppression (e.g., in transplant patients), or smoking, caffeine, alcohol, or other lifestyle variable affecting NK activity. Stress could be related to endometriosis. This concept is derived from the association of endometriosis and LUF syndrome, the relationship between a higher trait anxiety and LUF syndrome [111–114], and the hypothesis

10

Koninckx

that lower steroid hormone concentrations in peritoneal fluid might facilitate the implantation/development of endometriosis [115]. As there is no adequate animal model, this hypothesis cannot be tested. The best animal model is the baboon. It has been shown that baboons in captivity have more endometriosis than in the wild (probably through stress) [116]. Another argument to link endometriosis and stress is the widely held belief that endometriosis is a career women’s disease. This, however, can be explained by the delay of childbearing in this group of women, with the inevitable increase of infertility with age, and a higher prevalence of endometriosis at laparoscopy. Nulliparity could be a consequence of the disease but in a large study in Italy, the prevalence decreased with increasing parity [117]. Oral contraception use has been reported to be associated with a decreased prevalence [17]. Endometriosis was recently suggested to be associated with an increased risk in ovarian cancer (OR = 1.73, 95% CI: 1.10, 2.71) [118], and of nonHodgkin’s lymphoma [119]. Cystic Ovarian Endometriosis Cystic ovarian endometriosis increases with age [25]. Most reports confirmed that cystic ovarian endometriosis is clonal in origin [120–123]. Deep Endometriosis Deep endometriosis increases with age [25]. Rectovaginal endometriosis was known since the beginning of the century, but the high prevalence of deep endometriosis remained unsuspected until recently. The observations from Leuven from 1988 to 1991 [25], a period during which endoscopic surgery has not yet developed, showed that the prevalence of deep endometriosis was 10% to 20%. Referrals were only for infertility and pain and not for deep endometriosis. Assuming that laparoscopies for infertility are performed in 10% to 15% of the population and taking into account that Leuven is a tertiary referral center, the prevalence of deep endometriosis can be estimated to be 1% to 3%. No data are available to link deep endometriosis to a subgroup of women or to a potential causal factor. Endometriosis and Cancer Occasional reports describe cancer in cystic ovarian [124] or severe endometriosis [96,125]. We recently diagnosed an adenocarcinoma in a deep endometriotic lesion. The relationship between endometriosis and a possible increased risk of ovarian cancer, however, remains unclear.

Epidemiology

11

CONCLUSIONS AND DISCUSSION When evaluating reports on epidemiology of endometriosis, it is important to distinguish between subtle, typical, cystic, and deep endometriosis and to take into account the evolution of subtle endometriosis and deep endometriosis. A simple non invasive test of endometriosis is still not available. The prevalence of endometriosis is high, particularly in women with pain and infertility. Subtle endometriosis ranges from 5% to 50% in asymptomatic women to 50% to 80% in women with symptoms. For typical lesions, estimations are less than half of these figures, but the data came from reports before 1985. For severe endometriosis either cystic or deep, the prevalence is between 1% and 10%. A poorly addressed problem is the variability of prevalences by region and country. No systematic studies are available, but in my experience, it seems that the prevalence of very severe deep endometriosis is higher in Belgium than in the United Kingdom or south Italy. In Moscow, the prevalence of severe endometriosis is also high. In Middle Eastern countries, endometriosis seems to be rare. Although the evidence is anecdotal, it could be in agreement with the hypothesis of pollution. Endometriosis is a hereditary disease, particularly the typical and cystic ovarian types. Increased retrograde menstruation, for example, by outflow obstruction, will increase the prevalence of endometriosis. The role of nutrition, lifestyle, personality traits, the immune system, the peritoneal fluid, and other variables in endometriosis is unclear. Indirect evidence strongly suggests a modulating role. The question whether endometriosis represents normal endometrial cells or abnormal-modified endometrial cells remains unanswered. Until then, prevention of implantation, prevention of cellular damage, and the treatment of endometriosis will remain empirical.

REFERENCES 1.

2. 3. 4. 5.

Sampson JA. Peritoneal endometriosis due to the menstrual dissemination of endometrial tissue into the peritoneal cavity. Am J Obstet Gynecol 1927; 14: 422–469. Cullen TS. Adeno-myoma of the round ligament. J Hopkins Hosp Bull 1896; 7:112–114. Cullen TS. Adenoma-myoma uteri diffusum benignum. J Hopkins Hosp Bull 1896; 6:133–137. Lockyer C. Adenomyoma in the recto-uterine and recto-vaginal septa. Proc Royal Soc Med 1913; 6:112–120. Cullen TS. The distribution of adenomyomata containing uterine mucosa. Am J Obstet Gynecol 1919; 80:130–138.

12 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

20. 21. 22.

23. 24. 25.

26.

Koninckx Meighs V. An interest in endometriosis and its consequences. Am J Obstet Gynecol 1920; 79:625. Meyer R. Zur frage der Urnieren-genese van Adenomyomen. Zentralbl Gynakol 1923; 15:577–587. Russell WW. Aberrant portions of the Mullerian duct found in an ovary. Hopkins Hosp Bull 1899; 94–96:8–10. Latcher JW. Endometriosis of the umbilicus. Am J Obstet Gynecol 1953; 66:161. Gruenwald P. Origin of endometriosis from the mesenchyme of the celomic walls. Am J Obstet Gynecol 1942; 44:470–474. Scott RB, Telinde RW. External endometriosis-the scourge of the private patient. Ann Surg 1950; 131:697. Burgmeister RE, Fechner RE, Franklin RR. Endosalpingiosis of the peritoneum. Obstet Gynecol 1969; 34:310–317. Jansen RPS, Russel P. Nonpigmented endometriosis: Clinical, laparoscopic, and pathologic definition. Am J Obstet Gynecol 1986; 155:1154–1159. Stripling MC, Martin DC, Chatman DL, Vander Zwaag R, Poston WM. Subtle appearance of pelvic endometriosis. Fertil Steril 1988; 49:427–431. Stripling MC, Martin DC, Poston WM. Does endometriosis have a typical appearance? J Reprod Med Obstet Gynecol 1988; 33:879–884. Martin DC, Hubert GD, Van der Zwaag R, El Zeky FA. Laparoscopic appearances of peritoneal endometriosis. Fertil Steril 1989; 51:63–67. Mahmood TA, Templeton A. Prevalence and genesis of endometriosis. Hum Reprod 1991; 6:544–549. Rawson JMR. Prevalence of endometriosis in asymptomatic women. J Reprod Med Obstet Gynecol 1991; 36:513–515. Houston DE, Noller KL, Melton LJ, Selwyn BJ, Hardy RJ. Incidence of pelvic endometriosis in Rochester, Minnesota, 1970–1979. Am J Epidemiol 1987; 125: 959–969. Moen MH. Endometriosis in women at interval sterilization. Acta Obstet Gynecol Scand 1987; 66:451–454. Hull MGR, Glazener CMA, Kelly NJ. Population study of causes, treatment, and outcome of infertility. BMJ 1985; 291:1693–1697. Strathy JH, Molgaard CA, Coulam CB, Melton LJ III. Endometriosis and infertility: A laparoscopic study of endometriosis among fertile and infertile women. Fertil Steril 1985; 44:83–88. Nikanen V, Punnonen R. External endometriosis in 801 operated patients. Acta Obstet Gynecol Scand 1984; 63:699–701. Bitzer J, Korber HR. Laparoscopy findings in infertile women. Geburtshilfe Frauenheilkd 1983; 43:294–298. Koninckx PR, Meuleman C, Demeyere S, Lesaffre E, Cornillie FJ. Suggestive evidence that pelvic endometriosis is a progressive disease, whereas deeply infiltrating endometriosis is associated with pelvic pain. Fertil Steril 1991; 55:759– 765. Jansen RP. Tubal resection and anastomosis I. Sterilization-reversal. Aust N Z J Obstet Gynaecol 1986; 26:294–299.

Epidemiology

13

27. Martin DC. Is bloody fluid endometriosis? (II). Fertil Steril 1990; 54:1186–1187. 28. Bancroft K, Vaughan Williams CA, Elstein M. Minimal/mild endometriosis and infertility. A review [see comments]. Br J Obstet Gynaecol 1989; 96:454– 460. 29. Brosens IA, Cornillie FJ. Peritoneal endometriosis. Morphological basis of the laparoscopic diagnosis. Contrib Gynecol Obstet 1987; 16:125–137. 30. Nezhat F, Allan CJ, Nezhat C, Martin DC. Nonvisualized endometriosis at laparoscopy. Int J Fertil 1991; 36:340–343. 31. Brosens I, Puttemans P, Deprest J. Appearances of endometriosis. Baillieres Clin Obstet Gynaecol 1993; 7:741–757. 32. Vasquez G, Cornillie F, Brosens IA. Peritoneal endometriosis: Scanning electron microscopy and histology of minimal pelvic endometriotic lesions. Fertil Steril 1984; 42:696–703. 33. Murphy AA, Green WR, Bobbie D, dela Cruz ZC, Rock JA. Unsuspected endometriosis documented by scanning electron microscopy in visually normal peritoneum. Fertil Steril 1986; 46:522–524. 34. Ravinsky E. Cytology of peritoneal washings in gynecologic patients. Diagnostic criteria and pitfalls. Acta Cytol 1986; 30:8–16. 35. Redwine DB. Peritoneal blood painting: An aid in the diagnosis of endometriosis. Am J Obstet Gynecol 1989; 161:865–866. 36. Vernon MW, Beard JS, Graves K, Wilson EA. Classification of endometriotic implants by morphologic appearance and capacity to synthesize prostaglandin F. Fertil Steril 1986; 46:801–806. 37. Cornillie FJ, Vasquez G, Brosens I. The response of human endometriotic implants to the anti-progesterone steroid R 2323: A histologic and ultrastructural study. Pathol Res Pract 1985; 180:647–655. 38. Cornillie FJ, Brosens IA, Vasquez G, Riphagen I. Histologic and ultrastructural changes in human endometriotic implants treated with the antiprogesterone steroid ethylnorgestrienone (gestrinone) during 2 months. Int J Gynecol Pathol 1986; 5:95–109. 39. Brosens IA, Verleyen A, Cornillie F. The morphologic effect of short-term medical therapy of endometriosis. Am J Obstet Gynecol 1987; 157:1215–1221. 40. Cornillie FJ, Puttemans P, Brosens IA. Histology and ultrastructure of human endometriotic tissues treated with dydrogesterone (Duphaston). Eur J Obstet Gynecol Reprod Biol 1987; 26:39–55. 41. Brosens IA. The rationale for endocrine therapy. Acta Obstet Gynecol Scand Suppl 1989; 150:21–25. 42. Shaw RW. Endometriosis : Current evaluation of management and rationale for medical therapy. In: Brosens IA, Donnez J, eds. The Current Status of Endometriosis. New York: Parthenon Publishing, 1993:371–383. 43. Brosens IA, Cornillie F, Koninckx PR, Vasquez G. Evolution of the Revised American Fertility Society Classification of Endometriosis [letter]. Fertil Steril 1985; 44:714–716. 44. Moen MH, Halvorsen TB. Histologic confirmation of endometriosis in different peritoneal lesions. Acta Obstet Gynecol Scand 1992; 71:337–342.

14

Koninckx

45. D’Hooghe TM, Bambra CS, DeJonge I, Machai PN, Korir R, Koninckx PR. A serial section study of visually normal posterior pelvic peritoneum from baboons (Papio cynocephalus, Papio anubis) with and without spontaneous minimal endometriosis. Fertil Steril 1995; 63:1322–1325. 46. Walter AJ, Hentz JG, Magtibay PM, Cornella JL, Magrina JF. Endometriosis: Correlation between histologic and visual findings at laparoscopy. Am J Obstet Gynecol 2001; 184:1407–1411. 47. Jenkins JM, Anthony FW, Wood P, Rushen D, Masson GM, Thomas E. The development of functional ovarian cysts during pituitary down-regulation. Hum Reprod 1993; 8:1623–1627. 48. Alcazar JL, Laparte C, Jurado M, Lopez GG. The role of transvaginal ultrasonography combined with color velocity imaging and pulsed Doppler in the diagnosis of endometrioma. Fertil Steril 1997; 67:487–491. 49. Mais V, Guerriero S, Ajossa S, Angiolucci M, Paoletti AM, Melis GB. The efficiency of transvaginal ultrasonography in the diagnosis of endometrioma. Fertil Steril 1993; 60:776–780. 50. Outwater EK, Dunton CJ. Imaging of the ovary and adnexa: Clinical issues and applications of MR imaging. Radiology 1995; 194:1–18. 51. Guerriero S, Ajossa S, Paoletti AM, Mais V, Angiolucci M, Melis GB. Tumor markers and transvaginal ultrasonography in the diagnosis of endometrioma. Obstet Gynecol 1996; 88:403–407. 52. Guerriero S, Mais V, Ajossa S, Paoletti AM, Angiolucci M, Melis GB. Transvaginal ultrasonography combined with CA-125 plasma levels in the diagnosis of endometrioma. Fertil Steril 1996; 65:293–298. 53. Koninckx PR, Muyldermans M, Moerman P, Meuleman C, Deprest J, Cornillie F. CA 125 concentrations in ovarian ’chocolate’ cyst fluid can differentiate an endometriotic cyst from a cystic corpus luteum. Hum Reprod 1992; 7:1314–1317. 54. Koninckx PR. CA 125 in the management of endometriosis. Eur J Obstet Gynecol Reprod Biol 1993; 49:109–113. 55. Brosens IA, Puttemans PJ, Deprest J. The endoscopic localization of endometrial implants in the ovarian chocolate cyst. Fertil Steril 1994; 61:1034– 1038. 56. Martin DC, Demos Berry J. Histology of chocolate cysts. J Gynecol Surg 1990; 6:43–46. 57. Martin DC, Ahmic R, El Zeky FA, Vander Zwaag R, Pickens MT, Cherry K. Increased histologic confirmation of endometriosis. J Gynecol Surg 1990; 6: 275–279. 58. Gordts S, Boeckx W, Brosens I. Microsurgery of endometriosis in infertile patients. Fertil Steril 1984; 42:520–525. 59. Vercellini P, Bocciolone L, Crosignani PG. Is mild endometriosis always a disease? Hum Reprod 1992; 7:627–629. 60. Giorlandino C, Taramanni C, Muzii L, Santillo E, Nanni C, Vizzone A. Ultrasound-guided aspiration of ovarian endometriotic cysts. Int J Gynaecol Obstet 1993; 43:41–44.

Epidemiology

15

61. Aboulghar MA, Mansour RT, Serour GI, Rizk B. Ultrasonic transvaginal aspiration of endometriotic cysts: An optional line of treatment in selected cases of endometriosis. Hum Reprod 1991; 6:1408–1410. 62. Bruhat MA, Mage G, Chapron C, Pouly JL, Canis M, Wattiez A. Present day endoscopic surgery in gynecology. Eur J Obstet Gynecol Reprod Biol 1991; 41:4–13. 63. Donnez J, Nisolle M, Gillet N, Smets M, Bassil S, Casanas Roux F. Large ovarian endometriomas. Hum Reprod 1996; 11:641–646. 64. Brosens IA, Van Ballaer P, Puttemans P, Deprest J. Reconstruction of the ovary containing large endometriomas by an extraovarian endosurgical technique. Fertil Steril 1996; 66:517–521. 65. Hughesdon PE. Benign endometrioid tumours of the ovary and the mullerian concept of ovarian epithelial tumours. Histopathology 1984; 8:977–990. 66. Hughesdon PE. The endometrial identity of benign stromatosis of the ovary and its relation to other forms of endometriosis. J Pathol 1976; 119:201–209. 67. Brosens IA. New principles in the management of endometriosis. Acta Obstet Gynecol Scand Suppl 1994; 159:18–21. 68. Wood C. Endoscopy in the management of endometriosis. Baillieres Clin Obstet Gynaecol 1994; 8:735–757. 69. Canis M, Mage G, Wattiez A, Chapron C, Pouly JL, Bassil S. Second-look laparoscopy after laparoscopic cystectomy of large ovarian endometriomas [see comments]. Fertil Steril 1992; 58:617–619. 70. Fayez JA, Vogel MF. Comparison of different treatment methods of endometriomas by laparoscopy [see comments]. Obstet Gynecol 1991; 78:660–665. 71. Cornillie FJ, Oosterlynck D, Lauweryns JM, Koninckx PR. Deeply infiltrating pelvic endometriosis: Histology and clinical significance. Fertil Steril 1990; 53:978–983. 72. Koninckx PR, Meuleman C, Oosterlynck D, Cornillie FJ. Diagnosis of deep endometriosis by clinical examination during menstruation and plasma CA125 concentration. Fertil Steril 1996; 65:280–287. 73. Koninckx PR, Martin DC. Deep endometriosis: A consequence of infiltration or retraction or possibly adenomyosis externa? Fertil Steril 1992; 58: 924–928. 74. Redwine D. Was Sampson wrong? Fertil Steril 2002; 78:686. 75. Koninckx PR, Barlow D, Kennedy S. Implantation versus infiltration: The Sampson versus the endometriotic disease theory. Gynecol Obstet Invest 1999; 47(Suppl 1):3–9. 76. Holt VL, Weiss NS. Recommendations for the design of epidemiologic studies of endometriosis. Epidemiology 2000; 11:654–659. 77. Wheeler JM. Epidemiology of endometriosis-associated infertility. J Reprod Med Obstet Gynecol 1989; 34:41–46. 78. Redwine DB. Age-related evolution in color appearance of endometriosis. Fertil Steril 1987; 48:1062–1063. 79. Cramer DW, Missmer SA. The epidemiology of endometriosis. Ann NY Acad Sci 2002; 955:11–22.

16

Koninckx

80. Moen MH, Schei B. Epidemiology of endometriosis in a Norwegian county. Acta Obstet Gynecol Scand 1997; 76:559–562. 81. Matorras R, Rodriguez F, Pijoan JI, Etxanojauregui A, Neyro JL, Elorriaga MA, Rodriguez-Escodero F. Women who are not exposed to spermatozoa and infertile women have similar rates of stage I endometriosis. Fertil Steril 2001; 76:923–928. 82. D’Hooghe TM, Debrock S. Endometriosis, retrograde menstruation and peritoneal inflammation in women and in baboons. Hum Reprod Update 2002; 8:84–88. 83. Koninckx PR, Braet P, Kennedy SH, Barlow DH. Dioxin pollution and endometriosis in Belgium [see comments]. Hum Reprod 1994; 9:1001–1002. 84. Rier SE, Martin DC, Bowman RE, Dmowski WP, Becker JL. Endometriosis in Rhesus monkeys (Macaca-mulatta) following chronic exposure to 2,3,7,8tetrachlorodibenzo-p-dioxin. Fund Appl Toxicol 1993; 21:433–441. 85. Rier SE, Turner WE, Martin DC, Morris R, Lucier GW, Clark GC. Serum levels of TCDD and dioxin-like chemicals in Rhesus monkeys chronically exposed to dioxin: Correlation of increased serum PCB levels with endometriosis. Toxicol Sci 2001; 59:147–159. 86. Koninckx PR. The physiopathology of endometriosis: Pollution and dioxin. Gynecol Obstet Invest 1999; 47(Suppl 1):47–49. 87. Eskenazi B, Mocarelli P, Warner M, Samuels S, Vercellini P, Olive D. Serum dioxin concentrations and endometriosis: A cohort study in Seveso, Italy. Environ Health Perspect 2002; 110:629–634. 88. Tsutsumi O, Momoeda M, Takai Y, Ono M, Taketani Y. Breast-fed infants, possibly exposed to dioxins in milk, have unexpectedly lower incidence of endometriosis in adult life. Int J Gynaecol Obstet 2000; 68:151–153. 89. Fanton JW, Golden JG. Radiation-induced endometriosis in Macaca mulatta. Radiat Res 1991; 126:141–146. 90. Kennedy SH, Mardon H, Barlow DH. Familial endometriosis. J Assist Reprod Genet 1995; 12:32–34. 91. Hadfield RM, Mardon HJ, Barlow DH, Kennedy SH. Endometriosis in monozygotic twins. Fertil Steril 1997; 68:941–942. 92. Hadfield RM, Manek S, Nakago S, Mukherjee S, Weeks DE, Mardon HJ, Barlow DH, Kennedy SH. Absence of a relationship between endometriosis and the N314D polymorphism of galactose-1-phosphate uridyl transferase in a UK population. Mol Hum Reprod 1999; 5:990–993. 93. Hadfield RM, Manek S, Weeks DE, Mardon HJ, Barlow DH, Kennedy SH. Linkage and association studies of the relationship between endometriosis and genes encoding the detoxification enzymes GSTM1, GSTT1 and CYP1A1. Mol Hum Reprod 2001; 7:1073–1078. 94. Zondervan KT, Cardon LR, Kennedy SH. The genetic basis of endometriosis. Curr Opin Obstet Gynecol 2001; 13:309–314. 95. Treloar SA, Kennedy SH. Preliminary results from two combined genomewide scans in endometriosis. Fertil Steril 2002; 77(Suppl 1):S19–S20. 96. Kennedy S, Hadfield R, Mardon H, Barlow D. Age of onset of pain symptoms

Epidemiology

97. 98. 99. 100. 101.

102.

103.

104.

105.

106. 107.

108.

109.

110. 111. 112.

113.

17

in non-twin sisters concordant for endometriosis. Hum Reprod 1996; 11:403– 405. Moen MH, Magnus P. The familial risk of endometriosis. Acta Obstet Gynecol Scand 1993; 72:560–564. Moen MH. Endometriosis in monozygotic twins. Acta Obstet Gynecol Scand 1994; 73:59–62. Oosterlynck D. Immunosuppressive activity of peritoneal fluid in women with endometriosis. Obstet Gynecol 1993; 82:206–212. Oosterlynck, D. Natural killer cells and endometriosis. PhD thesis, 1983. Oosterlynck DJ, Meuleman C, Waer M, Koninckx PR. Transforming growth factor-beta activity is increased in peritoneal fluid from women with endometriosis. Obstet Gynecol 1994; 83:287–292. Oosterlynck DJ, Meuleman C, Waer M, Koninckx PR, Vandeputte M. Immunosuppressive activity of peritoneal fluid in women with endometriosis. Obstet Gynecol 1993; 82:206–212. Oosterlynck DJ, Cornillie FJ, Waer M, Koninckx PR. Immunohistochemical characterization of leucocyte subpopulations in endometriotic lesions. Arch Gynecol Obstet 1993; 253:197–206. Oosterlynck DJ, Meuleman C, Waer M, Vandeputte M, Koninckx PR. The natural killer activity of peritoneal fluid lymphocytes is decreased in women with endometriosis. Fertil Steril 1992; 58:290–295. Oosterlynck DJ, Cornillie FJ, Waer M, Vandeputte M, Koninckx PR. Women with endometriosis show a defect in natural killer activity resulting in a decreased cytotoxicity to autologous endometrium. Fertil Steril 1991; 56:45–51. Vinatier D, Dufour P, Oosterlynck D. Immunological aspects of endometriosis. Hum Reprod Update 1996; 2:371–384. Braun D, Ding J, Shen J, Rana N, Fernandez B, Dmowski W. Relationship between apoptosis and the number of macrophages in eutopic endometrium from women with and without endometriosis. Fertil Steril 2002; 78:830. Braun D, Ding J, Dmowski W. Peritoneal fluid-mediated enhancement of eutopic and ectopic endometrial cell proliferation is dependent on tumor necrosis factor-alpha in women with endometriosis. Fertil Steril 2002; 78:727. Ding J, Shen J, Braun DP, Rana N, Dmowski WP. Endometrial apoptosis, macrophage content, and TNFalpha expression in endometriosis. Fertil Steril 2002; 77(Suppl 1):S1–S2. Dmowski WP. Immunology of endometriosis. Arq Matern Dr Alfredo Da Costa 1992; 12:40–43. Koninckx PR. Pelvic endometriosis: A consequence of stress? Contrib Gynecol Obstet 1987; 16:56–59. Demyttenaere K, Nijs P, Evers-Kiebooms G, Koninckx PR. Coping and the ineffectiveness of coping influence the outcome of in vitro fertilization through stress responses. Psychoneuroendocrinology 1992; 17:655–665. Demyttenaere K, Nijs P, Evers-Kiebooms G, Koninckx PR. Coping, ineffectiveness of coping and the psychoendocrinological stress responses during invitro fertilization. J Psychosom Res 1991; 35:231–243.

18

Koninckx

114. Demyttenaere K, Nijs P, Evers-Kiebooms G, Koninckx PR. The effect of a specific emotional stressor on prolactin, cortisol, and testosterone concentrations in women varies with their trait anxiety. Fertil Steril 1989; 52:942–948. 115. Koninckx PR, Kennedy SH, Barlow DH. Pathogenesis of endometriosis: The role of peritoneal fluid. Gynecol Obstet Invest 1999; 47(Suppl 1):23–33. 116. D’Hooghe TM, Bambra CS, De JI, Lauweryns JM, Koninckx PR. The prevalence of spontaneous endometriosis in the baboon (Papio anubis, Papio cynocephalus) increases with the duration of captivity. Acta Obstet Gynecol Scand 1996; 75:98–101. 117. Parazzini F, Luchini L, Vezzoli F, Mezzanotte C, Vercellini P, Romanini C. Prevalence and anatomical distribution of endometriosis in women with selected gynaecological conditions: Results from a multicentric Italian study. Hum Reprod 1994; 9:1158–1162. 118. Ness RB, Cramer DW, Goodman MT, Kjaer SK, Mallin K, Mosgaard BJ. Infertility, fertility drugs, and ovarian cancer: A pooled analysis of casecontrol studies. Am J Epidemiol 2002; 155:217–224. 119. Olson JE, Cerhan JR, Janney CA, Anderson KE, Vachon CM, Sellers TA. Postmenopausal cancer risk after self-reported endometriosis diagnosis in the Iowa Women’s Health Study. Cancer 2002; 94:1612–1618. 120. Yano T, Jimbo H, Yoshikawa H, Tsutsumi O, Taketani Y. Molecular analysis of clonality in ovarian endometrial cysts. Gynecol Obstet Invest 1999; 47(Suppl 1):41–45. 121. Tamura M, Fukaya T, Murakami I, Uehara S, Yajima A. Analysis of clonality in human endometriotic cysts based on evaluation of X chromosome inactivation in archival formalin-fixed, paraffin-embedded tissue. Lab Invest 1998; 78:213–218. 122. Jimbo H, Hitomi Y, Yoshikawa H, Yano T, Momoeda M, Sakamoto A, Tsutsumi O, Taketani Y, Esumi H. Evidence for monoclonal expansion of epithelial cells in ovarian endometrial cysts. Am J Pathol 1997; 150:1173–1178. 123. Fujita M, Enomoto T, Wada H, Inoue M, Okudaira Y, Shroyer KR. Application of clonal analysis. Differential diagnosis for synchronous primary ovarian and endometrial cancers and metastatic cancer. Am J Clin Pathol 1996; 105:350–359. 124. Nishida M, Watanabe K, Sato N, Ichikawa Y. Malignant transformation of ovarian endometriosis. Gynecol Obstet Invest 2000; 50(Suppl 1):18–25. 125. Stern RC, Dash R, Bentley RC, Snyder MJ, Haney AF, Robboy SJ. Malignancy in endometriosis: Frequency and comparison of ovarian and extraovarian types. Int J Gynecol Pathol 2001; 20:133–139.

2 Pathogenesis of Endometriosis: Peritoneal Endometriosis, Ovarian Endometriosis, and Rectovaginal Adenomyosis Jacques Donnez and Jean Squifflet Catholic University of Louvain Brussels, Belgium

Pelvic endometriosis can be categorized into three different forms: peritoneal endometriosis, ovarian endometriosis, and endometriosis of the rectovaginal septum [1].

PERITONEAL ENDOMETRIOSIS Since its first detailed description by von Rokitansky in 1860 [2], several theories relating to the pathogenesis of endometriosis have been proposed. The most widely accepted is the transplantation theory that was proposed in 1927 by Sampson. He observed that endometrial cells regurgitated through the Fallopian tubes during menstruation [3]. Three essential conditions must be met to consider retrograde menstruation as the explanation for the pathogenesis of pelvic endometriosis [4]. First, endometrial cells must enter the peritoneal cavity through the Fallopian tubes. Second, cells within the menstrual debris must be viable and capable of 19

20

Donnez and Squifflet

being transplanted into pelvic structures. Third, the anatomical distribution of endometriosis in the pelvic cavity must correlate with the principles of transplantation for exfoliated cells. To explain the discrepancy between the occurrence of regurgitation and endometriosis, one must consider the volume of regurgitated menstrual debris, which can be determined by anatomic-mechanical predispositions. Ayers and Friedenstab [5] showed that uterotubal junction hypotonia occurred in patients with endometriosis, but not in (infertile) control subjects. Their findings seem to be corroborated by Bartosik et al [6], who found more endometrial cells in the abdominal cavity during flushing of the tubes in patients with endometriosis. Presence, Attachment, Invasion Numerous studies have demonstrated that reflux of endometrial cells into the peritoneal cavity during menstruation is a common physiologic condition in women with patent tubes [7,8]. The viability of endometrial cells was demonstrated by culture of menstrual blood or peritoneal fluid [9,10]. Experimental studies proved that endometriosis could be induced by exposure of the pelvis to increased amounts of menstrual discharge [11,12]. Implantation of endometrial tissue also induced endometriosis in rabbits [13,14]. Integrins are cell-surface glycoproteins that act as receptors for extracellular matrix (ECM) proteins. The expression of some integrins has been described in normal endometrium, where they are important in the interactions between glandular and stromal elements [15–17]. Foidart et al in 1993 first discovered these glycoproteins in endometriotic tissue [15]. Attachment of endometrial tissue fragments to the peritoneum could be explained by the detection of such cell adhesion molecules. After adherence of endometrial cells to the basement membrane (peritoneum), local degradation of the ECM is required. Recently, Marbaix et al [18,19] and Kokorine et al [20] demonstrated that the marked decline in progesterone (P) concentration at the end of the menstrual cycle would initiate the synthesis and activation of matrix metalloproteinases (MMPs), causing ECM breakdown, tissue collapse and menstruation. Thus, the presence of collagenases was proved during the menstrual period, and their probable presence in the peritoneal fluid, together with regurgitated endometrial cells, could be one of the elements in the local degradation of the peritoneal ECM. Kokorine et al [21] detected the presence of MMPs in red peritoneal lesions and ovarian endometriomas independent of the menstrual cycle, suggesting that such lesions could represent shedding independent of the decline in P concentration. This hypothesis was bolstered by the detection of

Pathogenesis

21

the aminoterminal propeptide of type III procollagen in peritoneal fluid, which is a sign of increase in the metabolism of the ECM [22]. In vitro studies demonstrated that the invasion index of endometriotic cells was similar to that of bladder metastatic cell lines, suggesting that the invasiveness of endometriotic cells may contribute to the pathogenesis of endometriosis [23]. The anatomic distribution of endometriosis in the pelvic cavity was described by Jenkins et al [24] and correlated with the principles of transplantation for exfoliated cells. These data suggested that any exposure of the pelvic peritoneum to menstrual reflux would result in an increased risk of endometriosis. Evolution of Red, Black, and White Peritoneal Lesions In red flame-like lesions (Figs. 1 and 2), biopsied together with the surrounding normal peritoneum, we observed an extensive vascular network between the stroma recently implanted onto the peritoneal surface and the peritoneal and subperitoneal layers, demonstrating the importance of angiogenesis in the early stages of development after implantation [25,26]. In our opinion, vascularization of endometriotic implants is probably one of the most important factors of growth and invasion of other tissue by endometrial glands [27,28]. It seems reasonable to say that angiogenic factors present in the peritoneal fluid of 58% of women with endometriosis facilitate the growth of endometriotic implants. Peritoneal macrophages, retrograde endometrial cells, or the ectopic endometriotic lesions could produce these angiogenic factors [29]. Data from our group [21] revealed the presence of MMPs in the stroma of red lesions throughout the menstrual cycle. However, in eutopic endometrium, MMPs are detected only during the marked decline in P. After this partial shedding, the remaining red lesion grows again until the next shedding, but menstrual shedding finally induces an inflammatory reaction, provoking a scarification process that encloses the implant. The enclosed implant becomes a ‘‘black’’ lesion because of the presence of intraluminal debris (Fig. 3). This scarification process is probably responsible for the reduction in vascularization, as proved by the significant decrease in the relative surface areas of the capillaries and stroma [27]. The role of trauma or inflammation of the mesothelium was suggested by van Der Linden et al [30], who cultured endometrial cells on amniotic membranes. Adherence of endometrial cells to the epithelial surface of the amniotic membranes was never observed when the epithelium was intact. These investigators proposed that an intact epithelium could be an important

22

Donnez and Squifflet

FIGURE 1 Evolution of red, black, and white peritoneal lesions. After attachment of endometrial cells to the basement membrane (peritoneum), local degradation of the extracellular matrix is required. Angiogenesis is one of the most important factors of growth and invasion (red lesions). Partial shedding occurs in red lesions, and the remaining red lesions constantly regrow until the next shedding. Menstrual shedding finally induces an inflammatory reaction provoking a scarification process that encloses the implant, which then becomes a ‘‘black’’ lesion because of the presence of intraluminal debris. White lesions are quiescent or inactive lesions with occasional glandular structures on histological examination.

Pathogenesis

23

FIGURE 2 (A) Red, flame-like lesion at laparoscopy. (B) Numerous glands with active epithelium and stroma are found on the peritoneal surface.

24

Donnez and Squifflet

FIGURE 3 Typical black lesion, surrounded by a white area of peritoneal fibrosis (white lesion).

defense mechanism in preventing initial adhesion of shed endometrial fragments to peritoneum. On the contrary, we have demonstrated that menstrual endometrium is able to adhere to normal mesothelium [31]. In agreement with Brosens [32], we regard red lesions as early endometriosis and black lesions as advanced endometriosis [27,33]. White lesions are believed to be healed endometriosis or quiescent or latent lesions [27]. This hypothesis corroborates the clinical findings of Redwine [34] and Goldstein et al [35] that red lesions precede the others and that, with time, their presence decreases, as they are replaced by black and ultimately white lesions. Red petechial lesions are found in adolescents [35]. The shedding and implantation of endometrial cells is a process that occurs during the initial phase of reproductive life. The fact that this period is characterized by anovulatory cycles is in accordance with the hypothesis that anovulation, with a low P concentration in peritoneal fluid, is the favored time for the implantation of endometriosis [36]. Black lesions rarely demonstrate typical progestational changes, although some investigators report that 70% of foci with a cyclic pattern undergo changes that are considered synchronous (F3 days) with eutopic endometrium [25,26,37]. This assumption is based on the observation of basal vacuoles in certain cells (<20%). However, to be considered a secretory

Pathogenesis

25

change, it has to be demonstrated in more than 50% of epithelial cells. Furthermore, basal vacuoles can also be observed in anovulatory cycles or in the absence of progesterone [38,39]. Oxidative Stress and Peritoneal Endometriosis Because retrograde menstruation can be considered a physiologic occurrence in menstruating women with patent tubes [7], how can we explain the fact that not all women are found to have endometriosis at laparoscopy? Endometriosis is a multifactorial disease associated with a general inflammatory response in the peritoneal cavity. Oxidative stress has been proposed as a potential factor involved in the pathophysiology of the disease (Fig. 4) [31]. Reactive oxygen species have been implicated in the pathogenesis of many human diseases and aging [40,41]. These species are intermediaries produced by normal oxygen metabolism. To protect themselves from the deleterious effects of reactive oxygen species, cells have developed a wide range of antioxidant systems to limit production of reactive oxygen species, inactivate them, and repair cell damage. However, oxidative stress may occur when the balance between reactive oxygen species production and antioxidant defense is disrupted. The increasing number of diseases associated with oxidative stress suggests that oxidative balance may be unstable [40]. Origin of Oxidative Stress in the Peritoneal Cavity Several hypotheses have been proposed to explain why oxidative stress is induced in case of pelvic endometriosis. Erythrocytes [42], apoptotic endometrial tissue, and cell debris [43] transplanted into the peritoneal cavity by menstrual reflux and macrophages [43] have been implicated as potential inducers of oxidative stress. Excessive production of reactive oxygen species may also result from exposure to environmental toxicants and heavy metals, which may disrupt the balance of pro-oxidants and antioxidants. Macrophages, endometriotic lesions, and peritoneal cells of patients with endometriosis show typical features of iron-overloaded tissue. Characteristically, these cells are heavily laden with hemosiderin [28,44,45]. Several authors have suggested that hemosiderin is formed when abundant ferritin cores come into close contact after partial digestion of their protein coat by lysosomal-siderosome enzymes [46,47]. In patients with endometriosis, the presence of tissue iron deposits in the peritoneum appears to be related to the presence of endometriotic lesions, because the rate of deposits encountered was found to be higher in peritoneum close to a lesion than in normal-looking peritoneum [48]. Compelling evidence suggests that oxidative stress is increased in women with pelvic endometriosis [31]. Oxidative stress occurs when there is

26

Donnez and Squifflet

FIGURE 4 Hypothesis explaining oxidative stress in the peritoneal cavity of women with endometriosis. Bold type indicates factors that have been studied specifically in relation to pelvic endometriosis. Carbon dioxide (CO); Heme oxygenase (HO); Nitric oxide (NO); Nitric oxide synthase (NOS).

an imbalance between production of reactive oxygen species (ROS) and antioxidant systems. ROS are intermediaries released by normal oxygen metabolism, and antioxidant systems control production of ROS, inactivate them, and repair cell damage. Production of ROS appears to be increased in women in whom endometriosis is developing. This is indicated by the increased release of ROS by macrophages, higher levels of oxidized low-density lipoproteins and their by-products in peritoneal fluid, and overexpression, by endometrial cells, of enzymes that produce reactive oxygen species, such as xanthine oxidase and nitric oxide synthase. Several studies indicate that antioxidant defenses may be altered in endometriosis, as suggested by the

Pathogenesis

27

aberrant expression of endometrial antioxidant enzymes and lower levels of the antioxidant vitamin E in peritoneal fluid. Further studies are warranted to assess the contribution of oxidative stress to the physiopathology of endometriosis. It would be particularly valuable to determine whether antioxidant treatment of endometriosis is effective. In fact, the use of mifepristone to treat endometriosis has had promising results [49]. This agent has been shown to inhibit endometrial cell growth, an effect that appears to be due to its antioxidant properties. Reactive oxygen species have also been increasingly studied as regulators of protein activity and inducers of gene expression [50]. As a potential inducer of NF-nB, which activates genes involved in cell adhesion, secretion of inflammatory cytokines, and recruitment of macrophages, oxidative stress may help to trigger the chain of events that leads to the development of endometriotic lesions. Further studies are needed to examine the potential involvement of oxidative stress in the pathophysiology of endometriosis. OVARIAN ENDOMETRIOSIS The pathogenesis of typical ovarian endometriosis is a source of controversy. The original paper by Sampson [51] on this condition indicated that perforation of the so-called chocolate cyst led to spillage of adhesions and the spread of peritoneal endometriosis. The findings of Hughesdon [52] contradicted Sampson’s [51] hypothesis and suggested that adhesions are not the consequence, but rather the cause of endometrioma formation. Hughesdon demonstrated, by serial section of ovaries containing an endometrioma, that 90% of typical endometriomas are formed by invagination of the cortex after the accumulation of menstrual debris from bleeding of endometrial implants, which are located on the ovarian surface and adherent to the peritoneum. The site of perforation, as described by Sampson [51], could represent the stigma of invagination. The observations of Brosens et al [53], based on ovarioscopy and in situ biopsies, were in agreement with Hughesdon’s hypothesis [52]. In 93% of typical endometriomas, the pseudocyst is formed by an accumulation of menstrual debris from the shedding and bleeding of active implants located by ovarioscopy at the site of inversion, resulting in progressive invagination of the ovarian cortex [53]. Other investigators have suggested that large endometriomas may develop as a result of the secondary involvement of functional ovarian cysts in the process of endometriosis [54]. We hypothesized that celomic metaplasia of invaginated epithelial inclusions could be responsible for the pathogenesis of ovarian endometriosis [28,55]. This hypothesis, based on the metaplastic potential of the pelvic mesothelium, is already a widely accepted theory on the pathogenesis of common epithelial ovarian tumors [56].

28

Donnez and Squifflet

Several papers and debates have tried to classify endometriomas, but there is still considerable uncertainty [53,54]. We believe that ovarian endometriosis is caused by metaplasia of invaginated celomic epithelium [55] (Fig. 5). Our arguments are as follows: 1. In our series, we found that 12% of endometriomas were not fixed to the broad ligament, and Hughesdon’s [52] theory cannot explain the formation of the endometrioma in such cases. 2. It was not unusual to find multilocular endometriomas that could not be explained by the theory of adhesions or by bleeding of active superficial implants adherent to the peritoneum. 3. The epithelium covering the ovary, which is the mesothelium, can invaginate into the ovarian cortex. Invaginations of the mesothelial layer covering the ovarian tissue were described by Motta et al [57] in animal and fetal ovaries and were also visualized in human adult ovaries [25,26]. In our serial sections of the ovary, we frequently observed mesothelial inclusions. Under the influence of unknown growth factors, these inclusions could be transformed into intraovarian endometriosis by metaplasia. 4. The fact that primordial follicles were found surrounding the endometriotic cyst is also in agreement with our hypothesis. When the mesothelium invaginates deep into the ovary, the follicles located at the invagination site are pushed concomitantly with the mesothelium (Fig. 6). 5. Our main argument is based on the presence of epithelial invaginations in continuum with endometrial tissue, proving the metaplasia theory [25,26,55] (Fig. 5). 6. Another major argument is related to the demonstration of the capacity of the endometrioma wall to invaginate secondarily into the ovarian cortex [25,26,55]. Such secondary invaginations were observed in 33% of our cases and represent the so-called deep ovarian endometriosis, which is actually just an extension of the endometrioma wall. 7. Arguments to support our hypothesis can also be found in the literature. First, endometriomas have been described in patients suffering from Rokitansky-Kuster-Hauser syndrome who do not have a uterus and, therefore, do not have retrograde menstruation [58]. Second, common epithelial tumors of the ovary are considered to be derived from the surface epithelium covering the ovary and from the underlying stroma [56]. Thus, our theory differs from the theories of Hughesdon [52] and Brosens et al [53]. They consider the pathogenesis of the typical ovarian

Pathogenesis

29

FIGURE 5 (A) Hypothesis on the histogenesis of ovarian endometriomas. (B) Continuum between the flat cells of the ovarian surface mesothelium and the endometrial-type epithelium of the endometrioma (stain, Gomori’s trichrome; original magnification, x410). (From Fertil Steril 1997;68:585–596. Courtesy of M. Nisolle and J. Donnez.)

30

Donnez and Squifflet

FIGURE 6 Biopsy of the endometrioma wall. Follicles located at the invagination site are pushed concomitantly with the mesothelium. They are visible just beneath the endometrioma wall (epithelium, stroma, and fibrosis).

endometrioma as a process originating from a free superficial implant that is in contact with the ovarian surface and is sealed off by adhesions. The menstrual shedding and bleeding of this small implant subsequently results in the progressive invagination of the ovarian cortex and the formation of the pseudocyst. In our opinion, the endometrioma must be considered as an invagination, but not the result of bleeding of a superficial implant. Metaplasia of the celomic epithelium invaginated into the ovarian cortex has been proven and might explain the formation of the endometrioma [55]. The deep-infiltrating ovarian endometriosis described by our group is only the consequence of the invagination of endometriotic tissue into the ovary and is probably responsible for the recurrence of ovarian endometriosis after cyst excision or vaporization [25,26,59–62]. These findings give us further arguments favoring the surgical technique already proposed in 1987, which consists of vaporization of the internal wall of the cyst [60]. The depth of vaporization is very shallow; only the glandular epithelium and the subjacent stroma have to be vaporized. There is no need to destroy the fibrotic capsule usually found surrounding the endometrioma. This technique avoids removal of oocytes observed when the endometriotic capsule is removed during ovarian cystectomy. Brosens et al [63] also confirmed that ovarian cystectomy was not mandatory for the management of large endometriomas. Their study and

Pathogenesis

31

our own strongly suggest that to minimize ovarian trauma and avoid the risk of premature ovarian failure, one should destroy only the internal lining of the endometrioma.

RETROCERVICAL OR RECTOVAGINAL ADENOMYOSIS, THE RETROPERITONEAL FORM OF THE DISEASE The concept of RAD (retroperitoneal adenomyotic disease) was born in 1998 and published in 2001 [64]. We propose to consider retroperitoneal space as the origin of adenomyotic disease, and to abandon the concept of deepinfiltrating endometriosis. We hypothesized that these lesions result from metaplasia of Mu¨llerian rests. These lesions are retroperitoneal and may extend laterally or anteriorly to the anterior rectal wall, provoking perivisceritis. This perivisceritis phenomenon, visible at radiography, is not a rectal endometriotic lesion or invasion of the rectal wall by the endometriotic process, as has been suggested by many investigators. In our opinion, it is only the consequence of serosal retraction caused by the inflammatory process or fibrosis of the anterior wall of the rectum due to an adenomyotic lesion. In recent studies, we described the differences between peritoneal and nodular lesions. We suggest that the nodule is not the consequence of deepinfiltrating endometriosis, but is the same as an adenomyotic nodule developed from Mu¨llerian rests by metaplasia (Fig. 7), [1,4,25,26,65,66]. Smooth muscle proliferation and fibrosis, consistently observed, are responsible for

FIGURE 7 Hypothesis on the histogenesis of rectovaginal endometriosis. (From Fertil Steril 1997;68:585–596. Courtesy of M. Nisolle and J. Donnez.)

32

Donnez and Squifflet

the nodular aspect of endometriosis located in the rectovaginal septum. The clinical diagnosis is made only when smooth muscle proliferation is palpated by vaginal examination. How Can It Be Diagnosed? The first step in the diagnosis of this pathology is the clinical examination, including a very thorough medical history. Its principal features are pelvic pain and infertility. Unfortunately, most women with adenomyotic lesions suffer for a long time. Adenomyotic nodules of the rectovaginal septum can involve the uterosacral ligaments, the posterior vaginal wall, the posterior fornix and, sometimes, extending into the anterior rectal wall or the distal part of the ureter. In case of severe lesions, the pouch of Douglas can be completely or partially obliterated. The symptoms are generally pelvic pain, deep dyspareunia on certain positions during sexual intercourse, and dyschezia. At least 50% of patients have a past history of one episode of pelvic surgery without correct diagnosis. A clinical examination is carried out to locate, with the help of a speculum, bluish lesions behind the cervix. Bimanual vaginal examination constitutes one of the most important diagnostic tools, including careful inspection of the posterior part of the cervix and posterior wall of the vagina. The size of the lesions and their location (medial or lateral to the uterosacral ligament) must be evaluated. Lateral localization of the pathology could indicate possible extension to the ureter. The aim of additional procedures is to confirm the diagnosis of adenomyotic nodules and other entities, such as endometriotic cysts, adenomyotic lesions of the uterus, or adenomyotic lesions of the bladder. Laparoscopic exploration could sometimes underestimate the presence and extension of the adenomyotic nodule if the preoperative diagnosis has not been made. Preoperative imaging is needed to establish the correct diagnosis and the extension of the adenomyotic nodule into the rectovaginal space. Bowel Barium Enema With double-contrast barium enema in a profile view of the rectosigmoid junction, we may be able to detect a mass effect or perivisceritis of the anterior wall of the rectum (Fig. 8). Thorough examinations of this profile view allow us to detect a mass effect in 54% of cases [67]. Decreased inflation of the rectal ampulla is the first indirect sign of adenomyotic lesions. In more extensive lesions, an irregular aspect of the anterior wall could reflect perivisceritis extended to the anterior wall. Barium enema also allows identification of upper bowel lesions as true endometriotic lesions of the

Pathogenesis

33

FIGURE 8 Barium enema. Perivisceritis of the anterior wall of the rectum is visible in 54% of cases of adenomyotic lesions.

sigmoid wall. These are not considered as adenomyotic lesions and require different treatment. Transrectal Ultrasonography In our department, transrectal ultrasonography was performed using a Corevision Pro (ToshibaR, Japan, Endocavitary Transducer PVL-715RT,

34

Donnez and Squifflet

linear and convey transducer head) ultrasound scanner with a biplane twodimensional longitudinal axial and sagital circumferential 7 MHz probe. A water-filled balloon was placed over the tip of the probe to obtain contrast to evaluate infiltration of the rectal wall. The transducer was inserted into the rectum and advanced until the midline image of the cervix was visualized, using the longitudinal view. Typical adenomyotic lesions look like pure hypoechogenic cystic lesions surrounded by irregular, less hypoechogenic structures. These small, focal, well-circumscribed lesions correspond to dilatation of the endometriotic glands surrounded by smooth muscle hyperplasia. Sometimes, these lesions produce a mass effect. All these retroperitoneal lesions were found either attached to the cervix, in the rectovaginal septum, or extending to the anterior wall of the rectum. In case of lateral extension and to evaluate the possibility of true ureteral lesions, Doppler ultrasound of the bladder was performed to demonstrate normal bilateral ureteral flow. Magnetic Resonance Imaging On MRI, endometriotic lesions are seen as hypointense areas surrounding hyperintense spots in the myometrium in T2-weighted spin echo images and as some hyperintense spots after intravenous injection of gadolinium agent in T1-weighted spin echo images. In addition, MRI may detect bladder and rectovaginal adenomyosis [68]. Intravenous Pyelography In case of rectovaginal adenomyotic nodules of >3 cm or developing laterally, patients should undergo preoperative intravenous pyelography (IVP). Intravenous pyelography is abnormal in more than 10% of these cases. It can detect small abnormalities of the ureter, such as medialization, substenosis, lack of distension after decompression, and may reveal the beginning of compression of the ureter by lateral extension of the nodule.

CLASSIFICATION: THE KEY TO PATHOGENESIS Koninckx proposed a classification divided into three types: T1, T2, and T3 that suggests adenomyosis (Fig. 9) [69]. Adamyan’s classification attempted to describe the lesions anatomically [70]. More recently, Vercellini et al [71] proposed that intraperitoneal endometriosis could explain the obliteration of the rectovaginal pouch and the subsequent infiltration of the rectovaginal septum by the lesions. That is why the term ‘‘deep-infiltrating disease’’ is

Pathogenesis

35

FIGURE 9 Classification (from left to right): (a) rectovaginal septum lesion (type I); (b) posterior vaginal fornix lesion (type II); (c) hourglass-shaped lesion (type III).

sometimes used to describe these lesions. This latter hypothesis, however, has neither anatomical nor histopathological support. In our theory, adenomyosis originates in the retrocervical area and involves the retroperitoneal space. We have proposed a classification, which takes into account the precise location of the retroperitoneal lesion, as defined by transvaginal ultrasonography and MRI [67]. 1. Rectovaginal septum These lesions are situated within the rectovaginal septum between the posterior wall of the vaginal mucosa and the anterior wall of the rectal muscularis. 2. Posterior vaginal fornix These lesions develop from the posterior fornix toward the rectovaginal septum. The posterior fornix is retrocervical and corresponds, in the attachment of the vaginal mucosa, to the posterior face of the posterior lip of the cervix. 3. Hourglass-shaped or diabolo-like These lesions occur when posterior fornix lesions extend cranially to the anterior rectal wall. The part of the adenomyotic lesion situated in the anterior rectal wall is the same size as the part of the lesion situated near the posterior fornix. A small but well-observed continuum exists between these two parts of the lesion. That is why we have termed these lesions diabolo-like or ‘‘hourglass’’ shaped. As noted before, these lesions always occur under the peritoneal fold of the recto-uterine pouch of Douglas. Hourglass-type lesions were also found to be large, their average size estimated to be 3 cm by clinical examination. The barium enema showed signs of perivisceritis in nearly all cases. In our experience, the most frequent types are lesions in the posterior vaginal fornix in 60% of cases with extension into the rectovaginal septum

36

Donnez and Squifflet

in 20% of all cases, followed by the hourglass-shaped or diabolo-like lesions in 25% of cases (Fig. 10). The infrequent type is the rectovaginal septum in 15% of cases. Accordingly, it is inappropriate to define all retroperitoneal lesions as rectovaginal septum adenomyosis. We suggest classifying the lesions into three types according to their location, clinical examination, transrectal echography and MRI. These three types are rectovaginal septum lesions, posterior vaginal fornix lesions, and hourglass-shaped or diabololike lesions.

FIGURE 10 Magnetic resonance imaging: hourglass-shaped or diabolo-like lesions. It is obvious that this type of lesion occurs under the peritoneal fold of the recto-uterine pouch of Douglas.

Pathogenesis

37

Smooth Muscle Proliferation At least three hypotheses may explain smooth muscle proliferation (Fig. 11). 1. Endometriotic foci involving smooth muscle are typically associated with prominent proliferation of the smooth muscle, creating an adenomyomatous appearance similar to that of adenomyosis in the endometrium [32]. 2. The endometriotic stroma may exhibit smooth muscle metaplasia, similar to that found within the wall of ovarian endometriotic cysts [72]. 3. It may be due to modified gene expression induced by toxic agents. When endometriotic glands affect the retroperitoneal space, smooth muscle proliferation can take place and a nodule develops. Sometimes, retroperitoneal space invasion provokes perivisceritis. This is not a rectal endometriotic lesion or invasion of the rectal wall by the endometriotic process, as has been suggested by many investigators. We believe it is the result of serosal retraction caused by the inflammatory process and fibrosis on the anterior wall of the rectum. The absence of evolution of the rectal lesion after removal of the nodule supports our hypothesis that it originates from the retroperitoneal space.

FIGURE 11 Smooth muscle proliferation in an adenomyotic nodule.

38

Donnez and Squifflet

This suggests that it is not necessary to excise the anterior wall of the rectum [65,66]. In any event, because of the contact between the endometrial glands and the vagina, excision of the nodule and the adjacent vaginal mucosa is essential. Secretory changes are frequently absent or incomplete during the second half of the menstrual cycle. This suggests that adenomyosis may not respond to physiologic levels of P. Similar histological findings can be seen in the endometriotic rectovaginal nodule. It looks like an adenomyoma with a circumscribed nodular aggregate of smooth muscle, endometrial glands and, often, endometrial stroma [28,65,73]. This lesion originates from the tissue of the retrocervical area or from the rectovaginal septum and consists mainly of smooth muscle with active glandular epithelium and scanty stroma. Invasion of the smooth muscle by active glandular epithelium without stroma proves that stroma is not mandatory for invasion. It also shows that the nodule is different from peritoneal endometriosis, where the epithelial glands are surrounded by endometrial-type stroma [25,26]. Because of the similarity between the endometriotic nodule of the rectovaginal septum and uterine adenomyoma, we agree with Brosens [42] that their origin is similar. Furthermore, the coexpression of vimentin and cytokeratin indicates a close relationship with its mesodermal Mu¨llerian origin [25,26,73]. SUMMARY The different aspects of peritoneal endometriosis (red, black, and white lesions) represent distinctive steps in the evolutionary process that can be explained by the transplantation theory. Red lesions are the most active and highly vascularized lesions and can be considered the early phase of peritoneal endometriosis. Based on our histological findings, celomic metaplasia of invaginated epithelial inclusions could be responsible for the development of ovarian endometriosis. The epithelium covering the ovary derives from the celomic epithelium, has great metaplastic potential, and provokes epithelial inclusion cysts by invagination. Under the influence of unknown growth factors, these inclusions could be transformed into intraovarian endometriosis by metaplasia. Retroperitoneal nodule is an adenomyotic nodule whose histopathogenesis is not related to the implantation of regurgitated endometrial cells but to metaplasia of Mu¨llerian remnants in the retroperitoneal space. Metaplastic changes of Mu¨llerian rests into adenomyotic glands of the rectovaginal septum and the retroperitoneal space are responsible for the striking proliferation of the smooth muscle, creating an adenomyomatous appearance similar to that of adenomyosis in the endometrium.

Pathogenesis

39

We conclude that intraperitoneal and retroperitoneal diseases are two different entities with specific histopathogenesis, evolution, and symptoms.

PRACTICAL POINT 

Intraperitoneal endometriosis and retroperitoneal endometriosis appear to be different entities.

REFERENCES 1.

2. 3.

4.

5. 6. 7.

8.

9. 10.

11. 12. 13.

Donnez J, Nisolle M, Casanas-Roux F, Brion P, Da Costa N. Stereometric evaluation of peritoneal endometriosis and endometriotic nodules of the rectovaginal septum. Hum Reprod 1995; 11:224–228. von Rokitansky C. Ueber Uterusdrusen-Neubildung in Uterus and Ovarialsarcomen. Zkk Gesellsch d Aerzte zu Wien 1860; 37:577. Sampson JA. Peritoneal endometriosis due to menstrual dissemination of endometrial tissue into the peritoneal cavity. Am J Obstet Gynecol 1927; 14:422– 469. Donnez J, Nisolle M, Casanas-Roux F, Grandjean P. Endometriosis: pathogenesis and pathophysiology. In: Shaw RW, ed. Endometriosis. Carnforth: Parthenon Publishing, 1990:11–29. Ayers JW, Friedenstab AP. Utero-tubal hypotonia associated with pelvic endometriosis [abstract]. Proc 41st Am Fertil Soc, 1985; 131. Bartosik D, Jacobs SL, Kelly LJ. Endometrial tissue in peritoneal fluid. Fertil Steril 1986; 46:796–800. Halme J, Hammond MG, Hulka JF, Raj S, Talbert LM. Retrograde menstruation in healthy women and in patients with endometriosis. Obstet Gynecol 1984; 64:151–154. Liu DTY, Hitchcock A. Endometriosis: its association with retrograde menstruation, dysmenorrhea and tubal pathology. Br J Obstet Gynecol 1986; 93:859–862. Keettel WC, Stein RJ. The viability of the cast-off menstrual endometrium. Am J Obstet Gynecol 1951; 61:440–442. Kruitwagen RF, Poels LG, Willemsen WN, De Ronde IJ, Jap PH, Rolland R. Endometrial epithelial cells in peritoneal fluid during the early follicular phase. Fertil Steril 1991; 55:297–303. Te Linde RW, Scott RB. Experimental endometriosis. Am J Obstet Gynecol 1950; 60:1147–1173. Scott RB, Te Linde RW, Wharton LR Jr. Further studies on experimental endometriosis. Am J Obstet Gynecol 1953; 66:1082–1103. Schenken RS, Asch RH, Williams RF, Hodgen GD. Etiology of infertility in monkeys with endometriosis: luteinized unruptured follicles, luteal phase defects, pelvic adhesions and spontaneous abortions. Fertil Steril 1984; 41:122– 130.

40

Donnez and Squifflet

14. Donnez J, Wayembergh M, Casanas-Roux F, Karaman Y, Willems T, Ferin J. Effect on ovulation of surgically induced endometriosis in rabbits. Gynecol Obstet Invest 1987; 24:131–137. 15. Foidart JM, Beliard A, Donnez J. Endometriosis and invasion. In: Brosens I, Donnez J, eds. The current status of endometriosis research and management. Carnforth: Parthenon Publishing, 1993:35–39. 16. Lessey BA, Damjanovich L, Coutifaris C, Castelbaum A, Albelda SM, Buck CA. Integrin adhesion molecules in the human endometrium: correlation with the normal and abnormal menstrual cycle. J Clin Invest 1992; 90:188–195. 17. Tabibzadeh S, Sun XZ. Cytokine expression in human endometrium throughout the menstrual cycle. Hum Reprod 1992; 7:1214–1221. 18. Marbaix E, Kokorine I, Henriet P, Donnez J, Courtoy PJ, Eeckhout Y. The expression of interstitial collagenase in human endometrium is controlled by progesterone and by estradiol and is related to menstruation. Biochem J 1995; 305:1027–1030. 19. Marbaix E, Kokorine I, Donnez J, Eeckhout Y, Courtoy PJ. Regulation and restricted expression of interstitial collagenase suggest a pivotal role in the initiation of menstruation. Hum Reprod 1996; 11:134–143. 20. Kokorine I, Marbaix E, Henriet P, Okada Y, Donnez J, Eeckhout Y, Courtoy PJ. Focal cellular origin and regulation of interstitial collagenase (matrixmetalloproteinase-1) are related to menstrual breakdown in the human endometrium. J Cell Sci 1996; 109:2151–2160. 21. Kokorine I, Nisolle M, Donnez J, Eeckhout Y, Courtoy PJ, Marbaix E. Expression of interstitial collagenase (matrix metalloproteinase-1) is related to the activity of human endometriotic lesions. Fertil Steril 1997; 68:246–251. 22. Spuijbroek MD, Dunselman GA, Menheere PP, Evers JL. Early endometriosis invades the extracellular matrix. Fertil Steril 1992; 58:929–933. 23. Gaetje R, Kotzian S, Hermann G, Baumann R, Starzinski-Powitz A. Invasiveness of endometriotic cells in vitro. Lancet 1995; 346:1463–1464. 24. Jenkins S, Olive DL, Haney AF. Endometriosis: pathogenetic implications of the anatomic distribution. Obstet Gynecol 1986; 67:335–358. 25. Nisolle M. Peritoneal, ovarian and rectovaginal endometriosis are three distinct entities. The`se d’Agre´gation de l’Enseignement Supe´rieur, Universite´ Catholique de Louvain, 1996. 26. Nisolle M, Donnez J, eds. Peritoneal, ovarian and rectovaginal endometriosis: the identification of three separate diseases. Carnforth: Parthenon Publishing, 1996. 27. Nisolle M, Casanas-Roux F, Anaf V, Mine JM, Donnez J. Morphometric study of the stromal vascularization in peritoneal endometriosis. Fertil Steril 1993; 59:681–684. 28. Donnez J, Nisolle M. L’endome´triose pe´ritone´ale, le kyste endometriotique ovarien et le nodule de la lame rectovaginale sont trois pathologies diffe´rentes (e´ditorial). Re´fe´rences en Gyne´cologie Obste´trique 1995; 3:121–123. 29. Halme PR. Angiogenic activity of peritoneal fluid from women with endometriosis. Fertil Steril 1993; 59:778–782.

Pathogenesis

41

30. van der Linden PJ, de Goeij AF, Dunselman GA, Erkens HW, Evers JL. Endometrial cell adhesion in an in vitro model using intact amniotic membranes. Fertil Steril 1996; 65:76–80. 31. van Langendonckt A, Casanas-Roux F, Donnez J. Oxidative stress and peritoneal endometriosis. Fertil Steril 2002; 5:861–870. 32. Brosens IA. Is mild endometriosis a progressive disease? Hum Reprod 1994; 9:2209–2211. 33. Donnez J, Casanas-Roux F, Nisolle M. Peritoneal endometriosis: new histological aspects. In: Brosens I, Donnez J, eds. Current status of endometriosis. Research and Management. Carnforth: Parthenon Publishing, 1993: 75–87. 34. Redwine DB. Age-related evolution in color appearance of endometriosis. Fertil Steril 1987; 48:1062–1063. 35. Goldstein MP, de Cholnoky C, Emans SJ, Leventhal JM. Laparoscopy in the diagnosis and management of pelvic pain in adolescents. J Reprod Med 1980; 44:251–258. 36. Brosens IA, Koninckx PR, Corveleyn PA. A study of plasma progesterone, oestradiol-17 beta, prolactin and LH levels and of the luteal phase appearance of the ovaries in patients with endometriosis and infertility. Br J Obstet Gynaecol 1978; 85:246–250. 37. Bergqvist A, Ljunberg O, Myrhe E. Human endometrium and endometriotic tissue obtained simultaneously: a comparative histologic study. Int J Gynecol Pathol 1984; 3:135–145. 38. Ferenczy A. Anatomy and histology of the uterine corpus. In: Kurman RJ, ed. Blaustein’s pathology of the female genital tract. New-York: Springer-Verlag, 1987:257–291. 39. Mazur T, Kurman RJ. Normal endometrium and infertility evaluation. In: Diagnosis of endometrial biopsies and curettings. A practical approach. New York: Springer-Verlag, 1995:7–32. 40. Halliwell B, Gutteridge JM. Role of free radicals and catalytic metal ions in human disease : an overview. Methods Enzymol 1990; 186:1–85. 41. Hippeli S, Elstner EF. Transition metal ion-catalyzed oxygen activation during pathogenic processes. FEBS Lett 1999; 443:1–7. 42. Brosens IA. New Principles in the management of endometriosis. Acta Obstet Gynecol 1994; 159:18–21. 43. Murphy A, Palinski W, Rankin S, Morales AJ, Parthasarathy S. Macrophage scavenger receptor(s) and oxidatively modified proteins in endometriosis. Fertil Steril 1998; 69:1085–1094. 44. Moen MH, Halvorsen TB. Histologic confirmation of endometriosis in different peritoneal lesions. Acta Obstet Gynecol Scand 1992; 71:337–342. 45. Stowell SB, Wiley CM, Perez-Reyes N, Powers CN. Cytologic diagnosis of peritoneal fluids. Applicability to the laparoscopic diagnosis of endometriosis. Acta Cytol 1997; 41:817–822. 46. Ponka P, Beaumont C, Richardson DR. Function and regulations of transferrin and ferritin. Semin Hematol 1998; 35:35–54.

42

Donnez and Squifflet

47. Fischbach FA, Gregory DW, Harrison PM, Hoy TG, Williams JM. On the structure of hemosiderin and its relationship to ferritin. J Ultrastruct Res 1971; 37:495–503. 48. Casanas-Roux F, Van Langendonckt A, Nisolle M, Donnez J. Iron deposits and oxidative stress in the peritoneal cavity of patients with endometriosis (abstract no. 10). In: Abstracts of the Endometriosis 7th Biennial World Congress, London, 2000. 49. Murphy A, Zhou MH, Malkapuram S, Santanam N, Parthasarathy S, Sidell N. RU486-induced growth inhibition of human endometrial cells. Fertil Steril 2000; 74:1014–1019. 50. Gue´rin P, El Mouatassim S, Me´ne´zon Y. Oxidative stress and protection against reactive oxygen species in the pre-implantation embryo and its surroundings. Hum Reprod Update 2001; 7:175–189. 51. Sampson JA. Perforating haemorrhagic (chocolate) cysts of the ovary. Arch Surg 1921; 3:245–323. 52. Hughesdon PE. The structure of endometrial cysts of the ovary. J Obstet Gynecol Br Emp 1957; 44:69–84. 53. Brosens IA, Puttemans PJ, Deprest J. The endoscopic localization of endometrial implants in the ovarian chocolate cyst. Fertil Steril 1994; 61:1034–1038. 54. Nezhat F, Nezhat C, Allan CJ, Metzger DA, Sears DL. A clinical and histological classification of endometriomas: implications for a mechanism of pathogenesis. J Reprod Med 1992; 37:771–776. 55. Donnez J, Nisolle M, Gillet N, Smets M, Bassil S, Casanas-Roux F. Large ovarian endometriomas. Hum Reprod 1996; 11:641–646. 56. Serov SF, Scully RE, Sobin LH. Histological typing of ovarian tumors. In: International Histological Classification of Tumors. No 9. Geneva: World Health Organization, 1973:17–21. 57. Motta PM, Van Blerkom J, Mekabe S. Changes in the surface morphology of ovarian germinal epithelium during the reproductive life and in some pathological conditions. Submicr Cytol 1992; 99:664–667. 58. Rosenfeld DL, Lecher BD. Endometriosis in a patient with Rokitansky-KusterHauser syndrome. Am J Obstet Gynecol 1981; 139:105. 59. Canis M, Wattiez A, Pouly JL, Bassil S, Bouquet J, Chapron C, Manhes H, Mage G, Bruhat MA. Laparoscopic treatment of endometriosis. In: Brosens I, Donnez J, eds. The Current status of endometriosis research and management. Carnforthe: Parthenon Publishing, 1993:407–417. 60. Donnez J. CO2 laser laparoscopy in infertile women with endometriosis and women with adnexal adhesions. Fertil Steril 1987; 48:390–394. 61. Donnez J, Nisolle M, Casanas-Roux F. Classification and endoscopic treatment of ovarian endometriosis. In: Popkin E, ed. Women’s health today. Carnforth: Parthenon Publishing, 1994. 62. Nisolle M, Donnez J. Conservative laparoscopic treatment of ovarian endometriosis. In: Shaw RW, ed. Endometriosis—current understanding and management. London: Blackwell Science, Ltd., 1995:237–247. 63. Brosens IA, Van Ballaer P, Puttemans P, Deprest J. Reconstruction of the ovary

Pathogenesis

64. 65. 66.

67. 68.

69. 70. 71.

72. 73.

43

containing large endometriomas by an extraovarian endosurgical technique. Fertil Steril 1996; 66:517–521. Donnez J, Donnez O, Squifflet J, Nisolle M. The concept of ‘‘adenomyotic disease of the retroperitoneal space’’ is born. Gynaecol Endosc 2001; 10:91–94. Sampson JA. Intestinal adenomas of endometrial type. Arch Surg 1922; 5:21–27. Donnez J, Nisolle M, Casanas-Roux F, Bassil S, Anaf V. Rectovaginal septum, endometriosis or adenomyosis: laparoscopic management in a series of 231 patients. Hum Reprod 1995; 2:230–235. Squifflet J, Feger C, Donnez J. Diagnosis and imaging of adenomyotic disease of the retroperitoneal space. Gynecol Obstet Invest 2002; 54:43–51. Balleyguier C, Chapron C, Duibuisson JB, Kinkel K, Fauconnier A, Vieira M, He´le´non O, Menu Y. Comparison of magnetic resonance imaging and transvaginal ultrasonography in diagnosing bladder endometriosis. J Am Assoc Gynecol Laparosc 2002; 9:15–23. Koninckx PR, Martin D. Deep endometriosis: a consequence of infiltration or retraction or possible adenomyosis externa? Fertil Steril 1992; 58:924–928. Adamyan L. Additional international perspectives. In: Nichols DH, ed. Gynecologic and Obstetrics Surgery. St. Louis: Mosby Year Book, 1993:1167–1182. Vercellini P, Aimi G, Panazza S, Vicentini S, Pisacreta A, Crosignani PG. Deep endometriosis conundrum: evidence in favor of peritoneal origin. Fertil Steril 2000; 73:1043–1046. Scully RE, Richardson GS, Barlow JF. The development of malignancy in endometriosis. Clin Obstet Gynecol 1966; 9:384–411. Donnez J, Nisolle M, Smoes P, Gillet N, Beguin S, Casanas-Roux F. Peritoneal endometriosis and ‘‘endometriotic’’ nodules of the rectovaginal septum are two different entities. Fertil Steril 1996; 66:362–368.

3 Anatomical Sites of Endometriosis Haya Al-Fozan and Togas Tulandi McGill University Montreal, Quebec, Canada

Endometriosis was described for the first time in 1860 by von Rokitansky as the presence of tissue resembling functioning endometrial glands and stroma outside the uterine cavity [1]. The prevalence of endometriosis among women of reproductive age was estimated to be as high as 10% [2]. However, the prevalence is higher in infertile women (38%) and in women suffering from pelvic pain (50%). The most common location of endometriosis is the pelvis. Occasionally, it is found in the skin, lungs, or gastrointestinal tract. PATHOGENESIS The possible etiology of endometriosis is discussed in Chapter 2. However, the exact pathogenesis of endometriosis remains unclear. In 1927, Sampson [3] postulated that endometriosis occurs because of retrograde menstruation, where fragments of endometrium reflux through fallopian tubes into the peritoneal cavity, then attach and grow on the peritoneum. This theory may explain the anatomical distribution of the endometriosis. Endometriosis is most commonly found close to the fimbriae and on the dependent portions of 45

46

Al-Fozan and Tulandi

the pelvis. Extrapelvic endometriosis, such as in the lung or brain, may be the result of lymphatic or hematogenic spread of endometriosis implants.

PELVIC ENDOMETRIOSIS Ovary and Posterior Cul de Sac The common sites of pelvic endometriosis are the ovaries and the posterior cul de sac [4–6]. Jenkins et al [5] reported that endometriosis affects both the ovary and the posterior cul de sac at equal frequency. In their report, the incidence of endometriosis on the posterior cul de sac, the left ovary, and the right ovary was 34%, 44%, and 31.3% respectively. They also found that endometriotic implants are found more frequently in the left hemipelvis. Their incidence of endometriosis on the left and right posterior broad ligaments was 25.2% and 21.4%, respectively. We found that 74.8% of endometriotic implants are in the posterior cul de sac [6]. Of interest, endometriosis is more frequently encountered in the left (64.3%) than in the right hemipelvis (Fig. 1) [6,7]. Similarly ovarian endometrioma is found more commonly in the left than in the right ovary. It is possible that this is related to decreased fluid movement in the left side of the

FIGURE 1 Black and whitish endometriotic implants on the left broad ligament.

Anatomical Sites

47

hemipelvis owing to the presence of sigmoid colon. In a study of patients with intraperitoneal malignant seeding using peritoneography, the author found that fluid from the pelvic cavity ascended both paracolic gutters. Compared with the right hemipelvis, the fluid flow in the left hemipelvis was weak and slow [8]. These findings may support the theory that the origin of endometriosis is from the regurgitated endometrial cells. Uterosacral ligaments are affected in 15.3% (right) and 20.8% (left) of the cases respectively [5]. Fallopian Tubes Fallopian tubes are infrequently involved with endometriosis (Fig. 2). The prevalence of tubal endometriosis is approximately 5%. Although endometriosis might not be found on the tube, it appears that tubal function is affected. Lyons et al [9] studied the effects of peritoneal fluid from women with endometriosis on the ciliary beat frequency of human fallopian tube epithelium. Compared to the control group, the ciliary beat frequency was significantly lower in the tube incubated with peritoneal fluid from women with endometriosis. The impairment of ciliary action in women with endometriosis might be one of the reasons for reduced fertility.

FIGURE 2 Bluish-black vesicles of endometriotic implants adjacent to the fallopian tube.

48

Al-Fozan and Tulandi

BOWEL The most common location of the extragenital endometriosis is the intestines [10,11]. It is found mainly in the rectum, sigmoid colon, cecum, appendix, and the terminal ileum [12]. In bowel endometriosis, the implants are usually seen on the serosa, but mural involvement may also occur [13]. Cyclic diarrhea, constipation, diarrhea alternating with constipation, cyclic rectal bleeding, cyclic abdominal distention, and partial or complete intestinal obstruction are symptoms suggestive of bowel involvement. Among all surgically treated endometriosis, intestinal obstruction on the level of ileum and sigmoid colon occurs in 0.8%, and 8% to 12%, respectively. However, complete obstruction of intestinal lumen is rare (<1%) [14]. Absence of pathognomonic symptoms makes the diagnosis difficult, especially in differentiating it from neoplasm. Varras et al [14] reported a case of extensive bowel obstruction resulting from sigmoid colon endometriosis, inadvertently treated with radical resection of descending and sigmoid colon as for bowel carcinoma.

BLADDER About 1% of women with endometriosis have involvement of the urinary tract, mostly involving the bladder (84%). Vesical endometriosis may present with variable symptoms and subtle onset, often mimicking recurrent cystitis. Early recognition is important to avoid prolonged morbidity and to limit further involvement of the bladder wall. Vercellini et al [15] reported eight patients with bladder detrusor endometriosis. The symptoms were mainly urinary frequency, urgency, and pain at micturition with vesical tenesmus of varying severity, all of which were limited to the menstrual time. Only one patient had gross hematuria. This is not surprising, as endometriosis rarely infiltrates and ulcerates the mucosal layer of the bladder. At vaginal examination, a painful nodule was palpated above the anterior fornix in seven patients. Cystoscopy showed endoluminal mass in the posterior bladder wall or dome in all cases. Bladder endometriosis should be suspected in perimenopausal women complaining of catamenial bladder symptoms with negative urine culture.

URETER The prevalence of ureteral endometriosis ranges from 0.01% to 1% in all women with endometriosis [16]. It is found more on the left side (63%) than on the right [17]. Because of concerns of silent loss of kidney function

Anatomical Sites

49

secondary to ureteral blockage, the authors suggest urinary tract imaging in women with severe endometriosis with parametrial infiltration or extensive rectovaginal lesions [17].

EXTRA PELVIC ENDOMETRIOSIS Extrapelvic endometriosis is endometriosis located outside the pelvic cavity. It could involve the gastrointestinal tract, urinary tract, lung, extremities, skin, central nervous system, and hepatic or retroperitoneal sites [18]. The prevalence of extrapelvic endometriosis is unknown because of the lack of epidemiologically well-defined studies. The different sites and variety of symptoms make the diagnosis of extrapelvic endometriosis difficult [16].

ENDOMETRIOSIS AFTER SURGICAL PROCEDURES Endometriosis has been reported after several surgical procedures, including hysterectomy, episiotomy, appendectomy, and laparoscopy. Perineal Endometriosis at the Site of Episiotomy Scar Endometriosis at the site of episiotomy scar is a rare occurrence. The incidence was estimated to be 15 of 2,028 consecutive deliveries [19]. In all cases, endometrial curettage was done as a routine preventive measure against postpartum hemorrhage. This suggests the relationship between curettage and subsequent endometriosis. Perineal endometriosis may present as an asymptomatic nodule or, classically, as a painful mass, particularly during menstruation when it becomes larger and more painful. It subsides several days after the cessation of menses. Perineal endometriosis most often presents after perineal trauma, but Pollack et al [20] described perineal endometriosis in the absence of prior perineal trauma. Endometriosis After Cesarean Section The incidence of endometriosis on the cesarean section scar is between 0.03% [21] and 1.7% [22]. Direct iatrogenic implantation of endometrial cells with subsequent growth is the most likely cause. Patients usually complain of cyclic pain within 1 to 2 years after a cesarean section. Rarely, a fistulous tract from the mass to the skin surface occurs, causing cyclic bleeding [23]. More than 100 cases of endometriosis after cesarean have been reported [21–25]. It rarely recurs [24].

50

Al-Fozan and Tulandi

PULMONARY ENDOMETRIOSIS Lung is an infrequent location of extrapelvic endometriosis. It is a rare cause of hemoptysis or pneumothorax. Treatment of pulmonary endometriosis includes a thoracotomy and decortication [26]. Bilateral oophorectomy might also be needed.

UMBILICAL ENDOMETRIOSIS The incidence of umbilical endometriosis is 0.5% to 1% [27]. The lesion can be painful or tender, especially before the onset of menstruation. In most cases, it is associated with other types of endometriosis or as the result of a laparoscopy. However, primary umbilical endometriosis has also been reported [28].

LATERAL PREDISPOSITION OF ENDOMETRIOSIS Other authors and we have reported a left predisposition of pelvic endometriosis [5–7]. In our study [6], the implants were found only in the left hemipelvis in 92 patients, in the right hemipelvis in 51, and bilaterally in 187. Among those with unilateral lesion, we found more frequent endometriosis in the left hemipelvis (64.3%) than in the right. A similar predisposition was found when we included those with bilateral lesions. Also, adhesionrelated endometriosis was found more frequently in the left than in the right hemipelvis. We also encountered more endometrioma on the left than on the right hemipelvis. This predisposition was not related to the size of the endometrioma [6].

STAGE OF ENDOMETRIOSIS AND SITES There is no correlation between the severity and the stage of endometriosis [29–31]. Studying 244 women with chronic pelvic pain, Vercellini et al [30] found that women with ovarian endometriosis had a lower prevalence of dysmenorrhea and deep dyspareunia compared with those with lesions elsewhere. These symptoms are more frequent in women with vaginal endometriosis. Fauconnier et al [32] described different types of pain associated with deeply infiltrating endometriosis. Deep dyspareunia is correlated with involvement of the uterosacral ligament, painful defecation with vaginal endometriosis, noncyclic pelvic pain with bowel and vaginal implants, and lower urinary tract symptoms with bladder involvement. Severe dysmenor-

Anatomical Sites

51

rhea was not correlated with deep infiltrating endometriosis, but with adhesions in the pouch of Douglas. SUMMARY Endometriosis is usually found in the pelvic cavity, mainly involving the ovary and posterior cul de sac. Extrapelvic endometriosis is uncommon, but its diagnosis is more difficult. This is because of its wide variety of symptoms. Pelvic endometriosis is usually found in the left hemipelvis. This left lateral predisposition of endometriosis could be related to decreased fluid movement in the left side of the pelvis because of the presence of sigmoid colon. These findings may support the theory that the origin of endometriosis is from the regurgitated endometrial cells. There is no correlation between the stage of endometriosis and symptoms. Symptoms are more related to the depth of infiltration than to the sites of endometriosis. PRACTICAL POINTS 

During surgery, pay more attention to the left hemipelvis. If you encounter endometrioma on the right ovary, the left ovary might also harbor an endometrioma.  Ovarian endometriosis is a sign of more extensive pelvic and intestinal disease. If you encounter a ovarian endometrioma, check for other sites. 

REFERENCES 1. 2. 3.

4.

5. 6.

von Rokitansky K. Uber uterusdrusen neubildung. Ztschr Gesselsch Aerzte Wein 1860; 16:577. Eskenazi B, Warner M. Epidemiology of endometriosis. Obstet Gynecol Clin North Am 1997; 24:235–258. Sampson JA. Peritoneal endometriosis due to menstrual dissemination of endometrial tissue in to the peritoneal cavity. Am J Obstet Gynecol 1927; 14:422– 469. Gruppo Italiano per to Studio dell Endometriosis. Prevalence and anatomical distribution of endometriosis in women with selected gynecological condition: results from a multicentric Italian study. Human Reprod 1994; 7:1158– 1162. Jenkins S, Olive DL, Haney AF. Endometriosis: pathogenetic implications of the anatomic distribution. Obstet Gynecol 1986; 67:335–338. Al-Fozan H, Tulandi T. Left lateral predisposition of endometriosis and endometrioma. Obstet Gynecol 2003; 101:164–166.

52

Al-Fozan and Tulandi

7.

Vercellini P, Aimi G, De Giorgi O, Maddalena S, Carinelli S, Crosignani PG. Is cystic ovarian endometriosis an asymmetric disease? Br J Obstet Gynecol 1998; 105:1018–1021. Meyers MA. Distribution of intra-abdominal malignant seeding: dependency on dynamics of the flow of ascitic fluid. Am J Roentgenol Radium Ther Nucl Med 1973; 119:198–206. Lyons R, Djahanbakhch O, Saridogan E, Naftalin A, Mahmood T, Weekes A, Chenoy R. Peritoneal fluid, endometriosis, and ciliary beat in the human fallopian tube. Lancet 2002; 360:1221–1222. Pillary S, Hardie I. Intestinal complications of endometriosis. Br J Surg 1980; 67:677. Bergqvist A. Different types of extragenital endometriosis: a review. Gynecol Endocrinol 1993; 3:207. Duleba A. Diagnosis of endometriosis. Obstet Gynecol Clin North Am 1997; 24:331–346. Livolsi V, Perzin K. Endometriosis of the small intestine producing intestinal obstruction or simulating neoplasm. Am J Dig Dis 1974; 19:100. Varras M, Kostopanagiotou E, Katis K, Farantes Ch, Angelidou-Manika Z, Antonio S. Endometriosis causing extensive intestinal obstruction simulating carcinoma of the sigmoid colon: a case report and review of the literature. Eur J Gynaecol Oncol 2002; 23:353–357. Vercellini P, Meschia M, De Giorgi O, Panazza S, Cortesi I, Crosignani PG. Bladder detrusor endometriosis: clinical and pathogenetic implications. J Urol 1995; 155:84–86. Jubanyik K, Comite F. Extrapelvic endometriosis. In: Olive DL, ed. Endometriosis. Obstet Gynecol Clin North Am 1997; 24:411–440. Vercellini P, Pisacteta A, Pesole S, Vicentini G, Stellato PG. Is ureteral endometriosis an asymptomatic disease? Br J Obstet Gynecol 2000; 107:559–561. Jeanes A, Murray D, Davidson B, Hamilton M, Watkinson F. Hepatic and retro-peritoneal presenting as obstructive jaundice with ascites: a case report and review of the literature. Clin Radiol 2002; 57:226–229. Paull T, Tedeschi LG. Perineal endometriosis at the site of episiotomy scar. Obstet Gynecol 1972; 40:28–34. Pollack R, Gordon Ph, Ferenczy A, Tulandi T. Perineal endometriosis: a case report. J Reprod Med 1990; 35:109–112. Chatterjee SK. Scar endometriosis: a clinicopathologic study of 17 cases. Obstet Gynecol 1980; 56:81–84. Wolf G, Singh K. Cesarean scar endometriosis: a review. Obstet Gynecol Surv 1989; 44:89–95. Kaloo P, Reid G, Wong F. Caesarean section scar endometriosis: two cases of recurrent disease and literature review. Aust N Z J Obstet Gynecol 2002; 42:218–220. Koger K, Shatney C, Hodge K, McClenathan J. Surgical scar endometriosis. Surg Gynecol Obstet 1993; 177:243–246. Dellon A, Grodin J, Ketchan A, Chretein P. Recurrent abdominal scar endo-

8.

9.

10. 11. 12. 13. 14.

15.

16. 17. 18.

19. 20. 21. 22. 23.

24. 25.

Anatomical Sites

26. 27.

28. 29. 30.

31.

32.

53

metriosis in patient with a fibrous mandibular neoplasm. Am J Obstet Gynecol 1974; 120:849–850. Moffatt S, Mitchell J. Massive pleural endometriosis. Eur J Cardiothorac Surg 2002; 22:321–323. Singh K, Lessells A, Adam D, Jordan C, Miles W, Macintyre I, Greig J. Presentation of endometriosis to general surgeons: a 10 year experience. Br J Surg 1995; 82:1349–1351. Sidani M, Khalil A, Tawil A, El-Hajj M, Seoud M. Primary umbilical endometriosis. Clin Exp Obstet Gynecol 2002; 29:40–41. Fedele L, Bianchi S, Bocciolone L, Di Nola G, Parazzini F. Pain symptoms associated with endometriosis. Obstet Gynecol 1992; 79:767–769. Vercellini P, Trespidi L, De Giorgi O, Cortesi I, Parazzini F, Crosignani PG. Endometriosis and pelvic pain: relation to disease stage and localization. Fertil Steril 1996; 65:299–304. Gruppo Italiano per to Studio dell Endometriosis. Relationship between stage, site and morphological characteristics of pelvic endometriosis and pain. Hum Reprod 2001; 16:2668–2671. Fauconnier A, Chapron C, Dubuisson JB, Vieira M, Dousset B, Breart G. Relation between pain symptoms and the anatomic location of deep infiltrating endometriosis. Fertil Steril 2002; 78:719–726.

4 Genetics of Endometriosis Stephen Kennedy University of Oxford, John Radcliffe Hospital Oxford, England

The cause of endometriosis remains unknown despite years of research. The difficulty of producing a unified theory to explain all disease types has led researchers to propose that different causes exist for the various manifestations [1]. Thus, although Sampson’s original theory may explain how peritoneal implants arise, it seems an unlikely explanation for ovarian endometriomas, which probably result from in situ metaplasia. Whichever mechanism is responsible, there must be additional factors influencing the probability of a woman becoming affected, and there is increasing evidence pointing to a complex interplay between susceptibility genes and the environment as being one of the most important effects. The study of the genetic basis of endometriosis is still in its infancy, and it remains unproven that disease susceptibility genes exist. However, there is now sufficient clinical and experimental evidence in the literature to put the case strongly for endometriosis being a genetic disease. The existing clinical evidence includes familial clustering in humans [2–4] and rhesus monkeys [5]; a founder effect in the Icelandic population [6]; concordance in monozygotic twins [7–9]; a similar age at onset of symptoms in affected, non-twin sisters [10]; a six to nine times increased prevalence among first degree relatives of 55

56

Kennedy

women compared with the general population [11–13], and a 15% prevalence of magnetic resonance imaging (MRI) findings suggestive of endometriosis in the first degree relatives of women with revised American Fertility Society (rAFS) stage III-IV disease [14]. In addition, a number of studies have implicated candidate disease susceptibility genes [15]. WHAT IS THE CLINICAL EVIDENCE THAT ENDOMETRIOSIS HAS A GENETIC BASIS? Twin studies provide the strongest clinical evidence, including case reports of concordance in monozygotic (MZ) twins [7,8] and, more importantly, data from a large study of MZ and dizygotic (DZ) twin pairs ascertained via the Australian National Health and Medical Research Council Twin Register [9]. Questionnaires were sent to 3298 women and confirmatory information was then sought from the physicians of twins who consented to participate in the study. Hence, endometriosis status was validated where possible by a pathology or surgical report, or both. Of the twins surveyed, 3096 (94%) responded, of whom 215 self-reported that they had endometriosis—a .07 prevalence rate among respondents. Including only those women for whom confirmatory reports were available, the MZ and DZ twin pair correlations were .52 F .08 and .19 F .16, respectively, suggesting that 51% of the variance of the latent liability to the disease may be attributable to additive genetic influences. There is also evidence that endometriosis clusters in families [2–4]. This can suggest a genetic basis, but other factors can explain familial clustering, such as exposure to environmental or infectious agents. Ascertainment bias is another explanation for such findings given that the diagnosis in a proband may increase the likelihood that other family members will be diagnosed. Bias can also occur if probands are not representative of affected individuals in the general population in terms of their symptoms or disease type, which can arise if they are ascertained through tertiary referral clinics. In clinical practice, such patients typically have more extreme manifestations of a disease, which may make a genetic basis more likely. In ideal circumstances, therefore, families should be ascertained through community- or population-based registers to reduce the possibility of introducing bias. This is essential because genetic and epidemiological studies in the field of endometriosis have been limited in terms of their number, scope, choice of study populations, and sample sizes. They have consequently left many unanswered questions as to the relative influence of genetic versus environmental factors, and possible gene-environment interaction. Given the large number of different environmental factors to which women may be exposed up to and during their reproductive years, studying such interactions in humans can be problematic. An animal model, such as the rhesus monkey, in

Genetics

57

which exposures are carefully controlled and recorded, would, therefore, be a valuable tool to investigate the genetic epidemiology of endometriosis. The advantage of rhesus monkeys is that they have many anatomical and physiological features in common with humans; they develop endometriosis spontaneously and the tissue is morphologically identical to their human counterparts [16]. The Wisconsin Regional Primate Research Center (WRPRC), University of Wisconsin-Madison, has consistently maintained a population of about 1,000 rhesus monkeys since 1971 for research and breeding. Comprehensive records of the animals’ experimental and environmental history, including all familial relationships, are available. Paternity is known for about 90% of the animals; some of the animals in the colony, however, are of feral origin and neither parent is known. Between 1981 and 2001, 142 animals with endometriosis were identified from necropsy and surgical records and with MRI. All the cases were used to construct a large, multigenerational pedigree and nine nuclear families consisting of 1602 females in total. By 2002, the pedigrees contained 124 cases diagnosed at necropsy, 17 at surgery, and 3 at MRI [17]. The prevalence of endometriosis among necropsied animals in the colony was 31.4% (95% CI: 26.9–35.9%); prevalence increased with rising age at death and calendar period. Familial aggregation was strongly suggested by a significantly higher average kinship coefficient among affected compared to unaffected animals ( P < 0.001), and a higher recurrence risk for full siblings (0.75, 95% CI: 0.45–1.0) compared to paternal halfsiblings (0.47, 95% CI: 0.42–0.52) and maternal half-siblings (0.26, 95% CI: 0.10–0.41). Similar studies are planned at the New England RPRC, at Harvard Medical School, and the California RPRC, at the University of California, Davis [18]. The same principles have been used in Iceland by researchers who have attempted to identify all the women with endometriosis diagnosed in the entire country over a defined time period [3] and then determine the extent to which the affected individuals are interrelated using a national genealogy database dating back 10 generations [6]. The 750 women identified with endometriosis were significantly more related to each other than the controls, with risk ratios for sisters and cousins of 5.20 and 1.56, respectively. The average kinship coefficient for the affected women was significantly higher than that for 1000 sets of 750 matched controls ( P < 0.001). The minimum number of ancestors to account for the group of women with endometriosis (n = 750) was compared with the minimum number needed to account for the control groups at different time points, using a technique known as the Minimum Founder Test. Taking the year 1880 as an example, the minimum number of ancestors (n = 635) for the group of 750 affected women was significantly less than that for the control groups.

58

Kennedy

In addition, there are four studies suggesting that the disease occurs more commonly in the first degree relatives of affected women than controls [11–14]. Simpson et al noted a sevenfold higher risk among the first degree relatives of North American patients compared with their husbands’ firstdegree relatives [13]. Coxhead & Thomas found a sixfold increased risk among the first degree relatives of UK patients compared with those of unaffected case controls [11]. Moen and Magnus reported a sevenfold increase in risk for endometriosis or adenomyosis, or both among the first degree relatives of Norwegian patients compared with those of case controls [12]; among the women with endometriosis, severe disease was significantly more common in those with an affected relative ( P < 0.01). Thus, the genetic influence may be more pronounced in the relatives of women with more severe disease. This was also demonstrated in a study of the 47 UK relatives of 29 probands with rAFS stage III-IV disease who underwent MRI as a diagnostic test. Endometriosis was suspected in 15 of 47 (32%) women as ovarian cysts, peritoneal lesions, or equivocal lesions less than 1 cm in diameter. Including equivocal findings, there was some evidence of endometriosis, adenomyosis, or both in 19 of 35 (54%) first degree relatives and 3 of 12 (25%) second degree relatives; the figures were 5 of 35 (14%) and 1 of 12 (8%) when equivocal findings and adenomyosis were excluded [14]. The extent to which endometriosis clusters within families can be estimated by the value of ES (e.g., the risk of the sister of an affected woman having the disease compared to the risk in the general population), which would appear to lie between 2 and 9 for all disease severities. However, in women with the most severe forms of endometriosis, ES may be as high as 15 based on the MRI data. Such a range of values is consistent with the hypothesis that endometriosis is inherited as a complex genetic trait [19], like diabetes, asthma, and hypertension, for which the phenotype is expressed as a result of interactions between allelic variations in a number of susceptibility genes and each other, and between those genes and environmental factors. WHAT IS THE EXPERIMENTAL EVIDENCE THAT ENDOMETRIOSIS HAS A GENETIC BASIS? A number of research groups are investigating candidate ‘‘functional’’ genes, which are chosen based on knowledge of their putative or actual role in mechanisms involved in disease development. Candidate genes are usually investigated in association studies by comparing the frequency of gene polymorphisms or mutations in affected individuals and controls. The subject has recently been reviewed [15], and up-to-date information about all relevant studies is maintained on a genetic epidemiology website (http://www.well.ox.

Genetics

59

ac.uk/fkrinaz/genepi_endo.htm) [20]. Results have been inconsistent often because the case-control studies have been underpowered or designed with inappropriate controls [21]. The genes investigated fall into four broad etiological areas, based upon theories relating to retrograde menstruation, the differential growth and differentiation of endometriotic cells, hormonal pathways, and detoxification mechanisms. The summary below is by no means comprehensive; a fuller account appears on the website. Retrograde Menstruation The N314D polymorphism in the GALT gene has been studied based on the known associations between (1) the polymorphism and Mu¨llerian anomalies, and (2) endometriosis and Mu¨llerian anomalies. The polymorphism causes reduced activity of the enzyme, galactose-1-phosphate uridyl transferase, which is involved in galactose metabolism. Cramer et al (1996) reported that 30% of 33 North American women with rAFS stage I–IV endometriosis carried at least one N314D allele, compared with 14% of 111 controls [21a]. Three other studies have failed to replicate the association [3,22,23], although in a subset of UK women with familial disease, Hadfield et al reported that 29% carried the N314D polymorphism, compared with only 6% of fertile women [22]. Differential Growth and Differentiation Two polymorphisms (G/R241) and (E/K469) in the gene, ICAM-1, encoding for intercellular adhesion molecule-1, a surface glycoprotein that promotes adhesion in immunological and inflammatory reactions, have recently been studied in 188 Italian women with endometriosis and 175 controls without disease [24]. The frequency of the R241 allele was slightly higher in the affected women compared with the controls (5.8% vs 2.9%), but the allele was found more commonly in those with rAFS stage IV disease (8.6% vs 2.8%). No statistically significant differences were noted for the E/K469 polymorphism. Chang et al compared the frequencies of the p53 codon 72 polymorphism between 118 Taiwanese women with rAFS stage III-IV disease and 140 unaffected women, and reported a possible protective effect for the Arg/Arg genotype [25]. Genotype frequencies for cases and controls were: Arg/Arg 10.2% versus 30.7%; Arg/Pro 89.9% versus 69.3%; Pro/Pro 22.9% versus 19.3%. The same group found no association for p21, an important regulator of the cell cycle that acts as a mediator of p53 [26]. Similarly, no evidence of association has been reported for polymorphisms in interleukin-1 beta (IL-1ß) and receptor antagonist (IL-1Ra) [27]; IL-4 [28]; IL-6 [29,30]; IL-10 [31], and tumor necrosis factor-alpha (TNF- a) [28,30,32].

60

Kennedy

Hormonal Pathways Genes involved in ovarian steroidogenesis have been investigated based on the well-recognized responsiveness of endometriotic tissue to hormones. Georgiou et al compared the frequency of the oestrogen receptor gene (ERa) Pvu II polymorphism in 57 Greek women with endometriosis and 57 controls. The Pvu II polymorphism was significantly more common in cases than controls (positive allele: 0.72 vs 0.49), seemingly because of a lower frequency of homozygous (-/-) negatives [33]. Similarly, Kitawaki et al reported an underrepresentation of the -/- Pvu II genotype (13% vs 24%) among 109 Japanese cases and 179 controls [34]. Wieser et al investigated the frequency of a 306-base pair insertion in intron G of the progesterone receptor gene in 95 affected Austrian women and 107 controls. Genotype frequencies in cases and controls were significantly different: T1/T1: 68.4% versus 85.0%; T1/T2 28.4% versus 14.0%; T2/T2: 3.2% versus 0.9%. The allele frequency of T2 among cases was 17.4% versus 7.9% in controls ( P = 0.005) [35]. No evidence of association, however, has been reported for polymorphisms in the variant luteinizing hormone (V-LH) gene [36]; catechol-Omethyltransferase (COMT) [37]; CYP1A1 [38], or CYP17 [39], although in the same study a significantly increased frequency of the D/D CYP19 genotype was found among cases compared to controls (genotype frequencies I/I: 43.6% vs 48.6%; I/II: 40.7% vs 45.2%; II/II: 15.7% vs 6.2%). Detoxification Mechanisms Genes encoding enzymes involved in detoxification such as the glutathione Stransferase (GST) family have been investigated based on the finding that the environmental pollutant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) induces endometriosis in the rhesus monkey [40]. Homozygotes for a null mutation in one of the GST family genes, GSTM1, were more common in affected French women compared with controls (86% vs 46%), although if only women with rAFS stage III-IV disease were included then more than 90% had the mutation [41]. In a Slavic population, 81% of women with endometriosis had the null mutation compared with 39% of healthy male and female controls [42]. Two UK studies, however, have failed to replicate the association [38,43], and no association has been found for a mutation in a similar gene, GSTT1 [38,44]. There are conflicting results regarding polymorphisms in the NAT 2 gene. This encodes an enzyme, N-acetyltransferase 2, involved in the initial biotransformation of aromatic amines and hydrazines. NAT 2 polymorphisms result in impaired enzyme activity: homozygotes for the NAT 2 *4 wild-type allele are fast acetylators; heterozygotes with a mutant NAT 2 *5, *6

Genetics

61

or *7 allele have reduced activity, and mutant-type homozygotes are slow acetylators. Baranova et al reported that a significant proportion of women with the slow NAT2 genotype among 49 French endometriosis cases (69%) compared with 72 controls (39%); however, this effect was limited to rAFS stage I-II cases [44]. Nakago et al, on the other hand, reported that a significantly higher proportion (57.4%) of 54 UK women with rAFS stage III-IV disease were fast acetylators compared with 99 male controls (32.3%) or 24 women with a normal pelvis (33.3%); this effect was caused by an overrepresentation of the *4/*6 genotype among cases [45]. POSITIONAL CLONING APPROACH A hypothesis-driven approach to identifying susceptibility genes by studying ‘‘functional’’ candidates chosen on the basis of their biological plausibility has a limited chance of being successful for the reasons outlined above. The alternative, non-hypothesis-driven method is known as ‘‘reverse genetics’’ or ‘‘positional cloning’’: it makes no assumptions about the mechanisms involved. This approach, which is proving so successful in other complex traits such as diabetes, has been adopted in endometriosis by six research groups in the world (in Australia, Iceland, India, Puerto Rico, UK/USA, and Utah). Two are sponsored by public funds (India and Puerto Rico) and four (in Australia, Iceland, UK/USA, and Utah) by biotechnology companies, which inevitably influences the amount of information, which enters the public domain. The approach broadly involves: 1. Recruitment of families with two or more affected sisters 2. Collection of DNA from the affected sisters and their parents 3. Performing a genome-wide screen, which involves localizing the chromosomal region linked to the disease locus, using DNA markers evenly spaced throughout the genome 4. Searching public databases to identify a candidate gene in that region—the so-called ‘‘positional candidate’’ approach 5. Identification of the gene and polymorphism(s) associated with the disease by showing more common occurrence in affected individuals than in controls 6. Determination of abnormal function associated with the polymorphism(s) The Oxford Group is collecting 200 sister-pair families for a genomewide screen, in collaboration with Dr. Mamta Deenadayal in Secunderabad, Professor Baidyanath Chakravarthy in Calcutta, and Dr. Sisinthy Shivaji at the Centre for Cellular & Molecular Biology, Hyderabad. The intention is also to collect 1000 triads for future association studies. In Puerto Rico,

62

Kennedy

Dr. Idhaliz Flores, Ponce School of Medicine, in collaboration with the National Institutes of Health (NIH)-National Human Genome Research Institute, is aiming to collect a large number of families from the island’s population for a genome-wide screen. In Iceland, Professor Reynir Geirsson, Landspı´ talinn University Hospital, is collaborating with DeCode Genetics, a company that has access to a unique genealogical database containing all individuals living in the country over the previous 10 generations. Blood samples have been obtained from 205 of the 750 women with endometriosis identified (see section above) by searching through the medical records of the central hospital in Reykjavı´ k and all outlying hospitals in the country. The 205 women comprised 64 families ranging in size from 2 to 13 affected members. To date, no genome-wide scan results have been reported, but 47 microsatellite markers have been genotyped across chromosome 9 without finding any evidence of linkage [3]. Icelandic families represent a unique resource for genetic studies and a genome-wide search in endometriosis may lead rapidly to the identification of disease susceptibility genes. However, there are certain limitations to performing genetic research in Iceland to identify susceptibility genes in complex traits. First, there may not be sufficient numbers of affected individuals in such a small population resulting in an underpowered study and an inability to detect small gene effects. Second, the results may only apply to Icelandic families, a very homogeneous population, and not to more heterogeneous groups elsewhere in the world. Similar strengths and weaknesses affect the collaboration between Dr. Ken Ward, University of Utah, and the biotechnology company EmerGen. This group has reported collecting 117 probands with endometriosis from whom three generational family histories have been taken [46]. The women had a mean number of 1.6 sisters of reproductive age, of whom 22% also had surgically confirmed disease. However, the largest single resource for a positional cloning approach has been collected by the International Endogene Study, which is a collaboration between the University of Oxford and the Australian Cooperative Research Centre for Discovery of Genes for Common Human Diseases (Gene CRC), and their respective commercial partners Oxagen Ltd. and Cerylid Biosciences Ltd. [4]. Their two projects, the OXEGENE (Oxford Endometriosis Gene) Study and the Genes Behind Endometriosis Study, led, respectively, by Dr. Stephen Kennedy and Dr. Sue Treloar, started independently in 1995 based on previous research [2,47,48]. Both groups have mainly collected affected sister-pairs using similar recruitment methods that have been described in detail elsewhere [49]. As of April 2002, the combined dataset consisted of more than 2500 families

Genetics

63

(Table 1). Of those women with disease that could be staged using the rAFS system, 2220 (67%) in the Australian study had stage A (the equivalent of rAFS stage I-II) and 1098 (33%) had stage B (the equivalent of rAFS stage III-IV) disease. In the UK study, 737 (33%) had stage A and 1470 (66%) had more severe or deeply infiltrating disease [4]. Suggestive linkage has been reported for one chromosomal locus based on the analysis of marker data (400 markers at f10 cM) generated for a total of 289 families from the complete dataset, containing 374 sister-pairs plus other affected relatives [50], but the results of the full genome-wide scan of all the families collected have not yet been published. TABLE 1

Composition of Families Recruited into the International Endogene

Study

Family composition

United Kingdom (N)

Families for ASP analysis At least 1 affected sibship 230 At least 1 affected sibship with 33 other affected relatives Total families with affected sibships 263 ASP families 2 affected sisters 245 2 affected pairs in different sibships 1 3 affected sisters 15 3 affected sisters in 1 sibship and 0 2 in another sibship 4 affected sisters 1 5 affected sisters 1 Families for association analysis trinds for TDTa 1 triad 358 1 triad and 1 other affected relative 48 1 triad and 2 other affected relatives 2 1 triad and 3 other affected relatives 1 Total families with z1 triad (some with an 409 additional affected relative) Individual cases for case-control studyb 248 Total families and cases recruited 920 a

Australia (N) 728 117

958 150

845

1,108

758 11 64 3

1,003 12 79 3

9 0

10 1

711 128 12 0 851

1,069 176 14 1 1,260

316 2,012

564 2,932

Triads consist of a case and both parents. Only one case per family can be used for case-control association testing. Source: Ref 4.

b

International Endogene Study (N)

64

Kennedy

SUMMARY Results emerging from the research described in this chapter may soon lead to a much clearer understanding of the molecular and cellular basis of endometriosis. Identifying the genes that predispose women to develop the disease and how environmental factors interact with those genes to produce the disease phenotype should radically change our clinical management. We may, in time, be able to develop new, disease-specific therapies based on knowledge of the underlying aberrant mechanisms and devise new, noninvasive diagnostic tests. New classification systems based on genetic information about individual patients may also lead to the development of targeted drugs, which in turn will produce more effective therapy and fewer side effects.

PRACTICAL POINT 

Identifying the gene for endometriosis may allow the development of new diagnostic tests and targeted drug therapy.

REFERENCES 1. 2. 3.

4. 5. 6. 7. 8.

Koninckx PR, Barlow D, Kennedy S. Implantation versus infiltration: the Sampson versus the endometriotic disease theory. Gynecol Obstet Invest 1999; 47(suppl 1):3–9. Kennedy S, Mardon H, Barlow D. Familial endometriosis. J Assist Reprod Genet 1995; 12:32–34. Stefansson H, Einarsdottir A, Geirsson RT, Jonsdottir K, Sverrisdottir G, Gudnadottir VG, Gunnarsdottir S, Manolescu A, Gulchar J, Stefansson K. Endometriosis is not associated with or linked to the GALT gene. Fertil Steril 2001; 76:5–22. Treloar SA, Hadfield RM, Montgomery G, Lambert A, Wicks J, Barlow DH. The International Endogene Study: a collection of families for genetic research in endometriosis. Fertil Steril 2002; 78:679–685. Hadfield RM, Yudkin PL, Coe CL, Scheffler J, Uno H, Barlow DH, Kemnitz JW, Kennedy SH. Risk factors for endometriosis in the rhesus monkey (Macaca mulatta): a case-control study. Hum Reprod Update 1997; 3:109–115. Stefansson H, Geirsson RT, Steinthorsdottir V, Jonsson H, Manolescu A, Kong A, Ingadottir G, Gulcher J, Stefansson K. Genetic factors contribute to the risk of developing endometriosis. Hum Reprod 2002; 17:3–9. Hadfield RM, Mardon HJ, Barlow DH, Kennedy SH. Endometriosis in monozygotic twins. Fertil Steril 1997; 68:941–942. Moen MH. Endometriosis in monozygotic twins. Acta Obstet Gynecol Scand 1994; 73:59–62.

Genetics 9. 10. 11. 12. 13. 14. 15. 16. 17.

18.

19. 20. 21. 21a. 22.

23. 24.

65

Treloar SA, O’Connor DT, O’Connor VM, Martin NG. Genetic influences on endometriosis in an Australian twin sample. Fertil Steril 1999; 71:701–710. Kennedy S, Hadfield R, Mardon H, Barlow D. Age of onset of pain symptoms in non-twin sisters concordant for endometriosis. Hum Reprod 1996; 11:403– 405. Coxhead D, Thomas EJ. Familial inheritance of endometriosis in a British population: a case control study. J Obstet Gynaecol 1993; 13:42–44. Moen MH, Magnus P. The familial risk of endometriosis. Acta Obstet Gynecol Scand 1993; 72:560–564. Simpson JL, Elias S, Malinak LR, Buttram-VC J. Heritable aspects of endometriosis. I. Genetic studies. Am J Obstet Gynecol 1980; 137:327–331. Kennedy S, Hadfield R, Westbrook C, Weeks DE, Barlow D, Golding S. Magnetic resonance imaging to assess familial risk in relatives of women with endometriosis. Lancet 1998; 352:1440–1441. Zondervan KT, Cardon LR, Kennedy SH. The genetic basis of endometriosis. Curr Opin Obstet Gynecol 2001; 13:309–314. MacKenzie WF, Casey HW. Animal model of human disease. Endometriosis. Animal model: endometriosis in rhesus monkeys. Am J Pathol 1975; 80:341– 344. Zondervan K, Weeks DE, Colman R, Cardon L, Hadfield RM, Schleffler J, Trainor AG, Golding S, Coe CL, Kemnitz JW, Kennedy SH. Familial aggregation of endometriosis in a large pedigree of rhesus macaques. Submitted for publication. Zondervan K, Cardon L, Desrosiers R, Hyde D, Kemnitz J, Mansfield K, Roberts J, Scheffler J, Weeks DE, Kennedy SH. The genetic epidemiology of spontaneous endometriosis in the rhesus monkey. Ann N Y Acad Sci 2002; 955:233–238. Kennedy S. Is there a genetic basis to endometriosis? Semin Reprod Endocrinol 1997; 15:309–318. Zondervan KT, Cardon LR, Kennedy SH. Development of a Web site for the genetic epidemiology of endometriosis. Fertil Steril 2002; 78:781. Zondervan KT, Cardon LR, Kennedy SH. What makes a good case-control study?—Design issues for complex genetic traits such as endometriosis. Hum Reprod 2002; 17:1415–1423. Cramer DW, Hornstein MD, Ng WG, Barbieri RL. Endometriosis associated with the N314D mutation of galactose-1-phosphate uridyl transferase (GALT). Mol Hum Reprod 1996; 2:149–152. Hadfield RM, Manek S, Nakago S, Mukherjee S, Weeks DE, Mardon HJ, Barlow DH, Kennedy SH. Absence of a relationship between endometriosis and the N314D polymorphism of galactose-1-phosphate uridyl transferase in a UK population. Mol Hum Reprod 1999; 5:990–993. Morland SJ, Jiang X, Hitchcock A, Thomas EJ, Campbell IG. Mutation of galactose-1-phosphate uridyl transferase and its association with ovarian cancer and endometriosis. Int J Cancer 1998; 77:825–827. Vigano P, Infantino M, Lattuada D, Lauletta R, Ponti E, Somigliana E,

66

25. 26. 27.

28.

29. 30. 31.

32. 33. 34.

35. 36. 37.

Kennedy Vignali M, DiBlasio AM. Intercellular adhesion molecule-1 (ICAM-1) gene polymorphisms in endometriosis. Mol Hum Reprod 2003; 9:1–52. Chang CC, Hsieh YY, Tsai FJ, Tsai CH, Tsai HD, Lin CC. The proline form of p53 codon 72 polymorphism is associated with endometriosis. Fertil Steril 2002; 77:1–5. Hsieh YY, Tsai FJ, Chang CC, Chen WC, Tsai CH, Tsai HD, Lin CC. p21 gene codon 31 arginine/serine polymorphism: non-association with endometriosis. J Clin Lab Anal 2001; 15:4–7. Hsieh YY, Chang CC, Tsai FJ, Wu JY, Shi YR, Tsai HD, Tsai CH. Polymorphisms for interleukin-1 beta (IL-1 beta)-511 promoter, IL-1 beta exon 5, and IL-1 receptor antagonist: nonassociation with endometriosis. J Assist Reprod Genet 2001; 18:506–511. Hsieh YY, Chang CC, Tsai FJ, Hsu Y, Tsai HD, Tsai CH. Polymorphisms for interleukin-4 (IL-4) -590 promoter, IL-4 intron3, and tumor necrosis factor alpha -308 promoter: non-association with endometriosis. J Clin Lab Anal 2002; 16:3–6. Wieser F, Fabjani G, Tempfer C, Schneeberger C, Sator M, Huber J, Wenzl R. Analysis of an interleukin-6 gene promoter polymorphism in women with endometriosis by pyrosequencing. J Soc Gynecol Investig 2003; 10:1–6. Lee MK, Park AJ, Kim DH. Tumor necrosis factor-alpha and interleukin-6 promoter gene polymorphisms are not associated with an increased risk of endometriosis. Fertil Steril 2002; 77:1304–1305. Kitawaki J, Obayashi H, Ohta M, Kado N, Ishihara H, Koshiba H, Kusuki I, Tsukamoto K, Hasegawa G, Nakamura N, Yoshikawa T, Honjo H. Genetic contribution of the interleukin-10 promoter polymorphism in endometriosis susceptibility. Am J Reprod Immunol 2002; 47:1–8. Wieser F, Fabjani G, Tempfer C, Schneeberger C, Zeillinger R, Huber JC, Wenzl R. Tumor necrosis factor-alpha promotor polymorphisms and endometriosis. J Soc Gynecol Investig 2002; 9:5–8. Georgiou I, Syrrou M, Bouba I, Dalkalitsis N, Paschopoulos M, Navrozoglou I, Lolis D. Association of estrogen receptor gene polymorphisms with endometriosis. Fertil Steril 1999; 72:164–166. Kitawaki J, Obayashi H, Ishihara H, Koshiba H, Kusuki I, Kado N, Tsukamoto K, Hasegawa G, Nakamura N, Honjo H. Oestrogen receptor-alpha gene polymorphism is associated with endometriosis, adenomyosis and leiomyomata. Hum Reprod 2001; 16:51–55. Wieser F, Schneeberger C, Tong D, Tempfer C, Huber JC, Wenzl R. PROGINS receptor gene polymorphism is associated with endometriosis. Fertil Steril 2002; 77:2–12. Gazvani R, Pakarinen P, Fowler P, Logan S, Huhtaniemi I. Lack of association of the common immunologically anomalous LH with endometriosis. Hum Reprod 2002; 17:1532–1534. Wieser F, Wenzl R, Tempfer C, Worda C, Huber J, Schneeberger C. CatecholO-methyltransferase polymorphism and endometriosis. J Assist Reprod Genet 2002; 19:7–8.

Genetics 38.

39.

40. 41.

42.

43. 44.

45.

46. 47. 48. 49. 50.

67

Hadfield RM, Manek S, Weeks DE, Mardon HJ, Barlow DH, Kennedy SH. Linkage and association studies of the relationship between endometriosis and genes encoding the detoxification enzymes GSTM1, GSTT1 and CYP1A1. Mol Hum Reprod 2001; 7:1073–1078. Kado N, Kitawaki J, Obayashi H, Ishihara H, Koshiba H, Kusuki I, Tsukamoto K, Hasegawa G, Nakamura N, Yoshikawa T, Honjo H. Association of the CYP17 gene and CYP19 gene polymorphisms with risk of endometriosis in Japanese women. Hum Reprod 2002; 17:897–902. Rier SE, Martin DC, Bowman RE, Dmowski WP, Becker JL. Endometriosis in rhesus monkeys (Macaca mulatta) following chronic exposure to 2,3,7,8tetrachlorodibenzo-p-dioxin. Fundam Appl Toxicol 1993; 21:433–441. Baranova H, Bothorishvilli R, Canis M, Albuisson E, Perriot S, Glowaczower E, Bruhat MA, Baranov V, Malet P. Glutathione S-transferase M1 gene polymorphism and susceptibility to endometriosis in a French population. Mol Hum Reprod 1997; 3:775–780. Baranov VS, Ivaschenko T, Bakay B, Aseev M, Belotserkovskaya R, Baranova H, Malet P, Perriot J, Mouraire P, Baskakov VN, Savitskyi GA, Gorbushin S, Deyneka SI, Michnin E, Barchuck A, Vakharlovsky V, Pavlov G, Shilko VI, Guembitzkaya T, Kovaleva L. Proportion of the GSTM1 0/0 genotype in some Slavic populations and its correlation with cystic fibrosis and some multifactorial diseases. Hum Genet 1996; 97:516–520. Baxter SW, Thomas EJ, Campbell IG. GSTM1 null polymorphism and susceptibility to endometriosis and ovarian cancer. Carcinogenesis 2001; 22:63–65. Baranova H, Canis M, Ivaschenko T, Albuisson E, Bothorishvilli R, Baranov V, Malet P, Bruhat MA. Possible involvement of arylamine N-acetyltransferase 2, glutathione S-transferases M1 and T1 genes in the development of endometriosis. Mol Hum Reprod 1999; 5:636–641. Nakago S, Hadfield RM, Zondervan KT, Mardon H, Manek S, Weeks DE, Barlow DH, Kennedy SH. Association between endometriosis and N-acetyl transferase 2 polymorphisms in a UK population. Mol Hum Reprod 2001; 7: 1079–1083. Hull DB, Gibson C, Hart A, Dowsett S, Meade M, Ward K. The heritability of endometriosis in large Utah families. Fertil Steril 2001; 77:S21. O’-Connor DT. Endometriosis. In: Singer A, ed. Current Reviews in Obstetrics and Gynaecology. Edinburgh: Churchill Livingstone, 1987. Treloar SA, Do KA, O’Connor VM, O’Connor DT, Yeo MA, Martin NG. Predictors of hysterectomy: an Australian study. Am J Obstet Gynecol 1999; 180:945–954. Kennedy S, Bennett S, Weeks DE. Affected sib-pair analysis in endometriosis. Hum Reprod Update 2001; 7:411–418. Treloar SA, Bahlo M, Ewen K, O’-Connor DT, Duffy DL, Wicking CA, Wainwright BJ, Montgomery G, Martin NG. Suggestive linkage for endometriosis found in genome-wide scan [abstr]. Am J Hum Genet 2000; 67(suppl 2): 318.

5 Research Aspects of Endometriosis Dan C. Martin University of Tennessee Memphis, Tennessee, U.S.A.

WHAT IS ENDOMETRIOSIS? One of the most difficult problems in discussing endometriosis is its interpretation. Endometriosis has many concepts, definitions, and meanings. Concerns include symptoms, signs, laboratory values, appearance, texture, and histology. The interpretations of patients or clinicians can conflict with those of pathologists or researchers. Definitions that are reasonable for a group may not be reasonable for others. Patients often discuss their endometriosis while their physicians discuss chronic pelvic pain and narcotic dependency. Several authors have discussed the treatment of endometriosis as one for chronic pelvic pain rather than to avoid chronic pelvic pain. There is a controversy regarding the degree of diagnostic certainty of endometriosis needed before treatment. These concerns make any discussion of endometriosis difficult. The most common definitions are, to some degree, based on the presence of endometrial glands and stroma with preperitoneal attachment, intraperitoneal invasion, and retroperitoneal infiltration. Definitions also include appearances such as red, dark, white, vesicular, or powder-burn lesions, chocolate cysts, raspberries or glandular appearance, peritoneal pockets, hemosi69

70

Martin

derin, blood, fibromuscular metaplasia, and adhesions. Other chapters in this text will discuss pain, infertility, and immune defects. HOW DO WE DEFINE ENDOMETRIOSIS? The diagnosis of endometriosis can be difficult because of the concepts involved. Different concepts involve attachment to the surface of the peritoneum, invasion of the peritoneum, and locations. Some authors consider peritoneum and retroperitoneal invasion as a continuum [1,2] whereas others consider them as distinct types of endometriosis at various locations [3]. It is difficult to study these various concepts, as there may be a 20-year gap between surface attachment and the findings of deep endometriosis [2]. Early concerns regarding attachment will be difficult to study in humans. In endometrial cell culture, endometrial stroma and epithelial cells attached to intact mesothelium within an hour in almost all cases. Early transmesothelial invasion involved the endometrial stroma cells. Within 18 to 24 hours of tissue culture, most of the implants did not have identifiable mesothelium beneath them. Most had intact mesothelium running up to the point of attachment. Approximately 10% of all implants had intact mesothelium at the site of attachment [4]. These types of implants appear to occur in early teens [2,5,6]. To study attachment of endometrial cells, teenage women will be the best model; however, one might face difficulty in creating and obtaining an informed consent. Brosens hypothesized that endometriosis is physiological unless recurrent bleeding in the ectopic implants causes progressive disease and symptoms. Implants of endometrial tissue in the pelvis are, to some extent, to be considered as a physiological process in menstruating women and, as such, do not constitute a disease any more than menstruation itself [7]. Because superficial endometriosis may occur in almost 100% of women, invasion may be a better endpoint for a definition of clinically relevant endometriosis [8]. Some authors proposed a retroperitoneal infiltration of more than 6 mm [2,9]. Donnez’s hypotheses of peritoneal endometriosis, ovarian endometriosis, and retroperitoneal adenomyosis are different [3]. He believes that transplantation theory can explain the occurrence of peritoneal endometriosis. Red lesions are the most active and highly vascularized lesions and are considered to be the first stage of peritoneal endometriosis. On the other hand, the retroperitoneal nodule is considered as an adenomyotic nodule related to metaplasia of Mu¨llerian remnants located in the rectovaginal septum. It is associated with the striking proliferation of the smooth muscle, creating an adenomyomatous appearance similar to that of adenomyosis in the endometrium.

Research Aspects

71

Rectovaginal Pouch Controversies involve the definition of the rectovaginal (RV) pouch. Some authors use the term ‘‘rectovaginal pouch’’ to imply any location in the pouch, rather than involvement of both the rectum and vagina [10]. Actually, the RV pouch covers part of the vagina and rectum, and its base is the upper limit of the RV septum. It is not the RV septum [11,12]. Retroperitoneal endometriosis [1] and posterior vaginal fornix endometriosis are behind the RV pouch and not within it. The Adamyan classification [13] demonstrates the retrocervical location of endometriosis (Figs. 1–3). The depth of the RV pouch extends to the middle one third of the vagina in 93% of women [14,15]. It has an average depth of 5.3 cm in nulliparous women and 5.4 cm in multiparous women, and it can descend 11% to 89% of the length of the vagina [16]. The RV septum is 2.1 cm in nulliparous women and 3.3 cm in multiparous women with normal anatomy [15]. Shortening of the RV pouch and elongation of the RV septum with RV involvement appears related to contraction of this area [12,17].

FIGURE 1 Rectovaginal Pouch of Douglas and Rectovaginal Septum. The rectovaginal (RV) pouch covers part of the vagina and rectum and its base is the upper limit of the RV septum. The RV pouch is not the RV septum. The RV septum extends from the depth of the pouch to the top of the perineal body. (Courtesy of www.danmartinmd.com.)

72

Martin

FIGURE 2 Adamyan’s staging system. Stage I: retrocervical endometriosis with no vaginal involvement. Stage II involves the vagina; stage III involves the vagina and rectum with cul-de-sac distortion, and stage IV includes cul-de-sac obliteration. (Courtesy of www.danmartinmd.com.)

Clinicians often describe the involvement of the RV septum erroneously. For example, illustrations in Cullen’s article [18] and magnetic resonance imaging (MRI) images in Chen’s publication [19] shows retrocervical RV pouch endometriosis with minimal or no involvement of the RV septum. It was described as cul-de-sac endometriosis [20] and retrocervical endometriosis [13,21,22]. Retrocervical endometriosis is a better term for RV pouch endometriosis and endometriosis in the retroperitoneal area and vaginal fornix behind or beneath the cervix with no rectal involvement. Rectovaginal endometriosis is used when there is involvement of both the vaginal and rectal areas of the pouch and may include involvement of the RV septum. These

Research Aspects

73

FIGURE 3 A, B Stage I endometriosis limited to the retrocervical area. This is confirmed by placing a probe in the vagina. The probe changes the light reflection and makes the endometriosis easier to see. (Courtesy of www.danmartinmd.com.)

74

Martin

distinctions are surgically important, as treatment of retrocervical endometriosis is less complex than treatment of endometriosis of the rectum and vagina [23–27]. Limiting extensive surgery to several centers is reasonable, as most gynecologists will encounter only a few cases of deep rectal endometriosis in their careers [17,28].

CAN WE SEE ENDOMETRIOSIS? There are many studies on recognition of endometriosis. They include both the pathologist’s confirmation of what gynecologist sees and the gynecologist’s recognition of endometriosis sent to pathology [29,30]. Confirmation is low and occurred in 64.5% at the Johns Hopkins Hospital [30], 57% to 99% in Memphis [29], 42% to 76% at Yale [31], and 45% in Phoenix [32]. Gynecologists have seen and documented 41% to 70% of the endometriosis that they remove and send to pathology [29,30]. The physician’s accuracy was directly associated with the number of resections of confirmed endometriosis [29]. A Belgian study quoted a 100% confirmation by experienced laparoscopists in their institution [33]. This wide range suggests that confirmation of lesions in clinical practice is not essential. In practice, biopsies and histology may be more valuable to rule out malignancy than to confirm endometriosis. This is particularly true with clear and opaque vesicles. The vesicles can be endometriosis, psammoma bodies, endosalpingiosis, or cancer [34]. On the other hand, endometrioid cancer also has been associated with classic appearance of endometriosis [35]. Concerns of cancer are increased in infertility patients with adhesions [36]. Histopathogical examination is, therefore, important with clear and vesicular lesions. Recognition is difficult because of the different sizes and colors [6,37– 41]. Recognition also can be a function of the distance of observation. When the lesions are observed at approximately 12 inches using 4 loop magnification, the incidence of microscopic endometriosis has been as high as 25% [42]. When observed at laparoscopy using standard techniques, this decreases to 13% [33]. This is as low as 0.5% using near-contact laparoscopy [43,44]. Near-contact laparoscopy [6] has been used to identify lesions as small as 180 A to 200 A [39,41]. Small lesions can be missed because of their size, whereas larger lesions [9,39,45,46] may be missed because of distortion by adhesions or retroperitoneal position. Deep lesions are often more palpable than visual. Palpation during menses increases the chance of detecting the nodules [9]. Deep lesions may have no surface abnormalities but often involve the bowel and ureter. These deep and difficult-to-see lesions can cause severe problems in patients. This includes hydronephrosis and rectal involvement [45].

Research Aspects

75

Visualization may be adequate for surgical purposes, but it may be inadequate for research studies. For example, drawing conclusions from studies with no confirmation in 66 endometriomas [47] is erroneous. Confirmation of ovarian endometriomas is generally reported at 86% [48,49]. Fayez’s observation that implants on the cyst lining can be ‘‘easily washed out’’ [50] were not confirmed in the pictures or photomicrographs in other articles [49]. The data do not support a call for histological documentation for a clinical diagnosis of endometriosis [32]. Instead, the problem with surgical recognition and confirmation reinforced the use of nonsurgical diagnosis. Nonsurgical diagnosis appears to be reasonable for ovarian endometriomas [51]. Further study may yield techniques adequate for diagnosis of peritoneal, retroperitoneal, bowel, and other areas [52]. IS TREATMENT NEEDED? The finding of endometriosis does not always mean that patients need treatment [53]. Asymptomatic endometriosis has been found in 61% of patients undergoing surgery for myomata, 50% of patients undergoing sterilization [54], and 25% of patients having tubal reanastomosis [39]. In addition, 93% of postmenopausal patients with endometriosis are relatively asymptomatic, and only 39 (29%) of 136 postmenopausal patients with endometriosis had disease that appeared to be clinically relevant [55]. However, the findings of asymptomatic endometriosis do not predict who will have progression of their disease and who may need subsequent treatment. Moen et al found that the odds ratio of diagnosing endometriosis increased to 4.5 at 10 years when compared with the first 5 years after delivery [56]. Even though these women were symptom-free, the prevalence of the disease increased with the length of voluntary infertility. This implies that endometriosis may be the result of infertility, whether voluntary or involuntary. With longer follow-up, some may progress and become symptomatic [9]. Cornillie [57] noted that the activities of superficial, intermediate, and deep lesions were 58%, 25%, and 68%, respectively. This is similar to the percent in-phase with the endometrium (57%, 38%, 74%). The activity of superficial lesions has been shown by several investigators [5,58–61] who suggest that these implants are important clinically. Evers [62] and Brosens [63] believe otherwise. The degree of activity shown by Cornillie [57] suggests that superficial and deep disease are more likely to be symptomatic than intermediate stages. This would be compatible with the hypothesis that intermediate lesions are commonly a residual disease. These intermediate lesions are less likely to require treatment than superficial or deep lesions. Research based on staging needs to consider this possibility.

76

Martin

IS EXCISION SUPERIOR TO COAGULATION? There are no prospective randomized trials comparing electrosurgical coagulation with sharp excision [64], but excision appears to be superior for deep lesions, lesions close to vital organs, and lesions of uncertain pathology. The ability to see the surgical planes is helpful for deep lesions. Prolonged electrosurgical coagulation might obscure the planes and increases the risk of thermal damage to the adjacent tissue. Small lesions of 2 mm or less can be adequately destroyed by coagulation (electrosurgical, thermal, or laser), vaporization, or excision. Lesions of 3 mm to 5 mm can be excised or vaporized; however, larger lesions require excision [9,45,53,65–67]. In most cases, this can be done by laparoscopy. Excision results in a lower recurrence and/or persistence rate; however, it is associated with increased risk compared with superficial coagulation or nonsurgical treatment [68]. The risks and benefits of each treatment modality should be tailored to the individual patient. CHRONIC PELVIC PAIN Chronic pelvic pain is a noncyclic pain that lasts for more than 6 months. This is in contrast to acute pain of underlying tissue damage. In chronic pelvic pain, the pain itself becomes the disease. Chronic pelvic pain is the diagnosis, and it impairs the patient’s defense mechanism and results in emotional behavioral changes. Prolonged chronic pelvic pain can result in a symptom complex called chronic pelvic pain syndrome. This pain is characteristically out of proportion to the physical findings. Patients appear depressed, have decreased family interactions, and limit their physical activities. Other body systems such as bowel and bladder do not function appropriately. All causes and associations must be diagnosed and treated. Conventional treatments yield little relief. These patients need more wide-ranging treatments than those for endometriosis only. Focusing on the pelvis often is not the answer. The objectives of treatments should be pain reduction, behavioral adjustments, and the patient’s ability to function. Research on endometriosis must take into account the possibility that the problem is chronic pelvic pain and not endometriosis. Endometriosis may have been the initiating factor or a cofactor. By the time chronic pelvic pain is present, endometriosis may play a minor role. Research studies should try to distinguish these two conditions. PRACTICAL POINT 

Endometriosis can cause pelvic pain, but not all pelvic pain is caused by endometriosis.

Research Aspects

77

REFERENCES 1.

2.

3.

4.

5. 6. 7. 8. 9.

10.

11.

12.

13. 14. 15. 16.

17.

Koninckx PR, Cornillie FJ. Infiltrating endometriosis: infiltration, retraction or adenomyosis externa? In: Martin DC, ed. Atlas of Endometriosis. London: Gower Medical Publishers, 1993:9.1–9.8. Koninckx PR, Meuleman C, Demeyere S, Lesaffre E, Cornillie FJ. Suggestive evidence that pelvic endometriosis is a progressive disease, whereas deeply infiltrating endometriosis is associated with pelvic pain. Fertil Steril 1991; 55:759–765. Donnez J, Van Langendonckt A, Casanas-Roux F, Van Gossum JP, Pirard C, Jadoul P, Squifflet J, Smets M. Current thinking on the pathogenesis of endometriosis. Gynecol Obstet Invest 2002; 54:52–62. Witz C, Dechaud H, Montoya-Rodriguez I, Thomas MR, Nair AS, Centonze VE, Schenken RS. An in vitro model to study the pathogenesis of the early endometriosis lesion. Ann N Y Acad Sci 2002; 955:296–307. Goldstein DP, De Cholnoky C, Emans SJ. Adolescent endometriosis. J Adolesc Health Care 1980; 1:37–41. Redwine DB. Age-related evolution in color appearance of endometriosis. Fertil Steril 1987; 48:1062–1063. Brosens IA. Endometriosis—A disease because it is characterized by bleeding. Am J Obstet Gynecol 1997; 176:263–267. Koninckx PR. Is mild endometriosis a condition occurring intermittently in all women? Hum Reprod 1994; 9:2202–2205. Koninckx PR, Martin DC. Deep endometriosis: a consequence of infiltration or retraction or possibly adenomyosis externa? Fertil Steril 1992; 58:924– 928. Anaf V, Simon P, El Nakadi I, Simonart H, Noel Jean-C, Buxant. Impact of resection of rectovaginal pouch of Douglas endometriotic nodule on pelvic pain and some elements of patient’s sexual life. J Am Assoc Gynecol Laparosc 2001; 8:55–60. Netter FH. Volume 2, Reproductive System. In: Oppenheimer E, ed. Ciba Collection of Medical Illustrations. Summit, NJ: Ciba Pharmaceutical Company, 1965. Vercellini P, Aimi G, Panazza S, Vicentini S, Pisacreta A, Crosignani PG. Deep endometriosis conundrum: evidence in favor of a peritoneal origin. Fertil Steril 2000; 73:1043–1046. Adamyan L. Additional international perspectives. In: Nichols DH, ed. Gynecologic and Obstetric Surgery. St. Louis: Mosby Year Book, 1993: 1167–1182. Batt RB, ed. Abdominopelvic diagnostic laparoscopy. 3d ed. Atlas of Female Surgery. St. Louis: Mosby, 1999:372–385. Kuhn R, Hollyock V. Observations on the anatomy of the rectovaginal pouch and septum. Obstet Gynecol 1982; 59:445–447. Baessler K, Schuessler B. The depth of the pouch of Douglas in nulliparous and parous women without genital prolapse and in patients with genital prolapse. Am J Obstet Gynecol 2000; 182:540–544. Martin DC, Batt RE. Retrocervical, rectovaginal pouch and rectovaginal septum endometriosis. J Am Assoc Gynecol Laparosc 2001; 8:12–17.

78

Martin

18. Cullen TS. Adenomyoma of the recto-vaginal septum. Johns Hopkins Hosp Bull 1917; 321:343–348. 19. Chen TF, Collier DSJ, Everett WG, Dixon AK. Endometriosis of the rectovaginal septum treated by anterior resection. Ann Chir Gynaecol 1989; 78:324– 326. 20. Martin DC. Laparoscopic and vaginal colpotomy for the excision of infiltrating cul-de-sac endometriosis. J Reprod Med 1988; 33:806–808. 21. Davis GD. Complications of laproscopy and hysteroscopy. In: Corfman RS, Diamond MP, DeCherney AH, eds. Endometriosis in the Cul-de-Sac: Complications of Treatment. 2d ed. Malden, Massachusetts: Blackwell Science, 1997:114–117. 22. Perry CP, Victoria MM. Occult retrocervical endometriosis. J Reprod Med 1995; 40:652–654. 23. Nezhat C, Nezhat F, Pennington E. Laparoscopic treatment of infiltrating rectosigmoid and rectovaginal septum endometriosis by the technique of videolaparoscopy and CO2 laser. Br J Obstet Gynaecol 1992; 99:664–667. 24. Possover M, Diebolder H, Plaul K, Schneider A. Laparoscopically assisted vaginal resection of rectovaginal endometriosis. Obstet Gynecol 2000; 96:304– 307. 25. Redwine DB. Laparoscopic en bloc resection for treatment of the obliterated cul-de-sac endometriosis. J Reprod Med 1992; 37:695–698. 26. Reich H, McGlynn F, Salvat J. Laparoscopic treatment of cul-de-sac obliteration secondary to retrocervical deep fibrotic endometriosis. J Reprod Med 1991; 36:516–522. 27. Smith T. Surgical treatment of endometriosis. Clin Obstet Gynaecol 1978; 5:557–570. 28. Martin DC. Research aspects of endometriosis surgery. Ann N Y Acad Sci 2002; 955:353–359. 29. Martin DC, Ahmic R, El-Zeky FA, Vander Zwaag R, Pickens MT, Cherry K. Increased histologic confirmation of endometriosis. J Gynecol Surg 1990; 6: 275–279. 30. Scott RB, TeLinde RW. External endometriosis—the scourge of the private patient. Ann Surg 1950; 131:697–720. 31. Pardanani S, Barbieri RL. The gold standard for the surgical diagnosis of endometriosis: visual findings or biopsy results? J Gynecol Techniques 1998; 4:121–124. 32. Walter AJ, Hentz JG, Magtibay PM, Cornella JL, Magrina F. Endometriosis: correlation between histologic and visual findings at laparoscopy. Am J Obstet Gynecol 2001; 184:1407–1413. 33. Nisolle M, Paindaveine B, Bourdon A. Histologic study of peritoneal endometriosis in infertile women. Fertil Steril 1990; 53:984–988. 34. Martin D, Khare V, Parker L. Clear and opaque vesicles: endometriosis, psammoma bodies, endosalpingiosis or cancer. In: Coutinho EM, Spinola P, de Moura LH, eds. Progress in the Management of Endometriosis. New York: Parthenon Publishing, 1994.

Research Aspects

79

35. Martin DC. Cancer and endometriosis: do we need to be concerned? Semin Reprod Endocrinol 1997; 15:319–324. 36. Lais CW, Williams TJ, Gaffey TA. Prevalence of ovarian cancer found at the time of infertility microsurgery. Fertil Steril 1988; 49:551–553. 37. Jansen RPS, Russell P. Nonpigmented endometriosis: clinical, laparoscopic, and pathologic definition. Am J Obstet Gynecol 1986; 155:1154–1159. 38. Karnaky KJ. Theories and known observations about hormonal treatment of endometriosis-in-situ, and endometriosis at the enzyme level. Arizona Med 1969; 26:37–41. 39. Martin DC, Hubert GD, Vander Zwaag R, El-Zeky FA. Laparoscopic appearances of peritoneal endometriosis. Fertil Steril 1989; 51:63–67. 40. Sampson JA. Benign and malignant endometrial implants in the peritoneal cavity, and their relation to certain ovarian tumors. Surg Gynecol Obstet 1924; 38:287–311. 41. Stripling MC, Martin DC, Chatman DL, Vander Zwaag R, Poston WM. Subtle appearance of pelvic endometriosis. Fertil Steril 1988; 49:427–431. 42. Murphy AA, Green WR, Bobbie D, Dela Cruz ZC, Rock JA. Unsuspected endometriosis documented by scanning electron microscopy in visually normal peritoneum. Fertil Steril 1986; 46:522–524. 43. Nezhat F, Allan CJ, Nezhat C, Martin DC. Nonvisualized endometriosis at laparoscopy. Int J Fertil 1991; 36:340–343. 44. Redwine DB, Yocum LB. A serial section study of visually normal pelvic peritoneum in patients with endometriosis. Fertil Steril 1990; 54:648–651. 45. Moore JG, Binstock MA, Growdon WA. The clinical implications of retroperitoneal endometriosis. Am J Obstet Gynecol 1988; 158:1291–1298. 46. Nesbitt RE, Rizk PT. Uterosacral ligament syndrome. Obstet Gynecol 1971; 37:730–733. 47. Fayez JA, Vogel MF. Comparison of different treatment methods of endometriomas by laparoscopy. Obstet Gynecol 1991; 78:660–665. 48. Bateman BG, Kolp LA, Mills S. Endoscopic versus laparotomy management of endometriomas. Fertil Steril 1994; 62:690–695. 49. Martin DC, Berry JD. Histology of chocolate cysts. J Gynecol Surg 1990; 6:43– 46. 50. Fayez JA. Comparison of different treatment methods of endometriomas by laparoscopy. (letter to editor). Obstet Gynecol 1992; 79:315–316. 51. Eskenazi B, Warner M, Bonsignore L, Olive D, Samuels S, Vercellini P. Validation study of nonsurgical diagnosis of endometriosis. Fertil Steril 2001; 76:929–935. 52. Eskenazi B. Challenges in studying the epidemiology of endometriosis [abstr]. In: Vogel D, ed. Endometriosis 2000. NIH: Bethesda, Maryland, 1995. 53. Martin D. Rationale for surgical treatment of endometriosis. In: Nezhat CR, Berger GS, Nezhat FR, et al., eds. Endometriosis, Advanced Management and Surgical Techniques. New York: Springer-Verlag, 1995:69–76. 54. Rawson JMR. Prevalence of endometriosis in asymptomatic women. J Reprod Med 1991; 36:513–515.

80

Martin

55. Kempers RD, Dockerty MB, Hunt AB, Symmonds RE. Significant postmenopausal endometriosis. Surg Gynecol Obstet 1960; 111:348–356. 56. Moen MH. Is a long period without childbirth a risk factor for developing endometriosis? Hum Reprod 1991; 6:1404–1407. 57. Cornillie FJ, Oosterlynck D, Lauweryns JM, Koninckx PR. Deeply infiltrating pelvic endometriosis: histology and clinical significance. Fertil Steril 1990; 53:978–983. 58. Davis GD, Thillet E, Lindemann J. Clinical characteristics of adolescent endometriosis. J Adolesc Health 1993; 14:362–368. 59. Perper MM, Nezhat F, Goldstein H, Nezhat CH, Nezhat C. Dysmenorrhea is related to the number of implants in endometriosis patients. Fertil Steril 1995; 63:500–503. 60. Vercellini P, Bocciolone L, Vendola N, Colombo A, Rognoni MT, Fedele L. Peritoneal endometriosis. Morphologic appearance in women with chronic pelvic pain. J Reprod Med 1991; 36:533–536. 61. Vernon MW, Beard JS, Graves K, Wilson EA. Classification of endometriotic implants by morphologic appearance and capacity to synthesize protaglandin F. Fertil Steril 1986; 46:801–806. 62. Evers JLH. Endometriosis does not exist; all women have endometriosis. Hum Reprod 1994; 9:2206–2209. 63. Brosens IA. Is mild endometriosis a progressive disease? Hum Reprod 1994; 9:2209–2211. 64. Farquhar C. Endometriosis. In: Barton S, ed. Clinical Evidence. 4th ed. London: BMJ Publishing Group, 2000:1058–1065. 65. Martin DC. Tissue effects of lasers. Semin Reprod Endocrinol 1991; 9:127–137. 66. Martin DC. Tissue effects of lasers and electrosurgery. In: Vitale GC, Sanfilippo JS, Perissat J, eds. Laparoscopic Surgery—An Atlas for General Surgeons. Philadelphia: J.B. Lippincott Company, 1995:65–73. 67. Martin DC, Diamond MP. Operative laparoscopy: comparison of lasers with other techniques. Curr Probl Obstet Gynecol Fertil 1986; 9:563–601. 68. Koninckx PR, Timmermans B, Meuleman C, Penninckx F. Complications of CO2 laser endoscopic excision of deep endometriosis. Hum Reprod 1996; 11: 2263–2268.

6 Animal Models for Research on Endometriosis* Thomas M. D’Hooghe and Sophie Debrock Leuven University Fertility Center Leuven, Belgium

Joseph A. Hill and Daniel C. Chai Fertility Center of New England Reading, Massachusetts, U.S.A.

Jason M. Mwenda Institute of Primate Research Nairobi, Kenya

* The content of this chapter is based on original research over the last 12 years. Funding for this research has been or is obtained from the Commission of the European Communities (DG VIII Development and DG XII Science, Research and Development); the Vlaamse Interuniversitaire Raad (VLIR, Flemish Interuniversity Council), Brussels, Belgium; the Collen Research Foundation, Faculty of Medicine and Research Council KU Leuven, University of Leuven, Belgium; the Fearing Research Laboratory Endowment, Department of Obstetrics and Gynecology, Brigham and Women’s Hospital, Harvard Medical School, Boston, USA; and the Commission for Educational Exchange between USA, Belgium, and Luxemburg. The first author has been or is sponsored as a Fulbright Fellow (1993–1995), NATO Fellow (1993–1995), and Fundamental Clinical Investigator (Fund for Scientific Research, Belgium, 1998–present).

81

82

D’Hooghe et al.

PROGRESS IN ENDOMETRIOSIS RESEARCH? Endometriosis is an important benign gynecological disease, pathologically defined by the ectopic presence of both endometrial glands and stroma, and clinically associated with pelvic pain and infertility. Our current knowledge of the pathogenesis, pathophysiology of related infertility, spontaneous evolution is still limited, although endometriosis has been described for many years. Furthermore, the diagnosis can still only be made by invasive tests (laparoscopy), and treatment either temporarily suppresses the disease (medical approach) or temporarily removes the disease (surgical excision). Recurrences of endometriosis after the cessation of medical treatment or after surgery are common, especially in cases of moderate to severe endometriosis. Several reasons contribute to this situation [1]. First, at the time of diagnosis most patients have had endometriosis for an unknown period. Therefore, it is impossible to undertake clinical research that would definitely determine the onset, etiology, or progression of the disease [2]. Second, an important reason for the lack of progress in endometriosis research is study design [3]: very few studies have been carried out so far using adequate control groups. When symptomatic patients with endometriosis are compared to women with a normal pelvis, adenomyosis, leiomyomata, adhesions, or other pelvic pathology, two factors are usually studied in a combined way: the pelvic condition (presence of endometriosis or other pathology) and symptoms (none, infertility, pain, other symptoms). To study the effect of endometriosis itself, it would be necessary to exclude patients with other possible causes of infertility or pain and compare patients with endometriosis and infertility to women with a normal pelvis and unexplained infertility, or compare patients with endometriosis and pain to women with a normal pelvis and pain [1,3]. To study the effect of endometriosis on infertility, the study group should include infertile patients with endometriosis and women with unexplained infertility, whereas the control group should include fertile women with endometriosis and with a normal pelvis (population available at interval tubal sterilization) [1,3]. Similarly, to study the effect of endometriosis on pain, the study group should include pain-experiencing patients with endometriosis and women with unexplained pain, whereas the control group should include asymptomatic and pain-free women with endometriosis and with a normal pelvis (population available at interval tubal sterilization) [1,3]. Clearly, it is hard to populate these adequately controlled studies with sufficient numbers of patients and, therefore, multicenter research is needed [1,3]. Third, endometriosis has been long considered as a surgical gynecological disease. Currently, there is a clear need for clinical management of

Animal Models for Research

83

endometriosis by multidisciplinary teams addressing medical, surgical, and psychological isues associated with endometriosis. Additionally, multidisciplinary research teams are needed to address the heterogeneous clinical, histological, immunological, endocrinological, toxicological, genetic, epidemiological, and psychosocial aspects of endometriosis [1]. Fourth, endometriosis occurs naturally in humans and nonhuman primates only. Because of ethical and practical considerations, properly controlled studies are very difficult, and invasive experiments cannot be performed in humans. It follows that there is a need for the development of a good animal model with spontaneous and induced endometriosis [1]. THE NEED FOR PRIMATE MODELS FOR THE STUDY OF ENDOMETRIOSIS The main advantage of rodents (rat and rabbit) models is the low cost relative to the monkey, but the disadvantages are numerous [3]. Both rodents lack a menstrual cycle and do not have spontaneous endometriosis. The rat is a spontaneous ovulator, but it has a shorter luteal phase than the human. The reproductive pattern of the rabbit even lacks a luteal phase. In addition to this, there is a wide phylogenetic gap between these two species and the human [3]. In both rodent models, induction is performed through the autotransplantation of endometrial fragments or uterine squares [4], which is not physiological, damages the uterus, and causes adhesions interfering with fertility. The resulting ‘‘endometriotic lesions’’ consist of cysts containing clear serous fluid in the rat, whereas vascularized hemorrhagic solid masses can also be found in the rabbit. This type of lesion in both species seems to be different from the variety of pigmented and nonpigmented lesions found in the human [5]. Recently, the use of nude mice [6] or SCID mice [7] has offered the advantage that these immunodeficient rodents do not reject xenographic human endometrial tissue, which can be introduced subcutaneously or into the peritoneal cavity, enabling the study of human endometrial-murine peritoneal interaction. However, the question remains how data from these rodent models can be extrapolated to the human situation, given the enormous species difference between mice and men [1]. Monkeys, although difficult and expensive to maintain in captivity, offer unique advantages in endometriosis research when compared to rodents, as published previously [1]. First, they are phylogenetically much closer to the human and have a comparable menstrual cycle. Second, nonhuman primates are known to be afflicted with spontaneous endometriosis: the rhesus monkey [8], the pigtailed macaque, the cynomolgus monkey, the De Brazza monkey [9], and the baboon [10,11]. It has been reported that irradiation is associated

84

D’Hooghe et al.

with an increased incidence of spontaneous endometriosis in rhesus monkeys, but only after at least 6 years [12]. In the same species, a positive correlation was found between dioxin dose and severity of endometriosis [13]. Thirdly, induced endometriosis in these monkeys results in macroscopic lesions that show similarity to the human disease [1,14–16]. The great apes (chimpanzee, gorilla, urang utang) are closest to humans in many anatomical and physiological aspects of reproduction. However, because they are all protected endangered species in the wild, they are not practical models for most studies [1]. Baboons are very intelligent animals with a well-studied and interesting social life. Hypotheses about the early evolution of human social behavior have been developed by carefully studying the behavior of baboon troops living on the grassy plains of Africa [17]. The baboon may offer clear advantages for the study of endometriosis when compared to the rhesus and cynomolgus monkey [1,3]. First, it is phylogenetically very close in that human (46 chromosomes) and baboon (42 chromosomes) karyotypes, evolving slowly, share many ancestral characters [18]. Second, detailed accounts of baboon reproductive anatomy and physiology, similar to the human, are available, including menstrual cycle characteristics, embryo implantation and fetal development [19]. Perineal skin inflation and deflation correspond with relative precision to follicular and luteal phase, offering external follow-up of the menstrual cycle without the need of serial blood samples for determination of estradiol and progesterone levels. Third, the baboon is a proven model for research in cardiovascular and endoscopic surgery, endocrinology, teratology, toxicology, testing of contraceptive agents, and placental development [1,3]. Fourth, the baboon is a continuous breeder with menstrual cycles throughout the year, also in captivity. Fifth, the baboon is a larger and stronger primate than rhesus or cynomolgus monkeys, allowing repetitive blood sampling and complex experimental surgery [20]. Sixth, specific advantages of the baboon model in gynecological research include the spontaneous presence of peritoneal fluid and the accessibility of the uterine cavity via the cervix, allowing endometrial sampling without hysterotomy [21]. For all these reasons, the baboon is considered to be a good model for research in reproduction [20]. Eighth, spontaneous endometriosis in the baboon has been found to be both minimal [11] and disseminated [10], similar to the different disease stages in women. Finally, more advanced stages of endometriosis can be induced after intrapelvic seeding of menstrual endometrium inside the pelvic cavity [21]. Experimental induction of endometriosis offers the opportunity to make serial observations in the same animal before and after induction, enabling investigators to identify factors in peripheral blood and peritoneal fluid as the consequence of endometriosis [1].

Animal Models for Research

85

Over the last 10 years, the baboon has been developed at the Institute of Primate Research as a model for the study of endometriosis and its clinical relevance has been reviewed extensively [1,3]. Prevalence of Macroscopic and Microscopic Endometriosis in Baboons Spontaneous minimal endometriosis occurs in baboons of proven fertility [3] with a prevalence of 25% and with laparoscopic appearances, pelvic localization [22], and microscopic aspects [23] similar to the human disease [24]. A significant association was observed between the prevalence of endometriosis and a previous hysterotomy [22], but the prevalence of endometriosis in baboons without previous hysterotomy was 8% in an initial study [25], comparable to the 7.5% prevalence of endometriosis in asymptomatic women undergoing tubal ligation [26]. The prevalence increased to 27% in animals that had been living in captivity for more than 2 years [27]. This trend could be explained, as in women [28], by more menstrual cycles uninterrupted by pregnancy in captive than in wild baboons, or by captivity-associated stress [3,27]. Endometriosis lesions were not often missed during systematic laparoscopic inspection [3], as demonstrated by the observation that microscopic endometriosis could be found only rarely (7%) in serial sections of large flaps of macroscopically normal peritoneum from female baboons [29]. The low prevalence of microscopic endometriosis in macroscopically normal peritoneum in both women [30] and baboons [29] suggests that the significance of microscopic endometriosis as a cause of disease recurrence after treatment remains to be established [3]. Prevalence of Spontaneous Retrograde Menstruation in Baboons In baboons, the hypothesis was tested that the incidence and recurrence of retrograde menstruation is higher in baboons with spontaneous endometriosis than in those without, [31]. Retrograde menstruation was defined by the presence of blood-(red or dark brown) stained peritoneal fluid (PF) during menses. Peritoneal fluid was 10 times more frequently blood stained during menses (62%) than during nonmenstrual phases (6%). Retrograde menstruation was observed more frequently in animals with spontaneous endometriosis (83%) than in primates with a normal pelvis (51%). Recurrence of retrograde menstruation was observed more frequently in baboons with (5/5) than in those without (3/8) spontaneous endometriosis [31]. The results of this study demonstrated that retrograde menstruation is common in baboons, with a higher prevalence and recurrence in animals with than in those without spontaneous endometriosis [3,31].

86

D’Hooghe et al.

Pathogenesis of Endometriosis: Retrograde Menstruation Experimental Retrograde Menstruation It is not known whether iatrogenically increased retrograde menstruation results in the development of endometriosis. Obstructed menstrual outflow, which possibly increases retrograde menstruation, has been associated with endometriosis in 77% of patients with functioning endometrium and patent tubes, and in up to 89% of those with hematocolpos/hematometra [32]. In female baboons, cervical occlusion was attempted to develop a primate model for the study of retrograde menstruation and endometriosis [33]. Supracervical ligation during laparotomy (n = 2) resulted in impeded uterine outflow as shown by a decreased duration of antegrade menstruation and increased retrograde menstruation, and both baboons developed endometriosis within 3 months after this procedure [3,33]. This observation is important when considering the increasing popularity of menstrual cups in the United States. These menstrual cups are removable cervical or vaginal obstructive devices that are permitted to be left in place for 12 hours during menstruation. Because many experimental and clinical data support that endometriosis is a consequence of cumulative retrograde menstruation [34, 35], the use of menstrual cups could be a risk factor for the development of this disease. Intraperitoneal Transplantation of Menstrual Endometrium In baboons, the Sampson hypothesis was tested by comparing the effect of intrapelvic injection of menstrual versus luteal endometrium on the incidence, peritoneal involvement, stage, and evolution of endometriosis [3,21]. Seventeen baboons were injected retroperitoneally with luteal (n = 6) or menstrual (n = 7) endometrium and intraperitoneally with menstrual endometrium (n = 4). Laparoscopies were performed after 2 months in all animals and after 5 and 12 months in six and five primates injected with luteal and menstrual endometrium, respectively. The peritoneal endometriosis surface area, number of implants, and incidence of typical and red subtle lesions were significantly higher after retroperitoneal injection of menstrual than of luteal endometrium. Using menstrual endometrium, intraperitoneal seeding was more successful in causing endometriosis than retroperitoneal injection. No significant changes in number or surface area of endometriotic lesions were observed in the six baboons induced with retroperitoneal injection of luteal endometrium after 5 months [3,21]. At repeat laparoscopy 12 months after intrapelvic injection of menstrual endometrium, progression was recorded in three of four regularly cycling animals, whereas regression was evident in one baboon that had become amenorrheic after induction [3,21]. In baboons with experimental endometriosis caused by intrapelvic

Animal Models for Research

87

injection of menstrual endometrium, the incidence of red lesions decreased and typical implants increased during follow-up [21]. These results indicate that intrapelvic injection of menstrual endometrium can cause peritoneal endometriosis and offers experimental evidence supporting the Sampson hypothesis [3,21]. Menstruation, Transplantation, and Inflammation In a recent study [37], we tested the hypothesis that menstruation and intrapelvic injection of endometrium for the induction of endometriosis affect inflammatory parameters in peritoneal fluid (PF) from baboons. During menstruation, the PF white blood cell (WBC) concentration was increased significantly as was the proportion of PF cells staining positive for tumor necrosis factor (TNF)-alpha, TGF-beta-1, and ICAM-1, and the PF concentration of TGF-beta-1 and interleukin (IL)-6, when compared to the follicular or luteal phase of the cycle. After intrapelvic injection of endometrium, a significant increase was also found in PF WBC concentration and in the proportion of PF cells staining positive for TNF-alpha, TGF-beta-1, CD3 and HLA-DR. Collectively, these data suggest that subclinical peritoneal inflammation occurs in baboons during menstruation and after intrapelvic injection of endometrium. Conclusion Many data from studies in women and in baboons reviewed previously [34] support the hypothesis that the process from retrograde menstruation to the establishment of endometriosis is determined by the quantity of retrograde menstruation, by the subclinical peritoneal fluid inflammation occuring during retrograde menstruation, and by local peritoneal factors such as TNF-alpha, MMPs, growth factors and potentially other substances that promote the adhesion of endometrium on the peritoneum. Future studies should quantify the amount of endometrial cells in the peritoneal fluid in women with and without endometriosis during menstruation in relation to uterine contractility and to menstrual characteristics, to determine the real role of retrograde menstruation in the pathogenesis of endometriosis. The adhesion potential of menstrual endometrial cells to pelvic peritoneum needs to be quantified in vitro in women with and without endometriosis, which may lead to the development of a a noninvasive test in the diagnosis of endometriosis. Finally, the attachment of menstrual endometrial cells to pelvic peritoneum needs to be assessed, stimulated, and blocked in the baboon, which provides an excellent in vivo culture model to test new preventative and therapeutic medical agents that may inhibit the development of endometriosis.

88

D’Hooghe et al.

Pathogenesis of Endometriosis: Immunological Aspects White Blood Cell Populations in Peritoneal Fluid and Peripheral Blood White blood cell populations in both peritoneal fluid and peripheral blood (PB) may be important in promoting ectopic endometrial growth and diminishing fertility by their activity [38], secretion of growth factors [39], and cytokines [40]. An increased concentration and total number of macrophages, lymphocytes, and their subsets has been reported in both PF and PB from women with endometriosis when compared to those without the disease, as reviewed recently [40,41]. However, it is not known whether changes in WBC subpopulations in PF and PB are the cause or the consequence of endometriosis. This aspect is difficult to study in women, because most patients with pain, infertility, and endometriosis have had the disease for some time at the moment of diagnosis [1]. In baboons, the percentages of PB WBC subsets, determined by mouse antihuman monoclonal antibodies CD2, CD4, CD8, CD11B, CD20, and CD68, are comparable to those reported in humans, showing that WBC subsets in baboons can be analyzed with commercially available monoclonal antibodies [42]. Furthermore, the immunobiology of the female reproductive tract in baboons also can be studied using other human antibodies against neutrophil elastase, CD45RA, HLA-DR, HML-1, TIA-1: CD3, IgA, IgG, IgM, J-chain, and secretory component [43]. In a previous study [42], the hypothesis was tested that PB and PF WBC populations are altered in baboons with spontaneous and induced endometriosis compared to animals without disease. In peripheral blood, the percentage of CD4+ and IL2 receptor-positive cells was increased in baboons with stage II to IV spontaneous or induced endometriosis, suggesting that alterations in PB WBC populations may be an effect of endometriosis. In PF the WBC concentration and percentages of CD68++ macrophages and CD8+ lymphocytes were only increased in baboons with spontaneous endometriosis and not in animals with induced disease, suggesting that alterations in PF WBC populations may lead to the development of endometriosis [3,42]. High-Dose Immunosuppression and Development of Endometriosis There is evidence that immune surveillance is altered in women with endometriosis [40,41], which may facilitate implantation of retrogradally shed menstrual endometrial cells. Whether immunosuppression facilitates the development of endometriosis is unknown. In baboons [44], the hypothesis was tested that immunosuppression can inhibit immune defense mechanisms proposed to prevent implantation of ectopic endometrial cells, thus allowing the development and progression of

Animal Models for Research

89

endometriosis. Thirty-two baboons (8 with normal pelvis, 10 with spontaneous endometriosis, and 14 with endometriosis induced by intraperitoneal seeding of menstrual endometrium) were studied. A daily injection was given with methylprednisolone 0.8 mg/kg and azathioprine 2 mg/kg for 3 months to 16 baboons (4 with normal pelvis, 5 with spontaneous and 7 with induced endometriosis). No treatment was given to the remaining 16 primates. The change in number and surface area (mm2) of endometriotic lesions was evaluated by laparoscopy. Immunosuppressed baboons with spontaneous endometriosis had a significantly higher number and larger surface area of endometriotic lesions than nontreated animals. One animal that had received azathioprine only developed 5-cm large bilateral endometriotic cysts. However, immunosuppressed and nontreated primates with induced endometriosis were comparable with respect to both number and surface area of implants. A transient decrease in typical lesions was noted during immunosuppression. Immunosuppression did not cause the development of endometriosis in baboons with previously documented normal pelvis [44]. The hypothesis that endometriosis is caused by immunosuppression was only partly supported by this study since immunosuppression did not affect the development of induced endometriosis nor did it cause disease in baboons with a normal pelvis [3,44]. Spontaneous Evolution of Endometriosis in Baboons The natural history of endometriosis is poorly understood. In baboons, the hypothesis was tested that spontaneous endometriosis is a progressive disease [3,45]. Serial laparoscopic observations were performed during up to 30 months in 13 baboons with spontaneous endometriosis. During each laparoscopy the pelvis was examined for the presence of endometriosis; the number, size, and type of endometriotic implants were noted on a pelvic map; the endometriosis score and stage was calculated according to the revised classification of the American Fertility Society [46]. Periods of development and regression were observed, resulting in overall disease progression [45], as indicated by a significant increase in AFS score and in both number and surface area of lesions [45,47]. Remodeling, defined by transition between typical, subtle, and suspicious implants, was observed in 23% of lesions [45]. Endometriosis did not undergo regression during the first and second trimester of pregnancy [48]. Subsequently, the incidence of spontaneous endometriosis in baboons with an initially normal pelvis was determined over a period of 32 months [49]. The cumulative incidence of minimal endometriosis (proven by histology) was 64% up to 32 months of follow-up. The 8 baboons that developed proven endometriosis were followed during a longer period of time and had undergone more serial laparoscopies than the animals that did not get the disease [50]. Remodelling of endometriotic

90

D’Hooghe et al.

implants was also observed in these baboons [47,49]. Collectively, these baboon data suggest that endometriosis is a dynamic and moderately progressive disease with periods of development and regression and with active remodeling between different types of lesions. This concept of remodeling was shown for the first time in baboons and has recently been confirmed in women with endometriosis [50]. It cannot be excluded that subclinical laparoscopy-associated inflammation was a cofactor in the development of endometriosis. Indeed, a transient increase in subclinical pelvic inflammation, characterized by a tenfold increase in PF volume, a threefold increase in WBC concentration, a tenfold increase in PF IL-6 concentration, and a twofold increase in PF TGF-beta-1 concentration, was observed 3 to 4 days, but not 30 days, after a diagnostic laparoscopy [51]. Endometriosis and Subfertility A causal relationship between endometriosis and infertility has not been definitely established. In women with moderate to severe endometriosis [46], pelvic adhesions may cause impairment of tubo-ovarian function and infertility. An inverse relationship between pregnancy rates and the degree of endometriosis has often been proposed, but this has not been substantiated in prospectively controlled fertility trials. Subfertility associated with minimal to mild endometriosis is even more controversial. In baboons, two independent prospective controlled studies [52,53] showed that animals with minimal endometriosis have a normal fertility. Subfertility was found in baboons with spontaneous or induced endometriosis AFS stages II, III, and IV. Ovarian endometriosis was not observed in baboons with either spontaneous or induced endometriosis participating in the fertility trials [52,53]. These results indicate that in baboons, minimal endometriosis is not associated with infertility and is probably not a disease but a physiological phenomenon caused by cyclic retrograde menstruation. In contrast, more extensive peritoneal endometriosis with (AFS stage III and AFS stage IV) or without (AFS stage II) adhesions is associated with subfertility, even in the absence of ovarian involvement [53]. These data, together with other data from women, strongly indicate that endometriosis is associated with subfertility, as reviewed recently [54]. Endometriosis, Subfertility, and Luteinized Unruptured Follicle Syndrome Reepithelialization of Ovulation Stigma in the Early Luteal Phase In baboons, serial laparoscopies were carried out to investigate the reepithelialization of the ovulation stigma by serial laparoscopies during the

Animal Models for Research

91

luteal phase in baboons [55]. If a fresh ovulation stigma was observed in baboons within 5 days after ovulation, it diminished in size but remained visible up to 8, 12, and 16 days after ovulation in 91%, 75%, and 50% of animals, respectively [55]. If the data obtained in baboons can be extrapolated to the clinical investigation of the infertile woman, it would appear that laparoscopies performed for the documentation of a fresh ovulation stigma should be done as early after ovulation as possible; however, they can be safely performed until 4 to 5 days after ovulation. The results from the baboon study [55] suggest that reepithelialization of the ovulation stigma takes time and explain why in clinical practice the ovulation stigma can be observed in the late luteal phase [3]. Luteinized Unruptured Follicle Syndrome The definition of luteinized unruptured follicle (LUF-) syndrome remains controversial. In most studies the LUF-syndrome has been defined as the visual absence of a fresh ovulation stigma (OS) on a recent corpus luteum (CL) during laparoscopy in the early luteal phase [3,56,57]. Using this definition, LUF-syndrome was found more frequently in infertile patients with endometriosis [58] and unexplained infertility [56] than in infertile women with tubal occlusion or male factor infertility. Other investigators, however, have failed to confirm an association between LUF-syndrome and endometriosis [57,59,60] and have reported an incidence of 47% in infertile women with ovulatory dysfunction [60] and between 33% and 47% in fertile women [59]. This clinical definition of LUF-syndrome has been criticized, because recognition of the OS may be directly related to the experience of the laparoscopist, the quality of the laparoscopic equipment, the presence of ovarian adhesions reducing the possibility to manipulate and thoroughly examine the ovaries, the optimum timing of laparoscopy during the luteal phase [3,60–62] and the fact that pregnancies have been reported in LUFcycles [60,62]. An undisputed definition of LUF-syndrome involves a histological demonstration of an entrapped oocyte within a morphological normal-appearing CL, but this definition is impractical for clinical practice. An alternative definition could be the absence of egg recovery from the female uterine tract when a recent CL without OS is found in the early luteal phase. For ethical reasons it is difficult to perform uterine flushes for egg recovery in women. However, nonhuman primates could be useful, as nonsurgical uterine flushing has been previously described [3,63]. In baboons [3], a recent CL with OS was found less frequently in baboons with endometriosis (67%) than in controls (85%) [64]. The incidence of a recent CL without OS was higher in animals with stage II–IV endometriosis (32%) than in those with stage I disease (20%) or controls (11%). The recurrence rate of a recent CL without OS was higher in primates with stage II

92

D’Hooghe et al.

endometriosis (5/6) than in animals with stage I disease (1/7) or in controls (0/ 6). The increased incidence and recurrence of a fresh CL without OS in baboons with mild endometriosis suggests that repetitive LUF-syndrome may be a cause of endometriosis-associated subfertility [64]. The egg recovery rate was lower (13%) in baboons with recent CL without OS than in animals with recent CL and OS (54%), suggesting that LUF-syndrome can be diagnosed by laparoscopy with an error rate of 13% [64]. In baboons, PF steroid levels were not significantly lower in cycles without fresh ovulation stigma when compared to cycles with a fresh ovulation stigma [65], as reported in women by some [59], but not all [66] investigators [3]. Baboon as a Preclinical Model for Prevention and Treatment of Endometriosis The baboon model for endometriosis can be used to test new drugs in the prevention or treatment of endometriosis and of endometriosis-associated subfertility [1,3]. Because induction of endometriosis is followed by moderate to severe endometriosis in most baboons [21,68], it is possible to either do prevention studies (prevent attachment of menstrual endometrium on the uterine peritoneum) or treatment studies (reduce extent of induced endometriosis after medical or surgical therapy). Treatment studies can also be done in baboons with spontaneous endometriosis, but it is very hard to have sufficient numbers of these [1,3]. Placebo-controlled randomized trials can be done to evaluate the effect of new anti-endometriosis drugs on endometriosis-associated subfertility with the possibility of complete standardization for the degree of endometriosis (after intrapelvic injection of menstrual endometrium), for the presence of ovulation (can be interpreted based on the perineal cycle), and for male factors (timed intercourse with male baboon of proven fertility, controlled by behavioral observation and poscoital test, as described before [1,3,52,53]. Intrapelvic injection of menstrual endometrium also allows the possibility to study early endometrial-peritoneal interaction at short-term intervals during in vivo culture and could give very important insight in the early development of endometriotic lesions [21,66]. This would be very important to assess the validity of the Sampson hypothesis [67]. In a recent prospective randomized study, we tested the hypothesis that r-hTBP-1 can inhibit the development of endo lesions and adhesions in the baboon, an established model for the study of endometriosis [68]. Endometriosis was induced with menstrual endometrium in 20 baboons with a normal pelvis, as reported previously [21]. In the first part of the study, the baboons were treated by subcutaneous (sc) injection [68]. Fourteen baboons were randomly assigned to treatment with either placebo (n = 4, 1 to 2 mL of PBS

Animal Models for Research

93

sc on days 0, 2, 4, 6, and 8 after induction), GnRH antagonist (n = 5, Antide 2 mg/kg sc on days 0, 3, 6, and 9 after induction) or TBP (n = 5, r-hTBP-1, 1 mg/kg sc on days 0, 2, 4, 6, and 8 after induction). In the second part of the study , endometrium was ex vivo exposed to PBS (n = 3) or TBP (n = 3) before intrapelvic seeding [68]. In the first part of the study [68], a lower endometriosis rAFS score ( P = 0.002) was observed in baboons treated with TBP (all with endo rAFS score 4) or with Antide (all with endo rAFS score 4) than in primates treated with PBS (median endo rAFS score 13.5, range 6–38). Endo related adhesions were completely absent in all baboons treated with TBP or Antide. Consequently, the number of baboons with endometriosis rAFS stage II, stage III, or stage IV was lower in baboons treated with TBP (all endo rAFS stage I, P = 0.008) or in primates treated with Antide (all endometriosis rAFS stage I, P = 0.008) than in animals treated with PBS (two with endometriosis rAFS stage II, two treated with PBS [472, range 98–1468]. In the second part of this study [68], the median surface area (mm2) of endometriosis lesions was lower ( P = 0.05) in TBP-treated baboons [25, range 14–49) than in PBS treated controls [86, range 58–95). Furthermore, less severe endometriosis was observed in the three TBP-treated baboons (all with endo rAFS stage I) than in the three PBS-treated baboons (n = two with endo rAFS stage IV, n = 1 with endo rAFS stage I). These data in baboons showed that r-hTBP-1 inhibited the development of endometriotic lesions and associated adhesions, and thus could be effective in the prevention/treatment of human endometriosis.

SUMMARY The baboon is the best animal model for the study of the pathogenesis and spontaneous evolution of endometriosis, for the evaluation of endometriosisassociated subfertility, and for preclinical evaluation of new methods to prevent the onset of endometriosis or to cure established endometriosis.

PRACTICAL POINT 

Baboon is the best animal model for studying endometriosis.

REFERENCES 1. 2.

Haney AF. Endometriosis: Pathogenesis and pathophysiology. In: Wilson EA, ed. Endometriosis. New York: AR liss, Inc. 1987:23–51. D’Hooghe TM. Clinical relevance of the baboon as a model for the study of endometriosis. Fertil Steril 1997; 68:613–625.

94

D’Hooghe et al.

3.

D’Hooghe TM. Clinical relevance of the baboon as a model for the study of endometriosis. Fertil Steril 1997; 68:613–625. Vernon MW, Wilson EA. Studies on the surgical induction of endometriosis in the rat. Fertil Steril 1985; 44:684–694. Jansen RPS, Russell P. Nonpigmented endometriosis: Clinical, laparoscopic and pathologic definition. Am J Obstet Gynecol 1986; 155:1160–1163. Bergqvist A, Jeppson S, Kullander S, Ljungberg O. Human uterine endometrium and endometriotic tissue transplanted into nude mice: Morphologic effects of various steroid hormones. Am J Pathol 1985; 121:337–341. Awwad JT, Sayegh RA, Tao XJ. The SCID mouse: An experimental model for endometriosis. Hum Reprod 1999; 14:3107–3111. McCann TO, Myers RE. Endometriosis in rhesus monkeys. Am J Obstet Gynecol 1970; 106:516–523. Binhazim AA, Tarara RP, Suleman MA. Spontaneous external endometriosis in a DeBrazza’s monkey. J Comp Pathol 1989; 101:471–474. Folse DS, Stout LC. Endometriosis in a baboon. Lab Anim Sci 1978; 28:217– 219. Merrill JA. Spontaneous endometriosis in the Kenya baboon. Am J Obstet Gynecol 1968; 101:569–570. Wood DH. Long-term mortality and cancer-risk in irradiated rhesus monkeys. Rad Research 1991; 126:132–140. Rier SE, Turner WE, Martin DC, Morris R, Lucier GW, Clark GC. Serum levels of TCDD and dioxin-like chemicals in rhesus monkeys chronically exposed to dioxin: Correlation of increased serum PCB levels with endometriosis. Toxicol Sci 2001; 59:147–159. Te Linde RW, Scott RB. Experimental endometriosis. Am J Obstet Gynecol 1950; 60:1147–1173. Mann DR, Collins DC, Smith MM, Kessler MJ, Gould KG. Treatment of endometriosis in Rhesus monkeys: Effectiveness of a gonadotropin-releasing hormone agonist compared to treatment with a progestational steroid. J Clin Endocrin Metab 1986; 63:1277–1283. Schenken RS, Asch RH, Williams RF, Hodgen GD. Etiology of infertility in monkeys with endometriois: Luteinized unruptured follicles, luteal phase defects, pelvic adhesions, and spontaneous abortions. Fertil Steril 1984; 41:122– 130. Strum SC. Almost human: A journey into the world of baboons. London: Elm Tree Books, 1987. Marks J. Evolutionary tempo and phylogenetic inference based on primate karyotypes. Cytogenet Cell Genet 1982; 34:261–264. Hendrickx AG. Reproduction. Methods. In: Hendrickx AG, ed. Embryology of the Baboon. Chicago and London: The University of Chicago Press, 1971:1– 44. Isahakia MA, Bambra CS. Primate models for research in reproduction. In: Gamete Interaction: Prospects for Immunocontraception. New York: WileyLiss, Inc., 1990: 487–500. D’Hooghe TM, Bambra CS, Raeymaekers BM, De Jonge I, Lauweryns JM,

4. 5. 6.

7. 8. 9. 10. 11. 12. 13.

14. 15.

16.

17. 18. 19.

20.

21.

Animal Models for Research

22.

23.

24. 25. 26. 27.

28. 29.

30. 31.

32. 33.

34. 35.

36. 37.

38.

95

Koninckx PR. Intrapelvic injection of menstrual endometrium causes endometriosis in baboons (Papio cynocephalus, Papio anubis). Am J Obstet Gynecol 1995; 173:125–134. D’Hooghe TM, Bambra CS, Cornillie FJ, Isahakia M, Koninckx PR. Prevalence and laparoscopic appearance of spontaneous endometriosis in the baboon (Papio anubis, Papio cynocephalus). Biol Reprod 1991; 45:411–416. Cornillie FJ, D’Hooghe TM, Bambra CS, Lauweryns JM, Isahakia M, Koninckx PR. Morphological characteristics of spontaneous endometriosis in the baboon (Papio anubis, Papio cynocephalus). Gynecol Obstet Invest 1992; 34:225–228. Donnez J, Nisolle M, Casanas-Roux F. Three-dimensional architectures of peritoneal endometriosis. Fertil Steril 1992; 57:980–983. D’Hooghe TM, Debrock S. Future directions in endometriosis research. Obstet Gynecol Clin North America March 2003. In press. Kirshon B, Poindexter AN, Fast J. Endometriosis in multiparous women. J Reprod Med 1989:215–217. D’Hooghe TM, Bambra CS, DeJonge I, Lauweryns JM, Koninckx PR. The prevalence of spontaneous endometriosis in the baboon increases with the time spent in captivity. Acta Obstet Gynecol Scand 1996; 75:98–101. Moen MH. Endometriosis in women at interval sterilization. Acta Obstet Gynecol Scand 1987; 66:451–454. D’Hooghe TM, Bambra CS, De Jonge I, Machai PN, Korir R, Koninckx PR. A serial section study of visually normal posterior pelvic peritoneum from baboons with and without spontaneous endometriosis. Fertil Steril 1995; 63:1322– 1325. Redwine DB, Yocom LB. A serial section study of visually normal pelvic peritoneum in patients with endometriosis. Fertil Steril 1990; 54:648–651. D’Hooghe TM, Bambra CS, Raeymaekers BM, Koninckx PR. Increased incidence and recurrence of retrograde menstruation in baboons with spontaneous endometriosis. Hum Reprod 1996; 11:2022–2025. Olive DL, Henderson DY. Endometriosis and Mullerian anomalies. Obstet Gynecol 1987; 69:412–415. D’Hooghe TM, Bambra CS, Suleman MA, Dunselman GA, Evers HL, Koninckx PR. Development of a model of retrograde menstruation in baboons (Papio anubis). Fertil Steril 1994; 62:635–638. D’Hooghe TM, Debrock S. Endometriosis, retrograde menstruation and peritoneal inflammation. Hum Reprod Update 2002; 8:84–88. D’Hooghe TM, Yankowitz J. Endometriosis, tampons and orgasm during menstruation: Science, press and patient organizations. Gynecol Obstet Invest 2003; 54:61–62. D’Hooghe TM, Bambra CS, Hill JA. A very sticky riddle. Letter-to-the-editor. Fertil Steril 1997; 67:182–183. D’Hooghe TM, Bambra CS, Ling Xiao, Hill JA. The effect of menstruation and intrapelvic injection of endometrium on peritoneal fluid parameters in the baboon. Am J Obstet Gynecol 2001; 184:917–925. Kauma S, Clark MR, White C, Halme J. Production of fibronectin by

96

39. 40.

41.

42.

43. 44.

45.

46. 47.

48.

49.

50.

51. 52.

53.

D’Hooghe et al. peritoneal macrophages and concentration of fibrobectin in peritoneal fluid from patients with or without endometriosis. Obstet Gynecol 1988; 72:13–18. Halme J. Release of tumor necrosis factor-alpha by human peritoneal macrophages in vivo and in vitro. Am J Obstet Gynecol 1989; 161:1718–1725. D’Hooghe TM, Hill JA. Immunobiology of endometriosis. In: Bronson RA, Alexander NJ, Anderson DJ, Branch DW, Kutteh WH, eds. Immunology of Reproduction. Cambridge: Blackwell Science, 1996:322–358. D’Hooghe TM, Hill JA. Is endometriosis an autoimmunological disease? In: Kurpisz M, Fernandez N, eds. Immunology of reproduction. Oxford University Press, 1995:133–162. D’Hooghe TM, Bambra CS, Hill JA, Koninckx PR. Effect on endometriosis and the menstrual cycle on white blood cell subpopulations in the peripheral blood and peritoneal of baboons. Hum Reprod 1996; 11:1736–1740. D’Hooghe TM, Pudney J, Alves L, Peixe K, Hill JA. Immunobiology of the female reproductive tract in the baboon. Am J Primatol 2001; 53:47–54. D’Hooghe TM, Bambra CS, Raeymaekers BM, Hill JA, Koninckx PR. Immunosuppression can increase progression of spontaneous endometriosis in baboons. Fertil Steril 1995; 64:172–178. D’Hooghe TM, Bambra CS, Raeymaekers BM, Koninckx PR. Serial laparoscopies over 30 months show that endometriosis is a progressive disease in captive baboons (Papio anubis Papio cynocephalus). Fertil Steril 1996; 65:645–649. American Fertility Society. Revised American Fertility Society Classification of Endometriosis. Fertil Steril 1985; 43:351–352. D’Hooghe TM, Bambra CS, Isahakia M, Koninckx PR. Evolution of minimal endometriosis in the baboon (Papio anubis, Papio cynocephalus) over a 12month period. Fertil Steril 1992; 58:409–412. D’Hooghe TM, Bambra CS, De Jonge I, Lauweryns JM, Raeymaekers BM, Koninckx PR. Pregnancy does not affect endometriosis in baboons (Papio anubis, Papio cynocephalus). Arch Gynecol Obstet 1997; 261:15–19. D’Hooghe TM, Bambra CS, Raeymaekers BM, Koninckx PR. The cumulative incidence rate of endometriosis in baboons (Papio anubis, Papio cynocephalus) with an initially normal pelvis is 70% after 30 months. Obstet Gynecol 1996; 88:462–466. Wiegerinck MAHM, Van Dop PA, Brosens IA. The staging of peritoneal endometriosis by the type of active lesion in addition to the revised American Fertility Society classification. Fertil Steril 1993; 60:461–464. D’Hooghe TM, Bambra CS, Raeymaekers BM, Hill JA. Pelvic inflammation induced by diagnostic laparoscopy in baboons. Fertil Steril 1999; 72:1134–1141. D’Hooghe TM, Bambra CS, Koninckx PR. Cycle fecundity in baboons of proven fertility with minimal endometriosis. Gynecol Obstet Invest 1994; 37:63– 65. D’Hooghe TM, Bambra CS, Raeymaekers BM, Riday AM, Suleman MA, Koninckx PR. A prospective controlled study over 2 years shows a normal monthly fertility rate (MFR) in baboons with stage I endometriosis and a decreased MFR in primates with stage II and stage III–IV disease. Fertil Steril 1996; 66:809–813.

Animal Models for Research 54.

55.

56. 57. 58.

59.

60. 61.

62. 63. 64.

65.

66.

67. 68.

97

D’Hooghe TM, Debrock S, Hill JA, Meuleman C. Endometriosis and subfertility: Is the relationship resolved? Sem Reprod Med March 2003. In press. D’Hooghe TM, Bambra CS, Raeymaekers BM, Koninckx PR. Disappearance of the ovulation stigma in baboons (Papio anubis, Papio cynocephalus) as determined by serial laparoscopies during the luteal phase. Fertil Steril 1996; 65:1219– 1223. Koninckx PR, Heyns WJ, Corveleyn PA, Brosens IA. Delayed luteinization as a cause of infertility. Fertil Steril 1978; 29:266–269. Marik J, Hulka J. Luteinized unruptured follicle syndrome: A subtle cause of infertility. Fertil Steril 1978; 29:270–274. Brosens IA, Koninckx PR, Corveleyn PA. A study of plasma progesterone, oestradiol-17beta, prolactin and LH levels and of the luteal phase appearances of the ovaries in patients with endometriosis and infertility. Br J Obstet Gynaecol 1978; 85:246–250. Dhont M, Serreyn R, Duvivier P, Vanluchene E, DeBoever J, Vandekerckhove D. Ovulation and concentration of progesterone and estradiol in peritoneal fluid: Relation with fertility and endometriosis. Fertil Steril 1984; 41:872–877. Dmowski WP, Rao R, Scommegna. The luteinized unruptured follicle syndrome and endometriosis. Fertil Steril 1980; 33:30–34. Scheenjes E, te Velde ER, Kremer J. Inspection of the ovaries and steroids in serum and peritoneal fluid at various time intervals after ovulation in fertile women: Implications for the luteinzed unrupured follicle syndrome. Fertil Steril 1990; 54:38–41. Portuondo JA, Augustin A, Herran C, Echanojauregui AD. The corpus luteum in infertile patients found during laparoscopy. Fertil Steril 1981; 36:37–40. Pope CE, Pope VZ, Beck LR. Nonsurgical recovery of uterine embryos in the baboon. Biol Reprod 1980; 23:657–662. D’Hooghe TM, Bambra CS, Raeymaekers BM, Koninckx PR. Increased incidence and recurrence of recent corpus luteum without ovulation stigma (luteinized unruptured follicle-syndrome?) in baboons (Papio anubis, Papio cynocephalus) with endometriosis. J Soc Gynecol Invest 1996; 3:140–144. D’Hooghe TM, Bambra CS, Koninckx PR. Peritoneal fluid volume and steroid concentration in baboons with and without endometriosis. Arch Gynecol Obstet 1995; 256:17–22. Koninckx PR, Ide P, Vandenbroucke W, Brosens IA. New aspects of the pathophysiology of endometriosis and associated fertility. J Reprod Med 1980; 24:257–260. Sampson JA. Peritoneal endometriosis due to menstrual dissemination of endometrial tissue into the pelvic cavity. Am J Obstet Gynecol 1927; 14:422–469. D’Hooghe TM, Nugent N, Cuneo S, Chai D, Deer F, Debrock S, Mwenda J. Recombinant human TNF binding protein (r-hTBP-1) inhibits the development of endometriosis in baboons: A prospective, randomized, placebo- and drug-controlled study. General Program Prize Winning Paper and oral presentation at the Annual Meeting of the American Society for Reproductive Medicine, Orlando, USA, October 22nd–24th 2001. Fertil Steril 2001; 76:O–2, S–1.

7 Immunology of Endometriosis and Immunotherapy Emre Seli, Murat Berkkanoglu, and Aydin Arici Yale University School of Medicine New Haven, Connecticut, U.S.A.

Neil G. Mahutte Dartmouth College School of Medicine Lebanon, New Hampshire, U.S.A.

INTRODUCTION Endometriosis is a common gynecologic disorder characterized by the presence of endometrial tissue outside the uterus. Various theories have been proposed to explain the pathogenesis of this disease. No single theory can explain all cases of endometriosis, but the most commonly accepted is Sampson’s theory of retrograde menstruation [1]. Sampson suggested that endometriotic implants reach their most common site of implantation, the peritoneal cavity, by traveling through the fallopian tubes during menstrual shedding. Sampson’s theory is supported by the finding of viable endometrial tissue in the peritoneal fluid that is capable of growth [2–5] and the dependent anatomical distribution of endometriotic implants [6]. 99

100

Seli et al.

Retrograde menstruation occurs in 76% to 90% of women [7,8]. The much lower prevalence of endometriosis, 6.2% to 8.2% [9,10] suggests that additional factors are involved in determining the susceptibility to endometriosis. Aberrant immune responses resulting in inadequate removal of refluxed menstrual debris have been proposed as possible causative factors in the development of endometriosis. Endometriosis is associated with changes in both cell-mediated and humoral immunity. The peritoneal fluid of women with endometriosis contains increased numbers of immune cells; however, these, at least in some respects, appear to facilitate rather than inhibit the development of endometriosis. Leukocytes that would be expected to clear endometrial cells from the peritoneal cavity may enhance endometrial proliferation by secreting growth factors and cytokines. Although it is unclear whether these immunological alterations induce endometriosis or are a consequence of its presence, they seem to play an important role in allowing endometriosis implants to persist and progress. The pelvic inflammation in women with endometriosis also may contribute to pain and infertility. Secretory products of immune cells in the peritoneal fluid such as cytokines and prostaglandins may trigger dysmenorrhea, dyspareunia or pelvic pain. Inflammation may also lead to adhesion formation and scarring that disrupt normal fallopian tube functioning. Similarly, the inflammatory environment may impair folliculogenesis, fertilization, and implantation. In this chapter, we will summarize the alterations in the immune parameters of women with endometriosis and describe how they may play a role in the pathogenesis of endometriosis. We will also discuss the immunological consequences of common hormonal treatments and highlight the potential of immunotherapy as a novel treatment for women with endometriosis.

IMMUNE MILIEU IN WOMEN WITH ENDOMETRIOSIS: IMPLICATIONS ON PATHOGENESIS Five critical steps have been postulated to explain the development of peritoneal endometriosis (Table 1). The two initial steps are attachment of refluxed endometrial cells to the peritoneal surface and the invasion of these cells into the mesothelium. After this, angiogenesis around the nascent implants, endometrial cellular proliferation, and recruitment of inflammatory cells subservient to the implants become important. The endometriotic tissue influences each of these steps, but they are also highly influenced by immune cells and inflammatory cytokines.

Immunology and Immunotherapy

101

TABLE 1 Critical Steps in the Pathogenesis of Peritoneal Endometriotic Implants Attachment of endometrial cells to the peritoneal surface Invasion of endometrial cells into the mesothelium Angiogenesis around nascent endometriotic implants Endometrial cell proliferation Recruitment of inflammatory cells subservient to the implant

Cellular Immune Response Macrophages Macrophages are the most abundant nucleated cells found in peritoneal fluid [11]. The number and activity of peritoneal fluid macrophages are increased in women with endometriosis [12–17], although the extent of endometriosis does not appear to correlate with the macrophage count [13]. The increased number and activity of peritoneal fluid macrophages in women with endometriosis would be expected to facilitate the clearance of ectopic endometrial cells and slow down or inhibit the development of endometriosis; however, this may not always be the case. The release of cytokines and growth factors from peritoneal macrophages [18] combined with impaired scavenger function may actually promote ectopic endometrial cell growth. One of the vital functions of macrophages is scavenger phagocytosis. Phagocytosis allows macrophages to eliminate invading foreign material as well as cellular debris and apoptotic cells [19,20]. A variety of surface receptors mediate this activity [19–22]. A number of substances regulate these receptors, including cytokines and growth factors [23–25]. Abnormal levels of these cytokines may impair scavenger function [26]. Additionally, the interaction between macrophages and extracellular matrix components may alter macrophage scavenger receptor function. When macrophages are not adherent to the extracellular matrix, they do not express type A scavenger receptors [27]. Hence, in comparison with tissue macrophages, peritoneal fluid macrophages may be less competent scavengers despite their differentiated status. Secretory products of peritoneal macrophages and circulating monocytes of women with endometriosis seem to promote growth and maintenance of ectopic endometrium [18]. Peritoneal fluid from women with endometriosis stimulates proliferation of cultured endometrial stromal cells [28]. Moreover, peripheral blood monocytes obtained from women with endometriosis enhance proliferation of cocultured autologous endometrial cells, whereas

102

Seli et al.

monocytes from fertile women show the opposite effect, suppressing endometrial cell proliferation [29]. Natural Killer Cells Natural killer (NK) cells are an important component of the innate immune system. They participate in host defenses against infections [30] and have antitumor effects [31]. Natural killer cells recognize their targets in one of two ways. Like many other immune cells, they have receptors that bind immunoglobulin G (IgG) and they kill IgG–coated target cells by antibody-dependent cellular cytotoxicity. A second recognition system characteristic of NK cells involves killer-activating receptors and killer-inhibitory receptors (KIR) [32]. When killer–activating receptors are occupied, NK cells show cytotoxic activity. Conversely when KIRs are stimulated, they send inhibitory signals that override the kill signal and suppress cytotoxic activity. It has been suggested that a decrease in NK cell number or activity may lead to impaired clearance of regurgitated endometrial cells from the peritoneal cavity. Initial studies investigating NK cell numbers in peritoneal fluid of women with endometriosis reported conflicting results. While some found a decrease in peritoneal NK cells [33], others reported no change [34] or an increase [17]. On the other hand, studies investigating NK cell activity in women with endometriosis have consistently showed a decrease in cytotoxic activity. Natural killer cells from both the peritoneal fluid and the peripheral blood of women with endometriosis have decreased cytotoxic activity against autologous and heterologous endometrium [34,35]. The decrease in NK cell cytotoxicity in the peritoneal fluid is more pronounced in moderate and severe stages of endometriosis [36]. Multiple mechanisms seem to be involved in the suppression of NK cell activity in women with endometriosis. Both sera [37] and peritoneal fluid [38,39] from women with endometriosis suppress NK cell cytotoxicity [37,38], suggesting that soluble factors are involved. Recently, Wu et al found that peritoneal NK cells of women with endometriosis have higher KIR expression [40]. More recently, Maeda et al identified KIR2DL1 as the subtype of KIR overexpressed in peripheral and peritoneal NK cells of women with endometriosis [41]. Because expression of KIR is related to NK cell activity, increased KIR expression in NK cells in women with endometriosis may contribute to the suppression of peritoneal NK cell activity. Lymphocytes Lymphocytes constitute the main cell type that mediates acquired immune response. B and T lymphocytes account for humoral and cellular acquired immune responses, respectively. There is significant cross-talk between the two systems. B cells secrete immunoglobulins, the antigen-specific antibodies responsible for eliminating extracellular microorganisms. T cells are differ-

Immunology and Immunotherapy

103

entiated into two major subclasses in the thymus: helper T cells assist B cells in the production of antibodies, whereas suppressor/cytotoxic T cells eradicate intracellular pathogens by activating macrophages and killing virally infected cells. More than 20 years ago, Dmowski et al showed that T cell-mediated immunity to autologous endometrium is suppressed in rhesus monkeys with spontaneous endometriosis [42]. Similarly, cytotoxic activity of peripheral blood lymphocytes against autologous endometrial cells is decreased in women with endometriosis [43]. These observations have led to speculation that endometriosis develops as a result of impaired cell-mediated immune response that is believed to be critical in clearing ectopic endometrial cells from the peritoneal cavity [44]. Quantitative analysis of T cells and their subgroups in the peripheral blood and peritoneal fluid of women with endometriosis followed these studies. Total lymphocyte numbers and helper/suppressor ratio in the peripheral blood are not markedly affected in women with endometriosis [17, 45], although one study reported an increase in helper/suppressor ratio [46]. Similarly, there is no change in total lymphocyte content or helper/suppressor ratios in the eutopic endometrium of women with endometriosis compared with eutopic endometrium from normal controls [47]. On the other hand, an increase in peritoneal fluid T lymphocyte numbers is observed in women with endometriosis [17,44]. Both helper and suppressor subtypes seem to be increased in numbers [17,44]. The number of T lymphocytes scattered in the stroma of ectopic endometrium as detected by immunohistochemistry is also elevated compared to proliferative and secretory eutopic endometrium from women without endometriosis [48]. An increase in both helper and suppressor subtypes contributes to the observed increase in T cell content of implants while their relative ratio seems to be unchanged [48]. Overall, it is still unclear if the alterations in the number, subgroup ratio, or function of lymphocytes play a role in the development or symptomatology of endometriosis. Humoral Immune Response Autoantibodies In addition to alterations in cell-mediated immunity, considerable evidence indicates that endometriosis is associated with polyclonal B cell activation and an increased incidence of autoantibodies. An increase in B-cell activity in women with endometriosis was recognized two decades ago. Weed et al demonstrated IgG and complement deposits in the eutopic endometrium of women with endometriosis associated with a reduction in the serum total complement level [49]. Mathur et al were the first to report an abnormal incidence of autoantibodies in women with endometriosis, and they presented

104

Seli et al.

the first evidence that patients with endometriosis and patients with established autoimmune diseases may exhibit similar autoantibody profiles [50]. They detected IgG and IgA autoantibodies to ovarian and endometrial cells in sera and in cervical and vaginal secretions of women with endometriosis [50]. Gleicher et al reported the presence of IgG, IgM, and IgA autoantibodies directed against cell-derived antigens such as phospholipids, histones, and polynucleotides in women with endometriosis and argued that ectopic endometrium might induce an autoimmune response and contribute to infertility associated with endometriosis [51]. An increased risk of pregnancy loss has been associated with the presence of abnormal non-organ-specific [52] and organ-specific autoantibodies [53]. The association between autoantibody abnormalities and endometriosis could play a role in endometriosisrelated infertility and recurrent pregnancy loss. In women with endometriosis undergoing IVF, the presence of autoantibodies may be associated with significantly lower pregnancy rates. In one study, treatment of women with endometriosis who had autoantibodies with corticosteroids resulted in a significant increase in pregnancy rates [54]. However, although autoantibodies may play a role in some cases of endometriosis-associated infertility, the relative importance of autoimmunity in the pathogenesis and pathophysiology of the disease is controversial. Cytokines and Growth Factors Cytokines are a large family of low-molecular-weight soluble proteins involved in regulating cellular activity. They act as paracrine and autocrine messengers both within the immune system and between the immune system and other systems of the body. Their action is mediated by specific cytokine receptors. Cytokines and growth factors play an important role in regulating chemotaxis, mitosis, angiogenesis, and differentiation (Table 2). Whereas impaired cellular immune response may act as a permissive factor in the survival of ectopic endometrial cells, cytokines and growth factors may actively promote implantation, proliferation, and angiogenesis. Moreover certain cytokines are also implicated in the attachment of endometrial cells to the peritoneal surface and invasion of these cells into the mesothelium. Interleukin-1. Interleukin-1 (IL-1) is a cytokine that plays an important role in inflammation and immune response. It is secreted mainly by activated monocytes and macrophages as well as T- and B-cells, and NK cells. It affects the activation of T cells and the differentiation of B cells. There are two distinct molecular forms of IL-1 (IL-1a and IL-1h) derived from two different genes. An IL-1 receptor antagonist (IL-1ra) has also been defined and acts as an endogenous receptor inhibitor.

Immunology and Immunotherapy

105

TABLE 2 Source and Function of Key Cytokines Elevated in the Peritoneal Fluid of Women with Endometriosis Cytokine IL-1

Source Activated macrophages Endometriosis implants

IL-8

MCP-1 RANTES

Mesothelial cells Macrophages Endometrial cells Fibroblasts and endothelial cells Macrophages Endometrial cells Endometrial stromal cells Hematopoietic cells

TNF-a

Leukocytes: . Macrophages . Neutrophils . Activated lymphocytes . NK cells Endometrial Cells

VEGF

Endometriotic implants Activated macrophages

Function Induces endometriotic stromal cells to secrete IL-6 and VEGF Increases IL-8, MCP-1 & sICAM-1 Activates T-lymphocytes Differentiates B-lymphocytes Stimulates adhesion of endometrial cells to fibronectin Stimulates angiogenesis Stimulates endometrial proliferation Chemoattractant and anti-apoptotic factor for neutrophils Recruits and activates macrophages Chemoattractant for: . monocytes . memory T-cells Promotes adherence of cultured stromal cells to mesothelial cells Increases endometrial epithelial cell prostaglandin production Initiates cytokine cascade and the inflammatory response Initiates cytokine cascade and the inflammatory response Kills certain cell lines Stimulates Angiogenesis Monocyte chemoattractant

Interleukin-1 has been isolated from the peritoneal fluid of women with endometriosis. Most researchers found increased levels in such women [55– 57], although others found no difference [58]. Endometrial stromal cells isolated from endometriotic lesions show two- to threefold increases in IL-1 receptor expression compared with those isolated from normal endometrium. Hence upregulation of IL-1 receptor expression may be another mechanism by which endometriotic tissue becomes more responsive to IL-1. Interleukin-1 may promote the development of endometriosis by upregulating the expression of other cytokines and growth factors. For example, IL-1h induces the expression of angiogenic factors such as vascular endothelial growth factor (VEGF) and IL-6 in endometriotic stromal cells [59]. Hence

106

Seli et al.

IL-1h may promote angiogenesis in endometriotic lesions. IL-1h also increases soluble intercellular adhesion molecule-1 (sICAM-1) shedding from endometrial cells and may interfere with peritoneal immune surveillance [60]. The effects of IL-1 on early reproductive events have been investigated in vitro [56,61,62]. Interleukin-1 inhibits mouse embryo development in vitro but only at very high concentrations. Interleukin-1 also impairs the oocyte penetrating capacity of the sperm in both the hamster and the human [63], whereas it does not appear to affect sperm motion parameters significantly [61]. Interleukin-8. Interleukin-8 is a chemokine that induces chemotaxis of neutrophils and T lymphocytes. Interleukin-8 is also a potent angiogenic factor. Potential sources of IL-8 include mesothelial cells, macrophages, endometrial cells, endothelial cells, and fibroblasts [64,65]. Normal eutopic endometrium expresses IL-8. Endometrial IL-8 expression is highest in the late secretory and early proliferative phase, around the time of retrograde menstruation [66]. Interleukin-8 is elevated in the peritoneal fluid of women with endometriosis and peritoneal fluid IL-8 levels correlate with the severity of disease [65,67]. Interleukin-8 is also expressed in ectopic endometrium in both stromal and epithelial compartments independent of menstrual cycle phase [68]. Moreover, IL-8 is expressed in cultured mesothelial cells and its expression is upregulated by IL-1h and tumor necrosis factor-alpha (TNF-a), suggesting that under inflammatory conditions mesothelium may be an important source of IL-8 [65]. Interleukin-8 seems to have multiple effects that would promote the implantation and growth of ectopic endometrium in the peritoneal cavity. Interleukin-8 stimulates the adhesion of endometrial stromal cells to fibronectin in a concentration-dependent manner [69]. Thus, IL-8 may facilitate the initial attachment of endometrial cells to the peritoneal surface. Moreover, in vitro attachment of endometrial stromal cells to extracellular matrix upregulates IL-8 gene expression [70]. After the initial attachment to the mesothelium, endometriotic cells invade the extracellular matrix of the peritoneum [71]. Secretion of metalloproteinases plays a key role in endometrial cell invasion of the extracellular matrix. In vitro metalloproteinases activity is upregulated when endometrial cells are exposed to IL-8, especially when those cells are also grown in the presence of extracellular matrix proteins [72]. Finally, IL-8 also increases endometrial stromal cell proliferation in vitro in a concentration-dependent manner, whereas anti-IL-8 antibody inhibits endometrial stromal proliferation [73]. Monocyte Chemotactic Protein-1. Endometriosis is associated with elevated numbers of activated macrophages in the peritoneal cavity. The products of these macrophages are thought to affect the growth of endo-

Immunology and Immunotherapy

107

metriotic implants. A possible mediator of macrophage recruitment into the peritoneal cavity in women with endometriosis is monocyte chemotactic protein-1 (MCP-1). A monocyte chemoattractant and activating cytokine, MCP-1 is produced by monocytes, T lymphocytes, fibroblasts, vascular smooth muscle, and endothelial cells as well as endometrial cells. Its production is upregulated by other cytokines such as interferon (IFN)-g, IL-1, and TNF-a. Concentrations of MCP-1 are elevated in the peritoneal fluid of women with endometriosis. Moreover, its levels correlate with the severity of the disease and tend to decrease with medical treatment [74,75]. Elevated MCP-1 levels in the peritoneal environment of women with endometriosis may be caused by increased MCP-1 expression in eutopic and ectopic endometrial cells. Monocyte chemotactic protein-1 is expressed in both eutopic and ectopic endometrium [76,77]. However, MCP-1 expression is increased in eutopic endometrium of women with endometriosis [76] and in ectopic endometrium [77]. Interleukin-1h stimulation upregulates MCP-1 expression in eutopic endometrial epithelial cells from women with endometriosis [78] as well as ectopic endometrial cells in culture [79]. Estrogen pretreatment markedly increases IL-1h induced MCP-1 expression in both cell types [77,80]. On the other hand, estrogen pretreatment does not cause a significant increase in MCP-1 expression in epithelial cell cultures from normal women or in stromal cell cultures [80]. RANTES. RANTES (regulated on activation, normal T-cell expressed and secreted) is another cytokine chemoattractant for monocytes, as well as memory T-cells, and eosinophils. It is secreted by T-cells, some epithelial cells, and mesenchymal cells. The concentration of RANTES is increased in the peritoneal fluid of women with endometriosis, and its level correlates with the severity of the disease [81]. Ectopic endometrial implants are the most likely source of elevated peritoneal fluid RANTES in women with endometriosis. In normal endometrium, RANTES is expressed in the stromal component [82]. Cultured endometrial stromal cells synthesize RANTES mRNA and secrete protein when stimulated by inflammatory proteins, whereas epithelial cells synthesize neither protein nor mRNA [82]. The discrepancy between basal RANTES expression in vivo and in vitro, with a requirement for exogenous cytokine stimulation for RANTES expression in cultured endometrial stromal cell, suggests that RANTES secretion is induced by other cytokines in the peritoneal cavity. In endometriotic implants, the pattern of RANTES protein distribution is similar to that found in the normal endometrium [82]. However, endometriotic cell cultures produce significantly higher amounts of RANTES

108

Seli et al.

when stimulated with cytokines, compared with cultures derived from normal endometrium. Increased RANTES production by ectopic endometrial implants in response to chemokines may explain its elevated levels in peritoneal fluid and contributes to the increased recruitment of macrophages into the peritoneal cavity in women with endometriosis. Tumor Necrosis Factor-a. Tumor necrosis factor-a is a cytokine that plays a key role in a multitude of inflammatory processes. Initially identified for its ability to kill certain cell lines, TNF-a was later found to be able to initiate an inflammatory response by activating a cascade of cytokines. It is produced by neutrophils, activated lymphocytes, macrophages, NK cells, and several other nonhematopoietic cells. Tumor necrosis factor-a has been implicated in the pathogenesis and pathophysiology of endometriosis. It is expressed in eutopic human endometrium, predominantly in epithelial cells, and during the secretory phase of the menstrual cycle [83]. Similar to some of the other cytokines previously discussed, TNF-a is expressed in cultured endometrial epithelial cells, and its expression is upregulated by IL-1 [84]. Tumor necrosis factor-a concentrations are increased in the peritoneal fluid of women with endometriosis, and its levels correlate with the stage of disease [85]. In addition to ectopic endometrial cells, peritoneal macrophages have been suggested as a possible source of elevated peritoneal fluid TNF-a in women with endometriosis [58]. Tumor necrosis factor-a causes an increase in the adherence of cultured stromal cells to mesothelial cells [86], suggesting that it may facilitate adherence of ectopic endometrial tissue to the peritoneum and allow implants to develop. Tumor necrosis factor-a may also be involved in endometriosisassociated infertility. At very high concentrations, TNF-a may adversely affect sperm motility [62], and show embryotoxic effects [61]. Vascular Endothelial Growth Factor. A key condition for the survival and growth of ectopic endometrial tissue after successful adhesion is the establishment of a new blood supply. Active endometriotic implants are markedly vascularized. An important mediator of local angiogenesis is VEGF. Vascular endothelial growth factor is a heparin-binding glycoprotein and a potent angiogenic protein produced by monocytes, macrophages, and smooth muscle cells. It is also a mitogen for endothelial cells, induces vascular permeability, and acts as a chemoattractant for monocytes. Vascular endothelial growth factor is localized predominantly in endometrial glands. Stromal staining is more diffuse [87,88]. Estradiol upregulates VEGF expression in cultured endometrial stromal cells [87] and peritoneal macrophages [89]. Hypoxia, IL-1, platelet derived growth factor (PDGF), and transforming growth factor beta (TGFh), epidermal growth factor (EGF), and prostaglandin E2 (PGE2) are other factors that upregulate VEGF expression [59,90,91].

Immunology and Immunotherapy

109

Vascular endothelial growth factor is elevated in the peritoneal fluid of women with endometriosis, and peritoneal fluid VEGF concentrations are significantly higher in women with moderate to severe endometriosis compared with women with minimal to mild endometriosis [89]. VEGF is expressed in endometriotic lesions. Its expression is more pronounced around red endometriotic lesions as compared with the more inactive black powderburn implants [89,92]. Like TNF-a, the cellular sources of VEGF in peritoneal fluid include activated peritoneal macrophages [89] and endometriotic lesions [87]. INFLAMMATORY PERITONEAL ENVIRONMENT IN WOMEN WITH ENDOMETRIOSIS: IMPLICATIONS ON FERTILITY AND PELVIC PAIN It is unclear whether immunological alterations induce endometriosis or are a consequence of its presence, but they may play an important role in the development of infertility and pelvic pain. Potential mechanisms by which the altered immune milieu may contribute to infertility (Table 3) and pelvic pain are summarized below. Impact of Inflammatory Milieu on Fertility The inflammatory milieu in women with endometriosis may impair folliculogenesis, especially in advanced-stage disease. Higher rates of apoptosis have been documented in granulosa cells obtained from women with endometriosis undergoing in vitro fertilization (IVF) compared with patients with tubal factor, male factor, or idiopathic infertility [93]. Granulosa cell apoptotic bodies have been shown to predict oocyte quality and the incidence of apoptotic bodies increases with advancing stages of endometriosis [94,95].

TABLE 3

Inflammatory Changes Contributing to EndometriosisAssociated Infertility

Compromised oocyte quality . Altered follicular fluid . Increased granulosa cell apoptosis Impaired fertilization . Increased sperm phagocytosis . Decreased sperm motility Implantation defects . Decreased integrin avh3 and LIF . Increased embryotoxicity

110

Seli et al.

Alterations within the follicular fluid of women with endometriosis may also compromise oocyte quality. In natural cycles follicular fluid VEGF levels are decreased while IL-6 levels are elevated [96]. Additionally, granulosa cell production of inflammatory cytokines, such as TNF-a, IL-1h, and IL-8 may be increased in women with endometriosis [97]. Follicular fluid TNF-a levels have been correlated with poor quality oocytes, and it has been speculated that the cytokine-induced proinflammatory milieu may disturb oocyte fertilization [98]. In natural cycles, reduced fertilization rates have been reported in women with endometriosis [99]. Spermatozoa incubated with follicular fluid from women with endometriosis demonstrate significantly lower zona binding than spermatozoa incubated in follicular fluid samples from women with tubal factor infertility. Additionally, sperm mixed with peritoneal fluid from women with endometriosis perform poorly on zona-free hamster egg sperm penetration assays [100]. Antibodies to transferrin and alpha 2-HS inhibit sperm motility in vitro and are commonly found in the peritoneal fluid of women with endometriosis [101,102]. In addition, peritoneal macrophages in women with endometriosis demonstrate higher sperm phagocytic activity than peritoneal macrophages in women without the disease [103]. Implantation defects also have been documented in women with endometriosis. In murine models, peritoneal fluid from infertile women with endometriosis decreases implantation rates and markers of endometrial receptivity such as leukemia inhibitory factor (LIF) and integrin avh3 [104]. In humans it has been reported that implantation window-specific integrins, such as avh3, are absent in women with endometriosis [105]. Moreover, aberrant expression of HOXA10 and HOXA11, homeobox genes essential for implantation, has been documented in midluteal phase endometrium of women with endometriosis [106]. Finally, it has been reported that serum and peritoneal fluid samples from infertile women with endometriosis are embryotoxic to two-cell mouse embryos [107]. Relationship of Inflammatory Milieu to Pelvic Pain Activated immune responses and accumulations of inflammatory cells may also contribute to pelvic pain. The prolonged survival and activation of macrophages and neutrophils may contribute to tissue damage and scarring. Such inflammation may also directly trigger sensory neurons or indirectly lead to pain through the formation of adhesions. Pelvic adhesions are a significant cause of morbidity. Adhesion formation classically involves three components: an acute inflammatory response, fibrinolysis, and metalloproteinases. Important proinflammatory cytokines identified in adhesion formation/reformation include IL-1, TNF-a, and IL-6

Immunology and Immunotherapy

111

[108]. All three may be elevated in the peritoneal fluid of women with endometriosis. The relationship between prostaglandins and pelvic pain is less clear. There is a significant increase in the release of prostaglandins E2 and F2-alpha from peritoneal macrophages in women with endometriosis [109]. Some investigators have correlated dysmenorrhea with prostaglandin production, but this has not been a consistent finding [110,111]. IMMUNE MODULATORS AS TREATMENT MODALITIES FOR ENDOMETRIOSIS As summarized above, substantial evidence indicates that immunological factors play a role in the pathogenesis and pathophysiology of endometriosis. Commonly used drugs in the medical treatment of endometriosis include estrogen-progesterone combinations, progestins, danazol, and GnRH analogs. Both steroidal and GnRH analog therapies induce a hypoestrogenic state by which they are believed to inhibit endometriotic implant growth. They also affect immune milieu indirectly by suppressing the growth of endometriotic implants and possibly by direct effects on immune cells. Danazol suppresses macrophage-dependent T lymphocyte activation [112] and spontaneous and activated NK cell cytotoxicity [113]. Moreover, treatment with Danazol suppresses the levels of autoantibodies associated with endometriosis [114] and downregulates T cells, macrophages, and expressed human leukocyte antigens (HLA) in eutopic endometrium of women with endometriosis [115]. Danazol treatment in women with endometriosis results in decreased peritoneal fluid IL-1h and TNF levels [75]. In vitro, Danazol suppresses IL-1h and TNF production by human monocytes [116]. Danazol also downregulates MCP-1 production by endometriotic cells [117] and eutopic endometrial epithelial cells from women with endometriosis in culture [118]. GnRH agonist treatment also affects the immune system. Similar to the effects of Danazol, treatment of women with endometriosis with the GnRH agonist buserelin results in downregulation of elevated IL-1 and TNF in the peritoneal fluid [75]. GnRH agonists also suppress the levels of autoantibodies associated with endometriosis [119]. On the other hand, GnRH agonist treatment causes a progressive increase in both NK cell activity [120] and numbers [121] in women with endometriosis. GnRH agonists may also enhance the mitogenic activity of peripheral blood lymphocytes [121]. Immunological effects of these treatments seem to contribute to the resulting improvement in the symptomatology of women with endometriosis. Unfortunately, these hormonal treatments are often associated with unwanted effects caused by a hypoestrogenic state. An ideal treatment for en-

112

Seli et al.

dometriosis would induce regression of the disease and its symptoms without any adverse effects associated with a hypoestrogenic state. In this respect, immune modulatory drugs may play a role in the treatment of endometriosis. Pentoxifylline, a phosphodiesterase inhibitor, is the first immune-modulator agent investigated for the treatment of endometriosis. Pentoxifylline inhibits phagocytosis and generation of toxic oxygen species and proteolytic enzymes by macrophages and granulocytes in vitro and in vivo [122]. Moreover, pentoxifylline inhibits both TNF-a production by macrophages [123], and the proinflammatory action of TNF-a and IL-1 on granulocytes in vitro [124]. In 1991, Steinleitner et al used pentoxifylline in a murine model of peritoneal inflammation. They showed that the transfer of activated peritoneal macrophages, but not basal state macrophages, into the peritoneal cavity of normal mice significantly inhibited fertilization. This was reversed by periovulatory pentoxifylline treatment [125]. Soon after their first report, they showed that the inhibition of fertilization by surgically induced endometriosis is also reversed by periovulatory pentoxifylline treatment [126]. Pentoxifylline was later found to inhibit endometriotic implant growth in an animal model without affecting circulating estradiol and progesterone levels [127]. In 1997 Balasch et al reported the first randomized clinical trial of pentoxifylline in the treatment of infertility associated with asymptomatic minimal or mild endometriosis [128]. Thirty women in each group were treated with 800 mg oral pentoxifylline for 12 months. The overall pregnancy rates were 31% and 18.5% in the pentoxifylline and placebo groups, respectively. However, the difference was not statistically significant because of the small study population. Recently, another treatment directed at downregulating TNF-a was reported [129]. In a prospective randomized placebo-controlled trial, D’Hooghe et al found that subcutaneous recombinant human TNF-binding protein-1 treatment inhibits the development of endometriosis in baboons. Because of the possible link between cell-mediated immune deficiencies and endometriosis, a different approach involving immune enhancers has also been attempted in animal models of endometriosis. Loxoribine, a guanosine analog, is an immune enhancer that stimulates NK cell activity, B-cell proliferation, macrophage-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity, and the differentiation of cytotoxic T-cells. Loxoribine also selectively enhances IL-1, TNF-a, TNF-h, IL-6, IFN-a, and IFN-g. Keenan et al used loxoribine in a rat model of endometriosis and showed that it caused regression of both stromal and epithelial components of implants [130]. SUMMARY Endometriosis is a common gynecological disorder characterized by the presence of endometrial tissue outside the uterus. No single theory can ex-

Immunology and Immunotherapy

113

plain all cases of endometriosis; however, the most commonly accepted theory is Sampson’s theory of retrograde menstruation. Retrograde menstruation occurs in 76% to 90% of women. The much lower prevalence of endometriosis suggests that additional factors determine susceptibility to endometriosis. Endometriosis is associated with changes in both cell-mediated and humoral immunity. Impaired NK cell activity resulting in inadequate removal of refluxed menstrual debris may play a role in the development of endometriotic implants. Moreover, although the peritoneal fluid of women with endometriosis contains increased numbers of immune cells, these seem to facilitate rather than inhibit the development of endometriosis. Macrophages that would be expected to clear endometrial cells from the peritoneal cavity appear to enhance their proliferation by secreting growth factors and cytokines. These immune mediators seem to promote implantation and growth of ectopic endometrium by inducing proliferation and angiogenesis. It is unclear whether these immunological alterations induce endometriosis or are a consequence of its presence, but they appear to play an important role in allowing endometriosis implants to persist and progress, and contribute to the development of infertility, adhesions, and pelvic pain. Danazol and GnRH agonists are commonly used for the medical treatment of endometriosis. Concomitant with their effect on endometriotic implants, these medications alter cellular and humoral immune response. Such immunomodulatory effects may contribute to the observed clinical improvement associated with their use. Finally, studies with novel immune system modulators suggest that they may prove to be useful in the treatment of women with endometriosis. PRACTICAL POINTS 

Danazol and GnRHa alter cellular and humoral immune response, improving endometriosis-related symptoms.  Studies with novel immune system modulators may prove to be useful in the treatment of women with endometriosis. REFERENCES 1.

2. 3.

Sampson JA. Peritoneal endometriosis due to the menstrual dissemination of endometrial tissue into the peritoneal cavity. Am J Obstet Gynecol 1927; 14: 422–469. Beyth Y, Yaffe H, Levij S, Sadovsky E. Retrograde seeding of endometrium: A sequela of tubal flushing. Fertil Steril 1975; 26:1094–1097. Bartosik D, Jacobs SL, Kelly LJ. Endometrial tissue in peritoneal fluid. Fertil Steril 1986; 46:796–800.

114

Seli et al.

4.

TeLinde RW, Scott RB. Experimental endometriosis. Am J Obstet Gynecol 1950; 60:1147–1173. Ridley JH, Edwards IK. Experimental endometriosis in the human. Am J Obstet Gynecol 1958; 76:783–790. Jenkins S, Olive DL, Haney AF. Endometriosis: Pathogenic implications of the anatomic distribution. Obstet Gynecol 1986; 67:335–338. Blumenkrantz JM, Gallagher N, Bashore RA, Tenckhoff H. Retrograde menstruation in women undergoing chronic peritoneal dialysis. Obstet Gynecol 1981; 57:667–670. Halme J, Hammond MG, Hulka JF, Raj SG, Talbert LM. Retrograde menstruation in healthy women and in patients with endometriosis. Obstet Gynecol 1984; 64:151–154. Houston DE, Noller KL, Melton LJ 3rd, Selwyn BJ, Hardy RJ. Incidence of pelvic endometriosis in Rochester, Minnesota, 1970–1979. Am J Epidemiol 1987; 125:959–969. Kjerulff KH, Erickson BA, Langenberg PW. Chronic gynecological conditions reported by US women: Findings from the National Health Interview Survey, 1984 to 1992. Am J Pub Health 1996; 86:195–199. vanFurth R, Raeburn JA, vanZwet TI. Characteristics of human mononuclear phagocytes. Blood 1979; 54:485–500. Haney AF, Muscato JJ, Weinberg JB. Peritoneal fluid cell populations in infertility patients. Fertil Steril 1981; 35:696–698. Olive DL, Weinberg JB, Haney AF. Peritoneal macrophages and infertility: The association between cell number and pelvic pathology. Fertil Steril 1985; 44:772–777. Halme J, Becher S, Hammond MG, Raj MHG, Rau S. Increased activation of pelvic macrophages in infertile women with mild endometriosis. Am J Obstet Gynecol 1983; 145:333. Zeller JM, Henig I, Radwanska E, Dmowski WP. Enhancement of human monocyte and peritoneal macrophage chemiluminescence activities in women with endometriosis. Am J Reprod Immunol 1987; 13:78–82. Dunselman GA, Hendrix MG, Bouckaert PX, Evers JL. Functional aspects of peritoneal macrophages in endometriosis of women. J Reprod Fertil 1988; 82: 707–710. Hill JA, Faris HMP, Schiff I, Anderson DJ. Characterization of leukocyte subpopulations in the peritoneal fluid of women with endometriosis. Fertil Steril 1988; 50:216–222. Lebovic DI, Mueller MD, Taylor RN. Immunobiology of endometriosis. Fertil Steril 2001; 75:1–10. Ramprasad MP, Fischer W, Witztum JL, Sambrano GR, Quehenberger O, Steinberg D. The 94- to 97-kDa mouse macrophage membrane protein that recognizes oxidized low density lipoprotein and phosphatidylserine-rich liposomes is identical to macrosialin, the mouse homologue of human CD68. Proc Natl Acad Sci U S A 1995; 92:9580–9584. Ottnad E, Parthasarathy S, Sambrano GR, Ramprasad MP, Quehenberger O,

5. 6. 7.

8.

9.

10.

11. 12. 13.

14.

15.

16.

17.

18. 19.

20.

Immunology and Immunotherapy

21.

22.

23.

24.

25.

26. 27.

28.

29.

30.

31.

32.

115

Kondratenko N, Green S, Steinberg D. A macrophage receptor for oxidized low density lipoprotein distinct from the receptor for acetyl low density lipoprotein: Partial purification and role in recognition of oxidatively damaged cells. Proc Natl Acad Sci USA 1995; 92:1391–1395. Stanton LW, White RT, Bryant CM, Protter AA, Endemann G. A macrophage Fc receptor for IgG is also a receptor for oxidized low density lipoprotein. J Biol Chem 1992; 267:22446–22451. Endemann G, Stanton LW, Madden KS, Bryant CM, White RT, Protter AA. CD36 is a receptor for oxidized low density lipoprotein. J Biol Chem 1993; 268:11811–11816. Moulton KS, Wu H, Barnett J, Parthasarathy S, Glass CK. Regulated expression of the human acetylated low density lipoprotein receptor gene and isolation of promoter sequences. Proc Natl Acad Sci USA 1992; 89:8102– 8106. Wu H, Moulton K, Horvai A, Parik S, Glass CK. Combinatorial interactions between AP-1 and ets domain proteins contribute to the developmental regulation of the macrophage scavenger receptor gene. Mol Cell Biol 1994; 14: 2129–2139. de Villiers WJ, Fraser IP, Gordon S. Cytokine and growth factor regulation of macrophage scavenger receptor expression and function. Immunol Lett 1994; 43:73–79. Sidell N, Han SW, Parthasarathy S. Regulation and modulation of abnormal immune responses in endometriosis. Ann NY Acad Sci 2002; 955:159–173. Kim JG, Keshava C, Murphy AA, Pitas RE, Parthasarathy S. Fresh mouse peritoneal macrophages have low scavenger receptor activity. J Lipid Res 1997; 38:2207–2215. Surrey ES, Halme J. Effect of peritoneal fluid from endometriosis patients on endometrial stromal cell proliferation in vitro. Obstet Gynecol 1990; 76:792– 797. Braun DP, Muriana A, Gebel H, Rotman C, Rana N, Dmowski WP. Monocyte-mediated enhancement of endometrial cell proliferation in women with endometriosis. Fertil Steril 1994; 61:78–84. Holmberg LA, Ault KA. Characterization of natural killer cells induced in the peritoneal exudates of mice infected with Listeria monocytogenes: A study of their tumor target specificity and their expression of murine differentiation antigens and human NK-associated antigens. Cell Immunol 1984; 89: 151–168. Voogt PJ, Falkenburg JH, Fibbe WE, Veenhof WF, Hamilton M, Van Krimpen BA, Bolhuis RL. Normal hematopoietic progenitor cells and malignant lymphohematopoietic cells show different susceptibility to direct cellmediated MHC-non-restricted lysis by T cell receptor-/CD3-, T cell receptor gamma delta+/CD3+ and T cell receptor-alpha beta+/CD3+ lymphocytes. J Immunol 1989; 142:1774–1780. Moretta A, Sivori S, Vitale M, Pende D, Morelli L, Augugliaro R, Bottino C, Moretta L. Existence of both inhibitory (p58) and activatory (p50) receptors

116

33.

34.

35.

36.

37.

38.

39. 40.

41.

42. 43. 44. 45. 46.

47.

48.

Seli et al. for HLA-C molecules in human natural killer cells. J Exp Med 1995; 182:875– 884. Kikuchi Y, Ishikawa N, Hirata J, Imaizumi E, Sasa H, Nagata I. Changes of peripheral blood lymphocyte subsets before and after operation of patients with endometriosis. Acta Obstet Gynecol Scand 1993; 72:157–161. Oosterlynck DJ, Cornillie FJ, Waer M, Vandeputte M, Koninckx PR. Women with endometriosis show a defect in natural killer activity resulting in a decreased cytotoxicity to autologous endometrium. Fertil Steril 1991; 56:45– 51. Wilson TJ, Hertzog PJ, Angus D, Munnery L, Wood EC, Kola I. Decreased natural killer cell activity in endometriosis patients: relationship to disease pathogenesis. Fertil Steril 1994; 62:1086–1088. Ho HN, Chao KH, Chen HF, Wu MY, Yang YS, Lee TY. Peritoneal natural killer cytotoxicity and CD25+ CD3+ lymphocyte subpopulation are decreased in women with stage III-IV endometriosis. Hum Reprod 1995; 10: 2671–2675. Kanzaki H, Wang H-S, Kariya M, Mori T. Suppression of natural killer cell activity by sera from patients with endometriosis. Am J Obstet Gynecol 1992; 167:257–261. Oosterlynck DJ, Meuleman C, Waer M, Koninckx PR, Vandeputte M. Immunosuppressive activity of peritoneal fluid in women with endometriosis. Obstet Gynecol 1993; 82:206–212. Ho HN, Wu MY, Yang YS. Peritoneal cellular immunity and endometriosis. Am J Reprod Immunol 1997; 38:400–412. Wu MY, Yang JH, Chao KH, Hwang JL, Yang YS, Ho HN. Increase in the expression of killer cell inhibitory receptors on peritoneal natural killer cells in women with endometriosis. Fertil Steril 2000; 74:1187–1191. Maeda N, Izumiya C, Yamamoto Y, Oguri H, Kusume T, Fukaya T. Increased killer inhibitory receptor KIR2DL1 expression among natural killer cells in women with pelvic endometriosis. Fertil Steril 2002; 77:297–302. Dmowski WP, Steele RW, Baker GF. Deficient cellular immunity in endometriosis. Am J Obstet Gynecol 1981; 141:377–383. Steele RW, Dmowski WP, Marmer DJ. Immunologic aspects of human endometriosis. Am J Reprod Immunol 1984; 6:33–36. Dmowski WP, Gebel HM, Braun DP. The role of cell-mediated immunity in pathogenesis of endometriosis. Acta Obstet Gynecol Scand 1994; 159:7–14. Gleicher N, Dmowski WP, Siegel I, Liu TL, Friberg J, Radwanska E, Toder V. Lymphocyte subsets in endometriosis. Obstet Gynecol 1984; 63:463–466. Badawy SZA, Cuenca V, Stitzel A, Tice D. Immune rosettes of T and B lymphocytes in infertile women with endometriosis. J Reprod Med 1987; 32:194–197. Mettler L, Volkov NI, Kulakov VI, Jurgensen A, Parwaresch MR. Lymphocyte subsets in the endometrium of patients with endometriosis throughout the menstrual cycle. Am J Reprod Immunol 1996; 36:342–348. Witz CA, Montoya IA, Dey TD, Schenken RS. Characterization of

Immunology and Immunotherapy

49. 50.

51. 52.

53.

54.

55.

56.

57.

58.

59.

60.

61.

62.

117

lymphocyte subpopulations and T cell activation in endometriosis. Am J Reprod Immunol 1994; 32:173–179. Weed JC, Arquembourg PC. Endometriosis: Can it produce an autoimmune response resulting in infertility? Clin Obstet Gynecol 1980; 23:885–893. Mathur S, Peress MR, Williamson HO, Youmans CD, Maney SA, Garvin AJ, Rust PF, Fudenberg HH. Autoimmunity to endometrium and ovary in endometriosis. Clin Exp Immunol 1982; 50:259–266. Gleicher N, el-Roeiy A, Confino E, Friberg J. Is endometriosis an autoimmune disease? Obstet Gynecol 1987; 70:115–122. Gleicher N, el-Roeiy A, Confino E, Friberg J. Reproductive failure because of autoantibodies: Unexplained infertility and pregnancy wastage. Am J Obstet Gynecol 1989; 160:1376–1380. discussion 1380–1385. Glinoer D, Soto MF, Bourdoux P, Lejeune B, Delange F, Lemone M, Kinthaert J, Robijn C, Grun JP, de Nayer P. Pregnancy in patients with mild thyroid abnormalities: Maternal and neonatal repercussions. J Clin Endocrinol Metab 1991; 73:421–427. Dmowski WP, Rana N, Michalowska J, Friberg J, Papierniak C, El-Roeiy A. The effect of endometriosis, its stage and activity, and of autoantibodies on in vitro fertilization and embryo transfer success rates. Fertil Steril 1995; 63:555– 562. Hill JA, Anderson DJ. Lymphocyte activity in the presence of peritoneal fluid from fertile women and infertile women with and without endometriosis. Am J Obstet Gynecol 1989; 161:861–864. Fakih H, Bagget B, Holtz G, Tsang K-Y, Lee JC, Williamson HO. Interleukin-1: Possible role in the infertility associated with endometriosis. Fertil Steril 1987; 47:213–217. Mori H, Sawairi M, Nakagawa M, Itoh N, Wada K, Tamaya T. Peritoneal fluid interleukin-1 beta and tumor necrosis factor in patients with benign gyecologic disease. Am J Reprod Immunol 1991; 26:62–67. Keenan JA, Chen TT, Chadwell NL, Torry DS, Caudle MR. IL-1h, TNF-a, and IL-2 in peritoneal fluid and macrophage conditioned media of women with endometriosis. Am J Reprod Immunol 1995; 34:381–385. Lebovic DI, Bentzien F, Chao VA, Garrett EN, Meng YG, Taylor RN. Induction of an angiogenic phenotype in endometriotic stromal cell cultures by interleukin-1beta. Mol Hum Reprod 2000; 6:269–275. Vigano P, Gaffuri B, Somigliana E, Busacca M, Di Blasio AM, Vignali M. Expression of intercellular adhesion molecule (ICAM)-1 mRNA and protein is enhanced in endometriosis versus endometrial stromal cells in culture. Mol Hum Reprod 1998; 4:1150–1156. Hill JA, Haimovici F, Politch J, Anderson DJ. Effect of soluble products of activated lymphocytes and macrophages (lymphokines and monokines) on human sperm motion parameters. Fertil Steril 1987; 47:460–465. Hill JA, Haimovici F, Anderson DJ. Products of activated lymphocytes and macrophages inhibit mouse embryo development in vitro. J Immunol 1987; 139:2250–2254.

118

Seli et al.

63. Sueldo CE, Kelly E, Montoro L, Subias E, Baccaro M, Swanson JA. Effect of interleukin-1 on gamete interaction and mouse embryo development. J Reprod Med 1990; 35:868–872. 64. Arici A, Head JR, MacDonald PC, Casey ML. Regulation of interleukin-8 gene expression in human endometrial cells in culture. Mol Cell Endocrinol 1993; 94:195–204. 65. Arici A, Tazuke SI, Attar E, Kliman HJ, Olive DL. Interleukin-8 concentration in peritoneal fluid of patients with endometriosis and modulation of interleukin-8 expression in human mesothelial cells. Mol Hum Reprod 1996; 2:40–45. 66. Arici A, Seli E, Senturk LM, Gutierrez LS, Oral E, Taylor HS. Interleukin-8 in the human endometrium. J Clin Endocrinol Metab 1998; 83:1783–1787. 67. Ryan IP, Tseng JF, Schriock ED, Khorram O, Landers DV, Taylor RN. Interleukin-8 concentrations are elevated in peritoneal fluid of women with endometriosis. Fertil Steril 1995; 63:929–932. 68. Akoum A, Lawson C, McColl S, Villeneuve M. Ectopic endometrial cells express high concentrations of interleukin (IL)-8 in vivo regardless of the menstrual cycle phase and respond to oestradiol by up-regulating IL-1-induced IL-8 expression in vitro. Mol Hum Reprod 2001; 7:859–866. 69. Garcia-Velasco JA, Arici A. Interleukin-8 stimulates the adhesion of endometrial stromal cells to fibronectin. Fertil Steril 1999; 72:336–340. 70. Garcia-Velasco JA, Arici A. Interleukin-8 expression in endometrial stromal cells is regulated by integrin-dependent cell adhesion. Mol Hum Reprod 1999; 5:1135–1140. 71. Spuijbroek MDEH, Dunselman GAJ, Menheere PPJA, Evers JLH. Early endometriosis invades the extracellular matrix. Fertil Steril 1992; 58:929–933. 72. Arici A. Local cytokines in endometrial tissue: The role of interleukin-8 in the pathogenesis of endometriosis. Ann NY Acad Sci 2002; 955:101–109. 73. Arici A, Seli E, Zeyneloglu HB, Senturk LM, Oral E, Olive DL. Interleukin-8 induces proliferation of endometrial stromal cells: A potential autocrine growth factor. J Clin Endocrinol Metab 1998; 83:1201–1205. 74. Arici A, Oral E, Attar E, Tazuke S, Olive DL. Monocyte chemotactic protein1 concentration in peritoneal fluid of women with endometriosis and its modulation of expression in mesothelial cells. Fertil Steril 1997; 67:1065–1072. 75. Taketani Y, Kuo TM, Mizuno M. Comparison of cytokine levels and embryo toxicity in peritoneal fluid in infertile women with untreated or treated endometriosis. Am J Obstet Gynecol 1992; 167:265–270. 76. Jolicoeur C, Boutouil M, Drouin R, Paradis I, Lemay A, Akoum A. Increased expression of monocyte chemotactic protein-1 in the endometrium of women with endometriosis. Am J Pathol 1998; 152:125–133. 77. Akoum A, Jolicoeur C, Boucher A. Estradiol amplifies interleukin-1-induced monocyte chemotactic protein-1 expression by ectopic endometrial cells of women with endometriosis. J Clin Endocrinol Metab 2000; 85:896–904. 78. Akoum A, Lemay A, Brunet C, Hebert J. Secretion of monocyte chemotactic protein-1 by cytokine-stimulated endometrial cells of women with endome-

Immunology and Immunotherapy

79.

80.

81.

82.

83.

84.

85.

86.

87.

88. 89.

90.

91.

119

triosis. Le groupe d’investigation en gynecologie. Fertil Steril 1995; 63:322– 328. Akoum A, Lemay A, Brunet C, Hebert J. Cytokine-induced secretion of monocyte chemotactic protein-1 by human endometriotic cells in culture. Am J Obstet Gynecol 1995; 172:594–600. Boucher A, Mourad W, Mailloux J, Lemay A, Akoum A. Ovarian hormones modulate monocyte chemotactic protein-1 expression in endometrial cells of women with endometriosis. Mol Hum Reprod 2000; 6:618–626. Khorram O, Taylor RN, Ryan IP, Schall TJ, Launders DV. Peritoneal fluid concentrations of the cytokine RANTES correlate with the severity of endometriosis. Am J Obstet Gynecol 1993; 169:1545–1549. Hornung D, Ryan IP, Chao VA, Vigne J-L, Schriock ED, Taylor RN. Immunolocalization and regulation of the chemokine RANTES in human endometrial and endometriosis tissues and cells. J Clin Endocrinol Metab 1997; 82:1621–1628. Philippeaux MM, Piguet PF. Expression of tumor necrosis factor-alpha and its mRNA in the endometrial mucosa during the menstrual cycle. Am J Pathol 1993; 143:480–486. Laird SM, Tuckerman EM, Saravelos H, Li TC. The production of tumour necrosis factor alpha (TNF-alpha) by human endometrial cells in culture. Hum Reprod 1996; 11:1318–1323. Eisermann J, Gast MJ, Pineda J, Odem RR, Collins JL. Tumor necrosis factor in peritoneal fluid of women undergoing laparoscopic surgery. Fertil Steril 1988; 50:573–579. Zhang R, Wild R, Ojago J. Effect of tumor necrosis factor-a on adhesion of human endometrial stromal cells to peritoneal mesothelial cells: an in vitro system. Fertil Steril 1993; 59:1196–1201. Shifren JL, Tseng JF, Zaloudek CJ, Ryan IP, Meng YG, Ferrara N, Jaffe RB, Taylor RN. Ovarian steroid regulation of vascular endothelial growth factor in the human endometrium: Implications for angiogenesis during the menstrual cycle and in the pathogenesis of endometriosis. J Clin Endocrinol Metab 1996; 81:3112–3118. Li XF, Gregory J, Ahmed A. Immunolocalisation of vascular endothelial growth factor in human endometrium. Growth Fact 1994; 11:277–282. McLaren J, Prentice A, Charnock-Jones DS, Millican SA, Muller KH, Sharkey AM, Smith SK. Vascular endothelial growth factor is produced by peritoneal fluid macrophages in endometriosis and is regulated by ovarian steroids. J Clin Invest 1996; 98:482–489. Brogi E, Wu T, Namiki A, Isner JM. Indirect angiogenic cytokines upregulate VEGF and bFGF gene expression in vascular smooth muscle cells, whereas hypoxia upregulates VEGF expression only. Circulation 1994; 90:649–652. Ben-Av P, Crofford LJ, Wilder RL, Hla T. Induction of vascular endothelial growth factor expression in synovial fibroblasts by prostaglandin E and interleukin-1: A potential mechanism for inflammatory angiogenesis. FEBS Lett 1995; 372:83–87.

120

Seli et al.

92. Donnez J, Smoes P, Gillerot S, Casanas-Roux F, Nisolle M. Vascular endothelial growth factor (VEGF) in endometriosis. Hum Reprod 1998; 13:1686– 1690. 93. Toya M, Saito H, Ohta N, Saito T, Kaneko T, Hiroi M. Moderate and severe endometriosis is associated with alterations in the cell cycle of granulosa cells in patients undergoing in vitro fertilization and embryo transfer. Fertil Steril 2000; 73:344–350. 94. Nakahara K, Saito H, Saito T, Ito M, Ohta N, Takahashi T, Hiroi M. The incidence of apoptotic bodies in membrana granulosa can predict prognosis of ova from patients participating in in vitro fertilization programs. Fertil Steril 1997; 68:312–317. 95. Nakahara K, Saito H, Saito T, Ito M, Ohta N, Takahashi T, Hiroi M. Ovarian fecundity in patients with endometriosis can be estimated by the incidence of apoptotic bodies. Fertil Steril 1998; 69:931–935. 96. Pellicer A, Albert C, Mercader A, Bonilla-Musoles F, Remohi J, Simon C. The follicular and endocrine environment in women with endometriosis: Local and systemic cytokine production. Fertil Steril 1998; 70:425–431. 97. Carlberg M, Nejaty J, Froysa B, Guan Y, Soder O, Bergqvist A. Elevated expression of tumour necrosis factor alpha in cultured granulosa cells from women with endometriosis. Hum Reprod 2000; 15:1250–1255. 98. Lee KS, Joo BS, Na YJ, Yoon MS, Choi OH, Kim WW. Relationships between concentrations of tumor necrosis factor-alpha and nitric oxide in follicular fluid and oocyte quality. J Assist Reprod Genet 2000; 17:222–228. 99. Cahill DJ, Wardle PG, Maile LA, Harlow CR, Hull MG. Ovarian dysfunction in endometriosis-associated and unexplained infertility. J Assist Reprod Genet 1997; 14:554–557. 100. Aeby TC, Huang T, Nakayama RT. The effect of peritoneal fluid from patients with endometriosis on human sperm function in vitro. Am J Obstet Gynecol 1996; 174:1779–1783. 101. Mathur SP. Autoimmunity in endometriosis: Relevance to infertility. Am J Reprod Immunol 2000; 44:89–95. 102. Pillai S, Zhou GX, Arnaud P, Jiang H, Butler WJ, Zhang H. Antibodies to endometrial transferrin and alpha 2-Heremans Schmidt (HS) glycoprotein in patients with endometriosis. Am J Reprod Immunol 1996; 35:483–494. 103. Jha P, Farooq A, Agarwal N, Buckshee K. In vitro sperm phagocytosis by human peritoneal macrophages in endometriosis-associated infertility. Am J Reprod Immunol 1996; 36:235–237. 104. Illera MJ, Juan L, Stewart CL, Cullinan E, Ruman J, Lessey BA. Effect of peritoneal fluid from women with endometriosis on implantation in the mouse model. Fertil Steril 2000; 74:41–48. 105. Lessey BA, Castelbaum AJ, Sawin SW, Buck CA, Schinnar R, Bilker W, Strom BL. Aberrant integrin expression in the endometrium of women with endometriosis. J Clin Endocrinol Metab 1994; 79:643–649. 106. Taylor HS, Bagot K, Kardana A, Olive DL, Arici A. HOX gene expression is altered in the endometrium of women with endometriosis. Hum Reprod 1999; 14:1328–1331.

Immunology and Immunotherapy

121

107. Martinez-Roman S, Balasch J, Creus M, Fabregues F, Carmona F, Vilella R, Vanrell JA. Immunological factors in endometriosis-associated reproductive failure: Studies in fertile and infertile women with and without endometriosis. Hum Reprod 1997; 12:1794–1799. 108. Cheong YC, Shelton JB, Laird SM, Richmond M, Kudesia G, Li TC, Ledger WL. IL-1, IL-6 and TNF-alpha concentrations in the peritoneal fluid of women with pelvic adhesions. Hum Reprod 2002; 17:69–75. 109. Karck U, Reister F, Schafer W, Zahradnik HP, Breckwoldt M. PGE2 and PGF2 alpha release by human peritoneal macrophages in endometriosis. Prostaglandins 1996; 51:49–60. 110. Koike H, Egawa H, Ohtsuka T, Yamaguchi M, Ikenoue T, Mori N. Correlation between dysmenorrheic severity and prostaglandin production in women with endometriosis. Prostaglandins Leukot Essent Fatty Acid 1992; 46:133–137. 111. Rapkin A, Bhattacherjee P. Peritoneal fluid eicosanoids in chronic pelvic pain. Prostaglandins 1989; 38:447–452. 112. Hill JA, Barbieri RL, Anderson DJ. Immunosuppressive effects of danazol in vitro. Fertil Steril 1987; 48:414–418. 113. Vigano P, Di Blasio AM, Busacca M, Sabbadini MG, Vignali M. Danazol suppresses both spontaneous and activated human lymphocyte-mediated cytotoxicity. Am J Reprod Immunol 1992; 28:38–42. 114. el-Roeiy A, Dmowski WP, Gleicher N, Radwanska E, Harlow L, Binor Z, Tummon I, Rawlins RG. Danazol but not gonadotropin-releasing hormone agonists suppresses autoantibodies in endometriosis. Fertil Steril 1988; 50: 864–871. 115. Ota H, Igarashi S, Hayakawa M, Matsui T, Tanaka H, Tanaka T. Effect of danazol on the immunocompetent cells in the eutopic endometrium in patients with endometriosis: A multicenter cooperative study. Fertil Steril 1996; 65: 545– 551. 116. Mori H, Nakagawa M, Itoh N, Wada K, Tamaya T. Danazol suppresses the production of interleukin-1 beta and tumor necrosis factor by human monocytes. Am J Reprod Immunol 1990; 24:45–50. 117. Jolicoeur C, Lemay A, Akoum A. Comparative effect of danazol and a GnRH agonist on monocyte chemotactic protein-1 expression by endometriotic cells. Am J Reprod Immunol 2001; 45:86–93. 118. Boucher A, Lemay A, Akoum A. Effect of hormonal agents on monocyte chemotactic protein-1 expression by endometrial epithelial cells of women with endometriosis. Fertil Steril 2000; 74:969–975. 119. Kennedy SH, Starkey PM, Sargent IL, Hicks BR, Barlow DH. Antiendometrial antibodies in endometriosis measured by an enzyme-linked immunosorbent assay before and after treatment with danazol and nafarelin. Obstet Gynecol 1990; 75:914–918. 120. Garzetti GG, Ciavattini A, Provinciali M, Muzzioli M, Di Stefano G, Fabris N. Natural cytotoxicity and GnRH agonist administration in advanced endometriosis: Positive modulation on natural killer activity. Obstet Gynecol 1996; 88:234–240. 121. Hsu CC, Lin YS, Wang ST, Huang KE. Immunomodulation in women

122

122.

123.

124.

125.

126.

127.

128.

129.

130.

Seli et al. with endometriosis receiving GnRH agonist. Obstet Gynecol 1997; 89:993– 998. Bessler H, Gilgal R, Djaldetti M, Zahavi I. Effect of pentoxifylline on the phagocytic activity, cAMP levels, and superoxide anion production by monocytes and polymorphonuclear cells. J Leu Biol 1986; 40:747–754. Strieter RM, Remick DG, Ward PA, Spengler RN, Lynch JP 3rd, Larrick J, Kunkel SL. Cellular and molecular regulation of tumor necrosis factor-alpha production by pentoxifylline. Biochem Biophys Res Commun 1988; 155:1230– 1236. Sullivan GW, Carper HT, Novick WJ Jr, Mandell GL. Inhibition of the inflammatory action of interleukin-1 and tumor necrosis factor alpha on neutrophil function by pentoxifylline. Infect Immun 1988; 56:1722–1729. Steinleitner A, Lambert H, Suarez M, Serpa N, Roy S. Immunomodulation in the treatment of endometriosis-associated subfertility: Use of pentoxifylline to reverse the inhibition of fertilization by surgically induced endometriosis in a rodent model. Fertil Steril 1991; 56:975–979. Steinleitner A, Lambert H, Roy S. Immunomodulation with pentoxifylline abrogates macrophage-mediated infertility in an in vivo model: A paradigm for a novel approach to the treatment of endometriosis-associated subfertility. Fertil Steril 1991; 55:26–31. Nothnick WB, Curry TE, Vernon MW. Immunomodulation of rat endometriotic implant growth and protein production. Am J Reprod Immunol 1994; 31:151–162. Balasch J, Creus M, Fabregues F, Carmona F, Martinez-Roman S, Manau D, Vanrell JA. Pentoxifylline versus placebo in the treatment of infertility associated with minimal or mild endometriosis: A pilot randomized clinical trial. Hum Reprod 1997; 12:2046–2050. D’Hooghe TM, Cuneo S, Nugent N, Chai D, Deer F, Mwenda J. Recombinant human TNF binding protein-1 (r-hTBP-1) inhibits the development of endometriosis in baboons: A prospective, randomized, placebo- and drug-controlled study. 57th Annual Meeting of the American Society for Reproductive Medicine, Orlando, FL, USA, October 20–25, 2001. Keenan JA, Williams-Boyce PK, Massey PJ, Chen TT, Caudle MR, Bukovsky A. Regression of endometrial explants in a rat model of endometriosis treated with the immune modulators loxoribine and levamisole. Fertil Steril 1999; 72:135–141.

8 Serum and Peritoneal Markers for Endometriosis Tommaso Falcone and Mohamed A. Bedaiwy The Cleveland Clinic Foundation Cleveland, Ohio, U.S.A.

Endometriosis is diagnosed by surgical intervention. Many attempts have been made to use clinical, imaging, and serum markers to arrive at a diagnosis without surgery. However these methods have a poor sensitivity and specificity when applied to a large number of unselected patients. There are many reports of serum, peritoneal fluid (PF), and tissue markers that are associated with endometriosis. The purpose of this chapter is to review these markers and assess their role in the diagnosis of endometriosis. It is unclear whether endometriosis is a local pelvic inflammatory process with altered function of immune-related cells in the peritoneal environment with a host response or a systemic disorder with a primary pelvic manifestation. Many studies have demonstrated that both serum and PF of women with endometriosis contain an increased number of activated macrophages that secrete various local products, such as growth factors, cytokines, and possibly free oxygen radicals. [1–3] Such a pathogenic ambiguity makes the diagnosis of endometriosis difficult. Currently laparoscopy and peritoneal tissue sampling are the stand123

124

Falcone and Bedaiwy

TABLE 1 Criteria of Ideal Screening Test 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Disease significantly impacts public health Intermediate probability of disease Detection occurs before a critical point High test sensitivity High test specificity Well tolerated by patients Affordable Applicable on mass screening Availability No side effects Disease has high enough prevalence to allow screening Medical care available if screening test positive

ard way for diagnosing endometriosis. Considerable effort has been invested in searching for noninvasive methods for predicting endometriosis. Ideally these markers would be used as a screening test for endometriosis. The criteria for an ideal screening test are listed in Table 1. Endometriosis as a disease process falls short of these criteria [4]. Many markers from different sources have been measured in endometriosis patients (Table 2). These have been investigated not as a screening test but mostly as a diagnostic test. Therefore it is uncertain if they would meet the criteria of an ideal screening test as listed

TABLE 2 Markers for Endometriosis Tumor markers and polypeptides A. CA-125 B. CA 19-9 C. SICAM-1 D. Glycodelin-A (PP 14) Immunological markers A. Cytokines B. Autoantibodies 1. Antiendometrial 2. Autoantibodies to markers of oxidative stress Genetic markers Tissue markers A. Aromatase P 450 B. Cytoskeratines C. Hormone receptors

Serum and Peritoneal Markers

125

in Table 1. This review will focus on the potential use of serum, PF, or tissue markers that may be used as a diagnostic test in symptomatic patients. Many markers have been proposed in serum, PF, and tissue (Table 2); however, none is satisfactory to be used on a wide scale in clinical practice. TUMOR MARKERS AND POLYPEPTIDES Serum CA-125 Serum CA-125—a 200,000 Dalton glycoprotein—concentration has been associated with the presence of many gynecologic disorders (Table 3) and endometriosis. [5] The CA-125 antigen is expressed in many normal tissues such as endometrium, endocervix, and peritoneum. The serum levels of CA125 may differ physiologically with age. [6] However the reports have been contradictory, with some authors showing decreasing levels and others showing increasing levels or no change with age. CA-125 levels are increased during

TABLE 3 CA 125 in Gynecologic Disease Malignant ovarian tumors Serous adenocarcinoma Mucinous adenocarcinoma Undifferentiated adenocarcinoma Endometroid adenocarcinoma Papillary carcinoma Dysgerminoma Clear cell carcinoma Ovarian cancer Borderline and benign ovarian tumors Adenoma Cysts Inflammatory disease Teratomas Granulosa cell tumor Thecoma Cervical and endometrial malignancy Cervical carcinoma Endometrial carcinoma Benign gynecologic conditions Uterine fibromyoma Endometriosis Cervical polyps

126

Falcone and Bedaiwy

menstruation in some women, which is thought to be due to reflux of the menstrual endometrium into the peritoneal cavity. [6] Pittaway and colleagues showed that mean CA-125 levels were higher during menses in both endometriosis and nonendometriosis patients. [7] It is, therefore, recommended that CA-125 levels not be drawn during a menstrual period. The most important clinical use of this serum marker has been in monitoring the course of ovarian cancer response to treatment. In a study at the Cleveland Clinic Foundation, [8] 213 consecutive patients with a CA125 of more than 65 IU/ml were assessed. In this group, gynecological cancers accounted for 74%, nongynecologic cancers accounted for 7%, and nonmalignant conditions accounted for 13% of the diagnosis. The majority of patients in the nonmalignant gynecological disorder group had endometriosis. If patients with a pelvic mass were excluded, 90% of patients with a CA125 greater than 65 IU/ml had a non-malignant condition. In premenopausal patients with a pelvic mass and a CA-125 level of greater than 65, Malkasian et al showed a positive predictive value for malignancy of only 49%. [9] The cut-off value for all these studies has been 65 and not the 35 that is reported as abnormal in standard laboratory reference values. Extremely high levels have been reported in endometriosis, tubo-ovarian abscess, and multivisceral tuberculosis. [6] The plausible explanation for the rise observed in endometriosis patients is that CA-125 membrane concentration is higher in ectopic compared to eutopic endometrial epithelial cells. Moreover, the endometriosisassociated inflammatory response leads to more CA-125 shedding into the peritoneal cavity. [5] Many studies have assessed the role of serum CA-125 measurement in the detection of endometriosis. [10–12] The main confounding variable in determining sensitivity and specificity of serum CA-125 is the stage of the disease. Typically, the majority of patients with advanced endometriosis and the minority of patients with early stage disease will have an elevated serum CA-125. This is similar to the observation of this marker in ovarian cancer. A recent meta-analysis was performed to assess the diagnostic performance of serum CA-125 in detecting endometriosis. [13] Twenty-three studies were included in the initial analysis, 16 were cohort studies and seven were case-control studies. The studies included women with infertility or pelvic pain. Sensitivity and specificity were presented as receiver operating characteristic (ROC) curves. Data were reported for the diagnosis of any form of endometriosis as well as advanced stages only. Sensitivity ranged from 4% to 100% and specificity ranged from 38% to 100% for the diagnosis of any stage of disease. The ROC curve (Fig. 1) showed a poor diagnostic performance. At a specificity of 90%, a sensitivity of 28% was reported. If the sensitivity was increased to 50%, the specificity dropped to 72%.

Serum and Peritoneal Markers

127

FIGURE 1 Receiver-operating characteristic (ROC) curve of serum CA-125 measurement in the diagnosis of any type of endometriosis. The solid circles represent point estimates as reported by single studies. Multiple cutoff points are represented by multiple solid circles. (From Ref. 13.)

For advanced disease, the sensitivity ranged from 0 to 100% and the specificity ranged from 44% to 95%. The ROC curve showed a better diagnostic performance (Fig. 2). For a specificity of approximately 90%, the sensitivity was 47%. If the sensitivity was increased to 60%, the specificity dropped to 81%. The main limitation of this meta-analysis is that it does not take into consideration the possible elements of a history (such as dysmenorrhea) or physical examination that may increase the sensitivity or specificity of the test. Studies that included patients who had pelvic mass on sonography were excluded from the analysis. If the purpose of a test is to identify the majority of patients with the disease, then the diagnostic accuracy of a serum CA-125 is inadequate. According to the analysis of the authors of this study, a negative result would delay the diagnosis in 70% of patients with endometriosis. The routine use of serum CA-125 cannot be advocated as a diagnostic tool to exclude the diagnosis of endometriosis in patients with chronic pelvic pain or infertility. A more useful role for CA-125 may be the evaluation of recurrent disease or the success of a surgical treatment. In a study to evaluate the prognostic value of serial CA-125 determinations, 342 women having a laparoscopy for infertility were evaluated. One hundred and twenty three (36%) had endometriosis and were surgically treated. [14] Fifty-six of 123 (45%) infertile women with endometriosis had preoperative CA-125 values more than or

128

Falcone and Bedaiwy

FIGURE 2 Receiver-operating characteristic (Roc) curve of serum CA-125 measurement in the diagnosis of endometriosis grade III/IV. The solid circles represent point estimates as reported by single studies. Multiple cutoff points are represented by multiple solid circles. (From Ref. 13.)

equal to 16 U/ml and were followed for 12 months with serial CA-125 determinations. The main outcome measure was the proportion of women achieving a pregnancy within 12 months from surgery. The authors found that preoperative CA-125 concentrations were not statistically different for women conceiving, but postoperative CA-125 values were significantly lower for women achieving a pregnancy. Univariate analyses indicated that preoperative CA-125 values between 16 and 25 U/ml and postoperative CA-125 values less than 16 U/ml were associated with significantly higher pregnancy rates. Multivariate analyses of confounding factors indicated only postoperative CA-125 concentrations to be associated with pregnancy even after controlling for all covariables. This study suggested that CA-125 levels have prognostic value for pregnancy in infertile women with surgically treated endometriosis. [14] CA-125 levels may also be useful in patients with initially elevated levels and advanced endometriosis. Several centers have reported high diagnostic accuracy for recurrent disease when elevated levels of CA-125 were observed after treatment. [15] This may be useful in symptomatic patients in whom repeat laparoscopy cannot be performed. Serum CA 19-9 CA19-9 is a high-molecular-weight glycoprotein. [16] Serum CA19-9 levels are elevated in patients with malignant and benign ovarian tumors [17] as well

Serum and Peritoneal Markers

129

as ovarian chocolate cysts. [18] It was also found that serum CA19-9 levels in women with endometriosis were significantly reduced after treatment for endometriosis compared with the basal levels before treatment. [19] There are a limited number of reports on the significance of serum CA19-9 levels in the diagnosis of endometriosis. In a recent study, 34 of the 101 patients with endometriosis (33.7%) had elevated serum CA19-9 levels (>37 IU/ml), but it was not elevated in any of the 22 control patients. [20] Serum CA19-9 levels were not elevated in 38 patients with stage I and II endometriosis but elevated in 34 of the 63 patients (54.0%) with stage III and IV endometriosis. On comparing the sensitivities of the CA19-9 and CA-125 tests for the diagnosis of endometriosis, the investigators found that the sensitivity of the CA19-9 test was significantly lower than that of the CA-125 test (0.34 and 0.49, respectively). Thus, the observed sensitivity of 0.34 limits the diagnostic value of the CA19-9 test, especially in the earlystage disease. [20] The same study showed that using a cutoff value of 37 IU/ml, the mean serum CA19-9 levels in patients with endometriosis increased in accordance with the stage of the disease. On the other hand, if a new cutoff value of the serum CA19-9 levels ranging from 20 to 25 IU/ml is used, the sensitivity of the CA19-9 test will be improved without change in specificity or positive and negative predictive values. However, this study concluded that the clinical utility of the CA19-9 measurement is not superior to that of the CA 125. Serum-Soluble Intercellular Adhesion Molecule-1 A soluble form of intercellular-adhesion molecule-1 (sICAM-1) is secreted from the endometrium and the endometriotic implants. [21] Moreover, endometrium from women with endometriosis secretes a higher amount of this molecule when compared with tissue derived from women without the disease. Consequently, a strong correlation exists between levels of sICAM-1 shed by endometrium and the number of endometriotic implants in the pelvis. [21] With this observation in mind, a role of sICAM-1 in the diagnosis of endometriosis has been hypothesized. A significant increase in serum concentration of sICAM-1 in endometriosis has been reported by many investigators. [22–25] In a recent prospective cohort study to evaluate the utility of sICAM-1 as a potential serum marker of endometriosis, Somigliana et al included a series of 120 consecutive women of reproductive age who underwent laparoscopy for benign gynecologic conditions. [26] They found that serum levels of sICAM-1 were slightly but not significantly elevated in women with endometriosis [71 women (stage I to II in 24 cases and stage III to IV in 47 cases)] compared with women without the disease. However, serum concentration of

130

Falcone and Bedaiwy

sICAM-1 in the 21 women who were found to have deep peritoneal endometriosis was significantly enhanced when compared with both women without the disease and those with superficial endometriosis. The sensitivity and specificity of sICAM-1 in detecting deep peritoneal endometriosis were 0.19 and 0.97, respectively. On comparing that to CA-125, the authors found that the measures of accuracy were 0.14 and 0.92, respectively. When both parameters were used concomitantly, the sensitivity and specificity were 0.28 and 0.92, respectively. They concluded that measurement of CA-125 and sICAM-1 may be helpful in specifically identifying women with deep infiltrating endometriosis. [26] Other Serum Polypeptides Serum placental protein 14 (PP14), currently known as glycodelin-A, [27] was found to be significantly more elevated in endometriosis patients than healthy controls. [28] Such high levels were significantly lowered by conservative surgery as well as by danazol and medroxy progesterone acetate treatment. The reliability of serum PP14 levels for the diagnosis of endometriosis is limited because of low sensitivity (0.59). Typically, the PF concentrations of PP14 are low. The levels are elevated in the luteal phase of endometriosis patients. Whether this is of any diagnostic importance is controversial. [29] Peritoneal Fluid CA 125 Peritoneal fluid is often seen in the vesicouterine cavity or the cul-de-sac during gynecologic surgery and bathes the pelvic cavity, uterus, fallopian tubes, and ovaries. It is believed to be a major factor controlling the peritoneal microenvironment that influences the development and progression of endometriosis and endometriosis-associated infertility. Peritoneal fluid is formed in part by the contribution of the follicular activity, corpus luteum vascularity, and hormonal production. The volume of PF is dynamic and phasedependent, peaking at the time of ovulation. [30] The PF ingredients are variable in normal menstrual cycles and different pathologic entities. [31,32] Women with endometriosis were found to have a greater PF volume than fertile controls, patients with tubal disease, or those with unexplained infertility. Moreover, an increased volume of PF may be commonly associated with endometriosis and with idiopathic infertility. CA-125 has been measured in PF of patients with and without endometriosis by many investigators. [33–35] Although PF levels of CA-125 were almost tenfold higher than the serum ones, no differences were found between women with and without endometriosis. [36] Measurement of CA-125 in other body fluids, such as menstrual discharge [37] and uterine fluid [38] have been tried but not found useful in clinical practice.

Serum and Peritoneal Markers

131

IMMUNOLOGICAL MARKERS A significant role of the immune system in the pathogenesis of endometriosis has been recently documented. [2] Based on these recent findings, the concept of treating endometriosis as an autoimmune disease is emerging. [39] Accumulating evidence suggests that systemic T-cell activity influences the pathogenesis of endometriosis. [40,41] Altered T-helper to T-suppressor ratio and concentration of both cells, respectively, have been reported in serum, PF, [42] and endometriotic tissue [43] in endometriosis patients. Moreover, such differences could be detected between eutopic endometrium from women with and without the disease. There is lack of consistency regarding the alterations in T-cells and their role in the pathophysiology of endometriosis. Natural killer (NK) cells are also altered in endometriosis. Both peripheral and peritoneal fluid NK cells from women with endometriosis showed different characteristics compared with those of the controls. [44] Additionally, NK cell cytotoxicity has been shown to be inversely correlated with the stages of the disease. [45] Consequently, altered NK cytotoxicity to endometrial tissue may be responsible in part for the initiation, propagation, and establishment of pelvic endometriosis. Sera and PF from women with endometriosis have been shown to reduce NK cell activity. [46] This is probably caused by monocyte or macrophage activity through their secretions that modulate immune and nonimmune cells. Besides the alterations of T-cell functions, many recent findings have shown alterations in B-cell function in endometriosis patients as evidenced by abnormal antigen-antibody reaction and increased B-cell function. Decreased C3 deposition in the endometrium and a corresponding reduction in the serum total complement levels has been shown in endometriosis patients. [47] Antiendometrial antibodies particularly IgG and IgA have been detected in sera, and vaginal and cervical secretions of endometriosis patients. [48] The presence of antiphospholipids and antihistones of IgG, IgM, and IgA have been documented by some investigators [49] and questioned by others. [50] The exact correlation between the stage of endometriosis and autoantibodies ranges from positive [51] to negative [4] to no relationship at all. [52] These observations of immune alterations have lead investigators to believe that markers of immune reactivity , particularly cytokines, may be potentially used as a diagnostic aid for endometriosis. Cytokines: Chemistry Cytokines are polypeptides or glycoproteins secreted into the extracellular compartment mainly by leukocytes. Upon secretion, they exert autocrine, paracrine, and sometimes endocrine effects. Moreover, cytokines may exist in cell-membrane-associated forms where they exert juxtacrine activity on

132

Falcone and Bedaiwy

adjacent cells. They are essential mediators of cell-cell communication in the immune system. They affect a wide variety of target cells exerting proliferative, cytostatic, chemoattractant, or differentiative effects. Their biological activities are mediated by coupling to intracellular signaling and secondmessenger pathways via specific high-affinity receptors on target cell membranes. The cytokine nomenclature reflects the historical description of these biological activities. Cytokines: Sources The main source of cytokines is macrophages, which originate in bone marrow, circulate as monocytes, and migrate to various body cavities. Chemoattractant cytokines, particularly regulated on activation, normal T-cell expressed and secreted (RANTES), and interleukin (IL)-8, facilitate macrophage recruitment into the peritoneal cavity. The second major source of cytokines is T-lymphocytes. Helper T-cells can be classified into two subsets: type 1 (Th1) and type 2 (Th2). Th1 cells produce IL-2, IL-12, and interferon-g, which are potent inducers of cell-mediated immunity. Th2 cells produce mainly IL-4, IL-5, IL-10, and IL-13, which are involved in suppression of cell-mediated immunity. There is alteration of cytokines secreted by Th1 and Th2 in endometriosis patients particularly in the balance of Th1 and Th2 cells toward the Th2. This may, in part, be responsible for the impaired immunological defense in endometriosis. [53] Tsudo et al hypothesized that cytokines are not only produced by immune competent cells but by endometriotic implants as well. [54] They demonstrated that endometriotic cells constitutively express IL-6 messenger RNA and produce IL-6 protein, and that adding TNF-a stimulated IL-6 gene and protein expression in a dose-dependent manner. On comparing IL-6 production by macrophages and endometriotic stromal cells in patients with endometriosis, they found that similar levels of IL-6 were produced in stromal cells derived from an endometrioma and by macrophages under basal- and TNF-a stimulated conditions. This finding supports the hypothesis that endometriotic tissue is another important source of cytokines. [54] Peritoneal Fluid Cytokines Peritoneal fluid is rich with variable cellular components including macrophages, mesothelial cells, lymphocytes, eosinophils, and mast cells. The normal concentration of PF leukocytes is 0.5 to 2.0  106/ml, of which approximately 85% are macrophages. [31,32] It has been hypothesized that peritoneal macrophage activation is a pivotal step in the disease initiation and progression. [55] Activated macrophages in the peritoneal cavity of women with endometriosis are potent producers of cytokines. [3] Thus, PF contains a

Serum and Peritoneal Markers

133

rich mixture of cytokines. Iron overload was also observed in the cellular and PF compartments of the peritoneal cavity of endometriosis patients, suggesting a role in its pathogenesis. [56] Individual Cytokines Tumor Necrosis Factors The tumor necrosis factors (TNF) are pleiotropic cytokines that exert an essential role in the inflammatory process. It is believed to be seminal in many physiological and pathological reproductive processes. The spectrum of its activity is very wide, with both beneficial and hazardous effects. The quantity of TNF produced is the main factor that controls its role in the disease process. The main TNF is TNF-a, which is produced by neutrophils, activated lymphocytes, macrophages, NK cells, and several nonhematopoietic cells. Little is known about TNF-h, which is produced by lymphocytes. The primary function of TNFs is their ability to initiate the cascade of cytokines and other factors associated with inflammatory responses. TNF-a helps to activate helper T-cells. In the human endometrium, TNF- a is a factor in the normal physiology of endometrial proliferation and shedding. Tumor necrosis factor-a is expressed mostly in epithelial cells, particularly in the secretory phase. [57] Stromal cells stain for TNF- a mostly in the proliferative phase of the cycle. These data suggest a hormonal control of this cytokine. [58] Peritoneal fluid TNF- a concentrations are elevated in patients with endometriosis, and some studies show higher concentrations correlate with the stage of the disease [59]. Our study did not observe any relationship between levels of TNF-a and stage of the disease. [3] The source of the elevated TNF-a concentration in the PF of endometriosis patients is variable. Some in vitro studies suggest that peritoneal macrophages [60] and peripheral blood monocytes [61] from these patients have up regulated TNF-a protein secretion. Activated macrophages play a critical role in the pathogenesis of endometriosis. The secreted TNF-a may play an important role in the local and systemic manifestations of the disease. Because of its importance in other inflammatory processes, it is likely that this cytokine plays a central role in the pathogenesis of endometriosis. [62] Moreover, its level in the PF can be used as a foundation for nonsurgical diagnosis of endometriosis. [3] Recently, the concept of using TNF-a blockers in treating endometriosis has been gaining popularity. [39] Interleukin-6 Interleukin-6 is a regulator of inflammation and immunity, which may be a physiological link between the endocrine and immune systems. It also

134

Falcone and Bedaiwy

modulates secretion of other cytokines, promotes T-cell activation and Bcell differentiation, and inhibits growth of various human cell lines. [39] Monocytes, macrophages, fibroblasts, endothelial cells, vascular smoothmuscle cells, and endometrial epithelial, stromal cells, and several endocrine glands, including the pituitary and the pancreas are all production sites for IL-6. [63] The role of IL-6 in the pathogenesis of endometriosis was extensively studied. Interleukin-6 response in the peritoneal macrophages, [64] endometrial stromal cells, [65] and peripheral macrophages [61] was dysregulated in patients with endometriosis. The level of IL-6 detected in the PF of patients with endometriosis was inconsistent. Some investigators have demonstrated elevated concentrations, [66,67] whereas others have found no elevation. [68] Some studies failed to demonstrate statistically significant differences in IL-6 levels between controls and endometriosis patients. [69] These inconsistent findings likely are related to antibody specificity of the assay. In our recent study, we found significant elevation of IL-6 in the sera of endometriosis patients but not in the PF as compared with patients with unexplained infertility and tubal ligation/reanastomosis. [3] Vascular Endothelial Growth Factor Many studies focused on the proliferation and neovascularization of the endometriotic implants. Vascular endothelial growth factor (VEGF) is one of the most potent and specific angiogenic factors. The main biochemical activity of VEGF when it binds to its targeted receptor is that VEGF-receptor activation leads to a rapid increase in intracellular Ca2+ and inositol triphosphate concentrations in endothelial cells. [70,71] The basic physiological function of VEGF is that VEGF-induced angiogenesis allows repair of the endometrium after menstruation. It also modulates the characters of the newly formed vessels by controlling the microvascular permeability, permitting the formation of a fibrin matrix for endothelial migration and proliferation. [72] This may be responsible for the local endometrial edema, which helps to prepare the endometrium for embryo implantation. [73] In endometriosis patients, VEGF was localized in the epithelium of endometriotic implants, [74] particularly in hemorrhagic red implants. [75] Moreover, there are increased concentrations of VEGF in PF of endometriosis patients. The exact cellular sources of VEGF in PF have not been precisely defined yet. Although evidence exists to suggest that endometriotic lesions themselves produce this factor, [74] activated peritoneal macrophages also have the capacity to synthesize and secrete VEGF. [76] Similar to the concept of using TNF-a blockers, antiangiogenic drugs are potential therapeutic agents in endometriosis.

Serum and Peritoneal Markers

135

RANTES RANTES belongs to the h or ‘‘C-C’’ chemokine family. It attracts monocytes and memory T-cells. RANTES is a secretory product of hematopoietic cells, epithelial and mesenchymal cells, and a mediator in both acute and chronic inflammation. [77] RANTES protein distribution in ectopic endometrium is similar to that found in a eutopic endometrium. [78] However, in vitro secretion of RANTES by endometrioma-derived stromal-cell cultures is significantly greater than in eutopic endometrium. In this way, PF concentrations of RANTES may be increased in patients with endometriosis. [79] Interleukin-1 Interleukin-1 is a key cytokine in the regulation of inflammation and immune responses. I+ affects the activation of T-lymphocytes and the differentiation of B-lymphocytes. There are two receptors for IL-1, namely IL-1a and IL-1h sharing only 18% to 26% amino acid homology. Both receptors are encoded by different genes but have similar biological activities. It was found that successful implantation in mice was blocked by the administration of exogenous IL-1 receptor antagonist. This illustrates their important role in the implantation of the ectopic endometrium. [80] Interleukin-1 has been isolated from the PF of patients with endometriosis. Results have been inconsistent, with some investigators demonstrating elevated concentrations in patients with endometriosis [81] and others finding no elevation. [3,60,82] Other Cytokines Highly sensitive enzyme-linked immunosorbent assay (ELISA) kits have made it easy to measure the entire battery of cytokines in the serum and PF of endometriosis patients. Other cytokines have been identified and include IL-4 [53]; IL-5 [66]; IL-8 [3,83]; IL-10 [84]; IL-12 [3,85]; IL-13 [86]; interferong [68]; monocyte chemotactic protein-1 (MCP-1) [87]; macrophage colony stimulating factor (MCSF), [88] and transforming growth factor (TGF)- h. [89] All these cytokines may regulate the actions of leukocytes or may act directly on ectopic endometrium, where they may play various roles in the pathogenesis and pathophysiology of endometriosis. However, their exact role needs further investigation. Cytokines as a Screening Tool The role of cytokines and growth factors in the pathophysiology of endometriosis is evident, as previously discussed. They are probably responsible for

136

Falcone and Bedaiwy

endometrial cell proliferation [90,91] and implantation of endometrial cells or tissue. [92] Moreover, cytokines increased tissue remodeling through their influence on the matrix metalloproteinases. [93] Increased angiogenesis of the ectopic endometrial tissue and neovasculariztion of the affected region are probably the most important effects of cytokines on ectopic endometrial tissue. Consequently cytokines play a major role in the initiation, propagation, and regulation of immune and inflammatory responses. Immune cell activation results in a burst and cascade of inflammatory cytokines. Besides their role in the pathogenesis of endometriosis, they might have a diagnostic role as well. To evaluate this hypothesis, we conducted a prospective controlled trial to investigate the ability of a group of serum and PF markers to nonsurgically predict endometriosis. [3] Serum and PF from 130 women were obtained while they underwent laparoscopy for pain, infertility, tubal ligation, or sterilization reversal. We measured the concentrations of six cytokines (IL-1h, IL-6, IL-8, IL-12, IL-13, and TNF-a) in serum and PF and reactive oxygen species (ROS) in PF, and compared the levels among women who were divided into groups according to their postsurgical diagnosis. Fiftysix patients were diagnosed with endometriosis, eight were diagnosed with idiopathic infertility, 27 had undergone tubal ligation or reanastomosis (control group), and 39 were excluded because of bloody PF. Only serum IL-6 and PF TNF-a were able to discriminate between patients with endometriosis and those without the disease with a high degree of sensitivity and specificity. The PF TNF-a had an exceptional 99% area under the curve (95% CI: 97% to 100%), indicating a very high discrimination ability (Fig. 3). A cutoff of 15 pg/ml provided 100% sensitivity and 89% specificity (positive likelihood ratio of 9.1 and negative likelihood ratio of 0). A cutoff of 20 pg/ml yielded 96% sensitivity and 95% specificity (positive likelihood ratio of 19.2 and negative likelihood ratio of 0.04). The serum IL-6 achieved a relatively high diagnostic value with an area under the curve of 87% (95% CI: 75% to 99%) [Fig. 3]. A serum IL-6 cutoff of 2 pg/ml provided a sensitivity of 90% and specificity of 67% (positive likelihood ratio of 2.7 and negative likelihood ratio of 0.14). A cutoff of 4 pg/ml provided sensitivity of 85% and specificity of 80% (positive likelihood ratio of 4.3 and negative likelihood ratio of 0.19), and a cut-off of 7.5 pg/ml provided sensitivity of 80% and specificity of 87% (positive likelihood ratio of 6.2 and negative likelihood ratio of 0.23). The positive and negative likelihood ratios of PF TNF-a are so good that, it is possible that ultrasonographically guided transvaginal aspiration of the PF from the cul-de-sac may serve as a basis for the nonsurgical diagnosis of endometriosis. However, the study had two main limitations. First, there may not be enough serum and PF to measure the cytokines. Second, all bloody PF samples were excluded because cytokine levels may be affected by

Serum and Peritoneal Markers

137

FIGURE 3 Receiver operating characteristic (Roc) curves of the endometriosis versus nonendometriosis groups. The area under the curve (AUC) for the peritoneal fluid TNF-a is 99%, and the AUC for the serum IL-6 is 87%. (From Ref. 3.)

blood contamination. Consequently, the study conclusions are not applicable to patients with blood-contaminated PF. This study showed that serum IL-6 and PF TNF-a might be potential markers for endometriosis, thereby allowing for nonsurgical diagnosis. Autoantibodies A variety of autoantibodies have been detected in endometriosis patients. The most commonly reported types are antiendometrial antibodies [47,94] and autoantibodies against oxidative stress parameters. [95] Antiendometrial Antibodies The antigens used to induce antiendometrial antibodies included sonicated endometrium of women with normal menstrual cycles, endometrial tissue of patients with endometriosis, endometriosis tissue, human endometrial carcinoma cells line, epithelial monolayers or endometrial glands, and stromal cells. The exact antigen is not known; consequently, there is no simple antigen-antibody assay as yet. [4] Serum Antiendometrial Antibodies. —Antiendometrial antibodies have been postulated to be a probable cause of infertility in endometriosis patients as shown by some investigators, [47,94] but not by others. [96] The of the assay techniques used are inconsistent, [97] as is the nature of the antigens used to elicit immune response.

138

Falcone and Bedaiwy

The sensitivity and the specificity of serum antiendometrial antibodies screening were reported by some investigators to be 0.84 and 1.00, respectively. [48] On comparing infertile women with endometriosis with unexplained infertility, Wild and Shivers, found a sensitivity of 0.71 and a specificity of 1.00. [52] Similarly, Meek et al [50] found a sensitivity of 0.75 and a specificity of 0.90, whereas in another study the values were 0.85 and 0.67, respectively. [98] Although serum antiendometrial antibodies match CA-125 regarding both sensitivity and specificity, it does not satisfy the criteria of an ideal screening test mentioned in Table 1. Despite this limitation, antiendometrial antibody was proposed as both a screening marker and as a follow-up marker of treatment results and recurrence. [99] Peritoneal Fluid Antiendometrial Antibodies. —Although antiendometrial antibodies were found in the PF of endometriosis patients, their sensitivity and specificity are variable. Halme and Mathur found a sensitivity of 0.23 and a specificity of 0.96 using a passive hemagglutination assay, [100] whereas the results were 0.75 and 0.90 using Ouchterlony immune diffusion. [50] Autoantibodies to Markers of Oxidative Stress There is increasing evidence of oxidative stress in the PF of women with endometriosis, and it has been shown that oxidatively modified lipid proteins exist in the PF. [1,3,101] In addition, oxidation-specific epitopes and macrophages are present in the endometrium and in endometriosis. [95] Lipid peroxides interact with proteins, resulting in several types of alterations, and such oxidatively modified proteins are themselves antigenic. Antigenicity is attributed to specific modified epitopes and not to the protein backbone. In a study to measure autoantibodies to oxidatively modified proteins in the sera of women with surgically proven endometriosis, Murphy et al included women undergoing surgery for endometriosis or tubal ligation. [95] They measured serum and PF autoantibody titers to malondialdehydemodified low-density lipoprotein, oxidized low-density lipoprotein, and lipid peroxide-modified rabbit serum albumin determined by enzyme-linked immunosorbent assay (ELISA). They correlated the autoantibody titers with the disease stage, symptoms, and morphological type of endometriosis. They found that autoantibodies to markers of oxidative stress were significantly increased in women with endometriosis without any correlation with the stage, symptoms, or morphological type of the disease. Peritoneal fluid did not contain autoantibodies to any of the three antigens. Given the fact that autoantibodies to Ox-LDL have been long considered as a screening tool for atherosclerosis, [102] a similar role might be claimed in endometriosis.

Serum and Peritoneal Markers

139

GENTIC MARKERS OF ENDOMETRIOSIS Given the fact that the etiology of endometriosis is complex and multifactorial, endometriosis is an ideal target for genome-wide scanning. Familial inheritance plays a role, and multiple candidate genes are involved. [103] Many recent technological approaches can help identify possible genetic markers of endometriosis. A gene-based diagnostic test for endometriosis may be the long-sought ideal screening test. [104] A number of technologies have emerged to facilitate progress in this direction. The potential geneticbased technologies that may be a suitable foundation for genetic markers of the disease include subtractive cDNA hybridization [105,106] and cDNA microarray techniques. [107–110] Based on the recent DNA technologies, an ongoing study in our center is trying to create a cDNA library from normal endometrial tissue as well as endometriotic implants. Based on the refined library, potential specific serum antibodies may be identified and subsequently used as a nonsurgical screening tool for endometriosis. ENDOMETRIAL TISSUE BIOCHEMICAL MARKERS Aromatase P450 Aromatase P450 is a catalyst of the conversion of androstenedione and testosterone to estrone. This enzyme is expressed in both eutopic and ectopic endometrium of endometriosis patients but not in eutopic endometrium of healthy controls. [111] Although endometrial aromatase P450 expression did not correlate with the disease stage, a recent study demonstrated that detection of aromatase P450 transcripts in the endometrium of endometriosis patients may be a potential qualitative marker of endometriosis. [112] Another retrospective, case-controlled study also reported that seven (25%) of 28 women without detectable levels of endometrial aromatase P450 protein, determined by immunohistochemical analysis, had either endometriosis, fibroids, adenomyosis, or a combination of these disorders. [111] The potential use of such markers as a clinically useful diagnostic tool of pelvic disease is limited by the observation that large numbers of women with endometriosis did not express aromatase P450 in their eutopic endometrium. However, the use of aromatase inhibitors as a potential treatment of endometriosis is gaining momentum based on these molecular facts. [113–116] Cytokeratines It has been reported that endometriosis cell lines exhibited an epithelial-like morphology and was immunoreactive for cytokeratins 8, 18, 19, vimentin, and human leukocyte class I antigens in culture. [117] However, the cultured

140

Falcone and Bedaiwy

cells were negative for a whole set of hematopoietic cell markers, including the lymphoid cell antigens CD3, CD20, and CD45, von Willebrand factor, carcinoembryonic antigen, and CA-125. [117] In another study, endometriotic tissues from various locations were immunostained for estrogen receptor, vimentin, Ber-EP-4, and cytokeratins. [118] Using immunofluorescence with monoclonal antibodies against cytoskeletal components and epithelial mucins, Matthews and associates studied the staining patterns for cytokeratins 18 and 19, vimentin, and three different epithelial mucins in endometriotic cell culture. They found that cytokeratins were located in epithelial cells, and vimentin was expressed in both stromal and epithelial cells. [119] No studies evaluated the potential use of these molecular markers as a potential diagnostic/screening tool in endometriosis. Hormone Receptors Given the fact that endometriosis is an estrogen-dependent condition, the expression of estrogen and progesterone receptors could be potentially useful as a screening marker of the disease. The contents of estrogen and progesterone receptors in eutopic endometrium are phase dependent and cyclic. [120] However, the eutopic endometrium of patients with endometriosis was very different from normal endometrium in apoptosis, [121] cytokines, and other characteristics. [65] Although cyclic changes were also detected in ectopic endometrium, they differed greatly from those of eutopic endometrium. The concentrations of steroid receptors in ectopic endometrium increased gradually as the cycle progressed. Compared with eutopic endometrium, estrogen and progesterone receptor concentrations were significantly lower in the proliferative phase, similar in the early secretory phase, and significantly higher in the late secretory phase. [122] The different patterns of receptor expression suggested different hormonal regulations between eutopic and ectopic endometrium. [120] There are two isoforms for E receptor, E receptor-a and E receptor-h, and two for P receptor, P receptor-A and P receptor-B. These isoforms exist in the endometrium and their function and content are different from each another. [123] The different concentration and biological activities of steroid receptor isoforms might lead to various hormonal responsiveness of ectopic endometrium. High concentrations of E and P receptors in the ectopic endometrium during the secretory phase could explain the high proliferative activity of endometriotic tissue in this phase. Conversely, a decrease in E and P receptor expression in ectopic implants during the secretory phase might lead to diminished proliferation. [122] The expression of estrogen and progesterone receptors may be regarded as an index of differentiation of the endometriotic implant. Consequently, E and P receptors may be used as markers of the activity of all subtypes of endometriotic lesions. [4]

Serum and Peritoneal Markers

141

SUMMARY One of the major challenges facing gynecologists is the inability to diagnose endometriosis because a definitive diagnosis of the disease can only be obtained by laparoscopy or laparotomy. At present, reliable markers of the disease are lacking. Serum concentration of tumor markers—particularly CA-125, the most extensively studied and used serum marker of endometriosis—has a limited diagnostic performance. This poses a problem for investigating the presence of early stages of the disease. The detection of endometriosis is critical in stage I to II in infertile women in whom the laparoscopic treatment of the lesions has been reported to almost double the rate of a spontaneous pregnancy. The high specificity of the CA-125 determination indicates its potential usefulness in disease monitoring and follow-up. Because medical treatment has been associated with temporary suppression of the disease activity, CA-125 may be important only in long-term monitoring of surgical therapy. Randomized clinical trials on the use of surgery for infertility or pain associated with endometriosis have shown a clear benefit. [124] This shows that early detection is critical part of the diagnosis and treatment of the disease. Immunological markers gained importance with the accumulating evidence regarding the immunological changes that occur during the evolution of the disease. The most promising diagnostic test is the use of peritoneal fluid and serum cytokines. [125,126] Large clinical trials will be needed to validate this hypothesis. Autoantibodies aim at detecting circulating antibodies raised against a variety of antigens. Their use as a screening tool is limited by their low sensitivity. A combination of the recent immunological discoveries and the recent advances in DNA technologies may provide the long-sought screening tool with the desirable accuracy for such a puzzling disorder.

PRACTICAL POINT .

CA-125 as a serum marker of endometriosis has a limited diagnosis value. It is only important in long-term monitoring after surgical therapy.

REFERENCES 1.

2. 3.

Wang Y, Sharma RK, Falcone T, Goldberg J, Agarwal A. Importance of reactive oxygen species in the peritoneal fluid of women with endometriosis or idiopathic infertility. Fertil Steril 1997; 68:826–830. Lebovic DI, Mueller MD, Taylor RN. Immunobiology of endometriosis. Fertil Steril 2001; 75:1–10. Bedaiwy MA, Falcone T, Sharma RK, Goldberg JM, ATTaran M, Nelson DR,

142

4. 5.

6. 7. 8.

9.

10. 11.

12.

13.

14.

15.

16.

17.

18.

Falcone and Bedaiwy Agarwal A. Prediction of endometriosis with serum and peritoneal fluid markers: A prospective controlled trial. Hum Reprod 2002; 17:426–431. Evers JL, Dunselman GA, van der Linden PJ. Markers for endometriosis. Baillieres Clin Obstet Gynaecol 1993; 7:715–739. Barbieri RL, Niloff JM, Bast RC Jr, Scaetzl E, Kistner RW, Knapp RC. Elevated serum concentrations of CA-125 in patients with advanced endometriosis. Fertil Steril 1986; 45:630–634. Meden H, Fattahi-Meibodi A. CA 125 in benign gynecological conditions. Int J Biol Markers 1998; 13:231–237. Pittaway DE, Fayez JA. Serum CA-125 antigen levels increase during menses. Am J Obstet Gynecol 1987; 156:75–76. Eltabbakh GH, Belinson JL, Kennedy AW, Gupta M, Webster K, Blumenson LE. Serum CA-125 measurements >65 U/mL. Clinical value. J Reprod Med 1997; 42:617–624. Malkasian GD Jr, Knapp RC, Lavin PT, Zurawski VR Jr, Podratz KC, Stanhope CR, Mortel R, Berek JS, Bast RC Jr, Ritts RE. Preoperative evaluation of serum CA 125 levels in premenopausal and postmenopausal patients with pelvic masses: Discrimination of benign from malignant disease. Am J Obstet Gynecol 1988; 159:341–346. Pittaway DE, Fayez JA. The use of CA-125 in the diagnosis and management of endometriosis. Fertil Steril 1986; 46:790–795. Moretuzzo RW, DiLauro S, Jenison E, Chen SL, Reindollar RH, McDonough PG. Serum and peritoneal lavage fluid CA-125 levels in endometriosis. Fertil Steril 1988; 50:430–433. Barbati A, Cosmi EV, Spaziani R, Ventura R, Montanino G. Serum and peritoneal fluid CA-125 levels in patients with endometriosis. Fertil Steril 1994; 61:438–442. Mol BW, Bayram N, Lijmer JG, Wiegerinck MA, Bongers MY, van der Veen F, Bossuyt PM. The performance of CA-125 measurement in the detection of endometriosis: A meta-analysis. Fertil Steril 1998; 70:1101–1108. Pittaway DE, Rondinone D, Miller KA, Barnes K. Clinical evaluation of CA125 concentrations as a prognostic factor for pregnancy in infertile women with surgically treated endometriosis. Fertil Steril 1995; 64:321–324. Fedele L, Arcaini L, Vercellini P, Bianchi S, Candiani GB. Serum CA 125 measurements in the diagnosis of endometriosis recurrence. Obstet Gynecol 1988; 72:19–22. Koprowski H, Steplewski Z, Mitchell K, Herlyn M, Herlyn D, Fuhrer P. Colorectal carcinoma antigens detected by hybridoma antibodies. Somatic Cell Genet 1979; 5:957–971. Ye C, Ito K, Komatsu Y, Takagi H. Extremely high levels of CA19-9 and CA125 antigen in benign mucinous ovarian cystadenoma. Gynecol Oncol 1994; 52:267–271. Imai A, Horibe S, Takagi A, Takagi H, Tamaya T. Drastic elevation of serum CA125, CA72-4 and CA19-9 levels during menses in a patient with probable endometriosis. Eur J Obstet Gynecol Reprod Biol 1998; 78:79–81.

Serum and Peritoneal Markers

143

19. Matalliotakis I, Panidis D, Vlassis G, Neonaki M, Goumenou A, Koumantakis E. Unexpected increase of the CA 19-9 tumuor marker in patients with endometriosis. Eur J Gynaecol Oncol 1998; 19:498–500. 20. Harada T, Kubota T, Aso T. Usefulness of CA19-9 versus CA125 for the diagnosis of endometriosis. Fertil Steril 2002; 78:733–739. 21. Vigano P, Somigliana E, Gaffuri B, Santorsola R, Busacca M, Vignali M. Endometrial release of soluble intercellular adhesion molecule 1 and endometriosis: Relationship to the extent of the disease. Obstet Gynecol 2000; 95:115–118. 22. De Placido G, Alviggi C, Di Palma G, Carravetta C, Matarese G, Landino G, Racioppi L. Serum concentrations of soluble human leukocyte class I antigens and of the soluble intercellular adhesion molecule-1 in endometriosis: Relationship with stage and non-pigmented peritoneal lesions. Hum Reprod 1998; 13:3206–3210. 23. Wu MH, Yang BC, Hsu CC, Lee YC, Huang KE. The expression of soluble intercellular adhesion molecule-1 in endometriosis. Fertil Steril 1998; 70:1139– 1142. 24. Daniel Y, Geva E, Amit A, Eshed-Englender T, Baram A, Fait G, Lessing JB. Do soluble cell adhesion molecules play a role in endometriosis? Am J Reprod Immunol 2000; 43:160–166. 25. Matalliotakis IM, Vassiliadis S, Goumenou AG, Athanassakis I, Koumantakis GE, Neonaki MA, Koumantakis EE. Soluble ICAM-1 levels in the serum of endometriotic patients appear to be independent of medical treatment. J Reprod Immunol 2001; 51:9–19. 26. Somigliana E, Vigano P, Candiani M, Felicetta I, Di Blasio AM, Vignali M. Use of serum-soluble intercellular adhesion molecule-1 as a new marker of endometriosis. Fertil Steril 2002; 77:1028–1031. 27. Mueller MD, Vigne JL, Vaisse C, Taylor RN. Glycodelin: A pane in the implantation window. Semin Reprod Med 2000; 18:289–298. 28. Telimaa S, Kauppila A, Ronnberg L, Suikkari AM, Seppala M. Elevated serum levels of endometrial secretory protein PP14 in patients with advanced endometriosis. Suppression by treatment with danazol and high-dose medroxyprogesterone acetate. Am J Obstet Gynecol 1989; 161:866–871. 29. Joshi SG. Progestin-dependent human endometrial protein: A marker for monitoring human endometrial function. Adv Exp Med Biol 1987; 230:167– 186. 30. Maathuis JB, VanLook PF, Michie EA. Changes in volume, total protein and ovarian steroid concentrations of peritoneal fluid throughout the human menstrual cycle. J Endocrinol 1978; 76:123–133. 31. Syrop CH, Halme J. Peritoneal fluid environment and infertility. Fertil Steril 1987; 48:1–9. 32. Syrop CH, Halme J. Cyclic changes of peritoneal fluid parameters in normal and infertile patients. Obstet Gynecol 1987; 69:416–418. 33. Fedele L, Vercellini P, Arcaini L, da Dalt MG, Candiani GB. CA 125 in serum peritoneal fluid, active lesions, and endometrium of patients with endometriosis. Am J Obstet Gynecol 1988; 158:166–170.

144

Falcone and Bedaiwy

34. Fisk NM, Tan CE. CA 125 in peritoneal fluid and serum of patients with endometriosis. Eur J Obstet Gynecol Reprod Biol 1988; 29:153–158. 35. Gurgan T, Kisnisci H, Yarali H, Aksu T, Zeyneloglu H, Develioglu O. Serum and peritoneal fluid CA-125 levels in early stage endometriosis. Gynecol Obstet Invest 1990; 30:105–108. 36. Kruitwagen RF, Thomas C, Poels LG, Koster AM, Willemsen WN, Rolland R. High CA-125 concentrations in peritoneal fluid of normal cyclic women with various infertility-related factors as demonstrated with two-step immunoradiometric assay. Fertil Steril 1991; 56:863–869. 37. Takahashi K, Nagata H, Musa AA, Shibukawa T, Yamasaki H, Kitao M. Clinical usefulness of CA-125 levels in the menstrual discharge in patients with endometriosis. Fertil Steril 1990; 54:360–362. 38. Abae M, Gibson M, Chapitis J, Riddick DH, Brumsted JR. CA-125 levels in human uterine fluid. Fertil Steril 1992; 57:531–534. 39. Nothnick WB. Treating endometriosis as an autoimmune disease. Fertil Steril 2001; 76:223–231. 40. Steele RW, Dmowski WP, Marmer DJ. Immunologic aspects of human endometriosis. Am J Reprod Immunol 1984; 6:33–36. 41. Badawy SZ, Cuenca V, Stitzel A, Tice D. Immune rosettes of T and B lymphocytes in infertile women with endometriosis. J Reprod Med 1987; 32:194– 197. 42. Dmowski WP, Gebel HM, Braun DP. The role of cell-mediated immunity in pathogenesis of endometriosis. Acta Obstet Gynecol Scand Suppl 1994; 159:7– 14. 43. Witz CA, Montoya IA, Dey TD, Schenken RS. Characterization of lymphocyte subpopulations and T cell activation in endometriosis. Am J Reprod Immunol 1994; 32:173–179. 44. Wilson TJ, Hertzog PJ, Angus D, Munnery L, Wood EC, Kola I. Decreased natural killer cell activity in endometriosis patients: Relationship to disease pathogenesis. Fertil Steril 1994; 62:1086–1088. 45. Ho HN, Chao KH, Chen HF, Wu MY, Yang YS, Lee TY. Peritoneal natural killer cytotoxicity and CD25+ CD3+ lymphocyte subpopulation are decreased in women with stage III-IV endometriosis. Hum Reprod 1995; 10: 2671– 2675. 46. Kanzaki H, Wang HS, Kariya M, Mori T. Suppression of natural killer cell activity by sera from patients with endometriosis. Am J Obstet Gynecol 1992; 167:257–261. 47. Weed JC, Arquembourg PC. Endometriosis: Can it produce an autoimmune response resulting in infertility? Clin Obstet Gynecol 1980; 23:885–893. 48. Mathur S, Peress MR, Williamson HO, Youmans CD, Maney SA, Garvin AJ, Rust PF, Fudenberg HH. Autoimmunity to endometrium and ovary in endometriosis. Clin Exp Immunol 1982; 50:259–266. 49. Gleicher N, Adelsberg BR, Liu TL, Cederqvist LL, Phillips RN, Siegel I. Immune complexes in pregnancy. III. Immune complexes in immune complexassociated conditions. Am J Obstet Gynecol 1982; 142:1011–1015.

Serum and Peritoneal Markers

145

50. Meek SC, Hodge DD, Musich JR. Autoimmunity in infertile patients with endometriosis. Am J Obstet Gynecol 1988; 158:1365–1373. 51. Taylor PV, Maloney MD, Campbell JM, Skerrow SM, Nip MM, Parmar R, Tate G. Autoreactivity in women with endometriosis. Br J Obstet Gynaecol 1991; 98:680–684. 52. Wild RA, Shivers CA. Antiendometrial antibodies in patients with endometriosis. Am J Reprod Immunol Microbiol 1985; 8:84–86. 53. Hsu CC, Yang BC, Wu MH, Huang KE. Enhanced interleukin-4 expression in patients with endometriosis. Fertil Steril 1997; 67:1059–1064. 54. Tsudo T, Harada T, Iwabe T, Tanikawa M, Nagano Y, Ito M, Taniguchi F, Terakawa N. Altered gene expression and secretion of interleukin-6 in stromal cells derived from endometriotic tissues. Fertil Steril 2000; 73:205–211. 55. Halme J, Becker S, Wing R. Accentuated cyclic activation of peritoneal macrophages in patients with endometriosis. Am J Obstet Gynecol 1984; 148:85– 90. 56. van Langendonckt A, Casanas-Roux F, Donnez J. Iron overload in the peritoneal cavity of women with pelvic endometriosis. Fertil Steril 2002; 78:712– 718. 57. Philippeaux MM, Piguet PF. Expression of tumor necrosis factor-alpha and its mRNA in the endometrial mucosa during the menstrual cycle. Am J Pathol 1993; 143:480–486. 58. Hunt JS, Chen HL, Hu XL, Tabibzadeh S. Tumor necrosis factor-alpha messenger ribonucleic acid and protein in human endometrium. Biol Reprod 1992; 47:141–147. 59. Eisermann J, Gast MJ, Pineda J, Odem RR, Collins JL. Tumor necrosis factor in peritoneal fluid of women undergoing laparoscopic surgery. Fertil Steril 1988; 50:573–579. 60. Keenan JA, Chen TT, Chadwell NL, Torry DS, Caudle MR. IL-1 beta, TNFalpha, and IL-2 in peritoneal fluid and macrophage-conditioned media of women with endometriosis. Am J Reprod Immunol 1995; 34:381–385. 61. Braun DP, Gebel H, House R, Rana N, Dmowski NP. Spontaneous and induced synthesis of cytokines by peripheral blood monocytes in patients with endometriosis. Fertil Steril 1996; 65:1125–1129. 62. Braun DP, Ding J, Dmowski WP. Peritoneal fluid-mediated enhancement of eutopic and ectopic endometrial cell proliferation is dependent on tumor necrosis factor-alpha in women with endometriosis. Fertil Steril 2002; 78:727– 732. 63. Laird SM, Li TC, Bolton AE. The production of placental protein 14 and interleukin 6 by human endometrial cells in culture. Hum Reprod 1993; 8:793– 798. 64. Rier SE, Parsons AK, Becker JL. Altered interleukin-6 production by peritoneal leukocytes from patients with endometriosis. Fertil Steril 1994; 61:294– 299. 65. Tseng JF, Ryan IP, Milam TD, Murai JT, Schriock ED, Landers DV, Taylor RN. Interleukin-6 secretion in vitro is up-regulated in ectopic and eutopic

146

66. 67.

68.

69.

70. 71.

72.

73.

74.

75.

76.

77.

78.

Falcone and Bedaiwy endometrial stromal cells from women with endometriosis. J Clin Endocrinol Metab 1996; 81:1118–1122. Koyama N, Matsuura K, Okamura H. Cytokines in the peritoneal fluid of patients with endometriosis. Int J Gynaecol Obstet 1993; 43:45–50. Harada T, Yoshioka H, Yoshida S, Iwabe T, Onohara Y, Tanikawa M, Terakawa N. Increased interleukin-6 levels in peritoneal fluid of infertile patients with active endometriosis. Am J Obstet Gynecol 1997; 176:593–597. Keenan JA, Chen TT, Chadwell NL, Torry DS, Caudle MR. Interferon-gamma (IFN-gamma) and interleukin-6 (IL-6) in peritoneal fluid and macrophageconditioned media of women with endometriosis. Am J Reprod Immunol 1994; 32:180–183. Taylor RN, Ryan IP, Moore ES, Hornung D, Shifren JL, Tseng JF. Angiogenesis and macrophage activation in endometriosis. Ann N Y Acad Sci 1997; 828:194–207. Lebovic DI, Mueller MD, Taylor RN. Vascular endothelial growth factor in reproductive biology. Curr Opin Obstet Gynecol 1999; 11:255–260. D’Angelo G, Struman I, Martial J, Weiner RI. Activation of mitogen-activated protein kinases by vascular endothelial growth factor and basic fibroblast growth factor in capillary endothelial cells is inhibited by the antiangiogenic factor 16-kDa N- terminal fragment of prolactin. Proc Natl Acad Sci U S A 1995; 92:6374–6378. Dvorak HN, Senger DR, Dvorak AM, Harvey VS, McDonagh J. Regulation of extravascular coagulation by microvascular permeability. Science 1985; 227: 1059–1061. Hornung D, Lebovic DI, Shifren JL, Vigne JL, Taylor RN. Vectorial secretion of vascular endothelial growth factor by polarized human endometrial epithelial cells. Fertil Steril 1998; 69:909–915. Shifren JL, Tseng JF, Zaloudek CJ, Ryan IP, Meng YG, Ferrara N, Jaffe RB, Taylor RN. Ovarian steroid regulation of vascular endothelial growth factor in the human endometrium: Implications for angiogenesis during the menstrual cycle and in the pathogenesis of endometriosis. J Clin Endocrinol Metab 1996; 81:3112–3118. Donnez J, Smoes P, Gillerot S, Casanas-Roux F, Nisolle M. Vascular endothelial growth factor (VEGF) in endometriosis. Hum Reprod 1998; 13:1686– 1690. McLaren J, Prentice A, Charnock-Jones DS, Smith SK. Vascular endothelial growth factor (VEGF) concentrations are elevated in peritoneal fluid of women with endometriosis. Hum Reprod 1996; 11:220–223. Ortiz BD, Krensky AM, Nelson PJ. Kinetics of transcription factors regulating the RANTES chemokine gene reveal a developmental switch in nuclear events during T-lymphocyte maturation. Mol Cell Biol 1996; 16:202–210. Hornung D, Ryan IP, Chao VA, Vigne JL, Schriock ED, Taylor RN. Immunolocalization and regulation of the chemokine RANTES in human endometrial and endometriosis tissues and cells. J Clin Endocrinol Metab 1997; 82: 1621– 1628.

Serum and Peritoneal Markers

147

79. Khorram O, Taylor RN, Ryan IP, Schall TJ, Landers DV. Peritoneal fluid concentrations of the cytokine RANTES correlate with the severity of endometriosis. Am J Obstet Gynecol 1993; 169:1545–1549. 80. Simon C, Frances A, Piquette GN, el Danasouri I, Zurawski G, Dang W, Polan ML. Embryonic implantation in mice is blocked by interleukin-1 receptor antagonist. Endocrinologym 1994; 134:521–528. 81. Fakih H, Baggett B, Holtz G, Tsang KY, Lee JC, Williamson HO. Interleukin1: A possible role in the infertility associated with endometriosis. Fertil Steril 1987; 47:213–217. 82. Taketani Y, Kuo TM, Mizuno M. Comparison of cytokine levels and embryo toxicity in peritoneal fluid in infertile women with untreated or treated endometriosis. Am J Obstet Gynecol 1992; 167:265–270. 83. Iwabe T, Harada T, Tsudo T, Tanikawa M, Onohara Y, Terakawa N. Pathogenetic significance of increased levels of interleukin-8 in the peritoneal fluid of patients with endometriosis. Fertil Steril 1998; 69:924–930. 84. Ho HN, Wu MY, Chao KH, Chen CD, Chen SU, Yang YS. Peritoneal interleukin-10 increases with decrease in activated CD4+ T lymphocytes in women with endometriosis. Hum Reprod 1997; 12:2528–2533. 85. Mazzeo D, Vigano P, Di Blasio AM, Sinigaglia F, Vignali M, Panina-Bordignon P. Interleukin-12 and its free p40 subunit regulate immune recognition of endometrial cells: Potential role in endometriosis. J Clin Endocrinol Metab 1998; 83:911–916. 86. McLaren J, Dealtry G, Prentice A, Charnock-Jones DS, Smith SK. Decreased levels of the potent regulator of monocyte/macrophage activation, interleukin13, in the peritoneal fluid of patients with endometriosis. Hum Reprod 1997; 12:1307–1310. 87. Arici A, Oral E, Attar E, Tazuke SI, Olive DL. Monocyte chemotactic protein-1 concentration in peritoneal fluid of women with endometriosis and its modulation of expression in mesothelial cells. Fertil Steril 1997; 67:1065–1072. 88. Fukaya T, Sugawara J, Yoshida H, Yajima A. The role of macrophage colony stimulating factor in the peritoneal fluid in infertile patients with endometriosis. Tohoku J Exp Med 1994; 172:221–226. 89. Oosterlynck DJ, Meuleman C, Waer M, Koninckx PR. Transforming growth factor-beta activity is increased in peritoneal fluid from women with endometriosis. Obstet Gynecol 1994; 83:287–292. 90. Hammond MG, Oh ST, Anners J, Surrey ES, Halme J. The effect of growth factors on the proliferation of human endometrial stromal cells in culture. Am J Obstet Gynecol 1993; 168:1131–1136; discussion 1136-1138. 91. Iwabe T, Harada T, Tsudo T, Nagano Y, Yoshida S, Tanikawa M, Terakawa N. Tumor necrosis factor-alpha promotes proliferation of endometriotic stromal cells by inducing interleukin-8 gene and protein expression. J Clin Endocrinol Metab 2000; 85:824–829. 92. Zhang RJ, Wild RA, Ojago JM. Effect of tumor necrosis factor-alpha on adhesion of human endometrial stromal cells to peritoneal mesothelial cells: An in vitro system. Fertil Steril 1993; 59:1196–1201.

148

Falcone and Bedaiwy

93. Osteen KG, Keller NR, Feltus FA, Melner MH. Paracrine regulation of matrix metalloproteinase expression in the normal human endometrium. Gynecol Obstet Invest 1999; 48:2–13. 94. Badawy SZ, Cuenca V, Stitzel A, Jacobs RD, Tomar RH. Autoimmune phenomena in infertile patients with endometriosis. Obstet Gynecol 1984; 63:271– 275. 95. Murphy AA, Palinski W, Rankin S, Morales AJ, Parthasarathy S. Evidence for oxidatively modified lipid-protein complexes in endometrium and endometriosis. Fertil Steril 1998; 69:1092–1094. 96. Dunselman GA, Bouckaert PX, Evers JL. The acute-phase response in endometriosis of women. J Reprod Fertil 1988; 83:803–808. 97. Wild RA, Shivers CA, Medders D. Detection of antiendometrial antibodies in patients with endometriosis: Methodological issues. Fertil Steril 1992; 58:518– 521. 98. Wild RA, Hirisave V, Podczaski ES, Coulam C, Shivers CA, Satyaswaroop PG. Autoantibodies associated with endometriosis: Can their detection predict presence of the disease? Obstet Gynecol 1991; 77:927–931. 99. Kennedy SH, Starkey PM, Sargent IL, Hicks BR, Barlow DH. Antiendometrial antibodies in endometriosis measured by an enzyme- linked immunosorbent assay before and after treatment with danazol and nafarelin. Obstet Gynecol 1990; 75:914–918. 100. Halme J, Mathur S. Local autoimmunity in mild endometriosis. Int J Fertil 1987; 32:309–311. 101. Murphy AA, Santanam N, Morales AJ, Parthasarathy S. Lysophosphatidyl choline, a chemotactic factor for monocytes/T-lymphocytes is elevated in endometriosis. J Clin Endocrinol Metab 1998; 83:2110–2113. 102. Salonen JT, Yla-Herttuala S, Yamamoto R, Butler S, Korpela H, Salonen R, Nyyssonen K, Palinski W, Witztum JL. Autoantibody against oxidised LDL and progression of carotid atherosclerosis. Lancet 1992; 339: 883–887. 103. Stefansson H, Geirsson RT, Steinthorsdottir V, et al. Genetic factors contribute to the risk of developing endometriosis. Hum Reprod 2002; 17:555–559. 104. Taylor RN, Lundeen SG, Giudice LC. Emerging role of genomics in endometriosis research. Fertil Steril 2002; 78:694–698. 105. Vaisse C, Atger M, Potier B, Milgrom E. Human placental protein 14 gene: Sequence and characterization of a short duplication. DNA Cell Biol 1990; 9:401–413. 106. Kao LC, Tulac S, Lobo S, Imani B, Yang JP, Germeyer A, Osteen K, Taylor RN, Lessey BA, Giudice LC. Global gene profiling in human endometrium during the window of implantation. Endocrinology 2002; 143:2119–2138. 107. Yan SF, Fujita T, Lu J, Okada K, Shan Zou Y, Mackman N, Pinsky DJ, Stern DM. Egr-1, a master switch coordinating upregulation of divergent gene families underlying ischemic stress. Nat Med 2000; 6:1355–1361. 108. Yan SF, Pinsky DJ, Mackman N, Stern DM. Egr-1: Is it always immediate and early? J Clin Invest 2000; 105:553–554. 109. Vidal F, Aragones J, Alfranca A, de Landazuri MO. Up-regulation of vascular

Serum and Peritoneal Markers

110. 111.

112.

113.

114.

115. 116. 117.

118. 119.

120.

121.

122.

123. 124.

149

endothelial growth factor receptor Flt-1 after endothelial denudation: Role of transcription factor Egr-1. Blood 2000; 95:3387–3395. Eyster KM, Boles AL, Brannian JD, Hansen KA. DNA microarray analysis of gene expression markers of endometriosis. Fertil Steril 2002; 77:38–42. Kitawaki J, Noguchi T, Amatsu T, Maeda K, Tsukamoto K, Yamamoto T, Fushiki S, Osawa Y, Honjo H. Expression of aromatase cytochrome P450 protein and messenger ribonucleic acid in human endometriotic and adenomyotic tissues but not in normal endometrium. Biol Reprod 1997; 57: 514–519. Dheenadayalu K, Mak I, Gordts S, Campo R, Higham J, Puttemans P, White J, Christian M, Fusi L, Brosens J. Aromatase P450 messenger RNA expression in eutopic endometrium is not a specific marker for pelvic endometriosis. Fertil Steril 2002; 78:825–829. Takayama K, Zeitoun K, Gunby RT, Sasano H, Carr BR, Bulun SE. Treatment of severe postmenopausal endometriosis with an aromatase inhibitor. Fertil Steril 1998; 69:709–713. Bulun SE, Zeitoun K, Takayama K, Noble L, Michael D, Simpson E, Johns A, Putman M, Sasano H. Estrogen production in endometriosis and use of aromatase inhibitors to treat endometriosis. Endocr Relat Cancer 1999; 6:293– 301. Zeitoun KM, Bulun SE. Aromatase: A key molecule in the pathophysiology of endometriosis and a therapeutic target. Fertil Steril 1999; 72:961–969. Bulun SE, Zeitoun KM, Takayama K, Simpson E, Sasano H. Aromatase as a therapeutic target in endometriosis. Trends Endocrinol Metab 2000; 11:22–27. Bouquet de Joliniere J, Validire P, Canis M, Doussau M, Levardon M, Gogusev J. Human endometriosis-derived permanent cell line (FbEM-1): Establishment and characterization. Hum Reprod Update 1997; 3:117–123. Mai KT, Yazdi HM, Perkins DG, Parks W. Pathogenetic role of the stromal cells in endometriosis and adenomyosis. Histopathology 1997; 30:430–442. Matthews CJ, Redfern CP, Hirst BH, Thomas EJ. Characterization of human purified epithelial and stromal cells from endometrium and endometriosis in tissue culture. Fertil Steril 1992; 57:990–997. Nisolle M, Casanas-Roux F, Donnez J. Immunohistochemical analysis of proliferative activity and steroid receptor expression in peritoneal and ovarian endometriosis. Fertil Steril 1997; 68:912–919. Dmowski WP, Gebel H, Braun DP. Decreased apoptosis and sensitivity to macrophage mediated cytolysis of endometrial cells in endometriosis. Hum Reprod Update 1998; 4:696–701. Jiang J, Wu R, Wang Z, Sun H, Xu Z, Xiu H. Effect of mifepristone on estrogen and progesterone receptors in human endometrial and endometriotic cells in vitro. Fertil Steril 2002; 77:995–1000. Oehler MK, Rees MC, Bicknell R. Steroids and the endometrium. Curr Med Chem 2000; 7:543–560. Marcoux S, Maheux R, Berube S. Laparoscopic surgery in infertile women with minimal or mild endometriosis. Canadian Collaborative Group on Endometriosis. N Engl J Med 1997; 337:217–222.

150

Falcone and Bedaiwy

125. Bedaiwy MA, Falcone T, Goldberg JF, Attaran M, Nelson DR, Sharma RK, Agarwal B. Asystemic cytokine production in endometriosis patients: Prospective controlled study. In: CCF 21st Annual Research Day, October 25, (2001): Abstract 115. 126. Bedaiwy MA, Falcone T, Goldberg JF, Attaran M, Nelson DR, Sharma RK. Local cytokine production and reactive oxygen species in the peritoneal fluid of endometriosis patients: Prospective controlled study. In: CCF 21st Annual Research Day, October 25, (2001): Abstract 116.

9 How Does Endometriosis Cause Infertility? Ariane Germeyer Stanford University School of Medicine Stanford, California, U.S.A. and University of Heidelberg Heidelberg, Germany

Linda C. Giudice Stanford University School of Medicine Stanford, California, U.S.A.

The association between infertility and endometriosis is well established [1]. However, mechanisms by which endometriosis causes infertility are complex and still largely not well defined [2]. One reason for the controversy surrounding the development of infertility in patients with endometriosis is that variation of disease characteristics and severity make study design more challenging. The fact that not all women with endometriosis experience infertility [3] and up to 50% of women with infertility are diagnosed with endometriosis [4], underscores the complexity of the mechanisms involved in the development of infertility in women with endometriosis. This benign disease, which affects 10% of women of reproductive age, can impair female reproduction in nearly every process during attempted conception. This has been shown in the meta-analysis of 22 studies performed by Barnhart et al, which com151

152

Germeyer and Giudice

pared the reproductive outcome after in vitro fertilization (IVF) of women with endometriosis to women with tubal obstruction from other causes. In this study women with endometriosis were found to have lower implantation, fertilization, and pregnancy rates, as well as fewer oocytes retrieved. The impact on those factors (with the exception of the fertilization rate) was more pronounced in women with severe endometriosis compared to those with mild disease [5]. The study emphasizes the multifactorial character of the underlying infertility problem in patients with endometriosis, including oocyte quality, embryo maturation, and interaction of the embryo with the endometrium. It is important to note that the prevalence of associated infer-

FIGURE 1 Factors with potential influence on reproductive functions in women with endometriosis.

Endometriosis and Infertility

153

tility factors in patients with endometriosis is similar to those without the disease [6,7], implying that minimizing endometriosis-related infertility factors may not necessarily lead to a viable pregnancy. It is known that fertility rates decrease with moderate to severe endometriosis, as defined by the revised classification of the American Fertility Society [8], as staging is related to anatomical changes in the pelvis. An observational study showed that advanced endometriosis has a higher incidence of infertility than early-stage endometriosis [9]. However, the pathogenesis of infertility in women with minimal to mild endometriosis is less obvious and remains a source of continued debate. Herein, we review the different factors involved in infertility in patients with endometriosis. These include known anatomical changes that lead to adhesions or tubal obstruction, potential negative effects of peritoneal and follicular fluid on egg maturation and sperm motility, potential oocyte maturational abnormalities and hormonal variations, and the effects of endometriosis on uterine receptivity for embryonic implantation (Fig. 1). ANATOMICAL CHANGES CONTRIBUTING TO INFERTILITY Endometriosis can lead to adhesions within the abdominal cavity caused by inflammatory reactions. In moderate to severe endometriosis [8], these adhesions occur between several intra-abdominal and pelvic structures and are usually visualized easily during laparoscopy or laparotomy. Peritubular and periovarian adhesions [10], as well as tubal wall fibrosis, can lead to decreased tubal motility and decreased access to the ovulated oocyte, interrupting ovum pickup [11]. Minor adhesions that are less obvious include intratubal adhesions that lead to blockage of the Fallopian tubes and the inability to transport oocytes to the uterine cavity. Unfortunately radiographic imaging is unreliable, and anatomical distortions can only be adequately diagnosed at the time of surgery [12]. Endometriomas can be diagnosed by ultrasound or MRI [13], but their associated effects on tubal structure and function are variable and effects on fertility cannot be predicted. IMMUNOLOGIC FACTORS Several components of peritoneal fluid (PF), such as growth factors, hormones, and cytokines, are known to be present at different levels in women with endometriosis compared to women without disease. These components are believed to contribute to development of disease by creating a more favorable environment for the ectopic endometrial tissue to implant on peritoneal and other surfaces [14–17]. The peritoneal environment may also affect sperm motility, oocyte maturation, fertilization, embryo survival, and tubal function (Figs. 1 and 2).

154

Germeyer and Giudice

FIGURE 2 Factors affecting fertility in women with endometriosis at the level of sperm/oocyte interaction and maturation (see text).

Macrophages in the Peritoneal Fluid Beside various proteins and other soluble factors, PF contains endometrial and immune cells, with a predominance of peritoneal macrophages (80% to 98%) and a few lymphocytes [18,19]. Increased macrophages in PF have been observed in endometriosis-associated infertility [20], as well as in unexplained infertility [21], compared to normal controls. One explanation would be the elevated level of macrophage migration inhibitory factor (MIF) in infertile women with endometriosis, as MIF promotes accumulation and activation of macrophages [22]. Therefore, it is not surprising that macrophages from infertile women with mild endometriosis have increased activity of acid phosphatase and neutral protease. This may lead to release of active substrates from enzymatic processing in the PF and may contribute to infertility in these patients [23]. It may also lead to higher phagocytotic activity against sperm [24] and a higher activity of nitric oxide (NO) synthase. The latter has been postulated to increase NO levels in the peritoneal cavity of women with

Endometriosis and Infertility

155

endometriosis [25], to contribute to infertility by toxicity to the embryo, inhibiting embryonic implantation (in a mouse model) [26], and to have a negative effect on sperm function [27–29]. Effects on Sperm Motility As with many other underlying factors of subfertility, conflicting data exist on the effect of PF from women with endometriosis on sperm motility. Several factors have been thought to contribute to a decrease in sperm motility after exposure of sperm to PF from patients with endometriosis. For example, high levels of NO decrease sperm motility [27–29]. Soldati et al showed that PF from fertile women increases sperm motility, whereas PF from endometriosis patients had less capability of enhancing sperm motility [30]. Transferrin and HS-glycoprotein and their autoantibodies are significantly elevated in serum and PF of women with endometriosis. These autoantibodies decrease sperm motility in vitro [16,31]. Elevated levels of iron have also been found in the PF of affected women, most likely as a result of compromised iron transport resulting from transferrin antibodies in the PF. This may lead to tissue damage and, therefore, de novo adhesions in the peritoneal cavity [32]. Studies have not been able to identify direct antisperm antibodies in PF [33] or a consistent effect of PF from endometriosis patients on sperm motility when fertile endometriosis patients were tested [34]. Sperm-Oocyte Interaction The peritoneal environment may also influence sperm-oocyte interactions. Studies in hamsters and mice have shown that PF from affected women decreases sperm-oocyte interaction in a dose-dependent and disease stagespecific manner [35–37]. It remains unclear which factors are responsible for decreased sperm binding to the oocyte. Nevertheless factors such as nitric oxide produced by macrophages [38] and endometrial glycodelin [39] have been proposed as mediators. Effect on Oocyte Maturation and Toxicity Transferrin affects follicle-stimulating hormone (FSH)-induced differentiation of granulosa cells. It can potentially contribute to ovarian dysfunction, including anovulation or inadequate luteal phase [16]. Women with severe endometriosis also have decreased vascular endothelial growth factor (VEGF) levels in granulosa cells. As VEGF is an angiogenic and mitogenic factor, its decreased levels in affected women can potentially contribute to a decreased oocyte maturation [40].

156

Germeyer and Giudice

Furthermore, in women with endometriosis, TNFa levels, which are elevated in ovarian granulosa cells, may affect fertilization capacity of the oocyte, perhaps by its activation of cytokine production, ovulation regulation and through metabolic and apoptotic pathways [41]. The action is at least partially mediated by the TNFa type II receptor, which was found in human cumulus cells and oocytes [42]. Effect on Endometrium Implantation Infertile women with stage I and II endometriosis have been reported to have higher antiphospholipid antibodies in their PF than women with stage III and IV endometriosis, potentially reflecting the activity of the lesions. It has been proposed that these antiphospholipid antibodies contribute to nidation defects in those women in whom anatomical anomalies are minimal [43]. Embryo Toxicity In a mouse model, PF has been shown to be embryotoxic in several studies. Barroso et al found a negative effect of nitric oxide on the development of the mouse embryo [26], whereas another group attributes the embryotoxic effect of the peritoneal fluid to elevated IL-6 levels found in women with endometriosis [44]. Fakih et al [45] demonstrated that PF from infertile patients with minimal or mild endometriosis has disease stage-dependent elevated levels of IL-1, a protein produced by macrophages, compared with fertile controls, in whom the protein was undetectable. In cultured macrophages, similar trends of the levels of protein were observed. An embryotoxic effect of IL-1 was proven on mouse embryos in an in vitro culture system, as degenerated embryos were observed after 24 hours of incubation with levels of IL-1 similar to the concentrations found in the PF during the follicular phase of affected women [45]. This is of major significance, as increased numbers of activated macrophages were found in tubal fluid—the physiologic environment for early embryo cleavage—of patients with endometriosis [46]. Exposure to this ‘‘toxic’’ environment can be minimized through assisted reproductive technologies.

ENDOCRINE DYSREGULATION Oocyte Development In patients with severe endometriosis undergoing IVF treatment, a decreased ovarian response to gonadotropins, with fewer developing follicles (and therefore lower E2 levels), has been reported. Granulosa cells from women with endometriosis express lower levels of VEGF and unchanged levels of

Endometriosis and Infertility

157

IL-6 compared with cells from unaffected women [40]. On the other hand, elevated IL-6 production in granulosa cells was noted by Pellicer et al [40a] and by Carlberg et al, although it did not reach statistical significance [41]. Vascular endothelial growth factor is thought to be important in the development of the oocyte due to increased angiogenesis, suggesting that patients with severe endometriosis experience compromised oocyte maturation [47]. This may also explain the observed decrease in ovarian reserve in patients with moderate to severe endometriosis [48], although other causes also may be operational, including oocyte toxicity by peritoneal fluid components. Harlow et al also demonstrated a decreased level of granulosa cell steroidogenesis in patients with endometriosis [49], presumably contributing to decreased oocyte maturation and, therefore, ovarian reserve. The effect of delayed follicular growth leading to asynchrony of oocyte maturation and ovulation was also observed by Doody et al [50]. Luteal Defect Hormonal abnormalities in patients with endometriosis, with a major emphasis on progesterone levels, are well documented. Early studies postulated a luteal defect in women with mild endometriosis [51]. Apart from abnormalities in luteal phase progesterone levels and corpus luteum development, Ayers et al found elevated progesterone to estradiol ratios in the follicular phase of the cycle of women with endometriosis to be a potential indicator for a defect in luteolysis [52]. Contributing to this change in the hormone ratio was an elevated level of progesterone compared to normal patients, as well as a decreased estradiol level, when the ratio in ovarian veins was examined. It has been suggested that this effect may contribute to asynchrony of endometrial development and folliculogenesis in affected women, despite normal basal body temperatures (BBT) of the subjects in this study [52]. However, luteal phase deficiency in the endometrium is not a consistent finding in women with endometriosis. Ayers et al also observed remnants of several corpora lutea on both ovaries in the early follicular phase in patients with endometriosis during laparotomy [52]. This finding is supported by an earlier study in which, during laparoscopy, fewer ovulation stigmata were noted in women with mild to severe endometriosis, compared with controls. In these patients, normal BBTs were observed. Nevertheless, hormonal dysregulation was noted, including a delayed increase of progesterone rise after the LH peak, and delayed estradiol decline after the LH surge in women with moderate to severe disease. A somewhat shorter and more variable luteal phase was also noted. The authors concluded a disturbance or failure of ovum release and in situ luteinization [53]. However, unruptured follicle syndrome is a controversial entity, and current assisted reproductive

158

Germeyer and Giudice

technologies would bypass such abnormalities. In concordance with this work, another study looking at ovarian dysfunction found a prolonged follicular phase in women with mild endometriosis, whereas normal LH levels arose. Only the LH level within the follicle appeared to be reduced, and affected women showed a significantly lower fecundability rate in unstimulated cycles [54]. IMPLANTATION DEFECT For women with minimal to mild endometriosis and infertility, the reasons for infertility are not as obvious, as the anatomical variations discussed above are not present. One possible reason for infertility in these patients is an abnormality during the attachment, intrusion, and invasive phases of implantation including decidualizing of the endometrium, as well as survival of the embryo proper. Even in the setting of minimal endometriosis, patent Fallopian tubes, regular cycles, and normal male fertility, a lower fertility rate is observed [5,55], potentially due to occult biochemical abnormalities in the endometrium. Despite normal histology of the endometrium, several investigators have identified molecular differences in the eutopic endometrium of women with endometriosis (Fig. 1). Bartosik et al found the expression of C3 complement in eutopic endometrium to be a negative predictor for a subsequent pregnancy, with a more pronounced expression of C3 complement in the endometrium of women with mild endometriosis compared to severe endometriosis [56]. It has been reported that HOX-A10 and HOX-A11, which are normally upregulated in secretory phase endometrium in women without disease, are not upregulated in women with endometriosis [57]. This decreased expression of HOX genes in eutopic endometrium during the secretory phase, which is not correlated to the stage of the disease, has been correlated with lower pregnancy/implantation rates in mice [58]. As HOX genes are transcription factors, it is not surprising that they regulate other genes as well. For example, a recent study has shown that HOXA10 directly regulates the h3-integrin subunit [59]. As avh3 integrin, a cell adhesion molecule of the endometrium expressed at the apical surface of the luminal epithelium, is important for embryonic attachment, its downregulation would negatively impact implantation [60,61]. In fertile women without endometriosis, both avh3 integrin and its ligand, osteopontin, are upregulated specifically during a brief period of uterine receptivity. In patients with endometriosis and patients with other infertility conditions, avh3 integrin is expressed to a lesser extent, whereas osteopontin expression is unchanged [62]. In a mouse model, Illera et al demonstrated subfertility after blocking the binding site of avh3 integrin [63]. Therefore, it seems reasonable to assume that changed levels of this integrin

Endometriosis and Infertility

159

during the window of implantation (WOI) have a negative impact on fertility in patients with endometriosis. Increased free radicals in the endometrium may also decrease the oocyte and embryo quality by creating a toxic environment for the developing embryo [64]. Glutathione peroxidase and catalase have been shown to be aberrantly expressed and to lose their typical fluctuation during the menstrual cycle in women with endometriosis [64,65]. This may affect the environment of the endometrium by changing the levels of free radicals. Other factors negatively affecting the free radical concentration in the uterine environment for the developing embryo are elevated levels of manganese and copper superoxide dismutase (SOD) within the endometrium of affected women [66]. On a protein level, changes in the endometrium of women with endometriosis were found for CA125 [67], soluble urokinase-type plasminogen activator receptor (suPA-R) [68], as well as the apoptosis related protooncogene B-cell lymphoma/leukemia-2 (Bcl-2) [69], potentially affecting uterine receptivity. Several genes are dysregulated in affected women. Gene expression studies have shown the following to be differentially regulated in eutopic endometrium of women with endometriosis: aromatase [70], endometrial bleeding-associated factor (EBAF) [71], hepatocyte growth factor and cMET receptor [72], thrombospondin-1 and VEGF [73], and endothelial nitric oxide synthase (eNOS) [74]. In addition in stromal cell cultures of affected women compared with women without disease, a downregulation of transducer of ErbB (Tob-1), a cell-cycle inhibitor, after IL-1h stimulation has been noted [75]. Using a global gene expression approach through microarray analysis, Kao et al detected more than 100 aberrantly regulated genes in eutopic endometrium of women with versus without endometriosis during the window of implantation that may contribute to implantation failure in women with this disorder on the level of embryonic attachment, survival and embryodecidua signaling. These changes affect cell adhesion molecules, endometrial epithelial secreted proteins, transporters, and immune modulators, etc. [76]. In particular, candidate genes like N-acetylglucosamine-6-O-sulfotransferase (GlcNAc6ST) were found, which are thought to be important in the embryo-decidua crosstalk during the attachment phase of human implantation [77]. Which of all the genes, discovered in such studies, contribute(s) to the underlying pathogenesis of endometrial-based infertility in women with endometriosis remains to be determined. It is also possible that a combination of factors may be responsible for the underlying subfertility. Interestingly, some of the better characterized biochemical markers for uterine receptivity are dysregulated in women with endometriosis compared with those without endometriosis. For example, the expression of glycodelin,

160

Germeyer and Giudice

which is upregulated during the WOI in the normal menstrual cycle [78], is lower in affected patients during this phase [76]. In contrast, some studies have shown no difference in implantation rates when oocytes are donated from healthy controls [79], indicating that normal implantation rates can be achieved in these women during artificial cycles or that the oocyte characteristics can overcome deficiencies in the endometrium of affected women [80,81]. A minor change in the receptivity of the endometrium, together with decreased oocyte quality, may have an additive effect on infertility [82], reflected in decreased fertility rates in natural cycles [83]. SUMMARY Endometriosis is a multifactorial disease with several entities affecting fertility. In addition to the obvious and well-established anatomical abnormalities in severe endometriosis, changes in the local environment of the abdominal cavity affecting sperm-oocyte interaction and oocyte maturation/ survival, hormonal changes, as well as abnormalities at the implantation site, contribute to subfertility in patients with minimal to mild disease. With new genomic and proteomic approaches, diagnosis, and perhaps, predicting fertility in women with endometriosis are soon to be realized. It is hoped that further investigation of these markers will lead to new treatment options to overcome the underlying infertility in women with endometriosis to realize their goals of having a family. PRACTICAL POINT 

New genomic and proteomic approaches might lead to easier diagnosis and new treatment of endometriosis-related infertility.

REFERENCES 1. 2. 3. 4. 5. 6.

Decker WH, Lopez H. Conservative surgical treatment of endometriosis and infertility. Infertility 1979; 2(2):155–164. Olive DL, Schwartz LB. Endometriosis. N Engl J Med 1993; 328(24):1759– 1769. Sensky TE, Liu DT. Endometriosis: Associations with menorrhagia, infertility and oral contraceptives. Int J Gynaecol Obstet 1980; 17(6):573–576. Houston DE. Evidence for the risk of pelvic endometriosis by age, race and socioeconomic status. Epidemiol Rev 1984; 6:167–191. Barnhart K, Dunsmoor-Su R, Coutifaris C. Effect of endometriosis on in vitro fertilization. Fertil Steril 2002; 77(6):1148–1155. McGoogan L. Sterility and Endometriosis. Arch Surg 1949; 59(3):437–444.

Endometriosis and Infertility 7. 8. 9.

10.

11. 12. 13. 14.

15. 16. 17.

18. 19. 20. 21.

22.

23.

24.

161

Grant A. Additional sterility factors in endometriosis. Fertil Steril 1966; 17(4): 514–519. The American Fertility Society. Classification of endometriosis. Fertil Steril 1979; 32:633. Thornton JG, Morley S, Lilleyman J, Onwude JL, Currie I, Crompton AC. The relationship between laparoscopic disease, pelvic pain and infertility, an unbiased assessment. Eur J Obstet Gynecol Reprod Biol 1997; 74(1):57–62. Acosta AA, Buttram VC Jr., Besch PK, Malinak LR, Franklin RR, Vanderheyden JD. A proposed classification of pelvic endometriosis. Obstet Gynecol 1973; 42(1):19–25. Kistner RW, Siegler AM, Behrman SJ. Suggested classification for endometriosis: Relationship to infertility. Fertil Steril 1977; 28(9):1008–1010. Frackiewicz EJ, Zarotsky V. Diagnosis and treatment of endometriosis. Expert Opin Pharmacother 2003; 4(1):67–82. Umaria N, Olliff JF. Imaging features of pelvic endometriosis. Br J Radiol 2001; 74(882):556–562. Harada T, Enatsu A, Mitsunari M, Nagano Y, Ito M, Tsudo T, Taniguchi F, Iwabe T, Tanikawa M, Terakawa N. Role of cytokines in progression of endometriosis. Gynecol Obstet Invest 1999; 47(suppl 1):34–39; discussion 39– 40. Halme J. Role of peritoneal inflammation in endometriosis-associated infertility. Ann N Y Acad Sci 1991; 622:266–274. Mathur SP. Autoimmunity in endometriosis: Relevance to infertility. Am J Reprod Immunol 2000; 44(2):89–95. Matarese G, Alviggi C, Sanna V, Howard JK, Lord GM, Carravetta C, Fontana S, Lechler RI, Bloom SR, De Placido G. Increased leptin levels in serum and peritoneal fluid of patients with pelvic endometriosis. J Clin Endocrinol Metab 2000; 85(7):2483–2487. Syrop CH, Halme J. Peritoneal fluid environment and infertility. Fertil Steril 1987; 48(1):1–9. Haney AF, Muscato JJ, Weinberg JB. Peritoneal fluid cell populations in infertility patients. Fertil Steril 1981; 35(6):696–698. Haney AF. Endometriosis, macrophages, and adhesions. Prog Clin Biol Res 1993; 381:19–44. Olive DL, Weinberg JB, Haney AF. Peritoneal macrophages and infertility: The association between cell number and pelvic pathology. Fertil Steril 1985; 44(6):772–777. Kats R, Collette T, Metz CN, Akoum A. Marked elevation of macrophage migration inhibitory factor in the peritoneal fluid of women with endometriosis. Fertil Steril 2002; 78(1):69–76. Halme J, Becker S, Hammond MG, Raj MH, Raj S. Increased activation of pelvic macrophages in infertile women with mild endometriosis. Am J Obstet Gynecol 1983; 145(3):333–337. Jha P, Farooq A, Agarwal N, Buckshee K. In vitro sperm phagocytosis by human peritoneal macrophages in endometriosis-associated infertility. Am J Reprod Immunol 1996; 36(4):235–237.

162

Germeyer and Giudice

25. Osborn BH, Haney AF, Misukonis MA, Weinberg JB. Inducible nitric oxide synthase expression by peritoneal macrophages in endometriosis-associated infertility. Fertil Steril 2002; 77(1):46–51. 26. Barroso RP, Osuamkpe C, Nagamani M, Yallampalli C. Nitric oxide inhibits development of embryos and implantation in mice. Mol Hum Reprod 1998; 4(5):503–507. 27. Weinberg JB, Doty E, Bonaventura J, Haney AF. Nitric oxide inhibition of human sperm motility. Fertil Steril 1995; 64(2):408–413. 28. Rosselli M, Dubey RK, Imthurn B, Macas E, Keller PJ. Effects of nitric oxide on human spermatozoa: Evidence that nitric oxide decreases sperm motility and induces sperm toxicity. Hum Reprod 1995; 10(7):1786–1790. 29. Zhang H, Zheng RL. Possible role of nitric oxide on fertile and asthenozoospermic infertile human sperm functions. Free Radic Res 1996; 25(4):347– 354. 30. Soldati G, Piffaretti-Yanez A, Campana A, Marchini M, Luerti M, Balerna M. Effect of peritoneal fluid on sperm motility and velocity distribution using objective measurements. Fertil Steril 1989; 52(1):113–119. 31. Pillai S, Rust PF, Howard L. Effects of antibodies to transferrin and alpha 2HS glycoprotein on in vitro sperm motion: implications in infertility associated with endometriosis. Am J Reprod Immunol 1998; 39(4):235–242. 32. VanLangendonckt A, Casanas-Roux F, Donnez J. Iron overload in the peritoneal cavity of women with pelvic endometriosis. Fertil Steril 2002; 78(4):712. 33. Gentry WL, Buster JE, Schriock ED, Carson SA. Failure to demonstrate significant antisperm antibodies in peritoneal fluid of patients with endometriosis. Fertil Steril 1989; 52(6):949–952. 34. Sharma RK, Wang Y, Falcone T, Goldberg J, Agarwal A. Effect of peritoneal fluid from endometriosis patients on sperm motion characteristics and acrosome reaction. Int J Fertil Womens Med 1999; 44(1):31–37. 35. Chacho KJ, Andresen PJ, Scommegna A. The effect of peritoneal macrophage incubates on the spermatozoa assay. Fertil Steril 1987; 48(4):694–696. 36. Coddington CC, Oehninger S, Cunningham DS, Hansen K, Sueldo CE, Hodgen GD. Peritoneal fluid from patients with endometriosis decreases sperm binding to the zona pellucida in the hemizona assay: a preliminary report. Fertil Steril 1992; 57(4):783–786. 37. Sueldo CE, Lambert H, Steinleitner A, Rathwick G, Swanson J. The effect of peritoneal fluid from patients with endometriosis on murine sperm-oocyte interaction. Fertil Steril 1987; 48(4):697–699. 38. Sengoku K, Tamate K, Yoshida T, Takaoka Y, Miyamoto T, Ishikawa M. Effects of low concentrations of nitric oxide on the zona pellucida binding ability of human spermatozoa. Fertil Steril 1998; 69(3):522–527. 39. Oehninger S, Coddington CC, Hodgen GD, Seppala M. Factors affecting fertilization: Endometrial placental protein 14 reduces the capacity of human spermatozoa to bind to the human zona pellucida. Fertil Steril 1995; 63(2): 377–383. 40. Yamashita Y, Ueda M, Takehara M, Yamashita H, Suzuki Y, Hung Y, Terai Y, Ueki M. Influence of severe endometriosis on gene expression of vascular

Endometriosis and Infertility

40a.

41.

42.

43. 44.

45.

46.

47. 48.

49.

50.

51. 52.

53.

54.

163

endothelial growth factor and interleukin-6 in granulosa cells from patients undergoing controlled ovarian hyperstimulation for in vitro fertilization-embryo transfer. Fertil Steril 2002; 78(4):865. Pellicer A, Albert C, Mercader A, Bonilla-Musoles F, Remohi J, Simon C. The follicular and endocrine environment in women with endometriosis: local and systemic cytokine production. Fertil Steril 1998; 70(3):425–431. Carlberg M, Nejaty J, Froysa B, Guan Y, Soder O, Bergqvist A. Elevated expression of tumour necrosis factor alpha in cultured granulosa cells from women with endometriosis. Hum Reprod 2000; 15(6):1250–1255. Naz RK, Zhu X, Menge AC. Expression of tumor necrosis factor-alpha and its receptors type I and type II in human oocytes. Mol Reprod Dev 1997; 47(2):127–133. Ulcova-Gallova Z, Bouse V, Svabek L, Turek J, Rokyta Z. Endometriosis in reproductive immunology. Am J Reprod Immunol 2002; 47(5):269–274. Gomez-Torres MJ, Acien P, Campos A, Velasco I. Embryotoxicity of peritoneal fluid in women with endometriosis. Its relation with cytokines and lymphocyte populations. Hum Reprod 2002; 17(3):777–781. Fakih H, Baggett B, Holtz G, Tsang KY, Lee JC, Williamson HO. Interleukin-1: A possible role in the infertility associated with endometriosis. Fertil Steril 1987; 47(2):213–217. Haney AF, Misukonis MA, Weinberg JB. Macrophages and infertility: Oviductal macrophages as potential mediators of infertility. Fertil Steril 1983; 39(3):310–315. Geva E, Jaffe RB. Role of vascular endothelial growth factor in ovarian physiology and pathology. Fertil Steril 2000; 74(3):429–438. Hock DL, Sharafi K, Dagostino L, Kemmann E, Seifer DB. Contribution of diminished ovarian reserve to hypofertility associated with endometriosis. J Reprod Med 2001; 46(1):7–10. Harlow CR, Cahill DJ, Maile LA, Talbot WM, Mears J, Wardle PG, Hull MG. Reduced preovulatory granulosa cell steroidogenesis in women with endometriosis. J Clin Endocrinol Metab 1996; 81(1):426–429. Doody MC, Gibbons WE, Buttram VCJ. Linear regression analysis of ultrasound follicular growth series: Evidence for an abnormality of follicular growth in endometriosis patients. Fertil Steril 1988; 49(1):47–51. Pittaway DE, Maxson W, Daniell J, Herbert C, Wentz AC. Luteal phase defects in infertility patients with endometriosis. Fertil Steril 1983; 39(5):712–713. Ayers JW, Birenbaum DL, Menon KM. Luteal phase dysfunction in endometriosis: Elevated progesterone levels in peripheral and ovarian veins during the follicular phase. Fertil Steril 1987; 47(6):925–929. Brosens IA, Koninckx PR, Corveleyn PA. A study of plasma progesterone, oestradiol-17beta, prolactin and LH levels, and of the luteal phase appearance of the ovaries in patients with endometriosis and infertility. Br J Obstet Gynaecol 1978; 85(4):246–250. Cahill DJ, Wardle PG, Maile LA, Harlow CR, Hull MG. Ovarian dysfunction in endometriosis-associated and unexplained infertility. J Assist Reprod Genet 1997; 14(10):554–557.

164

Germeyer and Giudice

55. Giudice LC, Telles TL, Lobo S, Kao LC. The molecular basis for implantation failure in endometriosis: On the road to discovery. Ann N Y Acad Sci 2002; 955:252–264; discussion 293–255, 396–406. 56. Bartosik D, Damjanov I, Viscarello RR, Riley JA. Immunoproteins in the endometrium: Clinical correlates of the presence of complement fractions C3 and C4. Am J Obstet Gynecol 1987; 156(1):11–15. 57. Taylor HS, Bagot C, Kardana A, Olive D, Arici A. HOX gene expression is altered in the endometrium of women with endometriosis. Hum Reprod 1999; 14(5):1328–1331. 58. Lim H, Ma L, Ma WG, Maas RL, Dey SK. Hoxa-10 regulates uterine stromal cell responsiveness to progesterone during implantation and decidualization in the mouse. Mol Endocrinol 1999; 13(6):1005–1017. 59. Daftary GS, Troy PJ, Bagot CN, Young SL, Taylor HS. Direct regulation of beta3-integrin subuni gene expression by HOXA10 in endometrial cells. Mol Endocrin 2002; 16(3):571–579. 60. Lessey BA, Arnold JT. Paracrine signaling in the endometrium: Integrins and the establishment of uterine receptivity. J Reprod Immunol 1998; 39(1–2):105– 116. 61. Ilesanmi AO, Hawkins DA, Lessey BA. Immunohistochemical markers of uterine receptivity in the human endometrium. Microsc Res Tech 1993; 25(3): 208–222. 62. Lessey BA, Castelbaum AJ, Sawin SW, Buck CA, Schinnar R, Bilker W, Strom BL. Aberrant integrin expression in the endometrium of women with endometriosis. J Clin Endocrinol Metab 1994; 79(2):643–649. 63. Illera MJ, Cullinan E, Gui Y, Yuan L, Beyler SA, Lessey BA. Blockade of the alpha(v)beta(3) integrin adversely affects implantation in the mouse. Biol Reprod 2000; 62(5):1285–1290. 64. Ota H, Igarashi S, Sato N, Tanaka H, Tanaka T. Involvement of catalase in the endometrium of patients with endometriosis and adenomyosis. Fertil Steril 2002; 78(4):804. 65. Ota H, Igarashi S, Kato N, Tanaka T. Aberrant expression of glutathione peroxidase in eutopic and ectopic endometrium in endometriosis and adenomyosis. Fertil Steril 2000; 74(2):313–318. 66. Ota H, Igarashi S, Hatazawa J, Tanaka T. Immunohistochemical assessment of superoxide dismutase expression in the endometrium in endometriosis and adenomyosis. Fertil Steril 1999; 72(1):129–134. 67. McBean JH, Brumsted JR. In vitro CA-125 secretion by endometrium from women with advanced endometriosis. Fertil Steril 1993; 59(1):89–92. 68. Sillem M, Prifti S, Monga B, Buvari P, Shamia U, Runnebaum B. Soluble urokinase-type plasminogen activator receptor is over-expressed in uterine endometrium from women with endometriosis. Mol Hum Reprod 1997; 3(12): 1101–1105. 69. Meresman GF, Vighi S, Buquet RA, Contreras-Ortiz O, Tesone M, Rumi LS. Apoptosis and expression of Bcl-2 and Bax in eutopic endometrium from women with endometriosis. Fertil Steril 2000; 74(4):760–766. 70. Kitawaki J, Noguchi T, Amatsu T, Maeda K, Tsukamoto K, Yamamoto T,

Endometriosis and Infertility

71.

72.

73.

74.

75.

76.

77.

78. 79.

80.

81.

82. 83.

165

Fushiki S, Osawa Y, Honjo H. Expression of aromatase cytochrome P450 protein and messenger ribonucleic acid in human endometriotic and adenomyotic tissues but not in normal endometrium. Biol Reprod 1997; 57(3): 514–519. Tabibzadeh S, Mason JM, Shea W, Cai Y, Murray MJ, Lessey B. Dysregulated expression of ebaf, a novel molecular defect in the endometria of patients with infertility. J Clin Endocrinol Metab 2000; 85(7):2526–2536. Khan KN, Masuzaki H, Fujishita A, Kitajima M, Sekine I, Ishimaru T. Immunoexpression of hepatocyte growth factor and c-Met receptor in the eutopic endometrium predicts the activity of ectopic endometrium. Fertil Steril 2003; 79(1):173–181. Tan XJ, Lang JH, Liu DY, Shen K, Leng JH, Zhu L. Expression of vascular endothelial growth factor and thrombospondin-1 mRNA in patients with endometriosis. Fertil Steril 2002; 78(1):148–153. Khorram O, Lessey BA. Alterations in expression of endometrial endothelial nitric oxide synthase and alpha(v)beta(3) integrin in women with endometriosis. Fertil Steril 2002; 78(4):860–864. Lebovic DI, Baldocchi RA, Mueller MD, Taylor RN. Altered expression of a cell-cycle suppressor gene, Tob-1, in endometriotic cells by cDNA array analyses. Fertil Steril 2002; 78(4):849–854. Kao LC, Germeyer A, Tulac S, Lobo S, Yang JP, Taylor RN, Osteen K, Lessey BD, Giudice LC. Expression profiling of endometrium from women with endometriosis reveals candidate genes for disease-based implantation failure and infertility. Endocrinology 2003 Jul; 144(7):2870–2881. Genbacev OD, Prakobphol A, Foulk RA, Krtolica AR, Ilic D, Singer MS, Yang ZQ, Kiessling LL, Rosen SD, Fisher SJ. Trophoblast L-selectin-mediated adhesion at the maternal-fetal interface. Science 2003; 299(5605): 405– 408. Seppala M, Koistinen H, Koistinen R. Glycodelins. Trends Endocrinol Metab 2001; 12:111–117. Simon C, Gutierrez A, Vidal A, de los Santos MJ, Tarin JJ, Remohi J, Pellicer A. Outcome of patients with endometriosis in assisted reproduction: Results from in-vitro fertilization and oocyte donation. Hum Reprod 1994; 9(4):725– 729. Diaz I, Navarro J, Blasco L, Simon C, Pellicer A, Remohi J. Impact of stage III–IV endometriosis on recipients of sibling oocytes: Matched case-control study. Fertil Steril 2000; 74(1):31–34. Sung L, Mukherjee T, Takeshige T, Bustillo M, Copperman A. Endometriosis is not detrimental to embryo implantation in oocyte recipients. J Assist Reprod Genet 1997; 14(3):152–156. Mahutte NG, Arici A. New advances in the understanding of endometriosis related infertility. J Reprod Immunol 2002; 55(1–2):73–83. Fedele L, Bianchi S, Marchini M, Villa L, Brioschi D, Parazzini F. Superovulation with human menopausal gonadotropins in the treatment of infertility associated with minimal or mild endometriosis: A controlled randomized study. Fertil Steril 1992; 58(1):28–31.

10 Medical Therapy for Endometriosis: An Overview Eric S. Surrey Colorado Center for Reproductive Medicine Englewood, Colorado, U.S.A.

INTRODUCTION The medical management of endometriosis represents a critical portion of the treatment used to attack this debilitating disorder. The primary goal of this approach is to eradicate the painful symptoms and to enhance fecundity. Reduction of the extent of endometriotic implants is a secondary goal. Medical therapy clearly represents a less invasive approach than surgical management of endometriosis. The majority of controlled trials evaluate the relative benefits of different medical interventions or of a medical intervention in comparison to placebo. Others evaluate the benefit of combined surgical and medical therapy in comparison to surgical therapy alone. Interestingly, there are no studies comparing primary surgical to primary medical therapy alone. One of the difficulties in evaluating these therapies is the great degree of variation among investigative designs. The absence of control groups in any study evaluating the effect of an intervention on either pelvic pain or infertility is clearly problematic given the subjective nature of pain and potential for placebo effect, as well as the very real although compromised potential for 167

168

Surrey

spontaneous conception. Outcome parameters also vary dramatically. Some investigators have assessed efficacy based on reduction of visible implants, which is an inherently flawed approach, given the lack of consistency in visual diagnosis of endometriosis and well-documented lack of correlation between extent of disease and symptoms. Similarly, the lack of a uniform instrument to evaluate painful symptoms makes interpretation of subjective improvement difficult. Lastly, few studies evaluate recurrence rates with long-term follow-up. Similarities in short-term pain relief resulting from several medical interventions may provide very different outcomes when long-term symptom recurrence is evaluated. In this chapter, we endeavor to provide an evidence-based overview of historic, current, and experimental modalities for the medical management of both symptomatic endometriosis and endometriosis-associated infertility (Table 1). Steroidal Manipulation of Endometriosis The theoretic basis for hormonal therapy of endometriosis has been the longheld assumption that endometriotic implants and the endometrium respond in a similar fashion to sex steroid hormone manipulation. Estrogens are believed to stimulate the disease and endometrium, whereas estrogen deficiency should theoretically result in atrophy of both tissue types [1]. However, there is conflicting evidence as to whether this assumption is truly valid. In a large series evaluating 443 implants in 196 patients, Metzger TABLE 1

Evaluated Medical Therapy for Endometriosis

Methyltestosterone Oral contraceptives (pseudopregnancy) Progestins Gestrinone Danazol GnRH agonists GnRH antagonistsa Aromatase inhibitorsa Mifepristonea Progesterone receptor modulatorsa Estrogen receptor modulatorsa Angiogenesis inhibitorsa Cytokine inhibitorsa Immunomodulatorsa a

Experimental

Overview of Medical Therapy

169

and colleagues demonstrated that histologic concordance between endometrium and endometriotic implants could be demonstrated in only 13% of cases [2]. Others have shown that the concentration of steroid receptors is significantly lower in endometriotic implants and that cyclic patterns could not be demonstrated in hormonally dependent secretory products of such lesions in culture [3–5]. In addition, these assumptions do not explain why hormonal manipulation would have an effect on the complex local peritoneal immunological changes, including enhanced cytokine concentrations, macrophage activation, and mesothelial proliferation associated with this disease (see Chaps. 2 and 7). Pseudo Pregnancy and Oral Contraceptives Pregnancy has been shown to have a beneficial effect on the symptoms associated with endometriosis, although long-term disease suppression is somewhat variable after delivery [6–8]. This concept led to the first hormonally induced ‘‘pseudo pregnancy regimens’’ proposed in 1958 by Kistner [9]. Highdose oral contraceptives were administered with the thought that elimination of cyclic changes in ovarian steroid secretion would inhibit development of endometriotic implants. In a comparative trial, Noble and Letchworth reported a 30% symptom improvement rate with this approach, with an extremely high incidence of side effects including mastalgia, abnormal distension, nausea, vomiting, and phlebitis [10]. Only 59% could complete more than 5 months of therapy and 41% required additional major surgery because of persistent symptoms. More recently, Vercellini and colleagues evaluated the effect of a lower dose cyclic oral contraceptive agent (ethinyl estradiol 20-30 mcg+ desogestrel 0.15 mg) [11]. Significant symptom relief with regard to dyspareunia, dysmenorrhea, and nonmenstrual pain was appreciated. Nevertheless, mean pain scores were not significantly different from baseline 6 months after completion of therapy. Oral contraceptive therapy has been associated with decidualization, necrobiosis, and enhanced apoptosis of endometriotic tissue [12,13]. There is little evidence to suggest that continuous oral contraceptive therapy is more beneficial than cyclic therapy, although the former may have an advantage in patients who present primarily with dysmenorrhea. Similarly, switching from one pill formulation to another is rarely beneficial. Progestins A variety of progestational agents (progestins) alone have been used in the medical management of endometriosis in an effort to reduce side effects associated with the estrogenic component of oral contraceptives (Table 2). As

170

Surrey

TABLE 2

Progestins Used in the Management of Symptomatic Endometriosis Medroxyprogesterone acetate (MPA) Megestrol acetate Dydrogestone Lynestrol Norethindrone acetate Levonorgestrel

previously described, it has been proposed that progestins inhibit endometriotic implants by causing an initial decidual reaction, which would presumably result in eventual atrophy. An inhibitory effect of medroxyprogesterone acetate (MPA) on in vitro endometrial stromal cell proliferation when used in therapeutic doses has been demonstrated [14]. More recently, Bruner et al demonstrated that progesterone may affect the expression of endometrial matrix metallproteinases, which may play a role in the establishment of endometriotic implants [15]. A relatively small number of randomized controlled trials have been reported with use of progestins alone. The majority of studies have been retrospective or observational in nature. In a placebo-controlled 6-month trial, Overton et al evaluated the effect of dydrogesterone 40 to 60 mg daily during the luteal phase for 12 days of each cycle [16]. Pain was significantly reduced with a 60-mg dose during treatment and up to 12 months thereafter. Vercellini et al compared MPA administered in an intramuscular depot preparation (150 mg every 90 days) for 12 months to a control group treated with low-dose danazol combined with an oral contraceptive and noted significant and equivalent improvement in symptom scores in both groups [17]. Dysmenorrhea was suppressed in the MPA group up to 1 year after therapy. One of the problems with MPA in depot preparations is the extremely slow return of menses and ovulation, which could have a negative impact on conception. The effect of high-dose oral MPA 100 mg daily was evaluated by Telimaa and coworkers in a 6-month trial with good efficacy noted up to 6 months after completion of therapy [18]. In an analysis of 14 trials using various progestins of which only four investigations were randomized, Vercellini reported that only 9% of symptomatic patients failed to respond [19]. The pooled incidence of recurrent pain was 50% (95% confidence interval [CI] 37.8–67.2%). It is important to note that, in general, rather high doses of progestins must be used to achieve beneficial effects. Medroxyprogesterone acetate and

Overview of Medical Therapy

171

megestrol acetate have been used in doses of 30 mg to 100 mg and 40 mg daily, respectively, to achieve efficacy [20–22]. Similarly, norethindrone acetate has been used in doses as high as 20 mg daily [23]. As a result, the incidence of side effects is not insignificant. Vercellini et al reported that approximately one third of patients experienced abnormal bleeding [19]. Other side effects reported in at least 10% of patients included weight gain, bloating, and edema. High progestin doses may also cause untoward changes in highdensity lipoprotein (HDL)-cholesterol and apolipoprotein A-1 [24]. In an effort to minimize side effects, Vercellini et al have reported the results of an uncontrolled prospective trial of a levonorgestrel-releasing intrauterine device [25]. A significant reduction in endometriosis-associated dysmenorrhea and menstrual flow was reported. A more detailed discussion of the role of progestins is presented in Chapter 12. Gestrinone Gestrinone is a 19-norsteroid derivative, that was originally designed as a weekly oral contraceptive, but has been used to treat estrogen-dependent disorders such as endometriosis. This agent acts in a somewhat complex fashion. It blocks follicular development and estradiol production, binds to androgen receptors as an agonist, and exhibits both agonistic and antagonistic effects after binding to the progesterone receptors [26,27]. This agent is administered orally in doses ranging from 1.25 mg to 2.5 mg twice weekly. Thomas and Cooke demonstrated that gestrinone 2.5 mg twice weekly for 6 months resulted in a significant improvement in visible endometriotic implants in comparison to placebo [28]. Hornstein et al reported equal efficacy when 2.5 mg was compared to 1.25 mg twice weekly [29], although others have suggested that the higher dose was more effective [30]. Fedele et al reported that gestrinone 2.5 mg was as effective as danazol with a similar recurrence rate [31]. Others have shown that this agent is as effective as a gonadotropin releasing hormone (GnRH) agonist with regard to pain relief and demonstrated a tendency toward decreased relative pain recurrence rates [32]. The primary side effects for this agent are decreases in HDLcholesterol, increases in low-density lipoprotein (LDL)-cholesterol, weight gain, hirsutism, seborrhea, and acne. This agent is not currently approved for use in the United States. Danazol Danazol is an isoxazol derivative of 17-a-ethinyl testosterone and had been the primary medication for the treatment of symptomatic endometriosis until the more recent general acceptance of GnRH agonists. This agent acts on many levels. From an endocrine viewpoint, it suppresses gonadotropin

172

Surrey

surges, lowers circulating estradiol and progesterone levels by steroidogenic enzyme inhibition, and interacts with both androgen and progesterone receptors to induce endometrial atrophy [33]. Danazol has been shown at therapeutic levels to directly inhibit endometrial stromal cell proliferation in vitro [14]. The agent also appears to act on the altered immunological state associated with endometriosis. Danazol has been shown to have an immunomodulatory effect on T-cell subsets and other immunocompetent cells in eutopic endometrium [34]. Danazol may also exhibit immunosuppressive activity. One investigative team has demonstrated that therapeutic doses of this agent suppress autoantibodies in endometriosis patients [35]. Peritoneal cytokine levels (interleukin-1 and tumor necrosis factor) are significantly suppressed after danazol therapy [36]. Elevated serum soluble C23 levels were also suppressed with this agent [37]. Danazol is typically administered in doses ranging from 400 mg to 800 mg daily in an effort to achieve amenorrhea. In a summary of 16 uncontrolled clinical trials involving 1,035 patients, Metzger and Luciano reported 88% symptomatic improvement and 77% clinical improvement [33]. The efficacy of danazol appears to vary with the dose. In a classic double-blind, 6-month trial, Dmowski et al compared the effects of daily doses ranging from 100 to 600 mg [38]. The improvement in clinical symptoms was 56% in patients receiving 100-mg daily doses and 83% in those receiving 600-mg daily doses. The greatest improvement was in those who developed amenorrhea during therapy. Similarly, the most regression of visible disease at follow-up laparoscopy was noted in patients receiving higher doses (74%) versus those receiving 100 mg to 200 mg daily doses (39%–42%). In a separate trial, Biberoglu and Behrman reported that symptom recurrence rates were also lowest in those receiving higher danazol doses [39]. In addition, recurrence rates seem to be highest in those with stage III to IV as opposed to stage I to II disease [40]. One of the greatest drawbacks of danazol is its side effect profile. The primary adverse effects are caused by the androgenic action of this agent and, at 800-mg daily doses, include relatively high incidences of weight gain (79%), muscle cramps (52%), atrophic breast changes (48%), vasomotor symptoms (42%), oily skin (37%), acne (27%), hirsutism (21%), and voice changes (7%) [41]. Adverse lipoprotein and lipid changes have been reported [24]. Should a patient ovulate and conceive while taking this drug, the potential for masculinization of a female fetus is of concern [42]. Several tactics have been taken to minimize side effects. Vercellini and colleagues treated 42 patients with a 50-mg daily dose of danazol for 9 months alone or for 6 months after an initial 3-month course of a GnRH agonist [43]. Although symptoms were similarly suppressed and side effects decreased in both groups, a significant incidence of ovulatory cycles was also noted in both

Overview of Medical Therapy

173

groups (55% and 45% of patients, respectively). It was recommended, therefore, that low-dose danazol be combined with an oral contraceptive to avoid inadvertent conception. Other small trials have minimized side effects while demonstrating efficacy with the use of either danazol suppositories or vaginal rings [44,45]. Needless to say, the side effect profile of this agent, and not its efficacy, has limited its usefulness in the face of newer and equally effective agents. Gonadotropin Suppression: GnRH Agonists Gonadotropin releasing hormone agonists have emerged as the primary medication for the medical treatment of endometriosis. These agents represent modifications of the native hypothalamic hormone GnRH, which stimulates pituitary gonadotropin follish stimulating hormone [FSH] and luteinizing hormone [LH] release in a pulsatile fashion. These agonists are more resistant to enzymatic breakdown and remain bound to pituitary GnRH receptors for prolonged times. The net agonistic effect is an initial stimulation and then suppression of gonadotropin, and subsequently, ovarian estrogen release. This hypoestrogenic state should lead to regression of endometriotic lesions. These agents may also act by altering other mechanisms that have been purported to enhance the development of endometriotic implants. Gonadotropin releasing hormone agonists have been shown to decrease peritoneal fluid leukocyte protease inhibitors [46], enhance apoptic change in endometrial culture [47], suppress cytokine expression in endometrial culture and peritoneal fluid [8,36,47], normalize aberrant expression of tissue inhibitors of metalloproteinase-1 [48], and suppress serum soluble CD 23 levels [37]. The efficacy of these agents has been well demonstrated with regard to both suppression of symptoms and regression of visible disease. The majority of prospective randomized trials are of 6 months’ duration, compare GnRH agonists to danazol, and demonstrate general equivalence in efficacy. Other studies compare the agents to placebo, gestrinone, or oral contraceptives (Table 3). These studies evaluated laparoscopically diagnosed endometriosis in the absence of surgical reduction. Waller and Shaw reported a cumulative recurrence rate of 53% after 5 years of an initial treatment course [58]. Interestingly, the incidence of recurrence was lower in those with minimal (36.9%) as opposed to severe (74.4%) disease at laparoscopy performed before initiation of therapy. Currently these agents are administered intranasally, subcutaneously, or intramuscularly. Dosing varies with the specific agonist and route of administration. One placebo-controlled trial evaluated the efficacy of a 3-month course of GnRH agonist therapy in patients with chronic pelvic pain and a diagnosis

174

Surrey

TABLE 3

Prospective Randomized Trials of GnRH Agonists as Therapy for Symptomatic Endometriosis Author [Ref]

Agonist

Patients

Dlugi [49] Henzl [50] Dmowski [51] Kennedy [52] Shaw [53] NEET [54] Wheeler [55] Rock [56] Wright [57] Berquist [58] Vercellini [11]

Leuprolide Nafarelin Buserelin Nafarelin Goserelin Nafarelin Leuprolide Goserelin Leuprolide Triptorelin Goserelin

28 143 19 50 204 206 128 208 22 24 29

Gestrinone Italian Study Group [32]

Leuprolide

28

Duration 6 6 6 6 6 6 6 6 3 6 6

months months months months months months months months months months months

6 months

Control Placebo Danazol 800 mg Danazol 800 mg Danazol 600 mg Danazol 600 mg Danazol 600 mg Danazol 800 mg Danazol 800 mg Danazol 800 mg Placebo Ethinyl Estradiol 0.02 mg + Desogestrel 0.15 mg Gestrinone

of clinically suspected endometriosis in the absence of surgical documentation [59]. Significantly greater pain reduction was noted in the group of 49 receiving the agonist in comparison to 46 receiving placebo ( P < 0.001). Endometriosis was appreciated at post-therapy laparoscopy in 82% of patients. The authors proposed that this agent can be safely and effectively used empirically in patients with chronic pelvic pain with clinically suspected disease without diagnostic laparoscopy after appropriate evaluation. The primary side effects associated with GnRH agonists are those that are secondary to the induced hypoestrogenic state. These include vasomotor symptoms, reduction in bone mineral density, vaginal dryness, decreased libido, depression, joint pain, mood swings, and fatigue. These symptoms may not only decrease the potential for long-term use of these agents, but can have a major impact on patient compliance. A variety of ‘‘add-back’’ regimens have been used in an effort to maintain the inherent efficacy of the GnRH agonists while minimizing side effects [60]. In patients treated for a standard 6-month therapeutic course, low-dose hormone replacement regimens using either conjugated equine estrogens (0.3– 0.625 mg) + MPA 5 mg or 17 h-estradiol 2 mg + norethindrone 1 mg daily have been shown to be beneficial [61,62]. An alternative approach that avoids administration of estrogens entirely is the use of norethindrone in a 2.5-mg daily dose [63]. In each of these studies, vasomotor and painful symptoms were suppressed, but some degree of reversible bone loss was appreciated.

Overview of Medical Therapy

175

Therefore, a different approach to add-back would be appropriate if treatment is to be extended beyond 6 months or retreatment considered. In a prospective, multi center, 12-month trial, Hornstein and colleagues demonstrated that pelvic pain and vasomotor symptoms remained suppressed and bone mineral density loss was prevented when the depot preparation of leuprolide acetate was combined with oral norethindrone acetate 5-mg daily [64]. (Fig. 1) Pelvic pain was suppressed for at least 12 months after completion of therapy [65]. Mean bone loss, which was noted to be progressive in patients receiving the agonist alone, did not return to baseline for 16 months after completion of therapy. This would suggest that a GnRH agonist can potentially be used for up to 12 months or for retreatment, but only so long as an appropriate add-back is administered. A more detailed discussion of GnRH agonists is provided in Chapter 13. Gonadotropin Suppression: GnRH Antagonists GnRH antagonists are a class of drug that binds to GnRH receptors, inhibits gonadotropin secretion, and creates a hypoestrogenic state. Their mechanism

FIGURE 1 Mean changes from baseline in bone mineral density (BMD) of the lumbar spine as measured by dual x-ray absorptiometry in endometriosis patients receiving a 12-month course of a depot preparation of the GnRH agonist leuprolide acetate alone or with add-back. Group A: GnRHa alone. Group B: GnRHa + norethindrone acetate (NEt) 5 mg daily. Group C: GnRHa + NEt 5 mg daily + conjugated equine estrogens (CEE) 0.625 mg daily. Group D: GnRHa + NEt 5 mg daily + CEE 1.25 mg daily. (From Ref. 64. Adapted with permission from the American College of Obstreticians and Gynecologists. Obstet Gynecol 1998; 91:61–24.)

176

Surrey

of action is different than the GnRH agonists in that they competitively inhibit GnRH binding to its own receptor. As a result, the initial flare associated with GnRH agonist is eliminated [66]. Whether this is of great clinical significance has not been clearly demonstrated. These agents have been shown to inhibit the growth of endometriosis in an animal model [67]. There are currently a series of ongoing trials evaluating the efficacy of these agents in treating endometriosis. It is logical that the long-term efficacy and side effect profile would at least be similar to that of the agonists. The need for add-back therapy or perhaps the intermittent use of shorter acting agents may be necessary to overcome side-effects [68]. GnRH antagonists are reviewed in detail in Chapter 13. Antiprogesterone, Antiestrogens, and Selective Progesterone and Estrogen Receptor Modulators Mifepristone (RU-486) is an antiprogesterone agent with antiestrogenic and antiglucocorticoid properties. Initial data had suggested efficacy on shrinking surgically induced endometriosis in an animal model [69,70]. Limited clinical data suggest a dose-dependent efficacy in humans, although large scale studies have not been performed [71]. Selective progesterone receptor modulators are agents that exhibit both progesterone agonistic and antagonistic effects based on the target tissue. Initial clinical trials suggest efficacy in suppressing pelvic pain associated with endometriosis with few side effects [72]. Phase III trials are currently ongoing. The antiestrogenic agent, raloxifene, has also been shown to reduce the severity of endometriosis in a monkey model [73]. Human trials have not been reported. Aromatase Inhibitors Traditional medical approaches to endometriosis have focused on suppression of ovarian hormones. However, recent evidence has suggested that endometriotic implants may synthesize estrogen locally as a result of inappropriate local expression of the enzyme aromatase [74]. Various aromatase inhibitors have been used in small series with success in treating severe and postmenopausal endometriosis [75–77]. This approach is reviewed in detail in Chapter 11. Immunomodulation Given the increasing evidence that the pathophysiology of endometriosis may be intimately related to alterations in immune factors, it is logical that immunomodulators may play some beneficial role in the treatment of this

Overview of Medical Therapy

177

disorder. A limited body of evidence primarily in animal models has been recently reported. Keenan et al documented regression of explants in rats with experimentally induced endometriosis administered through intraperitoneal loxoribine, an immunomodulatory agent that, among other actions, enhances the activity of a host of cytokines [78]. Interferon a 2b has been shown to inhibit endometrioma cell growth in culture [79]. In a prospective randomized trial, D’Hooghe and colleagues demonstrated that administration of recombinant human tumor necrosis factor binding protein-1 inhibited the development of experimentally induced endometriosis in the baboon model [80]. Inhibition of endometriosis symptoms was demonstrated in women with relapsing disease treated with thalidomide, an antiangiogenesis agent, in a small pilot study [81]. None of these agents has been evaluated in appropriately controlled human trials but all represent promising approaches for the future management of the disease.

MEDICAL MANAGEMENT OF ENDOMETRIOSIS-RELATED INFERTILITY As previously discussed, the efficacy of a variety of medications on the suppression of symptomatic endometriosis has been well established. In contrast, the efficacy of progestins, danazol, or GnRH agonists when used as primary therapy to enhance patients with endometriosis-associated infertility has not been demonstrated. Hughes et al evaluated data from nine trials comparing ovulation suppression with either danazol, gestrinone, or medroxyprogesterone acetate to no treatment or placebo which all failed to show any beneficial effect on enhancing pregnancy rates (common OR 0.85; 95% CI 0.95–1.22) [82]. In the same study, an additional six randomized trials comparing a GnRH agonist, gestrinone, or an oral contraceptive to danazol also failed to demonstrate any differences (common OR 1.07; 95% CI 0.71– 1.61). These findings were confirmed by Adamson and Pasta in a separate meta-analysis [83]. These investigators recommended that medical therapies should not be used as a treatment of infertility associated with asymptomatic endometriosis. There are several possible explanations for these findings. One could propose that minimal to mild endometriosis has no impact on fertility at all, given the proven efficacy of these agents in treating the underlying disease, but lack of efficacy in improving conception. A second explanation is that the mechanism of infertility associated with endometriosis is different from that associated with pelvic pain and is unaffected by these medications. Neither of these explanations can be supported by data. A third, and perhaps more

178

Surrey

plausible explanation, may be that by the time a patient resumes normal ovulatory patterns, which may be months after completion of therapy, the deleterious effects of the disease process on fertility that were initially suppressed by medications recur even if the patient remains asymptomatic. Thus, if a patient could attempt conception when the disease process is maximally suppressed, pregnancy rates would be heightened. This could only occur with the use of the assisted reproductive technologies. In a prospective randomized trial, Surrey et al have recently evaluated the effect of a 3-month course of a GnRH agonist administered immediately before in vitro fertilization (IVF) in patients with surgically confirmed endometriosis [84]. Significantly higher ongoing pregnancy rates with a trend toward higher implantation rates were appreciated in this group of 25 patients in comparison to 26 controls with endometriosis treated with standard controlled ovarian hyperstimulation techniques before oocyte aspiration in the absence of prolonged GnRH agonist (Fig. 2). These findings have been confirmed by others [85–88].

FIGURE 2 In vitro fertilization cycle outcomes for endometriosis patients pretreated with a GnRH agonist for 3 months (Group I) immediately prior to controlled ovarian hyperstimulation (COH) or undergoing standard COH (Group II). a: P < 0.05 versus Group I. (From Ref. 84. Reprinted with permission by the American Society for Reproductive Medicine. Fertil Steril 2002; 78:699–704.)

Overview of Medical Therapy

179

This effect may be a result of a beneficial effect of these agents on either peritoneal cytokine levels or endometrial markers of implantation [35,89].

COMBINED SURGICAL AND MEDICAL MANAGEMENT There are a limited number of prospective, randomized, placebo-controlled studies that evaluate the efficacy of medical therapy administered in conjunction with surgical management of symptomatic endometriosis. Telimaa et al evaluated the effects of a 6-month course of danazol (600 mg daily), MPA 100 mg daily, or placebo after surgical excision of endometriosis at laparoscopy [90]. A greater degree of postoperative pain relief and reduction of disease at follow-up laparoscopy were noted in both the danazol and MPA groups in comparison to placebo. Hornstein et al noted that the median time to initiation of alternative therapy was more than 24 months in patients receiving a 6-month postoperative course of GnRH agonist in comparison to 11.7 months in those randomized to receive post operative placebo [91] (Fig. 3). These findings were confirmed by Vercellini and coworkers [92]. Interestingly, other investigative teams did not demonstrate any difference in comparison to placebo when GnRH agonist or danazol was used post operatively [93–95]. These latter studies included only stage III–IV endometriosis and treated patients with a 3-month as opposed to a 6-month post

FIGURE 3 Time to initiation of alternative treatment for patients treated with the GnRH agonist nafarelin or placebo for 6 months post-laparoscopic ablation of endometriosis. Reprinted with permission by the American Society for Reproductive Medicine, Fertil Steril 1997, Vol. 68, p. 860–864.

180

Surrey

operative course of the GnRH agonist, which may be insufficient to achieve full efficacy. A 6-month preoperative course of GnRH agonists was compared to a 6-month postoperative course in a randomized trial [96]. Similar improvement in pelvic pain symptoms was appreciated, although endometriosis scores were more extensively suppressed when the agonist was given pre operatively. Shaw et al demonstrated that GnRH agonist therapy administered after endometrioma drainage resulted in less extensive recurrence and extent of endometriotic implants at follow-up laparoscopy [97]. In addition, postoperative adhesion formation and reformation after adhesiolysis were reduced in rats with surgically induced endometriosis and adhesions that had been treated preoperatively and postoperatively with a GnRH agonist [98]. The impact on conception of combining surgical resection or ablation with medical therapy administered either pre-or postoperatively has been evaluated. Unfortunately, the majority of studies are nonrandomized, which creates a high degree of selection and inclusion bias. Hughes et al evaluated five older cohort studies comparing laparoscopic surgery plus danazol to danazol alone [82]. The common odds ratio for this group was 1.42 (95% CI 0.94–2.14), suggesting no benefit of adjunctive danazol therapy. A similar finding was noted in patients undergoing laparotomy. Telimaa and colleagues reported the results of a placebo-controlled trial comparing post operative medroxyprogesterone acetate to danazol after conservative surgery [90]. Although only a small subset of patients in each group was attempting pregnancy, the conception rates were similar among the three treatment groups. Donnez et al prospectively evaluated 126 infertile women with ovarian endometriosis resected microsurgically at laparotomy who were treated with preoperative danazol, gestrinone, or the GnRH agonist buserelin, in a non randomized trial [99]. The cumulative pregnancy rates after 18 months of follow-up in patients treated with buserelin (58%) were significantly higher than those treated with danazol (45%) or gestrinone (47%) [ P < 0.05]. In the subsets of patients attempting conception in two randomized placebo-controlled trials that evaluated the effect of either 3 or 6 months of post operative GnRH agonist therapy, no difference in pregnancy rates was appreciated in either study [88,90]. It is important to note that the primary endpoint in these studies was symptom recurrence and not fertility, which may have created a degree of selection bias. Nevertheless, the preponderance of data would suggest that pre-or postoperative adjunctive medical therapy adds little to the benefit achieved with surgical intervention alone in overcoming endometriosis-associated infertility in the asymptomatic patient. In contrast, these approaches appear to be highly advantageous as an adjunct to the surgical management of symptomatic disease.

Overview of Medical Therapy

181

SUMMARY The medical management of endometriosis has evolved dramatically over the last 40 years and has greatly benefited from the result of well-designed trials. Nevertheless, the absence of a uniform instrument for evaluating outcomes makes comparisons between studies somewhat difficult. Although a host of medications have demonstrated efficacy in the management of symptomatic endometriosis, none appears to be valuable in the management of endometriosis-associated infertility. The administration of a prolonged course of a GnRH agonist prior to in vitro fertilization represents a possible exception. The use of adjunctive medical therapy appears to enhance surgical outcomes when administered for an appropriate time. Currently, GnRH agonists appear to represent the standard for medical management of endometriosis. However, new classes of agents, which attack the source of this disease and not solely its endocrine stimulation represent, exciting pursuits for the future.

PRACTICAL POINTS  

GnRHa is the standard medical treatment of endometriosis. Administration of a prolonged course of a GnRHa prior to in vitro fertilization may be beneficial.

REFERENCES 1. 2.

3.

4.

5.

6.

Dizerega G, Barber D, Hodger G. Endometriosis: Role of ovarian steroids in initiation, maintenance and suppression. Fertil Steril 1980; 33:649–653. Metzger DA, Olive DC, Haney AF. Limited hormonal responsiveness of ectopic endometriosis: histologic correlation with intrauterine endometrium. Hum Pathol 1988; 19:1417–1424. Berquist A, Ljungberg O, Mybrie E. Human endometrium and endometriotic tissue obtained simultaneously: A comparative histologic study. Int J Gynecol Pathol 1984; 3:133–145. Carlstrom K, Berquist A, Ljunberg O. Metabolism of estrone sulfate in endometriotic tissue and in uterine endometrium in proliferative and secretory cycle phase. Fertil Steril 1988; 49:229–233. Vierikko P, Kauppila A, Ronnberg L, Vihko R. Steroidal regulation of endometriosis tissue: Lack of induction of 17h-hydroxysteroid dehydrogenase activity by progesterone, medroxyprogesterone acetate, or danazol. Fertil Steril 1985; 43:218. Meigs JV. Endometriosis: Etiologic role of marriage, age, and parity. Conservative treatment. Obstet Gynecol 1953; 2:45–53.

182 7. 8. 9. 10. 11.

12. 13.

14.

15.

16.

17.

18.

19. 20. 21. 22. 23.

Surrey McArthur JW, Ulfelder H. The effects of pregnancy upon endometriosis. Obstet Gynecol Surv 1965; 20:709. Wheeler JM, Malinak LR. Recurrent endometriosis: Incidence, management, and prognosis. Am J Obstet Gynecol 1983; 146:247–250. Kistner RW. The use of progestins in the treatment of endometriosis. Am J Obstet Gynecol 1958; 75:264–278. Noble AD, Letchworth AT. Medical treatment of endometriosis: A comparative trial. Postgrad Med J 1979; 55(suppl 5):37–39. Vercellini P, Trespidi L, Colombo A, Vendola N, Marchini M, Crosignani PM. A gonadotropin releasing hormone agonist versus a low dose oral contraceptive for pelvic pain associated with endometriosis. Fertil Steril 1993; 30:75–79. Moghissi KS. Pseudo pregnancy induced by estrogen-progestogen or progestin alone in the treatment of endometriosis. Prog Clin Biol Res 1990; 323:221–232. Meresman G, Auge L, Bararro R, Lombardi E, Tesone M, Sueldo C. Oral contraceptives treatment suppresses proliferation and enhances apoptisis of eutopic endometrial tissue from patients with endometriosis. Fertil Steril 2001; 76:S47– S48. Surrey E, Halme J. Direct effects of medroxyprogesterone acetate, danazol, and leuprolide on endometrial stromal cell proliferation in vitro. Fertil Steril 1992; 58:273–278. Bruner K, Eisenberg E, Gorstein F, Osteen K. Progesterone and transforming growth factor-beta coordinately regulate suppression of endometrial matrix metalloproteinases in a model of experimental endometriosis. Steroids 1999; 64: 648–653. Overton C, Lindsay P, Johal B, Collins S, Siddle N, Shaw R, Barlow D. A randomized, double-blind, placebo-controlled study of luteal phase dydrogesterone (Duphaston) in women with minimal to mild endometriosis. Fertil Steril 1994; 62:701–707. Vercellini P, De Giorgi O, Oldavi S, Cortesi I, Panazza S, Crosignani PG. Depot medroxy-progesterone acetate versus an oral contraceptive combined with very low dose danazol for long-term treatment of pelvic pain associated with endometriosis. Am J Obstet Gynecol 1996; 175:396–401. Telimaa S, Puolakka J, Ronnberg L, Kaupilla A. Placebo-controlled comparison of danazol and high dose medroxyprogesterone acetate in the treatment of endometriosis. Gynecol Endocrinol 1987; 1:13–23. Vercellini P, Cortesi I, Crosignani PG. Progestins for symptomatic endometriosis: A critical analysis of the evidence. Fertil Steril 1997; 68:393–401. Luciano A, Turksoy N, Carleo J. Evaluation of oral medroxyprogesterone acetate in the treatment of endometriosis. Obstet Gynecol 1988; 72:323–327. Moghissi K, Boyce C. Management of endometriosis with oral medroxyprogesterone acetate. Obstet Gynecol 1976; 47:265–267. Schlaff W, Dugoff L, Damewood M, Rock J. Megestrol acetate for treatment of endometriosis. Obstet Gynecol 1990; 75:646–648. Muneyyirici-Delale O, Karacan M. Effect of norethindrone acetate in the treatment of symptomatic endometriosis. Int J Fertil 1998; 43:24–27.

Overview of Medical Therapy

183

24. Telimaa S, Penttila I, Puolakka J, Ronnberg L, Kauppila A. Circulating lipid and lipoprotein concentrations during danazol and high dose medroxyprogesterone acetate therapy of endometriosis. Fertil Steril 1989; 52:31–35. 25. Vercellini P, Aimi G, Panazza S, De Giorgi O, Pesole A, Crossignani PM. A levonorgestrel-releasing intrauterine system for the treatment of dysmenorrhea associated with endometriosis: a pilot study. Fertil Steril 1999; 72:505– 508. 26. Moguilewsky M, Philibert D. Dynamics of receptor interaction of danazol and gestrinone in the rat: Correlations with biologic activities. In: Raynard JP ed. Medical Management of Endometriosis. New York: Raven Press, 1984:163–181. 27. Chalbos D, Bardon S, Vignon F, Rocheford H. Use of hormone responsive cell lines to study the mechanism of action of progestins and antiprogestins. In: Raynaud J, ed. Medical Management of Endometriosis. New York: Raven Press 1984:53–71. 28. Thomas E, Cooke I. Impact of gestrinone on the course of asymptomatic endometriosis. BMJ 1987; 294:272–274. 29. Hornstein M, Gleason R, Barbieri R. A randomized double-blind trial of two doses of gestrinone in the treatment of endometriosis. Fertil Steril 1990; 53:237– 241. 30. Dawood M, Obasiolu C, Ramos J, Khan-Dawood F. Clinical, endocrine, and metabolic effects of two doses of gestrinone in treatment of pelvic endometriosis. Am J Obstet Gynecol 1997; 176:387–394. 31. Fedele L, Bianchi S, Viezzoli T, Arcaini L, Candiani G. Gestrinone versus danazol in the treatment of endometriosis. Fertil Steril 1989; 51:781–785. 32. The Gestrinone Italian Study Group. Gestrinone versus a gonadotropin-releasing hormone agonist for the treatment of pelvic pain associated with endometriosis: A multicenter, randomized, double-blind study. Fertil Steril 1996; 66:911–919. 33. Metzger D, Luciano A. Hormonal therapy of endometriosis. Obstet Gynecol Clin (N.A.) 1989; 16:105–122. 34. Ota H, Igarashi S, Hayakawa M, Matsui T, Tanaka H, Tanaka T. Effect of danazol on the immunocompetent cell in the eutopic endometrium in patients with endometriosis: A multicenter cooperative study. Fertil Steril 1996; 65:545– 551. 35. El-Roeiy A, Dmowski W, Gleicher N, Radwanska E, Harlow L, Binor Z, Tummon I, Rawlins RG. Danazol but not gonadotropin-releasing hormone agonists suppressed autoantibodies in endometriosis. Fertil Steril 1988; 50:864– 871. 36. Taketani Y, Kuo T-M, Mizuno M. Comparison of cytokine levels and embryo toxicity in peritoneal fluid in infertile women with untreated or treated endometriosis. Am J Obstet Gynecol 1992; 167:265–270. 37. Matalliotakis I, Neonalis M, Koumantaki Y, Goumeiou A, Kyriakou D, Koumantakis E. A randomized comparison of danazol and leuprolide acetate suppression of serum-soluble CD23 levels in endometriosis. Obstet Gynecol 2000; 95:810–813.

184

Surrey

38. Dmowski W, Kapetanakis E, Scommegna A. Variable effects of danazol on endometriosis at 4 low-dose levels. Obstet Gynecol 1982; 59:408–415. 39. Biberoglu K, Behrman S. Dosage aspects of danazol therapy in endometriosis: Short-term and long-term effectiveness. Am J Obstet Gynecol 1981; 139:645–654. 40. Barbieri R, Evans S, Kistner R. Danazol in the treatment of endoemtriosis: Analysis of 100 cases with a 4-year follow-up. Fertil Steril 1982; 37:737–746. 41. Buttram V, Reiter R, Ward S. Treatment of endometriosis with danazol: Report of a 6-year prospective study. Fertil Steril 1985; 43:353–360. 42. Kingsbury A. Danazol and fetal masculinization: A warning. Med J Aust 1985; 143:410–413. 43. Vercellini D, Trespidi L, Panazza S, Bramante T, Mauro F, Crosignani PG. Very low dose danazol for relief of endometriosis-associated pelvic pain: A pilot study. Fertil Steril 1994; 62:1136–1142. 44. Igarashi M, Cizuka M, Abe Y, Ibuki Y. Novel vaginal danazol ring therapy for pelvic endometriosis in particular deeply infiltrating endometriosis. Hum Reprod 1998; 13:1952–1956. 45. Janicki T. Treatment of the pelvic pain associated with endometriosis using danazol vaginal suppositories: Two year follow-up. Fertil Steril 2002; 77:S52. 46. Suzumori N, Sato M, Ikuta K, Suzumori K. Secretory leukocyte protease inhibitor in ovarian endometriomas following GnRH agonist therapy. Obstet Gynecol 2001; 97:561–566. 47. Meresman G, Bilotas M Jr, Lombardi E, Tesone M, Sueldo C. Effect of gonadotropin-releasing hormone analog on the apoptosis and release of Il-1 and VEGF in endometrial cultures from patients with endometriosis. Fertil Steril 2002; 78:S202–S203. 48. Sharpe-Timms K, Keisler L, McIntush E, Keisler D. Tissue inhibitor of metalloproteinase-1 concentrations are attenuated in peritoneal fluid and sera of women with endometriosis and restored in sera by gonadotropin-releasing hormone agonist therapy. Fertil Steril 1998; 69:1128–1134. 49. Dlugi A, Miller J, Knittle JLupron Study GroupLupron depot (leuprolide acetate in depot suspension) in the treatment of endometriosis: A randomized, placebo-controlled, double-blind study. Fertil Steril 1990; 54:419–427. 50. Henzl M, Corson S, Moghissi K, Buttram D, Berquist C, Jacobson J. Administration of nasal nafarelin as compared with oral danazol for endometriosis: A multicenter double-blind comparative clinical trial. N Engl J Med 1988; 318: 485–489. 51. Dmowski WP, Radwanska E, Binor Z, Tummon I, Pepping P. Ovarian suppression induced with Buserelin or danazol in the management of endometriosis: A randomized, comparative study. Fertil Steril 1989; 51:395–400. 52. Kennedy S, Williams I, Brodribb J, Barlow D, Shaw R. A comparison of nafarelin acetate and danazol in the treatment of endometriosis. Fertil Steril 1990; 53:998–1003. 53. Shaw RZoladex Endometriosis Study TeamAn open randomized comparative study of the effect of goserelin depot and danazol in the treatment of endometriosis. Fertil Steril 1992; 58:265–272.

Overview of Medical Therapy

185

54. The Nafarelin European Endometriosis Trial Group. Nafarelin for endometriosis: A large-scale, danazol-controlled trial of efficacy and safety, with 1-year follow-up. Fertil Steril 1992; 57:514–522. 55. Wheeler J, Knittle J, Miller J. Depot leuprolide versus danazol in treatment of women with symptomatic endometriosis. I. Efficacy results. Am J Obstet Gynecol 1992; 167:1367–1371. 56. Rock J, Truglia J, Caplan R, Zoladex Endometriosis Study Group. Zoladex (goserelin acetate implant) in the treatment of endometriosis: A randomized comparison with danazol. Obstet Gynecol 1993; 82:198–205. 57. Wright S, Valdes C, Dunn R, Franklin R. Short-term Lupron or danazol therapy for pelvic endometriosis. Fertil Steril 1995; 63:504–507. 58. Waller K, Shaw R. Gonadotropin-releasing hormone analogues for the treatment of endometriosis: Long-term follow-up. Fertil Steril 1993; 59:511–515. 59. Ling F, Pelvic Pain Study Group. Randomized controlled trial of depot leuprolide in patients with chronic pelvic pain and clinically suspected endometriosis. Obstet Gynecol 1999; 93:50–51. 60. Surrey E, Add-back Consensus Working Group. Add-back therapy and gonadotropin releasing hormone agonists in the treatment of patients with endometriosis: Can a consensus be reached? Fertil Steril 1999; 71:420–424. 61. Moghissi K, Schlaff W, Olive D, Skinner M, Yin H. Goserelin acetate (Zoladex) with or without hormone replacement therapy for the treatment of endometriosis. Fertil Steril 1998; 69:1056–1062. 62. Kiiholma P, Korhonen M, Tiumala R, Korhonen M, Hayman E. Comparison of the gonadotropin-releasing hormone agonist goserelin acetate alone versus goserelin combined with estrogen progestagen add-back therapy in the treatment of endometriosis. Fertil Steril 1995; 64:903–908. 63. Surrey E, Gambone J, Lu J, Judd H. The effects of combining norethindrone with a gonadotropin-releasing hormone agonist in the treatment of symptomatic endometriosis. Fertil Steril 1990; 53:620–626. 64. Hornstein M, Surrey E, Weisberg G, Casino L. Leuprolide acetate depot and hormonal add-back in endometriosis: A 12-month study. Obstet Gynecol 1998; 91:16–24. 65. Surrey E, Hornstein M. Prolonged GnRH agonist and add-back therapy for symptomatic endometriosis: Long-term follow-up. Obstet Gynecol 2002; 99: 709–719. 66. Martha P, Gray M, Campion M, Kuca B, Garnick M. Prolonged suppression of circulating estrogen levels without an initial hormonal flare using abarelixdepot, a pure GnRH antagonist, in women with endometriosis. Fertil Steril 1999; 72:S210–S211. 67. Jones RC. The effect of a luteinizing hormone-releasing hormone antagonist on experimental endometriosis in the rat. Acta Endocrinol 1987; 114:379–383. 68. Kupker W, Felberbaum R, Krapp M, Schill T, Malik E, Diedrich K. Oestradiol-threshold therapy using the GnRH antagonist, cetrorelix in women with endometriosis. Abstract O-O96. 16th Annual Meeting of the European Society for Human Reproduction and Embryology. Bologna, Italy 2000.

186

Surrey

69. Tjaden B, Galetto D, Woodruff J, Rock J. Time-related effects of RU486 treatment in experimentally induced endometriosis in the rat. Fertil Steril 1993; 59:437–440. 70. Grow D, Williams R, Hsiu J, Hodgen G. Antiprogestin and/or gonadotropinreleasing hormone agonist for endometriosis treatment and bone maintenance: A 1-year primate study. J Clin Endocrinol Metab 1996; 81:1933–1939. 71. Kettel L, Murphy A, Morales A, Ulmann A, Baulieu E, Yen S. Treatment of endometriosis with the antiprogesterone mifepristone (RU486). Fertil Steril 1996; 65:23–28. 72. Olive D. Role of progesterone antagonists and new selective progesterone receptor modulators in reproductive health. Obstet Gynecol Surv 2002; 57:S55– S63. 73. Fanning P, Kuehl T, Lee R, Pearson S, Wincek T, Pliego J, Spiekerman A, Bryant H, Rippy M. Video mapping to assess efficacy of an antiestrogen (raloxifene) on spontaneous endometriosis in the rhesus monkey. Fertil Steril 1997; 68:S38–S39. 74. Zeitoun K, Bulun S. Aromatase: A key molecule in the pathophysiology of endometriosis and a therapeutic target. Fertil Steril 1999; 72:961–969. 75. Scarpelini F, Sbracia M. Aromatase inhibitiors in the treatment of low responder women with severe endometriosis. Fertil Steril 1999; 72:S213. 76. Fang Z, Yang S, Tamura M, Gurates B, Bulun S. Treatment with aromatase inhibitor letrozole significantly reduced the size of endometriotic implants. Fertil Steril 2001; 76:S46. 77. Takayama K, Zeitoun K, Gumby R, Sasano H, Carr B, Bulun S. Treatment of severe postmenopausal endometriosis with an aromatase inhibitor. Fertil Steril 1998; 69:709–713. 78. Keenan J, Williams-Boyle P, Massey P, Chen T, Candle M, Bukovsky A. Regression of endometrial explants in a rat model of endometriosis treated with the immune modulators loxoribine and levamisole. Fertil Steril 1999; 721:135– 141. 79. Badawy S, Etman A, Cuenca V, Montante A, Kaufman L. Effect of interferon a-2b on endometrioma cells in vitro. Obstet Gynecol 2001; 98:417–420. 80. D’Hooghe T, Cuneo S, Nugent N, Chai D, Deer F, Mwenda J. Recombinant human TNF binding protein-1 (r-hTBP-1) inhibits the development of endometriosis in baboons: A prospective, randomized, placebo-and drug-controlled study. Fertil Steril 2001; 76:S1. 81. Scarpellini F, Sbracia M, Lecchini S, Scarpellini L. Anti-angiogenesis treatment with thalidomide in endometriosis: A pilot study. Fertil Steril 2002; 78: S87. 82. Hughes EG, Fedorkow DM, Collins JA. A quantitative overview of controlled trials in endometriosis-associated infertility. Fertil Steril 1993; 59:963–970. 83. Adamson GD, Pasta D. Surgical treatment of endometriosis-associated infertility: Meta-analysis compared with survival analysis. Am J Obstet Gynecol 1994; 171:1488–1505. 84. Surrey ES, Silverberg KM, Surrey MW, Schoolcraft WB. The effect of pro-

Overview of Medical Therapy

85.

86.

87.

88.

89. 90.

91.

92.

93.

94.

95.

96.

97.

187

longed GnRH agonist therapy on in vitro fertilization-embryo transfer cycle outcome in endometriosis patients: a multicenter randomized trial. Fertil Steril 2002; 78:699–704. Dicker D, Goldman GA, Ashkenazi J, Feldberg D, Voliovitz I, Goldman JA. The value of pretreatment with long-term gonadotropin-releasing hormone (GnRH) analogue in IVF-ET therapy of severe endometriosis. Hum Reprod 1990; 5:418–420. Marcus SF, Edwards RG. High rates of pregnancy after long-term downregulation of women with severe endometriosis. Am J Obstet Gynecol 1994; 171: 812–817. Nakamura K, Oosawa M, Kondou I, Inagaki S, Shibata H, Narita O, Suganuma N, Tomoda Y. Menotropin stimulation after prolonged gonadotropin releasing hormone agonist pretreatment for in vitro fertilization in patients with endometriosis. J Assist Reprod Genet 1992; 9:113–117. Curtis P, Jackson A, Bernard A, Shaw RW. Pretreatment with gonadotrophin releasing hormone (GnRH) analogue prior to in vitro fertilization for patients with endometriosis. Eur J Obstet Gynecol Reprod Biol 1993; 52:211–216. Lessey BA. Medical management of endometriosis and infertility. Fertil Steril 2000; 73:1089–1096. Telimaa S, Ronnberg L, Kaupilla A. Placebo-controlled comparison of danazol and high dose medroxyprogesterone acetate in the treatment of endometriosis after conservative surgery. Gynecol Endocrinol 1987; 1:363–371. Hornstein M, Hemmings R, Yuzpe A, Heinrichs W. Use of nafarelin versus placebo after reductive laparoscopic surgery for endometriosis. Fertil Steril 1997; 68:860–864. Vercellini P, Crosignani PG, Fadini R, Radici E, Belloni C, Sismondi P. A gonadotropin-releasing hormone agonist compared with expectant management after conservative surgery for symptomatic endometriosis. Br J Obstet Gynecol 1999; 106:672–677. Busacca M, Somigliane E, Bianchi S, De Marinis S, Cake C, Candiani M, Vignali M. Post-operative GnRH analogue treatment after conservative surgery for symptomatic endometriosis stage III-IV: A randomized controlled trial. Hum Reprod 1002; 16:2399–2402. Parazzini F, Fedele L, Busacca M, Falsetti L, Pellegrini S, Venturini PL, Stella M. Postsurgical medical treatment of advanced endometriosis: Results of a randomized clinical trial. Am J Obstet Gynecol 1994; 171:1205–1207. Bianchi S, Busacca M, Agnoli B, Candiani M, Calia C, Vignali M. Effects of 3 month therapy with danazol after laparoscopic surgery for stage III/IV endometriosis: A randomized study. Hum Reprod 1999; 14:1335–1337. Audebert A, Descamps D, Mariet H, Ory-Lavollee L, Bailleul F, Hamamah S. Pre- or post- operative medical treatment with nafarelin in stage III-IV endometriosis: A French multicenter study. Eur J Obstet Gynecol 1998; 79:145– 148. Shaw R, Garry R, McMillan L, Sutton C, Wood S, Harrison R, Das R. A prospective randomized open study comparing goserelin (Zoladex) plus surgery

188

Surrey

and surgery alone in the management of ovarian endometriomas. Gynecol Endosc 2001; 10:151–157. 98. Wright J, Sharpe-Timms K. Gonadotropin-releasing hormone agonist therapy reduces postoperative adhesion formation and reformation after adhesiolysis in rat models for adhesion formation and endometriosis. Fertil Steril 1995; 63: 1094–1100. 99. Donnez J, Nisolle-Pochet M, Casanas-Roux F. Endometriosis-associated infertility: Evaluation of preoperative use of danazol, gestrinone, and buserelin. Int J Fertil 1990; 35:297–301.

11 Treatment with Aromatase Inhibitors Serdar E. Bulun, Bilgin Gurates, Zongjuan Fang, Mitsutoshi Tamura, David Langoi, Gonca Imir, Sanober Amin, Santanu Deb, and Sijun Yang University of Illinois at Chicago Chicago, Illinois, U.S.A.

INTRODUCTION Estrogen causes proliferation of endometrial tissue. Aromatase P450 (P450arom) is the key enzyme for estrogen biosynthesis. Aromatase activity is not detectable in normal endometrium. In contrast, P450arom is expressed aberrantly in endometriosis and is stimulated by PGE2. This results in local production of estrogen, which induces PGE2 formation and establishes a positive feedback cycle. These molecular aberrations collectively favor accumulation of increasing quantities of estradiol and PGE2 in endometriosis. The clinical relevance of these findings was exemplified by the successful treatment of an unusually aggressive case of postmenopausal endometriosis using an aromatase inhibitor. We will discuss the molecular mechanisms that give rise to aromatase overexpression in endometriosis tissue. Endometriosis is the leading cause of pelvic pain [1,2]. Endometriosis is characterized by the presence of endometrial glands and stroma within the pelvic peritoneum and other extrauterine sites and is linked to pelvic pain 189

190

Bulun et al.

and infertility. It is estimated to affect 5% of women in the reproductive age group [1,2]. Endometriosis is a polygenically inherited disease of complex multifactorial etiology [3]. Sampson’s theory of transplantation of endometrial tissue on the pelvic peritoneum by retrograde menstruation is the most widely accepted explanation for the development of pelvic endometriosis because of convincing circumstantial and experimental evidence [4]. Because retrograde menstruation is observed in almost all cycling women, endometriosis is postulated to develop as a result of the coexistence of a defect in clearance of the menstrual efflux from pelvic peritoneal surfaces, possibly involving the immune system [5]. Alternatively, intrinsic molecular aberrations in pelvic endometriotic implants were proposed to contribute significantly to development of endometriosis. Aberrant expression of aromatase, certain cytokines and tissue metalloproteinases, deficiency of 17h-hydroxysteroid dehydrogenase (17h-HSD) type 2, and resistance to the protective action of progesterone are some of these molecular abnormalities [6–12]. Because endometriosis is an estrogen-dependent disorder, aromatase expression and 17h-HSD type 2 deficiency are of paramount importance in the pathophysiology of endometriosis. In this article, aberrant mechanisms of estrogen biosynthesis and metabolism in women with endometriosis are reviewed with emphasis on identifying targets for new treatment strategies. Estrogen Biosynthesis and Metabolism in Humans The conversion of androstenedione and testosterone to estrone and estradiol is catalyzed by aromatase, which is expressed in a number of human tissues and cells, such as ovarian granulosa cells, placental syncytiotrophoblast, adipose tissue and skin fibroblasts, and the brain. In the reproductive-age woman, the ovary is the most important site of estrogen biosynthesis, and this takes place in a cyclic fashion. Upon binding of follicle-stimulating hormone (FSH) to its G-protein-coupled receptor in the granulosa cell membrane, intracellular cyclic adenosine monophosphate (cAMP) levels rise and enhance binding of two critical transcription factors, that is, steroidogenic factor-1 (SF-1) and cAMP response element binding protein (CREB), to the classically located proximal promoter II of the aromatase gene [13,14]. This, in turn, activates aromatase expression and estrogen secretion from the preovulatory follicle [14,15]. On the other hand, in postmenopausal women, estrogen formation takes place in extraglandular tissues, such as the adipose tissue and skin [16– 18] (Fig. 1). In contrast to cAMP regulation of aromatase expression in the ovary, this is controlled primarily by cytokines (interleukin [IL]-6, IL-11, tumor necrosis factor [TNF]a) and glucocorticoids by the alternative use of promoter I.4 in adipose tissue and skin fibroblasts [15]. The major substrate

Treatment with Aromatase Inhibitors

191

FIGURE 1 Extraovarian estrogen synthesis in women. Estradiol in women is either directly secreted by the ovary or produced in extraglandular sites (adipose tissue and skin). The principal substrate for extraglandular aromatase activity in ovulatory women is androstenedione of adrenal and ovarian origins. In women receiving GnRH agonists or postmenopausal women, however, the adrenal remains as the primary source of androstenedione. Androstenedione is converted by aromatase to estrone in adipose tissue and skin fibroblasts. Estrone is further converted to estradiol by 17h-HSD type 1 activity in these peripheral tissues. Thus, peripheral aromatization is the major source for circulating estradiol in the postmenopausal period or during ovarian suppression.

for aromatase in adipose tissue and skin is androstenedione of adrenal origin. In postmenopausal women, approximately 2% of circulating androstenedione is converted to estrone, which is further converted to estradiol in these peripheral tissues. This may give rise to significant serum levels of estradiol capable of causing endometrial hyperplasia or even carcinoma [17,18]. Aromatase Expression in Normal and Abnormal Uterine Tissues Mu¨llerian tissues are known targets of estrogen action. Until recently, estrogen action has been classically viewed to occur only by an ‘‘endocrine’’ mechanism: in other words, it was thought that only circulating estradiol, whether secreted by the ovary or formed in the adipose tissue, could exert an estrogenic effect after delivery to target tissues through the bloodstream. Studies on aromatase expression in breast cancer demonstrated that paracrine mechanisms play an important role in estrogen action in this tissue [19]. Estrogen

192

Bulun et al.

produced by aromatase activity in breast adipose tissue fibroblasts was demonstrated to promote the growth of adjacent malignant breast epithelial cells [20]. Finally, we demonstrated an ‘‘intracrine’’ effect of estrogen in uterine leiomyomas and endometriosis: estrogen produced by aromatase activity in the cytoplasm of leiomyoma smooth muscle cells or endometriotic stromal cells can exert its effects by readily binding to its nuclear receptor within the same cell [6,21,22]. Disease-free endometrium and myometrium, on the other hand, lack aromatase expression [21,22]. Aromatase Expression in Endometriosis Among estrogen-responsive pelvic disorders, aromatase expression was studied in greatest detail in endometriosis [6,7,22,23]. First, extremely high levels of aromatase mRNA were found in extraovarian endometriotic implants and endometriomas. Second, endometriosis-derived stromal cells in culture incubated with a cAMP analog displayed extraordinarily high levels of aromatase activity comparable to that in placental syncytiotrophoblast [22]. These exciting findings led us to test a battery of growth factors, cytokines, and other substances that might induce aromatase activity via a cAMPdependent pathway in endometriosis. Prostaglandin E2 (PGE2) was found to be the most potent known inducer of aromatase activity in endometriotic stromal cells [22]. In fact, this PGE2 effect was found to be mediated through the cAMP-inducing EP2 receptor subtype (our unpublished observations). Moreover, estrogen was reported to increase PGE2 formation by stimulating cyclo-oxygenase type 2 (COX-2) enzyme in endometrial stromal cells in culture [24]. Thus, a positive feedback loop for continuous local productions of estrogen and PGs is established, favoring the proliferative and inflammatory characteristics of endometriosis (Fig. 2). Additionally, aromatase mRNA was also detected in the eutopic endometrial samples of women with moderate to severe endometriosis (but not in those of disease-free women), albeit in much smaller quantities compared with endometriotic implants [6]. This may be suggestive of a genetic defect in women with endometriosis, which is manifested by this subtle finding in the eutopic endometrium. We propose that when defective endometrium with low levels of aberrant aromatase expression reaches the pelvic peritoneum by retrograde menstruation, it causes an inflammatory reaction that exponentially increases local aromatase activity, for example, estrogen formation, induced directly or indirectly by PGs and cytokines [22]. It would be rather naive to propose that aberrant aromatase expression is the only important molecular mechanism in the development and growth of pelvic endometriosis. There may be many other molecular mechanisms that favor the development of endometriosis: abnormal expression of proteinase-type enzymes that remodel tissues or their inhibitors

Treatment with Aromatase Inhibitors

193

FIGURE 2 Origin of estrogen in endometriotic lesions. Estradiol in an endometriotic lesion arises from several body sites. In an ovulatory woman, estradiol is secreted directly from the ovary in a cyclic fashion. In the early follicular phase and after menopause, peripheral tissues (adipose and skin) are the most important sources to account for the circulating estradiol. Estradiol is also produced locally in the endometriotic implant itself in both ovulatory and postmenopausal women. The most important precursor, androstenedione of adrenal origin, becomes converted to estrone that is, in turn, reduced to estradiol in the peripheral tissues and endometriotic implants. We demonstrated significant levels of 17h-hydroxysteroid dehydrogenase type 1 expression in endometriosis, which catalyzes the conversion of estrone to estradiol [12]. Estradiol both directly and indirectly (through cytokines) induces COX-2 which gives rise to elevated concentrations of PGE2 in endometriosis [24]. Prostaglandin E2 is the most potent known stimulator of aromatase in endometriotic stromal cells [22]. This establishes a positive feedback loop in favor of continuous estrogen formation in endometriosis.

(matrix metalloproteinases, tissue inhibitor of metalloproteinase-1), certain cytokines (IL-6, RANTES), and growth factors (EGF) represent some of mechanisms [8–11]. Alternatively, a defective immune system that fails to clear peritoneal surfaces of the retrograde menstrual efflux has been proposed in the development of endometriosis [5,25]. The development of endometriosis in an individual woman probably requires the coexistence of a thresh-

194

Bulun et al.

old number of these aberrations. Nonetheless, aberrant aromatase expression is clinically relevant, as aromatase inhibitors suppress postmenopausal endometriosis [26]. Regulation of Aromatase Expression in Endometriotic Stromal Cells As emphasized earlier, PGE2 was found to be the most potent known inducer of aromatase activity by increasing cAMP levels in endometriotic stromal cells [22]. On the other hand, neither cAMP analogs nor PGE2 were capable of stimulating any detectable aromatase activity in eutopic endometrial stromal cells in culture. The obvious question became what are the molecular differences that give rise to aromatase expression in endometriosis and its inhibition in eutopic endometrium? To address this, we first determined that the cAMP-inducible promoter II was used for in vivo aromatase expression in endometriotic tissue [7]. Then, a stimulatory transcription factor, SF-1, and an inhibitory factor, chicken ovalbumin upstream promoter transcription factor (COUP-TF), were found to compete for the same binding site in aromatase promoter II. The COUP-TF was ubiquitously expressed in both eutopic endometrium and endometriosis, whereas SF-1 was expressed specifically in endometriosis but not in eutopic endometrium and binds to aromatase promoter more avidly than COUP-TF [7]. Thus, SF-1 and other transcription factors (e.g., CREB) activate transcription in endometriosis, whereas COUPTF, which occupies the same DNA site in eutopic endometrium, inhibits this process [7] (Fig. 3). In summary, one of the molecular alterations leading to local aromatase expression in endometriosis, but not in normal endometrium, is the aberrant production of SF-1 in endometriotic stromal cells,

Œ

FIGURE 3 Proposed mechanism of regulation of aromatase (P450arom) expression by SF-1 and COUP-TF in eutopic endometrium and endometriosis. (A) Binding of COUP-TF (dimer) to a specific DNA site (nuclear receptor half-site) upstream of aromatase promoter II in eutopic endometrial stromal cells. In the eutopic endometrium, COUP-TF binds to nuclear receptor half-site practically in the absence of any competition by SF-1, as SF-1 expression is not detected in the majority of eutopic endometrial samples. Thus, COUP-TF exerts its inhibitory effect on the complex of general transcription factors (GTFs) that bind to TATA box. (B) In the endometriotic stromal cell, where SF-1 is also present, SF-1 binds as a monomer to the nuclear receptor half-site with a higher affinity compared with that of COUP-TF, which binds to this site relatively loosely as a dimer. Upon replacing COUP-TF, SF-1 synergizes with cAMP response element binding protein (CREB, bound to upstream CRE) and other factors to activate transcription of the aromatase gene in response to cAMP [7].

Treatment with Aromatase Inhibitors

195

196

Bulun et al.

which overcomes the protective inhibition maintained normally by COUPTF in the eutopic endometrium. Inactivation of Estradiol in Endometrium and Endometriosis The primary substrate for aromatase activity in endometriosis is androstenedione of adrenal and ovarian origins in premenopausal women and adrenal androstenedione in postmenopausal women. The major product of aromatase activity in endometriosis, namely estrone, is only weakly estrogenic and must be converted to the potent estrogen estradiol to exert a full estrogenic effect. We demonstrated that the enzyme 17h-HSD type 1, which catalyzes the conversion of estrone to estradiol, is expressed in endometriosis [12,27]. In contrast, the enzyme 17h-HSD type 2 (encoded by a separate gene) inactivates estradiol by catalyzing its conversion to estrone in eutopic endometrial glandular cells during the luteal phase [27]. Progesterone actually induces the activity of this enzyme in endometrial glandular cells in culture, making inactivation of estradiol to estrone one of the antiestrogenic properties of progesterone [28]. The expression of 17h-HSD type 2 is absent from endometriotic glandular cells, as demonstrated in paired samples of eutopic endometrium and pelvic endometriosis obtained simultaneously during the luteal phase [12]. Consequently, this protective mechanism that lowers estradiol levels is lost in endometriotic tissue [12]. The aberrant expression of aromatase, the presence of 17h-HSD type 1, and the absence of 17h-HSD type 2 from endometriosis collectively give rise to elevated local levels of estradiol compared with eutopic endometrium. Additionally, 17h-HSD type 2 deficiency may also be viewed as a defective action of progesterone, which fails to induce this enzyme in endometriotic tissue (Fig. 4). Clinical Basis for Using Aromatase Inhibitors to Treat Endometriosis Endometriosis is successfully suppressed by estrogen deprivation with GnRH analogs or the induction of surgical menopause. Control of pelvic pain with GnRH agonists is usually successful during and immediately after the treatment, whereas pain associated with endometriosis returns in up to 75% of these women [29,30]. There may be multiple reasons for the failure of GnRH agonist treatment of endometriosis. One likely explanation is the presence of significant estradiol production that continues in the adipose tissue, skin, and endometriotic implant per se during the GnRH agonist treatment (Fig. 5). Therefore, blockage of aromatase activity in these extraovarian sites with an aromatase inhibitor may keep larger number of patients in remission for longer periods. The most striking evidence for the significance of extraovarian estrogen production is the recurrence of endometriosis after successfully

Treatment with Aromatase Inhibitors

197

FIGURE 4 Defective inactivation of estradiol in endometriosis. Estradiol (E2) reaches the endometriotic lesions via bloodstream (and possibly peritoneal fluid). Additionally, aromatase (P450arom) in the stromal cell catalyzes the conversion of androstenedione (A) to estrone (E1), which is further reduced to E2 by 17h-HSD type 1 in the endometriotic tissue. (At this time, the cell type that expresses 17h-HSD type 1 in endometriotic lesions is not known.) Estradiol is normally inactivated by conversion to E1 by 17h-HSD type 2 in epithelial cells of the eutopic endometrium during the secretory phase. In endometriotic tissue, however, E2 is not metabolized owing to the lack of 17h-HSD type 2 giving rise to increased local concentration of this potent estrogen. Elevated E2, in turn, will promote the growth of endometriotic tissue and local PGE2 formation in stromal cells [24]. Because PGE2 is the most potent known inducer of aromatase in endometriosis, this will complete the positive feedback cycle that favors increased levels of E2 in endometriosis through enhanced biosynthesis and deficient metabolism.

completed hysterectomy and bilateral salpingo-oophorectomy in a number of women [26,31]. Endometriotic tissue in one such aggressive case was found to express much higher levels of aromatase mRNA compared with premenopausal endometriosis [26]. We recently reported the treatment of a 57-year-old overweight woman who had recurrence of severe endometriosis after hysterectomy and bilateral salpingo-oophorectomy. Two additional laparotomies were performed owing to persistent severe pelvic pain and bilateral ureteral obstruction leading to left renal atrophy and right hydronephrosis. Treatment with megestrol acetate was ineffective. A large (3 cm) vaginal endometriotic lesion contained unusually high levels of aromatase mRNA. The patient was

198

Bulun et al.

FIGURE 5 Site of action of GnRH agonists and aromatase inhibitors to treat endometriosis. This figure depicts the origin of estrogen in women with endometriosis: (i) delivery from the ovary and adipose tissue/skin via circulation and (ii) local biosynthesis in endometriosis. GnRH agonists will eliminate estradiol secreted by the ovary by downregulating the pituitary hypothalamic unit. In cases resistant to treatment with GnRH agonists or in postmenopausal endometriosis, the use of aromatase inhibitors to block estrogen formation in the skin and adipose tissue as well as in endometriotic stromal cells may be critical in controlling the growth of endometriotic tissue. Recurrent endometriosis, especially after surgical removal of the ovaries, may represent lesions that are sensitive to extremely low levels of estradiol, thus suppression of estradiol production in the periphery (adipose tissue/skin) and in endometriotic tissue may be mandatory for successful treatment of endometriosis.

given anastrozole (an aromatase inhibitor) for 9 months. Despite the addition of calcium and alendronate (a nonsteroidal inhibitor of bone resorption), bone density in the lumbar spine decreased by 6.2%. The occurrence of significant bone loss in this particular case should be studied further. Dramatic relief of the pain and regression of the vaginal endometriotic lesion were observed within the first month of treatment. At the same time, circulating estradiol levels were reduced to 50% of the baseline value. Markedly high pretreatment levels of aromatase mRNA in the endometriotic tissue became undetectable in a repeat biopsy 6 months later, and the lesion nearly disappeared after 9 months of therapy. Two potential mechanisms may have accounted for this strikingly successful result. Firstly, there was evidence of suppression of peripheral (skin and adipose tissue) aromatase activity, giving

Treatment with Aromatase Inhibitors

199

rise to a significant decrease in serum estradiol level. Secondly, unusually high levels of aromatase expression in the endometriotic lesion disappeared after treatment with the aromatase inhibitor, anastrozole. Besides the expected direct inhibition of aromatase activity in endometriosis by anastrozole, the disappearance of aromatase mRNA expression in the lesion may be explicable by denial of estrogen that is known to stimulate local biosynthesis of PGE2, which, in turn, stimulates aromatase expression (Fig. 2). SUMMARY Endometriosis is an estrogen-dependent disease. The mechanisms and effectiveness of hormonal treatments for endometriosis should be re-evaluated in view of the new advances that increased our understanding of the body sites of estrogen production in a woman with endometriosis. In addition to ovarian secretion, estradiol is produced in peripheral sites such as skin, adipose tissue, and endometriotic lesions. We suggest that the intracrine and paracrine effects of estradiol produced in the target tissue amplify the estrogenic action of steroid hormones delivered through the circulation. Additionally, defective inactivation of estradiol in endometriosis in contrast to eutopic endometrium may further enhance this local effect. Aberrant aromatase activity and defective estradiol metabolism in endometriosis are consequences of specific molecular aberrations, such as inappropriate expression of a stimulatory transcription factor or progesterone resistance in this tissue. The clinical relevance of these findings was recently exemplified by the successful treatment of a severe case of recurrent postmenopausal endometriosis with an aromatase inhibitor. Understanding the unique transcriptional mechanisms responsible for aromatase expression will enable us to define specific targets and design treatments that will target only local estrogen biosynthesis in endometriosis. It remains to be seen whether such high-precision strategies will give rise to clinically successful treatments. The recently developed potent aromatase inhibitors are candidate drugs in the treatment of endometriosis resistant to standard regimens. In fact, the use of aromatase inhibitors may be the only available treatment for aggressive postmenopausal endometriosis. It remains to be seen whether aromatase inhibitors alone or together with present lines of therapy in premenopausal women will increase the pain-free interval and time to recurrence after discontinuation (Fig. 5). Studies are under way to address these questions. ACKNOWLEDGMENTS The preparation of this article was supported, in part, by NIH grants HD38691 and TW01339.

200

Bulun et al.

PRACTICAL POINT .

Aromatase inhibitors could be new drugs for treating endometriosis.

REFERENCES 1. 2.

3. 4.

5.

6. 7.

8.

9.

10.

11.

12.

13.

14.

Vessey MP, Villard-Mackintosh L, Painter R. Epidemiology of endometriosis in women attending family planning clinics. Br Med J 1993; 306:182–184. Kjerulff KH, Erickson BA, Langenberg PW. Chronic gynecological conditions reported by US women: Findings from the National Health Information Survey, 1984 to 1992. Am J Public Health 1996; 86:195–199. Olive DL, Schwartz LB. Endometriosis. N Engl J Med 1993; 328:1759–1769. Sampson JA. Peritoneal endometriosis due to the menstrual dissemination of endometrial tissue into the peritoneal cavity. Am J Obstet Gynecol 1927; 14:422– 425. Halme J, White C, Kauma S, Estes J, Haskill S. Peritoneal macrophages from patients with endometriosis release growth factor activity in vitro. J Clin Endocrinol Metab 1988; 66:1044–1049. Noble LS, Simpson ER, Johns A, Bulun SE. Aromatase expression in endometriosis. J Clin Endocrinol Metab 1996; 81:174–179. Zeitoun K, Takayama K, Michael MD, Bulun SE. Stimulation of aromatase P450 promoter (II) activity in endometriosis and its inhibition in endometrium are regulated by competitive binding of SF-1 and COUP-TF to the same cisacting element. Mol Endocrinol 1999; 13:239–253. Khorram O, Taylor RN, Ryan IP, Schall TJ, Landers DV. Peritoneal fluid concentrations of the cytokine RANTES correlate with the severity of endometriosis. Am J Obstet Gynecol 1993; 169:1545–1549. Sharpe-Timms KL, Penney LL, Zimmer RL, Wright JA, Zhang Y, Surewicz K. Partial purification and amino acid sequence analysis of endometriosis proteinII (ENDO-II) reveals homology with tissue inhibitor of metalloproteinases-1 (TIMP-1). J Clin Endocrinol Metab 1995; 80:3784–3787. Bruner KL, Matrisian LM, Rodgers WH, Gorstein F, Osteen KG. Suppression of matrix metalloproteinases inhibits establishment of ectopic lesions by human endometrium in nude mice. J Clin Invest 1997; 99:2851–2857. Osteen KG, Bruner KL, Sharpe-Timms KL. Steroid and growth factor regulation of matrix metalloproteinase expression and endometriosis. Semin Reprod Endocrinol 1996; 15:301–308. Zeitoun K, Takayama K, Sasano H, Suzuki T, Moghrabi N, Andersson S, Johns A, Meng L, Putman M, Carr B, Bulun SE. Deficient 17h-hydroxysteroid dehydrogenase type 2 expression in endometriosis: failure to metabolize estradiol-17h. J Clin Endocrinol Metab 1998; 83:4474–4480. Michael MD, Michael LF, Simpson ER. A CRE-like sequence that binds CREB and contributes to cAMP-dependent regulation of the proximal promoter of the human aromatase P450 (CYP19) gene. Mol Cell Endocrinol 1997; 134:147–156. Michael MD, Kilgore MW, Morohashi KI, Simpson ER. Ad4BP/SF-1 regu-

Treatment with Aromatase Inhibitors

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25. 26.

27.

201

lates cyclic AMP-induced transcription from the proximal promoter (PII) of the human aromatase P450 (CYP19) gene in the ovary. J Biol Chem 1995; 270: 13561–13566. Simpson ER, Mahendroo MS, Means GD, Kilgore MW, Hinshelwood MM, Graham-Lorence S, Amarneh B, Ito Y, Fisher CR, Michael MD, Mendelson CR, Bulun SE. Aromatase cytochrome P450, the enzyme responsible for estrogen biosynthesis. Endocr Rev 1994; 15:342–355. Ackerman GE, Smith ME, Mendelson CR, MacDonald PC, Simpson ER. Aromatization of androstenedione by human adipose tissue stromal cells in monolayer culture. J Clin Endocrinol Metab 1981; 53:412–417. MacDonald PC, Rombaut RP, Siiteri PK. Plasma precursors of estrogen. I. Extent of conversion of plasma D4-androstenedione to estrone in normal males and non-pregnant normal, castrate and adrenalectomized females. J Clin Endocrinol Metab 1967; 27:1103–1111. MacDonald PC, Edman CD, Hemsell DL, Porter JC, Siiteri PK. Effect of obesity on conversion of plasma androstenedione to estrone in postmenopausal women with and without endometrial cancer. Am J Obstet Gynecol 1978; 130: 448–455. Bulun SE, Price TM, Mahendroo MS, Aitken J, Simpson ER. A link between breast cancer and local estrogen biosynthesis suggested by quantification of breast adipose tissue aromatase cytochrome P450 transcripts using competitive polymerase chain reaction after reverse transcription. J Clin Endocrinol Metab 1993; 77:1622–1628. Yue W, Wang JP, Hamilton CJ, Demers LM, Santen RJ. In situ aromatization enhances breast tumor estradiol levels and cellular proliferation. Cancer Res 1998; 58:927–932. Bulun SE, Simpson ER, Word RA. Expression of the CYP19 gene and its product aromatase cytochrome P450 in human leiomyoma tissues and cells in culture. J Clin Endocrinol Metab 1994; 78:736–743. Noble LS, Takayama K, Zeitoun KM, Putman JM, Johns DA, Hinshelwood MM, Agarwal VR, Zhao Y, Carr BR, Bulun SE. Prostaglandin E2 stimulates aromatase expression in endometriosis-derived stromal cells. J Clin Endocrinol Metab 1997; 82:600–606. Bulun SE, Mahendroo MS, Simpson ER. Polymerase chain reaction amplification fails to detect aromatase cytochrome P450 transcripts in normal human endometrium or decidua. J Clin Endocrinol Metab 1993; 76:1458–1463. Huang JC, Dawood MY, Wu KK. Regulation of cyclooxygenase-2 gene in cultured endometrial stromal cells by sex steroids. Proceedings of the American Society For Reproductive Medicine, 1996. Meeting S5 [abstract]. Hill JA. Immunology and endometriosis. Fertil Steril 1992; 58:262–264. Takayama K, Zeitoun K, Gunby RT, Sasano H, Carr BR, Bulun SE. Treatment of severe postmenopausal endometriosis with an aromatase inhibitor. Fertil Steril 1998; 69:709–713. Andersson S, Moghrabi N. Physiology and molecular genetics of 17h-hydroxysteroid dehydrogenases. Steroids 1997; 62:143–147.

202

Bulun et al.

28. Satyaswaroop PG, Wartell DJ, Mortel R. Distribution of progesterone receptor, estradiol dehydrogenase, and 20a-dihydroprogesterone dehydrogenase activities in human endometrial glands and stroma: Progestin induction of steroid dehydrogenase activities in vitro is restricted to the glandular epithelium. Endocrinology 1982; 111:743–749. 29. Henzl MR, Corson SL, Moghissi K, Buttram VC, Berqvist C, Jacobson J. Administration of nasal nafarelin as compared with oral danazol for endometriosis. N Engl J Med 1988; 318:485–489. 30. Waller KG, Shaw RW. Gonadotropin-releasing hormone analogues for the treatment of endometriosis: Long-term follow-up. Fertil Steril 1993; 59:511– 515. 31. Metzger DA, Lessey BA, Soper JT, McCart KS Jr, Haney AF. Hormoneresistant endometriosis following total abdominal hysterectomy and bilateral salpingo-oophorectomy: Correlation with histology and steroid receptor content. Obstet Gynecol 1991; 78:946–950.

12 Endometriosis: Medical Treatment with Progestagens Robert Lahoud IVF Australia West, Westmead, Australia

Robert F. Harrison Rotunda Hospital, Dublin, Ireland

INTRODUCTION Endometriosis is a disorder defined by the presence of ectopic endometrial tissue. It is common and affects 1 in 7 of the female population of reproductive age [1]. It is more frequent in females presenting with chronic pelvic pain (70%) and in the subfertile population (30%–50%) [2]. The diagnosis is usually made at laparoscopy, hence, accurate prevalence estimates are difficult to obtain. Furthermore, the correlation between symptoms and visible disease is not linear [3]. In 25% of peritoneal biopsies of normal-appearing peritoneum, endometriosis was discovered [4]. Even though described by Rokitanski in 1860, it was only since the advent of laparoscopy that the large prevalence of this disorder was recognized. Endometrium is a tissue characterized by its regenerative potential, angiogenic properties, and hormone responsiveness. The hormones that particularly act on the endometrium include estrogen and progesterone. Medical treatments using danazol, progestagens, antiprogestagens and combined 203

204

Lahoud and Harrison

estrogen/progesterone preparations act on these hormones and have been used for many decades. Since the advent of the gonadotropin releasing hormone (GnRH) agonists, a new dimension of treatment choice has become available. The choice of medical treatment will depend on efficacy but is particularly based on the side effect profile, as long-term compliance with treatment is essential. Danazol, which was a particularly popular treatment for endometriosisrelated pain, has some very undesirable androgenic side effects (Table 1); hirsutism, mood changes, and adverse effects on the lipid profile as well as irreversible deepening of the voice [5,6]. These side effects have made clinicians and patients shy away from this treatment. On the other hand, danazol is effective in improving endometriosis-related pain [7] and is still a viable treatment option today. Progestional agents have been widely used to treat endometriosis. The decidualization and atrophy effect seen in endometriosis when using these agents also leads to irregular menstrual bleeding, which is one of the main reasons for noncompliance with this form of treatment (Table 1). Other side effects such as nausea, breast tenderness, fluid retention, and depression have been encountered [8,9]. High doses of medroxyprogesterone acetate (MPA) can adversely affect the lipoprotein profiles and may also cause suppression of gonadotropin production, leading to relative hypoestrogenism. Any shortterm loss in spinal bone mineral density is restored with time [10]. The commonly used dosages of MPA are 30-mg to 100 mg orally per day. It can also be administered parenterally using a 150-mg intramuscular depot preparation every 90 days. The advantages of progestagen treatment include its relative safety, wide availability, and low cost. Gestrinone is an antiprogestional steroid that causes a reduction in serum estradiol and sex hormone-binding globulin levels and reduces both estrogen and progesterone receptor concentrations in target tissues [11,12]. Like danazol, it is an effective treatment for the pain caused by endometriosis [13]. The androgenic side effects of gestrinone occur less frequently than with danazol. These include deepening of the voice and hirsutism. Another advantage of gestrinone is that it can be given as a once- or twice-weekly dose as compared with daily doses with Danazol. The combined estrogen/progestagen oral contraceptive (COC) has been used in the treatment of endometriosis for more then 40 years [14]. Many of the short-term side effects, such as weight gain and abnormal bleeding, and the more serious long-term effects, such as venous thrombosis, are well documented. However, COC is generally well tolerated [15], widely available, and inexpensive. Combined oral contraceptive treatment can be safely used for long-term treatment. The GnRH agonists induce a state of hypogonadotropic hypogonadism and the resultant hypoestrogenism is very effective in treating the pain of

Medical Treatment with Progestagens

205

TABLE 1 Medication Danazol

ProgestagensMedroxyprogesterone acetate Dydrogesterone Gestrinone

Combined oral contraceptive GnRH analoguesLeuprorelin acetate Goserelin Nafarelin acetate

GnRH analogues with add-back

Administration 200–800 mg/day orally for 6 months

30 to 100 mg/day (oral) 10 to 20 mg/day (oral) 2.5 mg twice a week

Various preparations in cyclical or continuous regimen 1 mg/day sc 3.6 mg sc per month 400–800 Ag/day intranasal

Side Effects Acne, weight gain, seborrhea, hirsutism, edema, hair loss, voice change, hot flushes and sweats, irregular uterine bleeding, muscle cramps, decrease in HDL cholesterol, increased insulin requirement in diabetic patients Irregular uterine bleeding, insomnia, depression, headache, nausea, acne, hirsutism, thromboembolism. Acne, seborrhea, hirsutism, voice change, weight increase, hot flashes, headaches, irritability, depression, nausea, irregular uterine bleeding Side effects estrogenic and progestagenic. Hot flashes, change in libido, vaginal dryness, headaches, emotional lability, acne, myalgia, decreased breast size, bone mineral density loss, functional ovarian cysts. Reduces hypoestrogenic symptoms associated with GnRH analogue use.

endometriosis. The menopause-like symptoms such as hot flushes, vaginal dryness, decreased libido, insomnia, depression, and irritability are a major drawback to this type of treatment (Table 1). All these effects are reversible. The long-term risk of GnRH agonist treatment reducing bone mineral density (BMD) and increasing the risk of osteoporosis is a major concern. It appears that the bone loss associated with treatment for no longer than 6 months does not have a long-term detrimental effect [16]. The concept of ‘‘add back’’ therapy (using estrogens, progestagens, or both) in conjunction with GnRH agonists can overcome many of these side effects and does not appear to reduce the efficacy of the treatment [17–22]. Gonadotropin releasing hormone analogs have the advantage that they can be administered as long-acting depot preparations and safety is now well established. The cost of this class of drug is greater than that of progestagens.

206

Lahoud and Harrison

One of the major adverse effects of all these treatments is that they all act as contraceptive agents. As fertility is not infrequently desired, it limits their use to patients not desiring fertility. In this chapter the concept of progesterone receptors, their agonists, and antagonists are discussed. The clinical evidence for the use of progestagens and antiprogestagens in the treatment of endometriosis-related pain and infertility is critically reviewed. Finally the concept of selective progesterone receptor modulators is described.

PATHOPHYSIOLOGY Receptor Talk By definition, a progestagen is a hormone that binds to progesterone receptors to give a progesterone-like action. There are two major forms of progesterone receptors, A and B. These receptors are expressed by a single gene. As a consequence of transcription from different promoters, the two isotypes exist. Each form of progesterone receptor is associated with additional proteins that make it unique. In the absence of hormone binding, the C terminal region exerts an inhibitory effect on transcription [23]. Receptor agonists induce a conformational change that overcomes this inhibitory function. On the other hand, receptor antagonists allow this inhibitory action to be maintained. As a general rule, the progesterone receptor B acts as a positive regulator. However, progesterone receptor A appears to inhibit the activity of the B receptor [24]. The progesterone receptor A (PRA) also inhibits estrogen, glucocorticoid, mineralocorticoid, and androgen receptor activity. The relative levels of the two receptors in the endometrium differ during the menstrual cycle. This also may be due partly to the fact that progesterone receptors are induced by estrogens at the transcriptional level and decreased by progesterones at both the transcriptional and translational level [25]. Furthermore predominant PRA expression in endometriotic tissue may play a role in the pathogenesis of endometriosis and may explain treatment failure in certain cases [26]. Hormones and Endometriosis Eutopic and ectopic endometrium (endometriosis) display some histological and biochemical differences. Furthermore differences in receptor levels have been noted. As a general rule, estrogen stimulates the growth of endometriotic implants. It has been observed that endometriosis usually regresses after menopause. Hence, inducing a hypoestrogenic state (pseudomenopause) should result in the reduction of endometriotic tissue. This is essentially the mode of action of GnRH agonists.

Medical Treatment with Progestagens

207

It is widely accepted that pregnancy, a state of high progesterone levels, is associated with an improvement in endometriosis. Progesterones are thought to cause decidualization and subsequent atrophy of endometrial tissue [27]. The pseudopregnancy concept was described as early as the 1950s [28]. The most commonly studied progesterone in the treatment of endometriosis is MPA. Progestagens for Endometriosis-Related Pain The pelvic pain of endometriosis commonly manifests as dysmenorrhea and dyspareunia. It can be extremely debilitating. The treatment options include surgery, analgesics (nonsteroidal anti-inflammatory drugs, for example) and medical therapy to suppress endometriosis. Many controversies remain in the field of endometriosis research. Progestagen use for endometriosis pain also remains somewhat controversial. The little evidence from placebo-controlled trials is often limited by low power studies and biases related to patient selection and cointerventions. Exploring some of this evidence, we evaluated several randomized controlled trials (RCTs). In 1987 a trial was performed [30] that evaluated the efficacy of continuous MPA in the treatment of endometriosis. Medroxyprogesterone acetate (100 mg orally) was compared to danazol at a dosage of 200 mg orally three times a day. A placebo group was included. Fifty-nine participants aged 26 to 38 with mild to moderate endometriosis diagnosed by laparoscopy were included. Twenty-seven percent of the patients had electrocoagulation of endometriotic implants at the initial diagnostic laparoscopy. Nine participants were lost to follow-up. A second-look laparoscopy was performed 6 months after completion of the treatment. The outcome measures included a change in the AFS (American Fertility Society) scores for endometriosis and a change in the reported patient pain symptoms. Medroxyprogesterone acetate was found to significantly reduce pelvic pain when compared with placebo. When compared to danazol, MPA showed no significant difference with regard to improvement in the pelvic pain score at 6-month’s follow-up. With regard to a change in AFS score at the secondlook laparoscopy, MPA did not show a significant improvement when compared with placebo, but there was certainly a trend toward an improvement in AFS score. When MPA was compared to danazol, no difference with regard to the AFS scores was noted. Overton published the results of a double-blind, randomized, multi center study in 1994 looking at the use of cyclical progestagen [31]. Sixty-two British women with minimal to mild endometriosis diagnosed at laparoscopy were followed up. Three intervention groups were created: first, 40 mg dydrogesterone (Duphaston) for 12 days starting 2 days after luteinizing hormone

208

Lahoud and Harrison

(LH) surge, second, 60 mg dydrogesterone given as above, third, placebo given as above. The duration of treatment was 6 months. The outcome measures included a change in AFS score and pain scores. At 12 months, the second-look laparoscopy was performed, 23 patients were lost to follow-up, and the remaining treatment groups were small. Dydrogesterone at a dose of 40 mg was not found to be efficacious in reducing pain as compared with placebo. This also held true for the higher dose of 60 mg. Furthermore, AFS scores were not significantly improved compared to placebo. In 1996, Vercellini performed a randomized trial comparing depot MPA (150 mg every 90 days) and a combination of the combined oral contraceptive pill and danazol (50 mg daily for 21 of 28 days) [32]. The outcome measures included pain scores. This study reported a significant improvement in pain scores from the baseline for both treatments. An increased reduction in dysmenorrhea was seen with the progesterone treatment. On the other hand, progesterinic therapy experienced a higher incidence of bloating and spotting. From the limited data, it would appear that continuous progestagens are no less effective in the treatment of endometriosis-related pain than other medical treatments, such as danazol, which has been shown to be effective [33]. On the other hand some questions about the overall efficacy of progestagens remain unanswered. The only study that used progesterone in a cyclical fashion (luteal phase) did use dydrogesterone, and this did not show significant improvement. From these results it is difficult to conclude whether continuous therapy is more effective than cyclical therapy. It is also interesting to note that the reduction in AFS scores seen at a second laparoscopy was not significant. This highlights the discrepancy between macroscopic disease and symptomatology and questions the validity of performing a second-look laparoscopy in patients treated for endometriosis unless further surgical treatment is warranted. Further research is required to evaluate the use of progestagens in the treatment of pain in patients with endometriosis. Larger RCTs are needed that look specifically at patients with chronic pain. Another area of interest would to investigate the use of progestagen-releasing intrauterine contraceptive devices in the treatment of this disorder. Progestagens and Infertility Treatment Medical treatment for endometriosis-related infertility has been accepted for many years. The concept that suppression of ovulation and resultant hypoestrogenism would result in regression of endometriosis, leading to increasing fecundability after the cessation of the treatment, was widely held. In the late 1980s and 1990s, several randomized trials demonstrated a significant lack of effectiveness for many of the medical treatments, such as danazol, progestagens, and GnRH analogues.

Medical Treatment with Progestagens

209

Telimaa [34] performed an RCT comparing MPA 100 mg/day for 6 months, danazol 600 mg per day, and placebo in cases of endometriosis. Seventeen patients were randomized to MPA treatment, 18 to danazol, and 14 to placebo. The outcome measure was pregnancy and the follow-up was up to 30 months. Some of the patients received cointerventions with surgery and clomiphene. The odds ratio for clinical pregnancy was 0.7 (0.2–2.8) for treatment with MPA compared with placebo and 1.1 (0.2–4.8) comparing danazol to placebo. This trial showed no benefit of using either MPA or danazol over using placebo in treating infertility in cases of endometriosis. Harrison performed a randomized, double-blind, placebo-controlled trial in Ireland [29]. One hundred women were randomized into either 3 months of treatment with MPA 50 mg/day or placebo. The diagnosis was made at diagnostic laparoscopy. Three months after the medical treatment, a second laparoscopy was performed during which surgical treatment was instituted for some patients. The main outcome measures were revised AFS scores, with symptomatic improvement and natural pregnancy forming secondary outcome measures. Of the 100 patients, 10 did not have a follow-up laparoscopy. With regard to rAFS scores, this study did not detect a benefit from using MPA over placebo. Also no significant improvement in pain symptoms was recorded. This may be in part because of the low incidence of pelvic pain in the MPA group at baseline. Overall well being was improved with MPA treatment. Pregnancies were achieved in both groups, with MPA showing no benefit. Further randomized trials compared medical treatments (danazol, GnRH agonist, and gestrinone) with either placebo or no treatment [33– 36]. The odds ratios for clinical pregnancy ranged from 0.6 to 1.1, with neither study showing statistical significance. Several other trials have compared danazol with GnRH agonists and have found neither medication to be more efficacious in achieving clinical pregnancies in cases of endometriosis [37–42]. Combining the results, one has to conclude that medical treatment for infertility in cases of endometriosis confers no notable benefit. No specific medical treatment is more effective than others. Hughes concludes that medical treatments for endometriosis related infertility could not be recommended, as there appears to be a lack of efficacy and as these treatments are associated with a significant period of amenorrhea [43]. Combined Medical and Surgical Treatment Studies have shown the effectiveness of surgical treatment of endometriosisrelated pain. Sutton performed a randomized trail comparing laparoscopic treatment of endometriosis with diagnostic laparoscopy and found a significant decrease in pain reported in the treatment group (62.5% vs 22.6%) [44]. This effect was still detectable at 12 months’ follow-up [45].

210

Lahoud and Harrison

The combination of surgical ablation or excision of endometriosis followed by medical treatment has been investigated. Sixty women with advanced endometriosis were given either danazol, MPA, or placebo [30]. Pain scores were significantly lower in the treatment groups as compared to placebo. A further trial compared 6 months’ postoperative GnRH analogue treatment with placebo in 109 women [46]. The percentage of patients requiring retreatment was lower in the GnRH analogue group (31% vs 57%). On the other hand, this same study found no difference in pain scores. In 1994 Parrazini reported similar findings, again showing no improvement in pain scores when postoperative GnRH agonist treatment was compared with placebo [47]. More recently Regidor reported the findings of a randomized trail comparing the postsurgical use of the GnRH agonist leuprorelin acetate (LAD) with the gestagen lynestrenol (LYN, 5 mg orally) [48]. Both groups were treated for 6 months and contained 26 and 22 patients, respectively. Improvements in dysmenorrhea and dyspareunia were more significant in the LAD group after 6 months of treatment, but significant improvements were noted in both groups. Again the data regarding post-surgical treatment of endometriosis is somewhat difficult to interpret. On balance there certainly may be a benefit to using medical treatment as long as fertility is not desired. Obviously treatment side effects may ultimately decide whether medical treatment is utilised.

RECURRENCE AFTER TREATMENT One of the major difficulties with treatment of endometriosis is recurrence of the disease. Follow-up studies of endometriosis have reported recurrence rates of 40% to 50% up to 4 years after danazol treatment [49], and 33% rates in another study [6]. With this in mind, many authorities would describe medical treatments as only suppressive and not curative. Whether endometriosis is a progressive disorder remains controversial. This leads us to the next dilemma—that of long-term treatment. Many of the side effects related to progestagen and antiprogestagen-only therapy make it difficult to achieve long-term compliance. The combined estrogen-progestagen contraceptive and GnRH agonists with add-back therapy offer the most viable long-term strategies. Antiprogesterones in the Treatment of Endometriosis Gestrinone (ethylnorgestrinone) is an antiprogestional steroid that causes a reduction in serum estradiol and sex hormone-binding globulin levels and

Medical Treatment with Progestagens

211

reduces both estrogen and progesterone receptor concentrations in target tissues [11,12]. Gestrinone can be given at a dose of 5 mg to 10 mg twice weekly and does have some androgenic and antiestrogenic side effects. Gestrinone was compared with danazol in two randomized controlled trials [50,51]. Fedele treated 39 patients with laparoscopic-diagnosed endometriosis with either 2.5 mg of gestrinone twice weekly or danazol 600–800 mg daily. The dosages of both drugs were increased if amenorrhea was not achieved at the low dose. Only 16 patients had a follow-up laparoscopy. The primary endpoint was infertility, but pain was also evaluated. The results of these studies showed no significant difference in improvement in dysmenorrhea or dyspareunia. The AFS scores were similar for both the gestrinone and danazol groups. The main differences observed are restricted to the side effect profile with androgenic side effects such as greasy skin and hirsutism being more common in the gestrinone group, and decreased breast size, muscle cramps, and hunger being more common in the danazol group. Bromham reports the findings of a randomized, double-blind, multicenter study comparing gestrinone 2.5 mg twice weekly for 6 months with danazol 200 mg twice daily for 6 months [51]. Two hundred sixty-nine British women with endometriosis were randomized into the two treatment arms. A follow-up laparoscopy was performed after the treatment. The outcome measures were AFS scores and pain scores during treatment and after 12 months. A large percentage of patients were lost to follow-up. The results of this study also show equivalent efficacy in improvement of dysmenorrhea or dyspareunia when comparing the two treatments. One further study compared gestrinone with the GnRH analogue leuproreline [52]. This randomized double-blind multicenter study included 55 Italian women aged 18 to 40 who had chronic pelvic pain and a laparoscopic diagnosis of endometriosis. The two intervention arms included gestrinone 2.5 mg twice weekly and placebo injections as well as leuproreline intramuscularly 3.75 mg once a month plus placebo tablets. Six patients withdrew from the treatment group, seven were lost to follow-up, and eight patients conceived. The conclusions of this study were that there appeared to be a small benefit with respect to dysmenorrhea when using the GnRH analogue. However by the end of the follow-up, this advantage was now with gestrinone. Both treatment arms showed significant reductions in pain scores from the baseline and appeared to be very similar in effectiveness. The side effect profile was again very similar apart from hot flashes being more significant than in the GnRH analogue group. A further study by Hornstein in 1990 compared two doses of gestrinone (1.25 mg and 2.5 mg twice weekly) [53]. No difference in efficacy was noted with regard to pain scores. The lower dose resulted in fewer side effects. Hence gestrinone appears to be as effective in the medical treatment of endome-

212

Lahoud and Harrison

triosis-related pain as danazol or GnRH analogues [54]. Gestrinone has also been studied in the treatment of endometriosis-related infertility and has not been found to be more effective than placebo or danazol [36,50]. RU486 (Mifepristone) is a synthetic steroid with antiprogesterone and antiglucocorticoid activity [55]. It has been used as an abortifacient and may have applications in the treatment of leiomyoma, breast cancer, and meningioma [56]. As this drug has been shown to inhibit ovulation and disrupt endometrial integrity, it was utilized by Kettel in six normally cycling women to treat endometriosis [55]. RU486 at a dose of 100 mg/day for 3 months resulted in amenorrhea in all patients. After cessation of treatment, regular menstruation resumed in all cases within 3 to 6 weeks. All patients treated with RU486 experienced an improvement in pelvic pain, with only one patient reporting a recurrence over the 12 to 24 months’ follow-up (two patients conceived after treatment). Symptoms such as hot flashes, anorexia, nausea, and fatigue were noted. Kettel performed a further trial using RU486 (50 mg/day) in nine women with endometriosis. Pelvic pain and uterine cramping decreased in all patients [57]. The endometriosis regressed by 55% in the absence of significant side effects. Randomized, controlled placebo trials will be required to further assess the use of RU486 in the treatment of endometriosis. Selective Progesterone Receptor Modulators The term ‘‘selective receptor modulators’’ excites most reproductive endocrinologists. The concept that hormonal receptors exist in more than one form within the same tissue, modulating each other’s actions, has raised the prospect of designing drugs with specific actions and side effect profiles. Selective estrogen receptor modulators, such as tamoxifen and raloxifene, are commonly used in clinical practice. Tamoxifen was used to treat recurrent endometriosis in two patients with good effect [58]. As discussed earlier, two types of progesterone receptors exist in differing concentrations in different tissues. These receptors modulate each other’s actions. Selective progesterone receptor modulators (SPRM) are a new class of progesterone receptor ligands that exhibit both agonistic and antagonistic actions [59]. There exists a spectrum of progesterone receptor ligands with pure agonistic actions such as those of progesterone and with the strongest antagonists being onapristone and ZK230211. Selective progesterone receptor modulators are situated in the middle of the spectrum. Most of the experience of these drugs comes from animal models. The best known SPRMs are J867, J956, J912, and J1042 [59]. They exhibit a high binding affinity for progesterone receptors (PR). It appears that in the absence of progesterone, the SPRMs act as weak progestins. In the presence of progesterone, they have weak progesterone antagonist actions, espe-

Medical Treatment with Progestagens

213

cially in the endometrium. In the pregnant guinea pig model, they have only minimal labor-inducing ability as compared to the strong action of PR antagonists [59]. With regard to other steroid hormone receptors, SPRMs have reduced glucocorticoid receptor-binding affinity when compared to RU486. In vivo, SPRMs show minimal binding to estrogen receptors and, in the rat at high doses, may display a mixed androgenic/antiandrogenic activity [59]. Studies using a monkey model showed the potential of SPRMs to suppress estrogen-dependent endometrial growth and to induce reversible amenorrhea [60]. This suppression appeared to be confined to the endometrium and may prevent the side effects of estrogen deprivation [61]. Breakthrough bleeding commonly seen with progestagen treatment is largely explained by progesterone-induced fragility of the endometrial blood vessels. Selective progesterone receptor modulators seem to have the spiral arterioles as a target and cause stabilization of the endometrial blood vessels [59]. These properties make SPRMs an exciting prospect in the treatment of endometriosis with the potential for effective treatment with an improved side effect profile. Clinical trials of these drugs are needed.

SUMMARY Progestagens have been used in the treatment of endometriosis for several decades. They appear to be effective in improving the pain related to endometriosis and as effective as other medical treatments, such as danazol and GnRH analogues. In the treatment of endometriosis-related infertility, medical therapy with progestagens plays no role. Antiprogestagens, such as gestrinone, also have been shown to be as effective as progestagens. Antiprogesterones such as RU486 and SPRMs need further clinical research before they can be recommended as mainstream medical therapy for endometriosis. The future must lie in the development of medications that effectively treat the pain of endometriosis and increase fertility while exhibiting a favorable side effect profile. PRACTICAL POINTS 

Progestagens can be used for endometriosis-related pain. It is as effective as danazol and GnRHa.  Progestagens have no role for endometriosis-related infertility. REFERENCES 1.

Montgomery Rice V. Conventional medical therapies for endometriosis. Ann N Y Acad Sci 2002; 955:352–434.

214 2.

3. 4.

5. 6. 7. 8.

9.

10.

11.

12.

13. 14. 15. 16.

17. 18.

19.

Lahoud and Harrison Koninckx PR, Meuleman C, Demeyere S, Lesaffri E, Cornillie FJ. Suggestive evidence that pelvic endometriosis is a progressive disease, whereas deeply infiltrating endometriosis is associated with pelvic pain. Fertil Steril 1991; 55:759–765. Wood A. Treatment of endometriosis. N Eng J Med 2001; 345:266–275. Murphy AA, Green WR, Bobbie D, dela Cruz ZC, Rack TA. Unsuspected endometriosis documented by scanning electron microscopy in visually normal peritoneum. Fertil Steril 1986; 46:522–524. Buttram VC Jr, Belue JB, Reiter R. Interim report of a study of danazol for the treatment of endometriosis. Fertil Steril 1982; 37:478–483. Barbieri RL, Evans S, Kistner RW. Danazol in the treatment of endometriosis: Analysis of 100 cases with a 4-year follow-up. Fertil Steril 1982; 37:737–746. Buttram VC Jr, Reiter RC, Ward S. Treatment of endometriosis with danazol: Report of a 6-year prospective study. Fertil Steril 1985; 43:353–360. Olive DL. Medical treatment: Alternatives to danazol. In: Schenken RS, ed. Endometriosis: Contemporary Concepts in Clinical Management. Philadelphia: JB Lippincott, 1989:189–211. Telimaa S, Poulakka J, Ronnberg L, Kauppila A. Placebo controlled comparison of danazol and high-dose medroxyprogesterone acetate in the treatment of endometriosis. Gynecol Endocrinol 1987; 1:13–23. Cundy T, Farquar CM, Cornish J, Reid JR. Short-term effects of high dose medroxyprogesterone acetate on bone density in premenopausal women. J Clin Endocrinol Metab 1996; 81:1014. Cornillie FJ, Brosens LA, Vasquez G, Riphagen I. Histologic and ultrastructural changes in human endometriotic implants treated with the antiprogesterone steroid ethylnorgestrienone (gestrionone) during 2 months. Int J Gynecol Pathol 1986; 5:95–109. Robyn C, Delogne-Desnoeck J, Bourdouz P, Copinschi G. Endocrine effects of gestrinone. In: Raynaud J-P, Ojasoo T, Martini L, eds. Medical Management of Endometriosis. New York: Raven Press, 1984:207–221. Thomas EJ, Cooke ID. Impact of gestrinone of the course of asymptomatic endometriosis. BMJ 1987; 294:272–274. Olive DL, Pritts EA. Treatment of endometriosis. N Eng J Med 2001; 345:266– 275. Noble AD, Letchworth AT. Medical treatment of endometriosis: A comparative trial. Postgrad Med J 1979; 55(suppl 5):37–39. Paoletti AM, Serra GG, Cagnacci A, et al. Spontaneous reversibility of bone loss induced by gonadotropin releasing hormone analog treatment. Fertil Steril 1996; 65:707. Moghissi KS. Medical treatment of endometriosis. Clin Obstet Gynecol 1999; 42:620–632. Kiilholma P, Korhonen M, Tuimala R, et al. Comparison of the gonadotropinestrogen-progestagen add-back therapy in the treatment of endometriosis. Fertil Steril 1995; 64:903–908. Berqvist A, Jacobson J, Harris S. A double-blind randomised study of the treat-

Medical Treatment with Progestagens

20.

21.

22.

23.

24.

25. 26.

27. 28. 29.

30.

31.

32.

33.

215

ment of endometriosis with nafarelin or nafarelin plus norethisterone. Gynecol Endocrinol 1997; 11:187–194. Makarainen L, Ronnberg L, Cauppila A, Judd HL. Medroxyprogesterone acetate supplementation diminishes the hypoestrogenic side effects of gonadotrophin-releasing hormone agonist without changing its efficacy in endometriosis. Fertil Steril 1996; 65:29–34. Surrey ES, Judd HL. Reduction of vasomotor symptoms and bone mineral density loss with combined norethindrone and long-lasting gonadotrophinreleasing hormone agonist therapy of symptomatic endometriosis: A prospective randomised trial. J Clin Endocrinol Metab 1992; 75:558–563. Surrey ES, Voigt B, Fournet N, et al. Prolonged gonadotrophin-releasing hormone agonist treatment of symptomatic endometriosis: The role of cyclic sodium etidronate and low dose norethindrone ‘‘add-back’’ therapy. Fertil Steril 1995; 63:747–755. Vegeto E, Allan GF, Schrader WT, Tsaims P, et al. Mechanism of RU486 antagonism is dependent on the conformation of the carboxyl-terminal tail of the human progesterone receptor. Cell 1992; 69:703. Speroff L, Glass RH, Kase NG. The progesterone receptor. In: Clinical Gynecologic Endocrinology and Infertility. 6th ed. Baltimore:Lippincout, Williams & Wilkins, 1999:66. Horwitz K, Tung L, Takimoto GS. Novel mechanism of antiprogestin action. J Steroid Biochem Mol Biol 1995; 53:9. Attia Gr, Zeitoun K, Edwards D, Johns A, Carr BR, Bulun SE. Progesterone receptor isoform A but not B is expressed in endometriosis. Hum Reprod Update 2000; 6:37–44. Vercellini P, Cortesi I, Crosignani PG. Progestins for synthetic endometriosis: A critical analysis of the evidence. Fertil Steril 1997; 68:393. Kistner RW. Treatment of endometriosis by inducing pseudo-pregnancy with ovarian hormones. Fertil Steril 1959; 10:539–554. Harrison RF, Barry-Kinsella C. A prospective randomized placebo-controlled study treating endometriosis in infertile women with medroxyprogesterone acetate. Fertil Steril 2000; 74:24–30. Telimaa S, Ronnberg L, Kauppila A. Placebo-controlled comparison of danazol and high dose medroxyprogesterone acetate in the treatment of endometriosis after conservative surgery. Gynecol Endocrinol 1987; 1:363–371. Overton CE, Lindsay PC, Johal B. A randomized, double blind, placebo controlled study of luteal phase dydrogesterone (Duphaston) in women with minimal to mild endometriosis. Fertility Steril 1994; 62:701–707. Vercellini P, De Giorgi O, Oldani S, Cortesti I, Panazza S, Crosignnani PG. Depot medroxyprogesterone acetate versus an oral contraceptive combined with very-low-dose danazol for long-term treatment of pelvic pain associated with endometriosis. Am J Obstet Gynecol 1996; 175:396–401. Bayer SR, Seibel MM, Saffan DS, Berger Mj, Taymor ML. Efficacy of danazol treatment for minimal endometriosis in infertile women: A prospective, randomised study. J Reprod Med 1988; 33:179–183.

216

Lahoud and Harrison

34. Telimaa S. Danazol and medroxyprogesterone acetate inefficacious in the treatment of infertility in endometriosis. Fertil Steril 1988; 50:872–875. 35. Fedele L, Parazzini F, Radici E, Boccoiolone L, Bianchi S, Bianchi C, Candiani GB. Buserelin acetate versus expectant management in the treatment of infertility associated with minimal or mild endometriosis: a randomized clinical trial. Am J Obstet Gynecol 1992; 166:1345–1350. 36. Thomas E., Cooke I. Successful treatment of asymptomatic endometriosis: Does it benefit infertile women? BMJ 1987; 294:1117–1119. 37. The Nafarelin European Endometriosis Trial Group (NEET)Nafarelin for endometriosis: A large-scale, danazol-controlled trial for efficacy and safety, with 1-year follow-up. Fertil Steril 1992; 57:514–522. 38. Dmowski WP, Tummon I, Pepping P, Radwanska E, Binor Z. Ovarian suppression induced with buserelin or danazol in the management of endometriosis: A randomized comparative study. Fertil Steril 1989; 51:395–400. 39. Fedele L, Bianchi S, Viezzoli T, Arcaini L, Candiani GB. Buserelin versus danazol in the treatment of endometriosis-associated infertility. Am J Obstet Gynecol 1989; 161:871–876. 40. Fraser IS, Shearman RP, Jansen RPS, Sutherland PD. A comparative treatment trial of endometriosis using the gonadotropin-releasing hormone agonist, nafarelin, and the synthetic steroid, danazol. Aust N Z J Obstet Gynaecol 1991; 31:158–163. 41. Henzl M, Corson SL, Moghissi K, Buttram VC, Berqvist C, Jacobsen J. Administration of nasal nafarelin as compared with oral danazol for endometriosis. A multicenter double-blind comparative clinical trial. N Engl J Med 1988; 318:485– 489. 42. Shaw RW. Zoladex Endometriosis Study Team. An open randomized comparative study of the effect of goserelin depot and danazol in the treatment of endometriosis. Fertil Steril 1992; 58:265–272. 43. Hughes E, Fedorkow D, Collins J, et al. Ovulation suppression for endometriosis (Cochrane Review), Cochrane library, Issue 3, 2002. 44. Sutton CJG, Ewen SP, Whitelaw N, Hanines P. Prospective, randomised, double-blind, controlled trial of laser laparoscopy in the treatment of pelvic pain associated with minimal, mild, and moderate endometriosis. Fertil Steril 1994; 62:696–700. 45. Sutton CJ, Pooley AS, Ewen SP, Hanines P. Follow-up report on a randomised controlled trial of laser laparoscopy in the treatment of pelvic pain associated with minimal to moderate endometriosis. Fertil Steril 1997; 68:1070–1074. 46. Hornstein MD, Hemmings R, Yuzpe AA, Heinrichs WL. Use of nafarelin versus placebo after reductive laparoscopic surgery for endometriosis. Fertil Steril 1997; 68:860–864. 47. Parazzini F, Fedele L, Busacca M, Falsetti L, Pellegrini S, Venturini PL, Stella M. Postsurgical medical treatment of advanced endometriosis: Results of a randomised clinical trial. Am J Obstet Gynecol 1994; 171:1205–1207. 48. Regidor PA, Regidor M, Schmidt M, Ruwe B, Lubben G, Fortig P, Kienle E, Schindler AE. Prospective randomized study comparing the GnRH-agonist

Medical Treatment with Progestagens

49.

50. 51.

52.

53.

54.

55.

56. 57.

58. 59.

60.

61.

217

leuprorelin acetate and the gestagen lynestrenol in the treatment of severe endometriosis. Gynecol Endocrinol 2001; 15:202–209. Dmowski WP, Cohen MR. Antigonadotropin (danazol) in the treatment of endometriosis: Evaluation of post treatment fertility and three-year follow-up data. Am J Obstet Gynecol 1978; 130:41–48. Fedele L, Bianchi S, Viezzoli T, Arcaini L, Candiani GB. Gestrinone versus danazol in the treatment of endometriosis. Fertil Steril 1989; 51:781–785. Bromham DR, Booker MW, Rose GL, Wardle PG, Newton JR. A multicentre comparative study of gestrinone and danazol in the treatment of endometriosis. J Obstet Gynecol 1995; 15:188–194. The Gestrinone Italian Study GroupGestrinone versus gonadotropin releasing hormone agonist for the treatment of pelvic pain associated with endometriosis: A multicenter, randomised, double-blind study. Fertil Steril 1996; 66:911– 919. Hornstein MD, Glaeson RE, Barbieri Rl. A randomized, double-blind prospective trial of two doses of gestrinone in the treatment of endometriosis. Fertil Steril 1990; 53:237–241. Bromham DR, Booker MW, Rose GL, Wardle PG, Newton JR. Updating the clinical experience in endometriosis—the European perspective. Br J Obstet Gynecol 1995; 102(suppl 12):12–16. Kettel Lm, Murphy AA, Mortola JF, Liu JH, Ulmann A, Yen SSC. Endocrine responses to long-term administration of the antiprogesterone RU 486 in patient with pelvic endometriosis. Fertil Steril 1991; 56:402–407. Mahajan DK, London SN. Mifepristone (RU486): A review. Fertil Steril 1997; 68:967–976. Kettel LM, Murphy AA, Morales AJ, Ulmann A, Baulieu EE, Yen SS. Treatment of endometriosis with antiprogesterone mifepristone (RU486). Fertil Steril 1996; 65:23–28. Haber GM, Behelak YF. Preliminary report on the use of tamoxifen in the treatment of endometriosis. Am J Obstet Gynecol 1987; 156:582–586. Chwalisz K, Garg R, Brenner RM, Schubert G, Elger W. Selective progesterone receptor modulators (SPRMs)—A Novel Therapeutic Concept in Endometriosis. Ann N Y Acad Sci 2002; 955:373–388. Elger W, Bartley J, Schneider B, Kaufmann G, Schubert G, Chwalisz K. Endocrine pharmacological characterization of progesterone antagonists and progesterone receptor modulators (PRMs) with respect to PR-agonistic and antagonistic activity. Steroids 2000; 65:713–723. Chwalisz K, Brenner RM, Fuhrmann U, Hess-Stumpp, Elger W. Antiproliferative effects of progesterone antagonists and progesterone receptor modulators on the endometrium. Steroids 2000; 65:741–751.

13 Gonadotropin Releasing Hormone Agonist and Antagonist for Endometriosis Robert L. Barbieri Brigham and Women’s Hospital Boston, Massachusetts, U.S.A.

CLINICAL BIOLOGY OF GONADOTROPIN RELEASING HORMONE The menstrual cycle is dependent on the integrated action of the hypothalamus, pituitary gland, ovarian follicle, and endometrium. The hypothalamus is a neuroendocrine transducer that integrates neural inputs from the cortex, amygdala, other hypothalamic nuclei, cranial nerves (light), and peripheral endocrine signals and transduces them into an endocrine output of pulsatile gonadotropin releasing hormone (GnRH). Like a metronome, the hypothalamus sets the beat for the menstrual cycle by the pulsatile release of GnRH. Gonadotropin releasing hormone pulses occur every 1 to 1.5 hours in the follicular phase of the cycle and every 2 to 4 hours in the luteal phase of the cycle. Pulsatile GnRH secretion stimulates the pituitary gland to secrete luteinizing hormone (LH) and follicle-stimulating hormone (FSH). The pituitary gland translates the tempo set by the hypothalamus to a signal, LH and FSH secretion, that can be understood by the ovarian follicle. In the ovarian follicle, LH stimulates the thecal cells to produce androstenedione, 219

220

Barbieri

and FSH stimulates granulosa cells to convert the androstenedione to estradiol. After ovulation, the follicle is transformed into the corpus luteum that secretes progesterone when stimulated by LH or human chorionic gonadotropin (hCG). Estradiol stimulates the endometrial epithelial cells to proliferate. Estradiol plus progesterone causes the endometrium to become differentiated into a secretory endometrium. During the midluteal phase of the menstrual cycle, when progesterone production is at its peak, the secretory endometrium is optimally prepared for the implantation of a pre-embryo. Native GnRH is a decapeptide with a short half-life of approximately 5 minutes (Fig. 1). The short half-life of GnRH results from the rapid degradation of the peptide by tissue and plasma endopeptidases, which cleave the 6–7 and 9–10 peptide bonds. Gonadotropin releasing hormone is secreted in a pulsatile fashion, and information is contained in the pulse interval and the pulse amplitude. The release of GnRH in neurosecretory bursts and the short half-life of GnRH ensure that each pulse is sharp and crisp. During the follicular phase of the cycle, GnRH pulses are characterized by high frequency, every 60 to 90 minutes, and low amplitude. During the luteal phase of the cycle, GnRH pulses are characterized by low frequency, every 120 to 240 minutes, and high amplitude. In classic experiments on the rhesus monkey, Knobil [1] demonstrated that if the arcuate nucleus is destroyed (loss of GnRH), the pituitary gland can no longer secrete gonadotropins, and amenorrhea ensues. Exogenous administration of hourly pulses of GnRH was demonstrated to restart the system and cause menstrual cycles. If GnRH was administered with abnormally low interpulse intervals, normal menstrual cycles were not re-established. These

FIGURE 1 The native decapeptide, GnRH, is secreted in pulses and the interpulse interval is much greater than the half-life of GnRH. This ensures that each pulse is clear and crisp. The short half-life of GnRH is caused by degradation of the peptide bonds 6–7 and 5–6 by endopeptidases.

GnRH Agonist and Antagonist

221

studies demonstrate the primacy of GnRH in regulating menstrual cycles in the primate. Early investigators hypothesized that if GnRH was given as a continuous infusion, superovulation could be achieved. Surprisingly, continuous infusion of GnRH resulted in the cessation of gonadotropin secretion and amenorrhea. The basis for this effect is that continuous stimulation of the GnRH receptor causes both downregulation and desensitization of the receptor resulting in the paradoxical suppression of LH and FSH secretion. This unexpected finding has been exploited by the creation of GnRH agonist analogues, which paradoxically initially stimulate LH and FSH secretion, but with prolonged administration (5 to 10 days) profoundly suppresses LH and FSH secretion. PHARMACOLOGY OF GNRH AGONISTS GnRH agonists are analogues of the native decapeptide with chemical changes in amino acids 6 and 10 (Table 1). The introduction of D-amino acids at position 6 of native GnRH results in analogues that are resistant to degradation by endopeptidases and have long half-lives, and high affinity for the pituitary GnRH receptor [2]. Paradoxically, initial treatment with a GnRH analogue stimulates pituitary secretion of LH and FSH, but prolonged treatment suppresses both LH and FSH secretion. The paradoxical suppression of LH and FSH observed with chronic GnRH agonist treatment is due to both downregulation and desensitization of the pituitary GnRH receptor. With GnRH agonist analogues, suppression of LH is greater than suppression of FSH. In turn, the decrease in LH and FSH secretion results in the suppression of ovarian follicular growth and ovulation resulting in low levels of circulating estradiol and progesterone secretion. In women chroni-

TABLE 1 Amino Acid Substitutions in Commonly Used GnRH Agonist Analogues GnRH agonist Leuprolide Nafarelin Goserelin Buserelin Histrelin Decapeptyl

Amino acid substitution D-Leu6-Pro9 D-Na1(2)6 D-Ser(tBu)6, (Aza-Gly-NH)10 D-Ser(tBu)6, Pro9 Imbzl-D-His6-Pro9 D-Trp6

222

Barbieri

cally treated with standard doses of parenteral GnRH agonists, the circulating estradiol concentration is in the range of menopausal women, approximately 15 pg/mL. THE CENTRAL DOGMA OF ENDOCRINE TREATMENT OF ENDOMETRIOSIS The central dogma that guides the hormonal therapy of endometriosis is the idea that sex steroid hormones, such as estrogen, progesterone, and androgen, are major regulators of the growth and function of the endometrium and endometriosis lesions. Both laboratory and clinical findings support this central dogma. Endometriosis lesions contain estradiol, progesterone, and androgen receptors and these sex steroids regulate cell function in both eutopic endometrium and endometriosis lesions. Using an immunohistochemistry technique, Bergqvist and colleagues [3] demonstrated that all eutopic endometrium and endometriosis lesions contained both estradiol and progesterone receptors. In endometriosis lesions, the concentration of estrogen and progesterone receptors tend to be approximately 50% to 70% lower than those observed in matched specimens of endometrium. Androgen receptors are present in similar concentrations in endometriosis lesions and matched endometrial specimens. Many laboratory findings support the central dogma that sex steroids regulate function of ectopic endometrial tissue. Bergqvist and colleagues [4] implanted human endometriosis lesions in the abdominal wall of nude mice. In a hypoestrogenic state, the implanted human endometriosis lesions atrophied. If estradiol was given to the mice, the endometriosis implants grew and retained their histological appearance. Sharpe and colleagues [5] autotransplanted rat endometrium to the intestinal mesentery. In animals with intact ovarian function, the autotransplanted endometrium grew into cystic structures lined with endometrial epithelium. In castrated animals, these lesions underwent regression. If either estradiol or progesterone was administered to castrated animals, the experimental endometriosis lesions continued to grow. The administration of estradiol plus progesterone was additive in supporting the growth of the transplanted endometrium compared to either hormone individually. Clinical observations that support the central dogma include: (1) endometriosis lesions rarely occur prior to menarche, (2) menopause, either surgical or natural, usually produces regression of endometriosis lesions, and (3) new cases of endometriosis occur very rarely after menopause unless estrogen is administered. Many investigators have reported the benefits of creating a hypoestrogenic effect resulting in a surgical menopause with bilateral salpingo-oophorectomy [6–8]. Interestingly, after bilateral salpingo-

GnRH Agonist and Antagonist

223

oophorectomy, replacement therapy with low-dose estrogen does not appear to be associated with recurrence of the disease. In contrast to estrogen, much less data are available concerning the effects of androgens on endometriosis lesions. Androgens are antiestrogenic in the endometrium and tend to cause atrophy of both eutopic endometrium and endometriosis lesions. The administration of the androgens testosterone proprionate, methyltestosterone, or danazol to women with endometriosis produces atrophy of both the eutopic endometrium and the endometriosis lesions [9,10]. Both low doses of danazol and methyltestosterone can cause regression of endometriosis lesions and atrophy of eutopic endometrium even without interrupting ovulation. This suggests that androgens can produce regression of endometriosis lesions in the presence of relatively normal concentrations of both estradiol and progesterone. Unfortunately, androgens are associated with significant side effects including weight gain from an increase in muscle mass, deepening of the voice, and adverse changes in lipids and liver function tests. Some synthetic progestins are derivatives of the androgen ethinyl testosterone. At high doses, these synthetic progestins have both progestational and androgenic effects, which can be effective in the treatment of endometriosis by suppressing LH and FSH secretion, estradiol secretion, and by a direct antiestrogenic effect in the endometrium and endometriosis lesions. At high doses, C-21 progestins also appear to be effective in the treatment of endometriosis [11]. These progestins probably act as antiestrogens directly in the endometrium by blocking the synthesis and action of the estrogen receptor. In addition, they may suppress LH and FSH, thereby reducing ovarian estrogen synthesis. In the most simplified terms, estrogen and physiological concentrations of progesterone support growth of endometriosis lesions. Androgens and pharmacological concentrations of androgenic progestins cause atrophy in endometriosis lesions. The polarity of the response of endometriosis lesions to androgens and estrogens is the ‘‘yin and yang’’ upon which most hormone therapy of endometriosis is based (Table 2). Gonadotropin releasing hormone agonists, such as leuprolide, nafarelin, and goserelin, are effective in the treatment of endometriosis because they produce low circulating concentrations of estradiol and progesterone.

PRESENTING PROBLEMS FOR WOMEN WITH ENDOMETRIOSIS Women with endometriosis typically present for treatment with one of three problems: (1) an adnexal mass (endometrioma), (2) infertility, or (3) pelvic pain. For women with an adnexal mass resulting from endometriosis, surgery

Danazol Methyltestosterone

Medroxyprogesterone Norethindrone Gestrinone

Hyperandrogenism

Synthetic Progestins

From Refs. 9 and 14.

Oophorectomy GnRH analogues

Hypoestrogenism

Methods available to produce hormonal state 80%

75%

85%

45%

50%

68%

Danazol 200 mg bid (Barbieri 1981)

Medroxyprogesterone acetate, 30 to 50 mg daily (Luciano 1988)

Nafarelin 200 Ag bid (Henzl 1988)

Typical regimen

Percent of patients with improvement in pain symptoms

Percent reduction in AFS endometriosis score

Steroid Hormone Manipulations Effective in the Treatment of Endometriosis

Hormonal state

TABLE 2

Vasomotor symptoms, bone loss, headache, dry vagina, decreased libido Increased body muscle mass, weight gain, hirsutism, oily skin, deepening of the voice Moliminal symptoms, bloating, mood changes, irregular uterine bleeding

Side effects

224 Barbieri

GnRH Agonist and Antagonist

225

is indicated to remove the mass [12]. Endometriomas are monoclonal tumors that arise from a somatic mutation in the DNA of a precursor cell. Monoclonal tumors are not likely to regress spontaneously. Surgery also allows for definitive determination of the histology of the adnexal mass, ensuring that cancer is not present. The approach to the treatment of infertility in women with endometriosis is dealt with in Chapters 9, 17, and 18. The remainder of this review will focus on the treatment of pelvic pain caused by endometriosis. GNRH AGONISTS FOR THE TREATMENT OF ENDOMETRIOSIS: THE KEY CLINICAL TRIALS Estrogen-dependent diseases often regress when estrogen production is reduced. Endometriosis is an estrogen-responsive disease, and the pelvic pain associated with it improves when estrogen production is reduced with bilateral oophorectomy or GnRH agonist treatment. Gonadotropin releasing hormone agonist treatment is the most reliable, widely available, nonsurgical method for suppressing ovarian estrogen production. Multiple clinical trials demonstrate that GnRH agonists: (1) suppress circulating estradiol concentration by suppressing pituitary secretion of LH and FSH, (2) reduce endometriosis disease activity as objectively measured by pretreatment and posttreatment surgical staging, and (3) decrease pelvic pain caused by endometriosis lesions. The GnRH agonists are the ‘‘gold standard’’ medical treatment of pelvic pain caused by endometriosis against which all other hormonal treatments are measured. In one randomized, placebo-controlled clinical trial, women with surgically proven endometriosis and pelvic pain were randomized to receive leuprolide acetate 3.75 mg IM every 4 weeks or a placebo injection every 4 weeks for 6 months. Of the women treated with leuprolide acetate, 85% reported a significant decrease in dysmenorrhea, pelvic pain, and dyspareunia. Of the women treated with placebo, 95% left the clinical trial because of continuing pelvic pain. Of the women treated with leuprolide, 82% completed the clinical trial. Women in the leuprolide group reported side effects such as vasomotor symptoms, headaches, and decreased libido [13]. In another clinical trial, Henzl and colleagues randomized women with endometriosis to one of two doses of nasal nafarelin (400 Ag or 800 Ag daily) or danazol (800 mg daily) [14]. Drug therapy was continued for 6 months. More than 80% of the patients treated with either dose of nafarelin or danazol had symptomatic relief of their pelvic pain and objective improvement of their endometriosis lesions as documented by pre-and posttreatment surgical staging. Nafarelin and danazol were observed to be similar in their efficacy. However the side effects of the two drugs differed greatly. The women receiving danazol reported weight gain, edema, and myalgia and had increased

226

Barbieri

circulating levels of serum aspartate aminotransferase and alanine aminotransferase and decreased levels of high-density lipoprotein cholesterol. In contrast, the principle side effects reported by women receiving nafarelin were hot flashes, decreased libido, and vaginal dryness. The women treated with nafarelin did not have notable decreases in high-density lipoprotein cholesterol or increases in serum aspartate aminotransferase and alanine aminotransferase. Bone density was not measured in this clinical trial. Many other clinical trials support the conclusion that GnRH agonists have similar efficacy to danazol in the treatment of pelvic pain caused by endometriosis, but the side effect profiles differ greatly between the two classes of hormone [15–19]. An important question that remains to be completely answered is, ‘‘What precise estradiol concentration is required to suppress the activity of endometriosis lesions?’’ A review of multiple studies suggest that GnRH agonist regimens that suppress estradiol concentrations to either 15 pg/ml or 30 pg/ml are both effective in the treatment of endometriosis (Table 3). However, suppressing estradiol to 15 pg/ml is associated with more symptoms of hypoestrogenism, such as vasomotor symptoms and increased bone loss. These observations form the basis for ‘‘add-back’’ regimens that contain estrogen.

GNRH AGONISTS PLUS STEROID ADD-BACK FOR THE TREATMENT OF ENDOMETRIOSIS An important concept in gynecology is that tissues vary in their sensitivity to estradiol. At very low estradiol concentrations (5 to 15 pg/ml), many estrogen responsive tissues are in a quiescent or atrophic state (e.g., the endometrium). In some tissues, an estradiol concentration in the range of 20 to 60 pg/ml will induce a significant tissue response (suppression of urinary calcium excretion, suppression of osteoclasts activity, suppression of vasomotor symptoms) [20– 22]. In other tissues, the estradiol concentration must be more than 80 pg/ml to induce a significant tissue response (stimulation of liver production of lipoproteins). The hierarchy of tissue response to various estradiol concentration is presented in Figure 2. In a manner that parallels the estradiol doseresponse relationships in normal tissues, different disease processes display different responsiveness to estradiol. For example, some breast cancer cell lines may be stimulated by estradiol concentrations as low as 1 to 10 pg/ml. Fibroids are stimulated by estradiol concentrations in the range of 15 to 25 pg/ ml. In contrast to breast cancer and fibroids, endometriosis lesions appear to require estradiol concentrations in the range of 20 to 40 pg/ml to begin to grow [23,24]. These considerations suggest that in the treatment of endometriosis, it is possible to find an estradiol target, in the range of 20 to 40 pg/ml,

Acetate leuprolide Goserelin

Nafarelin

Nafarelin

Buserelin

GnRH agonist 300 ug intranasal 3 times per day 200 Ag nasal spray bid 400 Ag nasal spray bid 3.75 mg depot IM every 4 weeks 3.6 mg implant for 6 months

Dose

15 13

40

15

28

28

Estradiol concentration (pg/ml)

52

79

77

40

Number of subjects studied

Low

Low

Moderate

High

High

Variability of patient response to treatment

Reference

Shaw 1990

Dlugi 1990

Henzl 1988

Henzl 1988

Cirkel 1986

TABLE 3 Effects of Gonadotropin Releasing Hormone Agonists on Serum Estradiol Concentration in Women with Endometriosis

GnRH Agonist and Antagonist 227

228

Barbieri

FIGURE 2 Estrogen dose-response hierarchy. Normal tissues vary in their sensitivity to estradiol stimulation. Menopause is associated with an estradiol concentration in the range of 5 to 20 pg/ml. Vasomotor symptoms are significantly suppressed by estradiol in the range of 30 pg/ml. Bone metabolism begins to respond to estrogen stimulation at low concentrations of estradiol (30 to 60 pg/ml). Estrogen stimulation of the synthesis of liver proteins, such as thyroxine-binding globulins, may require estradiol concentrations greater than 80 pg/ml. Estrogendependent disease processes also vary in their sensitivity to estradiol. Breast cancer cells may be stimulated to grow at estradiol concentration in the range of 1 to 10 pg/ml. Endometriosis lesions may require estradiol concentrations in the range of 20 to 40 pg/ml to grow. From Ref. 26.

that provides the best balance of treatment of endometriosis lesions while at the same time reducing hypoestrogenic side effects, such as vasomotor symptoms and bone loss [25–27]. In a seminal study, women with endometriosis and pelvic pain were randomized to receive either a GnRH agonist alone or a GnRH agonist plus low-dose estradiol (transdermal estradiol 25 Ag per day) plus low-dose

GnRH Agonist and Antagonist

229

progestin (oral medroxyprogesterone acetate 2.5 mg daily) [28]. The women who received the GnRH agonist alone had a circulating estradiol of approximately 15 pg/ml. The women who received the GnRH agonist plus the estrogen-progestin add-back had circulating estradiol levels of approximately 25 pg/ml. All the women underwent pretreatment and posttreatment surgical staging and bone density measurement. In both groups there was an equivalent reduction in pelvic pain symptom scores. In addition there was an equivalent reduction in endometriosis lesion activity as determined by surgical staging. The women who received the GnRH agonist alone reported more vasomotor symptoms and greater bone loss as measured by dual energy x-ray absorptiometry of the spine. This study suggests that an optimal balance of control of symptoms and disease activity versus induction of hypoestrogenic side effects can be achieved by administering a parenteral GnRH agonist plus steroid hormone add-back. Numerous clinical trials have demonstrated that add-back is a valid clinical treatment strategy [29]. Many trials have extended the concept to explore the relative efficacy of estrogen-progestin versus progestin-only addback and different doses of estrogen. For example, Hornstein and colleagues randomized women with pelvic pain and endometriosis to one of four treatment groups: (1) GnRH agonist alone (leuprolide acetate depot 3.75 mg IM every 4 weeks), (2) GnRH agonist plus progestin-only (norethindrone acetate 5 mg orally daily), (3) GnRH agonist plus low-dose estrogen (conjugated equine estrogen 0.625 mg orally daily) plus progestin (norethindrone acetate 5 mg orally daily), or (4) highdose estrogen (conjugated equine estrogen 1.25 mg orally daily) plus norethindrone acetate 5 mg orally daily [30]. Treatment was planned to continue for 1 year. The study demonstrated that add-back with high-dose estrogen was not as effective as add-back with low-dose estrogen. This conclusion was based on the observation that the group receiving GnRH agonist plus highdose estrogen had a significantly higher rate of treatment discontinuation due to pelvic pain than did any of the other treatment groups. The high-dose estrogen add-back probably stimulated the continued functioning and growth of the endometriosis implants. Consequently GnRH agonist plus high-dose estrogen add back is not recommended as a primary treatment option. The women in the other three groups had a similar decrease in their pelvic pain. Bone density decreased significantly in the women who received the GnRH agonist alone. Bone density was preserved in both the group that received low-dose estrogen plus progestin add-back and the group that received progestin only add-back (Fig. 3). Vasomotor symptoms were significantly reduced in the groups that received the steroid add-back. In longterm follow-up, at 12 and 24 months posttreatment, women in all treatment groups continued to have symptom scores that were below baseline for at least 8 months after completion of active therapy [31].

230

Barbieri

FIGURE 3 Effects of GnRH agonist on bone density in women with endometriosis and pelvic pain, who were treated with the GnRH agonist leuprolide depot for 52 weeks. Group A received GnRH agonist plus placebo pills. Group B received GnRH agonist plus norethindrone acetate 5 mg daily. Group C received GnRH agonist plus conjugated estrogen (0.625 mg) plus norethindrone (5 mg). Group D received GnRH agonist plus conjugated estrogen (1.25 mg) plus norethindrone (5 mg). No significant bone density loss occurred in the groups that received the GnRH agonist plus low dose steroid add-back. From Ref. 30.

This study and many others demonstrate that the optimal long-term treatment of pelvic pain caused by endometriosis may involve the use of GnRH agonists to suppress ovarian estrogen and progesterone production followed by the add-back low doses of estrogen plus progestin or progestin only [32,33]. Recent studies have suggested that GnRH agonist plus steroid add-back is clinically effective and safe for long-term use. For example, in one study women with pelvic pain and endometriosis were treated with a GnRH agonist plus estrogen-progestin add-back for up to a mean of 31 months. Pelvic pain was markedly reduced, compared to baseline, throughout the course of treatment. Bone mineral density was stable for the entire add-back treatment period [34]. Table 4 lists three add-back regimens that have been demonstrated to be effective in combination with a GnRH agonist for the treatment of pelvic pain caused by endometriosis.

GNRH AGONISTS: DOSE TITRATION In the treatment of pelvic pain caused by endometriosis, suppression of estrogen production reliably produces pain relief. However, low estrogen levels

GnRH Agonist and Antagonist

231

TABLE 4

GnRH Agonist Treatment Combined with Low-Dose Steroid ‘‘AddBack’’ Hormone Regimens Low-dose steroid hormone regimen Transdermal estradiol patch 25 Ag/day, plus medroxyprogesterone acetate 2.5 mg daily

Norethindrone acetate 5 mg/day

Conjugated equine estrogen 0.625 mg/day, norethindrone acetate 5 mg/day

Comments This regimen does not completely prevent bone loss. The estradiol concentration achieved is in the range of 30 pg/ml. This is a very high dose of progestin. This dose of progestin is associated with a decrease in HDL-cholesterol This regimen prevents bone loss and markedly reduces the vasomotor symptoms reported. Pain relief was excellent

Investigator Howell 1995

Hornstein 1997

Hornstein 1997

are associated with significant side effects, such as vasomotor symptoms and bone loss. One approach to this problem is to prescribe a GnRH agonist to suppress estrogen secretion and then add-back low doses of estrogen-progestin or progestin only. An alternative approach is to decrease the dose of GnRH agonist to reduce the magnitude of suppression of LH and FSH and allow some ovarian estrogen secretion. One method for decreasing the dose of the GnRH agonist is to initiate therapy with a standard dose of a daily GnRH agonist, such as nafarelin at a dose of 200 Ag bid. After 1 or 3 months, suppression of ovarian estrogen secretion and amenorrhea will have been achieved and pelvic pain should be improved. At that time, the dose of nafarelin can be decreased to two nasal sprays on the even calendar days and one nasal spray on the odd calendar days of the month. This will reduce the dose of GnRH agonist by about 25%. With this regimen, many women will note a decrease in vasomotor symptoms and continued amenorrhea and pelvic pain relief. If the patient remains amenorrheic on this dose, a small number of women can decrease the dose to nafarelin 200 Ag once daily. If menses or pain recurs, the dose can be increased to achieve more complete suppression of pituitary LH and FSH secretion, and in turn, ovarian estrogen secretion [35–37]. An alternative approach is to increase the time interval between depot injections of a GnRH agonist. For example, Tse and colleagues randomized

232

Barbieri

women with endometriosis and pelvic pain to triptorelin depot 3.75 mg IM every 4 weeks (standard therapy) or every 6 weeks (extended interval dosing) [38]. Women in both groups had similar improvement in pelvic pain. The extended interval dosing is also less expensive than the standard interval dosing. Body mass index is probably an important variable in predicting whether dose reduction or extended interval dosing will be effective. Women with body mass index (BMI) greater than 30 kg/m2 are less likely to achieve amenorrhea with reduced doses of GnRH agonists. Women with a BMI less than 25 kg/m2 who also exercise intensively are the most likely to reliably respond to reduced doses of GnRH agonist.

GNRH AGONIST ANALOGUES VERSUS THE ORAL CONTRACEPTIVE IN THE TREATMENT OF ENDOMETRIOSIS In 1959, Kistner, working at the Boston Free Hospital for Women (now the Brigham and Women’s Hospital) reported that the combination estrogenprogestin birth control pill, when used in a continuous (pseudopregnancy) fashion, was effective in the treatment of pelvic pain endometriosis [39]. Many clinicians have noted that some women with endometriosis and pelvic pain have improvement in their dysmenorrhea and pelvic pain when treated with combination estrogen-progestin pills. However, few randomized clinical trials have been completed that use oral contraceptives for the treatment of endometriosis. One randomized clinical trial compared the effects of continuous (pseudopregnancy) combination estrogen progestin oral contraceptive versus danazol in the treatment of pelvic pain caused by endometriosis [40]. The oral contraceptive regimen resulted in symptomatic improvement in 30% of the women. Danazol treatment resulted in symptomatic improvement in 86% of the women. Improvement in physical examination findings was demonstrated in 84% of the women treated with danazol and 18% of the women treated with continuous oral contraceptive. In this study, danazol was demonstrated to be superior to the oral contraceptive in the treatment of pain caused by endometriosis. In another randomized trial, an oral contraceptive was compared to a GnRH agonist in the treatment of pelvic pain caused by endometriosis. In this trial, the oral contraceptive and the GnRH agonist were not directly compared for their efficacy in relieving dysmenorrhea, because it was assumed that the GnRH agonist would be more effective in relieving dysmenorrhea. However, the GnRH agonist was superior to the oral contraceptive in the treatment of dyspareunia. A weakness of this study is that laparoscopy was not performed to assess the response of the endometriosis lesions to the hormonal treatment [41].

GnRH Agonist and Antagonist

233

In a recent clinical trial, women with endometriosis were treated with a GnRH agonist (3.75 mg leuprolide acetate depot) or a GnRH agonist plus a low-dose oral estrogen-progestin contraceptive(20 Ag ethinyl estradiol plus 0.15 mg desogestrel orally for 21 days every 4 weeks). The addition of the lowdose contraceptive reduced the efficacy of the GnRH agonist in the treatment of endometriosis [42]. Pre-and posttherapy surgical staging of the endometriosis lesions demonstrated that the GnRH agonist plus oral contraceptive treatment resulted in a 32% decrease (improvement) in the surgical staging score. In contrast, the GnRH agonist alone produced a superior, 76% decrease (improvement) in the surgical staging score. Both treatment regimens produced a similar improvement in dysmenorrhea. However, the GnRH agonist produced better relief of dyspareunia than the combination GnRH agonist plus oral contraceptives. These randomized studies suggest that oral contraceptives are probably not as effective as GnRH agonists or danazol in the treatment of pelvic pain caused by endometriosis. However, given the low cost of oral contraceptives, most clinicians treat women with pelvic pain with up to 3 months of oral contraceptives, either in a cyclic or pseudopregnancy manner before initiating treatment with a GnRH agonist or performing a laparoscopy. If oral contraceptive treatment is associated with a significant improvement in pain, the treatment can be continued for 6 to 12 months. If the patient has minimal improvement in pain, she is usually offered laparoscopy to definitively diagnose the cause of her chronic pelvic pain, or she is treated empirically with a GnRH agonist analogue based on a clinical diagnosis of endometriosis. It is important for the clinician to abandon oral contraceptive therapy if it is not effective in the treatment of pelvic pain. GNRH AGONISTS VERSUS PROGESTINS FOR THE TREATMENT OF PELVIC PAIN Prospective randomized clinical trials have suggested that GnRH agonists are boht superior to progestin treatment of pelvic pain caused by endometriosis and that the two therapies are similar in their efficacy. The reasons for the discrepancies are not clear. In one randomized clinical trial, women with surgically treated endometriosis and pelvic pain were randomized postoperatively to leuprolide acetate 3.75 mg IM every 4 weeks or lynestrenol, 5 mg orally twice daily for 6 months [43]. After 6 months of hormone treatment, the women underwent laparoscopy to measure the extent of the disease. The women treated with leuprolide had significantly better pain relief than the women treated with lynestrenol. In addition, at second look laparoscopy, the women treated with leuprolide had significantly more resolution of endometriosis lesions than the

234

Barbieri

women treated with lynestrenol. This study indicates that the GnRH agonist, leuprolide, is superior to the progestin, lynestrenol, for the treatment of endometriosis.

GNRH AGONISTS AS A SURGICAL ADJUVANT FOR THE TREATMENT OF ENDOMETRIOSIS Winkel and Bray [44] reported the results of a 24 month follow-up of 240 women with endometriosis and pelvic pain who had three different therapeutic interventions: (1) excision of lesions with the Nd:YAG laser, (2) laser ablation alone, or (3) laser ablation plus postoperative leuprolide treatment for 3 to 6 months. This was not a randomized trial. Ninety six percent and 69% of the women who had surgical excision of the lesions were pain-free at 12 and 24 months of follow-up, respectively. Sixty nine percent and 23% of the women who had laser ablation alone were pain-free at 12 and 24 months of follow-up, respectively. Ninety one percent and 70% of the women who had laser ablation plus postoperative leuprolide acetate were pain-free at 12 and 24 months of follow-up, respectively. These observations support the idea that surgical excision of endometriosis lesions is superior to ablation. In addition these data suggest that surgical ablation plus postoperative treatment with a GnRH agonist is as effective as the complete surgical excision of all lesions. Surgical ablation of lesions is technically easier to accomplish for the average gynecologic surgeon. These data suggest that for the average gynecologic surgeon who is most comfortable with ablation techniques, the use of postoperative GnRH agonist therapy can enhance the efficacy of the surgical procedure. Hornstein and colleagues have also reported that postoperative treatment with a GnRH agonist enhances the efficacy of surgical treatment of pelvic pain caused by endometriosis [45]. Other investigators have reported that postoperative treatment with a GnRH agonist for only 3 months did not result in long-term improvement in pelvic pain compared to postoperative expectant management [46]. Taken together, these studies suggest that for a postoperative GnRH agonist to be effective, it may need to be given for longer than 3 months.

GNRH AGONISTS: EMPIRICAL TREATMENT OF PELVIC PAIN Most authorities believe that the best approach to the evaluation and treatment of chronic pelvic pain that has not responded adequately to treatment with nonsteroidal anti-inflammatory drugs (NSAIDs) and oral contraceptives is to perform a laparoscopy. Conceptually, laparoscopy has the advant-

GnRH Agonist and Antagonist

235

age of providing a definitive diagnosis of the disease causing the pain and an opportunity to surgically treat the disease causing the pain. However, evidence suggests that laparoscopy often fails to identify cases of endometriosis caused by atypical and subperitoneal disease, and surgical treatment of visualized endometriosis is often suboptimal. An alternative approach to the treatment of chronic pelvic pain is to consider making a clinical diagnosis of endometriosis based on history, physical examination, and noninvasive laboratory testing [47,48]. Once a clinical diagnosis of endometriosis is made, treatment with a GnRH agonist could be initiated without the need for a laparoscopy after NSAIDs and oral contraceptive treatment have failed to treat the pain [49]. In a high quality, randomized, placebo-controlled trial, Ling and colleagues demonstrated the efficacy of this treatment algorithm [50]. The investigators randomized 100 women with chronic pelvic pain who had not had adequate relief of their pain with NSAIDs and doxycycline to receive either depot leuprolide acetate 3.75 mg IM every 4 weeks for 12 weeks or a placebo injection every 4 weeks for 12 weeks. At the completion of the 3month treatment regimen, all the women had a laparoscopy with videotaping of the findings and review of the videotapes by a single investigator. The study was completed by 49 women in the leuprolide group and 45 women in the placebo group. At the laparoscopy at the completion of the study, 78 of 95 women (82%) were demonstrated to have endometriosis lesions. This demonstrates the high frequency of endometriosis in women with chronic pelvic pain. The women treated with depot leuprolide had significantly more improvement in self-reported dysmenorrhea, pelvic pain, and dyspareunia than the women treated with placebo. The women treated with depot leuprolide had significantly more improvement in pelvic tenderness and pelvic induration than the women treated with placebo. Ling and colleagues emphasize that an extensive but non invasive pretreatment diagnostic workup, followed by the initiation of a treatment plan based on the test results, is important. The workup includes history, physical examination, pelvic ultrasound, complete blood count, urinalysis, and endocervical cultures for gonococci and Chlamydia organisms. Critical to the effectiveness of the protocol is the history of moderate to severe chronic pelvic pain unrelated to menstruation, lasting for at least 6 months and nonresponsive to nonsteroidal anti-inflammatory drugs or oral contraceptives. Gynecological causes of chronic pelvic pain such as pelvic inflammatory disease, leiomyomata uteri, and ovarian cysts should be have been identified by medical history, physical examination, and pelvic ultrasound. Health maintenance organizations that have implanted clinical protocols that use primary empiric hormonal treatment of pelvic pain have noted

236

Barbieri

reduced resource utilization compared with protocols that use a primary surgical approach to the diagnosis and treatment of pelvic pain [51]. GNRH ANTAGONISTS: THE FUTURE OF ENDOMETRIOSIS TREATMENT Gonadotropin releasing hormone agonist analogues have one serious problem; when first administered, they cause an increase in LH and FSH secretion and an increase in estradiol production. For women with pelvic pain, the increase in ovarian estrogen production is associated with increased pelvic pain and decreased quality of life [52]. However, the agonist phase is typically limited to the first 5 to 10 days of treatment. The GnRH antagonist analogues may be superior to GnRH agonists because they produce immediate suppression of LH and FSH secretion and avoid the agonist phase of GnRH agonist therapy. The GnRH analogues that possess antagonist properties can be generated by substitution of D-amino acids for native L-amino acids at positions 1, 2, and 3 (Table 5). The main mechanism of action of the GnRH antagonists is competitive receptor occupancy, which results in the blockade of GnRH receptor dimerization, a biochemical process that is required for receptor activation. Native GnRH elicits a gonadotrope response when only 1% to 10% of gonadotrope GnRH receptors are occupied. Consequently, an effective GnRH antagonist must possess high affinity for the receptor, have a prolonged duration of action, and be given in doses large enough to block almost all the pituitary GnRH receptors. A major challenge to the development of GnRH antagonists for general medical use is the cost inherent to manufacturing and administering large quantities of complex synthetic peptides. In addition, first generation GnRH antagonists were associated with histamine release from eosinophils, causing local skin reactions and in some cases systemic anaphylaxis [53]. Significant progress has been made in developing efficient processes for large scale peptide synthesis and GnRH antagonists with low allergic properties have been developed. In preliminary research studies, GnRH antagonists have been demonstrated to be effective in the treatment of endometriosis [54], uterine leiomyomata [55], premenstrual syndrome, polycystic ovary syndrome [56], ovulation induction for in vitro fertilization (IVF), and prostate cancer [57]. Gonadotropin rereasing hormone antagonists are currently approved and widely used to block premature LH surges during ovarian hyperstimulation for IVF. In many centers, GnRH antagonists are replacing GnRH agonists to block premature LH surges. In IVF, a major problem with the GnRH agonist analogues is that LH secretion is stimulated at the initiation of treatment. In some women, prolonged daily use of a GnRH agonist may

His D-Phe D-Phe D-Phe D-Phe

D-Nal D-Nal D-Nal D-Nal

2

pGlu

1

D-Pal D-Pal D-Pal D-Pal

Trp

3

Ser Ser Ser Ser

Ser

4

Tyr Tyr D-Glu Phe

Tyr

5

D-Cit D-hArg D-Glu D-hCit

Gly

6

Leu Leu Leu Leu

Leu

7

8

Arg hArg Arg Lys(iPr)

Arg

Amino Acid Composition of Native GnRH and Third Generation GnRH Antagonists

Pro Pro Pro Pro

Pro

9

D-Ala D-Ala D-Ala D-Ala

Gly-NH2

10

All the Antagonists Have Amino Acid Substitutions at Positions, 1, 2, 3, 6, and 10. In Contrast, the GnRH Agonists Typically Have Substitutions at Positions 6 and 10

GnRH Analogue Cetrorelix Ganirelix Nal-Glu Abarelix

Amino acid position

TABLE 5

GnRH Agonist and Antagonist 237

238

Barbieri

cause a small increase in LH secretion directly after the daily administration of the GnRH agonist. In turn, residual pituitary LH secretion stimulates ovarian androgen production, which may have detrimental effects on follicular development and endometrial function [58]. The GnRH antagonists offer the possibility of acutely suppressing LH secretion without an initial increase in LH secretion [59,60]. The GnRH antagonists have been used either as small daily doses (cetrorelix 0.25 mg daily subcutaneous injection) during the early or midfollicular phases of the stimulation cycle [61], or as a single dose (cetrorelix 3 mg subcutaneous) on approximately cycle day 8 [62]. Both regimens block the occurrence of spontaneous LH surges. The GnRH antagonists suppress LH secretion in a dose-dependent manner. At small doses, the suppression of LH is minimal. At large doses, near-complete suppression of LH can be achieved. In one study, the impact of six doses of the GnRH antagonist ganirelix on LH secretion and IVF outcomes was studied [63]. Ganirelix produced a dose-dependent suppression of LH. Ganirelix also produced a dose-dependent suppression of both androstenedione and estradiol. The larger doses of ganirelix were associated with a markedly reduced pregnancy rate. These data support the importance of both FSH and LH in the development of the ovarian follicle. Ovarian estradiol production requires the coordinated action of LH on the ovarian theca to stimulate the production of androstenedione and FSH on the granulosa cells to stimulate the aromatization of the androstenedione to estrogen. Large doses of GnRH antagonists can nearly ablate LH secretion, resulting in a reduction in follicular androstenedione and estradiol production. When LH secretion is completely blocked, pregnancy rates appear to be reduced. These findings support the hypothesis that both high LH levels (premature LH surge) and very low LH levels can be associated with low pregnancy rates in IVF-embryo transfer. The GnRH antagonists, because of their rapid onset of action and quick reversibility offer the opportunity to precisely target LH and FSH levels during ovarian stimulation for IVF-ET. It is likely that in the near future, GnRH antagonists will be approved for the treatment of prostate cancer. However, as of the date of writing this chapter, no GnRH antagonist has been approved for the treatment of endometriosis. In one preliminary study, women with endometriosis and pelvic pain were treated with the GnRH antagonist, cetrorelix 3 mg subcutaneous injection once weekly [64]. With cetrorelix treatment, circulating estradiol was suppressed to a mean of 50 pg/ml. Pelvic pain decreased and endometriosis lesions regressed as demonstrated by pre-and posttreatment surgical staging. Another GnRH antagonist, abarelix, has been demonstrated to be effective in the treatment of pelvic pain caused by endometriosis [54]. However, side effects may have slowed the progress of the development of this antagonist for treatment of endometriosis.

GnRH Agonist and Antagonist

239

SUMMARY The GnRH agonist analogues are the most reliable hormonal method of suppressing ovarian estrogen production. In turn, suppression of ovarian estrogen production is the most reliable method for improving the pelvic pain associated with endometriosis. Chronic suppression of ovarian estrogen production is associated with adverse side effects, such as vasomotor symptoms and accelerated bone loss. Low-dose steroid add-back has been demonstrated to avoid the adverse side effects of chronic GnRH agonist treatment without causing a recurrence of pelvic pain in most women with endometriosis.

PRACTICAL POINTS 

GnRHa is the most reliable method for improving the pelvic pain associated with endometriosis.  Low-dose steroid add-back avoids the adverse side effects of chronic GnRHa without causing recurrence of pelvic pain.

REFERENCES 1. 2. 3.

4. 5.

6. 7. 8.

9.

Knobil E. The neuroendocrine control of the menstrual cycle. Recent Prog Hormone Res 1980; 36:53. Conn PM, Crowley WF Jr. Gonadotropin releasing hormone and its analogues. N Engl J Med 1991; 324:93. Bergqvist A, Feno M. Steroid receptors in endometriotic tissue and endometrium assay with monoclonal antibodies. In: Genazzani AR, ed. Recent Research in Gynecologic Endocrinology. Vol. 1. Carnforth, U.K.: Panthenon, 1989:394. Bergqvist A, Jepsson S. Kullander. Human uterine and endometriotic tissue transplanted into nude mice. Am J Pathol 1985; 121:337. Sharpe K, Bertero MC, Muse KN. Spontaneous and steroid-induced recurrence of endometriosis after suppression by a gonadotropin releasing hormone antagonist in the rat. Am J Obstet Gynecol 1991; 164:187. Ranney B. Endometriosis III. Complete operations: reason, sequelae and treatment. Am J Obstet Gynecol 1971; 109:1137–1144. Gray LA. Endometriosis of the bowel. Ann Surg 1973; 177:580–587. Hammond CB, Rick JA, Parker RT. Conservative treatment of endometriosis: The effects of limited surgery and hormonal pseudopregnancy. Fertil Steril 1976; 27:756–766. Barbieri RL, Ryan KJ. Danazol: Endocrine pharmacology and therapeutic applications. Am J Obstet Gynecol 1981; 141:453.

240

Barbieri

10. Hammond MG, Hammond CB, Parker RT. Conservative treatment of endometriosis externa. The effects of methyltestosterone therapy. Fertil Steril 1978; 29:651. 11. Luciano AA, Turksoy RN, Carleo J. Evaluation of oral medroxyprogesterone acetate in the treatment of endometriosis. Obstet Gynecol 1988; 72:323. 12. Ahmed MS, Barbieri RL. Reoperation rates for recurrent ovarian endometriomas after surgical excision. Gynecol Obstet Invest 1997; 43:53–54. 13. Dlugi AM, Miller JS, Knittle J. Lupron study group. Lupron depot in the treatment of endometriosis: a randomized placebo controlled double blind study. Fertil Steril 1990; 54:419–426. 14. Henzl MR, Corson SL, Moghissi K, Buttram VC, Bergqvist C, Jacobson J, for the Nafarelin Study Group. Administration of nasal nafarelin as compared with oral danazol for endometriosis: A multicenter double-blind comparative clinical trial. N Engl J Med 1988; 318:485–489. 15. Barbieri RL. New therapy for endometriosis. N Engl J Med 1988; 318:512–514. 16. Barbieri RL. Comparison of the pharmacology of nafarelin and danazol. Am J Obstet Gynecol 1990; 162:581–855. 17. Prentice A, Deary AJ, Goldbeck-Wood S, Farquhar C, Smith SK. Gonadotropin releasing hormone analogues for pain associated with endometriosis. Cochrane Database of Systemic Reviews, CD000346. 18. Shaw RW. Goserelin depot preparation of LHRH analogue used in the treatment of endometriosis. In: Chadha DR, Buttram VC, eds. Current Concepts in Endometriosis. New York: Alan R. Liss, 1990:323–383. 19. Cirkel U, Schweppe KW, Ochs H. Effects of LHRH agonist therapy in the treatment of endometriosis. Chadha DR, Willemsen WNP, eds. Gonadotropin Down-regulation in Gynecological Practice, Vol 225. New York: Alan R. Liss, 1986:189. 20. Naessen T, Berglund L, Ulmsten U. Bone loss in elderly women prevented by ultra low doses of parenteral 17-estradiol. Am J Obstet Gynecol 1997; 177:115– 119. 21. Mizunuma H, Okano H, Soda M, Kagami I, Miyamoto S, Tokizawa T. Prevention of post-menopausal bone loss with minimal uterine bleeding using low dose continuous estrogen-progestin therapy: A 2-year prospective study. Maturitas 1997; 27:69–76. 22. Chetkowski RJ, Meldrum DR, Steingold KA, Randle D, Lu JK, Eggena P, Hershman JM, Alkjaersig NK, Fletcher AP, Judd HL. Biological effects of transdermal estradiol. N Engl J Med 1986; 314:1615–1620. 23. Barbieri RL, Gordon AMC. Hormonal therapy of endometriosis: The estradiol target. Fertil Steril 1991; 56:820–822. 24. Barbieri RL. Hormonal therapy of endometriosis. Infertil Reprod Med Clin North Am 1992; 3:187–200. 25. Barbieri RL. Gonadotropin releasing hormone agonists and estrogen-progestogen replacement therapy. Am J Obstet Gynecol 1990; 162:593–595. 26. Barbieri RL. Hormone treatment of endometriosis: The estrogen threshold hypothesis. Am J Obstet Gynecol 1992; 166:740–745.

GnRH Agonist and Antagonist

241

27. Barbieri RL. Endometriosis and the estrogen threshold theory. Relation to surgical and medical treatment. J Reprod Med 1998; 43(S):287–292. 28. Howell R, Edmonds D, Dowsett M. Gonadotropin releasing hormone analogue plus hormone replacement therapy for the treatment of endometriosis: A randomized clinical trial. Fertil Steril 1995; 64:474. 29. Surrey ES. Add-back therapy and gonadotropin releasing hormone agonists in the treatment of patients with endometriosis: Can a consensus be reached. Fertil Steril 1999; 71:420–424. 30. Hornstein MD, Surrey ES, Weinberg GW, Casino LA. Leuprolide acetate depot and hormonal add-back in endometriosis: A 12-month study. Lupron Add-Back Study Group. Obstet Gynecol 1998; 91:16. 31. Surrey ES, Hornstein MD. Prolonged GnRH agonist and add-back therapy for symptomatic endometriosis: Long-term follow-up. Obstet Gynecol 2002; 99: 709–719. 32. Ihahara M, Uemara H, Yasui T, Kinoshita H, Yamada M, Tezuka M, et al. Efficacy of every-other-day administration of conjugated equine estrogen and medroxyprogesterone acetate on gonadotropin-releasing hormone agonists treatment in women with endometriosis. Gynecol Obstet Invest 2001; 52:217–222. 33. Pierce SJ, Gazvani MR, Farquharson RG. Long-term use of gonadotropin releasing hormone analogs and hormone replacement therapy in the management of endometriosis: A randomized trial with a 6-year follow-up. Fertil Steril 2000; 74:964–968. 34. Mitwally MF, Gotlieb L, Casper RF. Prevention of bone loss and hypoestrogenic symptoms by estrogen and interrupted progestogen add-back in long-term GnRH agonist down-regulated patients with endometriosis and premenstrual syndrome. Menopause 2002; 9:236–241. 35. Hull ME, Barbieri RL. Nafarelin in the treatment of endometriosis: Dose management. Gynecol Obstet Invest 1994; 37:263–264. 36. Tahara M, Matsouka T, Yokoi T, Tasaka K, Kurachi H, Murata Y. Treatment of endometriosis with a decreasing dosage of a gonadotropin releasing hormone agonist (nafarelin): A pilot study with low-dose agonist therapy. Fertil Steril 2000; 73:799–804. 37. Uemura T, Shirasu K, Katagiri N, Asukai K, Suzuki T, Suzuki N. Low-dose GnRH agonist therapy for the management of endometriosis. J Obstet Gynecol Res 1999; 25:295–301. 38. Tse CY, Chow AM, Chan SC. Effects of extended-interval dosing regimen of triptorelin depot on the hormonal profile of patients with endometriosis: prospective observational study. Hong Kong Med J 2000; 6:260–264. 39. Kistner RW. The treatment of endometriosis by inducing pseudopregnancy with ovarian hormones: A report of 58 cases. Fertil Steril 1959; 10:539. 40. Noble AD, Letchworth AT. Medical treatment of endometriosis: A comparative trial. Postgrad Med J 1979; 55(suppl 5):37–41. 41. Vercellini P, Trespidi L, Colombo A. A gonadotropin releasing hormone agonist versus a low dose oral contraceptive for pelvic pain associated with endometriosis. Fertil Steril 1993; 60:75–79.

242

Barbieri

42. Freundl G, Godtke K, Gnoth C, Godehart E, Kienle E. Steroidal ‘‘add-back’’ therapy in patients treated with GnRH agonists. Gynecol Obstet Invest 1998; 45(suppl 1):22–30. 43. Regidor PA, Regidor M, Schmidt M, Ruwe B, Lubben G, Fortig P, Kienle E, Schindler AE. Prospective randomized study comparing the GnRH agonist leuprorelin acetate and the gestagen lynestrenol in the treatment of severe endometriosis. Gynecol Endocrinol 2001; 15:202–209. 44. Winkel CA, Bray M. Treatment of women with endometriosis using excision alone, ablation alone or ablation in combination with leuprolide acetate. In: Proceedings of the 4th World Congress on Endometriosis, Yokahama, 1996: 55. 45. Hornstein MS, Hemmings R, Yuzpe AA, Heinrichs WL. Use of nafarelin versus placebo after reductive laparoscopic surgery for endometriosis. Fertil Steril 1997; 68:860–864. 46. Busacca M, Somigliana E, Bianchi S, De Marinis S, Calia C, Candiani M, Vignali M. Post-operative GnRH analogue treatment after conservative surgery for symptomatic endometriosis stage III–IV: A randomized controlled trial. Hum Reprod 2001; 16:2399–2402. 47. Barbieri RL, Niloff JM, Bast RC, Shaetzl E, Kistner RW, Knapp RC. Elevated serum concentrations of CA-125 in patients with advanced endometriosis. Fertil Steril 1986; 45:630–634. 48. Hornstein MD, Thomas PP, Gleason RE, Barbieri RL. Menstrual Cyclicity of CA-125 in patients with endometriosis. Fertil Steril 1992; 58:279–283. 49. Barbieri RL. Primary gonadotropin releasing hormone agonist therapy for suspected endometriosis: A nonsurgical approach to the diagnosis and treatment of chronic pelvic pain. Am J Managed Care 1997; 3:285–290. 50. Ling FW, for the Pelvic Pain Study Group. Randomized controlled trial of depot leuprolide in patients with chronic pelvic pain and clinically suspected endometriosis. Obstet Gynecol 1999; 93:51–58. 51. Kephart W. Evaluation of Lovelace Health Systems chronic pelvic pain protocol. Am J Managed Care 1999; 5S:S309–S315. 52. Miller JD. Quantification of endometriosis-associated pain and quality of life during the stimulatory phase of gonadotropin releasing hormone agonist therapy: A double-blind, randomized placebo controlled trial. Am J Obstet Gynecol 2000; 182:1483–1488. 53. Karten MJ. An overview of GnRH antagonist development: Two decades of progress. In: Crowley WF Jr, Conn PM, eds. Modes of actions of GnRH and GnRH analogs. Amsterdam: Elsevier, 1992:277–297. 54. Martha PM, Gray ME, Campion M, Kuca B, Garnick MS. Initial safety profile and hormonal dose-response characteristics of the pure GnRH antagonist, abarelix-depot in women with endometriosis [abstract]. Gynecol Endocrinol 1999; 13(S):104. 55. Gonzalez-Barcena D, Alvarez RB, Ochoa EP, Cornejo IC, Comaru-Schally AM, Schally AV, Engel J, Resisman T, Riethmuller-Winzen H. Treatment of uterine leiomyomas with luteinizing hormone releasing hormone antagonist, cetrorelix. Hum Reprod 1997; 12:2028–2035.

GnRH Agonist and Antagonist

243

56. Hayes FJ, Taylor AE, Martin KA, Hall JE. Use of a gonadotropin releasing hormone antagonist as a physiological probe in polycystic ovary syndrome: Assessment of neuroendocrine and androgen dynamics. J Clin Endocrinol Metab 1998; 83:23443–23449. 57. Behre HM, Kliesch S, Puhse G, Reissman T, Nieschlag E. High loading and low maintenance doses of a gonadotropin releasing hormone antagonist effectively suppress serum luteinizing hormone, follicle stimulating hormone and testosterone in men. J Clin Endocrinol Metab 1997; 82:1403–1408. 58. Martin KA, Hornstein MD, Taylor AE, Hall JE, Barbieri RL. Exogenous gonadotropin stimulation is associated with increases in serum androgens in IVF-ET cycles. Fertil Steril 1997; 68:1011–1016. 59. Frydman R, Cornel C, de Ziegler D, Taieb J, Spitz IM, Bouchard P. Prevention of premature luteinizing hormone and progesterone rise with a gonadotropin releasing hormone antagonist, Nal-Glu, in controlled ovarian hyperstimulation. Fertil Steril 1991; 56:923–927. 60. Diedrich K, Diedrich C, Santos E, Zoll C, Al-Hasani S, Reissmann T, Krebs D, Klingmuller D. Suppression of the endogenous luteinizing hormone surge by the gonadotropin releasing hormone antagonist cetrorelix during ovarian stimulation. Human Reprod 1994; 9:788–791. 61. Albano C, Smitz J, Camus M, Riethmuller-Winzen H, Van Steirteghem A, Devroey P. Comparison of different doses of gonadotropin releasing hormone antagonist cetrorelix during controlled ovarian hyperstimulation. Fertil Steril 1997; 67:917–922. 62. Olivennes F, Alvarez S, Bouchard P, Franchin R, Salat-Baroux J, Frydman R. The use of a GnRH antagonist (cetrorelix) in a single dose protocol in IVFembryo transfer: A dose finding study of 3 versus 2 mg. Human Reprod 1998; 13:2411–2414. 63. The Ganirelix Dose Finding Study Group. A double-blind randomized dose finding study to assess the efficacy of the gonadotropin releasing hormone antagonist ganirelix (Org 37462) to prevent premature luteinizing hormone surges in women undergoing ovarian stimulation with recombinant follicle stimulating hormone (Puregon). Human Reprod 1998; 13:3023–3031. 64. Kupker W, Felberbaum RE, Krapp M, Schill T, Malik E, Diedrich K. Use of GnRH antagonists in the treatment of endometriosis. Reprod Biomed Online 2002; 5:12–16.

14 Management of Endometriosis After Hysterectomy and Bilateral Salpingo-Oophorectomy David Redwine Endometriosis Institute of Oregon Bend, Oregon, U.S.A.

INTRODUCTION For over a century, endometriosis has been a misunderstood disease. Evidence of this misunderstanding is easy to find in the medical literature, where countless authors have called it an enigmatic, mysterious disease, among other things. The intellectual confusion and contradictions that accompany the disease occur simply and only because much of what is accepted as conventional wisdom regarding the disease is not supported by evidence. Yet conventional wisdom is exceedingly slow to change, and so rational therapy for women with endometriosis lags even further behind. Why does change occur so slowly regarding endometriosis, whereas change occurs and is accepted so rapidly in unrelated fields such as astronomy or physics? One reason appears to be that many leaders in endometriosis are not actually experts in the study or treatment of endometriosis, but are merely parroting what others have said before, not believing that these echoes from the past may propagate ideas that are completely wrong. Worse, it is clear that 245

246

Redwine

pharmaceutical companies promoting medical treatment for endometriosis have tremendously influenced modern thought on the matter. Through an orchestrated approach using studies funded by drug companies, paid traveling speakers, and kickbacks to physicians for using medicines, the effort has been successful to popularize medical therapy given indefinitely for any undiagnosed pelvic pain syndrome. It should be obvious that the ship of endometriosis therapy is off course because its steerage is in the hands of a rogue crew using a distorted compass. However, these are just two of the latest problems in understanding endometriosis. One of the many long-standing errors of thought and therapy regarding endometriosis is that hysterectomy and bilateral salpingo-oophorectomy are ‘‘definitive’’ treatment for the disease. Women with ‘‘resistant’’ endometriosis undergo castration for cure. In this one preceding short sentence are crystallized many layers of identifiable misunderstanding that reach back into the last millennium, so this sentence will be examined sequentially and in detail to expose the multiple fallacies contained within it. THE CONCEPT OF ‘‘ ‘‘RESISTANT’’ ’’ ENDOMETRIOSIS There are only two treatment options for surgically diagnosed endometriosis: medicine or surgery. Observation alone would rarely, if ever, be a good choice for surgically diagnosed endometriosis, as this would put the clinician in the logically indefensible position of having to say to a patient, ‘‘I operated on you because I thought I might find a cause of your problem. I found something that might be the cause of your problem, but I chose not to treat it because it might not be the cause of your problem.’’ Medical therapy is based on hormonal suppression of ovarian function or of endometriosis itself. Although medical treatment can produce temporary reduction or relief of symptoms during therapy, it is clear that medical therapy does not eradicate endometriosis of any stage or disease site [1]. Ovarian suppression may relieve pain due to uterine fibroids, uterine adenomyosis, ovulation pain, dysmenorrhea, endometriosis, and some cases of idiopathic pelvic pain. The pain, which is reduced, may not be caused by endometriosis, and recurrent pain appears in most treated patients within a few weeks after the medicine wears off. If ‘‘treatment’’ is defined as something that destroys the origin of a disease process, like antibiotics destroy diseaseproducing bacteria, then medicines do not treat endometriosis. Because medicines do not treat endometriosis, experts agree that surgery is its only treatment. It thus becomes simply a matter of determining which type of surgical treatment destroys the disease most completely. First, though, we must consider what type of disease endometriosis is. It has long been known that endometriosis can be invasive for several centimeters be-

Management After Surgery

247

neath the visible surface [2]. It can occur in any pelvic location and can invade the bowel, bladder, ureter, or diaphragm. With this in mind, it immediately becomes clear that for a surgical technique to have a chance of completely destroying the disease, it must simultaneously be able to penetrate tissue up to several centimeters and allow for identification and repair of surgical defects left during treatment of endometriosis invading vital organs. Fortunately, it does not require a randomized controlled trial to complete this type of thought experiment and arrive at the correct conclusion, as common sense alone is sufficient. It is impossible to imagine that laser vaporization, electrocoagulation, harmonic scalpel coagulation, or endocoagulation would ever be able to destroy endometriosis completely. As well, none of these techniques has been validated in a sufficient number of reoperated patients as being effective in the eradication or reduction of disease extent. Although laser vaporization has been described in the literature in some detail, there is insufficient description of the other thermal ablation methods to allow their consistent application in human females. ‘‘Resistance’’ in medicine implies that a disease does not respond, either completely or at all, to any therapy. Resistance is a product of both the virulence of a disease as well as the efficacy of the applied therapy. The virulence of endometriosis can be manifest by impressive invasion or involvement of multiple vital organs. Regarding efficacy of surgical therapies, our thought experiment of the preceding paragraph has disqualified thermal ablation techniques from having much chance of complete eradication of disease. Thus, excision of endometriosis emerges as the only truly logical treatment for endometriosis of any stage, any location, and any symptomatology, in a patient of any age. It is also the only treatment that has proven curative ability [3,4]. Thus, the concept of resistant endometriosis is fueled primarily by the predictable results of ineffective therapies, which, together with Sampson’s theory of origin of endometriosis by reflux menstruation, have enjoyed a symbiotic relationship for many decades. Clinicians using ineffective treatments such as medical therapy or thermal ablation techniques could point to the notion of recurrence related to Sampson’s theory rather than to the inadequacy of their treatments. Sampson’s theory of origin is flying under an increasing burden of proof, which seems to make it unlikely to be the origin of any form of endometriosis [5]. As support for Sampson’s theory diminishes, realization of the waste associated with ineffective therapies will increase. REMOVAL OF THE UTERUS, TUBES, AND OVARIES FOR ‘‘ ‘‘CURE’’ ’’ OF ENDOMETRIOSIS Endometriosis is a disease that occurs primarily on peritoneal surfaces away from the uterus, tubes, and ovaries [6,7]. Removal of only the uterus, tubes,

248

Redwine

and ovaries with no further treatment to peritoneal endometriosis has been taught to generations of gynecologists as ‘‘definitive’’ therapy for the disease. Such surgery, however, will allow peritoneal disease to be left behind in almost 100% of patients, which is hardly a cure (Figs. 1–4). Many patients with endometriosis may have some associated pathology such as ovulation pain, adhesions, adenomyosis, fibroids, or primary dysmenorrhea that may be relieved by removal of the pelvic organs even if all endometriosis is allowed to remain in the body. If all such nonendometriotic sources of pain coming from the pelvic organs are erroneously ascribed to endometriosis, then surgeons removing the pelvic organs will certainly be impressed by the apparent efficacy of this surgery in relieving pain thought to be caused by endometriosis. Endometriosis appears to be the only disease that is treated by surgical ‘‘cure’’ with the removal of something else. Many reasons have been postulated to explain why removal of the pelvic organs may help symptoms of endometriosis. Apparently many physicians still believe that the hypoestrogenic state will cause endometriosis to disappear by some as yet unexplained cytocidal effect. Others believe that removal of the uterus is important in order to stop reflux menstruation to prevent new endometriosis from forming, although formation of new lesions does not appear to be the natural history of the disease in most patients [7].

FIGURE 1 A small vaginal lesion of endometriosis is seen at the vaginal apex in a woman with previous hysterectomy and bilateral salpingo-oophorectomy. Vaginal endometriosis is almost always an extension of a nodular lesion of a uterosacral ligament or of a nodular lesion associated with obliteration of the cul de sac. This lesion was an extension of a nodule of the right uterosacral ligament.

Management After Surgery

249

FIGURE 2 At laparoscopy, the right uterosacral ligament area looks thickened and nodular but is not particularly hemorrhagic.

FIGURE 3 Retroperitoneal dissection shows that the nodule is in the immediate vicinity of the stumps of the right uterine vessels as well as the ureter.

250

Redwine

FIGURE 4 The fibrosis resulting from the nodule also has incorporated posterior branches of the hypogastric artery. A small hole has been created in one of these branches.

Still others believe that endometriosis is symptomatic because of the monthly cycling of estrogen levels, so removal of the pelvic organs results in a steady state of menopausal levels of estrogen, or a steady state of estrogen due to estrogen replacement therapy in those women who are symptomatic without it. Whatever the reason for treatment of endometriosis by removal of the pelvic organs with retention of the disease, there is no question that some patients undergoing this therapy can continue to have symptoms. Where did this idea come from? How effective is this type of treatment?

THE ORIGIN OF DEFINITIVE SURGERY FOR ENDOMETRIOSIS The origin of the notion that menopause or low levels of estrogen can eradicate endometriosis is an interesting, albeit unfortunate, example of Berkson’s fallacy [8,9]. Berkson described the fallacy but did not commit it. Berkson observed that the nature of a disease process observed in hospitalized patients might not be an accurate reflection of that disease in unhospitalized patients. Such selection bias could lead to gross errors of understanding, which could lead to confusion and have dangerous effects on proposed treatments. Because endometriosis is a disease that has long required hospital-

Management After Surgery

251

ization and surgery to diagnose, it is a disease that could be subject to Berkson’s fallacy. Sampson’s early work was clearly affected by the unseen workings of Berkson’s fallacy. In 1921, writing about only 23 patients with chocolate cysts (and only 10 of those women had biopsy-proved endometriomas), he observed that he had never seen endometriosis in a menopausal patient [10]. This introduced the concept of menopause as treatment. Sampson fervently wanted to believe this and in 1922 he wrote ‘‘.. the implantations will usually, possibly always, atrophy after all ovarian tissue is removed. . . All of them probably cease to grow and actually atrophy after the menopause [11].’’ These multiple and hopeful adverbs notwithstanding, within 2 decades the oldest patient with endometriosis had been reported—age 78 [12]. The thoughts begun by Sampson were echoed in the literature over the years. According to Siegler, ‘‘After the menopause, endometriosis becomes only an asymptomatic relic [13].’’ Kistner wrote ‘‘. . . bilateral oophorectomy will almost always bring about permanent arrest of the disease’’ [14] and that ‘‘. . . functioning ovarian tissue is necessary for the continued activity of the disease [15].’’ Although many cases of endometriosis occurring after natural menopause have been reported in women taking or not taking estrogen [16– 24], even today it is commonly taught that menopause will eradicate endometriosis. If it is believed that natural menopause will eradicate endometriosis, then surely it is a natural extension to suppose that surgical menopause, ‘‘definitive’’ surgery, will do the same. Despite the widespread belief that pregnancy and menopause might eradicate endometriosis, unfortunately no one has gone to the trouble to do the simple studies that would validate this belief. HOW EFFECTIVE IS DEFINITIVE SURGERY FOR ENDOMETRIOSIS? Surgical efficacy can be measured in two ways: (1) eradication of disease (2) symptom reduction. Eradication of endometriosis requires reoperation to detect whether the disease is still present. There has never been a systematic study in reoperated patients validating the efficacy of definitive surgery for eradication of endometriosis. The efficacy of definitive surgery has been measured only by symptom response. Study of the response of symptoms requires a comparison of symptom levels pre- and postoperatively, although it must be borne in mind that not all pelvic pain is caused by endometriosis. Publications describing symptom reduction offer the only evidence of efficacy of definitive surgery. As symptoms may result from causes other than endometriosis, findings of such studies must be interpreted cautiously as they may not address directly the question of treatment of endometriosis symp-

252

Redwine

toms. It should also be remembered that in most studies of patients undergoing hysterectomy and castration as definitive therapy, the endometriosis was always left in place (other than disease that might rarely involve the ovaries, tubes, or uterus, or occasional confirmatory biopsies.) In 1951, Huffman [25] noted that among 77% of castrated women given estrogen replacement therapy, less than 5% had persistent symptoms. Schram [26] reported a single case of endometriosis after removal of the pelvic organs in a patient who had no endometriosis apparent at her previous surgeries. Venter et al. [27] described a woman who had undergone removal of the pelvic organs 15 years previously for whom estrogen was not prescribed postoperatively. Because of severe vasomotor symptoms, she was given oral estrogen and began vaginal bleeding 1 week later. Examination showed a polypoid lesion measuring 3 x 2 cm located at the vaginal apex. This was treated by cessation of estrogen therapy and vaginal removal and cautery by a surgical oncologist, but the lesion and bleeding recurred within 2 years despite a serum estradiol level of 66 pmol/L. The lesion did not respond to massive injections of 400 mg Depo-Provera weekly for 1 year but finally responded partially to external beam irradiation. Metzger et al. [28] reported a case of a 34-year-old black woman who had undergone previous removal of the pelvic organs for presumed pelvic inflammatory disease. A small focus of endometriosis on the uterine serosa was the only evidence of endometriosis at that surgery. Six years later she developed abdominal pain, constipation, urinary frequency, and nocturia. Bilateral hydronephrosis was noted on intravenous pyelography (IVP) as well as a 6-cm mass in the left lower abdominal area. At laparotomy, the mass was incompletely resected. The patient was given intramuscular injections of Depo-Provera 250 mg twice monthly. The mass increased to 15 cm in greatest dimension. Ultrasound-guided transvaginal drainage was done, but the patient ultimately required a repeat laparotomy with posterior exenteration, exploration of the left obturator fossa, and colostomy associated with 15 L of blood loss. The colostomy was later reversed. Immunohistochemical analysis showed that the endometriosis tissue had elevated levels of progesterone receptors, but not of estrogen receptors. Spence et al. [29] reported a single case of endometriosis after removal of the pelvic organs in a woman with no previous diagnosis of endometriosis. The patient had an excellent response to aggressive resection at laparotomy and was placed on oral estrogen immediately after surgery. The authors emphasized, ‘‘In most patients removal of the pelvic organs eradicates endometriosis.’’ Namnoun et al. [30] found that if the ovaries were left in at the time of hysterectomy, the rate of persistent or recurrent symptoms was 62% and the

Management After Surgery

253

rate of reoperation was 31%. In women with removal of the ovaries at the time of hysterectomy, the rate of recurrent/persistent symptoms was 10% and the rate of reoperation was 3.7%. The prevalence of invasive disease or ovarian disease in this population was not discussed. In a retrospective study of 95 patients who had undergone total abdominal hysterectomy/bilateral salpingo-oophorectomy (TAH/BSO) for endometriosis, Hickman et al. [31] found that symptom recurrence was 7% in women beginning estrogen therapy immediately postoperatively, compared with 20% when estrogen was begun 6 weeks or more postoperatively. The overall rate of symptom recurrence in women taking estrogen replacement therapy was 12%. Progesterone therapy did not appear to protect against symptom recurrence, and no case of cancer was observed. Matorras et al. [32] performed a prospective randomized trial in women undergoing castration for treatment of endometriosis. They compared postoperative combined continuous estrogen patch plus cyclic oral micronized progesterone with no therapy. In 57 women not receiving estrogen, there was no case of symptom recurrence, although the rate of vasomotor symptoms was not discussed. In 115 treated patients, there were five with symptomatic recurrence and two of these were confirmed histologically. All five recurrences were in women with impressive evidence of probable invasive endometriosis, including a woman with rectovaginal disease, another with a partially obstructing sigmoid nodule, two others with probable obliteration of the cul de sac who had required supracervical hysterectomy, and another woman with right ureteral obstruction resulting from dense retroperitoneal fibrosis leading to nephrectomy with retention of the pelvic fibrosis. Recurrence of symptoms among patients with supracervcal hysterectomy performed because of probable obliteration of the cul de sac was 22%. In an immunocytochemical study of postmenopausal ovarian endometriosis in 21 women who had never taken estrogen therapy [33], the ovarian disease showed robust biological activity even though the eutopic endometrium was atrophic. The continued activity of endometriosis may be the result of expression of aromatase enzyme by the lesions themselves, which can allow conversion of adrenal precursors into estrogen [34]. Several things seem clear from the evidence cited above. There is no scientific evidence that removal of the pelvic organs and low levels of estrogen eradicate endometriosis, and histologically active symptomatic endometriosis can certainly persist despite low levels of estrogen. However, symptomatic endometriosis occurs in less than 10% of patients after removal of all pelvic organs, especially if estrogen therapy is withheld. Furthermore, reoperation for recurrent symptoms was encountered in less than 5% of cases, although the rates of continuation of symptoms and reoperation seem higher if invasive disease is present at the time of hysterectomy and castration. It is also clear

254

Redwine

that an enormous number of women suffer from symptomatic endometriosis despite hysterectomy and castration. In the United States alone, several hundred thousand hysterectomies are performed annually for definitive treatment of endometriosis. For every 100,000 women undergoing surgery, 10,000 will remain symptomatic and a disproportionate number of these will have invasive disease. Fortunately for those who remain symptomatic because of their endometriosis, excision of persistent disease will result in pain relief [35–37]. MALIGNANT CHANGE OF RETAINED ENDOMETRIOSIS AFTER HYSTERECTOMY AND CASTRATION The possibility of malignant change of endometriosis during estrogen replacement therapy is of some concern. The adenomatous portion of endometriosis could respond to estrogen by developing hyperplastic changes, which could become atypical and then cancerous, similar to what occurs in eutopic endometrium. Maligant change of endometriosis has been reported to occur in the face of estrogen replacement therapy but seems very rare. Anderson et al. [38] reported the case of a 46-year-old woman who underwent hysterectomy and castration for endometriosis associated with ‘‘dense pelvic adhesions.’’ She was placed on postoperative estrogen replacement therapy and 6 months later underwent sigmoid colectomy for bowel obstruction due to endometriosis. By a year after her hysterectomy, a polypoid vaginal tumor was present and biopsies showed benign endometriosis. In response to oral medroxyprogesterone acetate, leuprolide acetate, and repeated biopsies, this tumor grew until it filled the vagina from its origin on a stalk on the right vaginal cuff. The mass was eventually removed by laparotomy almost 4 years after her hysterectomy, and adenosarcoma was diagnosed. The patient later had a recurrence in the right parametrium that was treated by radiation with good response. A similar case of adenosarcoma arising in endometriosis was reported in a 42-year-old woman by Judson et al. [39]. Modesitt et al. [40] reported that 13 of 15 cases of postmenopausal extraovarian cancers arose from endometriosis in the face of unopposed estrogen replacement therapy. Jones et al. [41] reported a case of a 52-year-old woman who had undergone hysterectomy and castration for treatment of deeply infiltrating rectovaginal endometriosis associated with complete obliteration of the cul de sac 12 years previously. Estrogen was administered by pellets for 12 years until she began to have bright red bleeding rectally. Barium studies and colonoscopy were normal except for a polyp of the distal sigmoid that showed atypical hyperplasia. At laparotomy, the lower rectosigmoid was involved with a hard fixed mass, and segmental bowel resection was performed. His-

Management After Surgery

255

tology showed adenocarcinoma arising in a field of endometriosis. The authors mention a literature review showing intestinal adenocarcinoma occurring in eight patients in the face of estrogen replacement therapy (ERT) and in another eight patients without ERT. Vara et al. [42] presented a case of bladder cancer arising from bladder endometriosis in a 62-year-old woman who had undergone TAH/BSO 10 years previously, although they did not mention whether she was receiving ERT. Endometriosis was not found elsewhere in the pelvis. In a review of medical records at M.D. Anderson Cancer Center [43], it was found that malignant change of nonovarian endometriosis was more common in postmenopausal women taking estrogen, whereas malignant change of ovarian endometriosis was not associated with estrogen therapy. It seems clear that there is a low rate of malignant transformation of endometriosis that was left in place at the time of hysterectomy and castration. Malignant transformation can occur without estrogen replacement therapy, although most cases have been associated with unopposed estrogen therapy. Importantly, most cases of malignant transformation of endometriosis after removal of the pelvic organs seem to arise in cases where there is severe invasive rectovaginal or intestinal disease. Some authors have called for consideration of combined ERT plus progesterone therapy. However, malignant change of residual endometriosis with unopposed ERT is rare and can occur even in the absence of ERT. Also, there is no evidence that progestins prevent malignant transformation of endometriosis, and there is some evidence that progesterone is associated with malignant change. Given that endometriosis is so different from native endometrium, it is simplistic and misleading to clinicians and patients to suggest that endometriosis would respond to progestins the way eutopic endometrium would and that progestins might prevent malignant change of endometriosis. For all these reasons, progesterone is currently not indicated in ERT for endometriosis patients after removal of the pelvic organs. An exception might be if a biopsy reveals atypical hyperplasia of the endometriosis in a patient in whom all disease was not removed. The best treatment is aggressive excision of all endometriosis in all patients undergoing surgery so that the issues of malignant transformation and continuing symptoms resulting from endometriosis are rendered moot. INDICATIONS FOR ERT IN PATIENTS WITH ENDOMETRIOSIS AFTER HYSTERECTOMY AND CASTRATION The indications for ERT in endometriosis patients are identical to the indications in any other woman: relief of bothersome symptoms. The primary symptoms of hot flashes or night sweats or symptoms of vaginal dryness are

256

Redwine

classic and easy to identify; however, other lesser symptoms also may occur, even in the absence of the primary symptoms. These include sleeping difficulty, fatigue or lack of energy, depression, mood swings, anxiety, or forgetfulness. These secondary symptoms may sound like a normal response to life in modern times, but the clinician should encourage women to report them. Because progestins are unnecessary, this simplifies things for the clinician and patient. When giving ERT to any patient, consideration should be given to bioavailability of the prescribed estrogen. The same dose of estrogen given to 10 different women might give 10 different levels of serum estradiol depending on intestinal absorption and estrogen metabolism. Additionally, the estrogen ‘‘thermostat’’ may be set higher in some women than in others, so these patients may not feel the full benefit of estrogen if their serum estradiol level is below their set point, even if it is within the normal range. Estrogen replacement therapy in any patient is simplified if the clinician focuses on two things: (1) symptoms and (2) serum estradiol levels. Clinicians should not focus on giving the lowest available dose of estrogen manufactured to all patients, as this ignores the simple truism that people are different for the reasons stated above. Giving the lowest possible dose to all patients will guarantee that many will be symptomatic and unhappy. It is important to be highly flexible and individualize treatment. There is no reason to withhold estrogen for any length of time after removal of the pelvic organs even if all endometriosis is left in place, because there is no evidence that the absence of estrogen is cytocidal to endometriosis and because there is evidence that withholding estrogen for several weeks or months is associated with a higher rate of recurrent painful symptoms in women with retained endometriosis. Estrogen replacement therapy can be started as early as the recovery room with patches, pellets, or injections because patients may be unable to tolerate oral intake for a day or two. When the patient is on a regular diet, pills can be started if that is the plan agreed to by the patient. Regardless of the form of estrogen given, a serum estradiol level should be obtained at the final 6- or 8-week postoperative checkup. By this time, the serum estradiol level will be stable on the patient’s daily dose. The serum estradiol level in young women with normally functioning ovaries ranges between 100 and 300 pg/ml. Estrogen replacement therapy should achieve a blood level in this range, although some patients may continue to have mild symptoms toward the low end of this range. It is popular in some practices for the clinician to be satisfied with a serum estradiol level of around 65, but many patients will feel better at a higher dose. Some might argue that giving estrogen until serum estradiol levels are in the normal range for women in their 20s is unphysiologic because older women have lower estrogen levels. This, of course, is not a valid argument because the end

Management After Surgery

257

result of such flawed logic would be the withholding or withdrawing of estrogen from all women because the inevitable physiologic level will be menopausal as women get older. Even if a patient is doing well on the initial dose of estrogen, some will have a change in absorption or metabolism that may result in a lower serum estradiol level and the appearance of symptoms. For this reason, the serum estradiol level should be checked as needed to help give the proper dose. Some women will have menopausal symptoms forever unless they take estrogen; others may have symptoms for a few years, which may eventually cease. A rare patient may never have symptoms. It may be necessary to intermittently discontinue ERT for 2 or 3 weeks to see if a patient still will benefit from it. Recurrent symptoms should reassure the clinician and the patient that ERT is indicated. Estrogen replacement therapy can be associated with activation of endometriosis in some patients, with worsening symptoms while on therapy. Patients with invasive disease involving parenchymal structures, such as the uterosacral ligaments or rectal wall, may remain symptomatic because of production of aromatase enzyme by the endometriosis with local conversion of adrenal precursors to estrogen. Regardless of whether a patient is on ERT or not, recurrent or continuing symptoms of endometriosis after removal of the pelvic organs are best treated by excision by an experienced endometriosis surgeon rather than by attempts at hormonal manipulation. The Women’s Health Initiative (WHI) [44] study from the United States gives reassuring information about the safety of estrogen replacement therapy in women, as only 19 women per 10,000 taking ERT had excess adverse events such as heart attacks, strokes, or breast cancer. The WHI study of 0.625 mg daily of conjugated estrogens combined with 2.5 mg daily of medroxyprogesterone indicated that this form of ERT will not prevent cardiovascular disease but can reduce colon cancer and hip fractures. Because women who have had removal of their pelvic organs do not need progesterone to prevent eutopic endometrial hyperplasia and as there is no evidence that progesterone has any role in medical treatment of endometriosis, the results of the WHI study may not be transferable to women taking only ERT in some other form. SUMMARY 1. Removal of the uterus, tubes, and ovaries does not make endometriosis go away, as the most common sites of involvement are peritoneal surfaces away from these organs. 2. Removal of the uterus, tubes, and ovaries can relieve most pain in most patients, even when endometriosis is left in.

258

Redwine

3. Some of the pain relieved by removal of the uterus, tubes, and ovaries may have originated in those organs and is mistakenly attributed to the endometriosis. 4. Invasive endometriosis is more likely to remain symptomatic even in the absence of estrogen. 5. Superficial endometriosis is less likely to remain symptomatic in the presence or absence of estrogen. 

Most women with endometriosis have superficial disease, which may explain why the majority do well with removal of the pelvic organs and retention of their disease.

6. When performing a hysterectomy and bilateral salpingo-oophorectomy, remove invasive endometriosis completely when possible to decrease the likelihood of persistent symptoms. 7. Estrogen therapy may be started immediately after removal of the pelvic organs.     



If all endometriosis is removed, there will be no problem. If only superficial endometriosis remains, it is unlikely that there will be a problem. If invasive endometriosis remains, it may be problematic with or without estrogen therapy. There is no evidence that withholding estrogen eradicates the disease. Withholding estrogen is associated with a higher rate of persistent or recurrent pain than if estrogen is begun immediately postoperatively. The serum estradiol level during therapy should be between 100 and 300 pg/ml.

8. Progesterone is not indicated for hormone replacement therapy after removal of the pelvic organs and retention of endometriosis, with the possible exception of atypical endometriosis having been left behind. 9. If endometriosis that was not removed at the time of hysterectomy and bilateral salpingo-oophorectomy is symptomatic with or without estrogen therapy, the patient should be referred to a qualified endometriosis surgical treatment program for excision of the disease. 10. Excision of symptomatic endometriosis persisting after castration cures the disease and will relieve pain caused by endometriosis. 11. Not all pelvic pain in women with endometriosis is necessarily caused by the disease, so it is possible to cure the disease yet still have a patient with pain.

Management After Surgery

259

PRACTICAL POINT .

During hysterectomy and bilateral salpingo-oophorectomy for pelvic pain, make sure to remove all endometriosis.

REFERENCES 1.

2. 3. 4.

5. 6. 7. 8. 9. 10. 11.

12. 13. 14. 15. 16. 17. 18.

Redwine DB. Treatment of endometriosis-associated pain. In: Olive DL, ed. Endometriosis: Infertility and Reproductive Medicine Clinics of North America. Philadelphia: WB Saunders, 1992:697–720. Cullen TS. Adenomyomas of the rectovaginal septum. JAMA 1914; 62:835–839. Wheeler JM, Malinak LR. Recurrent endometriosis. Contrib Gynecol Obstet 1987; 16:13–21. Redwine DB. Conservative laparoscopic excision of endometriosis by sharp dissection: Life table analysis of reoperation and persistent or recurrent disease. Fertil Steril 1991; 56:628–634. Redwine DB. Was Sampson wrong? Fertil Steril 2002; 78:686–693. Sampson JA. The development of the implantation theory for the origin of peritoneal endometriosis. Am J Obstet Gynecol 1940; 40:549–557. Redwine DB. The distribution of endometriosis in the pelvis by age groups and fertility. Fertil Steril 1987; 47:173–175. Berkson J. Limitations of the application of fourfold table analysis to hospital data. Biometrics 1946; 2:47–53. Berkson J. The statistical study of association between smoking and lung cancer. Proc Mayo Clinic 1955; 30:319–348. Sampson JA. Perforating hemorrhagic (chocolate) cysts of the ovary. Arch Surg 1921; 3:245–323. Sampson JA. Ovarian hematomas of endometrial type (perforating hemorrhagic cysts of the ovary) and implantation adenomas of the endometrial type. Bost Med Surg J 1922; 186:445–456. Haydon GB. A Study of 569 cases of endometriosis. Am J Obstet Gynecol 1942; 43:704–709. Siegler SL, Bisaccio JR. Endometriosis, clinical aspects and therapeutic considerations. Am J Obstet Gynecol 1951; 61:99. Kistner RW. Gynecology—Principles and Practice. 2d ed. Chicago: Year Book Publishers, Inc., 1971:432–457. Kistner RW. Management of endometriosis in the infertile patient. Fertil Steril 1975; 26, 1151–1166. Bennett ET. Endometriosis in the older age group. Am J Obstet Gynecol 1953; 65:100–108. Kempers RD, Dockerty MB, Hunt AB, Symmonds RE. Significant postmenopausal endometriosis. Surg Gynecol Obstet 1960; 3:348–356. Ranney B. Endometriosis. III. Complete operations. Am J Obstet Gynecol 1971; 109:1137–1143.

260

Redwine

19. Punonnen R, Klemi PJ, Nikkanen V. Postmenopausal endometriosis. Eur J Obstet Gynecol Reprod Biol 1980; 11:195–200. 20. Djursing H, Petersen K, Weberg E. Symptomatic postmenopausal endometriosis. Acta Obstet Gynecol Scand 1981; 60:529–530. 21. Madgar I, Ziv N, Many M, Jonas P. Ureteral endometriosis in postmenopausal women. Urology 1982; 20:174. 22. Vorstman B, Lynne C, Politano VA. Postmenopausal vesical endometriosis. Urology 1983; 22:540. 23. Goodman HM, Kredentser D, Deligdisch L. Postmenopausal endometriosis associated with hormone replacement therapy. A case report. J Reprod Med 1989; 34:231–233. 24. Habuchi T, Okagaki T, Miyakawa M. Endometriosis of bladder after menopause. J Urol 1991; 145:361–363. 25. Huffman JW. External endometriosis. Am J Obstet Gynecol 1951; 62:1243–1252. 26. Schram JD. Endometriosis after ‘‘pelvic cleanout.’’ South Med J 1978; 71:1419– 1420. 27. Venter PF, Anderson JD, Van Velden DJJ. Postmenopausal endometriosis: A case report. S Afr Med J 1979; 56:1136–1138. 28. Metzger DA, Lessey BA, Soper JT, McCarty KS, Haney AF. Hormone-resistant endometriosis following hysterectomy and bilateral salpingo-oophorectomy: Correlation with histology and steroid receptor content. Obstet Gynecol 1991; 78:946–950. 29. Spence MR, Maiese S, Amenta PS. Endometriosis occurring eight years after total abdominal hysterectomy and bilateral salpingo-oophorectomy. Am J Gynecologic Health 1992; 6:22–25. 30. Namnoun AB, Hickman TN, Goodman SB, Gehlbach DL, Rock JA. Incidence of symptom recurrence after hysterectomy for endometriosis. Fertil Steril 1995; 64:898–902. 31. Hickman TN, Namnoum AB, Hinton EL, Zacur HA, Rock JA. Timing of estrogen replacement therapy following hysterectomy with oophorectomy for endometriosis. Fertil Steril 1998; 91:673–677. 32. Matorras R, Elorriaga MA, Pijoan JI, Ramon O, Rodriguez-Escudero FJ. Recurrence of endometriosis in women with bilateral adnexectomy (with or without total hysterectomy) who received hormone replacement therapy. Fertil Steril 2002; 77:303–308. 33. Toki T, Horiuchi A, Li S-F, Nakayama K, Silverberg SG, Fujii S. Proliferative activity of postmenopausal endometriosis: A histopathologic and immunocytochemical study. Int J Gynecol Pathol 1996; 15:45–53. 34. Kitawaki J, Noguchi T, Amatsu T, Maeda K, Tsukamoto K, Yamamoto T, Fushiki S, Osawa Y, Honjo H. Expression of aromatase cytochrome P450 protein and messenger ribonucleic acid in human endometriotic and adenomyotic tissues but not in normal endometrium. Biol Reprod 1997; 57:514–519. 35. Redwine DB. Endometriosis persisting after castration: Clinical characteristics and results of surgical management. Obstet Gynecol 1994; 83:405–413. 36. Clayton RD, Hawe JA, Love JC, Wilkinson N, Garry R. Recurrent pain after

Management After Surgery

37. 38. 39. 40.

41.

42. 43.

44.

261

hysterectomy and bilateral salpingo-oophorectomy for endometriosis: Evaluation of laparoscopic excision of residual endometriosis. Br J Obstet Gynaecol 1999; 196:740–744. Finan MA, Kwark JA, Joseph GF Jr, Kline RC. Surgical resection of endometriosis after prior hysterectomy. J La State Med Soc 1997; 149:32–35. Anderson J, Behbackht K, De Geest K, Bitterman P. Adenosarcoma in a patient with vaginal endometriosis. Obstet Gynecol 2001; 98:964–966. Judson PL, Temple AM, Fowler WC, Novotny DB, Funkhouer WK. Vaginal adenosarcoma arising from endometriosis. Gynecol Oncol 2000; 76:123–125. Modesitt SC, Tortolero-Luna G, Robinson JB, Gershenson DM, Wolf JK. Ovarian and extraovarian endometriosis-associated cancer. Obstet Gynecol 2002; 100:788–795. Jones KD, Owen E, Berresford A, Sutton C. Endometrial adenocarcinoma arising from endometriosis of the rectosigmoid colon. Gynecologic Oncol 2002; 86:220–222. Vara AR, Ruzics EP, Moussabeck O, Martin DC. Endometrioid adenosarcoma of the bladder arising from endometriosis. J Urol 1990; 143:813–815. Modesitt SC, Tortolero-Luna G, Robinson JB, Gershenson DM, Wolf JK. Ovarian and extraovarian endometriosis-associated cancer. Obstet Gynecol 2002; 788–795. Rossouw JE, Anderson GL, Prentice RL, LaCroix AZ, Kooperberg C, Stefanick ML, Jackson RD, Beresford SA, Howard BV, Johnson KC, Kotchen JM, Ockene J, Writing Group for the Women’s Health Initiative Investigators. Risks and benefits of estrogen plus progestin in healthy postmenopausal women: Principal results From the Women’s Health Initiative randomized controlled trial. JAMA 2002; 288:321–333.

15 Treatment of Ovarian Endometrioma Haya Al-Fozan and Togas Tulandi McGill University Montreal, Quebec, Canada

INTRODUCTION Ovary is a common site of endometriotic cyst or endometrioma. Perhaps the irregular surface of the ovary allows endometrial tissue to burrow into its substance, predisposing the development of ovarian endometrioma. Sampson in 1927 [1] first described the term ‘‘chocolate cyst’’ for ovarian endometrioma. Similar to that of endometriotic implants, the left ovary is more commonly affected than the right [2].

PATHOGENESIS The exact pathophysiology of ovarian endometrioma remains unclear. Brosens [3] postulated that endometrioma develops by invagination of the endometriosis-affected ovarian cortex. Other theories include metaplasia of the coelomic epithelium covering the ovary [4–6] or secondary involvement of functional ovarian cyst [7]. Endometrioma is often associated with pelvic pain and infertility. The diagnosis is suspected on pelvic examination or ultrasound examination. It is 263

264

Al-Fozan and Tulandi

confirmed at the time of surgery and established by histopathological examination.

ENDOMETRIOMA AND IN VITRO FERTILIZATION The impact of endometrioma on the results of in vitro fertilization (IVF) is controversial. Some authors reported that endometrioma adversely affected the pregnancy outcome and suggested that ovarian endometrioma should be removed before IVF treatment [8,9]. Others found no effect of ovarian endometrioma on IVF outcome [10,11]. In a retrospective study, Isaacs et al. [11] evaluated the effects of aspiration of ovarian endometrioma before IVF. They found no difference in peak serum estradiol level, number of mature follicles, number of oocytes, number of embryos transferred, or clinical pregnancies between women with intact endometrioma and those whose endometrioma was aspirated before IVF [11]. Another study found higher rates of oocyte retrieval and clinical pregnancy in women whose endometrioma was aspirated before IVF [12].

ENDOMETRIOMA AND MALIGNANCY Malignancy rarely arises from endometriosis. The frequency of a malignant tumor arising from ovarian endometriosis varies between 0.3% and 0.8%, and most of these tumors are well-differentiated endometriotic carcinomas [13]. Medical Treatment Endometriosis can be treated medically or surgically. Medical treatment consists of estrogen-progestin combination, progestin only, gonadotropinreleasing hormone analog (GnRHa), or danazol. Recently, aromatase inhibitor has also been tried [14]. However, medical treatment for endometrioma is limited. Ovarian endometriomas larger than 1 cm do not respond favorably to medical treatment with hormonal suppression [15]. Also, Muzii et al. [16] reported that the preoperative use of GnRH-a offered no advantage to surgery or to the recurrence rate. Medical treatment is associated with symptomatic improvement in 50% of the patients, but the symptoms tend to recur 6 to 12 months after cessation of therapy [16–18]. It also has no effect on the pregnancy or on the recurrence rates.

Treatment of Ovarian Endometrioma

265

Aspiration of Endometrioma under Ultrasound Guidance Endometrioma can be aspirated under ultrasound or laparoscopic guidance. Drainage under ultrasound guidance is certainly less invasive [12,19–21], and it can be performed in the office setting under local anesthesia. However, the recurrence rate is high (28.5% to 100%) and the procedure carries the risk of infection and adhesions [22]. Garvey et al. [23] reported a case of ultrasound aspiration of bilateral ovarian endometrioma that was followed by severe pelvic adhesions. This could be caused by leakage of the content of the cyst creating inflammatory reaction and adhesions. Nevertheless, ultrasound aspiration of endometrioma might have a place prior to IVF [12]. Ultrasound-Guided Transvaginal Sclerotherapy To overcome the high recurrence rate of aspiration of ovarian endometrioma, Japanese physicians proposed instillation of a high concentration of ethanol (50%) for 5 minutes into the endometriotic cyst wall (ethanol sclerotherapy). Ethanol produces tissue dehydration [24,25]. In other organs such as thyroid, parathyroid, heart, liver and spleen, its efficacy has been demonstrated with a low rate of recurrence of the benign cyst [26]. Leakage of ethanol into the peritoneal cavity, however, can cause chemical irritation and adhesion formation. In theory, ethanol can cause damage to the ovarian cortex and the germ cells [26]. However, in a retrospective study of 45 women treated with transvaginal ethanol sclerotherapy, the rates of pregnancy, term deliveries, abortion, and retrieved oocytes or quality of embryos were similar to those of control women [25]. Systemic acute alcoholism has also been reported [27]. Laparoscopic Aspiration of Endometrioma Aspiration of endometrioma by laparoscopy allows the surgeon to perform a thorough irrigation and removal of its contents. Furthermore, evaluation of the whole abdominal cavity can be done. The diagnosis is more accurate, and the endometriosis can be staged and excised. However, similar to ultrasound drainage of ovarian endometrioma, laparoscopic aspiration is associated with a high recurrence rate. The reported recurrence rate is 21% to 88% at 6 months’ follow-up [28–30]. Donnez et al. [31] studied the effect of gonadotropin-releasing hormone-agonist (GnRH-a) after laparoscopic fenestration and drainage of endometrioma. At second-look laparoscopy 12 weeks later, the size of the

266

Al-Fozan and Tulandi

endometrioma was found to be reduced by 50%. However, all patients subsequently required further surgery for ablation of the cyst wall. Two-Step Eversion Technique Based on the hypothesis that ovarian endometrioma develops as a consequence of invagination of the ovarian cortex and that active endometriosis is often found on the ovarian surface, a two-step laparoscopic technique was proposed [32]. In most cases, the site of the invagination or inversion can be seen as a dimple on the surface of the endometrioma. The first step is biopsy of the endometriotic lesion, adhesiolysis, wide opening of the inversion site, and excision of the endometriotic rings. The second step is performed 2 to 3 months after the first laparoscopy. Laparoscopic adhesiolysis and coagulation of the neovascularization and the endometriotic implants are then performed. In 18 patients, Brosens et al. reported no recurrence [32]. The drawback of this technique is the need for two laparoscopic procedures. Laparoscopic Fenestration and Ablation of the Cyst Wall Several studies have shown that laparoscopic treatment of ovarian endometrioma is as effective or better than that by laparotomy [33,34]. However, the choice of laparoscopic techniques remains controversial [35]. In general, endometrioma is removed by excising the endometriotic cyst. Some authors prefer fenestration followed by ablation of the cyst wall using laser or electrocoagulation. The proponents of this technique believed it is associated with minimal loss of viable ovarian cortex and less adhesion formation. Laparoscopic Stripping or Excision of the Endometriotic Cyst Wall Metter and Semm [36] first performed laparoscopic treatment of endometrioma by removal of endometriotic cyst wall. First the content of the endometrioma is drained. The cyst wall is then stripped from the normal ovarian tissue. The recurrence rate after this procedure is 5% to 12%. All the procedures are done in a single laparoscopic setting. In our practice, we try to enucleate the whole endometrioma intact (Fig. 1). However, it often ruptures. In this case, stripping of the cyst wall is performed (Fig. 2). Results of Laparoscopic Fenestration and Ablation, and Excision There have been several studies on the results of different laparoscopic treatments of endometrioma. Most were observational studies; confounding fac-

Treatment of Ovarian Endometrioma

267

FIGURE 1 Laparoscopic enucleation of endometrioma.

tors of postoperative medical treatment were not taken into consideration and the follow-up period was short [29]. In a retrospective study, the authors concluded that laparoscopic ablation is as effective as excision of the cyst wall [37]. Contrary to their findings, we found that the recurrence rate after excision is significantly lower than that after ablation [38]. This could be attributed to several factors. We studied a large number of patients and one laparoscopic surgeon performed excision. Furthermore, instead of using a crude rate, our results are reported using Life Table Analysis, which takes into account patients who already had reoperation and were lost to follow-up at a given time. Our findings support a randomized prospective study that demonstrates the superiority of excision to fenestration and coagulation. The authors found that the recurrence rates of dysmenorrhea, dyspareunia, and pelvic pain were lower and the pregnancy rate was higher in the excision group than in the fenestration group. The recurrence rate of endometrioma was 6.2% in the excision group and 18.8% in the fenestration group. This is in agreement with our reoperation rate at 18 months’ follow-up. The concern of loss of viable

268

Al-Fozan and Tulandi

FIGURE 2 Laparoscopic stripping or excision of endometrioma (the whitish tissue is the wall of the cyst, the darker folded tissue is the inner side of the ovarian tissue).

ovarian tissue with excision is unfounded. Histopathology of the excised tissue in our series revealed absence of follicles in all specimens. Ovarian Response after Conservative Laparoscopic Surgery Some investigators feel that ovarian surgery might have a deleterious effect on the residual normal ovarian cortex. However, in a retrospective study of 820 cycles [39], the outcome of IVF-ET after ablation of ovarian endometrioma was similar to that of tubal factors. The clinical pregnancy rate was 37.4% in the endometrioma group and 34.6% in the tubal-factor group. The number of follicles and the number of mature oocytes were similar between the two

Treatment of Ovarian Endometrioma

269

groups. More importantly, the previously operated ovary had a similar response to the nonoperated contralateral ovary [39]. In a smaller series, Marconi et al. [40] reported similar findings.

SUMMARY Ovarian endometriomas larger than 1 cm do not respond favorably to medical treatment. Aspiration of endometrioma either by ultrasound or laparoscopy leads to a high recurrence rate in a short time. However, endometrioma aspiration before in vitro fertilization might be beneficial to the IVF outcome. The best surgical treatment of ovarian endometrioma is laparoscopic excision of the cyst wall. The procedure is technically more demanding than laparoscopic fenestration and ablation of the cyst wall, but is associated with prolonged symptomatic improvement, a lower recurrence of pelvic pain, and a lower reoperation rate. The pregnancy rate after excision is also higher. Concerns that excision of the endometrioma will result in more adhesion formation and the loss of ovarian follicles are not supported by the results of a randomized study. Studies to date have shown that laparoscopic excision of ovarian endometrioma results in a lower reoperation rate, a lower recurrence of symptoms, and a better improvement of pain compared with the ablation technique.

PRACTICAL POINTS 

Ovarian endometrioma larger than 1 cm do not respond to medical treatment.  Aspiration of endometrioma either by ultrasound or laparoscopy leads to a high recurrence rate in a short time.  Laparoscopic excision of the cyst wall is associated with prolonged symptomatic improvement, a lower recurrence of pelvic pain, a lower reoperation rate, and a higher pregnancy rate than after fenestration and ablation.

REFERENCES 1.

Sampson JA. Peritoneal endometriosis due to menstrual dissemination of endometrial tissue in to the peritoneal cavity. Am J Obstet Gynecol 1927; 14:422– 469.

270 2. 3. 4.

5.

6. 7.

8.

9.

10.

11.

12.

13. 14.

15. 16.

17. 18. 19.

Al-Fozan and Tulandi Al-Fozan H, Tulandi T. Left lateral predisposition of endometriosis and endometrioma. Obstet Gynecol 2002; 101:164–166. Brosens J, Puttemans P, Deprest J. Appearance of endometriosis. Bailliere’s Clin Obstet Gynecol 1993; 7:741–757. Nisolle M, Donnez J. Peritoneal endometriosis, ovarian endometriosis, and adenomyotic nodules of the rectovaginal septum are three different entities. Fertil Steril 1997; 68:585–596. Meyer R. Uber den stand der frage der adenomyositis und adenomyome im allgemeinen und insbesondere uber adenomyositis seroepithelialis und adenomyometritis sarcomatosa. Zentralbl Gynakol 1919; 36-:754. Meyer R. Zur Frage der Urnieren-Genese von Adenomyomen. Zentralbl Gynakol 1923; 15:577–587. Nezhat F, Nezhat C, Allan CJ, Metzger DA, Sears DL. Clinical and histologic classification of endometriosis implantation for a mechanism of pathogenesis. J Reprod Med 1992; 37:771–776. Dlugi AM, Loy RA, Dieterle S, Bayer S, Seibel M. The effect of endometriomas on in vitro fertilization outcome. J In-Vitro Fertil Embryo Transfer 1989; 6: 338–341. Wardle PG, McLaughlin EA, McDermont A, Mitchell JD, Ray BD, Hull MGR. Endometriosis and ovulatory disorder. Reduced fertilization in vitro compared with tubal and unexplained infertility. Lancet 1985; 2:236–239. Olivennes F, Feldberg D, Liu HC, Cohen J, Moy F, Rosenwaks Z. Endometriosis: A stage by stage analysis—the role of in vitro fertilization. Fertil Steril 1995; 64:392–398. Isaacs JD, Hines RS, Sopelak VM, Cowan BD. Ovarian endometriomas does not adversely affect pregnancy success following treatment with in vitro fertilization. J Assist Reprod Genet 1997; 12:2282–2285. Dicker D, Goldman JA, Feldberg D, Ashkenazi J, Levy T. Transvaginal ultrasonic needle-guided aspiration of endometriotic cysts before ovulation induction for in vitro fertilization. J In-Vitro Fertil Embryo Transfer 1991; 8:286–289. Heaps JM, Nieberg RK, Berek JS. Malignant neoplasm arising in endometriosis. Obstet Gynecol 1990; 75:1023–1028. Vignali M, Infantino M, Martrone R, Vignali M, Infantino M, Matrone R, Chiodo I, Semolina E, Busch M, Viand P. Endometriosis: Novel etiopathogenetic concept and clinical perspectives. Fertil Steril 2002; 78:665–678. Shaw RW. The role of GnRH analogues in the treatment of endometriosis. Br J Obstet Gynecol 1992; 99:9–12. Muzii L, Marana R, Carina P, Mancuso S. The impact of preoperative gonadotropin releasing hormone agonist treatment on laparoscopic excision of ovarian endometriotic cysts. Fertil Steril 1996; 65:1235–1237. Scheme RS. Gonadotropin-releasing hormone analogs in the treatment of endometrioma. Am J Obstet Gynecol 1990; 162:579–581. Shaw RT. Treatment of endometriosis. Lancet 1992; 340:1267–1271. Aboulghar MA, Mansour RT, Serour GI, Rizk B. Ultrasonic transvaginal aspiration of endometriotic cysts: an optional line of treatment in selected cases of endometriosis. Hum Reprod 1991; 6:1408–1410.

Treatment of Ovarian Endometrioma

271

20. Zanetta G, Lissoni A, Valle CD, Trio D, Pittelli M, Rangoni G. Ultrasoundguided aspiration of endometriomas: Possible applications and limitations. Fertil Steril 1995; 64:709–713. 21. Giorlandino C, Taramanni C, Muzii L, Santillo E, Nanni C, Vizzone A. Ultrasound-guided aspiration of ovarian cysts. Int J Gynecol Obstet 1993; 43:41–44. 22. Muzii L, Marana R, Caruana P, Catalano GF, Mancuso S. Laparoscopic findings after transvaginal ultrasound-guided aspiration of ovarian endometriomas. Hum Reprod 1995; 10:2902–2903. 23. Garvey T, Kazer R, Milad M. Severe pelvic adhesions following attempted ultrasound-guided drainage of bilateral ovarian endometriomas: Case report. Hum Reprod 1999; 14:2748–2750. 24. Noma J, Yoshida N. Efficacy of ethanol sclerotherapy for ovarian endometriomas. Int J Gynecol Obstet 2001; 72:35–39. 25. Koike T, Minakami H, Motoyama M, Ogawa S, Fujiwara H, Sato I. Reproductive performance after ultrasound-guided transvaginal ethanol sclerotherapy for ovarian endometriotic cysts. Eur J Obstet Gynecol 2002; 1005:39–43. 26. Okagaki R, Osuga Y, Momoeda M, Tsutsumi O, Taketani Y. Laparoscopic findings after ultrasound-guided transvaginal ethanol sclerotherapy for ovarian endometrial cyst. Hum Reprod 1999; 14:270. Letters to the editor. 27. Tei A, Ueki M, Yokono S, Ogli K. Acute alcoholism after ethanol fixation for ovarian chocolate cyst. Masui-Jap J Anesthesiol 1996; 45:496–499. 28. Marana R, Caruana P, Muzii L, Catalano GF, Mancuso S. Operative laparoscopy for ovarian cyst: Excision versus aspiration. J Reprod Med 1996; 41: 435– 438. 29. Fayez J, Vogel M. Comparison of different treatment methods of endometriosis by laparoscopy. Obstet Gynecol 1991; 78:660–665. 30. Vercellini P, Vendola N, Bocciolone L, Colombo A, Rognoni M, Bolis G. Laparoscopic aspiration of ovarian endometrioma: Effect of postoperative gonadotropin releasing hormone agonist treatment. J Reprod Med 1992; 37:577–580. 31. Donnez J, Nisolle M, Gillerot S, Anaf V, Clerckx-Braun F, Casanas-Roux F. Ovarian endometrial cyst: The role of gonadotropin-releasing hormone agonists and/or drainage. Fertil Steril 1994; 62:63–66. 32. Brosens I. Management of ovarian endometriomas and pregnancy. Fertil Steril 1999; 71:1166–1167. 33. Catalano GF, Marana R, Caruana P, Muzzi L, Mancuso S. Laparoscopy versus microsurgery by laparotomy for excision of ovarian cyst in patients with moderate or severe endometriosis. J Am Assoc Gynecol Laparosc 1996; 3:267– 270. 34. Milingos S, Loutradis D, Kallipolitis G, Liapi A, Drakakis P, Antsaklis A, Michalas S. Comparison of laparoscopy with laparotomy for the treatment of extensive endometriosis with large endometriomata. J Gynecol Surg 1999; 15: 131–136. 35. Jones KD, Sutton CJG. Endometriotic cysts: The case for ablative laparoscopic surgery. Gynaecol Endosc 2002; 10:1–8. 36. Metter L, Semm K. Three steps medical and surgical treatment of endometriosis. Isr J Med Sci 1993; 152:204.

272

Al-Fozan and Tulandi

37. Hemmings R, Bissonnette F, Bouzayen R. Result of laparoscopic treatments of ovarian endometriomas: Laparoscopic ovarian fenestration and coagulation. Fertil Steril 1998; 70:527–529. 38. Saleh A, Tulandi T. Reoperation after laparoscopic treatment of ovarian endometrioma by excision and by fenestration. Fertil Steril 1999; 72:322–324. 39. Donnez J, Wyns C, Nisolle M. Does ovarian surgery for endometriomas impair the ovarian response to gonadotropin? Fertil Steril 2001; 76:662–665. 40. Marconi G, Vilela M, Quintana R, Sueldo C. Laparoscopic ovarian cystectomy of endometrioma does not affect the ovarian response to gonadotropin stimulation. Fertil Steril 2002; 78:876–878.

16 Treatment of Endometriosis-Related Pelvic Pain Kevin Jones Great Western Hospital Swindon, Wiltshire, England

Christopher Sutton Royal Surrey County Hospital Guildford, Surrey, England

INTRODUCTION Painful symptoms of gynecological origin are most commonly due to endometriosis. Patients may present with chronic, nonmenstrual pelvic pain, dyspareunia, and dyschezia as well as cyclical menstrual pain, dysmenorrhea. These symptoms can be managed medically or surgically. The surgical management may be non conservative, where a total abdominal hysterectomy and bilateral salpingo-oophorectomy is performed, or conservatively, where the endometriotic deposits are ablated or excised laparoscopically leaving the reproductive organs in place. This may be combined with a denervation procedure to relieve painful symptoms. Three denervation procedures have been described: laparoscopic uterine nerve ablation, arcus taurinus, and presacral neurectomy. The objective of this chapter is to review the treatment 273

274

Jones and Sutton

of painful symptoms resulting from endometriosis and to discuss the controversies surrounding the management strategies.

EPIDEMIOLOGY The main limitation in trying to accurately measure the true prevalence of this disease is that the definitive diagnosis may only be made by laparoscopic examination. The variable appearance of endometriotic lesions at laparoscopy and the ability of gynecologists to recognize these lesions affect the reported incidence. Access to health care, to contraception, cultural patterns of childbearing, and the attitude toward menses also affects the estimated prevalence. The incidence of endometriosis also varies depending on the type of hospitalbased population being investigated. For example, the diagnosis of endometriosis in patients with pelvic pain ranges from 15% [1] to 71% [2], and in women with pelvic pain and infertility, it is as high as 84% [2]. Despite these limitations, population-based studies have been carried out in the United States and the UK, which have estimated the incidence and prevalence of chronic pelvic pain (CPP). An opinion poll-style telephone survey of women aged 18 to 50 years was carried out in the United States [3]. Pelvic pain for longer than 6 months was reported in 925 of 5325 (17.4%) women. After excluding participants who were pregnant or postmenopausal, and those with cyclical pain, 773 of 5263 (14.7%) CPP sufferers were identified. In the UK, a postal survey was conducted [4]. Women aged 18 to 49 years were randomly selected from the Oxfordshire Health Authority register, and those with CPP of longer than 6 months’ duration unrelated to menstruation, intercourse, or pregnancy were identified. The reported prevalence of CPP was 483 of 2016 (25%). Chronic pelvic pain represents a substantial public health care issue, which is frequently underreported. Among the 483 Oxfordshire women with CPP, 195 (40%) had not sought a medical consultation, 127 (26%) reported a past consultation and 139 (29%) reported a recent consultation for pain [4]. Even when CPP is reported, it is often left untreated or underinvestigated. Data from a national database recording contacts with family doctors found that 28% of women with CPP were not given a specific diagnosis and 60% were not referred to hospital [5]. The true incidence of dysmenorrhea and dyspareunia is also difficult to asses. In the group of women with CPP, dysmenorrhea was also reported by 81% and dyspareunia in 41%. Among women who did not have CPP in the Oxford study [4], dysmenorrhea was reported by 58% women and dyspareunia by 14%. Perhaps the best epidemiological study comes from a general practice survey carried out in Sweden. All 19-year-old girls from the town of Gothe-

Treatment of Pelvic Pain

275

burg were sent a questionnaire to investigate the prevalence of dysmenorrhea. The response rate was 90%, which is exceptionally high for such a survey [6]. As many as 73% of the young girls suffered from primary dysmenorrhea and 15% had severe dysmenorrhea that affected their working ability and could not be controlled adequately by analgesics or ovarian suppression. No studies have investigated the epidemiology of dyspareunia or dyschezia specifically.

MEASURING PELVIC PAIN Quantitative studies of pelvic pain are problematic because pain is a subjective phenomenon, and it is extremely easy for the investigator to influence the outcome of investigations where it is an endpoint. This is especially true if a subjective assessment of pain rather than an objective measure, such as a visual analogue score, is used. Pelvic pain is also a global term, made up of components such as dysmenorrhea, dyspareunia, dyschezia, and chronic nonmenstrual elements and it may have nongynecological causes, including psychological problems [7]. Whether a woman with CPP proves to have endometriosis or no evident disease, there is a high likelihood of a concomitant mood disorder [8]. Furthermore, studies that do not exclude sexually inactive and amenorrhoeic women when the assessment of dysmenorrhea and dyspareunia are made will tend to overreport positive results. Visual Analogue Scores There are many different methods for the measurement of chronic pain [9,10]. However, the most commonly used and well-validated technique for quantifying pelvic pain is a linear analogue score [11–13]. This involves the use of a 10-cm line on a piece of white paper, which represents the continuum of the patients’ opinion of the degree of pain. It is explained to the patient that the one extremity of the line represents ‘‘as much pain as she can possible imagine,’’ whereas the other represents ‘‘no pain at all’’. The patient rates the degree of pain by making a mark on the line. Scale values are then obtained by measuring the distance from zero to that mark. The individual scores for different pain symptoms can then be compared before and after treatment. The one disadvantage of this scoring system is that an especially painful incident can skew the result. Quality of Life Instruments Using quality of life measures (QOL) in clinical practice ensures that treatment focuses on the patient rather than the disease [14]. These psychometric instruments are increasingly being used in studies designed to assess treatment for pelvic pain caused by endometriosis. Quality of life indicators for patients

276

Jones and Sutton

with pelvic pain have been validated in a national audit [15] and for patients with dysmenorrhea [16], and to measure outcome after conservative surgery for stage III to IV endometriosis [17]. The impact of CPP on health-related QOL has been assessed by use of the SF-36 questionnaire in a populationbased study in Oxfordshire, UK [4] and among women referred by family doctors to gynecology clinics [18]. The SF-36 has eight subscales that can be compressed into two summary scales—the mental component and the physical component. It is best used to detect trends in mental and physical QOL. Staging Endometriosis and Painful Symptoms The r-AFS score [19] classification of endometriosis divides the disease into stages from minimal to severe (stages I to IV). It is generally agreed that the rAFS classification of disease severity is only of use as a prognostic index with regard to potential infertility. The disease stage does not correlate with the severity of painful symptoms [20,21]. However, the evidence is conflicting because other groups have suggested that endometriosis stage is directly related to the persistence of pelvic pain despite medical or conservative surgical therapy [22]. It is interesting to note that the r-AFS scores at laparoscopy were found to be significantly related to self-assignment into pain or no-pain groups; however, the extent of physical disease was not correlated with ratings of pain levels using standardized measures of behavioral and psychosocial factors [23]. Therefore, how pain is measured is of critical importance when interpreting clinical studies.

BASIC SCIENCE To understand how the treatment of pelvic pain resulting from endometriosis may be effective, it is necessary to understand the underlying etiology and pathophysiology of the common symptoms. Anatomy Pelvic pain is transmitted by the sympathetic and parasympathetic nerves to the pelvic organs. The sympathetic fibers leave the lumbar segments (L1–4) of the spinal cord, and the majority of these fibers form the hypogastric (presacral) plexus, which crosses the sacral promontory and divides into two major pelvic nerve bundles to form the deep pelvic plexus before innervating different pelvic organs. The parasympathetic fibers enter the pelvis through the sacral nerves (S2–S4) and travel along the pelvic nerves (nerve eregentes) to reach the Lee-Frankenhauser plexus in the anterior two-thirds of utero-

Treatment of Pelvic Pain

277

sacral ligaments and its attachment to the cervix. Selective division of these nerve pathways is carried out to relieve painful symptoms. Pathophysiology In the early stage of endometriosis, the release of mediators such as prostaglandins, bradykinins, interleukins, and inflammatory products of macrophages from the endometriotic implants cause painful symptoms, which alter the receptive properties of the nociceptor in the pelvis [24,25]. In the advanced stages, infiltrating endometriosis results in a direct mechanical compression of the nociceptors, particularly around the uterosacral ligaments [2,26]. In addition, the fibrosis, muscular hyperplasia, and scarring around the endometriotic implant can induce ischemic changes resulting in pain. C-type pain receptors, found in the peritoneum and viscera, are activated by chemical and mechanical stimuli. Endometriotic deposits that invade deeply into the underlying tissues may cause mechanical disruption of these fibers, leading to pain, or the surrounding local inflammatory response could affect receptor sensitivity. Ablation or excision of the endometriotic deposits would eliminate the mechanical activation of the receptors from infiltrating disease. They would also remove the stimulus for production of pain-mediating chemicals. The degree of stimulation of pain receptors would decrease, with relief of painful symptoms. Histopathology Deep retroperitoneal endometriosis causes pelvic pain [26], and the intensity of the pain is related to the depth to which the lesion penetrates [2]. However, there is evidence that even when there is no visible evidence of endometriosis at laparoscopy, microscopic deposits can be observed histological [27–29]. Deeply infiltrating endometriosis often involves the uterosacral ligaments [2,30,31]. Histological examination of 32 uterosacral ligament biopsies taken from patients with negative laparoscopies found that 6% of patients had endometriosis [27]. In another study, 15 patients had bilateral uterosacral ligament resection regardless of whether there was clinical suspicion of uterosacral ligament invasion by endometriotuc tissue [32]. Eight patients (54%) had histological involvement of the uterosacral ligaments, and all these patients had complete relief of their symptoms. It would appear that retroperitoneal muscular hyperplasia is responsible for much of the pain in these women. In a series of 51 women treated by radical excision of the uterosacral ligament complex, pain relief occurred in 46 of 50 (92%) with dysmenorrhea and 47 of 51 (92%) with dyspareunia; only 50% showed endometrial glands and stroma, the remainder showing fibromuscular hyperplasia alone [33].

278

Jones and Sutton

NONSURGICAL TREATMENT Symptomatic relief of pelvic pain may be achieved pharmacologically or with ‘‘alternative’’ medical therapies. Patient support groups and multidisciplinary teams also have an important role to play in the management of endometriosis sufferers. A detailed discussion of the medical treatment of endometriosis is given elsewhere in this book, and an overview of complementary therapies and the multidisciplinary approach to CPP is presented in this section.

Medical Treatments The introduction of the oral contraceptive pill in the early 1960s and the widespread use of nonsteroidal anti-inflammatory drugs revolutionized the treatment of dysmenorrhea. The development and introduction of danazol, gestrinone, progestagens, and, later, gnRH analogues, allowed menstruation to be abolished. This provided relief from pelvic pain and dysmenorrhea, particularly in cases associated with endometriosis [34]. Recently, aromatase inhibitors have been used to treat pelvic pain resulting from endometriosis [35,36]. All the drugs used to treat endometriosis are associated with unpleasant side effects, and the endometriosis tends to recur after the cessation of therapy [34]. Furthermore, they are contraceptive, and a significant proportion of the patients with pelvic pain are trying to conceive. Because of this, surgery is now regarded as the preferable first line treatment of endometriosis. However, it is worth noting that a course of 3 to 6 months of postoperative treatment with a GnRH has been shown to prolong the pain-free interval after conservative surgery for endometriosis, but failed at 1 to 2 years postoperatively [37]. A recent consensus statement from the chronic pelvic pain/ endometriosis working group in the United States, an expert panel composed of practicing gynecologists and experts in consensus in guide development, have issued a statement that is at variance with this [38]. They suggest that if endometriosis is the suspected cause of pelvic pain, laparoscopic confirmation of the diagnosis is unnecessary and a trial of medical therapy, including second line therapy such as danazol, GNRH antagonists, and progestins, is justified, providing there are no other indications for surgery such as the presence of a suspicious adnexal mass. This recommendation is in complete opposition to current clinical practice, which suggests that laparoscopy is the ‘‘gold standard’’ for the diagnosis of endometriosis, and laparoscopic surgery should be performed under the same anesthetic to eradicate all visible and palpable endometriotic disease either with a CO2 laser [39], electrosurgery and sharp dissection [40], or the ultrasonic scalpel. The end results of surgery are similar regardless of the energy source used and depend more on the experience and preference of the surgeon and careful patient selection [41].

Treatment of Pelvic Pain

279

In a randomized controlled trial conducted in the United States, women treated postoperatively with GNRH analogues had a better outcome than those treated by surgery alone plus placebo, and the effect lasted for up to 12 months [42]. The abolition of menses in the analogue-treated group would inevitably bias the results. Nevertheless a further study by Winkel and Bray [43] followed up 240 women with endometriosis and chronic pelvic pain over 2 years. They underwent excision alone, laser ablation alone, or laser ablation followed by leuprolide acetate for 3 to 6 months. In this nonrandomized trial, only 23% of the ablation group was pain-free at 24 months, whereas 70% of the ablation plus GNRH analogue-treated group remained pain-free after the same time interval [43]. Alternative Medical Treatments and Support Groups Acupuncture can relieve dysmenorrhea, but responses to treatment are variable. Relaxation can be achieved with massage, reflexology, aromatherapy, meditation, hypnotherapy, yoga, and tai chi, and these therapies may help to relieve muscle tension that increases pain. Naturopaths and nutritional therapists recommend a diet rich in omega-3 fatty acids, found in oily fish. Several herbs are traditionally used to relieve uterine and pelvic pain. These include black cohosh, dong quai (Chinese angelica), ginger, pulsatilla, chamomile, cramp bark, and wild yam. Medical herbalists may also include herbs believed to reduce menorrhagia like goldenseal, witch hazel, and dandelion root or milk thistle. For women who have not responded to ‘‘conventional’’ medical and surgical treatment, further management demands strategies beyond the capacity of a general gynecology clinic. Pain clinics offer a multidisciplinary approach and tend to focus on functional capacity and the provision of continuity of care. Services include direct pain-relieving interventions such as tricyclic antidepressants, anticonvulsants, or nerve blockade, as well as cognitive behavioral psychotherapy, physiotherapy, nursing support, and the use of complementary treatments. This ‘‘holistic’’ approach has been shown to be effective in a randomized trial [44]. There are also a number of patient selfhelp groups that play an important part in supporting endometriosis suffers. The National Endometriosis Society and the Endometriosis Association are two examples.

SURGICAL TREATMENT The surgical management of endometriosis may be nonconservative, in which a total abdominal hysterectomy and bilateral hysterectomy (TAH/BSO) is performed or conservative, in which the endometriotic deposits are ablated or excised laparoscopically, leaving the reproductive organs in place. These may

280

Jones and Sutton

be described as debulking operations, which may be combined with a denervation procedure to relieve painful symptoms. Three denervation procedures have been described, laparoscopic uterine nerve ablation (LUNA), arcus taurinus (AT), and presacral neurectomy (PSN). Debulking Operations Endometriotic implants may be removed from the pelvis by surgical excision or they may be ablated. Both excision and ablative surgery can be achieved using lasers or electrosurgical devices. Three areas of the pelvis are typically involved in the disease: the pelvic peritoneum, the rectovaginal septum, and the ovary. Endometriosis frequently coexists at all these sites, and different etiologies have been postulated for each. Peritoneal Endometriosis Endometriotic implants can occur throughout the pelvic peritoneum. They have variable appearances [45] and are typically superficial. When endometriosis is limited to the peritoneal surfaces, the r-AFS score is generally less than 40 (minimal–moderate disease). Both the CO2 laser and bipolar electrosurgery can be used to ablate these superficial deposits, which may also be excised with scissors and monopolar electrosurgery. By the early 1980s, it was widely reported that some 60% to 70% of patients experienced pain relief and amelioration of dysmenorrhea and dyspareunia with the ablation of the peritoneal endometriotic implants [46–51]. However these were uncontrolled studies. To obtain grade Ib evidence (Table 1), the Guildford Laser Laparoscopy Trial [52] was carried out. Seventy-four women were recruited, and at the time of laparoscopy were randomly allocated to laser treatment or expectant management. The laser

TABLE 1

Classification of Evidence Levels

Ia

Evidence obtained from meta-analysis of randomized controlled trials. Evidence obtained from at least one randomized controlled trial. Evidence obtained from at least one well-designed controlled study without randomization. Evidence obtained from at least one other type of well-designed quasi-experimental study. Evidence obtained from well-designed nonexperimental descriptive studies, such as comparative studies, correlation studies, and case studies. Evidence obtained from expert committee reports or opinions and/or clinical experience of respected authorities.

Ib IIa IIb III

IV

Treatment of Pelvic Pain

281

treatment included vaporization of all visible endometriotic implants, adhesiolysis, and uterine nerve transection. The patients in the sham arm had exactly the same incisions, but merely had a diagnostic laparoscopy. Patients were not informed of which treatment group they had been allocated to, and were followed up at 3 months and 6 months after surgery by an independent observer. Of 74 women who entered the study, 63 (32 laser, 31 expectant) completed the study to the 6-month follow-up visit. At 3 months after operation, there was very little difference between the two groups, but at 6 months the difference reached statistical significance and 62.5% of the patients who had the laser treatment had sustained pain relief. Only 22.6% of the patients who had no treatment said they were better. Patients who had not received any treatment but had an established diagnosis were followed up for 6 to 12 months, and the findings of second-look laparoscopies were reported, together with a comparison of the changes in their symptoms [53]. At second look laparoscopy, 10 (42%) patients had no change in the AFS score and (29%) patients had an increased score. One third of the patients (seven cases) had a reduced r-AFS score. These patients had improved or resolved symptoms, whereas in the others the symptomatology was the same or had become more severe. This study suggests that the disease does progress in the majority of patients, but in up to a third it can regress, and in a few of them it disappeared altogether. A long-term follow-up study of the cohort of patients who underwent laser laparoscopy has been reported [54]. Of the 56 laser-treated patients, 38 (67.9%) were contacted. At the time of follow-up, satisfactory symptom relief was reported in 21 of 38 (55.3%) patients. Rectovaginal Endometriosis Rectovaginal endometriosis with obliteration of the cul de sac involves the uterosacral ligaments, posterior cervix, frequently the rectovaginal septum, and the anterior bowel wall. Different surgical approaches to the laparoscopic management of rectovaginal endometriosis have been described. Donnez et al [55] have described a surgical technique based on the assumption that endometriotic involvement of the rectal muscularis or mucosa does not occur. Their technique is to stay within the uterosacral ligaments and dissect the cervix free from the rectum. Once the rectum is free, the vaginal adenomyoma is removed transvaginally and the defect sutured. Nezhat et al [56] and Reich et al [57] have reported laparoscopic dissection and partial proctectomy using a stapling technique. Redwine [58] describes radical en bloc dissection using unipolar electrodiathermy delivered through 3-mm scissors at high settings (90 watt cutting, 50 watt coagulation). Only when the rectum is mobilized completely and free along its length is the endometriotic area excised. Other groups have also reported variations of these techniques for excising [2,17,59] or ablating [60] deeply infiltrating endometriotic implants.

282

Jones and Sutton

There are currently no randomized controlled trials comparing surgery for severe endometriosis (stage IV) with placebo; therefore, we have to rely on IIa or b, III, and IV evidence (Table 1). In a series of 130 women [61] who had undergone aggressive surgical management by laparotomy of advanced colorectal endometriosis including low anterior resection, sigmoid resection, disc excision of the rectal wall, or right hemicolectomy, 90% of the patients reported good relief of their pelvic symptomatology. Resection of the uterosacral ligament at laparotomy has also been reported [32]. In this study, dysmenorrhea, dyspareunia, and dyschezia were relieved in 12 of 15 (80%), 7of 12 (58%), and 8 of 11 (77%) patients, respectively. Eight patients (54%) had histological involvement of the uterosacral ligaments, and all these patients had complete relief of their symptoms. However, a presacral neurectomy was also carried out in 12 of 15 (80%) of these patients. Therefore, this study was not able to separate the effect of debulking the endometriosis from radical denervation. A number of reports in the literature have documented the relief of painful symptoms following laparoscopic surgery for severe disease (r AFS score >40) [17,57,59,62–65]. Some of the studies concerned with stage III– IV endometriosis use pain questionnaires for analyses of dysmenorrhea, dyspareunia, and chronic nonmenstrual pain [17,64,65], and one group applied quality of life indicators to assess outcome as well [17]. Laparoscopic resection of deep endometriosis located in the uterosacral ligaments has also been described [59,66,67]. These authors are reporting the results of excision of the uterosacral ligaments rather than vaporization with the carbon dioxide laser. Laparoscopic bilateral uterosacral ligament resection with excision of all other endometriotic lesions was carried out in 21 patients [66]. Dysmenorrhea, dyspareunia, and chronic nonmenstrual pain were relieved in 16 of 19 (84%), 16 of 17 (94%), and seven of nine (77.7%) patients, respectively. The same group of authors reported a larger series of 85 patients undergoing the same operation [61]. In this study, dysmenorrhea and dyspareunia, were relieved in 46 of 50 (92%) and 47 of 51 (92%) patients, respectively. Furthermore, the efficacy of treatment did not differ according to whether the histological results were positive or negative. The r-AFS score or disease stage did not correlate consistently with the frequency and severity of painful symptoms either. The latest report of 110 patients from the same group continues to support the previous findings [59]. The efficacy of resecting the uterosacral ligament in uncontrolled studies has, therefore, been established. Only 50% of the excised specimens in these studies showed histological proof of endometriosis, and excellent pain relief can be obtained by removal of the fibromuscular hyperplasia; therefore, we have used the CO2 laser to vaporize the abnormal tissue rather than undertake the more lengthy pro-

Treatment of Pelvic Pain

283

cedure such as en-bloc excision with sharp dissection or needle electrosurgery. The CO2 laser has the ability to precisely vaporize tissue with the debris removed by suction in the laser plume or any residual carbon flushed away by the irrigation fluid. We use heparinized Ringers lactate solution at 40jC and leave one liter of 3% Icodextrin (Adeptk, Shire Pharmaceuticals, UK) at the end of the procedure to prevent adhesion formation. In fact, postoperative adhesions are rare after laser vaporization in the rectovaginal septum and uterosacral ligaments, but are more likely when the ovaries are involved. Vaporization must be very thorough and should continue until all the fibromuscular hyperplasia, endometrial glands, and stroma have been removed and there is no further release of hemosiderin. Sometimes it is necessary to continue vaporization to a considerable depth to achieve complete clearance of diseased tissue, but it is easy to see and palpate the endpoint when normal soft retroperitoneal fat has been reached. If the endometriotic nodule extends through to the vagina, the operation is completed by the CO2 laser transmitted by the colposcope, taking care to avoid injury to the rectum [68]. Our preoperative workup includes an air contrast barium enema and vaginogram looked at in the lateral view by an experienced radiologist. If there is evidence of a full thickness penetration, we prefer to advise that the patient undergoes an anterior bowel resection by laparotomy carried out by a colorectal surgeon. We followed up a cohort of 78 women with deep infiltrating endometriosis treated between September 1999 and August 2000. The mean age was 34 (20–51) years. The women were complaining of dysmenorrhea (82%), dyspareunia (88%), dyschezia (64%), and cyclical rectal bleeding (18%). Twelve (15%) had laser laparoscopy to the rectovaginal septum alone and 12 (15%) had vaporization to the uterosacral complex alone. Forty-eight (61%) had the arcus taurinus (uterosacral ligaments and rectovaginal septum) and six (9%) had anterior resection by laparotomy. At 12 months’ follow-up, 72 (92%) were pain-free, and six (8%) were not better. The length of the operation was 32 to 80 minutes (mean 48 minutes), and there were no serious complications, rectal perforations, or ureteric or bladder damage. Endometriotic Cysts The choice of which laparoscopic technique to use in the surgical management of endometriotic cysts remains controversial [69]. There are two main schools of thought. The cyst capsule may be excised (stripped out) or ablated (vaporized) with a laser, or coagulated with bipolar diathermy. A number of reports in the literature document the relief of painful symptoms after endoscopic surgery specifically on patients with endometrio-

284

Jones and Sutton

mas [70–74]. These represent IIa or b, III, and IV evidence (Table 1). There is one randomized, controlled trial comparing excision with ablation of endometriotic cysts, grade Ib evidence [75]. We have only been able to identify two studies of pain relief after laparoscopic surgery for endometriomas that analyze dysmenorrhea, dyspareunia, and chronic nonmenstrual pain individually using visual analogue scores [75,76]. In the randomized, controlled trial of Beretta et al, visual analogue scores were used to assess three types of pelvic pain during the followup period of 24 months [75]. This study reported the 24-month cumulative pain recurrence rates after surgery. These were 15.8% versus 52.9% for dysmenorrhea, 20% versus 75% for dyspareunia, and 10% versus 52.9% for chronic nonmenstrual pain in the excision and ablation groups, respectively. The results of the Guildford study [76] are better than those reported by Beretta et al [75] for their ablation group. There may be a number of reasons for this, including the surgical technique, instrument used for ablation, and cyst recurrence rate. A number of other papers have reported pain relief after laparoscopic surgery for endometriomas [70–74]. Unfortunately all these studies lack an objective measurement of pain, such as a visual analogue score. We would argue that these authors are reporting an expression of ‘‘patient satisfaction’’ with the procedure, rather than quantifiable, objective measurements of change. In the Guildford study [76], we found that 64 (87.7%) of our patients were satisfied or very satisfied with the treatment at 12 months, and this compares favorably with these other studies. Denervation Operations There are three denervation procedures described in the literature. They may be used in isolation to achieve relief of painful symptoms or as part of a debulking procedure. The denervation procedures have been the subject of prospective cohort studies, grade IIa or IIb, III, IV evidence (Table 1), and randomized controlled trials, as well as meta-analysis, grade Ia and Ib evidence (Table 1). Laparoscopic Uterine Nerve Ablation In 1954 Joseph Doyle from Massachusetts described the procedure of paracervical uterine denervation by transection of the uterosacral ligaments about 1 cm from their insertion into the posterior aspect of the cervix. He carried out this procedure in an attempt to interrupt the sensory parasympathetic fibers to the cervix and some of the sensory sympathetic fibers to the corpus contained in the cervical division of the Lee-Frankenhauser plexus. Doyle

Treatment of Pelvic Pain

285

reported an 86% success rate for complete pain relief from dysmenorrhea and a 95% success rate for complete or partial relief of pelvic pain in 73 patients followed up from 4 months to 4 years postoperatively [78,79]. In order to carry out a laparoscopic uterine nerve ablation (LUNA) procedure, the posterior leaves of the broad ligament are first examined to identify the course of the ureters and pelvic blood vessels. The uterosacral ligaments can be vaporized near the point of their attachments to the posterior aspect of the cervix with a CO2 laser. A crater about 2 cm in diameter and 1 cm deep is formed. The ligaments can also be excised using a combination of electrosurgery and sharp dissection [80]. Arcus Taurinus A refinement of the LUNA procedure is to laser the posterior aspect of the cervix between the insertions of the ligaments to interrupt fibers crossing to the contra lateral side [81]. If the crater between the uterosacral ligaments is extended, the rectovaginal septum is opened, and all abnormal tissue vaporized, including the entire uterosacral ligament complex, it becomes an arcus taurinus (AT), or ‘‘bull’s horn’’ procedure [82]. By definition, radical excisional or ablative surgery for endometriosis in the pouch of Douglas or rectovaginal septum includes the AT procedure [83–86]. The efficacy of LUNA in uncontrolled studies has been reviewed [80,81,85]. The Guildford study [85] found that 16 of 26 (73%) patients improved after a LUNA procedure. However, 15 of 18 (83%) of those having a complete procedure improved, compared to only 1 of 4 (25%) of those having an incomplete procedure. Furthermore, if the patients are undergoing laser vaporization of endometriosis at the same time [85], 86% of the patients improve. Similar results ranging from 75% to 87% have been reported by other workers [50,81]. In the same study, 26 of 35 (74%) women received a partial LUNA and reported an improvement in their symptoms [85]. The LUNA procedure has also been evaluated in prospective, randomized, and double-blind controlled studies [86–88], and these data have been the subject of a recent Cochrane Review [89]. The first of the studies found that in women with severe dysmenorrhea and no obvious pathology, 9 of 11 patients (81%) underwent LUNA and reported almost complete relief of pain at 3 months’ follow-up. This had fallen to 45% at 12 months, and none of those in the control group reported any benefit [86]. During the randomized, double-blind study carried out in Guildford, 24 women received laser vaporization alone, and 27 received a LUNA procedure in addition [87]. Comparisons were made between the two treatment groups for changes in pain scores at 3 and 6 months. The LUNA treatment group had less benefit from their operation than the group who had

286

Jones and Sutton

not had a LUNA procedure. These trends reached significance for dysmenorrhea at 3 months ( P = 0.003) and at 6 months ( P = 0.0217). These findings are in keeping with another recent study that also concluded that the addition of a LUNA gives no additional benefit to performing laser vaporization of endometriosis alone [88]. Presacral Neurectomy Presacral neurectomy (PSN) is another surgical technique that has been used to treat women experiencing midline pelvic pain and dysmenorrhea for almost 100 years [90–93]. The hypogastric plexus of nerves at the sacral promontory can be divided during a laparotomy or laparoscopically. The technique has been described in three recent review articles [81,93,94]. A transverse incision is made in the peritoneum overlying the sacral promontory, extending from the ureter on the right side to the inferior mesenteric and superior hemorrhoidal arteries and sigmoid colon on the left. Connective and fatty tissue containing the nerve plexus is dissected, avoiding the middle sacral artery, and isolated in small longitudinal bunches. These are cauterized in two places 1 cm to 2 cm apart, and removed. Some authors recommend a divisional neurotomy because it is quicker and because it appears to be as effective, rather than the traditional excisional neurectomy [95]. A meta-analysis of the summarized case reports from the literature calculated an overall success rate of 79% [92], and these findings have been substantiated more recently in a Cochrane Review [89]. Nonconservative Operations Painful symptoms resulting from endometriosis may be treated by performing a total abdominal hysterectomy (TAH) if there is no desire to conserve the reproductive organs. This is usually combined with a bilateral salpingooophorectomy (BSO) and postoperative hormone replacement (HRT) therapy in young women. There is no evidence that compared with women who had an oophorectomy for endometriosis, patients who undergo hysterectomy with ovarian conservation have a 6.1 times greater risk of developing recurrent pain and 8.1 times greater risk of reoperation [96]. In view of this, it is illogical to conserve unaffected ovaries because estrogen is a stimulus to growth of ectopic endometrial tissue, and if any ovarian tissue remains the disease is likely to recur in as many as 40% of women at 5 years [97]. If ovarian tissue is conserved at the time of hysterectomy for endometriosis, 25% of patients will require subsequent laparotomies, often more than once [98]. It has also been noted that residual ovarian fragments become functional and stimulate

Treatment of Pelvic Pain

287

recurrence of endometriosis [99]. Therefore it is important to explain to the patient that conservation of the ovary may result in recurrent disease, and that malignant transformation of residual endometriosis has been reported [100]. Women who undergo TAH/BSO for pelvic pain and endometriosis at less than 30 years of age are more likely than older women to have residual symptoms [101]. Furthermore, young patients undergoing a TAH/BSO for endometriosis frequently use an HRT preparation, where the risk of disease recurrence is in the order of 3.5% or 0.9% per year [102]. If endometriosis does recur with painful pelvic symptoms after a TAH/BSO, laparoscopic excision of residual disease is effective in relieving endometriosis-associated pain [103].

SUMMARY Painful symptoms resulting from endometriosis can be managed medically or surgically. Medical therapy provides symptomatic relief while the drugs are being taken, with no long-term cure. Surgical procedures have been shown to provide long-term relief of painful symptoms.

PRACTICAL POINT 

Treatment of endometriosis-related pelvic pain could be medical or surgical, but surgical treatment provides long-term relief.

REFERENCES 1. 2.

3.

4.

5.

Mahmood TA, Templeton A. Prevalence and genesis of endometriosis. Hum Reprod 1991; 6:544–549. Koninckx PR, Meuleman C, Demeyere S, Lesaffre E, Cornillie FJ. Suggestive evidence that pelvic endometriosis is a progressive disease, whereas deeply infiltrating endometriosis is associated with pelvic pain. Fertil Steril 1991; 55: 759–765. Mathias SD, Kuppermann M, Liberman RF, Lipschutz RC, Steege JF. Chronic pelvic pain: Prevalence, health-related quality of life, and economic correlates. Obstet Gynecol 1996; 87:231–237. Zondervan KT, Yudkin PL, Vessey MP, et al. The community prevalence of chronic pelvic pain in women and associated illness behaviour. Br J Gen Practice 2001; 51:541–547. Zondervan KT, Yudkin PL, Vessey MP, Dawes MG, Barlow DH, Kennedy

288

6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

16. 17. 18. 19. 20.

21.

22.

23. 24.

25.

Jones and Sutton SH. Patterns of diagnosis and referral in women consulting for chronic pelvic pain in UK primary care. Br J Obstet Gynaecol 1999; 106:1156–1161. Andersch B, Milsom I. An epidemiologic study of young women with dysmenorrhea. Am J Obstet Gynecol 1982; 144:655–658. McGurgan P, O’Donovan P. Chronic pelvic pain, presentation, investigation and management. Adv Obstet Gynaecol 1999; 17:2–9. Walker KG, Shaw RW. Endometriosis, pelvic pain, and psychological function. Fertil Steril 1995; 63:796–800. Chapman CR, Casey KL, Dubner R, Foley KM, Gracely RH, Reading AE. Pain measurement: An overview. Pain 1985; 22:1–31. Jensen MP, Karoly P, Bravers S. The measurement of clinical pain intensity: A comparison of six methods. Pain 1986; 27:117–126. Revill SI, Robinson JO, Rosen M, Hogg MIJ. The reliability of a linear analogue for evaluating pain. Anesthesia 1976; 31:1191–1198. Carlsson AM. Assessment of chronic pain. I. Aspects of the reliability and validity of the visual analogue scale. Pain 1983; 16:87–101. Scott J, Huskisson EC. Graphic representation of pain. Pain 1976; 2:175–184. Higginson IJ, Carr AJ. Using quality of life measures in the clinical setting. BMJ 2001; 322:1297–1300. Bodner CH, Garratt AM, Ratcliffe J, Macdonald LM, Penney GC. Measuring health-related quality of life outcomes in women with endometriosis: Results of the Gynaecological Audit Project in Scotland. Health Bull 1997; 55:109–117. Reading AE. The internal structure of the McGill pain questionnaire in dysmenorrhea patients. Pain 1979; 7:353–358. Garry R, Clayton R, Hawe J. The effect of endometriosis and its radical laparoscopic excision on quality of life indicators. BJOG 2000; 107:44–45. Stones RW, Price C. Health service for women with chronic pelvic pain. JRSM 2002; 95:531–535. The American Fertility Society. Revised American Fertility Society classification of endometriosis: 1985. Fertil Steril 1985; 43:351–352. Fukaya T, Hoshiai H, Yajima A. Is pelvic endometriosis always associated with chronic pain? A retrospective study of 618 cases diagnosed by laparoscopy. Am J Obstet Gynecol 1993; 169:719–722. Colwell HH, Mathias SD, Pasta DJ, Henning JM, Steege JF. A health-related quality of life instrument for symptomatic patients with endometriosis: A validation study. Am J Obstet Gynecol 1998; 179:47–55. Stovall DW, Bowser LM, Archer DF, Guzick DS. Endometriosis associated pelvic pain: Evidence for an association between the stage of the disease and a history of chronic pelvic pain. Fertil Steril 1997; 68:13–18. Stout AL, Steege JF, Dodson WC, Hughes LL. Relationship of laparoscopic findings to self-reporting of pelvic pain. Am J Obstet Gynecol 1991; 164:73–79. Vernon MW, Beard JS, Graves K, Wilson EA. Classification of endometriotic implants by morphologic appearance and capacity to synthesise prostaglandin F. Fertil Steril 1986; 46:801–806. Perper NM, Nezhat F, Goldstein H, Nezhat GH, Nezhat C. Dysmenorrhea is

Treatment of Pelvic Pain

26.

27.

28. 29. 30. 31. 32.

33. 34. 35.

36. 37.

38.

39.

40.

41.

289

related to the number of implants in endometriosis patients. Fertil Steril 1995; 63:500–503. Cornille FJ, Oosterlynck D, Lauweryns JM, Koninckx PR. Deeply infiltrating pelvic endometriosis; histology and clinical significance. Fertil Steril 1990; 53: 978–983. Nisolle M, Paindaveine B, Bourdon A, Berliere M, Casanas-Roux F, Donnez J. Histologic study of peritoneal endometriosis in infertile women. Fertil Steril 1990; 53:984–988. Redwine DB, Yocum LB. A serial section study of visually normal pelvic peritoneum in patients with endometriosis. Fertil Steril 1990; 54:648–651. Nezhat F, Allen CJ, Nezhat C, Martin DC. Nonvisualised endometriosis at laparoscopy. Int J Fertil 1991; 36:340–343. Redwine DB. The distribution of endometriosis in the pelvis by age groups and fertility. Fertil Steril 1987; 47:173–175. Jenkins S, Olive DL, Haney AF. Endometriosis: Pathogenetic implications of the anatomic distribution. Obstet Gynecol 1986; 67:335–338. Damario MA, Horowitz IR, Rock JA. The role of uterosacral ligament resection in conservative operations for recurrent endometriosis. J Gynecol Surg 1994; 10:57–61. Chapron C, Dubuisson JB. Management of deep endometriosis. Ann NY Acad Sci 1998; 943:276–280. Farquhr C, Sutton C. The evidence for the management of endometriosis. Curr Opin Obstet Gynaecol 1998; 10:321–332. Bulun SE, Zeitoun K, Takayama K, Noble L, Michael D, Simpson E, Johns A, Putman M, Sasano H. Estrogen production in endometriosis and use of aromatase inhibitors to treat endometriosis. Endocrine Related Cancer 1999; 6:293–301. Bulun SE, Zeitoun K, Takayama K, Sasano H. Molecular basis for treating endometriosis with aromatase inhibitors. Hum Reprod 2000; 6:413–418. Vercellini P, Crosignani PG, Fadini R, Radici E, Belloni C, Sismondi P. A gonadotrophin-releasing hormone agonist compared with expectant management after conservative surgery for symtomatic endometriosis. Br J Obstet Gynaecol 1999; 106:672–677. Gambone JC, Mittman BS, Munro NG, Scialli AR, Winkel CA, and the Chronic Pelvic Pain/Endometriosis Working Group. Consensus Statement for the Management of Chronic Pelvic Pain and Endometriosis: Proceedings of an expert—panel consensus process. Fertil Steril 2002; 78:961–972. Sutton CJG. Laser laparoscopy in the treatment of endometriosis. In: Thomas EJ, Rock J, eds. Modern Approach to Endometriosis. Lancaster UK: Kluiser Academic Publishers, 1991:199–220. Redwine D. Conservative laparoscopic excision of endometriosis by sharp dissection: Life table analysis of re-operation and persistent but recurrent disease. Fertil Steril 1991; 56:628–634. Tulandi T, Bugnah M. Operative laparoscopies: Surgical modalities. Fertil Steril 1995; 63:237–241.

290

Jones and Sutton

42. Hornstein MD, Heinrichs Le R, Yuzpe AA, Buttram VL, Burry KA, Orwoll ES. Prospective randomized double-blind trial of 3 versus 6 months of nafarelin therapy for endometriosis associated pelvic pain. Fertil Steril 1995; 65: 955–962. 43. Winkel CA, Bray M. Treatment of women with endometriosis using excision alone, ablation alone or ablation in combination with Leuprolide acetate (abstract 105). Proceedings of the 5th World Congress on endometriosis, October 21–24 1996. Pacifico, Yokohama Japan, 1996:55. 44. Peters AA, van Dorst E, Jellis, van Zuuren E, Hermans J, Trimbos IB. A randomized clinical trial to compare two different approaches in women with chronic pelvic pain. Obstet Gynecol 1991; 77:740–744. 45. Shaw R. Endometriosis and infertility. Update Postgraduate Centre Series, Infertility. Reed Healthcare Communications 1995; 38–43. 46. Daniell JF, Brown DH. Carbon dioxide laser laparoscopy: Initial experience in experimental animals and humans. Obstet Gynecol 1982; 59:761–764. 47. Davis GD. Management of endometriosis and its associated adhesions with the CO2 laser laparoscope. Obstet Gynaecol 1986; 68:422–425. 48. Sutton CJG. Initial experience with carbon dioxide laser laparoscopy. Laser Med Sci 1985; 1:25–31. 49. Donnez J. Carbon dioxide laser laparoscopy in infertile women with adhesions or endometriosis. Fertil Steril 1987; 48:390–394. 50. Feste JR. Laser laparoscopy: A new modality. J Reprod Med 1985; 30:413– 418. 51. Nezhat C, Crowgey SR, Garrison CP. Surgical treatment of endometriosis via laser laparoscopy. Fertil Steril 1986; 45:778–783. 52. Sutton CJG, Ewen SP, Whitelaw N, Haines P. Prospective, randomized, double-blind, controlled trial of laser laparoscopy in the treatment of pelvic pain associated with minimal, mild, and moderate endometriosis. Fertil Steril 1994; 62:696–700. 53. Sutton CJG, Pooley AS, Ewen SP, Haines P. Follow-up report on randomised controlled trial of laser laparoscopy in the treatment of pelvic pain associated with minimal to moderate endometriosis. Fertil Steril 1997; 68:1070–1074. 54. Jones KD, Haines P, Sutton C. A long-term follow-up report on controlled trial of laser laparoscopy for pelvic pain. JSLS 2001; 5:111–117. 55. Donnez J, Nisolle M, Gillerot S, Smets M, Bassil S, Csanas-Rou F. Rectovaginal septum adenomyotic nodules: A series of 500 cases. Br J Obstet Gynaecol 1998; 104:1014–1018. 56. Nezhat C, Nezhat F, Pennington E, Nezhat CH, Ambroze W. Laparocopic disk excision and primary repair of the anterior rectal wall for the treatment of full-thickness bowel endometriosis. Surg Endosc 1994; 8:682–685. 57. Reich H, McGlynn F, Salvat J. Laparoscopic treatment of cul-de-sac obliteration secondary to retrocervical deep fibrotic endometriosis. J Reprod Med 1991; 36:516–522. 58. Redwine DB. Laparoscopic en bloc resection for treatment of the obliterated cul-de-sac in endometriosis. J Reprod Med 1992; 37:695–698.

Treatment of Pelvic Pain

291

59. Chapron C, Dubuisson JB, Fritel X, Fernandez B, Poncelet C, Beguin S, Pinelli L. Operative management of deep endometriosis infiltrating the uterosacral ligaments. J Am Assoc Gynecol Laparosc 1999; 1:31–37. 60. Sutton CJG, Hill D. Laser laparoscopy in the treatment of endometriosis: A five year study. Br J Obstet Gynecol 1990; 97:901–905. 61. Randolph Bailey H, Ott MT, Hartendorp P. Aggressive surgical management for advanced colorectal endometriosis. Dis Col Rectum 1994; 37:747–753. 62. Busacca M, Bianchi S, Agnoli B, Candiani M, Calia C, De Marinis S, Vignali M. Follow-up of laparoscopic treatment of stage III–IV endometriosis. J Am Assoc Gynecol Laparos 1999; 6:55–58. 63. Nezhat C, Nezhat F, Pennington E. Laparoscopic treatment of infiltrative rectosigmoid colon and retovaginal septum endometriosis by the technique of videolaparoscopy and the CO2 laser. Br J Obstet Gynaecol 1992; 99:664–667. 64. Redwine DB, Wright JT. Laparoscopic treatment of complete obliteration of the cul-de-sac associated with endometriosis: Long-term follow-up of en bloc resection. Fertil Steril 2001; 76:358–365. 65. Vercellini P, Trespidi L, De Giorgi O, Cortesi I, Parazzini F, Crosignani PG. Endometriosis and pelvic pain: Relation to disease stage and localization. Fertil Steril 1996; 65:299–304. 66. Chapron C, Dubuisson JB. Laparoscopic treatment of deep endometriosis located on the uterosacral ligaments. Hum Reprod 1996; 11:868–873. 67. Chapron C, Dubuisson JB, Fritel X, et al. Retroperitoneal endometriosis and pelvic pain: Results of laparoscopic uterosacral ligament resection according to the rAFS classification and histopathologic results. Gynecol Surg 1998; 14:51–58. 68. Jones KD, Sutton CJG. The colposcopic approach to rectovaginal endometriotic nodules. Minimal Invasive Therapy & Allied Technology 2002; 10: 311–331. 69. Jones KD, Sutton CJG. Laparoscopic management of ovarian endometriomas: A critical review of current practice. Curr Opin Obstet Gynecol 2000; 12:309–315. 70. Sutton CJG, Ewen SP, Jacobs SA, Whitelaw N. Laser laparoscopic surgery in the treatment of ovarian endometriomas. J Am Assoc Gynecol Laparos 1997; 4:319–323. 71. Montanino G, Porpora MG, Montanino Oliva M, Gulemi L, Boninfante M, Cosmi EV. Laparoscopic treatment of ovarian endometrioma. One year follow-up. Clinical & Experimental Obstetrics and Gynaecology 1996; 23:70– 72. 72. Busacca M, Marana R, Caruana, Candiani M, Muzii I, Calia C, Bianchi S. Recurrence of ovarian endometrioma after laparoscopic excision. Am J Obstet Gynecol 1999; 180:519–523. 73. Bateman BG, Kolp LA, Mills S. Endoscopic versus laparotomy management of endometriomas. Fertil Steril 1994; 62:690–695. 74. Daniell JF, Kurtz BR, Gurley LD. Laser laparoscopic management of large endometriomas. Fertil Steril 1991; 55:692–695. 75. Beretta P, Franchi M, Ghezzi F, Busacca M, Zupi E, Bolis P. Randomized

292

76.

77. 78. 79. 80.

81.

82. 83.

84. 85.

86. 87.

88.

89.

90.

91.

Jones and Sutton clinical trial of two laparoscopic treatments of endometriomas: Cystectomy versus drainage and coagulation. Fertil Steril 1998; 70:1176–1180. Jones JD, Sutton CJG. Patient satisfaction and changes in pain scores after ablative laparoscopic surgery for stage III–IV endometriosis and endometriotic cysts. Fertil Steril 2003; 79:1086–1090. Jones KD, Sutton CJG. Does laparoscopic surgery for endometriomas really relieve painful symptoms? Gynaecol Surg 2002; 18:39–43. Doyle JB. Paracervical uterine denervation for dysmenorrhea. Trans New Eng Obstet Gynec Soc 1954; 8:143. Doyle JB. Paracervical uterine denervation by transection of the cervical plexus for the relief of dysmenorrhea. Am J Obstet Gynecol 1955; 70:1–16. Sutton CJG. Laparoscopic uterine nerve ablation for intractable dysmenorrhea. In: Sutton C, Diamond M, eds. Endoscopic Surgery for Gynaecologists. London: WB Saunders, 1998:249–260. Daniell JF, Lalonde CJ. Advanced laparoscopic procedures for pelvic pain and dysmenorrhea. Vol. 9. In: Sutton C, ed. Advanced Laparoscopic Surgery. London: Balliere Tindall, 1995:795–808. Jones KD, Sutton CJG. Arcus taurinus: The mother and father of all LUNAs. Gynaecol Endosc 2001; 10:83–89. Donnez J, Nisolle M, Casanas-Roux F, Bassil S, Anaf V. Rectovaginal septum, endometriosis or adenomyosis: Laparoscopic management in a series of 231 patients. Hum Reprod 1995; 10:630–635. von Theobald P, Barjot P, Levy G. Laparoscopic Douglasectomy in the treatment of painful uterine retroversion. Surg Endosc 1997; 11:639–642. Sutton CJG. Laser uterine nerve ablation. In: Donnez J, ed. Laser Operative Laparoscopy and Hysteroscopy. Leuven, Belgium: Nauwelaerts Publishing, 1989:43–52. Lichten EM, Bombard J. Surgical treatment of dysmenorrhea with laparoscopic uterine ablation. J Reprod Med 1987; 32:37–42. Sutton CJG, Pooley A, Jones KD, Dover RW, Haines P. Prospective randomised double-blind controlled trial of laparoscopic laser uterine nerve ablation in the treatment of pelvic pain associated with minimal, mild, and moderate endometriosis. Gynaecol Endosc 2001; 10:1–8. Vercellini P, Aimi G, Busacca M, Uglietti A, Viganali M, Crosignani PG. Laparoscopic uterosacral ligament resection for dysmenorrhea associated with endometriosis: Results of a randomised controlled trial [abstr]. Fertil Steril Oct; 1997 suppl:33. Wilson ML, Farquhar CM, Sinclair OJ, Johnson NP. Surgical interruption of pelvic nerve pathways for primary and secondary dysmenorrhea (Cochrane Review). The Cochrane Library, Issue 1. Oxford: Update Software, 2000. Fontaine R, Herrmann LG. Clinical and experimental basis for surgery of the pelvic sympathetic nerves in gynecology. Surg Gynecol Obstet 1932; 54:133– 163. Cotte G. Resection of the presacral nerve in the treatment of obstinate dysmenorrhea. Am J Obstet Gynecol 1937; 33:1034–1040.

Treatment of Pelvic Pain

293

92. Black WT. Use of presacral sympathectomy in the treatment of dysmenorrhea. Am J Obstet Gynecol 1964; 89:16–22. 93. Biggerstaff ED, Foster SN. Laparoscopic surgery for dysmenorrhea: Uterine nerve ablation and presacral neurectomy. In: Sutton C, ed. Gynecological Endoscopic Surgery. London: Chapman & Hall, 1997:63–83. 94. Biggerstaff ED. Laparoscopic surgery for pelvic pain. In: Sutton CJG, Diamond MD, eds. Endoscopic Surgery for Gynaecologists. 2d ed. London: WB Saunders, 1998:261–271. 95. Daniell JF, Kurtz BR, Gurley LD, Lalonde CJ. Laparoscopic presacral neurectomy vs neurotomy: Use of the argon beam coagulator compared to conventional technique. J Gynecol Surg 1993; 9:169–173. 96. Namnoum AB, Hickman TN, Goodman SB, Gehlbach DL, Rock JA. Incidence of symptom recurrence after hysterectomy for endometriosis. Fertil Steril 1995; 64:898–902. 97. Wheeler JH, Malinak LR. Recurrent endometriosis: Incidence, management and prognosis. Am J Obstet Gynaecol 1983; 146:247–253. 98. Henderson AF, Studd JWW, Watson N. The retrospective study of oestrogen replacement therapy following hysterectomy for the treatment of endometriosis. In: Shaw RW, ed. Advances in Reproductive Endocrynology: Endometriosis. Carnforth, UK: Parthenon Publishers, 1989:131–140. 99. Dmowski WP, Radwanska E, Rana N. Recurrent endometriosis following hysterectomy and oopherectomy: The role of residual ovarian fragments. Int J Gynecol Obstet 1988; 26:93–103. 100. Jones KD, Owen E, Berresford A, Sutton CJG. Endometrial adenocarcinoma arising from endometriosis of the rectosigmoid colon. Gynecol Oncol 2002. In press. 101. MacDonald SR, Klock SC, Magdy PM. Long-term outcome of nonconservative surgery (hysterectomy) for endometriosis-associated pain in women <30 years old. Am J Obstet Gynecol 1999; 180:1360–1363. 102. Matorras R, Elorriaga MA, Pijoan JI, Ramon O, Rodriguez-Escudero FJ. Recurrence of endometriosis in women with bilateral adnexectomy (with or without total hysterectomy) who received hormone replacement therapy. Fertil Steril 2002; 77:303–308. 103. Clayton RD, Hawe JA, Love JC, Wilkinson N, Garry R. Recurrent pain after hysterectomy and bilateral salpingo-oopherectomy for endometriosis: Evaluation of laparoscopic excision of residual endometriosis. Br J Obstet Gynaecol 1999; 106:740–744.

17 In Vitro Fertilization or Superovulation? Evidence, Flaws, and Advice Ian S. Tummon Mayo Clinic Rochester, Minnesota, U.S.A.

EVIDENCE Physicians Need Good Information Physicians need reliable, terse, cogent, numerical information [1]. The subject of this chapter is treatment of endometriosis-associated subfertility with either in vitro fertilization (IVF) or superovulation/insemination. Objectives of this chapter are to: 1. Categorize evidence according to the Oxford Centre for EvidenceBased Levels of Evidence [2] 2. Outline flaws of the evidence 3. Give advice regarding treatment of endometriosis-associated subfertility with either IVF or superovulation/insemination Search strategy was review of English-and French-language literature using Medline, EMBASE, and the Cochrane database of systematic reviews, performed on January 7, 2003. 295

296

Tummon

Goal: Healthy, Singleton Live Birth Healthy singleton live birth is the goal of fertility treatment. Untreated Prognosis Minimal/Mild Endometriosis: An Intermediate Prognosis In an inception cohort study [3], level of evidence 1b [2], untreated minimal/ mild endometriosis-associated subfertility was in an intermediate position between moderate/severe endometriosis (which was lower) and unexplained infertility (which was higher) in terms of prognosis (Figs. 1 and 2). For minimal/mild endometriosis-associated subfertility, cumulative live birth rates at 36 months were 20% [95% confidence intervals (CI) 7% to 33%] [3]. Confidence intervals of minimal/mild endometriosis overlapped with the trend to a better prognosis for unexplained infertility and the trend to a poorer prognosis for moderate/severe endometriosis [3]. Because confidence intervals overlapped, differences were not statistical. Clinical differences, however, appear likely. A similar overlap of confidence intervals between minimal/mild endometriosis and unexplained infertility was seen in a cohort study of 300

FIGURE 1 Prognosis of untreated minimal/mild endometriosis [3].

IVF or Superovulation

297

FIGURE 2 Prognosis of untreated moderate/severe endometriosis [3].

untreated women. The relative place of minimal/mild endometriosis was lower than unexplained subfertility. The untreated crude fecundity rate ratio for minimal/mild endometriosis compared to unexplained infertility was 0.7, with 95% (95% CI 0.5 to 1.3) [4], results that are not statistically different. A clinical difference, however, appears likely. Moderate/Severe Endometriosis: Untreated, A Starkly Poor Prognosis A starkly low prognosis was associated with untreated moderate/severe endometriosis [3], level of evidence 1b [2]. After 36 months of untreated observation, cumulative live birth rates were 5% (95% CI 0% to 15%). Confidence intervals of moderate/severe endometriosis and unexplained infertility did not cross, indicating the likelihood of a true difference. Primary treatment for moderate/severe endometriosis is surgical reduction [5]. Re-operation for recurrent moderate/severe endometriosis appears to offer little benefit [6], level of evidence 2b [2]. Comparing IVF to reoperation, the odds ratio for continuing pregnancy was 3.2 (95% CI 0.8 to 12.8) in favor of IVF.

298

Tummon

Prognosis after Surgical Reduction of Endometriosis Improved Fecundity for Minimal/Mild Endometriosis Endometriosis is usually diagnosed by laparoscopy and treated by surgery. Surgical reduction of minimal/mild endometriosis also improves fecundity, compared with diagnostic laparoscopy alone, OR 1.9 (95% CI 1.2 to 3.1) [7], level of evidence 1b [2]. Dismal Prognosis for Obliterated Cul de Sac At the other extreme, in even the best of hands, postsurgical fecundity is sometimes bleak. Given complete obliteration of the cul de sac, the 3-year cumulative life table pregnancy rate was 30% [8]. Efficacy of Treatments for Endometriosis-Associated Subfertility Clomiphene Citrate in Endometriosis-Associated Subfertility There is no systematic analysis of clomiphene for endometriosis-associated subfertility. In a cohort trial unstratified by stage of endometriosis, clomiphene was superior to no treatment, odds ratio 2.9 (95% CI 1.2 to 7.1) [9], level of evidence 2b [2]. Clomiphene Citrate and Endometriosis-Associated Subfertility In a randomized trial comparing clomiphene citrate/insemination with observation, the odds ratio for fecundity was 3.0 (95% CI 1.1 to 8.6) [10], level of evidence 2b [2] (Tables 1 and 2). Clomiphene Citrate/Insemination and Unexplained Subfertility Systematic analysis of empiric clomiphene for unexplained subfertility versus no treatment concluded in favor of clomiphene. The common odds ratio for pregnancy was 2.5 (95% CI 1.35–4.62) [11], level of evidence 1a [2]. Superovulation/Insemination For minimal/mild endometriosis-associated subfertility, randomized trials show superiority of superovulation/insemination compared to no treatment, with odds ratios for cycle fecundity ranging from 5.1 to 5.3, level of evidence 1b [2], as shown in Table 2. Systematic review of superovulation or insemination for persistent infertility found that compared to no treatment, the odds ratio for pregnancy with superovulation using follicle-stimulating hormone (FSH) was 2.3 (95%

IVF or Superovulation

TABLE 1

TT Oxford Centre for Evidence-Based Medicine Levels of Evidence [2]

Level 1a

1b 1c 2a

2b 2c 3a 3b 4 5

299

Prognosis

Treatment Systematic review of homogeneous randomized clinical trials

Systematic review of homogeneous inception cohort studies Inception cohort studies, follow-up z 80% All or none case series Systematic review with homogeneity of cohort series or control groups Retrospective cohort study

Individual randomized clinical trial with narrow confidence interval All or none Systematic review with homogeneity of cohort series Individual cohort study, low quality randomized clinical trial Outcomes research Systematic review with homogeneity of case-control studies Individual case-control study Cases series Expert opinion without explicit critical appraisal

Outcomes research

Cases series Expert opinion without explicit critical appraisal

CI 1.9 to 2.9), and the odds ratio for pregnancy using insemination was 2.8 (95% CI 2.2 to 3.7) [12], level of evidence 1a [2]. The adjusted odds ratio for pregnancy in association with a diagnosis of endometriosis, not stratified by stage, was 0.5 (95% CI 0.3 to 0.8). There are no randomized, controlled studies of superovulation/insemination for moderate/severe endometriosis. In superovulation/insemination, declining fecundity was associated with increasing female age [13] and decreasing ovarian reserve [14], both level

TABLE 2 TT Randomized Clinical Trials of Superovulation/Insemination for Minimal/ Mild Endometriosis Cycle Fecundity Author

Regimen

Test

Control

Odds Ratio (95% CI)

Deaton [10] Nulsen [49]

CC/insemination Gonadotropins/ insemination FSH/insemination

10 12

3 2

3.0 (1.1 to 8.6) 5.3 (1.1 to 22.5)

11

2

5.1 (1.7 to 15.1)

Tummon [50]

300

Tummon

of evidence 4 [2]. As is true for fertility treatments in general, fecundity after three cycles of superovulation/insemination diminishes in minimal/mild [15] and moderate/severe endometriosis [15], as well as ‘‘unexplained infertility’’ [16], both level of evidence 2b [2]. In Vitro Fertilization IVF for Endometriosis-Associated Subfertility Compared to No Treatment. —In a small, randomized comparison of IVF to control treatment for endometriosis-associated subfertility [17], pregnancy occurred in 5 of 15 subjects treated with IVF and zero of six control subjects (P= 0.11), level of evidence category 2b [2]. Endometriosis was not stratified by stage. IVF for Endometriosis-Associated versus Tubal Subfertility. —Systematic comparison of IVF results found an odds ratio for pregnancy of 0.6 (95% CI 0.4 to 0.7) in association with endometriosis compared to tubal subfertility [18]. Number of oocytes retrieved, fertilization rates, and implantation rates were all decreased. When endometriosis was stratified by stage, moderate/severe endometriosis had poorer prognosis than minimal/mild endometriosis, odds ratio 0.6 (95% CI 0.4 to 0.9), level of evidence 2a-[2]. In a case-control study [19], level of evidence 3b [2], FSH requirements were greater with ovarian endometriosis than tubal subfertility. Implantation in the Oocyte Donation Model. —Using the oocyte donation model, implantation was studied in endometriosis-associated subfertility [20,21]. In both a case-control study [20], level of evidence 3b [2], and a cohort study [21], level of evidence 2b [2], implantation was unimpaired. In a retrospective cohort study of 5,000 IVF cycles in one center [22], cumulative pregnancy rates were not lower for endometriosis than other diagnostic categories, level of evidence 2b [2]. In a review of 40,000 IVF cycles in 25,000 women, endometriosis was unassociated with a decreased chance of birth compared to other diagnostic categories [23], level of evidence 2b [2]. Central Registries IVF National Registries In vitro fertilization results by diagnostic category in national registries in the United States [24] and France [25] are shown in Figures 3 and 4. No decrease in results in association with endometriosis was seen. In the United States in 2000, endometriosis was the principal diagnosis in 10% of cycles and the live birth rate was 28% per cycle started. Stratification of results by stage of endometriosis is unavailable. In France, endometriosis was the principal diagnosis in 4% of cycles and the live birth rate was 23% per cycle started.

IVF or Superovulation

FIGURE 3 IVF results by primary diagnosis, United States, 2000 [24].

FIGURE 4 IVF results by primary diagnosis, France 1999 [25].

301

302

Tummon

Again, stratification of results by stage of endometriosis is unavailable. In the national IVF registry results of both the United States [24] and France [25], endometriosis did not have a poorer result than other diagnostic categories. Findings of noninferior IVF outcomes for a principal diagnosis of endometriosis are in contrast to the systematic review of IVF for endometriosis versus tubal subfertility [18]. Reasons for noninferiority may have included a preponderance of subjects with minimal/mild endometriosis (with a better prognosis than moderate/severe endometriosis) undergoing IVF. Additional factors may have been selection of the few best embryos for fresh transfer and individual adjustment of gonadotropic dosage according to patient needs [19]. No Registry for Superovulation/Insemination There is no central reporting registry for treatment with superovulation/ insemination. Efficacy—IVF Compared to Superovulation/Insemination Evidence from a Cohort Study In subjects with endometriosis-associated subfertility, there is no randomized comparative trial of IVF versus superovulation. In a cohort study of endometriosis-associated subfertility, the odds ratio for pregnancy after primary IVF was 7.4 (95% CI 4.9 to 11.2), compared to superovulation/insemination [15], level of evidence 2b [2]. Prior unsuccessful superovulation/insemination treatment did not adversely affect IVF outcome [15]. Evidence from a Randomized Trial No difference in fecundity was found between superovulation/insemination and IVF in a randomized comparative trial for unexplained subfertility. The primary endpoint was pregnancy resulting in at least one live birth [26], level of evidence 1b [2]. Subjects were treated for a maximum of six cycles. Superovulation was ‘‘gentle FSH’’ with 28% of responses monofollicular. Success rates were 12% with IVF and 9% with gentle FSH. The odds ratio for pregnancy leading to live birth was 1.4 (95% CI 0.8 to 2.9) in favor of IVF. See Section 2.7 for critique of the external validity of this trial, with IVF results well below the norm. IVF versus Superovulation/Insemination for Unexplained Subfertility Systematic review of IVF for unexplained subfertility [27] found no difference in live birth rates between IVF and superovulation/insemination, odds ratio 0.87 (95% CI 0.4 to 1.8). There was no difference in multiple pregnancy rates

IVF or Superovulation

303

between IVF and superovulation/insemination, odds ratio 1.59 (95% CI 0.7 to 3.7). Clinical heterogeneity was present among studies [27], level of evidence 2b [2]. Early Access to IVF National Institutes of Health are sponsoring a randomized trial of early access to IVF [28]. Subjects will be randomized to either 

Clomiphene/insemination for three cycles, then superovulation/ insemination for three cycles, then IVF  Clomiphene/insemination for three cycles, then IVF Study criteria do not include laparoscopy, and sufficient power for conclusions specific to endometriosis-associated subfertility is unlikely. Natural Cycle IVF Natural cycle IVF is a low-risk, low-cost alternative to traditional stimulated IVF. Live birth rates approximate 5% [29]. Outcomes specific for endometriosis are not available, level of evidence 2a [2]. Prolonged Use of Gonadotropin Releasing Hormone Agonist Before IVF Prolonged pretreatment with ovarian suppression may improve IVF results for endometriosis-associated subfertility. In a trial of young subjects with good ovarian reserve and moderate/severe endometriosis, the odds ratio for ongoing pregnancy was 3.4, (95% CI 0.98–11.9) in favor of prolonged pretreatment [30], level of evidence 2b [2]. Details of methodology are lacking, including allocation concealment and confounding factors such as stage of embryo development at transfer. Adverse Effects of IVF and Superovulation Multifetal Pregnancy Multifetal pregnancies are high risk for perinatal morbidity and mortality [31]. In superovulation/insemination, methods of primary prevention of multifetal pregnancy are direct: reduce ovarian stimulation and monitor follicular development fastidiously [32]. Figure 5 shows a 28% prevalence of multifetal pregnancy with superovulation/insemination in one cohort study, level of evidence 2b [2].

304

Tummon

FIGURE 5 Multifetal pregnancy after superovulation/insemination [33].

In IVF, primary prevention is by limiting the number of embryos transferred. Risk of multifetal pregnancy is a direct consequence of number of embryos transferred [23]. High Order Multifetal Pregnancy Perinatal morbidity and mortality are extreme in high-order multiple pregnancies (triplets or more).

FIGURE 6 Multifetal pregnancy and United Kingdom IVF registry 2000 [34].

IVF or Superovulation

305

FIGURE 7 Multifetal pregnancy and United States IVF registry 2000 [24].

As shown in Figure 5, the prevalence of high order multifetal pregnancy with superovulation/insemination in one cohort study was 8% [33], level of evidence 2b [2]. As shown in Figure 6, in the United Kingdom where number of embryos transferred is limited by statute (at most 3) [34], triplets are rare [34]. In contrast, in the United States, where number of embryos transferred is discretionary [24], the high order multiple pregnancy rate after IVF is 8%, as shown in Figure 7. Receiver operator characteristic curves identified thresholds for best prevention of high-order multiple pregnancy in superovulation/insemination treatment. The predictors: total number of follicles z 10 mm, serum estradiol > 862 pg/ml, and female age V 32 years [35], level of evidence 2b [2]. In another cohort study, risk factors were serum estradiol z 1385 pg/ml, odds ratio 1.9 (95% CI 1.3 to 2.8) and seven or more follicles z 16 mm, odds ratio 2.1 (95% CI 1.2 to 3.9) [33]. Ovarian Hyperstimulation Syndrome In the sole randomized comparison of superovulation and IVF, hospitalization for ovarian hyperstimulation syndrome occurred in 2 of 59 IVF subjects and 0 of 59 superovulation subjects [26], P= 0.25, level of evidence 1b [2]. Risk of ovarian hyperstimulation syndrome is likely proportional to the degree of aggressiveness of ovarian stimulation. Birth Defects Birth defects may be increased as a result of IVF. The prevalence of major birth defects in infants born after IVF was 9% compared to 4.2% for infants

306

TABLE 3

Tummon TT Approximate Relative Costs of Treatment [39]

Treatment

Pregnancy rate per cycle (%)

Estimated cost per pregnancy, $

1.3–4.1 8.3 17.1 20.7

10,000 17,000 50,000

No treatment Clomiphene/insemination Gonadotropins/insemination IVF

conceived without assisted reproduction, odds ratio 2.0 (95% CI 1.5 to 2.9) [36], level of evidence 1b [2]. There are no data with respect to risk of birth defects and superovulation. Economic Analyses Economic implications of treatment with superovulation/insemination or IVF require consideration, but economic analyses are ‘‘essentially parochial activities’’ [37] and results do not necessarily correlate with costs. When insurance coverage limits access to IVF, this may modify the way treatment is done. With reduced access to IVF, embryo numbers transferred may increase and tend to influence the rate of multifetal pregnancy [38]. Economic analyses are unavailable for treatment of endometriosisassociated subfertility using assisted conception. For unexplained subfertility, superovulation/insemination was more cost-effective than IVF [39], as shown in Table 3, level of evidence 2a [2], [26] level of evidence 1b [2], and [40], level of evidence 1b [2].

FLAWS Diagnosis of Endometriosis Laparoscopy—Reference Standard Laparoscopy is the reference standard for diagnosis and staging of endometriosis [41]. Neither wholly sensitive nor specific, laparoscopy is an invasive act, involving a 0.04% to 0.5% risk of major complications [42]. Laparoscopy also requires anesthesia. Although laparoscopy is a core diagnostic test for more than 85% of United States board-certified reproductive endocrinologists [43], it is a test (and treatment) applied selectively [44] and interpreted subjectively [45].

IVF or Superovulation

307

Gray Scale Ultrasound—Noninvasive Alternative for Diagnosis, if Ovarian Only if endometriosis is ovarian, which usually it is not, is there an accurate, useful, noninvasive alternative for diagnosis. This alternative is gray scale transvaginal ultrasound. Systematic review of the accuracy of gray scale ultrasound for diagnosis of ovarian endometriosis gives positive likelihood ratios ranging from 8 to 30 [46], indicating a useful test, level of evidence 3b [2]. Endometriosis is Not Found Because It is not Looked For When populations are defined as ‘‘unexplained infertility’’ but have not undergone laparoscopy, the prevalence of endometriosis is unknown [12,16]. Such populations represent a mix of subjects with and without endometriosis. Assessment Fails to Stratify by Stage of Endometriosis Without treatment [3,4], after surgery [8], and with superovulation/insemination or IVF [15]. the prognosis for endometriosis-associated subfertility varies with the stage of endometriosis. If the stage of endometriosis is not accounted for in assessment of treatment efficacy [12,33], the power of the study will be limited. Endometriosis is Considered ‘‘Unexplained Subfertility’’ Studies of unexplained subfertility have inclusion criteria with either minimal [26] or minimal/mild endometriosis [47] or surgically treated minimal/mild endometriosis [39]. Heterogeneity of Superovulation In a comparison of two protocols of superovulation/insemination, pregnancy rates were superior with gonadotropins compared with clomiphene citrate/ gonadotropins, relative risk 2.1 (95% CI 1.1 to 4.2) [48], level of evidence 1b [2]. Sample Size Limitations In systematic reviews, even large differences may be hidden owing to studies limited by small sample size and adverse effects such as multifetal pregnancies and ovarian hyperstimulation syndrome are unreported in most studies [27].

308

Tummon

External Validity Low IVF results limit the external validity of the only randomized comparison of superovulation/insemination versus IVF. In a population of young women likely to have a good prognosis, IVF cycle fecundity resulting in live birth was 12% [26]. This is well below current levels of endometriosis-specific live birth per treatment cycle 28% [24] and 23% [25] in national IVF registries. This study lacks external validity. Extensive Need for Extrapolation Extrapolation is use of data in situations with important differences from the original study situation. Extrapolations are essential in examining the question of efficacy of IVF or superovulation for endometriosis-associated subfertility. Endometriosis is often not diagnosed; if it is diagnosed, it is often not staged. If endometriosis is diagnosed, if it is staged, it is then sometimes categorized as unexplained subfertility. These circumstances limit our understanding of the prognosis and response to treatment for endometriosis-associated subfertility.

ADVICE Stage of Endometriosis Affects Prognosis Intermediate Prognosis for Minimal/Mild Endometriosis Untreated, the prognosis for minimal/mild endometriosis-associated subfertility is lower than untreated unexplained subfertility [3,4]. A clinical difference appears likely, although pregnancy and birth rates were not statistically different. Starkly Poor Prognosis for Moderate Severe Endometriosis Untreated, the prognosis for moderate/severe endometriosis-associated subfertility is starkly poor. Cumulative live birth rates are 5% (95% CI 0 to 15) after 36 months of untreated observation [3], level of evidence 1b [2]. Treatment Strategy by Stage of Endometriosis Minimal/Mild Endometriosis-Associated Subfertility  

Consider superovulation/insemination, if monitoring can be vigilant. Cancel cycle if more than five mature follicles.

IVF or Superovulation

309



Consider cancellation of cycle if more than three mature follicles and patient age less than 32 years.  Persist for four cycles if response appears normal.  If poor response or no success, advance to IVF. Surgical reduction of minimal/mild endometriosis is primary therapy to enhance fecundity [7]. Superovulation/insemination improves fertility for minimal/mild endometriosis-associated subfertility. Relative benefit of superovulation/insemination over no treatment is a three-to fivefold increase in fecundity, level of evidence 1b [2]. Expected fecundity per cycle, untreated, is 3% and approximately 9% with clomiphene/insemination, and 12% to 15% with gonadotropin/insemination. Danger lurks, however, and after treatment with gonadotropin/insemination, prevalence of high order multifetal pregnancy approximates 8% [33]. For primary prevention of high-order multifetal pregnancy in superovulation/insemination, vigilance in monitoring is essential. Treatment cancellation is strongly suggested if there are five or more mature follicles [35]. Risk of multifetal pregnancy is age dependent and proportionate to ovarian response. Estimates for the critical estradiol thresholds for multifetal pregnancy range from 860 to 1300 pg/ml [33,35]. Estimated crude fecundity per IVF cycle for minimal/mild endometriosis is 40% [15], level of evidence 2b [2], and is not diminished after prior treatment with superovulation/insemination. In national registries, live birth rates for endometriosis are not stratified by stage, and crude fecundity per cycle ranges from 23% to 28% [24,25]. Moderate/Severe Endometriosis-Associated Subfertility 

Consider IVF as first line treatment in assisted reproduction. Implantation rates are unimpaired in patients with endometriosis.  Do not adjust the number of embryos to be transferred on account of a diagnosis of endometriosis. 

Superovulation/insemination for subfertility associated with moderate/ severe endometriosis has a poor prognosis. Cumulative fecundity after six treatment cycles of superovulation/insemination was 10% [15], level of evidence 2b [2]. Using IVF for subfertility associated with moderate/severe endometriosis, cumulative fecundity after three cycles of IVF was approximately 50% [15], level of evidence 2b [2]. As indicated previously, in national registries, live birth rates for endometriosis are unstratified by stage. Live birth rates for endometriosis in toto range from 23% to 28% [24,25].

310

Tummon

Takes Two to Tango Fecundity is best if all subfertility factors are optimized simultaneously. The physician detective cannot limit the focus to endometriosis. PRACTICAL POINT 

Superovulation and insemination could be first considered for minimal and mild endometriosis-related infertility, whereas IVF is more effective in moderate and severe endometriosis.  Superovulation and insemination for minimal and mild endometriosis-related infertility.  IVF for moderate and severe endometriosis-related infertility.

REFERENCES 1.

Collins JA. Clinical research evidence and clinical practice. Hum Reprod 1997; 12:1847–1850. 2. Philips B. Oxford Centre for Evidence-based Medicine Levels of Evidence, 2003. http:/www.indigojazz.co.uk/cebm/levels_of_evidence.asp. 3. Collins JA, Burrows EA, Wilan AR. The prognosis for live birth among untreated infertile couples. Fertil Steril 1995; 64:22–28. 4. Berube S, Marcoux S, Langevin M, Maheux R. Fecundity of infertile women with minimal or mild endometriosis and women with unexplained infertility. The Canadian Collaborative Group on Endometriosis. Fertil Steril 1998; 69: 1034–1041. 5. Guzick DS, Silliman NP, Adamson GD, Buttram VC Jr, Canis M, Malinak LR, Schenken RS. Prediction of pregnancy in infertile women based on the American Society for Reproductive Medicine’s revised classification of endometriosis. Fertil Steril 1997; 67:822–829. 6. Pagidas K, Falcone T, Hemmings R, Miron P. Comparison of reoperation for moderate (stage III) and severe (stage IV) endometriosis-related infertility with in vitro fertilization-embryo transfer. Fertil Steril 1996; 65:791–795. 7. Marcoux S, Mheux R, Berube S. Laparoscopic surgery in infertile women with minimal or mild endometriosis. Canadian Collaborative Group on Endometriosis.[comment]. N Engl J Med 1997; 337:217–222. 8. Adamson GD, Pasta DJ. Surgical treatment of endometriosis-associated infertility: Meta-analysis compared with survival analysis. [Erratum appears in Am J Obstet Gynecol 1995 Jun;172:1937]. Am J Obstet Gynecol 1994; 171: 1488–1504. Discussion 1504–1505. 9. Simpson CW, Taylor PJ, Collins JA. A comparison of ovulation suppression and ovulation stimulation in the treatment of endometriosis-associated infertility. Int J Gynaecol Obstet 1992; 38:207–213. 10. Deaton JL, Gibson M, Blackmer KM, Nakajima ST, Badger GJ, Brumsted JR.

IVF or Superovulation

11. 12.

13.

14.

15.

16.

17.

18. 19.

20.

21.

22.

23. 24.

311

A randomized, controlled trial of clomiphene citrate and intrauterine insemination in couples with unexplained infertility or surgically corrected endometriosis. Fertil Steril 1990; 54:1083–1088. Hughes E, Collins J, Vandekerckhove P. Cochrane Database of Systematic Reviews 2002; 4. Hughes EG. The effectiveness of ovulation induction and intrauterine insemination in the treatment of persistent infertility: A meta-analysis. Hum Reprod 1997; 12:1865–1872. Sahakyan M, Harlow BL, Hornstein MD. Influence of age, diagnosis, and cycle number on pregnancy rates with gonadotropin-induced controlled ovarian hyperstimulation and intrauterine insemination. Fertil Steril 1999; 72:500–504. Burwinkel TH, Buster JE, Scoggan JL, Carson SA. Basal follicle stimulating hormone (FSH) predicts response to controlled ovarian hyperstimulation (COH)intrauterine insemination (IUI) therapy. J Assist Reprod Genet 1994; 11:24–27. Dmowski WP, Pry M, Ding J, Rana N. Cycle-specific and cumulative fecundity in patients with endometriosis who are undergoing controlled ovarian hyperstimulation-intrauterine insemination or in vitro fertilization-embryo transfer. Fertil Steril 2002; 78:750–756. Aboulghar M, Mansour R, Serour G, Abdrazek A, Amin Y, Rhodes C. Controlled ovarian hyperstimulation and intrauterine insemination for treatment of unexplained infertility should be limited to a maximum of three trials. Fertil Steril 2001; 75:88–91. Soliman S, Daya S, Collins J, Jarrell J. A randomized trial of in vitro fertilization versus conventional treatment for infertility. Fertil Steril 1993; 59:1239– 1244. Barnhart K, Dunsmoor-Su R, Coutifaris C. Effect of endometriosis on in vitro fertilization. Fertil Steril 2002; 77:1148–1155. Al-Azemi M, Bernal AL, Steele J, Gramsbergen I, Barlow D, Kennedy S. Ovarian response to repeated controlled stimulation in in-vitro fertilization cycles in patients with ovarian endometriosis. Hum Reprod 2000; 15:72–75. Diaz I, Navarro J, Blasco L, Simon C, Pellicer A, Remohi J. Impact of stage IIIIV endometriosis on recipients of sibling oocytes: Matched case-control study. Fertil Steril 2000; 74:31–34. Sung L, Mukherjee T, Takeshige T, Bustillo M, Copperman AB. Endometriosis is not detrimental to embryo implantation in oocyte recipients. J Assist Reprod Genet 1997; 14:152–156. Alsalili M, Yuzpe A, Tummon I, Parker J, Martin J, Daniel S, Rebel M, Nisker J. Cumulative pregnancy rates and pregnancy outcome after in-vitro fertilization:>5000 cycles at one centre. Hum Reprod 1995; 10:470–474. Templeton A, Morris JK. Reducing the risk of multiple births by transfer of two embryos after in vitro fertilization. N Engl J Med 1998; 339:573–577. Centers for Disease Control and Prevention. Assisted Reproductive Technology Reports 2000. Assisted Reproductive Technology Success Rates. National Summary and Fertility Clinic Reports, 2000:27 http://www.cdc.gov/nccdphp/drh/ ART00/PDF’s/ART2000.pdf.

312 25. 26.

27.

28. 29. 30.

31. 32.

33.

34.

35.

36.

37. 38. 39.

40.

41.

Tummon FIVNAT. Association FIVNAT, 2000. http://perso.wanadoo.fr/fivnat.fr/ bilan2000.htm Goverde AJ, McDonnell J, Vermeiden JP, Schats R, Rutten FF, Schoemaker J. Intrauterine insemination or in-vitro fertilisation in idiopathic subfertility and male subfertility: A randomised trial and cost-effectiveness analysis [comment]. Lancet 2000; 355:13–28. Pandian Z, Bhattacharya S, Nikolaou D, Vale L, Templeton A. In vitro fertilisation for unexplained subfertility. Cochrane Database of Systematic Reviews 2002: CD003357. NICHD. Conventional infertility therapy versus fast track to IVF, 2003. Pelinck MJ, Hoek A, Simons AH, Heineman MJ. Efficacy of natural cycle IVF: A review of the literature. Hum Reprod Update 2002; 8:129–139. Surrey ES, Silverberg KM, Surrey MW, Schoolcraft WB. Effect of prolonged gonadotropin-releasing hormone agonist therapy on the outcome of in vitro fertilization-embryo transfer in patients with endometriosis. Fertil Steril 2002; 78:699–704. The ESHRE Capri Workshop Group. Multiple gestation pregnancy. Hum Reprod 2000; 15:1856–1864. Child TJ, Barlow DH. Strategies to prevent multiple pregnancies in assisted conception programmes. Baillieres Clinical Obstetrics and Gynaecology 1998; 12:131–146. Gleicher N, Oleske DM, Tur-Kaspa I, Vidali A, Karande V. Reducing the risk of high-order multiple pregnancy after ovarian stimulation with gonadotropins. N Engl J Med 2000; 343:2–7. HFEA. Annual Report 2002: Human Fertilisation and Embryology Authority, Annual Report 2002. http://www.hfea.gov.uk/Downloads/Annual_Report/ HFEA_Annual%20Report2002.pdf Tur R, Barri PN, Coroleu B, Buxaderas R, Martinez F, Balasch J. Risk factors for high-order multiple implantation after ovarian stimulation with gonadotrophins: evidence from a large series of 1878 consecutive pregnancies in a single centre. Hum Reprod 2001; 16:2124–2129. Hansen M, Kurinczuk JJ, Bower C, Webb S. The risk of major birth defects after intracytoplasmic sperm injection and in vitro fertilization. N Engl J Med 2002; 346:725–730. Barlow DH. Cost-effectiveness modelling. Hum Reprod 2001; 16:2479–2480. Jain T, Harlow BL, Hornstein MD. Insurance coverage and outcomes of in vitro fertilization. N Engl J Med 2002; 347:661–666. Guzick DS, Sullivan MW, Adamson GD, Cedars MI, Falk RJ, Peterson EP, Steinkampf MP. Efficacy of treatment for unexplained infertility. Fertil Steril 1998; 70:207–213. Garceau L, Henderson J, Davis LJ, Petrou S, Henderson LR, McVeigh E, Barlow DH, Davidson LL. Economic implications of assisted reproductive techniques: A systematic review. Hum Reprod 2002; 17:3090–3109. Revised American Fertility Society classification of endometriosis: 1985. Fertil Steril 1985; 43:351–352.

IVF or Superovulation

313

42. Munro MG. Laparoscopic access: Complications, technologies, and techniques. Curr Opin Obstet Gynecol 2002; 14:365–374. 43. Glatstein IZ, Harlow BL, Hornstein MD. Practice patterns among reproductive endocrinologists: The infertility evaluation. Fertil Steril 1997; 67:443–451. 44. Tanahatoe SJ, Hompes PGA, Lambalk CB. Investigation of the infertile couple: Should diagnostic laparoscopy be performed in the infertility work up programme in patients undergoing intrauterine insemination? Hum Reprod 2003; 18:8–11. 45. Stripling MC, Martin DC, Chatman DL, Zwaag RV, Poston WM. Subtle appearance of pelvic endometriosis. Fertil Steril 1988; 49:427–431. 46. Moore J, Copley S, Morris J, Lindsell D, Golding S, Kennedy S. A systematic review of the accuracy of ultrasound in the diagnosis of endometriosis. Ultrasound Obstet Gynecol 2002; 20:630–634. 47. Guzick DS, Carson SA, Coutifaris C, Overstreet JW, Factor-Litvak P, Steinkampf MP, Hill JA, Mastroianni L, Buster JE, Nakajima ST, Vogel DL, Canfield RE. Efficacy of superovulation and intrauterine insemination in the treatment of infertility. National Cooperative Reproductive Medicine Network. N Engl J Med 1999; 340:177–183. 48. Ransom MX, Doughman NC, Garcia AJ. Menotropins alone are superior to a clomiphene citrate and menotropin combination for superovulation induction among clomiphene citrate failures. [comment]. Fertil Steril 1996; 65:1169–1174. 49. Nulsen JC, Walsh S, Dumez S, Metzger DA. A randomized and longitudinal study of human menopausal gonadotropin with intrauterine insemination in the treatment of infertility. Obstet Gynecol 1993; 82:780–786. 50. Tummon IS, Asher LJ, Martin JSB, Tulandi T. Randomized controlled trial of superovulation and insemination for infertility associated with minimal or mild endometriosis. Fertil Steril 1997; 68:8–12.

18 Does Endometriosis Affect the Results of In Vitro Fertilization? Simon M. Kelly, William M. Buckett, and Seang Lin Tan McGill University Montreal, Quebec, Canada

Endometriosis is commonly linked to infertility. As a disease entity it affects around 10% of all women of reproductive age but as many as 20 to 40% of women who seek help with infertility [1,2]. The exact mechanism by which endometriosis impairs fertility remains elusive and it is most likely a multifactorial effect. In moderate or severe endometriosis, patients may have anatomic distortion of the fallopian tubes or significant damage to the ovaries. In these situations, it is clear how fertility may be adversely affected. The mechanism of fertility impairment associated with milder forms of endometriosis is less apparent. Several factors have been implicated in the pathophysiology of endometriosis-related subfertility. These include various local paracrine chemicals, such as interleukins and other cytokines, autoimmune factors, or an altered inflammatory response [3,4]. In some animal and human studies, detrimental influence of endometriosis may be related to oocyte and early embryo quality [5–7]. 315

316

Kelly et al.

Many patients with endometriosis and infertility will require some assistance to conceive. Ultimately a good proportion may require assisted conception in the form of in vitro fertilization (IVF). The aim of this chapter is to review the evidence on whether endometriosis has an impact on the outcome of IVF treatment. This is a controversial issue and is confounded by a number of conflicting studies. INDICATIONS FOR IVF TREATMENT There is little doubt that IVF is a highly successful treatment for a range of fertility problems [8]. In women with endometriosis, the decision to proceed to IVF treatment will depend on a number of factors aside from the endometriosis itself. These will include the age of the female, duration of infertility, presence of any coexisting diagnosis, and a history of any previous fertility treatment. When these factors allow, more conservative therapies are generally instituted initially. These may include medical or surgical treatments aimed specifically at the endometriosis or simpler fertility treatments such as intrauterine insemination. In a study by Tummon et al. involving patients with mild endometriosis, superovulation treatment combined with intrauterine insemination proved superior to expectant management. The pregnancy rates were 11% and 2%, respectively, between the two groups [9]. Kodama et al. compared expectant management to IVF treatment in patients with mild to moderate endometriosis. There was a shorter duration to conception in the IVF group (20.6 months vs 27.1 months). In addition, the cumulative conception rate was significantly higher (62% vs 43%) [10]. In patients with more advanced endometriosis, IVF also appears to be more successful than a repeated surgical treatment [11]. In the series by Pagidas et al, the cumulative pregnancy rates at 3, 7 and 9 months after surgery were 5.9%, 18.1%, and 24.4%, respectively, whereas after one or two cycles of IVF, the respective cumulative pregnancy rates were 33.3% and 69.6%. The results of these studies and others demonstrate that assisted reproduction and, in particular IVF, is an effective treatment for endometriosisrelated infertility and in certain cases, limits will often be the treatment of choice. Surgical treatments will only lead to a modest improvement in fertility [12,13]. IVF OUTCOME IN ENDOMETRIOSIS-RELATED INFERTILITY The effect of endometriosis on the success rates of IVF treatment remains an issue of some debate. One of the main problems has been a variety of conflicting studies either demonstrating a negative impact of endometriosis or no impact at all.

In Vitro Fertilization Results

317

There have been two primary theories for the proposed poor outcome after IVF in patients with endometriosis. First, the oocyte quality is poor, resulting in lower fertilization rates. Second, implantation is impaired either as a result of endometrial dysfunction or combined with poor oocyte or embryo quality. Animal studies using serum from infertile women with endometriosis have shown a direct negative effect on both fertilization and early embryogenesis when compared to serum from fertile women without endometriosis [3]. The effect increased with the severity of endometriosis. This implies that any adverse effect of endometriosis is related to impaired oocyte or embryo quality. In addition, studies involving donor oocytes demonstrated similar implantation or pregnancy rates in recipients with endometriosis compared to recipients with other causes of infertility [4,5]. The initial studies on women with endometriosis undergoing IVF treatment suggested that fertilization rates were lower when compared with either tubal or unexplained infertility [13,14]. Today, many larger studies have shown similar fertilization rates in women with endometriosis compared with women with no endometriosis [15,16]. It has been suggested that the presence of endometriosis impairs implantation [17]. Arici et al. retrospectively studied 35 patients undergoing 89 cycles of IVF treatment. The implantation rate in women with a diagnosis of endometriosis was 3.9% compared with 7.2% and 8.1% in those with unexplained and tubal factor infertility, respectively [17]. The concept of impaired implantation was further supported by another study by Simon et al. that demonstrated reduced implantation and pregnancy rates [18]. Interestingly this study also showed that recipients of donor oocytes had lower implantation rates when the oocytes originated from women with endometriosis. To further confuse the issue, several subsequent studies have refuted the effect of endometriosis on implantation or pregnancy rates. In one of the largest series, Olivennes et al. compared 214 patients with endometriosis undergoing 360 cycles of IVF treatment to a control group of 111 patients with tubal factor infertility. There were no differences in the pregnancy rates between the two groups. The pregnancy rates among subgroups of patients with pure endometriosis, endometriosis, and tubal factor or endometriosis and other factors were also similar [19]. Geber et al. compared patients with endometriosis to those with male factor, unexplained, and tubal factor infertility. The implantation rates and clinical pregnancy rates were not statistically different between the four groups [15]. Of interest, the stage of endometriosis had no impact on the outcome [15,20,21]. It suggests that implantation and pregnancy rates are similar in women with minimal to mild disease as in those with moderate to severe endometriosis. It is interesting to note that whereas many of the earlier studies suggested negative impact of endometriosis on the IVF outcome, more recent

318

Kelly et al.

studies demonstrated minimal or no impact. This may be partly the result of the now almost universal use of gonaotropin-releasing hormone agonists (GnRH-a) before ovarian stimulation during IVF. Chedid et al. evaluated 145 patients with endometriosis and 174 IVF cycles. Compared with those not using GnRHa, the pregnancy rates in women pretreated with GnRH-a 3 weeks or 3 months before IVF were higher. There was no difference in the pregnancy rates between the two durations of GnRH-a [22]. Dicker et al. also demonstrated that prolonged treatment with GnRH-a before IVF was superior to IVF without GnRH-a therapy in patients with endometriosis [23]. Marcus and Edwards showed that pregnancy rates were higher when patients with endometriosis were pretreated with prolonged GnRH-a as opposed to the short or ultrashort GnRH-a treatment [24]. However, it is also known that regardless of the diagnosis, the IVF pregnancy rates with long protocol GnRH-a are superior to either not using GnRH-a or the short protocols [25,26]. Whether longer treatment with GnRH-a compared to the standard long protocol will improve the results remains unknown. In a small study, Surrey et al. suggested that a 3-month course of GnRH a before ovarian stimulation in patients with endometriosis led to improved pregnancy rates compared with a standard midluteal GnRH a protocol [27]. OVARIAN ENDOMETRIOMA AND IVF Ovarian endometriomas are relatively common in infertile women. They are unresponsive to medical therapy and often require surgical removal. It has been proposed that endometriomas in particular have a detrimental effect on the outcome of IVF. This is either as a result of the presence of the endometrioma itself or the damage to surrounding ovarian tissue after surgical treatment [28,29]. Nargund et al. showed that women who had had an ovarian cystectomy had lower pregnancy rates after IVF compared with a control group of women who had not had ovarian surgery. In addition the number of oocytes collected from the ovary that had been operated on was significantly lower than from the contralateral ovary. There was no difference in oocyte yield from either ovary in women who had not had surgery [30]. More recent evidence would suggest that the surgical method used might have an impact on subsequent response during IVF treatment. Donnez et al. evaluated 85 women undergoing IVF after fenestration and ablation of the endometrioma and a control group of 289 women with tubal factor infertility. The pregnancy rates were similar [31]. The effectiveness of this technique at minimizing any damage to the surrounding ovarian tissue and limiting any effect on subsequent IVF treatment also has been supported by a number of other studies [32–34].

In Vitro Fertilization Results

319

It should be remembered, however, that the presence of endometrioma increases the risk of infection after oocyte collection. This is especially the case when the endometrioma is punctured during the collection. Several case reports have shown that ovarian abscess formation and prophylactic antibiotics should be used in these situations [35,36]. From a practical point of view, the question of whether endometriosis has a significant effect on the outcome of IVF remains unclear. However a recent meta-analysis has perhaps given us the most comprehensive assessment of this issue [37]. This meta-analysis included 22 studies involving 2377 women with endometriosis and 4383 women without endometriosis undergoing IVF treatment (Table 1). The results suggest that the chance of achieving a pregnancy is lower for patients with endometriosis (odds ratio of 0.56 compared to tubal factor infertility). When compared to other diagnoses, endometriosis results in a 19% reduction in the chance of pregnancy (Fig. 1). Furthermore, multivariate analysis demonstrated reduced rates of fertilization and implantation for patients with endometriosis. The number of oocytes collected from women with endometriosis was significantly lower. Also, the pregnancy rates in women with severe endometriosis were lower than those with mild disease (odds ratio of 0.60). Accordingly, if endometriosis has an effect, it primarily impairs the developing follicle, oocyte, and early embryo rather than a direct effect on the uterine environment. One potentially confounding factor for many of these studies is the possibility that the control groups may have had endometriosis. It is not clear whether the control groups had endometriosis conclusively excluded by laparoscopy before IVF treatment. Whether endometriosis may have an adverse effect on the uterine environment, especially in the context of controlled ovarian hyperstimulation (COH) during IVF, is unclear. In a small study, Check et al. evaluated the outcomes from a shared oocyte program [38]. The pregnancy rates were similar in recipients receiving oocytes from donors with or without endometriosis. However, there was a trend to lower pregnancy rates in the donors with endometriosis [38]. Previous studies [4,5] had demonstrated that recipients with endometriosis had no reduction in pregnancy rates, suggesting that endometriosis may adversely effect the uterine environment when COH is used. An adverse effect on the uterine environment as a result of COH alone has also been implied from previous studies [39,40]. SUMMARY As a disease entity, endometriosis remains somewhat of an enigma. There are still many unanswered questions relating to its etiology, symptomatology, and treatment. Its relation to infertility is clearer, and it is particularly evi-

1984–1988 1988 1988–1990 1990–1993 1991–1993

1991 1992 1992 1994 1995 1995 1995 1995 1996 1996 1996 1997 1997 1998 1998 1998

Gerber et al (35) Olivennes et al (13) Tanbo et al (37) Arici et al (22) Cahill et al (20)a Padigas et al (14) Huang et al (33) Issacs et al (44) Bergendal et al (34) Pal et al (21) Yanushpolsky et al (24) 1990–1994 1993 1993–1997 1994–1997 1994–1997 1994–1995

1988–’91 1988–’92 1986–’94 1988–’94

Patient type

Endo, I-IV by stage Endo, all stages Endo, all stages Endo, all stages Endo, all stages + other infertility factors Endo, all stages Endo, all stages Endo, I Endo, I-II and Endo III-IV Endo, all stages Endo, II-III Endo, I-II and III-IV Endo, all stages and III-IV Endo, all stages Endo, I-II and III-IV Endo, III-IV

Endo, all stages, +/ tubal factor Endo treated, separated by stage I-II and III-IV Endo, all stages Endo, I-IV by stage Endo, II-III Endo, all stages

Excludes medroxyprogesterone acetate study group. Endo = endometriosis; N/A = not available. Barnhart. IVF in endometriosis-associated infertility. Fertil Steril 2002

a

1986 1986

1985 1986 1987 1987

Wardle et al (40) Matson and Yovitch (10) Devroey et al (42) Frydman and Belaisch-Allart (39) Tummon et al (43) Inoue et al (36) Mills et al (38) Simo´n et al (11) Dmowski et al (12)

1981–1984

1985

Chillik et al (23)

1981–1982

Study dates

1983

Publication date

Characteristics of 22 Studies Used in Meta-analysis

Mahadevan et al (41)

Reference

TABLE 1

129 236 265 89 22 37 75 147 65 85 37

240 309 62 96 119

17 154 16 53

39

14

No. of cycles

Tubal Tubal Tubal Tubal Tubal Tubal Tubal None Tubal None N/A

factor

factor factor factor factor factor factor factor

None All other infertility Tubal factor Tubal factor All other infertility

Tubal factor Tubal factor None Tubal factor

None

Tubal factor

Control type

1,139 160 331 147 48 414 60 0 98 0

0 372 122 96 118

47 40 0 933

0

261

No. of cycles

320 Kelly et al.

In Vitro Fertilization Results

321

FIGURE 1 Unadjusted meta-analysis of odds of pregnancy in endometriosis patients versus tubal factor controls. Barnhart. IVF in endometriosis-associated infertility. From Ref. 37.

dent when its effect on IVF outcome is considered. Many studies have, unfortunately, resulted in conflicting results, making it difficult for clinicians to counsel such patients adequately. The recent meta-analysis by Barnhart [37] is likely to represent the most up-to-date and detailed review of the impact of endometriosis. It implies a significant negative effect of endometriosis on the likelihood of achieving pregnancy after IVF treatment, compared to other infertility diagnoses. This should not detract from the fact that IVF remains a highly suitable and successful treatment for endometriosis related infertility. It offers a significantly better chance of conception than medical or surgical treatment and is superior to other forms of assisted conception. Patients should be offered IVF treatment with a minimum of delay. PRACTICAL POINT 

IVF is the most successful treatment for endometriosis related infertility.

322

Kelly et al.

REFERENCES 1. 2.

3.

4. 5.

6.

7.

8.

9.

10.

11.

12.

13. 14.

15.

16.

Eskenazi B, Warner ML. Epidemiology of endometriosis. Obstet Gynecol Clin North Am 1997; 24:235–238. Berube S, Marcoux S, Maheux R. Characteristics related to the prevalence of minimal or mild endometriosis in infertile women. Canadian Collaborative Group on Endometriosis. Epidemiology 1998; 9:504–510. Pellicer A, Oliveira N, Ruiz A, Remohi J, Simon C. Exploring the mechanism(s) of endometriosis related infertility: An analysis of embryo development and implantation in assisted reproduction. Hum Reprod 1995; 10:91–97. Lucena E, Cubillos J. Immune abnormalities in endometriosis compromising fertility in IVF-ET patients. J Reprod Med 1999; 44:458–464. Miller KA, Pittaway DE, Detain JL. The effect of serum from infertile women with endometriosis on fertilization and early embryonic development in a murine in vitro model. Fertil Steril 1995; 63:623–626. Simo´n C, Gutierrez A, Vidal A, del los Santos MJ, Tarin JJ, Remohi J, Pellicer A. Outcome of patients with endometriosis in assisted reproduction: Results from in-vitro fertilization and oocyte donation. Hum Reprod 1994; 9:725–729. Sung L, Mukherjee T, Takeshige T, Bustillo M, Cooperman A. Endometriosis is not detrimental to embryo implantation in oocyte recipients. J Asst Reprod Gen 1997; 14:152–156. Tan SL, Royston P, Campbell S, Jacobs HS, Betts J, Mason BA, Edwards RG. Cumulative conception and live birth rates after in vitro fertilization. Lancet 1992; 339:1390–1394. Tummon IS, Asher LJ, Martin JS, Tulandi T. Randomized controlled trial of superovulation and insemination for infertility associated with minimal or mild endometriosis. Fertil Steril 1997; 68:8–12. Kodama H, Fukuda J, Karube H, Matsui T, Shimizu Y, Tanaka T. Benefit of in vitro fertilization treatment for endometriosis-associated infertility. Fertil Steril 1996; 66:974–979. Pagidas K, Falcone T, Hemmings R, Miron P. Comparison of reoperation from moderate (stage III) and severe (stage IV) endometriosis related infertility with in vitro fertilization-embryo transfer. Fertil Steril 1996; 65:791–795. Marcoux S, Maheux R, Berube S. Canadian Collaborative Group on Endometriosis. Laparoscopic surgery in infertile women with minimal or mild endometriosis. N Engl J Med 1997; 337:217–222. Matson PL, Yovitch JL. The treatment of infertility associated with endometriosis by in vitro fertilization. Fertil Steril 1986; 46:432–434. Wardle PG, Mitchell JD, McLaughlin EA, Ray BD, McDermott A, Hull MGR. Endometriosis and ovulatory disorder: Reduced fertilization compared with tubal and unexplained infertility. Lancet 1985; ii:236–239. Geber S, Paraschos T, Atkinson G, Margara R, Winston RML. Results of IVF in patients with endometriosis: The severity of the disease does not affect outcome, or the incidence of miscarriage. Hum Reprod 1995; 10:1507–1511. Dmowski WP, Rana N, Michalowska J, Friberg J, Papierniak C, el-Roeiy A. The

In Vitro Fertilization Results

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28. 29.

323

effect of endometriosis, its stage and activity, and of autoantibodies on in vitro fertilization and embryo transfer success rates. Fertil Steril 1995; 63:555–562. Arici A, Oral E, Bukulmez O, Duleba A, Olive DL, Jones EE. The effect of endometriosis on implantation: Results from the Yale University in vitro fertilization program. Fertil Steril 1996; 65:603–607. Simon C, Gutierrez A, Vidal A, de los Santos MJ, Tarin JJ, Remohi J, Pellicer A. Outcome of patients with endometriosis in assisted reproduction: Results from in vitro fertilization and oocyte donation. Hum Reprod 1994; 9:725–729. Olivennes F, Feldberg D, Liu HC, Cohen J, Moy F, Rosenwaks Z. Endometriosis: A stage by stage analysis—the role of in vitro fertilization. Fertil Steril 1995; 64:392–398. Bukulmez O, Yarali H, Gurgan T. The presence and extent of endometriosis do not effect clinical pregnancy and implantation rates in patients undergoing intracytoplasmic sperm injection. Eur J Obstet Gynecol Reprod Biol 2001; 96: 102–107. Inoue M, Kobayashi Y, Honda I, Awaji H, Fujii A. The impact of endometriosis on the reproductive outcome of infertile patients. Am J Obstet Gynecol 1992; 167:278–282. Chedid S, Camus M, Smitz J, Van Steirteghem AC, Devroey P. Comparison among different ovarian stimulation regimens for assisted procreation procedures in patients with endometriosis. Hum Reprod 1995; 10:2406–2411. Dicker D, Goldman GA, Ashkenazi J, Feldberg D, Voliovitz I, Goldman JA. The value of pre-treatment with gonadotrophin releasing hormone (GnRH) analogue in IVF-ET therapy for severe endometriosis. Hum Reprod 1990; 5:418– 420. Marcus SF, Edwards RG. High rates of pregnancy after long-term down regulation of women with severe endometriosis. Am J Obstet Gynecol 1994; 171:812– 817. Antoine JM, Salat-Baroux J, Alvarez S, Cornet D, Tibi C, Mandelbaum J, Plachot M. Ovarian stimulation using human menopausal gonadotrophins with or without LHRH analogues in a long protocol for in vitro fertilization: A prospective randomised comparison. Hum Reprod 1990; 5:565–569. Tan SL, Kingsland C, Campbell S, Mills C, Bradfield J, Alexander N, Yovich J, Jacobs HS. The long protocol of administration of gonadotrophin-releasing hormone agonist is superior to the short protocol for ovarian stimulation for in vitro fertilization. Fertil Steril 1992; 57:810–814. Surrey ES, Silverberg KM, Surrey MW, Schoolcraft WB. Effect of prolonged gonadotropin-releasing hormone agonist therapy on the outcome of in vitro fertilization-embryo transfer in patients with endometriosis. Fertil Steril 2002; 78:699–704. Donnez J, Nisolle M, Gillet N. Large ovarian endometriomas. Hum Reprod 1996; 11:641–646. Loh FH, Tan A, Kumar J. Ovarian response after laparoscopic ovarian cystectomy for endometriotic cysts in 132 monitored cycles. Fertil Steril 1999; 72:316–321.

324

Kelly et al.

30. Nargund G, Cheng WC, Parsons J. The impact of ovarian cystectomy on ovarian response to stimulation during in-vitro fertilization cycles. Hum Reprod 1996; 11:81–83. 31. Donnez J, Wyns C, Nisolle M. Does ovarian surgery for endometriomas impair the ovarian response to gonadotropin? Fertil Steril 2001; 76:662–665. 32. Canis M, Pouly JL, Tamburro S, Mage G, Wattiez A, Bruhat MA. Ovarian response during IVF-embryo transfer cycles after laparascopic ovarian cystectomy for endometriotic cysts of >3 cm in diameter. Hum Reprod 2001; 16: 2583–2586. 33. Marconi G, Vilela M, Quintana R, Sueldo C. Laparascopic ovarian cystectomy of endometriomas does not affect the ovarian response to gonadotropin stimulation. Fertil Steril 2002; 78:876–878. 34. Jones KD, Sutton CJ. Pregnancy rates following ablative laparascopic surgery for endometriomas. Hum Reprod 2002; 17:782–785. 35. Padilla SL. Ovarian abscess following puncture of an endometrioma during ultrasound guided oocyte retrieval. Hum Reprod 1993; 8:1282–1283. 36. Yaron Y, Peyser MR, Samuel D, Amit A, Lessing JB. Infected endometriotic cysts secondary to oocyte aspiration for in-vitro fertilization. Hum Reprod 1994; 9:1759–1760. 37. Barnhart K, Dunsmoor-Su R, Coutifaris C. Effect of endometriosis on in vitro fertilization. Fertil Steril 2002; 77:1148–1155. 38. Check JH, Maze C, Davies E, Wilson C. Evaluation of the effect of endometriosis on oocyte quality and endometrial environment by comparison of donor and recipient outcomes following embryo transfer in a shared oocyte program. Fertil Steril 2002; 76:S201. 39. Check JH, Choe JK, Katsoff D, Summers-Chase D, Wilson C. Controlled ovarian hyperstimulation adversely effects implantation following in vitro fertilization-embryo transfer. J Assist Reprod Gen 1999; 16:416–420. 40. Sterzik K, Dallenbach C, Schneider V, Sasse V, Dallenbach-Hellweg G. In vitro fertilization: The degree of endometrial insufficiency varies with the type of ovarian stimulation. Fertil Steril 1988; 50:457–462.

Index

Add-back therapy, 226 Adhesion molecule, 129 Animal model, 81, 92 Antibody: antiendometrial antibody, 137, 138 antiphospholipid, 152 Antiprogesterone, 176 Appearance: black lesions, 21 deep endometriosis, 4, 10 macroscopic, 85 microscopic, 85 subtle endometriosis, 2, 8, 74 typical endometriosis, 3, 8 white lesions, 21 Aromatase: aromatase expression, 139, 192, 194

[Aromatase] aromatase inhibitor, 176, 189, 196 Aspiration of endometrioma, 265 Baboon, 85, 92 Barium enema, 32 CA-125, 124–125, 127, 130 CA19-9, 128 Cancer, 10 Cellular immune response, 101 Classification, 6, 34, 50, 72 Cloning, 61 Coagulation, 76 Cyst, 3, 10, 263, 283, 318 (see Endometrioma) Cytokeratines, 139 325

326 Cytokines: as a screening tool, 135 chemistry, 131 cytokines, 104, 105, 131, 132, 193 individual, 133 peritoneal fluid, 132 Danazol, 171, 224 Definition, 70 Denervation, 284 Detoxifixation mechanism, 60 Embryo toxicity, 156 Endometrioma 263, 318 Endometrium, 195 Epidemiology, 1, 274 Estrogen, 190, 191, 196, 227, 228, 231 Excision, 76 Fallopian tube, 47 Fenestration and ablation, 266 Genetics, 55, 139 Gestrinone, 171 GnRH: agonist, 173, 174, 198, 219, 221, 227, 237 antagonist, 175, 219, 237 Growth factors, 104, 193

Index Immunotherapy, 99 Implantation, 156, 158, 178 Infertility, 90, 109, 151, 178, 209, 315, 295 Inflammatory changes, 109 Interleukin, 104, 133, 135 Intravenous pyelography, 34 In-vitro fertilization 264, 295, 300, 315 Laparoscopic uterine nerve ablation, 284 Lateral predisposition of endometriosis, 50 LUNA, 285 Luteal defect, 157 Luteinized unruptured follicle, 90, 91 Lymphocytes, 102 Macrophages, 101, 152 Magnetic resonance imaging, 34 Malignancy, 254, 264 Markers: genetic, 139 immunologic marker, 131 peritoneal markers, 123 serum markers, 123 Metaplasia theory, 6 Monocyte chemotactic protein, 106 Natural killer cells, 101

Hormones: after surgery, 253, 255 dysregulation, 156 hormonal pathway, 60 hormonal therapy, 168, 219 hormone receptors, 140 hormones, 60, 140, 207 Humoral immune response, 102, 103 Hypothesis of evolution, 22 Hysterectomy, 245, 247 Immune modulators, 111, 176 Immunology, 88, 99, 131, 131, 154 Immunosuppression, 88

Oocytes: oocyte development, 156 oocyte maturation, 155 sperm-oocyte interaction, 152, 155 Oophorectomy, 245, 247 Oral contraceptives, 160 Oxidative stress, 25, 138 Pain, 76, 110, 207, 273 Pathophysiology, 6, 19, 50, 88, 100, 206, 277 Polypeptides, 125, 130, 193

Index Presacral neurectomy, 286 Prevalence, 8 Primate model, 83 Progestins, 169–170, 203, 224, 233 Pseudopregnancy, 169 Pulmonary endometriosis, 50 RANTES, 107, 135, 193 Receptor, 206 Rectovaginal endometriosis, 19, 31, 71 Recurrence, 211 Red lesions, 21 Research aspect, 69, 82 Resistant endometriosis, 246 Retrocervical endometriosis, 31, 73 Retrograde menstruation, 6, 50, 59, 85, 86 Sampson, 6 Sclerotherapy, 265 Screening, 124 Selective progestrerone receptor modulator, 212 Sites of endometriosis: anatomical sites, 45 bladder, 48 bowel, 48 cesarean section scar, 49 cul de sac, 46 episotomy scar, 49 extrapelvic, 49 ovarian endometriosis, 19, 27, 46, 263, 315 perineal endometriosis, 49 peritoneal endometriosis, 19 rectovaginal endometriosis, 19, 31, 71, 281 retrocervical endometriosis, 31, 73

327 [Sites of endometriosis] umbilical endometriosis, 50 vaginal endometriosis, 248 Sperm: sperm-oocyte interaction, 152, 155 motility, 155 Spontaneous evolution, 89 Stages, 6, 34, 50, 72, 276 Stripping of endometrioma, 266, 268 Superovulation, 295 Theories, 6, 22 Therapy, 75 (see Treatment) Treatment: add-back therapy, 226 alternative treatment, 279 aromatase inhibitor, 197 combination therapy, 210, 232 empirical treatment, 234 hormonal therapy, 168, 189, 219 medical therapy, 167, 177, 203, 264, 278 need of treatment, 75 progestagen, 203, 205 surgical 179, 250, 251, 265, 266, 279 Tumor necrosis factor, 108, 133, 152, 153 Transrectal sonography, 32 Ultrasonography, 32 Ureter, 48 Vascular endothelial growth factor, 108, 134, 152 White blood cells, 88