Lung Biology in Health & Disease Volume 151 Fetal Origins of Cardiovascular and Lung Disease

FETAL ORIGINS OF CARDIOVASCULAR AND LUNG DISEASE

LUNG BIOLOGY IN HEALTH AND DISEASE Executive Editor Claude Lenfant Director, National Heart, Lung and Blood Institute National Institutes of Health Bethesda, Maryland

1. Immunologic and Infectious Reactions in the Lung, edited by C. H. Kirkpatrick and H. Y. Reynolds 2. The Biochemical Basis of Pulmonary Function, edited by R. G. Crystal 3. Bioengineering Aspects of the Lung, edited by J. B. West 4. Metabolic Functions of the Lung, edited by Y. S. Bakhle and J. R. Vane 5. Respiratory Defense Mechanisms (in two parts), edited by J. D. Brain, D. F. Proctor, and L. M. Reid 6. Development of the Lung, edited by W. A. Hodson 7. Lung Water and Solute Exchange, edited by N. C. Staub 8. Extrapulmonary Manifestations of Respiratory Disease, edited by E. D. Robin 9. Chronic Obstructive Pulmonary Disease, edited by T. L. Petty 10. Pathogenesis and Therapy of Lung Cancer, edited by C. C. Harris 11. Genetic Determinants of Pulmonary Disease, edited by S. D. Litwin 12. The Lung in the Transition Between Health and Disease, edited by P. T. Macklem and S. Permutt 13. Evolution of Respiratory Processes: A Comparative Approach, edited by S. C. Wood and C. Lenfant 14. Pulmonary Vascular Diseases, edited by K. M. Moser 15. Physiology and Pharmacology of the Airways, edited by J. A. Nadel 16. Diagnostic Techniques in Pulmonary Disease (in two parts), edited by M. A. Sackner 17. Regulation of Breathing (in two parts), edited by T. F. Hornbein 18. Occupational Lung Diseases: Research Approaches and Methods, edited by H. Weill and M. Turner-Warwick 19. Immunopharmacology of the Lung, edited by H. H. Newball 20. Sarcoidosis and Other Granulomatous Diseases of the Lung, edited by B. L. Fanburg 21. Sleep and Breathing, edited by N. A. Saunders and C. E. Sullivan 22. Pneumocystis carinii Pneumonia: Pathogenesis, Diagnosis, and Treatment, edited by L. S. Young 23. Pulmonary Nuclear Medicine: Techniques in Diagnosis of Lung Disease, edited by H. L. Atkins 24. Acute Respiratory Failure, edited by W. M. Zapol and K. J. Falke 25. Gas Mixing and Distribution in the Lung, edited by L. A. Engel and M. Paiva

26. High-Frequency Ventilation in Intensive Care and During Surgery, edited by G. Carlon and W. S. Howland 27. Pulmonary Development: Transition from Intrauterine to Extrauterine Life, edited by G. H. Nelson 28. Chronic Obstructive Pulmonary Disease: Second Edition, edited by T. L. Petty 29. The Thorax (in two parts), edited by C. Roussos and P. T. Macklem 30. The Pleura in Health and Disease, edited by J. Chrétien, J. Bignon, and A. Hirsch 31. Drug Therapy for Asthma: Research and Clinical Practice, edited by J. W. Jenne and S. Murphy 32. Pulmonary Endothelium in Health and Disease, edited by U. S. Ryan 33. The Airways: Neural Control in Health and Disease, edited by M. A. Kaliner and P. J. Barnes 34. Pathophysiology and Treatment of Inhalation Injuries, edited by J. Loke 35. Respiratory Function of the Upper Airway, edited by O. P. Mathew and G. Sant’Ambrogio 36. Chronic Obstructive Pulmonary Disease: A Behavioral Perspective, edited by A. J. McSweeny and I. Grant 37. Biology of Lung Cancer: Diagnosis and Treatment, edited by S. T. Rosen, J. L. Mulshine, F. Cuttitta, and P. G. Abrams 38. Pulmonary Vascular Physiology and Pathophysiology, edited by E. K. Weir and J. T. Reeves 39. Comparative Pulmonary Physiology: Current Concepts, edited by S. C. Wood 40. Respiratory Physiology: An Analytical Approach, edited by H. K. Chang and M. Paiva 41. Lung Cell Biology, edited by D. Massaro 42. Heart–Lung Interactions in Health and Disease, edited by S. M. Scharf and S. S. Cassidy 43. Clinical Epidemiology of Chronic Obstructive Pulmonary Disease, edited by M. J. Hensley and N. A. Saunders 44. Surgical Pathology of Lung Neoplasms, edited by A. M. Marchevsky 45. The Lung in Rheumatic Diseases, edited by G. W. Cannon and G. A. Zimmerman 46. Diagnostic Imaging of the Lung, edited by C. E. Putman 47. Models of Lung Disease: Microscopy and Structural Methods, edited by J. Gil 48. Electron Microscopy of the Lung, edited by D. E. Schraufnagel 49. Asthma: Its Pathology and Treatment, edited by M. A. Kaliner, P. J. Barnes, and C. G. A. Persson 50. Acute Respiratory Failure: Second Edition, edited by W. M. Zapol and F. Lemaire 51. Lung Disease in the Tropics, edited by O. P. Sharma 52. Exercise: Pulmonary Physiology and Pathophysiology, edited by B. J. Whipp and K. Wasserman 53. Developmental Neurobiology of Breathing, edited by G. G. Haddad and J. P. Farber 54. Mediators of Pulmonary Inflammation, edited by M. A. Bray and W. H. Anderson 55. The Airway Epithelium, edited by S. G. Farmer and D. Hay

56. Physiological Adaptations in Vertebrates: Respiration, Circulation, and Metabolism, edited by S. C. Wood, R. E. Weber, A. R. Hargens, and R. W. Millard 57. The Bronchial Circulation, edited by J. Butler 58. Lung Cancer Differentiation: Implications for Diagnosis and Treatment, edited by S. D. Bernal and P. J. Hesketh 59. Pulmonary Complications of Systemic Disease, edited by J. F. Murray 60. Lung Vascular Injury: Molecular and Cellular Response, edited by A. Johnson and T. J. Ferro 61. Cytokines of the Lung, edited by J. Kelley 62. The Mast Cell in Health and Disease, edited by M. A. Kaliner and D. D. Metcalfe 63. Pulmonary Disease in the Elderly Patient, edited by D. A. Mahler 64. Cystic Fibrosis, edited by P. B. Davis 65. Signal Transduction in Lung Cells, edited by J. S. Brody, D. M. Center, and V. A. Tkachuk 66. Tuberculosis: A Comprehensive International Approach, edited by L. B. Reichman and E. S. Hershfield 67. Pharmacology of the Respiratory Tract: Experimental and Clinical Research, edited by K. F. Chung and P. J. Barnes 68. Prevention of Respiratory Diseases, edited by A. Hirsch, M. Goldberg, J.P. Martin, and R. Masse 69. Pneumocystis carinii Pneumonia: Second Edition, edited by P. D. Walzer 70. Fluid and Solute Transport in the Airspaces of the Lungs, edited by R. M. Effros and H. K. Chang 71. Sleep and Breathing: Second Edition, edited by N. A. Saunders and C. E. Sullivan 72. Airway Secretion: Physiological Bases for the Control of Mucous Hypersecretion, edited by T. Takishima and S. Shimura 73. Sarcoidosis and Other Granulomatous Disorders, edited by D. G. James 74. Epidemiology of Lung Cancer, edited by J. M. Samet 75. Pulmonary Embolism, edited by M. Morpurgo 76. Sports and Exercise Medicine, edited by S. C. Wood and R. C. Roach 77. Endotoxin and the Lungs, edited by K. L. Brigham 78. The Mesothelial Cell and Mesothelioma, edited by M.-C. Jaurand and J. Bignon 79. Regulation of Breathing: Second Edition, edited by J. A. Dempsey and A. I. Pack 80. Pulmonary Fibrosis, edited by S. Hin. Phan and R. S. Thrall 81. Long-Term Oxygen Therapy: Scientific Basis and Clinical Application, edited by W. J. O’Donohue, Jr. 82. Ventral Brainstem Mechanisms and Control of Respiration and Blood Pressure, edited by C. O. Trouth, R. M. Millis, H. F. Kiwull-Schöne, and M. E. Schläfke 83. A History of Breathing Physiology, edited by D. F. Proctor 84. Surfactant Therapy for Lung Disease, edited by B. Robertson and H. W. Taeusch 85. The Thorax: Second Edition, Revised and Expanded (in three parts), edited by C. Roussos

86. Severe Asthma: Pathogenesis and Clinical Management, edited by S. J. Szefler and D. Y. M. Leung 87. Mycobacterium avium—Complex Infection: Progress in Research and Treatment, edited by J. A. Korvick and C. A. Benson 88. Alpha 1–Antitrypsin Deficiency: Biology • Pathogenesis • Clinical Manifestations • Therapy, edited by R. G. Crystal 89. Adhesion Molecules and the Lung, edited by P. A. Ward and J. C. Fantone 90. Respiratory Sensation, edited by L. Adams and A. Guz 91. Pulmonary Rehabilitation, edited by A. P. Fishman 92. Acute Respiratory Failure in Chronic Obstructive Pulmonary Disease, edited by J.-P. Derenne, W. A. Whitelaw, and T. Similowski 93. Environmental Impact on the Airways: From Injury to Repair, edited by J. Chrétien and D. Dusser 94. Inhalation Aerosols: Physical and Biological Basis for Therapy, edited by A. J. Hickey 95. Tissue Oxygen Deprivation: From Molecular to Integrated Function, edited by G. G. Haddad and G. Lister 96. The Genetics of Asthma, edited by S. B. Liggett and D. A. Meyers 97. Inhaled Glucocorticoids in Asthma: Mechanisms and Clinical Actions, edited by R. P. Schleimer, W. W. Busse, and P. M. O’Byrne 98. Nitric Oxide and the Lung, edited by W. M. Zapol and K. D. Bloch 99. Primary Pulmonary Hypertension, edited by L. J. Rubin and S. Rich 100. Lung Growth and Development, edited by J. A. McDonald 101. Parasitic Lung Diseases, edited by A. A. F. Mahmoud 102. Lung Macrophages and Dendritic Cells in Health and Disease, edited by M. F. Lipscomb and S. W. Russell 103. Pulmonary and Cardiac Imaging, edited by C. Chiles and C. E. Putman 104. Gene Therapy for Diseases of the Lung, edited by K. L. Brigham 105. Oxygen, Gene Expression, and Cellular Function, edited by L. Biadasz Clerch and D. J. Massaro 106. Beta2-Agonists in Asthma Treatment, edited by R. Pauwels and P. M. O’Byrne 107. Inhalation Delivery of Therapeutic Peptides and Proteins, edited by A. L. Adjei and P. K. Gupta 108. Asthma in the Elderly, edited by R. A. Barbee and J. W. Bloom 109. Treatment of the Hospitalized Cystic Fibrosis Patient, edited by D. M. Orenstein and R. C. Stern 110. Asthma and Immunological Diseases in Pregnancy and Early Infancy, edited by M. Schatz, R. S. Zeiger, and H. N. Claman 111. Dyspnea, edited by D. A. Mahler 112. Proinflammatory and Antiinflammatory Peptides, edited by S. I. Said 113. Self-Management of Asthma, edited by H. Kotses and A. Harver 114. Eicosanoids, Aspirin, and Asthma, edited by A. Szczeklik, R. J. Gryglewski, and J. R. Vane 115. Fatal Asthma, edited by A. L. Sheffer 116. Pulmonary Edema, edited by M. A. Matthay and D. H. Ingbar 117. Inflammatory Mechanisms in Asthma, edited by S. T. Holgate and W. W. Busse 118. Physiological Basis of Ventilatory Support, edited by J. J. Marini and A. S. Slutsky

119. Human Immunodeficiency Virus and the Lung, edited by M. J. Rosen and J. M. Beck 120. Five-Lipoxygenase Products in Asthma, edited by J. M. Drazen, S.-E. Dahlén, and T. H. Lee 121. Complexity in Structure and Function of the Lung, edited by M. P. Hlastala and H. T. Robertson 122. Biology of Lung Cancer, edited by M. A. Kane and P. A. Bunn, Jr. 123. Rhinitis: Mechanisms and Management, edited by R. M. Naclerio, S. R. Durham, and N. Mygind 124. Lung Tumors: Fundamental Biology and Clinical Management, edited by C. Brambilla and E. Brambilla 125. Interleukin-5: From Molecule to Drug Target for Asthma, edited by C. J. Sanderson 126. Pediatric Asthma, edited by S. Murphy and H. W. Kelly 127. Viral Infections of the Respiratory Tract, edited by R. Dolin and P. F. Wright 128. Air Pollutants and the Respiratory Tract, edited by D. L. Swift and W. M. Foster 129. Gastroesophageal Reflux Disease and Airway Disease, edited by M. R. Stein 130. Exercise-Induced Asthma, edited by E. R. McFadden, Jr. 131. LAM and Other Diseases Characterized by Smooth Muscle Proliferation, edited by J. Moss 132. The Lung at Depth, edited by C. E. G. Lundgren and J. N. Miller 133. Regulation of Sleep and Circadian Rhythms, edited by F. W. Turek and P. C. Zee 134. Anticholinergic Agents in the Upper and Lower Airways, edited by S. L. Spector 135. Control of Breathing in Health and Disease, edited by M. D. Altose and Y. Kawakami 136. Immunotherapy in Asthma, edited by J. Bousquet and H. Yssel 137. Chronic Lung Disease in Early Infancy, edited by R. D. Bland and J. J. Coalson 138. Asthma’s Impact on Society: The Social and Economic Burden, edited by K. B. Weiss, A. S. Buist, and S. D. Sullivan 139. New and Exploratory Therapeutic Agents for Asthma, edited by M. Yeadon and Z. Diamant 140. Multimodality Treatment of Lung Cancer, edited by A. T. Skarin 141. Cytokines in Pulmonary Disease: Infection and Inflammation, edited by S. Nelson and T. R. Martin 142. Diagnostic Pulmonary Pathology, edited by P. T. Cagle 143. Particle–Lung Interactions, edited by P. Gehr and J. Heyder 144. Tuberculosis: A Comprehensive International Approach, Second Edition, Revised and Expanded, edited by L. B. Reichman and E. S. Hershfield 145. Combination Therapy for Asthma and Chronic Obstructive Pulmonary Disease, edited by R. J. Martin and M. Kraft 146. Sleep Apnea: Implications in Cardiovascular and Cerebrovascular Disease, edited by T. D. Bradley and J. S. Floras 147. Sleep and Breathing in Children: A Developmental Approach, edited by G. M. Loughlin, J. L. Carroll, and C. L. Marcus

148. Pulmonary and Peripheral Gas Exchange in Health and Disease, edited by J. Roca, R. Rodriguez-Roisen, and P. D. Wagner 149. Lung Surfactants: Basic Science and Clinical Applications, R. H. Notter 150. Nosocomial Pneumonia, edited by W. R. Jarvis 151. Fetal Origins of Cardiovascular and Lung Disease, edited by David J. P. Barker 152. Long-Term Mechanical Ventilation, edited by N. S. Hill

ADDITIONAL VOLUMES IN PREPARATION

Environmental Asthma, edited by R. K. Bush Asthma and Respiratory Infections, edited by D. P. Skoner Airway Remodeling, edited by P. H. Howarth, J. W. Wilson, J. Bousquet, and R. Pauwels Respiratory-Circulatory Interactions in Health and Disease, edited by S. M. Scharf, M. R. Pinsky, and S. Magder The Lung at High Altitudes, edited by T. F. Hornbein and R. B. Schoene The opinions expressed in these volumes do not necessarily represent the views of the National Institutes of Health.

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FETAL ORIGINS OF CARDIOVASCULAR AND LUNG DISEASE Edited by

David J.P. Barker University of Southampton Southampton, United Kingdom

ISBN: 0-8247-0391-X This book is printed on acid-free paper. Headquarters Marcel Dekker, Inc. 270 Madison Avenue, New York, NY 10016 tel: 212-696-9000; fax: 212-685-4540 Eastern Hemisphere Distribution Marcel Dekker AG Hutgasse 4, Postfach 812, CH-4001 Basel, Switzerland tel: 41-61-261-8482; fax: 41-61-261-8896 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 © 2001 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

INTRODUCTION

Is it nature or nurture? At any time in our lives, this question can, and probably should, be asked, especially when we are affected by some pathological state. During the past few years, we have witnessed the emergence of molecular medicine, which has brought, and will continue to bring, remarkable advances in the diagnosis, prevention, and treatment of inherited diseases. Genomics and genetics, the basic roots of molecular medicine, are undoubtedly contributing greatly to uncovering the determinants of what we are, and of what may happen to any of us. About four or five decades ago, the concept of risk factors was introduced, largely as a result of the work done in the Framingham Heart Study. Since that time, much research has been done to examine the role of risk factors in the development of cardiovascular and respiratory diseases. In addition, it has been established that one can successfully prevent, or at least control, a disease by reducing, if not eliminating, the risk factors. The new concept is that the expression of genes may, to some extent, iii

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be regulated by environmental factors or conditions. This is to say that, in effect, both nature and nurture are the determinants of our fate! The impact of genes begins at the time of conception—but when do the nurture determinants begin to influence and modulate gene expression? That is what this book is about. David Barker has been the leading proponent of the thesis that health is, in part, programmed by prenatal life. Surprisingly, this concept has been accepted only slowly in spite of significant biological observation and powerful societal comparison. However, today, the weight of evidence is so strong that it cannot be resisted. What was termed the “Barker hypothesis” has become a scientific movement that rallies basic and clinical researchers as well as experts in epidemiology, nutrition, and other disciplines. This volume, Fetal Origins of Cardiovascular and Lung Diseases, edited by David Barker, assembles what is known today about the prenatal programming of cardiovascular, pulmonary, and renal disease. The chapter authors are most distinguished for their scientific expertise. Moreover, the fact that they are from many continents attests to the universal interest in the subject of this book. That so many, from so many places, work on this subject is further testimony to the importance of David Barker’s foresight. The Lung Biology in Health and Disease series of monographs was conceived with the goal of informing, educating, and challenging those interested in important scientific, medical, and public health issues. This new volume is a major contribution to this goal. As the Executive Editor of this series of monographs, I am extremely grateful to Dr. Barker and to the contributors for the opportunity to present it. Claude Lenfant, M.D. Bethesda, Maryland

PREFACE

Living things are “plastic” in their early lives: their growth and development are molded by the environment. Biology provides us with many dramatic examples of developmental paths operated by environmental switches. Whether an American alligator develops as a male or female depends on the temperature at which the egg is incubated. Whether a male Gelada baboon adopts a rapid or slow growth trajectory at puberty depends on the number of other males in the area. There are many reasons why it may be advantageous, in evolutionary terms, for the body’s structure and function to remain plastic in early life and this is a general phenomenon of early development. Humans are not an exception. The human embryo does not contain a description of the person to whom it will give rise. Rather, it contains in its genes a generative program for making a person, a program that has been likened to a recipe for which the mother provides the ingredients. The plasticity of human development has been known for a long while. Only recently, however, has evidence appeared suggesting that the origins of important chronic diseases of adult life, including coronary heart disease, v

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stroke, and Type 2 diabetes, may lie in fetal responses to the intrauterine environment. The “fetal origins” hypothesis proposes that these disorders originate through adaptations that the fetus makes when it is undernourished. These adaptations permanently change the body’s structure, function, and metabolism. A feature of the early findings, which came mainly from epidemiological studies, was the strength of the associations between small body size at birth and later disease. Important biological effects must underlie such associations, and it soon became evident that these effects could be replicated experimentally in animals, usually by reducing food intake around the time of conception and during pregnancy. Through the efforts of a group of epidemiologists, clinicians, and basic scientists that includes many of the contributors to this book, the early epidemiological findings have been extensively replicated, and incorporated into a framework of ideas derived from fetal physiology, human metabolism, and endocrinology. The framework is described in this book. The first six chapters focus on the cardiovascular system. The substantial body of evidence showing that small size at birth is linked with later coronary heart disease, stroke, hypertension, and chronic renal failure is reviewed. New findings show that the mother’s body composition in pregnancy and particular paths of childhood growth are also associated with later cardiovascular disease. Chapter 3 reviews how fetal cardiovascular adaptations may have lifelong effects on the physiology and structure of the vascular system and thereby lead to hypertension. Chapter 5 examines the effects of fetal nutrition on cardiovascular development. The development of the cardiovascular system in embryonic and fetal life, and possible links with later diseases, are described in Chapter 6. Chapter 7 describes clinical and epidemiological studies of the links between reduced fetal growth and impaired glucose tolerance and Type 2 diabetes in later life. Insulin resistance may be viewed as the long-term price of successful metabolic adaptations in utero. Studies in animals have indicated that undernutrition for brief periods in early life produces a range of persisting metabolic alterations, and Chapter 8 suggests that these may prove disadvantageous if undernutrition in utero is followed by “overnutrition” in later life. Professor Joseph Hoet, a pioneer in this area of experimental science, died during the preparation of this book and an additional chapter (17) has been added in his honor. Clinicians studying the fetal origins of adult disease have to engage with the complexities of the fetus’ metabolic and endocrine responses to undernutrition and other influences. In Chapter 9 reduced fetal growth, with consequent

Preface

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small size at birth, is viewed as an appropriate adaptation to impaired nutrient supply in utero. This adaptation is partly mediated through hormones, which act as nutritional signals throughout fetal development (Chapter 10). Steroid hormones are powerful mediators of fetal programming and are known to be programmable by physiological stimuli during the course of development (Chapter 11). Chapter 12 shows that insights into the long-term effects of hormonal changes in utero may come from the clinic as well as the laboratory. Our understanding of fetal nutrition is currently too slight for us to change dietary advice to women before and during pregnancy. Fetal nutrition depends on the concentrations of nutrients in the maternal circulation, on utero-placental blood flow and transfer across the placenta. Nutrient concentrations in maternal blood are the product of the mother’s diet, body composition, and metabolism. Understanding these processes will be essential if we are to prevent chronic disease by improving fetal nutrition. Chapters 13 and 14 review the evidence in humans and animals. Chapter 15 extends the range of diseases linked to developmental plasticity to include asthma and gives an account of lung embryology and the ontogeny of host defense mechanisms. Chapter 16 examines the implications of the fetal origins hypothesis for the “nutritional transition,” during which societies move from chronic malnutrition to adequate nutrition. This transition exacts a heavy toll of rising epidemics of coronary heart disease and Type 2 diabetes. One of the two goals of the new agenda for medical research set out in this book is to lessen these epidemics by improving the body composition and nutrition of girls and young women and by protecting the growth of young children. There is already sufficient evidence to begin implementing new public health policies without further delay, but a deeper understanding of the biological processes is needed to refine these policies. The other goal of fetal origins research is earlier detection and better treatment of disease. To realize both goals, clinicians, public health physicians, epidemiologists, and basic scientists must join forces. My thanks go to the contributors to this book who have done just that in order to write it. Thanks also to my colleagues, Shirley Simmonds and Pamela Freeman, who helped with the editing, to Sandra Beberman and Paige Force of Marcel Dekker, Inc., who produced the book, and final thanks to Claude Lenfant, who conceived the book and was patient during its long gestation. David J. P. Barker

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CONTRIBUTORS

David J. P. Barker, M.D., Ph.D., F.R.S. Professor and Director, Medical Research Council Environmental Epidemiology Unit, University of Southampton, Southampton, United Kingdom Holly E. Bendall, M.Sc Statistician, Medical Research Council Environmental Epidemiology Unit, University of Southampton and Southampton General Hospital, Southampton, United Kingdom John R. G. Challis, Ph.D., D.Sc., F.R.S.C. Professor and Chair, Department of Physiology, University of Toronto, Toronto, Ontario, Canada David B. Cox, Ph.D. Department of Physiology, University of Toronto, Toronto, Ontario, Canada ix

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Francis de Zegher, M.D., Ph.D. Professor, Department of Pediatrics, University of Leuven, Leuven, Belgium Lisa Edwards, B.Sc. Department of Physiology, University of Adelaide, Adelaide, Australia Brent M. Egan, M.D. Department of Pharmacology, Medical University of South Carolina, Charleston, South Carolina Alison J. Forhead, B.Sc., Ph.D. Department of Physiology, University of Cambridge, Cambridge, United Kingdom Abigail L. Fowden, B.A., M.A., Ph.D. Department of Physiology, University of Cambridge, Cambridge, United Kingdom Inge Francois Department of Pediatrics, University of Leuven, Leuven, Belgium Kathryn L. Gatford, B.Agr.Sc., Ph.D. Department of Physiology, University of Adelaide, Adelaide, Australia Peter D. Gluckman, M.B.Ch.B., D.Sc., F.R.A.C.P., F.R.S.N.Z. Professor of Perinatal and Pediatric Biology, Research Centre for Development Medicine and Biology, Faculty of Medicine and Health Science, University of Auckland, Auckland, New Zealand Keith M. Godfrey, B.M., Ph.D., F.R.C.P. Clinical Scientist, Medical Research Council Environmental Epidemiology Unit, University of Southampton and Southampton General Hospital, Southampton, United Kingdom Stephen E. Greenwald, Ph.D. Reader in Cardiovascular Biomechanics, Histopathology and Morbid Anatomy Department, Royal London Hospital, London, United Kingdom C. Nicholas Hales, M.A., M.D., Ph.D., F.R.C.Path., F.R.C.P., F.Med.Sc., F.R.S. Professor and Head of Department, Department of Clinical Biochemistry, University of Cambridge, Cambridge, United Kingdom

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Mark A. Hanson, B.A., D.Phil. Professor, Department of Obstetrics and Gynecology, Royal Free and University College Medical School, University College London, London, United Kingdom Jane E. Harding, M.B.Ch.B., D.Phil. Professor, Research Centre for Developmental Medicine and Biology, Faculty of Medicine and Health Science, University of Auckland, Auckland, New Zealand Joseph J. Hoet* Professor, Laboratory of Cell Biology, Université Catholique de Louvain, Louvain-la-Neuve, Belgium Lourdes Ibáñez, M.D., Ph.D. Attending Physician, Pediatric Endocrinology, Endocrinology Unit, Hospital Sant Joan de Déu, University of Barcelona, Barcelona, Spain B. J. Jennings Professor, Department of Clinical Biochemistry, University of Cambridge, Cambridge, United Kingdom Catherine A. Jones, Ph.D. Allergy and Inflammation Sciences Division, School of Medicine, University of Southampton and Southampton General Hospital, Southampton, United Kingdom Karen Kind Department of Physiology, University of Adelaide, Adelaide, Australia Ilona Koupilová, M.D., Ph.D., M.Sc., Dr.Med.Sc. Clinical Lecturer in Epidemiology, Department of Epidemiology and Population Health, London School of Hygiene and Tropical Medicine, London, United Kingdom Daniel T. Lackland, Dr.P.H. Associate Professor, Department of Biometry and Epidemiology, Medical University of South Carolina, Charleston, South Carolina David A. Leon, Ph.D. Reader in Epidemiology, Department of Epidemiology and Population Health, London School of Hygiene and Tropical Medicine, London, United Kingdom __________________ * Deceased.

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S. J. Lye Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada Christopher N. Martyn, M.A., D.Phil., F.R.C.P. Senior Clinical Scientist, Medical Research Council Environmental Epidemiology Unit, University of Southampton and Southampton General Hospital, Southampton, United Kingdom Stephen G. Matthews, Ph.D. Assistant Professor, Department of Physiology, University of Toronto, Toronto, Ontario, Canada Roger B. McDonald, Ph.D. Professor, Department of Nutrition, University of California, Davis, California Caroline McMillen Department of Physiology, University of Adelaide, Adelaide, Australia Janna L. Morrison, M.Sc. Faculty of Medicine, University of British Columbia, Vancouver, British Columbia, Canada Clive Osmond, Ph.D. Reader in Medical Statistics and Senior Scientist, Medical Research Council Environmental Epidemiology Unit, University of Southampton and Southampton General Hospital, Southampton, United Kingdom Julie Owens Department of Physiology, University of Adelaide, Adelaide, Australia Susan Ozanne, Ph.D. Department of Clinical Biochemistry, University of Cambridge, Cambridge, United Kingdom David I. W. Phillips, Ph.D., F.R.C.P. Professor, Medical Research Council Environmental Epidemiology Unit, University of Southampton and Southampton General Hospital, Southampton, United Kingdom Barry M. Popkin, Ph.D. Professor, Department of Nutrition, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina

Contributors

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C. Remacle Department of Biology, Laboratory of Cell Biology, Université Catholique de Louvain, Louvain-la-Neuve, Belgium B. Reusens, Ph.D. Department of Biology, Laboratory of Cell Biology, Université Catholique de Louvain, Louvain-la-Neuve, Belgium Jeffrey S. Robinson, B.Sc., M.B., B.Ch., B.A.O., F.R.C.O.G., F.R.A.N.Z. C.O.G. Professor, Department of Obstetrics and Gynaecology, University of Adelaide, Adelaide, Australia Deborah M. Sloboda, M.Sc. Ph.D. Student, Department of Physiology, University of Toronto, Toronto, Ontario, Canada Eric Jackson Thomas, M.D., M.R.C.O.G., F.Med.Sci. Professor, Obstetrics and Gynecology Department, Princess Anne Hospital, Southampton, United Kingdom Kent L. Thornburg, Ph.D. Director, Heart Research Center, and Professor, Department of Physiology and Pharmacology, Oregon Health Sciences University, Portland, Oregon John O. Warner, M.D., F.R.C.P., F.R.C.P.C.H., F.Med.Sci. Professor of Child Health, and Director, Allergy and Inflammation Sciences Division, School of Medicine, University of Southampton and Southampton General Hospital, Southampton, United Kingdom E. Marelyn Wintour, Ph.D., D.Sc. Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne, Parkville, Victoria, Australia

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CONTENTS

Introduction Claude Lenfant Preface Contributors 1. Introduction David J. P. Barker I. The Undernourished Baby II. Other Diseases That Originate In Utero III. Mothers and Babies Today References

iii v ix 1 2 14 17 18

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xvi 2. Birth Weight, Blood Pressure, and Hypertension: Epidemiological Studies David A. Leon and Ilona Koupilová I. Birth Weight and Blood Pressure in Children and Adults II. Systolic Blood Pressure III. Diastolic Blood Pressure IV. Effect of Concurrent Body Size V. Is the Birth Weight–Blood Pressure Association Modified by Age? VI. Birth Weight and Hypertension VII. Studies of Births with Abnormal Obstetric or Neonatal Characteristics VIII. Socioeconomic and Behavioral Pathways IX. Maternal Influences X. Conclusion References 3. Mechanisms for In Utero Programming of Blood Pressure Christopher N. Martyn and Stephen E. Greenwald I. Structure of the Aorta and Large Conduit Arteries II. Summary III. Capillary Density and Microvascular Dilation IV. Renal Size and Function References 4. Low Birth Weight and the Emerging Burden of Renal Disease in the United States Daniel T. Lackland, Holly E. Bendall, Clive Osmond, and Brent M. Egan I. ESRD in South Carolina References 5. Intrauterine Nutrition: Its Importance During Critical Periods for Cardiovascular and Endocrine Development Joseph J. Hoet and Mark A. Hanson I. Summary II. Introduction

Contents

23 24 33 35 36 37 38 39 40 41 43 44 49 50 54 55 56 57 61 66 68 73 73 74

Contents III. IV. V. VI. VII. VIII. IX. X.

xvii Cardiovascular Development Endocrine Pancreas Insulin-Sensitive Tissues Kidney Brain Comparison of Dietary Restriction with Maternal Diabetes Worldwide Perspective Acknowledgments References

6. Physiological Development of the Cardiovascular System In Utero Kent L. Thornburg I. Introduction II. Heart Development in Embryonic Life III. Maturation of the Cardiomyocyte IV. Arrangement of the Fetal Circulation V. Metabolic Features and Cardiovascular Adaptations in the Fetus VI. Fetal Arterial Pressure VII. Regulation of Fetal Cardiac Output VIII. Differences Between the Right and Left Ventricles IX. Alterations in Myocardial Growth with Mechanical Stress X. Redistribution of Cardiac Output with Hypoxemia XI. Regulation of Fetal Coronary Flow XII. Fetal Cardiovascular Development and Adult Disease References 7. Non–Insulin-Dependent Diabetes and Obesity David I. W. Phillips I. Birth Weight and NIDDM II. Fetal Growth and Insulin Resistance III. Fetal Growth and Insulin Secretion IV. Mechanisms Linking Reduced Fetal Growth with Insulin Resistance V. Fetal Growth and Obesity in Adult Life

75 81 84 85 85 85 86 89 89 97 97 98 104 108 110 112 113 118 121 124 125 131 133 141 142 145 148 148 152

xviii VI. VII. VIII.

Contents Early Nutrition and Adult Body Weight Mechanisms Linking Early Environmental Influences with Adult Body Weight Conclusions References

8. Metabolic Alterations After Early Growth Retardation Susan Ozanne, B. J. Jennings, and C. Nicholas Hales I. Introduction II. Early Growth Retardation Consequent on Maternal Protein Deprivation III. Conclusions IV. Future Directions References

153 154 155 155 161 161 162 175 176 177

9. Growth, Metabolic, and Endocrine Adaptations to Fetal Undernutrition 181 Jane E. Harding and Peter D. Gluckman I. Introduction 181 II. Fetal Nutrition and Fetal Growth 182 III. Fetal Metabolic Adaptations to Undernutrition 184 IV. Endocrine Adaptations to Fetal Undernutrition 186 V. Cardiovascular Adaptations to Fetal Undernutrition 187 VI. Factors Influencing Fetal Adaptation to Undernutrition 188 VII. Fetoplacental Adaptations to Refeeding 190 VIII. Cardiovascular Adaptations to Refeeding 191 IX. Endocrine Adaptations to Refeeding 191 X. Conclusion 192 References 193 10. The Role of Hormones in Intrauterine Development Abigail L. Fowden and Alison J. Forhead I. Introduction II. Nutritionally Induced Hormonal Changes III. Endocrine Regulation of Fetal Growth and Development

199 199 200 204

Contents IV. V. VI.

xix Long-Term Consequences of the Nutritionally Induced Hormonal Changes In Utero Conclusions Acknowledgments References

11. The Hypothalamic-Pituitary-Adrenal and HypothalamicPituitary-Gonadal Axes in Early Life: Problems and Perspectives Stephen G. Matthews, David I. W. Phillips, John R. G. Challis, David B. Cox, Eric Jackson Thomas, Caroline McMillen, S. J. Lye, Roger B. McDonald, E. Marelyn Wintour, Janna L. Morrison, Deborah M. Sloboda I. Introduction II. Programming of the HPA Axis III. The HPG Axis IV. Conclusions References 12. Reduced Fetal Growth and Pediatric Endocrinopathies Francis de Zegher, Inge Francois, and Lourdes Ibáñez I. Somatotropic Axis II. Pronounced Adrenarche and Precocious Pubarche III. The Entities of Male Pseudohermaphroditism and Subfertility, Ovarian Hyperandrogenism, and Anovulation IV. Dyslipidemia and Insulin Resistance V. Conclusion References 13. Maternal Nutrition and Fetal Development: Implications for Fetal Programming Keith M. Godfrey I. Introduction II. Size at Birth III. Fetal Nutrient Demand IV. Maternoplacental Nutrient Supply

213 220 220 221

229

229 231 236 237 237 241 242 244 244 246 246 247 249 249 250 251 252

xx

Contents V. VI.

Fetal Adaptations and Developmental Changes An Integrated Framework and Future Research References

14. Maternal and Placental Influences that Program the Fetus: Experimental Findings Jeffrey S. Robinson, Caroline McMillen, Lisa Edwards, Karen Kind, Kathryn L. Gatford, and Julie Owens I. II. III. IV. V. VI.

Introduction Rat Guinea Pig Sheep Conclusion Acknowledgments References

15. Fetal Origins of Lung Disease John O. Warner and Catherine A. Jones I. II. III. IV. V. VI. VII. VIII. IX. X. XI. XII. XIII. XIV. XV.

Introduction Lung Embryology Molecular Basis of Lung Growth Ontogeny of Host Defense Congenital Lung Malformations Fetal Growth and Later Lung Function Infant Wheeze and Fetal Growth The Fetus and Allergic Sensitization Pregnancy as an Allergic Phenomenon Fetal and Maternal Influences on Atopy Maternal Atopy Timing and Concentration of Allergen Exposure and IgG Antibodies Maternal Nutrition and Atopy Conclusions Acknowledgments References

261 264 265

273

273 276 280 282 289 290 290 297 297 298 299 300 302 304 306 307 308 309 310 311 313 315 316 316

Contents 16. The Nutrition Transition and its Implications for the Fetal Origins Hypothesis Barry M. Popkin I. Introduction II. The United States: Immigrants and Other At-Risk Populations III. The Developing World IV. Conclusion References

xxi

323 323 325 327 336 336

17. Effects of Maternal Nutrition and Metabolism on the Developing Endocrine Pancreas: Experimental Findings B. Reusens and C. Remacle I. Introduction II. Programming the Endocrine Pancreas III. Short-Term Consequences IV. Long-Term Consequences V. Mechanism Involved in Altered Beta-Cell Mass VI. Changes in Other Organs VII. Prevention of Programmed Changes VIII. Conclusion References

339 340 341 342 347 349 350 353 353

Author Index Subject Index

359 387

339

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FETAL ORIGINS OF CARDIOVASCULAR AND LUNG DISEASE

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1 Introduction

DAVID J. P. BARKER University of Southampton Southampton, United Kingdom

The search for biological influences in the adult environment that determine the risk of cardiovascular disease has met with limited success. Obesity and cigarette smoking have been implicated and evidence on dietary fat has accumulated to the point where a public health policy of reduced intake is prudent, if not proven. Much, however, remains unexplained. For example, the steep rise in coronary heart disease in many western countries during the past century has been associated with rising prosperity. So why, in many countries, do the poorest people in the least affluent places now have the highest rates? Surprisingly, a clue to the possible importance of early development came from geographical studies. The geographical distribution of neonatal mortality (deaths before 1 month of age) in England and Wales at the beginning of the last century was found to closely resemble the distribution of death rates from cardiovascular disease today (Fig. 1) (1). At that time, most neonatal 1

2

Barker

Figure 1 Similarity between the distribution of neonatal mortality and cardiovascular disease mortality in England and Wales.

deaths were attributed to low birth weight. One interpretation of this geographical association was that harmful influences that act in fetal life, and slow fetal growth, permanently set or “program” the body’s structure and function in ways that are linked to cardiovascular disease. Because fetal growth is mainly determined by the supply of nutrients, undernutrition was an obvious possible harmful influence. As is described later in this book (Chs. 5, 8, 14), numerous studies in animals have shown that undernutrition during gestation permanently programs the body’s physiology and metabolism (2,3). 1. The Undernourished Baby In fetal life, the tissues and organs of the body go through what are called “critical” periods of development that may coincide with periods of rapid cell division. “Programming” describes the process whereby a stimulus or insult at a critical period of development has lasting effects. Rickets has for a long while served as a demonstration that undernutrition at a critical stage of early life leads to persisting changes in the body’s structure. Only recently

Introduction

3

have we realized that some of the persisting effects of early undernutrition become translated into pathology, and thereby determine chronic disease, including cardiovascular disease, in later life. That this has gone unremarked for so long is perhaps surprising, given the numerous animal experiments showing that undernutrition in utero leads to persisting changes in a range of metabolic, endocrine, and immune functions known to be important in human disease (3,4), and given that the heart and blood vessels are “plastic” during intrauterine development (Chs. 3, 4), ie, molded by physical and other influences to which they are subjected. Later chapters in this book (Chs. 9, 10, 11) describe how the human fetus adapts to undernutrition by metabolic changes, redistribution of blood flow, and changes in the production of fetal and placental hormones that control growth (5). Its immediate response to undernutrition is catabolism; it consumes its own substrates to provide energy (6). More prolonged undernutrition leads to a slowing in growth. This enhances the fetus’ ability to survive by reducing the use of substrates and lowering the metabolic rate. Slowing of growth in late gestation leads to disproportion in organ size because organs and tissues that are growing rapidly at the time are affected the most. For example, undernutrition in late gestation may lead to reduced growth of the kidney, which develops rapidly at that time (Ch. 4). Reduced replication of kidney cells in late gestation will permanently reduce cell numbers, because after birth there seems to be no capacity for renal cell division to catch up. Animal studies show that a variety of different patterns of fetal growth result in similar birth size. A fetus that grows slowly throughout gestation may have the same size at birth as a fetus whose growth was arrested for a period and then caught up. Different patterns of fetal growth will have different effects on the relative size of different organs at birth, although overall body size may be the same. Animal studies show that blood pressure and metabolism can be permanently changed by levels of undernutrition that do not influence growth. Preliminary observations point to similar effects in humans. Such findings emphasize the severe limitation of birth weight as a summary of fetal nutritional experience. While slowing its rate of growth, the fetus may protect tissues that are important for immediate survival, especially the brain. One way in which the brain can be protected is by redistribution of blood flow to favor it (7). This adaptation is known to occur in many mammals, but in humans it has exaggerated costs for other tissues, notably the liver and other abdominal viscera, because of the large size of the human brain. Metabolic fetal adaptations may result in the fetus sacrificing muscle growth and being born thin.

4

Barker

It is becoming increasingly clear that nutrition has profound effects on fetal hormones and on the hormonal and metabolic interactions between the fetus, placenta, and mother, on whose coordination fetal growth depends (6). Fetal insulin and insulin-like growth factors (IGFs) are thought to have a central role in the regulation of growth and to respond rapidly to changes in fetal nutrition. If a mother decreases her food intake, fetal insulin, IGF, and glucose concentrations fall, possibly through the effect of decreased maternal IGF. This leads to reduced transfer of amino acids and glucose from mother to fetus, and ultimately to reduced rates of fetal growth (8). In late gestation and after birth, the fetus’ growth hormone and IGF axis take over, from insulin, a central role in driving linear growth. Undernutrition leads to a fall in the concentrations of hormones that control fetal growth and a rise in cortisol, the main effects of which are on cell differentiation (5). One current line of research aims to determine whether the fetus’ hormonal adaptations to undernutrition tend, like many other fetal adaptations, to persist after birth and exert lifelong effects on homeostasis and hence on the occurrence of disease. A. Body Size at Birth and Cardiovascular Disease

The early epidemiological studies on the intrauterine origins of coronary heart disease and stroke were based on the simple strategy of examining men and women in middle and late life whose body measurements at birth were recorded. Sixteen thousand men and women born in Hertfordshire during 1911 to 1930 were traced from birth to the present day. Death rates from coronary heart disease fell twofold among those at the lower and upper ends of the birth weight distribution (Table 1) (9, 10). A study in Sheffield showed that Table 1 Death Rates From Coronary Heart Disease Among 15,726 Men and Women Birth weight (lbs [kg]) ≤5.5 [2.50] 5.5–6.5 [2.95] 6.5–7.5 [3.41] 7.5–8.5 [3.86] 8.5–9.5 [4.31] >9.5 [4.31] All

Standardized mortality ratio

Number of deaths

100 81 80 74 55 65 74

57 137 298 289 103 57 941

Introduction

5

people who were small at birth because they failed to grow, rather than because they were born early, were at increased risk of the disease (11). The association between low birth weight and coronary heart disease has been confirmed in studies in Uppsala, Sweden (12); Helsinki, Finland (13,14); Caerphilly, Wales (15); and among 80,000 women in the United States who took part in the American Nurses Study (16). An association between low birth weight and prevalent coronary heart disease has recently been shown in a study in south India (17). B. Body Proportions at Birth and Cardiovascular Disease The Hertfordshire records and the Nurses and Caerphilly studies did not include measurements of body size at birth other than weight. The weight of a newborn baby without a measure of its length is as crude a summary of its physique as is the weight of a child or adult without a measure of height. The addition of birth length allows a thin baby to be distinguished from a stunted baby with the same birth weight. With the addition of head circumference, the baby whose body is small in relation to its head, which may be a result of brain-sparing redistribution of blood flow, can also be distinguished. Thinness, stunting, and a low birth weight in relation to head size are the result of differing fetal adaptations to undernutrition and other influences, and have different consequences, both immediately and in the longterm. In Sheffield, death rates for coronary heart disease were higher in men who were stunted at birth (18). The mortality ratio for coronary heart disease in men who were 18.5 inches (47 cm) or less in length was 138 compared with 98 in the remainder (18). Similarly, coronary heart disease in south India was associated with stunting (17). Thinness at birth, as measured by a low ponderal index (birth weight/length3), is also associated with coronary heart disease. Table 2 shows findings among men born in Helsinki, Finland during 1924 to 1933. Death rates for coronary heart disease were related to low birth weight (13). Although this was not statistically significant, the strength of the association was similar to that in Hertfordshire and elsewhere. There was, however, a much stronger association with thinness at birth, especially in men born at term (Table 2). Men who were thin at birth, measured by a low ponderal index (birth weight/length3), had death rates that were twice those of men who had a high ponderal index. Interestingly, among women in the same cohort the association between low birth weight and coronary heart disease was similar to that in men, but there was a much stronger association with short body length at birth rather than thinness (14). An inference from this is

6

Barker

Table 2 SMRs for Coronary Heart Disease in 3302 Finnish Men Born During 1924–1933 Birth weight (kg [lbs])

≤2.5 [5.5]

2.5–3.0 [6.6] 3.0–3.5 [7.7] 3.5–4.0 [8.8] >4.0 [8.8] All p value for trend

Ponderal index at birth* (kg/m3) ≤25 25–27 27–29 >29 All p value for trend

SMR (number of deaths) 84 (11) 83 (44) 99 (124) 76 (80) 66 (27) 85 (286) 0.09 SMR (number of deaths)* 116 (59) 105 (88) 72 (64) 56 (33) 86 (244) < 0.0001

* Term babies only. Abbreviation: SMR, standardized mortality ratio.

that girls and boys have different paths of fetal growth at the same levels of maternal nutrition. Girls grow more slowly than boys. Slower-growing fetuses are, in general, less vulnerable to undernutrition, and one may speculate that the lower rates of coronary heart disease in women may originate in their slower rates of intrauterine growth. Trends in stroke, which in Sheffield have only been reported among men, are different from those in coronary heart disease. Although stroke has a similar association with birth weight, it is not related to stunting or thinness. Rather, high rates are associated with a low ratio of birth weight to head circumference. This finding has recently been confirmed in Finland (66). C. Infant Growth and Cardiovascular Disease Information routinely recorded in Hertfordshire included the infant’s weight at 1 year. In men, failure of weight gain during the first year of life predicted

Introduction

7

coronary heart disease and stroke independently of birth weight (18). Figure 2 shows that among men who weighed 17 lbs (7.7 kg) or less at 1 year of age, hazard ratios for coronary heart disease were three times greater than among men who weighed 27 lbs (12.2 kg) or more. The highest rates of the disease among men were in those who had both low birth weight and low weight at 1 year of age. In contrast, the highest rates among women were in those who had low birth weight but whose weight caught up before 1 year. The reasons for this are unknown, although it may reflect sex differences in the endocrine control of infant growth. Growth during infancy can be regarded

Figure 2 Hazard ratios for coronary heart disease in 10,141 men according to weight at 1 year.

8

Barker

as a postnatal continuation of the fetal phase of growth which is controlled by insulin and insulin-like growth factor I, and continues until growth hormone takes over at around the age of 1 year (19). Confounding Variables These findings suggest that influences linked to fetal and infant growth have an important effect on the risk of coronary heart disease and stroke. It has been argued, however, that people whose growth was impaired in utero and during infancy may continue to be exposed to an adverse environment in childhood and adult life, and it is this later environment that produces the effects attributed to programming (20–23). There is strong evidence against this. In three of the studies that have replicated the association between birth weight and coronary heart disease, data on lifestyle factors, including smoking, employment, alcohol consumption, and exercise, were collected (12, 15, 16). The associations between birth weight and coronary heart disease remained after allowing for them. D. Hypertension and Non–Insulin Dependent Diabetes In studies exploring the mechanisms underlying these associations, the trends in coronary heart disease with birth weight have been found to be paralleled by similar trends in two of its major risk factors: hypertension and non–insulin dependent diabetes mellitus (24,25). The extensive literature on these associations is described in detail in later chapters (Chs. 2, 7). Table 3 illustrates the Table 3 Prevalence of NIDDM and Impaired Glucose Tolerance in Men Aged 59–70 Years

Birth weight (lbs [kg]) ≤5.5 [2.50] 5.5–6.5 [2.95] 6.5–7.5 [3.41] 7.5–8.5 [3.86] 8.5–9.5 [4.31] >9.5 [4.31] All

Number of men

Percent with impaired glucose tolerance or NIDDM (plasma glucose ≥ 7.8 mmol/L)

20 47 104 117 54 28 370

40 34 31 22 13 14 25

Odds ratio adjusted for body mass index (95% confidence interval)

Abbreviation: NIDDM, non–insulin dependent diabetes mellitus.

6.6 (1.5–28) 4.8 (1.3–17) 4.6 (1.4–16) 2.6 (0.8–8.9) 1.4 (0.3–5.6) 1.0

Introduction

9

size of these trends. The prevalence of non–insulin dependent diabetes mellitus and impaired glucose tolerance in Hertfordshire men falls threefold across the range of birth weight (25). The associations between small size at birth and hypertension and non–insulin dependent diabetes are again independent of social class, cigarette smoking, and alcohol consumption. Influences in adult life, however, add to the effects of the intrauterine environment. For example, the prevalence of impaired glucose tolerance is highest in people who had low birth weight but became obese as adults. E. Cholesterol and Fibrinogen The published literature on the association between size at birth and the two other main biological risk factors for cardiovascular disease, raised plasma lipid and fibrinogen concentrations, is limited and smaller than that on the associations between size at birth and blood pressure or glucose-insulin metabolism. In studies in Hertfordshire, low birth weight was associated with low-serum high-density lipoprotein (HDL) cholesterol and raised triglyceride concentrations (26). Low birth weight was not related to low-density lipoprotein (LDL) cholesterol concentrations but was strongly related to the insulin resistance syndrome in which impaired glucose tolerance, hypertension, and raised serum triglyceride concentrations, together with other dyslipidemias, occur in the same patient (27). In a study in Sheffield where the birth measurements included abdominal circumference, it was this measurement that most strongly predicted plasma concentrations of total and LDL cholesterol and apolipoprotein B (28). A small abdominal circumference predicted raised lipid concentrations in both men and women (Table 4). Because abdominal circumferTable 4 Mean Serum Cholesterol Concentrations in Men and Women Aged 50–53 Years Abdominal circumference at birth (in [cm])

Number of people

Total cholesterol (mmol/L)

Low density lipoprotein cholesterol (mmol/L)

≤11.5 [29.2] 11.5–12.0 [30.5] 12.0–12.5 [31.8] 12.5–13.0 [33.0] >13.0 [33.0] All

53 43 31 45 45 217

6.7 6.9 6.8 6.2 6.1 6.5

4.5 4.6 4.4 4.0 4.0 4.3

10

Barker

ence at birth reflects, among other things, liver size, and cholesterol metabolism is regulated by the liver, an inference is that impaired liver growth in utero resets cholesterol concentration towards a more atherogenic profile. A small abdominal circumference also predicted raised plasma fibrinogen concentrations, a measure of blood coagulability that is also controlled by the liver (29). Recent studies suggest that differences in gestation will not distinguish low–birth weight children who are at special risk of disturbed lipid homeostasis (30). Among a group of 485 prepubertal Jamaican children, serum cholesterol concentrations were not related to birth weight but were inversely related to length at birth (31), further evidence that retarded trunk and visceral growth, including liver growth, may be associated with alterations in lipid metabolism (32). In a group of 517 20-year olds in France, the mean plasma lipid concentrations of those who were born small for gestational age were similar to those of a control group with normal birth weights (33). The small-for-gestational-age group were, however, insulin resistant. Perhaps disturbances of lipid homeostasis that are associated with insulin resistance do not manifest until adult life. F. Maternal Dietary Balance and Body Composition Indications that the balance of macronutrients in the mother’s diet can have important short- and long-term effects on offspring have come from a series of experimental studies on pregnant rats. It was found that maternal diets with a low ratio of protein to carbohydrate and fat alter fetal and placental growth, and result in lifelong elevation of blood pressure in the offspring (34). A follow-up study of 40-year-old men and women in Aberdeen, United Kingdom suggested that alterations in the maternal macronutrient balance during pregnancy could have similar adverse effects on the offspring (35); the relations with maternal diet were, however, complex. Among women with low intakes of animal protein, a higher carbohydrate intake was associated with a higher adult blood pressure in the offspring; among those with high animal protein intakes, a lower carbohydrate intake was associated with higher blood pressure. These increases in blood pressure were associated with decreased placental size (35). The effects of maternal dietary balance are described further in Chapter 13. As is described in Chapter 13, the availability of nutrients to the fetus is determined by the mother’s body size and composition at conception as well as by her diet in pregnancy. Size at birth, however, depends mainly on the mother’s body size; even extreme undernutrition during pregnancy has only modest effects on birth size. The size and composition of the mother’s

Introduction

11

body reflects her growth and nutrition from her own fetal life, through childhood, adolescence, and into adult life. All these stages of her life have to be embraced within maternal nutrition. As Mellanby wrote in 1933, “[I]t is certain that the significance of correct nutrition in childbearing does not begin in pregnancy itself or even in the adult female before pregnancy. It looms large as soon as a female child is born and indeed in its intrauterine life.” Evidence that maternal body composition has important effects on the offspring has come from studies showing that extremes of maternal body composition in pregnancy are associated with adverse long-term outcomes in the offspring. Follow-up of a group of Jamaican children showed that those whose mothers had thin skinfold thicknesses in pregnancy and a low pregnancy weight gain had higher blood pressure at the age of 11 years (36). A subsequent study of 11-year-old children in Birmingham, United Kingdom found similar associations (37). In Gambia, low pregnancy weight gain was associated with higher blood pressure in childhood (38). Studies in India have found that a low maternal weight in pregnancy is associated with an increased risk of coronary heart disease in the offspring in adult life (17). Among men and women who were in utero during the wartime famine in Holland, those whose mothers had low weight in pregnancy had the most impaired glucose tolerance and evidence of insulin resistance (39). In China, men and women whose mother had a low body mass index in pregnancy were insulin resistant (67). At the other extreme of maternal body fatness, evidence for long-term effects of maternal obesity has come from follow-up of a group of men in Finland born earlier this century (13). Markedly raised coronary heart disease rates were found in men whose mothers had a high body mass index in pregnancy (Table 5). This effect was independent of the association between thinTable 5 SMRs for Coronary Heart Disease in Finnish Men Body mass index of mother in late pregnancy (kg/m2) Ponderal index of baby (kg/m3) <24 –25 –27 –29 >29 All

–26

56 [6] 88 [12] 46 [5] 38 [2] 62 [25] *

134 87 76 61 89

[20] [21] [17] [7] [65]

–28 158 123 55 45 89

–30 [17] [26] [13] [7] [63]

131 104 98 68 97

[7] [11] [12] [6] [36]

>30 171 131 116 72 111

All

[7] 124 [57] [11] 104 [81] [16] 76 [63] [9] 58 [31] [43] 89 [232]

First number indicates standardized mortality ratio; number in brackets indicates number of deaths.

*

12

Barker

ness at birth and increased rates of adult coronary heart disease (Table 5), and it was confined to the offspring of short women (3,13). A study in India has also shown that the offspring of mothers who have a high body mass index in pregnancy are at increased risk of non–insulin dependent diabetes (40). G. Childhood Growth Studies in Helsinki have shown that the path of growth through childhood modifies the risk of disease associated with size at birth (41). The highest death rates from coronary heart disease occurred in men who were thin at birth but had accelerated weight gain in childhood (Table 6). We do not yet know whether this association is attributable to the pathological effects of a high fat mass persisting into adult life, deleterious effects of catch-up growth, or the intrauterine resetting of endocrine axes that control growth. It does suggest that although the primary prevention of coronary heart disease and non–insulin dependent diabetes may depend on changing the body composition and diets of young women, more immediate benefit may come from preventing imbalances between pre- and postnatal growth among today’s children. H. Chronic Obstructive Lung Disease In Hertfordshire, standardized mortality ratios for chronic bronchitis among men with birth weights of 5.5 lbs (2.5 kg) or less were twice those among men with birth weights of more than 9.5 lbs (>4.3 kg). There were even stronger trends with weight at 1 year of age. When the lung function of a sample of the men was measured, the mean forced expiratory volume (FEV1), which largely reflects airway size, rose between those with low and those with Table 6 Hazard Ratios for Death From Coronary Heart Disease, Adjusted for Length of Gestation Body mass index (kg/m2) at age 11 years Ponderal index at birth (kg/m3)

≤15.5 HR [ND]

≤25 25–27 27–29 >29

2.7 1.5 2.2 1.0

[21] [14] [17] [4]

15.5–16.5 HR [ND] 3.3 3.2 1.6 1.7

[26] [40] [18] [11]

Abbreviations: HR, hazard ratio; ND, number of deaths.

16.5–17.5 HR [ND] 3.7 [19] 4.0 [35] 1.8 [19] 1.5 [12]

17.5 HR [ND] 5.3 2.7 3.2 1.9

[14] [14] [21] [12]

Introduction

13

high birth weight. This association was independent of the subjects’ height, age, and smoking habits. FEV1 was not related to weight at 1 year, independent of birth weight, which suggests that it is linked to growth in utero rather than growth during infancy. In contrast to FEV1, the forced vital capacity (FVC) was not related to birth weight but was reduced in men with lower weights at 1 year of age. An interpretation of this is that aspects of lung physiology that determine FVC are programmed in infancy rather than intrauterine life. For many years there has been interest in the hypothesis that lower respiratory tract infection during infancy and early childhood predisposes to chronic airflow obstructions in later life (42–46). The large geographical differences in death rates from chronic bronchitis in England and Wales are closely similar to the differences in infant deaths from respiratory infection earlier in this century (47). Follow-up studies of individuals provide direct evidence that respiratory infection in early life has long-term effects. When the national sample of 3899 British children born in 1946 were studied as young adults, those who had had one or more lower respiratory infections before 2 years of age had a higher prevalence of chronic cough (48). A link between lower respiratory tract infection in early childhood and death from chronic bronchitis has been shown in follow-up studies in Hertfordshire and Derbyshire (49,50). Among 639 men in Hertfordshire, 59 were recorded as having had an attack of bronchitis or pneumonia during infancy. Table 7 shows Table 7 Mean FEV1 (Liters) Adjusted for Height and Age Among Men Aged 59–67 Years

Bronchitis or pneumonia in infancy Birth weight (lbs [kg])

Absent*

≤5.5 [2.5] 5.5–6.5 [2.9] 6.5–7.5 [3.4] 7.5–8.5 [3.9] 8.5–9.5 [4.3] >9.5 [4.3] All

2.39 (22) 2.40 (70) 2.47 (163) 2.53 (179) 2.54 (103) 2.57 (43) 2.50 (580)

Present* 1.81 2.23 2.38 2.33 2.36 2.36 2.30

Abbreviation: FEV1, forced expiratory volume in 1 sec. * Number in parentheses indicates number of people.

(4) (10) (25) (12) (5) (3) (59)

14

Barker

that at each birth weight their mean FEV1 values were lower than those of men not recorded as having had bronchitis or pneumonia. A total of 63 men were recorded as having had an attack of bronchitis or pneumonia between 1 and 5 years of age, but their mean FEV1 and FVC values were similar to those of all the other men. This suggests that infancy may be a critical period in which infection may change lung function. Further evidence of the longterm effects of respiratory infection in early life came from a study of 70year-old men in Derbyshire, England, which also made use of health visitors’ records (50). The FEV1 of men who had had pneumonia before the age of 2 years was 0.65 L less than that of other men, a reduction in FEV1 of approximately twice that associated with lifelong smoking. The simplest explanation of these observations is that infection of the lower respiratory tract during infancy has persisting deleterious effects which, added to the effects of poor airway growth in utero, predispose to the development of chronic bronchitis in later life. This issue is further discussed in Chapter 15. II. Other Diseases That Originate In Utero Although this book focuses on the fetal origins of cardiovascular and lung disease, there are a number of other common diseases for which there is evidence of in utero programming. They will be briefly reviewed in this section. They do not represent the extent and range of the effects of programming; fresh evidence linking in utero life with other diseases regularly appears. Because programming of hormonal release or tissue sensitivity to hormones is a central theme of this book, a chapter on reproductive endocrinopathies has been included (Ch. 12). A. Osteoporosis Bone mass at two common sites of osteoporotic fracture, the femoral neck and lumbar spine, has been shown to be positively associated with weight at 1 year in young women and in elderly men and women (51,68). This suggests that skeletal development tracks from early life and that growth retardation in prenatal and postnatal life may set in motion a series of pathological processes that lead eventually to osteoporotic fracture. Bone mass is a function of bone size and mineral density. Growth is the most important determinant of size, whereas density within the bony envelope is modified by a host of local factors, including hormonal status and physical activity. Studies of the 24hour growth hormone profiles of elderly men in Hertfordshire showed that

Introduction

15

peak growth hormone concentrations were related to bone density in the femoral neck (52), which suggests that this aspect of growth hormone secretion is one of the influences that modify bone density through life. Median growth hormone concentrations in old age were related to weight at the age of 1 year. This particular aspect of the growth hormone secretory profile may therefore be programmed in utero or during infancy, initiating changes that lead ultimately to hip fracture. B. Schizophrenia The advent of new imaging techniques has revolutionized ideas about the cause of schizophrenia. “Decades of scientifically unfounded psychological and social theories that blamed family and society have given way to increasingly compelling scientific evidence that schizophrenia is a brain disorder” (53). Subtle reductions in cortical volume revealed by neuroimaging, together with necropsy observations of altered architecture in the cortex, have led to the conclusion that the disorder originates through defective migration of cells into the cortex during the second trimester of gestation. Epidemiological evidence that schizophrenia originates in utero comes from the increased frequency of obstetric complications in the birth histories of patients (54,55), and delay in their motor and speech milestones during childhood (56). There is preliminary evidence that both infection and undernutrition in utero may initiate the defects in early brain development that are subtly manifest in childhood but become dramatically evident in early adult life. In a study of people who were at risk of in utero exposure to the 1957 influenza epidemic, those at risk during the second trimester had higher rates of hospital admission for schizophrenia than those at risk during other trimesters or those not at risk (57). The results of other similar studies have, however, been inconsistent (53,58). People exposed to the Dutch famine during the first 2 months of gestation had a twofold increase in risk of being hospitalized for schizophrenia (59). C. Depression For many years, depression in adult life has been thought to originate through parental indifference, abuse, and other adverse influences in childhood. A study in Hertfordshire found that men and women who committed suicide, which is commonly the result of depression, had low weight gain in infancy (60). Although this could be attributable to adverse psychosocial influences in infancy, there is nothing in the Hertfordshire records that supports this, and

16

Barker

it raises the possibility that adult depression is initiated by in utero programming of hormonal axes that influence growth in infancy and mood in later life. Patients with depression have been found to have abnormal secretion of growth hormone and abnormalities in the hypothalamic-adrenal and hypothalamic-thyroid axes (61). There is evidence that each of these axes are programmed in utero. D. Cancers of the Breast and Ovary Cancers of the breast have been linked to high birth weight. Among women in the Nurses’ Study in the United States, odds ratios for breast cancer doubled across the range of birth weight (69). This trend was little influenced by adjusting for other variables, such as body mass and family history of breast cancer. Other data support a link between high birth weight and breast cancer (62), and it has been suggested that high concentrations of estrogen in pregnancy may play a role (63). In the Hertfordshire study, rates of ovarian cancer increased, not with increasing birth weight, but with increasing weight at 1 year (64). The suggested explanation is that hormonal or nutritional influences acting in utero imprint an altered pattern of gonadotrophin release. Experiments in rats show that the hypothalamus is imprinted by androgens during a sensitive perinatal phase. Low concentrations of androgen lead to cyclical release of gonadotrophins (the female pattern); high concentrations result in continuous secretion of gonadotrophin (the male pattern). In humans, an altered pattern of gonadotrophin that increased estrogen release would also promote infant weight gain. It is suggested that in later life it might induce malignant change in the ovary. E. Polycystic Ovary Syndrome Direct evidence that events in utero may imprint an altered pattern of gonadotrophin release, and thereby program disorders of the female reproductive tract, comes from a study of polycystic ovaries, a common disorder associated with menstrual irregularities, subfertility, hirsutism, acne, and a spectrum of endocrine abnormalities including high plasma luteinizing hormone (LH) concentrations, a high ratio of LH to follicle stimulating hormone (FSH), and excessive androgen production. In one of the two common forms of the disorder, women are obese and androgenized, whereas in the other they are thin and have normal testosterone concentrations. The former group of women tends to have high birth weight and are born to heavy mothers (65). The latter

Introduction

17

group, however, tends to be born after term. A possible explanation of why this latter group develops polycystic ovaries is that they have an altered hypothalamic-pituitary “set point” for LH release as a result of their prolonged gestation. The human fetus produces large amounts of androgens, which are converted to estrogen by the placenta and pass to the maternal circulation. Placental failure associated with postmaturity could expose the fetal hypothalamus to increased concentrations of androgens or estrogens and reset its responses to them. III. Mothers and Babies Today The findings described in this book suggest that coronary heart disease, stroke, non–insulin dependent diabetes, and hypertension originate through undernutrition and other adverse influences in utero. Protecting the nutrition and health of young women and their babies must therefore be a priority. Today, even in the Western World, many babies are born thin, stunted, or short and fat. Encouraged by the fashion industry, many young women are unduly thin. Encouraged by sections of the food industry, other young women are unduly fat. Many have diets that are imbalanced according to established criteria. If we are to protect babies, we must also protect girls in childhood and adolescence. Body composition is established by childhood growth. Obesity and eating habits are entrained during childhood and adolescence. The path of fetal growth modifies responses to adverse influences encountered in later life. One such influence is obesity. In the Third World, and in less-affluent areas of the Western World, the transition from chronic malnutrition to good nutrition needs to be managed in such a way that people who were undernourished in utero do not become obese as adults (Ch. 16). As yet, we do not know the true impact of maternal nutrition on fetal development. The relatively disappointing effects of nutritional interventions in pregnancy on birth weight in humans have led to the view that fetal development is little affected by changes in maternal nutrition. It is, however, clear that birth weight alone is an inadequate summary measure of fetal experience. We need a more sophisticated view of optimal fetal development that takes account of the long-term sequelae of fetal adaptations to undernutrition. For effective interventions to prevent or arrest disease, we need to progress beyond the epidemiological associations to greater understanding of the

18

Barker

cellular and molecular processes that underlie them. We need to know what factors limit the delivery of nutrients and oxygen to the human fetus, how the fetus adapts to a limited supply, how these adaptations program the structure and physiology of the body, and by what molecular mechanisms nutrients and hormones alter gene expression. This book sets out an agenda for future research. We need to respond so that we can prevent disease in the next generation and offer better treatment to the present one. References 1. 2. 3. 4.

5. 6. 7. 8.

9. 10. 11.

12.

13.

Barker DJP, Osmond C. Infant mortality, childhood nutrition and ischaemic heart disease in England and Wales. Lancet 1986; 1:1077–1081. McCance RA, Widdowson EM. The determinants of growth and form. Proc R Soc Lond B 1974; 185:1–17. Barker DJP. Mothers, babies and health in later life. 2nd ed. Edinburgh: Churchill Livingstone; 1998. Lucas A. Programming by early nutrition in man. The Childhood Environment and Adult Disease. 1st ed. Bock GR, Whelen J, eds. Chichester: Wiley, 1991; 38–55. Fowden AL. Endocrine regulation of fetal growth. Reprod Fertil Dev 1995; 7: 351–363. Harding JE, Johnston BM. Nutrition and fetal growth. Reprod Fertil Dev 1995; 7:539–547. Rudolph AM. The fetal circulation and its response to stress. J Dev Physiol 1984; 6:11–19. Oliver MH. Harding JE, Breier BH, Evans PC, Gluckman PD. Glucose but not a mixed amino acid infusion regulates plasma insulin-like growth factor-1 concentrations in fetal sheep. Pediatr Res 1993; 34(1):62–65. Barker DJP, Osmond C, Winter PD, Margetts B, Simmonds SJ. Weight in infancy and death from ischaemic heart disease. Lancet 1989; 2:577–580. Osmond C, Barker DJP, Winter PD, Fall CHD, Simmonds SJ. Early growth and death from cardiovascular disease in women. BMJ 1993; 307:1519–1524. Barker DJP, Osmond C, Simmonds SJ, Wield GA. The relation of small head circumference and thinness at birth to death from cardiovascular disease in adult life. BMJ 1993; 306:422–426. Leon DA, Lithell H, Vagero D, et al. Biological and social influences on mortality in a cohort of 15,000 Swedes followed from birth to old age. J Epidemiol Community Health 1997; 51(abstr):594. Forsen T, Eriksson JG, Tuomilehto J, Teramo K, Osmond C, Barker DJP. Mother’s weight in pregnancy and coronary heart disease in a cohort of Finnish men: follow up study. BMJ 1997; 315:837–840.

Introduction

19

14. Forsen T, Eriksson JG, Tuomilehto J, Osmond C, Barker DJP. Growth in utero and during childhood among women who develop coronary heart disease: longitudinal study. BMJ 1999; 319:1403–1407. 15. Frankel S, Elwood P, Sweetnam P, Yarnell J, Davey Smith G. Birthweight, bodymass index in middle age, and incident coronary heart disease. Lancet 1996; 348:1478–1480. 16. Rich-Edwards JW, Stampfer MJ, Manson JE, Rosner B, Hankinson SE, Colditz GA, Willett WC, Hennekens CH. Birth weight and risk of cardiovascular disease in a cohort of women followed up since 1976. BMJ 1997; 315:396–400. 17. Stein CE, Fall CHD, Kumaran K, Osmond C, Cox V, Barker DJP. Fetal growth and coronary heart disease in South India. Lancet 1996; 348:1269–1273. 18. Martyn CN, Barker DJP, Osmond C. Mothers’ pelvic size, fetal growth, and death from stroke and coronary heart disease in men in the UK. Lancet 1996; 348:1264–1268. 19. Karlberg J. A biologically-oriented mathematical model (ICP) for human growth. Acta Paediatr Suppl 1989; 350:70–94. 20. Kramer MS, Joseph KS. Commentary: enigma of fetal/infant origins hypothesis. Lancet 1996; 348:1254–1255. 21. Paneth N, Susser M. Early origin of coronary heart disease (the “Barker hypothesis”). BMJ 1995; 310:411–412. 22. Elford J, Whincup P, Shaper AG. Early life experience and adult cardiovascular disease: longitudinal and case-control studies. Int J Epidemiol 1991; 20:833– 844. 23. Ben-Shlomo Y, Davey Smith G. Deprivation in infancy or in adult life: which is more important for mortality risk? Lancet 1991; 337:530–534. 24. Law CM, Shiell AW. Is blood pressure inversely related to birth weight? The strength of evidence from a systematic review of the literature. J Hypertens 1996; 14(8):935–941. 25. Hales CN, Barker DJP, Clark PMS, Cox LJ, Fall C, Osmond C, Winter PD. Fetal and infant growth and impaired glucose tolerance at age 64. BMJ 1991; 303:1019–1022. 26. Fall CHD, Osmond C, Barker DJP, Clark PMS, Hales CN, Stirling Y, Meade TW. Fetal and infant growth and cardiovascular risk factors in women. BMJ 1995; 310:428–432. 27. Barker DJP, Hales CN, Fall CHD, Osmond C, Phipps K, Clark PMS. Type 2 (non–insulin-dependent) diabetes mellitus, hypertension and hyperlipidaemia (syndrome X): relation to reduced fetal growth. Diabetologia 1993; 36:62–67. 28. Barker DJP, Martyn CN, Osmond C, Hales CN, Fall CHD. Growth in utero and serum cholesterol concentrations in adult life. BMJ 1993; 307:1524–1527. 29. Barker DJP, Meade TW, Fall CHD, Lee A, Osmond C, Phipps K, Stirling Y. Relation of fetal and infant growth to plasma fibrinogen and factor VII concentrations in adult life. BMJ 1992; 304:148–152.

20

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30. Decsi T, Erhardt E, Markus A, Burus I, Molnar D. Plasma lipids, phospholipid fatty acids and indices of glycaemia in 10-year-old children born as small-forgestational-age or preterm infants. Acta Paediatr 1999; 88:500–504. 31. Forrester TE, Wilks RJ, Bennett FL, Simeon D, Osmond C, Allen M, Chung AP, Scott P. Fetal growth and cardiovascular risk factors in Jamaican school children. BMJ 1996; 312:156–160. 32. Forrester TE, Wilks RJ, Bennett FI, Simeon D, Osmond C, Allen M, Chung AP, Scott P. Fetal growth and cardiovascular risk factors in Jamaican schoolchildren. BMJ 1996; 312:156–160. 33. Leger J, Levy-Marchal C, Bloch J, Pinet A, Chevenne D, Porquet D, Collin D, Czernichow P. Reduced final height and indications for insulin resistance in 20 year olds born small for gestational age: regional cohort study. BMJ 1997; 315:341–347. 34. Langley-Evans SC, Jackson AA. Increased systolic blood pressure in adult rats induced by fetal exposure to maternal low protein diets. Clin Sci 1994; 86:217– 222. 35. Campbell DM, Hall MH, Barker DJP, Cross J, Shiell AW, Godfrey KM. Diet in pregnancy and the offspring’s blood pressure 40 years later. Br J Obstet Gynaecol 1996; 103:273–280. 36. Godfrey KM, Forrester T, Barker DJP, Jackson AA, Landman JP, Hall JStE, Cox V, Osmond C. Maternal nutritional status in pregnancy and blood pressure in childhood. Br J Obstet Gynaecol 1994; 101:398–403. 37. Clark PM, Atton C, Law CM, Shiell A, Godfrey K, Barker DJP. Weight gain in pregnancy, triceps skinfold thickness and blood pressure in the offspring. Obstet Gynaecol 1998; 91:103–107. 38. Margetts BM, Rowland MGM, Foord FA, Cruddas AM, Cole TJ, Barker DJP. The relation of maternal weight to the blood pressures of Gambian children. Int J Epidemiol 1991; 20(4):938–943. 39. Ravelli ACJ, van der Meulen JHP, Michels RPJ, Osmond C, Barker DJP, Hales CN, Bleker OP. Glucose tolerance in adults after prenatal exposure to famine. Lancet 1998; 351:173–177. 40. Fall CHD, Stein CE, Kumaran K, Cox V, Osmond C, Barker DJP, Hales CN. Size at birth, maternal weight, and type 2 diabetes in South India. Diabet Med 1998; 15:220–227. 41. Eriksson JG, Forsen T, Tuomilehto J, Winter PD, Osmond C, Barker DJP. Catchup growth in childhood and death from coronary heart disease: longitudinal study. BMJ 1999; 318:427–431. 42. Holland WW, Halil T, Bennett AE, Elliott A. Factors influencing the onset of chronic respiratory disease. BMJ 1969; ii:205–208. 43. Reid DD. The beginnings of bronchitis. Proceedings of the Royal Society of Medicine 1969; 62:311–316. 44. Samet JM, Tager IB, Speizer FE. The relationship between respiratory illness

Introduction

45. 46. 47. 48.

49.

50.

51.

52.

53. 54.

55. 56.

57.

58. 59. 60.

21

in childhood and chronic air-flow obstruction in adulthood. Am Rev Respir Dis 1983; 127:508–523. Phelan PD. Does adult chronic obstructive lung disease really begin in childhood? Br J Dis Chest 1984; 78:1–9. Strachan DP. Do chesty children become chesty adults? Arch Dis Child 1990; 65:161–162. Barker DJP, Osmond C. Childhood respiratory infection and adult chronic bronchitis in England and Wales. BMJ 1987; 292:1271–1275. Mann SL, Wadsworth MEJ, Colley JRT. Accumulation of factors influencing respiratory illness in members of a national birth cohort and their offspring. J Epidemiol Community Health 1992; 46:286–292. Barker DJP, Godfrey KM, Fall C, Osmond C, Winter PD, Shaheen SO. Relation of birth weight and childhood respiratory infection to adult lung function and death from chronic obstructive airways disease. BMJ 1991; 303:671–675. Shaheen SO, Barker DJP, Shiell AW, Crocker FJ, Wield GA, Holgate ST. The relationship between pneumonia in early childhood and impaired lung function in late adult life. Am J Respir Crit Care Med 1994; 149:616–619. Cooper C, Cawley M, Bhalla A, Egger P, Ring F, Morton L, Barker DJP. Childhood growth, physical activity, and peak bone mass in women. J Bone Miner Res 1995; 10:940–947. Fall C, Hindmarsh P, Dennison E, Kellingray S, Barker D, Cooper C. Programming of growth hormone secretion and bone mineral density in elderly men: a hypothesis. J Clin Endocrinol Metab 1998; 83:135–139. Weinberger DR. From neuropathology to neurodevelopment. Lancet 1995; 346:552–557. Hultman CM, Ohman A, Cnattingius S, Wieselgren IM, Lindstrom LH. Prenatal and neonatal risk factors for schizophrenia. Br J Psychiatry 1997; 170:128– 133. Gunther-Genta F, Bovet P, Hohlfeld P. Obstetric complications and schizophrenia: a case-control study. Br J Psychiatry 1994; 164:165–170. Jones P, Rodgers B, Murray R, Marmot M. Child developmental risk factors for adult schizophrenia in the British 1946 birth cohort. Lancet 1994; 344:1398– 1402. Mednick SA, Machon RA, Huttunen MO, Bonnett D. Adult schizophrenia following prenatal exposure to an influenza epidemic. Arch Gen Psychiatry 1988; 45:189–192. Venables PH. Schizotypy and maternal exposure to influenza and to cold temperature: the Mauritius study. J Abnorm Psychol 1996; 105:53–60. Susser E, Neugebauer R, Hoek HW, Brown AS, Lin S, Labovitz D, Gorman JM. Schizophrenia after prenatal famine. Arch Gen Psychiatry 1996; 53:25–31. Barker DJP, Osmond C, Rodin I, Fall CHD, Winter PD. Low weight gain in infancy and suicide in adult life. BMJ 1995; 311:1203.

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61. Neuroendocrinology. Checkley S, Paykel ES, eds. Handbook of Affective Disorders. Edinburgh: Churchill Livingstone; 1992. 62. Ekbom A, Trichopoulos D, Adami HO, Hsieh CC, Lan SJ. Evidence of prenatal influences on breast cancer risk. Lancet 1992; 340:1015–1018. 63. Sanderson M, Williams MA, Malone KE, Stanford JL, Emanuel I, White E, Daling JR. Perinatal factors and risk of breast cancer. Epidemiology 1996; 7:34– 37. 64. Barker DJP, Winter PD, Osmond C, Phillips DIW, Sultan HY. Weight gain in infancy and cancer of the ovary. Lancet 1995; 345:1087–1088. 65. Cresswell JL, Barker DJP, Osmond C, Egger P, Phillips DIW, Fraser RB. Fetal growth, length of gestation and polycystic ovaries in adult life. Lancet 1997; 350:1131–1135. 66. Eriksson JG, Forsen T, Tuomileto J, Osmond C, Barker DJP. Early growth, adult income and risk of stroke. Stroke 2000 (in press). 67. Mi J, Law CM, Zhang K-L, Osmond C, Stein CE, Barker DJP. Effects of infant birthweight and maternal body mass index in pregnancy on components of the insulin resistance syndrome in China. Ann Intern Med 2000; 132:253–260. 68. Cooper C. Growth in infancy and bone mass in later life. Am Rheum Dis 1997; 56:17–21. 69. Michels KB, Trichopoulos D, Robins JM, Rosner BA, Manson JE, Hunter DJ, Colditz GA, Hankinson SE, Seizer FE, Willett WC. Birthweight as a risk factor for breast cancer. Lancet 1996; 348:1542–1546.

2 Birth Weight, Blood Pressure, and Hypertension Epidemiological Studies

DAVID A. LEON and ILONA KOUPILOVÁ London School of Hygiene and Tropical Medicine London, United Kingdom

This chapter reviews the human epidemiological evidence for an association between fetal factors and postnatal blood pressure in childhood and adult life. The past few years have seen a substantial expansion of the literature in this field. This chapter therefore draws on an evidence base up to January 1999 that goes beyond earlier reviews (1,2), which only covered the literature up to early 1996. Nearly all of the available human data addressing the link between fetal factors and later blood pressure and hypertension come from observational studies. This inevitably means that results need to be interpreted with caution. There is certainly no definitive study, and each set of findings must be regarded as contributing to the overall patchwork of evidence, which includes animal experimental models and mechanistic studies in humans. However, this chapter focuses on the epidemiological evidence and does not discuss issues of underlying biological mechanisms. 23

24

Leon and Koupilová

This chapter mainly deals with a systematic review and tabular summaries of epidemiological studies that have published quantitative estimates of the strength and direction of the association between birth weight and later blood pressure treated as a continuous variable. Unlike the review by Law and Shiell (2), we review and summarize the results for diastolic as well as systolic blood pressure. We also cover issues raised by adjustment for current body size; whether the strength of the birth-weight blood pressure association changes with age; studies of size at birth in relation to hypertension; how far associations between birth weight and later blood pressure involve socioeconomic and behavioral pathways in addition to the purely biological pathway of fetal programming; and whether any specific maternal factors (including maternal nutrition) have been implicated as driving the associations of fetal factors with later blood pressure. I.

Birth Weight and Blood Pressure in Children and Adults

None of the observational studies reported to date were set up as prospective investigations from birth specifically to investigate the association of birth weight with blood pressure in later life. In many cases, information about fetal characteristics has been collected retrospectively for study subjects whose blood pressure was initially measured for reasons unrelated to the issue of fetal programming. A. Criteria for Inclusion in Summary Tables

Tables 1 and 2 summarize the results of papers that have reported quantitative measures of the association between birth weight and blood pressure from age 1 year onwards for systolic and diastolic pressure, respectively. These papers were identified through searches of Medline and PubMed (ww.ncbi.nlm.nih.gov/PubMed), and from reference lists of papers known to the authors. To be included in the tables, a paper had to meet the following criteria: • The study reported had to be either cross-sectional or a prospective cohort. • Quantitative estimates of the association between birth weight and blood pressure had to be given. These could take the form of regression coefficients, correlation coefficients, or mean blood pressure levels across a range of birth weight categories.

Birth Weight, Blood Pressure, and Hypertension

25

• The study population was not selected with respect to either blood pressure or birth weight. • Estimates of effect were based on office blood pressure. • The language of the paper was English. Papers that included only qualitative statements about the association between birth weight and systolic blood pressure were excluded. Studies of binary outcomes such as hypertension, and follow-up studies of clinically abnormal births (eg, preterm or low birth weight) were also excluded from the summary tables, but are discussed in later sections. Papers that met the criteria and in addition included regression coefficients for the effect of birth weight on systolic blood pressure based on the same subjects at successive ages are summarized in Table 3. The studies included in this table should be distinguished from those summarized in Tables 1 and 2, which include estimates of the association between birth weight and blood pressure at different ages for different sets of subjects, along with papers reporting different analyses of the same data. B. Structure of Summary Tables

Tables 1 to 3 all have the same basic structure. Papers appear in ascending order of age at which blood pressure was measured. The first few columns identify each paper and indicate the age at which blood pressure was measured (mean and/or range), the year of birth of subjects, and sex. The column headed “Bwt” specifies which of three possible sources birth weight information was obtained from: (1) obstetric or other contemporaneous medical records (Obs); (2) other routine records such as birth registrations or medical birth registries (Rout); and (3) reported by study subjects or their parents (Recall). All estimates of the strength and direction of the association between birth weight and blood pressure in the tables were adjusted for age where appropriate. Estimates were included for males and females separately only when no combined estimate for both sexes together was available. The column in which an estimate was placed depended on the nature of statistical adjustment for other variables. If the estimate of effect was not adjusted for any factor (other than age and/or sex) it was placed in the column “Crude.” Estimates adjusted for body size only, measured at the time that blood pressure was taken, were placed in the next column in the table. Papers varied according to whether these adjustments were for weight, height, ponderal index, body mass index, or some combination thereof. The direction of the association between birth weight and blood pressure (adjusted for concurrent body size

26

Table 1 Systolic Blood Pressure in Children and Adults: Cohort and Cross-Sectional Studies with Casual BP as Outcome Effect* Paper: Setting (year of publication) and (reference number)

Age in years when examined

Japan Niigata cross-sectional study (1996) (49)

mean 38 (p = 021)

UK Salisbury cross-sectional study 1 (1991) (12)

4

1984– 1985

UK Farnborough Longitudinal study (1993) (8) India Pune cross-sectional study (1995) (13) UK Nine towns study (1989) (20)

4

1975– 1977 1987– 1989 1980– 1983

5–7

5–7 mean 65

NK

1982– 1985 1980s

7

1972– 1973

8

1975– 1976 1984 –1985 1984 –1986 1973 –1981

9 8–10 7–11

Source of birth weight

Sex

N

Rout

M+F

195

Obs

M+F

364

Obs

M+F

991

–28 (–41, –14)

Obs

M+F

200

–06 (–44, +31)

Recall

M+F

3524

–18 (–23, –13)

–17 (–23, –12)

Recall

M+F

3061

–21 (–27, –14)†

–19 (–27, –11)

Rout+ Recall Obs

M+F

576

–04 (–18, +11)

M+F

692

Pearson’s r = –006 (p < 005)

Obs

M+F

775

Obs

M+F

239

–05 (–26, +16)

Obs

M+F

52

–34 (–95, +27)†

Obs +Rout

M+F

1438

–004 (–07, +06)‡

Crude

Adjusted for concurrent body size (other adjustments) Adjusted for additional or other factors

Pearson’s r = +009

–62 (–110, –13)

Inverse

BMI, birth length, gestational age, pregnancy SBP, weight gain, oedema + proteinuria Weight, placental weight and gestational age

Weight, maternal age, birth rank, maternal history of hypertension BMI, height, age, sex gestational age

–17 (–33, –02)

–13 (–31, +05)

Weight, placental weight

Leon and Koupilová

UK 10 towns 1990 cross-sectional study (1997) (14) Zimbabwe Harare cross-sectional study (1998) (17) New Zealand Dunedin Child Development Study (1981) (50) Australia Adelaide birth follow-up study (1996) (51) UK Salisbury cross-sectional study 2 (1995) (13) France Nancy Family Study (1996) (52) US Bogalusa Heart Study (1997) (53)

4

Year of birth

Israel Jerusalem follow-up study (1997) (57)

17

1970

Recall

M F M+F

3010

Weak inverse Inverse +04 (–03, +12)†

Obs

M+F

838

–28 (–38, –18)†

Obs

M+F

332

Pearson’s r = –003 (p = 063)

Obs

M+F

77

–18 (–12, +49)†

Obs

M+F

1610

–26(–35, –16)†

1978– 1983 NK

Obs

M+F

105

–42 (–81, –03)†

M F

413 425

1974– 1976

Obs

1983– 1986 1980– 1983 NK 1979– 1981 NK

Obs

Recall

M F

Sweden Conscript study (1997) (16) Croatia Birth cohort study (1993) (58)

+04 (–15, +23) +16 (–02, +35)

6692 Pearson’s r = –001 (p ≥ 001) 4199 Pearson’s r = +001 (p ≥ 001) 130,842 –08 (–09, –07)

–15 (–22, –08)†

–13 (–30, +05) +05 (–13, 23)

–05 (–21, –12) +06 (–12, +25)

–09 (–15, –03) –07 (–15, +01)

18

1973–75

Rout

M

18–23 mean 20

NK

Recall

M

214

Inverse

–03 (p = 005)

F

251

Weak inverse

–05 (p = 002)

Obs

M+F

53

–35(–71, +01)†

Obs+ Recall

M+F

452

17–24 16–26 mean 21

1970– 1977 1959– 1970

–10 (–11, –09)

–22 (p = 006)

–08 (–09, –06)

–17 (p = 015)

BMI, weight height, mother’s height, season, s-e factors, smoking, drinking, exercise Weight, maternal prepregnancy weight, weight gain, BMI and ethnicity BMI, gestational age, maternal age, parity Father’s height, man ual activity, nutrition, s-e factors. BMI, skinfold thickness BMI, skinfold thickness, s-e factors

Weight, sex, parental SBP

27

France Nancy Family Study (1996) (52) Scotland Edinburgh cross-sectional study (1998) (59)

9921

Birth Weight, Blood Pressure, and Hypertension

UK 1970 birth cohort (1989) 10 (4) UK 10 towns 1994 cross-sec 8–11 tional study (1997) (15) mean 105 UK Guilford & Carlisle 9–11 cross-sectional study (1995) (23) Spain Valencia cross-sec6–16 tional study (1996) (54) mean 106 Jamaica Follow-up of mater10–12 nal nutrition study (1994) (47) Jamaica Kingston cross-sec6–16 tional study (1996) (55) France Nancy Family Study 11–16 (1996) (52) Scotland Longitudinal study 15 (1991) (56)

28

Table 1 Continued Effect* Paper: Setting (year of publication) and (reference number)

Age in years when examined

Year of birth

Source of birth weight

Sex

N

Recall

M+F

541

Obs

M F

1396 1553

M F M

1625 1634 1421

F

1409

US San Antonio Heart Study (1994) (60) UK 1946 birth cohort—1 (1985) (3)

32 36

1949– 1963 1946

UK 1946 birth cohort—2 (1989) (4) UK 1946 birth cohort—3 (1993) (5)

36

1946

Obs

36

1946

Obs

Crude

Adjusted for concurrent body size (other adjustments) Adjusted for additional or other factors

Inverse (in both ethnic groups)

Inverse Weak inverse –11 (–20, –03) per SDS bwt –11 (–20, –03) per SDS bwt

1947– 1964 1948– 1954 NK

Recall

F

92,940

Obs

M+F

253

Obs

M+F

620

1935– 1943

Obs

M+F

449

Inverse

UK Preston birth follow-up study—A2 (1992) (7)

1935– 1943

Obs

M+F

327

Inverse (p = 004)

46–54

–45 (p = 003)

–23 (p < 001) –20 (p < 005)

BMI, family history CVD, education, s-e status

–02 (–03, –01)

BMI, parental history of hypertension BMI, sex, alcohol consumption BMI, sex, parental NIDDM status BMI, placental weight, alcohol consumption, sex

–14 (–22, –05) per SDS bwt –12 (–21, –04) per SDS bwt

–48 (p = 001) –20 (p = 008) Inverse

Leon and Koupilová

US Nurses Health Study II 27–44 (1996) (30) mean 37 Scotland Aberdeen diet in mean 41 pregnancy study (1996) (45) Denmark Fredericia NIDDM 41–54 offspring study (1996) (61) mean 47 UK. Preston birth follow-up 46–54 study—A1 (1990) (6)

Inverse (p = 001) Sex and ethnicity

46–50

1935– 1943

Obs

Obs

M F M F M+F

123 116 117 103 139

1935– 1943 1920– 1924 1939–1940

–29 (–92, +37)

Obs

M

1333

–22 (–42, –03)

Obs

M–F

337

–59 (–119, –18)

1921–1946

Recall

F

71,100

–09 (–11, –08)

1911– 1946 1920– 1930

Recall

M

22,846

Obs

M

468

Inverse (p = 0001)

1920– 1930

Obs

1923– 1930

Obs

M F M F F

426 203 418 184 297

–30(–69, +09) –27 (–88, +34) –49 (–88, –10) –55 (–122, +12) –46 (–97, +04)

51–54 UK Preston birth follow-up study—B (1993) (9) Sweden Uppsala crosssectional study (1996) (18) UK Sheffield birth follow-up study (1995) (62) US Nurses Health Study I (1996) (30) US Health Professionals Cohort Study (1996) (31) UK Hertfordshire birth follow-up study—1 (1991) (10) 59–70 UK Hertfordshire birth follow-up study—2 (1993) (8) UK Hertfordshire birth follow-up study—3 (1995) (11)

47–56 49–51 50–53 44–69 mean 56 44–79 mean 60 59–70 mean 64 59–63 64–71 60–71 mean 64

–28 (–93, +37) –27 (–105, +51) –34 (–91, +23) –34 (–136, +68)

–31 (–50, –12)

Inverse

–07 (–10, –04)

BMI, sex, alcohol, gestational age BMI, parental history of hypertension BMI, parental history of hypertension

Birth Weight, Blood Pressure, and Hypertension

UK Preston birth follow-up study—A3 (1992) (8)

Unless otherwise stated, mmHg change per kg increase in birth weight adjusted for age where appropriate † Adjusted for sex ‡ Adjusted for sex and race Note: Shaded rows indicate study populations for which estimates are presented from more than one paper The papers reporting the results of studies from Preston of middle age men and women involve two independent populations (denoted as A and B). There are three papers based on the A population6-8 and one paper based on the B population9 A small number of cross-sectional or follow-up studies that meet the criteria specified in the main text are nevertheless excluded from these summary tables The paper by Seidman, et al6 which reported a study of 17-year-old Israeli conscripts, was excluded because of inconsistencies between text and tables in terms of the direction of association between birth weight and blood pressure: the paper by Launer, et al2 was excluded because it did not include a simple linear regression coefficient for the effect of birth weight on blood pressure at age 4 Abbreviations: BP, Blood pressure: N, Number of subjects: NK, Not known; BMI, Body mass index: SBP. Systolic blood pressure: Rout, Routine records; Obs, Obstetric medical records: s-e. Socio-economic; CVD, Cardiovascular disease; SDS bwt. Standard deviation score of birth weight *

29

30

Table 2 Diastolic Blood Pressure in Children and Adults: Cohort and Cross-Sectional Studies with Casual BP as Outcome Effect* Age in years when examined

Year of birth

Source of birth weight

Sex

N

Japan Niigata cross-sectional study (1996) (49) Zimbabwe Harare crosssectional study (1998) (17) Australia Adelaide birth follow-up study (1996) (51) UK 10 towns 1994 crosssectional study (1997) (15) UK Guilford & Carlisle cross-sectional study (1995) (23) Spain Valencia cross-sectional study (1996) (54) US Bogalusa Heart Study (1997) (53) Scotland Longitudinal study (1991) (56)

mean 38

NK

Rout

M+F

195

mean 65

1980s

Rout+ Recall

M+F

576

Israel Jerusalem perinatal follow-up study (1997) (57)

8

1975– 1976

Obs

M+F

775

8–11 mean 105

1983– 1986

Recall

M+F

3010

9–11

1980– 1983

Obs

M+F

838

Obs

M+F

332

1973– 1981 NK

Obs+ Rout Recall

M+F

1426

M F

413 425

1974– 1976

Obs

M

6692

F

4199

6–16 mean 106 7–11 15

17

NK

Crude

Adjusted for concurrent body size (other adjustments) Adjusted for additional or other factors

Pearson’s r = +011 (p = 013) –11 (–26, +04)

–10 (–24, +04)

Weight, placental weight

+12 (–04, +28) +07(–09, +24)

+06 (–10, +23) +07 (–13, +26)

Pearson’s r = +0003 (p ≥ 001) Pearson’s r = +0009 (p ≥ 001)

–03 (–07, +01) –03 (–08, +03)

BMI, weight, height, mother’s height, season. s-e factors, smoking, drinking, exercise Weight, maternal pre-pregnancy weight, weight gain, BMI and ethnicity

+02 (–02, +07)†

–03 (–08, +01)†

–14 (–21, –07)†

Pearson’s r = +007 (p = 020) –004 (–11, +03)‡

Leon and Koupilová

Paper: Setting (year of publication) and (reference number)

32 36

1949– 1963 1946

Recall

M+F

Obs

M F

US Nurses Health Study II (1996) (30) Scotland Aberdeen diet in pregnancy study (1996) (45) Denmark Fredericia NIDDM offspring study (1996) (61) UK Preston birth followup study—I (1990) (6) Sweden Uppsala cross-sectional study (1996) (18) UK Sheffield birth followup study (1995) (62) US Nurses Health Study I (1996) (30) US Health Professionals Cohort Study (1996) (31)

27–44 mean 37 41

41–54 mean 47 46–54 49–51 50–53 44–69 mean 56 44–79 mean 60

1947– 1964 1948– 1954 NK

1935– 1943 1920– 1924 1939– 1940 1921– 1946 1911– 1946

541 Inverse (in both ethnic groups) 1421 –05 (–12, +02) per SDS bwt 1409 –03 (–10, +04) per SDS bwt 92,940

Inverse (p = 0005) Sex and ethnicity –08 (–15, –01) per SDS bwt –04 (–10, +03) per SDS bwt

Recall

F

Obs

M+F

253

M+F

620

–10 (p = 005)

BMI, sex, parental NIDDM status

Obs

M+F

449

Inverse

Placental weight

Obs

M

1333

Obs

M+F

337

–42 (–64, –20)

Recall

F

71,100

–05 (–06, –03)

Recall

M

22,846 No clear association

–03 (–05, –01)

BMI, sex, alcohol, gestational age BMI, parental history of hypertension BMI, parental history of hypertension

Obs

–02 (–03, –01)

–36 (p = 002)

–10 (–22, +01)

–39 (p = 0006)

BMI, parental history of hypertension BMI, sex, alcohol consumption

–17 (–29, –06)

Birth Weight, Blood Pressure, and Hypertension

US San Antonio Heart Study (1994) (60) UK 1946 birth cohort—3 (1993) (5)

* Unless otherwise stated, mmHg change per kg increase in birth weight, adjusted for age where appropriate † Adjusted for sex ‡ Adjusted for sex and race Abbreviations: Bwt, Birth weight; N. Number of subjects; NK. Not known: BMI, Body mass index; Rout, Routine records: Obs, Obstetric medical records; s-e. Socio-economic: SDS. Standard deviation score

31

32

Table 3 Systolic Blood Pressure in Children and Adults: Longitudinal Studies with Repeat Measures of Casual Blood Pressure as Outcome Effect* Paper: Setting (year of publication) and (reference number)

Age in years when examined

Year of birth

UK Farnborough Longitudinal study (1993) (8) Restricted to births 38–42 weeks gestation

1 1985 2 3 4 5 6 7 8 9 UK Guilford & Carlisle longitudinal 5–7 1980– analysis (1995) (23) 9–11 1974 Finland, Longitudinal cardiovascular 6–18 1962– risk factor study (1996) (64) mean 11 1974 Three follow-up examinations at 6–21 3-year intervals 9–24 mean 16

5–9 10–14 15–19 20–24 25–29 30–37

1954– 1973

Obs

Obs Recall

Recall +Obs

Crude

Adjusted for concurrent body size (other adjustments) Adjusted for additional or other factors

Sex

N

M+F M+F M+F M+F M+F M+F M+F M+F M+F M+F M+F M F M F M F

1344 1313 1215 1147 994 1070 1066 1093 961 523 1132 1249 1323 1421 1079 1196

Pearson’s r = –004 Pearson’s r = +0.01 Pearson’s r = –004 Pearson’s r = +002 Pearson’s r = –005 Pearson’s r = –004

M+F M+F M+F M+F M+F M+F

94 208 316 298 213 112

–08 (–41, +2.6)† –05 (–29, +19)† +01 (–18, +21)† –0.7 (–27, +12)† –02(–24, +19)† –02 (–31, –25)†

–04 (–10, +18) –03 (–1.7, +11) –14 (–28, 00) –25 (–37, –13) –11 (–25, +0.3) –19 (–31, –07) –11 (–23, +01) –16 (–26, –06) –07 (–19, +0.5) –23 (–36, –10)† –40 (–57, –22)† –18 (–28, –09) –09 (–19, +00) –19 (–29, –10) –06 (–16, 04) –25 (–35, –1.5) –21 (–31, –10)

–1.7 (p = 001) (N = 967) –23 (p < 0.01) (N = 1047) –0.6 (–32, 20) –25 (–47, –04) –3.1 (–49, –12) –27 (–46, –09) –2.0 (–39, –0.01) –19 (–46, +07)

Weight, full-term birth, duration breast feeding, birth rank, maternal age, and pregnancy BP Weight, height, sex, use of alcohol, smoking, oral contraceptives

* Unless oth ise stated, mmHg change per kg increase in birth weight adjusted for age where appropriate † Adjusted for sex Note Forsen et al reported results of a longitudinal study of Finnish children from age 6 months to 7 years that was excluded from this table because of the absence of estimates of effect in the form of regression coefficients However, this study showed no evidence for the association changing in childhood, at all ages there being a suggestion of a very weak positive association between birth weight and systolic blood pressure Abbreviations: Obs, Obstetric medical records

Leon and Koupilová

Netherlands Zoetermeer longitudinal study of chronic disease risk (1997) (19) Annual follow-up of 483 peoplem with mean duration of follow-up 14 years (range 3–18)

Source of birth weight

Birth Weight, Blood Pressure, and Hypertension

33

alone) is given in the column headed “Type”: negative (or inverse) associations are indicated by a minus sign (–) and positive (or direct) associations are indicated by a plus sign (+). Estimates adjusted for any other factors alone or in addition to body size are given in the final column, together with a list of the variables adjusted for. Particular emphasis is placed on the estimates adjusted for concurrent body size, because these provide the most valid basis for comparison of effects between papers and hence across age groups. The estimates in the final column, which are adjusted for a heterogeneous set of variables, are for obvious reasons the least comparable. The protocols used to measure blood pressure varied between studies, although information about this is not provided in the summary tables. In most studies, blood pressure was measured (after a defined period of rest) more than once, and the mean taken of at least two of the readings. The measurement devices used differed, but most were either automated machines or research instruments such as the random zero sphygmomanometer. There was no consistency in the protocol with respect to cuff size, particularly in the studies of children. In terms of diastolic blood pressure, there were also differences in the Korotkoff phase sounds used. This lack of consistency in the measurement of blood pressure will have introduced varying degrees of misclassification of the main outcome, which may account for some small part of the variation in the strength of the association between birth weight and blood pressure. II. Systolic Blood Pressure The data in Table 1 are derived from 36 papers published from 1981 to 1998. Of these, four were published in the 1980s, 16 were published in 1990 to 1995, and 16 were published in 1996 to 1998. Some papers reported results from more than one study population, defined in terms of geography, birth cohort, sex, or age groups. Conversely, the results from the same study population were reported in several different papers. In summary, the 56 sets (rows) of estimates presented in Table 1 are based on 47 independent study populations. Analyses of the association between birth weight and blood pressure at age 36 from the UK 1946 birth cohort were reported in three papers (3–5); analyses of the UK Preston birth follow-up study at age 46 to 54 years were reported in three papers (6–8), a fourth paper from Preston (9) was based on a subgroup not included in these earlier papers; and analyses from the UK Hertfordshire birth follow-up study at age 59 to 71 years were presented in

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two studies for men (8,10) and in two studies for women (8,11). The UK Salisbury cross-sectional study population of children was examined on two separate occasions, 5 years apart, with the results being published separately (12,13). Finally, there were two UK “10 towns” cross-sectional studies conducted in 1990 (14) and 1994 (15). These two study populations were recruited in separate sampling exercises, and as such are regarded as providing independent estimates. Almost half of the 56 sets of estimates (rows) in Table 1 relate to children or young adults: 22 are for those aged less than 17 years, 15 are for those aged between 17 and 39 years, and the remaining 19 are for those aged between 40 and 71 years. The sex distribution of estimates is balanced, with 27 being for both sexes combined and 15 for males and 14 for females. The majority used birth weight data derived from obstetric, neonatal, or other routine records, with only 12 using birth weight data based wholly or in part on the recall or reporting of subjects or their relatives. There are 25 estimates of the crude (unadjusted) association between birth weight and systolic blood pressure, 20 of which indicate a negative or inverse association. Of the negative associations, seven are stated to be statistically significant (p < 0.05). The UK 1946 birth cohort study (4,5) and the UK Preston birth follow-up study (6,7) provide crude estimates based on the same population in more than one paper, all of which show an inverse association between birth weight and systolic blood pressure. If replicate estimates are removed, out of 22 independent crude estimates 17 indicate a negative or inverse association. There are 31 estimates of the association between birth weight and systolic blood pressure that are adjusted for concurrent body size only, of which 29 show negative or inverse relationships that in 14 instances are stated to be statistically significant (p < 0.05). The UK Hertfordshire cohort (8,10,11) provides data for more than one paper. If replicate estimates are removed, out of the total of 29 independent estimates 27 show a negative association between birth weight and systolic blood pressure. Two of the independent estimates in Table 1 present change in blood pressure per birth weight standard deviation score (5). For the remaining 27 independent estimates, there is a mean change in systolic blood pressure per 1 kg increase in birth weight (adjusted for concurrent body size) of –1.70 mmHg (–2.10, –1.30). This mean was calculated weighting each independent estimate directly according to the number of subjects—with the exception of the Swedish conscript’s estimate (16). This was given a nominal weight of 10,000, because otherwise the very substantial sample size (130,842) of this

Birth Weight, Blood Pressure, and Hypertension

35

Figure 1 Change in systolic blood pressure (mmHg) per 1 kg increase in birth weight adjusted for concurrent body size by age.

study would have made it far more influential than could be justified given the other sources of error that are likely to account for between-study variation, such as repeatability of birth weight and blood pressure measurements. The distribution of independent estimates of the association between birth weight and systolic blood pressure adjusted for concurrent body size by age is shown graphically in Figure 1. This shows very clearly that the vast majority of estimates show a negative association. There is no suggestion of any systematic difference in the size of effects according to whether they were for males, females, or both sexes in combination. III. Diastolic Blood Pressure The information available on the association between birth weight and blood pressure is far less extensive for diastolic compared with systolic blood pressure. Of the 36 papers summarized in Table 1 that provide quantitative information about the association of birth weight with systolic blood pressure,

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Leon and Koupilová

only 18 provide the equivalent for diastolic blood pressure, as summarized in Table 2. Of the other 18 papers that provide quantitative estimates for the association of birth weight with systolic but not diastolic blood pressure, over half included some qualitative remarks on the diastolic association in the text. These remarks are difficult to summarize, because they frequently fail to indicate anything about the direction or magnitude of the association of birth weight with diastolic blood pressure, a typical example being, “[T]here was no clear association between birth weight and diastolic blood pressure” (17). It is reasonable to suspect, in fact, that small or nonsignificant associations of birth weight with diastolic blood pressure were least likely to be reported in quantitative form. To this extent, Table 2 needs to be interpreted with caution, as reporting bias may favor larger effects that are in the same direction as with those for systolic blood pressure. Bearing these caveats in mind, Table 2 does suggest that the association between birth weight and diastolic blood pressure is consistently negative or inverse after adjustment has been made for concurrent body size. Among the 22 independent sets (rows) of estimates, there are seven quantitative estimates adjusted for body size, all of which are inverse, with three being statistically significant (p < 0.05). The strength of these adjusted associations is generally smaller than for systolic blood pressure, ranging from –0.3 to –1.7 mmHg per kg increase in birth weight. Of the 13 crude (unadjusted) estimates, seven indicate a positive association of birth weight with diastolic blood pressure, while five indicate a negative association. IV. Effect of Concurrent Body Size The negative association of birth weight with systolic blood pressure is not dependent on adjustment for concurrent body size. As already noted, three quarters of the crude (unadjusted) estimates shown in Table 1 are negative. This is not the case with diastolic blood pressure, where fewer than half of the crude associations are negative. However, the effect of adjustment for concurrent body size is the same for systolic and diastolic blood pressure. Crude (unadjusted) estimates could be compared with the estimates adjusted for concurrent body size in eight instances for systolic and four instances for diastolic blood pressure. In each case, adjustment for concurrent body size had the effect of strengthening the size of the crude negative association or (equivalently) reducing the size of the crude positive association. This effect

Birth Weight, Blood Pressure, and Hypertension

37

of adjustment for concurrent body size is not surprising because birth weight is positively correlated with body size, which in turn is positively correlated with diastolic and systolic blood pressure. There is evidence that the strength of the association of birth weight with later blood pressure increases directly with body mass index (18,19) and weight (20). This suggests that obesity potentiates the effect of impaired fetal growth on later blood pressure, as has been also reported with respect to fetal effects on insulin resistance (21) and coronary heart disease (22). V. Is the Birth Weight–Blood Pressure Association Modified by Age? It has been hypothesized that the strength of the negative association between birth weight and blood pressure increases or is amplified with age (8). Figure 1 supports this contention with respect to the association with systolic blood pressure adjusted for concurrent body size. The weighted regression line (using the same weights as for the weighted mean already described above) suggests that for every decade of age there is an increase in the slope of the birthweight–systolic blood pressure association of –0.35 mmHg (95% CI, –0.59, –0.11) per kg increase in birth weight. On this basis, at age 10 years the mean change is –1.45 mmHg per kg increase in birth weight, going up to –3.20 mmHg at 60 years of age. Interpreting this apparent progressive strengthening of the birth weight– blood pressure association with age needs to be done with caution, not least because we are lacking estimates of effect between the ages of 25 and 45 years as is evident from Figure 1. It should also be noted that estimates of the effect for those in late middle age are based on cohorts of men and women born prewar, many in the 1920s and 1930s. In contrast, most of the estimates for children and young adults are based on populations born in the 1970s or more recently. With the data we have been considering it is thus not possible to determine how far the effects seen in Figure 1 are attributable to birth cohort effects as distinct from those attributable to age per se. To do this, it is necessary to have data on individuals where the strength of the association between birth weight and blood pressure is estimated at different ages. Four longitudinal studies have provided data on the association of birth weight with systolic blood pressure at different ages. Their findings are summarized in Table 3. Only in the Guilford and Carlisle study (23) was it concluded that strength of the association increased with age (between ages 5–

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Leon and Koupilová

7 and 9–11 years). In fact, this is the only study in which the estimates at different ages are based on exactly the same subjects. Within the other three studies, the estimates at different ages are based on different subgroups of subjects, making interpretation of changes in estimates with age problematic. The regression line derived from the cross-sectional studies discussed above suggests that there is a change in the strength of association of only – 0.35 mmHg per decade. If such an amplification effect is real, then it is perhaps not surprising that the four studies summarized in Table 3 do not show much evidence to support the phenomenon, given that all of them are based on longitudinal follow-ups of only 10 years or less. The most established modification by age of the association of birth weight with blood pressure occurs in neonates. There is a considerable body of evidence that in the first few weeks of life the association is positive, ie, in the opposite direction to that found in children and adults. Hulman et al. (24) reported a significant positive correlation of birth weight with systolic blood pressure measured in the first day of life. Elevated neonatal blood pressure measured on day 3 to 4 of life has also been correlated with higher birth weight (25), whereas systolic and diastolic blood pressure in the first week of life increased with increasing birth weight (26) with a correlation coefficient of 0.20 (p < 0.001). Similar results were reported for systolic blood pressure by Launer et al. (27). Levine et al. (28) studied blood pressure patterns in twins during the first year of life. Both systolic and diastolic blood pressure at 4 days increased with birth weight. However, as infancy progressed, this positive association became attenuated, as the twins who were lightest at birth had a steeper rise in blood pressure than the heavier twins. A summary of other studies of the birth weight–blood pressure association in neonates, nearly all of which also show a positive association, is provided by Whincup et al. (14). The significance of this reversal of the association of birth weight with blood pressure from being positive in neonates to being negative in children and adults has not been adequately explored. It is likely to be informative with respect to potential biological mechanisms that underlie fetal programming. VI. Birth Weight and Hypertension A small number of studies have looked at the association between size at birth and the binary outcome of the presence or absence of hypertension. The first study to focus on the negative association between birth weight and later blood

Birth Weight, Blood Pressure, and Hypertension

39

pressure, by Gennser et al. (29), found a higher prevalence of low birth weight for gestational age among Swedish male conscripts who had a diastolic blood pressure greater than 99 mmHg compared with normotensive controls (odds ratio = 3.6, 95% CI, 1.1, 12.6). In the US Nurses’ Health Study (30), self-reported history of hypertension was greatest among women who were lightest at birth. In the older of the two nurses’ cohorts (44–69 years), there was a smooth negative association of hypertension with birth weight after adjustment for concurrent body mass index and parental history of hypertension, with an odds ratio of 1.6 between those weighing less than 5 lbs and those weighing greater than 10 lbs at birth. Similar, but slightly smaller associations were seen in the parallel study of male US health professionals (31). Negative associations between birth weight and prevalence of diagnosed hypertension were also seen in a longitudinal study of Swedish men at ages 60 and 70 years (32). VII. Studies of Births with Abnormal Obstetric or Neonatal Characteristics A range of studies have examined blood pressure in cohorts of children who were born preterm, low birth weight, or small for gestational age. Matthes et al. (33) found adolescents born at term weighing less than 2500 g to have a systolic blood pressure that was 1 mmHg (95% CI –1, +3) higher than term births weighing 3000 to 3800 g, having adjusted for concurrent body size. Leger et al. (34) found systolic and diastolic blood pressure adjusted for concurrent body size at age 20 to be greater in small-for-gestational age females born at term compared with those who were of appropriate weight for gestational age. The opposite effect was seen for males. Williams et al. (35) compared systolic and diastolic blood pressure at age 7 and 18 years in relatively small groups of children who had experienced intrauterine growth retardation, large-for-gestational age children, and normal children, adjusting for sex and current weight. No consistent differences in systolic or diastolic blood pressure were found. Morley et al. (36) found a positive association of birth weight for gestational age with both systolic and diastolic blood pressure in a group of 8-yearold children who were born at less than 34 weeks gestation weighing less than 1850 g. This association remained positive after adjustment for concurrent body size. Hack et al. (37) studied blood pressure in very low (< 1500 g) birth weight and normal birth weight children at age 8. Both systolic and diastolic

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Leon and Koupilová

blood pressures were significantly lower in the very low birth weight children. However, as the very low birth weight children were reported to have significantly lower mean current weight and height, and no adjustments for current weight were made, the results relating to the birth weight–blood pressure association are difficult to interpret. In contrast, Pharoah et al. (38) found higher systolic blood pressure in 15-year-old children of less than 1500 g birth weight than in a quasirandom comparison sample matched on age, sex, and school. A few studies have explored the birth weight–blood pressure association in offspring of women who were hypertensive in pregnancy. For instance, Ounsted et al. (39) examined children born to mothers who had undergone a trial for treatment of hypertension in pregnancy when they were 7 years old, and reported a negative correlation between birth weight and blood pressure. A small study of children born to hypertensive mothers in Sweden also showed a weak, nonsignificant negative correlation between birth weight and systolic and diastolic blood pressure at age 12 years (40). The findings of these studies of offspring of mothers with abnormal pregnancies and in individuals who were very small, small for gestational age, or born preterm are not consistent. Some find blood pressure to be higher in the abnormal group, and others find the reverse. Small sample size and other problems of design may explain some of this lack of consistency. It is also possible that being born at the extremes of the gestational age or birth weight distributions may have effects on later blood pressure that differ from those that would be predicted from studies of individuals spanning the normal range of birth outcomes. The biological mechanisms associated with preterm birth or low or very low birth weight are unlikely to be identical to those determining variation in size at birth within the normal range, and may thus have different programming effects on blood pressure. VIII. Socioeconomic and Behavioral Pathways One of the most commonly voiced concerns about the concept of fetal programming of later disease is that the associations reported from observational epidemiological studies could be attributable to confounding by socioeconomic and behavioral factors. In other words, socioeconomic disadvantage at birth is related to reduced birth weight, and also to socioeconomic disadvantage and hence increased disease risk in later life. This could conceivably be

Birth Weight, Blood Pressure, and Hypertension

41

through behavioral factors including diet, alcohol consumption, and smoking. Surprisingly few studies have attempted to deal with this possibility systematically. Koupilová et al. (41) examined whether the association between birth weight and systolic blood pressure in 50-year-old Swedish men could be explained by socioeconomic circumstances at birth and in adult life, and by behavioral factors such as alcohol consumption. They showed that adjustment for these factors reduced the strength of association only slightly, concluding that these factors could not explain the negative association between birth weight and systolic blood pressure. However, these analyses do suggest that a small part of the association could be so explained, a conclusion reinforced in more recent analyses (42) of the same data set and by others (38). A number of other studies have adjusted for socioeconomic and other factors, ranging from behavioral factors such as smoking and alcohol consumption to maternal characteristics such as blood pressure and height. These adjusted estimates are summarized in the tables. However, the combination and range of factors adjusted for vary substantially between studies. In most cases, estimates unadjusted for these factors are not given, making interpretation of their potential mediating or confounding role very difficult. What is apparent is that nearly all of the adjusted estimates indicate a negative association of birth weight with blood pressure, although where comparisons are possible with estimates adjusted for concurrent body size alone the effect of additional adjustment is to reduce the strength of association slightly.

IX. Maternal Influences One of the central unresolved questions with respect to fetal programming of blood pressure and adult disease is the contribution of maternal factors. Of particular interest is the potential role of maternal nutritional status. Stanner et al. (43) studied a group of men and women who were in utero during the Leningrad siege. The famine conditions resulted in substantial falls in mean birth weight. The mean blood pressure of this group in adult life was compared with that of people born at the same time who were not subject to famine conditions in utero. Neither systolic nor diastolic blood pressure were related to being in utero during the famine, although there was an association with indicators of abnormal endothelial function. In another study (44), one aspect

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Leon and Koupilová

of endothelial function, flow mediated dilatation, has been associated with low birth weight. Only one reported study has been able to directly assess the impact of maternal nutrition during pregnancy on an offspring’s blood pressure. Campbell et al. (45) took advantage of a survey of diet in late pregnancy conducted in Aberdeen, Scotland between 1948 and 1954. A total of 253 men and women were traced and examined 40 years later. The associations between pregnancy diet and offspring’s blood pressure were complex. In summary, blood pressure was raised among those people whose mothers’ diets had low protein but high carbohydrate intakes and among those whose mothers had a high protein but low carbohydrate intake. Maternal anthropometry has been related to offsprings’ blood pressure in several studies. Systolic blood pressure in children aged 8 to 9 years in the Gambia was negatively associated with maternal weight gain during pregnancy. At younger ages, maternal weight in the last trimester was positively associated with offsprings’ systolic blood pressure. Birth weight was unrelated to systolic blood pressure at any age. In a study of 11-year-old children in the United Kingdom, weight gain in pregnancy between 18 and 28 weeks gestation was also negatively associated with systolic blood pressure, but there was no association with diastolic blood pressure (46). Statistically significant negative associations between maternal weight gain and offsprings’ blood pressure were only found when a subgroup analysis was performed stratified by maternal skinfold thickness. There was a small nonsignificant negative association between birth weight and systolic blood pressure. A study in Jamaica of 10- to 12-year-old children showed negative associations between children’s blood pressure and maternal weight gain in pregnancy between 15 and 35 weeks gestation, maternal triceps skinfold thickness at 15 weeks, and maternal hemoglobin (47). These effects were independent of each other and were adjusted for concurrent weight of the children. Interestingly, this study found a nonsignificant positive association between birth weight and systolic blood pressure adjusted for sex and concurrent weight. Whincup et al. have looked at a range of maternal characteristics to see how far they are related to offsprings’ blood pressure. In a study of 5- to 7year-olds (14), maternal size was positively related to children’s blood pressure, but this association was eliminated on adjustment for the child’s own body build. Maternal social class, educational attainment, and smoking were not related to their children’s blood pressure. Birth weight was negatively associated with systolic blood pressure (see Table 1). Another study of 9- to 11-year-olds (48), found that the only maternal factors that showed a clear

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and consistent association with blood pressure were minimum maternal hemoglobin levels and change in mean corpuscular volume in pregnancy. In summary, the relatively few studies that have examined the role of maternal factors have failed to produce a consistent picture. Few of the studies have found clear effects in their main analyses, most of the associations reported being from subgroups. More strikingly, there have been almost no attempts to systematically examine how far birth weight mediates any effects of maternal characteristics on blood pressure in offspring. The hypothesis that aspects of maternal nutritional status or diet before or during pregnancy program offsprings’ blood pressure may be an attractive one, but good human evidence in support of it is lacking. This is a priority area for further work. X. Conclusion Our systematic review has shown that there is substantial and consistent evidence for a negative association between birth weight and systolic blood pressure from childhood through the eighth decade of life. This association is evident in crude, unadjusted data. It is strongest and most consistent when adjusted for concurrent body size. The mean effect of 27 independent estimates shows a change of –1.70 mmHg per 1 kg increase in birth weight. The strength of the negative association increases with age when assessed using estimates from cross-sectional studies. For every decade of age, the negative association of birth weight with systolic blood pressure increases by –0.35 mmHg per 1 kg increase in birth weight. However, true longitudinal data in support of this amplification effect are lacking. A negative association between birth weight and diastolic blood pressure is also apparent from our systematic review, particularly when concurrent body size is taken into account, although this assessment is based on far less evidence than for systolic blood pressure. It is plausible that the picture for diastolic blood pressure may be distorted because of reporting bias. Papers covered in this review focus mainly on systolic blood pressure. Investigators may have tended not to include quantitative estimates of the association of birth weight with diastolic blood pressure unless they were clear and consistent with those for systolic blood pressure. The negative association between birth weight and blood pressure found in our systematic review reflects an effect that is apparent across the entire normal birth-weight range. It is not driven by effects on blood pressure of those at the extremes of the birth weight or gestational age distribution. Indeed,

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studies of the effects on later blood pressure in preterm or those who were very light at birth do not show any consistent pattern of association. Only a small number of studies have addressed the issue of whether socioeconomic or behavioral factors can account for any of the negative association between birth weight and blood pressure. Those that have suggest that, although they may account for some of the effect, they cannot account for most of the negative association. The role of maternal factors in driving the negative association has also been underexplored. Although there are some intriguing reports of maternal nutritional status or diet in pregnancy affecting offsprings’ blood pressure, they are not conclusive. To date, the human evidence regarding the potential role of maternal nutritional status in the programming of later blood pressure must be considered as weak. Epidemiology will continue to play an important role in the research agenda on fetal programming of blood pressure. Important areas for future work include investigation of maternal characteristics on offsprings’ blood pressure, whether there is evidence of programming effects before 37 weeks, and how far measures of fetal growth other than birth weight per se may be more informative about the process of fetal programming. References 1.

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Lehingue Y. Fetal environment and coronary ischemia risk: review of the literature with particular reference to syndrome X. Rev Epidemiol Sante Publique 1996; 44:262–277. Law CM, Shiell AW. Is blood pressure inversely related to birth weight? The strength of evidence from a systematic review of the literature. J Hypertens 1996; 14:935–941. Wadsworth MEJ, Cripps HA, Midwinter RE, Colley JRT. Blood pressure in a national birth cohort at the age of 36 related to social and familial factors, smoking, and body mass. Br Med J 1985; 291:1534–1538. Barker DJP, Osmond C, Golding J, Kuh D, Wadsworth MEJ. Growth in utero, blood pressure in childhood and adult life, and mortality from cardiovascular disease. Br Med J 1989; 298:564–567. Holland FJ, Stark O, Ades AE, Peckham CS. Birth weight and body mass index in childhood, adolescence, and adulthood as predictors of blood pressure at age 36. J Epidemiol Community Health 1993; 47:432–435. Barker DJP, Bull AR, Osmond C, Simmonds SJ. Fetal and placental size and risk of hypertension in adult life. Br Med J 1990; 301:259–262.

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Barker DJP, Godfrey KM, Osmond C, Bull A. The relation of fetal length, ponderal index and head circumference to blood pressure and the risk of hypertension in adult life. Paed Perinat Epidemiol 1992; 6:35–44. Law CM, de Swiet M, Osmond C, et al. Initiation of hypertension in utero and its amplification throughout life. Br Med J 1993; 306:24–27. Godfrey KM, Barker DJP, Peace J, Cloke J, Osmond C. Relation of fingerprints and shape of the palm to fetal growth and adult blood pressure. Br Med J 1993; 307:405–409. Hales CN, Barker DJP, Clark PMS, et al. Fetal and infant growth and impaired glucose tolerance at age 64. Br Med J 1991; 303:1019–1022. Fall CHD, Osmond C, Barker DJP, et al. Fetal and infant growth and cardiovascular risk factors in women. Br Med J 1995; 310:428–432. Law CM, Barker DJP, Bull AR, Osmond C. Maternal and fetal influences on blood pressure. Arch Dis Child 1991; 66:1291–1295. Fall CH, Pandit AN, Law CM, et al. Size at birth and plasma insulin-like growth factor-I concentrations. Arch Dis Child 1995; 73:287–293. Whincup PH, Cook DG, Papacosta O. Do maternal and intrauterine factors influence blood pressure in childhood? Arch Dis Child 1992; 67:1423–1429. Taylor SJ, Whincup PH, Cook DG, Papacosta O, Walker M. Size at birth and blood pressure: cross sectional study in 8–11 year old children. Br Med J 1997; 314:475–480. Nilsson PM, Ostergren P-O, Nyberg P, Soderstrom M, Allebeck P. Low birth weight is associated with elevated systolic blood pressure in adolescence: a prospective study of a birth cohort of 147,378 boys. J Hypertens 1997; 15:1627– 1631. Woelk G, Emanuel I, Weiss NS, Psaty BM. Birthweight and blood pressure among children in Harare, Zimbabwe. Arch Dis Child Fetal Neonatal Ed 1998; 79:F119–F122. Leon DA, Koupilová I, Lithell HO, et al. Failure to realise growth potential in utero and adult obesity in relation to blood pressure in 50 year old Swedish men. Br Med J 1996; 312:401–406. Uiterwaal CS, Anthony S, Launer LJ, et al. Birth weight, growth, and blood pressure: an annual follow-up study of children aged 5 through 21 years. Hypertension 1997; 30:267–271. Whincup PH, Cook DG, Shaper AG. Early influences on blood pressure: a study of children aged 5–7 years. Br Med J 1989; 299:587–591. Lithell HO, McKeigue PM, Berglund L, Mohsen R, Lithell U-B, Leon DA. Relationship of birthweight and ponderal index to non–insulin-dependent diabetes and insulin response to glucose challenge in men aged 50–60 years. Br Med J 1996; 312:406–410. Frankel S, Elwood P, Sweetnam P, Yarnell J, Davey Smith G. Birth weight, body mass index in middle age, and incident coronary heart disease. Lancet 1996; 348:1478–1480.

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23. Whincup P, Cook D, Papacosta O, Walker M. Birth weight and blood pressure: cross sectional and longitudinal relations in childhood. Br Med J 1995; 311:773– 776. 24. Hulman S, Edwards R, Chen YT, Polansky M, Falkner B. Blood pressure patterns in the first three days of life. J Perinatology 1991; 11:231–234. 25. O’Sullivan MJ, Kearney PJ, Crowley MJ. The influence of some perinatal variables on neonatal blood pressure. Acta Paediatr 1996; 85:849–853. 26. Zinner SH, Lee YH, Rosner B, Oh W, Kass EH. Factors affecting blood pressure in newborn infants. Hypertension 1980; 2(suppl I):99–101. 27. Launer LJ, Hofman A, Grobbee DE. Relation between birth weight and blood pressure: longitudinal study of infants and children. Br Med J 1993; 307:1451– 1454. 28. Levine RS, Hennekens CH, Jesse MJ. Blood pressure in prospective population based cohort of newborn and infant twins. Br Med J 1994; 308:298–302. 29. Gennser G, Rymark P, Isberg PE. Low birth weight and risk of high blood pressure in adulthood. Br Med J 1988; 296:1498–1500. 30. Curhan GC, Chertow GM, Willett WC, et al. Birth weight and adult hypertension and obesity in women. Circulation 1996; 94:1310–1315. 31. Curhan GC, Willett WC, Rimm EB, Spiegelman D, Ascherio AL, Stampfer MJ. Birth weight and adult hypertension, diabetes mellitus, and obesity in US men. Circulation 1996; 94:3246–3250. 32. Koupilová I, Leon DA, Lithell HO, Berglund L. Size at birth and hypertension in longitudinally followed 50–70 year old men. Blood Pressure 1997; 6:223– 228. 33. Matthes JWA, Lewis PA, Davies DP, Bethel JA. Relation between birth weight at term and systolic blood pressure in adolescence. Br Med J 1994; 308:1074– 1077. 34. Leger J, Levy Marchal C, Bloch J, et al. Reduced final height and indications for insulin resistance in 20 year olds born small for gestational age: regional cohort study. Br Med J 1997; 315:341–347. 35. Williams S, St George IM, Silva PA. Intrauterine growth retardation and blood pressure at age seven and eighteen. J Clin Epidemiol 1992; 45:1257–1263. 36. Morley R, Lister G, Leeson-Payne C, Lucas A. Size at birth and later blood pressure. Arch Dis Child 1994; 70:536–537. 37. Hack M, Weissman B, Breslau N, Klein N, Borawski Clark E, Fanaroff AA. Health of very low birth weight children during their first eight years. J Pediatr 1993; 122:887–892. 38. Pharoah POD, Stevenson CJ, West CR. Association of blood pressure in adolescence with birthweight. Arch Dis Child Fetal Neonatal Ed 1998; 79:F114– F118. 39. Ounsted MK, Cockburn JM, Moar VA, Redman CW. Factors associated with the blood pressures of children born to women who were hypertensive during pregnancy. Arch Dis Child 1985; 60:631–635. 40. Himmelmann A, Svensson A, Hansson L. Relation of maternal blood pressure

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during pregnancy to birth weight and blood pressure in children. The Hypertension in Pregnancy Offspring Study. J Intern Med 1994; 235:347–352. Koupilová I, Leon DA, Vågerö D. Can confounding by socio-economic and behavioural factors explain the association between size at birth and blood pressure at age 50 in Sweden? J Epidemiol Comm Health 1997; 51:14–18. Leon DA. Fetal growth and later disease: evidence from Swedish cohorts. In: O’Brien PMS, Wheeler T, Barker DJP, eds. Fetal Programming. Influences on Development and Disease in Later Life. Proceedings of 36th RCOG Study Group. London: Royal College of Obstetricians and Gynaecologists, 1999: 12–29. Stanner SA, Bulmer K, Andrès C, et al. Does malnutrition in utero determine diabetes and coronary heart disease in adulthood? Results from the Leningrad siege study, a cross sectional study. Br Med J 1997; 315:1342–1349. Leeson CPM, Whincup PH, Cook DG, et al. Flow-mediated dilation in 9- to 11year-old children. Circulation 1997; 96:2233–2238. Campbell DM, Hall MH, Barker DJ, Cross J, Shiell AW, Godfrey KM. Diet in pregnancy and the offspring’s blood pressure 40 years later. Br J Obstet Gynaecol 1996; 103:273–280. Clark PM, Atton C, Law CM, Shiell A, Godfrey K, Barker DJ. Weight gain in pregnancy, triceps skinfold thickness, and blood pressure in offspring. Obstet Gynecol 1998; 91:103–107. Godfrey KM, Forrester T, Barker DJP, Jackson AA, Landman JP, Hall JStE, et al. Maternal nutritional status in pregnancy and blood pressure in childhood. Br J Obstet Gynecol 1994; 101:398–403. Whincup P, Cook D, Papacosta O, Walker M, Perry I. Maternal factors and development of cardiovascular risk: evidence from a study of blood pressure in children. J Hum Hypertens 1994; 8:337–343. Hashimoto N, Kawasaki T, Kikuchi T, Takahashi H, Uchiyama M. The relationship between the intrauterine environment and blood pressure in 3-year-old Japanese children. Acta Paediatr 1996; 85:132–138. Simpson A, Mortimer JG, Silva PA, Spears G, Williams S. Correlates of blood pressure in a cohort of Dunedin seven-year-old children. In: Onesti G, ed. Hypertension in the Young and Old. New York: Grune & Stratton, 1981: 153–163. Moore VM, Miller AG, Boulton TJ, et al. Placental weight, birth measurements, and blood pressure at age 8 years. Arch Dis Child 1996; 74:538–541. Zureik M, Bonithon Kopp C, Lecomte E, Siest G, Ducimetiere P. Weights at birth and in early infancy, systolic pressure, and left ventricular structure in subjects aged 8 to 24 years. Hypertension 1996; 27:339–345. Donker GA, Labarthe DR, Harrist RB, Selwyn BJ, Wattigney W, Berenson GS. Low birth weight and blood pressure at age 7–11 years in a biracial sample. Am J Epidemiol 1997; 145:387–397. Lurbe E, Redon J, Alvarez V, et al. Relationship between birth weight and awake blood pressure in children and adolescents in absence of intrauterine growth retardation. Am J Hypertens 1996; 9:787–794.

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55. Forrester TE, Wilks RJ, Bennett FI, Simeon D, Osmond C, Allen M, et al. Fetal growth and cardiovascular risk factors in Jamaican schoolchildren. Br Med J 1996; 312:156–160. 56. Macintyre S, Watt G, West P, Ecob R. Correlates of blood pressure in 15 year olds in the west of Scotland. J Epidemiol Community Health 1991; 45:143– 147. 57. Laor A, Stevenson DK, Shemer J, Gale R, Seidman DS. Size at birth, maternal nutritional status in pregnancy, and blood pressure at age 17: population based analysis. Br Med J 1997; 315:449–453. 58. Kolacek S, Kapetanovic T, Luzar V. Early determinants of cardiovascular risk factors in adults. B. Blood pressure. Acta Paediatr 1993; 82:377–382. 59. Walker BR, McConnachie A, Noon JP, Webb DJ, Watt GCM. Contribution of parental blood pressures to association between low birth weight and adult high blood pressure: cross sectional study. Br Med J 1998; 316:834–837. 60. Valdez R, Athens MA, Thompson GH, Bradshaw BS, Stern MP. Birthweight and adult health outcomes in a biethnic population in the USA. Diabetologia 1994; 37:624–631. 61. Vestbo E, Damsgaard EM, Froland A, Mogensen CE. Birth weight and cardiovascular risk factors in an epidemiological study. Diabetologia 1996; 39:1598– 1602. 62. Martyn CN, Barker DJP, Jespersen S, Greenwald S, Osmond C, Berry C. Growth in utero, adult blood pressure, and arterial compliance. Br Heart J 1995; 73:116– 121. 63. Seidman DS, Laor A, Gale R, Stevenson DK, Mashiach S, Danon YL. Birth weight, current body weight, and blood pressure in late adolesence. Br Med J 1991; 302:1235–1237. 64. Taittonen L, Nuutinen M, Turtinen J, Uhari M. Prenatal and postnatal factors in predicting later blood pressure among children: cardiovascular risk in young Finns. Pediatr Res 1996; 40:627–632. 65. Forsén T. Nissinen A, Tuomilehto J, Notkola IL, Eriksson J, Vinni S. Growth in childhood and blood pressure in Finnish children. J Hum Hypertens 1998; 12:397–402.

3 Mechanisms for In Utero Programming of Blood Pressure CHRISTOPHER N. MARTYN

STEPHEN E. GREENWALD

University of Southampton and Southampton General Hospital Southampton, United Kingdom

Royal London Hospital London, United Kingdom

The epidemiological evidence that individuals whose birth weight was low tend to have raised blood pressure in later life is, as Leon and Koupilová have shown in Chapter 2, substantial and consistent. Their review is an update of an earlier systematic review published in 1996 (1). Although more data are now available, the conclusions are little changed. In prepubertal children and adults there is a consistent negative relation between birth weight and current systolic blood pressure. There is a suggestion that the strength of the negative association increases with age, but true longitudinal data will be needed to confirm this. Rather less evidence is available about diastolic blood pressure, but what there is indicates that the size of the effect is smaller. Retarded fetal growth may therefore have a greater influence on pulse pressure (ie, systolic pressure minus diastolic pressure) than on mean blood pressure. Two questions immediately arise from these findings. The first concerns underlying biological mechanisms. What is the nature of the adaptations made by the fetus in response to influences that retard its growth that have such a permanent effect? The second question is about the relevance of the relation 49

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between blood pressure and birth weight to understanding why impairment of growth in fetal life is associated with increased risk of atherosclerosis (2), coronary heart disease, and stroke (3). This second question needs to be kept in mind when trying to answer the first because, although the results of studies linking blood pressure to birth weight are remarkably consistent, the absolute size of the effect is small. A change of 1 kg of birth weight corresponds to a change of only 2 or 3 mmHg in systolic blood pressure. By itself, the effect of birth weight on blood pressure can account for only a part of the observed increase in risk of vascular disease associated with low birth weight. Might it be that this small increase in blood pressure signals not just a process in the causal chain linking fetal growth to cardiovascular outcomes in adult life but widespread effects of intrauterine growth retardation on the physiology and structure of the vascular system? No doubt the answers to these questions will prove complex. The control of blood pressure is mediated and influenced by multiple structural, neural, endocrine, and renal mechanisms. Perturbations in one control mechanism inevitably lead to compensatory changes in others and dissecting out which is primary is likely to be difficult. In this chapter, we discuss three hypotheses about possible mechanisms, although we are sure further research will produce other candidates. I. Structure of the Aorta and Large Conduit Arteries Engineers go to a lot of trouble to ensure that the structures they design have the necessary properties of elasticity and resilience. Without these properties, the structure would be poor both in resisting an external force and in recovering its shape when the force is removed. In circumstances where a structure must endure repetitive loading, the materials that it is made of must possess the additional quality of being resistant to fatigue. Biological structures must meet the same requirements. Vertebrates have evolved a unique solution to the demand for a material with a high degree of elasticity and resilience; the rubber-like macromolecule, elastin. Elastin is a high–molecular weight, insoluble protein polymer formed extracellularly by the covalent cross-linking of tropoelastin monomers. The process by which elastin is laid down is complicated (4). Newly synthesized tropoelastin binds to a recycling 67 kDa galactolectin chaperone that prevents premature intracellular aggregation of the monomer. After excretion into the extracellular space, the galactolectin-tropoelastin complex remains on the cell

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surface until it comes into contact with a nascent elastin fiber. Here, the chaperone interacts with galactosugars of the microfibrillar component, releasing tropoelastin monomer to be aligned, rapidly modified by lysyl oxidase and incorporated into the elastic network by irreversible polymerization. In the vertebrate lineage, the first appearance of elastin coincides with the evolution of a closed circulatory system (5). This is surely more than accidental. An elastic reservoir is a fundamental requirement of any closed circulatory system powered by a pump whose output is pulsatile. In the adult human, the left ventricle ejects about 70 mL of blood with each contraction even at rest. This ejection takes place into a vessel filled with incompressible fluid at high pressure. Flow of blood out of the ventricle is only possible because the volume of the aorta is able to expand. Most of the energy of left ventricular contraction is stored briefly in the stretch of the aortic wall. In diastole, elastic recoil of the wall maintains the forward flow of blood against a closed aortic valve. The elastic properties of the aorta are important in reducing the tension that must be generated by the left ventricular wall and in limiting the rise in arterial pressure during systole. Further, blood flow in coronary circulation, which occurs almost exclusively in diastole, depends on the elastic recoil of the aortic wall. At a given cardiac output, loss of aortic elasticity results in a widening of pulse pressure and a rise in the circumferential stress in the walls of the aorta and the vessels that branch from it. At the same time, the work that must be done by the left ventricle is increased while the force driving coronary blood flow is lessened. Although these functions of the aorta have been understood by physiologists for many years, their clinical relevance has only recently been appreciated. With the development of noninvasive techniques for measurement of aortic compliance, decreased compliance has been shown to be associated with both systemic hypertension and left ventricular hypertrophy (6,7). Decreased aortic compliance has been recognized as a marker of cardiovascular disease (8), and pulse pressure is a powerful independent predictor of cardiovascular events, especially in patients with impaired left ventricular dysfunction after myocardial infarction (9). A. Molecular Basis of Aortic Compliance As has been mentioned already, the elastic properties of the aorta depend very largely on the presence of elastin in the vessel wall. In the thoracic aorta, elastin accounts for 40% of the dry weight of the tissue, and even in the abdom-

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inal aorta and carotid arteries elastin is a major component. Aortic elastin is arranged in multiple concentric lamellae interspersed with smooth muscle and collagen. The number of these elastic lamellae is greatest in the proximal part of the aorta. They begin to develop early in fetal life, and rates of elastin synthesis in blood vessels increase to a maximum in the perinatal period (10–12). Thereafter, rates of elastin synthesis fall rapidly. Mature cross-linked elastin is remarkable for its longevity. Its metabolic stability has been estimated in animals from changes in specific activity of incorporated radiolabels and in humans by measurement of racemization of L-aspartate and assessment of the prevalence of nuclear weapons–related radiocarbon (13–15). This work has shown both that turnover of elastin is extremely slow—its half-life is about 40 years—and that there is no appreciable synthesis of mature elastin in adult life. In the rat, a brief period of growth inhibition on day 15 of fetal life, when cellular growth in the developing aortic wall is rapid, induced persistent changes in the chemical composition of the aorta, including a reduction in the total content of elastin (16). It seems, therefore, that there is a critical period during development of the aorta and large arteries, and also perhaps other elastin-containing tissues, when elastin is laid down and that failure to synthesize adequate amounts of elastin at this time cannot be rectified later. B. Effects of Aging By middle age, the human aorta has undergone more than 1 billion cycles of expansion and contraction. The effect of cyclic mechanical stress on any material is gradually to reorganize its crystalline structure and cause it to fracture at a load that it was previously able to bear. In the aorta, the fatiguing effects of cyclic stress lead to fracture of elastin fibers and the transfer of stress to collagen fibers (17). This process is visible microscopically as a fragmentation and loss of regularity in the elastic layers of the tunica media. Collagen is about 100 times stiffer than elastin, and the gradual loss of elastin is inevitably accompanied by a reduction in vascular compliance. Loss of compliance leads to a rise in pulse pressure and an increase in the circumferential stress in the arterial wall. Vascular smooth muscle cells respond to mechanical stretch by synthesizing collagen (18,19), which results in thickening of the arterial wall and a further decrease in compliance. A feedback loop is established that tends to maintain higher levels of blood pressure (Fig. 1). The increasing stiffness of the aorta and large conduit arteries is at least part of the reason for the tendency of blood pressure to rise with age. But, by

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Figure 1 Inter-relations of arterial compliance, pulse pressure, and synthetic activity of vascular smooth muscle.

itself, it fails to account for the variability of the rise in blood pressure between individuals. Many longitudinal studies have shown that levels of blood pressure in individuals track over long periods of time (20,21). That is to say, within a cohort, people tend to maintain the same position in the rank order of blood pressures from one examination to the next. Tracking of systolic blood pressure is detectable from about 6 months of age (22), which indicates that mechanisms affecting both the distribution of blood pressure within populations and individual risk of adult hypertension are operating from very early in life. A deficiency in vascular elastogenesis during development of the aorta and large arteries may be one of these mechanisms. C. Regulation of Vascular Elastogenesis During Development Animal experiments have shown that the synthesis of elastin in the tunica media of developing arteries is influenced by local hemodynamic conditions. For example, at birth, when pulmonary and systemic pressures are similar, the ratios of elastin to collagen in the pulmonary artery and the aorta of the rabbit are the same. Two months after birth, by which time pulmonary pressure has decreased from 40 to 15 mmHg and systemic pressure increased to 80 mmHg, the ratio of elastin to collagen in the aorta is nearly twice that of the pulmonary artery (23). Human fetal arterial structure also adapts to abnormal levels of pressure and flow. These structural adapations persist, at least in some circumstances, in postnatal life. In fetuses with a single umbilical artery, the entire blood flow

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to the placenta passes through the common iliac artery on one side only. In autopsies of eight children, aged 2 days to 4 years, born with a single umbilical artery, the iliac vessel on the side of the umbilical artery was found to have a lamellar structure rich in elastin, whereas the contralateral artery, which had not been included in the placental circulation, was small in diameter and thin walled (24). A larger study of living children born with a single umbilical artery showed a striking asymmetry in the compliance of the iliac arteries at ages 5 to 9 years (25). As yet, there is no direct evidence that elastin synthesis is impaired in the developing aorta of human fetuses whose growth is retarded. It is known, however, that in such fetuses, Doppler blood flow velocity waveforms are altered in several vascular beds, including the descending aorta. These alterations represent a redistribution of fetal cardiac output that maintains the supply of oxygen and nutrients to the developing brain at the expense of other organs (26,27). At their most extreme, the cardiovascular adaptations made by the growth-retarded fetus result in a reversal of the normal direction of blood flow in the umbilical arteries and in the aorta at the end of diastole (28). It would not be surprising if these hemodynamic changes, which are occurring at a time of rapid vascular development, influenced rates of synthesis of elastin. Indirect evidence comes from the finding in a study of 50-year-old men and women that aortic pulse wave velocity, a measure of the elasticity of the aorta, was related to size at birth (29). People with higher pulse wave velocities, and therefore stiffer aortas, tended to have been smaller babies. The relation persisted after adjustment for current blood pressure, which is consistent with the idea that reduced aortic compliance was a primary event rather than simply a consequence of raised blood pressure. II. Summary One possible explanation of the association of low birth weight with raised blood pressure in adult life is that in fetuses whose growth is retarded, there is impairment in the synthesis of elastin during a critical period of blood vessel development. This impairment may be a consequence of hemodynamic changes in fetal circulation that accompany intrauterine growth retardation although, because expression of the elastin gene is regulated by, among other things, IGF-1 and glucocorticoids, other pathways may be involved. As a result of the relative deficiency in elastin, the compliance of the aorta and large

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arteries is reduced. This in turn leads to higher pulse pressures. Over time, the gradual loss of elastin that accompanies aging and its replacement with collagen will tend to amplify the increase in blood pressure and may also predispose to left ventricular hypertrophy and cardiovascular disease. This hypothesis is currently being tested both experimentally in animals and in observational studies in humans. III. Capillary Density and Microvascular Dilation The anatomical correlate of increased vascular resistance in essential hypertension is mainly in microvessels with luminal diameters of less than 100 ìm. In several tissues, capillary density has been found to correlate inversely with blood pressure and peripheral resistance in both hypertensive and normotensive subjects (30). Dermal vessels in men with a familial predisposition to raised blood pressure show impaired microvascular dilation to a heat stimulus and capillary rarefaction (31). A decrease in capillary density may contribute to an increase in vascular resistance, and it has been suggested that defective angiogenesis may be a causal component in the inheritance of raised blood pressure. One does not have to make a large leap to consider the additional possibility that angiogenesis may be impaired in people who experienced intrauterine growth retardation. Empirical evidence on the matter, however, is largely lacking. No associations were found between birth measurements and forearm blood flow, muscle capillary density, or capillary basement membrane thickness in a group of 27 women who were investigated as part of a larger study of the relations between fetal growth, blood pressure, and diabetes in adult life (32). On the other hand, strong relations between microvascular function and both blood pressure and insulin sensitivity have been recently reported in a small study of normotensive glucose-tolerant subjects (33). Eighteen men and women, selected to show a wide range of insulin sensitivity as assessed by the hyperinsulinemic euglycemic clamp technique, had blood pressure measured by 24-hour ambulatory monitoring. Videomicroscopy was used to measure skin capillary density and capillary recruitment after arterial occlusion. Skin blood flow responses after iontophoresis of acetylcholine and sodium nitroprusside were evaluated by laser Doppler fluximetry. A substantial part of the variation in both blood pressure (r2 = 0.38) and insulin sensitivity (r2 = 0.71) could be explained by variation in microvascular function.

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In view of the strong associations between thinness at birth and insulin resistance in adult life, it would be of great interest to carry out a similar investigation in a population who had been measured in detail at birth. IV. Renal Size and Function The kidney, of course, plays a crucial part in the long-term regulation of intravascular fluid volume and pressure (34). It undergoes a period of rapid development in late fetal life, and the complement of nephrons is determined before birth. Nephron number in the human kidney varies from under 300,000 to more than 1,100,000, and there is good evidence both in animal experiments (35) and in observational studies in humans that nephron number is permanently reduced by fetal growth retardation. The idea that nephron endowment at birth influenced risk of developing essential hypertension in later life was first proposed by Brenner more than a decade ago (36,37). He argued that maintenance of raised levels of blood pressure must involve a renal factor favoring sodium retention, thereby preventing a pressure-induced natriuresis from restoring blood pressure toward normal levels. He postulated that this renal factor was a restricted capacity for sodium excretion imposed by a congenital deficit of nephrons. A reduction in renal mass, and therefore in glomerular filtration surface area, tends to produce a rise in blood pressure in the systemic arterial circulation and in the glomerular capillaries. This rise can be viewed as compensatory because it increases the glomerular filtration rate and promotes fluid excretion. However, sustained exposure of nephrons to higher glomerular perfusion pressures gradually causes the development of focal and segmental glomerular sclerosis. This in turn leads to further glomerular loss, a reduction in ability to excrete sodium, and a self-perpetuating cycle of rising blood pressure and progressive glomerular injury. There is already some evidence to link renal function in adult life with fetal growth. In a study of 50-year-old men and women, plasma renin concentrations were related to size at birth (38). In another group of similar age, microalbuminuria was more common in those whose weight or ponderal index at birth had been low (39). Unpublished observations from a recent study of 70-year-old men and women show that plasma creatinine concentrations were higher and creatinine clearance lower in those who had been light at birth. More extensive study of the possible effects of fetal adaptations and renal function would be worthwhile. Meanwhile findings from South Carolina pro-

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vide early evidence linking impaired fetal growth with the later development of renal failure.

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Law CM, Shiell AW. Is blood pressure inversely related to birth weight? The strength of evidence from a systematic review of the literature. J Hypertension 1996; 14:935–941. Martyn CN, Gale CR, Jespersen S, Sheriff SB. Impaired fetal growth and atherosclerosis of carotid and peripheral arteries. Lancet 1998; 352:173–178. Martyn CN, Barker DJP, Osmond C. Mothers pelvic size, fetal growth and death from stroke in men. Lancet 1996; 48:1264–1268. Debelle L, Tamburro AM. Elastin: molecular description and function. Int J Biochem Cell Biol 1999; 31:261–272. Sage H. The evolution of elastin: correlation of functional properties with protein structure and phylogenetic distribution. Comp Biochem Physiol 1983; 74B:373–380. Relf IRN, Lo CS, Myers KA, Wahlqvist ML. Risk factors for changes in aortoiliac arterial compliance in healthy men. Atherosclerosis 1986; 6:105–108. Girerd X, Laurent S, Pannier B, Asmar R, Safar M. Arterial distensibility and left ventricular hypertrophy in patients with sustained essential hypertension. Am Heart J 1991; 122:1210–1214. Lehmann ED. Pulse wave velocity as a marker of vascular disease. Lancet 1996; 348:744. Mitchell GF, Moye LA, Braunwald E, et al. Sphygmomanometrically determined pulse pressure is a powerful independent predictor of recurrent events after myocardial infarction in patients with impaired left ventricular function. Circulation 1997; 96:4254–4260. Davis EC. Elastic lamina growth in the developing mouse aorta. J Histochem Cytochem 1995; 43:1115–1123. Berry CL, Looker T, Germain J. Nucleic acid and scleroprotein content of the developing human aorta. J Pathol 1972; 108:265–274. Bendeck MP, Langille BL. Rapid accumulation of elastin and collagen in the aortas of sheep in the immediate perinatal period. Circulation Res 1991; 69:1165– 1169. Lefevre M, Rucker RB. Aorta elastin turnover in normal and hypercholesterolemic japanese quail. Biochimica et Biophysica Acta 1980; 80:519–529. Powell JT, Vine N, Crossman M. On the accumulation of D-aspartate in elastin and other proteins of the ageing aorta. Atherosclerosis 1992; 97:201–208.

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15. Schapiro SD, Endicott SK, Province MA, Pierce JA, Campbell EJ. Marked longevity of human lung parenchymal elastic fibres deduced from prevalence of D-aspartate and nuclear weapons–related radiocarbon. J Clin Invest 1991; 87:1828–1834. 16. Berry CL, Looker T. An alteration in the chemical structure of the aortic wall induced by a finite period of growth inhibition. J Anat 1973; 114:83–94. 17. Nichols WW, O’Rourke M. Aging, high blood pressure and disease in humans. In: McDonald’s Blood Flow in Arteries. London: Edward Arnold, 1990:398– 420. 18. Folkow B. Structure and function of the arteries in hypertension. Am Heart J 1987; 114:938–947. 19. Leung DYM, Glagov S, Mathews MB. A new in vitro system for studying cell response to mechanical stimulation. Different effects of cyclic stretching and agitation on smooth muscle cell biosynthesis. Experimental Cell Research 1977; 109:285–298. 20. Voors W, Webber LS, Berenson GS. Time course studies of blood pressure in children: the Bogalusa heart study. Am J Epidemiol 1979; 109:320–334. 21. Clarke WR, Schrott H, Leaverton PE, Connor WE, Laver RM. Tracking of blood lipids and blood pressure in school age children: the muscatine study. Circulation 1978; 58:626–634. 22. Labarthe DR, Eissa M, Varas C. Childhood precursors of high blood pressure and elevated cholesterol. Annual Review of Public Health 1991; 12:519–541. 23. Leung DYM, Glagov S, Mathews MB. Elastin and collagen accumulation in rabbit ascending aorta and pulmonary trunk during postnatal growth. Circ Res 1977; 41:316–323. 24. Meyer WW, Lind J. Iliac arteries in children with a single umbilical artery: structure, calcifications, and early atherosclerotic lesions. Arch Dis Child 1974; 49:671–679. 25. Berry CL, Gosling RG, Laogun AA, Bryan E. Anomalous iliac compliance in children with a single umbilical artery. Br Heart J 1978; 40:709–717. 26. Al-Ghazali W, Chita SK, Chapman MG, Allan LD. Evidence of redistribution of cardiac output in asymmetrical growth retardation. Br J Obs Gynae 1989; 96:697–704. 27. Rizzo G, Arduini D. Fetal cardiac function in intrauterine growth retardation. Am J Obstet Gynecol 1991; 165:876–882. 28. Fouron JC, Teyssier G, Shalaby L, Lessard M, van Doesburg NH. Fetal central blood flow alterations in human fetuses with umbilical artery reverse diastolic flow. Am J Perinatol 1993; 10:197–207. 29. Martyn CN, Barker DJP, Jespersen S, Greenwald S, Osmond C, Berry C. Growth in utero, adult blood pressure, and arterial compliance. Br Heart J 1995; 73:116– 121. 30. Prasad A, Dunhill GS, Mortimer PS, MacGregor GA. Capillary rarefaction in the forearm skin in essential hypertension. J Hypertens 1995; 13:265–268.

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31. Noon JP, Walker BR, Webb DJ, Shore AC, Holton DW, Edwards HV. Impaired microvascular dilatation and capillary rarefaction in young adults with a predisposition to high blood pressure. J Clin Invest 1997; 99:1873–1879. 32. Thompson CH, Sanderson AL, Sandeman D, et al. Fetal growth and insulin resistance in adult life: role of skeletal muscle morphology. Clin Sci 1997; 92:291–296. 33. Serne EH, Stehouwer CD, ter Maaten JC, et al. Microvascular function relates to insulin sensitivity and blood pressure in normal subjects. Circulation 1999; 99:896–902. 34. Guyton AC. Blood pressure control—special role of the kidneys and body fluids. Science 1991; 252:1813–1816. 35. Merlet-Bénichou C, Gilbert T, Muffat-Joly M, Lelièvre-Pégorier M, Leroy B. Intrauterine growth retardation leads to a permanent nephron deficit in the rat. Pediatric Nephrology 1999; 8:175–180. 36. Brenner BM, Chertow GM. Congenital oligonephropathy and the etiology of adult hypertension and progressive renal injury. Am J Kidney Dis 1994; 23:171– 175. 37. Mackenzie HS, Brenner BM. Fewer nephrons at birth: a missing link in the etiology of essential hypertension? Am J Kidney Dis 1995; 91:98. 38. Martyn CN, Lever AF, Morton JJ. Plasma concentrations of inactive renin in adult life are related to indicators of foetal growth. J Hypertens 1996; 14:881– 886. 39. Yudkin JS, Phillips DIW, Stanner S. Proteinuria and progressive renal disease: birthweight and microalbuminuria. Nephrology, Dialysis and Transplantation 1997; 12:10–13.

4 Low Birth Weight and the Emerging Burden of Renal Disease in the United States

DANIEL T. LACKLAND and BRENT M. EGAN

HOLLY E. BENDALL and CLIVE OSMOND

Medical University of South Carolina Charleston, South Carolina

University of Southampton and Southampton General Hospital Southampton, United Kingdom

The geographic variation in cardiovascular disease risks in the United States has been an enigma for many years. The southeastern region of the United States has been on record for over 6 decades as an area of unusually high death rates from stroke and has been described as the “stroke belt” (1,2). The rates of stroke and cardiovascular disease are particularly high in South Carolina, where stroke incidence and mortality rates are significantly greater than in other areas of the country (Fig. 1) (2,3). Likewise, within a specific geographic area, significant racial differences in risk are observed, with blacks having a twofold stroke risk compared with whites (Fig. 2). However, the geographic variation in disease is not explained by the demographics of the population as the race-sex specific stroke rates remain the highest in South Carolina (3). In addition to the higher overall disease rates in this population, the rates are greater in younger age groups, suggesting an earlier onset of disease in South Carolina compared with other populations (3–8). In fact, the age-specific rates in South Carolina are similar to the rates in the Framingham (4), Rochester (Minnesota) (5), Norway (6), Copenhagen (7), and Minnesota 61

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Figure 1 Age-adjusted stroke mortality rates per 100,000 population and 95% CI for males and females in the United States and South Carolina, 1989– 1991.

Figure 2 Age-adjusted stroke mortality rates per 100,000 population by racesex groups. South Carolina, 1989–1991.

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(8) cohorts 10 years of age older than the South Carolina population (3). Adding to the excess risks puzzle for this population is the recent finding that South Carolina residents who were born in the state have significantly higher rates of stroke than residents who were born out of the Southeast (9). The higher “nativity” stroke risks for South Carolinians are consistent for each of the demographic groups (9). Hypertension is a long-recognized risk factor for stroke, and this relationship is particularly evident in young black males where nearly 50% of the strokes for this group are classified as intracerebral hemorrhage (3,10). Hypertension affects nearly one third of the adult population in South Carolina. As seen in Figure 3, a significant percentage of men and women are hypertensive at early ages, with higher rates among blacks (11). Similar to hypertension, diabetes also affects a significant number of residents in the region, again with higher rates in the black population (12). Although stroke has been the most evident and recognized icon for the southeastern United States, end-stage renal disease (ESRD) is rapidly emerging as another major public health concern for the region. The rates of ESRD, as defined by individuals with renal failure requiring dialysis, are higher for South Carolina compared with the United States, age-race-sex standardized incidence of ESRD in the state being 345 per 1,000,000 compared with an overall rate of 268 per 1,000,000 in the United States (13). Adding to the burden is the incidence of ESRD in South Carolina, which is increasing in dramatic fashion and actually doubled from 1987 to 1996 (13). Similarly high rates and trends are seen in the neighboring states in the southeastern United States (13,14). Typically, ESRD is considered a late complication of chronic diseases, including diabetes and hypertension (15,16). However, in South Carolina 40% of the prevalent cases of ESRD are under 55 years of age (14). Similar to stroke, the disease burden is greater among blacks who have fivefold higher rates of ESRD than the white population even after adjusting for differences in socioeconomic status (15–18). Whereas blacks account for approximately 30% of the South Carolina population, over two thirds (69%) of the ESRD cases are black (Fig. 4) (14). As identified in other populations and areas in the United States, hypertension and diabetes account for 71% of the ESRD cases in South Carolina, with the remainder being attributed to glomerulonephritis, polycystic disease, and other renal disorders (Fig. 5) (14). As previously indicated, hypertension and diabetes are significantly more prevalent among blacks in South Carolina (11,12).

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Figure 3 Prevalance of hypertension among white and black (a) men and (b) women in South Carolina, 1987. Hypertension was defined as blood pressure 140/90 mmHg or greater and/or if the patient was medically treated for high blood pressure.

The reasons for the excess ESRD risks in this population remain unknown. However, events occurring early in life may play a role. One possible assessment could use low birth weight as an index for impaired development in utero. As described in other chapters, individuals with low birth weight have been found to have increased rates of hypertension and non–insulin dependent diabetes (NIDDM) (19–24), although the mechanisms underlying these asso-

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Figure 4 End-stage renal disease cases in South Carolina by race, 1997.

Figure 5 Specific diagnoses for end-stage renal disease cases, South Carolina, 1997.

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Table 1 Low Birth Weight Rates* by Race for South Carolina and United States, 1987

South Carolina United States *

White

Black

Total

6.8% 6.5%

13.5% 13.0%

9.2% 7.5%

Defined as the percent of births less than 2500 g.

ciations remain unclear. As described in the previous chapter, Brenner and colleagues have proposed that chronic renal failure is associated with a reduced number of nephrons in the kidneys of people who had low birth weight (25–28). In humans, 60% of the normal complement of nephrons are laid down during the last trimester and development of kidneys ceases at around week 35 (29). The rate of low birth weight is higher in South Carolina than is the average for the United States, with rates for blacks twice those for whites (Table 1) (30). The increased disease risks, population characteristics, and prevalence of hypertension, diabetes, and low birth weight suggested that events in utero might be linked to ESRD in the high-risk geographic area of the southeastern region of the United States. I.

ESRD in South Carolina

ESRD and birth weight were studied in 1230 young, black and white dialysis patients born in South Carolina after 1950 and still living in the state (31,42). The cases of ESRD were identified from the Southeast Kidney Council dialysis registry and matched to birth certificates in the South Carolina birth registry. The birth registry was also used to select two age, sex, and race-matched controls, of known birth weight, for each case. Birth weights were abstracted from the birth certificates and categorized into five groups: less than 2500 g, 2500 to 2999 g, 3000 to 3499 g, 3500 to 3999 g, and at least 4000 g. The median age of the 1230 cases was 34 years; 70% (858) were black, 73% (892) were male. For 20% (233), the nephrologist’s diagnosis of primary cause of renal failure was diabetes, 29% (359) had hypertension, 46% (571) were recorded as “other,” and for 67 (5%) the primary cause was recorded

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as “unknown.” The mean birth weight, for cases and controls combined, was 3210 g for blacks and 3361 g for whites, and higher in men (3295 g) than in women (3149 g). Table 2 shows odds ratios for renal failure from all causes according to birth weight. The odds ratio for ESRD is highest in the lowest birth weight group (<2500 g), compared with the 3000 to 3499 g reference group (OR 1.4, 95% CI 1.1–1.8), but there is evidence of a U-shaped trend in the odds ratios (p = 0.02 for quadratic trend) (42). Similar patterns were observed in men and women, and in blacks and whites, analyzed separately. The odds ratios for renal failure in relation to birth weight according to primary cause are shown in Table 2. There are elevated odds ratios in the lowest birth weight group compared with the reference category irrespective of primary cause (odds ratios ranging from 1.3–1.4) (42). The U-shaped trend is evident among subjects with diabetes (p = 0.02 for quadratic trend), but it is not present in people with hypertension or other causes of renal failure. One interpretation of these findings is that reduced fetal growth is associated with defects in the development of the kidney that make it more vulnerable to a number of pathological processes. As indicated previously, the number of nephrons in the normal population varies widely, from 300,000 to 1,100,000 or more (32). Animal and human studies have shown that low rates of intrauterine growth are associated with reduced numbers of nephrons (33). Brenner has suggested that retarded fetal growth leads to reduced numbers of nephrons, which in turn lead to increased hydrostatic pressure in the glomerular capillaries, glomerular hyperfiltration, and the development of glomerular sclerosis

Table 2 Odds Ratios for Renal Failure by Primary Cause and Birth Weight Group* Birth weight group (g) Diabetes

Hypertension

<2500 1.3 2500–2999 3000–3499 3500–3999 ≥4000

1.4 (0.9, 1.2 (0.8, 1.0 0.6 (0.4, 1.0 (0.6,

*

(0.7, 2.4) 1.0 (0.6, 1.6) 1.0 1.3 (0.8, 2.0) 2.4 (1.3, 4.2)

2.3) 1.7) 0.9) 1.6)

Other 1.3 0.9 1.0 0.8 0.8

Odds ratio (95% CI). Data from Lackland et al. (42).

(0.9, 1.9) (0.7, 1.3) (0.6, 1.1) (0.6, 1.2)

Unknown 2.1 1.6 1.0 1.1 1.3

(0.7, 5.7) (0.7, 3.6) (0.5, 2.6) (0.4, 4.1)

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(34). The sclerosis that develops from this process could lead to even further loss of nephrons. Further support for an effect of fetal development on renal function is seen in the Aborigines, among whom low birth weight is associated with high urinary albumin/creatinine ratios (35). The association between birth weight and diabetic ESRD was U-shaped and may reflect a mixture of insulin-dependent, childhood-onset, and noninsulin dependent diabetes (NIDDM). The risk of ESRD is four times greater in patients with insulin-dependent diabetes (36), which is associated with high birth weight (37). In contrast, development of NIDDM is associated with low and high birth weight (38). Prevalent in the South Carolina population, maturity-onset diabetes of the young (MODY), described as the clinical characteristics of early-onset NIDDM, should also be considered in this assessment (39). Although somewhat different in physical characteristics and demographics from the black-white population in our study, an investigation of early-onset Japanese NIDDM patients (diagnosed before age 30) found a high incidence of diabetic nephropathy consistent with Pima Indian NIDDM and Caucasian IDDM patients (40). In similar fashion, a U-shaped association between birth weights and elevated urinary albumin excretion was identified in Pima Indians with NIDDM (41). The excess rates of ESRD among young people in South Carolina seem to be associated with and originate through an adverse environment in utero, which may impair the development of the kidney and make it more vulnerable to damage by a range of pathologies. The associations between low birth weight and ESRD extend across race and gender lines, and could provide one explanation for the high rates of chronic renal failure in the southeastern region of the United States as well as the excess rates of ESRD among the black population. Likewise, the higher disease rates of renal failure in young blacks with diabetes and hypertension, when compared with whites, may reflect an accelerated progression of these diseases in blacks and/or a greater vulnerability of the kidney to their effects. References 1. 2. 3.

Perry HM, Roccella EJ. Conference report on stroke mortality in the southeastern United States. Hypertension 1998; 31:1206–1215. Lackland DT, Moore MA. Hypertension-related mortality and morbidity in the Southeast. Southern Med J 1997; 90:191–198. Lackland DT, Bachman DL, Carter TD, et al. The geographic variation in stroke

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5.

6.

7. 8.

9. 10.

11.

12.

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14. 15.

16. 17. 18. 19.

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incidence in two areas of the southeastern stroke belt: the Anderson and Pee Dee Stroke Study. Stroke 1998; 29:2061–2068. Wolf PA, Cobb JL, D’Agostino RB. Epidemiology of stroke. In: Barnett HJM, Stein BM, Mohr JP, Yatsu FM, eds. Stroke: Pathology, Diagnosis, and Management. New York: Churchill-Livingstone, 1992; 3–27. Brown RD, Whisnant SJ, Sicks JD, O’Fallon WM, Wiebers DO. Stroke incidence, prevalence and survival secular trends in Rochester, Minnesota, 1986 through 1989. Stroke 1996; 27:373–380. Ellekjaer H, Holmen J, Indredovik B, Lerent A. Epidemiology of stroke in Innherred, Norway, 1994 to 1996: incidence and 30-day care fatality rate. Stroke 1997; 28:2180–2184. Truelson T, Prescott E, Gronbuck M, Schnohr P, Boysen G. Trends in stroke incidence: the Copenhagen City Heart Study. Stroke 1997; 28:1903–1907. McGovern PG, Burke GL, Sprafka JM, Xue S, Folsom AR, Blackburn H. Trends in mortality, morbidity, and risk factor levels for stroke from 1966 through 1990: the Minnesota Heart Survey. JAMA 1992; 268:753–759. Lackland DT, Egan BM, Jones PJ. Impact of nativity and race on ‘Stroke Belt’ mortality. Hypertension 1999; 34:57–62. Lackland DT, Carter TD, Bachman DL. Intracerebral Hemorrhage in Young African Americans in the Stroke Belt. Proceedings of the American Heart Association International Conference on Stroke and Cerebral Circulation, 1999. Lackland DT, Orchard TJ, Keil JE, et al. Are race differences in the prevalence of hypertension explained by body mass and fat distribution?: a survey in a biracial population. Int J Epidemiol 1992; 21:236–246. Eberhardt MS, Lackland DT, Wheeler FC, German RR, Teutsch SM. Is race related to glycemic control? An assessment of glycosylated hemoglobin in two South Carolina communities. J Clin Epidemiol 1994; 47:1181–1189. US Renal Data System, USRDS 1998. Annual Data Report, National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland, April 1998. ESRD Network 6 1997 Annual Report, Southeastern Kidney Council, Inc. Raleigh, North Carolina, 1998. Brancati FL, Whittle JC, Whelton PK, Seidler AJ, Klag MJ. The excess incidence of diabetic end-stage renal disease among blacks. JAMA 1992; 268:3079– 3084. McClellan W. Hypertensive end-stage renal disease in blacks: the role of endstage renal disease surveillance. Am J Kidney Dis 1993; 21:25–30. Perneger TV, Whelton PK, Klag MJ. Race and end-stage renal disease. Arch Intern Med 1995; 155:1201–1208. Rostand SG. US minority groups and end-stage renal disease: a disproportionate share. Am J Kidney Dis 1992; 19:411–413. Hales CN, Barker DJP, Clark PMS, et al. Fetal and infant growth and impaired glucose tolerance at age 64. Br Med J 1991; 303:1019–1022.

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20. Lithell HO, McKeigue PM, Berglund L, Mohsen R, Lithell UB, Leon DA. Relation of size at birth to non–insulin dependent diabetes and insulin concentrations in men aged 50–60 years. Br Med J 1996; 312:406–410. 21. Barker DJP, Osmond C, Gloding J, Kuh D, Wadsworth MEJ. Growth in utero, blood pressure in childhood and adult life, and mortality from cardiovascular disease. Br Med J 1989; 298:564–567. 22. Nilsson PM, Ostergren PO, Nyberg P, Soderstrom M, Allebeck P. Low birth weight is associated with elevated systolic blood pressure in adolescence: a prospective study of a birth cohort of 149,378 Swedish boys. J Hypertens 1997; 15:1627–1631. 23. Barker DJP. Fetal origins of coronary heart disease. Br Med J 1995; 311:171– 174. 24. Law CM, Shiell AW. Is blood pressure inversely related to birth weight? The strength of evidence from a systematic review of the literature. J Hypertens 1996; 14:935–941. 25. Brenner BM, Chertow GM. Congenital oligonephropathy and the etiology of adult hypertension and progressive renal injury. Am J Kidney Dis 1994; 23:171– 175. 26. Brenner BM, Milford EL. Nephron underdosing: A programmed cause of chronic renal allograft failure. Am J Kidney Dis 1993; 21:66–72. 27. Brenner BM, Chertow GM. Congenital iligonephropathy: an inborn cause of adult hypertension and progressive renal injury? Current Opinions in Nephrology and Hypertension 1993; 2:691–695. 28. Brenner BM, Cohen RA, Milford EL. In renal transplantation, one size may not fit all. J Am Soc Nephrol 1992; 3:162–169. 29. Hinchcliffe SA, Lynch MRJ, Sargent PH, Howard CV, Van Velzen D. The effect of intrauterine growth retardation on the development of renal nephrons. Br J Obstet Gynaecol 1992; 99:296–301. 30. Ventura SJ, Anderson RN, Martin JA, Smith BL. Births and deaths; preliminary data for 1997. National Vital Statistics Report, 1998; 47:1–44. 31. Lackland DT, Bendall HE, Osmond C, Egan BM, Barker DJP. Low birth weights contribute to the high rates of early-onset chronic renal failure in the southeastern United States. Archives of Internal Medicine 2000; 160:1472–1476. 32. Mackenzie HS, Brenner BM. Fewer nephrons at birth: a missing link in the etiology of essential hypertension? Am J Kidney Dis 1995; 26:91–98. 33. Merlet-Benichou C, Leroy B, Gilbert T, Lelievre-Pegorier M. Intrauterine growth retardation and inborn nephron deficit. Medicine/Sciences 1993; 9:777–780. 34. Brenner BM, Mackenzie HS. Nephron mass as a risk factor for progression of renal disease. Kidney International 1997; 52(suppl 63):124–127. 35. Hoy WE, Rees M, Kile E, et al. Low birthweight and renal disease in Australian aborigines. Lancet 1998; 352:1826–1827. 36. Perneger TV, Brancati FL, Whelton PK, Klag MJ. End-stage renal disease attributable to diabetes mellitus. Ann Intern Med 1994; 121:912–918.

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37. Dahlquist G, Sandberg Bennich S, Kallen B. Intrauterine growth pattern and risk of childhood onset insulin dependent (type 1) diabetes: population based case-control study. Br Med J 1996; 313:1174–1177. 38. Barker DJP. The fetal origins of Type 2 diabetes mellitus. Ann Intern Med 1999; 130:322–324. 39. Hattersley AT. Maturity-onset diabetes of the young: clinical heterogeneity explained by genetic heterogeneity. Diabetic Medicine 1998; 15:15–24. 40. Yokoyama H, Okudaira M, Otani T, et al. High incidence of diabetic nephropathy in early-onset Japanese NIDDM patients. Risk analysis. Diabetes Care 1998; 21:1080–1085. 41. Nelson RG, Morgenstern H, Pennet PH. Birth weight and renal disease in Pima Indians with Type 2 diabetes mellitus. Am J Epidemiol 1998; 148:650–656. 42. Lackland DT, Bendall HE, Osmond C, Egan BM, Barker DJP. Low birthweights contribute to the high rates of early onset chronic renal failure in the Southeast United States. Archives of Internal Medicine (in press).

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5 Intrauterine Nutrition Its Importance During Critical Periods for Cardiovascular and Endocrine Development

JOSEPH J. HOET†

MARK A. HANSON

Université Catholique de Louvain Louvain-la-Neuve, Belgium

University College London London, United Kingdom

I.

Summary

Experimental investigations in animals highlight the role of early reducedcalorie and protein nutrition on fetal cardiovascular development, and the occurrence of a transition from a low fetal arterial blood pressure in late gestation to a high arterial blood pressure postnatally. These observations may explain the correlation between health, including appropriate nutrition, in pregnant women and the outcome of their pregnancies. Emphasis has been placed on low–birth weight infants who have an increased risk of developing cardiovascular diseases, including hypertension, coronary heart disease, and stroke in adulthood. †

Sadly, Joseph J. Hoet died shortly after publication of this article in the Journal of Physiology (Lond) 1999; 514:617–627. It is reprinted here in memory of him.

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Vascular pathology in adults is not always associated with low birth weight, and animal experiments indicate that substantial changes in cardiovascular and endocrine function can result from maternal or fetal undernutrition without impairing fetal growth. Experimental investigation on organogenesis shows the pivotal role of adequate protein availability as well as total caloric intake. Amino acid metabolism in the fetomaternal unit appears to have key influence on the development of organs involved in chronic degeneration disease in the adult. Experimental investigation has also highlighted the role of carbohydrate metabolism and its effect on the fetus in this respect. Either restriction of protein intake or diabetes in pregnant rats has intergenerational effects at least on the endocrine pancreas and the brain. Further investigation is needed to clarify the mechanisms involved and lead to a new understanding of the importance of nutrition during pregnancy. This will provide an important approach to the primary prevention of diabetes and chronic degenerative diseases. II. Introduction The developing mammal needs to establish a degree of autonomy during fetal life in order to achieve independent survival after birth. It therefore develops homeostatic mechanisms necessary to guarantee its existence. But it passes through critical periods that may be influenced by aspects of the intrauterine environment dependent on maternal nutrition and metabolism. These have been clearly shown for implantation, organogenesis, and parturition, all of which are influenced by maternal health, including nutritional intake. But apart from the striking effects on survival (eg, in relation to implantation) or overt anatomical structure (organogenesis), recent evidence reveals that disturbances during critical periods can also affect homeostatic mechanisms. The effects may be subtle during development, but can nonetheless have longlasting deleterious effects on health in adult life. This review concentrates primarily on how nutritional intake during pregnancy affects cardiovascular (especially arterial blood pressure) and blood glucose homeostasis in the offspring. These two areas are increasingly studied in humans (1,2) and, although superficially distinct, they share some common causal features. To date, most of the studies in humans have been epidemiological so that, although they identify phenomena, they do not give insight into mechanisms. Awareness of this has recently shifted emphasis to prospective studies of smaller groups of people and to animal studies, the latter providing

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the more direct approach to understanding the processes involved. We concentrate on these animal studies in this brief review. In some of them, an isocaloric low-protein diet has been used to explore the mechanisms by which protein metabolism affects developing organs. Other studies have used varying degrees of global reduction in nutrition. It is noteworthy that in some of these studies, effects on homeostatic development were produced even in the absence of body growth restriction. It is therefore possible to envisage a spectrum of health problems in adult life, deriving from the influence of the intrauterine environment and maternal/placental/fetal compensatory responses to a diet mildly to severely altered in its composition or its volume. It is also noteworthy that even if an isocaloric low-protein diet does not affect birth weight significantly in the first generation, it may reduce it in subsequent generations. These observations are discussed further below. III. Cardiovascular Development It is well established in a variety of species that altered nutrition can have permanent effects if it occurs at a sensitive period during development (3–8). The effects of a period of early undernutrition on cardiovascular function may not be manifest until a much later stage in life. Attempts to understand the underlying mechanisms have been made in a variety of animals, from rodents to sheep and nonhuman primates. One of the major issues that emerges from recent work is that birth weight does not have to be significantly reduced for the physiological effects of reduced nutrition in pregnancy to be manifest. This raises the possibility that the placental or fetal compensatory mechanisms, which occur in response to reduced maternal nutrition, preserve normal fetal growth and hence birth weight, but have postnatal consequences that become important in later life. Thus, the adoption of a biological “strategy” needed for development in utero results in an organism in which the strategy for well-being in adulthood is not achieved. In sheep, global undernutrition in early pregnancy produces reduction of birth weight/placental weight ratio (9) and in early to mid-gestation produces increased placental size (10,11), whereas global undernutrition in late gestation only reduces fetal growth (12). Early gestation undernutrition alters the distribution of placentome types in favor of those with a predominant fetal vascular pattern (13). This pattern is reminiscent of the increased fetal vascularization reported in human, guinea pig, and sheep placentae at high altitude

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(14–18) and some forms of intrauterine growth retardation (IUGR) (19), possibly occurring as a placental compensatory response. Early undernutrition affects the development and responses of the hypothalamic-pituitary-adrenal (HPA) axis in late gestation. Both the pituitary response to an arginine vasopressin/corticotrophin releasing hormone (AVP/ CRH) challenge and the adrenal cortical response to an adrenocorticotropic hormone (ACTH) challenge are reduced (20,21). These fetuses have a lower basal plasma cortisol concentration, which may account for their lower arterial blood pressure (Fig. 1) because cortisol and other glucocorticoids are known to elevate arterial blood pressure (22). Whether the suppression of basal cortisol concentration and of HPA axis responses are attributable to prior exposure of the fetus to cortisol is not known. A hypothesis has been proposed to relate the suppression of the HPA axis function to prior exposure of the fetus to elevated corticosteroids (23,24) and it is already known that dexamethasone administration suppresses HPA axis function in the sheep (25). Cardiovascular and HPA axis development are also affected postnatally. Lambs born after periconceptual undernutrition have higher arterial blood pressure and an exaggerated arterial blood pressure response to an HPA axis challenge (21). Their ACTH and cortisol responses are now also greater. The mechanism for this switching from blunted responses in utero to enhanced responses postnatally is not yet known. The effects of undernutrition are manifest not only in terms of changes in reflex and endocrine control of the cardiovascular system, but also at the level of the local vasculature. Ozaki et al. (26) found that the responses of small resistance arteries, especially to endothelium-dependent vasodilator agonists, are altered in ewes and their fetuses subjected to an early gestation nutritional challenge. The causal role of such changes in arterial blood pressure development remains to be determined. Similar effects have also been reported in experimental diabetes and with a high-fat diet in the rat (27,28). Besides undernutrition in the ewe, the removal of placental caruncles produces fetuses with lower mean arterial blood pressure, higher fetal heart rate, and altered responses to an episode of acute hypoxia in late gestation (29). Fetal heart rate and the rise in arterial blood pressure in acute hypoxia are greater in carunclectomized fetuses, suggesting greater chemoreflex or endocrine responses although such fetuses are not necessarily smaller than controls. These carunclectomized sheep fetuses, being hypoxemic and hypoglycemic, have higher plasma levels of adrenaline and noradrenaline, suggesting greater sympathetic activity. Their higher levels of cortisol without increased

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Figure 1 Basal mean arterial pressure (left panel) and ACTH response to administration of CRH + AVP (right panel) in control (white column) and nutrient-restricted (black column) fetuses (113–127 days gestation) and lambs (84 ± 4.4 days). Values are mean ± SEM. Arterial pressure is shown as the average of measurements made over a 2-week period in the fetus, and as the average of measurements made on a single day in the lamb. ACTH responses to CRH + AVP challenge were measured for 180 min after drug administration in the fetus, and for 60 min in the lamb; ACTH data are shown as the cumulative response over the first 15 min after drug administration, with samples taken every 5 min. Blood pressure data were compared by unpaired t test, and ACTH data by two-way analysis of variance followed by Dunnett’s post-hoc test (*p < 0.05). Fetal basal blood pressure, and ACTH responses to CRH + AVP were significantly reduced after maternal nutrient restriction. Postnatally, basal blood pressure and ACTH responses were significantly greater in lambs of undernourished mothers. (From Ref. 20.)

ACTH and suppressed pituitary pro-opimelanocortin (POMC) expression (30), suggest that HPA axis feedback may have been altered. In another context, alteration of the HPA axis affects maturation of organ systems, and may even initiate parturition (see Ref. 31 for discussion). Studies in rats have shown that undernutrition in utero can lead to lifelong elevation of blood pressure. For example, an isocaloric low-protein diet

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(8%) during pregnancy reduces fetal:placental weight ratio (32), produces elevated arterial blood pressure in the offspring (33) via changes in angiotensin-converting enzyme activity (34), and produces changes in glutathione metabolism and glucose tolerance as well as insulin secretion (33,34). In subsequent experiments, it was shown that induction of hypertension by reduced maternal protein was abolished if fetal cortisol synthesis was inhibited. This led to the hypothesis that maternal protein restriction programs lifelong changes in the fetal HPA axis, which in turn resets homeostatic mechanisms controlling blood pressure. Maternal protein restriction attenuates activity of 11-β HSD (hydroxysteroid dehydrogenase) Type 2, and an alternative explanation is that fetal blood pressure is altered through increased exposure to maternal glucocorticoids (24). In this species, placental 11-β HSD activity is correlated with birthweight and inversely correlated with placental weight (23), and inhibition of 11-β HSD with carbenoxolone in pregnancy produces offspring with a higher arterial blood pressure (35). A third possibility is that placental activity of the enzyme plays a crucial role in the development of the fetal adrenal, and hence may determine patterns of glucocorticoid secretion throughout life. There may also be effects on the kidney (see following discussion) and on vascular growth and metabolism. The effects of severe total dietary restriction have also been examined in the rat. Woodall et al. (36) reduced diet by 75% and reported elevated blood pressure. However, Holemans et al. (37) found that severe restriction in late gestation did not produce hypertensive offspring. It appears that the timing of the insult in gestation is all-important, with insults occurring earlier having greater effects on cardiovascular development in the offspring. The experiments on the rat appear to differ from those on the sheep in that cardiovascular and endocrine changes are associated with growth retardation. Methodological differences and the role of undernutrition versus isocaloric protein restriction have, however, to be noted. In the rat, the level of reduction in maternal carbohydrate and/or protein intake has usually been based on requirements at the time of conception, and maintained throughout pregnancy. Because dietary requirement increased throughout pregnancy, this may constitute a challenge of increasing severity. Recently, in a series of studies in which intake was reduced by 30% of the appropriate daily requirement throughout gestation, only male pups were not smaller than control, at least at the first generation. Nonetheless, they showed perturbed vascular development, particularly in arterial blood pressure and in responses of small vessels to vasodilator agonists (26) (Fig. 2). In addition, a recent report on the growth of the spontaneously hypertensive rat (SHR) (38) (Fig. 3) shows that the pups

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Figure 2 Effects of a 30% restriction in global diet during pregnancy on the mean arterial pressure (top panel) and responses of small resistance arteries to the thromboxane A2 mimetic U46619 in vitro (lower panel) of male offspring. Offspring of nutritionally restricted dams (NR) show higher arterial blood pressure at both 100 and 200 days postnatally, compared with controls (C). In addition, small artery contractile responses, measured as maximal tension (E max) to U46619 as a percentage of maximal K+induced tension, are greater in NR than in C at both ages. All data are mean ± SEM * p < 0.05 NR vs. C by Mann-Whitney U test. (From Ref. 26.)

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Figure 3 Weight (mean ± SD) of the fetus (upper panel) and placenta (lower panel) in late gestation in spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto controls (WKY). The lower fetal weight in midgestation in SHR is not seen in late gestation, and this effect is associated with greater placental weight. Note that gestation is slightly longer in SHR than WKY. (From Ref. 38.)

of this strain were not smaller than normotensive pups at birth, although as fetuses they were smaller at days 16 to 20 of gestation. Their greater lategestation growth than controls is associated with a significantly larger placenta. Thus, whatever the mechanisms for the earlier impaired growth, fetal growth in late gestation was accelerated by a greater placental mass. However, the latter has consequences for subsequent postnatal cardiovascular function. Spontaneously hypertensive rat pups had larger hearts and kidneys, which may be associated with the cardiovascular effects. Somewhat similar observations have been made in the rat using iron deficiency anemia. This reduces birth weight and neonatal mean arterial blood pressure, but the pups become hypertensive relative to controls when postnatal “catch-up” growth occurs and their heart size increases (39). Blood pressures of SHR are permanently lowered if they are suckled by dams of normotensive strains for 2 weeks after birth. Milk of SHR differs from milk of other strains in protein and electrolyte concentrations (40–42). Other studies have shown that the intrauterine environment of the SHR is different, because the amniotic fluid has high osmolarity and sodium concen-

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trations (43). Altering the intrauterine environment experimentally by inducing maternal diabetes also affects blood pressure in the SHR. Finally, in the guinea pig, maternal undernutrition impairs placental development and fetal growth (44), and it has also been shown that unilateral ligation of the uterine artery during pregnancy indeed leads to reduced birth weight in the offspring and raised blood pressure after birth. The mechanism underlying this phenomenon is unknown, but increased circulating catecholamines have been proposed (45). IV. Endocrine Pancreas Besides the vasculature, the development of other key tissues may be affected in the offspring of ewes or dams having global nutritional restriction. However, a specific limitation of protein in a normocaloric diet that prevents an energy constraint has been explored widely in pregnant animals and found to affect the offspring’s islet cells (7), insulin-sensitive tissues such as liver (46), muscle (47,48), and adipose tissue (49) as well as the kidney (50) and brain (51). With this experimental dietary approach, particular mechanisms operating in the fetomaternal unit that are dependent on specific nutritional intake of the mother might be explored. The latter part of this chapter deals with the consequences for the offspring of dams receiving a moderate protein restriction in a normocaloric diet. In vitro, fetal β cell differentiation, multiplication, and insulin secretion are amplified more by an increase in essential amino acid than by an increase in glucose concentration (52). This suggests a specific role for appropriate protein availability on the development of fetal β cells, acting via changes in amino acid metabolism. This raises the possibility of abnormal features being acquired by the developing β cell, which may lead to pathological events postnatally as a result of a protein-deficient diet of the dam. With an isocaloric low-protein (8%) diet during gestation, the profile of amino acids is changed in maternal and fetal plasma as well as in amniotic fluid. The total, essential, and nonessential amino acids concentrations were not modified, nor were glucose and insulin levels (53). More specifically, áamino butyric acid, phosphoserine, taurine, and valine were reduced in maternal as well as in fetal plasma. The weight of the fetus was normal at 19.5 days but slightly lower at birth compared with the controls. The endocrine pancreas was abnormal in that islet cell proliferation (not evenly distributed in head and tail of the endocrine pancreas), islet cell size,

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pancreatic insulin content, and islet vascular density were all reduced at birth (7). Furthermore, the ontogeny of the endocrine pancreas of offspring from these dams was perturbed when the low-protein diet was maintained during the suckling period. At each fetal and postnatal day analyzed, the number of apoptotic cells in these islets was increased whereas the number of cells positive for insulin-like growth factor 2 (IGF-2), considered to be a survival factor that prevents apoptosis (54), was decreased (55). The function of these fetal â cells was affected with insulin secretion being diminished by 50% in vitro compared with controls (56). With a normal diet postnatally, insulin secretion in vitro remained impaired when stimulated by arginine and leucine (57). In vivo the adult offspring showed normal basal plasma glucose and insulin levels as well as a normal amino acid profile. During a glucose challenge, the insulin level was abnormally low in adult nonpregnant females and it remained low during pregnancy, associated with higher-thannormal plasma glucose levels (Table 1). When the isocaloric low-protein diet was maintained postnatally, the adult offspring showed an abnormal amino acid profile that was associated with a reduced volume of the endocrine pancreas and pancreatic insulin content (56). Islet blood vessel density (58) as well as pancreatic and islet blood flow (59) were also diminished. It was of particular interest that reduced activity of mitochondrial glycerophosphate dehydrogenase (mGPDH) was observed in these islets (60). A similar reduction is observed in islet cells of human subjects with type 2 diabetes. Special emphasis should be placed on the sensitivity of fetal islets to taurine, which is an indispensable amino acid during fetal and neonatal development in rats, cats, and baboons (61). Plasma taurine was significantly reTable 1 Effects of Protein Deficiency During Pregnancy on the Endocrine Pancreas of the Adult Offspring Low diet maintained postnatally Islet cell mass ↓ Pancreatic insulin content ↓ β cell sensitivity to glucose and amino acids in vitro ↓ Plasma insulin level ↓ Insulin response to oral glucose challenge ↓

Normal diet given postnatally

β cell sensitivity to amino acids in vitro ↓ Insulin response to oral glucose only in female ↓

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duced in dams fed low protein in gestation and in their fetuses. Insulin secretion by normal fetal islets was stimulated in vitro by taurine and, when added to the culture medium, it enhanced insulin secretion in response to other secretagogues (62). Islets of fetuses from dams on a low-protein diet did not secrete insulin in response to taurine, and in the culture medium it did not restore a normal secretory response to other secretagogues. However, when taurine was added to the drinking water of rats on a low-protein isocaloric diet to reestablish plasma taurine levels in dams and fetuses, the insulin secretion in response to taurine and other secretagogues was restored to normal (63). Thus, current observations point to the need for normal protein availability in the diet and identify taurine as a necessary amino acid for the normal functional development of fetal β cells. A low-protein isocaloric diet also has an intergenerational effect on birth weight and the endocrine pancreas. This diet reduced the birth weight of pups and it reduced it further in subsequent generations, producing an increased number of growth-restricted pups (64). As noted above, the female offspring showed an abnormally low plasma insulin and high plasma glucose levels in response to an oral glucose challenge when they were pregnant. Their pups had lower plasma insulin and lower insulin content along with reduced volume density of the endocrine pancreas (Table 2). When the low protein intake was maintained postnatally as well as during gestation, the mother and the pups showed a higher plasma glucose and lower plasma insulin levels than normal. Alterations of endocrine pancreas and its insulin content in these pups were Table 2 Effects of Protein Deficiency During Pregnancy on Insulin Glucose Homeostasis in 1st and 2nd Generation Offspring First generation pregnant female offspring Low protein postnatal diet Plasma glucose Plasma insulin Pancreatic insulin content Insulin response to OGTT Islet cell mass

+ – – – —

+, increase; –, decrease; n.e., no effect; —, not measured.

Second generation late gestation fetus

Normal postnatal diet

Low protein postnatal diet

Normal postnatal diet

n.e. n.e. – — —

+ – – — –

n.e. – – — –

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even more marked than in the previous generation. Therefore, chronic isocaloric protein deprivation, which lowered the fetomaternal levels of amino acids such as taurine, initiates intergenerational effects on glucose homeostasis and the development of the endocrine pancreas. A similar intergenerational effect of a protein-restricted diet has already been observed on brain development and tryptophan metabolism (65). V.

Insulin-Sensitive Tissues

Many aspects of liver function, including cholesterol synthesis and fibrinogen production, are differentially expressed in the periportal and perivenous zones. The livers of pups born to mothers on an isocaloric low-protein diet underwent changes in zonation and enzyme activity, including a reduction in glucokinase and an increase in phosphenol pyruvate carboxykinase activity. These were not restored in adulthood even when the animals were nourished with a normal diet (66), and were responsible for changes in the regulation of hepatic glucose output (47). Altered zonation is likely to be linked to other important changes in hepatic function, although these are as yet unknown. There are, however, indications that manipulations during gestation up-regulated cholesterol synthesis (67,68). The number of insulin receptors was increased in the liver, in skeletal muscle (47,48), and white fat adipocytes (49). In addition, the adipocytes were smaller and did not show changes in Glut 4 expression, although this was increased in the plasma membrane of skeletal muscle (48). The adipocytes of adults had a greater glucose uptake and a higher phosphatidyl inositol 3 kinase activity (69). Adipose tissue of offspring was also affected by global dietary restriction, which comprises low protein availability in the dams. In this instance, white adipose tissue increased and brown adipose tissue decreased in the adult, possibly indicating lower sympathetic activity. Rats whose mothers had restricted food during the first 2 weeks of pregnancy indeed became obese, but depending on the strain and the diet used it was either the males or the females that were affected (70,71). Thus, limited protein intake during gestation leads to alterations in glucose output by the liver as well as in the sensitivity of tissues to insulin. Glucose transporters in muscles and the expression of key components of insulin signalling pathways in adipocytes are also altered. In addition, studies have suggested that maternal dietary restriction during gestation and lactation as well as transient dietary protein restriction after weaning may permanently alter growth-hormone secretion in offspring (72).

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Kidney

In nonhuman primates and rats, neonatal kidney weights were reduced by an isocaloric low-protein diet given to the mother. Hence, there appears to be specific effect of protein limitation on the kidney (50,73). In rats, a severe low-protein restricted isocaloric diet during pregnancy is associated with low birth weight and a significant reduction in the number of mature glomeruli (50), with an increase in the number of immature glomeruli (74) in the progeny. Kidney weight, as well as the final number of mature glomeruli, remained reduced at 14 days postnatally even when the pups were fed a normal diet postnatally. Therefore, an isocaloric low-protein diet during pregnancy leads to permanent changes in the kidney that are not reversible postnatally. The number of renal glomeruli is also reported to be reduced in infants who are malnourished in early life (75,76) as well as in adults with hypertension (77,78). Lastly, the number of nephrons in humans is known to be correlated with birth weight (79). VII.

Brain

In the brain of offspring of dams exposed experimentally to an isocaloric low-protein (8%) diet before and during pregnancy, anatomical and physiological aspects were altered (51). The distribution and levels of biogenic amines were changed and there were modifications of tryptophan metabolism. Although a normal diet postnatally restores some of these features, the changes in biogenic amines persist (51). These alterations became even more severe when the same low-protein isocaloric diet was given to subsequent generations (65). Thus, for brain development, a low-protein isocaloric diet initiates an intergenerational effect similar to that shown for the endocrine pancreas. In addition, brain blood-vessel density was reduced in pups born to dams on an isocaloric low-protein diet (80) and remained reduced when a normal diet was given postnatally. This contrasts with the restoration of bloodvessel density in the endocrine pancreas, which was restored by giving a normal diet postnatally (58). The mechanisms involved in the effects of nutrition on vasculogenesis in various tissues are not yet known. VIII.

Comparison of Dietary Restriction with Maternal Diabetes

Besides maternal nutritional deprivation, with protein reduction or global dietary reduction, maternal diabetes is also known to affect fetal tissue develop-

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ment in humans and experimental animals. Streptozotocin-induced diabetes in a mild form induced a higher birth weight than normal, an increased percentage of pancreatic endocrine tissue with cell hyperplasia, and an increase in the number of degranulated cells (81). Hence, the proliferative capacity of the islet was increased in vivo, and it remained elevated when cultured in a normal medium for 7 days (82). In the gut, villous and microvillous surface area was increased in the duodenum, jejunum, and ileum, and mucosal bloodvessel density was also increased (92). However, when dams had severe diabetes, the fetal weight, islet size, and cell mass were decreased (81), and atrophy of the fetal intestinal tract occurred (92). In such maternal diabetes, induced experimentally by streptozotocin, the pups of a second generation showed a reduction in birth weight together with permanent changes in endocrine pancreatic structure and function (81). Pups from moderately as well as severely diabetic dams became diabetic in adulthood. Pancreatic insulin depletion in the former and insulin resistance in the latter were causal in initiating the diabetes in the offspring postnatally (83). There are, in addition, effects of maternal streptozotocin-induced diabetes on the responses of small resistance arteries in the pups (28). Both the experimental conditions of maternal protein restriction and diabetes stress the impact of maternal nutritional limitation or poor health in producing effects on the offspring. Several tissues are affected, including the vasculature, endocrine pancreas, insulin-sensitive tissues, kidney, and brain. Some of the pathological changes can develop after a delay, and there are intergenerational effects. The effects may cause degenerative diseases in adults and they can occur without major changes in birth weight. Therefore, birth weight may only be a poor proxy for intrauterine events.

IX. Worldwide Perspective In considering sources of variation in fetal growth, the focus in this chapter has been on nutrition, with particular reference to the nutritional programming hypothesis. There are, however, many factors affecting fetal growth and size at birth: the fetal genotype, the maternal genotype, the mother’s prepregnancy nutritional status, her metabolism and physiology, her diet during pregnancy, and the resultant hormonal and circulatory milieu that sustains fetal growth. At least as far as the protein-restriction model is concerned, we have stressed the importance of the quantity and quality of amino acids available to the

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fetus. But for all the factors involved, there is a need to determine the specific mechanisms responsible for the alterations in fetal development. Birth weight is a crude measure of fetal growth: babies of the same weight may, for example, be short and fat or long and thin, and may be markedly different in organ size and structure, physiology, and metabolism. In humans, low birth weight is the result of multifactorial processes operating during pregnancy. It is confounded by the influence of ethnic origin, low socioeconomic status, and poor nutrition, which may affect the mother and her offspring concomitantly. Data from developing countries indicate that in some areas of Asia 20% of women have stunted growth with an adult height of 1.45 m, and 65% have an adult body weight below 45 kg. More than 35% of mothers deliver infants with birth weights under 2.500 kg (84). Much of the situation stems from a poverty-related deficiency of protein and energy intake, as well as of key micronutrients, such as vitamin A, iodine, and iron, from which women and children suffer disproportionately. Inadequate diets are also associated with infectious diseases which, when occurring during pregnancy, increase the energy, protein, and micronutrient needs and are associated with low birth weight. In infants up to 5 years of age, protein/energy malnutrition contributes 12.7% of the total burden of diseases. Malnutrition is also sex-linked, and severe malnutrition affects seven times more female than male infants in the developing world (85). Vitamin A, iodine, or iron deficiency during pregnancy and early life each have a specific and immense impact on offsprings’ health, contributing 11.7%, 7.2%, and 14% respectively to the total disease burden throughout the developing world. Recently, an epidemic of diabetes and cardiovascular disease in the younger age group has become apparent in these countries. In western countries, epidemiological surveys highlight the importance of low birth weight in increasing the risk of developing diabetes and cardiovascular disease in adulthood (2,86). A study of 15,000 Swedish men and women provides by far the most convincing evidence of a true association between size at birth and mortality from ischemic heart disease, and it strongly suggests that it is variation in fetal growth rate rather than size at birth that is an important causal factor (87). Low birth weight (<2.500 kg) and poor early growth (<8 kg at age 1) are associated with a high incidence (>45%) of diabetes, impaired glucose tolerance, and/or myocardial infarction at 65 years of age. However, in twin pregnancies, the baby with the lower birth weight is more likely to develop type 2 diabetes than its twin, indicating that the intrauterine environment of the individual fetus is more important in the origin of this disease than genetics. Thinness at birth is also associated with insulin resistance later (88).

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Epidemiological studies of degenerative diseases should now focus on nutritional conditions across generations to relate events occurring early in life to the time span for pathological conditions to appear. Recently, the first studies on this topic have been published. Examination of maternal diet during pregnancy and health of the offspring suggests that, in humans, nutrition during pregnancy does indeed have long-term consequences for the health of the offspring. For example, in Aberdeen the blood pressures of men and women were found to be related to the balance of animal protein and carbohydrate in their mother’s diet during late pregnancy (89). This association did not depend on birth weight. The protein intake of the mother is also related to the offspring’s glucose-insulin metabolism (1). Low protein intake was found to be associated with insulin resistance, although the association was weaker than that with low maternal body mass. Recently, it has been shown that people exposed to the Dutch famine in utero had higher plasma glucose and insulin concentration after a standard glucose load, suggesting that their poor glucose tolerance was mainly determined by insulin resistance (90). The effects of famine were independent of size at birth. These findings provide direct evidence that undernutrition in utero is a key factor in the cause of non– insulin dependent diabetes mellitus, and show that the mother’s dietary intake during pregnancy can program metabolism without altering size at birth. It is thus clear from a range of human and animal studies that poor maternal health and/or nutritional deficiencies affect key tissues during their development, and can be responsible for pathological changes in the offspring. Specific protein malnutrition during human pregnancy and children’s health has not been explored extensively, although experimental observations suggest new approaches to establishing a correlation. In the human, neonatal hypercysteinemia and hypercysteinuria are associated with vascular damage in early life (91). Increased plasma levels of homocysteine have been found to be associated with increased risk of myocardial infarction in adulthood, possibly because of enzymatic alterations associated with vitamin B6 or vitamin B12 activity. One may surmise that perturbed amino acid homeostasis during pregnancy may initiate fetal developmental abnormalities, leading to pathological events in later life. We suggest that extensive animal studies are now needed to discover specific mechanisms by which altered fetomaternal nutrition and amino acid metabolism lead to degenerative diseases in the offspring. Only when the causes in early life of these human diseases are established, will it be pos`sible to devise ways for their primary prevention.

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X. Acknowledgments We would like to thank Professor C. Remacle and Dr. B. Reusens for their helpful comments, as well as W. Rees for preparing the manuscript. References 1. 2.

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14. Bacon BJ, Gilbert RD, Kaufmann P, Smith AD, Trevino RT, Longo LD. Placental anatomy and diffusing capacity in guinea pigs following long-term maternal hypoxia. Placenta 1984; 5:475–488. 15. Scheffen I, Kaufmann P, Philippens L, Leiser R, Geisen C, Mottaghy K. Alternations in the fetal capillary bed in the guinea pig placenta following long-term hypoxia. Adv Exper Med Biol 1990; 277:779–789. 16. Burton G, Reshetnikova OS, Milovanov AP, Teleshova OV. Stereological evaluation of vascular adaptations in human placental villi to differing forms of hypoxic stress. Placenta 1996; 17:49–55. 17. Krebs C, Longo LD, Leiser R. Term ovine placental vasculature comparison of sea level and high altitude conditions by corrosion cast and histomorphometry. Placenta 1997; 18:43–51. 18. Penninga L, Longo ID. Ovine placentome morphology: effect of high altitude, long-term hypoxia. Placenta 1998; 19:187–193. 19. Kingdom JCP, Kaufmann P. Oxygen and placental villous development: origins of fetal hypoxia. Placenta 1997; 18(8):613–621. 20. Hawkins P, Crowe C, Calder NA, Saito T, Ozaki T, Stratford LL, Noakes DE, Hanson MA. Cardiovascular development in late gestation fetal sheep and young lambs following modest maternal nutrient restriction in early gestation. J Physiol 1997; 505:18P. 21. Hawkins P, Crowe C, McGarrigle HHG, Saito T, Ozaki T, Stratford LL, Noakes DE, Hanson MA. Effect of maternal nutrient restriction in early gestation on hypothalamic pituitary adrenal axis responses during acute hypoxaemia in late gestation fetal sheep. J Physiol 1998; 507:50P. 22. Tangalakis K, Roberts FE, Wintour EM. The time-course of ACTH stimulation of cortisol synthesis by the immature ovine foetal adrenal gland. J Ster Biochem Molec Biol 1992; 42(5):527–532. 23. Benediktsson R, Lindsay RS, Noble J, Seckl JR, Edwards CRW. Glucocorticoid exposure in utero: new model for adult hypertension. Lancet 1993; 341:339– 341. 24. Edwards CRW, Benediktsson R, Lindsay RS, Seckl JR. Dysfunction of placental glucocorticoid barrier: link between fetal environment and adult hypertension? Lancet 1993; 341:355–357. 25. Norman LJ, Challis J. Synergism between corticotropin-releasing factor and arginine vasopressin and adrenocorticotrophin release in vivo varies as function of gestational age in the ovine fetus. Endocrinology 1987; 120:1052–1058. 26. Ozaki T, Nishina H, Hawkins P, Poston L, Hanson MA. Isolated systemic resistance vessel function in hypertensive male rat offspring of mild nutritionally restricted dams. J Physiol 1998; 513:118. 27. Gerber RT, Holemans K, Van Assche FA, Poston L. Female offspring from pregnant diabetic rats demonstrate cardiovascular dysfunction. Fetal and Neonatal Physiology Symposium, Cambridge, United Kingdom, 1997. 28. Koukkou E, Lowry C, Poston L. The offspring of diabetic rats fed a high satu-

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57. Dahri S, Cherif H, Reusens B, Remacle C, Hoet JJ. Effect of an isocaloric low protein diet during gestation in the rat on in vitro insulin secretion by islets of the offspring. Diabetologia 1994; 37(Suppl 1):A80. 58. Dahri S, Snoeck A, Reusens B, Remacle C, Hoet JJ. Low protein diet during gestation in rats: its relevance to human non–insulin dependent diabetes. J Physiol 1993; 467:292. 59. Iglesias-Barreira V, Ahn MR, Reusens B, Remacle C, Hoet JJ. Pre- and postnatal low protein diet affect pancreatic islets blood flow and insulin release in adult rats. Endocrinology 1996; 137:3797–3801. 60. Rasschaert J, Reusens B, Dahri S, Sener A, Remacle C, Hoet JJ, Malaisse WJ. Impaired activity of rat pancreatic islet mitochondrial glycerophosphate dehydrogenase in protein malnutrition. Endocrinology 1995; 136:2631–2634. 61. Sturman GA. Taurine in development. Physiol Revs 1993; 73:119–147. 62. Cherif H, Reusens B, Dahri S, Remacle C, Hoet JJ. Stimulatory effect of taurine on insulin secretion by fetal rat islets, cultured in vitro. J Endocrinol 1996; 151:501–506. 63. Cherif H, Reusens B, Ahn MT, Hoet JJ, Remacle C. Effect of taurine on the insulin secretion of islets of fetus from dams fed a low protein diet. J Endocrinol 1998; 159:341–348. 64. Stewart RJ, Preele RF, Sheppart HG. Twelve generations of marginal protein deficiency. Br J Nutrition 1975; 33:233–253. 65. Resnick O, Morgan PJ. Generational effect of protein malnutrition in the rat. Develop Brain Res 1984; 15:219–227. 66. Desai M, Crowther NJ, Ozanne SE, Lucas A, Hales CN. Adult glucose and lipid metabolism may be programmed during fetal life. Biochem Soc Trans 1995; 23:331–335. 67. Innis SM. The role of diet during development on the regulation of adult cholesterol homeostasis. Can J Physiol Pharmacol 1985; 63:557–564. 68. Innis SM. Influence of the maternal cholestyramine treatment on cholesterol and bile acid metabolism in adult offspring. J Nutrition 1983; 113:24264–24270. 69. Ozanne SE, Nave BT, Wang CL, Shepherd PR, Prins J, Smith GD. Poor fetal nutrition causes a long term change in expression of insulin signalling components in adipocytes. Am J Physiol Endocrine Metabol 1997; 36:E46–E51. 70. Anguita RM, Sigulem DM, Sawaya AL. Intrauterine food restriction is associated with obesity in young rats. J Nutrition 1993; 123:1421–1428. 71. Jones AP, Friedman MI. Obesity and adipocyte abnormalities in offspring of rats undernourished during pregnancy. Science 1982; 215:1518–1519. 72. Harel Z, Tannenbaum GS. Long-term alterations in growth hormone and insulin secretion after temporary dietary protein restriction in early life in the rat. Pediatr Res 1995; 38:747–753. 73. Cheek DB, Hill DE. Changes in somatic growth after placental insufficiency and maternal protein deprivation. In: Check DB, ed. Fetal and Postnatal Cellular Growth Hormones and Nutrition. New York: J. Wiley & Sons, 1975:299–310.

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74. Zeman J. Effect of maternal protein restriction on the kidney of newborn young of rats. J Nutrition 1968; 94:111–116. 75. Naeye RL. Malnutrition, probable cause of growth retardation. Archiva Pathologica 1965; 79:264–291. 76. Hinchliffe SA, Lynch MR, Sargent PH, Howard CV, Van Velzen D. The effect of intrauterine growth retardation on the development of renal nephrons. Br J Obstet Gynaecol 1992; 99(4):296–301. 77. Hayman JM, Martin J, Miller M. Renal function and the number of glomeruli in the human kidney. Arch Int Med 1939; 64:69–83. 78. Mackenzie HS, Brenner BM. Fewer nephrons at birth. A missing link in the etiologic of essential hypertension. Am J Kidney Dis 1995; 26:91–98. 79. Merlet-Benichou C, Leroy B, Gilbert T, Leliévre-Pégorier M. Retard de croissance intra utérin et déficit en néphrons. Médicine Science 1993; 9:777– 780. 80. Bennis-Taleb N, Remacle C, Hoet JJ, Reusens B. A low protein diet during gestation affects brain development and alters permanently cerebral cortex blood vessels in rat offspring. J Nutr 1999; 129:1613–1619. 81. Aerts L, Holemans K, Van Assche FA. Maternal diabetes during pregnancy: consequences for the offspring. Diabetes and Metabolism Review 1990; 16:147– 197. 82. Reusens-Billen B, Remacle C, Daniline J, Hoet JJ. Cell proliferation in pancreatic islets of rat fetus and neonates from normal and diabetic mothers. An in vitro and in vivo study. Hormone Metabol Rev 1984; 16:565–571. 83. Holemans K, Aerts L, Van Assche FA. Evidence for an insulin resistance in the adult offspring of pregnant streptozotocin diabetic rats. Diabetologia 1991; 34:81– 85. 84. Galloway R, Anderson MA. Prepregnancy nutritional status and its impact on birth weight. SCN (Subcommittee on Nutrition), News United Nations. Administrative Committee on Coordination. World Health Organization Ch. 1211 Geneva 27, Switzerland 1994; 11:6–10. 85. World Development Report 1993. Investing in Health. Published for the World Bank. Washington, DC: Oxford University Press, 1993:72–81. 86. Hales CN. Fetal nutrition and adult diabetes. Sci Am Sci Med 1996; 1:54–63. 87. Leon DA, Lithell HO, Vagero D, Koupilova I, Mohsen R, Berglund L, Lithell UB, McKeigue PM. Reduced fetal growth rate and increased risk of death from ischaemic heart disease: a cohort study of 15,000 Swedish men and women born 1915–1929. Br Med J 1998; 4:241–245. 88. Phillips DW, Barker DJP, Hales CN, Hirst S, Osmond C. Thinness at birth and insulin resistance. Diabetologia 1994; 37:150–154. 89. Campbell DM, Hall MH, Barker DJP, Cross J, Shiell AW, Godfrey KM. Diet in pregnancy and the offspring’s blood pressure 40 years later. Br J Obstet Gynaec 1996; 103:273–280. 90. Ravelli ACJ, Van der Meulen JHP, Michels RPJ, Osmond C, Barker DJP, Hales

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CN, Bleker OP. Glucose tolerance after prenatal exposure to famine. Lancet 1998; 35:173–177. 91. McCully KS. Vascular pathology of homocysteinemia. Implications for the pathogenesis of arteriosclerosis. Am J Pathol 1969; 56(1):111–128. 92. Reusens B, Remacle C, Hoet JJ. The development of the fetal rat intestine and its relation to maternal diabetes: effect of mild and severe maternal diabetes. Diabetes Res Clin Practice 1989; 6:213–219.

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6 Physiological Development of the Cardiovascular System In Utero

KENT L. THORNBURG Oregon Health Sciences University Portland, Oregon

I.

Introduction

As the millenium turns, Western medical scientists are pleased to tell their public that there has been a recent overall reduction in death rate from cardiovascular diseases. In the United States, the public is generally aware of the tremendous progress in treating coronary disease and stroke through techniques introduced in the last few decades. These include the coronary graft, the enzymatic dissolution of clots in coronary or cerebral vessels, and the angioplasty balloon used in opening clogged coronary arteries, among many others. In the public’s mind, there is further relief in believing that even the failing heart can be replaced by transplantation when all else fails. Yet, despite all these advances, cardiovascular disease is still the number-one killer in Western countries and is on the rise in developing countries. Even in the United States, where the decline began in the 1960s, the rate of decline in cardiovascular disease is less for women than for men (1). As the population ages, the absolute number of deaths attributable to cardiovascular 97

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disease in women is actually increasing (2). Based on recent trends, one can estimate that the real cost to society from cardiovascular disease and stroke will exceed some $300 billion in the United States at the turn of the century (2). Thus, cardiovascular disease is an enormous health problem in the United States where the mortality, morbidity, and emotional toll brought on by cardiovascular disease are greater than for any other disease entity. These sobering facts raise the questions, “Why do some individuals develop cardiovascular pathologies during the active years of their life, and why are others spared?” Scientific research has brought partial answers to these questions. We have become aware of the lifestyle factors that place adults at risk for cardiovascular disease. The American Heart Association lists smoking tobacco, sedentary lifestyle, high blood cholesterol, and hypertension as modifiable risk factors for cardiovascular disease, and increasing age, male sex, and genetic background as risk factors that are beyond an individual’s control. Yet even these answers, backed by thousands of studies, are not satisfactory in explaining the facts worldwide, where some populations have much lower rates of heart disease with apparently similar risk factors (3). During this decade, a number of epidemiological studies have shown that suboptimal development in early life is associated with a high risk for cardiovascular disease through a process known as “programming” (4). These findings bring a new urgency for understanding the development of the cardiovascular system and its relationship to adult disease. We now stand at the threshold of opportunity to link the processes of cardiovascular development with heart and blood vessel pathology. Such a link would provide the promise of reversing the “program” of the underdeveloped infant that apparently leads to premature loss of life through heart and vessel disease. With this in mind, this chapter will cover the basic features of embryonic and fetal heart/ vessel development and will point to specific hypotheses relating cardiovascular development to a high risk for disease in adult life. II. Heart Development in Embryonic Life Unlike the liver, the heart does not develop as a miniature version of the adult organ. The morphological changes have been known for about 80 years (5,6). In the early postgastrulation stage of the embryo, three early cardiac lineages are thought to arise from the cardiac mesoderm, which lies laterally and rostrally to Henson’s Node. These lineages give rise to three cell types: (1) endothelial cells, which become endocardium and line the inner heart chambers;

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(2) ventricular myocytes; and (3) atrial myocytes (7). In the early embryo, the two wing-like regions on each side of the primitive streak fold inwardly and form the primitive foregut. As the folding is in process, separate cardiac tubes are formed inside the wings, and these fuse beneath the embryo to form a single tubular heart with outer muscle (myocardium) and inner endocardium layers. Between these two layers, a thick gelatinous material known as cardiac jelly is formed. It is not known whether the numbers of cells that are allocated to each of these layers is important at this stage in determining the final cell numbers and the functional capability of the heart in later life. The human heart begins to beat during the late tubular stage, some 24 days after conception (6). Figure 1 shows in diagrammatic form that the tubular heart is already regionalized and that these regions give rise to specific adult structures in the working heart (8). At this stage, blood flows through the heart from the caudal to the cephalic ends of the tube. The most caudal structure is the sinus venosus, followed by the atrium, the ventricle, the bulbus cordis, and then the truncus arteriosus. Figure 2 shows a computer reconstruction of the lumen of the tubular heart in the human embryo. The tubular heart continues to contract rhythmically while going through a series of shape changes that cause the heart tube to bulge rightward and form a Cshaped structure like a hairpin loop. This process is called looping. Specific regions of the looping heart synthesize site-specific cardiac proteins, including a number of growth factors, transcription factors, and cytokines. Very little is known about how this regionalization affects the overall structure and function of the adult heart (9). During the looping process, the sinus venosus, which begins as the most caudal heart structure, migrates toward the head behind (dorsal to) the bulbus cordis and ventricle (Fig. 1) (8). At the completion of the looping process, the primitive atria sit above (cephalic to) the ventricle. The inflow of the looped heart is found behind the right portion of the common atrium, and blood flows into a common atrial chamber, which is relatively large, through an atrioventricular canal, into the ventricular chamber, and then through the bulbus cordis and the truncus arteriosus. Once looping is complete and the appropriate cardiac structures are apposed, the heart transforms from a complex loop where blood flows through a single channel into a four-chambered heart where the right and left heart pumps can operate independently. Septation is the process of partitioning the primitive heart into four chambers and includes partitioning of the atria and the atrioventricular canal, absorption of the sinus venosus and pulmonary veins into the right and left atria, separation of the truncus arteriosus into aortic and

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Figure 1 Development of the embryonic heart. A, B, and C are transverse sections of the early embryo. D, E, and F show looping processes and the arrangement of the chambers just before septation. C = celomic cavity; E = endocardial tubes; F = foregut; ME = mesoderm; MY = myocardium; P = pericardial cavity; NC = neural crest; NT = neural tube; Y = yolk sac; A = atrium; B = bulbus cordis; SV = sinus venosus; T = truncus arteriosus; AVV = atrio-ventricular valve; SAV = sino-atrial valve; TV = truncal valve. (From Ref. 8.)

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Figure 2 Showing computer representation of the embyronic human heart from the Carnegie Collection specimen 5874, a stage 13 embryo. Heart was reconstructed from serial sections of human embryo by methods previously described. This heart is in the early septation stage so that blood flows through the heart as a single tube. RA = right atrium; TA = truncus arteriosus, BC = bulbus cordis, V = primitive ventricle. (Courtesy of J.O. Pentecost, B.L. Thornburg, and K.L. Thornburg.)

pulmonary roots, creation of the ventricular chambers from a single large ventricle, and development of complex membranous valves. Figure 3 (8) shows, in diagrammatic form, some of the changes that occur in the septation process during the fourth to fifth week of human life. The common atrium is divided into individual atria through the formation of two independent septa. The first, which grows from the “roof” of the atrium, is called the septum primum. It would block the flow of blood between the atria if it were not for the sudden appearance of a hole that appears in the septum as it forms. About a week later, the secondary septum, the septum secundum, grows from the roof of the right atrium alongside the primary septum. However, it only grows until it reaches about three fourths of the way toward the floor of the atrium, at which point its growth ceases. The appropriate positioning of the septum primum and secundum form a flap-valve that allows right-to-left blood flow through the atria. The establishment of the two separate ventricular chambers depends on the development of the interventricular septum and the septation of the outflow

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Figure 3 Cardiac septation. (a) Early atrial and ventricular septation. (b) Atrial osmium primum closed and osmium secundum forming. Ventricular septation incomplete. (c) Septation complete and membranous septum formed. Atrioventricular valves formed by cushion tissue and myocardial delamination. AS1 = atrial septum primum; AS2 = atrial septum secundum; AVC = atrio-ventricular cushion; LA = left atrium; LV = left ventricle; MS = membranous septum; MV = mitral valve; 01 = atrial ostium primum; 02 = atrial ostium secundum; P = papillary muscle; RA = right atrium; RV = right ventricle; SV = sinus venosus; TV = tricuspid valve; VSD = ventricular septal defect; VV = venous valves. (From Ref. 8.) tract to form the aorta and pulmonary artery. By the time septation begins, the working myocardium has changed its form so that the outer layer becomes typical compact myocardium. The cardiac jelly is invaded by migrating cardiomyocytes that form a sponge-like layer with numerous blood-filled pockets called trabeculae. Some of these trabeculae fuse to form the papillary muscles that attach to the atrioventricular valves. Others form the muscular portion of the interventricular septum. The valves are formed from thickenings, called endocardial cushions, that align themselves to divide the atrio-ventricular (A-V) canal into the right and left flow channels. The upper part of the interventricular septum is formed when cushion tissue grows in a caudal direction to meet the muscular septum arising from the floor of the ventricular chamber. The aorta itself and the pulmonary trunk are formed as two longitudinal spiral ridges formed within the truncus arteriosus into the bulbotruncal region of the heart. These new vessels must position themselves properly over appropriate regions of the AV canal in order for the ventricles to eject through the appropriate vessel. The septation occurs in the truncus arteriosus with the infiltration of neural crest cells derived from the tissue of the closing neural tube early in embryogenesis. “Cardiac neural crest cells” migrate through branchial arches 3, 4, and 6

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where their presence supports the structural formation of the great vessels (10). Although it is known that neural crest cells are needed for normal development, the mechanisms that underlie the neural crest’s role in heart development are unknown. Could inadequate numbers of neural crest cells lead to subtle deficiencies in outflow vessels and valves in adulthood? The beating heart of the late postseptation embryo finally becomes a miniaturized version of the adult heart in appearance; one might be tempted to think that a normal fetal heart would inevitably arise from it. However, at this early stage the myocyte numbers have yet to be set, the extracellular matrix has not yet been fully formed, and the coronary tree has not become fully established. The enormous growth that must take place during embryonic and fetal life begins at this stage. The heart is responsible for circulating blood for the distribution of oxygen and nutrients and the gathering of wastes for disposal by the embryo and placenta. At the earliest stages of the beating heart, it is able to generate pressure and to move blood through the circulatory system. The function of the embryonic heart can be analyzed via pressure dimension loops (11–13). Blood pressure can be measured in the early rat embryo as young as 11 days postconception, where an arterial pressure of approximately 0.2 mmHg can be measured. Figure 4 (14) shows the increase in blood pressure that occurs in the very early stages of embryogenesis in both the chick and the rat.

Figure 4 Changes in blood pressure in chick and rat embryos with respect to wet weight of embryo. Stage 18 is equivalent to 3 days; Stage 21, 3.5 days; Stage 24, 4.5 days; and Stage 27, 5.5 days. (From Ref. 14.)

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The fully mature heart muscle (myocardium) is highly adapted to function as a pump that is continually active over the lifetime of an individual, which may be up to some 100 years. It is likely that most of the heart cells, even in an older person, have been functioning since birth (15). In addition to heart muscle cells (cardiomyocytes), the myocardium contains a number of other cell types. These include capillary endothelial cells, macrophages, fibroblasts, neurons, blood cells, and many others. When all the cell numbers are accounted for, the mature myocardium contains fewer than half that are myocytes. The myocytes themselves come in many forms, including those specialized as atrial working cells, ventricular working myocardial cells, Purkinje cells, and foramen ovale cells. The mature cardiomyocyte is particularly striking, with its alternating bands that are formed by aligned myofibrils (16). Mitochondria are sandwiched between myofibrillar layers (Fig. 5) (17). The myocytes contain a flattened central nucleus (one or more) around which many of the functional organelles are found, including Golgi, smooth and rough endoplasmic reticulum, centrioles, lipid droplets, microtubules, and in atrial cells, atrial naturetic peptide-containing granules. The myofibrils are composed of individual contractile units known as sarcomeres. Under the electron microscope, the sarcomere sits between the characteristic dark lines known as Z lines. Between the Z lines are the light isotropic and dark anisotropic bands derived from the overlapping actin and myosin contractile proteins that give the striated appearance to cardiac muscle. The plasma membrane of the myocyte (sarcolemma) invaginates regularly to form T-tubules at the Z lines. T-tubules greatly extend the surface area of the myocyte, and carry the action potential to the cellular interior where it sets off excitation-contraction reactions. In addition, the mature cardiomyocyte contains the membranous sarcoplasmic reticulum that stores calcium ion for release and uptake during the cardiac cycle. Once a primitive mesodermal cell has committed to becoming a cardiomyocyte, it continually remodels itself until it becomes the mature cell previously described (18,19). It is interesting that the primitive cardiomyocyte is able to contract even when the cell is so immature that myofibrillar material is not easily seen within the cell by light microscopy. Figure 6 (20) shows an electron micrograph of a previously beating chick cardiomyocyte, showing nascent sarcomeres with Z material and contractile protein in between. In the newborn cat, the myocytes are much smaller and have only peripheral myofi-

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Figure 5 Electron micrographs of heart muscle from immature cat. In neonatal cat (a) five cells are seen separated by sarcolemma (SL). In the adult heart (b) one cell is seen. Contractile material is found at the periphery in neonate cells, but throughout the cytoplasm in the adult cell. Nucleus (N) in the neonate is centered within the cell, and mitochondria are scattered throughout the central cytoplasm. In the adult, mitochondria are sandwiched between the layers of myofibrillar material. (From Ref. 17.)

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Figure 6 Showing electron micrograph of an embryonic chick heart cell from postfusion stage. This cell is in early stages of sarcomerogenesis but is capable of contraction (z) Z line. (M) Contractile protein forming a myofibril. (From Ref. 20.) brils (Fig. 5) (17). By late gestation in sheep, the myocytes are characterized by well-organized myofibrils, abundant glycogen particles, and scattered groups of mitochondria (Fig. 7) (21–23). There is wide species variation in the rate of maturation of the excitation-contraction apparatus. Some mammals like the sheep are without T tubules at midgestation, but T-tubules, sarcoplasmic reticulum, and coupling structures are already in place before birth. Other mammals like rats and cats have virtually no T-tubules or sarcoplasmic reticulum at birth (23). In mammals, myocytes receive an unknown signal to exit the cell cycle, just before or soon after birth, depending on species. In rats and sheep, most myocytes become binucleate as they undergo this terminal differentiation process, and are generally unable to divide thereafter. There is evidence that a few myocytes are able to divide well into adult life and to increase proliferation in endstage ischemic heart disease (24). However, it is well known that the surrounding myocardium is not able to replace myocytes lost to myocardial infarction. Instead, dead myocytes are generally replaced by connective tissue and “scarring” (25).

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Figure 7 Electron micrograph from 135-day-old fetal sheep heart. Myocyte boundaries are shown by large arrows. Note myofibril along cell periphery. Mytochondria are scattered among myofibril and cytoplasm. Pepper-like background cytoplasm shows presence of glycogen particles. Magnification 6500×. (From Ref. 21.)

It is not known how many myocytes are optimal for a heart of any given size (26). Because heart cell size in the mature heart is roughly the same for all mammals, the number of myocytes in very large hearts must be much greater than for small hearts. It follows, then, that the number of cells in the heart at birth must generally reflect the expected size of the heart in adulthood. In the adult heart of the human, the left ventricular myocyte increases in volume by some 30- to 40-fold during the neonatal to adolescent period (15). Well-trained athletes have cardiac hypertrophy (27) and their heart masses may be 50% greater than those of the normal population, but the resulting architecture is dependent on the type of training (27). When cells enlarge according to such a physiological need, this is called physiological hypertrophy, and is considered normal. Hearts also gain mass in response to pathological changes (hypertension). In such hearts, hypertrophy is accompanied by a general deterioration of the myocardium, including fibrosis and biochemical failure of the myocyte (28). The regulation of myocyte dimension and volume before birth is not

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Figure 8 Myocyte cross-sectional area during development of sheep heart. During fetal life, right ventricular myocytes have larger cross-sectional area than do left ventricular myocytes. During the early postnatal period, left ventricular myocytes become larger than right ventricular myocytes. (From Ref. 23.) well understood (15). Figure 8 (23) shows that right ventricular myocytes are larger in cross-sectional area than are the left in sheep and that this difference is maintained until after birth, whereupon with increasing systolic systemic pressure the myocytes of the left ventricle become larger. The prenatal myocyte size difference in the separate ventricles is not seen in all species. IV. Arrangement of the Fetal Circulation A circulatory system allows an organism to exceed in size the distance over which oxygen can diffuse from the environment to the tissues consuming oxygen (29). The circulatory system is designed to carry large quantities of oxygen from the environmental oxygen source to the individual cells consuming oxygen and to pick up wastes and return them to the appropriate exit site. In the fetus, oxygen is acquired through a complex cascade of gas exchange that begins in the mother’s lungs where desaturated hemoglobin acquires oxygen that is distributed through the arterial system to the uterine circulation and

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Figure 9 Circulation of the mature fetal lamb. Numbers indicate mean oxygen saturation (%). RV, right ventricle; LV, left ventricle; SVC, superior vena cava; BCA, brachiocephalic artery; FO, foramen ovale; DA, ductus arteriosus; DV, ductus venosus. (From Ref. 30.)

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placenta. Because the placenta is the organ of fetal gas exchange, the fetal circulatory system must be designed to take oxygen from the placenta and distribute it to growing organs and especially the crucial organs, the heart and brain. As organogenesis, the period of organ-specific differentiation, comes to a close by 8 weeks in the human, the embryo becomes a fetus. At that point, the general circulatory arrangement remains until birth. The circulatory pattern of the fetus differs from that of the adult in that the two fetal ventricles pump blood in parallel rather than in series (Fig. 9) (30). This is possible because of the presence of four shunts that persist throughout fetal life but disappear soon after birth (31). The shunts include the ductus venosus, the foramen ovale, the ductus arteriosus, and the umbilical circulation. The ductus venosus allows oxygen-rich placental blood coming from the umbilical vein to flow directly into the inferior vena cava toward the heart. There is evidence that this oxygen-rich stream is shunted preferentially across the foramen ovale into the left atrium where that blood can then flow into the left ventricle to be distributed to the coronary circulation as well as the upper body and brain of the fetus. Blood returning from the upper and lower body via the vena cavae is collected in the right atrium and empties into the right ventricle, whereupon it is ejected into the main pulmonary artery. Most of the flow coming from the right ventricle is carried through the third shunt, the ductus arteriosus, to the descending aorta. In fact, the right ventricle contributes more flow to the dorsal aortic flow than does the left ventricle. Blood flowing down the dorsal aorta is then diverted at the terminal aorta to the placental umbilical arteries, where this blood then travels to the placenta for reoxygenation. V.

Metabolic Features and Cardiovascular Adaptations in the Fetus

During fetal life in sheep, oxygen consumption is about 8 mL·min-1 · kg-1 body weight (32). This is about 1 1/2 times higher than that found in the adult sheep, but only 1/2 of that found in the immediate newborn period (33–35). Because the fetal oxygen consumption is higher than for the adult, fetal cardiac output must be higher per kilogram, considering the fact that fetal blood oxygen content is lower than for adults. While the resting oxygen consumption for the fetus is lower than for the adult, the fetal body carries out very few activities in utero. It does not expend extra oxygen for feeding or for true limb exercise. Occasional bouts of muscle movement and respiratory activity are about the only oxygen expensive activity undertaken by the fetus. Even when fetal skeletal muscle activity is reduced by paralysis, oxygen consumption is

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decreased by less than 20%. Because the obvious purpose of fetal life is to develop a body prepared to face environmental and reproduction stresses successfully, growth is the most important outcome of fetal life. Oxygen consumption of the fetal body increases roughly linearly with body weight over the period of gestation and across species (36). Therefore, blood flow to the body must also increase with body weight. Because the cardiac output is the product of heart rate and the combined stroke volumes of the ventricles, the volume of the heart must increase with fetal weight to keep up with fetal growth. Therefore, as fetal weight increases, from about 1 kg to 5 kg over the duration of days 100 to 140 of gestation, cardiac volume and output must also increase about fivefold. Thus, the large daily increase in cardiac output found in the mature fetus is accomplished entirely by cardiac growth. The adult heart prefers long-chain fatty acids, with carbohydrates being secondary, as a fuel under aerobic conditions (37). The immature myocardium of the fetus is hardly able to metabolize free fatty acids primarily because of a deficiency of the mitochondrial enzyme carnitine palmitoyl transferase-1, which is required for shuttling long-chain free fatty acids into the mitochondria where it undergoes beta oxidation. The immature myocardium prefers lactate over other fuels, and lactate may account for most of the energy consumption with glucose and pyruvate providing the remainder (34). This is illustrated in Figure 10 (35). Within a

Figure 10 Showing normal full use by left ventricle in near-term fetus, neonate, and adult sheep. (Adapted from Ref. 35.)

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few hours of birth, the myocardium of most mammals is able to use free fatty acids as a fuel, whereupon they remain the preferred fuel throughout life (37). It is not clear whether the maturation of the biochemical constituents for free fatty acid metabolism are important in the maturation process of the myocyte itself. This raises the question of whether the heart of an undernourished newborn will suffer lifelong metabolic consequences. VI. Fetal Arterial Pressure The two fetal heart ventricles have about the same filling pressures and eject against approximately equal arterial pressures. Table 1 (38) shows the central arterial pressures in a series of fetal sheep. Two of the great mysteries in fetal physiology is how the fetus maintains its arterial pressure and whether that pressure is predictive of the arterial pressure set point after birth. Arterial pressure increases over gestation and is about half of the normal adult value in sheep and in humans just before birth. The fetal systemic circulation is characterized by its low-resistance umbilical circulation. This shunt is lost at birth and systemic arterial pressure increases immediately upon its loss. The mechanisms used by the fetal kidney to maintain arterial pressure before birth and in setting adult arterial pressure after birth are not known. Although the fetal kidneys are capable of generating a severe hypertension in the fetus when their inflow is down-regulated (39), it is known that removal of the fetal kidneys will cause a hypotension that worsens in severity over time (40). Table 1 Normal Fetal Hemodynamic Values in Sheep Output (ml · min · kg-1) Right ventricle Left ventricle Heart rate (beats/min) Pressure (mmHg) Brachiocephalic artery Pulmonary artery Abdominal aorta Right atrium Left atrium Source: Adapted from Ref. 38.

277 185 162 45 48 43 2.4 2.9

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The driving pressure for flow through the fetal systemic circulation is the difference between the mean arterial pressure and the mean atrial pressure. The systemic resistance to flow is the ratio of driving pressure to flow. By regulating relative individual organ resistances, the fetal body is able to distribute cardiac output to provide oxygen for tissues as needed. The lung is highly vasoconstricted during fetal life, and increases in oxygen content bring about large increases in flow. Thus, the lung does not appear to autoregulate in the traditional sense. The heart is very sensitive to decreases in oxygen content and is able to dilate to high levels in order to provide oxygen for the working myocardium. This is also true, but to a lesser degree, for the cerebral circulation. On the other hand the placenta does not appear to autoregulate (39). The mean systemic filling pressure is the pressure that would be found at equilibrium throughout the circulatory system if the heart were stopped. This pressure is an indication of the relative degree of filling of the circulatory system and is the upstream venous pressure driving flow back to the heart. The mean systemic filling pressure is higher in the fetus than in the adult (41). This may be an important signal for the appropriate filling of the heart. Because vascular filling also affects the filtration pressure of the fetal placental capillary, the acquisition of water by the placenta and circulatory filling must be exquisitely regulated. Fetuses that acquire too little water may have a decrease in this pressure with negative growth consequences for the circulatory system.

VII.

Regulation of Fetal Cardiac Output

A. Vocabulary

A number of terms are used by cardiac physiologists to explain the physical parameters that underlie heart function (42). These terms include the following: preload, the force per unit cross-sectional area at end diastole (just before contraction); afterload, the force per unit cross-sectional area in the heart muscle during contraction; and contractility, the intrinsic strength of contraction of the myocardium that is independent of preload and afterload. Stroke volume is the volume of blood ejected each beat and is the difference between the volumes at end-diastole and end-systole. Ejection fraction is the portion of the end-diastolic volume that is ejected each beat. Filling pressure, the transmural ventricular pressure at end diastole, is often approximated by mean atrial pressure and is an important determinant of preload. Vascular impedance is the resistance of the vascular tree offered to pulsatile ventricular output; it includes

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vascular resistance and compliance. Wall stress is the equilibrium force per unit cross-sectional area of myocardium that counteracts transmural pressure. B. Stroke Volume Determinants Cardiac output is the volume of blood pumped by the heart per minute and thus depends on stroke volume and heart rate. Because the fetal ventricles pump into the circulation in parallel, fetal cardiac output is the sum of both ventricles, the biventricular output. Preload, afterload, and contractility are the mechanisms by which stroke volume can be altered acutely. However, growth of the ventricular chambers is a more powerful determinant of output than these short-term regulators over the gestational period. In order to understand preload and afterload, it is important to know the basic physiological features of cardiac muscle, which is a specialized striated muscle (42). The first feature is the length-tension relationship. As a strip of striated muscle is stretched from its resting length, it will generate more and more active tension when stimulated until it reaches the length where its active tension generation capability can no longer increase. This length is known as Lmax. The second property of striated muscle is the forcevelocity relationship. A strip of cardiac muscle will contract at maximum velocity when it contracts against no retarding tension (eg, no load “to lift” at all). As the load that the muscle strip is required to contract against is increased, its contraction velocity slows. At some finite increasing load, the muscle will be unable to contract, and only isometric tension will be generated. C. Preload In a ventricular chamber, the tension in the muscle wall of the chamber will increase as fluid is forced into the chamber. In other words, as end diastolic pressure goes up, the muscle fibers in the wall of the ventricle will be stretched much like the stretching of the muscle strip. As the end diastolic pressure increases, the tension that can be generated by the heart muscle also increases, as predicted by the length-tension relationship, and the volume of ejected blood goes up. The preload-stroke volume relationship is known as the FrankStarling Mechanism (42). Therefore, preload is one way in which stroke volume can be regulated on a beat to beat basis. D. Afterload The load-velocity relationship also applies to heart function. In the intact ventricular chamber, this effect is seen during contraction. For example, as the

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pressure in the arterial circulation is increased, the load on the heart muscle during ejection (afterload) is also increased, the velocity of shortening decreases, and the heart ejects a smaller stroke volume. Stroke volume goes down as afterload goes up. Thus, increased afterload is a negative regulator of stroke volume. E. Contractility The inherent capability of muscle to generate tension, contractility, is the third regulator of stroke volume. The ability of the myocardium to generate increased tension (inotropy) without changing rate, preload, or afterload is, by definition, an increase in contractility. The biochemical determinants of contractility include the enzymatic speed and power of myosin, the sensitivity of the contractile proteins that are activated by calcium, and the availability of cytosolic calcium. It has long been known that contractility is partly related to ATPase activity of the contractile protein, myosin. There are two primary myosin isoforms that have either high (α or V1) or low (β or V3) ATPase activity (37). The isoforms change with development in small rodents but the ventricle in large mammals has the slow isoform throughout life. Regardless of isoform status, there is evidence for an increase in contractility over the period of perinatal life (43,44). This improvement in cardiac function of the developing myocardium appears to be attributable to the amount and orientation of myofibrils within the myocyte, and important maturation of biochemical mechanisms. F. How Does the Normal Fetus Regulate its Cardiac Output? Output can be easily changed by changes in either heart rate or stroke volume. It is not possible, at present, to measure actual preload, or the force per unit cross-sectional area in the myocardium of a fetal heart, so other indicators must be used. Mean atrial pressure, or better, ventricular end diastolic pressure (or volume) are the commonly used estimates of preload. The relationship between mean atrial pressure and stroke volume (known as the cardiac function curve) is often used for understanding the role of preload in determining stroke volume. Figure 11 (45) shows a ventricular function curve in the nearterm sheep fetus and adult sheep. Note that the fetal function curve has a similar shape to that of the adult sheep. It is clear that fetal stroke volume increases along with right atrial pressure until a plateau is reached—at which point further filling brings little increase in stroke volume. This was first shown in the fetus by Gilbert (46). The regulatory question then arises, “Does the fetus

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Figure 11 The relationship between stroke volume and mean left atrial pressure is shown for a near-term fetal lamb (a) and adult sheep (b). Both studies were performed in chronically instrumented animals with electromagnetic flow probes on the ascending aorta. Both function curves show a steep ascending limb and a plateau, although the breakpoint of the adult animal is at a higher filling pressure. (From Ref. 45.)

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have significant preload reserve with which it can increase its stroke volume when needed?” Experiments from a number of different laboratories have shown that the fetus operates at the “breakpoint” of the function curve where end-diastolic dimension, and thus stroke volume, cannot be easily increased (47). This means that although the fetus has an active length-tension relationship (48), it has very little preload reserve. The same appears to be true for the adult sheep. However, humans are different in that they operate below the break point of the function curve while upright but near the break point when supine. Figure 12 (49) shows that the stroke volume of the two ventricles decreases differentially in response to increased arterial pressure, which increases afterload. As arterial pressure is increased, the stroke volume of the

Figure 12 The simultaneous average responses of the right and left ventricles to increased arterial pressure are shown for nine fetuses. Stroke volume is expressed as a percent of control value and arterial pressure as the increment above control. The linear regression coefficient for each ventricle was calculated, the average slope forced through 100% on the Y axis, and the lines extended through the pressure range studied. The right ventricular pressure sensitivity (–2.5 ± 1.4% stroke volume · torr-1) was more than five times the left ventricular pressure sensitivity (–0.5 ± 0.7% stroke volume · torr-1) (p < 0.001). (From Ref. 49.)

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ventricles decrease. The differences will be discussed shortly. However, the take-home message is that whenever arterial pressure increases in the fetus, stroke volume will be depressed. In the normal fetus, heart rate is highly variable and filling pressure is always changing along the bend in the break point of the function curve. Thus, both heart rate and stroke volume are in a constant state of flux on a beat to beat basis. Nevertheless, cardiac output over minutes of time is relatively constant, as the product of stroke volume and rate is constant. Although there is good evidence that contractility is augmented in the perinatal period, there does not appear to be a measurable variation in beat to beat regulation of contractility. VIII.

Differences Between the Right and Left Ventricles

Over most of recorded history, it has been held that the right and left ventricular outputs of the fetus are equal because the chambers appear to have about the same volume and because filling pressures, chamber volumes, and arterial pressures in the fetus are similar (31). In the human, autopsy data as well early ultrasound images supported this notion (50,51). However, experiments in fetal sheep over the last 20 years have shown that right ventricular stroke volume exceeds left ventricular stroke volume (52,38). Early experiments showed that right ventricular stroke volume was 60 to 70% of the total cardiac output. Detailed anatomical and physiological studies have shown the following (53,54,49): 1. Right ventricular stroke volume exceeds left ventricular stroke volume because at the common filling pressure, which fills both chambers simultaneously, the right ventricular chamber is larger than the left chamber (55). 2. The pressure volume curves (Fig. 13) (56) show that the right ventricular volume is larger at any transmural pressure compared with the left, and that both chambers are effected by a pressure differential across the septum. 3. Because of its larger size, the right ventricle operates on an elevated function curve compared with the left ventricle (Fig. 14) (49). Thus, at any filling pressure, right ventricular stroke volume exceeds left ventricular stroke volume. Because both ventricles operate at about

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Figure 13 Right and left ventricular pressure-volume relationships are shown for a potassium-arrested, near-term fetal lamb heart. Each ventricle is totally emptied and then filled by means of a syringe pump, with the contralateral ventricle at 10 or 0 mmHg and the pericardium in place. Right ventricular volumes are always greater than left ventricular volumes at common filling pressures. Both ventricular volumes are importantly affected by contralateral ventricular pressure. (From Ref. 56.) the “break point” of their respective function curves, neither ventricle has a great preload reserve. 4. The right ventricle has a larger radius to wall thickness ratio than the left ventricle. This is due to the fact that the two ventricles have about the same wall thickness when measured at a distance halfway between the base of the heart and the apex. However, because the right ventricle is larger, its radius of curvature is also much larger. This places the right ventricle at a mechanical disadvantage

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Figure 14 Average function curves are shown for the right and left ventricle of the chronically instrumented fetal lamb. The break points are not significantly different from control values of right atrial pressure and right ventricular stroke volume, and left atrial pressure and left ventricular stroke volume, respectively. Thus, at filling pressures less than control, mean atrial pressure has a powerful effect on stroke volume, but a lesser effect at filling pressures above control. (Adapted from Ref. 49.)

compared with the left. This finding is also true for the human fetus, although the effect is more pronounced in the sheep. The law of Laplace predicts that wall stress (Sw) is dependent on the radius-towall thickness ratio (r/h). Sw = Pw/2 · r/h

(Eq. 1)

This simple formula indicates that as the radius of curvature (r) increases, for a constant transmural pressure (P) and wall thickness (h), wall stress (Sw) will increase. Thus, we can predict that the right ventricle will have the higher stress and more difficulty in generating pressure against an arterial pressure load than will the left ventricle. Figure 12 (49) shows that left ventricular stroke volume is only mildly affected with increases in aortic pressure. On the other hand, the right ventricle is very sensitive to increases in pulmonary arterial pressure so that stroke volume decreases rapidly as pulmonary arterial pressure is increased. This finding has great physiological significance because it indicates that whenever systemic arterial pressure is increased in the fetus, both ventricles will be affected but right ventricular stroke volume will suffer most.

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The right ventricle is also different from the left in other ways (23). Right ventricular myocytes are larger and the total cross-sectional area of its capillaries are also larger. And, as will be discussed, the blood flow to the right ventricle, even under resting conditions, is considerably larger than for the left ventricle. IX. Alterations in Myocardial Growth with Mechanical Stress Adult hearts adapt to volume of pressure overload stress by altering the architecture of the ventricle. Pressure overload leads to concentric hypertrophy, and volume overload to eccentric hypertrophy (57). However, the nature of adaptations to mechanical stress in the immature myocardium is not well understood. It is known that a number of congenital cardiac adaptations appear to be related to alterations in hemodynamic forces. For example, the neonatal hypoplastic left heart syndrome is associated with reduced left ventricular inflow as with the closure of the foramen ovale and/or mitral atresia. It is also known that cerebral arterial venous fistulas are known to cause congenital cardiomegaly in newborns, and recent studies suggest that the cardiomegaly found in severe intrauterine growth retardation is attributable to cerebral vasodilation of the cerebral circulation in response to hypoxemia. When the fetal right ventricle is subjected to an increased pressure load independent of the left, its wall becomes thicker (Fig. 15) (53). This thickened wall decreases the radius to wall thickness ratio, and allows the right ventricle to become a more powerful pump similar to the left ventricle in its natural state (Fig. 16) (54). It is also interesting to note that the right ventricle in the loaded condition shifts its pressure volume curve to the left, which means that for a given transmural pressure, the volume in the ventricle will be smaller. This means that as the wall thickens, it does so at the expense of the ventricular chamber. Pressure overload also produces a small-chambered, hypoplastic left ventricle (58). This is not unlike the clinical anomaly bearing the same name. The hypoplastic ventricle is usually defined by its gross reduction in chamber volume, without regard to cell number or size. It may be that the so-called hypoplastic ventricle actually develops via hyperplastic growth with the free wall growing to obliterate the chamber. In 1978, Fishman (58) showed a doubling of left ventricular mass after an experimental outflow obstruction without increasing myofiber cross-section area, suggesting hyperplastic growth. Recent studies have shown that loading the

Figure 15 Showing the ratio of circumferential radius of curvature (r) to ventricular free wall thickness (h) in fetal hearts with no constriction of the pulmonary artery (control) or 10 days of constriction. Mean pulmonary arterial pressure was increased by 10 mmHg for the duration of the study. After loading, the wall thickness of the right ventricle was thicker and the radius of curvature smaller. The r/h was not different between the ventricles after loading. (Adapted from Ref. 53.)

Figure 16 Showing percent change in stroke volume of control fetal left ventricular (LV), right ventricular (RV), and right ventricular after 10 days of pulmonary arterial constriction (RV load). The right ventricular radius to wall thickness ratios decreased from 4.5 under control conditions to 3.1 after loading (see Fig. 15). The architectural and physiological changes allowed the right ventricle to decrease its sensitivity to increases in arterial pressure. (Adapted from Ref. 54.)

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right ventricle causes both an increase in cell number as well as an increase in myocyte length. This means that for the near-term sheep fetus, the socalled cardiac hypertrophy is attributable to true hypertrophy (increase in cell volume) as well as hyperplastic growth (increase in cell numbers). There is increasing evidence that large mammals adapt to pressure loading conditions in utero differently than do small mammals like mice, rats, and rabbits. In rats it appears that myocardial mass enlargement during chronic hypoxia is primarily attributable to increases in cell number without cell enlargement (18). Clubb and Bishop (59) were the first to show that rat myocytes are mononucleate during fetal life, and that during postnatal life they become mostly binucleate over the first 3 weeks after birth. Figure 17 (59) shows the changes in binucleation with maturation in the rat. Their evidence indicated binucleate cells did not proliferate thereafter, and that the myocytes present at the completion of the binucleate conversion process were the myocytes that would remain as the working myocardium for the rest of the life of the individual. Unfortunately, we know very little about human myocyte maturation. We know that some human cardiomyocytes become polyploid as they mature (15), and that they get larger with maturation before birth (60).

Figure 17 Showing percentage of cells with two nuclei as a function of postnatal age in rats. (Adapted from Ref. 59.)

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The immature sheep heart is different. Loading the fetal right ventricle causes the percentage of cells that are binucleate to increase (61). Thus, the maturation program for the myocyte is altered by hemodynamic signals. These data indicate that a fetal heart that has experienced a high systolic wallstress load before birth will have a smaller, more efficient ventricle with myocytes that have an augmented maturation cycle. X. Redistribution of Cardiac Output with Hypoxemia Rudolph and Heymann (62) measured blood flow distribution in the fetal sheep at several gestational ages. For the near-term fetus, they found that of the 500 mL/min biventricular cardiac output, 3% is delivered to the brain, 4% to the heart, 2% to the kidneys, and some 40% to the fetal body. About 40% of the cardiac output is delivered to the placenta. These data were collected from normoxic fetuses that had an arterial oxygen tension of about 25 torr, a CO2 tension of 45 torr, and a pH of 7.35. In 1974, Cohn et al. (63) allowed pregnant ewes to breathe a gas mixture that lowered fetal arterial PO2 to about 12 torr

Figure 18 Changes in blood flow and oxygen delivery to selected sheep fetal organs during an acute fetal hypoxemia episode induced by allowing the ewe to breathe air containing a reduced fraction of oxygen. (Adapted from Ref. 64.) Oxygen delivery was estimated from published relationships between organ flow and blood content for various organs and the estimated contents from similar experiments in other laboratories. (From Refs. 63–66.)

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without changing the CO2 tension. In response to the lowered oxygen tension, the heart, brain, and adrenal vasodilated to maintain oxygen delivery to normal or above at the expense of oxygen delivery to the kidney, gut, and carcass (Fig. 18) (64). Others have studies the distribution of flow under conditions of hemorrhage (67), umbilical occlusion (68), reduced uterine flow (69), and fetal placental microsphere embolization (70). All of these show a reduced carcass flow and an “attempt” to spare brain and heart. Nevertheless, even the relatively spared heart may be affected for life. XI. Regulation of Fetal Coronary Flow The prenatal development of the cardiac capillary bed has not been studied extensively. It appears that the myocyte to capillary ratio, which may be as high as six in the newborn, decreases with maturation where it is believed to be one to one in the adult human, rabbit, and rat. During that same period, the intercapillary distance and the average capillary diameter decrease (23). In sheep, the capillary lumenal area is larger for the right ventricle than for the left ventricle during fetal life, but capillary density is greater for the left ventricle than the right ventricle. It is not known to what degree the environment in which the myocardium grows determines capillary size and density. However, it is known that capillary density can change in response to ventricular loading and chronic hypoxia (71–74). Coronary flow regulation in the fetus is much different than that found in the adult (75–77). Therefore, it is not possible to automatically apply mechanisms from adult coronary physiology directly to the immature heart. In the fetus, coronary flows can be measured by the microsphere method (78) with a Doppler flow sensor that has been calibrated by the microsphere method, or by an electromagnetic or transonic flow sensor alone. Fisher et al. showed that total myocardial flow in the fetus is about twice that of the adult level, and right ventricular flow is higher than for the left ventricle (Fig. 19) (79). They also showed that when the oxygen content of blood supplying the myocardium is reduced by about 50%, fetal myocardial oxygen consumption is not changed but coronary blood flow doubles— increasing from ~ 180 to 340 mL · min 1 · 100g-1. Under these conditions, oxygen extraction increases so that coronary sinus blood oxygen content is decreased. Pyruvate and lactate consumptions also increase slightly under these circumstances. The increase in coronary flow under hypoxemic conditions shows the degree to which the fetal myocardium is protected from

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Figure 19 Showing myocardial oxygen deliveries for the right and left heart ventricles in fetal, newborn, and adult sheep. Oxygen delivery is the product of ascending aortic oxygen content and blood flow as measured by the microsphere technique. (Adapted from Ref. 79.)

moderate hypoxemia by autoregulation. However, during newborn life, right ventricular blood flow decreases and left ventricular flow increases as the workload of the two ventricles is reversed. The concept of coronary flow reserve is often used to evaluate changes in coronary regulation. Figure 20 (80) shows the concept of flow reserve where coronary blood flow is plotted as a function of mean aortic pressure. The straight line labeled D shows that when the coronary vessels are fully dilated with a pharmacological agent (like adenosine), flow will increase linearly as a function of increased arterial pressure. However, under nondrug conditions, flow will increase with pressure only until it reaches a plateau phase (Curve A) where the autoregulation mechanism causes a compensatory vasoconstriction that keeps flow relatively constant over a wide range of pressure. When pressure exceeds the autoregulatory range, flow again increases. At any pressure, the difference in flow between the dilated curve and the autoregulatory curve is the flow reserve. The problem with interpreting

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Figure 20 Diagram of pressure-flow relationships in normal left ventricle during autoregulation (A) and maximal vasodilatation (D). R1 and R2 are the coronary flow reserves at mean coronary perfusing pressures of 75 and 100 mmHg when aortic pressure and heart rate are constant. (From Ref. 80.)

flow reserve values is clear from Figure 20 (80). A different flow reserve value would be found at any different pressure in the autoregulatory range. Therefore, flow reserve is only valuable as a concept if the perfusion pressure is taken into account. Figure 21 (81) shows changes in right ventricular blood flow of the nearterm fetus under control conditions, as pulmonary arterial pressures are increased up to the maximum and while the coronary vessels are fully dilated with adenosine. The figure shows that right ventricular flow increases with increasing workload and that even at maximal workload, flow is less than during adenosine infusion. During systolic work, the fetal right ventricle cannot take full advantage of its potential flow reserve. Figure 21 (81) also

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Figure 21 Showing myocardial flow of right and left ventricles in fetal sheep under baseline pulmonary arterial pressure (53 mmHg) and during acute increases in arterial pressure (57, 63, 71 mmHg) obtained by inflating an occluder around the pulmonary artery. Seventy-one mmHg is the maximum load that can be generated by the right ventricle. Myocardial flows in right and left ventricles increase with increasing pressure load but do not reach the levels obtained during the administration of the vasodilator, adenosine (Ad-47 mmHg). (Adapted from Ref. 81.)

shows that left ventricular flow increases as right ventricular workload goes up, even though left ventricular work is little increased. Because left ventricular and right ventricular flow stay in a constant ratio, it raises the question of whether there are common signaling mechanisms for the entire coronary tree. Although it is known that coronary flow increases with decreasing oxygen content, it is not known to what degree coronary flow can increase under severe hypoxic conditions. Figure 22 (82) shows that basal coronary flow decreases in the presence of the nitric oxide synthase antagonist Nω-nitro-Larganine (L-NINA). This figure also shows that in the presence of severe hypoxemia, coronary flow equals or even exceeds the flow obtained during adenosine administration. This figure also indicates that coronary flow cannot exceed

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Figure 22 Myocardial flow to the left ventricle of the sheep fetus. Note that flow is higher with hypoxemia (hypox) than during adenosine administration (Ad) or with hypoxemia plus L-NNA administration (H + LNN). Also, note that flow was reduced with LNNA compared with control. All flows are significantly different except that Ad and H+LNNA are not different. (Adapted from Ref. 82.)

the flow obtained during chemical dilation when nitric oxide synthase is inhibited. The coronary flow reserve has been believed to be the maximal increase in flow that could be expected under any physiological condition. In adults, maximal cardiac work is not able to use the full flow reserve. However, in the fetus, coronary flow may exceed the level during chemical dilation while under conditions of severe hypoxemia. Figure 23 (83) shows that chronic hypoxemia may stimulate growth of the coronary bed. In a group of fetuses that were spontaneously hypoxemic and hypercapnic, the resting coronary flow exceeded that which is found in normoxic fetuses under dilated conditions. This would suggest that the coronary reserve had been “used up” in response to chronic hypoxemia. Surprisingly, when adenosine was administered to these fetuses, their coronary reserve was not used up; in fact, their maximal flow far exceeded that possible in the normoxemic fetus.

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Figure 23 Right ventricular (RV) and left ventricular (LV) myocardial blood flow using radiolabeled microsphere technique. Myocardial flows were measured in normoxemic fetuses at baseline (Control), during acute right ventricular pressure loading (Load), and during adenosine administration (adenosine) (n = 7) (12). Myocardial blood flow was later measured in a group of chronically hypoxemia fetuses (n = 4) at baseline (Control), and with adenosine. Maximal myocardial flow with adenosine in the hypoxemia fetuses was significantly greater than any other measured flow. Baseline (Control) hypoxemia myocardial blood flow was not different from maximal myocardial blood flow in normoxemic fetuses. (From Ref. 83.)

The regulation of coronary flow can be summarized as follows: 1. The coronary reserve in the fetus far exceeds the coronary reserve found in the adult. 2. The fetus is not able to access its full coronary reserve under high workload conditions, but is able to under extreme hypoxemia. 3. The coronary bed is very plastic and new arteriolar level vessels can be formed as an adaptation to chronic hypoxemia. Recent studies of chronically anemic fetuses show that the coronary bed can be greatly increased, and that this is accompanied by a sudden increase in expression of vascular endothelial growth factor (73,84). It should be noted, however, that fetuses raised at altitude are able to fully compensate for the low ambient oxygen tension and do raise coronary flow above normal (85,86).

The Cardiovascular System in Utero XII.

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Fetal Cardiovascular Development and Adult Disease

To say that the regulation of the development of the cardiovascular system is very complex is an understatement. Yet it appears that there is a genetic expression cascade that is the basic driving force for patterning, differentiation, and maturation of the heart and vascular tree. This genetic plan is sensitive on a number of levels to environmental alterations in the form of chemical signals, nutrient over- and undersupply, and mechanical signals. There is evidence that every cell type that contributes to the heart and vessels is uniquely sensitive to various environmental signals and, further, each organ seems to have critical windows of time when one or more cell types are particularly sensitive to stress, be it from nutrient deficit, hypoxia, acidosis, or perhaps an overproduction of a growth active cytokine. Presented here are a number of non–mutually exclusive hypotheses that may each explain, in part, the propensity for an underdeveloped fetus to suffer cardiovascular pathology as an adult. Those listed here are meant to stimulate new ideas and strategies. Hypothesis 1. Alteration of the Adult Arterial Set Point Before Birth

It is now well known that maternal protein deficit leads to abnormal function of the fetal renin angiotensin system (RAS), abnormal natal kidney development (87,88), and lifelong hypertension of offspring in rats (89). However, the link between nutrition deprivation and the establishment of the arterial set point is not known. Most animal models of hypertension have defects in the complex roles of corticosteroids, vascular structure, and circulating vasoactive substances. All of these mechanisms must be studied in animal models of protein deprivation during pregnancy. Abnormal expression of endothelial-derived vasoactive compounds is another likely outcome of nutrition deficit. If the endothelial vasorelaxation regulatory mechanisms are inadequate, this would not only lead to a volatile systemic microcirculatory bed, but also to a coronary bed susceptible to vasoconstriction and perhaps platelet aggregation. Hypothesis 2. Inadequate Placentation

The placenta is the key organ for fetal gas and nutrient exchange with the mother, for fetal steroid and protein hormone production as well as water

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acquisition. Furthermore, the umbilical circulation provides a low-resistance shunt for proper function of the parallel fetal circulation. An inadequate placenta can lead to diminished nutrient transfer, which limits the rate at which carbon is acquired and used by the fetus for metabolic and tissue-growth needs. An abnormal placenta can fail to protect the fetus against deleterious transplacental flux of steroid hormones, catecholamines, and growth factors. Lastly, a high placental vascular resistance leads to systolic pressure loading of the fetal heart, right ventricular hypertrophy, and abnormal coronary tree development. An increased systolic load may augment maturation of cardiac myocytes, which might lead to a precocial myocyte maturation and diminished cell numbers. Uncompensated hypoxemia caused by poor placental gas exchange has long-term consequences. The autoregulatory brain and heart sparing effect leads to asymmetrical growth with a relatively underdeveloped body. Furthermore, severe hypoxemia leads to extreme vasodilation of the cerebral vasculature to the extent that it may form a low-resistance shunt. This can lead to a fetal volume-overload cardiomegaly. It may also lead to abnormal regulation of endothelial vasodilators (90) and a host of systemic endocrine effects. Therefore, an abnormal umbilical arterial pulsatility attributable to increased vascular impedance would be expected to lead to ventricular wall thickening at the expense of chamber size. However, an increased ventricular wall thickness and chamber volume (cardiomegaly) would be expected when accompanied by severe hypoxemia and cerebral shunting. Hypothesis 3.

Inadequate Composition of the Cardiovascular Extracellular Matrix

The extracellular matrix is under construction throughout fetal life, but collagen and elastin are deposited in the large vessels at an especially high rate during the immediate preterm period. Because elastin is a stable insoluble molecule, its prenatal deposition may be important in determining vascular compliance in adult life. The accumulation of elastin appears to be affected by blood flow and cortisol levels during the perinatal period (91). Furthermore, it has been suggested that the deposition of elastin may be meager in newborn infants with low birthweight (92), as suggested by increased pulsewave velocity increases in adults who were born small at term (93). In addition, the extracellular matrix of the heart is augmented as cardiomyocytes become mature and the coronary bed is expanded (94). The matrix has a different composition in immature hearts that are subjected to pressure

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(94,61). Although elastin is known to have a long biological halflife, there is evidence that it can undergo degradation by special proteases. How the heart, blood vessels, and uterus are able to remodel and change compliance is not known (95,96). References 1. 2.

3. 4. 5. 6. 7. 8.

9. 10. 11. 12.

13.

14.

American Heart Association. 1997 Heart and Stroke Facts: Statistical Update. American Heart Association, Dallas, TX, 1996. Cardiovascular Health Branch, Division of Chronic Disease Control and Community Intervention, National Center for Chronic Disease, Prevention and Health Promotion, CDC. Trends in ischemic heart disease mortality. United States, 1980–1988. Morb Mortal Week Rep 1992; 41:548–556. Ravnskov U. The questionable role of saturated and polyunsaturated fatty acids in cardiovascular disease. J Clin Epidemiol 1998; 51(6):443–460. Barker DJP. Mothers, Babies and Health in Later Life. Edinburgh: Churchill Livingstone, 1998. Streeter GL. The “Miller” ovum—the youngest normal human embryo thus far known. Contrib Embryol Carneg Instn 1926; 18:31–48. Arey LB. Developmental Anatomy. Philadelphia; W.B. Saunders Co., 1966. Mikawa T. Cardiac lineages, In: Harvey RP, Richard P, Rosenthal N, eds. Heart Development. New York: Academic Press, 1999:19–33. Reller MD, McDonald RW, Gerlis LM, Thornburg KL. Cardiac embryology: basic review and clinical correlations. J Am Soc Echocardiograph 1991; 4:519– 532. Kelly RG, Zammit PS, Buckingham ME. Cardiosensor mice and transcriptional subdomains of the vertebrate heart. Trends Cardiovasc Med 1999; 9:3–10. Kirby ML, Waldo KL. Neural crest and cardiovascular patterning. Cir Res 1995; 77:211–215. Taber LA, Keller BB, Clark EB. Cardiac mechanics in the stage-16 chick embryo. J Biomech Eng 1992; 114:427–434. Keller BB. Overview: functional maturation and coupling of the embryonic cardiovascular system. In: Clark EB, Markwald RR, Takao A, eds. Developmental Mechanisms of Heart Disease. Armonk, NY: Futura, 1995:367–385. Keller BB, Yoshigi M, Tinney JP. Ventricular–vascular uncoupling by acute conotruncal occlusion in the stage 21 chick embryo. Am J Physiol 1997; 273:H2861–2866. Nakazawa M, Miyagawa S, Ohno T, Miura S, Takao A. Developmental hemodynamic changes in rat embryos at 11 to 15 days of gestation: normal data of blood pressure and the effect of caffeine compared to data from chick embryo. Pediatr Res 1988; 23(2):200–205.

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15. Rakusan K. Cardiac growth, maturation, and aging. In: Zak R, ed. Growth of the Heart in Health and Disease. New York: Raven Press, 1984:131–164. 16. Simpson FQ, Rayns DG, Ledingham JM. The ultrastructure of ventricular and atrial myocardium. In: Challice CE, Viragh S, eds. Ultrastructure of the Mammalian Heart. New York: Academic Press, 1973:1–41. 17. Maylie JG. Excitation-contraction coupling in neonatal and adult myocardium of cat. Am J Physiol 1982; 242(5):H834–H843. 18. Hirakow R, Gotoh T. A quantitative ultrastructural study on the developing rat heart. In: Lieberman M, Sano T, eds. Perspectives in Cardiovascular Research, Vol. 1: Developmental and Physiological Correlates of Cardiac Muscle. New York: Raven Press, 1976:37–50. 19. Challice CE, Viragh S. The embryologic development of the mammalian heart. In: Challice CE, Viragh S, eds. Ultrastructure of the Mammalian Heart. New York: Academic Press, 1973:91–126. 20. Thornburg KL. Development of the cardiovascular system. In: Rodeck CR, Whittle M, eds. Fetal Medicine, Basic Science and Clinical Practice. Edinburgh: Churchill Livingstone, 1999; 141–154. 21. Hanson MA, Spencer JA, Rodeck CH. Fetus and Neonate: Physiology and Clinical Applications, Volume One, The Circulation. Cambridge: Cambridge University Press, 1993. 22. Brook WH, Connell S, Cannata J, Maloney JE, Walker AM. Ultrastructure of the myocardium during development from early fetal life to adult life in sheep. J Anat 1983; 137:729–741. 23. Smolich JJ, Walker AM, Campbell GR, Adamson TM. Left and right ventricular myocardial morphometry in fetal, neonatal, and adult sheep. Am J Physiol 1989; 257:H1–H9. 24. Kajstura J, Leri A, Finato N, DiLoreto C, Beltrami CA, Anversa P. Myocyte proliferation in end-stage cardiac failure in humans. Proc Natl Acad Sci USA 1998; 95:8801–8805. 25. Hao J, Ju H, Zhao S, Junaid A, Scammell-La Fleur T, Dixon IM. Elevation of expression of Smads 2, 3, and 4, decorin and TGF-beta in the chronic phase of myocardial infarct scar healing. J Mol Cell Cardiol 1999; 31(3):667–678. 26. Zak R. Growth of the Heart in Health and Disease. New York: Raven Press, 1984. 27. Colan SD, Sanders SP, Borow KM. Physiologic hypertrophy: effects on left ventricular systolic mechanics in atheletes. J Am Coll Cardiol 1987; 9(4):776– 783. 28. Grossman W, Carabello BA, Gunther S, Fifer MA. Ventricular wall stress and the development of cardiac hypertrophy and failure. In: Alpert NR, ed. Myocardial Hypertrophy and Failure. New York: Raven Press, 1983:1–18. 29. Weibel ER. The Pathway for Oxygen: Structure and Function in the Mammalian Respiratory System. Cambridge: Harvard University Press, 1984.

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30. Born GVR, Dawes GS, Mott JC, Widdicombe JG. Changes in the heart and lungs at birth. Cold Spring Harb Symp Quant Biol 1954; 19:102–108. 31. Dawes GS. Foetal and Neonatal Physiology: A Comparative Study of the Changes at Birth. Chicago: Year Book Medical Publishers, Inc., 1968. 32. Parer JT. Fetal oxygen uptake and umbilical circulation during maternal hypoxia in the chronically catheterized sheep. In: Longo LD, Reneau DD. Fetal and Newborn Cardiovascular Physiology, Volume 2, Fetal and Newborn Circulation. New York: Garland STPM Press, 1978:231–247. 33. Klopfenstein SH, Rudolph AM. Postnatal changes in the circulation and responses to volume loading in sheep. Circ Res 1978; 42:839–845. 34. Fisher DJ, Heymann MA, Rudolph AM. Myocardial oxygen and carbohydrate consumption in fetal lambs in utero and in adult sheep. Am J Physiol 1980; 238:H399–H405. 35. Fisher DJ, Heymann MA, Rudolph AM. Myocardial consumption of oxygen and carbohydrates in newborn sheep. Pediatr Res 1981; 15:843–846. 36. Battaglia FC, Meschia G. An Introduction to Fetal Physiology. New York: Academic Press, 1986. 37. Katz AM. Physiology of the Heart. 2nd ed. New York: Raven Press, 1992. 38. Anderson DF, Bissonnette JM, Faber JJ, Thornburg KL. Central shunt flows and pressures in the mature fetal lamb. Am J Physiol 1981; 241:H60–H66. 39. Anderson DF, Parks CM, Faber JJ. Arterial pressure after chronic reductions in suprarenal aortic flow in fetal lambs. Am J Physiol 1987; 253:H838–844. 40. Anderson DF, Barbera A, Faber JJ. Substantial reductions in blood pressure after bilateral nephrectomy in fetal sheep. Am J Physiol 1994; 266:H17–20. 41. Gilbert RD. Venous return and control of fetal cardiac output. In: Longo LD, Reneau DD, eds. Fetal and Newborn Cardiovascular Physiology, Volume 1: Developmental Aspects. New York: Garland STM Press, 1978:299–316. 42. Braunwald E, Ross J, Sonneblick EH. Mechanisms of the Normal and Failing Heart. 2nd ed. Boston: Little, Brown and Company, 1976. 43. Anderson PAW. Myocardial development. In: Long WA, ed. Fetal and Neonatal Cardiology. Philadelphia: Saunders, 1990:17–38. 44. Colan SD, Parness IA, Spevak PH, Sanders SP. Developmental modulation of myocardial mechanics: age- and growth-related alterations in afterload and contractility. J Am Coll Cardiol 1992; 19(3):619–629. 45. Thornburg KL, Morton MJ. Development of the cardiovascular system. In: Thorburn GD, Harding R, Patrick J, eds. Textbook of Fetal Physiology. Oxford: Oxford University Press, 1994. 46. Gilbert RD. Control of fetal cardiac output during changes in blood volume. Am J Physiol 1980; 238:H80–H86. 47. Thornburg KL, Morton MJ. Filling and arterial pressures as determinants of left ventricular stroke volume in unanaesthetized fetal lambs. Am J Physiol 1986; 251:H961–968.

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48. Friedman WF. The intrinsic physiologic properties of the developing heart. Progress in Cardiovascular Disease 1972; 15:87–111. 49. Reller MD, Morton MJ, Reid DL, Thornburg KL. Fetal Lamb ventricles respond differently to filling and arterial pressures and to in utero ventilation. Pediatr Res 1987; 22:621–626. 50. Wladimiroff JW, Vosters R, McGhie JS. Normal cardiac ventricular geometry and function during the last trimester of pregnancy and early neonatal period. Br J Obstet Gynecol 1982; 89:839–844. 51. St. John Sutton MC, Raichlen JS, Reichek N, Huff DS. Quantitative assessment of right and left ventricular growth in the human fetal heart: a pathoanatomic study. Circ 1984; 70:935–941. 52. Heymann MA, Creasy RK, Rudolph AM. Quantitation of blood flow pattern in the foetal lamb in utero. In: Foetal and Neonatal Physiology, Proceedings of the Sir Joseph Barcroft Centenary Symposium. Cambridge: Cambridge University Press, 1973:129–135. 53. Pinson CW, Morton MJ, Thornburg KL. An anatomic basis for fetal right ventricular dominance and arterial pressure sensitivity. J Dev Physiol 1987; 9:253– 269. 54. Pinson CW, Morton MJ, Thornburg KL. Mild pressure loading alters right ventricular function in fetal sheep. Circ Res 1991; 68:947–957. 55. Romero T, Covell J, Friedman WF. Comparison of pressure-volume relations in the fetal, newborn and adult heart. Am J Physiol 1972; 222:1285–1290. 56. Thornburg KL, Morton MJ, Pinson CW, Reller MD, Reid DL. Anatomic and functional distinctions between the fetal heart ventricles. In: Lipshitz J, Maloney J, Nimrod C, Carson G. Perinatal Development of the Heart and Lung: Proceedings of the 1st International Christie Conference. Ithaca: Perinatology Press 1987:49–71. 57. Grossman W, Jones D, McLaurin LP. Wall stress and patterns of hypertrophy in the human left ventricle. J Clin Invest 1975; 56:56–64. 58. Fishman NH, Hof RB, Rudolph AM, Heymann MA. Models of congenital heart disease in fetal lambs. Circulation 1978; 58:345–364. 59. Clubb FJ, Bishop SP. Formation of binucleated myocardial cells in the neonatal rat: an index for growth hypertrophy. Lab Invest 1984; 50:571–577. 60. Mandarim-de-Lacerda CA, das Santos MB, Le Floch-prigent P, Narcy F. Stereology of the myocardium in human foetuses. Early Hum Dev 1997; 48(3):249– 259. 61. Giraud GD, Barbera A, Reller M, Morton M, Wu D, Thornburg KL. Right ventricular pressure load accelerates myocyte maturation in fetal sheep. J Soc Gynecol Invest 1995; 2:338. 62. Rudolph AM, Heymann MA. Control of the foetal circulation. In: Foetal and Neonatal Physiology. Cambridge: Cambridge University Press, 1973. 63. Cohn HE, Sachs EJ, Deymann MA, Rudolph AM. Cardiovascular response to hypoxemia and acidemia in fetal lambs. Am J Obstet Gynecol 1974; 120:817– 824.

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64. Thornburg KL. Fetal response to intrauterine stress [review]. In: Bock GF, Whelan J. Childhood Environment and Adult Disease. New York: Wiley (Ciba Foundation Symposium 156), 1991:17–37. 65. Longo LD, Wyatt JF, Hewitt CW, Gilbert RD. A comparison of circulatory responses to hypoxic hypoxia and carbon monoxide hypoxia in fetal blood flow and oxygenation. In: Longo LD, Reneau DD, eds. Fetal and Newborn Cardiovascular Physiology, Volume 2. New York: Garland STPM Press, 1978:259– 287. 66. Peeters LLH, Sheldon RE, Jones MD JR, Makowski EL, Meschia G. Blood flow to fetal organs as a function of arterial oxygen content. Am J Obstet Gynecol 1979; 135:637–646. 67. Itskovitz J, LaGamma F, Rudolph AM. Effects of hemorrhage on umbilical venous return and oxygen delivery in fetal lambs. Am J Physiol 1982: 242:H543–H548. 68. Itskovitz J, LaGamma F, Rudolph AM. Effects of cord compression on fetal blood flow distribution and Os delivery. Am J Physiol 1987; 252:H100–H109. 69. Bocking AD, Gagnon R, White SE, Homan J, Milne KM, Richardson BS. Circulatory responses to prolonged hypoxemia in fetal sheep. Am J Obstet Gynecol 1988; 159:1418–1424. 70. Murotsuki J, Challis JR, Han VK, Fraher LJ, Gagnon R. Chronic fetal placental embolization and hypoxemia cause hypertension and myocardial hypertrophy in fetal sheep. Am J Physiol 1997; 272:R201–207. 71. Vlahakes GJ, Turley K, Uhlig PN, Verrier ED, Hoffman JIE. Experimental model of congenital right ventricular hypertrophy created by pulmonary artery banding in utero. Surg Forum 1981; 32:233–236. 72. Vlahakes GJ, Turley K, Hoffman JIE. Increased myocardial vascularity in conscious lambs with right ventricular hypertrophy acquired early in life. Surg Forum 1983; 34:276–279. 73. Martin CA, Yu Y, Jiang BH, Davis L, Kimberly D, Hohimer AR, Semenza GI. Cardiac hypertrophy in chronically anemic fetal sheep: increased vascularization is associated with increased myocardial expression of vascular endothelial growth factor and hypoxia-inducible factor 1. Am J Obstet Gynecol 1998; 178: 527–534. 74. Davis LE, Hohimer AR, Morton MJ. Myocardial blood flow and coronary reserve in chronically anemic fetal lambs. Am J Physiol 1999; 277:R306–R313. 75. Archie JP, Fixler DE, Ullyot DJ, Buckberg GD, Hoffman JIE. Regional myocardial blood flow in lambs with concentric right ventricular hypertrophy. Circ Res 1974; 34:143–154. 76. Archie JP, Fixler DE, Hoffman JIE. Coronary reserve and right ventricular function in awake newborn lambs with persistent right ventricular hypertension. Pediatr Res 1977; 11:867–870. 77. Thornburg KL, Reller MD. Coronary flow regulation in the fetal heart ventricles. Am J Physiol 1999; 277:R1249–R1260.

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78. Heymann MA, Payne BD, Hoffman JIE, Rudolph AM. Blood flow measurements with radionuclide labeled microspheres. Prog Cardiovasc Dis 1977; 20: 55–77. 79. Fisher DJ, Heymann MA, Rudolph AM. Regional myocardial blood flow and oxygen delivery in fetal, newborn, and adult sheep. Am J Physiol 1982; 243: H729–H731. 80. Hoffman JIE. Maximal coronary flow and the concept of coronary vascular reserve. Circ 1984; 70:153–159. 81. Reller MD, Morton MJ, Giraud GD, Wu DE, Thornburg KL. Severe right ventricular pressure loading in fetal sheep augments global myocardial blood flow to submaximal levels. Circ 1992; 86:581–588. 82. Reller MD, Burson MA, Lohr JL, Morton MJ, Thornburg KL. Nitric oxide is an important determinant of coronary flow at rest and during hypoxemic stress in fetal lambs. Am J Physiol 1995; 269:H2074–H2081. 83. Reller MD, Morton MJ, Giraud GD, Wu DE, Thornburg KL. Maximal myocardial blood flow is enhanced by chronic hypoxemia in late gestation fetal sheep. Am J Physiol 1992; 263:H1327–H1329. 84. Tomanek RJ, Ratajska A, Kitten GT, Yue X, Sandra A. Vascular endothelial growth factor expression coincides with coronary vasculogenesis and angiogenesis. Dev Dyn 1999; 215:54–61. 85. Kamitomoto M, Alonso JG, Takashi O, Longo LD, Gilbert RD. Effects of longterm, high altitude hypoxemia on ovine fetal cardiac output and blood flow distribution. Am J Obstet Gynecol 1993; 169:701–707. 86. Kamitomoto M, Ohtsuka T, Gilbert RD. Effects of isoproterenol on the cardiovascular system of fetal sheep exposed to long-term high altitude hypoxemia. J Appl Physiol 1995; 78:173–179. 87. Woods LL. Neonatal uninephrectomy causes hypertension in adult rats. Am J Physiol 1999; 276:R974–978. 88. Woods LL, Rasch R. Perinatal ANG II programs adult blood pressure, glomerular number, and renal function in rats. Am J Physiol 1998; 275:R1593–1599. 89. Langley-Evans SC. Hypertension induced by foetal exposure to a maternal lowprotein diet, in the rat, is prevented by a pharmacological blockage of maternal glucocorticoid synthesis. J Hypertens 1997; 15(5):537–544. 90. Aguan K, Murotsuki J, Gagnon R, Thompson LP, Weiner CP. Effect of chronic hypoxemia on the regulation of nitric-oxide synthase in the fetal sheep brain. Dev Brain Res 1998; 111(2):271–277. 91. Bendeck MP, Keeley FW, Langille BL. Perinatal accumulation of arterial wall constituents: relation to hemodynamic changes at birth. Am J Physiol 1994; 267:H2268–H2279. 92. Martyn CN, Greenwald SE. Impaired synthesis of elastin in walls of aorta and large conduit arteries during early development as an initiating event in pathogenesis of systemic hypertension. Lancet 1997; 350:953–955. 93. Martyn CN, Barker DJ, Jesperson S, Greenwald S, Osmond C, Berry C. Growth

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in utero, adult blood pressure, and arterial compliance. Br Heart J 1995; 73:116– 121. 94. Engelmann GL, Campbell SE, Rakusan K. Immediate postnatal rat heart development modified by abdominal aortic banding: analysis of gene expression. Mol Cell Biochem 1996; 163–164:47–56. 95. Sharrow L, Tinker D, Davidson JM, Rucker RB. Accumulation and regulation of elastin in the rat uterus. Proc Soc Exp Biol Med 1989; 192:121–126. 96. Wells SM, Langille BL, Adamson SL. In vivo and in vitro mechanical properties of the sheep thoracic aorta in the perinatal period and adulthood. Am J Physiol 1998; 274:H1749–H1760.

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7 Non–Insulin-Dependent Diabetes and Obesity DAVID I. W. PHILLIPS University of Southampton and Southampton General Hospital Southampton, United Kingdom

Although non–insulin-dependent diabetes (NIDDM) is by far the most common form of diabetes, its causal origins are still poorly understood. Whereas there is general agreement that obesity is important for the development of the disease, it is also clear that obesity only leads to diabetes in susceptible individuals. Relatively little progress has been made in understanding the nature of this susceptibility. Observations that the disease clusters in families, along with the results of twin studies that show higher concordance rates for NIDDM in identical as compared with nonidentical twins, have suggested a genetic basis. This view is also encouraged by the recent identification of a number of genetic mutations that cause rare, inherited forms of diabetes. However, research over the past decade has suggested that developmental factors may be important determinants of the susceptibility to diabetes. This research had its origins in the observation that low birth weight is associated with a higher prevalence of glucose intolerance and NIDDM in adult life. 141

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Birth Weight and NIDDM

The epidemiological studies that pointed to the possible importance of low birth weight as a causal factor for NIDDM were based on studies of samples of men and women in middle and later life whose measurements at birth had been routinely recorded. These records came to light as a result of a systematic search of archives and record offices in Britain—a search that led to the discovery of three important collections of birth records in Hertfordshire, Preston, and Sheffield. From 1911 onwards, all babies born in the county of Hertfordshire, whether at home or in hospital, were weighed at birth and again at 1 year. These weights were recorded in ledgers. The health visitor records used during this period have survived and have been used to trace men and women born 60 years ago. In 1991, a group of 370 men aged 59 to 70 years who were born and were still living in East Hertfordshire agreed to attend a clinic for glucose tolerance tests (1). The percentage of men with impaired glucose tolerance (IGT) or NIDDM fell progressively with increasing birth weight from 40% in those who weighed at least 5.5 lb (2.5 kg) at birth to 14% in those who weighed 9.5 lb (4.3 kg) or more (Table 1). After allowing for the effect of adult obesity, men who were small babies were six times more likely to develop diabetes or impaired glucose tolerance than men who had been Table 1 Percentage of Men Aged 64 Years with Impaired Glucose Tolerance or Diabetes According to Birth Weight Percentage of men with 2hr glucose (mmol/L) of: Birth weight (lb)* ≥ 5.5 5.6–6.5 6.6–7.5 7.6–8.5 8.6–9.5 >9.5 All *

Number of men

7.8–11.0†

≥ 11.1‡

≥ 7.8

Odds ratio adjusted for body mass index (95% CI)

20 47 104 117 54 28 370

30 21 25 15 4 14 18

10 13 6 7 9 0 7

40 34 31 22 13 14 25

6.6 (1.5–28) 4.8 (1.3–17) 4.6 (1.4–16) 2.6 (0.8–8.9) 1.4 (0.3–5.6) 1.0 p value for trend <0.001

1 lb = 0.454 kg.

† Impaired glucose tolerance. ‡ Diabetes.

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143

large at birth. Similar trends were seen between the weight at 1 year of age and subsequent diabetes. A study carried out in a population of 50-year-old men and women born in a maternity hospital in Preston, Lancashire showed similar strong relationships between birth size and the prevalence of glucose intolerance (2). However, this study also had information on the gestational age of the baby estimated from the date of the mother’s last menstrual period. Analysis of these data showed that the association between low birth weight and glucose intolerance was independent of the baby’s gestational age, suggesting that low birth weight and impaired glucose metabolism are associated through reduced rates of fetal growth rather than prematurity. Studies in North America, Europe, and in the developing world have broadly confirmed these findings by showing that people who were term babies but small at birth are at greater risk of NIDDM or glucose intolerance in adult life (Table 2). Although the strength of the association is greater in some studies than others, so far no published studies have failed to find an association. A consistent feature of these studies is the continuous relationship between birth weight and glucose intolerance, suggesting that babies whose birth weights are within the normal range are affected as well as severely undersized babies. In a number of studies, babies had not only their birth weight recorded, but other measurements of body size at birth as well. Analysis of these measurement suggests that diabetes is not just related to low birth weight but also to unusual patterns of fetal growth. Two patterns of growth appear to be related to disease in adult life. Babies who are thin at birth or disproportionately short subsequently develop glucose intolerance or NIDDM (2–4). It has long been known that obesity, particularly abdominal obesity, is associated with an increased risk of diabetes. Analysis of the effects of obesity, assessed by the body mass index, shows that its diabetogenic effect adds to that of birth or infant weight (Table 3). The mean 2-hr glucose concentration in the Hertfordshire men ranged from 5.8 mmol/L in those who had been above the highest tertile of weight at 1 year but were at or below the lowest tertile of current body mass index (=25.4 kg/m2), to 7.7 mmol/L in men below the lowest tertile of weight at 1 year and above the highest tertile of current body mass index (>28.4 kg/m2) (1). Other studies, however, suggest that the effect of low birth weight interacts with that of adult obesity so that the diabetogenic effect of obesity is more marked among people who were of low birth weight than those of high birth weight (3). Several studies have shown a higher prevalence of diabetes in the offspring of women who had diabetes or glucose intolerance during pregnancy.

144

Table 2 Studies Showing the Association Between Low Birth Weight and Impaired Glucose Tolerance Reference

Birth year

Geographical location

Sex

Hales et al. (1) Fall et al. (49) Lithell et al. (3) Curhan et al. (50) Phipps et al. (2)

1920–1930 1923–1930 1920–1924 1911–1946 1935–1943

Hertfordshire, UK Hertfordshire, UK Uppsala, Sweden United States Preston, UK

Valdez et al. (7)

1949–1963

San Antonio, Texas

McCance et al. (5) Robinson et al. (51) Whincup et al. (54) Law et al. (52)

1940–1972 1966–1973 1979–1983 1984–1985

Phoenix, Arizona Southampton, UK UK schoolchildren Salisbury, UK

Yajnik et al. (53)

1987–1989

Pune, India

Male Female Male Male Male Female Male Female Male and female Male Male and female Male Female Male Female

*

n 370 297 1333 22,846 140 126 251 290 1179 42 1398 101 105 201 178

Age (years) 59–70 60–71 60 40–75 46–54 31–32 20–39 18–25* 10–11* 7* 4

Abbreviated (30-min) glucose tolerance test.

Phillips

NIDDM and Obesity

145

Table 3 Mean Plasma Glucose 2 hr after 75 g Oral Glucose Load in Men Aged 64 Years Weight at 1 year (lb)* Adult body mass index (kg/m2)

≤ 21.5

≤25.4 –28 >28 All

6.6 6.7 7.7 7.0

*

(45) (47) (39) (131)

–23.5 6.1 6.9 7.4 6.8

(39) (44) (43) (126)

>23.5 5.8 5.9 6.6 6.1

(36) (36) (41) (113)

All 6.2 6.5 7.2 6.6

(120) (127) (123) (370)

Numbers of men in parentheses.

As gestational diabetes is associated with high birth weight, a link between high birth weight and the subsequent development of glucose intolerance or diabetes would be expected. Therefore it is of interest that in the Pima Indians, who have a very high incidence of diabetes and among whom gestational diabetes is relatively common, the relationship of birth weight to diabetes was U shaped with highest diabetes prevalence in those with the lowest and highest birth weights (5). This was not observed in the Northern European studies, probably because there were relatively few diabetic pregnancies and their survival 60 or more years ago would have been poor. It is possible that the very few examples of IGT or NIDDM detected in adults in Hertfordshire who had high birth weight were the outcome of pregnancies complicated by hyperglycemia. In this case, the elimination of such pregnancies would greatly strengthen the underlying protective effect of normal fetal growth. II. Fetal Growth and Insulin Resistance NIDDM is the culmination of metabolic abnormalities that have taken several years to develop. Insulin resistance is an early metabolic defect that both precedes and predicts IGT and NIDDM. There is increasing evidence that insulin resistance plays an important role in the link between low birth weight and diabetes. Low birth weight is associated with a higher prevalence of the metabolic syndrome (the coexistence of raised blood pressure, glucose intolerance, and dyslipidemia), which in turn is known to be associated with insulin resistance. In the Hertfordshire study, 56 of the 407 men had the syndrome, defined as a 2 hr plasma glucose concentration of 7.8 mmol/L or more, a systolic blood pressure of 160 mmHg or more, or currently receiving antihypertensive

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Table 4 Prevalence of the Insulin-Resistance Syndrome* in Men Aged 64 Years, According to Birth Weight Birth weight (lb)† ≥5.5 –6.5 –7.5 –8.5 –9.5 >9.5 Total *

Total number of men

Percentage with syndrome X

Odds ratio adjusted for body mass index (95% CI)

20 54 114 123 64 32 407

30 19 17 12 6 6 14

18 (2.6–118) 8.4 (1.5–49) 8.5 (1.5–46) 4.9 (0.9–27) 2.2 (0.3–14) 1.0 p value for trend <0.001

Type 2 diabetes, hypertension, and hyperlipidemia.

† 1 lb = 454 g.

treatment and a serum triglyceride concentration equal to or above the median value of 1.4 mmol/L (6). The percentage fell progressively from 30% in those who had birth weights of 5.5 lb or less to 6% in those who weighed 9.5 lb or more (Table 4). The corresponding odds ratio, adjusted for Body Mass Index (BMI), was increased 18-fold in the group with the lowest birth weight. A similar relationship was shown in Preston, and the finding has also been confirmed in a study of 30-year-old Mexican-Americans and non-Hispanic white people in San Antonio, Texas (7). Because these studies suggested a link between reduced fetal growth and insulin resistance, a study was carried out to determine whether there was an association between body size at birth and insulin resistance in a sample of the men and women who took part in the Preston study. Insulin resistance was measured using a short insulin tolerance test in a sample of 81 normoglycemic and 22 glucose-intolerant subjects. The results suggested that men and women who were thin at birth as indicated by a low ponderal index were insulin resistant in adult life (8). The association was independent of current BMI. The relationship between reduced fetal growth and insulin resistance has now been confirmed by a variety of techniques, including the euglycemic clamp and the intravenous glucose tolerance test with minimal modeling (Table 5) (9–12). Together with the evidence from the relationship between birth weight and the prevalence of the metabolic syndrome, these findings suggest that insulin resistance may originate through impaired development in fetal life.

Reference

Geographical location

Sex

n

McKeigue et al. (9) Phillips et al. (8)

Uppsala, Sweden Preston, UK

Clausen et al. (11)

Copenhagen, Denmark

Flanagan et al. (12)

Adelaide, South Australia

Hofman et al. (10)

Auckland, New Zealand

Male Male Female Male Female Male Female Male Female

709 53 50 162 169 85 78 18 9

Age (years)

Technique

6 47–55

Euglycemic clamp Short insulin tolerance test

18–32

Intravenous glucose tolerance test

20–21

Intravenous glucose tolerance test

6–10

Intravenous glucose tolerance test

NIDDM and Obesity

Table 5 Studies Showing Association Between Birth Size and Insulin Resistance

147

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III. Fetal Growth and Insulin Secretion Impaired pancreatic β-cell function is also predictive of NIDDM. However, it is currently uncertain whether low birth weight is associated with a defect in insulin secretion as well as insulin resistance. There is a body of evidence based on studies in experimental animals and some human evidence suggesting that defective β-cell growth and function can result from undernutrition in early life. Thus, for example, rats exposed to a low-protein diet during pregnancy produce offspring with a marked defect in glucose-stimulated insulin secretion (13,14). In human populations, attempts to show the presence of a β-cell defect in people who were small at birth have been contradictory. Some studies show no relationship between birth weight and insulin secretion (3,15), suggest insulin secretion is reduced (16,51), while others suggest that it is increased (10,12). There is evidence from these studies, however, that in childhood and young adult life, people with low birth weight hypersecrete insulin to compensate for the increased level of insulin resistance, but manage to maintain normal glucose tolerance (10,12). In contrast, the studies carried out during later life suggest that the hyperinsulinemia after a glucose challenge is less marked in people who were small at birth. This together with the development of hyperglycemia suggests that although insulin secretion is maintained, it is inappropriately low for the level of insulin resistance and indicates the possibility of an underlying pancreatic defect associated with low birth weight (3,15). IV. Mechanisms Linking Reduced Fetal Growth with Insulin Resistance The processes that could explain the link between reduced fetal growth and glucose intolerance in adult life are not understood but are currently under intensive investigation. It could be argued that the observations have a genetic basis. It has long been known that insulin has a central role in fetal growth, ensuring that growth rates are commensurate with the nutrient supply. Experimental manipulations that reduce insulin secretion in utero also reduce fetal growth (17). Hence, any gene reducing insulin secretion would be expected to lower birth weight and predispose to diabetes in adult life. Recently it has shown that diabetes caused by the rare mutation of the pancreatic glucosesensing, glucokinase gene that leads to reduced insulin secretion is associated with 500 g birth weight reduction (18). Genetically-determined insulin resis-

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tance would also impair fetal growth. This phenomenon has been demonstrated in transgenic mice lacking key intermediates in the insulin receptor signalling mechanism (19). McCance et al. have taken this idea further by suggesting that genes that lead to insulin resistance would enable the survival of a fetus exposed to undernutrition in utero. In conditions of undernutrition, a genotype conferring insulin resistance would be selected for as it would increase survival among small babies who have a high perinatal mortality— ”The surviving small baby hypothesis” (5). Dungar has studied common allelic variation at the variable number of tandem repeats (VNTR) locus in the insulin-promoter region of the insulin gene. In a prospective study of 758 children born in Avon, the III/III genotype, which has a prevalence of about 9%, was found to be linked with a 200 g increase in birth weight and larger body size at birth (20). Other studies have previously shown associations between this genotype and insulin resistance or NIDDM. On the basis of this

Figure 1 Fetal undernutrition resulting from maternal undernutrition or reduced nutrient transfer to the fetus may result in long-term changes in physiology and metabolism that predispose to insulin resistance and diabetes.

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finding the investigators suggested that this gene, which increased birth weight, might enhance perinatal survival but at the price of increasing susceptibility to NIDDM. It is difficult, however, to see how this gene could explain the association of low birth weight with NIDDM. Indeed, a recent analysis suggested that the influence of this polymorphism was much smaller and operated separately from the stronger birth size effect in the Hertfordshire cohort (21). Although genes may contribute to the association between birth size and diabetes, several sources of evidence suggest that it is the maternal environment rather than genetic factors that is the dominant influence on birth size. The stronger influence of the mother is shown by half-sibling studies, which show a higher correlation between the birth weights of half-siblings who share the same mother (r = 0.58) than among half-siblings who share a father (r = 0.1) (22). In an analysis of the familial aggregation of birth weight, Penrose concluded that 62% of the variation in birth weight was attributable to the intrauterine environment, 20% resulted from maternal genes, and 18% from fetal genes (23). The importance of the maternal environment is also supported by animal cross-breeding experiments and by embryo transfer experiments; a fetus transferred to a larger uterus will attain a larger birth size (24). Recent important evidence that the diabetogenic effect of small birth size is not genetic has come from an analysis of the relationship between birth size and diabetes in the Danish Twin Registry. Poulsen et al. found 14 pairs of monozygous twins discordant for NIDDM. The twins with NIDDM had significantly lower birth weights compared with the nondiabetic twins (2571 vs. 2841 g, p < 0.0001). Because of the genetic identity of monozygous twins, this finding suggests that the association between low birth weight and diabetes is independent of genetic factors (see Figure 1) (25). A nongenetic explanation is also supported by animal experiments that show that poor nutrition may both impair growth during critical periods of fetal life and permanently affect the structure and physiology of a range of organs and tissues. For example, a reduced nutrient supply to the fetal guinea pig as a result of unilateral uterine artery ligation or a low protein maternal diet during gestation in rats causes lifelong elevation of blood pressure in the offspring (26,27). These represent examples of programming, whereby stimuli applied at a critical point in early development have lifelong effects. These experiments have led to the hypothesis that the association between reduced fetal growth and insulin resistance or glucose intolerance in adult life results from fetal undernutrition. This hypothesis is supported by a follow-up study of babies born in the Netherlands during the “Dutch Hunger Winter” of 1944.

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This study of men and women aged 50 years who were born around the time of the Dutch famine, shows that undernutrition at any stage of gestation is linked with reduced glucose tolerance and evidence of insulin resistance in the offspring (28). A wide variety of factors may cause undernourishment in the fetus. These include a poor maternal diet, poor nutritional reserves in the mother, inadequate uterine blood flow, or defects in the passage of nutrients across the placenta. Fetal undernutrition could cause insulin resistance by directly influencing the growth and differentiation of insulin-sensitive tissues such as liver or muscle. Recent experiments in a rat model have shown that undernutrition in utero can permanently alter the structure and function of the liver by altering hepatic zonation (29) (see Ch. 8). The activities of enzymes such as glucokinase and glutamine synthetase associated with the anabolic, perivenous zone of the liver are reduced, whereas catabolic, periportally situated enzymes such as phosphoenolpyruvate carboxykinase and carbamyl phosphate synthetase are increased. Because hepatic glucose production is periportal, the liver would appear to be biased towards the production rather than the consumption of glucose. However, detailed human studies of body composition and the structure of key insulin-sensitive tissues such as skeletal muscle have failed to explain the increased insulin resistance of subjects who were thin at birth, suggesting that the alterations in carbohydrate metabolism in low-birth-weight subjects are functional rather than structural (30). Nor is the insulin resistance explained by alterations in the vascular structure or blood flow of these tissues (31). There is, however, increasing evidence that early growth has its effects by permanently resetting major hormonal axes that control growth and metabolism. Because the placenta is impermeable to many hormones, especially the polypeptide hormones and growth factors, the fetus and placenta form a substantially autonomous unit in endocrine terms. In response to undernutrition, the fetus reduces insulin secretion and increases the levels of several hormones that modulate fetal and placental metabolism to increase fuel availability, redistribute blood flow, and alter the rate and pattern of fetal growth. Enhanced adrenal growth, together with elevated cortisol levels and an earlier prepartum rise in fetal plasma cortisol, are associated with increased levels of catecholamines and beta-endorphins. The levels of other hormones or growth factors, including thyroxine, insulin, and IGF-1, may fall (32). Many of these hormones have well-characterized programming or imprinting effects that occur during specific developmental windows and persist throughout life. Recent experimental studies suggest that the hypothalamic-pituitary-adrenal axis

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(HPAA) may play a particularly important role. In rats, fetal growth retardation induced by dexamethasone leads to permanently increased activity of the HPAA, with increased circulating concentrations of cortisol (33). In accordance with the known effects of glucocorticoids, the rats become hypertensive and glucose intolerant (34). Because programmed increases in HPAA activity in humans and consequent elevated circulating cortisol concentrations could contribute to the pathogenesis of the insulin-resistance syndrome and link the syndrome with low birth weight, we recently measured fasting cortisol levels in the 370 men who took part in the Hertfordshire study. Men of low birth weight had high plasma cortisol concentrations, the concentrations falling progressively from 408 nmol/L among men who weighed 5.5 lb or less at birth to 309 nmol/L among those who weighed 9.5 lb or more (35). Higher cortisol concentrations were related to higher systolic blood pressure and to higher fasting and 2-hr plasma glucose concentrations. We investigated a subset of the men and showed that those who were small at birth had greater adrenocortical responses to adrenocorticotropic hormone (ACTH), suggesting that the increased cortisol secretion was resulting from increased activity of the HPAA (36). These preliminary findings suggest that intrauterine programming of the HPAA may play an important part in mediating the association between low birth weight and reduced glucose tolerance in adult life. V.

Fetal Growth and Obesity in Adult Life

Although the effects of adult obesity appear to add to those of poor intrauterine growth in producing diabetes or glucose intolerance, there is increasing evidence that body weight or body fat distribution may be influenced by early growth. Several longitudinal studies have shown that anthropometric measurements in infancy or early childhood tend to predict body size in later life. This obviously implies that factors that influence body weight in the first few years of life may exert long-lasting effects. Although there are weak associations between birth weight and overall body mass index, there is evidence that the pattern of body fat distribution is also associated with birth size. People who had low birth weight tend to accumulate fat on the trunk and abdomen, a pattern of adiposity found in the insulin-resistance syndrome and associated with an increased risk of diabetes and cardiovascular disease. Elderly men in Hertfordshire who had low birth weight and low weight at 1 year had high ratios of waist to hip circumference, an index of central obesity (37). Among young adult Mexican and non-Hispanic Americans, low birth weight was not

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associated with abdominal fat deposition, as indicated by a high waist to hip ratio, but rather was associated with fat storage on the trunk, reflected in a high ratio of subscapular to triceps skinfolds (7). These investigators suggest that at younger ages, skinfolds may be better indicators of regional fat distribution than the waist to hip ratio. Similar associations between low birth weight and the ratio of the subscapular to triceps skinfolds have been found in teenagers in the United States and United Kingdom (38). VI. Early Nutrition and Adult Body Weight Evidence that early undernutrition may influence adult body weight also derives from animal experiments and human observations. In a colony of rats maintained on a low-protein diet for 10 generations, Stewart showed that if the rats were given adequate protein from the fourteenth day of gestation, the offspring became obese (39). A diet with adequate protein begun at birth or weaning had no effect on their likelihood of becoming obese. Later experiments tended to confirm these findings. Pregnant rats that were severely undernourished during the first 2 weeks of gestation but fed on a normal diet for the last week of gestation had offspring who became markedly obese (40). However, these and other studies have suggested that the effects of undernutrition may be sex-specific. Depending on the strain of rat and the diet used, it is either the male or the female offspring who become obese. The most widely known human example of the effect of maternal starvation on adult obesity are the studies of children born to women after the Dutch winter famine of 1944 to 1945. People born around the time of the famine were studied when they were 19 years of age (41). Those exposed to the famine during the first two trimesters of pregnancy had approximately double the prevalence of obesity, whereas those exposed during the third trimester had a 40% reduction in the rate of obesity. There are, however, some difficulties in interpreting the data because many mothers, presumably the thinner ones, would have been infertile during the famine. Furthermore the second half of gestation of the babies conceived during the famine would have occurred after liberation when food had become abundant. Nevertheless, these data, along with the animal experiments, suggest that nutritional deprivation during embryonic or early fetal life can result in increased body weight in adult life. In contrast, nutritional excess during later pregnancy or early infancy appears to be associated with higher obesity rates in later life. Evidence for an effect of prenatal overnutrition on adult body weight derives from studies

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linking exposure to hyperglycemia during pregnancy and the subsequent development of obesity. The offspring of rats made hyperglycemic in the last week of pregnancy are heavier at birth and during infancy (42). The human parallel is found in children born to diabetic mothers. Woman who are hyperglycemic in the last two trimesters of pregnancy have heavier babies who remain fatter through childhood, and in whom the tendency to fatness persists into adulthood (43). This effect remains after taking into account the mother’s size and her own birth weight. Because there was no obesity in the offspring of mothers who did not have diabetes during pregnancy but developed diabetes later or in subsequent pregnancies, the effect is not likely to be genetic but rather attributable to the metabolic environment during pregnancy. Animal experiments also suggest that overnutrition in early postnatal life is associated with subsequent development of obesity. The experiments by Aubert (1979) and Plagemann (1992) show that preweaned rat and mice pups overfed with milk had larger and more numerous adipocytes in adult life (44,45). Studies of primates support these findings, but again emphasize the importance of timing and gender specificity of the effects. Baboons overfed in infancy increase their body weight above control-fed animals as expected, but after weaning their body weight normalized. However, at adolescence, only the female baboons had increased body weight (46).

VII.

Mechanisms Linking Early Environmental Influences with Adult Body Weight

What then, are the mechanisms that would link events occurring in very early life with adult body weight? The quantity of adipose tissue reflects both the number and average volume of the component adipose cells. Brook has suggested that the basic complement of adipose cells is determined during a critical period from 30 weeks of gestation to the first year of life (47). When this period has ended, growth in adipose tissue mass depends on an increase in cell size rather than number. Studies of animals exposed to overnutrition during intrauterine and early postnatal life show that they have more numerous adipocytes. Thus it has been suggested that nutritional abundance in late gestation or infancy, eg, that occurring in the offspring of gestationally diabetic women, may increase adipocyte mass by means of an increase in cell numbers. Recent studies, however, suggest that the number of adipocytes in adult life is not fixed. Rather, fat cell acquisition seems to occur throughout life in a process

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involving the expansion and differentiation of preadipocytes (48). Moreover, there is increasing evidence that adipocytes can be lost by apoptosis. It is now known that adipose cell number is regulated by a number of hormones including insulin, corticosteroids, and cytokines. Furthermore, the recent data suggesting that the set point of many of these hormonal systems can be permanently changed or imprinted during the course of development provide a possible mechanism by which the effects of early growth could influence adult obesity (35). VIII.

Conclusions

There is now increasing evidence that the pathogenesis of NIDDM and disorders related to NIDDM begins in utero. It is suggested that these disorders arise as a result of fetal undernutrition. As a result of undernutrition, the fetus makes metabolic adaptations that benefit it in the short term by increasing fuel availability but become permanently programmed, persisting throughout life and causing insulin resistance (Fig. 1). Insulin resistance may thus be viewed as the price of a short-term successful adaptation to undernutrition in utero. An increasing body of evidence suggests that alterations in the setpoint of major hormonal axes controlling growth and development, particularly the hypothalamic-pituitary adrenal axis, may be important mechanisms. Although these early changes determine susceptibility, additional factors such as obesity, aging, and physical inactivity further increasing insulin resistance must also play a part in determining the time of onset and severity of NIDDM. These findings offer the prospect of new approaches to prevent NIDDM by optimizing maternal diet and improving health in early childhood. However, effective prevention will not be possible until we understand more about the mechanisms by which adverse influences early in life predispose to NIDDM.

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33. Levitt NS, Lindsay RS, Holmes GE, Seckl JR. Dexamethasone in the last week of pregnancy attenuates hippocampal glucocorticoid receptor gene expression and elevates blood pressure in the adult offspring of rats. Neuroendocrinol 1996; 64:412–418. 34. Lindsay RS, Lindsay RM, Waddell B, Seckl JR. Programming of glucose tolerance in the rat; role of placental 11 â-hydroxysteroid dehydrogenase. Diabetologia 1996; 39:1299–1305. 35. Phillips DIW, Barker DJP, Fall CHD, Seckl JR, Whorwood CB, Wood PJ, Walker BR. Elevated plasma cortisol concentrations; a link between low birth-weight and the insulin resistance syndrome? J Clin Endo Metab 1998; 83:757–760. 36. Reynolds RM, Bendall HE, Walker BR, Wood PJ, Phillips DIW, Whorwood CB. Hyperactivity of the hypothalamic-pituitary-adrenal axis may mediate the link between impaired fetal growth and the insulin resistance syndrome. J Endocrinol 1998; 159:OC5. 37. Law CM, Barker DJP, Osmond C, Fall CHD, Simmonds SJ. Early growth and abdominal fatness in adult life. J Epidemiol Community Health 1992; 46:184– 186. 38. Barker M, Robinson S, Osmond C, Barker DJP. Birth weight and body fat distribution in adolescent girls. Arch Dis Child 1997; 77:381–383. 39. Stewart RJ, Sheppard H, Preece R, Waterlow JC. The effect of rehabilitation at different stages of development of rats marginally malnourished for ten to twelve generations. Br J Nutr 1980; 43:403–412. 40. Jones AP, Friedman MI. Obesity and adipocyte abnormalities in offspring of rats undernourished during pregnancy. Science 1982; 215:1518–1519. 41. Ravelli G-P, Stein ZA, Susser MW. Obesity in young men after famine exposure in utero and early infancy. N Engl J Med 1976; 295:349–353. 42. Proietto J, Thorburn AW. Animal models of obesity—theories of aetiology. Ballière’s Clin Endocrin Metabol 1994; 8:509–513. 43. Pettitt DI, Aleck KA, Baird HR, Carraher MJ, Bennett PH, Knowler WC. Congenital susceptibility to NIDDM; role of intrauterine environment. Diabetes 1988; 37:622–628. 44. Aubert R, Suquet JP, Lemmonier D. Long-term morphological and metabolic effects of early under and over nutrition in mice. J Nutr 1979; 110:649–661. 45. Plagemann A, Heidrich I, Gotz F. Obesity and enhanced diabetes and cardiovascular risk in adult rats due to early postnatal overfeeding. Exp Clin Endocrinol 1992; 99:154–158. 46. Lewis DS, Bertrand HA, McMahon HC, McGill HC Jr, Carey KD, Mason EJ. Preweaning food intake influences the adiposity of young baboons. J Clin Invest 1986; 78:899–905. 47. Brook CG. Evidence for a sensitive period in adipose-cell replication in man. Lancet 1972; 2:624–627.

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48. Prins JB, O’Rahilly SP. Regulation of adipose cell number in man. Clin Science 1997; 92:3–11. 49. Fall CHD, Osmond C, Barker DJP, Clark PMS, Hales CN, Stirling Y, Meade TW. Fetal and infant growth and cardiovascular risk factors in women. Br Med J 1995; 310:428–431. 50. Curhan GC, Willett WC, Rimm EB, Spiegelman D, Ascherio AL, Stampfer MJ. Birth weight and adult hypertension, diabetes mellitus and obesity in US men. Circulation 1996; 94:3246–3250. 51. Robinson SM, Walton RJ, Clark PMS, Barker DJP, Hales CN, Osmond C. The relation of fetal growth to plasma glucose in young men. Diabetologia 1992; 35:444–446. 52. Law CM, Barker DJP, Hales CN, Shiell AW. Insulin resistance in 7-year old children who were thin at birth. Pediatr Res 1994; 35:263. 53. Yajnik CS, Fall CHD, Vaidya U, Pandit AN, Bavdekar A, Bhat DS, Osmond C, Hales CN, Barker DJP. Fetal growth and glucose and insulin metabolism in four-year-old Indian children. Diabetic Med 1995; 12:330–336. 54. Whincup PH, Cook DG, Adshead F, Taylor SJC, Walker M, Papacosta O, Alberti KGMM. Childhood size is more strongly related than size at birth to glucose and insulin levels in 6–11 year old children. Diabetologia 1997; 40:319–326.

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8 Metabolic Alterations After Early Growth Retardation

SUSAN OZANNE, B. J. JENNINGS, and C. NICHOLAS HALES University of Cambridge Cambridge, United Kingdom

I. Introduction Our epidemiological observations linking indices of poor early human growth to an increased risk of Type 2 diabetes and the insulin-resistance syndrome (reviewed in Ch. 7) led us to propose the “thrifty phenotype” hypothesis (1). We have subsequently revisited and reviewed the status of the hypothesis (2–4). This hypothesis states that poor fetal nutrition, as a consequence of poor maternal (and possibly, we now recognize, poor grandmaternal) nutrition, or an altered ability to deliver nutrients and hormones to the fetus for other reasons, has at least two important consequences for the fetus: 1. A selective redistribution of nutrients alters the relative growth rates of different organs according to priorities that (we now know) differ between male and female fetuses (5). 2. A permanent alteration of the metabolic setting of key tissues involved in glucose homeostasis in a direction that is beneficial to 161

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Ozanne et al. postnatal survival in conditions where access to nutrition is poor or intermittent. An important feature of the hypothesis is that although under these circumstances survival is enabled, the superimposition of obesity on such a metabolically adapted organism is highly detrimental. We propose that it is this conflict between the early programming of metabolism and the actuality of abundant nutrition that lead to Type 2 diabetes and the insulin-resistance syndrome.

In addition to testing this hypothesis in a variety of further epidemiological studies (and most directly in terms of the consequence of the Dutch Famine (6), we have undertaken a series of studies in experimental animals to define more precisely what the nature and mechanisms of the adaptations may be. It is the purpose of this chapter to review the progress that has been made with this research, summarize the present state of our understanding, and to suggest important new questions for further research.

II. Early Growth Retardation Consequent on Maternal Protein Deprivation In proposing the thrifty phenotype hypothesis, we reviewed the considerable literature going back many years showing that both in humans and animals protein restriction led to a loss of glucose tolerance and poor insulin secretion. Evidence was also forthcoming that such changes could be irreversible despite the removal of the restriction. We therefore drew attention to the critical role of the protein supply in fetal life in determining the development and function of β cells. However, in doing this it was not and still is not our intention to suggest that this is likely to be the only nutritional deprivation (or even imbalance) that might be of importance for the subsequent development of glucose intolerance. It remains, as will be discussed shortly, of major importance to evaluate systematically, alone and in combination, which nutritional deficiencies or imbalances are capable of inducing long-term metabolic consequences that may be detrimental to the health of the adult animal. Also, while emphasizing the importance of insulin production for both fetal growth and adult glucose homeostasis, we hypothesized that insulin resistance would be a long-term consequence of poor fetal nutrition and growth. In human epidemiological studies, it has been proven easier to show links between poor early growth and insulin resistance (7–10) than links between poor early growth and a reduction in insulin secretion (11), although some evidence has been found in studies of young adults (12,13).

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As a consequence of the substantial literature linking protein deficiency to glucose intolerance, we have adopted a protein-deficient model with which to test, in the first instance, the thrifty phenotype hypothesis. This model involves feeding pregnant and/or lactating rats a diet containing 8% protein. Offspring of such dams are growth retarded compared with offspring of rats fed a 20% protein diet. Although initially referring to this as a “low protein” model, we have recently begun to refer to it as “reduced protein.” The reasons for this are as follows: 1. The reduction to just under half the normal protein intake is relatively modest compared with some regimes studied by others. 2. There is no evidence from either reduced fertility or litter size that the diet is detrimental to reproductive performance or the health of the dam. 3. We do not know what the protein intake is of wild rats, and it may well be the case that the reduced-protein diet that we use would be “normal” for the wild animal and that laboratory diets in this context may be “high protein.” In our studies of this model, our prime focus has been on glucose homeostasis, but we have also monitored other key features of the insulin-resistance syndrome such as blood pressure and plasma lipids. A. Changes In Vivo

Longevity Maternal protein restriction in the rat has been shown to have striking effects on the longevity of male offspring. The exact time frame of the protein restriction appears to be critical in determining the nature of the effect on longevity. The offspring of rats who were fed a reduced-protein diet during pregnancy but who were subsequently cross-fostered to and nursed by dams being fed a control diet, died at a significantly younger age than control offspring. In contrast, control pups who were nursed by dams being fed a reduced-protein diet lived significantly longer than control offspring (14). In a subsequent study of male offspring (15) we have reproduced these findings in a more exaggerated form by increasing the contrast of the postnatal nutrition of these two groups of animals. “Catch-up” or recuperation of the former group was enhanced by culling the litter size to four, whereas the poor nutrition of the latter group previously culled to eight animals was further exacerbated by retaining the full litter (average number of pups: 13). Signifi-

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cant differences in longevity were already apparent by 13 months (when the experiment was terminated), and in groups of 10 to 12 animals compared with the 30 animals in the original experiment. There is now convincing evidence that the longevity of nontransformed cells grown in tissue culture in vitro is usually determined by the shortening of telomeres. These nucleoprotein structures at the ends of each chromosome, in the absence of the enzyme telomerase which can elongate telomeres, shorten at each cell division. When telomeres reach a critically short length (thought to be in the region of 1–4 Kb) cell cycling is arrested, the cells senesce and may then die (16). Although shortening of telomeres with age in vivo has been shown in humans (17) and in mice (18), this has not been studied in rats and there is no convincing evidence that telomere length may determine longevity in vivo. We decided therefore to determine whether rat telomeres shorten with age, whether they reach a critical degree of shortness around the time of death, and, if so, whether differences in the rate of shortening of telomeres in a critical organ might underlie the differences in longevity of the two groups of rats previously described. Telomere lengths at different ages were measured in the liver, kidney, and brain of control male rats (15). Significant shortening by the age of 13 months was observed in the liver and kidney but not the brain, such that approximately 50% of telomeres were in the smallest size range examined (<4.36 Kb). When telomere lengths were measured in the two groups of rats in which changes in longevity were observed, it was found that in the kidney, but not the liver, telomeres were significantly shorter in the animals with shorter life spans. Because male rats frequently die of kidney disease, these findings are consistent with the hypothesis that more rapid telomere shortening, leading to senescence of kidney cells and failure of this critical organ, is an important determinant of longevity in these studies. These findings also open up the possibility that a similar phenomenon in other critical cells, such as the β cells of the islets of Langerhans or the endothelium, may link changes in glucose tolerance or ischemic heart disease to poor early growth. Clearly much further research is needed to explore these possibilities. It is curious that a period of poor nutrition as short as 3 weeks, induced by suckling a protein-restricted rat dam, can lead to a lifelong restriction of growth. Detailed analysis of the rat dam’s food intake and weight gain during pregnancy (Fig. 1) as well as lactation (Fig. 2) revealed a somewhat complex pattern. Early in pregnancy, the reduced-protein dams ate significantly more, as previously reported (5). However, the reduced food intake of dams immediately before delivery occurred earlier in the reduced-protein than in the control

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Figure 1 (a) Daily food intakes and (b) body weights of (

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) control (n = 16 ) reduced-protein (n = 10 litters) mother rats fed the 20% protein litters) and ( (control) diet and the 8% protein (reduced-protein) diet, respectively, throughout pregnancy. Data are mean ± standard error of the mean.

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Figure 2 (a) Daily food intakes and (b) body weights of (

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) control (n = 16 litters) and ( ) reduced-protein (n = 10 litters) mother rats fed the 20% protein (control) diet and the 8% protein (reduced-protein) diet, respectively, throughout the suckling period. Data are mean ± standard error of the mean.

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dams. Reduction in weight gain during late pregnancy was also observed in the reduced-protein dams. After delivery, these dams initially ate normal quantities of food and maintained their body weight. However, after a week their food intake dropped and they lost weight. By the end of lactation their food intake had returned to normal and their weights had stabilized but were considerably reduced. It is reasonable to conclude that the nutrition available to the pups suckled by the reduced-protein dams was considerably reduced by virtue of the reduced protein in the diet and the reduced food consumption. This was further borne out by the poor weight gain of the pups (15). After weaning onto a normal diet fed ad lib the weight gain of the recuperated animals was very similar to that of controls whereas animals that had been suckled by reduced-protein dams remained at a consistently lower weight despite free access to a normal diet (Fig. 3). At 3 and 13 months of age, the body lengths of the postnatal low-protein offspring, both male and female, were significantly shorter than the controls. There was no difference in their body mass indices (kg/m2). Thus, poor nutrition during lactation had programmed a reduced growth trajectory. Examination of the food consumed by these animals showed it closely paralleled their gain in weight (Fig. 4). Thus a simple explanation of the programming of growth and weight gain is that it is secondary to a programming of appetite. However, with this data alone we cannot exclude the possibility that a programming of growth leads to an appropriate and secondary adjustment of appetite. Evidence that the former is more likely to be the explanation comes from studies of the effects of litter size during lactation on postweaning food consumption and weight gain. Increased or decreased food consumption during lactation by small- or large-sized litters, respectively, was shown to be followed by increased or decreased food intake after weaning, respectively (19). Further experiments indicated that body size is not determined by, but rather is a function of, voluntary food intake (20). In addition, Cohn and Joseph (1962) (21) reported that after a 3-month period of forced feeding, grossly overweight rats delayed ad libitim feeding or consumed food sparingly for as long as 2 to 3 weeks after treatment, thus providing evidence that voluntary food intake was not simply a function of body size. Therefore, the increased longevity of male rats suckled by rat dams fed a reduced-protein diet is attributable to a downward programming of appetite followed by a lifelong voluntary restriction of food intake. It has been repeatedly shown over many years that the imposition of reduced-calorie consumption increases longevity (22). The exciting difference in our experiments is that this effect can be achieved in the narrow window of the 3 weeks of lactation by

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Figure 3 (a) Male and (b) female growth curves of (

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) control, ( ) postnatal reduced-protein, and ( ) recuperated groups up to 56 weeks of age. Data are mean (no error bars are shown for clarity).

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Figure 4 (a) Male and (b) female food intakes of (

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) control, ( ) postnatal reduced-protein, and ( ) recuperated groups up to 10 weeks of age. Data are mean ± standard error of the mean.

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programming appetite at this critical time. It will be important to determine whether human appetite is also programmable, and, if so, when the critical period of development is when this occurs. The cause of reduced longevity of the recuperated male animals showing catch-up growth is less clear. One possibility is that it is attributable to excessive telomere shortening consequent on the increased number of cell divisions needed to achieve normal body dimensions starting from a reduced number of cells at birth. There is evidence from human epidemiological studies that an increased gain in weight (23) or height (24) from a reduced birth weight is detrimental in terms of coronary artery disease and hypertension, respectively. Glucose Tolerance and Insulin Sensitivity The glucose tolerance of offspring of normally fed dams showed an age-dependent deterioration of a fairly modest nature between the ages of 3 and 15 months (14). Offspring of reduced-protein fed dams exhibited an increased glucose tolerance at ages up to 3 months (14,25). At 1 year of age there was no difference in the glucose tolerance of these animals compared with controls (26). However, by the age of 15 months reduced-protein offspring showed a greater age-dependent loss of glucose tolerance such that they were then worse than controls (14). The mechanism(s) by which the glucose tolerance of the reduced-protein offspring switches from better to worse with age is not clear. Measurements of plasma-insulin concentrations at these different time points indicate that the reduced-protein offspring were more insulin sensitive than controls when young (27). The glucose intolerance of older animals seems to have a different basis depending on the sex of the animals; male rats appear more insulin resistant and female animals more insulin deficient than controls (14). There is some evidence that the age-dependent loss of glucose tolerance in the reduced-protein offspring may be accelerated by sucrose (28) or high-fat feeding (28,29) during adult life. Blood Pressure In agreement with the findings of others (30), we have observed that the blood pressure of offspring of reduced-protein fed dams is the result of an interaction between the early growth retardation resulting from this restriction and the postnatal diet. Most commonly in our model, we observe a reduction in blood pressure using our particular maternal and postweaning diet. However, in experiments in which we prolonged the reduced-protein exposure to the age of

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70 days and used a different laboratory chow or a highly palatable diet to induce obesity, hypertension resulted (26). In these experiments it was clear that both early growth retardation (produced by protein restriction) and obesity (produced by high-fat feeding) could independently elevate blood pressure. Furthermore, the combination of the two led to an additive effect such that animals that were growth retarded in early life and became obese in later life had very high blood pressures. The precise features of the early diet during pregnancy and the postweaning diet that lead to hypo- or hypertension are far from clear. One constituent that has been shown to be important is the fat content of the diet (31). It has been reported that feeding rats a diet rich in saturated fatty acids increases systolic blood pressure. In contrast, the same group have reported that a high intake of n-6 fatty acids and, in particular, linoleic acid lowers blood pressure (31). Further research is needed to clarify this point. The question of the interaction between pre- and postweaning nutrition is of particularly practical importance with regard to the prevention of human disease. From the results of work on animal models there is a clear indication that although the effects of early growth restriction are profound and permanent, they interact with the postweaning diet. There is, therefore, the real prospect that if we can unravel these interactions, possibilities will emerge for postnatal or even adult intervention strategies that will greatly ameliorate the deleterious effects of early growth retardation. Plasma Lipids Studies in our animal models have consistently shown that this type of early growth retardation has led to neither hypercholesterolemia nor hypertriglyceridemia. In fact, most of our data indicated an actual reduction or a tendency to a reduction in these parameters (32). Again, as with blood pressure, it is probable that the constituents of the postweaning diet are of great importance in determining the outcome. It will be necessary to determine what the susceptibility is of early–growth-retarded animals to the hyperlipidemic effects of weaning diets with a different quality or quantity of dietary fat. Acute Phase Response Our studies of the livers of the offspring of reduced-protein fed dams have indicated profound and permanent changes in this organ. A possible explanation of these changes is that it has a cellular basis such that functions located predominantly in the periportal zone of the liver are enhanced at the expense of those in the perivenous zone. There is considerable current interest in the

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role of inflammation and the inflammatory and acute phase response in the origins of atheroma and ischemic heart disease (33). The acute phase response is largely mediated through the enhanced production of proteins such as C-reactive protein and fibrinogen, which are both synthesized in the periportal zone of the liver. We therefore considered that if there was overactivity of the periportal zone of the livers of our reduced-protein offspring, they might have an enhanced acute phase response. To test this possibility, animals were injected with lipopolysaccharide, and the response of the major acute phase protein, α2 macroglobulin, in the rat was determined. Consistent with this proposal we found significant increases in the á2 macroglobulin response in both male and female rats (O’Connell DA, Fall CHD, Jennings G, Doherty C, Barker DJP, Hales CN. Unpublished data). These findings in turn led us to wonder whether the production of the major human acute phase protein, C-reactive protein, might be programmed by growth in early life. Sensitive measurements of this protein in plasma from people of mean age in Hertfordshire revealed that in men but not women birth weight was significantly negatively related to it. Thus it is an intriguing possibility that at least part of the process linking low birth weight to an increased risk of death from ischemic heart disease springs from an early alteration of hepatic differentiation, leading to an enhanced production of acute phase proteins. B. Changes In Vitro Pancreas Early studies on the fetuses of mothers who were fed an 8% protein diet during pregnancy showed that at 21.5 days gestation such fetuses had impaired structural development of the endocrine pancreas compared with that of control fetuses (34). The endocrine pancreas was less well vascularized and contained less insulin, islets were smaller, and β-cell proliferation was reduced in the 8% protein group. Further studies by this group showed that fetal islets from the reduced-protein group had impaired insulin secretion in response to amino acids such as arginine and leucine (35). More recently it has been shown that a defect in glucose-stimulated insulin secretion from islets of adult lowprotein offspring was only observed when an additional dietary insult such as high-fat or sucrose feeding is introduced postnatally (28). This provides more evidence that it is some kind of imbalance between fetal/neonatal and later nutrition that may lead to adult diseases such as Type 2 diabetes, and thus make nutritional intervention programs a realistic possibility. Indeed, recent studies

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by Cherif et al. (36) have shown that supplementing the mothers’ diet with taurine prevents the impaired insulin secretion normally observed in fetuses of low protein-fed dams. The precise time window available for such an effect of taurine requires further investigation. Liver The offspring of dams fed a reduced-protein diet during pregnancy and lactation have been shown to have large structural and functional changes in their liver (37). The liver lobule size is increased in the reduced-protein offspring. These livers also appear to be less responsive to the ability of glucagon to stimulate hepatic glucose output (38). This apparent glucagon resistance was related to a reduction in the expression of hepatic glucagon receptors. In contrast there was an increase in the expression of hepatic insulin receptors and a paradoxical response to the hormone in its effect on glucagon-stimulated hepatic glucose output. Addition of insulin initially stimulated the hepatic glucose output before inhibition (at a level comparable to controls) was seen. The mechanistic basis of this effect is not known. However, there is evidence that a similar paradoxical response to insulin occurs in individuals with Type 2 diabetes (39) and Aborigines (who have a high probability of developing Type 2 diabetes) (40). A number of studies have suggested that some people with Type 2 diabetes and malnutrition-related diabetes are relatively unable to produce the ketone body β hydroxybuhydrate (41,42). The precise molecular basis of this apparent ketosis resistance is unknown; however, it has been suggested that it may in part result from glucagon resistance, glucagon being a key regulator of ketone production by the liver (43). In light of the observed reduction in hepatic glucagon receptors in male reduced-protein offspring, more recent studies have assessed hydroxybuhyrate levels in these animals. These studies have shown that male reduced-protein offspring have lower plasma levels of the ketone body β-hydroxybutyrate when fed and after 24 and 48 hours of starvation (27). It is not clear if this results from increased utilization and/or decreased production of the ketone body. It is not a result of decreased availability of nonesterified fatty acids because plasma levels of these were increased when fed and tended to be higher after a 24-hour starvation in the reduced-protein group (27). Muscle In early adult life, reduced-protein offspring had an improved glucose tolerance compared with control offspring. This appears to be associated with

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increased insulin sensitivity because reduced-protein offspring have lower plasma-insulin concentrations both when fed and after a 24-hour fast. Consistent with these observations, experiments using isolated muscle strips from 3-month-old male reduced-protein and control offspring have shown that at submaximal concentrations of insulin, the hormone has a greater ability to stimulate methyl-glucose uptake, suggesting the tissue is more sensitive to insulin (44). Maximum insulin-stimulated methyl-glucose uptake was similar in both groups. In addition, it has been shown that under fasting conditions, the reduced-protein offspring had more GLUT 4 in their plasma membrane compared with controls. In contrast, after administration of insulin to attain supraphysiological concentrations of the hormone, GLUT 4 content in the plasma membrane was similar in the two groups. These observations are consistent with muscle from reduced-protein offspring being more sensitive to insulin at low concentrations of the hormone but having similar maximal responses. This increased insulin sensitivity appears at least in part to result from increased expression of insulin receptors. It was not related to any change in the fiber composition of the muscle (44). Adipose Tissue Epididymal adipocytes from 3-month-old reduced-protein offspring had persistently elevated basal rates of glucose uptake (45). In addition, insulinstimulated glucose uptake was also elevated. This was not related to any changes in total expression of GLUT 4. Similar to findings in liver and muscle, the reduced-protein adipocytes had increased insulin receptors. Further analyses revealed that adipocytes from reduced-protein offspring had increased basal and insulin-stimulated insulin receptor substrate (IRS)-1 associated phosphatidyl inositol (PI) 3-kinase activity. This enzyme is thought to play a key role in mediating many of the metabolic actions of insulin (46). Studies using the inhibitor wortmannin have shown that PI 3-kinase activation is required both for the antilipolytic action of insulin and the ability of the hormone to stimulate glucose uptake (47). However, despite having elevated PI 3-kinase activity, adipocytes from 3-month-old reduced-protein offspring were resistant to the antilipolytic action of insulin (48). The molecular basis of this discrepancy is not clear. It may be related to a difference in expression of isoforms of the catalytic subunit of PI 3-kinase (45). Expression of the p110 á isoform of the catalytic subunit of PI 3-kinase was similar in control and reduced-protein offspring adipocytes but there was a large reduction in the expression of the p110β isoform in the reduced-protein group (45).

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The implications of these findings are not yet clear. Indeed it is generally not understood why many proteins that are involved in intracellular cascades mediating hormonal control often exist in many isoforms. We have speculated (48) that one possible role of such isoforms is to provide spatial and/or metabolically specific signals in a single cell from a single agonist. The biological advantage of this otherwise apparently wasteful diversity is that it allows these routes of signalling to be subject to other regulatory inputs. In this particular example, we speculated that p110 α may transmit the insulin-stimulated glucose transport signal, whereas p110 β serves this function for the antilipolytic action of insulin. In an animal programmed to survive poor and intermittent nutrition, the subtlety of being able to independently modulate insulin’s control of glucose metabolism from that of antilipolysis could well have survival advantages. The consequences of our findings would be an organism that avidly stores glucose as fat but freely releases it in the form of nonesterified fatty acid as an alternative fuel to glucose oxidation (49). III. Conclusions These data relate to a major theme of the Thrifty Phenotype Hypothesis, which is that the changes mediated by maternal malnutrition on the offspring subserve the function of aiding the offspring’s survival postnatally. It appears that by mechanisms yet to be elucidated, the mother can communicate to the fetus the critical elements of the external environment to which the fetus is shortly to be exposed. By some means, fetal metabolism is then programmed in an appropriate direction. Clearly a maternal ability to adapt the fetus to the postnatal nutritional environment would have advantages in terms of survival. Theoretically this seems such an obviously biologically advantageous strategy that one is led to ask whether it could not be a more general principle of biological behavior. Another critical environmental characteristic in terms of postnatal survival is the ambient temperature. There is now a considerable body of evidence that indicates that the temperatures to which the mother is exposed has important consequences for the development of the fetus. An example is in the finding that exposing pregnant sheep to cold by early winter shearing changes fetal development and accelerates the generation of nonshivering thermogenesis (50). Thus, interpreted more broadly, it would seem that our own and other research opens up a more general and basically important physiological in-

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sight. This is that the environment experienced by the pregnant mammal is by mechanisms yet to be defined, communicated to the fetus in such a way that the latter is induced to develop in a way that maximizes the ability of the offspring to survive in the environment in question. It remains to be seen what factors other than nutrition and temperature impact on this process. One obvious candidate for investigation is maternal infection. In identifying this novel aspect of mammalian physiology, we also wish to add another component. This is the pathophysiological consequence of the postnatal environment “contradicting” the “instructions” that the developing fetus received. We propose that this is a fundamental process whereby fetal development and the adult environment collide with detrimental consequences for health and longevity. IV. Future Research Directions There is clearly a great deal of data collection to be undertaken before we have an overview of exactly what can happen in relation to “nutritionally”* altered early development and programming, and their interaction with the postnatal environment. In more specific terms, questions to be answered pertain to: 1. Phenotypic consequences of different types and timings of early nutritional deprivations. 2. Definition of markers that in early postnatal life identify these deprivations. 3. Elucidation of appropriate postnatal strategies that ameliorate the consequences of these adaptations. The realization of this research requires a large effort. However, in the hope of stimulating such an effort we would point out the following: 1. Very important “postgenomic” and practical strategies of improving health will emerge. 2. Fascinating insights into how the environment directs the genome will be discovered. ___________________________

“Nutritional” is used here to include factors other than material nutrition, eg, grandmaternal nutrition, problems of placenta, etc., and changes in maternal endocrine status.

*

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3. We shall accelerate the process of going from the understandings of molecules to the understanding of the whole organism and its longterm diseases. In case this optimistic evaluation of what might be achieved by addressing these issues is considered misplaced, we draw attention to the consequences of a “natural” experiment carried out this century on the island of Nauru in the Pacific Ocean. This island, population approximately 5000 with relatively little migration or racial admixture, has undergone a huge environmental transformation during the second half of this century. Having experienced poor nutrition up to the end of the Second World War, its economic basis was transformed by phosphate mining thereafter. As a consequence, what was described as an epidemic of diabetes was discovered in 1975/1976 such that 30 to 40% of the adult population had Type 2 diabetes. Of course, postwar prosperity brought better nutrition. Therefore, according to our view of the pathogenesis of Type 2 diabetes, one might expect the products of postwar pregnancies to exhibit the benefits of this. Entirely in keeping with this it was observed in 1987 that the prevalence of impaired glucose tolerance has decreased sharply from 21.1% in 1975/1976 to 8.7% in 1987" (51). Thus, although it is of undoubted intellectual and practical interest to define in great detail the nutritional and other environmental conditions relevant to optimizing health and longevity, it is probably not necessary to wait much longer before starting to do something about the problem in populations where clear and severe deficiencies exist. References 1. 2. 3.

4. 5. 6.

Hales CN, Barker DJP. Type 2 (non–insulin-dependent) diabetes mellitus: the thrifty phenotype hypothesis. Diabetologia 1992; 35:595–601. Hales CN, Desai M, Ozanne SE. The thrifty phenotype hypothesis: how does it look after 5 years? Diabetic Medicine 1997; 14:189–195. Hales CN. Fetal and infant growth and impaired glucose tolerance in adulthood: the “thrifty phenotype” hypothesis revisited. Acta Paediatr Suppl 1997; 422:73–77. Ozanne SE, Hales CN. The thrifty phenotype hypothesis. Diabetes Rev Internat 1998; 7:5–7. Desai M, Crowther NJ, Lucas A, Hales CN. Organ-selective growth in the offspring of protein-restricted mothers. Br J Nutrition 1996; 76:591–603. Ravelli ACJ, van der Meulen JHP, Michels RPJ, Osmond C, Barker DJP, Hales

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CN, Bleker OP. Glucose tolerance in adults after prenatal exposure to the Dutch famine. Lancet 1998; 351:173–177. 7. Barker DJP, Hales CN, Fall CHD, Osmond C, Phipps K, Clark PMS. Type 2 (non–insulin-dependent) diabetes mellitus, hypertension and hyperlipidaemia (syndrome X): relation to reduced fetal growth. Diabetologia 1993; 36:62–67. 8. Phillips DIW, Barker DJP, Hales CN, Hirst S, Osmond C. Thinness at birth and insulin resistance in adult life. Diabetologia 1994; 37:150–154. 9. Law CM, Gordon GS, Shiell AW, Barker DJP, Hales CN. Thinness at birth and glucose tolerance in seven-year-old children. Diab Med 1995; 12:24–29. 10. Yajnik CS, Fall CHD, Vaidya U, Pandit AN, Bavdekar A, Bhat DS, Osmond C, Hales CN, Barker DJP. Fetal growth and glucose and insulin metabolism in four year old Indian children. Diab Med 1995; 12:330–336. 11. Phillips DIW, Hirst S, Clark PMS, Hales CN, Osmond C. Fetal growth and insulin secretion in adult life. Diabetologia 1994; 37:592–596. 12. Robinson S, Walton RJ, Clark PM, Barker DJP, Hales CN, Osmond C. The relation of fetal growth to plasma glucose in young men. Diabetologia 1992; 35:444–446. 13. Wills J, Watson JM, Hales CN, Phillips DIW. The relation of fetal growth to insulin secretion in young men. Diab Med 1996; 13:773–774. 14. Hales CN, Desai M, Ozanne SE, Crowther NJ. Fishing in the stream of diabetes: from measuring insulin to the control of fetal organogenesis. Biochem Soc Trans 1996; 24:341–350. 15. Jennings BJ, Ozanne SE, Dorling MW, Hales CN. Early growth determines longevity in male rats and may be related to telomere shortening in the kidney. FEBS Letters 1999; 448(1):4–8. 16. Faragher RGA, Kipling D. How might replicative senescence contribute to human ageing? BioEssays 1998; 20:985–991. 17. Hastie ND, Dempster M, Dunlop MG, Thompson AM, Green DK, Allshire RC. Telomere reduction in human colorectal carcinoma and with ageing. Nature 1990; 346:866–868. 18. Coviello-McLaughlin GM, Prowse KR. Telomere length regulation during postnatal development and ageing in Mus spretus. Nucleic Acids Res 1997; 25:3051– 3058. 19. Oscai LB, McGarr JA. Evidence that the amount of food consumed in early life fixes appetite in the rat. Am J Physiol 1978; 235:R141–R144. 20. Oscai LB. Evidence that body size does not determine voluntary food intake in the rat. Am J Physiol (Endocrinol Metab 1) 1980; 238:E318–E321. 21. Cohn C, Joseph D. Influence of body weight and body fat on appetite of “normal” lean and obese rats. Yale J Biol Med 1992; 34:598–607. 22. Masoro EJ. The biological mechanism of aging: is it still an enigma? Age 1996; 19:141–145. 23. Eriksson JG, Forsen T, Tuomilehto J, Winter PD, Osmond C, Barker DJP. Catchup growth in childhood and death from coronary heart disease: longitudinal study. Br Med J 1999; 318:427–431.

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24. Leon DA, Koupilova I, Lithell HO, Bergland L, Mohsen R, Vagero D, Lithell U-B, McKeigue PM. Failure to realise growth potential in utero and adult obesity in relation to blood pressure in 50 year old Swedish men. Br Med J 1996; 312:401–406. 25. Shepherd PR, Crowther NJ, Desai M, Hales CN, Ozanne SE. Altered adipocyte properties in the offspring of protein malnourished rats. Br J Nutrition 1997; 78:121–129. 26. Petry CJ, Ozanne SE, Wang CL, Hales CN. Early protein restriction and obesity independently induce hypertension in year old rats. Clin Science 1997; 93:147– 152. 27. Ozanne SE, Wang CL, Petry CJ, Smith JM, Hales CN. Ketosis resistance in the male offspring of protein malnourished rat dams. Metabolism 1998; 47(12):1450– 1454. 28. Wilson MR, Hughes SJ. The effect of maternal protein deficiency during pregnancy and lactation on glucose tolerance and pancreatic islet function in adult rat offspring. J Endocrinol 1997; 154:(1)177–185. 29. Holness MJ. The influence of sub-optimal protein nutrition on insulin hypersecretion evoked by high-energy/high-fat feeding in rats. FEBS Lett 1996; 392(1):53–56. 30. Langley-Evans SC, Gardner DS, Welham SJM. Intrauterine programming of cardiovascular disease by maternal nutritional status. Nutrition 1998; 14:39– 47. 31. Langley-Evans SC, Clamp AG, Grimble RE, Jackson AA. Influence of dietary fats upon systolic blood pressure in the rat. Internatl J Food Sci Nutr 1996; 47:417–425. 32. Lucas A, Baker BA, Desai M, Hales CN. Nutrition in pregnant or lactating rats programs lipid metabolism in the offspring. B J Nutrition 1996; 76:605–612. 33. Liuzzo G, Biasucci LM, Gallimore JR, Grillo RL, Rebuzzi AG, Pepys MB, Maseri A. The prognostic value of C-reactive protein and serum amyloid A protein in severe unstable angina. N Engl J Med 1994; 331:417–424. 34. Snoeck A, Remacle C, Reusens B, Hoet JJ. Effect of a low protein diet during pregnancy on the fetal rat endocrine pancreas. Biol Neonate 1990; 57:107–118. 35. Dahri SA, Snoeck B, Reusens-Bill B, Remacle C, Hoet JJ. Islet function in offspring of mothers on low-protein diet during gestation. Diabetes 1991; 40:115– 120. 36. Chierif H, Reusens B, Ahn MT, Hoet JI, Remacle C. Effects of taurine on the insulin secretion of rat fetal islets from dams fed a low-protein diet. J Endocrinol 1998; 159(2):341–348. 37. Burns SP, Desai M, Cohen RD, Hales CN, Iles RA, Germain JP, Going TCH, Bailey RA. Gluconeogenesis, glucose handling, and structural changes in livers of the adult offspring of rats partially deprived of protein during pregnancy and lactation. J Clin Invest 1997; 100:1768–1774. 38. Ozanne SE, Smith GD, Tikerpae J, Hales CN. Altered regulation of hepatic glucose output in the male offspring of protein-malnourished rat dams. Am J Physiol 1996; 270(33):E559–E564.

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9 Growth, Metabolic, and Endocrine Adaptations to Fetal Undernutrition JANE E. HARDING and PETER D. GLUCKMAN University of Auckland Auckland, New Zealand

I. Introduction There are multiple causes of impaired fetal growth in utero. Some—such as infective, toxic, and chromosomal disorders—may have their major effects in the embryonic phase of development before completion of organogenesis. However, the majority are nutritional in origin, that is, fetal growth is limited by nutrient supply, although it is important to note that this does not necessarily imply maternal undernutrition. Under circumstances of fetal undernutrition, it is useful to think of impaired growth, particularly in late gestation, as simply a fetal adaptation to inadequate nutrient supply, and to consider some of the other adaptations that occur in parallel with this change in growth. Much of our recent laboratory work has focused on the metabolic and endocrine adaptations that are associated with limited fetal nutrient supply and impaired fetal growth. In this chapter we will discuss how such adaptations occur in utero, and how the long-term legacy of these intrauterine adaptations may result in altered structure and function in postnatal life. 181

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It has frequently been shown in a variety of animal species that maternal undernutrition during pregnancy is associated with reduced size of the offspring at birth. Similarly, in chronically catheterized fetal sheep, it is possible to show that fetal growth usually slows abruptly within a few days of the onset of severe maternal undernutrition, and resumes upon maternal refeeding (1–4). However, it is widely believed that, except under famine conditions, maternal nutrition has little influence on the growth of the human fetus. This is largely because studies of maternal diet during pregnancy have shown little correlation with birth weight of the offspring, and because maternal dietary supplements during pregnancy have had very little effect on birth weight unless the mother was severely undernourished before pregnancy (5,6). Nevertheless, it is reasonable to assert that the relationship between fetal nutrition and fetal growth is maintained in human pregnancy, provided it is remembered that fetal nutrition is not equivalent to maternal nutrition. Fetal nutrient supply is dependent on a series of physiological functions, including uterine blood flow, placental transport capacity and metabolism, and fetal nutrient uptake. In normal pregnancy all of these functions have considerable reserve capacity, so that even quite major changes in maternal nutritional uptake may not influence fetal nutrient supply. Conversely, minimal change in maternal nutritional uptake may be associated with critical changes in fetal nutrient supply if placental nutrient delivery or transport are affected. This is perhaps most obvious in discrepant growth in twins, where fetal nutrition has clearly been unequal between twins although maternal nutrition was identical. Fetal nutrient supply is allocated by the fetus to a variety of activities, including movement, growth, and “basal” metabolic maintenance. The relative size of these allocations depends on the species and gestational age, but in late gestation in most mammalian species growth consumes a large proportion of available nutrients (Table 1). The fetus faced with inadequate nutrient Table 1 Allocation of Oxidative Substrates to Different Activities in the Late Gestation Mammalian Fetus Basal metabolism Movement Growth Source: From Ref. 63.

20–60% 15–30% 25–50%

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supply in late gestation, therefore, has limited options. Assuming that “basal” metabolic rate is essentially fixed, then reductions of movement and growth are the only mechanisms available to the fetus to reduce substrate demand in the face of inadequate substrate supply. The fetus can and does stop moving in response to acute substrate deprivation, and indeed this is used as a test of fetal well-being in clinical practice. However, this is not a good long-term strategy for the fetus. Limited fetal breathing movements are associated with impaired lung growth and pulmonary hypoplasia in utero, whereas limited limb movements are associated with impaired limb growth and limb deformities. Thus, if fetal movements are inhibited for more than a relatively brief period of time, function may be compromised. This imperative for the fetus to resume movement promptly to avoid functional compromise is perhaps reflected in the observation that, if chronic hypoxemia is imposed on fetal sheep, fetal breathing movements are inhibited for only a relatively brief period of time and return to normal within 24 hours despite continued hypoxemia (7,8). For the fetus faced with chronically reduced nutrient supply, the most obvious alternative adaptation is to reduce growth. In this way, metabolic demand is reduced and the fetus may be able to withstand its intrauterine limitations long enough to mature and survive into extrauterine life. Thus, limited fetal growth can be thought of as an appropriate fetal adaptation to limited intrauterine nutrient supply, rather than the end result of fetal undernutrition. Such adaptation may result in disproportionate changes in the growth of different fetal tissues and organs, depending on factors such as the time in gestation at which nutrient limitation occurs relative to the growth of that organ, the nature of the nutrient limitation, and the other adaptations that the fetus is simultaneously required to make. For example, limitation of fetal nutrient supply at the end of gestation in the human fetus is unlikely to alter neurogenesis, which is largely complete, but may markedly limit growth of adipocytes, which is proceeding rapidly at this time. Limitation of fetal oxygen supply may require fetal cardiovascular adaptations (see Section V), which include redistribution of cardiac output to favor blood flow to the brain and heart at the expense of peripheral tissues, resulting in altered growth of these organs. Similarly, limitation of fetal glucose supply may result in preferential growth of organs that can use alternative substrates, such as lactate, which is taken up by the fetal heart and liver (9,10). It may also result in endocrine adaptations (see Section IV), which include decreased circulating fetal insulin and insulin-like growth factor 1 (IGF-1) concentrations, thus resulting in preferential growth of organs, such as the brain, that do not require insulin for glucose uptake.

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The major metabolic substrates for the late gestation mammalian fetus are glucose, lactate, and amino acids, although the proportion of these substrates required for oxidative metabolism varies between species and over time in late gestation. Many other micronutrients are obviously also essential for fetal growth, and in some species lipids may also be fetal macronutrients, although these will not be discussed further in this chapter. We have studied fetoplacental metabolic adaptations to acute severe maternal undernutrition in late-gestation fetal sheep. This provides an acute reduction in the delivery of glucose and amino acids to the placenta, with no limitation of oxygen supply. Over the first 24 to 48 hours of maternal undernutrition, uterine glucose delivery is approximately halved, as is fetal glucose uptake. Fetal amino acid uptake is also approximately halved, although at the same time fetal amino acid oxygenation is doubled. Thus, the fetus becomes catabolic, burning endogenous amino acid substrates to replace the deficit in

Figure 1 Changes in amino acid metabolism in fetal sheep exposed to acute maternal undernutrition. Fetal amino acid oxidation (open bars) increases over the first day of undernutrition. However, fetal amino acid uptake from the placenta (solid bars) becomes negative, indicating that the fetus has become catabolic and is losing amino acids back to the placenta. Values are mean ± SE for 28 fetuses.

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delivery from the placenta (Fig. 1). Fetal wasting has also been shown by ultrasound in the late-gestation human fetus (11). Clearly, such an adaptation can only be a short-term solution, because catabolism cannot persist if the fetus is to survive. Nevertheless, over a period of 10 days of undernutrition in sheep, fetal amino acid oxidation replaces all of the deficit in glucose oxidation. Thus, by altering the proportions of different substrates consumed, the fetus is able to maintain its metabolic activities in the face of severely limited nutrient supply (Table 2). The placenta may also alter the proportions of different substrates it consumes. During 10 days of maternal undernutrition, placental glucose uptake is halved, whereas lactate production persists. The resulting deficit in metabolic substrates is made up in the sheep by placental consumption of ketones from the mother. This remarkable adaptation means that approximately 40% of placental oxidative metabolism could be accounted for by ketone uptake during undernutrition in sheep (12). Because human fetal tissues have also been shown to have the capacity to metabolize ketones (13), and ketones and lipids cross the human placenta much more readily than they do in sheep, this raises the interesting possibility that the human fetus may substitute lipid substrates for glucose supply during fetal nutrient limitation. A further fetoplacental metabolic adaptation to limited nutrient supply is to redistribute substrates between fetus and placenta. Under normal circumstances, the sheep placenta consumes more than half of the glucose and oxygen but only a very small proportion of the amino acids taken up from the uterine circulation. When fetal nutrient supply is limited by limitation of placental implantation sites (carunclectomy), placental glucose and oxygen consumption are reduced to less than one third of uterine uptake, so that a larger proportion of these substrates is available to the fetus (14). On the other hand, the Table 2 Fetoplacental Adaptations to Limitations in Nutrient Supply Short term Reduced demand (reduced movement) Catabolism Long term Reduced demand (reduced growth) Altered metabolic patterns Use of alternative substrates, eg, ketones Redistribution of substrates between fetus and placenta

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placenta may also consume substrates from the fetal circulation to maintain its own metabolic requirements. When the supply of glucose to the placenta from uterine circulation is reduced in sheep, the placenta takes up an increasing proportion of glucose from the fetal circulation (15). The placenta has also been shown to take up large amounts of amino acids from the fetal circulation when fetoplacental substrate supply is limited in the carunclectomy studies (14). IV. Endocrine Adaptations to Fetal Undernutrition The major endocrine axes influencing fetal growth in late gestation appear to be the insulin/insulin-like growth factor (IGF)/growth hormone (GH) axis and the hypothalamic/pituitary/adrenal (HPA) axis. In some species, the fetal thyroid axis may also be important. Because these axes are markedly influenced by nutrition in utero, it seems likely that the slowing of fetal growth in response to fetal undernutrition is mediated via changes in these endocrine axes. Insulin levels are regulated by circulating glucose and amino acid levels in the late-gestation fetus, as they are in postnatal life (16). Fetal insulin deficiencies are associated with impaired growth, whereas replacement of insulin appears to influence fetal growth largely by mediating nutrient uptake into fetal tissues (17,18). Thus, in the face of impaired fetal nutrient supply, a fall in insulin concentrations will result in reduced fetal nutrient uptake and reduced fetal growth. IGF-1 appears to be an important regulator of fetal growth in late gestation. Gene deletion experiments in mice have shown that IGF-1 is essential for late-gestation fetal growth (19,20), and deletions of the IGF-1 gene are also associated with extremely small size at birth in humans (21). Circulating IGF1 levels in late gestation are regulated by fetal glucose and insulin levels (22,23), and thus once again reduced fetal nutrient supply will be associated with a fall in glucose and insulin levels, resulting in a fall in fetal IGF-1 levels and reduced fetal growth. In addition, the balance of circulating IGF-1 levels in mother and fetus may regulate the distribution of substrates between mother, placenta, and fetus. In sheep, acute infusion of IGF-1 to the mother increases placental substrate uptake from the uterine circulation and placental lactate production (24), whereas IGF-1 infusion to the fetus increases fetal substrate uptake from the placenta and decreases placental lactate production (25). Thus, altered glucose and insulin levels in mother or fetus are, by altering circulating IGF-1 levels, likely to alter the nature of the substrates available and their

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distribution between fetus and placenta, and hence contribute to the fetoplacental metabolic adaptations to nutrient limitation previously described. Fetal GH concentrations are higher than those observed in postnatal life, although the rise in GH concentrations in response to undernutrition in utero is similar to that observed postnatally (26). However, GH appears to have only a small role in the regulation of late-gestation linear growth under normal circumstances (27). GH receptors are present in many fetal tissues but receptor concentration in the fetal liver is much lower than postnatally, and this may explain why GH has little effect on fetal IGF-1 concentrations in utero (28,29). However, GH may play an important role in substrate distribution within the fetus, particularly fat deposition, which is likely to be altered with impaired fetal nutrition (30). Impaired nutrition is associated with activation of the HPA axis and a rise in circulating cortisol levels in a number of experimental circumstances, and infusion of cortisol inhibits growth of the late-gestation fetal sheep (31). Acute undernutrition in pregnant sheep results in approximate doubling in maternal cortisol concentrations with little change in those of the fetus (32). However, much circulating fetal cortisol is of maternal origin at least over a limited period in late gestation, and mimicking the rise in maternal cortisol concentrations by low-dose maternal cortisol infusion also impairs growth in fetal sheep (33). Thyroid hormones appear to be important regulators of fetal oxidative metabolism, and thus reduced fetal thyroid hormone levels may result in reduced fetal oxygen uptake (34). However, their role in regulating fetal growth and the relative contribution of maternal thyroid hormone to fetal levels varies between species. Undernutrition is associated with reduced thyroid activity in postnatal life that is reversible on refeeding (35), and this may also be true in the fetus because circulating fetal thyroid hormone levels are reduced in both animal and human fetuses that are growing poorly (36,37). Thus, a variety of fetal endocrine responses to impaired nutrient supply may all be part of the adaptive mechanisms whereby fetal growth is reduced in order to reduce substrate demand in the face of limited substrate supply. V.

Cardiovascular Adaptations to Fetal Undernutrition

Fetal cardiovascular responses to impaired nutrient delivery will depend to a large extent on the degree and severity of the associated reduction in oxygen supply. Fetal hypoxemia induces a variety of cardiovascular responses.

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including elevated blood pressure and redistribution of cardiac output in favor of the vital organs. Even in the absence of hypoxemia, reduced substrate delivery may induce cardiovascular responses. Maternal undernutrition in late-gestation sheep is associated with a failure of the normal gestation-associated increase in uterine blood flow, and a similar failure of the normal rise in umbilical blood flow. The mechanisms whereby these changes occur are not clear. However, they do raise the possibility that the cardiovascular changes may themselves lead to reduced fetal availability of a variety of micronutrients, and thus contribute to limitation in fetal growth. The endocrine changes induced by undernutrition may also contribute to cardiovascular alterations. Elevated maternal cortisol concentrations are associated with fetal hypertension and marked myocardial hypertrophy in fetal sheep (33), whereas insulin, IGF, and growth hormone all have important cardiovascular actions that may be altered in the face of impaired nutrient supply. VI. Factors Influencing Fetal Adaptation to Undernutrition A. Timing of the Insult

Although the fetal adaptations to impaired nutrient supply in late gestation may be relatively easily understood, they appear in turn to be influenced by events that occur around the time of conception. We have found that when faced with acute severe maternal undernutrition for 10 days in late gestation, most fetal sheep show an abrupt slowing of growth. However, some continue to grow in the face of this insult (1). These fetuses appear to be growing slowly before the beginning of the insult, and have increased placental capacity for simple and facilitated diffusion. We hypothesized that this slow growth and altered placental function may represent adaptations made to a nutritional insult earlier in gestation, and that these adaptations in turn altered the response to a late gestational insult. We tested this hypothesis by exposing animals to moderate undernutrition for a period of 60 days before to 30 days after conception. This periconceptual undernutrition resulted in fetuses that in late gestation were growing slowly and were able to continue to grow in the face of a late gestational insult (2). Thus, the fetal adaptation to a given insult in late gestation may depend in part on adaptations made earlier in pregnancy. These may include changes in placental size and function. Undernutrition in the first part of pregnancy in rats alters placental glucose transporter activity (GLUT 1) at the end of pregnancy (38). Changes in nutrition in early gestation are

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also associated with increased placental size in a number of experimental paradigms (39) and potentially, therefore, with altered fetal nutrient supply in the face of a given nutritional insult. Periconceptual undernutrition may also result in altered “tempo” of development in late gestation. The fetal sheep that have been exposed to periconceptual undernutrition, and subsequently grow slowly in late gestation, appear to show catch-up growth after a late nutritional insult and do not show the normal slowing of fetal growth shortly before delivery (2). This raises the possibility that one fetal adaptation to undernutrition is an altered tempo of maturation, which may affect a variety of organ systems and have immediate survival advantages as well as long-term consequences. B. Balance of Nutrients

Little experimental work has yet been performed on the influence of the balance of different nutrients on fetal growth and adaptations to limited supply. It seems likely from human studies that the balance of protein and carbohydrate, at least, is important. High-protein dietary supplements may be associated with impaired fetal growth, particularly in mothers who are well nourished (5,6). There also appears to be an interaction between the carbohydrate intake of women in early pregnancy and their protein intake in late pregnancy in determining the size of offspring (40). In rats, it appears that the proportion of protein in the diet determines relative growth of fetus, placenta, and different fetal organs in late gestation (41). The type and concentration of lipid in the diet may also be of critical importance. C. Experience of Previous Generations Fetal growth in response to a given nutritional environment may be influenced by the prenatal nutritional experience of the mother. In humans, this is reflected by the marked influence, even in well-nourished populations, of maternal birth weight on the size of offspring at birth (42). Rats that are protein undernourished for multiple generations do not resume normal postnatal growth on normal feeding (43). In this experiment, refeeding of the mothers during pregnancy resulted in offspring that were relatively obese, and three generations were required before adult size of the offspring returned to that of controls. Similar influence of previous generations on offspring size is seen in the results of studies of humans exposed to famine during the Dutch hunger winter (44,45). These intergenerational phenomena are likely to be mediated at least in part by the nutritional environment provided by the mother at the

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time of conception and early embryogenesis. However, once again these mechanisms have yet to be investigated. D. Prematurity

One question of considerable clinical importance concerns the differences, if any, between the adaptations to nutritional limitation made by a baby in the extrauterine, as opposed to the intrauterine, environment. Limited studies of preterm babies have suggested that size at birth and early nutrition have little impact on later blood pressure, in contrast to the situation in term babies (46,47). This raises the interesting possibility that the adaptations to limited nutrition that are thought to underlie these associations are unique to the intrauterine environment. Fetoplacental cardiovascular, metabolic, and endocrine interactions all obviously cannot occur ex utero. Whether premature delivery, by removing the fetus from such intrauterine adaptations, will prevent some of the long-term consequences remains to be explored. VII. Fetoplacental Adaptations to Refeeding A. Metabolic Adaptations

It is our primary thesis that the metabolic and endocrine adaptations made by the fetus to undernutrition in utero are critical to survival but may not be entirely reversible. If this is the case, then some evidence of the long-term consequences of these adaptations should be seen after the nutritional insult is removed. There is now some experimental evidence that this is indeed the case. Fetal undernutrition is associated with altered carbohydrate metabolism, both prenatally and postnatally. In late gestation, as in postnatal life, acute undernutrition results in upregulation of gluconeogenic enzymes in liver and kidney, presumably in an attempt to maintain glucose supply (48). However, when late-gestation fetal sheep are undernourished for 10 days and then refed for 10 days, activities of gluconeogenic enzymes in fetal liver and kidney are markedly suppressed. This may be because when substrate supply is restored, tissue growth is restored at the expense of maturation of enzyme function. There is other evidence that fetal undernutrition is associated with long-term disturbance of carbohydrate metabolism. Offspring of rats who experience protein undernutrition in pregnancy have normal regulation of their glycolytic and gluconeogenic enzymes in response to subsequent obesity, but the regulation is around new set points (49). There is also evidence of altered glycolysis

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in adult humans who were small at birth (50). Mechanisms by which such changes occur may include intrauterine endocrine changes, because cortisol exposure in fetal sheep induces early expression of gluconeogenic enzymes (51) and premature onset of gluconeogenesis (52). Altered clonal selection of hepatocytes may also result in altered hepatic function postnatally (53). There is also evidence of altered protein metabolism in postnatal life after intrauterine undernutrition. In our fetal sheep studies, fetal undernutrition results in increased fetal amino acid oxidation (see Section III), but during the refeeding period the fetal amino acid oxidation is markedly suppressed. It is not clear if this is an adaptive response to refeeding, allowing catch-up growth to occur, or a maladaptive response with impaired protein turnover. However, a possibly parallel situation is observed in human infants who were small at birth, and in whom normal protein gain during the postnatal growth phase is achieved as a balance between reduced protein synthesis and reduced protein breakdown (54). VIII.

Cardiovascular Adaptations to Refeeding

The cardiovascular adaptations to fetal undernutrition may be of relatively little significance until the undernutrition is reversed. In fetal sheep, fetal blood pressure changes little in response to acute maternal undernutrition, but is elevated on maternal refeeding (55). Similarly, in rats the offspring of undernourished mothers develop elevated blood pressure in postnatal life (56,57), and this may be markedly aggravated by a high-fat diet. These observations raise the issue of the role of postnatal catch-up growth in the development of disease risk after impaired fetal growth.

IX. Endocrine Adaptations to Refeeding As previously described, fetal undernutrition is associated with reduced circulating levels of insulin, IGF-1, and thyroid hormones, and elevated levels of cortisol and growth hormone. It appears that many of these changes are not completely reversible on refeeding, and that fetal undernutrition may result in a syndrome of relative multihormone resistance. Short children who were small at birth show marked insulin resistance in childhood compared with similarly short children of normal birthweight (58). In fetal sheep that are growing slowly in late gestation, which is thought to reflect periconceptual undernutrition, insulin levels are elevated above those of control fetuses, sug-

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gesting a degree of insulin resistance. In fetuses exposed to periconceptual undernutrition, insulin levels fall normally in response to late-gestation undernutrition but fail to return to normal on refeeding. These effects also appear to persist into postnatal life. In another experiment, groups of sheep were either well nourished or exposed to 10 or 20 days of undernutrition in late gestation. When the offspring were studied at 5 months of age, glucose tolerance was inversely related to birth weight in these lambs, but there was no strong nutritional group effect. When studied at 30 months, however, ewes that had been exposed to 20 days of undernutrition in utero showed glucose intolerance and insulin resistance. Thus, even a relatively brief period of adaptation to undernutrition in utero may have long-term metabolic consequences, and these consequences may become more significant with increasing postnatal age. The IGF axis also appears to be influenced by periconceptual nutrition. When studied in late gestation, fetuses that had been exposed to periconceptual undernutrition had normal circulating IGF-1 concentrations, but elevated insulin-like growth factor binding protein (IGFBP)-1 and reduced IGFBP-3 levels (59). When undernourished in late gestation, these fetuses showed larger changes in circulating IGF-1 and binding proteins than did fetuses with good periconceptual nutrition, and on refeeding, circulating IGF-1 levels failed to return to normal. Muscle from growth-restricted fetal and neonatal rats also appears to be resistant to the metabolic effects of IGF-1 (60,61). Thus, the IGF axis may also be perturbed in the long term as a result of intrauterine nutritional events. The GH axis may also be influenced in this way. Rats exposed to maternal undernutrition in utero showed altered tempo of maturation of the GH/ IGF axis postnatally. These animals have no growth response to GH stimulation when prepubertal, at the time when control rats respond normally. However, by the time they reach adulthood, GH responsivity is restored (62). Thus, the normal postnatal maturation of the GH axis is influenced by the prenatal nutritional environment. X. Conclusion Impaired fetal growth is commonly an appropriate adaptation to impaired fetal nutrient supply in utero. This growth adaptation is mediated largely by the accompanying metabolic and endocrine adaptations that are required for fetal survival in the face of inadequate nutrient supply. Such adaptations may be influenced by a number of factors, as yet poorly understood, including the timing, severity, and duration of the nutrient limitation, the balance of nutri-

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ents, and the experience of previous generations. Such adaptations may have a number of long-term metabolic, endocrine, and cardiovascular consequences. Their legacy may be seen in altered homeostatic and homeorhetic functions that may mediate the observed associations between impaired fetal growth and predisposition to adult disease. References 1.

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28. Klempt M, Bingham B, Breier BH, Baumbach WR, Gluckman PD. Tissue distribution and ontogeny of growth hormone receptor messenger ribonucleic acid and ligand binding to hepatic tissue in the midgestation sheep fetus. Endocrinology 1993; 132:1071–1077. 29. Breier BH, Ambler GR, Sauerwein H, Surus A, Gluckman PD. The induction of hepatic somatotrophic receptors after birth in sheep is dependent on parturition-associated mechanisms. J Endocrinol 1994; 141:101–108. 30. Stevens D, Alexander G. Lipid deposition after hypophysectomy and growth hormone treatment in the sheep fetus. J Dev Physiol 1986; 8:139–145. 31. Fowden AL, Szemere J, Hughes P, Gilmour RS, Forhead AJ. The effects of cortisol on the growth rate of the sheep fetus during late gestation. J Endocrinol 1996; 151:97–105. 32. Bassett JM, Madill D. The influence of maternal nutrition of plasma hormone and metabolite concentrations of foetal lambs. J Endocrinol 1974; 61:465–477. 33. Jensen EC, Harding JE. The effects of a maternal cortisol infusion in the late gestational fetal sheep. Proceedings of the Second Annual Congress of the Perinatal Society of Australia & New Zealand. 1998; 191 (abstr). 34. Fowden AL, Silver M. The effects of thyroid hormones on oxygen and glucose metabolism in the sheep fetus during late gestation. J Physiol 1995; 482:203– 213. 35. Danforth E Jr. The impact of nutrition on thyroid hormone physiology and action. Ann Rev Nutr 1989; 9:201–227. 36. Thorpe-Beeston JG, Nicolaides KH, Snijders RJM, Felton CV, McGregor AM. Thyroid function in small for gestational age fetuses. Obstet Gynecol 1991; 77:701–706. 37. Harding JE, Jones CT, Robinson JS. Studies on experimental growth retardation in sheep. The effects of a small placenta in restricting transport to and growth of the fetus. J Dev Physiol 1985; 7:427–442. 38. Bassett NS, Currie MJ, Breier BH, Woodall SM, Gluckman PD. Regulation of placental glucose transporter gene expression. Fetal and Neonatal Physiology Symposium. IUPS Satellite, Cambridge, 1997; Abstract. 39. Kelly RW, Newnham JP. Nutrition of the pregnant ewe. In: Oldham CM, Martin GB, Purvis IW, eds. Reproductive Physiology of Merino Sheep, Concepts and Consequences. The University of Western Australia: School of Agriculture (Animal Science), 1990:161–168. 40. Godfrey K, Robinson S, Barker DJP, Osmond C, Cox V. Maternal nutrition in early and late pregnancy in relation to placental and fetal growth. BMJ 1996; 312:410–414. 41. Levy L, Jackson AA. Modest restriction of dietary protein during pregnancy in the rat fetal and placental growth. J Dev Physiol 1993; 19:113–118. 42. Godfrey KM, Barker DJ, Robinson S, Osmond C. Maternal birthweight and diet in pregnancy in relation to the infant’s thinness at birth. Br J Obstet Gynaecol 1997; 104:663–667.

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43. Stewart RJC, Sheppard H, Preece R, Waterlow JC. The effect of rehabilitation at different stages of development of rats marginally malnourished for ten to twelve generations. Br J Nutr 1980; 43:403–412. 44. Lumey LH. Decreased birthweights in infants after maternal in utero exposure to the Dutch famine of 1944–1945. Paediatr Perinat Epidemiol 1992; 6:240– 253. 45. Ravelli G-P, Stein ZA, Susser MW. Obesity in young men after famine exposure in utero and early infancy. N Engl J Med 1976; 295:349–353. 46. Morley R, Payne CL, Lister G, Lucas A. Maternal smoking and blood pressure in 7.5 to 8 year old offspring. Arch Dis Child 1995; 72:120–124. 47. Lucas A, Morley R. Does early nutrition in infants born before term programme later blood pressure? BMJ 1994; 309:304–308. 48. Lemons JA, Moorehead HC, Hage GP. Effects of fasting on gluconeogenic enzymes in the ovine fetus. Pediatr Res 1986; 20:676–679. 49. Desai M, Byrne CD, Meeran K, Martenz ND, Bloom SR, Hales CN. Regulation of hepatic enzymes and insulin levels in offspring of rat dams fed a reduced-protein diet. Am J Physiol 1997; 273:G899–G904. 50. Taylor DJ, Thompson CH, Kemp GJ, et al. A relationship between impaired fetal growth and reduced muscle glycolysis revealed by 31P magnetic resonance spectroscopy. Diabetologia 1995; 38:1205–1212. 51. Fowden AL, Mijovic J, Silver M. The effects of cortisol on hepatic and renal gluconeogenic enzyme activities in the sheep fetus during late gestation. J Endocrinol 1993; 137:213–222. 52. Townsend SF, Rudolph CD, Rudolph AM. Cortisol induces perinatal hepatic gluconeogenesis in the lamb. J Dev Physiol 1991; 16:71–79. 53. Burns SP, Desai M, Cohen RD, et al. Gluconeogenesis, glucose handling, and structural changes in livers of the adult offspring of rats partially deprived of protein during pregnancy and lactation. J Clin Invest 1997; 100:1768–1774. 54. Cauderay M, Schutz Y, Micheli J-L, Calame A, Jéquier E. Energy-nitrogen balances and protein turnover in small and appropriate for gestational age low birthweight infants. Eur J Clin Nutr 1988; 42:125–136. 55. Harding JE, Johnston BM. Nutrition and fetal growth. Reprod Fertil Dev 1995; 7:539–547. 56. Langley SC, Jackson AA. Increased systolic blood pressure in adult rats induced by fetal exposure to maternal low protein diets. Clin Sci 1994; 86:217– 222. 57. Woodall SM, Johnston BM, Breier BH, Gluckman PD. Chronic maternal undernutrition in the rat leads to delayed postnatal growth and elevated blood pressure of offspring. Pediatr Res 1996; 40:438–443. 58. Hofman PL, Cutfield WS, Robinson EM, et al. Insulin resistance in short children with intrauterine growth retardation. J Clin Endocrinol Metab 1997; 82:402– 406. 59. Gallaher BW, Breier BH, Harding JE, Gluckman PD. Periconceptual undernutrition resets plasma IGFBP levels and alters the response of IFGBR-1, IGFBP-3

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and IGF-1 to subsequent maternal undernutrition in fetal sheep. In: Blum WF, Hall K, eds. Progress in Growth Factors Research. Vol. 6, No. 2–4. Great Britain: Elsevier Science Ltd, 1995:189–195. Frampton RJ, Jonas HA, MacMahon RA, Larkins RG. Failure of IGF-1 to affect protein turnover in muscle from growth-retarded neonatal rats. J Dev Physiol 1990; 13:125–133. Simmons RA, Flozak AS, Ogata ES. The effect of insulin and insulin-like growth factor-1 on glucose transport in normal and small for gestational age fetal rats. Endocrinology 1993; 133:1361–1368. Woodall SM, Breier BH, Johnston BM, Gluckman PD. Reduced maternal nutrition during gestation in the rat leads to temporary growth hormone resistance during the neonatal period. Proceedings of the 79th Annual Meeting of the Endocrine Society, 1997; Abstract. Fowden AL. Fetal metabolism and energy balance. In Thorburn GD, Harding R, eds. Textbook of Fetal Physiology. New York: Oxford University Press, 1994; 70–82.

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10 The Role of Hormones in Intrauterine Development ABIGAIL L. FOWDEN and ALISON J. FORHEAD University of Cambridge Cambridge, United Kingdom

I.

Introduction

Growth in utero is critical in determining life expectancy. It affects not only neonatal viability but also adult rates of mortality and morbidity in man. The prognosis is worse for fetuses that are growth retarded in utero. These infants are less likely to survive at birth and are at greater risk of developing adultonset degenerative diseases, such as hypertension and Type 2 diabetes (1). Similarly, in domestic and other species, it is the small neonates, or runts in the litter, that fail to thrive after birth (2). Detailed morphometric analyses of the human epidemiological data has shown that certain patterns of intrauterine growth can be related to specific adult diseases (1). For instance, it is the thin baby with a low ponderal index, rather than the symmetrically small infant, that is more prone to Type 2 diabetes in adult life (3). Therefore, the mechanisms regulating the rate and pattern of development in utero have an important role in the intrauterine programming of adult disease. 199

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The supply of oxygen and nutrients to the fetus is one of the major determinants of the fetal growth rate (2,4). It affects the accretion of new structural tissues and fuel reserves, as well as the differentiation of fetal tissues in preparation for extrauterine life. Fetal nutrient availability has also been shown to influence postnatal cardiovascular and metabolic function in several different experimental animals (5–7). Even relatively mild nutritional insults during pregnancy with little, if any, effect on fetal growth have longterm postnatal sequelae (7). The effects that restriction of the oxygen or nutrient supply have on fetal growth and development depend on a number of factors, including the specific nature of the nutrient deficiency and the duration, severity, and gestational age at onset of the nutritional insult (4). Therefore, there must be mechanisms to signal qualitative, quantitative, and temporal information about nutrient availability to the fetal tissues. Hormones can act as these nutritional signals in the fetus because their concentrations in utero vary with the fetal nutritional state (8). They affect both tissue accretion and differentiation, and ensure that the fetal growth rate is appropriate to the nutrient supply (2,8). Yet, by altering the balance between tissue accretion and differentiation early in development, hormonal changes in utero may predispose tissues for subsequent pathophysiology (9). The aim of this chapter is threefold: first, to discuss the hormonal changes that occur in the fetus in response to variations in its nutrient supply; second, to examine the effects that these hormonal changes have on fetal growth and development; and third, to consider the mechanisms by which the hormonal changes in utero may increase susceptibility to disease later in life. II. Nutritionally Induced Hormonal Changes Changes in the fetal hormonal environment occur in response to nutritional challenges induced by a wide range of factors, including alteration in maternal nutritional state (2,4), placental size and function (10,11), and in the blood flow through the uterine and umbilical circulations (12–14). In general, nutritional challenges that reduce fetal nutrient availability lower anabolic hormone (eg, insulin, insulin-like growth factors, thyroxine) and increase catabolic hormone concentrations (eg, cortisol, catecholamine, growth hormone), whereas challenges that increase the fetal nutrient supply raise anabolic and reduce catabolic hormone levels in the fetal circulation (Table 1). However, the particular pattern of endocrine changes is determined by the specific nature of the nutritional abnormality and its duration (Table 1).

Hormonal changes Fetal nutritional state

Causes

Decrease

No change

Increase

Hypoglycemia alone

Reduced maternal dietary intake Maternal fasting Maternal insulin infusion Fetal insulin infusion

Insulin IGF-I/IGF-II Placental lactogen

Glucagon Adrenaline T4/T3

Hypoxemia alone

Maternal inhalation hypoxia High altitude

Insulin IGF-I

PGE2 IGF-II

Combined hypoglycemia and hypoxemia

Carunclectomy Placental embolization

Insulin IGF-I/IGF-II T4/T3 Prolactin

Adrenaline ACTH

Cortisol ACTH GH PGE2/PGF2α Noradrenaline ACTH Cortisol Noradrenaline Adrenaline AVP Erythropoietin Glucagon ANP Adenosine AII Cortisol Noradrenaline PGE2

Hormones and Intrauterine Development

Table 1 Fetal Hormonal Changes in Response to Fetal Hypoglycemia or Hypoxemia Alone or Combined Fetal Hypoglycemia and Hypoxemia in Sheep During Late Gestation (=80% Gestation)

Abbreviations: IGF, Insulin-like growth factor; ACTH, Adrenocorticotrophic hormone; GH, Growth hormone; PGE2, Prostaglandin E2; PGF2α, Prostaglandin F2γ; AVP, Arginine vasopressin; ANP, Atrial natriuretic peptide; AII, Angiotensin II; T4, Thyroxine; T3, Triiodothyronine. Source: Refs. 4, 10, 12, 14–18, 29, 41, 106, 107.

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In the sheep fetus, hypoglycemia produces different hormonal changes from those seen in response to oxygen deprivation alone or combined hypoglycemia and hypoxemia (Table 1). In particular, the catabolic and vasoactive hormones show nutrient-specific changes in concentration. For instance, fetal adrenaline and glucagon levels are raised in response to hypoxemia but not hypoglycemia, whereas prostaglandin E2 (PGE2) concentrations are elevated during fetal hypoglycemia but not hypoxemia alone (Table 1). Similarly, fetal plasma adrenocorticotrophic hormone (ACTH) is raised by either hypoglycemia or hypoxemia alone, but not when hypoglycemia and hypoxemia are combined in fetuses that are growth retarded by carunclectomy before conception (10,11,15). In contrast, anabolic hormones, such as insulin and IGF-I, are invariably reduced in concentration irrespective of the nutrient deficit (Table 1). The endocrine changes can therefore act as both general and specific signals of nutrient availability. The time course of the endocrine responses to nutrient restriction also differs between the individual hormones. For instance, the increases in plasma adrenaline, arginine vasopressin (AVP), atrial natriuretic peptide (ANP), and adenosine induced by fetal hypoxemia are transient and are not maintained even when the hypoxemia is sustained (13,16). On the other hand, the hypoxemia-induced increases in cortisol and noradrenaline are maintained and become more pronounced after 24 h of low pO2 (14,16). In addition, some hormone concentrations vary continuously with nutrient availability while others only change when nutrient levels reach a critical value. For example, changes in fetal PGE2 levels are known to be correlated linearly with fetal glycemia throughout the range of glucose levels observed in utero (17,18). By contrast, fetal glucagon levels are unresponsive to changes in the fetal pO2 and glucose levels within the normal range but increase rapidly when fetal pO2 falls by 50% or more (8). The set point and sensitivity of these nutritionally induced endocrine changes also vary with gestational age. The slope of the relationship between the changes in fetal PGE2 and glucose levels increases towards term, which leads to higher PGE2 levels for a smaller fall in plasma glucose late in gestation (17). Similarly, the increments in ACTH, cortisol, and adrenaline that occur in response to hypoglycemia are elevated as term approaches in the fetal foal (19). Therefore, hormones are sensors of the duration of nutrient deprivation and appear to progressively amplify the nutritional signal as the nutrient demands of the fetus increase towards term. The nutritionally induced endocrine changes in the fetus mainly arise in three ways. First, they may be attributable to altered hormone secretion by the fetal endocrine glands. The fetal pancreas, thyroid, pituitary, and adrenal me-

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dulla are functional from early in gestation, whereas the fetal adrenal cortex becomes more responsive to stimuli during late gestation (8,17). Secondly, the changes in fetal hormone concentrations may be attributable to alterations in the production and metabolism of hormones by the uteroplacental tissues (17). These tissues synthesize peptide and steroid hormones including prostaglandins, progestagens, estrogens, corticotrophic-releasing hormone, adenosine, and placental lactogen (20). They also metabolize prostaglandins and steroid hormones to their inactive forms and remove catecholamines and other hormones from the umbilical circulation. Several of these processes are known to be regulated directly by the uteroplacental nutrient supply (17,21). Thirdly, the hormones in the fetal circulation may be derived from the mother. Steroids readily cross the placenta and, in many species, their transplacental passage is facilitated by significant concentration differences between maternal and fetal plasma (21). In the sheep, which has a small transplacental cortisol gradient compared with other species, 90% of the cortisol in the fetal circulation is of maternal origin before the fetal adrenal cortex begins to secrete cortisol close to term (22). Overexposure of the fetus to maternal and placental hormones is normally prevented by several mechanisms. The levels of binding proteins in fetal plasma are high and regulated by the nutritional and endocrine environments in utero (4,15). The placental and fetal tissues also contain enzymes, such as 11α hydroxysteroid dehydrogenase (HSD), prostaglandin dehydrogenases, and catecholamine-O-methytransferase, which convert active hormones to their inactive metabolites (9,20). The increase in fetal plasma glucocorticoids that occurs in response to maternal undernutrition may therefore be attributable to decreased activity of placental 11β HSD and to increased glucocorticoid secretion by both the fetal and maternal adrenal glands. The contribution and relative importance of the three different sources of circulating hormones in the fetus are also modified by nutritional state. Undernutrition during pregnancy in rats has been shown to lower placental 11β HSD activity (23) and could thereby increase fetal glucocorticoid exposure without any changes in fetal or maternal glucocorticoid secretion. Prolonged undernutrition as opposed to short-term changes in nutrient availability also influences the functioning of the maternal endocrine glands and alters the development of many of the fetal endocrine axes. In the fetal pancreas, pituitary and adrenal, chronic nutrient restriction changes the number, type, and secretory capacity of the endocrine cells as well as their innervation and vascularization (11,19,24). Therefore, long-term changes in the fetal nutritional state affect not only the quantity of hormone available for release, but also the mechanisms by which hormone secretion is stimulated.

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For hormones to act as nutritional signals, appropriate receptors must be present in the fetal tissues. Receptors for most of the hormones involved in regulating fetal growth and metabolism (Table 1) have been identified in several fetal tissues from early in gestation (2,21,25). Their abundance generally increases toward term and can be regulated by both the hormonal and nutritional environments in utero (26). A change in the level of cortisol is known to alter the density of receptors for other hormones (eg, GH, catecholamines) involved in signaling nutritional status (21,25). Hormones can also directly influence the functioning of other endocrine glands. In the sheep fetus, for instance, catecholamines regulate secretion of the pancreatic hormones and are, in turn, regulated by thyroid hormones (8,15). Hormones can therefore accentuate or attenuate the nutritional signal that they convey by altering the sensitivity of other endocrine axes or by changing binding protein levels, tissue receptor abundance, and their own clearance from the fetal circulation. III. Endocrine Regulation of Fetal Growth and Development Most of the hormones altered in concentration by nutrient availability are known to affect fetal growth and development (Table 2). They act on tissue accretion and differentiation either directly via the genes or indirectly through changes in fetal metabolism, cardiovascular function, and/or production of growth factors. The key hormones involved in these processes are insulin, the thyroid hormones, the IGFs, the catecholamines, and the glucocorticoids. A.

Insulin

Before birth, insulin has a major role in promoting tissue accretion and is required throughout late gestation for normal fetal growth (Fig. 1). Its plasma concentration in utero varies with the fetal glucose level and is positively correlated to birthweight and to the rates of glucose utilization and linear growth in the sheep fetus (27). Insulin deficiency in utero leads to growth retardation as well as reductions in bodyweight and crown rump length (CRL) at term of 30 to 50% and 10 to 20%, respectively (Table 2). The daily increment in CRL fell by 40 to 50% immediately after pancreatectomy and remained uniformly low throughout the remaining 20 to 30 days of gestation (Fig. 1). These changes were accompanied by reductions in limb lengths and in the weights of most of the individual fetal organs (27). Fetal insulin deficiency therefore affects the growth of both bone and soft tissue and leads to a propor-

Hormone

Experimental procedure

Insulin

Deficiency Pancreatectomy Streptozotocin Excess Fetal infusion Deficiency Thyroidectomy Deficiency Hypophysectomy

Thyroid hormones Pituitary hormones

IGF-I Catecholamines

Glucocorticoids

Pituitary Stalk Section Excess Fetal infusion Deficiency Pharmacological blockade Adrenal demedullation Excess Fetal infusion Deficiency Adrenalectomy Excess Fetal administration Maternal administration

CRL

Specific tissue developmental changes

↓ 30% ↓ 50%

↓ 15% ↓ 25%

None—proportionate growth retardation None—proportionate growth retardation

↑ 0–10%

No ∆

Fat deposition

↓ 20–30%

↓ 10%

Skeleton, skin, muscle, nervous system

↓ 20–30%

↓ 10%

↓ 15%

No ∆

Skeleton, muscle, liver, lungs, gut, adrenal fat deposition Adrenals, other tissues?

No ∆

↑ 0–5%

Increased growth of visceral organs

No ∆ No ∆

No ∆ No ∆

None Kidneys enlarged

↓ 20–30%

↓ 10%

None—proportionate growth retardation

↑ 10–15%

No ∆

Lungs, liver, gut, kidney

↓ 0–5% ↓ 10–20%

↓ 0–5% ↑ 0–5%

Lungs, liver, gut, kidney Proportionate growth retardation Enlarged liver

205

Abbreviation: CRL, crown rump length. Source: Refs. 4, 8, 27, 40, 108, 109.

Body weight

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Table 2 The Effects of Hormones on the Sheep Fetus During Late Gestation (≥85% Gestation)

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Figure 1 The mean (± SEM) increment in crown rump length (CRL) with respect to days before and after either pancreatectomy (l, n = 6) or sham operation (ˆ, n = 5) of sheep fetuses during late gestation. (From Ref. 28.)

tionate type of growth retardation (28). Fetal hyperinsulinemia, on the other hand, appears to produce a more disproportionate pattern of fetal growth with increased fat deposition but little, if any, linear growth (Table 2). Insulin stimulates fetal growth primarily by increasing cell proliferation and appears to have little effect on the differentiation or maturation of fetal tissues before birth (28). It controls growth via its anabolic actions on fetal metabolism and by stimulating the production of plasma IGF-I (27,29). In the sheep fetus, insulin has been shown to enhance glucose utilization, reduce amino acid catabolism, and stimulate the preferential use of glucose for oxida-

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tive metabolism (8,27). Therefore, it increases the rate of fetal carbon and nitrogen accretion both as fuel reserves and new structural tissue. Hence, insulin is a signal of a plentiful nutrient supply and principally matches the rate of fetal glucose utilization to its umbilical supply. B. Thyroid Hormones

Thyroid hormones also stimulate fetal growth. They affect both tissue accretion and differentiation and act, at a cellular level, to increase cell size and number (30). In fetal sheep, thyroid hormone deficiency induces a symmetrical type of growth retardation with a relative reduction in muscle mass (8). It also alters development of the central and peripheral nervous system and delays maturation of the skin, skeleton, and pulmonary and neuromuscular systems (Table 2). Thyroid hormones stimulate growth by metabolic and nonmetabolic mechanisms. Their main metabolic effect is on fetal O2 uptake. Thyroid hormone deficiency induced by fetal thyroidectomy reduces fetal O2 consumption by 20 to 30%, whereas raising fetal thyroxine (T4) and triiodothyronine (T3) levels by exogenous infusion increases umbilical O2 uptake (30). By altering fetal O2 consumption, thyroid hormones can control the amount of energy available for growth. Thyroid hormones also affect the circulating levels and tissue content of the IGFs and their binding proteins (2,31). There are reductions in plasma IGF-I, but not IGF-II, in hypothyroid fetal sheep and pigs that can be restored to normal values by T4 replacement (32,33). The normal prepartum rise in plasma T3 has also been shown to have an important role in the ontogenic changes in IGF gene expression that occur in fetal liver and muscle towards term (21,32). Abolition of this prepartum T3 surge by fetal thyroidectomy may also account, in part, for the other developmental delays observed in these fetuses (34). Many of the genes involved in growth and maturation have thyroid hormone response elements and, hence, can be regulated directly by thyroid hormones at a molecular level. Thyroid hormones are primarily signals of nutrient sufficiency and act to match oxygen consumption to its availability in the fetus. C. Insulin-like Growth Factors

These growth factors have a central role in regulating growth and development in utero (2,35). They are produced by a wide variety of fetal tissues to act both locally and systemically (26). Plasma concentrations and tissue gene expression of the IGFs are positively correlated with the glucose and PO2 levels

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in the sheep fetus (36). They are also regulated by the other key hormones involved in the control of fetal development, namely insulin, thyroid hormones, catecholamines, and the glucocorticoids (15,25,29,31). In mice, disruption of the genes for IGF-I, IGF-II, or their receptors leads to severe growth retardation with up to 60% reduction in body weight and abnormalities in muscle and bone development (35). Administration of either IGF-I or IGF-II to fetal rats has also been shown to enhance intrauterine growth (35). By contrast, infusion of IGF-I for 10 days into fetal sheep during late gestation has little effect on bodyweight but does increase the growth of certain bones and visceral organs (Table 2). Like the thyroid hormones, the IGFs stimulate growth by both metabolic and nonmetabolic effects. They act as progression factors in the cell cycle, prevent apoptosis, and increase synthesis of DNA and protein in fetal tissues and cell lines in vitro (26,37). They also have anabolic actions on fetal metabolism similar to those of insulin (2). In the sheep fetus, IGF-I reduces amino acid catabolism and oxidation and may also increase glucose uptake by the fetal tissues (38,39). During late gestation, IGF-I production appears to be more sensitive to changes in fetal nutrient availability than IGF-II and may have a more prominent role in modulating cell proliferation in relation to the specific nutritional conditions in utero (36). Fetal IGF-II appears to provide a more general stimulus to cell growth in utero as its tissue abundance is greater than that of IGF-I in utero or of IGF-II after birth. Fetal IGF-II may also be responsible for the developmental and tissue-specific changes in cell differentiation seen close to term and during undernutrition. D. Catecholamines

These hormones inhibit fetal growth (Table 2). Infusion of adrenaline and noradrenaline into fetal sheep for 10 days during late gestation reduces bodyweight and CRL by 20% and 10% respectively, and has severe effects on the accretion of skeletal muscle (40). These growth inhibitory effects are mediated, partially, by redistribution of cardiac output away from muscle (7) and, partially, by the reduction in the circulating levels of anabolic hormones, such as insulin and IGF-I (15,40). Catecholamines also increase the fetal levels of GH, glucagon, and the IGF binding proteins, which all antagonize the actions of the anabolic hormones (15,40). In late gestation, catecholamines can stimulate fetal glucose production which will increase glucose delivery to key fetal tissues and ameliorate, in part, any reduction in the umbilical glucose supply (41). However, prolonged elevation in the fetal catecholamine concentrations

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lowers the glycogen content of several fetal tissues, and thereby reduces the capacity of the fetus to respond to subsequent nutritional challenges (13,42). Because catecholamine concentrations can change rapidly, these hormones are good signals of acute changes in the nutrient or oxygen supply. E. Glucocorticoids

In normal conditions, the glucocorticoids appear to have a relatively minor role in the control of tissue accretion compared with the other endocrine secretions (8). Their concentration in utero is low for most of gestation, and fetal adrenalectomy has little effect on bodyweight before term (19). However, in the sheep fetus, there is a rise in glucocorticoid concentrations close to term that is responsible for reducing the fetal growth rate in this period immediately before birth (43). Abolition of this cortisol surge by adrenalectomy increases bodyweight at term (Table 2), whereas raising cortisol levels to prepartum values early in gestation prematurely reduces the CRL increment to a value similar to that seen in fetuses close to term (Fig. 2). Early fetal overexposure to glucocorticoids by maternal administration of synthetic glucocorticoids has also been shown to retard fetal growth in rats (44), rabbits (45), sheep (46), monkeys (47), and man (48). Glucocorticoids therefore inhibit tissue accretion in the fetus when their concentrations in utero are raised. Glucocorticoids also have major effects on differentiation and prepartum maturation of fetal tissues essential for adaptation to extrauterine life (Table 3). They are particularly important in the development of tissues— such as the lungs, liver, and gut—that take over the functions of the placenta immediately after birth (34). The prepartum rise in glucocorticoid concentrations induces structural and functional changes in these tissues and activates many of the biochemical processes that have little or no function in fetal life (Table 3). For example, the establishment of pulmonary gas exchange and gastrointestinal absorption after birth are both dependent on the prepartum rise in fetal glucocorticoids (34). Close to term, these hormones act as maturational as well as nutritional signals, and ensure an adequate supply of oxygen and nutrients to the neonatal tissues immediately after birth. The actions of the glucocorticoids on tissue accretion and differentiation occur by direct and indirect mechanisms. Metabolically, glucocorticoids depress umbilical glucose uptake and alter amino acid uptake and utilization by the fetal tissues (49). They also influence the abundance of glucose transporters in the placenta and fetal tissues (50). The availability of glucose carbon for tissue accretion may, therefore, be changed as a consequence of glucocorticoid

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Figure 2 The mean (±SEM) increment in crown rump length (CRL) in fetal sheep with respect to days before and after cortisol infusion (l, 2–3 mg/kg/day, n = 8) or in age-matched untreated controls (ˆ, n = 6). (From Ref. 43.) *Significantly different from values in controls. exposure. In the sheep fetus, tissue expression of both IGF genes is directly related to the circulating cortisol concentration in utero. Cortisol suppresses IGF-II mRNA abundance in the fetal liver, skeletal muscle, and adrenal, and reduces IGF-I gene expression in fetal skeletal muscle (21,25,51). In part, these effects of cortisol on IGF status may be mediated by the actions of T3. Cortisol induces deiodination of T4 to T3 (Table 3) and leads to a rise in fetal plasma T3 close to term (34). In turn, T3 may alter tissue IGF gene expression and lead to other maturational changes in the fetal tissues (31,34). But, whatever the mechanisms involved, the cortisol-induced changes in IGF gene expression will reduce the drive for fetal growth and trigger cell differentiation. At a molecular level, glucocorticoids can act on transcription, mRNA stability, translation, and/or on the posttranslational processing of the protein

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Table 3 Maturational Changes Essential for Neonatal Survival Normally Induced by Cortisol in Fetal Ovine Tissues Tissue Maturational change Lung

Liver

Kidney

Surfactant production and release Collagen and elastin synthesis β adrenoreceptor induction Lung liquid reabsorption Structural maturation of alveoli ACE induction Glycogenolysis Glycogen deposition Gluconeogenic enzyme induction IGF-II gene downregulation IGF-I gene upregulation β adrenoreceptor induction Prolactin receptor induction GH receptor induction CBG synthesis Angiotensinogen gene downregulation Increased GFR Tubular Na reabsorption Ion-exchange pump induction Erythropoietin gene downregulation AT1 receptor gene downregulation Renin gene downregulation Gut Villi and smooth muscle growth Stomach acid secretion Digestive enzyme induction

Abbreviations: ACE. Angiotensin converting enzyme; IGF, insulin-like growth factor; GH, Growth hormone; CBG, Corticosteroid binding globulin; GRF, Glomerular filtration rate; AT1, Angiotensin type 1. Source: Refs. 21, 34, 111.

products (52). Several of the genes known to be regulated by glucocorticoids in utero have the necessary glucocorticoid and/or thyroid hormone response elements to allow direct transcriptional control by these hormones. In genes that have multiple transcripts, the effects of the glucocorticoids may be specific to certain leader exons in the untranslated regions of the gene. In fetal liver, cortisol has been shown to modifyx use of selective leader exons in the

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IGF-I, IGF-II, and GH receptor genes (25,51,53). Hence, part of the maturational and growth inhibitory actions of the glucocorticoids may be to initiate use of specific promotors, which in turn alters the translatability of the gene product. An increase in fetal glucocorticoid exposure induced by maternal or fetal undernutrition therefore switches the cell cycle from proliferation to differentiation and triggers permanent changes in gene expression. Because these molecular changes occur relatively slowly, glucocorticoids produce a delayed response to nutrient restriction and, hence, act as a good signal of long-term nutrient insufficiency. F. Other Hormones

In addition to the hormones listed in Table 1, there are other newly identified hormones in the fetal circulation that may signal nutritional status to the fetal tissues (54). Leptin is present in fetal plasma and is an index of adipose tissue mass in the fetus (55). Its expression is also known to be upregulated by insulin and glucocorticoids in adult animals (56). Leptin is expressed in the placenta and fetal adipose tissue of rodents and sheep and its concentration in utero is positively correlated with birthweight in the human infant (55,56). Nutritionally induced changes in fetal plasma leptin of either placental or fetal origin could also have long-term effects on the postnatal regulation of nutrient intake and fat deposition by programming the hypothalamus in utero (54). G. Integrated Hormonal Changes

During oxygen and/or nutrient restriction, the changes in the fetal hormonal environment are designed to limit the oxygen and nutrient requirements of the peripheral and visceral tissues while preserving a supply of oxygen and nutrients to more essential fetal tissues such as the brain, heart, and placenta. The fall in anabolic hormone levels reduces carbohydrate and amino acid uptakes into fetal muscle and visceral tissues while the rise in catabolic hormone concentrations provides gluconeogenic substrates and activates glucose production by the liver and kidneys. The rise in vasoactive hormones facilitates this redistribution of nutrients by directing fetal cardiac output away from the periphery toward the brain and placenta (7). These metabolic and cardiovascular changes, together with the fall in IGFs (Table 1), reduce the growth rate and thereby lower the fetal demand for oxygen and nutrients. In addition, hormone changes, such as the rise in PGE2, help reduce the nutrient demand by lowering the incidence of breathing movements in the fetus (18). Furthermore, the rise in fetal cortisol levels stimulates tissue differentiation, which

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would improve neonatal viability should the nutrient restriction be sufficiently severe to precipitate early delivery (34). The hormonal changes during nutrient restriction therefore adapt fetal development to the nutrient supply and maximize the chances of survival both in utero and at birth. IV. Long-Term Consequences of the Nutritionally Induced Hormonal Changes In Utero Undernutrition during pregnancy has been shown to cause hypertension and abnormalities in the metabolism of glucose and lipid in the adult offspring of a variety of experimental animals, including rats, sheep, guinea pigs, and pigs (6,7,24,55). Similarly, fasting of pregnant women during late gestation has been shown to increase the incidence of Type 2 diabetes in the adult offspring (57). In rats, adult hypertension and glucose intolerance have been related more specifically to the protein than calorific uptake during pregnancy (23,58). Feeding a low-protein diet for either the whole of gestation or for shorter periods in late gestation leads to an increase in blood pressure in the offspring by 14 weeks of postnatal age that then persists throughout adult life (59). Because hormones have a critical role in the nutritionally induced changes in fetal development, they are probably also responsible, at least in part, for the subsequent abnormalities in tissue function. Of all the hormones changed in concentration by fetal undernutrition, it is the glucocorticoids that are most likely to cause tissue programming (59– 61). Their effects are permanent and, prenatally, they affect development of all the tissues and organ systems that are at increased risk of adult pathophysiology when growth is impaired in utero (21,34). They also increase blood pressure in fetal and adult animals and their hypertensive effects are known to persist after cessation of treatment in sheep and rat fetuses (9,62–64). In addition, treatment of ewes with the synthetic glucocorticoid, dexamethasone, early in pregnancy has been shown to elevate blood pressure in the offspring throughout adult life (22). Similarly, in rats, fetal glucocorticoid overexposure induced either by maternal dexamethasone administration or by inhibition of placental 11β HSD activity leads to hypertension and glucose intolerance in the adult offspring (63,65). Increased glucocorticoid exposure in utero probably also accounts for the hypertension and glucose intolerance observed in the adult offspring of rats protein derived during pregnancy, because these changes are prevented by maternal adrenalectomy or metyrapone treatment (59). Inappropriate intrauterine exposure to glucocorticoids therefore appears

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to be associated with adult hypertension and glucose intolerance irrespective of whether the glucocorticoids are of fetal or maternal origin. A. Cardiovascular Function

There are several mechanisms by which glucocorticoid overexposure in early life could affect the subsequent functioning of the cardiovascular system (Table 4). First, glucocorticoids can alter the development of the central nervous system and, in particular, those areas of the brain involved in regulating blood pressure (7,61). They have been shown to reduce neuron number as well as synaptic contacts and myelination in the hypothalamus, hippocampus, neocortex, and brain stem both before and after birth (66–68). They also alter neuronal enzyme activities, hormone receptor density and the tissue content, and gene expression of various neuropeptides in these brain areas (67,69– 71). In addition, increased activities of central noradrenergic, cholinergic, and serotonergic pathways have been observed in neonatal rats exposed to dexamethasone in utero (71). Furthermore, dexamethasone treatment in fetal sheep reduces the permeability of the blood-brain barrier, which may prevent access of bloodborne factors to the brain at critical periods of development (72). However, it is the glucocorticoid-induced changes in the homeostatic functions of the hypothalamus that are likely to have the most important long-term effects on blood pressure control. Several studies have suggested that resetting of the hypothalamic regulatory axes occurs after prenatal glucocorticoid exposure (23,61,73). The set point and gain of the baroreflexes are known to be altered in newborn lambs and adult sheep treated with dexamethasone in utero (61,74). Similarly, the set point of the hypothalamic-pituitary-adrenal (HPA) axis appears to be elevated both by prenatal dexamethasone treatment (23,67) and by adverse intrauterine conditions that raise cortisol levels in utero (11). Sensitivity of the HPA feedback mechanisms may be altered in these circumstances by the changes in glucocorticoid receptor density induced in the hippocampus and other brain regions by early intrauterine glucocorticoid exposure (70). Certainly, increased basal and stress-induced levels of glucocorticoids are observed in adult rats and monkeys treated with glucocorticoids in utero (67,68) and in adult men who were small at birth (75). Raised endogenous glucocorticoid levels in adult life may directly induce hypertension and antagonize the actions of insulin (6). Changes in the regulatory functions of the hypothalamus after prenatal glucocorticoid exposure are also consistent with other recent epidemiological and experimental findings that show impaired growth in utero is associated with altered energy balance and reproductive function in later life (76,77).

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Secondly, the glucocorticoids may affect cardiovascular function by altering the structure and function of the heart and blood vasculature. In sheep and rat fetuses, glucocorticoid exposure stimulates growth of the heart relative to other fetal tissues (62,64,78). In rats, these changes were accompanied by alternations in the expression of the Na+, K+-ATPase and myosin heavy chain isoforms in the neonatal heart (78,79). In addition, changes in cardiac autonomic control, hormone receptor density, and receptor coupling have been observed after prenatal glucocorticoid exposure, although the specific nature of these changes appears to depend on the dose and duration of glucocorticoid treatment (80–82). Increases in gene expression for AT1 receptors, but not β1 adrenoreceptors, have been observed in fetal cardiac ventricles in response to glucocorticoid administration (80,83). Activity of myocardial adenylate cyclase is also increased in neonatal rats after prenatal glucocorticoid treatment, which may enhance the sensitivity of postreceptor signaling for both adrenergic and nonadrenergic stimuli such as PGE2 (80). In fetal sheep, both increases and decreases in heart rate have been observed in response to cortisol and betamethasone, respectively (62,84). However, newborn rats treated with dexamethasone in utero have a reduced intrinsic heart rate but show no change in the balance between sympathetic and vagal tone (82). By contrast, increased pulse rates have been observed in adult men who were small at birth (85). Taken together, these glucocorticoid-induced changes in cardiac function are likely to enhance cardiac output and thereby raise blood pressure. Certainly, increases in cardiac output have been observed in newborn lambs and adult sheep treated with glucocorticoids in utero (61,86). Glucocorticoid-induced hypertension may also be attributable to increased peripheral resistance induced by changes in the blood vasculature. Glucocorticoid administration to sheep fetuses has been shown to increase femoral vascular resistance and alter the vascular sensitivity to angiotensin II (AII), bradykinin, and acetylcholine, but not to noradrenaline (62,64,87). Small resistance branches of the femoral artery from betamethasone-treated fetal sheep also showed increased sensitivity to depolarizing potassium solutions, which suggests that the excitation contraction coupling as well as endothelial function may be altered in these circumstances (87). Glucocorticoids may therefore induce changes in the ion channels in smooth muscle as occurs in the heart and kidney (79,88). The altered vasoreactivity after glucocorticoid administration may also reflect changes in the number and/or contractile protein content of the smooth muscle cells or in the activity of the enzymes involved in the local production of AII or nitric oxide (7,89,90). Changes in local exposure to glucocorticoids as a result of alterations in vascular 11β

Organ system Central nervous system

Major adaptations Neuronal structure Neuropeptides

Hormone receptor density Blood-brain barrier Enzyme activities Behavioral change Homeostatic function

Morphology

Decreased neuron number and myelination Changes in neuropeptide expression in neocortex and hippocampus Increased activity of noradrenergic, cholinergic, and peptidergic neurones Glucocorticoid receptor downregulation in hippocampus Decreased permeability Increased levels of glucocorticoid inducible enzymes Hyperexcitability Control of autonomic nervous system, pituitary function, and baroreceptor and chemoreceptor reflexes by the hypothalamus Increased cardiac mass Changes in contractile proteins and ion channels Increased vascular smooth muscle cell number

References 66, 68 67, 71 67, 71 69, 70 72 69 68 15, 61, 74, 82

62, 112 78, 79 7, 87

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Heart and vasculature

Specific changes

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Table 4 Mechanisms by Which Glucocorticoid Exposure In Utero May Program Cardiovascular Function in Later Life

Hormone receptor density Vasoreactivity

Kidney

Morphology Glomerulotubular mechanisms

Renin-angiotensin system

Changes in the balance of sympathetic/ 82 vagal tone Increased intracellular coupling of receptors 78, 80 through adenylate cyclase Changes in cardiac sensitivity to β 80, 83 agonists and PGE1 Increased cardiac and vascular AT1 receptor 89, 90, 98 mRNA expression Increased sensitivity to AII, bradykinin, and 64, 87 acetycholine Decreased sensitivity to bradykinin 87, 92 Decreased nephron number and telomere ch. 8 this volume, 93 length Decreased GFR and renal blood flow 93 Increased ion exchangers 88, 95 Increased fractional Na reabsorption 93, 94 Decreased renal renin and AT1 receptor mRNA expression 81, 98 Changes in plasma/tissue ACE 21

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Innervation

Abbreviations: PGE1, Prostaglandin E1; GFR, Glomerular filtration rate; ACE, Angiotensin converting enzyme; AT1, Angiotensin type I: AII, Angiotensin II.

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HSD or glucocorticoid receptor density may also contribute to the adult hypertension observed after intrauterine growth retardation. The third mechanism by which glucocorticoids may affect cardiovascular function is via changes in renal structure and function (Table 4). The kidney has an important role in the control of extracellular fluid volume and blood pressure in adult animals, and its development in utero is known to be affected by the glucocorticoids (7,61). In sheep and man, nephrogenesis is complete at birth, and hence changes in renal development in utero are likely to have long-term consequences for the control of blood pressure (7). Low nephron number is a recognized risk factor for hypertension and is commonly associated with intrauterine growth retardation in both man and experimental animals (91,92). There are also reductions in nephron number, cell proliferation, and in the thickness of the nephrogenic zone in kidneys from adult offspring of rats treated with dexamethasone during pregnancy (93). These glucocorticoid-induced changes in nephrogenesis are accompanied by alterations in glomerulotubular function and a lower fractional sodium excretion in adult rats (93). Acute administration of dexamethasone in utero has also been shown to increase glomerular filtration rate, renal blood flow, renal sympathetic nerve activity, and tubular absorptive capacity in the newborn preterm lamb (74,94). The increases in sodium reabsorption induced by glucocorticoids may be attributable, in part, to the increased activities of the sodium-hydrogen exchanger and Na+, K+-ATPase observed in kidney tubules in these circumstances (88,95). Increased sodium retention may therefore contribute to the adult hypertension observed after prenatal glucocorticoid treatment. The effects of glucocorticoids on renal development may occur secondarily to local changes in the activity of 11β HSD and the renin-angiotensin system (RAS). Normally, the fetal kidney is protected from the effects of cortisol by 11β HSD, but activity of this enzyme is low early in gestation and is downregulated by adverse intrauterine conditions late in gestation (96,97). The fetal kidney may therefore be more vulnerable to the effects of the glucocorticoids early in gestation and during conditions that lead to fetal growth retardation. Similarly, there are local changes in the renal RAS during cortisol infusion and fetal undernutrition (81,98). In particular, there are decreases in the mRNA levels for renin and AT1 receptors in the fetal kidney (61,81,98). Glucocorticoids may also induce more widespread changes in the RAS by altering pulmonary angiotensin converting enzyme (ACE), hepatic angiotensinogen gene expression, and the number and relative density of AT1 and AT2 receptors in extrarenal tissues including the brain (21). Tissue-specific changes

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in the activity of the RAS may therefore have an important role in mediating the hypertensive effects of the glucocorticoids. Certainly, antagonism of the RAS with either ACE inhibitors or AII receptor blockers in the period immediately after birth has long-term effects on adult blood pressure both in normal rats and in those deprived of protein in utero (99,100). B. Metabolic Function Adult glucose intolerance can arise from abnormalities in glucose production, insulin secretion, and/or the effectiveness of insulin in stimulating glucose uptake in the insulin-sensitive tissues. All three of these processes are known to be altered in adult animals undernourished before birth (6,7,24). In particular, protein deprivation during pregnancy in rats has been shown to lead to increased hepatic gluconeogenic enzyme levels, reduced pancreatic β cell number (6), and increased peripheral insulin resistance in the adult offspring (101,102). More specifically, these changes are associated with alterations in the insulin-signaling pathways as well as in the metabolic handling of glucose and fatty acids by the liver, adipose tissue, and skeletal muscle of adult animals (103–105). In addition, protein deprivation in utero leads to reductions in the vascularity and innervation of the islets of Langerhans in adult life (24). However, the extent to which these prenatally determined metabolic changes can be attributed to specific hormonal changes in utero remains unknown. In rats, glucocorticoid exposure in utero is known to cause glucose intolerance and hyperactivity of the hepatic gluconeogenic enzymes in the adult offspring (63,101). Glucocorticoids have also been shown to affect expression of several glucoregulatory genes (eg, glucose transporters, GLUT 1 and 4, GH receptor) in the liver and skeletal muscle before birth, which may make a major contribution to insulin-sensitive glucose disposal if the changes in gene expression persist after birth (21). In contrast, glucocorticoids appear to have relatively little effect on basal or stimulated insulin secretion in utero (27). Direct changes in the nutritional supply may therefore be more important than the endocrine environment in controlling pancreatic development (24). Certainly, in vitro experiments have shown that amino acid availability has a key role in the growth and secretory capacity of fetal pancreatic β cells in culture (7). It is possible that the transition from fetal to adult β cells that normally occurs in the pancreas during late gestation is glucocorticoid sensitive. The process of apoptosis of the fetal β cells and their replacement by adult cells is known to depend on downregulation of pancreatic IGF-II gene expression which, in other fetal tissues, is regulated by the glucocorticoids (21,37). Gluco-

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corticoid exposure in utero may therefore predispose tissues for Type 2 diabetes by effects on glucose production, insulin secretion, and insulin sensitivity.

V.

Conclusions

Hormones act as nutritional signals throughout fetal development. Their concentrations in utero reflect the type and quantity of nutrients available to the fetus and the duration of any nutritional insult. They change both the accretion and differentiation of fetal and placental tissues, and thereby adapt the pattern of growth and development to the prevailing nutritional conditions in utero. Although these changes ensure survival both before and at delivery, they may have consequences for tissue function long after birth. Early cessation of tissue accretion in response to fetal undernutrition may permanently reduce cell numbers and the functional reserve of tissues to cope with aging. Similarly, premature induction of tissue differentiation may switch organ systems from the fetal to the adult modes of functioning before delivery actually occurs, with adverse sequelae for both fetal and postnatal development. The glucocorticoids, in particular, appear to have a key role in the nutritional programming of adult disease. They act as general signals of nutrient insufficiency and activate many of the molecular switches responsible for the successful adaptation to nutrient restriction in utero. However, by resetting homeostatic and growth regulatory mechanisms, early exposure to glucocorticoids may reduce the capacity of tissues to adapt to subsequent nutritional and other homeostatic challenges. The hormone-induced adaptations in growth and development designed to ensure survival in early life may therefore increase mortality in adult life. However, a reduction in total life span may be an acceptable quid pro quo for surviving long enough to reproduce and pass on genes to the next generation.

VI. Acknowledgments We would like to thank the many members of the Department of Physiology, past and present, who contributed to these studies and the preparation of this manuscript. We are also indebted to the Biotechnology and Biological Research Council, The Birth Defects Foundation, the Horserace Betting Levy Board, and the Wellcome Trust for their financial support.

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90. Sato A, Suzuki H, Nakazato Y, Shibata H, Inagami T & Saruto T (1994). Increased expression of vascular angiotensin II type IA receptor gene in glucocorticoid induced hypertension. J Hypertension 12:511–516. 91. Brenner BM, Garcia DL and Anderson A (1988). Glomeruli and blood pressure: Less of one, more of the other? Am J Hypertens 1:335–347. 92. Hinchliffe SA, Lynch MR, Sarget PH, Howard CV, Van Velzen D (1992). The effect of intrauterine growth retardation on the development of renal nephrons. Brit J Obstet Gynec 99:296–301. 93. Celsi G, Kistner A, Aizman R, Eklof AC, Ceccatelli S, de Santiago A & Jacobson SH (1998). Prenatal dexamethasone causes oligonephronia, sodium retention and higher blood pressure in the offspring. Ped Res 44:317–322. 94. Stonestreet BS, Hansen NB, Laptook AR & Oh W (1983). Glucocorticoid accelerates renal functional maturation in fetal lambs. Early Hum Dev 8:331– 341. 95. Guillery EM, Karniski LP, Mathew MS, Pope WV, Orlowski J, Jose PA & Robillard JE (1995). Role of glucocorticoids in the maturation of renal corticol Na+/H+ exchanger activity during fetal life in sheep. Am J Physiol 268:F710– F717. 96. Murotsuki J, Gagnon R, Pu X & Yang K (1998). Chronic hypoxemia selectively downregulates 11â hydroxysteroid dehydrogenase type 2 gene expression in the fetal sheep kidney. Biol Reprod 58:234–239. 97. Wood CE & Srun R (1995). Ontogeny of 11â hydroxysteroid dehydrogenase in ovine fetal kidney and lung. Reprod Fert Dev 7:1329–1332. 98. Segar JL, Bedell K, Page WV, Mazursky JE, Nuyt A-M & Robillard JE (1995). Effect of cortisol on gene expression of the renin-angiotensin system in fetal sheep. Ped Res 37:741–746. 99. Sherman RC & Langley-Evans SC (1998). Early administration of angiotensinconverting enzyme inhibitor, captopril, prevents the development of hypertension programmed by intrauterine exposure to a maternal low-protein diet in the rat. Clin Sci 94:373–381. 100. Woods LL & Rasch R (1998). Perinatal ANG 11 programs adult blood pressure, glomerular number and renal function in rats. Am J Physiol 275:R1539– R1599. 101 Lindsay RS, Lindsay RM, Waddell BJ & Seckl JR (1996). Prenatal glucocorticoid exposure leads to offspring hyperglycaemia in the rat: studies with the 11â hydroxysteroid dehydrogenase inhibitor carbonoxolone. Diabetologia 39:1299– 1305. 102. Ozanne SE, Wang CL, Coleman N & Smith GD (1996). Altered muscle insulin sensitivity in the male offspring of protein-malnourished rats. Am J Physiol 271:E1128–1134. 103. Ozanne SE, Martensz ND, Petry CJ, Loizou CL & Hales CN (1998). Maternal low protein diet in rats programmes fatty acid desaturase activities in the offspring. Diabetologia 41:1337–1342. 104. Ozanne SE, Nave BT, Wang CL, Shepherd SR, Prins J & Smith GD (1997).

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11 The Hypothalamic-Pituitary-Adrenal and Hypothalamic-Pituitary-Gonadal Axes in Early Life Problems and Perspectives STEPHEN G. MATTHEWS, JOHN R. G. CHALLIS, DAVID B. COX, and DEBORAH M. SLOBODA University of Toronto Toronto, Ontario, Canada

ERIC JACKSON THOMAS Princess Anne Hospital Southampton, United Kingdom

CAROLINE McMILLEN

S. J. LYE

University of Adelaide Adelaide, Australia

Mount Sinai Hospital Toronto, Ontario, Canada

ROGER B. McDONALD

E. MARELYN WINTOUR

University of California Davis, California

University of Melbourne Parkville, Victoria, Australia

JANNA L. MORRISON

DAVID I. W. PHILLIPS

University of British Columbia Vancouver, British Columbia, Canada

University of Southampton and Southampton General Hospital Southampton, United Kingdom

I.

Introduction

Epidemiological studies in several populations have shown that small size at birth is associated with an increased risk of developing hypertension, non– 229

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insulin-dependent diabetes, dyslipidemia, and insulin resistance in adult life (1). This combination is known as the insulin resistance or metabolic syndrome, and it predisposes to cardiovascular disease. It is strikingly similar to the biological effects of excessive circulating glucocorticoids whether attributable to Cushing’s disease or exogenous glucocorticoid administration. This similarity has led Björntorp and others to propose that elevated glucocorticoids, possibly as a result of increased stressful stimuli in adult life, could underlie the syndrome (2). However, there is now a substantial body of evidence from animal studies and preliminary human evidence that events in early life may be a more potent cause of a lifelong increase in adrenal glucocorticoid secretion. This occurs because the set point of hypothalamic-pituitary-adrenal (HPA) function can be influenced by a variety of adverse environmental stimuli during development (Fig. 1). The increased glucocorticoid secretion throughout adult life increases the risk of the insulin resistance syndrome. Other evidence suggests that the hypothalamicpituitary-gonadal (HPG) axis may also be reset during development and contribute to these disorders.

Figure 1 Animal models show that a variety of stressors or exposure of the mother to synthetic glucocorticoids program the development of the fetal brain such that the offspring have a lifelong increase in HPA activity. This increased HPA activity is, in turn, associated with metabolic, structural, behavioral, and reproductive effects.

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II. Programming of the HPA Axis Animal models show that exposure to a variety of stressors during pregnancy, including low-protein diets, maternal restraint, ethanol, morphine, and non-abortive maternal infections, result in the birth of offspring with elevated basal or stress-induced glucocorticoid secretion (3–7). These effects can be also reproduced by administration of the synthetic glucocorticoid, dexamethasone, to rats in the last week of pregnancy (8). However, these effects were the result of fairly large doses of steroid given in late gestation. Recently, Dodic and colleagues have reported experiments in which pregnant sheep were treated with dexamethasone (0.28 mg/kg) given over 2 days, either at day 22 to 29 or at day 59 to 66 of pregnancy (term 145 days) (9). At 4, 10, 19, and 40 months postnatally, lambs from mothers who had received dexamethasone at day 22 to 29 of gestation but not at day 59 to 66 of gestation had elevated blood pressures. These animal experiments suggest that the timing of the experimental manipulations is critical for the outcome. The brain is particularly sensitive during rapid neurogenesis, but the timing of this differs greatly in different species. Many of these studies have been undertaken in rats, which—compared with other species, including man—give birth to neuroendocrinologically immature young (10). In rats, maximal brain growth does not occur until after birth (11). It has been shown in rats that the neonatal period is a particularly sensitive period, and that neonatal handling or the lack of maternal contact during this time has long-term effects of the HPA axis (12). However, the rat may be less appropriate as a model of human programming because the most rapid phase of brain growth in human infants occurs before birth (11,13). Hence, there is a need for studies in species with more similar neuroendocrine development to man. In most models, programming of the HPA axis has been linked to permanent changes in hippocampal corticosteroid receptor populations (8,12). Negative feedback at the level of the hippocampus inhibits HPA responses and is thought to act as a brake on the magnitude of stress responses (Fig. 2) (14). Hence, reduced glucocorticoid feedback in this area would increase the magnitude of stress-induced glucocorticoid secretion. However, the mechanisms involved are not completely understood. Meaney and colleagues have shown that the hippocampal changes may be mediated by modification of central serotoninergic activity (15). An increase in serotonergic activity in the hippocampus, during specific windows of development, permanently increases glucocorticoid receptor levels (16). Glucocorticoids are also implicated in aging of the hippocampus (17). Glucocorticoids have been shown to endanger hippo-

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Figure 2 Negative feedback at the level of the hippocampus inhibits the HPA axis. Fetal programming of the HPA axis is linked with permanent changes in hippocampal corticosteroid receptor populations and alterations in the feedback control of the axis.

campal neurons such that the combination of a metabolic insult (such as hypoglycemia) with altered glucocorticoids leads to neuron death (18,19). This would result in decreased glucocorticoid feedback at the levels of the hippocampus. Hence, the elevated circulating glucocorticoids after adverse prenatal events combined with exposure to stressful events and aging in adult life may amplify the prenatal programming of the hippocampus, resulting in a progressive increase in glucocorticoid secretion throughout life. One might also consider how synthetic steroids work on the developing central and peripheral nervous systems. There is good evidence that neurosteroids are particularly important in developing nervous systems, and that their synthesis may be altered by synthetic steroids such as dexamethasone (20). For example, allopregnanolone is a potent neurosteroid that can affect areas of the brain involved in bloodpressure control (21). For the synthesis of allopregnanolone, two enzymes (5α reductase, type 2 and 3α dehydrogenase) are required. The gene for 5α reductase type 2 is transiently expressed in rat brain in late gestation (22). The 3á hydroxysteroid dehydrogenases (HSDs) are inducible by dexamethasone working on elements unrelated to classical glucocorticoid response elements (23), probably via the recently de-

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scribed pregnane X receptor (24). Thus it is possible that synthetic steroids may affect brain development in critical periods, in ways in which natural glucocorticoids would not. Human evidence for HPA axis programming so far is limited. However, small babies have high umbilical cord cortisol concentrations, elevated cortisol metabolite excretion in childhood, and in a recent study in Hertfordshire, 64-year-old men who were small at birth had high fasting plasma glucocorticoid concentrations and increased adrenal responsiveness to synacthen (see Chapter 7) (25,26). A. Mother, Fetus, or Placenta? If HPA programming is an important mechanism linking reduced fetal growth with the metabolic syndrome in adult life, a number of problems remain unanswered. Because fetal growth is dependent largely on nutrient and oxygen availability, it has been suggested that the long-term consequences of small size at birth are attributable to the persistence of fetal adaptations to undernutrition or underoxygenation (27). However, it is not clear the extent to which fetal undernutrition depends on the maternal diet during pregnancy, maternal nutritional reserves, or local factors including placental blood flow or placental nutrient transfer. In arguing for a “nutritional” cause for the long-term adverse effects of reduced fetal growth, Barker has pointed to animal models of maternal undernutrition and the effects of starvation during pregnancy (eg, Dutch Hunger Winter) (28–30). Although these interventions are associated with programmed increases in blood pressure or glucose intolerance, it is not clear whether the effects are attributable to fetal nutrient deficiency or are mediated by increased maternal stress hormones gaining access to the fetus and programming the HPA. There are relatively limited data on whether acute or chronic stressors such as maternal undernutrition result in significant fetal exposure to maternal glucocorticoids, and it appears that this is an important area for investigation in both human subjects and animal studies. The effects are likely to be complex. They may be in different stages of pregnancy and may depend on the mother’s pre-existing nutritional status. Exposure to other nonnutritional, “stressful” life events during pregnancy may also program the fetus. Currently, little is known about this possibility in humans despite the wealth of animal evidence that a variety of maternal stressors, including restraint, may program the HPA axis of the offspring (3–7). One particularly important gap in our knowledge is whether the repeated administration of synthetic glucocorticoids to pregnant women has long-term

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adverse effects on the offspring. The human fetus may be exposed to exogenous synthetic glucocorticoids in at least two situations. In the comparatively rare cases of congenital adrenal hyperplasia, dexamethasone is administered to the mother, and crosses the placenta to the fetus, for long periods in order to suppress the release of excess adrenocorticotrophin (ACTH) by the fetal pituitary. The rationale is that suppression of the excess ACTH release that occurs in the absence of normal cortisol production by the fetal adrenal will suppress the formation of excessive adrenal androgens, which would masculinize a female fetus. In this respect, it is generally quite effective. However, treatment must be started before the sex of the fetus can be determined, and before it is possible to know if the defective gene is actually carried by the fetus (31). Thus, seven out of eight fetuses exposed to dexamethasone from weeks 5 to 12 are subsequently found to be male or not to carry the defective gene for cortisol biosynthesis, and thus reap no benefit. The question as to whether this steroid exposure might have some detrimental side effects is a very real concern. So far there have been two studies in which children so treated have been examined at some time after birth (32,33). It is clearly important that ongoing follow-up occurs particularly with respect to the potential for development of cardiovascular and/or metabolic disease. In the second situation where the human fetus is exposed to exogenous steroids, a much larger population of babies are treated with steroids later in development. These are the fetuses of women threatening to deliver prematurely. Antenatal treatment with glucocorticoids is undoubtedly effective at reducing the risk of respiratory distress syndrome in the newborn. However, it is relatively common practice for more than one treatment to be administered when the threatened delivery does not occur after the first treatment (each treatment consists of exposure to a synthetic steroid—usually betamethasone, for 48 h), without any scientific proof that multiple treatments are more efficacious than single treatments (34). The potential detrimental consequences of multiple exposures to synthetic glucocorticoids also requires systematic investigation. There is an urgent need for properly controlled follow-up studies, which should include tests of HPA axis function, cardiovascular physiology, and glucose tolerance. However, in terms of fetal long-term health, it may not matter whether the elevated fetal glucocorticoids are derived from endogenous production in the fetus, the mother, or from synthetic glucocorticoid administration. The differences are likely to be dose-dependent depending on the activity of the placental enzyme 11β hydroxysteroid dehydrogenase (11βOHSD) and perhaps also cortisol-binding globulin concentrations in the fetal and maternal compartments.

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It is also possible that the HPA axis activity could account for some of the transgenerational effects of low birthweight. It has long been known that the birthweight of a child is associated with the maternal and grandmaternal (but not paternal or grandpaternal) birthweight. If low birthweight in a woman was associated with increased HPA activity during her pregnancies, this could program the HPA axis of her offspring and by this means affect subsequent generations. If maternal stress is to affect the fetus, then her circulating stress hormones must either cross the placenta or affect placental function in some other way. The extent to which glucocorticoids are able to cross the placenta, however, is not clear. The enzyme 11βOHSD type 2 normally acts as a barrier to maternal glucocorticoid by converting it to inactive metabolites (35). Thus, in most species, fetal glucocorticoid concentrations are maintained at about one tenth of maternal levels (36). Yet there is increasing evidence that maternal cortisol does reach the fetus (37) as it has been shown that the circadian rhythm of fetal androgens are inversely related to the maternal circadian cortisol rhythm (38,39). The efficiency of the placental 11βOHSD may depend on a multiplicity of factors, including the stage of gestation, the species involved, and the concentration gradient, and may also be directly affected by undernutrition and other maternal insults. If we are to understand the nature of HPA programming, the nature of the placental 11βOHSD barrier in humans needs to be characterized. There are also some early experimental data which indicate that an increase in glucocorticoids may alter structural and functional features of the placenta (40). Even less is known about fetal HPA responses. Although animal studies suggest that both fetal undernutrition and fetal hypoxia result in increased fetal glucocorticoid levels, little is known about the extent to which fetal cortisol concentrations must be elevated in order to reprogram the HPA axis. The available evidence suggests that the stimulus to increase glucocorticoid production may originate within the fetal brain, but it may also be derived from the placenta. Again, despite differences in fetal adrenal histology and in placental steroidogenic capacity between humans, lower primates, and other animal species, there is no reason to presume a priori that the mechanism of glucocorticoid effects in the fetus will be fundamentally different between the species. The capacity of the fetus to measure and respond to a mismatch between its energy requirement and the ability of the mother and/or placenta to supply the energy to meet the demand requires further investigation. In addition, the extent to which fetal insulin, IGF, leptin, and cortisol concentrations are signals of fetal energy stores needs to be defined. Clearly, human fetal studies can only provide limited information, but there is a need for better

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characterization of the endocrinology of the human growth-retarded neonate or infant. III. The HPG Axis There is substantial evidence that the HPG axis can be programmed by gonadal steroids. Thirty years ago, Barraclough showed that the developing hypothalamus was plastic, and exposure of female rats to testosterone at a critical window in the neonatal period resulted in permanent masculinization of the animal (41). These and similar experiments have now been reported in a variety of different species with the same dramatic effects. The critical windows for programming of HPG function are similar to those for HPA programming and are highly species-specific. The ability of testosterone to alter hypothalamic development depends on the initial intraneural conversion to estradiol that occurs by aromatization by a cytochrome P450-dependent aromatase (42). There are close links between gonadal hormones and the development of the HPA axis and HP-thyroid axis. As a result, altered programming of one system will affect the other indirectly. This is illustrated by the sex differences in HPA function (43). Gonadal steroids are known to have profound influences on hippocampal corticosteroid receptor expression, leading to sex differences in both glucocorticoid and mineralocorticoid expression (44). Further, prenatal stress (which increases fetal glucocorticoid exposure) leads to male offspring with reduced testosterone and female offspring with elevated plasma testosterone levels (45). Recent human evidence suggests that events in early life are linked with programming of gonadotrophin hormones and reproductive function. Thus, for example, birth size is linked with several cancers of the reproductive system, including ovary and breast (46,47). Small size at birth is associated with earlier menarche (48). Finally, polycystic ovary syndrome, the most common endocrine disorder in women, is known to be linked to patterns of early growth (49). Women who were born postmature and those who had high birthweight had a markedly higher prevalence of this syndrome. Although these results need confirmation in another study, they suggest that studies of the relationship between the early environment and gonadotrophin or gonadal steroid secretion in men and women will help in the understanding of the pathophysiology of several reproductive diseases.

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IV. Conclusions Steroid hormones are powerful mediators of fetal programming and are known to be programmable by physiological stimuli during the course of development. Programming is a biological mechanism present in many longlived species, adding a survival advantage that enables a developing organism to adapt its physiology to the prevailing environment to a greater extent than is possible in the adult organism. Increasing evidence suggests that programming of the HPA and HPG axes may play an important role in several human diseases, including the metabolic syndrome and a number of disorders of reproduction. The challenge for researchers is to identify and characterize these mechanisms, because they may offer new therapeutic opportunities in these diseases.

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25. Clark PM, Hindmarsh PC, Sheill AW, Law CM, Honour JW, Barker DJP. Size at birth and adrenocortical function in childhood. Clin Endocrinology 1996; 45:721– 726. 26. Phillips DIW, Barker DJP, Fall CHD, et al. Elevated plasma cortisol concentrations; a link between low birthweight and the insulin resistance syndrome? J Clin Endo Metab 1998; 83:757–760. 27. Barker DJP, Gluckman PD, Godfrey KM, Harding JE, Owens JA, Robinson JS. Fetal nutrition and cardiovascular disease in adult life. Lancet 1993; 341:938– 941. 28. Snoek A, Remacle C, Reusens B, Hoet JJ. Effect of a low protein diet during pregnancy on the fetal rat endocrine pancreas. Biol Neonate 1990; 57:107–118. 29. Langley SC, Jackson AA. Increased systolic blood pressure in adult rats induced by fetal exposure to maternal low protein diets. Clin Science 1994; 86:217–222. 30. Ravelli ACJ, van der Meulen JHP, Michels RPJ, et al. Glucose tolerance in adults after prenatal exposure to famine. Lancet 1998; 351:173–177. 31. Forest MG, Morel Y, David M. Prenatal treatment of congenital adrenal hypoplasia. Trends Endocrinol Metab 1998; 9:284–289. 32. Trautman PD, Meyer-Bahlburg HFL, Postelnek J, New MI. Effect of early prenatal dexamethasone on the cognitive and behavioral development of young children: results of a pilot study. Psychoneuroendocrinology 1995; 18:343–354. 33. Lajic S, Wedell A, The-Hung B, Ritzez EM, Holst M. Long-term somatic followup of prenatally treated children with congenital adrenal hyperplasia. J Clin Endo Metab 1998; 83:3872–3880. 34. Crowley P, Chalmers I, Keirse MJN. The effects of corticosteroid administration before preterm delivery: an overview of the evidence from controlled trials. Br J Obstet Gynacol 1990; 97:11–25. 35. Seckl JR, Chapman KE. Medical and physiological aspects of the 11-betahydroxysteroid dehydrogenase system. Eur J Biochem 1997; 249:361–364. 36. Cadet R, Pradier P, Dalle M, Delost P. Effects of prenatal maternal stress on the pituitary adrenocortical reactivity in guinea pig pups. J Dev Physiol 1986; 8:467– 475. 37. Gitau R, Cameron A, Fisk NM, Glover V. Fetal exposure to maternal cortisol. Lancet 1998; 352:707–708. 38. Patrick JE, Challis J, Campbell K, Carmichael L, Natale R, Richardson B. Circadian rhythms in maternal plasma cortisol and estriol at 30 to 31, 34 to 35 and 38 to 39 weeks’ gestation. Am J Obstet Gynecol 1980; 136:325–334. 39. Patrick JE, Challis JRG, Natale R, Richardson B. Circadian rhythms in maternal plasma cortisol, estrone, estradiol and estriol at 34 to 35 week’s gestation. Am J Obstet Gynecol 1979; 135:791–798. 40. Sun K, Yang K, Challis J. Glucocorticoid action and metabolism in pregnancy: implications for placental function and fetal cardiovascular activity. Placenta 1998; 19:353–360. 41. Barraclough CA. Modifications in the CNS regulation of reproduction after expo-

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12 Reduced Fetal Growth and Pediatric Endocrinopathies FRANCIS de ZEGHER and INGE FRANCOIS

LOURDES IBÁÑEZ

University of Leuven Leuven, Belgium

University of Barcelona Barcelona, Spain

Over the past few years, pediatric endocrinology has witnessed the unfolding of a novel concept based on four solid cornerstones. The concept associates reduced prenatal growth to a series of postnatal endocrinopathies. It is currently unknown whether the latter are sequelae of the former or whether both are the result of a more fundamental defect that remains to be defined. The first cornerstone for this concept—as for all medical paradigms—consists of a series of carefully documented clinical observations. The second cornerstone is the principle of the “critical window,” originally derived from undernutrition studies in early life (1,2), and nowadays established not only within the cascades of genetic switches directing tissue differentiation but also within other developmental events that proceed rapidly and are therefore vulnerable. The third cornerstone is the recognition that some components of the endocrine system (eg, the anterior pituitary, the gonads, and the inner zone of the adrenal cortex) are more active in early life than in childhood or adulthood, implying that modulation of pronounced prenatal activity might account for 241

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some of the postnatal variations that had remained unexplained. The fourth cornerstone was provided by Barker and colleagues (3), who compiled evidence consolidating the two aforementioned principles as indeed applicable to endocrine-related disorders in adulthood and senescence. Convincing evidence linking reduced prenatal growth to pediatric endocrinopathies is accumulating. This chapter summarizes available data on these relationships in regards to the emerging implications for clinical practice. In particular, we focus on some aspects of the somatotropic axis, adrenarche and pubarche, sexual differentiation and gonadal function, lipid metabolism, and insulin sensitivity. Most of the discussed observations concern children with either no dysmorphologic condition or with a Silver-Russell morphotype. I.

Somatotropic Axis

By definition, approximately 3% of human infants are born small for gestational age (SGA), or less than the third percentile. The vast majority of these infants experience an early and rapid catch-up growth and reach normal stature by 2 years of age (4). Approximately 10% maintain a height below –2 standard deviations (SD) at least throughout childhood (5,6). The mechanisms underlying the growth failure in this small percentage remain incompletely understood. In short SGA children, there is an increased incidence of growth hormone deficiency (GHD), either in the classic form, which is detectable by conventional stimulation tests, or in the more subtle neurosecretory form, which is detectable by growth hormone (GH) profile studies (7). However, the majority of short SGA children appear to be neither GHD- nor GH-resistant, as indicated by basal serum levels of insulinlike growth factor 1 (IGF-1) and IGF-binding protein 3 (IGFBP-3) that are within the normal range, and/or by normal IGF-1 and IGFBP-3 responses to exogenous GH, and/or by relatively low serum leptin concentrations (8,9). Thus, available evidence points towards some form of IGF-1 resistance as the predominant factor responsible for the persistent growth failure in this group. The pathophysiology of this type of IGF-1 resistance remains to be clarified. It may have been induced by an adverse prenatal environment that programmed the growth plates at a lower level of responsiveness within a critical window of time (10–12). In addition, GHD in SGA children is commonly associated with IGF-1 resistance, which is thought to account for an average reduction of 20% in the growth-promoting efficacy of GH therapy when given to these children (12). When a supraphysiological dose of GH is administered (Fig. 1), the growth response of short SGA children

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Figure 1 Epianalysis results (mean ± SEM) of height gain (left panel) and weight gain (right panel) from three independent, randomized, controlled, multicenter studies in short, prepubertal, non–GH-deficient SGA children treated with three doses of GH over 2 years. The study population (n = 146) had a mean birthweight of –2.9 SDS, birth length of –3.6 SDS, chronological age of 4.9 years (range 2–8 years), actual height of –3.6 SDS, and weight of –6.3 SDS. GH-induced catch-up growth is dose-dependent. Note the lack of spontaneous height gain in the untreated control group. (Adapted from Ref. 12.)

(regardless of their secretory GH status) readily matches that of other GHD children, indicating that the IGF-1 resistance towards growth can be overcome and that a normal height and weight can be obtained at least throughout childhood (12). Therefore, it is anticipated that the indications and doses for GH therapy in children will become increasingly interlinked with the emerging principles of endocrine programming in early life. Growth-hormone–deficient children are candidates for continuous GH therapy using a physiological dose if they were not born SGA, and a higher dose if they were SGA. For non-GHD SGA children, the two major determinants of the growth response to exogenous GH are (1) the dose of GH administered and (2) the age of the child. The higher the GH dose and the younger the child, the more pronounced the response (7,12). Two treatment avenues are being explored. The more conventional strategy is to treat these SGA children as if they were GHD, ie, with a continuous GH regimen using a slightly supraphysiological dose (0.033 mg/kg/d) throughout childhood. A more innovative strategy aims at restoring the altered responsiveness within the growth plate by administering a high dose (0.067–0.100 mg/kg/d) of GH treatment early in life and over only 2 years (to stay within the presumed critical window). This approach results in an early and rapid normalization of body size, thus mimicking, albeit

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at a later age, the spontaneous catch-up growth that failed to occur in these children. More importantly, there are now preliminary data up to 6 years after GH withdrawal indicating that early and high-dose GH treatment may have the capacity to reprogram the growth pattern at a higher level in the majority of these children. If these findings are confirmed, they ultimately may result in the development of an early and brief treatment regimen with fewer injections and a greatly improved cost–benefit ratio compared with the more conventional strategy. Finally, we emphasize that, to date, the different GH-treatment regimens applied to SGA children have a reassuring safety profile and do not lead to inappropriate acceleration of bone maturation or to disproportionate growth, eg, within the craniofacial complex (12,13). II. Pronounced Adrenarche and Precocious Pubarche Almost 80% of the fetal adrenal consists of the so-called “fetal adrenal zone,” which is located in the inner area of the cortex and virtually disappears in early infancy. It has been known for 25 years that reduced fetal growth is associated with reduced size of the fetal adrenal zone and reduced concentrations of dehydroepiandrosterone sulfate (DHEAS) in fetal serum. “Adrenarche” refers to the endocrine process that results in rising serum concentrations of adrenal androgens produced by the zona reticularis within the inner part of the adrenal cortex. Adrenarche occurs normally between 6 and 8 years of age. “Pubarche” refers to the appearance of public hair. By definition, pubarche before the chronological age of 8 years is considered to be precocious. Two independent studies recently indicated that adrenarche is more pronounced in children born SGA as compared with appropriate-for-gestational age (AGA) controls, particularly if the SGA children had experienced spontaneous catch-up growth (14,15). These biochemical findings are already becoming of clinical relevance as idiopathic precocious pubarche, a frequent clinical expression of early adrenarche, has also been associated with reduced fetal growth (16). III. The Entities of Male Pseudohermaphroditism and Subfertility, Ovarian Hyperandrogenism, and Anovulation The embryonic gonads play a crucial role in sexual differentiation. Subsequently, during fetal and neonatal life the human gonads present a high profile

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of secretory activity. In turn, this phase is followed by a decade of low activity under neuroendocrine inhibition, and then by gonadal steroid hormone production and pubertal development, which leads to a fully reproductive status. Androgen insensitivity, a form of male pseudohermaphroditism (17), was found to result in a slightly reduced birth weight (–0.4 SD on average) (18). Even more intriguing is the recent finding that unexplained forms of male pseudohermaphroditism appear to be associated with a markedly reduced birth weight (–2 SD on average) (19). Although reduced fetal growth has been known to be associated with subsequent testicular dysfunction for half a century (20,21), only recently has a specific link with male subfertility been recognized (22). Reduced fetal

Figure 2 Birth weight SD scores of postmenarcheal control girls (–, – and –) and postmenarcheal girls with a history of precocious pubarche without ovarian hyperandrogenism and without hyperinsulinemia (+, – and –), or with ovarian hyperandrogenism and without hyperinsulinemia (+, + and –), or with both ovarian hyperandrogenism and hyperinsulinemia (+, + and +). The diagnosis of ovarian hyperandrogenism was based on the 17-alpha-OH-progesterone response to LHRHagonist; hyperinsulinemia refers to an excessive insulin response during a standardized oral glucose tolerance test. (Adapted from Ref. 16.)

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growth is thought to be accompanied by an early decrease in the number and/or function of Sertoli cells which, in turn, appear to be one of the key determinants of adult spermatogenesis (22). Reduced fetal growth has also been associated with a reduced fraction of primordial follicles in fetal ovaries (23), with ovarian hyperandrogenism in adolescent girls (Fig. 2) (16), and with anovulation, particularly dating from late adolescence. The timing of menarche and menopause, however, are quite independent of birth weight (24,25). IV. Dyslipidemia and Insulin Resistance Five years ago, Barker et al. identified the relation between reduced fetal growth and syndrome X in senescent men (26). Asymptomatic insulin resistance has now been documented in SGA children with and without spontaneous catch-up growth, and is often detectable before puberty (16,27). In a sequence of pediatric endocrinopathies linked to reduced fetal growth, insulin resistance was found to be associated with the lowest birth weights (Fig. 2). V.

Conclusion

There is increasing evidence that reduced prenatal growth is associated with a modulation in the function of multiple components within the postnatal endocrine system. A minority of children born SGA experience a persistent reduction of postnatal growth, which has been attributed to either classic or neurosecretory GHD and/or to some form of IGF-1 resistance. The short stature of most prepubertal SGA children can be normalized with a variety of GH treatment regimens; however, on average, a slightly higher-than-conventional dose seems to be required. Adrenarche appears to be amplified in SGA children, and idiopathic precocious pubarche has been associated with relatively low birth weights. Similar associations have been found for male pseudohermaphroditism and subfertility and for ovarian hyperandrogenism and anovulation. Finally, insulin resistance has been documented in both prepubertal and pubertal SGA children with and without spontaneous catch-up growth. Thus, children born small appear to be at increased risk for experiencing endocrine and metabolic dysfunction; in addition, the lower the birth weight, the more prone the child seems to be for developing a broad spectrum of endocrine and metabolic anomalies. The challenge now is to identify the molecular and cellular mechanisms underlying these associations so that preventive strategies and

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more than merely symptomatic treatments can be designed. A new chapter in pediatric endocrinology has begun. References 1. 2.

3. 4.

5.

6. 7.

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14.

McCance RA, Widdowson EM. Proc R Soc Med (Lond) 1962; 156:326–337. Widdowson EM, McCance RA. The effect of finite periods of undernutrition at different ages on the composition and subsequent development of the rat. Proc R Soc Med (Lond) 1963; 158:329–342. Barker DJP, Mothers, Babies and Diseases in Later Life. New York: Churchill Livingstone, 1998. Hokken-Koelega ACS, De Ridder MAJ, Lemmen RJ, Den Hartog H, De Muinck Keizer-Schrama SMPF, Drop SLS. Children born small for gestational age: do they catch up? Pediatr Res 1995; 38:267–271. Albertsson-Wikland K, Karlberg J. Natural growth in children born small for gestational age with and without catch-up growth. Acta Paediatr Scand 1994; 399(suppl):64–70. Karlberg J, Albertsson-Wikland K. Growth in full-term small-for-gestational-age infants: from birth to final height. Pediatr Res 1995; 38:733–739. de Zegher F, Francois I, van Helvoirt M, Van den Berghe G. Small as fetus and short as child: from endogenous to exogenous growth hormone. J Clin Endocrinol Metab 1997; 82:2021–2026. de Zegher F, Maes M, Gargosky SE, et al. High-dose growth hormone treatment of short children born small for gestational age. J Clin Endocrinol Metab 1996; 81:1887–1892. Boguszewski M, Albertsson-Wikland K, Aronsson S, et al. Growth hormone treatment of short children born small-for-gestational-age: the nordic multicentre trial. Acta Paediatr 1998; 87:257–263. Balsamo A, Tassoni P, Cassio A, et al. Response to growth hormone therapy in patients with growth hormone deficiency who at birth were small or appropriate in size for gestational age. J Pediatr 1995; 126:474–477. Chatelain PG, Cauderay MC, de Zegher F, et al. Growth hormone secretion and sensitivity in children born small for gestational age. Acta Paediatr Suppl 1996; 417:15–16. de Zegher F, Francois I, van Helvoirt M, Beckers D, Ibanez L, Chatelain P. Growth hormone treatment of short children born small for gestational age. Trends Endocrinol Metab 1998; 9:233–237. Van Erum R, Mulier M, Carels C, de Zegher F. Craniofacial growth and dental maturation in short children born small for gestational age: effects of growth hormone treatment. Horm Res 1998; 50:141–146. Clark PM, Hindmarsh PC, Shiell AW, Law CM, Honour JW, Barker DJP. Size

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de Zegher, Francois, Ibáñez at birth and adreno-cortical function in childhood. Clin Endocrinol 1996; 45:721– 726. Francois I, de Zegher F. Adrenarche and fetal growth. Pediatr Res 1997; 41:440– 442. Ibanez L, Potau N, Francois I, de Zegher F. Precocious pubarche, hyperinsulinism and ovarian hyperandrogenism in girls: relation to reduced fetal growth. J Clin Endocrinol Metab 1998; 83:3558–3562. French FS, Lubahn DB, Brown TR, et al. The molecular basis of androgen insensitivity. Prog Horm Res 1990; 46:1–38. de Zegher F, Francois I, Boehmer A, et al. Androgens and fetal growth. Horm Res 1998; 50:243–244. Francois I, van Helvoirt M, de Zegher F. Male pseudohermaphroditism related to complications at conception, in early pregnancy and in prenatal growth. Horm Res 1999; 51:91–95. Silver HK, Kiyasu N, George J, Deamer WC. Syndrome of congenital hemihypertrophy, shortness of stature and elevated urinary gonadotrophins. Pediatrics 1953; 12:368–372. Angehrn V, Zachmann M, Prader A. Silver-Russell syndrome: observations in 20 patients. Helv Paediatr Acta 1979; 84:297–308. Francois I, de Zegher F, Spiessens C, D’Hooghe T, Vanderschueren D. Low birth weight and subsequent male subfertility. Pediatr Res 1997; 42:899–901. de Bruin JP, Dorland M, Bruinse HW, Spliet W, Nikkels PGJ, Te Velde ER. Fetal growth retardation as a cause of impaired ovarian development. Early Hum Dev 1998; 51:39–46. Cooper C, Kuh D, Egger P, Wadsworth M, Barker DJP. Childhood growth and age at menarche. Br J Obstet Gynaecol 1996; 103:814–817. Cresswell JL, Egger P, Fall CH, Osmond C, Fraser RB, Barker DJP. Is the age of menopause determined in utero? Early Hum Dev 1997; 49:143–148. Barker DJP, Hales CN, Fall CH, Osmond C, Phipps K, Clark PM. Non-insulin dependent diabetes mellitus, hypertension and hyperlipidaemia (syndrome x): relation to reduced fetal growth. Diabetologia 1993; 36:62–67. Hofman PL, Cutfield WS, Robinsom EM, Bergman RN, Menon RK, Sperling MA, Gluckman PD. Insulin resistance in short children with intrauterine growth retardation. J Clin Endocrinol Metab 1997; 82:402–406.

13 Maternal Nutrition and Fetal Development Implications for Fetal Programming KEITH M. GODFREY University of Southampton and Southampton General Hospital Southampton, United Kingdom

I.

Introduction

The demonstration that people who were small or thin at birth are at an increased risk of developing coronary heart disease, hypertension, and non– insulin-dependent diabetes during adult life raises the important question of the influences acting during fetal growth and development that underlie the long-term associations. The recent observation that maternal diet and body composition in pregnancy can have long-term consequences for the offspring without necessarily affecting size at birth has indicated the need for a fundamental reevaluation of the regulation of fetal growth and development. This has revealed many unanswered question about the effect of maternal nutrition on human fetal development. Until such issues are addressed, it is unlikely that we will understand the true impact of maternal diet and body composition. 249

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Although the fetal genome determines growth potential in utero, the weight of evidence suggests that it plays a subordinate role in determining the growth that is actually achieved (1,2). Rather, it seems that the dominant determinant of fetal growth is the nutritional and hormonal milieu in which the fetus develops, and in particular the nutrient and oxygen supply (3,4). Evidence supporting the importance of the intrauterine environment comes from animal cross-breeding experiments (5) and from studies of half-siblings related either through the mother or the father (6). For example, among half-siblings related through only one parent, those with the same mother have similar birthweights, the correlation coefficient being 0.58; the birthweights of half-siblings with the same father are dissimilar, the correlation coefficient being only 0.1 (6). A recent study of babies born after ovum donation illustrates how birth size is essentially controlled by the mother’s body and the nutritional environment it affords (7). The birthweights of the babies were strongly related to the weight of the recipient mother, heavier mothers having larger babies. Birthweights were, however, unrelated to the weights of the women who donated the eggs. Although maternal cigarette smoking is known to also restrict fetal growth, initial followup studies have found that it is not related to levels of cardiovascular risk factors in the offspring in childhood (8,9). Conceptually, size at birth may be thought of as reflecting the product of the fetus’s trajectory of growth and the maternoplacental capacity to supply sufficient nutrients to maintain that trajectory. Experimental studies in animals and recent observations in humans have shown that a mother’s own fetal growth and her dietary intakes and body composition can exert major effects on the balance between the fetal demand for nutrients and the maternoplacental capacity to meet that demand (10). Failure of the maternoplacental supply line to satisfy fetal nutrient requirements results in fetal undernutrition and leads to a range of fetal adaptations and developmental changes. Although these may confer an initial survival advantage to the fetus, it is thought that they may lead to permanent alterations in the body’s structure and metabolism, and consequently to cardiovascular and metabolic disease in adult life (10,11). Figure 1 shows a framework illustrating this hypothesis. The hypothesis suggests that understanding the causes and consequences of an imbalance between fetal nutrient demand and maternoplacental supply will provide insights into the influences that underlie cardiovascular and metabolic disease in later adult life. Indeed, it could be argued that the 40 to 70

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Figure 1 The regulation of fetal development—a conceptual framework. year time scale required to observe the full effects of an adverse intrauterine environment dictates that identification of interventions that confer longterm benefits is likely to require characterization of the fetal adaptations and developmental changes that underlie programming. Such characterization would provide important end points for future interventional studies. III.

Fetal Nutrient Demand

A rapid trajectory of growth increases the fetus’s demand for nutrients. This reflects effects on both maintenance requirements, greater in fetuses that have achieved a larger size as a result of a faster growth trajectory, and on requirements for future growth. Although the fetal demand for nutrients is greatest late in pregnancy, the magnitude of this demand is thought to be primarily

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determined by genetic and environmental effects on the trajectory of fetal growth set at an early stage in development. Experimental studies of pregnant ewes have shown that, although a fast growth trajectory is generally associated with larger fetal size and improved neonatal survival, it does render the fetus more vulnerable to a reduced maternoplacental supply of nutrients in late gestation. Thus, maternal undernutrition during the last trimester adversely affected the development of rapidly growing fetuses with high requirements, while having little effect on those growing more slowly (12). Rapidly growing fetuses were found to make a series of adaptations in order to survive, including fetal wasting and placental oxidation of fetal amino acids to maintain lactate output to the fetus (12). Although the identity of the major genes determining growth potential and the fetal growth trajectory is unknown, animal studies indicate that insulinlike growth factors (IGF) and their receptors may be important. While the glucose-insulin-IGF-1 axis is currently thought to play a central role in the nutritional regulation of fetal growth and anabolism, constitutively expressed IGF-2 is thought to play an important role in determining background rates of fetal growth from early pregnancy onwards (13,14). Experiments in animals have shown that periconceptional alterations in maternal diet and plasma progesterone concentrations can alter gene expression in the preimplantation embryo to change the fetal growth trajectory (15,16). Environmental effects have been shown on both embryonic growth rates and on cell allocation in the preimplantation embryo. Maternal progesterone treatment can, for example, permanently alter the trajectory of fetal growth by changing the allocation of cells between the inner cell mass that develops into the fetus and the outer trophectoderm that becomes the placenta (15,16). The trajectory of fetal growth is thought to increase with improvements in periconceptional nutrition, and is faster in male fetuses (17). These observations challenge the view that fetal size in early pregnancy simply reflects the duration of time since conception. They raise the possibility that the greater vulnerability of such fetuses on a fast growth trajectory could contribute to the rise in coronary heart disease with Westernization and the higher death rates in men. IV. Maternoplacental Nutrient Supply Maintenance of the fetal growth trajectory is dependent on an adequate maternoplacental supply of nutrients. Recent observations suggest that a woman’s diet, body composition, and her own fetal growth all influence the supply

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of nutrients to the fetus. These effects may act either directly on the availability and partitioning of nutrients between plasma in the maternal and fetal microvasculature, or indirectly through effects on the size and transfer capabilities of the placenta and on uteroplacental blood flow. A. Maternal Dietary Intakes

In early pregnancy, a woman’s intakes of macronutrients are unlikely to have direct effects on fetal tissue accretion rates as the fetus’s absolute nutrient requirements are low. Macronutrient intakes in early pregnancy may, however, have indirect effects on later fetal growth as a result of alterations in embryonic development, placental growth, and uteroplacental blood flow. In late pregnancy, fetal requirements increase markedly towards term, and although maternal intakes could then limit fetal growth directly, randomized controlled trials of maternal macronutrient supplementation in Western communities have had relatively small effects on birthweight (18). To consider absolute macronutrient intakes may, however, be oversimplistic, and the source of protein, micronutrient content and balance of nutrients in the diet may be more important. Indications that the source of dietary protein may be important have come from observational studies of maternal nutrition. Among a group of pregnant women in Southampton, low intakes of meat protein in late pregnancy were associated with lower birthweight and low intakes of dairy protein with low placental weight (19). Analyses of a survey of maternal diet in Aberdeen similarly showed that low intakes of animal protein in relation to carbohydrate in late pregnancy were associated with low placental weight (20); this survey, however, did not differentiate meat and dairy protein. Further studies are needed to confirm the importance of dairy protein intake, and to identify the critical nutrient(s) associated with it that might alter placental growth and development. The differing effects of meat, dairy, and cereal protein found in these studies could reflect differences in the amino acid composition of the proteins or parallel differences in the micronutrient content of the different sources of dietary protein. An effect of calcium intakes in dairy products is, for example, supported by a follow-up study of children whose mothers took part in a trial of calcium supplementation in pregnancy (21); this found that maternal supplementation was associated with lowering of the offspring’s blood pressure in childhood. Indications that macronutrient balance may be important have come from a follow-up study of men and women whose mothers took part in the Aberdeen survey of diet in pregnancy (20). Alterations in placental weight at

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birth and raised blood pressure in adult life were found at either extreme of the balance of maternal animal protein to carbohydrate intake in pregnancy (20). Support for adverse effects of a high ratio of animal protein to carbohydrate comes from a review of 16 trials of protein supplementation showing that supplements with a high protein density were consistently associated with lower birthweight (22). Support for adverse effects of a diet with a low ratio of animal protein to carbohydrate comes from studies in pregnant rats showing that a low protein density diet altered fetal and placental development, and was associated with lifelong elevation of blood pressure in the offspring (23). B. Maternal Body Composition

Follow-up studies of children and adults for whom maternal data were recorded have shown that maternal thinness is associated with adverse effects in the offspring. Initial support for the hypothesis that maternal thinness could program levels of cardiovascular risk factors in the offspring came from a follow-up study of children in Jamaica (24). Children whose mothers had thin skinfold thicknesses in pregnancy and a low pregnancy weight gain had higher blood pressure at the age of 11 years (Fig. 2). A subsequent study of 11-year-old children in Birmingham found similar associations (25). Follow-up of a group of men and women in Mysore, India has shown that a low maternal weight in pregnancy is associated with insulin resistance and an increased risk of coronary heart disease in the offspring in adult life (26,27). Short, thin mothers were found to produce small, thin babies that had an increased risk of hypertension, insulin resistance, and coronary heart disease in adult life (26,27). Adverse long-term effects in the offspring of short, thin women may reflect diminished nutrient availability and supply to the fetus. A follow-up study of men in Finland born earlier this century suggests, however, that interventions that simply increase maternal fatness are unlikely to improve long-term outcomes (28). Markedly raised coronary heart disease death rates were found in men whose mothers had a high body mass index in pregnancy. This effect was independent of an association between thinness at birth and increased rates of adult coronary heart disease. Modeling the data to derive contour lines of similar coronary heart disease death rates indicated that increasing maternal body mass index had little effect on the offspring’s death rates in tall women, but strong effects in short women (11,28). One interpretation of these findings is that greater maternal body fatness may increase fetal growth, and hence the fetal demand for nutrients; short women may not be able to meet this increased demand as a result of a constrained

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Figure 2 Systolic pressure of 11-year-old Jamaican children according to the mother’s triceps skinfold thickness in pregnancy. (From Ref. 24.) nutrient supply capacity determined during their own intrauterine development (28). C. Intergenerational Effects

Experimental an have cumulative effects on reproductive performance over several generations. Thus, feeding rats a protein-deficient diet over 12 generations resulted in progressively greater fetal growth retardation over the generations; after refeeding with a normal diet, it took three generations to normalize growth and development (29). Strong evidence for major intergenerational effects in humans has come from studies showing that a woman’s birthweight influences the birthweight of her offspring (30,31). We have also found that whereas low-birthweight mothers tend to have thin infants with a low ponderal index, the father’s birthweight was unrelated to ponderal index at birth (Fig. 3); crown-heel

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Figure 3 Ponderal index at birth according to the mother’s and father’s birthweights. length at birth was, however, more strongly related to the father’s birthweight than to the mother’s (32). The effect of maternal birthweight on thinness at birth is consistent with the hypothesis that the maternoplacental supply line may be unable to satisfy fetal nutrient demand in low-birthweight mothers. Potential mechanisms underlying this effect include alterations in the uterine or systemic vasculature, programmed changes in maternal metabolic status, and impaired placentation. The strong effect of paternal birthweight on crownheel length may reflect paternal imprinting of genes important for skeletal growth, such as those regulating the concentrations of insulin-like growth factors (33). D. Nutrient Availability and Partitioning

The maternofetal exchange of hydrophilic solutes, such as glucose and amino acids, is influenced by their concentrations in maternal and fetal plasma (34). Nutrient concentrations in maternal plasma vary according to effects of the mother’s metabolism, body composition, and diet. A woman’s metabolism and body composition, particularly her fat mass, have major effects on both glucose tolerance and maternal protein metabolism (35), and much work re-

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mains to be done to gain an integrated understanding of the regulation of glucose and amino acid availability in the maternal circulation. Understanding the regulation of maternal glucose availability is of particular importance because fetal soft-tissue overgrowth and macrosomic changes occur even at raised maternal glucose concentrations within the normal range (36). This could have long-term implications for the fetus because studies in rats have shown that maternal hyperglycemia during pregnancy can lead to lifelong impairment of insulin secretion and diabetes in the offspring (37). Such effects could underlie the observation that, in some populations, those at the high end of the distribution of birthweight have increased rates of impaired glucose tolerance in adult life (26,38). Thus, the North American Pima Indians have a high incidence of gestational diabetes, and high rates of adult non–insulin-dependent diabetes mellitus (NIDDM) have been reported in the offspring of mothers who were diabetic during pregnancy (38). Although it has been suspected that maternal hyperglycemia may damage pancreatic development in the fetus, the hypothesis has received little attention. Gestational diabetes is common among women from the Indian subcontinent, and it has been proposed that this may underlie the high rates of adult NIDDM in India (26). Although little is known about the partitioning of nutrients between the mother, the placenta, and the fetus, IGF-1 in the maternal circulation may play an important role. During pregnancy, the placenta becomes a major regulator of many aspects of the mother’s endocrine system, including her somatotropic axis. Experimental data suggest that the secretion of placental lactogen and placental growth hormone variant into the maternal circulation could act to increase nutrient availability in maternal plasma and to alter the partitioning of nutrients in a manner that favors nutrient transfer to the fetus (39,40). Placental lactogen is lipolytic and may mobilize free fatty acids for use as a source of energy by the mother, maintaining glucose availability for fetal consumption (40). Maternal IGF-1 concentrations increase in pregnancy, perhaps as a result of placental growth hormone variant secretion. This may be of importance because infusion of IGF-1 into pregnant ewes increases glucose availability to the fetus (39). In humans, the physiological regulation of maternal placental lactogen, placental growth hormone variant, and IGF-1 concentrations is poorly understood. Preliminary observations suggest that maternal serum IGF-1 concentrations may influence later weight gain in thin women (41). A low pregnancy weight gain in thin women is associated with raised blood pressure in childhood (24,25,42).

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The transfer of glucose across the placenta is by facilitated diffusion and therefore largely dependent on maternal and fetal plasma concentrations. In contrast, the transfer of amino acids is by active transport using transport proteins located in the maternal- and fetal-facing membranes of the transporting epithelium of the placenta, the syncytiotrophoblast (43). This transport is determined by hormonal influences in maternal plasma that affect nutrient partitioning between mother and fetus, and by the surface area and transfer capabilities of the syncytiotrophoblast. A variety of mechanisms mediate solute transfer across microvillous (maternal-facing) and basal (fetal-facing) plasma membranes of the syncytiotrophoblast (34,43). Although few of these have been related to fetal size, the activity in the microvillous plasma membrane of one amino acid transporting mechanism, the system A transporter, is reduced in placentas from severely growth-retarded fetuses (44). Such fetuses tend to have placentas with gross morphological abnormalities, and little is known about how normal fetal growth relates to the transfer capabilities of the syncytiotrophoblast. An initial study using a microvillous membrane vesicle technique found that, within the normal range of birthweight, smaller babies with a lower abdominal circumference had higher placental system A activity (Fig. 4) (45). Within the normal range of fetal and placental size, this may reflect a tendency towards compensatory upregulation of the placental system A transporter in smaller babies. Vesicles prepared from the placentas of smaller fetuses were also found to have a higher diffusive permeability to short side-chain neutral amino acids. This may reflect a more “leaky” plasma membrane that, in vivo, would result in greater diffusion of amino acids back from the placenta to the maternal circulation (45). Confirmation of such an effect is important, particularly because diffusive permeability was found to be higher in placentas from women with a low body mass index (45). Although the size of the placenta gives only an indirect measure of its capacity to transfer nutrients to the fetus, it is nonetheless strongly associated with fetal size at birth (46). Experiments in sheep have shown that maternal nutrition in early pregnancy can exert mVajor effects on the growth of the placenta, and thereby alter fetal development (47). The effects produced depended on the nutritional status of the ewe in the periconceptional period. In ewes poorly nourished around the time of conception, high nutrient intakes in early pregnancy increased the size of the placenta. Conversely, in ewes

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Figure 4 Placental system A activity (Na+-dependent MeAIB uptake) in microvillous membrane vesicles prepared from 61 term pregnancies.

well nourished around conception, high intakes in early pregnancy resulted in smaller placental size (47). Our observational studies of pregnant women in a well-nourished Western community have shown that those who reported high dietary intakes in early pregnancy, especially of carbohydrates, had smaller placentas, particularly if this was combined with low intakes of dairy protein in late pregnancy (19). This was the same pattern of dietary intakes in early and late pregnancy that we found to be associated with thinness at birth (32). The effect of maternal diet was independent of the mother’s body size, social class, and smoking (19). Support for an effect of maternal diet on human placental growth has subsequently come from analyses of the Dutch Hunger Winter famine, where increased placental weight was associated with the combination of famine exposure in early pregnancy and high food intakes in mid to late pregnancy (48). Nutritional effects on placental growth alter the ratio of placental weight to birth weight (placental ratio) (19,47), and may be of importance because

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there is a U-shaped relation between the placental ratio and later coronary heart disease (49). Babies with a disproportionately small placenta may suffer as a result of an impaired placental supply capacity; those with a disproportionately large placenta may experience fetal catabolism and wasting to supply amino acids for placental consumption (4). Our studies have also shown that placental growth is strongly influenced by a woman’s own birthweight (19). This suggests that the effect of low maternal birthweight on thinness at birth may operate partly through constraint of placental growth determined during the woman’s own fetal life. An interpretation of this is that in female fetuses, restricted growth in utero may alter their own uterine vascular development; consequences of this in their reproductive life could include impaired placentation, resulting in a reduced supply of nutrients for fetal soft-tissue deposition and fetal wasting. F. Uteroplacental Blood Flow

The net transfer of small lipophilic solutes, such as O2 and CO2, is primarily dependent on uterine and umbilical blood flow, and impairment of the uteroplacental and/or fetoplacental circulations is associated with restricted fetal growth. During pregnancy, the maternal cardiovascular system undergoes functional changes that allow increased uteroplacental blood flow. Cardiac output rises as early as the fifth week to peak at midpregnancy; this reflects increases in both heart rate and stroke volume resulting from peripheral vasodilation combined with increased left ventricular contractility (50). Recent work has shown that estrogen upregulates the production of vascular endothelial growth factor (VEGF) by vascular smooth muscle cells, and that VEGF stimulates release of the endogenous vasodilator nitric oxide by endothelial cells (51,52). This may play a key role in initiating cardiovascular adaptations to pregnancy. Plasma volume expansion results from secondary activation of volume-retaining mechanisms and peaks around 34 weeks of gestation. Concentrations of albumin and hemoglobin fall because the increases in circulating protein and red cell mass are proportionately less than the increase in fluid volume. As yet, no study has systematically examined either the influences that determine cardiovascular function in human pregnancy or its effects on fetal growth and development. Production of nitric oxide requires arginine as a substrate. Studies in animals have shown that alterations in dietary arginine intake can influence vascular function (53), raising the possibility that alterations in maternal nutrition and arginine availability may also impair the vas-

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cular response to pregnancy. Recent studies have shown that, in later childhood and adult life, low-birthweight infants tend to have impaired nitric oxide–mediated vasodilatation (54), a hyperdynamic circulation (55), and decreased arterial compliance (56). In pregnant women whose own fetal growth was retarded, consequences of these effects could include a diminished rise in cardiac output, reduced plasma volume expansion, and constrained physiological remodeling of the uterine vasculature. A consequent decrease in uteroplacental blood flow could contribute to the intergenerational effects of low maternal birthweight on fetal growth. V.

Fetal Adaptations and Developmental Changes

Although a variety of paternal and maternal influences have been linked with variations in fetal size and proportions at birth, few studies have as yet examined whether these influences have long-term consequences for the fetus. Those that have suggest that a woman’s diet and body composition in pregnancy can have long-term effects on the offspring’s risk of raised blood pressure and coronary heart disease without necessarily altering size at birth (20,21,24–26,28). These observations have provided impetus to identifying the fetal adaptations and developmental changes that underlie the programming of cardiovascular and metabolic disease. Experimental studies in animals suggest that alterations in fetal body composition and organ growth, in fetal endocrine status, and in the fetal cardiovascular system may be involved (Fig. 5). A. Fetal Body Composition and Organ Growth

Following the initial observation of an association between thinness at birth (reflected in a low ponderal index) and raised adult blood pressure (57), subsequent studies in Western populations have shown a consistent association between a low ponderal index at birth and adult insulin resistance and impaired glucose tolerance (58–61). A low neonatal ponderal index results from reduced fat or lean mass in relation to skeletal length (62). Detailed studies of rural Indian babies have shown that they are characterized by relative sparing of head and skeletal growth as well as subcutaneous fat, along with a marked deficit in skeletal muscle and a reduced abdominal circumference (63). Although fat stores may confer an advantage for immediate postnatal survival, if they persist into adult life they may add to the tendency to insulin resistance. In the rural Indian babies studied by Fall and colleagues, the babies with the

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Figure 5 Fetal programming framework. fattest skinfold thicknesses were born to mothers who were short and fat (63). Short, fat women can be thought of as being in “nutritional transition,” having been undernourished in early life (stunted) and better nourished (fat) as adults. This has led to the proposal that these mothers produce babies who have the worst of all worlds: reduced lean body mass and disproportionate fat deposition. It has also led to the hypothesis that increasing fatness in stunted mothers could be responsible for the rise in NIDDM in urban Indian populations (26). Impaired fetal muscle development may also play a role, because it appears to lead to permanent alterations in skeletal muscle metabolism, thereby contributing to adult insulin resistance (59). Experimental observations in animals have shown that impaired development of specific fetal organs, most notably the fetal kidneys and liver, can have long-term effects that may be of relevance to the programming of human cardiovascular and metabolic disease (64–66). Impaired renal and hepatic development may be a consequence of changes in fetal nutrition or endocrine status, or of cranial redistribution of blood flow to spare brain growth at the expense of the trunk and abdominal viscera (67). The likely importance of changes in fetal kidney growth, for example, is supported by the observation that men and women who had a small abdominal circumference at birth have

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elevated blood pressure and alterations in the plasma concentrations of inactive renin in adult life (68). Ultrasound studies have shown that growthretarded fetuses have impaired renal growth between 26 and 34 weeks gestation (69), and future research will need to examine the effects of alterations in the maternoplacental supply of nutrients on fetal liver and kidney growth. B. Fetal Endocrine Status

Studies in pregnant rats have directly implicated fetal glucocorticoid excess as a mechanism that can program cardiovascular parameters. These studies have shown that maternal dexamethasone treatment in pregnancy leads to raised adult blood pressure and impaired glucose tolerance in the offspring, and that the activity of the placental glucocorticoid barrier, 11β-hydroxysteroid dehydrogenase-2, correlates with fetal and placental growth (70–72). At present, the role of glucocorticoids in determining normal variations in human fetal size at birth is unclear, particularly because the delivery process is associated with acute perturbations in cord blood glucocorticoid concentrations (73). Future studies will need to relate markers of fetal glucocorticoid status to size at birth and to the mother’s diet and body composition. Current concepts suggest a central role for the fetal glucose-insulinIGF-1 axis in the endocrine regulation of fetal growth and anabolism. Fetal plasma IGF-1 levels have been studied extensively in the sheep fetus, and have been shown to be regulated primarily by nutrient supply and fetal insulin release (13). Babies who are of low weight, or who were short or thin at birth, have low concentrations in cord plasma of insulin and 32-33 split proinsulin (74). The strong, graded effects across the range of neonatal size and thinness found in a normal population support a major physiological role for the glucose-insulin-IGF-1 axis in matching fetal growth to substrate supply. Lower cord plasma split proinsulin concentrations have been found in male babies, in those with a small placenta and in those who had a primiparous mother; those who remained in utero beyond term had lower cord plasma insulin concentrations (74,75). These findings support the view that failure of the maternoplacental supply of nutrients to match fetal demands preferentially occurs in these groups. Although cord plasma split proinsulin and insulin concentrations were not related to maternal height, body mass index, or smoking, they were lower in babies whose mothers had high energy intakes in early pregnancy and low protein intakes in late pregnancy (75). The effect of diet in early pregnancy on fetal split proinsulin and insulin concentrations may reflect the suppression of placental growth by high dietary intakes; the effect

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of diet in late gestation may be attributable to differences in amino acid or micronutrient availability, or an indirect effect of maternal endocrine changes. Animal studies show that maternal nutrition can influence fetal growth and development through effects on the fetal glucose-insulin-IGF-1 axis; these data provide evidence that similar effects occur in humans. Insulin and IGF-1 have major effects on the growth of fetal blood vessels (76), pancreatic beta cells (77), and skeletal muscle (14,78), and nutritional effects on fetal concentrations of these hormones could provide one mechanism underlying long-term effects of the intrauterine environment on susceptibility to cardiovascular disease and NIDDM in adult life. C. Fetal Cardiovascular Adaptations

Experimental and clinical studies have documented that fetal growth retardation is associated with a fall in cerebral vascular resistance and an increase in the diastolic component of the cerebral arterial blood velocity waveform resulting from vasodilatation in the cerebral circulation (79). Vasodilatation in the cerebral vasculature and vasoconstriction in other vascular beds leads to cranial redistribution of cardiac output. It has been proposed that this cranial redistribution may tend to increase blood flow to the upper limbs, leading to fingertip swelling and an increased prevalence of whorl patterns in people who had raised adult blood pressure and had been thin at birth (80). The prevalence of whorls is greater on the right hand than the left, and it was argued that this could be a reflection of the right subclavian artery arising from the brachiocephalic trunk, whose other branch is the right common carotid artery, whereas the left subclavian artery is a branch of the fetal dorsal aorta. Direct support for the hypothesis that cranial redistribution of blood may preferentially increase blood flow in the right subclavian artery has since come from Doppler ultrasound studies of growth-retarded fetuses (81). Fetal growth retardation is also associated with increased placental vascular impedance, and consequently with increased right ventricular afterload. Fetal echocardiography has shown that, compared with control fetuses, growth-restricted fetuses have an increase in left ventricular cardiac output and a decrease in right ventricular cardiac output (82); this could be of major importance in programming adult left ventricular hypertrophy and inability of the coronary circulation to meet myocardial oxygen demand. VI.

An Integrated Framework and Future Research

Suggestions that normal variations in the maternoplacental supply of nutrients may exert important effects on the fetus contrast with the view that regulatory

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mechanisms in the maternal and placental systems act to ensure that the growth and development of fetuses in Western communities are largely independent of the mother’s nutritional state. This view partly arose from studies of famine showing that extreme dietary restriction in pregnancy may exert only relatively small effects on size at birth (83), and partly from the disappointing results of human interventional studies of maternal nutrition during pregnancy (18). Recent observations challenge the view that the fetus is little affected by changes in maternal nutrition except in circumstances of famine. The observations support the hypothesis that cardiovascular and metabolic disease in adult life may in part be the result of fetal adaptations and developmental changes invoked when the maternoplacental supply of nutrients fails to match the fetal demand for them. Growth is a major goal for the fetus, and although many fetuses continue to grow despite a suboptimal intrauterine environment, this growth may be at the expense of adaptive changes that exact a long-term price. The complexities of fetal growth and development are such that currently available data form only a limited basis for changing dietary recommendations to pregnant women. Previous studies of maternal nutrition have adopted a far too simplistic approach to assessing the true impact on fetal development, and have failed to address the possibility of long-term effects on the health of the offspring in adult life. A strategy of interdependent clinical, animal, and epidemiological investigations is essential to identify the factors that set the trajectory of fetal growth, as well as the influences that limit the maternoplacental delivery of nutrients to the fetus. We also need to define how the fetus adapts to a limited nutrient supply, how these adaptations program the structure and physiology of the body, and by what molecular mechanisms nutrients and hormones alter gene expression. Without this, we are unlikely to define the extent to which a woman’s own fetal growth, along with her diet and body composition before and during pregnancy, play a major role in programming the future health of her children. References 1. 2.

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Barker DJP, Gluckman PD, Godfrey KM, Harding JE, Owens JA, Robinson JS. Fetal nutrition and cardiovascular disease in adult life. Lancet 1993; 341:938– 941. Walton A, Hammond J. The maternal effects on growth and conformation in Shire horse–Shetland pony crosses. Proc Roy Soc Land B 1938; 125:311–335. Morton NE. The inheritance of human birth weight. Ann Hum Genet 1955; 20:123–134. Brooks AA, Johnson MR, Steer PJ, Pawson ME, Abdalla HI. Birth weight: nature or nature? Early Hum Dev 1995; 42:29–35. Law CM, Barker DJP, Bull AR, Osmond C. Maternal and fetal influences on blood pressure. Arch Dis Child 1991; 66:1291–1295. Whincup PH, Cook DG, Papacosta O. Do maternal and intrauterine factors influence blood pressure in childhood? Arch Dis Child 1992; 67:1423–1429. Godfrey KM. Maternal regulation of fetal development and health in adult life. Eur J Obstet Gynecol Reprod Biol 1998; 78:141–150. Barker DJP. Mothers, Babies and Health in Later Life. 2nd ed. Edinburgh: Churchill Livingstone, 1998. Harding JE, Liu L, Evans P, Oliver M, Gluckman P. Intrauterine feeding of the growth-retarded fetus: can we help? Early Hum Dev 1992; 29:193–197. Gluckman PD. The endocrine regulation of fetal growth in late gestation: the role of insulin-like growth factors. J Clin Endocrinol Metab 1995; 80:1047–1050. Baker J, Liu J-P, Robertson EJ, Efstratiadis A. Role of insulin-like growth factors in embryonic and postnatal growth. Cell 1993; 75:73–82. Kleeman DO, Walker SK, Seamark RF. Enhanced fetal growth in sheep administered progesterone during the first three days of pregnancy. J Reprod Fertil 1994; 102:411–417. Walker SK, Hartwich KM, Seamark RF. The production of unusually large offspring following embryo manipulation: concepts and challenges. Theriogenology 1996; 45:111–120. Leese HJ. The energy metabolism of the pre-implantation embryo. In: Heyner S, Wiley L, eds. Early Embryo Development and Paracrine Relationships. New York: Alan R. Liss, 1990:67–78. Kramer MS. Effects of energy and protein intakes on pregnancy outcome: an overview of the research evidence from controlled clinical trials. Am J Clin Nutr 1993; 58:627–635. Godfrey K, Robinson S, Barker DJP, Osmond C, Cox V. Maternal nutrition in early and late pregnancy in relation to placental and fetal growth. BMJ 1996; 312:410–414. Campbell DM, Hall MH, Barker DJP, Cross J, Shiell AW, Godfrey KM. Diet in pregnancy and the offspring’s blood pressure 40 years later. Br J Obstet Gynaecol 1996; 103:273–280.

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21. Belizan JM, Villar J, Bergel E, Del Pino A, Di Fulvio S, Galliano SV, Kattan C. Long term effect of calcium supplementation during pregnancy on the blood pressure of offspring: follow up of a randomised controlled trial. BMJ 1997; 315:281–285. 22. Rush D. Effects of changes in maternal energy and protein intake during pregnancy, with special reference to fetal growth. In: Sharp, Fraser, Milner, eds. Fetal Growth. London: Royal College of Obstetricians and Gynaecologists, 1989:203– 229. 23. Langley-Evans SC, Jackson AA. Increased systolic blood pressure in adult rats induced by fetal exposure to maternal low protein diets. Clin Sci 1994; 86:217– 222. 24. Godfrey KM, Forrester T, Barker DJP, Jackson AA, Landman JP, Hall JStE, Cox V, Osmond C. Maternal nutritional status in pregnancy and blood pressure in childhood. Br J Obstet Gynaecol 1994; 101:398–403. 25. Clark PM, Atton C, Law CM, Shiell A, Godfrey K, Barker DJP. Weight gain in pregnancy, triceps skinfold thickness and blood pressure in the offspring. Obstet Gynaecol 1998; 91:103–107. 26. Fall CHD, Stein CE, Kumaran K, Cox V, Osmond C, Barker DJP, Hales CN. Size at birth, maternal weight, and type 2 diabetes in South India. Diabet Med 1998; 15:220–227. 27. Stein CE, Fall CHD, Kumaran K, Osmond C, Cox V, Barker DJP. Fetal growth and coronary heart disease in South India. Lancet 1996; 348:1269–1273. 28. Forsen T, Eriksson JG, Tuomilehto J, Teramo K, Osmond C, Barker DJP. Mother’s weight in pregnancy and coronary heart disease in a cohort of Finnish men: follow up study. BMJ 1997; 315:837–840. 29. Stewart RJC, Sheppard H, Preece R, Waterlow JC. The effect of rehabilitation at different stages of development of rats marginally malnourished for ten to twelve generations. Br J Nutr 1980; 43:403–412. 30. Klebanoff MA, Meirik O, Berendes HW. Second-generation consequences of small-for-dates birth. Pediatrics 1989; 84:343–347. 31. Emanuel I, Filakti H, Alberman E, Evans SJW. Intergenerational studies of human birthweight from the 1958 birth cohort. I. Evidence for a multigenerational effect. Br J Obstet Gynaecol 1992; 99:67–74. 32. Godfrey KM, Barker DJP, Robinson S, Osmond C. Maternal birthweight and diet in pregnancy in relation to the infant’s thinness at birth. Br J Obstet Gynaecol 1997; 104:663–667. 33. DeChiara T, Efstratiadis A, Robertson EJ. A growth-deficiency phenotype in heterozygous mice carrying an insulin-like growth factor II gene disrupted by targeting. Nature 1999; 345:78–80. 34. Morris FH, Boyd RDH, Mahendran D. Placental Transport. In: Knobil E, Neil JD, eds. The Physiology of Reproduction, Vol. 1. 2nd ed. New York: Raven Press Ltd, 1994:813–862. 35. Child SC, Soares MJ, Reid M, Persaud C, Forrester T, Jackson AA. Urea kinetics

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49. Martyn CN, Barker DJP, Osmond C. Mothers pelvic size, fetal growth and death from stroke in men. Lancet 1996; 348:1264–1268. 50. Robson SC, Hunter S, Boys RJ, Dunlop W. Serial study of factors influencing changes in cardiac output during human pregnancy. Am J Physiol 1989; 256:H1060–H1065. 51. Karas RH, Bieber HE, Baur WE, Mendelsohn ME. Estrogen enhances vascular endothelial growth factor (VEGF) gene expression in human vascular smooth muscle cells. Circulation 1996; 94:I-595. 52. Tsurumi Y, Murohara T, Krasinski K, Chen D, Witzenbichler B, Kearney M, et al. Reciprocal relation between VEGF and NO in the regulation of endothelial integrity. Nature Med 1997; 3:879–886. 53. Hutchison SJ, Reitz MS, Sudhir K, Sievers RE, Zhu B-Q, Sun Y-P, et al. Chronic dietary L-arginine prevents endothelial dysfunction secondary to environmental tobacco smoke in normocholesterolemic rabbits. Hypertension 1997; 29:1186– 1191. 54. Leeson CPM, Whincup PH, Cook DG, Donald AE, Papacosta O, Lucas A, Dean-field JE. Flow-mediated dilation in 9- to 11-year-old children: the influence of intrauterine and childhood factors. Circulation 1997; 96:2233–2238. 55. Phillips DIW, Barker DJP. Association between low birthweight and high resting pulse in adult life: is the sympathetic nervous system involved in programming the insulin resistance syndrome. Diabetic Med 1997; 14:673–677. 56. Martyn CN, Barker DJP, Jespersen S, Greenwald S, Osmond C, Berry C. Growth in utero, adult blood pressure and arterial compliance. Br Heart J 1995; 73:116– 121. 57. Barker DJP, Godfrey KM, Osmond C, Bull A. The relation of fetal length, ponderal index and head circumference to blood pressure and the risk of hypertension in adult life. Paediatr Perinat Epidemiol 1992; 6:35–44. 58. Barker DJP, Hales CN, Fall CHD, Osmond C, Phipps K, Clark PMS. Type 2 (non–insulin-dependent) diabetes mellitus, hypertension and hyperlipidaemia (syndrome X): relation to reduced fetal growth. Diabetologia 1993; 36:62–67. 59. Phillips DIW. Insulin resistance as a programmed response to fetal undernutrition. Diabetologia 1996; 39:1119–1122. 60. Lithell HO, McKeigue PM, Berglund L, Mohsen R, Lithell U-B, Leon DA. Relation of size at birth to non–insulin-dependent diabetes and insulin concentrations in men aged 50–60 years. BMJ 1996; 312:406–410. 61. Ravelli ACJ, van der Meulen JHP, Michels RPJ, Osmond C, Barker DJP, Hales CN, Bleker OP. Glucose tolerance in adults after prenatal exposure to famine. Lancet 1998; 351:173–177. 62. Widdowson EM, Crabb DE, Milner RDG. Cellular development of some human organs before birth. Arch Dis Child 1972; 47:652–655. 63. Fall CHD, Yajnik CS, Rao S, Coyaji KJ, Shier RP. The effects of maternal body composition before pregnancy on fetal growth: the Pune Maternal Nutrition and Fetal Growth Study. In: O’Brien PMS, Wheeler T, Baker DJP, eds. Fetal Pro-

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14 Maternal and Placental Influences that Program the Fetus Experimental Findings

JEFFREY S. ROBINSON, CAROLINE McMILLEN, LISA EDWARDS, KAREN KIND, KATHRYN L. GATFORD, and JULIE OWENS University of Adelaide Adelaide, Australia

I.

Introduction

More than 10 years ago, Barker and colleagues described the association between indices of poor fetal growth and the early onset of common adult diseases, including hypertension, ischemic heart disease, and non–insulindependent diabetes mellitus. Associations with obesity and abnormal lipid concentrations are also evident, and that between “the metabolic syndrome” and poor fetal and infant growth is particularly strong. Barker and colleagues (1) have suggested that poor maternal nutrition could underpin these associations (see Chapter 1). Therefore, it has been hypothesized that poor nutrition during development programs the setting of physiological mechanisms. Lucas (2) has suggested several ways in which an early event could have permanent biological effects, including the following: 1. Direct damage (eg, loss of a limb because of vascular accident or trauma) 273

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Robinson et al. 2. Induction, deletion, or impaired development of a permanent somatic structure as the result of a stimulus or insult operating at a critical period 3. Physiological setting of control set points by an early stimulus or insult at a sensitive period, resulting in long-term consequences for function; the effects could be immediate or deferred

Lucas confined the definition of programming to encompass the second and third examples. However, the definition of programming is often restricted to the physiological setting by an early stimulus or insult operating at a sensitive or critical period, with long-term consequences for function. Waterland and Garza (3) have recently reviewed the potential mechanisms that may underlie the programming of physiological systems. These included variations in organ structure, alterations in cell number, clonal selection, metabolic differentiation, and hepatic polyploidization during ontogeny. These investigators preferred the term “metabolic imprinting” to “programming,” and recognized that molecular mechanisms would underlie each or all of their categories. To date, the implication of epidemiological and experimental studies is that environmental perturbation of the embryo or fetus attributable to maternal or placental factors can lead to programming, and may have significant consequences for later function. Other important implications are that therapeutic attempts to improve the immediate or short-term outcome for the fetus may potentially have later adverse outcomes. Thus, treatment with glucocorticoid before preterm birth may have immediate beneficial effects (e.g., lung maturation). However, there are concerns that fetal glucocorticoid exposure may have, in the long-term, adverse consequences attributable to delay in myelination of the optic nerve (4,5). Such an effect may be ameliorated by subsequent catch-up before the critical period for myelination of the optic nerve is completed (5). Nevertheless, the demonstrated roles of glucocorticoids and the hypothalamo-pituitary-adrenal axis during fetal life in programming cardiovascular and metabolic homeostasis in postnatal life suggest that other adverse consequences may ensue and will only be detected in long-term follow-up programs. Historically, adverse prenatal events have long been considered to damage the fetus, with adverse consequences appearing later and persisting into adult life. For example, Little (6) in 1862 proposed that lack of oxygen at the time of birth caused structural change in the brain that resulted in the

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later development of cerebral palsy. From these early observations, it has frequently been concluded that the majority of cases of cerebral palsy were attributable to intrapartum asphyxia. Osler (7) also noted that “in a large proportion of cases the trouble dates from birth, and is the result of injury to the child during its passage into the world.” However, Freud (8) urged caution in this interpretation, noting that “difficult birth in itself in certain cases is merely a symptom of deeper effects that influenced the development of the fetus.” The notion that there are critical periods in development where irreversible changes in subsequent functional and structural maturation may be wrought was first clearly defined by Stockard (9), and emphasized in relation to brain development by Dobbing (10). The classic experiments of Hubel and Weisel (11,12) showed the effects of brief stimuli on the permanent structure of the visual cortex. Levine (13) extended this concept of critical periods to the hypothalamo-pituitary-adrenal axis by showing that handling neonatal rats permanently alters adrenal steroid response to stress in adult life. Exploration of maternal behavior and handling continues to unravel the setting of the control of neuroendocrine systems in the offspring (14). It was also shown that exposure of neonatal rats to androgens alters sexual behavior for life (15). Furthermore, the temperature at which eggs are incubated determines both sexual differentiation and lifelong rate of growth of reptiles (16). In mammals, the level of nutrition in both prenatal and early postnatal life can determine final body size of adults (17). The epidemiological findings (1) linking different patterns of fetal growth, as indicated by body size and shape at birth, to different common adult diseases has extended interest in prenatal programming of body systems, in particular, in relation to cardiovascular and metabolic homeostasis. This has also created an increased sense of excitement among fetal and developmental physiologists, setting a new research agenda. This chapter is largely concerned with events during pregnancy that may program body systems, many of which may partly depend on programming via common mechanisms relating to endocrine and neural control systems. It will concentrate on recent experimental findings that underpin the concept of the fetal origins of adult disease. We have chosen to discuss studies in various species separately, to emphasize that although programming occurs in several species, the type or nature of effective prenatal stimuli and the timing or age of onset of any postnatal outcomes varies, possibly attributable in part in species differences in rates of development before birth.

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Cardiovascular Homeostasis In rats, maternal isocaloric protein restriction restricts fetal growth and increases the ratio of placental weight to fetal weight. Langley-Evans and colleagues (18–28) have widely published on the postnatal consequences of maternal protein restriction (6, 9, or 12% compared with a control diet containing 18% protein as casein). Body weight at birth is reduced in proportion to the reduction of protein intake, and this effect persists to adulthood (21 weeks of age) (18). Between 9 and 21 weeks of age, blood pressure is higher in the offspring exposed to low-protein diets in utero. The magnitude of the increase in blood pressure in offspring is negatively related to the percentage of protein in the maternal diet. A sustained rise in postnatal blood pressure also results from severe food restriction. When mothers are fed 30% of an ad libitum diet, their offspring have higher blood pressure throughout the first year of life (29). Recently, radiotelemetry has been used to avoid the potential confounder of stress (30). Many of the studies evaluated blood pressure changes by using a tail cuff (18,27), which can expose the animals to handling stress. Maternal protein restriction was associated with a small and significant rise in diastolic blood pressure and an increase in heart rate in offspring, compared with those from mothers consuming a normal diet. These changes were confined to the wake phase and were not found in the sleep phase of the 24-hour cycle in the telemetric recordings. After an olfactory stress, there was an augmented increase in both systolic and diastolic blood pressure in the offspring of protein-restricted dams. Tonkiss and colleagues (30) argued for a reassessment of the meaning of the large elevations of blood pressure as measured by the tail cuff technique. In human populations, however, postnatal stress may be important, interacting with prenatal experience to determine the extent of hypertension that results from an early insult. In a human study in which some of the stresses of normal everyday living may have been avoided. Timio et al. (31) followed the difference in the rise in blood pressure in a secluded order of nuns and women from the same social background for a period of 20 years. The rise in blood pressure with age was much less in the secluded order, and this difference was attributed to the benefits of a quiet, meditative (nonstressful) life. Increased activity of angiotensin-converting enzyme in the lungs at 9 weeks of age in offspring exposed to the lowest maternal protein intake implicates the renin-angiotensin system in the generation or maintenance of high

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blood pressure (1). Postnatally, there is a small and significant increase in the maximum pressor response to angiotensin II in the offspring of proteinrestricted mothers (28). Short-term treatment of these offspring of restricted mothers soon after birth with captopril normalized blood pressure for a prolonged period, suggesting that maintenance of high blood pressure is dependent on the renin-angiotensin system (20). Another important endocrine axis is also implicated in the prenatal programming of postnatal cardiovascular regulation. Maternal protein restriction influences the development of the hypothalamo-pituitary adrenal axis in the rat. Weanling rats from protein-restricted mothers have a low amplitude diurnal pattern of variation of plasma adrenocorticotrophic hormone (ACTH) compared with controls (18% protein in the maternal diet) (22). In addition, type II glucocorticoid receptor number and binding capacity in the hippocampus from offspring of protein-restricted mothers are apparently elevated (21,26). From these observations, Langley-Evans et al. (22) inferred that programming of the hypothalamo-pituitary-adrenal axis occurred in utero with protein restriction. Treatment with metyrapone (an 11-â hydroxylase inhibitor) of the protein-restricted mother during pregnancy inhibits corticosterone synthesis and prevents the development of postnatal hypertension in offspring, indicating that it is dependent on glucocorticoids (24). This is also supported by the findings that administration of carbenoxolone, which blocks 11-â hydroxysteroid dehydrogenase and therefore increases exposure of the fetus to maternal corticosterone, at any stage of pregnancy to the mother elevates blood pressure in the offspring (23,25). Postnatally, the high blood pressure is dependent on glucocorticoids because it can be abolished by adrenalectomy of the young rats, a procedure that did not lower blood pressure in controls (23,26,27). Maternal anemia in humans is associated with an altered ratio of fetal weight to placental weight and may be a marker of poor maternal nutrition. The human studies have shown that low birth weight and high placental weight are associated with the highest blood pressure (1). Maternal iron deficiency in rats reduced both fetal and placental weights (32). The placental weight to fetal weight ratio was also increased. At 20 days of postnatal age, the offspring of anemic mothers had larger hearts and lower blood pressure than controls. This was reversed by 40 days of age, when blood pressure in the offspring of anemic mothers was higher than in controls. It is clear from these studies that high blood pressure can be induced in rats by a variety of prenatal perturbations. The induction of high blood pressure is most effective when the insult is present in the last third of pregnancy, and in many of the studies is amplified by postnatal handling stress,

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both of which implicate the hypothalamo-pituitary-adrenal axis in prenatal programming of postnatal cardiovascular homeostasis. Metabolic Homeostasis A detailed description of the postnatal consequences of maternal protein restriction in the rat for glucose metabolism and regulation of insulin can be found in Chapter 8. Although a range of perturbations, such as moderate to severe maternal undernutrition or maternal protein restriction, have been imposed in studies using this species and consistently produced elevations in blood pressure postnatally, more variable consequences for postnatal glucose metabolism and its regulation by insulin have been reported. In the rat, moderate maternal feed restriction or protein restriction have resulted in improved (19), unaltered (33), or impaired (34,35) glucose tolerance or insulin sensitivity in progeny. Thus, a 50% restriction of feed intake in rats during the second half of pregnancy decreases insulin sensitivity of wholebody glucose uptake and impairs hepatic but not peripheral insulin sensitivity in female adult progeny (34,35). A similar level of restriction during the first two thirds of pregnancy had no effect on glucose tolerance, insulin secretion, or insulin sensitivity of whole-body glucose utilization or endogenous glucose production in adult male progeny (33). Some of these findings may reflect differences in timing of the perturbation imposed or in gender susceptibility, but postnatal insulin-regulated glucose metabolism does appear to be sensitive to maternal feed restriction, particularly in late gestation. Maternal protein restriction in rats improves glucose tolerance in offspring when young but not in mature adults (19). Maternal protein restriction imposed during pregnancy only in the rat actually increases insulin sensitivity in skeletal muscle, liver, and adipose tissue in adult offspring, consistent with the observed lack of impairment of glucose tolerance (40). Only when restriction is extended through the immediate postnatal period do offspring appear to exhibit impaired glucose homeostasis as adults. As for blood pressure, prenatal perturbation may also affect postnatal metabolic homoeostasis in part through the HPA axis. In rats, administration of carbenoxolone throughout pregnancy to block placental inactivation of endogenous and maternal glucocorticoids reduces birthweight (36), results in hypertension as described earlier (36,37), and impairs glucose tolerance in the progeny as adults (38). Exposure by treatment of pregnant rats with dexamethasone in the third, but not the first or second third of pregnancy, also impairs glucose tolerance in the adult offspring and elevates blood pressure (39).

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Transgenerational Effects Many generations of moderate malnutrition reduces adult size and has a selective effect on organ size. After 10 to 12 generations of moderate malnutrition, brain weight is reduced less than liver, lung, kidney, or body size (41). The malnourished mothers have a high incidence of small pups and stillbirths compared with ad libitum fed mothers in a control colony. The effect of returning the malnourished colony to an ad libitum diet on adult body size depends on when in the life cycle the rats received the new ad libitum diet. If diet is restored after weaning, it takes three generations for adult body size to match that of the control, ad libitum, fed colony. If the diet is restored during pregnancy, however, fetal weight at birth is the same as in the controls, but the small maternal size is likely to be responsible for the reported injuries to the pups and the mothers that occur during birth. Some of these surviving pups carried neurological injuries into adult life. Overall, it took three generations to restore body size and neurological outcome to the norm of the control colony (42). In rats, induction of maternal diabetes with streptozotocin alters carbohydrate metabolism in the offspring. The pups are smaller at birth and remain smaller throughout life than those from control mothers. They have normal glucose tolerance although they are insulin resistant. The third generation has impaired glucose tolerance (43). Islet cell transplants, which normalize glucose tolerance in the first generation, prevent these transgenerational effects (44). B. Placental Restriction of Fetal Growth

Placental weight at term can account for much of the variation in size at birth in mammalian species. Normal fetal growth is dependent on an interaction between the genome and the supply of nutrients and oxygen. Restriction of placental growth creates a mismatch between the genetic drive to grow and the supply of both oxygen and glucose. Therefore, several methods have been devised to restrict placental growth and function experimentally. Fetal growth restriction attributable to impaired placental function can be induced in rats by ligation of a uterine artery in late pregnancy (45). The offspring are disproportionately grown with relative preservation of brain weight. Postnatally, these pups tend to grow along lower centiles than controls. Restriction of fetal growth by uterine artery ligation of rats alters the expression and activity of enzymes involved in energy metabolism. After prenatal restriction, increased expression of genes associated with the generation of ATP and NADH persists into postnatal life (46,47). Longer-term studies

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are required, however, to assess the impact of this perturbation on homeostasis and function in adult life. III. Guinea Pig The guinea pig offers several advantages for studies of growth and development compared with other small laboratory animals, including rats and mice. Placentation in the guinea pig has a number of similarities to that in the human, including trophoblast invasion of maternal uterine arteries. In addition, like the human fetus, the guinea pig deposits white fat stores before birth. The guinea pig pup at birth is comparatively precocious and can be weaned just a few days later, enabling rapid transfer to an unrestricted diet without cross-fostering. Its use has been criticized because of the different settings of endocrine axes compared with humans (48), although other laboratory species also vary substantially in their absolute abundance of and sensitivity to key hormones. Nevertheless, the major endocrine axes in the guinea pig respond to stressors in a qualitatively but not quantitatively similar way to many species. A. Maternal Undernutrition

Growth Moderate maternal food restriction from 30 days before pregnancy in the guinea pig can alter the earliest stages of development (49). Mild maternal undernutrition (85% ad libitum), and severe (70%), do not alter the total number of cells in the blastocyst. However, with 85%, a greater proportion of these cells are allocated to the inner cell mass and fewer to the trophectoderm with maternal undernutrition. In ad libitum fed animals, the total number of cells in the blastocyst and in the inner cell mass on day 6 of pregnancy positively correlates with free fatty acid concentrations in maternal plasma. In the 70% food-restricted group, total cell numbers were negatively correlated with serum triglycerides, which are indices within the individual animals of their response to undernutrition. These findings, together with the altered distribution of cells in the moderate food-restricted group, indicate that maternal undernutrition during the pre- and peri-implantation periods can alter embryonic development. Both mild and moderate food restriction from before and throughout pregnancy reduce fetal weight, length, and abdominal circumference in late gestation, without altering ponderal index (weight/length3) (50). Placental

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weight relative to fetal weight is increased (50). Intriguingly, fat stores in the fetuses from the food-restricted mothers are relatively preserved, and increase as a proportion of body weight, as has been reported previously when maternal food intake is restricted (51,52). Widdowson (51) suggested that there may have been a comparative excess of placental transfer of carbohydrate compared with nitrogen. In pigs, as in guinea pigs, muscle mass is also reduced. In addition, fat to body weight ratio is increased (51). In pigs, Widdowson and McCance (17) showed that there was a critical period before and after birth for the determination of final body size. Restoration of nutrition during this critical period results in catch-up growth. However, if refeeding is delayed until after this period there is a reduction in final body size. Cardiovascular Homeostasis Blood pressure has been measured in offspring of food-restricted and control mothers in the guinea pig. In young adult offspring of ad libitum fed mothers, there is a negative correlation between systolic blood pressure and birth weight. Systolic blood pressure is also higher in the male offspring of food-restricted mothers compared with male offspring of ad libitum fed mothers. Metabolic Homeostasis Postnatally, in the guinea pig there is a period of increased sensitivity to insulin in the neonate followed by the emergence of insulin resistance in adulthood accompanied by reduced glucose tolerance in association with low birth weight. In weanlings (30 days of age), insulin sensitivity is negatively related to birth weight. At this age, body fat is reduced as a proportion of body weight. However, by young adult life (120 days postnatally), insulin sensitivity is reduced in those pups that were light at birth. In these young adults, the percentage of body fat correlated negatively with birth weight. In male guinea pig offspring of the mothers subjected to food restriction from before and throughout pregnancy, plasma total cholesterol before and during dietary cholesterol loading (0.25%) was significantly higher than that in offspring of ad libitum fed mothers (53). Dietary cholesterol loading for 6 weeks increased plasma total cholesterol more in the offspring of the foodrestricted mothers. The influence of birth weight on total and low density lipoprotein (LDL) cholesterol was examined by dividing the pups into those above and below median birth weight. Plasma total and LDL cholesterol concentrations before cholesterol loading did not differ. Both total and LDL cho-

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lesterol were higher after cholesterol loading in the low–birth-weight group. These observations suggest that maternal nutrition and small size at birth permanently impair cholesterol metabolism after birth in the guinea pig (53). These findings were not observed in the smaller number of females that were available for study. Postmortem examination of these animals showed an increase in the area of fatty streaks in selected sites of the aorta that correlated with plasma total cholesterol concentrations achieved with loading in the offspring of undernourished compared with ad libitum fed mothers (53). Thus, moderate maternal food restriction in the guinea pig produces in the offspring many of the components of the “metabolic syndrome” found in humans. Moderate maternal food restriction reduces insulin sensitivity, impairs glucose tolerance, increases blood pressure and obesity, and increases plasma total and LDL cholesterol concentrations as well as the propensity to develop fatty streaks in the aorta when challenged with dietary cholesterol. Furthermore, the onset of these occurs at a relatively young age compared with total life span of the guinea pig. B. Placental Restriction

The epidemiological associations between low birth weight and high blood pressure prompted Persson and Jansson (54) to examine the effects of experimental intrauterine growth restriction induced by ligation of a single uterine artery on blood pressure in young adult guinea pigs. In a small study of littermates, direct measurement of arterial blood pressure under anesthesia showed that it was higher in the lighter-born pups. IV. Sheep A. Maternal Undernutrition

Early studies in sheep show that fetal growth is reduced and phenotype altered by moderate undernutrition (55–57). Most studies showed that maternal undernutrition from early in pregnancy or confined to late pregnancy reduces placental size. However, a few indicated that moderate maternal food restriction can be associated with an increase in the size of the placenta in mid- or late gestation (58). We have extended these observations to maternal food restriction from before and throughout pregnancy or for defined periods during pregnancy (Fig. 1, 2). Continuous moderate food restriction beginning 60 days before pregnancy that reduces maternal prepregnancy weight by ~15%, reduces fetal weight and length at 140 days (0.93) of gestation by the same

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Figure 1 Continuous maternal food restriction beginning before and in mid or late pregnancy. Results are expressed as mean ± SEM.

proportion as restriction beginning after 90 days of pregnancy. Interestingly, restriction beginning in midpregnancy and continuing in late pregnancy has a smaller effect. Similar effects are seen in internal organs, including the heart, kidney, and liver of the fetus (Fig. 3). At 140 days, fetal weight is reduced when maternal food restriction is confined to late pregnancy but not when it is only present before and in early

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Figure 2 Maternal food restriction confined to prepregnancy and early, mid, or late pregnancy. Results are expressed as mean ± SEM.

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Figure 3 Continuous food restriction beginning before and in mid or late pregnancy. Results are expressed as mean ± SEM.

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pregnancy or midpregnancy. However, fetal organ size may still be altered by these perturbations. Fetal heart, liver, and kidney weights are altered differently in absolute and relative size by maternal food restriction confined to one phase of pregnancy (Fig. 4) (Owens and De Barro, unpublished observations). In sheep with good nutritional stores, maternal food restriction confined to the periods immediately before, and in early and mid pregnancy, increased placental weight but did not increase the placental surface area for exchange (Tania De Barro, unpublished observation). Cardiovascular Homeostasis Severe maternal food restriction for a brief period in late pregnancy in sheep did not increase fetal blood pressure. However, restoration of maternal food intake was rapidly followed by an increase in fetal blood pressure (59). Periconceptional undernutrition of the mother is associated with lower blood pressure in the fetus (60). It has been suggested that the lower blood pressure in these fetuses from food-restricted mothers may be attributable to their lower cortisol concentrations (61,62). These investigators also reported that this maternal food restriction is associated with increased blood pressure in the lambs after birth (60). Postnatally, the offspring have higher blood pressure and cortisol in response to challenge with hypoxia (60). B. Placental Restriction

Growth Placental growth can be readily restricted in sheep by prepregnancy surgical limitation of the number of attachment sites. We have characterized the consequences of restriction of placental growth for growth of the fetus in sheep (63). Briefly, the placentally restricted fetus is disproportionately grown with less restriction of brain size than carcass, or particularly spleen and thymus. However, there are significant histological changes in the brain of the placentally restricted fetus that are consistent with altered function (64). We have also characterized the metabolic and endocrine status in relation to this placental restriction of fetal growth (63). Briefly, the concentrations of anabolic hormones, including insulin. IGFs, and thyroid hormones, are reduced. Conversely, the abundance of stress hormones (cortisol and catecholamines) is increased, and this is accompanied by an earlier preparturient growth of the adrenal gland in the growth-restricted fetus.

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Figure 4 Maternal food restriction confined to prepregnancy and early, mid, or late pregnancy. Results are expressed as mean ± SEM.

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Blood pressure of the placentally restricted fetus is normal in late gestation. The regulation of blood pressure in growth-restricted animals is different from the controls, however, because they lack the normal positive association between fetal oxygen tension and blood pressure in late gestation. The maximal diastolic blood pressure response to angiotensin II also increased with gestational age in controls, but not in the growth-restricted fetus. The blood pressure response to angiotensin II is greater at the beginning of late gestation in the growth-restricted fetus compared with controls. The accelerated growth of the fetal adrenal in placental restriction is accompanied by higher cortisol concentrations in fetal plasma between days 127 and 141 of gestation (67). It is possible that this early rise in cortisol may induce an early sensitivity to angiotensin II in sheep. This would be analogous to the effects of maternal or fetal corticosterone inducing the rise in blood pressure in offspring from protein-restricted rats. In relation to this, it is particularly interesting that intrafetal infusion of cortisol increases the pressor response to angiotensin II in young fetal sheep (68). Captopril administration decreased diastolic blood pressure in the growth-restricted fetuses but not in the control group in late gestation near term (65). This suggests a more significant role for the renin-angiotensin system in the maintenance of blood pressure in growth-restricted than in control fetuses. However, these experiments do not differentiate whether this regulation of blood pressure occurs centrally or peripherally. In the kidneys from growth-restricted fetuses, the expression of angiotensinogen mRNA is reduced compared with that of controls. In the fetal kidney, the expression of renin was positively correlated with that of angiotensinogen. It is suggested that placental restriction suppresses the intrarenal renin angiotensinogen system, which may impair growth and development of the kidney with long-term adverse consequences (66). The altered settings of the fetal cardiovascular system in the growthrestricted sheep fetus are unlikely to be attributable to an increased afterload, as is frequently observed with human fetal growth restriction by finding increased umbilical vascular resistance. There is no increase in placental vascular resistance when placental growth is restricted by excision of endometrial caruncles (69). In contrast, placental vascular resistance is increased with placental embolization of the umbilical arteries (70). In this chronic form of placental damage, Murotsuki and colleagues (71) reported higher blood pressure and myocardial hypertrophy in the fetuses. No long-term, follow-up studies

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of lambs after birth from this experimental restriction of fetal growth have been completed as yet, and it not known if this hypertension persists or amplifies after birth. V.

Conclusion

It is now clear that restriction or perturbation of nutrient supply to the fetus in a variety of species can program the set points of physiological control mechanisms in the fetus. The studies described in this chapter also show that the regulation of physiological systems might already be altered in fetal life. The extent to which various components of the metabolic syndrome emerge postnatally after prenatal perturbation in different species may relate to the maturity of the species at birth. Many body systems undergo sensitive or critical periods after birth in altricial species and before birth in precocial ones. In an altricial species, eg, the rat, although hypertension evolves early in postnatal life after prenatal perturbation, glucose intolerance may only emerge late in life, if at all. In contrast, all components of the metabolic syndrome are present in low birth weight guinea pigs in early adult life. Sheep and humans are intermediate in their stage of maturity at birth compared with rats and guinea pigs, with sheep being more precocial than humans at birth. A theoretical framework is proposed for further examination (Fig. 5). This figure additionally emphasizes that postnatal events or lifestyle can modify the time of onset of various components of the metabolic syndrome. The experimental studies also emphasize that it is unlikely that small size and subsequent development of hypertension, diabetes, and cardiovascular disease in humans is simply a genetic predisposition. Rather, the emergence of these adverse outcomes is likely to be attributable to an interaction between the environment before and after birth working on the genetic background of an individual or population. The potential for reversibility of the development of high blood pressure, insulin resistance, and abnormal glucose tolerance has been investigated in only a few studies to date (20,72). Reversibility of blood pressure with captopril has been noted earlier. There may also be dietary options because it has been shown that altering the fat content of the diet reversed the high blood pressure in rats (20,72). A diet high in saturated fat increases blood pressure. In contrast, a diet high in unsaturated fat lowers blood pressure. However, the high– unsaturated-fat diet did not reduce blood pressure in offspring of mothers who had been subjected to a low-protein diet in pregnancy (73). Determina-

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Figure 5 Theoretical diagram illustrating the age of onset of the metabolic syndrome in altrical or precocial species. Lifestyle factors, diet, and exercise move the age of onset of the syndrome.

tion of the importance of catch-up or catch-down (74) growth on future cardiovascular and metabolic homeostasis will be a significant priority. VI. Acknowledgments We gratefully acknowledge generous financial assistance from the National Health and Medical Research Council of Australia, National Heart Foundation, Women’s and Children’s Hospital Foundation, and the University of Adelaide. We are particularly indebted to Frank Carbone for excellent technical assistance. References 1. 2.

Barker DJP. Mothers, Babies and Health in Later Life. Edinburgh, Scotland: Churchill Livingstone, 1998. Lucas A. Programming by early nutrition in man. In: Bock GR, Whelan J, (eds).

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48. Keigthley M-C, Fuller PJ. Anomalies in the endocrine axes of the guinea pig: relevance to human physiology and disease. Endocr Rev 1996; 17:30–44. 49. Erwich JHM, Robinson JS. Factors in early pregnancy and fetal growth and development. Contemp Rev Obs Gynae 1997; 9:5–9. 50. Schloström A, Katsman A, Kind KL, Roberts CT, Owens PC, Robinson JS, Owens JA. Food restriction alters pregnancy-associated changes in IGF and IGFBP in the guinea pig. Am J Physiol 274, 1998. (Endocrinol Metab 37), E410– E416. 51. Widdowson EM. Immediate and long-term consequences of being large or small at birth: a comparative approach. In: Elliott K, Knight J, eds. Size at Birth. Ciba Foundation Symposium 27. Amsterdam, North Holland: Elsevier, 1974:65–76. 52. Ashwell M, Purkins L, Cowen T, Day KC. Pre- and postnatal development of adipose tissue at four sites in the guinea pig: effect of maternal diet restriction during the second half of pregnancy. Ann Nutr Metab 1987; 31:197–210. 53. Kind KL, Clifton PM, Katsman AI, Tsounis M, Robinson JS, Owens JA. Restricted fetal growth and the response to dietary cholesterol in the guinea pig. Am J Physiol (Regulatory Integrative Comp Physiol) 1999; 277:R1675–R1682. 54. Persson E, Jansson T. Low birth weight is associated with elevated adult blood pressure in the chronically catheterized guinea-pig. Acta Physiol Scand 1992; 145:195–196. 55. Wallace LR. The growth of lambs before and after birth in relation to the level of nutrition. Part I. J Agri Sci 1948; 38:93–153. 56. Wallace LR. The growth of lambs before and after birth in relation to the level of nutrition. Part II. J Agri Sci 1948; 38:243–302. 57. Wallace LR. The growth of lambs before and after birth in relation to the level of nutrition. Part III. J Agri Sci 1948; 38:367–401. 58. McCrabb GJ, Hosking BJ, Egan AR. Changes in the maternal body and fetoplacental growth following various lengths of feed restriction during mid-pregnancy in sheep. Aust J Agri Sci 1992; 43:1429–1440. 59. Harding JE, Johnston BM. Nutrition and fetal growth. Reprod Fertil Dev 1995; 7:539–547. 60. Hoet JJ, Hanson MA. Intrauterine nutrition: its importance during critical periods of cardiovascular and endocrine development. J Physiol (Lond) 1999; 514:617– 627. 61. Hawkins P, Crowe C, Calder NA, Saito T, Ozaki T, Stratford LL, Noakes DE, Hanson MA. Cardiovascular development in late gestation fetal sheep and young lambs following modest maternal nutrient restriction in early gestation. J Physiol (Lond) 1998; 505P:18P. 62. Hawkins P, Crowe C, McGarrigle HHG, Saito T, Ozaki T, Stratford LL, Noakes DE, Hanson MA. Effect of maternal nutrient restriction in early gestation on hypothalamic pituitary adrenal axis responses during acute hypoxaemia in late gestation fetal sheep. J Physiol (Lond) 1998; 507P:50P. 63. Owens JA, Owens PC, Robinson JS. Experimental fetal growth retardation: metabolic and endocrine aspects. In: Nathanielsz PW, Cluckman PD, Johnston BM,

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15 Fetal Origins of Lung Disease JOHN O. WARNER and CATHERINE A. JONES University of Southampton and Southampton General Hospital Southampton, United Kingdom

I.

Introduction

The lung is unusual in that its development is incomplete at birth. There is, therefore, a significant potential for postnatal insults to have a long-term influence on respiratory health. Furthermore, function must undergo a very rapid and dramatic change at birth. Professor Simon Godfrey described this event in very graphic terms: “Man begins his existence as an aquatic parasite living in a stable friendly environment. Quite suddenly, he is ejected into the air and must thereafter rapidly adapt his physiology to that of a terrestrial mammal. This involves a drastic redirection of his regional blood flow and the calling into service of his lungs which have so far been semi-collapsed, fluid filled sacks” (1). It is perhaps not surprising, then, that most research into the early origins of lung disease have focused on postnatal events. However, it is becoming clear that very subtle influences on fetal lung and immune function development can have a profound impact on the risks of respiratory disease 297

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host defence mechanisms is required to appreciate the potential outcome of perturbations during fetal life.

II. Lung Embryology In the fourth week of gestation, a ventral diverticulum arises from the foregut of the embryo. As the epithelium pushes out from the pharyngeal floor, it is surrounded by mesenchymal tissue from the splanchnic mesoderm. This condenses around the developing bronchial tree and differentiates into cartilage, muscle, and connective tissue. It is from this tissue that the pulmonary blood vessels and lymphatics develop. Within 1 week, the airway has grown and divided to the level of lobar bronchi. Airways continue to branch such that by the sixteenth week of gestation, the bronchial tree is fully formed. The pulmonary vessels, both arteries and veins, follow the development of airways with a similar pattern of branching but with a slight time lag (2). Alveolar development does not begin until airway growth is complete. Between 4 and 6 months of gestation, there is a transformation of the last airway generation to form respiratory bronchioles, and arising from them, thinwalled saccules which are the primitive alveoli. Further generations of saccules appear with the progressive formation of alveoli. However, alveolar development progresses through gestation and continues after birth until 8 to 10 years of age. The alveolar number at birth has been quoted as being anything between 8 and 50% of the eventual adult number (2,3). As with bronchial tree development, the intra-acinar vessels follow the development of alveoli. In the early stages of bronchial bud development, the vascular supply is from a systemic capillary plexus that normally is lost with continuing lung development. Persistence of these primitive arteries that arise from the aorta will give rise to one form of lung malformation, known as sequestration. As the primitive aorta grows cordally, the communication often eventually arises from the abdominal section of the aorta. Bronchial arteries develop from the aorta at a much later stage and independent of pulmonary artery development. Bearing in mind this basic developmental progress, it is clear that different components of the lung develop at different rates and reach functional maturity at remarkably diverse time points, both ante- and postnatally. Thus, any insults to growth and development that could take the form of localized trauma, ischemia, infection, inadequate nutrition, or hormonal aberration will have very differing impacts depending on the timing of the insults. Indeed, it

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has been proposed that it is not the nature of the insult but the timing and severity that determines the morphology of the eventual lesion (4).

III. Molecular Basis of Lung Growth There is currently only a relatively sketchy understanding of the molecular basis of lung development, which has been more easily elucidated in simple organisms. However, organ formation in the embryo is under the influence of a set of controlling genes known as homeotic or Hox genes (5). The Hox genes encode nuclear proteins that bind to DNA and thereby modulate the transcription of sets of target genes. The temporal and spacial distribution of the nuclear transcription factors in turn control the expression of other Hox genes, which in turn influence many downstream genes encoding other transcription and growth factors. Two transcription factors have been particularly highlighted as being of importance in lung development, namely hepatocyte nuclear factor-3β (HNF-3β) and thyroid transcription factor– one (TTFI). These are involved with cell commitment and differentiation as well as influencing the expression of specific genes. TTFI is also critical in regulating the expression of surfactant proteins (SP), which in turn have effects not only on surfactant phospholipid production but also host defense (6). Complex intercellular signaling between the mesenchyme and developing respiratory epithelium also has a profound influence on cell behavior. Furthermore, autocrine and paracrine interactions regulate cell proliferation and differentiation and almost certainly have an influence postnatally on repair and remodeling. The mesenchyme produces a wide array of peptide growth factors, which influence lung morphogenesis. There are, in addition, mechanical factors of importance such as the volume of the thoracic cavity, fetal lung liquid positive pressure, and amniotic fluid volume. Even fetal breathing movements have an impact. Thus pulmonary hypoplasia has been associated with space-occupying lesions in the chest such as congenital diaphragmatic hernia and pleural effusions. It also occurs where there are congenital malformations of the chest such as occurs with congenital scoliosis but also with oligohydramnios, which may result from renal agenesis, urinary outflow obstruction, or prolonged premature rupture of membranes. Neuromuscular disorders that prevent normal fetal breathing movements have also sometimes been associated with pulmonary hypoplasia (7). At a molecular level, a congenital malformation known as acinar dysplasia has been associated with decreased or absent levels of TTFI, HNF3β, and surfactant proteins. The lungs

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of infants in this situation are very hypoplastic with reduced peripheral airway development (6). IV. Ontogeny of Host Defense Host defense may be divided into innate and adaptive, and clearly has immense importance in relation to the unique postnatal exposure of respiratory epithelium directly to the environment and all its noxious constituents. However, maturation of the host defense system during fetal life may be profoundly influenced by variations in the health of the mother and her own environmental experience which, in turn, will influence susceptibility to a range of respiratory diseases, not the least being atopic asthma. There has been a commonly held misconception that the newborn baby is predominantly immunologically naive. However, it is clear that the capacity to mount a significant immune response is present from very early in gestation. Stem cells are present in the human yoke sac at 21 days of gestation, with the first lymphocytes seen in the thymus at the end of the ninth week of gestation. B lymphocytes can be seen in a range of organs, including the lungs and gut from 14 weeks of gestation, and by 19 to 20 weeks, circulating B cells with surface immunoglobulin M can be detected. This implies that the full sensitization process must have occurred from antigen presentation through T-cell proliferation to B-cell stimulation and antibody production. That there is the potential for immune response well before birth is best evidenced by the presence of IgM rubella antibodies in fetuses infected via their mothers with this virus. However, studying neonatal immune responses, there is a clear impression that the system is predominantly naïve (8). To what extent this naiveté is truly a function of lack of sensitization and to what extent it is late gestational suppression of immune responses remains to be established, but increasing evidence would suggest that it is the latter. Normal pregnancy is characterized by a suppression of maternal cellmediated responses to fetopaternal antigens, predominantly effected by a switch to a dominant humoral immune response (9). Initially in studies of mouse fetoplacental tissues and more recently in humans it has been apparent that a range of tissues in the fetoplacental unit secrete cytokines, which have a profile similar to that associated with a T-helper lymphocyte type 2 phenotype, which is associated with atopy. These cytokines shift the balance away from cell-mediated activity towards humoral immune responses. Such cytokines almost certainly also have additional properties in promoting fetal growth. It

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has, for instance, been shown that granulocyte macrophage–colony stimulating factor GM-CSF has a critical effect on surfactant homeostasis by stimulating differentiation and proliferation of the type 2 pneumonocytes (6). Implicit in the demonstration that the Th-2 cytokine bias of normal pregnancy promotes a successful outcome is that a Th-1 bias will be associated with pregnancy loss. There is ever increasing evidence that this is indeed the case (10). Furthermore, a number of additional Th-2 cytokines, such a IL-4 and IL-10, will have an influence on cell maturation and therefore, directly and indirectly, like GM-CSF, affect lung structure and function. IL-10 is worthy of attention because of its Th-1 immunosuppressive properties, by diminishing the production of IL-12 and consequently IFN-γ. Is this cytokine contributing to the apparent immunological naivity of the newborn? Maternally derived decidual tissue is a source of IL-1 α and β, IL-6, GM-CSF, IL-4, 10, and 13 (11). We have recently identified IL-13 immunoreactivity in the fetal placenta, which is exclusively generated between 16 and 27 weeks of gestation. From 27 to 28 weeks of gestation onwards until 34 weeks, IL-13 can be found released spontaneously from fetal mononuclear cells, and from 37 weeks to term, spontaneous release ceases and production can only be induced by mitogenic stimulation of the fetal cells. Thus, there must be a very subtle regulation of the production of this cytokine with an interaction between the mother, the placenta, and the fetus, which leads to changes in production from different tissues at different times. It is very likely that this regulation will extend to other cytokines (12). The cytokines generated by gestation-associated tissues may be transported to the fetus either via the trophoblasts into the fetal circulation or alternatively, and perhaps more likely, via the amniotic fluid. Certainly we can detect high levels of the Th-2-associated cytokines in amniotic fluid. The protein turnover in amniotic fluid occurs at a rate of 70% each day with much of this removal being via fetal swallowing (11). Furthermore, the fetus aspirates amniotic fluid into the respiratory tract, and in addition, the highly permeable skin is exposed directly. Fetal gut is currently best studied. We have found many HLA-DR positive cells comprising macrophages, B cells, and dendritic cells in the lymphoid follicles of rudimentary Peyers patches from fetuses very early in the second trimester of pregnancy. Surface markers identified on these cells suggest that necessary costimulatory signals are available to facilitate antigen presentation in the development of an antigen-specific response. Interestingly, amniotic fluid also contains IgG and IgE, putatively of maternal origin, from as early as 16 weeks gestation. We can detect IgG receptors (CD16, CD32, and CD64) and IgE receptors (CD23 and high-affinity receptor) on cells within the lamina

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propria of the fetal gut. There is, therefore, the potential for these immunoglobulins to produce antigen focusing in antigen-presenting cells, which might lead either to sensitization or tolerance, depending on which immunoglobulin and which receptor is involved (13). Although we have not yet shown allergen in the amniotic fluid, there is good evidence that fetal allergic priming does occur. At birth, it is possible to show antigen-specific Th-2-biased immune responses in cord blood mononuclear cells. This Th-2-biased response is universal among atopic and nonatopic populations (14), but in those infants destined to develop atopic disease, the Th-2 bias, at least in terms of suppression of IFN-γ production, is greater (15). We have been able to show that the specific responses occur from as early as 22 weeks gestation (16). This has been further elaborated in a study identifying that birch and timothy grass pollen exposure only sensitized fetuses if it took place in the first 6 months of pregnancy. Exposure in later pregnancy appeared to result in either immune suppression or tolerance (17). It is interesting to speculate why such sophisticated immune responses should be in place early in gestation. We have proposed that this could be to facilitate neonatal host responses to the obligate exposure it will have to maternal helminths (18). Certainly, infants born to helminth-infected mothers have specific Th-2–biased immune responses to helminth antigen and IgE antibodies (19). In this era of low parasite infestation, it is likely that molecules present in sensitizing allergens that have counterparts in parasites leads to stimulation of the same immune response (20). V.

Congenital Lung Malformations

The frequency of congenital lung anomalies has not been accurately ascertained, but ranges between 7.5 and 19% of all congenital malformations (21). Recent data from England and Wales suggest a rate of approximately 80 cases per million births (22). These figures are probably a significant underestimate of true frequency because many lesions are not recognized. Major malformations of the respiratory tract are commonly associated with other congenital anomalies, particularly involving the cardiovascular system. However, minor malformations, such as defects in bronchial branching, are more common and may go unrecognized even when they predispose to poor drainage and recurrent infection. Once infection has occurred, the distortion created by inflammatory responses may mask the primary malformation that was the cause of the problem. Although it is not the purpose of this chapter to describe gross

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congenital lung malformation in detail, there are lessons to be learned from some of the malformations in relation to influences on susceptibility to particular respiratory problems. A. Vitamin A and Congenital Malformation

It has long been known that vitamin A, or retinol, plays a major role in lung development, such that deprivation during pregnancy results in profound disturbance of lung organogenesis (23). However, even mild vitamin A deficiency may be associated with some effects on fetal lung maturation. Studies in rats suggest that reduction in blood retinol levels, between 30 and 60% of normal, results in reduced surfactant phospholipid generation which, in turn, is probably attributable to an effect on the generation of surfactant proteins which then regulates phospholipid generation. This appears to be through an effect on gene expression and has a greater impact on SP-A than SP-B and SP-C (24). It is worthwhile noting that SP-A also has important host defense functions serving as an opsonizing protein within the airway. Thus, if this effect is operative in humans, then there may not only be susceptibility to idiopathic respiratory distress syndrome in the neonatal period but also a greater risk of infection. Earlier studies on lung tissue cultures from fetal rats have also shown that exogenous retinoic acid has remarkable effects on airway branching and lung epithelial cell differentiation. It interferes in a dose-dependent fashion with the expression of epithelial genes found in the distal segment of the fetal lung, such that in very high concentrations, it suppresses distal epithelial buds and favors growth of proximal airway tissues (25). This is exactly the pattern of abnormality one sees in pulmonary hypoplasia (7). Extrapolation from these observations would suggest that subtle differences in Vitamin A intake through pregnancy could have minor but nevertheless relevant effects on lung development, which could increase susceptibility to neonatal respiratory problems particularly if coexisting with prematurity, as well as to infection and its consequences. B. Congenital Cystic Adenomatoid Malformation

This is a rare defect of the non–cartilage-containing airways, probably occurring in the mid to late stages of lung development. Basically, there is a haphazard proliferation of terminal bronchiolar elements interspersed with mesodermal elements. Histological features of the lesion depend partly on the timing of the disruption and the humoral influences from the mesenchyme on

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subsequent growth. Lesions can be exceedingly large and incompatible with survival, even in utero. Some, however, have been detected on ultrasound in utero and have subsequently resolved completely (26). It is reported that such lesions may present in later life with recurrent infection (27), or occasionally can be associated with malignant change (28). C. Bronchiectasis and Congenital Malformation

As previously intimated, abnormalities in bronchial branching might be associated with abnormal drainage and thereby increase susceptibility to infection and eventually bronchiectasis. Certainly, bronchiectasis has been associated with a range of malformations, including congenital cystic adenomatoid malformation and so-called intralobar sequestration, where there is a systemic arterial supply to a lung segment (21). D. Wheezing and Congenital Malformation

The degree to which congenital malformation might contribute to nonatopic wheezing, particularly in infancy, remains to be established. However, infants with tracheo- and bronchomalacia, mediastinal cysts, and vascular rings present with wheeze, with or without stridor. There is some suggestion that such abnormalities occur more frequently than is normally appreciated and can account for chronic persistent wheeze, often misdiagnosed as asthma and consequently often grossly overtreated. The long-term consequences are, therefore, not only of the primary disorder but also of the side-effects of inappropriate therapy (29). VI. Fetal Growth and Later Lung Function After insults to the lung postnatally, inflammation can be followed by repair and restitution of normal function, and there does appear to be a consistent tracking of respiratory function from infancy to late childhood (30). It has long been appreciated that impaired lung growth may lead to chronic obstructive airways disease in late adult life because of a failure to attain maximum potential lung function as a young adult (31). Thus, linking these two observations suggests that abnormal lung function in early infancy will be associated with persistent impairment of lung function through childhood, resulting in reduced maximal lung function as a young adult. Even if the subsequent rate of decline in function with age is normal, significant impairment likely to lead

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to symptoms will occur at an earlier age in later life. The superimposed increase in attrition of lung function that occurs with additional lung insults, such as environmental tobacco smoke exposure, will inevitably lead to a greater prevalence of disease (32). Fetal growth retardation has been associated with increased risks of lower respiratory tract infection in early childhood and impaired lung growth. Thus, newborn infants with evidence of intrauterine growth retardation who are born small for gestational age have reduced lung function in childhood (33). Furthermore, there is an increased frequency of early respiratory illnesses, whether this be pneumonia (34) or bronchiolitis (35). To what extent the impaired lung growth in utero affects adult lung function, independent of any intervening acute severe infection, is difficult to establish. One recent study has suggested that impaired fetal growth alone is not associated with impaired lung function in adults, whereas those who have an early significant acute respiratory illness do have deficits in flow and volume (36). However, a historical cohort study of men born in the first quarter of this century in the United Kingdom revealed a strong association between lower birth weight and reduced lung function, even after controlling for age, height, smoking, and social class. The effect, however, was much greater in those who also had had bronchitis, pneumonia, or whooping cough in infancy (37). It is unlikely that these latter findings would have been confounded by maternal smoking in pregnancy because very few women smoked in the first quarter of this century. However, more recent studies may well be confounded by this effect. It is now very clear that maternal smoking in pregnancy has a significant influence on the newborn infant’s lung function. This has been shown by using a number of measurement techniques in several different countries; United Kingdom (38), Norway (39); and the United States (40). Furthermore, such reduced lung function, caused by maternal smoking in pregnancy, was also associated with an increased frequency of wheezing illnesses in early life in all of the studies (38,39,41). The mechanism by which poor fetal growth affects lung function and thereby susceptibility to respiratory infection remains in the realm of speculation. However, as suggested from studies of congenital malformation, it seems possible that subtle dietary influences could have a significant impact, of which perhaps the most obvious might be vitamin A intake. With regard to tobacco smoke, animal studies suggest that exposure to nicotine in utero reduces alveolar number and surface area but not total lung volume because of compensation by increased alveolar size (42). It is also possible that effects on fetal respiratory movements might impact lung development.

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Infant Wheeze and Fetal Growth

Wheeze is an extremely common symptom in infancy. Thirty-four percent of children up to 3 years of age will have experienced at least one wheezing episode, and by 6 years, it is 49% (43). However, not all children who wheeze in infancy continue to do so in later life. Approximately half of those wheezing before the age of 3 are asymptomatic by 6 (43). The percentage that is eventually shown to have asthma varies between studies being dependent on size and type of cohort and the length of follow-up. Some would suggest that 80% of these infants outgrow their problem (44), and many such transient wheezers have reduced premorbid lung function attributable to maternal smoking

Figure 1 Factors influencing infant wheezing and its outcome.

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(38,39,41,43). Thus, nonatopic wheezing in infancy has a tendency to remit in early childhood (Fig. 1). It is, however, intriguing that the risk factors for early wheezing are rather similar to those associated with later-onset adult chronic obstructive pulmonary disease (45). Thus, to what extent infant wheeze and late-onset adult chronic obstructive lung disease with a nonasthmatic basis can be linked remains to be established by long-term cohort studies. However, there is strong circumstantial evidence to link the two. It is clear that there are other influences on persistence of wheeze from infancy into later childhood and early adult life, of which allergic sensitization is the most important. It is for this reason that the remainder of the chapter will concentrate on the fetal origins of allergy. VIII. The Fetus and Allergic Sensitization Many studies have now shown that the neonate is not immunologically naïve but is capable of mounting significant immune responses to environmental antigen. This can only have occurred as a result of antenatal sensitization. Indeed, the presence of peripheral blood mononuclear cell sensitivity to allergens in neonatal blood samples has predicted the subsequent development of atopic disease (14,46,47). From the Prescott study (14), proliferative responses of cord blood cells to house dust mite occurred in 46% of samples, to the purified major allergen of house dust mite in 73%, and to ovalbumin from hen’s egg in 42%. Any suggestion that this is a consequence of maternal cross-contamination of the neonatal blood sample has now been adequately answered by genotyping of T-cell clones from cord blood samples, showing that they are definitely of fetal origin (14). Furthermore, the allergen-specific reactivity develops in the peripheral circulation of the fetus from as early as 22 weeks gestation and onwards (16). Thus, we have evidence that there is a virtually universal priming to environmental antigens occurring before birth, as well as evidence that those destined to be allergic can in some way be distinguished by altered responsiveness to allergens (15). Future research, therefore, must identify the mechanisms by which normal priming occurs and to elaborate which factors upset homeostasis, leading to enhanced risk of atopic disease. There is a strong indication that one component upsetting this balance is the presence of maternal atopy. Thus, infants born to mothers with allergic disease are far more likely to express early onset atopic disease themselves than those born to fathers with atopy (48,49).

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In murine models, it is known that the fetoplacental unit produces interleukins 4, 5, and 10 throughout pregnancy (9). Interleukin-4 has also been shown to be produced by human amnion epithelium both in the first and third trimesters of pregnancy (no specimens tested from the second trimester) (50), and mRNA coding for IL-10 has also been shown in term human placental tissue (51). Our group has very recently shown IL-13 production by the placenta predominantly occurring during the second trimester of pregnancy (12). It is well established both in murine models and humans that the allergic immune response leading to immunoglobulin-E production is associated with a bias of cytokine production towards IL-4, 5, 10, and 13. Thus, pregnancy itself may be viewed as an allergic phenomenon. Indeed, it has been suggested that this inhibits maternal Th-1 production of the cytokines IL-2, IFN-β, and tumor-necrosis factor â. These, in turn, would be likely to compromise the continuation of pregnancy (10). Interleukin-2 increases proliferation of uterine large granular lymphocytes, creating lymphokineactivated killer cells with enhanced cytotoxic activity (52). Furthermore, in vitro IL-2 increases the proportion of decidual large granular lymphocytes that express CD 16, which in turn is associated with increased killing of cultured trophoblast cells (53). Thus, IL-2 expression may compromise the invasiveness of trophoblasts during implantation and placental growth. IFN-γ is an abortifactant whose effect is probably mediated through activation of NK cells, this effect being inhibited by the production of IL-10 (54). Thus, a Th-1–biased environment at the maternofetal interface is pregnancy compromising via activation of cytotoxic lymphocytes and NK cells. It may, therefore, be assumed that the Th-2 cytokines manufactured by placental tissues will downregulate such responses and thereby protect the pregnancy. However, such activity will universally bias the fetus towards a Th-2– allergy-promoting response, which explains the findings of Prescott et al. (14). There is, however, an important role for the Th-1 cytokine IFN-γ in regulating early fetal immunological development. This cytokine stimulates the expression of HLA-G by trophoblasts, which allows these cells to avoid cytolysis by decidual large granular lymphocytes. The trophoblast populations do not express MHC Class II molecules or the highly polymorphic classical HLA-A, B, and C antigens. HLA-G is a nonclassical, nonpolymorphic product which, therefore, does not activate maternal immune responses. Our own preliminary investigations have shown that most fetuses spontaneously release IFN-γ from cultured unstimulated peripheral blood mononuclear cells during

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the second trimester of pregnancy. Furthermore, other groups have shown that fetal plasma concentrations of IFN-γ are higher in the first compared with the third trimester (55). Such IFN-γ production will also, to a certain extent, counterbalance the effects of the Th-2 cytokines from the placenta, thereby protecting the fetus from overcommitment to an allergic response. IL-4, on the other hand, is known to stimulate cell growth and differentiation. It also upregulates expression of MHC Class II molecules, thereby assisting activation of antigen-presenting cells and thus promoting the fetus’ ability to respond to antigens (56). There is, therefore, a remarkably fine balance of cytokine production between the mother, placenta, and fetus orchestrating a downregulation of maternal immune responses to fetopaternal antigens while simultaneously encouraging normal growth development and immunological responsiveness in the fetus. Thus, it is not surprising that relatively minor influences can have an impact either increasing or decreasing the risk of subsequent disease in the resulting infant. X. Fetal and Maternal Influences on Atopy A. G enetic Diversity

Recent worldwide comparisons of prevalences of allergic disease have shown that all are significantly higher in English-speaking communities in Australia, New Zealand, the United Kingdom, and North America (57). This cannot be explained by any of the hypotheses hitherto mounted to elucidate differences in prevalences (58). English-speaking communities have a far broader genetic diversity in terms of ethnic backgrounds than those communities with low prevalences of atopy. Such genetic diversity may facilitate the presentation of a wider range of antigens, which in the increasingly diverse environment could provide the real explanation for varying and changing prevalences. However, this genetic diversity may well have its major effects in utero. As previously indicated, downregulation of Th-1 responses in the mother to fetopaternal antigens is essential to fetal well being. That this downregulation occurs in human pregnancy is well substantiated by the clinical observation that rheumatoid arthritis, a Th-1–mediated disease, often improves appreciably during pregnancy only to relapse or indeed appear for the first time in the postpartum period (59). However, not all women with rheumatoid arthritis improve during pregnancy. One group has suggested that improvement is dependent on the degree

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of mismatching in major histocompatibility complex genes. The maternofetal disparity in alleles for HLA DR and DQ antigens is more common in cases where the arthritis remits or improves compared with cases of those with continuing active disease (60). This would suggest that the Th-2–biased cytokine production by the placenta is actually switched on by the tissuetype disparity between mother and fetus. It, therefore, seems equally credible to propose that the greater the genetic disparity between mother and fetus, the greater the immunological drive towards allergy. Thus, hybrid vigor may have a downside! XI. Maternal Atopy Although a large number of regions of the genome have now been identified as having possible linkage to atopy, bronchial hyperresponsiveness, and/or asthma (61), this does not explain the predominance of atopy in infants born to atopic mothers compared with those born to atopic fathers (48,49). Indeed, there is one study showing inheritance primarily through the maternally derived alleles for polymorphisms on the long arm of chromosome 11, which codes for a component of the high-affinity IgE receptor and is linked to atopy (62). This phenomenon may either be attributable to genomic imprinting, which seems unlikely, or a manifestation of intrauterine environmental differences when the mother is atopic. We have evidence that the IL-10 levels in amniotic fluid of mothers who are allergic are higher than those in nonallergic mothers (13). This IL-10 will, in turn, suppress IFN-γ production (54). Thus, it is not surprising that studies have shown impaired IFN-γ generation by newborn babies’ peripheral blood mononuclear cells when born to mothers who are atopic (63). Furthermore, this reduced capacity to generate IFN-γ on stimulation in the newborn is associated with a subsequent higher risk of developing atopy and atopic disease (47,64,65). IL-10 has, of course, generally immuno-suppressive activity, and it is perhaps not surprising that the newborn infant destined to be atopic has a reduced capacity to generate a wide range of cytokines from stimulated cord blood mononuclear cells. Although IgE is not transported across the placenta into the fetal circulation, it is possible to detect IgE in the amniotic fluid from as early as 13 weeks gestation, with a good correlation between the amniotic fluid and maternal circulating levels throughout pregnancy (18). Thus, the atopic mother with high IgE levels will in turn bathe her fetus in higher IgE. For amniotic fluid IgE to influence fetal immune development, receptors for this immunoglobulin must be expressed within tissues, particularly in the gastrointestinal tract be-

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cause fetal swallowing results in a continuous circulation of proteins to the fetus. We have been able to show both high- and low-affinity IgE receptors in samples of fetal gut from as early as 11 weeks gestation. There is, therefore, the potential for IgE bound to antigen-presenting cells in the fetal gut to facilitate antigen presentation by a phenomenon known as IgE-mediated antigen focusing. This means that responses can occur with a hundred- to a thousandfold less antigen than that normally required for priming. This focusing of responses need not be specific for the particular IgE antibody but may facilitate sensitization by a bystander effect (66). Thus, raised IgE in the mother will facilitate allergen sensitization in the fetus and account for higher proliferative responses to allergens in newborn babies that are destined to develop allergy (47). It is sometimes possible to show raised cord-blood IgE in newborns, which is to a certain extent predictive of subsequent development of atopic sensitization and disease. There are a very large number of studies showing that raised cord IgE is specific for subsequent atopy but very insensitive because the majority of infants destined to have allergy have not had a sufficient antigenic stimulus to raise the IgE antenatally. However, those in whom it has occurred are almost certainly going to develop atopy within the first year or two of life (67). XII.

Timing and Concentration of Allergen Exposure and IgG Antibodies

Several groups have shown that priming to aeroallergen can occur during fetal life (14,16,68). Interestingly, this exposure to allergen antenatally may lead to sensitization provided it occurs in early and mid pregnancy but does not occur if the exposure is only the latter half of the third trimester (17). Immunoglobulin-G is actively transported across the placenta to the fetus and serves a very important role in passive protection of the infant for the first few months of life against infection (69). IgG crosses the placenta as early as 20 weeks gestation with preferential transfer of IgG1 and IgG3 compared with G4 and G2 (69). Old studies of animal models had suggested that passively acquired IgG antibody could protect from IgE sensitization. Thus, rat pups immunized with ovalbumin while maternal antibody was still present in the circulation did not develop IgE sensitivity, and remained nonresponsive to secondary exposures even after the maternal antibody had disappeared (70). This suggests that IgG not only acted as a blocking antibody preventing anti-

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gen from gaining access to antigen-presenting cells, but it also had a longterm immunomodulatory effect. Dendritic cells, which are the main antigen-presenting cells for naïve T cells, have both high- and low-affinity IgG receptors (71). Binding of IgG to the low-affinity receptor facilitates uptake of antigen-bearing complexes through the endocytic pathway. This results in, among other things, release of IL-12. This cytokine promotes IFN-ã production which, in turn, will promote Th-1 responsiveness and suppress the ability to develop a Th2 allergy-associated response. This, once established, nearly always persists such that the perinatal T-cell response can be shown to be a significant determinant of the response in adulthood (72). Hitherto, prospective studies in humans have failed to show a protective effect of IgG food protein antibodies in the cord blood against the subsequent development of atopic disease, and consequently the idea that IgG might be protective had until recently been ignored (73). There are a few human studies that would support the hypothesis that maternally derived IgG antibodies are protective. The children of mothers who had undergone rye-grass immunotherapy during pregnancy and consequently had high IgG antibody levels, compared with children born to untreated mothers, had fewer positive skin tests to the grass at between 3 and 12 years of age (74). High cord-blood IgG antibody levels to â-lactoglobulin from cow’s milk has been associated with less subsequent cow’s milk allergy in the offspring (75), but not all studies are able to show this (73). However, high levels of IgG anti-IgE antibodies in the cord blood are associated with less atopic symptoms over the first 18 months of life, and this particularly operates in babies with a strong family history of atopic disease (76). Unfortunately, none of these studies have quantitated the IgG antibody levels to establish whether there is a dose-response effect, and this may account for the discrepant results from different studies. The level of IgG antibody to particular allergens in the mother will be directly related to the maternal exposure to allergen. Thus, exceedingly low exposure will be associated with very low IgG antibody levels, but will also be associated with a much-reduced chance of the fetus being exposed to the antigen, and therefore a lower probability of sensitivity. Exceedingly high exposure of the mother, as occurs in immunotherapy, will produce high IgG antibodies and may, by the mechanisms previously described, be protective against sensitization by inducing tolerance. Thus, it could be hypothesized that only in those pregnancies where the dose of exposure is moderate will sensitization occur. We have preliminary evidence in relation to egg sensitivity that this may indeed be the case. Thus, very low or very high maternal IgG

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antiovalbumin antibodies were associated with a low probability of positive skin-test responses to egg by 6 months of age. The highest probability of sensitivity to egg in the offspring occurred when the mother had only moderately raised levels of ovalbumin IgG antibody. Thus, these data point to there being a critical timing and dose of exposure during pregnancy in relation to outcome. XIII. Maternal Nutrition and Atopy One hypothesis proposed to explain the progressive increase in asthma and allergic disease in affluent communities has been that this has paralleled a significant change in diet as well as lifestyle. There has been a declining rate of consumption of fresh fruit and vegetables in the United Kingdom that has paralleled the rise in prevalence of atopic disease (77). Such foods are associated with antioxidant activity and might be implicated as protecting against the development of airway inflammation but may also influence IL-4–dependent IgE production, at least in murine models (78). To what extent this has its impact antenatally rather than postnatally remains to be established by controlled clinical study. However, a recent British survey showed that after accounting for the effect of cigarette smoking, a low fresh-fruit intake was associated with frequent wheeze and speech-limiting asthma attacks in 33-year olds, which suggests that diet certainly influences severity of disease but has not necessarily had any effect on prevalence (79). It is possible that other dietary factors, such as fatty acids, might be involved. No studies have yet been conducted through pregnancy. However, there is compelling evidence that manipulation of the composition of polyunsaturated fatty acids in the diet has an impact on established atopic disease. Thus, dietary supplementation with evening primrose oil, rich in gamma linolenic acid, produces moderate clinical improvement in patients with atopic dermatitis (80,81). Supplementation with fish oils, rich in eicosapentaenoic acid and docosahexaenoic acid, produces modest improvement in lung function in asthmatics (82). Most studies have shown a far greater effect on in vitro immune responsiveness, such as decreased production of tumor-necrosis factor α and leukotrienes rather than clinical effects (83,84). That trials of dietary supplementation during pregnancy are urgently required is further amplified by the intriguing observation of an association with large head circumference at birth, suggesting altered fetal growth in utero and therefore nutrition, with circulating total IgE levels at birth (85), in child-

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hood (86), and adulthood (87). Large head circumference at birth was also strongly associated with severe symptomatic asthma (86). The hypothesis proposed to explain these observations is that good maternal nutrition at conception programs the fetus to adopt a rapid growth trajectory. The nutritional requirements of a rapidly growing fetus cannot be so easily sustained, particularly in the third trimester of pregnancy. At this point, a brainsparing reflex allows continuing growth of the head at the expense of the body. Thus, the newborn baby is disproportionate with a relatively large head but normal body size. The relatively poor nutrition of the body will affect rapidly growing tissues, such as those in the immune system, which could subtly alter the balance of Th-1 and Th-2 activity and thereby be associated with a higher risk of atopy (Fig. 2).

Figure 2 Hypothesis linking affluence, good nutrition, large birth head circumference, and allergy.

Fetal Origins of Lung Disease XIV.

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Conclusions

Although postnatal insults clearly do have a greater effect on later lung disease because of the unique continuing lung growth and development that occurs postnatally, it is clear that there are major factors in fetal life that impact on later lung disease (Table 1) The embryology of lung growth and development is well established, although the molecular basis for this is only sketchily understood. Subtle alterations in nutrition, most studied in relation to vitamin A, and the effects of maternal smoking in pregnancy have clearly been shown to affect lung growth and development and thereby increase susceptibility to later lung disease. There are also strong associations between poor fetal growth in general and increased susceptibility to abnormal lung function and a greater risk of adult respiratory disease. However, what factors are involved in this association have yet to be elucidated. The ontogeny of the immune response in the fetus is now beginning to be understood. There is a complex interaction between the mother, the placenta, and the fetus aimed at maintaining the pregnancy and facilitating fetal growth and development. An allergic bias to the maternal response to fetopaternal antigens is orchestrated by the generation of allergy-promoting cytokines such as IL-4, -10, and -13 by the placenta. This is balanced by the production of non–allergy-promoting cytokines, such as IFN-γ, by the fetus. Very subtle perturbations during pregnancy can upset this balance, either increasing the chances of fetal growth retardation and spontaneous miscarriage or of allergy that will manifest itself postnatally. There are genetic and environmental factors that have been identified to influence this outcome. Maternal atopy itself increases the risk of atopy in the offspring. Additional factors may include maternal nutrition, with a current focus of attention being on polyunsatuTable 1 Perinatal Factors Influencing Three Respiratory Syndromes Virus-associated wheeze Low birth weight Preterm delivery Chronic lung disease of prematurity Pregnancy smoking

Asthma Affluence Maternal atopy Birth head circumference Maternal nutrition and allergen exposure

Chronic obstructive pulmonary disease Low birth weight Smoking Respiratory illness in infancy

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rated fatty acids. The concentration of exposure to allergen via the mother during pregnancy also has a profound effect on probabilities of sensitization. Elaboration of the key factors involved could facilitate new intervention strategies that will reduce the prevalence of allergy, which is associated with a high risk of developing the extremely common allergic airway disease, namely asthma, which now affects up to 20% of the population, at least in developed countries worldwide. XV.

Acknowledgments

We thank Mrs. Wendy Willcocks for preparing the manuscript, and the National Institutes of Health, who are funding a major research program in our Department (Project No. 1R01HL61858, Ontogeny of fetal sensitization to allergens and asthma).

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16 The Nutrition Transition and its Implications for the Fetal Origins Hypothesis

BARRY M. POPKIN University of North Carolina at Chapel Hill Chapel Hill, North Carolina

I. Introduction In the earlier chapters of this book, the fetal origins or programming hypothesis is laid out and the evidence is evaluated. This work shows how the presence of risk factors for cardiovascular disease and other comorbidities will interact with the physiological adaptations caused by fetal and infant nutritional insults to significantly increase the risk of these diseases. The health effects of these insults will rapidly rise over the next several decades because the populations in the world most at risk of these fetal and infant nutritional insults are undergoing a rapid nutrition transition. This shift in the level and structure of their diets in conjunction with rapid reductions in physical activity and increases in inactivity pose the potential to significantly accelerate the negative effects of this transition. The key pathways through which the fetal origins hypothesis works related to reduced growth during the second and third trimester of pregnancy 323

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(1). Insults at various times in this process can lead to low birth weight, stunted or thin babies, or stunted fat babies. In addition, there may be added insults that continue to occur during infancy. As Barker has posited with limited evidence and my research group has tested, there is evidence that stunting during infancy will lead to the same type of insults as stunting at birth, but this pathway remains to be tested systematically (2). Low weight during infancy and stunting in early childhood are also risk factors for subsequent obesity. In some cases, infant and early childhood growth retardation result from the same underlying factors that cause intrauterine growth (IUGR). Poverty is related to poor maternal nutrition, poor weaning diet, and increased exposure to infections (3,4). IUGR of itself increases the risk of stunting in infancy and later childhood (5). Independent of IUGR, many children in developing countries, as well as poor children in the United States and other developed nations, become stunted during infancy as the result of inappropriate weaning practices, repeated infections, and poor diet—all in the context of poverty (5,6). The highest incidence of stunting occurs in the weaning period and soon after. Early childhood stunting is not readily reversible when children remain in the same poor environments. After age 3, children in developing countries typically have growth increments comparable to those of U.S. children, but having begun with such large deficits, their attained size remains smaller. When environments change, as might be the case when socioeconomic status improves, the metabolic adaptations that promoted survival during a phase of undernutrition may now predispose stunted children to obesity. Whatever the set of preconditions that establish the increased sensitivity to environmental insults, the focus in this chapter is on the nutrition transition itself in an attempt to describe the population that is and will be at risk from the adverse effects of the fetal origins hypothesis. The main focus is on the dynamic circumstances, to provide some sense of the way that at-risk populations in the United States and the developing world will be faced with an increasing likelihood of adverse effects. For example, as Erickson and his collaborators in Finland have shown, the path of growth through childhood modifies the risk of disease associated with size at birth (7). In particular, among men, being overweight in childhood increases the risk of coronary heart disease associated with thinness at birth. In the United States, evidence of the rapid shift in obesity and related factors linked with the acculturation of the huge Hispanic and Asian immigrant population points to great potential for increased likelihood of greater cardiovascular disease risk among those who are currently first- or second-generation immigrants. Similarly, the very rapid shifts in diet and activity in the devel-

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oping world pose the same risks. Evidence on each of these pathways is presented in this chapter. II.

The United States: Immigrants and Other At-Risk Populations

A. Background

Although a substantial number of studies attribute racial and ethnic differences in body dimensions and patterns of physical growth to genetics (8), there is also ample evidence from migrant studies that show dramatic increases in stature and fatness in subsequent generations of immigrants born in the United States, suggesting a strong role of the environment. For example, Mayan and other Hispanic “short” populations catch up rapidly to the height profiles of other Hispanic and racial-ethnic groups when they are provided with higher quality environments (9). In 1990, one in seven children in the United States (13%) lived in an immigrant family (10). Immigrants tend to cluster by country of origin in selected states. For instance, immigrants account for 38% of the children in California. California is the home of the majority of immigrant children from Central America, Southeast Asia, and the Philippines. Over 80% of U.S. immigrant children speak a language other than English at home. Firstgeneration children and their native-born parents are less educated than second-generation children (10). The immigrant populations are so diverse that we are finding many paradoxes in the classic assimilation model for immigrant adjustment (11,12). For example, second-generation Mexicans might have poorer health outcomes than the first generation (13–17). Parental socioeconomic backgrounds differ widely among different immigrant groups—in particular, among Asian, Latin American, and Caribbean groups (18). B. National Studies on U.S. Adolescents

Our research suggests a rapid assimilation of eating and activity patterns from the first to the second generation of Americans, evidenced by markedly higher rates of obesity in second-generation immigrants. Popkin and Udry use a huge nationally representative survey, the National Longitudinal Study of Adolescent Health, to examine how immigrant status affects adolescent obesity. A sample of 13,783 adolescents is studied. Measurements of weight and height collected in the second wave of the survey are used to study adolescent obesity. There are representative subsamples of

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Puerto Ricans, Cubans, and Chinese included among the 4400 non-Hispanic blacks, 3400 Hispanics, and 1400 non-Hispanic Asian-Americans. Body mass index is used as the measurement of overweight status. The smoothed version of the National Health and Nutrition Examination Survey I (NHANES I) 85th percentile cutoff is used for the measure of obesity in this paper (these National Center for Health Statistics standards are currently under revision (19,20). For the total sample, 26.5% are obese. The rates were 24.2% for white non-Hispanics and 30.9% for black non-Hispanics, 30.4% for all Hispanics, and 20.6% for all Asian-Americans. There are important variations within the Hispanic and Asian-American subpopulations. The Chinese (15.3%) and Filipino (18.5%) samples show substantially lower obesity than non-Hispanic whites. All groups show more obesity among males than among females, except for blacks (27.4% for males and 34.0% for females). Asian-American and Hispanic adolescents born in the United States are more than twice as likely to be obese than the first-generation residents of the 50 states. First-generation American children are those who were not born in the United States nor to U.S. citizens abroad, and thus migrated to the United States as children (in most cases with their immigrant parents). Second generation are the “children of immigrants” born in the United States but who have at least one foreign-born parent. Third generation or higher are those born in the United States to native-born parents. Third or higher generation children may have grandparents or great grandparents who were immigrants. These children and their families have had a much longer period of acculturation in the United States. For Asians other than Chinese, Filipinos, and Koreans, along with Hispanics from Cuba, Central, and South America, there are few children in the third generation. The likelihood of obesity increases dramatically between the first and second generation in both groups, with the greatest increase among Asian Americans. Multivariate logit techniques are used to provide an understanding of the ethnic, age, gender, and intergenerational patterns of adolescent obesity. The third-generation effects were not significantly different from the second generation in either group. Analysis of subgroups within these broad categories show that Asian-American females are considerably less likely to be obese than males, and Asians from Korea, Japan, Southeast Asia, and India were more likely to be overweight than Chinese-American youth. The generation effect in Asians is primarily attributable to those from countries other than China. A more extensive description of our Add Health obesity work is found in Popkin and Udry (21). Figure 1 presents this pattern. Gordon-Larsen et al. have been investigating the role that generation

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Figure 1 Proportion of adolescent obesity categorized by ethnicity and generation of birth. (From National Longitudinal Study of Adolescent Health, Wave 2. Adjusted for gender and age. Ref. 21.)

plays in explaining the activity/inactivity and dietary patterns for these adolescents (37,38). In work to date, there are consistent effects of generation of residence on shifts in activity and inactivity patterns toward reduced energy expenditure. III. The Developing World There is limited population research on the epidemiology of CVD and key comorbidities such as obesity and diabetes. The literature that does exist poses a stark picture of a major surge in obesity and adult-onset diabetes among most populations of the developing world. We focus here on the nutrition transition as it relates to diet, activity/inactivity, and obesity. Zimmet and others have done extensive work on adult-onset diabetes (22–24). A. Underlying Factors Although low birth weight and stunting are linked with a complex interplay between a range of behaviors affecting maternal diet, energy expenditure, and

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infant feeding, behaviors that seem to change after many other changes linked with socioeconomic development occur, and obesity seems to be more rapidly linked with shifts in the overall structure of diet and activity. There is no clear consensus on why Asian and African countries, particularly those in South Asia, continue to have such high rates of low birth weight. It is clear, however, that their children are increasingly facing a prospect of becoming obese. The underlying sociodemographic and technological factors are the rapid changes in incomes, technology, dissemination of new knowledge, and urbanization (25,26). Here, we focus on the more proximate shifts in activity and diet to provide some sense of the dynamic shifts that will ultimately enhance the adverse effects of poor fetal growth. B. Activity Patterns and Energy Expenditures are Rapidly Changing

The factors that produce the rapid shifts in activity and inactivity and the level and structure of diet are becoming increasingly understood. Some of these relate to vast shifts in the structure of occupations, labor-saving technologies linked with electricity, and newer and more powerful technologies. The shifts in occupations, activities in occupations, and leisure are vast (26,27). The sectoral distribution of the labor force toward industry and service has accelerated around the world. Figure 2 presents data on this pattern for higher- and lower-income countries. It shows for all lowerincome countries a pronounced movement away from agriculture toward manufacturing and service employment. As has often been shown, the most labor-intensive agricultural work also requires the greatest amount of energy expenditure. One of the most inexorable shifts with modernization and industrialization is the reduced use of human energy to produce more capital-intensive manufacturing, goods, and services. The result is obviously a marked shift in activity patterns at work, a trend particularly associated with our shift into increasingly capital-intensive production and increasingly sedentary manufacturing, service, and commercial work. This withinoccupation shift in energy expenditure cannot readily be shown with national data. It requires individual-level information. Unfortunately, few longitudinal studies attempt to measure physical activity and energy expenditures. One quite simple measure of overall activity, an assessment of overall activity level into nine categories of energy expenditure, has been collected in each survey from 16,000 Chinese as part of the China Health and Nutrition Surveys (CHNS). We present in Table 1 the distribution of these activities into shifts in the proportion of Chinese adults in-

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Figure 2 Shifts in the distribution of occupation (1972–1995). (From World Bank. 53 countries over 23-year period.)

volved in low, middle, and higher levels of physical activity at work. In particular, this table shows the remarkably rapid shift of urban residents in all income groups into more sedentary activity patterns at work. Elsewhere, we have linked this activity pattern shift with significant increases in Body Mass Index (BMI) and obesity (28). In contrast, this pattern was not seen in the rural areas. In fact, rural residents, particularly low-income ones, showed a significant change from low and moderate activity patterns toward a high physical activity pattern, and related to that, an increase in chronic energy deficiency measured by a BMI below 18.5. In the past, home production and leisure activities were typically fairly active. There were few energy-saving or time-saving technologies. People washed clothes by hand, gathered their own cooking materials and water, and performed a wide range of labor-intensive activities. One of the major revolutions in the past 2 to 3 decades has been the introduction of electricity to populations in all lower-income countries who never had this. Herrin’s classic study on the impact of electrification on the lives of families in poorer regions has shown the profound shift in the use of time related to the use of electricity (29).

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Table 1 Distribution of Physical Activity of Chinese Aged 20–45 Years, by Tertile of Household Income and Residence, CHNS 1989, 1991, and 1993

Urban residence Lowest-level activity Middle-level activity Highest-level activity Rural residence Lowest-level activity (%) Middle-level activity (%) Highest-level activity (%)

Household income per capita tertiles Low Middle 1989 1991 1993 1989 1991

1993

23.7b,d 49.6b,c 26.7b

34.3b 30.1 35.6b,c

42.6 30.2 27.2

35.5c 46.1c 18.4

45.4 39.7a 14.9

2.8 40.6 16.6

15.3c 22.2b,d 62.5b,d

3.9b 5.3b 90.8b

4.8 b 7.9 b 87.3b

16.2c 28.9c 54.9c

12.3 14.1 73.6

12.6 13.3 74.1

The proportion differs significantly from middle- and high-income groups within same year (p < 0.05). b The proportion differs significantly among three income groups within same year (p < 0.05). c The proportion differs significantly from corresponding value in other 2 years (p < 0.05). d The proportion differs significantly from corresponding value among the 3 years (p < 0.05). Source: Ref. 26. a

Among leisure activities, the most profound change in activity may relate to the ownership of television. Television-ownership patterns are one way to comprehend the shift that is occurring. We present in Figure 3 the shifts in television ownership in China during the 1989 to 1993 period. The increase in television ownership in China is probably more rapid than that found in most lower-income countries; however, in all regions of the world, TV ownership is increasingly rapidly. For children, the combination of TV availability, a wide range of new and lower-cost hand-held video games, and the increasing likelihood that they reside in polluted, crowded cities have combined to increase the sedentary component of their day. However, adequate data and analysis on child physical activity in the lower-income world are unavailable. C. The Rapidly Changing Structure of Diet

As remarkable as the shifts in television ownership and reduced physical activity are, we find equally rapid shifts in the composition of food consumed. Most of the lower-income world has changed to a situation where adequate food intake is available and the diet has become more varied with a higher

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Figure 3 The proportion of television ownership in China (1989–1993). (From China Health and Nutrition Surveys, 1989–1993.)

fat intake (30). Although the increase in variety is probably an important link with improved health and reduced growth retardation, the shift in the proportion of the diet from fat and refined carbohydrate is most rapid and can be linked with increased obesity. The data displayed in Figure 4 show that the income—fat relationship had undergone a dramatic change from 1962 to 1990. This figure is based on a nonparametric regression of aggregate food availability and real gross national product (GNP) valued in 1992 dollars. Most significantly, by 1990 even the poor nations now had access to a relatively highfat diet. Whereas in 1962 a diet deriving 20% of energy from fat was associated with a GNP of $1475, the same diet was now associated with a GNP of only $750. This dramatic difference was largely accounted for by a major increase in the consumption of vegetable fats by poor and rich nations alike. The proportion of energy from vegetable fats is now much higher, accounting for up to 13% of total energy, compared with 10% in 1962. The growing proportion of energy from animal fats was now better described by a quadratic function, as richer nations consumed less but the poorest nations consumed more animal products and fats. As a result, total fat consumption was less dependent on the GNP than previously, resulting in a flatter curve. These trends in the income–fat relationship are documented in regression analyses (without the urbanization variable)

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Figure 4 Relationship between the percentage of energy from fat and GNP per capita (1962 and 1990). in Figure 4. GNP per capita is expressed in constant 1993 dollars. The data show that the percentage of energy from fat in 1990 is now less dependent on income than in 1962. Although the availability of animal fats continued to be linked to income, albeit less strongly then before, vegetable fats now accounted for a greater proportion of dietary energy, and their availability was virtually independent of income. As a result of this adjustment, the lowest-income countries now had access to an additional 4 to 5% of energy from fat. The net result is a considerable weakening of the relationship between changes in GNP per capita and energy from all sources of fat over time. That means that lower-income countries are much more likely to consume more fat today than previously. This issue and the historical reasons for this change are discussed elsewhere (30). The food-pattern shifts responsible for this overall change and the net effect on overall consumption are part of a most remarkable shift in diet that has occurred over 3 to 4 decades in Latin America and has occurred in just a decade in many Asian, North African/Mideast, and Southern African countries. Again, we use the Chinese longitudinal survey to illustrate these trends. In Table 2 we present the overall shift from a lower-fat to higher-fat diet. Just during 4 years, one can see the equivalent of what would have taken decades to change in diets in the past. The classic low-fat Chinese diet is disappearing very rapidly, and is being replaced by one where over 30% of energy is ob-

China (1989–1993 by the 1989 Income Tertile)

Mean Food group

Total fat % Consuming

Mean

Saturated fat Cholesterol % Consuming Mean % Consuming

≥30%

g/day

%E

≥10%

g/day

≥300g

29.6 15.8 9.7

11.6 16.2 9.6

13.3 16.8 18.1

4.7 5.9 6.3

8.6a 12.9 13.5

128.9 167.6 220.8

12.6 17.1 26.1

22.3 26.5 30.2

12.2 4.4 0.8

26.5 37.7 54.6

17.1 21 23.5

5.6 6.9 7.8

10.5 17.3 22.2

164.1 222.1 273.5

18.9 26.3 35.3

23.4 28 32

11.5a 3.7 2.3

26.9 40.3 57.8

17.4 21.5 24.3

6 7.5 8.5

15.1 23.5 31.1

175.4 246.3 292.9

21.3 29.5 39.8

g/day

%E

Low income Medium income High income

47.9 58.6 63.2

17 20.5 22

Low income Medium income High income

67.6 80.1 90.2

Low income Medium income High income

67.8 80.7 90.9

<10%

1989

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Table 2 Adjusted Intake of Fat and Fat Components and the Proportion of Population Consuming Different Fat Components in

1991

1993

Note: Mean fat intakes and proportions are adjusted for age, sex, residence, region, and educational level. All means are significantly different across income tertiles at same year, p < 0.001. All proportions except those marked with a superscript are significantly different across income tertiles at same year, p < 0.001. a Proportion in the low-income group is significantly different from those in other income groups at same year, p < 0.01.

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tained from dietary fat intake. Behind this change is a shift from a diet dominated by grains and vegetables to one where edible oil, meats, and refined carbohydrates are becoming the dominant foods. We present trends on this on a worldwide basis and for selected low-income countries in other studies (30–33). D. Changes in Obesity Levels

In all regions of the world, particularly in Latin America, Northern Africa, and the Near East, obesity levels are reaching rates that are as high or higher than most higher-income countries. Body mass index (BMI) is the standard population-based measure of overweight and obesity status. For adults, the cutoffs used to delineate obesity are less than 18.5 for thinness (chronic energy deficiency), 18.5 to 24.99 for normal, 25.0 to 29.99 for overweight/ preobesity, and 30.0 and above for obesity (20). The data presented here come from larger and more representative samples of adults. Our selection criteria for presenting data from other surveys were size, sampling design, and geographic area. If a study was representative of a region or country, it was always used (34). Because there are few studies of trends in obesity, those that provide reasonably comparable measurement and sampling criteria were selected. Elsewhere we present in detail the data on obesity and overweight status in the regions of the developing world (26,34). We summarize these results here and present only one figure for North Africa and the Near East. Latin America We find high levels of overweight status in all reported surveys from the Latin America countries (eg, Cuba, Mexico, Brazil) for which we have either largescale or smaller representative survey data. One recent study of ours found a decrease in adult obesity among Brazilian women from middle and upper income groups and increased obesity among all Brazilian men and lower income women (35). Middle East The limited data for oil-exporting countries such as Kuwait and Saudi Arabia indicate over a third of the adult population are overweight or obese (see Figure 5). The data for Jordan and Egypt reflect a serious problem in these countries, and in other Northern African countries the levels of obesity are also large. Female obesity is higher in all countries where data were available

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Figure 5 Obesity patterns in North Africa and the Middle East. (From Ref. 34.) for both genders. The data for this region indicate a very high prevalence of obesity. Sub-Saharan Africa Aside from Mauritius, there are no nationally representative surveys in subSaharan Africa. The scattered data from South Africa, Mali, and the Congo indicate high levels of obesity in urban sub-Saharan Africa. There are few data on rural areas, but what exists shows a minimal problem. South Africa might be the exception: limited studies on Africans, particularly women, indicate the possibility of high levels of obesity in both urban and rural areas (36). For many sub-Saharan African countries obesity and overweight status is high in urban areas but is rarely high in rural areas. Asia Aside from a few exceptions, there is very little serious obesity; however, there are rapid increases in overweight status in all Southeast Asian countries. These are remarkable because 15 years ago there were no Asian countries where the prevalence of overweight status was considered a public health problem. Today, close to a third of Malaysians and even 12 to 16% of Filipino and Chinese adults are overweight (34).

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We have excellent data on trends in body composition for a small number of lower- and middle-income countries. We have nationally representative or large nationwide datasets for Brazil in Latin America; China and India in Asia; Mauritius in Africa; Nauru and Western Samoa in the South Pacific; and Russia. These provide some sense of trends in adult obesity. In each of these countries, the 10-year percentage point increase in the overweight prevalence among adults is a 5% or higher. All of our research on this topic would lead us to conclude that these past rates of increase in the overweight prevalence are very low compared with what we will find in the future. IV. Conclusion The central theme of this chapter is that the nutrition transition and the resultant obesity epidemic will interact with the persistence of poor fetal growth and lead to a worsening of morbidity from type 2 diabetes and cardiovascular disease. The potential effects of stunting in early childhood will only add to the adverse affects. Both in the lower-income world and among immigrants to the United States, there are rapid increases in inactivity and changes in diet toward higher fat intake and more refined carbohydrate. These are reflected in rapid increases in obesity. The fetal origins hypothesis would lead us to predict that the health costs of the energy imbalance reflected in these nutrition transition–related trends will lead to a much greater increase in Type 2 diabetes and cardiovascular disease in the next several decades. References 1. 2.

3. 4. 5.

Barker DJP. Mothers, Babies and Disease in Later Life. 2nd ed. London: British Medical Journal Publishing, 1998. Popkin BM, Richards MK, Monteiro C. 1996. Stunting is associated with overweight in children of four nations that are undergoing the nutrition transition. J Nutr 1996; 126:3009–3016. Cebu Study Team. Underlying and proximate determinants of child health: the Cebu Longitudinal Health and Nutrition Study. Am J Epidemiol 1991; 133:185. Guilkey DK, Popkin BM, Akin JS, Wong E. Prenatal care and pregnancy outcome in the Philippines. J Devel Econ 1989; 30:241–272. Adair LS, Guilkey DK. 1997. Age-specific determinants of stunting in Filipino children. J Nutr 1997; 127:314–320.

The Nutrition Transition 6. 7. 8. 9. 10.

11. 12. 13.

14. 15. 16. 17. 18.

19. 20. 21.

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Weicha JL, Casey VA. 1994. High prevalence of overweight and short stature among Head Start children in Massachusetts. Pub Health Rep 1994; 109:767. Eriksson JG, Forsen T, Tuomilehto J, Winter PD, Osmond C, Barker DJP. 1999. Catch-up growth in childhood and death from coronary heart disease: longitudinal study. Br Med J 1999; 318:427–431. Eveleth PB, Tanner JM. Worldwide Variation in Human Growth, Second Edition. Cambridge: Cambridge University Press, 1990. Bogin B, Louchy J. 1997. Plasticity, political economy, and physical growth status of Guatemala Maya children living in the United States. Am J Phys Anthr 1997; 102:17–32. Hernandez DJ, Darke K. Socioeconomic and demographic risk factors and resources among children in immigrant and native-born families: 1910, 1960, and 1990. In: Hernandez D, ed. Children of immigrants—health, adjustment, and public assistance. Washington, DC: National Academy Press, 1999. Park RE. Race and Culture. Glencoe, IL: Free Press, 1950. A Portes, ed. The New Second Generation. New York: Russell Sage Foundation, 1996. Guendelman S. Sociocultural factors in Hispanic pregnancy outcome. In: Morton CJ, Hirsch RG, eds. Developing Public Health Social Work Programs to Prevent Low Birthweight and Infant Morality: High Risk Populations and Outreach. Maternal and Child Health Program, School of Public Health, University of California at Berkeley. 1988:31–39. Guendelman S, Abrams B, Dietary intake among Mexican-American women: generational differences and a comparison with white non-Hispanic women. Am J Pub Health 1995; 85:20–25. Scribner R, Dwyer JH. Acculturation and low birthweight among Latinos in the Hispanic HANES. Am J Pub Health 1989; 79:1263–1267. Scribner R. Editorial: Paradox as health outcomes of Mexican Americans. Am J Pub Health 1996; 86:303–305. Harris KM. The health status and risk behavior of adolescents in immigrant families. In: Hernandez D, ed. Children of immigrants-health, adjustment, and public assistance. Washington, DC: National Academy Press, 1999. Landale N, Oropesa RS, Gorman BK. Immigration and infant health: birth outcomes of immigrant and native women. In: Hernandez D, ed. Children of immigrants—health, adjustment, and public assistance. Washington, DC: National Academy Press, 1999. Himes JH, Dietz WH. Guidelines for overweight in adolescent preventive services: recommendations from an expert committee. Am J Clin Nutr 1994; 59: 307–316. WHO Expert Committee. Physical Status: The Use and Interpretation of Anthropometry. WHO Technical Report Series 854. Geneva: World Health Organization, 1995. Popkin BM, Udry JR. Adolescent obesity increases significantly in second and third generation U.S. immigrants: The National Longitudinal Study of Adolescent Health. J Nutr 1998; 128:701–706.

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22. Hodge AM, Dowse GK, Toelupe P, Collins VR, Zimmet PZ. The association of modernization with dyslipidaemia and changes in lipid levels in the Polynesian population of Western Samoa. Int J Epidemiol 1997; 26:297–306. 23. Zimmet PZ. Kelly West Lecture. Challenges in diabetes epidemiology—from west to the rest. Diabetes Care 1991; 15:232–252. 24. Zimmet PZ, McCarty DJ, de Courten MP. The global epidemiology of non– insulin-dependent diabetes mellitus and the metabolic syndrome. J Diabetes Complication 1997; 11(2):60–68. 25. Popkin BM. Urbanization, lifestyle changes and the nutrition transition. World Development 1999; 27:1905–1916. 26. Popkin BM. The nutrition transition and its health implications in lower income countries. Pub Health Nutr 1998; 1:5–21. 27. Levin S, Ainsworth BE, Kwok CW, Addy CL, Popkin BM. Patterns of physical activity among Russian youth: The Russian Longitudinal Monitoring Survey. Eur J Pub Health 1999; 9(3):166–173. 28. Paeratakul S, Popkin BM, Ge K, Adair LS, Stevens J. Changes in diet and physical activity affect the body mass index of Chinese adults. Int J Obesity 1998; 22:424–432. 29. Herrin AN. Rural electrification and fertility change in the Southern Philippines. Popul Devel Rev 1979; 5:61–86. 30. Popkin BM, Drewnowski A. 1997. Dietary fats and the nutrition transition: new trends in the global diet. Nutr Rev 1997; 55:31–43. 31. Monteiro CA, Mondini L, de Souza ALM, Popkin BM. The nutrition transition in Brazil. Eur J Clin Nutr 1995; 49:105–113. 32. Kim S, Moon S, Popkin BM. The nutrition transition in South Korea. Am J Clin Nutr 2000; 71:44–53. 33. Popkin BM, Ge K, Zhai F, Guo X, Ma H, Zohoori N. The nutrition transition in China: a cross-sectional analysis. Eur J Clin Nutr 1993; 47:333–346. 34. Popkin BM, Doak C. The obesity epidemic is a worldwide phenomenon. Nutr Rev 1998; 56:106–114. 35. Monteiro CA, Benicio MHD’A, Mondini L, Popkin BM. 1999. Shifting obesity trends in Brazil. Eur J Clin Nutr 2000; 54:1–5. 36. Bourne LT, Walker ARP. The nutrition transition in the Republic of South Africa. In: Proceedings of the International Congress of Nutrition. Fitzpatrick DW, Anderson JE, L’Abbe ML, eds. Ottawa: Canadian Federation of Biological Sciences, 1998; 268–269. 37. Gordon-Larsen P, McMurray RG, Popkin BM. Adolescent physical activity and inactivity vary by ethnicity: The national longitudinal study of adolescent health. J Pediatrics 1999; 135:301–306. 38. Gordon-Larsen P, McMurray RG, Popkin BM. Environmental and sociodemographic determinants of adolescent physical activity and inactivity: The national longitudinal study of adolescent health. Pediatrics (In press).

17 Effects of Maternal Nutrition and Metabolism on the Developing Endocrine Pancreas Experimental Findings B. REUSENS and C. REMACLE Université Catholique de Louvain Louvain-la-Neuve, Belgium

I. Introduction Nutrition during the perinatal period is of major importance for proper tissue development and functional maturation. Epidemiological findings described elsewhere in this book suggest that fetal malnutrition, even over a brief period, may induce irreversible changes in the offspring that lead to non–insulin dependent diabetes, obesity, hypertension, and cardiovascular diseases in adult life. The “thrifty phenotype” hypothesis, which suggests that the fetus diverts nutrients to critical organs at the expense of others during times of nutritional deprivation, was thought to be the cause of syndrome X, including hypertension, obesity, hyperglycemia, hyperlipidemia, and the propensity to Type 2 diabetes (1). Poor nutrition in fetal and early life could be detrimental to the development and the function of the beta cell in the islets of Langerhans and may predispose to later development of Type 2 diabetes. The question is how the memory of these events is stored and later expressed. Although the current __________________ This chapter was commissioned in memory of Professor Joseph J. Hoet after his death in 1999. His pioneering studies gave new insights into the links between maternal metabolism and diabetes in the offspring.

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epidemiological observations identify phenomena, they do not give adequate insight into the mechanisms that establish their causal link. It has been postulated that nutrient environment may permanently alter cellular mechanisms. Gene expression can be modulated (2) as well as permanently modified by nutrients. Fetal malnutrition may reduce cell number and alter cell function. For instance, the lower beta cell mass can lead to improper insulin action and then to decreased fetal growth. Experimental studies in animals are needed to identify specific alterations of structure and function in fetal organs, and thereby elucidating the link between maternal health and the occurrence of diabetes and cardiovascular diseases in humans. Although fetal or neonatal malnutrition in animals may not always represent the human situation, it will help to identify key nutrients and provide the basis for further epidemiological studies. Ten years ago, we developed a model of fetal malnutrition in the rat in order to investigate the consequences for development of the endocrine pancreas, the critical organ involved in diabetes. Protein deprivation, along with an adequate calorie intake, was selected because amino acids play a key role in the differentiation and function of beta cells. This chapter will describe the consequences for the endocrine pancreas when the mother is either malnourished or suffers from metabolic disturbances such as diabetes in pregnancy. Early and long-term consequences will be related in order to evaluate early imprinting. Some mechanisms involved in the abnormal development of the endocrine pancreas will be examined. Last, but not least, prevention will be discussed. II. Programming the Endocrine Pancreas In the model of protein deprivation, pregnant rats were fed either a control diet containing 20% protein or a low-protein diet (LP) containing 8% protein throughout gestation, both diets being isocaloric. Protein restriction in the mother induced a disturbance in the fetomaternal metabolic environment. Although plasma glucose and insulin levels were normal in the LP mother and her fetuses (3), plasma, amino acid profiles were altered (4). Branched amino acids, taurine, and α-aminobutyric acid were reduced the most. At birth, the LP pups featured a small (– 5.5%) reduction of body weight and development of the endocrine pancreas was shown to be altered (5). In the newborn animals, the pancreatic insulin content and the proliferation of islet cells were lower.

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The islets were smaller and less vascularized. The cerebral cortex was also less vascularized (6). In another rat model of maternal malnutrition, reduction of caloric intake by 50% from day 15 of gestation resulted in a more severe growth retardation in the offspring than protein restriction. At day 1 postnatally, beta-cell mass and insulin content were significantly decreased (7). The reduction in beta-cell mass was attributed to a reduced number of islets rather than to lower beta-cell proliferation. Maternal diabetes is also known to affect fetal tissue development. Maternal diabetes can be induced in animals by injection of streptozotocin on the first day of gestation, the severity of diabetes depending on the dose of streptozotocin injected. Total amino acid concentration was normal in mildly diabetic rats, but was decreased in severely diabetic rats. It was also decreased in the fetal plasma irrespective of the severity of the maternal diabetes (8). Amino acid profiles were altered in the mother and the offspring in both mild and severe maternal diabetes. As in the LP model, branched amino acids and taurine were most affected in the fetus of the mildly diabetic mother. The development of the endocrine pancreas was enhanced by the moderate increase in glucose concentration in maternal plasma, which resulted in hypertrophy and hyperplasia of fetal islets from day 20 of gestation until birth. Pancreatic insulin content and insulin secretion in response to glucose were raised in fetuses from mildly diabetic rats (9). Islet-cell proliferation was enhanced by 42% at birth (10). In these conditions, the progeny was macrosomic. When maternal hyperglycemia was severe, fetuses were small for gestational age. The beta cells of these fetuses degranulated because of an overstimulation by the excessive glucose concentration. This led to a decrease in pancreatic and plasma insulin concentrations (11). Severe hyperglycemia induced a lower fetal pancreatic insulin content and insulin secretion (9). This was confirmed in fetuses of spontaneously diabetic BB rats (12). The experimental conditions in dams presented here emphasize, the impact of the intrauterine environment on the development of the endocrine pancreas. The following question arises: Are such alterations permanent, or can normal metabolic conditions after birth reverse them? III. Short-Term Consequences Short-term consequences of the LP diet were evaluated in fetal beta cells after 7 days of culture. During the 7 days in the same culture medium, beta cells

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of both control and LP fetuses multiplied to form pseudoislets mainly constituted by beta cells (13). However, islets of LP fetuses featured a lower beta-cell proliferation after tritiated thymidine incorporation than control islets (published data). When these LP islets were challenged for their insulin secretion, they released 50% less insulin in response to leucine and arginine with or without theophylline (14). Therefore, even after withdrawal from the abnormal metabolic maternal milieu and cultured during 7 days in the same conditions, fetal beta cells maintained a reduced insulin secretion and proliferation rate. The same short-term consequences were also shown in beta cells of fetuses from mildly diabetic rats. Indeed, after 7 days of culture, beta cells originating from fetuses of mildly diabetic rats showed a 60% increase of proliferative capacity (10%). IV. Long-Term Consequences A. Youth

Long-term effects of the LP diet during gestation were investigated at 3 months in progeny suckled by normal mothers and fed a normal protein diet after weaning. The LP diet administered only during gestation did not permanently affect the body weight of the offspring; LP pups regained normal body weight immediately after birth (14). However, the structure and function of the endocrine pancreas were not restored (3). At 3 months, islets were bigger whereas the pancreatic insulin content was decreased, which suggests an increased proliferation rate but fewer islets as a result of reduced neogenesis during fetal development. When these islets were challenged in vitro with glucose, they released an appropriate quantity of insulin. However, when they were challenged with leucine or arginine, their insulin secretion was depressed (15). Low-protein fed female, but not male, offspring had lower plasma insulin concentrations after an oral glucose challenge (3). When a LP diet was maintained throughout lactation and replaced by a normal diet after weaning, the “recuperated” female and male offspring both exhibited a lower growth rate (Fig. 1). Again, although plasma glucose concentrations were normal, plasma insulin concentrations were lower in females. Insulin secretion in response to an oral glucose challenge was markedly reduced in both sexes, but glucose tolerance was better than in controls (Fig. 2). This normal glucose tolerance, alongside a reduced insulin release, may

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Figure 1 Male and female growth curves in control (C) and recuperated (R) male (M) and female (F) adult offspring. be explained by adaptation of the peripheral tissues through an increased number of insulin receptors in the liver, adipose tissue, and hepatocytes, as well as increased levels of Glut 4 in adipocytes, leading to an increase in wholebody insulin sensitivity (16–18). Long-term consequences are also readily apparent in the offspring of mothers on caloric restriction during gestation and lactation (19,20). At 3 months of age, male offspring had fewer beta cells and secreted less insulin in response to an oral glucose challenge, but again they did not show glucose intolerance. Adult offspring of severely diabetic rats maintained a lower bodyweight. They had a morphologically normal endocrine pancreas and a normal plasma glucose concentration in basal conditions (21). After 3 hours of glucose infusion, they were able to maintain glucose concentrations within the control

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Figure 2 Plasma glucose and insulin values during oral glucose tolerance test.

range, but at the expense of high insulin levels (22). Euglycemic hyperinsulinemic clamp showed that offspring of diabetic rats were resistant to the action of insulin at the hepatic and peripheral levels (23). In summary, the endocrine pancreas is affected at birth in the three experimental models: low-protein, low-calorie, and streptozotocin-induced diabetes. As a consequence, insulin secretion is abnormal at 3 months despite the withdrawal of the primitive metabolic insult. However, glucose tolerance does not develop because adaptations seem to occur. B. Pregnancy

Increased insulin demand attributable to obesity, pregnancy, or aging may be necessary to better reveal vulnerability of the beta cell acquired early in life.

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During pregnancy, the mother’s endocrine pancreas has to adapt to meet the higher requirement for insulin. This adaptation in normal pregnancy is reversible and consists of a lower threshold for glucose-stimulated insulin secretion, increased insulin synthesis, and a strongly increased beta-cell proliferation rate (24). In rats, the beta-cell mass is nearly doubled at the end of gestation (25). The lower beta-cell mass resulting from protein or caloric deprivation early in life could be a limiting factor for this adaptation. Female offspring from dams fed a LP diet during gestation only were not glucose intolerant in pregnancy. However, after an oral glucose challenge performed at day 18.5 of gestation, enhanced insulin secretion was almost absent and plasma glucose concentrations were significantly higher than in controls (3). Adaptation of the endocrine pancreas was incomplete because the pancreatic insulin content was significantly lower in LP mothers than in controls on the last day of gestation (Fig. 3). Incomplete adaptation of the maternal pancreas to gestation will result in the fetus developing in an abnormal intrauterine milieu with consequent alterations to its endocrine pancreas. During the last day of gestation, the fetuses tended to be hyperglycemic and had lowelasma insulin concentrations. Pancreatic insulin content (Fig. 4) and volume density of the fetal

Figure 3 Absolute (µg) and relative (µg/100g BW) pancreatic insulin content of the pregnant control (C) and recuperated (R) offspring.

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Figure 4 Absolute (µg) and relative (µg/g BW) pancreatic insulin content of the 21.5-day-old fetus (second generation, see text).

beta-cell mass (Fig. 5) were reduced. Among female offspring of dams fed a low-calorie diet, adaptations to gestation were normal when the offspring were young, around 4 months of age, but at 8 months the offspring were no longer able to increase their beta-cell mass (26). The mechanisms underlying this blunted increase of beta-cell mass are not known. The possibilities include lower beta-cell proliferation, an increased rate of apoptosis, a lack of growth hormone, or placental lactogen or their receptors. When the female offspring from streptozotocin diabetic rats became pregnant, they developed gestational diabetes. The disturbed maternal metabolism again affected the fetuses, which displayed islet hyperplasia, beta-cell degranulation (11), hyperinsulinemia (27), and macrosomia (11). The studies by Grill (28) and Ktorza (29) strongly suggest that the diabetic or hyperglycemic intrauterine environment must be responsible for these alterations. C. Old Age

In the LP models, not only did gestation precipitate the offspring into glucose intolerance but aging (30) or a high-fat diet (31) also led LP offspring to a loss of glucose tolerance compared with controls. By 15 months of age, these

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Figure 5 Volume density of the β-cell mass in the pancreas of the 21.5 days old fetus (second generation, see text).

animals had a significantly worse glucose tolerance than controls (30). Aging in rats deprived of calories during fetal life and lactation led to severe insulopenia and glucose intolerance. Beta-cell growth, which should continue normally with age, ceased at 3 months. This did not seem to be attributable to a reduced beta-cell proliferation, but rather to an increased rate of apoptosis (20). V. Mechanism Involved in Altered Beta-Cell Mass

The beta-cell mass of LP fetuses was inadequate at birth. The number of beta cells as well as insulin secretion were reduced. Why do LP fetal beta cells proliferate less? The mechanisms involved in such alterations need to be elucidated because they will help to establish a causal relationship between events in early life and disease later. The endocrine pancreas develops from the primitive gut (32,33). Rotation of the polarity of the mitotic division in specific epithelial duct cells gives rise to cells committed to endocrine differentiation. These cells will multiply and vascularization will appear. Finally, the islets will detach from the duct and become dispersed between the exocrine tissue. At birth, the endocrine tissue represents around 4% of the total pancreas. The

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beta-cell mass is achieved by a balance of the multiplication of stem cells, neoformation from the ducts, multiplication of already differentiated beta cells, and beta-cell death either through apoptosis or necrosis. The beta-cell mass increases rapidly during the last 3 days of gestation. At birth, the glucidic and peptidic nature of the diet of the fetus changes into the high-fat diet of the sucking rat. Plasma insulin concentrations, which were high during fetal life, decrease rapidly to adult values. Postnatally, the amount of the beta-cell mass does not increase further between day 5 and 20 (34). During lactation, the endocrine pancreas undergoes a reorganization of the cell mass. A wave of apoptosis occurs in neonatal rat islets between 1 and 2 weeks of age (35). It has been suggested that this postnatal apoptosis allows replacement of fetal beta cells, which are weakly sensitive to glucose in a stable metabolic environment, with adult beta cells capable of rapidly releasing insulin under nutritional, endocrine, and neural control. Not only is the fetal period critical for determining an appropriate betacell mass but the neonatal period too could be a sensitive period for nutritional insult. How can an LP diet during fetal life until weaning alter the balance between beta-cell birth and death? The proliferative capacity of beta cell estimated after BrdU incorporation was calculated at the fetal stage and at several days postnatally. It was decreased in LP offspring compared with control offspring (36). Beta cells that are positive for BrdU represent cells that have synthesized DNA. Cyclins are proteins expressed and destroyed in order to allow the cell to proceed through the cell division cycle. To further delineate cell cycle events in LP islet cells, two proteins were analyzed: cyclin D1, which is associated with the G1 phase, and NEK2, which has a maximal expression in G2 and during mitosis. More LP fetal and neonatal beta cells contained cyclin D1 and fewer contained NEK2 when compared with control beta cells. This suggests that the beta-cell cycle is lengthened in the LP offspring (36). The apoptotic rate was also calculated at the fetal and various postnatal stages. The wave of apoptosis described at 14 days in normal neonatal islets was also present in LP offspring. However, fetal and neonatal LP islets had an increased rate of programmed cell death compared with control islets (36). It has been proposed that the low level of survival factors, such as IGFs, after birth is responsible for the increased apoptotic rate at 14 days (37). Protein restriction in the mother was associated with a reduction of plasma IGFs in the fetus (38). We calculated the number of islet cells positive for IGF-2 and IGF1 during development. IGF-2, which was higher at fetal stage, decreased after birth whereas the IGF-1 level increased in neonatal life. This

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was true in control and LP islets, but the number of cells positive for IGF-2 and IGF-1 was about 50% lower in the LP animals at each age (36). A LP diet given during development thus changes the balance between beta-cell replication and apoptosis, possibly through an alteration of the cell cycle. A reduced expression of IGFs in the islets may be implicated in the lower beta-cell proliferation and the increased apoptotic rate. This may contribute to the smaller islet size and beta-cell mass, which leads to impaired insulin secretion at adult life. VI. Changes in Other Organs

Other tissues and organs, which have different periods of rapid cell divisions and of cell commitment, are also sensitive to nutritional insults. Vascularization of the islets and cerebral cortex was reduced at birth when the mother was fed a LP diet during gestation. Although the normal diet after birth restored the normal vascularization in the islets, brain vascularization remained lower at adulthood (6). These data emphasize the sensitivity of brain vessels to metabolic alterations in the intrauterine milieu induced by protein deprivation. The mechanisms by which a LP diet in early life induces decreased vascularization are not known. Altered amino acid, IGF, and vascular endothelial growth factor (VEGF) levels, as well as higher glucocorticoid levels are candidates that need to be examined. It is important, however, to mention that increased homocysteine plasma levels in men have been shown prior to the development of cardiovascular disease (39). Exposure to maternal diabetes during the fetal and perinatal periods induce disturbance in cardiovascular function in the offspring at 3 months (40). Although the offspring of severely diabetic rats were normotensive, they had profound bradycardia. When reactivity of the blood vessels was tested in vitro, a reduced relaxation to endothelium-dependent dilators and an enhanced constriction in response to norepinephrin in small mesenteric arteries were found (41). These changes observed early in adulthood in the offspring of LP or diabetic mothers may predispose to later overt cardiovascular disease. Anatomical and physiological variables in the brains of the offspring were also altered when dams were given a LP diet before and during pregnancy and were not restored with a normal diet postnatally (42,43). At birth, the number of dendrites was reduced. The sensory corticocortical and thalamocortical-evoked potentials were lower and the brain tissue contained elevated biogenic amines and a modified tryptophan metabolism (42).

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A LP diet during gestation also impairs the development of the kidney. The number of nephrons was reduced in the neonate (44) and was not restored by a normal diet after birth. Experimental intrauterine growth retardation produced by partial intrauterine ligation also caused a reduction of nephron numbers (44). Such a disorder in kidney development is likely to have long-term consequences. A hereditary deficit of nephron numbers in rats has been shown to be associated with functional alterations, that enhance susceptibility to hypertension in later life (45). VII. Prevention of Programmed Changes The central aim of experimental and epidemiological studies of the fetal origins of adult disease is prevention. Therefore, we have examined mechanisms of prevention in the different animal models presented here. Taurine is a normal constituent of the human diet and is contained in animal food sources (46). This sulphur amino acid is found in almost all mammalian tissues and constitutes more than 50% of free amino acids in many tissues (47). Taurine is not an essential amino acid in the usual sense, but is an important nutrient for developing tissue (48). Some observations suggest that taurine in some way affects glucose utilization (49) and interacts with insulin receptors (50). The importance of an adequate amount of taurine for normal development and function was shown by the numerous pathological consequences that occur when transport of taurine into the cells is curtailed by dietary deficiency or diseases; growth and development are altered (51), and blindness (52) and cardiac dysfunction may occur (53). Taurine was recently reported to be a secretagogue for fetal beta cells (54). In the adult pancreas, taurine was 10 times more abundant in the islets than in the exocrine tissue (55). As described earlier in this chapter, when a LP instead of a normal diet was given throughout gestation, taurine concentrations were lower in the serum of both dams and their fetuses at 21.5 days (4). It was also lower in mothers and fetuses of diabetic rats (8). Plasma and platelets of diabetic patients have also been shown to have low taurine concentrations (56). These observations encouraged us to evaluate the role of lower taurine concentrations in the altered insulin secretion of the LP islets. The experiments were performed through taurine supplementation in vitro as well as in vivo (57). Taurine, which is a secretagogue for normal fetal beta cells (54), was unable to stimulate any insulin release from LP fetal islets. The addition of taurine during the last 2 days of culture allowed an enhanced insulin release

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from normal fetal beta cells but not in fetal LP islets. Taurine supplementation to the culture medium was thus unable to restore the sensitivity of islets from the LP group to amino acid secretagogues. The other strategy was to supplement the LP diet of the mother with 2.5% taurine in the drinking water throughout pregnancy and to test fetal insulin secretion in vitro after 7 days of culture. Addition of taurine in vivo completely restored the insulin secretion of the LP islets to normal. The discrepancy between the absence of prevention of the in vitro treatment and the positive prevention after in vivo supplementation could be explained by the fact that islets from fetuses from LP dams supplemented with taurine originated, differentiated, and proliferated in an intrauterine environment in which there was an adequate amount of taurine. These results emphasize the need for an appropriate plasma taurine concentration at least during fetal development in order to develop normal fetal insulin secretion. To our knowledge, this was the first time that very early deleterious events could be prevented by taurine. However, other cases of pathological occurrences prevented by taurine supplementation should be pointed it out. Diabetic patients who exhibit a lower plasma and platelet taurine concentration and an increased platelet aggregation are normalized by oral taurine supplementation (56). In rats, oral treatment with taurine before the injection of streptozotocin suppressed the hypoglycemia induced by the drug (58). Taurine supplementation attenuated the development of hypertension and could prevent stroke in spontaneously hypertensive rats (59). Taurine not only attenuates hypertension but suppresses relative hypercholesterolemia in spontaneously hypertensive rats (60). Recently, a clinical trial was conducted in 17 Tibetans from a population who never eat fish and suffer from hypertension. After 2 months of 3 g taurine per day, both systolic and diastolic BPs were significantly reduced (61). Pathologies later in life can also be prevented by other intrauterine interventions. The prevention of hypertension in spontaneously hypertensive rats can be achieved by early treatment of the mother with the angiotensin-converting enzyme inhibitor. Primary prevention was possible only in utero and the normalizing effect was maintained after withdrawal of the inhibitor from the offspring after birth. The second generation was also devoid of hypertension (62). Long-term consequences of the hyperglycemic intrauterine milieu can be prevented by normalizing the maternal glycemia during the last trimester. Transplantation of isolated islets from healthy rat neonates to severely diabetic mothers at day 15 of gestation eliminated hyperinsulinemia and insulin resistance during glucose infusion in the adult offspring (23,63).

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Could the data obtained in animal models be extrapolated to the human situation? There is no mechanistic demonstration in humans that malnutrition in utero reduces beta-cell growth and permanently alters beta-cell function. However, growth-retarded newborn infants, which may be a consequence of fetal undernutrition, have reduced beta-cell numbers and insulin secretion (64), and recent research in the United Kingdom, Sweden, and the United States has shown that insulin resistance is increased in men and women who had a lower birthweight (65–67). There is little evidence in human beings that maternal nutrition during gestation affects insulin-glucose metabolism later in life. However, a recent epidemiological study of people born around the time of the famine in the Netherlands in 1944 to 1945 revealed that prenatal exposure to famine, especially during late pregnancy, is linked to decreased glucose tolerance in adulthood. Thus, poor nutrition in utero may lead to permanent changes in insulin-glucose metabolism even if the effect on growth is small (68). The thrifty phenotype hypothesis, which proposes that insulin resistance results from persistence of fetal adaptation to inadequate intrauterine nutrition, is supported by recent findings in 8-year-old Indian children (69). In this study, lower birth weight was associated with increased insulin resistance in children, who later were heavy. Endocrine and metabolic adaptations associated with reduced intrauterine growth may limit nutrient utilization in later life, leading to fat deposition and increased insulin resistance. Pancreatic beta-cell function did not seem to be affected in this young population (69). There is now a general consensus that the number and total area of islets are reduced in Type 2 diabetes (70). The concept that stress situations, such as overnutrition superimposed on a limited beta-cell mass, results in diabetes may be applicable to humans. Ethiopian Jews who emigrated to Israel have a high prevalence of diabetes (71) (see Chap. 7). Amino-acid profiles may also be involved in the picture of the fetal origin of adult diseases in humans (see Chap. 13). Amino acids are actively transported by the placenta against a concentration gradient and provide the nitrogen source for growing (72).* Taurine was found to be deficient in premature infants (73), probably through an insufficiency of the hepatic cysteine sulfinic acid decarboxylase (74). _______________ * A deficiency in amino acids transport in the placenta was observed in small-forgestational-age human fetuses (78).

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Maternal diabetes still compromises the development of the fetus and its endocrine pancreas even if it is well controlled. The volume density of the endocrine pancreas and percentage of insulin-producing beta cells are reduced in these fetuses. When diabetes is poorly controlled, degranulation of the fetal islets may also be observed (75). The risk of non–insulin dependent diabetes is increased among the offspring of women with gestational diabetes (76). In infants who develop neonatal diabetes, fetal beta cells are immature. These infants need insulin and a majority recover, but about 25 to 30% of them become diabetic in childhood (77). This could be attributable to the inability of altered beta cells to meet the requirements of growth. VIII. Conclusion The information obtained from a combination of human epidemiological and animal studies reveals the long-term impact that fetal malnutrition can have on the health of the offspring. The identification of critical windows of susceptibility is a challenge, because susceptible periods are likely to differ between different organs and according to the nature of the insult. However, animal models are tools for determining these critical windows, and more importantly will pave the way for the prevention of adult disease such as diabetes, hypertension, and cardiovascular diseases. Data emerging from experimental models will help prospective epidemiological investigations to search for specific proxies for fetal malnutrition, including amino acid concentrations and possibly taurine concentrations in maternal and cord blood. References 1. 2.

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AUTHOR INDEX

Italic numbers give the page on which the complete reference is listed.

Abbas A, 309, 319 Abbott DH, 209, 224 Abdalla HI, 250, 266 Abrams B, 325, 337 Adair LS, 324, 329, 337, 338 Adam PAJ, 185, 194 Adami HO, 16, 22 Adamson SL, 133, 139 Adamson TM, 106, 108, 121, 125, 134 Addy CL, 328, 338 Ades AE, 28, 31, 32, 33, 34, 44 Adshead F, 155, 159 Aerts L, 86, 94, 257, 268, 279, 293, 341, 343, 352, 353, 354, 355, 357, 358 Aguan K, 132, 138 Ahmed ML, 149, 157 Ahn MR, 82, 85, 93 Ahn MT, 173, 179, 351, 357 Ainsworth BE, 328, 338 Aitken DH, 231, 238 Aizman R, 218, 227

Akesujarvi G, 215, 226 Akin JS, 324, 337 Al-Ghazali W, 54, 58 Alberman E, 255, 267 Albertsson-Wikland K, 242, 247, 290, 295 Aleck KA, 154, 158 Alexander G, 187, 195, 288, 295 Allan LD, 54, 58 Allebeck P, 27, 34, 45, 64, 70 Allen M, 10, 20, 27, 48, 143, 156 Allshire RC, 164, 178 Alonso JG, 130, 138, 219, 228 Alonson FJ, 350, 357 Alvarez V, 27, 30, 47 Ambler GR, 187, 195 Anderson A, 218, 227 Anderson DF, 112, 135 Anderson HR, 313, 321 Anderson MA, 87, 94 Anderson PAW, 115, 135 Anderson RN, 66, 70 Anderson S, 350, 356 359

360 Andres C, 41, 47 Andrew R, 236, 240 Angehrn V, 244, 248 Anguita RM, 84, 93 Angus GE, 298, 316 Anichini R, 350, 351, 357 Annesi-Maesano I, 313, 321 Anthony S, 32, 37, 45 Anversa P, 106, 134 Anwar MA, 215, 226 Aperia A, 215, 226 Appleton M, 148, 156 Arany E, 208, 219, 223, 348, 356 Archer MA, 214, 225, 274, 291 Archie JP, 125, 137 Ardini D, 54, 58 Arey LB, 97, 98, 133 Arm JP, 313, 321 Arnay E, 82, 92 Aronsson S, 242, 247 Arudini D, 264, 271 Ascherio AL, 28, 29, 31, 39, 46, 155, 159 Ash M, 212, 224 Ashwell M, 281, 294 Asmar R, 51, 57 Aste-Amezaga M, 308, 319 Astrom A, 232, 238 Athens MA, 28, 30, 48, 146, 156, 352, 358 Atton C, 11, 20, 42, 47, 254, 257, 267 Aubert R, 154, 158 Baber FM, 290, 295 Bachman DL, 61, 63, 69 Bacon BJ, 76, 90 Bailey RA, 173, 179 Baird HR, 154, 158 Baker BA, 171, 179 Baker E, 214, 225 Baker J, 186, 194, 207, 208, 223, 252, 264, 266 Bakketeig L, 305, 318 Balbe D, 278, 292

Author Index Ballantyne E, 148, 156 Ballard PL, 219, 228 Ballard RA, 219, 228 Balsamo A, 242, 247 Bannis-Taleb N, 340, 353 Bapat S, 352, 358 Barbazanges A, 231, 233, 237 Barbera A, 112, 124, 135, 136 Barker D, 15, 21, 236, 240, 263, 270 Barker DJ, 28, 31, 42, 47, 132, 138, 189, 195 Barker DJP, 1, 3, 4, 5, 7, 8, 9, 10, 11, 12, 12, 13, 14, 16, 18, 19, 20, 21, 22, 27, 28, 29, 31, 32, 33, 34, 42, 44, 45, 47, 48, 50, 54, 57, 58, 64, 66, 68, 70, 71, 74, 87, 88, 89, 94, 97, 133, 142, 143, 146, 151, 152, 153, 155, 155, 156, 157, 158, 159, 161, 162, 170, 177, 178, 189, 195, 199, 200, 209, 213, 214, 215, 218, 221, 224, 226, 227, 230, 233, 236, 237, 239, 240, 242, 244, 246, 247, 248, 250, 253, 254, 255, 256, 257, 259, 260, 261, 262, 264, 266, 267, 268, 269, 271, 273, 290, 305, 307, 314, 318, 319, 321, 324, 336, 337, 339, 352, 353, 357, 358 Barker J, 264, 271 Barker M, 153, 158, 214, 226 Barraclough CA, 236, 239 Barth S, 209, 224 Bassett JM, 187, 195, 208, 223 Bassett NS, 78, 80, 91, 188, 195 Batchelor DC, 78, 80, 91 Battaglia FC, 111, 135 Bauer MK, 187, 194, 219, 228 Baulieu EE, 232, 238 Bauman JE, 75, 89 Baumbach WR, 187, 195 Baur WE, 260, 269 Bavdekar A, 155, 159, 162, 178, 352, 358 Baxter RC, 207, 223 Beards F, 148, 156

Author Index Beazley LD, 214, 225, 274, 275, 291 Beck-Nielsen H, 150, 157 Beckers D, 242, 243, 244, 247 Bedell K, 218, 227 Belizan JM, 253, 267 Bell GI, 279, 293 Bell SC, 263, 270 Beltrami CA, 106, 134 Ben-Shlomo Y, 8, 19 Bencio MHDA, 335, 338 Bendall HE, 66, 70, 152, 158 Bendeck MP, 52, 57, 132, 138 Benediktsson R, 76, 78, 90, 203, 213, 214, 222, 225, 263, 270, 276, 277, 289, 292 Bennardini F, 350, 351, 357 Bennet L, 80, 91, 200, 202, 221 Bennett FI, 10, 20, 27, 48, 143, 156 Bennett L, 277, 292 Bennett PH, 145, 154, 156, 158, 173, 180, 257, 268 Bennett ST, 150, 157 Bennion LJ, 173, 180 Bennis-Taleb N, 81, 82, 85, 92, 341, 354 Bereck KM, 351, 357 Berendes HW, 255, 267 Berenson GS, 26, 30, 47, 53, 58 Bergel E, 253, 267 Berglund L, 10, 20, 37, 39, 45, 46, 64, 70, 87, 94, 143, 148, 156, 170, 178, 261, 269, 352, 357 Bergman RN, 146, 148, 156, 246, 248 Berk BC, 218, 227 Bernet F, 231, 237 Bernstein KE, 211, 218, 224, 227 Berry C, 27, 31, 48, 54, 58, 132, 138, 261, 269 Berry CL, 52, 54, 57, 58 Berry LM, 209, 215, 224, 226 Berthault MF, 346, 355 Bertrand HA, 154, 158 Beslagic D, 209, 224 Bethel JA, 39, 46

361 Bhalla A, 14, 21 Bhat DS, 155, 159, 162, 178 Bheeka R, 311, 320 Bian X, 215, 226 Biasucci LM, 172, 179 Bichisao E, 276, 292 Bieber HE, 260, 269 Biggs CS, 209, 224 Bihoreau MT, 346, 355 Bingham B, 187, 195 Bird IM, 209, 224 Bishop SP, 121, 136 Bjorntorp P, 230, 237 Blackburn H, 63, 69 Blair GK, 304, 318 Bleker OP, 11, 14, 16, 20, 88, 95, 162, 177, 209, 224, 261, 269, 352, 358 Bloch J, 10, 20, 39, 46 Blondeau B, 346, 355 Blondel O, 278, 292 Bloom SR, 190, 196 Bocking AD, 125, 137, 200, 202, 204, 221, 286, 295 Boehmer A, 244, 248 Bogin B, 325, 337 Boguszewski M, 242, 247 Boksa P, 231, 238 Bone AJ, 345, 355 Bonithon Kopp C, 26, 27, 47 Bonner-Weir S, 348, 356 Bonnett D, 15, 21 Borch-Johnson K, 146, 156 Born GVR, 110, 135 Borrow KM, 107, 134 Borthwick AC, 151, 157 Bouillon R, 341, 354 Boulton TJ, 26, 30, 47 Bourne LT, 335, 338 Bovet P, 15, 21 Bower S, 264, 271 Bowsher RR, 208, 223 Boyd RDH, 258, 268 Boyd RH, 256, 259, 267

362 Boyle DW, 208, 223 Boys RJ, 260, 269 Boysen G, 61, 69 Brace RA, 215, 226 Bradley L, 288, 295 Bradshaw BS, 28, 30, 48, 146, 156, 352, 358 Bramich C, 81, 92 Brancat FL, 68, 71 Brancati FL, 63, 69 Braunwald E, 51, 57, 113, 114, 135 Breant B, 341, 343, 346, 354, 355 Breier BH, 4, 18, 78, 91, 186, 187, 188, 191, 192, 194, 195, 196, 197, 206, 219, 222, 228, 257, 268, 276, 292 Brelje TC, 345, 355 Brenner BM, 56, 59, 66, 68, 70, 71, 85, 94, 218, 227, 262, 270, 350, 356 Breslau N, 39, 46 Briggs MM, 215, 226 Bristow J, 183, 193 Bronzino J, 81, 85, 92, 349, 350, 356 Brook CG, 154, 158 Brook WH, 106, 134 Brooks AA, 250, 266 Brown A, 15, 21 Brown KM, 313, 321 Brown RD, 61, 69 Brown TJ, 236, 240 Brown TR, 244, 248 Browne CA, 207, 223 Browne RF, 276, 278, 292 Bruijnzeel-Koomen CAFM, 311, 320 Bruinse HW, 246, 248 Bryan E, 54, 58 Bryant MJ, 175, 180 Buckberg GD, 125, 137 Buckingham ME, 98, 101, 133 Buggins AGS, 309, 319 Bull A, 28, 29, 32, 33, 34, 45, 261, 269

Author Index Bull AR, 28, 29, 31, 32, 33, 34, 44, 250, 266 Bulmer K, 41, 47 Buonono FC, 207, 223 Burchell A, 214, 225, 278, 293 Burge DM, 302, 304, 317 Burgmann KE, 311, 320 Burgmann RL, 311, 320 Burke GL, 63, 69 Burns SP, 173, 179, 191, 196 Burson MA, 128, 138 Burton G, 76, 90 Burton JL, 313, 321 Burton SL, 313, 321 Burus I, 10, 20 Bustamante J, 350, 357 Butland BK, 313, 321 Bynner J, 306, 319 Byrne CD, 190, 196 Cadet P, 308, 319 Cadet R, 235, 239 Cahill CJ, 348, 356 Calame A, 191, 196 Calder NA, 76, 77, 90, 286, 294 Caldji C, 275, 291 Camacho-Hubner C, 186, 194 Cameron A, 235, 239 Camilleri M, 173, 180 Campagna D, 313, 321 Campbell DM, 10, 20, 28, 31, 42, 47, 88, 94, 250, 253, 265, 266 Campbell EJ, 52, 58 Campbell GR, 106, 108, 121, 125, 134 Campbell K, 235, 239 Campbell SE, 133, 139 Cannata J, 106, 134 Carabello BA, 107, 134 Carbone GMR, 215, 226 Cardoso WV, 303, 317 Carels C, 244, 247 Carey KD, 154, 158 Carlsen KH, 305, 318

Author Index Carmichael L, 183, 193, 235, 239 Carr-Hill R, 250, 265 Carraher MJ, 154, 158 Carter TD, 61, 63, 69 Casey VA, 324, 337 Casimir CJA, 312, 320 Cassio A, 242, 247 Cattingius S, 15, 21 Cauderay M, 191, 196 Cauderay MC, 242, 247 Cawley M, 14, 21 Ceccatelli S, 218, 227 Celotti F, 232, 238 Celsi G, 215, 218, 226, 227 Chailley-Heu B, 303, 317 Challice CE, 104, 134 Challis J, 76, 90, 183, 193, 200, 202, 212, 221, 222, 235, 239 Challis JR, 125, 137 Challis JRG, 202, 222, 235, 239, 288, 295 Chalmers I, 234, 239 Chamberlain PF, 185, 193 Chandorkar AK, 279, 293 Chapman KE, 235, 239 Chapman MG, 54, 58 Chatelain P, 242, 243, 244, 247 Chatelain PG, 242, 247 Cheek DB, 85, 93 Chelly N, 303, 317 Chen D, 260, 269 Chen YT, 38, 46 Cherif H, 82, 83, 93, 342, 350, 351, 354, 357 Chertow GM, 28, 29, 31, 39, 46, 56, 59, 66, 70 Cheung CY, 215, 226 Chevenne D, 10, 20 Chez RA, 209, 224 Chidzanja S, 258, 259, 268 Chierif H, 173, 179 Child SC, 256, 267 Chinn S, 305, 318 Chita SK, 54, 58

363 Chung AP, 10, 20 Cidlowski JA, 211, 224 Ciuti M, 350, 351, 357 Clamp AG, 171, 179, 289, 295 Clapp JF, 212, 224 Clark AJL, 186, 194 Clark EB, 103, 133 Clark PM, 11, 20, 42, 47, 233, 239, 244, 246, 247, 248, 254, 257, 267 Clark PMS, 8, 9, 19, 29, 45, 64, 70, 142, 143, 146, 148, 155, 155, 156, 158, 159, 162, 178, 261, 269 Clarke IJ, 77, 91, 200, 212, 214, 221, 224, 288, 295 Clarke L, 75, 89, 175, 180 Clarke WR, 53, 58 Clausen JO, 146, 156 Clements BS, 302, 304, 317 Clewlow F, 183, 193 Clifton PM, 262, 270, 281, 282, 294 Cloke J, 29, 45, 264, 271 Clubb FJ, 121, 136 Cobb JL, 61, 69 Cockburn JM, 40, 41, 46 Cockington R, 146, 148, 156 Coelho C, 348, 356 Coghlan JP, 203, 214, 215, 218, 222, 225, 231, 238 Cohen MP, 352, 358 Cohen RA, 66, 70 Cohen RD, 173, 179, 191, 196 Cohn C, 168, 178 Cohn HE, 124, 136 Cok D, 30, 32, 37, 46 Colan SD, 107, 115, 134, 135 Colciago A, 232, 238 Colditz GA, 5, 8, 19, 74, 87, 89 Cole TJ, 11, 20, 257, 268 Coleman N, 81, 84, 92, 174, 180, 219, 228, 343, 354 Colley JRT, 13, 21, 28, 32, 33, 44 Collin D, 10, 20 Collins MH, 305, 318 Collins VR, 177, 180, 327, 338

364 Comline RS, 186, 194, 206, 222 Conelly A, 288, 295 Connell S, 106, 134 Connor WE, 53, 58 Considine RV, 212, 224 Cook D, 42, 47 Cook DG, 26, 27, 29, 30, 37, 38, 41, 42, 44, 45, 47, 155, 159, 250, 261, 266, 269 Cook JTE, 148, 156 Cookson WOCM, 310, 320 Cooper C, 14, 15, 21, 236, 240, 246, 248, 257, 268 Coulter CL, 202, 222 Coviello-McLaughlin R, 164, 178 Cowen T, 281, 294 Cox LJ, 8, 19, 142, 155 Cox V, 5, 11, 12, 19, 20, 189, 195, 253, 254, 255, 257, 260, 262, 266, 267 Coyaji KJ, 261, 262, 269 Crabb DE, 261, 269 Creasy RK, 118, 136 Cresswell JL, 16, 22, 214, 226, 236, 240, 246, 248 Cripps HA, 28, 32, 33, 44 Crocker FJ, 13, 14, 21 Crook AR, 81, 92 Cross J, 10, 20, 28, 31, 42, 47, 88, 94, 253, 266 Crossman M, 52, 57 Crowe C, 75, 76, 77, 80, 89, 90, 91, 277, 286, 292, 294 Crowley MJ, 38, 46 Crowley P, 234, 239 Crowther N, 81, 84, 92, 343, 354 Crowther NJ, 81, 84, 92, 93, 161, 163, 170, 177, 178, 179, 262, 270, 346, 347, 355 Cruddas AM, 11, 20, 257, 268 Cubiyashi Y, 307, 319 Curhan GC, 28, 29, 31, 39, 46, 155, 159 Currie MJ, 188, 195

Author Index Cutfield WS, 146, 148, 156, 191, 196, 246, 248 Cuvelier P, 312, 320 Czernichow P, 10, 20, 341, 343, 354, 355 D’Hooghe T, 244, 246, 248 D’Souza SW, 258, 268 Dahlquist G, 68, 71 Dahri S, 81, 82, 83, 85, 92, 93, 148, 156, 203, 213, 219, 222, 340, 342, 350, 351, 353, 354, 357 Dahri SA, 172, 179 Daling JR, 16, 22 Dalle M, 235, 239 Dallman MF, 230, 237 Damsgaard EM, 28, 31, 48 Dandekar P, 80, 91, 277, 292 Danforth E, 187, 195 Daniline J, 86, 94, 341, 354 Danon UL, 29, 48 Darby CJ, 175, 180 Darke K, 325, 337 das Santos MB, 121, 136 Dauncey MJ, 210, 212, 224 Davey Smith G, 5, 8, 19, 37, 45 David M, 233, 239 Davidson JM, 133, 139 Davies DP, 39, 46 Davies G, 298, 316 Davis EC, 52, 57 Davis L, 125, 130, 137 Davis LE, 125, 137 Dawes GS, 110, 135, 183, 193 Day KC, 281, 294 De Barro T, 258, 259, 268 De Barro TM, 75, 89 de Bruin JP, 246, 248 De Chazal R, 263, 270 de Courten MP, 327, 338 De Gasparo M, 81, 85, 92 De Prins FA, 353, 358 De Ridder MAJ, 242, 247 de Santiago A, 218, 227

Author Index de Souza ALM, 331, 338 de Swiet M, 26, 29, 32, 33, 34, 45, 264, 271 de Zegher F, 242, 243, 244, 246, 247, 248 Deamer WC, 244, 248 Deanfield JE, 261, 269 Dearden LC, 209, 224 Deayton JM, 202, 222 Debelle L, 50, 57 Declerck F, 78, 91, 213, 224, 349, 356 Decsi T, 10, 20 Degani S, 264, 271 DeJesus O, 214, 225 Del Pino A, 253, 267 deLancey E, 212, 224 Delost P, 235, 239 Dempster M, 164, 178 Den Hartog H, 242, 247 Denne J, 263, 270 Denne SC, 208, 223 Dennison E, 15, 21 Dequeker J, 278, 293 Derks JB, 214, 225 Desai M, 81, 84, 92, 93, 161, 163, 170, 171, 177, 178, 179, 190, 196, 200, 213, 219, 221, 262, 270, 343, 354 Desaye G, 209, 224 Deshpande V, 352, 358 Deymann MA, 124, 136 Dezateux C, 304, 305, 307, 318 Dhingra K, 277, 292 Di Andrea A, 308, 319 Di Fulvio S, 253, 267 Di Iulio J, 81, 92 Di Nicolantonio R, 80, 81, 91, 92 Dietz WH, 326, 337 DiLoreto C, 106, 134 Dinneen S, 173, 180 Diorio J, 231, 238, 275, 291 Divon MY, 185, 193 Dixon IM, 106, 134 Doak C, 334, 338

365 Dobbing J, 231, 238, 275, 291 Dodic M, 203, 213, 214, 215, 218, 222, 225, 231, 238 Dohr G, 209, 224 Donald AE, 261, 269 Donker GA, 26, 30, 47 Donnai P, 258, 268 Dorland M, 246, 248 Dorling MW, 163, 164, 167, 178 Dostal K, 212, 224 Doull I, 314, 321 Dowse GK, 177, 180, 327, 338 Drewnowski A, 331, 332, 334, 338 Driscoll SG, 209, 224 Drop SLS, 242, 247 Dry J, 313, 321 Du V, 305, 318 Duchateau J, 312, 320 Ducimetiere P, 26, 27, 47 Dundas I, 305, 307, 318 Dunger DB, 149, 150, 157 Dunhill GS, 55, 58 Dunlop MG, 164, 178 Dunlop SA, 214, 225, 274, 275, 291 Dunlop W, 260, 269 Dupouy JP, 231, 237 Dutriez-Casteloot I, 231, 237 Dwyer CM, 81, 92 Dwyer JH, 325, 337 Earl CR, 75, 89 Eberhardt MS, 63, 69 Ecob R, 27, 30, 48 Edenharter G, 311, 320 Edwards CR, 214, 225 Edwards CRW, 76, 78, 90, 213, 225, 263, 270, 276, 277, 278, 289, 292, 293 Edwards HV, 55, 59 Edwards LJ, 286, 295 Edwards R, 38, 46 Efstratiadis A, 186, 194, 207, 208, 223, 252, 264, 266, 271 Egan AR, 75, 89, 282, 294

366 Egan BM, 63, 66, 69, 70 Egger O, 14, 21 Egger P, 16, 22, 214, 226, 236, 240, 246, 248 Eisele S, 214, 225 Eisenberg S, 218, 227 Eissa M, 53, 58 Ekbom A, 16, 22 Eklof AC, 218, 227 Eley DW, 350, 357 Elford J, 8, 19 Ellard S, 148, 156 Ellekjaer H, 61, 69 Elliott A, 13, 20 Elliott J, 236, 240 Elwood P, 5, 8, 19, 37, 45 Emanuel I, 16, 22, 26, 30, 36, 45, 255, 267 Endicott SK, 52, 58 Engelmann GL, 133, 139 Engelmann M, 209, 224 Epstein MF, 209, 224 Erhardt E, 10, 20 Eriksson J, 32, 48 Eriksson JG, 5, 11, 12, 18, 19, 170, 178, 254, 255, 267, 324, 337 Eriksson UY, 346, 355 Erkadisu E, 81, 92 Ervin MG, 209, 215, 224, 226 Erwich JHM, 280, 294 Eswards CRW, 203, 214, 222 Evans P, 252, 266 Evans PC, 4, 18, 185, 186, 193, 194, 208, 219, 223, 257, 268 Evans SF, 274, 275, 291 Evans SJW, 255, 267 Eveleth PB, 325, 337 Everhart J, 353, 358 Eyles JP, 214, 225 Faber JJ, 112, 135 Faichney GJ, 75, 89 Faktor JH, 308, 319 Fal CH, 200, 221

Author Index Falkkner B, 38, 46 Fall C, 8, 13, 15, 19, 21, 142, 150, 155, 157, 305, 318 Fall CH, 246, 248 Fall CHD, 4, 5, 9, 10, 11, 12, 18, 19, 20, 29, 34, 45, 143, 146, 152, 155, 155, 156, 158, 159, 162, 178, 214, 226, 233, 239, 254, 257, 261, 262, 267, 269, 352, 358 Falth-Magnusson K, 312, 320 Fanaroff AA, 39, 46 Fanger NH, 312, 320 Faragher RGA, 164, 178 Farmer G, 257, 268 Farrell PM, 209, 224 Farstad M, 349, 356 Faure A, 343, 355 Fawcett A, 288, 295 Felton CV, 187, 195 Ferguson MWJ, 275, 291 Feytons V, 341, 354 Fields-Okotcha C, 80, 91 Fifer MA, 107, 134 Filakti H, 255, 267 Finato N, 106, 134 Finch CF, 177, 180 Finegood DT, 348, 356 Finlay-Jones JF, 308, 319 Finnage VL, 212, 224 Fishel RS, 218, 227 Fisher DJ, 110, 111, 125, 135, 138 Fishman NH, 121, 136 Fisk NM, 235, 239, 264, 271 Fixler DE, 125, 137 Flanagan DEH, 146, 148, 156 Fleming JV, 340, 353 Fletcher ME, 305, 307, 318 Florey C, 305, 318 Flozak AS, 192, 197, 279, 293 Folkow B, 52, 58 Folsom AR, 63, 69 Foord FA, 11, 20, 257, 268 Forbes W, 81, 85, 92, 349, 350, 356 Forest MG, 233, 239

Author Index Forhead AJ, 187, 195, 203, 204, 207, 208, 209, 210, 219, 222, 223 Forrester T, 11, 20, 27, 42, 47, 254, 256, 257, 267 Forrester TE, 10, 20, 27, 48, 143, 156 Forsen T, 5, 11, 12, 18, 19, 32, 48, 170, 178, 254, 255, 267, 324, 337 Fouron JC, 54, 58 Fowden AL, 3, 4, 18, 148, 156, 186, 187, 191, 192, 194, 195, 196, 197, 200, 202, 203, 204, 206, 207, 208, 209, 210, 212, 219, 221, 222, 223, 224 Fox M, 80, 91, 277, 292 Fraher L, 202, 212, 222 Fraher LJ, 125, 137, 200, 202, 204, 221, 288, 295 Frampton RJ, 192, 197 Francis D, 275, 291 Francis J, 231, 238 Francois I, 242, 243, 244, 246, 247, 248 Franconi F, 350, 351, 357 Francucci B, 276, 292 Frank JW, 173, 180 Frankel S, 5, 8, 19, 37, 45 Fraser GC, 304, 318 Fraser RB, 16, 22, 214, 226, 236, 240, 246, 248 Freedman A, 275, 291 French FS, 244, 248 Freud S, 274, 275, 291 Friedman MI, 84, 93, 153, 158 Friedman WF, 117, 136 Friedrichs D, 309, 319 Froland A, 28, 31, 48 Fuller PJ, 280, 294 Gagnon R, 125, 132, 137, 138, 200, 202, 212, 218, 221, 222, 227, 288, 295 Gale CR, 50, 57 Gale R, 27, 29, 30, 48 Gallaher BW, 192, 196

367 Galler JR, 276, 292 Galliano SV, 253, 267 Gallimore JR, 172, 179 Galloway R, 87, 94 Garcia DL, 218, 227, 350, 356 Gardner DS, 170, 179, 203, 214, 222, 225, 276, 277, 289, 292 Gargosky SE, 242, 247 Garland PB, 175, 180 Garofano A, 341, 343, 354, 355 Garza C, 274, 291 Gaugier D, 346, 355 Gaull G, 350, 356 Ge K, 329, 334, 338 Geisen C, 76, 90 Gennser G, 39, 46 Gentili S, 276, 292 George J, 244, 248 Gerber PP, 173, 180 Gerber R, 78, 91, 213, 224 Gerber RT, 76, 90, 349, 356 Gerlis LM, 98, 101, 133 Germain J, 52, 57 Germain JP, 173, 179 German RR, 63, 69 Ghekiere L, 312, 320 Ghisolfi J, 352, 358 Gilles WB, 288, 295 Gilbert RD, 76, 90, 113, 115, 125, 130, 135, 137, 138, 219, 228 Gilbert T, 56, 59, 68, 71, 81, 85, 92, 94, 350, 356 Gilmour RS, 187, 195, 204, 207, 208, 209, 210, 212, 222, 223, 224 Gine E, 350, 357 Giraud GD, 124, 127, 129, 136, 138 Girerd X, 51, 57 Gironi A, 350, 351, 357 Gitau R, 235, 239 Giussani DA, 214, 225 Glagov S, 52, 53, 58 Glazier JD, 258, 268 Gleason CA, 183, 193 Glemons GK, 219, 228

368 Gloding J, 64, 70 Glover V, 235, 239 Glovsky MM, 312, 320 Gluckman P, 252, 266 Gluckman PD, 4, 18, 78, 91, 186, 187, 188, 191, 192, 194, 195, 196, 197, 206, 208, 219, 222, 223, 228, 233, 239, 246, 248, 250, 257, 260, 266, 268, 276, 292 Godden DJ, 313, 321 Godfrey K, 11, 20, 189, 195, 253, 254, 255, 257, 260, 266, 267 Godfrey KM, 10, 11, 20, 27, 28, 29, 31, 32, 33, 34, 42, 45, 47, 88, 94, 189, 195, 233, 239, 250, 253, 254, 256, 257, 258, 259, 260, 261, 264, 266, 267, 268, 269, 271, 305, 314, 318, 321 Godfrey S, 297, 316 Godsland IH, 146, 148, 156 Going TCH, 173, 179 Golding J, 27, 28, 32, 33, 34, 44, 150, 157 Goodman HO, 350, 357 Gordon GS, 162, 178 Gorman BK, 325, 337 Gorman J, 15, 21 Gosling RG, 54, 58 Gotoh T, 104, 134 Gotz F, 154, 158 Grant PA, 208, 223 Grat R, 209, 224 Green DK, 164, 178 Greenwald S, 27, 31, 48, 54, 58, 132, 138, 261, 269 Greenwald SE, 132, 138 Gregory A, 314, 321 Grill V, 346, 355 Grillo RL, 172, 179 Grimble RE, 171, 179 Grimble RF, 289, 295 Grino M, 231, 237 Grobbee DE, 38, 46 Gronbuck M, 61, 69

Author Index Gross CG, 275, 291 Grossman W, 107, 121, 134, 136 Gu W, 186, 194 Guendelman S, 325, 337 Guilbert L, 300, 308, 316 Guilkey DK, 324, 337 Guillaume M, 341, 354 Guillery EM, 218, 227 Guilliford MC, 305, 318 Gunn AJ, 187, 194 Gunther S, 107, 134 Gunther-Genta F, 15, 21 Guo X, 334, 338 Guyton AC, 56, 59 Habich K, 352, 358 Hack M, 39, 46 Hage GP, 190, 196 Hales CN, 8, 9, 11, 14, 16, 19, 20, 29, 45, 64, 70, 81, 84, 87, 88, 92, 93, 94, 95, 142, 143, 146, 148, 151, 155, 155, 156, 157, 158, 159, 161, 162, 163, 164, 167, 170, 171, 173, 175, 177, 178, 179, 180, 190, 196, 200, 209, 213, 218, 219, 221, 224, 227, 228, 246, 248, 254, 257, 261, 262, 263, 267, 269, 270, 279, 293, 339, 343, 346, 347, 352, 353, 354, 355, 357, 358 Hales JRS, 288, 295 Halil T, 13, 20 Hall IP, 310, 320 Hall JStE, 11, 20, 27, 42, 47, 254, 257, 267 Hall MH, 10, 20, 28, 31, 42, 47, 88, 94, 250, 253, 265, 266 Hall R, 81, 85, 92, 349, 350, 356 Halonen M, 307, 319 Hamilton-Nicol DR, 257, 268 Hammond J, 250, 266 Han VK, 125, 137, 204, 222 Han VKM, 200, 202, 204, 221, 288, 295

Author Index Hankinson SE, 5, 8, 19, 74, 87, 89 Hann T, 209, 224 Hanrahan JP, 305, 318 Hansen NB, 218, 227 Hanson C, 208, 223 Hanson MA, 75, 76, 77, 78, 79, 80, 89, 90, 91, 106, 134, 200, 202, 214, 218, 219, 221, 277, 286, 292, 294 Hanson RL, 145, 156, 257, 268 Hansona GJ, 207, 223 Hansson L, 40, 41, 46 Hao J, 106, 134 Harding JE, 3, 4, 18, 182, 185, 186, 187, 188, 191, 192, 193, 194, 195, 196, 200, 202, 206, 208, 219, 221, 222, 223, 228, 233, 239, 250, 252, 257, 260, 266, 268, 282, 294 Harding R, 200, 202, 203, 221, 222, 286, 295 Harel Z, 84, 93 Harries DN, 340, 353 Harris GW, 275, 291 Harris KM, 325, 337 Harrist RB, 26, 30, 47 Hart HA, 308, 319 Hartwich KM, 252, 266 Harty PH, 308, 319 Harvey R, 148, 156 Hashimoto N, 26, 30, 47 Hassink SG, 212, 224 Hastie ND, 164, 178 Hattersley AT, 68, 71, 148, 156 Hawkins P, 76, 77, 78, 79, 90, 286, 294 Hay SM, 340, 353 Hayakawa T, 149, 157 Hayman JM, 85, 94 Hayward AR, 300, 316 Hazeki O, 174, 180 Heesch CM, 232, 238 Heidrich I, 154, 158 Heitz PU, 352, 358 Hendrick SK, 209, 224

369 Hennekens CH, 5, 8, 19, 38, 46, 74, 87, 89 Henquin JC, 342, 354 Henry BA, 212, 224 Hernandez DJ, 325, 337 Herrera VL, 276, 292 Herrin AN, 329, 338 Hertzler JH, 304, 318 Hewitt CW, 125, 137 Hey EN, 305, 318 Heymann MA, 110, 111, 118, 121, 124, 125, 135, 136, 138 Hildy J, 310, 320 Hill D, 348, 356 Hill DE, 85, 93 Hill DJ, 82, 92, 208, 219, 223, 257, 268 Himes JH, 326, 337 Himmelmann A, 40, 41, 46 Hinchcliffe SA, 66, 70, 85, 94, 218, 227 Hindmarsh P, 15, 21 Hindmarsh PC, 233, 239, 244, 247 Hintz RL, 207, 223 Hirakow R, 104, 134 Hirst S, 87, 94, 146, 148, 156, 162, 178, 218, 227, 352, 357 Hodge AM, 327, 338 Hodge L, 313, 321 Hoeak W, 15, 21 Hoet JJ, 75, 81, 82, 83, 85, 86, 89, 92, 93, 94, 148, 156, 172, 173, 179, 200, 203, 213, 214, 218, 219, 221, 222, 233, 239, 286, 294, 340, 341, 342, 348, 350, 351, 353, 354, 356, 357 Hoffman JIE, 125, 126, 127, 137, 138 Hofman A, 38, 46 Hofman PL, 146, 148, 156, 191, 196, 246, 248 Hohimer AR, 125, 130, 137 Hohlfeld P, 15, 21 Hokken-Koelega ACS, 242, 247

370 Holden J, 214, 225 Holemans K, 76, 78, 86, 90, 91, 94, 213, 224, 257, 268, 278, 279, 293, 341, 344, 349, 353, 354, 355, 356, 358 Holgate ST, 13, 14, 21 Holland FJ, 28, 31, 32, 33, 34, 44 Holland WW, 13, 20 Hollingshead P, 186, 194 Holmen J, 61, 69 Holmes GE, 152, 157, 231, 237, 263, 270 Holmes MC, 214, 225 Holness MJ, 170, 179, 346, 355 Holsboer R, 209, 224 Holst M, 234, 239 Holt BJ, 302, 307, 311, 317 Holt PG, 312, 320 Holton DW, 55, 59 Homan J, 125, 137 Honour JW, 233, 239, 244, 247 Hooper SB, 200, 202, 204, 209, 221, 222, 223 Hopkin JM, 310, 320 Horie R, 351, 357 Horneff G, 310, 320 Horrobin DF, 313, 321 Horton CE, 313, 321 Hosking BJ, 75, 89, 282, 294 Hou QC, 215, 226 Howard CV, 66, 70, 85, 94, 218, 227 Hoy WE, 68, 71 Hsieh CC, 16, 22 Hubel DH, 275, 291 Huff DS, 118, 136 Hughes JM, 313, 321 Hughes KA, 310, 319 Hughes P, 186, 187, 194, 195, 206, 209, 222, 223 Hughes SJ, 170, 172, 179 Hulman S, 38, 46 Hultman CM, 15, 21 Hunter S, 260, 269 Huss JM, 232, 238

Author Index Hutchison JB, 236, 240 Hutchison SJ, 260, 269 Huttunen MO, 15, 21 Huxtable RJ, 350, 356 Huxtable SJ, 149, 157 Ibanez L, 242, 243, 244, 246, 247, 248 Iglesias-Barreira V, 82, 85, 93 Ikeda K, 351, 357 Ikegami M, 209, 215, 224, 226 Iles RA, 173, 179 Imaki T, 231, 237 Inagami T, 218, 227 Indredovik B, 61, 69 Innis SM, 84, 93 Isberg PE, 39, 46 Ito H, 264, 270 Itskovitz J, 125, 137, 183, 193 Jackson A, 258, 268 Jackson AA, 10, 11, 20, 27, 42, 47, 78, 91, 150, 157, 171, 179, 189, 191, 195, 196, 203, 214, 222, 225, 233, 239, 254, 256, 257, 267, 276, 277, 278, 289, 291, 292, 295 Jacobson L, 231, 238 Jacobson SH, 218, 227 Jacobsson LTH, 145, 156, 257, 268 Jakobsen JG, 350, 356 Jalkanen P, 346, 355 Janson RA, 209, 224 Jansson T, 81, 92, 150, 157, 282, 294 Jarrett EEE, 311, 320 Jenkins SL, 214, 225 Jennings BJ, 163, 164, 167, 178 Jensen EC, 187, 188, 195 Jequier E, 191, 196 Jespersen S, 27, 31, 48, 50, 54, 57, 58, 132, 138, 261, 269 Jesse MJ, 38, 46 Jiang BH, 125, 130, 137 Jobe AH, 209, 215, 224, 226 Joglekar C, 352, 358

Author Index Johannson B, 346, 355 Johnson MR, 250, 266 Johnston BM, 3, 4, 18, 78, 80, 91, 191, 192, 196, 197, 219, 228, 276, 282, 292, 294 Johnston L, 202, 212, 222 Jonas HA, 192, 197 Jones AC, 302, 307, 310, 311, 317, 319 Jones AP, 84, 93, 153, 158 Jones CA, 301, 302, 308, 310, 317, 319 Jones CT, 76, 91, 186, 187, 194, 195, 200, 202, 219, 221, 228 Jones D, 121, 136 Jones MD, 125, 137 Jones P, 15, 21 Jones PJ, 63, 69 Jose PA, 218, 227 Joseph D, 168, 178 Joseph KS, 8, 19 Jost A, 341, 354 Ju H, 106, 134 Junaid A, 106, 134 Kadowaki T, 149, 157 Kajstura J, 106, 134 Kallen B, 68, 71 Kamei K, 214, 225 Kamitomoto M, 130, 138 Kanayasu T, 264, 270 Kapetanovic T, 27, 30, 48 Karas RH, 260, 269 Karlberg J, 8, 19, 242, 247, 290, 295 Karniski LP, 218, 227 Karwo WG, 218, 227 Kashiwai KT, 215, 226 Kasper CB, 232, 238 Kass EH, 38, 46 Katsman A, 280, 294 Katsman AI, 262, 270, 281, 282, 294 Kattan C, 253, 267 Katz AM, 111, 112, 115, 135 Kaufmann P, 76, 90

371 Kaung HLC, 347, 355 Kawano Y, 174, 180 Kawasaki T, 26, 30, 47 Kearney M, 260, 269 Kearney PJ, 38, 46 Keeley FW, 132, 138 Keigthley MC, 280, 294 Keil JE, 63, 69 Keirse MJN, 234, 239 Kekomaki M, 185, 194 Keller BB, 103, 133 Keller U, 173, 180 Kellingray S, 15, 21 Kellingray SD, 352, 358 Kelly RG, 98, 133 Kelly RW, 189, 195 Kemeny DM, 307, 319 Kemp AS, 310, 320 Kemp GH, 191, 196 Kemper T, 81, 85, 92, 349, 350, 356 Kenyon CJ, 214, 225, 278, 293 Kergoat M, 278, 292 Kervan A, 341, 354 Ketelslegers JM, 348, 356 Kiess W, 212, 224 Kietera K, 214, 225 Kihara M, 351, 357 Kikuchi T, 26, 30, 47 Kilburn SA, 301, 317 Kile E, 68, 71 Kim S, 334, 338 Kimberly D, 125, 130, 137 Kind KL, 208, 223, 262, 270, 280, 281, 282, 294 King A, 308, 319 Kingdom JCP, 76, 90 Kingston EJ, 76, 91 Kipling D, 164, 178 Kirby ML, 103, 133 Kistner A, 218, 227 Kitamaka T, 219, 228 Kitten GT, 130, 138 Kiyasu N, 244, 248 Kjellman NIM, 312, 320

372

Klag MJ, 63, 68, 69, 70, 71 Klebanoff MA, 255, 267 Kleeman DO, 252, 264, 266 Klein N, 39, 46 Kleinerman J, 305, 318 Klempt M, 187, 195 Kliewer SA, 232, 238 Klopper G, 352, 358 Knowler W, 353, 358 Knowler WC, 145, 154, 156, 158, 257, 268 Kolacek S, 27, 30, 48 Kolpfenstein SH, 110, 135 Kondo N, 307, 319 Konje JC, 263, 270 Koukkou E, 76, 86, 91 Koupilova I, 10, 20, 29, 31, 32, 37,39, 41, 45, 46, 47, 87, 94, 170,178 Kozuma S, 200, 202, 221 Kramer JH, 350, 357 Kramer MS, 8, 19, 182, 193, 253,265, 266 Krasinski K, 260, 269 Krebs C, 76, 90 Krey LC, 232, 238 Ktorza A, 346, 355 Kuh D, 27, 28, 32, 33, 34, 44, 64, 70,236, 240, 246, 248 Kuhn P, 311, 320 Kumaran K, 5, 11, 12, 19, 20, 254,257, 262, 267 Kunzelman CL, 353, 358 Kuriyama K, 351, 357 Kuziora M, 299, 316 Kwok CW, 328, 338 Kyvic KO, 150, 157 La Mear NS, 219, 228 Labarthe DR, 26, 30, 47, 53, 58 Laberge JM, 304, 317 Lackland DT, 61, 63, 66, 69, 70 LaGamma F, 125, 137 Lagecrantz H, 219, 228 Laiprasert JD, 232, 238

Author Index Lajic S, 234, 239 Lake N, 350, 357 Lampson WG, 350, 357 Lan SJ, 16, 22 Landale N, 325, 337 Landman JP, 11, 20, 27, 42, 47, 254, 257, 267 Lane RH, 279, 293 Langille BL, 52, 57, 132, 133, 138, 139 Langley SC, 78, 91, 150, 157, 191, 196, 233, 239, 276, 278, 291, 292 Langley-Evans SC, 10, 20, 78, 91, 131, 138, 170, 171, 179, 203, 213, 214, 219, 222, 225, 227, 231, 237, 254, 267, 276, 277, 278, 289, 292, 293, 295 Laogun AA, 54, 58 Laor A, 27, 29, 30, 48 Lappi SE, 214, 225 Laptook AR, 218, 227 Larkins RG, 192, 197 Latimer AM, 207, 223 Launer LJ, 32, 37, 38, 45, 46 Laurent S, 51, 57 Laver RM, 53, 58 Law CM, 8, 11, 19, 20, 23, 24, 26, 29, 32, 33, 34, 42, 44, 45, 47, 49, 57, 64, 70, 152, 155, 158, 159, 162, 178, 233, 239, 244, 247, 250, 254, 257, 266, 267 Le Floch-Prigent P, 121, 136 Le Moal M, 231, 233, 237 Leaverton PE, 53, 58 Lechner AJ, 75, 89 Lecomte E, 26, 27, 47 Ledingham JM, 104, 134 Lee A, 10, 19 Lee JH, 80, 91 Lee S, 231, 237 Lee WH, 208, 223 Lee YH, 38, 46 Leef K, 212, 224 Leese HJ, 252, 266

Author Index Leeson CPM, 41, 47, 261, 269 Leeson-Payne C, 39, 46 Lefevre M, 52, 57 Leger J, 10, 20, 39, 46 Lehingue Y, 23, 44 Lehmann ED, 51, 57 Leiser R, 76, 90 Lelievre-Pegorier M, 56, 59, 68, 71, 81, 85, 92, 94, 303, 317, 350, 356 Lemmen RJ, 242, 247 Lemmonier D, 154, 158 Lemons JA, 190, 196 Leon DA, 5, 8, 10, 18, 20, 29, 31, 32, 37, 39, 41, 45, 46, 47, 64, 70, 87, 94, 143, 146, 148, 156, 170, 178, 261, 269, 352, 357 Lerent A, 61, 69 Leri A, 106, 134 Leroy B, 56, 59, 68, 71, 81, 85, 92, 94, 350, 356 Lesage J, 231, 237 Lessard M, 54, 58 Leung DYM, 52, 53, 58 Lever AF, 56, 59, 263, 270 Levine RS, 38, 46 Levine S, 275, 291, 328, 338 Levitt NS, 152, 157, 214, 225, 231, 237, 263, 270 Levy JC, 148, 156 Levy L, 78, 91, 189, 195 Levy Marchal C, 10, 20, 39, 46 Lewis DS, 154, 158 Lewis PA, 39, 46 Lewis RM, 78, 80, 91 Lewis S, 306, 319 Li J, 203, 204, 207, 208, 210, 212, 219, 222, 223, 224 Liechty EA, 208, 219, 223 Liggins GC, 207, 209, 210, 213, 223 Lin H, 300, 308, 316 Lin S, 15, 21 Lind J, 54, 58 Lindsay RM, 152, 158, 214, 219, 225, 228, 263, 270, 278, 293

373 Lindsay RS, 76, 78, 90, 152, 157, 158, 213, 214, 219, 225, 228, 231, 237, 263, 270, 278, 293 Lindsay S, 76, 78, 90 Lindstrom LH, 15, 21 Lister G, 39, 46, 190, 196 Lithell H, 5, 8, 18, 146, 156 Lithell HO, 10, 20, 29, 31, 32, 37, 39, 45, 46, 64, 70, 87, 94, 143, 148, 156, 170, 178, 261, 269, 352, 357 Lithell U, 352, 357 Lithell UB, 10, 20, 64, 70, 87, 94, 143, 148, 156, 170, 179, 261, 269 Little WJ, 274, 275, 291 Liu D, 275, 291 Liu JP, 186, 194, 207, 208, 223, 252, 264, 266, 271 Liu L, 186, 194, 208, 219, 223, 252, 266 Liuzzo G, 172, 179 Lo CS, 51, 57 Lobo MV, 350, 357 Lodrup Carlsen KC, 305, 318 Lohr JL, 128, 138 Lohr M, 352, 358 Loizou CL, 219, 228 Lok F, 219, 228, 258, 259, 268 Loky YW, 308, 319 Lomax MA, 175, 180 Longo LD, 76, 90, 125, 130, 137, 138, 219, 228 Looker T, 52, 57, 58 Louchy J, 325, 337 Lovell CR, 313, 321 Lovenberg W, 351, 357 Low LC, 290, 295 Lowry C, 76, 86, 91 Lubahn DB, 244, 248 Lucas A, 39, 46, 75, 84, 89, 93, 161, 171, 177, 179, 190, 196, 261, 262, 269, 270, 273, 290 Lucente F, 81, 92 Lumbers ER, 214, 215, 225, 226, 288, 295

374 Lumey LH, 189, 196, 259, 268 Lurbe E, 27, 30, 47 Luzar V, 27, 30, 48 Lynch MR, 85, 94, 218, 227 Lynch MRJ, 66, 70 Ma H, 334, 338 Maccari S, 231, 233, 237 MacGilvray SS, 219, 228 MacGregor GA, 55, 58 Machon RA, 15, 21 Macintyre S, 27, 30, 48 Mackenzie HS, 56, 59, 68, 70, 71, 85, 94, 262, 270 MacLusky NJ, 236, 240 MacMahon RA, 192, 197 Madgwick AJA, 81, 92 Madill D, 187, 195 Maes M, 242, 243, 247 Magness RR, 209, 224 Mahendran D, 256, 258, 259, 267, 268 Maiter D, 348, 356 Makowski EL, 125, 137 Malaisse WJ, 82, 93 Malek A, 311, 320 Malik N, 350, 357 Mallone KE, 16, 22 Maloney JE, 106, 134 Mandarim-de-Lacerda CA, 121, 136 Mann SL, 13, 21 Manning FA, 185, 193 Manson JE, 5, 8, 19 Manson JEM, 74, 87, 89 Margetts B, 4, 18 Margetts BM, 11, 20, 257, 268 Markus A, 10, 20 Marmot M, 15, 21 Marolla F, 265, 271 Martensz ND, 219, 228 Martenz ND, 190, 196 Martin CA, 125, 130, 137 Martin del Rio R, 350, 357 Martin J, 85, 94

Author Index Martin JA, 66, 70 Martinez FD, 306, 307, 318 Martini L, 232, 238 Martyn CN, 5, 7, 9, 19, 27, 31, 48, 50, 54, 56, 57, 58, 59, 132, 138, 260, 261, 263, 269, 270 Maseri A, 172, 179 Mashiach S, 29, 48 Mason EJ, 154, 158 Masoro EJ, 168, 178 Matheson IC, 182, 193 Mathew MS, 218, 227 Mathews MB, 52, 53, 58 Matsuda Y, 183, 193 Mattana A, 350, 351, 357 Matthes JWA, 39, 46 Matthew SG, 200, 202, 221 Matthews N, 258, 268 May CN, 203, 222, 231, 238 Maylie JG, 104, 106, 134 Mazursky JE, 218, 227 McCance DR, 145, 156, 257, 268 McCance RA, 2, 18, 241, 247 McCarthy AL, 349, 356 Mccarty DJ, 327, 338 McCarty R, 80, 91 McClellan W, 63, 70 McConnachie A, 27, 30, 48 McCook EC, 214, 225 McCorbus C, 302, 307, 310, 311, 317, 320 McCrabb GJ, 282, 294 McCrabb GJM, 75, 89 McCully KS, 88, 95 McCusker RH, 207, 223 McDonald RW, 98, 101, 133 McDonald TJ, 82, 92, 200, 202, 204, 208, 219, 221, 223, 348, 356 McEwen BS, 232, 238 McGarr JA, 168, 178 McGarrigle HHG, 76, 90, 200, 202, 221, 286, 294 McGhie JS, 118, 136 McGill HC, 154, 158

Author Index McGinnis W, 299, 316 McGovern PG, 63, 69 McGregor AM, 187, 195 McKeigue PM, 10, 20, 37, 45, 64, 70, 87, 94, 143, 146, 148, 156, 170, 179, 261, 269, 352, 357 McKeown T, 258, 268 McLaurin LP, 121, 136 McMahon HC, 154, 158 McMillen IC, 77, 91, 200, 214, 221, 286, 288, 295 McQuiddy P, 232, 238 Meade TW, 9, 10, 19 Meaney MJ, 231, 238, 275, 291 Mecacci F, 311, 320 Mednick SA, 15, 21 Meeran K, 190, 196 Meirik O, 255, 267 Melhotra I, 302, 317 Mellor DJ, 75, 89, 182, 193, 200, 221 Mendelsohn ME, 260, 269 Menon RK, 146, 148, 156, 246, 248 Meredith A, 250, 265 Merlet-Benichou C, 56, 59, 68, 71, 81, 85, 92, 94, 350, 356 Meschia G, 111, 125, 135, 137 Mesiano S, 207, 223 Mestyan J, 352, 358 Meurens K, 278, 293 Meurrens K, 78, 91, 213, 224, 349, 356 Meyer WW, 54, 58 Meyer-Bahlburg HFL, 234, 239 Mian M, 350, 351, 357 Miceli M, 350, 351, 357 Micheli JL, 191, 196 Michels KB, 236, 240 Michels RPJ, 11, 14, 16, 20, 88, 94, 151, 157, 162, 177, 209, 224, 233, 239, 261, 269, 352, 358 Midwinter RE, 28, 32, 33, 44 Mijoric J, 208, 223 Mijovic J, 191, 196 Mikawa T, 98, 133

375 Mikuni M, 214, 225 Miles EA, 301, 302, 307, 310, 311, 317, 319 Milford EL, 66, 70 Miller AG, 26, 30, 47 Miller M, 81, 85, 92, 94, 349, 350, 356 Milley JR, 209, 224 Milne KM, 125, 137 Milner GR, 81, 85, 92 Milner RDG, 81, 85, 92, 261, 269 Milovanov AP, 76, 90 Ming AM, 215, 226 Mitchell GF, 51, 57 Mitchell JB, 231, 238 Mitsialis SA, 303, 317 Miura S, 103, 133 Miyagawa S, 103, 133 Mizushima S, 351, 357 Moar VA, 40, 41, 46 Moessinger AC, 305, 318 Mohsen R, 10, 20, 37, 45, 64, 70, 87, 94, 170, 178, 261, 269 Moller-Jensen D, 150, 157 Molnar D, 10, 20 Mondini L, 331, 335, 338 Montanari M, 276, 292 Monteiro C, 324, 336 Monteiro CA, 331, 335, 338 Montel V, 231, 237 Moon S, 334, 338 Moore JT, 232, 238 Moore MA, 61, 69 Moore V, 146, 148, 156 Moore VM, 26, 30, 47 Moorehead H, 208, 223 Moorehead HC, 190, 196 Morel Y, 233, 239 Morgan T, 81, 92 Morgane PJ, 81, 84, 85, 92, 93, 349, 350, 356 Morgensen CE, 28, 31, 48 Morgenstern H, 68, 71 Morita I, 264, 270

376 Morley R, 39, 46, 190, 196 Morris GH, 256, 259, 267 Mortimer JG, 26, 47 Mortimer PS, 55, 58 Mortiz KM, 214, 215, 225, 288, 295 Morton JJ, 56, 59, 263, 270 Morton L, 14, 21 Morton M, 124, 136 Morton MJ, 115, 117, 118, 120, 121, 125, 127, 128, 129, 135, 136, 137, 138 Morton NE, 150, 157, 250, 266 Moshen R, 143, 148, 156, 352, 357 Mosier HD, 209, 224 Mosmann T, 300, 308, 316 Mott JC, 110, 135 Mottaghy K, 76, 90 Mourmeaux JL, 342, 354 Moye LA, 51, 57 Muaku SM, 348, 356 Mudde GC, 311, 320 Muffat-Joly M, 56, 59, 81, 85, 92, 350, 356 Muirray L, 75, 89 Mulier M, 244, 247 Munday L, 208, 223 Mundy L, 219, 228 Muneoka K, 214, 225 Murohara T, 260, 269 Murota S, 264, 270 Murotsuki J, 125, 132, 137, 138, 200, 202, 218, 221, 227, 288, 295 Murphy JJ, 304, 318 Murray R, 15, 21 Myers KA, 51, 57 Myers TF, 219, 228 Naeye RL, 85, 94 Nafstad P, 305, 318 Nakanishi K, 313, 321 Nakao-Hayash J, 264, 270 Nakazato Y, 218, 227 Nakazawa M, 103, 133

Author Index Nankervis AJ, 173, 180 Napier J, 78, 80, 91 Nara Y, 351, 357 Narcy F, 121, 136 Natale R, 183, 193, 235, 239 Nathaniels PW, 214, 215, 225, 226 Nave BT, 84, 93, 174, 180, 219, 228 Neilson IR, 304, 317 Nelson JL, 310, 319 Nelson RG, 68, 71 Nergi-Cesci P, 232, 238 Neugebauer R, 15, 21 New MI, 234, 239 Newnham JP, 189, 195, 214, 225, 274, 275, 291 Newsholme EA, 175, 180 Nichols WW, 52, 58 Nicolaides KH, 187, 195 Nicolaides P, 264, 271 Nikkels PGJ, 246, 248 Nilsson PM, 27, 34, 45, 64, 70 Nishina H, 76, 78, 79, 90 Nissinen A, 32, 48 Noakes DE, 76, 77, 90, 286, 294 Noble A, 75, 89 Noble J, 76, 78, 90, 213, 225 Nodrehaug JE, 349, 356 Noon JP, 27, 30, 48, 55, 59 Noordam J, 264, 271 Norman LJ, 76, 90 Norris P, 81, 85, 92 Notkola IL, 32, 48 Nuutinen M, 32, 48 Nuyt AM, 214, 218, 226, 227 Nwagwu M, 276, 277, 292 Nyberg P, 27, 34, 45, 64, 70 Nygard O, 349, 356 Nyirenda MJ, 214, 225, 278, 293 O’Connor CM, 212, 224 O’Dea K, 173, 180 O’Fallon WM, 61, 69

Author Index O’Rahilly SP, 155, 158 O’Rourke M, 52, 58 O’Sullivan MJ, 38, 46 Oberholzer M, 352, 358 Odelram H, 312, 320 Ogata ES, 192, 197, 279, 293 Ogawa T, 214, 225 Ogenbede HO, 257, 268 Oh W, 38, 46, 218, 227 Ohman A, 15, 21 Ohno T, 103, 133 Ohtsuka T, 130, 138 Okada T, 174, 180 Okudaira M, 68, 71 Oliver M, 252, 266 Oliver MH, 4, 18, 186, 194, 206, 222, 257, 268 Olsson T, 231, 238 Ong KKL, 149, 150, 157 Opertanova I, 212, 224 Orchard TJ, 63, 69 Orlowski J, 218, 227 Oropesa RS, 325, 337 Oryszczyn MP, 313, 321 Oscai LB, 168, 178 Osler W, 274, 275, 291 Osmond C, 1, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 16, 18, 19, 20, 21, 22, 26, 27, 28, 29, 31, 32, 33, 34, 44, 45, 48, 50, 54, 57, 58, 64, 66, 70, 87, 88, 94, 132, 138, 142, 143, 146, 148, 151, 153, 155, 155, 156, 157, 158, 159, 162, 170, 177, 178, 189, 195, 209, 214, 218, 224, 226, 227, 236, 240, 246, 248, 250, 253, 254, 255, 257, 260, 261, 262, 263, 264, 266, 267, 269, 270, 271, 314, 321, 324, 337, 352, 357, 358 Osofsky HJ, 75, 89 Ostergren PO, 27, 34, 45, 64, 70 Otani T, 68, 71 Ouma J, 302, 317 Ounsted C, 250, 265 Ounsted M, 250, 265

377 Ounsted MK, 40, 41, 46 Owens JA, 75, 77, 89, 91, 151, 157, 185, 194, 200, 204, 208, 210, 214, 219, 221, 222, 223, 228, 233, 239, 250, 258, 259, 260, 262, 266, 268, 270, 280, 281, 282, 286, 288, 294, 295 Owens PC, 151, 157, 186, 194, 204, 208, 210, 219, 222, 223, 228, 280, 286, 294 Ozaki T, 76, 77, 78, 79, 90, 286, 294 Ozanne SE, 81, 84, 92, 93, 161, 163, 164, 167, 170, 171, 173, 174, 175, 177, 178, 179, 180, 218, 219, 227, 228, 262, 270, 279, 293, 343, 346, 347, 354, 355 Padbury JF, 214, 215, 225, 226 Paeratakul S, 329, 338 Page RCL, 148, 156 Page WV, 218, 227 Pandit AN, 29, 34, 45, 155, 159, 162, 178, 352, 358 Paneth N, 8, 19 Pannier B, 51, 57 Papacosta O, 27, 29, 30, 32, 37, 38, 42, 44, 45, 46, 47, 155, 159, 250, 261, 266, 269 Parer JT, 110, 135 Parke RE, 325, 337 Parks CM, 112, 135 Parness IA, 115, 135 Patlak CS, 214, 226 Patrick J, 183, 193 Patrick JE, 235, 239 Pawson ME, 250, 266 Payne BD, 125, 138 Payne CL, 190, 196 Peace J, 29, 45, 264, 271 Pearce N, 314, 321 Pearson D, 275, 291 Pearson DM, 257, 268 Peckham CS, 28, 31, 32, 33, 34, 44

378 Pedersen O, 146, 156 Peers A, 214, 215, 218, 225 Peeters LLH, 125, 137 Peeters TL, 341, 354 Pennet PH, 68, 71 Penninga I, 76, 90 Penrose LS, 150, 157 Pepe G, 209, 224 Pepys MB, 172, 179 Perkins AS, 186, 194, 264, 271 Perneger TV, 63, 68, 70, 71 Perry HM, 61, 69 Perry I, 42, 47 Persaud C, 256, 267 Persson E, 81, 92, 150, 157, 282, 294 Peters JH, 309, 319 Petersson KH, 214, 226 Petrick J, 208, 219, 223 Petrik A, 82, 92 Petrik J, 348, 356 Petry CJ, 170, 171, 173, 179, 219, 228 Pettigrew KD, 214, 226 Pettitt DJ, 145, 154, 156, 158, 257, 268, 353, 358 Pharoah POD, 40, 41, 46 Phelan PD, 13, 21 Phernetton T, 209, 224 Philiport AI, 304, 318 Philippens L, 76, 90 Phillips DIW, 16, 22, 56, 59, 146, 148, 150, 152, 155, 156, 157, 158, 162, 178, 200, 215, 218, 221, 226, 227, 233, 236, 239, 240, 261, 262, 269, 352, 357 Phillips DW, 77, 87, 91, 94 Phillips GJ, 203, 214, 222, 276, 277, 289, 292 Phillips ID, 200, 214, 221, 288, 295 Phipps K, 9, 10, 19, 143, 146, 155, 156, 162, 178, 246, 248, 261, 269 Piazza PV, 231, 233, 237 Piccinni MP, 311, 320 Picon L, 346, 355

Pictet R, 347, 355

Author Index Pierce JA, 52, 58 Pinet A, 10, 20 Pinson CW, 118, 121, 136 Plagemann A, 154, 158 Platt LD, 185, 193 Plotsky PM, 275, 291 Polansky M, 38, 46 Poletti A, 232, 238 Polk DA, 215, 226 Pollard I, 236, 240 Pope WV, 218, 227 Popkin BM, 324, 326, 328, 329, 331, 332, 334, 335, 336, 336, 337, 338 Porquet D, 10, 20 Porter HJ, 299, 303, 316 Portha B, 278, 292 Postelnek J, 234, 239 Poston L, 76, 78, 79, 86, 90, 91, 213, 215, 224, 226, 349, 356 Potau N, 244, 246, 248 Poulsen P, 150, 157 Poulton J, 150, 157 Powell JT, 52, 57 Powell-Braxton L, 186, 194 Prader A, 244, 248 Pradier P, 235, 239 Prasad A, 55, 58 Pratt L, 209, 224 Preece R, 153, 158, 189, 196, 255, 267, 279, 293 Prescott E, 61, 69 Prescott SL, 302, 307, 310, 311, 317, 320 Price JF, 307, 319 Prins J, 84, 93, 174, 180, 219, 228 Prins JB, 155, 158 Proietto J, 154, 158, 173, 180 Province MA, 52, 58 Prowse KR, 164, 178 Psaty BM, 26, 30, 36, 45 Pu X, 218, 227 Pugh WW, 349, 356 Pullan CR, 305, 318 Purkins L, 281, 294

Author Index Quinlivan JA, 214, 225, 274, 275, 291 Quinn KJ, 208, 223 Rabuffetti M, 232, 238 Radda GK, 151, 157 Rady PL, 308, 319 Raghupathy R, 301, 308, 317 Rahiala EL, 185, 194 Raichlen JS, 118, 136 Raiha N, 185, 194 Raiha NCR, 352, 358 Rakusan K, 104, 107, 108, 133, 134, 139 Ralph M, 200, 202, 203, 221, 222 Randle PJ, 175, 180 Rao S, 261, 262, 269 Rasch R, 131, 138, 219, 227 Rassachaert J, 82, 93 Rassin DK, 350, 356 Ratajska A, 130, 138 Ravelli ACJ, 11, 14, 16, 20, 88, 94, 151, 157, 162, 177, 209, 224, 233, 239, 261, 269, 352, 358 Ravelli GP, 153, 158, 190, 196 Ravnskov U, 97, 133 Rayns DG, 104, 134 Rebuzzi AG, 172, 179 Record RG, 258, 268 Redde RA, 218, 227 Redman CW, 40, 41, 46 Redon J, 27, 30, 47 Rees M, 68, 71 Rees S, 286, 295 Rees WD, 340, 353 Refsum H, 349, 356 Reichek N, 118, 136 Reid DD, 13, 20 Reid DL, 117, 118, 120, 121, 136 Reid LM, 298, 316 Reid M, 256, 267 Reinisch JM, 218, 227 Reitz MS, 260, 269 Rejzek E, 312, 320 Relf IRN, 51, 57

379 Reller M, 124, 136 Reller MD, 98, 101, 117, 118, 120, 121, 125, 127, 128, 129, 133, 136, 137, 138 Remacle C, 75, 81, 82, 83, 85, 86, 89, 92, 93, 94, 148, 156, 172, 173, 179, 203, 213, 219, 222, 233, 239, 340, 341, 342, 348, 350, 351, 353, 354, 356, 357 Renl JNHM, 209, 224 Reshetnikova OS, 76, 90 Resnick O, 81, 85, 92, 349, 350, 356 Reul JNHM, 231, 237 Reusens B, 75, 81, 82, 83, 85, 86, 89, 92, 93, 94, 172, 173, 179, 203, 213, 219, 222, 233, 239, 340, 341, 342, 348, 350, 351, 353, 354, 356, 357 Reusens-Billen B, 86, 94, 148, 156, 172, 179, 341, 342, 354 Reynolds RM, 152, 158 Rich-Edwards J, 74, 87, 89 Rich-Edwards JW, 5, 8, 19 Richards D, 306, 319 Richards MK, 324, 336 Richardson B, 183, 193, 235, 239 Richardson BS, 125, 137 Rimm EB, 28, 29, 31, 39, 46, 155, 159 Rinas U, 310, 320 Ring F, 14, 21 Ritzez EM, 234, 239 Rivier C, 231, 237 Rizza RA, 173, 180 Rizzo G, 54, 58, 264, 271 Roberts CT, 280, 294 Roberts FE, 76, 90 Roberts RC, 209, 224 Robertson EJ, 186, 194, 207, 208, 223, 252, 256, 259, 264, 266, 267, 271 Robillard JE, 214, 218, 226, 227 Robins JM, 236, 240 Robinson EM, 146, 148, 156, 191, 196, 246, 248

380 Robinson J, 146, 148, 156 Robinson JS, 75, 76, 77, 86, 89, 91, 151, 157, 186, 187, 194, 195, 200, 202, 208, 214, 219, 221, 223, 228, 233, 239, 250, 258, 259, 260, 262, 266, 268, 270, 280, 281, 282, 286, 288, 294, 295 Robinson PC, 286, 294 Robinson S, 153, 158, 162, 178, 189, 195, 214, 226, 253, 255, 260, 266 Robinson SM, 155, 159 Robson SC, 260, 269 Roccella EJ, 61, 69 Rodeck CH, 106, 134 Rodgers B, 15, 21 Roebuck MM, 219, 228 Rogers RC, 232, 238 Rombauts W, 341, 354 Rona RJ, 305, 318 Ronconi M, 276, 292 Rose JC, 215, 226 Rosela G, 173, 180 Rosmond R, 230, 237 Rosner B, 5, 8, 19, 38, 46, 74, 87, 89 Ross IS, 257, 268 Ross J, 113, 114, 135 Rostand SG, 63, 70 Roth CB, 303, 317 Rowe W, 231, 238 Rowland MGM, 11, 20, 257, 268 Rucker RB, 52, 57, 133, 139 Rudolph AM, 3, 4, 18, 110, 111, 118, 121, 124, 125, 135, 136, 137, 138, 183, 191, 193, 196, 262, 270 Rudolph CD, 183, 191, 193, 196 Ruiz Opazo N, 276, 292 Ruiz RGG, 307, 319 Rupert J, 309, 319 Rusecki Y, 352, 358 Rush D, 182, 193, 254, 267 Russell G, 257, 268 Russo P, 304, 317 Rutter WJ, 347, 355 Rymark P, 39, 46

Author Index Sachs EJ, 124, 136 Sadowska GD, 214, 226 Saenger G, 265, 271 Safar M, 51, 57 Sage H, 51, 57 Sager R, 311, 320 Saito T, 76, 77, 90, 286, 294 Sakai A, 214, 225 Sakakibara T, 174, 180 Sakura H, 149, 157 Salome CM, 313, 321 Samet JM, 13, 20 Sampognaro S, 311, 320 Sandberg Bennich S, 68, 71 Sandeman D, 55, 59 Sanders SP, 107, 115, 134, 135 Sanderson AA, 151, 157 Sanderson AL, 55, 59 Sanderson DD, 151, 157 Sanderson M, 16, 22 Sandford AJ, 310, 320 Sandra A, 130, 138 Sands J, 231, 238 Sapolsky RM, 231, 232, 238 Sargent PH, 66, 70, 85, 94, 218, 227 Saruto T, 218, 227 Saslow SB, 173, 180 Sato A, 218, 227 Sauerwein H, 187, 195 Saunders JC, 204, 210, 212, 222,224 Savage MO, 186, 194 Savage PJ, 173, 180 Sawaya AL, 84, 93 Scaglia L, 348, 356 Scammell-La Fleur T, 106, 134 Schachat FH, 215, 226 Schafer SW, 350, 357 Scheffen I, 76, 90 Schnell H, 173, 180 Schnohr P, 61, 69 Scholstrom A, 280, 294 Schrott H, 53, 58 Schutz Y, 191, 196 Schwab M, 215, 226

Author Index Scott P, 10, 20 Scribner R, 325, 337 Seamark RF, 252, 266 Seaton A, 313, 321 Seckl JR, 76, 78, 90, 152, 155, 157, 158, 200, 213, 214, 219, 221, 225, 228, 231, 235, 237, 238, 239, 263, 270, 276, 277, 278, 289, 292, 293 Segal MR, 305, 318 Segar JL, 214, 218, 226, 227 Seghieri G, 350, 351, 357 Seidler AJ, 63, 69 Seidler FJ, 214, 215, 225, 226 Seidman DS, 27, 29, 30, 48 Sekl JR, 203, 214, 222, 226 Selvais PL, 348, 356 Selwyn BJ, 26, 30, 47 Semenza GI, 125, 130, 137 Sener A, 82, 93 Sepuldeva W, 264, 271 Serne EH, 55, 59 Shaheen SO, 13, 14, 21, 305, 318 Shai SY, 218, 227 Shalaby L, 54, 58 Shaper AG, 8, 19, 26, 37, 45 Shapiro SD, 52, 58 Sharma S, 231, 238, 275, 291 Sharrow L, 133, 139 Shaw JAG, 148, 156 Sheikh AU, 215, 226 Sheill AW, 233, 239 Sheldon RE, 125, 137 Shelton S, 214, 225 Shemer J, 27, 30, 48 Shen LL, 312, 320 Shepherd PR, 81, 84, 92, 93, 170, 174, 179, 180, 219, 228, 343, 354 Sheppard H, 153, 158, 189, 196, 255, 267, 279, 293 Sherman RC, 219, 227, 289, 295 Sherriff A, 149, 157 Sherriff SB, 50, 57 Sheslow DDV, 212, 224 Shibata H, 218, 227

381 Shiell A, 11, 20, 254, 257, 267 Shiell AW, 8, 10, 13, 14, 19, 20, 21, 23, 24, 28, 31, 42, 44, 47, 49, 57, 64, 70, 88, 94, 155, 159, 162, 178, 244, 247, 253, 266, 305, 318 Shier RP, 261, 262, 269 Shihabi ZK, 350, 357 Shinoda S, 307, 319 Shohat B, 308, 319 Shohat M, 308, 319 Shore AC, 55, 59 Sibley C, 258, 268 Sibley CP, 258, 268 Sicard RE, 183, 193 Sicks JD, 61, 69 Siddle K, 174, 180 Siest G, 26, 27, 47 Sievers RE, 260, 269 Sigulem CM, 84, 93 Silva PA, 26, 39, 46, 47 Silver HK, 244, 248 Silver M, 186, 187, 191, 194, 195, 196, 200, 202, 207, 208, 221, 222, 223 Simeon D, 10, 20, 143, 156 Simmonds SJ, 4, 5, 18, 28, 29, 31, 32, 33, 34, 44, 152, 158 Simmons RA, 192, 197, 279, 293 Simon NG, 218, 227 Simonetta G, 77, 91, 200, 214, 221, 286, 288, 295 Simpson A, 26, 47 Simpson FQ, 104, 134 Sipos L, 185, 193 Siu BL, 219, 228 Skinner SJM, 78, 80, 91 Slotkin TA, 214, 215, 225, 226 Smallacombe T, 310, 320 Smith AD, 76, 90 Smith AG, 310, 319 Smith BL, 66, 70 Smith GD, 81, 84, 92, 93, 173, 174, 179, 180, 218, 219, 227, 228, 343, 354

382 Smith JM, 170, 173, 179 Smith JRLH, 350, 356 Smith OJ, 214, 226 Smith-Kiswic SM, 212, 224 Smolich JJ, 106, 108, 121, 125, 134 Snijders RJM, 187, 195 Snoeck A, 75, 81, 82, 85, 89, 92, 93, 148, 156, 172, 179, 233, 239, 340, 342, 353, 354 Snow MHL, 150, 157, 250, 265 Soares JM, 256, 267 Soderstrom M, 27, 34, 45, 64, 70 Solis JM, 350, 357 Soltesz G, 352, 358 Sonneblick EH, 113, 114, 135 Sonnenberg GE, 173, 180 Sorenson RI, 345, 355 Sparks JW, 209, 224 Spear ML, 212, 224 Spears G, 26, 47 Speigelman D, 28, 29, 31, 39, 46 Speizer FE, 13, 20 Speizer PE, 304, 318 Spencer JA, 106, 134 Sperling MA, 146, 148, 156, 246, 248 Spevak PH, 115, 135 Spiegelman D, 155, 159 Spiessens C, 244, 246, 248 Spitz B, 78, 91 Spitzer AR, 212, 224 Spliet W, 246, 248 Sprafka JM, 63, 69 Spurr BW, 313, 321 Srun R, 218, 227 St George IM, 39, 46 St. John Sutton MC, 118, 136 Stampfer MJ, 5, 8, 19, 28, 29, 31, 39, 46, 74, 87, 89, 155, 159 Stanford JL, 16, 22 Stanner S, 56, 59 Stanner SA, 41, 47 Stark O, 28, 31, 32, 33, 34, 44 Stauffacher W, 173, 180 Stec I, 231, 237

Author Index Steer PJ, 250, 266 Stehouwer CD, 55, 59 Stein CE, 5, 11, 12, 19, 20, 151, 157, 254, 257, 262, 267 Stein Z, 265, 271 Stein ZA, 153, 158, 190, 196 Stephens DN, 75, 89 Stern D, 307, 319 Stern E, 352, 358 Stern MP, 28, 30, 48, 146, 156, 352, 358 Stern WC, 349, 356 Sterne JAC, 305, 318 Stevens D, 187, 195, 288, 295 Stevens J, 329, 338 Stevenson CJ, 40, 41, 46 Stevenson DK, 27, 29, 30, 48 Stewart GA, 302, 317 Stewart RJ, 84, 85, 93, 153, 158 Stewart RJC, 189, 196, 255, 267, 279, 293 Stirling Y, 9, 10, 19, 155, 158 Stockard CR, 275, 291 Stocks J, 304, 305, 307, 318 Stonestreet BS, 214, 218, 226, 227 Stovin PGI, 299, 316 Strachan DP, 13, 21, 313, 321 Stratford L, 209, 223 Stratford LL, 76, 77, 90, 286, 294 Streeter GL, 97, 133 Strickland NC, 81, 92 Sturman GA, 82, 84, 93, 350, 357 Sturman JA, 350, 356 Sudhir K, 260, 269 Sugden MC, 346, 355 Sultan HY, 236, 240 Sun K, 202, 222, 235, 239 Sun YP, 260, 269 Suquet JP, 154, 158 Surus A, 187, 195 Susser E, 15, 21 Susser M, 8, 19, 265, 271 Susser MW, 153, 158, 190, 196 Sutter-Dub MT, 343, 355

Author Index Suzuki H, 218, 227 Svensson A, 40, 41, 46 Swaab DF, 231, 238 Sweetnam P, 5, 8, 19, 37, 45 Swenne I, 264, 270 Symonds M, 75, 89 Symonds MR, 175, 180 Szekely E, 304, 318 Szemere J, 187, 195 Szemese J, 209, 223 Taber LA, 103, 133 Tager IB, 13, 20, 304, 305, 318 Taittonen L, 32, 48 Takahashi H, 26, 30, 47 Takamashi K, 214, 225 Takao A, 103, 133 Takashi O, 130, 138 Takigawa M, 214, 225 Takla TY, 75, 89 Tamarit-Rodriguez J, 350, 357 Tamburro AM, 50, 57 Tamemoto H, 149, 157 Tang MLK, 310, 320 Tangalakis K, 76, 90, 214, 215, 225, 288, 295 Tannenbaum B, 275, 291 Tannenbaum GS, 84, 93 Tanner JM, 325, 337 Tasoni P, 242, 247 Taussig LM, 306, 307, 318, 319 Taylor DJ, 191, 196, 263, 270 Taylor KP, 263, 270 Taylor KW, 345, 355 Taylor PD, 349, 356 Taylor SJ, 27, 30, 44, 45 Taylor SJC, 155, 159 Tayyeb MI, 214, 225 Te Velde ER, 246, 248 Teleshova OV, 76, 90 Tennett AEB, 13, 20 ter Keurs HEDJ, 350, 357 ter Maaten JC, 55, 59

383 Teramo K, 5, 11, 12, 18, 254, 255, 267 Teutsch SM, 63, 69 Teyssier G, 54, 58 The-Hung B, 234, 239 Thian S, 200, 221 Thiliginathan B, 309, 319 Thomas CR, 349, 356 Thomforde GM, 173, 180 Thompson AM, 164, 178 Thompson CH, 55, 59, 151, 157, 191, 196 Thompson GA, 352, 358 Thompson GH, 28, 30, 48, 146, 156 Thompson LP, 132, 138 Thompson PJ, 302, 317 Thompson RS, 288, 295 Thorburn AW, 154, 158 Thorburn GD, 202, 203, 207, 222, 223 Thorburn J, 310, 320 Thornburg KL, 98, 101, 104, 115, 117, 118, 120, 121, 124, 125, 127, 128, 129, 133, 134, 135, 136, 137, 138 Thorpe-Beeston JG, 187, 195 Thorstensen EB, 185, 193 Thurlbeck WM, 298, 316 Tikerpae J, 81, 84, 92, 173, 179, 218, 227, 343, 354 Tinker D, 133, 139 Tobe K, 149, 157 Todd JA, 150, 157 Toelupe P, 327, 338 Tokunaga H, 351, 357 Tokuyama H, 313, 321 Tokuyama Y, 313, 321 Tomanek RJ, 130, 138 Tonge HM, 264, 271 Tonkiss J, 276, 292 Tosteson TD, 305, 318 Townsend SF, 191, 196 Towstoless MK, 214, 215, 225, 288, 295 Traianedes K, 173, 180

384 Trautman PD, 234, 239 Treiman LJ, 275, 291 Trevino RT, 76, 90 Trichopoulos D, 16, 22, 236, 240 Trudinger BJ, 288, 295 Truelson T, 61, 69 Trzcinska M, 276, 292 Tseng YT, 215, 226 Tsiounis M, 262, 270 Tsounis M, 281, 282, 294 Tsurumi Y, 260, 269 Tucker JS, 305, 318 Tucker MA, 215, 226 Tuomilehto J, 5, 11, 12, 18, 19, 32, 48, 170, 178, 254, 255, 267, 324, 337 Turley K, 125, 137 Turner RC, 148, 156 Turtinen J, 32, 48 Tyring SK, 308, 319 Uchiyama M, 26, 30, 47 Udry JR, 326, 338 Ueland PM, 349, 356 Uhari M, 32, 48 Uhlig PN, 125, 137 Ui M, 174, 180 Uiterwaal CS, 32, 37, 45 Ullyot DJ, 125, 137 Underwood LE, 348, 356 Uno H, 214, 225 Vaag AA, 150, 157 Vagero D, 5, 8, 10, 18, 20, 41, 47, 87, 94, 170, 178 Vaidya U, 155, 159, 162, 178 Valdez R, 28, 30, 48, 146, 156, 352, 358 Vale W, 231, 237 Valiante NM, 308, 319 Van Assche FA, 76, 78, 86, 90, 91, 94, 213, 224, 257, 268, 278, 279, 293, 341, 343, 349, 352, 353, 354, 355, 356, 357, 358

Author Index Van Brec R, 278, 293, 341, 354 Van den Berghe G, 242, 243, 247 Van der Meulen JHP, 11, 14, 16, 20, 88, 94, 151, 157, 162, 177, 209, 224, 233, 239, 261, 269, 352, 358 van Doesburg NH, 54, 58 Van Erum R, 244, 247 van Eyck J, 264, 271 van Helvoirt M, 242, 243, 244, 247, 248 Van Veltzen D, 66, 70, 85, 94, 218, 227 Vandeputte M, 341, 354 Vanderschueren D, 244, 246, 248 Varas C, 53, 58 Vassella CC, 312, 320 Veldhuis JD, 187, 194, 219, 228 Venables PH, 15, 21 Ventura SJ, 66, 70 Verdecchia P, 276, 292 Verhaeghe J, 278, 293, 341, 353, 354, 358 Verrier ED, 125, 137 Vestbo E, 28, 31, 48 Vhave S, 352, 358 Viau V, 231, 238 Villar J, 253, 267 Vincent D, 313, 321 Vine N, 52, 57 Vinni S, 32, 48 Viragh S, 104, 134 Vis HL, 312, 320 Visser GHA, 214, 225 Vlahakes GJ, 125, 137 Vollset SE, 349, 356 Voors W, 53, 58 Vosters R, 118, 136 Wada N, 209, 224 Waddell B, 152, 158, 263, 270 Waddell BJ, 219, 228, 278, 293 Wade L, 232, 238 Wadsworth M, 236, 240, 246, 248

Author Index Wadsworth MEJ, 13, 21, 27, 28, 32, 33, 34, 44, 64, 70 Wahlqvist ML, 51, 57 Wahn V, 310, 320 Waldo KL, 103, 133 Walker AM, 106, 108, 121, 125, 134 Walker ARP, 335, 338 Walker BR, 27, 30, 48, 55, 59, 152, 158, 200, 221, 236, 240 Walker DW, 219, 228 Walker M, 27, 30, 32, 37, 42, 44, 45, 46, 47, 155, 159 Walker SK, 252, 266 Wallace LR, 282, 294 Walton A, 250, 266 Walton PE, 208, 223 Walton RJ, 155, 159, 162, 178 Wamachi A, 302, 317 Wang CL, 84, 93, 170, 171, 173, 174, 179, 180, 219, 228, 343, 354 Wang H, 351, 357 Wang SI, 232, 238 Wang ZM, 215, 226 Want CL, 81, 84, 92 Warburtonn C, 186, 194 Wardwell K, 312, 320 Warkany J, 303, 317 Warner JA, 301, 302, 307, 310, 311, 317, 319 Warner JO, 301, 302, 304, 309, 310, 317, 319 Waschek JA, 215, 226 Waterland RA, 274, 291 Waterlow JC, 153, 158, 189, 196, 255, 267, 279, 293 Watson JM, 162, 178 Watt G, 27, 30, 48 Watt GCM, 27, 48 Wattigney W, 26, 30, 47 Webb DJ, 27, 48, 55, 59 Webber LS, 53, 58 Wedell A, 234, 239 Wegmann T, 300, 308, 316 Weibel ER, 108, 134

385 Weicha JL, 324, 337 Weinberger DR, 15, 21 Weiner CP, 132, 138 Weisel TN, 275, 291 Weiss NS, 26, 30, 36, 45 Weiss ST, 305, 318 Weissman B, 39, 46 Welham SJM, 170, 179 Wells SM, 133, 139 Werner JC, 183, 193 Wert SE, 299, 300, 316 West CR, 40, 41, 46 West P, 27, 30, 48 Westworth RA, 214, 225 Wharwood CB, 200, 221 Wheeler FC, 63, 69 Whelton PK, 63, 68, 69, 70, 71 Whie SE, 200, 202, 204, 221 Whincup P, 8, 19, 27, 30, 32, 37, 46 Whincup PH, 26, 27, 29, 30, 37, 38, 41, 42, 44, 45, 47, 155, 159, 250, 261, 266, 269 Whisnant SJ, 61, 69 White E, 16, 22 White GA, 75, 89 White SE, 125, 137 Whitsett JA, 299, 300, 316 Whittle JC, 63, 69 Whorwood CB, 152, 155, 158 Widdicombe JG, 110, 135 Widdowson EM, 2, 18, 241, 247, 261, 269, 275, 281, 291, 294 Wiebers DO, 61, 69 Wiegers GJ, 231, 237 Wield GA, 5, 13, 14, 18, 21 Wieselgren IM, 15, 21 Wigglesworth JS, 279, 293 Wijngaard JAGW, 264, 271 Wilder RL, 309, 319 Wilks RJ, 10, 20, 27, 48, 143, 156 Willett WC, 5, 8, 19, 28, 29, 31, 39, 46, 74, 87, 89, 155, 159 Williams KA, 308, 319 Williams MA, 16, 22

386 Williams MC, 303, 317 Williams S, 26, 39, 46, 47 Williams TJ, 301, 317 Wills J, 162, 178 Wilman C, 258, 268 Wilson JCG, 303, 317 Wilson MR, 170, 172, 179 Wimeon D, 27, 48 Wincup P, 42, 47 Winick M, 75, 89 Winston CC, 75, 89 Winter PD, 4, 8, 13, 16, 18, 19, 21, 22, 170, 178, 236, 240, 324, 337 Wintour EM, 76, 90, 203, 214, 215, 222, 225, 231, 238, 288, 295 Wintour M, 214, 215, 218, 225 Withers DJ, 174, 180 Witzenbichler B, 260, 269 Wladimiroff JW, 118, 136, 264, 271 Woelk G, 26, 30, 36, 45 Wolf PA, 61, 69 Wolf SA, 304, 318 Wong E, 324, 337 Wood CE, 215, 218, 226, 227 Wood PJ, 152, 155, 158, 200, 221 Woodall SM, 78, 91, 188, 191, 192, 195, 196, 197, 276, 292 Woods KA, 149, 157, 186, 194 Woods LL, 131, 138, 219, 227 Wray A, 187, 194 Wright AL, 306, 307, 318 Wright S, 313, 321 Wu D, 124, 136 Wu DE, 127, 129, 138 Wu JJ, 288, 295 Wu JN, 351, 357 Wultan HY, 16, 22 Wyatt JF, 125, 137

Author Index Xu H, 309, 319 Xue S, 63, 69 Yagi T, 149, 157 Yajnik CS, 155, 159, 162, 178, 261, 262, 269, 352, 358 Yamori Y, 351, 357 Yang K, 202, 218, 222, 227, 235, 239 Yarnell J, 5, 8, 19, 37, 45 Yeung CY, 290, 295 Yokoyama H, 68, 71 Yoneda Y, 351, 357 Yoshigi M, 103, 133 Young IR, 207, 223 Young RP, 310, 320 Yu Y, 125, 130, 137 Yuan H, 236, 240 Yudkin JS, 56, 59 Yue X, 130, 138

Zachmann M, 244, 248 Zak R, 107, 134 Zammit PS, 98, 133 Zehnder T, 215, 226 Zeidler A, 352, 358 Zeman J, 85, 94 Zhai F, 334, 338 Zhang DY, 288, 295 Zhao S, 106, 134 Zhu BQ, 260, 269 Zimmet PZ, 177, 180, 327, 338 Zinner SH, 38, 46 Zohoori N, 334, 338 Zureik M, 26, 27, 47

SUBJECT INDEX

ACTH, 202 Acute phase response, maternal protein deprivation and, 171– 172 Adenosine, 202 Adipose tissue, maternal protein deprivation and, 174–175 Adolescents, obesity, 325–326, 327 Adrenalin, 202 Adrenal zone, fetal and, 244 Adrenarche, 244 Adults arterial set point prenatal alteration, 133 body weight early nutrition, 153–154 environmental influences, 154– 155 Afterload, 118 fetal cardiac output, 114–115 Aged low-protein diet pancreas, 346–347 Aging, aorta and, 52–53

Allergic phenomenon, pregnancy and, 308–309 Allergic sensitization, 307 Allopregnanolone, nervous system and, 232–233 Alveoli, embryology and, 298 Ambient temperature, 175 Amino acids, 184, 212, 352 maternofetal exchange, 256–257 metabolism maternal undernutrition, 184– 185 Androgen insensitivity, 245 Angiotensin-converting enzyme rat low-protein diet, 276–277 Aorta aging effects, 52–53 structure, 50–54 Aortic compliance, molecular basis and, 51–52 Appropriate-for-gestational (AGA), adrenarche and, 244 Arginine, 260–261 Arterial compliance, 53

387

388 Arterial pressure fetal, 112–113 mean, 115 sheep, 120 Arterial set point adult prenatal alteration, 133 Arteries, structure and, 50–54 Asia, obesity and, 335–336 Asthma birth head circumference, 314, 314 perinatal factors, 315 Atopy fetal influences, 309–310 genetic diversity, 309–310 maternal, 309–311 maternal nutrition, 313–314 ATPase, 115 β-cell, pancreatic and, 148 Beta-cell mass low-protein diet, 347–349 stress, 352 Birth rate low race, 66 Birth weight, 24–33 blood pressure age factors, 37–38 diabetic ESRD, 68 diastolic blood pressure, 35–36 hypertension, 38–39 impaired glucose tolerance, 144 insulin resistance, 147 insulin-resistance syndrome, 145–146 low developing world, 328 HPA, 235 lung function, 305 renal disease, 61–68 western countries, 87 NIDDM, 142–145

Subject Index [Birth weight] systolic blood pressure, 33–35, 42–43 socioeconomics, 41 Blood pressure abnormal births, 39–40 aging effects, 52–53 aorta, 50–54 birth weight age factors, 37–38 capillary density, 55–56 diastolic birth weight, 35–36 studies, 30–31 embryonic heart, 103–104 glucocorticoids, 214 maternal nutrition, 10–11, 41–43 maternal protein deprivation, 170–171 microvascular dilation, 55–56 rats intrauterine nutrition, 80–91 low-protein diet, 276–277 refeeding, 191 renal size and function, 56–57 systolic birth weight, 33–35, 42–43 socioeconomics, 41 concurrent body size, 36–37 studies, 26–29, 32 in utero programming, 49–57 vascular elastogenesis regulation, 53–54 Body proportions neonatal cardiovascular disease, 5–6 Body size neonatal cardiovascular disease, 4–5 Body weight adult early nutrition, 153–154 11βOHSD, 235 Bone mass, 14–15

Subject Index Brain intrauterine nutrition, 85 low-protein diet, 349 Breast cancer, 16 Bronchiectasis, congenital lung malformations and, 304 Bronchitis, malnourished infants and, 12–13 Capillaries density, 55–56 luminal area sheep, 125 Carbenoxolone, 278 Carbohydrates, 212 maternal dietary intake, 253, 259 metabolism refeeding, 190–191 Carbon dioxide, uteroplacental circulation and, 260 Cardiac capillary bed, prenatal development and, 125 Cardiac function curve, 115, 116 Cardiac jelly, 99 Cardiac muscles force-velocity relationship, 114 length-tension relationship, 114 Cardiac output, 114 fetal, 113–118 afterload, 114–115 contractility, 115 preload, 114 regulation, 115–118 stroke volume determinants, 114 vocabulary, 113–114 hypoxemia, 124–125 Cardiomegaly, 132 Cardiomyocytes, maturation and, 104–108 Cardiovascular disease, geographic variation and, 61–62 infant growth, 6–8 neonatal body proportions, 5–6 neonatal body size, 4–5

389 Cardiovascular extracellular matrix, inadequate composition and, 132–133 Cardiovascular homeostasis maternal undernutrition guinea pigs, 281 rats, 276–278 sheep, 286–289 Cardiovascular mortality, 2 Cardiovascular system development, 75–81 adult disease, 131–133 cardiac output, 124–125 cardiomyocyte maturation, 104–108 fetal arterial pressure, 112–113 fetal cardiac output, 113–118 fetal circulation, 108–110 fetal coronary flow, 125–130 fetal metabolism, 110–112 heart, 98–104 mechanical stress, 121–124 rats, 77–80 sheep, 75–77 ventricles, 118–121 glucocorticoids, 216–217 Cat, embryonic heart and, 105 Catecholamines, fetal growth and development and, 205, 208– 209 Central nervous system, glucocorti coids and, 214 Chicken, embryonic heart and, 106 China fat (dietary), 333 televisions, 331 China Health and Nutrition Surveys (CHNS), 328–331 Chinese, physical activity and, 328–330 Cholesterol, malnourished infants and, 9–10 Chronic obstructive pulmonary disease malnourished infants, 12–14 perinatal factors, 315

390 Circulation fetal, 108–110 umbilical, 110 Concentric hypertrophy, pressure overload and, 121 Congenital cystic adenomatoid malformation, 303–304 Congenital lung malformations, 302–304 bronchiectasis, 304 vitamin A, 303 wheezing, 304 Contractility, 118 fetal cardiac output, 115 Coronary flow, 127–130 fetal regulation, 125–130 vs. adult, 125–126 Coronary flow reserve, 126–127 Coronary heart disease death rates, 4, 11 hazard ratios, 12 infant growth, 7–8 malnourished infants mortality, 5–6 Cortisol, 204, 209–211 HPA, 235–236 C-reactive protein, 172 Critical window, 241 Cystic adenomatoid malformation, congenital and, 303–304 Degenerative disease, epidemio logical studies and, 88 Dehydroepiandrosterone sulfate (DHEAS), 244 Depression, 15–16 Developing countries malnutrition, 87 nutrition transition, 327–336 obesity, 334–336 Dexamethasone, 214 kidneys, 218 pregnancy, 234, 263

Subject Index Diabetes gestational, 257 maternal pancreas, 341, 352–353 vs. dietary restriction, 85–86 Diabetic end-stage renal disease (ESRD), birth weight and, 68 Diastolic blood pressure birth weight, 35–36 studies, 30–31 ventricular end, 115 Diet changing structure, 330–334 maternal malnourished infants, 10–12 restriction vs. maternal diabetes, 85–86 Ductus arteriosus, 110 Ductus venosus, 110 Dust mite, 307 Dyslipidemia, pediatric endocrino pathies and, 246 Early growth retardation maternal protein deprivation, 162–175 metabolic alterations, 161–177 Early nutrition, adult body weight and, 153–154 Eccentric hypertrophy, volume overload and, 121 Ejection fraction, 118 Elastin, 50–51 Elderly low-protein diet pancreas, 346–347 Embryonic heart, 105–106 blood pressure, 103–104 cat, 105 chicken, 106 physiological development, 98–104 sheep, 107–108 Endocardial cushions, 102

Subject Index End-stage renal disease (ESRD) diabetic birth weight, 68 South Carolina, 62–63, 65–68 Energy expenditure, developing world and, 328–329 Excitation-contraction apparatus, maturation rate and, 106 Extracellular matrix cardiovascular inadequate composition, 132– 133 Fat (dietary) China, 333 developing world, 331–332 Fatty acids, atopy and, 313 Fetal origins hypothesis, nutrition transition and, 323–336 Fetal programming, 2 behavioral pathways, 40–41 blood pressure, 49–57 framework, 262 guinea pig, 280–282 maternal undernutrition, 280– 282 placental restriction, 282 maternal and fetal influences, 273–275 prevention, 350–353 rat maternal undernutrition, 276– 280 placental restriction, 279–280 sheep, 282–289 maternal undernutrition, 282– 286 placental restriction, 286–289 socioeconomic pathways, 40–41 Fetus adrenal zone, 244 arterial pressure, 112–113 body composition maternal nutrition, 261–263 cardiac output, 113–118 afterload, 114–115 contractility, 115

391 [Fetus] preload, 114 stroke volume determinants, 114 vocabulary, 113–114 cardiovascular adaptations maternal nutrition, 264 circulation, 108–110 coronary flow regulation, 125–130 cortisol, 235–236 endocrine status maternal nutrition, 263–264 growth and development endocrine regulation, 204–213 HPA, 235–236 hypoxemia, 187–188 islets taurine, 82–83 metabolism, 110–112 nutrient demand, 251–252 nutrition fetal growth, 182–185 undernutrition cardiovascular adaptations, 187–188 endocrine adaptations, 187 insult timing, 188–189 nutrient balance, 189 prematurity, 190 previous generations experi ence, 189–190 Fibrinogen, 172 malnourished infants, 9–10 Filling pressure, 118 Follicle stimulating hormone (FSH), 16–17 Foramen ovale, 110 Forced vital capacity (FVC), malnourished infants and, 13 Fruit, atopy and, 313 Genetic diversity, atopy and, 309– 310 Gestational diabetes, 257 Glucagon, 202

392 Glucocorticoid-induced hyperten sion, 215–218 Glucocorticoids blood pressure, 214 cardiovascular system, 216–217 central nervous system, 214 fetal growth and development, 205, 209–212 heart, 215 kidneys, 218–219 metabolic function, 219–220 pregnancy, 233–234 respiratory distress syndrome, 234 Glucose, 184, 352 maternofetal exchange, 256–258 Glucose tolerance birthweight, 144 maternal protein deprivation, 171 maternal undernutrition rats, 278 GLUT 1, 219 GLUT 4, 174, 219 Gonadal steroids, HPG and, 236 Growth childhood malnourished infants, 12 fetal endocrine regulation, 204–213 malnourished infants, 12 myocardial mechanical stress, 121–124 retardation maternal protein deprivation, 162–175 metabolic alterations, 161–177 Growth hormone deficiency (GHD), SGA and, 242–243 Growth hormone (GH), 187 bone density, 14–15 Guinea pigs, 280–282 maternal undernutrition, 280–282 cardiovascular homeostasis, 281 growth, 280–281 metabolic homeostasis, 281–282

Subject Index Heart embryonic (see Embryonic heart) glucocorticoids, 215 Hepatocyte nuclear factor-3β(HNF3β), 299 High blood pressure (see Hyperten sion) High-density lipid (HDL) choles terol, 9 Hippocampus, HPA and, 231–232 Homeostasis cardiovascular (see Cardiovascu lar homeostasis) insulin glucose isocaloric low-protein diet, 83 Homeotic genes, 299 Hormones, 199–220 integrated changes, 212–213 nutritionally induced changes, 200–205 cardiovascular function, 214– 219 long-term consequences, 213– 220 metabolic function, 219–220 Host defense, lung disease and, 300–302 Hox genes, 299 Hydrophilic solutes, maternofetal exchange and, 256–257 Hydroxybuhyrate, 173 Hypertension, 63 birth weight, 38–39 glucocorticoid-induced, 215–218 malnourished infants, 8–9 rats, 277–278 South Carolina, 64 Hypertrophy eccentric volume overload, 121 Hypoglycemia, fetal hormone changes and, 202, 210 Hypothalamic-pituitary-adrenal axis (HPA), 151–152, 186–187 dexamethasone, 214 mothers, 233–236

Subject Index [Hypothalamic-pituitary-adrenal axis (HPA)] programming, 231–236 rat low-protein diet, 277 undernutrition, 76 Hypothalamic-pituitary-gonadal axis (HPG), 236 Hypoxemia cardiac, output, 124–125 fetal, 187–188 fetal hormone changes, 210 IFN-γ, 308–309 IgE, 310–311 IgG antibodies maternal, 312 timing and concentration, 311– 313 IL-2, 308 IL-4, 308 IL-10, 308 IL-13, 308 Immigrants, nutrition transition and, 325–327 Inactivity, developing world and, 328 Infants cardiovascular disease body proportions, 5–6 body size, 4–5 growth cardiovascular disease, 6–8 malnourished (see Malnourished infants) mortality, 2 wheeze, 306–307 Inotropy, 115 Insulin, 352 fetal growth, 204–207 Insulin deficiencies, fetal growth and, 186 Insulin deprivation, maternal protein deprivation, and, 171 Insulin glucose homeostasis, isocaloric low-protein diet and, 83

393 Insulin growth factor 1 (IGF-1), 186–187, 192, 252 fetal growth and development, 205, 207–208, 243, 257, 263 Insulin growth factor 2 (IGF-2), 252 Insulin/insulin-like growth factor (IGF)/growth hormone (GH) axis, 186–187 Insulin-like growth factors (IGFs), 4 Insulin resistance birthweight, 147 fetal growth, 145–146, 148–152 pediatric endocrinopathies, 246 refeeding, 191–192 Insulin-resistance syndrome, birthweight and, 145–146 Insulin secretion, fetal growth and, 148 Insulin-sensitive tissues, intrauterine nutrition, and, 84 Intrauterine nutrition, 73–88 brain, 85 cardiovascular development, 75– 81 rats, 77–80 sheep, 75–77 dietary restriction vs. maternal diabetes, 85–86 endocrine pancreas, 81–84 insulin-sensitive tissues, 84 kidneys, 85 In utero programming (see Fetal programming) Iron deficiency maternal rats, 277 Islets fetal taurine, 82–83 Isocaloric low-protein diet brain, 85 endocrine pancreas, 81–84 insulin glucose homeostasis, 83 kidneys, 85 liver, 84

394 Kidneys blood pressure, 56–57 dexamethasone, 218 glucocorticoids, 218–219 intrauterine nutrition, 85 Lactate, 184 Lamb, fetal circulation and, 109 Latin America, obesity and, 334 Leptin, 212 Liver isocaloric low-protein diet, 84 maternal protein deprivation, 173 Longevity, maternal protein deprivation and, 163–170 Looping, 99 Low birth rate, race and, 66 Low birth weight developing world, 328 HPA, 235 lung function, 305 renal disease, 61–68 western countries, 87 Low-density lipid (LDL) choles terol, 9 Lower respiratory tract infection, 305 Low-protein diet beta-cell mass, 347–349 brain, 349 early growth retardation, 162–175 isocaloric (see Isocaloric lowprotein diet) pancreas, 340–341 long-term consequences, 342– 347 short-term consequences, 341–342 pregnancy pancreas, 344–346 rat blood pressure, 276–277 Lung disease, 297–316 allergic sensitization, 307 congenital, 302–304 host defense, 300–302 infant wheeze, 306–307

Subject Index Lungs embryology, 298–299 function fetal growth, 304–305 low birth weight, 305 growth molecular basis, 299–300 Luteinizing hormone (LH), 16–17 Male pseudohermaphroditism, 244– 245 Male subfertility, 245–246 Malnourished infants, 2–14 cardiovascular disease, 4–8 childhood growth, 12 cholesterol, 9–10 chronic obstructive lung disease, 12–14 fibrinogen, 9–10 hypertension, 8–9 maternal dietary balance, 10–12 NIDDM, 8–9 Malnutrition, developing countries and, 87 Maternal atopy, 310–311 Maternal diabetes pancreas, 341, 352–353 vs. dietary restriction, 85–86 Maternal dietary balance, malnour ished infants and, 10–12 Maternal IgG antibodies, 312 Maternal iron deficiency, rats and, 277 Maternal nutrition, 249–265 blood pressure, 41–43 fetal adaptation, 261–264 fetal body composition, 261– 263 fetal cardiovascular adapta tions, 264 fetal endocrine status, 263–264 fetal nutrient demand, 251–252 future research, 264–265 maternoplacental nutrient supply, 252–261

Subject Index [Maternal nutrition] intergenerational effects, 255– 256 maternal body composition, 254–255 maternal dietary intakes, 253– 254 nutrient availability, 256–257 placental size, 258–260 uteroplacental blood flow, 260–261 pancreas, 339–354 size at birth, 250–251 Maternal protein deprivation. See also Low-protein diet early growth retardation, 162–175 acute phase response, 171–172 adipose tissue, 174–175 blood pressure, 170–171 future research, 176–177 glucose tolerance, 170 insulin sensitivity, 170 liver, 173 longevity, 163–170 muscles, 173–174 pancreas, 172–173 plasma lipids, 171 Maternal undernutrition, 233 amino acid metabolism sheep, 184–185 fetal growth, 182–185 fetal metabolic adaptations, 184– 186 guinea pigs, 280–282 rats, 276–279 sheep, 282–287 Mean arterial pressure, 115 sheep, 120 Mean systemic filling pressure, 113 Mechanical stress, myocardial growth and, 121–124 Metabolic homeostasis maternal undernutrition guinea pigs, 281–282 rats, 278 sheep, 286

395 Metabolic imprinting, 274 Metabolic syndrome, 273 age of onset, 290 Metabolism amino acids maternal undernutrition, 184– 185 carbohydrates, 190–191 early growth retardation, 161–177 fetal, 110–112 protein, 191 Metyrapone, 277 Microvascular dilation, 55–56 Middle East, obesity and, 334–335 Mortality, cardiovascular and, 2 Muscles, maternal protein depriva tion and, 173–174 Myocardial flow, 127–130 fetal regulation, 125–130 total fetal vs. adult, 125–126 Myocardium, 104 growth mechanical stress, 121–124 Myocytes (see Cardiomyocytes) Myofibrils, 104, 105 Myosin isoforms, 115 National Longitudinal Study of Adolescent Health, 325–326 Neonates (see Infants) Nervous system, neurosteroids and, 232–233 Neural crest cells, 102–103 Neurosteroids, nervous system and, 232–233 Non-growth hormone deficiency (GHD)/small for gestational age (SGA) children GH, 243– 244

396 Non-insulin dependent diabetes (NIDDM), 141–152, 257 birthweight, 142–145 insulin resistance, 145–146, 148– 152 insulin secretion, 148 malnourished infants, 8–9 prevalence, 8 Nutrient balance, fetal undernutri tion and, 189 Nutrition early adult body weight, 153–154 fetal fetal growth, 182–185 hormonal changes, 200–204 maternal (see Maternal nutrition) Nutrition transition fetal origins hypothesis, 323–336 developing world, 327–336 immigrants, 325–327 Obesity, 324 adolescents, 325–327 adult fetal growth, 152–153 developing world, 334–336 fetal growth, 152–153 Occupations, developing world and, 328, 329 Organogenesis, 110 Osteoporosis, 14–15 Ovarian cancer, 16 Ovarian hyperandrogenism, 246 Oxidative substrates, late gestation and, 182–183 Oxygen consumption, 110–112 uteroplacental circulation, 260 Pancreas intrauterine nutrition, 81–84 low-protein diet, 81–84, 340–341

Subject Index [Pancreas] long-term consequences, 342– 347 short-term consequences, 341–342 maternal nutrition, 339–354 maternal protein deprivation, 173 programming, 340–341 Pediatric endocrinopathies, 241– 247 adrenarche, 244 dyslipidemia, 246 male pseudohermaphroditism, 244–245 male subfertility, 244–245 ovarian hyperandrogenism, 246 pubarche, 244 somatotropic axis, 241–244 Physical activity Chinese, 328–330 developing world, 328–329 PI 3-kinase, 174 Pituitary hormone, fetal growth and development and, 205 Placenta fetal programming guinea pig, 282 rat, 279–280 sheep, 286–289 HPA, 233–236 nutritional effects, 259–260 sheep cardiovascular homeostasis, 288–289 growth, 286 Placentation, inadequate and, 133– 134 Plasma lipids, maternal protein deprivation and, 171 Pneumonia, malnourished infants and, 13 Polycystic ovary syndrome, 16–17 Ponderal index, 256, 261–262 Precocious pubarche, 244 birth weight, 245 Pregnancy allergic phenomenon, 308–309

Subject Index [Pregnancy] cardiovascular adaptations, 260 dexamethasone, 234, 263 glucocorticoids, 233–234 low-protein diet pancreas, 344–346 stress, 233 Preload, 118 fetal cardiac output, 114 Prematurity, fetal undernutrition and, 190 Pressure overload, concentric hypertrophy and 121 Primordial follicles, 246 Programming, fetal and (see Fetal programming) Protein deprivation (see Maternal protein deprivation) Protein metabolism, refeeding and, 191 Proteins, maternal dietary intake and, 253 Pseudohermaphroditism, male and, 244–245 Pubarche precocious, 244 birth weight, 245 Rats cardiovascular development intrauterine nutrition, 77–80 maternal undernutrition cardiovascular homeostasis, 276–278 metabolic hemostasis, 278 placental restriction, 279–280 transgenerational effects, 279 myocytes, 106 Reduced protein diet (see Lowprotein diet) Refeeding cardiovascular adaptations, 191 endocrine adaptations, 191–192 fetoplacental adaptations, 190–191

397 Renal disease, low birth weight and, 61–68 Renal failure fetal growth, 68 odds ratios, 67 Renin angiotensin system (RAS), 218–219 Respiratory distress syndrome, glucocorticoids and, 234 Respiratory tract infection, lower and, 305 Sarcolemma, 104, 105 Schizophrenia, 15 Septation, 99–100, 102 Septum primum, 101 Septum secundum, 101 Sertoli cells, 246 Sheep amino acid metabolism maternal undernutrition, 184–185 capillary lumenal area, 125 cardiovascular development intrauterine nutrition, 75–77 catecholamines, 208–209 embryonic heart, 107–108 fetal arterial pressure, 112–113 fetal circulation, 109 glucocorticoids, 209–212 hypoglycemia fetal hormone changes, 202, 210 hypoxemia cardiac output, 124–125 IGF-1, 207–208 maternal undernutrition, 282–286 mean arterial pressure, 120 myocardial flow hypoxemia, 130 myocytes, 106 placental restriction, 286–289 thyroid hormones, 207 total myocardial flow fetal vs. adult, 125–126

398 [Sheep] ventricles right vs. left, 118–121, 124 ventricular pressure-volume relationships, 119 Small for gestational age (SGA), incidence and, 242 Smoking, neonatal lung function and, 305 Somatotropic axis, 241–244 South Carolina ESRD, 62–63, 65–68 hypertension, 64 low birth rate race, 66 stroke, 61–63 Streptozotocin-induced diabetes, 86 Stress, pregnancy and, 233 Stroke malnourished infants, 6 mortality rates, 62 South Carolina, 61–63 Stroke volume, 115, 117–118 determinants fetal cardiac output, 114 Subfertility, male and, 245–246 Sub-Saharan Africa, obesity and, 335 Systemic filling pressure, mean and, 113 Systolic blood pressure birth weight, 33–35, 42–43 socioeconomics, 41 concurrent body size, 36–37 studies, 26–29, 32 Taurine, 350–351 fetal islets, 82–83 Televisions, China and, 331 Telomeres, 164 Temperature, ambient and, 175 Testosterone, HPG and, 236 Th-1, 308 Th-2, 302

Subject Index Thrifty phenotype hypothesis, 161– 162, 175, 339, 352 Thyroid hormone, 187 fetal growth and development, 205, 207 Thyroid transcription factor-one (TTF1), 299 Total myocardial flow, fetal vs. adult and, 125–126 T-tubules, 104 Tubular heart, 99 Umbilical circulation, 110 Undernutrition fetal (see Fetus, undernutrition) maternal (see Maternal undernu trition) Variable number of tandem repeats (VNTR), 149 Vascular elastogenesis, regulation and, 53–54 Vascular endothelial growth factor (VEGF), 260 Vascular impedence, 118–119 Vegetables, atopy and, 313 Ventricles, right vs. left and, 118– 121 Ventricular end diastolic pressure, 115 Ventricular pressure-volume relationships, sheep and, 119 Vitamin A, congenital lung malformations and, 303 Volume overload, eccentric hyper trophy and, 121 Wall stress, 114 Western countries, low birth weight and, 87 Wheezing congenital lung malformations, 304 infant, 306–307 perinatal factors, 315