Clinical Reproductive Medicine and Surgery E-Book
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Clinical Reproductive Medicine and Surgery E-Book

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1905 pages
English

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Description

Clinical Reproductive Medicine and Surgery is the new, definitive resource in reproductive medicine. This unique text offers detailed discussion on both the medical and surgical management of reproductive disorders, as well as coverage of associated imaging modalities. Included are chapters on Reproductive Genetics, Management of Endometriosis (including interventional radiology), Ultrasonography and Sonohysterography, Preservation of Fertility, and Recurrent Pregnancy Loss.  A resource every practitioner interested in Reproductive Endocrinology and Infertility needs!
  • Offers detailed discussion of medical and surgical management of reproductive disorders ... No other text offers coverage of both medical and surgical management in one resource.
  • Covers gynecologic disorders that impact fertility--an important aspect of identifying fertility issues, not included in major competition
  • Section on basic reproductive biology ... Not overly detailed -- Written for a clinician to understand how to practice reproductive medicine
  • Section on reproductive imaging ... Unique to this text - includes US and MRI of the reproductive organs
  • Algorithm in each chapter ... 4-color throughout ... Demonstrates the appropriate clinical investigation and management ... Offers attractive layout and best views of surgical procedures

Sujets

Ebooks
Savoirs
Medecine
Septo
Derecho de autor
Lesión
Gametogénesis
Genoma mitocondrial
Vasoepididymostomy
Intrauterine device
Puberty
Endometriosis of ovary
Oncology
Ejaculation
Pregnancy rate
Sexually transmitted disease
Birth control
Acne
Gonorrhea
Surgical suture
Fallopian tube obstruction
Endocrine disease
Medical procedure
Adhesion (medicine)
Reproductive medicine
Bone density
Ovulation induction
Type 1
Pelvic pain
Gynecological surgery
Disease management
Premature ovarian failure
Endometrial ablation
Electrosurgery
Research design
Metrorrhagia
Female infertility
Glutaric aciduria
Male infertility
Hysterosalpingography
Hypothalamic-pituitary-gonadal axis
Blood?testis barrier
Reproductive health
IUD with progestogen
Myoma
Azoospermia
Pregnancy
Antrum
Salpingectomy
Hysteroscopy
Varicocele
Ovarian hyperstimulation syndrome
Embryo transfer
Hyperandrogenism
Habitual abortion
Clomifene
Prolactinoma
Insemination
Hypopituitarism
Hypogonadism
Skin grafting
Gonadotropin
Hot flash
Septa
Precocious puberty
Random sample
Oogenesis
Oocyte
Sterility
Review
Hypercalcaemia
Congenital adrenal hyperplasia
Physician assistant
Dysmenorrhea
Hysterectomy
Cytogenetics
Ovarian cyst
Laparotomy
Steroid hormone
Lesion
Bowel obstruction
Follicle-stimulating hormone
Congenital disorder
Hyperprolactinaemia
Gametogenesis
Fertility
Progestin
Antiphospholipid syndrome
Androgen
Amenorrhoea
Medical imaging
Premenstrual syndrome
Prolactin
Artificial insemination
Ascites
Cyst
Urinary incontinence
Infertility
List of surgical procedures
Bleeding
Miscarriage
Medical ultrasonography
Hirsutism
Cushing's syndrome
Menstrual cycle
Tubal ligation
Laparoscopy
Hypothyroidism
Coeliac disease
In vitro fertilisation
Insulin resistance
Ectopic pregnancy
Polycystic ovary syndrome
Endometriosis
Obstetrics and gynaecology
Philadelphia
Menopause
Turner syndrome
Surgery
Diabetes mellitus
Infection
Urology
Spermatozoon
Pelvic inflammatory disease
Physiology
Pediatrics
Ovary
Estrogen
Osteoporosis
Magnetic resonance imaging
Laparoscopic surgery
General surgery
Genome
Major depressive disorder
Chemotherapy
Chlamydia infection
Carbon dioxide
Contraception
Allele
Anxiety
Abortion
Fractures
Divine Insanity
Spironolactone
Clomiphène
Anovulation
Ménopause
Human
Pathology
Acupuncture
Gene
Septum
Lésion
Varicocèle
Méthotrexate
Pelvis
Lactation
Ovulation
Attitude
Lévonorgestrel
Progestérone
Estradiol
Éjaculation
Acné
Chirurgie
Maladie infectieuse
Philadelphie
Death
Allèle
Contrôle des naissances
Ostéoporose
Copyright
Génome

Informations

Publié par
Date de parution 26 avril 2007
Nombre de lectures 1
EAN13 9780323076593
Langue English
Poids de l'ouvrage 14 Mo

Informations légales : prix de location à la page 0,0614€. Cette information est donnée uniquement à titre indicatif conformément à la législation en vigueur.

Exrait

Clinical Reproductive Medicine and Surgery
First Edition

Tommaso Falcone, MD
Professor and Chairman, Department of Obstetrics and Gynecology, Cleveland Clinic, Cleveland, Ohio

William W. Hurd, MD, MS
Professor of Obstetrics and Gynecology and Community Health, Department of Obstetrics and Gynecology, Wright State University School of Medicine, Dayton, Ohio
MOSBY
Copyright
MOSBY ELSEVIER
1600 John F. Kennedy Blvd.
Ste 1800
Philadelphia, PA 19103-2899
CLINICAL REPRODUCTIVE MEDICINE AND SURGERY
ISBN-13: 978-0-323-03309-1
ISBN-10: 0-323-03309-1
Copyright © 2007 by Mosby, Inc., an affiliate of Elsevier Inc.
All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher.
Permissions may be sought directly from Elsevier’s Health Sciences Rights Department in Philadelphia, PA, USA: phone: (+1) 215 239 3804, fax: (+1) 215 239 3805, e-mail: healthpermissions@elsevier.com . You may also complete your request on-line via the Elsevier homepage ( http://www.elsevier.com ), by selecting “Customer Support” and then “Obtaining Permissions.”


Notice
Knowledge and best practice in this field are constantly changing. As new research and experience broaden our knowledge, changes in practice, treatment and drug therapy may become necessary or appropriate. Readers are advised to check the most current information provided (i) on procedures featured or (ii) by the manufacturer of each product to be administered, to verify the recommended dose or formula, the method and duration of administration, and contraindications. It is the responsibility of the practitioner, relying on his or her own experience and knowledge of the patient, to make diagnoses, to determine dosages and the best treatment for each individual patient, and to take all appropriate safety precautions. To the fullest extent of the law, neither the Publisher nor the Editors/Authors assume any liability for any injury and/or damage to persons or property arising out or related to any use of the material contained in this book.
Library of Congress Cataloging-in-Publication Data
Clinical and reproductive medicine and surgery/[edited by] Tommaso Falcone,
William W. Hurd
p.; cm.
ISBN-10: 0-323-03309-1
ISBN-13: 978-0-323-03309-1
1. Generative organs—Diseases. 2. Generative organs, Female—Diseases. 3. Infertility. 4. Human reproduction. I. Falcone, Tommaso. II. Hurd, William W.
[DNLM: 1. Genital Diseases, Female. 2. Genital Diseases, Female–surgery. 3. Gynecologic Surgical Procedures–methods. 4. Reproductive Medicine–methods. WP 140 C6403 2007]
RC875.C555 2007
616.65—dc22
2006048168
Acquisitions Editor: Rebecca Schmidt Gaertner
Developmental Editors: Jennifer Ehlers and Christine Oberle
Project Manager: Mary B. Stermel
Design Direction: Louis Forgione
Marketing Manager: Matt Latuchie
Printed in Hong Kong
Last digit is the print number: 9 8 7 6 5 4 3 2 1
Contributors

Cynthia Abacan, MD , Fellow, Department of Endocrinology, Diabetes, and Metabolism, Cleveland Clinic, Cleveland, Ohio

Ashok Agarwal, PhD, HCLD , Director, Andrology Laboratory and Reproductive Tissue Bank, Director, Reproductive Research Center, Professor, Cleveland Clinic Lerner College of Medicine, Case Western Reserve University, Glickman Urological Institute and Departments of Obstetrics and Gynecology, Anatomic Pathology, and Immunology, Cleveland Clinic, Cleveland, Ohio

Raedah Al-Fadhli, MD , Fellow, Reproductive Endocrinology and Infertility, McGill University, Montreal, Quebec, Canada

Shyam S.R. Allamaneni, MD , Resident, Department of General Surgery, Saint Vincent Catholic Medical Center, Jamaica, New York

Lawrence S. Amesse, MD, PhD , Department of Obstetrics and Gynecology, Wright State University School of Medicine, Dayton, Ohio

Aydin Arici, MD , Professor, Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, Connecticut

Francisco Arredondo, MD, MPH , Department of Obstetrics and Gynecology, University Hospitals of Cleveland, Cleveland, Ohio

Khalid Ataya, MD , Professor, Department of Obstetrics and Gynecology, MetroHealth Medical Center, Cleveland, Ohio

Marjan Attaran, MD , Head, Section of Pediatric Gynecology, Department of Obstetrics and Gynecology, Cleveland Clinic, Cleveland, Ohio

Cynthia Austin, MD , Department of Obstetrics and Gynecology, Cleveland Clinic, Cleveland, Ohio

Jaswant S. Bal, MD, FRCOG, FACOG , Assistant Clinical Professor, Department of Obstetrics and Gynecology, SUNY Health Science Center, Syracuse, New York

Sheela Barhan, MD , Department of Obstetrics and Gynecology, Wright State University School of Medicine, Dayton, Ohio

Kurt Barnhart, MD, MSCE , Associate Professor of Obstetrics and Gynecology and Epidemiology, Penn Fertility Care, University of Pennsylvania, Philadelphia, Pennsylvania

Kshonija Batchu, MD , Research Assistant, Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, California

Mohamed A. Bedaiwy, MD , Fellow, Department of Obstetrics and Gynecology, Cleveland Clinic, Cleveland, Ohio

Sarah L. Berga, MD , Professor and Chair, Department of Obstetrics and Gynecology, Emory University School of Medicine, Atlanta, Georgia

Charles V. Biscotti, MD , Department of Anatomic Pathology, Cleveland Clinic, Cleveland, Ohio

Linda D. Bradley, MD , Department of Obstetrics and Gynecology, Cleveland Clinic, Cleveland, Ohio

Ronald T. Burkman, MD , Chairman, Department of Obstetrics and Gynecology, Baystate Medical Center, Springfield, Massachusetts, Deputy Chair and Professor, Department of Obstetrics and Gynecology, Tufts University School of Medicine, Boston, Massachusetts

John Carey, MD , Department of Rheumatic and Immunologic Disease, Cleveland Clinic, Cleveland, Ohio

Allison M. Case, MD , Department of Obstetrics and Gynecology, Royal University Hospital, Saskatoon, Saskatchewan, Canada

Robert F. Casper, MD , Professor, Toronto Center for A.R.T., Toronto, Ontario, Canada

SuYnn Chia, MD , Department of Endocrinology, Cleveland Clinic, Cleveland, Ohio

Gregory M. Christman, MD , Associate Professor of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, University of Michigan Medical School, Associate Professor, Reproductive Sciences Program, Department of Obstetrics and Gynecology, University of Michigan Health System, Ann Arbor, Michigan

Brian Clark, MD, PhD , Professor, Department of Obstetrics and Gynecology, Magee-Womens Hospital, Center for Medical Genetics, Pittsburgh, Pennsylvania

Damon Davis, MD , Resident, Department of Urology, University of Michigan Medical School, Ann Arbor, Michigan

Miriam Delaney, MD , Department of Rheumatic and Immunologic Disease, Cleveland Clinic, Cleveland, Ohio

Nina Desai, PhD , Assistant Professor, Director, In Vitro Fertilization Laboratories, Department of Obstetrics and Gynecology, Cleveland Clinic, Cleveland, Ohio

Anne S. Devi Wold, MD , Reproductive Research Center, Lexington, Massachusetts

Michael P. Diamond, MD , Associate Chair and Kamran S. Moghissi, Professor of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Wayne State University, Detroit, Michigan

Richard L. Drake, PhD , Professor, Department of Education, Cleveland Clinic, Cleveland, Ohio

Janice Duke, MD , Department of Obstetrics and Gynecology, Wright State University School of Medicine, Dayton, Ohio

Kristin A. Englund, MD , Department of Infectious Disease, Cleveland Clinic, Cleveland, Ohio

Navid Esfandiari, DVM, PhD, ELD, HCLD , Director, IVF, Andrology and Research Laboratories, Toronto Center for A.R.T., Toronto, Ontario, Canada

Charles Faiman, MD , Department of Endocrinology, Cleveland Clinic, Cleveland, Ohio

Tommaso Falcone, MD , Professor and Chairman, Department of Obstetrics and Gynecology, Cleveland Clinic, Cleveland, Ohio

Stephanie Fisher, MD, FRCS(C) , Assistant Professor Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, British Columbia, Canada

Maria Fleseriu, MD , Assistant Professor, Division of Endocrinology, Diabetes, and Clinical Nutrition, Oregon Health and Sciences University, Portland, Oregon

Margo Fluker, MD, FRCS(C) , Co-Director, Genesis Fertility Center, Clinical Professor Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, British Columbia, Canada

Gita Gidwani, MD , Department of Obstetrics and Gynecology, Cleveland Clinic, Cleveland, Ohio

Jeffrey M. Goldberg, MD , Department of Obstetrics and Gynecology, Cleveland Clinic, Cleveland, Ohio

James Goldfarb, MD, MBA , Clinical Professor, Beachwood Family Health Center, Cleveland Clinic, Beachwood, Ohio

Dorothy Greenfeld, MSW, LCSW , Associate Professor, Department of Obstetrics, Gynecology, and Reproductive Sciences, Yale University Fertility Center, Yale University School of Medicine, New Haven, Connecticut

Manjula K. Gupta, PhD , Department of Clinical Pathology, Cleveland Clinic, Cleveland, Ohio

Robert Hemmings, MD , OVO Clinic, Montreal, Quebec, Canada

Melissa Hiner, BS , Embryologist, Department of Obstetrics and Gynecology, University of Michigan Medical School, Ann Arbor, Michigan

Gary M. Horowitz, MD , Associate Professor, Department of Obstetrics and Gynecology, Southern Illinois School of Medicine, Springfield, Illinois

Elizabeth M. Hurd, RN, BSN , Lactation Consultant, Miami Valley Hospital, Dayton, Ohio

William W. Hurd, MD, MS , Professor of Obstetrics and Gynecology and Community Health, Department of Obstetrics and Gynecology, Wright State University School of Medicine, Dayton, Ohio

Shahryar K. Kavoussi, MD, MPH , Fellow, Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, University of Michigan Health System, Ann Arbor, Michigan

Elizabeth Ann Kennard, MD , Ohio Reproductive Medicine, Columbus, Ohio

Harry J. Khamis, PhD , Director and Professor, Statistical Consulting Center, Wright State University Dayton, Ohio

Peter N. Kolettis, MD , Division of Urology, University of Alabama at Birmingham, Birmingham, Alabama

Layne Kumetz, MD , House Officer, Department of Obstetrics and Gynecology, Cedars-Sinai Medical Center, Los Angeles, California

William H. Kutteh, MD, PhD, HCLD , Division of Reproductive Endocrinology, University of Tennessee, Memphis, Tennessee

Steven R. Lindheim, MD , Department of Obstetrics and Gynecology, University of Wisconsin, Madison, Wisconsin

Hanna Lisbona, MD , Department of Obstetrics and Gynecology, Cleveland Clinic, Cleveland, Ohio

James H. Liu, MD , Arthur H. Bill Professor and Chair, Department of Obstetrics and Gynecology, University Hospitals/MacDonald Women’s Hospital, Department of Reproductive Biology, Case School of Medicine, Cleveland, Ohio

J. Ricardo Loret de Mola, MD , Department of Obstetrics and Gynecology, University Hospitals of Cleveland, Cleveland, Ohio

Tammy L. Loucks, MPH , Director of Clinical Research, Department of Obstetrics and Gynecology, Emory University School of Medicine, Atlanta, Georgia

Andrea Magen, MD , Department of Radiology, Cleveland Clinic, Cleveland, Ohio

Neal Gregory Mahutte, MD , Assistant Professor, Reproductive Endocrinology and Infertility, Dartmouth Medical School, Lebanon, New Hampshire

Beth A. Malizia, MD , Fellow, Reproductive Endocrinology and Fertility, Beth Israel Deaconess Medical Center Boston, Massachusetts

Mohamed F. Mitwally, MD , Clinical Assistant Professor, Department of Obstetrics and Gynecology, Wayne State University, Detroit, Michigan

Dana A. Ohl, MD , Professor of Urology, Department of Urology, Head, Division of Andrology and Microsurgery, University of Michigan, Ann Arbor, Michigan

Sophia Ouhilal, MD , Assistant Professor, Reproductive Endocrinology and Infertility, Dartmouth Medical School, Lebanon, New Hampshire

Kelly Pagidas, MD , Department of Reproductive Medicine Women and Infants Hospital Providence, Rhode Island

John K. Park, MD , Division of Reproductive Endocrinology and Fertility, Department of Obstetrics and Gynecology, Emory University School of Medicine, Atlanta, Georgia

Pasquale Patrizio, MD, MBe , Yale Fertility Center, Yale University, New Haven, Connecticut

Teresa Pfaff-Amesse, MD , Assistant Professor, Departments of Pathology and Neuroscience, Cell Biology, and Physiology, Wright State University School of Medicine, Dayton, Ohio

Barry Peskin, MD , Department of Obstetrics and Gynecology, Cleveland Clinic, Cleveland, Ohio

Susanne A. Quallich, NP , Nurse Practitioner, Division of Andrology and Microsurgery, Department of Urology, University of Michigan, Ann Arbor, Michigan

S. Sethu K. Reddy, MD , Department of Endocrinology, Diabetes, and Metabolism, Cleveland Clinic, Cleveland, Ohio

Robert L. Reid, MD , Department of Obstetrics and Gynecology, Queen’s University, Kingston General Hospital, Kingston, Ontario, Canada

Ellen S. Rome, MD , Department of Pediatric and Adolescent Medicine, Cleveland Clinic, Cleveland, Ohio

Jonathan Ross, MD , Glickman Urological Institute, Cleveland Clinic, Cleveland, Ohio

Joseph S. Sanfilippo, MD, MBA , Professor, Obstetrics-Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Vice Chairman, Reproductive Sciences, Division Director, Reproductive Endocrinology, Residency Program Director, Magee-Womens Hospital, Pittsburgh, Pennsylvania

Erin J. Saunders, MD , Clinical Professor Department of Obstetrics and Gynecology, Vanderbilt University, Nashville, Tennessee

Timothy G. Schuster, MD , Assistant Professor, Department of Urology, Division of Andrology and Microsurgery, University of Michigan, Ann Arbor, Michigan

Beata Seeber, MD , Department of Obstetrics and Gynecology, University of Pennsylvania, Philadelphia, Pennsylvania, Department of Gynecologic Endocrinology and Reproductive Medicine, Medical University of Innsbruck, Innsbruck, Austria

Rakesh K. Sharma, PhD , Center for Advanced Research in Human Reproduction, Infertility, and Sexual Function Glickman Urological Institute, Department of Obstetrics and Gynecology Cleveland Clinic Cleveland, Ohio

Howard T. Sharp, MD , Department of Obstetrics and Gynecology, University of Utah Medical Center, Salt Lake City, Utah

Cristine Silva, BS , Embryologist, Department of Obstetrics and Gynecology, University of Michigan Medical School, Ann Arbor, Michigan

Gary D. Smith, MD , Department of Obstetrics and Gynecology, University of Michigan Medical School, Ann Arbor, Michigan

Jonathon M. Solnik, MD , Director, Minimally Invasive Gynecologic Surgery, Department of Obstetrics and Gynecology, Cedars-Sinai Medical Center, Assistant Professor, Department of Obstetrics and Gynecology, The David Geffen School of Medicine at UCLA, Los Angeles, California

Michael P. Steinkampf, MD , Director, Alabama Fertility Specialists, Birmingham, Alabama

Thomas G. Stovall, MD , Women’s Health Specialists, Germantown, Tennessee

Holly L. Thacker, MD, FACP , Director, Women’s Health Center, Departments of Internal Medicine and Obstetrics and Gynecology, Cleveland Clinic, Cleveland, Ohio

Geoffrey D. Towers, MD , Assistant Professor, Department of Obstetrics and Gynecology, Wright State University, Dayton, Ohio

Togas Tulandi, MD, MHCM , Professor and Chief of Obstetrics and Gynecology, JGH, Department of Obstetrics and Gynecology, Milton Leong Chair in Reproductive Medicine, McGill University, Montreal, Quebec, Canada

Meike L. Uhler, MD , Clinical Associate Professor, Department of Obstetrics and Gynecology, Loyola University School of Medicine, Maywood, Illinois, Fertility Centers of Illinois, Chicago, Illinois

Joseph C. Veniero, MD , Department of Radiology, Cleveland Clinic, Cleveland, Ohio

Gary Ventolini, MD , Associate Professor, Department of Obstetrics and Gynecology, Wright State University, Dayton, Ohio

James L. Whiteside, MD , Department of Obstetrics and Gynecology, Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire

Mylene W.M. Yao, MD , Assistant Professor, Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, California
Preface
Reproductive medicine and surgery have together evolved into a distinctive field of modern gynecology. The focus of this field is on the identification and restoration of medical and anatomic abnormalities affecting the human reproductive system. Replacement therapy and more extensive surgery are now used only when required, since natural reproductive function is often more effective and safer than the best artificial replacement techniques.
The knowledge gained in reproductive sciences over the last half of the 20th century is truly remarkable, and encyclopedic volumes have been written as a result. These distinguished works, however, deal primarily with the medical aspects of reproduction, and little has been written about the surgical aspects. This book is unique in that it includes both medical and surgical treatments, an approach we believe to be more reflective of the clinical practice of reproductive medicine.
When compiling this text, we focused on two goals. The first was to provide a comprehensive review of all facets of clinical reproductive medicine and surgery. This review encompasses basic science and pathophysiology, clinical diagnosis and imaging, and medical and surgical treatment approaches. The second, and perhaps more challenging goal, was to create a book designed to be read rather than a reference work to be reserved for consultation. To this end, we diligently organized information provided by many experts into 53 understandable and consistent chapters. We hope this book serves as a concise knowledge base for those in training and in the early years of practice, especially those planning to take comprehensive examinations on this information.
With these goals in mind, we divided the book into seven parts. The first deals with basic science, covering the classic topics of neurophysiology, gametogenesis, fertilization and genetics, as well as anatomy, histology, statistics, and bioethics. The clinical medicine section has three parts that cover pediatric, adolescent and adult clinical reproductive medicine and infertility for both men and women. There are separate sections that cover the broad areas of female contraception and imaging. The final section deals with reproductive surgery performed to maintain fertility. Since visualization is an important part of learning surgery, a number of instructive video presentations on compact disk are included to reinforce the text.
We hope that this book weaves together the elements of basic, clinical, and surgical science in such a way that the reader emerges with both a broad understanding of the basic science of reproductive medicine and a comprehensive approach to the reproductive patient.

TOMMASO. FALCONE, BILL. HURD
Table of Contents
Copyright
Contributors
Preface
Section 1: Basic Science
Chapter 1: The Hypothalamic-Pituitary-Ovarian Axis and Control of the Menstrual Cycle
Chapter 2: Ovarian Hormones: Structure, Biosynthesis, Function, Mechanism of Action, and Laboratory Diagnosis
Chapter 3: Oogenesis
Chapter 4: Physiology of Male Gametogenesis
Chapter 5: Reproductive Genetics
Chapter 6: Normal Fertilization and Implantation
Chapter 7: Surgical Anatomy of the Abdomen and Pelvis
Chapter 8: Pathology of Reproductive Endocrine Disorders
Chapter 9: Statistics for the Clinical Scientist
Chapter 10: Ethics of Reproduction
Section 2: Pediatric and Adolescent Disorders
Chapter 11: Normal Puberty and Pubertal Disorders
Chapter 12: Congenital Anomalies of the Female Reproductive Tract
Chapter 13: Pediatric Gynecology
Chapter 14: Reproductive Disorders in the Adolescent Patient
Section 3: Adult Reproductive Endocrinology
Chapter 15: Polycystic Ovary Syndrome
Chapter 16: Amenorrhea
Chapter 17: Lactation and Galactorrhea
Chapter 18: Hirsutism
Chapter 19: Anovulation and Ovulatory Dysfunction
Chapter 20: Premature Ovarian Failure
Chapter 21: Abnormal Uterine Bleeding
Chapter 22: Management of Pituitary, Adrenal, and Thyroid Disease
Chapter 23: Premenstrual Syndrome and Menstrual-Related Disorders
Chapter 24: Menopause
Chapter 25: Osteoporosis
Section 4: Contraception
Chapter 26: Hormonal Contraception
Chapter 27: Modern Concepts in Intrauterine Devices
Chapter 28: Surgical Sterilization
Section 5: Reproductive Imaging
Chapter 29: Hysterosalpingography
Chapter 30: Pelvic Ultrasonography and Sonohysterography
Chapter 31: Magnetic Resonance Imaging
Section 6: Infertility and Recurrent Pregnancy Loss
Chapter 32: Fertility Preservation in Cancer Patients
Chapter 33: Infections and Infertility
Chapter 34: Female Infertility
Chapter 35: Evaluation of Male Infertility
Chapter 36: Artificial Insemination
Chapter 37: Induction of Ovulation
Chapter 38: Assisted Reproductive Technology: Clinical Aspects
Chapter 39: Assisted Reproductive Technology: Laboratory Aspects
Chapter 40: Complications of Assisted Reproductive Technologies
Chapter 41: Recurrent Pregnancy Loss
Section 7: Reproductive Surgery
Chapter 42: Diagnostic and Operative Hysteroscopy: Polypectomy, Myomectomy, and Endometrial Ablation
Chapter 43: Hysteroscopic Management of Intrauterine Adhesions and Uterine Septa
Chapter 44: Gynecologic Laparoscopy
Chapter 45: Complications of Laparoscopic and Hysteroscopic Surgery
Chapter 46: Uterine Leiomyomas
Chapter 47: Tubal Disease
Chapter 48: Ectopic Pregnancy
Chapter 49: Endometriosis
Chapter 50: Laparoscopic Management of Adnexal Masses
Chapter 51: Surgical Techniques for Management of Anomalies of the Müllerian Ducts and External Genitalia
Chapter 52: Adhesion Prevention
Chapter 53: Surgery for Male Infertility
Index
Section 1
Basic Science
Chapter 1 The Hypothalamic-Pituitary-Ovarian Axis and Control of the Menstrual Cycle

Neal Gregory Mahutte, Sophia Ouhilal

INTRODUCTION
The hypothalamus and pituitary gland form a unit that exerts control over a wide range of endocrine organs, including the gonads. This chapter describes the hypothalamic-pituitary-ovarian axis and control of the menstrual cycle, which is modulated by the central nervous system, other endocrine systems, and the environment. Key hormones in the hypothalamic-pituitary-ovarian axis include gonadotropin-releasing hormone (GnRH), follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol, and progesterone ( Table 1-1 ). Supporting roles are also played by inhibin, activin, follistatin, and endorphins.

Table 1-1 Major Hormones of the Hypothalamic-pituitary-ovarian Axis

THE HYPOTHALAMUS
The hypothalamus forms the lower part of the lateral wall and the floor of the third ventricle, and weighs approximately 10 grams. The hypothalamus is typically divided into eight specific nuclei (consistently clustered groups of neurons) and three areas (less clustered, less distinctly demarcated neurons), as illustrated in Figure 1-1 . From a reproductive standpoint, the most important of these are the arcuate nucleus and the preoptic area, the principal sites of GnRH-producing neurons. 1 The arcuate nucleus is located in the medial basal hypothalamus and is the most proximal of all the hypothalamic nuclei to the optic chiasm and the pituitary stalk. The arcuate nucleus is also the site of dopamine-secreting neurons that function to inhibit pituitary prolactin secretion and neurons that secrete growth hormone-releasing hormone.

Figure 1-1 Illustration of the hypothalamus, pituitary, sella turcica, and portal system. The arcuate nucleus is the primary site of neurons that produce gonadotropin-releasing hormone (GnRH). GnRH is released from the median eminence into the portal system. The blood supply of the pituitary gland derives from the internal carotid arteries. In addition to the arcuate nucleus, the other hypothalamic nuclei are SO, supraoptic nucleus; SC, suprachiasmatic nucleus; PV, paraventricular nucleus; DM, dorsal medial nucleus; VM, ventromedial nucleus; PH, posterior hypothalamic nucleus; PM, premammillary nucleus; LM, lateral mammillary nucleus; MM, medial mammillary nucleus. The three hypothalamic areas are PA, preoptic area; AH, anterior hypothalamic area; DH, dorsal hypothalamic area.
Neurosecretory cell products from the hypothalamus, including GnRH, are released into the portal system from the median eminence, a prominence in the pituitary stalk at the floor of the third ventricle. The portal system serves as the major route of communication between the hypothalamus and the anterior pituitary. In contrast, the pituitary stalk (infundibulum) directly connects neuronal cell bodies in the hypothalamus to the posterior pituitary. The pituitary stalk lies immediately posterior to the optic chiasm.

GnRH
GnRH is the primary hypothalamic regulator of pituitary reproductive function. Two human forms of GnRH (GnRH-I and GnRH-II) have been identified. 2, 3 Both are decapeptides and are the products of different genes. At least 20 other types of GnRH have been identified in fish, amphibians and protochordates, but none of these are believed to be present in humans. 4, 5
GnRH-I was first characterized and synthesized in 1971 by Andrew Schally and Roger Guillemin, an accomplishment for which both men ultimately received the Nobel prize. 6 - 9 The structure of GnRH-I is common to all mammals, and its action is similar in males and females ( Fig. 1-2 ). GnRH-I is synthesized from a much larger, 92-amino acid precursor peptide that contains GnRH-associated peptide. 10 GnRH-I then travels along an axonal pathway called the tuberoinfundibular tract to the median eminence of the hypothalamus, where it is released into the portal circulation in a pulsatile fashion. The half-life of GnRH-I is very short (2 to 4 minutes) because it is rapidly cleaved between amino acids 5 and 6, 6 and 7, and 9 and 10. Because of its short half-life and rapid dilution in the peripheral circulation, serum GnRH-I levels are difficult to measure and do not correlate with pituitary action.

Figure 1-2 Structure of GnRH-I.
GnRH-I has three principal actions on anterior pituitary gonadotrophs: (1) synthesis and storage of gonadotropins, (2) movement of gonadotropins from the reserve pool to a point where they can be readily released, and (3) direct secretion of gonadotropins. GnRH-I pulses occur in response to intrinsic rhythmic activity within GnRH neurons in the arcuate nucleus. Pulsatile release of GnRH-I from the median eminence within a critical frequency and amplitude results in normal gonadotropin secretion. 11, 12 Continuous, rather than pulsatile, exposure to GnRH-I results in suppression of FSH and LH secretion and suppression of gonadotropin gene transcription. 13, 14
In the absence of gonadal feedback, the GnRH pulse frequency is approximately once per hour. 15 During the menstrual cycle the frequency and amplitude of GnRH pulses vary in response to hypothalamic feedback ( Table 1-2 ). 16 In general, the follicular phase is characterized by high-frequency, low-amplitude pulses, and the luteal phase is characterized by lower-frequency, higher-amplitude pulses. 17, 18 However, considerable variability exists both between and within individuals. 19 In humans, GnRH-I pulse frequency and amplitude are best approximated by measuring LH pulse frequency and amplitude.
Table 1-2 Menstrual Cycle Variation in LH Pulse Frequency and Amplitude Cycle Phase Mean Frequency (minutes) Mean Amplitude (mIU/mL) Early follicular 90 6.5 Mid-follicular 50 5 Late follicular 60–70 7 Early luteal 100 15 Mid-luteal 150 12 Late luteal 200 8
GnRH-II is most highly expressed outside of the brain, in tissues that include kidneys, bone marrow, and prostate. This is in contrast to GnRH-I, which is not expressed in high levels outside the brain. Although GnRH-II can induce release of both FSH and LH, it appears to have a wide array of physiologic functions outside the brain including regulation of cellular proliferation and mediation of ovarian and placental hormone secretion. 20
Initial attempts in the mid-1990s to identify estrogen receptors in GnRH neurons were unsuccessful. 21, 22 However, subsequent use of more sophisticated techniques identified estrogen receptors α and β in the arcuate nucleus. 23 - 26 Both receptors mediate estrogen action on GnRH neurons in vivo. 27, 28 The GnRH gene contains a hormone response element for the estrogen–estrogen receptor complex. 29 Transcription of GnRH-I and GnRH-II is differentially regulated by estrogen. 30 The regulatory role of estradiol on GnRH is complex. Estrogen inhibits GnRH gene expression/biosynthesis, but secretion of GnRH may be increased, decreased, or unaffected. 31, 32
The activity of the hypothalamus is further modulated by nervous stimuli from higher brain centers. GnRH neurons exhibit many connections to each other and to other neurons. Some of the neurotransmitters that modulate GnRH secretion are outlined in Table 1-3 . The effects of these neurotransmitters help explain the mechanism by which certain physical or clinical conditions may affect the menstrual cycle ( Table 1-4 ).
Table 1-3 Neurotransmitter Effects on GnRH Release Neurotransmitter Effect Dopamine Inhibits GnRH release Endorphin Inhibits GnRH release Serotonin Inhibits GnRH release Norepinephrine, epinephrine Stimulates GnRH release
Table 1-4 Mechanisms for Oligo/amenorrhea in Various Clinical Conditions Hyperprolactinemia Elevated dopamine suppresses GnRH Hypothyroidism Elevated TRH increases prolactin, which in turn increases dopamine which, then suppresses GnRH Stress Increased corticotropin (ACTH) results in increased endorphins (both are derived from the same peptide precursor); endorphins suppress GnRH Exercise Increased endorphins suppress GnRH
TRH = thyrotropin-releasing hormone; GnRH = gonadotropin-releasing hormone.
Cells that produce GnRH originate embryologically from the olfactory area. 33 GnRH neurons, like olfactory epithelial cells of the nasal cavity, have cilia. 34 During embryogenesis GnRH neurons migrate from the medial olfactory placode to the arcuate nucleus of the hypothalamus. 35 The common origin of GnRH and olfactory neurons is demonstrated by Kallmann’s syndrome, where GnRH deficiency is associated with anosmia. Kallmann’s syndrome is believed to be caused by a variety of gene defects that affect neuronal cell migration. 36
The common origin of GnRH and olfactory neurons is also suggestive of the relationship between pheromones and menstrual cyclicity. Pheromones are small airborne chemicals that when secreted externally by one individual may be perceived by other individuals of the same species, producing a change in sexual or social behavior. It is well recognized that women who work or live together often develop synchrony of their menstrual cycles. 37 Moreover, it has been shown that odorless compounds from the axillae of cycling women can alter the cycle characteristics of recipient women. 38 Presumably, these alterations occur through olfactory GnRH-mediated mechanisms.

The GnRH Receptor
The GnRH-I receptor is a G-protein receptor that utilizes inositol triphosphate and diacylglycerol as second messengers to stimulate protein kinase, release of calcium ions, and cyclic adenosine monophosphate (cAMP) activity. The GnRH-I receptor is encoded by a gene on chromosome 14q21.1 and is expressed in many parts of the body outside of the brain, including ovarian follicles and the placenta. In humans it appears that GnRH-II signaling occurs through the GnRH-I receptor. 5 Although a GnRH-II receptor is present in many mammalian species, its functional capacity is limited due to a frame shift and a stop codon. GnRH receptors are regulated by many substances, including GnRH itself, inhibin, activin, estrogen, and progesterone.
Changes to the amino acid sequence of GnRH can extend its half-life to hours or days and can change its biologic activity from an agonist to an antagonist. All of the GnRH agonists currently available extend their half-life by substitutions at amino acid 6 and sometimes amino acid 10 of native GnRH ( Table 1-5 ). Continuous activation of the GnRH receptor results in desensitization due to phosphorylation and conformational change of the receptor, uncoupling from G proteins, internalization of the receptor via endocytosis, and decreased receptor synthesis. 39, 40 On administration all GnRH agonists increase gonadotropin secretion (the flare effect). However, after 7 to 14 days GnRH receptor desensitization occurs and pituitary suppression is achieved.

Table 1-5 Properties of Commercially Available GnRH Agonists
In contrast, GnRH antagonists directly inhibit gonadotropin secretion. Structurally, GnRH antagonists are characterized by multiple amino acid substitutions to the natural GnRH decapeptide. The commercially available GnRH antagonists cetrorelix and ganirelix have large amino acid additions to position 1 of native GnRH. GnRH antagonists compete for and occupy pituitary GnRH receptors, thus competitively blocking endogenous GnRH–GnRH receptor binding. In contrast to GnRH agonists, there is no flare effect with GnRH antagonists. Because receptor loss does not occur, a constant supply of antagonist is necessary to ensure that all GnRH receptors are continuously occupied. Thus, the therapeutic dosage range for antagonists is typically higher than that for agonists (mg versus μg).

THE PITUITARY
The pituitary gland measures approximately 15 × 10 × 6 mm and weighs approximately 500 to 900 mg. The pituitary gland lies immediately beneath the third ventricle and just above the sphenoidal sinus in a bony cavity called the sella turcica (Turkish saddle). It consists of anterior and posterior lobes, each having different embryologic origins, functions, and control mechanisms ( Fig. 1-3 ).

Figure 1-3 X-ray and T1-weighted magnetic resonance images (MRI) of the pituitary gland. A, Lateral skull film with the sphenoidal sinus and sella turcica. B, Sagittal section demonstrating the relationship between the sphenoidal sinus and the pituitary gland. The normal posterior pituitary is brighter on MRI compared to the anterior pituitary. The sella turcica is not well seen on MRI. C, Sagittal section after administration of gadolinium contrast. Because the pituitary lies outside the blood-brain barrier, both the anterior and posterior pituitary are illuminated with the contrast. D, Coronal section demonstrating the relationship of the pituitary to the optic chiasm and the pituitary stalk. E, Coronal section after gadolinium contrast demonstrating the close proximity of the pituitary to the internal carotid arteries.
The secretion of pituitary hormones is controlled primarily by the hypothalamus. However, the activity of the hypothalamus and pituitary are modulated by nervous stimuli from higher brain centers and feedback from circulating hormones. There is no direct nervous connection between the hypothalamus and the anterior pituitary gland. Instead, communication occurs via the hypothalamic-pituitary portal system.
The arterial blood supply of the pituitary is derived from branches of the internal carotid artery. The anterior pituitary gland is the most richly vascularized of all mammalian tissues. The blood supply of the anterior pituitary comes from the superior hypophyseal artery. The superior hypophyseal artery forms a capillary network in the median eminence of the hypothalamus that recombines in long portal veins draining down the pituitary stalk to the anterior pituitary, where they break up into another capillary network. The blood supply of the posterior pituitary (neurohypophysis) comes from the middle and inferior hypophyseal arteries. Veins from these arteries drain into the cavernous sinuses, from which they ultimately reach the petrosal sinuses and then the jugular veins.
The posterior pituitary is best understood as a continuation of the hypothalamus. The posterior pituitary is a direct downgrowth of nervous tissue from the hypothalamus through the pituitary stalk. The posterior pituitary is composed of glial tissue and axonal termini that secrete oxytocin and arginine vasopressin (AVP, also known as antidiuretic hormone ). Oxytocin is synthesized in the paraventricular nucleus of the hypothalamus, whereas AVP is synthesized in the supraoptic nucleus, just above the arcuate nucleus. Oxytocin and AVP pass down the length of the pituitary stalk and are stored in terminal parts of axons in the posterior pituitary. Both oxytocin and AVP consist of nine amino acid residues (nonapeptides). Release of oxytocin and AVP is controlled directly by nervous impulses passing down the axons from the hypothalamus.

The Anterior Pituitary
The anterior pituitary arises from an epithelial upgrowth from the roof of the primitive oral cavity (Rathke’s pouch). The anterior pituitary wraps around the posterior pituitary and constitutes two thirds of the volume of the pituitary gland. The portion of Rathke’s pouch in direct contact with the posterior pituitary develops less extensively than the rest of the anterior pituitary and is termed the pars intermedia.
The major cell types of the anterior pituitary are outlined in Table 1-6 . The anterior pituitary is a classic endocrine gland in that it is composed of secretory cells of epithelial origin supported by connective tissue rich in blood and lymphatic capillaries. In accordance with their active synthetic function the endocrine cells are characterized by prominent nuclei and prolific mitochondria, endoplasmic reticulum, Golgi bodies, and secretory vesicles. Synthesis of gonadotropins takes place in the rough endoplasmic reticulum, after which the hormones are packaged within the Golgi apparatus and stored as secretory granules. In response to GnRH the secretory granules are extruded from the cell membrane. The endothelial lining of capillary sinusoids is fenestrated, facilitating the passage of pituitary hormones into the sinusoids.

Table 1-6 Major Cell Types of the Anterior Pituitary Gland

Gonadotropins
The anterior pituitary secretes two hormones that stimulate the growth and activity of the gonads, FSH and LH. These glycoprotein hormones, termed gonadotropins, work in conjunction to stimulate secretion of steroid hormones from the ovary.

FSH
FSH is a glycoprotein with a molecular weight of approximately 29,000 daltons. It consists of both an α and β subunit. The α subunit consists of 92 amino acids stabilized by 5 disulfide bonds and is identical to the α subunit of LH, thyroid-stimulating hormone (TSH) and human chorionic gonadotropin (hCG). The β subunit contains 118 amino acids and 5 sialic acid residues. Neither subunit has any intrinsic biologic activity by itself.
The sialic acid content of FSH, LH, TSH, and hCG varies, and these differences are largely responsible for variations in half-life of the glycoprotein hormones. The liver is the major site of clearance for gonadotropins. Sialic acid prevents hepatic clearance; thus, the greater the sialic acid content, the longer the half-life. 41 hCG, with 20 sialic acid residues, has the longest half-life (about 24 hours), whereas LH (1 to 2 sialic acid residues) has the shortest half-life (20 to 30 minutes). Addition of sialic acid residues in urinary-derived commercially available gonadotropins (e.g., hMG) is responsible for their longer half-life (30 hours).
In gonadotrophs of the anterior pituitary, GnRH signaling leads to transcription of the α and β subunits for both FSH and LH. The GnRH-dependent availability of the β subunits is the rate-limiting step in gonadotropin synthesis. Although both FSH and LH require GnRH stimulation, synthesis of the FSH β subunit also requires the presence of activin. 42, 43
Follicle-stimulating hormone plays a crucial role in follicle recruitment and selection of the dominant follicle. FSH has a trophic effect on granulosa cells in antral follicles, including the induction of aromatase activity, inhibin synthesis, and expression of LH receptors. A certain amount of FSH (the FSH threshold ) is required to induce these changes in a given follicle. FSH must then remain above that threshold for folliculogenesis to continue.
In the normal menstrual cycle serum concentrations of FSH begin to rise a few days before the onset of menstruation. FSH levels plateau in the midfollicular phase and decline in the late follicular phase in response to the rise in estrogen and inhibin B. This decline contributes to the dominance of selected follicles over others. FSH levels then peak briefly during the ovulatory gonadotropin surge, after which they decline to their nadir in the luteal phase.

LH
LH is a glycoprotein with a molecular weight of 29,000 daltons. Like FSH, TSH, and hCG, it consists of both an α and β subunit. The α subunit is identical to the α subunit of FSH, TSH, and hCG. The β subunit of LH has 121 amino acids and 1 to 2 sialic acid residues.
Luteinizing hormone is synthesized in gonadotrophs of the anterior pituitary gland. Because it contains fewer sialic acid residues than FSH, LH is rapidly cleared from the circulation by the liver and kidney. For this reason LH is rapidly biosynthesized, and LH pulses occur at higher amplitude than those of FSH. It is believed that the pituitary content of LH is turned over 1 to 2 times per day.
Serum LH levels begin to rise a few days before the onset of menstruation. They increase very gradually during the follicular phase. Unlike FSH, serum LH values rise in the late follicular phase, surpassing FSH values around cycle day 9 or 10. The LH surge at midcycle is followed by a steady decline to their nadir in the midluteal phase.

FSH and LH Receptors
The actions of both LH and FSH are mediated by G protein receptors on the cell membrane. LH receptors are found exclusively on theca cells in the ovary. Stimulation of these receptors by increased cytochrome P450c17 enzyme activity (17-hydroxylase and 17,20-lyase) in theca cells, resulting in activation of both adenylate cyclase and cAMP-dependent protein kinases and leading to increased production of androstenedione and testosterone.
In contrast, FSH receptors are found exclusively on granulosa cells in the ovary. FSH binds to receptors on the cell surface of granulosa cells in antral follicles. Like LH, FSH acts via the cAMP-dependent protein kinase pathway. 44 In response to FSH the androgens produced as a result of LH stimulation are then aromatized to estrogens in granulosa cells.

Opioid Modulation of Pituitary Hormones
Opioids (i.e., endogenous opiates) are natural occurring sedative narcotics produced in the brain whose structure and function are similar to opium. Opioids include enkephalins, endorphins, and dynorphins; they modulate every pituitary hormone by acting on the hypothalamus. An important action of opioids is to inhibit gonadotropin secretion by suppressing GnRH release. 45
Opioid tone is an important regulator of menstrual cyclicity. 46 - 49 Endorphins are at a nadir in the early follicular phase (menstruation) and gradually rise to peak levels in the luteal phase in response to the rise in estrogen and progesterone. It is believed that opioids mediate the negative feedback of ovarian steroids on gonadotropin release, particularly in the luteal phase. 50
Endogenous opioids appear to play a central role in hypothalamic amenorrhea. Treatment of women suffering from this condition with an opioid receptor antagonist (e.g., naltrexone) results in the return to ovulatory menstrual patterns and even conception in some cases. 51, 52 Women with stress-related amenorrhea demonstrate increased hypothalamic corticotropin-releasing hormone and pituitary corticotropin, which manifests as hypercortisolism. 53 The corticotropin precursor peptide, pro-opiomelanocortin, is also the precursor for endorphin synthesis. It is hypothesized that stress-related amenorrhea is the result of GnRH inhibition secondary to increased production of endogenous opioids. Opioids also rise during exercise (“runners’ high”), and this may contribute to hypothalamic amenorrhea in athletes. 54, 55

Ovarian Peptide Hormone Feedback on Gonadotropin Secretion
The ovary secrets two polypeptide hormones that inhibit or stimulate FSH secretion by the anterior pituitary. Inhibin and activin act as opposing nonsteroidal gonadal hormones that regulate FSH synthesis and secretion by the pituitary. They also have paracrine effects within the ovary, where they modulate follicle growth and steroidogenesis. Follistatin is a binding protein that modulates the effects of activin but not inhibin.

Inhibin and Activin
Inhibin and activin are members of the transforming growth factor-β (TGF-β) superfamily of ligands, which includes müllerian inhibiting substance (MIS; see Chapter 2 ). Like the gonadotropins, inhibin and activin are comprised of two subunits. Inhibin is comprised of an α and β subunit and has been isolated in two forms containing different β subunits, Inhibin-A and Inhibin-B. Activin is comprised of two beta subunits identical to those found in inhibin.
Inhibin is secreted by granulosa cells in response to FSH. 56 However, mRNA for inhibin has also been found in pituitary gonadotrophs. Inhibin selectively inhibits FSH but not LH secretion. 57 Thus, a negative feedback loop is created where FSH stimulates inhibin and in turn inhibin suppresses FSH.
Inhibin B is predominantly secreted in the follicular phase of the menstrual cycle, whereas inhibin A is predominantly secreted in the luteal phase. 58 Peak levels of inhibin B in the follicular phase are in the range of 50 to 100 pg/mL. Peak levels of inhibin A in the luteal phase are between 40 and 60 pg/mL.
Activin is also secreted by granulosa cells. Activin augments the secretion of FSH by the pituitary by enhancing GnRH receptor formation. Activin is also required for synthesis of the FSH β subunit.
In the ovary, activin augments FSH action and stimulates production of follistatin by a paracrine effect. These effects of activin are blocked by both inhibin and follistatin. With increased GnRH stimulation, activin is increasingly antagonized by inhibin and bound by follistatin.

Follistatin
Follistatin is an activin-binding protein that sequesters activin. Follistatin is produced in the same tissues as activin, including the pituitary, and regulates activin’s local paracrine and autocrine actions. In the circulation, follistatin binds the majority of activin, thereby inhibiting FSH secretion. This suggests that activin’s primary actions are paracrine in nature. Follistatin does not bind inhibin, thus allowing it to act as a conventional endocrine hormone between the ovary and the pituitary in addition to its local ovarian effects.

OVARIAN STEROIDOGENESIS DURING THE MENSTRUAL CYCLE
Ovarian steroidogenesis during the menstrual cycle occurs in granulosa and theca cells ( Table 1-7 and Fig. 1-4 ). Before ovulation, theca cells are separated from granulosa cells in the same follicle by a basal membrane. Thus, granulosa cells of preovulatory follicles do not have a blood supply. However, at the time of the LH surge the preovulatory follicle undergoes luteinization with disappearance of the basal membrane and capillary invasion of the granulosa cells. Theca cells become theca-lutein cells, and granulosa cells become granulosa-lutein cells.
Table 1-7 Site of Synthesis of Major Steroidogenic Products of the Ovary Cell Type Major Steroid Hormone Products Theca cells Androgens (androstenedione, DHEA, testosterone) * Granulosa cells Estrogens (estradiol, estrone) Theca-lutein cells Progestogens (progesterone, 17-hydroxyprogesterone) ** Granulosa-lutein cells Estrogens (estradiol, estrone)
* Mostly via Δ 5 pathway
** Via Δ 4 pathway

Figure 1-4 The Δ 5 and Δ 4 pathways. The rate-limiting step in steroidogenesis is the conversion of cholesterol to pregnenolone via side chain cleavage (P450scc). In the follicular phase pregnenolone is preferentially converted to androstenedione via the Δ 5 pathway involving 17-hydroxypregnenolone and dehydroepiandrosterone (DHEA). In contrast, the corpus luteum preferentially converts pregnenolone to progesterone (Δ 4 pathway) via 3β-hydroxysteroid dehydrogenase (3βHSD).
If pregnancy does not occur, the lifespan of the corpus luteum is fixed at approximately 14 days. After 12 to 14 days luteolysis and apoptosis are initiated. The corpus luteum involutes, and menstruation occurs. Ovarian steroidogenesis then shifts to a new cohort of follicles with their granulosa and theca cells.

Two-Cell Theory
The two-cell, two-gonadotropin theory of ovarian steroidogenesis holds that follicular estrogen/androgen production is compartmentalized. 59 Ovarian theca cells produce androgens in response to LH. These androgens may then be aromatized to estrogens in granulosa cells appropriately stimulated by FSH. FSH receptors are present only on granulosa cells, and early in the follicular phase LH receptors are present only on theca cells. 60 The enzyme P450c17 (17-hydroxylase and 17,20-lyase) is only present in theca cells. Thus, only theca cells have the ability to convert 21-carbon steroids to 19-carbon steroids. In contrast, aromatase is only present in granulosa cells. Thus, in the ovary only granulosa cells have the ability to aromatize androgens to estrogens ( Table 1-8 and Fig. 1-5 ). Supporting evidence for the two-cell theory includes the fact that women with hypogonadotropic hypogonadism may develop follicles in response to treatment with recombinant FSH, but do not significantly elevate androgen or estrogen levels unless LH is added to the stimulation regimen. 61
Table 1-8 Location Specificity of P450c17 and Aromatase Enzyme Location Function P450c17 Theca cells only Converts 21-carbon steroids (progesterone/pregnenolone) to 19-carbon steroids (androstenedione, DHEA) Aromatase Granulosa cells only Converts 19-carbon steroids (androstenedione/testosterone) to 18-carbon steroids (estrone, estradiol)

Figure 1-5 The two-cell theory and follicular phase steroidogenesis. Binding of luteinizing hormone (LH) to LH receptors on ovarian theca cells stimulates conversion of cholesterol to androstenedione. Binding of follicle-stimulating hormone (FSH) to FSH receptors on granulosa cells then stimulates the aromatization of androgens to estrogens. StAR, steroidogenic acute regulatory protein; scc, side chain cleavage; HSD, hydroxysteroid dehydrogenase.

Estrogens
Estrogens are 18-carbon steroids that include estradiol (i.e., 17β-estradiol), estrone, and estriol. The most potent estrogen, estradiol, is predominantly secreted by the ovary. Estrone, which is one twelfth as potent as estradiol, is also secreted by the ovary. However, the principal source of estrone is from peripheral conversion from androstenedione. Estriol, which is one eightieth as potent as estradiol, is the principal estrogen formed by the placenta during pregnancy. Estriol is also formed by metabolism of estradiol and estrone by the liver and is the most abundant estrogen found in urine.
Estrogen is largely bound to carrier proteins in serum. Approximately 60% of estradiol is bound to albumin, 38% is bound to sex hormone-binding globulin (SHBG), and 2% to 3% is free. It had previously been thought that only the free hormone was active and could enter cells, but recent evidence suggests that hormone transport and hormone availability may be more complex. 62

Estrogen Receptors
There are two known estrogen receptors, ERα and ERβ. Both contain a steroid-binding domain, a DNA-binding domain, a hinge region, and a transcription-activation functional domain. The negative feedback of estradiol on FSH secretion is a direct effect of estradiol coupled to its receptor repressing transcription of the FSH β subunit. 63 Negative feedback of estradiol on FSH may also be modulated by the estrogen-associated decline in pituitary expression of activin. 64
Estrogens can enter any cell, but only cells with estrogen receptors will respond to its presence. When estrogens enter susceptible cells, estrogen associates with its receptor in the cell nucleus. This binding then activates the receptor. The DNA-binding domain of the estrogen–ER complex then associates with specific promoter sequences (DNA response elements) and activates gene transcription.

Estrogen Metabolism
Serum concentrations of estradiol are less than 50 pg/mL in the early follicular phase. In response to follicle development estradiol levels rise, typically peaking at 200 to 300 pg/mL just before ovulation. Estradiol levels drop at the time of ovulation, then rise again, with a second peak in the midluteal phase reflecting estrogen secretion from the corpus luteum.
The liver conjugates estrogens to form glucuronides and sulfates, about 80% of which are then excreted into the urine and 20% of which are excreted in the bile. In the liver, circulating estradiol is rapidly converted to estrone by 17β-hydroxysteroid dehydrogenase. Estrone may then be further metabolized in the liver to 16α-hydroxyestrone and then estriol. Estriol is then converted to estriol 3-sulfate-16-glucuronide before excretion by the kidney.
Some 16α-hydroxyestrone may also be converted to catechol estrogens (i.e., 2-hydroxy or 4-hydroxyestrone). Biologically active catechol estrogens may then be converted to 2-methoxy and 4-methoxy compounds. Catechol estrogens are important because they have been implicated in carcinogenesis. Metabolism of catechol estrogens may generate oxygen free radicals.

Progesterone
Progesterone, a 21-carbon steroid, is the principle secretory steroid of the corpus luteum. Progesterone is responsible for the induction of secretory changes that prepare estrogen-primed endometrium for implantation. If implantation occurs, continued progesterone production is necessary for maintenance of the pregnancy.
The release of FSH and LH require the continuous pulsatile release of GnRH. The coordinated secretion of FSH and LH control follicle growth, ovulation, and maintenance of the corpus luteum. The release of FSH and LH is both positively and negatively influenced by estrogen and progesterone. Whether estrogen and progesterone stimulate or inhibit gonadotropin release depends on the quantity and duration of exposure to the steroid.
At high concentrations progesterone inhibits both FSH and LH secretion by negative feedback on both the hypothalamus and pituitary. 65 Progesterone also slows the GnRH pulse generator; hence the decline in GnRH pulse frequency in the luteal phase. However, at low concentrations, and only after previous exposure to estrogen, progesterone stimulates LH release. 66

Progesterone Receptors
Progesterone receptors are similar to estrogen receptors in that they contain a steroid-binding domain, a DNA-binding domain, a hinge region, and a transcription-activation functional domain. There are two progesterone receptors, A and B. Receptor B is the positive regulator of progesterone-responsive genes. Binding of progesterone to progesterone receptor A inhibits activity of receptor B. Progesterone causes a depletion of estrogen receptors. This is the mechanism by which progestins protect against endometrial hyperplasia.

Progesterone Metabolism
Throughout the follicular phase serum progesterone concentrations are less than 1 ng/mL. Much of this progesterone is believed to result from adrenal production of pregnenolone and progesterone as a by-product of cortisol and aldosterone synthesis. In the late follicular phase progesterone levels begin to rise as the ovary starts to contribute to progesterone production. The rise in progesterone accelerates markedly after ovulation, and progesterone levels reach 10 to 20 ng/mL in the midluteal phase. If pregnancy does not occur, progesterone levels fall in the late luteal phase and return to levels below 1 ng/mL just before the onset of menstruation.
In the circulation, approximately 80% of progesterone is bound to albumin. Another 18% is bound to corticosteroid-binding globulin. About 2.5% is freely circulating, and only about 0.5% is bound to SHBG.
Progesterone is rapidly cleared from the circulation by the liver. The liver converts progesterone to pregnanediol. Pregnanediol is then conjugated to glucuronic acid, and pregnanediol glucuronide is excreted in the urine. Measurement of urinary pregnanediol glucuronide can be used as an index of progesterone production.

Androgens
Ovarian theca cells secrete a variety of androgens (i.e., 19-carbon steroids), including androstenedione, testosterone, and dehydroepiandrosterone (DHEA). Androstenedione is the principal androgen secreted by ovarian theca cells. Theca cells also possess the enzyme 17β-hydroxysteroid dehydrogenase, which may convert androstenedione to testosterone. In premenopausal women, at least 60% of circulating testosterone is derived from the ovary, by either this conversion or direct secretion.
Androstenedione and testosterone can then be aromatized to estrogens in granulosa cells under the influence of FSH. Androstenedione can also be converted to estrone or testosterone in peripheral tissues. Unlike testosterone and dihydrotestosterone, androstenedione does not have high affinity for the androgen receptor.
Side chain cleavage of cholesterol to pregnenolone is the starting point and rate-limiting step in steroidogenesis. In the ovary cholesterol side chain cleavage is regulated by LH. Low-density lipoprotein (LDL) cholesterol is the principal source of cholesterol for steroidogenesis in the human ovary. 67 Increased cAMP production due to LH stimulation of adenylate cyclase increases transcription of LDL receptor mRNA and LDL uptake. cAMP-activated steroidogenic acute regulatory protein then increases the intracellular transport of cholesterol to the inner mitochondrial membrane, where side chain cleavage occurs. 68
In the preovulatory follicle the preferred pathway for androgen/estrogen synthesis involves conversion of pregnenolone to 17-hydroxypregnenolone, the so-called Δ 5 pathway (see Fig. 1-4 ). Ovarian theca cells have the enzymatic capability to convert pregnenolone to androgens, but lack the ability to aromatize androstenedione or testosterone into estrogens. Only granulosa cells, under the influence of FSH, can aromatize androgens to estrogens. In contrast to the preovulatory follicle, the corpus luteum prefers the Δ 4 pathway, the initial conversion of pregnenolone to progesterone.

Androgen Receptors
The androgen receptor is similar to the estrogen receptor. It activates target gene expression via a similar sequence of ligand binding, nuclear translocation, DNA binding, and complex formation with coregulators and general transcription factors. Although the androgen receptor is known to play a central role in the development of male sex organs and secondary sexual characteristics, its physiologic roles in female reproduction remain unclear. Recent studies in androgen-deficient animals suggest that androgen receptors play an important role in granulosa cell development. 69 These animals also exhibit reduced fertility with defective folliculogenesis, reduced corpus luteum formation, and reduced uterine response to gonadotropins.

Androgen Metabolism
Androstenedione is produced in equal amounts by the ovaries and adrenal glands. The serum concentrations mirror estradiol levels and range from 0.5 to 3 ng/mL. Androstenedione levels increase in the mid- to late follicular phase and are maximal just before the LH surge. They decline at the time of ovulation, reach a second peak in the midluteal phase, and are lowest around the time of menstruation. Serum testosterone concentrations, in contrast, vary to a much lesser extent during the menstrual cycle and exhibit only a transient periovulatory increase. Testosterone concentrations range from 1.5 to 60 ng/dL.

CONTROL OF THE MENSTRUAL CYCLE
Normal menstrual cycles, termed eumenorrhea, normally range in length between 24 to 35 days, with menstrual bleeding lasting 3 to 7 days. The average amount of blood loss is approximately 30 mL. 70 Heavy, prolonged, or irregular menses are referred to as abnormal uterine bleeding and are considered in length in Chapter 21 .
Normal menstrual cycles result from a relatively precise interaction of the hypothalamus, pituitary, and ovaries. Under the influence of pituitary gonadotropins, the ovary undergoes cyclic changes providing for the development and release of a mature oocyte and production of ovarian hormones that prepare the endometrial lining for implantation. LH stimulates androgen production in theca cells; FSH promotes follicle development and aromatization of androgens to estrogens in granulosa cells (see Fig. 1-5 ). In turn, estrogens lead to proliferation of the endometrial lining and the induction of endometrial receptors for both estrogen and progesterone. 71
To understand the menstrual cycle it is helpful to divide it into four phases: the follicular phase, the ovulatory phase, the luteal phase, and the luteal–follicular transition. We concentrate on changes in pituitary and ovarian hormones and the effects that these hormones have on the hypothalamus, pituitary, and ovary.

Follicular Phase
The purpose of the follicular phase is to develop a single mature follicle to release a mature oocyte at ovulation. The presence of sufficient FSH also leads to expression of LH receptors on mature granulosa cells of preovulatory follicles. Thus, in the late follicular phase LH can sustain follicular endocrine activity, even in the absence of FSH. 72 The follicular phase is variable in duration, but the other three phases are relatively constant, averaging 14 ± 2 days.

GnRH
Throughout most of the follicular phase GnRH pulses occur every 60 to 90 minutes. Just before the onset of the LH surge both GnRH pulse frequency and amplitude increase.

Gonadotropins
FSH levels rise in the early follicular phase due to the lack of negative inhibition from estradiol and inhibin ( Fig. 1-6 ). FSH stimulates follicle growth and estrogen production. 73 Through binding of FSH to its receptor, granulosa cells in developing follicles attain the ability to aromatize androstenedione to estrone and testosterone to estradiol. Importantly, receptors for FSH are not detected on granulosa cells until the preantral stage. 74 Moreover, both in vitro and in vivo administration of FSH to granulosa cells can cause upregulation or downregulation of granulosa cell FSH receptors. 75 Without functional FSH follicle growth and ovarian estrogen production cannot occur. 76

Figure 1-6 Hormone fluctuations during the menstrual cycle. A, Mean values of follicle-stimulating hormone and luteinizing hormone throughout the cycle. B, Mean values of estradiol and inhibin. C, Mean values of progesterone during the menstrual cycle.
The steady decline in FSH beginning in the midfollicular phase serves to inhibit development of all but the dominant follicle. The dominant follicle remains dependent on FSH and must complete its development in spite of declining FSH levels. Because it has the largest cohort of granulosa cells, it has the largest cohort of FSH receptors and thus is able to grow in the face of insufficient FSH for smaller follicles.
Luteinizing hormone levels are stable in the first half of the follicular phase. However, in the second half LH levels rise in response to positive feedback from increasing estrogen.

Ovarian Hormones
Estradiol levels rise as the dominant follicle emerges. FSH and estrogen synergistically exert a mitogenic effect on granulosa cells, stimulating their proliferation. This in turn increases the FSH receptor content of the follicle, enhancing the ability of the follicle to respond to FSH and produce estrogen. Curiously, not every granulosa cell must express FSH receptors to respond to the gonadotropin signal. Gap junctions between cells allow cells with receptors to transmit protein kinase activation to their neighbors. 77
Within the follicular fluid of follicles greater than 8 mm in diameter, the concentration of FSH, estradiol, and progesterone are all extremely high. Within smaller antral follicles, androgens predominate in the follicular fluid. The role of androgens in the follicle is dose-dependent. At low levels androgens provide a substrate for aromatization. However, at higher levels androgens are converted in granulosa cells by 5α-reductase to more active forms such as dihydrotestosterone (DHT) that cannot be aromatized to estrogens. 78, 79 Granulosa cells have androgen receptors. 80 Activation of granulosa cell androgen receptors inhibits aromatase activity and also inhibits FSH induction of granulosa LH receptors. 81 Follicles exposed to excessive androgens eventually become atretic. 82, 83 In contrast, follicles with the highest estrogen-to-androgen ratios and the highest estrogen concentrations are most likely to contain a competent oocyte. 84
Rising estradiol levels have a dual role for the follicle. Within the follicle, estradiol promotes granulosa cell growth, aromatization of androgens to estrogens, and, in combination with FSH, induction of development of LH receptors on granulosa cells. However, outside the follicle rising serum estradiol levels have an inhibitory effect on FSH secretion. The resulting decline in FSH levels in the mid- to late follicular phase limits aromatase activity in smaller follicles, leading to higher androgen levels and follicular atresia. Indeed, a reduction in granulosa cell expression of FSH receptors is one of the first signs of follicular atresia.
Inhibin B levels begin to rise almost immediately after the rise in FSH levels. By the midfollicular phase the rise in estradiol and inhibin B levels causes FSH levels to decline. Inhibin B levels peak approximately 4 days after the FSH peak. 47 In the late follicular phase inhibin B levels fall, mirroring the decline in FSH levels.
Progesterone and inhibin A levels are low throughout most of the follicular phase, but both begin to rise in the days immediately preceding ovulation. The rise in progesterone in the late follicular phase mirrors the rise in LH.

Ovulatory Phase
The ovulatory phase begins with the midcycle LH surge, which disrupts contacts between granulosa cells and the cumulus oophorus (specialized granulosa cells surrounding the oocyte), causing the oocyte to detach from the follicle wall. The LH surge also induces the resumption of meiosis within the oocyte and release of the oocyte–cumulus complex from the follicle (ovulation).

GnRH
GnRH plays a supporting role for the midcycle gonadotropin surge, but it does not trigger the surge. 85 There is no change in GnRH pulse frequency during the midcycle gonadotropin surge. 86 Rather, ovarian steroid feedback to the primed anterior pituitary triggers the LH surge. 87 Whereas estrogen inhibits the secretion of pituitary gonadotropins, it facilitates their synthesis and storage. Estrogen also increases the expression of GnRH receptors. 88, 89 Thus, in the mid- to late follicular phase each pulse of GnRH is met with a greater gonadotropin response. 90, 91 When the estradiol level in the circulation meets a critical level for a sufficiently long period of time, the inhibitory action of estradiol on LH secretion changes to a stimulatory one. The LH surge is accompanied by a surge of GnRH in both portal and peripheral blood. 92 However, as demonstrated by women with hypogonadotropic hypogonadism treated with a pulsatile GnRH pump, ovulation and pregnancy may occur in the absence of any change in GnRH pulse frequency or amplitude. 93 Moreover, the LH surge ends before there is a decline in the GnRH signal. 94

Gonadotropins
Both FSH and LH levels peak just before ovulation. The initiation of the gonadotropin surge is dependent on attaining serum estradiol levels of at least 200 pg/mL for at least 2 days. 95 In natural cycles this level of estradiol is typically not attained until the dominant follicle reaches a mean diameter of 15 mm. 96
During the gonadotropin surge serum LH levels increase tenfold over a period of 2 to 3 days, while FSH levels increase fourfold. Within the dominant follicle the LH surge induces detachment of the oocyte–cumulus complex from the follicle wall. The release of the oocyte–cumulus complex from its follicular wall attachments is accompanied by the resumption of meiosis and release of the first polar body.
The LH surge also induces luteinization of the periovulatory follicle. Luteinization refers to functional and morphologic changes within the theca–granulosa cell complex associated with accumulation of a yellow pigment called lutein . The function of the FSH surge is less clearly known, but it is believed to ensure an adequate number of LH receptors on the granulosa layer and to increase production of plasminogen activator, thus increasing the concentration of the proteolytic enzyme plasmin.
Ovulation typically occurs from mature follicles 34 to 36 hours after the onset of the LH surge. 97 The peak of LH and FSH occurs 10 to 12 hours before ovulation. 98 The LH surge usually lasts 48 to 50 hours and must be maintained for at least 14 hours for full maturation of the oocyte to occur. 99 The mechanism that turns off the LH surge is unknown. It may simply reflect the depletion in pituitary LH content.

Ovarian Hormones
Just prior to ovulation the follicle becomes vascularized. 100 Angiogenesis is mediated by LH and a variety of other factors, including vascular endothelial growth factor. 101 - 103 Prostaglandins reach peak levels in the follicular fluid. 104 Proteolytic enzymes digest collagen in the follicular wall, resulting in distensibility and thinning just before ovulation. 105 Progesterone rises in the follicular fluid. FSH, LH, and progesterone all serve to increase the activity of proteolytic enzymes. There is a rapid increase in follicular fluid volume, but due to increased elasticity there is little to no change in intrafollicular pressure. Finally, a protrusion (stigma) appears on the follicular wall, and it is at this site that ovulation ultimately occurs.
Interestingly, spontaneous lueteinization occurs in the absence of LH when granulosa cells are removed from follicles and cultured in vitro. Similarly, cumulus-enclosed oocytes removed from developing follicles before the LH surge will spontaneously resume meiosis. 106, 107 These findings have led to speculation that substances functioning as oocyte maturation inhibitors or luteinization inhibitors must exist within each follicle. Further support for this hypothesis lies in the fact that cumulus cells lack LH receptors.
Estradiol levels fall beginning with the onset of the LH surge. Progesterone and inhibin A levels continue to rise at the time of ovulation. Inhibin B levels surge at the time of ovulation. Luteinization of the follicle begins.

Luteal Phase
The luteal phase is the time when the ovary secrets progesterone, resulting in endometrial receptivity for embryo implantation. LH receptors on granulosa cells result in luteinization, driving the postovulatory follicle to become a corpus luteum. Granulosa cells become granulosa-lutein cells with their own blood supply and begin secretion of both estrogen and progesterone. Release of large amounts of progesterone from the corpus luteum results in secretory endometrial changes and the final development of endometrial receptivity.

GnRH and Gonadotropins
GnRH pulse frequency declines but pulse amplitude increases in the luteal phase. Changes in GnRH pulse frequency correlate with the duration of exposure to progesterone; changes in pulse amplitude correlate with progesterone levels. 17
Luteinizing hormone and FSH levels reach a nadir during the luteal phase in response to the elevation in estrogen, progesterone, and inhibin A. Nevertheless, function of the corpus luteum is dependent on continued low-level pituitary gonadotropin secretion throughout the luteal phase. 108 LH pulses stimulate pulses of progesterone secretion from the corpus luteum. 17, 109 Moreover, reducing LH pulse frequency and amplitude with a GnRH agonist in the luteal phase shortens the luteal phase itself. 110

Ovarian Hormones
After ovulation granulosa and theca cells of the dominant follicle are luteinized. Luteinization involves both chemical and morphologic changes. Granulosa and theca cells hypertrophy and increase steroidogenesis. Moreover, breakdown of the basal membrane that previously separated granulosa and theca cells leads to capillary invasion around granulosa-lutein cells.
Granulosa-lutein cells can now make progesterone directly from LDL via side chain cleavage and 3β-hydroxysteroid dehydrogenase. Levels of mRNA for side chain cleavage and 3β-hydroxysteroid dehydrogenase are maximal at the time of ovulation and in the early luteal phase. 111 The induction of LDL receptor expression in granulosa cells occurs in response to the LH surge and is an early feature of luteinization. 112 Progesterone secretion correlates with the number of LH receptors and adenylate cyclase activity. 113 Progesterone levels peak during the midluteal phase.
Granulosa-lutein cells cannot make estrogens directly from cholesterol because they lack the enzyme P450c17. However, granulosa-lutein cells continue to be able to aromatize theca-lutein produced androgens to estrogens, and estrogen levels remain high throughout most of the luteal phase.
In granulosa-lutein cells inhibin production switches from inhibin B to inhibin A. Thus inhibin B levels decline to their nadir during the luteal phase, whereas inhibin A levels reach their peak. Secretion of inhibin A by granulosa-lutein cells is controlled by LH. 114 Inhibin A, like inhibin B, suppresses FSH levels. 115

Luteal–Follicular Transition
As the luteal phase continues, progesterone inhibits LH release via a negative feedback loop on the anterior pituitary. During the luteal–follicular transition, subsequent decline in LH causes the corpus luteum to involute unless the corpus luteum is rescued by production of hCG from an implanting embryo.
Involution of the corpus luteum leads to a fall in both estrogen and progesterone production. The endometrium can no longer be maintained and menstruation occurs. The fall of estrogen production then reactivates FSH secretion, initiating a new cycle of follicular development with estrogen secretion and renewed proliferation of the endometrial lining.

GnRH
A progressive and rapid increase in GnRH pulse frequency occurs during the luteal–follicular transition. GnRH pulse frequency, as estimated by LH pulse frequency, increases from 3 pulses per 24 hours (midluteal phase) to 14 pulses per 24 hours. 18

Gonadotropins
FSH and LH levels rise from their nadir due to the decline in negative feedback from estradiol and inhibin and the rise in activin. 116 The rise in FSH initiates recruitment of gonadotropin-responsive follicles for the next menstrual cycle. This recruitment of antral follicles actually begins at least 2 days before the onset of menstrual bleeding. In fact, an increase in FSH bioactivity can be measured back to the midluteal phase. 117 During the luteal–follicular transition both inhibin A and B levels are at a nadir. 118 In contrast, activin levels begin to increase in the late luteal phase and peak at the time of menstruation. 119 Activin plays an important role as gonadotropin responses to GnRH require the presence of activin. 43

Ovarian Hormones
In the absence of pregnancy corpus luteum function declines approximately 10 days after ovulation. The exact mechanisms for luteolysis are unclear. Luteolysis involves apoptosis and expression of matrix metalloproteinases. 120, 121 Luteolysis may also be mediated by nitric oxide. 122 Nitric oxide induces apoptosis in the human corpus luteum. 123 One of the final signs of luteolysis is ovarian production of prostaglandin F 2α , which inhibits luteal steroidogenesis. Thus, unless the corpus luteum is rescued by the hCG of pregnancy, estrogen, progesterone, and inhibin levels fall as the luteal–follicular transition occurs.


PEARLS

• GnRH-secreting neurons are found predominantly in the arcuate nucleus of the hypothalamus. GnRH is released into the portal circulation at the median eminence in a pulsatile fashion and binds to cell membrane receptors in the anterior pituitary.
• GnRH pulse frequency and amplitude vary during the menstrual cycle. The follicular phase is characterized by high-frequency (q60–90 minutes), low-amplitude pulses; the luteal phase is characterized by lower-frequency (q2–4 hours), higher-amplitude pulses.
• The activity of GnRH-releasing neurons is modified by a variety of factors, including neurotransmitters, glycoprotein hormones, and steroid hormones.
• GnRH has an extremely short half-life (2 to 4 minutes). GnRH agonists and antagonists are characterized by modifications to the native GnRH decapeptide structure that extend its half-life.
• The pituitary gland lies in the sella turcica and is connected to the hypothalamus via the pituitary stalk. The blood supply to the pituitary gland comes from the internal carotid artery. Specialized cells within the anterior pituitary (gonadotrophs) secrete FSH and LH in response to GnRH. Gonadotrophs comprise only 5% of all the cells in the anterior pituitary gland.
• FSH and LH are glycoproteins that share the same α subunit. The half-life of LH (20 to 30 minutes) is much shorter than that of FSH (1 to 4 hours), and LH pulse frequency/amplitude correlates closely with GnRH pulse frequency and amplitude.
• FSH and LH bind to cell membrane receptors that activate adenylate cyclase, thus raising intracellular cyclic AMP. FSH plays a crucial role in folliculogenesis, as well as inducing aromatization of androgens to estrogens. LH plays a crucial role in ovulation and steroidogenesis.
• Certain steroidogenic enzymes are specific to certain ovarian cell types. Only theca cells express P450c17, the enzyme that allows conversion of progestins (21-carbon steroids) to androgens (19-carbon steroids). In turn, only granulosa cells, stimulated by FSH, have the ability to aromatize androgens to estrogens (18-carbon steroids).
• Granulosa cells of preovulatory follicles do not have a direct blood supply; they are separated from theca cells by a basal membrane. In contrast, almost immediately after ovulation capillaries invade the granulosa layer.
• In theca cells of the preovulatory follicle pregnenolone is preferentially converted to 17-hydroxypregnenolone (Δ 5 pathway) on the way to synthesis of androstenedione.
• In the corpus luteum pregnenolone is preferentially converted to progesterone (Δ 4 pathway). Progesterone, 17-hydroxyprogesterone, and inhibin A levels all peak in the luteal phase.
• The lifespan of the corpus luteum is fixed at approximately 14 days. If the corpus luteum is not rescued by hCG, luteolysis and apoptosis are initiated.

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89 Bauer-Dantoin AC, Weiss J, Jameson JL. Roles of estrogen, progesterone, and gonadotropin-releasing hormone (GnRH) in the control of pituitary GnRH receptor gene expression at the time of the preovulatory gonadotropin surges. Endocrinology . 1995;136:1014-1019.
90 Adams TE, Norman RL, Spies HG. Gonadotropin-releasing hormone receptor binding and pituitary responsiveness in estradiol-primed monkeys. Science . 1981;213:1388-1390.
91 Menon M, Peegel H, Katta V. Estradiol potentiation of gonadotropin-releasing hormone responsiveness in the anterior pituitary is mediated by an increase in gonadotropin-releasing hormone receptors. Am J Obstet Gynecol . 1985;151:534-540.
92 Xia L, Van Vugt D, Alston EJ, et al. A surge of gonadotropin-releasing hormone accompanies the estradiol-induced gonadotropin surge in the rhesus monkey. Endocrinology . 1992;131:2812-2820.
93 Leyendecker G, Wildt L, Hansmann M. Pregnancies following chronic intermittent (pulsatile) administration of Gn-RH by means of a portable pump (“Zyklomat”)—a new approach to the treatment of infertility in hypothalamic amenorrhea. J Clin Endocrinol Metab . 1980;51:1214-1216.
94 Caraty A, Antoine C, Delaleu B, et al. Nature and bioactivity of gonadotropin-releasing hormone (GnRH) secreted during the GnRH surge. Endocrinology . 1995;136:3452-3460.
95 Young JR, Jaffe RB. Strength–duration characteristics of estrogen effects on gonadotropin response to gonadotropin-releasing hormone in women. II. Effects of varying concentrations of estradiol. J Clin Endocrinol Metab . 1976;42:432-442.
96 Cahill DJ, Wardle PG, Harlow CR, Hull MG. Onset of the preovulatory luteinizing hormone surge: Diurnal timing and critical follicular prerequisites. Fertil Steril . 1998;70:56-59.
97 Hoff JD, Quigley ME, Yen SS. Hormonal dynamics at midcycle: A reevaluation. J Clin Endocrinol Metab . 1983;57:792-796.
98 Pauerstein CJ, Eddy CA, Croxatto HD, et al. Temporal relationships of estrogen, progesterone, and luteinizing hormone levels to ovulation in women and infrahuman primates. Am J Obstet Gynecol . 1978;130:876-886.
99 Zelinski-Wooten MB, Hutchison JS, Chandrasekher YA, et al. Administration of human luteinizing hormone (hLH) to macaques after follicular development: Further titration of LH surge requirements for ovulatory changes in primate follicles. J Clin Endocrinol Metab . 1992;75:502-507.
100 McClure N, Macpherson AM, Healy DL, et al. An immunohistochemical study of the vascularization of the human graafian follicle. Hum Reprod . 1994;9:1401-1405.
101 Wulff C, Wilson H, Wiegand SJ, et al. Prevention of thecal angiogenesis, antral follicular growth, and ovulation in the primate by treatment with vascular endothelial growth factor Trap R1R2. Endocrinology . 2002;143:2797-2807.
102 Wulff C, Wilson H, Largue P, et al. Angiogenesis in the human corpus luteum: Localization and changes in angiopoietins, tie-2, and vascular endothelial growth factor messenger ribonucleic acid. J Clin Endocrinol Metab . 2000;85:4302-4309.
103 Dickson SE, Fraser HM. Inhibition of early luteal angiogenesis by gonadotropin-releasing hormone antagonist treatment in the primate. J Clin Endocrinol Metab . 2000;85:2339-2344.
104 Lumsden MA, Kelly RW, Templeton AA, et al. Changes in the concentration of prostaglandins in preovulatory human follicles after administration of hCG. J Reprod Fertil . 1986;77:119-124.
105 Yoshimura Y, Santulli R, Atlas SJ, et al. The effects of proteolytic enzymes on in vitro ovulation in the rabbit. Am J Obstet Gynecol . 1987;157:468-475.
106 Edwards RG. Maturation in vitro of human ovarian oocytes. Lancet . 1965;2(7419):926-929.
107 Edwards RG. Maturation in vitro of mouse, sheep, cow, pig, rhesus monkey and human ovarian oocytes. Nature . 1965;208:349-351.
108 Hutchison JS, Zeleznik AJ. The rhesus monkey corpus luteum is dependent on pituitary gonadotropin secretion throughout the luteal phase of the menstrual cycle. Endocrinology . 1984;115:1780-1786.
109 Filicori M, Butler JP, Crowley WFJr. Neuroendocrine regulation of the corpus luteum in the human. Evidence for pulsatile progesterone secretion. J Clin Invest . 1984;73:1638-1647.
110 Sheehan KL, Casper RF, Yen SS. Luteal phase defects induced by an agonist of luteinizing hormone-releasing factor: A model for fertility control. Science . 1982;215:170-172.
111 Bassett SG, Little-Ihrig LL, Mason JI, Zeleznik AJ. Expression of messenger ribonucleic acids that encode for 3 β-hydroxysteroid dehydrogenase and cholesterol side-chain cleavage enzyme throughout the luteal phase of the macaque menstrual cycle. J Clin Endocrinol Metab . 1991;72:326-362.
112 Brannian JD, Shiigi SM, Stouffer RL. Gonadotropin surge increases fluorescent-tagged low-density lipoprotein uptake by macaque granulosa cells from preovulatory follicles. Biol Reprod . 1992;47:355-360.
113 Rojas FJ, Moretti-Rojas I, Balmaceda JP, Asch RH. Regulation of gonadotropin-stimulable adenylyl cyclase of the primate corpus luteum. J Steroid Biochem . 1989;32:175-182.
114 McLachlan RI, Cohen NL, Vale WW, et al. The importance of luteinizing hormone in the control of inhibin and progesterone secretion by the human corpus luteum. J Clin Endocrinol Metab . 1989;68:1078-1085.
115 Molskness TA, Woodruff TK, Hess DL, et al. Recombinant human inhibin-A administered early in the menstrual cycle alters concurrent pituitary and follicular, plus subsequent luteal, function in rhesus monkeys. J Clin Endocrinol Metab . 1996;81:4002-4006.
116 le Nestour E, Marraoui J, Lahlou N, et al. Role of estradiol in the rise in follicle-stimulating hormone levels during the luteal-follicular transition. J Clin Endocrinol Metab . 1993;77:439-442.
117 Christin-Maitre S, Taylor AE, Khoury RH, et al. Homologous in vitro bioassay for follicle-stimulating hormone (FSH) reveals increased FSH biological signal during the mid- to late luteal phase of the human menstrual cycle. J Clin Endocrinol Metab . 1996;81:2080-2088.
118 Roseff SJ, Bangah ML, Kettel LM, et al. Dynamic changes in circulating inhibin levels during the luteal-follicular transition of the human menstrual cycle. J Clin Endocrinol Metab . 1989;69:1033-1039.
119 Muttukrishna S, Fowler PA, George L, et al. Changes in peripheral serum levels of total activin A during the human menstrual cycle and pregnancy. J Clin Endocrinol Metab . 1996;81:3328-3334.
120 Shikone T, Yamoto M, Kokawa K, et al. Apoptosis of human corpora lutea during cyclic luteal regression and early pregnancy. J Clin Endocrinol Metab . 1996;81:2376-2380.
121 Young KA, Hennebold JD, Stouffer RL. Dynamic expression of mRNAs and proteins for matrix metalloproteinases and their tissue inhibitors in the primate corpus luteum during the menstrual cycle. Mol Hum Reprod . 2002;8:833-840.
122 Friden BE, Runesson E, Hahlin M, Brannstrom M. Evidence for nitric oxide acting as a luteolytic factor in the human corpus luteum. Mol Hum Reprod . 2000;6:397-403.
123 Vega M, Urrutia L, Iniguez G, et al. Nitric oxide induces apoptosis in the human corpus luteum in vitro. Mol Hum Reprod . 2000;6:681-687.
Chapter 2 Ovarian Hormones: Structure, Biosynthesis, Function, Mechanism of Action, and Laboratory Diagnosis

Manjula K. Gupta, SuYnn Chia

INTRODUCTION
The main function of the ovaries, maturation and release of oocytes, is accomplished via the production of several steroidal and nonsteroidal hormones that locally modulate a series of complex events. Peripherally, these hormones act on various target organs, including the uterus, vagina, fallopian tubes, mammary glands, adipose tissue, bones, kidneys, and liver, leading to the female phenotype.
The secretion of the ovarian hormones in turn is precisely regulated by the hypothalamic-pituitary axis. The complex interactions and regulations of the hypothalamic, pituitary, and ovarian hormones are collectively responsible for the regular and predictable ovulatory menstrual cycle and fertility in females.

The Ovary as an Endocrine Organ
A single ovarian follicle is regarded as the basic endocrine/reproductive unit of the ovary. It is composed of one germ cell that is surrounded by a cluster of endocrine cells, which are organized in two layers separated by a basal membrane. The inner layer surrounding the oocyte is composed of granulosa cells, and the outer layer is composed of thecal cells. These two cell types provide the basic machinery that is responsible for producing ovarian hormones. These cells are also differentially regulated by the gonadotropins (i.e., luteinizing hormone [LH] and follicle-stimulating hormone [FSH]) and produce distinctly different steroid hormones.
The two-cell theory describes the sequence of events that occurs during ovarian follicular growth and steroidogenesis. According to this theory, LH primarily stimulates thecal cells to produce androstenedione and testosterone, both 19-carbon steroids. In contrast, FSH primarily stimulates granulosa cells to aromatize these 19-carbon steroids into estrogens. 1, 2
The ovarian production of steroid hormones is regulated both within the ovary, by paracrine (intercellular) and autocrine (intracellular) mechanisms, and by endocrine regulation of FSH secretion by the pituitary. Central to this regulation are several nonsteroidal hormones and factors produced by the ovary. 3 This chapter focuses on these aspects of the ovary and discusses the biochemistry, biosynthesis, regulation, and actions of both steroidal and peptide ovarian hormones.

STEROIDOGENESIS AND STEROID HORMONES OF THE OVARY
The ovary contains multiple distinctive steroid-producing cells, including stromal cells, theca cells, granulosa cells, and luteinized granulosa cells. Each cell type contains all the enzymes necessary for synthesis of androgens, estrogens, and progesterone. However, the types of hormones produced vary according to the cell type and the expression of steroidogenic enzymes. Other factors that influence which steroid hormone is synthesized in a given cell include the level and expression of gonadotropin and the availability of low-density lipoprotein (LDL) cholesterol. (As discussed below, steroid hormone synthesis occurs via one of two pathways: the Δ 5 [3β-hydroxysteroid] pathway or the Δ 4 [3 ketone] pathway.)

Steroidogenesis
The ovary, like the adrenal gland, produces all three classes of steroid hormones from cholesterol—estrogens, progesterone, and androgens. In contrast to the adrenal gland, the ovary cannot produce glucocorticoids or mineralocorticoids because it lacks the enzymes 21-hydroxylase and 11β-hydroxylase.
Steroid hormone formation in the steroid-producing endocrine glands follows the same fundamental pathway and mainly relies on exogenous (or plasma) cholesterol, with the exception of the liver and intestinal mucosa, which are capable of synthesizing cholesterol endogenously from acetyl-coA. The primary source of cholesterol for steroidogenesis in the ovary is derived from the uptake of plasma LDL. 4 The rate-limiting step in steroidogenesis is transfer of cholesterol from the cytosol to the inner membrane of the mitochondria. 5 This is mediated by an LH-induced mitochondrial enzyme called steroidogenic acute regulatory ( StAR ) protein . 6 The StAR gene is located on chromosome 8p11.2 and codes for a 285-amino acid precursor protein, of which 25 amino acids are cleaved off after transport to the mitochondria. 7, 8 Nonsense mutations of the StAR gene that result in premature stop codons have been identified as a cause of congenital lipoid adrenal hyperplasia, which is characterized by the presence of intracellular lipid deposits that destroy steroidogenesis. 7
Ovary steroid hormones are synthesized in both interstitial and follicular cells. The basic structure of cholesterol is three hexagonal carbon rings and a pentagonal carbon ring to which a side chain is attached ( Fig. 2-1 ). Two important methyl groups are also attached at positions 18 and 19. Progestins and corticosteroids (pregnane series 21-carbon steroids) are produced by partial cleavage of the side chain (i.e., the desmolase reaction ). Androgens (androstane series 19-carbon steroids) are produced by the total removal of the side chain. Estrogens (estrane series 18-carbon steroids) are produced by aromatization of one of the three hexagonal carbon rings to a phenolic structure with loss of the 19-methyl group.

Figure 2-1 Structures of cholesterol (27 carbons) and of the three major classes of ovarian steroids: the progestins (21 carbons), androgens (19 carbons), and estrogens (18 carbons).
The first step in steroidogenesis is the conversion of cholesterol to pregnenolone via hydroxylation at the carbon 20 and 22 positions, which is followed by cleavage of the side chain ( Fig. 2-2 ). From pregnenolone, steroid hormones are produced by one of two general pathways. 5 The pregnenolone (Δ 5 ) pathway produces androgens and estrogens (pregnenolone→17OH-pregnenolone →dehydroepiandrosterone [DHEA]→testosterone→estrogen). The progesterone (Δ 4 ) pathway produces androgens and estrogens (pregnenolone→progesterone→17OH-progesterone→androgen →estrogen). In the adrenal gland, the Δ 4 pathway produces mineralocorticoids and glucocorticoids.

Figure 2-2 Steroid hormone biosynthesis pathways in the ovary. The Δ 4 pathway converts pregnenolone to progesterone and is the main pathway in the corpus luteum. The Δ 5 pathway converts pregnenolone to androgens and subsequently estrogens and is the preferred pathway in the thecal cells. Also shown are the conversion of 17OH-progesterone to aldosterone and cortisol as a result of CYP21 and CYP11, which occurs exclusively in the adrenal gland. Irreversible reactions are denoted by a single arrow , and reversible reactions are denoted by double arrows .
The enzymes involved in the intracellular synthesis of steroid hormones include five hydroxylases, two dehydrogenases, a reductase, and an aromatase. The hydroxylases and aromatase belong to the cytochrome P450 (CYP) supergene family ( Table 2-1 ). These enzymes exist on both the mitochondria and endoplasmic reticulum.
Table 2-1 Enzyme Reaction and Cellular Location of Steroidogenic Enzymes Enzyme Reaction Gene (Enzyme) Cellular Location/Tissue Location Cholesterol side chain cleavage CYP11A (P450scc) Mitochondria (theca; granulosa) 17α-hydroxylase CYP17 (P450c17) ER (theca) 17,20-hydroxylase (lyase) CYP17 (P450c17) ER (theca) Aromatase CYP19 (P450arom) ER (granulosa) 3β-hydroxysteroid dehydrogenase 3βHSD ER (theca; granulosa) 17β-hydroxysteroid dehydrogenase 17βHSD ER (granulosa) 21-hydroxylase CYP21 (P450c21) ER (adrenal) 11β-hydroxylase CYP11B1 (P450c11) Mitochondria (adrenal)
ER, endoplasmic reticulum
Of these nine enzymes, four key enzymes regulate the main steps of steroidogenesis (see Fig. 2-2 ): CYP11A (P450scc), a side chain cleavage enzyme that catalyzes the conversion of cholesterol to pregnenolone; 3βHSD, or 3 βa-hydroxysteroid dehydrogenase, which converts pregnenolone to progesterone; CYP17 (P450c17), an hydroxylase that converts pregnenolone to androgens; and CYP19 (P450arom), an aromatase that converts androgens to estrogens. Most reactions are irreversible (denoted by a single arrow in Fig. 2-2 ). The few reversible reactions (denoted by double arrows) are dependent on cofactor availability (e.g., NADP/NADPH ratio).
The kind of hormone produced depends on the nature of the cell and the presence or absence of the inherent steroidogenic enzymes in the tissue. The adrenal cortex lacks 17βHSD; hence, adrenal androgen production is limited to DHEA and androstenedione. In the testes, LH controls 17βHSD activity and testosterone production. The steroid-producing cells of the ovary (granulosa, theca, corpus luteum) contain the full enzymatic complement for steroid hormone synthesis. In the thecal cells, LH also controls 17βHSD activity and androstenedione production, whereas CYP19 (P450arom) activity in the granulosa cells is controlled by FSH and hence estradiol production. These relationships are the basis for the two-cell, two-gonadotropin system ( Fig. 2-3 ). Aromatization occurs in the endoplasmic reticulum.

Figure 2-3 Differential regulation by luteinizing hormone (LH) and follicle-stimulating hormone (FSH) of ovarian estrogen, progesterone, and androgen production, the basis of the two-cell, two-gonadotropin system. LH acts on both thecal and granulosa cells; FSH acts only on granulosa cells. FSH and LH stimulate adenylate cyclase via G protein-coupled receptors. Cyclic adenosine monophosphate (cAMP) generated from adenosine triphosphate (ATP) activates protein kinase A, which in turn stimulates steroidogenic enzymes. Gs, G protein; GDP, guanosine diphosphate; GTP, guanosine triphosphate.
In each of the two cell types, the amount of the various enzymes differs depending on the stage of follicle development. CYP11A (P450scc) and 3βHSD are expressed in both thecal and granulosa cells of antral and preovulatory follicles and in the luteinized granulosa and thecal cells of the corpus luteum. In contrast, CYP17 (P450c17) is expressed only in the thecal cells of antral and preovulatory follicles and of the corpus luteum (see Fig. 2-3 ).

Steroid Hormones of the Ovary
On the basis of chemical structure and biologic function, the major steroid hormones synthesized and secreted by the ovaries can be classified into three major types: estrogens, progesterone, and androgens.

Estrogens

Physiologic Role
Estrogens are essential in the development and maintenance ofthe female phenotype, germ cell maturation, and pregnancy. In addition to their reproductive effects, estrogens also have many other nonreproductive systemic effects, such as bone metabolism/remodeling, nervous system maturation, and endothelial responsiveness. 9
At puberty, estrogen stimulates breast development and enlargement and maturation of the uterus, ovaries, and vagina. 10, 11 Estrogen also works in concert with growth hormone and insulin-like growth factor I (IGF-I) to produce a growth spurt and stimulates the maturation of chondrocytes and osteoblasts, which ultimately leads to epiphyseal fusion. 12, 13 After midpuberty, estrogen begins to exert a positive feedback on gonadotropin-releasing hormone (GnRH) secretion, leading to the progressive increase of LH and FSH production, culminating in the LH surge, ovulation, and the initiation of the menstrual cycle.
In the adult female, estrogen plays a critical role in maintaining the menstrual cycle. 14 The cyclical changes in estradiol, progesterone, and pituitary hormones are illustrated in Figure 2-4 . In the early follicular phase of the menstrual cycle, FSH stimulates granulosa cell aromatase activity, resulting in increased follicular concentrations of estrogen. The rising estrogen level further increases the sensitivity of the follicle to FSH and estrogen by increasing the number of estradiol receptors on the granulosa cells. Follicular growth and antral formation is also promoted by estrogen. This sets up a positive feedback cycle, which culminates in one dominant follicle producing an exponential rise in estrogen levels. This exerts a negative feedback on FSH so that falling FSH levels contribute to atresia of other nondominant follicles. The dominant follicle secretes large quantities of estrogen; estradiol levels must be greater than 200 pg/mL for approximately 50 hours before a positive feedback on LH release is achieved. 13, 15 Once the LH surge is initiated, luteinization of the granulosa cells and progesterone production occurs. In pregnancy, estrogen augments uterine blood flow, although it is not required in itself for the maintenance of pregnancy. 16

Figure 2-4 Plasma hormone concentrations (mean ± standard error) during the female menstrual cycle. Graph A, inhibins; Graph B, progesterone and estradiol; Graph C, LH and FSH.
(Data from Groome NP, et al: Measurement of dimeric inhibin B throughout the human menstrual cycle. J Clin Endocrinol Metab 81:1401–1405, 1996.)
In the central nervous system, estrogen withdrawal at menopause has been associated with reduced libido, altered mood, and cognitive disturbances. These effects have been attributed to estrogen’s ability to modulate the synthesis, release, and metabolism of many neuropeptides and neurotransmitters. 17 Estrogen acts as a serotoninergic agonist by increasing serotonin synthesis in the brain, which may positively influence mood. 18 Although prospective observational studies in postmenopausal women have suggested that estrogen replacement therapy might protect against cognitive decline 19 and the development of dementia, 20 randomized trials of estrogen in the treatment of Alzheimer’s disease have shown no evidence of benefit. 21 - 24
In the skeletal system, estrogen antagonizes the effect of parathyroid hormone by directly inhibiting the function of osteoclasts, which decreases the rate of bone resorption and diminishes bone loss. The Postmenopausal Estrogen/Progestin Interventions (PEPI) trial was a prospective, placebo-controlled trial designed to study the effects of hormone replacement on bone density in postmenopausal women. After 12 months of treatment with estrogen, bone mineral density increased by 1.8% at the hip and by 3% to 5% at the spine. 25 The Women’s Health Initiative (WHI) showed that estrogen reduced the risk of both hip and vertebral fractures by 30% to 39%. 26
In the cardiovascular system, there is strong evidence that estrogen has a natural vasoprotective role. At a cellular level, estrogen receptors are found on the smooth muscle cells of coronary arteries 27 and the endothelial cells of various sites. 28 Estrogen causes short-term vasodilation by increasing nitric oxide and prostacyclin release in endothelial cells. 29 Several large observational studies, including the Framingham study and the Nurses Health Study, have shown that cardiovascular incidence rates are lower in premenopausal than postmenopausal women. 30 There was also a significant association between a younger age at menopause and a higher risk of coronary artery disease. 31 These studies led to the conviction that estrogen replacement therapy would consequently prevent the progression of atherosclerosis and coronary heart disease. However, the WHI study and the Heart and Estrogen/progestin Replacement Study (HERS), both large randomized, prospective trials designed to specifically address this issue, have not shown any benefit of estrogen for either the primary or secondary prevention of coronary artery disease, respectively. 26, 32

Biosynthesis and Metabolism
Estrogens are 18-carbon steroids derived from cholesterol (see Fig. 2-1 ). The three forms of naturally occurring estrogen include estrone, 17β-estradiol, and estriol. In nonpregnant females, estrone and estradiol are the main biologically active estrogens secreted by the ovary. Estradiol is almost 2 to 5 times more potent than estrone. 33 The circulating levels of estradiol are 2 to 4 times higher than those of estrone in premenopausal women. Estradiol concentrations in postmenopausal women are one tenth of those in premenopausal women. Estrone concentrations do not differ with menopausal status; thus, over time, the premenopausal estradiol-to-estrone ratio is reversed. 34 In contrast, estriol is not the secretory product of the ovary but is the peripheral metabolite of estrone and estradiol.
The main estrogen in premenopausal women is estradiol, which is produced primarily by the granulosa cells of the ovary. Androstenedione is converted to testosterone via 17βHSD, which is rapidly demethylated at the C-19 position and aromatized to estradiol. Estradiol is also generated to some degree from androstenedione via estrone. Estrone is also a secreted product of the ovary. It constitutes the remaining circulating estrogen (40%) and is mainly derived from the extragonadal peripheral aromatization of adrenal androstenedione. 35 Peripheral conversion of androgens to estrogens occurs in skin, muscle, and adipose tissue and in the endometrium. 36
In the normal adult female, the production of estradiol varies according to the phase of the menstrual cycle. During the mid luteal phase, for example, the production rate is about 100 to 270 μg/day. In comparison, the production rate for androstenedione is about 3 mg/day, and with its peripheral conversion rate to estrone of about 1.5%, it accounts roughly for about 10% to 30% of estrone production per day. Secondary increases in estrone formation occur in patients with polycystic ovaries or with ovarian cancer characterized by increased androgen production. In such patients, the increased estrogen can disturb the menstrual cycle. In postmenopausal women, the ovarian contribution shrinks, leaving estrone, derived from adrenal androstenedione, as the main source of circulating estrogen. 37
In the pregnant woman, the placenta becomes the main source of estrogen in the form of estriol. The placenta is unable to synthesize steroids de novo and depends on circulating precursors from both fetal and maternal steroids. Most of the placental estrogens are derived from fetal androgens (e.g., DHEA sulfate), produced by the fetal adrenal gland. 38 Fetal DHEA sulfate is converted to free DHEA by placental sulfatase and then to androstenedione and testosterone before being aromatized to estrone and estradiol. Finally, it is hydroxylated to form estriol.
Estradiol is rapidly converted in the liver to estrone by 17βHSD. Estrone can be further metabolized via three pathways. First, it can be hydroxylated to 16α-hydroxyestrone, which is then converted to estriol. Estriol is further metabolized by sulfation and glucuronidation, and the conjugates are excreted into the bile or urine. Secondly, estrone can be conjugated to form estrone sulfate, which occurs primarily in the liver. Estrone sulfate is biologically inactive and is present in concentrations that are 10-fold to 20-fold higher than concentrations of estrone or estradiol. 39 Estrone sulfate can be hydrolyzed by sulfatases present in various tissues to estrone and may serve as a reserve of estrogen in an inactive form. Estrone sulfate may be of some importance in assessing estrogenicity in women and can be detected in serum as well as in urine. 40 Thirdly, estrone can also be metabolized by hydroxylation to form 2-hydroxyestrone and 4-hydroxyestrone, which are known as catechol estrogens. These are then converted to the 2-methoxy and 4-methoxy compounds by catechol-O-methyltransferase.

Progesterone

Physiologic Role
Progesterone plays a critical role in reproduction. It inhibits further endometrial proliferation mediated by estrogen and converts the endometrium into a secretory type, preparing it not only to receive the blastocyst for implantation, but also to maintain the pregnancy. It inhibits uterine contractions and increases the viscosity of cervical mucus. Progesterone also inhibits the action of prolactin so that lactation occurs only after delivery. It raises the basal body temperature by about 0.5°C (0.9°F) and increases the sensitivity of the respiratory center to carbon dioxide (CO 2 ), leading to hyperventilation.
During pregnancy, progesterone increases insulin resistance in concert with the production of the other placental counterregulatory hormones, including placental growth hormone, placental lactogens, placental corticotropin-releasing hormone, and cortisol. 41

Biosynthesis and Metabolism
Progesterone is part of the group of 21-carbon steroids, which also includes pregnenolone and 17OH-progesterone. Progesterone is responsible for all the progestational effects, whereas pregnenolone is the precursor for all steroid hormones. 17OH-progesterone has little biologic activity. Progesterone and 17OH-progesterone are mainly produced by the corpus luteum in the luteal phase of the menstrual cycle and by the placenta if pregnancy occurs. Circulating levels of progesterone at concentrations greater than 4 to 5 ng/mL (12.7 to 15.9 nmol/L) are indicative of ovulation. 42
Progesterone is rapidly metabolized by the liver and has a half-life of approximately 5 minutes. It is converted to pregnanediol and conjugated to glucuronic acid in the liver. Pregnanediol glucuronide is excreted in the urine. Pregnanetriol is the main urinary metabolite of 17OH-progesterone.

Androgens

Physiologic Role
In women, androgens originate as 19-carbon steroids from the adrenals and ovaries. The major androgens produced in the ovary, primarily by the thecal cells and to a lesser degree by the ovarian stroma, include DHEA, androstenedione, and a small amount of testosterone. Both DHEA and androstenedione serve as precursors to estrogen synthesis and have little, if any, androgenic activity. However, these biologically inactive androgens are converted by extraglandular metabolism to biologically active androgens such as testosterone and dihydrotestosterone (DHT). Normally, the levels of these potent androgens are low in females and have no significant physiologic function. Excessive production of androgens by the ovary or adrenals has been implicated as the cause of hirsutism and virilization in women. 43 In contrast, in the male the androgens, of which testosterone and DHT are the most crucial, are of primary importance.

Biosynthesis and Metabolism
Androgens are 19-carbon steroids derived from cholesterol. The rate-limiting step in androgen synthesis is the conversion of cholesterol to pregnenolone, which is mediated by the action of LH on the ovary and testes. In a normal ovulatory woman, the ovaries secrete approximately 1 to 2 mg of androstenedione, 1 mg of DHEA, and approximately 0.1 mg of testosterone. The majority (≈0.2 mg) of circulating testosterone is derived from peripheral metabolism of DHEA and androstenedione. Overall, testosterone production in women is about 0.3 mg/day; roughly 50% of this is derived from peripheral conversion whereas the remaining 50% is secreted equally by the ovary and the adrenals. 44
In the male, more than 95% of circulating testosterone is secreted by the testicular Leydig cells. The testes also secrete small amounts of DHT and the weak androgen DHEA and androstenedione. In most androgen target cells, testosterone is converted to the biologically more potent DHT by the enzyme 5α-reductase. In the female, androgens are derived either from the adrenal cortex in the form of DHEA and androstenedione or from the peripheral conversion of these androgen precursors to testosterone and DHT.
Most of the circulating testosterone is metabolized in the liver into androsterone and etiocholanolone, which are conjugated with glucuronic acid or sulfuric acid and excreted in the urine as 17-ketosteroids. Of note, only 20% to 30% of urinary 17-ketosteroids are derived from testosterone metabolism; the rest originate from the metabolism of adrenal steroids.

Transport of Ovarian Steroid Hormones in Plasma
Steroid hormones are not water-soluble and require transport proteins to be carried to their target tissues. The two types of transport proteins are general carrier proteins such as albumin and transthyretin and specific carrier proteins such as thyroxine-binding globulin, sex hormone-binding globulin (SHBG), and transcortin. Both types of proteins are produced in the liver. Less than 2% of ovarian steroid hormones are free in the circulation; the remainder are mostly bound to SHBG and albumin. 45, 46
Sex hormone-binding globulin, a β-globulin of 95 kDa, is synthesized in the liver. Its gene is localized on the short arm of chromosome 17 (p12-13). 47 It is a homodimer composed of two polypeptide chains and has a single binding site for androgens and estrogens. Dimerization is a necessary step in the binding process. 48 The bound and free fractions appear to exist in a steady state of equilibrium. The amount of free fraction depends on the concentration of steroid hormone and on the levels and binding affinities of the binding proteins.
Of all the steroid hormones, DHT has the highest affinity for SHBG. Approximately 98% of testosterone circulates bound to SHBG (≈65%) and albumin (≈33%). Estradiol is primarily bound to albumin (≈60%) but also to SHBG (38%); about 2% circulates as the free fraction. 49 Progesterone, on the other hand, is mainly bound to albumin (≈80%) but also to transcortin (≈18%). Only approximately 0.6% of progesterone is bound to SHBG and about 2% exists in the free state.
The metabolic clearance of these steroids is inversely related to their binding affinity to SHBG. Thus, conditions that affect levels of SHBG (e.g., pregnancy, oral contraceptives) directly affect the levels of free hormone. Because estrogens increase SHBG synthesis and androgens decrease its synthesis, SHBG levels are twice as high in women compared to men. Several other hormones and other factors are known to influence SHBG levels. Thyroid hormones increase its synthesis and release by the liver. 46 Insulin, IGF-I, and prolactin have been shown to inhibit SHBG production in cultured cells. 50, 51 Furthermore, serum concentrations of SHBG are increased in many disease states, including hyperthyroidism and liver cirrhosis. Certain drugs, including estrogen, tamoxifen, and phenytoin, can also increase serum SHBG concentrations. Carrier protein levels are decreased by hypothyroidism, obesity, and acromegaly and by administration of exogenous androgens, glucocorticoids, and growth hormones.
For many years, only the free fraction of testosterone was regarded as the biologically active component. However, researchers noted that steroid hormones bind with greater affinity to their specific carrier proteins and with much less affinity to albumin. In addition, studies of tissue delivery in vivo showed that the dissociation of albumin-bound testosterone can occur rapidly in a capillary bed so that the active fraction can be larger than the free fraction measured under equilibrium conditions in vitro. 52 Thus, unconjugated steroids that are bound to albumin may be treated as free and biologically available. 53, 54
As mentioned above, SHBG levels can be influenced by numerous disease states. As such, changes in SHBG concentrations can result in large shifts in the free and SHBG-bound fractions. Hence, measurement of SHBG is of great clinical interest because it allows more accurate assessment of free hormones. SHBG is measured by a technique called saturation analysis, in which specific binding capacity of 3 H-labeled testosterone is detected. 55, 56 With modifications, this method can also measure the non-SHBG bound fraction (bioavailable). 57 Recently, specific nonisotopic two-site immunoassays for SHBG have become available and are used in most clinical laboratories.

Measurement of Steroid Hormones in Circulation
The technique responsible for the accurate measurement of low concentrations of various steroid hormones and metabolites is competitive inhibition immunoassay or radioimmunoassay (RIA), which was originally described in 1960 by Yalow and Berson. 58 However, the development of steroid immunoassays presented several problems. First, they are not immunogenic and have a similar structure—they all have a same cyclopentahaptene nucleus with only minor structural variations—which makes it difficult to generate specific antibodies. Steroids can be made immunogenic via chemical coupling to a carrier protein known as hapten, and antibodies can be raised by immunization with haptens. 59 However, the site of the steroid where the protein is conjugated has a significant impact on the specificity of the resulting antibody. 59 Antibodies raised to conjugates of BSA coupled at the 19th position show higher specificity than those coupled at the 3rd or 17th positions. 60 For accurate clinical interpretation, it is important to know the cross-reactivity data on each antibody that is selected for a given assay. Most commercial assay reagent manufacturers provide cross-reactivity data, but it may not always be reliable and must be evaluated in the clinical laboratories performing the assay. 61
Second, the high-affinity binding proteins such as SHBG in the serum compete with the antibody and thus interfere with the measurement of steroid molecules by RIA. This makes direct measurement difficult and necessitates a preassay extraction procedure with organic solvents and often a chromatographic separation of the steroid. Alternatively, the use of certain chemicals, such as 8-anilinonaphthalene sulfonic acid can inhibit the binding of steroids to proteins, which allows the direct measurement of steroid hormones without the extraction step. Direct assays are fast and can be automated. Several automated platforms for measuring estradiol, progesterone, and testosterone are commercially available and are used in most clinical laboratories. These assays, however, have a low sensitivity and, when used to measure very low concentrations, have poor reliability. 62 Therefore, they may not be the best choice for clinical applications that require the ability to measure very low hormone concentrations such as estradiol measurement in children and in men (<100 pg/mL), 63 testosterone measurement in children and women(<1.5 ng/mL), 64 or progesterone measurement during ovarian stimulation (<1 ng/mL). 65 To overcome this problem, serum can be extracted with hexane-ethyl acetate (3:2 by volume), dried, and reconstituted in steroid-free serum, which can then be assayed in the automated platform. 66
Gas chromatography combined with mass spectrometry (GC-MS) addresses many of the shortcomings of immunoassays and is considered more reliable and accurate than immunoassays. However, this technique requires multiple steps, including solvent extraction, chromatographic fractionation, and chemical dramatization before instrumental analysis, and it is often less sensitive than some immunoassays. This technique has now been superseded by liquid chromatography combined with tamdem mass spectrometry. This newer technique has a higher sensitivity and throughput than GC-MS and is considered a reference methodology. 67, 68 The technique has been used to simultaneously measure estradiol and estrone in human plasma with no cross-reactivity. 69 These methods are recommended as reference methods and can be used to standardize and validate immunoassays, which provide the simplicity and rapid throughput needed for clinical use.

Estrogens
Measurement of estradiol is important in the assessment of female reproductive function. It can be used as an aid in the diagnosis of infertility and oligomenorrhea and to determine menopausal status ( Table 2-2 ). In addition, measurement of estradiol is widely used to monitor ovulation induction and in vitro fertilization protocols. 70, 71 Estrone levels are of limited clinical value in nonpregnant women because their levels closely parallel those of estradiol, except in the postmenopausal woman, in whom estrone becomes the main form of circulating estrogen. Also, as mentioned above, a specific RIA for the measurement of estrone sulfate has been described and made available commercially. Because estrone sulfate has a large circulating pool, it can serve as a marker of estrogenicity, especially in women on estrogen replacement therapy, in whom estradiol measurements are of little value due to the variable cross-reactivity of conjugated estrogens in the estradiol assays. 72, 73 In pregnant women, estriol is the main form of estrogen produced, and the amount of estrogen secreted increases from microgram quantities to milligram quantities.
Table 2-2 Assay Techniques and Clinical Applications of Ovarian Steroid Hormones Hormone Assay Techniques Clinical Application Estradiol
Radioimmunoassay
Nonisotopic immunoassays
Evaluation of ovarian function
Assessment of menopausal status
Monitoring ovulation induction and in vitro fertilization cycle Estrone sulfate Radioimmunoassay Marker of estrogenicity in women on hormone replacement therapy Progesterone
Radioimmunoassay
Nonisotopic immunoassays
Marker for ovulation
Detection of luteal phase defects
Marker for threatened abortion in early pregnancy Total testosterone
Radioimmunoassay
Nonisotopic immunoassays
Evaluation of patients with hirsutism.
Male infertility Free testosterone
Equilibrium-dialysis
Diafiltration
Evaluation of patients with hirsutism.
Polycystic ovarian syndrome

Progesterone
Although GC-MS has been recommended as the reference method for the measurement of progesterone, 74 immunoassays using steroid-specific antibodies are again the preferred mode of measurement in most clinical laboratories. 75, 76 Both RIA and nonisotopic immunoassays for progesterone are available for commercial use. Progesterone measurement is routinely used to detect ovulation and luteal phase defects (see Table 2-2 ). 77 In the follicular phase, progesterone levels are low (<5 nmol/L or 1.5 ng/mL). In the luteal phase, they range between 9 and 79 nmol/L (3 to 25 ng/mL). As noted, progesterone is required to maintain pregnancy, and progesterone measurement in early pregnancy can be valuable in the diagnosis of defects or threatened abortion. 78

Androgens
The measurement of androgens, including androstenedione and total testosterone or DHT, can also be accomplished using immunoassays. 79 - 81 The one main drawback of these assays is the cross reactivity with other steroid hormones. Interference with cross-reactive testosterone in the androstenedione assay has been overcome by the use of specific testosterone antiserum to neutralize plasma testosterone. 81 Direct measurement of androstenedione and testosterone without extraction is now possible with new commercial assays. However, commercial assays demonstrate high variability, which is greatest in samples from females. 82 Total testosterone in hirsute women overlaps significantly with levels seen in normal women, and measurement of free testosterone correlates better with disease. 83 Free testosterone has been measured by equilibrium-dialysis, which is a time-consuming and difficult technique for most clinical laboratories to perform. An ultrafiltration technique can be used instead, which depends on MPS-1 centrifugal gel filtration devices and correlates well with the equilibrium-dialysis method 83, 84 as well as with GC-MS. 85 Direct, single-step, nonextraction immunoassay methods using 125 I-labeled testosterone analog have been developed commercially and are used in a number of laboratories. The accuracy and validity of this direct assay has been questioned. 86, 87 Alternatively, an indirect parameter of free testosterone—FAI—can be calculated as a ratio of testosterone to SHBG. 88 FAI is a better discriminator of hirsutism than either total testosterone or SHBG levels. 87 - 89 When bound with albumin and transcortin, testosterone dissociates more quickly than when bound with SHBG. This loosely bound teatosterone may be biologically available through dissociation during capillary transit. Cumming and Wall provided evidence for this hypothesis and suggested that this non-SHBG bound testosterone is a marker of hyperandrogenism. 57, 90

Saliva Measurements
SHBG is either undetectable or minimally present in saliva. Thus, this biologic fluid may reflect the free fraction of plasma steroids. Therefore, measurement of steroid hormones in saliva has attracted considerable attention. 91 - 93 The ease of noninvasive collection combined with the simplicity of measurement makes salivary measurement a promising and attractive alternative to measurement of steroids in plasma. In the future, salivary assays may become useful adjuncts to those performed in plasma. 94 - 97

PEPTIDE HORMONES AND INTRAOVARIAN GROWTH FACTORS OF OVARY
The role of gonadotropins and gonadal steroids in the process of folliculogenesis in the ovary is well recognized. However, multiple other phenomena within the ovary suggest the presence of other intraovarian factors that may fine-tune the effects of gonadotropins and gonadal steroids. For example, the initiation and arrest of meiosis and the different rates of follicular growth leading to the selection of a dominant follicle point toward the existence of an intraovarian modulatory system. The concept of a gonadal factor with endocrine action at the pituitary level can be traced back more than 70 years to Mottram and Cramer. 98 The first moiety was identified and named inhibin for its inhibitory effect on the pituitary. 99 Since then, there has been an explosion of information regarding multiple and potential intraovarian regulators and their physiology, biochemistry, and biosynthesis as well as the identification of their receptors. Some of these compounds, which have been the subject of intense investigation, include peptide hormones/growth factors, cytokines, and neuropeptides. These factors may act in either an endocrine, autocrine, or paracrine fashion.

Peptide Hormones of the Ovary: Inhibins, Activins, and Follistatins
The first water-soluble peptide hormone in testicular extracts that exerted selective inhibitory activity at the pituitary level was described in 1932 and termed inhibin . 99 It was not until 1985 (almost 50 years later) that inhibin was isolated and characterized. 100, 101 This was followed by the identification and characterization of two other related peptide factors (i.e., activin and follistatin). 102, 103 With the cloning of inhibin α and β subunit genes, 104 it was recognized that inhibin and activin belonged to the transforming growth factor-β (TGFβ) superfamily of growth and differentiation factors. Both inhibin and activin are of particular clinical interest and have been extensively reviewed 3, 105, 106 in recent years. They have been found to exert numerous different regulatory functions in a wide variety of both normal and neoplastic cells. Follistatin, a glycoprotein that is structurally distinct but functionally closely linked to inhibin and activin, is also discussed here. The site of production and areas of clinical interest of these peptide hormones are listed in Table 2-3 .

Table 2-3 Role of Inhibins and Activins in Reproduction

Inhibins
The primary sources of inhibin production are the granulosa cells of the ovary and the Sertoli cells of the testis. Inhibin is also produced during pregnancy by the fetus, placenta, decidua, and fetal membranes. The main role of inhibin is to selectively suppress the production of FSH by the pituitary. 107, 108 This is achieved by modulating FSH biosynthesis through two main mechanisms: by reducing steady-state FSH mRNA in pituitary gonadotropes 109 and decreasing the stability of FSH mRNA. 110
Inhibin is a 32 kDa glycoprotein heterodimer consisting of 2 subunits, α and β, linked by disulfide bonds. 111 There is a common α subunit but also two types of β subunits known as β A or β B . Thus, the two isoforms of inhibin are denoted inhibin A and inhibin B. As shown in Figure 2-5 , each subunit derives from a separate precursor molecule called prepro-inhibin α (364 amino acid residues), prepro-inhibin β A (424 amino acid residues) and prepro-inhibin β B (407 amino acid residues). These are processed by proteolytic cleavage to yield the mature forms. 104 In addition to the fully mature forms (αβ dimers, Mr∼32,000), larger forms of dimeric inhibins with amino terminally extended α and/or β subunits have been identified in follicular fluid, which also possess FSH-suppressing bioactivity. 112, 113 Furthermore, monomeric forms of both α and β subunits and certain fragments (αN and proαN-αC) generated during subunit processing are present in follicular fluid (see Fig. 2-1 ) and have intrinsic biologic activities distinct from classical inhibin-like bioactivity. 114, 115

Figure 2-5 Activins, inhibins, follistatin, and TGFβ. A schematic representation of the formation of different dimeric proteins from three basic subunits, α, β A , and β B . Inhibins are heterodimers of α and β subunits (α-β A , α-β B ); activins are homodimers of the β subunits (β A β A , β A β B , β B β B ). Links between the units represent disulfide bridges.
Because the circulation contains multiple molecular forms of inhibin, it can be difficult to accurately measure inhibin levels using conventional RIAs. Most lack specificity for dimeric inhibin due to the variable cross-reactivity of monomeric forms and of various fragments with the antibody used in the assay. 112, 116, 117 Also, conventional inhibin bioassays based on FSH suppression or release by cultured pituitary cells lack specificity due to the FSH-regulating activities of follistatin and activins. In addition, bioassays lack the sensitivity necessary to measure inhibin levels in the circulation. The development of two-site immunoassays utilizing αβ dimer specific antibodies overcame these problems and allowed specific measurement of the two forms of inhibin dimers (A and B). 118 Use of these novel two-site assays allows measurement of inhibin levels throughout the normal menstrual cycle. 119
Synthesis of the two isoforms of inhibin differ during the various phases of the menstrual cycle (see Fig. 2-4 ). Inhibin B levels are highest during the luteal–follicular transition and the early follicular phase, and studies on its presence in follicular fluid and basal granulosa cell secretion suggest that it is secreted by small developing antral follicles. 120 In contrast, inhibin A levels in the early and midfollicular phase reflect the sum of FSH- and LH-stimulated inhibin A secretion from all antral follicles. Levels of inhibin A during the late follicular phase mainly reflect secretion from the dominant follicle. Hence, inhibin A and B levels can be used as markers of follicular development. Inhibins have also been investigated as prognostic markers for women undergoing assisted reproductive technologies. In particular, it was suggested that measuring inhibin B levels during the early stages of FSH stimulation for ovulation induction could predict the number of oocytes retrieved and may be useful in monitoring ovarian stimulation for in vitro fertilization. However, because there is a large overlap between normal and subnormal ovarian responses in terms of inhibin B levels, it may be just as effective to obtain Day 3 FSH or perform a clomiphene challenge test. 121, 122 Women in early perimenopause show significant decrease in inhibin B (no significant change in inhibin A and estradiol), which correlates with a mild increase in FSH levels. This perimenopausal decrease in inhibin B precedes a decrease in inhibin A, suggesting that inhibin B may serve as a sensitive marker for the onset of menopause. 123 Studies investigating the role of inhibins in the pathophysiology of polycystic ovary syndrome (PCOS) show conflicting results. Serum inhibin B levels in early follicular phase show significant increase in some studies or no change in others 124, 125 —this awaits future studies to confirm its role in PCOS.
During pregnancy, inhibin A is produced primarily by the fetoplacental unit, whereas inhibin B levels remain low throughout the pregnancy. 126 Because there is a twofold elevation of circulating inhibin A levels in the second trimester of Down syndrome pregnancies, measurement of inhibin A is a clinically useful and important test. 127 When measurement of inhibin A level is added to alpha-fetoprotein, maternal age, and β-hCG (quad screen), the detection rate for Down syndrome increases from 53% to 75%. 128
Inhibin A has also recently been used as a tumor marker for ovarian sex cord tumors. This heterogeneous group of tumors accounts for 7% of all malignant primary ovarian neoplasms. They are composed of granulosa cells, thecal cells, Sertoli cells, Leydig cells, and other nonspecific stromal cells. It is important to distinguish this group of tumors from carcinomas and sarcomas because the former are low-grade tumors with a better prognosis. Inhibin A and its α-subunit have been found to be sensitive immunohistochemical markers of most ovarian sex cord-stromal tumors. 129 Inhibin B, however, has been found to be elevated in both sex cord-stromal and epithelial tumors and hence is of limited value in differentiating between the two entities. In addition, low levels of inhibin A in the cyst fluid of epithelial ovarian tumors has recently been reported to be associated with a worse prognosis. 130
Inhibin B has been found to be the predominant inhibin secreted in males and is produced by the Sertoli cells of the testes. It also has a negative feedback role on FSH from the pituitary, and its production is regulated by spermatogenesis. Inhibin B levels correlate with sperm count and testicular volume 131 - 133 but cannot distinguish between spermatid arrest and obstructive azoospermia—a condition in which sperm counts are normal. 134 As such, it is unlikely to replace testicular biopsy in the evaluation of male infertility.

Activins
Activins are made up of dimers of the inhibin β subunit (β A β A, β A β B , or β B β B ) and have a molecular weight of approximately 25 kDa (see Fig. 2-5 ). 135 They are predominantly produced by the granulosa cells of the ovary. Activin/inhibin mRNA and protein have also been detected in extragonadal sources, including the placental trophoblast and decidua, testes, adrenal cortex, brain, spinal cord, and anterior pituitary. This implies that activin has diverse physiologic roles that are not confined to the reproductive system.
Acting either alone or with FSH, activin exerts an autocrine effect on granulosa cells to promote and maintain granulosa cell differentiation. It promotes FSH receptor expression on small undifferentiated granulosa cells 136 , enhances their response to FSH and LH, and hence increases aromatase activity and estrogen production. 137 This may explain how small preantral follicles progress from a gonadotropin-independent to a gonadotropin-dependent stage of development. After the granulosa cells acquire FSH receptors, further growth and differentiation of those cells to a preovulatory stage would be driven by activin acting in concert with FSH. Activin also inhibits both spontaneous and LH/hCG-induced increases in progesterone output by human follicles, 137 implying that it plays a role in delaying the onset of premature luteinization.
Activin also has a paracrine effect on thecal steroidogenesis by inhibiting thecal androgen output. It has been proposed that at earlier stages of follicular development, when the androgen requirements are low, thecal androgen synthesis is kept in check due to the relative excess of activin over inhibin and follistatin. However, as dominant follicles approach preovulatory status, increasing granulosa cell expression of inhibin and follistatin upregulates thecal androgen synthesis and ensures that the granulosa cells receive an adequate supply of aromatase substrate for conversion to estradiol. Activin stimulates pituitary FSH production, acting as a functional antagonist to inhibin ( Fig. 2-6 ). 102 It achieves this effect by increasing FSH mRNA synthesis as well as by increasing the stability of produced mRNA. Its actions are intimately modulated by intrapituitary concentrations of follistatin, which bind to activin to limit its bioavailability.

Figure 2-6 A diagrammatic presentation of the hypothalamic-pituitary-ovarian axis. Developed follicles secrete steroid hormones (estradiol and progesterone) and peptide hormones (inhibin, activin, and follistatin); all collectively control secretion of gonadotropins. Estradiol and progesterone, depending on concentration, have either positive or negative feedback and can alter the frequency and/or amplitudes of pulses at the level of both the hypothalamus and pituitary.
Free activin levels as measured by competitive protein binding assay, using follistatin as binding protein, show very little change over the menstrual cycle. 138 However, activin levels were elevated throughout the cycle in older versus younger women, suggesting that activin may play an endocrine role in maintaining FSH elevation in reproductive aging. 139 Lower levels of activin are detected in PCOS patients with a simultaneous increase in inhibins and follistatin, suggesting that an imbalance in these hormones may contribute to an abnormal LH/FSH ratio. 125, 140

Follistatin
In the ovary, follistatin is produced by the granulosa cells in antral follicles as well as by luteinized granulosa cells, which are under the positive regulation of FSH. It modulates the function of granulosa cells in favor of luteinization or atresia by neutralizing the effects of activin. It may also directly modulate progesterone metabolism by granulosa cells. 141
Follistatin is a single-chain polypeptide (315 amino acids) that functions as the binding protein for activin, thereby neutralizing it. Most of its biology is explained by its antagonism with activin. Follistatin exists in two forms; as full-length follistatin (FS 315) in the circulation and as processed follistatin (FS 288) in follicular fluid 142 and the pituitary. It is part of an intrapituitary negative feedback loop where activin promotes FSH biosynthesis and the increased expression of follistatin limits its bioavailability for binding to the activin receptor on target cell membranes. It has been shown that the processed isoform of follistatin (FS 288) binds to cell-surface heparan sulfate proteoglycans with a higher affinity than FS 315. 143 Because proteoglycans are anchored to cell membranes, this suggests that this would limit follistatin diffusion from the site of release, leading to high local concentrations of follistatin being maintained. The membrane-anchored follistatin would be able to compete with activin receptors on nearby cells, thus modulating the biologic effects of activin. Once bound to activin, follistatin is able to accelerate the endocytotic internalization and lysosomal degradation of activin by pituitary cells. 144
Follistatin levels as measured by sensitive and specific two-site enzyme immunoassay are significantly higher in women with PCOS in comparison to controls. Higher follistatin levels combined with lower activin levels in these patients suggest its role in the lack of pre-ovular follicle development and FSH suppression. 125

Growth Factors/Cytokines As Intraovarian Regulators
Certain growth factors have been implicated in cellular communications within the ovary, including insulin-like growth factors IGF-I and IGF-II, epidermal growth factor (EGF), transforming growth factor-α (TGFα), basic fibroblast growth factor (bFGF), cytokines such as interleukins (IL-1 and IL-6), and tumor necrosis factor (TNFα). Growth factors and cytokines that are important as intraovarian regulators are outlined in Table 2-4 .

Table 2-4 Autocrine and Paracrine Growth Factors and Cell–cell Signaling in the Ovary

Insulin-like Growth Factors
IGF-I and IGF-II promote cellular mitosis and differentiation in a variety of systems and play an important role in modulating folliculogenesis in an autocrine/paracrine fashion. 145 - 147 Insulin-like growth factors consist of two single-chain polypeptide growth factors that are structurally and functionally similar to proinsulin. The IGF autocrine/paracrine system includes IGFs, their specific receptors in target cells, and a family of IGF-binding proteins that regulate their bioavailability. Both IGF-I and IGF-II are produced in the ovary and augment the effects of the gonadotropins, although the main IGF in human follicles is IGF-II. 148 In small antral follicles, both IGF-I and IGF-II are expressed but restricted to thecal cells. However, IGF-I receptor mRNA has been detected only in granulosa cells. 146 It serves as an autocrine regulator in thecal cells and as a paracrine regulator in granulosa cells. In dominant follicles, however, no IGF-I mRNA has been detected either in thecal or in granulosa cells, and IGF-II expression is restricted to granulosa cells only. IGF-I receptors are present in granulosa cells only, and IGF-II receptors are expressed in both cell types. 146 Thus, in the dominant follicle, IGF-I functions mainly as a paracrine regulator 149, 150 and IGF-II acts as an autocrine factor. This suggests that IGF-II has an important role in coordinating differential follicular development within the ovary.

Epidermal Growth Factor (EGF), Transforming Growth Factor-α (TGFα), and Basic Fibroblast Growth Factor (bFGF)
A number of other peptide growth factors have been implicated as regulators of follicular development and steroidogenesis (see Table 2-4 ). 151 These include EGF, TGFα, and bFGF. EGF is a single-chain polypeptide of 53 amino acids with three disulfide bonds and has mitogenic effects in a variety of ectodermal and mesodermal tissues. TGFα is a 50-amino acid peptide with 30% to 40% homology with EGF. The EGF receptor is a 170-kDa glycoprotein with tyrosine kinase activity. TGFα binds to EGF receptors with the same affinity as EGF.
The presence of EGF/TGFα and bFGF as well as their receptors in the ovary has been shown both at the protein and mRNA levels. The presence of immunoreactive EGF as well as EGF receptors has been identified in the preovulatory follicles 152 and corpus luteum. 153, 154 Furthermore, studies have shown the presence of TGFα 155 and bFGF 156 mRNAs in follicular cells; studies have also shown that the TGFα message is upregulated by FSH in vivo. FSH plus TGFα or FSH plus EGF resulted in significantly elevated progesterone and 20α-hydroxyprogesterone levels in granulosa cells in culture. 157 The presence of TGFα message in cultured granulosa cells and the fact that it mediates its action via binding to EGF receptor all point to its autocrine role in granulosa cell differentiation, follicle development, and selection. Expression of bFGF and its receptor mRNA has been detected in fetal ovaries and in granulosa cells. 158, 159 bFGF has been shown to be mitogenic for granulosa cells and to cause an inhibitory action on granulosa cell differentiation and thecal cell steroidogenesis. 160, 161 It also has potent angiogenic activity. 162

Cytokines
Cytokines primarily produced by white blood cells modulate various cellular functions. The cytokines in the ovary are secreted both by the immune cells that are recruited from the circulation in the ovarian stroma and by the thecal and granulosa cells. A number of cytokines have been linked to modulation of ovarian function, including interleukins IL-1 and IL-6 and TNFα. Both IL-1 and IL-6 have been found in significant amounts in follicular fluid. 163, 164 Granulosa cells accounted for the majority of immunostaining for IL-1 and IL-6 in follicular aspirates, which suggests that these cytokines are produced in granulosa cells 164, 165 and that they affect granulosa function. 166 During folliculogenesis, IL-I promotes proliferation and suppresses differentiation. In the ovulatory process, it promotes ovulation by increasing production of chemokines, steroids, ecosanoids, and vasoactive substances. 167 IL-6 demonstrates inhibitory effects on both estradiol and progesterone secretion by FSH-stimulated granulosa cells. 168 Elevated IL-6 levels during genital infections may provide a possible link to reproductive dysfunction. TNFα expression has also been detected in granulosa cells of human antral and atretic follicles by immunohistochemistry. 169 Also, in vitro treatment with TNFα enhanced steroidogenesis in both healthy and atretic follicles, 170 suggesting that TNFα has a paracrine and/or autocrine role. Nonetheless, the physiologic implications of these actions remain unclear and require further investigation.

Neuropeptides
Some evidence suggests that an independent ovarian-central nervous system axis exists. 171, 172 Electrical stimulation of the hypothalamus in hypophysectomized and adrenalectomized rats produced a change in ovarian steroidal synthesis that was independent of changes in ovarian blood flow. 173 In addition, murine thecal cells can produce androgens under adrenergic stimulation. 174 Adrenergic innervation of the ovary acts primarily on the thecal-interstitial cells through β 2 receptors, synergizing with the effects of gonadotropins in the production of ovarian androgens. 174 This may in turn play a role in the regulation of estrogen production by granulosa cells, thereby influencing follicle recruitment and selection.

REGULATION OF OVARIAN HORMONES
The hypothalamic-pituitary axis plays a key role in the regulation of hormonal synthesis by the ovaries. The hypothalamus is connected to the pituitary gland via a portal vascular system that allows transport of hypothalamic releasing factors from the brain to the pituitary (see Fig. 2-6 ). The hypothalamus, being the coordinating center, provides precise signals via pusatile release of GnRH to the gonadotrophs, which in turn secrete LH and FSH. Any interruption in this connection results in low gonadotropin levels, leading to failure of ovarian hormone secretion.

Hypothalamic Regulation

Gonadotropin-releasing Hormone
The gonadotropin-releasing activity in hypothalamic extracts was first demonstrated in the late 1950s. In 1971, almost 15 years later, GnRH was first isolated and characterized from a hypothalamic extract. This was followed by its synthesis and its clinical availability. However, early clinical studies proved disappointing until the nature of its pulsatile secretion was recognized. In their classic experiment in the rhesus monkey, Knobil and colleagues showed that normal LH and FSH release required the pulsatile infusion of GnRH at approximately 60-minute intervals 175 Furthermore, a change in pulse frequency (lesser or greater) or continuous infusion resulted in failure of LH and FSH secretion and release ( Fig. 2-7 ). 176 These observations were confirmed in humans. Pulsatile GnRH infusion reproduces appropriate patterns of hormonal changes, resulting in ovulation and fertility in women with hypothalamic amenorrhea.

Figure 2-7 Change in luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels in gonadotropin-releasing hormone (GnRH) deficient monkeys, according to the mode of delivery of GnRH. A continuous infusion decreased both LH and FSH levels, which are restored by pulsatile GnRH administration.
(Data from Belchetz PE, et al: Hypophysial responses to continuous and intermittent delivery of hypopthalamic gonadotropin-releasing hormone. Science 202:631–633, 1978.)

Physiologic Role of GnRH
GnRH is produced by secretory neurons located in the arcuate nucleus of the medial basal hypothalamus and the preoptic area of the anterior hypothalamus. The nerve terminals are found in the lateral portions of the external layer of the median eminence adjacent to the pituitary stalk. 177 GnRH has an intrinsically pulsatile pattern of secretion, which is under the control of a hypothalamic pulse generator in the arcuate nucleus. 178, 179 The frequency and amplitude of the pulsatile rhythm of GnRH secretion are crucial in regulating gonadotropin secretion and hence gonadal activity. 180, 181 Physiologic frequency (approximately hourly pulses) tends to upregulate GnRH receptors, enhancing pituitary responsiveness to subsequent stimulation by GnRH. This leads to a “self-priming” effect, whereby LH levels have been shown to increase sequentially with sequential GnRH pulses. A longer frequency causes anovulation and amenorrhea; a shorter frequency or constant exposure to GnRH downregulates the GnRH receptors, inducing refractory gonadotropin responses. 176, 182 - 184
The “pulse generator” is subject to modification by two main inputs: (1) hormone-mediated signals and (2) neural signals. Hormone signals include negative and positive feedback from the gonadal steroids (e.g., estrogen and progesterone) as well as gonadal protein hormones. Neural signals may come from a wide variety of sources and are mediated by neurotransmitters, including acetylcholine, catecholamines, serotonin, opioids, and γ-aminobutyric acid. 185 Norepinephrine is believed to stimulate GnRH release whereas opioids exert inhibitory effects. Dopamine can produce both inhibitory and excitatory GnRH responses, depending on the physiologic state. 186

Biochemistry and Biosynthesis
GnRH is a linear decapeptide, derived from the post-translational processing of a large precursor molecule, prepro-GnRH. The prepro-GnRH molecule consists of 92 amino acids in a tripartite structure ( Fig. 2-8 ). It begins with a signal peptide of 23 amino acids, which is followed by the decapeptide and then a Gly-Lys-Arg sequence needed for proteolytic processing and C-terminal amidation of GnRH molecules. The last 56 amino acid residues are collectively known as the GnRH-associated peptide ( GAP ), which may have prolactin-inhibiting activity. 187, 188 Knowledge of GnRH structure led to the development of many clinically important long-acting GnRH agonists, including buserelin, leuprolide, and nafareline (see Fig. 2-8 ).

Figure 2-8 The structures of native gonadotropin-releasing hormone (GnRH) and some analogs currently in clinical use.
Gonadotropin-releasing hormone is encoded from a single gene on the short arm of chromosome 8p21-p11. The human gene contains 4 exons; exon 2 encodes pro-GnRH, exon 3 and part of exon 2 and 4 encode the GAP protein, and exon 4 encodes a long 3′ untranslated region. Molecular processing occurs primarily in the nucleus of the cell body (soma). After transcription, the mRNA is transported to the cytoplasm where translation takes place and it is converted into the decapeptide. GnRH and its cleavage products, GAP and pro-GnRH, are then transported to the nerve terminals where they are secreted in tandem into the portal circulation. 187, 189, 190

Measurement
GnRH has a short half-life (2–4 min) due to its degradation by peptidases in the hypothalamus and pituitary gland. These peptidases cleave the molecule at the Gly-Leu bond and at position 10. Due to the large dilutional effect, peripheral GnRH serum levels are too low for pulse characteristics to be determined reliably in humans. However, the simultaneous measurement of circulating levels of LH in hypophysial-portal and peripheral blood samples in other species has been shown to correlate closely with GnRH release. 191, 192 As such, frequent measurements of LH pulses can be used as an accurate indicator of GnRH secretion patterns. FSH also correlates with GnRH secretion, but is less useful due to its long half-life.

Regulation by Pituitary Hormones
As mentioned above, GnRH action on gonadotrophs stimulates gonadotropin production and release in a pulsatile fashion. In addition, LH and FSH release from the pituitary is also affected in both a positive and negative manner by estrogen and progesterone as well as by the protein hormones secretion by the ovaries (see Fig. 2-6 ). The positive effect of estrogen and the negative effect of progesterone on the gonadotropins depend on the level of steroid hormone and the duration of exposure to the gonadotrophs. On the other hand, both LH and FSH are required for ovarian estrogen synthesis and the level of estrogen production depends on the time of exposure and the level of gonadotropins. 193, 194 Nonetheless, a disordered signal from the pituitary gland may result in infrequent ovulation ( oligo-ovulation ) or absent ovulation ( anovulation ).

Regulation by Luteinizing Hormone and Follicle-stimulating Hormone

Physiologic Role
As discussed above, LH regulates ovarian steroidogenesis. The surge of LH in the middle of the menstrual cycle is also responsible for inducing ovulation. The surge occurs as a result of a dramatic rise in estradiol produced by the preovulatory follicle, which produces a positive feedback on LH. The midcycle surge stimulates the resumption of meiosis and the completion of reduction division in the oocyte with release of the first polar body. Proteolytic enzymes and prostaglandins are increased in response to LH, leading to a release of the oocyte from the ovary. 195 Finally, the continued secretion of LH after ovulation converts the remaining follicular cells in the ovary to the corpus luteum and stimulates the corpus luteum to produce progesterone by enhancing the conversion of cholesterol to pregnenolone.
Follicle-stimulating hormone regulates ovarian estrogen synthesis by binding to the FSH receptor on the surface of the granulosa cell and is required for follicular maturation and growth. 196, 197 This results in elevated cyclic adenosine monophosphate (cAMP) levels and the induction of aromatase, which converts androstenedione from the neighboring thecal cells to estrone. FSH also induces expression of 17βHSD type 1, which converts estrone to estradiol. Increased secretion of estradiol leads to further proliferation of granulosa cells and follicular growth and an increase in the number of estradiol receptors. 196, 198, 199 In the mature follicle, FSH and estradiol increase the LH receptors’ expression in granulosa cells, making these cells responsive to LH and augmenting progesterone secretion. Progesterone then increases FSH release in midcycle.

Biochemistry and Biosynthesis
The anterior pituitary produces three glycoprotein hormones: LH, FSH, and thyrotropin-stimulating hormone (TSH), all of which share a similar biochemical structure. Each consists of a heterodimer of two noncovalently linked protein subunits, αβ. Each subunit is cysteine-rich and contains multiple disulfide linkages ( Fig. 2-9 ). There are also multiple carbohydrate moieties on both subunits that are important in the metabolism and biologic activity of the hormones. 200, 201 The α subunit is common to all three hormones, whereas the β subunit is unique.

Figure 2-9 A schematic presentation of the gonadotropin subunits, showing the sizes, locations of carbohydrate side chains, and currently known mutations and polymorphisms: The symbols Y and O represent the locations of the N-linked and O-linked carbohydrate side chains, respectively. The symbol S represents disulfide bonds. The arrows indicate the positions of point mutations/polymorphisms. The numbers below the right end of the bars indicate the number of amino acids in the mature subunit protein. Cα, common α subunit; LHβ, luteinizing hormone β subunit; FSHβ, follicle-stimulating hormone β subunit; HCGβ, human chorionic gonadotropin β subunit.
(Adapted from Huhtaniemi I: Functional consequences of mutations and polymorphisms in gonadotropin and gonadotropin resistant genes. In Leung PCK, Adashi E (eds): The Ovary, 2nd ed. London, Elsevier Academic Press, 2004, p 56.)

α Subunit
The human α subunit gene is located on chromosome 6p21.1-23 and is composed of 4 exons. The first exon is noncoding. The gene encodes a 14-kDa polypeptide that consists of a 24-amino acid signal peptide and the mature α subunit of 92 amino acids with 10 cysteine residues and two N-linked oligosaccharide groups. 202 The cysteine residues participate in the intrasubunit disulfide linkages. The α subunit is more abundant than the β subunit, and unassociated, or “free,” α subunits are present in the serum and pituitary. They have little known biologic activity. Hence, only the αβ heterodimer possesses biologic activity.

β Subunit
The β subunits for LH and FSH are encoded by separate genes and are located on different chromosomes. The gene coding for the LHβ subunit consists of three exons and is present in a complex gene cluster on human chromosome 19q13.3. 203 The cluster includes six chorionic gonadotropin β (CGβ) genes that are presumably derived from single ancestral LHβ gene by gene duplication. 204 Both LHβ and CGβ proteins are structurally and functionally similar. They are approximately 80% homologous in amino acid sequence. Both propeptides contain a 20-amino acid signal sequence. The mature LHβ and CGβ subunits consist of 121 and 145 amino acids, respectively. The major difference between LHβ and CGβ proteins is the presence of a 24-amino acid C-terminal peptide in CGβ, which is heavily glycosylated, with four O-linked carbohydrate moieties ( Fig. 2-10 ).

Figure 2-10 Basic structure and general mechanism of action for cytoplasmic/nuclear steroid hormone receptors. A, Basic structure of steroid hormone receptor. B, Mechanism of action involving steps 1–6 as described in the text. HSP90, heat shock protein; HRE, hormone response element; CA, coactivators.
The gene for the FSHβ subunit is located on the short arm of chromosome 11p13 and, like the gene for LH, it consists of three exons. 205 The molecular size of FSH is 33 kDa, consisting of a signal peptide of 18 amino acids and the mature FSHβ protein of 111 amino acids. Like α subunits, both LHβ and FSHβ subunits contain 10 cysteines for disulfide formation. Compared to FSHβ and CGβ subunits, LH does not have a terminal sialic acid on its carbohydrate side chain. This results in a shorter metabolic clearance time for LH, compared to FSH and HCGβ subunits. HCGβ has the highest content of sialic acid and has the longest half-life. 206, 207 Deglycosylation of gonadotropins has no effect on receptor binding but abolishes signal transduction. 208

Mutations in Gonadotropin Genes
Although rare, mutations in gonadotropin genes can result in clinical disorders, which have been described in the literature. In fact, the subject has been recently reviewed extensively. 201, 209 There are several reports of neutral polymorphisms, but no activating mutations have been reported so far in the α subunit gene. 209
Examples of mutations in the LHβ subunit include a single amino acid substitution (Glu to Arg) at codon 54 210 that is associated with hypogonadism in homozygous males and with a high incidence of infertility in heterozygotes. In addition two point mutations—Trp to Arg at codon 8 and Ile to Thr at codon 15—have been described in five women with immunologically anomalous LH but with hyperbioactivity. 211 However the direct relationship of these mutations with a specific pathogenesis remained elusive. The other variant of LHβ (polymorphism) with substitution of Serine to Glycine at codon 102 in exon 3 has been found to be population-specific in 4% of women in Singapore with menstrual disorders.
Several inactivating mutations have been described in the FSHβ subunit gene (see Fig. 2-9 ). The first mutation reported was a homozygous 2bp deletion in a codon 61 (Valine) in a woman who presented with delayed puberty, amenorrhea, and infertility. 212 The mutation was a nonsense mutation leading to altered amino acid sequence after codon 60, leading to a stop codon at residue 87. The mutated protein was altered and was thus unable to associate with the α subunit and was nonfunctional. The patient was treated with exogenous FSH, leading to follicle maturation and pregnancy. Another case report on FSH mutation with a similar phenotype was found to be heterozygote for two FSH mutations: the first was a nonsense mutation at codon 61 and the second was a substitution of Cysteine to Glycine at codon 51. The loss of the cysteine residue was likely responsible for the altered conformation change and loss of function leading to the phenotype. 213 An inactivating mutation leading to the substitution of Cysteine to Arginine residue at codon 82 was described in an infertile man. 214

Measurement of LH and FSH
It has been difficult to develop highly specific immunoassays for the gonadotropins due to the high homology of the glycoprotein hormones as well as the need to distinguish between free α subunits and intact hormones. Cross-reactivity with the α subunit has made it difficult to accurately measure LH and FSH with RIAs that use polyclonal antibodies. 215 - 217 In addition, there is microheterogeneity in both pituitary and circulating gonadotropins due to the degree of glycosylation. Variation in oligosaccharide content and structure also causes charge microheterogeneity, resulting in serum isoforms that separate over the pH range of 6.5 to 10 on electrofocusing. 218 Although the clinical significance of this microheterogeneity of circulating isoforms remains unknown, it affects immunoreactivity as detected by various antibodies, all of which lead to variability among different immunoassays. Microheterogeneity also affects the biologic activity and may be primarily responsible for the discrepant results generated by immunoassays versus bioassays.
The other problem stems from a lack of a suitable reference preparation for both immunoassays and bioassays. The international reference preparation (IRP) of purified human urinary menopausal gonadotropins (2nd IRP-hMG) was established by the World Health Organization (WHO) and has been widely used in most immunoassays and bioassays. The unitage assigned to IRP was defined by bioassays and formed the basis of all subsequent purified pituitary preparations as, for example, the WHO international standard (2nd IS). Most commercial assays are calibrated against 2nd IS. Although the biologic potency estimates are given for the IRP or IS, the immunoreactivity varies in different immunoassays depending on the antibody specificity.
In recent years, two-site directed immunoradiometric (IRMA) or immunochemiluminometric (ICMA) assays have been developed and are based on the use of two monoclonal antibodies. These have helped overcome most limitations of RIAs. 219 These assays are automated and show exquisite sensitivity approaching 0.1 mIU/mL. Furthermore, these assays are not affected by the presence of free α subunits and correlate better with bioassays. The high sensitivity of these assays also allows detection of low levels seen in early puberty.
Luteinizing hormone bioassays utilize dispersed mouse or rat Leydig cells or a Leydig cell tumor cell line (MA-10) in culture 220, 221 and measure testosterone production in vitro. These assays measure the biologic activity of circulating LH under physiologic conditions. This is important because the biologic activity of LH changes with alterations in glycosylation and the tertiary structure of the molecule. The combination of bioassay and RIA permits the calculation of bioactive/immunoreactive ratios, which can provide a useful index of qualitative changes of the LH molecule. 219, 222 Although the bioactive and immunoactive LH profiles are generally well correlated during physiologic changes, significant discrepancies can occur in some pathologic states. For example, inactivating mutations in the LHβ gene result in elevated levels of immunoreactive LH but a marked loss of bioactivity. In vitro bioassays for FSH activity using rat granulosa cells or Sertoli cells measure production of cAMP or aromatase activity in response to FSH. The sensitivity of these assays is about 2.5 mIU/mL. Alhough in vitro bioassays have been valuable in elucidating the physiology, they remain cumbersome and time consuming and are not practical for routine clinical use.
Measurement of LH and FSH is useful in the diagnosis of gonadal function disorders ( Table 2-5 ). Elevated levels of FSH generally indicate ovarian failure but may be seen in some patients with viable ovarian follicles. 223 Although rare, high gonadotropin levels associated with gonadotropin-secreting pituitary tumors or ectopic gonadotropin-producing tumors. In an amenorrheic patient, an elevated LH level with normal FSH and LH-to-FSH ratio typically (but not invariably) of greater than 2 is suggestive of PCOS. Low levels of these hormones are indicative of pituitary or hypothalamic dysfunction and occur together with low serum estradiol levels. For further assessment of pituitary reserve, provocative GnRH testing is required. LH and FSH responses to intravenous injection of 100 μg GnRH are measured at 20 and 60 minutes. Lack of response may suggest the likely diagnosis of hypogonadotropic hypogonadism; however, its sensitivity and specificity are low in patients receiving exogenous sex steroids. 224
Table 2-5 Role of Pituitary Hormones in Assessment of Female Infertility Hormone Hormone Levels Interpretation Prolactin ↑Prolactin Evaluate for prolactinoma after excluding hypothyroidism, pregnancy, macroprolactinemia as cause. LH and FSH ↓ LH, ↓ FSH Hypothalamic or pituitary disease ↑ LH, ↑ FSH Premature ovarian failure ↑ LH, ↓ or normal FSH Polycystic ovary syndrome

Prolactin

Physiologic Role of Prolactin
The main role of prolactin is the stimulation of lactation in the postpartum period. Its effect is blunted by estrogen and progesterone; the decrease in both these hormones after parturition allows lactation to occur. Prolactin also acts at the hypothalamus to suppress gonadotropin production, primarily by inhibiting the pulsatile secretion of GnRH. Thus, conditions associated with hyperprolactinemia can cause hypogonadism, which leads to shortened luteal phases, decreased numbers of granulosa cells, anovulation, and amenorrhea in women. In men, excess prolactin can lead to decreased spermatogenesis, decreased libido, impotence, and infertility.

Biochemistry and Biosynthesis
Prolactin is a single polypeptide with a molecular weight of 22 kDa. It consists of 198 amino acids and is folded into a globular shape connected by disulfide bonds. It is remarkably homologous to human growth hormone (hGH) and human placental lactogen (hPL). The gene for prolactin is found on chromosome 6, and seems to have been evolutionarily derived from a common somatomammotropic (hGH-hPRL-hPL) precursor.
It is produced by lactotrophs in the pituitary, which make up almost 50% of the total pituitary cell population. Its production is under tonic inhibition by dopamine, produced by the tuberoinfundibular cells, and the hypothalamic tuberohypophyseal dopaminergic system. Prolactin is extremely heterogeneous and exists in at least four different molecular forms 225 - 227 : (1) little prolactin, molecular weight (MW) 23 kDa, a nonglycosylated monomeric hormone with high receptor binding and bioactivity; (2) G, or glycosylated prolactin, MW 25 kDa, which has reduced immunoreactivity; (3) big prolactin, MW 50 kDa, consisting of a mixture of both dimeric and trimeric forms of G prolactin; and (4) big-big prolactin, MW 100 kDa, consisting of G prolactin covalently coupled with an immunoglobulin, also known as macroprolactin. The big and big-big forms have lower receptor-binding affinity, but may be converted to little prolactin by reduction of the disulfide bonds. As such, discrepancies can exist between measured prolactin levels and the clinical effects.

Measurement
Prolactin levels are measured by use of IRMA and ICMA. These methods give excellent reproducibility, sensitivity, and assay efficiency; however, they vary in their abilty to react with biologically inactive macroprolactin. Hence, a measured serum immunoreactive prolactin level often does not correlate with expected clinical effects. A polyethylene glycol precipitation method should be used to detect the macroprolactinemia. 228 The other caveat is that these samples are usually assayed at a single dilution. As such, extremely high levels of prolactin (≈1000 ng/mL), such as those seen in macroprolactinomas, may saturate both capture and localizing antibodies, leading to a falsely low value in some one-step sandwich immunoassays. This has been known as the hook effect . Thus, in patients with macroadenomas, a 1:100 serial dilution should be performed if the assay is prone to this hook effect.

MECHANISMS OF ACTION OF HORMONES
Ovarian/pituitary hormones circulate in very low concentrations in the extracellular fluid, generally in the range of 10 −15 to 10 −9 mol/L. To exert their biologic effects on the target cells, special recognition mechanisms are required. Target cells are able to discriminate between the different hormones at low concentrations by depending on cell-associated recognition molecules called receptors.
Hormones exert their biologic effects by interacting with these high-affinity receptors, which in turn trigger one or more effector systems within the cell. The high affinity, specificity, and receptor expression level together define the nature and degree of biologic response of a hormone. All receptors have at least two functional domains, a recognition domain and a signal-generating domain. The recognition domain binds to the hormone, and the second domain generates a signal that couples hormone recognition to some intracellular function. This coupling of hormone binding to signal transduction, or receptor–effector coupling , provides the first step in the amplification of a hormonal response and distinguishes the target cell receptor from the plasma carrier proteins that bind hormone but do not generate a signal.
Just on the basis of the location of the hormone receptor (i.e., intracellular/nuclear or cell surface), two distinct mechanisms of hormone actions can be classified. These mechanisms further differ by the nature of the signal transduction pathway or second messenger responsible for mediating hormone action ( Table 2-6 ). Examples of nuclear receptors include steroid hormones that are lipophilic and pass through the cell membrane to interact with receptors located either within the cytoplasm or the nucleus. This in turn affects gene transcription within the nuclear compartment. Polypeptide hormones (i.e., LH, FSH, hCG, GnRH, inhibins, and activins) and growth factors that are hydrophilic interact with cell-surface receptors that are located on the plasma membrane. They trigger a plethora of signaling activity in the membrane and cytoplasmic compartments as well as exerting parallel effects on the transcriptional apparatus in the nuclear compartment. These cell surface receptors can be further classified based on the second messenger into four major subgroups, as listed in Table 2-6 .
Table 2-6 Classification of Receptors for Steroid and Peptide Hormones Hormones that Bind to Intracellular/Nuclear Receptors Hormones that Bind to Cell Surface Receptors
Type 1 receptor, classical steroid hormone receptor
Requires ligand binding for activation
Homodimerization pattern
Estrogen receptor
Progesterone receptor
Androgen receptor
Glucocorticoid receptor
Mineralocorticoid receptor
Seven-transmembrane domain receptors
Second messenger is cAMP
LH
FSH
Second messenger is calcium and/or phosphatidylinositols
GnRH
TRH
Second messenger is cGMP
Nitric oxide
Atrial natriuretic factor
Type 2 receptors
Able to bind DNA in the absence of ligand, exerting repressive effect
Heterodimerization pattern
Thyroid Hormone
Retinoic Acid Receptor
Vitamin D receptor
Retinoid X receptor
Orphan receptor
A. Ligand unknown
Single-transmembrane domain receptors
Intrinsic kinase activity
Second messenger is tyrosine kinase
IGF
Insulin
EGF
Second messenger is serine kinase
Activin
TGFβ
Aquired kinase activity by interaction with transducer molecules
Growth hormone
Prolactin

Steroid Hormone Action

Nuclear Receptors Superfamily
Steroid hormone nuclear receptors (estrogen receptor [ER], progesterone receptor, and androgen receptors) are ligand-inducible transcription factors that regulate the expression of target genes involved in reproduction and metabolism. They belong to the superfamily of nuclear hormone receptors and share many structural and functional features. 229 Other members of the superfamily include receptors for glucocorticoids, mineralocorticoids, thyroid hormones, 1,25-dihydroxy vitamin D 3 , retinoic acid, and an ever-increasing number of orphan receptors, which show structural similarity but for which ligands are not known.
Within this nuclear receptor superfamily, three main groups have been identified based on the differences in their functional and recognition characteristics 230 : type 1, or steroid receptor subclass; type 2, or thyroid/retinoid/vitamin D 3 receptor subclass; and a third subclass of orphan receptors.

Type 1 (“Steroid” or “Classical”) Receptor Subfamily
This includes the ER as well as the progesterone, androgen, glucocorticoid, and mineralocorticoid receptors. These receptors cannot bind to DNA in the absence of ligand and thus remain functionally silent. They exist as cytoplasmic/nuclear, multimeric complexes that are in association with heat shock proteins (e.g., HSP90, HSP70, and HSP56). The association of the ligand with the receptor and dissociation of the heat shock proteins are required for activation of the receptor.

Type 2 Receptor Subfamily
This includes the thyroid hormone, vitamin D 3 , and retinoic acid receptors, as well as the retinoid X receptor. In the absence of ligand, these receptors can bind to DNA and exert the repressive effect or silence their respective promoters. Unlike steroid receptors, type 2 receptors bind constitutively to response elements and are capable of forming heterodimers with the retinoid X receptor. These interactions may serve to modulate the amplitude of the transcriptional response to the ligand.
Most of the early knowledge about steroid hormone mechanism of action has been derived from in vitro and in vivo binding studies that used radiolabeled estradiol as a ligand. 231 The role of ERs in the regulation of breast cancer growth has been well studied over the past four decades; it was the first steroid receptor to be discovered in the early 1960s. This led to the subsequent elucidation of a general pathway for steroid hormone action. In general, nuclear receptors share a common protein architecture consisting of five functional domains 232 as illustrated in Figure 2-10A .

Amino-Terminal Transactivation Domain (A/B Region)
This is the most variable domain in terms of sequence and length in the family of nuclear receptors. It can range in size from 20 amino acids in the vitamin D 3 receptor to 600 amino acids in the mineralocorticoid receptor. It usually contains a transcription activation function (TAF-1), which interacts with other core transcriptional machinery (e.g., coactivators) to activate target gene transcription.

Central DNA-binding Domain (C Region)
This region is essential for activation of transcription. It encodes two zinc finger motifs and has a very high degree of homology among all types of nuclear receptors. Hormone binding to the receptor induces specific conformational changes in this region, allowing receptor binding to the hormone-responsive element (HRE) of the target gene. The amino acid sequence that lies between the first and second zinc fingers (i.e., the recognition helix ) is responsible for establishing specific contact with the DNA. The second zinc finger stabilizes the contact and increases the affinity of the receptor for the DNA.

Hinge Region (D Region)
As the designation hinge indicates, this region is the site of rotation that allows the protein to alter its conformation after ligand binding. It provides the localization signal that is important for moving the recptor to the nucleus in the absence of ligand and contains a nuclear localization domain (glucocorticoid and progesterone receptors) and/or a transactivation domain (thyroid hormone or glucocorticoid receptors).

Carboxy-Terminal Ligand-Binding Domain (E Region)
This region is responsible for binding of the relevant ligand and is responsible for receptor dimerization or heterodimerization. It also contains the binding site for heat shock proteins and has a transactivation function (TAF-2) that can drive transcriptional activity. Unlike TAF-1, its transcriptional activity depends on hormone binding. Conformational change that occurs after ligand binding is responsible for interaction with coactivators or corepressors.

Cellular Mechanism of Action of Steroid Hormones
The steroid hormone nuclear receptors are known as ligand-dependent transcription factors. Binding with their ligands is a necessary step for their function as transcriptional regulators. Unliganded receptors may be localized to either the cytoplasm (e.g., glucocorticoid receptor) or the nucleus (e.g., estrogen, progesterone, and thyroid hormone receptors). For most steroid hormones the unliganded receptors exist in the cell nucleus as large molecular weight oligomers (≈300K; 7-10S sedimentation rate) 233 and can be isolated in cytosolic fraction from cells or tissues disrupted in hypotonic media. The oligomers are formed by noncovalent association of a monomeric receptor protein with a dimer of heat shock protein (HSP90, HSP70, or HSP56). 234
The general features of the mechanism of action of these hormones are depicted in Figure 2-10B . Steroid hormones that freely diffuse through the cell membrane bind to the specific receptors in the nucleus. Ligand binding to receptor initiates the receptor transformation, or so-called activation process. During this process the receptor undergoes conformational changes that primarily occur as a result of its dissociation from HSP, which exposes the DNA-binding site. Nuclear translocation and dimerization of the activated receptor then occurs. Most evidence suggests that this process is thermodynamically irreversible. The hormone receptor complex then binds to a specific region of DNA, the HRE, which is located upstream of the gene. The first HRE was identified for the glucocorticoid receptor. Later the HREs for the progesterone, androgen, estrogen, and mineralocorticoid receptors were shown to be similar to that of the glucocorticoid receptor. 235 - 237 The steroid HREs in the target genes are a palindromic (inverted-repeat) DNA sequence of 15 base pairs ( Table 2-7 ). This interaction leads to the recruitment of a host of ancillary factors known as coregulators (coactivators or corepressors), creating a transcriptionally permissive or nonpermissive environment at the promoter, as well as communicating with other general transcription factors and RNA polymerase II. Coactivators function as adaptors in a signal transduction pathway. The binding of these coregulators modulates the resulting transcription (i.e., the activation and inactivation of specific genes). Hormone antagonists, for example, induce a different conformation in the TAF-2 that hinders the coactivator-binding site and recruits a corepressor instead, and inhibits gene expression. The availability of these coregulators in different tissues plays an important role in defining the biologic response to both steroid hormone agonists and antagonists. 238
Table 2-7 Sequence of DNA Recognition Elements for Steroid Hormone Receptors Steroid Hormone Receptor Element DNA Recognition Sequence Estrogen receptor ERE
AGGTCAsssTGACCT

Progesterone receptor
Androgen receptor
Glucocorticoid receptor
Mineralocorticoid receptor HRE
AGAACAsssTGTTCT

Sequences read 5′-3′ direction indicated by arrows. S indicates spacer nucleotides (A, G, C, or T).
The biologic activity of a hormone is determined by at least four factors intimately related to the structure of the hormone receptor in question. The first factor is the affinity of the hormone for the hormone-binding domain of the receptor. The second factor is the differential expression of receptor subtypes in the target tissue, altering the response to the same hormone. The third factor is the conformational shape of the ligand–receptor complex and its consequent effects on dimerization and the modulation of adaptor proteins. The last factor is the differential expression of target tissue adaptor proteins and phosphorylation. A higher concentration of coactivators or corepressors in the target tissue can affect the cellular response of that tissue to the same ligand. Phosphorylation of the receptor by protein kinases increases the transcriptional activity of the receptor.

The Estrogen Receptor

Structure and Function
The structure of ER (now known as ERα) was reported in 1986. 239 It consists of five components or domains that are divided into six regions, referred to as A-F ( Fig. 2-11 ), instead of the five regions seen in most steroid receptors. The F region is a C-terminal segment of 42 amino acids that influences the conformational changes that occur after estrogen/antiestrogen binding. Thus, it modulates the level of transcriptional activities, most likely by affecting the interaction with coregulator proteins. It has a molecular weight of 66,000 and contains 595 amino acids. ER mRNA is 6.8 kilobases and contains 8 exons derived from a gene located on the long arm of chromosome 6. More recently a second form of ER has been discovered and named ERβ; it is encoded by a gene located on chromosome 14 240 and is in close proximity to the genes that are related to Alzheimer’s disease. 241

Figure 2-11 A schematic diagram of two estrogen receptor (ER) isoforms. Different domains (A-F) and their corresponding functions are illustrated. ER-α, estrogen receptor α; ER-β, estrogen receptor β.
The two receptors show a high degree of homology in the DNA-binding domain (97%) and ligand-binding domain (59%) but less so in hinge (30%), regulatory (17%), and F regions (17.9%) 241, 242 (see Fig. 2-11 ). Hence, the binding characteristics of these two receptors are similar, although they differ significantly in their ability to activate gene transcription by regulatory domain TAF-1, which is minimal or absent in ERβ. Both ERα and ERβ are required for normal ovarian function, as shown by specific receptor knockout studies in mice. 16 ERα is primarily responsible for estrogenic effects in other tissues, including the uterus.
17β-estradiol binds to the estrogen receptor with a much higher affinity than estrone or estriol. In addition, the binding of estradiol to its receptor and its subsequent activation also enhances cooperativity , meaning that the action of estradiol binding to one site increases the affinity for it to bind to another site, enabling the receptors to respond to small changes in hormone concentration. Estrogen’s relatively long duration of action is also due in part to the high-affinity state achieved by its receptor. On the other hand, clomiphene exerts its antiestrogenic effects by negative cooperativity , preventing the transition of the estrogen receptor from its low-affinity state to its high-affinity state.
The two receptors (ERα and ERβ) are differentially expressed in different tissues, leading to the differences in the response to the same hormone subtypes. 243, 244 The α receptors are predominantly expressed in breast cancer tissue, ovarian stroma, and endometrium. ERβ receptors, on the other hand, are expressed in several nonclassic target tissues, including the kidney, intestinal mucosa, lung, bone, brain, endothelial cells, and the prostate gland. 17β-estradiol and estrone have a higher affinity for α receptors and thus exert their effects predominantly on target tissue with α receptor expression. Phytoestrogens such as genistein and coumestrol, on the other hand, bind predominantly to β receptors 245 and would be expected to exert their effects on target tissues expressing these receptors.
The conformational change of the ligand-binding domain also differs in both α and β estrogen receptors, depending on which ligand has been bound to the receptor. 244 This distinct conformational change is the major factor that determines the receptor’s ability to interact with coactivators or corepressors. For example, estradiol activates transcription when it binds to ERα but inhibits transcription when bound to ERβ. Raloxifene and tamoxifen, on the other hand, inhibit transcription when forming complexes with ERα and activate transcription when bound to ERβ.
The differential expression of target tissue adaptor proteins and phosphorylation also affect gene transcription. A higher concentration of coactivators or corepressors in the target tissue can affect the cellular response of that tissue to the same ligand. Phosphorylation of the receptor by protein kinases increases the transcriptional activity of the receptor. For example, growth factors such as epidermal growth factor and IGF-I can stimulate protein kinase phosphorylation, activating the estrogen receptor—even in the absence of estrogen.
Somatic mutations in ERα are described and may be associated with certain disease states. A nonsense mutation (premature stop codon) in ERα has been described in a patient with decreased bone mineral density, increased bone turnover, and incomplete closure of bone epiphyses, which demonstrates the role of the ERα in bone growth and homeostasis. 246 ER mutations have also been detected in patients with breast cancer. Such mutations include exon 5 deletion within the ligand-binding domain, leading to a constitutively active receptor, and exon 7 deletion, displaying dominant negative activity and inhibition of ER function. 247

Antiestrogens and ER
Compounds with antiestrogenic activity can be classified into two categories: those with pure antiestrogenic activity and those with both agonist and antagonist properties. Tamoxifen—an antiestrogen that is used both as a chemopreventive agent and as a hormonal therapeutic agent for breast cancer—inhibits ER action. 248
Paradoxically, tamoxifen acts as an estrogen in uterine tissue, and this tissue-specific estrogenic effect is the reason why prolonged tamoxifen therapy increases the risk of uterine cancer. 249 Raloxifene, a related benzothiophene analog, retains its antiestrogenic effect in breast and uterine tissue. Both tamoxifen and raloxifene have estrogen-like effects on nonreproductive tissues such as bone and heart and lung tissue. 250
Tamoxifen acts by competing with estrogen for receptor binding. Because estrogen-binding affinity is of higher magnitude than tamoxifen, severalfold higher concentrations of tamoxifen are required to inhibit estrogen action. The agonistic or antagonistic effect of tamoxifen is determined by the presence of different promoter elements in the specific cell type. 251 Estrogen binding to receptors activates both transcription domains (i.e., TAF-1 and TAF-2). Tamoxifen’s agonistic activity is due to activation of TAF-1, and its antagonistic activity is due to its ability to inhibit the estrogen-dependent activation of TAF-2. The ligand-binding sites for estrogen and antiestrogen are not identical, and tamoxifen binding on receptors induces conformational changes that alter interaction with estrogen-associated proteins and modulates transcriptional activity. 251, 252 Tamoxifen also activates ER-mediated induction of promoters that are regulated by the TAF-1 site, which explains why it has estrogenic effects on the endometrium—a tissue with significant TAF-1 transcription function. In other cell types, such as those in the breast, TAF-1 has weak transcriptional activity. Hence, antiestrogens have no effect on TAF-1 mediated transcription. 253 Raloxifene, on the other hand, may activate estrogen-responsive genes through a response element that is distinct from HRE. 254
Pure antiestrogens are derivatives of estradiol that have a long hydrophobic side chain at position 7. Examples of pure antiestrogens include ICI 164,384 and ICI 182,780 (Fulvestrant). Binding of pure antiestrogens may sterically interfere with the dimerization process and thus inhibit DNA binding. Furthermore, these compounds increase the rate of receptor degradation and also inhibit ER-mediated transcription by preferential binding to corepressors, which contributes to their antiestrogenic activity. 255, 256

Progesterone Receptor

Structure and Function
As with the estrogen receptor, there are two major forms of progesterone receptors— PR-A and PR-B—which are derived from the same gene. PR-A and PR-B are identical except that PR-B contains an additional 164-amino acid sequence at the N-terminal end, which is referred to as the B-upstream segment ( BUS ) ( Fig. 2-12 ). PR-A has a molecular weight of 94 kDa and contains 768 amino acids; PR-B is 114 kDa with 933 amino acids. The two forms derive from two distinct estrogen-regulated promoters. 257 The transcription function domain (TAF-1) in the progesterone receptor is located in the 91-amino acid segment of the regulatory region, and TAF-2 is located in the hormone-binding domain. In PR-B, the BUS contains a third activation domain, TAF-3, which can synergize the actions of other TAFs or autonomously activate transcription. 258 TAF-3 recruits and allows a separate subset of coactivators to bind with PR-B that do not interact efficiently with PR-A. Thus, PR-A and PR-B display different transactivation properties that are both cell specific and target gene promoter specific. 259 The two isoforms PR-A and PR-B have distinct cellular localization and in the absence of ligand binding, PR-A is predominantly localized in the nucleus and PR-B is present in the cytoplasm. 260

Figure 2-12 A schematic diagram of progesterone receptors isoforms. Different domains (A-E) and their corresponding functions are shown. PR-A, progesterone receptor-A; PR-B, progesterone receptor-B.
The role of progesterone receptor isoforms is not yet fully elucidated. Selective ablation of PR-A expression in mice resulted in severe abnormalities in ovarian and uterine function that led to infertility but did not affect progesterone responses in the mammary gland or thymus. 261 In contrast PR-B ablation does not affect ovarian, uterine, and thymic responses to progesterone and manifests as reduced mammary ductal morphogenesis. Thus, PR-A is essential for female fertility; in the absence of PR-A, PR-B functions in a tissue-specific manner and mediates some of the progesterone receptor actions in the mammary gland. 261 However, transgenic mice carring an extra copy of the PR-A gene have abnormal mammary gland development, indicating that overexpression of PR-A may have physiologic significance.
The relative levels of the two isoforms differ in the endometrium during the menstrual cycle. 262 In the uterus, progesterone action downregulates cell-cycle arrest proteins but upregulates growth factors and their receptors and other regulators 263 and is also essential for the initiation and maintenance of pregnancy. Progesterone antagonist RU-486 (mifepristone) initially was synthesized as a glucocorticoid receptor antagonist 264 but later was found to display marked antiprogesterone activity. 265 It binds to the glucocorticoid recptor with threefold higher affinity than dexamethasone and to the progesterone receptor with a fivefold higher affinity than natural progesterone. 266 Unlike progesterone, the RU-486–progesterone receptor complex inhibits transcription because it has a slightly different conformational change in the TAF-2 domain. 267 If implantation occurs, it downregulates progesterone-induced genes and results in decidual necrosis and detachment of the conception products. 251, 266

Androgen Receptor

Structure and Function
The androgen receptor gene was cloned in 1988 and was localized on the human X chromosome between the centromere and q13. 268 Like the progesterone receptor, the androgen receptor also exists in two forms: a full-length B form and a shorter A form (molecular weights ≈110 kDa and 87 kDa); both are encoded by the same gene. 269 The 87-kDa isoform (AR-A) contains the intact C-terminus but lacks 188 amino acid residues in the N-terminus of the 110-kDa isoform (AR-B) ( Fig. 2-13 ). The ratio of AR-B to AR-A in genital skin fibroblasts from healthy subjects is 10:1. It is not known whether there are functional differences between these isoforms. 269 The DNA-binding domain or TAF-2 of the androgen receptor is similar to the TAF-2 regions of other steroid hormone receptors (progesterone, estrogen, glucocorticoid, and mineralocorticoid receptors) but is related most closely to the progesterone receptor. 270 Progesterone shows cross-reactivity with androgen receptors, to a degree that becomes clinically relevant only at pharmacologic doses.

Figure 2-13 A schematic diagram of androgen receptor isoforms. The androgen receptor is very similar to the progesterone receptor and exists in a shorter A form and a full-length B form.
In most tissues, testosterone is converted to DHT by the action of the enzyme 5α-reductase. DHT binds to the androgen receptor with a higher affinity than testosterone, leading to greater stabilization of the receptor and more efficient signaling, which amplifies the androgen action. Hence, the efficiency of local conversion of testosterone to DHT is an important intracellular step in the androgen response.
A large number of androgen receptor mutations that can alter receptor function have also been described 271, 272 ; for example inactivating point mutations in the hormone-binding domain of the androgen receptor can generate different phenotypes that lead to partial to complete androgen insensitivity. A point mutation at residue 689 that substitutes Proline to Histidine may alter the conformation of the ligand-binding domain, which reduces the androgen receptor’s affinity for DHT and abolishes its ability to transactivate the HRE. 273 A Serine to Proline substitution at residue 865 eliminates androgen binding and transactivation; thus it also causes complete androgen insensitivity. 274 At residue 807, a substitution of Threonine for Methionine causes partial androgen insensitivity by reducing—but not abrogating—the androgen binding to the receptor. However, a Valine or Arginine substitution at the same site totally abrogates androgen binding and causes complete androgen insensitivity syndrome. 275

Nongenomic Actions of Steroids
Some steroid hormone actions are independent of their classic genomic actions, as mediated via nuclear receptors—these are called nongenomic actions. 276 These effects are rapid, occur within seconds, and are not affected by inhibitors of gene transcription such as dactinomycin or inhibitors of protein synthesis such as cycloheximide. These rapid actions include sodium and calcium ion transport and some neural and cardiovascular effects. 277 The messenger and effector system vary with the steroid and the cell type. Thus far, studies have suggested that specific binding sites or receptors are present on the cell membrane and that steroid binding triggers rapid changes in the electrolyte transport system. For example, estrogens have been shown to modulate some cardiovascular effects by inducing Ca 2+ flux and vasodilation in the coronary artery. 278 Furthermore, steroids can activate second-messenger pathways, generating second messengers capable of altering gene transcription independently of their classical receptor-mediated gene transcription. 279, 280 Thus, steroid hormones have nongenomic effects as well as genomic effects that are mediated by both classical steroid receptor-mediated pathways and second-messenger pathways.

Tropic Hormone Action
Tropic hormones (i.e., gonadotropins and GnRH) are primarily hydrophilic and depend on their interactions with receptors scattered on the plasma membrane at the cell surface. These receptors, in turn, can be classified into four groups based on their structural homology and type of intracellular messenger involved in activating the signal transduction pathway (see Table 2-6 ).

G Protein-Coupled Receptors
The gonadotropin and GnRH receptors belong to the large family of receptors that are known as serpentine or seven-transmembrane domain receptors because each contains three domains: an amino-terminal extracellular domain (ectodomain) followed by seven hydrophobic amino acid segments; the transmembrane domains, which span the membrane bilayer (or endodomain); and a hydrophilic carboxyl-terminal domain, which resides within the cytoplasmic compartment. Because they depend on G protein transducers to execute their biologic effects, they are also known as G protein-coupled receptors ( GPCRs ). G proteins are heterotrimeric proteins associated with these receptors and are so-called because they bind the guanine nucleotides guanosine diphosphate (GDP) and guanosine triphosphate (GTP). Each G protein consists of three subunits: α, β, and γ. They are a heterogeneous family, and at least 16 α subunit genes, 6 β subunit genes, and 12 γ subunit genes have been identified. Various combinations of these provide a large number of possible αβγ complexes. The identity of the individual G protein is determined by the nature of the α subunit. As such, G proteins can be divided into four subfamilies (G s , G i , G q , and G i2 ) based on their protein sequence homology. They act as transducers, linking the receptors with the effector proteins that are responsible for producing changes in cellular function. The large variety of G proteins allows for flexibility and diversity in the response of the target cell, depending on the level of G protein expression.
There are many types of possible effector molecules, including adenylyl cyclase, calcium channels, potassium channels, cGMP, and phospholipase. Each effector molecule in turn produces a large quantity of second messengers such as cAMP, Ca 2+ , and phosphatidylinositols, which activate an even larger number of downstream molecules. This is an important factor that explains why the endocrine system is sensitive to such low concentrations of circulating ligand and why only a small percentage of cell membrane receptors need to be occupied to generate a response.
Other examples of receptors and their ligands that fall into this family include the TSH receptor and ACTH receptor. The structure of the LH receptor and FSH receptor are similar to each other and show close homology with the TSH receptor. A comparison of the LH receptor with the FSH receptor shows about 70% homology in the transmembrane domain, but only 42% in the ectodomain and 48% in the endodomain regions.

Gonadotropin Receptors
The gonadotropin receptors are primarily expressed in the gonadal tissues and demonstrate a unique expression pattern that determines their cell-specific roles. LH receptors are present in ovarian thecal and luteal cells and in testicular Leydig cells. FSH receptors are found in ovarian granulosa cells and in testicular Sertoli cells. Furthermore, FSH induces LH receptors in the granulosa cells that are expressed in mature follicles. These receptors are essential in mediating the gonadotropin actions in steroidogenesis and in gonadal growth and differentiation. Their structure, function, and regulation as well as molecular biology have been extensively covered in recent reviews. 281, 282

Structure and Function
Genes for both LH and FSH receptors are located on chromosome 2p21. The LH receptor gene is about 70 kb and consists of 11 exons and 10 intervening introns. 281 The FSH receptor gene is about 54 kb and contains 10 exons and 9 intervening introns. 282 As shown in Figure 2-14 , the structural similarities between the two receptors are remarkable with the exception of an additional exon in the LH receptor. The long exon 11 in LH receptor and exon 10 in FSH receptor encodes for the seven-transmembrane domains and the intracellular tail as well as the C-terminal end of the hinge region of ectodomain. The remaining 9 exons in the FSH receptor and 10 exons in the LH receptor encode for the entire ectodomain. Both receptors have multiple splice sites, leading to transcription of multiple mRNA splice variants and expression in the ovary and testis. 281

Figure 2-14 A schematic representation of the FSH ( A )/LH ( B ) receptor gene and localization of identified mutations. The structural organization of the FSH/LH receptor protein is provided below it. The extracellular domain of the protein is shown as a straight line, followed by the seven-transmembrane domains and the intracellular domain. In the FSH receptor gene exon 10 and in the LH receptor gene exon 11 codes for the seven-transmembrane signal transduction domains.
The LH receptor protein contains a 24-amino acid signal peptide (17 in FSH receptor) and the mature protein consists of 675 amino acid residues (678 in FSH receptor) (see Fig. 2-14 ). The molecular mass is approximately 85 to 90 kDa for both glycosylated LH and FSH receptor proteins. The ectodomains of these receptors are composed of several leucine-rich repeats (LRRs) of about 24 amino acid residues each (9 in LH receptor and 10 in FSH receptor) that form a half donut-shaped structure and are essential for the binding of gonadotropins. The ectodomain is connected to the transmembrane region by the hinge region with conserved sequences. The transmembrane region consists of seven helical structures and is followed by the cytoplasmic C-terminal tail, both of which are important for interaction with intracellular proteins. The binding of LH and FSH to their specific receptors leads to receptor activation and downstream signaling.

Mechanism of Action
The intracellular messenger for LH and FSH is cAMP, which is derived from adenosine triphosphate (ATP) through the action of the enzyme adenylate cyclase. The basic steps involved in the receptor-mediated action of gonadotropins are illustrated in Figure 2-15 . The regulation of adenylate cyclase is mediated by a GTP-dependent regulatory G-protein, each of which is composed of three subunits—α, β, and γ—and is associated with the receptor in an inactive GDP-bound form. Gonadotropic binding to their respective receptors induces a conformational change in the receptor and activation of the G-protein complex that leads to release of GDP and binding of the α subunit to GTP. 283 Subsequently, the GTP-bound form of the α subunit disassociates from the receptor as well as from the stable β/γ dimer and activates the adenylate cyclase. This in turn leads to an increase in the levels of intracellular cAMP, which in turn activates protein kinase A (PKA) by binding to the inhibitory regulatory subunit of PKA and causing it to dissociate from the complex. Activated PKA phosphorylates many cellular substrates, including several nuclear transcription factors (e.g., cAMP response binding protein [CREB]). 281 The binding of CREB to the cAMP response element activates many genes. The fact that PKA activation does not account for all the actions of gonadotropins and that LH can stimulate steroid hormone synthesis without significant changes in cAMP indicated that another pathway may be activated. There is now evidence that the phosphatidylinositol 3,4,5-triphosphate (IP 3 ) pathway is also activated by the LH receptor 284 as well as by the FSH receptor, 285 although it is not clear whether the IP 3 pathway activates the same or different responses in the target cells. The third signaling pathway, the MAPK pathway, has also been shown to be activated by the LH receptor 286 (see Fig. 2-15 ).

Figure 2-15 A diagrammatic representation of the interlinked signaling pathways between FSH/LH G-protein coupled receptors (GPCRs) and insulin/IGF-I receptor tyrosine kinases in ovarian cells. Activation of GPCR by hormone binding stimulates the Gα subunit to bind guanosine triphosphate (GTP) instead of guanosine diphosphate (GDP), leading to its dissociation from β/γ subunits to activate downstream signaling factors such as adenylyl cyclase that synthesizes second-messenger cyclic adenosine monophosphate (cAMP). Binding of cAMP in turn activates protein kinase A (PKA), leading to DNA binding and downstream cellular response. Also illustrated here is the IGF-I receptor signaling pathway. IGF-I/Insulin binding to the receptor initiates autophosphorylation and tyrosine phosphorylation of insulin receptor substrates (IRS), leading to activation of phosphatidylinositol-3-kinase (PI3K) and generation of 3-phosphorylated-inositol (IP3) from phosphoinositol (PIP2), which activates PI-dependent protein kinase-1 (PDK-1). PDK-1 in turn activates Akt/protein kinase B (Akt/PKB), leading to biologic effects. The activation of the insulin receptor substrates (IRS) also allows the docking and activation of small adaptor molecules with SH-2 domains (e.g., growth factor receptor binding protein-2 [Grb-2[] and Shp2). Activated Grb-2 recruits SOS-1, which activates the RAS pathway and gene transcription. (Main pathways are in bold, interlinked signaling pathways are in broken lines). SOS-1, son-of-sevenless; ERK, extracellular signal regulated kinase; FSH, follicle stimulating hormone; LH, luteinizing hormone.
Continuous stimulation of receptors can lead to a decrease in response, a phenomenon known as downregulation. For example, pulsatile LH secretion maintains LH receptors and steroidogenesis in the gonads. However, persistent endogenous elevation of LH or hCG levels downregulates LH receptors and leads to desensitization to the hormonal signal. 287 This involves receptor phosphorylation, which uncouples the receptor from the G protein, ending the response. The LH/hCG receptor undergoes desensitization in response to LH or hCG by the phosphorylation of the C-terminal cytoplasmic tail of the receptor. 288 Another mechanism of downregulation is the uncoupling of the regulatory and catalytic subunits of the adenylate cyclase enzymes. For example, LH stimulates steroidogenesis by coupling the stimulatory to the catalytic units of adenylate cyclase. Prostaglandin F 2α , on the other hand, inhibits the action of LH by an inhibitory regulatory unit that uncouples the catalytic unit to interfere with gonadotropin action.

Mutations in Gonadotropin Receptors
Both activating and inactivating gene mutations have been described in LH and FSH receptors and have been the subject of recent review. 209 Interestingly, the activating mutations in the LH receptor gene result in altered testosterone production, and these mutations are known to affect the phenotype of men only, whereas inactivating mutations affect sexual differentiation and fertility in both men and women.
To date, a total of 15 activating mutations have been identified in the LH receptor, all localized in the transmembrane region or in the intracellular loops (see Fig. 2-14A ). Transmembrane 6 and the third intracellular loop are recognized as mutational hot spots, and 10 of 14 mutations are localized to these regions. These gain-of-function mutations lead to male-limited precocious puberty characterized by Leydig cell hyperplasia, premature puberty, and onset of spermatogenesis in boys as young as age 3 years. 289, 290 These patients have blood testosterone levels in the pubertal range, with low or undetectable LH levels. A novel activating LH receptor mutation associated with Leydig cell tumors and nonfamilial precocious puberty has also been described. 291
An equal number of inactivating mutations in LHR that cause partial to complete inactivation of receptor have been described. 209 These inactivating mutations in LH receptors cause Leydig cell hypoplasia in men and amenorrhea in women. In contrast, loss-of-function mutations are scattered throughout the LH receptor protein. Depending on the mutation, the severity of the male phenotype varies from primary hypogonadism to male pseudohermaphroditism with sexual ambiguity.
Conversely, activating mutations of the FSH receptor are rare. Only one mutation has been identified this far (located in the third intracellular domain of exon 10) in a hypophysectomized patient who was fertile despite the lack of FSH and LH production. 292 Likewise, fewer inactivating mutations have been identified in the FSH receptor (see Fig. 2-14B ). Most of these are located in the extracellular domain and show reduced ligand binding and signal transduction. They are implicated in hypergonadotropic ovarian dysgenesis in females and variable degrees of spermatogenic failure in males. Recently, two heterozygous missense mutations in the exon 10 encoding the transmembrane region have been described, and both were associated with the familial spontaneous ovarian hyperstimulation syndrome. Although there were no significant differences in the cAMP responses between the wild-type and mutated receptor, the mutated receptor gained sensitivity to hCG. 293, 294 Low-affinity binding of hCG to the FSH receptor most likely triggered signal transduction that led to hyperstimulation of ovaries.

Gonadotropin-releasing Hormone (GnRH) Receptor
GnRH binds to specific membrane receptors in pituitary gonadotrophs and stimulates LH and FSH secretion ( Fig. 2-16 ). Besides gonadotrophs, GnRH receptors have also been expressed on the gonads, placenta, and brain 295 - 297 Specific GnRH receptors are also characterized in immortalized αT3-1 gonadotroph cells as well as in GnRH-secreting GT1 hypothalamic cells. 296, 298 In the ovary, GnRH receptors are expressed in granulosa and luteal cells. In the testis, these receptors are expressed in Leydig cells but not Sertoli cells. 299 In cultured granulosa cells, the receptor activation stimulates progesterone and prostaglandin synthesis, oocyte maturation, and ovulation 300, 301 but inhibits FSH-induced steroidogenesis, follicular development, and maturation as well as inhibin secretion. 302 - 304 In luteal cells, it inhibits LH receptor expression and thus LH action. 305

Figure 2-16 A schematic representation of the gonadotropin-releasing hormone (GnRH) receptor gene and localization of identified mutations. The structural organization of the GnRH receptor protein is provided below it. The short extracellular domain of the protein is indicated by the straight line, followed by the seven-transmembrane domains. The GnRH receptor lacks a carboxyterminal cytoplasmic tail. Exon 1 codes for transmembrane domains 1-3, exon 2 codes for transmembrane domains 4-5, and exon 3 codes for transmembrane domains 6-7. 5′UTR, 5′’ untranslated region.
The structure function and intracellular signaling pathways involved in GnRH action have been extensively reviewed in recent years. 306 - 308 The GnRH receptor gene, located on chromosome 4q21.2, consists of three exons 309 and encodes a 327-amino acid protein with an approximate molecular weight of 50 to 60 kDa. The receptor has the characteristic features of GPCRs ( Fig. 2-17 ). It consists of an aminoterminal domain, followed by seven transmembrane domains that are connected by three extracellular and three intracellular loops. Unlike other GPCRs, the GnRH receptor lacks the characteristic C-terminal cytoplasmic domain. GnRH binds to the transmembrane domain and the extracellular loop in the hairpin structure and requires partial entry of its N- and C-terminal regions into the transmembrane core of the receptor. 310

Figure 2-17 A schematic representation of GnRH signaling through GPCR. Binding of GnRH to its receptor acts through G q protein. Activated G q binds ATP and activates its downstream target phospholipase C (PLC) and hydrolyzes PIP2, producing IP 3 and diacylglycerol (DAG). IP 3 diffuses through the cytoplasm to the endoplasmic reticulum (ER) and opens a calcium channel releasing calcium stores. Calcium and DAG activate protein kinase C (PKC), leading to cellular response.
The nature of the intracellular signaling mechanism by the GnRH receptor has been primarily studied in a gonadotroph-derived αT3-1 cell line, as depicted in Figure 2-18 . The effector system in the GnRH receptor is the phospholipase C β (PLCβ) and the second messengers are IP 3 and 1,2 diacylglycerol (DAG). Its mechanism of action is dependent on calcium. Activation of the receptor by ligand binding initiates a series of steps that lead to transduction of signals. The first step is G protein (G q/11 )-mediated activation of enzyme PLCβ, leading to hydrolysis of phosphoinositides (PIP 2 ) and resulting in the production of IP 3 and DAG. The IP 3 interacts with a receptor on the endoplasmic reticulum to promote the oscillatory or biphasic release of Ca 2+ from intracellular stores, which is known to be an important trigger for gonadotropin secretion. The increased Ca 2+ and DAG in turn activate a series of protein kinase C subspecies (PKC). PKC then induce various downstream signal transduction cascades, including the extracellular signal regulated kinase and Jun N-terminal kinase signaling pathways.

Figure 2-18 The TGF-β/activin signaling pathway. Binding of a TGF-β family member (e.g., activin) to its type 2 receptor forms a ligand–receptor complex and activation of the type 1 receptor by phosphorylation. The activated type 1 receptor then phosphorylates receptor-regulated SMAD (R-SMAD). This allows R-SMAD to associate with Co-SMAD and move into the nucleus. In the nucleus, the SMAD complex associates with a DNA-binding partner, such as FAST-1. This complex then binds to specific enhancers in the target gene, activating gene transcription. (In the TGF-β/activin pathway, R-SMAD is formed by SMAD2 and SMAD3. Co-SMAD is formed from SMAD4.)

Mutations in GnRH Receptor Gene
A functional GnRH receptor is a prerequisite for normal LH and FSH production. Hence, it is critical for normal pubertal development and reproduction. Several inactivating mutations have been identified in the GnRH receptor gene that are associated with variable phenotypes ranging from modest to complete insensitivity to GnRH. These mutations are present in a number of patients with idiopathic hypogonadotropic hypogonadism (IHH) with an autosomal recessive pattern of inheritance as well as in some patients with sporadic IHH. 311 Most identified mutations are heterozygous missense mutations and are scattered throughout the GnRH receptor gene. A total of 14 mutations have been identified to date 308 and are listed in Figure 2-16 . In vitro studies have shown that some of these mutations make the receptor totally nonfunctional; others have some ability to elicit a response to GnRH.

Peptide Hormone/Growth Factor Action
Pituitary gonadotropin signaling plays a key role in follicular growth, ovulation, and luteinization. However, it is increasingly recognized that their actions are also dependent on their interaction with peptide/growth factor signaling pathways, including IGFs, EGF, and members of the TGFβ family (see Fig. 2-15 ). Understanding these pathways provides insight into how these factors interact and complement the FSH/LH pathway to control follicular growth.

Family of Receptors with Tyrosine Kinase Activity
Insulin, IGF, and EGF receptors belong to a distinct group of receptors and differ from the GPCRs both structurally and functionally. Unlike the GPCRs, they only span the membrane once and acquire their signaling ability through the activation of tyrosine kinase, which is intrinsic to these individual receptor molecules. Hence, they are known commonly as tyrosine kinases. Their main ligands include hormones such as insulin and IGF, as well as paracrine and autocrine regulators such as platelet-derived growth factor (PDGF), bFGF, and EGF. Thus, via tyrosine phosphorylation, a number of physiologic processes such as cell proliferation, cell migration, cell differentiation, and apoptosis are mediated by these receptors. This explains why this group of receptors has been the target of much oncogenic research.
All tyrosine kinase receptors have a similar structure: an extracellular domain for ligand binding, a single transmembrane domain, and a cytoplasmic domain. Ligand specificity is determined by the unique amino acid sequences making up the extracellular domain, which determines the three-dimensional conformation of the receptor. The transmembrane domains are heterogeneous, whereas the cytoplasmic domains are fairly homologous. They respond to ligand binding by undergoing conformational changes and autophosphorylation. The structure of the IGF-I receptor is strikingly similar to that of the insulin receptor, with two transmembrane domains linked by disulfide bridges, formed by two α and β subunits. 312 The IGF-I receptor gene is located on chromosome 15 at bands q25-26 and contains 21 exons. 313
The steps involved in signal transduction by IGF-I/insulin have been extensively reviewed in recent years 314, 315 and are illustrated in Figure 2-15 . The association of the ligand (e.g., insulin) with the receptor’s extracellular domain triggers receptor dimerization. This results in the phosphorylation of tyrosine residues on both the receptor and nonreceptor substrates. The phosphorylation of receptor tyrosine residues occurs at specific locations, which causes these sites to associate with a variety of accessory proteins that have independent signaling capabilities. These accessory proteins include phospholipase Cγ, PI3 kinase (PI3K), GAP, and growth factor receptor-bound protein-2 (GRB2). These interactions are mediated by the presence of highly conserved type 2 src homology domains (SH2) in each accessory molecule, thus named based on their sequence homology to the src proto-oncogene. Each SH2 domain is specific for the amino acids surrounding the phosphotyrosine residues in the receptor molecule. PI3K also produces a second messenger, such as IP 3 , which in turn activates kinase AKT, also known as protein kinase B (PKB). Proteins phosphorylated by PKB promote cell survival.
Although these associations may trigger immediate signaling events, other accessory proteins (e.g., GRB2) may serve to construct the scaffolding for a more complex signaling apparatus, such as that seen in the RAS-RAF-MEK pathway, This pathway recruits multiple other proteins, resulting in the activation of nuclear transcription and protein synthesis (see Fig. 2-15 ).
Growth hormone and prolactin fall into another group of receptors that also have a single transmembrane-spanning segment and a short cytoplasmic tail. However unlike IGF-I/insulin receptors they do not possess intrinsic tyrosine kinase activity but interact with other soluble transducer molecules (e.g., Janus kinase 2) that do have tyrosine kinase activity.

Mechanism of Action of Activins and Inhibins
Activins and inhibins are members of the TGFβ superfamily and use a common general mechanism for signal transduction through serine/threonine-specific protein kinases rather than tyrosine kinases. The mechanisms involved in signal transduction by the serine/threonine kinases has been extensively reviewed in recent years. 316, 317 The activin receptor was first to be cloned and was later followed by other receptors. 318, 319 They are glycoproteins of approximately 55 kDa and consist of a 500-amino acid sequence. Two types of activin membrane receptors have been identified: type I (ActR-I) and type II receptors (ActR-II). Each contains an intracytoplasmic serine/threonine kinase domain. The steps involved in the mechanism of action of activin are depicted in Figure 2-18 . Activin directly interacts with and binds to the relatively short extracellular region of ActR-II when expressed alone or in concert with ActR-I. It can also bind to other TGFβ family members (e.g., bone morphogenic proteins [BMPs] 2, 4, and 7) in concert with BMP type I receptor, which suggests that these receptors have the ability to cross-talk. Activin binding brings together two type II receptors and two type I receptors, which form a receptor complex. One of the receptor kinases phosphorylates and activates the other, which in turn phosphorylates their substrates—the SMAD proteins [the term SMAD is derived from the combination of names of two genes, the C. elegans gene called Sma and the Drosiphila gene Mad]. SMADs are a novel family of signal transducers that can be divided into three groups that include receptor-regulated SMADs (R-SMAD) and a single common SMAD (C-SMAD or SMAD4). The third group includes inhibitory SMADS that antagonize signaling. In activin/TGFβ signaling, the activated type I receptor phosphorylates ligand-specific R-SMADs (SMAD2 and SMAD3), allowing these proteins to associate with SMAD4. The complex then translocates to the nucleus as transcription cofactor. In the nucleus, the action of SMAD complex is modulated by a variety of transcription factors (DNA-binding proteins) at target DNA promoters, which leads to gene transcription.
Inhibin is a heterodimer formed between an inhibin α chain and an activin β chain, and its biologic activity is opposite that of activin. A separate receptor for inhibin has not been identified. The mechanisms by which inhibin antagonizes activin actions are not clearly understood. It has been shown that inhibin competes with activin for binding to the activin receptors but is unable to trigger signaling. 320 This may be one of the mechanisms by which inhibins antagonize activin actions. Other actions of inhibin may be mediated by as-yet unidentified inhibin receptors.

SUMMARY
The ovary is a dynamic endocrine organ. The follicle cells interact in a highly integrated manner to produce several steroid and peptide hormones. Steroidogenesis requires effective delivery, uptake, and use of sterol by an array of steroidogenic enzymes. Virtually all steps in steroid biosynthesis require the action of LH and FSH and are influenced by endocrine, autocrine, and paracrine actions of several intraovarian peptide hormones, growth factors, cytokines, and neuropeptides. In recent years, research has shown that these growth factors affect various cell processes, such as cytodifferentiation, mitogenesis, and apoptosis in a variety of ways. They act in concert with LH/FSH via a complex network of intracellular signaling to mediate their actions.
A functional hypothalamic-pituitary axis is essential for ovarian hormone production. GnRH secretion in a synchronized pulsatile fashion is a key feature in the control of LH/FSH secretion, and new insights have made it a prime drug target for the treatment of infertility. Both GnRH and gonadotropin actions are transmitted through G protein-coupled receptors to target cells via multiple signaling mechanisms. Several mutations and polymorphisms have been identified in their genes and their receptors. These mutations have deleterious effect on reproduction. Although rare, this study has allowed a better understanding of receptor structure and function relationships and helped clarify the molecular pathogenesis of conditions associated with altered gonadotropin secretion and actions.
Steroid hormones play a central role in the reproductive system. Physiologic effects of steroid hormones are mediated via their nuclear receptors, which belong to a superfamily of ligand-dependent transcription factors. The two isoforms of ER and the progesterone receptor are differentially expressed in different tissues, leading to tissue-specific responses. Furthermore, the differences in gene expression depend on interactions with protein cofactors, the coactivators and corepressors. A better understanding of the effect that the cell environment has on nuclear receptors and their coregulators led to the discovery and understanding of the mechanism of action of antiestrogens and selective receptor modulators.


PEARLS

• There is a specific cellular location for many steroidogenic enzymes.
• Sex steroids can be produced thtough two pathways: Δ 5 (the 3β-hydroxysteroid pathway) or Δ 4 (the 3-ketone pathway).
• Hydroxylases and aromatase enzymes belong to the cytochrome P450 family.
• The two-cell theory explains the production of steroid hormones of the ovary.
• Thecal cells respond to LH; granulosa cells respond to both FSH and LH.
• Theca cells produce androgens that are aromatized by the granulosa cells to estrogens.
• Estradiol is the main estrogen in premenopausal women.
• Progesterone and 17 OH-progesterone are produced mainly by the corpus luteum in the luteal phase.
• The majority of testosterone is derived from the peripheral metabolism of DHEA and androstenedione.
• Very little ovarian steroid hormone circulates free in the circulation but rather is bound to proteins.
• Inhibin A and B are heterodimers produced by the granulosa cells. Inhibin B is highest during the luteal–follicular transition and early follicular phase. Inhibin A rises in the luteal phase.
• Inhibin A is produced primarily by the fetoplacental unit during pregnancy.
• Activins are dimers of the inhibin β subunit and are produced by the granulosa cells.
• Follistatin is produced by the granulosa cells and acts mostly as as an antagonist of activin.
• Steroid hormones bind to intracellular receptors located within the cytoplasm or nucleus.
• Polypeptide hormones and growth factors interact with cell- surface receptors located on the plasma membrane.

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Chapter 3 Oogenesis

Mylene W.M. Yao, Kshonija Batchu

INTRODUCTION
Oogenesis is an area that has long been of interest in medicine, as well as biology, economics, sociology, and public policy. Almost four centuries ago, the English physician William Harvey (1578–1657) wrote ex ovo omnia —“all that is alive comes from the egg.” Oogenesis exemplifies many fundamental biologic processes, such as mitosis, meiosis, and intracellular and intercellular signaling. The process of oogenesis begins with migratory primordial germ cells (PGCs) and results in an ovulated egg containing genetic material, proteins, mRNA transcripts, and organelles that are essential to the early embryo.
Although our understanding of these basic mechanisms has increased significantly over the past few decades, we are still limited in our ability to treat female infertility due to “poor oocyte quality” or to identify women at risk for this condition to allow more patient-specific family planning. Currently, poor oocyte quality refers to a broad spectrum of clinical conditions, including poor response to exogenous gonadotropins, recurrent pregnancy loss or congenital anomalies due to aneuploidy, and abnormalities of oocyte maturation, fertilization, and early embryo development that are observed during in vitro fertilization (IVF) treatment. Because these conditions are more prevalent in older women, they also carry serious social implications for women and their partners in family planning.
Many aspects of oogenesis and meiosis are well conserved across sexually reproducing species. Experimental work on the nematode Caenorhabditis elegans , the fruit fly Drosophila melanogaster , the frog Xenopus laevis , and the fission and budding yeasts Saccharomyces cerevisiae and S. pombe, respectively, has provided us with much insight into these processes. However, there are key differences in oogenesis between mammalian and nonmammalian species. As with many processes in development, oogenesis is extremely similar between mice and humans. Thus, in vivo gene-targeted and in vitro oocyte maturation models in the mouse have been most informative and relevant to our understanding of mammalian oogenesis.
The generation of healthy eggs with the correct genetic complement and the ability to develop into viable embryos requires that oocyte development and maturation be tightly regulated. In an effort to describe in detail our current understanding of the basic biologic mechanisms behind this process, this chapter discusses mammalian oogenesis as it is understood in the mouse model ( Fig. 3-1 ) and highlights clinical conditions that are related to defects in oogenesis.

Figure 3-1 Stages of oocyte maturation. Oocytes and their corresponding chromosomes at various stages of maturation, as visualized by differential interference contrast (DIC) [A, C, E, G, I, K] and 4′6-diamidino-2-phenylindole (DAPI) stain [B, D, F, H, J, L], respectively. DAPI stain binds to natural double-stranded DNA in the chromosomes and allows chromosomes to be visualized in live cells. A, An oocyte at the germinal vesicle (GV) stage (center). GV, germinal vesicle; N, nucleolus; ZP, zona pellucida. B, The same oocyte under milliseconds of exposure to UV light, upon which DAPI-stained chromosomes are visualized as fluorescent structures. The chromosomes (arrow) are localized to the periphery of the nucleolus and assume the “surrounding nucleoli” configuration indicative of meiotic competence (see text for detailed explanation). C, D, An oocyte that has undergone germinal vesicle breakdown and is in pro-metaphase I, as indicated by complete disappearance of both GV and nucleoli on DIC and the presence of condensed bivalents (arrow). E, F, An oocyte in metaphase I. The chromosomes (arrow) are aligned at the metaphase plate and individual bivalents cannot be distinguished. G, H, An oocyte that has been cultured in media containing the proteasome inhibitor MG132 (2.5 μM) for 24 hours. Proteasome inhibition causes this oocyte to be arrested at metaphase I, and congression of chromosomes (arrow) remains at the metaphase plate.
I, J, An oocyte (top) in the metaphase–anaphase transition, as indicated by chromosomes that are in the process of segregating. The bottom oocyte has extruded the first polar body, which is indicated by the condensed chromosomes (single spot). (The chromosomes in the oocyte proper are not seen on this plane.) K, L, An oocyte (center) that is in metaphase arrest, which is indicated by extrusion of the first polar body. Note a very faint fluorescent spot, which represents very condensed chromosomes that are tightly localized in the first polar body. Chromosomes in this metaphase II-arrested oocyte are aligned at the metaphase plate. These chromosomes appear fainter and not in optimal focus because their focal planes are slightly different. (Due to the thickness of the oocyte [≈80–100 μm], optimal visualization of both sets of chromosomes may not be possible if they are in different focal planes.)

OVERVIEW OF OOGENESIS
Oogenesis begins in the early embryo with PGCs, which migrate from the yolk sac to the urogenital ridge to establish the germline. Upon sexual differentiation, PGCs give rise to oogonia, which undergo mitosis to expand the population in the developing female gonad. Oogonia become oocytes as they enter meiosis I.
During prophase of meiosis I, oocytes undergo DNA replication, homologous chromosome pairing, synopsis, and recombination, but then remain arrested at the diplotene stage of prophase I until sexual maturity. While the oocyte remains in meiotic arrest, it continues to increase in size coordinately with follicular growth. Before meiotic resumption, the fully grown oocyte is characterized by the presence of the germinal vesicle (GV), which is the meiotic counterpart of the nuclear envelope in somatic cells.
Gonadotropin-dependent follicular growth is followed by oocyte maturation, which refers to the resumption and completion of meiosis I in the oocyte. This process occurs at each estrus cycle in mice and at each menstrual cycle after the onset of puberty in humans. On mating in an estrus cycle in the mouse, luteinizing hormone (LH) and cumulus–oocyte signaling result in decreased cyclic adenosine monophosphate (cAMP) in a cohort of oocytes. These oocytes resume meiosis and undergo GV breakdown (GVBD). Condensation of chromosomes is followed by congression of homologous chromosomes at metaphase I and segregation at anaphase I. Extrusion of the first polar body signifies exit from meiosis I and concomitant entry into meiosis II, whereupon the eggs are arrested at metaphase II until fertilization with sperm.
After fertilization, they exit meiosis II and transition into one-cell embryos. In the human menstrual cycle, a cohort of follicles is similarly recruited to resume meiosis, but typically only one dominant follicle develops, so that only one oocyte completes meiosis I and is released during the endocrine- and paracrine-mediated process of ovulation.

PRIMORDIAL GERM CELLS
Primordial germ cells are the founder cells of the gametes. In many species, including Drosophila , Danio rerio (zebrafish), and C. elegans , germ cells are derived from germ plasm, which is maternally derived cytoplasm that contains germ cell determinants. In contrast, mammalian PGC fate is thought to be induced by cell–cell interaction requiring the adhesion molecule E-cadherin and signaling from surrounding tissues. 1, 2
The migration of PGCs from the base of allantois to the genital ridges has been mapped out by alkaline phosphatase staining; more recently, their migration in vivo has been visualized by green fluorescent protein (GFP) expressed under the control of a truncated Oct-4 promoter (GFP:Oct-4) acting specifically in PGCs. 3, 4 Primordial germ cells are derived extragonadally from yolk sac endoderm and part of the allantois that arises from the posterior part of the primitive streak.
Primordial germ cells can be distinctly identified at 7.5 days post-coitum (dpc, a measure of embryonic age) during gastrulation by their high expression of alkaline phosphatase or GFP:Oct-4. At 7.5 dpc, PGCs are seen at the root of the allantois, from which they migrate along the endoderm via passive transfer mechanisms, to arrive at the epithelial layer of the hindgut at 8 dpc. Then, with the acquisition of motility via long pseudopodia, they traverse through the wall of the hindgut at 9 dpc. 3
At 9.5 to 11.5 dpc, they migrate along the dorsal mesentery toward the genital ridges in the roof of the coelomic cavity. PGCs remain sexually undifferentiated until 12.5 dpc, when they coalesce with the mesodermally derived somatic components of the gonads to form ovaries or testes. During the process of migration, PGCs undergo proliferation by mitosis at approximately every 18 hours, so that their population increases from fewer than 100 founder cells to approximately 3,000 cells by 11.5 dpc and approximately 25,000 cells by 13.5 dpc.

Specific Genes Expressed for Migration of Primordial Germ Cells
Several genes are known to be required for PGC cell fate determination, migration, and survival in the mouse embryo. Bone morphogenetic protein 4 (Bmp4) and Bmp8b are maximally expressed in the extraembryonic ectoderm adjacent to the proximal epiblast, where precursors of PGCs are localized at 6.5 dpc. 5, 6 These two proteins are thought to have essential roles in the differentiation of PGC precursors into PGCs because PGCs are absent in both Bmp4 and Bmp8b homozygous null mutant mouse models. 6, 7
Bone morphogenetic proteins are members of the transforming growth factor β (TGFβ) superfamily of growth factors, which also includes the TGFβ family and activins. These signaling molecules have diverse and often critical functions during embryonic development and in adult tissue homeostasis. They signal via formation of heterodimers or homodimers of the highly conserved carboxyl-terminal domain, which is characterized by six or seven cysteine residues that are critical to the structure, stability, and function of these proteins. TGFβ superfamily members signal by binding to serine/threonine kinase cell membrane receptors that leads to phosphorylation of proteins of the Smad family (named after the C. elegans gene sma and Drosophila gene Mad , reviewed in Shi and Massague 8 ).
Smad complexes can bind DNA and mediate transcriptional activation or repression. Smad1 and Smad5 are known to act downstream of the Bmp signaling pathway and may have important functions in PGC development because loss of these proteins results in decreased size of the PGC population. 9 - 11
Migration of PGCs through the hindgut and along the mesentery is mediated by the binding of c-Kit, a tyrosine kinase receptor expressed in PGCs, to its ligand, stem cell factor (Scf). Scf is expressed by somatic cells and its gradient along the migratory path is thought to direct the migration of PGCs. Mutations in the dominant white spotting ( W ) and Steel loci, which encode c-Kit and Scf, respectively, result in defects in gametogenesis. Although PGCs form, they do not proliferate, migrate to ectopic sites, and aggregate prematurely, so that the genital ridges are poorly populated by germ cells and the animals are sterile. 12, 13
Interestingly, these mutant mouse models also exhibit defects in melanogenesis and hematopoiesis because these genes are also required for the migration of melanocytes and mast cells. 14 PGCs also express integrin subunits, which may potentially interact with laminin and fibronectin produced by cells in the genital ridges during the formation of the gonads. For example, integrin β 1 is required for PGC colonization of the gonads, as the migration of PGCs is arrested at the gut endoderm in integrin β 1 null mutants. 15

OOGONIA AND THEIR MEIOTIC ENTRY TO BECOME OOCYTES
Colonization of the genital ridges by PGCs occurs concomitantly with the formation of the ovaries, at 12 to 13 dpc. Postmigratory PGCs, together with somatic epithelial cells that have derived from the coelomic epithelium and mesenchyme, form the ovarian cortical sex cords. Germ cell survival beyond 13 dpc requires the C2H2-type zinc-finger transcription factor Zpf148, which may activate tumor suppressor protein p53 in germ cells at this stage of development. 16 Around the same time, PGCs differentiate into oogonia, which are sexually differentiated germ cells that would undergo the last rounds of mitotic divisions to establish the germ cell population at 11 to 13 dpc. 17
Our understanding of mammalian oogonium function has been limited, perhaps due to their transient existence. Incomplete cytokinesis during synchronous, mitotic divisions of the oogonia results in the formation of clusters, or cysts, that are connected by intercellular bridges 0.5 to 1.0 μm in diameter. 17 Oogonia become oocytes by entering meiosis I at 14 dpc; by 17 dpc, most oogonia have transitioned to oocytes. In the perinatal period, these germ cell cysts undergo breakdown, after which only a third of the oocytes survive and become enclosed in a single layer of granulosa cells to form primordial follicles. 17 Thus, perinatal germ cell apoptosis appears to be a developmental process that is differentially regulated compared to the loss of oocytes during follicular atresia that occurs in the sexually mature animal. Pepling and Stradling suggested that cyst formation and subsequent breakdown may serve to ensure the incorporation of mitochondria from dying oocytes into those that survive. 17

Oocyte Development
The female germ cell becomes an oocyte when it enters prophase of meiosis I. The signals that instruct the germ cell to enter meiosis are not known. Further, we do not know whether this decision is cell-autonomous or whether it is dependent on signaling from adjacent somatic cells. Nevertheless, we do know that all oogonia have entered prophase to become oocytes by 17 dpc. DNA replication, chromosome pairing, and recombination, which are hallmarks of sexual reproduction ensuring that all eggs contain a unique haploid complement of DNA, occur in prophase.

Prophase of Meiosis I
The four stages of prophase I are leptotene, zygotene, pachytene, and diplotene. DNA replication is finalized in preleptotene, and in leptotene, sister chromatids search for their homologous counterparts. Formation of recombination nodules also commences at this time to facilitate interaction between homologous chromosomes.
In zygotene, homologous chromosomes pair and begin to synapse. Their synapses are maintained by the synaptonemal complex, which is formed by many protein subunits, including synaptonemal complex protein 3 (Scp3) in the axial elements, Scp1 in the central element, and Scp2. 18 Synapsis is completed in pachytene and continues to be maintained until diplotene, when homologous chromosomes are held together mainly at sites of chiasmata. The crossing-over and recombination of chromosomes occur over 4 days in the pachytene stage, prior to the formation of ovarian follicles.
Many proteins, including those with functions in DNA mismatch repair, recombination, and DNA damage checkpoint, have essential roles during prophase I. Gene-targeted mouse models deficient in these proteins exhibit female meiosis phenotypes ranging from premature ovarian failure due to perinatal oocyte loss, to defects that become apparent only later during oocyte maturation. These proteins are also required in spermatogenesis, which demonstrates mechanisms of prophase I that are common to both sexes, although the meiosis phenotypes tend to be more severe and occur in slightly earlier stages of development in males. Our discussion focuses on the requirement of these proteins in oogenesis.

MutL and MutS Families of Proteins
DNA mismatch recognition and repair by the MutL and MutS families of proteins are well conserved from yeast to humans. 19 MutL and MutS heterodimeric protein complexes are thought to interact to activate DNA mismatch repair. Although the MutL and MutS vertebrate homologs (Mlh and Msh, respectively) are generally known for their roles in maintaining genomic stability against tumor formation, gene targeting in mice revealed that Mlh1, Mlh3, Msh4, and Msh5 have essential functions in meiosis in both males and females. 20 - 24
Mlh1 and Mlh3 homozygous mutant females have similar infertility phenotypes, in which the newborn ovaries appear normal with a normal number of follicles at various stages of development. However, there is a significant decrease in the number of oocytes that can complete meiosis I, meiosis II, and undergo subsequent development to two-cell embryos. 21, 23 Both Mlh1 and Mlh3 colocalize to chromosomes in pachytene and are thought to have critical functions in meiotic recombination, as supported by the decreased number of chiasmata formed in the oocytes of these mutant animals. 21, 23, 25
Although oocytes seem to enter and arrest at diplotene, chromosomes are unpaired and cannot stably attach to the bipolar spindle, which leads to abnormal meiotic I spindle formation, abnormal or incomplete meiosis I, and fertilization failure. 25 Further, in both human and mouse pachytene oocytes, MLH1 and MLH3 serve as molecular markers of recombination nodules, which are necessary for the crossover structures that are present in the diplotene stage. 26
Similarly, Msh4 and Msh5 have essential functions in pachytene of prophase I, although their phenotypes were more severe. 20, 22, 24 Female null mutants of Msh4 and Msh5 have significant loss of oocytes by postnatal days 2 to 4, before meiotic arrest at the diplotene stage. 20, 22, 24 Their oocytes enter leptotene to form synaptonemal complexes but fail to undergo complete pairing at zygotene and fail to enter diplotene. These oocytes undergo apoptosis, which results in atretic follicles, loss of follicular architecture, and premature ovarian failure. Both Msh4 and Msh5 localize to chromosomes, can form heterodimers in vitro, and are thought to function at the same time point during chromosomal synapsis. 22 The relevance of these findings from the mouse model to human reproduction is further supported by the expression of MSH4 and MSH5 in the human testes and ovaries. 27
We do not yet know the exact mechanisms by which these Mlh and Msh proteins act or how they bind to chromosomes. The oocyte apoptosis phenotype has been attributed to failure of chromosome pairing or formation of synapses. However, recent genetic analyses based on the Spo11/Msh or Spo11/Mlh double null mutants indicate that oocyte apoptosis is a consequence of failure to repair double-strand breaks. 28 Spo11 initiates double-strand breaks, which are required for recombination. In the absence of Spo11, the phenotype of Mlh1 and Msh5 was less severe and resembled that of Spo11. Similarly, Dmc1, another protein involved in recombination, and ataxia-telengiectasia mutated (Atm), a DNA damage checkpoint protein, are both required for the repair of double-strand breaks; in their absence, persistent double-strand breaks lead to oocyte death via apoptosis. 28
The functional mechanism of a protein is often elucidated by identifying proteins with which it associates and the nature of their interactions. The colocalization of Mlh1 proteins with Scp3 and cyclin-dependent kinase 2 (Cdk2) may suggest interaction or related functions, especially since Scp3 and Cdk2 have also been shown to be required in meiosis. 29, 30 Scp3 is a key protein subunit in the synaptonemal complex, which holds homologous chromosome pairs to facilitate synapsis. Female mice lacking Scp3 are subfertile, because many of their oocytes contain univalents (unpaired chromosomes), exhibit abnormal chromosomal segregation, and produce a decreased number of viable embryos. Interestingly, their fertility worsens with age, so that this model is thought to be potentially powerful in studying the age-related subfertility and aneuploidy that are major problems in human reproduction. 30

Cdk Family of Proteins
Cyclin-dependent kinase (Cdk2) belongs to the family of Cdk proteins, which are considered master regulators of the cell cycle. 31 However, Cdk2 -null mutants are unexpectedly viable; thus, Cdk2 is not required for mitosis during development in vivo to be required for prophase I of meiosis in the oocytes instead. Oocyte loss and premature ovarian failure occur by the first few postnatal days. Although the precise molecular defects differ, a similar phenotype is seen in the null mutants of Dmc1, Msh4, Msh5, and Atm, presumably because these oocytes fail to enter or complete pachytene, and enter the common apoptotic pathway by default. In contrast, by postnatal day 5, all oocytes in wild type mice have progressed to the diplotene stage, where they remain arrested until peri-ovulation in the sexually mature adult, when they resume and complete meiosis.

OOCYTE DEVELOPMENT AND EARLY STAGES OF FOLLICULOGENESIS
The transition from pachytene to diplotene marks the beginning of folliculogenesis, which is closely associated with subsequent oocyte development. Whereas no pachytene oocytes are found within follicles, approximately 80% of diplotene oocytes are enclosed in primordial or more developmentally advanced follicles. 32 Primordial follicles are formed by the encapsulation of a diplotene-arrested oocyte by a single flat layer of granulosa cells. Subsequently, the granulosa cells, still in a single layer, assume a cuboidal shape to form primary follicles.

Transcription Factors
Our understanding of primordial and primary follicular development has recently been enlightened by the discovery of the requirement of two transcription factors at these stages of development ( Fig. 3-2 ). Figα, a β-loop-helix-loop transcription factor, is required for oocyte survival into the primordial follicle stage. 33 Based on its known role in the transcription of oocyte-specific genes zona pellucida 1 ( zp1), zp2, and zp3 , its role in primordial follicle formation is presumed to be the transcription of other oocyte-specific transcription factors. 33

Figure 3-2 Schematic representation of oogenesis and folliculogenesis during development in the mouse model. This figure shows and correlates the developmental stages of the oocyte and follicle, and highlights examples of essential regulators at each stage. The “Types 1-8” nomenclature, as described by Pedersen and Peters 191 , is also shown to facilitate interpretation of the literature. PGCs, primordial germ cells; dpc, number of days postcoitum; dpp, number of days postpartum.
Another transcription factor, Nobox, which is an oocyte-specific homeobox protein, has a critical role in the transition of primordial to primary follicles. 34 In the absence of Nobox, mouse ovarian follicles do not develop beyond the primordial stage, which results in massive oocyte loss in the early postnatal period. 34 Identification of novel downstream target genes of Figα and Nobox in the future should provide insight into these early stages of folliculogenesis.

Zona Pellucida
The growing oocyte synthesizes zona pellucida glycoproteins, which comprise approximately 17% of the total cellular protein content. The zona pellucida proteins zp1, zp2, and zp3 are encoded by distinct genes and are coordinately expressed under the control of Figα during oocyte growth. 33 These zona proteins form the zona pellucida or zona matrix that surrounds the growing oocyte, but each one has a critical role in proper oocyte and embryo development. zp1, the only zp protein that forms intermolecular disulfide bonds, contributes to 10% to 15% of the zona mass and is thought to provide structural integrity to the zona pellucida. zp3 serves as the receptor for sperm binding and the subsequent acrosome reaction; zp2 functions as a secondary receptor. Together, zp2 and zp3 mediate sperm binding and species specificity during fertilization.
These three glycoproteins are essential for fertilization and viability of growing oocytes and early embryos. Mice lacking zp3 cannot form the zona matrix and are sterile, 35 and zp1 knockout mice have decreased fecundity and precocious hatching of early embryos from the structurally compromised zona matrix. 36 Further, ectopic granulosa cells are seen between the oolemma and zona pellucida, and may increase the perivitelline space prior to ovulation. 36 It is important to note that these may be four zona pellucida genes that are expressed in humans ( ZP1, ZP2, ZP3, and zPB ). Interestingly, abnormalities and lack of zona pellucida in human oocytes have been observed during IVF; however, the cause and implication of this defect in human reproduction is not well understood.

Regulation of Folliculogenesis by Oocyte Morphogens
Although bidirectional communication between the oocyte and follicular cells is required for the development of the meiotically competent oocyte, there is increasing support to view the oocyte as autonomous in determining its own fate by regulating follicular development via the secretion of various growth factors. 37, 38 The establishment of a morphogen gradient by the oocyte regulates follicular development via differential gene expression and function in granulosa cells. In the absence of these morphogens, follicle-stimulating hormone (FSH) induces all cumulus cells, which are directly adjacent to the oocyte and have distinct functions, to differentiate into membrane granulosa cells. Currently, several members of the TGFβ superfamily are the most well-understood candidate oocyte morphogens.

Transforming Growth Factor β
The mammalian oocyte expresses at least three TGFβ superfamily members: growth differentiation factor 9 (Gdf9), Bmp15, and Bmp6. 38 Although Gdf9 is expressed throughout the reproductive tract and bone marrow, 39 its oocyte expression is restricted to oocytes in follicles at the primary (type 3a), preantral, or more advanced stages of development. 39, 40 After fertilization, the level of Gdf9 transcripts decreases to low or undetectable in preimplantation embryos. 41
Gdf9 is a critical regulator of follicular development. In Gdf9-deficient female mice, primordial and primary follicles appear normal, but development beyond type 3a follicles is blocked and the animals are sterile. 42 Further, in the absence of Gdf9, theca cell precursors are absent around the follicles, and the expression of kit ligand and inhibin-α is upregulated in the granulosa cells of the primary follicles. 43 The increased levels of kit ligand are thought to mediate excessive oocyte growth via interaction with the c-kit tyrosine kinase receptor on the oocyte, leading to abnormally large oocytes and their cell death. 43 Interestingly, Gdf9 deficiency also causes failure of granulosa cells to proliferate or undergo cell death, which presumably leads to their abnormal differentiation into clusters of steroidogenic cells reminiscent of corpora lutea. 43 Therefore, the oocyte can be viewed as the master regulator of early folliculogenesis via its secretion of Gdf9.
The oocyte expression pattern of Gdf9 is shared by its family member Bmp15, which is encoded by an X-linked gene. 44, 45 Bmp15 homozygous null mutant females are subfertile due to decreased rates of ovulation and early embryo survival. Further, an effect of gene dosage is observed in Gdf9 +/− Bmp15 −/− mice, which have a more severe phenotype than Bmp15 −/− mice. 46 In addition, cumulus–oocyte reconstitution and RNA interference (RNAi) experiments demonstrated that both proteins regulate cumulus expansion in vitro. Therefore, Gdf9 and Bmp15 are thought to have synergistic roles in the regulation of folliculogenesis.
Interestingly, naturally occurring mutations in GDF9 and BMP15 are common in certain strains of domestic sheep, perhaps as a result of breeding practices. Although sheep carrying mutations in both alleles of GDF9 or BMP15 are sterile, those carrying a single mutant allele have increased ovulation rates and are “super fertile.” Superovulation in the presence of heterozygosity of either of these genes is thought to be caused by decreased inhibin production by granulosa cells, which in turn leads to increased pituitary FSH secretion, development of more than one dominant follicle, and ultimately superovulation. Thus, the paradoxical increase in fertility when one allele is mutated exemplifies the impact of gene dosage on ovarian follicular function. 45

RECRUITMENT OF OVARIAN FOLLICLES
Nongrowing mouse oocytes within primordial follicles have a diameter of approximately 12 μm and comprise the resting pool. Cohorts from the resting pool are continuously induced by ovarian paracrine signals to undergo coordinated oocyte and follicular growth during a process called initial recruitment ( Fig. 3-3 47 ). The recruited, growing follicles are called primary follicles, but this growth phase is very protracted so that primordial and primary follicles may not be easily distinguishable. These growing follicles subsequently develop into secondary and antral follicles. (The timeline of different stages of follicular development in humans and rodents are shown in Fig. 3-4 and 3-5 . 47 )

Figure 3-3 Life history of ovarian follicles: endowment, maintenance, initial recruitment, maturation, atresia or cyclic recruitment, ovulation, and exhaustion. A fixed number of primordial follicles are endowed during early life, and most of them are maintained in a resting state. Growth of some of these dormant follicles is initiated before and throughout reproductive life (initial recruitment). Follicles develop through primordial, primary, and secondary stages before acquiring an antral cavity. At the antral stage most follicles undergo atresia; however, under the optimal gonadotropin stimulation that occurs after puberty, a few of them are rescued (cyclic recruitment) to reach the preovulatory stage. Eventually, depletion of the pool of resting follicles leads to ovarian follicle exhaustion and senescence.
Rights were not granted to include this figure in electronic media. Please refer to the printed book.
(From McGee EA, Hsueh AJW: Initial and cyclic recruitment of ovarian follicles. Endocrine Reviews 21:200–214, 2000; with permission. Copyright 2000, The Endocrine Society. 47 )

Figure 3-4 Duration of follicle recruitment and selection in human and rat ovaries. Primordial follicles undergo initial recruitment to enter the growing pool of primary follicles. Due to its protracted nature, the duration required for this step is unknown. In the human ovary, more than 120 days are required for the primary follicles to reach the secondary follicle stage, whereas 71 days are needed to grow from the secondary to the early antral stage. During cyclic recruitment, increases in circulating follicle-stimulating hormone (FSH) allow a cohort of antral follicles (2 to 5 mm in diameter) to escape apoptotic demise. Among this cohort, a leading follicle emerges as dominant by secreting high levels of estrogens and inhibins to suppress pituitary FSH release. The result is a negative selection of the remaining cohort, leading to its ultimate demise. Concomitantly, increases in local growth factors and vasculature allow a positive selection of the dominant follicle, thus ensuring its final growth and eventual ovulation. After cyclic recruitment, it takes only 2 weeks for an antral follicle to become a dominant graafian follicle. In the rat, the duration of follicle development is much shorter than that needed for human follicles. The time required between the initial recruitment of a primordial follicle and its growth to the secondary stage is more than 30 days, whereas the time for a secondary follicle to reach the early antral stage is about 28 days. Once reaching the early antral stage (0.2–0.4 μm in diameter), the follicles are subjected to cyclic recruitment, and only 2 to 3 days are needed for them to grow into preovulatory follicles.
Rights were not granted to include this figure in electronic media. Please refer to the printed book.
(From McGee EA, Hsueh AJW: Initial and cyclic recruitment of ovarian follicles. Endocrine Reviews 21:200–214, 2000; with permission. Copyright 2000, The Endocrine Society. 47 )

Figure 3-5 Landmarks of follicular development during fetal and neonatal life in humans and rodents. In the human ovary, primordial follicles are present by 20 weeks of fetal life, whereas primary follicles are found by 24 weeks. By 26 weeks, some follicles have progressed to the secondary stage. Antral follicles develop in the third trimester and are also seen postnatally when follicle-stimulating hormone (FSH) levels are elevated. After puberty, cyclic increases in serum gonadotropins stimulate the antral follicles to become preovulatory follicles during each menstrual cycle. In the rat ovary, primordial follicles are formed by 3 days after birth when the first wave of follicles begins growth. These follicles progress to the early antral stage during the third week of life when serum gonadotropin levels are elevated. After early antral follicles are formed, ovarian cell apoptosis increases. FSH receptors are found by day 7 of age, when secondary follicles are present, followed closely by the formation of luteinizing hormone (LH) receptors in the thecal cells. Cyclic ovarian function begins around day 35 of age. The neonatal rodent model allows analysis of early follicle development in a synchronized population of growing follicles. Pmd, primordial.
Rights were not granted to include this figure in electronic media. Please refer to the printed book.
(From McGee EA, Hsueh AJW: Initial and cyclic recruitment of ovarian follicles. Endocrine Reviews 21:200–214, 2000; with permission. Copyright 2000, The Endocrine Society. 47 )
The oocyte grows from approximately 12 μm to approximately 80 μm in the fully grown state, which is achieved prior to formation of the antral follicle in sexually mature mice. In general, antral follicles tend to undergo follicular atresia, which is initiated by apoptosis in the granulosa cells, unless they are rescued by increased levels of FSH in the estrus or menstrual cycles in mice and humans, respectively. This cyclic recruitment of antral follicles results in the rescue and continued development of a few follicles, but usually only one dominant follicle prevails and develops to the periovulatory stage in the human menstrual cycle. 47 Although cyclic recruitment is operative after puberty in humans, most antral follicles ultimately undergo atresia, which results in oocyte death.
Primordial follicles that have not been recruited are thought to remain dormant and be available for recruitment at a later time. Therefore, maintenance of the pool of nongrowing oocytes is necessary to sustain a normal reproductive life span. Müllerian inhibiting substance or antimüllerian hormone, a TGFβ superfamily member well-known for its regulatory role in the sexual differentiation of the reproductive tract during development, is expressed in granulosa cells of growing follicles starting in the perinatal period. 48 During folliculogenesis, it inhibits the recruitment of primordial follicles to the pool of growing follicles by decreasing the responsiveness of granulosa cells to FSH. 49 Antimüllerian hormone is recognized as an important clinical marker of the ovarian reserve of viable follicles.

Regulation of Folliculogenesis by Gonadotropins
Preantral follicle development, which involves oocyte growth with limited granulosa cell proliferation, is regulated predominantly by paracrine and autocrine signals. Although gonadotropins are not required for early follicle development in vivo, these follicles can develop in response to FSH in vitro. 47 In contrast, gonadotropins are required for antrum formation and the rapid granulosa cell proliferation that results in the graafian or antral follicle.
Follicular growth from the primary and secondary follicles to antral stages takes several weeks in the mouse and several months in many larger mammals, including humans. The Graafian follicle can exceed 600 μm and contains more than 50,000 granulosa cells.
Granulosa cells fall into two distinct subtypes—cumulus and mural granulosa cells—that exhibit differential morphologic features and functions. Mural granulosa cells, located in the periantral and outer membrane regions, express FSH receptor (FSHR) and are responsive to FSH.
FSHR comprises a large extracellular domain that binds the FSH hormone with high affinity, and a seven membrane-spanning domain that activates downstream cyclic adenylate cyclase signaling via the heterotrimeric G proteins. 50 Mice deficient in the FSH β subunit, which normally forms heterodimers with the α subunits, are infertile due to arrested follicular development prior to formation of the antrum. 51
Consistent with the critical role of FSH signaling via its receptor, follitropin receptor knockout (FORKO) mice are also infertile. 52 At postnatal day 2, there are fewer nongrowing but more growing follicles, but by postnatal day 24, the numbers of resting and growing follicles are both decreased. Therefore, postnatal recruitment of resting follicles into the growing phase appears to be aberrantly accelerated, followed by arrest in further follicular recruitment later on.
Further, no antral follicles are observed. Although these mouse models confirm that FSH signaling is required for follicular development beyond the preantral stage, the phenotype of the FORKO mice also suggests that FSHR signaling is essential in the regulation of the timing and rate of follicular recruitment. 52 In contrast, luteinizing hormone receptor knockout mice (LuRKO) females are infertile, presumably due to defects in the later stages of folliculogenesis because follicles up to the early antral stage are present, but preovulatory follicles and corpora lutea fail to form. 53
FSH and FSHR-mediated downstream intracellular signaling results in gene and protein expression that exemplifies granulosa cell function. For example, antimüllerian hormone, which is implicated in primordial follicle recruitment, exhibits an aberrant expression pattern in FORKO mice. 52 Although recruitment of theca cells and their expression of the LH receptor and P450 aromatase genes are independent of FSH signaling, expression of these genes in granulosa cells is dependent on FSH signaling. 54 Similarly, the genes encoding for inhibin and activin subunits, whose homodimers and heterodimers are thought to be important paracrine and autocrine mediators of follicular growth, have decreased expression levels in granulosa cells in the FSHβ knockout model. 54 Other genes that are dysregulated as a result of FSHβ deficiency include those encoding for androgen receptor ( AR ), estrogen receptor β ( ER β), and cyclin D2, all of which have been shown to be essential in the final stages of follicular development. 54

Estrogen Receptors
Two estrogen receptors, ERα and ERβ, are expressed in granulosa cells in the antral follicle. Different knockout models have been generated for these two receptors based on different strategies of gene targeting, but they produced similar phenotypes. 55 ERαKO females are infertile; ERβKO females are subfertile. In both ERαKO and ERβKO mice, folliculogenesis progresses normally until the large antral stage, when ERβ is critical for ER-mediated granulosa cell proliferation and subsequent ovulation.
In contrast, ERα is not critical for follicular growth but is required for ovulation. The ERαβKO females clarify the question of functional redundancy between the two ERs by demonstrating that in the absence of ERs, antral follicles only have single layers of granulosa and theca cells, multiple small pockets of antral fluid, and oocytes that are dissociated from cumulus cells. 55

D-type Cyclin
Granulosa cell proliferation in late folliculogenesis also requires cyclin D2, which belongs to the D-type cyclin family of cell cycle regulators that promote the transition from G 1 to S phases via binding to Cdk4 or Cdk6. 56 Cyclin D2–deficient females have decreased FSH-mediated granulosa cell proliferation, which results in antral follicles that have significantly fewer layers of granulosa cells. This defect in granulosa cell proliferation also results in ovulation failure on stimulation by LH. However, the number of follicles is not decreased, luteinization of theca cells proceeds, and follicles differentiate to become corpora lutea.
We learn from the cyclin D2–deficient mutants that although these final stages of folliculogenesis and ovulation require cyclin D2 via FSHR-mediated activation, specifically of protein kinase A (PKA), oogenesis per se does not require these last rounds of granulosa cell proliferation. In fact, the oocyte not only appears normal, but it is also competent to undergo meiotic resumption and fertilization to produce viable embryos. 56 Therefore, although oogenesis and folliculogenesis are closely intertwined, oocyte development is a cell-autonomous process to a certain extent, especially in the advanced stages and at periovulation.

THE ROLE OF CUMULUS CELLS IN OOGENESIS
Cumulus cells, also called corona radiata, are specialized granulosa cells that directly line the oocyte. In addition to their supportive role in cytoplasmic maturation of the oocyte, they have important functions in oocyte development, including the maintainenance of meiotic arrest and induction of ovulation. Ovulation is a broad term that encompasses the processes of follicular luteinization, follicle rupture, and meiotic resumption in the oocyte.
A critical step during LH-induced ovulation is the cumulus cell-mediated cumulus expansion. Mural granulosa cells express LH receptor, which allows them to respond to LH by secreting proteins of the epidermal growth factor (EGF) family—amphiregulin, epiregulin, and betacellulin—which are thought to act as paracrine signals that lead to cumulus expansion. 57 During this process, cumulus cells disperse in the extracellular matrix, which comprises hyaluronic acid, tumor necrosis factor-stimulated gene 6 ( TSG6 ), and serum-derived inter-α-inhibitor, all of which are essential for follicular rupture. 58
The cumulus expression of Tsg6 and several other proteins implicated in cumulus expansion is regulated by prostaglandin E 2 (PGE 2 ) via its receptor EP 2 . 58 Consistent with the critical role of PGE 2 in cumulus expansion and ovulation, mice deficient in either EP 2 or cycloxygenase-2 (Cox-2), which is the rate-limiting enzyme for PGE 2 , are infertile due to their inability to ovulate. 58 - 60

Connexin and Gap Junctions
Cumulus cells mediate many of their important functions by communicating through gap junctions among themselves as well as with the oolemma. These gap junctions are intercellular channels formed by proteins of the connexin family for the diffusion of sugars, amino acids, lipid precursors, nucleotides, metabolites, and signaling molecules. Connexin family members share the same protein domains, including four membrane-spanning domains, two extracellular loops, a cytoplasmic loop, and cytoplasmic N- and C-terminals. 61 In mice, there are at least 17 connexin proteins, whose distinct sequence or length in the cytoplasmic loops and C-terminal tails, as well as heterodimeric and homodimeric coupling, allow functional diversity.
In mice, connexin (Cx) 32, 37, 43, 45, and 57 are expressed in the cumulus–oocyte complex, where they are located between cumulus cells, on cumulus transzonal projections that anchor cumulus cells into the zona pellucida, or on the microvilli or plasma membrane of the oocyte. 61
The critical role of gap junctions in oogenesis is exemplified by the sterility phenotype found in Cx37-deficient mice. Cx37 is made by both the oocyte and granulosa cells, but it may be the only connexin member in cumulus–oocyte gap junctions that is contributed by the oocyte. 61 In the absence of Cx37, follicular development fails at the preantral-to-antral transition, so that most follicles are arrested at the primary follicle stage, and only a few small antral follicles are present. Further, ovulation does not occur despite the formation of numerous corpora lutea. 62
Similarly, in vitro experiments show that Cx43, which is also expressed in granulosa cells, is required for folliculogenesis beyond the primary follicle stage. 63 Most importantly, oocytes from these two mutant mouse models are meiotically incompetent, which may reflect the requirement of cumulus cell communication in the acquisition of meiotic competence.
These connexin-deficient experimental models demonstrate the critical role of gap junctions in folliculogenesis, but they do not explain the role of cumulus cells in the regulation of meiotic arrest and resumption in the oocyte. Numerous models and hypotheses attempt to explain how and to what extent cumulus cells mediate the high oocyte intracellular cAMP levels that are required to maintain meiotic arrest even after the oocyte has acquired meiotic competence. 64 Although the complete story remains to be told, several key players have been identified.

MAINTENANCE OF MEIOTIC ARREST
The oocyte is maintained in meiotic arrest via developmental stage-specific mechanisms. Before reaching the fully grown stage, the oocyte is intrinsically incompetent, but once the fully grown oocyte acquires meiotic competence, cumulus cells are thought to contribute at least partially to the maintenance of meiotic arrest.
It is well known that fully grown oocytes isolated from surrounding cumulus cells spontaneously undergo meiotic resumption independent of LH signaling. Although the signal that confers cumulus regulation of arrest has not been identified, the signaling cascade within the oocyte compartment has become much better understood.

GTP-binding Protein
The stimulatory heterotrimeric GTP-binding protein (G s )-linked receptor 3 (GPR3) is expressed in the oocyte plasma membrane and is thought to mediate cumulus control of meiotic arrest. However, GPR3 is capable of activity independent of LH or cumulus cells also. The role of GPR3 is exemplified by ovarian sections from prepubertal GPR 3 −/− females in which meiotic stages beyond prophase I are seen in oocytes in intact early antral follicles in the absence of LH signaling or cumulus expansion. 65
Similarly, inactivation of G s by microinjection of an antibody specifically targeting G s into the oocyte within an intact follicle allows the oocyte to resume meiosis even if cumulus cells remain adherent to the oocyte. 65 GPR3 maintains activity of G s in stimulating adenylyl cyclase to produce high cAMP levels, which in turn maintain PKA activity. 65 Specifically, the isoform adenylyl cyclase 3 expressed by the oocyte is critical in generating sufficient cAMP. 66 Acting downstream of the GPR3→G s →cAMP→PKA signaling cascade, PKA-dependent phosphorylation of substrate proteins presumably inhibits meiotic resumption through as-yet unknown mechanisms that include suppression of M-phase promoting factor (MPF) activity; this represents an area of intense investigation. 64

MEIOTIC RESUMPTION
Whereas LH triggers ovulation by the cumulus cell compartment and release of the oocyte from meiotic arrest, the resumption and completion of meiosis may be regulated intrinsically to a large extent by the fully grown oocyte. Resumption of meiosis requires both decreased levels of intracellular cAMP and activation of MPF. 64 Decreased production and increased degradation of cAMP in the oocyte appear to contribute to meiotic resumption.
Decreased cAMP production is mediated by the inhibitory G protein, which in turn is activated by an oocyte-expressed G protein-coupled receptor called leucine-rich repeat-containing G protein-coupled receptor 8 ( LGR8 ). 67 Interestingly, the role of LH in meiotic resumption is demonstrated by this LGR8-mediated decrease in cAMP levels, because LGR8 is stimulated by insulin-like 3, which is expressed and secreted by ovarian theca cells in response to LH. 67

Phosphodiesterase (PDE)
On the other hand, the degradation and inactivation of cAMP in the oocyte are mediated by the oocyte-expressed cyclic nucleotide phosphodiesterase family member, PDE3A. GV oocytes from PDE3A −/− mice fail to resume meiosis in vivo and remain in the GV stage even after ovulation. In addition, meiosis does not resume even when fully grown GV oocytes from these mice are isolated from cumulus cells and cultured in vitro. 68 Thus, the GPR3 and PDE3A knockout mouse models demonstrate their critical and opposing roles in the regulation of meiotic arrest and resumption, respectively. More importantly, these models also prove that ovulation and meiotic resumption, though usually occurring synchronously, are independent processes that can be differentially regulated.

Meiotic Competence and Oocyte Maturation
Meiotic progression requires activation of MPF, which is composed of cyclin B and its protein kinase Cdk1 (also called p34 cdc2 ). 69 The requirement of MPF for the G→M transition in mitosis and meiosis is highly conserved across species. Activation and inactivation of many cell-cycle regulators are mediated via phosphorylation, a form of post-translational protein modification of specific residues. The dual phosphatase, Cdc25b, is required for the dephosphorylation of residues 14 and 15 of p34 cdc2 , which results in activating p34 cdc2 . Oocytes from Cdc25b −/− mice are unable to resume meiosis and remain arrested in prophase unless rescued by microinjection of Cdc25b mRNA. 70 Active p34 cdc2 accumulates rapidly through an autoamplification loop. Therefore, the rate-limiting step of MPF activation is thought to be the synthesis and accumulation of cyclin B. During oocyte growth, the level of active MPF increases and eventually reaches the threshold above which the oocyte becomes meiotically competent, which refers to its ability to undergo meiotic resumption.
In general, meiotic competence is related to oocyte size, presumably because cytoplasmic volume reflects the accumulation of synthesized proteins, including cyclin B, p34 cdc2 , and Cdc25b, which are essential for meiosis. 71 Oocytes that are smaller than 70 to 80 μm have not reached their full growth potential and have low rates of meiotic competence, whereas those approximately 100 μm are considered fully grown and tend to be able to undergo meiosis. Another predictor of meiotic competence is the presence of a surround nucleolar (SN) chromosomal localization pattern that can be visualized in the live oocyte with culture media containing Hoescht stain. 72, 73

Germinal Vesicle Breakdown (GVBD)
The first observable morphologic change of meiotic resumption is GVBD, which is characterized by the sequential disappearance of GV and nucleolus that is frequently concurrent with a shift from central to ectopic localization of the GV. After GVBD, bivalents start to become organized at prometaphase and then align at the metaphase plate in metaphase I.
At the anaphase–metaphase transition, chromosomes begin to segregate, and a cytoplasmic protrusion signifying late anaphase may be visible. MPF activity increases as the oocyte progresses from GV, GVBD, to metaphase I, but then declines transiently between metaphase I and metaphase II. 74, 75 However, progress beyond metaphase I may not occur in some oocytes if molecular prerequisites are not met. 76

Metaphase–Anaphase Transition
The metaphase–anaphase transition is regulated by decreased MPF activity, suppression of protein kinase C (PKC) activity, and the activity of many other proteins. 77 - 79 Decreased MPF activity is achieved by proteasome degradation of cyclin B, which is required for polar body extrusion (metaphase I–metaphase II) and at metaphase II exit on fertilization. 80 - 83 Like many mitotic and meiotic cell cycle regulators, the proteolysis of cyclin B is mediated by ubiquitin and the anaphase-promoting complex (APC), which are well conserved and required for proteolysis of many proteins across species.
The anaphase-promoting complex is an E3 ubiquitin ligase that transfers ubiquitin “tags” from E2 ubiquitin conjugating enzymes to substrates, such as cyclin B, thereby “tagging” them for recognition and proteolysis by the 26S proteasome. 82 The essential role of the proteasome in meiosis is further supported by the metaphase I arrest phenotype that is seen in mouse and rat oocytes cultured in media containing chemical inhibitors of the proteasome. 84, 85

Meiotic Spindle
Achievement of metaphase I depends on the formation of an intact meiotic spindle, which requires the cell cycle regulator, cell division cycle-6 homologue (Cdc6). Insight into this process is provided by oocytes in which Cdc6 expression is abrogated by the RNA interference technique. 86 These oocytes undergo GVBD, but the meiotic spindle is not formed, and they fail to reach metaphase I. In the absence of the meiotic spindle, condensed chromosomes fail to form bivalents and do not align to form the metaphase plate. Instead, the chromosomes form a halolike structure and meiosis I is arrested with no cell division or extrusion of polar body. 86
In the presence of a normal meiotic spindle, the oocyte will reach metaphase I and then undergo the metaphase–anaphase transition, which is an important event because defective chromosome segregation at this stage can lead to aneuploidy in the resulting egg and embryo.
In mitosis, the centromeres of sister chromatids are held together by cohesin until the onset of anaphase, when cohesin cleavage by separase allows separation of sister chromatids. 87 - 89 Inhibition of separase activity by securin prior to anaphase onset, and its subsequent activation at anaphase upon securin degradation via the APC, are critical in ensuring correct chromosomal segregation. 90, 91 The APC→securin→separase cascade is also required to ensure correct spindle function and homologous chromosome disjunction during the metaphase–anaphase transition in meiosis in the oocyte. 92, 93

Spindle Assembly Checkpoint (SAC)
Another mechanism that the oocyte has in common with mitotic cells to ensure the correct segregation of chromosomes is the spindle assembly checkpoint (SAC). SAC proteins detect whether chromosomes are properly aligned and whether kinetochores are attached to the spindle. SAC proteins control the onset of anaphase I through interactions with one another, the kinetochores of unaligned chromosomes, and Cdc20, which is a protein that mediates APC/C proteolysis. ( Kinetochores are the sites of attachment of centromeres of the chromosomes to the microtubules of the spindle. Homologous chromosomes migrate to opposite poles through tension exerted by the spindle on the kinetochores.) If chromosome alignment is not complete, the SAC protein would inhibit APC/C to prevent precocious sister chromatid separation, which can cause aneuploidy in the resulting egg and embryo.
Mitotic SAC proteins that also have essential spindle checkpoint functions during maturation of the mouse oocyte include Mad2, BubR1, and Bub1. Interference of the function of these proteins results in accelerated completion of meiosis I, presumably because anaphase occurs prematurely. 94 Further, the abrogation of Mad2 expression has specifically been shown to increase the rate of aneuploidy, whereas its overexpression inhibits disjunction of homologous chromosomes. 95 Therefore, the expression and function of these cell-cycle regulators are candidates to be investigated for their potential involvement in age-related aneuploidy in human oocytes.
At the end of anaphase I, the first polar body is extruded, which is followed by reaccumulation of cyclin B levels. MPF reactivation is required for the synthesis of c-mos, which leads to activation of MAP kinase kinase and MAP kinase. 96 - 100 This c-mos-mediated MAP kinase activity is critical for the maintenance of metaphase II arrest, as shown by the c-mos −/− mouse model. Unfertilized eggs from c-mos −/− mice fail to arrest at metaphase II, and undergo parthenogenetic activation, during which the second polar body is extruded in the absence of fertilization. 101, 102

NUCLEAR AND CYTOPLASMIC CHANGES DURING OOCYTE GROWTH
The growing oocyte undergoes nuclear and cytoplasmic changes that pertain to nuclear organization, epigenetic regulation, transcription, translation, ultrastructural morphology, and organelle function. These changes are sometimes referred to as nuclear or cytoplasmic maturation, broadly defined based on their association with oocyte maturation, subsequent fertilization, or embryo development. Many of the morphologic changes of the oocyte have been elegantly described at the cellular and ultrastructural levels. 103 Further, expression, translational modification, and function of cell-cycle regulators, also considered indices of cytoplasmic maturation, are discussed in this chapter under “Meiotic Competence and Oocyte Maturation.” In this section, we highlight some biochemical features characteristic of the oocyte around the time of maturation.

Transcriptional Regulation
The growing oocyte is characterized by a high level of transcriptional activity that increases its total RNA content from approximately 0.2 ng in a small oocyte to approximately 0.6 ng in a fully grown oocyte. 104 In growing oocytes, gonadotropin stimulation of follicles increases the proportion of transcriptionally inactive oocytes; thus granulosa cells are thought to have an important role in the regulation of transcriptional activity in the growing oocyte. 105 The fully grown oocyte undergoes global transcriptional silencing, which is a prerequisite for meiotic resumption, oocyte maturation, and subsequent embryo development. 106 Therefore, messenger RNAs (mRNAs) that are synthesized during oocyte growth are not only utilized for the translation of proteins during the growth phase, but are also stored for protein synthesis in the fully grown stage, during oocyte maturation, or later in the early embryo. Thus, precise control of translation is vital to the function and ultimate developmental potential of the oocyte and embryo.

Regulation of Translation
Translation requires modification of the mRNA transcript such as polyadenylation at the 3′ end (addition of a poly-A tail ) and cap binding at the 5′ end. Such modifications are controlled by cis and trans elements. For example, the translation and accumulation of cyclin B1 is regulated via polyadenylation and cis -regulatory mechanisms in the 3′ untranslated region (3′UTR) of the cyclin B mRNA transcript. 107
Cis elements in the 3′UTR consist of a highly conserved U-rich sequence (CPE) in conjunction with a downstream hexanucleotide cytoplasmic polydenylation element, AAUAAA. Cis elements mediate polyadenylation via binding to the phosphorylated form of the CPE binding protein (CPEB), which is a trans- acting factor specific to the oocyte. The critical function of CPEB in the oocyte was demonstrated in CPEB knockout mice. 108 In the absence of CPEB oogenesis arrested at the pachytene stage on 16.5 dpc, presumably because translation of SCPs 1 and 3 did not occur, which led to death and resorption of the oocytes. 108, 109 However, the requirement of CPEB at this early stage also precluded the function of this protein during oocyte maturation to be ascertained.
Other regulatory mechanisms of translation involve inhibition of translation if the presence of certain proteins would interfere with oocyte maturation. For example, some mRNAs can be stored in the form of ribonuclear particles (RNPs), which do not support translation and prevent the misexpression of certain proteins. 110
The importance of this mechanism is shown by the relative abundance of the Y-box protein family member, MSY2, which has an essential role in mediating translational silencing by RNPs and comprises 2% of the total protein content in the fully grown oocyte. 111, 112 In addition, poly-A-specific ribonuclease (PARN) mediates widespread deadenylation and subsequent degradation of mRNAs encoding for a variety of proteins, including zona pellucida proteins, actin, alpha tubulin, and Cx43, at the time of meiotic resumption. 110 In contrast, the polyadenylation of certain cell-cycle proteins required during oocyte maturation (i.e., cyclin B1) is enhanced at meiotic resumption. 113

Epigenetic Regulation
In addition to regulation at the levels of transcription and translation, epigenetic regulatory mechanisms in the oocyte, sperm, and early embryo affect the expression of specific genes through the process of genomic imprinting, as well as the entire molecular program through global changes in methylation status and structural chromatin remodeling. Structural remodeling of chromatin is associated with significant changes in the nuclear architecture during oocyte growth and maturation.
Chromosomes in the live mouse oocyte can be viewed using culture media containing Hoescht stain, a chemical that chelates with the minor groove of DNA and emits blue fluorescent light upon UV light absorption. Centromeres and pericentromeric heterochromatin are initially located at the periphery of the nucleus in oocytes in primordial and primary follicles. 106 Then during oocyte growth, they are spread within the nucleus, followed by localization to the periphery of the nucleolus. This perinucleolar heterochromatin rim, or karyosphere, appears as a bright rim around the nucleolus and is also known as the surround nucleoli (SN) pattern. 73, 114
Although the SN pattern is associated with global transcriptional repression and high potential for meiotic competence and embryo development, we now know that chromatin remodeling and transcriptional repression are distinct processes controlled by different mechanisms. 106 As in somatic cells, histone deacetylases were also found to be critical for the large-scale chromatin remodeling in the fully grown oocyte because their inhibition resulted in a breakdown of the SN architecture and aberrant configuration of the meiotic chromosomes and spindle. 106
Another important epigenetic mechanism that has emerged is control of transcriptional activation and repression via demethylation and methylation, respectively. Whereas the genome of somatic cell precursors undergoes remethylation before gastrulation at 6.5 dpc, the genome of PGC precursors remains demethylated at 12.5 dpc. Then partial methylation occurs at 15.5 dpc and methylation is completed by 18.5 dpc. 32 The embryonic genome is inactivated until the two-cell stage in mice (eight-cell stage in humans), when it becomes activated by global demethylation.
These global changes in methylation status are distinct from X-inactivation and genomic imprinting. Briefly, X-inactivation is a complex and random process in which one of the X chromosomes in XX mammalian females is inactivated to ensure an equivalent dosage of X-linked genes in XX females and XY males. 115 Initiation of this process is controlled by a locus called X-inactivation center (Xic) at Xq13. This locus contains X-inactive specific transcript ( Xist ), which encodes a non-coding mRNA that coats the X-chromosome in cis to trigger inactivation 115 (see Chapter 5 ).

Genomic Imprinting
Finally, a more gene-specific mechanism of epigenetic regulation is genomic imprinting, which mediates predesignated, differential silencing of the maternal and paternal alleles. 116 During gametogenesis, certain genes on autosomes become hypermethylated, or silenced, based on their parental origin. Once established, these imprints are thought to be protected from genome-wide demethylation.
Imprinting possibly occurs at different time points in oogenesis, spermatogenesis, and embryogenesis for different genes. Congenital syndromes or cancer can result from a failure of the allele from the designated parental origin to be silenced or an effective null mutation based on a heterozygous mutation in the nonimprinted allele. These disease mechanisms and syndromes are further discussed in Chapter 5 .
The mechanism of imprinting is an area of research that is particularly relevant to reproductive endocrinology and infertility treatment because of reports associating assisted reproductive technologies (ART) with possible increased risk of imprinting defects. 117 In addition, it has been suggested that in the mouse model, oocyte maturation and embryo culture in vitro may interfere with imprinting. 118, 119 However, because defects in imprinting are rare in the general population and in children conceived through ART, it is difficult to determine the statistical significance in these studies. Consequently, whether there is a true association or a causal link remains an open question.
If ART is indeed associated with imprinting defects, one would have to examine all possible causes, including controlled ovarian hyperstimulation (COH), clinical embryology, and the cause of infertility itself, because imprinting defects may be the cause of infertility that becomes unmasked by infertility treament. Therefore, this area warrants intense investigation to determine whether and how mechanisms of imprinting impact on future directions in ART.

THE OOCYTE–EMBRYO TRANSITION
In addition to the maternal genome, the oocyte contributes specific gene products that are required for early embryo development prior to zygote gene activation. In the mouse model, zygote genome activation occurs at the two-cell stage, while in the human it occurs at the four- to eight-cell stage.

Maternal Effect Genes
Because the oocyte does not synthesize new mRNA transcripts after meiotic resumption and the embryonic genome is not activated until the two-cell stage, the oocyte must store transcripts and proteins for all of its requirements during the oocyte–embryo transition. Genes encoding oocyte proteins that persist and have critical functions in the early stages of embryogenesis are called maternal effect genes.
It is not surprising, therefore, that several proteins encoded by maternal effect genes—oocyte-specific linker histone H1 (H1F00), nucleoplasmin 2 (Npm2), DNA methyltransferase1 oocyte isoform (Dnmt1o)—are involved in mechanisms of epigenetic gene regulation; these processes are initiated in the oocyte, but continue during early embryo development. 120 - 124 All of these maternal effect genes, and potentially ones that have yet to be discovered, serve as critical links to demonstrate how the unfertilized oocyte can affect subsequent embryo development, besides contributing to the embryonic genome.
H1FOO is an oocyte-specific H1 linker histone that associates with chromatin during the GV and maturation stages; this association continues until the late two-cell to four-cell embryo stages when they are gradually replaced by somatic H1. Because somatic H1 linker histones are important in the regulation of chromatin function, the oocyte- and early embryo-specific expression of H1FOO suggests that it may be critical in chromatin remodeling during the oocyte–embryo transition. 120, 121
In contrast, Npm2, a protein that is present in the nuclei of oocytes and peri-implantation embryos, is proven to have an essential role in chromatin structure. Embryos of Npm2 −/− mutant females lack Npm2, and die before implantation because Npm2 is required for the maintenance of normal nucleolar structures. Specifically, heterochromatin and deacetylated histone H3, which are usually localized to the rim of the nucleolus in oocytes and early embryos, are absent. 122 Therefore, this mouse model also demonstrates the importance of chromatin regulation and nuclear architecture in early embryo development.
Another maternal effect gene that is important in epigenetic regulation is Dnmt1 . The oocyte variant, Dnmt1o, is an isoform of the Dnmt1 protein that is expressed only in the growing oocyte and early embryo, under an oocyte-specific promoter. Embryos derived from Dnmt1o-deficient oocytes are also deficient in Dnmt1o and develop into fetuses that die during gestation. 123 These embryos have defective imprinting, as shown by inappropriate methylation at certain gene loci and loss of allele-specific gene expression. The critical function of Dnmt1o is thought to occur at the eight-cell stage, when the protein transiently translocates from its usual location in the cytoplasm to the nucleus. 123
Zar1 -null mutant females appear to have normal oogenesis and fertilization appears to be initiated, but the two pronuclei remain separated and most embryos arrest at the one-cell stage. 125 Further, of the small percentage of embryos lacking Zar1 that survive to the two-cell stage, there is decreased expression of proteins in the transcription-requiring complex, which indicates a defect in embryonic genome activation. Whereas Zar1 is critical for progression to the two-cell stage, Mater, another protein encoded by a maternal effect gene, is expressed in oocytes and preimplantation embryo and is critical for embryonic development beyond the two-cell stage. 126 Investigation into the functions of these proteins will contribute to our understanding of mechanisms that control the merging of paternal and maternal genomes, embryonic genome activation, and early embryo development.

Polarity and Cytoplasmic Reorganization
Unlike other model organisms such as the fruit fly, the determination and establishment of cell polarity, or asymmetric distribution of cytoplasmic contents, in the mammalian oocyte and embryo is not well understood. Part of the difficulty is that although polarity in the form of an eccentric GV is observed in oocytes of some mammalian species, such as human, Rhesus monkey, bovine, and porcine, GV oocytes isolated from mouse and rat ovarian follicles do not demonstrate polarity, perhaps due to artificial effects of the in vitro culture system. 127
The growing oocyte has one dominant microtubule organizing center (MTOC). The MTOC comprises centrosome, which organizes microtubules in an astral configuration. In mitotic cells, centrosomes are made up of a pair of centrioles, which are cylindrical structures containing nine triplets of microtubules that form the astral fibers required for cell division. However, centrosomes in the oocyte do not have centrioles and are thus called MTOCs. The fully grown, meiotic competent oocyte has multiple MTOCs that mediate migration of the GV from the center to an eccentric, cortical position during oocyte maturation. The eccentric position of the GV is thought to be related to or to determine the location of the meiotic spindle, which in turn determines the site of polar body extrusion. 127
The eccentric position of the meiotic spindle defines the animal–vegetal axis, in which the animal pole contains the spindle-associated cortex, and the vegetal pole is characterized by clusters of endoplasmic reticulum. This asymmetric organization of the cytoplasm is thought to be critical in ensuring that both cell divisions at meiosis I and meiosis II will produce polar bodies, rather than daughter cells of equal size. This asymmetry in the generation of the egg is well conserved across species and is thought to be nature’s way of maximally conserving resources for the resulting embryo.
It is controversial whether the meiosis II cell division further determines the point of fusion between sperm and egg, the position of the first mitotic spindle, and even the spatial organization of the first mitotic division. 128 The mos/MAP kinase pathway, which regulates the metaphase II arrest, is also required for transitioning the meiotic spindle from a cortical location back to the central location of a mitotic spindle. 129 Therefore, determinants of oocyte polarity likely have as-yet unrecognized impact on the development and survival of the early embryo. Indeed, the cytoplasm undergoes drastic reorganization from the asymmetric oocyte and egg, to become an embryo that is capable of symmetric cell divisions.

APOPTOSIS: THE ULTIMATE CELL FATE FOR MOST OOCYTES
The oocyte is perhaps the most limiting factor in human reproduction because only about 300 to 400 eggs are ovulated during a woman’s entire reproductive lifespan. In other words, more than 99.9% of all oocytes undergo programmed cell death, or apoptosis, without ever having the opportunity to become fully grown, ovulate, or fertilize.
The first wave of apoptosis in the mouse model is thought to occur when germline cysts containing oogonia break down, and a similar process likely occurs in the human female fetus. The number of oocytes reaches a peak of approximately 7 million by midgestation in humans. 32, 130 - 133 Subsequently, oocytes that do not become encapsulated by granulosa cells undergo apoptosis. Further, growing follicles undergo atresia as they reach the antral stage, so that there are approximately 300,000 to 400,000 follicles at birth and about 200,000 follicles remaining by puberty. 132, 134, 135
Although apoptosis is the most common cell fate, the initiating factor and the mechanism may differ depending on when it occurs during development. For example, the apoptosis that affects primary oocytes before their incorporation into primordial follicles is thought to be oocyte-autonomous, whereas oocytes in growing preantral and antral follicles die secondary to follicular atresia that is caused by apoptosis of the granulosa cells.
Many hypotheses have been raised in an attempt to explain the high attrition rate, including that it is advantageous for the species to eliminate germ cells containing chromosomal or other errors. However, no hypothesis has been rigorously tested and the question remains open.

Environmental Factors
In addition to apoptosis that is part of “normal” development, environmental factors and genetic susceptibility can exacerbate depletion of the oocyte pool, which is often manifested clinically as premature ovarian failure or early menopause. Environmental factors that have demonstrated oocyte toxicity include organochlorine chemicals from pesticides and industrial processes, 136 polycyclic aromatic hydrocarbons from tobacco smoke or fuel combustion, 137, 138 and chemotherapeutic agents (i.e., doxorubicin) that activate the apoptotic pathway via ceramide, a lipid that is generated by acid sphingomyelinase. 139 The harmful effect of ceramide is further supported by the ability of its metabolite and antagonist, sphingosine-1-phosphate, to confer protection in preventing oocytes from undergoing apoptosis when exposed to chemotherapy. 140, 141

Genetic Factors
Genetic mouse models of oocyte apoptosis may exhibit phenotypes varying from subfertility (i.e., small litter size or infrequent litters), premature ovarian failure, or infertility due to congenital ovarian atresia. Broadly, genetic defects may primarily be due to structural or aneuploid chromosomal abnormalities; defective X chromosome inactivation; mutation in single genes that are essential in oogenesis, folliculogenesis, or gonadogenesis; or single-gene defects affecting apoptotic pathways in the ovary. Examples of the latter are highlighted here; the other genetic abnormalities are discussed elsewhere in this book. Most importantly, apoptosis should be recognized as a common final pathway that will follow defects interfering with oogenesis.
As in somatic cells, appropriate expression of pro-apoptotic proteins and those protecting against apoptosis is required to maintain the “normal” rate of attrition. Bcl-2 and Bcl-x proteins are expressed in the oocyte and granulosa cells and “protect” the oocyte from apoptosis. Although Bcl-2 -deficient mice are fertile with normal litter size, there is a decrease in the number of primordial follicles as well as an increase in the number of abnormal primordial follicles that contain no oocyte or a dying oocyte. 142 Similarly, Bcl-x hypomorphic mutant mice have significantly decreased numbers of primordial and primary follicles, but their phenotype is more severe and fertility is impaired. 143 Therefore, these two genes are required for the maintenance of both the oocyte pool and follicular architecture. In contrast, targeted overexpression of Bcl-2 in granulosa cells results in enhanced folliculogenesis, larger litter size, and an increased incidence of germ cell tumors with age. 144
Interestingly, female mice that are deficient in Bax are fertile and have significantly more primordial follicles than wild type mice even with advancing age. Further, they are able to superovulate in response to exogenous gonadotropins even at an advanced age, and these oocytes develop into viable embryos. 145 Similar results were seen in the mouse model in which the pro-apoptotic gene caspase-2 is deleted. In addition, in the absence of caspase-2, oocytes do not undergo drug-induced apoptosis on exposure to the chemotherapeutic agent doxorubicin. 146
These data support the relevance of this research area in understanding and potentially extending the female reproductive lifespan. Most importantly, understanding the regulation of apoptosis in the mammalian oocyte is vital to the development of oocyte- or follicle-sparing chemotherapeutic and other pharmaceutical agents.

CLINICAL RELEVANCE AND FUTURE DIRECTIONS
Many key challenges in clinical reproductive medicine today relate to the availability and function of human oocytes and the follicles that enclose them. However, we are very far from understanding what proportion of “unexplained infertility” is due to an oocyte-specific factor and even further from having any oocyte-specific treatment. 147, 148 Significant advances in our management of oocyte-related infertility will require a better understanding of the mechanisms through which maternal age, environmental effects, and patient-specific genetic susceptibility impact on oocyte function.

Human Gene Mutations and Oocyte Phenotypes
Human and mouse genetic models of oocyte apoptosis may exhibit phenotypes varying from subfertility, premature ovarian failure, or infertility due to congenital ovarian atresia. Although these conditions are usually considered distinct clinical entities that are managed differently, their genetic basis may be closely related. Broadly, genetic defects may primarily be due to structural or aneuploid chromosomal abnormalities involving the X chromosome (i.e., Turner’s syndrome), defective X chromosome inactivation, and mutation or epigenetic defects involving single genes that are essential for oogenesis, folliculogenesis, or gonadogenesis. In particular, familial or de novo single-gene mutations affecting any stage of oogenesis can theoretically result in a spectrum of conditions varying from congenital ovarian dysgenesis, to premature ovarian failure, to “poor oocyte quality” identified during COH or IVF treatment.
Although gene targeting in experimental mouse models has contributed significantly to our understanding of oogenesis, it is critical that we increase our effort to relate human phenotypes encountered in clinical reproductive endocrinology and infertility treatment, such as “poor ovarian response” and developmental abnormalities of oocytes and embryos, to our understanding of the mechanisms regulating oogenesis.

Premature Ovarian Failure
Of all oocyte- or ovarian follicle-related conditions, identification of human gene mutations has thus far largely focused on familial premature ovarian failure. Using cytogenetic and fluorescent in situ hybridization techniques, familial cases of premature ovarian failure associated with balanced translocation could be studied to identify candidate genes that are disrupted by the translocation breakpoint. For example, many autosomal dominant mutations in FOXL2 , a transcription factor that is expressed only in granulosa and eyelid cells, have been identified as the cause of a syndrome of premature ovarian failure and eyelid malformation called blepharophimosis ptosis epicanthus inversus syndrome. 149 - 153
Another genetic mutation that has been linked to familial hypergonadotropic gonadal failure in humans is localized to the X-linked gene BMP15. 45, 154 Further, the “critical region” on the long arm of the X chromosome (Xq13-Xq26) is proposed to contain several genes that are required for oogenesis or ovary development. A familial case of premature ovarian failure has been linked to a gene located in this critical region, DIA , which is the human homolog of the Drosophila diaphanous gene that is critical for oogenesis in Drosophila. 155 Because mouse homologs of FOXL2 and BMP15 , as well as the D. diaphanous gene, have critical functions in oogenesis or folliculogenesis, these examples underscore the value of connecting what we learn from animal models and scenarios encountered in the clinic.
Familial hypergonadotropic ovarian dysgenesis has also been identified in patients with autosomal recessive, inactivating point mutations in the FSHR gene, 156 - 158 which presumably abrogated follicular response to FSH, resulting in atresia and premature ovarian failure. Conversely, there are also reports of various FSHR mutations that increase the sensitivity of the FSH receptor to FSH, human chorionic gonadotropin (hCG), and thyroid-stimulating hormone (TSH), which led to spontaneous ovarian hyperstimulation syndrome and spontaneous superovulation. 159 - 165
These human gene mutations serve as spontaneous gene targeting models that demonstrate the role of these genes in human reproduction in vivo. Further, biochemical investigations of proteins encoded by these genes may provide insight into the pathogenesis of, and potential therapy for poor ovarian response to COH and the prevention of premature ovarian failure and ovarian hyperstimulation syndrome.
In the distant (or not so distant) future, it is conceivable that as more human genetic mutations causing ovarian dysfunction are identified, genetic screening panels combined with proper counseling may help predict risks of premature “oocyte aging” to help young women with their family planning. Similarly, poor responders and patients at risk for ovarian hyperstimulation syndrome may be identified before their COH/intrauterine insemination or IVF treatment. Establishment of reliable predictors will not only help optimize cycle outcomes, but will also be valuable in counseling couples regarding their treatment options and risks of multiple gestation and ovarian hyperstimulation syndrome.

Aneuploidy
Abnormal chromosomal segregation in meiosis results in aneuploidy, which increases in incidence with advancing maternal age in humans. Aneuploidy, considered “the most important problem in human reproduction,” affects an estimated 25% to 50% of human eggs, accounts for at least 35% of spontaneous abortions and approximately 4% of stillbirths, and is the leading genetic cause of congenital mental retardation and developmental disabilities. 166 - 172
The key risk factors for maternally derived aneuploidy in humans are maternal age and susceptible patterns of meiotic recombination, such as recombination sites that are close to the centromere or the telomere (reviewed in Lamb and colleagues 173 ). Compared to the mouse model, there is an extreme variability in the location of recombination nodules in human oocytes, 26 which may explain the high rates of aneuploidy in our eggs and embryos. However, although susceptible locations of recombination nodules are thought to be the main risk factor in young women, they do not explain the high aneuploidy rates seen with increasing maternal age. 173 In fact, the association between these recombination patterns and aneuploidy decreases with increasing age. Therefore, as-yet undefined recombination-independent factors are proposed to be risk factors for older women. 173

IN VITRO FERTILIZATION
IVF allows us to see the entire developmental process from GV, GVBD, to metaphase II arrest, formation of the one-cell embryo with two pronuclei, all the way to the blastocyst stage. Although we expect to see some abnormal oocytes or embryos during IVF treatment, when an unusual oocyte or embryo phenotype is consistently observed in the same couple, especially if they have “unexplained infertility,” we are alerted to the possibility that those abnormalities may be related to the underlying cause of the couple’s infertility.
Repeated IVF treatment failure in these patients would further identify them as the patient population that may benefit from having a molecular diagnosis and nonempirical treatment that specifically targets the molecular diagnosis. For example, recurrent or familial cases of zona pellucida abnormalities, 174 maturation arrest, 175 - 179 parthenogenetic activation, 180 triploidy, 181 abnormal fertilization, 182 abnormal cell division in meiosis II, 183 and empty follicle syndrome 184, 185 have been reported. Indeed, the development of molecular diagnosis and treatment for this subset of patients is a major challenge in reproductive medicine.

FUTURE DIRECTIONS
Outcome-based, systematic surveys of human oocyte and embryo phenotypes encountered in IVF treatment are critical in guiding clinicians and scientists to focus on conditions that are prevalent and relevant in patients seeking infertility treatment. Further, although mammalian oocyte biology research has traditionally been based on the candidate gene approach, complementary experimental strategies by more unbiased, forward genetic methods such as random chemical mutagenesis of the mouse genome followed by screening for infertility phenotype has begun to uncover novel genes that have critical functions in oogenesis. 186 Newly identified genes with potentially critical functions in oogenesis can then be further studied by various gene targeting techniques.
Another forward genetic approach is chemical genetics in the form of high throughput small molecule (SM) library screening of processes such as oocyte maturation and follicular development. SM library screening followed by functional experiments have led to the identification of lead compounds in drug discovery and the identification of novel proteins in other areas of research. 187 - 189 Although the technical challenges involved in the high throughput experimentation of the oocyte or follicle may seem quite overwhelming, they may not be truly insurmountable. We can look beyond medicine and biology, and engage bioengineers to bring microfluidics and other microsystems-based technologies to help us accelerate discovery in the field of oocyte biology and infertility. 190 Such interdisciplinary effort will be instrumental in giving us the ability to treat or prevent oocyte-related reproductive problems. It will also facilitate the development of oocyte- or fertility-sparing pharmaceuticals and the identification of environmental aneugens to direct preventive strategies to preserve reproductive health.
New findings on functions and human mutations of ovarian genes can be searched online on several databases, including the Ovarian Kaleiodoscope ( http://ovary.stanford.edu/ ) and Online Mendelian Inheritance in Man (OMIM) ( http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM ), 191 OMIM and Online Mendelian Inheritance in Man are trademarks of the Johns Hopkins University.


PEARLS

• During prophase of meiosis I, oocytes undergo DNA replication, homologous pairing, synapse, and recombination, but then remain arrested at the diplotene stage of prophase I until sexual maturity.
• Primordial germ cells are derived extragonadally from the yolk sac endoderm. They migrate from the base of the allantois to the genital ridges.
• Members of the TGF superfamily of growth factors are important signaling molecules for the migration and survival of primordial germ cells.
• Primordial germ cells differentiate into oogonia, which are sexually differentiated germ cells.
• Oogonia become oocytes by entering into prophase of meiosis I.
• Primordial follicles are diplotene-arrested oocytes surrounded by a single layer of flattened granulosa cells.
• Primary follicles are characterized by a single layer of cuboidal granulosa cells.
• Prophase I has four stages: leptotene, zygotene, pachytene, and diplotene.
• The transition from pachytene to diplotene marks the beginning of folliculogenesis.
• Oocytes arrest at the diplotene stage, when homologous chromosomes are held together at chiasmata.
• The oocyte is surrounded by the zona pellucida, which is composed of four proteins (ZP) in the human.
• ZP2 and ZP3 mediate sperm binding.
• Members of the family of TGF proteins are critical regulators of follicular development.
• The recruited growing follicles are primary follicles.
• Antral follicles tend to undergo atresia unless rescued by FSH.
• Antimüllerian hormone is expressed in granulosa cells of growing follicles.
• Preantral follicle growth is regulated predominantly by paracrine and autocrine signals.
• Gonadotropins are required for antrum formation.
• Follicular growth from primary and secondary follicles to antral stages takes several months.
• The cumulus cells are specialized granulosa cells that line the oocyte and have important functions in oocyte development, including meiotic arrest and induction of ovulation.
• LH triggers ovulation by the cumulus cell compartment and release of the oocyte from meiotic arrest.
• The first observable change of resumption of meiosis is germinal vesicle breakdown.
• Achievement of metaphase I and the metaphase–anaphase transition depends on the formation of an intact meiotic spindle.
• Genomic imprinting mediates pre-designated, differential silencing of the maternal and paternal alleles.
• More than 99% of all oocytes undergo programmed cell death (apoptosis).

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Chapter 4 Physiology of Male Gametogenesis

Rakesh K. Sharma

INTRODUCTION
The contribution of the male to the biology of reproduction is to produce a genetically intact spermatozoa that will fertilize an oocyte. The end product of male gametogenesis, the mature spermatozoa, is designed for one purpose: to deliver the male contribution of the genetic makeup to the embryo. The biology of gamete production is different in males compared to females. Gamete production in females is intimately part of the endocrine responsibility of the ovary. If there are no gametes, then hormone production is drastically curtailed. Depletion of oocytes implies depletion of the major hormones of the ovary. In the male this is not the case. Androgen production will proceed normally without a single spermatozoa in the testes.
This chapter reviews some of the basic structural morphology of the testes and the process of development to obtain mature spermatozoa.

ORGANIZATION OF THE TESTES
The testes are ellipsoid in shape, measuring 2.5 × 4 cm in diameter and engulfed by a capsule (tunica albuginea) of strong connective tissue. 1 Along its posterior border, the testis is loosely connected to the epididymis, which gives rise to the vas deferens at its lower pole. 2 The testis has two main functions: it produces hormones, in particular testosterone, and it produces the male gamete—the spermatozoa. The testis is incompletely divided into a series of lobules. Most of the volume of the testis is made up of seminiferous tubules, which are looped or blind-ended and packed in connective tissue within the confines of the fibrous septa ( Fig. 4-1 ). The fibrous septae divide the parenchyma into about 370 conical lobules consisting of the seminiferous tubules and the intertubular tissue. The seminiferous tubules are separated by groups of Leydig cells, blood vessels, lymphatics, and nerves. The seminiferous tubules are the site of sperm production (see Fig. 4-1 ). The wall of each tubule is made up of myoid cells of limited contractility and also of fibrous tissue. Each seminiferous tubule is about 180 μm in diameter; the height of the germinal epithelium measures 80 μm, and the thickness of the peritubular tissue is about 8 μm. 3 The germinal epithelium consists of cells at different stages of development located within the invaginations of Sertoli cells, namely spermatogonia, primary and secondary spermatocytes, and spermatids. Both ends of the seminiferous tubules open into the spaces of the rete testis 4 (see Fig. 4-1 ). The fluid secreted by the seminiferous tubules is collected in the rete testis and delivered in the ductal system of the epididymis.

Figure 4-1 This cross-section through the testicle shows the convoluted seminiferous tubules and interstitial tissue. The epithelium of the convoluted tubules has sperm in various stages of maturation (inset) and the Sertoli cells. Mature but immobile sperm are released into the lumen. A magnified view of a mature spermatozoon is seen. Diagrammatic representation of the human spermatozoon showing the acrosome, the nucleus and nuclear envelopes, the mitochondrial sheath of the midpiece, the principal piece, and the end piece.

SUPPORTING CELLS
The supporting cells of the testes refer to cells that are part of the cellular development that leads to a mature sperm. They are, however, extremely important to sperm production and spermatogenesis would be impossible without them. The two most important cells are the Leydig and Sertoli cells.

Leydig Cells
The Leydig cells are irregularly shaped cells with granular cytoplasm present individually or more often in groups within the connective tissue. 5, 6 Leydig cells are the prime source of the male sex hormone testosterone. 7 - 9 The pituitary hormone luteinizing hormone (LH) acts on Leydig cells to stimulate the production of testosterone. This acts as a negative feedback on the pituitary to suppress or modulate further LH secretion. 8 Compared with the testosterone levels in the blood, the intratesticular concentration of testosterone is many times higher, especially near the basement membrane of the seminiferous tubule.

The Sertoli Cell
The seminiferous tubules are lined with highly specialized Sertoli cells that rest on the tubular basement membrane and extend into the lumen with a complex ramification of cytoplasm ( Fig. 4-2 ). Spermatozoa are produced at puberty but are not recognized by the immune system that develops during the first year of life. The seminiferous tubule space is divided into basal (basement membrane) and luminal (lumen) compartments by strong intercellular junctional complexes called tight junctions. These anatomic arrangements, complemented by closely aligned myoid cells that surround the seminiferous tubule, form the basis for the blood–testis barrier. The blood–testis barrier provides a microenvironment for spermatogenesis to occur in an immunologically privileged site. Sertoli cells serve as “nurse” cells for spermatogenesis, nourishing germ cells as they develop. These also participate in germ cell phagocytosis. Multiple sites of communication exist between Sertoli cells and developing germ cells for the maintenance of spermatogenesis within an appropriate hormonal milieu. Follicle-stimulating hormone (FSH) binds to the high-affinity FSH receptors found on Sertoli cells, signaling the secretion of androgen-binding protein. High levels of androgens are also present within the seminiferous tubule.

Figure 4-2 Section of the germinal epithelium in the seminiferous tubule. Semithin section drawing × 900. Sertoli cells divide the germinal epithelium into basal and luminal compartments.
(Adapted from Holstein A-F, Schulze W, Davidoff M: Understanding spermatogenesis is a prerequisite for treatment. Reproductive Biology and Endocrinology. BioMed Central Ltd., 2003).
The two most important hormones secreted by the Sertoli cells are antimüllerian hormone and inhibin. Antimüllerian hormone is a critical component of embryonic development and is involved in the regression of the müllerian ducts. Inhibin is a key macromolecule in pituitary FSH regulation. Some of the functions of the Sertoli cell are (1) maintenance of integrity of seminiferous epithelium, (2) compartmentalization of seminiferous epithelium, (3) secretion of fluid to form tubular lumen to transport sperm within the duct, (4) participation in spermiation, (5) phagocytosis and elimination of cytoplasm, (6) delivery of nutrients to germ cells, (7) steroidogenesis and steroid metabolism, (8) movement of cells within the epithelium, (9) secretion of inhibin and androgen-binding protein, (10) regulation of spermatogenic cycle, and (11) provision of a target for hormones LH, FSH, and testosterone receptors present on Sertoli cells.

SPERMATOGENESIS
The process of differentiation of a spermatogonium into a spermatid is known as spermatogenesis. 4 It is a complex, temporal event whereby primitive, totipotent stem cells divide to either renew themselves or produce daughter cells that become specialized testicular spermatozoa over a span of weeks. Spermatogenesis involves both mitotic and meiotic proliferation as well as extensive cell remodeling. Spermatogenesis can be divided into three major phases: (1) proliferation and differentiation of spermatogonia, (2) meiosis, and (3) spermiogenesis, a complex metamorphosis that transforms round spermatids arising from the final division of meiosis into a complex structure called the spermatozoon. In humans, the process of spermatogenesis starts at puberty and continues throughout the entire lifespan of the individual. It takes place in the lumen of the seminiferous tubules. In fact, 90% of the testis volume is determined by the seminiferous tubules and their constituent germ cells at various stages of development. Once the gonocytes have differentiated into fetal spermatogonia, an active process of mitotic replication is initiated very early in embryonic development. This appears to be under FSH control and develops the baseline number of precursor cells of the testicle.

Proliferation and Differentiation of Spermatogonia
Within the seminiferous tubule, germ cells are arranged in a highly ordered sequence from the basement membrane to the lumen (see Fig. 4-2 ). Spermatogonia lie directly on the basement membrane, followed by primary spermatocytes, secondary spermatocytes, and spermatids as they progress toward the tubule lumen. The tight junction barrier supports spermatogonia and early spermatocytes within the basal compartment and all subsequent germ cells within the luminal compartment.

Types of Spermatogonia
Spermatogonia represent a population of cells that divide by mitosis, providing both a renewing stem cell population as well as spermatogonia that are committed to enter the meiotic process. Germ cells are staged by their morphologic appearance; there are dark type A (A dark ) and pale type A (A pale ) and type B spermatogonia, primary spermatocytes (preloptotene, leptotene, zygotene, and pachytene), secondary spermatocytes, and spermatids (Sa, Sb, Sc, Sd 1 , and Sd 2 ) ( Fig. 4-3 ). Other proliferative spermatogonia include A paired (A pr ), resulting from dividing A isolated (A is ), and subsequently dividing to form A aligned (A al ). Differentiated spermatogonia include type A1, A2, A3, A4, intermediate, and type B, each a result of the cellular division of the previous type. In humans, four spermatogonial cell types have been identified; these are A long , A dark , A pale , and B. 10 - 12 In the rat, type A is is believed to be the stem cell 13, 14 ; however, it is not clear which human type A spermatogonia is the stem cell. Some investigators have proposed that the type A dark spermatogonia represent the reserve or nonproliferative spermatogonial population that gives rise to A pale . 11, 15, 16 Spermatogonia do not separate completely after meiosis but remain joined by intercellular bridges, which persist throughout all stages of spermatogenesis and are thought to facilitate biochemical interactions, allowing synchrony of germ cell maturation. 17

Figure 4-3 A diagrammatic representation of spermatogenesis, spermiogenesis, and spermiation during which the developing germ cells undergo mitotic and meiotic division to reduce the chromosome content.
(Adapted from Huckins C: Adult spermatogenesis. Characteristics, kinetics, and control. In Lipshultz LI, Howards SS (eds): Infertility in the Male. New York, Churchill Linvingstone, 1975, p 108.)
Type B spermatogonia possess considerably more chromatin within the inner nuclear envelope than intermediate or type A spermatogonia. Type B spermatogonia represent the cells that differentiate and enter into the process of meiosis during which they are called primary spermatocytes . 11 They are the differential precursors to preleptotene spermatocytes. This last mitotic division helps maintain a pool of stem cells so the process can continue indefinitely.

Spermatocytogenesis
The purpose of spermatogenesis is to produce genetic material necessary for the replication of the species through mitosis and meiosis. Spermatocytogenesis takes place in the basal compartment. Primary spermatocytes enter the first meiotic division to form secondary spermatocytes. Prophase of the first meiotic division is very long, and the primary spermatocyte has the longest lifespan. Secondary spermatocytes undergo the second meiotic division to produce spermatids. Secondary spermatocytes are short-lived (1.1 to 1.7 days).
The meiotic phase involves primary spermatocytes until spermatids are formed; during this process, chromosome pairing, crossover, and genetic exchange is accomplished until a new genome is determined. In turn, a postmeiotic phase involving spermatids all the way up to spermatozoa develops, ending in the formation of the specialized cell. The process by which spermatids become mature spermatozoa can take several weeks and is one of the most elaborate differentiation events of any mammalian cell. This process requires the synthesis of hundreds of new proteins and the assembly of unique organelles. Within the purview of the Sertoli cell, several events occur during this differentiation of spermatid to sperm.

Mitosis
Mitosis involves proliferation and maintenance of spermatogonia. It is a precise, well-orchestrated sequence of events involving duplication of the genetic material (chromosomes), breakdown of the nuclear envelope, and equal division of the chromosomes and cytoplasm into two daughter cells. 18, 19 DNA is also spatially organized into loop domains on which specific regulatory proteins interact during cellular replication. 19 - 24 The mitotic phase involves spermatogonia (types A and B) and primary spermatocytes (spermatocytes I). Developing germ cells interconnected by intracellular bridges produce the primary spermatocyte through a series of mitotic divisions. Once the baseline number of spermatogonia is established after puberty, the mitotic component will proceed in order to continue to provide precursor cells and to start the process of differentiation and maturation.

Meiosis
Meiosis is a complex process with specific regulatory mechanisms of its own. 25 The process commences when type B spermatogonia lose their contact with the basement membrane to form preleptotene primary spermatocytes. Thus, each primary spermatocyte can theoretically yield four spermatids, although fewer actually result, because some germ cells are lost due to the complexity of meiosis. The primary spermatocytes are the largest germ cells of the germinal epithelium. Meiosis is characterized by prophase, metaphase, anaphase, and telophase. In this, two successive cell divisions yield four haploid spermatids from one diploid primary spermatocyte. As a consequence, the daughter cells contain only half of the chromosome content of the parent cell. After the first meiotic division (reduction division), each daughter cell contains one partner of the homologous chromosome pair, and they are called secondary spermatocytes . These cells rapidly enter the second meiotic division (equational division), in which the chromatids then separate at the centromere to yield haploid early round spermatids. Meiosis assures genetic diversity and involves primary and secondary spermatocytes, which give rise to spermatids.

Spermiogenesis
Spermiogenesis is a process during which the morphologic changes occur during the differentiation of the spermatid into the spermatozoon. It begins once the process of meiosis is completed. Six different stages have been described in the process of spermatid maturation in humans: S a-1 and S a-2 , S b-1 and S b-2 , and S c-1 and S c-2 (see Fig. 4-3 ). Each of these stages can be identified by morphologic characteristics. During the S a-1 stage, both the Golgi complex and mitochondria are well developed and differentiated, the acrosomal vesicle appears, the chromatid body develops in one pole of the cell opposite from the acrosomal vesicle, and the proximal centriole and the axial filament appears. During S b-1 and S b-2 stages, acrosome formation is completed, the intermediate piece is formed, and the tail develops. This process is completed during the S c stages. During the postmeiotic phase, progressive condensation of the nucleus occurs with inactivation of the genome, the histones are converted into transitional proteins, and finally protamines are converted into well-developed disulfide bonds.

Spermiation
The process whereby a mature spermatid frees itself from the Sertoli cell and enters the lumen of the tubule as a spermatozoon is known as spermiation. The spermatids originating from the same spermatogonia remain connected by bridges to facilitate the transport of cytoplasmic products. Spermiation involves the active participation of the Sertoli cell. This may also involve actual cell movement as the spermatids advance toward the lumen of the seminiferous tubules. 26 The mature spermatids close their intracellular bridges, disconnect their contact to the germinal epithelium, and become free cells called spermatozoa. Portions of the cytoplasm of the Sertoli cell known as the cytoplasmic droplet may remain as part of the spermatozoon during the process of spermiation. This is a morphologic feature present on immature sperm in semen. 27

The Cycle or Wave of Seminiferous Epithelium
A cycle of spermatogenesis involves the division of primitive spermatogonial stem cells into subsequent germ cell types through the process of meiosis. Type A spermatogonial divisions occur at a shorter time interval than the entire process of spermatogenesis. Therefore, at any given time, several cycles of spermatogenesis coexist within the germinal epithelium. In humans, spermatocyte maturation takes 25.3 days, spermiogenesis 21.6 days, and the total estimated time for spermatogenesis is 74 days. Spermatogenesis is not random throughout the seminiferous epithelium. Germ cells are localized in spatial units referred to as stages and represent consistent associations of germ cell steps. 28 - 31 In rodent spermatogenesis, one stage can be found in a cross-section of seminiferous tubule.
Each stage is recognized by development of the acrosome, meiotic divisions and shape of the nucleus, and release of the sperm into the lumen of the seminiferous tubule. A stage is designated by Roman numerals. Each cell type of the stage is morphologically integrated with the others in its development process. Each stage has a defined morphologic entity of spermatid development, called a step, designated by an Arabic number. Several steps occur together to form a stage, and several stages are necessary to form a mature sperm from immature stem cells. Within any given cross-section of the seminiferous tubule there are four or five layers of germ cells. Cells in each layer comprise a generation; a cohort of cells that develop as a synchronous group and each has a similar appearance and function. Stages I to III have four generations comprising type A spermatogonia, two primary spermatocytes, and an immature spermatid. Stages IV to VIII have five generations: type A spermatogonia, one generation of primary spermatocytes, one generation of secondary spermatocytes, and one generation of spermatids.
The cycle of spermatogenesis can be identified for each species, but the duration of the cycle varies. 11 The stages of spermatogenesis are sequentially arranged along the length of the tubule. This arrangement of the stages of spermatogenesis is such that it results in a “wave of spermatogenesis” along the tubule. This wave is in space but the cycle is in time. 31 Along the length of the seminiferous tubule there are only certain cross-sections where spermatozoa are released. In the rat, all stages are involved in spermatogenesis, but spermatozoa are released only in stage VIII.
Although it appears that the spatial organization is lacking or poor in the human seminiferous tubule, mathematic modeling indicates that these stages are tightly organized in an intricate spiral pattern. 32 In addition to the steps being organized spatially within the seminiferous tubule, the stages are organized in time. 31 Thus a position in the tubule that is occupied by cells comprising stage I will become stage II, followed by stage III, until the cycle repeats. In humans, the duration of the cycle is 16 days and the progression from spermatogonia to spermatozoa takes 70 days, or four and a half cycles of the seminiferous cycle. During spermatogenesis, cytoplasmic bridges link cohorts of germ cells that are at the same point in development, and these cells pass through the process together. Groups of such cells at different stages can be observed histologically on cross-section and many germ cell cohorts are seen only in association with certain other germ cells. This has led to the description of six stages of the seminiferous tubule epithelium in men. To add another level of complexity, the steps of the spermatogenic cycle within the space of the seminiferous tubules demonstrate a specific organization, termed spermatogenic waves. In humans, this wave appears to describe a spiral cellular arrangement as one progresses down the tubule. This spatial arrangement probably exists to ensure that sperm production is a continuous rather than a pulsatile process.

Efficiency of Spermatogenesis
Spermatogenetic efficiency varies between different species but appears to be relatively constant in man. The time for the differentiation of a spermatogonium into a mature spermatid is estimated to be 70 ± 4 days. 33 In comparison to animals the spermatogenetic efficiency in man is poor. The daily rate of spermatozoa production is calculated at 3 to 4 million per gram of testicular tissue. 34 A higher number of spermatozoa should be expected in the ejaculate than the 20 million/mL described by the World Health Organization. 35 A majority of the cells developed (>75%) are lost as a result of apoptosis or degeneration; of the remaining, more than half are abnormal. Therefore, only about 12% of the spermatogenetic potential is available for reproduction. 36 An age-related reduction in daily sperm production in men, which is associated with a loss of Sertoli cells, is also seen. This may result from an increase in germ cell degeneration during prophase of meiosis or from loss of primary spermatocytes. There is also a reduction in the number of Leydig cells, non-Leydig interstitial cells, myoid cells, and Sertoli cells.
The process of spermiation and the journey of a sperm through the ductus deferens of the testis to a site where it can be included in the ejaculate are thought to take a further 10 to 14 days. It is a lengthy process, and quantitative changes in sperm production may take some time to become apparent in a sample of semen. The nucleus progressively elongates as its chromatin condenses. The head is characterized by a flattened and pointed paddle shape, specific to each species. This process involves the Golgi phase, cap phase, acrosomal phase, and maturation phase.

Golgi Phase
The Golgi complex forms a caplike structure called the acrosome. The centrioles migrate from the cytoplasm to the base of the nucleus. The proximal centriole becomes the implantation apparatus to anchor the flagellum to the nucleus. The distal centriole becomes the axoneme.

Cap Phase
The acrosome forms a distinct cap over nucleus. The acrosome is formed from the Golgi complex. As the Golgi complex moves away from the nucleus, a flagellum is constructed from the centriole.

Acrosomal Phase
The formation of the acrosome starts with the coalescence of a series of granules from the Golgi complex, which migrates to come into contact with the nuclear membrane, where it covers like a caplike structure over 30% to 50% of the nuclear surface. 37 The acrosome covers the nucleus and contains the hydrolytic enzymes necessary for fertilization. The manchette is formed, and the spermatids are embedded in Sertoli cells.

Maturation Phase
Mitochondria migrate toward the segment of the growing tail to form the mitochondrial sheath. Dense outer fibers and a fibrous sheath are formed and this completes assembly of the tail. The bulk of the spermatid cytoplasm is eventually discarded as a residual body. The spermatid is now called a spermatozoon . As spermiogenesis proceeds, the spermatid moves toward the lumen of the seminiferous tubule. With completion of spermatid elongation, the Sertoli cell cytoplasm retracts around the developing sperm, stripping it of all unnecessary cytoplasm and extruding it into the tubule lumen. The mature sperm has remarkably little cytoplasm left after extrusion. The mature spermatozoon is an elaborate, highly specialized cell produced in massive quantity, up to 300 per g of testis per second.

SPERMATOZOA
Spermatozoa are highly specialized and condensed cells that do not grow or divide. A spermatozoon consists of the head, containing the paternal material (DNA), and the tail, which provides motility (see Fig. 4-1 ). The spermatozoon is endowed with a large nucleus but lacks the large cytoplasm characteristic of most body cells. Men are unique in the morphologic heterogeneity of the ejaculate. 38 - 40

Head
The heads of stained spermatozoa are slightly smaller than heads of living spermatozoa in the original semen. 41 The normal head is oval, measuring about 4.0 to 5.5 μm in length and 2.5 to 3.5 μm in width. The normal length-to-width ratio is about 1.50 to 1.70. 41 Under bright field illumination, the most commonly observed aberrations include head shape/size defects, including large, small, tapering, piriform, amorphous, vacuolated (>20% of the head surface occupied by unstained vacuolar areas), and double heads, or any combination of these defects. 42

Acrosome
The acrosome is represented by the Golgi complex and covers about two thirds of the anterior head area. 39, 40, 42 The apical thickening seen in many other species is missing; however, the acrosome shows a uniform thickness/thinning toward the equatorial segment and covers about 40% to 70% of the sperm head. When observed under the scanning electron microscope, a furrow that completely encircles the head (i.e., acrosomal and postacrosomal regions) divides the sperm head unequally. An equatorial segment that is followed by the postacrosomal region is not very clearly visible on scanning electron microscope. The maximal thickness and width of the spermatozoon is seen in the postacrosomal region.
Under the electron microscope the sperm head is a flattened ovoid structure consisting primarily of the nucleus. The acrosome is a caplike structure covering the anterior two thirds of the sperm head, which arises from the Golgi complex of the spermatid as it differentiates into a spermatozoon. The acrosome contains several hydrolytic enzymes, including hyaluronidase and proacrosin, which are necessary for fertilization. 38
During fertilization of the egg, the fusion of the outer acrosomal membrane with the plasma membrane at multiple sites releases the acrosomal enzymes at the time of the acrosome reaction. The anterior half of the head is devoid of plasma and an outer acrosomal membrane and is covered only by the inner acrosomal membrane. 43 The posterior region of the sperm head is covered by a single membrane called the postnuclear cap. The overlap of the acrosome and the postnuclear cap results in an equatorial segment, which does not participate in the acrosome reaction. The nucleus, comprising 65% of the head, is composed of DNA conjugated with protein. The chromatin is tightly packed, and no distinct chromosomes are visible. The genetic information, including the sex-determining X or Y chromosome, is coded and stored in the DNA. 38

Neck
This forms a junction between head and tail. It is fragile and the presence of decapitated spermatozoa is a common abnormality.

Tail
The sperm tail arises at the spermatid stage. The centriole during spermatogenesis is differentiated into three parts: midpiece, the main or principal piece, and endpiece. The mitochondria reorganize around the midpiece. An axial core comprising of two central fibrils is surrounded by a concentric ring of nine double fibrils, which continue to the end of the tail. The additional outer ring is comprised of nine coarse fibrils. The principal piece is comprised of nine coarse outer fibrils diminishing in thickness and finally only the inner 11 fibrils of the axial core surrounded by a fibrous sheath. The mitochondrial sheath of the midpiece is relatively short but slightly longer than the combined length of the head and neck. 38

Endpiece
The endpiece is not distinctly visible by light microscopy. Both tail sheath and coarse filaments are absent. The tail, containing all the motility apparatus, is 40 to50 μm long and arises from the spermatid centriole. It propels by waves that are generated in the neck region and pass distally along like a whiplash. Under bright field illumination, common neck and midpiece aberrations include their absence, bent tails, distended or irregular/ bent midpiece, abnormally thin midpiece (no mitochondrial sheath), or any of these combinations. 42 Tail aberrations include short, multiple, hairpin, and broken tails; tails of irregular width; coiling tails with terminal droplets; or any of these combinations. 42 Cytoplasmic droplets greater than one third the area of a normal sperm head are considered abnormal. They are usually located in the neck/midpiece region of the tail, although some immature spermatozoa may have a cytoplasmic droplet at other locations along the tail. 40, 42
Under scanning electron microscopy the tail can be subdivided intro three distinct parts (i.e., midpiece, principal piece, and endpiece. In the midpiece the mitochondrial spirals can be clearly visualized. This ends abruptly at the beginning of the midpiece. The midpiece narrows toward the posterior end. A longitudinal column along with transverse ribs is visible. The short endpiece has a small diameter due to the absence of the outer fibers. 38 Under transmission electron microscopy, the midpiece possesses a cytoplasmic portion and a lipid-rich mitochondrial sheath that consists of several spiral mitochondria, surrounding the axial filament in a helical fashion. The midpiece provides the sperm with the energy necessary for motility. An additional outer ring of nine coarser fibrils surrounds the central core of 11 fibrils. Individual mitochondria are wrapped around these fibrils in a spiral manner to form the mitochondrial sheath, which contains the enzymes needed in the oxidative metabolism of the sperm. The mitochondrial sheath of the midpiece is relatively short, slightly longer than the combined length of the head and neck. 38
The principal piece, the longest part of the tail, provides most of the propellant machinery. The coarse nine fibrils of the outer ring diminish in thickness and finally disappear, leaving only the inner fibrils in the axial core for most of the length of the principal piece. 44 The fibrils of the principal piece are surrounded by a fibrous tail sheath, which consist of branching and anastomosing semicircular strands or ribs, held together by their attachment to two bands that run lengthwise along opposite sides of the tail. 38 The tail terminates in the endpiece with a length of 4 to 10 μm and diameter of less than 1 μM. The small diameter is due to the absence of the outer fibrous sheath and a distal fading of the microtubules.

REGULATION OF SPERMATOGENESIS
The spermatogenic process is maintained by different intrinsic and extrinsic influences.

Intrinsic Regulation
Leydig cells secrete hormone (testosterone), neurotransmitters (neuroendocrine substances), and growth factors to neighboring Leydig cells, blood vessels, lamina propria of the seminiferous tubules, and Sertoli cells. 5, 36, 45 They help maintain the nutrition of the Sertoli cells and the cells of the peritubular tissue and influence the contractility of myofibroblasts, thereby regulating the peristaltic movements of the seminiferous tubules and transportation of the spermatozoa. Leydig cells also help in the regulation of blood flow in the intertubular microvasculature. 1 In addition, different growth factors are delivered from Sertoli cells and various germ cells participating in a complicated regulation of cell functions and developmental processes of germ cells. Altogether these factors represent an independent intratesticular regulation of spermatogenesis.

Extrinsic influences
The local regulation of spermatogenesis is controlled by the hypothalamus and hypophysis. Pulsatile secretion of gonadotropin-releasing hormone of the hypothalamus initiates the release of LH from the hypophysis; in response the Leydig cells produce testosterone. Testosterone not only influences spermatogenesis, but is also distributed throughout the body. It thus provides feedback to the hypophysis that regulates the secretory activity of Leydig cells. Stimulation of Sertoli cells by FSH is necessary for maturation of the germ cells. Complete qualitative spermatogenesis requires both FSH and LH. The interaction between the endocrine and paracrine mechanisms determines the functions within the testis. 46 - 48 Inhibin secreted by Sertoli cells functions in the feedback mechanism directed to the hypophysis. These extratesticular influences are necessary for the regulation of intratesticular functions. Thus growth and differentiation of testicular germ cells involve a series of complex interactions between both somatic and germinal elements. 47, 49, 50

IMMUNE STATUS OF THE TESTIS
The spermatozoa, late pachytene spermatocytes, and spermatids express unique antigens. These antigens are not formed until puberty; therefore, immune tolerance is not developed. The blood–testis barrier develops as these autoantigens develop. The testis is considered to be an immune-privileged site (i.e., transplanted foreign tissue can survive for a period of time without immunologic rejection). An immune surveillance is present in the testis and the epididymis, which shows an active immunoregulation to prevent autoimmune disease. 51, 52

DISTURBANCES OF SPERMATOGENESIS
Both proliferation and differentiation of the male germ cells and the intratesticular and extratesticular mechanisms regulating spermatogenesis can be disturbed. These disturbances may occur as a result of environmental influences or due to diseases that directly or indirectly affect spermatogenesis. 53, 54 Additionally, nutrition, therapeutic drugs, hormones and their metabolites, increased scrotal temperature, toxic substances, or x-radiation may reduce or destroy spermatogenesis. All these events can result in reduced spermatogenesis.

SPERM TRANSPORT IN THE EPIDIDYMIS
The epididymis lies along the dorsolateral border of each testis. It is made up of the efferent ductules, which emanate from the rete testis, and the epididymal ducts (see Fig. 4-1 ). The epididymis opens into the vas deferens, which then passes through the inguinal canal into the peritoneal cavity and opens into the urethra adjacent to the prostate. The primary function of the epididymis is post-testicular maturation and storage of spermatozoa during their passage from the testis to the vas deferens. The epididymal epithelium is androgen-dependent and has both absorptive and secretory functions.
The epididymis is divided into three functionally distinct regions: head, body, and tail or caput epididymis, corpus epididymis, and cauda epididymis. Their functions can be described simplistically as increasing the concentration, maturation, and storage of the spermatozoa. Much of the testicular fluid that transports spermatozoa from the seminiferous tubules is resorbed in the caput, increasing the concentration of the spermatozoa by 10-fold to 100-fold. The epididymal epithelium secretes the epididymal plasma in which the spermatozoa are suspended. As the newly developed spermatozoa pass through these regions of the epididymis, many changes occur, including alterations in net surface charge, membrane protein composition, immunoreactivity, phospholipid and fatty acid content, and adenylate cyclase activity. Many of these changes are thought to improve the structural integrity of the sperm membrane and also increase the fertilization ability of the spermatozoa. The capacities for protein secretion and storage within the epididymis are known to be extremely sensitive to temperature and reproductive hormone levels, including estrogens. It is a complex fluid whose composition changes along the length of the epididymis; and spermatozoa experience a series of sophisticated microenvironments that regulate their maturation.

Epididymal Sperm Storage
As many as half of the spermatozoa released from the testis die and disintegrate within the epididymis and are resorbed by the epididymal epithelium. The remaining mature spermatozoa are stored in the cauda epididymis, which contains about 70% of all spermatozoa present in the male tract. The cauda epididymis stores sperm available for ejaculation. The ability to store functional sperm provides a capacity for repetitive fertile ejaculations. The capacity for sperm storage decreases distally; spermatozoa in the vas deferens may only be motile for a few days. The environment of the cauda epididymis is adapted for storage in laboratory animals. In humans it is not a perfect storage organ, and the spermatozoa do not remain in a viable state indefinitely. After prolonged sexual activity, caudal spermatozoa first lose their fertilizing ability, followed by their motility and then their vitality; they finally disintegrate. Unless these older, senescent spermatozoa are eliminated from the male tract at regular intervals, their relative contribution to the next ejaculate(s) increases, thereby reducing semen quality, even though such ejaculates do have a high sperm concentration. The vas deferens are not a physiologic site of sperm storage and contain only about 2% of the total spermatozoa in the male tract. The transit time of sperm through the fine tubules of the epididymis is thought to be 10 to 15 days in humans.

Role of the Epididymis in Sperm Maturation
Early on, the nucleus of the round spermatid is spherical and is located in the center of the cell. Subsequently, the shape changes from spherical to asymmetric as chromatin condenses. It is theorized that many cellular elements contribute to the reshaping process, including chromosome structure, associated chromosomal proteins, the perinuclear cytoskeletal theca layer, the manchette of microtubules in the nucleus, subacrosomal actin, and Sertoli cell interactions.
As spermatozoa traverse the epididymis, they are exposed to a continuously changing milieu of the luminal fluid derived from the rete testis and modified by the secretory and absorptive activity of the epididymal epithelium. In nonhuman mammals there is compelling evidence that the epididymal epithelium does provide essential factors for sperm maturation. 55 - 58 In humans most of the information is obtained from treatment of pathologic cases rather than from normal fertile men. Sperm maturation occurs outside the testis. Spermatozoa within the testis have very poor or no motility and are incapable of fertilizing an egg. Both epididymal maturation and capacitation is necessary before fertilization. Capacitation, the final step required to acquire the ability to fertilize, may be an evolutionary consequence of the development of a storage system for inactive sperm in the caudal epididymis. Preservation of optimal sperm function during this period of storage requires adequate testosterone; levels in the circulation.
The epididymis is limited to a storage role because spermatozoa that have never passed through the epididymis and that have been obtained from the efferent ductules in men with congenital absence of vas deferens can fertilize the human oocyte in vitro and result in pregnancy with live birth (as well as with intracytoplasmic sperm injection with sperm obtained after testicular biopsy). A direct involvement of the epididymis comes from the results of epididymovasostomy bypass operations for epididymal obstruction. However, these results are not consistent, whereas significantly reduced fertility was reported in anastomosis of vas deferens to the proximal 10 mm of the duct compared to anastomosis of the more distal region of the tract 59 ; others have reported pregnancies in anastomosis of the vas deferens to the efferent ductules 60 or from spermatozoa retrieved from the efferent ductules and proximal caput regions of men with congenital absence of vas deferens and used for successful in vitro fertilization. 60 More direct evidence of epididymal involvement comes from experiments in which immature epididymal sperm recovered from fertile men were incubated in the presence of human epithelial cell cultures. 61 These spermatozoa showed improved motility and a significant increase in their capacity to attach to human zona in vitro.

SPERM ENTRY INTO CERVICAL MUCUS
At the moment of ejaculation, spermatozoa from the cauda epididymis are mixed with secretions of the various accessory glands in a specific sequence and deposited around the external cervical os and in the posterior fornix of the vagina. Spermatozoa in the first fraction of the ejaculate have significantly better motility and survival than those in the later fractions. The majority of the spermatozoa penetrate cervical mucus within 15 to 20 minutes of ejaculation. 62, 63 The ability to migrate across the semen–mucus interphase is highly dependent on the specific movement pattern of the spermatozoa. 64 At the time of sperm penetration into the cervical mucus, further selection of the spermatozoa occurs based on the differential motility of normal versus abnormal spermatozoa. This is further modified once the vanguard spermatozoon is within the cervical mucus. 65 The receptivity of the cervical mucus to penetration by the spermatozoa is cyclic, increasing over a period of about 4 days before ovulation and decreasing rapidly after ovulation. Maximum receptivity is seen the day before and on the day of the LH peak. 66 Spermatozoa enter the uterine cavity from the internal cervical os by virtue of their own motility. 67 From here the spermatozoa traverse to the site of fertilization in the ampulla of the fallopian tube or the oviduct.

CAPACITATION AND THE ACROSOME REACTION
Animal studies in rats and rabbits indicate that spermatozoa that are stored in the female tract are unable to penetrate the ova. They have to spend time in the female tract before they acquire this ability. Capacitation is a series of cellular or physiologic changes that spermatozoa must undergo to fertilize. 68, 69 It is characterized by the ability to undergo the acrosome reaction, to bind to the zona pellucida, and to acquire hypermotility. Capacitation may be an evolutionary consequence of the development of a storage system for inactive sperm in the caudal epididymis.
Capacitation per se does not involve any morphologic changes even at the ultrastructural level. It represents a change in the molecular organization of the intact sperm plasmalemma that then confers on spermatozoa the ability to undergo the acrosome reaction in response to induction of the stimulus. Capacitation involves the removal of seminal plasma factors that coat the surface of the sperm; modification of the surface charge; modification of the sperm membrane and of the sterols, lipids, and glycoproteins and the outer acrosomal membrane lying immediately under it. It also involves an increase in intracellular free calcium. 70, 71 Changes in sperm metabolism, increase in 3′, 5′-cyclic monophosphate, and activation of acrosomal enzymes are believed to be components of capacitation. Sperm capacitation may be initiated in vivo during migration through cervical mucus. 72 This is finally followed by the acquisition of hypermotility, displayed by an increase in velocity and flagellar beat amplitude. This may be necessary for avoiding attachment to the tubal epithelium and penetrating the cumulus and zona pellucida.
Capacitation can also be achieved by culture medium supplemented with appropriate substrates for energy and in the presence of protein or biologic fluid such as serum or follicular fluid. Usually it takes about 2 hours for sperm to undergo capacitation in vitro. Further modifications occur when capacitated sperm reach the vicinity of the oocyte.
The acrosome reaction confers the ability to penetrate the zona pellucida and also confers the fusogenic state in the plasmalemma overlying the nonreactive equatorial segment, which is needed for interaction with the oolemma. There are distinct fusion points between the outer acrosomal membrane and the plasma membrane. The fusion begins posteriorly around the anterior border of the equatorial segment, which is always excluded from the reaction. The changes termed as acrosome reaction prepare the sperm to fuse with the egg membrane. The removal of cholesterol from the surface membrane prepares the sperm membrane for the acrosome reaction. 73, 74 In addition D-mannose-binding lectins are also involved in the binding of human sperm to the zona pellucida. 75, 76
Thus, these series of changes are necessary to transform the stem cells into fully mature, functional spermatozoa equipped to fertilize the egg.


PEARLS

• The testis is an immune-privileged site. The blood–testis barrier provides a microenvironment for spermatogenesis to occur. The seminiferous tubules are the site of sperm production.
• The process of differentiation of a spermatogonium into a spermatid is known as spermatogenesis. It involves both mitotic and meiotic proliferation as well as extensive cell remodeling.
• In humans, the process of spermatogenesis starts at puberty and continues throughout the entire life.
• Spermatogenesis produces genetic material necessary for the replication of the species. Meiosis assures genetic diversity.
• Several cycles of spermatogenesis coexist within the germinal epithelium.
• Along the length of the seminiferous tubule, there are only certain cross-sections where spermatozoa are released.
• Sperm production is a continuous and not a pulsatile process.
• Spermatozoa are highly specialized cells that do not grow or divide.
• The spermatogenic process is maintained by different intrinsic and extrinsic influences.
• Spermatozoa undergo a series of cellular or physiologic changes such as capacitation and the acrosome reaction before they can fertilize.
• The epididymis is limited to a storage role because spermatozoa that have never passed through the epididymis can fertilize the human oocyte in vitro.
• Nutrition, therapeutic drugs, hormones and their metabolites, increased scrotal temperature, toxic substances, or x-radiation may reduce or destroy spermatogenesis.

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Chapter 5 Reproductive Genetics

Brian A. Clark

INTRODUCTION
We learn quickly in life that variability exists between individuals. For many of us the differences are as simple as hair or eye color. For others, the differences are profound and can take the form of a severe birth defect or syndrome. As a whole, these differences add up and cause significant morbidity and mortality. A Canadian study has found that approximately 12% of individuals suffer health problems related to or caused by genetic disease from birth to early adulthood. 1 Genetics is the study of traits and inherited differences between individuals; as Gregor Mendel demonstrated, we were able to learn the principles of inheritance without knowing about or understanding DNA and its organization in the genome. The traditional study of medicine and medical genetics is, however, about to be revolutionized. In this technological revolution of genomic medicine, we see that a fundamental understanding of genetics and the principles of inheritance are still required.
The practice of clinical medical genetics has been significantly transformed over the past 15 years, from a somewhat arcane specialty dealing with prenatal diagnosis, chromosome abnormalities, and dysmorphology to a challenging and cutting edge specialty taking the lead in introducing genomic medicine to the broader field of medicine. In 2001, the Human Genome Project announced that a rough sequence of the human genome had been completed. 2, 3 In its original iteration the Human Genome Project was an attempt to sequence and determine the linear sequence of 3 billion base pairs and map the individual genes encoded by this sequence. Genomics is the study of our complete set of genes, their functions and interactions with themselves and their environment, and how genetic variation contributes to disease risk and response to treatment (i.e., pharmacogenomics). This sequencing and mapping effort has now spawned functional genomics, which seeks to identify gene function, regulation, and gene interaction.
The knowledge and technologies learned from this effort have, and will have, as profound an impact on the field of reproductive medicine as any other. The physician in obstetrics and gynecology will not only have to have an understanding of genetics and the principles of inheritance, but he or she will also have to understand genomics and its derivative technologies and how these will affect the risk, diagnosis, and treatment of medical disorders. Physicians will not only be practicing the traditional paradigm of diagnosis and treatment of disease, but will also be recognizing and treating genomically derived disease risk prior to disease manifestation. They must be ready to deal with all the ethical, legal, and social implications of this revolution. Although the era of genomic medicine will bring new tools to individualize disease risk, emphasize prevention, and treat disease, the simple basic recording and understanding of the family history will still be a fundamental component of this new era. 4

THE HUMAN GENOME
The Human Genome Project has resulted in some surprising revelations. 2, 3, 5 Less than 2% of the human genome has genes that code for proteins. This was a surprising finding because it was thought that the human genome would reflect the complexity of human development and it was estimated that the human genome would contain up to 120,000 genes. The first report of the Human Genome Project estimated that the human genome contained only 30,000 to 35,000 genes. Drosophila melanogaster had been found to have 14,000 genes and the mustard plant Arabidopsis thaliana 26,000 genes. 6, 7 It is likely now that the complexity of human growth and development and its relation to disease is not going to be explained by just new gene discovery. The previous dogma that one gene produces one protein has now been replaced by the theory of alternative splicing, in which a gene’s exons or coding regions are shuffled to produce alternative forms of a related protein ( Fig. 5-1 ).

Figure 5-1 Alternative splicing. A single gene with multiple exons can produce multiple proteins (isoforms) through the mechanism of alternative splicing. A, All 6 exons are translated in the protein. B, The last exon is not translated, producing a truncated protein. C, Exons 4 and 5 are not translated, altering the basic structure of the protein.
Exons are sequences of a gene that are transcribed into a protein. Introns are regions of a gene that do not code for a protein. When the mRNA of a gene is translated, exons are spliced together. Exons have been found to comprise only 1% of the human genome and introns about 25%. Typically more than 25 times the amount of DNA in a gene is not associated with protein structure and function. This has significant implications for DNA testing and interpretation of a test’s sensitivity and specificity. Regions of genes on chromosomes have been described as existing in gene-rich oases separated by gene-sparse deserts.
Over 10% of the genome is composed of repeated sequences of DNA that may be related to chromosome structure. Long interspersed repetitive elements and short interspersed repetitive elements have been described, including Alu repeat sequences. Alu sequences have been found in gene-rich regions and may play a role in genetic recombination.

Genetic Variation
The human genome contains just over 3 billion base pairs and the sequence is about 99.9% identical in all individuals. It is a surprising finding that human genetic variability may be due to just 0.1% difference in the human genome sequence. A single-nucleotide polymorphism, or SNP, is the most common DNA variant in the genome, with about 10 million occurrences. SNPs are single-base substitutions and occur on average in 1 in every 1250 nucleotides. SNPs are polymorphisms in that a base substitution does not cause a change in the phenotype; they are found in more than 1% of the population. Because of their ubiquity in the human genome SNPs have become invaluable in identifying sequence changes associated with disease risk through traditional linkage studies and population-based association studies. Although not thought to alter protein coding, SNPs have been associated with diseases by their presence in regulatory control regions or introns. SNPs may also determine a disease’s response to treatment, as with the angiotensin-II type 1 receptor polymorphism and its association with congestive heart failure. 8

GENES
With the recent advances of the Human Genome Project and the rapid adoption of these findings into clinical medicine, the clinician will need to understand the organization of the human genome. The average size of a gene is about 3000 base pairs. Chromosome 1, the largest chromosome in a karyotype, has about 2968 genes; the Y chromosome, the smallest, has 231 genes. Genes are distributed in random areas of the chromosome, with some chromosomes gene-rich and some gene-poor. Chromosomes 17, 19, and 22 are gene-dense. Chromosomes 4, 8, 13, 18, and Y are gene-sparse. It is not surprising then that chromosomes 13, 18, and 21, which are the common trisomies that can survive to birth, have the fewest genes.
Genes can be altered and cause human disease through several mechanisms. Point mutations, in which there is a single change of a DNA base in the sequence, can have multiple effects on gene function. Missense mutation is the substitution of a single base in the DNA sequence, incorporating a different amino acid in the protein sequence. These mutations may have little effect on protein function if the amino acid substituted is similar to the original one. If the substitution incorporates a very different amino acid, the protein structure and function may be significantly affected. A silent mutation is the substitution of a single base in the DNA sequence that does not change the specific amino acid in the sequence. Nonsense mutations are the substitution of a single DNA base in the sequence, causing the premature termination or truncation of a protein. In addition to point mutations, frame-shift mutations can also lead to an altered or truncated protein. Frame-shift mutations are deletions or additions of DNA bases that are not multiples of three. These changes lead to a downstream change in the reading frame, often truncating a protein. In general, these types of mutations result in loss of function of a protein and alter the phenotype by decreasing the activity of a protein.

CHROMOSOMAL BASIS OF INHERITANCE
Since Gregor Mendel’s work with the garden pea elucidating the mechanisms of inheritance, genetics has focused on single-gene defects. Modes of inheritance have now been identified for thousands of conditions and catalogued in the online compendium Online Mendelian Inheritance in Man, OMIM at http://www.ncbi.nlm.gov/omim/ .
Typically an obstetrician-gynecologist will confront chromosome abnormalities under two specific clinical scenarios. The first is in pregnancy where there is an association between chromosome abnormalities and advanced maternal age. There is also an association between miscarriage and chromosome abnormalities; approximately half or more of all first-trimester losses are associated with a chromosome abnormality. Obstetricians and gynecologists will also manage patients with chromosome abnormalities when they present for recurrent pregnancy loss and a history of infertility.

Normal Karyotype
Our cells contain 23 pairs of chromosomes; 22 pairs are autosomes and shared between males and females, and one pair is the sex chromosomes, XX and XY. The ordered arrangement of chromosomes is a karyotype. Routine chromosome analysis involves drawing blood and stimulating lymphocytes in culture into rapid division. Cell division is then inhibited at metaphase and the chromosomes fixed on glass slides. The chromosomes are then stained and photographed for analysis.

CHROMOSOME ABNORMALITIES
Chromosome abnormalities fall into two general categories: numerical and structural. In numerical abnormalities the total number varies from the normal, 46. With structural abnormalities, the chromosome structure has been physically rearranged.

Chromosome Structure
Chromosomes are composed of short arms, p, and long arms, q. The chromosome has a primary constriction, or centromere, where the microtubules attach for cell division. The telomeres, or tips of the chromosomes, are capped with a repeating sequence TTAGGG that is critical for the maintenance of chromosome integrity. The relative position of the centromere further delineates the structure of the chromosome. In humans the acrocentric chromosomes 13, 14, 15, 21, and 22 are characterized by stalks and satellites where genes for ribosomal RNA are located ( Fig. 5-2 ).

Figure 5-2 Human chromosomes. These chromosomes represent the pattern seen with G banding or Giemsa staining. The different types of chromosomes and elements of chromosome structure are noted. The bands for chromosome 17 are numbered.

Numerical Abnormalities
Abnormalities of chromosome number are the most commonly recognized clinical chromosome abnormalities. Structural chromosome anomalies contribute significantly to birth defects, infertility, and recurrent pregnancy loss. Abnormalities in chromosome number generally arise from mistakes at cell division where there is either gain or loss, or both, of chromosomes in daughter cells, or nondisjunction. Nondisjunction can occur either during meiosis or mitosis. Cells resulting from this occurrence are aneuploid because their chromosome number is not a multiple of the haploid number 23. Nondisjunction can occur during either meiosis I or meiosis II. Either can produce an aneuploid conception ( Fig. 5-3 ). Nondisjunction or anaphase lag can occur at mitosis, causing mosaicism, the presence of two or more cell lines in an individual. Mosaicism is often seen with the sex chromosome abnormality Turner’s syndrome, where up to 50% of cases have some form of mosaicism. 9 The common numerical chromosome abnormalities are listed in Table 5-1 . Numerical chromosome abnormalities can also involve multiples of the haploid number of 23 chromosomes. Triploidy, with 69 chromosomes, usually arises from the fertilization of a single egg by two sperm. Triploid conceptions are seen in about 15% of chromosomally abnormal miscarriages and occasionally survive to term. Tetraploidy has a modal number of 92 chromosomes and occurs in a small percentage of spontaneous losses.

Figure 5-3 Nondisjunction. A, Nondisjunction can occur at meiosis I, producing an aneuploid gamete and trisomic conception. The polar bodies are represented by empty circles. B, Postzygotic or mitotic nondisjunction can occur, producing somatic mosaicism. This is seen frequently in Turner’s syndrome. C, Nondisjunction in meiosis II can produce normal gametes, along with those with 22 and 24 chromosomes.
Table 5-1 Chromosome Abnormalities Chromosome Clinical Findings Trisomy 13 Severe CNS abnormalities, holoprosencephaly, microphthalmia, coloboma, cleft lip and palate, abnormal auricles, polydactyly, cardiac defects Trisomy 18 CNS malformations, prominent occiput, micrognathia, small mouth, low-set ears, hypoplastic nails, overlapping fingers, cardiac defects Trisomy 21 Microcephaly, flat occiput, Brushfield spots, epicanthal folds, simian crease, conotruncal cardiac defects, redundant skin on nape of neck, hypotonia XXX Normal female phenotype, tall stature, may have some learning and developmental disabilities, normal fertility 45,X Turner’s Short stature, gonadal failure, absent secondary sex characteristics, webbed neck, low-set hairline, coarctation of the aorta, horseshoe kidney, may have some spatial learning disabilities XXY Klinefelter’s May be tall, infertile, small testis, learning disabilities, gynecomastia

Structural Chromosome Abnormalities
Structural chromosomal rearrangements occur when chromosomes break and the original architecture is not restored. Chromosome rearrangements are balanced when the diploid genetic state is maintained. When rearrangements are unbalanced, they result in aneuploidy for one or more chromosome segments. These structural rearrangements may segregate and be termed familial, or they may occur as a first or new event, de novo. Balanced familial chromosome rearrangements are in most cases truly balanced and represent little risk of birth defects or mental retardation. De novo chromosome rearrangements that appear balanced carry a small risk of aneuploidy at the molecular level of about 5% for birth defects and developmental delay.
Translocations involve the exchange of chromosome arms between two different chromosomes. Reciprocal translocations occur when there is breakage within two arms and reciprocal exchange of the distal segments, creating a derivative chromosome ( Fig. 5-4 ). In most cases balanced translocation carriers are phenotypically normal but are at risk for producing unbalanced gametes during gametogenesis. In a balanced translocation carrier the types of chromosome segregation can be complex, resulting in a normal segregation pattern, a balanced translocation pattern, and an unbalanced pattern producing a partial trisomy and partial monosomy for the chromosomes involved (see Fig. 5-4 ).

Figure 5-4 Reciprocal translocation between two nonhomologous chromosomes. A, Germ line chromosomes of a balanced translocation carrier. B, Alternate segregation produces gametes with normal chromosomes and balanced translocation products. C, Adjacent-1 segregation produces unbalanced gametes, resulting in partial monosomy and partial trisomy conceptions.
When the short arms of two acrocentric chromosomes are involved in a translocation, the long arms are joined in the centromeric region of one chromosome, with the loss of the short arms of the acrocentric chromosomes producing Robertsonian translocations. Because the short arms of acrocentric chromosomes contain redundant ribosomal genetic material, the loss of this material is of no phenotypic consequence. As with balanced translocations, the products of meiotic segregation can be either balanced or unbalanced ( Fig. 5-5 ).

Figure 5-5 Robertsonian translocation. A translocation has occurred between two acrocentric chromosomes. A, Normal and balanced segregants will produce genetically-balanced offspring. B, The unbalanced segregants will produce translocation Down syndrome and the nonviable monosomy 21.
Other structural abnormalities can produce pregnancy loss and birth defects. When two breaks occur in a single chromosome with the interstitial segment flipped 180 degrees at the time of repair, an inversion can occur. If this involves each arm of the chromosome a pericentric inversion is produced. If only a single arm of the chromosome is involved in the inversion, a paracentric inversion is produced ( Fig. 5-6 ). Each has unique and different implications for gamete production and pregnancy loss. With chromosome duplication a chromosome segment of varying size can be duplicated, causing a partial trisomy for this segment. With a deletion, a segment of varying size is missing, causing a genetic imbalance or partial monosomy. This condition, in which a second copy of a gene or segment of chromosome is missing, resulting in an abnormal phenotype or clinical presentation, is known as haploinsufficiency.

Figure 5-6 The inversion on the left is a pericentric inversion involving the centromere. The inversion on the right is a paracentric inversion involving a single chromosome arm.

Fluorescent In Situ Hybridization
Traditional cytogenetics has always been limited by the band level or resolution of the karyotype. Even with high-resolution banding that could elongate chromosomes and resolve an increasing number of bands per chromosome, one could never be certain that a chromosome was intact at the molecular level. With advances in DNA technology and cytogenetics it is now possible using fluorescent in situ hybridization (FISH) to analyze chromosomes at the molecular level for changes in the DNA. Molecular cytogenetics has now revolutionized cytogenetics by permitting (1) the analysis of DNA structure within a chromosome down to within 10 to 100 kb and (2) the diagnostic analysis of nondividing interphase cells, producing a significant impact on the field of prenatal diagnosis and that of preimplantation genetic diagnosis. 10
FISH technology uses DNA probes that can bind or anneal to specific DNA sequences within the chromosome. A denatured probe is incubated with native DNA from a cell that has also been denatured to the single-strand state. The probe substitutes biotin-dUTP or digoxigenin-UTP for thymidine. After the probe has annealed to native DNA, the probe–DNA complex can be detected by adding fluorochrome-tagged avidin that binds to biotin or fluorochrome-labeled antidigoxigenin. This signal can be additionally amplified by adding antiavidin and the complex visualized by fluorescence microscopy. Using several different fluorochromes tagged to different DNA probes, different chromosomes or chromosome segments can be simultaneously visualized within a cell as different colored signals. The ability to detect specific gene segments that are either present or missing has permitted the diagnosis of contiguous gene syndromes at the DNA level as well as translocations in interphase nuclei, often in single cells.
Material for FISH can be either metaphase chromosomes obtained from dividing cells or interphase nuclei from cells that are not dividing. Slides are pretreated with RNAse and proteinase to remove RNA that may cross-hybridize with the probe and chromatin. The slides are heated in formamide to denature the DNA and then fixed in cold ethanol. The probe is then prepared for hybridization by heating. The probe and chromosome preparation are then mixed and sealed with a coverslip at 37°C for hybridization. By varying the incubation temperature or the salt composition of the hybridization solution, the stringency of the binding can be increased and the background labeling reduced.

Applications of FISH
The technique of in situ hybridization first proved useful for localizing genes to chromosomes. With the introduction of fluorescence labeling, in situ hybridization proved invaluable in identifying chromosome abnormalities that could not be identified by traditional banding methods. FISH also played a key role in one of the most unusual discoveries of modern genetics, that of genomic imprinting.
FISH technology has been developed in three forms. Centromeric or alpha-satellite probes are relatively chromosome specific and have had probably the broadest application in interphase genetics. 11, 12 These probes produce somewhat diffuse signals near the centromere with adequate strength, but do not cross-hybridize with chromosomes that have similar centromeric sequences. Single copy probes have now been developed that give discrete signals from a specific band on a chromosome and avoid the issue of cross-hybridization. These can also be used to detect copy number and specific chromosome regions known to be associated with syndromes. Single copy probes and centromeric probes for chromosomes 13, 18, 21, X, and Y have been developed for use in prenatal diagnosis. It is also possible to “paint” whole chromosomes using FISH. Using spectral karyotyping technology that combines mixtures of fluorochromes, it is now possible to produce a unique fluorescent pattern for each individual chromosome in 24 different colors. This technology permits the detection of complex chromosome rearrangements that cannot be seen with traditional cytogenetic techniques ( Fig. 5-7 ).

Figure 5-7 Patterns of FISH probes in interphase cell preparations. The first figure shows a hypothetical locus-specific probe with a discrete signal. The middle figure shows a centromeric repeat probe with a larger, more diffuse signal. The figure on the right shows a whole chromosome painting probe with the diffuse pattern of overlapping domains making chromosome enumeration less reliable.

Prenatal Diagnosis
For an older woman, pregnancy may not be a time of joy but a time of anxiety. Advanced maternal age has a long-known association with an increasing risk of fetal chromosome abnormalities. Amniocentesis done at 16 weeks’ gestation followed by traditional karyotype analysis may take from 10 days to 2 weeks. The use of FISH for preliminary results can expedite a diagnosis and reduce the waiting time for results. Most geneticists and laboratories recommend that FISH not be used alone to make a management decision in pregnancy. FISH studies should always be confirmed with a final karyotype or at minimum correlated with abnormal ultrasound findings or an abnormal maternal serum analyte screen.

Contiguous Gene Syndromes
Contiguous gene syndromes are also known as microdeletion syndromes, or segmental aneusomy. 10 These are deletions of a contiguous stretch of chromosome that usually involve multiple genes. The contiguous gene syndromes were first described in 1986 using classical cytogenetic methodologies. Using FISH, submicroscopic deletions can now be identified at the level of DNA; this has permitted the characterization of the smallest deleted region consistently associated with a syndrome, known as the critical region . By identifying the critical region of a syndrome, it is often possible to identify the specific genes that, when missing, are associated with the syndrome ( Fig. 5-8 ). A recent compendium of deletion syndromes has reported 18 deletion and microdeletion syndromes spread over 14 chromosomes. 13 Some of the more common deletion and mirodeletion syndromes and their clinical findings are shown in Table 5-2 .

Figure 5-8 DiGeorge probe. This photo shows a peripheral blood sample from a patient tested for DiGeorge syndrome. The Tuple 1 probe binds to both copies of chromosome 22 as does the control. The child did not have the deletion causing DiGeorge syndrome.
Table 5-2 Deletion and Contiguous Gene Syndromes Deletion Syndrome Clinical Description 4p– Wolf-Hirschhorn Microcephaly, hypertelorism, frontal bossing, cleft lip and palate, “Greek helmet” face, mental retardation, hypotonia, cardiac anomalies 5p– Cri du chat Microcephaly, characteristic “cat cry,” hypotonia, mental retardation 7q11.23 Williams Round face, full lips, stellate iris, supravalvular aortic stenosis, mental delay, “cocktail personality” 11p13 WAGR Wilm’s tumor, aniridia, genital abnormalities, mental retardation 15q11-13 Angelman Blonde hair, prognathism, seizures, ataxia, laughter, hypotonia, mental retardation 15q11-13 Prader-Willi Birth hypotonia, hyperphagia, obesity, short stature, hypogonadism, mental retardation 16p13 Rubinstein-Taybi Characteristic facies, beaked nose, microcephaly, mental retardation 17p11 Smith-Magenis Brachycephaly, prominent chin, short stature, mental retardation, behavioral phenotype 17p13 Miller-Dieker Microcephaly, lissencephaly, growth retardation, seizures, mental retardation 20p12 Alagille Cholestasis, heart defects, ocular findings, skeletal defects 22q11 DiGeorge/CATCH 22 Thymic and parathyroid hypoplasia, calcium abnormalities, conotruncal heart defects, short stature, behavioral and learning problems

Telomeres
Telomeres are structures that cap the tips of the long and short arms of chromosomes. They are composed of repeating sequences of TTAGGG and effectively prevent the ends of the chromosomes from fusing together. Telomere probes can be important in sorting out complex translocations that cannot be determined by traditional cytogenetic means. In addition, one of the findings of the Human Genome Project was that the chromosome regions next to telomeres are gene-rich. It has now been shown that submicroscopic subtelomeric deletions are responsible for a significant proportion of genetic morbidity. 14

GENERAL PRINCIPLES OF MENDELIAN GENETICS

Single-Gene Disorders
In humans as with all diploid organisms, genes come in pairs on related or homologous chromosomes. The exception to this is the sex chromosomes, the X and the Y in the male. Single-gene disorders are described on the basis of the interaction of these pairs or alleles in the genotype and on how the interaction is expressed in the effect or the phenotype. When the alleles are not identical, the genotypes are heterozygous. When the alleles are identical, the genotype is described as homozygous. Gregor Mendel recognized that single-gene disorders are often recognizable and that these single-gene disorders segregate in families with predictable proportions; they have thus been referred to as mendelian traits. 15 If a single-gene disorder manifests itself in the heterozygous state, the condition is inherited in a dominant manner. If the single-gene disorder is only inherited when both alleles are affected or altered, the condition is inherited in a recessive fashion. In addition, single-gene disorders are further characterized according to whether they are transmitted on the autosomes or the sex chromosomes.

Autosomal Dominant Inheritance
The following features characterize autosomal dominant inheritance:
1. In most cases an affected individual has an affected parent.
2. Males and females are equally likely to be affected.
3. Affected individuals have a 50:50 chance of passing on the condition to an offspring. Unaffected individuals will in general not pass on the condition.
4. Within a family, about half the individuals are affected and half unaffected.
5. Within a pedigree the overall pattern is that of vertical transmission, with the disorder appearing in each successive generation.
An example of an autosomal dominant pedigree is seen in Figure 5-9 . Autosomal dominant inheritance patterns have additional characteristics that the clinician must recognize. Not all individuals in a family who have inherited a dominant condition are affected to the same extent or have the same organ systems affected. This characteristic is known as variable expressivity. This is one reason why it is important in clinical genetics to examine other family members when a proband, or referred patient, is initially evaluated. An autosomal dominant condition is considered fully penetrant if all individuals who inherit the single-gene mutation manifest its characteristics. Many autosomal dominant conditions have reduced penetrance, and not all individuals with the dominant gene manifest its phenotype. For women who inherit a BRCA1 or BRCA2 gene mutation, the lifetime risk of developing a breast cancer is 85%. Therefore, some families that are segregating BRCA1 and BRCA2 mutations will appear to “skip generations” when in fact an individual will carry a mutation but will not develop a breast cancer in her lifetime.

Figure 5-9 Pedigree: Autosomal dominant inheritance. Filled-in symbol signifies affected individual. Transmission occurs to sons and daughters.
There are in addition other characteristics of autosomal dominant inheritance. Autosomal dominant conditions are rarely found in the homozygous condition and almost always result in embryonic lethality. Finally, autosomal dominant conditions will occur in families with no previous family history. This does not imply nonpaternity, which the clinician must be aware of. For example, new mutations occur in Marfan syndrome in about 25% of cases and about 80% of cases in achondroplasia. 16 Parents of such an affected child are unlikely to be at risk for having another affected child; however, for the child, the risk of passing the condition on to an offspring is 50%. Geneticists have all been faced with the clinical scenario where a dominant, sporadic condition occurred in a family with a negative family history. Traditional genetic counseling would consider the recurrence risk negligible. However, most geneticists have seen these conditions recur in families, and empirically the recurrence risk has been as high as 3% to 5%. This phenomenon is usually attributed to germ cell mosaicism caused by a postzygotic nondisjunctional chromosome event or nondisjunctional mutational event. Presumably one of the parents in such a case has a small population of germ cells carrying the mutation. Recurrence risks must therefore be revised upward.

Autosomal Recessive Inheritance
Autosomal recessive inheritance has features distinct from autosomal dominant inheritance. Autosomal recessive inheritance is characterized by the following conditions:
1. Parents are usually unaffected, and affected individuals do not have affected parents.
2. Males and females are equally affected.
3. The condition usually occurs in siblings, with a horizontal pattern of inheritance within a generation.
4. Recurrence risks for subsequent pregnancies is 25%.
5. Consanguinity or relatedness is frequently observed in recessively inherited pedigrees.
An example of an autosomal recessive pedigree is shown in Figure 5-10 . Autosomal recessive inheritance requires an affected individual to be homozygous; therefore, these traits tend to occur less frequently than dominantly inherited disorders. For some conditions, such as cystic fibrosis, in which many of the mutations causing the disorder are known, carrier status can be determined by DNA testing. In other conditions in which the gene product is known, the level of an enzyme, approximately 50% of normal, can determine carrier status.

Figure 5-10 Pedigree: Autosomal recessive inheritance. The individuals with central dots indicate obligate carriers. Filled-in symbol signifies affected individual.

X-linked Inheritance
Because there are relatively few genes on the Y chromosome, sex-linked disorders are primarily X-linked disorders. Because males have only one copy of the X chromosome, they are hemizygous for X-linked genes and are in general affected by any mutation on the X chromosome. Because of this differential expression in males, X-linked disorders (e.g., hemophilia) were the earliest recognized human genetic disorders. Characteristics of X-linked inheritance are:
1. There is no male-to-male transmission of the condition.
2. Males are much more frequently affected than females. That some females can be affected is explained by X inactivation.
3. Affected males have obligate carrier daughters.
4. Mothers of affected males are obligate carriers. Half of their sons will be affected, and half of their daughters are carriers.
An example of X-linked recessive inheritance is shown in Figure 5-11 . Complete androgen insensitivity (testicular feminization) syndrome is an X-linked recessive disorder that presents as primary amenorrhea. These patients have a 46,XY genotype with a mutation in the androgen receptor gene located on the long arm of the X chromosome (Xq11-12). Mutations of this gene can also cause partial androgen insensitivity with genital androgenization or a milder form with abnormalities of spermatogenesis. These patients have normal-appearing female phenotype but with sparse pubic and axillary hair, short vagina, no müllerian derivatives, and normal testes. The testes can be in the abdomen or inguinal canal. The testes are removed after completion of spontaneous puberty. The serum androgen levels are in the male range.

Figure 5-11 Pedigree: X-linked recessive inheritance. The individuals with central dots indicate obligate carriers. All daughters of affected males are carriers. Male-to-male transmission does not occur. Filled-in symbol signifies affected individual.
X-linked dominant conditions are less common disorders than X-linked recessive disorders, with vitamin D-resistant rickets an example of this type of inheritance. Characteristics of this type of inheritance are:
1. Affected males have affected daughters, not sons.
2. The mutation may be lethal in males for some conditions.
3. Affected females tend to be twice as common as affected males in the pedigree.
4. Affected females on average transmit the disorder to half their daughters and sons.
An example of X-linked dominant inheritance is shown in Figure 5-12 .

Figure 5-12 Pedigree: X-linked dominant inheritance. More females than males are affected. Filled-in symbol signifies affected individual.

X Chromosome Inactivation
Some females might be affected by X-linked recessive disorders through X chromosome inactivation. Only one X-linked gene and one X chromosome are active in the somatic tissues of women. For all autosomal genes there are two copies; in terms of gene dosage and transcriptional activity, there is an equal amount in the somatic cells of men and women. To keep transcriptional activity and gene dosage equal for the X chromosome between men and women, the X chromosome would have to be twice as active in males or one X chromosome could be inactivated in females. Transcriptional equality is maintained by the inactivation of most but not all of the genes on the X chromosome in female somatic tissues. Reversible X inactivation is accomplished by methylation.
For a woman who is heterozygous for an X-linked disorder, only one gene is active in each cell; she is functionally a mosaic of cells expressing either the normal or abnormal gene product. If random X chromosome inactivation is skewed in the direction of inactivating the normal gene in most cells, a woman may manifest the signs of an X-linked disorder. This can be seen in women who are carriers for hemophilia who may have the bleeding tendencies of their affected sons. This variability in X chromosome gene expression has another significant clinical effect. Skewed X chromosome inactivation favoring the normal gene product will cause an overlap in the level of the gene product for a carrier woman and a noncarrier woman. This will not only make it difficult to identify a carrier from a noncarrier female, but will also mean that an inherited disorder cannot be distinguished from a new mutation.

NONTRADITIONAL INHERITANCE
Mendel laid the foundation of modern genetics in 1865 by describing dominant and recessive inheritance. For some time inherited diseases were considered to result from single-gene mutations that followed his classic principles of inheritance. Now with new genetic technologies and discoveries from the Human Genome Project, we know that not all inherited diseases follow these traditional principles of inheritance. Several new forms of nontraditional inheritance have now been described.

Multifactorial Disorders
There are many birth defects, genetic conditions such as congenital heart disease and spina bifida, and adult onset disorders that cannot be explained by single genes or the traditional patterns of inheritance. Yet, many of these conditions are clearly familial. These conditions are considered to be the result of additive genetic and environmental factors that trigger a threshold for expression in development or adulthood. Multifactorial inheritance attributes disease risk to a combination of multiple genetic factors that interact with an environmental stimulus. Polygenic traits are those diseases attributable to the additive effect of several genes. Often, these two concepts are used interchangeably.
Evidence for multifactorial or familial inheritance has led to a threshold model of genetic liability. This model assumes a continuous normal distribution of genetic risk in the population in the form of a bell-shaped curve. Affected individuals are considered to fall to the far right of the genetic liability curve. Affected first-degree relatives (siblings, parents, and children) would also fall to the right of the curve, but not as far. As the degree of relatedness falls off, the genetic liability falls back to that of the general population. 15 Evidence for multifactorial inheritance can be found in the following:
1. Some disorders are found more commonly in certain racial or ethnic groups, such as neural tube defects in the Irish.
2. Higher frequencies of birth defects are found in the relatives of affected individuals than the population at large. The incidence of birth defects falls off with the degree of relatedness. For example, the incidence of cleft lip with or without cleft palate is 4% in first-degree relatives, 0.8% in second-degree relatives, and 0.3% in third-degree relatives.
3. Single-gene disorders are found to be 100% concordant in monozygotic twins. For multifactorial inheritance in identical twins concordance is expected to be less than 100%, but much higher than in nonidentical twins. Concordance for cleft lip/palate in identical twins is 40%, whereas it drops to 4% in nonidentical twins.
Multifactorial inheritance also has specific implications for recurrence risks. Unlike recurrence risks for mendelian disorders, recurrence risks for multifactorial inheritance are empirically derived. General guidelines for multifactorial inheritance are:
1. Recurrence risks are empirically derived and may vary among families. The recurrence risk decreases with decreasing degree of relationship.
2. Within a family the risk increases with the number of affected family members. The risk to a sibling increases with each affected sibling.
3. The risk increases with the severity of the malformation. The more severe the condition, the greater the risk to the sibling.
4. When there is a predilection for the disorder in one sex over the other, there is often a greater risk to the offspring of the least affected sex versus the other. Presumably the lesser-affected sex has a higher genetic liability and a greater number of affected genes, putting a greater risk on their offspring.

Mitochondrial Inheritance
Genes located on chromosomes in a cell nucleus exhibit mendelian patterns of inheritance. However, there is an extranuclear source of DNA found in the mitochondria in the cytoplasm of cells. The mitochondria are a major source of adenosine triphosphate (ATP) for the energy requirements of a cell. Through the electron transport chain producing large amounts of NADH, ATP is generated in a process known as oxidative phosphorylation. Mitochondria have their own DNA, mtDNA, in the form of a circular 16,596 base pair chromosome. This chromosome contains the genes for 13 polypeptides of the oxidative phosphorylation system, 22tRNAs, and 2 rRNAs. However, most of the proteins in the mitochondria are encoded by nuclear genes, transcribed in the cytosol, and transferred into the mitochondria via molecular chaperones. Each cell contains thousands of mitochondria and each mitochondrion contain from 2 to 20 mtDNAs.
Unlike nuclear genes, mtDNA follows a maternal line of transmission. Each oocyte contains from 200,000 to 300,000 mtDNA molecules. At fertilization the sperm have few mitochondria, located in the tails, and their contribution of mitochondria to the conception is negligible. An example of mitochondrial or maternal inheritance is shown in Figure 5-13 . Both males and females can be affected, but inheritance only occurs from an affected mother. The phenotype of a mitochondrial disorder may be quite variable because of the nature of the mutation itself and its distribution within the population of mitochondria. At fertilization the oocyte contains thousands of mitochondria, both mutant and normal. At cell division, the proportion of mutant and normal mitochondria distributed to each daughter cell is never even. In addition, as the embryo grows new mutations accumulate in the mtDNA. Therefore, any cell or organ system may have proportionately few or many mutant mitochondria. This characteristic of mitochondrial inheritance is known as heteroplasmy. Within a single family there may be different manifestations of the same mitochondrial disease and within an individual the manifestations may change over time. As predicted with disorders of the oxidative phosphorylation system, those organ systems affected are those with the highest energy demands. Examples of mitochondrial disorders include Leber hereditary optic neuropathy, myoclonic epilepsy with red ragged fibers, and mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes.

Figure 5-13 Pedigree: Mitochondrial inheritance. Both males and females can be affected, but inheritance only occurs from an affected mother.

Imprinting
Pronuclear transplantation studies in mice have shown a reversible effect of the sex of the parent on the embryo. Mice can be conceived with both haploid chromosome complements coming from either the father or the mother. Mouse zygotes with paternally derived genes have arrested embryonic development but near-normal placental development. Zygotes with maternally derived genes have embryonic development but arrested placental development. Thus it became apparent that specific genes are differentially marked, or imprinted, at gametogenesis depending on whether they are inherited from the mother or father. This process is known as genomic imprinting. An example of this has long been familiar to the obstetrician/gynecologist. Hydatidiform moles are placental tumors without embryonic tissue with a genome derived from two paternal sets of haploid chromosomes. Ovarian teratomas are embryonic tumors without placental tissue derived from two maternal sets of haploid chromosomes. Triploidy also demonstrates examples of genomic imprinting. Android embryos with two sets of paternal chromosomes and a single set of maternal chromosomes occur as partial moles. Gynoid embryos with two sets of maternal chromosomes and a single set of paternal chromosomes occur as growth-restricted fetuses with abnormal placentas. Imprinting is accomplished by the reversible process of methylation.
The role of imprinting in human genetic syndromes was revealed by a cytogenetic paradox. Prader-Willi syndrome is a syndrome of hypotonia at birth, moderate developmental delay, hypogonadotrophic hypogonadism, small hands and feet, uncontrolled appetite, and obesity. In most cases it is associated with a chromosome 15 deletion at 15q11-13. The phenotype of Angelman syndrome, quite different from that of Prader-Willi, is associated with severe mental retardation, inappropriate laughter, ataxic movements, and seizures. It is also associated with a deletion of 15q11-13. The dilemma of how two very distinct syndromes could be caused by the same cytogenetic deletion was solved when molecular studies showed that the deletion of Prader-Willi syndrome was always paternally derived and the deletion associated with Angelman syndrome was maternally derived. 17 Individuals who inherit the 15q11-13 deletion paternally have Prader-Willi syndrome because a critical region that is needed for normal development is inactivated or imprinted during maternal meiosis and the corresponding region on the paternal chromosome, which must remain active for normal development, is lost in the deletion. Infants with Angelman syndrome have a maternally derived deletion because the critical region needed for normal development is inactivated or imprinted in the paternally derived chromosome and lost in the maternal deletion.

Uniparental Disomy
For a subset of patients with Prader-Willi syndrome and Angelman syndrome no cytogenetic deletion can be detected, nor were submicroscopic deletions detected by FISH. Molecular studies did however reveal that both copies of the chromosome were inherited from the same parent. For Prader-Willi syndrome without a deletion both copies of chromosome 15 were maternal in origin. 18 For Prader-Willi syndrome the critical region of chromosome 15 is maternally imprinted, and if no paternally derived copy is present, there are no functioning paternal genes for this critical region. Uniparental inheritance of two copies of the same chromosome from one parent is termed uniparental disomy and has been shown to cause genetic morbidity and specific syndromes.
Genomic imprinting and uniparental disomy have been reported for a number of chromosomes, including 6, 7, 14, 15, and 16. The most common mechanism causing uniparental disomy is believed to be trisomic rescue (i.e., the loss of a chromosome after a trisomic conception that reestablished the diploid number of 46 chromosomes). On average, two thirds of the time the chromosome lost will be that from the parent who contributed the extra chromosome. However, one third of the time the chromosome lost will be from the other parent, resulting in uniparental genetic syndromes; it can also be a cause of more common genetic disease. If a parent is a carrier for a recessive genetic disorder and conceives an infant with uniparental disomy, the infant can be affected with the recessive condition if two identical copies of the same chromosome are inherited from the same parent. Several cases of cystic fibrosis have been reported in individuals with uniparental disomy for chromosome 7. 19

Trinucleotide Repeat Disorders
Fragile X syndrome is one of the most common inherited mental retardation syndromes, occurring in about 1 in 12,000 males and 1 in 2500 females. Fragile X males also have behavioral problems and characteristic features, including a long face with prognathia, large ears, and macroorchidism. Individuals are often diagnosed or labeled with autism. Affected females have a similar but milder phenotype and are developmentally less delayed. 20 Fragile X syndrome was originally identified as a fragile site on the X chromosome at Xq27.3 under cell culture conditions deficient in folate.
Fragile X syndrome is one of a number of trinucleotide repeat disorders resulting from trinucleotide repeat expansion. The human genome was found to have several types of repetitive DNA. Tandem repeats of trinucleotide sequences can be found in both the coding and noncoding regions of the genome. During replication a trinucleotide repeat sequence can either expand or contract; if expanded beyond a critical copy number, gene function is compromised. Triplet repeat expansion is now recognized as a cause of several human diseases and as a mechanism of inheritance to explain the phenomenon of anticipation. It had been recognized for years that some inherited diseases seem to get more severe in each successive generation. Anticipation had always been considered a bias of ascertainment because each successive generation of an affected family was put under increased scrutiny. Anticipation due to triplet repeat expansion has now been documented for several genetic conditions. Triplet repeat disorders also have another nonmendelian characteristic, dynamic inheritance. Traditionally with single-gene disorders, mutations were considered immutable and passed down through each generation unchanged. We now know that mutations are much more dynamic and can alter when passed through each generation and that the phenotype may also be unstable.
Fragile X syndrome is caused by expanded number of CGG repeats in the promoter region of the FMR-1 gene. The inheritance and its consequences can be complex, as shown in Table 5-3 . Normal X chromosomes have between 6 and 54 CGG repeats. Individuals with 40 to 54 repeats are in a gray zone with the possibility of expansion. Males and females with between 55 and 230 repeats carry premutations; males are called transmitting males because they can pass the premutation on to daughters, who are then at risk of expanding the allele to their offspring. In general males only transmit premutations to their daughters. This change of normal CGG repeat number to full mutation is likely a multistep process occurring over several generations. There is now evidence in the literature to suggest that the premutation state in a male carrier may not be harmless. Reports have suggested that a neurologic syndrome can develop with cerebellar and parkinsonian features and decline of cognitive function. 21 Although women who are carriers for the fragile X premutation are not cognitively affected, new concerns have also arisen over their long-term functioning. Premutation carriers have shown subtle changes in psychological and cognitive functioning. For women premutation carriers there is also a concern for premature menopause, with a 4% risk by age 30 and a 25% risk for ovarian failure by age 40. 22 These authors also reported an imprinting effect for premature ovarian failure with paternally inherited premutations responsible for ovarian failure.

Table 5-3 Fragile X Syndrome and Trinucleotide Repeat Lengths
Two mechanisms have been proposed to cause the fragile X phenotype when the CGG repeats are expanded. The repeat sequence is located in the 5′ untranslated region of the gene. When this region expands beyond 230 copies of CGG it is hypermethylated and the FMR-1 allele with the expansion is inactivated. 23 Not all fragile X chromosomes are methylated. It has also been proposed that the expanded CGG transcript is unstable, such that the mRNA is not transcribed, leading to an absence of the gene product.
Other trinucleotide repeat disorders show similar inheritance characteristics as fragile X. These include myotonic dystrophy, which is characterized by myotonia, cataracts, muscular dystrophy, and cardiac arrhythmias. The severity of the disease is directly related to the number of CTG repeats; a severe infantile form of the disease can occur if the expanded repeats are inherited from a mother. Huntington disease is a severe neurologic degenerative disorder caused by CAG repeats in the Huntington disease gene. Individuals are usually affected with 38 or more CAG repeats. Normal individuals have 11 to 34 copies, and premutation individuals carry 34 to 37 copies. The disease is inherited in a dominant fashion and is characterized by progressive dementia, chorea, personality changes, and psychiatric changes. Onset is usually around age 40; it may run a 15-year course to completion. This is usually a tragic situation for families, because most affected individuals have had families and grandchildren before they become aware of the disease.

Somatic Mosaicism
In cases of infertility involving Turner’s syndrome and at the time of prenatal diagnosis it is not uncommon to get a mosaic test result. Mosaicism is defined as the presence of two or more cell populations within the same individual. Chromosomal mosaicism results from postzygotic nondisjunction or anaphase lag of a chromosome. Because it is a postzygotic event, it would not be expected to have a recurrence risk beyond that of the random population risk. The significance of mosaicism to an individual or fetus depends on at what time of development the nondisjunction occurred. Individuals with a low level of mosaicism may never be ascertained. A young girl who is short with a webbed neck and female secondary sexual characteristics may have a significant population of 46,XX cells amid a background of 45,X cells.
Mosaicism is not an uncommon finding during cytogenetic analysis of prenatal samples. Mosaicism may arise as an artifact of the culture conditions; this is termed pseudomosaicism. Or, it may arise from the extraembryonic chorion or amnion and represent a true mosaicism. If the mosaicism is confined to the placenta and extraembryonic tissues with a normal diploid embryo, this is termed confined placental mosaicism ; the majority of these events, discovered at chorionic villus sampling, result in normal fetuses. Mathematical models have been developed and applied to laboratory techniques to separate true mosaicism from pseudomosaicism.

Germ Cell Mosaicism
In prenatal counseling the clinician can be faced with following scenario. An unaffected couple has had a child with an obvious dominantly inherited disorder and want to know the recurrence risk. In most cases the condition is sporadic and the recurrence risk is minimal, the same as the population risk. However, there have been a number of reports of couples having a second child with a dominant condition that cannot be explained as two rare spontaneous mutations. These cases are likely due to germ cell mosaicism. These are thought to be due to spontaneous mutations in the development of the germline in the early gonad. This has been demonstrated for a number of conditions, including osteogenesis imperfecta. Recurrence risks are empirically given up to 3%.

GENETICS OF SEX DETERMINATION AND SEXUAL DIFFERENTIATION
With advances in molecular methodologies and the Human Genome Project, it is now possible to identify the molecular mechanisms of sex determination and sexual differentiation. It is also possible to identify specific clinical disorders with abnormalities of these underlying molecular mechanisms. The determination of sex is a sequential process. Chromosomal sex is determined at conception and determines gonadal sex. Gonadal sex then determines sexual differentiation and our secondary sexual characteristics. At any point along this developmental pathway sex determination and sexual differentiation can be altered by mutation.
Primordial germ cells initially migrate from the ectodermal layer of the embryo to the urogenital ridge, where the early gonad develops. Genetic sex is determined at the time of fertilization. If conceived with a Y chromosome the primordial gonad develops into a testis. If conceived with two X chromosomes, the primordial gonad develops into an ovary. Sexual differentiation is determined at the time of gonadal development. The bipotent gonads have both Wolffian and müllerian ducts. The bipotent or indifferent gonad will develop into the Sertoli and Leydig cells of the testis in the presence of a Y chromosome and the testis-determining gene SRY . With an XX chromosome complement, the indifferent gonad will develop into the follicular and theca cells of the ovary.
This development takes place on the background of a common, undifferentiated genital blueprint. The blueprint of the undifferentiated primordial genitalia includes paired gonads, paired internal ducts, a genital sinus, labial scrotal folds, and a genital tubercle. The default blueprint for gonadal and genital development is female. In the absence of a Y chromosome and SRY expression the primordial gonad develops into an ovary. The underlying molecular mechanisms for ovarian development are not as well understood as the molecular biology of testicular development. Abnormalities of sex determination and sexual differentiation appear to be determined mostly by mutations in male sexual determination in the background of a female blueprint.
The testis, differentiated under the influence of the SRY gene, secrete testosterone, müllerian inhibiting substance (MIS), and an insulin-like growth factor. 24 Testosterone induces Wolffian duct differentiation into the vas deferens, epididymis, and seminal vesicles of the male reproductive tract. The MIS inhibits the differentiation of the müllerian ducts. In the absence of the SRY gene and the production of male hormones and in the presence of a female gonad, the müllerian ducts persist and differentiate into the fallopian tube, uterus, cervix, and the proximal portion of the vagina. In the presence of an SRY- determined testis secreting male hormones, the undifferentiated external genitalia develop into a penis and scrotum and male secondary sexual characteristics. In the absence of the SRY gene and the presence of female hormones secreted by the ovary, the undifferentiated external gonad develops into the distal vagina, vulva, clitoris, and female secondary sexual characteristics. This process is depicted in Figure 5-14 .

Figure 5-14 Process for determination of sex.

Gene Disorders Causing Abnormal Gonadal Development

SRY
With the discovery of Klinefelter’s males having an 47,XXY karyotype and the description of Turner’s females as having a 45,X karyotype, it appeared that the presence of a Y chromosome and not the number of X chromosomes resulted in male differentiation in mammalian embryos. The gene on the Y chromosome responsible for male sex determination was named TDF, or testis-determining factor but was not easily identified. Because the X and the Y chromosome pair and segregate during meiosis just like the autosomes, it had been proposed that the X and Y chromosomes have autosomal-like regions in their distal chromosome arms, or pseudoautosomal regions allowing for pairing and segregation during meiosis. Magenis and coworkers in 1982 reported that 46,XX males have translocations of the Yp region to one of their Xp regions, demonstrating one mechanism for primary sex reversal. 25 The occurrence of synapsis and crossovers between the Yp pseudoautosomal regions more proximal to the centromere resulted in the transfer of TDF to the X chromosome and resulted in the construction of deletion maps of the Y chromosome that led to the identification of SRY as the TDF. 26, 27 The discovery of mutations in the SRY gene in XY pure gonadal dysgenesis confirmed SRY as the candidate gene for TDF. 28, 29

WT1, Frasier, and Denys-Drash Syndromes
The genes that control the development of the early indifferent gonad have been more difficult to define. These genes may also be involved in renal development, and many potential regulatory pathways may be involved. WT1, or Wilms’ tumor gene, is a complex gene expressed in early intermediate mesoderm formation prior to urogenital development and when mutated can lead to malformation in renal and gonadal development. Streak gonads, renal abnormalities, and nephrotic syndrome characterize Frasier syndrome. It is caused by mutations in the WT1 gene. This gene codes for a DNA-binding transcription factor with 24 different isoforms due to alternative splicing. Alternative splice donor sites at the end of exon 9 lead to the insertion (+) or deletion (–) of three amino acids—lysine, threonine, and serine (KTS)—between the third and fourth zinc fingers of the transcription factor. XY patients with Frasier syndrome have loss of the +KTS isoform of WT1, causing decreased production of the SRY protein in the urogenital ridge and XY sex reversal. The phenotype is that of a female with gonadal dysgenesis with streak gonads, müllerian duct structures, and nephropathy.
The WT1 gene is also associated with the WAGR syndrome and the Denys-Drash syndrome. The WAGR syndrome consists of Wilms’ tumor, aniridia, genitourinary abnormalities of undescended testis and hypospadias, and mental retardation. It is caused by a deletion in the 11p13 region and hemizygosity for the WT1 gene.
Individuals with Denys-Drash syndrome are 46,XY with some testosterone production and MIS production. Müllerian duct regression occurs, as does partial testicular development. They have a male phenotype but may have hypospadias and undescended testis and male pseudohermaphroditism. These individuals are +KTS and have mutations outside the KTS region that are believed to affect gonads later in development, causing a less severe phenotype. 30 These individuals are at risk for Wilms’ tumors and nephropathy that can lead to end-stage renal disease.

SF1
SF1, a steroidogenic factor, is a zinc-finger DNA-binding transcription factor involved with steroid biogenesis. It is expressed during development in the adrenals, gonads, and the pituitary. It appears to have a role in growth and maintenance of the indifferent gonad. SF1 mutations have now been reported with 46,XX sex reversal and adrenal insufficiency shortly after birth. 31

DAX1 Gene Mutations and Duplications
DAX1 is an X-linked gene that is expressed in part of the developing embryo, specifically the hypothalamus, pituitary, adrenal, and the gonads. 32 Alterations in DAX1 expression are associated with sex reversal and adrenal hypoplasia congenita. DAX1 is a nuclear receptor protein named for d osage-sensitive sex reversal, a drenal hypoplasia congenita, critical region on the X chromosome, and gene 1 . DAX1 appears to be part of a class of genes that antagonize male development. Mutations or deletions of the DAX1 gene cause adrenal hypoplasia congenita. Duplications of this gene cause sex-reversed XY females. Mutations in DAX1 cause adrenal hypoplasia congenita with adrenal insufficiency; abnormal pituitary development causes hypogonadotropic hypogonadism.

XY Sex Reversal
A duplication of the DAX1 region can cause XY sex reversal and the failure of the testis to develop, or gonadal dysgenesis. The gonad without sperm or oocytes has also been termed a streak gonad. Partial gonadal dysgenesis can occur if some functional testicular tissue remains; the external genitalia may be ambiguous. Sex-reversed males will also have an absence of MIS and müllerian structures will persist, thus distinguishing XY females with androgen resistence, who will not have residual mullerian structures. Patients with XY pure gonadal dysgenesis are at an increased risk of gonadoblastoma, which occurs in 10% to 30% of individuals. Most cases of pure gonadal dysgenesis are caused by mutations of SRY. The workup should include a karyotype looking for X chromosome deletions, analysis of SRY for mutations, and molecular or FISH testing for DAX1 duplications. Treatment includes estrogen replacement therapy for delayed puberty.

Campomelic Dysplasia and SOX9
SOX9 was identified in 1994 as a downstream regulator of male sexual differentiation and was also implicated in skeletal development. 33, 34 The SOX9 gene is located on chromosome 17q and shares some homology with the SRY gene; hence its name SOX for Sex-related-box . It functions as a transcription factor and in humans is expressed in the testis, kidney, chrondrocytes, liver, and brain. SOX9 plays a significant dose-dependent role in male sex determination, as shown by haploinsufficiency for SOX9 causing either sex reversal or ambiguous sexual differentiation. Increased dosages of SOX9 from duplication have been shown to cause autosomal sex reversal in XX individuals. 35 SOX9 also plays a role in MIS activation. In addition SOX9 plays a role in bone and cartilage development and may regulate COL2A1 expression, as seen in the skeletal abnormalities of campomelic dysplasia.
Campomelic dysplasia is a sporadic condition characterized by short stature, short limbs, bowed lower limbs, abnormal facies, cleft palate, and often congenital heart disease. It is often a lethal disorder caused by mutations in the SOX9 gene. Inheritance is thought to be autosomal dominant with a significant incidence of new mutations because of the incidence of lethality of this condition. As with any condition with suspected gonadal mosaicism in a parent, the recurrence risk is considered about 3%.

ABNORMALITIES OF THE FEMALE REPRODUCTIVE TRACT
Abnormalities of the female reproductive tract are thought to occur in up to 3% of live births. 36 Abnormalities of the female reproductive tract affect the fallopian tubes, uterus, cervix, or vagina. The spectrum of abnormalities includes agenesis, atresia, and septations. With a female conception and the differentiation of the bipotential gonad into the ovary, the Wolffian ducts degenerate and the müllerian ducts differentiate into the female reproductive system. The molecular basis for female reproductive tract development in humans is not well described. More is known about the development of the female reproductive tract in mice through knockout studies. The process of development of the müllerian ducts involves apoptosis, or programmed cell death. This process is highly regulated and involves many genes, including the bcl-2 gene, which protects cells from apoptosis. If expression of this gene is absent at a critical time when cells of the fused ducts were to undergo resorption, the septum may persist. A number of human genetic syndromes have been described that affect female reproductive tract formation, differentiation, and regression.
Human genetic syndromes involving abnormalities of female reproductive tract formation include Kaufman-McKusick syndrome, Mayer-Rokitansky-Küster-Hauser syndrome, and maturity-onset diabetes of the young type V. Kaufman-McKusick syndrome is an autosomal recessive syndrome originally described in the Amish population with polydactyly, congenital heart disease, and hydrometrocolpos. 37 Males can be affected but have only the hand and cardiac findings. The failure to cannalize the uterovaginal plate during embryogenesis produces a transverse vaginal septum, causing uterine distension and organ compression. These septa tend to occur in the upper one third of the vagina. This condition is related to Bardet-Biedl syndrome, which is made up of retinopathy, learning disabilities, obesity, polydactyly, renal anomalies, and male hypogonadism. 38 The MKKS gene codes for a protein that functions as a chaperonin, a group of proteins that assist in protein folding as the protein is produced from the ribosomes. 39 How mutations in the MKKS gene cause Kaufman-McKusick syndrome or the more variable Bardet-Biedl syndrome is not clearly understood.
Mayer-Rokitansky-Küster-Hauser syndrome is also known as Rokitansky-Küster-Hauser syndrome. This is a syndrome of müllerian aplasia with an absence of uterus, cervix, and upper vagina. Most cases are 46,XX females with ovaries and normal external genitalia. Renal anomalies and occasional skeletal anomalies are associated with this syndrome. Inheritance is considered autosomal recessive, but a gene for this disorder has not been identified. Treatment consists of creating a neovagina with dilators. MURCS association consists of müllerian aplasia, renal agenesis, vertebral anomalies, Klippel-Feil anomaly, and short stature. It is a sporadic condition and the underlying genetic anomaly has not been identified. Fraser syndrome and Meckel-Gruber syndrome are also associated with müllerian aplasia. Maturity-onset diabetes of the young type V is an autosomal dominant condition due to mutations in the hepatic nuclear factor 1 β (HNF1 β) that is associated with renal anomalies and müllerian aplasia. Mutations in this gene can cause müllerian aplasia in the absence of juvenile diabetes.
MIS, also known as antimüllerian hormone (AMH), is a member of the transforming growth factor β (TGFβ) family produced by the gonad. Produced by the gonad as testicular sex is determined, müllerian regression is one of the earliest signs of male sexual differentiation. Two types of persistent müllerian duct syndrome have been identified, type I and type II. Type I is caused by mutations in the AMH gene located on chromosome 19. A mutation in this gene is found in about 47% of persistent müllerian duct syndrome families and is an autosomal recessive condition. Type II is caused by mutations in the AMH type II receptor gene, found in about 38% of persistent müllerian duct syndrome families. These individuals have normal AMH levels. Type II is also an autosomal recessive condition. Clinically these individuals are 46,XY with normal male external genitalia. They may have cryptorchidism associated with inguinal hernias; the condition is frequently identified at surgery.
Hand-foot-genital syndrome is an autosomal dominant disorder characterized by shortening of the thumbs and great toes, fifth finger clinodactyly, incomplete müllerian fusion with or without a double cervix and longitudinal vaginal septum, and renal anomalies. Males may have hypospadias. Hand-foot-genital syndrome is caused by mutations in the HOXA13 gene and is inherited as an autosomal dominant condition. 40 Hand-foot-genital syndrome is caused by any mutation producing haploinsufficiency in the HOXA13 gene. HOXA13 is a member of the HOX group of transcription factors expressed in distal structures of the limb and the müllerian ducts. Its exact role in müllerian duct fusion and differentiation is unclear. Fryns’ syndrome is a multiple malformation syndrome consisting of cleft lip and palate, CNS abnormalities, renal tract malformations, cardiac defects, and müllerian fusion abnormalities. It is usually lethal and is inherited in an autosomal recessive manner.
Most well-recognized genetic disorders associated with reproductive tract abnormalities are due to deficiencies of key enzymes in the adrenal or testes that are autosomal recessive disorders or an androgen receptor defect. Deficiencies in adrenal enzymes such as 21-hydroxylase will result in a female pseudohermaphrodite (XX karyotype). Deficiencies in enzymes for testosterone synthesis or androgen receptor deficiency will result in male pseudohermaphroditism (XY karyotype).

GENETICS OF MALE INFERTILITY
Oligospermia (concentrations <5 million/mL) or azoospermia can be attributed to chromosome abnormalities in approximately 3% to13% of cases. 41 The chromosome abnormalities include sex chromosome abnormalities such as 47,XXY, 47,XYY, sex chromosome mosaics, and structural Y chromosome abnormalities. Autosomal chromosome abnormalities include reciprocal translocations, Robertsonian translocations, inversions, and other structural abnormalities. The most common chromosomal abnormality associated with nonobstructive azoospermia is 47,XXY, Klinefelter’s syndrome. The most common gene disorders associated with congenital obstructive azoospermia are mutations of the cystic fibrosis transmembrane conductance regulator gene.

Klinefelter’s Syndrome
Klinefelter’s syndrome is the most common cause of hypergonadotropic hypogonadism in the male (high FSH, low testosterone). Although most have a 47,XXY karyotype, some are mosaics. This syndrome is characterized by progressive germ cell and steroid cell depletion and gonadal fibrosis. These patients usually present at puberty with phenotypic abnormalities such as taller than predicted height, small genitalia, and scanty body hair. Androgen therapy is usually initiated. Although these patients are mostly azoospermic, rare immature or mature spermatozoa may be found in the testes. Most mature sperm obtained for in vitro fertilization (IVF) have a normal haploid karyotype so that the offspring should also have a normal karyotype.

Azoospermic Factor (AZF) Microdeletions
SRY, the testis-determining gene, is found proximal to the pseudoautosomal region of the short arm of the Y chromosome. There are three azoospermia factor (AZF) regions located on the long arm of the Y chromosome. AZFa, AZFb, and AZFc are found in the proximal long arm of the Y chromosome, as is the DAZ gene. Yq microdeletions of these azoospermia factors account for approximately 10% to 20% of cases of azoospermia and 3% to 10% of severe oligospermia. AZFa and AZFb deletions appear to be more severe than AZFc deletions, which are the most commonly reported deletion. Some men with Yq deletions are fertile. These conditions can be treated with IVF and intracytoplasmic sperm injection (ICSI); however, males conceived from fathers with Yq deletions would be expected to also be infertile (i.e., carry the same microdeletion).

PREIMPLANTATION GENETIC DIAGNOSIS
For couples who have had a child die from a congenital genetic disorder or who have conceived and then terminated a wanted but affected pregnancy, the option of prenatal diagnosis and pregnancy termination is not attractive. These couples have welcomed an alternative to traditional prenatal diagnosis and pregnancy termination. Preimplantation genetic diagnosis (PGD) is a prepregnancy form of prenatal diagnosis that combines routine IVF with embryo biopsy to identify affected pregnancies before embryo transfer. PGD has been used in the clinical setting now for almost 15 years. It is now used in the prevention and treatment of three groups of genetic disorders.
First, it has been used in the prevention of single-gene recessive disorders when the parents are carriers and the recurrence risk is 25%. Cystic fibrosis was the first such condition to be identified by PGD. 42 Individuals affected with autosomal dominant disorders have a recurrence risk of 50%. Examples of autosomal dominant disorders identified by PGD include Marfan syndrome and Huntington disease. Because PGD requires the analysis of DNA from single cells, polymerase chain reaction (PCR) has been the primary method used for the analysis. Most analyses have been done with nested multiplex and fluorescent multiplex PCR.
Preimplantation genetic diagnosis has also been used in the diagnosis and prevention of recurrent miscarriages when one partner is a reciprocal translocation or Robertsonian translocation carrier. Two techniques have been used for treating couples who are carriers. For women who are translocation carriers, the first polar body can be biopsied shortly after retrieval, prior to fertilization, and can be evaluated by whole chromosome FISH. Because this technique can only be used with female carriers, the more commonly used technique is that of embryo biopsy. For Robertsonian translocations two FISH probes are required, each located at the telomeric end of the chromosomes involved. For reciprocal translocations three probes with different reporter fluorochromes are required. Two probes must be for the centromeric region of each chromosome, and one probe must be at the telomeric end of one chromosome. Embryos with an unbalanced chromosome complement can be distinguished from those with a balanced complement, either normal or balanced carriers. Unfortunately studies have shown that embryos from translocation carriers have a high incidence of chromosome abnormalities and that pregnancy rates from these couples are low. 43, 44
There is a long-known relation between maternal age and chromosome abnormalities. It is also recognized that about 60% of pregnancies that miscarry have chromosome abnormalities. Therefore, it is not surprising that PGD has been used in combination with IVF when a couple is at risk of aneuploidy by virtue of advanced maternal age. IVF success rates may be improved when PGD and aneuploidy screening are used in women age 35 to 40. 45 Aneuploidy screening has now become the most common indication for PGD.
Routine IVF protocols are used for ovarian stimulation and oocyte retrieval. Oocytes are fertilized in vitro or by ICSI and the embryo grown to the six-cell or eight-cell stage. The embryo is then held in culture by a pipette and the zona pellucida opened by either an acid solution or laser beam; a biopsy pipette is inserted to aspirate one or two blastomeres for genetic analysis. An advantage of embryo biopsy is that more than one cell can be biopsied and analyzed. An alternative to embryo biopsy is polar-body biopsy of an oocyte for carrier status. The group from Chicago has pioneered this technique and shown it to be effective in the identification of single-gene disorders. 46
With an estimated 1 in 10 individuals infertile, assisted reproductive technologies (ART) are now responsible for up to 3% of annual births in some Western countries. 47 Although ART has been considered safe, registries have been monitoring the children conceived through these technologies. A possible association between ART and Beckwith-Wiedemann syndrome has now been reported by three registries around the world. 48 Caused by a defect located at chromosome 11p15, Beckwith-Wiedemann syndrome is characterized by organ overgrowth, macroglossia, and abdominal wall defects. These studies reported about a fourfold increased risk of Beckwith-Wiedemann syndrome with ART. An association between ART and Angelman syndrome has also been reported. Both of these syndromes are associated with imprinted contiguous gene clusters. About 10% of sporadic cases of Angelman syndrome and about 50% of sporadic Beckwith-Weidemann syndrome are due to epigenetic defects in imprinting. In all cases of Angelman syndrome reported and in 13 of 19 cases of Beckwith-Weidemann syndrome reported, there was loss of methylation of the maternal allele for these genes. This has raised speculation that imprinted genes may be at risk for loss of methylation during preimplantation and that ART may be responsible, whether due to ICSI or the culture conditions in vitro. These reports suggest the need for longitudinal studies of children conceived by ART and long-term evaluation of their development.


PEARLS

• With the advent of the Human Genome Project, we are now able to identify and in some cases treat individuals prior to the onset of a hereditary disorder.
• The Human Genome Project has revealed new complexities of gene structure and expression. Physicians will need to understand how new technologies identify gene mutations and their impact on human disease.
• Imprinting, uniparental disomy, mitochondrial inheritance, and trinucleotide repeat disorders have been identified as non-traditional forms of inheritance causing human disease.
• SRY, SOX9, SF1, WT1, and DAX have been identified as genes critical in gonadal differentiation.
• The important genes for expression of normal male phenotype that can reproduce are the androgen receptor gene located on the long arm of the X chromosome, the SRY gene on the short arm of the Y chromosome that determines development of the testes, and the AZF genes for spermatogenesis.
• Klinefelter’s syndrome (XXY) is the most common genetic cause of non-obstructive azoospermia.
• Microdeletions of AZF region account for about 10% to 20% of cases of azoospermia and 3% to 10% of severe oligospermia. These deletions are passed on to all male progeny.
• Preimplantation genetic diagnosis can provide prepregnancy diagnosis for single-gene defects and chromosome abnormalities, but there are concerns that congenital disorders associated with imprinting may be increased with PGD.

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12 Pinkel D, Straume T, Gray J. Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization. Proc Natl Acad Sci USA . 1986;85:2934-2938.
13 Gardner R, Sutherland G. Chromosome abnormalities and genetic counseling. In: Motulsky A., et al, editors. Oxford Monographs on Medical Genetics . 3rd ed. Oxford: Oxford University Press; 2004:577.
14 Knight S, Flint J. Perfect endings: A review of subtelomeric probes and their use in clinical diagnosis. J Med Genet . 2000;37:401-409.
15 Dickerman L, Park V, Clark BA. Genetic aspects of perinatal disease and prenatal diagnosis. In: Fanaroff A, Martin R, editors. Neonatal-Perinatal Medicine: Diseases of the Fetus and Infant . 6th ed. New York: Mosby Year Book; 1997:26.
16 Harper P. Practical Genetic Counseling, 5th ed. Oxford: Butterworth Heinemann, 1988.
17 Knoll J, et al. Angelman and Prader-Willi syndromes share a common chromosome 15 deletion but differ in parental origin of deletion. Am J Med Genet . 1989;32:285.
18 Nicholls R, et al. Genetic imprinting suggested by maternal heterodisomy in nondeletion Prader-Willi syndrome. Nature . 1989;342:281-285.
19 Spence J, et al. Uniparental disomy as a mechasnism for human genetic disease. Am J Hum Genet . 1988;42:217-226.
20 Hagerman R, et al. Fragile X Syndrome: Diagnosis, Treatment, and Research. Baltimore: Johns Hopkins University Press, 1991.
21 Leehey M, et al. The fragile X premutation presenting as essential tremor. Arch Neurol . 2003;60:117-121.
22 Hundscheid R, et al. Imprinting effect in premature ovarian failure confined to paternally inherited fragile X premutations. Am J Hum Genet . 2000;66:413-418.
23 Verkerk A, et al. Identification of a gene ( FMR-1 ) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome. Cell . 1991;65:905-914.
24 Nef S, Parada L. Hormones in male sexual development. Genes Dev . 2000;14:3075-3086.
25 Magenis R, et al. Translocation (X;Y)(p22.33;p11.2) in XX males: Etiology of male phenotype. Hum Genet . 1982;62:271-276.
26 Vergnaud G, et al. A deletion map of the Y chromosome based on DNA hybridization. Am J Hum Genet . 1986;38:109-124.
27 Sinclair A, et al. A gene from the human sex-determining region encodes a protein with homology to a conserved DNA-binding motif. Nature . 1990;346:240-244.
28 Berta P, et al. Genetic evidence equating SRY and the testis-determining factor. Nature . 1990;348:448-450.
29 Jager R, et al. A human XY female with a frame shift mutation in the candidate testis-determining gene SRY. . Nature . 1990;348:452-454.
30 Little M, et al. DNA binding capacity of the WT1 protein is abolished by Denys-Drash syndrome WT1 point mutations. Hum Mol Genet . 1995;4:351-358.
31 Achermann J, et al. A mutation in the gene encoding steroidogenic factor-1 causes XY sex reversal and adrenal failure in humans. Nat Genet . 1999;22:125-126.
32 McCabe E. Adrenal hypoplasias and aplasias. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The Metabolic and Molecular Bases of Inherited Diseases . 8th ed. New York: McGraw-Hill; 2001:4263-4274.
33 Foster J, et al. Campomelic dysplasia and autosomal sex reversal caused by mutations in an SRY-related gene. Nature . 1994;372:525-530.
34 Wagner T, et al. Autosomal sex reversal and campomelic dysplasia are caused by mutations in and around the SRY -related gene SOX9. . Cell . 1994;79:1111-1120.
35 Huang B, et al. Autosomal XX sex reversal caused by duplication of SOX9. . Am J Med Genet . 1999;87:349-353.
36 Gidwani G, Falcone T. Congenital Malformations of the Female Genital Tract: Diagnosis and Management. Philadelphia: Lippincott Williams & Wilkins, 1999.
37 Kaufman R, Hartmann A, McAlister W. Family studies in congenital heart disease, II: A syndrome of hydrometrocolpos, postaxial polydactyly, and congenital heart disease. Birth Defects Orig Artic Ser . 1972;8:85-87.
38 Beals P, et al. New criteria for improved diagnosis of Bardet-Biedl syndrome: Results of a population survey. J Med Genet . 1999;36:4237-4246.
39 Stone D, et al. Genetic and physical mapping of the McKusick-Kaufman syndrome. Hum Mol Genet . 1998;7:475-481.
40 Mortlock D, Innis J. Mutation of HOXA13 in hand-foot-genital syndrome. Nat Genet . 1997;15:179-180.
41 Dohle G, et al. Genetic risk factors in infertile men with severe oligospermia and azoospermia. Hum Reprod . 2002;17:13-16.
42 Handyside A, et al. Birth of a normal girl after in vitro fertilization and preimplantation diagnositic testing for cystic fibrosis. NEJM . 1992;327:905-909.
43 Committee of the E.P.C.S. ESHRE Preimplantation Genetic Diagnosis Consortium: Data collection III. Hum Reprod . 2002;17:233-246.
44 Iwarsson E, et al. Highly abnormal cleavage divisions in preimplantation embryos from translocation carriers. Prenat Diagn . 2000;20:1038-1047.
45 Wilton L. Preimplantation genetic diagnosis for aneuploidy screening in early human embryos: A review. Prenat Diagn . 2002;22:312-318.
46 Rechitsky S, et al. Accuracy of preimplantation diagnosis of single-gene disorders by polar body analysis of oocytes. J Assist Reprod Genet . 1999;16:192-198.
47 Evers J. Female subfertility. Lancet . 2002;360:151-159.
48 Gosden R, et al. Rare congenital disorders, imprinted genes, and assisted reproductive technology. Lancet . 2003;361:1975-1977.
Chapter 6 Normal Fertilization and Implantation

Navid Esfandiari

INTRODUCTION
Fertilization is a complex process in which sperm interacts with the homologous oocyte to create a new individual. The bringing together of two gametes in mammals starts with cells moving through the reproductive tracts of both the male and female organisms until they arrive close to each other in the female reproductive tract. The subsequent interaction between the two gametes requires several steps that result in gamete fusion to produce a zygote, including (1) binding of the spermatozoon to the oocyte coat, (2) oocyte activation, (3) formation of male and female pronuclei, and (4) initiation of cell division and early development.
In the past two decades, considerable efforts have been made to identify molecules and pathways utilized during gamete interaction. The interaction and communication between two completely foreign cells is influenced by numerous biologic, physiologic, and genetic factors. Most of what is known about gamete interactions has been derived from animals, and the vast majority of our understanding rests on a murine model. Although many of the molecules involved in fertilization processes that have been identified in the mouse model are conserved in humans, whether this information can be extrapolated to fertilization in humans is a matter of debate. The main experimental method used to study the cellular and molecular mechanisms of spermoocyte interactions has been the in vitro fertilization (IVF) technique. With IVF in laboratory animals and humans now a routine procedure, many of the critically important key points in fertilization have been identified.
This chapter summarizes the frontiers of knowledge regarding the molecular and cellular mechanisms of fertilization and focuses on basic understanding of the cell biologic processes and molecular events underlying implantation and its relevance to clinical reproductive medicine.

HISTORICAL PROSPECTIVE
Before the initiation of modern reproductive and developmental biology in the 17th century, the theory of “seeds” belonging to the pluralistic current of the Pythagorean School led by Anaxagoras of Clazomenae and Empedocles of Akragas was the most believed (5th century B.C. ). 1 In the field of human reproduction, pluralism means that a fetus results from the mixing of two parental seeds. Hippocrates (c. 460–370 B.C. ) believed that “seeds” are produced in all parts of the body, each containing both the masculine and feminine principle; when transmitted to offspring at the time of conception, they cause certain parts of the offspring to resemble their parents. A century later, Aristotle (384–322 B.C. ) rejected Hippocrates’ theory. For him, only the male’s seed contributes to form the fetus; the female’s role in reproduction is to contribute menstrual blood. He had noticed that offspring sometimes resemble their grandparents rather than their parents. It was difficult to understand how seed of tissue and blood could be maintained unnoticed in the parents only to be revealed again in the children. Aristotle proposed that male semen was a mix of ingredients that was not blended perfectly so that materials from previous generations could sometimes get through. Most of Aristotle’s ideas were presented in his treatise, On Generation of Animals . This was the very first complete embryologic work ever written. Moreover, he was the first to illustrate his treatises, which significantly helped to clarify his writings.
Galen (130–201 A.D. ), who is considered the greatest Greek physician after Hippocrates and founder of experimental physiology, supported Hippocrates’ theory that the seeds of both man and woman contribute to reproduction, but that each contains only one principle. 1 In the 17th century, several outstanding findings fueled new scientific developments in modern reproductive biology. William Harvey (1578–1657) was the first to suggest that humans and other mammals reproduced via the fertilization of an oocyte by sperm. However, many authors see Regnier de Graaf (1641–1673) as the founder of modern reproductive biology. 2 De Graaf is the one who discovered (1672) the source of oocytes in the testes of women, which we now call ovaries. Five years later the initial discovery of spermatozoa was made by a medical student named John Ham, who told Anton van Leeuwenhoek of the presence of what was termed animalcules in human seminal fluid, thought to have arisen from putrefaction. Leeuwenhoek (1632–1723) was the first to make detailed and accurate identification of sperm as a normal constituent of semen. 3 Van Leeuwenhoek proposed that fertilization occurs when the sperm enters the oocyte, but this could not actually be observed for another 100 years because of the quality of microscopes available.
Another discovery that revolutionized scientific thinking was by the Italian priest and physiologist Lazaro Spallanzani in 1779. Until that time, our understanding of reproduction was based on our knowledge of how plants grow. It was believed that the embryo was the “product of male seed, nurtured in the soil of the female.” Spallanzani’s experiment established for the first time that for an embryo to develop there must be actual physical contact between the oocyte and the sperm. Spallanzani successfully inseminated frogs, fish, and dogs. The first successful artificial insemination of a woman was recorded just 11 years after Spallanzani’s experiment. In 1790, the renowned Scottish anatomist and surgeon, Dr. John Hunter, reported successful insemination of the wife of a linen draper using her husband’s sperm. These discoveries led to the emergence of modern reproductive technologies, by which the world’s first IVF baby was born just before midnight on 25 July 1978 as a result of continued efforts of Drs. Edwards and Steptoe.

SPERM TRANSPORT IN THE MALE REPRODUCTIVE TRACT
Spermatogenesis refers to the complex process of transformation of germline stem cells into sperm cells within the seminiferous tubules of the testes. Spermatogenesis is regulated through endocrine interactions between the pituitary gland and Sertoli cells. This endocrine system is referred to as the hypothalamic-pituitary-gonadal axis and involves a series of signaling mechanisms. Two hormones, follicle-stimulating hormone (FSH) secreted by the pituitary, and androgens (i.e., testosterone) produced by the Leydig (interstitial) cells in the testis, control Sertoli cell functions. Once formed within the seminiferous tubules, the immotile spermatozoa are released into the luminal fluid and transported to the epididymis, where they gain the ability to move and fertilize the oocyte. 4 The testicular spermatozoa are transported passively to the rete testis, which is a branched reservoir of the openings of the seminiferous tubules. From the rete testis, the spermatozoa are transported to the epididymis via the efferent ductules. 5 In mammals, the transit of spermatozoa through the epididymis usually takes 10 to 13 days; in humans the estimated transit time is 2 to 6 days. 6 The epididymal segment where most spermatozoa attain their full fertilizing capacity appears to be the proximal cauda. The spermatozoa from that region are capable of moving progressively, which is characteristic of spermatozoa preceding fertilization, and bind to zona-free hamster ova in vitro at a higher percentage than spermatozoa obtained from more proximal locations. 7
To attain the capacity to fertilize an oocyte, sperm undergo many maturational changes during its transit in the epididymal duct. 4 These include, for instance, changes in plasma membrane lipids, proteins, and glycosylation; alterations in the outer acrosomal membrane; gross morphologic changes in acrosome in some species; and cross-linking of nuclear protamines and proteins of the outer dense fiber and fibrous sheath. The cauda epididymidis (and proximal ductus deferens) are the regions where spermatozoa are stored before ejaculation. 7 When ejaculation occurs, the stored spermatozoa with the surrounding fluid are mixed with the alkaline secretions of the male accessory sex glands and deposited in the vagina.

SPERM TRANSPORT IN THE FEMALE REPRODUCTIVE TRACT
Spermatozoa are actively transported from the vagina via the cervical canal and the uterine cavity to the ampulla of the oviducts, where fertilization occurs. In the human vagina, the ejaculated semen is deposited near the external cervical opening, where the environment is very acidic due to lactic acid and thus is hostile to spermatozoa. 8 The alkaline pH of the ejaculate protects spermatozoa in this acidic environment. 9 This protection is, however, temporary, and most spermatozoa only remain motile in the vagina for a few hours. Thus, the human vagina does not serve as an effective reservoir for spermatozoa and the role of the vagina in sperm transport is transitory at best. There is significant sperm loss in the vagina, and the proportion of ejaculated sperm that enter the cervical mucus in vivo is not known. The spermatozoa are transported into the cervical canal by pressure alterations in the vagina due to the female orgasm, assisted by the normal motility of sperm. Migration of spermatozoa through the cervical canal is thought to be dependent at least in part on sperm concentration, motility, and morphology and is modulated by the physiochemical characteristics of cervical mucus. The interactions between sperm and mucus and the motility of spermatozoa during transport are important; one cause of infertility is presumably impaired sperm movement through the cervical mucus. 9 The change in the composition of cervical mucus at midcycle also affects the passage of sperm. 10 In ovulatory mucus, sperm penetration and forward progression occur in an efficient and directional manner. In this phase of the cycle, mucin molecules are arranged in a parallel position, directing the spermatozoa into the uterus and also to the cervical crypts where they are stored. 11
It is not clear whether the human female reproductive tract has the capacity to establish sperm reservoirs as do the resproductive tracts of other mammals. However, it has been shown that the release of spermatozoa from human cervical crypts may continue for several days. 4 In a small percentage of women displaying no sperm in the cervical mucus, however, motile sperm can be recovered from the uterine cavity. 12 The uterus seems to be a conduit to sperm transport. The transport of spermatozoa from the cervix to the uterotubal junction is mainly attributable to the myometrial contractility, ciliary movements on the surface of the endometrial cells, and intrinsic sperm motility, although the latter has been shown not to be critical. 4 The human endometrium prepares for ovulation by secreting a unique kind of fluid into the uterine lumen. The fluid has a different protein pattern, ionic composition, and volume than at other stages of the cycle. 13 This fluid serves to suspend spermatozoa and to keep them viable during the transport process and to remove the coating from the sperm surface as one facet of capacitation. It also contains macrophages that remove dead and nonviable spermatozoa, which in mice is probably the most important physiologic mechanism for the disposal of sperm from the uterus. 14 The passage of sperm from the uterus to the fallopian tube is apparently modulated by the uterotubal junction. This segment prevents the entry of nonmotile cells and therefore acts as a selective barrier. The isthmus functions as a sperm reservoir and only a few sperm pass along the fallopian tube to the site of fertilization at any given time. 11 The ampulla of the oviduct is the site of fertilization, and motile spermatozoa can be found there up to 85 hours after intercourse. The transport of spermatozoa through the oviducts is a combination of sperm motility, fluid flow, and the contractive movements of the oviduct walls. 8

Sperm Capacitation
Immediately after deposition in the female genital tract, besides active motility, the sperm are not able to fertilize the oocyte. The physiologic changes that occur to sperm during their transport in the female reproductive tract are collectively referred as capacitation. Capacitation, first described in 1951 (independently by Chang in the United States and Austin in Australia), was later shown to be a requirement for fertilization. It seems that the initiation and completion of capacitation in human spermatozoa takes place in the cervix. 4, 9 The molecular events that initiate capacitation include removal of cholesterol from the sperm plasma membrane, increase in membrane fluidity, ion influxes resulting in alteration of sperm membrane potential, hyperpolarization of sperm membrane, increase in tyrosine phosphorylation, and changes in the adenylate cyclase-cAMP system, nucleus, and acrosome. 4, 15 In humans, capacitation can be mimicked in vitro in defined culture media, the composition of which is based on the electrolyte concentration of oviductal fluid. In most cases capacitation media contain energy substrates such as pyruvate, lactate, and glucose; a cholesterol receptor (such as serum albumin); NaHCO 3, calcium, potassium, and physiologic sodium concentrations.

Hyperactivation and Acrosome Reaction
Capacitation, as a molecular event, occurs both in the sperm head (i.e., the acrosome reaction) and in the tail (i.e., changes in the sperm motility pattern designated as sperm hyperactivation). Sperm hyperactivation occurs before the acrosome reaction. 4 The vigorous pattern of movement observed in hyperactivation is a result of physiologic changes in spermatozoa. Compared to sperm in seminal plasma, the speed, velocity, rate of flagellar beating or beat frequency, and mean beat width (lateral head displacement) are increased in capacitated sperm. This pattern of movement enables the spermatozoa to swim in the viscous oviduct fluid and to overcome the physical resistance of the three vestments—cumulus oophorus, corona radiata, and the zona pellucida—which surround the oocyte. 16, 17 Hyperactivation is a prognostic indicator for IVF; low levels are associated with male infertility and decreased binding to the zona pellucida.
The endpoint of capacitation is the acrosome reaction, which occurs when spermatozoa come into close contact with the oocyte in the ampullae of the oviduct. The acrosome reaction is an exocytotic event and enables spermatozoa to penetrate through the zona pellucida and fuse with the oocyte plasma membrane. Many artificial stimuli are reported to trigger the acrosome reaction, either by driving extracellular Ca 2+ into the sperm cell (Ca 2+ ionophores) or by acting through intracellular second messengers that are involved in the cascade leading to acrosomal exocytosis. The anterior region of the sperm head is covered by the acrosome, containing several hydrolytic enzymes, including proteases, phosphatases, arylsulfatases, and phospholipases. Hyaluronidase and acrosin have been the most extensively studied of these enzymes. The membranes surrounding the nucleus of spermatozoa are the nuclear membrane, the inner acrosomal membrane, the outer acrosomal membrane, and the plasma membrane. In this reaction, the plasma membrane and the outer acrosomal membrane fuse, enabling release of the acrosomal contents important for the events preceding fertilization. 4 The acrosome reaction may facilitate recognition, adhesion, and fusion with oocytes by at least three different mechanisms: externalization of ligand proteins (e.g., CD46 on the inner acrosomal membrane); protein migration through the fluid membrane to reach binding sites, such as that for PH-20; or conformational changes of preexisting membrane proteins. 18
A knowledge of the capacitation process from the time sperm are deposited in the female reproductive tract until the final penetration of the oocyte suggests clinical applications in several areas of human fertility. The fact that once sperm have been capacitated their fertilizable life is shortened in the oviduct is of interest from the standpoint of fertility enhancement. In vitro capacitation of sperm, by removing the seminal plasma and incubating washed sperm in culture media in optimum temperature and a carbon dioxide incubator, allows a precise control of the time of conception and has application for treating some cases of infertility. There are several ways to evaluate acrosomal status in human sperm: (1) methods using transmission electron microscopy, allowing ultrastructural examination of the acrosome; (2) methods using light microscopy based on staining sperm with specific fluorescein plant lectins or various dyes; and (3) methods using a fluorescence-activated cell sorter and fluorescein monocloned antibodies or lectins.

OOCYTE TRANSPORT
The transport of oocytes from the ovarian follicles to the site of implantation has been long recognized as a fundamental step in the reproductive process in the female. 19 The fallopian tubes provide the path and means of transport for the ovum from the ovary to the uterus; the duration of ovum transport is the time from its discharge from the follicle until it reaches the normal site of implantation. 20 At midmenstrual cycle, approximately day 14 of an idealized 28-day cycle, the surge of pituitary luteinizing hormone (LH) results in the maturation of the oocyte by resumption of meiosis and completion of the first meiotic division. The oocyte nucleus or germinal vesicle undergoes a series of changes that involve germinal vesicle breakdown. The oocyte then enters into the second meiotic division and arrests in the second metaphase or first polar body stage. Meiosis will proceed no further unless the oocyte is fertilized. Meiotic maturation is a vital event in ovulation because it is obligatory for normal fertilization. During the process of meiotic maturation, the cumulus granulosa cells undergo mucification followed by expansion. 21 The preovulatory surge of FSH appears to initiate the process of cumulus expansion. The onset of mucification is marked by a dramatic increase in the secretion of mucopolysaccharides into the extracellular spaces. This leads to the dispersal of the cumulus cells and causes the oocyte–cumulus complex to expand tremendously.
After ovulation and discharge of the follicular fluid, the oocyte–cumulus complex, consisting of an oocyte surrounded by a zona pellucida, noncellular porous layers of glycoprotein secreted by the oocyte, and granulosa cells (cumulus oophorus), is ovulated from ovarian follicles into the peritoneal cavity and is picked up by the infundibulum of the oviduct. 8 The infundibulum is a highly specialized, funnel-shaped portion of oviduct (up to 10 mm in diameter) that bears long, fingerlike extensions of the mucosa (i.e., fimbriae), which sweep over the surface of the ovary; the oocyte–cumulus complex is transported by means of ciliary action along the surface of the fimbriae toward the opening of the oviduct. Cilia that cover the exterior surface of the infundibulum beat in the direction of the ostium and are important in moving the oocyte–cumulus complex into the oviduct. Oocyte pickup is not dependent on a suction effect secondary to muscle contractions, and ligation of the tube just proximal to the fimbriae in the rabbit does not interfere with pickup. 22 The mucosal layer of the fallopian tube is lined with a single layer of columnar epithelium that undergoes cyclic changes in response to hormonal changes of the menstrual cycle. There are two distinct types of oviductal epithelium—ciliated and nonciliated secretory epithelium—and the ratio of these two fluctuate in response to ovarian steroids. There are fewer ciliated cells in the isthmus than in the ampullary portion of the tube, whereas they are most prominent in the fimbriated infundibulum. Ciliary activity is responsible for the pickup of oocytes by the fimbrial ostium and for movement through the ampulla, as well as the distribution of the tubal fluid, which supports gamete maturation and fertilization and facilitates gamete and embryo transport. Under the influence of the estrogens of the follicular phase of the menstrual cycle, the cilia of the isthmus beat in the direction of the ovary. At the same time, the cilia of the fimbria and the cilia of the ampulla next to the fimbria beat in the direction of the uterus. In this fashion, the ovulated oocyte will be sequestered within the middle of the ampulla. If sperm enter the uterus at this time, they will be directed into the oviduct and propelled to the awaiting oocyte.
It is within the ampulla that fertilization takes place; in humans the oocyte spends about 90% of its stay in the ampulla. After surgical reversal of tubal ligation where a large part of the ampulla was removed, excellent pregnancy rates result along with mildly increased rates of ectopic pregnancies. Pregnancy is possible after reversal of tubal ligation where complete excision of the entire ampula or fimbria was performed, but pregnancy rates are poor. After ovulation and in the presence of an increasing concentration of progesterone, the cilia and myosalpingeal contractions all pulse from the ovary toward the uterus. 23 In most species, transport of the fertilized oocyte through the tube requires approximately 3 days. 19 Tubal fluid is rich in mucoproteins, electrolytes, and enzymes. This fluid is abundant in midcycle when gametes or embryos are present and may play an important role during fertilization and early cleavage. Fluid in the tubes is believed to be formed by selective transudation from the blood and active secretion from the epithelial lining.

SUCCESSIVE FERTILIZATION STEPS
Fertilization is a series of steps that require immense coordination and communication between the two principal participants, the oocyte and sperm. Each gamete must undergo a series of changes before the final event of union can occur ( Figs. 6-1 and 6-2 ).

Figure 6-1 Fertilization process. (1) Sperm penetration of cumulus cells, (2) attachment to zona, (3) exocytosis of acrosomal contents, (4) penetration to the zona pellucida, (5) entry into perivitelline space, (6) binding and fusion with the oocyte plasma membrane, (7) cortical reaction, and (8) block to polyspermy.

Figure 6-2 Schematic drawing showing the major events from ovulation to the implantation of blastocyst during the first week of human life.

Adhesion of Spermatozoa to Oocyte

Sperm Penetration of the Cumulus Oophorus
The cumulus oophorus is a specialized layer of granulosa cells that surrounds the ovulated mammalian oocyte. During oocyte maturation, the cumulus cell mass undergoes expansion and mucification, resulting in a well-expanded mass with an extensive amount of extracellular matrix material binding the cells together. The cumulus cells and their matrix are probably involved in several reproductive processes, including pickup of the oocyte–cumulus complex by the oviduct and increasing the chances of an encounter with one of the few sperm that have reached the ampulla of the tube. 24 The extracellular matrix consists of a variety of glycoproteins and proteoglycans, with hyaluronic acid being a major component. The mechanisms of sperm penetration through the cumulus mass may involve both mechanical and chemical forces. Hyperactivated sperm and membrane-bound hyaluronidase are necessary, and perhaps sufficient, to digest a path through the extracellular matrix of the cumulus cells. The role of hyaluronidase in cumulus mass penetration is controversial because sperm remain largely acrosome-intact during in vitro penetration of the cumulus; sperm with no hyaluronidase can penetrate the cumulus mass and reach the zona pellucida. 25 The role of the cumulus cell mass in fertilization is not fully understood. In human fertilization, however, a body of evidence has shown that the presence of the cumulus oophorus is beneficial for fertilization, partly by stimulation of proacrosin conversion to acrosin and initiation of the acrosome reaction. However, removal of the cumulus does not prevent sperm penetration and fertilization.

Sperm Interaction with the Zona Pellucida
Eventually after a long journey and penetration of the cumulus mass, the sperm encounters the zona pellucida, the cell type-specific extracellular matrix or coat of the oocyte where species-specific gamete recognition is believed to occur. The mammalian zona pellucida is composed of the transcripts of three highly conserved gene families named ZPA, ZPB, and ZPC. The mature products of the genes based on their deduced amino acid sequence are classified as ZP1, ZP2, and ZP3. They are synthesized during oogenesis as a product of the oocyte itself and exhibit heterogeneity because of extensive post-translational modification, including glycosylation. 26 Determining the function of individual ZP proteins in humans has been difficult because of the limited availability of native human ZP, and much of the research on the zona pellucida and sperm recognition of the zona pellucida has been carried out using the mouse as a model system. In this species, the ZP3 protein serves as a sperm receptor molecule, mediating binding of the sperm to the intact zona pellucida. The currently favored model is that O-linked oligosaccharide chains on ZP3 act as a binding epitope for sperm. ZP3 is the primary ligand for sperm, whereas ZP2 in the mouse acts as a secondary sperm receptor that binds acrosome-reacted sperm and ZP1 is considered as a scaffold-like protein that appears to cross-link the ZP2 and ZP3 proteins. 27 The complexity of the sperm membrane architecture, along with the difficulty of isolating and purifying it, have made the identification of the spermatozoon counterpart of the oocyte coat ligand a difficult task.
Sperm proteins thought to participate in zona binding have been identified by a range of approaches, including inhibitory monoclonal antibodies. Some of these sperm proteins have been specifically implicated as primary sperm receptors for ZP3, whereas others are thought to function as generalized adhesive proteins for the zona matrix. Thus far, among the sperm proteins thought to bind ZP3 oligosaccharides in particular, the sperm surface enzyme β1,4-galactosyltransferase-I (GalT) and two sperm membrane proteins called sp56 and sp95 are the main candidates; GalT satisfies virtually all the criteria expected of a ZP3 receptor. 25, 28 It presumably recognizes and binds specifically to N-acetylglucosamine residues on ZP3, an enzyme–substrate reaction in which ZP3 serves as substrate. The location of GalT, on the plasma membrane covering the dorsal aspects of the anterior region of the sperm head, is consistent with the inability of acrosome-reacted sperm to bind the zona pellucida because the plasma membrane bearing GalT is lost during the acrosome reaction. 29 The binding of ZP3 to sperm activates a range of intracellular signal cascades that culminate in fusion of the plasma membrane and underlying outer acrosomal membrane (i.e., the acrosome reaction). 30 The ability of ZP3 to act as an acrosome reaction inducer depends on its polypeptide chain, whereas its ability to act as a sperm receptor rests entirely on its oligosaccharide side chains. ZP3-induced exocytosis of the acrosomal contents proceeds through two sperm-signaling pathways. In the first, ZP3 binding to GalT and other potential receptors results in activation of a heterotrimeric GTP-binding protein and phospholipase C, thus elevating the concentration of cytoplasmic calcium. In the second pathway, ZP3 binding to the same receptor(s) stimulates a transient influx of calcium through T-type channels. In a later phase of the signaling, these initial ZP3-induced events produce additional calcium entry through transient receptor potential proteins, candidate subunits of the ion channels, resulting in a sustained increase in cytoplasmic calcium concentration that triggers exocytosis. 24, 31

Sperm Penetration of the Zona Pellucida
Penetration of the zona pellucida likely involves several factors, including physical forces (e.g., hyperactivated motility of sperm, which involves an increase in flagellar bend amplitude and usually beat asymmetry) and chemical forces (e.g., proteases and glycosidases). The proteases could be sperm surface, membrane-anchored proteases or soluble proteases from the acrosomal contents. The spermatozoon penetrates through the thick zona, cutting a penetration slit that is just as wide as the sperm head.

Sperm–Oocyte Membrane Fusion
After sperm bind to the zona pellucida and the acrosome reacts, they penetrate the zona and enter the perivitelline space, the extracellular region between the zona and the oocyte plasma membrane where the final adhesion in the fertilization process occurs (i.e., adhesion of the sperm plasma membrane to the oocyte plasma membrane). Spermatozoa entering the perivitelline space approach the oocyte surface at an angle that lies somewhere between the vertical and the horizontal. Mammalian oocytes are spherical cells covered with microvilli before fertilization. The sperm head is like a flat dish, and the thickness of the head (∼0.2 μm) is a little less than the average distance between oocyte microvilli. 32 Spermatozoa, therefore, just pass between the microvilli and make contact with the oocyte close to the cortex through the equatorial segment of the head. In the search for sperm surface proteins that function in this process, most attention has recently been given to the cysteine-rich secretory protein 1 (Crisp1) and members of the ADAM family of metallopeptidase domains (fertilin α [ADAM1], fertilin β [ADAM2], and cyritestin [ADAM3]) that function as cell adhesion molecules. The ADAM family molecules have an adhesion module, the disintegrin domain, which leads directly to the idea that oocytes have an appropriate plasma membrane adhesion partner (i.e., an integrin). 33 To better assess the requirement for specific members of the ADAM family in binding (and possibly fusion), knockout mice were constructed that were null for fertilin β, cyritestin, or both (double knockout). Sperm from each of these mouse lines showed dramatically (90%) reduced binding to zona-free oocytes. 34, 35 Because other sperm proteins may be lost in these knockouts, this reduction in total sperm binding could possibly be caused by loss of an unidentified protein. Initial attachment of the sperm to the oocyte membrane are reversible and appear to require sperm motility. Sperm tail movement decreases or stops within a few seconds of sperm–oocyte fusion. Electron microscopy shows that the inner acrosomal membrane is later engulfed by the oocyte through a process that appears similar to phagocytosis. 33 The sperm tail is also eventually incorporated into the oocyte. The adhesion of sperm to oocyte is frequently cited as likely to be analogous to the mechanism by which leukocytes interact with endothelial cells in a stepwise fashion; sperm–oocyte fusion shares common features with another classical, well-known membrane fusion event between cells and virus particles. Fusion of the sperm and oocyte membrane is followed by the cortical reaction and metabolic activation of the oocyte.

Oocyte Activation
As a consequence of successful sperm–oocyte fusion, the oocyte undergoes a series of well-defined morphologic and biochemical endpoints, some of which occur within seconds or minutes of sperm–oocyte plasma membrane interaction and some that occur over the course of several hours. 4 These modifications can collectively be termed oocyte activation. One of the earliest events of oocyte activation is an increase in the level of intracellular free calcium in a periodic and oscillatory pattern known as calcium signal. It is clear that this calcium signal induces the release of meiosis, cortical granule exocytosis, zygote formation, and first cell mitosis. The oocyte possesses receptors for sperm glycoproteins in its membrane, and binding the sperm to the receptor alters the receptor so that it might trigger the G-protein cascade or activate tyrosine kinase pathways that then lead to activation of phospholipase C and the production of inositol triphosphate, which mobilizes internal calcium. Alternatively, the sperm may activate the oocyte by direct injection into the oocyte cytoplasm of a sperm-derived protein or factor, called oscillin, which may be the signaling agent for the critical calcium oscillation. This theory helps to explain the clinical success of intracytoplasmic sperm injection (ICSI), in which sperm is introduced directly into the human oocyte cytoplasm and bypasses the activation of the oocyte through binding to a receptor. 36 Regardless of the regulatory mechanism, the end results are the resumption and completion of the second meiotic division and ultimately the formation of the female pronuclei.

Cortical Reaction and Block to Polyspermy
It is essential that only one spermatozoon fuses and enters the oocyte; therefore, establishment of the block to polyspermy is one of the most critical sequelae of oocyte activation. Entry of more than one sperm into the oocyte cytoplasm at fertilization represents a pathologic condition and leads to the breakdown of development. Under in vivo conditions, sperm–oocyte interaction is regulated in part by the fact that relatively few sperm reach the site of fertilization in the fallopian tube. However, the oocyte must possess mechanisms that can rapidly and precisely eliminate the likelihood of penetration by supernumerary sperm. Fusion of the sperm with the oocyte triggers a release of calcium ions from calmodulin, which results in depolarization of the oocyte plasma membrane, by which fusion of another sperm is prevented. 29 The rapid depolarization of the oocyte plasma membrane during fertilization provides an early (fast) block to polyspermy that is only transient and returns to normal within a few minutes. The initiation of the block to penetration of the zona by other sperm is mediated by the cortical reaction, an exocytotic discharge of the oocyte’s cortical granules that are found just below the oocyte surface and is also apparently triggered by the increase in calcium. Cortical granules contain a variety of hydrolytic enzymes, and these enzymes presumably result in the hardening of the zona pellucida by cross-linking of structural proteins and inactivation of ligands for sperm receptors. 37 This process, which is collectively known as the zona reaction, results in the lost of zona ability to bind sperm. The loss of sperm-binding activity is due, at least in part, to the release of β-N-acetylhexosaminidase from the oocyte cortical granules, which destroys the GalT-binding sites on ZP3. 38

Male Pronucleus Formation and Genomic Union
Fertilization requires accurate cytoplasmic events mediated by the centrosome, which is the cell’s microtubule organizing center. 39 The centrosome is a complex organelle composed of many different proteins such as γ-tubulin. Shortly after sperm penetration, maternal γ-tubulin is drawn to the sperm centrosome to assist in the formation of the sperm aster, which is found adjacent and affixed to the sperm nucleus. The continuing elongation of the sperm astral microtubules throughout the cytoplasm allows them to come into contact with the female pronucleus, which is then translocated toward the male pronucleus resulting in pronuclear migration and apposition. Defects in centrosome function during microtubule elongation may result in the failure of normal fertilization. The transformation of the sperm centrosome into a functional zygotic centrosome capable of aster formation suggests that centrosome inheritance in humans is paternal in origin. 40 Remodeling of the sperm chromatin into a male pronucleus is guided by the oocyte-produced reducing peptide glutathione and a number of molecules required for the reconstitution of the functional nuclear envelope and nuclear skeleton. After migration and union of the pronuclei, the centrosome duplicates and splits, with each centrosome serving as one pole for the first mitotic spindle. Once the two pronuclei are closely apposed, the nuclear envelope dissolves. The DNA undergoes replication and the chromosomes from each pronuclei are now paired, aligned on the newly formed mitotic spindle, and ready for initiation of first mitotic division through a new cascade of cell cycle-mediating events. Although some of the sperm structures are transformed into zygotic components, the elimination of others is vital to the early stages of embryonic development. The accessory structures of the sperm axoneme, including the fibrous sheath, microtubule doublets, outer dense fibers, and the striated columns of the connecting piece, are discarded in an orderly fashion. During ICSI, which bypasses multiple steps of natural fertilization by introducing an intact spermatozoon into oocyte cytoplasm, the sperm accessory structures that would normally be eliminated before or during the entry of sperm into the oocyte cytoplasm persist and may interfere with early embryonic development, thus decreasing the success rate of assisted reproductive technologies (ART) and possibly causing severe embryonic anomalies. 41

IMPLANTATION
The molecular events of embryonic attachment to the endometrial epithelium and subsequent invasion into the stroma have long been of interest for reproductive scientists and clinicians. In most successful human pregnancies, the conceptus implants 7 to 10 days after ovulation. A successful pregnancy in the human requires a receptive endometrium, a functionally normal embryo at the blastocyst stage, a dialogue between the maternal and embryonic tissues, transformation of the endometrium to deciduas, and finally formation of the definitive placenta. Hatching of the blastocyst from the zona pellucida is a key event in mammalian development. The zona pellucida initially prevents the blastocyst from adhering to the oviduct wall, in theory preventing a tubal or ectopic pregnancy. Because the embryo is covered by the zona pellucida until immediately before implantation, all embryo–maternal signaling has to pass the zona and be detectable within it. When the embryo reaches the uterus it must hatch from the zona pellucida; then it can adhere to the uterine wall to establish a pregnancy. On differentiation at the early blastocyst stage, it would appear that trophectoderm is responsible for secreting the zona lysine required for hatching. A trypsinlike protease, termed strypsin, released by the trophectoderm has been proposed to be the zona lysine. On the other hand, impaired uterine receptivity is one of the major reasons for the failed implantation and failure of ART. 42, 43 The phenomena of implantation and trophoblast invasion are currently considered as the major limiting factor for the establishment of pregnancy. 44

Role of Ovarian Hormones
In animals, ovarian hormones regulate the development of endometrial receptivity. To prepare for implantation, the endometrium undergoes a precise developmental progression, starting at the proliferative phase under the control of follicular phase estradiol. Estrogen triggers the expression of a unique set of genes in the preimplantation endometrium that in turn control implantation. Expression of the estrogen receptor amplifies the estrogen effects required for uterine cell proliferation and/or differentiation during implantation. Following ovulation, the endometrium is transformed into a specialized secretory structure under the direction of progesterone. 45 The cellular actions of progesterone are mediated through intracellular progesterone receptors, which are well-studied gene regulators. It is postulated that hormone-occupied progesterone receptors trigger the expression of specific gene networks in different cell types within the uterus, and the products of these genes mediate the hormonal effects during early pregnancy.

Embryonic Contribution to Implantation
In addition to hormonal regulation, increasing evidence demonstrates that embryonic regulation also has a significant impact by inducing reciprocal embryo–uterine interactions that change throughout the implantation process. The preimplantation embryo produces several factors during its development to signal its presence to the maternal organism. The appropriate interaction between the preimplantation embryo and maternal endometrium is at least partly controlled by paracrine cytokines. 46, 47 Cytokines and growth factors and their corresponding receptors have, on the mRNA level, been detected in blastomeres and in preimplantation embryos from different species as well as in the human endometrium throughout the menstrual cycle. Most of our knowledge of the physiology of implantation has come from animal studies; for obvious ethical reasons, it is not possible to study the process of implantation in humans in detail. However, we focus on biochemical and molecular events that are relevant to humans.

APOSITION AND ADHESION

Endometrial Receptivity and the Window of Implantation
Endometrial receptivity can be defined as the capacity of the uterine mucosa to facilitate successful embryonic implantation. The human endometrium is receptive to blastocyst implantation only during a very short and precise period in the luteal phase. It is the so-called nidation or implantation window, which can be defined as the period of maximum uterine receptivity for implantation. 48 In the implantation window, changes occur in endometrial epithelial morphology characterized by the appearance of membrane projections called pinopodes. The time of maximal endometrial receptivity is thought to happen on cycle days 20 to 24 and is manifested by the expression of peptides and proteins that can serve as biomarkers of uterine receptivity. 45 Additional information derives from ART cycles in which the window of endometrial receptivity was tested by performing embryo transfer at different times after luteinizing hormone (LH) peak. It has been recently shown that ongoing pregnancy is significantly higher for embryos that implant between cycle days 22 and 24 (postovulation days 8 to 10) than those embryos that are implanted 11 days or beyond after ovulation. Also, the risk of early pregnancy loss increases with later implantation, from 13% among conceptuses that implanted by the day 9 to 52% on day 11, and to 82% after day 11. 49

Pinopodes
During the receptive phase, the apical plasma membranes of the epithelial cells lining the uterine cavity lose their microvilli and develop large and smooth apical projections called pinopodes. Pinopodes are progesterone-dependent organelles, appearing as apical cellular protrusions that become visible between days 20 and 21 of the natural menstrual cycle, as shown by scanning electron microscopy in sequential endometrial biopsies. 50 The cellular and molecular functions of pinopodes in humans are still unclear; however, tracer experiments in the rat and mouse have shown that these structures perform pinocytosis. They may be also prevent the cilia from sweeping off the blastocyst and may promote withdrawal of uterine fluid, facilitating adhesion of the blastocyst to molecules of the pinopodes, drawing the uterus closely around the embryo. 51, 52 The disappearance of pinopodes was shown to occur at the time of downregulation of the progesterone receptors. 53 Pinopodes last for less than 2 days in all cases, and the timing of their formation depends both on the hormone treatment applied and the patient’s individual response in an IVF cycle. On average, they form on day 20 to 21 in a natural cycle, day 19 to 20 in controlled ovarian stimulation, and day 21 to 22 in a hormone-controlled cycle. Such short duration and discrete timing of the window of receptivity could significantly affect the outcome of ART. Pinopode appearance, loss of steroid receptors, and maximal expression of α v β 3 integrin, osteopontin, and leukemia inhibitory factor (LIF) and receptor have been demonstrated in the same biopsy, showing a consistent association of pinopode appearance and other receptivity changes. 54

Adhesion Molecules

Integrins
Attachment of the embryo may involve temporary adhesion between exposed surface receptors and ligands on the embryonic and endometrial epithelium. The role of integrins in the processes of adhesion and migration makes them attractive potential participants in the complex events of embryo implantation and placentation. Integrins are glycoproteins that serve as cell-surface receptors for the extracellular matrix and connect extracellular cell adhesion proteins to cytoskeletal components. Certain integrin subunits appear to be regulated within the cyclic endometrium, 55 and specific alterations were shown in the preimplantation period, suggesting a possible role for α 4 , β 3 , and α v β 3 subunits in the establishment of endometrial receptivity. The apical surface of the luminal epithelium expresses the α v β 3 integrin, which localizes on the pinopodes. The loss of α v β 3 is highly associated with endometriosis, retarded endometrial development, and infertility. 45, 55 The α v β 3 integrin recognizes extracellular matrix ligands that contain the 3-amino acid sequence that has been implicated in trophoblast attachment and outgrowth, and blockade of either the integrin or its ligand-binding sequence reduces implantation in the mouse. 56, 57 Osteopontin is an endometrial glycoprotein that is recognized by α v β 3 integrin and is present in glandular and luminal epithelium and appears approximately 7days after ovulation on pinopodes. 45

Trophinin
Trophinin is an intrinsic membrane protein that is important in trophoblast cell adhesion; bystin and tastin are cytoplasmic proteins that associate with trophinin by presumably forming active adhesion machinery. 58 Both trophoblasts and endometrial epithelial cells express trophinin, which mediates apical cell adhesion through homophilic trophinin-trophinin binding. The expression patterns of these molecules are suggestive of their involvement in the initial blastocyst attachment to the uterus as well as in subsequent placental development.

Mucins
Mucins are glycosylated molecules found on a great number of tissues, including the endometrial epithelial cells. Large-molecular-weight mucin glycoproteins, including MUC1 through MUC7 and the sialomucin ASGP are present at the apical surface of the uterine epithelium and are secreted into gland lumens. These mucins appear to protect the mucosal surface from infection and the degrading action of enzymes. MUC1, the prominent component, may also play a role in forming a protective barrier in the upper genital tract, preventing infections, and modulating sperm access, but the actual role of MUC1 in human implantation remains to be determined. 59 MUC1 has been shown experimentally to inhibit cell-to-cell interactions by steric hindrance of binding interactions mediated by receptors, including integrins. These mucins represent a barrier to embryo attachment and their expression persists in the human uterus during the proposed receptive phase. Pinopodes may function to elevate the implantation surface toward the embryo, free of antiadhesion MUC1. It is also possible that mucin loss is localized to the implantation sites in humans. 59, 60

Cytokines and Growth Factors
The endometrium of most species is now recognized as an important site of production of cytokines and their receptors. The cellular origin of the cytokines varies, but many predominate in the uterine glandular or luminal epithelium or in the decidualized stromal cells. 61 Cytokines and growth factors are cell-derived polypeptides and proteins that have the capacity to bind to specific cell-surface receptors and may act as potent intercellular signals, regulating functions of endometrial cells. They regulate cell proliferation, differentiation, and apoptosis by autocrine, paracrine, and endocrine mechanisms. 62, 63 Cytokines produced by the uterine mucosa and the embryo may play a role in maternal–embryonic interaction, enhancing endometrial receptivity by controlling the expression of adhesion and antiadhesion proteins. 64

Interleukins
There is evidence that the interleukin-1 (IL-1) system is important to endometrial and embryonic cross-talk during human implantation. 65 The IL-1 receptor is expressed in the endometrium of various species; antagonizing the biologic effects of IL-1 leads to implantation failure in mice. This has been shown to be due to an endometrial rather than an embryonic effect. IL-1 has a possible influence on other systems involved in embryonic implantation, including invasion and angiogenesis, therefore suggesting a role for this cytokine family during early embryonic development. 66 Members of the IL-6 family of cytokines include LIF, IL-6, IL-11, cardiotrophin, ciliary neurotropic growth factor, oncostatin M, and the recently discovered cardiotropin-like cytokine novel neurotrophin-1. Gene targeting in mice provided the first indication of a role for the IL-6 family of cytokines in implantation with the generation of mice with a null mutation of the gene encoding LIF. LIF-null female mice were infertile because of failure of blastocyst implantation. More recently, IL-11 signaling has been shown to be required for the uterine decidualization response. 67

Vascular Endothelial Growth Factor (VEGF)
VEGF has been shown to increase the proliferative ability of vascular endothelial cells in vitro by acting as a highly specific mitogen for these cell types. After the embryo invades the maternal endometrium, its development is characterized by a dramatic growth of blood vessels coincident with decidualization, development of vascular membranes, and placenta formation. 66 Insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1) is highly expressed in the rat uterus around the time of implantation and modulates the proliferation of rat uterine cells and their production of prostacyclin (PGI 2 ) during the peri-implantation period. Furthermore, IGFBP-rP1 significantly stimulated PGI 2 synthesis and cyclo-oxygenase-2 mRNA expression in myometrial cells, both of which are essential molecules for successful implantation. 68 Known biologic effects of the epidermal growth factor (EGF), VEGF, and fibroblast growth factor in the oviduct and endometrium during the estrous cycle and early pregnancy in pigs are related to cellular differentiation and angiogenesis. This suggests their involvement in the transformation of the endometrium into a decidua, subsequently leading to successful establishment of pregnancy. 69

Prostaglandins
Prostaglandins, either maternally or embryo derived, have been thought to be involved in the initial phase of implantation. The principal role of prostaglandins may be to create a mild inflammatory reaction and increase vascular permeability in the endometrium during implantation. 70 Cyclo-oxygenase (COX) is the rate-limiting enzyme in the synthesis of prostaglandins and exists in two isoforms: COX-1 and COX-2. COX-1-deficient mice are fertile; mice lacking COX-2 are infertile due to both anovulation and impaired implantation. 71 The candidate prostaglandins that participate in these processes and their mechanism of action remain undefined. Using COX-2-deficient mice, it has been demonstrated that COX-2-derived prostacyclin is the primary prostaglandin that is essential for implantation and decidualization. 72 Immunostaining for COX-1 found it to be present mainly in the glandular and luminal epithelium; COX-2 was localized to the luminal epithelium and perivascular cells. Treatment with mifepristone significantly reduced the expression of COX-1 in glandular epithelium and COX-2 in luminal epithelium and has been shown to reduce immunostaining for 15-hydroxyprostaglandin dehydrogenase within endometrial glands. 73

Human Chorionic Gonadotropin
Recent evidence suggests that human chorionic gonadotropin (hCG), in addition to its well-known endocrine effects on the corpus luteum, may act as a growth and differentiation factor during pregnancy. According to experimental results, its mode of action may be divided into three sequential phases. During the first phase, which begins at the blastocyst stage and lasts until hCG is seen in the serum, hCG acts preferentially in a juxtacrine manner. Administration of hCG may provoke profound effects on paracrine parameters of differentiation and implantation. VEGF, which is important for neoangiogenesis, is stimulated by hCG, suggesting a role for hCG in the control of endometrial vascularization and placentation. The second, endocrine, phase of hCG action is marked by the appearance of hCG in the maternal serum. Rising systemic hCG levels cause a very rapid elevation of serum progesterone, reflecting the rescue of the corpus luteum. Other endocrine functions of hCG include its intrinsic thyrotropic activity as well as modulation of fetal testicular, ovarian, and adrenal function. The third phase may be characterized by the expression of full-length hCG/LH receptors on the trophoblasts themselves. hCG seems to have a variety of local and systemic functions in and outside the embryo–endometrial microenvironment. 74

HOX Genes
HOX genes are highly evolutionarily conserved and act as regulators of embryonic morphogenesis and differentiation. Mammalian species have at least 39 HOX genes arranged in four clusters, termed HOXA, HOXB, HOXC, and HOXD. 75 specific HOXA genes are important in the development of the müllerian tract. HOXA-10 and HOXA-11 expression rises during the menstrual cycle, increasing dramatically in the midluteal phase, the time of implantation. Recently, studies using targeted mutagenesis have revealed that mice homozygous for HOXA-10 deficiency show implantation failure and embryonic resorption in the early postimplantation period. 76 The regulation of a HOX gene by sex steroids may provide a mechanism to allow differential HOX gene expression in the reproductive tract. Estrogen and progesterone each upregulate HOX-10 expression. The sex steroid regulation pattern of expression is consistent with HOX genes having a role in implantation. HOX genes are necessary for implantation; they activate the endometrial downstream target genes in each menstrual cycle that are necessary for implantation. 75

INVASION AND PLACENTATION

Endometrial Decidualization
To protect the mother from the attack of invasive trophoblasts migrating toward the uterine spiral arteries, and in response to ovarian hormones, the endometrial stroma transforms itself into a dense cellular matrix known as the decidua. 77 During the process of decidualization, the fibroblast-like mesenchymal cells in the uterine stroma differentiate to epitheloid-like cells. This morphologic change in humans is initiated in the luteal phase under the influence of estradiol, progesterone, and relaxin. The other changes include appearance within the tissue of a specialized lymphocyte subset characterized by an abundant expression of CD56. These cells have been defined in both women and rodents as members of the natural killer (NK) cell lineage and are called uterine (u) NK cells. 78 In women, the cells are also called decidual CD56 bright cells; a term previously used in mice was granulated metrial gland cells. 79 These CD56-positive cells comprise over 90% of the leukocyte population at the time of implantation. The precise function of CD56 cells in the developing decidua is unknown, although they have been proposed to be important in implantation and pregnancy maintenance. 80 The mechanism is unclear; however, recognition by NK cells and their receptors and receptor inhibitors of HLA class I molecules, specifically HLA-G on trophoblasts, has been proposed to protect the implanting blastocyst from NK cell lysis. 81 Dysregulation of these potentially important events may be involved in either failed implantation or pregnancy failure. Decidual cytokine levels have been characterized primarily at the mRNA level. Among the many genes upregulated during the implantation window, the most prominent include IL-1, CSF, LIF, EGF, and TBF-β. 61, 64 The secretion of these factors can be modified by gonadal steroids and the blastocyst itself. The blastocyst expresses receptors for these same factors, providing a communicative link from maternal tissue to embryo.
The sequence of biochemical and molecular events associated with decidualization is not completely understood. In the baboon, the sequential changes during this period in vivo are characterized by the downregulation of β-smooth actin followed by induction of COX-2 at the implantation site and the expression of insulin growth factor binding protein-1 (IGFBP-1). IGFBP-1 is the predominant protein in decidualized cells and is considered to be a biochemical marker of decidualization. In addition IL-1β, as a possible conceptus-mediated factor, can induce IGFBP-1 expression in the presence of hormones after 3 days of incubation. Current data suggest that IL-1β can activate multiple signaling pathways that either positively (no exogenous cAMP) or negatively (in presence of exogenous cAMP) regulate IGFBP-1 gene expression and decidualization in vitro. Signaling pathways activated by IL-1β following 10 minutes of stimulation result in the phosphorylation of mitogen-activated protein kinase (MAPK, specifically p38 MAPK) and also lead to NF-κB activation. The expression of COX-2 and matrix metalloproteinase-3 (MMP-3) genes follows after 4 to 6 hours. The steroid hormones, particularly progesterone, which are critical for IGFBP-1 expression, modulate the activity of IL-1β by downregulating MMP-3 activity. IL-1β-induced MMP-3 may upregulate IGFBP-1 by initiation of cytoskeletal reorganization through degradation of extracellular matrix. 82

Trophoblast Invasion
During the invasive phase of implantation, several events occur, including cytotrophoblast adhesion to the extracellular matrix via cell adhesion molecules, local extracellular matrix proteolysis by MMPs, cellular migration, and inhibition of these processes. 63 It is difficult to obtain human material in early gestation; therefore, much of our understanding of the earliest phases of trophoblast invasion has been extrapolated from monkey material. 83 Examination of monkey implantation sites has revealed that trophoblasts begin to migrate down into the maternal spiral arteries as early as 10 days after fertilization, and at 14 days, many of the spiral arteries beneath the conceptus are totally occluded. 84 Cytotrophoblastic cells are derived from the trophectodermal cells of the blastocyst and represent a heterogeneous population during early pregnancy. Cytotrophoblastic cells follow one of two existing differentiation pathways: Villous cytotrophoblastic cells (vCTBs) form a monolayer of polarized epithelial stem cells that proliferate and fuse to form syncytiotrophoblasts covering the entire surface of the vCTBs. Cytotrophoblastic cells can also break through the syncytium at selected sites (anchoring villi) to form multilayered columns of nonpolarized cytotrophoblastic cells. These motile and highly invasive extravillous cytotrophoblastic cells (evCTBs) are found as cytokeratin-positive cells in the decidua, the intima of the uterine spiral arteries, and the proximal third of the myometrium. 85
Trophoblast invasion, like tumor invasion, is due to the active secretion of proteolytic enzymes capable of digesting the different extracellular matrix of the host’s tissues. Serine proteases, cathepsins, and metalloproteinases have been implicated in invasive processes. 86 MMPs, also called matrixins, form a family of at least 17 human zinc-dependent endopeptidases collectively capable of degrading essentially all components of the extracellular matrix. According to their substrate specificity and structure, members of the MMP gene family can be classified into four subgroups: gelatinases (digest type IV collagen, the major constituent of basement membranes and denatured collagen), collagenases (digest types I, II, III, VII, and X collagens), stromelysins (digest type IV, V, and VII collagens as well as laminin, fibronectin, elastin, proteoglycans, and gelatin), and membrane-type MMPs. The substrate of the membrane metalloproteinases (MMP-14, MMP-15, MMP-16) is essentially proMMP-2, and these enzymes allow activation of proMMP-2 at the cell surface on the invasive front. 87 They are thus appropriately designed for digesting the collagens of the extracellular matrix of the interstitium. Several enzymes are capable of activating the promatrixins, the most well-known being plasmin. The activity of MMPs in the extracellular space is specifically inhibited by tissue inhibitor of metalloproteinases (TIMP), which binds to the highly conserved zinc-binding site of active MMPs at molar equivalence. 85 TIMP and broad-spectrum protease inhibitors (such as α 2 -macroglobulin) of primarily decidual and cytotrophoblast origin are important in limiting cytotrophoblast invasion and in endometrial stromal receptivity. 63 Further penetration and survival depend on factors that are capable of suppressing the maternal immune response to paternal antigens. During implantation, the uterine decidua is invaded by extravillous trophoblast cells that express an unusual combination of HLA class I molecules HLA-C, HLA-E, and HLA-G. The decidua is infiltrated by a population of NK cells that are particularly numerous in the decidua basalis at the implantation site, where they come into close contact with invading extravillous trophoblast cells. It is believed that interaction between these NK cells and extravillous trophoblast cells provides the controlling influence for implantation. On the other hand the increased activity of NK cells has been related to an increased risk of recurrent miscarriage. It seems that the endometrial-derived glycoprotein with immunosuppressive properties, glycodelin-A, may locally inhibit NK cell activity and contribute to protection of the embryonic semi-allograft. Glycodelin-A is the major progesterone-regulated glycoprotein secreted into the uterine luminal cavity by secretory/decidualized endometrial glands. 88

Angiogenesis and Vasculogenesis
After the embryo invades the maternal endometrium, its development is characterized by a dramatic growth of blood vessels coincident with decidualization, development of vascular membranes, and placenta formation. These active processes involve both angiogenesis, the growth of blood vessels by sprouting from a preexisting endothelium, and vasculogenesis, the in situ formation of primordial vessels from hemangioblasts. 89 Human placenta is a rich source of angiogenic substances, and these may play an important role in the regulation of placental vessel formation as well as in maternal vascular adaptation to pregnancy. VEGF expression has been described in villous trophoblasts and fetal macrophages within villous stroma. VEGF increase the proliferative ability of the vascular endothelial cells in vitro by acting as a highly specific mitogen for this cell type. It induces angiogenesis and increases the permeability of blood vessels. 90
Another member of the VEGF family, placental growth factor, has been also detected in the villous trophoblast and in the tunica media of larger stem vessels. 91 Basic fibroblast growth factor is expressed in the villous trophoblast and can be detected in the conditioned medium of villous tissue explants. 92 Proliferin and proliferin-related protein, two prolactin-related peptides expressed by trophoblast giant cells, have been identified as potent regulators of angiogenesis in the murine placenta, although the role of these placental proteins has yet to be evaluated in humans. 93 Another recently described angiogenic factor, leptin, is expressed in the placenta and serum during pregnancy and may play a role in vascular development during pregnancy. 94

CLINICAL RELEVANCE
Clinical evidence indicates the existence in humans of a narrow window of uterine receptivity, which opens during the midluteal phase. At the same time, formation of pinopodes on the apical membrane of the endometrial epithelial cells occurs. Detection of pinopodes may be extremely useful for the assessment of receptivity in women with history of multiple implantation failure. 95 Also it is important to note that in normal fertile women, pinopodes are present on day 6 to 8 postovulation, whereas pinopode formation is observed 1 to 2 days earlier in patients undergoing controlled ovarian hyperstimulation. In addition to the individual variations in the timing of pinopode formation, the number of pinopodes also varies between patients, some showing plentiful and others only sparse pinopodes. More interestingly, the number of pinopodes correlated strongly with implantation success after embryo transfer in a subsequent cycle. 54
The contemporary approach to ovarian stimulation for IVF treatment results in supraphysiologic concentrations of steroids during the follicular and luteal phases of the menstrual cycle. These sex steroids act directly and indirectly to mature the endometrium, influencing receptivity for implantation. Altered endometrial development has been demonstrated with most of the protocols used for ovarian stimulation. 96 A series of studies shows that high estradiol concentrations on the day of hCG administration are detrimental to uterine receptivity. 97 Corpus luteum function is distinctly abnormal in IVF cycles, and therefore luteal support is widely used. Since the introduction of gonadotropin-releasing hormone agonists, it has been observed that pregnancy rates increased significantly with luteal-phase supplementation. 98 The development of an endometrium receptive to embryo implantation is a complex process that may be altered by inappropriate exposure to sex steroids in terms of timing, duration, and magnitude. 99
Embryo transfer has received little clinical attention and has been, until recently, the most inefficient step in IVF. Factors such as contamination of the catheter tip with cervical bacteria, stimulation of uterine contractions during the procedure, the type of catheter, ultrasound guidance during the transfer, and the position of the embryos in the uterine cavity appear to influence implantation rates. Easy and atraumatic transfer is essential for successful implantation; the embryos need to be placed in the middle of the cavity, away from the fundus. Standardization of the transcervical intrauterine transfer of embryos in a large randomized study is needed before definitive conclusions can be drawn. 100
There is an evident decline of female fertility with age. This decline is mainly due to increased risk of pregnancy termination either after conception or after embryo implantation. The observation that very good clinical results could be obtained in aged female patients using oocytes donated by younger women has led to the conclusion that the reproductive aging of the women is solely due to a decline of oocyte quality rather than uterine aging and lack of receptivity. Despite some macroscopic possible causes that may play a role in the reduction of age-related endometrial receptivity, many endometrial factors possibly related to its receptivity need to be further studied, especially in older women. 101


PEARLS: FERTILIZATION

• The natural fertilization process in vivo is not yet completely understood; however, the recent advances in assisted reproductive technologies have provided new avenues to the understanding of normal fertilization.
• Gametes in mammals move through the reproductive tracts of both the male and female until they arrive close to each other in the ampulla of the fallopian tube. The subsequent interaction between the two gametes requires several steps that result in formation of a zygote, including binding and penetration of the spermatozoon to the oocyte coat, oocyte activation, formation of male and female pronuclei, and initiation of cell division and early development.
• Cellular and molecular events after IVF and ICSI differ from those that occur in vivo in several aspects. Despite these differences, normal IVF and ICSI embryos have been produced and have led to numerous healthy births after transfer.
• Complete fertilization failure is a disappointing experience in IVF cycles. Little is known about the mechanisms of fertilization failure following the IVF or ICSI procedures, but they can be categorized as impaired sperm capacitation and acrosome reaction; failure to attach, bind, and penetrate into the zona pellucida; or failure in signal transduction, oocyte activation, pronuclei formation and apposition, and arrest in the metaphase plate of the embryo’s first mitotic division.


PEARLS: IMPLANTATION

• The early embryo remains in the tubal ampulla for approximately 80 hours after ovulation, travels through the isthmus for approximately 10 hours, and then enters the uterus in the 8-cell or 16-cell stage (morula). The embryo develops into a blastocyst while freely floating in the endometrial cavity 90 to 150 hours after conception.
• Implantation begins with hatching from the zona pellucida 2 to 3 days after the morula enters the uterine cavity.
• The endometrium is receptive to the blastocyst during a very short and precise period of time; this occurs as a result of complex action of sex hormones, especially progesterone; embryonic regulatory mechanisms that induce reciprocal embryo–uterine interactions; and the activity of several cytokines and growth factors.
• Once in contact with the endometrium, the blastocyst becomes surrounded by an outer layer of syncytiotrophoblast, a multinucleate mass with no discernible cell boundaries, and an inner layer of cytotrophoblast made up of individual cells. The syncytiotrophoblast erodes the endometrium, and the blastocyst implants into it. This process is mediated by cytokines and envolves adhesion molecules, mainly integrins.
• The inability of the embryo to properly implant in the uterus is a significant cause of pregnancy failure after in vitro or in vivo fertilization.

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Chapter 7 Surgical Anatomy of the Abdomen and Pelvis

Tommaso Falcone, Richard L. Drake, William W. Hurd

INTRODUCTION
The goal of reproductive surgery is to treat medical conditions using procedures and techniques least likely to compromise fertility. The goal, whenever possible, is to restore normal anatomy. To reach this goal and minimize complications, every reproductive surgeon requires a thorough knowledge of pelvic anatomy. Knowledge of abdominal wall anatomy is important to ensure safe placement of primary and secondary laparoscopic ports. This chapter reviews practical surgical anatomy of the anterior abdominal wall and pelvis important to gynecologists regardless of surgical approach.

ANTERIOR ABDOMINAL WALL
The abdominal wall is made up of four structural layers beneath the skin: (1) subcutaneous tissue and superficial fascial layers, (2) muscles and transversalis fascia, (3) deep fascia of the rectus sheath and the extraperitoneal fascia, and (4) parietal peritoneum ( Fig. 7-1 ). Interspersed among these layers are several important nerves and blood vessels.

Figure 7-1 The round ligament is seen entering the deep inguinal ring to course in the inguinal canal and exit through the superficial inguinal ring. The ureter is in close proximity to the ovarian vessels. At laparoscopy, the ureter is seen anterior to the internal iliac for a short distance.
(From Drake RL, Vogl W, Mitchell AWM: Gray’s Anatomy for Students. Philadelphia, Elsevier, 2005, p 410, Fig. 5-50.)

Subcutaneous Tissue
The layer collectively referred to by most surgeons as subcutaneous tissue is actually made up of superficial (Camper’s) and deep (Scarpa’s) fascia. These loose, fatty connective tissue layers contain the superficial abdominal wall vessels and are the most common site of postoperative wound infections.

Muscles and Transversalis Fascia
The abdominal wall is made up of five pairs of muscles. In the midline, the rectus abdominis muscles extend along the whole length of the front of the abdomen from the xiphoid process and costal cartilages of the fifth through the seventh ribs to the pubic crest and pubic symphysis. This broad strap muscle is divided into four segments by three fibrous intersections attached to the anterior, but not the posterior, rectus sheath. This allows the inferior (deep) epigastric vessels to pass along the posterior surface of the muscle without encountering a barrier.
The pyramidalis muscle is a small triangular muscle that lies in front of the rectus abdominis at the lower part of the abdomen and is contained within its fascial sheath. It arises from the front of the pubic symphysis and the anterior pubic ligament bilaterally and inserts into the linea alba, between the umbilicus and pubic symphysis. This muscle is commonly absent on one or both sides.
There are three sets of lateral muscles. The external oblique muscle is the most external and arises from the lower eight ribs. The fibers run downward and forward to form aponeuroses that extend anteriorly. Aponeuroses are fibrous membranes resembling flattened tendons that bind muscles to each other or bones. Beneath the external oblique muscle, the internal oblique muscle arises from the lumbar fascia, the iliac crest, and the lateral two thirds of the inguinal ligament and runs upward and forward to form aponeuroses. The most internal of the lateral muscles is the transversus abdominis muscle. It arises from the lateral third of the inguinal ligament, from the anterior three fourths of the iliac crest, from the costal cartilages of the sixth through eighth ribs, interdigitating with the diaphragm, and from the lumbodorsal fascia and ends in front in a broad aponeurosis. Deep to the transversus abdominis muscle is a continuous layer of specialized investing fascia that lines the abdominal cavity and continues into the pelvic cavity, the transversalis fascia.

Deep Fascia of the Rectus Sheath and Extraperitoneal Fascia
The rectus abdominis muscle is enclosed anteriorly and posteriorly by fascia known as the rectus sheath. This sheath is formed from fusion of the aponeuroses of all three lateral abdominal muscles. These aponeuroses fuse lateral to the rectus abdominis muscles as the linea semilunares and again in the midline as the linea alba, which extends from the xiphoid process to the pubic symphysis. The arcuate line is a tranverse line midway between the umbilicus and pubic symphsis. Above this line, the aponeuroses of the lateral muscles split to enclose the rectus muscle both anterior and posterior; below this line these aponeuroses all pass anterior to the rectus muscle. Inferiorly, the aponeuroses of the external oblique inserts into the anterior superior iliac spine and stretches over to the pubic tubercle, forming the inguinal ligament.
The inguinal canal is about 4 cm long and runs parallel to the inguinal ligament. The inguinal canal has an anterior wall formed by the aponeurosis of the external oblique, an inferior wall formed by the inguinal ligament, a superior wall formed by arching fibers of the internal oblique and transversus abdominis muscles, and a posterior wall formed by the transversalis fascia. A defect, or more precisely a tubular evagination, of the transversalis fascia forms the deep inguinal ring, through which the round ligament enters the inguinal canal. This ring lies midway between the anterior superior iliac spine and the pubic symphysis. Medial to the deep inguinal ring are the inferior epigastric vessels. The opening of the aponeurosis of the external oblique superior to the pubic tubercle is the superficial inguinal ring. Through it the round ligament, the terminal part of the ilioinguinal nerve, and the genital branch of the genitofemoral nerve exit the inguinal canal (see Fig. 7-1 ).
Deep to the transversalis fascia and the rectus sheath is a layer of connective tissue separating the transversalis fascia from the parietal peritoneum, the extraperitoneal fascia. This layer contains varying amounts of fat, lines the abdominal cavity and is continuous with a similar layer lining the pelvic cavity. Viscera in the extraperitoneal fascia are referred to as retroperitoneal.

Parietal Peritoneum
The parietal peritoneum is a one-cell thick membrane that lines the abdominal cavity and in certain places reflects inward to form a double layer of peritoneum called the mesentery.

Nerves
There are four categories of nerves that supply the anterior abdominal wall, each of which contain both motor and sensory fibers. The thoracoabdominal nerves originate from T7–T11, travel anteroinferiorly between the internal oblique and transversus abdominis muscles, and have the following distribution:
• T7–T9: superior to the umbilicus
• T10: at level of umbilicus
• T11: inferior to umbilicus
The subcostal nerves originate from T12 and travel anteroinferiorly between the internal oblique and transversus abdominis muscles to innervate the abdominal wall inferior to the umbilicus.
The iliohypogastric nerve and ilioinguinal nerve both originate from L1. Like the thoracoabdominal and subcostal nerves, these nerves begin their course anteroinferiorly between the internal oblique and transversus abdominis muscles. However, at the anterior superior iliac spine, they both pierce the internal oblique muscle to travel between the internal and external oblique muscles. The iliohypogastric nerves innervate the abdominal wall lateral and inferior to the umbilicus. The ilioinguinal nerve enters the inguinal canal and emerges from the superficial inguinal ring and is sensory to the labia majora, inner thigh, and groin.
These nerves are particularly at risk in lower abdominal incisions, which are the most common causes of abdominal wall pain as a result of nerve entrapment by suture or scar tissue. 1 For this reason, knowledge of the course of the ilioinguinal and iliohypogastric nerves in the anterior abdominal wall can help avoid injury during laparotomy and laparoscopic surgery. Data from cadaveric studies suggest that injury to these nerves can be minimized during laparoscopy by making transverse skin incisions and placing laparoscopic trocars at or above the level of the anterior superior iliac spine. 2 In cases of chronic abdominal pain caused by these nerves, an injection of local anesthetic at a site approximately 3 cm medial to the anterior superior iliac spine will often provide relief.

Blood Vessels
The major vessels in the anterior abdominal wall can be divided into deep and superficial vessels ( Fig. 7-2 ). 3 The superficial vessels include the superficial epigastric and the superficial circumflex iliac vessels. These vessels are branches of the femoral artery and vein. They course bilaterally through the subcutaneous tissue of the abdominal wall, branching as they proceed toward the head of the patient.

Figure 7-2 Anterior abdominal wall blood vessels.
(Modified from Hurd WW, Bude RO, DeLancey JOL, Newman JS: The location of abdominal wall blood vessels in relationship to abdominal landmarks apparent at laparoscopy. Am J Obstet Gynecol 171:642–646, 1994.)
To avoid vessel injuries, these superficial vessels can often be seen before secondary laparscopic port placement by transillumination of the abdominal wall using the intra-abdominal laparoscopic light source. 3 Injury to these vessels during trocar placement can result in a palpable hematoma that will be found to be located anterior to the fascia on computed tomography (CT) scan. 4 In unusual cases, the hematoma can dissect down into the labia majora.
The deep vessels consist of the inferior epigastric artery and vein, which are also bilateral. These vessels originate from the external iliac artery and vein and course along the peritoneum until they dive deeply into the rectus abdominis muscles midway between the pubic symphysis and the umbilicus. The inferior epigastric vessels are the lateral border of an inguinal triangle called Hesselbach’s triangle. This triangle is bound medially by the rectus abdominis muscle and inferiorly by the inguinal ligament.
The course of the inferior epigastric vessels can often be visualized at laparoscopy as the lateral umbilical fold because of the absence of the posterior rectus sheath below the arcuate line ( Fig. 7-3 ). 5 Injury to these vessels can result in life-threatening hemorrhage that must be quickly controlled by occluding the lacerated vessels with electrosurgery or precisely placed sutures.

Figure 7-3 Laparoscopic view of peritoneal landmarks and inferior epigastric vessels. The left round ligament is seen entering the deep inguinal ring. Medial to the round ligament at the deep inguinal ring are the inferior epigastric vessels. The peritoneal fold is referred to as the lateral umbilical fold. The peritoneal fold seen medial to the vessels has the obliterated umbilical artery and is referred to as the medial umbilical fold.
If these vessels cannot be visualized (usually because of excess tissue), trocars should be place approximately 8 cm lateral to the midline and 8 cm above the pubic symphysis. 3 On the right side of the abdomen, this point approximates McBurney’s point, located one-third the distance from the anterior superior iliac spine to the umbilicus. The corresponding point on left is sometimes referred to as Hurd’s point.

Peritoneal Landmarks

Peritoneal Folds
Several useful landmarks can be used to guide the laparoscopic surgeon to avoid injury to important retroperitoneal structures. Two midline and two bilateral pairs of peritoneal folds can usually be seen on the anterior abdominal wall at laparoscopy ( Fig. 7-4 ). The falciform ligament, which is the remnant of the ventral mesentery and contains the obliterated umbilical vein in its free edge, can be seen in the midline above the umbilicus extending to the liver. The median umbilical fold, which contains the urachus, can usually be seen in the midline below the umbilicus extending to the bladder. Although the urachus normally closes before birth, it should be avoided during secondary trocar placement, both because it can be difficult to penetrate and in rare cases can remain patent to the bladder.

Figure 7-4 The peritoneal reflections onto the urachus (median umbilical ligament), umbilical artery (medial umbilical ligament), and inferior epigastric vessels (lateral umbilical fold) are seen. The two important peritoneal pouches are seen between pelvic organs.
(From Drake RL, Vogl W, Mitchell AWM: Gray’s Anatomy for Students. Philadelphia, Elsevier, 2005, p 416, Fig. 5-58A.)
On each side of the urachus lie the medial umbilical folds. These landmarks contain the obliterated umbilical arteries and extend from the umbilicus to the anterior division of the internal iliac artery. Lateral to these, the lateral umbilical folds can be seen in 82% of patients. 5 These are the most important structures to the laparoscopist, because they contain the inferior epigastric vessels and knowing their location can help the laparoscopist avoid injury to these large vessels during placement of secondary laparoscopic ports.
Peritoneal pouches normally exist between the pelvic organs (see Fig. 7-4 ). The vesico-uterine pouch is located anteriorly between the uterus and bladder. The ventral margin of the bladder can be visualized in approximately half of patients behind the anterior abdominal wall peritoneum and is important for secondary trocar placement, especially after previous abdominal surgery. 5 The dorsal bladder margin can often be visualized on the anterior uterus and is used as a landmark during dissections during hysterectomy.
The recto-uterine pouch (pouch of Douglas) is located between the anterior surface of the rectum and the posterior surface of the vagina, cervix, and uterus. Endometriosis often involves the recto-uterine pouch and in severe cases, completely obliterates it. Inferiorly, an extraperitoneal fascial plane called the rectovaginal septum extends from the recto-uterine pouch to the perineal body. It lies between the posterior wall of the vagina and anterior wall of the rectum, and when involved with endometriosis can be felt on pelvic examination as nodularity.

UPPER ABDOMEN
In the past, the reproductive surgeon had little need to understand the anatomy of the upper abdomen. However, for laparoscopists who utilize the left upper quadrant primary trocar placement for laparoscopy, an understanding of the anatomy of this area becomes important.
For the left upper quadrant technique, the Verres needle and primary trocar are placed into the abdomen 2 cm below the subcostal arch at the midclavicular line. It is important to know what anatomic structures lie close to this area to avoid injury during insertion of the primary cannula. The anatomic structures at risk of injury in this area include (from posterior to anterior) the spleen, splenic flexure of the colon, stomach, and left lobe of the liver. Although relatively few series using the left upper quadrant approach have been reported, it appears that the colon might be the organ at greatest risk of injury using this technique. 6 Table 7-1 lists the common body structures and distances from the left upper quadrant point from CT scan data. 7

Table 7-1 Distance from Left Upper Quadrant Insertion Site to Common Structures

POSTERIOR ABDOMINAL WALL AND PELVIC SIDEWALLS
Structures of the posterior abdominal wall anterior to the vertebral column and the pelvic sidewalls are of interest to the reproductive surgeon for several diverse reasons. First, retroperitoneal dissection can be required in these areas during some gynecologic procedures, such as treatment of deep endometriosis and removal of pelvic masses adherent to the peritoneum. Secondly, an understanding of the course of retroperitoneal nerves is a useful reminder to the surgeon to be aware of the position of self-retaining retractor blades during laparotomy, because permanent nerve injury can result from prolonged pressure on these structures. Finally, with the use of closed techniques for primary laparoscopic trocar placement, injury to the retroperitoneal structures can occur, and knowledge of this anatomy is essential for effective and expedient management.
Like the anterior abdominal wall, the posterior abdominal wall and pelvic sidewalls contain multiple, well-defined muscles. Other important structures include several large nerves and blood vessels and the ureters.

Muscles
Several muscles of importance in the posterior abdominal wall lateral to the vertebral column can be seen lateral to the pelvic inlet. The diaphragm forms the roof of the abdomen and extends down to form the most superior aspect of the posterior abdominal wall. The psoas major muscle runs longitudinally from the transverse processes of the upper lumbar vertebrae to the lesser trochanter of the femur and makes up a large part of the posterior and medial wall. The tendon of the psoas minor muscle can be seen anterior to the psoas major muscle during dissection near the external iliac vessels. The quadratus lumborus muscle runs lateral and posterior to the psoas major muscle from the transverse process of the lumbar vertebrae and ribs to the iliac crest. The iliacus muscle spans the iliac fossa. Finally, the piriformis muscle begins at the anterior surface of the sacrum and passes through the greater sciatic foramen to attach to the greater trochanter of the femur. It lies immediately beneath the internal iliac vessels.

Nerves
Multiple nerves enter or transverse the pelvic sidewalls. Deep nerves of the pelvis, such as the superior and inferior gluteal nerves, supply several of the pelvic muscles but are not visible during reproductive surgery. Likewise, the obturator nerve traverses the pelvis. The obturator nerve originates at spinal cord levels L2–L4 and descends in the psoas major muscle until the pelvic brim, when it emerges medially to lie on the obturator internus muscle lateral to the internal iliac artery and its branches ( Figs. 7-5 and 7-6 ). It descends on the obturator internus muscle to enter the obturator canal and exits in the thigh. It is sensory to the medial side of the thigh and motor to the muscles in the medial compartment of the thigh (the adductor muscles). It can easily be seen during pelvic sidewall dissections for endometriosis or lymph node dissection.

Figure 7-5 The obturator nerve is seen coursing over the pelvic brim when it emerges medially to lie on the obturator internus muscle lateral to the internal iliac artery and its branches. The anterior trunk of the internal iliac artery gives off several branches: a common trunk that typically divides into the umbilical artery and uterine artery, the obturator artery, the internal pudendal artery, the vaginal artery, and the middle rectal artery. The anterior trunk terminates as the inferior gluteal artery. The umbilical artery gives off the superior vesical artery.
(From Drake RL, Vogl W, Mitchell AWM: Gray’s Anatomy for Students. Philadelphia Elsevier, 2005, p 428, Fig. 5-64.)

Figure 7-6 Laparoscopic view. Two branches of the anterior trunk of the left internal iliac artery, the uterine artery and the umbilical artery, are seen medial to the left obturator nerve. The nerve lies on the obturator internus muscle. The relationship of the vessels with the ureter (retracted by the instrument) is seen.
The genitofemoral nerve (from spinal cord levels L1 and L2) lies on the anterior surface of the psoas muscle ( Fig. 7-7 ). It has two branches, the femoral and genital. The femoral branch enters the thigh under the inguinal ligament, and the genital branch enters the inguinal canal. The genitofemoral nerve is sensory to the skin over the anterior surface of the thigh. Injury to this nerve is seen after appendectomy or when the fold of peritoneum from the sigmoid colon to the psoas muscle is incised.

Figure 7-7 The peritoneum over the left external iliac artery and psoas muscle has been removed. The instrument is on the left external iliac artery and points to the left genitofemoral nerve that is splitting into the two branches. Lateral to the nerve is the left psoas minor tendon.
The femoral nerve (spinal cord levels L2–L4) is not usually seen during pelvic surgery but may be injured by compression at laparotomy. It is a branch of the lumbar plexus and descends within the substance of the psoas major muscle, emerging at its lower lateral border. The nerve continues between the psoas and iliacus muscles and then passes posterior to the inguinal ligament to supply the skin of the anterior thigh region as well as many of the muscles in the anterior compartment of the thigh. Prolonged pressure on the psoas muscle may cause temporary or permanent damage to the femoral nerve. For this reason, caution must be taken when using self-retaining retractors to make certain that the lateral blades do not put pressure on the pelvic sidewalls.
The sacral and coccygeal nerve plexuses are located anterior to the piriformis muscle beneath the branches of the internal iliac artery. The most important nerves in this area are the sciatic and pudendal nerves. The sciatic nerve (from spinal cord levels L4–S3) lies anterior to the piriformis muscle and exits the pelvic cavity through the greater sciatic foramen inferior to the muscle. Anterior to the sciatic nerve are many branches of the internal iliac artery. The pudendal nerve (from spinal cord levels S2–S4) is also found anterior to the piriformis muscle and exits the pelvic cavity inferior to the piriformis muscle through the greater sciatic foramen. It courses around the sacrospinous ligament and ischial spine, through the lesser sciatic foramen, and into the perineum. Endometriosis may involve the sciatic nerve at this level and cause pain syndrome related to the course of this nerve.

Blood Vessels
The major blood vessels are perhaps the most important structures of the pelvis (see Fig. 7-5 ). Successful pelvic surgery requires a thorough understanding of their anatomy. The aorta bifurcates at the level of L4 into the left and right common iliac arteries. The common iliac artery passes laterally, anterior to the common iliac vein to the pelvic brim. At the lower border of L5, the common iliac artery divides into internal and external iliac branches. The external iliac artery gives off only two branches, the inferior epigastric artery and the deep circumflex iliac artery, and then, after passing under the inguinal ligament, becomes the femoral artery, which is the primary blood supply to the lower limb.
The internal iliac artery supplies all of the organs within the pelvis and sends branches out through the greater sciatic foramen to supply the gluteal muscles. A branch also exists through the greater sciatic foramen and re-enters the lesser sciatic foramen to supply the perineum. After passing over the pelvic brim, the internal iliac arteries divide into anterior and posterior trunks. The posterior trunk consists of three branches: the ilolumbar artery, the lateral sacral artery, and the superior gluteal artery. These vessels are closely related to the nerve plexus on the piriformis muscle. The superior gluteal artery is the largest branch of the internal iliac artery and supplies the muscles and skin of the gluteal region. Accidental occlusion of this artery during uterine fibroid embolization can result in necrosis of the gluteal region.
The anterior trunk of the internal iliac artery has several branches that are routinely seen during laparoscopic surgery. The obliterated umbilical artery is a fibrous band seen on the anterior abdominal wall as the medial umbilical fold and can be traced back to its juncture with the internal iliac artery. At this point, the uterine artery can be identified as it arises from the medial surface of the internal iliac. The superior vesical artery also arises near this point and courses medially and inferiorly to supply the superior aspect of the bladder and the distal ureter.
The uterine artery is of particular importance to the reproductive surgeon. After the umbilical artery emerges from the anterior trunk of the internal iliac artery, the uterine artery runs parallel to and then crosses over the ureter at the level of the uterine cervix in the base of the broad ligament.
The vaginal artery most commonly originates from the uterine artery, but may arise independently from the internal iliac artery. The uterine, vaginal, and ovarian arteries anastomose with each other, with branches of the internal pudendal artery, and with the corresponding contralateral arteries.
The other important branches of the anterior trunk of the internal iliac artery are the obturator artery, which courses laterally and anteriorly toward the obturator canal, and the middle rectal, internal pudendal, and inferior gluteal arteries. The inferior gluteal artery is the largest branch of the anterior trunk.

Ureters
The ureters measure approximately 25 to 30 cm from kidney to bladder and are occasionally duplicated on one or both sides for all or part of this course. Their abdominal segment lies behind the parietal peritoneum on the medial part of the psoas major muscle and crosses the common iliac vessels at the level of their bifurcation at the pelvic brim.
The pelvic segment of the ureters runs down on the lateral wall of the pelvic cavity, along the anterior border of the greater sciatic notch immediately beneath the parietal peritoneum. Here the ureters form the posterior boundary of the ovarian fossae. They then run medial and forward between the two layers of the broad ligaments. It is here that the ureters run parallel to the uterine arteries for about 2.5 cm before crossing under the arteries and ascending on the lateral aspects of the uterine cervix and upper part of the vagina to reach the bladder.
The average distance between the ureters and cervix is greater than 2 cm. 8 However, the reproductive surgeon should remember that this distance can be less than 0.5 cm in approximately 10% of women, which explains in part the relatively common occurrence of ureteral injury during hysterectomy ( Fig. 7-8 ).

Figure 7-8 Schematic representation of the proximity of the ureter to the cervix. A, Sagittal view of pelvic organs. B, Transverse view of pelvic organs at the level of the cervix.
(From Hurd WW, Chee SS, Gallagher KL, et al: Location of the ureters in relation to the uterine cervix by computed tomography. Am J Obstet Gynecol 184:336–339, 2001.)
On reaching the base of the bladder, the ureters run obliquely through the wall for approximately 2 cm and open by slitlike orifices at the angles of the trigone. When the bladder is distended during cystoscopy, these orifices are approximately 5 cm apart. When the bladder is emptied, this distance decreases by 50%.

MUSCLES OF THE PELVIC FLOOR
The pelvic floor consists of two closely related muscle layers: the pelvic diaphragm and the deep perineal pouch. Damage to these muscles or their innervation is common during vaginal delivery. A thorough understanding of this anatomy is imperative for anyone doing vaginal surgery for prolapse or urinary incontinence.

Pelvic Diaphragm
The pelvic diaphragm forms the muscular floor of the pelvis and is made up of the levator ani and coccygeus muscles that are all attached to the inner surface of the minor pelvis ( Fig. 7-9 ). 9 The levator ani is composed of three muscles. The innermost puborectalis muscle is attached to the pubic symphysis and encircles the rectum. The thicker, more medial pubococcygeus muscle runs from the pubic symphysis to the coccyx. This muscle is attached laterally to the obturator internus muscle by a thickened band of dense connective tissue called the arcus tendineus. The fusion of these bilateral muscles in the midline is called the levator plate and forms a shelf on which the pelvic organs rest. When the body is in a standing position, the levator plate is horizontal and supports the rectum and upper two thirds of the vagina above it. The thinner, more lateral iliococcygeus muscle runs from the arcus tendineus and ischial spine to the coccyx. The posterolateral margin of the pelvic diaphragm is the coccygeus muscle, which extends from the ischial spine to the coccyx and lower sacrum.

Figure 7-9 Laparoscopic view of the levator ani muscle. The rectum has been transected. The hysterectomy is complete and sutures are seen in the vagina. The ureters are seen laterally entering the pelvis. Most of the levator ani muscle seen here is iliococcygeus. The fibers are directed toward the sacrum and coccyx as well as toward the opposite muscle to form a midline raphe.
Weakness or damage to parts of the pelvic diaphragm may loosen the sling behind the anorectum and cause the levator plate to sag. Women with prolapse have been shown to have an enlarged urogenital hiatus on clinical examination. 10

Perineal Membrane, Deep and Superficial Perineal Pouches
The deep perineal pouch bridges the gap between the inferior pubic rami and the perineal body. It closes the urogenital hiatus and has a sphincter-like effect at the distal vagina. It provides structural support for the distal urethra and also contributes to continence because it is attached to periurethral striated muscles. Knowledge of the anatomy of this area is necessary for surgical procedures that involve creation of a neovagina in patients with androgen insensitivity syndrome or correction of ambiguous genitalia.
The perineal membrane and the deep perineal pouch are musculofascial structures located over the anterior pelvic outlet below the pelvic diaphragm. The perineal membrane is a fascial layer attached to the ischiopubic rami. There are openings in this fascia for the urethra and vagina. The deep perineal pouch is located superior to this membrane and has several contiguous striated muscles: the compressor urethrae and sphincter urethrovaginalis muscles, the external urethral sphincter, and the deep transverse perineal muscles.
The superficial perineal pouch contains several muscles, the ischiocavernosus, bulbospongiosis, and superficial transverse perineal muscles as well as the greater vestibular glands (Bartholin’s glands).

PRESACRAL SPACE
Reproductive surgeons are sometimes required to operate in the presacral space when performing a presacral neurectomy to control chronic pelvic pain. In this space lies the superior hypogastric plexus that contains both sympathetic and parasympathetic nerve fibers. In reality this plexus is more prelumbar than presacral. The superior hypogastric plexus divides into two branches at about the level of the bifurcation of the aorta near L4. In addition to visceral afferent fibers from the uterus, these nerve trunks also carry parasympathetic fibers that stimulate bladder contraction and modulate activity of the distal colon. It is for this reason that presacral neurectomy can result in both bladder and bowel dysfunction.
Another risk of presacral neurectomy is blood vessel injury. The left common iliac vein makes up the left superior margin of the presacral area as it lies inferior to the bifurcation of the aorta and anterior to the vertebra; it can be injured during this dissection. The median sacral vessels originate from the aortic bifurcation and descend in the midline into the presacral area. Bleeding from these vessels and the presacral venous plexus can be potentially life-threatening.

PELVIC VISCERA
The pelvic viscera includes the rectum, urinary organs, and the internal genitalia, including the vagina, uterus, uterine tubes, and ovaries.

The Rectum
The rectum of the adult is approximately 12 to 15 cm in length. It begins at the rectosigmoid junction in front of S3 and ends at the anorectal junction at the level of the tip of the coccyx. Unlike the colon, it lacks taeniae coli, haustra, and omental appendices.
The upper third of the rectum projects into the peritoneal cavity anteriorly and laterally. At its midpoint, the anterior peritoneum of the rectum extends onto the fornix of the vagina to form the rectouterine pouch. The distal one third is completely retroperitoneal.
The blood supply to the rectum includes the superior rectal artery, a branch from the inferior mesenteric artery; the middle rectal artery, a branch from the internal iliac artery; and the inferior rectal artery, a branch from the internal pudendal artery. The rectum is innervated by sympathetic fibers from the inferior hypogastric plexus, parasympathetic fibers that leave the sacral spinal cord (S2–S4) as pelvic splanchnic nerves and enter the inferior hypogastric plexus before passing to the rectum, and sensory fibers from the rectum that join the inferior hypogastric plexus.

Vagina
The vagina is a musculomembranous sheath 7 to 9 cm in length that extends anteroinferiorly from the uterine cervix to the vestibule. Because the cervix enters the vagina along the anterior wall, the posterior wall is about 1 cm longer than the anterior wall. Anterior and posterior to the cervix are vaginal fornices. Intraperitoneally, the vagina is separated from the rectum by the rectouterine pouch and from the bladder by the vesicouterine pouch (see Fig. 7-4 ).
The vagina receives its blood supply from the uterine, vaginal, and middle rectal arteries, which form an extensive anastomotic network. Innervation of the vagina is from the inferior hypogastric plexus and pelvic splanchnic nerves.

Uterus
The uterus is a fibromuscular organ that varies in size and weight according to life stage and parity. The uterus is divided into a body (corpus) and cervix. The part of the body of the uterus above the uterine tube is called the fundus. In a nulliparous woman the uterus is about 8 cm in length from the external os to the fundus, 5 cm in width at the fundus, and 2 to 3 cm deep anteroposteriorly. It weighs between 40 and 100 g.
The uterine cavity is triangular in shape, and the anterior and posterior walls approximate each other. Because the uterine cavity is a virtual space, ultrasound assessment of the uterus will only show a cavity when it is distended with fluid.
The length of the uterine cavity changes according to life stage, in part because of the profound effect of hormones on uterine size. In premenarchal girls the uterine length from the external os to the fundus is 1 to 3 cm. The cervix occupies two thirds of the uterine length in prepubertal girls and only one third after menarche. During the reproductive years, the expected length of the cavity is about 6 to 7 cm, which is important to remember when introducing instruments into the uterine cavity during endometrial biopsy, hysteroscopy, or embryo transfer. In postmenopausal women the uterus decreases to about 3 to 5 cm long.
The uterine wall is made up of three layers: endometrium, myometrium, and serosa. The endometrium is hormonally responsive and varies in thickness from 5 to 15 mm during a single menstrual cycle during the reproductive years. After menopause, it should be less than 5 mm in thickness as measured by ultrasound.
The myometrium consists of three to four indistinct layers of smooth muscle approximately 1 to 1.25 cm thick during the reproductive years. It is thickest in the midportion of the uterine corpus and thinnest near the opening of the uterine tube. The outermost layer consists of mostly longitudinal fibers. The middle layer consists of circular and oblique fibers and includes most of the blood vessels and loose connective tissue. The innermost layer is composed of mostly longitudinal fibers that are continuous with the uterine tubes and ligaments that surround the uterus.
The uterine blood supply is from the uterine artery, a branch of the internal iliac artery. The uterine artery courses along the lateral border of the uterus and forms extensive anastomoses with the ovarian and vaginal arteries. Approximately 6 to 10 blood vessels penetrate the uterus from the uterine artery and run circumferentially as the anterior and posterior arcuate arteries. Vessels from the sides anastomose in the midline, but no large blood vessels are found in the midline. Doppler studies have shown that the arcuate arteries are at the periphery of the uterus. The arcuate arteries supply the radial branches that penetrate deeply the myometrium to reach the endometrium. These radial arteries give rise to the spiral arteries of the endometrium.
Incisions on the uterus made during myomectomy should take into consideration the anatomy of the uterus. An incision made vertically in the midline is less likely to divide the large uterine vessels on the lateral border of the uterus. However, vertical uterine incisions may transect several arcuate arteries.

Uterine Tubes
The uterine tubes are contained within the uppermost margin of the broad ligament and measure about 10 to 12 cm (see Fig. 7-1 ). Each tube is divided into several distinct anatomic segments: intramural (or interstitial), isthmic, ampullary, and infundibulum. The internal diameter of the tube ranges from less than 1 mm at the intramural portion to up to 10 mm at the infundibulum.
The intramural portion of the tube is usually 1.5 cm long and may be tortuous. The tubal ostium where it opens into the uterine cavity can be seen at hysteroscopy at each angle of the fundus.
The isthmic portion is often the site of tubal ligation and therefore the site of tubal anastomosis. The lumen is approximately 0.5 mm. Although magnification is required for anastomosis, the subsequent pregnancy rates are highest for procedures done in this area.
The ampulla comprises two thirds of the length of the tube and has four to five longitudinal ridges. It is the site of fertilization and thus the most common site of ectopic pregnancy. Although the lumen is much larger here, the pregnancy rates after anastomosis are lower.
The infundibulum is the distal section of the tube. It is not attached by peritoneum and is open to the peritoneal cavity. Its delicate finger-like projections are called fimbriae.
The tubal wall consists of three layers: mucosa, muscularis, and serosa. The muscular layer has a somewhat indistinct external longitudinal layer and inner circular layer of smooth muscle. The intramural portion has no anatomic sphincter, but closure is sometimes seen during hysteroscopy. The blood supply to the tube runs in the mesosalpinx and consists of branches from the uterine and ovarian arteries.

Ovaries
The ovaries are ovoid structures suspended from the posterior aspect of the broad ligament by the mesovarium (see Fig. 7-1 ). This fold of peritoneum contains an extensive complex of blood vessels. The infundibulopelvic ligament (suspensory ligament of the ovary) enters the ovary along its superior pole and carries the ovarian vessels, lymphatics, and nerves. These vessels are in close proximity to the ureter at the pelvic brim (see Fig. 7-1 ). The ovarian ligament is located on the inferior pole of the ovary. The ovary is attached to the broad ligament by the mesovarium. It has a rich vascular supply. An arcade of vessels is formed from the anastomoses of the uterine and ovarian vessels. Theses vessels, called helicine because of their highly coiled structure, course through the mesovarium into the medulla. Veins then drain the medulla from a plexus seen in the mesovarium. Dissection around this area during a lysis of tubal adhesions or cystectomy should be performed with great care to avoid bleeding.
The ovarian volume is dependent on both the life stage and the germ cell population. During the reproductive years, ovaries without functional cysts weigh approximately 20 to 35 g and measure approximately 4 × 2 × 1 cm. Before menarche and after menopause, the ovaries will be smaller.

PELVIC FASCIAS AND LIGAMENTS
The pelvic viscera are attached to the pelvic sidewalls by (1) peritoneal folds, (2) condensations of pelvic fascia, and (3) remnants of embryonic structures. In the past, it was believed that these structures supported the uterus and prevented genital prolapse. Thus many of these structures were designated as ligaments. However, it has become clear that none of these structures provide significant support for the pelvic viscera in the presence of pelvic floor defects.

Peritoneal Folds
The broad ligament is a double-layered transverse fold of peritoneum that encloses the uterus and uterine tubes and extends to the lateral walls and pelvic floor (see Figs. 7-1 and 7-4 ). It is made up of the mesometrium lateral to the uterus that encloses the uterine vessels and the ureters, the mesovarium that attaches the ovary to the broad ligament posteriorly, and the mesosalpinx that connects the uterine tube near the base of the mesovarium.
The suspensory ligament of the ovary, often referred to as the infundibulopelvic ligament, is a lateral continuation of the broad ligament beyond the uterine tube that connects the ovary to the pelvic brim and contains the ovarian artery and vein. The ureter crosses beneath these vessels near the point where this ligament joins the pelvic sidewall.

Fascial Ligaments
The cardinal ligament is a connective tissue condensation lateral to the cervix that is bordered anteriorly and posteriorly by the leaves of the broad ligament and inferiorly by the pelvic floor. It is continuous with the paracervix, a dense fibrous sheath around the lower cervix and the upper vagina, and is attached to the pelvic walls laterally. It usually contains major branches of the uterine vessels.
The uterosacral ligament is a connective tissue band arising from the posterior paracervix that fans out to attach to the sacrum and rectum. It contains some smooth muscle fibers.

Gubernacular Ligaments
The ovarian ligament runs in the broad ligament and attaches the medial pole of the ovary to the posterior-lateral uterine surface beneath the uterine tube. The round ligament is the forward continuation of the uterine attachment of the ovarian ligament. This fibromuscular structure traverses the deep inguinal ring and terminates as fibrous strands in the connective tissue of the labium majus.

REFERENCES

1 Stultz P. Peripheral nerve injuries resulting from common surgical procedures in the lower abdomen. Arch Surg . 1982;117:324-327.
2 Whiteside J, Barber M, Walters M, Falcone T. Anatomy of ilioinguinal and iliohypogastric nerves in relation to trocar placement and low transverse incisions. Am J Obstet Gynecol . 2003;189:1574-1578.
3 Hurd WW, Bude RO, DeLancey JOL, Newman JS. The location of abdominal wall blood vessels in relationship to abdominal landmarks apparent at laparoscopy. Am J Obstet Gynecol . 1994;171:642-646.
4 Hurd WW, Pearl ML, DeLancey JO, Quint EH, et al. Laparoscopic injury of abdominal wall blood vessels: A report of three cases. Obstet Gynecol . 1993;82:673-676.
5 Hurd WW, Amesse LS, Gruber JS, et al. Visualization of the epigastric vessels and bladder before laparoscopic trocar placement. Fertil Steril . 2003;80:209-212.
6 Patsner B. Laparoscopy using the left upper quadrant approach. J Am Assoc Gynecol Laparosc . 1999;6:323-325.
7 Tulikangas PK, Nicklas A, Falcone T, Price LL. Anatomy of the left upper quadrant for cannula insertion. J Am Assoc Gynecol Laparosc . 2000;7:211-214.
8 Hurd WW, Chee SS, Gallagher KL, et al. Location of the ureters in relation to the uterine cervix by computed tomography. Am J Obstet Gynecol . 2001;184:336-339.
9 Herschorn S. Female pelvic floor anatomy: The pelvic floor, supporting structures, and pelvic organs. Rev Urol . 2004;6(Suppl 5):S2-S10.
10 Delancey JO, Hurd WW. Size of the urogenital hiatus in the levator ani muscles in normal women and women with pelvic organ prolapse. Obstet Gynecol . 1998;91:364-368.
Chapter 8 Pathology of Reproductive Endocrine Disorders

Charles V. Biscotti, Tommaso Falcone

INTRODUCTION
An appreciation of the relationship between form and function is important for understanding of female reproduction. An awareness of histologic changes associated with both the normal ovulatory cycle and reproductive diseases allows the physician a better understanding of pathophysiology and potential treatment.
This chapter begins with an examination of the histologic changes in the endometrium associated with a normal ovulatory cycle. This is followed by an illustrated survey of common gynecologic diseases of the reproductive organs that are most likely to present to the reproductive surgeon.

UTERUS

Endometrium
The endometrium is functionally divided into two layers: the basalis and the functionalis. Both layers are composed of stroma and glands. The stroma is composed of stromal cells, vessels, and white blood cells thought to be lymphocytes or macrophages. Cyclic changes occur in both endometrial glands and stroma in response to a changing endocrine environment.

Endometrial Dating
Endometrial dating refers to the determination of how closely the histologic characteristics of the endometrium match what is expected on the corresponding day of the menstrual cycle. In the past, this approach was one of the standard tests in an investigation of causes of infertility and pregnancy loss. However, the accuracy of this test has been questioned because abnormal results can be observed in cycles that eventually prove to result in a viable pregnancy.
Endometrial dating can be performed both before and after ovulation. Preovulatory phase (proliferative phase) endometrial dating is described as menstrual days, early follicular, midfollicular, and late follicular, but is not very precise. Postovulatory phase (secretory phase) endometrial dating has become the standard and is usually reported within a 2-day range, although the accuracy of this dating methodology is the subject of some debate.
For secretory phase endometrial dating, ovulation is used as the primary reference point. Originally, ovulation was assumed to occur on day 14 of the menstrual cycle and the days after ovulation numbered accordingly. Some pathologists refer to ovulation as “day 0” and report the postovulation day as the number of days after ovulation.
Most clinicians perform endometrial biopsy in the midluteal phase at about the time implantation is thought to occur. However, the original reports on secretory endometrial dating recommend a biopsy 3 days before the expected menses. Histologic criteria are then used to determine where the endometrial response would be in relation to ovulation ( Table 8-1 and Figs. 8-1 and 8-2 ).
Table 8-1 Criteria for Histologic Dating Gland mitosis Pseudostratification of nuclei Subnuclear vacuoles Edema Stromal mitosis Decidual reaction in stroma Leukocyte infiltration Secretion

Figure 8-1 Midluteal phase endometrium has early spiral arteriolar formation (center) arising in edematous stroma. Endometrial glands are tortuous with basal nuclei, absence of mitotic activity, and intraluminal secretions.

Figure 8-2 Late luteal phase endometrium has large areas of coalescent predecidua and a conspicuous lymphoid infiltrate, giving the stroma a dimorphic appearance.
Several assumptions in the original concept of dating the endometrium besides the assumption of ovulation on day 14 increased the variation found with this method. For example, another assumption was that the length of the luteal phase is 14 days. 1 In reality, there is a normal variation in luteal phase length of several days. In addition, the original description fixed the time of ovulation with the onset of the period after the biopsy. The onset of ovulation can be more accurately determined using current modalities, such as determination of the midcycle urinary luteinizing hormone surge or ultrasonic identification of the collapse of a follicle. The accuracy of the former is approximately 85%; of the latter, 95%. Additional intrinsic inaccuracies of dating resulted from intraobserver and interobserver variation. This variation is typically about 2 days. For these reasons, a 2-day difference between the histologic estimation and the actual interval since ovulation is considered within normal limits.
Recently a detailed analysis on endometrial dating demonstrated that the histologic criteria used are not as temporally distinct as originally thought, and thus do not provide an accurate method to detect a luteal phase defect. In one study, approximately 20% of fertile couples had a delay of more than 2 days. 2 A between-cycle variation of more than 2 days was found in 30% to 60% of patients if the biopsy was performed between day 6 and day 13 after ovulation.

Endometrial Response to Exogenous Hormones
The endometrium responds to exogenous hormones with specific morphologic changes. With oral contraceptives, the progestin effect dominates because progestins decrease estrogen receptors. As a result, endometrial glands atrophy over time. The appearance of the stroma depends on the dose of progestin. Typically, a weak pseudodecidual response will occur ( Fig. 8-3 ).

Figure 8-3 The microscopic appearance of the endometrium exposed to oral contraceptives varies, depending on the dose of progestin. Typically, stroma having a weak progestin effect surrounds simple, straight, widely spaced, and inactive-appearing endometrial glands.
Exposure to progestins only (e.g., medroxyprogesterone acetate) will show similar gland pattern but usually a more prominent stromal pseudodecidual response due to higher progestin dose. The selective estrogen receptor modulator tamoxifen is anti-estrogenic at some sites but has a weak estrogenic agonist effect on the endometrium. The endometrium usually atrophies, but variable hyperplasia and, rarely, adenocarcinoma can occur due to the weak estrogen agonist effect.

Endometritis
Chronic endometritis is sometimes demonstrated in endometrial biopsies for infertility or recurrent pregnancy loss. This is characterized by infiltration of plasma cells ( Fig. 8-4 ). Chronic pelvic inflammatory disease, intrauterine devices, and postabortion complications are also associated with chronic endometritis. Although rare in the United States, endometrial tuberculosis may sometimes be found during a biopsy for infertility and is characterized by granulomas ( Fig. 8-5 ).

Figure 8-4 The presence of plasma cells defines chronic endometritis. Typically, the endometrium resembles a proliferative-type endometrium. The stroma often has a spindled appearance.

Figure 8-5 Tuberculosis endometritis causes granulomatous inflammation. In reproductive age women, granulomas tend to be cellular but not markedly necrotic. The case illustrated here has a well-formed nonnecrotizing granuloma (right) adjacent to an endometrial gland. In postmenopausal patients, tuberculous endometritis causes striking necrotizing granulomatous endometritis.

Abnormal Uterine Bleeding
The biopsy of an endometrium for dysfunctional uterine bleeding in women may show a range of abnormalities of the endometrium ( Fig. 8-6 ). In the reproductive age group organic lesions such as polyps and leiomyomas or a normal endometrium are often found. Pregnancy-related endometrial changes or premalignant or malignant changes may be found in this age group.

Figure 8-6 This endometrial biopsy specimen illustrates gland–stromal asynchrony. The endometrial glands have consistent and conspicuous subnuclear vacuoles corresponding to approximately postovulatory day 3 (cycle day 17). In contrast, the endometrial stroma has striking stromal edema corresponding to approximately postovulatory day 8 (cycle day 22). This appearance of gland–stromal asynchrony is the most common secretory phase morphologic abnormality.
In adolescent and perimenopausal age patients, anovulatory cycles are one of the most common causes of abnormal uterine bleeding. During anovulatory cycles, the endometrium is proliferative and may exhibit some breakdown. Thrombosis of vascular sinusoids also commonly occurs. Anovulatory cycles create a milieu of unopposed estrogen; therefore, hyperplasia and carcinoma can result.

Pregnancy-related Endometrial Changes
Early pregnancy, both intrauterine and ectopic, is characterized by hypersecretory endometrium ( Fig. 8-7 ). However, hypersecretory endometrium is not specific for pregnancy, and similar changes can be seen with persistent corpus luteum cyst, double corpora lutea, or rarely as a drug effect. By the end of the first trimester, endometrial glands involute and the stroma shows a prominent decidual reaction ( Fig. 8-8 ). Other histologic changes associated with gestation include the Arias-Stella reaction ( Fig. 8-9 ) and optically clear nuclei ( Fig. 8-10 ).

Figure 8-7 Hypersecretory endometrium has closely packed endometrial glands with marked intraglandular budding (ferning) and ongoing glandular secretions with secretory vacuoles. Hypersecretory endometrium characterizes early pregnancy; however, this change is not specific for gestation.

Figure 8-8 By the end of the first trimester, the hypersecretory glandular changes resolve. Subsequently, gestational endometrium has mostly simple widely spaced endometrial glands, separated by stroma with a prominent progestin effect (true decidual change), as seen in this example.

Figure 8-9 This photomicrograph illustrates the Arias-Stella reaction. The Arias-Stella reaction is a cellular and nuclear change characterized by nuclear enlargement and hyperchromatisma resulting from polyploidy. The enlarged hyperchromatic nuclei are typically arranged in an apical portion of the endometrial glandular cells. This nuclear change can be mistaken for malignancy; however, the Arias-Stella reaction, unlike adenocarcinoma, typically affects only scattered cells within the endometrial glands and mitotic figures are absent or at most infrequent.

Figure 8-10 This endometrial gland has the optically clear nuclear appearance seen with pregnancy (center top).
The Arias-Stella reaction refers to cytonuclear changes, including voluminous, vacuolated cytoplasms surrounding enlarged, hyperchromatic, polyploid nuclei that includes massively enlarged forms. The Arias-Stella reaction occurs almost exclusively in gestational endometrium associated with pregnancy or gestational trophoblastic disease; however, this reaction can rarely occur as a response to hormonal therapy in nonpregnant patients. The optically clear nuclei associated with gestation can resemble herpes virus inclusions.

Endometriosis
Endometriosis is defined as the presence of endometrial glands and stroma at an extrauterine site. Although most commonly located in the pelvis, endometriosis can occur at a remarkable variety of extrapelvic sites ( Table 8-2 ). Endometriotic lesions can invade neural tissue. The stromal component can undergo smooth muscle metaplasia. Hyperplasia of smooth muscle, especially the bowel, is characteristic.
Table 8-2 Reported Sites of Endometriosis Implants
Pelvic Locations
Ovaries, uterine ligaments, vagina, cervix
Pelvic peritoneum
Rectovaginal septum
Urinary system
Bladder
Ureters
Gastrointestinal
Rectum
Rectosigmoid colon
Nerves
Sciatic nerve Lymph nodes
Extrapelvic Locations
Gastrointestinal, including colon, appendix, and small bowel
Inguinal hernia sacs
Skin of umbilicus and inguinal area
Surgical incisions, especially following cesarean delivery
Lung and pleura
Kidney
Liver
Endometriotic lesions are sensitive to sex hormones and undergo cyclic changes. The appearance of endometriosis on visual inspection at laparoscopy or laparotomy is quite variable and ranges from blue nodules to red or white lesions. The microscopic appearance also varies, depending on secondary changes of fibrosis and hemorrhage ( Figs. 8-11 and 8-12 ). Endometriotic lesions should be differentiated microscopically from the benign serous tubules of endosalpingiosis.

Figure 8-11 In this example, endometriosis, characterized by well-preserved endometrial glands and stroma, involves the muscularis of the uterine tube.

Figure 8-12 Over time, secondary changes predominate due to repeated cycles of hemorrhage and fibrosis. In this example, dense fibrous tissue borders a more recent hemorrhage (top).
Importantly, endometriosis must also be distinguished from well-differentiated adenocarcinoma. The latter lacks endometrial stroma and has malignant cellular features. However, endometriosis can rarely undergo malignant change.
Estimates for malignant transformation of endometriosis range from 0.3% to 0.8% for surgical series of ovarian endometriosis. 3 In one study almost 80% of endometriosis-associated malignancies arose in the ovary. 4 Malignancies arising in endometriosis are usually adenocarcinomas, almost always endometroid or clear cell type. Rarely sarcomas such as endometrial stromal sarcoma or müllerian adenosarcoma occur. 5

Adenomyosis
Adenomyosis refers to endometrial glands and stroma located deep within the myometrium. The etiology and pathogenesis of adenomyosis are poorly understood. Smooth muscle cell hyperplasia and hypertrophy are commonly found. Many cases of adenomyosis are associated with leiomyomas, and it is difficult to ascertain which pathology is responsible for the symptoms. Histologically, thickened rims of myometrial smooth muscle surround benign endometrial glands and stroma ( Fig. 8-13 ).

Figure 8-13 Benign endometrial glands and stroma deep within the myometrium characterize adenomyosis. These adenomyotic foci are often associated with hyperplasia of the adjacent smooth muscle.

Leiomyoma
Leiomyomas are benign monoclonal tumors of smooth muscle. These tumors commonly have cytogenetic abnormalities, such as rearrangements of 6p or deletion of 7q, which are identified in 40% of uterine leiomyomas. The myometrial tissue adjacent to the leiomyoma is cytogenetically normal.
Grossly, leiomyomas appear as circumscribed masses of whorled, bulging, rubbery, usually light tan masses ( Fig. 8-14 ). Microscopically, fascicles of bland spindled cells characterize leiomyomas. Mitotic figures are infrequent, and the nuclei are uniform ( Fig. 8-15 ).

Figure 8-14 This leiomyoma well illustrates the usual gross appearance, characterized by sharp circumscription and a gray tan homogeneous cross-section.

Figure 8-15 Microscopically, leiomyomas have fascicles of uniform spindle-shaped cells with elongated, spindle-shaped, blunt-ended nuclei. Note the lack of nuclear pleomorphism. Importantly, necrosis, other than infarct-type necrosis, is absent and mitoses are infrequent.
Leiomyoma variants ( Table 8-3 ) include cellular, apoplectic, bizarre ( Fig. 8-16 ), mitotically active, hyaline, cystic, myxoid, and infarcted. All of these variant appearances can occur in benign tumors, but some, especially myxoid, mitotically active, and bizarre, should prompt a thorough search to exclude malignancy.
Table 8-3 Leiomyoma Variants Cellular Apoplectic Bizarre Mitotically active Hyaline degeneration Cystic Myxoid Infarcted

Figure 8-16 Markedly enlarged, pleomorphic, and hyperchromatic nuclei characterize bizarre (symplastic) leiomyomas. This nuclear change has not been associated with malignant behavior in the absence of other features of malignancy.
A common treatment for leiomyomas is gonadotropin-releasing hormone (GnRH) agonists. A 50% decrease in uterine volume is seen over a 3-month period, although leiomyomas rapidly enlarge again after a GnRH agonist is stopped. The effects of these drugs on leiomyoma histopathology are unpredictable. Both hyaline degeneration and decreased vessel lumen size have been observed.
Another treatment modality for symptomatic uterine leiomyomas is arterial embolization. Histopathologic evaluation of the effects of this treatment are obtained from leiomyomas expelled from the uterus after therapy or from hysterectomy specimens. The primary features are thrombosis, necrosis, and presence of embolic foreign material ( Fig. 8-17 ). Acute features are coagulation necrosis and acute inflammation. Chronic features are hyaline necrosis and dystrophic calcification.

Figure 8-17 This infarcted leiomyoma has well-developed coagulation necrosis characterized by loss of nuclei and prominent cytoplasmic eosinophilia.

Leiomyosarcoma
Leiomyosarcomas are encountered in the treatment of presumed leiomyomas in approximately 0.1% of cases. Leiomyosarcomas typically have tumor cell necrosis ( Fig. 8-18 ), many mitoses (almost always >4 mitotic figures/10 high magnification fields [hpf] and usually ≥10/10 hpf), and nuclear atypia ( Fig. 8-19 ). These histologic features are reflected grossly as soft, variegated tumors with necrosis ( Fig. 8-20 ). Table 8-4 summarizes the principal predictors of malignancy.

Figure 8-18 A uterine leiomyosarcoma with prominent tumor cell necrosis. Tumor cell necrosis, as illustrated here, is an important diagnostic criterion for leiomyosarcoma. Tumor cell necrosis is characterized by a sharply circumscribed focus of necrosis lacking a marginal healing zone.

Figure 8-19 This uterine leiomyosarcoma has striking nuclear atypia and many mitotic figures. An atypical mitotic figure is present here (center left).

Figure 8-20 Grossly, leiomyosarcomas have more variable coloration than seen with benign leiomyomas. This example has multiple pale yellow foci indicative of tumor cell necrosis.
Table 8-4 Histologic Predictors of Malignancy Tumor cell necrosis Mitotic activity Nuclear atypia (pleomorphism and nuclear hyperchromatism)

FALLOPIAN TUBES

Acute Salpingitis
The tubal mucosa is arranged in longitudinal branching folds called plicae. The epithelium of the mucosa has three different cell types: secretory, ciliated, and “peg” cell (intercalated). Damage to the epithelium of the tube is associated with infertility and ectopic pregnancy.
Acute salpingitis is an ascending infection that is usually associated with a sexually transmitted disease. The lumen contains pus (pyosalpinx) and may become distended. In chronic salpingitis there is a marked fibrosis of the tubal wall, usually associated with luminal dilatation as well as tubo-ovarian adhesions ( Fig. 8-21 ). Xanthogranulomatous salpingitis is a histologic variant of chronic salpingitis resulting from necrosis and obstruction ( Fig. 8-22 ).

Figure 8-21 Grossly, chronic salpingitis manifests with thickening and gray-white discoloration of the tubal wall. This is associated with edema in this example.

Figure 8-22 Xanthogranulomatis salpingitis has a mixed chronic inflammatory infiltrate dominated by foamy hystiocytes. Xanthogranulomatis inflammation results from tissue necrosis and obstruction. This pattern of inflammation is identical to that seen with malakoplakia except for the presence of Michaelis-Gutmann bodies in malakoplakia.
Hydrosalpinx is thought to be the result of end-stage salpingitis. Grossly, the tube is dilated and often thin-walled ( Fig. 8-23 ). The lumen has a plasma transudate that is embryotoxic. Microscopically, the tubal wall is thin and fibrotic. A flattened epithelium lines the attenuated wall. However, scattered relatively normal-appearing tubal mucosal plicae usually remain.

Figure 8-23 A dilated, thin-walled uterine tube with agglutinated fimbria characterizes the gross pathology of hydrosalpinx.

Ectopic Pregnancy
Ectopic pregnancies can occur in several nonuterine locations. The most common site is the uterine tube. Hematosalpinx is the typical gross appearance. Trophoblastic tissue can grow predominantly intraluminally or invade the tubal wall ( Fig. 8-24 ).

Figure 8-24 Hematosalpinx is the most consistent gross and microscopic feature of tubal pregnancy. In this example, chorionic villi are identified within the tubal lumen (center).
Morphologically, trophoblasts and chorionic villi are usually visible at the implantation site. However, the diagnostic tissue (products of conception) may be confined to the intraluminal blood clot. Thus, the blood clot should be carefully examined in these cases.
Ovarian ectopic pregnancies account for 3% of all ectopic gestations. An ectopic pregnancy found in the ovary is believed to be the result of an aborted tubal pregnancy in many cases. In 1878, Spiegelburg suggested four criteria to distinguish a primary ovarian pregnancy from a distal tubal pregnancy that has secondarily involved the ovary: (1) the fallopian tube with its fimbriae should be intact and separate from the ovary, (2) the gestational sac should occupy the normal position of the ovary, (3) the gestational sac should be connected to the uterus by the ovarian ligament, and (4) ovarian tissue must be present in the specimen attached to the gestational sac.

Tuberculosis Salpingitis
Tuberculosis can hematogeneously spread to the pelvic organs to cause chronic salpingitis. The majority of cases of tuberculosis of the uterine tubes simultaneously involve the endometrium. Other uncommon causes of granulomatous tubal inflammation are sarcoidosis and Crohn’s disease.

OVARIES

Ovarian Cysts

Functional Cysts
The most common cysts found in reproductive age women are functional cysts such as follicular or corpus luteum cysts. These cysts are most commonly lined with granulosa cells. Some functional cysts may cause disruption of the menstrual cycle or symptoms. However, most will spontaneously regress without surgical intervention. Occasionally a corpus luteum cyst may be confused grossly at the time of surgery with an endometriotic cyst. Microscopically, fibrosis and hemorrhage around the cyst can make it difficult to differentiate these cysts from endometriomas.

Gonadal Dysgenesis
Patients with gonadal dysgenesis usually have streak gonads ( Fig. 8-25 ). Dysgenetic gonad (i.e., abnormally developed gonads) can harbor gonadoblastoma ( Fig. 8-26 ). Gonadoblastomas are mixed germ cell-sex cord stromal tumors characterized by nests of immature sex cord cells surrounding germ cells. Gonadoblastomas almost always affect dysgenetic gonads in patients with a karyotype containing Y chromosome. Importantly, gonadoblastomas can give rise to invasive germ cell neoplasms, most commonly dysgerminoma ( Fig. 8-27 ). Patients with genotypes that include a Y chromosome are at risk to develop a gonadoblastoma. In these patients the gonad should be removed.

Figure 8-25 The microscopic appearance of streak gonads varies over time. Disorganized germ cells, sex cord cells, and stroma, seen initially, give way to a nonspecific fibrous stroma. In this example of a streak gonad removed from an adult, a focus of lutein cells (top center) is surrounded by nonspecific fibrous stroma.

Figure 8-26 This gonadoblastoma is dominated by masses of dystrophic calcium associated with fibrosis. The constituent germ cells and sex cord cells are inconspicuous. Gonadoblastomas are tumors composed of nests of germ cells associated with sex cord cells. Calcification is a consistent feature. Gonadoblastomas almost always arise in dysgenetic gonads associated with a karyotype containing Y chromosome material. Gonadoblastomas can give rise to invasive malignant germ cell tumors, usually dysgerminoma.

Figure 8-27 Delicate fibrous septa containing lymphocytes separate nests of germinoma cells in this characteristic example of an ovarian dysgerminoma.

Pregnancy-related Cysts
Pregnancy causes prominent luteinization of the theca layer of atretic follicles. Multiple atretic follicles with theca luteinization are designated theca-lutein cysts. Theca-lutein cysts result from high levels of or increased sensitivity to human chorionic gonadotropin. This condition has been called hyperreactio luteinalis. Multiple theca-lutein cysts often occur with hydatidiform mole and choriocarcinoma but occasionally in other clinical settings, including normal single pregnancy. Grossly, the process is typically bilateral with greatly enlarged multicystic ovaries ( Fig. 8-28 ). Pregnancy luteomas are non-neoplastic solid masses of lutein cells occurring in pregnant patients ( Fig. 8-29 ). 6 Multiple nodules occur in half of patients, and bilaterality occurs in at least one third of cases. The nodules spontaneously regress postpartum. Pregnancy luteomas are easily confused with sex cord neoplasms.

Figure 8-28 Cystic atretic follicles with luteinization of the theca characterize theca-lutein cysts. Theca-lutein cysts can result in massive cystic enlargement of the ovaries, as seen here. Theca-lutein cysts are associated with excessive levels of human chorionic gonadotropin. Therefore, typically, theca-lutein cysts are found with hydatidiform mole (in one third to one half of cases) and choriocarcinoma. However, theca-lutein cysts do occur with other gestations, including normal single pregnancies.

Figure 8-29 As illustrated here, pregnancy luteomas contain markedly luteinized cells having abundant, usually eosinophilic cytoplasm surrounding round nuclei with conspicuous nucleoli. A follicular space is identified (center).

Polycystic Ovaries
Polycystic ovary syndrome (PCOS) is an endocrine diagnosis rather than a pathologic one. The pathologic findings include characteristic gross and microscopic features. Grossly, the ovaries are referred to as sclerocystic ( Fig. 8-30 ).

Figure 8-30 Sclerocystic ovaries result from chronic anovulation. Multiple cystic follicles underly thickened fibrotic superficial stroma. Note the absence of gross stigmata of prior ovulation, such as involuting corpora lutea.
Sclerocystic ovaries are enlarged with cysts underlying thickened fibrotic superficial stroma. Stigmata of prior ovulation are inconspicuous or absent. The cysts are relatively small, usually ranging in size from 0.5 to 1.5 cm. Histologically, luteinized follicular cells line the cysts.

Stromal Hyperthecosis
Stromal hyperthecosis is defined histologically as the presence of luteinized cells within the ovarian stroma. Stromal hyperthecosis can cause clinical signs of hyperandrogenism, and with peripheral conversion, hyperestrogenism. These patients are typically postmenopausal. Stromal hyperthecosis also occurs commonly in pregnancy. The ovaries are enlarged bilaterally with no clear appearance of cysts. Microscopically nests and individual lutein cells occur in the stroma ( Fig. 8-31 ). The associated spindle cell ovarian stroma is typically hyperplastic, characterized by increased cellularity and a loss of the normal corticomedullary demarcation.

Figure 8-31 The presence of luteinized cells within the ovarian stroma, as illustrated here, defines stromal hyperthecosis. The luteinized stromal cells have abundant cytoplasm that might stain eososinophilic or clear and vacuolated, as seen in this example. Stromal hyperthecosis commonly affects postmenopausal and pregnant patients and also causes a virilizing syndrome in younger nonpregnant patients.

Sex Cord-Stromal Tumors
Sex cord-stromal tumors represent approximately 5% of ovarian neoplasms. They are usually unilateral and occur in all age groups but are more common in postmenopausal women. Sex cord-stromal tumors often secrete hormones and are thus often associated with endocrine-related symptoms ( Table 8-5 ). Both Leydig and theca cells secrete androgens ( Table 8-6 ). They should be considered as part of the differential diagnosis of severely hyperandrogenic patients. However, many of these tumors have no endocrine manifestations.
Table 8-5 Types of Sex Cord-Stromal Tumors of the Ovary
Granulosa-stromal cell tumors
Granulosa cell tumors
Tumors of the thecoma-fibroma group Sertoli-Leydig cell tumors: Androblastomas Steroid cell tumors Sex cord tumor with annular tubules Gynandroblastoma
Table 8-6 Typical Clinical Features of Sex Cord-Stromal Tumors Can occur in any age group May be hormonally active Usually unilateral Solid or cystic solid tumors
The stroma of the embryonic gonad has the potential to differentiate into cell types that have specific endocrine functions in both the male (Sertoli and Leydig cells) and the female (granulosa and theca cells) gonad. The Sertoli and granulosa cells both secrete inhibin, which can be used as a tumor marker for these patients. Elevated inhibin levels usually precede clinical recurrence of disease by months to years.

Granulosa Cell Tumors
Granulosa cell tumors are mostly found in postmenopausal women. The symptoms are related to estrogen secretion and can include postmenopausal bleeding with endometrial pathology such as endometrial hyperplasia or carcinoma. The tumors also occur in prepubertal girls and may produce precocious sexual development. In some cases, androgen secretion results in symptoms of hyperandrogenism.
Granulosa cell tumors are usually unilateral and characteristically have both cystic and solid components ( Fig. 8-32 ). Histologically, the tumor architecture varies, but the cells often have “coffee bean” nuclei described as pale, angular, and cleaved ( Fig. 8-33 ). 7

Figure 8-32 This adult-type granulosa cell tumor has a characteristic hemorrhagic cystic and solid gross appearance.

Figure 8-33 The pale angular grooved appearance of nuclei in adult-type granulosa cell tumors is the key diagnostic criterion.
Granulosa cell tumors in prepubertal girls and young adults often have microscopic features differing from adult-type granulosa cell tumors. The differences are sufficient to permit recognition of these tumors as a distinctive subset with different behavior that is designated juvenile granulosa cell tumor. 8, 9

Thecomas
Thecomas are usually unilateral (>90%) and typically occur in postmenopausal women. Thecomas may be hormonally active tumors. Grossly, thecomas typically are yellow and have a solid uniform appearance. Histologically, plump vacuolated spindle cells predominate ( Fig. 8-34 ).

Figure 8-34 Ovarian thecomas often have a distinctive gross color, often yellowish tan, as seen in this example. Also note the homogeneous appearance and sharp circumscription.

Fibromas
Fibromas are usually unilateral tumors. Large fibromas (>6 cm) may be associated with ascites (40% of cases) or hydrothorax (Meigs’ syndrome). Other ovarian tumors may also cause these clinical signs (pseudo-Meigs’ syndrome). Fibromas are also associated with basal cell nevus syndrome (Gorlin’s syndrome). Fibromas have a uniform, pale, tan, solid gross appearance ( Fig. 8-35 ) and benign spindle cells microscopically.

Figure 8-35 In contrast to the typical yellow color of a thecoma, ovarian fibromas, as illustrated here, typically have a pale tan gross appearance.

Sertoli-Leydig Cell Tumors
Sertoli-Leydig cell tumors are hormonally active tumors that occur in reproductive age women. These tumors are mostly unilateral. These tumors are classically associated with severe masculinization, with hirsutism, balding, clitoral hypertrophy, and voice changes.
Sertoli-Leydig cell tumors have remarkably diverse histologic appearances. 10 The fundamental components are a variable spindle cell stroma and variably immature tubules. Often, an alternating hypercellular and hypocellular zonation characterizes Sertoli-Leydig cell tumors. The presence of heterologous tissue contributes to the array of microscopic appearances. Histologic grading (well, intermediate, and poorly differentiated) correlates well with survival ( Fig. 8-36 ). Other hormonally active tumors occur that have lipid-laden cells associated with hyperandrogenism and are variably referred to as steroid cell tumors .

Figure 8-36 Sertoli-Leydig cell tumors have a broad spectrum of microscopic appearances resulting in part from variable mixtures of Sertoli tubules and nonspecific cellular stroma. In this example, well-formed Sertoli tubules arise in a background of densely cellular tissue.


PEARLS

• A luteal phase endometrial biopsy lacks the accuracy required to make the diagnosis of luteal phase defect.
• Endometrial tuberculosis may be found on endometrial biopsy.
• Most common cysts found in reproductive age women are functional cysts.
• Sex cord-stromal tumors are more common in postmenopausal women.
• Fibromas can be associated with ascites or hydrothorax (Meigs’s syndrome).

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