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n i n f e r t i l i t y a n d a s s i s t e d r e p r o d u c t i o n n

Infertility and Assisted Reproduction presents, in detail, thetechniques and philosophies behind medical procedures ofinfertility and assisted reproduction. Assisted reproductivetechnology is available to two-thirds of the world’s popu-lation, and world-class experts in their field, representingresearch from 18 different countries, have contributed to thisgroundbreaking textbook. This is one of the most rapidlychanging and hotly debated fields in medicine. Different coun-tries have different restrictions on the research techniques thatcan be applied to this field, and, therefore, experts fromaround the world bring varied and unique authorities to dif-ferent subjects in reproductive technology. This compre-hensive textbook encompasses the latest research into thephysiology of reproduction, infertility evaluation and treat-ment, and assisted reproduction and concludes with perspec-tives on the ethical dilemmas faced by clinicians andprofessionals. This book is designed to be a definitive resourcefor those working in the areas of reproductive medicine theworld over.

Dr. Botros R. M. B. Rizk is Professor of Obstetrics andGynecology, Head of the Division of Reproductive Endocrinologyand Infertility, and Medical and Scientific Director of USA ARTprogram at the University of South Alabama, College ofMedicine, Alabama, USA.

Dr. Juan A. Garcia-Velasco is an Assistant Professor of Obstet-rics and Gynecology at Rey Juan Carlos University School ofMedicine, Madrid, Spain, and the Director of the InstitutoValenciano de Infertilidad in Madrid.

Dr. Hassan N. Sallam is Professor in Obstetrics and Gynecol-ogy, Vice-Dean and Director of Research in the Faculty ofMedicine, and the Director of the Suzanne Mubarak RegionalCenter for Women’s Health and Development at the Universityof Alexandria, Egypt.

Dr. Antonis Makrigiannakis is Professor of Obstetrics andGynecology at the University of Crete Medical School.

infertility andassisted reproduction

e d i t e d b y

botros r. m. b . rizkUniversity of South Alabama, College of Medicine

juan a. garcia-velascoRey Juan Carlos University School of Medicine

hassan n. sallamUniversity of Alexandria

antonis makrigiannakisUniversity of Crete

nn

CAMBRIDGE UNIVERSITY PRESS

Cambridge, New York, Melbourne, Madrid, Cape Town, Singapore, São Paulo

Cambridge University Press

The Edinburgh Building, Cambridge CB2 8RU, UK

First published in print format

ISBN-13 978-0-521-87379-6

ISBN-13 978-0-511-45546-9

© Cambridge University Press 2008

Every effort has been made in preparing this publication to provide accurate and

up-to-

date information that is in accord with accepted standards and practice at the time

of publication. Nevertheless, the authors, editors, and publisher can make no

warranties that the information contained herein is totally free from error, not least

because clinical standards are constantly changing through research and

regulation. The authors, editors, and publisher therefore disclaim all liability for

direct or consequential damages resulting from the use of material contained in

this publication. Readers are strongly advised to pay careful attention to

information provided by the manufacturer of any drugs or equipment that they

plan to use.

2008

Information on this title: www.cambridge.org/9780521873796

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Published in the United States of America by Cambridge University Press, New York

www.cambridge.org

eBook (EBL)

hardback

Dr. Botros R. M. B. RizkTo Mary, Christopher, Christine, David, Gabrielle, and Danielle for their love and inspiration

Dr. Juan A. Garcia-VelascoTo Carmela, Marı́a, and Jaime, my daily inspiration

Dr. Hassan N. SallamTo my wife, Magda; my daughters, Yasmine and Shahira; and my son, Nooman,

whose sacrifice for my work can never be repaid

n c o n t e n t s n

Contributors page xi

Foreword xixHoward W. Jones, Jr.

Preface xxiSamuel J. Strada

Introduction xxiiiBotros R. M. B. Rizk, Juan A. Garcia-Velasco,Hassan N. Sallam, Antonis Makrigiannakis

P A R T I : P H Y S I O L O G Y O F R E P R O D U C T I O N

1. Folliculogenesis: From Preantral Follicles toCorpus Luteum Regression 3Antonis Makrigiannakis, A. Rolaki

2. Mechanisms of Follicular Development:The Role of Gonadotrophins 10Ioannis E. Messinis

3. Human Follicle Culture In Vitro 25Jean Clair Sadeu, Claire Mazoyer, Johan Smitz

4. Endometrial Receptivity 38Dominique de Ziegler, Timothee Fraisse,Charles Chapron

5. Molecular Mechanisms of Implantation 46F. Domı́nguez, Carlos Simón, Juan A. Garcia-Velasco

P A R T I I : IN F E R T I L I T Y E V A L U A T I O N A N D

T R E A T M E N T

6. Evaluation of the Infertile Female 55Timothy N. Hickman, Rafael A. Cabrera,Laurie J. McKenzie, Hany F. Moustafa,Botros R. M. B. Rizk

7. Fertiloscopy 70A. Watrelot

8. Microlaparoscopy 76Botros R. M. B. Rizk, Hany F. Moustafa,Mary George Nawar, Christopher B. Rizk,Christine B. Rizk, David B. Rizk, Nicole Brooks,Craig Sherman, Stephen Varner

9. Pediatric and Adolescent Gynecologic Laparoscopy 82Suketu M. Mansuria, Joseph S. Sanfilippo

10. Laparoscopic Tubal Anastomosis 91Carlos Rotman, Nasir Rana, Jonathan Y. Song,Edgardo Yordan, Carlos E. Sueldo

11. Tubal Microsurgery versus Assisted Reproduction 99Shawky Z. A. Badawy, Frances Shue, Jennifer Shinners

12. The Future of Operative Laparoscopyfor Infertility 107Camran Nezhat, Bulent Berker

13. Operative Hysteroscopy for UterineSeptum 115Mohamed F. M. Mitwally, Mostafa Abuzeid

14. Laser in Subfertility 132John Erian, Anastasios Pachydakis

15. Ultrasonography of the Endometriumfor Infertility 137Richard Palmer Dickey

16. Ultrasonography of the Cervix 143Mona Aboulghar, Botros R. M. B. Rizk

17. Transrectal Ultrasonography in Male Infertility 152Levent Gurkan, Andrew C. Harbin,Wayne J. G. Hellstrom

18. The Basic Semen Analysis: Interpretation andClinical Application 157T. F. Kruger, S. C. Oehninger

19. Evaluation of Sperm Damage: Beyond theWHO Criteria 161Nabil Aziz, Ashok Agarwal

20. Male Factor Infertility: State of the ART 178Frank Comhaire, Ahmed Mahmoud

21. Diagnosis and Treatment of MaleEjaculatory Dysfunction 187Gordon Lucas Fifer, Levent Gurkan,Wayne J. G. Hellstrom

22. Ovulation Induction 193Evert J. P. Van Santbrink, Bart C. J. M. Fauser

23. Clomiphene Citrate for Ovulation Induction 202Richard Palmer Dickey

v i i

24. Aromatase Inhibitors for AssistedReproduction 220Mohamed F. M. Mitwally, Robert F. Casper

25. Pharmacodynamics and Pharmacokineticsof Gonadotrophins 228A. Michele Schuler, Jonathan G. Scammell

26. The Future of Gonadotrophins: Is There Roomfor Improvement? 235Marc Princivalle

27. Ovarian Hyperstimulation Syndrome 243Cristiano E. Busso, Juan A. Garcia-Velasco,Raúl Gomez, Claudio Alvarez, Carlos Simón,Antonio Pellicer

28. Reducing the Risk of High-Order MultiplePregnancy Due to Ovulation Induction 258Richard Palmer Dickey

29. Hyperprolactinemia 270Hany F. Moustafa, Ahmet Helvacioglu,Botros R. M. B. Rizk, Mary George Nawar,Christopher B. Rizk, Christine B. Rizk,Caroline Ragheb, David B. Rizk,Craig Sherman

30. Medical Management of Polycystic OvarySyndrome 286Nadia Kabli, Togas Tulandi

31. Surgical Management of PolycysticOvary Syndrome 294Hakan Yarali, Gurkan Bozdag,Ibrahim Esinler

32. Endometriosis-Associated Infertility 302Cem S. Atabekoglu, Aydin Arici

33. Medical Management of Endometriosis 309Botros R. M. B. Rizk, Mary George Nawar,Christine B. Rizk, David B. Rizk

34. Reproductive Surgery forEndometriosis-Associated Infertility 318Alexis H. Kim, G. David Adamson

35. Congenital Uterine Malformationsand Reproduction 327Theodore A. Baramki

36. Unexplained Infertility 332Juan Balasch

37. ‘‘Premature Ovarian Failure’’:Characteristics, Diagnosis, and Management 339Robert W. Rebar

P A R T I II : A SS I ST E D R E P R O D U C T IO N

38. Medical Strategies to Improve ART Outcome:Current Evidence 349C. M. Boomsma, N. S. Macklon

39. Surgical Preparation of the Patient forIn Vitro Fertilization 361Eric S. Surrey

40. IVF in the Medically Complicated Patient 371Botros R. M. B. Rizk, Christopher B. Rizk,Sameh Mikhail, Christine B. Rizk,Hany F. Moustafa, James Hole,Sheri Owens, Susan Baker, Kathy B. Porter

41. Polycystic Ovary Syndrome and IVF 375Timur Gurgan, Aygul Demirol

42. Endometriosis and AssistedReproductive Technology 381Juan A. Garcia-Velasco, Alfredo Guillén,Guillermo Quea, Antonio Requena

43. Evidence-Based Medicine Comparing hMG/FSHand Agonist/Antagonist and rec/UrinaryhCG/LH/GnRH to Trigger Ovulation 386Mohamed Aboulghar, Hesham Al Inany

44. Luteal Phase Support in Assisted Reproduction 395Luciano G. Nardo, Tarek A. Gelbaya

45. Thrombophilia and Implanation Failure 407Sameh Mikhail, Botros R. M. B. Rizk,Mary George Nawar, Christopher B. Rizk

46. Intrauterine Insemination 416Norman F. Angell, Hany F. Moustafa,Botros R. M. B. Rizk, Mary George Nawar,Christopher B. Rizk, Chris A. Huff,Ruth Kennedy, Scherri B. Holland, Julie Hazelton,Juan A. Garcia-Velasco, Hassan N. Sallam

47. The Prediction and Management of PoorResponders in ART 428Hassan N. Sallam, Botros R. M. B. Rizk,Juan A. Garcia-Velasco

48. Oocyte Donation 443E. Bosch, S. Reis, J. Domingo, J. Remohı́

49. In Vitro Maturation of Human Oocytes 448Ezgi Demirtas, Hananel Holzer, Shai Elizur,Yariv Gidoni, Ri-Cheng Chian, Seang Lin Tan

50. Oocyte and Embryo Freezing 456Eleonora Porcu, Patrizia Maria Ciotti,Giuseppe Damiano, Maria Dirodi,Stefano Venturoli

51. Cryopreservation of Male Gametes 466Amjad Hossain, Manubai Nagamani

52. The Management of Azoospermia 478S. Friedler, A. Raziel, D. Strassburger, M. Schachter,O. Bern, E. Kasterstein, D. Komarovsky, R. Ron-El

53. Spermatid Injection: Current Status 493Rosália Sá, Nieves Cremades, Joaquina Silva,Alberto Barros, Mário Sousa

54. Optimizing Embryo Transfer 506Hassan N. Sallam

55. Single Embryo Transfer 518Jan Gerris, Petra De Sutter

56. Blastocyst Transfer 531David K. Gardner

v i i i n C O N T E N T S n

57. Clinical Significance of Embryo Multinucleation 540Carlos E. Sueldo, Florencia Nodar,Mariano Lavolpe, Vanesa Y. Rawe

58. Quality and Risk Management in theIVF Laboratory 548David Mortimer, Sharon T. Mortimer

59. The Nurse and REI 562Ruth Kennedy

60. Understanding Factors That Influence theAssessment of Outcomes in AssistedReproductive Technologies 570Brijinder S. Minhas, Barry A. Ripps

61. The Revolution of Assisted ReproductiveTechnologies: How Traditional ChineseMedicine Impacted Reproductive Outcomesin the Treatment of Infertile Couples 576Paul C. Magarelli, Diane K. Cridennda,Mel Cohen

62. Complications of Assisted ReproductiveTechnology 604Gamal I. Serour

63. Ectopic and Heterotopic Pregnancies Followingin Vitro Fertilization 619Ziad R. Hubayter, Suheil J. Muasher

64. The Impact of Oxidative Stress on FemaleReproduction and ART: An Evidence-BasedReview 629Sajal Gupta, Lucky Sekhon, Nabil Aziz,Ashok Agarwal

65. PGD for Chromosomal Anomalies 643Anna Pia Ferraretti, Luca Gianaroli,M. Cristina Magli, Valeria Farfalli,Michela Lappi, Santiago Munnè

66. Preimplantation Genetic Diagnosis forSingle-Gene Disorders 657Hany F. Moustafa, Botros R. M. B. Rizk,Zsolt Peter Nagy

67. Epigenetics and ART 677Martine De Rycke

68. Congenital Anomalies and AssistedReproductive Technology 684Amutha Anpananthar, Alastair Sutcliffe

P A R T I V : E T H I C A L D IL E M M A S I N F E R T I L I T Y

A ND A S S IS T E D R E P R O D U C T I O N

69. Stem Cell Research 695M. E. Poo, Carlos Simón

70. Fertility Preservation in Female andMale Cancer Patients 706Mohamed A. Bedaiwy, Tommaso Falcone

71. Ethical Dilemmas in ART: Current Issues 717Francxoise Shenfield

72. Infertility Treatment in PerimenopausalWomen: Ethical Considerations 723Hyacinth N. Browne, Alicia Armstrong,Alan DeCherney

73. Religious Perspectives of Ethical Issues inInferility and ART 728Botros R. M. B. Rizk, Sherman J. Silber,Gamal I. Serour, Michel Abou Abdallah

74. The Future of Assisted Reproduction 747Biljana Popovic Todorovic, Paul Devroey

Index 759

Color plates follow page 24

n C O N T E N T S n i x

n c o n t r i b u t o r s n

editors

Botros R. M. B. Rizk, M.D., M.A., F.R.C.O.G.,F.R.C.S.(C), H.C.L.D., F.A.C.O.G., F.A.C.S.

Professor and Head, Division of ReproductiveEndocrinology and Infertility,Department of Obstetrics and Gynecology,University of South Alabama, Medical andScientific Director USA ART program,Mobile, Alabama, USA

Juan A. Garcia-Velasco, M.D., Ph.D.Director, IVI-Madrid, Assistant Professor,

Rey Juan Carlos University, Madrid, Spain

Hassan N. Sallam, M.D., F.R.C.O.G., Ph.D. (London)Director, The Suzanne Mubarak Regional Centre

for Women’s Health and Development,Professor, Obstetrics and Gynaecology,and Vice Dean, University of Alexandria,Alexandria, Egypt

Antonis Makrigiannakis, M.D.Professor of Obstetrics and Gynecology,

Laboratory of Human Reproduction,Department of Obstetrics and Gynecology,University of Crete, Crete, Greece

authors

Michel Abou Abdallah, M.D.Executive Director, Middle East Fertility Society,

Beirut, Lebanon

Mohamed Aboulghar, M.D.Professor, Department of Obstetrics and Gynaecology,

Cairo University, Medical Director,Egyptian IVF-ET Centre, Cairo, Egypt

Mostafa Abuzeid, M.D., F.A.C.O.G., F.R.C.O.G.Director of the Division of Reproductive Endocrinology,

Department of Obstetrics and Gynecology,Hurley Medical Center, Flint, Michigan, USA

Practice Director, IVF Michigan,Rochester Hills, Michigan, USAProfessor, Department of Obstetrics and Gynecology,Michigan State University College of Human Medicine,East Lansing, Michigan, USA

G. David Adamson, M.D., F.R.C.S.C., F.A.C.O.G., F.A.C.S.Director, Fertility Physicians of Northern California,

Palo Alto, California, USADirector, Fertility & Reproductive Health Instituteof Northern California, San Jose, California, USAClinical Professor, Stanford University,Associate Clinical Professor,U.C. San Francisco School of Medicine,San Francisco, California, USA

Ashok Agarwal, Ph.D., H.C.L.D.Professor and Director,

Reproductive Research Centre,Glickman Urological Institute,The Cleveland Clinic Foundation,Cleveland, Ohio, USA

Claudio AlvarezInstituto Valenciano de Infertilidad, Santiago, Chile

Amutha Anpananthar, M.B.B.S., B.Sc.University College London,

Department of Community Child Health,London, UK

Aydin Arici, M.D.Associate Professor and Head, Division of Reproductive

Endocrinology & Infertility, Department of Obstetricsand Gynecology, Yale University School of Medicine,New Haven, Conneticut, USA

Alicia Armstrong, M.D., M.H.S.C.R.Reproductive Biology and Medicine Branch, National

Institute of Child Health & Human Development,Bethesda, Maryland, USA

Cem S. Atabekoglu, M.D.Department of Obstetrics and Gynecology, Ankara

University School of Medicine, Ankara, Turkey

x i

Nabil Aziz, M.B., Ch.B., M.R.C.O.G., M.D.Consultant Gynecologist, Liverpool Women’s Hospital,

Lecturer, The University of Liverpool, Liverpool, UK

Shawky Z. A. Badawy, M.D.Professor and Chair, Department of Obstetrics and

Gynecology, Division of Reproductive Endocrinologyand Infertility, State University of New York, UpstateMedical University, Syracuse, New York, USA

Susan BakerAssociate Professor, Division of Maternal Fetal Medicine,

Department of Obstetrics and Gynecology,University of South Alabama, Mobile, Alabama, USA

Juan Balasch, M.D.Professor and Chairman, Institute Clinic Gynecology,

Obstetrics and Neonatology,Hospital Clinic-Institut d’InvestigacionsBiomediques August Pi I Sunyer (IDIBAPS),Faculty of Medicine – University of Barcelona,Barcelona, Spain

Theodore A. Baramki, M.D., F.A.C.O.G.Department of Gynecology and Obstetrics,

Johns Hopkins University School of Medicine,Baltimore, Maryland, USA

Alberto BarrosCentre for Reproductive Genetics, Porto, Portugal

Mohamed A. Bedaiwy, M.D.Department of Obstetrics and Gynecology, The Cleveland

Clinic Foundation, Cleveland, Ohio, USA

Bulent Berker, M.D.Ankara University School of Medicine, Department of

Obstetrics & Gynecology, Division of ReproductiveEndocrinology & Endoscopic Surgery, Ankara, Turkey

O. BernInfertility and IVF Unit, Assaf Harofeh Medical Center,

Zerifin, Sackler School of Medicine, Tel-AvivUniversity, Tel-Aviv, Israel

C. M. Boomsma, Ph.D. StudentDivision Perinatology and Gynaecology,

University Medical Center Utrecht,Utrecht, The Netherlands

E. BoschInstituto Universitario IVI, Madrid, Spain

Gurkan Bozdag, M.D.Hacettepe University Faculty of Medicine, Department of

Obstetrics and Gynecology, Ankara, Turkey

Hyacinth N. Browne, M.D.Reproductive Biology and Medicine Branch,

National Institute of Child Health & HumanDevelopment, Bethesda, Maryland, USA

Cristiano E. BussoInstituto Valenciano de Infertilida, Valencia, Spain

Rafael A. Cabrera, M.D.HOUSTON IVF,

Memorial Hermann Memorial City Hospital,Houston, Texas, USA

Robert F. Casper, M.D., F.R.C.S.(C)Professor, Division of Reproductive Sciences, Fran and

Lawrence Bloomberg Department of Obstetrics &Gynecology, Senior Scientist, Samuel LunenfeldResearch Institute, Mount Sinai Hospital,The University of Toronto, Toronto,Ontario, Canada, President, CAREM(Canadian American Reproductive Medicine),Windsor, Ontario, Canada

Charles Chapron, M.D.Reproductive Endocrinology and Infertility,

Department of Obstetrics and Gynecology,Hôpital Cochin, Paris, France

Ri-Cheng Chian, Ph.D.McGill University, Department of Obstetrics and

Gynecology, Royal Victoria Hospital, Montreal,Quebec, Canada

Patrizia Maria Ciotti, B.Sc.Infertility and IVF Centre, University of Bologna,

Bologna, Italy

Mel Cohen, Ph.D.Executive Director, Reproductive Medicine & Fertility

Centers, Colorado Springs, Colorado, USACorona Institute for ReproductiveMedicine & Fertility, Corona, California, USANew Mexico Gynecology & Fertility Institute,Santa Fe, New Mexico, USA

Frank Comhaire, M.D., Ph.D.Professor Emeritus, Endocrinology-Andrology,

Ghent University Hospital, Gent, Belgium

Nieves CremadesIVF Lab, Department of Gynecology and Obstetrics,

Academic Hospital of Alicante, Spain

Diane K. Cridennda, L.Ac.Owner/Director, East Winds Acupuncture, Inc.,

Colorado Springs, Colorado, USA

Giuseppe Damiano, M.D.Infertility and IVF Centre, University of Bologna,

Bologna, Italy

Alan H. DeCherney, M.D.Branch Chief of Reproductive Biology and

Medicine Branch, National Institute ofChild Health & Human Development,Bethesda, Maryland, USA

x i i n C O N T R I B U T O R S n

Aygul Demirol, M.D.Medical Director, CLINIC Women Health,

Infertility and IVF Center, Ankara, Turkey

Ezgi Demirtas, M.D.McGill University,

Department of Obstetrics and Gynecology,Royal Victoria Hospital, Montreal, Quebec, Canada

Martine De Rycke, Ph.D.Research Centre for Reproduction and Genetics,

Academisch Ziekenhuis, Vrije Universiteit Brussel,Brussels, Belgium

Petra De Sutter, M.D., Ph.D.Center for Reproductive Medicine,

Research Fellow of the Flemish Foundation forScientific Research (FWO), Women’s Clinic,Ghent University Hospital, Ghent,Belgium

Paul Devroey, M.D., Ph.D.Clinical Director,

Research Centre for Reproduction and Genetics,Academisch Ziekenhuis, Vrije Universiteit Brussel,Brussels, Belgium, The Center for ReproductiveMedicine, Brussels, Belgium

Dominique de ZieglerJoint Division of Reproductive Endocrinology and

Infertility, Department of Obstetrics and Gynecology,University Hospitals of Geneva and Lausanne,Switzerland

Richard Palmer Dickey, M.D., Ph.D., F.A.C.O.G.Section of Reproductive Endocrinology,

Department of Obstetrics and Gynecology,Louisiana State University School of Medicine,New Orleans, Louisiana, USA, Medical Director,The Fertility Institute of New Orleans,Mandeville, Louisiana, USA

Maria Dirodi, M.D.Infertility and IVF Centre, University of Bologna,

Bologna, Italy

J. DomingoInstituto Universitario IVI, Spain

F. Domı́nguezFundacion IVI, Instituto Universitario IVI, Universidad

de Valencia, Valencia, Spain

Shai Elizur, M.D.McGill University, Department of Obstetrics and

Gynecology, Royal Victoria Hospital, Montreal,Quebec, Canada

John Erian, M.B., B.Ch., F.R.C.O.G.Consultant Gynaecologist and Minimal Access Surgeon,

Kent, UK

Ibrahim Esinler, M.D.Hacettepe University Faculty of Medicine,

Department of Obstetrics and Gynecology,Ankara, Turkey

Tommaso Falcone, M.D.Professor and Chair,

Department of Obstetrics & Gynecology,The Cleveland Clinic Foundation,Cleveland, Ohio, USA

Valeria Farfalli, M.D.S.I.S.Me.R., Reproductive Medicine Unit, Bologna, Italy

Bart C. J. M. Fauser, M.D., Ph.D.Professor of Reproductive Medicine, Chair,

Department of Reproductive Medicine & Gynecology,Head, Division of Perinatology & Gynecology,University Medical Center, Utrecht, The Netherlands

Anna Pia Ferraretti, M.D., Ph.D.Scientific Director, S.I.S.Me.R. Reproductive Medicine

Unit, Bologna, Italy

Gordon Lucas Fifer, M.D.Department of Urology, Tulane University, School of

Medicine, New Orleans, Louisiana, USA

Timothee Fraisse, M.D.Joint Division of Reproductive Endocrinology and

Infertility, Department of Obstetrics and Gynecology,University Hospitals of Geneva and Lausanne,Switzerland

S. Friedler, M.D.Infertility and IVF Unit, Assaf Harofeh Medical Center,

Zerifin, Sackler School of Medicine,Tel-Aviv University, Tel-Aviv, Israel

Juan A. Garcia-Velasco, M.D.Director, IVI Madrid,

Assistant Professor of Obstetrics and Gynecology,Rey Juan Carlos University, Madrid, Spain

David K. Gardner, Ph.D.Chair of Zoology, University of Melbourne, Australia,

Scientific Director, Colorado Center forReproductive Medicine, Englewood, Colorado, USA

Tarek A. Gelbaya, M.D.Department of Obstetrics and Gynaecology,

Royal Bolton Hospital, Bolton, UK

Jan Gerris, M.D., Ph.D.Professor, Division of Gynecology,

Center for Reproductive Medicine,Women’s Clinic, University Hospital,Ghent, Belgium

Luca Gianaroli, M.D.S.I.S.MeR. Centre, Bologna, Italy

n C O N T R I B U T O R S n x i i i

Yariv Gidoni, M.D.McGill University, Department of Obstetrics and

Gynecology, Royal Victoria Hospital, Montreal,Quebec, Canada

Raúl Gomez, M.D.Universidad de Valencia, Valencia, Spain

Alfredo GuillénIVI-Madrid, Spain

Timor Gurgan, M.D.Professor, Hacettepe University,

Department of OB/GYN, Reproductive Endocrinologyand IVF Unit, Academic Director,CLINIC Women Health, Infertility and IVF Center,Ankara, Turkey

Levent Gurkan, M.D.Department of Urology, Tulane University,

School of Medicine, New Orleans,Lousiana, USA

Julie HazeltonDivision of Reproductive Endocrinolgy and Infertility,

Department of Obstetrics and Gynecology,University of South Alabama, Mobile,Alabama, USA

Wayne J. G. Hellstrom, M.D., F.A.C.S.Professor, Department of Urology, Tulane University,

School of Medicine, New Orleans, Lousiana, USA

Ahmet Helvacioglu, M.D., F.A.C.O.G.Obstetrics and Gynecology, Fairhope, Alabama, USA

Timothy N. Hickman, M.D.Medical Director, Houston IVF, Memorial Hermann

Memorial City Hospital, Houston, Texas, USA

James Hole, D.O., F.A.C.O.G.Division of Maternal Fetal Medicine,

Department of Obstetrics and Gynecology,University of South Alabama, Mobile, Alabama, USA

Scherri B. Holland, R. N.Division of Reproductive Endocrinolgy and Infertility,

Department of Obstetrics and Gynecology,University of South Alabama, Mobile,Alabama, USA

Hananel Holzer, M.D.McGill University, Department of Obstetrics and

Gynecology, Royal Victoria Hospital, Montreal,Quebec, Canada

Amjad Hossain, Ph.D., H.C.L.D.Assistant Professor and Director, Andrology and ART

Laboratory Services, Division of ReproductiveEndocrinology and Infertility, Department ofObstetrics and Gynecology, University of TexasMedical Branch, Galveston, Texas, USA

Ziad R. Hubayter, M.D.Department of Gynecology and Obstetrics,

Division of Reproductive Endocrinology and Infertility,The Johns Hopkins University, Baltimore,Maryland, USA

Chris A. HuffDivision of Reproductive Endocrinology and Infertility,

Department of Obstetrics and Gynecology,University of South Alabama, Mobile,Alabama, USA

Hesham Al Inany, M.D.Department of Obstetrics and Gynaecology,

Cairo University, Egyptian IVF-ET Centre,Cairo, Egypt

Howard W. Jones, Jr.Emeritus Professor, Eastern Virginia Medical School,

Founder of the Jones Institute,Norfolk, Virginia, USA

Nadia Kabli, M.D.Fellow of Reproductive Endocrinology and Infertility,

McGill University, Montreal, Quebec, Canada

E. KastersteinInfertility and IVF Unit,

Assaf Harofeh Medical Center, Zerifin,Sackler School of Medicine, Tel-AvivUniversity, Tel-Aviv, Israel

Ruth Kennedy, C.R.N.P.IVF Coordinator, Division of Reproductive

Endocrinology and Infertility,Department of Obstetrics and Gynecology,University of South Alabama, Mobile,Alabama, USA

Alexis H. KimFertility Physicians of Northern California, Palo Alto,

California, USA

D. KomarovskyInfertility and IVF Unit, Assaf Harofeh Medical Center,

Zerifin, Sackler School of Medicine, Tel-AvivUniversity, Tel-Aviv, Israel

T. F. Kruger, M.D.Reproductive Biology Unit, Department of

Obstetrics and Gynecology,University of Stellenbosch and TygerBerg Hospital,TygerBerg, South Africa

Michela LappiMedicine via pazzani, Bologna, Italy

Mariano Lavolpe, M.Sc.Associate Director IVF Laboratory,

Center for Studies in Gynecology andReproduction (CEGYR), Buenos Aires,Argentina

x i v n C O N T R I B U T O R S n

N. S. Macklon, M.B.Ch.B., M.D.Department of Reproductive Medicine and Gynecology,

University Medical Center, Utrecht, The Netherlands

Paul C. Magarelli, M.D., Ph.D.Owner/Medical Director, Reproductive Medicine &

Fertility Centers, Colorado Springs, Colorado, USAAssociate Professor, University of New Mexico,Department of Obstetrics & Gynecology,Albuquerque, New Mexico, USA

M. Cristina Magli, M.Sc., S.I.S.Me.R.Reproductive Medicine Unit, Bologna, Italy

Ahmed Mahmoud, M.D., Ph.D.Laboratory of Andrology, Ghent University Hospital,

Gent, Belgium

Antonis Makrigiannakis, M.D.Professor of Obstetrics and Gynecology,

Laboratory of Human Reproduction,Department of Obstetrics and Gynaecology,University of Crete, Crete, Greece

Suketu M. Mansuria, M.D.Assistant Professor of Obstetrics, Gynecology and

Reproductive Sciences, Division of Minimally InvasiveGynecologic Surgery, University of Pittsburgh MedicalCenter, Magee-Womens Hospital, Pittsburgh,Pennsylvania, USA

Claire Mazoyer, M.D.Laboratory of Hormonology and Tumor Markers,

Department of Clinical Chemistry/Anatomopathology,University Hospital Free University Brussels,Brussels, Belgium

Laurie J. McKenzie, M.D., F.A.C.O.GHouston IVF, Houston, Texas, USA

Ioannis E. MessinisDepartment of Obstetrics and Gynecology,

University of Thessalia, Larissa, Greece

Sameh Mikhail, M.D.Department of Medicine, Rochester General Hospital,

Rochester, New York, USA

Mohamed F. M. Mitwally, M.D.Clinical Assistant Professor,

Division of Reproductive Endocrinology & Infertility,Department of Obstetrics and Gynecology,University of New Mexico, Albuquerque,New Mexico, USAReproductive Endocrinologist,RMFC (Reproductive Medicine and Fertility Center),Colorado Springs, Colorado, USA

David Mortimer, Ph.D.President, Oozoa Biomedical, Inc.,

West Vancouver, British Columbia,Canada

Sharon T. MortimerOozoa Biomedical, Inc., West Vancouver,

British Columbia, Canada

Hany F. Moustafa, M.D.Fellow, Division of Reproductive Endocrinology

and Infertility, Department of Obstetricsand Gynecology, University of South Alabama,Mobile, Alabama, USA,Alabama Lecturer, Suez Canal University,Ismailia, Egypt

Suheil J. Muasher, M.D.Department of Gynecology and Obstetrics,

Division of Reproductive Endocrinology and Infertility,Johns Hopkins University,Baltimore, Maryland, USA, Director,The Muasher Center for Fertility and IVF,Fairfax, Virginia, USA, Department of Obstetrics andGynecology, Johns George Washington University,Washington, DC, USA, Department of Obstetrics andGynecology, Virginia Commonwealth University,Richmond, Virginia, USA

Santiago Munnè, Ph.D.Reprogenetics, Paramus, New Jersey, USA

Manubai Nagamani, M.D.Professor and Chief, Division of Reproductive

Endocrinology and Infertility,Department of Obstetrics and Gynecology,University of Texas Medical Branch,Galveston, Texas, USA

Zsolt Peter Nagy, M.D., Ph.D., H.C.L.D.Scientific and Laboratory Director,

Reproductive Biology Associates, Atlanta,Georgia, USA

Luciano G. Nardo, M.D.Department of Reproductive Medicine, St Mary’s Hospital,

Division of Human Development,University of Manchester, Manchester, UK

Mary George Nawar, M.D., M.R.C.Oph.Department of Obstetrics and Gynecology,

University of South Alabama, Mobile,Alabama, USA

Camran Nezhat, M.D., F.A.C.O.G., F.A.C.S.Fellowship Director,

Center for Special Minimally Invasive Surgery,Past President, Society of Laparoendoscopic Surgeons,Clinical Professor of OB/GYN(Adj),Clinical Professor of Surgery(Adj), StanfordUniversity Medical School, Stanford University,Palo Alto, California, USA

Florencia Nodar, M.Sc.Director, IVF Laboratory, Center for Studies in

Gynecology and Reproduction (CEGYR),Buenos Aires, Argentina

n C O N T R I B U T O R S n x v

Anastasios Pachydakis, M.D., D.F.F.P., M.R.C.O.G.Specialist Registrar Obstetrics and Gynaecologist,

Princess Royal University Hospital UK,Sidcup, UK

Antonio PellicerProfessor, IVI, Instituto Valenciano de Infertilidad,

Valencia, Spain

M. E. Poo, C., M.D.Spanish Stem Cell Bank, Prince Felipe Research Center,

University of Valencia, IVI Foundation, University ofValencia, Valencia, Spain

Eleonora Porcu, M.D.Director, Infertility and IVF Center, University of Bologna,

Bologna, Italy

Kathy B. Porter, M.D., M.B.A.Professor and Chair, Department of Obstetrics and

Gynecology, University of South Alabama, Mobile,Alabama, USA

Marc Princivalle, Ph.D.Target Validation, Ferring Research Ltd, Southampton, UK

Guillermo QueaIVI-Madrid, Spain

Caroline RaghebUniversity of Alabama, School of Medicine, Birmingham,

Alabama, USA

Nasir Rana, M.D., M.P.H., F.A.C.O.G.Associate Director, Oak Brook Institute of Endoscopy,

Oak Brook, Illinois, USA, Assistant Professor,Rush Medical College, Chicago, Illinois, USA

Vanesa Y. Rawe, M.Sc., Ph.D.Director, Basic Research Laboratory,

Center for Studies in Gynecology and Reproduction(CEGYR), Buenos Aires, Argentina

A. RazielInfertility and IVF Unit, Assaf Harofeh Medical Center,

Zerifin, Sackler School of Medicine, Tel-AvivUniversity, Tel-Aviv, Israel

Robert W. Rebar, M.D.Executive Director, American Society for Reproductive

Medicine, Volunteer Professor, Department ofObstetrics and Gynecology, University of Alabama,Birmingham, Alabama, USA

S. ReisInstituto Universitario IVI, Madrid, Spain

J. Remohı́Instituto Universitario IVI, Madrid, Spain

Antonio RequenaIVI-Madrid, Spain

Botros R. M. B. Rizk, M.D., M.A., F.R.C.O.G., F.R.C.S.(C),H.C.L.D., F.A.C.O.G., F.A.C.S.

Professor and Head, Division of ReproductiveEndocrinology and Infertility,Department of Obstetrics and Gynecology,University of South Alabama,Medical and Scientific Director, USAART program, Mobile, Alabama, USA

Christine B. RizkJohn Emory Scholar, Emory University, Atlanta,

Georgia, USA

Christopher B. RizkRice University, Houston, Texas, USA

David B. RizkResearch Assistant, Department of Obstetrics and

Gynecology, University of South Alabama,College of Medicine, Mobile,Alabama, USA

A. RolakiLaboratory of Human Reproduction,

Department of Obstetrics and Gynaecology,Medical School, University of Crete, Crete,Greece

R. Ron-El, M.D.Professor and Head of Fertility and IVF Unit,

Assaf Harofe Medical Center, Tel-Aviv University,Zerifin, Israel

Carlos Rotman, M.D., F.A.C.O.G.Director, Oak Brook Institute of Endoscopy,

Oak Brook, Illinois, USA,Associate Professor, Rush Medical College,Chicago, Illinois, USA

Rosália SáLab Cell Biology, ICBAS, University of Porto,

Porto, Portugal

Jean Clair Sadeu, M.D.Follicle Biology Laboratory, Vrije Universiteit Brussel

(VUB), Brussels, Belgium

Hassan N. Sallam, M.D., F.R.C.O.G., Ph.D. (London)Director, The Suzanne Mubarak Regional Centre for

Women’s Health and Development, Professor,Obstetrics and Gynaecology, University of Alexandria,Alexandria, Egypt

Joseph S. Sanfilippo, M.D., M.B.A.Professor of Obstetrics,

Gynecology and Reproductive Sciences,Division of Reproductive Endocrinology &Infertility & Minimally Invasive Gynecologic Surgery,Vice Chairman, Division of Reproductive Sciences,University of Pittsburgh Medical Center,Magee-Womens Hospital, Pittsburgh,Pennsylvania, USA

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Jonathan G. Scammell, Ph.D.Professor, Pharmacology and Chair Comparative

Medicine, University of South AlabamaCollege of Medicine, Mobile, Alabama, USA

M. SchachterInfertility and IVF Unit,

Assaf Harofeh Medical Center,Zerifin, Sackler School of Medicine,Tel-Aviv University, Tel-Aviv, Israel

A. Michele Schuler, D.V.M., Ph.D.Department of Comparative Medicine,

University of South Alabama, Mobile, Alabama, USA

Gamal I. Serour, F.R.C.O.G., F.R.C.S.Professor of Obstetrics and Gynecology,

Director, International Islamic Center of PopulationStudies and Research, Al-Azhar University, FormerDean of Al-Azhar University, Clinical Director,The Egyptian IVF & ET Center, Maadi, Cairo, Egypt

Francxoise Shenfield, L.R.C.P., M.R.C.S., M.A.Reproductive Medicine Unit,

University College Hospital,London, UK

Jennifer Shinners, M.D.Department of Obstetrics and Gynecology,

State University of New York,Upstate Medical University, Syracuse,New York, USA

Frances Shue, M.D.Department of Obstetrics and Gynecology,

State University of New York,Upstate Medical University, Syracuse,New York, USA

Sherman J. Silber, M.D.Infertility Center of St. Louis, St. Luke Hospital,

St. Louis, Missouri, USA

Joaquina SilvaCentre for Reproductive Genetics, Porto, Portugal

Carlos Simón, M.D.Spanish Stem Cell Bank, Prince Felipe Research Center,

University of Valencia, IVI Foundation,University of Valencia, Valencia, Spain

Johan Smitz, M.D., Ph.D.Laboratory of Hormonology and Tumor Markers,

Department of Clinical Chemistry/Anatomopathology,University Hospital Free University Brussels,Brussels, Belgium

Jonathan Y. Song, M.D., F.A.C.O.G.Oak Brook Institute of Endoscopy, Oak Brook,

Illinois, USA, Assistant Professor,Rush Medical College, Chicago,Illinois, USA

Mário Sousa, M.D., Ph.D.Specialist of Laboratorial Medicine of Reproduction,

Director, Lab Cell Biology,Inst. Biomedical Sciences Abel Salazar,University of Porto, Porto,Portugal

Samuel J. Strada, Ph.D.Dean, College of Medicine,

Professor of PharmacologyUniversity of South Alabama,Mobile, Alabama, USA

D. StrassburgerInfertility and IVF Unit,

Assaf Harofeh Medical Center,Zerifin, Sackler School of Medicine,Tel-Aviv University, Tel-Aviv, Israel

Carlos E. Sueldo, M.D., F.A.C.O.G.Clinical Professor, University of California

San Francisco-Fresno, Fresno,California, USASenior Consultant,Oak Brook Institute of Endoscopy,Oak Brook, Illinois, USACenter for Studies in Gynecology and Reproduction,Buenos Aires, Argentina, USA

Eric S. Surrey, M.D.Medical Director,

Colorado Center for Reproductive Medicine,Englewood, Colorado, USA

Alastair Sutcliffe, M.D., M.R.C.P., F.R.C.P.C.H.Senior Lecturer in Child Health,

Honorary Consultant, Institute of Child Health,Royal Free and University of Medical School,University of College London,Department of Community Child Health,London, UK

Seang Lin Tan, M.D., M.B.B.S., F.R.C.O.G., F.R.C.S.C.,M.med. (O&G), M.B.A.

Professor and Chair, McGill University,Department of Obstetrics and Gynecology,Royal Victoria Hospital Montreal, Quebec, Canada

Biljana Popovic TodorovicThe Center for Reproductive Medicine, Brussels,

Belgium

Togas Tulandi, M.D., M.H.C.M.Professor of Obstetrics and Gynecology,

Milton Leong Chair in Reproductive Medicine,McGill University, Montreal, Quebec, Canada

Evert J. P. Van Santbrink, M.D., Ph.D.Division of Reproductive Medicine,

Department of Obstetrics and Gynecology,Dijkzigt Academic Hospital, Rotterdam,The Netherlands

n C O N T R I B U T O R S n x v i i

Stefano Venturoli, M.D.Infertility and IVF Centre,

University of Bologna,Bologna, Italy

A. Watrelot, M.D.CRES (Centre de Recherche et d’Etude de la Sterı́lı́te),

Lyon, France

Hakan Yarali, M.D., Ph.D.Hacettepe University Faculty of Medicine,

Department of Obstetrics and Gynecology,Ankara, Turkey

Edgardo YordanOak Brook Institute of Endoscopy, Oak Brook,

Illinois, USA

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n f o r e w o r d n

Howard W. Jones, Jr.

This text on infertility and assisted reproduction is truly inter-national. There are authors from 18 nations. This reflects thewidespread availability of assisted reproductive technology(ART) and indicates that experts are found worldwide. Indeed,in a compilation of legislation and guidelines concerning ART,sponsored by the International Federation of Fertility Societiesand published as a supplement to Fertility and Sterility in 2007under the title ‘‘Surveillance 07,’’ it was noted that two-thirdsof the world’s population lived in countries where ART is avail-able. To be sure, it needs to be added that accessibility withinvarious countries varies widely.

‘‘Surveillance 07’’ also found that there was a wide variationamong nations in what society really wished to survey andcontrol. Indeed, this variation has had some interesting andunexpected consequences. For example:

n In Italy, cryopreservation of fertilized eggs is prohibited.This has stimulated investigation into the improvementof cryopreservation or vitrification for oocytes (see chapterby Porcu).

n In Belgium, among those qualified for insurance coverageprovided by the government, no more than a single fertil-ized egg can be transferred in the first IVF attempt, pro-vided the patient is younger than 36 years of age. This hasprovided and given stimulus to the evaluation of electivesingle embryo transfer (eSET). It turns out that eSET offers

a good pregnancy rate and largely eliminates multiples (seechapter by Gerris).

The complete physician does not practice medicine likea cook who depends on a cookbook to prepare a special dish.

To be sure, the physician needs practical knowledge, butthe complete physician also must know the underlying phys-iology and pathology of the problem at hand in order to selectthe best solution. Thus, this new text can make the physiciancomplete. For example, as to the matter of implantation aftertransfer of a fertilized egg, this text addresses the practical (seechapter by deZiegler) aspects and the molecular aspect (seechapter by Simon).

To understand controlled ovarian hyperstimulation priorto IVF, it is necessary to understand the basic physiology (seechapters by Makrigiannakis and Messinis). Only with thisinformation can one understand the ins and outs of the varioustypes of stimulation and the response thereto (see chapters byFauser, Macklon, Aboulghar, and Nardo).

Reproductive medicine in contrast to other subspecialtiesdeals with hot-button ethical and public policy issues simplybecause it deals with reproduction, which society regards asspecial and different. The complete physician must understandthe patient’s concerns in this area (see chapter by Shenfeld).

All in all, the physician can become complete by having thistext at hand.

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n p r e f a c e n

Samuel J. Strada, Ph.D.

The readers of this text will encounter a comprehensive andperspicacious view of the discipline of reproductive medicineas revealed by leaders in their respective fields. The editorshave assembled a talented group of authors spanning six geo-graphic continents. Some sections of the book – for example,its second section on infertility – are comprehensive enoughto stand on their own. The section begins with the evolutionof the female and covers in detail the roles of endoscopy,hysteroscopy, and laparoscopy in the management of infertil-ity. The following section deals with ultrasonography of boththe female and male, which is followed by a cutting-edge sec-tion on the evaluation of the infertile male, beginning withstandards of current clinical management and ending withfuture implications for developing DNA technology. Subse-quent sections focus on the physiology of ovulation andthe pharmacological agents prescribed by gynecologists today,in addition to a discussion of future pharmacological advan-ces that have the potential to significantly alter treatment

strategies. Other clinical areas discussed include the medicalmanagement of endometriosis, the relationship of polycysticovaries to infertility, and the status of premature ovarianfailure. A comprehensive assessment on the state of the artin assisted reproduction is detailed in the third section of thebook. This is the expertise of one of its editors, Dr. BotrosR. M. B. Rizk, so it is perhaps not too surprising that this topicdid receive significant attention. More esoteric topics, or atleast topics that are not usually found in texts of this genre,include cryobiology, herbal Chinese medicine and assistedreproductive technology (ART), various congenital malfor-mations and chromosomal abnormalities associated withART, and a concluding section entitled ‘‘Ethical Dilemmasin Fertility and Assisted Reproduction,’’ which evaluates stemcell research from scientific, legal, and religious perspectives.In other words, there is something in this textbook for every-one interested in reproductive biology, whether a novice or anexpert in the field.

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n i n t r o d u c t i o n n

Botros R. M. B. Rizk, Juan A. Garcia-Velasco, Hassan N. Sallam, Antonis Makrigiannakis

The past three decades have witnessed a transformation inreproductive medicine from science fiction to one of the mostadvanced medical disciplines. Our textbook is a confirmation ofthe tremendous achievements in scientific research that changedthe course of our clinical practice. Thirty years of in vitro fertil-ization (IVF) was celebrated this year in Alexandria, honoringRobert Edwards specifically. More than four million IVF babieshave been born worldwide. The doors that were opened byRobert Edwards and Patrick Steptoe lead to many miracles.Intracytoplasmic sperm injection, in vitro maturation, oocytevitrification, pre-implantation genetic diagnosis, and ovariantransplantation are dreams that were fulfilled. Many couples stillhave no hope of having their own families and demand us tokeep moving forward.

The authors of the chapters of this book have lead the worldfor the three decades. They contributed their finest and most

advanced research. We find in every one of them a sinceredesire to uphold the ethics and the respect in our society.The friendship and cordiality between them has been amazing.They represented the six continents truly, and many of themhave worked in more than one continent. It is not surprisingthat they worked together in such an elegant and a unique way.The different chapters are individualized in style, but the spiritof the book has united them. From reproductive physiology tosurgery and assisted reproduction, the authors move with greatelegance. The ethical and moral issues have been thoughtfullyconsidered. To every author we acknowledge his or her exper-tise and admire and enjoy their friendship. We place in yourhands, our readers, a text that covers the present state of the artand gives you a glimse of the future, confirming your instinctsand stimulating your desire for knowledge. We hope that youenjoy reading it as we have enjoyed editing it.

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physiology of reproduction

n 1 n

folliculogenesis: from preantral follicles tocorpus luteum regression

Antonis Makrigiannakis, A. Rolaki

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

The most common function of the female gonad is to producegametes, the oocytes, and sex hormones, such as estrogens andprogesterone, which control the development of the femalesecondary sexual characteristics and support pregnancy. Thesetwo functions are exerted cyclically between puberty and themenopause, and they are regulated by diverse endocrine andparacrine factors acting on many cell types situated in theovary. Ovarian functions result from the evolution of a mor-phological unit, the ovarian follicle, which consists of a centraloocyte surrounded by granulosa cells and other layers of so-matic theca cells (1). The maturation of the follicle proceedsthrough primordial, primary, and secondary stages of develop-ment and is controlled by various factors produced in theovary. The main physiological stimulants for differentiationand luteinization of granulosa cells, which are a main cellularcomponent of the follicle, are the gonadotropin hormones,follicle-stimulating hormone (FSH), and luteinizing hormone(LH). Throughout the reproductive life span of the female, onlylimited number of follicles will reach the stage of Graafian fol-licle and will ovulate, whereas the vast majority is graduallyeliminated through a process called atresia. In every menstrualcycle, only one follicle, named the dominant follicle, is destinedto complete maturation and ovulate, and thus, the formation ofthe multiple embryos during pregnancy is prevented.

Degeneration of the old corpus luteum is a process essentialfor maintaining the normal production of progesterone in ev-ery menstrual cycle. The complexity of the interrelation of theevents that control oocyte growth and ultimate acquisition ofdevelopmental competence is under continuous investigation(2). It is generally believed that follicular atresia and luteolysisoccur by mechanisms that accompany a highly organized typeof cell death, called programmed cell death or apoptosis (3).The present review reports a variety of factors involved in thedifferent stages of follicular development. Elucidation of themechanisms that regulate follicular development may lead tothe prevention of female reproductive disorders or other path-ological conditions and to the development of new culturemethods for oocytes for in vitro fertilization.

P R E O V U L A T O R Y F O L L I C L E

The development of preantral follicles involves oocyte enlarge-ment, zona pellucida formation, extensive granulosa cell

proliferation, formation of a basal lamina, condensation ofstromal cells around the basal lamina to form the theca layer,and the development of fluid-filled spaces that gradually co-alesce to form the antral cavity (4, 5). In the absence of appro-priate gonadotropic stimulation, follicles develop until theearly antral stage and atresia occurs.

The early stages of follicular development, including theearly antral follicle, are independent of the FSH and the lutei-nizing hormone (LH). In agreement with these findings is thestudy of Touraine et al., which shows that inactivation of FSHreceptor does not disrupt the follicular growth to the largepreantral stages (6). The low responsiveness of antral folliclesto gonadotropins results presumably by the low number ofgonadotropin receptors on follicle cells at this stage of devel-opment, although it is believed that anti-Mullerian hormone(AMH) reduces the FSH responsiveness of preantral and smallantral follicles (7). However, the preantral follicle is affected byother nongonadotropic factors, such as members of the TGF-bfamily, estrogens, androgens, insulin, and insulin-like growthfactor-1 (6, 1). The follicles at the preantral stage are shown toproduce very low amounts of progesterone, and androstene-dione and no estradiol production is detectable (8) and pos-sesses only faint aromatizing capacity.

A Graafian or antral follicle measures 0.4–2.3 mm in di-ameter and is characterized by a cavity of antrum containinga fluid termed follicular fluid. The development of the antralcavity begins with the formation of a cavity on the one pole ofthe oocyte. After antrum formation, the development of thefollicle precipitates, and in sixty days, the follicle reaches thepreovulatory stage. The size of an antral follicle is mainly de-termined by the size of the antral cavity and the proliferationrate of the follicle cells. In a dominant follicle, for example,extremely rapid proliferation of granulosa and theca cellsoccurs, and therefore, the dominant follicle is correlatively big-ger in size than any other follicle during the follicular phase ofthe cycle.

During antrum development, the follicles acquire capillarynetworks, located in the theca interna and externa. The bloodvessels increase in number and size as follicular developmentproceeds but do not penetrate the basal membrane (9). It isbelieved that VEGF, a mitogenic factor, is involved in angio-genesis process and thus in antral cavity formation, throughVEGF receptors. It has been shown that inhibition of VEGFresults in decreased follicle angiogenesis, reduced recruitment,and growth of antral follicles in the primate. VEGF is also

3

thought to be involved in the ovulatory process, as other stud-ies correlate the VEGF with local factors involved in ovulation(10). Furthermore, suppression of VEGF in the developing fol-licle is associated with inhibition of follicular angiogenesis andantral follicular development, which results in the inhibition ofovulation (11, 12).

FSH is considered to be the fundamental driver of folliculo-genesis. During the normal menstrual cycle, elevated FSH levelsin the early follicular phase stimulate recruitment and growthof preantral and small antral follicles. In the mid- and latefollicular phases, however, the decline of FSH concentrationsand a progressive rise of LH levels are associated with the se-lection and growth of the dominant follicle destined for ovu-lation. Gonadotropins are even used in controlled ovarianstimulation (COS), which is an important component of assis-ted reproduction technology (ART). Particularly, exogenousFSH administration, alone or with variable amounts of LHactivity, causes a rise of FSH concentrations throughout thefollicular phase, so that the development of multiple ovarianfollicles and oocytes is achieved (13). However, recent studieshave shown that selective addition of LH activity, in the form oflow-dose hCG, can replace mid- or late follicular phases� FSHadministration (14).

C O R P U S L U T E U M

Following ovulation, under the influence of luteogenic hor-mones, the corpus luteum (CL) develops from the remnantsof the ovulated ovarian follicle. This process called luteiniza-tion and the stimulus for its initiation, the preovulatory LHsurge, are common among species. The morphological eventsunderlying this process involve intense reorganization ofconstituent cells, particularly granulosa cells, phenomenathat includes varying cell-matrix interactions. These events,however, are poorly characterized. After expulsion of theoocyte, the blood capillaries of the theca rapidly invade thegranulosa, thereby provoking the transformation of these cells(luteinization) and the formation of the CL. The blood vesselscompletely traverse the granulosa and open up in the follicu-lar cavity. The granulosa cells are transformed into large lutealcells whose ultrastructure is the same as that of steroidogeniccells. The main hormone product of the CL is progesterone,which induces the necessary endometrial modificationsrequired for the acquisition of a receptive state, an anticipa-tion of embryo implantation. The life span of the CL islimited. In a nonfertile cycle, corpora lutea regress at theend of the menstrual cycle and are eliminated by a processcalled luteolysis. If pregnancy does occur, regression must beinhibited since the CL is the main source of steroidogenesis,supporting the establishment and maintenance of a success-ful pregnancy. Although some of the biochemical and endo-crine events characterizing the formation and regression of CLhave been well established, the molecular aspects underlyingluteinized granulosa cell (GC) migration and survival andthe endocrine/paracrine mechanisms by which LH and hCGact on GCs to transform the ruptured follicle into the CL arenot well characterized. A number of studies have shown thatcell-cell adhesion is strongly correlated with maturation andintegrity of CL (15). It is also believed that VEGF and its receptorFlt-1, which is expressed on luteinizing GCs (Figure 1.1), areinvolved in CL development. Recent studies, in a rat model, haveshown that suppression of VEGF resulted in nearly complete

suppression of CL formation (16). Our unpublished data ex-tend these observations and support the idea that hCG pro-motes the migration and survival of human luteinized GCs,through a VEGF-dependent mechanism. Particularly, the fol-lowing model is proposed (Figure 1.2): The binding of luteo-genic hormone (LH or hCG) to GCs triggers their release ofVEGF and induces the surface expression of VEGFR on thesecells. The released VEGF (and possibly VEGF from other sour-ces) in turn binds to the newly expressed VEGFR on the GCs,stimulating the secretion of FN into the surrounding matrix andupregulating the surface expression of at least two FN-binding

Figure 1.1. In situ staining of ovarian tissue for expression of Flt-1.Ovarian tissue was stained with antibodies against Flt-1 to determineits expression in developing follicles and during CL formation and

regression. Flt-1 was not detected in developing follicles (A, arrows)but was present on the GCs of early luteal phase (B), expression thatwas not evident during the late luteal phase (C). (magnification: 3400).

4 n A . M A K R I G I A N N A K I S , A . R O L A K I n

integrins, a5b1 and avb3. Subsequent interactions between FNand these integrins trigger adhesive events and intracellularsignaling cascades involved in promoting the migration, sur-vival, and differentiation of GCs, activities that contributeultimately to the formation and/or persistence of the CL.Relative to atresia, little is known about luteolysis and themechanisms that are involved in this process. Apoptosis seemsto be the mechanism of CL regression in humans (17). Whileapoptosis is present already in the early CL, it is significantlyincreased in the late CL when luteal regression takes place (18).The Bcl-2 family members have been shown to play a centralrole in this process (19, 20).

Apoptosis and Apoptosis-Related Genes

It has been mentioned before that apoptosis or programmedcell death is an essential process in maintaining ovarian homeo-stasis in mammals and plays a prominent role in the develop-ment of fetal ovaries and in the postnatal ovarian cycle (21). Itensures that in every estrus/menstrus cycle, only one or veryfew follicles will ovulate. This process minimizes the possibilityof multiple embryos during pregnancy. The rest of the folliclesare gradually eliminated during the fertility period of thefemale. The apoptotic process of the old corpora lutea is essentialfor preserving the cyclicity and for ensuring the release of pro-gesterone during the estrus/menstrus cycle (22). Furthermore,

recent studies have shown that apoptosis of granulosa cellsaffects the conception in ovulation induction cycle (23) andthat might explain the etiology of unexplained infertility (24).As is the case with other major organ systems, an evolutionarilyconserved framework of genes and signaling pathways has beenimplicated in determining whether or not ovarian germ cellsand somatic cells will die in response to either developmentalcues or pathological insults. Therefore, it has been suggestedthat some apoptosis-related genes may have a role in ovarianfollicular growth and atresia. The p53 gene is one of the mosthighly investigated tumor suppressor genes, and it seems to bea key player in apoptosis (25). The basic action of p53 is toprotect the cellular genome from a variety of deleterious stim-uli, such as reactive oxygen species and ionizing radiation. p53is a transcriptional factor, which has the ability to alter theactivity of target genes, an action that can be modulated byanother antioncogenic protein, the product of the Wilms�tumour suppressor gene (WT1) (26). Regarding p53, nuclearaccumulation of this tumor suppressor protein has beendocumented in GCs of follicles destined for atresia in the ratovary, whereas in vivo gonadotropin priming inhibits granu-losa cell apoptosis with a concomitant suppression of p53immunoreactivity (27). These initial investigations have sincebeen confirmed and extended by a number of laboratories, col-lectively supporting the hypothesis that nuclear translocation ofp53 in GCs heralds their demise during follicular atresia (28).

Figure 1.2. Proposed mechanism for the involvement of fibronectin and two of its integrin ligands in CL formation and their regulation by hCGthrough VEGF in this process.

n F O L L I C U L O G E N E S I S n 5

That p53 serves a similar function in the human ovary is sug-gested by the findings of p53 expression in the human ovaryand isolated granulosa cells (29), as well as by recent studies onthe ability of overexpressed p53 to induce apoptosis in trans-formed human granulosa cell lines (30). However, the spatiallocalization of p53 in the human ovary during follicular devel-opment and the regulation of tumor suppressor gene expres-sion in nontransformed human GCs remain to be determined.Evidence linking these two important members of the tumorsuppressor gene family, p53 and WT1, to ovarian folliculargrowth and atresia has been provided (31). It was shown thatthe p53 gene is expressed in GCs and is closely related to theirsurvival. This apoptosis-inducing gene is reduced by treatmentwith exogenous gonadotropin in vivo (27), suggesting that p53may play an important role in regulating follicular survivalduring gonadotropin-dependent stages of follicular life or

maintenance of luteal cells during pregnancy. WT1, a knowntranscriptional regulator of p53, is also expressed in humanGCs (31). Data that we have published indicate that this geneis constitutively expressed in human granulosa cells and itsexpression remains strong during early stages of developmentbut progressively declines during gonadotrophin-dependentfollicular maturation. These data suggest a possible implicationof WT1 in the mechanisms responsible for the maintenance ofa quiescent state in follicles during gonadotropin-independentstages of follicular life (31) (Figure 1.3). Many p53-regulatedtarget genes have been identified, including the bcl-2 prosur-vival gene, the bax proapoptotic gene, and several reduction-oxidation genes. In particular, it is believed that interactionamong pro- and antiapoptotic members of the bcl-2 familyin the mitochondrion determines whether or not the apoptoticpathways are activated. This interaction regulates release of

Figure 1.3. Expression of p53 and WT1 in the human ovary in situ. (A, B, and C) Immunolocalization of WT1 in isolated preantral human follicles.Human preantral follicles were isolated from ovarian biopsy specimens and immunostained for WT1. Under low magnification (A), multiplepreantral follicles of varying sizes (arrows) can be seen surrounded by WT1-positive GCs. Under high magnification (B, phase contrast), remaining

adherent GCs stain intensely for WT1 (C, arrow). Immunohistochemical detection of p53 (D) and WT1 (E) in fetal human ovary in situ. Note thatp53 staining is absent (D) in contrast to the very strong staining of WT1 in almost all GCs (E, arrows). Immunohistochemical detection of p53 inan atretic follicle (F) and at a higher magnification (G); WT1 staining (H) and at a higher magnification (I) in an atretic follicle. Note that in therepresentative atretic follicle, p53 nuclear staining and some cytoplasmic staining were present in many GCs (G, arrow) in contrast to the absence

of WT1 (H and I) in an antrum. Magnification: A and F, 33100; B and C, 33600; D, E, G, and I, 33400; H, 33200.

6 n A . M A K R I G I A N N A K I S , A . R O L A K I n

cytochrome c from the mitochondrion into the cytosol, whichactivates caspase-9 and downstream caspases, including cas-pase-3 (32). Bcl-2 gene was first discovered by its involvementin B-cell malignancies. A growing number of studies have im-plicated this proto-oncogene in apoptotic events of ovarianfollicles in the different developmental stages. In the recentyears, it has been demonstrated that Bcl-2 is an important fac-tor in regulating apoptosis of human GCs (33), but the role ofBcl-2 in ovarian function remains to be fully elucidated. Baxgene is another member of the bcl-2 gene family, which caninduce apoptosis through inactivation of bcl-2 in cells. Targe-ted disruption of the bax gene in mice leads to a defect in theability of GCs to undergo apoptosis during follicular atresia(34), while a reduction in bax levels was correlated withgonadotropin-mediated follicular survival in rat GCs (27).Bax has also been detected in human apoptotic GCs, but notin healthy follicles (29), even though its precise role in the de-velopment and apoptosis of human follicles is not yetunderstood.

Role of Steroids

Sex steroids play an important role in the development of theovarian follicle and in the preservation of fertility in women(35). In the ovary, steroids are produced by theca and gran-ulosa cells, and this process is induced by gonadotropins. Thewell-known ‘‘two-cell, two-gonadotrophin’’ theory emphasi-zes the fact that stimulation of both theca cells by LH andgranulosa cells by FSH is required for estradiol synthesis. Pro-gesterone (P4) is one of the major steroids secreted by theovary, and it is synthesized by preantral follicles. The rate ofsecretion is increased while follicular development proceeds,and many studies have shown the importance of these high P4levels in regulating ovulation (36). It has been suggested thatprogesterone, and other ovarian steroids, may act upon theovary in part through influences on cGMP concentrations(37). Progesterone production is induced by LH as it has beenshown in rat and porcine cultured GCs, whereas progesteroneantagonist, RU486, inhibits luteinization of GCs coming frompreovulatory follicles (38). Moreover, it has recently been in-dicated that progesterone is involved in stimulating ovulationin human ovarian follicles (39), probably by inducing pro-duction of proteolytic enzymes, important for ovulating pro-cess. Progesterone also acts through the progesteronereceptor to inhibit GC/luteal cell apoptosis (40). We have alsoshown that RU486 triggers apoptosis in human GC. Andro-gens are produced by theca cells in response to LH and exerttheir actions via receptors localized to secondary and domi-nant follicles in human. Prenatal testosterone treatment inewes indicates an enhancement in follicular development(41), whereas treatment with androgens in primate ovariesled to an increased number of small antral and preantral fol-licles (42). Moreover, androgens are thought to play a role inoocyte maturation by enhancing granulosa cells� differentia-tion (43). In PCOS patients, abnormal androgen production,by theca cells, leads to hyperandrogenism, which is thought tobe responsible for anovulation (44). This hypothesis is sup-ported by reports from therapies with the antiandrogens, flu-tamide, or cyproterone acetate, where ovulation was restoredand the rates of pregnancy were extremely higher (45). How-ever, the precise mechanisms that lead to anovulation ina large number of PCOS patients are very complex and need

to be elucidated in the future. Estrogens are also produced inthe ovary by aromatization of androgens. In particular,androgens are the substrate of P450 aromatase, an enzymethat mediates the conversion to estrogens. Estradiol exertsits actions via two forms of receptors, ERa and ERb. Bothisoforms are important for the female reproductive ability,but ERb receptor has shown to be implicated in folliculargrowth (46). In normal ovaries, increased estrogen produc-tion by the dominant follicles leads to a decrease in FSH se-rum concentration due to negative feedback effects ofestradiol on the hypothalamipituitary axis. The reduction inFSH levels inhibits the development of less mature follicles,but whether estrogens are essential for follicular growth andoocyte maturation remains unclear yet.

Role of Adhesion Molecules

Follicular development is established by gap junctions andadhesion-type junctions between GCs. The degeneration ofthe follicles and the CL regression are associated with the lossof adherence between GC. Previous studies have also revealedthat a single large GC is twice as likely to be apoptotic afterculture, as an aggregated GC (47). This observation impliesthat cell contact may inhibit GC apoptosis. Despite the factthat aggregated GCs are more steroidogenic, there is evidencethat progesterone production is not associated with the mech-anism by which cell contact prevents apoptosis of GCs. It wasalso been found that gap junctions are not involved with thesurvival of GCs, and this observation supported the idea thatthe adhesion-type junctions convey the protective effects ofcell contact. Cell-to-cell contact is mediated by a great diver-sity of cell adhesion molecules including some integrins, theimmunoglobulin supergene family, selectins, and cadherins.The expression of these adhesion molecules is cell specific,with cadherins involved in mediating calcium-dependentcell-to-cell adhesion in virtually all solid tissues of multicel-lular organisms (48). Cadherins are a rapidly expanding familyof calcium-dependent CAMs and have been shown to regulateepithelial, endothelial, neural, and cancer cell adhesion, withdifferent CADs expressed on different cell types. The adhesionjunctions are formed between adjacent cells through a homo-philic interaction of N-cadherin molecules, which is expressedin primordial, primary, and early secondary follicles, as well asin healthy antral follicles (47) and is located at the junctionalinterface between aggregating cells. Luteal cells are also stronglypositive for N-cadherin in the early luteal and midluteal phase,whereas there is only weak N-cadherin staining during lateluteal phase. As the follicle degenerates, the expression of N-cadherin decreases, and GCs ultimately dissociate (47). Inaddition, apoptosis does not occur in preantral follicles and isvery low in the early luteal phase, whereas it increases signifi-cantly in the late luteal phase (47). These observations suggest(a) that the expression of N-cadherin is regulated in humanGCs in vivo during follicular maturation and CL formationand (b) that there is a direct correlation between the presenceof the N-cadherin molecule and the absence of features char-acteristic of cellular apoptosis. The mechanism through whichN-cadherin exerts the survival effects on GCs is not yetcompletely understood. Recent studies have shown that N-cadherin interacts with the FGF receptor, which is requiredfor cell contact to prevent apoptosis (49). Additionally, bFGFinduces the tyrosine phosphorylation of its own receptor by

n F O L L I C U L O G E N E S I S n 7

inducing PKC activity, a process that is involved in stimulatingcalcium uptake into the cytoplasmic stores (50). Taking thesedata together, it is likely that N-cadherin interaction with FGFreceptor promotes cell survival by enhancing the activity ofPKC and thereby maintaining calcium homeostasis (51). More-over, it have been demonstrated that cell-cell aggregation pro-motes survival of GCs and that loss of N-cadherin from the cellsurface induces apoptosis in these cells, supporting a major roleof this adhesion molecule in the GC life cycle. N-cadherin pos-sesses an extracellular domain with five tandemly arrangedrepeats. The N-terminal repeat contains the adhesive domainthat is involved in cadherin-specific adhesions. Cleavage of theextracellular domain by metalloproteinases (MMP) is followedby loss of the adhesive ability of N-cadherin and cell death.It has been shown that inhibition of cleavage by MMP inhib-itor decreases the rate of apoptosis in granulosa cells (47).Other studies have indicated cAMP-dependent pathways thatinduce downregulation of N-cadherin in a dose-dependentmanner.

E-cadherin is also an important member of the cadherinsuperfamily, and it is expressed in spontaneously immortalizedgranulosa cells (SIGC) (52). E-cadherin connects adjacent cells,and a disruption in calcium-dependent cell contacts, withEGTA or an E-cadherin antibody, results in an increase in cas-pase-3 activity, in both the cytoplasm and nuclei of SIGCs.There have been detected cleavage products of b-catenin, whichis an E-cadherin-associated protein, in apoptotic SIGCs. Pre-vious studies have revealed that b-catenin and E-cadherin aresubstrates for caspase-3. These findings strengthened the ideathat the increase of the cytoplasmic caspase-3 activity is asso-ciated with the degradation of b-catenin and E-cadherin. It isthus presumed that promotion of cell survival by E-cadherin isregulated by a signal transduction pathway that inhibits theactivation of caspase-3.

C O N C L U S I O N S

Follicular development and CL formation and regression inhuman ovaries are strongly correlated with female reproductivecapacity. Production of steroids and apoptosis of ovarian cellsseems to play an active and important role in ovarian physio-logical functions. Disruption of the normal activity of the ovarymay lead to reproductive disorders or even malignancies.Therefore, the mechanisms that control the normal life cycleof the dominant follicle, from folliculogenesis to luteolysis,must be elucidated.

K E Y P O I N T S

n Development of multiple ovarian follicles and oocytes isachieved in controlled ovarian stimulation, which is animportant component of assisted reproduction technology.

n The main hormone product of the CL is progesterone,which induces the endometrial modifications required forembryo implantation.

n Progesterone is also involved in stimulating ovulation inhuman ovarian follicles.

n In PCOS patients, abnormal androgen production, by thecacells, leads to hyperandrogenism, which is thought to beresponsible for anovulation.

n Estrogen production by the dominant follicle leads to a de-crease in FSH levels, which inhibits the development of lessmature follicles.

n Apoptosis or programmed cell death ensures that in everyestrus/menstrus cycle, only one or very few follicles willovulate. This process minimizes the possibility of multipleembryos during pregnancy.

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3. Markström E, Svensson EC, Shao R, et al. 2002. Survival factorsregulating ovarian apoptosis—dependence on follicle differenti-ation. Reproduction 123:23–30.

4. Zeleznik JA. 2004. The physiology of follicle selection. ReprodBiol Endocrinol 2:31–7.

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6. Touraine P, Beau I, Gougeon A, et al. 1999. New natural inacti-vating mutations of the follicle-stimulating hormone receptor:correlations between receptor function and phenotype. MolEndocrinol 13:1844–54.

7. Visser JA, Themmen AP. 2005. Anti-Mullerian hormone andfolliculogenesis. Mol Cell Endocrinol 234:81–6.

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12. Filicori M, Cognigni EG. 2001. Roles and novel regimens ofluteinizing hormone and follicle stimulating hormone in ovula-tion induction. J Clin Endocrinol Metab 86:1437–41.

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14. Filicori M, Cognigni EG, Tabarelli C, et al. 2002. Stimulation andgrowth of antral ovarian follicles by selective LH activity admin-istration in women. J Clin Endocrinol Metab 87:1156–61.

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16. Senger DR, Claffey KP, Benes JE, et al. 1997. Angiogenesis pro-moted by vascular endothelial growth factor: regulation througha1b1 and a2b1 integrins. Proc Natl Acad Sci USA 94:13612–17.

17. Vaskivuo TE, Ottander U, Oduwole O, et al. 2002. Role of ap-optosis, apoptosis-related fectors and 17beta-hydroxysteroiddehydrogenases in human corpus luteum regression. Mol CellEndocrinol 30:191–200.

18. Vaskivuo TE, Tapanainen JS. 2003. Apoptosis in the humanovary. Reprod BioMed Online 6(1):24–35.

19. Rodger FE, Fraser HM, Krajewski S, et al. 1998. Production ofthe proto-oncogene Bax does not vary with changing in lutealfunction in women. Mol Hum Reprod 4:27–32.

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20. Sugino N, Suzuki T, Kashida S, et al. 2000. Expression of Bcl-2and Bax in the human corpus luteum during the menstrual cycleand in early pregnancy: regulation by human chorionic gonad-otropin. J Clin Endocrinol Metabol 85:4379–86.

21. Rolaki A, Drakakis P, Millingos S, et al. 2005. Novel trends infollicular development, atresia and corpus luteum regression:a role for apoptosis. Reprod Biomed Online 11:93–103.

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23. Oosterhuis GJE, Michgelsen HW, Lambalk CB, et al. 1998.Apoptotic cell death in human granulosa-lutein cells: a possibleindicator of in vitro fertilization outcome. Fertil Steril 4:747–9.

24. Idil M, Cepni I, Demirsoy G, et al. 2004. Does granulosa cellapoptosis have a role in the etiology of unexplained infertility?Eur J Obstet Gynecol Reprod Biol 112:182–4.

25. Kaelin WG Jr. 1999. Cancer. Many vessels, faulty gene. Nature399:203–4.

26. Davies R, Moore A, Schedl A, et al. 1999. Multiple roles for theWilms� tumor suppressor, WT1. Cancer Res 59:1747–50.

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28. Kim JM, Yoon YD, Tsang BK. 1999. Involvement of the Fas/Fasligand system in p53-mediated granulosa cell apoptosisduring follicular development and atresia. Endocrinology 140:2307–17.

29. Kugu K, Ratts VS, Piquette GN, et al. 1998. Analysis of apoptosisand expression of bcl-2 gene family members in the human andbaboon ovary. Cell Death Differen 5:67–76.

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34. Knudson CM, Tung KSK, Tourtellotte WG, et al. 1995. Bax-deficient mice with lymphoid hyperplasia and male germ celldeath. Science 270:96–99.

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36. Robker RL, Russell DL, Espey LL, et al. 2000. Progesterone-regulated genes in the ovulation process: ADAMTS-1 andcathepsin L proteases. Proc Natl Acad Sci USA 97:4689–94.

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38. Natraj U, Richards JS. 1993. Hormonal regulation, localisationand functional activity of the progesterone receptor in granulosacells of rat preovulatory follicles. Endocrinology 133:761–9.

39. Zalanyi S. 2001. Progesterone and ovulation. Eur J Obstet Gyne-col Reprod Biol 98:152–9.

40. Makrigiannakis A, Coukos G, Christofidou-Solomidou M, et al.2000. Progesterone is an autocrine/paracrine regulator of humangranulosa cell survival in vitro. Ann N Y Acad Sci 900:16–25.

41. Steckler T, Wang J, Bartol FF, et al. 2005. Fetal programming:prenatal testosterone treatment causes intrauterine growth re-tardation, reduces ovarian reserve and increases ovarian follicu-lar recruitment. Endocrinology 3185–93.

42. Vendola KA, Zhou J, Adesanya OO, et al. 1998. Androgensstimulate early stages of follicular growth in the primate ovary.J Clin Investig 101:2622–9.

43. Hillier SG, De Zwart FA. 1981. Evidence that granulosa cell aro-matase induction/activation by follicle-stimulating hormone isan androgen receptor-regulated process in-vitro. Endocrinology109:1303–5.

44. Abbott DH, Dumesic DA, Franks S. 2002. Developmental originof polycystic ovary syndrome—a hypothesis. J Endocrinol 174:1–5.

45. De Leo V, Lanzetta D, D�Antona D, et al. 1998. Hormonal effectsof flutamide in young women with polycystic ovary syndrome. JClin Endocrinol Metab 83:99–102.

46. Hegele-Hartung C, Seibel P, Peters O, et al. 2004. Impact ofisotype-selective oestrogen receptor agonists on ovarian func-tion. Proc Natl Acad Sci USA 101:5129–34.

47. Makrigiannakis A, Coukos G, Christofidou-Solomidou M, et al.1999 N-cadherin mediated human granulosa cell adhesion pre-vents apoptosis: a role in follicular atresia and luteolysis? Am JPathol 154:1391–406.

48. Knudsen KA, Soler AP, Johnson KR et al. 1995. Interaction ofa-actinin with the cadherin cell-cell adhesion complex viaacatenin. J Cell Biol 130:67–77.

49. Trolice MP, Pappalardo A, Peluso JJ. 1997. Basic fibroblastgrowth factor and N-Cadherin maintain rat granulosa cell andovarian surface epithelial cell viability by stimulating the tyrosinephosphorylation of the fibroblast growth factor receptors. Endo-crinology 138:107–13.

50. Fewtrell C. 1993. Ca+ oscillations in non-excitable cells. AnnuRev Physiol 55:427–54.

51. Peluso JJ. 1997. Putative mechanism through which N-Cadherin-mediated cell contact maintains calcium homeostasis and therebyprevents ovarian cells from undergoing apoptosis. BiochemPharmacol 54:847–53.

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mechanisms of follicular development:the role of gonadotrophins

Ioannis E. Messinis

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

Folliculogenesis in women is a dynamic and uninterrupted pro-cess from fetal life until menopause. Following pubertal matu-ration of the reproductive axis, all types of follicles from theprimordial to the preovulatory stage are present in the humanovary. Over the past twenty years, it has become clear that thesefollicles represent sequential forms of the developmental pro-cess classified into eight categories, based on the size and thenumber of the granulosa cells (Gougeon, 1986). For example,class 1 corresponds to a secondary preantral follicle and class 8to a large preovulatory follicle.

Folliculogenesis is a lengthy process (Figure 2.1). Based onthe calculation of the doubling time of granulosa cells, it isestimated that the time spent from the primordial to the pre-ovulatory stage is approximately one year (Gougeon, 1986).However, maturation of a follicle from class 1 to class 8 isachieved within eighty-five days (Gougeon, 1986). At the be-ginning, proliferation of the granulosa cells on several layerstakes place and the primordial follicle becomes preantral. Fol-lowing this, the theca interna develops and the antral cavity isformed. The rate at which follicles leave the primordial pool isnot known. However, it seems that the departure follows anordered sequence, so that follicles formed first leave the poolearlier (Hirshfield, 1991).

It remains unclear which factors are responsible forthe initiation of maturation of a primordial follicle or what isthe trigger for the passage of a follicle from the preantral to theantral stage (Figure 2.1). In humans, this part of folliculogen-esis is gonadotrophin independent. The growth of a folliclefrom class 1 to class 5 is to some extent affected by gonadotro-phins, while from class 5 to class 8, that is, during the last fifteendays of follicle maturation that correspond to the follicularphase of the normal menstrual cycle, gonadotrophins arethe only determinants of follicle growth (Gougeon, 1986). Inother words, follicle maturation to the preovulatory stage isnot feasible without the presence of follicle-stimulatingharmone (FSH) and luteinizing harmone (LH).

I N I T I A L R E C R U I T M E N T : P R E A N T R A L F O L L I C L EG R O W T H

The term ‘‘recruitment’’ refers to a cohort of follicles that leave aparticular developmental stage for further growth (Figure 2.1).At the primordial stage, the term ‘‘initial recruitment’’ has been

proposed (McGee and Hsueh, 2000). Similarly, the term ‘‘cyclicrecruitment’’ has been proposed for the cohort of antral folli-cles from which ‘‘selection’’ of the dominant follicle takes placeduring the early follicular phase of the cycle (McGee andHsueh, 2000). It has been established that FSH is the principalhormone that promotes follicle maturation, especially at moreadvanced stages of development. Although receptors of FSHare expressed in the granulosa cells of preantral follicles (Royet al., 1987), evidence has been provided that in humans thishormone is not required for follicle maturation up to the antralstage. A logical explanation is that primordial follicles are lo-cated in an avascular part of the ovary, and therefore, they canbe easily reached by locally produced but not by systemic fac-tors (van Wezel and Rodgers, 1996). There are several examplesof follicle maturation up to the early antral stage in womeneither in the presence of negligible amounts of FSH in thecirculation, such as before puberty (Peters et al., 1978), duringpregnancy (Westergaard et al., 1985), and in cases of hypogo-nadotrophic hypogonadism (Rabin et al., 1972), or in the ab-sence of FSH activity, such as in mutations of FSHb (Matthewset al., 1993) and inactivating mutations of the FSH receptor(Touraine et al., 1999).

The situation is different in certain species in which FSHparticipates in the control of preantral follicle development, al-though it is still unclear whether this hormone is involved in themechanism that triggers initial recruitment. In vitro data havedemonstrated that FSH is a growth and differentiation factor forrat preantral follicles in the presence of a cGMP analog thatsuppresses apoptosis (McGee et al., 1997). However, FSH alonedid not prevent apoptosis in these follicles. Also, preantral folliclegrowth, number of cells, and cell differentiation were promotedby FSH, and these effects were enhanced by the addition ofantimullerian hormone (AMH or MIS) or activin (McGeeet al., 2001). In contrast to rats, in mice, AMH, produced bypreantral follicles, inhibited initial recruitment as well as the stim-ulatory effect of FSH on the growth from the primary to the earlyantral stage (Visser and Themmen, 2005). Although still unclear,species variability may account for these opposite actions ofAMH. Recently, AMH was able to inhibit initiation of humanprimordial follicle growth in vitro (Carlsson et al., 2006).

Several genes encoding specific proteins and growth factorsare expressed in the granulosa cells of small follicles. Thesefactors including epidermal growth factor (EGF), transforminggrowth factor-a (TGF-a), TGF-b and insulin-like growthfactor-1 (IGF-1) may be involved in the initiation of growth

1 0

of primordial follicles (May et al., 1990; Adashi, 1998; Knightand Glister, 2006). A group of such factors, members of theTGF-b superfamily, are shown in Figure 2.2. Another factor,produced particularly in primordial and primary follicles, isWilms tumor suppressor gene (WT1) that may have a suppres-sive role in the expression of genes of various growth factors,maintaining thus the follicles at the resting stage (Hsu et al.,1995). Similarly, retinoblastoma protein (pRb) may also have asuppressive effect by inhibiting proliferation of granulosa cellseven in humans (Bukovsky et al., 1995a, 1995b). A role in thetransition from the primordial to the primary stage may be alsoplayed by the oncogene myc (Piontkewitz et al., 1997), the steellocus encoding the kit ligand (Huang et al., 1993), and theplatelet-derived growth factor (Nilsson et al., 2006). Finally,neurotrophin molecules, such as nerve growth factor (NGF)

may also play a role in mice for early follicular development(Dissen et al., 2001).

During follicle growth, the oocyte undergoes functionalchanges in order to become fertilizable at ovulation. However,various factors derived from the oocyte seem to play a crucialrole for preantral follicle development (Erickson and Shimasaki,2001). For example, growth differentiation factor-9 (GDF-9),a member of TGF-b superfamily, is expressed by oocytes(McGrath et al., 1995) and stimulates granulosa cel