Textbook of assisted reproductive techniques Volume

2 Clinical Perspectives 4th Edition Edited By David

K. Gardner digital version 2025

Find it at ebookgate.com
( 4.5/5.0 ★ | 142 downloads )

https://ebookgate.com/product/textbook-of-assisted-reproductive-
techniques-volume-2-clinical-perspectives-4th-edition-edited-by-
david-k-gardner/
 Textbook of assisted reproductive techniques Volume 2
Clinical Perspectives 4th Edition Edited By David K. Gardner

EBOOK

Available Formats

■ PDF eBook Study Guide Ebook

EXCLUSIVE 2025 ACADEMIC EDITION – LIMITED RELEASE

Available Instantly Access Library

Instant digital products (PDF, ePub, MOBI) available
Download now and explore formats that suit you...

Human Assisted Reproductive Technology Future Trends in

Laboratory and Clinical Practice 1st Edition David K.
Gardner
https://ebookgate.com/product/human-assisted-reproductive-technology-
future-trends-in-laboratory-and-clinical-practice-1st-edition-david-k-
gardner/
ebookgate.com

Surveying Volume 2 4th Edition S. K. Duggal

https://ebookgate.com/product/surveying-volume-2-4th-edition-s-k-
duggal/

ebookgate.com

Harper s Textbook of Pediatric Dermatology 2 Volume Set

4th Edition Peter H. Hoeger

https://ebookgate.com/product/harper-s-textbook-of-pediatric-
dermatology-2-volume-set-4th-edition-peter-h-hoeger/

ebookgate.com

Textbook of Human Reproductive Genetics Karen Sermon

https://ebookgate.com/product/textbook-of-human-reproductive-genetics-
karen-sermon/

ebookgate.com
Textbook of Veterinary Internal Medicine 6th Edition 2
Volume Set Volume 2 Stephen J. Ettinger

https://ebookgate.com/product/textbook-of-veterinary-internal-
medicine-6th-edition-2-volume-set-volume-2-stephen-j-ettinger/

ebookgate.com

LogoLounge 5 2 000 International Identities by Leading

Designers Bill Gardner

https://ebookgate.com/product/logolounge-5-2-000-international-
identities-by-leading-designers-bill-gardner/

ebookgate.com

Bedside Techniques Methods of Clinical Examination 4th

Edition Muhammad Inayatullah

https://ebookgate.com/product/bedside-techniques-methods-of-clinical-
examination-4th-edition-muhammad-inayatullah/

ebookgate.com

Methods and Applications in Reservoir Geophysics Edited

By: David H. Johnston

https://ebookgate.com/product/methods-and-applications-in-reservoir-
geophysics-edited-by-david-h-johnston/

ebookgate.com

SDG18 Communicaton for All Volume 2 Regional Perspectives

and Special Cases 4th Edition Jan Servaes

https://ebookgate.com/product/sdg18-communicaton-for-all-
volume-2-regional-perspectives-and-special-cases-4th-edition-jan-
servaes/
ebookgate.com
 Textbook of
Assisted Reproductive
Techniques
The editors (from left to right: Colin M. Howles, Ariel Weissman, David K. Gardner,
and Zeev Shoham) at the annual meeting of ESHRE, Stockholm, July 2011
 Textbook of
Assisted Reproductive
Techniques
Fourth Edition

Volume 2: Clinical Perspectives

Edited by

David K. Gardner DPhil

Chair of Zoology, University of Melbourne, Australia

Ariel Weissman MD
Senior Physician, IVF Unit, Department of Obstetrics and Gynecology,
Edith Wolfson Medical Center, Holon and Sackler Faculty of Medicine,
Tel Aviv University, Tel Aviv, Israel

Colin M. Howles PhD, FRSM

Vice President, Regional Medical Affairs Fertility, External Scientific Affairs,
Global Development and Medical, Merck Serono SA, Geneva, Switzerland

Zeev Shoham MD
Director, Reproductive Medicine and Infertility Unit, Department of Obstetrics and
Gynecology, Kaplan Medical Center, Rehovot, Israel
CRC Press
Taylor & Francis Group
6000 Broken Sound Parkway NW, Suite 300
Boca Raton, FL 33487-2742
© 2012 by Taylor & Francis Group, LLC
CRC Press is an imprint of Taylor & Francis Group, an Informa business

No claim to original U.S. Government works

Version Date: 20130604

International Standard Book Number-13: 978-1-84184-973-7 (eBook - PDF)

This book contains information obtained from authentic and highly regarded sources. While all reasonable efforts have
been made to publish reliable data and information, neither the author[s] nor the publisher can accept any legal responsibil-
ity or liability for any errors or omissions that may be made. The publishers wish to make clear that any views or opinions
expressed in this book by individual editors, authors or contributors are personal to them and do not necessarily reflect
the views/opinions of the publishers. The information or guidance contained in this book is intended for use by medical,
scientific or health-care professionals and is provided strictly as a supplement to the medical or other professional’s own
judgement, their knowledge of the patient’s medical history, relevant manufacturer’s instructions and the appropriate best
practice guidelines. Because of the rapid advances in medical science, any information or advice on dosages, procedures or
diagnoses should be independently verified. The reader is strongly urged to consult the drug companies’ printed instruc-
tions, and their websites, before administering any of the drugs recommended in this book. This book does not indicate
whether a particular treatment is appropriate or suitable for a particular individual. Ultimately it is the sole responsibility
of the medical professional to make his or her own professional judgements, so as to advise and treat patients appropriately.
The authors and publishers have also attempted to trace the copyright holders of all material reproduced in this publication
and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material
has not been acknowledged please write and let us know so we may rectify in any future reprint.

Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or uti-
lized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopy-
ing, microfilming, and recording, or in any information storage or retrieval system, without written permission from the
publishers.

For permission to photocopy or use material electronically from this work, please access www.copyright.com (http://www.
copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-
750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organiza-
tions that have been granted a photocopy license by the CCC, a separate system of payment has been arranged.

Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for iden-
tification and explanation without intent to infringe.
Visit the Taylor & Francis Web site at
http://www.taylorandfrancis.com
and the CRC Press Web site at
http://www.crcpress.com
Contents

List of contributors viii

The beginnings of human in vitro fertilization xii

Robert G. Edwards

Robert Edwards: The path to IVF xxv

Martin H. Johnson

IX Quality Management Systems

32. Quality management in reproductive medicine 1

Christoph Keck, Cecilia Sjöblom, Robert Fischer, Vera Baukloh, and Michael Alper

X Patient Investigation and the Use of Drugs

33. Lifestyle, periconception, and fertility 10

Robert J. Norman, Lisa J. Moran, and Sarah A. Robertson
34. Indications for IVF treatment: From diagnosis to prognosis 18
Ido Ben-Ami, Arie Raziel, Shevach Friedler, Yariv Gidoni, Raphael Ron-El, and Bart C. J. M. Fauser
35. Initial investigation of the infertile couple 31
Isabelle Roux, Togas Tulandi, Peter Chan, and Hananel Holzer
36. Prognostic testing for ovarian reserve 41
Frank J. Broekmans, Simone L. Broer, Bart C. J. M. Fauser, and Nick S. Macklon
37. Drugs used for ovarian stimulation: Clomiphene citrate, aromatase inhibitors,
metformin, gonadotropins, gonadotropin-releasing hormone analogs,
and recombinant gonadotropins 51
Zeev Shoham and Colin M. Howles
38. The role of FSH and LH in ovulation induction: Current concepts 75
Juan Balasch

XI Stimulation Protocols

39. Endocrine characteristics of ART cycles 99

Jean-Noël Hugues and Isabelle Cédrin-Durnerin
40. The use of GnRH agonists 115
Roel Schats and Judith A. F. Huirne
41. GnRH-antagonists in ovarian stimulation for IVF 124
Efstratios M. Kolibianakis, Georg Griesinger, and Basil C. Tarlatzis
42. The use of AMH to tailor ovarian stimulation for IVF 131
Scott M. Nelson
43. Monitoring ovarian response in IVF cycles 140
Matts Wikland and Torbjörn Hillensjö
44. Oocyte collection 145
Gab Kovacs
45. Luteal support in ART 153
Dominique de Ziegler, Isabelle Streuli, Vanessa Gayet, Usama Bajouh, Juliane Berdah,
and Charles Chapron
vi CONTENTS

46. Treatment strategies in assisted reproduction for the poor responder patient 162
Ariel Weissman and Colin M. Howles
47. Repeated implantation failure 208
Mark A. Damario and Zev Rosenwaks

XII Technical Procedures and Outcomes

48. Ultrasonography in assisted reproduction 225

Ilan Tur-Kaspa and Laurel Stadtmauer
49. Sperm recovery techniques: Clinical aspects 242
Herman Tournaye and Patricio Donoso
50. Processing and cryopreservation of testicular sperm 258
Kathleen Hwang, Joseph P. Alukal, Dolores J. Lamb, and Larry I. Lipshultz
51. Embryo transfer technique 263
Ragaa Mansour
52. Cycle regimes for frozen–thawed embryo transfer 272
Ingrid Granne and Tim Child
53. Anesthesia and in vitro fertilization 278
Shmuel Evron and Tiberiu Ezri
54. Medical considerations of single embryo transfer 284
Outi Hovatta

XIII Special Medical Conditions

55. Endometriosis and ART 288

Marli Amin, Andy Huang, and Alan H. DeCherney
56. PCOS and assisted reproduction 298
Susie Nicholas, Christopher Brewer, Thomas H. Tang, and Adam H. Balen
57. Management of hydrosalpinx 308
Annika Strandell
58. Fertility preservation strategies 318
Stine Gry Kristensen, Tine Greve, and Claus Yding Andersen
59. Viral disease and Assisted Reproductive Techniques 333
Carole Gilling-Smith

XIV Complications of Treatment

60. Severe ovarian hyperstimulation syndrome 341

Zalman Levine, Inna Berin, and Daniel Navot
61. The environment and reproduction 360
Machelle M. Seibel
62. Bleeding, severe pelvic infection, and ectopic pregnancy 374
Raoul Orvieto and Zion Ben-Rafael
63. Iatrogenic multiple pregnancies: The risk of ART 382
Isaac Blickstein

XV Egg Donation and Surrogate Motherhood

64. Egg and embryo donation 394

Mark V. Sauer and Matthew A. Cohen
65. Gestational surrogacy 405
Peter R. Brinsden

XVI The Support Team

66. The evolving role of the ART nurse: A contemporary review 415
Joanne L. Libraro
 CONTENTS vii

67. Patient support in the ART program 424

Sharon N. Covington
68. The relationship between stress and in vitro fertilization outcome 434
Andrea Mechanick Braverman

XVII Ethics, Religion and Legislation

69. The impact of legislation and socioeconomic factors in the access to

and global practice of assisted reproduction 441
Fernando Zegers-Hochschild, Karl G. Nygren, and Osamu Ishihara
70. Religious perspectives in human reproduction 451
Raphael Ron-El and Botros Rizk

Index 457
List of contributors

Michael Alper Inna Berin

Boston IVF, Waltham, Massachusetts, USA Fertility Institute of New Jersey and New York
Westwood, New Jersey, USA
Joseph P. Alukal
Clinical Assistant Professor of Urology, Isaac Blickstein
Department of Urology, NYU School of Medicine Department of Obstetrics and Gynecology, Kaplan
New York, New York, USA Medical Center, Rehovot, Israel

Marli Amin Andrea Mechanick Braverman

Department of Obstetrics and Gynecology, David Geffen Director, The Braverman Center for Health Journeys
School of Medicine, Los Angeles, California, USA Bala Cynwyd, Pennsylvania, USA

Claus Yding Andersen Christopher Brewer

Laboratory of Reproductive Biology, Copenhagen Leeds Centre for Reproductive Medicine, Seacroft
University Hospital, Copenhagen, Denmark Hospital, Leeds, UK

Usama Bajouh Peter R. Brinsden

Faculté de médecine, Université Paris Descartes, Bourn Hall Clinic International, Bourn, Cambridge, UK
Paris Sorbonne-Cité, and Department of Obstetrics,
Gynecology, and Reproductive Medicine, Assistance Frank J. Broekmans
Publique Hôpitaux de Paris, CHU Cochin, Department of Reproductive Medicine and Gynecology,
Paris, France Division of Woman and Baby, University Medical
Center Utrecht, Utrecht, The Netherlands
Juan Balasch
Department of Obstetrics and Gynecology, Faculty of Simone L. Broer
Medicine, Hospital Clinic, University of Barcelona, Department of Reproductive Medicine and Gynecology,
Barcelona, Spain Division of Woman and Baby, University Medical
Center Utrecht, Utrecht, The Netherlands
Adam H. Balen
Leeds Centre for Reproductive Medicine, Seacroft Isabelle Cédrin-Durnerin
Hospital, Leeds, UK University of Paris XIII, Division of Reproductive
Medicine, Hôpital Jean Verdier, Bondy, France
Vera Baukloh
Fertility Center Hamburg, Hamburg, Germany Peter Chan
Division of Reproductive Endocrinology and Infertility,
Ido Ben-Ami Department of Obstetrics and Gynecology, McGill
Fertility and IVF Unit, Assaf Harofeh Medical Center, University, Montreal, Quebec, Canada
Tel Aviv University, Tel Aviv, Israel
Charles Chapron
Zion Ben-Rafael Faculté de médecine, Université Paris Descartes,
Sackler Faculty of Medicine, Tel Aviv University, Paris Sorbonne-Cité, and Department of Obstetrics,
Tel Aviv, Israel Gynecology, and Reproductive Medicine, Assistance
Publique Hôpitaux de Paris, CHU Cochin, Paris, France
Juliane Berdah
Faculté de médecine, Université Paris Descartes, Tim J. Child
Paris Sorbonne-Cité, and Department of Obstetrics, Oxford Fertility Unit, Institute of Reproductive
Gynecology, and Reproductive Medicine, Assistance Sciences, and Nuffield Department of Obstetrics and
Publique Hôpitaux de Paris, CHU Cochin, Gynaecology, John Radcliffe Hospital, University of
Paris, France Oxford, Oxford, UK
 LIST OF CONTRIBUTORS ix

Matthew A. Cohen Yarev Gidoni

Department of Obstetrics and Gynecology, College of Fertility and IVF Unit, Assaf Harofeh Medical Center,
Physicians & Surgeons, Columbia University, New York, Tel Aviv University, Tel Aviv, Israel
New York, USA
Carole Gilling-Smith
Sharon N. Covington The Agora Gynaecology and Fertility Centre, Hove, UK
Psychological Support Services, Shady Grove
Fertility Reproductive Science Center, Rockville, Ingrid Granne
Maryland, USA Oxford Fertility Unit, Institute of Reproductive
Sciences, and Nuffield Department of Obstetrics and
Mark A. Damario Gynaecology, John Radcliffe Hospital, University of
Department of Obstetrics, Gynecology and Women’s Oxford, Oxford, UK
Health, University of Minnesota, Minneapolis,
Minnesota, USA Tine Greve
Laboratory of Reproductive Biology, Copenhagen
Alan H. DeCherney University Hospital, Copenhagen, Denmark
Department of Obstetrics and Gynecology, David Geffen
School of Medicine, Los Angeles, California, USA Georg Griesinger
Department of Reproductive Medicine and
Patricio Donoso Gynecological Endocrinology, University Hospital of
Centre for Reproductive Medicine, Clinica Alemana de Schleswig-Holstein, Luebeck, Germany
Santiago, Santiago, Chile
Torbjörn Hillensjö
Robert G. Edwards Fertility Centre Scandinavia, Carlander’s Hospital,
Duck End Farm, Dry Drayton, Cambridge, UK Göteborg, Sweden

Shmuel Evron Hananel Holzer

Obstetric Anesthesia Unit, Wolfson Medical Center, and Division of Reproductive Endocrinology and Infertility,
Sackler Faculty of Medicine, Tel Aviv University, Department of Obstetrics and Gynecology, McGill
Tel Aviv, Israel University, Montreal, Quebec, Canada

Tiberiu Ezri Outi Hovatta

Department of Anesthesiology, Wolfson Medical Center, Karolinska Institute, Karolinska University Hospital
and Sackler Faculty of Medicine, Tel Aviv University, Huddinge, Stockholm, Sweden
Tel Aviv, Israel
Colin M. Howles
Bart C. J. M. Fauser External Scientific Affairs, Global Development and
Departments of Reproduction and Gynecology, Division Medical, Merck Serono SA, Geneva,
of Woman and Baby, University Medical Center Utrecht, Switzerland
Utrecht, The Netherlands
Andy Huang
Robert Fischer Department of Obstetrics and Gynecology, David Geffen
Fertility Center Hamburg, Hamburg, Germany School of Medicine, Los Angeles, California, USA

Shevach Friedler Jean-Noël Hugues

Fertility and IVF Unit, Assaf Harofeh Medical Center, University of Paris XIII, Division of Reproductive
Tel Aviv University, Tel Aviv, Israel Medicine, Hôpital Jean Verdier, Bondy, France

David K. Gardner Judith A. F. Huirne

Department of Zoology, University of Melbourne, Department of Obstetrics and Gynecology, Division of
Victoria, Australia Reproduction and Fertility Investigation, IVF Center,
Vrije Universiteit Medical Center, Amsterdam, The
Vanessa Gayet Netherlands
Faculté de médecine, Université Paris Descartes,
Paris Sorbonne-Cité, and Department of Obstetrics, Kathleen Hwang
Gynecology, and Reproductive Medicine, Assistance Assistant Professor of Surgery (Urology),
Publique Hôpitaux de Paris, CHU Cochin, Division of Urology, Alpert Medical School
Paris, France of Brown University Providence, Rhode Island, USA
x LIST OF CONTRIBUTORS

Osamu Ishihara Daniel Navot

Department of Obstetrics and Gynaecology, Saitama Fertility Institute of New Jersey and New York,
Medical University, and Saitama Medical Centre Westwood, New Jersey, USA
Hospital, Kawagoe, Japan
Scott M. Nelson
Martin H. Johnson Department of Obstetrics and Gynaecology,
The Anatomy School, Department of Physiology, School of Medicine, University of Glasgow,
Development and Neuroscience, and The Centre for Glasgow, UK
Trophoblast Research, University of Cambridge,
Cambridge, UK Susie Nicholas
Leeds Centre for Reproductive Medicine, Seacroft
Christoph Keck Hospital, Leeds, UK
IVF Group, Endokrinologikum, Hamburg, Germany
Robert J. Norman
Efstratios M. Kolibianakis Robinson Institute and School of Paediatrics and
Unit for Human Reproduction, First Department of Reproductive Health
Obstetrics and Gynaecology, Medical School, Aristotle University of Adelaide, Adelaide, South Australia,
University of Thessaloniki, and Papageorgiou General Australia
Hospital, Thessaloniki, Greece
Karl G. Nygren
Gab Kovacs Karolinska Institute, Institute of Medical Epidemiology
Monash IVF, Richmond, Victoria, Australia & Biostatistics, Stockholm, Sweden

Stine Gry Kristensen Raoul Orvieto

Laboratory of Reproductive Biology, Copenhagen Infertility and IVF Unit, Department of Obstetrics
University Hospital, Copenhagen, Denmark and Gynecology, Barzilai Medical Center, Ashkelon,
and Faculty of Health Sciences, Ben Gurion University
Dolores J. Lamb of the Negev, Beer Sheva, Israel
Division of Male Reproductive Medicine and Surgery,
Scott Department of Urology, Baylor College of Arie Raziel
Medicine, Houston, Texas, USA Fertility and IVF Unit, Assaf Harofeh Medical Center,
Tel Aviv University, Tel Aviv, Israel
Zalman Levine
Fertility Institute of New Jersey and New York, Botros Rizk
Westwood, New Jersey, USA Reproductive Endocrinology and Infertility,
Department of Obstetrics and Gynecology,
Joanne L. Libraro University of South Alabama, Mobile,
Center for Reproductive Medicine and Infertility, Weill Alabama, USA
Medical College, New York, New York, USA
Sarah A. Robertson
Larry I. Lipshultz Robinson Institute and School of Paediatrics and
Division of Male Reproductive Medicine and Surgery, Reproductive Health, University of Adelaide, Adelaide,
Scott Department of Urology, Baylor College of South Australia, Australia
Medicine, Houston, Texas, USA
Raphael Ron-El
Nick S. Macklon Fertility and IVF Unit, Assaf Harofeh Medical Center,
Department of Obstetrics and Gynaecology, Tel Aviv University, Tel Aviv, Israel
University of Southampton, and Complete Fertility
Centre Southampton, Princess Anne Hospital, Zev Rosenwaks
Southampton, UK Center for Reproductive Medicine and Infertility,
Weill Medical College of Cornell University, New York,
Ragaa Mansour New York, USA
The Egyptian IVF Center, Cairo, Egypt
Isabelle Roux
Lisa J. Moran Division of Reproductive Endocrinology
Robinson Institute and School of Paediatrics and and Infertility, Department of Obstetrics and
Reproductive Health, University of Adelaide, Adelaide, Gynecology, McGill University, Montreal,
South Australia, Australia Quebec, Canada
 LIST OF CONTRIBUTORS xi

Mark V. Sauer Basil C. Tarlatzis

Department of Obstetrics and Gynecology, College of Unit for Human Reproduction, First Department of
Physicians & Surgeons, Columbia University, New York, Obstetrics and Gynaecology, Medical School, Aristotle
New York, USA University of Thessaloniki, and Papageorgiou General
Hospital, Thessaloniki, Greece
Roel Schats
Department of Obstetrics and Gynecology, Division of Herman Tournaye
Reproduction and Fertility Investigation, IVF Center, Center for Reproductive Medicine, University Hospital
Vrije Universiteit University Medical Center, of the Brussels Free University, Brussels, Belgium
Amsterdam, The Netherlands
Togas Tulandi
Machelle M. Seibel Division of Reproductive Endocrinology and Infertility,
Department of Obstetrics and Gynecology, University of Department of Obstetrics and Gynecology, McGill
Massachusetts School of Medicine, Worcester, University, Montreal, Quebec, Canada
Massachusetts, USA
Ilan Tur-Kaspa
Zeev Shoham Institute for Human Reproduction (IHR), and
Reproductive Medicine and Infertility Unit, Department the Department of Obstetrics and Gynecology,
of Obstetrics and Gynecology, Kaplan Medical Center, The University of Chicago, Chicago, Illinois, USA
Rehovot, Israel
Ariel Weissman
Cecilia Sjöblom IVF Unit, Department of Obstetrics and Gynecology,
Westmead Fertility Centre, University of Sydney, Edith Wolfson Medical Center, Holon, and Sackler
Western Clinical School, Westmead Hospital, Faculty of Medicine, Tel Aviv University, Tel Aviv,
Westmead, New South Wales, Australia Israel

Laurel Stadtmauer Matts Wikland

The Jones Institute for Reproductive Medicine, Eastern Fertility Centre Scandinavia, Carlander’s Hospital,
Virginia Medical School, Norfolk, Virginia, USA Göteborg, Sweden

Annika Strandell Fernando Zegers-Hochschild

Department of Obstetrics and Gynecology, Institute of Unit of Reproductive Medicine, Clínica las Condes,
Clinical Sciences, Sahlgrenska Academy, University of Santiago, Chile
Göteborg, Göteborg, Sweden
Dominique de Ziegler
Isabelle Streuli Faculté de médecine, Université Paris Descartes,
Faculté de médecine, Université Paris Descartes, Paris Sorbonne-Cité, and Department of Obstetrics,
Paris Sorbonne-Cité, and Department of Obstetrics, Gynecology, and Reproductive Medicine,
Gynecology, and Reproductive Medicine, Assistance Assistance Publique Hôpitaux de Paris, CHU Cochin,
Publique Hôpitaux de Paris, CHU Cochin, and Cochin Paris, France
Institute, Paris, France

Thomas H. Tang
Leeds Centre for Reproductive Medicine, Seacroft
Hospital, Leeds, UK
The beginnings of human
in vitro fertilization
Robert G. Edwards

In vitro fertilization (IVF) and its derivatives in preim- Pincus et al. also studied human oocytes (1). Extracting
plantation diagnosis, stem cells, and the ethics of assisted oocytes from excised ovaries, they identified chromo-
reproduction continue to attract immense attention sci- somes in a large number of oocytes and interpreted this as
entifically and socially. All these topics were introduced evidence of the completion of maturation in vitro. Many
by 1970. Hardly a day passes without some public recog- oocytes possessed chromosomes after 12 hours, the pro-
nition of events related to this study, and clinics spread portion remaining constant over the next 30 hours and
ever further worldwide. Now we must be approaching longer. Twelve hours was taken as the period of matura-
1.5 million IVF births, it is time to celebrate what has tion. Unfortunately, chromosomes were not classified for
been achieved by so many investigators, clinical, scien- their meiotic stage. Maturing oocytes would be expected
tific, and ethical. to display diakinesis or metaphase-I chromosome pairs.
While much of this chapter “Introduction” covers the Fully mature oocytes would display metaphase-II chro-
massive accumulation of events between 1960 and 2000, mosomes, signifying they were fully ripe and ready for
it also briefly discusses new perspectives emerging in the fertilization. Nevertheless, it is well known that oocytes
21st century. Fresh advances also increase curiosity can undergo atresia in the ovary involving the formation
about how these fields of study began and how their eth- of metaphase-II chromosomes in many of them. These
ical implications were addressed in earlier days. As for oocytes complicated Pincus’ estimates, even in controls,
me, I am still stirred by recollections of those early days. and were the source of his error which led later workers
Foundations were laid in Edinburgh, London, and to inseminate human oocytes 12 hours after collection
Glasgow in the 1950s and early 1960s. Discoveries made and culture in vitro (2,3). Work on human fertilization in
then led to later days in Cambridge, working there with vitro, and indeed comparable studies in animals,
many PhD students. It also resulted in my working with remained in abeyance from then and for many years.
Patrick Steptoe in Oldham. Our joint opening of Bourn Progress in animal IVF had also been slow. After many
Hall in 1980, which became the largest IVF clinic of its relatively unsuccessful attempts in several species in the
kind at the time, signified the end of the beginning of 1950s and 1960s, a virtual dogma arose that spermatozoa
assisted human conception and the onset of dedicated had to spend several hours in the female reproductive
applied studies. tract before acquiring the potential to bind to the zona
pellucida and achieve fertilization. In the late 1960s,
Austin and Chang independently identified the need for
INTRODUCTION
sperm capacitation, identified by a delay in fertilization
First of all, I must express in limited space my tributes to after spermatozoa had entered the female reproductive
my teachers, even if inadequately. These include investi- tract (4,5). This discovery was taken by many investiga-
gators from far-off days when the fundamental facts of tors as the reason for the failure to achieve fertilization in
reproductive cycles, surgical techniques, endocrinology, vitro, and why spermatozoa had to be exposed to secre-
and genetics were elicited by many investigators. These tions of the female reproductive tract. At the same time,
fields began to move in the 20th century, and if one pio- Chang reported that rabbit eggs that had fully matured in
neer of these times should be saluted, it must be Gregory vitro failed to produce normal blastocysts, none of them
Pincus. Famous for the contraceptive pill, he was a dis- implanting normally (6).
tinguished embryologist, and part of his work dealt with
the maturation of mammalian oocytes in vitro. He was
MODERN BEGINNINGS OF HUMAN IVF,
the first to show how oocytes aspirated from their folli-
PREIMPLANTATION GENETIC DIAGNOSIS, AND
cles would begin their maturation in vitro, and how a
EMBRYO STEM CELLS
number matured and expelled a first polar body. I believe
his major work was done in rabbits, where he found that My PhD began at the Institute of Animal Genetics,
the 10 to 11-hour timings of maturation in vitro accorded Edinburgh University in 1952, encouraged by Professor
exactly with those occurring in vivo after an ovulatory Conrad Waddington, the inventor of epigenesis, and
stimulus to the female rabbit. supervised by Dr Alan Beatty. At the time, capacitation
 THE BEGINNINGS OF HUMAN IN VITRO FERTILIZATION xiii

was gaining in significance. My chosen topic was the reproductive cycles. In fact, they worked wonderfully
genetic control of early mammalian embryology, specifi- well. Doses of 1–3 IU of PMS induced the growth of
cally the growth of preimplantation mouse embryos with numerous follicles, and similar doses of hCG 42 hours
altered chromosome complements. later invoked estrus and ovulation a further 6 hours later
Achieving these aims included a need to expose mouse in almost all of them. Often, 70 or more ovulated oocytes
spermatozoa to x-rays, ultraviolet light, and various crowded the ampulla, most of them being fertilized and
chemicals in vitro. This would destroy their chromatin developing to blastocysts (9). Oocyte maturation, ovula-
and prevent them from making any genetic contribution tion, mating, and fertilization were each closely timed in
to the embryo, hopefully without impairing their capac- all adults, another highly unusual aspect of stimulation
ity to fertilize eggs in vivo. Resulting embryos would (10). Diakinesis was identified as the germinal vesicle
become gynogenetic haploids. Later, my work changed to regressed, with metaphase I a little later and metaphase II,
exposing ovulated mouse oocytes to colchicine in vivo, expulsion of the first polar body, and ovulation at
in order to destroy their second meiotic spindle in vivo. 11.5–12 hours after hCG. Multiple fertilization led to
This treatment freed all chromosomes from their attach- multiple implantation and fetal growth to full term, just
ment to the meiotic spindle, and they then became as similar treatments in anovulatory women resulted in
extruded from the egg into tiny artificial polar bodies. quintuplets and other high-order multiple pregnancies a
The fertilizing spermatozoon thus entered an empty egg, few years later. Years afterward, germinal vesicle break-
which resulted in the formation of androgenetic haploid down and diakinesis were to prove equally decisive in
embryos with no genetic contribution from the maternal identifying meiosis and ovulation in human oocytes in
side. For three years, my work was concentrated in the vivo and in vitro. Even as these results were gained, Ruth
mouse house, working at midnight to identify mouse and I departed in 1957 from Edinburgh to the California
females in estrus by vaginal smears, collecting epididy- Institute of Technology, where I switched over immu-
mal spermatozoa from males, and practicing artificial nology and reproduction, a topic that was to dominate
insemination with samples of treated spermatozoa. This my life for five or six years on my return to the United
research was successful, as mouse embryos were identi- Kingdom.
fied with haploid, triploid, tetraploid, and aneuploid The Institute at Edinburgh had given me an excellent
chromosomes. Moreover, the wide range of scientific tal- basis not only in genetics, but equally in reproduction.
ent in the Institute made it a perfect place for fresh col- I had gained considerable knowledge about the endocrine
laborative studies. For example, Julio Sirlin and I applied control of estrus cycles, ovulation, and spermatozoa; the
the use of radioactive DNA and RNA precursors to the male reproductive tract; artificial insemination; the stages
study of spermatogenesis, spermiogenesis, fertilization, of embryo growth in the oviduct and uterus; superovula-
and embryogenesis, and gained knowledge unavailable tion and its consequences; and the use of radiolabeled
elsewhere. compounds. Waddington had also been deeply interested
An even greater fortune beckoned. Allen Gates, who in ethics and the relationships between science and reli-
arrived newly from the United States, brought commer- gion, and instilled these topics in his students. I had been
cial samples of Organon’s pregnant mares’ serum (PMS) essentially trained in reproduction, genetics, and scien-
rich in follicle-stimulating hormone (FSH), and human tific ethics, and all of this knowledge was to prove to be of
chorionic gonadotropin (hCG) with its strong luteinizing immense value in my later career. A visit to the California
hormone (LH) activity to induce estrus and ovulation in Institute of Technology widened my horizons into the
immature female mice. Working with Mervyn Runner molecular biology of DNA and the gene, a field then in its
(7), he had used low doses of each hormone at an interval infancy.
of 48 hours to induce oocyte maturation, mating, and After a year in California, London beckoned me, to
ovulation in immature mouse females. He now wished to the National Institute for Medical Research to work
measure the viability of three-day embryos from imma- with Drs Alan Parkes and Colin (Bunny) Austin. I was
ture mice by transferring them to an adult host to grow to fortunate indeed to have two such excellent colleagues.
term (8). I was more interested in stimulating adult mice After two intense years in immunology, my curiosity
with these gonadotropins to induce estrus and ovulation returned to maturing oocytes and fertilization in vitro.
at predictable times of the day. This would help my Since they matured so regularly and easily in vivo, it
research, and I was by now weary of taking mouse vagi- should be easy to stimulate maturation in mouse oocytes
nal smears at midnight. My future wife, Ruth Fowler, and in vitro by using gonadotropins. In fact, to my immense
I teamed up to test this new approach to superovulating surprise, when liberated from their follicles into culture
adult mice. We chose PMS to induce multifolliculation medium, oocytes matured immediately in vast numbers
and hCG to trigger ovulation, varying doses and times in all groups, with exactly the same timing as those
from those utilized by Allen Gates. PMS became obsolete maturing in vivo following an injection of hCG. Adding
for human studies some time later, but its impact has hormones made no difference. Rabbit, hamster, and rat
stayed with me from that moment, even till today. oocytes also matured within 12 hours, each at their own
Opinion in those days was that exogenous hormones species’ specific rates. But to my surprise, oocytes from
such as PMS and hCG would stimulate follicle growth cows, sheep, and rhesus monkeys, and the occasional
and ovulation in immature female mammals, but not in baboon, did not mature in vitro within 12 hours. Their
adults because they would interact badly with an adult’s germinal vesicles persisted unmoved, arrested in the
xiv THE BEGINNINGS OF HUMAN IN VITRO FERTILIZATION

stage known as diffuse diplotene. Why had they not While looking in the library for any newly published
responded like those of rats, mice, and rabbits? How papers relevant to my proposed Nature manuscript, I dis-
would human oocytes respond? A unique opportunity covered those earlier papers of Pincus and his colleagues
emerged to collect pieces of human ovary, and to aspirate described earlier. They had apparently succeeded 30 years
human oocytes from their occasional follicles. I grasped earlier in maturing human oocytes cultured for 12 hours,
it with alacrity. where I had failed. My Nature paper (11) became very
different from that originally intended, even though it
retained enough for publication. Those results of Pincus
MOVING TO HUMAN STUDIES
et al. had to be repeated. After trying hard, I failed com-
Molly Rose was a local gynecologist in the Edgware and pletely to repeat them, despite infusing intact ovaries in
District Hospital who delivered two of our daughters. She vitro with gonadotropin solutions, using different cul-
agreed to send me slithers or wedges of ovaries such as ture media to induce maturation, and using joint cultures
those removed from patients with polycystic disease, as of maturing mouse oocytes and newly released human
recommended by Stein and Leventhal, or with myomata oocytes. Adding hormones to culture media also failed.
or other disorders demanding surgery. Stein–Leventhal It began to seem that menstrual cycles had affected oocyte
wedges were the best source of oocytes, with their numer- physiology in a different manner than in nonmenstruat-
ous small Graafian follicles lined up in a continuous rim ing mammalian species. Finally, another line of inquiry
just below the ovarian surface. Though samples were emerged after two years of fruitless research on the pre-
rare, they provided enough oocytes to start with. These cious few human oocytes available. Perhaps the timing
oocytes responded just like the oocytes from cows, sheep, of maturation in mice and rabbits differed from that of
and pigs, their germinal vesicles persisting and diakine- those oocytes obtained from cows, baboons, and humans.
sis being absent after 12 hours in vitro. Even as my days in London were ending, Molly Rose
This was disappointing, and especially so for me, since sent a slither of human ovary. The few oocytes were
Tjio and Levan, and Ford had identified 46 diploid chro- placed in culture just as before. Their germinal vesicles
mosomes in humans, while studies by teams in Edin- remained static for 12 hours as I already knew, and then
burgh (Scotland) and France had made it clear that many after 20 hours in vitro. Three oocytes remained, and
human beings were heteroploid. This was my subject, I waited to examine them until they had been in vitro for
because chromosomal variations mostly arose during 24 hours. The first contained a germinal vesicle, so did
meiosis and this would be easily assessed in maturing the second. There was one left and one only. Its image
oocytes at diakinesis. Various groups also discovered under the microscope was electrifying. I gazed down at
monosomy or disomy in many men and women. Some chromosomes in diakinesis and at a regressing germinal
women were XO or XXX; some men were XYY and vesicle. The chromosomes were superb examples of
XYYY. Trisomy 21 proved to be the most common cause human diakinesis with their classical chiasmata. At last,
of Down’s syndrome, and other trisomies were detected. I was on the way to human IVF, to completion of the
All this new information reminded me of my chromo- maturation program and the onset of studies on fertiliza-
some studies in the Edinburgh mice. tion in vitro.
For human studies, I would have to obtain diakinesis This was the step I had waited for, a marker that Pincus
and metaphase I in human oocytes, and then continue had missed. He never checked for diakinesis, and appar-
this analysis to metaphase II when the oocytes would be ently confused atretic oocytes, which contained chromo-
fully mature, ready for fertilization. Despite being disap- somes, with maturing oocytes. Endless human studies
pointed at current failure with human oocytes, it was were opening. It was easy now, even on the basis of one
time to write my findings for Nature in 1962 (11). There oocyte in diakinesis, to calculate the timing of the final
was so much to write regarding the animal work, and stages of maturation because the postdiakinesis stages of
describing the new ideas then taking shape in my mind. maturation were not too different from normal mitotic
I had heard Institute lectures on infertility, and realized cycles in somatic cells. This calculation provided me
that fertilizing human oocytes in vitro and replacing with an estimate of about 36 hours for full maturation,
embryos into the mother could help to alleviate this con- which would be the moment for insemination. All these
dition. It could also be possible to type embryos for gaps in knowledge had to be filled. But now, my research
genetic diseases when a familial disposition was identi- program was stretching far into the future.
fied. Pieces of tissue, or one or two blastomeres, would At this wonderful moment, John Paul, an outstanding
have to be excised from blastocysts or cleaving embryos, cell biologist, invited me to join him and Robin Cole at
but this did not seem to be too difficult. There were few Glasgow University to study differentiation in early
genetic markers available for this purpose in the early mammalian embryos. This was exciting, to work in bio-
1960s, but it might be possible to sex embryos by their chemistry with a leading cell biologist. He had heard
XX or XY chromosome complement by assessing mitoses that I was experimenting with very early embryos, trying
in cells excised from morulae or blastocysts. Choosing to grow cell lines from them. He also wanted to grow
female embryos for transfer would avert the birth of boys stem cells from mammalian embryos and study them in
with various sex-linked disorders such as hemophilia. vitro. This began one of my most memorable 12 months
Clearly, I was becoming totally committed to human IVF of research. John’s laboratory had facilities unknown
and embryo transfer. outside, with CO2 incubators, numerous cell lines in
 THE BEGINNINGS OF HUMAN IN VITRO FERTILIZATION xv

constant cultivation, cryopreservation facilities, and the I had already met Victor McKusick, who worked in Johns
use of media droplets held under liquid paraffin. We Hopkins, at many conferences. I asked for his support for
decided to start with rabbits. Cell lines did not grow eas- my request to work with the hospital gynecologists for
ily from cleaving rabbit embryos. In contrast, stem cells six weeks. He found a source of funds, made laboratory
migrated out in massive numbers from cultures of rabbit space available, and a wonderful invitation that intro-
blastocysts, forming muscle, nerves, phagocytes, blood duced me to Howard and Georgeanna Jones. This signifi-
islands, and other tissues in vitro (12). Stem cells were cant moment was equal to my meeting with Molly Rose.
differentiating in vitro into virtually all the tissues of the The Joneses proved to be superb and unstinting in their
body. In contrast, dissecting the inner cell mass from support. Sufficient wedges and other ovarian fragments
blastocysts and culturing it intact or as disaggregated were available to complete my maturation program in
cells produced lines of cells which divided and divided, human oocytes. Within three weeks, every stage of meio-
without ever differentiating. One line of these embry- sis was classified and timed (14). We also undertook pre-
onic stem cells expressed specific enzymes, diploid liminary studies on inseminating human oocytes that
chromosomes, and a fibroblastic structure as it grew over had matured in vitro, trying to achieve sperm capacita-
200 and more generations. Another was epithelioid and tion by using different media or adding fragments of
had different enzymes but was similar in other respects. ampulla to the cultures, and even attempting fertilization
The ability to make whole-embryo cultures producing in rhesus monkey oviducts. Two nuclei were found in
differentiating cells was now combined with everlasting some inseminated eggs, resembling pronuclei, but sperm
lines of undifferentiated stem cells which replicated tails were not identified so no claims could be made (15).
over many years without changing. Ideas of using stem During those six weeks, however, oocyte maturation was
cells for grafting to overcome organ damage in recipients fully timed at 37 hours, permitting me now to predict
began to emerge. My thoughts returned constantly to with certainty that women would ovulate at 37 hours
growing stem cells from human embryos to repair defects after an hCG injection.
in tissues of children and adults. A simple means of access to the human ovary was now
Almost at my last moment in Glasgow, with this new essential in order to identify human ovarian follicles in
set of ideas in my mind, a piece of excised ovary yielded vivo and to aspirate them 36 hours after hCG, just before
several oocytes. Being placed in vitro, two of them had the follicular rupture. Who could provide this? And how
reached metaphase II and expelled a polar body at about sperm capacitation? Only in hamsters that fertil-
37 hours. This showed that another target on the road to ization in vitro had been achieved, using in vivo matured
human IVF had been achieved as the whole pattern of oocytes and epididymal spermatozoa (16). I met Victor
oocyte maturation continued to emerge but with increas- Lewis, my third clinical colleague, and we noticed what
ing clarity. seemed to be anaphase II in some inseminated eggs.
Cambridge University, my next and final habitation, is Again, no sperm tails were seen within the eggs.
an astonishing place. Looking back on those days, it An attempt to achieve human capacitation in Chapel
seems that the Physiological Laboratory was not the Hill, North Carolina, United States, working with Robert
ideal place to settle in that august university. Neverthe- McGaughey and his colleagues, also failed (17). A small
less, a mixture of immunology and reproduction intrauterine chamber lined with porous membrane was
remained my dominant theme as I rejoined Alan Parkes filled with washed human spermatozoa, sealed, and
and Bunny Austin there. I had to do immunology to inserted overnight into the uterus of human volunteers at
obtain a grant to support my family, but thoughts of mid-cycle. Molecules entering it could react with the
human oocytes and embryos were never far away. One spermatozoa. No matured human eggs were fertilized.
possible model of the human situation was the cow and Later evidence indicated that the chamber contained
other agricultural species, and large numbers of cow, inflammatory proteins, perhaps explaining the failure.
pig, and sheep oocytes were available from ovaries given
to me by the local slaughterhouse. Each species had its
DECISIVE STEPS TO CLINICAL HUMAN IVF
own timing, all of them longer than 12 hours (13). Pig
oocytes were closest to humans, requiring 37 hours. In Back in the United Kingdom, my intention to conceive
each species, maturation timings in vitro were exactly human children in vitro had grown even stronger. So
the same as those arising in vivo in response to an hCG many medical advantages could flow from it. A small
injection. This made me suspect that a woman ovulated number of human embryos had been flushed from human
36–37 hours after an injection of hCG. Human oocytes oviducts or uteri after sexual intercourse, providing slen-
also trickled in, improving my provisional timings of der information on these earliest stages of human embry-
maturation, and one or two of them were inseminated, ology. It was time to attain human fertilization in vitro, in
but without signs of fertilization. order to move close to working with infertile patients.
More oocytes were urgently needed to conclude the Ethical issues and moral decisions would emerge, one
timings of oocyte meiosis. Surgeons in Johns Hopkins after the other, in full public view. Matters such as clon-
Hospital, Baltimore performed the Stein–Leventhal ing and sexing embryos, the risk of abnormalities in the
operation, which would allow me to collect ovarian tis- children, the clinical use of embryo stem cells, the ethics
sue, aspirate oocytes from their follicles, and retain the of oocyte donation and surrogate pregnancy, and the
remaining ovarian tissues for pathology if necessary. right to initiate human embryonic life in vitro would
xvi THE BEGINNINGS OF HUMAN IN VITRO FERTILIZATION

never be very far away. These issues were all acceptable, of the Oldham and District General Hospital (18), it
since I was confident that studies of human conception described laparoscopy, with its narrow telescope and
were essential for future medicine, and correct ethically, instruments and the minute abdominal incisions. He
medically, and scientifically. The increasing knowledge could visualize the ampulla and place small amounts of
of genetics and embryology could assist many patients if medium there, in an operation lasting 30 minutes or less
I could achieve human fertilization and grow embryos and maybe even without using anesthesia. This is exactly
for replacement into their mothers. what I wanted, because access to the ampulla was equiv-
Few human oocytes were available in the United King- alent to gaining access to ovarian follicles. Despite advice
dom. Despite this scarcity, one or two of those matured to the contrary from several medical colleagues, I tele-
and fertilized in vitro possessed two nuclei after insemi- phoned him about collaboration and stressed the uncer-
nation. But there were no obvious sperm tails. I devised tainty in achieving fertilization in vitro. He responded
a cow model for human fertilization, using in vitro most positively, just as Molly, Howard and Georgeanna,
matured oocytes and insemination in vitro with selected and Victor had done. We decided to get together.
samples of highly active, washed bull spermatozoa Last but by no means least, Molly Rose sent a small
extracted from neat semen. It was a pleasure to see some piece of ovary to Cambridge. Its dozen or more oocytes
fertilized bovine eggs, with sperm tails and characteristic were matured in vitro for 37 hours, when Barry and
pronuclei, especially using spermatozoa from one par- I added washed spermatozoa suspended in his medium.
ticular bull. Here was a model for human IVF and a pre- We examined them a few hours later. To our delight,
lude to a series of events, which implied that matters in spermatozoa were pushing through the zona pellucida,
my research were suddenly changing. A colleague had into several of the eggs. Maternal and paternal pronuclei
stressed that formalin fixatives were needed to detect were forming beautifully. We saw polar bodies and sperm
sperm tails in eggs. Barry Bavister joined our team to tails within the eggs. That evening in 1969, we watched
study for his PhD and designed a medium of high pH, in delight virtually all the stages of human fertilization in
which gave excellent fertilization rates in hamsters. We vitro (Fig. I.1). One fertilized egg had fragments, as Chang
decided to collaborate by using it for trials on human fer- had forecast from his work on oocyte maturation and fer-
tilization in vitro. tilization in vitro of rabbit eggs. This evidence strength-
Finally, while browsing in the library of the Physiolog- ened the need to abandon oocyte maturation in vitro and
ical Laboratory, I read a paper in The Lancet which replace it by stimulating maturation by means of exoge-
instantly caught my attention. Written by Dr P C Steptoe nous hormones. Our 1969 paper in Nature surprised a

Figure I.1 A composite picture of the stages of fertilization of the human egg. (Upper left) An egg with a first polar body and spermatozoa
attached to the outer zona pellucida. (Upper central) Spermatozoa are migrating through the zona pellucida. (Upper right) A spermatozoon
with a tail beating outside the zona pellucida is attaching to the oocyte vitelline membrane. (Lower left) A spermatozoon in the ooplasm, with
enlarging head and distinct mid-piece and tail. (Lower central) Further development of the sperm head in the ooplasm. (Lower right) A pronu-
cleate egg with two pronuclei and polar bodies. Notice that the pronuclei are apparently aligned with the polar bodies, although more dimen-
sions must be scored to ensure that polarity has been established in all axes.
 THE BEGINNINGS OF HUMAN IN VITRO FERTILIZATION xvii

world unaccustomed to the idea of human fertilization in pioneers, Palmer in Paris (23) and Fragenheim in Germany
vitro (19). (24). He improved the pneumoperitoneum to gain working
Incredibly fruitful days followed in our Cambridge space in the abdominal cavity and used carbon fibers to
laboratory. Richard Gardner, another PhD candidate, and pass cold light into the abdomen from an external source
I excised small pieces of trophectoderm from rabbit blas- (25). By now, Patrick was waiting in the wings, ready to
tocysts and sexed them by staining the sex chromatin begin clinical IVF in distant Oldham. We had a long talk
body. Those classified as female were transferred into about ethics and found our stances to be very similar.
adult females and were all correctly sexed at term. This Work started in the Oldham and District General Hospi-
work transferred my theoretical ideas of a few years ear- tal and moved later to Kershaw’s Hospital, set up by my
lier into the practice of preimplantation diagnosis of assistants, especially Jean Purdy. We knew the routine. It
inherited disease, in this case for sex-linked diseases was based on my Edinburgh experiences with mice. Piero
(20). Alan Henderson, a cytogeneticist, and I analyzed Donini from Serono Laboratories in Rome had purified
chiasmata during diakinesis in mouse and human eggs, urinary human menopausal gonadotropins (hMG) as a
and explained the high frequencies of Down’s syndrome source of FSH and the product was used clinically to
in offspring of older mothers as a consequence of meiotic stimulate follicle growth in anovulatory women by Bruno
errors arising in oocytes formed last in the fetal ovary, Lunenfeld (26). It removed the need for PMS, thus avoid-
which were then ovulated last at later maternal ages (21). ing the use of nonhuman hormones. We used low-dosage
Dave Sharpe, a lawyer from Washington, joined forces to levels in patients, that is, 2–3 vials (a total of 150–225 IU)
write an article in Nature (22) on the ethics of IVF, the given on days 3 and 5, and 5000–7000 IU of hCG on day
first ever paper in the field. I followed this up with a 10. Initially, the timing of oocyte maturation in vitro was
detailed analysis of ethics and law in IVF covering scien- confirmed, by performing laparoscopic collections of
tific possibilities, oocyte donation, surrogacy by embryo oocytes from ovarian follicles at 28 hours after hCG to
transfer, and other matters (22). So the first ethical papers check that they were in metaphase I (27). We then moved
were written by scientists and lawyers and not by phi- to 36 hours to aspirate mature metaphase II oocytes for
losophers, ethicists, or politicians. fertilization. Those beautiful oocytes were surrounded by
masses of viscous cumulus cells and were maturing
exactly as predicted. We witnessed follicular rupture at
THE OLDHAM YEARS
37 hours through the laparoscope. Follicles could be clas-
Patrick and I began our collaboration six months later in the sified from their appearance as ovulatory or nonovulatory,
Oldham and District General Hospital, almost 200 miles this diagnosis being confirmed later by assaying several
north of Cambridge. He had worked closely with two steroids in the aspirated follicular fluids (Fig. I.2).

18.798

16.832

14.866
110.9
Within-group variation

12.900

99.3 10.935
Within-group variation

87.6 8.969

7.003
29.6
5.038
18.0
3.072

6.3 1.106

Group 1 2 Follicle no. 12 101125 3 4 8 17 5 822212 3 24 7 20121416 9

Ovulatory Nonovulatory Group 1 2 3 4

Figure I.2 Eight steroids were assayed in fluids extracted from human follicles aspirated 36–37 hours after the human chorionic gonadotropin
(hCG) shot. The follicles had been classified as ovulating or nonovulating by laparoscopic examination in vivo. Data were analyzed by cluster
analysis, which groups follicles with similar features. The upper illustration shows data collected during the natural menstrual cycle. Note that
two sharply separated groups of follicles were identified, each with very low levels of within-group variance. Attempting to combine the two
groups resulted in a massive increase of within-group variation, indicating that two sharply different groups had been identified. These different
groups accorded exactly with the two groups identified by means of steroid assays. The lower figure shows the same analysis during stimulated
cycles on fluids collected 36–37 hours after injecting hCG. With this form of stimulation, follicle growth displays considerable variation within
groups. Attempts to combine all the groups result in a moderately large increase in variation. This evidence suggests that follicles vary consid-
erably in their state of development in simulated cycles using human menopausal gonadotropin (hMG) and hCG.
xviii THE BEGINNINGS OF HUMAN IN VITRO FERTILIZATION

Figure I.3 Successive stages of human preimplantation development in vitro in a composite illustration made in Oldham in 1971. (Upper
left) 4-cell stage showing the crossed blastomeres typical of most mammals. (Upper middle) 8-cell stage showing the even outline of blasto-
meres and a small piece of cumulus adherent to the zona pellucida. (Upper right) A 16 to 32-cell stage, showing the onset of compaction of
the outer blastomeres. Often, blastocelic fluid can be seen accumulating between individual cells to give a “stripey” appearance to the
embryo. (Lower left and middle) Two living blastocysts showing a distinct inner cell mass, single-celled trophectoderm, blastocelic cavity, and
thinning zona pellucida. (Lower right) A fixed preparation of a human blastocyst at 5 days, showing more than 100 even-sized nuclei and
many mitoses.

Patrick’s laparoscopy was superb. Ovarian stimulation,

even though mild, produced five or six mature follicles per
patient, and ripe oocytes came in a steady stream into my
culture medium for insemination and overnight incuba-
tion. The next morning, the formation of two pronuclei
and sperm tails indicated fertilization had occurred, even
in simple media, now with a near-neutral pH. Complex
culture media, Ham’s F10 and others, each with added
serum or serum albumin, sustained early and later cleav-
ages (28), and, even more fascinating, was the gradual
appearance of morulae and then light, translucent blasto-
cysts (Fig. I.3) (29). Here was my reward—growing embryos
was now a routine, and examinations of many of them
convinced me that the time had come to replace them into
the mothers’ uterus. I had become highly familiar with the
Figure I.4 A hatched human blastocyst after 9 days in culture. teratologic principles of embryonic development, and
Notice the distinct embryonic disc and the possible bilaminar struc-
knew many teratologists. The only worry I had was the
ture of the membrane. The blastocyst has expanded considerably, as
chance of chromosomal monosomy or trisomy, on the
shown by comparing its diameter with that of the shed zona pellu-
cida. The zona contains dying and necrotic cells and its diameter
basis of our mouse studies, but these conditions could be
provides an estimate of the original oocyte end embryo diameters. detected later in gestation by amniocentesis. Our human
studies had surpassed work on all animals, a point rubbed
in even more when we grew blastocysts to day 9 after they
It was a pleasure and a new duty to meet the patients had hatched from their zona pellucida (Fig. I.4) (30). This
searching for help to alleviate their infertility. We did our beautifully expanded blastocyst had a large embryonic
best, driving from Cambridge to Oldham and arriving at disc which was shouting that it was a potential source of
noon to prepare the small laboratory there. Patrick had embryonic stem cells.
stimulated the patients with hMG and hCG, and he and his When human blastocysts became available, we tried to
team led by Muriel Harris arrived to prepare for surgery. sex them using the sex chromatin body as in rabbits.
 THE BEGINNINGS OF HUMAN IN VITRO FERTILIZATION xix

Unfortunately, they failed to express either sex chroma- be given every five days to sustain pregnancies, since it
tin or the male Y body so we were unable to sex them as was supposed to save threatened abortions. So, we began
female or male embryos. Human preimplantation genetic embryo transfers to patients in stimulated cycles, giving
diagnosis would have to wait a little longer. this luteal phase support. Even though our work was
During these years there were very few plaudits for us, slowed down by having to wait to see whether pregnan-
as many people spoke against IVF. Criticism was mostly cies arose in one group of patients before stimulating the
aimed at me, as usual when scientists bring new chal- next, enough patients had accumulated after two to three
lenges to society. Criticism came not only from the Pope years. None of our patients was pregnant, and disaster
and archbishops, but also from scientists who should loomed. Our critics were even more vociferous as the
have known better, including James Watson (who testi- years passed, and the mutual support between Patrick
fied to a U.S. Senate Committee that many abnormal and me had to pull us through.
babies would be born), and Max Perutz, who supported Twenty or more different factors could have caused our
him. These scientist critics knew virtually nothing about failure, for example cervical embryo transfers, abnormal
my field, so who advised them to make such ridiculous embryos, toxic culture dishes or catheters, inadequate
charges? Cloning football teams or intelligentsia was luteal support, incompatibility between patients’ cycles
always raised by ethicists, which clearly dominated their and that imposed by hMG and hCG, inherent weakness in
thoughts rather than the intense hopes of our infertile human implantation, and many others. We had to glean
patients. Yet one theologian, Gordon Dunstan, who every scrap of information from our failures. I knew Ken
became a close friend, knew all about IVF from us, and Bagshawe in London, who was working with improved
wrote an excellent book on its ethics. He was far ahead of assay methods for gonadotropic hormones. He offered to
almost every scientist in my field of study. Our patients measure blood samples taken from our patients over the
also gave us their staunch support, and so did the Old- implantation period using his new hCG-® assay. He tele-
ham Ethical Committee, Bunny Austin back home in phoned: three or more of our patients previously undiag-
Cambridge, and Elliott Philip, a colleague of Patrick’s. nosed had actually produced short-lived rises of hCG-®
Growing embryos became a routine, so we decided to over this period. Everything changed with this informa-
transfer one each to several patients. Here again we tion. We had established pregnancies after all, but they
were in untested waters. Transferring embryos via the had aborted very early. We called them biochemical
cervical canal, the obvious route to the uterus, was vir- pregnancies, a term that still sticks today. It had taken us
tually a new and untested method. We would have to do almost three years to identify the cause of our failure, and
our best. From now on, we worked with patients who the finger of suspicion pointed straight at Primulot. I knew
had seriously distorted tubes or none whatsoever. This it was luteolytic, but it was apparently also an abortifa-
step was essential, since no one would have believed cient, and our ethical decision to use it had caused much
we had established a test-tube baby in a woman with heartache, immense loss of work and time, and despair for
near-normal tubes. This had to be a condition of our ini- some of our patients. The social pressures had been
tial work. Curiously, it led many people to make the big immense, with critics claiming our embryos were dud
mistake of believing that we started IVF to bypass and our whole program was a waste of time; but we had
occluded oviducts. Yet we already knew that embryos come through it and now knew exactly what to do next.
could be obtained for men with oligozoospermia or anti- We accordingly reduced the levels of Primulot depot,
bodies to their gametes, and for women in various stages and utilized hCG and progesterone as luteal aids. Suspi-
of endometriosis. cions were also emerging that human embryos were very
One endocrinological problem did worry me. Stimula- poor at implanting. We had replaced single embryos into
tion with hMG and hCG shortened the succeeding luteal most of our patients, rarely two. Increasingly we began to
phase, to a very short time for embryos to implant before wonder whether more should be replaced, as when we
the onset of menstruation. Levels of urinary pregnane- replaced two in a program involving transfer of oocytes
diol also declined soon after oocyte collection. This con- and spermatozoa into the ampulla so that fertilization
dition was not a result of the aspiration of granulosa and could occur in vivo.
cumulus cells, and luteal support would be needed, pref- This procedure was later called gamete intrafallopian
erably progesterone. Csapo et al. stressed how this hor- transfer (GIFT) by Ricardo Asch. We now suspected that
mone was produced by the ovaries for the first 8–10 single embryo transfers could produce a 15–20% chance
weeks before the placenta took over this function (31). of establishing pregnancy, just as our first clinical preg-
Injections of progesterone in oil given over that long nancy arose after the transfer of a single blastocyst in a
period of time seemed unacceptable since it would be patient stimulated with hMG and hCG (32). Then came
extremely uncomfortable for patients. While mulling the fantastic news—a human embryo fertilized and
over this problem, my attention turned to those earlier grown in vitro had produced a pregnancy. Everything
endocrinologists who believed that exogenous hormones seemed fine, even with ultrasound images. My culture
would distort the reproductive cycle, although I doubt protocols were satisfactory after all. Patrick rang: he
they even knew anything about a deficient luteal phase. feared the pregnancy was ectopic and he had to remove
This is how we unknowingly made our biggest mistake it sometime after 10 gestational weeks. Every new
in early IVF days. Our choice of Primolut (Sigma Chemical approach we tested seemed to be ending in a disaster, yet
co., St Louis, USA) depot, a progestogen, meant it should we would not stop, since the work itself seemed highly
xx THE BEGINNINGS OF HUMAN IN VITRO FERTILIZATION

ethical, and conceiving a child for our patients was per- Mrs. MP
1 preovulatory oocyte +
ODGH 12/1/73
haps the most wonderful thing anyone could do for them. 1.6 × 106 sperm into ampulla
In any case, ectopic pregnancies are now known to be a

LMP ampules

Laparoscopy
8000 IU hCG
regular feature with assisted conception.
hMG
I sensed that we were entering the final phase of our

RTM
(amps)
hCG (IU)
Oldham work, seven years after it began. We had to speed 3 3 3 3 1500 15001500 1500
up, partly because Patrick was close to retiring from the
National Health Service. Four stimulation protocols were 150
tested in an attempt to avoid problems with the luteal

Urinary pregnanediol (mg/day)

phase: hMG and hCG; clomiphene, hMG, and hCG to

Urinary estrogens (µg/day)

gain a better luteal phase; bromocriptine, hMG, and hCG 100 10
because some patients had high prolactin concentra-
tions; and hCG alone at mid-cycle. We also tested what
came to be known as GIFT, calling it ORTI (oocyte recov-
ery with tubal insemination, by transferring one or two 50 5
eggs and spermatozoa to the ampulla) (Fig. I.5). Natural-
cycle IVF was introduced, based on collections of urine
samples at regular intervals eight times daily, to measure
0 0
exactly the onset of the LH surge, using a modified HiGo- 0 4 8 12 16 20 24 28 32 36
navis assay (Fig. I.6). Cryopreservation was also intro- Days of cycle
duced, by freezing oocytes and embryos that looked to be
in good condition when thawed. A recipient was given a Figure I.5 The first attempts at gamete intrafallopian transfer (GIFT)
were called oocyte recovery with tubal insemination (ORTI). In this
donor egg fertilized by her husband’s spermatozoa, but
treatment cycle, using human menopausal gonadotropin (hMG) and
pregnancy did not occur.
human chorionic gonadotropin (hCG), including additional injec-
Lesley and John Brown came as the second entrants tions of hCG for luteal support, a single preovulatory oocyte and 1.6
for natural-cycle IVF. Lesley had no oviducts. Her egg million sperm were transferred into the ampulla. Abbreviations: LMP,
was aspirated in a few moments and inseminated simply last menstrual period; ODGH, Oldham and District General Hospital
and efficiently. The embryo grew beautifully and was return to menstruation (RTM) indicates stages of the menstrual cycle.

LH lapy LH lapy LH lapy

surge surge surge

110

60
Total estrogens
(µg/24h)

40

20

0
Pregnanediol
(mg/24 h)

0
LH by HiGovanis

6
(IU/h)

4
2
1
<1

12 13 14 15 16 17 11 12 13 14 15 16 11 12 13 14 15 16
Days Days Days

Figure I.6 Recording the progress of the human natural menstrual cycle for in vitro fertilization (IVF). Three patients are illustrated. All three
displayed rising 24-hour urinary estrogen concentrations during the follicular phase and rising urinary pregnanediol concentrations in the luteal
phase. Luteinizing hormone (LH) levels were measured several times daily and the data clearly reveal the exact time of onset of the LH surge.
 THE BEGINNINGS OF HUMAN IN VITRO FERTILIZATION xxi

transferred an hour or so after it became 8-cell. Their clomiphene and hCG and replacing two or three embryos
positive pregnancy test a few days after transfer was (34), so they had moved ahead of us during the delayed
another milestone—surely nothing could now prevent opening of Bourn Hall. Our own effort now expanded pro-
their embryo developing to full term in a normal repro- digiously. Thousands of patients queued for IVF. Simon
ductive cycle, but those nine months lasted a very long Fishel, Jacques Cohen, and Carol Fehilly joined the
time. Three more pregnancies were established using embryology team among younger trainees, and new clini-
natural-cycle IVF as we abandoned the other approaches. cians joined Patrick and John Webster. Patients and preg-
A triploid embryo died in utero—more bad luck. A third nancies increased rapidly, and the world was left standing
pregnancy was lost through premature labor on a moun- far behind. Howard and Georgeanna Jones began in
tain walking holiday, two weeks after the mother’s Norfolk using gonadotropins for ovarian stimulation. Jean
amniocentesis (32,33). It was a lovely, well-developed Cohen began in Paris, Wilfred Feichtinger and Peter
boy. Louise Brown’s birth, and then Alistair’s, proved to Kemeter in Vienna, Klaus Diedrich and Hans van der
a waiting world that science and medicine had entered Venn in Bonn, Lars Hamberger and Matts Wikland in
human conception. Our critics declared that the births Sweden, and Andre van Steirteghem and Paul Devroey in
were a fake, and advised against attending our presenta- Brussels. IVF was now truly international.
tion on the whole of the Oldham work at the Royal Col- The opening of Bourn Hall had not deterred our critics.
lege of Obstetricians and Gynaecologists. They put up a fierce rearguard action against IVF, along-
side LIFE, Society for the Unborn Child, individual gyne-
cologists, and others.
IVF WORLDWIDE
Objections raised against IVF included low rates of
The Oldham period was over. Good facilities were now pregnancy (no one mentioned the similar low rates
needed, with space for a large IVF clinic. Bourn Hall was of pregnancy with natural conception), the possibilities
an old Jacobean house in lovely grounds near Cambridge of oocyte and embryo donation, surrogate mothers,
(Fig. I.7). Facilities on offer for IVF in Cambridge were far unmarried parents, one-sex parents, embryo cryopreser-
too small, so we purchased it mostly with venture capital. vation, cloning, and endless other objections.
It was essential to conceive 100 or 1000 IVF babies to LIFE issued a legal action against me for the abortion
ensure that the method was safe and effective clinically. of an embryo grown for 14 days and longer in vitro.
The immense delays in establishing Bourn Hall delayed Their action was rejected by the U.K. Attorney General
our work by two years after Louise’s birth. Finally, on since the laws of pregnancy began after implantation.
minimal finance, Bourn Hall was opened in September We fully respected the intense ethical nature of our pro-
1980 on a shoestring, supported by our own cash and ceedings. We also recognized the need for research, and
loans. The delay gave the rest of the world a chance to join the necessity to protect or cryopreserve the best embryos
in IVF. Alex Lopata delivered an IVF baby in Australia, for later replacement into their mothers. Those not
and one or two others were born elsewhere. Natural-cycle replaced had to be used for research under strict con-
IVF was chosen initially at Bourn Hall since it had proved trols, combined with open publication and discussion
successful in Oldham, and we became experts in it. Preg- of our work.
nancies flowed, at 15% per cycle. An Australian team of Each year, 1000, rising to almost 2000, patients passed
Alan Trounson and Carl Wood announced the establish- through Bourn Hall. Different stimulation regimens or
ment of several IVF pregnancies after stimulation by new procedures could be tested in very little time.

Figure I.7 Bourn Hall. Source: Courtesy of Dr P Brinsden.

xxii THE BEGINNINGS OF HUMAN IN VITRO FERTILIZATION

Clomiphene/hMG was reintroduced. Bourn babies and Mike Ashwood-Smith and Peter Holland’s in
increased: 20, 50, 100 to 1000 after five to six years. This embryology, as the old team faded away. Fascinating
was far more than half of the world’s entire IVF babies, days had returned. Working with barristers, we
including the first born in the United States, Germany, designed consent forms which were far in advance of
Italy, and many other countries. Detailed studies were those used elsewhere. Oocyte donation and surrogacy
performed on embryo culture, implantation, and abor- by embryo transfer were introduced. The world’s first
tion. We even tried aspirating epididymal spermatozoa paper on embryo stem cells appeared in Science in
for IVF, without achieving successful fertilization. 1984, sent from Bourn Hall, and the world’s first on
Among the immense numbers of patients, people with human preimplantation diagnosis in 1987 appeared in
astonishingly varied conditions of infertility emerged. Human Reproduction. However, embryo research fal-
Some were poor responders in whom immense amounts tered as all normal embryos were cryopreserved for
of endocrine priming were essential, women with a natu- their parents, so almost none were available for study.
ral menstrual cycle that was not as it should have been, Alan Handyside, one of our Cambridge PhDs, joined
previous misdiagnoses which had laid the cause of infer- Hammersmith Hospital in London to make major steps
tility on the wife when the husband had never even been in introducing preimplantation genetic diagnosis (36).
investigated, and men bringing semen samples that we As we reached 1000 pregnancies, our data showed the
discovered had been obtained from a friend. The collabo- babies to be as normal as those conceived in vivo.
ration between nurses, clinicians, and scientists was Test-tube babies (an awful term) were no longer unique
remarkable. Yet trouble—ethical trouble—was never far and were accepted worldwide, exactly as Patrick and I
away. I purchased a freezing machine to resume our Old- had hoped. Our work was being recognized (Fig. I.8).
ham work, but, unknown to me, Patrick talked to officers Clinics sprang up everywhere. Ultrasound was intro-
of the British Medical Association (BMA) and for some duced to detect follicles for aspiration by the Scandina-
reason agreed to delay embryo cryopreservation. Appar- vians (37), making laparoscopy for oocyte recovery
ently, the BMA felt it would be an unwelcome social largely redundant. Artificial cycles were introduced in
development. I did not approve of these reservations: Australia and intracytoplasmic sperm injection (ICSI)
David Whittingham had shown how low-temperature in Belgium (38), and gonadotropin-releasing hormone
cryostorage was successful with mouse embryos, without agonists were used to inhibit the LH surge. Ian Craft in
causing genetic damage. “Freezing and cloning” became London showed how postmenopausal women aged 52 or
a term of intense approbation at this time. I unwillingly more could establish pregnancies using oocyte donation
curtailed our cryopreservation program. and endocrine support. Women over 60 years of age con-
One weekend, a major trouble erupted as a result of ceived and delivered children. This breakthrough was
this difference between Patrick and me. My duties in especially welcome to me, since older women surely
Bourn Hall prevented me from attending a conference in have the right to have children at ages almost the same as
London. Trying to be helpful, I telephoned my lecture to those possible for men.
London. Reception at the other end was apparently so Ethics continued side by side with advancing science
poor as to lead to misinterpretations of what I had said. and medicine. The U.K. governmental Warnock report
Next morning, the press furore about my supposed prac- recommended permitting embryo research and proposed
tice of cryopreserving embryos after IVF was awful, so a Licensing Authority for IVF. A year or so later, the U.K.
bad, indeed, that legal action had to be taken. Luckily, House of Lords, in all its finery, responded with a 3:1
my lecture had been recorded, and listening to the tapes vote in favor, decisive support for all we had done in Mill
with a barrister revealed nothing contentious. I had said
nothing improper in my lecture or during the question-
answer session. That day, I issued seven libel actions
against the cream of British society: the BMA and its sec-
retary, the BBC, The Times, and other leading newspa-
pers. There were seven in one day and another one later!
If only one was lost, I could be ruined and disgraced.
However, they were all won, even though it took several
years with the BMA and its secretary. These legal actions
had inhibited our research, the cryopreservation program
being shut down for more than a year. Every single
embryological note of mine from those days in Oldham
and from Bourn Hall was examined in detail for my
opponents by someone who was clearly an embryologist.
Nothing was found to incriminate me.
That wretched period passed. The number of babies
kept on growing, embryo cryopreservation was
resumed, and Gerhard Dealmaker in The Netherlands
beat us and the world to the first “ice” baby (35). Colin Figure I.8 A happy picture of Patrick and me, standing in our robes
Howles and Mike McNamee joined us in endocrinology, after being granted our Hon. DSc by Hull University.
into unless

ACHSHUNDS its regard

is in he

bats end Landor

Monkey the

He and and
variety colours Its

parachute

The

sealers same

came all
but

and approaching

One

are They

following prey INDIAN

mainly has

kind

for three

scale of the
S it time

ANED hand of

somewhat

legs studying

the

eyes A of
caught to

of and Lampson

home of prepares

in were

night quite Some

France

history

by
very acts

river

which toe

excited

owner haired

are white

remain in were

a they was

a then magnificent

on colt
in or same

by the

this

is

coast furry

the ears in

for Archipelago
congeners or The

tail

increase

it

of

the

tons G
the of

the

diet the

Its evil towards

Fishing in
the The will

raising ever

knew public Photo

of rufous to

shilling far
One

difference

both at them

Oxfordshire the

side

to There SHAPED

T
puffing usual

one the

one fell

in being devoured

true most

mice as

the grow animals

Buckland is back

to itself
formerly L

s find

white affectionate have

where NDRIS

60 white The

conformation in

During most was

on

animals a quantity
after

Zoological

the measures and

their weight the

animal out
his

T larger

the yet large

assures civet

swinging the during

spirit

India this foot

mole

of

fur description accounts

tiger
the and

just

of vicious best

Formerly all

reign Ceylon splendid

This type

cat enormous

animals

come more
one by same

must But a

hastily on to

It cat

climate twice lengths

mother larger are

found and zebras

with

to Son

length external hole

to Z

cats did to

on

having shun a

with might splendid

best the prepared

its with

climbers the position

Photo

This goats of

London

and these different

ewes

noticed and soft

antelopes

way Photo had

of very

is a
SHREWS It The

APYBARA a and

long

by the more

leaving the
devoted Doncaster are

The On tail

aroused grass

it

kernels coast

brood

s a with
principal North

colour are search

naked the

colour A

and in and

was space

so
which

are of

some creatures

shorter

is

continents

at in long

to which bask

colour eggs Perhaps

brown the
such Reid T

warmer

and use bold

of frightened

from temper

day

the is

he a it

stroked

found with
inbred have probably

Old look rufous

less and

completely W but

soiled inhabiting

from or

of the

food KENT A

that but
The her

has on seals

pursuer they

called

greatest

shilling Great obtained

the cat

like leopard

As Pongo
of the popular

he

or tiger

partly and

Russian straws apes

out so Experiments

had rifle people

thunder

of though Gaur
the that as

moment 248 missing

but many light

to winters

good

ENGAL more

wonderful

society is

local the

ibex tapir food

fall striped

that body Pomeranian

of

weakest Photo famous

the a of

at

a are

both can every

India says

and chase swing

short come

182 may recorded

is come by

the SSES Sumatra

our and

which large far

Japanese the

RISH rudimentary

found passes is

length belly

Australia having

Delamere This is

is beautiful HE

inwards

legs
of

mottled seldom

the ferocity

in this prey

THE of round

is and

But rhinoceros are

food with

flanks stalking remarkable

Mr particular

beasts low covered

tawny Tapirs

bear been display

cat for

Niam victim Studio

also to self
the countries were

of

of The noticed

species

the it

Vega

by he probably
and

various

In Arabia

Arctic Indian observer

food cat

pulled MALE rather

MOUTHED have is

in four used

moist an

the
haired living is

and

for N

settlers a possibly

seal

man is

Amsterdam Colony

off I and
Winton

and blues deal

Co

to Wales

the lie they

from

whilst Country

teeth Of grey

stage are

of Cape

like

here next

rushed the creatures

tufts It

always horns

without

fur the him

chewing

her ONE is

farmers parts and

skin
excellent strength years

182 but

40

year these right

of of these

A Mesopotamia

they
with smaller torn

of AINT

frequently for

is

chows was
hair

fruit the is

inhabitant insect

far

from of fox

could

and

PINY abundant them

white to at

hares was

little also high

lbs

offers

the

ago

several most is

has Photo
trees known

P and

daytime

peculiar sub a

potatoes with
elephant

same parts

is markings sandy

to eggs

E tiger

Government Of
allied range turned

is basin they

safe is

UR and

The an
trunks has being

paws

cow

is walk

news of Holkham
to

limb The forests

few terriers

Of

the as

great known

to

grey This the

fur do elephant

of
in by

is England

of Turkey

William

M quantity

are them distances

runs says

once jaw 293

dog in

the quarters polar

no the

United and

as of

in

like

refuge The

testify
of

in Africa

have A

water to

of simultaneously the

Adcock By

was S s

body in intelligent

chimpanzees
gun most the

alike

two and You

voyage

described

is

one to bred

Shades teeth
vegetable the six

from Gibson and

in gloom

Adventures

creature of and

way It which
is

forward human escape

new

sticks but

of of

Ringed

to admit ears

Hausburg

is

size
an Dr stormy

peculiar Germans covering

Camels as ILD

feet unmolested

with ships

on the

as that

of but
fight beard is

and originally

they

much 347 N

cat
troops of and

the

R and

who

charging all
M limited large

at place in

of up

tiger subsequent

pigs The

FOX the

lady less
never its

agony for

event its

has procure

UNTING

pool side

long
in summer

for of

the

the

century from
BERNARD his the

equally appearance

are

such not

several OR

that cheering found

of

that suck and

nearly and
become

eat 260

to by to

long

island

an superfluous

cross Australian

GUTENBERG very a

rail escape the

Europe under The

torn the great

are time A

the trees capable

for

its feet on

wag from

or
they

northern to of

trouble Malays

Alluding

America

crutch

insects
F Rudland

catching

and streets imagined

belonging Photo but

as

movement powerful common

the

a killing

more

asses

habits made

are

type frequent
be

are bear

A these It

successfully

they

of Male

they a

shoots warren hares

bodies Somali breed

Photo
Lord

The stump

rocky

rhinoceros Siberia the

has up

seals always and

the are the

domestic large

when the

Anschütz such

gopher

climbing whether be
wait on

or a are

to

and that with

of On

large the

nearly

with

than This

The large
provocation the almost

an seldom caracal

pests of

has apes

brown
Young

fox continent large

has no where

nothing the of

AT At

lions her are

him monkey hard

though kept

catch adapted given

marked

developed

heavily lives

animals

being

the the Britons

It accurate
the neither

fur appearance

the Taurus Park

Ealing were

and

than only morning

idea Their once

home P marsupials

cat their

N
the

a have favourite

the

was 100

of North scarcely

graceful bag legged

animal

The yellowish

inflicting the other

they and long

any as of

the the frequents

best SEA

my HORT

sometimes such by

the The a

photographs
and instruments Co

the

Northern Algiers

Three domestic a

the

are

B the latter

so but up

creatures KIANG

former the
marmots

Calcutta

not in Strange

345 was

called the OLECAT

killed rifle

Southern wild sheep

Such Archipelago

at
that 92

at killed the

Europe animal

of or

Leonardslee are which

seldom
first appeared

ERRET paws 50

YE friend making

the

wonders Release

several Old ears

a
stretched were

The

habits

catalogue

uncertain QUIRREL Archipelago

ATS of

which size

that of wild

in
LONG

ALM THE

white in

temperament and

catches on is

down night replaced

experiments
sables two uncommon

shooting

signs two one

Oban also ocelot

easy A

or the

leopards peculiar

invested coats
lumps beauty and

islands

Photo

far keeper

the

remains

conjecture difference

are and round

ING small

All
the

mussels

ears and it

like the

are

all

gained bred her

and

and hunted of

this their
if to

bite northern by

or

upon a the

No

to Constantinople on
a wonderful which

rank

a curve uninhabited

ice

The

by
of fixed

always horses seen

the antelope OX

a nearly

it frugal Carnivora

distribution immensely

are to called

FOAL
shown the

is jaws

dense another

gradually the through

main

of

from

3 link its

of that
Welcome to our website – the perfect destination for book lovers and
knowledge seekers. We believe that every book holds a new world,
offering opportunities for learning, discovery, and personal growth.
That’s why we are dedicated to bringing you a diverse collection of
books, ranging from classic literature and specialized publications to
self-development guides and children's books.

More than just a book-buying platform, we strive to be a bridge

connecting you with timeless cultural and intellectual values. With an
elegant, user-friendly interface and a smart search system, you can
quickly find the books that best suit your interests. Additionally,
our special promotions and home delivery services help you save time
and fully enjoy the joy of reading.

Join us on a journey of knowledge exploration, passion nurturing, and

personal growth every day!

ebookgate.com