A Manual of Cryobiology for Assisted Reproduction and Stem Cells
Editor
Pankaj Talwar Associate Professor
Department of Obstetrics and Gynecology Assisted Reproductive Technologies Specialist Trained in Human Embryonic Stem Cell Biology Army Hospital (Research and Referral)
Delhi Cantt 110 010, India
Published by
Jitendar P Vij
Jaypee Brothers Medical Publishers (P) Ltd
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Frozen Life
© 2009, Jaypee Brothers Medical Publishers
All rights reserved. No part of this publication should be reproduced, stored in a retrieval system, or transmitted in any form or by any means: electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of the editor and the publisher.
First Edition: 2009
9788184486810
Typeset at JPBMP typesetting unit
Printed at Ajanta Press
5Dedicated to
My father Late MP Talwar and my mother Madhu Talwar without their blessings such monumental work would not have been possible.
To
My wife Dr Neetu Talwar and sons Pratik and Arjun for giving unfailing support and encouragement during compilation of the book. It was their constant motivation which made me move ahead in trying times.
To
All my unknown patients who visited me for treatment with utmost faith and gave me opportunity to learn from their illness6
7Contributors
- A Camus
- IMV Technologies
- 10, rue Clemenceau
- 61300 L'Aigle, France
- A Maertens
- Laboratoire de Bacteriogie Virologic
- GIMAP, University Hospital, Saint-Etienne 420 55
- Saint-Etienne, France
- Akanksha Mishra
- Lilavati Hospital IVF Centre, Mumbai, Maharashtra, India
- Fortis Bloom IVF Centre, New Delhi, India
- DY Patil Fertility Centre, Navi Mumbai, Maharashtra, India
- Alain Ehrsam
- Cryo Bio System
- 29 rue Tronchet
- 75008 Paris, France
- A-L Van Kappel
- Cryo Bio System
- 29, rue Tronchet
- 75008 Paris, France
- Arti Sarin
- Professor
- Department of Radio Therapy
- Army Hospital (Research and Referral)
- Delhi Cantt 110 010, India
- Ashok Agarwal
- Center for Reproductive Medicine
- Cleveland Clinic, Cleveland
- Ohio, USA
- Aviva Devaux
- Department of Histo-embryology
- HoApital Bichat, 75018 Paris
- Axel Deroubaix
- Air Liquide, Cryogenic Division
- Parc Gustave Eiffel-8 Avenue, Gutenburg
- France 77 607, Bussy Saint George, France
- B Lejeune
- GIGA GIGA Transgenic Platform
- Universite de Liege, Belgique
- B Pozzetto
- Laboratoire de Bacteriogie Virologic
- GIMAP, University Hospital, Saint-Etienne 420 55
- Saint-Etienne, France
- Beatrice Ledos
- Cryo Bio System
- 29 rue Tronchet, 75008 Paris, France
- Bharati Dhorepatil
- Crescent Exclusee
- Behind Simla Office
- 116/3, Shivaji Nagar
- Pune 411 005, Maharashtra, India
- Bharti Bansal
- Lilavati Hospital IVF Centre, Mumbai, India
- Fortis Bloom IVF Centre, New Delhi, India
- DY Patil Fertility Centre, Navi Mumbai, Maharashtra, India
- C Staub
- IMV Technologies
- 10, rue Clemenceau
- 61300 L'Aigle, France
- C Vijayakumar
- Scientist
- Department of Radio Diagnosis
- Armed Forces Medical College
- Pune 410 040, Maharashtra, India
- Cathy Boutin
- CRi, 35 Cabot Road, Woburn, MA 01801
- Chandra Viswanathan
- Sr. Vice President (Regenerative Medicine)
- Reliance Life Sciences, R-282, DALSC
- Thane Belapur Road, Rabale
- Navi Mumbai, Maharashtra, India
- Christophe Sifer
- Department of Histo-embryology
- HoApital Bichat, 75018 Paris
- David Mortimer
- Oozoa Biomedical, Box 93012
- Caulfeild Village RPO, West Vancouver
- BC, Canada V7W 3G4
- DK Mishra
- Senior Adviser (Pathology and Haematology)
- Department of Pathology and Molecular Medicine
- Army Hospital (Research and Referral)
- Delhi Cantt, New Delhi 110 010, India
- E Balaji Prasath
- Chief Scientific Officer
- KKIVF, KK Women's and Children's Hospital, Singapore
- Eleonora Bedin Pasqualotto
- CONCEPTION - Center for Human Reproduction
- Caxias do Sul, RS, Brazil
- F Ectors
- Centre Hospitalier Inter Regional Cavell (CHIREC)
- Braine l'alleud - Bruxelles, Belgique
- Fabio Firmbach Pasqualotto
- Institute of Biotechnology
- University of Caxias do Sul, RS, Brazil
- Francoise Brun-Vezinet
- Department of Virology Hospital Bichat, 75018 Paris
- Frederique Kuttenn
- Cecos, Hospital Necker, 75015 Paris
- G Savic
- IMV Technologies
- 10, rue Clemenceau
- 61300 L'Aigle, France
- GA Ramaraju
- Krishna IVF Clinic
- ZP Junction, Krishna Nagar Maharanipeta
- Visakhapatnam-530 002
- Andhra Pradesh, India
- Gabor Vajta
- PIVET Medical Centre
- Leederville, Perth, Western Australia
- Gerard Feldmann
- Department of Histo-embryology
- HoApital Bichat, 75018 Paris
- Gille Collin
- Department of Virology
- Hospital Bichat, 75018 Paris
- GS Joneja VM
- Consultant Obstetrics and Gynaecology
- Office of Director Gen Medical Service (Air), New Delhi
- Guruprasad Kalthur
- Division of Reproductive Medicine
- Kasturba Medical College, Manipal University
- Manipal - 576 104, Karnataka, India
- Helene Letur-Konirsch
- Cecos, Hospital Necker, 75015 Paris
- Centre de Fertilite, Institut Mutualiste Montsouris 75014
- Paris
- Hrishikesh Pai
- Lilavati Hospital IVF Centre, Mumbai, India
- Fortis Bloom IVF Centre, New Delhi, India
- DY Patil Fertility Centre, Navi Mumbai, Maharashtra, India
- J Greindl
- Centre Hospitalier Inter Regional Cavell (CHIREC)
- Braine l'alleud - Bruxelles, Belgique
- Jack YZ Huang
- Division of Reproductive Biology
- Department of Obstetrics and Gynecology
- McGill University, Montreal, Quebec, Canada H3A 1A1
- Jaffar Ali PhD
- CTHM Reproductive Medicine Unit
- Women's Specialized Hospital
- King Fahad Medical City
- P.O. Box 59046, Riyadh 11525
- Kingdom of Saudi Arabia, Saudi Arabia
- Jaideep Malhotra
- Malhotra Nursing and Maternity Home (P) Ltd
- 84, MG Road, Agra 282 010 (UP), India
- Janavi Sanghvi
- Lilavati Hospital IVF Centre, Mumbai, Maharashtra, India
- Fortis Bloom IVF Centre, New Delhi, India
- DY Patil Fertility Centre, Navi Mumbai, Maharashtra,
- India
- Jaya Gogate
- Lilavati Hospital IVF Centre, Mumbai, India
- Fortis Bloom IVF Centre, New Delhi, India
- DY Patil Fertility Centre, Navi Mumbai, Maharashtra,
- India
- Jean-Louis Benifla
- Department of Obstetrics and Gynecology
- HoApital Bichat, 75018 Paris
- Department of Obstetrics and Gynecology
- HoApital Rothschild, 75012 Paris, France
- John Morris
- Asymptote Ltd
- St Johns Innovation Center, Cowley Road
- Cambridge CB4 0WS, England
- John Yovich
- PIVET Medical Centre
- Leederville, Perth, Western Australia
- Krishan Kapur
- Professor
- Gynecocolgical Endoscopist
- Army Hospital (Research and Referral)
- Delhi Cantt 110 010, India
- Leena Wadhwa
- Department of Assisted Reproductive Technologies and
- Reproductive Biology
- Maulana Azad Medical College
- New Delhi, India
- Mala Arora
- Noble Hospital Sector-14
- Faridabad 121007, Haryana, India
- Fortis La Femme, S-549
- Greater Kailash 2, New Delhi, India
- Manisha Taktani
- Lilavati Hospital IVF Centre, Mumbai, Maharashtra, India
- Fortis Bloom IVF Centre, New Delhi, India
- DY Patil Fertility Centre, Navi Mumbai, Maharashtra,
- India
- Meena Chimote
- Medical Director
- Vaunshdhara Clinic and Assisted Conception Centre
- Nagpur, Maharashtra, India
- Mrinalini Chaturvedi md
- Medical Director
- Cord Blood Bank
- Cryobanks International India
- N Plotton
- Laboratoire de Bacteriogie Virologic
- GIMAP, University Hospital
- Saint-Etienne 420 55
- Saint-Etienne, France
- N Zech
- Institute for Reproductive Medicine and Endocrinology
- Bregenz, Austria
- Nandita Palshetkar
- Lilavati Hospital IVF Centre, Mumbai, India
- Fortis Bloom IVF Centre, New Delhi, India
- DY Patil Fertility Centre, Navi Mumbai
- Maharashtra, India
- Narendra Malhotra
- Malhotra Nursing and Maternity Home (P) Ltd
- 84, MG Road
- Agra 282 010 (UP), India
- Natachandra Chimote
- Reproductive Endocrinologist and Embryologist
- Scientific Director
- Vaunshdhara Clinic and Assisted Conception Centre
- Nagpur, Maharashtra, India
- P Vanderzwalmen
- Institute for Reproductive Medicine and Endocrinology
- Bregenz, Austria
- Centre Hospitalier Inter Regional Cavell (CHIREC)
- Braine l'alleud - Bruxelles, Belgique
- Pankaj Talwar
- Associate Professor
- Department of Obstetrics and Gynecology
- Assisted Reproductive Technologies Specialist
- Trained in Human Embryonic Stem Cell Biology
- Army Hospital (Research and Referral)
- Delhi Cantt 110 010, India
- Patrick Madelenat
- Department of Obstetrics and Gynecology
- HoApital Bichat, 75018 Paris
- Philippe Clairaz
- Cryo Bio System
- 29 rue Tronchet
- 75008 Paris, France
- Pratap Kumar
- Division of Reproductive Medicine
- Kasturba Medical College, Manipal University
- Manipal - 576 104, Karnataka, India
- Praveen Pandaredattil
- IMV India Udyog Vihar
- Gurgoan, Haryana, India
- Preeti Kabra
- Sr. Vice President (Regenerative Medicine)
- Reliance Life Sciences, R-282, DALSC
- Thane Belapur Road, Rabale
- Navi Mumbai, Maharashtra, India
- R Levy
- Laboratoire de Biologie de la
- Reproduction, GIMAP
- University Hospital of Saint-Etienne, 42055
- Saint-Etienne, France
- Randhir Puri
- Professor
- Obstetrics and Gynaecology
- Military Hospital, Belgaun, Karnataka, India
- Rashmi Sharma
- Southend Fertility and IVF Centre
- Holy Angels Hospital, Vasant Vihar
- New Delhi, India
- Ri-Cheng Chian msc, phd
- Division of Reproductive Biology
- Department of Obstetrics and Gynecology
- McGill University, Montreal, Quebec, Canada H3A 1A1
- Rima Dada
- Associate Professor
- Department of Anatomy
- All India Institute of Medical Sciences
- New Delhi, India
- Rishma Pai
- Lilavati Hospital IVF Centre, Mumbai, India
- Fortis Bloom IVF Centre, New Delhi, India
- DY Patil Fertility Centre, Navi Mumbai, Maharashtra, India
- S Bhargava
- Professor and Head Department of Radio Diagnosis
- Armed Forces Medical College
- Pune 410 040, Maharashtra, India
- Satish Kumar Adiga
- Division of Reproductive Medicine
- Kasturba Medical College
- Manipal University, Manipal-576 104
- Karnataka, India
- Shan Liu
- Reproductive Medical Center
- Institute of Stem Cell Shandong University, PR, China
- Shashi Sareen
- Trained in ART and Reproductive Sonology
- Office of Dir Gen Armed Forces Medical Services
- Delhi, India
- Shehbaaz Daruwala
- Crescent Exclusee
- Behind Simla Office
- 116/3, Shivaji Nagar
- Pune 411 005, Maharashtra, India
- Sonia Mallik
- Southend Fertility and IVF Centre
- Holy Angels Hospital, Vasant Vihar, New Delhi, India
- SPS Kochar
- Professor
- Obstetrics and Gynaecology Department
- Armed Forces Medical College
- Pune -40, India
- Sudha Prasad
- Department of Assisted Reproductive Technologies and
- Reproductive Biology
- Maulana Azad Medical College
- New Delhi, India
- Surveen Ghumman Sindhu
- ART Specialist and Clinical Embryologist
- Department of Obst. and Gynae
- Vardhaman Mahavir Medical College and
- Safdarjang Hospital, New Delhi, India
- Suryakant Hayatnagar
- Medical Director, Cryogenie India Limited
- Sushma Ved
- Southend Fertility and IVF Centre
- Holy Angels Hospital, Vasant Vihar, New Delhi, India
- T Bourlet
- Laboratoire de Bacteriogie Virologic
- GIMAP, University Hospital, Saint-Etienne 420 55
- Saint-Etienne, France
- T Mukaida
- Hiroshima HART Clinic
- 5-7-10 Ohtemachi, Naka-ku, Hiroshima, 730-0051, Japan
- Usha Ahluwalia
- Dewsbury District Hospital
- Halifax Road Dewsbury, WF13 4HS, UK
- Vandana Bhatia
- Southend Fertility and IVF Centre
- Holy Angels Hospital, Vasant Vihar, New Delhi, India
- Ved Prakash
- Southend Fertility and IVF Centre
- Holy Angels Hospital, Vasant Vihar, New Delhi, India
- Velu Nair, VSM**
- Senior Adviser (Medicine and Clinical Haematology) Professor and Head
- Department of Clinical Haematology and BMT
- Army Hospital (Research and Referral)
- Delhi Cantt, New Delhi 110 010, India
- Vijay Mangoli
- Laboratory Director
- Fertility Clinic and IVF Centre
- 101-Shanti Niketan
- V Gandhi Road, Gamdevi, Mumbai 400 007,
- Maharashtra, India
- Vrinda
- Lilavati Hospital IVF Centre, Mumbai, Maharashtra, India
- Fortis Bloom IVF Centre, New Delhi, India
- DY Patil Fertility Centre, Navi Mumbai
- Maharashtra, India
- William F Rall
- Physiologist and Head of Embryo Cryopreservation and
- Rederivation Species
- Preservation and Special Breeding Section
- Veterinary Resources Program
- Office of Research Services
- National Institutes of Health, Bethesda
- Zi Jiang Chen
- Vice President
- Shandong Provincial Hospital
- Vice Dean, School of Medicine, Shandong University
- Chair and Professor
- Department of Obs and Gyn
- Director, Reproductive Medical Center
- Director, Institute of Stem Cell Shandong University, PR
- China
It is indeed a great pleasure to write a foreword for this book on cryobiology. Human reproductive cryobiology is rapidly developing subspecialty of Assisted Reproductive Technologies.
Since the first report of human pregnancy and the first live birth following cryopreservation two decades ago, the techniques has developed as a reliable science and is carried out regularly at all ART laboratories.
Combined with tailored patient preparation protocols, thawed embryo transfer cycles have become relatively successful. The transfer of cryopreserved embryos represents around 20% of all transfers worldwide.
Recently, the focus is shifting to the vitrification technique which involves ultrarapid cooling of the reproductive cells resulting in glass formation. A lot of researches are going on in development of ideal oocyte cryofreezing protocols. If perfected such technique would be an important technology to help unmarried women who are suffering from malignancy and require treatment.
The book has covered these topics at length and would be ideal compendium for any ART manual.
I am happy that Lt Col Pankaj Talwar has taken this initiative to edit the textbook of Frozen Life. The book has been written in a concise and systemic manner and readers can easily assimilate the subject and practise it.
Punita Arora pvsm, sm, vsm
Consultant, Obstetrics and Gynecology (Armed Forces)
Ex-Director General Medical Services and Ex-Commandant and Director
Armed Forces Medical College
15Foreword
I am happy to write a foreword to this textbook of cryobiology by my student Lt Col Pankaj Talwar. This is probably the 1st book ever published dealing entirely with the principles of cryobiology and its application in Assisted Reproductive Technologies.
The main aim of cryopreservation for human oocytes and embryos is to reduce damage caused by intracellular/extracellular ice crystal formation. To achieve this, it is important to consider the behavior of intracellular water at subzero temperatures. Two basic techniques have been developed for reproductive cells cryopreservation: controlled slow-rate freezing protocols and rapid freezing protocols such as vitrification.
These techniques may be applied to cryofreezing of oocytes, spermatozoa, embryos, genital tissue, umbilical cord blood cells and embryonic stem cells. All these techniques and protocols are thoroughly covered in this book. The protocols have been well elucidated and deliberated.
The editor has covered the process of cryobiology step by step at length in the last section of the book. This will reduce the learning curve of the embryologists and clinicians. These protocols encompass the techniques being followed at Army Hospital (Research and Referral), New Delhi, India.
I congratulate the editor for bringing so many researchers, clinicians and reproductive biologists under the one roof and compiling this textbook. I am sure this book will help all the medical personnels working in the field of Assisted Reproductive Technologies and related specialties to understand the topic better and put it to practical use.
Professor and Head
Department of Obstetrics and Gynecology
Director, Assisted Reproductive
Technologies Centre, Army Hospital
(Research and Referral)
Delhi Cannt 110 010
17Preface
Cryobiology is a rather interesting but complex subspecialty of Assisted Reproductive Technologies. This branch has been always considered as an appendage of ART and clinicians have been somewhat sceptical about accepting the concept of creating icecream babies. The protocols are easy but the physiology and thermodynamics of the procedure are not clear to majority of us. This scares us from accepting this technology.
I am endeavoring to simplify the gray areas of cryobiology with lucid articles and videos pertaining to all aspects of reproductive medicine and stem cell biology.
As the embryo culture conditions are improving, more number of embryos are available to the embryologist for cryofreezing. With advent of single embryo transfer, the Assisted Reproductive Technology centres have been encouraged to adopt cryobiology techniques in their day-to-day practice.
The advent of oocyte freezing for fertility preservation along with the advances in the fields of ovarian tissue and testicular tissue banking has generated enormous interest in this field.
Till date there has been a scare in minds of reproductive biologists who prefer fresh embryo transfer in the IVF cycles. Prior experiences with poor cryosurvival of the gametes and embryos and compromised implantation rates were not encouraging.
Better protocols and development of less toxic cryoprotectant solutions have led to resurgence of the subspecialty which had taken backseat due to poor outcomes in past few years.
Cryobiology has played pivotal role in cryopreservation of hematopoietic stem cells and embryonic stem cells. Reproductive biologists are slowly shifting the focus of research toward vitrification which is cheaper, quicker and easier technology.
The aim of the book has been to bring together researchers, clinicians and embryologists on one platform to share their views on various aspects of cryobiology.
A wealth of information pertaining to basics of cryobiology, thermodynamics of cryofreezing, cryoprotecants along with newer protocols for vitrification of embryos, oocytes, etc. have been thoroughly covered. We have also covered at length umbilical cord blood banking and cryobiology of embryonic stem cells.
Efforts have been made to present to the readers the protocols using lots of pictures so that they can closely associate with the techniques and easily assimilate them.
The subject is vast and has been covered in 53 Chapters and Appendices. Some amount of repetition is unavoidable while compiling such monumental work.
Please feel free to communicate with me if you need any clarifications or have any suggestions.
19Acknowledgements
I would like to thank all my teachers, colleagues for their constant motivation and encouragement.
My sincerest thanks to Col RK Sharma and Lt Gen (Mrs) Punita Arora who constantly held my hand in this humble spiritual journey and gave me strength to move on in the face of all adversities.
I am grateful to Lt Gen Y Singh PVSM, VSM, PHS, DGAFMS and Sr Col Comdt, Lt Gen NK Parmar AVSM, VrC, VSM, PHS, DGMS Army and Col Comdt, Lt Gen OP Mathew AVSM, SM, Col Comdt and Commandant Army Hospital (Ramp;R), for permitting me to take on this monumental work. Without their constant support the book would not have been in our hands today.
I am also grateful to Prof Itskovitz-Eldorj or Amit M and team from technion, Israel, for teaching me the intracacies of embryonic stem cell biology.
I would like to thank my colleagues Col BS Duggal and Col BK Goyal for the guidance and constant motivation.
The work would not have been possible without unflinching support of Shri Jitendar P Vij (Chairman and Managing Director), Mr Tarun Duneja (Director-Publishing), Mr Subroto Adhikari, Mr KK Raman, Mr DC Gupta, Mr Manoj Pahuja, Mrs Y Kapoor and Mrs Niti of M/s Jaypee Brothers Medical Publishers (P) Ltd., New Delhi.
I thank Trivector Scientific Pvt. Ltd., Dr H Ingolf Nilesen and Dr Lars Johanson, consultant embryologists (MediCult, Denmark) for their suggestions and constant support in compilation of the manuscript.
I would like to thank IMV India Ltd., Alex Deroubaix of Air Liquid, Alain Ehrsam, Philippe Clairaz, Ms Beatrice Ledos and Ms Melanie lelogof CBS France, Intermedic and Cook International (Australia), FertiPro (Belgium), vitrolife for support and permission to utilize the videos on vitrification for educational purposes.
I am thankful to Dr Sarabpreet Singh, Mr Parikshit Singh and Mr GS Pillai who spent late nights with me while compiling pictures for of this book.
I humbly acknowledge that this dream would not have been possible until all the contributors rendering me their unflinching support and motivation. The journey from inception of the book to its publication was rough but I was always supported by my friends and my patients.
20Disclaimer
This manual presents an understanding and an outlook of the current scientific advances in the field of reproductive and stem cell cryobiology. This is an emerging field and is changing every day. The information presented in this book may not be considered as exclusive methodology of cryobiology techniques; rather, it is intended to be a basic guideline for the clinicians and embryologists.