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Vitrification in Assisted Reproduction
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REPRODUCTIVE MEDICINE & ASSISTED REPRODUCTIVE TECHNIQUES SERIES Series Editors David K Gardner DPhil Colorado Center for Reproductive Medicine, Englewood, CO, USA Jan Gerris MD PhD Professor of Gynecology, University Hospital Gheni, Ghent, Belgium Zeev Shoham MD Director, Infertility Unit, Kaplan Hospital, Rehovot, Israel Published Titles Gerris, Delvigne and Olivennes, Ovarian Hyperstimulation Syndrome ISBN 978 1842143285 Sutcliffe, Health and Welfare of ART Children ISBN 9780415379304 Tan, Chian and Buckett, In-vitro Maturation of Human Oocytes ISBN 978 1842143322 Keck, Tempfer and Hugues, Conservation Infertility Management ISBN 9780415384513 Pellicer and Simon, Stem Cells in Human Reproduction ISBN 978 0 415 397 773 Elder and Cohen, Human Preimplantation Embryo Solution ISBN 978 0415399739 Forthcoming Titles Aplin, Fazleabas, Glasser, Giudice, The Endometrium, second edition ISBN 978 0415385831
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Vitrification in Assisted Reproduction A User’s Manual and Trouble-shooting Guide Edited by
Michael J Tucker
PhD FIBiol HCLD
Scientific Director Georgia Reproductive Specialists Atlanta, GA USA
Juergen Liebermann Scientific Director Fertility Centers of Illinois Chicago, IL USA
PhD HCLD
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© 2007 Informa UK Ltd First published in the United Kingdom in 2007 by Informa Healthcare, Telephone House, 69–77 Paul Street, London EC2A 4LQ. Informa Healthcare is a trading division of Informa UK Ltd. Registered Office: 37/41 Mortimer Street, London W1T 3JH. Registered in England and Wales number 1072954. Tel: +44 (0)20 7017 5000 Fax: +44 (0)20 7017 6699 Website: www.informahealthcare.com All rights reserved. No part of this publication may 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 permission of the publisher or in accordance with the provisions of the Copyright, Designs and Patents Act 1988 or under the terms of any licence permitting limited copying issued by the Copyright Licensing Agency, 90 Tottenham Court Road, London W1P 0LP. Although every effort has been made to ensure that all owners of copyright material have been acknowledged in this publication, we would be glad to acknowledge in subsequent reprints or editions any omissions brought to our attention. A CIP record for this book is available from the British Library. Library of Congress Cataloging-in-Publication Data Data available on application ISBN-10: 0 415 40882 2 ISBN-13: 978 0 415 40882 0 Distributed in North and South America by Taylor & Francis 6000 Broken Sound Parkway, NW, (Suite 300) Boca Raton, FL 33487, USA Within Continental USA Tel: 1 (800) 272 7737; Fax: 1 (800) 374 3401 Outside Continental USA Tel: (561) 994 0555; Fax: (561) 361 6018 Email:
[email protected] Distributed in the rest of the world by Thomson Publishing Services Cheriton House North Way Andover, Hampshire SP10 5BE, UK Tel: +44 (0)1264 332424 Email:
[email protected] Composition by C&M Digitals (P) Ltd, Chennai, India Printed and bound in India by Replika Press Pvt Ltd
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Dedication
Michael J Tucker would like to thank his wife Megan and three children who make this all worthwhile. Juergen Liebermann gives most sincere thanks to his wife Maike and his sons Richard, Lennart Martin, and Tobias Georg for providing unfailing support and encouragement to bring this work to reality.
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Contents
List of Contributors Preface Acknowledgments 1A.
Vitrification: an overview Gregory M Fahy and William F Rall
1B.
Vitrification in small quenched volumes with a minimal amount of, or without vitrificants: basic biophysics and thermodynamics Igor I Katkov, Vladimir Isachenko and Evgenia Isachenko
ix xiii xv 1
21
2.
Disadvantages and benefits of vitrification Gábor Vajta, Masashige Kuwayama and Pierre Vanderzwalmen
33
3.
Development of vitrification solutions Jaffar Ali and James Shelton
45
4A.
4B.
Vitrification in animal reproduction: vitrification of embryos using open pulled straws Gábor Vajta
65
Vitrification in animal reproduction: vitrification of embryos using conventional straws with ethylene glycol-based solutions Magosaburo Kasai and Keisuke Edashige
75
5.
Cryoprotectant-free vitrification of spermatozoa Evgenia Isachenko, Vladimir Isachenko, Igor I Katkov, Raul Sanchez, Hans van der Ven, Markus Montag and Frank Nawroth
6.
Potential developmental consequences of cryopreservation of mammalian oocytes and embryos Gary D Smith and Luis G Villa-Diaz
107
Vitrification of oocytes: general considerations and the use of the Cryotop method Masahige Kuwayama, Ana Cobo and Gábor Vajta
119
7A.
87
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CONTENTS
7B. Vitrification of oocytes: various procedures Shee-Uan Chen and Yu-Shih Yang
129
7C. Vitrification of oocytes using gold grid and slush nitrogen Tae Ki Yoon, Dong Ryul Lee and Kwang Ryul Cha
145
7D. Vitrifying and warming of oocytes using cryotop Koichi Kyono, Yukiko Nakajo, Shima Kumagai, and Chikako Nishinaka
153
8. Vitrification of pronuclear embryos: research basis for aseptic technology and its application to oocytes and blastocysts Hans van der Ven, Vladimir Isachenko, Evgenia Isachenko Markus Montag and Frank Nawroth
163
9. Vitrification of day 2–3 human embryos: using various techniques (Cryoloop, Cryotop and conventional cryostraw) Tetsunori Mukaida and Katsushiko Takahashi
183
10A. One decade of experience with vitrification of human embryos in straws, hemi-straws, and high security vitrification straws Pierre Vanderzwalmen, Thomas Ebner and Nicolas Zech
195
10B. Vitrification of blastocysts using the Cryoloop technique Tetsunori Mukaida and Katsuhiko Takahashi
219
10C. Vitrification of blastocysts using the electron microscope grid Weon-Young Son and Jin-Ho Lim
239
10D. Vitrifying and warming of human blastocysts using the Cryotop Juergen Liebermann and Michael J Tucker
253
11A Vitrification of ovarian tissue Frank Nawroth, Vladimir Isachenko, Evgenia Isachenko and Gohar Rahimi
261
11B. Vitrification of ovarian tissues Ying C Song, Zhenzhen Chen, Carol Journey, Adelina M Emmi, Xiayang Xie and Rosemary L Song
273
12.
293
Vitrification of human embryonic stem cells Yoel Shufaro, Gábor Vajta, Alan O Trounson and Benjamin E Reubinoff
299
Index
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Contributors
Jaffar Ali PhD IVF Unit Maternity Hospital King Fahad Medical City Riyadh Saudi Arabia
Keisuke Edashige PhD Laboratory of Animal Science College of Agriculture Kochi University Kochi Japan
Kwang Ryul Cha MD Fertility Center of CHA General Hospital, CHA Research Institute Pochon Cha University University College of Medicine Seoul Korea
Adelina M Emmi Medical College of Georgia Augusta, GA USA
Shee-Uan Chen MD Department of Obstetrics and Gynecology National Taiwan University Hospital Taipei Taiwan Zhenzhen Chen Organ Recovery Systems Inc Charleston, SC USA Ana Cobo Institute of Infertility Valencia University School of Medicine Valencia Spain Thomas Ebner PhD Landes-Frauen- und Kinderklinik IVF Unit Linz, Austria
Gregory M Fahy PhD 21st Century Medicine Inc Rancho Cucamonga, CA USA Evgenia Isachenko PhD Department of Endocrinology and Reproductive Medicine University of Bonn Bonn Germany Vladimir Isachenko PhD Department of Endocrinology and Reproductive Medicine University of Bonn Bonn Germany Magosaburo Kasai PhD Kochi Laboratory of Animal Science College of Agriculture Kochi University Nankoku Japan
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LIST OF CONTRIBUTORS
Igor I Katkov PhD Department of Pediatrics, University of California San Diego, CA USA
Markus Montag MD PhD Department of Gynaecological Endocriniology and Reproductive Medicine University of Bonn Bonn-Venusberg Germany
Carol Journey Medical College of Georgia Augusta, GA USA Masashige Kuwayama Kato Ladies Clinic Tokyo Japan
Tetsunori Mukaida MD Hiroshima Hart Clinic Hiroshima Japan
PhD
Yukiko Nakajo Kyono ART Clinic Japan Frank Nawroth MD PhD Zentrum für Hormon- und Stoffwechselerkrankungen, Reproduktionsmedizin und Gynäkologische Endokrinologie Hamburg Germany
Koichi Kyono Kyono ART Clinic Japan Dong Ryul Lee PhD Fertility Center of CHA General Hospital CHA Research Institute Pochon Cha University University College of Medicine Seoul Korea Juergen Liebermann PhD Fertility Centers of Illinois Chicago, IL USA
Chikako Nishinaka Kyono ART Clinic Japan Gohar Rahimi Department of Obstetrics and Gynecology University of Cologne Cologne Germany
HCLD
William F Rall PhD National Center for Research Resources National Institutes of Health Bethesda, MD USA
Jin-Ho Lim MD President Maria Infertility Hospital Seoul South Korea
Benjamin E Reubinoff MD PhD Department of Obstetrics and Gynecology Hadassah Embryonic Stem Cell Research Center Hadassah University Hospital Jerusalem Israel
Peter Mazur PhD Fundamental and Applied Cryobiology Group The University of Tennessee Knoxville, TN USA
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LIST OF CONTRIBUTORS
Ying C Song MD PhD Director of Research Xytex Research Augusta, GA USA
Raul Sanchez PhD MD University of Temuco Chile Shima Kumagai Kyono ART Clinic Japan James Shelton Scarborough Queensland Australia
Katsuhiko Takahashi MD Hiroshima HART Clinic Hiroshima Japan
DVSc PhD FACVSc
Alan O Trounson BSc MSc Monash Immunology and Stem Cell Laboratories Monash Medical Center Clayton, Victoria Australia
Yoel Shufaro MD IVF Unit Department of Obsetrics and Gnecology The Hadassah Human Embryonic Stem Cells Research Center Hassadah Ein Kerem University Hospital Jerusalem Israel
PhD
Michael J Tucker PhD FIBiol HCLD Georgia Reproductive Specialists Atlanta, GA USA
Gary D Smith PhD HCLD Department of Obstetrics and Gynecology University of Michigan Ann Arbor, MI USA
Gábor Vajta MD PhD DVSc Department of Genetics and Biotechnology Danish Institute of Agricultural Sciences Research Center Foulum Tjele Denmark
Weong-Yong Son PhD McGill Reproductive Center Royal Victoria Hospital Montreal, Quebec Canada
Hans van der Ven MD Zentrum für Geburtshilfe u. Frauenheilkunde Rheinische Friedrich-WilhelmUniversität Bonn Bonn Germany
Weon-Young Son PhD Lead Embryologist McGill Reproductive Center Department of Obstetrics and Gynecology Royal Victoria Hospital McGill University Montreal Canada
Pierre Vanderzwalmen PhD Institute for Reproductive Medicine and Endocrinology Bregenz Austria and Centre Hospitalier Inter Regional Cavell (CHIREC) Brussels Belgium
Rosemary L Song Medical College of Georgia Augusta, GA USA
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LIST OF CONTRIBUTORS
Luis G Villa-Diaz PhD University of Michigan Ann Arbor, MI USA
Tae Ki Yoon MD Fertility Center of CHA General Hospital CHA Research Institute Pochon CHA University Seoul Korea
Xiayang Xie Medical College of Georgia Augusta, GA USA
Nicolas Zech MD Institute for Reproductive Medicine and Endocrinology Bregenz Austria
Yu-Shih Yang MD PhD National Taiwan University Hospital and College of Medicine Taipei Taiwan
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Preface
The impact of cryopreservation on the growth and improved efficiency of assisted reproduction in humans is becoming increasingly appreciated, with close to one-fifth of all births following in vitro fertilization and embryo transfer worldwide arising from cryopreservation of supernumerary embryos. As culture techniques and related embryo quality improve, it is inevitable that this ratio of fresh to cryopreserved embryo babies will become more equal, indicative of an increased reliance on the use of embryos following cryostorage. This will be a consequence of the improved efficiencies inherent in this approach, including possibly improved uterine receptivity in unstimulated uterine transfer cycles, in conjunction with the recognition of the need for more routine single embryo transfers, to reduce multiple implantations often seen with cycles where multiple embryos have been transferred, as is a common current practice. Although initially reported in 1985 as a successful cryopreservation approach for mouse embryos, vitrification has taken a backseat to the much more widely adopted conventional freezing technology applied to both gametes and embryos in animal and human assisted reproduction. Recent years have seen a resurgence of interest in this ultrarapid cryopreservation technology, as the limitations of slow-rate freezing have become more evident in the clinical arena. Frustrations with mediocre cryosurvival, development, and ultimately compromised implantation rates have fuelled clinical embryologists in particular to seek alternative strategies to improve outcomes. Vitrification, an ice-free form of cryopreservation, offers a level of consistency of performance, once mastered, that may achieve clinical results that rival outcomes using fresh material. Additionally, vitrification offers certain benefits in the ease of its application that make current conventional freezing technology appear unpredictable, costly, and inflexible. This book makes no pretence to be the definitive text on vitrification. It nonetheless attempts to present in a straightforward manner the current and breaking vitrification technology available to those in the animal reproduction industry, and to clinical practitioners in human assisted reproduction. This includes discussion and guidance for cryoprotectant-free sperm cryostorage, highly consistent oocyte cryopreservation, as well as consideration and explanation of successful protocols for vitrification of embryos at all stages of preimplantation development, with particular emphasis on in vitro derived human embryos. These various protocols are discussed in the clear context of the type of vitrification carrier device used, with comment on how and why they were developed, and the relevance of cooling and warming rates that are central to achieving the vitrified state. A wealth of easily understood background material is presented, as well as the extremely useful comparative discussion of vitrification protocols applied to mammalian oocytes and
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PREFACE
embryos, that enable greater appreciation of the nuances of vitrification technology, and the crucial need in vitrification for close adherence to protocol per cell and tissue type. We are proud to have been able to have worked with so many of the pivotal researchers in this area of cryobiology, and trust our collaboration here might prove as useful to others interested in vitrification and assisted reproduction as it has done for us. Michael J Tucker Juergen Liebermann
xiv
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Acknowledgments
The editors Michael J Tucker and Juergen Liebermann would like to thank the following individuals for their efforts to bring this publication to fruition: Nick Dunton (formerly of Informa Healthcare) who had the vision to propose and initiate this project; also Lindsay Campbell Robert Peden, and Alexa Chamay Berrier who as current editors at Informa Healthcare, have been extremely helpful in keeping this publication on track throughout the vagaries of the process to provide a well-focused and timely book. All contributing authors are thanked for their commitment to providing current and comprehensive chapters in this increasingly recognized area of cryobiology. Their enthusiasm and professionalism in their work is evident in their writings, and we thank them enormously for allowing us to act as coordinating editors to put this book together on a subject that we think is both interesting and important. We hope that this book is enjoyed by, and of benefit to, all who read it. We are also grateful to all the IVF laboratory staff at the Fertility Centers of Illinois, River North: Jill Matthews, Elissa Pelts, Mandy Erman, Becky Brohammer, Sara Sanchez, Yuri Wagner, and Andrew Barker whose clinical skills facilitated clinical application of routine vitrification within that laboratory – to all we say a very special thank you. We also extend thanks to all the beautiful people out there who have ever been nice to us.
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1A
Vitrification: an overview Gregory M Fahy and William F Rall
vitrification or the range of present applications of vitrification. Instead, our goal is to provide a broader background context for the chapters that follow by briefly considering the place of vitrification within the natural world, the historical origins of the modern concept of vitrification as a means of cryopreservation, and the fundamental theoretical basis of all methods of natural and applied cryopreservation by vitrification.
INTRODUCTION Vitrification is an increasingly popular method of cryopreservation (Table 1A.1; Figure 1A.1). In the realm of reproduction, vitrification is currently used for the routine cryopreservation of human4,5 and animal5 embryos, and has been used successfully for the cryopreservation of ovarian tissue,6 ova,7–9 and possibly even whole ovaries.10 In addition to solving an increasing variety of basic and applied biological problems, vitrification has attained much of its appeal by making cryopreservation both simpler and more convenient than conventional freezing methods for many living systems. Our present overview is not intended to recapitulate the scope of specific methods of Table 1A.1
VITRIFICATION IN NATURE The lowest terrestrial temperature ever recorded was apparently −89.3°C, at the Russian Vostok station in Antarctica.11 This is well above the glass transition temperature
Some successes in cell, tissue, organ, and organism cryopreservation by vitrification
Cells
Tissues
Organs and organisms
Ova (cow, human, mouse) Oocyte cytoplasts Embryonic stem cells (human) Spermatozoa? (no cryoprotectant) Hematopoietic progenitors Hepatocytes (rat, microencapsulated) Islet substitute cells Monocytes (human) Osteoblasts Plant cells (many varieties) Red blood cells (human)
Embryos (buffalo, cow, fly, human, llama, mouse, etc.) Ovarian tissue (various) Embryonic kidney tissue? Corneas (rabbit, human) Heart valves (human) Organ slices (hippocampus, liver, kidney) Islets, pancreatic (man, monkey, mouse) Peripheral nerves Cartilage (rabbit, pig) Plant tissues (many types)
Ovaries? (mouse) Embryonic hearts (chicken) Embryonic brains (chicken) Veins (jugular, rabbit) Arteries (rabbit) Kidney (rabbit) Skin? (human) Schistosomes Tetrahymena
The information, opinions, data, and statements contained herein are not necessarily those of the US Government or the National Institutes of Health (NIH) and should not be interpreted, acted on or represented as such.
1
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The high cytoplasmic glass transition temperature may result from high concentrations of intracellular protein, raffinose, and stachyose.20 Similarly, Sakai concluded that for several other superhardy plant species:21 ‘the most appropriate interpretation is that completely hardy plants form aqueous glasses intracellularly.’ Many polar species are ‘poikilohydric’, meaning that their moisture content varies with the ambient humidity, which can become low enough to induce cytoplasmic vitrification.11,14,19,21–23 This strategy of freeze avoidance by vitrification may actually be more common than the ability to survive freezing.11,24 Crowe et al. showed that soil nematodes dried to below 0.3 g of water per gram of dry weight did not exhibit any evidence of freezing below −30°C and survived cooling in liquid nitrogen, whereas nematodes with higher water contents were killed.25 Similarly, Holmstrup et al. reported that several arctic soil invertebrates dehydrated at −14 to −17°C contained no freezable water when subsequently cooled to and rewarmed from −60°C.24 Certain Alaskan beetles dehydrate sufficiently to generate concentrations of up to 10 mol/L of endogenous glycerol,26 which is enough to vitrify aqueous solutions under laboratory conditions.27,28 The mean winter supercooling points for these insects were −35 to −42°C, but at some times of the year they were able to cool to −80°C without freezing.26 Whether any of these beetles actually vitrify in nature was not clear, but they are perhaps the closest natural analog to laboratory vitrification. They tolerate the same high concentrations of permeating cryoprotectant used by the cryobiologist and do not require virtually complete drying to attain vitrifiability. Supercooling points as low as −66°C have also been reported for dipterans.18 A particularly striking natural illustration of the importance of avoiding extracellular ice formation in organs is the convergent evolution of mechanisms for controlling the
500 400 300 200
Total citations
Cumulative PubMed entries for vitrification
600
8 6 4 2 0 1982
1984 1986 Year
100 0 1970 1975 1980 1985 1990 1995 2000 2005 Year of publication
Figure 1A.1 Cumulative citations to vitrification as a method of cryopreservation in PubMed since 1966. Inset: onset of the curve.1,2 The smooth line drawn through the data from 1984 to 2005 is a double exponential curve fit. Modified from an earlier version.3
(Tg) range for aqueous solutions of permeating cryoprotectants, but well below the Tg of concentrated aqueous solutions of natural non-penetrating cryoprotectants such as glucose,12 sucrose,12 trehalose,13,14 sugar mixtures containing fructose,15 and concentrated general tissue solutes.16 In fact, intracellular glass transition temperatures above − 50°C have been documented in several species,17–19 and it is not atypical for temperatures in polar regions to fluctuate between values as low as −50°C or below in the colder months and highs of +20°C,11 so overwintering in a partially or completely vitreous state is not an implausible adaptation.15,18 The ultrahardy tree, Populus balsamifera, has twigs that can survive direct immersion in liquid nitrogen during the winter months. Freeze-fracture and differential scanning calorimetric studies published by Allen Hirsh in 198517 demonstrated that dehydration of the cytoplasm in this species during slow extracellular freezing is arrested at about −30°C by limiting intracellular solute concentrations, and that further cooling results in supercooling into a glassy phase at approximately −45°C.
2
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location of ice in frogs and the silver fir, Abies sachalinensis. In the terrestrial frog, ice forms first in strategic locations within the relatively hardy peripheral tissues, such as between the skin and the muscle and in the abdominal cavity.29 This ensures long freezing plateaus and sufficient time for water to distill over macroscopic distances from the sensitive abdominal, thoracic, and intracranial organs onto ice crystals present external to these organs. The liver, heart, brain, intestines, and kidneys visibly shrink as adjacent ice masses expand, thus allowing them to behave like single cells shrinking in response to extracellular ice formation rather than suffering injury from extensive invasion by ice.29 In the fir tree, the apical meristem survives by essentially the same mechanism. Ice is nucleated in a safe place adjacent to the apical meristem, and this results in distillation of meristem water through a special barrier through which water can diffuse but ice cannot grow in the opposite direction to invade the meristem (Sakai, personal communication). Other plants appear to have evolved essentially the same mechanism.30 Several other very different and fascinating freeze avoidance strategies have also evolved in both plants and animals. For our present purposes, we simply note the ‘take home’ message of these many diverse and elaborate examples, which is that ice avoidance is a winning natural strategy for the cryopreservation of complex living systems. Therefore, the experience of nature provides reassuring support for the pursuit of vitrification in the laboratory.
for cryopreservation came from Walter Stiles in 1930.32 Although his suggestion could have been more clearly stated, he nevertheless definitely proposed that biological vitrification might be both possible and desirable: ‘In general . . . protoplasm . . . is similar to . . . non-living colloidal systems . . . if a hydrosol or hydrogel is frozen very rapidly . . . the whole will set into a finely crystalline or even amorphous mass . . . Such a solid in thawing, might be expected to give again the original system without change.’
Luyet and colleagues Father Basile J Luyet independently envisioned and is widely acknowledged as being the first to take to heart the idea of vitrification of living cells.33 Luyet and his associates expended a vast amount of effort in the direct pursuit of vitrification over the period from 1937 to 195834 and much subsequent indirect work that would later prove to be more important for vitrification than his original direct efforts.34 Luyet’s fascination with the nature of life35 led him to seek clues about the living state by attempting to arrest life and then restart it, and vitrification seemed to be a promising means to this end. Both Stiles and Luyet were inspired36 by Tammann’s theory37 that it should be possible to vitrify any liquid through the use of very high cooling rates. As Luyet stated in his first paper on vitrification in 1937:33 ‘There are 2 intrinsic factors which control the production of the vitreous state, they are the velocity of crystallization and the size of the zone of crystallization temperatures. A third factor is extrinsic and depends on the method employed, it is the cooling velocity. The essential problem of the vitrification technique consists of . . . obtaining a cooling velocity sufficient to prevent the formation of crystals.’ The freezing of water leads to the evolution of a great deal of heat that slows the cooling of the remaining unfrozen water, and the
THE HISTORICAL DEVELOPMENT OF VITRIFICATION Stiles Although the possibility of vitrified water was proposed by Brayley as long ago as 1860,31 the earliest suggestion we are aware of that vitrification might be an appropriate strategy
3
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Luyet successfully vitrified human red blood cells almost by accident in 1968 in the course of examining the relationship between ice formation and hemolysis,43 but seemed not to recognize the significance of this accomplishment. In 1969, Luyet characterized his attempts to achieve vitrification in retrospect as ‘mostly negative’.28 A more detailed review of Luyet’s views and experiments has appeared elsewhere.34
attainable cooling rate depends also on the volume of the material to be vitrified. Accordingly, Luyet and his colleagues often dried or dehydrated their biological samples prior to rapid cooling, sometimes using brief exposure to solutes such as ethylene glycol or glycerol to withdraw water just before cooling.34,36 This approach is very different from the modern method of deliberately allowing intracellular uptake of high concentrations of cryoprotectant to slow the cooling rates needed for vitrification, but was a reasonable approach to vitrification at the time. Despite some triumphs,38,39 Luyet’s claims of successful vitrification were directly and powerfully challenged by Smith in 1954.36 This challenge probably prompted two specific experiments that, as reported in 1958, directly contradicted Luyet’s conclusion that his methods had generally achieved vitrification. The first experiment was to microscopically examine putatively vitreous films of gelatin gels using crossed polarizing filters. This examination showed the macroscopically transparent films to be full of ice crystals in the form of ‘evanescent spherulites’.40 The second experiment, by Meryman, was to examine the same type of film using the parallel technique of X-ray crystallography. This study showed an X-ray diffraction pattern characteristic of the presence of ice, although only one peak was observed instead of the expected three.41 This suggested to Meryman the presence of incompletely formed ice crystals (reflecting growth primarily in the direction of the 001, or basal, plane of ice). However, Dowell et al.42 later disagreed, concluding, in reference to Meryman’s results: ‘it seems quite evident in the light of our studies that this was really . . . cubic ice and that a scan in the angular region 40–48° (2 θ) would have shown the other two cubic lines.’ After 1962, all deliberate attempts by Luyet and his colleagues to vitrify living systems ceased,34 and all of their subsequent cryopreservation methods were referred to as freezing methods. Ironically, Rapatz and
The electron microscopists Tokio Nei44 essentially reproduced the 1968 Rapatz and Luyet result for vitrified red blood cells in 1976, for very similar reasons. He showed no ice inside or outside human red cells vitrified in 4.1 mol/L (30%) glycerol and cooled at 105°C/min and obtained over 95% survival of the cells after rewarming. However, Nei never referred to the concept of vitrification or its desirability for cryopreservation, saying only that ‘as a cryotechnique for electron microscopy, the addition of 30% glycerol and ultrarapid freezing at 105°C/min are minimum requirements for the inhibition of ice formation and the prevention of the corresponding artifacts in erythrocytes.’ Nei’s goal was to achieve cryofixation without ice artifacts, which was a serious objective for electron microscopists at the time. However, the real goal of the field was to accomplish this without the use of cryoprotectants. Franks and Skaer took a major step away from the use of permeating cryoprotectants for morphological vitrification in the same year, claiming vitrification or quasivitrification of a cell in the center of a 1 mm thick cockroach brain by infiltrating the specimen with 50% w/w polyvinylpyrrolidone (PVP) and quenching in melting freon 22.45 In 1980, Bruggeller and Mayer published the first reproducible demonstration of the vitrification of pure liquid water and a 0.1 mol/L CuCl2 solution.46 They achieved an estimated cooling rate of 105–106°C/s. One year later, Dubochet and McDowall claimed
4
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vitrification of layers up to 1 micron thick in small water droplets based on electron microscopy.47 These methods, and others that added brief exposure to very high hydrostatic pressures, produced impressive results, achieving fields of view that were devoid of discernible ice crystals even in the electron microscope. However, there was no way to connect the kind of vitrification sought by electron microscopists to the problem of preserving and recovering the viability of living systems.
high cooling rates were generally required for complete vitrification and, even more problematically, astronomical warming rates were required in theory to prevent devitrification. The combination of these two factors seemed to confirm Luyet’s conclusion that for all practical purposes, complete vitrification was untenable, and relegated the practical significance of Boutron’s initial work to the improvement of the outcomes of freezing and thawing as he himself said was his objective.48,49 Third, Boutron’s experiments on solutions that were actually concentrated enough to demonstrably vitrify and remain amorphous on warming merely confirmed that enormous concentrations were needed that were, in his words, ‘toxic for most cells’.49 A key discovery of Boutron was the relatively miraculous behavior of propylene glycol (PG, or 1,2-propanediol), which vitrifies at concentrations as low as ~30–40% w/w, depending on the cooling rate.50 The critical warming rates to prevent devitrification of 35 and 40% w/w solutions of PG were calculated to be nearly 109 and 105°C/min, respectively. Nevertheless, in 1984, Boutron and Arnaud were able to show that at cooling rates sufficient to vitrify 30% and 35% PG, high levels of survival of human erythrocytes were obtainable despite the high critical warming rates for these concentrations and the existence of homogeneous nucleation on cooling.51 Survival was explained on the basis of initial formation of cubic ice and the inability of cubic ice to damage cells until it is converted to hexagonal ice, which was not expected to happen at the 5000°C/min warming rates employed.51 Although these experiments recapitulated the prior studies of Rapatz and Luyet in 1968 and of Nei in 1976, they took place in a much more useful conceptual context in which cryopreservation in the amorphous state was clearly recognized as being valuable in its own right, and not just valuable as a means to some other end. However, there was still no demonstration that nucleated cells could be vitrified and rewarmed successfully.
Boutron A major development in the history of vitrification began in the late 1970s, when Pierre Boutron decided to give up his work on magnetism and devote his considerable skills as a physical scientist to the problems of cryobiology. His first paper in 197848 explained his aims as follows: ‘It should be interesting to see if the more stable the amorphous state of a cryoprotective solution is, the better will be its protection of cells against freezing . . . In the extreme case of a solution which remains entirely amorphous even at very slow cooling or warming rates, all cells should be protected . . . It should then be interesting to find cryoprotective solutions of very low toxicity . . . or, in other words, to find a very stable amorphous state of the whole solution even for diluted solutions.’ As this quote illustrates, Boutron clearly understood the ability of cryoprotectants to enable vitrification and to do so even at low cooling rates, but there were three significant problems with vitrification as Boutron pursued it. First, it was assumed that low concentrations were necessary to avoid toxicity, which harkens back to the approaches of Luyet and the electron microscopists. Second, it was abundantly clear from Boutron’s path-breaking studies of the kinetics of glass formation on cooling and devitrification on warming48 that even at concentrations well above those normally used in cryobiology at the time, very
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liquid to form a metastable glass during rapid cooling’.56 Although this insight did not suggest an alternative to freezing as a means of cryopreservation, the realization that embryos survive cryopreservation in the final analysis as a result of at least intracellular vitrification, even when the cells are said to be ‘frozen’ due to the presence of extracellular ice, was to be a key impetus toward the first truly influential demonstration of the complete vitrification of living cells.
Rall Peter Mazur’s classic 1963 paper showing that the survival of slowly frozen cells is attributable to the avoidance of intracellular ice formation52 assumed that the remaining unfrozen intracellular water at very low temperatures is essentially unfreezable ‘bound water’. The concept that significant freezable water could remain at the end of slow cooling but fail to freeze upon subsequent transfer into liquid nitrogen due to intracellular vitrification simply had not ‘crystallized’ at the time. Vitrification had always been associated with rapid cooling, not slow cooling, and, 6 years after Mazur’s seminal paper, Luyet28 noted that ‘the reaction of most cryobiologists, when the notion of amorphous or glassy state is mentioned to them, is that it may be a subject of academic interest but not one of practical significance, since to obtain the amorphous state one needs to use extremely high cooling rates difficult to attain.’ Even Boutron did not explicitly state that slowly frozen cells survive by virtue of intracellular vitrification. In 1980, Rall and colleagues53 reported their cryomicroscopic observations of mouse embryos during slow freezing, subsequent rapid cooling, and rewarming. They described, for the first time, intracellular devitrification upon rewarming, which clearly implied previous intracellular vitrification.54,55 Rall, Reid, and Polge56 later used differential scanning calorimetry to demonstrate that the extracellular solution in contact with the embryos at the time they were rapidly cooled does in fact vitrify upon rapid cooling and devitrify upon slow warming, as predicted from their cryomicroscopic observations. They thereby convincingly supported their view that slow freezing followed by rapid cooling causes the cytoplasm to vitrify, and generalized their observation by concluding: ‘other cryopreservation methods that employ a protective additive and rapid cooling from an intermediate subzero temperature may rely on the ability of the residual
Fahy In 1965, John Farrant57 showed that whole guinea pig uteri could fully recover after previous cooling without freezing to −79°C in the presence of 55% dimethyl-sulfoxide (DMSO). Elford and Walter,58 in 1972, successfully extended Farrant’s observations to intestinal smooth muscle. Inspired by these successes, Fahy began attempts to apply the same principles to other systems in 1972 as an undergraduate student59 and continued this effort in graduate school. However, his graduate studies showed that even 50% DMSO was overwhelmingly toxic to kidney tissue.60 Discouraged by this result, Fahy returned to freezing as a way of preserving kidneys in the late 1970s, but soon found that frozen kidneys appeared to be severely damaged structurally.61,62 Searching for an alternative to both Farrant’s equilibrium freezing point depression method and freezing, he turned to deep supercooling as a way of avoiding both ice formation and Farrent’s high concentrations of cryoprotectant, hoping to be able to preserve kidneys for a week at −80°C. He was disappointed to find that supercooled cryoprotectant systems stored at −80°C were too prone to freeze after one or more days,61 but in contemplating this situation, a key inspiration dawned: it might be possible to extend supercooling all the way down to the glass transition temperature, enabling the
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same concentrations attempted for preservation by supercooling to allow vitrification and thereby stability against ice formation for indefinite periods of time. Farrant did not connect his method to vitrification, stating that cooling to lower temperatures would result in freezing,57 but, as a graduate student, Fahy observed that such high concentrations do vitrify upon such cooling. Fahy realized that in order to pursue his new vision of the possibility of preserving whole organs indefinitely by vitrification he would have to face the problem of the toxicity of vitrifiable concentrations of cryoprotectant head on.27,62–66 To control cryoprotectant toxicity, Fahy combined several different leads and new ideas, including the use of ‘toxicity neutralizers’;63,67b Boutron’s 1,2-propanediol;50,61,63 the use of elevated hydrostatic pressure;27,61,63 non-penetrating cryoprotectants to reduce intracellular permeating cryoprotectant requirements;1 mild osmotic dehydration to facilitate intracellular vitrification, reduce toxicity, and facilitate cryoprotectant washout;1 exponential addition and washout of cryoprotectants;1,66 introduction of the highest cryoprotectant concentrations at reduced temperatures;1,65 and determination of the minimum amount of cryoprotectant needed to vitrify at the cooling rates applicable to organs from both an empirical and a theoretical point of view, and restriction of the concentrations of cryoprotectant used to those minimum levels.61,63–64 By 1984 Fahy and his colleagues were able to publish the first full and generally available description of his new approach to vitrification, and to describe a solution that was both capable of permitting vitrification at 10°C/min and compatible with a 90% functional recovery of renal tissue.1 This paper also demonstrated the avoidance of fracturing in a whole vitrified rabbit kidney. The stage was thereby set for the first proof of principle of the new method and a demonstration that the results obtained using the rabbit kidney slice model
were not so idiosyncratic as to be inapplicable to other important biological systems.
Rall and Fahy In 1985, we were fortunate enough to be able to close the gap between theory and practice, and confirm the universality of the fundamental principles of vitrification by successfully applying lessons learned from adult rabbit kidneys and the freezing of mouse embryos to the vitrification of 8-cell mouse embryos.2 Mouse embryos vitrified in plastic straws using a wide range of cooling rates and rewarmed over a wide range of warming rates survived in high proportions provided that warming was fast enough to prevent devitrification. Our paper coined the term ‘vitrification solution’ and demonstrated that the first vitrification solution attempted, VS1, while toxic to embryos at a concentration permitting vitrification at a cooling rate of only 10°C/min and after longer exposure periods, permitted successful vitrification without appreciable toxicity when slightly diluted. Success was attained using a protocol for addition and washout of VS1 that facilitated diffusion and cellular uptake of lower concentrations of the permeating cryoprotectants of VS1 at room temperature and inhibited toxicity and facilitated embryo dehydration by the highest concentrations near 0°C. Survival was judged initially based on in vitro development to expanded blastocysts,2 but Rall et al. soon demonstrated that these embryos were capable of developing to live young following transfer to recipient females.68 The survival rates reported were equivalent to those obtained using optimized slow freezing methods, but the time required to carry out the vitrification procedure was much less than the time required for freezing and thawing, and no expensive controlled rate freezing device was required. Vitrification also eliminated the usual need to search for the optimum cooling and warming rates when freezing and thawing. All of these practical
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vitrification ultimately related to the now widespread use of vitrification as a practical and successful method of cryopreservation long after he, himself, had abandoned this approach.
advantages of vitrification soon caught the attention of many embryology laboratories, which proceeded to investigate the technique for their own purposes and in their own ways (Figure 1A.1). As even less toxic vitrification solutions were developed for selected systems,21,69 interest in vitrification continued to accelerate.
THE PHYSICAL BASIS OF VITRIFICATION The physics of ice formation and of the glass transition have been summarized in depth in several excellent and cryobiologically oriented reviews to which the interested reader is referred for a more complete treatment than can be presented here.73,74 Here we focus on the essential issues of why the glass transition takes place, how it stabilizes living systems, and what the limiting conditions are for vitrification and survival.
The ghost of Luyet Although the modern advent of vitrification arose from the realization that ultrarapid cooling is not required for vitrification and that solutions concentrated enough for vitrification at slow cooling rates need not inevitably be extremely toxic, once the feasibility of vitrification became widely appreciated there was no longer any need to limit vitrification to low cooling rates for those systems that could be cooled and warmed rapidly, and lingering problems with the toxicity of many alternative vitrification solutions, and particularly with chilling injury in some systems,70–72 accordingly inspired more and more efforts to reduce damage by using lower concentrations and faster cooling rates. There is insufficient space in this short overview to recount in any adequate detail the many ingenious approaches that have emerged, and the reader must be referred to later chapters for further information within the sphere of reproductive cryobiology. Here we can only note the irony that, having been launched by breaking free of Luyet’s tyranny of ultrarapid cooling, vitrification methods have now essentially turned back closer to Luyet’s original idea of cooling as quickly as possible with minimal intracellular exposure to cryoprotectants, albeit this time using at least marginally adequate concentrations of intracellular solutes. The ghost of Luyet lives on in the form of this ongoing methodological evolution, and we think he would have been pleased to see how his ideas about
The thermodynamic necessity of vitrification Vitrification is ultimately the result of the fact that a liquid cannot have more order than its corresponding crystal. As the temperature of a liquid substance is lowered, its entropy is reduced more rapidly than the entropy of the corresponding crystalline form of the substance. If the liquid does not freeze, a thermodynamic conundrum is approached as the entropy of the liquid approaches the entropy of the crystal. Kauzmann75 recognized that to maintain thermodynamic consistency, some event must prevent the continued reduction of entropy of the liquid from creating a liquid whose entropy is less than the entropy of the crystal (a situation referred to as Kauzmann’s paradox). That event is the glass transition, and it must occur at a temperature above the Kauzmann temperature, TK, at which the laws of thermodynamics would be violated by the attainment of a liquid state with the same entropy as the crystalline state of the same material. The glass transition prevents the paradox by eliminating the translational and
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Log viscosity (Po)
VITRIFICATION: AN OVERVIEW
1e +14 1e + 13 1e +12 1e +11 1e +10 1e + 9 1e + 8 1e + 7 1e + 6
Glycerol77 B = 891.3; T0 = −138.3 Tg–T0 = 51.3°C 66.7% Glycerol76 B = 121.9; T0 = −140.6 Tg –T0 = 31.1°C 40% DAP10 + 6% PEG (Fahy, unpublished) B = 118.7; T0 = −154.5; Tg –T0 = 30.5
1e + 5 1e + 4 1e + 3 1e + 2 1e + 1 1e + 0 1e −1 1e − 2
50% DMSO + 2.7% Dextran + 2.25% glucose78 B = 906.9; T0 = −159.3; Tg–T0 = 24.3 40
20
0
−20
−40
−60
−80
−100 −120
Temperature (°C) Figure 1A.2 Effect of temperature on viscosity for representative vitrifiable solutions of cryobiological interest (pure glycerol;77 66.7% w/w glycerol in water;76 40% w/v DAP10 +6% w/v PEG 6000 in a carrier solution of RPS-2 minus calcium and magnesium (a high pressure vitrification solution, Tg estimated; Fahy, unpublished data.) and a DMSO-based solution) (Tg estimated),78 with derived values for T0, the lowest temperature at which translational movements are possible in the glass, and the difference between Tg and T0, indicated at the right. B and T0 values were derived by fitting the logarithmic form of the VTF equation, In(η(T2)) = a + B/(T2 − T0) to the data using SigmalPlot’s Regression Wizard.
curve fits of the Vogel–Tammann–Fulcher (VTF) equation:
rotational degrees of freedom that characterize the liquid state and are responsible for the greater dependence of the entropy of a liquid on temperature in comparison to that of the crystal, which retains primarily vibrational degrees of freedom. Another way to view the glass transition is that the molecular mobility of a liquid has an activation energy, and as the glass transition temperature is approached, that activation energy becomes unavailable.
η(T2) = η(T1)exp[B/(T2 − T0)] where η(T1) is a known reference viscosity at temperature T1, η(T2) is the viscosity at temperature T2, and T0 is the limiting temperature for structural change, which approximates TK. Figure 1A.2 shows several curve fits that can be assembled for cryobiologically relevant solutions from literature viscosity data and from unpublished data of Fahy on an actual vitrification solution. The curve fit makes the simplifying assumption that the known glass transition temperature for each solution corresponds to a viscosity of 1013 poise (Po).77 Based on this assumption, the difference between Tg and T0 increases with the initial viscosity of the solution, the difference being
The kinetic basis of vitrification As the activation energy for translational molecular motions vanishes with continued cooling, the time scale for structural reorganizations within the liquid stretches out toward infinity. The temperature at which structural relaxation becomes impossible in principle, T0, can be empirically inferred, for example, from
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where D is the diffusion coefficient of the substance. At temperatures above Tg, the Stokes–Einstein equation states that D = kT/(6πη(T)), where η(T) is the viscosity at temperature T. Combining the equations for diffusion, the temperature dependence of the diffusion coefficient, and the VTF equation, one obtains
51°C for pure glycerol but only 24°C for a comparatively fluid dimethyl sulfoxide-based solution. In the parlance of glass physicists, cryoprotectant-water solutions form ‘fragile’ glasses (glasses with steep slopes in such plots), and Tg appears to come closer and closer to T0 as the fragility of the glass increases. Stated another way, the higher the glass transition temperature, the farther below Tg one needs to go to reach T0. In still other words, Tg as measured in the laboratory is an artifact of the time scale of typical laboratory measurements. If it were possible to cool test solutions more and more slowly without limit, Tg would be seen at lower and lower temperatures until, in principle, Tg would approach TK as closely as possible as the cooling rate approached zero.
t = t1(T1/T)exp[B[(1/(T−T0)) – 1/(T1 − T0)]] where t is the time required to diffuse distance d at temperature T and t1 is the time required to diffuse the same distance at reference temperature T1. If diffusion times are related to biological changes in or near the vitreous state, and if we take the injury onset time for the on set of injury as being 1 minute at −20°C (based arbitrarily on the observation80 that perfusing a rabbit kidney with the M22 vitrification solution at −22°C requires an equilibration time that approaches the time associated with significant M22 toxicity), then we can derive the relationship shown in Figure 1A.3 between storage temperature and the time required for storage injury to accumulate, using the curve fit parameters for the vitrification solution described in Figure 1A.2. According to Figure 1A.3, storage times already begin to reach extreme values even at temperatures above Tg, which indicates that normal diffusional sources of injury are not likely to be important for systems stored comfortably below Tg regardless of the approximations underlying the projections in Figure 1A.3. On the other hand, ice nucleation is known to take place on short time scales between − 90°C and about − 135°C in the VS41A/VS55 vitrification solution (Tg approximately − 125°C).81 Nucleation can take place over a very short distance scale and evidently requires very little diffusion since the temperature optimum for nucleation is very much below the temperature optimum for ice crystal growth.34,81 At the present time, the time and temperature dependence of ice nucleation in
Optimal storage below Tg From the point of view of cryopreservation, storage below TK (or T0) should be unnecessary. Further, storage in liquid nitrogen may be undesirable if sample contamination or sample fracturing is an issue,56,79 as might occur for vitrified ovaries intended for vascular transplantation. Given that in many situations T0 and TK will be unknown and that in many cases the risk of fracturing will increase as these critical temperatures are approached, we would like to have a clearer general idea of how the safe period of storage depends on temperature below Tg, particularly given the potential need for very long-term storage of precious genetic resources. On the assumption that biological change is generally diffusion limited, we can get a useful feeling for the time dependence of diffusion-mediated changes from the following simplified treatment. Although the kinetics of relaxational processes are nominally slower below Tg than the simpler VTF equation would indicate, we will see that the difference may well be academic. The time t required for a substance to diffuse a distance d is equivalent to d2/6D,
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1e + 8
Year to equal 1 min of diffusion at −20 °C
1e + 7 1e + 6 1e + 5 1e + 4
10 000 Years
1e + 3 100 Years
1e + 2
Ten Years
1e + 1
One Year
1e + 0 One Month
1e − 1 1e − 2
One Week One Day
1e − 3 1e − 4 −70
−80
−90
−100
−110
−120
−130
Strorage temperature (°C) Figure 1A.3 Estimated time required at low temperatures for the same amount of diffusion to take place as can occur in 1 minute at −20°C. The latter is used as a benchmark for an acceptable amount of diffusion-dependent change based on the effects of prolonged exposure of rabbit kidneys to 9.3 mol/L cryoprotectant.80
vitrification solutions below Tg is a virtually unexplored area of research and deserves more careful scrutiny for cases in which devitrification may be a limiting factor for successful recovery of stored systems. The significance of enthalpy relaxations well below the Tg of aqueous ethylene glycol solutions73 also deserves more clarification.
dominant concern is homogeneous nucleation, or the self-nucleation of water, which is a very rapid process. In region II, Th becomes harder to define (gray line) near Tg due to increased viscosity, but its existence becomes obvious indirectly upon rewarming due to strong crystallization on rewarming (devitrification). In region III, homogeneous nucleation is nominally avoided on cooling because Th lies below Tg, and the boundary between regions II and III has been called Cv, or the minimum concentration needed to vitrify without forming visible ice on slow cooling.1,80 Devitrification remains a problem in region III, probably largely because of cubic ice nucleation along the extension of the Th curve below Tg, but at the concentration marked Cndv (concentration allowing no devitrification), ice does not form even during slow warming, marking the onset of region IV.
The role of cryoprotective agents in vitrification and quasivitrification Figure 1A.4 sets forth the overall relationships between the total concentration of cryoprotectant and the glass transition temperature, homogeneous nucleation temperature Th, devitrification temperature Td, and melting point Tm that are generic for all cryoprotectant systems that do not form precipitates.1,80 In region I of this diagram, the
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I
II
III
place even at rapid rates of temperature change, the nucleation density of the sample can reach truly astronomical levels83 (≥ 2500 nuclei/µm3). Each such nucleus must by necessity be so small as to have doubtful directly damaging effects, and almost all will tend to be relatively uniform in size,73 which may greatly inhibit recrystallization on warming given that recrystallization is driven by the existence of a significant size distribution of ice crystals in the sample64 and may be possible only for hexagonal and not for cubic ice.51 These factors may explain why many cells have been successfully ‘rescued’ by rapid warming after extensive intracellular ice formation.84 Therefore, although vitrification is an appropriate goal, absolutely perfect vitrification and the complete avoidance of devitrification are not mandatory for the successful recovery of small living systems. It follows that survival after rapid cooling and warming is not equivalent to a demonstration that vitrification was achieved and that devitrification was avoided.36
V
IV
0
Tm
Temperature (°C)
−20
Cv
Cndv
−40
Th
−60
Cu
−80 −100
Td
−120
Tg 0
20
40
60
80
100
Concentration (% w/w)
Figure 1A.4 Relationships between the melting point (Tm) the glass transition temperature (Tg) the homogeneous nucleation (ice self-nucleation) temperature (Th) the devitrification temperature (Td), and the concentration of glycerol in solutions of glycerol and water (modified from prior representations).1,80 Point on the (Tm) curve signifies the eutectic composition, which usually does not crystallize in practice. For discussion, see text. In region I, ice nucleation is likely even above the homogeneous nucleation temperature owing to the universal presence of heterogeneous nucleating agents. At the opposite extreme, in regions IV and V, ice formation is absent during both slow cooling and slow warming, although ice formed during slow freezing of lower concentrations can continue to grow until region V is reached and the ‘unfreezable concentration’ Cu is attained. Traditionally, region IV has been inaccessible due to overwhelming problems with cryoprotectant toxicity, but this problem has recently been essentially overcome for at least some living systems.80,82
The cooling rate needed for vitrification Figure 1A.5 shows the projected cooling rate needed to induce vitrification as concentration varies between 0% and high concentrations of cryoprotectant based primarily on the calculations of Toner,85 who extended Boutron’s mathematical models of ice crystallization48,88 to the case of very dilute solutions. Toner’s projections include the weak glass-forming agent, glycerol, and the strong glass-forming agent, propylene glycol, and are put into the context of Bruggeller and Mayer’s46 and William Bald’s86 experimentally based estimates of the cooling rates needed to vitrify pure water on the one hand, and directly observable critical cooling rates for ethylene glycol–water solutions and propylene glycol–water solutions from Baudot and Odagescu87 on the other. The agreement shown between all of these estimates allows Figure 1A.5 to be used to
Ice can still grow in region IV if it is introduced, but cannot grow in region V, which is above Cu, a concentration that is unfreezable for practical purposes. For the small samples often used in reproductive cryobiology, it becomes important that Th, Tg, and Td are all rate dependent (i.e. Th will go down, Tg will go up, and Td will go up as the rate of change of temperature increases) because extremely high cooling and warming rates are feasible. However, as the concentration becomes more and more dilute and crystallization takes
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1010
Critical cooling rate ( °C/min )
109 Bald’s estimate
108
Bruggeller and Mayer’s estimate
107 106 105 104 103
EG
102 PG
101 100
0
10
20
30
G
40
50
60
Concentration (% w/w) Figure 1A.5 Cooling rate required to vitrify samples ranging in concentration from 0% to 60% w/w cryoprotectant. white points, unpublished calculations of Mehmet Toner85 (published here by permission); star, estimate of Bald;86 filled circle, high estimate of Bruggeller and Mayer (their low estimate is one order of magnitude lower);46 gray diamonds, corrected observed critical cooling rates for the prevention of ice in propylene glycol (PG) – water solutions (from Baudot and Odagescu);87 G = glycerol hexagons, observed critical cooling rates for ethylene glycol (EG) – water solutions.87
cryoprotected systems in general. It therefore provides a useful frame of reference against which to judge claims of vitrification made for a wide variety of different protocols.
evaluate the tradeoff between concentration and the critical cooling rate. Most permeating cryoprotectants, like ethylene glycol, are expected to give results that lie between the behaviors of PG and glycerol. However, Figure 1A.5 is based on pure permeating cryoprotectants in water and does not take into account the effects of the carrier solution (see below) or additives such as serum or sucrose, nor does it take into account the effect of concentrated intracellular protein in shrunken cells or the naturally low water concentration of cells like spermatozoa. However, given the similarity of the projections for PG and glycerol, with and without carriers (see below), it may be possible to estimate the effects of all of these factors based on the total solute concentration of the system in question. In the meantime, Figure 1A.5 permits a general order of magnitude estimate for the overall behavior of
The warming rate needed after vitrification Figure 1A.6 plots the calculated critical warming rate for the suppression of devitrification (white points) against the calculated or directly measured critical cooling rates of aqueous solutions of glycerol, propylene glycol, and ethylene glycol. The critical warming rates are about 103 to 108 times higher than the critical cooling rates even when the critical cooling rates are less than 1000°C/min. However, as noted above, Boutron and Arnaud51 showed that red blood cells can survive vitrification even when the warming rate
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1011 1010
EG
9
Warming rate (°C/min)
10
108 107
Glycerol survival
PG
106
G
5
10
104
PG survival
103 102 101 100 101
102
103
104
Critical cooling rate (°C/min) Figure 1A.6 Warming rates required to prevent appreciable ice formation in solutions of the stated critical cooling rates (white points), and warming rates known to permit survival after cooling in similar solutions (gray points).84,87 The survival data are simply known examples and do not represent the minimum warming rates known to be compatible with good survival; the latter are likely to be lower than depicted. G, glycerol; EG, ethylene glycol; PG, propylene glycol.
is insufficient to suppress devitrification. Plotting examples of warming rates that allow survival84 on the same graph indicates that these rates are only 10–103 times higher than the critical cooling rate. The discrepancy between the theoretical critical warming rate and warming rates that are compatible with survival is a factor of 102 to at least 104 and probably far more than this for the more dilute solutions of glycerol, and calls for an explanation. One possible explanation is that the presence of a carrier solution, which is always present in experiments with living cells, lowers the critical warming rate dramatically. Figure 1A.7 shows that if the theoretical critical warming rate of a solution containing both carrier and cryoprotectant is plotted against the critical cooling rate for the same solution, there is little difference between the behavior of solutions containing carrier solutions and cryoprotectant solutions lacking carriers. In
fact, the presence of phosphate buffered saline (PBS) and St Thomas solution has no effect in 2,3-(levo)-butanediol (BD) solutions but actually seems to raise rather than lower the critical warming rate for a given critical cooling rate by nearly two orders of magnitude in PG solutions because salt forms a less stable amorphous state than PG. Euro-Collins solution (EC) has no apparent effect on the critical warming rate in BD solutions. If one looks at the effect of carriers at given concentrations of cryoprotectant rather than at given critical cooling rates (Figure 1A.8), it can be seen that PBS and St. Thomas solution can lower the critical warming rate by about 1–2 orders of magnitude in BD but not in PG solutions. EC, on the other hand, can lower the critical warming rate by more than a factor of 104 in the more dilute BD solutions, evidently by increasing the total solute concentration of these dilute solutions and thereby reducing the critical cooling rates (Figure 1A.7).
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1011
Critical warming rate (°C/min)
2,3-BD in PBS
109
2,3-BD in EC
PG in PBS
1010
108 PG in St Thomas
107
PG in water
106 105 104 2,3-BD in water
103 102 101
10
1000
100 Critical cooling rate (˚C/min)
Figure 1A.7 (a) Lack of effect of carrier solution on the critical warming rate versus critical cooling rate relationship. Abbreviations: BD = butanediol; PG = propylene glycol; EC = Euro-collins; PBS = phosphate-buffered saline; St. Thomas = St. Thomas cardioplegia solution. Data assembled from several sources.89,90,91
1011
Critical warming rate (°C/min)
1010
2,3-BD in water
9
10
108
PG in PBS or St Thomas
107
2,3-BD in EC
6
10
105 104 PG in water
2,3-BD in PBS or St Thomas
103 102 101 20
25
30
35
40
45
50
55
Concentration (% W/W) Figure 1A.8 Effect of carrier solution on the critical warming rate at a given concentration of permeating cryoprotectant. PBS, phosphate buffered saline; 2,3-BD, 2,3-(levo)-butanediol; EC, Euro-Collins solution. Abbreviations as in Figure 1A.7.88–90
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matical models of cellular dehydration and rehydration kinetics (although in practice such models are often dispensable for familiar systems). 2 Cell shrinkage before vitrification renders the cytoplasm more stable.1,94 Therefore, the final step of cryoprotectant exposure need not be longer than the time required for the cells of interest to shrink osmotically, resulting in reduced intracellular permeating cryoprotectant to cause toxicity before vitrification and to cause osmotic damage during cryoprotectant washout. 3 Cells are more permeable to cryoprotectants at higher temperatures, but more resistant to toxic effects at lower temperatures.2,94 Therefore, it is frequently advantageous to introduce and remove lower, less toxic concentrations at higher temperatures and to use lower temperatures for the safe dehydration of the cell by higher concentrations of cryoprotectants the full permeation of which is less critical for and may even be counterproductive for the vitrification of osmotically concentrated cytoplasm. 4 Cellular toxicity is time dependent.1,2,94 Therefore, variations in exposure time from one cell to another, as can occur with manual methods for vitrification of ova in which ova are sequentially pipetted into a common vitrification solution before vitrification is done, may contribute to variations in outcome and should be minimized. In addition, many reported procedures are unclear about the time spent pipetting the ova or the time between the end of pipetting and vitrification, which may make reported results difficult to reproduce. 5 Cellular toxicity is concentration dependent.2 Therefore, the dilution caused by pipetting cells into a nominally vitrifiable medium must be taken into account and reported if injury is to be linked to either the toxicity of the vitrification solution or the stability of the amorphous state of the solution after dilution with the cell suspension. Other biological principles governing the success of vitrification are less well established
The calculated critical warming rate is a warming rate sufficient to reduce ice formation to a very small volume fraction of the solution. It is apparent that cells can at least sometimes tolerate a much larger volume fraction of ice than the presumptive value upon which the critical warming rate is calculated.56,84 In the case of erythrocytes, the cytoplasm is known to be more resistant to ice formation than the extracellular milieu,43,44 perhaps due to the high intracellular hemoglobin concentrations. On the other hand, in slowly frozen embryos, killing during slow warming was associated with an invisible event that took place after devitrification but before recrystallization and ice melting,53 suggesting that at least in this case the traditional explanations for devitrification injury may be invalid. Whether other explanations such as enrichment of cryoprotectant concentrations to damaging levels92 could be involved remains to be seen. In summary, experimentally determining and theoretically understanding the effect of devitrification on living cells and tissues deserves much more attention than it has received. Excellent previous studies on the physics of devitrification73,74,89 should facilitate understanding of the biology of devitrification in future investigations.
BIOLOGICAL PHENOMENA RELATED TO SUCCESSFUL VITRIFICATION The biological requirements for vitrification are the subject of this book, and only a few general points can be touched on here. We start by listing a few simple biological principles that are easy to state and easy to accommodate methodologically, but the implications of which are not always kept firmly in mind. 1 Cells have limited tolerance to shrinkage and swelling.93 Therefore, due care must be taken not to exceed their osmotic limits during the introduction and removal of cryoprotective agents, preferably using the guidance provided by appropriate mathe16
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and straightforward, such as chilling injury and cellular toxicity exacerbated by strong glass-forming agents. 1 A minority of living systems experience serious ‘chilling injury’, 70,71,80 or injury caused by cooling per se, independent of ice formation. Separating chilling injury from other sources of injury may be necessary if satisfactory results are not otherwise obtained. Because chilling injury has recently been linked to the tonicity of the vitrification solution in the case of kidney slices and whole kidneys,80 unbridled shrinkage of the cell before vitrification may be counterproductive for cells whose resistance to chilling injury is dependent on their degree of shrinkage.80 2 Cellular toxicity may be exacerbated by strong glass-forming agents.82 A recent analysis of the toxicities of 21 different vitrification solutions showed that toxicity was linearly dependent on the ratio of the molarity of water in each solution to the molarity of hydrogen bonding groups required to vitrify the solution under standardized conditions.82 This ratio, called qv* for short, goes up as the mean glass-forming tendency of the vitrification solution components increases, and directly correlates with injury after removing the cryoprotectants. Fortunately, very low toxicity solutions have been developed on the basis of this observation and may have some advantages in reproductive cryobiology. For example, mouse ova vitrified with a solution
known as 90% VM3 were able to be fertilized and develop to blastocysts at 80% of the rate of untreated control ova without the need for ICSI.82
SUMMARY AND CONCLUSIONS Vitrification is a viable approach to cryopreservation of a wide range of living systems. Its physical and biological principles are continuing to become better understood, and this is leading to more numerous and more successful applications. Although the history of the concept goes back more than threequarters of a century, the field is probably still in the infancy of its full potential. As always, nature may have preceded biologists in discovering viable approaches to vitrification, but for the most part nature’s examples remain both recondite and difficult to emulate directly. Nevertheless, reproductive cryobiologists have ample means and ample incentive to follow nature’s lead and develop their own approaches to answering one of biology’s most interesting challenges, the goal of arresting life in a state of suspended animation and restarting it at the right time to enable new lives to begin.
ACKNOWLEDGMENTS We thank Stanley Leibo for his valued advice and for his friendship over more than two decades.
References 4. Michelmann HW, Nayudu P. Cryopreservation of human embryos. Cell Tissue Bank 2006; 7: 135–41. 5. Kasai M, Mukaida T. Cryopreservation of animal and human embryos by vitrification. Reprod Biomed Online 2004; 9: 164–70. 6. Kagabu S, Umezu M. Transplantation of cryopreserved mouse, chinese hamster, rabbit, Japanese monkey and rat ovaries into rat recipients. Exp Anim 2000; 49: 17–21.
1. Fahy GM, MacFarlane DR, Angell CA, et al. Vitrification as an approach to cryopreservation. Cryobiology 1984; 21: 407–26. 2. Rall WF, Fahy GM. Ice-free cryopreservation of mouse embryos at −196oC by vitrification. Nature 1985; 313: 573–5. 3. Fahy GM, Wowk B, Wu J. Cryopreservation of complex systems: the missing link in the regenerative medicine supply chain. Rejuvenation Res 2006; 9: 279–91.
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22. Burke MJ. The glassy state and survival of anhydrous biological systems. In Leopold AC, ed. Membranes, Metabolism, and Dry Organisms. Ithaca: Cornell University Press, 1986: 358–64. 23. Crowe JH, Crowe LM, Tablin F, et al. Stabilization of cells during freeze-drying: the trehalose myth. In Fuller BJ, Lane N, Benson EE, eds. Life in the Frozen State. Boca Raton: CRC Press, 2004: 581–601. 24. Holmstrup M, Bayley M, Ramlov H. Supercool or dehydrate? An experimental analysis of overwintering strategies in small permeable arctic invertebrates. Proc Natl Acad Sci USA 2002; 99: 5716–20. 25. Crowe JH, Jackson S, Crowe LM. Nonfreezable water in anhydrobiotic nematodes. Mol Physiol 1983; 3: 99–105. 26. Bennett VA, Sformo T, Walters K, et al. Comparative overwintering physiology of Alaska and Indiana populations of the beetle Cucujus clavipes (Fabricius): roles of antifreeze proteins, polyols, dehydration and diapause. J Exp Biol 2005; 208: 4467–77. 27. Fahy GM, Hirsh A. Prospects for organ preservation by vitrification. In Pegg DE, Jacobsen IA, Halasz NA, eds. Organ Preservation, Basic and Applied Aspects. Lancaster: MTP Press, 1982: 399–404. 28. Luyet B. On the amount of water remaining amorphous in frozen aqueous solutions. Biodynamica 1969; 10: 277–91. 29. Storey KB, Storey JM. Physiology, biochemistry, and molecular biology of vertebrate freeze tolerance: the wood frog. In Fuller BJ, Lane N, Benson EE, eds. Life in the Frozen State. Boca Raton: CRC Press, 2004: 243–74. 30. Wisniewski M, Fuller M. Ice nucleation and deep supercooling in plants: new insights using infared thermography. In Margesin R, Schinner F, eds. Cold-Adapted Organisms – Ecology, Physiology, Enzymology and Molecular Biology. Berlin: Springer, 1999: 105–18. 31. Brayley E. Note on the apparent universality of a principle analogous to regelation, on the physical nature of glass, and on the probable existence of water in a state corresponding to that of glass. Proc R Soc 1860; 10: 450–60. 32. Stiles W. On the cause of cold death of plants. Protoplasma 1930; 9: 459–68. 33. Luyet B. The vitrification of organic colloids and of protoplasm. Biodynamica 1937; 1(29): 1–14. 34. Fahy GM. Vitrification. In McGrath JJ, Diller KR, eds. Low Temperature Biotechnology: Emerging Applications and Engineering Contributions. New York: American Society of Mechanical Engineers, 1988: 113–46.
7. Koutlaki N, Schoepper B, Maroulis G et al. Human oocyte cryopreservation: past, present and future. Reprod Biomed Online 2006; 13: 427–36. 8. Kuwayama M, Vajta G, Kato O et al. Highly efficient vitrification method for cryopreservation of human oocytes. Reprod Biomed Online 2005;11: 300–8. 9. Leibo SP. Cryopreservation of mammalian oocytes. In: Tulandi T, Gosden RG, eds. Preservation of Fertility. London: Taylor & Francis, 2004: 141–55. 10. Migishima F, Suzuki-Migishima R, Song S-Y, et al. Successful cryopreservation of mouse ovaries by vitrification. Biol Reprod 2003; 68: 881–7. 11. Elster J, Benson EE. Life in the polar terrestrial environment with a focus on algae and cyanobacteria. In Fuller BJ, Lane N, Benson EE, eds. Life in the Frozen State. Boca Raton: CRC Press, 2004: 111–50. 12. Luyet B, Rasmussen D. Study by differential thermal analysis of the temperatures of instability of rapidly cooled solutions of glycerol, ethylene glycol, sucrose, and glucose. Biodynamica 1968; 10: 167–91. 13. Chen T, Fowler A, Toner M. Literature review: supplemented phase diagram of the trehalose-water binary mixture. Cryobiology 2000; 40: 277–82. 14. Acker JP, Chen T, Fowler A, et al. Engineering desiccation tolerance in mammalian cells: tools and techniques. In Fuller BJ, Lane N, Benson EE, eds. Life in the Frozen State. Boca Raton: CRC Press, 2004: 563–80. 15. Lee RE Jr, Denlinger DL. Insects at Low Temperature. New York: Chapman and Hall, 1991. 16. Rasmussen D. A note about ‘phase diagrams’ of frozen tissues. Biodynamica 1969; 10: 333–9. 17. Hirsh AG, Williams RJ, Meryman HT. A novel method of natural cryoprotection: intracellular glass formation in deeply frozen populus. Plant Physiol 1985; 79: 41–56. 18. Leather SR, Walters KFA, Bale JS. The Ecology of Insect Overwintering. Cambridge, UK: Cambridge University Press, 1993. 19. Sun WQ, Leopold AC. Cytoplasmic vitrification and survival of anhydrobiotic organisms. Comp Biochem Physiol 1997; 117A: 327–33. 20. Hirsh AG. Vitrification in plants as a natural form of cryoprotection. Cryobiology 1987; 24: 214–28. 21. Sakai A. Plant cryopreservation. In Fuller BJ, Lane N, Benson EE, eds. Life in the Frozen State. Boca Raton: CRC Press, 2004: 329–45.
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53. Rall WF, Reid DS, Farrant J. Innocuous biological freezing during warming. Nature 1980; 286: 511–4. 54. Rall WF. The role of intracellular ice in the slow warming injury of mouse embryos. In Zeilmaker GH, ed. Frozen Storage of Laboratory Animals. New York: Gustav Fischer Verlag, 1981: 33–44. 55. Lehn-Jensen H, Rall WF. Cryomicroscopic observations of cattle embryos during freezing and thawing. Theriogenology 1983; 19: 263–77. 56. Rall WF, Reid DS, Polge C. Analysis of slowwarming injury of mouse embryos by cryomicroscopical and physicochemical methods. Cryobiology 1984; 21: 106–21. 57. Farrant J. Mechanism of cell damage during freezing and thawing and its prevention. Nature 1965; 205: 1284–7. 58. Elford BC, Walter CA. Effects of electrolyte composition and pH on the structure and function of smooth muscle cooled to −79oC in unfrozen media. Cryobiology 1972; 9: 82–100. 59. Fahy G. The effects of low temperatures and DMSO on the frog sciatic nerve. J Undergrad Res Biol Sci 1972; 2: 411. 60. Fahy GM. Analysis of ‘solution effects’ injury: rabbit renal cortex frozen in the presence of dimethyl sulfoxide. Cryobiology 1980; 17: 371–88. 61. Fahy GM. Vitrification as an approach to organ cryopreservation: past, present, and future. In Smit Sibinga CT, Das PC, Meryman HT, eds. Cryopreservation and Low Temperature Biology in Blood Transfusion. Boston: Kluwer, 1990: 255–68. 62. Fahy GM. Analysis of ‘solution effects’ injury: cooling rate dependence of the functional and morphological sequelae of freezing in rabbit renal cortex protected with dimethyl sulfoxide. Cryobiology 1981; 18: 550–70. 63. Fahy GM. Prospects for vitrification of whole organs. Cryobiology 1981; 18: 617. 64. MacFarlane DR, Angell CA, Fahy GM. Homogeneous nucleation and glass formation in cryoprotective systems at high pressures. Cryo Letters 1981; 2: 353–8. 65. Fahy GM. Prevention of toxicity from high concentrations of cryoprotective agents. In Pegg DE, Jacobsen IA, Halasz NA, eds. Organ Preservation, Basic and Applied Aspects. Lancaster: MTP Press, 1982: 367–69. 66. Fahy GM. Cryoprotectant toxicity: biochemical or osmotic? Cryo Letters 1984; 5: 79–90. 67. Baxter S, Lathe G. Biochemical effects on kidney of exposure to high concentrations of dimethyl sulphoxide. Biochem Pharmacol 1971; 30: 1079–91.
35. Luyet BJ. Working hypotheses on the nature of life. Biodynamica 1934(1); 1: 1–7. 36. Smith AU. Effects of low temperatures on living cells and tissues. In Harris RJC, editor. Biological Applications of Freezing and Drying. New York: Academic Press, 1954: 1–53. 37. Tammann G. Kristallisieren und Schmelzen. Leipzig, 1903. 38. Gonzales F, Luyet B. Resumption of heart beat in chick embryo frozen in liquid nitrogen. Biodynamica 1950; 7: 1–5. 39. Luyet BJ, Gonzales F. Growth of nerve tissue after freezing in liquid nitrogen. Biodynamica 1953; 7: 171–4. 40. Luyet B, Rapatz G. Patterns of ice formation in some aqueous solutions. Biodynamica 1958; 8: 1–68. 41. Meryman HT. X-ray analysis of rapidly frozen gelatin gels. Biodynamica 1958; 8: 69–72. 42. Dowell LG, Moline SW, Rinfret AP. A lowtemperature X-ray diffraction study of ice structures formed in aqueous gelatin gels. Biochim Biophys Acta 1962; 59: 158–67. 43. Rapatz G, Luyet B. Electron microscope study of erythrocytes in rapidly cooled suspensions containing various concentrations of glycerol. Biodynamica 1968; 10: 193–210. 44. Nei T. Freezing injury to erythrocytes. I. Freezing patterns and post-thaw hemolysis. Cryobiology 1976; 13: 278–86. 45. Franks F, Skaer HLB. Aqueous glasses as matrices in freeze-fracture elecron microscopy. Nature 1976; 262: 323–5. 46. Bruggeller P, Mayer E. Complete vitrification in pure liquid water and dilute aqueous solutions. Nature 1980; 288: 569–71. 47. Dubochet J, McDowall AW. Vitrification of pure water for electron microscopy. J Microsc 1981; 124: RP3–4. 48. Boutron P, Kaufmann A. Stability of the amorphous state in the system water-glyceroldimethylsulfoxide. Cryobiology 1978: 15: 93–108. 49. Boutron P, Kaufmann A. Stability of the amorphous state in the system water-glycerolethylene glycol. Cryobiology 1979; 16: 83–9. 50. Boutron P. Stability of the amorphous state in the system water-1,2-propanediol. Cryobiology 1979; 16: 557–68. 51. Boutron, P, Arnaud F. Comparison of the cryoprotection of red blood cells by 1,2-propanediol and glycerol. Cryobiology 1984; 21: 348–58. 52. Mazur P. Kinetics of water loss from cells at subzero temperatures and the likelihood of intracellular freezing. J Gen Physiol 1963; 47: 347–69.
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82. Fahy GM, Wowk B, Wu J, et al. Improved vitrification solutions based on predictability of vitrification solution toxicity. Cryobiology 2004; 48: 22–35. 83. Dupuy J, Jal JF, Ferradou C, et al. Controlled nucleation and quasi-ordered growth of ice crystals from low temperature electrolyte solutions. Nature 1982; 296: 135–40. 84. Fahy GM. Biological effects of vitrification and devitrification. In Pegg DE, Karow AM, Jr, eds. The Biophysics of Organ Cryopreservation. New York: Plenum Press, 1987: 265–93. 85. Toner M, Cravalho EG, Chiang YM. Vitrification of biological cell suspensions: the importance of ultrarapid cooling and warming. Cryobiology 1988; 25: 551. 86. Bald WB. Quantitative Cryofixation. Bristol: Hilger, 1987. 87. Baudot A, Odagescu V. Thermal properties of ethylene glycol and aqueous solutions. Cryobiology 2004; 48: 283–94. 88. Boutron P. Comparison with the theory of the kinetics and extent of ice crystallization and of the glass-forming tendency in aqueous cryoprotective solutions. Cryobiology 1986; 23: 88–102. 89. Boutron P, Mehl P. Theoretical prediction of devitrification tendency: determination of critical warming rates without using finite expansions. Cryobiology 1990; 27: 359–77. 90. Baudot A, Peyridieu JF, Boutron P, et al. Effects of saccharides on the glass-forming tendency and stability of solutions of 2,3butanediol, 1,2-propanediol, or 1,3-butanediol in water, phosphate-buffered saline, Euro-Collins solution, or Saint Thomas cardioplegic solution. Cryobiology 1996; 33: 363–75. 91. Boutron P. Glass-forming tendency and stability of the amorphous state in solutions of a 2,3-butanediol containing mainly the levo and dextro isomers in water, buffer, and Euro-Collins. Cryobiology 1993; 30: 86–97. 92. Fahy GM. The relevance of cryoprotectant ‘toxicity’ to cryobiology. Cryobiology 1986; 23: 1–13. 93. Meryman HT. Cryopreservation of living cells: principles and practice. Transfusion 2007; in press. 94. Rall WF. Factors affecting the survival of mouse embryos cryopreserved by vitrification. Cryobiology 1987; 24: 387–402.
67b. Fahy GM. Cytoprotectant toxicity neutralizers reduce freezing damage. Cyro-letters 1983; 4: 309–314. 68. Rall WF, Wood MJ, Kirby C, et al. Development of mouse embryos cryopreserved by vitrification. J Reprod Fertil 1987; 80: 499–504. 69. Rall WF, Wood MJ. High in vitro and in vivo survival of day 3 mouse embryos vitrified or frozen in a non-toxic solution of glycerol and albumin. J Reprod Fertil 1994; 101: 681–8. 70. Steponkus PL, Myers SP, Lynch DV, et al. Cryopreservation of Drosophila melanogaster embryos. Nature 1990; 345: 170–2. 71. Mazur P, Schneider U, Mahowald AP. Characteristics and kinetics of subzero chilling injury in Drosophila embryos. Cryobiology 1992; 29: 39–68. 72. Mazur P, Cole KW, Hall JW, et al. Cryobiological preservation of Drosophila embryos. Science 1992; 258: 1896–7. 73. Mehl PM. Crystallization and vitrification in aqueous glass-forming solutions. Adv Low Temp Biol 1996; 3: 185–255. 74. MacFarlane DR, Forsyth M, Barton CA. Vitrification and devitrification in cryopreservation. Adv Low Temp Biol 1991; 1: 221–77. 75. Kauzmann W. The nature of the glassy state and the behavior of liquids at low temperatures. Chem Rev 1948; 43: 219–56. 76. Segur JB. Physical properties of glycerol and its solutions. In Miner CS, Dalton NN, eds. Glycerol. New York: Reinhold Publishing Corporation, 1953: 238–334. 77. Angell CA, Sichina W. Thermodynamics of the glass transition: empirical aspects. Ann NY Acad Sci 1976; 279: 53–67. 78. Sherwood GJ, Flower JR. Engineering aspects of equipment design for subzero organ preservation. In Pegg DE, editor. Organ Preservation. London: Churchill Livingstone, 1973: 152–74. 79. Rall WF, Polge C. Effect of warming rate on mouse embryos frozen and thawed in glycerol. J Reprod Fertil 1984; 70: 285–92. 80. Fahy GM, Wowk B, Wu J, et al. Cryopreservation of organs by vitrification: perspectives and recent advances. Cryobiology 2004; 48: 157–78. 81. Mehl P. Nucleation and crystal growth in a vitrification solution tested for organ cryopreservation by vitrification. Cryobiology 1993; 30: 509–18.
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Vitrification in small quenched volumes with a minimal amount of, or without vitrificants: basic biophysics and thermodynamics
1B
Igor I Katkov, Vladimir Isachenko and Evgenia Isachenko
process (CP), which in the majority of cases requires the use of permeable and impermeable cryoprotective agents (CPAs). While it remains the mainstream method of current cryobanking, particularly of cryopreserved cell suspensions and some tissues, it holds its own limitations described in other parts of this book (see, for example, Introduction by Leibo and Mazur). There are interesting scientific questions related mostly to this way of vitrification and thawing, such as aging of vitrified solutions, devitrification of overcooled samples during slow warming, annealing, overshoot, etc.; however, these are not covered in this chapter. Lyophilization involves slow freezing to moderately low (around −40°C) temperatures, sublimation of the bulk of ice at very high vacuum, and secondary drying of the ‘cake’ at elevated (up to +30°C) temperatures until a state close to vitrification is achieved (note, that in practical terms, one can not dry a sample higher than temperature Tg − 10°C.4,5 Thus enabling stable long-term storage available at a temperature above 0°C, but one has to ensure that it is still lower than the Tg of the sample.5 This method is widely used in food production, microbiology, and the pharmaceutical industry, but so far it has had very limited applications for preservation of the majority of animal eukaryotic cells (see our recent review).3
INTRODUCTION: FIVE BASIC METHODS OF VITRIFICATION OF CELLS It is well established that storage of cells in a liquid milieu leads to degradation processes, and to eventual loss of cell viability. At the same time, intracellular ice is lethal for the majority of cells.1 Thus, the only stage in which the cells can be stabilized would be a solid-like phase in which intracellular ice crystals are not formed, or have not grown to the ‘critical lethal size’. Such a vitreous (‘glassy’) state has elevated viscosity (in the range of 1012 Pa.s at the glass transition temperature Tg and up to 1013.5 Pa.s at the so called ‘strain point),2 so processes of chemical and physical degradation are essentially stopped for the duration of experiments or storage. We can distinguish five basic methods to achieve intracellular vitrification; all of which lead to a drastic decrease of the water activity.3 Equilibrium (slow) freezing causes the bulk of intracellular water to form ice outside the cell, while dehydrating the cell slowly enough to prevent intracellular ice formation, and thus leading to vitrification of the intracellular space. This is usually followed by storage at extremely low temperatures, usually −196°C, and more recently −130°C to −80°C. This is currently the conventional cryopreservation
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devitrification during warming, and does not require the use of potentially toxic high concentrations of vitrificants. For simplicity, we call this method fast rate-low concentrations vitrification (FR-LC VF). Although historically this was the first method of vitrification of cells (see below), it has not found wide application until recent work, mostly by the Isachenko group.20–25 The practical aspects of this method are discussed in Chapters 5, 8, and 11a. In this chapter, we concentrate on the basic aspects of biophysics and thermodynamics of this method, with substantial overview of the historical background of early ‘fast rate-low concentration vitrification’ work in 1930s and 1940s when this method was first introduced.
‘High temperature’ (HT) vitrification of a highly dehydrated sample and its stabilization by air/vacuum drying at temperatures >0°C.6–16 is similar to lyophilization but does not require the very damaging slow freezing step to the cake formation temperatures, so it holds great potential for the future of longterm stabilization of mammalian cells at ambient temperatures.3 However, so far both lyophilization and ice-free HT drying of mammalian cells have proved to be formidable tasks and need further investigation. Ice-free vitrification of cell suspensions, tissues, and organs at very low temperatures and relatively slow to moderate rates of freezing 17,18 requires the use of a high concentration of vitrificants (historically but erroneously referred to as ‘cryoprotectants’ in analogy with the slow freezing, see below), which elevates the viscosity of the milieu and prevents ice formation during cooling and devitrification during warming. We will refer to this method as relatively slow to moderate rate of freezing–thawing with high concentrations of the vitrificants (SR–HC VF). It has had limited but notable successes in preserving animal oocytes, embryos, and organs (see Chapters 4, 6, 7, 11, 12), as well as plant specimens. Moreover, it will probably be the method of choice for cryopreservation of larger samples, tissues, or even entire organs (see Chapter 1A and below). However, both chemical toxicity of the vitrification solutions and osmotic damage associated with this method have necessitated another approach for vitrification in situations when possible, for example for vitrification of sensitive reproductive cells. It could also be useful in the future for cryopreservation of certain types of stem cells and their therapeutically important derivatives such as embryoid bodies, differentiated progenitor lines, etc. (see Chapter 12 and references 3 and 19). Ice-free vitrification of small sizes of the samples at extremely high rates of cooling and especially warming (105 to 106°C/min) prevents ice formation during cooling and
VITRIFICATION OF CELLS: HISTORICAL BACKGROUND AND BASIC DEFINITIONS The idea of using freezing and drying to preserve food and other perishable materials goes back thousands of years to prehistoric times. However, long-term preservation of living objects, and particularly of cells of animals such as vertebrates, mollusks, and insects, is a novel approach. Pioneering work on cryopreservation was performed by Italian priest and scientist Lazzaro Spallanzani in 1776, when he ‘froze’ stallion sperm in snow, noting the recovery of sperm motility upon warming.26 Later, in the 19th century and the first half of the 20th century, scientists elucidated some aspects of the mechanisms of cold adaptation of living matter, particularly those used by plants and fungi. The most important contribution was made by Father Luyet, who has been rightfully called the founder of the science of cryobiology. From the outset, he recognized that ice damage must be avoided, and that vitrification could be a method for preservation of cell viability.27 In 1938, Luyet and Hodapp achieved survival of frog spermatozoa vitrified with liquid nitrogen.28
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It should be mentioned that at almost the same time several western European and American groups reported their experiences with attempts at cryopreservation of fowl,29 human,30–32 and rabbit30 spermatozoa. In contrast to the widely cited work of Father Luyet, these efforts did not receive the recognition they deserved, hindered by the variability of the reported results and also by the Second World War. Hoagland and Pincus,30 and Jahnel31 used a microbiological loop for vitrification of cells. At approximately the same time the research of another pioneer in the field vitrification of cells began in the USSR. Emmanuil Yakovlevich Graevsky started his work in 1946 by confirming in his PhD dissertation the observations of Luyet;33 he then went on to explore the mechanisms of vitrification and devitrification.34–36 In particular, he reported33,34 that following the Luyet’s technique frog semen could be frozen with even better results than in the original Luyet’s report in 1938. Moreover, Graevsky and Medvedeva were two of the first cryobiologists to use a microbiological loop to vitrify suspensions of bacteria.35,36 Only in the past few years has this ‘cryo-loop’ method been reinvented and applied to the vitrification of mammalian cells, particularly to the cryoprotectant-free vitrification of human spermatozoa, which is discussed in more detail below. The approach to vitrification developed by Luyet and the other cryobiologists mentioned above was based on the application of relatively fast cooling rates. Yet the sample viability was low and variable, mostly because, as the authors recognized, sufficiently rapid cooling and warming which must be very high for low concentrations of vitrificants were not achievable. One of the reasons for this was that very low temperature efficient cooling agents such as liquid nitrogen and liquid oxygen were not used in their research. As a result, the mainstream of cryobiology turned to preservation methods based on slow freezing.
This method was facilitated by the discovery of the protective properties of glycerol on human and animal sperm when spermatozoa were frozen to extremely low temperatures, made independently by Igor Smirnov in the USSR37 and by Alan Parkes et al. in the UK.38,39 (Parkes also deserves mention for coining the term ‘cryobiology’, the study of ‘frosty life’.) The cryoprotective qualities of glycerol and other low molecular weight, mostly non-electrolytic substances had been described before. For example, Bernstein and Petropavlovski in 1937 used 0.3–0.5 mol/L glycerol and other electrolytes for freezing of bull, ram, stallion, boar, and rabbit spermatozoa to a temperature of −21°C.40 However, it was only after development of stable longterm cryopreservation at very low temperatures (−196°C and below), initiated by Smirnov’s and by Parkes’s groups, that the era of practical applications of cryobiology, particularly for animal breeding, began in the West and in the Soviet Union. During the following decades, the slow-freeze method dominated the field of cryopreservation, and it remains the basis of the majority of cryobiological techniques in use in production facilities and research laboratories. Progressively more sophisticated understandings of the mechanisms underlying cell damage during slow to moderate freezing, as well as methods to prevent it, were contributed particularly by Lovelock,41 Mazur,1,42 and Meryman,43 and by many others in the later generations of cryobiologists. In particular, Mazur developed a set of equations that allow calculation of the optimal (equilibrium) regimens of freezing cells, which avoid formation of intracellular ice. These equations also permit estimation of the probability of intracellular ice formation if the freezing has been done at higher than equilibrium rates. Despite the fact that equilibrium freezing has been widely used and drawn a lot of attention, there are many interesting scientific questions that remain. Particularly, the central dogma that ‘intracellular ice always
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kills’ has recently undergone scrutiny by McGann et al.44,45 Moreover, even the author of this postulate has recently questioned its absolute applicability to all scenarios: when it is applied to spermatozoa, even the author of the equations has admitted that the ice formation might not be damaging (or, as we think, may even not exist at all!) at freezing rates much higher than his theory predicts.46 While ‘conventional’ slow freezing has given satisfactory results for many suspended cells, such as blood, sperm, and embryos, issues of ice formation and non-homogeneity of thermal profiles of samples have made slow freezing and thawing a formidable task for tissues and organs. The breakthrough came when Fahy et al. vitrified an entire organ – a kidney.17 The presence of high concentrations of vitrificants, particularly glycerol, allowed an ice-free bulk glassy state to be achieved both inside the organ (by perfusion) and in the external milieu. By tradition, glycerol and other vitrificants were still called ‘cryoprotective agents’ (CPAs) as for slow freezing, probably because the same chemicals were used for both applications. However, one has to remember that for icefree vitrification, they play a completely different role as the glass-formers than they do for the slow freezing. In the latter case, especially at suboptimal low rates of freezing, lower than the equilibrium, the CPAs (particularly, the permeable ones) act mostly as osmotic buffers and ‘water holders’ preventing osmotic damage, rehydration, increase of concentration of the ions, and other events collectively defined as the ‘solution effects’.1 However, some changes in the ice structure of the intracellular milieu might also play a positive role at superoptimal rates of ice content freezing as the viscosity of CPAs such glycerol or dimethylsulfoxide (DMSO) increase at subzero temperatures, so the probability of the lethal amount of intracellular ice decreases.47,48 Thus, while used interchangeably, we think one has to be careful when equating the terms ‘CPA’ with ‘vitrificants’, particularly, in the
case of small molecular weight permeable substances such as glycerol, DMSO, ethylene glycol (EG), or propylene glycol (PG). Furthermore, it is important to note that they all have glass transition temperatures (Tgs) much lower than 0°C, thus, by definition, in the case of HT vitrification they would act as plasticizers by effectively decreasing the Tg of the blend. Thus making them unsuitable for stable storage of vitrified samples above − 20°C. At the same time, they are widely used for vitrification and following stable long-term storage at ultra-low temperatures of liquid nitrogen (LN2) and industrial freezers (−120°C and below). Vitrification eliminated many of the problems related to the slow freezing of liquid water to an ice phase, particularly extensive rehydration and osmotic damage, the increased ionic strength of concentrated eutectic solutions, and shifts in pH, etc. However, vitrification in high concentrations of vitrificants has introduced its own set of problems. Notable among them are osmotic damage during addition and removal of vitrificants, CPA toxicity, mechanical cracking of glasses, and devitrification due to inadvertent thermal cycling during storage, etc. (see Chapter 1A). Nevertheless, practical cryobiological applications of vitrification with the use of high concentrations of vitrificants has continued to grow, especially after Rall and Fahy reported the successful vitrification of mouse embryos in 1985.18 However, more widespread use of vitrification was slowed by the inherent pitfalls of high-CPA methods, and also by the lack of understanding of the mechanisms of vitrification and devitrification by most practitioners of the craft. By the 1980s, the fundamental work of Luyet and the other pioneers of vitrification had been largely forgotten. Unfortunately, the pioneering work of the cryobiologists of the 1930s, 1940s, and early 1950s is still under appreciated today. In the 1990s, vitrification was applied to new areas such as oocytes and ovarian tissues
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0
Tm
III
II
I
human sperm without any conventional CPAs using simply constructed cryonic copper loops.49 In this case, they applied a very rapid rate of cooling (up to hundred of thousands °C/min,23 see below) in a film (pellicle). Essentially, they independently rediscovered the method used by Luyet and the others more than a half century ago. It has become evident that the rate of warming was even more important, so that as soon as almost instant warming was secured, the rate of freezing did not play such a crucial role. This suggests that intracellular vitrification can be achieved at relatively low rates of cooling without the use of CPAs and preliminary dehydration, presumably due to the low water content of human spermatozoa.20,23–25 The next section concentrates on the specifics of the FR-LC method of vitrification.
IV
Temperature (°C)
X −40 X
Th
−80
Tg2 Td Tg
−120
20
Doubly unstable
40
60
Concentration (%w/w) Figure 1B.1 Phase diagram of a hypothetical ‘cryoprotectant’ (vitrificant) originally proposed by Fahy et al.17 and supplemented by us.3 The dotted line (Tg2) is a hypothesized vitrificant with a much higher glass transition temperature than glycerol or DMSO. The left most arrow is a new border between zones I and II, at which vitrification is achievable at a much lower concentration of the vitrificant and much higher rates of cooling and warming than was proposed by Fahy et al.17 Reproduced from3 with permission of Elsevier.
VITRIFICATION BY VERY FAST FREEZING AND WARMING BY INSTANT THAWING The main process involved in crystallization and conditions for ice-free vitrification can be summarized in the supplemented phase diagram proposed by Fahy et al.,17 and modified by us3 as shown in Figure 1B.1. The larger that the difference between the homogenous ice crystallization curve Th (the top left part of Figure 1B.1) and the glass transition curve Tg is the higher the probability that crystallization of the solution will occur during freezing (shown as crosses on the left part of Figure 1B.1). In zone I, Fahy et al. considered the probability of vitrification at the rate of cooling they used (up to thousands °C/min) to be very low, while the probability of ice crystallization would be almost 100%. As the concentration of the ‘CPA’ (vitrificant) increases, the temperature of homogeneous ice crystallization drops, while Tg rises. At the point when the Th meets the Tg curve, zone III begins, and there is a probability of achieving
(see Chapters 6, 7, 11). However, the high osmotic sensitivity of small germ and blood cells, such as sperm or erythrocytes mostly due to the osmotically active and (sometimes) chemically cytotoxic vitrification agents has made this approach unattractive despite the fact that vitrification can be simple, fast, and cheap. Instead, practitioners, farmers, and blood banks are committed to the slowfreezing route, with its attendant need for expensive, programmable cryo-equipment, and skilled technical personnel. All this recent development did little to resolve the problems related to high concentrations of ‘CPAs’ used in LR-HC VF developed by Fahy and Rall. A new breakthrough came in 2002 when Nawroth et al. vitrified
25
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vitrification during appropriately fast cooling. However, the devitrification curve Td indicates that there would be a high possibility of devitrification during warming. The devitrification curve Td drastically decreases with higher percentages of ‘CPA’ (vitrificant) due to a dramatic rise of viscosity, and when Td meets the non-homogeneous equilibrium crystallization (melting) curve Tm zone IV begins. At this point and higher, crystallization and devitrification will never occur at any reasonable rate of cooling and warming. Note, however, that for ‘conventional’ vitrificants such as glycerol, this concentration is in the range of 65–70%, which is extremely high, and few known mammalian cells would withstand such enormous (tens of Osm) osmotic pressure. Thus, vitrification should be performed in a kinetic way, playing with concentration of the vitrificant and rates of cooling and warming. Fahy et al. defined the zone when practical vitrification is achievable as from 45% of ‘CPA’ (vitrificant) marking this as zone II. The critical speed of cooling and warming, however, is negatively related to the concentration of the CPA: the higher is the speed of cooling/warming, the lower is the concentration of the vitrificant needed, so that the border between ‘non-achievable’ and ‘achievable’ vitrification (zones I and II) is arbitrary. This means that if the speed of cooling and warming are high enough, then the concentration of the glass-formers can be very low (the dashed line is a new border between zone I and zone II). Even pure water can vitrify if special conditions, such as superquenching are met (although there is still a debate on the exact value of its Tg).50,51 While there is still discussion among scientists about the minimal cooling rate that can be achieved to vitrify water it must be at least millions of °C/min.52–54 In any case, lowering the amount of vitrificants by increasing speed of cooling and warming (as defined
above HR-LC VF), as well as by partial dehydration using impermeable osmotically active compounds such as sucrose has been the main direction for the past decade in vitrification cryobiology, particularly for reproductive cells. Note that the positive effect of the addition of sucrose is sometimes erroneously attributed to ‘lowering toxicity’ of glycerol or DMSO, while it is mostly due to the osmotic and kinetic effects. Several types of devices, such as open pulled and hemi-straws, microscopic grids, and cryoloops and cryotops have been investigated to increase the speed of cooling and warming and, thus, to decrease the CPA concentrations to values of 25–30% (see details in Chapters 4A, 10, 11, 12). While it has been working well for oocytes and embryos, small cells like sperm and erythrocytes have proved to be practically intolerant even to such ‘moderate’ concentrations of CPA (e.g. 30% of most widely used CPAs such as glycerol, ethylene glycol, and propylene glycol translate to 15–20 times the isotonic value). However, a major breakthrough came in 2001, when a group from Germany managed successfully to vitrify sperm without any conventional cryoprotectants or vitrificants. The spermatozoa were cooled in very thin films (pellicles) in copper cryoloops.49 Basically, they rediscovered the original Luyet–Graevsky approach, but with the additional knowledge that had been accumulated since the pioneering works from 1939 to 1948. We have collaborated with this group, and recently published several joint papers,20,23–25,49 in which we suggested that the extremely high rate of cooling (several hundred thousand °C/min) and almost instant warming by dissolving in a warm media made vitrification during freezing without devitrification during rewarming practically feasible. Taking in to account that the culture medium contained proteins (serum albumin), and the internal cellular milieu is abundant in high glass transition temperature (much above 0°C) components such as proteins, polysaccharides, nucleic
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2
1
3
4 2
Figure 1B.2 Images and scheme of warming a microdroplet in aseptic conditions using the cut standard straw (CSS) container for vitrification: (1) closed 0.5 ml straw, (2) CSS, (3) microdroplet of vitrification medium with the embryo, (4) tube with solution for warming and removal of the vitrificant.
22
acids, and other biopolymers, then kinetic vitrification can be achieved at very low concentrations of non-osmotically active glass formers, so no conventional osmotically damaging and toxic ‘CPAs’ are needed (the far left part of Figure 1B.1). The typical glass transition temperature of such intracellular components lies much higher than for glycerol or other ‘conventionally used’ vitrificants (shown as Tg2 in Figure 1B.1), so the probability of vitrification is increased dramatically when high rates of cooling and very high rates of warming are combined. In experiments with cryonic loops, we estimated that the rate of cooling could be as high as a hundred thousand °C/min, so we speculated that the border between zones I and II could be shifted to very low values (Figure 1B.1, left arrow). In these conditions of very fast cooling and practically instant warming the cell’s natural high molecular weight intracellular vitrificants and presence of albumin in the media would be enough to ensure both intra- and (probably) extracellular vitrification, as we have suggested.23 Even
cooling rates 100-fold slower than in cryonic loops immersed in LN2, e.g. in vapor of liquid nitrogen, were sufficient to ensure the cell survival at the same level as for direct plunging to LN2.25 Moreover, the use of pellicles in cryonic loops is not mandatory: the cells can be cryopreserved in an aseptic manner in small droplets in plastic straws without direct contact with LN2.20 One such modification is shown in Figure 1B.2. The crucial points are the small volume of the liquid to be cooled and fast warmed with intensive agitation of the surrounding warm medium. For small cells such as human spermatozoa no vitrificants were needed, while larger and probably ‘more watery’ cells need a certain amount of vitrificants (see Chapters 5, 8, and 11). Application of fast cooling in combination with relatively small amounts of vitrificants can be applied to human embryonic cells obtained from cultured embryonic cell lines, which are proven to lose pluripotent ability after conventional slow freezing with DMSO, so vitrification seems a more favorable
27
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approach55–61 (see also Chapter 12). We have also initiated thorough studies of the difference in human embryonic stem cell (hESC) pluripotency with respect to the mode of cryopreservation: slow freezing versus vitrification, which has confirmed that slow freezing suppressed expression of pluripotency marker Oct-4.19
∂2 T ∂2 T ∂2 T + + ∂x 2 ∂y2 ∂z2 ∂T 1 ∂2 T 1 ∂ ∂2 T r + 2 2 + 2 ∇ 2T = r ∂r ∂r r ∂ϕ ∂z ∂T 1 ∂ r2 ∇ 2T = 2 r ∂r ∂r 1 ∂ ∂T + 2 sin θ r sin θ ∂θ ∂θ 2 1 ∂T + 2 2 r sin θ ∂ϕ2 ∇ 2T =
CALCULATION OF THE RATE OF COOLING/WARMING OF SMALL SAMPLES For small objects such as a thin pellet within a cryoloop, or a very small droplet of cell suspension shown in Figure 1B.2, it is practically impossible to measure directly the cooling or warming rate with a thermometer or a thermocouple. Accurate measurement by visual observation is also impossible because both vitrification and warming occur very quickly. The only practical estimation of the range of the rate of cooling and warming can be done by calculation. Here we give an example of the calculations in general, and the rate of warming of the droplet depicted in Figure 1B.2. The heat transfer in a thermal conductor is described by the Biot-Fourier equation (also called the heat conduction equation) describing the variation of temperature T (heat flux) with position and time t in a thermal conductor, and in the general form for isotropic propagation with no internal heat sources it can be written as follows:62,63
are the operators for Cartesian, cylindrical, or spherical coordinates, respectively. For a simple system such as a rod where its side surface is thermo-isolated, so that the heat flow is in one direction, the equation can be simplified to a one-dimensional form that in Cartesian coordinates can be written as follows:
∂T ∂ 2T = DT 2 ∂x ∂t If the initial temperature of the sample is T0 and both its sides are kept at (immersed to) the same temperature Tf (i.e. in a thin film we have estimated before),23 the solution of this equation can be expressed as follows:
4 (Tf − T0 ) T (x, t) = π ∞ sin(2m + 1)π dx −[(2m+1)π]2 ( t ) τ e 2m + 1 m=1
∂T = DT ∇ 2 T ∂t
where τ = d2/DT is the characteristic time after which all the processes are practically equilibrated, x is the distance from the middle to the surface, and d is the thickness of the film. Thus, the rate of temperature change B can be determined as (Tf −T0) / T. For a homogeneous sphere with initial temperature T0 immersed into a highly thermoconductive medium with temperature Tf, the heat
where DT =
κ cρ
is the temperature diffusivity, κ is the thermal conductivity, c is the specific heat capacity, ρ is the density of the conductor, and 28
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flow distribution will be dependent on only the distance from the center to the position in consideration (r) but will be independent of the spherical angles θ and ϕ, so a one-dimensional form in the spherical coordinates is:
DT ∂ ∂T = 2 ∂t r ∂r
After less than a half of the characteristic time, practically the whole droplet is in equilibrium with the warming surroundings, so even the center of the droplet is warmed practically to 243K at time q = 0.4 τ. After the temperature of the droplet reaches 0°C (Tf = 273K), it devitrifies, detaches from the surface, and dissolves in the surrounding medium. Now we can estimate the characteristic time for a frozen spherical droplet immersed in a hot solution. Taking into account that for ice κ = 0.005 cal/s/cm/K, c = 0.49 cal/g/K, and ρ= 0.92 g/cm3 64 this gives the temperature diffusivity DT to be equal to 0.011 cm2/s. A spherical droplet of volume 0.75 µL will have a radius rdr = 0.056 cm, so the characteristic time τ can be estimated as 0.29 s. In reality, the shape of the sample is not a spherical droplet but rather a spherical segment (cap) due to the low surface tension of the DMSO. The spherical surface of the cap is opened to the warming solution, while the base surface can be considered to be thermoisolated for a short time due to contact with the plastic. We estimated the height of the cap h is about half of its base radius a. The volume of this cap is still 0.75 µL, so it can be shown that h = 0.048 cm, and c = 0.096 cm. The spherical surface of the cap would be 0.036 cm2, while the total surface of the spherical droplet discussed above would be 0.040 cm2. In this case, the process of total equilibrium ends in 0.135 s. In such a scenario, the characteristic time τ of the process is about 0.27 s, and the process of total equilibrium occurs in 0.135 s. That means the rate of warming is in the range of 87 000°C/ min. The presence of ‘cryoprotectants’ – vitrificants such as DMSO and sucrose would affect viscosity, and thus the probability of ice formation, but c, κ, and ρ would not be significantly different, so the time τ and the rate of warming would be in the same range. A similar approach applied to a thin pellicle on the cryoloop gave us an estimation of cooling at an order of magnitude higher,23 while warming occurs in this case almost instantly. In comparison, the use of the commonly used open-pulled straws (calculated as a
2 ∂T r ∂r
By substituting v = rT this can be simplified to:
∂ν ∂ 2ν = DT 2 ∂t ∂r This equation is completely analogous to the equation for a one-dimensional case discussed above, but the thickness d must be substituted by the radius of the sphere R, and r instead of x would be the current distance from the core. Thus, both for a thin film, and a sphere, the mathematical solution is equivalent and all considerations are the same. Practical difference is due to the fact that v = rT, all processes in the center of the sphere are equilibrated at a time of about 0.5 τ, while for a pellicle they are in range of its characteristic time. Assuming for simplicity that the droplet shown in Figure 1B.2 is a sphere. The kinetics of warming as the function of the relative distance from the center of the sphere λ = x/r and the relative time q = t/τ is shown in Figure 1B.3. At the first moment (q = 0) in all points of the droplet except its surface (λ =1), the temperature is equal to the temperature of the liquid nitrogen (T0 = 77K), while we assume for the sake of simplicity that the temperature of the surface is equal to 0°C (Tf = 273K). So no warming above zero (warming and dissolving) occurs until the whole droplet is warmed to 0°C. At time τ/20, the core of the droplet is still very cold, so the temperature at the center (λ = 0) is far below (122K) the freezing point. At the same time, close to the surface the temperature of the layer T is substantially higher (243K). The volume of more distant layers is the quadratic function of the radius that means a larger portion of the droplet is warmed (in comparison with the thin film, where all layers have the same volumes regardless of their distance from the center). 29
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300 I
Temperature (K)
X
X
X
I X
X
X
X
X
I
I
I
I
I
250
200
150
100
50 0
0.2
0.4
0.6
0.8
1
lambda = x/r ( along radius) q-0
q-0.05
q-0.20
q-0.10
q-0.25
I
X
q-0.15
q-0.40
Figure 1B.3 Spatial and temporal distribution of the temperature of a spherical droplet as shown in Figure 1B.2 as a function of dimensionless time q = t/τ and distance from the center λ = x/r, where τ is the characteristic time of the droplet, and r is the radius. Initial temperature of the droplet is the temperature of liquid nitrogen (77K), while the final warming temperature of the frozen droplet is 0°C (273K). At such conditions, the characteristic time τ of the 0.75 µl droplet (r = 0.056 cm) can be estimated as 0.29 s, that gives the rate of warming in the range of 87 000°C/min. After the temperature of the droplet reached 0°C, it devitrifies, detaches from the surface, and dissolves in the surrounding medium.
vitrification can occur at a rate of 105–106°C/min without the use of conventional external vitrificants such as glycerol, DMSO, EG, etc. (2) Larger and more watery cells such as oocytes need a certain amount of exogenous vitrificants, although this is much less than for ‘traditional modes’ of vitrification. (3) The suggestion that intracellular ice might not be damaging for mouse spermatozoa, and therefore they do not require the use of glycerol as an intracellular protector, yet can survive high rates of cooling may indirectly indicate that vitrification-like processes can occur in spermatozoa at relatively low cooling rates.46 (4) The recent observation by Morris who reported no presence of intracellular ice in human spermatozoa in the presence of 10% of glycerol even at cooling rates as low as 3000°C/min.65
cylinder with the sample volume approximately 3 µL) would produce a warming rate in the range of 20 000°C/min, thus making this less effective than the droplet, not to mention that the latter allows practically aseptic cooling without direct contact with LN2. In conclusion, at such fast processes that occur during cooling and warming of a pellicle or a microdroplet depicted in Figure 1B.2, it is practically impossible to observe actual vitrification or measure it by, for example, differential scanning calorimetry (DSC). However, several independent bodies of evidence suggest that it is a vitrification process. Among them, in our opinion, are: (1) A large amount of high molecular weight intracellular vitrificants, mostly polymers, in small cells like spermatozoa, the inactive osmotic volume of which can be as high as 56%, suggests that at least intracellular 30
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49. Nawroth F, Isachenko V, Dessole S, et al. Vitrification of human spermatozoa without cryoprotectants. Cryo Letters 2002; 23: 93–102. 50. Yue Y, Angell CA. Clarifying the glass-transition behaviour of water by comparison with hyperquenched inorganic glasses. Nature 2004; 427: 717–20. 51. Kohl I, Bachmann L, Mayer E, et al. Water behaviour: glass transition in hyperquenched water? Nature 2005; 435(7041): E1; discussion E1–2. 52. Angell CA. Amorphous water. Annu Rev Phys Chem 2004; 55: 559–83. 53. Johari GP, Hallbrucker A, Mayer E. Two calorimetrically distinct states of liquid water below 150 Kelvin. Science 1996; 273: 90–2. 54. Velikov V, Borick S, Angell CA. The glass transition of water, based on hyperquenching experiments. Science 2001; 294: 2335–8. 55. Fujioka T, Yasuchika K, Nakamura Y, et al. A simple and efficient cryopreservation method for primate embryonic stem cells. Int J Dev Biol 2004; 48: 1149–54. 56. Heng BC, Kuleshova LL, Bested SM, et al. The cryopreservation of human embryonic stem cells. Biotechnol Appl Biochem 2004. 57. Ji L, de Pablo JJ, Palecek SP. Cryopreservation of adherent human embryonic stem cells. Biotechnol Bioeng 2004; 88: 299–312. 58. Reubinoff BE, Pera MF, Vajta G, et al. Effective cryopreservation of human embryonic stem cells by the open pulled straw vitrification method. Hum Reprod 2001; 16: 2187–94. 59. Richards M, Fong CY, Tan S, et al. An efficient and safe xeno-free cryopreservation method for the storage of human embryonic stem cells. Stem Cells 2004; 22: 779–89. 60. Solberg S, Laerum OD. Cryobiology – freeze preservation and storage of living cells and tissues. Tidsskr Nor Laegeforen 2004; 124: 2607–9. [in Norwegian] 61. Zhou CQ, Mai QY, Li T, et al. Cryopreservation of human embryonic stem cells by vitrification. Chin Med J (Engl) 2004; 117: 1050–5. 62. Zhou X. The Fourier Heat Flux Model and Relevant Associated Equations. 1999:http:// www.msi.umn.edu/~xiangmin/paper/hhc97/ node6.html. 63. Aramanovich IG, Levin VI. Equations of Thermal Conductivity. In: Equations of Mathematical Physics, 2nd edn. Moscow: Nauka, 1969: 145–216. [in Russian] 64. Nave CR. HyperPhysics. 2005: http:// hyperphysics.phy-str.gsu.edu/hbase/hph.html. 65. Morris GJ. Rapidly cooled human sperm: no evidence of intracellular ice formation. Hum Reprod 2006; 21: 2075–83.
32. Parkes AS. Preservation of human spermatozoa at low temperatures. Br Med J 1945; 2: 212–213. 33. Graevsky EY. Studies on deep cooling of the protoplasm. PhD Thesis. Leningrad: Leningrad State University, 1946: 112. [in Russian] 34. Graevsky EY. Living matter and low temperatures. Priroda (Rus) 1948; 5: 13–25. [in Russian] 35. Graevsky EY. Glassy state of protoplasm in conditions of deep cooling. Uspehi Sovr Biol (Rus) 1948; 14: 186–202. [in Russian] 36. Graevsky EY, Medvedeva YA. Causes of damage of protoplasm during deep freezing. Zh Obschey Biologii (Rus) 1948; 9: 436–469. [in Russian] 37. Smirnov IV. Preservation of domestic animal semen by deep cooling. Sovetskaja Zootechnia (Rus) 1949; 4: 63–65. [in Russian] 38. Polge C, Smith AU, Parkes AS. Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature 1949; 164: 666–676. 39. Smith AU, Polge C, Parkes AS. Survival of spermatozoa at low temperatures. Nature 1950; 166: 668–9. 40. Bernstein AD, Petropavlovski VV. Influence of non-electrolytes on viability of spermatozoa. Buleten’ eksperimentalnoi biologii i medicini 1937; III: 21–25. [in Russian] 41. Lovelock JE. The mechanism of the protective action of glycerol against haemolysis by freezing and thawing. Biochim Biophys Acta 1953; 11: 28–36. 42. Mazur P. Kinetics of water loss from cells at subzero temperatures and the likelihood of intracellular freezing. J Gen Physiol 1963; 47: 347–69. 43. Meryman HT. Freezing injury and its prevention in living cells. Annu Rev Biophys Bioeng 1974; 3: 341–63. 44. Acker JP, McGann LE. Innocuous intracellular ice improves survival of frozen cells. Cell Transplant 2002; 11: 563–71. 45. Acker JP, McGann LE. Protective effect of intracellular ice during freezing? Cryobiology 2003; 46: 197–202. 46. Mazur P, Koshimoto C. Is intracellular ice formation the cause of death of mouse sperm frozen at high cooling rates? Biol Reprod 2002; 66: 1485–90. 47. Karlsson JO, Cravalho EG, Borel Rinkes IH, et al. Nucleation and growth of ice crystals inside cultured hepatocytes during freezing in the presence of dimethyl sulfoxide. Biophys J 1993; 65: 2524–36. 48. Rall WF, Mazur P, McGrath JJ. Depression of the ice-nucleation temperature of rapidly cooled mouse embryos by glycerol and dimethyl sulfoxide. Biophys J 1983; 41: 1–12.
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2
Gábor Vajta, Masashige Kuwayama and Pierre Vanderzwalmen
cells, in the cytoplasm and nucleus is regarded as the major source of injury. The phenomenon may occur between −5 and −80°C (predominantly between −5 and −40°C) and has deleterious, mostly mechanical effects on all structures. Both major groups of successful cryopreservation strategies try to minimize these effects. The main elements are the same, using cryoprotectant solutions and controlling the rate of temperature changes. Most important (but not the only) effect of cryoprotectants is to minimize ice crystal formation by either removing water from the cytoplasm with osmotic effect (non-permeable cryoprotectants), or interfering with ice nucleation and growth inside the cytoplasm or in the extracellular space closely surrounding the cells (extracellular cryoprotectants). Unfortunately the use of cryoprotectants introduces a new source of injuries into the process. None of the known cryoprotectants is entirely harmless. The permeable ones may have a considerable toxic effect, while the non-permeable cryoprotectants may induce osmotic damage. The borders are, however, not sharply defined and most cryoprotectants result in both toxic and osmotic injury. Toxicity is usually proportional to the concentration of the substance and to the time of exposure and can be decreased by lowering the temperature, while the osmotic effect in the solution phase depends mostly on the concentration, and also partially on the time of exposure. Between −50 and −150ºC, the mechanical effect of the solidified and occasionally broken
CURRENT PROBLEMS, SHORTCOMINGS, AND POTENTIAL PROBLEMS WITH VITRIFICATION METHODS All cryopreservation methods expose mammalian tissues and cells to an environment that they would not normally experience and have no intrinsic genetically coded capacity to survive. The art of the work is to establish a situation where the injuries are minimal and defensive-regenerative capacities are supported. The most important known mechanisms of damage that occur during cooling to low subzero temperatures (without any external intervention to increase chances of survival) include chilling injury, ice crystal formation, and fracture damage. Chilling occurs between around +15 and −5°C, induces at least partially irreversible changes in certain structures including lipid droplets, lipid-rich membranes, and microtubuli of the mitotic or meiotic spindle.1–4 Oocytes and embryos from different species and developmental phases contain different amounts of lipids that may partially explain their different sensitivities to chilling injury. According to our present knowledge, there are very limited possibilities to avoid this damage including the mechanical or chemical removal of lipid droplets from the cytoplasm,5–11 or to pass oocytes and embryos extremely quickly through the critical temperature zone both at cooling and warming. Ice crystal formation both in the medium directly surrounding the cells, and inside the
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trypsinized monolayer cultures of some mammalian cells. From this point of view, embryology is in a privileged situation, as the early phase of development offers a possibility to stop the biological time of life in mammals. The size, however, matters even in the case of relatively small objects, for example, oocytes, zygotes, and early stage embryos are generally more sensitive to injuries than blastocysts that consist of cells with a size comparable with a tissue culture monolayer. However, shortly after hatching, with the increasing overall size and complexity, the chances of successful cryopreservation decrease radically. During the past decades, from the various approaches, two major strategies have emerged that may fulfill the requirements for successful cryopreservation of mammalian oocytes and embryos. Chronologically the first was the strictly controlled, slow rate freezing,18–24 where the initial toxic and osmotic injury is minimal, and the induced ice crystal formation (seeding) results in a slow, stepwise concentration of the cryoprotectants around and in the cytoplasm. This increasing concentration occurs in parallel with the decreasing temperatures and can cause relatively low levels of harm to the cells. When the temperature drops to the level that induces solidification in the whole solution (around −30 or −40°C), solidification occurs with minimal or no ice formation in and around the cells, i.e. in the microenvironment and in the cytoplasm the dominant phenomenon is the extremely increased viscosity, i.e. vitrification. The other strategy is much more radical: it is an approach to avoid ice crystal formation in the entire solution containing the embryos. It is usually achieved with a drastic elevation of cryoprotectant concentration, and an increased cooling rate. Basically, this latter approach seems to be more straightforward, as it eliminates totally one source of injury; however, it exposes cells to a considerably elevated toxic and osmotic effect. Both
solution may cause fracture damage, especially in relatively large biological objects such as oocytes and embryos.12 By applying cryopreservation methods developed in the past 30 years, this type of damage may occur more often during the warming procedures, and may be minimized by decreasing slightly the rate of temperature changes in this temperature zone, although this approach is rather empirical and is without clear scientific explanation. It is quite unlikely that zona fracture could occur as a simple consequence of osmotic stress as suggested by Smith and Silva.13 Storage at −196°C is probably the least harmful part of the cryopreservation process, although accidental warming may destroy the stored samples. The effect of the background (cosmic) radiation is probably less harmful than previously supposed.14 On the other hand, an existing – although frequently overestimated – danger is the possibility of liquid nitrogen-mediated cross contamination of samples. At warming, the same types of injuries may occur as at cooling, obviously in a reverse order. Apart from these processes, there are some partially understood injuries including damage of intracellular organelles, cytoskeleton, and cell-to-cell contacts.15–17 For successful cryopreservation, an attempt should be made to establish an optimal balance, i.e. to keep the toxic and osmotic injury low while profiting maximally from the ice preventing effects of the cryoprotectants. In spite of the considerable efforts in the past century including analyses of physical, chemical, and biological factors, and the establishment of mathematical models and introduction of sophisticated equipment, only limited success has been achieved. In general, the size of the biological object is critical: the smaller is the sample, the better are the chances of survival. Viruses and bacteria can be cryopreserved with relatively simple methods, and good survival rates have been achieved with masses of single cells, for example, suspensions of
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strategies have disadvantages and limitations. The subject of this chapter is to analyze those related to vitrification.
cryoprotectants in a mixture; to add cryoprotectants stepwise, in an increasing concentration to the solutions; and to decrease the temperature when the oocytes and embryos are exposed to the concentrated, final vitrification solution.25–28 Except for the latter strategy (that may be beneficial in several situations, but may also increase the risk of chilling injury), almost all listed approaches are now indispensable parts of a successful vitrification method. However, even the best cryoprotectants and the most sophisticated strategies in combining and providing them may be insufficient if the final required concentration is high. As mentioned above, according to our present knowledge the only practical approach to keep this concentration at a level that is tolerated by oocytes and embryos is to increase the cooling and warming rates. Traditional tools of cryopreservation are insufficient for this purpose, as for example the maximum achievable cooling rate with direct plunging of a standard 0.25 mL insemination straw into liquid nitrogen is 2500°C/min29 due to the relatively large amount of solution (> 5 µL) required to form a stable column in the straw without danger of dispersion during the pressure changes caused by cooling and warming, and also due to the relatively thick plastic wall of the sealed straw that presents a considerable thermoinsulating layer. On the other hand, even this relatively low rate was still hazardous to perform as direct immersion into liquid nitrogen at cooling, and transfer to a water bath at warming induced extreme pressure changes in the closed system, and frequently led to the collapse or explosion of the straws and loss of the sample. Obviously, the achievable cooling rate is even lower in cryovials. Interestingly, however, for a relatively long period of time scientists have respected the limits determined by these old tools. Even in 2005 and 2006, there are many publications dealing with vitrification experiments performed in 0.25 mL straws, i.e. disregarding completely
ELIMINATION OF ICE FORMATION AND THE REQUIRED COST When we talk about the benefits of vitrification, the first and unquestionable one is the total elimination of the major source of injuries at cryopreservation, that is ice crystal formation. However, to ensure this benefit, we have to establish technically difficult and potentially harmful conditions. To achieve vitrification of solutions a radical increase of both the cooling rates and the concentration of cryoprotectants is required. The higher the cooling rate, the lower is the required cryoprotectant concentration, and vice versa. However, none of the extremes can be used under practical conditions. Although with the use of drastically high cryoprotectant concentrations vitrification may be achieved, with relatively moderate rates of temperature change, this approach would expose the samples to unreasonable toxic and osmotic injury. On the other hand, although vitrification can also be achieved in pure water with a cooling rate of approximately 107°C/s,25 this theoretical limit cannot even be approached with the usual technology of an embryology laboratory. The art of establishment of an efficient vitrification method is to find a balance, i.e. to keep the toxic-osmotic injury as low as possible while providing safely the required speed of cooling and warming. There are several approaches to keep toxic and osmotic injury low, including (evidently) the application of cryoprotectants with low toxicity and high permeability (from this point of view ethylene glycol or propylene glycol is almost indispensable for successful vitrification); or to use two or more cryoprotectants to decrease the specific toxicity of each (provided there is no synergistic toxic effect between the different cryoprotectants); to use both permeable and non-permeable
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the wide range of possibilities to overcome the above-mentioned limitations. The main principles of these innovative approaches are extremely simple and understandable at an elementary level. The two clear possibilities to increase cooling and warming rates are to decrease the volume of the solution, and to minimize or completely eliminate the thermoinsulating layer that separates the solution containing the sample from the cooling substance, in most cases liquid nitrogen. Apart from the high cooling rate, the small solution volume offers another benefit as it decreases the chances of heterogeneous ice formation,25 and provides the opportunity to decrease further the cryoprotectant concentration required for safe and complete vitrification of the sample. The most logical way to achieve these goals seems to be to drop the solution directly into the liquid nitrogen.30–34 However, the actual result may not be as good as the idea itself. To form a drop, a relatively large amount of solution (approximately 5 µL) is required; accordingly the rate of cooling is limited. Moreover, the warm drop induces an extensive evaporation on the surface of the liquid nitrogen, and the vapor coat not only isolates the sample from the rest of the liquid nitrogen but keeps it floating on the surface for a relatively long period of time (4–8 s) before sinking and reaching the final −196°C temperature; accordingly the cooling rate is far from what is optimal. Finally, based on successful attempts in Drosophila melanogaster embryo cryopreservation35 copper grids manufactured to hold the ultrathin sections at electron microscopic investigations were used as carrier tools to vitrify bovine oocytes.36–38 The method has some benefits that are almost unsurpassable: the solution surrounding the sample can be minimized by touching a filter paper before submerging into liquid nitrogen, only a thin, film-like layer remains that protects the sample from drying. Accordingly, the cooling and warming rate is close to the theoretical maximum achievable with direct
immersion into liquid nitrogen. Moreover, the oocytes and embryos remain fixed safely to the surface of the grid during the whole cryopreservation procedure, but are separated immediately at warming, when the grid is simply immersed into the warming solution. Unfortunately, there are some drawbacks of this approach, as well. Electron microscopic grids are tiny little tools, and require delicate handling during cooling, warming, and especially storage. Although a possible solution for the latter problem has been published by placing vitrified grids into cryovials filled with liquid nitrogen,39 this application is strongly opposed by producers of all cryovials because of the potential danger of explosion during accidental or intentional warming. Additionally, the direct contact between liquid nitrogen and the solution containing the sample may raise some concerns regarding the possibility of disease transmission. As this problem is common in almost all recent vitrification techniques, it will be discussed in a separate section below. As a result of these problems, the use of the copper grids as carrier tools has been mostly restricted to experimental purposes, and new tools had to be developed for practical application. These tools are discussed in detail in other chapters of this book, so here we provide only a short and incomplete list beginning with the open pulled straw (OPS) technique40 and continuing with all its later subclones: glass micropipettes, GMP;41 super-finely pulled OPS, SOPS;42 gel-loading tips;43 sterile stripper tip;44 flexipet denuding pipettes, FDP;45 fine diameter plastic micropipettes;46 100 µL pipetting tip;47 etc. The principles in all these methods were the same: a narrow, thin walled plastic capillary that is usually filled with a tiny (< 1 µL) amount of solution containing the sample by using the capillary effect, and direct immersion of the tool into liquid nitrogen. The achievable cooling and warming rates with these tools may be as high as 20 000°C/min.
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Table 2.1 Examples in mammalian embryology where the first success with cryopreservation was achieved by vitrification. Embryos and oocytes were not treated mechanically or chemically to prepare them for the vitrification. Full term developments were reported except where otherwise indicated. Species, stage, system
Reference
Bovine immature oocytes for IVF Bovine in vitro matured oocytes for IVF Bovine in vitro matured oocytes for somatic cell nuclear transfer Bovine cytoplasts for embryonic cell nuclear transfer Bovine early stage IVF embryos Bovine zona-included blastocysts generated by somatic cell nuclear transfer Bovine zona-free blastocysts generated by somatic cell nuclear transfer Bovine transgenic blastocysts generated by somatic cell nuclear transfer Ovine zona included embryos generated by nuclear transfer Porcine immature oocytes for ICSI Porcine in vitro matured oocytes for ICSI Porcine in vivo derived blastocysts Porcine in vivo derived morulae Porcine in vitro produced blastocysts Equine in vivo matured oocytes European polecat in vivo derived morulae and blastocysts Siberian Tiger in vivo derived embryos Minke whale immature oocytes for maturation
Vieria et al., 200280 Martino et al., 1996;87 Vajta et al., 1998a82 Hou et al., 200583 Booth et al., 199984 Vajta et al., 1998a, in vitro study40 French et al., 200285 Tecirlioglu et al., 200486 French et al., 200387 Peura et al., 200388 Fujihira et al., 2004, in vitro study89 Fujihira et al., 2005, in vitro study90 Kobayashi et al., 199891 Berthelot et al., 200191 Men et al., 2005, in vitro study92 Maclellan et al., 200293 Piltty et al., 200494 Crichton et al., 2003, in vitro study95 Iwayama et al., 2004, in vitro study96
ICSI, intracytoplasmic sperm injection.
a rapid movement that also continues under the level to remove the vapor coat that forms around the tube, which may slow down the cooling rate. At warming, in open systems the tube should be simply immersed into the appropriate warming solution containing an osmotic buffer to counterbalance the effect of the cryoprotectants accumulated in the cytoplasm. The vitrified solution becomes liquid almost immediately after immersion, and at the same moment, as the result of the capillary effect, the warming solution enters the straw and dilutes the concentrated cryoprotectants. Eventually, as a result of gravity, oocytes and embryos slowly float out of the straw and can be subjected to stepwise dilution of both the cryoprotectant and the osmotic buffer of the warming medium. Closed tubes are usually warmed in a water bath, then the end of the tube is opened with scissors or a blade, and from this point the dilution process is almost identical to that of the open systems; however,
This is almost an order of magnitude higher than the limits of the standard 0.25 mL insemination straw, but still considerably lower than that obtained with other advanced vitrification methods. Other versions of the original OPS idea including the closed pulled straws (CPS),48 sealed open pulled straws,49 and the more professional and sophisticated form, the Cryotip system50 hermetically isolated the sample from the liquid nitrogen, consequently eliminating the danger of cross contamination. However, this modification does result in a decrease in the cooling and warming rates, and limits the efficiency of the technique in cases of certain sensitive biological materials such as human oocytes. The main benefit of application of these narrow tubes is the easy manipulation. Loading is performed by either using the capillary effect, or attaching the tool to a small volume automatic pipette. The immersion into the liquid nitrogen should be performed with
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the required manipulation and the lack of the direct contact considerably slows down the warming rate and the dilution of the cryoprotectants. A third practical approach to increase the cooling and warming rates is the Cryoloop technique,51–54 a small loop formed from a thin nylon fiber. At vitrification, a thin solution film bridging the hole of the loop is formed, and the oocytes and embryos are loaded onto this film. Although the system seems to be very fragile and sensitive, it has been widely acknowledged and used in human embryology, and its application seems to be easy and practical. The film remains intact during immersion into liquid nitrogen, the solution volume is negligible, accordingly the cooling and warming rate may reach the estimated level of 700 000°C/min,55 and the storage may be performed in cryovials. According to the latest data, cooling may also be performed in the vapor of liquid nitrogen without compromising results.56 The disadvantages of this technique are that the tool is unusual, and the method seems to be technically difficult to perform, although those who have routinely tried it praise its simplicity and applicability. Apart from these approaches a fourth group of methods has also been developed stepwise during the past 15 years using a common principle, but entirely different tools. First described by Arav57 as minimum drop size (MDS) technique, a very small (< 0.5 or even 0.1 µL) droplet containing the sample is placed onto a solid surface and immersed into the liquid nitrogen. Depending on the tool onto which the drop is placed, different vitrification methods have been developed including the minimum volume cooling (MVC),58 the hemistraw system,59 or the latest and probably most practical approach, the Cryotop technique.60,61 Loading, cooling, warming, and dilution procedures are quite similar to those applied with the electron microscopic grids, but instead of touching a filter paper, the excess solution is removed with a capillary.
Furthermore, these newer tools were developed for the given purpose, accordingly their handling and storage is easier and requires less technical skill than the grid approach; therefore standardization and widespread practical application is possible. The achievable cooling and warming rates are very difficult to measure, however, according to our estimation they should be between those achievable with the OPS analogs and the Cryoloop, i.e. between 20 000 and 700 000°C/min. Apart from the small volume and/or direct contact approach, there are some other attempts to increase the cooling rates. A logical approach is the elimination of the vapor coat that arises around the sample in the liquid nitrogen at cooling, for example by using liquid nitrogen slush instead of liquid nitrogen for cooling (VitMaster),62–64 or to place small drops on pre-cooled metal surfaces instead of liquid nitrogen for cooling. Originally, a metal block immersed into liquid nitrogen was used,65 but eventually a commercially available technique has also been produced (CMV, Cryologic, Australia). The few comparative data do not provide entirely convincing evidence regarding the superiority of these vapor-minimizing or vapor-free approaches compared with the other vitrification procedures, although the use of metal surfaces instead of liquid nitrogen for cooling may decrease potential contamination problems (see below). When talking about advantages and drawbacks of vitrification, we also have to mention that the broad choice of solutions, tools, and techniques is actually a considerable disadvantage that hampers the widespread application of vitrification. A routine practitioner both in domestic animals and especially in the human field needs a highly standardized, commercially available technology, a kit with ready to use solutions and tools, and also a broad independent reference regarding the chances of successes and failures. In contrast to traditional freezing, very few (if any) of the current vitrification methods offer this
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in detail in a recent review,68 and here we summarize the arguments and facts to give an authentic picture about the real risks. The potential of infection in current reproductive techniques is inherent, as semen and embryo collection protocols are not sterile procedures.69 Consequently, the contents of virtually all stored straws and cryovials may be sources of infection when transferred to recipients. In case an infection occurs in the future, it may be difficult to localize the source as either infection before the cryopreservation or cross-contamination during storage. On the other hand, in embryology practice there are many other sources for contaminating liquid nitrogen. In the everyday work, the surface of straws, cryovials, racks, and other tools are not handled fully aseptically. According to our knowledge, the systematic and regular cleaning of containers and samples in liquid nitrogen containers is not part of routine in any embryology laboratory, and it seems to be technically very demanding, maybe even impossible. Moreover, seemingly sterile containers may not be as safe as supposed, infection may occur through incomplete sealing, pores of the plastic walls of most commonly used straws (except for some special ones produced to eliminate this problem). As the result, all storage tanks may contain a number of potentially pathogenic environmental microorganisms.69 Liquid nitrogen-mediated disease transmission in other areas of human medicine or food industry is a documented fact, although a very rare event.70–72 Also, the potential for transfer of infectious agents between open tools used for vitrification has been proven under experimental conditions.73 Accordingly, the theoretical danger of liquid nitrogen-mediated disease transfer exists, although it is not restricted to open vitrification methods, as most traditional tools and methods of cryopreservation (probably at a lower level) may also be vulnerable. However, it should be noted that the few published disease transmissions happened between blood specimens and carcasses,
option, the choices are abundant, but the availability in most cases is difficult. Kits (if they exist) are incomplete, and most of the available data originate from the (potentially commercially interested) creators of the method. Accordingly, this confusing situation discourages practitioners from entering this new field. Very recently, there have been signs of more professional handling of the matter by some commercial ventures: if this trend continues, it may be a decisive factor in the common acceptance of vitrification, to find its well deserved place among cryopreservation techniques in reproductive biology.
DECREASED CHILLING INJURY The other unquestionable benefit of vitrification is the possibility to decrease dramatically chilling injury. Compared with slow-rate freezing, the rate samples pass through the dangerous temperature zones at vitrification are extremely high, and the very short exposure to dangerous temperatures (15 to −5°C) radically decreases the injury of the sensitive structures. Accordingly, the only successful strategy that is currently available for cryopreservation of intact porcine embryos (containing extremely high amounts of chill-sensitive lipid droplets) is vitrification.66,67
THE POTENTIAL DANGER OF DISEASE TRANSMISSION According to our knowledge, no infection related to liquid nitrogen-mediated transmission of infective agents has been described after application of embryo technologies including all forms of cryopreservation, traditional slow freezing, or vitrification, although these techniques are applied in huge numbers worldwide in both humans and domestic and experimental animals. However, the most emphasized argument against the use of the new and efficient vitrification techniques is the potential risk of liquid nitrogen-mediated disease transmission. The subject was discussed
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between volumes approximately 103 to 104 larger than samples in embryology, as a result of huge leaks of wrapping or when no wrapping was present at all. The fact that infection following embryo transfer (with or without cryopreservation) is a rare event may also prove that the oviduct and uterus have an appropriate defense system to eliminate infectious agents in the quantities in which they may be transferred during routine embryo transfer. Both human and animal medicine uses methods that may result in infections in different ways. Our duty is to eliminate all possible sources of infections, but there are some rational limitations to this approach. For example, nobody states that the face masks of surgeons eliminate 100% of the expired bacteria and viruses; however, according to the routine experience the risk is minimal, and reasonable compromises are made, e.g. astronaut’s ‘scaphanders’ (spacesuits) are fortunately rarely used by surgeons performing routine surgeries. On the other hand, our duty is to use systems that are resistant even to theoretical hazards. In the past few years, a considerable effort has been performed in this direction, and as the result of the advancements many vitrification techniques now offer equally high or even a higher level of aseptic handling of samples than routine slow freezing procedures. One approach is to separate the cooling and warming phase from storage: to use a relatively low amount of clean liquid nitrogen for cooling, then to wrap the sample into a precooled sterile container and seal it hermetically before placing it into the common storage tanks. Liquid nitrogen that is low or free of infective agents can be obtained from some producers, or can also be obtained by UV irradiation,62 or by filtration through some 0.2 µm filters.74 Although the latter manipulation does not remove viral particles from the solution, the same is also applicable to most filter sterilized media. The probability that factory-derived, separately stored liquid nitrogen will contain pathogenic
viruses is extremely low. The effect of UV sterilization may depend on the actual situation (volume, thickness, intensity, wavelength, and duration of irradiation) and can only be regarded as reliable under standardized and controlled conditions. The choice of storage container depends on the vitrification tool. For the OPS analogs, as well as for the Cryotop and some other tools, the best solution is probably to use large (0.5 or 1 mL) plastic straws produced from material impermeable to any pathogenic agents.45,74–76 For safe loading, commercial kits are available including the VitSet produced by Minitube. The safety of the OPS-vitset technique has been recently confirmed by an independent investigation.77 Cryoloop is equipped with a special cryovial, and other tools including electron microscope grids can also be stored in common cryovials, but for safe application, producers’ suggestions should be strictly followed. The same is applicable for approaches where the cooling is performed on metal surfaces (SSF technique), or by dropping the sample into the liquid nitrogen. The application of a sterile container for storage of vitrified samples may offer another benefit. Compared with the samples frozen by using the slow rate method, or vitrified in 0.25 mL insemination straws, the small size of samples used in recent vitrification methods makes them extremely fragile to temporary warming including transfer of the sample from one container to other. The wrapping applied for sterility measures may also serve as a buffer to avoid accidental damage caused by such transitional warmings. Based on an earlier description,77 recently a new combination of OPS vitrification and double wrapping was applied by Isachenko et al.78 (discussed in detail in another chapter of this book). Very recently, an aseptic vitrification approach based on the hemi-straw principle has also been developed. This High Security Vitrification kit (HSV kit)79 makes it possible to place a microdroplet (< 0.5 µL) of
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cryoprotectant containing the embryos in the gutter of a capillary before inserting it in a mini-straw. It is heat-sealed using a special welder which ensures a leak-proof seal and is resistant to pressures of up to 150 kg/cm2
before immersion in the liquid nitrogen. Future experiments are required to prove if the achievable cooling rates in the two latter systems are appropriate for cryopreservation of chill-sensitive structures.
References 1. Aman RR, Parks JE. Effects of cooling and rewarming of the meiotic spindle and chromosomes of in vitro matured bovine oocytes. Biol Reprod 1994; 50: 103–10. 2. Leibo SP, Martino A, Kobayashi S, Pollard JW. Stage-dependent sensitivity of oocytes and embryos to low temperatures. Anim Reprod Sci 1996; 42: 45–53. 3. Martino A, Pollard JA, Leibo SP. Effect of chilling bovine oocytes on their developmental competence. Mol Reprod Dev 1996; 45: 503–12. 4. Zenzes MT, Bielecki R, Casper RF et al. Effects of chilling to 0°C on the morphology of meiotic spindles in human metaphase II oocytes. Fertility and Sterility 2001; 75: 769–77. 5. Nagashima H, Kashiwazaki N, Ashman RJ et al. Removal of cytoplasmic lipid enhances the tolerance of porcine embryos to chilling. Biol Reprod 1994; 51: 618–22. 6. Nagashima H, Cameron R, Kuwayama M. Survival of porcine delipated oocytes and embryos after cryopreservation by freezing or vitrification. J Reprod Dev 1999; 45: 167–76. 7. Dobrinsky JR, Nagashima H, Pursel VG et al. Cryopreservation of swine embryos with reduced lipid content. Theriogenology 1999; 51: 164. 8. Beebe LFS, Cameron RDA, Blackshaw AW et al. Piglets born from centrifuged and vitrified early and peri-hatching blastocysts. Theriogenology 2002; 57: 2155–65. 9. Esaki R, Ueda H, Kurome M et al. Cryopreservation of porcine embryos derived from in vitro-matured oocytes. Biol Reprod 2004; 71: 432–7. 10. Du Y, Kragh PM, Zhang X et al. Sucessful vitrification of parthenogenetic porcine blastocysts produced from delipated in vitro matured oocytes. Reprod Fertil Dev 2006; 18: 153. 11. Men H, Agca Y Riley LK, Critser JK Improved survival of vitrified porcine
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38. Cho HJ, Son WY, Yoon SH et al. An improved protocol for dilution of cryoprotectants from vitrified human blastocysts. Hum Reprod 2002; 17: 2419–22. 39. Son WY, Lee SY, Chang MJ et al. Pregnancy resulting from transfer of repeat vitrified blastocysts produced by in vitro matured oocytes in patient with polycystic ovary syndrome. Reprod BioMed Online 10, 398–401. 40. Vajta G, Holm P, Kuwayama M, Booth PJ et al. Open Pulled Straw (OPS) vitrification: a new way to reduce cryoinjuries of bouine ova and embryos. Mol Reprod Dev 1998; 51(1) 53–8. 41. Kong IK, Lee SI, Cho SG et al. Comparison of open pulled straw (OPS) vs glass micropipette (GMP) vitrification in mouse blastocysts. Theriogenology 2000; 53: 1817–26. 42. Isachenko V, Alabart JL, Vajta G et al. Double cryopreservation of rat embryos at different developmental stages with identical vitrification protocol: the not properly understood phenomenon. In: Abstracts of the Winter Meeting of Society for the Study of Fertility. Utrecht, Holland. J Reprod Fertility 2000; 26 (Abstract Series): 10. 43. Tominaga K, Hamada Y. Gel-loading tips as container for vitrification of in vitro-produced bovine embryos. J Reprod Dev 2001; 47: 259–65. 44. Kuleshova LL, Lopata A. Vitrification can be more favorable than slow cooling. Fertil Steril 2002; 78: 449–54. 45. Liebermann J, Tucker MJ, Graham JR et al. Blastocyst development after vitrification of multipronuclear zygotes using the Flexipet denuding pipette. Reprod BioMed Online 2002; 4: 146–50. 46. Cremades N, Sousa M, Silva J. Experimental vitrification of human compacted morulae and early blastocysts using fine diameter plastic micropipettes. Hum Reprod 2004; 19: 300–5. 47. Hredzak R, Ostro A, Zdilova V. Clinical experience with a modified method of human embryo vitrification Ceska Gynekologica 2005; 70: 99–103. [in Slovakian] 48. Chen SU, Lien YR, Cheng YY et al. Vitrification of mouse oocytes using closed pulled straws (CPS) achieves a high survival and preserves good patterns of meiotic spindles, compared with conventional straws, open pulled straws (OPS) and grids. Hum Reprod 2001; 11: 2350–56. 49. Lopez-Bejar M, Lopez-Gatius F. Nonequilibrium cryopreservation of rabbit embryos using a modified (sealed) open
22. Willadsen SM, Polge C, Rowson LE et al. Deep freezing of sheep embryos. J Reprod Fertil 1976; 46: 151–54. 23. Trounson A, Mohr L. Human pregnancy following cryopreservation, thawing, and transfer of an eight-cell embryo. Nature 1983; 305: 707–9. 24. Zeilmaker G, Alberda A, van Gent I et al. Two pregnancies following transfer of intact frozen-thawed embryos. Fertility and Sterility 1984; 42: 293–96. 25. Rall WF. Factors affecting the survival of mouse embryos cryopreserved by vitrification. Cryobiology 1987; 24: 387–402. 26. Vanderzwalmen P, Touati K, Ectors FJ et al. Vitrification of bovine blastocysts. Theriogenology 1989; 31: 270. 27. Saha S, Takagi M, Boediono A et al. Direct rehydration of in vitro fertilised bovine embryos after vitrification. Vet Rec 1994; 134: 276–77. 28. Széll A, Shelton NJ. Survival of vitrified sheep embryos in vitro and in vivo. Theriogenology 1994; 42: 881–9. 29. Palasz AT, Mapletoft RJ. Cryopreservation of mammalian embryos and oocytes: recent advances. Biotechnol Adv 1996; 14: 127–49. 30. Landa V, Tepla O. Cryopreservation of mouse 8-cell embryos in microdrops. Folia Biologica (Praha) 1990; 36: 153–8. 31. Riha J, Landa V, Kneissl J et al. Vitrification of cattle embryos by direct dropping into liquid nitrogen and embryo survival after nonsurgical transfer. Zivoc Viroba 1994; 36: 113–20. 32. Yang BS, Leibo SP. Viability of in vitroderived bovine zygotes cryopreserved in microdrops. Theriogenology 1999; 51: 178. 33. Papis K, Shimizu M, Izaike Y. The effect of gentle pre-equilibration on survival and development rates of bovine in vitro matured oocytes vitrified in droplets. Theriogenology 1999; 51: 173. 34. Papis K, Shimizu M, Izaike Y. Factors affecting the survivability of bovine oocytes vitrified in droplets. Theriogenology 2000; 54: 651–58. 35. Steponkus PL, Myers SP, Lynch DV et al. Cryopreservation of Drosophila melanogaster embryos. Nature 1990; 10: 345, 170–2. 36. Martino A, Songsasen N, Leibo SP. Development into blastocysts of bovine oocytes cryopreserved by ultra-rapid cooling. Biol Reprod 1996; 54: 1059–69. 37. Choi DH, Chung HM, Lim JM et al. Pregnancy and delivery of healthy infants developed from vitrified blastocysts in an IVF-ET program. Fertil and Steril 2000; 74: 838–39.
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63. Arav A, Yavin S, Zeron Y et al. New trends in gamete’s cryopreservation. Mol Cell Endocrinol 2002; 187: 77–81. 64. Huang et al., Successful pregnancy following blastocyst cryopreservation using super-cooling ultra-rapid vitrification. Hum Reprod 2005; 20(1): 122–8. 65. Dinnyes A, Dai Y, Jiang S et al. High developmental rates of vitrified bovine oocytes following parthenogenetic activation, in vitro fertilization, and somatic cell nuclear transfer. Biol of Reprod 2000; 63: 513–8. 66. Berthelot F, Martinat-Botté F, Perreau C et al. Birth of piglets after OPS vitrification and transfer of compacted morula stage embryos with intact zona pellucida. Reprod Nutr Dev 2001; 41: 267–72. 67. Cuello C, Gil MA, Parrila I et al. In vitro development following one-step dilution of OPS vitrified porcine blastocysts. Theriogenology 2004; 62: 1144–52. 68. Vajta G and Nagy ZP. Are programmable freezers still needed in the embryo laboratory? Review on vitrification. Reprod Biomed Online 2006; 12(6): 779–96. 69. Bielanski A, Bergeron H, Lau PCK et al. Microbial contamination of embryos and semen during long-term banking in liquid nitrogen. Cryobiology 2003; 46: 146–52. 70. Tedder RS, Zuckerman MA, Goldstone AH, et al. Hepatitis-B transmission from contaminated cryopreservation tank. Lancet 1995; 346: 137–40. 71. Fountain DM, Ralston M, Higgins N et al. Liquid nitrogen freezers: a potential source of microbial contamination of hematopoietic stem cell components. Transfusion 1997; 37: 585–91. 72. Berry ED, Dorsa WJ, Siragusa GR et al. Bacterial cross-contamination of meat during liquid nitrogen immersion freezing. J Food Prot 1998; 61: 1103–8. 73. Bielanski A, Nadin-Davis S, Sapp T et al. Viral contamination of embryos cryopreserved in liquid nitrogen. Cryobiology 2000; 40: 110–16. 74. Vajta G, Lewis IM, Kuwayama M, Greve T, Callesen H. Sterile application of the Open Pulled Straw (OPS) vitrification method. Cryo Letters 1998b; 19: 389–392. 75. Jelinkova L, Selman HA, Arav A et al. Twin pregnancy after vitrification of 2-pronuclei human embryos. Fertil and Steril 2002; 77: 412–14. 76. Vanderzwalmen P, Bertin G, Debauche C et al. Vitrification of human blastocysts with the hemi-straw carrier: application of
pulled straw procedure. Theriogenology 2002; 58: 1541–52. Kuwayama M, Vajta G, Ieda S, Kato O. Vitrification of human embryos using the CryoTip™ method. Reprod BioMed Online 2005; 11: 608–14. Lane M, Forest KT, Lyons EA et al. Live births following vitrification of hamster embryos using a novel containerless technique. Theriogenology 1999; 51: 167. Lane M, Schoolcraft WB, Gardner DK. Vitrification of mouse and human blastocysts using a novel cryoloop container-less technique. Fertil Steril 1999; 72: 1073–78. Mukaida T, Nakamura S, Tomiyama T et al. Successful birth after transfer of vitrified human blastocysts with use of a cryoloop containerless technique. Fertil Steril 2001; 76: 618–20. Mukaida T, Takahashi K, Kasai M. Blastocyst cryopreservation: ultrarapid vitrification using cryoloop technique. Reprod BioMed Online 2003; 6: 221–25. Isachenko E, Isachenko V, Katkov II et al. Vitrification of mammalian spermatozoa in the absence of cryoprotectants: from past partial difficulties to present success. Reprod BioMed Online 2003; 6: 191–200. Larman MG, Sheenan CB, Gardner D. Vitrification of mouse pronuclear oocytes with no direct liquid nitrogen contact. Reprod Biomed Online 2006; 12: 66–69. Arav A. Vitrification of oocytes and embryos. In: Lauria A, Gandolfi F, eds. New Trends in Embryo Transfer. Cambridge, UK: Portland Press, 1992: 255–64. Hamawaki A, Kuwayama M, Hamano S. Minimum volume cooling method for bovine blastocyst vitrification. Theriogenology 1999; 51: 165. Vanderzwalmen P, Bertin G, Debauche V et al. In vitro survival of metaphase II oocytes (MII) and blastocysts after vitrification in an hemi-straw (HS) system. Fertil Steril 2000; 74: S215–16. Kuwayama M, Kato O. All-round vitrification method for human oocytes and embryos. J Assist Reprod Genet 2000; 17: 477. Kuwayama M, Vajta G, Kato O et al. Highly efficient vitrification method for cryopreservation of human oocytes. Reprod BioMed Online 2005; 11: 300–8. Arav A, Zeron Y, Ocheretny A. A new device and method for vitrification increases the cooling rate and allows successful cryopreservation of bovine oocytes. Theriogenology 2000; 53: 248.
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88. Peura TT, Hartwich KM, Hamilton HM et al. No differences in sheep somatic cell nuclear transfer outcomes using serum starved or actively growing donor granulosa cells. Reproduction, Fertility and Development 2003; 15, 157–165. 89. Fujihira T, Kishida R, Fukui Y. Developmental capacity of vitrified immature porcine oocytes following ICSI: effects of cytochalasin B and cryoprotectants. Cryobiology 2004; 49, 286–290. 90. Fujihira T, Nagai H, Fukui Y. Relationship between equilibration times and the presence of cumulus cells, and effect of Taxol treatment for vitrification of in vitro matured porcine oocytes. Cryobiology 2005; 51(3): 339–43. 91. Kobayashi S, Takei M, Kano M et al. Piglets produced by transfer of vitrified porcine embryos after stepwise dilution of cryoprotectants. Cryobiology 1998: 36: 20–31. 92. Men H, Agca Y, Critser E et al. Beneficial effects of serum supplementation during in vitro production of porcine embryos on their ability to survive cryopreservation by the open pulled straw vitrification. Theriogenology 2005; 64, 1340–1349. 93. Maclellan LJ, Carnevale EM, Coutinho da Silva MA et al. Pregnancies from vitrified equine oocytes collected from superstimulated and non-stimulated mares. Theriogenology 2002; 58: 911–919. 94. Piltty K, Lindeberg H, Aalto J et al. Live cubs born after transfer of OPS vitrified-warmed embryos in the farmed Europen polecat (Mustela putorius). Theriogenology 2004; 61: 811–820. 95. Crichton EG, Bedows E, Miller-Lindholm AK et al. Efficacy of porcine gonadotropins for repeated stimulation of ovarian activity for oocyte retrieval and in vitro embryo production and cryopreservation in Siberian tigers (Panthera tigris altaica). Biology of Reproduction 2003; 68: 105–113. 96. Iwayama H, Hochi S, Kato M et al. 2004 Effects of cryodevice type and donor’s sexual maturity on vitrification of minke whale (Balaenopter bonaerensis) oocytes at germinal vesicle stage. Zygote 12, 333–338.
assisted hatching after thawing. Hum Reprod 2003; 18: 1501–11. Bielanski A. Hanniman A.Non-crosscontamination of bovine embryos with microbes using the OPS vitrification system. Reprod Fertil Dev 2007 19 (1): 232–233. Isachenko V, Montag M, Isachenko E et al. Aseptic technology of vitrification of human pronuclear oocytes using open-pulled straws. Hum Reprod 2005; 20(2): 492–6. Camus A, Clairaz P, Ersham A et al. (The comparison of the process of five different vitrification devices) Gynecol Obstel Fertil 2006; 34(9): 737–45. French. Vieria AD, Mezzalira A, Barieri DP et al. Calves born after open pulled straw vitrification of immature bovine oocytes. Cryobiology 45, 91–94. Martino A, Songsasen N, Leibo SP. Development into blastocysts of bovine oocytes cryopreserved by ultra-rapid cooling. Biology of Reproduction 1996b; 54: 1059–1069. Vajta G, Kuwayama M, Holm P et al. Open pulled straw vitrification; a new way to reduce cryoinjuries of bovine ova and embryos. Molecular Reproduction and Development 1998a; 51: 53–58. Hou YP, Dai YP, Zhu SE et al. Bovine oocytes vitrified by the open pulled straw method and used for somatic cell cloning supported development to term. Theriogenology 2005; 64: 1381–91. Booth PJ, Vajta G, HØj A, Holm P, Jacobsen H, Greve T, Callesen H. Full-term development of nuclear transfer-calves produced from Open Pulled Straw (OPS) vitrified cytoplasts. Theriogenology 1999; 51: 413. French AJ, Hall VJ, Korfiatis NT et al. Viability of cloned bovine embryos following OPS vitrification. Theriogenology 2002; 57, 413. Tecirlioglu RT, French AJ, Lewis JM et al. Birth of a cloned calf derived from a vitrified cloned embryo. Reproduction, Fertility and Development 2004; 15: 361–366. French AJ, Lewis IM, Ruddock NT et al. Generation of aS1 casein gene transgenic calves by unclear transfer. Biology of Reproduction 2003; 68: 240.
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3
Jaffar Ali and James Shelton
the procedure. Temperature has a large effect on toxicity. Furthermore, it has been observed that crystallization of water to form ice can occur during the cooling as well as during the warming of embryos. It is clear that a cryoprotectant for use in vitrification of embryos must have the following characteristics:
INTRODUCTION The development of the science and technology of vitrification of embryos and oocytes is described elsewhere in this manual. The essential requirements include a high concentration of cryoprotectant, non-toxicity at this concentration, and a rapid rate of cooling. The obvious practical way to obtain a rapid rate of cooling is by plunging into liquid nitrogen and this has been adopted almost universally as the cooling method in vitrification. As researchers began to realize that vitrification may offer a simplified technique of embryo and oocyte cryopreservation they utilized the cryoprotectants that had proved successful for conventional preservation by slow cooling. It is clear now that the requirements of the cryoprotectant for successful vitrification of embryos are more demanding than they are for slow cooling. Practitioners of conventional cryopreservation, in the main, choose the cryoprotectant arbitrarily and mainly use glycerol and dimethylsulfoxide. The work of various groups1–5 indicated that the common cryoprotectants vary in the rate of permeation of embryos, and in toxicity6–8 to embryos. Rate of permeation is also dependent on temperature.3,9 There are also species differences in the rate of permeation of cryoprotectants.3–5 With the necessity to use high concentrations of cryoprotectants for vitrification it became obvious that toxicity was now an important consideration in the selection of cryoprotectants for this purpose. Thus, a cryoprotectant suitable for use in vitrification of embryos must vitrify at a concentration that is not toxic to embryos during the cooling process and at the temperature of
• It must enable vitrification when cooled by plunging into liquid nitrogen • It must not devitrify to form ice or fracture on cooling or warming • It must be non-toxic to embryos. Toxicity is temperature dependent; thus the selection of a cryoprotectant must be undertaken at the temperature at which embryos and media are held during the preparatory stages of cryopreservation. A scientific approach to the design of vitrification solutions (VSs) requires an understanding of the biophysical principles of cryopreservation. It should take into consideration the cytotoxicity of the cryoprotectant; the role or effect of temperature; the interactions between various intracellular biomolecules, organelles, cytoskeleton, and the membrane with the cryoprotectant; and other supplements to the cryoprotectant solution that promote vitrification and/or protect the cell against cryoinjury such as serum proteins, osmolytes (to maintain membrane integrity), and buffers (to maintain pH of the cryoprotectant solution). An appreciation of these factors and the physicochemical forces inside the cell during dehydration, cooling, warming, and rehydration will be particularly useful in designing efficacious VSs. These
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Table 3.1 Minimum concentrations of cryoprotectants in 0.25 mL straws that will vitrify when cooled by plunging into liquid nitrogen
interactions and the biophysical principles of cryopreservation are discussed elsewhere in this book. A review of the literature on the various VSs devised to cryopreserve embryos and oocytes revealed that most authors, with notable exceptions, did not indicate how they had arrived at a particular formulation. It is not clear whether such formulations are products of scientific enquiry. Capricious selection of cryoprotectants does not conform to the standard norms of scientific practice. A ‘quick-fix’ approach could have long-term negative implications because the chosen cryoprotectant solution may not be the ideal solution and it may be deficient in many respects. It could prove disastrous, especially in the healthcare industry, to utilize products that have been developed without thorough investigation. The objective of this chapter is to describe ways of assessing the effects of cryoprotectant properties and vitrification methodology on the formulation of VSs.
Vitrification concentration Cryoprotectant
Molarity (mol/L)
Percentage
Butylene glycol Propylene glycol Dimethyl sulfoxide Glycerol Ethylene glycol Methanol
3.0 4.0 5.0 5.0 6.5 Crystallizes at high concentrations
27.04 30.44 39.07 46.05 40.35 Crystallizes at 99.8%
Much work remains to be done investigating ternary, quaternary, and higher combinations of cryoprotectant solutions that may prove more efficacious and less toxic than the currently used binary cryoprotectant solutions. High concentrations of cryoprotectants are needed to achieve vitrification by plunging into liquid nitrogen (Table 3.1). Because such concentrations can be toxic (Table 3.2), mixtures of cryoprotectants have been used with a view to reducing toxicity of the cryoprotectant solution. The first requirement in testing cryoprotectants is to determine the concentration at which they will vitrify. Plunging into liquid nitrogen has been adopted as the method of cooling because of the simplicity of this method and the ready availability of liquid nitrogen. There have been some experiments12–18 in which lower temperatures have been used to reduce cryoprotectant chemotoxicity. Single cryoprotectants or mixtures of cryoprotectants may be tested for their ability to vitrify on plunging and to remain free of ice on both cooling and warming as shown in Tables 3.1 and 3.3. Table 3.1 shows the minimum concentration of cryoprotectants that will vitrify under the conditions employed by Ali and Shelton7 (loaded into 0.25 mL straws and plunged into liquid nitrogen).
CRYOPROTECTANTS Types of cryoprotectants Cryoprotectants can be divided into two main types: permeating and non-permeating. Both types have been used in VSs. The cryoprotectants used to date in vitrification are those that have been previously used in conventional freezing of embryos. When used at an adequate concentration many will enable vitrification without formation of ice crystals. There are various types of VSs. In the field of assisted reproductive technology (ART) the common VSs are usually binary or consist of two main cryoprotectants. The purpose of having more than one cryoprotectant in a VS is to lower the overall molarity at which vitrification occurs. Lowering the molarity is a strategy to reduce the toxicity of the vitrification solution.10,11 Other less common and less investigated VSs consist of three (ternary), four (quaternary), or possibly more cryoprotectants.
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Table 3.2 The durations of exposure (5, 10, or 20 min) at which molar concentrations of cryoprotectant are toxic to day-4 mouse embryos Molar concentration 2.0 3.0 4.0 5.0 6.0 7.0 8.0
Cryoprotectant EG
> 20 > 20 5 5
Gly
DMSO
PG
20 20
> 20 > 20 20 5 5
> 20 10 5 5
10 5
Meth
BG 5 5
> 20 > 20 > 20 10
EG, ethylene glycol; Gly, glycerol; DMSO, dimethyl sulfoxide; PG, propylene glycol; Meth, methanol; BG, butylene glycol.
Table 3.3 Appearance of straws containing different concentrations of ethylene glycol and glycerol after direct plunging into liquid nitrogen (C) and subsequent warming by plunging into 25° C waterbath (W) Glycerol (molar concentration) 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0
Ethylene glycol (molar concentration) Mode
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
C W C W C W C W C W C W C W C W C W
M M Mi M Mi M Mi M T M T M T M T Ti T Ti
Mi M i M i M T M T M T M T i T Ti TK T
I M i M T M T M T M T Mi T Ti T Ti T Ti
Ti M i M T M T M T M T T T Ti T Ti T Ti
Ti M T M T M T Mi TK Ti T T T Ti T Ti TK Ti
T M T M T M T Ti T TK T TK TK Ti TK T TK Ti
T M T M T Ti T T T TK T TK TK Ti T Ti T Ti
TK M T i T T T TK T TK TK TK T Ti TK Ti TK T
T Ti T T T T T TK TK TK T TK TK Ti TK Ti T T
TK Ti T TK T TK T T TK TK T TK TK Ti TK Ti T Ti
M, milky; T, transparent; i, intermediate; K, fractured.
butylene glycol, dimethylsulfoxide, ethylene glycol, glycerol, methanol, and propylene glycol, as well as DL-alanine, dextran (mw 70 000), Ficoll (mw 70 000), polyethylene glycol (mw 80 000), and polyvinylpyrrolidone (mw 10 000). The authors used concentrations of permeating cryoprotectants ranging from 0.5 mol/L to 6.0 mol/L except when solubility
GLASS FORMING CAPABILITIES OF CRYOPROTECTANTS AND THEIR MIXTURES Ali and Shelton7,19 tested over 3000 combinations of cryoprotectants for the abovementioned characteristics. The cryoprotectants tested included permeating compounds:
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limited the concentration that could be used. The high molecular weight polymers, Ficoll, dextrose, and polyvinylpyrrolidone were used at concentrations of 5–20%. Polyethylene glycol was used at a maximum concentration of 10% as higher concentrations were found to be extremely toxic to embryos. The authors loaded the cryoprotectant solutions to be tested into 0.25 mL plastic straws by suction with a syringe at room temperature (25oC). The straws were heat-sealed at both ends then plunged into liquid nitrogen. The straw is suitable for this procedure as vitrification can be easily distinguished from crystallization (ice formation) within the straw. With vitrification on plunging the contents of the straw are clear and transparent, whereas they are white and opaque (milky) when ice formation occurs. With some solutions the glass (vitrified solution) will fracture during vitrification. This produces a crackling noise within the straw and is easily detected. Both ice formation and fracturing of the vitrified medium can be lethal to embryos contained therein. Ice formation and fracture of the glass can occur on cooling and warming of the VS. It has been found that it is more difficult to avoid ice formation on warming than on cooling. Very small crystals that are not easily visible and are not harmful may form on cooling and these can act as nuclei for the formation of larger crystals during the warming process. Fractures formed during cooling may be readily visible but those formed during warming often are not visible to the naked eye. However, small bubbles often form at the fracture site and with crackling sounds are indicative of fracture formation. Solutions of high concentrations of cryoprotectant appear to be more brittle and tend to fracture. Unfortunately good glass formers have a greater tendency to fracture than poor glass formers. Kroener and Luyet20 demonstrated the factors responsible for the formation of fractures during vitrification procedures. Fractures may be an indication of excess
solute concentration. A slight reduction in solute concentration may reduce fracture formation without compromising the vitrification properties of the solution. The capacity to warm without devitrifying is tested by plunging the straws of vitrified solution into a water bath. Ali and Shelton7,19 chose to use a water bath at 25oC for warming of straws as this is similar to the room and field temperatures in most countries and thus obviates the need for an incubator and facilitates work under field conditions. During warming, solutions that do not devitrify are transformed from the clear glass state to the liquid state without evidence of a milky appearance. Results of these tests for vitrification and devitrification revealed the behavior of individual cryoprotectants and a number of combinations of cryoprotectants. Several combinations of cryoprotectants provided satisfactory vitrification without crystallization or fracturing during cooling or warming. Table 3.3 is adapted from Ali and Shelton7 and shows results from one of the many combinations of cryoprotectants tested. They tested ethylene glycol and glycerol combinations up to a combined concentration of 12 mol/L. For each concentration of cryoprotectants the table shows the appearance of straws during cooling (C) by plunging into liquid nitrogen and warming (W) by plunging the cooled straw into water at 25oC. From these and similar observations with other cryoprotectants and combinations thereof Ali and Shelton7 selected solutions for further testing. For example, it is obvious in Table 3.3 that 3.0 mol/L or 3.5 mol/L ethylene glycol with 4.5 mol/L glycerol is a solution that vitrifies on cooling and does not crystallize on warming. It is evident also that whilst vitrification is achieved with higher concentrations of cryoprotectant, cracking of the glass also becomes a common occurrence. Another observation was that brittleness and fracture formation appear to be more pronounced
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in solutions containing butylene glycol, propylene glycol, and dimethylsulfoxide, and to a lesser degree in those containing glycerol and methanol. While the first three were shown to be good glass formers, vitrified solutions of these compounds are prone to brittleness and fracture readily during cooling or warming. Ethylene glycol proved to be the cryoprotectant least prone to fracture. The presence of high molecular weight polymers, Ficoll, dextrose, polyethylene glycol, and polyvinylpyrrolidone did not significantly reduce the vitrification concentration of solutions.
warming. For instance the cooling rate for a 0.25 mL straw when plunged directly into liquid nitrogen from room temperature is about 2500oC/min.13 The warming rate for a 0.25 mL straw from − 196oC to 25oC is about 1000oC/min.21 If the volume is reduced to about 1 µL it will be possible to increase the cooling rate to about 15 000–30 000oC/min.22–24 Higher cooling rates will allow VSs of lower solute concentration to vitrify during cooling and warming. Also, the cytotoxicity of VSs with lower solute concentrations will be lower. The same rule applies to warming rates. Higher warming rates will prevent the formation and growth of ice nuclei during warming. A warming rate in the order of 4460oC/min (when the specimen is warmed from −196oC to 37oC) used by some workers has resulted in acceptable survival of embryos.
Rate of cooling and warming The rate of cooling is dependent on the volume of liquid to be cooled and also its surface area. Therefore it is important that VSs be tested in the container and at the same volume that will be used for embryo vitrification. Whilst the results of testing in 0.25 mL straws are a very good indicator of the capacity of cryoprotectant solutions to vitrify and subsequently warm without ice formation or cracking, and thus are potentially useful for vitrification of embryos and oocytes, it is important to be aware that many variables may affect the behavior of any putative VS. In addition to concentration of cryoprotectant, factors that influence vitrification include cooling rate, warming rate, volume of solution, and shape of container. Ali19 compared direct plunging into liquid nitrogen with plunging after pre-cooling in a boat on the surface of the nitrogen for 2 minutes. The latter method required the concentration of the solution to be increased by at least 0.5 mol/L to allow vitrification. Furthermore, the straw vitrified by direct plunging did not devitrify on warming, whereas the straw vitrified by indirect plunging devitrified on warming. It is also clear that volume of solution and the shape of the container have a large influence on rate of cooling and
Choice of carriers The experiments of Ali and Shelton7,25,26 used 0.25 mL straws as the container in which vitrification was tested. Recently a number of novel containers have been trialed for vitrification of embryos.22,23,27–33 Because of the influence of volume, shape, and surface area on cooling and warming rates it is important that any new cryoprotectant solution be assessed in the container intended for use in embryo vitrification. The smaller the container or carrier, the lower the solute concentration required to vitrify. For instance, a concentration of solution that barely allows vitrification when loaded in a 0.25 mL straw, devitrifies when loaded into a 0.5 mL straw. This is probably due to the creation of unequal thermal gradients at the surface of the vehicle that is in direct contact with the cooling or warming agent and the core of the carrier. The cooling or warming rates at the surface of the carriers are faster than at the core. This imbalance induces ice nucleation in the VS and subsequently ice crystallization. Crystallization could prove
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hazardous for the viability of cells. The direct implication of this observation is that it may be possible to vitrify embryos and gametes with VSs of significantly lower solute concentration if carriers that are capable of carrying very low volumes, in the order of microliters, of cryoprotectants can be devised. The influence of volume and shape on the incidence of fracture formation is unclear but it is likely that smaller volumes are less prone to fracture because of smaller temperature gradients within the solution. The use of VSs of significantly reduced solute concentrations is advantageous because such solutions may be non-toxic or of insignificant toxicity to embryos and gametes. The use of various carriers that allow the use of low or ultralow volumes of VSs for vitrification, such as pulled straw, electron microscope copper grids, etc. are discussed elsewhere in this book. It must, however, be borne in mind that some microscopic carriers may not afford protection against cross-infection by dangerous pathogens during storage because these carriers are not sealed and may expose the cryopreserved specimen to the liquid nitrogen in storage tanks. Various strategies to prevent cross-contamination during storage are also discussed elsewhere in this book.
contents vitrified during cooling and remained ice-free during warming. The cell will dehydrate immediately after exposure to the high osmolar cryoprotectant solution with the rapid passage of water out of and shrinkage of the cell. The reduction in volume of the embryo immediately after exposure to the high osmolar solution could be as high as 85–88%. Water leaves the cell at about 5000 times faster than the macromolecules and other solutes present in the cytoplasm9 and as a consequence, the intracellular solute concentration will increase many times, often sufficient to promote intracellular vitrification should cooling occur. This high solute concentration can, however, be toxic. In the mouse the increase in intracellular solute concentration after exposure to the VS appears to allow most preimplantation stages to vitrify and survive the ultrarapid cooling and warming. Some higher preimplantation stages of the mouse embryo can be cryopreserved by vitrification without prior partial equilibration with the cryoprotectant. This is not the case for embryos of most higher mammals which seem to need prior partial equilibration. It is evident from the preceding paragraphs that high concentrations of cryoprotectant are required for vitrification at the cooling rate that can be achieved by plunging into liquid nitrogen. Obviously the VS used for embryo vitrification must be non-toxic to embryos at the temperature and exposure time employed throughout the whole procedure. In the testing of cryoprotectants for capacity to vitrify, many combinations were tested as it is commonly believed that the high concentration attained by combination of cryoprotectants may be less toxic than a single cryoprotectant at the same concentration. Ali and Shelton7 conducted a number of trials to determine the toxicity of individual cryoprotectants and combinations thereof. In their initial tests they used day-4 Swiss outbred mouse embryos to screen for toxicity. Extracellular ice formation or devitrification during cooling or warming should
Toxicity of cryoprotectants and their mixtures Most cryoprotectants are cytotoxic. A thorough investigation with various preimplantation stages of the mouse embryo will provide some basic information on the cytotoxicity of individual cryoprotectants and mixtures of cryoprotectant.25 Cytotoxicity is dependent on the operating temperature. Based on this information, the most promising VSs can be selected for further investigation and if necessary further refinements can be made. The study can then be extended to embryos of higher mammals and, finally, to human embryos and possibly gametes. Viability of embryos is dependent on whether intracellular
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3.1 and 3.2 it is apparent that to achieve vitrification with the commonly used cryoprotectants it is necessary to use concentrations which at best (e.g. ethylene glycol) approach a level that is toxic to mouse morulae. From examination of the data on vitrification of mixtures of cryoprotectants it is possible to select mixtures that will vitrify on cooling and not devitrify on warming. Thus, Ali and Shelton7 selected for toxicity testing 13 mixtures that had proven to be good vitrifiers. These selections took into consideration the individual cryoprotectant’s toxicity and the mixture’s capacity to vitrify. The data previously generated suggested that the molar concentration required to vitrify and to be of low toxicity might be achieved by using ethylene glycol (low toxicity) to achieve the necessary molar concentration in combination with glycerol, dimethylsulfoxide, or propylene glycol that are good vitrifiers but of greater toxicity. Butylene glycol, a good vitrifier, could be used only at 1.0 mol/L concentration in mixtures because of toxicity at higher concentrations (Table 3.4).
theoretically not harm the specimen. However, should the growing ice nuclei continue to grow rapidly in size, the potential to damage the cryopreserved cell is high. For this reason VSs that do not devitrify during cooling and warming are selected for further investigation. The temperature and duration of exposure are variables which influence the toxic effects of cryoprotectants on embryos. For each molar concentration of cryoprotectant 60–78 morulae in 3–6 replicates were exposed for 5, 10, or 20 min at 25oC. Following exposure for the specified duration, the morulae were exposed to 1.0 mol/L sucrose for 10 min at 25oC to dilute the cryoprotectant in the morulae. The morulae were then placed in Hepes buffered Whitten’s medium34 for 5 min at the same temperature after which they were cultured overnight in Whitten’s medium in an atmosphere of 5% CO2 in air at 37oC. The following day the number of embryos that had developed to the blastocyst stage was recorded. Table 3.2 presents a summary of the tests for toxicity of six cryoprotectants for day-4 mouse morulae. It is clear that ethylene glycol is the least toxic of the cryoprotectants tested. The order of toxicity from least to most toxic was shown to be ethylene glycol, methanol, dimethyl sulfoxide, glycerol, propylene glycol, and butylene glycol. In the previous section it was shown that butylene glycol is a good vitrifier (see Table 3.1), but its high toxicity to embryos renders it unsuitable for embryo cryopreservation. It must be noted that the butylene glycol studied by Ali and Shelton7 was a racemic mixture of isomers of butylene glycol, and the results may not apply to each of the isomers. On the other hand, methanol is of relatively low toxicity but does not vitrify even at very high concentrations. Glycerol and dimethylsulfoxide, both commonly used cryoprotectants, were quite toxic to mouse morulae after exposure for 5 min. Propylene glycol, also commonly used as a cryoprotectant showed toxicity to morulae after 10 min at a concentration of 3.0 mol/L. On comparing Tables
Table 3.4 Composition of vitrification solutions designated VS1 to VS13 Mixture
Composition
VS1 VS2
2.5 mol/L glycerol + 5.5 mol/L ethylene glycol 3.0 mol/L methanol + 6.0 mol/L ethylene glycol 1.5 mol/L propylene glycol + 6.0 mol/L ethylene glycol 2.0 mol/L propylene glycol + 5.5 mol/L ethylene glycol 3.5 mol/L glycerol + 4.5 mol/L ethylene glycol 3.0 mol/L glycerol + 3.0 mol/L propylene glycol 3.0 mol/L ethylene glycol + 4.0 mol/L dimethylsulfoxide 1.0 mol/L butylene glycol + 6.0 mol/L ethylene glycol 1.8 mol/L glycerol + 6.1 mol/L ethylene glycol 1.5 mol/L glycerol + 6.5 mol/L ethylene glycol 1.8 mol/L glycerol + 6.0 mol/L ethylene glycol 1.5 mol/L glycerol + 6.3 mol/L ethylene glycol 8.0 mol/L ethylene glycol
VS3 VS4 VS5 VS6 VS7 VS8 VS9 VS10 VS11 VS12 VS13
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100 VS14
Percentage survival
80
60
VS11
VS1 40
20
0 0
5
10
20
Duration of exposure (min) Figure 3.1 Percentage survival of mouse embryos exposed to VS1, VS11, and VS14 at 25oC for 5, 10, or 20 min. Adapted from Ali and Shelton.7
Another VS (VS14) was also tested. This consisted of 5.5 mol/L ethylene glycol and 1.0 mol/L sucrose. This formulation is similar to VS1 which consisted of 5.5 mol/L ethylene glycol and 2.5 mol/L glycerol.7 In VS14 2.5 mol/L glycerol was replaced with 1.0 mol/L sucrose. The rationale for this mixture was that whilst 5.5 mol/L ethylene glycol will not vitrify it is non-toxic. The addition of 1.0 mol/L sucrose may provide sufficient increase in solute concentration and intracellular dehydration to permit vitrification. Glycerol is a permeating cryoprotectant and was found to be more toxic than ethylene glycol7 (Table 3.2), whereas sucrose is nonpermeating and is therefore less likely to cause intracellular damage. Szell and Shelton35 had previously established that 1.0 mol/L sucrose is not toxic to embryos. The formulation of VS14 was a strategy to reduce the toxicity of the VS.
Day-4 mouse morulae were exposed for 5, 10, or 20 min to the 14 VSs formulated as above under the same conditions as used for the individual cryoprotectants. The data for three of the less toxic VSs (VS1, VS11, and VS14) are summarized in Figure 3.1 Duration of exposure to cryoprotectant can be expected to have an influence on toxicity to embryos.
Selection of vitrification solutions The solutions that vitrified and proved nontoxic or less toxic to mouse embryos can be further tested for their ability to allow the survival of embryos after vitrification, initially in vitro and then in vivo. VSs that support high rates of in vivo development can then be tested on embryos of higher mammals. From this study it is possible to select the most promising VS for routine application.
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Table 3.5
Percentage survival of mouse embryos exposed to or vitrified with VS1, VS11, or VS14 Stage of embryo development
VS
Treatment
SO blastocysts, early blastocysts and morulae
SO 8 cell
SO 4 and 2 cell
SO 1 cell
F1 1 cell
VS1
Control Exposed Vitrified
89.6 91.9 92.6
87.2 36.8 21.6
85.3 8.0 0
40.0 7.3 0
87.1 78.1 66.7
VS11
Control Exposed Vitrified
100 98.8 94.4
94.6 75.4 91.2
89.7 41.8 19.0
40.0 37.2 16.3
90.9 86.4 0
VS14
Control Exposed Vitrified
99.2 99.2 97.5
89.9 97.1 87.3
83.9 83.4 70.2
46.0 40.5 19.4
91.3 95.7 91.7
SO, Swiss outbred; F1, C57BL/6J × SJL/J females mated with SO males.
As an illustration, the previous experiments by the authors on vitrification and toxicity led to the selection of three solutions for testing in vitrification experiments with mouse embryos. These solutions were VS1 (5.5 mol/L ethylene glycol and 2.5 mol/L glycerol), VS11 (6.0 mol/L ethylene glycol and 1.8 mol/L glycerol), and VS14 (5.5 mol/L ethylene glycol and 1.0 mol/L sucrose). They were tested with all developmental stages of Swiss outbred embryos and 1-cell F1 embryos. In these experiments the embryos were subjected to one of two treatments: either exposure to the VS for the designated period of time or exposure followed by vitrification. Both groups of embryos were then cultured to determine survival rates. This approach enabled Ali and Shelton7 to distinguish between the effects of toxicity and the effects of vitrification on survival of embryos. In all cases the survival of exposed and vitrified embryos was compared with survival of untreated embryos of the same developmental stage. Table 3.5 summarizes the data. Vitrification with VS1 resulted in good survival of blastocysts, early blastocysts, and compacted morulae of outbred mice. Survival of 8-cell embryos was poor and there was no survival of 4-, 2-, and 1-cell embryos.
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However, 1-cell embryos of F1 mice showed good survival from vitrification with VS1. With VS11 survival from vitrification was good for blastocysts, early blastocysts, compacted morulae, and 8-cell embryos of outbred mice. Some of the 4-, 2-, and 1-cell embryos survived but at a rate significantly below that of control embryos. None of the 1-cell embryos of F1 mice survived vitrification with VS11. With VS14 survival of vitrified outbred embryos was the same as that of control embryos for all developmental stages except the 1-cell stage. However, there was good survival of 1-cell embryos of F1 mice. From the data it is clear that toxicity of the VS was the cause where survival of the vitrified embryos was reduced. In all but one group (1-cell outbred embryos) poor survival of vitrified embryos was accompanied by poor survival of embryos exposed to the VS but not vitrified. The data also illustrate that the choice of cryoprotectant is dependent not only on strain but also the developmental stage of the embryo. The results suggest that the glycerol content of the solution (VS1) may be responsible for the toxicity. When glycerol is replaced by 1.0 mol/L sucrose (VS14) survival of exposed
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affected by the concentration of cryoprotectant in the embryo and the rate at which the particular cryoprotectant permeates the cell membrane. Whilst Szell et al.5 demonstrated that ethylene glycol rapidly permeates cattle embryos, the concentration of ethylene glycol in VS11 is far greater than the 1.5 mol/L used by those workers. Therefore, it is important to investigate whether sucrose is helpful in removing VS11 or other vitrifying solution from embryos of different stages of development that have been exposed for specific durations. Ali and Shelton7 exposed mouse embryos to VS11 for 1, 5, 10, and 20 min and subsequently diluted the cryoprotectant from the embryos with or without 1.0 mol/L sucrose for 10 min. After this treatment embryos were cultured to the blastocyst stage to determine rates of survival. The data in Table 3.6 indicate that VS11 is well tolerated for short durations of exposure by all stages of mouse embryos except the 1- and 2-cell stages. Similar tests of VS11 toxicity on sheep embryos showed that compacted morulae are much more tolerant of exposure to VS11 than uncompacted morulae (Table 3.7). These data (Tables 3.6 and 3.7) demonstrate the effect of species and stage of development on response of embryos to exposure to VSs. Both these factors must be considered when selecting or testing VSs. To extrapolate directly between species or between stages of development of embryos is fraught with probabilities of disappointment.
and vitrified embryos is greatly enhanced. The beneficial effect of non-permeating sucrose also suggests that dehydration is an important contributor to conditions suitable for vitrification. Furthermore, the short duration of exposure to VS would not permit equilibration of the embryos before vitrification. Ali and Shelton7,19,25,36 showed that embryos are in a very shrunken state when vitrified after brief exposure to VSs. This observation leads to the suggestion that concentration of the solute within the embryonic cells is an important contributor to vitrification. Sucrose has been used extensively in dilution of cryoprotectant from embryos thawed after conventional cryopreservation. It may also be advantageous to use sucrose in dilution of cryoprotectant after thawing of vitrified embryos. Ali and Shelton7,19,25 compared dilution of VS11 from all stages of Swiss outbred embryos directly in medium with dilution in 1.0 mol/L sucrose for 10 min. Survival in vitro was not affected by the method of VS dilution.
The effects of species, stage of development, and dilution procedure on toxicity Testing on day-4 mouse morulae can be considered as a screening test to eliminate cryoprotectants and mixtures thereof that are toxic. The susceptibility of embryos to the toxic effects of cryoprotectants varies between species and between developmental stages of embryos. From these data VS can be selected for further testing with embryos of different stages and of different species. Ali and Shelton7 selected VS11 for further testing and proceeded to test its toxicity on a range of developmental stages of mouse embryos. The other important consideration is the method of dilution of the cryoprotectant from the embryos after warming. Non-permeating sucrose has been used successfully to remove cryoprotectant from embryos prior to their return to physiological medium. This can be
Preparation of vitrification solutions Cryoprotectant and VSs are normally prepared in phosphate buffered saline. When mixing cryoprotectant solutions, calculation of the amount (volume) of cryoprotectant to be added to the buffer solution should be based on the specific gravity of the cryoprotectant. The pH of the solution must be adjusted to about 7.25 prior to making up volume. This is crucial as the pH will drop during cooling and this could affect subsequent viability of the
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Table 3.6 sucrose
Survival of mouse embryos after exposure to VS11 and its subsequent removal with or without 1.0 mol/L Percentage of embryos surviving
Stage of development
Duration of exposure (min)
Removal without sucrose
Removal with sucrose
Blastocyst
Control 1 5
100.0 100.0 82.9
— 100.0 —
Early blastocyst
Control 2 5
100.0 100.0 93.0
— 100.0 —
Compacted morula
Control 3
94.7 93.0
— 94.6
8-cell
Control 1 5
100.0 97.5 87.5
— 100.0 77.1
2-cell
Control 1 5
93.8 0 0
— 25.0 0
1-cell
Control 1 5
41.9 39.4 0
— 27.3 6.1
Table 3.7 Survival of sheep compacted and uncompacted morulae after exposure to VS11 and its subsequent removal with or without out 1.0 mol/L sucrose Morulae surviving % Developmental stage Compacted morulae
Uncompacted morulae
Duration of exposure (min)
Without sucrose
With sucrose
Control
84.6
61.5
3 5
100.0 87.5
81.8 60.0
Control
—
100.0
3 5
— —
0 0
cooled cell. The final solution usually contains about 5% or more fetal calf serum depending on the type of VS. Other protein sources such
55
as bovine serum albumin, human maternal serum, etc, are also used. It may be difficult to dissolve high concentrations of solid chemicals such as sucrose in VSs. Mild warming in an incubator with gentle agitation may be useful in dissolving solid chemicals. Once prepared it may be pertinent to let the VS stand in the incubator in a closed container for about 2 hours to drive out the dissolved air. During cooling air goes rapidly into solution. The reverse is true during warming. Dissolved air could prove disastrous in ultrarapid cooling and warming procedures. During rapid warming air comes out of solution rapidly damaging the intracellular organelles of the cell irreversibly. We have observed that a further ‘curing’ of the VS in a refrigerator for 1 week or longer seems to enhance efficacy of the VS. The longer the VS is held the more efficacious it becomes. It is not clear how this happens and it remains to be explained. Ethylene glycol and sucrose are highly hygroscopic. It is therefore
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important to ensure storage containers of VS are airtight to prevent progressive reduction of the solute concentration as a consequence of hygroscopic dilution.
and 3.3) as seen with an image analyzer. If allowed to rest in its most stable position the shrunken embryo appeared approximately spherical but on closer examination was seen to resemble a hollow ball that had been partially evacuated. One side had become concave and the other remained convex or was slightly flattened. To calculate the approximate volume of the shrunken embryos Ali and Shelton7,19,25,36 used two solid geometry models. One was the difference between the volumes of two half spheres, and the other was the difference between the volumes of the segments of two spheres (Figure 3.2). They conducted these experiments with three sheep and three mouse embryos. Here we describe the experiment performed on sheep embryos. Embryos of both species behaved in a similar manner. The sheep embryos were observed at 0, 3, 5, 10, 15, and 20 min. At 3 min the embryos were 24.0%, 30.0%, and 15.7% of original volume. Figure 3.2 shows the responses of the individual embryos. Because of the irregularities in the solid geometry of individual embryos the calculated volumes can be considered as approximations only. In view of the estimate37 that 80–85% of the volume of ova consists of water, it is apparent that a high degree of dehydration was induced immediately after exposure to VS11 and there had been very little inflow of ethylene glycol. It is clear that, when plunged into liquid nitrogen after brief exposure to VS, embryos are in a shrunken state when they vitrify. The concentration of cryoprotectant within the embryos is unknown but in combination with the high concentration of cell solutes it is sufficient to permit vitrification without ice formation.
OSMOTIC EFFECTS OF VITRIFICATION SOLUTIONS ON EMBRYOS In the experiments described above and in published reports on vitrification of embryos of a number of species7,10–18,19,25,34 several cryoprotectant combinations have been used. A common component of these procedures is a short duration of exposure to the VS before plunging in liquid nitrogen. As Ali and Shelton7,19,25 explored a range of cryoprotectants they reduced the exposure time so as to minimize toxicity, and good survival of transferred vitrified/warmed embryos was obtained when the embryos were plunged into liquid nitrogen after exposure to the VS for only a few minutes. It is obvious that equilibration of VS concentrations intra- and extracellularly could not occur in this time. This is in contrast to the equilibration which is considered desirable for conventional cryopreservation of embryos. To explore the degree of cryoprotectant equilibration that occurs when embryos are subjected to vitrifying concentrations of cryoprotectants Ali and Shelton conducted a series of experiments to measure the volume of embryos exposed to VS11 (6.0 mol/L ethylene glycol and 1.8 mol/L glycerol) for varying durations.7,19,25,36 Immediately after the embryo was immersed in the VS, microscopic observations were commenced and were continually recorded by a video camera connected to a recorder and a monitor screen. Measurements for calculation of volume were made with the aid of an image analyzer. Study of the images showed that when subjected to the osmotic stress resulting from immersion in a high concentration of cryoprotectant, embryos shrank immediately and did not retain a spherical shape (Figures 3.2
SURVIVAL OF VITRIFIED EMBRYOS Survival of vitrified mouse embryos There appeared to be little difference between VS1, VS11, and VS14 when the endpoint was in vitro survival after vitrification and warming. The crucial test, however, is
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r3
a
h
r2
r1
Volume of segment
Volume of embryo Vol. of embryo (shaded) = Vol. of segment of a sphere (striped & shaded) − Vol. of unshaded (striped) segment
V = πh (3r22 + 3r32 + h2 ) = 0.52359878h (3r22 + 3r32 + h2 ) 6
b
100 90 Relative volume (%)
80 70 60
A3
50 A1
40 30
A4
20 10 0 0
3
5
10
15
20
Duration of exposure (min)
Figure 3.2 (a) Mathematical models for the calculation of changes in the volume of the compacted morula after exposure to VS. (b) Shows the volumetric changes of compacted day-6 sheep morulae calculated by the above mathematical model following exposure to VS11. A1, A3 and A4 are individual day 6 sheep compacted morulae. These three embryos were employed to determine volume changes in the embryo when challenged with the vitrification solution. Adapted from Ali36 with permission from the Middle East Fertil Soc J.
survival of vitrified and warmed embryos when transferred to surrogates (in vivo survival). VS1 and VS11 were used for vitrification of Swiss outbred mouse blastocysts, early blastocysts, and morulae. The vitrified embryos were then warmed, subjected to cryoprotectant dilution with or without sucrose and transferred to surrogates. It became clear
57
that VS11 is superior to VS1 and that sucrose dilution is beneficial. This was not apparent when the endpoint was in vitro survival. The in vivo survival rate of embryos that had been vitrified in VS11 did not differ from the survival rate of transferred untreated embryos. A further trial also showed that VS14 also gave excellent results. It may be concluded that
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VS11 and VS14 are highly suited to vitrification of mouse embryos.
Survival of vitrified sheep embryos It is important to know whether the results with mouse embryos can be extrapolated to other species. Ali and Shelton25 chose to examine the response of sheep embryos to the VSs found to be effective for mouse embryos. Day6 sheep embryos were used as this is the developmental stage most commonly used in sheep embryo transfer and cryopreservation. At day 6 sheep embryos are a mixture of blastocysts, early blastocysts, and morulae. Initially the vitrified and warmed embryos were cultured in vitro. None of the embryos vitrified with VS1 survived. There was no loss of viability in vitro of sheep early blastocysts and morulae vitrified with VS11 but viability of blastocysts was poor. The next step was to test the survival of vitrified and warmed embryos after transfer to surrogates. Only VS11 and VS14 were used in these tests of in vivo survival because VS1 had failed in vitro. The in vivo survival of embryos vitrified in VS11 was very poor. It seems that sheep embryos are more susceptible to the osmotic stress and/or solute effects incurred by immersion in VS11 than are mouse embryos. A means of reducing this stress may be to partially equilibrate the embryo with VS before immersing it in the VS. To examine this approach Ali and Shelton25 tested in vitro survival of sheep embryos exposed for 5 min to 20%, 30%, 40%, or 50% VS11 before exposure to 100% VS and vitrification. Subsequently 30% VS for 5 min was selected as the first step in a two-step procedure. VS11 was tested for in vivo survival of day-6 sheep embryos using one- and two-step procedures and including dilution of the cryoprotectant after warming with or without 1.0 mol/L sucrose. Embryos vitrified with VS14 in a one-step procedure followed by sucrose dilution on warming
a
b
c Figure 3.3 The following three images show a day-6 compacted sheep morula before and immediately after (~1–3 min) exposure to VS11. (a) Untreated fresh sheep day-6 compacted morula. (b) This image shows the same morula at its most stable position immediately after exposure to vitrification solution. It has assumed the appearance of an ‘ashtray’ due to severe dehydration after exposure to the vitrification solution. Instead of the ashtray appearance some morulae assumed the appearance of a deflated ball. The volumes of morulae that assume the appearance of a deflated ball can be calculated by the mathematical Model 1 while those that assume the ashtray appearance can be calculated using Model 2 given in Figure 3.2a. (c) This image shows the same morula at its least stable position immediately after exposure to VS11. Note how the zona pellucida and the morula have folded in due to intense dehydration after exposure to the VS. Adapted from Ali 36 with permission from the Middle East Fertil Soc J.
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Table 3.8
Viability of day-6 sheep embryos vitrified with VS11 or VS14 and transferred to surrogates
Vitrification procedure
Dilution procedure
Stage of development
One-step VS11
With sucrose
Morulae Early blastocysts Blastocysts Morulae Early blastocysts Blastocysts
Without sucrose
Percentage developed to live fetuses
Percentage of surrogates pregnant
7.9 2.4 0 13.3 0 0
15.8 4.4 0 26.7 0 0
Total one-step VS11 Two-step VS11
With sucrose
Morulae Early blastocysts Blastocysts Expanded blastocysts
(8/158) 5.1 55.2 10.0 62.1 50.0
(8/79) 10.1 78.6 20.0 78.6 50.0
Total two-step VS11 One-step VS14
With sucrose
Morulae Blastocysts Expanded blastocysts
(37/72) 51.4 50.0 100.0 0
(25/35) 71.4 100.0 100.0 0
(5/10) 50.0
(3/5) 60.0
Total one-step VS14
earlier had shown that 5.5 mol/L ethylene glycol is well tolerated by embryos, and Szell and Shelton35 have shown that 1.0 mol/L sucrose is not toxic to mouse embryos. It is interesting that neither 5.5 mol/L ethylene glycol nor 1.0 mol/L sucrose will vitrify on its own but clearly the combination will vitrify. The results with vitrification of sheep embryos suggest that embryos of some species might be more sensitive to cryoprotectants and may require partial equilibration with the cryoprotectant before cooling. It is apparent also that the necessity for this can differ between cryoprotectant solutions.
were also included in this experiment. It was considered that a two-step procedure was not necessary with VS14 as it had shown no evidence of toxicity when applied to the embryos in one step. The results indicate that VS14 can be used to vitrify day-6 sheep embryos using the onestep procedure with little loss of viability (Table 3.8). VS11 (6.0 mol/L ethylene glycol and 1.8 mol/L glycerol) was selected for vitrification experiments after intensive investigation of cryoprotectants and mixtures of cryoprotectants for capacity to vitrify and for toxicity to mouse and sheep embryos. Observations had suggested that in the ethylene glycol/glycerol combination toxicity was largely attributable to the glycerol. When the glycerol is replaced with 1.0 mol/L sucrose embryo viability on vitrification is improved particularly when the embryos are transferred to surrogates for development. Furthermore, this was achieved with a lower concentration (5.5 mol/L) of ethylene glycol. Tests described
Vitrification of oocytes, embryos, tissues, and cell lines of several species with ethylene glycol and sucrose Subsequent to the publication of the above results with VS147,25,26 numerous workers22,38–56 have used this combination of ethylene glycol
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and sucrose for successful vitrification of embryos and oocytes of several species, including human, with live births. Moreover, immature oocytes, ovarian tissue, and cell lines have also been cryopreserved with VS14.40,48,56 A minor modification27,57,58 (5.75 mol/L ethylene glycol + 0.6 mol/L sucrose) of VS14 (5.5 mol/L ethylene glycol +1.0 mol/L sucrose)7,25,26 has also been used to vitrify human zygotes and 8-cell embryos with resultant live births. VS14 has also been called 5.5EG.22
blastomeres were irreversibly damaged during the inferior procedure with the result that subsequent survival was affected in vivo but not in vitro. These data did not differentiate between trophoblastic and inner cell mass cells. It may be desirable also to count the number of the inner cell mass cells by differential staining technique60,61 which will indicate the viability of the embryo.
Development of vitrified embryos
Compounds other than those discussed here show promise as good vitrifiers. Butanediol has been shown to be a good vitrifier6 but is also quite toxic, at least in some of its isomeric forms. Chemical substitutions in conventional cryoprotectants may have an effect on critical cooling rate and critical warming rate necessary for avoidance of ice formation. Methoxylation of 35% propylene glycol and 45% glycerol reduced the critical cooling rate for vitrification from approximately 500oC to 50oC/min.62 The usefulness of such a methoxylated compound in embryo vitrification would depend on its toxicity to embryos. While the mechanisms of cryoprotectant toxicity may not be fully understood, it is clear that in the case of embryos they vary between species and between developmental stages of the embryos. Identification of compounds with superior vitrification performance might permit the use of slower cooling rates and/or lower concentration of vitrifying solution. The latter would be beneficial in reducing toxic effects of the cryoprotectant solution. The former might allow the use of solid carbon dioxide (dry ice) as the cooling agent in vitrification. It may be questionable whether this would be advantageous. The efficacy of some novel compounds in maintaining the integrity of the cytoskeleton of the egg and embryo53 during vitrification remains to be demonstrated conclusively. The investigation of novel cryoprotectants for their
NOVEL AND POTENTIAL CRYOPROTECTANTS
It is clear that merely demonstrating postwarming survival of vitrified embryos in culture does not warrant an assumption that equally good survival of embryos will follow transfer to surrogates. An examination of embryos cultured after vitrification and postwarming dilution procedures provides a possible clue to differences between embryos that are not evident on conventional examination but may influence survival when transferred to surrogates. Ali and Shelton19,26 cultured untreated and vitrified sheep embryos for 24–48 h and then counted the nuclei after fluorescent staining. The method of Pursel et al.59 can be used for fluorescent staining of the nuclei. The technique employs Hoescht 33342 stain with trypan blue as a counter stain. The fluorescing nuclei are counted on excitation with wavelengths between 351 and 364 nm. There was a strong trend for the treatment that provided the better in vivo survival to result in embryos that had a greater number of nuclei. Day-6 sheep early blastocysts cultured after vitrification by the one-step procedure had an average of 60 nuclei, whereas controls had 138 nuclei. Similar embryos cultured after vitrification by the two-step procedure had 184 nuclei compared with 147 for the control embryos (unpublished work). It may be concluded that a significant number of
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potential in cryopreservation of embryos by vitrification must follow sound experimental procedures taking into account the effect of species and stage of embryonic development as outlined above. They must also be assessed in combination with other cryoprotectants to arrive at VSs that result in optimal embryo survival. Researchers and clinicians must not neglect to study the possible genetic effects, on embryos and gametes of vitrification procedures. Whilst there have been some reports of genetic damage from freezing,63,64 as discussed elsewhere in this book, it is generally accepted that genetic anomalies subsequent to cryopreservation do not occur at detectable rates. Vitrification with VS14 does not appear to cause chromosomal damage to human oocytes, cleavage stage embryos, and immature oocytes.45,65,66 There is, however, some evidence of long-term effects of embryo freezing in mice.67 Possible subtle behavioral effects of cryopreservation must be investigated in greater detail. Studies of the genetic effects of embryo vitrification should
consider not only the effect of vitrification per se but also the effect of the cryoprotectants that are used in the VS.
ACKNOWLEDGMENTS The authors are grateful to The Australian National University, Canberra, Australia, for allowing the authors the use of their library facilities and to Dr Basim Abu-Rafea and Mrs Sandra A. Al-Abdulmunem of the King Fahad Medical City, Riyadh, Kingdom of Saudi Arabia, for their assistance in literature search. Jaffar Ali is grateful to the management of the King Fahad Medical City for allowing him to undertake this task. The authors gratefully acknowledge the assistance of Ms Genesis I Salao and Mr Khalid Al Houte of the Audio Visual Department of the King Fahad Medical City for their assistance in the preparation of the mathematical models and graphics given in Figure 3.2.
References 8. Emiliani S, Van den Bergh M, Vannin AS et al. Comparison of ethylene glycol, 1,2-propanediol, and glycerol for cryopreservation of slowcooled mouse zygotes, 4-cell embryos, and blastocysts. Hum Reprod 2000; 15: 905–10. 9. Jackowski S, Leibo SP, Mazur P. Glycerol permeabilities of fertilized and unfertilized mouse ova. J Exp Zool 1980; 212: 329–41. 10. Scheffen B, Van der Zwalmen P, Massip A. A simple and efficient procedure for preservation of mouse embryos by vitrification. Cryo Letters 1986; 7: 260–9 11. Massip A, Van der Zwalmen P, Scheffen B, et al. Pregnancies following transfer of cattle embryos preserved by vitrification. Cryo Letters 1986; 7: 270–3 12. Rall WF, Fahy GM. Ice-free cryopreservation of mouse embryos at −196 degrees C by vitrification. Nature 1985; 313: 573–5. 13. Rall WF. Factors affecting the survival of mouse embryos cryopreserved by vitrification. Cryobiology 1987; 24: 387–402.
1. Whittingham DG, Wales RG. Storage of twocell mouse embryos in vitro. Aust J Biol Sci 1969; 22: 1065–8. 2. Miyamoto H, Ishibashi T. The protective action of glycols against freezing damage of mouse and rat embryos. J Reprod Fertil 1978; 54: 427–32. 3. Schneider U, Mazur P, Leibo S. The permeability of bovine embryos to DMSO or glycerol. Cryobiology 1983; 20: 741 abstr. 4. Leibo SP. A one-step method for direct nonsurgical transfer of frozen-thawed bovine embryos. Theriogenology 1984; 21: 767–90. 5. Szell A, Shelton JN, Szell K. Osmotic characteristics of sheep and cattle embryos. Cryobiology 1989; 26: 297–301. 6. Bourton P. Levo- and dextro-2,3-butanediol and their racemic mixture: very efficient solutes for vitrification. Cryobiology 1990; 27: 55–9 7. Ali J, Shelton JN. Design of vitrification solutions for the cryopreservation of embryos. J Reprod Fertil 1993; 99: 471–7.
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28. Lane M, Gardner DK. Vitrification of mouse oocytes using a nylon loop. Mol Reprod Dev 2001; 58: 342–7. 29. Papis K, Shimizu M, Izaike Y. Factors affecting the survivability of bovine oocytes vitrified in droplets. Theriogenology 2000; 15: 651–8 30. Vanderzwalmen P, Bertin G, Debauche Ch., et al. “In Vitro” survival of metaphase ii oocytes (mii) and blastocysts after vitrification in a hemi-straw (hs) system. Fertil Steril 2000: 74: S215–S216 31. Matsumoto H, Jiang JY, Tanaka T, et al. Vitrification of large quantities of immature bovine oocytes using nylon mesh. Cryobiology 2001; 42: 139–44 32. Liebermann J, Tucker M, Graham J, et al. Blastocyst development after vitrification of multipronucleate zygotes using the flexipet denuding pipette (FDP). Reprod Biomed Online 2002; 4: 148–52 33. Momozawa K, Fukuda Y. Vitrification of bovine blastocysts on a membrane filter absorbing extracellular vitrification solution. J. Mammalian Ova Res 2006; 23: 63–6 34. Ali J, Whitten WK, Shelton JN. Effect of culture systems on mouse early embryo development. Hum Reprod 1993; 8: 1110–4. 35. Szell A and Shelton JN. Sucrose dilution of glycerol from mouse embryos frozen rapidly in liquid nitrogen vapour. J Reprod Fertil 1986; 76: 401–8. 36. Ali J. Intense dehydration during cryopreservation by vitrification of the mammalian embryo is essential for subsequent embryo survival and viability: a preliminary report. Middle East Fertil Soc J 2001; 6: 50–8 37. Leibo SP. Cryobiology: preservation of mammalian embryos. Basic Life Sci 1986; 37: 251–72. 38. Papis K, Avery H, Holm P, et al. The effect of vitrification solution, equilibration time, and direct dilution method on survivability of equilibrated or vitrified bovine in vitro matured oocytes. Theriogenology 1995; 43; 293 (abstr) 39. Ali J. Developmental competence of unipronuclear and triploid day-2 human embryos after vitrification with VS14. Med Sci Res 1996; 24: 377–8. 40. Ali J. Highly efficient ultrarapid cryopreservation of established cell lines by vitrification with VS14. Med Sci Res 1996; 24: 837–8. 41. Hong SW, Hyung MS, Chung HM, et al. (1999) Improved human oocyte development after vitrification: a comparison of thawing methods. Fertil Steril 1999; 72: 142–6. 42. Chen SU, Lien YR, Chen HF, et al. Open pulled straws for vitrification of mature mouse oocytes preserve patterns of meiotic spindles
14. Nakagata N. Survival of mouse morulae and blastocysts derived from in vitro fertilization after ultra rapid freezing. Jikken Dobutsu 1993; 42: 229–31 15. Shaw PW, Bernard AG, Fuller BJ, et al. Vitrification of mouse oocytes using short cryoprotectant exposure: effects of varying exposure times on survival. Mol Reprod Dev 1992; 33: 210–4. 16. Nakao K, Nakagata N, Katsuki M. Simple and efficient vitrification procedure for cryopreservation of mouse embryos. Exp Anim 1997; 46: 231–4 17. Nakao K , Nakagata N, Katsuki M. Production of chimeric mice from cryopreserved blastocysts. Exp Anim 1998; 47: 167–71 18. Anzai M, Nishiwaki M, Yanagi M, et al. Application of laser-assisted zona drilling to in vitro fertilization of cryopreserved mouse oocytes with spermatozoa from a subfertile transgenic mouse. J Reprod Dev 2006; 52: 601–6. 19. Ali J. Factors affecting the ultrarapid vitrification and cryopreservation of embryos. PhD Thesis Canberra: Australian National University, 1992. 20. Kroener C, Luyet B. Formation of cracks during the vitrification of glycerol solutions and disappearance of the cracks during rewarming. Biodynamica 1966; 10: 47–52. 21. Rall WF, Meyer TK, Leibo SP. Effect of warming conditions on the survival of mouse embryos cryopreserved and diluted by a one-step straw procedure. Theriogenology 1986; 25: 186 (abstr) 22. Martino A, Songsasen N, Leibo SP. Development into blastocysts of bovine oocytes cryopreserved by ultra-rapid cooling. Biol Reprod 1996; 54: 1059–69. 23. Vajta G, Booth PJ, Holm P, et al. Successful vitrification of early stage bovine in vitro produced embryos with the open pulled straw (OPS) method. Cryo Lett 1997; 18: 191–5 24. Arav A, Zeron Y. Vitrification of bovine oocytes using modified minimum droplet size technique (MDS) is affected by the composition and concentration of the vitrification solution and by the cooling conditions. Theriogenology 1997; 47: 341 (abstr) 25. Ali J, Shelton JN. Vitrification of preimplantation stages of mouse embryos. J Reprod Fertil 1993; 98: 459–65. 26. Ali J, Shelton JN. Successful vitrification of day-6 sheep embryos. J Reprod Fertil 1993; 99: 65–70. 27. Rama Raju GA, Haranath GB, Krishna KM, et al. Vitrification of human 8-cell embryos, a modified protocol for better pregnancy rates. Reprod Biomed Online 2005; 11: 434–7
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43.
44.
45.
46.
47.
48.
49.
50. 51.
52.
53.
54.
55. Hong S, Kim T, Lee S, et al. Cryopreserved blastocysts using vitrification protocol give excellent pregnancy and implantation rates after thawing. Fertil Steril 2005: 84: Suppl.1, pp. S178–S179 (abstr) 56. Martins RD, Costa EP, Chagas JSC, et al. Effects of vitrification of immature bovine oocytes on in vitro maturation. Anim Reprod 2005; 2: 128–34 57. El-Danasouri I, Selman HA. Successful pregnancies and deliveries after a simple vitrification protocol for day 3 human embryos. Fertil Steril 2001; 76: 400–2. 58. Selman HA, El-Danasouri I. Pregnancies derived from vitrified human zygotes. Fertil Steril 2002; 77: 422–3. 59. Pursel VG, Wall RJ, Rexroad CE Jr, et al. A rapid whole-mount staining procedure for nuclei of mammalian embryos. Theriogenology 1985; 24: 687–91. 60. Papaionnou VE, Ebert KM. The preimplantation pig embryo: cell number and allocation to trophectoderm and inner cell mass of the blastocyst in vivo and in vitro. Development 1988: 102: 793–803. 61. Iwasaki S, Yoshiba N, Ushijima H, et al. Morphology and proportion of inner cell mass of bovine blastocysts fertilized in vitro and in vivo. J Reprod Fertil 1990; 90: 279–84. 62. Wowk B, Darwin M, Harris SB, et al. Effects of solute methoxylation on glass-forming ability and stability of vitrification solutions. Cryobiology 1999; 39: 215–27 63. Shaw JM, Kola I, McFarlane DR, et al. An association between chromosomal abnormalities in rapidly frozen 2-cell mouse embryos and the ice-forming properties of the cryoprotective solution. J Reprod Fertil 1991; 91: 9–18 64. Bouquet M, Selva J, Auroux M. Cryopreservation of mouse oocytes: mutagenic effects in the embryo? Biol Reprod 1993; 49: 764–9 65. Ali J, Bongso A, Ratnam SS. Chromosomal analysis of day-2 human embryos vitrified with VS14. Med Sci Res 1995; 23: 539–40. [Erratum: Introduction section, paragraph 5, lines 2-3: the sentence should read: ‘VS14 has been shown to be non-teratogenic.’] 66. Park SE, Hong SW, Lee SH, et al. Chromosome and spindle configurations of human oocytes matured in vitro after vitrification at the germinal vesicle stage in stimulated cycle. Fertil.Steril 2004; 82: Suppl. 2, pp. S114 67. Dulioust E, Toyama K, Busnel MC, et al. Long-term effects of embryo freezing in mice. Proc Natl Acad Sci USA 1995; 92: 589–93
and chromosomes better than conventional straws. Hum Reprod 2000; 15: 2598–603. Chen SU, Lien YR, Chao KH, et al. Cryopreservation of mature human oocytes by vitrification with ethylene glycol in straws. Fertil Steril 2000; 74: 804–8. Choi DH, Chung HM, Lim JM, et al. Pregnancy and delivery of healthy infants developed from vitrified blastocysts in an IVFET program. Fertil Steril 2000; 74: 838–9. Chung HM, Seung WH, Hong MS, et al. In vitro blastocyst formation of human oocytes obtained from unstimulated and stimulated cycles after vitrification at various maturational stages. Fertil Steril 2000; 73: 545–51. Yoon TK, Chung HM, Lim JM, et al. Pregnancy and delivery of healthy infants developed from vitrified oocytes in a stimulated in vitro fertilization-embryo transfer program. Fertil Steril 2000; 74: 180–1. Yoon TK, Kim TJ, Park SE, et al. Live births after vitrification of oocytes in a stimulated in vitro fertilization-embryo transfer program. Fertil Steril 2003; 79: 1323–6. Yin H, Kim SS, Fisher J, et al. Investigation of optimal conditions for equilibrating ovarian tissue with ethylene glycol prior to vitrification. Fertil Steril 2001; 76(Suppl 1): S101 (abstr). Kim, TJ, Hong SW, Park, SE, et al. Pregnancy after vitrification of human oocytes and blastocysts using same cryoprotectant solution, ethylene glycol, and sucrose. Fertil Steril 2003; 80 (Suppl 3): 143 abstr. Kim T, Hong S, Cha K. Pregnancies from cryopreserved oocytes using vitrification protocol. Fertil Steril 2005; 84: Suppl. 1, pp. S179 (abstr) Kim SH, Ku SY, Sung KC, et al. Simplified EM grid vitrification is a convenient and efficient method for mouse mature oocyte cryopreservation. Yonsei Med J 2006; 30; 47: 399–404. Kim, TJ, Hong SW, Chung HM, et al. Pregnancy and delivery after vitrification of human oocytes. Fertil Steril 2005; 83: Suppl. 5, pp. S13 (abstr) Park SE, Chung HM, Cha KY, et al. Cryopreservation of ICR mouse oocytes: improved post-thawed preimplantation development after vitrification using Taxol, a cytoskeleton stabilizer. Fertil Steril 2001; 75: 1177–84. Park SE, Kim TJ, Hong SW, et al. Vitrification of human mature oocytes in a straw to prevent the risk of liquid nitrogen contamination during storage. Fertil Steril 80: Suppl. 3, pp. 64–5 (abstr)
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Vitrification in animal reproduction: vitrification of embryos using open pulled straws
4A
Gábor Vajta
an increasing number of successful oocyte vitrification) results achieved in the world’s largest human IVF clinic are particularly attractive to human embryologists, and motivate them to attempt to replicate the published achievements. This distribution of applications seems to be established, and may remain for some years to come, although very few convincing statistics show the superiority of either of the two methods for any given purpose, provided the technique is applied strictly according to the established guidelines.
THE USE OF OPEN PULLED STRAWS IN ANIMAL REPRODUCTION Among various high-rate cooling approaches used for vitrification of mammalian oocytes and embryos (discussed in detail in Chapter 2) two techniques have obtained relatively wide acceptance among embryologists: the Cryotop with extensive and rapidly growing application in the human field, and the open pulled straw (OPS) in animal reproductive biology. Although there is considerable overlap, with the first baby born after oocyte vitrification using OPS being reported, followed by high pregnancy rates being achieved recently after OPS use,1,2 and excellent results also being achieved with the Cryotop in animal reproduction,3,4 the field is more or less evenly distributed. The reasons are partially understandable, as domestic animal embryologists prefer to work with straws, and the more robust and simpler OPS also permits a kind of semi-direct transfer after warming,5 an approach that is not only very simple and practical, but also has not been found to compromise pregnancy and calving rates at all in cattle after transfer of in vitro produced or somatic cell cloned, zona intact or zona free embryos. This fact may provide considerable benefits for on-farm applications (see below). On the other hand, the beauty of the delicate approach of Cryotop, and even more the extremely convincing, and burgeoning statistics based on blastocyst cryopreservation (and
THE OPEN PULLED STRAW METHOD Principles of the OPS method are described in Chapter 2, the device is commercially available, and the technique has been published several times and is now used in many laboratories worldwide. Accordingly, here only a short description of the technical procedure is provided, along with some illustrations. The term OPS refers to the preparation technology and its application. Standard 0.25 ml insemination straws are warmed and pulled just like glass capillaries, to reduce wall thickness and diameter to approximately half of the original (Figure 4A.1). Straws are then cut at the narrowest point with a sharp razor blade. Sterilization can be performed with gas or irradiation. The process seems to be quite easy, however, according to our past experience, the toolmaking is still the most difficult
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part of the whole OPS vitrification. From this point of view it is helpful that the straws are now produced by several companies (including Minitüb, Germany), and may be purchased worldwide.The ‘openness’ mentioned in the name permits extremely simple loading and expelling, which is, to date, unique among cryopreservation technologies. In contrast to the standard 0.25 ml insemination straws, the narrow end of the OPS straw predisposes it for loading by the capillary effect. After equilibration (according to the specific requirements of the oocyte or embryo) the sample is loaded into a small (approximately 1 µl) droplet, which is immediately touched with the narrow end of the OPS straw (Figure 4A.2a). As a consequence of the capillary effect, most of the solution including the sample will be loaded into the straw, which
a
b
Figure 4A.1 An open pulled straw (a) with approximately 50% of the diameter of a standard 0.25 ml insemination straw (b) Reproduced with permission from Vajta and Nagy.6
a
b
c
Figure 4A.2 The process of loading, cooling, warming, and expelling at OPS vitrification. (a) Oocytes or embryos are placed into an approximately 1 µl droplet of cryoprotectant medium; the drop is touched with the OPS straw; as a result of the capillary effect, the medium with the oocytes or embryos enters the straw. (b) Subsequently, straws are immersed with a continuous rapid movement into the liquid nitrogen. (c) At warming, straws are immersed into the warm medium: the solid column melts immediately, and as the result of gravity, embryos or oocytes leave the straw. This latter process can be facilitated by closing the wide end of the straw approximately 1s after immersion: the warming and expanding air trapped in the straw will expel the solution column. Reproduced with permission from Vajta and Nagy.6
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can subsequently be immersed immediately into the liquid nitrogen (Figure 4A.2b). At warming, the straws are simply immersed into the warming medium preferably containing an osmotic buffer to avoid excessive swelling, the vitrified column melts, and is also diluted immediately (again, as the result of the capillary effect), and, finally, embryos or oocytes slowly descend into the medium as the result of gravity. This latter process can be facilitated by closing the wide top end of the OPS straw approximately 1 s after immersion: the warming and expanding air trapped in the straw will expel the solution column into the warming medium. Exactly the same approach should be used at semi-direct transfer, but the warming medium has to be placed into a 0.25 ml standard insemination straw, and the narrow end of the OPS straw is inserted into this straw. Although expelling occurs almost always automatically, it may also be facilitated by closing the other end of the OPS straw. Control with the naked eye is satisfactory: if the solution column disappears from the OPS straw, the embryo will always be in the 0.25 ml straw and can be transferred immediately into the recipient animal. With this application, the two traumatic events (warming and transfer) occur at the same time, which may explain the high pregnancy rates seen when using this technique.
Figure 4A.3 Calves born after OPS vitrification of in vitro matured oocytes, in vitro fertilization, embryo culture, and another OPS vitrification cycle at the blastocyst stage. Reproduced with permission from Vajta et al.9
only was their in vitro survival impressively high (at and after day 3 vitrification, there was no difference in developmental competence compared with the control embryos), but also the day 3 vitrified embryos 24 hours after warming were successfully used as donors for embryonic cell nuclear transfer.7,8 Even more convincing was the 25% blastocyst rate that was achieved by OPS vitrification of matured oocytes, followed by in vitro fertilization and embryo culture to day 7.9 A second OPS vitrification of these blastocysts, then transfer of the warmed embryos into recipients, resulted in three calves being born (Figure 4A.3). OPS vitrified and warmed oocytes10 and cytoplasts11 were also used as recipients for nuclear transfer, and development to term was achieved. However, by far the greatest success has been achieved by Vieira et al.,12 who used OPS vitrification of the extremely cryosensitive immature bovine oocyte, and obtained offspring after in vitro maturation, fertilization, and embryo culture, even after another OPS vitrification at the blastocyst stage (Figure 4A.4). For logistical reasons, the possibility of being able to cryopreserve cloned embryos
APPLICATION IN CATTLE, SHEEP, AND THE GOAT As successful vitrification of high quality in vitro produced bovine blastocysts can be successfully performed by using relatively low cooling rates, high cryoprotectant concentrations, and 0.25 ml standard insemination straws, the new vitrification methods in this field may not offer any real breakthrough. Accordingly, the first convincing evidence regarding the usefulness of the OPS method was provided with cryopreservation of early stage in vitro produced bovine embryos. Not
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Figure 4A.4 Calves born after OPS vitrification of immature oocytes, in vitro maturation, fertilization, embryo culture, and (for the two calves on left) another OPS vitrification cycle at the blastocyst stage. Reproduced with permission from Vieira et al.12
Figure 4A.5 Handmade somatic cell cloned calf born after OPS vitrification at the blastocyst stage followed by semi-direct transfer. Reproduced with permission from Tecirlioglu et al.15
may have even more importance. OPS vitrification resulted in the first non-transgenic and transgenic calves born after somatic cell nuclear transfer, after cryopreservation of the blastocysts before transfer.13,14 Offspring were also obtained after vitrification of zona-free blastocysts made with a different nuclear technology, handmade cloning (i.e. without micromanipulators)15 (Figure 4A.5). In sheep, live births have been achieved after OPS vitrification of zona-included, somatic cell cloned blastocysts (Peura, personal communication) (Figure 4A.6).
All these applications in the cow are mainly in the experimental and biotechnological area, as cryopreservation of transferable-stage bovine in vivo derived embryos is an established business, and pregnancy rates achievable with fresh versus conventionally frozen embryos are less than 10%, a gap that is difficult to narrow. Moreover, as emphasized in Chapter 2, the often mentioned benefits of vitrification in commercial cattle embryo transfer are not too significant. Vitrification of a single embryo can be performed in minutes, compared with the hours
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genetic material. The main reason for this is probably the high lipid content. Porcine embryos are extremely sensitive to chilling injury that is unavoidable during traditional freezing, and survival rates are extremely poor, so commercial application is almost entirely out of the question. Vitrification was seen as a reasonable opportunity to overcome the problem. However, in spite of many attempts and some promising achievements including cytoskeleton relaxants and delipidation with centrifugation followed by vitrification in normal straws, the in vivo developmental rates remained moderate, and the cumulative costs of the technology were not commensurate with the achievements. Even initial approaches with OPS vitrification were controversial because while the in vitro survival and developmental rates were excellent, no pregnancies were achieved. Finally, with a small modification of parameters, e.g. extension of the equilibration in the concentrated cryoprotectant solution has resulted in the long-expected breakthrough. With these improved parameters,18 herds of pigs have been born after OPS vitrification of in vivo derived embryos, remarkable survival and offspring per transferred embryo rates were achieved, and the technology is efficient for different developmental stages, from morulae to expanded blastocysts.19 Offspring have even been reported after OPS vitrification and one-step dilution combined with non-surgical transfer20,21 (Figure 4A.7). This latter achievement indicates the potential of this compact technology similar to that widely applied in cattle, although the overall efficiency still can be increased further. Regarding the experimental application of OPS vitrification in pigs, the first surviving embryos produced in vitro were first reported by Men et al.22 Subsequently, results were further improved after chemical delipidation.23 A significant breakthrough from both the somatic cell nuclear transfer and vitrification point of view, was the production of healthy piglets after delipidation of oocytes with
Figure 4A.6 Somatic cell cloned sheep born after OPS vitrification at the blastocyst stage. Published with permission of T. T. Peura.
required for traditional freezing. However, in most commercial situations freezing is not restricted to one or two, but performed with tens of embryos, grouped in a maximum of two. Vitrification requires individual repeats for all (pairs of) embryos, while for the most part of the procedure in traditional freezing, it is performed in one run for an almost unlimited number of (groups of) embryos. The other benefit, the price of equipment required for traditional freezing may not be a decisive factor for an established cattle embryo transfer practice. Roughly the same is applicable to sheep,16 although the commercial aspects of embryo transfer in that species are less significant. However, it should be mentioned that in the goat, OPS vitrification has significantly increased pregnancy rates compared with traditional freezing.17
VITRIFICATION OF PORCINE EMBRYOS AND OOCYTES WITH THE OPS METHOD In pigs, however, the lack of an efficient embryo cryopreservation system profoundly limits possibilities, regarding both local commercial embryo transfer and especially transportation including international exchange of
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methods may be equally efficient for any given purpose.
OTHER DOMESTIC AND EXPERIMENTAL ANIMALS In the rabbit, OPS vitrification of in vivo derived morulae was found to be superior compared with traditional freezing, and vitrification performed in 0.25 ml insemination straws in terms of in vitro survival and births of kittens.25 Similar results were achieved earlier with a modified version of the OPS, the ‘sealed open pulled straw procedure’, when the method was compared with vitrification in 0.25 ml straws.26 In the horse, both immature and mature oocyte vitrification was successfully performed with the OPS method, although the survival rates were lower than those in cattle.27 Later observations have revealed serious spindle damage of matured oocytes after OPS vitrification, although for immature oocytes, the resumption of meiosis was higher after OPS than after traditional freezing.28 For equine embryos, traditional slow rate freezing and OPS vitrification were found to be equally efficient with respect to in vitro survival rates.29 In contrast to the equine results, in the mouse a high proportion of oocytes preserved patterns of meiotic spindles and chromosomes after OPS vitrification. The results were significantly better than those achieved with vitrification in normal insemination straws.30 Survival rates with OPS were lower, but could be improved by modification of the applied technique (closed pulled straws).31 For mouse blastocysts, high survival and in vitro developmental rates were reported both with the OPS and its modification, the glass micropipette (GMP) method.32 With further optimization of parameters for OPS vitrification, a method was established that was suitable to cryopreserve mouse embryos from the 4-cell to the early blastocyst stage.33 An even more impressive
Figure 4A.7 Piglets born after OPS vitrification of in vivo derived embryos and non-surgical transfer. Reproduced with permission from Cuello et al.
Figure 4A.8 Piglets born after OPS vitrification of somatic cell cloned blastocysts produced from delipated oocytes. Reproduced with permission from Li et al.
centrifugation, micromanipulation-based nuclear transfer, and OPS vitrification (Figure 4A.8).24 Similar results have been achieved by Du et al. (unpublished) by using handmade cloned embryos, reconstructed from oocytes delipidated by using a simplified procedure. As the latter work was performed by using the Cryotop vitrification method, the achievements provide further evidence that the two
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species is, however, the first reported birth after OPS vitrification of in vivo derived carnivore embryos, achieved in the European polecat (Figure 4A.9).37
REASONS TO USE THE OPS VITRIFICATION METHOD As mentioned in the introduction, the OPS technique – compared with the other highrate cooling vitrification procedures – is relatively robust and is based on a simple modification of the 0.25 ml insemination straw that is the most common tool for cryopreservation in reproductive biology. Moreover, loading and expelling of samples is simple, does not require any special or delicate tools, and can be learned in minutes. The technique, if properly applied, ensures appropriate cooling and warming rates (as shown above) for a wide variety of mammalian oocytes and embryos of different developmental stages and origin. Storage and sample identification do not require any special approach as they are based on the traditional tools and methods of cryopreservation. A unique benefit of OPS vitrification for application in the animal field is the possibility of one-step dilution and semi-direct transfer. This option was described first for cattle embryo vitrification,5 and was used afterwards for various purposes including transfer of in vitro fertilized, embryonic and somatic cell cloned embryos, both with and without their zona pellucida. Although no direct comparison is available, according to our experience the semidirect transfer results in at least as high pregnancy and calving rates as the stepwise dilution and delayed transfer. This is an obvious benefit, and may open a wide scale practical application of OPS in cattle. Moreover, the method for successful one-step dilution and transfer of OPS vitrified embryos has also been published for small ruminants,16 and the mouse,38 widening the possibilities for practical application in many mammalian species.
Figure 4A.9 European polecat pups born after vitrification of in vivo derived embryos with the OPS method. Reproduced with permission from Piltti et al.37
achievement was reported for rat embryos, where double vitrification was applied by using a super-fine variation of the OPS straws, cryopreserving the embryos first at the early morula, then at various subsequent developmental stages.34
NON-DOMESTIC SPECIES Although the low costs and the very simple equipment predispose vitrification for cryopreservation of oocytes and embryos of wild and endangered species permitting work under compromised conditions, surprisingly few papers deal with this area, probably reflecting the low level of attention given to, and the even lower funding of, this very important field. Among the few published works successful experiments involving three mammals should be mentioned. In Siberian tigers, OPS vitrification of early stage embryos resulted in further development, in contrast to traditional freezing.35 Minke whale oocytes were successfully vitrified with both the OPS and the Cryotop method, although results were slightly better with Cryotop.36 The greatest promise for endangered carnivore
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Additionally, OPS vitrification offers a solution for disease transmission problems that are a major concern regarding the new vitrification procedures. With the application of filtered, separately stored liquid nitrogen for cooling and using a hermetically isolated double-straw technique for storage,39 the possibility
of cross-contamination is eliminated (as confirmed by independent investigation),40 while all the previously mentioned benefits (high cooling rate, low chilling injury, low cryoprotectant concentration, and consequently low toxic and osmotic damage, easy and safe handling, possibility for direct transfer) are still preserved.
References 1. Kuleshova L, Gianaroli L, Magli C, Trounson A. Birth following vitrification of a small number of human oocytes. Hum Reprod 1999; 14, 3077–79. 2. Selman et al., 2006 3. Chian RC, Kuwayama M, Tan L et al. High survival rate of bovine oocytes matured in vitro following vitrification. J Reprod Dev 2004; 50: 685–96. 4. Fujihira T, Nagai H, Fukui Y. Relationship between equilibration times and the presence of cumulus cells, and effect of Taxol treatment for vitrification of in vitro matured porcine oocytes. Cryobiology 2005; 51: 339–43. 5. Vajta G, Murphy C, Macháty Z et al. In straw dilution of in vitro produced bovine blastocysts after vitrification with the Open Pulled Straw (OPS) method. Vet Rec 1999; 144: 180–81. 6. Vajta and Nagy, 2006. 7. Vajta G, Booth PJ, Holm P, Jacobsen H, Greve T, Callesen H. The use of vitrified Day 3 embryos as donors in bovine nuclear transfer. Cryo Letters 1997; 18: 355–58. 8. Peura TT, Lane MW, Vajta G, Trounson AO. Post-thaw in vitro survival of vitrified cloned bovine embryos. Vet Rec 1997; 140: 404. 9. Vajta G, Kuwayama M, Holm P et al. Open pulled straw vitrification: a new way to reduce cryoinjuries of bovine ova and embryos. Mol Reprod Dev 1998; 51, 53–58. 10. Hou YP, Dai YP, Zhu SE et al. Bovine oocytes vitrified by the open pulled straw method and used for somatic cell cloning supported development to term. Theriogenology 2005; 64, 1384–91. 11. Booth PJ, Vajta G, Høj A et al. Full-term development of nuclear transfer calves produced from Open Pulled Straw (OPS) vitrified cytoplasts. Theriogenology 1999; 51: 999–1006 12. Vieira AD, Mezzalira A, Barieri DP et al. Calves born after open pulled straw vitrification of
13. 14.
15.
16.
17. 18. 19.
20.
21. 22.
23. 24.
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immature bovine oocytes. Cryobiology 2002; 45, 91–94. French AJ, Hall VJ, Korfiatis NT et al. Viability of cloned bovine embryos following OPS vitrification. Theriogenology 2002; 57, 413. French AJ, Lewis IM, Ruddock NT et al. Generation of aS1 casein gene transgenic calves by nuclear transfer. Biol Reprod 2003; 68, 240 Tecirlioglu RT, French AJ, Lewis IM et al. 2003 Birth of a cloned calf derived from a vitrified cloned embryo. Reprod Fertil Dev 15, 361–66. Isachenko V, Alabart JL, Dattena M et al. New technology for vitrification and field (microscope free) warming and transfer of small ruminant embryos. Theriogenology 2003; 59, 1209–18 El-Gayar and Holtz, 2001 Berthelot et al. 2000. Berthelot F, Martinat-Botté F, Perreau C et al. Birth of piglets after OPS vitrification and transfer of compacted morula stage embryos with intact zona pellucida. Reprod Nutr Dev 2001; 41: 267–72. Cuello C, Gil MA, Parrila I et al. In vitro development following one-step dilution of OPS vitrified porcine blastocysts. Theriogenology 2004; 62, 1144–52. Cuello et al., 2005 Men H, Agca Y, Critser E et al. Beneficial effects of serum supplementation during in vitro production of porcine embryos on their ability to survive cryopreservation by the open pulled straw vitrification. Theriogenology 2005; 64, 1340–49. Men et al., 2006. Li R, Lai L, Wax D, Hao Y, et al. Cloned transgenic swine via in vitro production and cryopreservation. Biol Reprod. 2006; 75: 226–30.
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33. Zhou GB, Hou YP, Jin F, et al. Vitrification of mouse embryos at various stages by openpulled straw (OPS) method. Anim Biotechnol 2005; 16, 153–63. 34. Isachenko V, Alabart JL, Vajta G et al. Double vitrification of rat embryos at different developmental stages using an identical protocol. Theriogenology 2002; 60, 445–52. 35. Crichton EG, Bedows E, Miller-Lindholm AK et al. Efficacy of porcine gonadotropins for repeated stimulation of ovarian activity for oocyte retrieval and in vitro embryo production and cryopreservation in Siberian tigers (Panthera tigris altaica). Biol Reprod 2003; 68, 105–13. 36. Iwayama H, Hochi S, Kato M et al. Effects of cryodevice type and donor’s sexual maturity on vitrification of minke whale (Balaenopter bonaerensis) oocytes at germinal vesicle stage. Zygote 2004; 12, 333–38. 37. Piltti K, Lindeberg H, Aalto J et al. Live cubs born after transfer of OPS vitrified-warmed embryos in the farmed European polecat (Mustela putorius). Theriogenology 2004; 61, 811–20. 38. Yang QE, Hou YP, Zhou GB, Yang ZQ, Zhu SE. Stepwise in-straw dilution and direct transfer using open pulled straws (OPS) in the mouse: a potential model for field manipulation of vitrified embryos. J Reprod Dev; in press. 39. Vajta G, Lewis IM, Kuwayama M, Greve T, Callesen H. Sterile application of the Open Pulled Straw (OPS) vitrification method. Cryo Letters 1998; 19, 389–92. 40. Bielanski et al. in press 41. El-Gayar
25. Naik BR, Rao BS, Vagdevi R, Gnanprakash M, Amarnath D, Rao VH. Conventional slow freezing, vitrification and open pulled straw (OPS) vitrification of rabbit embryos. Anim Reprod Sci 2005; 86, 329–38. 26. Lopez-Bejar M, Lopez-Gatius F. Nonequilibrium cryopreservation of rabbit embryos using a modified (sealed) open pulled straw procedure. Theriogenology 2002; 58, 1541–52. 27. Hurtt AE, Landim-Alvarenga F, Seidel GE Jr, Squires EL. Vitrification of immature and mature equine and bovine oocytes in an ethylene glycol, ficoll and sucrose solution using open-pulled straws. Theriogenology 2000; 54: 119–128, 2000. 28. Tharasanit T, Colenbrander B, Stout TAE. Effect of maturation stage at cryopreservation of post-thaw cytoskeleton quality and fertilizability of equine oocytes. Mol Reprod Dev 2006; 73, 627–37. 29. Moussa M, Bersinger I, Doligez P, et al. In vitro comparisons of two cryopreservation techniques for equine embryos: slow-cooling and open pulled straw (OPS) vitrification. Theriogenology 2005; 64, 1619–32. 30. Chen SU, Lien YR, Chen HF et al. Open pulled straws for vitrification of mature mouse oocytes preserve patterns of meiotic spindles and chromosomes better than conventional straws. Hum Reprod 2000; 15: 2598–603. 31. Chen SU, Lien YR, Cheng YY et al. Vitrification of mouse oocytes using closed pulled straws (CPS) achieves a high survival and preserves good patterns of meiotic spindles, compared with conventional straws, open pulled straws (OPS) and grids. Hum Reprod 2001; 11: 2350–6. 32. Kong IK, Lee SI, Cho SG et al. Comparison of open pulled straw (OPS) vs glass micropipette (GMP) vitrification in mouse blastocysts. Theriogenology 2000; 53, 1817–26.
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Vitrification in animal reproduction: vitrification of embryos using conventional straws with an ethylene glycol-based solutions
4B
Magosaburo Kasai and Keisuke Edashige
sample could be directly cooled in liquid nitrogen from 0°C or above. Based on this idea, Rall and Fahy2 developed a simple method of cryopreservation called vitrification, in which samples are cooled in liquid nitrogen without a slow cooling stage. Their idea was proven effective3 and vitrification has markedly simplified the cooling process. In addition, the method has the potential to increase rates of embryonic survival because ice does not form during cooling. Although extracellular ice can form during warming in less concentrated solutions, its effect could be minimized by warming the sample rapidly, which would help prevent the formation of intracellular ice (devitrification). On the other hand, vitrification has a serious disadvantage in that the chemical toxicity of the solution is quite high, because the concentration of cryoprotectant(s) is quite high (5–8 mol/L), and the temperature at which embryos are exposed to the solution is high.
VITRIFICATION With conventional slow freezing, a process first developed by Whittingham et al.,1 embryos suspended in a solution containing a low concentration (1–2 mol/L) of permeating cryoprotectant are cooled slowly (0.3–0.5°C/min) before the sample is cooled with liquid nitrogen. This method, derived deductively by Peter Mazur, was proven effective and is now used routinely with the aid of a programmable freezer. However, it requires a long cooling stage and a machine for controlling the cooling rate. In addition, the formation of ice in the preservation solution can lead to intracellular ice forming, probably the biggest obstacle to the successful cryopreservation of embryos. With slow freezing, embryos cooled slowly to subzero temperatures between − 30°C and − 70°C before cooling with liquid nitrogen are placed in a very concentrated solution, which constitutes small channels among ice. Under such conditions, ice forms in neither the solution channel nor the embryo even after the cooling in liquid nitrogen. Therefore, even with slow freezing, embryos and the small concentrated channels where they are located are vitrified. Vitrification is the solidification of a solution without crystallization. Therefore, if a solution similar to the one in the unfrozen channel in slowly frozen samples is composed using a very high concentration of cryoprotectant(s) and embryos are equilibrated in it, the
VITRIFICATION USING AN ETHYLENE GLYCOL-BASED SOLUTION The first vitrification solution, named VS1,2 contained three permeating cryoprotectants, dimethylsulfoxide (DMSO), acetamide, and propylene glycol. This solution, or a modification
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Table 4B.1
Successful vitrification of mammalian embryos using EFS40 Pregnancy rate
Species
Embryo stage
Fertilization
Mouse Rabbit Cow Horse Sheep Human Mastomys Rat Mongolian gerbil
Morula Morula Blastocyst Blastocyst Blastocyst 4-cell Blastocyst Morula Blastocyst
In vivo In vivo In vitro In vivo In vivo In vitro In vivo In vivo In vivo
Young†
Step*
n
%
n
%
Reference
1 1 1 2 2 2 2 2 2
11/16 11/12 8/10 2/5 12/24 1/7 5/9 6/6 3/3
69 92 90 40 50 14 56 100 100
57/167 60/120 6/20 2/5 8/24 2/6 31/144 51/83 20/34
34 50 30 40 33 33 22 61 59
7 11 12 13 14 15 16 17 18
Treatment with cryoprotectant; †no. live young/no. embryos transferred.
*
thereof, was successfully used for the cryopreservation of mouse embryos,4 and has been shown to be effective for oocytes5 and ovaries6 in the mouse. In 1990, Kasai et al.7 showed that ethylene glycol is an effective permeating cryoprotectant for vitrification of mouse morulae. Since then, mostly ethylene glycol has been used for vitrifying mammalian embryos, either alone or in combination with another permeating agent.8 Kasai et al.7 developed EFS solution as an ethylene glycol-based vitrification solution. This solution was later renamed EFS40, after the concentration of ethylene glycol in it. When mouse morulae were vitrified in straws with EFS40, the rate of survival was quite high and no appreciable loss of viability was observed if the period of exposure to the solution and the room temperature for handling embryos were suitable. Then, Kasai’s group demonstrated that the survival of vitrified embryos was significantly influenced by the stage of development9 and by the temperature at which embryos are treated with the vitrification solution before rapid cooling.10 To date, EFS solution has been proven effective for the cryopreservation of embryos of various mammalian species, especially in the later stages of development (8-cell embryos, morulae, and blastocysts), if the embryos are treated under suitable conditions. Reports on the successful
vitrification of mammalian embryos using EFS solution, resulting in the production of live young, are listed in Table 4B.1. Among the nine species listed, embryos of mastomys and Mongolian gerbil were first cryopreserved by vitrification with EFS solution. Horse embryos have been vitrified only with EFS solution. A protocol for vitrifying mouse embryos is described below.
PROTOCOL FOR VITRIFYING MOUSE EMBRYOS The procedure for vitrifying mouse embryos has been described elsewhere.19–21 A brief outline is described below. (1) Solutions PB1 medium (modified phosphate-buffered saline)22 as a basic isotonic solution; S-PB1 medium, which is PB1 medium containing 0.5 mol/L sucrose; EFS40, which consists of 40% v/v ethylene glycol and 60% v/v FS solution; FS solution is PB1 medium containing 30% w/v Ficoll 70 and 0.5 mol/L sucrose (e.g. 1.5 g Ficoll + 0.856 g sucrose + 3.51 ml PB1 medium; to prevent bubbling, it would be better to add bovine serum albumin (BSA) after other solutes were dissolved thoroughly). Load EFS40 in a 1 ml syringe with an 18 G needle. This makes it possible to
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(2)
(3)
(4)
(5)
use a minimal amount of fresh solution for each sample (evaporation will make the solution concentrated). Liquid nitrogen Pour liquid nitrogen into a dewar flask with a large mouth or into a Styrofoam box, and float a thin Styrofoam boat on it. Vitrification of mouse morulae Into a 0.25 ml insemination straw (13 cm, including the cotton plug), load 65 mm S-PB1, 15 mm air, 3 mm EFS40, 4 mm air, and 13 mm EFS40, successively. Place the straw horizontally at 25°C. Place mouse morulae in the 13 mm EFS40 in the straw directly with a minimal amount of PB1 medium using a fine pipette. If this is technically difficult, embryos can be washed in EFS40 quickly before being loaded in straw. Aspirate the straw to seal the powder end and then seal the open end with a heat sealer. After 30–60 s of exposure of embryos to EFS40, place the straw on the Styrofoam boat on liquid nitrogen horizontally to cool the sample in the gas phase (this prevents fracture damage.)23 Leave it for 3 min or more before the straw is stored in liquid nitrogen. Vitrification of mouse 2-cell embryos and blastocysts For vitrifying 2-cell embryos and blastocysts, embryos need to have been pretreated with PB1 medium containing 10% v/v ethylene glycol for 5 min (or with EFS20, which is a mixture of 20% v/v ethylene glycol and 80% v/v FS solution, for 2 min) before being transferred to EFS40 in straw. In this case, the exposure time in EFS40 at room temperature should be limited to 30 s. Warming For warming, remove the straw from the liquid nitrogen and keep it in air at room temperature for 10 s (this also prevents fracture damage.)23 Then, immerse it into a 20–25°C water bath. After ~7 s, when the S-PB1 medium begins to melt, remove the straw, and wipe it quickly. Keeping the straw horizontal, cut off both
ends. Tilt the straw slightly with the EFS side down, and perfuse the straw slowly with 1 ml of S-PB1 medium using a 1 ml syringe into a watch glass. Shake the watch glass gently to dilute the EFS40. Recover the embryos with a pipette and place them in S-PB1 medium prepared in a culture dish. About 5 min after the perfusion, transfer the embryos to PB1 medium.
COMPONENTS OF THE VITRIFICATION SOLUTION During the cryopreservation process, embryos are at risk of injury from chilling (this applies to certain types of embryos, depending on the species and the developmental stage), the toxicity of the cryoprotectant, extracellular ice, intracellular ice, fracture damage, osmotic swelling, and osmotic shrinkage.24 To obtain high rates of survival, all these problems must be circumvented. Among the injuries, the damage caused by the formation of intracellular ice during cooling or warming is surely the greatest obstacle to overcome. In addition, injury from osmotic swelling is also a major factor, which can occur during removal of the permeated cryoprotectant after warming.25 For preventing intracellular ice from forming, rapidly permeating cryoprotectants are suitable because permeation by the cryoprotectant is essential to vitrify the embryo. To prevent osmotic swelling during removal of the cryoprotectant(s) after warming, rapidly permeating agents are also suitable, because the faster the diffusion of the intracellular cryoprotectant out of the cell, the lower the risk of osmotic over-swelling. We examined the permeability of mouse embryos to various cryoprotectants from the apparent volume change in cryoprotectant solutions.26 We found the permeability to vary depending on the developmental stage of the embryo and on the cryoprotectant (Figure 4B.1). In mouse morulae, ethylene glycol permeates quite rapidly. We have obtained
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Relative volume
(a)
1
0.8
0.6
0.4
0.2
(b)
1
(c)
0.8
0.6
0.4
0.2
(d)
1
(e)
0.8
0.6
0.4
0.2 0
1
2
3
4
5
0
1
2
3
4
5
Exposure time (min) Oocyte 2- cell Morula
1- cell 8- cell
Figure 4B.1 The change in volume of mouse oocytes (at metaphase II), and embryos at the 1-cell, 2-cell, 8-cell, and morula stages during exposure to (a) 1.5 mol/L acetamide, (b) 10% v/v DMSO, (c) 10% v/v ethylene glycol, (d) 10% v/v glycerol, and (e) 10% v/v propylene glycol, dissolved in PB1 medium, for 5 min at 25°C. Reproduced with permission from Pedro et al.26
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Table 4B.2 Survival of mouse morulae exposed to 30% v/v cryoprotectants for 20 min at 20° C. Data from Kasai28 Permeating cryoprotectant Ethylene glycol Glycerol DMSO Propylene glycol Acetamide
EFS solution contains not only ethylene glycol and Ficoll, but also sucrose as a nonpermeating small sugar which has considerable osmotic effect. The inclusion of sucrose is quite effective at reducing the apparent toxicity of ethylene glycol, probably because sucrose promotes the shrinkage of embryos, which would restrict excess permeation of ethylene glycol and thus reduce its toxic effect.7 In addition, sucrose helps prevent over-swelling during the removal of ethylene glycol after warming.29 Like Ficoll, sucrose is virtually non-toxic.28,30
Survival rate n
%
48/49 44/50 41/60 8/48 0/60
98* 88* 68† 17‡ 0**
Values with different superscripts differ significantly (P < 0.05, χ2 analysis or Fisher’s probability test).
THE PATHWAY FOR THE MOVEMENT OF WATER AND CRYOPROTECTANTS IN EMBRYOS
similar results for bovine embryos,27 and rabbit embryos (unpublished observation). Therefore, it is suggested that the permeability of embryos to cryoprotectants is less species specific and more stage specific. In addition to the injuries caused by intracellular ice and osmotic swelling, the chemical toxicity of the permeating cryoprotectant is a major obstacle to vitrification. The sensitivity of embryos to chemical toxicity would depend on the cryoprotectant and the species and developmental stage of the embryos. Kasai28 examined the toxicity of five major permeating cryoprotectants to mouse morulae and showed that ethylene glycol is the least toxic (Table 4B.2). Therefore, in terms of ability to permeate and chemical toxicity, ethylene glycol is the most suitable cryoprotectant, at least for mouse morulae. Rall and Fahy2 suggested that the inclusion of a macromolecule promotes vitrification of a solution, and used polyethylene glycol. In EFS solution, Ficoll is included as a non-permeating polymer, because it has high solubility, low viscosity, and low toxicity. It has been shown that even a large amount of Ficoll is virtually non-toxic.28 A large amount of polymer occupies a significant proportion of the solution volume. Therefore, its inclusion must increase the proportion of permeating cryoprotectant per water volume. This may be one mechanism that promotes vitrification of the solution.
For the successful cryopreservation of embryos, the smooth movement of water and cryoprotectants through the plasma membrane is essential. In most types of cells, water moves through the plasma membrane with limited permeability by simple diffusion through the lipid bilayer. However, the plasma membrane of some cells, e.g. human red blood cells and cells in renal proximal tubules, is extremely permeable by water. In the 1990s, small intrinsic membrane proteins that act as water channels, called aquaporins (AQPs), were discovered and characterized.31 The channels occur in two groups: one is highly selective for water and the other transports not only water but also neutral solutes with a small molecular weight, such as cell-permeating cryoprotectants. We have shown that mRNAs of AQP3 and AQP7 are present in metaphase II oocytes, 4-cell embryos, morulae, and blastocysts (Figure 4B.2), and that mRNAs of AQP8 and AQP9 are expressed in blastocysts in the mouse.32 Offenberg et al.33 also detected mRNAs of AQPs, and Barcroft et al.34 detected AQP proteins in mouse embryos. However, it is important to know whether such a channel pathway actually plays a significant role in the movement of water and cryoprotectants in embryos.
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(bp)
Ms
C
O
4
M
B
603 GAPDH 310
603 AQP3 234
603 AQP7 234
603 AQP8 234
603 AQP9 234 Figure 4B.2 Expression of mRNA of aquaporins in mouse oocytes at metaphase II (O) and embryos at the 4-cell (4), morula (M), and blastocyst (B) stages. Ms, molecular size marker; C, polymerase chain reaction (PCR) products amplified from cDNA of the kidney (GAPDH and aquaporin-3), testis (aquaporin-7), and liver (aquaporin-8 and aquaporin9) as a control. The predicted molecular sizes of PCR products are indicated by arrows (modified from Edashige et al).32
to water and its temperature dependency, which is expressed by Arrhenius activation energy (Ea). In general, higher permeability to water with a lower Ea value is suggestive of movement by facilitated diffusion through channel processes, whereas lower permeability with a higher Ea value is suggestive of movement via a channel-independent process, i.e. by simple diffusion. Verkman et al.35 suggested
THE MOVEMENT OF WATER THROUGH THE PLASMA MEMBRANE: VIA SIMPLE DIFFUSION OR CHANNEL PROCESSES? The involvement of water channels in the movement of water across the plasma membrane can be deduced from the permeability
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Relative volume
1.0
0.8
0.6
0.4 0.0 0
0.5
1.0
2.0
Time (min) Figure 4B.3 Changes in volume of mouse oocytes at metaphase II (circles) and embryos at the 4-cell (triangles), morula (squares), and blastocyst (diamonds) stages in PB1 medium containing sucrose (0.8 osmol/kg) at 25oC. Data are indicated as means of relative volume ± SD. Modified from Edashige et al.43
that a permeability to water higher than 4.5 µm/min/atm (or Pf ≥ 0.01 cm/s) and an Ea value lower than 6 kcal/mol are suggestive of the movement of water principally through channels, whereas an Ea higher than 10 kcal/mol with low water permeability is suggestive of movement principally via channelindependent diffusion. Applying these criteria to mouse oocytes at the metaphase II stage would suggest that channels do not play a role in the movement of water.36–43 Therefore, the expression of channels in embryos could be detected by comparing the permeability with that of oocytes. In mouse embryos at early cleavage (2–4cell) stages, channels may not be the major pathway for the movement of water across the plasma membrane, because 2–4-cell embryos have low permeability to water similar to oocytes (Figure 4B.3),36,41,43 and high Ea values,36,43 in spite of the fact that these cells express mRNAs of AQP3 and AQP7.32 Thus, most water molecules are assumed to move across the plasma membrane of 2–4-cell embryos predominantly by simple diffusion, and AQPs would be expressed at negligible
levels if at all. On the other hand, morulae and blastocysts have high water permeability (3.6–4.5 µm/min/atm) (Figure 4B.3) and low Ea values (5.1–6.3 kcal/mol),43 suggesting that the movement is dependent on a channel pathway. Thus, most water molecules would move across the plasma membrane predominantly through channel processes in mouse morulae and blastocysts. Since AQP3 is expressed abundantly in mouse morulae (Figure 4B.4)34,43 and blastocysts,34 and mRNA of AQP3 is much more abundantly expressed in mouse blastocysts than the mRNA of other AQPs,44 AQP3 would be the major contributor to the rapid movement of water in mouse morulae and blastocysts.
THE MOVEMENT OF CRYOPROTECTANTS THROUGH THE PLASMA MEMBRANE It would be possible to deduce the pathway of a cryoprotectant’s movement from the permeability and Ea value, as in the case of permeability to water, although few reports are available on a quantitative evaluation.
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a
by the change in volume of morulae in the ethylene glycol solution, in which the initial shrinkage is quite small (Figure 4B.1). Since AQP3 is also permeable to other major cryoprotectants, i.e. propylene glycol, acetamide, and DMSO (Table 4B.3),48–52 AQP3 expressed in mouse morulae and blastocysts may contribute extensively to the permeation by various cryoprotectants. As described above, changes in the pattern of permeation of embryos by cryoprotectants were stage specific rather than species specific. Therefore, it is suggested that a marked increase in the expression of AQP3 in the later stages of embryonic development occurs in various mammalian species, and that the expression affects the permeability of the embryos to various cryoprotectants. If so, cryopreservation techniques developed for embryos of one species would be applicable to those of various species at the same stage, in terms of permeability.
b
Figure 4B.4 The expression of aquaporin-3 in mouse oocytes at metaphase II (a) and morulae (b). Expression of aquaporin-3 protein was detected by an immunofluorescence technique with antiaquaporin-3 antibody. Bar = 20 µm. Modified from Edashige et al.43
In mouse embryos, the permeability to glycerol and its Ea value changes as in the case of permeability to water. The permeability to glycerol of 2–4-cell mouse embryos is quite low (Figure 4B.1)26,43,45,46 like that of oocytes, and the Ea for the permeability is remarkably high.43 On the other hand, the permeability to glycerol of mouse morulae and blastocysts is markedly high and the Ea value is much lower than that of oocytes.43 Therefore, glycerol permeates mouse morulae and blastocysts via channels and AQP3, recognized as an aqua-glyceroporin,31,47 may play an important role in the process. AQP3 is permeable to various neutral solutes with a low molecular weight, including ethylene glycol (Table 4B.3).48–50 For the permeability to ethylene glycol of mouse embryos, no quantitative study has been conducted. However, from the data shown in Figure 4B.1, it is clear that ethylene glycol permeates mouse morulae more rapidly than glycerol. Considering that these two agents belong to the same polyalcohol group, ethylene glycol may permeate faster because of its smaller molecular weight (62 vs. 92). Alternatively, the movement of ethylene glycol through AQP3 competes with and suppresses that of water, thus promoting the permeation by ethylene glycol. This is suggested
MEMBRANE PERMEABILITY AND CRYOPRESERVATION Facilitated diffusion via channel processes not only increases membrane permeability but also decreases the temperature dependency of the permeability. Therefore, the expression of water/cryoprotectant-permeable channels affects the procedure for the cryopreservation of cells. Especially for vitrification, the time and temperature for exposure of embryos to the vitrification solution are critical, because embryos could be easily injured by a high concentration of cryoprotectant(s), although embryos need to be permeated by the cryoprotectant to prevent intracellular ice from forming. For mouse morulae, the period of exposure to the cryoprotectant solution should be shorter than for embryos at earlier stages, because water and cryoprotectants permeate morulae rapidly, and excess exposure to high concentrations of cryoprotectant(s) is toxic to the embryos. The temperature for exposure of mouse morulae to cryoprotectant solutions
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Table 4B.3 Permeability to cryoprotectants (Ps; 10−3 cm/min) of AQP3 cRNA-injected Xenopus oocytes in cryoprotectant solutions Cryoprotectant dissolved in Barth’s solution Oocytes
10% Glycerol
8% EG
10% PG
0.2 Osmol/kg AA
Injected with water Injected with AQP3 cRNA
0.04 ± 0.01 36.13 ± 7.63*
0.11 ± 0.03 33.50 ± 2.75*
0.10 ± 0.02 31.45 ± 5.17*
6.50 ± 1.98 24.60 ± 9.90*
0.2 Osmol/kg DMSO 0.79 ± 0.40 6.33 ± 2.76*
*Significantly different from water-injected oocytes (Student’s t test, P < 0.01). Modified from Yamaji et al.50 EG, ethylene glycol; PG, propylene glycol; AA, acetamide; DMSO, dimethylsulfoxide
and for the dilution of cryoprotectants would not be a very important factor from the viewpoint of membrane permeability, because the channel processes are less affected by temperature. However, in terms of the toxicity of cryoprotectants, the exposure of embryos to cryoprotectant solutions at high temperature should be avoided, because cryoprotectants are more toxic at higher temperatures. Blastocysts are also highly permeable to water and cryoprotectants like morulae, but they have a blastocoel. In mouse blastocysts, it has been shown that the distribution of AQPs in the plasma membrane is not homogeneous among the apical and basolateral sides of the trophectoderm and inner cell mass.34 This suggests that blastocysts have compartments differing in membrane permeability, although overall they are highly permeable to water and cryoprotectants. Moreover, expanded blastocysts contain a large amount of water, most of which has to diffuse out before cryopreservation. At the same time, the cryoprotectant(s) has to permeate the blastocoel through trophoblastic cells. To minimize the toxic effect of the cryoprotectant while promoting dehydration and permeation, a modified procedure is required
for the cryopreservation of blastocysts having a large blastocoel, in which ice forms easily. Usually, blastocysts are exposed to the cryoprotectant in a stepwise manner: first in a solution containing a low concentration of cryoprotectant for permeation, and then in vitrification solution to cause the embryos to shrink via rapid dehydration. In some cases, puncturing the blastocoel or ultrarapid vitrification would be effective. Such approaches will be described further in other chapters in this book. In mouse embryos at early cleavage stages, most water and cryoprotectant molecules permeate slowly by simple diffusion across the plasma membrane. Thus, the stepwise treatment of embryos, as in the case of blastocysts, is effective. The temperature for the treatment is very important, because the movement of water and cryoprotectants by simple diffusion is greatly affected by temperature. After warming, the temperature at which embryos are diluted is also important to prevent osmotic overswelling during the recovery of cryopreserved embryos. Thus, permeability and the pathway by which water and cryoprotectants move in embryos are closely related to the protocol used for cryopreservation.
References 1. Whittingham DG, Leibo SP, Mazur P. Survival of mouse embryos frozen to −196°C and −269°C. Science 1972; 178: 411–4.
2. Rall WF, Fahy GM. Ice-free cryopreservation of mouse embryos at −196°C by vitrification. Nature 1985; 313: 573–5.
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3. Rall WF, Wood MJ, Kirby V et al. Development of mouse embryos cryopreserved by vitrification. J Reprod Fertil 1987; 80: 499–504. 4. Nakao K, Nakagata N, Katsuki M. Production of chimeric mice from cryopreserved blastocysts. Exp Anim 1998; 47: 167–71. 5. Nakagata N. High survival rate of unfertilized mouse oocytes after vitrification. J Reprod Fertil 1989; 87: 479–83. 6. Migishima F, Suzuki-Migishima R, Song SY, et al. Successful cryopreservation of mouse ovaries by vitrification. Biol Reprod 2003; 68: 881–7. 7. Kasai M, Komi JH, Takakamo A, et al. A simple method for mouse embryo cryopreservation in a low toxicity vitrification solution, without appreciable loss of viability. J Reprod Fertil 1990; 89: 91–7. 8. Ishimori H, Saeki K, Inai M, et al. Vitrification of bovine embryos in a mixture of ethylene glycol and dimethyl sulfoxide. Theriogenology 1993; 40: 427–33. 9. Miyake T, Kasai M, Zhu SE, et al. Vitrification of mouse oocytes and embryos at various stages in an ethylene glycol-based solution by a simple method. Theriogenology 1993; 40: 121–34. 10. Kasai M, Nishimori M, Zhu SE, et al. Survival of mouse morulae vitrified in an ethylene glycol-based solution after exposure to the solution at various temperatures. Biol Reprod 1992; 47: 1134–9. 11. Kasai M, Hamaguchi Y, Zhu SE, et al. High survival of rabbit morulae after vitrification in an ethylene glycol-based solution by a simple method. Biol Reprod 1992; 46: 1042–6. 12. Tachikawa S, Otoi T, Kondo S, et al. Successful vitrification of bovine blastocysts, derived by in vitro maturation and fertilization. Mol Reprod Dev 1993; 34: 266–71. 13. Hochi S, Fujimoto T, Braun J, et al. Pregnancies following transfer of equine embryos cryopreserved by vitrification. Theriogenology 1994; 42: 483–8. 14. Martinez AG, Matkovic M. Cryopreservation of ovine embryos: slow freezing and vitrification. Theriogenology 1998; 49: 1039–49. 15. Mukaida T, Wada S, Takahashi K, et al. Vitrification of human embryos based on the assessment of suitable conditions for 8-cell embryos. Hum Reprod 1998; 13: 2874–9. 16. Mochida K, Matsuda J, Noguchi Y, et al. Birth of pups by transfer of mastomys embryos cryopreserved by vitrification. In: Proc 31st Annual Meeting Soc Study Reprod 1998:
17. 18.
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180–1, abstr. Society for the Study of Reproduction, Inc, Madison, WI, USA. Han M-S, Niwa K, Kasai M. Vitrification of rat embryos at various developmental stages. Theriogenology 2003; 59: 1851–63. Mochida K, Wakayama T, Takano K, et al. Birth of offspring after transfer of Mongolian gerbil (Meriones unguiculatus) embryos cryopreserved by vitrification. Mol Reprod Dev 2005; 70: 464–70. Kasai M. Cryopreservation of mammalian embryos: vitrification. Methods Mol Biol 1995; 38: 211–9. Kasai M. Cryopreservation of mammalian embryos. Mol Biotechnol 1997; 7: 173–9. Shaw JM, Kasai M. Embryo cryopreservation for transgenic mouse lines. Methods Mol Biol 2001; 158: 397–419. Whittingham DG. Survival of mouse embryos after freezing and thawing. Nature 1971; 233: 125–6. Kasai M, Zhu SE, Pedro PB, et al. Fracture damage of embryos and its prevention during vitrification and warming. Cryobiology 1996; 33: 459–64. Kasai M, Ito K, Edashige K. Morphological appearance of the cryopreserved mouse blastocyst as a tool to identify the type of cryoinjury. Hum Reprod 2002; 17: 1863–74. Pedro PB, Zhu SE, Makino N, et al. Effects of hypotonic stress on the survival of mouse oocytes and embryos at various stages. Cryobiology 1997; 35: 150–8. Pedro PB, Yokoyama E, Zhu SE, et al. Permeability of mouse oocytes and embryos at various developmental stages to five cryoprotectants. J Reprod Dev 2005; 51: 235–46. Pedro PB, Kasai M, Mammaru Y, et al. Change in the permeability to different cryoprotectants of bovine oocytes and embryos during maturation and development. In: Proc 13th Int Congr Anim Reprod 1996; 3: abstr P15–9. The University of Sydney Printing Servece Sydney Australia. Kasai M. Cryopreservation of mammalian embryos by vitrification. In: Mori T, Aono T, Tominaga T, Hiroi M, eds. Perspectives on Assisted Reproduction (Frontiers in Endocrinology) Rome, Ares-Serono Symposia Publications, 1994; 4: 481–7. Kasai M, Niwa K, Iritani A. Survival of mouse embryos frozen and thawed rapidly. J Reprod Fertil 1980; 59: 51–6. Kasai M, Niwa K, Iritani A. Protective effect of sucrose on the survival of mouse and rat
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31. 32.
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42. Toner M, Cravalho EG, Armant DR. Water transport and estimated transmembrane potential during freezing of mouse oocytes. J Membrane Biol 1990; 115: 261–72. 43. Edashige K, Tanaka M, Ichimaru N, et al. Channel-dependent permeation of water and glycerol in mouse morulae. Biol Reprod 2006; 74: 625–32. 44. Offenberg H, Thomsen PD. Functional challenge affects aquaporin mRNA abundance in mouse blastocysts. Mol Reprod Dev 2005; 71: 422–30. 45. Mazur P, Rigopoulos N, Jackowski SC, et al. Preliminary estimates of the permeability of mouse ova and early embryos to glycerol. Biophys J 1976; 16: 232a. 46. Jackowski S, Leibo SP, Mazur P. Glycerol permeability of fertilized and unfertilized mouse ova. J Exp Zool 1980; 212: 329–41. 47. Ishibashi K, Sasaki S, Fushimi K, et al. Molecular cloning and expression of a member of the aquaporin family with permeability to glycerol and urea in addition to water expressed at the basolateral membrane of kidney collecting duct cells. Proc Natl Acad Sci USA 1994; 91: 6269–73. 48. Zeuthen T, Klaerke DA. Transport of water and glycerol in aquaporin 3 is gated by H+. J Biol Chem 1999; 274: 21631–6. 49. Valdez DM Jr, Hara T, Miyamoto A, et al. Expression of aquaporin-3 improves the permeability to water and cryoprotectants of immature oocytes in the medaka (Oryzias latipes). Cryobiology 2006; 53: 160–8. 50. Yamaji Y, Valdez DM Jr, Seki S, et al. Cryoprotectant permeability of aquaporin-3 expressed in Xenopus oocytes. Cryobiology 2006; 53: 258–67. 51. Meinild AK, Klaerke DA, Zeuthen T. Bidirectional water fluxes and specificity for small hydrophilic molecular in aquaporins 0–5. J Biol Chem 1998; 273: 32446–51. 52. Tsukaguchi H, Shayakul C, Berger UV, et al. Molecular characterization of a broad selectivity neutral solute channel. J Biol Chem 1998; 273: 24737–43.
embryos stored at 0°C. J Reprod Fertil 1983; 68: 377–80. King LS, Kozono D, Agre P. From structure to disease: the evolving tale of aquaporin biology. Nat Rev Mol Cell Biol 2004; 5: 687–98. Edashige K, Sakamoto M, Kasai M. Expression of mRNAs of the aquaporin family in mouse oocytes and embryos. Cryobiology 2000; 40: 171–5. Offenberg H, Barcroft LC, Caveney A, et al. mRNAs encoding aquaporins 1–9 are present during murine preimplantation development. Mol Reprod Dev 2000; 57: 1–8. Barcroft LC, Offenberg H, Thomsen P, et al. Aquaporin proteins in murine trophectoderm mediate transepithelial water movements during cavitation. Dev Biol 2003; 256: 342–54. Verkman AS, van Hoek AN, Ma T, et al. Water transport across mammalian cell membranes. Am J Physiol 1996; 270: C12–30. Leibo SP. Water permeability and its activation energy of fertilized and unfertilized mouse ova. J Membr Biol 1980; 53: 179–88. Hunter J, Bernard A, Fuller B, et al. Measurements of the membrane water permeability (LP) and its temperature dependence (activation energy) in human fresh and failedto-fertilize oocytes and mouse oocytes. Cryobiology 1992; 29: 240–9. Benson CT, Critser JK. Variation of water permeability (LP) and its activation energy (Ea) among unfertilized golden hamster and ICR murine oocytes. Cryobiology 1994; 31: 215–23. Gao DY, Benson CT, Liu C, et al. Development of novel microperfusion chamber for determination of cell membrane transport properties. Biophys J 1996; 71: 443–50. Litkouhi B, Marlow D, McGrath JJ, et al. The influence of cryopreservation on murine oocyte water permeability and osmotically inactive volume. Cryobiology 1997; 34: 23–35. Pfaff RT, Liu J, Gao D, et al. Water and DMSO membrane permeability characteristics of invivo and in-vitro derived and cultured murine oocytes and embryos. Mol Hum Reprod 1998; 4: 51–9.
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5
Evgenia Isachenko, Vladimir Isachenko, Igor I Katkov, Raul Sanchez, Hans van der Ven and Frank Nawroth
the addition and removal of cryoprotectants, and the possibility of its negative influence on the genetic apparatus has, as yet, not been solved,5,6 and cellular cryodamage can arise during slow thawing.7 At present the freezing procedures for many species including human show acceptable results, but cryoprotectant solutions and freezing equipment are necessary. Most IVF laboratories prefer programmable freezing devices. The whole freezing procedure (equilibration, freezing, and dilution of cryoprotectant) takes around 30–60 minutes. Cryopreservation by direct plunging into liquid nitrogen (vitrification) could be beneficial when compared with the ‘slow’ method, because it does not need any expensive equipment and takes only a few seconds for freezing and warming. The vitrification technique arose as an alternative to slow conventional freezing, in order to avoid the crystallization.8 The successful vitrification of frog9 and fowl spermatozoa10 has supported Luyet’s proposal. However, the subsequent attempts to vitrify mammalian spermatozoa using this technique resulted in low or no survival.11,12 Based on this, the vitrification technique was initially unacceptable for routine work. While this method of vitrification using high concentrations of permeable cryoprotectants was successfully applied in 1985 for mouse embryos,13 nevertheless it was impossible to perform this technique for spermatozoa cryopreservation because of the resulting osmotic and cytotoxic effects.5,14
INTRODUCTION Cryopreservation and low-temperature storage of different cell types and tissues, including male and female gametes and embryos, are used worldwide and have become an integral part of most human IVF programs. Since the late 1930–1940s1,2 spermatozoa of several mammalian species, especially bovine and human, have been cryopreserved effectively. The empirical methods of cryopreservation developed in the 1950s are still used today, and are very important as they allow the preservation of male fertility before radiotherapy and/or chemotherapy.3 Such treatments and some kinds of surgical procedures may lead to testicular failure or ejaculatory dysfunction.4 During conventional freezing, water precipitates as ice and thus separates from dissolved substances. Both intracellular ice crystal formation and the high concentration of dissolved substances pose problems. Slow cooling rates aim to maintain a very delicate balance between these factors, yet often lead to cell damage mostly because of ice crystallization, but also due to osmotic and chilling injury, cytoplasm fracture, or even effects on the cytoskeleton or genome-related structures. Due to damage associated with freezing, the motility of cryopreserved spermatozoa after thawing is significantly reduced in comparison with the motility before and shows a wide interindividual variability.5 To date, the problem of cryoprotectant toxicity due to osmotic stress during
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with a loss of sperm motility.22,23 As previously suggested,23 the injury of human spermatozoa induced by conventional cryopreservation occurs mainly during thawing and has been related to diminished antioxidant defense activity during cooling, and/or structural damage to the cytoskeleton, and/or antioxidant enzymes during cryopreservation.23 All these findings suggest that slow cooling, and especially thawing of spermatozoa, quite apart from ice crystal formation, is intrinsically deleterious. The use of egg yolk as a non-permeable cryoprotectant provides indirect support for this viewpoint. Egg yolk, a natural complex of lipids, proteins, lipoproteins, cholesterol, phospholipids, and antioxidants, has been used in sperm preservation for many years to reduce the negative influence of freezing/thawing, as well as cold and osmotic shock at temperatures above 0°C. How this protective effect is exerted is not entirely clear, but all the components of egg yolk may play a pivotal role in reducing the deleterious effects of hyperosmotic salt solutions, membrane lipid phase transition, and peroxidation by changing the composition of membranes, making them stronger (mechanism of membrane fortification) and less susceptible to peroxidation damage.18,24 To prevent excessive cell shrinkage during slow cooling, permeable cryoprotective agents (CPAs) are used. However, the effectiveness (prevention of intracellular ice formation) of permeable and non-permeable cryoprotectants during conventional freezing can only be achieved with a low cooling rate,20 which as we indicated above can be damaging itself. In addition to this, the introduction (during freezing) and removal (after thawing) of CPAs can produce damage per se, even at room temperature in the absence of freezing/thawing. The main mechanisms of CPAs’ toxicity have been discussed by us elsewhere,25 and include osmotic damage as well as chemical cell and membrane toxicity.5,19,26 All these negative effects of conventional slow
CRYOPRESERVATION OF SPERMATOZOA (CONVENTIONAL METHOD) AND CELL DAMAGE DURING FREEZING AND THAWING The beneficial effects of glycerol and nonpermeable cryoprotectants, including sucrose, on plant cryostability were described as early as 1908.15 In 1937 this was followed by the demonstration of the positive effect of 1 mol/L glycerol on rabbit, guinea pig, bull, ram, stallion, and boar sperm1 frozen to −21°C. In the late 1940s, the results of experiments based on the use of glycerol in the UK by Polge, Smith and Parkes2 as well as in the USSR by Smirnov16 and Milovanov17 were published. These empirical methods, which were subsequently developed in the 1950s for use in many species, are still applied today. The motility of cryopreserved/thawed spermatozoa normally drops to about 50% of their pre-freezing value, with considerable intersample fluctuation.5 The question of diminished spermatozoal motility after cryopreservation is crucial since this variable is known to be the first affected,18 although the mechanism of sperm impairment and its mechanical and/or physical–chemical etiology remain unclear. The mechanical cell injury by conventional (ice-equilibrium) freezing is a consequence of intracellular or extracellular ice crystal formation, and osmotic damage due to extensive cell shrinkage. Subsequent re-warming and thawing of the cells can further deteriorate their viability through possible excessive osmotic swelling.19,20 As a result, average velocity in terms of the percentage of motile spermatozoa drops significantly after cryopreservation when compared with that of fresh sperm.18 Conventional slow freezing may also cause extensive chemical and physical damage to sperm cell membranes due to changes in lipid phase transition and/or increased lipid peroxidation. It is well established that the production of reactive oxygen species leads to an increase in lipid peroxidation after cryopreservation21 and that this event is associated
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(secondary drying).29 Neither method necessarily implies a need for high initial concentration of solutes. However, eventually the solute concentration dramatically increases due to dehydration, that leads to a very low content of residual water. The next approach is vitrification of the bulk solution, which is free of a sizable portion of ice during cooling and thawing, performed substantially faster than with equilibrium freezing (during which ice is formed). This can be done either by cooling at relatively slow to moderate speed, but using high concentrations of CPAs/vitrificants such as dimethylsulfoxide (DMSO), ethylene glycol, propylene glycol, or glycerol, or by solidification of the bulk solution by abrupt cooling at very high speed to temperatures below the glass transition temperature of the solution (Tg, the temperature at which the transition to the vitreous condition begins) by plunging the specimen directly into liquid nitrogen. According to our experience, this can be achieved in the absence of the ‘conventional’ CPAs, provided that the cooling/warming speed is high enough to ensure both intra- and extracellular vitrification. In general, the rate of cooling/warming and the concentration of cryoprotectants required to achieve vitrification are inversely related. In other words, the higher the CPA concentration, the lower the critical speed of cooling and warming needed to avoid ice formation. Conversely, the faster cooling and warming is undertaken, the lower the critical solute concentration necessary to obtain icefree vitrification. Although the pioneer of the idea of vitrification, Luyet, initially emphasized the need for fast cooling and warming of relatively dilute solutions,8 it is clear from the literature, that the use of highly concentrated vitrification solutions soon became mainstream practice. Given the biological and physiochemical effects of cryoprotectants and the high concentrations used in vitrification, it is therefore not surprising that cryoprotectant toxicity has been described as a key limiting factor in
(ice-equilibrium) freezing and thawing on cells can also lead to chromatin damage, and as has recently been noted,27 the chromatin abnormalities have repercussions on sperm quality and male fertility status.
VITRIFICATION AS AN ALTERNATIVE TO CONVENTIONAL FREEZING The vitrification technique has its own peculiarities. A decisive factor of cryopreservation is the incompatibility of crystallization with living systems.8 It is possible to eliminate ice formation and create instead a glass-like (vitreous) state by cooling of small liquid suspensions or just water at ultra-high speeds of cooling.9 According to Fahy28 the physical definition of vitrification is the solidification of a solution at low temperature, not by ice crystallization, but by extreme elevation in viscosity during cooling that provides stability and invariability of the entire solution, because the water does not precipitate, so no ice crystals are formed. However, the glass-like solidification of water requires a very high cooling rate (> 300 000° C/min). Thus, to preserve living organisms stably for a long period, they have to be placed in an environment where the viscosity of both intra- and extracellular water are raised to levels that result in the essential arrest of diffusion, so practically all chemical processes including degradation and aging are effectively stopped. There are several ways to achieve vitrification. One approach is to slowly (equilibrium) freeze out the pure water as ice so that the remaining solution becomes more and more concentrated until it reaches its point of vitrification, at a temperature usually referred to as Tg, or the glass transition temperature at equilibrium freezing. Another method is to remove excess water either by sublimation of the frozen portion of ice (lyophilization or freeze-drying), which is performed at subzero temperatures, or by evaporation from the liquid phase by direct vacuum/air drying, or by softening and liquefying the glassy material left after lyophilization
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the cryobiology of vitrification. In an attempt to avoid this toxicity, we achieved ultra-rapid cooling and warming rates in the range of 0.5–1 ×106K/min using a relatively dilute vitrification medium (around 12% total solute content), of similar solute concentration to semen or blood; thus, ‘resurrecting’ the original vitrification approach of Luyet.
pressure suffices for subsequent storage. Moreover, it has been possible to lower the critical CPA concentration (Cv) by also including non-permeable polymers, which are unable to enter the cells.28 Cryoprotectant toxicity can also be reduced by combining two cryoprotectants, and/or exposing the tissue to a graded series of pre-cooled concentrated solutions. These modifications of the conventional method reduce the toxic and osmotic effects of cryoprotectants. In addition, if we increase the rate of cooling and warming, the cryoprotectant concentration can be reduced even further, diminishing its toxicity. It was recently shown that the higher cooling rate provided by the nylon loop, permits an effective reduction in cryoprotectant concentration such that 5.5 mol/L ethylene glycol (EG) can be substituted by a 3.2 mol/L EG/3.2 mol/L DMSO mixture.14 The size of the sample to be cryopreserved should be minimized such that most of the specimen will be immersed in liquid rather than vapor leading to the maximal cooling rate. To achieve even higher cooling rates, the volume of vitrification solution should also be kept to a minimum through the use of specially designed carriers such as open pulled straws (OPS),35 the flexipet-denuding pipette,14,36 micro drops,37 electron microscope copper grids,38 the hemi-straw system,39 small nylon coils,40 or nylon meshes,41 and the Cryoloop.36 These all have been used as carriers or vessels to achieve higher cooling rates and have provided good results for the vitrification of embryo species that are particularly susceptible to freeze damage42 along with the highly sensitive human oocytes.43 There have even been reports of the successful vitrification of human embryonic stem cells using the OPS method.44 A further factor to consider is the changes in the properties of ethylene glycol-based vitrification solutions after the addition of sugars (sucrose, glucose, fructose, sorbitol, trehalose, or raffinose).45 High molecular weight additives such as disaccharides e.g. sucrose or
VITRIFICATION USING HIGH CRYOPROTECTIVE AGENT CONCENTRATIONS: ACHIEVEMENTS AND PITFALLS In contrast to the conventional slow freezing ice-forming techniques, the protocols of vitrification currently used for the cryopreservation of oocytes, embryos, and tissues as a rule involve the use of very high concentrations (3.5–8 mol/L) of permeating cryoprotectants and relatively high cooling rates (up to 105°C/min) compared with rates associated with conventional slow freezing. According to the literature, the critical cooling speed for the vitrification of pure water varies drastically, depending on the method used, from 106 to 1010°C/min (see Figure 9 in Karlsson30 for references). Kanno et al.31 were able to demonstrate that the temperature of homogeneous crystallization (Th) can be reduced through an increase in the hydrostatic pressure. Later, MacFarlane et al.32 observed that Tg rises with increasing pressure, allowing glass transition at lower cryoprotectant concentrations. For example, for a 35% liquid DMSO solution at a hydrostatic pressure of 1300 atm, Th is −80°C and the solution vitrifies.33 A downside of this is that the increased pressure can cause damage to the biological system. For example, dog kidneys can survive 20 min of exposure to 1000 atm,34 while rabbit kidneys show signs of severe damage after only 20 min at 500 atm. This high pressure, however, is only needed during the vitrification process itself and atmospheric
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trehalose, cannot pass through the cell membrane, but can significantly reduce the amount of cryoprotectant required as well as the toxicity of ethylene glycol by decreasing the concentration needed to efficiently cryopreserve human oocytes and embryos. Adding non-permeating compounds to the vitrifying solution as well as pre-incubation in this solution before vitrification to withdraw more water from the cells, shortens the time of exposure of the cells to the toxic effects of the cryoprotectants. Non-permeating sucrose also acts as an osmotic buffer, reducing the osmotic shock induced by diluting the cryoprotectant after cryopreservation. However, even molecules as large as sucrose (MW = 342), trehalose (MW = 378), or raffinose (MW = 504) can produce substantial osmotic damage when used at high concentrations. A further option is to add high molecular weight polymers, both synthesized (polyvinyl pyrrolidone, polyethylene glycol) and of a biological origin (proteins). This approach is discussed below.
methods used for other types of mammalian cells.46 Classical vitrification requires a high proportion of permeable cryoprotectants in the medium (from 3.5 to 8 mol/L along or with carbohydrates in combination with a cooling rate from 2000 to 30 000°C/min achieved by direct plunging into liquid nitrogen (LN2) compared with 5–7% for slow-freezing with 0.3°C/min cooling rate), and seems to be unsuitable for the vitrification of spermatozoa due to the lethal osmotic effects and possible chemical alterations. In the opinion of some authors, this is the main reason for the lack of any significant practical results so far (see Holt47 for a comprehensive analysis of the background). Indeed, using the ‘conventional’ methods of vitrifying human spermatozoa, our survival rates have been extremely low or even lacking. This prompted our idea of exploring vitrification methods that would not require high concentrations of potentially toxic CPAs.
Do we always need cryoprotectants for successful vitrification?
BACKGROUND OF SPERM VITRIFICATION
Still Luyet, in his vast number of papers on cryopreservation by vitrification, mentioned that a small specimen cooled very rapidly could be vitrified without substantial loss of viability.48 Observations of Jahnel,49 who performed cryoprotectant-free cryopreservation of human spermatozoa cooled in liquid nitrogen and liquid helium (−269.5°C), and Parkes who also published in 194550 on freezing of human spermatozoa without cryoprotectants. Large volumes (milliliters) of sperm were cooled in glass or metal tubes. Probably due to the absence of quick thawing, motility of spermatozoa after thawing was reduced. In 194211 the freezing of human and rabbit spermatozoa using a bacteriological loop to rapidly cool small specimens was described. These authors obtained up to 40% of viable human spermatozoa after cooling of a sperm film in LN2 followed by quick warming of these microvolumes. Later, however, they reported
After the successful vitrification of frog spermatozoa by Luyet and Hodapp in 1938,9 4 years later Shaffner10 vitrified fowl spermatozoa. He modified Luyet’s sperm vitrification technique which was considered an attractive alternative to conventional slow freezing. However, early attempts at vitrifying mammalian spermatozoa using this approach had very low or no survival11,12 mostly because, as shown below, critical speeds of freezing and warming were unachievable at that time; the low CPA concentration tolerated by sperm requiring high speeds. In spite of this, in the early 1980s, Rall and Fahy13 managed to successfully vitrify embryos using high CPA concentrations and a relatively low speed of cooling and warming and since then, the main approach to vitrification of spermatozoa has been considered the same as the
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low or no viability of mammalian sperm after vitrification.11,12 Some years later, when investigators turned their attention to the vitrification of large cells, tissues, and even organs, the dogma was established that vitrification could only be achieved using high concentrations of combinations of permeable (PCP) and impermeable (PVP) cryoprotectants.48 The total concentration of these protecting agents had to be at least 50% w/w (if performed at atmospheric pressure) to achieve stable vitrification and enable the use of a relatively low speed of cooling and warming. Such high CPA concentrations can be very damaging to cells, causing both biochemical alterations and lethal osmotic injury.51 It has been possible to assess the relative contributions of osmotic damage and true biochemical alterations to apparent ‘CPA toxicity’.26 Very often, some of the deleterious effects of exposure to cryoprotectants can be avoided by optimizing regimens for the addition and removal of the CPAs5,19,26 for human and animal sperm; we dedicated a set of papers to this particular problem.52 However, these methods also have their limitations. Regardless of the mechanism of damage, we could summarize that in the majority of species, sperm cannot tolerate the high concentrations of cryoprotectants conventionally used for vitrification. Therefore the obvious alternative is to use very rapid cooling and warming rates on a very small sample size. Recently, Bischof ’s group52 predicted that one of the optimal cooling rates for spermatozoa theoretically lies between 5000 and 7000°C/min. According to other data53 the form and size of sperm head could be the factors which define the cryosensitivity of cells. Comparative investigation54 of cryoproperties from different mammalian species (boar, bull, ram, rabbit, cat, dog, horse, human) has shown a negative correlation between size of sperm head and cryostability. Among the above-mentioned mammalian species, the human spermatozoa have minimal size parameters and maximal cryostability.20
Taking into account all these data, we have assumed that vitrification of human spermatozoa without permeable cryoprotectants can be successful.
VITRIFICATION OF SPERMATOZOA: OUR EXPERIENCE Our results agreed with the above-mentioned data. In our preliminary investigation,25,55 the study performed in Italy and Germany which was approved by the University Review Board (Italy) and University Ethics Committees (Germany), we have shown that the successful vitrification of spermatozoa is possible without permeable cryoprotectants. It was necessary to investigate not only the influence of vitrification technique (vitrification using Cryoloops) on physiological spermatozoa parameters, but also to compare results of this technique with routinely used conventional programmable freezing in French straws.56 Earlier it was shown that the simple ‘swim up’ procedure allows selection of spermatozoa with progressive motility and normal morphology before freezing.6,57,58 Taking this into account we have investigated the role of such treatment on the physiological parameters of spermatozoa after both slow freezing and vitrification. We found a 1.6-fold increase in sperm motility and a 1.9-fold increase in the percentage of morphologically normal spermatozoa in swim up prepared spermatozoa compared with original samples. However, the negative influence of the routinely used cryoprotectant medium (TEST-egg-yolk-glycerol, TEYG) after only 10 min exposure before freezing led to a significantly reduced motility by 12.6% in the original semen sample and by 13.8% in swim up prepared spermatozoa, as well as to a reduced percentage of living cells by 11.4% for the original sample and by 13.1% for swim up prepared spermatozoa compared with non-treated samples (P < 0.05). The programmable freezing reduced the percentage of living cells by 24.5% in the ejaculated
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semen and by 22.2% in swim up prepared samples (P < 0.05). The percentage of acrosomereacted cells after TEYG-treatment before cooling was increased by 10.9% in semen and by 15.7% for swim up prepared spermatozoa. After programmable freezing with cryoprotectant compared with non-frozen samples the motility was reduced by 16.4% for semen and by 35.6% for swim up prepared spermatozoa; also the rate of spermatozoa with normal morphology was decreased by 11.6% for semen samples and by 12.3% for swim up prepared ones (P < 0.05). Nevertheless, beneficial effects of cryoprotectant could be shown when comparing with sperm parameters after conventional freezing of TEYGtreated and non-treated spermatozoa. Slow freezing without cryoprotectant reduced sperm motility dramatically for original semen samples (by 43.2%) and for swim up prepared spermatozoa (by 86.1%) compared with the values before freezing. It was shown that slow freezing without cryoprotectant reduced the rate of normal sperm morphology by 7.3% for semen and by 25.0% for swim up prepared spermatozoa compared with the same method after treatment with cryoprotectant. In contrast to spermatozoa conventionally frozen with cryoprotectant the percentage of motile spermatozoa recovery frozen without cryoprotectant was 1.3% for semen and 0.2% for swim up prepared spermatozoa (P < 0.05). Completely opposite results were obtained after vitrification of spermatozoa on Cryoloops. To perform the vitrification procedure, we manufactured our own cryoloops55 with a 5 mm diameter and a loaded volume of 20 ± 2 µl (Figure 5.1). The warming was performed by plunging the Cryoloops into a centrifuge tube containing 10 ml of sperm preparation medium (SPM; Scandinavian IVF Science, Gothenburg, Sweden) at 37°C under intense agitation. The same concentration of cryoprotectant as for conventional freezing had a highly toxic (possibly osmotic) effect. This is questionable because we do not know
what kind of effect glycerol has on the sperm membrane and intracellular structures, because the typical sperm osmotic reaction of ‘coiled tail’ was not noted. Vitrification without permeable cryoprotectant (only 1% human serum albumin in preparation medium – medium + 1% HSA) has shown the best results with swim up prepared spermatozoa. In comparison with the best post-thaw data after conventional freezing with obligatory use of permeable cryoprotectants, vitrification resulted in a significant increase of sperm motility after thawing (by 11.6%; P < 0.05). However, the difference in morphology, motile sperm recovery, viability after freezing, and acrosome reacted cells between the two cryopreservation methods and sperm treatments (with or without cryoprotectant) was not statistically significant (P > 0.05). At present, it is not clear why all these parameters for ejaculated spermatozoa after vitrification are lower than those of spermatozoa vitrified after swim up. We have found that, in contrast to vitrification, conventional freezing of ejaculated and swim up prepared spermatozoa without cryoprotectant resulted in 34.4% and 25.2% living cells (P < 0.05), respectively, with nearly all of them being non-motile.
FREEZING IN LIQUID NITROGENS VAPOR AND INTRACELLULAR VITRIFICATION To investigate the role of warming velocity for successful viability of vitrified samples we designed an interesting experiment,60 and developed an original technique (Figure 5.2). The experiment was conducted as follows. A pre-loaded Cryoloop with spermatozoa suspension was placed without agitation in a horizontal position into an N2 vapor at −160°C. A thin (27 gauge) injection needle was then used to periodically (at ~1 s intervals) transfix the film at different locations on the loop (center, near the copper ring at the periphery). When the film is liquid, it is possible to punch through it many times without disruption of
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3
4
5 3
6 2
1
Figure 5.1 Scheme of ‘Cryoloop’ vitrification and warming of spermatozoa. (1) suspension of spermatozoa, (2) Petri dish, (3) Cryo-loop, (4) film of sperm suspension, (5) tube for warming, (6) warming medium. With permission from Isachenko et al.59
calculations of this scenario are very complex. However, we were able to estimate the average rate of cooling, by first measuring the time of solidification of the pellicle near the ring, and then estimating the time taken for the surface of the film to completely solidify at the center. This gave an initial cooling rate in the range of 270°C/min near the copper ring to 162°C/min at the center of the film. It is clear that at this rate of cooling and in the absence of any viscous vitrificants in the SPM (sperm preparation medium) medium, the extracellular milieu of the cells will not vitrify, but will start freezing with the initiation of ice crystal formation. However, human spermatozoa contain large amounts of proteins, sugars, and other components that make the intracellular matrix highly viscous and compartmentalized. The quantity of high molecular weight macromolecules and polymers diluted in the cytosol can be estimated from the fraction of the osmotically inactive volume, which is about 20–25% for embryos and oocytes, and much higher (45–77%) for spermatozoa.21 As
the film, and after the needle is removed the film remains intact. Upon freezing, the film solidifies (starting from the copper ring area towards the center) and transfixion without disruption of the film becomes impossible and so the ring begins to move. The time elapsed (visually indicated) from placing the loop in the box at room temperature (+23°C) to the beginning of solidification of the suspension (−4°C) allowed us to calculate the speed of cooling of the spermatozoa. It was from 6 s near the metal ring to 10 s at the center of it. Thus, the rate of cooling of spermatozoa in N2 vapor was in the range of 162–270° C/min. When the specimen is kept above the surface of the LN2 in its vapor at a temperature of −160OC, given that the thermal conductivity of the vapor is substantially lower than that of the LN2; the cooling rate can be several orders of magnitude lower. The main cooling front will extend from the surface of the copper ring in a radial direction towards the center of the pellicle. The mathematical
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Table 5.1 Fertilization of oocytes with vitrified spermatozoa and subsequent development of embryos
5
Rate of fertilization and development
2
1
4
3
Pronuclear formation 4–6 blastomeres Blastocysts formations
Vitrification with ‘rapid’ cooling (n = 23) (%)
Vitrification with ‘slow’ cooling (n = 22) (%)
79 63 47
86 64 41
From Isachenko et al.60
Figure 5.2 Scheme of estimation of spermatozoa cooling rate in vapor of liquid nitrogen. (1) Copper loop with film of spermatozoa suspension, (2) foam box, (3) liquid nitrogen, (4) foot for loop, (5) needle. With permission from Isachenko et al.60
vapor. It was shown that both regimens of vitrification gave about a 40% reduction of motility of spermatozoa (P < 0.05) when compared with swim-up treated controls. No statistically significant difference was found in this parameter between the two regimens of vitrification. The results from IVF, show approximately equal fertilization potential for fresh human spermatozoa samples compared with swim up prepared CPA-free samples vitrified by direct plunging into LN2 or into N2 vapor (Table 5.1).
a result, we can speculate that we were able to achieve intracellular vitrification of the human spermatozoa even at such a low rate of cooling. A further factor to consider is the small size and high degree of compartmentalization of the sperm head, such that even if small (non-lethal) crystals start to form during this relatively ‘slow’ cooling, there would be insufficient time for substantial growth during cooling. It is known that a major problem for such metastable systems can be the re-growth of crystals and devitrification during warming. The cooling rate of 160–250°C/min is 5–10 times higher than the rate of conventional slow freezing with the use of permeable cryoprotectants. At such a slow freezing speed, water has time to escape from the cells upon freezing. As a result, the cell shrinks and can be osmotically damaged unless an osmotic buffer (permeable CPA) is used to prevent hydration and excessive volume loss. In our case, however, the freezing rate is much faster, so the cell maintains its volume without the need for a conventional cryoprotectant (permeable osmotic buffer or non-permeable CPA). Our data indicate essentially similar results, in terms of sperm motility observed after ‘instant vitrification’ by direct plunging into LN2 and relatively ‘slow’ cooling in N2
ROLE OF DEVITRIFICATION AND IMPORTANCE OF FAST RATE OF WARMING The founder of modern cryobiology emphasized that devitrification and the growth of ice crystals formed during cooling could be a key factor promoting cell damage during rewarming and thawing procedures.9 Therefore, we directly placed the specimens in a warm solution, ensuring a very high rate of warming. In this process, the probability of substantial devitrification (recrystallization) of the vitrified intracellular solution and regrowth of large lethal intracellular crystals is low, due to the high speed and very short time of warming. Our estimations showed that, in general, during cooling and especially during warming, the small specimen size, high viscosity of the freezing medium and intracellular matrix, very high speed of
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warming, small size of the specimen and cells, their low water content, and high degree of compartmentalization would ensure that devitrification (especially intracellular) would not occur.30,61
viscosity of the solution, and high speed of cooling and warming25 would ensure that devitrification (especially intracellularly) would not occur.61 Seemingly, the presence of relatively high concentrations of albumin and some amount of sugars substantially raised the viscosity of the solution, especially at lower temperatures, as well as the small amount of cells and the small specimen size, made vitrification stable during both cooling and warming, and showed positive results after warming. Using such an extreme cryoprotocol for sperm preservation it is reasonable to assume that sperm DNA may be damaged. However, Evenson et al. found no difference in sperm chromatin structure assay results for both non- and cryopreserved sperm, and for slowly or flash-frozen specimens.67 Duty et al.68 have confirmed these results, and found that flash freezing in LN2 without cryoprotectants most closely reproduced the results obtained with freshly ejaculated human spermatozoa. They suggest that the unique packaging of sperm DNA protects it from intracellular fluid shifts, and formation of nuclei of crystallization during the cooling–warming cycle. Thus preventing DNA damage from intracellular fluid shifts and ice crystal formation during cryopreservation.68 The substantial compartmentalization of their intracellular components may also contribute to the successful survival of human spermatozoa. It is also known that the amount of osmotically inactive water is higher in spermatozoa than in oocytes or embryos, since it is bound to several macromolecular structures such as DNA, histones, hyaluronidase, etc. According to our calculations25 the amount of high molecular weight components can be 6–8 times higher than in embryos, and this will probably affect the subsequent increased viscosity and glasstransition temperature of the intracellular cytosol in sperm, yet the risk of lethal ice formation during cooling is likely to be higher for embryos. This hypothesis is supported by
INTEGRITY OF CHROMOSOME APPARATUS OF SPERMATOZOA All the negative influences of freezing on cells, that have been described earlier, can also lead to chromatin damage and are strongly correlated with mutagenic effects.62 It was shown that freezing/thawing in fertile and infertile men resulted not only in significantly changed sperm morphology and membrane integrity, but also in significant chromatin damage. Futhermore, it was shown that the fragmented DNA was negatively correlated with fertilization rates in IVF63 and ICSI.64 Although some deleterious cryoprotectantexposure effects on mammalian sperm can be avoided using optimal regimens of addition and removal of CPA.26,65 However, such methods do not work with very high concentrations of cryoprotectants. With the cooling parameters involved in vitrification such as very high speed (up to 10 000°C/min at the initial phase of cooling), short time (5–8 s), and low specimen size (20 µl), it seems that not many crystallization nuclei were formed, and their size was not large enough to damage human spermatozoa. The probability of substantial devitrification (recrystallization) of the vitrified solution is also low due to the high speed, very short time of warming, and the small size of the specimen (extracellular recrystallization) and the cells (intracellular recrystallization). To investigate the occurrence of apoptosis in spermatozoa DNA after vitrification we performed the comet assay (Figure 5.3). Our estimations66 for albumin showed that, in general, both during cooling and especially during rewarming/resuscitation, the small amount of the specimen and the cells, high
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Damaged DNA (control)
Non-damaged DNA after warming
Comet tail Figure 5.3 Example of damaged and non-damaged DNA of spermatozoa ‘Comet’ assay. With permission from Isachenko et al.66
thawed spermatozoa.66 The DNA integrity (P > 0.05)60 was found to be unaffected by the vitrification method.
at least three findings, two of which were reported by other authors. The first is the successful vitrification of frog spermatozoa, achieved in the absence of permeable CPAs by Luyet and Hodapp in 1938.9 The second is that there are reports of an effective relatively slow freezing protocol for mouse spermatozoa requiring no permeable CPAs.69,70 In both cases, spermatozoa even larger than human spermatozoa were used, therefore for small human spermatozoa the beneficial effect of cell size would be even more pronounced. The third finding supporting our hypothesis is the fact that we were not able to achieve cell survival after vitrification without CPAs using large cells such as embryos and oocytes (unpublished data). All these data were confirmed by our results.60,66 It was shown that no significant difference in DNA integrity was found independent of the cooling rate and presence of cryoprotectants (P > 0.05).66 We found that the DNA integrity of vitrified spermatozoa is comparable with standard slow-frozen and
Factors that may enhance vitrification Several investigations69–71 confirmed the beneficial cryoprotective role of sugars for low temperature preservation of mouse spermatozoa. It is known that human spermatozoa contain large amounts of proteins, sugars, and other components that make the intracellular matrix highly viscous and compartmentalized, and may act as natural cryoprotectants since spermatozoa, which are as osmotically as fragile as those of the mouse,26,29 can be successfully frozen in the absence of permeable CPAs, using protein- and sugar-rich skimmed milk and raffinose as extracellular nonpermeable cryoprotectants.70 To confirm such a stance and improve our vitrification results, we decided to investigate the mitochondrial stability and ability of artificial capacitation induction due to vitrification. The leading
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b
a
b
a
c
e
d
Figure 5.4 Examples of non-capacitated (a), capacitated (b), acrosome reacted (c), and non-viable (d) spermatozoa. (e) Example of healthy non-apoptotic (a) and damaged (b) mitochondria.
detection of mitochondrial function (initiation of cellular apoptosis) we have measured the changes in the membrane potential (∆ψ). A unique fluorescent cationic dye, 5,5′,6,6′tetachloro-1-1′,3,3′-tetraethyl-benzamidazolocarbocyanin iodide, commonly knows as JC-174 was used for detection of the change in mitochondrial membrane potential in apoptotic cells (Figure 5.4e). All investigated swim up prepared ejaculates were divided into three groups and diluted with subsequent media as follows: group 1 with only spermwash medium; group 2 with the sperm-wash medium and 1% HSA; and group 3 with the sperm-wash medium and 1% HSA with 0.25 mol/L sucrose. Our data showed that the increase of the percentage of capacitated spermatozoa after warming was not significantly different to controls in both groups (HSA: 7.2 ± 0.5%; HSA + sucrose: 10.7 ± 2.6%, and control: 8.9 ± 1.1%; P > 0.1). The acrosome-reacted spermatozoa in the group with both supplements (HSA and sucrose) were detected in 5.9 ± 1.9%, in the group with one supplement (HSA) in 11.1 ± 3.9%, and in the
role of mitochondria in different metabolic processes in cells is well known.72 At present a lot of information about the influence of slow conventional freezing on artificial spermatozoa capacitation and mitochondrial function exist. It was shown that sperm capacitation can significantly optimize the acrosome reaction,58 and can possibly induce the acrosome reaction. It was discovered that mitochondrial activities and viability are equally susceptible to cryopreservation-induced damage.73 However, at present, routinely used conventional freezing for human sperm cryopreservation does not induce the capacitation process, acrosome reaction, but cannot preserve the mitochondrial integrity sufficiently.23,73 Thus it was necessary to investigate the influence of vitrification on these physiological processes, and on mitochondrial sensitivity. For this purpose we performed the double fluorescence CTC (5-cyano-2,3-di-(ppolyl)trazolium chloride)-Hoechst 33258 staining technique to evaluate the capacitation and acrosome reaction ability of spermatozoa after vitrification (Figure 5.4a–d). For
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5
4 1
3
6 2 7 7 5
Figure 5.5 Scheme of ‘droplet’ vitrification and warming of spermatozoa. (1) Foam box, (2) liquid nitrogen, (3) foot for aluminum foil, (4) aluminum foil, (5) suspension of spermatozoa, (6) tube for warming, (7) warming medium. With permission from Isachenko et al.59
control in 9.7 ± 1.4% (P > 0.05). It was observed that supplements such as HSA alone (28.6 ± 4.7%) or in combination with sucrose (65.2 ± 2.6%) significantly preserved the mitochondrial membrane integrity compared with controls (7.7 ± 0.7%, P < 0.05). However, the combination of two supplements had a stronger cryoprotective effect (P < 0.05) compared with HSA alone.
with the same concentration as for slow freezing was used for vitrification with cryoprotectant. Samples of spermatozoa were located onto copper loops of 5 mm diameter (volume of drops 20 ± 2 µL), and then the loops were plunged into LN2 (Figure 5.1). The Cryoloops with samples, which were cooled for 3 min in N2 vapor (at −160°C), were placed into pre-cooled cryovials after 5 min and stored in LN2 until the time of use. (2) Cooling in N2 vapor at −160°C or by direct plunging into LN2 using droplets. Spermatozoa were cooled as shown in Figure 5.5. Thirty microliters of sample were dropped onto aluminum foil previously cooled in vapor of N2 to −160°C or direct into LN2. The temperature of foil was determined using an electrical thermometer. After 5 min of cooling, the solidified droplets of SPS were placed into pre-cooled in LN2 cryovials and stored in LN2 until the time of use. (3) Aseptic cooling in LN2 using open pulled straws. Five microliters of SPS were drawn inside the end of open pulled straws by capillary effect.35 Straws were placed inside a sterile 0.5 mL insemination straw
VITRIFICATION METHODS On the basis of these investigations, four different vitrification techniques using rapid warming due to a small amount of cooling/ warming medium, or due to a combination of a relatively large volume of cooled suspension and high warming rates in agitated medium were investigated. In this way, an aseptic technology for spermatozoa vitrification was developed, and the following vitrification techniques were tested. (1) Cooling by direct plunging into LN2 or by cooling in N2 vapor using Cryoloops. The samples of both spermatozoa-prepared groups were divided into the same four subgroups as for the slow protocol. TEYG
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5
5
4
1 7 8
2
2
2 3
5
5
Figure 5.6 Scheme of the method and photograph of container for ‘open-pulled straw’ vitrification and warming of spermatozoa. (1) Open-pulled straw, (2) suspension of spermatozoa, (3) meniscus of suspension, (4) 90 mm straw, (5) heat sealed end of 90 mm straw, (6) marked end of open-pulled straw, (7) tube for warming, (8) warming medium. With permission from Isachenko et al.59
hermetically sealed using an ultrasound hand-held sealer (Figure 5.6) and plunged into LN2. It should be noted that there was no contact between the wall of the 90 mm straw and the suspension of spermatozoa inside the open pulled straws due to the presence of a meniscus of the spermatozoa sample. (4) Aseptic cooling in LN2 using insemination straws. One microliter of sample, using a micropipette, was drawn inside the end of an insemination straw. The straw was
placed inside a sterile 0.5 mL insemination straw and sealed the same way as for the open pulled straw method (Figure 5.7).75 The warming of Cryoloops and droplets was performed by plunging the Cryoloops and droplets into a centrifuge tube with 10 mL of sperm-preparation medium (SPM) at 37°C under intense agitation. The open pulled or insemination straws were rapidly warmed by plunging into 1.5 mL microcentrifuge tubes
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5
5
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2
2
1
7
8
3 3 3
2
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5
5
5
Figure 5.7 Scheme of the method and photograph of container for ‘open straw’ vitrification and warming of spermatozoa. (1) Tip of pipettor, (2) open straw, (3) drop of spermatozoa, (4) 90 mm straw, (5) heat sealed end of 90 mm straw, (6) marked end of open straw, (7) tube for warming, (8) warming medium. With permission from Isachenko et al.59
containing 1 mL of SPM at 37°C after being expelled from their packaging. Comparing these four vitrification techniques we have found that all the regimens of cryopreservation gave about 40% reduction of spermatozoa motility (P < 0.05) in comparison with non-treated swim up control. The quality of spermatozoa increased dramatically 2–5 h later, and decreased after 24 h of culture (Figure 5.8). No statistically significant difference was found in these parameters between all the regimens of cryopreservation tested.
CONCLUSIONS (1) The cryoprotectant-free cryopreservation of human spermatozoa by fast or relatively
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slow cooling, by either direct plunging into LN2 or freezing in N2 vapor beforehand, followed in both cases by rapid thawing is feasible. (2) The speed of warming plays a decisive role in vitrification. (3) The DNA integrity of vitrified spermatozoa is comparable with that of standard slow-frozen spermatozoa and that of fresh sperm. (4) The use of a non-permeable cryoprotectant mixture (HSA and sucrose) can significantly enhance the mitochondrial integrity and prevent initiation of capacitation and the acrosome reaction process.
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25 a
Motility (%)
a, d
a, d
20
d
d
15 b
b, e
b, e e
10
e
b, e
b, e e
c
5
0 Fresh control
Cryoloop
Droplets
Open pulled straw
Open straw
Method of cooling Just after
2 hours
5 hours
24 hours
Figure 5.8 Viability of human spermatozoa after cryoprotectant-free vitrification using different methods of cooling and warming. Different superscripts indicate significant differences (P < 0.05). With permission from Isachenko et al.59
(5)
The vitrification of human spermatozoa without permeable cryoprotectants by direct plunging into LN2 is effective. In contrast to conventional freezing, the method is quick and simple and does not require special cryobiological equipment. The evaluation of motility and
long-term survival of spermatozoa allows us to conclude that all four investigated methods can be used successfully. However, the ‘straw in straw’ vitrification method can be recommended, because it minimizes the potential risk of microbial contamination.
References 5. Critser JK, Huse-Benda AR, Aaker D, Arneson BW, Ball GD. Cryopreservation of human spermatozoa. III. The effect of cryoprotectants on motility. Fertil Steril 1988; 50: 314–20. 6. Perez-Sanchez F, Cooper TG, Yeung CH, Nieschlag E. Improvement in quality of cryopreserved human spermatozoa by swim-up before freezing. Int J Androl 1994; 17: 115–20. 7. Mazur P, Rall WF, Rigopoulos N. Relative contribution of the fraction of unfrozen water and of salt concentration to the survival of slowly frozen human erythrocytes. Biophys J 1981; 36: 653–75. 8. Luyet BJ. The vitrification of organic colloids and of protoplasm. Biodynamica 1937; 1: 1–14.
1. Bernschtein AD, Petropavlovski VV. Influence of non-electrolytes on viability of spermatozoa. Bull Exp Biol Med 1937; III: 21–5. [in Russian] 2. Polge C, Smith AU, Parkes AS. Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature 1949; 164: 666–76. 3. Sanger WG, Oslon JH, Sherman JK. Semen cryobanking for men with cancer – criteria change. Fertil Steril 1992; 58: 1024–7. 4. Donnelly ET, Steele EK, McClure N, Lewis SEM. Assessment of DNA integrity and morphology of ejaculated spermatozoa from fertile and infertile men before and after cryopreservation. Hum Reprod 2001; 16: 1191–9.
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9. Luyet BJ, Hoddap A. Revival of frog’s spermatozoa vitrified in liquid air. Proc Meet Soc Exp Biol 1938; 39: 433–4. 10. Schaffner CS. Longevity of fowl spermatozoa in frozen condition. Science 1942; 96: 337. 11. Hoagland H, Pincus G. Revival of mammalian sperm after immersion in liquid nitrogen. J Genet Physiol 1942; 25: 337–44. 12. Smith AU. Biological Effects of Freezing and Supercooling. London: Edward Arnold Ltd, 1961: 196. 13. Rall WF, Fahy GM. Ice-free cryopreservation of mouse embryos at –196 °C by vitrification. Nature 1985; 313: 573–5. 14. Liebermann J, Tucker M, Graham J et al. Blastocyst development after vitrification of multipronucleate zygotes using the flexipet denuding pipette (FDP). Reprod Biomed Online 2002; 4: 148–52. 15. Maksimov NA. Chemical protection of plants from being killed by frost. Influence of solutions of anorganic electrolytes of the anorganic salts and salts of organic acids. Importance of eutectic point of solutions protection. In: About being killed by frost and cold-resistant of plants. Experimental and critical investigations. Sankt-Petersburg, printing-house of Frolova, Galernaja 6, 1913: 242–278. [in Russian] 16. Smirnov IV. Preservation of domestic animals’ semen by deep cooling. Sovetskaja Zootechnia 1949; 4: 63–5. [in Russian] 17. Milovanov VK. Reproductive Biology and Artificial Insemination of Animals. Moskau, Publishers of domestic animals’ literature, journals and posters, MOSKau, udUSSR, 1962: 696. [in Russian] 18. Watson PF. Recent developments and concepts in the cryopreservation of spermatozoa and the assessment of their post-thawing function. Reprod Fertil Dev 1995; 7: 871–91. 19. Gao DY, Liu C, McGann LE, Watson PF, Kleinhans FW, Mazur P et al. Prevention of osmotic injury to human spermatozoa during addition and removal of glycerol. Hum Reprod 1995; 10: 1109–22. 20. Gao D, Mazur P, Critser J. Fundamental cryobiology of mammalian spermatozoa. In: Karow AM, Critser JK, eds. Reproductive Tissue Banking. London: Academic Press, 1997: 263–328. 21. Aitken RJ, Clarkson JS, Hargreave TB, Irvine DS, Wu FC. Analysis of the relationship between defects sperm function and the generation of reactive oxygen species in cases of oligospermia. J Androl 1989; 10: 214–20.
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22. Alvarez JG, Storey BT. Evidence for increased lipid peroxidative damage and loss of superoxide dismutase activity as a mode of sublethal cryodamage to human sperm during cryopreservation. J Androl 1992; 13: 232–41. 23. O’Connell M, McClure N, Lewis SEM. The effect of cryopreservation on sperm morphology, motility and mitochondrial function. Hum Reprod 2002; 17: 704–9. 24. Holt WV, Morris GJ, Coulson G, North RD. Direct observation of cold shock effects in ram spermatozoa with use of a programmable cryomicroscope. J Exp Zool 1988; 246: 305–14. 25. Isachenko E, Isachenko V, Katkov II, Nawroth F. Vitrification of human spermatozoa without cryoprotectants: review of problems and practical success. Reprod Biomed Online 2003; 6: 191–200. 26. Katkov II, Katkova N, Critser JK, Mazur P. Mouse spermatozoa in high concentrations of glycerol: chemical toxicity vs osmotic shock at normal and reduced oxygen concentration. Cryobiology 1998; 37: 235–8. 27. Sakkas D, Tomlinson M. Assessment of sperm competence. Semin Reprod Med 2000; 18: 133–9. 28. Fahy GM. Vitrification: a new approach to organ cryopreservation. In: Meryman HT editors. Transplantation: Approaches to Graft Rejection. New York: Alan R Liss, 1986: 305–35. 29. Kusakabe H, Szczygiel MA, Whittingham DG, Yanagimachi R. Maintenance of genetic integrity in frozen and freeze-dried mouse spermatozoa. Proc Natl Acad Sci USA 2001; 98: 13501–6. 30. Karlsson JOM. A theoretical model of intracellular vitrification. Cryobiology 2001; 42: 154–69. 31. Kanno H, Speedy RJ, Angell CA. Supercooling of water to –90°C under pressure. Science 1975; 189: 880–1. 32. MacFarlane DR, Scheirer J, Smedley SI. Pressure coefficient of conductance and of glass transition temperatures in concentrated aqueous LiCl, LiI and AlCl3 solutions. J Phys Chem 1986; 90: 2168–73. 33. MacFarlane DR. Physical aspects of vitrification in aqueous solutions. Cryobiology 1987; 24: 181–95. 34. Karow AM, Liu WP, Humphries AL. Survival of dog kidneys subjected to high pressures: necrosis of kidneys after freezing. Cryobiology 1970; 7: 122–8. 35. Vajta G, Booth PJ, Holm P, Callesen H. Successful vitrification of early stage bovine in vitro produced embryos with the Open Pulled
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36.
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Straw (OPS) method. Cryo Letters 1997; 18: 191–5. Oberstein N, O’Donovan MK, Bruemmer JE. Cryopreservation of equine embryos by open pulled straws, cryoloop, or conventional cooling methods. Theriogenology 2001; 15: 607–13. Papis K, Shimizu M, Izaike Y. Factors affecting the survivability of bovine oocytes vitrified in droplets. Theriogenology 2000; 15: 651–8. Martino A, Pollard JA, Leibo SP. Effect of chilling bovine oocytes on their developmental competence. Mol Reprod Dev 1996; 45: 503–12. Kuwayama M, Kato O. Successful vitrification of human oocytes. Fertil Steril 2000; 74: 49 abstr. 127. Kurokawa T, Kinoshita T, Ito T, Sato H, Hotta T. Detection of minimal residual disease B cell lymphoma by PCR mediated RNase protection assay. Leukemia 1996; 10: 1222–31. Matsumoto H, Jiang JY, Tanaka T, Sasada H, Sato E. Vitrification of large quantities of immature bovine oocytes using nylon mesh. Cryobiology 2001; 42: 139–44. Lane M, Bavister BD, Lyons EA, Forest KT. Containerless vitrification of mammalian oocytes and embryos. Nat Biotechnol 1999; 17: 1234–6. Chen SU, Lien YR, Chao KH, Lu HF, Ho HN, Yang YS. Cryopreservation of mature human oocytes by vitrification with ethylene glycol in straws. Fertil Steril 2000; 74: 804–8. Reubinoff BE, Pera MF, Vajta G, Trounson AO. Effective cryopreservation of human embryonic stem cells by the open pulled straw vitrification method. Hum Reprod 2001; 16: 2187–94. Kuleshova LL, MacFarlane DR, Trounson AO, Shaw JM. Sugars exert a major influence on the vitrification properties of ethylene glycolbased solutions and have a low toxicity to embryos and oocytes. Cryobiology 1999; 38: 119–30. Rall WF. Prospects of the Cryopreservation of mammalian spermatozoa by vitrification. In: Johnson LA, Katz D, eds. Boar Semen Preservation. Berlin-Hamburg: Paurey Scientific Publishers, 1991: 125–56. Holt WV. Alternative strategies for the longterm preservation of spermatozoa. Reprod Fertil Dev 1997; 9: 309–19. Fahy GM. Vitrification. In: McGrath JJ, Diller KR, eds. Low Temperature Biotechnology: Emerging Applications and Engineering Contributions. Chicago: American Society of Mechanical Engineering 1988: 113–46.
49. Jahnel F. Über die Widerstandsfähigkeit von menschlichen Spermatozoen gegenüber starker Kälte. Klin Wochenschrift 1938; 37: 88–9. [in German] 50. Parkes AS. Preservation of human spermatozoa at low temperatures. Br Med J 1945; 2: 212–3. 51. Fahy GM, McFarlane DR, Angell CA, Meryman HT. Vitrification as an approach to cryopreservation. Cryobiology 1984; 21: 407–26. 52. Devireddy RV, Swanlund DJ, Roberts KP, Pryor JL, Bishof JC. The effect of extracellular ice and cryoprotective agents on the water permeability parameters of human sperm plasma membrane during freezing. Hum Reprod 2000; 15: 1125–35. 53. Watson PR, Plummer JM. In deep freezing of boar semen: the responses of boar sperm membranes to cold shock and cooling, Johnson LA, Larsson K editors. Swedish Univ. of Agricultural Sciences, Uppsala, Sweden; 1985: 113–28. 54. Nauk VA. Structure and function of spermatozoa from farm animal by cryopreservation. Furdui FI editor. Shtiinca, Kishineu, Modova; 1991: 198. 55. Nawroth F, Isachenko V, Dessole S, Rahimi G, Farina M, Vargiu et al. Vitrification of human spermatozoa without cryoprotectants. Cryo Letters 2002; 23: 93–102. 56. Giraud MN, Motta C, Boucher D, Grizard G. Membrane fluidity predicts the outcome of cryopreservation of human spermatozoa. Hum Reprod 2000; 15: 2160–4. 57. Holt WV. Fundamental aspects of sperm cryobiology: the importance of species and individual differences. Theriogenology 2000; 53: 47–58. 58. Esteves SC, Sharma RK, Thomas AJ Jr, Agarwal A. Effect of in vitro incubation on spontaneous acrosome reaction in fresh and cryopreserved spermatozoa. Int J Fertil Womens Med 1998, 43: 235–42. 59. Isachenko V, Isachenko E, Montag M et al. Clean technique for cryoprotectant-free vitrification of human spermatozoa. Reprod Biomed Online 2005; 10: 350–4. 60. Isachenko V, Isachenko E, Katkov II, Montag M, Dessole S, Nawroth F et al. Cryoprotectantfree cryopreservation of human spermatozoa by vitrification and freezing in vapor: effect on motility, DNA integrity, and fertilization ability. Biol Reprod 2004; 71: 1167–73. 61. Karlsson JOM, Cravalho EG. A model of diffusion-limited ice growth inside biological cells during freezing. J Appl Phys 1994; 75: 4442–55.
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62. Hammadeh ME, Askari AS, Georg T, Rosenbaum P, Schmidt W. Effect of freezingthawing procedure on chromatin stability, morphological alteration and membrane integrity of human spermatozoa in fertile and subfertile men. Int J Androl 1999; 22: 155–62. 63. Sun JG, Juriscova A, Casper RF. Detection of deoxyribonucleic acid fragmentation in human sperm: correlation with fertilization in vitro. Biol Reprod 1997; 56: 602–7. 64. Lopes S, Sun JG, Juriscova A, Meriano J, Casper RF. Sperm deoxyribonucleic acid fragmentation in increased in poor-quality semen samples and correlated with failed fertilization in intracytoplasmic sperm injection. Fertil Steril 1998; 69: 528–32. 65. Katkov II. The point of maximum cell water volume excursion in case of presence of an impermeable solute. Cryobiology 2002; 44: 193–203. 66. Isachenko E, Isachenko V, Katkov II, Rahimi G, Schondorf T, Mallmann P et al. DNA integrity and motility of human spermatozoa after standard slow freezing versus cryoprotectant-free vitrification. Hum Reprod 2004; 19: 932–9. 67. Evenson DP, Jost LK, Baer RK, Turner TW, Schrader SM. Individuality of DNA denaturation patterns in human sperm as measured by the sperm chromatin structure assay. Reprod Toxicol 1991; 5: 115–25. 68. Duty SM, Singh NP, Ruan L, Chen Z, Lewis C, Huang T et al. Reliability of the comet assay in cryopreserved human sperm. Hum Reprod 2002; 17: 1274–80.
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69. Nakagata N, Takeshima T. High fertilizing ability of mouse spermatozoa diluted slowly after cryopreservation. Theriogenology 1992; 37: 1263–91. 70. Koshimoto C, Gamliel E, Mazur P. Effect of osmolality and oxygen tension on the survival of mouse sperm frozen to various temperatures in various concentrations of glycerol and raffinose. Cryobiology 2000; 41: 204–31 71. Nakagata N. Cryopreservation of mouse spermatozoa. Mamm Genome 2000; 11: 572–6. 72. Van Blerkom J, Sinclair J, Davis P. Mitochondrial transfer between oocytes: potential applications of mitochondrial donation and the issue of heteroplasmy. Hum Reprod 1998; 12: 2857–68. 73. Meseguer M, Garrido N, Martinez-Conejero JA, Simon C, Pellicer A, Remohi J. Role of cholesterol, calcium, and mitochondrial activity in the susceptibility for cryodamage after a cycle of freezing and thawing. Fertil Steril 2004; 82: 514–5. 74. Smiley ST, Reers M, Motolla-Hartshorn C, Lin M, Chen A, Smith TW et al. Intracellular heterogeneity in mitochondrial membrane potentials revealed by a J-aggregate-forming lipophilic cation JC-1.Proc Natl Acad Sci USA 1991; 88: 3671–5. 75. Isachenko V, Isachenko E, Montag M, Zaeva V, Krivokharchenko A, Nawroth F, et al. Clean technique for cryoprotectant-free vitrification of human spermatozoa. Reprod Biomed Online 2005; 10: 350–4.
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Potential developmental consequences of cryopreservation of mammalian oocytes and embryos
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Gary D Smith and Luis G Villa-Diaz
INTRODUCTION The objective of prolonged storage of tissues that could be revitalized following suspended animation in cryostorage was envisioned by Dr John Hunter over two centuries ago. Since then significant advances have been made in cryopreservation of living cells, especially in the 1940s when it was discovered that glycerol greatly enhanced survival of cryopreserved living cells.1 Around that time investigations by Chang on low temperature storage of rabbit oocytes, zygotes, and embryos2,3 paved the way for studies on cryopreservation of female gametes and embryos. Subsequent experiments in the 1950s by Lin and Sherman4–6 demonstrated that mouse oocytes could also be cooled in glycerol, stored, and, subsequently, fertilized in recipients, resulting in embryos that support pregnancies. In the 1960s early investigations by Mazur7,8 formed the foundation for understanding cellspecific optimal cooling and warming rates which today remain as pivotal keys to successful mammalian gamete and embryo cryopreservation. Then in the 1970s the combined strengths of Mazur, Leibo, and Whittingham resulted in successful cryopreservation of mouse embryos.9 In 1977 the first successful IVF with live offspring from cryopreserved mouse oocytes was reported,10 followed by the first human pregnancies after embryo cryopreservation.11,12 Finally in 1986, Chen reported the first pregnancy after human oocyte cryopreservation.13
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OOCYTE CRYOPRESERVATION METHODS AND CRYODAMAGE Currently mammalian oocyte cryopreservation is performed in two different ways either ‘slow-rate’ freezing or vitrification.14–16 ‘Slowrate’ freezing attempts to minimize adverse cellular events through control of the biophysical properties of freezing, such as the cooling and warming rates in conjunction with cryoprotectants. With ‘slow-rate’ freezing, cells are cooled to very low temperatures while minimizing intracellular ice crystal formation, and at the same time it reduces the detrimental influences caused by increased solute concentrations and osmotic stress.17 Therefore with ‘slow-rate’ freezing, cells are dehydrated through an equilibrium process where extracellular ice is formed. Alternatively, vitrification, a non-equilibrium approach to cryopreservation, utilizes high concentrations of cryoprotectants that solidify without forming ice crystals, which are a major cause of intracellular cryodamage. The vitrified solids therefore contain the normal molecular and ionic distributions of the original liquid state and can be considered as an extremely viscous, supercooled liquid.18 During vitrification, cells are dehydrated by brief exposure to a concentrated solution of cryoprotectants before plunging the samples directly into liquid nitrogen. Vitrification comes from the Latin word vitrum, which means glass or resembling glass, and the technique was originally developed for cryopreservation of
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mammalian embryos.19 Currently vitrification of oocytes and embryos is an area of focus for many clinical, rodent, and domestic animal production laboratories. Cryopreservation of both germinal vesicle (GV)-stage20–22 and metaphase II (MII)23–25 oocytes has been reported in numerous species. With the use of ‘slow-rate’ freezing Eroglu et al.22 found that 72% of GV-stage mouse oocytes survived the freezing process, 74% of those that survived progressed to MII, and 81% of MII oocytes underwent pronuclear formation following insemination. Correspondingly, vitrification of GV-stage mouse oocytes resulted in similar rates of surviving oocytes and maturation to MII.26,27 Live offspring produced from vitrified GV-stage mouse oocytes have also been reported.27 Successful cryopreservation of human GVstage oocytes has also been reported using ‘slow-rate’ freezing, resulting in oocytes capable of in vitro maturation, fertilization,28 and supporting viable births.13,29 However, most reported pregnancies after oocyte cryopreservation have been achieved with MII female gametes. In spite of the fact that GV-intact oocyte cryopreservation is feasible, the inefficiency of human oocyte in vitro maturation and subsequent ability to generate embryos with embryonic developmental competence and capacity to establish livebirths is a major stumbling block in GV-intact oocyte cryopreservation.30 Regardless of the methodology used for cryopreservation, effects on oocyte cellular functions can compromise the oocyte’s ability to develop normally following the cryopreservation process. These compromised cellular events can be collectively and generally termed ‘cryodamage’. Experience in cryopreservation of various cell types led to the appreciation that as cell size increases, difficulty in cryopreservation also increases.8 During cryopreservation, cells are exposed to numerous stresses including mechanical, thermal, and chemical,8,31 which can lead to compromised cell function and cell death.
This review addresses documented and theoretical specific cellular structures and functions that are/may be compromised by cryopreservation and subsequent effects on oocyte developmental competence. The biophysics of cryopreservation are not discussed here; however, such information is available elsewhere.32 Moreover, we do not attempt to delineate methodology-induced cryodamage, but view cryodamage from a cellular structural/functional level, with attention directed toward intracellular organelles and extracellular structures susceptible to cryodamage. Data from numerous mammalian species are considered concomitantly without differentiation between species.
INTRACELLULAR DAMAGE Intracellular organelles within the nucleus and cytoplasm can be damaged during oocyte cryopreservation; therefore we consider their normal developmental and homeostatic functions, and the potential developmental consequences of compromising their functions with cryodamage.
Nucleus The nucleus in the oocyte, also known as the germinal vesicle, is composed of a nuclear envelope (NE), and nuclear material such as chromatin, and the nucleolus. The nucleus is present during oocyte development and once oocytes resume the first meiotic division, the NE is disintegrated allowing mixing of the nuclear material into the cytoplasm.
Nuclear envelope When present the NE surrounds the nucleus and ensures a temporal and spatial separation of events that take place within the nucleus and cytoplasm. Processes such as DNA replication, transcription, RNA processing, and ribosomal subunit assembly occur within
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the nucleus, whereas most protein synthesis takes place in the cytoplasm.33–35 The NE is composed of the outer and inner nuclear membranes, nuclear pore complexes, and lamina. Facing the cytoplasm is the outer nuclear membrane, which is a specialized functional domain of the endoplasmic reticulum. The inner nuclear membrane is important both structurally and functionally since it contains proteins that provide attachment sites for both heterochromatin and the nuclear lamina. Both membranes are lipid bilayers that are connected to, and fenestrated by, nuclear pore complexes which span the nuclear membrane.36 The nuclear pore complexes are multiprotein assemblies displaying octagonal symmetry and form channels that transport substrates with a diameter of approximately 26 nm.37 The nuclear pore complexes also contain peripheral structures projecting into both the nucleoplasm and cytoplasm,38 and it is believed that these filamentous projections contain docking sites for transport substrates in association with specific soluble receptors. Appropriate NE assembly with associated pore complexes provides the correct nuclear and cytoplasmic substrates and signal trafficking necessary for normal intracellular communication and function. Finally, the lamina is a cage-like structure located within the nucleoplasm and in close association with the inner nuclear membrane and nuclear pore complexes. Lamins, which are type-V intermediate filaments, are the major protein components of the lamina. The importance of lamina in determining nuclear shape, size, and ability to resist deformation and/or resume its initial shape after deformation have been demonstrated in studies using lamin knockout mice,39 dominant-negative mutants,40 and RNA interference (RNAi) ‘knock-down’.41 In addition, it has been suggested that the lamina may also be important regulators of gene expression,34 since it has been found associated with heterochromatin at sites of DNA replication, RNA processing, replication proteins, and RNA polymerases.42
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Therefore it could be hypothesized that compromising the structural integrity of the NE, and its associated lamina might influence subsequent DNA replication, transcription, and normal cell function, and that both ‘slowrate’ freezing and vitrification could disrupt and/or distort the NE. Although the nucleus has the ability to reassemble morphologically following oocyte cryopreservation,43 the future development of the cell could be suboptimal. Recently it was demonstrated that reconstructed oocytes utilizing vitrified/warmed GVs were able to mature up to MII;44 however, their potential for further development was not tested.
Nucleolus The nucleolus is the most prominent nuclear organelle. In oocytes the nucleolus is present in periods when protein synthesis is particularly important, such as during the oocyte growth phase, and after the major activation of the embryonic genome following fertilization. These periods of profound stage-specific need for protein synthesis also correlate well with elevated transcription; therefore adequate cytoplasmic pools of ribosomes must exist during this time. Ribosomal subunits are formed in the nucleolus and subsequently transferred from the nucleus to the cytoplasm through the nuclear membrane pores of the NE, and associate to form cytoplasmic ribosomes in conjunction with translation of mRNAs into proteins.45 Van Blerkom et al.,20 using time-lapse photography in vitrified/warmed oocytes, observed that the number and nuclear position of nucleoli as well as the majority of nuclear structures returned to a normal state following warming and rehydration. However, in some cases it was observed that nucleolar bodies were present in the cytoplasm after oocyte warming and re-formation of the NE. Whether this aberrant intracellular nucleoli localization influences subsequent development is unknown. Although these experiments
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Figure 6.1 Representative micrographs of an oocyte immediately following vitrification and warming (a) or after a subsequent 2 h culture at 37°C (b). Chromatin is stained blue and β-tubulin is stained green by immunocytochemistry. This is an example of the spindle’s ability to depolymerize, yet repolymerize under proper conditions. PB, polar body.
PB
a
PB
b
provide key morphological observations, proper enzymatic regulatory protein–fine structure interactions were not assessed. It is possible that nucleolar functional/structural interactions like the association of RNA polymerase I with the nucleolus may be affected as well. Tethering of enzymes in close association with their substrates (i.e. RNA polymerase I and rRNA genes) is an important regulatory mechanism. It has been suggested that inadequate focal localization of RNA polymerase I with nucleoli in porcine in vitro-produced embryos may contribute to inefficient offspring production.46 Whether subsequent function of the nucleolus is influenced by cryopreservation needs to be assessed, as well as whether ‘slow-rate’ freezing would cause aberrant nucleoli localization.
Cytoplasm The potential effects of cryodamage on the oocyte cytoplasmic function will be inherently related to stage of the cell cycle, since the ultrastructural configuration in the cytoplasm is continually changing dependent upon the stage of meiosis. This is especially true with respect to cytoskeleton distribution and/or function, which is essential for normal segregation of mitochondria (see below), chromosomes, spindle rotation, cytokineses, and pronuclei/nuclei formation.47,48
Microtubules/spindles Microtubules consist of polymerized tubulin and are one of the predominant components of the cytoskeleton. The major microtubular structure in oocytes is the spindle, which is responsible for spatial organization and subsequent migration of chromosomes during meiotic divisions. Disturbance of the microtubular network within the oocytes may result in scattering and/or displacement of the spindle and thus changes in the chromosomal complement within the cell. It has been demonstrated that exposure of oocytes to cooling,49–51 cryoprotectants,52,53 or the freeze/thaw process54 can cause depolymerization and disorganization of spindle microtubules (Figure 6.1). Recently, polscope microscopy studies have shown that the oocyte spindle transiently disappears during cryopreservation, but reorganizes after 2–5 h of culture;55,56 however, the spindle microtubular organization of the re-organized spindles was not analyzed in detail. In another study using confocal microscopy it was demonstrated that the spindle and chromosome configurations of cryopreserved/thawed human oocytes were severely compromised.57 Disruption of the microtubular network could ultimately lead to aneuploidy, if repolymerization does not occur properly following thawing/warming.
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PB
Cleavage Furrow
a
b
Actin/microfilaments Microfilaments are the other main component of the cytoskeleton and are composed of polymerized actin derived from free actin. The equilibrium of polymerized actin (microfilaments) and free actin is dynamic, just as with microtubules and tubulin. In human oocytes microfilaments have been found organized in a uniform layer enveloping the cortex58 (Figure 6.2), while in mouse oocytes microfilaments are located in a similar cortexassociated fashion, yet appear to be concentrated in the region adjacent to the meiotic spindles.59 During oocyte meiosis microfilaments play important roles in spindle rotation, polar body extrusion, pronuclear migration, intracellular trafficking of molecules and organelles, and cytokinesis.60–63 Numerous developmental perturbations can occur if actin polymerization and microfilament function are negatively influenced by or during cryopreservation such as inappropriate intracellular organelle migration in the oocytes, premature cortical granules release (see below), and interference with polar body extrusion. It has been reported that at 37°C 1,2-propanediol causes ‘blebbing’ on oocytes, which were found to be lacking microfilaments.64 Cooling of oocytes has not been demonstrated to alter grossly microfilament polymerization;14 however, there has
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Figure 6.2 Representative micrographs of an oocyte (a) and 2-cell embryo (b) fixed and stained to recognize the intracellular location of polymerized actin. One can recognize the peri-oolemma location of oocyte polymerized actin and its presence in the 2-cell embryo in cortical regions and in the cleavage furrow. PB, polar body.
been some suggestion that cooling effects on microtubule depolymerization in the spindle may also alter adjacent microfilaments.65 Moreover, dimethylsulfoxide (DMSO) and 1,2-propanediol have both been shown to disrupt polymerized cortical actin.53,62
Mitochondria Oocytes contain a pool of non replicating yet functional mitochondria from which all cells of the resultant embryos directly inherit. Mitochondria play a vital role in the metabolism of energy-containing compounds in the oocyte cytoplasm to provide adenosine triphosphate (ATP) for fertilization and preimplantation embryo development. One current view of mitochondrial function is that reduced meiotic competence and fertilizability of oocytes, and developmental failure in the preimplantation embryo could result from pre-existing oocyte mitochondrial DNA (mtDNA) defects,66 from age-related accumulation of mtDNA mutations,67 and/or abnormal distributions of mitochondria in the oolema.68,69 Abnormal distribution of oocyte mitochondria may be caused by compromised microfilament function after oocyte cryopreservation. Mitochondria swelling has been observed after both oocyte cryopreservation by ‘slow-rate’ freezing,70 and vitrification.71 Whether mitochondria can recover
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from swelling, or what are its developmental consequences, has yet to be tested. Recently, Thouas et al.72 demonstrated that sublethal mitochondrial injury in mouse oocytes is heritable by resultant embryos, and results in postimplantation pathologies similar to those reported for clinically subfertile women, including recurrent implantation failure or miscarriage, and decreased live birth weight.
EXTRACELLULAR DAMAGE Zona pellucida The zona pellucida is a glycoprotein membrane surrounding the plasma membrane of oocytes and preimplantation embryos. The zona pellucida is composed of three glycoproteins termed ZP1, ZP2, and ZP3,73 and it is known to play a critical role in the entire fertilization process and in blockage of polyspermy following initial penetration by one spermatozoon. During the fertilization process the spermatozoon binds to the zona pellucida and undergoes the acrosome reaction, it then penetrates the zona pellucida and binds to a specific site on the oolemma in a ligand-receptor fashion.74 Binding of sperm to the oolemma is believed to trigger the ‘cortical reaction’, which involves exocytosis of cortical granules from the oocyte cortex into the perivitelline space, and thus release of cortical granule enzymes. These released enzymes result in a block to polyspermy by modifying the zona pellucida (zona reaction), the oolemma, or both. The degree of zona reaction and plasma membrane block to polyspermy has been found to differ between species.75 Cortical granules are diffusely localized throughout the GV-intact cytoplasm, and migrate to the oocyte cortex during maturation.76 Translocation and release of cortical granules involves proper cytoskeleton function and plasma membrane organization. It has been reported that DMSO77,78 and 1,2-propanediol79 exposure during the cooling
process in oocyte cryopreservation cause premature cortical granule release and zona hardening, compromising sperm penetration and fertilization. Recently, Gardner et al.80,81 demonstrated that the cryoprotectants mentioned above, as well as ethylene glycol, cause transient calcium increases in mouse MII oocytes, which were sufficient to cause zona hardening, presumably through triggering cortical granule exocytosis, which is a calcium-dependent event.82 The authors also observed that removal of calcium from the vitrification medium facilitated fertilization by conventional insemination and development to the 2-cell stage at a rate approaching that of control (non-vitrified) oocytes. Zona hardening after oocyte cryopreservation can also be avoided with the use of bovine fetal serum;62,83 however, human serum albumin has been shown not to provide the same protection.84 Another negative effect of cryopreservation on oocytes is the rapid changes in cell configuration. Such is the case when oocytes are vitrified.43 This cellshape alteration is observed as the cell folding in upon itself and forming a crescent moon or concave appearance. This can result in fracture of the zona pellucida,85,86 and most likely contributes to polyspermic fertilization following oocyte cryopreservation.
OOCYTE CRYOSURVIVAL AND PARTHENOGENETIC ACTIVATION Obviously the future developmental competency of a cryopreserved oocyte will be truncated if it does not survive, or if it becomes parthenogenetically activated after thawing/warming. Although oocyte cryosurvival has yielded rates of ~90%,87,88 its variability represents a major obstacle for oocyte cryopreservation, especially with ‘slow-rate’ freezing. Gardner and Lane have demonstrated that ‘slow-rate’ freezing has detrimental effects on oocyte metabolism, and on subsequent embryo development and viability.88 Due to a
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significant rise in intracellular calcium, the authors, in a separate set of experiments, observed low survival rates in oocytes cryopreserved using ‘slow-rate’ freezing and 1,2-propanediol.80 The correlation between elevated and sustained increases of intracellular calcium, degeneration, and apoptosis in mature oocytes has been previously documented.89,90 Elevated intracellular levels of calcium induced by slow-rate freezing with 1,2-propanediol may also result in parthenogenetic activation of oocytes.91–94 Advances in technologies, such as mass spectrometry, have allowed the ability to generate protein profiles and identifying biomarkers from oocytes and embryos. Proteonomic analysis of MII oocytes following cryopreservation revealed that ‘slow-rate’ freezing has a significant effect on protein expression when compared with non-cryopreserved MII oocytes. In contrast, vitrification had a minimal impact in protein expression.80 Now that this technology is available, it will be pertinent to study proteins and factors present in the nucleus and cytoplasm that affect oocyte maturation or its future development. For example, it is known that factors present in the oocyte nucleus are required for male pronucleus formation.95 Whether cryopreservation would damage those nuclear factors remains unknown; however, one could speculate about its consequences if such damage occurs.
CONCLUSION Oocyte cryopreservation has evolved significantly since the first achieved human pregnancy reported by Chen13 in 1986. Accumulation of knowledge in basic biology and physiology of mammalian oocytes and cryobiology has resulted in high survival rates of cryopreserved oocytes, and moderate rates of embryo development after fertilization. Healthy children have been born after both slow-rate freezing and vitrification. Continued accumulation of knowledge regarding the intricacies of cryopreservation and related cell biology, such as the importance of protein structural/functional relationships to normal gene expression, protein translation, intracellular trafficking, epigenetic modifications, and cell development is necessary. This knowledge will translate into a better understanding of the potential developmental consequences of cryopreserved oocytes, and will likely lead to the optimization of oocyte cryopreservation.
ACKNOWLEDGMENTS The authors would like to express their appreciation to Jeni Chapman for critical review of this manuscript. We apologize to those whose work we have not cited owing to space limitation.
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22. Eroglu A, Toner M, Leykin L, Toth TL. Cytoskeleton and polyploidy after maturation and fertilization of cryopreserved germinal vesicle-stage mouse oocytes. J Assist Reprod Genet 1998; 15: 447–54. 23. Martino A, Songsasen N, Leibo S. Development into blastocysts of bovine oocytes cryopreserved by ultra-rapid cooling. Biol Reprod 1996; 54: 1059–69. 24. Eroglu A, Toth T, Toner M. Alterations of the cytoskeleton and polyploidy induced by cryopreservation of metaphase II mouse oocytes. Fertil Steril. 1998; 69: 944–57. 25. Hong S, Chung H, Lim J, Ko J, Yoon T, Yee B, et al. Improved human oocyte development after vitrification: a comparison of thawing methods. Fertil Steril 1999; 72: 142–6. 26. Isachenko E, Nayudu P. Vitrification of mouse germinal vesicle oocytes: effect of treatment temperature and egg yolk on chromatin and spindle normality and cumulus integrity. Hum Reprod 1999; 14: 400–8. 27. Aono N, Abe Y, Hara K, Sasada H, Sato E, Yoshida H. Production of live offspring from mouse germinal vesicle-stage oocytes vitrified by a modified stepwise method, SWEID. Fertil Steril 2005; 84 (Suppl 2): 1078–82. 28. Toth T, Baka S, Veeck L, Jones HJ, Muasher S, Lanzendorf S. Fertilization and in vitro development of cryopreserved human prophase I oocytes. Fertil Steril 1995; 61: 891–4. 29. Tucker M, Morton P, Wright G, Sweitzer C, Massey J. Clinical application of human egg cryopreservation. Hum Reprod 1998; 13(11): 3156–9. 30. Smith G. In vitro maturation of oocyte. Curr Womens Health Rep 2001: 143–51. 31. Meryman H. Cryoprotective agents. Cryobiology 1971; 8: 173–83. 32. Mazur P. Equilibrium, quasi-equilibrium, and nonequilibrium freezing of mammalian embryos. Cell Biophysics 1990; 17: 53–92. 33. Buendia B, Courvalin J, Collas P. Dynamics of the nuclear envelope at mitosis and during apoptosis. Cell Mol Life Sci 2001; 58: 1781–89. 34. Hutchison C. Lamins: building blocks or regulators of gene expression. Nature 2002; 3: 848–58. 35. Rzepecki R. The nuclear lamins and the nuclear envelope. Cell Mol Biol Lett 2002; 7: 1019–35. 36. Hinshaw J, Carragher B, Milligan R. Architecture and design of the nuclear pore complex. Cell 1992; 69: 1133–41. 37. Feldherr C, Kallenbach E, Schultz N. Movement of a karyophilic protein through
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62. Vincent C, Johnson M. Cooling cryoprotectants and the cytoskeleton of the mammalian oocyte. Ox Rev Reprod Biol 1992; 14: 73–100. 63. Wang W, Abeydeera L, Prather R, Day B. Polymerization of nonfilamentous actin into microfilaments is an important process for porcine oocyte maturation and early development. Biol Reprod 2000; 62: 1177–83. 64. Joly C, Bchini O, Boulekbache H, Testart J, Maro B. Effects of 1,2-propanediol on the cytoskeletal organization of the mouse oocyte. Hum Reprod 1992; 7: 374–8. 65. Webb M, Howlet S, Maro B. Parthogenesis and cytoskeletal organization in ageing mouse eggs. J Embryol Exp Morphol 1986; 95: 131–45. 66. Perez GI, Trbovich AM, Gosden RG, Tilly JL. Mitochondria and the death of oocytes. Nature 2000; 403: 500–1. 67. Keefe DL, Niven-Fairchild T, Powell S, Buradagunta S. Mitochondrial deoxyribonucleic acid deletions in oocytes and reproductive aging in women. Fertil Steril 1995; 64: 577–83. 68. Nagai S, Mabuchi T, Hirata S, Shoda T, Kasai T, Yokota S, et al. Correlation of abnormal mitochondrial distribution in mouse oocytes with reduced developmental competence. Tohoku J Exp Med 2006; 210: 137–44. 69. Van Blerkom J, Davis P, Alexander S. Differential mitochondrial distribution in human pronuclear embryos leads to disproportionate inheritance between blastomeres: relationship to microtubular organization, ATP content and competence. Hum Reprod 2000; 15: 2621–33. 70. Valojerdi MR, Salehnia M. Developmental potential and ultrastructural injuries of metaphase II (MII) mouse oocytes after slow freezing or vitrification. J Assist Reprod Genet 2005; 22: 119–27. 71. Hochi S, Kozawa M, Fujimoto T, Hondo E, Yamada J, Orugi N. In vitro maturation and transmission electron microscopic observation of horse oocytes after vitrification. Cryobiology 1996; 33: 300–10. 72. Thouas GA, Trounson AO, Jones GM. Developmental effects of sublethal mitochondrial injury in mouse oocytes. Biol Reprod 2006; 74: 969–77. 73. Moos J, Faundes D, Kopf G, Schultz R. Composition of the human zona pellucida and modifications following fertilization. Hum Reprod 1995; 10: 2467–71. 74. Wassarman P. Profile of a mammalian sperm receptor. Development 1990; 108: 1–17.
75. Wolf D. The mammalian egg’s block to polyspermy. In: Mastroianni L, Biggers JD, eds. Fertilization and Embryonic Development in Vitro. New York: Plenum Press, 1981: 183–97. 76. Ducibella T, Anderson E, Albertini D, Aalberg J, Rangarajan S. Quantitative studies of changes in cortical granule number and distribution in the mouse oocyte during meiotic maturation. Dev Biol 1988; 130: 184–97. 77. Johnson M. The effect on fertilisation of exposure of mouse oocytes to dimethyl sulfoxide: an optimal protocol. J In Vitro Fert Embryo Transf 1989; 6: 168–75. 78. Pickering S, Braude P, Johnson M. Cryoprotection of human oocytes: inappropriate exposure to DMSO reduces fertilization rates. Hum Reprod 1991; 6: 142–43. 79. Schalkoff M, Oskowitz S, Powers R. Ultrastructural observations of human and mouse oocytes treated with cryopreservation. Biol Reprod 1989; 40: 379–93. 80. Larman MG, Katz–Jaffe MG, Sheehan CB, Gardner DK. 1,2-propanediol and the type of cryopreservation procedure adversely affect mouse oocyte physiology. Hum Reprod 2007; 22: 250–9. 81. Larman MG, Sheehan CB, Gardner DK. Calcium-free vitrification reduces cryoprotectant-induced zona pellucida hardening and increases fertilization rates in mouse oocytes. Reproduction 2006; 131: 53–61. 82. Kline D, Kline JT. Repetitive calcium transients and the role of calcium in exocytosis and cell cycle activation in the mouse egg. Dev Biol 1992; 149: 80–9. 83. Carroll J, Wood M, Whittingham D. Normal fertilisation and development of frozen-thawed mouse oocytes: protective action of certain macromolecules. Biol Reprod 1993; 48: 606–12. 84. George MA, Johnson MH. Use of fetal bovine serum substitutes for the protection of the mouse zona pellucida against hardening during cryoprotectant addition. Hum Reprod 1993; 8: 1898–900. 85. Dhali A, Manik R, Das S, Singla S, Palta P. Post-vitrification survival and in vitro maturation rate of buffalo (Bubalus bubalis) oocytes: effect of ethylene glycol concentration and exposure time. Anim Reprod Sci 2000; 63: 159–65. 86. Wu C, Rui R, Dai J, Zhang C, Ju S, Xie B, et al. Effects of cryopreservation on the developmental competence, ultrastructure and cytoskeletal structure of porcine oocytes. Mol Reprod Dev 2006; 73: 1454–62.
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Vitrification of oocytes: general considerations and the use of the Cryotop method
7A
Masashige Kuwayama, Ana Cobo and Gábor Vajta
WHY DO WE NEED TO CRYOPRESERVE HUMAN OOCYTES? In most papers dealing with this topic, a long list of simple and obvious reasons to answer this question can be found. These answers are also listed below. However, this review starts with a more general and rarely mentioned argument: the handicapped situation of women from the point of reproduction. Although gender-based discrimination is less and less acceptable in most human societies, nature still preserves the right to make a seemingly unfair but strong distinction between males and females in certain areas including – obviously – reproduction. In most mammalian species, females hold most of the weight of reproduction, including discomfort related to reproductive cycles and pregnancy, pain of labor, and nursing of babies. In humans, even with the best intentions, male partners cannot share most of these sacrifices, and most women accept them as an inevitable part of their full life, that is at least partially compensated by the most intimate relationship with their babies. However, there are additional, even more frustrating differences between males and females regarding the possibility to distribute and preserve genetic material. A healthy male can produce many millions of sperm cells every day, while the number of oocytes is highly restricted, in many mammals including human to one or
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two per month. Moreover, males normally may preserve their reproductive ability for their whole life, while the time for women is rather limited. Theoretically they may become pregnant and have babies before the menopause – which also means almost halving the available time compared with males – but in practice, even this period is drastically shortened by the fact that the quality of oocytes decreases sharply after the age of 35, restricting the real freedom to reproduce without concern and fear to approximately 15 years, coinciding exactly with the time which is the most critical to establish a professional career for a lifetime. We may be referring to the inevitable order or laws of nature, but aging, loss of teeth or hair may also be referred to as such, not to mention blindness, deafness, or other serious handicaps. Considerable and fully justified efforts are made to eliminate these unfair differences created by life and nature, and legislative and financial help are provided to ensure equal opportunity in many fields. More attention should probably be focused on this half of humankind to alleviate their handicapped situation in the field of reproduction. Unfortunately, although not at all according to the original intentions, modern reproductive technologies in the human have made the situation even worse. With the application of intracytoplasmic sperm injection (ICSI), one gamete from both genders may be enough to
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produce an offspring. Compared with the almost infinite possibility, the 10-, sometimes 20-fold artificial increase of the number of available oocytes (as the result of an expensive, painful, and risky medical intervention) does not seem to be a real compensation. It is even more embarrassing than with ICSI, incompletely capacitated or matured, handicapped, immotile, or dying spermatozoa can still be successfully used for fertilization, while the slightest deviation in morphology, function of oocytes (including those caused by the supportive medical intervention itself), or the smallest inaccuracy of the stage of maturation may seriously compromise their further developmental competence. Finally, when we consider storage possibilities of male and female gametes of mammals, the difference is even more frustrating. In many domestic species large commercial networks are dealing with collection, deepfreezing, and distribution of sperm of valuable animals, by using highly standardized procedures and with an efficiency that almost completely eliminates the need for natural mating or artificial insemination with fresh semen. The establishment of the ‘cryoprotectant-free vitrification’ method1 for spermatozoa also proves the extreme tolerance of male germ cells towards cryoinjuries, although the term itself may raise some concerns. In the mouse, a technology has also been developed enabling sperm transportation in a sealed envelope by ordinary mail. On the other hand, the recovery, laboratory handling, and especially cryopreservation of mammalian oocytes from live animals is still regarded as a challenge and restricted (with a few exceptions) to the experimental field. The situation is no better in humans: sperm can be easily collected, frozen, stored, and utilized in small aliquots, creating the commercial distribution of this supposedly valuable male genetic ‘stuff ’ is a prosperous business in some countries. In contrast, storage and use of the female gamete is seriously restricted by the above-mentioned biological, technical, and
psychological problems related to collection; by legal measures restricting experimental use and/or donations in many countries; and by the poor and inefficient technologies available for their cryopreservation. Until recently, due to the cumulative effect of these factors, the efficiency of the whole procedure was so low that practically every baby born after oocyte cryopreservation deserved a scientific publication. Establishment and widespread application of an efficient and safe cryopreservation method for oocytes would not eliminate differences in reproductive flexibility between females and males, but may provide a solution for many specific problems and eventually reverse the actual trend of this unacceptable artificial widening of the gap between the two genders. Accordingly, this area deserves special attention, and should be regarded as more than just the subject of scientific ambition of a few, accidentally selected scientists; making this an area that is benignly and respectfully disregarded by the vast majority of reproductive specialists. The need for change in the general attitude towards oocyte cryopreservation is even more justified in view of the recent rapid advancement in technology now offering the prospect of a real breakthrough, and a definite and currently available solution in many important fields including the following. • Malignant diseases where systemic anticancer treatment is required2 • Surgical procedures resulting in loss of ovarian function3 • Treatment of patients with polycystic ovarian syndrome4,5 • Patients with ovary hyperstimulation syndrome3 • Poor responders to ovarian stimulation3 • Patients at risk of ovarian function loss through premature menopause3 • In cases of male factor infertility or problems associated with difficulty of sperm collection, inadequate seminal samples, or
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non-viable spermatozoa at the time of oocyte retrieval3 • To overcome ethical concerns and legal restrictions in several countries associated with embryo cryopreservation6 • Cryobanking oocytes for young women who wish to delay motherhood for various reasons (career, lack of appropriate partner, etc.)7 • Cryobanking oocytes for egg donation programs or for research purposes.8
WHY IS CRYOPRESERVATION OF OOCYTES DIFFICULT? Some of the reasons why cryopreservation of oocytes is difficult, including the size, shape, and cell number, are quite obvious. It is well known that oocytes are the largest cells of the human body. In cryobiology, the size or rather the mass is a decisive factor. Suspensions of somatic cell cultures can be cryopreserved with high efficiency and without any sophisticated approach by using simple media, a refrigerator, and a deep freezer or liquid nitrogen. For the even smaller bacteria and viruses, we meet the frustrating evidence of this every day: they are present in almost every liquid nitrogen tank, and preserve their viability without any protection and in spite of our best intention (although apart from the size, some other factors, for example, the simple structure may also play a role in this resistance). In reproductive biology we just refer to the above-mentioned differences between cryotolerance of spermatozoa and oocytes that can also at least partially be attributed to the differences in volume. Quite controversially, the cumulative mass of cells decreases exponentially during the first week of embryo development, and at the expanded blastocyst stage the mass may become as low as 1/10 to 1/100 of that of the oocyte, with an obvious similar decrease in the water content. Although the solution accumulated in the blastocoel may be a potential source of damage either by ice
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crystal formation, or through the accumulation and slow dilution of toxic cryoprotectants,9–11 these mechanisms obviously cause more harm when they occur intracellularly in the oocyte. Apart from the size, the shape of the oocyte is also most unfortunate. The almost perfect sphere slows down formation of an equal distribution of any substance, including permeable cryoprotectants coming from outside or released from the oocyte. Accordingly, for a relatively long period of time a continuous concentration gradient from the periphery to the center or vice versa exists, resulting in toxic damage in one part while providing less than optimal protection in the other. From this point of view, the change in shape caused by the osmotic effect at equilibration may offer some kind of benefit, but may also contribute to the damage of the cytoskeleton (see below). The third major factor is the lowest possible cell number. From this point of view the oocyte resembles a gambler who puts all his money on the very first bet: all or nothing. Multicellular embryos can survive and compensate for as much as 50% loss of their cells (and supposedly also some level of injury in the remaining ones) as demonstrated by biopsies, bisection of embryos, or just the less than optimal culture conditions apart from the cryopreservation experiences. The oocyte has only one chance, and there is no backup to regenerate from a serious injury. We have to use an extremely careful approach to get out of the game as winners. Unfortunately, apart from the factors listed above, there are still many other factors that contribute to the sensitivity of oocytes to cryoinjuries. Chilling injury – that occurs at relatively high temperatures and induces irreversible damage of the cytoplasmic lipid droplets, lipid-rich cell membranes, and microtubules – affects mostly the latter two structures in the human oocyte, as (in contrast for example to pigs) in humans cytoplasmic lipid droplets are less abundant. On the other hand, the membranes are extremely
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sensitive and rapidly undergo a transition from the liquid state to the gel state, an irreversible process that is detrimental for future development. For unknown reasons, just one step further on, after fertilization the membranes of zygotes are much less sensitive to this type of injury.12 The depolymerization of microtubules, misalignment of the chromosomes, and the possible increased risks of aneuploidy are frequently emphasized and have a wide experimental background,13–16 although in the human comparative examinations may not entirely confirm the seriousness of these problems,17 and the supposed beneficial effect of some agents (cytoskeleton relaxants or stabilizers) is not fully proven. Similar to somatic cell nuclear transfer, spindle reorganization may occur surprisingly efficiently, and the number of chromosomal abnormalities in children born after oocyte vitrification does not seem to show a significant increase. A strange and not completely understood phenomenon is the change in cryosensitivity of oocytes during the maturation process. Although there is only a minimal difference between the size and shape, immature oocytes are usually more sensitive to cryopreservation than mature (metaphase II (MII) oocytes.12,18,19 The contrary might be supposed, based on the known sensitivity of the meiotic spindle to chilling. More research is needed to understand the reasons for this difference; the alteration of sensitivity of membranes may be one of the possible explanations. The osmotic shock at equilibration may result in shrinking and misshaping of the oocytes, supposedly damaging the cytoskeleton. However, the effect of other agents (for example pronase digestion of the zona pellucida) induces much more serious deformation, followed by surprisingly rapid recovery and maintenance of developmental competence. On the other hand, the osmotic shock that can occur during dilution may result in extensive swelling, rupture of the membrane, lysis, and immediate death of the oocytes.
Hardening of the zona pellucida, attributed by some authors to premature cortical granule release may cause decreased rates of fertilization.6 Fracture is a common consequence of all cryopreservation procedures20 and does not seem to occur more frequently in oocytes than in embryos. However, while the consequence for zona fracture may be similar for both, embryos may survive some level of cell membrane damage, while for the oocyte, any injury at this level is evidently fatal.
WHAT IS THE BEST APPROACH? Based on the points listed above, the principles of a successful cryopreservation strategy can be outlined. Although infrequent in biological study, theory is mostly justified by practice; however, it should be confessed that the sequence of events was (as usual) inverted: the empirically established methods were retrospectively supported by the subsequent detailed theoretical analyses of events. First, we need a method that minimizes chilling injury. So far, in mammalian embryos and oocytes two approaches have been successfully applied for this purpose: the removal of the lipid droplets (by high-speed centrifugation and micromanipulation, although the latter step is not required with the use of some recent techniques21) and by radically increasing the cooling and warming rate to minimize the duration of exposure to the dangerous temperatures. As human oocytes contain a relatively low amount of lipids, centrifugation does not significantly improve survival chances. On the other hand, all forms of traditional slow-rate freezing are obviously less appropriate to avoid chilling than high-rate cooling vitrification strategies. The large cell mass and spherical shape of the oocyte necessitate the use of highly permeable cryoprotectants with low toxicity. As in many areas of vitrification in mammalian embryology, ethylene glycol is the candidate
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of choice for this purpose. According to earlier investigations in rabbits,22 the permeability of ethylene glycol is facilitated by dimethylsulphoxide (DMSO). Further studies have also demonstrated that DMSO may have a beneficial effect on spindle polymerization, and consequently a protective effect at oocyte vitrification.3 Although various proportions of DMSO and ethylene glycol were extensively tested for vitrification of bovine oocytes and embryos, the best results were always achieved with a 1:1 mixture (Vajta, unpublished). To facilitate dehydration, thus decreasing the chances of intracellular ice formation, the addition of non-permeable cryoprotectants is also required. Various substances including polymers with low toxicity were suggested for the purpose, however, the traditionally used sugars, i.e. sucrose or trehalose, seem to be more appropriate. Curiously, although trehalose has been reported many times to be superior, in the past few years it has gradually disappeared from the list of frequently used cryoprotectants. In the past few years, two basically different strategies of equilibration before cooling were applied. Martino et al.23 suggested that dehydration may even be more important than cryoprotectant concentration for prevention of ice crystal formation, and suggested extremely short equilibrations for both the diluted and concentrated cryoprotectant solutions. Subsequently, this strategy was successfully applied by many others for domestic animal oocytes and embryos. However recently another approach has received more attention, and seems to be more efficient for mammalian oocytes: an extended equilibration in a rather diluted first cryoprotectant solution, followed by a short, but slightly prolonged incubation in the second, relatively concentrated vitrification solution also containing a non-permeable cryoprotectant.24–26 Although the time of the exposure is significantly increased, the cumulative toxic effect (as a result of the lower concentration) may be the same or even lower, and the prolonged
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equilibration may ensure proper penetration of cryoprotectant providing appropriate protection to the entire oocyte. The other way to minimize toxic and osmotic effects of cryoprotectant is to decrease the required concentration while maintaining the ice-free solidification pattern. Currently the only practical way to achieve this goal is with the extreme increase in cooling rates. Among the various tools applied for the purpose, electron microscopic grids, Cryoloops, and Cryotops seem to be the most appropriate ones; although very recently similar results were achieved with the open pulled straw technique.27 Either directly by the higher rate of cooling, or indirectly by the decreased toxic and osmotic effect, Cryotop and Cryoloop vitrification with a mixture of relatively low concentrations of DMSO and ethylene glycol do not seem to cause serious anomalies in the spindle structure, and may ensure relatively high developmental rates.26,28 As mentioned above, fracture damage is not specific to oocyte cryopreservation, although the consequences may be more detrimental in oocytes. Fortunately, the open vitrification systems have drastically reduced the occurrence of this type of damage. Retrospectively, it may be supposed that in a closed system, the extreme pressure changes caused by rapidly cooling or warming air bubbles induce dislocations in the partially solidified solution, and with a scissor-like effect cut the zona pellucida or the cell membranes. In the open systems, such mechanical forces are almost completely avoided. The extremely small volume of solutions used also minimizes the chance of fractures. Accordingly, this type of damage is almost entirely eliminated by the application of the ultrarapid open vitrification systems. Finally, the problem of zona hardening and subsequent low level of fertilization has been eliminated in humans with the discovery and subsequent widespread application of ICSI. Although not included in the original
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c
a
b
d Figure 7A.1 The Cryotop vitrification device. The polypropylene strip (a) is attached to a hard plastic handle (b). After vitrification, a hard plastic cover (c) is attached to protect the strip during storage in liquid nitrogen (d). Reproduced with permission from Kuwayama et al.7
goals, the application of ICSI after cryopreservation has contributed much to the increase of efficiency, and has opened the gate for widespread application of oocyte cryopreservation. A road, that seems to be open now, although, to date, very few human embryologists have the courage to enter!
THE CRYOTOP TECHNIQUE The technique and the tool for the Cryotop technique were briefly described in Chapter 2. Here we provide some technical details required for successful cryopreservation of MII phase human oocytes. The commercially available kit (Kitazato, Tokyo, Japan) contains the Cryotop device, a filmstrip attached to a plastic handle also equipped with a cap to cover the filmstrip for safe handling and storage (Figure 7A.1), and all media required for washing, equilibration, vitrification, warming, and dilution. These solutions are based on TCM 199 medium supplemented with synthetic serum substitute (SSS), and containing ethylene glycol, DMSO, and sucrose as permeable and non-permeable cryoprotectants, respectively (see exact concentrations and
sources in Kuwayama et al.7). All media and manipulations should be performed at 25–27°C, except for thawing where medium should be warmed to 37°C. A pulled, fire polished glass pipette with 140–150 µm inner diameter is suggested for all the manipulations. Oocytes can be vitrified 2–6 hours after the ovum pick-up, immediately after denudation. A stepwise, very mild initial equilibration procedure can be carried out by making 20 µL droplets of washing and equilibration solutions (1 and 2 droplets, respectively) close to each other, and unifying droplets when oocytes seem to have completely recovered from the osmotic effect (a total of approximately 6 min). Finally, oocytes should be placed into an equilibration drop and incubated until they are completely recovered (approximately in an additional 9 min). Subsequently, one oocyte should be placed into a large volume (4.5 mL) of vitrification solution, mixed well, and after 60 s loaded on the film strip of the Cryotop. All excess media should be removed leaving only the oocyte covered with a thin layer of vitrification solution. Then the film part should be submerged into liquid nitrogen with a quick and continuous vertical movement to ensure the maximum cooling rate (23 000°C/min). Finally, under the liquid nitrogen, the cap should be fixed on the Cryotop with forceps to protect the film part from mechanical damage during transfer to the container and storage. At warming, the film part of the Cryotop should be submerged quickly into the 37°C thawing solution to achieve the required 42 000°C/min warming rate. After 10 s, the oocyte can be gently removed from the surface of the Cryotop with a pipette and kept submerged in the thawing solution. After 1 min, the dilution should be continued in dilution solution and washing solution 1 and 2 for 3, 5, and 5 min, respectively. Oocytes should be cultured for an additional 2 hours before the
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Before vitrification
PN stage (day 1)
2 h after culture
Immediately after thawing
4-cell stage (day 2)
Blastocyst stage (day 5)
ICSI. Images of oocyte morphology before and after vitrification, ICSI and embryo culture are shown on Figure 7A.2.
OOCYTE VITRIFICATION RESULTS ACHIEVED WITH THE CRYOTOP TECHNIQUE The Cryotop is now used in an increasing number of laboratories worldwide for oocyte vitrification. Almost all these laboratories indicate survival, in vitro developmental, and eventually pregnancy rates much higher than those achieved previously with traditional freezing. However, due to the recent international acknowledgment of the technique, published reports are still sporadic. Here we summarize only documented achievements. It is strongly expected that reports with similar results will be published soon from other groups, as well, confirming the value of the technology for human oocyte cryopreservation. Updated results achieved in the Kato Ladies’ Clinic, Tokyo are shown on Figure 7A.3. Almost all oocytes survived vitrification and ICSI, and the cleavage rate did not differ from that of controls in our laboratory. When blastocyst transfer was applied, 45% of
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Figure 7A.2 Inverted microscopic images of oocytes before and after vitrification, ICSI and embryo culture. PN, pronuclei.
vitrified oocytes developed to healthy babies. So far, more than 50 babies have been born after oocyte vitrification using the Cryotop method, and none of them have had any developmental abnormality. Lucena et al.3 reported 89.2% survival rates after Cryotop vitrification of oocytes, and a total pregnancy rate of 56.5% (13 of 23 patients) with an average of 4.63 embryos transferred to each patient. The Colombian group has also achieved the first baby born after oocyte vitrification in South America (Lucena, unpublished). Ruvalcava et al.29 from Mexico have reported 401/445 (90.1%) survival and 34.1% pregnancy rates after Cryotop vitrification. In Valencia, Spain, Cobo et al. (submitted) have vitrified a total of 225 MII oocytes, of which 217 (96.5%) survived cryopreservation, and of those surviving cryopreservation 165 (76.0%) were normally fertilized after ICSI, which was not different from the controls. Of zygotes 93.9% underwent cleavage on day 2, and blastocyst per fertilized oocyte rates (22.4%) did not differ from the controls. Twenty-one embryo transfers were performed with vitrified oocytes resulting in 13 pregnancies (61.9% pregnancy and 37.2% implantation
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100 90
94
96 90 92 86
80
Percentage
70
73
60 50
50 45
45
40 39 30 28 20 10 0 Surv
2PN
2cell
BL
Day 2 ET
Preg
Deliv
Day 5 ET
Figure 7A.3 Results of oocyte cryopreservation performed at the Kato Ladies’ Clinic with Cryotop vitrification. Columns refer to percentages of vitrified oocytes surviving vitrification (Surv), developing to the two pronuclei stage (2PN), proceeding to cleavage (2cell), developing to blastocyst stage (BL), resulting in pregnancy (Preg), and delivery of healthy babies (Deliv). The total number of oocytes used for day 2 and day 5 embryo transfer (ET) was 86 and 25, respectively. Finally, 18 and 11 embryos were transferred on day 2 and 5, respectively (2.9 and 1 embryos per recipient).
rates). At the time of going to press, 11 patients are pregnant (52.4%). In total, more than 200 healthy babies have been obtained from vitrified oocytes using the Cryotop method so far.
CONCLUSION According to the high number of ongoing pregnancies listed here and confirmed by personal communication in other laboratories, the number of babies born after Cryotop vitrification of oocytes may soon exceed the
total number of babies born after other cryopreservation methods worldwide. All data obtained from different laboratories (including survival, fertilization, embryo development, pregnancy rates) suggest that oocytes vitrified with this technology are highly viable, and their developmental competence is comparable with that of fresh oocytes. The increasing evidence proves that Cryotop vitrification may offer solutions for women with various fertility problems, and may participate in compensation of the handicap of women from the standpoint of reproduction.
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References 1. Isachenko V, Isachenko E, Montag M, et al. 2005c Clean technique for cryoprotectant-free vitrification of human spermatozoa. Reprod Biomed Online 10: 350–3544. 2. Meirow D 2000 Reproduction post-chemotherapy in young cancer patients. Mol Cell Endocrinol 169, 123–131. 3. Lucena E, Bernal DP, Lucena C, Rojas A, Moran A, Lucena A 2006 Successful ongoing pregnancies after vitrification of oocytes. Fertility and Sterility 85: 108–11. 4. Cha KY, Han SY, Chung HM et al. Pregnancies and deliveries after in vitro maturation culture followed by in vitro fertilization and embryo transfer without stimulation in women with polycystic ovary syndrome. Fertil Steril 2000; 73: 978–83. 5. Chung HM, Hong SW, Lim JM, et al. In vitro blastocyst formation of human oocytes obtained from unstimulated and stimulated cycles after vitrification at various maturational stages. Fertil Steril 2000; 73: 545–51. 6. Stachecki JJ, Cohen J 2004 An overview of oocyte cryopreservation. Reproductive Biomedicine Online 9, 152–63. 7. Kuwayama M, Vajta G, Kato O et al. Highly efficient vitrification method for cryopreservation of human oocytes. Reproductive BioMedicine Online 2005; 11: 300–308. 8. Paynter SJ. A rational approach to oocyte cryopreservation. Reprod BioMed Online 2005; 10: 578–86. 9. Vanderzwalmen P, Bertin G, Debauche Ch et al. Births after vitrificaton at morula and blastocyst stages: effect of artificial reduction of the blastocoelic cavity before vitrification. Human Reproduction 2002; 17: 744–51. 10. Son WY, Yoon SH, Yoon, HJ. Pregnancy outcome following transfer of human blastocysts vitrified on electron microscopy grids after induced collapse of the blastocoele. Human Reproduction 2003; 18: 137–9. 11. Hiraoka K, Hiraoka K, Kinutani M et al. Blastocoele collapse by micropipetting prior to vitrification gives excellent survival and pregnancy outcomes for human day 5 and 6 expanded blastocysts. Human Reproduction 2004; 19: 2884–8. 12. Ghetler Y, Yavin S, Shalgi R et al. The effect of chilling on membrane lipid phase transition
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24. 25.
in human oocytes and zygotes. Human Reproduction 2005; 20: 3385–9. Magistrini M, Szollosi D. Effects of cold and of isopropyl N-phenylcarbamate on the second meiotic spindle of mouse oocytes. Eur J. Cell Biol 1980; 22: 699–707. Sathananthan, 1998. Pickering S, Braude P, Hohnson M et al. Transient cooling to room temperature can cause irreversible disruption of the meiotic spindle in the human oocyte. Fertil Steril 1990; 54:102–8. Fabbri R, Porcu E, Marsella T et al. Human oocyte cryopreservation: new perspectives regarding oocyte survival. Hum Reprod 2001; 16: 411–6. Stachecki JJ, Munne S, Cohen J. Spindle organization after cryopreservation of mouse, human, and bovine oocytes. Reproductive BioMedicine Online 2004; 8: 664–72. Leibo SP, Martino A, Kobayashi S, Pollard JW. Stage-dependent sensitivity of oocytes and embryos to low temperatures. Anim Reprod Sci 1996 ; 42: 45–53. Men H, Monson RL, Rutledge JJ. Effect of meiotic stage and maturation protocols on bovine oocyte’s resistance to cryopreservation. Theriogenology 2002; 57: 1095–103. Kasai M, Zhu SE, Pedro PB et al. Fracture damage of embryos and its prevention during vitrification and warming. Cryobiology 1996; 33: 459–64. Esaki R, Ueda H, Kurome M et al. Cryopreservation of porcine embryos derived from in vitro-matured oocytes. Biol Reprod 2004; 71: 432–7. Vicente JS, Garcia-Ximenez F. Osmotic and cryoprotective effects of a mixture of DMSO and ethylene glycol on rabbit morulae. Theriogenology 1994; 42: 1205–15. Martino A, Songsasen N, Leibo SP. Development into blastocysts of bovine oocytes cryopreserved by ultra-rapid cooling. Biol Reprod 1996; 54: 1059–69. Papis K, Shimizu M, Izaike Y. Factors affecting the survivability of bovine oocytes vitrified in droplets. Theriogenology 2000; 54: 651–8. Dinnyes A, Dai Y, Jiang S et al. High developmental rates of vitrified bovine oocytes following parthenogenetic activation, in vitro
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fertilization, and somatic cell nuclear transfer. Biol Reprod 2000; 63: 513–8. 26. Kuwayama M. Vitrification of human oocytes and embryos (Japanese). In: IVF Update. Tokyo: Medical View, 2001: 230–4. 27. Selman H, Angelini A, Barnocchi N, Brusco GF, Pacchiarotti A, Aragona C. Ongoing pregnancies after vitrification of human oocytes
using a combined solution of ethylene glycol and dimethyl sulfoxide. Fertility and Sterility 2006; 86: 997–1000. 28. Liebermann et al. 2003. 29. Ruvalcaba L, García M, Martínez R et al. Oocytes vitrification success: first ongoing Pregnancy in Mexico. Reprod Hum 2005; 3: 7–10.
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7B
Shee-Uan Chen and Yu-Shih Yang
INTRODUCTION Cryopreservation of mammalian and human oocytes has been significantly improved by the refined slow-freezing methods and new vitrification techniques.1–12 The slow-freezing method using a programmed cryo-machine is traditionally employed for the cryopreservation of oocytes.13 This procedure usually take several hours. Vitrification is an important alternative method. With high concentrations of cryoprotectants and a fast cooling rate, it transforms cells into an amorphous glassy state, instead of ice crystal formation.14,15 Vitrification is time-saving and does not require special equipment. The vitrification methods may replace the slow-freezing method for cryopreservation of oocytes.16–19 The recent improvements of vitrification include the concepts of reduction of concentration of cryoprotectants, increase of cooling and warming rates, recovery of meiotic spindle, and timely fertilization.
REDUCTION OF CONCENTRATION AND TOXICITY OF VITRIFICATION SOLUTION Rall and Fahy14 first successfully vitrified mouse 8-cell embryos using conventional straws with the medium consisting of 20.5% w/v dimethylsulfoxide (DMSO), 15.5% w/v acetamide, 10% w/v propylene glycol, and 6% w/v polyethylene glycol, which required a low temperature of 4°C during the treatment. Subsequent investigators made a significant
improvement by adjustment of cryoprotectants to reduce toxicity, thus permitting the equilibration steps to be performed at room temperature or 35–37°C.20,21 Ali and Shelton21 undertook a systematic and extensive investigation involving various combinations of cryoprotectant solutions. They developed an ethylene glycol (EG)-based solution consisting of 5.5 mol/L EG and 1.0 mol/L sucrose which was less toxic. This vitrification solution was used to cryopreserve all preimplantation stages of in vivo generated mouse22 and day-6 sheep embryos23 without significant loss of viability in vitro or in vivo. Chen et al.24 used this formulation of vitrification solution for human oocytes and attained high survival rates in conventional straws. Recently, the concentration of vitrification solution has been further decreased because the minimum volume method with increased cooling and warming rates achieves vitrification with less concentrated cryoprotectants (Table 7B.1). The vitrification solution consisting of 15% (v/v) EG, 15% (v/v) DMSO or 1,2-propanediol (PROH), and 0.5 mol/L sucrose can be vitrified with the minimum volume method.11,26,27 EG, with the characteristics of low toxicity and rapid permeation of the cell, is an important component of vitrification solutions. Some authors have mixed other permeating agents, such as DMSO or PROH, to reduce the concentration of this single cryoprotectant in order to decrease the individual specific toxicity.26–29 Non-permeable cryoprotectants can facilitate dehydration and vitrification, and can be applied in combination to reduce
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Table 7B.1
Various regimens of cryoprotectants, exposure time, and temperature for vitrification and their devices
Equilibration solution
Vitrification solutions
Warming and dilution ο
Devices
Authors
25% VS1, 15 min, 20ο C; 50% VS1, 10 min, 4ο C
VS1: 20.5% w/v DMSO, 15.5% w/v acetamide, 10% w/v propylene glycol, 6% w/v polyethylene glycol, 4ο C
50% VS1, 4 C
Straw
Rall and Fahy, 198514 (mouse embryos)
7.5% EG, 7.5% DMSO, 3 min, 34–36 oC
16.5% EG, 16.5% DMSO, 0.5 mol/L sucrose, 25 s, 34–36ο C
0.25, 0.15 mol/L sucrose, 37ο C
OPS
Vajta et al., 19987 (bovine oocytes and embryos)
1.5 mol/L EG, 5 min, RT
5.5 mol/L EG, 1.0 mol/L sucrose, 60 s, RT
0.5, 0.25, 0.125 mol/L sucrose, RT
Straw
Chen et al., 200024 (human oocytes)
1.5 mol/L EG, 2.5 min, 37ο C
5.5 mol/L EG, 1.0 mol/L sucrose, 20 s, 37ο C
1.0, 0.5, 0.25, 0.125 mol/L sucrose, 37ο C
Grid
Yoon et al., 20039 (human oocytes)
7.5% EG, 7.5% DMSO, 2 min, 37oC
15% EG, 15% DMSO, 10 mg/mL Ficoll, 0.65 mol/L sucrose, 25–30 s, 37ο C
0.33, 0.2 mol/L sucrose, 37ο C
Cryoloop
Mukaida et al., 200325 (human blastocysts)
1.6 mol/L EG, 5–15 min, 22ο C
5.0 mol/L EG, 1.0 mol/L sucrose, 30 s, 22ο C
1.0, 0.5 mol/L sucrose, 37ο C
Cryotop
Kuwayama et al., 200510 (human oocytes)
7.5% EG, 7.5% PROH, 5 min, RT
15% EG, 15% PROH, 0. 5 mol/L sucrose, 45–60 s, RT
1.0, 0.5, 0.25 mol/L sucrose, 37ο C
Cryoleaf
Chian et al., 200511 (human oocytes)
VS, vitrification solution; RT, room temperature.
the required concentration of permeable cryoprotectants. This strategy further reduces the toxicity of the vitrification solution. Sucrose has become almost a standard component of vitrification solutions. Other investigators have added other macromolecules, such as Ficoll, in vitrification solutions that are thought to stabilize the glass formation and form a protective coating around embryos or oocytes.20,25,30 Sera of various origins, serum albumin preparations, recombinant albumin, or synthetic serum substitute (SSS) are common additives to vitrification solutions. Another important strategy to reduce toxic effects from vitrification solution is the stepwise
equilibration of cryoprotectants. A two-step strategy is most commonly used. The pretreatment (equilibration) solution contains 20–50% concentrations of permeating cryoprotectants of the vitrification solution. The lower concentration of permeating cryoprotectants in the equilibration solution is much less toxic than the vitrification solution. Oocytes in the pretreatment solution shrink initially and gradually re-expand to their original volume. This observation indicates the entry of the permeating cryoprotectants into the oocytes that may facilitate intracellular vitrification in the subsequent procedures. It reduces the time needed for exposure to the vitrification solution that is more toxic for oocytes. This approach may
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provide better protection for oocytes that have a low surface/volume ratio. In the vitrification of human oocytes, it has been demonstrated that oocytes pretreated with equilibration solution had a significantly higher survival rate than those without pretreatment.24 One-step vitrification without pretreatment has the possibility of insufficient permeation of the cryoprotectants that may result in intracellular ice formation during cooling or warming.
EQUILIBRATION AND VITRIFICATION For oocyte vitrification, some investigators perform exposure to cryoprotectants at room temperature,10,11,24 but other investigators operate the procedures at 35–37°C7,9 (Table 7B.1). The higher temperature enhances the passage of the permeating cryoprotectants across the cell membrane, but the toxicity is also increased. Therefore, at 37°C, 2–3 min are usually used for the pretreatment solution, and 20–30 s for exposure to the vitrification solution.7,9 In contrast, at room temperature, 5–15 min are commonly used for the pretreatment solution, and 30–60 s for exposure to the vitrification solution.10,11,24 Prolonged exposure to the concentrated cryoprotectants may induce toxic effects. The human oocytes treated in vitrification solution for 120 s had a poorer fertilization outcome than those vitrified in 60 s.24 Yoon et al.9 used electron microscope grids to perform vitrification of human oocytes. At 37°C, oocytes were pre-equilibrated for 2.5 min in Dulbecco’s phosphate buffered saline supplemented with 1.5 mol/L EG and 10% (v/v) fetal bovine serum. Oocytes were then placed for the final equilibration in 5.5 mol/L EG and 1.0 mol/L sucrose vitrification solution for 20 s. They achieved a survival rate of 69% (325/474) and a pregnancy rate of 21% (6/28). Kuwayama et al.10 used Cryotops to carry out human oocyte vitrification. At 22°C, oocytes were pretreated with 1.6 mol/L EG in tissue culture medium (TCM) for 5–15 min until the
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oocytes had completely recovered their original volume. Oocytes were then treated with a vitrification solution of 6.8 mol/L EG and 1.0 mol/L sucrose in TCM medium for 30 s. They achieved a survival rate of 90% (58/64) and a pregnancy rate of 41% (12/29). Chian et al.27 used Cryotops to vitrify bovine oocytes. At room temperature, oocytes were pretreated in equilibration solution of 7.5% (v/v) EG and 7.5% (v/v) PROH for 5 min. Then the oocytes were transferred to a vitrification solution of 15% (v/v) EG, 15% (v/v) PROH, and 0.5 mol/L sucrose for 45–60 s. They found that 15% EG, 15% PROH, and 0.5 mol/L sucrose had a higher rate of blastocyst development than 15% EG, 15% DMSO and 0.5 mol/L sucrose. This may be due to lower toxicity with PROH than DMSO. The formula has been used for human oocytes by the same group, and they accomplished good results with a survival rate of 94% (169/180) and a pregnancy rate of 47% (7/15).11 For vitrification, therefore, using minimum volume methods instead of conventional straws achieves higher cooling and warming rates, and leads to the reduction in concentration of cryoprotectants vitrified. The newly formulated cryoprotectants allow the performance of equilibration at room temperature or 37°C. The duration of exposure for equilibration or vitrification solutions should depend on operating temperature and concentration of cryoprotectants.
WARMING AND DILUTION The vitrified oocyte is sensitive to osmotic changes after warming. Stepwise dilution in an osmotic buffer is commonly used to prevent excessive swelling and lysis of the oocyte as the permeating cryoprotectants are removed. Two- to four-step dilutions with sucrose solutions have usually been used (Table 7B.1). Chen et al.24 found that there were no differences in survival and fertilization for vitrified human oocytes diluted by three or four steps. Therefore, the four-step
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dilutions may not be necessary for human oocytes. Although, one-step dilution without significant compromise of survival was reported in vitrification of bovine blastocysts31 and porcine blastocysts.32 The speed of warming is important to prevent devitrification. For the conventional straw, warming is performed by direct immersion of the straw into a water bath. Holding the straw in air for 5 seconds before immersion can avoid bursting. After cutting the straw, the oocytes are expelled into the dilution solution. Minimum volume methods such as electron microscope grid, open pulled straws (OPS), Cryoloop, or Cryotop can be directly submerged into the dilution medium. The oocytes come into contact with the dilution medium immediately. Most investigators performed warming and dilution at 37°C7,9–11 (Table 7B.1). For thawing of oocytes vitrified by the Cryoleaf method, Chian et al.11 used three-step dilutions and performed thawing with 1.0 mol/L sucrose solution for 1 min at 37°C. The thawed oocytes were transferred to 0.5 mol/L and 0.25 mol/L sucrose solutions for 3 min respectively, and then washed twice with culture medium. Kuwayama et al.10 used two-step dilutions and performed thawing using 1.0 mol/L sucrose for 1 min at 37°C. Oocytes were transferred into 0.5 mol/L sucrose for 3 min, and then washed twice with culture medium. It has been shown that the meiotic spindle of human oocytes depolymerized when cooled to room temperature for 10–30 min, and that the damage was time dependent.33 Therefore, dilution for vitrified oocytes with sucrose solutions at 37°C may reduce spindle damage during the procedures.
INCREASED COOLING AND WARMING RATES BY MINIMUM VOLUME METHODS A 0.25 mL conventional straw was initially, commonly used for vitrification of mammalian oocytes or embryos.14,20,24 The cooling rate was around 2500°C/min and the warming
rate was 1300°C/min.14 The limited speed of thermal change of the conventional straw needs more concentrated cryoprotectants to achieve vitrification during cooling and to prevent devitrification during warming. The higher concentrated cryoprotectants are more toxic to oocytes or embryos. A higher cooling rate can facilitate vitrification and a higher warming rate will prevent devitrification with less concentrated cryoprotectants. This can reduce toxicity of vitrification solutions. Minimum volume methods can prevent less concentrated cryoprotectants from ice crystal formation that had been observed microscopically.34 In addition, the high speed of cooling and warming of minimum volume methods can rapidly pass through the damaging temperature zone liable to cause chilling injury, between 15 and − 15°C.6 Chilling injury harms mainly the cytoplasmic lipid droplets and meiotic spindle of oocytes. Minimum volume vitrification may also avoid fracture injury. Therefore, scientists have made great efforts to find new methods using minimum volume techniques to increase thermal change.
EVOLUTION OF MINIMUM VOLUME METHODS Landa and Tepla35 performed vitrification by dropping mouse embryos directly into the liquid nitrogen. However, to form a drop requires a relatively large amount of solution (approximately 5 µL). When the drop reaches the liquid nitrogen, it will not sink immediately but will float on the surface for several seconds. The drop induces a strong evaporation at its surface that decreases the cooling rate. Furthermore, it is difficult to find the embryos and to perform thawing. Speculative advantages in maximizing cooling and warming rates cannot be attained by this method. Further refinements using various carriers to minimize the volume of vitrification solution and to submerge the sample quickly into the liquid nitrogen have been comprehensively studied. The innovative devices for supporting
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oocytes or embryos for minimum volume vitrification include the electron microscope grid, minimum drop size on a solid surface, open pulled straw (OPS), Cryoloop, hemistraw system, or Cryotop, etc. (Table 7B.2). Steponkus et al.15 first utilized electron microscope copper grids (Figure 7B.1) as a carrier for the minimum volume-direct contact approach. They successfully cryopreserved chill-sensitive Drosophila embryos by vitrification. The method overcame the problem of chilling injury at subzero temperatures associated with conventional slow-freezing procedures for Drosophila embryos. Martino et al.6 applied this technique for bovine oocytes, and achieved higher growth potential than using conventional straws. Hong et al.8 and other authors9 achieved successful pregnancies from vitrified human oocytes using the grid method. Arav36 developed the minimum drop size (MDS) method with a small droplet of vitrification solution containing the oocyte or embryo placed on a solid surface that was then immersed into liquid nitrogen. The approach was used later with some modifications called the minimum volume cooling (MVC).37 Hamawaki et al.37 placed the minimum vitrification volume containing bovine blastocysts on the outside wall of a conventional straw and directly submerged it into liquid nitrogen. They found that the survival rate was higher for MVC than conventional vitrification in straws. Vanderzwalmen et al.41 developed the hemi-straw system (HSS) using a cut open straw as the carrier. Dinnyes et al.40 designed a pre-cooled metal surface (solidsurface vitrification; SSV) to vitrify bovine oocytes. A steel cube is partially submerged into liquid nitrogen. Microdrops of vitrification solution containing oocytes are dropped onto the cold surface of aluminum foil on the metal cube for vitrification. Kuwayama and Kato42 developed the Cryotop method from a modification of MVC procedure. Oocytes were placed on the top of a fine polypropylene strip attached to a hard plastic handle. Chian et al.11 modified the Cryotop to create
Table 7B.2 embryos
Various vitrification methods for oocytes or
Vitrification method
Authors
Straw Direct dropping into liquid nitrogen Electron microscopic grids Minimum drop size (MDS) Open pulled straw (OPS) Minimum volume cooling (MVC) Cryoloop Glass micropipettes (GMP) Solid surface vitrification (SSV) Hemi-straw system (HSS)
Rall and Fahy, 198514 Landa and Tepla, 199035
Cryotop Nylon mesh Closed pulled straw (CPS) Flexipet denuding pipette (FDP) Cryotip Cryoleaf Direct cover vitrification (DCV) (for ovarian tissues)
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Steponkus et al., 199015 Arav, 199236 Vajta et al., 19987 Hamawaki et al., 199937 Lane et al., 199938 Kong et al., 200039 Dinnyes et al., 200040 Vanderzwalmen et al., 200041 Kuwayama and Kate, 200042 Matsumoto et al., 200143 Chen et al., 200144 Liebermann et al., 200245 Kuwayama et al., 200546 Chian et al., 200511 Chen et al., 200647
the Cryoleaf with a different method of applying the protective sheath. Both Cryotop and Cryoleaf achieved high success rates in vitrification of human oocytes.10,11 Vajta et al.7 developed the open pulled straw (OPS) (Figure 7B.2) to hold bovine oocytes with a very small amount of vitrification solution. The idea was to reduce the volume of the sample by diminishing the diameter of the conventional straw. Straws were heated and pulled by hand, then cut at the tapering end with a razor blade. As a result, the diameter and the wall thickness of the straw decreased to approximately half of the original. The volume for keeping oocytes is reduced from 25 µL to 1 µL. They found that the OPS achieved better results than conventional straw vitrification. Kuleshova et al.48 applied the OPS for vitrification of human oocytes and achieved a successful pregnancy. With a similar design, Kong et al.39 used glass micropipettes (GMP) to perform vitrification
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Figure 7B.1 The image shows an electron microscope copper grid. Oocytes are transferred onto the grid. To reduce the volume of vitrification solution, the underside of the grid is blotted on a filter membrane. The grid supporting oocytes is plunged into liquid nitrogen using a fine forceps. Scale bar = 0.3 cm.
Figure 7B.2 The image shows an open pulled straw (OPS). To produce a pulled straw, the 0.25 mL plastic straw is heat-softened over a hot plate and pulled manually. The pulled straw is cut at the tapered end. Oocytes are loaded into the tip (arrow) of the OPS by simply touching a microdrop (1–2 µL) of vitrification solution containing oocytes. Scale bar = 1 cm.
of mouse blastocysts. Liebermann et al.45 applied the Flexipet denuding pipette (FDP) for human pronuclear zygotes. Chen et al.44 modified the loading of the OPS turning it into a closed system, called closed pulled straw (CPS). CPS has the beneficial characteristics of the OPS such as a rapid thermal change method, and of conventional straws in that it is a non-contact mode. The vitrification medium containing the oocytes was isolated by two small segments of air and medium. Through this closed loading system of CPS, the oocytes do not directly contact the liquid nitrogen, which may occur with the OPS. Kuwayama et al.46 developed the Cryotip method. Principally, it is a heat-sealed pulled straw technique. The speed of cooling and warming is slower with the Cryotip than with the Cryotop. However, the thermal speed accompanied by the Cryotip is still high enough to obtain adequate vitrification. Kuwayama et al.46 reported no difference for supporting blastocyst survival and pregnancies between the Cryotip and the Cryotop. The use of the closed Cryotip system may eliminate the
potential of contamination and maintain the efficacy. Lane et al.38 used Cryoloops for vitrifying mouse and human blastocysts. The Cryoloop consists of a small nylon loop attached to a holder and is equipped with a vial as a container (Figure 7B.3). Oocytes or embryos are suspended on a film of vitrification solution bridging the hole of the loop that is then plunged into liquid nitrogen. The Cryoloop was originally designed for mounting of crystals for macromolecular cryo-crystallography. 49,50 Cryoloops have been confirmed to be highly successful for human blastocyst vitrification by Mukaida et al.25 With these minimum volume methods, the achievable cooling rate and warming rate were significantly increased (Table 7B.3). The cooling and warming rates were 23 000°C/ min and 42 100°C/min, respectively, for the Cryotop (Figure 7B.4).10 The cooling and warming rates are higher than those achievable with the OPS (16 700°C/min and 13 900°C/min, respectively).7 Even for the noncontact method of the Cryotip, the cooling and warming rates are around 12 000 °C/min
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Table 7B.3 Various procedures for vitrification of oocytes and volumes of vitrification solution with estimated cooling and warming rates in the literature Vitrification method Straw (Rall and Fahy, 1987)14 OPS (Vajta et al., 1998)7 Cryotop (Kuwayama et al., 2005)10 Cryotip (Kuwayama et al., 2005)46 Figure 7B.3 The image shows a Cryoloop. The Cryoloop consists of a nylon loop (small arrow) mounted on a stainless steel rod that is inserted into the lid of a cryovial. The Cryoloop is dipped into vitrification solution to make a filmy layer of solution on the nylon loop. The oocytes are transferred onto the filmy layer. It is then stored in a cryovial (large arrow) filled with liquid nitrogen. Scale bar = 1 cm.
and 24 000°C/min, respectively.46 Rapid thermal changes are important to achieve vitrification during cooling and to prevent devitrification during warming. Therefore, we can use less concentrated cryoprotectant to avoid toxicity from chemicals. With the principle of minimum volume of vitrification solution and direct contact with liquid nitrogen, Chen et al.47 developed an innovative method to vitrify ovarian tissues by direct cover vitrification (DCV). They used the less concentrated cryoprotectants (15% EG, 15% DMSO, and 0.5 mol/L sucrose). After twostep equilibration with cryoprotectants, ovarian tissues were placed on a piece of gauze to remove the surrounding vitrification solution. The ovarian tissues were put in a cryovial. Liquid nitrogen was directly poured onto the ovarian tissue for vitrification. The cooling rate was maximized because there was no thermoinsulating layer between the tissues and the liquid nitrogen. The cap of the cryovial was then closed. The vial was placed into a liquid nitrogen tank for storage. For thawing, the ovarian tissues were moved into dilution solutions.
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Cooling rate (° C/min)
Warming rate (° C/min)
25
2 500
1 300
1
16 700
13 900
0.1
23 000
42 100
1
12 000
24 000
Volume (µL)
In the mice models of ovarian tissue cryopreservation, the percentages of follicle viability and pregnancy potential from DCV were found to be significantly greater than for conventional vitrification or slow freezing.
COMMON VITRIFICATION METHODS Following pretreatment with the equilibration solution and treatment with the vitrification solution, the steps of loading oocytes for cooling are described for common vitrification methods. The steps of warming with release of oocytes are also included in the following.
Conventional straw For the conventional straw method24 oocytes are loaded in a 0.25 mL plastic straw. The straw is filled with 1 cm of vitrification medium, 0.5 cm of air, 2 cm of vitrification medium containing the oocytes, 0.5 cm of air, and 3.5 cm of vitrification medium. The straw is then plunged into liquid nitrogen. For thawing, the straw is taken out and held in the air for 5 s, then it is plunged into water for 10 s. The vitrification solution in the straw should remain transparent in the liquid
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Open pulled straw
Figure 7B.4 The image shows a Cryotop. The Cryotop consists of a fine polypropylene strip (small arrow), attached to a plastic holder. Oocytes are loaded on the strip with minimal vitrification solution. The Cryotop is then immersed into liquid nitrogen. The strip is covered with the plastic tube (large arrows) in liquid nitrogen to protect it during storage. Scale bar = 1 cm.
For making an OPS7 (Figure 7B.2), a 0.25 mL plastic straw is heat-softened over a hot plate and pulled manually. The pulled straw is cut at the tapered end. The inner diameter of the tip is approximately 0.8 mm with a wall thickness of 0.07 mm. Commercial sterile pulled straws have recently become available. Oocytes are loaded into the tip of the pulled straw through the capillary effect by simply touching a microdrop (1–2 µL) of vitrification solution containing the oocytes. Plunging the OPS into liquid nitrogen performs the cooling. For thawing, the tip of OPS is put into the dilution solution, and the oocytes are expelled spontaneously by the pressure of the warming gas in the straw.
Cryoloop nitrogen, air, and water. Otherwise, a whitish discoloration of vitrification medium in the straw indicates ice crystal formation or devitrification. The straw is cut by scissors, and the contents containing the oocytes are expelled into the dilution solution.
Electron microscope copper grids For the electron microscope grid procedure6 oocytes are transferred onto the electron microscope copper grid (Figure 7B.1). To reduce the volume of vitrification solution, the underside of the grid is blotted on a filter membrane. A small volume is critical to prevent fracture injury. The grid supporting the oocytes is plunged into liquid nitrogen using fine forceps, and stored in a cryovial filled with liquid nitrogen. The solidified vitrification solution fixes the oocytes to the grid during cooling and storage. The vials are attached onto standard canes and stored in liquid nitrogen. At warming, the grid is picked up and put directly into the dilution solution.
The Cryoloop38 consists of a nylon loop (0.5–0.7 mm diameter) mounted on a stainless steel rod that is inserted into the lid of a Cryovial (Hampton Research, Laguna Niguel, CA) (Figure 7B.3). While the oocytes are suspended in pretreatment solution, the Cryoloop is dipped into vitrification solution to make a filmy layer of solution on the nylon loop by surface tension. The oocytes are then treated in vitrification solution and transferred onto the filmy layer on the nylon loop. The volume of vitrification solution in the Cryoloop method is especially confined. The Cryoloop is plunged into liquid nitrogen. It is then sealed in a cryovial, which has been previously submerged in liquid nitrogen. When warming the oocytes, the vial is opened, and the Cryoloop is placed directly into the dilution solution. Oocytes are immediately released from the loop into the solution.
Solid surface vitrification For SSV40 groups of five to ten oocytes are dropped on the surface of aluminum foil on a steel cube that is cooled to around −150 to −180oC
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by partial immersion in liquid nitrogen. The drop size is 1–2 µL. The oocytes are instantaneously vitrified on the pre-cooled metal surface, and do not contact liquid nitrogen directly. This method also avoids nitrogen vapor. The vitrified droplets are moved with a nitrogen-cooled forceps into 1 ml cryovials. For thawing, they are dropped into a 39oC dilution solution. A commercially available device has also been manufactured (CMV; Cryologic, Australia).
Japan), attached to a plastic holder and equipped with a protective plastic tube (Figure 7B.4). Oocytes are loaded on the strip with minimal solution, and the remaining solution is almost completely removed by aspiration. The Cryotop is immersed into liquid nitrogen. Then, the strip is covered with the plastic tube in liquid nitrogen to protect it during storage. For warming, the protective cover is removed from the Cryotop while it is still submerged in liquid nitrogen. The strip is immersed directly into the dilution solution.
Hemi-straw system For the HSS method41,51 an estimated volume of 0.3 µl of vitrification solution containing the oocytes is placed on the tip of the trough of the hemi-straw (CBS; Cryo Bio System, France). The hemi-straw is put into liquid nitrogen. It is then inserted in a larger pre-cooled straw with the aid of forceps. For thawing, the hemi-straw is pulled out of the larger straw under liquid nitrogen using forceps. The tip of the hemistraw holding the samples is immediately placed into the dilution solution.
Closed pulled straw In the CPS method44 the oocytes are loaded into the tip of a pulled straw with 2 mm of vitrification medium, 2 mm of air, 2 mm of vitrification medium containing the oocytes, 2 mm of air, and 2 mm of vitrification medium using a syringe. They are then plunged into liquid nitrogen for cooling and storage. For thawing, the CPS is removed from the liquid nitrogen. The tip is submerged into the dilution solution. The opposite end of the pulled straw is sealed using the index finger. The contents are then expelled by using the increase in air pressure in the tube caused by the thermal change.
Cryotop The Cryotop10 has a fine polypropylene strip (0.4 mm wide × 20 mm long × 0.1 mm thick) (Kitazato Bio Pharma Co, Ltd, Shizuoka-ken,
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Cryotip The Cryotip46 consists of a plastic straw with a narrow part (250 µm inner diameter, 20 µm wall thickness, and 3 cm length) connected to a wide part. It is equipped with a movable protective metal sleeve. Oocytes are loaded in approximately 1 µL vitrification solution into the narrow part of the Cryotip by aspiration with a connected syringe. The straw is heatsealed at both ends. The Cryotip is plunged into liquid nitrogen. For warming, the Cryotip is taken out from liquid nitrogen and placed into a 37°C water bath for 3 s. It is then decontaminated with ethanol, and the sealed ends are cut with a pair of sterile scissors. The contents are expelled into the dilution solution.
Devices for vitrification of large numbers of oocytes The number of oocytes per supporting device such as electron microscope grid, OPS, Cryoloop, and Cryotop is limited. The electron microscope grid may hold 10–15 oocytes, while an OPS can hold four to six, and a Cryoloop one to three. Using a Cryotop, Chian et al. reported that a maximum of 10–15 denuded oocytes can be loaded, however, a smaller number of cumulus–oocyte complexes (COC) can be loaded. For vitrification of large numbers of immature COCs in domestic animals, Matsumoto et al.43 developed a nylon mesh technique. The nylon mesh design was a
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modification of the electron microscope grid. The larger area of the supporting device allowed a large amount of oocytes to be held for vitrification at the same time; it could hold 40 COCs. The thawed COCs were able to continue maturation in vitro and development.52 For the same purpose, Chian et al.27 used a thin plastic sticker (20 × 5 × 0.2 mm), a modification of the Cryotop, to vitrify bovine oocytes. It allowed 30–50 COCs to be held, and achieved a survival rate of 90%.
INCREASED COOLING RATE USING LIQUID NITROGEN SLUSH Three main factors influence the possibility of ice crystal formation during vitrification including the volume of the sample, the viscosity of the solution, and the cooling rate. For enhancing the cooling rate, one approach is to employ liquid nitrogen slush instead of liquid nitrogen for cooling. Nitrogen slush can be produced from liquid nitrogen by using a vacuum so that part of the liquid nitrogen evaporates and the remainder cools down. The mixture of nitrogen slush and cooled liquid nitrogen reaches about −205°C. The sample immersed into liquid nitrogen slush has less evaporation, and the cooling rate is relatively faster. The approach had been used very early on by Steponkus et al.15 It is commercially available in the form of the VitMaster (IMT, Israel), a device producing a vacuum in the toughened liquid nitrogen container.53 However, the theoretical advantages for enhancing vitrification using the VitMaster have not gained it popularity. Most investigators achieve satisfactory results with vitrification of human oocytes or embryos using liquid nitrogen only.10–12,35 Huang et al.54 used Cryoloops and the VitMaster to perform vitrification of human blastocysts. They achieved a survival rate of 77% (74/96), a pregnancy rate of 54% (7/13), with an implantation rate of 23% (14/60). Their results did not prove the advantage of the VitMaster, compared with the results from
Mukaida et al.25 or Hiraoka et al.26 who did not use the VitMaster. Cuello et al.55 used the OPS method for vitrifying porcine embryos with the VitMaster, or liquid nitrogen only. They found that, with the same vitrification solution, using the VitMaster did not enhance the efficiency of in vitro development of vitrified porcine embryos. In contrast, Cai et al.56 vitrified rabbit oocytes with Cryoloops and studied meiotic spindles with immunofluorescent stain and confocal microscopy. They found that the faster cooling rate with the VitMaster had fewer adverse effects on the spindle configuration and embryo development than using liquid nitrogen alone. The superiority of the VitMaster compared with liquid nitrogen alone still needs further investigation.
PREVENTING POTENTIAL CONTAMINATION FROM LIQUID NITROGEN Recent vitrification methods with a minimum volume-direct contact technique significantly improved success rates. However, a major concern should be mentioned regarding the potential risk to human oocytes or embryos from contaminated liquid nitrogen at the time of vitrification or storage. Yet, no reports have documented cases of liquid nitrogen-mediated disease transmission through embryo transfer of frozen-thawed cycles. Nonetheless, based on the experiments of Bielanski et al.57 crosscontamination may take place during storage among open systems. In order to avoid contamination from vitrification of oocytes or embryos, efforts should be made to reduce the risk with the procedures of cooling and storage. Cooling can be performed in aseptically treated liquid nitrogen. The liquid nitrogen is filtered through a 0.2 µm pore-size filter. UV illumination can also be applied. Another strategy is to place the open carrier into a container that isolates it from the liquid nitrogen during storage. For aseptic storage of OPS, after
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cooling the pulled straws can be wrapped in pre-cooled 0.5 mL straws.7 For storage of grids, they can be placed into vials.9 Some minimum-volume vitrification techniques were developed to avoid direct contact with the liquid nitrogen.40,44,46 These modified techniques may have reduced thermal change compared with direct contact methods. The method of SSV using metal surfaces for cooling does not expose samples to liquid nitrogen.40 Chen et al.44 modified the loading of OPS into a closed system, called closed pulled straws (CPS). Kuwayama et al.46 developed the Cryotip method using a heat-sealed pulled straw technique. These procedures have been described above.
DYNAMICS OF MEIOTIC SPINDLES OF FROZEN–THAWED OOCYTES The meiotic spindles of oocytes consist of microtubules that are constructed by polymerization of tubulin dimers of α- and βtubulin. Microtubules start from microtubular organizing centers at both poles and anchor chromosomes at the kinetochores, forming a barrel shape. The chromosomes align at the equatorial plane of the meiotic spindles. The tubulin dimers polymerize and depolymerize at various stages of a cell cycle. The meiotic spindles are crucial for the events following fertilization at completion of meiosis, second polar body formation, migration of the pronuclei, and formation of the first mitotic spindle.58 The spindle is very sensitive to cryoprotectants and low temperature. Oocytes analyzed immediately after thawing displayed severe disorganization or disappearance of spindles using either slow-freezing or vitrification methods.59–61 Incubation for 1–5 h at 37oC resulted in recovery of spindles to varying degrees dependent on the time interval after thawing, methods of freezing and thawing, and species.59–62 Eroglu et al.59 found complete recovery of spindles for slow-freezing mouse oocytes after incubation for 1 h post-thawing.
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With different distribution of pericentriolar materials and cytoplasmic asters, mouse oocytes are very distinct from human oocytes.41,62 The former are stronger than the latter with recuperation of temperatureinduced microtubule disorganization. Gook et al.62 noticed that 60% of human oocytes with slow cryopreservation were comprised of normal spindles after 1 h of incubation, compared with 81% of control specimens. Rienzi et al.61 used a computer-assisted polarization microscopy (Polscope) to observe meiotic spindle changes. Immediately after thawing, the spindle was visible in 36% of oocytes, but it disappeared in all of the thawed oocytes during the subsequent washing steps. The spindle reappeared in all surviving thawed oocytes, by 3 hours of incubation at 37°C. Bianchi et al.63 slowly froze human oocytes with a 1.5 mol/L PROH and 0.3 mol/L sucrose solution and evaluated meiotic spindle with immunofluorescent stain and confocal microscopy. Immediately after thawing, only 22.9% of oocytes showed a weak birefringence signal, while only 1.2% of oocytes exhibited a high signal. Three hours after thawing, the proportion of oocytes displaying a weak or high intensity signal was 49.4% and 18.1%, respectively. After culture for 5 hours, a weak birefringence signal was detected in 51.8% of oocytes, while 24.1% showed a high signal. There was a significant increase in signal recuperation after 3–5 h of culture. Coticchio et al.64 performed human oocyte cryopreservation using a slow-freezing method with 1.5 mol/L PROH and 0.1 or 0.3 mol/L sucrose. After thawing, oocytes were cultured for 3 h before examination. Spindle and chromatin organization were significantly affected after cryopreservation using 0.1 mol/L sucrose concentration, while these parameters were unchanged using 0.3 mol/L. Protocols with the higher sucrose concentration (0.3 mol/L) in the freezing solution preserved an intact chromosome segregation apparatus comparable with that of freshly collected oocytes.
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In vitrified mouse oocytes, Chen et al.44,60 observed that post-thawing incubation for 1 h allowed recovery of normal spindle and chromosomes to varying degrees. The OPS, CPS, and electron microscope grids preserved the spindle morphology and chromosomal pattern better than in conventional straws. The rapid thermal change of minimum volume methods could traverse the temperature range damaging to the spindle (assumed to be 15 to –15°C) more quickly.6 Moreover, oocytes in minimum volume methods (< 1 µL) are directly warmed in the dilution solution and immediately diluted (~1 s), reducing exposure of oocytes to inappropriate temperatures and concentrated cryoprotectants. In contrast, the conventional straw is warmed in water and then cut with scissors. The oocytes in vitrification medium (25 µL) are expelled into the dilution solution and then placed into another dilution solution. Therefore, it takes more time to pass through the unfavorable conditions (~45 s). Chen et al.44 further demonstrated that incubation for 2 or 3 h resulted in higher incidences of normal spindles than incubation for 1 h. Minimum volume methods for vitrification of oocytes such as electron microscope grids, OPS, Cryotop, and Cryoleaf have been developed to improve fertilization, development, and pregnancy potential.6–12 With more rapid cooling and warming rates, oocytes vitrified in a very small amount of vitrification solution may better preserve the meiotic spindles and expedite the recovery compared with conventional straws.44–60 These modifications could be important for cold-sensitive oocytes such as bovine and human oocytes, and may partly explain why the developmental competence of vitrified bovine oocytes can be enhanced using OPS or grids, compared with conventional straws.6,7 Recent reports of vitrified human oocytes with minimum volume methods such as the Cryotop or Cryoleaf have shown significantly improved success rates.10–12
SPINDLE STATUS OF FROZEN–THAWED OOCYTES AND FERTILIZATION OUTCOME The oocyte freezing and thawing by slow protocol or vitrification inevitably leads to meiotic spindle injury. The changes and recovery of the spindles have been linked to the functional effects of oocytes on fertilization and development.30,44,59 Eroglu et al.59 observed that slowly cryopreserved mouse oocytes inseminated immediately after thawing exhibited impairment of the spindle rotation, second polar body formation, pronuclear migration, and formation of the mitotic spindle. They found an increased rate of digyny that was attributed to disorganization of the spindles, rather than to a primary malfunction of the microfilaments, for the failure of second polar body extrusion. Insemination of slowly cryopreserved mouse oocytes after 1 h of incubation led to normal fertilization dynamics. Park et al.30 reported that vitrified mouse oocytes inseminated at 2 h after warming had a lower fertilization rate than the control specimens. Chen et al.44 found that the percentages of fertilization and blastocyst formation of vitrified mouse oocytes inseminated at 1 h of incubation were significantly lower than the control specimens, but they were improved when inseminated after 2 or 3 h of incubation.
TIME SCHEDULE FOR VITRIFICATION OF OOCYTES AND FERTILIZATION: CONSIDERING ASPECTS OF BOTH OOCYTE AGING AND SPINDLE RECOVERY During ovulation of a dominant follicle, the oocyte at the diplotene stage of prophase resumes meiosis and extrudes the first polar body. The oocyte enters into metaphase of meiosis II and stays at this stage with an organized spindle system. After fertilization by entry of a spermatozoon, the intracellular calcium increases, and the cytostatic factor decreases.65 The oocyte completes meiosis II and extrudes the second polar body. After
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Table 7B.4
Survival of vitrified human oocytes and pregnancy using various procedures
Method
Cumulus
OPS Grid Cryotop Cryoleaf Cryotop
Removed Not removed Partially removed Removed Removed
Survival of oocytes 11/17 325/474 58/64 169/180 120/143
(65%) (69%) (90%) (94%) (84%)
ovulation the oocyte must be fertilized at the appropriate time, and then continue development; otherwise, it will undergo apoptosis. Delayed insemination of mature oocytes results in compromised embryos and development failure.66 Dozortsev et al.67 found that the optimal time for ICSI of human oocytes was from 37 to 41 h after administration of human chorionic gonadotropin (hCG). These fertilized oocytes achieved the highest implantation rate. However, inseminating oocytes shortly after thawing when there is serious spindle disorganization adversely affects fertilization outcome and increases the rate of digyny.44,59 Choosing the optimum time interval between oocyte thawing and insemination is critical for normal fertilization and subsequent development.68 Therefore, considering the competing aspects of oocyte aging and spindle recovery is essential for a successful oocyte cryopreservation program. We usually perform oocyte retrieval 34 h post-hCG. Cryopreservation of oocytes is performed at 2 h after oocyte retrieval. The duration of vitrification and dilution is about 10 min which is negligible. ICSI is performed after 3 h post-thaw (post-hCG 39 h). Therefore, the timing of insemination for frozen–thawed oocytes is within an optimal time frame. In the recent reports regarding vitrified human oocytes, ICSI is usually performed at 2 or 3 h after thawing and incubation.9–12
REMOVING CUMULUS CELLS IS BETTER FOR VITRIFICATION OF MATURE OOCYTES For vitrification of mature human oocytes, some authors have removed cumulus cells
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Pregnancy 1/3 6/28 12/29 7/15 13/23
(33%) (21%) (41%) (47%) (57%)
Authors Kuleshova et al., 199948 Yoon et al., 20039 Kuwayama et al., 200510 Chian et al., 200511 Lucena et al., 200612
before exposure to cryoprotectants,11,12,48 but others have not denuded the oocytes9 (Table 7B.4). Some authors partially removed cumulus cells.10 Chian et al.27 reported that bovine oocytes vitrified without cumulus cells had a higher survival rate after thawing, and a superior embryonic developmental capacity compared with oocytes vitrified with cumulus cells. For vitrification of immature oocytes, the cumulus cells should remained intact. The cumulus cells may help the in vitro maturation of oocytes after thawing of COCs.
SUPERIORITY OF VARIOUS VITRIFICATION METHODS Several studies compared superiority of vitrification techniques regarding oocyte or embryo survival and development. In vitrification of mouse oocytes, Chen et al.44 found that significantly more oocytes cryopreserved by the CPS or OPS methods survived than those by the grid method. It is more difficult to attain the skill required to reach a consistent result with the grid method. Using bovine oocytes, Kuwayama et al.10 found that more oocytes vitrified by the Cryotop method cleaved and developed into blastocysts than those by conventional straw or OPS. With reference to the reports in the literature, for vitrification of human oocytes, the Cryotop or Cryoleaf methods appear to have a higher survival rate than the grid method9–12 (Table 7B.4). Kuwayama et al.46 applied the Cryotip method for human embryo vitrification. They found that the Cryotip had the same efficiency as the Cryotop, although the cooling rates of the Cryotip are slightly lower than those of the Cryotop. The
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Cryotip method has the advantage of better aseptic conditions.
CONCLUSIONS The new vitrification techniques have significantly improved cryopreservation of mammalian and human oocytes. These techniques transform cells into an amorphous glassy state, instead of ice crystal formation, thus preventing freezing–thawing injury during cryopreservation. The minimum volume methods facilitate increased rates of cooling and warming, reduction of concentration of cryoprotectants, and recovery of the meiotic spindle. A time schedule for vitrification of
oocytes and fertilization should refer to aspects of both oocyte aging and spindle recovery. Vitrification is time-saving and does not require special equipment. With increases in survival, fertilization, and pregnancy rates, the vitrification of human oocytes would make a significant contribution to infertility treatments, including its use for oocyte donation and for patients about to lose ovarian function due to advancing age or prior to cancer therapy.5,69
ACKNOWLEDGMENTS The authors would like to thank Ms Li-Jung Chang, Ms Yi-Yi Tsai, and Ms Li-Ting Lin for their technical assistance.
References 1. Porcu E, Venturoli S. Progress with oocyte cryopreservation. Curr Opin Obstet Gynecol 2006; 18: 273–9. 2. Porcu E, Fabbri R, Damiano G et al. Clinical experience and applications of oocyte cryopreservation. Mol Cell Endocrinol 2000; 169: 33–7. 3. Fabbri R, Porcu E, Marsella T et al. Human oocyte cryopreservation: new perspectives regarding oocyte survival. Hum Reprod 2001; 16: 411–6. 4. Boldt J, Cline D, McLaughlin D. Human oocyte cryopreservation as an adjunct to IVF-embryo transfer cycles. Hum Reprod 2003; 18: 1250–5. 5. Chen SU, Lien YL, Chen HF et al. Observational clinical follow-up of oocyte cryopreservation using a slow-freezing method with 1,2-propanediol plus sucrose followed by ICSI. Hum Reprod 2005; 20: 1975–80. 6. Martino A, Songsasen N, Leibo SP. Development into blastocysts of bovine oocytes cryopreserved by ultra-rapid cooling. Biol Reprod 1996; 54: 1059–69. 7. Vajta G, Holm P, Kuwayama M et al. Open pulled straw (OPS) vitrification: a new way to reduce cryoinjuries of bovine ova and embryos. Mol Reprod Dev 1998; 51: 53–8. 8. Hong SW, Chung HM, Lim JM et al. Improved human oocyte development after vitrification: a comparison of thawing methods. Fertil Steril 1999; 72: 142–6.
9. Yoon TK, Kim TJ, Park SE et al. Live births after vitrification of oocytes in a stimulated in vitro fertilization-embryo transfer program. Fertil Steril 2003; 79: 1323–6. 10. Kuwayama M, Vajta G, Kato O et al. Highly efficient vitrification method for cryopreservation of human oocytes. Reprod Biomed Online 2005; 11: 300–8. 11. Chian RC, Son WY, Huang JY et al. High survival rates and pregnancies of human oocytes following vitrification: preliminary report. Fertil Steril 2005; 84 (Suppl 1): S36. 12. Lucena E, Bernal DP, Lucena C et al. Successful ongoing pregnancies after vitrification of oocytes. Fertil Steril 2006; 85: 108–11. 13. Chen C. Pregnancy after human oocyte cryopreservation. Lancet 1986; 1: 884–6. 14. Rall WF, Fahy GM. Ice-free cryopreservation of mouse embryos at −196°C by vitrification. Nature 1985; 313: 573–5. 15. Steponkus PL, Myers SP, Lynch DV et al. Cryopreservation of Drosophila melanogaster embryos. Nature 1990; 345: 170–2. 16. Kuleshova LL, Lopata A. Vitrification can be more favorable than slow cooling. Fertil Steril 2002; 78: 449–54. 17. Liebermann J, Dietl J, Vanderzwalmen P, Tucker MJ. Recent developments in human oocyte, embryo and blastocyst vitrification: where are we now? Reprod Biomed Online 2003; 7: 623–33.
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18. Oktay K, Cil AP, Bang H. Efficiency of oocyte cryopreservation: a meta-analysis. Fertil Steril 2006; 86: 70–80. 19. Vajta G, Nagy ZP. Are programmable freezers still needed in the embryo laboratory? Review on vitrification. Reprod Biomed Online 2006; 12: 779–96. 20. Kasai M, Komi JH, Takakamo A et al. A simple method for mouse embryo cryopreservation in a low toxicity vitrification solution, without appreciable loss of viability. J Reprod Fertil 1990; 89: 91–7. 21. Ali J, Shelton JN. Design of vitrification solutions for the cryopreservation of embryos. J Reprod Fertil 1993; 99: 471–7. 22. Ali J, Shelton JN. Vitrification of preimplantation stages of mouse embryos. J Reprod Fertil 1993; 98: 459–65. 23. Ali J, Shelton JN. Successful vitrification of day-6 sheep embryos. J Reprod Fertil 1993; 99: 65–70. 24. Chen SU, Lien YL, Chao KH et al. Cryopreservation of mature human oocytes by vitrification with ethylene glycol in straws. Fertil Steril 2000; 74: 804–8. 25. Mukaida T, Nakamura S, Tomiyama T et al. Vitrification of human blastocysts using cryoloops: clinical outcome of 223 cycles. Hum Reprod 2003; 18: 384–91. 26. Hiraoka K, Hiraoka K, Kinutani M et al. Blastocoele collapse by micropipetting prior to vitrification gives excellent survival and pregnancy outcomes for human day 5 and 6 expanded blastocysts. Hum Reprod 2004; 19: 2884–8. 27. Chian RC, Kuwayama M, Tan L et al. High survival rate of bovine oocytes matured in vitro following vitrification. J Reprod Dev 2004; 50: 685–96. 28. Ishimori H, Saeki K, Inai M et al. Vitrification of bovine embryos in a mixture of ethylene glycol and dimethyl sulfoxide. Theriogenology 1993; 40: 427–33. 29. Yokota Y, Sato S, Yokota M et al. Successful pregnancy following blastocyst vitrification: case report. Hum Reprod 2000; 15: 1802–3. 30. Park SE, Chung HM, Cha KY et al. Cryopreservation of ICR mouse oocytes: improved post-thawed preimplantation development after vitrification using Taxol, a cytoskeleton stabilizer. Fertil Steril 2001; 75: 1171–84. 31. Vajta G, Holm P, Greve T et al. Survival and development of in vitro produced bovine blastocysts following assisted hatching, vitrification and in-straw direct rehydration. J Reprod Fertil 1997; 111: 65–70. 32. Cuello C, Gil MA, Parrila I et al. In vitro development following one-step dilution of
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OPS vitrified porcine blastocysts. Theriogenology 2004; 62: 1144–52. Pickering SJ, Cant A, Braude PR et al. Transient cooling to room temperature can cause irreversible disruption of the meiotic spindle in the human oocytes. Fertil Steril 1990; 54: 102–8. Arav A, Yavin S, Zeron Y et al. New trends in gamete cryopreservation. Mol Cell Endocrinol 2002; 187: 77–81. Landa V, Tepla O. Cryopreservation of mouse 8-cell embryos in microdrops. Folia Biologica (Praha) 1990; 36: 153–8. Arav A. Vitrification of oocytes and embryos. In: Lauria A, Gandolfi F, eds. New Trends in Embryo Transfer. Cambridge: Portland Press, 1992: 255–4. Hamawaki A, Kuwayama M, Hamano S. Minimum volume cooling method for bovine blastocyst vitrification. Theriogenology 1999; 51: 165. Lane M, Schoolcraft WB, Gardner DK. Vitrification of mouse and human blastocysts using a novel cryoloop container-less technique. Fertil Steril 1999; 72: 1073–8. Kong IK, Lee SI, Cho SG et al. Comparison of open pulled straw (OPS) vs. glass micropipette (GMP) vitrification in mouse blastocysts. Theriogenology 2000; 53: 1817–26. Dinnyes A, Dai Y, Jiang S et al. High developmental rates of vitrified bovine oocytes following parthenogenetic activation, in vitro fertilization, and somatic cell nuclear transfer. Biol Reprod 2000; 63: 513–8. Vanderzwalmen P, Benin G, Debauche Ch et al. In vitro survival of metaphase II oocytes and blastocysts after vitrification in a hemistraw (HS) system. Fertil Steril 2000; 74: S215–6. Kuwayama M, Kato O. All-round vitrification method for human oocytes and embryos. J Assist Reprod Genet 2000; 17: 477. Matsumoto H, Jiang JY, Tanaka T et al. Vitrification of large quantities of immature bovine oocytes using nylon mesh. Cryobiology 2001; 42: 139–44. Chen SU, Lien YL, Cheng YY et al. Vitrification of mouse oocytes using closed pulled straws (CPS) achieves a high survival and preserves good patterns of meiotic spindles, compared with conventional straws, open pulled straws (OPS), and grids. Hum Reprod 2001; 16: 2350–6. Liebermann J, Tucker MJ, Graham JR et al. Blastocyst development after vitrification of multi-pronuclear zygotes using the Flexipet denuding pipette. Reprod Biomed Online 2002; 4: 146–50.
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46. Kuwayama M, Vajta G, Ieda S et al. Comparison of open and closed methods for vitrification of human embryos and the elimination of potential contamination. Reprod Biomed Online 2005; 11: 608–14. 47. Chen SU, Chien CL, Wu MY et al. Novel direct cover vitrification for cryopreservation of ovarian tissues increases follicle viability and pregnancy capability in mice. Hum Reprod 2006; 21: 2794–800. 48. Kuleshova L, Gianaroli L, Ferraretti A et al. Birth following vitrification of a small number of human oocytes: case report. Hum Reprod 1999; 14: 3077–9. 49. Teng TY. Mounting of crystals for macromolecular cryocrystallography in a free-standing thin film. J Appl Crystallogr 1990; 23: 387–91. 50. Parkin S, Hope H. Macromolecular Cryocrystallography: cooling, mounting, storage and transportation of crystals. J Appl Crystallogr 1998; 31: 945–53. 51. Vanderzwalmen P, Bertin G, Debauche Ch et al. Vitrification of human blastocysts with the Hemi-Straw carrier: application of assisted hatching after thawing. Hum Reprod 2003; 18: 1504–11. 52. Abe Y, Hara K, Matsumoto H et al. Feasibility of a nylon-mesh holder for vitrification of bovine germinal vesicle oocytes in subsequent production of viable blastocysts. Biol Reprod 2005; 72: 1416–20. 53. Arav A, Zeron Y, Ocheretny A. A new device and method for vitrification increases the cooling rate and allows successful cryopreservation of bovine oocytes. Theriogenology 2000; 53: 248. 54. Huang CC, Lee TH, Chen SU et al. Successful pregnancy following blastocyst cryopreservation using super-cooling ultra-rapid vitrification. Hum Reprod 2005; 20: 122–8. 55. Cuello C, Gil MA, Parrilla I et al. Vitrification of porcine embryos at various developmental stages using different ultra-rapid cooling procedures. Theriogenology 2004; 62: 353–61. 56. Cai XY, Chen GA, Lian Y et al. Cryoloop vitrification of rabbit oocytes. Hum Reprod 2005; 20: 1969–74. 57. Bielanski A, Nadin-Davis S, Sapp T et al. Viral contamination of embryos cryopreserved in liquid nitrogen. Cryobiology 2000; 40: 110–6. 58. Schatten G, Simerly C, Schatten H. Microtubule configurations during fertilization, mitosis, and early development in the mouse and the requirement for egg microtubule-mediated motility during mammalian fertilization. Proc Natl Acad Sci USA 1985; 82: 4152–6.
59. Eroglu A, Toth TL, Toner M. Alterations of the cytoskeleton and polyploidy induced by cryopreservation of metaphase II mouse oocytes. Fertil Steril 1998; 69: 944–57. 60. Chen SU, Lien YL, Chen HF et al. Open pulled straws for vitrification of mature mouse oocytes preserve patterns of meiotic spindles and chromosomes better than conventional straws. Hum Reprod 2000; 15: 2598–603. 61. Rienzi L, Martinez F, Ubaldi F et al. Polscope analysis of meiotic spindle changes in living metaphase II human oocytes during the freezing and thawing procedures. Hum Reprod 2004; 19: 655–9. 62. Gook DA, Osborn SM, Johnston WIH. Cryopreservation of mouse and human oocytes using 1,2-propanediol and the configuration of the meiotic spindle. Hum Reprod 1993; 8: 1101–9. 63. Bianchi V, Coticchio G, Fava L et al. Meiotic spindle imaging in human oocytes frozen with a slow freezing procedure involving high sucrose concentration. Hum Reprod 2005; 20: 1078–83. 64. Coticchio G, De Santis L, Rossi G et al. Sucrose concentration influences the rate of human oocytes with normal spindle and chromosome configurations after slow-cooling cryopreservation. Hum Reprod 2005; 21: 1771–6. 65. Edwards RG, Brody SA. Oocyte growth, maturation, and fertilization. In: Principles and Practice of Assisted Human Reproduction. Philadelphia: WB Saunders Company, 1995: 322–3. 66. Gook DA, Osborn SM, Bourne H et al. Fertilization of human oocytes following cryopreservation: normal karyotypes and absence of stray chromosomes. Hum Reprod 1994; 9: 684–91. 67. Dozortsev D, Nagy P, Abdelmassih S et al. The optimal time for intracytoplasmic sperm injection in the human is from 37 to 41 hours after administration of human chorionic gonado- tropin. Fertil Steril 2004; 82: 1492–6. 68. Chen SU, Lien YL, Chao KH et al. Effects of cryopreservation on meiotic spindles of oocytes and its dynamics after thawing: clinical implications in oocyte freezing- a review article. Mol Cell Endocrinol 2003; 202: 101–7. 69. Li XH, Chen SU, Zhang X et al. Cryopreserved oocytes of infertile couples undergoing assisted reproductive technology could be an important source of oocyte donation: a clinical report of successful pregnancies. Hum Reprod 2005; 20: 3390–4.
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7C
Tae Ki Yoon, Dong Ryul Lee and Kwang Ryul Cha
OVERVIEW Cryopreservation is a process of long-term lowtemperature storage of biological samples without morphological and functional destruction such that the samples can be recovered in an intact state. Recent advances in reproductive biology have resulted in the cryopreservation of human oocytes, zygotes, early cleavage-stage embryos, and blastocysts becoming an integral part of the human in vitro fertilization embryo transfer (IVF-ET) program. The establishment of an egg bank using oocyte cryopreservation techniques would provide a number of benefits. First, it could prevent ethical and legal problems associated with embryo freezing, particularly in certain countries where embryo freezing is banned or limited by law. Second, the age at which people marry is rising, resulting in infertility issues, and an egg bank would provide an option for older women to have children later in life. Third, it would allow for a quarantine period for genetic and infectious disease screening in donor oocyte programs. Oocyte freezing also increases the convenience for synchronization of procedures. Finally, oocyte freezing gives the option of fertility preservation for patients who receive anticancer treatments or oophorectomy. Although cryopreservation of human oocytes has been carried out successfully, clinical outcomes remain unsatisfactory due to low pregnancy and implantation rates resulting from decreased survival rates and high levels of chromosome abnormalities in
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thawed oocytes. Many researchers have introduced several changes to improve the viability and quality of oocytes after thawing. However, oocyte cryopreservation remains one of the most elusive tasks in the field of assisted reproductive technology (ART).
APPLICATION OF VITRIFICATION FOR OOCYTE CRYOPRESERVATION Vitrification is the solidification of a solution into a glassy, vitrified state from the liquid phase due to an extreme elevation in viscosity while cooling at low temperature.1 During vitrification the solution remains unchanged and the water does not crystallize, and hence no ice crystals are formed.2 In the early 1970s, this technique was used for long-term preservation of various cell types,3,4 and was also applied to mammalian embryos as it avoided ice crystallization during cooling and warming.5 Although the first successful cryopreservation of unfertilized mammalian oocytes was achieved in 1977,6 the efficiency was extremely low until the mid-1990s because oocytes contain a large amount of water in the cytoplasm resulting in ice crystal formation when using conventional freezing protocols. Critser et al.7 were the first to apply the vitrification method to oocyte cryopreservation. Subsequently, new cryoprotective agents and/ or cryovehicles have resulted in improved survival and developmental rates of oocytes after vitrification.8–12
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CLINICAL APPLICATION OF VITRIFICATION FOR HUMAN OOCYTES Following the first reported pregnancy from frozen human mature oocytes by the application of slow cooling,13 numerous studies have investigated optimal methods for oocyte cryopreservation. The studies utilized surplus mature oocytes from patients undergoing in vitro fertilization embryo transfer (IVF-ET), or all the oocytes from patients who might lose their gonadal function during therapy.14–21 The oocytes were stored for future use by slow cooling or vitrification methods. When the patients wanted to have babies after their fresh IVF-ET cycles or therapies, stored oocytes were recovered and provided for an additional IVF-ET. A meta-analysis of reports in the literature up to June 2005 gave the number of clinical pregnancies as 117 and ten for cryopreservation using slow cooling and vitrification, respectively, and the number of live births as 85 and ten, respectively. These resulted in the birth of 107 and 11 children, respectively, for slow cooling and vitrification.22 In that report, the fertilization rate, live-birth rate per injected oocyte, and live-birth rate per ET for vitrified oocytes were 70.6%, 4.5%, and 29.4%, respectively. Up to 2002, our group achieved six pregnancies from 28 ET cases using vitrified/warmed oocytes, and the pregnancies resulted in the delivery of seven healthy babies.19 However, the clinical outcomes for the thawed oocyte cycles using surplus eggs remain inferior to those for IVF using unfrozen oocytes. Hence, many researchers have introduced new techniques and tools in order to improve the viability and quality of oocytes after warming.
ELEVATING COOLING SPEED CAN IMPROVE THE CLINICAL RESULTS Generally, vitrification requires a high concentration of cryoprotectants and an elevated
cooling speed to avoid ice crystal formation, which is a major cause of cryoinjury. A variety of cryo-containers and cryo-mediators were recently introduced to improve the results of oocyte and embryo freezing. The use of a high cooling rate during vitrification using an electron microscope (EM) grid has resulted in the freezing of chillingsensitive Drosophila embryos and bovine oocytes.10,23 This system has been successfully applied to the human IVF-ET program and several pregnancies have resulted.18,19 The use of EM grids may provide rapid heat conductivity from the outside into the oocytes. In particular, the gold grid may provide low toxicity with extremely high heat transmission, and may reduce the damage caused by long exposure to high cryoprotective agent (CPA) concentrations. Immersion of a sample into liquid nitrogen (LN2) results in boiling and the formation of gas bubbles around the specimen, which in turn results in poor heat transfer. By applying negative pressure with a vacuum, LN2 will freeze and convert into a slush state (SN2). SN2 has a lower internal temperature of −210°C without vaporization.23 Thus, SN2 may offer high-speed cooling rates, which may increase the oocyte survival rate. In our preliminary studies, the survival of mouse metaphase II (MII) oocytes and human fertilization-failed oocytes was highly improved after vitrification using SN2 and warming. The analysis of cumulus complex (CC) obtained from immature oocytes showed that the number of apoptotic cells was lower in the super-rapid cooling group (using SN2) compared with the rapid cooling group (using LN2) (unpublished data). However, whether the short-term exposure of apoptotic CC after freezing/thawing influences fertilization or further embryonic development of the warmed oocytes remains to be investigated. In clinical trials involving 30 cycles using a gold grid and SN2, the fertilization rate of vitrified/warmed oocytes was 77.4 ± 3.5% (168/218), the cleavage rate on day 2 was
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94.3% ± 2.1% (158/168), and ET was successful in all patients. The pregnancy rate per ET and the implantation rate for SN2-vitrified IVF-ET were 43.3% (13/30) and 14.2% (17/120), respectively. Moreover, higher pregnancy (72.7% (8/11)) and implantation (22.2% (12/54)) rates were observed after SN2-vitrification in patients who did not undergo ET for various reasons, even though there was no difference in the indication for IVF-ET.24 These data suggest that the use of the super-rapid cooling vitrification procedure was not limited to specific patients, and may be an option for fertility preservation and avoiding the ethical problems of embryo freezing or the risk of ovarian hyperstimulation syndrome (OHSS). During two periods of time (phase I: October 1997 to December 2002; phase II: December 2003 to August 2005), the pregnancy rates in fresh cycles of conventional IVF-ET for patients who had similar indications (with more than 15 retrieved oocytes) at our center were 50.3% (731 pregnancies/1454 cycles), and 47.2% (376 pregnancies/797 cycles); there was no significant difference between the two periods. In order to analyze the efficiency of super-rapid cooling, we compared these clinical results (phase II)24 with data from our previous oocyte cryopreservation program performed in phase I using LN2 vitrification.19 The survival and fertilization rates after warming of oocytes using SN2 were improved when compared with those using LN2. Furthermore, the embryonic development, pregnancy, and implantation rates after warming and ICSI were significantly improved (Figure 7C.1).
CRYOPROTECTIVE AGENT While CPAs are essential for cell cryopreservation, they are usually damaging at high concentrations. As such, researchers have used a low toxicity CPA such as ethylene glycol (EG)16,19 or a combination of several CPAs.25 EG has a rapid diffusion rate into the cell
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through the zona pellucida and the cellular membrane, and equilibrates rapidly.26,27 Also, combinations of permeating and non-permeating CPAs (sucrose, trehalose, or Ficoll) have been used for human oocyte cryopreservation.
STORAGE DEVICES USING NITROGEN (N2) VAPOR Vitrification has several advantages compared with slow cooling, including control of solute penetration, dehydration rate, and exposure time to outside environmental conditions. While the LN2 system equipment and running costs are inexpensive,28 this system is based on direct contact with LN2, and there is a potential risk of disease transmission through contaminated LN2 during storage.29 Thus, various sealing methods have been introduced to eliminate the potential risk of contamination.30–32 In 2005, Bielanski et al.33 reported that N2 vapor could be a useful tool since no transmission of bacterial or viral microbes to embryos and semen stored in the vapor phase of LN2 in dry shippers had been observed. Indeed, our experience is that over 6 months the survival and development rates of embryos stored in N2 vapor were similar to those in LN2 (unpublished data). The survival and developmental capacity of oocytes stored in the vapor phase of LN2 remains under investigation.
PROCEDURE OF OOCYTE VITRIFICATION USING AN ELECTRON MICROSCOPE GRID Yoon et al.24 described the vitrification of surplus oocytes from patients who yielded more than 15 oocytes, or all oocytes from patients who did not undergo fertilization for various reasons. Recovered cumulus–oocyte complexes (COCs) were briefly incubated for 10 with 80 IU/mL hyaluronidase to remove excess cumulus cells, and were then pre-equilibrated
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100
*
Rate (%)
80 60
94.7
*
82.9 71.7
68.6
94.0
77.1
40 20 0 Survival
Fertilization Phase I
a
Cleavage
Phase II
100
Rate (%)
80 69.5
60
*
40
31.9
20 0
50.8
16.1
11.7 Good
Poor Phase I
b
15.8
Bad Phase II
Rate (%)
* 45 40 35 30 25 20 15 10 5 0
43.3
*
21.4
14.2 6.4 Pregnancy
c
Implantation Phase I
Phase II
Figure 7C.1 Comparison of clinical outcomes for vitrified mature human oocytes after stimulation cycles involving either liquid N2 (LN2) or slush N2 (SN2) cooling methods. Phase I was from October 1997 to December 2002 when vitrification using conventional LN2 was performed for clinical oocyte cryopreservation. Phase II was from November 2003 to August 2005 when vitrification using SN2 was performed for clinical oocytes cryopreservation. (a) Comparison of survival, fertilization, and cleavage rates between the Phase I and II after warming of human oocytes. (b) Embryonic morphology before embryo transfer on day 3. Good, embryos at ≥ 8-cell stage and less than 20% cytoplasmic fragmentation; Poor, embryos at ≥ 6-cell or ≤ 8-cell stage, and 20–50% cytoplasmic fragmentation; Bad, embryos with < 6cell stage and > 50% cytoplasmic fragmentation. (c) Comparison of pregnancy and implantation rates between the Phase I and II after embryo transfer.* P < 0.05.
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b
a
c
d
Figure 7C.2 The oocyte vitrification procedure using the electron microscope (EM) gold grid. (a) Equilibrated cumulus–oocyte complexes (COCs) mounted on the EM gold grid. (b) Mounted COCs (arrows). (c) Plunging into LN2. (d) Plunging into SN2. (e) Intracytoplasmic sperm injection of warmed human metaphase II oocyte.
e
for 2.5 min in 2 mL Dulbecco’s phosphatebuffered saline (DPBS) supplemented with 1.5 mol/L ethylene glycol and 10% synthetic serum substitute (SSS) at 37oC. For the final equilibration, COC with 2–3 layers of CC were then placed in the same volume of DPBS supplemented with 5.5 mol/L EG, 1.0 mol/L sucrose, and 10% SSS for 20 s. Two to five partially denuded COC were mounted on an EM gold grid using a fine glass pipette (Figure 7C.2). Excess cryoprotectant solution was removed using underlying sterilized paper. The gold grids containing COC were immediately plunged into slush nitrogen (SN2) which was generated in a rapid-cooling LN2 chamber by applying negative pressure (Vit-MasterTM, IMT, Israel). For long-term storage, a cryovial cap and goblet were used for placement of the grid. For warming, the gold grids were sequentially transferred to culture dishes containing 2 mL DPBS supplemented with 1.0, 0.5, 0.25, 0.125, or 0 mol/L sucrose and 10% (vol/ vol) SSS at intervals of 2.5 min at 37oC. After being washed 4–6 times, CCs were removed by mechanical pipetting and then transferred into culture medium. ICSIs were performed 3–6 h after oocyte warming (day 0). The number of pronuclei (PN) in the cytoplasm was counted to verify normal fertilization of
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vitrified/warmed oocytes at 16–19 h after ICSI. The embryos from 2PN zygotes were cultured for 2–3 days and then transferred into the patient’s uterus. For the artificial preparation of endometrium, estradiol (valerate 4–6 mg/day) was administered, and after reaching 8 mm in thickness, progesterone injections (100 mg/ day) were given. In a natural cycle, 50 mg/day progesterone supplementation was commenced after ovulation (day 0).
CONCLUSION Increasing the freezing rate via the use of SN2 may not improve cryopreservation of mouse pronuclear stage embryos.34 Also, applying SN2 did not increase the fertilization rate or further embryonic development of immature bovine oocytes despite improved survival rates and morphology after vitrifying/warming.35 These data suggest that the effect of the cooling rate in vitrification may be species specific or dependent on the cell stage (i.e. oocytes, zygotes, embryos, or blastocysts). However, survival of the cryopreserved oocytes has been a major obstacle to successful execution of human clinical trials. A highly improved survival rate, embryonic development, and pregnancy rate were
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obtained by increasing the cooling rate using SN2 and a gold grid, which may have a significant positive effect on the undertaking of clinical trials. Such advances may lead to oocyte cryopreservation being a valuable tool in human ART.
ACKNOWLEDGMENTS The authors thank Ms Soo Kyung Cha for technical support, and staff at the Fertility Center of CHA General Hospital, Seoul, Korea.
References 1. Fahy GM, MacFarlane DR, Angell CA et al. Vitrification as an approach to cryopreservation. Cryobiology 1987; 24: 387–402. 2. Fahy GM. Vitrification: a new approach to organ cryopreservation. In: Meryman HT, ed. Transplantation: Approaches to Graft Rejection. New York: Alan R Liss, 1986: 305–35. 3. Wilmut I. The effect of cooling rate, warming rate, cryoprotective agent and stage of development on survival of mouse embryos during freezing and thawing. Life Sci II 1972; 11: 1071–9. 4. Leibo SP, Mazur P. Methods for the preservation of mammalian embryos by freezing. In: Daniel JC Jr, ed. Methods in Mammalian Reproduction. New York: Academic Press, 1978: 179–201. 5. Rall WF, Fahy GM. Ice-free cryopreservation of mouse embryos at –196°C by vitrification. Nature 1985; 313: 573–5. 6. Whittingham D. Fertilization in vitro and development to term of unfertilized mouse oocytes previously stored at –196oC. J Reprod Fertil 1977; 49: 89–94. 7. Critser JK, Arneson BW, Aaker DW et al. Cryopreservation of hamster oocytes: effects of vitrification or freezing on human sperm penetration of zona-free hamster oocytes. Fertil Steril 1986; 46: 277–84. 8. Nakagata N. High survival rate of unfertilized mouse oocytes after vitrification. J Reprod Fertil 1989; 87: 479–83. 9. Hotamisligil S, Toner M, Powers RD. Changes in membrane integrity, cytoskeletal structure, and developmental potential of murine oocytes after vitrification in ethylene glycol. Biol Reprod 1996; 55: 161–8. 10. Martino A, Songsasen N, Leibo SP. Development into blastocysts of bovine oocytes cryopreserved by ultrarapid cooling. Biol Reprod 1996; 54: 1059–69.
11. Vajta G, Holm P, Kuwayama M et al. Open pulled straw (OPS) vitrification: a new way to reduce cryoinjuries of bovine ova and embryos. Mol Reprod Dev 1998; 51: 53–8. 12. Isachenko V, Alabart JL, Nawroth F et al. The open pulled straw vitrification of ovine GVoocytes: positive effect of rapid cooling or rapid thawing or both? Cryo Letters 2001; 22: 157–62. 13. Chen C. Pregnancy after human oocyte cryopreservation. Lancet 1986; 1: 884–6. 14. Tucker M, Wright G, Morton P et al. Preliminary experience with human oocyte cryopreservation using 1,2-propanediol and sucrose. Hum Reprod 1996; 11: 1513–5. 15. Porcu E, Fabbri R, Seracchioli R et al. Birth of healthy female after intracytoplasmic sperm injection of cryopreserved human oocytes. Fertil Steril 1997; 68: 724–6. 16. Kuleshova L, Gianoroli L, Magli C et al. Birth following vitrification of small number of human oocytes. Hum Reprod 1999; 14: 3077–9. 17. Porcu E, Fabbri R, Damiano G et al. Clinical experience and applications of oocyte cryopreservation. Mol Cell Endocrinol 2000; 169: 33–7. 18. Yoon TK, Chung HM, Lim JM et al. Pregnancy and delivery of healthy infants developed from vitrified oocytes in a stimulated in vitro fertilization-embryo transfer program. Fertil Steril 2000; 74: 180–1. 19. Yoon TK, Kim TJ, Park SE et al. Live birth after vitrification of oocytes in a stimulated in vitro fertilization-embryo transfer program. Fertil Steril 2003; 79: 1323–6. 20. Borini A, Bonu MA, Coticchio G et al. Pregnancies and births after oocyte cryopreservation. Fertil Steril 2004; 82: 601–5. 21. Chen ZJ, Li M, Li Y et al. Effects of sucrose concentration on the developmental potential of human frozen-thawed oocytes at different
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22. 23.
24.
25.
26.
27. 28.
stages of maturity. Hum Reprod 2004; 19: 2345–9. Oktay K, Cil AP, Bang H. Efficiency of oocyte cryopreservation: a meta-analysis. Fertil Steril 2006; 86: 70–80. Steponkus PL, Caldwell S. An optimized procedure for the cryopreservation of Drosophila melanogaster embryos. Cryo Letters 1993; 14: 375–80. Yoon TK, Lee DR, Cha SK et al. Survival rate of human oocytes and pregnancy outcome after vitrification using slush nitrogen in assisted reproductive technology. Fertil Steril 2007; in press. (electronic version published Mar 8, 20) Katayama KP, Stehlik J, Kuwayama M et al. High survival rate of vitrified human oocytes results in clinical pregnancy. Fertil Steril 2003; 80: 223–4. Zhu SE, Kasai M, Otoge H et al. Cryopreservation of expanded mouse blastocysts by vitrification in ethylene glycol based solutions. J Reprod Fertil 1993; 98: 139–45. Yokota Y, Sato S, Yokota M et al. Successful pregnancy following blastocyst vitrification. Hum Reprod 2000; 15: 1802–3. Kuleshova LL, Lopata A. Vitrification can be more favorable than slow cooling. Fertil Steril 2002; 78: 449–54.
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29. Bielanski A, Nadin-Davis S, Sappi T et al. Viral contamination of embryos cryopreserved in liquid nitrogen. Cryobiology 2000; 40: 110–6. 30. Kuleshova LL, Shaw JM. A strategy for rapid cooling of mouse embryos within a double straw to eliminate the risk of contamination during storage in liquid nitrogen. Hum Reprod 2000; 15: 2604–9. 31. Isachenko V, Montag M, Isachenko E et al. Aseptic technology of vitrification of human pronuclear oocytes using open-pulled straws. Hum Reprod 2005; 20: 492–6. 32. Kuwayama M, Vajta G, Kato O et al. Highly efficient vitrification method for cryopreservation of human oocytes. Reprod Biomed Online 2005; 11: 300–8. 33. Bielanski A. Non-transmission of bacterial and viral microbes to embryos and semen stored in the vapour phase of liquid nitrogen in dry shippers. Cryobiology 2005; 50: 206–10. 34. Nowshari MA, Brem G. Effect of freezing rate and exposure time to cryoprotectant on the development of mouse pronuclear stage embryos. Hum Reprod 2001; 16: 2368–73. 35. Khanna S, Lee DR, Parks JE. Effect of cooling rate on vitrification of immature bovine oocytes. Biol Reprod 2002; 67(Suppl 1): 338.
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7D
Vitrifying and warming of oocytes using Cryotop Koichi Kyono, Yukiko Nakajo, Shima Kumagai and Chikako Nishinaka
INTRODUCTION As of March 2006, a total of 51 pregnancies (28 live births, 23 ongoing, and 40 children born) after vitrification of a human mature oocyte had been reported worldwide.1 We reported the first delivery in Japan following cryopreservation of a human mature oocyte in 2001.2 This was achieved by using the slow freezing method (SFM) with 1,2-propanediol (PROH) and mature oocytes. We also reported the first successful pregnancy and delivery after transfer of a single blastocyst derived from a vitrified human oocyte in 2005.3
PROCEDURES Intracellular osmotic pressure at −30°C in the SFM is about 13 000 mOsmol/L, compared with 8000 mOsmol/L for the vitrification method (VM). Intracellular condition during cooling and warming is thus better with the VM than with the SFM. Table 7D.1
Kuleshova7 Yoon8 Katayama9 Kyono3 Kuwayama6 Chian11 Mukaida10 Sun
VMs have been greatly improved over the years. Initially, 30 or 40% ethylene glycol (EG) and 1 or 0.5 mol/L sucrose were used as cryoprotectants for vitrification of human mature oocytes. In 2000, Kuwayama and Kato reported the first pregnancy of a mature human oocyte using this protocol, although unfortunately the pregnancy did not persist.4 Currently, 15% EG and 15% dimethylsulfoxide (DMSO) are used as cryoprotectants in vitrification solutions (VSs),3,5,6 as the toxicity of these lower concentration VSs is reduced and permeability is more prompt. Cryotop, Cryotip, Cryoloop, Cryoleaf, electron microscopy (EM) grid, open or closed pulled straw, and the like have all been used as cryotools3,5–11 (Table 7D.1). Cooling and warming procedures have also differed between reporters (Tables 7D.2, and 7D.3). Further accumulation of outcome data at an international level is needed. However, the Cryotop devised by Kuwayama offers numerous advantages, including that it is a simple and easy procedure (cooling and warming);
Delivery after vitrification of mature human oocytes Year
Cryotool
Country
1999 2003 2003 2005 2005 2005 2005 2006
OPS EM grid Cryotop Cryotop Cryotop Cryoleaf Cryoloop Cryoloop
Italy South Korea USA Japan Japan Canada Japan USA
OPS, open pulled straw; EM, electron microscope.
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Table 7D.2
Vitrification methods for mature human oocytes (cooling) Vitrification solution volume
Equilibration solution
Vitrification solution
10% EG, 40 s; 20% EG, 30 s 1.5 mol/L EG, 2.5 min
<0.1 µL <0.1 µL
Kuleshova Yoon Katayama Kyono
7.5% EG + 7.5% DMSO, 5–20 min
40% EG + 0.6 mol/L sucrose, 60 s 5.5 mol/L EG + 1.0 mol/L sucrose, 20 s 15% EG + 15% DMSO + 0.5 mol/L sucrose 15% EG + 15% DMSO + 0.5 mol/L sucrose, 45–60 s
Kuwayama
7.5% EG + 7.5% DMSO, 5–20 min
15% EG + 15% DMSO + 0.5 mol/L sucrose, 45–60 s
7.5% EG + PROH, 5 min 0.625, 1, 25, 2.5, 5, and 10% (EG + DMSO), every 2–3 min 7.5% EG + 7.5% DMSO, 3 min
15% EG + PROH + 0.5 mol/L sucrose, 45–60 s 20% EG + 20% DMSO + 1% Ficol l + 0.65 mol/L sucrose, 30 s 15% EG + 15% DMSO + 0.5 mol/L sucrose, 30 s
Chian Mukaida Sun
1–2 µL
EG, ethylene glycol; DMSO, dimethylsulfoxide; PROH, 1, 2-propanediol.
Table 7D.3
Kuleshova Yoon Katayama Kyono Kuwayama Chian Mukaida Sun
Vitrification methods for mature human oocytes (warming) Thawing protocol
Culture medium
Time to ICSI
0.4 mol/L sucrose, 2–3 min; 0.25 mol/L sucrose, 2–3 min; 0.125 mol/L sucrose, 3–6 min 1.0, 0.5, 0.25, 0.125, and 0 mol/L sucrose every 2–3 min
IVF 50
4h
P-1 medium
3h
Universal IVF medium (MediCult, Denmark) Modified TCM199
2h
1.0 mol/L sucrose, 1 min; 0.5 mol/L sucrose 3 min; 3 min; 0 mol/L sucrose, 5 min; 0 mol/L sucrose, 5 min; 1.0 mol/L sucrose, 1min; 0.5 mol/L sucrose, 3 min; 0 mol/L sucrose 5 min; 0 mol/L sucrose, 5 min 1.0 mol/L sucrose, 5 min; 0.5 mol/L sucrose, 3 min; 0.25 mol/L sucrose, 3 min; 0 mol/L sucrose, 0 mol/L sucrose 1.0, 0.75, 0.5, 0.25, and 0.125 mol/L sucrose, every 2–3 min 1.0, 0.5, and 0 mol/L sucrose
2h
2h
ICSI, intracytoplasmic sperm injection; IVF, in vitro fertilization; TCM, tissue culture medium.
the possibility of vitrifying more oocytes per cane; and higher survival, fertilization, cleavage, blastulation, and pregnancy rates. The keys to optimal outcomes from VMs using a vitrification kit and Cryotop are low concentration of VS (15% EG and 15% DMSO), the ultra-high speed of cooling (−23 000°C/min) and warming (+42 000°C/min), minimal VS volume (<0.1 µL) on Cryotop, and minimal change in volume (within 70–100% of normal volume during shrinking and within 100–130% of normal volume during swelling).
MATERIALS AND METHODS Cryopreservation of human oocytes: Cryotop Mature human oocytes from patients who provided informed consent were vitrified by the minimum volume cooling (MVC) method with VS using Cryotop. There were one or two oocytes on each Cryotop. A total of five Cryotops were able to undergo cryopreservation per cane in the liquid nitrogen (LN2) tank, compared with two Cryoloops per cane
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LN2
Figure 7D.1
Two Cryoloops or five Cryotops per cane in liquid nitrogen container.
(Figure 7D.1). Mature human denuded oocytes after removal of cumulus cells by pipetting were cryopreserved. A pipette bore equal to the diameter of the oocyte was used for pipetting with constant monitoring to ensure a minimum volume of solution. The vitrification procedure was primarily performed at room temperature. The Vitrification Kit was obtained from Kitazato Biopharma (Mitojima, Japan) at 4–8°C within 24 hours of ordering. The Vitrification Kit (Figure 7D.2) included • Base medium (BM): modified tissue culture medium (TCM) 199 (10.5 mmol/L Hepes + 9.5 mmol/L Na-Hepes + 5 mmol/L NaHCO3 + TCM199) • Equilibration solution (ES): BM + 7.5% EG + 7.5% DMSO + 20% Serum Substitute Supplement (SSS; Irvine Scientific, Santa Ana, USA)
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• VS: BM + 15% EG + 15% DMSO + 20% SSS + 0.5 mol/L sucrose • Thawing solution (TS): BM + 20% SSS + 1.0 mol/L sucrose • Diluent solution (DS): BM + 20% SSS + 0.5 mol/L sucrose • Washing solution (WS): BM + 20% SSS. TS, DS, and WS had densities of 1.12, 1.06, and 1.0 g/cm3, respectively.
Freezing at Kyono ART Clinic (KAC) (Figure 7D.3) Materials The materials used for freezing at KAC included • Vitrification kit: ES and VS • Cryotop
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Figure 7D.2 Equilibration solution (ES), vitrification solution (VS), thawing solution (TS), diluent solution (DS), washing solution 1(WS1), and WS2.
~15 min
5 min
WS
ES
ES
WS + ES
WS + ES
ES
ES
One oocyte 20 µL Two or more oocytes 50 µL 1 min
VS
VS
VS
LN2
Figure 7D.3 Vitrification procedure for mature human oocytes at Kyono ART Clinic (KAC) using Cryotop (cooling). WS, washing solution, ES, equilibration solution; VS, vitrification solution.
• Stereoscopic microscope • 35-mm plastic dish × 2 (Falcon 1008) (VS, ES); 50-mm plastic dish (Falcon 1006) • Pasteur pipette (bore same size as oocyte diameter)
• A pair of tweezers • LN2 container (volume 1–2 L, depth 8–9 cm) • LN2 tank (cane, cane holder, tank, LN2, etc.) • Timer.
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Procedure (1) ES, VS, and WS were kept for more than 30 min at room temperature, and then poured onto a 35-mm dish (Falcon 1008) from the tube. On the bottom of 50-mm dish were written one ‘WS’ and two ‘ES’s. One 20 µL drop of WS and two 20 µL drops of ES were placed onto the 50-mm dish in that order. In case there were more than two oocytes, one 50 µL drop was needed. The LN2 was poured into the container (vessel or bottle, volume 1–2 L) at the 90% level. The sterilized pouch of the Cryotop was opened and the date of cryopreservation, name, and grade of oocytes were noted. (2) Culture dishes with denuded oocytes from the incubator (atmosphere: 6% CO2, 5% O2, 89% N2 at 37°C) were placed in air at room temperature for 5 min. Oocytes were judged by rolling using the pipette microscopically. Oocytes with a poor grade, irregular contours, or dark coloration were excluded from cryopreservation. (3) Denuded oocytes with minimum volume of previous medium were moved into the center of the WS drop. (4) WS including denuded oocytes was mixed with the adjacent first drop of ES and then placed in mixed medium at room temperature for 5 min. (5) Denuded oocytes were then placed on the surface of the second drop of ES. We then waited for ES to permeate into the inside of the oocyte and for perfect recovery of size and shape within 15 min. Recovery time is dependent on the viability of each oocyte. During this time, three ‘VS’s were written on the bottom of 50-mm dish. Three 20 µL drops of VS were placed in the marked postions onto the 50-mm dish. (6) Perfectly recovered oocytes with minimum volume of ES after the end of equilibration were placed on the surface of the first VS drop. ES in the pipette
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(7)
(8)
(9)
(10)
was discarded in the dish and the inside of the pipette was washed in the VS drop. After 5 s, floating oocytes were sunk in a second VS drop to exchange water with cryoprotectants (EG and DMSO) inside the oocyte. The procedure was repeated twice in a new third VS drop. Oocytes that had shrunk due to dehydration after perfect exchange of oocytes were confirmed. Perfectly exchanged oocytes were picked up with a minimum volume (<0.1 µL) of VS, then placed on the end of the Cryotop. The tip of Cryotop loading oocytes was immediately plunged into LN2. The time required from steps (6) to (9) was 45–60 s. Cooling rate was 23 000°C/min. The tip of the Cryotop was covered by the cap and cryopreserved in LN2.
Thawing at KAC (Figure 7D.4) Materials The materials used for thawing at KAC (Figure 7D.5) included • Vitrification kit: TS, DS, WS1, and WS2 • Culture medium for recovery (Universal IVF medium; MediCult, Denmark) • Stereoscopic microscopy • 35-mm dish × 4 (TS, DS, WS1, WS2) (Falcon 1008) • LN2 • LN2 container (volume 1–2 L; depth 8–9 cm) • Timer • Pasteur pipette • A pair of tweezers.
Procedure (1) TS tube with closed cap and 35-mm dish on which ‘TS’ was written on the bottom were warmed in an incubator at 37°C overnight. (2) Cryotop was taken from the cane in the LN2 tank and promptly moved into the container 90% filled with new LN2.
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For warming 1 min 37°C
5 min × 2 RT
3 min RT
LN2 TS (1.0 mol/L sucrose)
DS (0.5 mol/L sucrose)
WS1, WS2
2h
37°C, CO2 6%, O2 5%, N2 89
ICSI
Figure 7D.4 Vitrification procedure for mature human oocytes at KAC using Cryotop (warming). TS, thawing solution; RT, room temperature; DS, diluent solution, WS, washing solution; ICSI, intracytoplasmic sperm injection; RT, room temperature.
LN2 container
Figure 7D.5
(3) On the bottom of the three 35-mm dishes was labeled ‘DS’, ‘WS1’, and ‘WS2’. (4) The TS tube and the 35-mm dish were taken from the incubator. The TS tube
158
Preparation for thawing procedure.
was mixed by rotating twice, and then the contents were poured over the 35-mm dish, which was then placed on the microscope stage. Focus was adjusted to the
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bottom of the 35-mm dish at minimum magnification. (5) The cap of the Cryotop in the LN2 container was removed using the pair of tweezers, and the tip of the Cryotop was dipped into 37°C TS within 1 s. Warming speed was +42 000°C/min. The Cryotop with oocyte was placed in TS at the center of the dish for 1 min. Oocytes spontaneously separated from the end of the Cryotop. Oocytes were found by focusing on the tip of the Cryotop and aspirated by the pipette tip after separation from the tip of the Cryotop, and placed in the center of the 35-mm dish. During this time, we confirmed that the shrunken oocyte increased in volume due to the return of water into the inside of the oocyte after thawing. (6) Oocytes were moved to the bottom center of the DS dish and kept at room temperature for about 3 min. The most important point was to aspirate the oocyte with a TS volume in accordance with the 3 mm length of tip of pipette and to make a TS layer in accordance with the 2 mm length of tip of pipette on the bottom of the 35mm dish, followed by placement of the oocyte into a TS layer in the DS. All TS remaining in the pipette was poured over the oocyte. Allowing the oocyte to adapt gradually from TS to DS is very important. Oocytes start to shrink after briefly swelling. A DS layer in accordance with the 2 mm length of tip of pipette as above was made in WS1 on the bottom of a 35mm dish and the oocyte was placed into the DS layer in the WS1 drop. DS remaining in the pipette was poured over the oocyte and kept at room temperature for 5 min. (7) The oocyte was aspirated by pipette and placed on the surface of the center of the WS2 dish. Minimizing the volume of WS1 is important when moving the oocyte from WS1 to WS2 at the end of the pipette. The oocyte was warmed from
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room temperature to 37°C by turning the hot plate ‘on’ for 5 min. Re-expanded oocytes were considered to have survived. (8) Intracytoplasmic sperm injection (ICSI) was performed after culturing in media (Universal IVF medium; MediCult, Denmark) in an atmosphere of 6% CO2, 5% O2, 89% N2 at 37°C for 2 hours.3,6
INDICATIONS FOR HUMAN OOCYTE CRYOPRESERVATION Indications for human oocyte preservation include • • • •
Oocyte donation Preservation of own fertile ability for aging Malignant disease (leukemia, etc.) Premature ovarian failure (Turner syndrome, etc.)
OUTCOME OF VITRIFICATION Our data showed that survival (82.2% = 60/73 vs. 100% = 12/12), fertilization (63.3% = 38/60 vs. 41.7% = 5/12), cleavage (97.4% = 37/38 vs. 100% = 5/5) and blastulation rates (26.7% = 4/15 vs. 33.3% = 1/3) differed significantly between SFM and vitrification using cryotop (VF), respectively.13 The survival rate was 50–80% for SFM, compared with more than 80% for VF in some studies. Fertilization rate, clinical pregnancies per thawed oocyte, clinical pregnancies per injected oocyte, clinical pregnancies per transfer, and implantation rate for SFM were 64.9% (2478/3818), 2.3% (153/6720), 4.0% (153/3818), 2.1% (153/7429), and 10.1% (185/1828), respectively. Fertilization rate, clinical pregnancies per thawed oocytes, clinical pregnancies per transfer, live births per transfer, and implantation rate for VF were 74.2% (637/859), 4.5% (61/1354), 45.5% (61/134), 36.6% (49/134), and 17.1% (81/473), respectively.1 Pregnancy rates with VF thus appear to have improved.
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SEX RATIO We examined sex ratio in all references introduced by Oktay et al.1 More females than males have been born following cryopreservation of human mature oocytes (female : male, 52 : 28).
SPINDLE DAMAGE Some studies have suggested spindle distruption.14–16 Several studies have shown that the spindle reforms after thawing and rewarming.17–1 In particular, some studies have suggested that vitrification forms morphologically normal metaphase spindles with properly aligned chromatin.19–21.
Outcome for children born from slow frozen oocytes
damages the cytoplasm. The use of in vitro maturation of oocytes from germinal vesicle (GV) to metaphase II has not been established, as associated problems of cytoplasmic maturation have been identified. TaxolR, a cytoskeletal stabilizer, appears effective in the cryopreservation of GV oocytes. Park et al.25 reported that addition of Taxol significantly improved post-thaw development of cumulus-enclosed ICR mouse oocytes vitrified at the mature stage. We have reported that addition of Taxol to vitrification solution greatly promoted post-thaw preimplantation development of human immature oocytes.26 Damage to the cytoskeleton and alteration of gene/protein expression or mitochondrial function due to oocyte freezing will be investigated in the future.
CONCLUSIONS
A total of 52 pregnancies have been reported in two studies,22,23 with 32 live births. Followup has been reported for 30 of the 32 live births, with abnormalities limited to ventricular septal defect (VSD) in one case and triploidy in one case. In our clinic, the one child born following SFM in 2001 and the child born following vitrification in 2004 were normal as of November 2006. An international registry is needed to compile data regarding pregnancy/birth outcomes especially from vitrified oocytes.
Oocyte cryopreservation remains experimental, but the techniques utilized are rapidly being refined and improved, particularly in vitrification. Further follow-up data are needed on children born after oocyte cryopreservation.
ACKNOWLEDGMENTS The authors would like to thank Ms Chiharu Onuma and Ms Wakako Funakoshi.
REFERENCES IMMATURE OOCYTE CRYOPRESERVATION Immature oocytes have been suggested to be more suitable than mature oocytes for cryopreservation, as the chromatin and spindles of immature oocytes are not damaged by cryopreservation. However, only one case of pregnancy and delivery achieved using cryopreserved immature human oocytes has been reported.24 Recent reports have suggested that cryopreservation of immature oocytes
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1. Oktay K, Cil AP, Bang H. Efficiency of oocyte cryopreservation: a meta-analysis. Fertil Steril 2006; 86: 70–80. 2. Kyono K, Fukunaga N, Haigo K et al. Pregnancy and delivery of a healthy female infant after intracytoplasmic sperm injection into cryopreserved human oocytes. Jpn J Fertil Steril 2001; 46: 171–7. 3. Kyono K, Fuchinoue K, Yagi A et al. Successful pregnancy and delivery after transfer of a single blastocyst derived from a vitrified mature human oocyte. Fertil Steril 2005; 84: 1017. 4. Kuwayama M, Kato O. Successful vitrification of human oocytes. Fertil Steril 2000; 74: S49.
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5. Okimura T, Kato K, Zhan Q et al. Update on clinical efficiency of the vitrification method for human oocytes in an in vitro fertilization program. Fertil Steril 2005; 84: S174. 6. Kuwayama M, Ieda S. Vitrification of mouse oocytes. J Mamm Ova Res 2005; 22: 71–5. 7. Kuleshova K, Gianaroli L, Magli C et al. Birth following vitrification of a small number of human oocytes: case report. Hum Reprod 1999; 14: 3077–9. 8. Yoon TK, Kim TJ, Park SE et al. Live births after vitrification of oocytes in a stimulated in vitro fertilization-embryo transfer program. Fertil Steril 2003; 79: 1323–6. 9. Kayatama KP, Stehlik J, Kuwayama M et al. High survival rate of vitrified human oocytes results in clinical pregnancy. Fertil Steril 2003; 80: 223–4. 10. Mukaida T, Matsubara T, Takahashi K et al. Birth after vitrified oocytes using cryoloop technique. Fertil Steril 2005; 84: S454. 11. Chian RC, Son WY, Huang JY et al. High survival rates and pregnancies of human oocytes following vitrification: preliminary report. Fertil Steril 2005; 84: S36. 12. Personal Communication Sun 2006. 13. Kyono K, Fuchinoue K, Nakajo Y et al. Comparing vitrification and slow freezing procedures in cryopreservation of mature human oocytes. Fertil Steril 2004; 82: S135. 14. Boiso I, Marti M, Santalo J et al. A confocal microscopy analysis of the spindle and chromosome configurations of human oocytes cryopreserved at the germinal vesicle and metaphase II stage. Hum Reprod 2002; 17: 1885–91. 15. Pickering SJ, Braude PR, Johnson MH et al. Transient cooling to room temperature can cause irreversible disruption of the meiotic spindle in the human oocyte. Fertil Steril 1990; 54: 102–8. 16. Wang WH, Meng L, Hackett RJ et al. Limited recovery of meiotic spindles in living human oocytes after cooling-rewarming observed using polarized light microscopy. Hum Reprod 2001; 16: 2374–8.
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17. Gook DA, Osborn SM, Johnston WI. Cryopreservation of mouse and human oocytes using 1,2-propanediol and the configuration of the meiotic spindle. Hum Reprod 1993; 8: 1101–9. 18. Stachecki JJ, Munne S, Cohen J. Spindle organization after cryopreservation of mouse, human, and bovine oocytes. Reprod Biomed Online 2004; 8: 664–77. 19. Silva CA, Swain J, Acevedo N et al. Influence of vitrification on metaphase II spindle dynamics, spindle morphology, and chromatin alignment. Fertil Steril 2004; 82: S111. 20. Chen CK, Wang CW, Tsai WJ et al. Evaluation of meiotic spindles in thawed oocytes after vitrification using polarized light microscopy. Fertil Steril 2004; 82: 666–72. 21. Huang JY, Chen HY, Wang Y et al. Meiotic spindle and chromosome alignment of in vitro matured oocytes following vitrification. Fertil Steril 2006; 86: S66. 22. Porcu E, Fabbri R, Seracchioli R et al. Obstetric, perinatal outcome and follow up of children conceived from cryopreserved oocytes. Fertil Steril 2000; 74: S48. 23. Winslow KL , Yang D, Blohm PL et al. Oocyte cryopreservation/a three year follow up of sixteen births. Fertil Steril 2001; 76: S120–1. 24. Tucker MJ, Wright G, Morton PC et al. Birth after cryopreservation of immature oocytes with subsequent in vitro maturation. Fertil Steril 1998; 70: 578–9. 25. Park SE, Chung HM, Cha KY et al. Cryopreservation of ICR mouse oocytes: improved post-thawed preimplantation development after vitrification using Taxol, a cytosleleton stabilizer. Fertil Steril 2001; 75: 1177–84. 26. Kyono K, Haigo K, Fukunaga N et al. Comparison of two freezing methods for human immature oocytes with cumulus or cumulus free: Slow freezing versus vitrification (including the effect of Taxol). Fertil Steril 2002; 76: S13.
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8 Vitrification of pronuclear embryos: research basis for aseptic technology and its application to oocytes and blastocysts Hans van der Ven, Vladimir Isachenko, Evgenia Isachenko, Markus Montag and Frank Nawroth
Development of a refined method for cryopreservation of human pronuclear embryos is an important topic, as it is illegal to cryopreserve an oocyte after fusion of the pronuclei due to ethical difficulties in some countries. To date, in some centers, numerous babies have been born after transfer of embryos developed from frozen pronuclear embryos.1 However, with regard to the low efficacy of the oocyte freezing technique, the ‘frozen oocyte’ pregnancy rate is lower than that resulting from fresh pronuclear embryos.2 Conventional (slow) freezing of human pronuclear and developing embryos has been the most widely used method of preservation to date.2,3 However, there have been several reports of the successful cryopreservation of human pronuclear embryos by direct plunging into liquid nitrogen (so called vitrification).5–13 This method is now the object of intensive investigation in numerous laboratories taking into account that the protocol of vitrification includes two parameters: the vitrification process occupies only a few minutes in contrast to the long time-consuming conventional method, and this method needs no special equipment compared with conventional freezing. Our protocol of vitrification of human pronuclear embryos includes the following parameters: step-wise saturation by and removal of permeable cryoprotectants, using a
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small amount of vitrification medium that allows quick warming of cells; full isolation of vitrification medium from contact with liquid nitrogen; and short contact of cells with permeable cryoprotectant dimethylsulfoxide (DMSO) at decreased temperature for reduction of parthenogenetic and toxic effects. The research basis of the technology is described below.
STEP-WISE SATURATION BY AND REMOVAL OF PERMEABLE CRYOPROTECTANTS According to the most common protocols of vitrification, human oocytes and embryos, once thawed, are placed in hypertonic disaccharide solution (normally sucrose) to remove permeable cryoprotectants before transferring the cells to an isotonic culture medium. This rehydration process is generally conducted by gradual dilution of the permeable cryoprotectants through exposure in three steps to 0.5, 0.25, and 0.125 mol/L sucrose5,6 or to 1.0, 0.5, and 0.25 mol/L sucrose.9 Fourstep dilution protocols involving exposure to 1.0, 0.5, 0.25, and 0.125 mol/L sucrose have also been described.8,10 In contrast, it has recently been possible to directly rehydrate vitrified ovine and bovine oocytes after thawing without the need for gradual dilution of the cryoprotectants.14–16
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v
CG
PN M No
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aV
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M V1
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b M LV
CG V1 L
V LFB
LV
LV
M V
M L
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Figure 8.1 Electron micrographs of pronuclear embryos: cooled without cryoprotectants (a) and (b), and vitrified with step-wise dilution of cryoprotectants (c). M, mitochondria; CG, cortical granules; PN, pronucleus; Nc, nucleolus; V, small vesicles of smooth endoplasmic reticulum encircled by flattened mitochondria; L, lipids, LV, large vesicles of smooth endoplasmic reticulum, associated with mitochondria; LFB, lipofuscin bodies inside of circle; arrows, deformations of nuclear membrane; arrowheads, disruption of nuclear membrane. Scale bar = 1 µm.
Viability of vitrified human pronuclear embryos after step-wise removal of cryoprotectants versus direct rehydration (with intense osmotic processes) has been studied.11–13 Embryo viability was assessed by
their subsequent in vitro survival, post-transfer implantation rate, and ultrastructural features. Following step-wise rehydration, embryos showed a high viability after vitrification/warming. Cytoplasm of embryos after vitrification/warming followed by direct rehydration showed a dark, granular appearance, and none of these oocytes developed to the two blastomere stage. The ultrastructure of both fresh and vitrified/ step-wise rehydrated pronuclear embryos has been described.11–13 The smooth endoplasmic reticulum (SER) of a fresh pronuclear oocyte is associated with mitochondria.11 The presence of small vesicles not associated with mitochondria was observed. Others demonstrated that intracellular structures of pronuclear embryos are very sensitive to cooling17 and vitrification.18 The negative effects of the cryopreservation process on human pronuclear embryos can be distributed into three groups: cooling, osmosis and toxicity. According to Sathananthan et al.,17 human cells seem less sensitive to cooling than oocytes of other mammalian species because they contain a smaller volume of lipids. However, our investigations show that the sensitivity of organelles in fertilized oocytes to cooling to 4°C changes with respect to the presence or absence of cryoprotectants (Figure 8.1). The aim of our investigations was to examine the peculiarities of ultrastructural changes in pronuclear embryos cooled to low positive temperatures in the presence or absence of the cryoprotectant ethylene glycol.11,12 We investigated the basic cell organelles, such as mitochondria, and three types of smooth endoplasmic reticulum (SER): tubular, small vesicular, and large vesicular aggregates. The connections between SER vesicles and mitochondria, as well as the intensity of these aggregates (absence of connection with mitochondria) were determined. Cryoprotectants prevent the severe damage to intracellular organelles
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such as multiple vacuolizations, clustering of mitochondria, and deformation and disruption of nucleolar and plasma membranes. After vitrification, thawing, and 1 h of exposure in CO2 incubator, the presumably viable pronuclear embryos showed large SER vesicles associated with mitochondria, and small vesicles close to flattened, crescent-shaped mitochondria (Figure 8.1). Human pronuclear embryos are highly sensitive to cooling and low positive temperatures. Adding the cryoprotectant provides significant protection against damage of the intracellular organelles (Figure 8.1). We believe that osmosis plays a central role throughout all negative effects of the cryopreservation process of human pronuclear embryos. Figure 8.2 shows images of human pronuclear embryos that were vitrified, warmed, and directly rehydrated with intense osmotic processes. The cytoplasm was seen to be filled with a finely granulated substance with dark inclusions. Note the remains of membrane structures and a dense dark pulp with transparent (low density) membrane-bearing and membrane-free vesicles. The dark pulp is enveloped by membrane and is characteristic of the deformation and destruction provoked by the intense osmotic effects of direct rehydration. The cytoplasm shows transparent (low density) spots which are probably the result of the disruption of lysosomes, that release their proteolytic contents, inducing lysis of the cytoplasmic matrix. The image shows the disruption of the pronucleus followed by rupture of the membranes first and the nucleoli later. The method of direct post-thaw rehydration induces lethal osmotic effects in human pronuclear embryos. Accordingly, the vitrification protocol for these embryos must include the step-wise dilution of the cryoprotectant. Taking into account that the saturation by cryoprotectants is also accompanied by osmotic processes, our aseptic technology includes a step-wise method of saturation by cryoprotectants.
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FULL ISOLATION OF A SMALL AMOUNT OF VITRIFICATION MEDIUM FROM CONTACT WITH LIQUID NITROGEN (ASEPTIC MODE) Rall and Fahy,19 and others have published intensely on vitrification including the influence of cooling and warming rates on survival of vitrified objects. Investigations of these authors provided evidence that the rate of cooling during vitrification is secondary as long as it is fast enough to prevent crystallization. For example, for vitrification of 8-cell mouse embryos using solutions of different combinations of DMSO, acetamide, propylene glycol, and glycerol in total concentrations from 5.5 to 6.54 mol/L plus 6% polyethylene glycol, equally high survival rates were obtained at cooling rates ranging from about 15 to 2500°C/min.20 Differential scanning calorimetry compared the appearance of the solutions using phase-contrast cryomicroscopy. No crystallization was observed when these solutions were cooled at rates greater than 10° C/min.21 High survival rates of mouse embryos after vitrification with a cooling rate of at least 10° C/min using vitrification solution 6.5 glycerol plus 6% polyethylene glycol were reported.20 In practice there are two different technologies of cooling of cells before storage in liquid nitrogen: cooling by direct plunging into liquid nitrogen (decreasing of temperature from some thousands to some tens of thousands °C/min;15,22–27 and cooling in the vapor above liquid nitrogen before long-term storage in liquid nitrogen (at about 200°C/min).28–34 This methodology of ‘slow’ cooling of cells before storage in liquid nitrogen is evidence for the secondary role of the parameter ‘speed of cooling’ for vitrification of oocytes and embryos. Another important parameter of vitrification is the speed of warming after storage in liquid nitrogen. Formation of crystals in vitrified
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M M
M
M M P
V
C C
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S RPN
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N
Figure 8.2 Electron micrographs of vitrified pronuclear embryo after thawing and direct rehydration. Note the deformation and disruption of the cytoplasmic membranes (M). RPN, remains of the pronucleus; S, low density spots; C, cavity; P, dark pulp; V, vesicle; N, nucleus; arrowheads, pronuclear membrane remnants (envelope). Bar = 1 µm.
solution during warming (so called devitrification) is associated with the death of embryos after conventional cryopreservation.21 The same negative effect of ‘slow’ warming can be observed with vitrification. For example, a stable glassy state of vitrified warmed solution as well as a good viability of mouse embryos was obtained using moderate cooling and warming rates ( ≥ 20 and ≥ 100°C/min, respectively).20 All combinations of ‘slow’ or
‘quick’ cooling with ‘slow’ warming were linked to decreasing viability of vitrified mouse embryos.19,30 We showed the importance and efficacy of elevated warming rate during vitrification of porcine germinal vesicle (GV)-oocytes,35 and absence of visible crystal formation when the warming/cooling proportion was higher than 1.3.36 The majority of authors have noted that the high level of effectiveness of different protocols
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for the vitrification of oocytes and embryos with decreased volumes of cooled medium, can be explained by the combination of high speeds of cooling and warming.22,23,33,37–40 The results of our investigations with human oocytes do not support this point of view. For human GVoocytes and pronuclear embryos a relatively slow cooling rate in combination with rapid warming is an efficient ‘conventional’ vitrification process with respect to survival rates and embryo development.41–43 We have shown that effective vitrification of ovine GV-oocytes is dependent on a combination of high speed of cooling and warming.24 However, using the regimen ‘slow cooling – rapid warming’ we observed no development of oocytes but excellent expansion of cumulus of these oocytes, in contrast to ‘slow cooling – slow warming’ when oocytes as well as cumulus cells were dead after warming.24 This phenomenon can be explained by differences of cryoproperties (cryostability) of oocytes and cumulus cells within of the same mammalian species. Vitrification of human embryos using fine diameter plastic micropipettes, which were cooled in the vapor above liquid nitrogen before their placement into a precooled cryotube, was reported. Similarly to previous findings, this methodology also included the parameter ‘slow cooling’ and ‘rapid warming’.44 ‘Slow’ cooling of biological objects can also be used for cooling of relatively large volumes of cooled solution. To prevent the 0.25 mL straw from coming into direct contact with liquid nitrogen and eliminating the potential contamination risk associated with storage in liquid nitrogen, Kuleshova and Shaw45 reported about ‘straw-in-straw’ vitrification of mouse embryos. A standard 0.25 mL straw with column of vitrification medium was located into a 0.5 mL straw, which was hermetically closed before plunging into liquid nitrogen. However, this method, in contrast with the one described in this chapter, does
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not allow rapid warming of small volumes of vitrification medium with simultaneous removal of cryoprotectant. Our conclusion about the minor importance of the speed of cooling for vitrifying oocytes and embryos, can probably be applied to all reproductive human cells including spermatozoa. We recently reported that vitrification of human spermatozoa by fast (20 000°C/min) or relatively slow (200°C/min) cooling with fast warming, results in similar post-thaw characteristics.46 In many publications about vitrification of cells in a small amount of vitrification medium, the described technique includes the direct contact between liquid nitrogen and the cells. However, any other technology in reproductive biology, and especially in medicine, must ensure and guarantee full isolation of biological objects from microorganisms.47 Liquid nitrogen, which is used for storage of frozen materials, can be a source of contamination by these microorganisms.47,48 Filtration or ultraviolet treatment of liquid nitrogen cannot guarantee the absence of contamination of biological materials by viruses including the HIV and mycoplasma. For example, the contamination of blood probes by hepatitis virus during the time of storage of probes in liquid nitrogen has been reported.48 Different types of viruses, which are simple and very cryostable structures, may increase their virulence after direct plunging and storage in liquid nitrogen: hepatitis virus,49 papova (?) virus,50 vesicular stomatitis virus,51 and herpes virus.52 The aim of our investigations was to test whether the method of cryopreservation of human pronuclear embryos in straws, which are placed inside a hermetically closed container (larger straw), guarantees complete isolation of embryos from liquid nitrogen and avoids potential contamination by pathogenic microorganisms. We found that vitrification of human pronuclear embryos (Figure 8.3) in open straws, which are placed inside a
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1
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Figure 8.3 The same two-pronuclear embryo before vitrification (1), and after warming for 5 min (same (2) and (3) with different focus), 6 h (4), 18 h (5), 30 h (6), 42 h (7), 66 h (8), 78 h (9), 96 h (10), and 96 h stained by Hoechst 33342 (11). Bar = 30 µm.
hermetically closed container before plunging into liquid nitrogen, allows reliable isolation of oocytes from liquid nitrogen and avoids contamination by pathogenic microorganisms. Although this technique is associated with a relatively slow cooling rate, the developmental potential of these pronuclear embryos is not compromised if the thawing process involves rapid warming and simultaneous removal of cryoprotectants.41 For human
pronuclear embryos and GV-oocytes the relatively slow cooling rate in combination with rapid warming is an efficient ‘conventional’ vitrification method with regard to survival rates and embryo development.41–43 A new container for aseptic vitrification with simultaneous quick warming and removal of cryoprotectants was tested. Therefore, cooling and warming of oocytes and embryos was performed in cut standard straws (CSS)
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2
1
3
4 2
22
Figure 8.4 Photographs and scheme of warming using the cut standard straw (CSS) container for vitrification: (1) closed 0.5 mL straw, (2) CSS, (3) vitrification medium with embryo, and (4) tube with solution for warming and removal of cryoprotectant.
(Figure 8.4). CSS were produced from standard insemination 0.25 mL (so called French) straws, which are cut at an angle of approximately 45°. Vitrification medium holding the embryo is placed onto the cut part of the straw. After exposure to vitrification medium, one embryo with 0.25–0.75 µL of this medium is aspirated into the tip of a pipettor and transferred to CSS (Figure 8.4). The CSS are loaded into 0.5 mL straws, which are closed at both sides and plunged into liquid nitrogen with a cooling speed of 600°C/min. For rapid warming, the CSS is first removed from the larger straw, which is still half submerging in liquid nitrogen, prior to plunging
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into sucrose solution (Figure 8.4). This method allows for the simultaneous removal of cryoprotectant and rapid warming.
SHORT CONTACT OF CELLS WITH PERMEABLE CRYOPROTECTANT DIMETHYLSULFOXIDE AT DECREASED TEMPERATURE The cryoprotectant DMSO is widely used for vitrification of human oocytes and embryos. It was reported that vitrification of blastocysts using ethylene glycol and DMSO resulted in a 33.3% pregnancy rate and the birth of a healthy baby.53 Later, in the same clinic two
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Maturation rate (%)
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100 90 80 70 60 50 40 30 20 10 0
a b
b
b
Aseptic vitrification with long exposure in EG + DMSO
Aseptic vitrification with the long exposure in EG and short exposure
c
Fresh control
Direct into LN vitrification with long exposure in EG + DMSO
Aseptic vitrification without DMSO
Figure 8.5 Effect of the pre-cooling treatment and cooling method of germinal vesicle (GV)-oocytes on maturation rate and post-warm parthenogenesis. LN, liquid nitrogen; DMSO, dimethylsulphoxide; EG, ethylene glycol. Soild columns indicate the groups in which the cases of parthenogenesis were observed. Different superscripts indicated significant differences (P < 0.05).
morula-stage embryos were vitrified using the same protocol (with DMSO) and transferred, which resulted in the birth of two healthy twins.34 Up to March 2005, 86 healthy babies were born after transfers of 332 embryos (birth rate 26%) after vitrification using DMSO (Yokota, personal communication). The possibility and necessity of introducing DMSO into the vitrification medium for human reproductive cells is supported by data from Vanderzwalmen et al.39 and Huang et al.,54 who have used this cryoprotectant for vitrification of blastocysts. The high pregnancy rate after warming of blastocysts (41% by Vanderzwalmen et al.39 and 54% by Huang et al.54 gives evidence about the efficiency of vitrification media composed in both cases of 20% DMSO + 20% ethylene glycol + sucrose + other compounds. Later the mentioned pregnancies resulted in the birth of healthy babies (about 98 healthy births including 28 twins per 414 warmed and transferred embryos (Vanderzwalmen, personal communication); about ten healthy births per 14 implantations with three spontaneous abortions, and one reduction from triplet (Huang and Lee, personal communication). Also, using DMSO for vitrification of oocytes was successful and resulted in a high
survival rate of warmed oocytes, ongoing clinical pregnancies,55 and birth of healthy babies after embryo transfers (Katayama, personal communication). We specifically investigated the effect of DMSO on the vitrification process as well as its impact on further maturation in vitro of human GV-oocytes.43 Furthermore, the aim of our investigations was to test the method of aseptic vitrification of these oocytes in small straws, which are placed inside a hermetically closed container that guarantees complete isolation of oocytes from liquid nitrogen and avoids potential contamination by pathogenic microorganisms. The presence of DMSO in vitrification medium with regard to the duration of contact of this cryoprotectant with the cells was studied. Cryopreservation solutions in this as well as in all other experiments were prepared in Dulbecco phosphate buffered saline (DPBS) with 0.75 mol/L sucrose, 20% serum substitute supplement, and 10% polysaccharide Ficoll-70. Four experimental groups were formed (Figure 8.5): ‘direct’ vitrification with long exposure in ethylene glycol (20%) and DMSO (20%), aseptic vitrification without DMSO (when only 40% ethylene glycol was used), aseptic vitrification with long exposure in ethylene glycol (20%) and
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a
b
c
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Figure 8.6 The same GV-oocyte vitrified with long (5 min) contact with DMSO at 37°C: (a) fresh before vitrification, (b) just after warming, after maturation for 12 h (c), for 24 h (d), for 48 h (e), and stained by Hoechst 33342 (f). Insets are magnified and contrasted images of germinal vesicle ((a) and (b)) and pronucleus ((d) and (e)).
DMSO (20%), and aseptic vitrification with long exposure in ethylene glycol and short exposure in ethylene glycol (20%) and DMSO (20%). After warming and step-wise removal
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of cryoprotectants in sucrose, we found, that slow cooling/rapid warming can also be applied to human GV-oocytes. Using aseptic technology a maturation rate of GV-oocytes after
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vitrification of approximately 85% was observed. The short-term presence of DMSO at room temperature in the vitrification medium can improve the maturation rate of GV-oocytes and does not cause spontaneous parthenogenesis.43 In one experiment we studied the possibility of using a vitrification solution which was completely free of DMSO. One reason to do so was that DMSO was reported to affect the organization of microfilaments in mouse oocytes,56 as well as to induce chromosomal abnormalities (increase in the rates of degeneration and digynic polyploid embryos) after cryopreservation of mouse oocytes in the presence of DMSO.57 However, in our experiments the absence of DMSO in the vitrification medium caused a significant decrease in maturation rates. On the other hand, a 5 min contact of oocytes with DMSO at 37°C was enough to cause spontaneous oocyte activation and parthenogenetic development. The protocol which we found to be recommendable for use in medical practice is a compromise between the presence of DMSO in vitrification medium and parthenogenetic activation. Based on our results we propose to use short (1 min) contact with DMSO which is only included in the final portion of vitrification medium, and contact with DMSO at room temperature that theoretically can decrease the negative effect on oocytes. The high maturation rates and the absence of parthenogenesis are a direct proof that this way of using DMSO (decreasing time and temperature of contact) is suitable for vitrification of human oocytes and embryos.
USE OF ASEPTIC TECHNOLOGY FOR LATE HUMAN EMBRYOS AND PRONUCLEAR EMBRYOS OF OTHER SPECIES In most human in vitro fertilization programs embryo transfer is routinely performed on
day 2 or 3. However, implantation rates are relatively low.58 One attempt to increase this rate might be the prolonged culture of human embryos up to the blastocyst stage and to select the best embryos at that stage for transfer. To achieve this goal, sequential culture media were developed which fulfill the differential requirements of the embryo during early preimplantation development.59 Consequently, these media enabled blastocyst formation rates of up to 50% and due to extended culture those embryos with the best developmental potential were recognized more readily on day 5.60 Based on this approach, several studies reported higher implantation rates following transfer of selected blastocysts. Comparative investigations on freezing and vitrification of human late blastocysts were performed. Liebermann and Tucker61 have evaluated implantation of day 5 and day 6 vitrified and conventionally (slowly) frozen blastocysts. Day 5 and day 6 blastocysts were vitrified or frozen and transferred after warming or thawing. In 508 transfer cycles, embryonic implantation rates for day 5 and day 6 vitrified blastocysts were 33% and 26%, respectively, and after conventional freezing were 30% and 28%, respectively. Blastocysts with laser opening of zona pellucida were the object of our investigations. They are more difficult to cryopreserve than early blastocysts or blastocysts with intact zona pellucida for two reasons. It was reported that the survival rate of expanded blastocysts after vitrification is relatively low.62 These authors explain this fact by the presence of a large volume of water in the blastocoel which, in addition, can be crystallized on cooling and these crystals can destroy the embryo. Also, the laser opening of the zona pellucida decreases the cryostability of blastocysts and reports about vitrification of this type of blastocyst are limited.63 The aim of this study was to compare the viability of vitrified human blastocysts, using decreased concentrations of cryoprotectants,
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by direct submerging of cut standard straws (CSS) into liquid nitrogen, with that after vitrification by cooling of CSS loaded inside a 0.5 mL straw (aseptic system). Six-day blastocysts were obtained after culture of pronuclear embryos and laser assisted hatching. These blastocysts were cryopreserved in CSS by vitrification in ethylene glycol + DMSO + 0.3 mol/L sucrose. In the first experiment, embryos were vitrified using vitrification medium with 20% ethylene glycol + 20% DMSO and 15% ethylene glygol + 15% DMSO. In the second experiment, when vitrification medium included 15% ethylene glycol + 15% DMSO + sucrose, embryos were cooled rapidly and slowly (with isolation from liquid nitrogen). Embryos in both experiments (four groups) were rapidly thawed at a speed of 30 000– 85 000°C/min using an identical protocol. Embryos of all experimental groups had the same rate of reexpansion (80–85%) after in vitro culture (Figure 8.7). It was noted that the method involving vitrification of expanded blastocysts with decreased concentrations of permeable cryoprotectants and complete isolation of embryos from liquid nitrogen in CSS is efficient. The described aseptic technology can be applied to pronuclear embryos of other species including mouse. Pronuclear mouse embryos attracted special interest for cryoinvestigation for two reasons. First, this stage is used for production of transgenic mouse, which is a promising model for present and future biological investigation. Second, in some European countries, cryopreservation of zygotes after fusion of pronuclei or later stage embryos is prohibited. For these two reasons, cryopreservation of mouse pronuclear embryos by direct plunging into liquid nitrogen is a good model for investigation. The aim of our investigations was to test whether the method of cryopreservation of biopsied mouse pronuclear embryos in small straws, which are placed inside an
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hermetically closed container, guarantees complete isolation of oocytes from liquid nitrogen and avoids potential contamination by pathogenic microorganisms. It was shown that the developmental potential of mouse pronuclear oocytes is not compromised if the thawing process involves rapid warming and simultaneous removal of cryoprotectants.42 Aseptic vitrification can be applied to human pronuclear embryos and oocytes as well as for embryos of any developmental stage; the possibility of using this technology for other mammalian species is promising. Recentely, several reports about vitrification of human embryos using cooling with isolation from liquid nitrogen were published. Kuwayama et al.64 performed 16 000 human embryo cryopreservations and proved that vitrification is a good alternative to conventional freezing and resulted in high survival and in vitro developmental rates for pronuclear, multicellular, and blastocyst stage human embryos. The authors described a vitrification method that eliminates the risk of contamination of oocytes and embryos in liquid nitrogen.64 This method includes isolation of the vitrification medium from liquid nitrogen at cooling. Vanderzwalmen et al.65 proposed the method of small volume vitrification in hemi-straws, tested two methods of cooling 3- and 5-day embryos using direct plunging into liquid nitrogen (non-aseptic conditions) and cooling with isolation from liquid nitrogen (aseptic conditions). The results show that post-thaw survival rates of morulae and blastocysts vitrified at a lower cooling rate inside a hermetic sealed straw are comparable with those of the control nonaseptic group. Recently, it was shown that the previously reported method of vitrification using CryoloopTM can be used to vitrify and store mouse embryos without direct liquid nitrogen contact (during cooling and storage). When such vitrified embryos are warmed, they are capable of subsequent development comparable with that of non-vitrified embryos.66
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a
b
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Figure 8.7 Six-day blastocysts from pronuclear embryo after polar body biopsy: fresh (a), after 30 exposure in 20% vitrification medium (b), 6 min after warming (c), 6 h after warming (d), and 12 h after warming (e) and (f) (stained by Hoechst 33342). Bar = 30 µm.
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We believe that these investigations of aseptic vitrification arose following our own results.11–13,26,27,41,43
DIFFERENCES WITH HUMAN AND ANIMAL PRONUCLEAR EMBRYOS DURING CRYOPRESERVATION: INTRACELLULAR LIPIDS As a rule, cryoinvestigations in human embryos are based on successfully used protocols emerging from similar experiments performed on embryos from laboratory animals. Before it was possible to vitrify human pronuclear embryos, animal embryos at this stage had been successfully cryopreserved using the method of direct plunging into liquid nitrogen, and an effective protocol for vitrification of mouse pronuclear embryos was developed. These data proved useful in developing protocols for the vitrification of human pronuclear embryos. Aseptic technology of vitrification of human oocytes and embryos can be successfully used for cryopreservation of mammalian oocytes of other species. However, it is necessary to take into account that human cells have their own peculiarities. One such attribute is the presence of intracellular lipids. Intracellular lipids are a ‘stumbling block’ for cryopreservation including vitrification. Data demonstrating the role of these intracellular structures during cryopreservation have been published.67 The method proposed involves polarization and removal of cytoplasmic lipids from oocytes or embryos before vitrification. Nagashima et al.67 were the first to successfully grow embryos from GV-porcine oocytes that were vitrified following delipidization. Using this method the authors avoided negative effects caused by cooled intracellular lipids. According to the data provided by the authors, removal of intracellular lipids does not lead to a worsening of
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further development of oocytes and embryos. Successful oocyte vitrification after removal of cytoplasmic lipids leads to the question of possible changes in the physiochemical properties of cytoplasmic membrane lipids arising at low temperatures68 which were discussed as a significant cause of cryobiological problems during experiments. We believe that it is impossible to dismiss classic data about the role of intracellular lipids as energetic materials of oocytes and building materials for membranes of future embryos. The fact that the volume of mitochondria as well as lipid vesicles increases during oocyte development to metaphase II (MII) stage69 indirectly confirms this point. Moreover, Sathananthan et al.70 have shown that in the cell complex called ‘smooth endoplasmic reticulum–lipid globules–mitochondria’ reticulum–globules–mitochondria connections do exist. They have also shown that these connections may be damaged after oocyte cooling or freezing. In the overwhelming majority of work studying the effect of cooling on mammalian oocytes and embryos, a negative cryo-influence is explained in terms of the effect on cytoskeletal elements. For example, cooling of human oocytes causes depolymerization of cytoskeletal protein structures and most mouse oocytes cooled to 25°C for 10 min had an abnormal cytoskeleton.71 After exposure to 4°C for 20 min, completely disassembled spindles were observed. This process of depolymerization is, however, reversible. Spindles of mouse oocytes returned to normal appearance after warming at 37°C for 60 min. Spindles of about half of human oocytes exposed to room temperature for 10 min returned to normal after 4 h of cultivation at 37°C.71 The negative effect of cooling is also explained as depolymerization of microtubules and microfilaments in other works performed on human oocytes. Bovine oocytes are also sensitive to decreasing temperatures.72 It has been shown
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that 56% of oocytes exposed to 25°C and 90% of oocytes exposed to 4°C for 1 min had abnormal spindles. Data on the sensitivity of porcine oocytes, which probably are a more ‘difficult’ entity to cryopreserve at low temperatures, are limited. Didion et al.73 who examined the viability of pig GV-oocytes following cooling or freezing by conventional methods found that the cumulus-intact GV-porcine oocytes do not survive cooling to temperatures at or below 15°C. As the authors noted, this was not surprising considering that porcine embryos from the 8-cell to blastocyst stage were killed when cooled below 15°C. Publications on problems of mammalian oocyte and embryos cryopreservation contain information on the negative effects of low temperature including the cytoskeletal depolymerization by permeable cryoprotectants.74 We suppose that the negative effect of cooling on oocytes can be explained by the effect of cooling lipids on cytoskeletal structures. Whilst performing our investigations on porcine oocytes, we found that following centrifugation, redistribution of lipids occurs within 48 h of in vitro culture in oocytes not exposed to vitrification/warming (data not published). However, when polarized oocytes are vitrified/warmed, the lipid polarization is irreversible. This, in our opinion, suggests that the vitrification/warming process induces an alteration to the physiochemical properties of intracellular lipids. It is known that MII oocytes are more resistant to freeze damage than GV-stage oocytes. We consider that this may be due to differences in the properties of cytoskeletal elements. One important difference is that the configuration of microtubules and microfilaments is different at these two stages of oocyte maturation. Cytoskeletal elements in GV-oocytes appear straight and rigid, while the appearance of microfilaments and microtubules in MII stage oocytes is undulating and flexible. Based on the hypothesis of a complex interaction between the lipid phase of cells and the
elements of the cytoskeleton, hardening of these lipids might cause deformation and disruption of the cytoskeleton. In the case of the rigid GV-oocyte cytoskeleton this apparently results in permanent damage while in the more flexible MII-oocyte cytoskeleton, permanent damage is absent. Cytochalasins have a specific, reversible effect on cytoskeletal elements making them more flexible and less susceptible to the effect from cooled lipids. Testing this substance for vitrification of GVporcine oocytes in combination with elevated temperature was effective.35 For the successful vitrification of GV-porcine oocytes,35 the following points could be important. An optimal protocol of vitrification must prevent the alterations of the physiochemical properties of cooled lipids; avoid irreversible damage to the lipid globules membranes; and protect the cooling reticulum-lipids connection from destruction. Further investigations are needed to verify these assumptions. It is known that bovine oocytes are to a considerable extent more cryostable than porcine oocytes. There is also information suggesting that the diameters of bovine and porcine intracellular lipid vesicles are different. The characteristics of the intracellular lipid granule membranes are also a topic of discussion. We compared the ultrastructure of lipid droplets, and the effect of cooling on intracellular lipid vesicles of bovine and porcine GV-oocytes.75 It was shown that lipid droplets of bovine GV-oocytes have a homogenous structure. The utilization of lipids takes place directly from these vesicles without formation of interim lipid compounds. In contrast, there are two kinds of lipid droplets in porcine GV-oocytes: ‘dark’ and homogenous vesicles next to ‘gray’ vesicles with electronlucent streaks. Vesicles of each specific group are connected to each other. After 12 hour culture, the formation of the cisternal smooth endoplasmic reticulum layer is always associated with ‘gray’ lipid vesicles. This is evidence that during oogenesis lipolysis takes place only in ‘gray’ vesicles. It is
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L L L L L L
L
L
a1
a
L
L
L
b1
b
Figure 8.8 Electron micrographs of the intracellular lipid vesicles of fresh (a) and vitrified (a1) human, and fresh (b) and vitrified (b1) ovine pronuclear oocytes. L, lipid vesicle. Bar = 0.5 µm.
supposed that cytoplasmic lipolysis has two stages: ‘dark’ vesicles change into a ‘gray’ form followed by a utilization of these ‘gray’ lipids. Furthermore, both types of lipid droplets in porcine oocytes changed morphologically during cooling: they turned from a round into a spherical form with lucent streaks. Lipid droplets in bovine GVoocytes revealed no visible morphological changes after cooling.75 In order to compare intracellular lipids and the cryostability of ovine and human pronuclear embryos we have vitrified embryos of both species and evaluated the ultrastructure of intracellular lipids before and after vitrification. Cryopreservation of
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embryos was performed according to the method previously described for ovine GVoocytes24 with two different methods of removal of cryoprotectant: step-wise and direct rehydration. We noted, that in contrast to human pronuclear embryos, where direct rehydration has a mortal effect after thawing, ovine pronuclear embryos after in vitro culture show high developmental rates (31–34%). Fresh lipid droplets in both species are homogenous in structure. It was noted that after vitrification the intracellular lipids in cryopreserved human embryos underwent no visible morphological changes while distinct changes were observed in the lipid droplets of the sheep embryos. These alterations,
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attributable to the vitrification process, reflect changes in the physical and chemical properties of the lipids such as hardening. Figure 8.8 shows intracellular lipid droplets in both human and sheep pronuclear embryos and illustrates the differences between the intracellular lipids. The protocols used for vitrifying human pronuclear embryos are based on methods developed for animal embryos at this stage. In animal models, vitrification may sometimes be followed by effective post-thaw rehydration, without the need for step-wise dilution of permeable cryoprotectants in hypertonic solution. Thus, the vitrified and thawed cells are directly plunged into isotonic (culture) medium. In our investigations we used a vitrification/direct post-warm rehydration protocol that had been previously tested in our laboratory on GV-ovine oocytes24 and, after slight modification, in rat early morulae, early blastocysts, and expanded blastocysts.25 With the subsequent step-wise dilution of cryoprotectants, the same vitrification protocol was found to be highly efficient for human pronuclear embryos yet was completely ineffective when these human embryos were directly rehydrated after warming. Thus, it appears that the ideal protocol for vitrification of pronuclear embryos with direct rehydration may have to be a compromise between the two conditions of maximal binding of intracellular water by permeable cryoprotectants and the presence of minimal post-thaw quantities of these cryoprotectants in the cytoplasm. Prolonged exposure to the cryoprotectant seems to require extrarehydration due to the increased quantity of intracellular cryoprotectant. In contrast, an insufficient amount of intracellular cryoprotectant during pre-cooling exposure may result in insufficient binding of intracellular water. The step-wise dilution procedure that we used after warming indicates that both exposure times to the permeable cryoprotectant are sufficient for intracellular water binding. We,
therefore, also tested the two time periods of 10 s or 1 min exposure in vitrification solution when the vitrified embryos were directly rehydrated after thawing. All attempts to vitrify human pronuclear oocytes with direct rehydration were unsuccessful. We suggest that the specificity of human pronuclear embryos, reflected by their high sensitivity to osmotic processes, is related to the specificity of both intracellular lipids and cytoplasmic and organelle membranes. Lipids are the most cryo-labile intracellular compounds of oocytes and embryos. Indeed, the specific nature of intracellular lipids in pig oocytes makes them practically unsuitable for cryopreservation, particularly vitrification. Figure 8.8 shows the appearance of intracellular lipids of fresh human pronuclear embryos before and after vitrification in which no changes can be observed. We compared these lipids with those of pronuclear ovine embryos subjected to the same protocol which was used for human oocytes with direct rehydration in our previous investigations. These lipids of viable embryos showed ultrastructural changes after vitrification not noted in the human oocytes (Figure 8.8). Bearing in mind the resistance of ovine pronuclear embryos to direct post-thaw rehydration, we were able to observe hardening (increase in density) and morphological alterations in the intracellular lipids of all cooled ovine oocytes. These alterations were absent in all human oocytes, which were clearly unable to tolerate direct rehydration. It may be assumed that within the same cell, the structure of intracellular and membrane lipids are the same. Given the detrimental role of lipids during cryopreservation, the lipid cryostability yet osmotic instability of pronuclear human embryos is still far from being fully understood. Direct post-thaw rehydration induces lethal osmotic effects in human pronuclear oocytes but is successful for ovine oocytes. A correlation between the cryostability of mammalian oocytes and the ultrastructure of intracellular lipids is proposed for further investigations. Taking into account that
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the ultrastructure of intracellular lipids of laboratory and agricultural animals are different from human intracellular lipids, we believe that using animal oocytes as a model for human oocytes in cryo-investigations is questionable.
In conclusion, aseptic vitrification can be successfully applied to human pronuclear embryos, oocytes, and embryos of any developmental stage; the possibility of using this technology for other mammalian species is promising.
References 1. Porcu E, Fabbri R, Seracchioli et al. Birth of a healthy female after intracytoplasmic sperm injection of cryopreserved human oocytes. Fertil Steril 1997; 68: 724–6. 2. Mohr LR, Trounson AO. Cryopreservation of human embryos. Ann N Y Acad Sci 1985; 442: 536–43. 3. Siebzehnrübl ER. Cryopreservation of gametes and cleavage stage embryos. Hum Reprod 1989; 4: 105–10. 4. Al-Hasani S, Demirel LC, Schöpper B et al. Pregnancies achieved after frozen-thawed pronuclear oocytes obtained by intracytoplasmic sperm injection with spermatozoa extracted from frozen-thawed testicular tissues from non-obstructive azoospermic men. Hum Reprod 1999; 14: 2031–5. 5. Park SP, Kim EY, Oh JH et al. Ultra-rapid freezing of human multipronuclear zygotes using electron microscope grids. Hum Reprod 2000; 15: 1787–90. 6. Liebermann J, Tucker MJ, Graham JR, Han T, Davis A, Levy MJ. Blastocyst development after vitrification of multipronuclear zygotes using the Flexipet denuding pipette. Reprod Biomed Online 2002; 4: 148–52. 7. Liebermann J, Nawroth F, Isachenko V, Isachenko E, Rahimi G, Tucker M. Potential importance of vitrification in reproductive medicine. Biol Reprod 2002; 67: 1671–80. 8. Liebermann J, Tucker MJ. Effect of carrier system on the yield of human oocytes and embryos as assessed by survival and developmental potential after vitrification. Reproduction 2002; 124: 483–9. 9. Selman HA, El-Danasouri I. Pregnancies derived from vitrified human zygotes. Fertil Steril 2002; 77: 422–3. 10. Jelinkova L, Selman HA, Arav A, Strehler E, Reeka N, Sterzik K. Twin pregnancy after vitrification of 2-pronuclei human embryos. Fertil Steril 2002; 77: 412–4.
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11. Isachenko V, Selman H, Isachenko E, Montag M, Al-Danasuri I, Nawroth F. Modified vitrification of human pronuclear oocytes: efficacy and effect on ultrastructure. Reprod Biomed Online 2003; 7: 211–6. 12. Isachenko V, Selman H, Isachenko E, Montag M, El-Danasouri I, Nawroth F. Effect of cryoprotectants on the ultrastructure of cooled human pronuclear oocytes. Fertil Steril 2004; 81: 720–2. 13. Isachenko V, Montag M, Isachenko E, Nawroth F, Dessole S, Van der Ven H. Developmental rate and ultrastructure of vitrified human pronuclear oocytes after step-wise versus direct rehydration. Hum Reprod 2004; 19: 660–5. 14. Papis K, Shimizu M, Izaike Y. Factors affecting the survivability of bovine oocytes vitrified in droplets. Theriogenology 2000; 54: 651–8. 15. Isachenko V, Isachenko E, Ostashko F, Grishchenko V. Ultra-rapid freezing of rat embryos with rapid dilution of permeable cryoprotectants. Cryobiology 1997; 34: 157–64. 16. Isachenko V, Alabart JL, Isachenko E, Michelmann HW, Bezugly N. Ultra-rapid freezing and storage of rat embryos in an electric refrigerator at –130°C without liquid cryoagents with ultra-short exposure in the freezing medium and direct rehydration after thawing. Cryo Letters 2000; 21: 13–8. 17. Sathananthan AH, Trounson A, Freemann L, Brady T. The effects of cooling human oocytes. Hum Reprod 1988; 3: 968–77. 18. Fuku E, Xia L, Downey BR. Ultrastructural changes in bovine oocytes cryopreserved by vitrification. Cryobiology. 1995; 32: 139–56. 19. Rall WF, Fahy GM. Ice-free cryopreservation of mouse embryos at −196 °C by vitrification. Nature 1985; 313: 573–5. 20. Rall WF. Factors affecting the survival of mouse embryos cryopreserved by vitrification. Cryobiology 1987; 24: 387–402. 21. Rall WF, Reid DS, Polge C. Analysis of slowwarming injury of mouse embryos by cryo-
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microscopical and physiochemical methods. Cryobiology 1984; 21: 106–21. Martino A, Songansen N, Leibo SP. Development into blastocysts of bovine oocytes cryopreserved by ultra-rapid cooling. Biol Reprod 1996; 54: 1059–69. Vajta G, Kuwayama M, Holm P, Booth PJ, Jacobsen H, Greve T et al. Open pulled straw (OPS) vitrification: a new way to reduce cryoinjuries of bovine ova and embryos. Mol Reprod Dev 1998; 51: 53–8. Isachenko V, Alabart JL, Nawroth F, Isachenko E, Vajta G and Folch J. The open pulled straw vitrification of ovine GV-oocytes: positive effect of rapid cooling or rapid thawing or both? Cryo Letters 2001; 22: 157–62 . Isachenko V, Folch J, Nawroth F, Krivokharchenko A, Vajta G, Dattena M, et al. Double vitrification of rat embryos at different developmental stages using an identification protocol. Theriogenology 2003; 60: 445–52. Isachenko V, Selman H, Isachenko E, Montag M, El-Danasouri I, Nawroth F. Modified vitrification of human pronuclear oocytes: efficacy and effect on ultrastructure. Reprod Biomed Online 2003; 7: 211–6. Isachenko V, Montag M, Isachenko E, Nawroth, Dessole S, Van der Ven H. Developmental rate and ultrastructure of vitrified human pronuclear oocytes after step-wise versus direct rehydration. Hum Reprod 2004; 19: 660–5. Szell A, Zhang J, Hudson R. Rapid cryopreservation of sheep embryos by direct transfer into liquid nitrogen vapour at –180°C. Reprod Fertil Dev 1990; 2: 613–8. Rall WF. Advances in the cryopreservation of embryos and prospects for application to the conservation of salmonid fishes. In: Cloud JG, Thorgaard GH, eds. Genetic Conservation of Salmonid Fishes. New York: Plenum Press, 1993: 137–58. Rall WF, Wood MJ. High in vitro and in vivo survival of day 3 mouse embryos vitrified in a non-toxic solution of glycerol and albumin. J Reprod Fertil 1994; 101: 681–8. Dinnyes A, Dai Y, Jiang S, Yang X. High developmental rates of vitrified bovine oocytes following parthenogenetic activation, in vitro fertilization, and somatic cell nuclear transfer. Biol Reprod 2000; 63: 513–8. Vajta G, Holm P, Greve T, Callesen. Factors affecting survival rates of in vitro produced bovine embryos after vitrification and direct in-straw rehydration. Anim Reprod Sci 1996; 45: 191–200.
33. Mukaida T, Nakamura S, Tomiyama T et al. Wada S, Oka C, Kasai M et al. Vitrification of human blastocysts using cryoloops: clinical outcome of 223 cycles. Hum Reprod 2003; 18: 384–91. 34. Yokota Y, Yokota H, Yokota M, Sato S, Araki Y. Birth of healthy twins from in vitro development of human refrozen embryos Fertil Steril 2001; 76: 1063–5. 35. Isachenko V, Perez-Sanchez F, Isachenko E, Grishchenko V, Soler C. Vitrification of GVporcine oocytes with intact intracellular lipids: effect of the cryoprotectant saturation/dilution stepping, elevated temperature and cytoskeletal inhibitor. Cryobiology 1998; 36: 250–3. 36. Isachenko V, Gorbunov L, Isachenko E, Ostashko F, Bezugly N. Some physical and technological aspects of GV-porcine oocyte vitrification. Cryobiology 1999; 39: 35. 37. Lane M, Bavister BD, Lyons EA, Forest KT. Containerless vitrification of mammalian oocytes. Nat Biotechnol 1999; 17: 1234–6. 38. Lane M, Gardner DK. Vitrification of mouse oocytes using a nylon loop. Mol Reprod Dev 2001; 58: 342–7. 39. Vanderzwalmen P, Bertin G, Debauche Ch, Standaert V, Bollen N, Rosendaal van E et al. Vitrification of human blastocysts with hemistraw carrier: application of assisted hatching after thawing. Hum Reprod 2003; 18: 1504–11. 40. Son WY, Yoon SH, Yoon HJ, Lee SM, Lim JH. Pregnancy outcome following transfer of human blastocysts vitrified on electron microscopy grids after induced collapse of the blastocoele. Hum Reprod 2003; 18: 137–9. 41. Isachenko V, Montag M, Isachenko E, Zaeva V, Krivokharchenko I, Shafei R, et al. Aseptic technology of vitrification of human pronuclear oocytes using open-pulled straws. Hum Reprod 2005; 20: 492–6. 42. Isachenko V, Montag M, Isachenko E, Van der Ven H. Vitrification of mouse pronuclear embryos after polar body biopsy without direct contact with liquid nitrogen. Fertil Steril 2005; 84: 1011–6. 43. Isachenko V, Montag M, Isachenko E, Dessole S, Nawroth F, Van der Ven H. Aseptic vitrification of human germinal vesicle oocytes using dimethyl sulfoxide as a cryoprotectant Fertil Steril 2006; 85: 741–7. 44. Cremades N, Sousa M, Silva J, Viana P, Sousa S, Olivera C, et al. Experimental vitrification of human compacted morulae and early blastocysts using fine diameter plastic micropipettes. Hum Reprod 2004; 19: 300–5.
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45. Kuleshova LL, Shaw JM. A strategy for rapid cooling of mouse embryos within a double straw to eliminate the risk of contamination during storage in liquid nitrogen. Hum Reprod 2000; 15; 2604–9. 46. Isachenko V, Isachenko E, Katkov II, Montag M, Dessole S, Nawroth F, et al. Cryoprotectantfree cryopreservation of human spermatozoa by vitrification and freezing in vapor: effect on motility, DNA integrity, and fertilization ability. Biol Reprod 2004; 71: 1167–73. 47. Bielanski A, Nadin-Davis S, Sapp T, LutzeWallace C. Viral contamination of embryos cryopreserved in liquid nitrogen. Cryobiology 2000; 40: 110–16. 48. Tedder RS. Hepatitis B nucleotide sequence analysis: linking an outbreak of acute hepatitis B to contamination of a cryopreservation tank. J Virol Methods 1996; 60: 818. 49. Hawkins AE, Zuckerman MA, Briggs M, Gilson RJ, Goldstone AH, Brink NS et al. Hepatitis B nucleotide sequence analysis: linking an outbreak of acute hepatitis B to contamination of a cryopreservation tank. J Virol Methods 1996; 60: 81–8. 50. Charles GN, Sire DJ. Transmission of papova virus by cryotherapy applicator. JAMA 1971; 218: 1435. 51. Schaffer TW, Everett J, Silver GH, Came PE. Biohazard potential: recovery of infectious virus from the liquid nitrogen of a virus repository. Health Lab Sci. 1976; 13: 23–4. 52. Jones SK, Darville JM. Transmission of virusparticles by cryo-therapy and multi-use caustic pencils: a problem to dermatologist? Br J Dermatol 1989; 121: 481–6. 53. Yokota Y, Sato S, Yokota M, Araki Y. Birth of a healthy baby following vitrification of human blastocysts. Fertil Steril 2001; 75: 1027–9. 54. Huang CC, Lee TH, Chen SU, Chen HH, Cheng TC, Liu CH et al. Successful pregnancy following blastocyst cryopreservation using super-cooling ultra-rapid vitrification. Hum Reprod 2005; 20: 122–8. 55. Katayama KP, Stehlik J, Kuwayama M, Kato O, Stehlik E. High survival rate of vitrified human oocytes results in clinical pregnancy. Fertil Steril 2003; 80: 223–4. 56. Vincent C, Pickering SJ, Johnson MH, Quick SJ. Dimethylsulphoxide affects the organisation of microfilaments in the mouse oocytes. Mol Reprod Dev 1990; 26: 227–35. 57. Bouquet M, Selva J, Auroux M. Effect of cooling and equilibration in DMSO, and cryopreservation of mouse oocytes, on the rates of in vitro fertilization, development and
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chromosomal abnormalities. Mol Reprod Dev 1995; 40: 110–5. Edwards RG, Brody SA. Principles and Practice of Assisted Human Reproduction. Philadelphia: WB Saunders 1995: 425–518. Jones GM, Trounson AO, Gardner DK. Evolution and culture protocol for successful blastocyst development and pregnancy. Hum Reprod 1998; 13: 169–77. Gardner DK. Development of serum-free media for the culture and transfer of human blastocysts. Hum Reprod 1998; 13: 218–85. Liebermann J, Tucker MJ. Comparison of vitrification and conventional cryopreservation of day 5 and day 6 blastocysts during clinical application. Fertil Steril 2006; 86: 20–6. Vanderzwalmen P, Bertin G, Debauche Ch, Standaert V, van Roosendaal E, Vandervorst M et al. Births after vitrification at morula and blastocyst stages: effect of artificial reduction of the blastocoelic cavity before vitrification. Hum Reprod 2002; 17: 744–51. Zech NH, Lejeune B, Zech H, Vanderzwalmen P. Vitrification of hatching and hatched human blastocysts: effect of an opening in the zona pellucida before vitrification. Reprod Biomed Online 2005; 11: 355–61. Kuwayama M, Vajta G, Ieda S, Kato O. Comparison of open and closed methods for vitrification of human embryos and the elimination of potential contamination. Reprod Biomed Online 2005; 11: 608–14. Vanderzwalmen P, Lejeune A, Stecher N, Zech N, Delval A, Zech H. Survival of day 3 and day 5 embryos following vitrification in aseptic and non-aseptic conditions: a prospective randomised analysis. Hum Reprod 2005; 84 (Suppl 1): 185. Larman MG, Sheehan CB, Gardner DK.Vitrification of mouse pronuclear oocytes with no direct liquid nitrogen contact. Reprod Biomed Online 2006; 12: 66–9 Nagashima H, Kashiwazaki N, Ashman R J, Grupen CG, Nottle MB. Cryopreservation of porcine embryos. Nature 1995; 374: 416. Quinn P. Principles of membrane stability and phase behaviour under extreme conditions. J Bioenerg Biomembr 1989; 21: 3–19. Dvorak M. Ultrastructure and quantitative analysis of mouse and human oocytes. In: Liss AR, ed. Developments in Ultrastructure of Reproduction. New York: Academic Press, 1989: 273–280. Sathananthan AH, Kirby C, Peura A, Trounson A. Mouse oocyte cooling. J Assist Reprod Genet 1992; 9: 139–48.
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71. Pickering SJ, Braude PR, Johnson MH, Cant A, Currie J. Transient cooling to room temperature can cause irreversible disruption of the meiotic spindle in the human oocyte. Fertil Steril 1990; 54: 102–8. 72. Aman RR, Parks JE. Effects of cooling and rewarming on the meiotic spindle and chromosomes of in vitro-matured bovine oocytes. Biol Reprod 1994; 50: 103–10. 73. Didion BA, Pom D, Martin MJ, Homanics GE, Markert CL. Observation on the cooling and cryopreservation of pig oocytes at the germinal vesicle stage. J Anim Sci 1990; 68: 2803–10.
74. Johnson MH, Pickering SJ. The effect of dimethylsulfoxide on the microtubular system of the mouse oocyte. Development 1987; 100: 313–24. 75. Isachenko V, Michelmann HW, Alabart JL, Vazquez I, Isachenko E, Bezugly N, et al. Lipolyse and ultra-structural changes of an intracellular lipid vesicles after cooling of bovine and porcine GV-oocytes. Anat Histol Embryol 2001; 30: 333–8.
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Vitrification of day 2–3 human embryos: using various techniques (Cryoloop, Cryotop, and conventional cryostraw)
9
Tetsunori Mukaida and Katsuhiko Takahashi
INTRODUCTION The definition of vitrification is the solidification of a solution at a low temperature without the formation of ice crystals, by increasing the viscosity using high cooling rates.1 The rapid cooling process can minimize chilling injury and osmotic shock to the embryos. Vitrification, with recent improvements, has become a reliable strategy, not only because it is very simple but also because it can lead to high survival rates. To induce vitrification in liquid nitrogen, the solution must contain a high concentration of cryoprotectant.2 In vitrification, the selection of cryoprotectants requires extreme care because their concentration can be as high as 6 mol/L, which can make the toxicity of these compounds a key limiting factor in cryobiology. The most appropriate characteristics of a penetrating cryoprotectant are low toxicity and high permeability. For cryopreservation of human embryos, 1,2-propanediol (PROH) and dimethylsulfoxide (DMSO) have been used as the dominant cryoprotectants, although glycerol is used when embryos are frozen at the blastocyst stage.3 As a less toxic cryoprotectant, ethylene glycol is commonly and widely used.2 However, few comparative studies have examined the effect of the cryoprotectant on the survival of vitrified embryos. In 1998, we performed an investigation to find a suitable cryoprotectant and suitable conditions for
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exposing embryos to the vitrification solution using 8-cell mouse embryos.4 The survival rates of 8-cell embryos vitrified in various solutions after exposure to the solutions for 0.5 and 2 min at 20 and 25°C are summarized in Figure 9.1. The highest levels of survival were obtained, regardless of the time and temperature, with ethylene glycol-based solutions. Although none of the vitrified embryos were morphologically normal when embryos were vitrified after 0.5 min exposure to any mixture of 30% cryoprotectant, the survival rate was over 90% when embryos were treated for a longer time (2 min) at a higher temperature (25°C), or when embryos were treated with a higher concentration of ethylene glycol (EFS40) at a higher temperature (25°C). In addition, a small saccharide (e.g. sucrose) and a macromolecule (e.g. Ficoll 70, bovine serum albumin (BSA), or polyvinylpyrrolidone (PVP)) are frequently included in vitrification solutions. These non-permeating agents are much less toxic, and are known to promote vitrification of the solution.5 Therefore, their inclusion can reduce the toxicity of the solution by decreasing the concentration of the permeating agent required for vitrification. In addition, inclusion of a saccharide promotes shrinkage of embryos, and thus reduces the amount of intracellular cryoprotectant, which will also reduce the toxic effect of the permeating cryoprotectant.5 At the same time, the osmotic action of a saccharide plays an
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100
*
**
P: PROH D: DMSO
Survival rate (%)
80
G: Glycerol E: Ethylene Glycol
60
A: Acetamide
40
20
**
**
0 PFS
DFS
GFS
EFS
30%
AFS
40%
Figure 9.1 Survival of vitrified 8-cell mouse embryos, assessed by their ability to develop into expanded blastocysts, expressed as the percentage of morphologically normal embryos at recovery for each vitrification solution, regardless of the time and temperature of exposure. Vitrification solutions contain 30% (closed bar) or 40% (hatched bar) permeating cryoprotectant. All the concentrations were diluted with FS solution (PB1 medium containing 30% Ficoll + 0.5 mol/L sucrose). AFS, acetamide-based solution; PFS, propanediol-based solution; DFS, dimethylsulphoxide-based solution; GFS, glycerol-based solution; EFS, ethylene glycol-based solution. **P<0.01, *P<0.05; significantly different within solutions containing the same permeating cryoprotectant. Reproduced with permission from Mukaida et al.4
important role in minimizing the swelling of embryos during dilution, since a quick dilution is necessary to prevent the toxic effect of the solution. The basic procedure for vitrification is simple. Embryos are suspended in a vitrification solution and then plunged in liquid nitrogen. The sample is warmed rapidly and diluted quickly with a sucrose solution. The most important process is the exposure of embryos to the vitrification solution before cooling. To prevent intracellular ice from forming, a longer period of exposure is desirable. However, if the exposure is too long, cells suffer from the toxicity of the solution. Therefore, the optimal exposure time for successful vitrification must be a compromise between preventing the formation of intracellular ice and preventing toxic injury. Actually, however, embryos may be injured by the toxicity of the cryoprotectant before sufficient
cryoprotectant can permeate the cells. Therefore, a two-step procedure is commonly adopted, in which embryos are first equilibrated in a dilute (e.g. 10%) cryoprotectant solution followed by a brief (30–60 s) exposure to a vitrification solution before the sample is cooled with liquid nitrogen. The optimal exposure time in the vitrification solution depends not only on the cryoprotectant solution but also on the temperature, since both the permeability of embryos and the toxicity of the cryoprotectant are largely influenced by the temperature.6,7
GENERAL METHODOLOGIES FOR VITRIFICATION For vitrification, a solution must be prepared which does not form ice in liquid nitrogen. Vitrification solutions are made by adding 30–50% (v/v) cryoprotectant in a physiological
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solution such as Hepes HTF or phosphate buffered saline (PBS). In a few cases, embryos are suspended in a vitrification solution directly from a physiological solution (one-step method).5 In most cases, however, embryos are first suspended in a solution containing a lower concentration (10–20%) of permeating cryoprotectant to promote permeation of the cryoprotectant under less toxic conditions; then they are suspended in the vitrification solution (two-step method). Embryos are loaded in a container, cooled with liquid nitrogen, and preserved in it. The time embryos are suspended in the vitrification solution before cooling is critical. During the suspension, embryos need to be dehydrated and concentrated, but this usually occurs quickly. Thus the exposure time must be minimized (~30 s) to prevent injury from the toxicity of the cryoprotectant. As the container, 0.25 mL cryostraws have been conventionally used.4 However, to intensify the rates of cooling and warming, various minute tools have been used, such as small grids,8 thin capillaries,9 tiny loops,10 and small plastic sticks.11 For embryo recovery, vitrified embryos are warmed rapidly and the embryos are suspended in a solution containing sucrose. In solution, sucrose prevents over-swelling of embryos as the permeated cryoprotectant diffuses out. After this diffusion has been confirmed by embryo shrinkage, the embryos are recovered in a physiological solution. In this chapter, three different approaches are described including the conventional Cryostraw, Cryotop, and Cryoloop techniques.
PROTOCOLS AND RESULTS Vitrification using conventional cryostraws A two-step protocol for straw vitrification using ethylene glycol-based solutions, EFS20 and EFS40, is described.12 This method has been proven available for human embryos on day 2–34 (Mukaida et al., unpublished data).
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The two solutions (EFS20 and EFS40) are used for pretreatment and vitrification, respectively. The base medium used for vitrification of embryos is modified phosphate-buffered saline (PB1), in which BSA is replaced with human serum albumin (HSA). Ethylene glycol is diluted to 20% (v/v) or 40% (v/v) with Ficollsucrose (FS) solution; the components of the FS solution are 30% (w/v) Ficoll 70 (average molecular weight 70 000; Amersham Pharmacia Biotech, Buckinghamshire, UK), and 0.5 mol/L sucrose in PB1 medium. The respective vitrification solutions are designated EFS20 and EFS40. The final concentrations of Ficoll 70 and sucrose are 24% (w/v) and 0.4 mol/L, respectively, in EFS20, and 18% (w/v) and 0.3 mol/L, respectively, in EFS40. For dilution, PB1 medium containing 0.5 mol/L sucrose (S-PB1) is prepared. All the solutions are placed in a room at 25–27°C, at which temperature embryos are manipulated. A 0.25 mL plastic straw (~132 mm including the cotton plug) is prepared for embryo loading by drawing S-PB1 medium up to a depth of ~60 mm, followed by air (~25–30 mm), EFS40 (~5 mm), another volume of air (~5 mm), and finally more EFS40 (~12 mm). First, embryos are pretreated by being suspended in a drop of EFS20 in the lid of a culture dish (or a dish) for 2 min. Then, embryos are transferred into the larger column of FES40 near the mouth of the straw. The contents of the straw are aspirated until the first column of S-PB1 medium is in contact with the cotton plug, and the straw is sealed with the heat-sealer. After exposure of embryos to EFS40 for ~30 s, the straw is positioned in the liquid nitrogen vapor phase by placing it horizontally on a ~1 cm thick Styrofoam boat floating on the surface of the liquid nitrogen in a Dewar vessel (inner diameter, 140 mm). After 3 min or more, the straw is placed in a canister and stored in liquid nitrogen. EFS40 and EFS20 are prepared in 1 mL syringes equipped with 18 G needles, and new small drops are placed on the lid of a dish just
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before use for each sample, to prevent concentration of the solution by evaporation. For embryo recovery, the straw is kept in air for 10 s and then immersed in water at 25–28°C. When the crystallized S-PB1 medium in the straw begins to melt (after about 7 s), the straw is removed from the water, quickly wiped dry, and cut at both ends. The contents of the straw are expelled into a watch glass (or a culture dish) by flushing the straw with 0.8 mL of S-PB1 medium using a 1 mL syringe attached with an 18 G needle. After gently agitating the watch glass to promote mixing of the contents, the embryos are pipetted into fresh S-PB1 medium. About 5 min after being flushed out, the embryos are transferred to fresh PB1 medium. Embryos are further washed with fresh PB1 medium, and are transferred to a culture medium for culture until transfer. In 1998, Mukaida et al.4 reported the effectiveness of this vitrification method for day 2–3 human embryos, more trials on this straw vitrification were performed in the HART Clinic group (Hiroshima HART Clinic, Osaka HART Clinic, and Tokyo HART Clinic), and its effectiveness was confirmed. In our unpublished data for day 2–3 embryos, a total of 661 embryos were vitrified, and 486 (74%) of them had 50% or more morphologically intact blastomeres after warming which was confirmed by further development of these vitrified embryos on the day after warming. A total of 335 vitrified embryos were transferred in 127 cycles at 1 or 2 days after warming, resulting in 34 (26.8%) women becoming pregnant, and 22 (17%) women delivering babies.
Vitrification using the Cryoloop Here we describe an ultrarapid vitrification approach using the Cryoloop.13–15 This method is available not only for embryos on day 2–3, but also for blastocysts, for which straw vitrification was found to be less effective. The protocol for vitrification using the
Cryoloop can be found in the chapter of vitrification of blastocysts (Chapter 10B), which is basically the same. The Cryoloop consists of a tiny nylon loop (20 µm wide, 0.5–0.7 mm in diameter) mounted on a small stainless steel tube inserted into the lid of a cryovial (Hampton Research, Laguna Niguel, CA, USA).16 A metal insert on the lid enables the use of a stainless steel handling rod with a small magnet (CrystalWand With Tab; Hampton Research) for manipulation of the loop at low temperature (liquid nitrogen). Cryoprotectant solution I and cryoprotectant solution II are used for pretreatment and vitrification, respectively. The cryoprotectant solutions are made with a base medium consisting of HEPES-buffered human tubal fluid (HTF) medium containing 5 mg/mL of HSA. Cryoprotectant solution I consists of base medium containing 1.07 mol/L DMSO and 1.36 mol/L ethylene glycol, and cryoprotectant solution II consists of base medium containing 2.08 mol/L DMSO, 2.64 mol/L ethylene glycol, 10 mg/mL Ficoll 70, and 0.65 mol/L sucrose. Both cryoprotectant solutions (1–1.2 mL) are placed in a 4-well culture dish and warmed in an incubator at 37°C for ~30 min. Embryos are manipulated on a plate warmed at 37°C in a room at 25–27°C, and thus presumably at ~37°C. Initially, embryos are suspended in cryoprotectant solution I. At 2 min after suspension, the embryos are washed quickly in small drops of solution II on the lid of a culture dish. Quickly, a Cryoloop is dipped into cryoprotectant solution II to create a thin filmy layer of the solution on the nylon loop, and the embryos are transferred onto the thin filmy layer on the nylon loop using a micropipette. Within 30 s of suspension in solution II, the Cryoloop is plunged into liquid nitrogen. The time the embryos are kept on the loop with the thin layer of solution in the air should be as short as possible, because evaporation will make the solution more concentrated. Using the stainless steel rod, the loop containing the
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Table 9.1 Summary of the protocols regarding concentration, time, and properties of the vitrification solution for day 2–3 human embryo cryopreservation Cryostraw
Cryoloop17
Cryoloop18
Cryotop
Temperature
Room (25–27°C)
Warm stage (37°C)
Warm stage (37°C)
Room (25–27°C)
Equilibration step
EGFS 20: 20% EG + F + S (2 min)
7.5% EG + 7.5% DMSO (2 min)
10% EG (5 min)
7.5% EG + 7.5% DMSO (5–10 min*)
Vitrification step
EGFS 40: 40% EG + F + S (1 min)
15% EG + 15% DMSO + F + S (35 s)
40% EG + S (30 s)
15% EG + 15% DMSO + S (1 min)
Cooling system
Vapor phase above LN2 (3 min), then plunged into LN2
Plunged into LN2 directly (ultra-rapid cooling)
Plunged into LN2 directly (ultra-rapid cooling)
Plunged into LN2 directly (ultra-rapid cooling)
Warming step
One step: 0.5 mol/L S (5 min)
Two steps: Four steps: 0.25 mol/L S (2 min) 1 mol/L S (2.5 min), 0.12 mol/L S (3 min) 0.5 mol/L S (2.5 min), 0.25 mol/L S (2.5 min), 0.125 mol/L S (2.5 min)
Two steps: 1 mol/L S (1 min) 0.5 mol/L S (3 min)
*
Duration of equilibration is adjusted according to the time needed for re-expansion of the vitrified embryos. EG, ethylene glycol; F, Ficoll; S, sucrose. Data from Desai et al.17 and Rama Raju et al.18
embryos is sealed in a cryovial, which has been previously submerged in liquid nitrogen. The vial is attached to a standard cane and stored in liquid nitrogen. The whole procedure is completed within 5 min. For recovery, the cryovial is opened with the aid of the stainless steel rod. Then the loop containing the embryos is removed from the liquid nitrogen and placed quickly and directly into a well of the base medium containing 0.33 mol/L sucrose at ~35°C. The embryos never fail to float off the loop into the solution; thus, the embryos are almost always successfully recovered. After 2 min, embryos are transferred to the base medium containing 0.2 mol/L sucrose. After an additional 3 min, embryos are suspended in base medium for 5 min. Finally, they are transferred into culture medium for further culturing until transfer. At the HART Clinic group, Cryoloop vitrification is adopted for cryopreservation of supernumerary human embryos on day 2–3 for a short period of time, and mainly for
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blastocysts on day 5–6 that were obtained from culture in sequential media. Available data show that for embryos on day 2–3, a total of 269 embryos have been vitrified, and 188 (70%) of them had 50% or more morphologically intact blastomeres which was confirmed by further development on the day after warming. A total of 112 vitrified embryos were transferred into 44 patients, and 14 (32%) of these women conceived (Mukaida et al., unpublished data). In 2007, Desai et al.17 reported the postvitrification development, pregnancy outcomes, and live birth rates for Cryoloop vitrification of human day 3 cleavage-stage embryos. Table 9.2 includes their results that represent consecutive vitrification-warming cycles performed over a 2.5-year interval. A total of 236 embryos were warmed, and the average number of embryos transferred per patient was 2.66 ± 0.86. They reported that the clinical pregnancy rate was 44% (34/77), and the implantation rate was 20% (40/201). The postwarming survival rate was 85% (201/236).
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Table 9.2
Summary of the clinical results in each vitrification approach for day 2–3 embryos Cryostraw
Age No. of cycles
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Survival rate Cleavage rate* Pregnancy rate Implantation rate Delivery rate**
486/661 76% 34/127 26.8%
Cryoloop (1)
Cryoloop (2)
Cryotop
34.1 ± 4.5 77
31.3 ± 4.5 40
201/236 85% 184/236 78% 34/77 44.2% 40/201 19.9%
121/127 95%
35.0 ± 4.5 604 346 patients. 1701/1774 95.9% 1289/1774 72.7% 164/604 27.2% 192/1442 13.3% 118/604 19.5%
22/127 17%
14/40 35.0% 18/121 14.9% 13/40 32.5%
*
Including survival and further cleavage rate. Including on-going pregnancy. Cryoloop (1): Reported by Desai in 2007. Cryoloop (2): Reported by Raju in 2005. **
They also reported that 78% (184/236) of warmed embryos showed signs of embryonic compaction and/or blastulation by the time of transfer, indicating that the surviving embryos were confirmed as potentially viable. Theoretically, vitrification does not involve ice crystal formation, and survival judged by the morphological appearance may not always be related to viability. Therefore, confirmation of further development is necessary and is an important factor to evaluate after warming. In 2005, Rama Raju et al.18 reported a modified protocol for vitrification of human 8-cell embryos using the Cryoloop technique. They reported using 10% ethylene glycol (EG) for 5 min at 37° C as an equilibration phase, and 40% EG in 0.6 mol/L sucrose for 30 s as a vitrification phase. Human 8-cell embryos were loaded onto a nylon loop made in their biomechanical department at the Krishna IVF Clinic. The nylon loop with a thin filmy layer of vitrification solution and vitrified embryos were directly placed into a cryovial containing liquid nitrogen. Loading and storage steps were similar to our Cryoloop protocol described above, however, the protocol including the type of cryoprotectant and
duration of exposure were different. Initially embryos were suspended in a 10% ethylene glycol solution for 5 min at 37°C, and transferred to a 40% ethylene glycol in 0.6 mol/L sucrose solution for 30 s. For warming, vitrified embryos were passed through four different concentrations of sucrose solution, 1, 0.5, 0.25, and 0.125 mol/L, for 2.5 min at each step at 37°C. Table 9.2 includes their results. Mean age of patients was 31.3 ± 4.5 years, the post-thaw survival rate of embryos was 95.3%, and the clinical pregnancy rate and implantation rates were 35.0% and 14.9%, respectively.
Vitrification using the Cryotop Since in our center (Hiroshima HART clinic) the Cryoloop system has only been applied for the cryopreservation of embryos at the blastocyst stage, the protocol and data of vitrification using Cryotops in this chapter were adopted from the study carried out at the IVF Nagata Clinic, Hakata, Japan. Their study was originally presented as an abstract at the 2006 American Society of Reproductive Medicine meeting in New Orleans, USA, and is now
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being prepared for publication including their intensive analysis.19 Their protocol and data of vitrification were based on 346 women who needed to have their embryos cryopreserved on day 2 of development to avoid either ovarian hyperstimulation syndrome (OHSS), or as supernumerary embryos for subsequent transfer attempts, who agreed to cryopreservation of the embryos by vitrification. A total of 1774 day-2 embryos were vitrified with equilibration solution, an equal mixture of 7.5% DMSO and 7.5% ethylene glycol (EG) in HTF supplemented with 20% HSA, and a vitrification solution, using a mixture of 15% DMSO, 15% EG, and 0.5 mol/L sucrose in HTF/HSA. Initially, embryos were exposed to the equilibration solution for 5–10 min, and to the vitrification solution for 1 min at room temperature (25–27°C). The duration of the equilibration time was adjusted by morphological changes that indicated shrinkage for dehydration and re-expansion for cryoprotectant (CPA) permeation, and was individually recorded for further analysis. Embryos were then loaded onto a minute nylon sheet (Cryotop), and plunged into liquid nitrogen immediately. For warming, vitrified embryos on the tip of a Cryotop were dipped and kept in 1 mol/L sucrose solution for 1 min and then diluted in 0.5 mol/L sucrose solution for 3 min. Embryos with 70% or more intact blastomeres were considered as indicative of survival and were kept in culture until transfer on the following day. Table 9.2 includes the results from the use of the Cryotops at Nagata Clinic. Briefly, 346 patients with day-2 embryos that were to be cryopreserved to avoid either OHSS, or as supernumerary embryos for subsequent transfer attempts, were entered in this investigation. A total of 1774 day 2 embryos were warmed in 604 transfer cycles. Of the vitrified embryos 1701 survived, and 1442 of them were transferred. Pregnancy was confirmed in 164 cycles and 192 embryos implanted as confirmed by the presence of a gestational
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sac. Forty six cycles ended in miscarriage and 67 deliveries were achieved; other cases are ongoing pregnancy. Total survival and cleavage rates were 95.9% (1701/1774) and 75.8% (1289/1701), respectively. The mean number of embryos transferred was 2.4 ± 0.7, the pregnancy rate was 27.2% (164/604), the implantation rate was 13.3% (192/1442), and the abortion rate was 28.0% (46/164). Ninety nine healthy babies were born following 84 deliveries (49 boys and 50 girls).
TROUBLE SHOOTING Conventional vitrification (1) Room temperature other than 25°C. The temperature for handling embryos in a cryoprotectant solution has significant effects. When the room temperature is higher, the duration of exposure of embryos to EFS40 must be shortened and vice versa. Elevating the temperature is preferable for promoting the permeation of the cryoprotectant, but the optimal range of the exposure time decreases because the toxicity of the cryoprotectant increases considerably. For the protocol described (at 25°C), keep the lights off so as not to elevate the temperature of the microscope stage when embryos are not being manipulated under the microscope. Avoid warming the EFS40 column of the straw with one’s fingers. (2) Cooling in a small dewar flask. If only a small dewar flask is available, immerse half of the straw containing the embryos in liquid nitrogen first, and then cool the rest of the straw slowly in nitrogen vapor. If the whole straw is immersed in liquid nitrogen in one step, it may rupture because of the rapid increase in the volume of the freezing sucrose solution. By this method, a small percentage of vitrified embryos may suffer fracture damage.
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side of the tip should be clearly marked in order to identify its side at the time of warming.
Ultrarapid vitrification: Cryoloop (1) During the step of equilibration, morphological change can be an important factor to understand the degree of permeation of cryoprotectant and the degree of dehydration. Two minutes duration is approximate, and the duration of equilibration may need to be adjusted depending on the morphological changes similar to when using the Cryotop protocol. (2) Preventing evaporation of the vitrification solution. Because the amount of vitrification solution on the Cryoloop is very small, the solution is liable to be concentrated by evaporation, which increases its toxicity. To prevent this, the Cryoloop should be loaded with the vitrification solution just before the loading of embryo(s) on the loop. The amount of vitrification solution on the loop need not be minimal, as long as the drop is kept on the loop by surface tension. A dewar flask containing liquid nitrogen should be placed nearby to enable quick cooling. (3) Quick warming. At warming, the loop containing the embryo(s) should be soaked in the sucrose solution as quickly as possible, making sure the sample is never held in the air. Again, a dewar flask containing liquid nitrogen and the sucrose solution should be placed nearby. When the loop is dipped in the sucrose solution, the steel pipe portion should not be immersed to prevent bubbling. (4) Morphology of the recovered blastocyst. At recovery in an isotonic solution, the blastocoel of the blastocyst will be collapsed and the embryo may look like a ‘morula’. However, the blastocoelic cavity should reform within 1–3 h of culture.
Ultrarapid vitrification: Cryotop (1) When the Cryotop system is used to load the embryos for vitrification, the loaded
(2) Tip of the Cryotop should be capped tightly into the capping part in a dewar flask containing liquid nitrogen, and confirmation of tight capping should always be made before transferring back to the storage tank. (3) Size and volume of vitrification on the tip of Cryotop should be minimized, however, if the volume is too small, the vitrified embryo may occasionally be stucked on the surface of the tip. (4) At the step of warming, the tip of the Cryotop should always be inside the warming solution. (5) Prevention of evaporation of the vitrification solution on the tip. The drop with the embryos should be loaded on the tip of Cryotop just before plunging into liquid nitrogen. This is the same precaution as for Cryoloop vitrification. Because the amount of vitrification solution on the tip of Cryotop is very small, evaporation of the solution increases its toxicity. A dewar flask containing liquid nitrogen should be placed nearby to enable quick cooling and warming.
DISCUSSION Numerous protocols for the cryopreservation of mammalian embryos have been reported. The protocols can be classified into four methods, original slow freezing, conventional slow freezing, conventional vitrification using the conventional cryostraw, and ultrarapid vitrification using a minute tool. Although strategies to circumvent various injuries (especially from the formation of intracellular ice) are different, the principle of cryopreservation is the same. The most suitable protocol should be adopted for each case. For certain types of embryos, such as
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human blastocysts and bovine embryos at earlier stages, ultrarapid vitrification will be the preferred choice, because the survival rates of embryos cryopreserved by other methods have been low. For other embryos, e.g. mouse embryos, bovine blastocysts, and human embryos at 2–8-cell stages, both slow freezing and conventional vitrification have been proven effective. However, vitrification has a potential advantage in that higher survival rates can be obtained if conditions, such as temperature and duration of exposure of embryos to the cryoprotectant, as well as the skill of pipetting, are optimized. Therefore, vitrification would be a preferred method of cryopreservation compared with the slowcooling method because of the lack of ice crystal formation and its convenience. In this chapter, three different approaches of vitrification were compared for the cryopreservation of human day 2–3 embryos. Table 9.1 summarizes the differences in characteristics with each vitrification approach. In the Cryoloop approach, temperature is always set at 37° C using a stage warmer. On the other hand, the cryostraw and Cryotop techniques are performed at room temperature. Higher temperatures can stimulate the plasma membrane permeability to permeating cryoprotectants and water. For the equilibration and vitrification phase, either EG only or an EG + DMSO mixture is used as the permeating cryoprotectant. The cryostraw approach had the highest concentration of cryoprotectant (20%, 40%) compared with other approaches, because the cooling rate in the vapor phase of liquid nitrogen might not be high enough to avoid ice crystal formation with relatively low concentrations of cryoprotectants. Theoretically, the flexible time needed according to the morphological changes during the equilibration and vitrification steps may not be realistic, as it might become highly subjective due to the individual judgment of each embryologist, and may not achieve adequate full equilibration. Furthermore, in clinical ART laboratories consistent and reproducible
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protocols are important. This might be one of the reasons why fixed durations are commonly applied. Figure 9.1 shows that EG-based solutions obtained the highest level of survival, regardless of the time and temperature. However, this result was not obtained with human embryos, but with mouse 8-cell embryos. The equilibration phase (10% EG for 5 min) of Rama Raju’s group18 approach to vitrification may be the most appropriate strategy based on that study. The cryostraw has a disadvantage in terms of cooling rate because of the thermal insulating layer of the cryostraw, and the larger volume of vitrification solution required. However, direct contact of liquid nitrogen with minimal volume of vitrification solution as with the Cryoloop and Cryotop system can create extremely high cooling rates that can avoid ice crystal formation, even with a relatively lower level of concentration of cryoprotectant that would cause devitrification in classical cooling steps. Warming is the step for removing the permeating cryoprotectant inside the vitrified embryos and rehydrating the embryos. In order to facilitate this step, an osmotic gradient of sucrose concentration is necessary. Rama Raju’s vitrification18 using a Cryoloop has four steps of sucrose concentration. The concentration of sucrose at 1.0 mol/L is relatively high compared with other protocols that have been reported. However, a higher concentration of sucrose would be better in order to prevent osmotic swelling during the warming steps, unless warmed embryos start collapsing. Ideally, the duration of the warming steps can also be recommended to adjust the morphological appearance of the vitrified embryos, and the sucrose concentration should be maintained until the embryos start to shrink. We believe more frequent gradient steps of sucrose with adjustment of the duration according to individual evaluation can improve the survival and viability after warming; however, this may not be practical during daily routine clinical ART work. Table 9.2 shows the comparison of clinical results from each vitrification protocol. The
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protocols of Rama Raju et al.18 using the Cryoloop and Cryotop vitrification had similar high survival rates (95%). However, vitrification theoretically does not involve ice crystal formation, and morphological intactness (survival) is not equivalent to viability. With vitrification, vitrified embryos with sufficient equilibration of high concentration of cryoprotectant generally show a clear and transparent appearance because no ice crystal formation has occurred, and confirmation of further development is an important factor to evaluate after warming. Therefore, cleavage rate is related to real viability after warming. All of the protocols except for that of Rama Raju et al.18 using the Cryoloop showed 72–78% survival rates which are clinically acceptable. In the protocol of Rama Raju et al.18 using the Cryoloop, vitrified embryos after warming were cultured only for 3–4 hours before transfer. In this case, it is difficult to evaluate further growth of embryos through cleavage rate. The pregnancy rate varied from 27% to 44% depending on the embryo scoring, and criteria for vitrification and patient background. It seems that the pregnancy rates using the Cryoloop were higher than those using the cryostraw and Cryotop. Implantation and delivery rates were similar among Cryoloop and Cryotop protocols except for the cryostraw protocol. This suggests that ultrarapid cooling with direct contact of liquid nitrogen seems to be more effective than conventional cooling with use of the vapor phase of liquid nitrogen with the cryostraw as a carrier. An extremely high cooling rate is one of the most important factors for improving the effectiveness of vitrification, because it allows a reduction in the concentration of cryoprotectants to even lower levels that will create ice crystal formation (devitrification) during conventional cooling. In this way, lowering the concentration of the cryoprotectants will reduce their toxicity. The volume of the vitrification solution should be minimized by using special carriers such as the Cryoloop, Cryotop, and
other comparable carriers in order to achieve this extremely high cooling rate. We have already reported4 a simple vitrification method using an ethylene glycol-based solution for 4–8-cell human embryos frozen in conventional cryostraws. Moreover, the success of vitrification procedures has recently been increased by techniques that substantially reduce the volume of the vitrification solution. Among such techniques, the Cryoloop and Cryotop are the most refined strategies. A major difference between the Cryoloop and Cryotop, and the conventional cryostraw for vitrification is the cooling/ warming rate. The Cryoloop and Cryotop enable ultrarapid cooling and warming, and this may prevent intracellular ice formation more consistently, since we have observed that human embryos are dehydrated and concentrated more slowly than other types of embryos, suggesting that intracellular ice is more likely to form. The difference between the Cryoloop and Cryotop is only in the way that vitrified embryos are held. In the Cryoloop system, the vitrified embryo is almost floating in the thin filmy layer of the droplet on the nylon loop, and heat conduction to the embryos becomes homogenous. In the Cryotop, the vitrified embryo is placed on the surface of a nylon sheet, and heat conduction might not be so homogenous especially from the sides of the tip. For the cryopreservation of multiple cells or small sizes of tissue, this may cause uneven heat conduction, however, for a smaller number of eggs or embryos, it does not seem to make any difference. In conclusion, for day 2–3 human embryos, vitrification through ultrarapid cooling achieved by direct contact with liquid nitrogen seems preferable, and either EG only or EG + DMSO as cryoprotectants are acceptable. For equilibration prior to vitrification, a two-step approach is enough to obtain acceptable clinical results. Moreover, individual adjustment of cryoprotectant exposure times depending on the morphological change is always better for equilibration compared with the fixed duration
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protocol. Theoretically, the Rama Raju et al.18 protocol for equilibration and warming, adding individual adjustment in each step, will be most appropriate approach based on our review. Finally, maximizing the cooling rate and minimizing the concentration of
cryoprotectants is critical to establish the protocol for vitrification with any stage of embryo, and vitrification of day 2–3 human embryos is more effective than using the slow cooling approach.
References 1. Rall WF, Fahy GM. Ice-free cryopreservation of mouse embryos at −196°C by vitrification. Nature 1985; 313: 573–5. 2. Kasai M. Simple and efficient methods for vitrification of mammalian embryos. Anim Reprod Sci 1996; 42: 67–75. 3. Menezo Y, Sakkas D, Veiga A. Cryopreservation of blastocysts. In: Gardner DK, Lane M, eds. ART and the Human Blastocyst. New York: Springer-Verlag, 2000: 188–95. 4. Mukaida T, Takahashi K, Kasai M et al. Vitrification of human embryos based on the assessment of suitable conditions for 8-cell mouse embryos. Hum Reprod 1998; 13: 2874–9. 5. Kasai M, Komi JH, Machida T et al. A simple method for mouse embryo cryopreservation in a low toxicity vitrification solution, without appreciable loss of viability. J Reprod Fertil 1990; 89: 91–7. 6. Miyake T, Kasai M, Machida T et al. Vitrification of mouse oocytes and embryos at various stages of development in an ethylene glycol-based solution by a simple method. Theriogenology 1993; 40: 121–34. 7. Zhu SE, Kasai M, Machida T et al. Cryopreservation of expanded mouse blastocysts by vitrification in ethylene glycol-based solutions. J Reprod Fertil 1993; 98: 139–45. 8. Martino A, Songsasen N, Leibo SP. Development into blastocysts of bovine oocytes cryopreserved by ultra-rapid cooling. Biol Reprod 1996; 54: 1059–69. 9. Vajta G, Kuwayama M, Callesen H et al. Open Pulled Straw (OPS) vitrification: a new way to reduce cryoinjuries of bovine ova and embryos. Mol Reprod Dev 1998; 51: 53–8. 10. Lane M, Bavister BD, Forest KT et al. Containerless vitrification of mammalian oocytes and embryos. Nat Biotechnol 1999; 17: 1234–36.
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11. Kuwayama M, Vajta G, Kato O et al. Comparison of open and closed methods for vitrification of human embryos and the elimination of potential contamination. Reprod Biomed Online 2005; 11: 300–8. 12. Kasai M. Vitrification: refined strategy for the cryopreservation of mammalian embryos. J Mamm Ova Res 1997; 14: 17–28. 13. Mukaida T, Kasai M, Takahashi K et al. Successful birth after transfer of vitrified human blastocysts with use of a cryoloop containerless technique. Fertil Steril 2001; 76: 618–20. 14. Mukaida T, Oka C, Takahashi K et al. Vitrification of human blastocysts using cryoloops: clinical outcome of 223 cycles. Hum Reprod 2003; 18: 384–91. 15. Mukaida T, Takahashi K, Kasai M et al. Blastocyst cryopreservation: ultrarapid vitrification using cryoloop technique. Reprod Biomed Online 2003; 6: 221–5 16. Lane M, Schoolcraft WB, and Gardner DK. Vitrification of mouse and human blastocysts using a novel cryoloop container-less technique. Fertil Steril 1999; 72: 1073–78. 17. Desai N, Blackmon H, Goldfarb J et al. Cryoloop vitrification of human day 3 cleavage-stage embryos: post-vitrification development, pregnancy outcomes and live births. Reprod Biomed Online 2007; 14: 208–13. 18. Rama Raju GA, Haranath GB, Madan K et al. Vitrification of human 8-cell embryos, a modified protocol for better pregnancy rates. Reprod Biomed Online 2005; 11: 434–37. 19. Tomari Y, Nagata K, Honjo K, Takahara K. Kunitake. IVF Nagata Clinic, Fukuoka, Japan. Data presented to American Society for Reproductive Medicine 2006 annual meeting at New Orleans, LA.
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One decade of experience with vitrification of human embryos in straws, hemi-straws, and high security vitrification straws
10A
Pierre Vanderzwalmen, Thomas Ebner and Nicolas Zech
WHY IS THERE A RENEWED INTEREST IN CRYOPRESERVATION? In the past few decades assisted reproductive technologies (ART) have been used to help couples with infertility problems. With this in view, many techniques have been developed or improved, including ovary stimulation methods, criteria and selection processes to assess the quality and viability of gametes and embryos, as well as culture conditions, and culture media. This has led to being able to obtain significantly more viable and transferable embryos nowadays. The result has been that the proportion of viable and transferable embryos has increased significantly in the past few years. Moreover, awareness of risks linked to multiple pregnancies has required the implementation of standards limiting the number of embryos transferred at any one time. One of these standards, namely cryopreservation of embryos between the zygote and blastocyst stages has become common practice when attempting ART. In 1984, Zeilmaker et al.1 reported the first birth of a healthy child following slow freezing of a 4-cell embryo. Since then, slow-rate freezing techniques have become widely used for the cryopreservation of zygotes and early cleavage stage embryos. For blastocysts, however, the situation looks quite different. In 1985, Cohen
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et al.2 reported the first pregnancy after controlled slow freezing of blastocysts using glycerol as a permeable cryoprotectant in a series of ten increasing solution concentrations. This initial protocol modified by Ménézo et al.3 by adding sucrose as a non-permeable cryoprotectant was considered popular for slow cooling of blastocysts obtained after co-culture.4 In the mid-1990s, interest in blastocyst cryopreservation emerged due to the introduction of commercial sequential and non-sequential media allowing more and more fresh embryo transfers on a large scale at the blastocyst stage. Even though several manuscripts report acceptable implantation rates ranging from 16% to 34%,5–8 it became apparent that, in spite of the fact that high implantation and pregnancy rates were advocated after transfer of fresh blastocysts, slow-freezing strategies for blastocysts had limited applications and were rather inconvenient due to the relatively poor survival rates reported by several ART units.9–11 The decrease of blastocysts available after the slow-freezing procedure resulted in a reduction in the cumulative pregnancy rate per retrieval following the transfer of both fresh and frozen blastocysts. This is one of the main reasons why embryo culture to the blastocyst stage has never become popular. Such variable results with conventional slow-freezing methods justified investigations into alternative approaches.
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VITRIFICATION: AN ALTERNATIVE TO SLOW FREEZING During the various stages of a cryopreservation process, including exposure to cryoprotectants, cooling, storage in liquid nitrogen, re-warming, and return to a physiological solution, it is solely the skill of being able to prevent ice crystal formation that determines the viability of an embryo after re-warming. It is obvious that crystallization is incompatible with any living organism and must be avoided as far as possible. As a consequence, the primary factor which influences whether a cell or an embryo survives cryopreservation is the prevention of intracellular ice formation. There are two general approaches to achieve this aim: the slow-rate freezing12 and vitrification.13 Before initiating slow cooling in a programmable controlled system (0.3°C/min), embryos have to be equilibrated in a solution containing a low concentration (1.5 mol/L) of permeable cryoprotectant. During the following cooling step, a progressive dehydration, shrinkage, and increase in intra-cellular solute concentration occurs due to the rising osmolarity in the extracellular solution as water freezes out. In the most favorable situation, the intracellular concentration of cryoprotectant becomes high enough so that the remaining intracellular unfrozen fraction will vitrify, preventing intracellular ice formation. However, during the cooling process, the formation of intracellular ice crystals is frequent and is thought to be the major cause of ‘cryoinjury’ during re-warming due to aggregation of small ice crystals that can induce irreversible cell damage. In this context vitrification is another cryopreservation strategy which, unlike programmed slow freezing, does not theoretically involve the formation of ice crystals either in the intracellular or in the extracellular space, because the whole sample turns directly to ‘glass’. In contrast to slow freezing, the embryos are exposed to high concentrations of cryoprotectant solutions before being
plunged into liquid nitrogen (LN2) at very high cooling rates (2000–20 000°C/min). As a consequence, the physical injuries caused by the formation of extracellular as well as intracellular ice crystals during the controlled slow-freezing procedure are eliminated. Moreover, and in addition to the absence of ice formation, this technique is carried out quickly and thus makes it possible to avoid problems caused by transitions between non-physiological temperature zones, known as ‘chilling’. However, the toxicity caused by the high concentration of cryoprotectants is considered by many scientists to be a drawback of this technique. Another beneficial aspect of this technique is that it does not require the use of programmed freezing equipment.
WHAT ARE THE REQUIRED CONDITIONS TO ACHIEVE A VITRIFIED STATE? The combination of high concentration of cryoprotectants and an extremely fast cooling and warming rate are two conditions that support the formation of a glassy state. Other parameters that influence the formation of an amorphous state include temperature conduction, the concentration of the cryoprotectant, the volume of the solution, and the biophysical parameters of the embryos.
First condition: high concentration of cryoprotectants in the final solution The type of cryoprotectants Since the development of the first vitrification solution,13 which contained dimethylsulfoxide (DMSO), acetamide, and propylene glycol, numerous cryoprotectant solutions have been tested and reported to be effective. At the present time, the cryoprotectant solutions most widely used for vitrification are composed of agents that penetrate the cell (ethylene glycol (EG), DMSO, 1,2-propanediol
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(PROH), or glycerol), and non-penetrating agents of low molecular weight (sucrose or trehalose). In some cases, polymers with high molecular weights such as polyethylene glycol (PEG, MW 8000), Ficoll (MW 70 000 or 400 000; Sigma), or polyvinyl pyrrolidone (MW 360 000) are also added to the vitrification medium.14
The concentration of cryoprotectants Usually, with vitrification, embryos are exposed to increasing concentrations of permeable and non-permeable cryoprotectants in two steps. When the embryos are immersed in the liquid nitrogen the medium, which has a high viscosity, solidifies so quickly that the molecules do not have time to rearrange themselves into a crystalline structure. It is this that induces the formation of an intra- and extracellular vitreous state. Furthermore, the extreme cytotoxicity resulting from the high concentration of required cryoprotectants with higher membrane permeability can be reduced by addition of an appropriate concentration of non-permeable cryoprotectant.15,16
Exposure to or equilibration of the cryoprotectants The success of cryopreservation protocols depends on an optimal dehydration process when cells are exposed to hypertonic conditions and an optimal penetration rate of cryoprotectant. This is determined by several biophysical factors such as the properties of the membrane (cellular permeability), the type and concentration of cryoprotective additives (permeability property, concentration, and temperature of the cryoprotectants), and the surface–volume ratio of the cells. Nearly all vitrification methods (cooling without equilibrium) consist of exposing the embryos for short periods and in two steps to increasing concentrations of cryoprotectants. In the first step (Figure 10A.1), the embryos are exposed to a non-vitrifying solution (VS1)
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of permeable cryoprotectants (2.3–3.2 mol/L) for a period of from 2 to 4 min depending on the stage of embryonic growth, each cell of which has a well-determined surface to volume ratio and permeability coefficient. Following a rapid dehydration phase (< 30 s) at the beginning of exposure to the solution, a certain quantity of cryoprotectants (e.g. only about 30% of the initial cryoprotectant concentration will enter the cytoplasm of a zygote) penetrates the cells during the 2–4 min exposure. Another pre-incubation strategy17,18 consists of exposing the embryos to the first solution until the moment when the embryo regains its original volume. This prolonged exposure in the first bath may last between 5 and 15 min, but this increases the risk of inducing toxicity problems. However, as suggested by Vatja and Nagy,19 this approach, even though it may increase slightly the toxic effect, could provide much better protection for the whole cell, and may be especially beneficial in the case of large objects with a low surface/volume ratio including oocytes or early stage embryos such as zygotes. In the second step (Figure 10A.1) the embryos are put in contact with a vitrifying (VS2) solution of permeable and non-permeable cryoprotectants (4.8–6.4 mol/L) for a short period lasting 30–45 s before being placed on the carrier maximum of two embryos and plunged into liquid nitrogen. During this whole step a second phase of dehydration occurs. Applying these two steps, an intracellular vitrifying state is obtained as a result of dehydration of the embryos in VS2, which concentrates the intracellular solutions of salts, proteins, and cryoprotectant that have penetrated the cell in the course of exposure to VS1. The extracellular vitrifying state is obtained by the high concentration of VS2 cryoprotectants that encapsulate the embryo in a vitrifying sheath. Consequently, cell dehydration is carried out before cooling, as is the achieving of an intra- and extracellular
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Volume
Volume
a
H20
CP + H20
H20
CP + H20
Plunge in LN2 Plunge in LN2
2– 4 min
45 s
5–15 min
45 s
Dehydration Dehydration + equilibration
Dehydration Dehydration + exposure
Low intracellular concentration of CP
Extracellular vitrification solution 2 High intracellular concentration of CP
b
Step 1 Solution: not vitrified Exposition Dehydration and partial penetration of CP
10% 10%
7.5% 7.5%
2– 4 min
2– 4 min
Equilibration
7.5% 7.5%
5–15 min
Total penetration of CP
Step 2
20% 20%
15% 15%
15% 15%
Vitrification solution Concentration of CP that enter during step 1
30– 45 s
30– 45 s
60 – 90 s
Figure 10A.1 (a) Different strategies for vitrification of embryos and cell volume changes in response to addition of cryoprotectant (CP). (b) Concentration and time of contact of different cryoprotectant solutions with oocytes or embryos.
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vitrifiable state. When the embryos are immersed in LN2 the whole fluid (intra- and extracellular) has gained such a high viscosity that it solidifies so quickly that the molecules do not have time to rearrange themselves into a crystalline structure. Thus, an intraand extracellular vitreous state can be achieved.
Addition in one step or several steps The method of addition of cryoprotectant is also an important step to consider especially for oocytes. The cytoskeleton and the metaphase plate are particularly sensitive to osmotic fluctuation and excessive variation in volume. For this reason, addition of the cryoprotectants in several steps or by dilution is proposed.17
Second condition: fast cooling and warming The first successful results were obtained using a 0.25 mL French mini-straw (IMV-France) as the storage method for mouse embryos.13 The thickness of the straw walls, the volume of liquid therein (250 µL), and the formation of a thermal insulating screen (Leidenfrost effect) around the straw during immersion in the liquid nitrogen made it impossible to obtain cooling speeds of more than 2000°C/min. Thereafter, particular attention has been paid to the cooling rate. Martino et al.,20 Vajta et al.,21,22 and Lane et al.23 have shown the beneficial effect of a significant increase of cooling speed on the survival rate of cattle oocytes and hamster blastocysts. Such increases in the cooling rates up to 20 000°C/ min, make it possible to reduce the probability of ice crystal formation during cooling to even under insufficient permeation of cryoprotectant. In particular, a high cooling rate helps to reduce problems linked to ‘chilling’ (mainly observed with cryopreservation of
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oocytes). The speed of cooling depends on the type of carrier as well as on the volume of the sample. The smaller volume a sample has, the less surface area will hinder contact with the LN2. In ultrarapid vitrification techniques the smallest possible volumes (< 1 µl) of vitrification solutions are deposited on special supports, giving cooling speeds of around 20 000°C/min. With the aim of reducing the thermal gradient, one of the approaches consists of using open pulled straws, the walls of which are made thinner,21,24,25 or to encourage direct contact of the whole microdroplet of cryoprotectant solution with LN2.20,23,26–30 One important step that has not to be underestimated, is the rate of warming. Devices allowing a direct contact of the droplet with LN2 permit also to attain very high warming rates (> 20 000°C) because the small droplet is directly placed in contact with the dilution medium solution.
DESCRIPTION OF DIFFERENT VITRIFICATION STRATEGIES DEVELOPED DURING THE PAST DECADE IN OUR ART UNITS Rall and Fahy13 reported on the first ‘in vitro’ development of mice embryos after vitrification in a solution of DMSO, PROH, acetamide, and PEG. The following year, the first births after vitrification of mice and calf morulae and blastocysts treated with a mixture of glycerol and PROH were reported.31,32 Based on our previous experiences in animal models,31,32 and referring to several reports from mammalian species,33–35 in 1996 we decided to apply vitrification as an alternative technique for handling day 4 and day 5 human embryos.36 Later a whole spate of papers on the subject were published, and this technique is now considered to be the best method for preserving embryos of certain species of mammal (e.g. those threatened with extinction) or embryos produced from transgenic strains.16 In spite of a certain coldness with
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which the idea is received in some ART centers, nevertheless, mounting interest is being shown for this technique among some units and companies. Randomized studies comparing slow freezing with vitrification have concluded that the vitrification technique is better in terms of embryo survival rates and pregnancy in mice37 as well as in humans.11,38,39 Over the past decade, several embryo supports have been used and designed with the aim of first improving the survival rate of vitrified embryos and second to achieve cooling and storage in aseptic conditions.
First approach: vitrification in 0.25 mL French mini-straw as embryo carrier Our first vitrification approach36,40 for cryopreservation of morulae and blastocysts was based on a known and already published method for vitrification of mouse33 and cattle embryos.35
Description of the carrier system As mentioned above, the 0.25 mL French ministraw was the first device that we utilized to vitrify morulae and human blastocysts in an aseptic manner. However, the drawback of such an approach concerns the low cooling and warming rates due to the amount of liquid in the straw, the volume of air inside the straw, and the large surface area of the straw creating formation of a thermal insulating vapor coat. One possibility to eliminate the vapor coat that arises around the sample is to use a system (VitMaster) that decreases the temperature of LN2 to as low as −210°C, thereby eliminating the formation of vapor around the straw. Moreover, during the direct immersion into LN2 and/or during the warming step in a water bath collapsing or explosion of the straws and loss of the sample can occur because of extreme pressure changes in poorly sealed straws.
Methodology of vitrification and warming After an exposure of 3 min at room temperature to a solution of EG (20%) in PBS + 20% HSA, the embryos were placed in the vitrification solution consisting of 40% (v/v) EG + 18% (w/v) Ficoll (MW 70 000), and 0.3 mol/L sucrose. A maximum of two embryos were loaded directly into a 0.25 mL French straw containing vitrification medium. The time interval between exposure of the embryos to the vitrification solution and plunging the straw into a container filled with LN2 ranged between 45 and 60 s. After storage, the straws were taken out of the LN2 tank and warmed in a water bath at 40°C. The contents of the straw were expelled into a Petri dish containing 3 mL of 0.5 mol/L sucrose solution at room temperature. Five minutes after being flushed out, the embryos were transferred to 0.25 mol/L sucrose solution before washing and subsequent culture for 24 h.
Second approach: ultrarapid vitrification using the hemi-straw as embryo carrier Description of the carrier system According to different documents reporting the beneficial effect of a significant increase of the cooling speed, a method using the small volume–direct contact principle was developed. Therefore, in order to circumvent the problem of intracellular ice formation in less-permeable embryos such as with blastocysts, we developed an embryo carrier device (hemi-straw) that allows extremely fast cooling and warming rates30 (Figure 10A.2). The tip of the hemi-straw is designed to hold the embryos (maximum of two) in a very small volume of vitrification solution (< 0.5 µL) thus reducing the thermoinsulating effect of a conventional straw. The advantage of the hemi-straw is that the microdrop of cryoprotectant is loaded in a gutter and
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Hemi-straw
Outer CBS straw
Identification
Cryoprotectant microdrop – blastocyst
Hemi-straw
CBS straw
Blastocyst b
a
Figure 10A.2 Scheme of the hemi-straw ‘Vitroplug’: (a) loading the blastocyst on the tip of the hemi-straw; and (b) insertion of the hemi-straw into a larger Cryo Bio System (CBS) straw.
as a consequence is protected during its insertion inside the protective large high security Cryo Bio System straw (CBS; Cryo Bio System, France). By dropping the tip of the hemi-straw in the dilution solution, the microdrop is instantly warmed and expelled into the dilution solution reducing cytotoxic and osmotic effects. The cooling and warming rates with such carrier device are dramatically faster compared with those achieved with the 0.25 mL plastic straws. On a practical level, the use of the hemi-straw vessel reduces the space required for storage inside a cryotank. A similar carrier system but without protective gutter named ‘the Cryotop’ was developed by Kuwayama et al.17,18,41 The carrier tool consists of a thin film attached to a plastic holder and after plunging the tip in LN2, the Cryotop is inserted into a protective plastic tube.
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Methodology of vitrification and warming Ultrarapid vitrification using the hemi-straw has been our standard protocol since 2000. According to their stage of development, embryos were exposed for different periods of time and temperatures to a solution of cryoprotectants (VS1) (Fertipro NV, Beernem, Belgium) containing 10% (V/V) EG + 10% (V/V) DMSO in PBS + 20% (V/V) HSA. The embryos were exposed to VS1 solution according to their stage of development. The vitrification solution (VS2) (Fertipro NV, Beernem, Belgium) was composed of 20% (V/V) EG + 20% (V/V) DMSO + Ficoll 400 (10 mg/mL), and 0.75 mol/L sucrose in PBS + 20% (V/V) HSA. The blastocysts were then exposed to the vitrification solution for 30–45 s. An estimated volume of < 0.5 µL of cryoprotectant solution containing embryos was deposited
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using a fine pipette into the tip of the trough of the hemi-straw. The hemi-straw was then instantly plunged into a dewar of LN2 and with the aid of forceps inserted in a larger pre-cooled straw (CBS) before closing it (Figure 10A.3). Before warming, a dewar of LN2 containing the hemi-straw was placed close to the microscope. Under LN2 and with forceps, the hemi-straw was pulled out of the larger straw and the tip of the straw holding the embryos was immediately immersed and stirred into a Petri dish containing 3 mL of 0.5 mol/L sucrose at 37°C. After 2–3 min, the blastocysts were transferred to different bath of 0.25 mol/L sucrose at intervals of 3–4 min (Table 10A.1). The blastocysts were then washed several times in PBS + HSA solution, then placed in culture medium (Figure 10A.3B).
Third approach: aseptic vitrification using High Security Vitrification carrier Problems linked with an open system Vitrification has suffered from different drawbacks that have hampered its wider application. Although successful results were reported with the previous carrier systems, one of the main remaining drawbacks concerns the possible risk of contamination by bacteria, fungi, and viruses from the LN2, or by contamination with another sample even if the probability of the latter is low. The different methods do not provide a leak proof environment for cryopreserved embryos. Liquid nitrogen in storage tanks probably contains several pathogenic environmental micro-organisms.42 These could be acquired at the time of distribution of LN2, or through storage by deposition of ice and microbes from the air, through inadequate asepsis of surfaces of straws or cryovials, or as a result of crosscontamination with unprotected samples.42–45
For clinical application, it is therefore mandatory to cool and store the embryos in aseptic conditions, to minimize the likelihood of microbial transmission during storage, while at the same time retaining acceptable survival rates. Various aseptic approaches such as the use of sterile LN2, storage in nitrogen vapor, and hermetically sealed and isolated containers have been proposed and designed.46 Mouse embryos have been vitrified using Cryoloops which were put into the vapor of liquid nitrogen. However, there is an obvious risk of accidental warming and the greater fluctuations in storage temperature should not be underestimated. Various aseptic closed systems such as the ‘open pulled straw (OPS) in straw’,47 or the sealed OPS ‘Cryotip’18 have been recently developed.
Description of the High Security Vitrification kit In response to the demand stated above, with respect to European regulation, our ART unit, in collaboration with Cryo Bio System Company, developed an aseptic vitrification kit based on the hemi-straw principle (Figure 10A.4). The system is composed of two distinct parts, a high security thermal autogenic sealed clear mini-straw, and a split capillary tube with its extremity in the form of a gutter. This High Security Vitrification kit (HSV kit) makes it possible to place a microdroplet (< 0.5 µL) of cryoprotectant containing the embryos in the gutter of a small capillary tube before inserting it in a mini-straw. It is heatsealed using a special welder which ensures a leak-proof seal, before immersion in the LN2. Cooling rates of 1627°C/min have been recorded.48
Methodology of vitrification and warming The methodology is similar to that applied with the hemi-straw carrier system. The embryos
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a
10% / 10% Loading maximum two embryos in the gutter of the hemi-straw
Vitroplug: HS
Plunge in LN2 and insert in CBS straw 20% / 20%
LN2 CBS straw
b
0.25 mol / L sucrose
4 min
0.5 mol/L sucrose 2 min PBS + 20% HSA
If less than 4 min in VS1
2 × 2 min
0.5 mol/ L sucrose
4 min
1 mol/L sucrose
2 min PBS + 20% HSA
If more than 4 min in VS1
2 × 2 min Figure 10A.3 (a) Hemi-straw (HS) vitrification procedure. (b) Hemi-straw warming procedure. VS, vitrification solution; CBS, Cryo Bio System; PBS, phosphate buffered saline; HSA, human serum albumin.
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Table 10A.1
Protocols for vitrification of embryos at different stages of development with the hemi-straw. Before cooling VS1
Zygotes 6–8 cells Morulae Early blastocysts Blastocysts Full blastocyst + AS Expanded blastocyst + AS Intact full blastocysts Intact expanded blastocysts
Warming dilution VS2
Sucrose (mol/L)
10/10% (min)
Temperature
20/20% (s)
Temperature
1 (min)
0.5 (min)
0.25 (min)
6–8 3 2 2 3 2–3 2–3 7–10 7–10
RT RT RT RT 37° C 37° C 37° C RT RT
45 45 45 45 45 45 45 45 45
RT RT RT RT 37° C 37° C 37° C RT RT
2–3
3–4 2–3 2–3 2–3 2–3 2–3 2–3 3–4 3–4
3–4 3–4 3–4 3–4 3–4 3–4 3–4 3–4 3–4
2–3 2–3
AS, artifical shrinkage before vitrification; RT, room temperature ~ 25° C; Cryoprotectant, ethylene glycol + DMSO.
4 3 2 1 Figure 10A.4 High Security Vitrification Kit: 1 Mini hemi-straw, 2 250ml high security straw; 3 mini hemi-straw inside the high security straw; and 4 complete sealed system (VHS).
were vitrified after exposure to VS1 solutions as presented in Table 10A.1. After 15 s in the vitrification solution (VS2), an extremely small amount (< 0.5 µL) of solution containing the
embryos was deposited at the tip of the trough of a HSV using a fine pipette. The HSV was inserted inside a small straw, sealed, and then plunged into LN2. For warming, the
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Table 10A.2 Survival and hatching blastocysts (n (%) after vitrification of mice zygotes exposed for different times to VS1 (10% EG + 10% DMSO) solution and plunged directly in LN2 or after a period of 5 min 10 cm above LN2. For each experiment two replicates of ten zygotes were used. Time in VS1 solution (min) Cooling conditions
1
2
3
5
10
Survival rate
Directly in LN2 10 cm above LN2 level for 5 min
14/20 (70) 6/20 (30)
18/20 (90) 8/20 (40)
20/20 (100) 17/20 (85)
20/20 (100) 19/20 (95)
19/20 (95) 18/20 (90)
Hatch blastocysts
Directly in LN2 10 cm above LN2 level for 5 min
8/20 (40) 1/20 (5)
12/20 (60) 5/20 (25)
16/20 (80) 13/20 (65)
15/20 (75) 16/20 (80)
16/20 (80) 17/20 (85)
upper part of the outer straw was removed from LN2 before cutting. After cutting, the tip of the HSV holding the embryos was immediately immersed into 3 mL of 1 mol/L or 0.5 mol/L sucrose at 37°C. The embryos were instantly dispersed into the sucrose solution. After 2 min, the embryos were transferred to decreasing concentrations of sucrose solutions (0.5 mol/L and 0.25 mol/L sucrose) at intervals of 3–4 min at room temperature (Table 10A.1). The embryos were then washed several times in HTF-HEPES-HSA solution.
Problems related with aseptic carrier system Direct contact with LN2 makes it possible to cool the embryo at very high speeds. Conversely an aseptic system that isolates the embryo from the LN2 may have an adverse effect on survival after warming and decrease the efficacy of the vitrification protocol. This is because the slower the cooling, the greater the concentration of cryoprotectants, and the greater must be the warming rate.
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With the aim of analyzing whether there was a correlation between the time of exposure to cryoprotectants and the rate of cooling, we assessed mouse zygotes (FVB strain) to determine the survival rate and the potential of blastocysts to hatch according to different times of exposure to cryoprotectants (1, 2, 3, 5, and 10 min in VS1) before rapid (plunged directly in LN2), or slow (5 min in the vapor before dropping into LN2) cooling. The results show (Table 10A.2) that in the case of a reduced cooling rate, more time of contact with the cryoprotectant solution is necessary to protect the cells. Therefore to limit problems of toxicity while ensuring an acceptable survival rate it is essential to find the correct balance between the amount of intracellular cryoprotectant and the rate of cooling and this varies in relation to the different types of embryos that have their own biophysical properties. Such observations reinforce the concept that one of the key objectives is to find the balance between optimal cooling rate and sufficient intracellular concentration of cryoprotectants.19
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CLINICAL APPLICATION OF VITRIFICATION OF HUMAN EMBRYOS – WHERE DO WE STAND? In spite of a certain reluctance in some ART centers as mentioned above, nevertheless, mounting interest is being shown for this technique among some units and companies.19,49,50
Vitrification of zygotes High rates of survival (81–100%), and growth up to the blastocyst stage have been recorded for zygotes exposed for short periods to cryoprotectant solutions and using non-hermetically sealed supports such as the flexible denuding pipette,51 copper grid,52 OPS,53,54 and Cryotop.18,41 Using aseptic supports (straw in straw) Isachenko et al.47 reported a survival rate of 75%, and 25% of the zygotes went on to grow to the blastocyst stage. They concluded that reducing the cooling speed does not have any major negative effect on the later growth of an embryo. Zygotes like oocytes are the largest cell in the body, have a high water content, and one single membrane. Such large cells have a low surface to volume ratio, hence they are less efficient at taking up cryoprotectant and at losing water. Isachenko et al. reported that out of 5881 vitrified zygotes using the Cryotop as embryo support, that were equilibrated in 7.5% EG + 7.5% DMSO, 93% survived and 52% developed to the blastocyst stage.
Vitrification of day 3 embryos The first pregnancy following vitrification of embryos at the 8-cell stage was reported by Mukaida et al.55 after exposure of the embryos to a mixture of ethylene glycol + Ficoll and sucrose, and loading them into 0.25 mL french mini-straws as support. El Danasouri and Selman56 using the OPS as carrier system, vitrified 215 embryos on day
3 of culture. Survival rates of 79% and 40% were observed following vitrification of embryos with 8 and 6–7 blastomeres, respectively. After transfer, a pregnancy rate of 30% and implantation rate of 10% were observed. More recently, Gottumukkala et al.57 cryopreserved day 3 embryos both by vitrification using a Cryoloop, and by slow freezing in a randomized manner. The study showed significantly higher rates of survival (95% vs. 60%), pregnancies (37% vs. 17%), and implantation (15% vs. 4%) following vitrification as compared with slow freezing. After vitrifying more than 890 4-cell embryos using a Cryotop, Kuwayama et al.18 recorded significantly higher rates of survival and pregnancies compared with slow cryopreservation (98% vs. 91%). Liebermann and Tucker24 reported that the hemi-straw support was easy to use and successful for vitrification of different stages of development such as day 3 embryos.
Our experience with vitrification of day 3 embryos Table 10A.3 shows the results of vitrification of day 3 embryos using different cryoprotectant solutions and the hemi-straw as embryo support. We observed that the results were in relation to the concentration of the vitrification solution (VS2) suggesting that the vitrifcation state is probably not fully obtained when loading the embryos in the 15% EG + 15% DMSO solution. It may be that an incubation time of 2–3 min in the first solution is too short to allow sufficient intra-cellular permeation of cryoprotectant that has to be concentrated during the second dehydration phase in the vitrification VS2 solution. Using a more concentrated cryoprotectant VS2 solution such as 20% EG + 20% DMSO allows more rapid dehydration of the cells, and as a consequence a threshold level of solute is reached that has the property of an amorphous state.
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Table 10A.3 Vitrification and warming cycles of day 3 embryos in three different solutions of cryoprotectants using the hemi-straw as embryo carrier (2005–2006). Time in VS1 2–3 min at room temperature and 45 s in VS2 EG/DMSO (%/%)
No. of vitrification cycles No. of day 3 embryos Survival at 24 h No. of transfers No. of ongoing pregnancies after vitrification cycles Implantation rates
7.5/15
7.5/20
10/20
16 40 12 (30%) 6 (38%) 0 (0%)
14 30 18 (60%) 10 (71%) 6 (43%)
36 84 54 (64%) 30 (83%) 14 (39%)
33%
26%
0%
Vitrification of morulae Cremades et al.25 assessed the survival rate of excess human compacted morulae that were experimentally vitrified with the OPS method using a plastic micropipette tip. After vitrification of morulae on day 4 and day 5 they observed satisfactory survival rates of 73.7% and 72.7%, respectively.
Our experience with vitrification of morulae Over the past 18 months, vitrification of morulae has been routinely implemented with success in our vitrification program (Table 10A.4). As with day 3 embryos, we also have observed with our vitrification protocol a better outcome for morulae in terms of pregnancy and implantation when more concentrated cryoprotectant solutions have been used.
Table 10A.4 Vitrification and warming cycles of morulae in two different solutions of cryoprotectants using the hemi-straw as embryo carrier (2005–2006) Time in VS1 2–3 min at room temperature EG/DMSO (%/%) 7.5/15 No. of vitrification cycles No. of morulas Survival at 24 h No. of transfers No. of ongoing pregnancies after vitrification cycles Implantation rates
10/20
27
32
64 42 (66%) 25 (93%) 4 (15%)
76 46 (61%) NS 26 (81%) NS 12 (38%)*
14%
26%
P < 0.05
*
Vitrification of blastocysts Many papers dealing with vitrification of blastocysts have been published in the past few years. Different approaches in term of combinations and concentration of cryoprotectants, exposure time with the cryoprotectant solutions, temperature of exposure to the cryoprotectant solutions, methods and steps for dilution of the cryoprotectants, and the types of support tools used to vitrify the blastocysts
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have been evaluated and proposed (Table 10A.5). Therefore, to date it has been difficult to draw firm conclusions about which methods work best. The inconsistent results with all the different vitrification protocols reflect the fact that a blastocyst is an embryo with a complex structure, and therefore more difficult to cryopreserve compared with earlier stages of embryos. Mukaida et al.58,59 presented a large series of 1150 ultrarapid vitrification and warming cycles of blastocysts (using a Cryoloop as support) exposed for a maximum period of 2 min 30 s to a mixture of DMSO and EG.
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Hemi-straw Not aseptic
Cryotip Aseptic
VHS Aseptic
60
18
61
Zona pellucida opening before vitrification
Blastocoel shrinkage
Blastocoel shrinkage
Blastocoel shrinkage
EG/DMSO 10/10% 2 min 3 min 37° C
EG/DMSO 7.5/7.5% 5 min 15 min RT
EG/DMSO 10/10% 2 min 3 min 37° C
EG/DMSO 10/10% 2 min 3 min 37° C
EG/DMSO 7.5/7.5% 5 min 15 min RT
EG/DMSO 7.5/7.5% 3 min 37° C
EG 20% 1.5 min RT
EG 20% 3 min RT
EG/DMSO 7.5/7.5% 2 min
EG/DMSO 20/20% sucrose Ficoll 30 s 37° C
EG/DMSO 15/15% sucrose max 90 s 27° C
EG/DMSO 20/20% sucrose Ficoll 30 s 37° C
EG/DMSO 20/20% sucrose Ficoll 30 s 37° C
EG/DMSO 15/15% sucrose 45 s 37° C
EG/DMSO 15/15% 25 s 37° C
EG 40% sucrose Ficoll 30 s RT
EG 40% sucrose Ficoll 30 min RT
EG/DMSO 15/15% sucrose Ficoll 30 s 37° C
EG 5.5 mol/L sucrose 1 mol/L 20 s 37° C
3 steps 2 min 37° C
3 steps 4 min 27° C
3 steps 2 min 37° C
3 steps 2 min 37° C
2 steps B 3 min 37° C
2 steps A 5 min
5 steps 1.5 min
2 steps C 5 min RT
2 steps A 3 min
4 steps 2.5 min
2 steps C 5 min RT
Warming dilution of cryoprotectant
S 22/30 (73%)
S 30/54 (56%) B/V 5/22 (23%)
S 15/21 (71%)
Morulae
S 18/19 (95%) P/V 2/5 (40%)
S 27/33 (82%)
S 24/41 (59%) N/V 4/17 (24%)
S 497/569 (87%)
S 39/46 (83%) N/V (17%)
Early blastocyst
S 41/48 (85%) P/V 2/6 (33%)
S 82/88 (93%) P/V 42/80(51%)
S 31/38 (82%) B/V 7/20 (35%)
S 188/281 (67%) B/V 25/93 (27%)
S 5695/6328 (90%) P/V (53%)
S 81/90 (90%) P/V 12/25 (48%)
S 53/75 (71%) B/V 8/39 (20%)
S 14/69 (20%) B/V 1/22 (5%)
S 124/216 (57%) P/V 18/61 (30%)
S 48/93 (52%) P/V 5/20 (25%)
Full blastocyst/ expanded blastocyst
S, survival; P, one pregnancy; B, births per vitrification cycle; EG, ethylene glycol; RT, room temperature; DMSO, dimethylsulfoxide.
Dilution methods of the cryoprotectants: 2 steps A sucrose 0.33 mol/L 0.2 mol/L; 2 steps B sucrose 1 mol/L, 0.5 mol/L; 2 steps C sucrose 0.5 mol/L, 0.25 mol/L; 3 steps sucrose 0.5 mol/L, 0.25 mol/L, 0.125 mol/L; 4 steps sucrose 1 mol/L, 0.5 mol/L, 0.25 mol/L, 0.125 mol/L; 5 steps sucrose 0.5 mol/L, 0.4 mol/L, 0.3 mol/L, 0.2 mol/L, 0.1 mol/L.
Hemi-straw Not aseptic
EM grids Not aseptic
62
30
Straw 0.25 mL Aseptic
40
Cryotop Not aseptic
Straw 0.25 mL Aseptic
40
18
Cryoloop Not aseptic
28, 29
EG 1.5 mol/L 2.5 min
EG 40% sucrose Ficoll 30 s RT
Solution 2 (VS2)
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OPS Aseptic
EM grids Not aseptic
27
EG 20% 3 min RT
Solution 1 (VS1)
Results
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25
Straw 0.25 mL Aseptic
36
Reference
Specific treatment
Exposure to the cryoprotectants
Different approaches for vitrification of human blastocysts
Embryo carrier
Table 10A.5
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Table 10A.6 Overall results after vitrification and warming of morulae (n = 22) and blastocysts (n = 78) in 0.25 mL French mini-straw: effect of the developmental stage and artificial shrinkage of the blastocoel. Without artificial shrinkage
No. of vitrification–warming cycles No. of vitrified–warmed embryos Survival after 24 hours (%) Mean embryos transferred Births per vitrification–warming cycle (n (%)) Implantation per (%) transferred embryo vitrified embryos
With artificial shrinkage
Morulae
Early blastocysts
Blastocysts expanded blastocysts
Blastocysts expanded blastocysts
22 55 54.5 1.7 5 (22.7%)
17 41 58.5 1.5 4 (23.5%)
22 71 20.3* 2.0 1 (4.5%)
39 75 74.6 1.4 8 (20.5%)
20.0 10.9
16.7 9.8
7.1 1.4
18.4 12.0
P < 0.001 (χ2 test)
After warming 2749 blastocysts they recorded a survival rate of 89.7%. Out of a total of 543 early pregnancies (49.1%), 174 developed gestations and 247 deliveries (295 babies including 153 boys and 142 girls) were recorded. An implantation rate of 34.5% was observed. It should be noted, as we describe below, that the blastocoelic cavity of blastocysts and expanding blastocysts were systematically artificially collapsed. Also, presenting a large series of vitrification using a different approach, Kuwayama et al.18 obtained pregnancy rates of 59% and 51%, using a Cryotop (open, non-aseptic system) and Cryotip (sealed, aseptic system) supports, respectively. In their protocol the blastocysts were no longer exposed to cryoprotectant solutions, but instead equilibrated for an extended period of time (ranging between 5 and 15 min) in a DMSO + EG solution until the moment when they return to almost their original volume.
Our experience with vitrification of blastocysts In 1995 prolonged embryo culture to day 5 was implemented in our ART activities. As a consequence, for one decade, the greater part
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of our experience with vitrification has been with embryos that have developed in culture for 5 days. During these past 10 years, a total of 664 vitrification and warming cycles of blastocysts have been undertaken using the 0.25 mL French straw (n = 78), the hemi-straw (n = 554), and recently the aseptic HSV kit (n = 22) as embryo support systems.
Vitrification of blastocysts in 0.25 mL French straw Our results showed that application
of the vitrification method using EG + Ficoll + sucrose solution and loading the embryos in a 0.25 mL French straw was effective for the cryopreservation of day 4 and day 5 early blastocysts. With this method we obtained the first pregnancy after vitrification of a human blastocyst.36 However, it was questionable whether this simple aseptic method was applicable to blastocysts and expanded blastocysts. In fact, our preliminary results showed that the efficiency of the vitrification method was dependent on the stage of embryo development, and was negatively correlated to the expansion of the blastocoel (Table 10A.6). Only 20% of the blastocysts and expanded blastocysts survived the vitrification and warming procedure with the
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French 0.25 mL straw as compared with a 50% survival rate for day 4 morulae and day 5 early blastocysts.
Vitrification of blastocysts with the hemi-straw support Between the years 2000 and 2006, a total of 554 vitrification and warming cycles with the hemi-straw carrier system were undertaken in a group of women aged between 21 and 44 years. After 505 transfers 3 h or 24 h after the warming step of an average of 2.1 embryos, a total of 179 patients became pregnant, out of which 141 ongoing pregnancies resulted (with 101 deliveries at the time of going to press). The ongoing pregnancy rates were 25.4% and 27.9% per vitrification and warming cycle, and embryo transfer attempts, respectively. The implantation rates per transferred embryo and per vitrified and warmed blastocysts were 15.8% and 10.2%, respectively.
Vitrification of blastocysts with the High Security Vitrification kit A prospective randomized analysis of the survival rates on sibling day 5 embryos vitrified at a lower cooling rate inside a hermetically sealed straw demonstrated comparable results with the control non-aseptic group in hemistraws.61 With both techniques, the warming rate is extremely fast because the tip of both systems is plunged directly into a bath of sucrose at 37°C/min. This reinforces the concept that the warming rate is as important as the cooling rate especially in case of low cooling rate or low exposure time with the cryoprotectant. Twenty-two vitrification and warming cycles of blastocysts in the HSV kit have been performed. After warming of 106 blastocysts, 91 survived. Fifty-eight were transferred in a first group to 18 patients after 3 h of additional culture thereby establishing four (22.2%) ongoing pregnancies. In a second group, 13 blastocysts re-expanded after 24 h in culture, and after four transfers of ten
embryos, two ongoing pregnancies were obtained.
Factors affecting the outcome of blastocyst vitrification Several factors whether or not they are associated with vitrification may influence the outcome, and may explain differences in the results between published studies.
Time of embryo transfer: 3 h or 24 h after warming Significantly higher pregnancy rates were observed when embryo transfers were performed 24 h as compared with embryo transfers 3 h after warming. During a period of 14 months, 64 and 95 blastocyst transfers were performed at 3 or 24 h after warming, respectively. The percentages of blastocysts showing signs of survival in the 3 h group (blastocoel cavity starting to develop) and in the 24 h culture group (full re-expansion or hatching) were 86.1% and 79.4%, respectively (no significant difference). Nevertheless, the pregnancy rate per vitrified and warming cycle in the 3 h culture group was 14.7% (14/95) compared with 29.7% (19/64) when transfers occurred 1 day later. The implantation rate per transferred embryo in the 3 h culture group was 8.9% (16/179) compared with 20.9% (19/91) after an additional 24 h of in vitro culture. Such results are very intriguing, but it is difficult to find a good explanation. After 24 h culture, it is possible to detect more accurately which embryos have the higher capacity to further develop. Immediately after warming and dilution of the cryoprotectant, it is difficult to assess exactly the viability of the embryos at the stereomicroscopic level even though some signs of re-expansion may occur. Under high magnification (× 400), if only minor morphological changes are detectable, a prolonged culture period may
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Table 10A.7 Overall results after vitrification and warming of blastocysts exhibiting different quality before vitrification. Embryo transfers 24 h after warming step. Quality of blastocysts before vitrification using the hemi-straw
No. of vitrification–warming cycles No. of vitrified–warmed embryos Survival after 24 h (n) Survival after 24 h (%) Transfers (n (%)) No. of embryos transferred Mean no. of embryos transferred Birth/Ongoing pregnancy per vitrification–warming cycle (n (%)) transfers (%) Implantation per (%) transferred embryos vitrified embryos Twins
Bad quality
Good quality
Mixed quality
Total
113 361 194 53.7* 88 (77.9) 194 2.2
211 537 414 77.1 205 (97.1) 393 1.9
135 481 320 66.5* 121 (89.6) 261 2.2
459 1379 928 67.3 414 (90.2) 848 2.0
12 (10.6) 13.6
76 (36.0) 37.1
36 (26.6) 30.0
124 (27.0) 30.0
6.2 3.3 0
24.0 17.5 18
15.3 8.3 4
17.2 10.6 22
*P < 0.001.
be necessary to evaluate the re-expansion and viability of the embryos.
Selection of blastocysts for vitrification An important parameter to consider before starting the vitrification process concerns embryo selection. We can conclude from our data (Table 10A.7) that the quality of the blastocysts selected before vitrification has an important impact on the capacity of the blastocyst to survive the vitrification and to develop further. If poor quality blastocysts are vitrified, then the probability for them to survive the vitrification and warming procedure is rather low. When compared with day 3 embryo selection, the selection of blastocysts is more difficult to perform. In fact, classification of blastocysts between ‘good’ and ‘moderate’ can vary between biologists. It is not always an easy task to judge the quality of a blastocyst according to the type and the quality of either the inner cell mass or the trophectoderm
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cells. This part of the work is vital for the outcome of the embryo after warming. Moreover, impaired embryonic development may also contribute to a reduction in the capacity to produce an optimal blastocyst that will survive the vitrification procedure. After vitrification of blastocysts that originate from good quality early stage embryos, the survival rates observed immediately after thawing, the percentage of blastocysts that hatch and re-expand after 24 h culture, and the proportion of blastocysts available for embryo transfer, are all considerably higher when compared with those already exhibiting poor embryo quality during cleavage stage. We observed a reduced likelihood of success after vitrification of morphologically normallooking blastocysts that originated from cleavage stage embryos with suboptimal morphology. The post-thaw survival rates depend on the cleavage characteristics during the 5 days of culture. Only those developing from good quality cleavage stage embryos could be cryopreserved and thawed with good results.30
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This underlines the need for good scoring and selection of embryos through all whole stages of in vitro development. Only with such an approach, can the best embryos be identified and selected, not only for a fresh embryo transfer but also for future cryopreserved cycles.
Degree of blastocoelic expansion The surface to volume ratio of blastomeres in the blastocysts is higher and even increases with an increase in the degree of expansion of the blastocyst compared with that of blastomeres from embryos in their early stage of development. As a consequence, we may expect a faster rate of entry of the permeating cryoprotectant, and more rapid intracellular protection in the expanded blastocyst. In addition, the cytoplasm of the blastomeres contains various intrinsic macromolecules that increase during the dehydration phase contributing to the formation of an amorphous state. Nevertheless, our experience is that the ability of blastocysts to survive the vitrification procedure depends on the degree of expansion of the blastocoel, and was negatively correlated with the expansion of the blastocoel (Table 10A.6). Full blastocysts and expanded blastocysts are more likely to survive the vitrification procedure. We postulated40 that a large blastocoel might disturb cryopreservative potential due to ice crystal formation during the cooling step. Due to the short exposure time to the cryoprotectant solutions (maximum exposure time in VS1 4 min), the large blastocoelic cavity is not sufficiently protected against formation of ice crystals. Therefore, in order to verify whether the blastocoel is the source of the problem, we analyzed in the first instance the effect of reducing the cavity, and subsequently the effect of a longer exposure to the cryoprotectant.
Artificial shrinkage of the blastocoel
We investigated the impact that a reduction in blastocoel fluid before vitrification has on
post-thaw embryo survival, and demonstrated an increase in the survival rates after artificial shrinkage of the blastocoel cavity using a microneedle.40 The beneficial effect of this procedure prior to vitrification was later confirmed by other ART units by applying a 29-gauge needle,62 a hand-drawn Pasteur pipette,63 and more recently also by using a laser pulse.64 They reported survival and pregnancy rates of almost 90% and 45%, respectively. Artificial shrinkage prior vitrification has shown no adverse effects on the development of blastocysts and on the health status on babies born to date.
Full equilibration with the cryoprotectant solution If the reduced viability of blastocysts is truly related to an insufficient permeation of cryoprotectant inside the cavity, other approaches such as increasing the time of exposure to cryoprotectant solutions can be proposed to prevent ice crystal formation. With conventional slow-freezing procedures, the blastocysts are equilibrated first in a solution of cryoprotectant for a period ranging from 10 to 20 min. If blastocysts still survived after slowcooling steps to –30°C to –40°C, and then plunging in LN2, we could postulate that the reason for those embryos to survive was because an amorphous state in the blastomeres and inside the cavity had formed. Based on this deduction, recently we started to equilibrate blastocysts for a longer time period in a low concentration of cryoprotectants in order to allow full equilibration inside the cavity. The low concentration of cryoprotectants is more beneficial when considering the cytotoxic effect of high concentration of cryoprotectants at prolonged exposure time. After this initial equilibration step, blastocysts were exposed for a shorter period to VS1 and VS2, before loading them on to the hemi-straw or HSV, and plunging into LN2. The preliminary results demonstrate, after vitrification and warming of 40 intact full and expanded blastocysts, a re-expansion rate of 97.5% 24 h after warming.
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In vitro culture conditions: reduction of cryotolerance One factor that can have an effect on the viability of blastocysts lies in the in vitro culture conditions. The introduction of sequential culture media allows for production of blastocysts without the need for co-culture using feeder cells. Notwithstanding the fact that fresh blastocysts give good implantation rates, it has been shown65 that cryopreserved IVF human embryos obtained in sequential media are less viable than cryopreserved blastocysts after co-culture. According to Massip et al.66 inappropriate in vitro culture conditions can also explain the low pregnancy rates obtained after blastocyst freezing. It is increasingly confirmed that culture conditions influence the cryotolerance of the embryo, and that a high survival rate of cryopreserved, in vitro cultured embryos requires improvement in the techniques of maturation and culture, rather than simple changes in cryopreservation methods.67,68
WHY HAS THERE BEEN A LACK OF INTEREST IN VITRIFICATION? Several articles have been published claiming that vitrification is an optimal method for the cryopreservation of embryos of several mammalian species.11,16,50,69 Yet despite the publication of encouraging results,18,28,39 there has been a notable lack of enthusiasm for introducing this technique to ART programs. The lack of an aseptic embryo support system has been one reason for this lack of interest. Another cause of concerns is the high concentrations of cryoprotectants utilized; but is this the reality? The need to expose embryos to high concentrations of cryoprotectants (30–50% v/v) has aroused a certain mistrust in many ART centers about using this method of cryopreservation for humans. Yet there are various methods of limiting the toxicity of cryoprotectants. Use of a solution containing two different cryoprotectants lowers the relative
213
toxicity of each one. Another alternative consists of adding macromolecules to the cryoprotectant solution. These polymers, with high molecular weights, are generally less toxic and can protect embryos against the effects of cold by increasing the viscosity of the extracellular solution.14 It is also possible to reduce the toxicity of vitrification solutions by lowering the temperature of cryoprotectants or the exposure time before cooling. Penetration speed of the cryoprotectant is affected by temperature, and it has been shown that prior equilibration of mouse blastocysts with a mixture of PROH and glycerol at 4°C has a beneficial effect.70 A disadvantage of this approach lies in the difficulty of maintaining the solutions at 4°C under the microscope. The crucial question remains about the quantity of cryoprotectant that may jeopardize embryonic survival and growth. It is essential to determine a balance between prevention of intracellular ice crystal formation and prevention of toxic effects. As in most vitrification methods, exposures to cryoprotectant solutions are of short duration (Table 10A.1), and the quantity of intracellular cryoprotectants is probably similar or even lower than those present in slow freezing. Indeed we have observed that one can achieve an acceptable survival rate by placing the vitrified embryos directly into a sucrose solution at 0.33 mol/L, and even directly into the PBS. Following immersion of blastocysts in a 0.5 mol/L sucrose solution (63 vitrification cycles) or directly into the PBS (55 vitrification cycles) survival rates of 60% and 71% were obtained, respectively. Following transfer of on average 1.7 embryos, pregnancy rates of 27% and 24%, and implantation rates of 18% and 16% were obtained in each group, respectively. Observations of similar survival rates in the absence or presence of sucrose, suggest that low levels of cryoprotectants are present in the cell. This is because rapid penetration of the cell by water, due to a high intracellular concentration of cryoprotectants, would make the cell swell beyond its critical volume before
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the cryoprotectants are removed. Such a strategy cannot be used after slow freezing, because direct dilution in an isotonic solution would lead to immediate cell lysis following too rapid hydration of the cell well before removal of the cryoprotectants. Over 15 years ago Kasai et al.71 showed that EG can be considered to be a cryoprotectant of very low toxicity in terms of embryo survival, and is therefore now considered as a reference cryoprotectant for vitrifying oocytes and embryos. On the other hand, a study by Klug et al.72 recorded an embryo-toxic effect of EG and more particularly of its metabolites in contact with rat embryos (on days 9.5–11.5 of gestation). According to Vatja and Nagy19 no study has shown any growth abnormality in animal or human embryos following vitrification using EG. Despite these findings one must remain cautious. Other paths remain to be explored, such as the use of other permeable cryoprotectants (PROH), or addition of polymers at higher concentration in order to reduce the amount of permeating cryoprotectants.14 In a recent study Takahashi et al.73 did not record any difference in terms of malformations after the birth of children produced by transfers of fresh and vitrified blastocysts.
CONCLUSIONS AND PROSPECTS Are programmable freezers still needed in the embryo laboratory? It seems evident that vitrification methods will continue to improve in the coming years, thanks to a resurgence of
interest of scientists in this field.19 It may be that in future vitrification will prove to be effective for all stages of embryo development. However, in spite of the promising results obtained so far, the procedure will have to be adapted first to each type of embryo carrier system. The higher is the cooling rate, the lower is the required cryoprotectant concentration and vice versa.19 Also, it will be necessary to adjust our methods according to the different stages of embryo development in order to find the most favorable conditions of exposure, in terms of time and temperature, to cryoprotectant solutions in relation to the rate of cooling that depends on the type of embryo carrier device used. For zygotes, or blastocysts with a large blastocoel, a longer exposure time with the cryoprotectant solution seems a better approach to obtain acceptable survival and pregnancy rates. For day 3 embryos, morulae, and blastocysts with an artificial reduced cavity shorter exposure to the cryoprotectant will be sufficient to protect the embryos. In order to optimize the technique, vitrification has undergone several changes. The use of ultrarapid cooling has made it possible to improve results, but because of the ever present risk of contamination, alternative systems of embryo supports, such as the Cryotip or the high security vitrification straw, that eliminate contact with LN2, have recently been developed. The results, expressed in terms of embryo survival rates and pregnancies, do not seem to be adversely affected in any way by this security adjustment.
References 3. Ménézo Y, Nicollet B, Herbaut N et al. Freezing cocultured human blastocysts. Fertil Steril 1992; 58: 977–80. 4. Kaufman R, Ménézo Y, Hazout A et al. Cocultured blastocysts cryopreservation; experience of more than 500 transfer cycles. Fertil Steril 1995; 64: 1125–9.
1. Zeilmaker G, Alberda A, van Gent I et al. Two pregnancies following transfer of intact frozenthawed embryos. Fertil Steril 1984; 2: 293–6. 2. Cohen J, Simons R, Fehilly CB et al. Birth after replacement of hatching blastocyst cryopreserved at expanded blastocyst stage. Lancet 1985; 1: 647.
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5. Langley M, Marek D, Gardner D et al. Extended embryo culture in human assisted reproduction treatment. Hum Reprod 2001; 16: 902–8. 6. Behr B, Gebhardt J, Lyon J et al. Factors relating to a successful cryopreserved blastocyst transfer program. Fertil Steril 2002; 77: 697–9. 7. Veeck L, Bodine R, Clarke R et al. High pregnancy rates can be achieved after freezing and thawing human blastocysts. Fertil Steril 2004; 82: 1418–27. 8. Virant- Klun I, Tomazevic T, Bacer-Kermavner L et al. Successful freezing and thawing of blastocysts cultured in sequential media using a modified method. Fertil Steril 2003; 79: 1428–33. 9. Ludwig M, Al-Hasani S, Felderbaum R et al. New aspect of cryopreservation of oocytes and embryos in assisted reproduction and future perspectives. Hum Reprod 1999; 14 (Suppl 1): 162–85. 10. Plachot M, Belaisch-Allart J, Mayenga J et al. Blastocyst stage transfer: the real benefits compared with early embryo transfer. Hum Reprod 2000; 15 (Suppl 6): 24–30. 11. Stehlik E, Stehlik J, Katayama K et al. Vitrification demonstrates significant improvement versus slow freezing of human blastocysts. Reprod Biomed Online 2005; 11: 53–7. 12. Whittingham D, Leibo S, Mazur P. Survival of mouse embryos frozen to −196° and −269°C. Science 1972; 178: 411–14. 13. Rall W, Fahy G. Ice-free cryopreservation of mouse embryos at −196°C by vitrification. Nature 1985; 313: 573. 14. Kuleshova L, Shaw J, Trounson A. Studies on replacing most of the penetrating cryoprotectant by polymers for embryo cryopreservation. Cryobiology 2001; 43: 21–31. 15. Rall W. Factors affecting the survival of mouse embryos cryopreserved by vitrification. Cryobiology 1987; 24: 387–402. 16. Kuleshova L, Lopata A Vitrification can be more favorable than slow cooling. Fertil Steril 2002; 78: 449–54. 17. Kuwayama M, Vajta G, Kato O et al. Highly efficient vitrification method for cryopreservation of human oocytes. Reprod Biomed Online. 2005; 11: 300–8. 18. Kuwayama M, Vajta G, Leda S et al. Comparison of open and closed methods for vitrification of human embryos and the elimination of potential contamination. Reprod Biomed Online. 2005b; 11: 608–14. 19. Vajta G, Nagy Z. Are programmable freezers still needed in the embryo laboratory? Review
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on vitrification. Reprod Biomed Online 2006; 7: 623–33. Martino A, Songsasen N, Leibo S. Development into blastocysts of bovine oocytes cryopreserved by ultra-rapid cooling. Biol Reprod 1996; 54: 1059–69. Vajta G, Holm P, Greve T et al. Vitrification of porcine embryos using the Open Pulled Straw (OPS) method. Acta Vet Scand 1997; 38: 349–52. Vajta G, Kuwayama M, Holm P et al. Open pulled straw vitrification: a new way to reduce cryoinjuries of bovine ova and embryos. Mol Reprod Dev 1998; 51: 53–8. Lane M, Schoolcraft WB, Gardner D. Vitrification of mouse and human blastocysts using a novel cryoloop container-less technique. Fertil Steril 1999; 72: 1073–78. Liebermann J, Tucker M. Effect of carrier system on the yield of human oocytes and embryos as assessed by survival and developmental potential after vitrification. Reprod 2002; 124: 483–9. Cremades N, Sousa M, Silva J et al. Experimental vitrification of human compacted morulae and early blastocysts using fine diameter plastic micropipettes. Hum Reprod 2004; 19: 300–5. Hamawaki A, Kuwayama M, Hamano S. Minimum volume cooling method for bovine blastocyst vitrification. Theriogenology 1999; 51: 165. Choi D, Chung H, Lim J et al. Pregnancy and delivery of healthy infants developed from vitrified blastocysts in an IVF-ET program. Fertil Steril 2000; 74: 838–39. Mukaida T, Nakamura S, Tomiyama T et al. Vitrification of human blastocysts using cryoloops: clinical outcome of 223 cycles. Hum Reprod 2003a; 18: 384–91. Mukaida T, Nakamura S, Tomiyama T et al. Successful birth after transfer of vitrified human blastocysts with use of a cryoloop containerless technique. Fertil Steril 2001; 76: 618–20. Vanderzwalmen P, Bertin G, Debauche C et al. Vitrification of human blastocysts with the Hemi-Straw carrier: application of assisted hatching after thawing. Hum Reprod 2003; 18: 1504–11. Scheffen B, Vanderzwalmen P, Massip A et al. A simple and efficient procedure for preservation of mouse embryos by vitrification. Cryo Letters; 1986; 7: 260–9. Massip A, Vanderzwalmen P, Scheffen B et al. Pregnancies following transfer of cattle embryos preserved by vitrification. Cryo-Letters 1986; 7: 270–3.
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33. Kasai M, Komi JH, Takakamo A. A simple method for mouse embryo cryopreservation in a low toxicity vitrification solution, without appreciable loss of viability. J Reprod Fertil 1990; 89: 91–7. 34. Kasai M, Nishimori M, Zhu SE et al. Survival of mouse morulae vitrified in an ethylene glycol-based solution afer exposure to the solution at various temperatures. Biol Reprod 1992; 47: 1134–39. 35. Mahmoudzadeh A, Van Soom A, Ysebaert M et al. Comparison of two-step vitrification versus controlled freezing on survival of in vitro produced cattle embryos. Theriogenology, 1994; 42: 1387–97. 36. Vanderzwalmen P, Delval A, Chatziparasidou A et al. Pregnancies after vitrification of human day 5 embryos. Hum Reprod 1997; 12 (Suppl): 98. 37. Walker D. Vitrification versus programmable rate freezing of late stage murine embryos: a randomised comparison prior to application in clinical IVF. Reprod Biomed Online 2004; 8: 558–68. 38. Zheng W, Zhuang G, Zhou C et al. Comparison of the survival of human biopsied embryos after cryopreservation with four different methods using non-transferable embryos. Hum Reprod 2005; 20: 1615–18. 39. Liebermann J, Tucker MJ. Comparison of vitrification and conventional cryopreservation of day 5 and day 6 blastocysts during clinical application. Fertil Steril 2006; 86: 20–6. 40. Vanderzwalmen P, Bertin G, Debauche C et al. Births after vitrification at morula and blastocyst stages: effect of artificial reduction of the blastocoelic cavity before vitrification. Hum Reprod 2002; 17: 744–51. 41. Kuwayama M. Highly efficient vitrification for cryopreservation of human oocytes and embryos: the Cryotop method. Theriogenology 2006; 67: 73–80. 42. Bielanski A, Bergeron H, Lau PCK et al. Microbial contamination of embryos and semen during long-term banking in liquid nitrogen. Cryobiology 2003; 46: 146–52. 43. Tedder R, Zuckerman M, Goldstone A et al. Hepatitis-B transmission from contaminated cryopreservation tank. Lancet 1995; 346: 137–40. 44. Bielanski A, Nadin-Davis S, Sapp T et al. Viral contamination of embryos cryopreserved in liquid nitrogen. Cryobiology 2000; 40: 110–1. 45. Fountain D, Ralston M, Higgins N et al. Liquid nitrogen freezers: a potential source of microbial contamination of hematopoietic
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stem cell components. Transfusion 1997; 37: 585–91. Larman M, Sheenan C, Gardner D. Vitrification of mouse pronuclear oocytes with no direct liquid nitrogen contact. Reprod Biomed Online 2006; 12: 66–9. Isachenko V, Montag M, Isachenko E et al. Aseptic technology of vitrification of human pronuclear oocytes using open-pulled straws. Hum Reprod 2005; 20: 492–6. Camu A, Clairaz P, Ersham A et al. The comparison of the process of five different vitrification devices. Gynécol Obstét Fertil 2006; 34: 737–45. Kasai M, Mukaida T Cryopreservation of animal and human embryos by vitrification. Reprod Biomed Online 2004; 9: 164–70. Lieberman J, Dietl J, Vanderzwalmen P et al. Recent developments in human oocytes, embryo and blastocyst vitrification: where are we now? Reprod Biomed Online 2003; 7: 623–33. Liebermann J, Tucker M, Graham J et al. Blastocyst development after vitrification of multipronucleate zygotes using the Flexipet denuding pipette. Reprod Biomed Online 2002; 4: 146–50. Park S, Kim E, Oh J et al. Ultra-rapid freezing of human multipronuclear zygotes using electron microscope grids. Reprod 2000; 15: 1787–90. Jelinkova L, Selman H, Arav A et al. Twin pregnancy after vitrification of 2 pronuclei human embryos. Fertil Steril 2002; 77: 412–4. Selman H, El-Danasouri I. Pregnancies derived from vitrified human zygotes. Fertil Steril 2002; 77: 422–3. Mukaida T, Wada S, Takahashi K et al. Vitrification of human embryos based on the assessment of suitable conditions for 8-cell mouse embryos. Hum Reprod 1998; 13: 2874–9. El-Danasouri I, Selman HA. Successful pregnancies and deliveries after a simple vitrification protocol for day 3 human embryos. Fertil Steril 2001; 76: 400–2. Gottumukkala A, Geddam B, Kota K et al., Vitrification of human 8-cell embryos, a modified protocol for better pregnancy rates. Reprod Biomed Online 2005; 11: 434–7. Mukaida T, Takahashi K, Kasai M. Blastocyst cryopreservation: ultrarapid vitrification using cryoloop technique. Reprod Biomed Online 2003; 6: 221–5. Mukaida T, Takahashi K, Goto T et al. Ultrarapid vitrification using cryoloop technique
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for human blastocyst cryopreservation. Fertil Steril 2005; 84 (Suppl) S454. Zech N, Lejeune B, Zech H et al. Vitrification of hatching and hatched human blastocysts: effect of an opening in the zona pellucida before vitrification. Reprod Biomed Online 2005; 11: 355–61. Vanderzwalmen P, Lejeune B, Stecher A et al. Survival of day 3 and day 5 embryos following vitrification in aseptic and non-aseptic conditions: a prospective randomized analysis. Fertil Steril 2005; 84 (Suppl), S175. Son W, Yoon S, Yoon H et al. Pregnancy outcome following transfer of human blastocysts vitrified on electron microscopy grids after induced collapse of the blastocoele. Hum Reprod 2003; 18: 137–9. Hiraoka K, Hiraoka K, Kinutani M, Kinutani K. Blastocoele collapse by micropipetting prior to vitrification gives excellent survival and pregnancy outcomes for human day 5 and 6 expanded blastocysts. Hum Reprod 2004; 19: 2884–8. Mukaida T, Oka C, Goto T et al. Artificial shrinkage of blastocoeles using either a microneedle or a lazer pulse prior to the cooling steps of vitrification improves survival rate and pregnancy outcome of vitrified human blastocysts. Hum Reprod 2006; 21: 3246–52. Dumont-Hassan, M, Aubriot, F, Cohen, J et al. Viability of cryopreserved blastocysts: sequential medium versus coculture. 11th World Congress on In Vitro Fertilization and Hum Reprod Genetics 1999; 129: 230, abstr. Massip, A, Mermillod P. Dinnyes A. Morphology and biochemistry of in-vitro produced bovine
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embryos: implications for their cryopreservation. Hum Reprod 1995; 10: 3004–11. Rizos D, Gutierrez-Adan A, Perez-Garnelo S et al. Bovine embryo culture in the presence or absence of serum: implication for blastocysts development, cryotolerance, and messenger RNA expression. Biol Reprod 2003; 68: 236–43. Mucci N, Aller J, Kaiser G et al. Effect of estrous cow serum during bovine embryo culture on blastocyst development and cryotolerance after slow freezing or vitrification Theriogenology 2006; 65: 1551–62. Shaw J, Jones G. Terminology associated with vitrification and other cryopreservation procedures for oocytes and embryos. Hum Reprod Update 2003; 9: 583–605. Vanderzwalmen P, Gaurois B, Ectors F et al. Some factors affecting successful vitrification of mouse blastocysts. Theriogenology 1988; 30: 1177–83. Kasai M, Nishimori M, Zhu S et al. Survival of mouse morulae vitrified in an ethylene glycolbased solution after exposure to the solution at various temperatures. Biol Reprod 1992; 47: 1134–39. Klug S, Merker H, Jackh R. Effects of ethylene glycol and metabolites on in vitro development of rat embryos during organogenesis. Toxicology In Vitro 2001; 15: 635–42. Takahashi K, Mukaida T, Goto T et al. Perinatal outcome of blastocyst transfer with vitrification using cryoloop: a 4-year follow-up study. Fertil Steril 2005; 84: 88–92.
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Vitrification of blastocysts using the Cryoloop technique
10B
Tetsunori Mukaida and Katsuhiko Takahashi
INTRODUCTION In assisted reproductive technology (ART), cryopreservation of embryos has proved important to enable the best use of supernumerary embryos. In the cryopreservation of embryos, there is a risk of various types of injury.1,2 Among them, the formation of intracellular ice appears to be the most damaging. The first strategy to prevent intracellular ice from forming was to adopt a lower concentration of cryoprotectant and a long slow-cooling stage. This slow freezing method has proved effective for embryos of a wide range of mammalian species. Unlike embryos of laboratory animals and domestic animals, in which dimethylsulfoxide (DMSO), glycerol, or ethylene glycol (EG) is commonly used as the cryoprotectant, human embryos at early cleavage stages have most often been frozen in a solution of propanediol supplemented with sucrose,3 although those at the blastocyst stage have more frequently been frozen with glycerol and sucrose.4–6 With slow freezing, however, it is difficult to eliminate completely injuries occurring from ice formation. Furthermore, the slow freezing method requires a long period of time before embryos are stored in liquid nitrogen. In 1985, Rall and Fahy reported7 an innovative approach called vitrification, in which injuries related to ice formation are minimized by using very high concentrations of cryoprotectant. This approach simplifies the cooling process, because embryos can be rapidly cooled directly in liquid nitrogen. Although embryos subjected to vitrification
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are liable to be injured by the toxicity of the high concentration of cryoprotectant, the method has been refined and proved effective for the cryopreservation of embryos at various stages of development in laboratory and domestic species. In 1998, we showed that vitrification using an EG-based vitrification solution (EFS20–408) with conventional cryostraws is effective for human embryos at the 4–8-cell stage.9 The effectiveness of vitrification was confirmed for human embryos at the 8–16-cell stage10 and the morula stage,11 also using EG-based solutions. Recent advances in culture systems with sequential media have made it possible to develop human IVF embryos to the blastocyst stage quite easily. Because the blastocyst is better suited to the uterine environment, and blastocyst formation is a form of selection for more viable embryos, blastocyst transfer has become a promising option to raise the overall pregnancy rate.12,13 Accordingly, the need to cryopreserve human blastocysts is increasing. Menezo et al.14 cryopreserved human blastocysts which were developed in a co-culture system using the slow freezing method with glycerol and obtained reasonable clinical results (27% pregnancy rate, 17% implantation rate). However, results reported by other clinics have not been consistent.15–17 Menezo et al.14 speculated that the cryopreservation outcome is influenced by the culture conditions, such as the co-culture system. Recently, human blastocysts were successfully vitrified in straws.18 However, our own attempts to vitrify human blastocysts using straws resulted in only 45% survival (39/86,
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unpublished data). Vanderzwalmen et al.19 also reported a low pregnancy rate with human blastocysts vitrified in straws. This is probably because human blastocysts are much less permeable to cryoprotectant and water, since we have observed that they shrink more slowly than mouse and bovine blastocysts in the cryoprotectant solution. This suggests that human blastocysts are more likely to be injured by intracellular ice. Increased rates of cooling and warming can help circumvent the problem of intracellular ice formation in less permeable embryos. Faster rates of cooling and warming can be achieved by minimizing the volume of the solution with which embryos are vitrified, i.e. by using minute tools such as electron microscope grids,20 open pulled straws,21 or Cryoloops22 (see Kasai2 for a review). We demonstrated the first successful birth of a baby following transfer of human blastocysts vitrified using Cryoloops.23 Since our original report, we have continued to use this vitrification approach in our group of two clinics (Tokyo, Osaka, Hiroshima HART clinic) for the cryopreservation of blastocysts on day 5 and day 6. This chapter includes our summary of the clinical outcomes from our blastocyst vitrification program for the past 6 years, which confirms the effectiveness of the Cryoloop technique for the cryopreservation of human blastocysts. As previously mentioned, the first successful outcome of blastocyst vitrification using a Cryoloop was originally reported in 2001, and in 2003 the summary of the clinical results with 223 warming cycles confirmed the effectiveness of the Cryoloop technique for the cryopreservation of human blastocysts. However, the report in 2003 revealed the survival rates were dependent on the developmental stage of blastocysts, and were negatively correlated with the expansion of the blastocoel.24 The survival rate of early blastocysts with a smaller blastocoelic cavity, which was scored 1 and 2 according to Gardner’s criteria25 were 87% (48/55) and 97% (62/64), respectively. Also, full blastocysts
lacking an expanded blastocoelic cavity, which were scored 3, had a survival rate of 89% (99/111). The total survival rate of blastocysts scored 1–3 was 91% (209/230). However, the survival rate of both expanded and hatching blastocysts, scored 4 and 5, respectively, was 85.0% (288/339), which was significantly lower than that of the score 1–3 group (P < 0.05). We therefore postulated that a large blastocoel might lessen cryopreservative potential due to ice crystal formation during the rapid cooling phase of vitrification. In order to overcome this problem, shrinkage of the blastocoel was thought to be the appropriate approach. Several studies reported an increase in the survival rate of blastocysts when the volume of the blastocoel was artificially reduced with glass microneedle,26 29-gauge needle,27 or micropipetting with a hand-drawn Pasteur pipette.28 Since 2003, therefore, we have added artificial shrinkage (AS) after puncturing the blastocoel with a microneedle, or laser pulse using a Cryoloop technique prior to vitrification to improve the survival rate and clinical outcome of vitrified blastocyst transfer programs. In 2006, we reported the effectiveness of AS prior to vitrification, including the confirmation of the safety of this procedure.29
VITRIFICATION OF BLASTOCYSTS The protocol for the Cryoloop vitrification of blastocysts was adopted from a previous report,22,30 albeit with slight modifications, and has been described previously (Figures 10B.1 and 10B.2).22,23,28 The Cryoloop consisted of a nylon loop (20 µm wide; 0.5–0.7 mm in diameter) mounted on a stainless steel pipe inserted into the lid of a cryovial (Hampton Research, Laguna Niguel, CA, USA). A metal insert on the lid enables the use of a stainless steel handling rod with a small magnet (Crystalwand, Hampton Research) for manipulation of the loop at low temperature.31 One to three blastocysts were vitrified in a Cryoloop after a two-step procedure to load the
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Freeze loops
a
Cap
b
c Figure 10B.1 (a) Capping portion of the cryovial, which consists of a minute nylon loop (20 µm wide, 0.5–0.7 mm in diameter) mounted on a stainless steel tube inserted into the lid of a cryovial. (b) Container part of the cryovial. The shape and the size are similar to those of Nunc Cryovials used for semen cryopreservation. (c) The capping portion attached to a stainless steel handling rod with a small magnet for manipulation of the loop at low temperature.
blastocysts with cryoprotectants at ~37°C. As the base medium, HEPES-buffered modified HTF medium containing 5 mg/mL human serum albumin (HSA) was used. Initially, blastocysts were placed in the base medium containing 7.5% (v/v) DMSO and 7.5% (v/v) EG (cryoprotectant solution I). After 2 min, the blastocysts were transferred into cryoprotectant solution II, which is the base medium containing 15% (v/v) DMSO, 15% (v/v) EG, 10 mg/mL of Ficoll 70 (average molecular weight 70 000; Pharmacia Biotech, Uppsala, Sweden), and 0.65 mol/L sucrose (cryoprotectant solution II). Both cryoprotectant solutions had been warmed briefly in an incubator at 37°C and blastocysts were handled on the stage warmer of a dissecting microscope at 37°C. While the blastocysts were suspended in cryoprotectant solution II, a Cryoloop was dipped into cryoprotectant solution II in order to create a thin, filmy layer of solution, by surface tension, on the nylon loop. The blastocysts were then washed quickly in solution II and transferred onto the filmy layer on the nylon loop using a micropipette.
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Immediately after the loading of blastocysts, the Cryoloop was plunged into liquid nitrogen. The time blastocysts were exposed to solution II before cooling was limited to 25–30s. Using a stainless steel rod, the loop containing the blastocysts was sealed in a cryovial, which was previously submerged in liquid nitrogen. The vials were attached to standard canes and stored in liquid nitrogen. The entire procedure was completed within 5 min. Vitrified blastocysts were kept in the liquid nitrogen tank for from 1 month to 5 years depending on the patient’s background.
WARMING OF BLASTOCYSTS, ASSISTED HATCHING, AND ASSESSMENT OF SURVIVAL In a 4-well multidish, ~1 mL of base medium containing 0.33 mol/L sucrose in no. 1 well, base medium containing 0.2 mol/L sucrose in no. 2 well, and base medium in no. 3 well were warmed briefly in an incubator at 37°C and then placed on the stage warmer of a
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b
a
c
d
Figure 10B.2 (a) A minute nylon loop (20 µm wide, 0.5–0.7 mm in diameter) mounted on a stainless steel pipe under × 100 magnification. (b) A thin layer of the vitrification solution on the nylon loop after dipping the loop into the solution. (c) Under a dissecting microscope, the capping portion of cryovial with the Cryoloop attached with a stainless steel handling rod for manipulation is held by the left hand, and a pulled Pasteur pipette with blastocysts is held by the right hand. Prior to loading, blastocysts are rinsed several times in small drops of final vitrification solution (solution II, see text for formulation) on the lid of a culture dish. (d) Blastocysts on the loop with thin layer of vitrification solution.
dissecting microscope. With the cryovial submerged in liquid nitrogen, the vial was opened with the aid of the stainless steel rod, and the loop containing blastocysts was removed from the liquid nitrogen, and placed directly and quickly into the well containing the 0.5 mol/L sucrose solution (no.1 well). Blastocysts immediately fell from the loop into the solution. Thus blastocysts were warmed and diluted instantly at around 37°C adjusted by the stage warmer. After 2 min, the blastocysts were transferred to the 0.25 mol/L sucrose solution in no. 2 well. After an additional 3 min, blastocysts were washed and kept in the base medium in no. 3 well for 5 min.
During this 5 min, assisted zona hatching was always performed on warmed blastocysts with either acidic tyrodes solution (PH 2.3) as previously described,32,33 or laser pulse29 on warmed blastocysts (Figure 10B.3). Then blastocysts were returned to Blast Assist Medium 2 (Medicult) for further culture until transfer. About 2–3 h after warming, the appearance of the blastocysts was examined on an inverted microscope at 400 times magnification, and survival was assessed based on the morphological integrity of the blastomeres, inner cell mass, and trophectoderm, and re-expansion of the blastocoel. The surviving blastocysts were scored as to developmental stage and were
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Zona hatching by laser pulse
After 2–3 hr
Figure 10B.3
Assisted hatching using laser pulses on warmed blastocyst prior to transfer.
graded according to quality as described in the section on grading of blastocysts.
ARTIFICIAL SHRINKAGE OF EXPANDED BLASTOCYSTS When we cryopreserve human blastocysts using the above vitrification technique at full or more advanced stages including expanded and hatching blastocysts, the artificial shrinkage (AS) procedure is always performed prior to vitrification (Figures 10B.4 and 10B.5). The technique of shrinkage using microneedle puncture has been described previously.25 Briefly, about 10 min before the vitrification, the expanded blastocysts were placed in 50 µL drops of pre-equilibrated Blast Assist Medium 2. The expanded blastocyst was held with a holding pipette connected to the micromanipulator, and the inner cell mass (ICM) was placed at the 6 or 12 o’clock position; a glass microneedle was pushed through the cellular junction of the trophectoderm into the blastocoel cavity until it shrank (Figure 10B.4). After removing the microneedle, contraction of the blastocoel was observed within a few minutes. After complete shrinkage of the
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blastocoel, the blastocyst was vitrified and stored in the liquid nitrogen tank. Since September of 2004, a laser pulse generated by laser system ZILOS-tkTM (Hamilton Thorn Bioscience Inc., Beverly, MA, USA) has been introduced to perform the artificial shrinkage, instead of microneedle puncture. The ICM should be located away from the targeted point of the laser pulse. One single laser pulse (200 ms) targeted at the cellular junction of the trophectoderm creates a hole to induce collapsing of the blastocoelic cavity. The blastocoel of the expanded blastocyst shrank immediately (Figure 10B.5). With the use of this laser system, it is not necessary to hold and locate the expanded blastocyst with a holding pipette connected to micromanipulator. The laser technique makes the procedures simple and convenient.
LABORATORY PROTOCOL In Appendix 10B.1 of this chapter, our laboratory manual of vitrification for human blastocysts is detailed in order to help establish this technique in the clinical ART laboratory.
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a
b
d
e
c
f
Figure 10B.4 Artificial shrinkage of expanded blastocyst with the microneedle: (a) holding the expanded blastocyst with holding micropipette connected to micromanipulator; (b) insertion of the microneedle inside the blastocoel at a point away from the inner cell mass; (c) puncture through the blastocoel and removing the microneedle gradually; (d) beginning of shrinkage 10 s after puncture; (e) partial shrinkage 30 s after puncture; and (f) complete shrinkage 1 min after puncture.
a
b
d
e
c
f
Figure 10B.5 Artificial shrinkage of expanded blastocyst with a single laser pulse: (a) prior to the artificial shrinkage; (b) a single laser pulse at the point of the cellular junction of trophectoderm cell at a point away from the inner cell mass (circled); (c) beginning of shrinkage 5 s after laser shooting, arrows indicate formation of perivitelline space because of contraction; (d) shrinkage 10 s after laser shooting; (e) shrinkage 20 s after laser shooting; and (f) almost complete shrinkage 30 s after laser shooting.
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TROUBLESHOOTING (1) Warming solutions: Instead of warming solutions after preparation in a 4-well multidish, it is possible to warm the solutions and then prepare them in the dish. (2) Preventing evaporation of the vitrification solution: Because the amount of vitrification solution on the Cryoloop is very small, the solution is liable to be concentrated by evaporation, which increases its toxicity. To prevent this, the Cryoloop should be loaded with the vitrification solution just before the loading of embryo(s) on the loop. The amount of vitrification solution on the loop need not be minimal, as long as the drop is kept on the loop by surface tension. A dewar flask containing liquid nitrogen should be placed nearby to enable quick cooling. (3) Quick warming: At warming, the loop containing embryo(s) should be soaked in the sucrose solution as quickly as possible, and to ensure that the loop with vitrified embryo(s) is never held in the air. Again, liquid nitrogen and the sucrose solution should be placed nearby. When the loop is dipped in the sucrose solution, the steel pipe portion should not be immersed to prevent bubbling. (4) Morphology of the recovered blastocyst: At recovery in an isotonic solution, the blastocoel of the blastocyst will be collapsed and the embryo may look like a ‘morula’. However, the blastocoel should reform within 1–3 h of culture. (5) Cryoprotectant equilibration: With this vitrification protocol complete equilibration of cryoprotectant is not expected. Whenever the blastocoelic cavity is large, it is recommended to collapse the cavity in order to improve blastocoel survival. (6) Temperature equilibration: With this vitrification protocol equilibration time is just 2 min and exposure of vitrification solution is 30 sec. This means the status inside the vitrified blastocyst may not be fully
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equilibrated and will be metastable situation. Therefore, vitrified embroys should always be kept under the phase transition (liquid/ solid) tempertature (approximately −1300C) in order to avoid fracture damage. When the samples are transferred between tanks, an unexpected rising in temperature can occur; it is critical to avoid such transient temperature fluctuations.
PATIENTS, EMBRYO CULTURE, AND GRADING OF BLASTOCYSTS We perform blastocyst transfer programs on patients who have had previous multiple failures of conventional day 2 or day 3 embryo transfer and who have agreed to use of the Cryoloop vitrification method to cryopreserve their supernumerary blastocysts obtained 5 or 6 days after oocyte retrieval. Women were treated with gonadotropin releasing hormone (GnRH) agonist and human menopausal gonadotropin (hMG) using either a long or a short treatment protocol. An injection of human chorionic gonadotropin (hCG) was given when dominant follicles reached a diameter of ~18 mm. Oocytes were collected 36 h after hCG administration using the vaginal ultrasound-guided procedure. The oocytes were inseminated by either conventional IVF or intracytoplasmic sperm injection (ICSI), and were incubated in HTF medium containing 5 mg/mL of HSA or Blast Assist Medium 1, respectively, in a 4-well multidish under mineral oil in a CO2 incubator at 37°C. Fertilization was assessed 15–18 h after insemination by the presence of two pronuclei. Zygotes were washed well and cultured in groups of two or three in Blast Assist Medium 1 for 48 h, and then in Blast Assist Medium 2 for another 48–72 h. All culturing of embryos was performed in a CO2, O2, N2 environment (6:5:89%, respectively). In conventional cases, one or two 4–8-cell embryos were transferred to the patient after 24–48 h of culture, and only supernumerary embryos were extended to culture in Blast Assist Medium 2.
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On day 5 after the oocyte pick up, blastocyst development was examined. Only on day 5, each embryo developed to the blastocyst stage was scored depending on the developmental stage and graded according to quality criteria25, 34 with slight modifications.24 Briefly, blastocysts were first given a numerical score from 1 to 6 on the basis of their degree of development. Second, the blastocysts were graded in three ranks based on morphological appearance. For example, the ICM was graded as A (many tightly packed cells), B (several loosely grouped cells), or C (few cells); and the trophectoderm was graded as A (many cells forming a cohesive epithelium), B (fewer cells forming a loose epithelium), or C (very few large cells). When patients had their fresh embryos transferred on day 2–3, all the remaining embryos were cultured to allow those that developed into blastocysts to be vitrified. Patients who received transfers of fresh blastocysts had all their remaining supernumerary blastocysts vitrified. On day 5, if at least one supernumerary blastocyst was graded as A or B, all the blastocysts of the patient were vitrified regardless of the developmental stage and the grading. In a few cases, compacted morulae were also vitrified with the blastocysts. If all the blastocysts of the patient were graded C, they were not cryopreserved. On day 6, if at least one blastocyst had a large blastocoel (i.e. scored as 3–6) and was graded as A or B, all the developed blastocysts scored as 3–6 were vitrified.
TRANSFER OF VITRIFIED BLASTOCYSTS AND ASSESSMENT OF PREGNANCY All women received transdermal estradiol (Estrana 0.4 mg/day; Hisamitsu, Tokyo, Japan) with GnRH agonists for preparation of the endometrium. Administration of both progesterone (50 mg in oil, daily) and hCG 10 000 IU was initiated when the endometrial thickness achieved more than 10 mm. On day 5 after the initiation of progesterone treatment, the
Table 10B.1 Clinical outcome of vitrified blastocyst transfer program (November 1999–April 2006) Attempted cycles (n) Warmed vitrified blastocysts (n) Survived blastocysts (n) Survival rate (%) Transferred cycles (n) Average no. of blastocysts transferred Clinical pregnancies/blastocyst transfer (n (%) Implantation (n %) Ongoing pregnancies (n) Miscarriages (n %)
1549 3496 3176 90.8 1496 1.82 750 (50.1) 945 (38.5) 213 174 (23.2)
blastocysts were warmed and surviving blastocysts were transferred into the patient’s uterus. Most patients received one or two blastocysts; occasionally three blastocysts were transferred depending on the patient’s background (i.e. multiple failures of ART). In potential pregnancy, serum hCG levels were assessed 14 days after oocyte pick up (OPU), then implantation and clinical pregnancy was confirmed by the presence of fetal heart activity or a gestational sac.
CLINICAL RESULTS OF VITRIFIED BLASTOCYST TRANSFER Table 10B.1 summarizes the clinical results of a vitrified blastocyst transfer program using the Cryoloop technique between November 1999 and April 2006. A total of 3496 blastocysts originating from 1549 cycles of oocyte collection from 1258 patients were vitrified and warmed. Mean age was 35.2 years. Vitrified blastocysts were generated in three categories of patient groups. Group 1 had their fresh embryos transferred on day 2–3, and all the remaining embryos were cultured to allow those that developed into blastocysts to be vitrified. Group 2 received transfers of fresh blastocysts and had all their remaining supernumerary blastocysts vitrified. Group 3 had no fresh embryo transfer due to either ovarian hyperstimulation syndrome (OHSS) symptoms or attempting vitrified blastocyst transfer intentionally along with controlled endometrial cycle supplemented by exogenous
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female hormones, in order to overcome multiple implantation failures, because uterine receptivity under controlled endometrial cycles was indicated as better than that under stimulated cycles. In HART clinics, fresh blastocyst transfer is intentionally avoided after two or three failures of fresh transfer. One of the reasons why fresh transfer is intentionally avoided is that ovarian hyperstimulation does not always create a suitable uterine receptivity and environment for implantation compared with controlled endometrial preparation using exogenous hormone. Group 1 included 580 cycles (37.4% of total), group 2 included 540 cycles (34.9% of total), and group 3 included 429 cycles (27.7% of total). After warming of vitrified blastocysts for transfer, 3176 (90.8%) of the embryos survived. In 45 cycles, no blastocysts survived and embryo transfer was not conducted. In five cycles, viable blastocysts were obtained but embryo transfer was cancelled because the number of cells that survived and the quality of the embryos were low. A total of 2722 blastocysts were transferred into 1213 patients in 1496 cycles. The mean number of blastocysts transferred per cycle was 1.8. Of 1496 transfers, 750 resulted in clinical pregnancy (confirmed by gestational sac in the uterus); the pregnancy rate was 48.4% per warming cycle, and 50.1% per transfer. A total of 174 pregnant cycles ended in miscarriage (23.2%), with 455 healthy babies being born in 361 deliveries, and with 213 pregnancies ongoing. No bias in the sex ratio was observed since 232 babies were boys and 223 were girls. In comparison of 935 pregnancies established from fresh blastocyst transfer in our group of clinics during the same period, 178 (19%) resulted in miscarriages.
and/or hatching blastocysts vitrified were retrospectively evaluated. The average age of the patients was 35.6 years (range 27–41). In total 266 cycles had vitrified blastocyst transfer with artificial shrinkage. In four cycles, no blastocysts survived and embryo transfer was cancelled. There were 502 vitrified blastocysts warmed for transfer, and 488 survived. Survival rate was 97.2%. Of the vitrified blastocysts 448 were transferred, and the mean number of blastocysts transferred per cycle was 1.7. Of 266 transfers, 160 resulted in clinical pregnancy, with a pregnancy rate of 59.3% per warming cycle (160/270) and 60.2% per transfer (160/266). Results of vitrified expanded and hatching blastocysts in our previous study reported in 2003 served as a control group. Survival rate of both expanded and hatching blastocysts, scored 4 and 5, respectively, was 85.0% (288/339). A statistical difference was noted between the study and control groups (P< 0.05). When the pregnancy rate of the study group was compared with the control group, a statistically significant improvement was noticed in the AS group (60.2% vs. 34.1%; P < 0.01). Furthermore, we performed a preliminary comparisons between the results achieved by using microneedle or laser pulse for blastocoel shrinkage to show the differences between methodologies for AS. AS using a microneedle was performed in 240 cycles with 462 blastocysts, and AS using a laser pulse was performed in 26 cycles with 40 blastocysts. The survival rates achieved with the two methods were similar (microneedle 97.2% vs. laser pulse 97.5%). The mean number of survived blastocysts transferred was also similar. Clinical pregnancy, implantation, and miscarriage rates were also similar. No statistical difference was observed in the results achieved with the two methods (Table 10B.3).
CLINICAL RESULTS OF ARTIFICIAL SHRINKAGE PROCEDURES
PERINATAL OUTCOME OF VITRIFIED BLASTOCYSTS
In order to show the effectiveness of AS, 270 cycles in 245 patients who had only expanded
We reported the perinatal outcome of our vitrified blastocyst transfer program in 2004.35
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Table 10B.2 Perinatal outcome of infants conceived after vitrified blastocyst transfer (November 1999–April 2006) n Live born Male Female Vaginal delivery Cesarean section Mean gestational age (weeks) Preterm birth (< 37 weeks) Mean birth weight (g) < 1500 1500–2500 >2 500 Twins Triplets
%
455 (361 deliveries) 232 50.9 223 49.1 164 45.4 197 54.6 37.5 ± 3.4 74 2663 ± 779 15 180 260 76 10
20.5
21.0 2.7
Table 10B.3 Clinical outcome of artificial shrinkage using either a microneedle or a laser pulse Microneedle Cycles with vitrified blastocyst transfer (n) Blastocysts vitrified (n) Survived blastocyst (n) Survival rate (%) Mean no. of blastocysts transferred Clinical pregnancies (%) Implantation (n) Implantation rate (%) Cycles miscarried (%)
Laser pulse
240
26
462 449 97.2 1.6
40 39 97.5 1.4
144 60.0 191 46.5 32 22.2%
16 61.5 18 48.6 3 18.8
This report summarized the comparison between perinatal outcome after fresh blastocyst transfer and that of vitrified blastocysts transfer. Between April 2000 and June 2003, we performed 435 cycles of vitrified-warmed blastocyst transfer in our clinics. Of which, 138 were IVF and 297 were ICSI. During the same period, we performed 662 fresh blastocyst transfers. Of which, 184 were IVF and 478 were ICSI. Many patients were included
in both groups because they received vitrified blastocyst transfer due to failure of conception, or were having their second baby after a successful birth following fresh blastocyst transfer. The mean age of women was 34.5 ± 4.2 years (range 25–40) in the vitrified group, and 35.7 ± 4.4 years (range 24–43) in the fresh group. The vitrified group was significantly younger (P< 0.05), indicating that younger women had a high number of good quality supernumerary blastocysts vitrified. There were 34 patients in the vitrification group (23.9%) and 43 in the fresh group (21.6%) lost to follow-up (either no responses or changed address). The survival rate of vitrified blastocysts was 85.7% (967/1129). We transferred 716 warmed-vitrified blastocysts in 413 cycles out of 435 attempted cycles (mean number of embryo transfers 1.7). Of the cycles transferred, 182 resulted in pregnancy (44.1% per transfer), and the implantation rate was 29.0%. Of which, 40 resulted in miscarriage (22.0%) and 108 deliveries were reported. In fresh blastocyst transfer, of 662 cycles, transfer was carried out in 602 cycles with 1252 blastocysts (mean number of embryos transferred 2.1) and 267 became pregnant (44.4% per transfer), and the implantation rate was 23.4%. Of the pregnancies, 68 resulted in miscarriage (25.5%) and 153 deliveries were reported. There were no statistical differences between the groups. We performed retrospective analysis of the data obtained from questionnaires sent to the parents, who were asked to send us information after delivery, and/or from referring physicians in cases of abnormal outcome. We obtained 108 responses out of 142 parents (76.1%). Of 108 deliveries, we evaluated 147 live-born infants, and compared these with 205 live-born infants born from 153 (78.1%) responding parents out of 196 who conceived after fresh blastocyst transfer during the same period. From 108 deliveries with vitrified blastocysts, 147 babies (74 boys and 73 girls) were born. The
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mean gestational age was 38.1 weeks and the mean birth weight was 2601 ± 709 g. There were 20 preterm deliveries (18.5%); 40 sets of twins (27.8%); and four sets of triplets (5.1%). From 153 deliveries following fresh blastocyst transfer, 205 babies (120 boys and 85 girls) were born. The male to female ratio was significantly higher (P < 0.05). The mean gestational age was 37.9 weeks and the mean birth weight was 2593 ± 629 g. There were 19 preterm deliveries (12.4%); 40 sets of twins (26.1%); and six sets of triplets (3.9%). No statistical differences were seen between the groups except for the sex ratio difference. With respect to congenital birth defects, two were observed following vitrification (1.4%), and four in the fresh transfer group (2.0%). In the vitrification group, one dizygotic twin had Treacher–Collins syndrome or mandibulofacial dystosis, and the other had patent ductus arteriosus (PDA). In the fresh transfer group, a baby from a 39-year-old mother died with 21 trisomy at 2 days postpartum, and three minor defects, including anal atresia, PDA, and defect of 4th lumbar spine were noted. The latest results arising between November 1999 and April 2006 for perinatal outcome are shown in Table 10B.2. A total of 455 children were born in 361 deliveries. Of which, 232 babies were male, and 223 babies were female, therefore no bias in the sex ratio was observed. Cesarean section was performed in 197 deliveries and the mean gestational age was 37.5 weeks. Mean birth weight was 2663 g. There were 86 (23.7%) multiple pregnancies and 74 births were preterm (20.5%). Twelve babies in 12 deliveries had either congenital birth defects or neonatal complication (2.7%), including four chromosomal abnormalities (one trisomy 18, three trisomy 21), two multiple anomalies, one death of unknown cause during delivery, one malformation of the skull, two congenital heart malformations, and two minor anomalies.
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DISCUSSION Numerous protocols for the cryopreservation of mammalian embryos have been reported. The protocols can be classified into four methods: original slow freezing, conventional slow freezing, conventional vitrification, and ultra rapid vitrification. Although the principles of cryopreservation are the same, strategies to circumvent various injuries (especially from the formation of intracellular ice) are different. The most suitable protocol should be adopted for each case. For certain types of embryos such as human blastocysts and bovine embryos at earlier stages, ultrarapid vitrification would be a preferable choice, because the survival rates of embryos cryopreserved by other methods have been low. For other embryos, such as mouse embryos, bovine blastocysts, and human embryos at 2–8-cell stages, both slow freezing and conventional vitrification has proved effective. Slow freezing produces more consistent results, because embryos are less likely to be injured by the toxicity of the cryoprotectant and thus can be handled under less strict conditions. However, vitrification has a potential advantage in that higher survival rates can be obtained if conditions, such as temperature and period of exposure of embryos to the cryoprotectant, as well as the skill of pipetting, are optimized. Therefore, vitrification would be a preferred method of cryopreservation to the slow-cooling method because of both the lack of ice crystal formation and the convenience. We have already reported a simple vitrification method using an ethylene glycol-based solution for 4–8cell human embryos frozen in conventional cryostraws.8 Moreover, the success of vitrification procedures has recently been increased by techniques that substantially reduce the volume of the vitrification solution. Among such techniques, the Cryoloop can be considered to be the most refined strategy.21,22 A major difference between the Cryoloop and the conventional straw for vitrification are the cooling/warming rates.
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With the development of sequential culture media, based upon the physiology of the human reproductive tract and the changing physiology of the developing embryos, it is possible to grow viable blastocysts easily in vitro; and because of the resulting high implantation rates, blastocyst transfer will necessarily be a good approach in ART. Accordingly, the need to cryopreserve human blastocysts is increasing. The Cryoloop enables ultrarapid cooling and warming, and this may consistently prevent intracellular ice formation, since we have observed that human blastocysts are dehydrated and concentrated more slowly than in earlier stage embryos, suggesting that intracellular ice is more likely to form in blastocysts. Furthermore, the technique using the Cryoloop is easier and simpler than that using the cryostraw. In this chapter, we have shown that vitrification using the Cryoloop is a clinically useful method for the cryopreservation of human blastocysts.
ADDING ARTIFICIAL SHRINKAGE AS PRIOR TO VITRIFICATION Achievement of pregnancy is related to many different factors. Obviously one of the most important factors to achieve pregnancy is embryo viability. Developing more blastocysts by day 5 should indicate that these have better potential developmental competency and higher viability. Theoretically, the more developed blastocysts should give a better pregnancy rate than that of early stage blastocysts. However, we have observed that the survival rate of expanded (86%) and hatching (82%) blastocysts were significantly lower than that of early stage blastocysts (97%), even after we had established the vitrification steps. This is in accordance with the observation of Vanderzwalmen et al.,19,26 that the efficiency of vitrification of human blastocysts was negatively correlated with the expansion of the blastocoel. Increase in survival rate might contribute to the improvement of the
viability of survived blastocysts in the AS group, and may facilitate implantation. Other reports showing the effectiveness of blastocoel shrinkage contained 25 cycles,26 29 cycles,27 and 49 cycles of transfers. However, in our study (above), 270 cycles of transfer with artificial shrinkage were used to confirm the clinical value of this technique. A possible explanation for the lower survival rate is that the large blastocoel of more developed blastocysts may disturb the efficacy of vitrification due to inappropriate dehydration and permeation of cryoprotectant, which might cause ice crystal formation in the rapid cooling and warming steps during vitrification. A study on mouse blastocysts also reported that survival rates of vitrified blastocysts after a one-step exposure to EFS40 decreased as the volume of the blastocoelic cavity increased.36 In mouse blastocysts, a two-step loading of cryoprotectant was effective in preventing the decrease in post-warming survival of fully expanded blastocysts.37 Bovine blastocysts were also successfully vitrified after a two-step loading of cryoprotectant.38 However, human blastocysts appear to be different25 and are thought to have specific characteristics related to their lower cryoresistance. Such characteristics could be attributable to the permeability of the membrane, which would decrease as the blastocyst develops. Therefore, it seems preferable to cryopreserve human blastocysts on day 5 before the blastocoel fully develops, or even earlier on day 4. Recently, it has been suggested that mechanical damage caused by ice crystal formation can be avoided by reducing the fluid content of the blastocoel of more developed stage blastocysts.25 One of the strategies reported was reducing the size of the blastocoel mechanically. Vanderzwalmen et al.26 reported making a small hole in the trophectoderm with a needle, so causing the blastocoel to shrink. They vitrified shrunken blastocysts in straws in EFS40, and reported that this artificial shrinkage dramatically raised the post-warming survival rate, which resulted
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in eight pregnancies from 35 transfers. Son et al.27 reported using a 29-gauge needle for artificial shrinkage of the blastocoele. Hiraoka et al.28 reported collapsing the blastocoel by micropipetting without puncturing the zona and trophectoderm. An alternative method is to increase the concentration of, and exposure to, the cryoprotectant. The use of a high concentration and the increase in the duration of exposure to the cryoprotectant might be effective for preventing injuries from intracellular ice while allowing sufficient dehydration. However, the risk of an injury from the chemical toxicity of the cryoprotectant increases as the concentration and the duration of exposure is increased. For these reasons, reducing the fluid content of the blastocoel of more developed stage blastocysts is preferred to instead of increasing the concentration of the cryoprotectant. Thus, these authors have reported that the rate of survival was improved after artificial shrinkage of the blastocoel. Similarly, we applied the artificial shrinkage technique in our vitrification protocol using the Cryoloop for human blastocyst cryopreservation. Initially, we collapsed the blastocoel by puncturing it with a fine needle using micromanipulation procedures. Occasionally, we came across cases where collapsing of the blastocoel occurred partially or incompletely, because the elastic plasma membrane of the trophectoderm cells adhered to the opening site of puncture with a microneedle. In those cases, puncturing was performed repeatedly at a different site of the trophectoderm until complete collapse of the blastocoel was achieved. However, since we have applied laser pulse for artificial shrinkage since September 2004, incomplete collapse of the blastocoel has seldom been observed. We speculated that the single laser pulse creates an instant heat effect, that causes cellular degeneration of the trophectoderm, and allows a large enough hole to be collapsed. Also, collapsing the blastocoel by laser pulse
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did not require time for preparation prior to the procedure, such as setting the holding pipette and microneedle for puncturing needle pipette, as well as holding the blastocyst on the micromanipulation stage. It is only necessary to locate the peripheral junction of trophectoderm cells of the expanded blastocyst, and administer a single laser pulse. The safety of the laser application to human embryos and blastocysts such as for blastomere biopsy has already been reported.39,40 When we compared the various techniques of artificial shrinkage in other reports, blastocoel collapse by micropipetting,27 and 29-gauge needle26 do not require the micromanipulation technique. Both techniques can be performed under the dissecting microscope. Micropipetting is much the same as removal of cumulus cells from oocytes for ICSI. However, the size of human oocytes is quite similar to blastocysts depending on the size of the blastocoel relative to the developmental stage of the blastocyst. Thus, for micropipetting it is necessary to prepare several sizes of hand-drawn Pasture pipettes according to the expansion of the blastocoel. Also, shrinkage by 29-gauge needle requires a very precise hand movement, because the edge of the 29-gauge needle is quite large compared with a glass microneedle. The final goal of this technique is complete collapse of the blastocoel in order to facilitate the vitrification steps. Among these techniques, laser pulse is the simplest and most convenient for artificial shrinkage. The only drawback is that it is expensive to purchase the equipment initially. According to our observation, the average time for re-expansion of surviving blastocysts in the AS group tends to be shorter than for the control group. This also might be related to the viability of the surviving blastocyst. In conclusion, we have shown that artificial shrinkage of human expanded and hatching blastocysts prior to vitrification statistically improves the survival and pregnancy rate, and that delivery of healthy babies confirms the safety of this technique.
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SUMMARY For embryo cryopreservation, the vitrification method has many advantages over the slow freezing method: injuries related to ice are less likely to occur; survival of embryos can be maintained at a higher level if conditions for embryo treatment are optimized; and embryos can be cryopreserved by a simple method in a short period without a programmable freezer. Therefore, vitrification is suitable for human embryos, in which a small number of embryos
are cryopreserved frequently. Human embryos at early cleavage stages can be cryopreserved by conventional vitrification using cryostraws, or by ultrarapid vitrification using Cryoloops. Human blastocysts are more efficiently cryopreserved by the ultrarapid approach. Our clinical outcomes show that vitrification of blastocysts using the Cryoloop technique results in high survival and high pregnancy rates, and confirms the safety of this procedure as seen in our perinatal evaluation.
References 1. Kasai M. Simple and efficient methods for vitrification of mammalian embryos. Anim Reprod Sci 1996; 42: 67–75. 2. Kasai M. Advances in the cryopreservation of mammalian oocytes and embryos: development of ultra rapid vitrification. Reprod Med Biol 2002; 1: 1–9. 3. Lassalle B, Testart J, Renard JP. Human embryo features that influence the success of cryopreservation with the use of 1,2 propanediol. Fertil Steril 1985; 44: 645–51. 4. Fehilly CB, Cohen J, Edwards RG et al. Cryopreservation of cleaving embryos and expanded blastocysts in the human: a comparative study. Fertil Steril 1985; 44: 638–44. 5. Hartshorne GM, Elder K, Edwards RG et al. The influence of in-vitro development upon post-thaw survival and implantation of cryopreserved human blastocysts. Hum Reprod 1991; 6: 136–41. 6. Menezo Y, Nicollet B, Andre D, et al. Freezing cocultured human blastocysts. Fertil Steril, 1992; 58: 977–80. 7. Rall WF and Fahy GM. Ice-free cryopreservation of mouse embryos at −196°C by vitrification. Nature 1985; 313: 573–5. 8. Kasai M, Komi JH, Machida T, et al. A simple method for mouse embryo cryopreservation in a low toxicity vitrification solution, without appreciable loss of viability. J Reprod Fertil 1990; 89: 91–7. 9. Mukaida T, Takahashi K, Kasai M, et al. Vitrification of human embryos based on the
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assessment of suitable conditions for 8-cell mouse embryos. Hum Reprod 1998; 13: 2874–9. Saito H, Kaneko T, Hiroi M, et al. Application of vitrification to human embryo freezing. Gynecol Obstet Invest 2000; 49: 145–9. Yokota Y, Yokota H, Araki Y, et al. Birth of healthy twins from in vitro development of human refrozen embryos. Fertil Steril 2001; 76: 1063–5. Gardner DK, Schoolcraft WB, Stevens J, et al. A prospective randomized trial of blastocyst culture and transfer in in vitro fertilization. Hum Reprod 1998; 13: 3434–40. Cruz JR, Dubey AK, Gindoff PR, et al. Is blastocyst transfer useful as an alternative treatment for patients with multiple in vitro fertilization failures? Fertil Steril 1999; 72: 218–20. Menezo Y, Sakkas D, and Veiga A. Cryopreservation of blastocysts. In: Gardner DK, Lane M, eds. ART and the Human Blastocyst. New York: Springer-Verlag 2000: 188–95. Troup SA, Matson PL, Lieberman BA, et al. Cryopreservation of human embryos at the pronucleate, early cleavage, or expanded blastocyst stages. Eur J Obstet Gynecol Reprod Biol 1990; 38: 133–9. Nakayama T, Goto Y, Noda Y, et al. Developmental potential of frozen-thawed human blastocysts. J Assist Reprod Genet 1995; 12: 239–43. Ludwig M, Al-Hasani S, Diedrich K, et al. New aspects of cryopreservation of oocytes and
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embryos in assisted reproduction and future perspectives. Hum Reprod 1999; 14: (Suppl 1): 162–85. Yokota Y, Sato S, Araki Y, et al. Birth of a healthy baby following vitrification of human blastocysts. Fertil Steril 2001; 75: 1027–9. Vanderzwalmen P, Zech H, Van Roosendaal E, et al. Pregnancy and implantation rates after transfers of fresh and vitrified embryos on day 4 or 5. J Assist Reprod Genet 1999; 16: 147. Martino A, Songsasen N. and Leibo SP. Development into blastocysts of bovine oocytes cryopreserved by ultra-rapid cooling. Biol Reprod 1996; 54: 1059–69. Vajta G, Kuwayama M, Callesen H, et al. Open pulled straw (OPS) vitrification: a new way to reduce cryoinjuries of bovine ova and embryos. Mol Reprod Dev 1998; 51: 53–8. Lane M, Bavister BD, Forest KT, et al. Containerless vitrification of mammalian oocytes and embryos. Nat Biotechnol 1999; 17: 1234–6. Mukaida T, Kasai M, Takahashi K, et al. Successful birth after transfer of vitrified human blastocysts with use of a cryoloop containerless technique. Fertil Steril 2001; 76: 618–20. Mukaida T, Oka C, Takahashi K, et al. Vitrification of human blastocysts using cryoloops: clinical outcome of 223 cycles. Hum Reprod 2003; 18: 384–91. Gardner DK, Schoolcraft WB. In-vitro culture of human blastocyst. In Jansen R. Mortimer D, eds, Towards Reproductive Certainty: Infertility and Genetics Beyond 1999 Carnforth: Parthenon Publishing, 1999: 378–88. Vanderzwalmen P, Mukaida T, Schoysman R, et al. Births after vitrification at morula and blastocyst stages: effect of artificial reduction of the blastocoelic cavity before vitrification. Hum Reprod 2002; 17: 744–51. Son WY, Yoon SH, Lim JH, et al. Pregnancy outcome following transfer of human blastocysts vitrified on electron microscopy grids after induced collapse of the blastocoele. Hum Reprod 2003; 18: 137–9. Hiraoka K, Hiraoka K, Kinutani K, et al. Blastocoele collapse by micropipetting prior to vitrification gives excellent survival and pregnancy outcomes for human day 5 and 6 expanded blastocysts. Hum Reprod 2004; 19: 2884–8. Mukaida T, Takahashi K, Oka C, et al. Artificial shrinkage of blastocoele using
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either micro–needle or laser pulse prior to the cooling steps of vitrification improves survival rate and pregnancy outcome of vitrified human blastocysts. Hum Reprod 2006; 21: 3246–52. Lane M, Schoolcraft WB, Gardner DK. Vitrification of mouse and human blastocysts using a novel cryoloop container-less technique. Fertil Steril 1999; 72: 1073–8. Mukaida T, Takahashi K, Kasai M, et al. Blastocyst cryopreservation: ultra rapid vitrification using cryoloop technique. Reprod BioMed Online 2003; 6: 221–5 Cohen J, Alikani M, Rosenwaks Z, et al. Implantation enhancement by selective assisted hatching using zona drilling of human embryos with poor prognosis. Hum Reprod 1992; 7: 685–91. Obruca A, Strohmer H, Sakkas D. Use of lasers in assisted fertilization and hatching. Hum Reprod 1994; 9: 1723–6. Gardner DK, Lane M, Schoolocraft WB, et al. Blastocyst score affects implantation and pregnancy outcome: towards a single blastocyst transfer. Fertil Steril 2000; 73: 1155–8. Takahashi K, Mukaida T, Oka C, et al. Perinatal outcome of blastocyst transfer with vitrification using cryoloop: a 4 year follow-up study. Fertil Steril 2005; 84: 88–92. Miyake T, Kasai M, Machida T, et al. Vitrification of mouse oocytes and embryos at various stages of development in an ethylene glycol-based solution by a simple method. Theriogenology 1993; 40: 121–34. Zhu SE, Kasai M, Machida T, et al. Cryopreservation of expanded mouse blastocysts by vitrification in ethylene glycolbased solutions. J Reprod Fertil 1993; 98: 139–45. Mahmoudzadeh AR, Van Soom A, de Kruif A, et al. Optimization of a simple vitrification procedure for bovine embryos produced in vitro: effect of developmental stage, two-step addition of cryoprotectant and sucrose dilution on embryonic survival. J Reprod Fertil 1995; 103: 33–39. Veiga A, Sandalinas M, Menezo Y, et al. Laser blastocyst biopsy for preimplantation diagnosis in the human. Zygote 1997; 5: 351–4 Ebner T, Moser M, Tews G. Possible applications of non-contact 1.48 micron wavelength diode laser in assisted reproduction technologies. Hum Repord Update 2005; 11: 425–35.
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Appendix 10B.1
Laboratory manual of vitrification for human blastocyst vitrification
Protocol: Ultra-rapid vitrification for HUMAN BLASTOCYSTS using cryoloop PRINCIPLE: Cryopreserved embryos will be vitrified and warmed at the request of the patient and physician for the purpose of embryo transfer. Cryoprotectants will be added and removed stepwise and post warmed embryos will be cultured for a period of two to four hours depending on the embryo stage at the time of freezing. All embryos will be evaluated prior to transfer to the patient. The words ‘Vitrified and Warmed’ are used in this protocol, instead of ‘freezing and thawing,’ because no ice formation is involved in this approach. MATERIALS AND CHEMICALS: – Blastocyst vitrification media Ethylene Glycol: SIGMA E-9129 (250 ml), DMSO: SIGMA D-2650 (5 × 5 ml glass vial) – Blastocyst warming media Ficoll: Ficoll 70 Amersham Pharmacia Biotech AB 17-0410-01, 100 g – pulled 9″ Pasteur pipet – 1 ml pipet – Culture media for blastocyst stage (Ex. G2.2, Blast Assist, KSBM, Life Global, etc.) – 10 × 35 Primaria dish – Mineral oil for embryo culture grade (Minoil, Ovoil, etc) – 4 well Nunc plate – Mounted crystal loops: Catalog No. HR4 –963 (20 microns, 0.5-0.7mm, 25 pieces) – Crystal cap: Catalog No. HR4 – 913 (60 pieces), HR4 – 911 (Sampler pack) – Crystal wand with Tab: Catalog No. HR4 – 619 www.hamptonresearch.com To Prepare the Cryoloop by yourself: Use forceps to place a mounted cryoloop into the cap of Crystal Cap manually. Not necessary to use glue for this. If that is loose and easy to come off, heat the forceps with the alcohol or Gas burner and squeeze the stainless pipe with the loop at the base of the cap until it is tight. EQUIPMENT: – – – – – –
Olympus Inverted microscope (IX-71) Nikon stereo microscope or Wild dissecting microscope Forma or Heracell Incubator Forma hood Thermometer Stage warmer (Continued)
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Appendix 10B.1 (Continued) VITRIFICATION PROCEDURE: There are two ways to prepare the solutions for vitrifying and warming: (A) Preparing the solution A,B,C just for use once. Do not keep it more than 3 days. (B) Making stock solutions 1,2,3 every two weeks and adding DMSO and Ethylene glycol when you vitrify. Either (A) or (B) can be chosen depending on the volume of the cases. (A) Prepare the vitrification solution: (Vitrification solution B: VB, Vitrification solution C: VC) Solution A
Pipet 29 ml Hepes (with penicillin) into a 50 ml flask. Add 1 ml H.S.A. or SSS, mix.
Solution VB
Pipet 8.5 ml solution A into a 50 ml flask, add 0.75 ml DMSO, 0.75 ml ethylene glycol, mix. Filter sterilize with 0.2 µ syringe filter into a 50 ml flask. Wrap with foil.
Solution VC
Weigh 2.4 g sucrose into a tube. Weigh 0.07 g ficoll into a tube. Add 7 ml solution A to the ficoll tube, vortex to completely dissolve. Combine with the sucrose, allow to dissolve for a while. Add 1.5 ml DMSO and 1.5 ml ethylene glycol, mix. Filter sterilize with 0.2 µ syringe filter into a 50 ml flask. Wrap with foil.
(A) Prepare the warming solution: (Warming solution B: WB, Warming solution C: WC) Solution WB
Weigh 1.14 g sucrose into a 17 × 100 tube. Add 10 ml solution A. Filter sterilize with 0.2 µ syringe filter into a 50 ml flask.
Solution WC
Weigh 0.68 g sucrose into a 17 × 100 tube. Add 10 ml solution A. Filter sterilize with 0.2 µ syringe filter into a 50 ml flask The day before the warming preparing & incubate rinse and culture dishes with blastocyst culture medium. (Continued)
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Appendix 10B.1 (Continued) (B) Another way to prepare the stock solution and media for cryoloop vitrification
Base Medium Hepes HTF (Hepes G1.2 & G2.2 or any other hepes medium)
Solution 1, To 10 ml of base medium add 5 mg/ml of H.S.A
Solution 2, To 10 ml of base medium add 3.4 g of sucrose (dissolve) 5 mg/ml of H.S.A
Solution 3, To 10 ml of base medium 0.1 g of Ficoll (dissove) 3.4 g of sucrose (dissolve) 5 mg/ml of H.S.A Solution for Vitrification (Vitrification solution B: VB, Vitrification solution C: VC)
Solution A is equal to Solution 1
Solution VB 850 µl of Solution 1 75 µl of DMSO 75 µl of Ethylene glycol
Solution VC 700 µl of Solution 3 150 µl of DMSO 150 µl of Ethylene glycol (Continued)
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Appendix 10B.1 (Continued) Solution for warming (Warming solution B: WB, Warming solution C: WC) Solution WB 800 of Solution 1 400 of Solution 2 Solution WC 800 of Solution 1 200 of Solution 2 Solution 1, 2, 3 will be kept as a stock solution for two weeks. At the day of vitrification/ warming, solution VB, VC, WB, WB will be prepared with Solution 1, 2, 3. VITRIFICATION STEPS: Fill liquid nitrogen dewar (Styro-foam Box is enough) 1. At 370 C temperature, add 0.7 ml of A, VB, and VC in each of the well of a 4 well plate. 2. Place the dish in an incubator at 370 C for ~30 min 3. Check under the microscope that the loop is not broken. 4. Attach an open cryovial (without loop portion) to the labeled cane and immerse it in the liquid nitrogen in a dewer flask. 5. Take the warmed 4-well dish out of the incubator and place it on the stage warmer at 370 C 6. Use a large pulled pipet to move the blast to A for 1-2 minutes. And ready to cryopreserve it. < Transfer it to VB for 2 minutes. > 7. Empty pipet into the empty well, load with solution VB, pick the blast up to transfer them at the bottom of the solution VB well. They will collapse in solution VB. 8.
At the almost end of this 2 minutes, prepare two ~20 µl droplets of solution VC on the rid of petri dish.
9.
Attach the crystal wand to cap and carefully remove the cap with loop. Just before the 2 minutes timer dip the loop portion into VC to create a thin, filmy layer of solution on the nylon loop by surface tension.
< Transfer it to VC for 30 seconds. Be careful of time > (Continued)
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Appendix 10B.1 (Continued) 10. Empty pipet into the empty well, load with solution VC, pick up the blasts from solution VB well to transfer to one of the droplets of solution VC. They will more collapse in solution C. 11. Pick up and release the blasts several times in the droplet of solution VC and move to another droplet. 12. At the almost end of this 30 seconds, pick the blast up in the droplet of solution VC. < Loading it onto the loop > 13. Load the blast into the tip of the pipet and pipet it onto the loop. In order to confirm if all the blasts are in the loop, expel out the media in the pipet to check whether nothing is left behind. You do not need to force the blasts on the loop, they will move almost automatically by surface tension. (Do practice with any discarded embryos plenty times to feel confident this step prior to clinical application. This is easy step but really critical step!) 14. Immediately after loading the blastocyst, plunged the loop portion into the LN2 by holding the cap with crystal wand (at 30 s) 15. Screw and seal tightly the cap with the loop into cryovial with a labeled cane by crystal wand under LN2. Place on the cane and move into the storage tanks. 16. If there are more blastocyst to freeze, empty the pipet and fill with A and repeat above.
WARMING STEPS: 1.
At 370 C temperature, add 0.7 ml of A, WB, and WC in each of the well of a 4 well plate. (make 2 wells of A)
2.
Place the dish in an incubator at 37°C for ~30 min
3.
Take the warmed 4-well dish out of the incubator and place it on the stage warmer at 37°C
4.
Remove the cap with a loop from cryovial under LN2, immediately dip the loop directly into solution WB well. The vitrified blastocyst will sink to the bottom of the well, leave for 2 minutes.
5.
Transfer it to WC then leave in WC for 3 minutes.
6.
Rinse it through first well of A then leave in 2nd A well for 5 minutes.
7.
After 5 min, transfer the embryo to culture medium, which has been equilibrated in a CO2 incubator at 370 C overnight, for further culture until transfer.
8.
About 2 h after warming, observe the appearance of the blastocyst under a dissecting microscope to assess the survival.
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10C
Weon-Young Son and Jin-Ho Lim
INTRODUCTION Cryopreservation of human embryos has become a routine procedure to increase cumulative pregnancy rates, to help avoid the risk of multiple pregnancies after the transfer of many embryos, and to avoid unnecessary additional stimulation procedures. Recently, blastocyst transfer based on an improved culture system has been proven effective for increasing the pregnancy rate in assisted reproductive technology (ART).1,2 Therefore, a reliable procedure for the cryopreservation of supernumerary blastocysts is needed because usually only a small number of blastocysts after transfer are likely to be available for cryopreservation. Freezing of human blastocysts has been carried out with the slow-cooling method, but clinically satisfactory results have not been obtained.3,4 In addition, the slow-freezing method requires expensive equipment, and is time-consuming. Therefore, it is essential to establish a simple, fast, and reliable procedure to optimize clinical outcomes of cryopreservation at the blastocyst stage. Vitrification has become more widely used, and is now regarded as a potential alternative to conservative slow freezing. Vitrification takes only a few seconds to cool embryos, and there is no extracellular crystallization, which is one of the major causes of cell injury.5 It also offers lower osmotic and toxic effects, and less severe chilling injury resulting from the rapid passage through the ‘dangerous’ temperature zone.6 Furthermore, it does not require specialized programmable
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freezers. Initially, most vitrification methods used standard French mini-straws for holding the embryos during cooling, storage, and thawing. Recently, to overcome the disadvantages of straws that have low cooling and warming rates, techniques using either electron microscope (EM) grids,7,8 Cryotop,9 hemi-straw,10 Cryoloop,11,12 or cryotip13 that substantially increase the cooling rate have been applied, and significant improvements in the success rate of human blastocyst vitrification have been reported.7–13 Among them, it has been shown that an ultrarapid freezing method using EM grids was efficient for the cryopreservation of bovine oocytes.14 Since then, several researchers have applied EM grids successfully for the vitrification of human oocytes15 and blastocysts.7,8,16 Embryo cryopreservation using EM grids was originally designed for the vitrification of exceedingly chill-sensitive Drosophila embryos.17,18 The EM grid has about 3-fold higher cooling rates than those obtained with straws. It is known that the increased rate of cooling and thawing may considerably decrease the chilling injury of embryos. In addition, the EM grid is much cheaper than another apparatus that is being used regularly for vitrification. In this article, we discuss our experience of 1000 cycles of human blastocyst vitrification using EM grids from 1999–2006. The human blastocyst vitrification system using EM grids was set up in different periods. In period I: ‘vitrification of human blastocysts on EM grids’, we examined the possibility of clinical application of human blastocyst
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vitrification using EM grids based on the experience of bovine blastocysts,19 and we examined the effect of a six-step serial dilution after vitrification and thawing using EM grids. In period II: ‘artificial shrinkage of expanded blastocysts before vitrification’, we applied ‘artificial shrinkage’ (i.e. induced collapse of the blastocoel) before vitrification of human blastocysts and examined the effects on blastocyst survival and subsequent pregnancy rate. In period III: ‘modified two-step dilution method’, we modified dilution method to a two-step method after thawing vitrified blastocysts using EM grids where blastocysts were subjected to artificial shrinkage. Finally, we applied the established vitrification method to blastocysts derived from immature oocytes in our in vitro maturation (IVM) program.
METHODOLOGY Patients and IVF treatment Women were treated with gonadotropin releasing hormone (GnRH) agonist and human menopausal gonadotropin (hMG) in either a long- or a short-treatment protocol. When two and more follicles reached 18 mm in diameter, a dose of 10 000 IU human chorionic gonadotropin (hCG) (IVF-C; LG Chemical, Seoul, Korea) was administered. Oocytes were retrieved transvaginally 36–38 h after hCG injection, and the oocytes were inseminated by either conventional in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Fertilization was examined 17–19 h after insemination for the appearance of two distinct pronuclei (PN) and two polar bodies.
Embryo culture procedure The procedure used to culture fertilized oocytes was the same as described in a previous study.2 All zygotes were co-cultured with cumulus cells in a 10 µl YS medium supplemented
with 10% human follicular fluid (hFF) for 5 or 6 days at 37°C in an atmosphere of 5% CO2, 5% O2, and 90% N2.2 The hFF was prepared using a previously reported method.20 The cumulus cells for co-culture were prepared using the method reported by Yoon et al.2 The embryos were transferred on either day 3 or day 5. The date of the embryo transfer was determined by the number of zygotes and the quality of the embryo on day 2, according to established criteria.2 After transferring the embryos, surplus embryos were further cultured until day 6, regardless of the embryo transfer date. The developed blastocysts were classified according to their degree of expansion (Figure 10C.1). In early blastocysts (ErB) the blastocoel is less than half the volume of the embryo and diameter < 140 µm; in early expanding blastocysts (EEB) the blastocoel is greater than or equal to half of the volume of the embryo and diameter 140–160 µm; in middle expanding blastocysts (MEB) the blastocoel completely fills the embryo and diameter 160–180 µm; and in expanded blastocysts (EdB) the blastocoel volume is larger than that of the early embryo with thinning zona and diameter > 180 µm. The blastocysts were assigned one of four grades: grade A blastocysts have a clear inner cell mass (ICM) and trophectoderm cells; grade B a clear ICM but poor trophectoderm development; grade C a poor ICM but good trophectoderm cells; and grade D a poor ICM and poor trophectoderm cells. Only the embryos that developed to the expanded blastocyst stage (diameter is > 160 mm and grade A/B) were cryopreserved by vitrification on EM grids.
Vitrification of blastocysts on electron microscope grid and warming procedure Materials Chemicals were obtained from Sigma Chemical Company (St Louis, MO, USA) and
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a
b
c
d
Figure 10C.1 Developmental stages of human blastocysts cultured in vitro for 5 or 6 days from zygotes. (a) Early blastocyst (ErB), (b) early expanding blastocyst (EEB), (c) middle expanding blastocyst (MEB), and (d) expanded blastocyst (EdB). Scale bar = 50 µm.
media from GIBCO (Grand Island, NY, USA), unless otherwise stated. All of the manipulation procedures for freezing and warming were conducted at 25°C (room temperature). Cryoprotective agents were intimately related to embryo survival.21 Since 1990, EFS (ethylene glycol, Ficoll, sucrose) solution has been widely used to cryopreserve the various embryo stages of several species; ethylene glycol permeates the cell rapidly
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and has low toxicity. The materials used are shown in Table 10C.1.
Blastocyst vitrification method on electron microscope grid (1) Blastocysts prepared for vitrification were equilibrated in EG20 for 1.5 min at room temperature.
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Table 10C.1
Materials used for blastocyst vitrification using EM grids
• • • • • • • • • • • • • •
Four hundred mesh copper EM grids (IGC 400; Pelco International, CA, USA) Filter paper (sterilized) Modified-Dulbecco’s phosphate-buffered saline (m-DPBS; GIBCO, Grand Island, NY, USA) Serum (human follicular fluid (hFF) or human serum albumin (HSA)) Ethylene glycol (EG; Sigma Chemical Co, St Louis, USA) Ficoll (Ficoll 70, average MW 70 000 Da; Pharmacia Biotech, Uppsala, Sweden) Sucrose (Sigma Chemical Co, St Louis, USA) 4-well nunc dish Cryovial Cane Liquid nitrogen (LN2) Storage LN2 Tank Pretreatment solution (EG20): 20% (v/v) EG in m-DPBS containing 10% hFF (or HSA) Vitrification solution (EFS40): 40% (v/v) EG, 18% (w/v) Ficoll, and 0.3 mol/L sucrose in m-DPBS containing 10% hFF (or HSA) • Dilution solutions: 0.5 mol/L, 0.4 mol/L, 0.3 mol/L, 0.2 mol/L, 0.1 mol/L, and 0.0 mol/L sucrose in m-DPBS containing 10% hFF (or HSA)
(2) Blastocysts were incubated in EFS40 at room temperature, loaded onto the EM grid using a Pasteur pipette, and excess cryoprotectant was removed using sterilized filter paper (Figure 10C.2a). Mean number of blastocysts loaded on one grid was 2–3. (3) EM grid containing blastocysts was directly plunged in LN2 within 30 s. (4) EM grid was scaled in a cryovial that had previously been submerged under LN2 (Figure 10C.2b).
(3) Blastocysts were transferred into DPBS containing 10% serum for 1.5 min at room temperature. Modified two-step dilution method: (1) EM grids stored in LN2 were transferred directly into 1 mL of 0.5 mol/L sucrose solution in a 4-well dish as soon as possible, and the blastocysts were recovered (Figure 10C.2d and e).
(5) Cryovial was attached on canes and stored in LN2 storage tank (Figure 10C.2c).
(2) Blastocysts were quickly transferred into fresh 0.5 mol/L sucrose and incubated for 5 min at room temperature (Figure 10C.2f).
Warming methods after thawing of vitrified blastocyst on electron microscope grid
(3) Blastocysts were transferred into DPBS containing 10% serum for 5 min at room temperature.
Two-step dilution method:
Six-step serial dilution method:
(1) EM grids stored in LN2 were transferred directly into 1 mL of 0.3 mol/L sucrose solution in a 4-well dish as soon as possible, and the blastocysts were recovered.
(1) EM grids stored in LN2 were transferred directly into 1 mL of 0.5 mol/L sucrose solution as soon as possible, and the blastocysts were recovered.
(2) Blastocysts were quickly transferred into fresh 0.3 mol/L sucrose and incubated for 1.5 min at room temperature.
(2) Blastocysts were quickly transferred into fresh 0.5 mol/L sucrose and incubated for 3 min at room temperature.
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a
b
c
d
e
f
Figure 10C.2 Vitrification and warming process of blastocysts on electron microscope (EM) grids. (a) Blastocysts were loaded onto the EM grid on sterilized filter paper using a Pasteur pipette, (b) directly plunged the EM grid containing blastocysts in LN2, (c) the cryovial was attached in canes and stored in LN2 storage tank, (d) EM grid stored in LN2 was transferred into 0.5 mol/L sucrose solution, (e) the blastocysts on EM grid were recovered, (f) blastocysts were tranferred into fresh 0.5 mol/L sucrose.
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(3) Blastocysts were transferred sequentially into 1 mL of 0.4, 0.3, 0.2, and 0.1 mol/L sucrose at intervals of 1.5 min at room temperature.
RESULTS
(4) Blastocysts were transferred into m-DPBS containing 10% serum for 1.5 min at room temperature.
Previously, Park et al. reported that higher survival (78.1%) and hatching (65.4%) rates of vitrified–thawed bovine blastocysts could be obtained using EM grids and a two-step cryoprotectant dilution.19 Initially, we applied the same protocol to human blastocysts, but this resulted in disappointing survival rates (30%, 6/20). Cryopreserved blastocyst survival usually depends on the freezing–thawing procedure. Thus, the low percentage of blastocysts that survived after thawing in a two-step dilution method might have been due to the dramatic osmotic shock. Following some trial experiments, we obtained a good survival rate by changing the first concentration of sucrose to 0.5 mol/L in cryoprotectant dilution after thawing. Thus, we compared a thawing protocol using a sixstep serial dilution with the two-step dilution used for bovine blastocysts19 on the survival of vitrified human blastocysts (n = 293) derived from 3PN before trying to apply the method clinically. Figure 10C.3 shows the survival rate of blastocysts after thawing. As shown in Figure 10C.3, the survival rate of blastocysts following the six-step dilution method was significantly higher than following the two-step dilution method (P < 0.01). It could be speculated that the higher survival rate of human blastocysts in a six-step cryoprotectant dilution was due to a decrease in the osmotic shock. Based on the above results, we applied the six-step thawing method clinically. Table 10C.2 shows the clinical results. A total of 92 patients (mean age ± SD = 34.2 ± 3.5) and 382 blastocysts were included in this study. Out of 287 (75.1%) survived blastocysts, 42 (14.6%) hatched at the time of the embryo transfer. After transfer, 29 clinical pregnancies (31.5%) were achieved. Although we obtained a reasonable clinical pregnancy rate (31.5%) following transfer, this pregnancy outcome was lower
Assessment of survival after thawing After thawing the blastocysts were washed three times in culture medium and co-cultured with cumulus cells in 10 µL YS medium containing 10% hFF. The post-thawing survival of blastocysts was observed ~18–20 h after warming under a microscope, and blastocysts with a morphologically intact ΙCM and trophectoderm, and re-expanding blastocoel were judged to have survived.
Embryo transfer and endometrium preparation Vitrified day 5 and 6 blastocysts were thawed in the afternoon of the day before embryo transfer. Embryo transfer was scheduled on day 5 after ovulation in the spontaneous cycles, or on days 19–20 in artificial cycles prepared with exogenous estrogen and progesterone. One to three surviving blastocysts were transferred into the patient’s uterus. Pregnancy was first assessed by serum βhCG 9 days after blastocyst transfer, and then clinical pregnancy was determined by the presence of fetal heart activity 30 days after blastocyst transfer.
Statistical analysis The significance of difference between treatment groups in each experiment was compared with the χ2 test using the Statistical Analysis System (SAS) Institute software package (SAS Institute Inc, 1985).
Vitrification of human blastocysts on electron microscope grid
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100
**
** Survival (%)
**
**
75
50
25
0 ErB
EEB
MEB
EdB
Developmental stage of blastocysts vitrified Figure 10C.3 Survival rate of vitrified blastocysts derived from three pronuclei (n = 293) after a two-step (open bar) or a six-step (solid bar) dilution method. ** P< 0.01, compared with other group. Erb, early blastocyst ( < 140 µm); EEB, early expanded blastocysts (140–160 µm); MEB, middle expanded blastocyst (160–180 µm); EdB, expanded blastocyst (> 180 µm).
than that obtained from the transfer of fresh blastocysts in our hospital.2
Table 10C.2 Clinical results for patients receiving blastocysts surviving after a six-step cryoprotectant dilution in vitrification using EM grids Variable
Artificial shrinkage of expanded blastocysts before vitrification on electron microscope grid As shown in Figure 10C.3, it was observed that the survival rate of blastocysts at late stage (MEB, EdB) was lower than that of blastocysts at early stage (ErB, EEB) in both twoand six-step dilution methods. It has been reported that the survival rate of expanded blastocysts after vitrification is increased significantly when the blastocoel is artificially shrunk with a glass microneedle,22 two 29gauge needles,16 pipetting,23 or a laser pulse,24 which is thought to reduce ice crystal formation. Similarly, we applied clinically the artificial shrinkage technique in our vitrification system, using EM grids and a six-step thawing procedure, and examined the effect on survival and hatching of vitrified human blastocysts. Artificial shrinkage of expanded blastocysts (≥ 160 µm) was performed with two
No. No. No. No. No. No. No.
245
of of of of of of of
cycles blastocysts warmed blastocysts survived (%) blastocysts hatched (%) blastocysts transferred (mean) blastocysts implanted (%) clinical pregnancies (%)
92 382 287 42 245 39 29
(75.1) (14.6) (2.7) (15.9) (31.5)
29-gauge needles (Figure 10C.4b). After holding the expanded blastocyst with the flat side of a needle and placing the ICM at the 12 or 6 o’clock position, a needle was pushed through the trophectoderm cell into the blastocoel cavity until it shrank (Figure 10C.4c). Contraction of the blastocysts was then observed after 30 s to 1 min. After complete shrinkage of the blastocoel, the blastocysts were vitrified on EM grids (Figure 10C.4d). In a preliminary experiment, we evaluated the effect of artificial shrinkage on blastocyst
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a
b
c
d
e
f
Figure 10C.4 Freezing and warming of human expanded blastocysts on electron microscope (EM) grids after artificial shrinkage. Human expanded blastocyst (a) before artificial shrinkage, (b) during artificial shrinkage, (c) after artificial shrinkage, (d) loaded onto EM grids, (e) a hatching blastocysts 18 h after thawing, and (f) a hatched blastocysts. Original magnification × 200.
survival and the further development after thawing of poor quality vitrified expanded blastocysts, which had been donated by consenting patients. The rates of survival (90.0%, 81/90) and hatching (56.8%, 46/81) in the artificial shrinkage group were significantly higher than those of the control group (70.8%, 68/96; 11.8%, 8/68) (P < 0.01). No harmful effect of the procedure was noted. Based on the above result, we applied the artificial shrinkage technique clinically. Table 10C.3 shows the clinical results of human blastocyst vitrification after artificial shrinkage. A total of 206 expanded blastocysts were vitrified and warmed from 64 patients (mean age ± SD = 34.5 ± 3.7). In all, 179 blastocysts (86.9%) were re-expanded after warming. Of the 179 blastocysts that survived, 145 had hatched (81.0%) at the time of transfer. A total of 166 blastocysts were transferred into 64 patients. The implantation rate was 25.9%
(43/166) and the pregnancy rate was 46.9% (30/64). Therefore, we found that artificial shrinkage is a useful technique for the vitrification of expanded human blastocysts. Furthermore, the high percentage of hatching (81.0%) observed at the time of embryo transfer might be due to the effect of assisted hatching caused by the formation of a large hole in the zona pellucida produced by using a 29-gauge needle. These clinical outcomes were similar to those obtained from the transfer of fresh blastocysts in our hospital.2
Application of the modified two-step dilution method after thawing of vitrified blastocysts subjected to artificial shrinkage In this current study we examined whether the step-wise dilution of cryoprotectants after thawing was still needed for human
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Table 10C.3 Clinical results following six-step dilution of human blastocysts vitrified on EM grids after artificial shrinkage Variable of of of of of of of
cycles blastocysts warmed blastocysts survived (%) blastocysts hatched (%) blastocysts transferred (mean) blastocysts implanted (%) clinical pregnancies (%)
64 206 179 145 166 43 30
(86.9) (81.0) (2.6) (25.9) (46.9)
blastocysts that were vitrified after artificial shrinkage. This study was performed to optimize the dilution method appropriate for our vitrification system using artificial shrinkage and an EM grid. In our preliminary experiments we tried to determine the optimal exposure time of cryoprotectant dilution in 0.5 mol/L sucrose, after thawing of poor quality expanded blastocysts, which had been donated by consenting patients and then vitrified. Figure 10C.5 shows the results of the treatments. We observed that the highest survival rate was obtained when blastocysts were treated with
a
b
100
87.4
85.7
79.2 76
Survival (%)
Survival (%)
100
**
**
86.7
75
c
100
75
69.6
Percentage
No. No. No. No. No. No. No.
0.5 mol/L sucrose for 5 min (86.7%, 26/30) rather than when treated for 3 min (60.9%, 14/23), 4 min (79.2%, 19/24), or 6 min (76.0%, 19/25) (P < 0.01) (Figure 10C.5a). In addition, when we compared the survival rate after dilution in 0.5 mol/L or 0.3 mol/L for 5 min, the survival rate of blastocysts treated with 0.5 mol/L (85.7%, 12/14) was significantly higher than with 0.3 mol/L (69.6%, 16/23) (P < 0.01) (Figure 10C.5b). Finally, we compared the modified two-step dilution method, in which blastocysts were treated with 0.5 mol/L sucrose for 5 min, with the six-step dilution method (Figure 10C.5c). There was no difference in survival and hatching rates of blastocysts between the twostep method (85.6%, 77/90; 68.8%, 53/77) and the six-step method (87.4%, 76/87; 72.4%, 55/76). This study indicates that the modified two-step dilution method is appropriate for diluting human blastocysts that were vitrified on an EM grid after artificial shrinkage. In addition, the method is simple and effective. Based on these results, we applied the twostep dilution method to clinical treatment. In all, 7315 blastocysts from 2019 patients
85.6
72.4
75
68.8
60.9
50 3 4 5 6 Time exposed in 0.5 mol/L sucrose (min)
50
50 0.5 0.3 Sucrose concentration (mol/L)
Survival
Hatching
Figure 10C.5 Effects of dilution time and sucrose concentration on survival and hatching after thawing in expanded blastocysts vitrified after artificial shrinkage. (a) Survival rates of blastocysts exposed to different times in 0.5 mol/L sucrose, (b) survival rates of blastocysts diluted in different concentrations of sucrose for 5 min, and (c) survival and hatching rates in blastocysts diluted with a six-step (open bar) or a modified two-step method (solid bar). **P< 0.01, compared with other group.
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(mean age ± SD = 34.8 ± 4.2) were vitrified on EM grids between January 1999 and December 2005. For 4 years, from March 2002 to December 2005, we vitrified and warmed 2626 blastocysts from 884 cycles using the modified two-step dilution method, 2366 of the vitrified blastocysts (90.1%) survived and 2066 of the surviving blastocysts (87.3%) hatched at the time of transfer. A total of 2287 survived blastocysts were then transferred into patients. The implantation rate was 26.8% (612/2287) and the pregnancy rate was 49.7% (439/884) (Table 10C.4). The live birth rate was 40.3% (356/884). There were no triplet pregnancies. Birth weights of the infants were within the range of 1540–4220 g, and at the time of going to press all delivered infants had normal physical profiles.
Vitrification of blastocysts derived from in vitro maturation of immature human oocytes using the established vitrification method In vitro maturation (IVM) is an attractive option to eliminate several problems associated with controlled ovarian hyperstimulation (COH) used for conventional in vitro fertilization (IVF). Recent studies have shown an improved pregnancy rate per embryo transfer in small numbers of cycles.25,26 In addition, several IVF centers have reported that acceptable rates of oocyte maturation and pregnancies were achieved in patients with polycystic ovary syndrome (PCOS).26 However, they reported that the implantation rate was less than 15%. Therefore, more embryos in an IVM program have been transferred to obtain acceptable pregnancy rates than in COH cycles. In addition, although cryopreservation is now a routine procedure in human IVF, there have been only a few case reports in which embryos generated from an IVM program were frozen at 2PN27 and cleavage stage28 using the slow-cooling
Table 10C.4 Clinical results following a modified two-step dilution of human blastocysts vitrified on EM grids after artificial shrinkage Variables No. No. No. No. No. No. No.
of of of of of of of
cycles blastocysts warmed blastocysts survived (%) blastocysts hatched (%) blastocysts transferred (mean) blastocysts implanted (%) clinical pregnancies (%)
884 2626 2366 2066 2287 612 439
(90.1) (87.3) (2.6) (26.8) (49.7)
method. One of the reasons is that considerable differences of efficiency exist depending on the origin of mature oocytes (in vivo or in vitro produced). Generally, in vitro produced embryos were much more sensitive to freezing than their in vivo derived counterparts.29 Suikkari et al. also found that the cryosurvival of in vitro matured zygotes and cleaved embryos was very poor compared with embryos generated from in vivo matured oocytes using the slow-cooling method.30 In their study, eight out of 24 cleaved embryos and 14 out of 25 zygotes survived after thawing, suggesting that cryopreservation of in vitro matured embryos might not be an optimal procedure. Therefore, clinically satisfactory results from embryos generated from IVM oocytes have not yet been obtained.30 Recently, we found that the oocytes retrieved after hCG priming in women with high risk of OHSS in the IVM program can develop to blastocyst stage, and pregnancies can be established by transfer of these blastocysts.31–34 Therefore, a reliable procedure for the cryopreservation of supernumerary blastocysts generated from IVM oocytes is also needed. Although we have already reported that successful pregnancies can be achieved after vitrification of blastocyst stages generated from IVM cycles,35,36 it is still unclear whether the established vitrification method can be applied to the blastocysts produced
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by in vitro matured oocytes. Therefore, we observed the clinical outcome after vitrification and warming of the blastocysts produced from IVM cycles using the method established in our IVF program. A total of 233 blastocyst-stage embryos produced from 66 IVM cycles were vitrified through 2005. Among them 32 cycles that had transfers after warming were examined. Out of the 125 vitrified blastocysts a total of 100 expanded blastocysts were warmed from 32 cycles. Ninety-two blastocysts (92.0%) were reexpanded after warming. Of the 92 blastocysts that survived, 68 had hatching or hatched blastocysts (73.9%) at the time of transfer. A total of 89 blastocysts were transferred in 32 cycles, 14 clinical pregnancies (43.8%, 14/32) and a 23.1% implantation rate (21/89) were established.
DISCUSSION After being warmed, the vitrified human embryos must be separated from the permeable cryoprotectants, which were used in the cooling process. This is usually achieved by immersing embryos in a graded series of sucrose solutions until isotonic conditions are met. This gradual replacement of cryoprotectants with sucrose solution serves to rehydrate the thawed embryos, and helps to reduce osmotic pressure during rehydration.9,37 Generally, three- or four-step dilution procedures have been used in this process.10,22,23,38,39 As shown in previous results, we also improved the success rate of human blastocyst vitrification by increasing the number of cryoprotectant dilution steps after thawing by using a six-step dilution method. However, we found that there was a relatively poor survival of the expanded blastocysts after vitrification. It was thought that late blastocysts consist of a well-developed blastocoel, which may disturb effective cryopreservation due to ice crystal formation, and that this may be caused by inadequate permeation of the cryoprotectants during the cool-
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ing step. Therefore, we introduced an artificial shrinkage technique into our vitrification system, and we have noted a dramatic increase in the survival and pregnancy rates of human expanded blastocysts.16,40 After the potential for intrablastocoelic ice formation was eliminated in expanded blastocysts by such an artificial shrinkage technique, we questioned whether the step-wise dilution of cryoprotectants after thawing is still needed for human blastocysts that were vitrified after artificial shrinkage. Finally, we found that the two-step dilution method could be a simpler and more effective protocol for human expanded blastocysts that are vitrified using EM grids following artificial shrinkage, than the six-step method. When the blastocyst is vitrified after artificial shrinkage, the fast exchange of cryoprotectant seems to be more important than reducing the osmotic stress during the cryoprotectant dilution step. To date, we have been achieving reasonable clinical outcomes in our IVF and IVM programs using our established vitrification system. Choi and her colleagues observed that only 51.6% (48/93) of human blastocysts survived after warming in an EM grid vitrification program.8 The vitrification and warming methods were different from our methods. Therefore, the difference in survival rates of blastocysts after warming may depend on the concentrations and times they are exposed to cryoprotectant solutions, rather than on the use of the same EM grids. Another possible explanation could be the different culture conditions used for producing the blastocysts, and the quality of the expanded blastocysts cryopreserved. New culture systems and media are currently being developed for IVF programs, which are helping in the production of good quality blastocysts. Variations caused by different media batches can be minimized by using qualified products that are commercially controlled. Therefore, blastocyst culture and transfer are becoming routine procedures in ART programs. This implies that the
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current culture system is relatively good to culture blastocysts from embryos generated in IVF cycles. However, it is still questionable whether the commercially available media can produce blastocysts from embryos derived from in vitro matured oocytes. Although, Barnes et al. were the first to report successful development and pregnancy to the blastocyst stage from in vitro matured oocytes in sequential culture medium designed specifically to optimize blastocyst development,41 since then, there have not been any reports about the successful clinical application following transfer of blastocysts in IVM programs using a sequential culture system similar to that used in conventional IVF. Therefore, there may still be suboptimal conditions for support of later development of the embryos generated in IVM cycles. The other option for culturing embryos generated from IVM cycles is a co-culture system. It has been shown that co-culture systems significantly enhance the percentage of embryos developing to the blastocyst stage in IVF42,43 and IVM44,45 cycles. These reports imply that to date the co-culture method is the best option to obtain blastocysts in an IVM program. An improved survival rate after cryopreservation of in vitro produced blastocysts may be attributed to the improved culture conditions, as well as changes in the cryopreservation technology. We have previously reported high clinical pregnancy (51.9%, 55/106) and implantation (26.8%, 84/313) rates without assisted hatching of blastocysts produced from IVM cycles.34 After vitrification of blastocysts produced from IVM cycles we observed a clinical pregnancy of 43.8%, and an implantation rate of 23.6% similar to that obtained in fresh cycles. These results imply that this vitrification method is also an efficient cryopreservation method for blastocysts produced from IVM programs. In addition, these results highlight the use of three advanced assisted reproduction techniques, namely
IVM, blastocyst culture, and vitrification, to overcome infertility problems.
CONCLUSION This study showed that vitrification of human blastocysts at the expanded blastocyst stage using EM grids and artificial shrinkage technique is a clinically useful cryopreservation method. Additionally, the modified two-step dilution method could be a simpler and more effective protocol for human expanded blastocysts that are vitrified using EM grids following artificial shrinkage, when compared with the six-step method. Furthermore, blastocyst-stage embryos produced from in vitro matured human oocytes can be safely cryopreserved by the established vitrification method, and successful pregnancy can be achieved following embryo transfer. However, a disadvantage of vitrification on EM grids is the risk of contamination by pathogens such as viruses, prions, and bacteria caused when the vitrification solution comes into direct contact with LN2 during cooling or storage.46 Although to date there are no cases of contamination occurring via LN2 in our vitrification system, the development of safety strategies for reducing the risk of contamination by larger pathogens may be necessary. Nevertheless, our results, based on more than 500 live births, prove that the vitrification method using an EM grid is a simple, inexpensive, and efficient technique for cryopreservation of expanded human blastocysts.
ACKNOWLEDGMENTS We owe great thanks to the physicians and embryologists working at the Maria Infertility Hospital, Korea. We are very grateful to Belen Herrero and Jin-Tae Chung, Department of Obstetrics and Gynecology, McGill University, Montreal, Quebec, Canada, for critical review of this manuscript.
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References 1. Gardner DK, Vella P, Lane M et al. Culture and transfer of human blastocysts increases implantation rates and reduces the need for multiple embryo transfers. Fertil Steril 1998; 69: 84–8. 2. Yoon HG, Yoon SH, Son WY et al. Alternative embryo transfer of day 3 or day 5 for reducing the risk of multiple gestations. J Assist Reprod Genet 2001; 18: 262–7. 3. Ménézo Y, Nicollet B, Herbaut N et al. Freezing cocultured human blastocysts. Fertil Steril 1992; 58: 977–80. 4. Kaufman RA, Ménézo Y, Hazout A et al. Cocultured blastocyst cryopreservation: experience of more than 500 transfer cycles. Fertil Steril 1995; 64: 1125–9. 5. Rall WF, Fahy GM. Ice-free cryopreservation of mouse embryos at –196°C by vitrification. Nature 1985; 313: 573–5. 6. Vajta G, Holm P, Kuwayama M et al. Open pulled straw (OPS) vitrification: a new way to reduce cryoinjuries of bovine ova and embryos. Mol Reprod Dev 1998; 51: 53–8. 7. Cho HJ, Son WY, Yoon SH et al. An improved protocol for dilution of cryoprotectants from vitrified human blastocysts. Hum Reprod 2002; 17: 2419–22. 8. Choi DH, Chung HM, Lim JM et al. Pregnancy and delivery of healthy infants developed from vitrified blastocysts in an IVF–ET program. Fertil Steril 2000; 74: 838–44. 9. Kuwamaya M, Hamano S, Nagai T. Vitrification of bovine blastocysts obtained by in vitro culture of oocytes matured and fertilized in vitro. J Reprod Fertil 1992; 96: 187–93. 10. Vanderzwalmen P, Bertin G, Debauche CH et al. Vitrification of human blastocysts with the Hemi-Straw carrier: application of assisted hatching after thawing. Hum Reprod 2003; 18: 1504–11. 11. Yokota Y, Sato S, Yokota M et al. Successful pregnancy following blastocyst vitrification: case report. Hum Reprod 2000; 15: 1802–3. 12. Mukaida T, Nakamura S, Tomiyama T et al. Successful birth after transfer of vitrified human blastocysts with use of a cryoloop containerless technique. Fertil Steril 2001; 76: 618–20. 13. Kuwayama M, Vajta G, Ieda S et al. Comparison of open and closed methods for vitrification of human embryos and the elimination of potential contamination. RBM Online 2005; 11: 608–14.
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14. Martino A, Songsasen N, Leibo SP. Development into blastocysts of bovine oocytes cryopreserved by ultra-rapid cooling. Biol Reprod 1996; 54: 1059–69. 15. Yoon TK, Chung HM, Lim JM et al. Pregnancy and delivery of healthy infants developed from vitrified oocytes in a stimulated in vitro fertilization–embryo transfer program. Fertil Steril 2000; 74: 180–1. 16. Son WY, Yoon SH, Yoon HJ et al. Pregnancy outcome following transfer of human blastocysts vitrified on electron microscopy grids after induced collapse of the blastocoele. Hum Reprod 2003; 18: 137–9 17. Mazur P, Cole KW, Hall JW et al. Cryobiological preservation of Drosophila embryos. Science 1992; 258: 1932–5. 18. Steponkus PL and Caldwell S. An optimized procedure for the cryopreservation of Drosophila melanogaster embryos. CryoLetters 1993; 14: 375–80. 19. Park SP, Kim EY, Kim DI et al. Simple, efficient and successful vitrification of bovine blastocysts using electron microscope grids. Hum Reprod 1999; 14: 2838–43. 20. Chi HJ, Kim DH, Koo JJ et al. The suitability and efficiency of human follicular fluid as a protein supplement in human in vitro fertilization programs. Fertil Steril 1998; 70: 871–7. 21. Tachikawa S, Otoi T, Kondo S et al. Successful vitrification of bovine blastocysts derived by in vitro maturation and fertilization. Mol Reprod Dev 1993; 34: 266–71. 22. Vanderzwalmen P, Bertin G, Debauche CH et al. Birth after vitrification at morula and blastocyst stage: effect of artificial reduction of the blastocoelic cavity before vitrification. Hum Reprod 2002; 17: 744–51. 23. Hiraoka K, Kiraoka K, Kinutani M et al. Blastocoele collapse by micropipetting prior to vitrification gives excellent survival and pregnancy outcomes for human day 5 and 6 expanded blastocysts. Hum Reprod 2004; 19: 2884–8. 24. Mukaida T, Oka C, Goto T et al. Artificial shrinkage of blastocoeles using either a microneedle or a laser pulse prior to the cooling steps of vitrification improves survival rate and pregnancy outcome of vitrified human blastocysts. Hum Reprod 2006; 21: 3246–52. 25. Mikkelsen AL, Smith SD, Lindenberg S. In-vitro maturation of human oocytes from regularly menstruating women may be successful without
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26. 27.
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follicle stimulating hormone priming. Hum Reprod 1999; 14: 1847–51. Chian RC. In-vitro maturation of immature oocytes for infertile women with PCOS. RBM Online 2004; 8: 547–52. Chian RC, Gülekli B, Buckett WM et al. Pregnancy and delivery after cryopreservation of zygotes produced by in-vitro matured oocytes retrieved from a woman with polycystic ovarian syndrome. Hum Reprod 2001; 16: 1700–2. Godin PA, Gaspard O, Thonon F et al. Twin pregnancy obtained with frozen-thawed embryos after in vitro maturation in a patient with polycystic ovarian syndrome. J Assist Reprod Genet 2003; 20: 347–50. Leibo SP. Piglets produced by transfer of vitrified porcine embryos after stepwise dilution of cryoprotectants. Cryobiology 1998; 36: 20–31. Suikkari AM, Tulppala M, Tuuri T et al. Luteal phase start of low-dose FSH priming of follicles results in an efficient recovery, maturation and fertilization of immature human oocytes. Hum Reprod 2000; 15: 747–51. Son WY, Park SJ, Hyun CS et al. Successful birth after transfer of blastocysts derived from oocytes of unstimulated woman with regular menstrual cycle after IVM Approach. J Assist Reprod Genet 2002; 19: 541–3. Son WY, Yoon SH, Lee SW et al. Blastocyst development and pregnancies after IVF of mature oocytes retrieved from unstimulated patients with PCOS after in-vivo HCG priming. Hum Reprod 2002; 17: 134–6. Son WY, Lee SY, Lim JH. Fertilization, cleavage and blastocyst development according to the maturation timing of oocytes in in vitro maturation cycles. Hum Reprod 2005; 20: 3204–7. Son WY, Lee SY, Yoon SH et al. Pregnancies and deliveries after transfer of human blastocysts derived from in vitro matured oocytes in IVM cycles. Fertil Steril 2007; in press. Son WY, Yoon SH, Park SJ et al. Ongoing twin pregnancy after vitrification of blastocysts produced by in vitro matured oocytes retrieved from a woman with polycystic ovary syndrome. Hum Reprod 2002; 17: 2963–6. Son WY, Lee SY, Chang MJ et al. Pregnancy resulting from transfer of repeat vitrified
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blastocysts produced by in-vitro matured oocytes in patient with polycystic ovary syndrome. RBM online 2005; 10: 398–401. Isachenko V, Montag M, Isachenko E et al. Developmental rate and ultrastructure of vitrified human pronuclear oocytes after stepwise versus direct rehydration. Hum Reprod 2004; 19: 660–5. Mukaida T, Nakamura S, Tomiyama T et al. Vitrification of human blastocysts using cryoloops: clinical outcome of 223 cycle. Hum Reprod 2003; 18: 384–91. Takahashi K, Kukaida T, Goto T et al. Perinatal outcome of blastocyst transfer with vitrification using cryoloop: a 4-year follow-up study. Fertil Steril 2004; 84: 88–92. Lee SY, Kim HJ, Park SJ et al. Optimization of a dilution method for human expanded blastocysts vitrified using EM grids after artificial shrinkage. J Assist Reprod Genet 2006; 23: 87–91. Barnes FL, Crombie A, Gardner DK et al. Blastocyst development and birth after in-vitro maturation of human primary oocytes, intracytoplasmic sperm injection and assisted hatching. Hum Reprod 1995; 10: 3243–7. Menezo YJR, Guerin JF, Czyba JC. Improvement of human early embryo development in vitro by co-culture on monolayers of Vero cells. Biol Reprod 1990; 42: 301–7. Bongso A, Ng SC, Fong CY et al. Cocultures: a new lead in embryo quality improvement for assisted reproduction. Fertil Steril 1991; 56: 179–91. Hwu YM, Lee RK, Chen CP et al. Development of hatching blastocysts from immature human oocytes following in vitro maturation and fertilization using a co-culture system. Hum Reprod 1998; 13: 1916–21. Cobo AC, Requena A, Neuspiller F et al. Maturation in vitro of human oocytes from unstimulated cycles: selection of the optimal day for ovum retrieval based on follicular size. Hum Reprod 1999; 14: 1864–8. Bielanski A, Nadin-Davis S, Sapp T et al. Viral contamination of embryos cryopreserved in liquid nitrogen. Cryobiology 2000; 40: 110–6.
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10D
Juergen Liebermann and Michael J Tucker
clearly an optimization of the assisted reproductive technologies (ART) process.
PURPOSE Cryopreservation of human oocytes, embryos, or blastocysts allows us to maximize the potential for conception from any one in vitro fertilization cycle, and prevents wastage of embryos. Furthermore, this enables best utilization of a patient’s supernumerary oocytes after retrieval, and maximization of the use of embryos from a single stimulation cycle. In other words, at completion of a cycle a patient’s supernumerary embryos may be used beyond the fresh transfer, and can lead to later opportunities for pregnancy. Cryopreservation has been shown to increase pregnancy rates while allowing for further selection of embryos. In addition, it is possible to achieve implantation and pregnancy rates with frozen–thawed embryos close to those achieved with fresh embryos. Blastocysts have been shown to increase pregnancy rates while allowing for improved selection of potentially viable embryos. At this late stage of development lower numbers of embryos are transferred, resulting in fewer high-order multiple pregnancies and increased implantation rates. Decreased numbers of embryos are transferred which results in lower multiple implantations and the potential for more blastocysts to be placed in frozen storage with good potential postthaw, also this reduces the overall number of oocyte retrieval procedures to which patients are exposed. Blastocyst cryopreservation is
MATERIALS AND METHODS The materials and reagents used are shown in Tables 10D.1 and 10D.2, respectively. Table 10D.1 Materials used for blastocyst vitrification using the Cryotop • Cryotop (Kitazato BioPharma Co, Ltd, Japan) • Polycarbonate micropipettes, 175 and 150 µm internal diameter (MidAtlanticDiagnostics) • 60 × 15 mm Tissue culture dish (Nunclon 150362) • 60 × 15 mm Center-well organ culture dish (Falcon 353037) • Weigh paper (Fisher 09-898 12A) • 0.22 µm Syringe filter (Millipore Millex SLGP033RS) • 1 mL Serological pipets (Falcon 7521) • 5 mL Serological pipets (Falcon 7543) • 10 mL Serological pipets (Falcon 7551) • 50 mL Tissue culture flasks (Falcon 3014) • Indelible marker (Sharpie pen, Sanford 33000) • 9” Glass Pasteur pipettes (Sigma S6143) • 10 mL Syringe (Airtite A-10) • 5 mL cryovial (Nalgene 5000-0050)
Table 10D.2 The reagents used for blastocyst vitrification using the Cryotop • Synthetic serum substitute (SSS) (Irvine Scientific 99193P) • Modified human tubal fluid (mHTF) (Sage) • Ethylene glycol (EG) (Sigma E-9129) • Dimethylsulfoxide (DMSO) (Sigma D-2650) • Sucrose (Sigma #S1888)
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Table 10D.3 Formulations of equilibration and vitrification solutions • Stock solution A: 40 mL mHTF + 10 mL SSS (mHTF + 20% SSS) – final total volume of 50 mL • Stock solution B: 6.0 ml EG + 6.0 mL DMSO + 28 mL A (15% EG/DMSO) − final total volume of 40 mL • Equilibration solution: 15 mL A + 15 mL B (7.5% EG/DMSO) − final total volume of 30 mL • Vitrification solution: 5 mL B + 1.71 g sucrose (15% EG/DMSO + 0.5 mol/L sucrose) – bring to a final total volume of 10 mL with solution B* *
Always consider 10 mL as the final total volume allowing for total dissolution of sucrose.
Solutions were prepared as follows: (1) All solutions were filtered to ensure sterility using a 60 mL syringe and a 0.2 µm syringe filter into clean labeled flasks. The first few drops of solution were discarded to help remove residual contamination in the filters. (2) Aliquots of both solutions were dispensed into 1.0 mL vials and labeled appropriately with data. (3) All solutions were refridgerated when not in use. (4) All quality control procedures for the use of in-house prepared media were followed.
(5) The Cryotop was labeled using a cryomarker with the patient’s last name, first name, the date of vitrifying, accession number, and the number and developmental stage of blastocysts (place no more than two blastocysts per Cryotop). Note: Cryotops were pre-labeled on the same side on which the blastocysts were placed. Before vitrification, a micropipette with a diameter of 175–200 µm (‘Stripper’ tip) was used to load the blastocysts on the top. (6) A styroform container was filled with liquid nitrogen (LN2). (7) Procedures for each patient that had blastocysts vitrified were performed in separate hoods and all details were verified by a second embryologist before proceeding. Good expanded/hatching blastocysts were vitrified on day 5/6. (8) Blastocysts were removed from their culture dish using a 200 µm Stripper tip and placed into the drop of solution A, any traces of culture media were removed by gentle aspiration. (9) Blastocysts were pipetted from mHTF to the 7.5% ES, and the 15% VS as follow: (a) The blastocysts were placed in solution A (#1 droplet) and this was connected with droplet #2 (ES) at room temperature. The blastocysts were allowed to dehydrate appropriately. (b) After shrinkage and re-expansion droplet #2 was connected with droplet #3 of ES, and then with droplet #4 of ES to close the ‘circle’. In detail: Exposure to ES occurred until blastocysts re-expanded to ~80% of their original volume. Blastocysts were placed, one to three at a time, into a single drop of ES (see Figure 10D.1), with three drops of ES (A) on a plain 60 mm dish lid surface. The drops were connected 1–2, 2–3, 3–4, and finally 4–1 approximately every 1–2 min, so exposing the blastocysts to a gently increasing concentration
The step-wise vitrification procedure was performed as follows: (1) The benchwarmer was turned off for 2 min to cool closer to room temperature (∼25oC). (2) Aseptic techniques were required at all stages. Equilibration and vitrification were performed at room temperature of 22–25°C. (3) Reagents were removed from the refrigerator and allowed to warm to room temperature. (4) A tissue culture dish lid (upside down) was labeled with the patient’s name as follows: stock solution A, ES, and VS. Aliquots of 1 × 50 µL of solution A, 4 × 50 µL of ES, and 4 × 50 µL of VS were prepared (see Figure 10D.1).
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ES
Stock solution A (Merge drops) 1.
2.
4.
3. (Transfer to drop)
ES 5.
6. 7.
VS
8. 9.
Figure 10D.1 Setup for the vitrification procedure on a plain 60 mm dish lid surface ES, equilibration solution; VS, vitrification solution.
gradient until they were in nearly ‘pure’ equilibration solution (ES). Note that the blastocysts remained in the original drop #1 area, and were influenced by influx of higher concentrations of ES over a period of 5–7 min. These stages should not be rushed, and care was taken not to introduce increasing ES concentrations until the blastocysts were partially re-expanded. (c) Blastocysts were placed into the top of drop #5 and allowed to sink to the bottom for 1–2 min depending on re-expansion of the blastocysts which should have returned to ~75–85% of original volume by which time they were ready to pass into the vitrification solution (VS). (10) The blastocysts were loaded in a new VS back loaded Stripper tip, and rinsed through the four droplets of VS, between each droplet the tip of the pipette was rinsed. In detail: With four drops (#6–9) of the VS aligned next to the ES drop #5 (see Figure10D.1), the re-expanded blastocysts were moved into the first of the four drops (#6–9), and up through the VS drops of
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15% EG + 15% DMSO plus 0.5 mol/L sucrose by a process of joining the first three drops (#6–8), so that the blastocysts ‘rose’ up incrementally through into the purest VS droplet; then were moved into drop #9. Blastocysts shrinkage was quite extreme as was to be expected at this high concentration of vitrificants. Re-expansion should not be allowed. (11) Placement into the VS and loading of the Cryotop took less than 1 min, preferably the total exposure time in VS was approximately 30 s. After 30 s, they were gently transferred to the tip of the Cryotop by using a Stripper tip to load the blastocysts in as small volume (0.5 µL) as possible onto the edge of the Cryotop (the ‘loading’ side, which is the side labeled with ‘CRYOTOP’ plus the patient information, was used). The tip of the Cryotop was marked with black marker to help visualize the tip under the liquid nitrogen. (12) Placement of the blastocyst(s) was visually confirmed. (13) The loaded Cryotop was plunged directly and vertically in liquid nitrogen (LN2). Under LN2 the Cryotops were capped with the flared blue straw cover
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Table 10D.4 solutions
Summary of vitrification and warming
Equilibration solution (ES) Vitrification solution (VS) Thawing solution (TS) Diluent solution (DS) Holding solution (HS)
Ethylene glycol
DMSO
Sucrose
7.5%
7.5%
0.0 mol/L
15%
15%
0.5 mol/L
0.0% 0.0% 0.0%
1.0 mol/L 0.5 mol/L 0.0 mol/L
0.0 mol/L 0.0 mol/L 0.0 mol/L
(3) All solutions were refridgerated when not in use. (4) All quality control procedures for the use of in-house prepared media were followed. The step-wise warming procedure was performed as follows:
Modified human tubal fluid (mHTF) (commercially available) + 20% SSS (Holding solution – HS)
Table 10D.5
Warming solutions used for blastocyst
• Holding solution (HS): 16 mL mHTF + 4 mL SSS (mHTF + 20% SSS) • Thawing solution (TS): Initial starting with 5 ml HS + 3.423 g sucrose (1.0 mol/L sucrose + HS) – after dissolution of sucrose add an additional HS to final total volume of 10 mL. • Diluent solution (DS): 5 mL HS + 5 ml TS (0.5 mol/L sucrose + HS)
before they were placed in an enclosed labeled cane holder. The Cryotop was placed on a precooled aluminum cane for further storage. (14) The cane was stored in a LN2 dewar. (15) The cane location was recorded on the freezing worksheet and in the cryoinventory log. The warming solutions used are shown in Table 10D.5 (see also Table 10D.4). Solutions were prepared as follows: (1) All solutions were filtered to ensure sterility using a 60 mL syringe and a 0.2 µm syringe filter into clean labeled flasks. The first few drops of solution were discarded to remove residual contamination from the filters. (2) Aliquots of both solutions were dispensed into 1.0 mL vials and labeled appropriately with data.
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(1) Reagents were removed from the refrigerator and allowed to warm to room temperature. All cryoprotectants were removed at 25°C. (2) 1 mL of TS was placed into an organ culture dish and warmed for 5 min at 37°C. (3) A 60 mm Petri dish was labeled with the patient’s name under the lid as follows: TS, DS, and HS. Aliquots of 1 × 50 µL of TS, 4 × 50 µL of DS, and 6 × 50 µL of HS were prepared (see Figure 10D.2). (4) Before warming, a micropipette with a 200 µm bore was used for removing the blastocysts from the Cryotop. (5) A styroform container was filled with LN2. (6) The location and identification of blastocysts was verified with a second embryologist before warming any Cryotop. One Cryotop was warmed at a time. (7) A separate hood was used for each patient for whom Cryotops are to be warmed, and verification by a second embryologist occurred before proceeding. (8) With the Cryotop under LN2, the tip was opened by removing the cap. (9) The Cryotop was submerged direct in the pre-warmed organ culture dish (~33oC) containing ~0.5 mL TS. As soon as the Cryotop contents liquefied (within 15 s), the blastocysts were located before being removed with a Stripper pipette. Blastocysts were located as they floated free from the Cryotop surface, and moved into drop A (see Figure 10D.2). Immediately drops B and A were connected together, allowing the 0.5 mol/L sucrose solution
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TS 60 mm Organ culture dish with 1.0 mL TS
DS
(Merge drops)
A. D. DS
(Transfer to drop)
B. C.
I. J.
(Merge drops)
HS
K.
E.
F.
H.
G.
Figure 10D.2 Setup for the warming procedure on a plain 60 mm dish lid surface. TS, thawing solution; DS, diluent solution; HS, holding solution.
(DS) at room temperature to wash into drop A (TS) for ~2 min. (10) Drops were connected C–B, D–C, and then finally drop D directly with A where the blastocysts still sat. All connections were performed such that blastocyst over-expansion was limited, to ensure that they were not allowed to re-expand to their full 100% state, reconnecting occurred when approximately 75–85% of full size. Blastocysts were moved to drop E, and the gradual dilution repeated by connecting drops F–E, G–F, H–G, and finally H directly to E (‘the circle of life’). The benchwarmer was turned on, and finally dilution occurred through a series of three wash drops of HS (I–K). (11) Blastocysts were moved into culture compatible media and put in the incubator for subsequent culture. (12) Survival and appearance of all blastocysts were recorded. The log was updated with data following warming of blastocyst(s), and the physician notified of results.
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Definitions A positive pregnancy was defined as a positive βhCG of ≥ 20 IU 10 days after blastocyst transfer. Implantation rate was defined by the number of gestational sacs per embryo number transferred. Clinical pregnancy refers to the identification of a pregnancy sac in the uterus, whereas ongoing/delivered pregnancy decribes pregnancies that continued beyond 20 weeks. Statistical analysis was carried out by means of a χ2 test using Microsoft Excel 2001 for Mac (Redmond, WA, USA). Statistical significance was defined as P < 10.05.
RESULTS Table 10D.6 shows the mean age and clinical outcome of patients who completed the vitrified blastocyst transfer program. The mean age of the women was 34.1 ± 5.1 years in the vitrified group. The blastocyst post-warming survival rate after using the vitrification technique is shown in Table 10D.2. A total of 1140 vitrified blastocysts (day 5 and day 6) were
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Table 10D.6 Retrospective data from the blastocyst cryopreservation program at the Fertility Centers of Illinois, Chicago, where vitrification technology was applied from January 2004 to December 2006 Technique Patient’s age (years) No. of thawed cycles No. of transfers No. of blastocysts thawed No. of blastocysts survived (%) No. of blastocysts transferred Mean no. of blastocysts transferred No. of implantations (%) No. of positive pregnancy/thaw (%) No. of positive pregnancy/FET (%) No. of clinical pregnancy/thaw (%) No. of clinical pregnancy/FET (%) Ongoing pregnancies (%) No. of livebirths
34.1 ± 5.1 548 541 1140 1085(95.2) 1073 2.0 310 (28.9) 270 (49.3) 270 (50.0) 228 (41.6) 228 (42.1) 198 (73.3) 117 (65 girls and 52 boys)
pregnancy rates per transfer were 28.9% and 42.1%, respectively. To date, 93 deliveries have occurred with no reported abnormalities (117 babies: 65 girls and 52 boys). When the vitrified warmed blastocysts were divided into blastocysts vitrified on day 5 and day 6 groups, 95.5% (611/640) of day 5 blastocysts and 94.8% (474/500) of day 6 blastocysts survived after warming (Table 10D.7), but this difference was not significant. As shown in Table 10D.7, implantation and clinical pregnancy rates per transfer occurring in the day-5 blastocyst group were 32.3% and 45.8%, respectively, which were significantly higher (P < 0.01) than the day-6 blastocyst group (24.6% and 37.8%, respectively).
CONCLUSIONS
FET, frozen embryo transfer.
warmed, of which 1085 survived warming (95.2%). In the vitrified group 1073 vitrified warmed blastocysts were transferred in 541 cycles out of 548 attempted cycles (mean 2.0 blastocysts per frozen embryo transfer). Overall the implantation and clinical
In conclusion, although some problems remain to be fully addressed regarding vitrification as a routine cryopreservation technique, we believe that it shows much promise as a successful alternative to conventional freezing technology. Even without significant clinical improvement, the evident advantages of vitrification are that cryosurvival seems
Table 10D.7 A comparison of retrospective data from the blastocyst cryopreservation program at the Fertility Centers of Illinois, Chicago of vitrified on day 5 and day 6 from January 2004 to December 2006 Day of development
Patient’s age (years) No. of thawed cycles No. of transfers No. of blastocysts thawed No. of blastocysts survived (%) No. of blastocysts transferred Mean no. of blastocysts transferred No. of implantations (%) No. of positive pregnancy/thaw (%) No. of positive pregnancy/FET (%) No. of clinical pregnancy/thaw (%) No. of clinical pregnancy/FET (%) Ongoing/delivered pregnancies (%) P < 0.01
*
FET, frozen embryo transfer.
258
Day 5
Day 6
34.0 ± 5.0 297 295 640 611 (95.5) 601 2.0 194 (32.3)* 164 (55.2) 164 (55.6)* 135 (45.5) 135 (45.8)* 120 (73.2)
34.2 ± 5.2 251 246 500 474 (94.8) 472 1.9 116 (24.6)* 106 (42.2) 106 (43.0)* 93 (37.0) 93 (37.8)* 78 (73.6)
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more consistent, allowing greater ease of patient management with transfers being almost certain to occur. Concerns with vitrification are well defined and limited in number, and to our way of thinking easily surmountable. In general, with much shorter protocols, vitrification is able to be undertaken on a more flexible basis by laboratory staff, allowing for the potential reduction in personnel time needed during the entire vitrification process, simplifies laboratory techniques for cryopreservation in human ART, and may enable more optimal timing of embryo cryopreservation, e.g. individual blastocysts may be cryopreserved at their optimal stage of development and expansion. Interest levels will inevitably rise given the potential benefits of vitrification. This in turn will drive its development to higher levels of clinical efficiency and utilization.
SPECIAL NOTES FOR THE PRACTITIONER Special care must be given to the selecting of the carriers. It is necessary to use types of carrier or vessel material with rapid heat transfer that also support the process of uniform heat exchange to achieve higher cooling rates. To minimize the toxicity of the cryoprotectant a step-wise exposure of cells to pre-cooled concentrated solutions is recommended. Higher concentrations of cryoprotectant should be utilized that allow shorter exposure
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times to the cryoprotectant – but be careful – the toxicity of the cryoprotectants will be raised with their concentration. Because almost all cryoprotectants are toxic, it is important to watch the duration of exposure to the final cryoprotectant very carefully before plunging into liquid nitrogen. To facilitate vitrification by even higher cooling rates, it is also necessary to minimize the volume of the vitrification solution as much as practical. From this point of view it is very important to use a micropipette with a small diameter (< 150 µm). Furthermore, by collecting the embryos on one place, and loading not more than two embryos at the same time in the pipette it is possible to keep the volume small. To make sure that the cells have been loaded on the carrier, perform the loading process under a stereo-microscope. Check the number of loaded embryos, and check the pipette is empty after loading. Submerge the carrier loaded with the cells directly in liquid nitrogen by passing quickly through the vapor phase (nitrogen gas). Before moving the carrier quickly from the LN2 in to the warming solution, prepare a micropipette. Fill the pipette with a small amount of the first warming solution. Even when switching the embryos between different concentrations of warming solutions, fill up the pipette tip with the next lower concentration of warming solution before picking up the embryos for moving in to the following concentration.
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11A
Frank Nawroth, Vladimir Isachenko, Evgenia Isachenko and Gohar Rahimi
INTRODUCTION Secondary ovarian failure could for a variety of reasons be an indication to cryopreserve ovarian tissue. Such incidences have increased since malignant diseases can now be successfully treated, but often result in irreversible fertility loss. At the moment, for later fertility preservation the extraction and storage of larger amounts of ovarian cortex using unilateral ovarectomy is favored.1 The protocols used for the cryopreservation of ovarian tissue are based on those for mature oocytes.2 However, additional problems exist with the cellular heterogeneity of ovarian tissue, giving rise to different diffusion rates of the cryoprotectants throughout the tissue. The optimal protocol with regard to suitable cryoprotectant and freezing technique for ovarian tissue has yet to be found,2 but at present ‘slow cooling and rapid thawing’ is favored.3 Ovarian tissue can be auto- or xenotransplanted after thawing. At the time of ovarian tissue harvest careful examination sometimes reveals the presence of free immature oocytes that can be gathered during tissue preparation before freezing, and can additionally be frozen at the immature stage or after in vitro maturation (IVM).4–6 The conventional cryopreservation of human ovarian tissue is developing from an experimental procedure into daily practice. Although further research must focus on investigating current options and new alternatives to identify the best way of using ovarian tissue after thawing, a consultation on the possibility of this procedure for different patients
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(for example before chemotherapy/radiation) is now more commonly recommended.7,8 Several pregnancies after conventional slow freezing of human ovarian tissue have been reported since 2004 (Table 11A.1). At the same time, the method should be routinely performed with minimal equipment, staff, and time requirements. This is the reason for the increased interest in vitrification not only of cells but also of tissue.13 The problems related to successful cryopreservation increase with the complexity of the sample intended for vitrification (cell–tissue–entire organ). A renaissance of interest in vitrification started in 1980. At this time the American Red Cross initiated studies investigating the potential of vitrification as an alternative to classic cryopreservation. Viability assays have demonstrated successful application of vitrification in a variety of single cells or small cell aggregates including human islets of Langerhans, monocytes, red blood cells, liver cells in culture, certain plants and plant tissues, a variety of animal embryos, and rodent egg cells.14 It has been performed partially successfully with human corneas and rabbit kidney slices. When vitrification is used with tissue, due to the high concentration of cryoprotectants, the heterogeneity of the cells with long diffusion times and the resulting potential toxicity can be problematic. The main problems with vitrification of large samples are fracturing as well as crystallization during cooling and/or warming. Fracturing can mostly be prevented through careful handling of the sample, so that crystallization remains the more serious problem.15
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Table 11A.1 Published pregnancies after autotransplantation of cryopreserved/thawed human ovarian tissue (up to November 2006)
Reference
Age before freezing (years)
Indication
Location of transplantation
Reproductive outcome
Donnez et al., 20049
25
Hodgkin’s lymphoma
Orthotopic: peritoneum of the ovarian fossa
Spontaneous pregnancy, live birth
Meirow et al., 200510
28
Non-Hodgkin’s lymphoma
Orthotopic: ovary
Modified menstrual cycle, IVF-ICSI, ET, live birth
Demeestere et al., 200611
24
Hodgkin’s lymphoma
Orthotopic: ovary + peritoneum of the ovarian fossa combined with Heterotopic: abdominal wall subcutaneously
Spontaneous pregnancy, abortion at 7 weeks
Rosendahl et al., 200612
28
Hodgkin’s lymphoma
Orthotopic: ovary + peritoneum of the ovarian fossa combined with Heterotopic: subperitoneal tissue beneath the abdominal fascia between the umbilicus and the pubic bone
FSH stimulation, IVF-ICSI after oocyte retrieval from the heterotopic graft, ET, biochemical pregnancy
IVF-ICSI, in vitro fertilization – intracytoplasmic sperm injection; ET, embryo transfer; FSH, follicle stimulating hormone.
VITRIFICATION OF OVARIAN TISSUE IN ANIMALS Various research groups have reported the successful vitrification of ovarian tissue from mice, rats, Chinese hamsters, rabbits, Japanese apes, monkey, cows, sheep, dog, and human fetuses.15–24 The problem of the transfer of data from animal studies to the human was the focus of a study dealing with the determination of the best experimental model for developing new protocols of equilibrium cooling and vitrification.25 The authors compared adult cow and pig with human ovaries and found that human and bovine follicles responded in the same way to the two equilibrium cooling protocols used, whereas pig tissue was more cryoresistant. Both vitrification protocols caused extensive damage to the tissue of all species. Human tissue showed a response to vitrification that
was different from that of both cow and pig. Their conclusion was the cow is a good animal model for the development of equilibrium cooling procedures but at present, neither cow nor pig can be considered relevant animal models for the vitrification of human ovarian tissue.
Specific experiences with vitrification of ovarian tissue in some animal species Vitrification of ovarian tissue in rats and mice Miyamoto and Sugimoto16 vitrified rat ovaries and removed the cryoprotectant step-wise. Histological examination of the follicles yielded positive results regarding surface area, but revealed degenerative changes, such as pyknosis, vacuolization, and cell swelling, in
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the remaining tissue. Therefore, ‘slow cooling’ was considered to be superior, even though the tissue showed partial vitality. Vital follicles were still detected 4 days after the warming of vitrified fetal rat ovaries.18 Sugimoto et al.19 vitrified infantile rat ovaries (left side) and assessed their viability after warming and autotransplantation under the kidney capsule. The right ovary of each rat was removed. For the control animals, the left ovary was dissected and immediately transplanted without prior vitrification. The time course of development of endocrine function of cryopreserved grafts was similar to that of fresh grafts. In ovarian transplants recovered on postnatal day 84, antral follicles and corpora lutea were observed in addition to small follicles, although the number of antral follicles in cryopreserved grafts was smaller than in the fresh grafts. The authors concluded that vitrification of ovarian tissue can be used for the preservation of fertility and endocrine function of ovaries. Salehnia et al.26 vitrified ovaries from 8–10week-old mice using a vitrification solution containing 30% (w/v) Ficoll 70, 0.5 mol/L sucrose, 10.7% (v/v) acetamide, and 40% (v/v) ethylene glycol (EG). After warming with 1 mol/L sucrose solution the vitrified and fresh ovarian tissues were fixed and histological and electron microscopic investigations were performed. Under the electron microscope, the integrity of cell organelles, nuclei, and microvilli of oocyte and follicular cells were well preserved but some mitochondria were swollen and their cristae had partially disappeared. Vitrification did not cause any harmful damage to follicular cells and oocytes. The same group27 reported interesting results if mouse ovaries, vitrified/warmed using the same solutions, were autografted intraperitoneally and recovered after one and two estrous cycles. Light microscope studies after 5 days showed that the grafted ovarian tissues were invaded by many fat and fibrous cells. Many large preantral and antral follicles were degenerated;
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however, after 10 days the stroma of transplanted ovaries was devoid of necrotic cells and contained normal primordial and primary follicles. Non-frozen ovaries (control) had normal follicles at all developmental stages. Kim et al.28 investigated whether oocytes within preantral follicles isolated from vitrified mouse ovaries are viable and can be rescued to undergo growth, maturation, fertilization, and embryo development in vitro. EG, dimethylsulfoxide (DMSO), and sucrose were used as cryoprotectants. Equilibration time was either 5 or 10 min. Survival and maturation rates were significantly higher in the 5 min compared with the 10 min exposure groups. This led to the conclusion that mouse oocytes within preantral follicles isolated from the vitrified ovary can achieve full maturation and normal fertilization and embryo development. Takahashi et al.29 reported the first successful vitrification of adult mouse ovaries using the method of Rall and Fahy30 with VS1, a combination of 20.5% (w/v) DMSO, 15.5% (w/v) acetamide, 10% (w/v) propylene glycol (PG) and 6% (w/v) polyethylene glycol (PEG). Migishima et al.31 developed a new method of cryopreservation of whole mouse ovaries by vitrification using DAP213 (2 mol/L DMSO, 1 mol/L acetamide, and 3 mol/L PG) as a cryoprotectant. Cryopreserved or fresh ovaries were orthotopically transplanted (experimental or control group). Histologically, normal development of follicles and formation of corpora lutea were observed in frozen/thawed grafts. However, estimated number of follicles decreased in frozen/thawed ovaries compared with fresh ovaries showing that further studies are required to overcome the possible inhibitory effects of this method on the growth of the ovarian graft.31 Segino et al.32 isolated and cultured cumulusenclosed oocyte complexes (COC) and preantral folicles from vitrified mouse ovarian tissue. The survival rate of the follicles obtained from the cooled/warmed ovaries was 66.4%. Comparison of the follicles isolated from fresh
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and frozen/thawed ovaries after 12 days of culture showed significant differences regarding the diameter of the isolated follicles (620.2 ± 11.3 µm vs. 518.7 ± 15.1 µm) and estradiol concentrations (3474.2 ± 159 pg/mL vs. 1508.2 ± 134 pg/mL). After in vitro ovulation, metaphase II (MII) stage oocytes were observed in 84.5% of the fresh group and 60.5% of the cooled/warmed group, while the fertilization rates were 74.2% and 53.5%, respectively. The study demonstrated that vitrification of mouse ovarian tissues did not affect the oocyte’s ability to undergo meiosis. Follicles isolated from vitrified/warmed mouse ovarian tissues reached the mature follicle stage on the 12th day of culture. However, the developmental ability was lower than that of fresh ovarian tissue. The same group33 cultured preantral follicles isolated from vitrified/warmed ovarian tissues for 12–16 days. Then the follicles were stimulated with human chorionic gonadotropin. They developed slowly compared with the freshly prepared preantral follicles. However, during prolonged culture from 12 to 16 days, these follicles obtained the potential to fertilize and develop to the blastocyst stage. This confirmed the results from another study published 1 year earlier.34 COC were retrieved from vitrified mouse ovaries by enzymatic treatment and in vitro fertilized after in vitro growth (IVG) and IVM. EG and DMSO were used as cryoprotectants. Following the procedure including IVM, 75.9% of oocytes in COC matured to the MII stage compared with 75.2% from fresh ovaries. After in vitro fertilization (IVF) the fertilization rate of these oocytes was 57.5% as compared with 69.5% for fresh ovaries. Both differences were not statistically significant. The conclusion was that oocytes enclosed in preantral follicles from vitrified/warmed mouse ovaries preserved capacity for fertilization and development to preimplantation embryos. Recently an interesting paper was published dealing with a new vitrification method using less concentrated cryoprotectants and direct application of liquid nitrogen to the ovarian
tissue in mice (direct cover vitrification, DCV).35 In the first step the ovary (size of an ovary approximately 1.2 × 1.5 × 1.5 mm) was pretreated with an equilibration solution (0.8 mL) consisting of 7.5% (v/v) EG and 7.5% DMSO in Dulbecco’s phosphate-buffered solution (DPBS) with 20% fetal bovine serum (FBS) for 10 min at room temperature. It was then transferred to a vitrification solution (0.8 mL) consisting of 15% EG, 15% DMSO, and 0.5 mol/L sucrose for 2 min. After removal of the surrounding vitrification medium the ovary was placed on a piece of gauze, put in a 1.8 mL plastic standard cryovial (Nunc, Roskilde, Denmark), and liquid nitrogen was directly applied onto the ovary. Following this procedure the vial was placed into a liquid nitrogen tank. For thawing, the ovary was moved into 1 mL of 1 mol/L sucrose, kept there for 5 min and then put into 0.5 mol/L followed by 0.25 mol/L sucrose and DPBS medium for 5 min each. One of the results of the study was a significantly higher pregnancy rate of DCV compared with conventional vitrification after orthotopic transplantation (P < 0.01). Hani et al.36 showed that vitrification of mouse ovaries is possible for the preservation of female germ cells from mice of various ages. They used the above mentioned DAP213 as vitrification solution31 and compared the viability of cryopreserved adult mouse ovaries with that of immature ovaries. Both were viable and able to produce young after orthotopic transplantation to 4- or 10week-old mice.
Vitrification of bovine ovarian tissue The comparison of conventional freezing and vitrification of bovine ovarian tissue demonstrated that a vitrification protocol using EG and equilibration with 5.5 mol/L EG at 22°C for 20 min may be just as effective as ‘slow freezing’.15
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Vitrification of ovarian tissue in sheep Al-Aghbari and Menino37 removed and cut ovaries from 15 pubertal ewes. They used equilibration medium consisting of 4% (v/v) EG and 20% (v/v) FBS in TCM-199 on ice for 30 min and transferred the pieces to vitrification solution (35% EG, 5% polyvinylpyrrolidone, 0.4 mol/L trehalose, and 20% FBS in TCM-199) for 5 min. They also collected and vitrified COC. After 2–3 weeks storage in liquid nitrogen, ovarian tissues and COC were thawed at 37°C. Vitrified COC and freshly collected (mechanically and enzymatically isolated from vitrified tissue after thawing) COC were cultured for 23–25 h. Significantly fewer (P < 0.05) oocytes obtained from vitrified ovarian tissue (70%) reached MII compared with vitrified oocytes (88%) and non-vitrified control oocytes (90%). In contrast, when oocytes with at least 3–5 layers of cumulus cells were considered from each of the three groups, no significant differences were observed due to treatment in the percentages of oocytes developing to MII. Hence, these authors demonstrated that sheep oocytes can be successfully cryopreserved by vitrification of ovarian tissue and exhibit in vitro maturation rates similar to those of vitrified and non-vitrified oocytes. A further step was the attempt to vitrify whole sheep ovaries with vascular pedicle.38 Their study based on results from a rabbit model,39 where whole rabbit kidneys were perfused with a vitrification solution containing 2.75 mol/L DMSO, 2.76 mol/L formamide, and 1.97 mol/L PG as cryoprotectants (VS4). The kidneys were evaluated using an autograft model with immediate contralateral nephrectomy. Using the protocol with the highest concentrations and perfusion at about −3°C, the survival rate was 100%, serum creatinine returned to a normal baseline after transient elevation, other clinical chemistry results normalized, and no histological damage was apparent 3 weeks after autografting.
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Courbiere et al.38 used the same VS4 in comparison with VS1 (2.62 mol/L DMSO, 2.60 mol/L acetamide, 1.31 mol/L PG, and 0.0075 mol/L PEG as cryoprotectants diluted in BM1 medium (Eurobio, Les Ulis, France)) in their sheep model. They described no statistically significant differences in follicle viability or normal primordial follicle rates between ovaries exposed or not exposed to cryoprotectant solutions, before and after vitrification with the two cryoprotectant solutions. VS4 was beneficial regarding nuclear anomalies and general follicular anomalies. However, vascular pedicle fractures occurred in most ovaries during thawing (11/15).38 In a later published study the same authors24 focused on the question of whether whole sheep ovaries can really be totally vitrified using VS4. The difference of follicle viability for ovaries exposed to VS4, without vitrification and for ovaries vitrified with VS4 was not statistically significant (70.6% ± 4.7% vs. 61.4% ± 2.2%). However, they showed that the critical cooling rate for the impregnated ovarian cortex exceeded −300°C/min, suggesting that, under the studied experimental conditions, the ovarian tissue is unlikely to be totally vitrified at the end of cooling. After vitrification using VS1, warming and orthotopic autotransplantation of hemiovaries into sheep three pregnancies occurred. One of the four lambs that were born had a malformation of the left leg and the esophagus, with a questionable link to the vitrification.40
Vitrification of ovarian tissue in monkey Co-culture of vitrified/warmed or slowfrozen monkey ovarian tissue on mouse fetal fibroblast monolayers supplemented with follicle stimulating hormone (FSH), insulin, transferrin, and selenium resulted in a significantly increased rate of viable follicles in vitrified/warmed as well as slow-frozen tissue. Co-culture could therefore be a beneficial approach to improve graft survival after
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vitrification, just as it can be as effective as after slow freezing.23
Vitrification of ovarian tissue in rabbit Ishijima et al.41 vitrified canine ovaries. Warmed tissue which showed normal morphology in histology was transplanted into the ovarian bursa of the severe combined immune deficiency (SCID) mice and recovered 4 weeks after the operation. Antral follicle formation did not occur after grafting but proliferating cell nuclear antigen immunoreactivity was detectable in many of the granulosa cells in the primary follicles of the grafts. The authors concluded that vitrification has the potential to restore endocrine function and ovulation potential in canine.
VITRIFICATION OF OVARIAN TISSUE IN HUMAN It is known from other areas of human research that the vitrification of cornea42 and vessels14,43 is possible. Practical knowledge regarding virtification of human ovarian tissue is limited. Nevertheless, some initial studies have dealt with vitrification of human ovarian tissue.44–46
Vitrification of human ovarian tissue and investigation/in vitro culture after warming Human fetal17,22,47 and adult ovarian tissue samples20 have been vitrified successfully using EG and sucrose. In a computerized picture analysis of the cell nuclei the results were comparable after vitrification or slow freezing of human ovarian tissue.44 They removed ovarian cortical slices (10 × 2 × 1 mm) from six consenting women (aged 29–36 years) during second Cesarean section and cut each slice into five pieces. The aim was to compare a fresh sample (scraped with a scalpel, smeared on a microscopic slide, and air-dried at room
temperature), a necrotic sample (put on a gauze moisturized with 0.9% NaCl for 24 h), a slow-frozen sample (automated freezer in cryovials in α-MEM + 10% patient serum +1.5 mol/L DMSO), and two vitrified samples (vitrified in cryovials in 0.5 mL of PBS + 20% fetal cord blood serum + 5.5 mol/L EG + 1 mol/L sucrose for 1 and 5 min, respectively, and then plunged into liquid nitrogen). After thawing, 15 nuclear parameters including morphometric descriptors (nuclear area, perimeter, form factor) and nuclear texture features (contrast, entropy, variance, and Markovian features 4, 5, 6, 7, 10, 11, 16, 17, and 18) were assessed for each nuclear image. Preliminary results of computerized image analysis of nuclear features including texture indicated a promising model for post-thaw viability evaluation. In their study, 1 min vitrification in 5.5 mol/L EG appeared to be comparable with slow freezing for human ovarian tissue. During our own first histological studies of vitrified human adult ovarian tissue samples (maximum size, 1 mm3) (unpublished data) we found that freezing and warming with EG + saccharose + egg yolk in combination with direct plunging of straws or grids in liquid nitrogen did not significantly influence the ovarian tissue morphology or the follicle morphology. In combination with suitable long-term cultures of human ovarian tissue, the subsequent IVM could complement treatment for example, in planned transplants. In a long-term culture of native human ovarian tissue, we were already able to show that no significant increase in apoptosis occurred after 6 weeks compared with control tissues on day 1.48 We further evaluated the effect of different vitrification protocols on reactive oxygen species (ROS) and apoptosis in human ovarian tissue.46 Ovarian tissue pieces (1 ± 0.5 mm3) were randomly distributed into three treatment groups and exposed at 0–1°C to different vitrification solutions as follows: group 1,
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control (without treatment); group 2, 40% EG (v/v) + 0.35 mol/L sucrose + 10% egg yolk extract (v/v)49 for 6 min; and group 3, 40% EG + 18% Ficoll 70 (w/v) + 0.35 mol/L sucrose50 for 6 min. Egg yolk was used as cryoprotectant.51 All exposures in vitrification solutions started with 1 min shaking at 5 Hz. After equilibration with the cryoprotectants, ovarian tissue pieces were placed with a 1cm column of vitrification solution in 0.25 mL standard insemination straws (IMV, L’Aigle, France) and plunged into liquid nitrogen, inserted into the metallic powder cooled previously in liquid nitrogen,52 inserted into liquid nitrogen vapor (−120°C) for 15 min and subsequently plunged into liquid nitrogen, or plunged into liquid nitrogen after being transferred onto copper grids.53 Very rapidly cooled tissue (plunged directly into liquid nitrogen in straws or on grids, or plunged directly into metal filings precooled to −196°C) showed no statistically significant increase in either tissue ROS levels or the number of apoptotic cells after warming. In contrast, cooling using a less rapid method (nitrogen vapor at −120°C) resulted in significantly elevated ROS levels and apoptosis after warming. There were no significant differences between the two vitrification solutions. This indicates that human ovarian tissue pieces should be vitrified using very rapid cooling rates.46 Our results indicated that vitrification of human ovarian tissue should involve only permeable cryoprotectants and substances that prevent ice formation. The addition of the disaccharide Ficoll 70 resulted in an increased osmotic injury.54 In another study, we tried to further develop the vitrification protocol. Human ovarian biopsies from 20 patients (cut into ~ 0.5 mm3 pieces) were exposed to 40% EG + 0.35 mol/L sucrose + 5% egg yolk, 40% EG + 18% Ficoll 70 + 0.35 mol/L sucrose, or 20% EG + 20% DMSO. Cryopreservation of pieces was accomplished by plunging 0.25 mL straws or copper grids into liquid nitrogen, or 0.25 mL straws into precooled (−196°C)
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metallic powder. Thawed pieces were transferred to sucrose solution for incremental dilution of cryoprotectants. Histological observation of the tissue was performed after cryopreservation, and in vitro culture was undertaken to study the ability to produce hormones after cryopreservation. The only vitrification solution which protected both follicles and stroma was 40% EG + 0.35 mol/L sucrose + 5% egg yolk using standard 0.25 mL straws or copper grids, with direct plunging into liquid nitrogen.45 Recently a modified method of in vitro culture of vitrified human ovarian tissue was published.55 Different groups of in vitro culture after warming during 2 and 6 weeks were compared with respect to follicle growth: in 2 mL of culture medium which was regularly renewed (group 1), in 30 mL of culture medium without agitation (group 2), and in 30 mL of culture medium with agitation (75 oscillations/min using a rotation shaker) (group 3). After 2 weeks of culture, the mean number of non-degenerated follicles per mm2 of tissue was significantly higher in group 3 when compared with groups 1 and 2 (P < 0.05). The conclusion was that agitation during culture of ovarian tissue is beneficial (Figures. 11A.1 and 11A.2). Kagawa et al.56 vitrified human ovarian tissue using different sample sizes (0.5 × 0.5 × 0.5 mm (small), 10 × 10 × 1 mm or 20 × 10 × 1 mm (large)) with the Cryotop as carrier. After incubating the tissues in the equilibration solution (7.5% EG + 7.5% DMSO) at room temperature, they transferred the samples to five different vitrification solutions (EG and DMSO: 15 + 15%; 17.5 +17.5%; 20 + 20%; 22.5 + 22.5% and 25 + 25%, respectively), all supplemented with 0.5 mol/L sucrose for 5, 10, 15, 20, 25, 30, 35, and 40 min, respectively. Warming was performed by immersing in a 37°C thawing solution containing 1 mol/L sucrose for 3 min, followed by 0.5 mol/L sucrose solution for 5 min, and 30 min incubation in isotonic solutions. The authors reported that both large and
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0.5 mm
70 µm b
a
0.5 mm
1 mm
c
d
Figure 11 A.1 Follicles developed in vitrified/warmed human ovarian tissue after 3 weeks of culture: (a) primordial, (b) and (c) early antral, and (d) antral.
small samples could be successfully vitrified: the large tissues with > 15 min incubation and in concentrations of cryoprotectants of 17.5 + 17.5% or higher in the vitrification solution, and the small tissues with a 91% survival rate after 10 min incubation in the 15 + 15% group. Survival rates of oocytes were in the ranged between 0 and 91%, and negatively correlated with the size of the ovarian tissue.
Vitrification of human ovarian tissue and xenografting after warming We investigated and compared the necrotic areas after subcutaneous transplantation
of vitrified/warmed and slow cryopreserved/ thawed human ovarian tissue into SCID mice for 6 weeks.57 Solutions for vitrification were prepared in DPBS medium supplemented with 15% fetal calf serum. The cryoprotectants used were glycerol, EG, and the synthetic ice blocking agent Supercool® X100 (Cooltechnica, New York, USA). The study showed that the size of the necrotic areas in human ovarian tissue were comparable between the different methods (Figure 11A.3). In a further study of ours (unpublished data) in SCID mice, we observed neovascularization of human ovarian tissue after slow
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*
* * *
a
b
c
d
e
f
400 µm
Figure 11A.2 Vitrified/warmed human ovarian tissue after 2-week (a)–(d) and 6-week (e), and (f) culture in a large volume of medium by agitation. Scale bars in (a)–(e) = 50 µm. Note: formation of blood vessels with erythrocytes (arrows) and adipose tissue (fat cells) (asterisks).
b
a
Figure 11A.3 Detection of necrotic areas with Lucifer Yellow CH staining in the human ovarian tissue after (a) slow freezing and (b) vitrification. Scale bar = 200 µm.
freezing/transplantation, as well as after vitrification/transplantation using the same vitrification protocol as in the above mentioned study. There was no significant difference in
269
the neovascularization between the groups. With increased time of transplantation we found an increase in the area of platelet endothelial cell adhesion molecule (PECAM)-1
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a
Figure 11A.4 The platelet endothelial cell adhesion molecule (PECAM)-1 immunofluorescence in the human ovarian tissue after vitrification and transplantation for 1 week (a) and 4 weeks (b). Scale bar = 20 µm.
b
positive blood vessels in the transplanted human ovarian tissue. The area increased from 2.85 ± 0.27 mm² (slow freezing), 2.19 ± 0.48 mm² (vitrification), and 1.96 ± 0.89 mm² (control = fresh transplanted tissue) after 1 week, to 92.5 ± 24 mm², 104.1 ± 22 mm² and 72.3 ± 17 mm², respectively, after 4 weeks (Figure 11A.4).
Vitrification of human ovarian tissue and autografting after warming To our knowledge there have been no published papers dealing with vitrification of
human ovarian tissue and autografting until now.
CONCLUSION At the moment the conventional cryopreservation of human ovarian tissue seems to be favored due to considerably greater experience. Vitrification, nevertheless, could become a realistic alternative, however, further studies regarding optimization of cryoprotectant solutions and protocols are necessary to achieve the same results as with equilibrium methods.
References 6. Isachenko E, Rahimi G, Isachenko V et al. Invitro maturation of germinal-vesicle oocytes and cryopreservation in metaphase I/II: a possible additional option to preserve fertility during ovarian tissue cryopreservation. Reprod Biomed Online 2004; 8: 553–7. 7. Lightman A, Werner-Kimel N, Solt I et al. A program of ovarian tissue cryopreservation for women with malignant disease: lessons from 5 years’ experience. Fertil Steril 2001; 76(Suppl 1): 81. 8. Donnez J, Martinez-Madrid B, Jadoul P et al. Ovarian tissue cryopreservation and transplantation: a review. Hum Reprod Update 2006; 12: 519–35. 9. Donnez J, Dolmans MM, Demylle D et al. Livebirth after orthotopic transplantation of cryopreserved ovarian tissue. Lancet 2004; 364: 1405–10.
1. Kim SS, Battaglia DE, Soules MR. The future of human ovarian cryopreservation and transplantation: fertility and beyond. Fertil Steril 2001; 75: 1049–56. 2. Newton H, Fisher J, Arnold JRP et al. Permeation of human ovarian tissue with cryoprotective agents in preparation for cryopreservation. Hum Reprod 1998; 13: 376–80. 3. Gosden RG. Low temperature storage and grafting of human ovarian tissue. Mol Cell Endocrinol 2000; 163: 125–9. 4. Revel A, Koler M, Simon A et al. Oocyte collection during cryopreservation of the ovarian cortex. Fertil Steril 2003; 79: 1237–9. 5. Revel A, Safran A, Benshushan A et al. In vitro maturation and fertilization of oocytes from an intact ovary of a surgically treated patient with endometrial carcinoma: case report. Hum Reprod 2004; 19: 1608–11.
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10. Meirow D, Levron J, Eldar-Geva T et al. Pregnancy after transplantation of cryopreserved ovarian tissue in a patient with ovarian failure after chemotherapy. N Engl J Med 2005; 353: 318–21. 11. Demeestere I, Simon P, Buxant F et al. Ovarian function and spontaneous pregnancy after combined heterotopic and orthotopic cryopreserved ovarian tissue transplantation in a patient previously treated with bone marrow transplantation: case report. Hum Reprod 2006; 21: 2010–14. 12. Rosendahl M, Loft A, Byskov AG et al. Biochemical pregnancy after fertilization of an oocyte aspirated from a heterotopic autotransplant of cryopreserved ovarian tissue: case report. Hum Reprod 2006; 21: 2006–9. 13. Liebermann J, Nawroth F, Isachenko V et al. Potential importance of vitrification in reproductive medicine. Biol Reprod 2002; 67: 1671–80. 14. Brockbank KGM, Song YC, Khirabadi BS et al. Storage of tissues by vitrification. Transplant Proc 2000; 32: 3–4. 15. Yin H, Kim SS, Fisher J et al. Investigation of optimal conditions for equilibrating ovarian tissue with ethylene glycol prior to vitrification. Fertil Steril 2001; 76(Suppl 1): S101. 16. Miyamoto H, Sugimoto M. Assessment of follicle development and histocytological examination of neonatal rat ovaries cryopreserved by vitrification technique. Cryobiology 1994; 31: 614–5. 17. Zhang J, Liu J, Xu KP et al. Extracorporal development and ultrarapid freezing of human fetal ova. J Assist Reprod Genet 1995; 12: 361–8. 18. Sugimoto M, Miyamoto H, Kabasawa T et al. Follicle survival in neonatal rat ovaries cryopreserved by vitrification. Cryo Letters 1996; 17: 93–8. 19. Sugimoto M, Maeda S, Manabe N et al. Development of infantile rat ovaries autotransplanted after cryopreservation by vitrification. Theriogenology 2000; 53: 1093–103. 20. Lee SH, Shin CS, Ko JJ et al. In vitro culture of the human adult ovarian tissues after vitrification: comparison among detection methods of the culture effect. Fertil Steril 2000; 74(Suppl 1): 161. 21. Kagabu S, Umezu M. Transplantation of cryopreserved mouse, Chinese hamster, rabbit, Japanese monkey, and rat ovaries into rat recipients. Exp Anim 2000; 49: 17–21. 22. Van den Broecke R, Liu J, Handyside A et al. Follicular growth in fresh and cryopreserved human ovarian cortical grafts transplanted to immunodeficient mice. Eur J Obstet Gynecol Reprod Biol 2001; 97: 193–201.
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23. Yeoman RR, Wolf DP, Lee DM. Coculture of monkey ovarian tissue increases survival after vitrification and slow-rate freezing. Fertil Steril 2005; 83(Suppl 1): 1248–54. 24. Courbiere B, Odagescu V, Baudot A et al. Cryopreservation of the ovary by vitrification as an alternative to slow-cooling protocols. Fertil Steril 2006; 86(Suppl 4):1243–51. 25. Gandolfi F, Paffoni A, Papasso Brambilla E et al. Efficiency of equilibrium cooling and vitrification procedures for the cryopreservation of ovarian tissue: comparative analysis between human and animal models. Fertil Steril 2006; 85(Suppl 1): 1150–6. 26. Salehnia M, Abbasian Moghadam E, Rezazadeh Velojerdi M. Ultrastructure of follicles after vitrification of mouse ovarian tissue. Fertil Steril 2002; 78: 644–5. 27. Salehnia M, Moazzeni SM. Autograft of vitrified mouse ovarian tissue using ethylene glycol as cryoprotectant. Hum Reprod 2001; 16(Suppl 1):159. 28. Kim DH, Lee HC, Ko DS et al. In vitro growth and maturation of preantral follicles isolated from vitrified mouse ovaries. Fertil Steril 2002; 78(Suppl 1): S268. 29. Takahashi E, Miyoshi I, Nagasu T. Rescue of a transgenic mouse line by transplantation of a frozen-thawed ovary obtained postmortem. Contemp Top Lab Anim Sci 2001; 40: 28–31. 30. Rall WF, Fahy GM. Ice-free cryopreservation of mouse embryo at −196 degrees C by vitrification. Nature 1985; 313: 573–5. 31. Migishima F, Suzuki-Migishima R, Song SY et al. Successful cryopreservation of mouse ovaries by vitrification. Biol Reprod 2003; 68: 881–7. 32. Segino M, Ikeda M, Aoki S et al. In vitro culture of mouse GV oocytes and preantral follicles isolated from ovarian tissues cryopreserved by vitrification. Hum Cell 2003; 16: 109–16. 33. Segino M, Ikeda M, Hirahara F et al. In vitro follicular development of cryopreserved mouse ovarian tissue. Reproduction 2005; 130: 187–92. 34. Hasegawa A, Hamada Y, Mehandjiev T et al. In vitro growth and maturation as well as fertilization of mouse preantral oocytes from vitrified ovaries. Fertil Steril 2004; 81(Suppl 1): 824–30. 35. Chen SU, Chien CL, Wu MY et al. Novel direct cover vitrification for cryopreservation of ovarian tissues increases follicle viability and pregnancy capability in mice. Hum Reprod 2006; 21: 2794–800.
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36. Hani T, Tachibe T, Shingai S et al. Fertility of mice receiving vitrified adult mouse ovaries. Reproduction 2006; 131: 681–7. 37. Al-Aghbari AM, Menino AR. Survival of oocytes recovered from vitrified sheep ovarian tissues. Anim Reprod Sci 2002; 71: 101–10. 38. Courbiere B, Massardier J, Salle B et al. Follicular viability and histological assessment after cryopreservation of whole sheep ovaries with vascular pedicle by vitrification. Fertil Steril 2005; 84(Suppl 2): 1065–71. 39. Kheirabadi BS, Fahy GM. Permanent life support by kidneys perfused with a vitrifiable (7.5 molar) cryoprotectant solution. Transplantation 2000; 70: 51–7. 40. Bordes A, Lornage J, Demirci B et al. Normal gestations and live births after orthotopic autograft of vitrified-warmed hemi-ovaries into ewes. Hum Reprod 2005; 20: 2745–8. 41. Ishijima T, Kobayashi Y, Lee DS et al. Cryopreservation of canine ovaries by vitrification. J Reprod Dev 2006; 52: 293–9. 42. Armitage WJ, Rich SJ. Vitrification of organized tissues. Cryobiology 1990; 27: 483–91. 43. Song YC, Khirabadi BS, Lightfoot F et al. Vitreous cryopreservation maintains the function of vascular grafts. Nat Biotechnol 2000; 18: 296–9. 44. Duru NK, Öngürü Ö, Celasun B et al. Postthaw texture analysis of slowly frozen and vitrified human ovarian cortex. Hum Reprod 2001; 16(Suppl 1):180. 45. Isachenko EV, Isachenko E, Rahimi G et al. Cryopreservation of human ovarian tissue by direct plunging into liquid nitrogen. Eur J Obstet Gynecol Reprod Biol 2003; 108: 187–94. 46. Rahimi G, Isachenko E, Sauer H et al. Effect of different vitrification protocols for human ovarian tissue on reactive oxygen species and apoptosis. Reprod Fertil Dev 2003; 15: 343–9. 47. Lee KA, Lee SH, Yoon SJ et al. Resumption of the human primordial follicle growth in xenografts after vitrification of the ovarian tissues. Fertil Steril 2000; 74(Suppl 1): 214. 48. Rahimi G, Isachenko E, Sauer H et al. Measurement of apoptosis in long-term cultures
49.
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of human ovarian tissue. Reproduction 2001; 122: 657–63. Isachenko E, Nayudu PL. Vitrification of mouse germinal vesicle oocytes: effect of treatment temperature and egg yolk on chromatin and spindle normality and cumulus integrity. Hum Reprod 1999; 14: 400–8. Miyake T, Kasai M et al. Vitrification of mouse oocytes and embryos at various stages of development in an ethylene glycol-based solution by a simple method. Theriogenology 1993; 40: 121–34. Hallak J, Sharma RK, Wellstead C et al. Cryopreservation of human spermatozoa: comparison of TEST-yolk buffer and glycerol. Int J Fertil Womens Med 2000; 45: 38–42. Isachenko V, Alabart J, Isachenko E et al. Ultrarapid freezing and storage of rat embryos in an electric refrigerator at −130ºC without liquid cryo-agents, with ultra-short exposure in the freezing medium and direct rehydration after thawing. Cryo Letters 2000; 21: 13–8. Steponkus PL, Myers SP, Lynch DV et al. Cryopreservation of Drosophila melanogaster embryos. Nature 1990; 345: 170–2. Isachenko V, Isachenko E, Rahimi G et al. Cryopreservation of human ovarian tissue by direct plunging into liquid nitrogen: negative effects of disaccharides in vitrification solution. Cryo Letters 2002; 23: 333–44. Isachenko V, Montag M, Isachenko E et al. Effective method for in-vitro culture of cryopreserved human ovarian tissue. Reprod Biomed Online 2006; 13: 228–34. Kagawa N, Kuwayama M, Silber SJ et al. Vitrification may be a promising approach for cryopreservation of human ovarian tissue for auto- and xenotransplantation. Fertil Steril 2006; 86(Suppl 2): S403. Rahimi G, Isachenko E, Isachenko V. et al. Comparison of necrosis in human ovarian tissue after conventional slow freezing or vitrification and transplantation in ovarectomized SCID mice. Reprod Biomed Online 2004; 9: 187–93.
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Ying C Song, Zhenzhen Chen, Carol Journey, Adelina M Emmi, Xiayang Xie and Rosemary L Song
INTRODUCTION It was estimated that by the year 2006 1.4 million people would be diagnosed with cancer in the United States, of which 49% would be women.1 It was estimated that 8% of these women would be under the age of 40.1–3 With modern improvements in treatment regimens, which include aggressive chemo- and radiotherapy, as well as bone marrow transplantation, cure rates can exceed 90%.4 However, alkylating agents commonly used in chemotherapy regimens, as well as ionizing radiation, induce premature ovarian failure in a majority of these patients. This is a significant consequence of cancer treatment as patients are not only rendered infertile but also undergo premature menopause.5 Ovarian tissue banking and transplantation, and other means of fertility preservation are promising prophylactic options for these patients. Patients with a low to intermediate probability of ovarian involvement are good candidates for an autografting procedure.6 Ovarian tissue banking allows time to screen tissue samples to avoid reseeding cancer cells to the patients. For patients with a high risk of ovarian metastasis, ovarian tissue banking, in vitro follicle maturation, and in vitro fertilization could be an option for preserving their fertility.7,8 The opportunities in the area of human infertility are immense for cryopreservation technologies.8,9 At present, techniques used for ovarian cryopreservation have a serious limitation. Extracellular ice formation directly damages cells and tissues through mechanical disruption
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and high salt concentration. This damage precludes normal cellular organization in both the maturing oocyte and the surrounding cell layers (theca and granulosa), conditions which are necessary for fertilization. Secondary injury to microvasculature by mechanical disruption is detrimental to revascularization of cryopreserved implants. Ischemic damage, induced by delayed revascularization, is caused by free radicals and apoptosis. There is clear evidence to show that only about 50% of the follicular population survives after re-implantation. Therefore, cryopreserved ovarian tissue is limited by the current technology. Several groups have demonstrated the competence of cryopreserved ovarian grafts in ewes,10 non-human primates,11 and 12–14 humans. Each of the human studies resulted in a live birth, demonstrating the concept of ovarian autograft implantation using cryopreserved tissues, but the live birth rate is extremely low (only three live births). One of the problems is the conventional cell cryopreservation protocols used. These conventional approaches to cryopreservation cannot be successfully extrapolated to more complex multicellular tissues primarily due to the destructive effect of extracellular ice formation. Tissues are much more than simple aggregates of different cell types; they have a highly organized and often complex structure, which undoubtedly influences their response to freezing and thawing. The formation of extracellular ice, in particular, which is generally innocuous for cells in suspension, is a prime hazard to structured tissues and organs. There are many questions that have
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not yet been answered in ovarian tissue cryopreservation. Whether sufficient primordial and growing follicles survive in the ovaries after cryopreservation, and whether surviving oocytes resume meiosis, fertilize, and develop to the blastocyst stage after in vitro maturation and fertilization have yet to be investigated. Communication between an oocyte and its surrounding granulosa cells is essential for the physiological function and development of the follicle.15 Extracellular matrix is very important for the survival of primordial and primary follicles in long-term culture.16 Gosden et al. suggested that microvasculature damage by mechanical disruption during ice formation is a critical problem in ovarian tissue cryopreservation.17 Poor development rates (37%) of frozen and thawed oocytes have been observed after in vitro maturation.18 The reason for the poor results of conventional cryopreservation has been linked to spindle damage,19 chromosomal abnormalities,20 pathogenesis of oocytes, zona hardening, and low oocyte survival rates after thawing. Post-thaw recovery of follicles in cryopreserved mouse ovarian tissue has ranged from 5%21 to 30%22 with vitrification. Extremely low post-thaw survival rates of human oocytes necessitate banking of ovarian tissues for patients who will soon receive radiotherapy or chemotherapy.23 In this regard, better survival may be expected from an ice-free cryopreservation method.17 Restricting the amount and size of ice crystal formation during cryopreservation can be achieved by using sufficiently high concentrations of cryoprotectants to promote amorphous solidification rather than crystallization, that is vitrification rather than freezing.24–27 Vitrification is an alternative to conventional freezing of living biological materials with ubiquitous applications in cell, tissue, and organ storage. Employing a rabbit jugular vein model, a comparison of the effects of vitrification and conventional cryopreservation upon venous contractility showed that the maximum
contractions achieved by the vitrified blood vessel rings, in response to a panel of four agonists, were greater than 80% of fresh matched controls. In contrast, the maximum contractions achieved by conventionally cryopreserved frozen veins were less than 30% of fresh matched controls.28 During a 4-week follow-up of vitrified and fresh grafts in an autologous transplant model, there were no significant differences between the fresh and vitrified groups with patency rates of ~90%.29 Furthermore, there was no evidence of tunica media disruption, loss of endothelial integrity, aneurysm development, or graft stenosis in either group.29 Feasibility studies using vitrification have been reported for the preservation of arteries,30 heart valves,31,32 articular cartilage,33–36 tissue engineered pancreas substitutes,37,38 and tissue engineered blood vessels.39,40 The results combine to demonstrate feasibility of vitrification as a storage method for living tissue implants.41 Cryopreservation technologies represent a potential long-term storage method to preserve tissues. Ovary banking provides the following options and advantages: it allows the banking of the patient’s own ovarian tissues for future autologous grafting and long-term fecundity; it allows time for adequate quality assurance and safety measures to be applied to avoid reseeding cancer cells via transplanted tissue; and it provides the means for banking ovarian tissues for future in vitro follicle maturation for patients with high risk of ovarian metastasis. If the current cryopreservation method can be improved and incorporated within fertility restoration programs, it will be possible to create various options by which fertility can be successfully preserved. In this chapter, the current trend in fertility preservation and the current methods for cryopreservation of ovarian tissues are given in introductory sections. The introductory parts are followed by a section that explains how to avoid ice formation using vitrification including properties of vitrification solutions, critical cooling and warming rates, ice crystal
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growth, cryosubstitution, and vitrification solution design. The final part deals with the transplantation of vitrified ovarian tissues in an animal model, follicle growth in explant tissues, and cell proliferation and apoptosis.
CRYOPRESERVATION OPTIONS FOR FERTILITY PRESERVATION Chemotherapy and radiotherapy can severely deplete the follicular store, often compromising ovarian function. Ovarian damage will result in loss of fertility, premature menopause, loss of sexual interest, and in the long term, an enhanced risk of osteoporosis and arterial vascular disease. While in younger patients amenorrhea is often temporary, hormone replacement therapy decreases the severity of the symptoms. However, restoration of fertility is more difficult, and at present lies with embryo cryopreservation prior to treatment. Although in some cases embryo cryopreservation may be considered before chemotherapy and radiotherapy, this is a far from optimal solution, because many of the patients are premenarchal girls, women without male partners, and patients for whom chemotherapy and radiotherapy cannot be delayed. Embryo preservation is indeed not an option for these women, because delaying treatment for at least 2 months of in vitro fertilization cycles is inappropriate or life-threatening. Another option is oocyte cryopreservation. Cryopreservation of oocytes can be performed in single women who can undergo a stimulation cycle, although the effectiveness of this technique is very low, with pregnancy and delivery rates ranging from 1 to 5% per frozen oocyte.42–44 There are two main reasons for these poor results: zona pellucida hardening during freezing,45–48 and spindle apparatus damage by intracellular ice formation.49 Ovarian cryopreservation may overcome these problems. In addition, ovarian tissue can be collected simply by laparoscopy at any time during the menstrual cycle. It is possible
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to bank ovarian tissue from cancer patients prior to therapy and re-implant the cryopreserved tissue once the patients are cured to restore fertility. In the most idealized situation, ovarian tissue transplantation would restore both the synthesis of sex steroids to maintain phenotype and the cyclical production of oocytes. Steroidogenic function would be restored in these patients, thereby eliminating the need for exogenous hormone replacement therapy. The most reliable and pressing need appears to be for those women with premature sterility as a result of certain cancer treatments, including high-dose chemotherapy and abdominal irradiation. If ovarian tissue could be cryopreserved before patients received cancer treatment and then returned successfully after remission, the natural state could potentially be restored and conception could even be achieved. Although whole ovaries from mice and rats survive freezing,17 successful cryopreservation of whole ovaries from other mammalian species (human and non-human primates, and livestock species) is difficult due to their large size and low survival rate of vasculature. Effective cryopreservation would enable transplantation of intact ovaries with preserved vasculature and immediate revascularization by vascular anastomosis. In vitro maturation of ovarian tissue is an alternative for patients with high risk of ovarian metastasis. Success has been demonstrated in fresh tissue.7 However, successful in vitro maturation using cryopreserved ovarian tissue has not yet been reported.50,51 Communication between the oocyte and surrounding granulosa cells is essential for correct function and development of the follicle.15 Extracellular matrix is very important for the survival of primordial and primary follicles in long-term culture.16 The destructive effects of extracellular ice formation caused by conventional cryopreservation (freezing) and ischemia after transplantation are the major obstacles for the development of in vivo maturation using cryopreserved tissue. We anticipate that
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these hurdles can be overcome using ice-free cryopreservation techniques.
CONVENTIONAL CRYOPRESERVATION Cryopreservation is a complex process of coupled heat and mass transfer, generally executed under non-equilibrium conditions.52 Simply freezing cells or tissues results in dead, non-functional materials. During slow freezing, water is removed from cells and tissues as ice begins to form in the extracellular environment. Widespread formation of intracellular ice in the cell has been uniformly associated with lethal cell injury.53,54 For most cell types, optimal cooling rates exist that result in maximum cell viability. These cooling rates provide sufficient cellular dehydration to prevent the formation of intracellular ice without causing excessive water loss that may lead to cell injury via ‘solution effects’. Thawing cells from cryopreserved states is typically performed with maximal warming rates to prevent the recrystallization of ice from smaller ice crystals as the sample temperature is raised from cryogenic levels to the melting point of the solution. Recrystallization during rewarming is deleterious and results in lower cell viability; exact mechanisms of injury are not fully understood. Viability is greater when cells are thawed rapidly through the conditions in which recrystallization occurs.55,56 Advances in the field of cryopreservation had been modest until Polge et al,57 discovered the cryoprotective properties of glycerol. Subsequent research by Lovelock and Bishop58 showed that dimethylsulfoxide (DMSO) was also a cryoprotectant agent (CPA). These chemicals are usually divided into two classes: intracellular cryoprotectants with low molecular weights that permeate into cells; and, extracellular cryoprotectants with relatively high molecular weights (greater than or equal to sucrose e.g. 342 Da) which do not penetrate cells.59 The primary mode of protection
for permeating cryoprotectants is the displacement of intracellular water by the cryoprotectant. Regulated removal of intracellular water is essential to inhibiting the lethal formation of intracellular ice. Intracellular cryoprotectants, such as glycerol and DMSO at concentrations from 0.5 to 3.0 mol/L, are effective in minimizing cell damage in many small biological systems frozen with slow cooling rates. Extracellular cryoprotective agents such as polyvinylpyrrolidone or hydroxyethyl starch are more effective at protecting biological systems cooled at rapid rates. Such agents are often large macromolecules that affect the properties of the solution to a greater extent than would be expected from their colligative properties. The primary mechanism of action appears to be the induction of extracellular glass formation. However, non-penetrating cryoprotectants also condition the intracellular compartment by causing the osmotic efflux of intracellular water, thereby preventing the formation of intracellular ice. Some of these non-permeating cryoprotective agents are also thought to have direct protective effects on the cell membrane. When cryoprotectants are used in extremely high concentrations, ice formation can be eliminated during cooling to and warming from cryogenic temperatures. As discussed above, ovarian tissue has been cryopreserved and transplanted into rodents, rabbits, sheep, and marmoset monkeys.60–63 In a recent review of their experimental studies, Baird et al.64 observed a significant decrease of primordial follicles in conventionally cryopreserved ovarian tissues. Experimental studies have indicated that the fall in the number of primordial follicles in grafted tissue is due to hypoxia and the delay that occurs before reimplanted cortical tissue becomes revascularized. The loss of primordial follicles in cryopreserved ovarian tissue after transplantation was estimated to be 50–65% in some studies65,66 and > 90% in one study.67,68 Kim et al.69 showed that a correlation exists between ischemic tissue damage
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and the duration of ischemia, and found that the ovarian cortex can tolerate ischemia for 3 h at 4°C. Significant ischemic damage was evidenced by decreased oxygen consumption and increased apoptosis. Moreover, it appeared that stromal cells were more vulnerable to ischemia than primordial follicles. The apoptosis rate of stromal cells was higher in the frozen–thawed group than in the fresh group, regardless of the duration of incubation, which may reflect a degree of ice damage to the ovarian cortex.
ICE-FREE CRYOPRESERVATION – VITRIFICATION Introduction When a sufficiently high concentration of CPA is used, the formation of ice is avoided completely.52 The rate of cooling and warming is then unimportant because there is no driving force for trans-membrane water movement and no ice to recrystallize during warming. The concentration of CPA necessary to avoid freezing is very high (typically ~60%) and ‘compatibility’ (the absence of deleterious effects of the solute itself) is the essential problem: the concentration of solute required is unattainable at temperatures above 0°C. When materials are vitrified, ice formation is prevented, even at cryogenic temperatures, by the presence of high concentrations of chemicals that interact strongly with water and, therefore, prevent water molecules from interacting to form ice. Depressing the homogeneous nucleation temperature until it equals the glass transition temperature permits vitrification of macroscopic biological systems. Prevention of freezing means that the water in a tissue remains liquid during cooling. As cooling proceeds, however, the molecular motions in the liquid permeating the tissue decrease. Eventually, an ‘arrested liquid’ state known as a glass is achieved. A glass is a liquid that is too cold or viscous to flow. A vitrified liquid is essentially a liquid in
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molecular stasis. Vitrification does not have any of the biologically damaging effects associated with freezing because no appreciable degradation occurs over time in living matter trapped within a vitreous matrix. It provides effective preservation for a number of cells, including monocytes, ova, and early embryos, and pancreatic islets.27,70–75 Vitrification is potentially applicable to all biological systems. Cryopreservation by complete vitrification of the tissue suspension offers several important advantages compared with procedures that allow or require crystallization of the suspension. First, complete vitrification eliminates concerns for the known damaging effects of intra- and extracellular crystallization. Second, tissues cryopreserved by vitrification are exposed to less concentrated solutions of CPAs for shorter periods of time. For example, during a typical cryopreservation protocol involving slow freezing to −40°C, or −70°C, cells are exposed to solutions the concentration of which increases gradually to 21.5 and 37.6 osmol/L, respectively. In contrast, cells dehydrated in vitrification solutions are exposed for much shorter periods of time to < 18 osmol/L, solution, although the temperature of exposure is higher.71 Third, unlike conventional procedures that employ freezing, vitrification does not require controlled cooling and warming at optimum rates – cooling and warming need only be rapid enough to prevent crystallization, and this can generally be achieved without the need for specialist equipment. Vitrification offers a number of practical advantages that will be attractive in tissue processing, as indeed they have been for embryo banking.71
Critical cooling rates and critical warming rates The development of ice-free cryopreservation techniques applicable to ovarian tissues and oocytes requires careful attention to avoid damaging ice growth during both cooling
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0.0
−122.68°C
Heat flow (w/g)
−0.1
−61.43°C
Td
−0.2 −39.71°C
Tg −0.3 −118.69°C
Tm
−0.4
−0.5 −140
−42.30°C
−120
−100
−80
−60
−40
−20
Temperature (°C) Figure 11B.1 Differential scanning calorimeter thermogram for two vitrification solutions. VS55 (top trace) and VS83 (bottom trace) showing heat flow resulting from phase transitions during warming at 5°C/min after rapid cooling. Both solutions exhibit anticipated glass transitions (Tg). However, the VS55 solution shows significant ice recrystallization during warming as evidenced by the devitrification peak (Td) and subsequent ice melt (Tm). The VS83 formulation remains completely ice free.
and warming of vitrified samples. Ironically, the attainment of sufficiently rapid warming to avoid destabilization of the glassy state with resultant ice growth can be significantly more challenging than achieving and maintaining the vitreous state during cooling. The critical cooling rates and critical warming rates, defined as threshold values to avoid ice crystallization, can be identified by calorimetry using a differential scanning calorimeter (DSC) (Figure 11B.1). More specifically, we have determined that using both visual examination techniques and preliminary calorimetric measurements, clinically relevant sized samples can be vitrified at practically achievable cooling rates through use of vitrification solutions.28–30,33–35,37,40 While we have confidence that such systems do not result in detectable freezing, it is not possible to be absolutely sure that microcrystalline ice nuclei do not exist. If present, these would be undetectable by the conventional techniques and would be innocuous to the integrity of
the tissue during the cooling phase of preservation. However, during storage and rewarming, ice nuclei provide sites for ice growth by the process of recrystallization. This is a temperature dependent phenomenon in which small ice crystals, or nuclei, having a high surface energy, seek to attain a lower energy state by growing or amalgamating into larger crystals. Furthermore, the vitreous, or glassy component of a vitrified sample, is metastable and vulnerable to a phase change (glassy to crystalline) during warming (devitrification) if heating is not rapid enough to avoid this event. The objective is to heat vitrified samples at a sufficiently rapid rate (critical warming rate) that would prevent both devitrification and recrystallization, and the sample reaches the point of glassmelting without ice crystallization. Obviously, the geometry, size, and thermal properties of the vitrified tissue dictate the maximum rate of heat transfer that can be uniformly achieved in the sample.
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0 sec
30 sec
1 min
Figure 11B.2 Ice crystals formed from vitrification solution with and without ice control at −60°C. Ice crystal growth can be recorded by video-cryomicroscopy. Kinetics of linear ice crystallization growth in vitrification solutions can be calculated as a function of temperature. Bar = 100 µm.
Ice crystal growth A variety of microscopic and macroscopic techniques are applied to measure the physical parameters associated with ice nucleation and growth (Figure 11B.2), the efficacy of a range of prospective ice control molecules have been evaluated. These include natural antifreeze proteins, existing (and newly synthesized) synthetic ice blocker molecules,76 and other macromolecules such as polyethylene glycol (PEG) known to promote a vitreous state.77 The selection of CPAs was based upon our extensive experience and knowledge of the choice and efficacy of CPAs in other biological systems. For example, DMSO has been widely used as a cryophylactic agent and is still regarded as the single most effective CPA available. Attempts to understand the requirements of vitrification solutions at the molecular level have led to the identification of new solutes with physical properties that would promote the vitreous state during cooling.
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One promising class of compounds is the polyalcohols such as 1,2-propanediol (PROH) and butane-2,3-diol, the optical isomers of which have been shown to vitrify at significantly lower concentrations than any other known CPA.78,79 This is important to minimize the toxic effects of high concentrations of CPAs. Moreover, it is now established that both the concentration needed to vitrify, and the toxicity can be reduced by incorporating a wide variety of non-permeating disaccharides, or polymeric compounds.79,80 Individual ice control molecules were combined in the baseline vitrification medium, VS55, or the alternative DP6 (3 mol/L DMSO and 3 mol/L PROH) medium and examined for ice nucleation and growth during cooling to, and warming from, the glass transition temperature.76,77 Critical cooling and warming rates were determined by DSC (Figure 11B.1). Ice growth kinetics can be measured by video-cryomicroscopy (Fig 11B.2). The presence of ice in bulk samples (20 mL glass
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vials; transparent polyester freezer bags that can be fabricated to any desired form; and the 75 mL plexiglass cassette) was determined initially by visual inspection and quantified in a custom-built plexiglass cassette using digital photography and image analysis.77
Design of a vitrification solution using bulk samples The effect of these compounds on the modulation of ice recrystallization kinetics during the thawing of deliberately frozen solutions can be determined by cryomicroscopy. Compounds that promote ice nucleation are commercially available as products used principally for snowmaking (Snowmax). This product can be used as a tool to test the efficacy of the best combinations of ice-control molecules emerging from these physical studies. This will be achieved by determining the threshold concentration of Snowmax necessary to promote crystallization in the vitrification cryoprotectant cocktails. The effects of sample volume and geometry on critical cooling and warming rates can be determined using the best ice-control cocktails as a prelude to attempting vitrification of biological tissues suspended in samples of similar dimensions and materials. The vitrification solution design will then define the combinations of CPA type and CPA concentration of vitrification solutions with cooling and heating conditions that will guarantee ice-free systems. The objective is to optimize the interaction of the solution formulations with the cooling and heating rates to determine the conditions necessary to maintain stable vitreous systems during cooling, storage and warming of bulk samples. After physical measurement, the bulk vitrification solution can be tested in biological samples to evaluate ice formation using a cryosubstitution technique. The most favorable systems will finally be evaluated for biocompatibility and efficacy in sustaining
biological integrity (structure and function) during vitrification of bulk samples.
Vitrification process To achieve optimal cryoprotection, it is essential that the protocols allow uniform penetration of CPAs throughout the ovarian tissue. Thus, the rate of CPA permeation is an important determining factor in developing better cryopreservation protocols for ovarian tissues. Some studies used the more sophisticated technique of 1H nuclear magnetic resonance (NMR) spectroscopy to measure DMSO permeation in human and porcine ovarian tissue (3–5 mm3). Newton et al.81 have investigated DMSO, PROH, 1,2-ethanediol (ED), and glycerol diffusion into human tissue at both 4ºC and 37ºC, and have observed that at 4ºC, PROH and glycerol penetrate the tissue significantly more slowly than either ED or DMSO. At the higher temperature (37ºC), however, all four CPAs penetrate at a faster rate. They have shown that bathing the tissue (4 mm in diameter and 2 mm in thickness) for 30 min at 4ºC in a 1.5 mol/L solution of DMSO produced a mean tissue CPA concentration approaching 80% of that in the bathing medium. Thomas et al.82 reported that by the end of the 20 min exposure at 0–2ºC, the mean tissue concentration reaches 0.68 mol/L and 0.76 mol/L in the porcine and human ovarian tissue, respectively, indicating that CPA permeation into both tissues was incomplete using their protocols. A method was adapted from vitrification of vein segments, in which a baseline vitrification medium, designated VS55 to reflect that it comprises 55% (w/v) total cryoprotective solute, was used to replace at least 50% of the tissue water with a combination of CPAs. The VS55 solution consisted of 3.1 mol/L DMSO, 3.1 mol/L formamide, and 2.2 mol/L PROH in EuroCollins solution, and VS70 and VS83 are concentrated VS55 solutions.35 The full strength of the vitrification solutions were added and removed in incremental steps.28–30 Based on the NMR study on ovarian tissue81,82
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and the experience in blood vessels,28–30 a protocol used for blood vessel (15 min at each step at 4ºC) was adapted for addition and removal of cryoprotectant for ovarian tissue. This protocol has shown minimal toxicity to biological materials and optimal viability in blood vessel, heart valve, cartilage and tissue engineered constructs.28–30,30–35,37,40,83 The vitrification procedure employed the use of a closed sample container alongside a dummy sample that had been fitted with a thermocoupler for temperature monitoring. The procedure has been used on blood vessels, other tissues, and engineered constructs.28–30,33–35,37,40,83 The packaging should eliminate excessive contamination of the sample during the vitrification process. As discussed above, steps for loading and unloading the VS55 and VS83 solutions were derived from the kinetics of CPA permeation studies.81,82 After addition of the final vitrification solution, ovarian tissues were cooled rapidly (43°C/min) to −100°C, followed by slow cooling (3°C/min) to −135°C, and finally stored in a freezer at −135°C for a minimum of 24 h. These samples were then either warmed for further study, or cryosubstituted to determine the location and distribution of ice, if any, within the ovaries. A thermocouple was inserted into a separate dummy sample of the same vitrification solution, and its output was monitored via a digital thermometer. Vitrified samples were rewarmed in two stages: first, slow warming to −100°C (30°C/min) and then rapid warming to melting (225°C/min). A slow warming rate was achieved by moving the sample to the top of the −135°C freezer. The fast warming rate was generated by placing the glass vial in a mixture of 30% DMSO/water at room temperature. This technique prevents ice from forming on the outside surface of the glass vial, thereby allowing visualization of the melting process. After rewarming, the vitrification solution was removed in a step-wise manner using a mannitol solution for osmotic buffering.28–30
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A clinical procedure (1.5 mol/L DMSO)84 has been used as a classic protocol for human ovarian tissue cryopreservation in the field. However, a less toxic CPA, 1.5mol/L PROH and 0.2 mol/L sucrose85 has been selected by others as the conventional cryopreservation method. The protocol used by Gosden et al. was selected as a ‘freezing’ control to compare with the ice-free cryopreservation methods. It has to be noted that there will be ice formation in conventional cryopreservation protocols regardless of CPA type and concentration used. The study focused on comparing the protocols with and without ice formation on post-thaw cell survival. For conventional cryopreservation, ovaries were equilibrated for 30 min at 0ºC in a cryogenic container containing Leibovitz medium with 10% bovine calf serum and 1.5 mol/L DMSO. The container was transferred to a programmable freezer (Planar products) and cooled at 2ºC/min to −9ºC for seeding. The second cooling ramp was cooled at 0.3ºC/min to −40ºC, and subsequently at 10ºC/min to −140ºC. Finally, the containers were plunged into liquid nitrogen and stored in vapor phase liquid nitrogen (less than −160ºC). The tissues were thawed rapidly by swirling in a water bath at room temperature. They were immediately transferred to fresh medium and washed three more times to remove the cryoprotectant.
Cryosubstitution Cryosubstitution can be used to identify ice formation in cryopreserved tissues. Cryosubstitution is a technique in which tissue is preserved at subzero temperatures using organic solvents to dissolve ice.28,29,40,86 In this way the profile of any ice domains can be revealed in the frozen or vitrified tissue samples. Cryosubstitution of the cryopreserved ovarian tissues took place at −90°C using a substitution media of methanol and osmium tetroxide. Vials containing samples and the substitution mixture were placed in a heat sink to maintain a constant temperature. The substitution
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a
b
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Figure 11B.3 Cryosubstitution of ovarian tissues. (a) Fresh sheep ovarian tissues. (b) Cryosubstituted, frozen sheep ovarian tissues demonstrating the presence of ice crystals (white spaces) and matrix distortion. (c) Cryosubstituted vitrified sheep ovarian samples devoid of large ice domains with normal tissue morphology. Toluidine blue, × 20.
media was changed three times over a 5-day period. After 5 days the heat sink with samples was transferred to −20°C for 24 h. This was followed by 4°C for 2 hours and finally brought to room temperature. Samples were transferred to 100% acetone followed by infiltration of Araldite resin, embedded and polymerized for routine examination in the light and electron microscopes. Sections were cut at 0.75 µm and stained with toluidine blue. All histology specimens were analyzed using an Olympus IX 70 light microscope and ImagePro Plus software. Quantitative analysis of cryosubstituted specimens was conducted to determine the size of ice crystal domains, thus determining the space occupied by ice. Cryosubstitution of fresh and vitrified ovaries demonstrated that there was very little, if any, ice formation in vitrified ovaries. Intact ovaries cryopreserved using the baseline vitrification method with 55% cryoprotectants (V/V) resulted in macroscopic ice formation. However, increasing the concentration of the cryoprotectant to 83% (V/V) resulted in little, if any, ice formation, fresh and cryosubstituted vitrified ovaries are shown in Figure 11B.3. Vitrification was effective in preventing ice formation in the ovaries, and both 70% and 83% formulations were effective compared with traditional cryopreservation by freezing with DMSO (Figure 11B.3).
Evaluation of biocompatibility of vitrification formulations An in vitro tissue culture-based assay with the metabolic indicator, Alamar blue in microtiter plates, was used as a test for the effect of a wide range of cryobiological variables on the viability of a variety of cell lines. This established assay can be used to evaluate the biological outcome of exposure to useful vitrification formulations emerging from the physical studies. This high throughput screening method enables multiple variables to be screened expeditiously and economically. Moreover, the same assay can be applied to ovarian tissues for correlation with the more rigorous function test described below. The purpose of the screening assay is to define the toxic limits of the individual and combined CPAs or ice-control molecules for prospective use in the bulk tissue sample studies. Following vitrification using the formulas of VS55 and VS83, or cryopreservation by conventional freezing, the viability of the sheep ovarian tissues was assessed using an Alamar blue assay, and the frozen and vitrification methods were compared. The initial study showed a protocol that used V55/VS83 combinations (VS83 was used in
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Secondary
17 weeks
FSH Independent
Preantral
9 weeks
Antral
3 weeks
Preovulatory
FSH dependent
Figure 11B.4 Timeline of follicular growth. The timeline shows the stages of follicular growth, beginning with the secondary stage, along with the approximate number of weeks to reach each stage. FSH dependency is also shown. Reproduced with permission from Gougeor.89
the last minute before vitrification) produced an adequate result. The specimens vitrified using this protocol had a mean of 66.55% cell viability compared with 52.8% for the frozen tissue. Statistical analysis using the Kruskal–Wallis, non-parametric Dunn’s post test demonstrated that the vitrified group (n = 42) was significantly different compared with the frozen group (n = 28) (P < 0.01). However, cell viability in both the vitrified and frozen groups was significantly less than in the fresh group (P < 0.05 and P < 0.001, respectively) where n = 6. The ovary samples were obtained from eight sheep (16 ovaries). Therefore, further optimization of vitrification in ovarian tissues is required to achieve a result equivalent to that of fresh tissues.
Aspects of reproductive biology and physiology that relate to ovarian tissue preservation and transplantation Before discussing the results on transplantation of ovarian tissues, it is necessary to discuss some aspects of reproductive biology and physiology that are directly related to the protocol of ovarian tissue transplantation. As we know, gonadotropin releasing hormone (GnRH) is synthesized in the hypothalamus and released from neurons in pulses. These pulses, which increase in frequency as a female begins puberty, stimulate the biosynthesis and release of luteinizing hormone (LH) and follicle stimulating hormone (FSH).87 As the concentration of these two hormones rises, primordial follicles (in which the oocytes were arrested
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in meiosis at the female’s birth) begin to grow.88 Growth from the primordial to the secondary stage has been shown to be FSH independent, while follicle maturation from the preantral stage is FSH/LH dependent. Figure 11B.4 below shows the timeline of follicular growth as stated by Gougeon et al.89 in 1986. Not shown is the 21-week interval between primordial and secondary follicle. However, as the timeline of follicle growth is currently under debate, we should take into account other investigators’ procedures regarding the timing of gonadotropin stimulation. Several investigators use injections of FSH in an effort to stimulate the growth of antral follicles in ovarian tissue xenografts. There are many different doses and time intervals that have been used by various groups. Van den Broecke et al. 90 found that a 5 IU daily injection of FSH for 2 weeks beginning 14 weeks after grafting resulted in a ‘significant shift from primordial to primary follicles’. Van den Broecke et al. 90 also used another procedure in which SCID mice were injected every other day with 5 IU of FSH/LH for 3 months. In this instance, antral stage development was seen in the xenografted tissues. Kim et al.91 stimulated with 4 IU pregnant mare’s serum gonadotropin (PMSG) every other day for 4 weeks beginning 16 weeks after xenografting. Then in 2005, Kim et al. 92 stimulated with 5 IU PMSG every other day for 2 weeks beginning 20 weeks after grafting. In both instances, Kim et al. also stimulated with 10 IU human chorionic gonadotropin (hCG) and this resulted
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in antral follicle development. Finally, Weismann et al. 93 used 4 IU FSH/LH daily for 2 weeks beginning 12 weeks after grafting. Results from this study showed preantral or larger follicle development. Oocytes are surrounded by one or more layers of granulosa cells (GC) depending on the stage of the follicle in which it resides. These granulosa cells promote oocyte growth through junctions, which allow cell–cell communication,87 thus enabling the oocyte to receive signals from growth factors. The oocytes, in turn, synthesize proteins such as growth differentiation factor (GDF)-9 and GDF-9b (members of the tranforming growth factor (TGF-β) superfamily), which allow the granulosa cells to proliferate.87,94–96 Oocytes produce proliferating cell nuclear antigen (PCNA) during follicle growth, therefore immunostaining of PCNA has been used to assess the growth of follicles in human ovarian explants.97 PCNA is an auxiliary protein to DNA polymerase delta, which is involved in DNA synthesis and repair. Ki-67 is another indicator of cell viability.98 Unlike PCNA, it is utilized only if nuclear DNA is functioning at the time the stain is incorporated, but Ki-67 has a very short life span.
Animal model Although it is important to evaluate whether ovarian tissues and follicles are viable and competent after the cryopreservation procedure, the current in vitro models used to study preantral follicle growth are not well established.99 One way to evaluate the function of cryopreserved tissues is to transplant the grafts and then monitor the developmental competence in host animals. However, to avoid immune rejection of the graft in hetero- or xenotransplantation, allogeneic transplantation into a recipient with a suitable histocompatibility complex or into an immunodeficient recipient is necessary. Immunodeficient mice have been used effectively as in vivo research models for studying
xenogeneic follicle development.68 Currently two of these animal models, athymic nude (nu/nu) mice, which are lacking mature T cells, and severe combined immunodeficient (SCID) mice, which are lacking both mature B and T cells, are being used to assess in vivo xenogeneic follicular development. Immunodeficient rodents are promising research models for ovarian cortex xenografting. Ovarian grafts appeared to take better in castrated or hypogonadal mice.65,93,97 Several groups92,93,100,101 working with xenografts of cryopreserved human ovarian tissue use male SCID mice instead of female SCID mice as graft recipients, as this results in not only a higher frequency of developing follicles (13 of 17 vs. 6 of 20),93 but also in follicles of increased diameter (15 mm).101 Aubard et al. demonstrated a high follicular survival rate in grafts placed under the kidney capsule.68 Transplants grafted subcutaneously above the flanks (several on each side) of the recipient have shown results equivalent to kidney capsule grafts.91,93,101 A very recent study in mice concluded that the graft site affects the number and quality of oocytes produced from ovarian grafts.102 The study used a mouse ovarian grafting model to investigate whether the graft site (bursal cavity or kidney capsule) influences the number, fertilization rate, and developmental potential of oocytes recovered from grafts. The number of 2-cell embryos produced was significantly higher with oocytes from grafts to the bursa, compared with grafts to the other sites. Fresh, vitrified, or frozen sheep ovarian tissues have been implanted beneath the renal capsule or in an orthotopic location. The renal capsule was used as a graft location due to its highly vascularized nature and its ability to hold a graft in place. However, not only does the size of the kidneys limit the amount of tissue that can be transplanted, but the surgery is also difficult and highly invasive for the animal. An optimal alternative graft site that would allow an increased tissue sample number as well as a less difficult
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and less invasive surgery is a subcutaneous graft site. Many investigators have used a subcutaneous graft site above the flanks in SCID mouse models, as discussed above.101 This site has been used to graft up to six 1–2 mm3 tissue blocks on dorsal areas of the mouse. This site has also been shown to allow for larger follicle growth due to its expanding nature.
Logistics of a research design for ovarian tissue vitrification Human ovarian tissues could be obtained from a hospital or a tissue compository agency. The tissues can be shipped to the vitrification facility overnight at 4ºC. Tissue acquisition begins in the operating room where a section of ovarian tissue is removed. The tissues are then transported to the laboratory on ice in a covered container containing Leibovitz-15 (L-15) medium. The warm ischemia time is limited to 30 min and cold ischemia time to 24 h. Once in the laboratory, manipulation of the tissue including trimming and vitrification can be conducted under sterile conditions inside a tissue culture hood. After the tissues are transported to the laboratory, the gross anatomy of the acquired tissue is assessed for retrieving tissue blocks. Cauliflower-like protrusions (which are known to contain follicles) are trimmed out of the stroma bridges and further cut into 3 mm3 blocks. This is a way to ensure there are follicles in tissue samples before vitrification and implantation. There have been concerns about low follicle density in a 1 mm3 tissue block that is used for in vivo or in vitro maturation. Hovatta et al. published a study in 2004 in which they counted approximately 100 primordial and primary follicles in a 1–2 mm3 tissue block.103 Figure 11B.5 shows follicles that were seen in human ovarian tissue following the tissue dissection procedure. Oktay et al.104 developed an isolation technique for human primordial follicles using
Figure 11B.5 Follicle pool of human ovarian tissue. Human ovarian tissue from a 32-year-old patient showing primordial follicles in 1 mm3 cortex block. H&E, × 25.
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enzymatic digestion and microdissection, and obtained high follicular viability rates with both fresh and frozen ovarian tissues. However, for clinical utility of vitrified ovarian tissue blocks, it may be not practical for surgical handling during transplantation. Serum anti-Müllerian hormone levels have been found to reflect the size of the primordial follicle pool.105,106 Incubation of ovarian tissue blocks before vitrification and/or transplantation and detection of anti-Müllerian hormone levels may be useful for selection of follicle-rich blocks for vitrification and subsequent transplantation. After trimming, the vitrification process begins. The tissue blocks are evenly distributed into small, glass specimen vials (approximately ten blocks per vial) and the first of the vitrification solutions is added. After the vitrification process has begun, the sample vials are kept on ice at all times and shaken on an orbital shaker under the tissue culture hood. The addition and removal of vitrification solutions and vitrification process have been described in previous sections. After the last rewarming solution (pure medium) is added, the samples are kept in L-15 medium on ice
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b
a
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Figure 11B.6 Follicle growth in sheep ovarian tissue and explants. (a) Un-implanted control; (b) frozen, 5-week explant; and (c) vitrified, 6-week explant. H&E, × 10.
a
b
Figure 11B.7 Cell proliferation by proliferating cell nuclear antigen (PCNA) staining of human ovarian tissue (a) and vitrified sheep ovarian explants (b). PCNA (brown color) appears in oocytes and other cells. × 200.
and transported to the surgical room for implantation or used for in vitro maturation.
Evaluation of follicle growth, cell proliferation, and apoptosis Fresh or cryopreserved ovarian tissue from sheep xenografted under the kidney capsule of castrated SCID mice developed apparently normal antral follicles.65,84 In this study, sheep ovarian cortex xenografting was used to assess follicle survival in SCID mice. The number of follicles surviving and the degree of apoptosis were analyzed by light microscopy, and selected samples before and after vitrification and implantation were evaluated by electron microscopy. An additional technique included the use of PCNA
immunostaining to assess the growth of follicles in ovarian explants.97 Follicle growth in both conventionally cryopreserved and vitrified ovarian tissues was detected without follicle stimulation (Figure 11B.6). Because of the difficultly in determining whether a follicle is progressively maturing or decaying using only hematoxylin and eosin (H&E) staining techniques, PCNA and apoptosis (caspase-3 and CD95) analyses can be applied to supplement evaluation (Figures 11B.7 and 11B.8). PCNA is actively expressed by cells in the G1/S-phase and can be used as a growth marker for developing follicles (Oktay, 1995).108 Although there is follicle growth in frozen–thawed tissue (Figure 11B.6), apoptotic oocytes are also present in the explanted frozen tissues (Figure 11B.8).
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a
b
Figure 11B.8 Apoptosis assay in frozen sheep ovarian explants. Immunocytochemistry of (a) Caspase-3 and (b) CD95 showing apoptotic oocytes in brown. There was no apoptosis in other cells. × 200.
CONCLUSION The reliability of tissue transport logistics in both animal and human ovarian tissue as well as tissue viability after exposure to vitrification solutions have been discussed. The feasibility of vitrification of ovarian tissue has been demonstrated in vivo in the SCID mouse model. More large-size follicles can be produced through use of FSH stimulation and longer term maturation. Immunocytochemical techniques used to supplement morphological analysis for better evaluations of vitrified explants help distinguish between proliferated cells and apoptotic cells. In conclusion, vitrification has no apparently negative effect on follicle formation or follicle growth. The main problem of using a high concentration of vitrification solutions, as opposed to low concentration vitrification solutions in egg vitrification, is the chemical toxicity of CPAs. Low concentrations may cause problems with recrystalization and devitrification during rewarming. This chapter deals with a challenging problem, namely the vitrification of threedimensional tissue in comparison with conventional, ice-forming cryopreservation processes. Vitrification involves several key parameters, including chemical identity and concentration of CPA(s), and rates of cooling
and warming. Ovarian tissues are also complex systems in respect to reproductive biology and physiology. This chapter, therefore, can not possibly represent a comprehensive evaluation and discussion of the entire parametric space of such a problem. For instance, oocyte competence, aspects of reproductive physiology, and surgical model cannot be dealt with in a comprehensive fashion. Oocyte retrieval from the matured ovarian explants has not been covered in the chapter. Grafting locations, male or female recipients, castrated or non-castrated animals, and exogenous follicle stimulation protocols (timing and dose) are mentioned, but not in detail. Nevertheless, the study offers a multitude of opportunities for further clinical investigations, such as cancer cell detection in ovarian tissue biopsies, in vitro maturation of vitrified tissues, and many other topics, which will all bring contributions from various disciplines to the field.
ACKNOWLEDGMENTS Partly supported by US Public Health Grant, R43HD047060. The authors would like to thank John Wash and Fred G Lightfoot for their work on the physical measurements of ice growth kinetics and cryosubstitution of ovarian tissues.
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39. Elder EN, Chen Z, Ensley AE, Nerem RM, Brockbank KGM, Song YC. Enhanced tissue strength in cryopreserved collagen-based blood vessel constructs. Transplantation Proc 2005; 37: 4625–9. 40. Dahl S, Chen Z, Solan A, Brockbank KGM, Niklason LE, Song YC. Feasibility of vitrification as a storage method for tissue engineered blood vessels. Tissue Eng 2006; 12: 291–300. 41. Taylor MJ, Song YC, and Brockbank KGM. Vitrification in tissue preservation: new developments. In: Benson E, Fuller B, and Lane N, (eds.), Life in the Frozen State. Taylor and Francis: London, 2004; pp. 603–41. 42. Stachecki JJ, Cohen J. An overview of oocyte cryopreservation. Reprod Biomed Online 2004; 9: 152–63. 43. Borini A, Sciajno R, Bianchi V, Sereni E, Flamigni C, Coticchio G. Clinical outcome of oocyte cryopreservation after slow cooling with a protocol utilizing a high sucrose concentration. Hum Reprod 2006; 21: 512–7. 44. Levi Setti PE, Albani E, Novara PV, Cesana A and Morreale G. Cryopreservation of supernumerary oocytes in IVF/ICSI cycles. Hum Reprod 2006; 21: 370–5. 45. Gook DA, Osborn SM, Johnston WI. Cryopreservation of mouse and human oocytes using 1,2-propanediol and the configuration of the meiotic spindle. Hum Reprod 1993; 8: 1101–9. 46. Gook DA, Schiewe MC, Osborn SM, Asch RH, Jansen RP, Johnston WI. Intracytoplasmic sperm injection and embryo development of human oocytes cryopreserved using 1,2-propanediol. Hum Reprod 1995; 10: 2637–41. 47. Porcu E, Fabbri R, Seracchioli R, Ciotti PM, Magrini O, Flamigni C. Birth of a healthy female after intracytoplasmic sperm injection of cryopreserved human oocytes. Fertil Steril 1997; 68: 724–6. 48. Fabbri R, Porcu E, Marsella T, Rocchetta G, Venturoli S, Flamigni C. Human oocyte cryopreservation: new perspectives regarding oocyte survival. Hum Reprod 2001; 16: 411–16. 49. Pickering SJ, Braude PR, Johnson MH, Cant A, Currie J. Transient cooling to room temperature can cause irreversible disruption of the meiotic spindle in the human oocyte. Fertil Steril 1990; 54: 102–8. 50. Paynter SJ, Cooper A, Fuller BJ, Shaw RW. Cryopreservation of bovine ovarian tissue: structural normality of follicles after thawing
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64. Baird DT, Campbell BK, Souza C, Telfer EE. Long-term ovarian function in sheep after ovariectomy and autotransplantation of cryopreserved cortical strips. Eur J Obstet Gynecol Reprod Biol 2004; 113: 55–9. 65. Baird DT, Webb R, Cambell K, Harkness LM, Gosden RG. Long-term ovarian function in sheep after ovarectomy and transplantation of autografts stored at −196oC. Endocrinology 1999; 140: 462–71. 66. Nisolle M, Godin PA, Casanas-Roux F, Qu J, Motta P, Donnez J. Histological and ultrastructural evaluation of fresh and frozenthawed human ovarian xenografts in nude mice. Fertil Steril 2000; 74: 122–9. 67. Aubard Y, Piver P, Cogni Y, et al. Orthotopic and heterotopic autografts of frozen-thawed ovarian cortex in sheep. Hum Reprod 1999; 14: 2149–54. 68. Aubard Y. Ovarian tissue xenografting. Eur J Obstet Gynecol Reprod Biol 2003; 108: 14–8. 69. Kim SS, Yang HW, Kang HG, et al. Quantitative assessment of ischemic tissue damage in ovarian cortical tissue with or without antioxidant (ascorbic acid) treatment. Fertil Stertil 2004; 82: 679–85. 70. Bodziony et al., 1994 71. Rall WF. Factors affecting the survival of mouse embryos cryopreserved by vitrification. Cryobiology 1987; 24: 387–402. 72. Jutte NHPM, Heyse P, Jansen HG, Bruining GJ, Zeilmaker GH. Vitrification of mouse islets of Langerhans: comparison with a more conventional freezing method. Cryobiology 1987; 24: 292–302. 73. Jutte NHPM, Heyse P, Jansen HG, Bruining GJ, Zeilmaker GH. Vitrification of human islets of Langerhans. Cryobiology 1987; 24: 403–11. 74. Takahashi T, Hirsh AG, Erbe EF, Bross JB, Steere RL, Williams RJ. Vitrification of human monocytes. Cryobiology 1986; 23: 103–15. 75. Rall WF, Fahy GM. Ice-free cryopreservation of mouse embryos at –196°C by vitrification. Nature 1985; 313: 573–5. 76. Brockbank KGM, Song Ying C, Walsh JR, Taylor MJ. Vitrification, the new frontier in preservation of tissues. In: Vossoughi J, ed. Biomedical Engineering: Recent Developments. Washington, DC: Medical and Engineering Publishers, Inc., 2002; 197–8. 77. Brockbank KGM, Walsh JR, Song YC, Taylor MJ. Vitrification: preservation of cellular implants. In: Ashammakhi, N. and Ferretti, P. (eds.), Topics in Tissue Engineering, Published on the Web: www.
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tissue-engineering-c.com/ebook_topics_ in_t_e/ contents.html, 2003; Chapter 19, 1–26. Boutron P. Levo- and dextro-2,3butanediol and their racemic mixture: very efficient solutes for vitrification. Cryobiology 1990; 27: 55–69. Sutton RL. Critical cooling rates for aqueous cryoprotectants in the presence of sugars and polysaccharides. cryobiology 1992; 29: 585–98. Boutron P, Peyrdieu J-F. Reduction in toxicity for red blood cells in buffered solutions containing high concentrations of 2,3-butanediol by trehalose, sucrose, sorbitol, or mannitol. Cryobiology 1994; 31: 367–73. Newton H, Fisher J, Arnold JRP, Pegg DE, Faddy MJ, Gosden RG. Permeation of human ovarian tissue with cryoprotective agents in preparation for cryopreservation, Hum Reprod 1998; 13 pp. 376–80. Thomas N, Busza A, Cooper A, et al. Measurement of permeating levels of cryoprotectant during ovarian tissue cryopreservation using 1H NMR spectroscopy in human porcine ovaries. Cryo Letters 1997; 18: 179–84. Song Ying C, Chen ZZ, Taylor MJ, Brockbank KGM. Successful vitrification of articular cartilage in a large animal model. Presented at the Sixth Annual TESi International Conference and Exposition, Orlando FL, 2003, 11–13. Gosden GR, Baird DT, Wade JC, Webb R. Restoration of fertility in oophorectomised sheep by ovarian autografts stored at −196ºC. Hum Reprod 1994; 9: 597–603. Fabbri R, Pasquinelli G, Bracone G, Orrico C, Tommaso BD, Venturoli S. Cryopreservation of human ovarian tissue. Cell Tissue Bank 2006; 7: 123–33. Brockbank KGM, Lightfoot FG, Song YC, Taylor MJ. Interstitial ice formation in cryopreserved homografts: a possible cause of tissue deterioration and calcification in vivo. J Heart Valve Dis 2000; 9: 200–206 Fauser BCJM. Reproductive Medicine: Molecular, Cellular and Genetic Fundamentals. Lancaster, UK: Parthenon Publishing, 2003. Erickson GF. Female Reproductive Endocrinology: Morphology and Physiology of the Ovary. www.endotext.com. Gougeon A. Dynamics of follicular growth in the human: a model from preliminary results. Hum Reprod 1986; 1: 81–87. Van den Broecke R, Liu J, Handyside A, et al. Follicular growth in fresh and cryopreserved
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human ovarian cortical grafts transplanted to immunodeficient mice. Eur J Obs Gynecol Reprod Biol 2001; 97: 193–201. Kim SS, Soules MR, Battaglia DE. Follicular development, ovulation, and corpus luteum formation in cryopreserved human ovarian tissue after xenotransplantation. Fertil Steril 2002; 78: 77–82. Kim SS, Kang HG, Kim NH, Lee HC, Lee HH. Assessment of the integrity of human oocytes retrieved from cryopreserved ovarian tissue after xenotransplantion. Hum Reprod 2005; 20: 2502–8. Weissman A, Gotlieb L, Colgan T, Juriscova A, Greenblatt EM, Casper RF. Preliminary experience with subcutaneous human oavrian cortex transplantationin the NONSCID mouse. Biol Reprod 1999; 60: 1462–7. Juengel JL, Hudson NL, Heath DA, et al. Growth differentiation factor 9 and bone morphogenetic protein 15 are essential for ovarian follicular development in sheep. Biol Reprod 2002; 67: 1777–89. McNatty KP, Juengel JL, Reader KL, et al. Bone morphogenic protein 15 and growth differentiation factor 9 co-operate to regulate granulose cell function in ruminants. Reproduction 2005; 129: 481–7. Bodensteiner KJ, Clay CM, Moeller CL, Sawyer HR. Molecular cloning of the ovine growth/differentiation factor-9 gene and expression of growth/differentiation factor-9 in ovine and bovine ovaries. Biol Reprod 1999; 60: 381–6. Oktay K, Newton H, Mullan J, Gosden RG. Development of human primordial follicles to antral stages in SCID/hpg mice stimulated with follicle stimulatiing hormone. Hum Reprod 1998; 13: 1133–8. Nubani R, Hughes FF, Virdi A, Leven R Wood-Molo M and Rawlins RG. Viability testing of cryopreserved ovarian tissue. Fertil Steril 86 (Suppl 2): S209, 2006 Donnez J, Martinez-Madrid B, Jadoul P, Van Langendonckt A, Demylle D, Dolmans MM. Ovarian tissue cryopreservation and transplantation: a review. Hum Reprod Update 2006; 5: 519–35. Van den Broecke R. Timing of FSH-stimulation and follicular development in cryopreserved human ovarian grafts. Reproductive BioMedicine Online 2001; 4: 21–6. Hernandez-Fonseca H, Bosch P, Sirisathien S, Wininger JD, Massey JB, Brackett BG. Effect of site of transplantation on follicular development of human ovarian tissue
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transplanted into intact or castrated immunodeficient mice. Fertil Steril 2004; 81: 888–92. 102. Yang HY, Cox SL, Jenkin G, Findlay J, Trounson A, Shaw J. Graft site and gonadotrophin stimulation influences the number and quality of oocytes from murine ovarian tissue grafts. Reproduction 2006; 131(Suppl): 851–9. 103. Zhang P, Henna L, Timo T. In vitro effect of cyclic adenosine 3′, 5′-monophosphate (cAMP) on early human ovarian follicles. J Assist Reprod Genet 2004; 21: 301–6. 104. Oktay K, Nugent D, Newton H, Salha O, Chatterjee P, Gosden RG. Isolation and characterization of primordial follicles from fresh and cryopreserved human ovarian tissue. Fertil Steril 1997; 67: 481–6.
105. Kevenaar ME, Mohamed F, Meerasahib Piet Kramer, et al. Serum anti-Müllerian hormone levels reflect the size of the primordial follicle pool in mice Endocrinology 2006; 147: 3228–34. 106. Visser JA, Frank H de Jong, Joop S E et al. Anti-Müllerian hormone: a new marker for ovarian function. Reproduction 2006; 131: 1–9. 107. Fabbri R, Venturoli S, D’Errico A, et al. Ovarian tissue banking and fertility preservation in cancer patients: histological and immunohistochemical evaluation. Gynecologic Oncology 2003; 89: 259–66. 108. Oktay K, Schenken R, Nelson J. Proliferating cell nuclear antigen marks the initation of follicular growth in the rat. Biol Reprod 1995; 53: 295–301.
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Vitrification of human embryonic stem cells
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Yoel Shufaro, Gábor Vajta, Alan O Trounson and Benjamin E Reubinoff
BACKGROUND Human embryonic stem cells (hESCs), are derived from early embryos and typically the blastocyst’s inner cell mass, can be propagated indefinitely in vitro, have a normal karyotype, and are pluripotent. Thus, these stem cells can differentiate into progeny from all three germ layers both in vitro and in vivo. When engrafted into immune deficient mice, hESCs develop into teratoma tumors with mesoderm, endoderm, and ectoderm tissue components.1,2 Given their unique properties, hESCs can be utilized for the study of early human development, drug discovery, and as a renewable source of human cells for transplantation therapy.3 Safe and robust cryopreservation is required for the development and clinical application of hESC lines. Cryopreservation enables the preservation of stocks of early passage cells, and is insurance for situations in which the culture is lost or damaged. Early passage stocks may be needed after prolonged cultivation in order to return to the properties of the original cells, which might be genetically or permanently altered for development due to prolonged culture and repeated passaging. Efficient freezing and thawing methods are also required for the conservation of specific ESCs such as genetically modified clones, or patient and disease specific autologous hESC lines originating from embryos created by somatic nuclear transplantation. Moreover, these technologies will be essential if hESC banks are to be established.3,4 Finally, effective
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cryopreservation technologies will allow efficient transfer of cells between research centers, promoting scientific collaboration, and facilitating widespread use of the hESCs for research and clinical applications.
FREEZING METHODS Slow cooling rate freezing and rapid thawing methods are most commonly used for the cryopreservation of cell lines.5 While these standard methods are efficient for the cryopreservation of mouse ESCs,6 the survival of undifferentiated hESCs following slow cooling rate freezing are variable. They are particularly poor when the hESCs are cultured in the presence of serum, where after thawing most of the cells differentiate or die.7 Since embryonic stem cells originate from the pluripotent cells of the blastocyst and in culture retain the properties of these cells such as morphology, gene expression, and pluripotence, it is plausible that methods which have been specifically developed for the cryopreservation of human blastocysts may be also efficient for hESCs. Vitrification, which is solidification of solutions at low temperature without ice formation, has been extensively studied as a method of cryopreservation of embryos8 and oocytes.9–11 It was found to be highly efficient in cattle12,13 as well as in other species such as pigs and hamsters,14,15 in which slow cooling freezing methods usually cause lethal cryoinjuries to most embryos. In addition,
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vitrification methods may also be beneficial for the cryopreservation of human oocytes and blastocysts.11,15–17 With the slow cooling conventional freezing methods, the cells are transferred into a standard cryovial containing freezing medium supplemented with a cryoprotectant. The vials are slowly cooled (~1°C/min) in a freezing container or in a programmed freezer to −80°C and then plunged into and stored in liquid nitrogen. Thawing is conventionally performed by immersion of the vials in a water bath at 37°C, followed by a gradual dilution and replacement of the freezing medium by the culture medium and transfer of the cells into the appropriate culture conditions.5 With the vitrification approach, a glass-like solidification of solutions is achieved by using high solute concentrations of cryoprotectant ingredients and rapid cooling. While this approach can eliminate cell injury due to ice crystal formation, the high concentration of cryoprotectants may induce significant toxic and osmotic damage. The concentrations of cryoprotectants that are required to achieve vitrification are inversely related to the rate of cooling. Therefore, an increased speed of cooling can lessen the cryoprotectant induced toxicity, as it minimizes the time of exposure to these toxic compounds at liquid concentrations. Increasing the cooling rate has been achieved by plunging samples in small volume held on electron microscope grids,9 thinwalled open pulled straws, (OPS)12 and small nylon loops15 directly into liquid nitrogen. Vitrification is simple to perform; cells are transferred into thin straws which are plunged directly into liquid nitrogen. OPS are most commonly used.7,12–14,18 Common 0.25 mL insemination straws are heat-softened and then pulled manually to a thin diameter. The hESC clusters, incubated briefly in the freezing medium are then loaded into the narrow end of the straw by capillary action. For vitrification of hESCs we used two types of vitrification solutions (VS1 and VS2), both
based on a holding medium (HM) which included DMEM containing HEPES buffer supplemented with 20% fetal bovine serum (FBS). The cryoprotectants used are 10% dimethylsulfoxide (DMSO) and 10% ethylene glycol (EG) for VS1, and 20% DMSO, 20% EG, and 0.5 mol/L sucrose for VS2. All procedures are performed on a heated stage at 37°C. Four to six clusters of hESCs are first incubated in VS1 for 1 min followed by incubation in VS2, containing a higher concentration of cryoprotectants, for 25 s. They are then washed in a 20 µL droplet of VS2 and placed within a droplet of 1–2 µL of VS2. The small hESC clusters are loaded into the narrow end of the straw from the droplet by capillary action (Figure 12.1a). The narrow end is immediately submerged into liquid nitrogen. Warming is performed on a heated stage at 37°C. Three seconds after removal from liquid nitrogen, the narrow end of the straw is submerged into the holding medium with 0.2 mol/L sucrose. When the drop of medium at the tip of the straw has liquified, and medium begins to fill up the straw, the wide opening of the straw is sealed by the operator’s finger. The expansion of cold air in the straw caused by the gradual rise of its temperature leads to the expulsion of the hESC clusters into the dish (Figure 12.1b). After 1 min of incubation the clusters are transferred to and further incubated for 5 min in holding medium with 0.1 mol/L sucrose, followed by two 5 min washout incubations in holding medium without sucrose before being plated on a fresh feeder layer.7
RESULTS OF VITRIFICATION OF HUMAN EMBRYONIC STEM CELLS The efficacy of hESC vitrification can be assessed by several in vitro and in vivo quantitative and qualitative parameters. A primary parameter is the rate of recovery, i.e. the rate of hESC clusters successfully recovered from the vitrification straws after warming. Two
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a
b
37 °C Figure 12.1 (a) Vitrification by the open pulled straw method. The cells to be vitrified, which are held at this stage in a cryoprotectant supplemented medium, are pulled into the thin end of an open pulled straw by capillary action. The straw is then plunged into liquid nitrogen. (b) Warming of vitrified cells. The straw is plunged in holding medium supplemented with sucrose heated to 37°C. The vitrified-warmed cell clusters are expelled out of the straw once the temperature rises and the large open end is sealed by the operator's finger. The cells are then washed out of the sucrose before further culture.
parameters are growth rate and the level of background spontaneous differentiation. In addition to quantitative criteria, preservation of the embryonic stem cell chacteristic state has to be evaluated by morphological criteria, karyotyping, immunophenotyping, and fluorescent activated cell sorting (FACS) analysis of the expression of markers of pluripotent cells, such as the cell surface markers stage specific embryonic antigen 3 (SSEA-3), SSEA4, TRA 1–60, TRA 1–81, etc., and analysis of the expression of transcriptional factors characteristic of hESCs such as Oct4, Nanog, and Sox2. The pluripotent potential of the thawed hESCs should be confirmed by demonstrating their capability to differentiate into progeny representing the three embryonic germ layers both in vitro and in vivo within teratoma tumors after xenografting the devitrified cells into severe combined immunodeficiency (SCID) mice.7,19 Human ESCs from various cell lines can be successfully recovered and propagated after cryopreservation with conventional slow rate
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freezing and rapid thawing methods.5 However, in two comparative analyses, vitrification was superior to slow freezing.7,19 The efficiency of the standard slow freezing methods with hESCs was relatively low. It was possible to recover only 23–70% of the hESC clusters after freezing by slow cooling and thawing, and only 16% developed further after plating into hESC colonies; these were undersized compared with controls. In contrast, an improved outcome post-warming was observed when the OPS vitrification method was used. A total of 82–100% of the vitrified hESC cell clusters were recovered after warming and all generated colonies after plating.7,19 Vitrification has been reported to be associated with some cell death at the first day after plating and with a significantly reduced mean area of the colonies at day 2 and 7 after plating, compared with control non-frozen thawed colonies.7 However, an additional day in culture was sufficient to overcome the vitrification induced cell deficit, and at day 8 after
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plating the area of the vitrified warmed colonies was similar to that of the control colonies at day 7.7 Vitrification was also associated with a significant increase in the level of background differentiation as evaluated at day 7 compared with controls.7,19 Nevertheless, the morphological appearance of the colonies improved with additional time in culture, probably due to the proliferation of undifferentiated cells.7 After vitrification hESCs retain their characteristic morphology and other key properties of human pluripotent cells.7,19 Vitrified warmed hESCs can be propagated for prolonged periods, express alkaline phosphatase activity, and retain their normal karyotype.2,7,19 Immunophenotyping of the vitrified ESCs can be carried out using a series of antibodies that detect cell surface carbohydrates and associated proteins found on human pluripotent cells.1,2 The vitrified warmed hESCs remain immunoreactive in indirect immunofluorescence assays with antibodies against the SSEA-4 and TRA 1-60 carbohydrate epitopes, and with the monoclonal antibody GCTM-2, which detects an epitope on the protein core of a keratan sulfate/chondroitin sulfate pericellular matrix proteoglycan found in human embryonal carcinoma cells.7,19–21 Devitrified hESC colonies express Oct4, an essential factor for the establishment of the pluripotent stem cell population of the inner cell mass, which governs the fate of ESCs.1,2,22 A critical level is required to sustain stem cell self-renewal. Increased expression of Oct4 induces differentiation into primitive endoderm and mesoderm, while down regulation of Oct4 levels results in dedifferentiation to trophectoderm.22 Oct4 is expressed in hESCs and its expression is down regulated when these cells differentiate.2 Reverse transcriptase polymerase chain reaction (RT-PCR) analysis of mRNA isolated from colonies consisting mainly of devitrified stem cells, showed that these hESCs retained the expression of Oct4 after vitrification.7,19 The
retention of pluripotency after vitrification and warming of hESCs was also evaluated in vivo by xenografting the cells into SCID mice. Benign teratomas containing tissues representative of all three germ layers were formed, and embryonal carcinoma was not observed in any lesion.7,19
CONCLUDING REMARKS The data published in the literature indicate that vitrification by the OPS method is a very simple and effective approach for the cryopreservation of hESCs. A high proportion of the vitrified hESC clusters can be recovered after warming, and develop into hESC colonies after plating. The vitrified warmed hESCs retain the key properties of pluripotent cells as demonstrated by their normal karyotype, marker expression, and the potential to differentiate to derivatives of the three germ layers in xenografts.7 Nevertheless, it should be noted that vitrification of hESCs is associated with some cell injury. A significant increase in the levels of cell death and spontaneous differentiation after warming do occur, however, an additional proliferation of the ES cells in culture for a further 2 days is sufficient to overcome these effects.7 Therefore, it seems that for the purpose of cryopreservation of cell lines, the practical significance of this cryoinjury is probably negligible. It is possible that alteration of the composition of the vitrification solution in favor of low toxicity cryoprotectants such as ethylene glycol,23 use of nylon loops instead of the OPS,15 and increasing the rate of cooling may further reduce the vitrification induced cryoinjury. Increased cooling rate requires direct contact between the cell containing medium and liquid nitrogen.9,12,15 This direct contact may carry a potential hazard for transmission of infective agents.24 The potential hazard of contamination may be eliminated by utilizing the OPS vitrification method under sterile conditions.13 While vitrification is a highly effective method for hESC cryopreservation, and is an
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ideal method for the efficient storing of new hESC lines and clones and for their safe and expeditious transfer between research laboratories, it has limitations with regard to the number of cells that are cryopreserved. Current methods of vitrification do not allow freezing of large cell pools or volumes. This is a significant limitation since cryopreservation of large scale bulk cultures may be required in the future when clinical grade hESC lines are to be developed for clinical purposes. Additional methodologies will be required to allow the exploitation of vitrification methods for the preservation of hESC bulk cultures for clinical purposes. The currently published successful results of hESC vitrification may further enhance the cryopreservation of human blastocysts by vitrification, since it has been demonstrated that human pluripotent inner cell mass-like cells retain a normal karyotype and pluripotent state following vitrification and warming.
Cryopreservation of hESCs may serve as an in vitro model to test and compare the efficacy of various cryopreservation protocols before exploring their use for cryopreservation of human blastocysts in clinical infertility. Nevertheless, while cryopreservation of hESCs may serve as a reliable model for the pluripotent cells of the inner cell mass, this model will not be totally predictive of the outcome of cryopreservation of the total blastocyst which has a critical layer of trophectoderm cells as well. In conclusion, the results published in the literature indicate that hESCs may be effectively cryopreserved by using the vitrification method. The key properties of pluripotent cells were maintained after warming and the associated cryoinjury was mild and transient. Cryopreservation by vitrification improves the handling of hESC lines and may be used for storage of stocks of cells and for the establishment of ESC banks, facilitating efficient interlaboratory transfer of cells.
References 1. Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem cell lines derived from human blastocysts. Science 1998; 282: 1145–7. 2. Reubinoff BE, Pera MF, Fong CY et al. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 2000; 18: 399–404. 3. Shufaro Y, Reubinoff BE. Therapeutic applications of embryonic stem cells. Best Pract Res Clin Obstet Gynaecol 2004; 18: 909–27. 4. Gearhart J. New potential for human embryonic stem cells. Science 1998; 282: 1061–2. 5. Freshnny R. Culture of Animal Cells; a Manual of Basic Technique. New York, Wiley-Liss Inc, 1994: 255–65. 6. Robertson EJ. Embryo derived stem cell lines. In: Robertson EJ, ed. Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. Oxford: IRL Press, 1987: 71–112. 7. Reubinoff BE, Pera MF, Vajta G, Trounson AO. Effective cryopreservation of human embryonic stem cells by the open pulled straw vitrification method. Hum Reprod 2001; 16: 2187–94.
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8. Rall WF, Fahy GM. Ice-free cryopreservation of mouse embryos at −196 degrees C by vitrification, in Nature. 1985; 313: 573–5. 9. Martino A, Songsasen N, Leibo S. Development into blastocysts of bovine oocytes cryopreserved by ultra-rapid cooling. Biol Reprod 1996; 54: 1059–69. 10. Chen SU, Lien YR, Chen HF et al. Open pulled straws for vitrification of mature mouse oocytes preserve patterns of meiotic spindles and chromosomes better than conventional straws. Hum Reprod 2000; 15: 2598–603. 11. Kuleshova L, Gianaroli L, Magli C et al. Birth following vitrification of a small number of human oocytes: case report. Hum Reprod 1999; 14: 3077–9. 12. Vajta G, Holm P, Greve T, Callesen H. Vitrification of porcine embryos using the open pulled straw (OPS) method Acta Vet Scand 1997; 38: 349–52. 13. Vajta G, Holm P, Kuwayama M et al. Open Pulled Straw (OPS) vitrification: a new way to
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reduce cryoinjuries of bovine ova and embryos. Mol Reprod Dev 1998; 51: 53–8. Vajta G, Hyttel P, Callesen H. Morphological changes of in-vitro-produced bovine blastocysts after vitrification, in-straw direct rehydration, and culture. Mol Reprod Dev. 1997; 48: 9–17. Lane M, Schoolcraft WB, Gardner DK. Vitrification of mouse and human blastocysts using a novel cryoloop container-less technique. Fertil Steril. 1999; 72: 1073–8. Lane M, Bavister BD, Lyons EA, Forest KT. Containerless vitrification of mammalian oocytes and embryos. Nat Biotechnol 1999; 17: 1234–6. Yokota Y, Sato S, Yokota M et al. Successful pregnancy following blastocyst vitrification: Case report. Hum Reprod 2000; 15: 1802–3. Karlsson JO. Cryopreservation: freezing and vitrification. Science 2002; 296: 655–6. Zhou CQ, Mai QY, Li T et al. Cryopreservation of human embryonic stem cells by vitrification. Chin Med J (Engl) 2004; 117: 1050–5.
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20. Pera MF, Blasco-Lafita MJ, Cooper S et al. Analysis of cell-differentiation lineage in human teratomas using new monoclonal antibodies to cytostructural antigens of embryonal carcinoma cells. Differentiation 1988; 39: 139–49. 21. Badcock G, Pigott C, Goepel J, Andrews PW. The human embryonal carcinoma marker antigen TRA-1-60 is a sialylated keratan sulfate proteoglycan. Cancer Res 1999; 59: 4715–9. 22. Niwa H, Miyazaki Smith AG. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet 2000; 24: 372–6. 23. Palasz AT, Mapletoft RJ. Cryopreservation of mammalian embryos and oocytes: recent advances. Biotechnol Adv 1996; 14: 127–49. 24. Tedder RS, Zuckerman MA, Goldstone AH et al. Hepatitis B transmission from contaminated cryopreservation tank. Lancet 1995; 346: 137–40.
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actin 111 American Red Cross 261 American Society of Reproductive Medicine 188 animal reproduction, vitrification using EG-based solutions 75–85 apoptosis 266, 286vit assay 287 in spermatozoa 96 aqua-glyceroporin 82 aquaporin-3 in mouse oocytes 82 aquaporins (AQPs) 79, 80–81 bacteria cryopreservation 34 banking ovarian tissue 273 ovary 274 biocompatibility, BS evaluation 282–6 Biot-Fournier equation 28 blastocoels 83 artificial shrinkage 212 expansion degree 212 fluid content reduction 230 shrinkage effectiveness 230 volume reduction 220 blastocysts Cryoloop vitrification 220–1 cryopreservation 65, 195 early expanding (EEB) 240, 245 EM grid vitrification, six-step dilution, clinical results 245 expanded artificial shrinkage 223–4 before EM vitrification 245–6 effects of dilution and sucrose concentration 247 survival and pregnancy rate increase 249 full equilibrium with cryoprotectant solution 212 hatching using laser pulses 223 human developmental stages 241 EM grid vitrification 239–252 materials 242
embryo transfer 244 freezing with glycerol and sucrose 219 and vitrification 172 survival 244–5 assessment after thawing 244 transfer 239 vitrification, manual of 234–8 warming methods after thawing 242, 243–4 human expanded, EM grid freezing and warming 246 middle expanding (MEB) 240, 245 mouse survival and hatching 205 survival rates 230 permeability 83 recovered, morphology 225 selection for vitrification 211 transfer 153 vitrification 207, 208–210 factors affecting outcome 210–213 vitrified infant outcome 228 perinatal outcome 227–9 survival rate after dilution methods 245 transfer 226 outcome 226–7 in vitro culture, cryotolerance reduction 213 warming, hatching and survival assessment 221–3 Boutron, Pierre 5 bovine serum albumin (BSA) 183 butanediol 60 butane-2,3–diol 279 butylene glycol 49, 51 calorimeter differential scanning 278 thermogram 278 calorimetry 165
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calves born after OPS vitrification 67 of immature oocytes 68 somatic cell cloned, born after OPS process 68 capillary effect 36 carrier selection 259 solution, lack of effect on warming vs cooling rates 15 system 200–201 aseptic, problems 205 see also container cattle, OPS method 67–9, 71 cattle embryos, cryoprotectant toxic effects 54 cells concentration dependent toxicity 16–17 cooling techniques 165 cryodamage 87 cryopreservation by vitrification 1 cryosensitivity 92 damage in freezing and thawing 88–9 dehydration 197, 198–9 glass-forming agents toxicity 17 ice formation 33 osmotic limits 15–16 permeability 197 to cryoprotectants 16 proliferation by PCNA staining of human ovarian tissue 286 shrinkage 16 during cooling 88 surface-volume ratio 197 thawing 276 time dependent toxicity 16 vitrification 22–5 children born from slow frozen oocytes, outcome 160 chilling injury 17, 77, 87, 121, 122 avoidance 196 decrease 39 EM copper grid use for avoidance 133 chromatin damage 89
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chromosomes 139–140 spermatozoal 96–9 closed pulled straw (CPS) 134, 137 congenital birth defects 229 container aseptic 168–9 for OPS vitrification and spermatozoa warming 100–1 see also carrier controlled ovarian hyperstimulation (COH) 248 cooling rate 49 calculation of small samples 28–30 critical 277–8 fast 196, 199 high, improved clinical results 146–7 increased 138 liquid nitrogen 45 ’slow’ 165, 167 for vitrification 13 Cryo Bio System 201–3 cryo-injury 196 cryodamage 107–8 Cryoleaf method superiority 141 thawing 132 vitrification 133, 140 Cryoloop 90, 92–3, 123, 155, 202 advantages 229–230 blastocysts 207 human, cryopreservation 231 vitrification 219–238 day 3 embryos 206 manipulation 222 modified protocol 188 with spermatozoa 93–5 use without LN contact 173 vitrification 133–135, 136, 186–8, 191–2 ultrarapid 190 vitrification scheme 94 cryomicroscopy, phase-contrast 165 cryopreservation 33–4 animal model 284–5 conventional 276–7 early work 22–4 embryo and oocyte successes 37 ice-free 277–286 mammalian developmental consequences 107–117 mammalian embryo protocols 190–3 process (CP) 21 see also vitrification
cryoprotectant, hypothetical 25 cryoprotectant equilibration 56 cryoprotectants 29 causing osmotic damage 33 combinations 48 concentration 197 effect on embryos 213 equilibration 225 exposure to or equilibration of 197 glass forming capabilities 47–56 for hESCs 29 increased concentration of and exposure to 231, 276–7 intracellular concentration 196 mouse embryo toxicity 47 movement through plasma membrane 81–2 regimens 130 required characteristics 45 saturation by and removal of 163–5 selection 259 stepwise equilibration 130 temperature rate change control 33 toxicity 33, 50–2, 89, 90 types 46, 196–7 for vitrification 91 vitrification concentrations 46 cryoprotective agents (CPAs) 21, 24–5, 26–7, 88, 92 combination 147 ice crystal growth 279–282 role 11–12 cryostability 167 cryostorage 107 cryostraws 185–6 disadvantage 191 cryosubstitution 281–2 Cryotip 37, 202 blastocysts 209 method 134, 137 cryotolerance reduction 213 Cryotop 123, 155, 201 advantages 153–4 blastocysts 209 human, vitrifying and warming 253–9 definitions 257 equilibrium solution 254–6 materials used 253 reagents used 253 storage container choice 40
300
vitrification 124–125, 133–4, 188–9, 191–2 device 124, 136 human application 65 mouse and human blastocysts 134 oocytes 131–2 procedure 137, 254, 255–6 superiority 141 ultrarapid 190 vitrification solution 254–6 warming procedure 256–7 solutions 256 zygotes 206 cryovial capping portion 221 cumulus cells removal 141 cumulus complex 146 cumulus-oocyte complexes (COC) 137–8, 147, 149, 263–4 cut standard straws (CSS) 168–9, 173 cytochalasins 176 cytoplasm 110–112 fracture 87 dehydration 197 depolymerization 175 devitrification 11–12 and fast warming rate 95–6 dewar flask, cooling in 189 differential scanning calorimetry 30 dilution method six-step, human blastocysts EM vitrified clinical results 247–8 two-step 240, 242, 244 after thawing of vitrified shrunk blastocysts 246–8 EM vitrified human blastocysts 248–9 dimethylsulfoxide (DMSO) 24, 29, 45, 49, 123 cell contact at decreased temperature 169–172 as CPA 276 direct cover vitrification (DCV) 135 disease transmission liquid nitrogen-mediated 40 potential danger 39–41 DNA damaged and non-damaged 97 fragmented 96–7 droplet, spherical, temperature distribution 30
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egg bank 145 egg yolk 88 electron microscope (EM) grids 36, 146 blastocyst vitrification 239–252 copper 133–4, 136 high cooling rate 239 human blastocyte vitrification, results 244–9 oocyte vitrification procedure 147, 149 electron microscopists 4–5 embryos animal, vitrification using EG-based solutions 75–85 carrier 200–1 cryoprotectant toxic effects 54 day 3 vitrification 206–7 and warming cycles 207 dehydration 198 freezing, legal problems 145 human cryopreservation negative effects 164–5 culture for EM grid procedure 240 frozen with propanediol and sucrose 219 transfer 172 viability 164 mammalian, cryopreservation developmental consequences 107–117 mouse 8–cell, vitrification 183–4 pronuclear blastocysts 174 human and animal differences 175–9 human intracellular lipids 178 micrographs 164, 168 vitrification 163–182 vitrified 166 shrinkage in cryoprotectant 56 transfer, time after warming 210–211 vitrification with EG and sucrose 59–60 methods 133 using OPS 65–73 vitrified development 60 survival 56–60 rate 213 water and cryoprotectants movement pathway 79–80
endometrium preparation 149, 244 equilibration solution 156 strategies 123 and vitrification 131 ethylene glycol 35, 122–3, 129 with glycerol 48, 53 low toxicity 214, 219 and sucrose embryo vitrification 59–60 oocyte vitrification 59–60 toxicity 91 vitrification 47–48 ethylene glycol-based solutions, animal embryo vitrification 75–85 evanescent spherulites 4 extracellular damage 112 extracellular vitrifying state 197, 199 Fahy John 6–8, 24, 165 embryo vitrification 91 Fertility Centers of Illinois, Chicago, retrospective data 258 fertility preservation 273–4 cryopreservation options 275–6 fertilization assessment 225–6 Flexipet denuding pipette (FDP) 134 fluorescent activated cell sorting (FACS) 295 follicle stimulating hormone (FSH) 283–4 follicles development 284 growth evaluation 286 in sheep ovarian tissue 286 timeline 283 human ovarian tissue 285 survival 274, 276 fracture in cryopreservation 122–3 formation 48 freezers, industrial 24 freezing at Kyono ART Clinic 155–9 conventional 87 genetic damage 61 for hESCs 293–4 in liquid nitrogen vapor 93–5 prevention 277 slow 21, 23–24, 89, 219, 229 method (SFM) 153
301
French mini-straw 199, 200 blastocyst vitrification 209 French straws 92 glass, fracture 48 glass micropipette method (GMP) 70, 133 glass transition temperatures 24, 89 glycerol 23–24, 26, 45, 60 cryoprotective properties 276 vitrification 47–8 goat, OPS method 67–69 gold grid 146, 149–150 Graevsky, Emmanuil Y 23 granulosa cells 284 growth differentiation factor (GDF) 284 HART Clinic group 186–7, 227 heat conduction equation 28 hemi-straw system 133, 137, 200–2 blastocyst vitrification 210 vitrification procedure 203 vitrification protocols 204 warming procedure 203 High Security Vitrification (HSV) kit 41, 202, 204 for blastocysts 210 horse, OPS vitrification 70 human embryonic stem cells (hESCs) 28, 90 vitrification 293–7 results 294–6 human serum albumin (HSA) 185 hydrostatic pressure 90 ice avoidance 34 crystallization 49 dry 60 formation 48, 50–1 elimination 35–39 extracellular 273–5, 276–7 intracellular 30 seeding 34 ice crystals 279 growth 279–280 in vitro fertilization embryo transfer (IVF-ET) 146–7 in vitro fertilization (IVF) 248–9, 250 in vitro maturation (IVM) 248–9, 250, 261, 266 ovarian tissue 275 program 240 infections elimination 40
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intracellular damage in oocyte cryopreservation 108–112 intracytoplasmic sperm injection (ICSI) 119–120, 123–4, 149 IVF Nagata Clinic, Japan 188–9 Kato Ladies’ Clinic, Tokyo, Cryotop results 125–6 Kauzman’s paradox 8 laser pulse 231 for shrinkage 223–4 lipids 164 cryostability 178 droplet removal 122 gray vesicles 176 intracellular 175–9 micrographs 177–8 liquid nitrogen 24, 36–38, 39, 89, 205 contamination 167 prevention 138–9 cooling 45 vapor, freezing in 93–5 liquid nitrogen slush (SN2) 138, 146–7 luteinizing hormone (LH) 283 Luyet BJ 3–6, 8, 22–23, 24–5, 48 frog spermatozoa vitrification 97 sperm vitrification technique 89–90, 91 lyophilization 21, 89 magnetic resonance spectroscopy (NMR) 280 meiotic spindles 139 injury 140 melting point relationships 12 membrane permeability and cryopreservation 82–3 microdroplet, aseptic warming 27 microfilaments 111 microneedle for shrinkage 223, 224 micropipettes 167 micropipetting 231 microtubules 110, 139 microvacular damage 273–4 minimum drop size (MDS) 133 technique 38 minimum volume cooling (MVC) 38, 133, 154 minimum volume vitrification 132 evolution 132–5 techniques 139
Minke whale oocytes, OPS vitrification 71 mitochondria 98, 111–112 monkey, ovarian tissue vitrification 265–6 morulae vitrification and warming cycles 207 results 209 mouse OPS vitrification 70–1 ovarian tissue vitrification 263–4 mouse embryos survival after exposure to VS 52, 53 after VS11 55 vitrification protocol 76–7 volume change with VS 78 mouse morulae, survival after VS 79 mouse oocytes volume change with PB1 81 volume change with VS 78 mRNA in AQPs 79, 80–1 necrosis 268–29 Nei, Tokio 4 nitrogen (N2) vapor, storage devices 147 non-vitrifying solution (VS1) 197 nuclear envelope 108–9 nucleation, ice 10 nucleolus 109–110 nucleus, in oocyte 108 oocytes aging 140–1 bovine, lipid granules 176 cryopreservation 145, 275 methods 107–8 problems 121–122 reasons for 120–1 cryoprotectant addition 199 cryosensitivity change 122 cryosurvival and parthenogenetic activation 112–113 frozen-thawed fertilization outcome 140 meiotic spindle dynamics 139–140 GV after long contact with DMSO 171 cooling treatment effects 170 cytoskeletal elements 176
302
human clinical outcomes 148 cryopreservation indications 159 Cryotop 154–5 delivery after vitrification 153 vitrification 146 at Kyono ART Clinic 156, 158 methods 154 immature, cryopreservation 160 inverted microscopic images 125 large numbers, vitrification devices 137–8 mammalian, cryopreservation developmental consequences 107–117 meiosis 140 micrograph 110–111 pig, sensitivity 176 vitrification and Cryotop method 119–128 Cryotop results 125–6 with EG and sucrose 59–60 methods 133 time schedule 140–1 vitrified, survival and pregnancy 141 open pulled straw (OPS) 66, 90 animal reproduction use 65–73 application in cattle, sheep and goat 67–9 aseptic 202 storage 138–9 for hESCs 294 method 65–7 glass micropipette (GMP) 70 reasons to use 71–2 vitrification 295 technique 36–8, 40–1 vitrification 133–4, 136 process 66 organs, cryopreservation by vitrification 1 osmosis 165 osmotic shock 122 osmotic stress 34, 87 ovarian autograft implantation 273 ovarian hyperstimulation syndrome 147, 189, 226 ovarian tissues autotransplantation, pregnancies 262 bovine, vitrification 264 cryopreservation, limitations 273–4
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Cryotop vitrified human 267 human follicles in 268 vitrification and autografting after warming 270 and xenografting after warming 268, 269–270 reproductive biology and physiology 283–4 sheep cryosubstitution 282 implantation 284–5 transplanting 261 vitrification 261–272, 273–292 in animals 262–6 in human 266–8 in monkeys 265–6 in rabbit 266 in rats and mice 262–4 research design logistics 285–6 in sheep 265 ovary banking 274 cryopreservation 275–6 Parkes, Alan 23, 88, 91 patients and IVF treatment 240 preparation 225–6 pellicles 26–7, 29–30 permeability property 197 pig embryos and oocytes, vitrification with OPS method 69–70 piglets born after OPS vitrification of cloned blastocysts 70 born after OPS vitrification of in vivo embryos 70 plasma membrane cryoprotectant movement through 81–2 water movement through 80–81 platelet endothelial cell adhesion molecule (PECAM) 269 polecat pups born after OPS vitrification of in vivo embryos 71 Polscope 139 polycystic ovary syndrome 248 polyethylene glycol (PEG) 279 polyvinylpyrrolidone 183 pregnancy assessment 226 multiple 195
pregnancy rate after DMSO vitrification 169–170 Cryoloop vitrification 187–8 Cryotop vitrification 189 proliferating cell nuclear antigen (PCNA) 284, 286 1,2–propanediol 153, 279 propylene glycol 35, 49, 60 quarantine 145 quasivitrification, cryoprotective agents and 11–12 rabbit OPS vitrification 70 ovarian tissue vitrification 266 Raju, Rama 191–3 Rall Peter 6, 7–8, 24, 129, 165 embryo vitrification 91 rat, ovarian tissue vitrification 262–3 reactive oxygen species (ROS) 266–7 rehydration post-thaw 165 osmosis 176 RNA interference 109 room temperature 189 severe combined immunodeficiency (SCID) mice 268, 284, 295–6 sex ratio after cryopreservation 160 sheep OPS method 67–9 ovarian tissue vitrification 265 sheep embryos cryoprotectant toxic effects 54–5, 57–8 vitrified survival 58–9 viability 59 shrinkage artificial 220, 223, 240 before EM grid vitrification 245–6 before vitrification 230–1 clinical results 227–8 Siberian tigers, OPS vitrification 71 Smirnov, Igor 23, 88 smooth endoplasmic reticulum (SER)-lipid globules-mitochondria 175 smooth endoplastic reticulum (SER) 164–5 solid-surface vitrification (SSV) 133, 136–7 Spallanzani, Lazzaro 22
303
spermatozoa chromosome apparatus integrity 96–9 cooling rate estimation 95 cryopreservation 88–9 diminished motility 88 examples 98 human viability after cryoprotectant-free vitrification 102 vitrification 25, 26 ‘swim up’ selection 92 vitrification background 91 cryprotectant-free 87–105 vitrified, oocyte fertilization and embryo development 95 warming 94 and ’droplet’ vitrification 99 and OPS vitrification container 100–1 spindles 110, 139–140 damage after cryopreservation 160 by ice formation 275 recovery 141 SSF technique 41 statistical analysis 244, 283 stereo-microscope 259 Stiles, Walter 3 Stokes-Einstein equation 9 storage containers 40 optimal, below Tg 9–11 strain point 21 straw vitrification method, conventional 135–6 straw-in-straw vitrification 167 method 102 sucrose 53–4, 130, 139, 163, 183 gradient steps 191 as osmotic buffer 91 sugars 123 cryoprotective role 97 Supercool X-100268 Tammann’s theory 3 Taxol 160 temperature distribution in spherical droplet 30 effect on viscosity 10 equilibration 225 low, diffusion time 11 and toxicity 45
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TEST-egg-yolk-glycerol (TEYG) 92–3 thawing at Kyono ART Clinic 157 preparation for 158 preparation procedure 158 six-step method 243–4 tissue, cryopreservation by vitrification 1 toxicity 197 cryoprotectants 50–2 dilution procedure effects 54 species effects 54 transplantation, ischemia after 275 trehalose 123 trophectoderm cells 240 viability assays 261 virus cryopreservation 34 VitMaster 138, 200 vitreous state 21 vitrificants 22, 24 vitrification achievement 89 as alternative to freezing 89–90 animal embryos with EG-based solutions 75–85 method 75 using EFS40 76 in animal reproduction 65–73 aseptic 173, 202, 204 cryoprotectant intracellular concentration 196 cumulative citations 2 day 2–3 embryos, clinical results 188 definition 89 enhancement factors 97–9 and equilibration 131 fast rate-low concentration (FR-HC VF) 22, 25–8
high temperature 22 historical development 3–8 human blastocyst, laboratory manual 234–8 human embryonic stem cells (hESC) 293–8 ice-free 22 kinetic basis 9 kits 39 medium isolation from liquid nitrogen contact 165–9 methods 99–101, 184–5, 280–1 minimum volume methods 132–5 in nature 1–3 outcome 159 ovarian tissue 261–272 overview 1–20 problems 24–5 procedures with cooling and warming rates 135 strategies 199–205 successful, biological phenomena 15–17 superior methods 141–2 testing container choice 49–50 thermodynamic necessity 8–9 ultrarapid 200–202 using EG-based solutions 75–6 using high CPA concentrations 90–1 warming rate after 13–15 see also cryopreservation Vitrification Kit 155 vitrification solutions (VS) 45–6 biocompatibility evaluation 282–6 components 77, 79 composition 51 concentration reduction and toxicity 129–131 design using bulk samples 280 for hESCs 294
304
osmotic effects on embryos 56 preparation 54–6 protocols 187 regimens 130 selection 52–4 toxicity reduction 213 vitrification solutions (VS2) 197 day 3 embryos 206 vitrified solutions (VS), evaporation prevention 225 Vitroplug 201 VitSet 40 Vogel-Tammann-Fulcher equation 9, 10 warming rate 49 calculation of small samples 28–30 critical 277–8 and dilution 131–2 fast 196, 199, 225 and devitrification 95–6 increase 35 ‘rapid’ 167 ‘slow’ 166 to prevent ice formation 14 warming solutions 225 water critical cooling speed 90 diffusion 80–1 evaporation 89 water channels 80–1 Xenopus oocytes, permeability to cryoprotectants 83 zona pellucida 112, 122–3 hardening 275 laser opening 172 zygotes, vitrification 206
