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Highly efficient vitrification for cryopreservation of human oocytes and embryos: The Cryotop method
Theriogenology 67 (2007) 73–80 www.theriojournal.com
Highly efficient vitrification for cryopreservation of human oocytes and embryos: The Cryotop method Masashige Kuwayama * Kato Ladies’ Clinic, 7-20-3 Nishishinjuku, Shinjuku, Tokyo 160-0023, Japan
Abstract Vitrification is frequently referred to as a novel technology of cryopreservation in embryology, although some young embryologists were born after its first successful application. Unfortunately, in spite of the accumulated evidence regarding its enormous potential value, most domestic animal and human laboratories use exclusively the traditional slow-rate freezing with its compromised efficiency and inconsistency. The purpose of this paper is to clarify terms and conditions, to summarize arguments supporting or disapproving the use of vitrification, and to outline its role among assisted reproductive technologies. To provide evidence for the potential significance of vitrification, achievements with the Cryotop technology, an advanced version of the ‘‘minimal volume approaches’’ is analyzed. This technology alone has resulted in more healthy babies after cryopreservation of blastocysts than any other vitrification technique, and more successful human oocyte vitrification resulting in normal births than any other cryopreservation method. The value of this method is also demonstrated by achievements in the field of domestic animal embryology. A modification of the technique using a hermetically sealed container for storage may help to eliminate potential dangers of disease transmission and open the way for widespread application for cryopreservation at all phases of oocyte and preimplantation embryo development in mammals. # 2006 Elsevier Inc. All rights reserved. Keywords: Cryodevice; Blastocyst; Pregnancy
1. Introduction One of the eternal ambitions of humankind is to overcome limits created by dimensions. After initial signs of successes to overcome horizontal barriers with railways almost 200 years ago, a real breakthrough only occurred in the 20th century, accompanied by expansion also into the third dimension, i.e. into the air and also into space. Today, the once demanding 42 km horizontal distance presents a challenge only for a marathon runner; practically all points of the globe can be reached in less than 48 h, while traveling at most
* Tel.: +81 3 3366 3777; fax: +81 3 5332 7373. E-mail address: [email protected]. 0093-691X/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2006.09.014
at 10 km above the surface of the earth. In parallel, the astonishing advancement of information technology has helped us to eliminate virtually all remaining distances by enabling communication between continents exactly as easily as between neighboring offices. Compared to that, almost nothing has happened with the fourth, maybe even more important dimension: time. We cannot travel forwards nor backwards, cannot slow down our limited available time, and cannot probably even use it better than previous generations. Among the very limited achievements in this debate we may list the cooling and later deep-freezing of biological materials, mainly food to slow down post mortem degradation; and hibernation to expand the lifespan of living tissues and organs under temporarily unfavorable conditions.
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However, the second half of the last century has resulted in considerable advances allowing the biological clock of live objects to be stopped for an unlimited (or practically unlimited) period, made possible by cooling them to extremely low temperatures, below 150 8C. This approach was first applied to simple structures including bacteria or single cells, and then advanced to some multicellular organisms and organs. Currently, major limitations are size and complexity: generally the smaller the sample and the simpler its structure, the better the chances for successful cryopreservation. Accordingly, embryology is in a rather privileged situation because of the relatively small size of the biological samples we work with, although even seemingly negligible variations in size may cause considerable differences in survival rates. Since the middle of the last century, in parallel with the development of in vitro reproductive technologies, remarkable successes have been achieved in cryopreservation of spermatozoa, embryos and oocytes, enabling us to stop and restart time both shortly before and after fertilization. Apart from the biological success, this possibility may have profound philosophic and moral consequences, especially when human material is concerned. Among other manipulations, stopping of time seem to be tolerable in the case of gametes, but it has been forbidden for embryos in many countries for ethical and legal reasons. As a curious consequence, to avoid restrictions with serious consequences, developmental stages have been re-defined in some of these countries by replacing the name ‘zygotes’ or ‘one-cell embryos’ with the term ‘pronuclear stage oocytes’, an expression that is rather paradoxical for experimental and domestic animal embryologists, but one that permits more legal freedom in assisted reproduction in humans. Another example is the euphemistic category of ‘‘pre-embryo’’ in human assisted reproduction. In spite of these controversial situations, the need for efficient cryopreservation of oocytes and embryos is enormous both for theoretical and practical reasons. However, in spite of the vast efforts invested, advances are rather slow. The main problems are the lack of consistency [1] as well as differences in survival and developmental rates after warming between species, developmental stages and quality. While the latter is easy to understand, we have only partial and not entirely supported information regarding the reasons for the lack of consistency in results. On the other hand, basic research in this field follows only empirically obtained advances: instead of indicating future directions for improvements, its main role is restricted to explaining retrospectively the achievements (a very recent example
for this situation is the history of the discovery of somatic cell nuclear transfer. In contrast to spermatozoa, where slow-rate freezing is almost exclusively used for cryopreservation and alternative methods are applied only at an experimental level [2], in cryopreservation of mammalian embryos and especially oocytes, vitrification has become a viable and promising alternative to traditional approaches. The convincing evidence that has been accumulated regarding the huge potential importance of vitrification has been reviewed recently [3–6]. Consequently, this paper will focus only on the latest achievements, and will discuss in detail the Cryotop vitrification method that has resulted in the highest number of babies born after vitrification of human embryos and after cryopreservation of human oocytes worldwide, and is now also successfully applied in various areas of animal biotechnology. 2. Vitrification In spite of the fact that mammalian embryo and oocyte vitrification was the subject of more than 500 publications in the past 10 years, and no comparison in any systems has proved its efficiency to be inferior to that of traditional freezing, vitrification is still regarded as experimental [3,4] and its practical use is restricted to a very few human and domestic animal embryology laboratories. Apart from the theoretical and practical problems, many misunderstandings also hamper its large-scale application. There are problems even with the definition. Vitrification is just a vitreous, transparent, ice-free solidification of water-based solutions at subzero temperatures [7]. Accordingly, even the first paper describing successful cryopreservation of mammalian spermatozoa used this term in a context that is not applicable today [4,8]. In some papers, vitrification is described as the result of extremely high cryoprotectant concentrations and extremely high cooling rates. However, vitrification does not necessarily require high cryoprotectant concentrations, because even pure water can be vitrified if the cooling rate is high enough ( 107 8C/s) [9] and, on the other hand, with concentrated cryoprotectant solutions, vitrification can also be achieved with a moderate or even slow cooling rate [10]. Other papers regard direct contact between liquid nitrogen and the embryo-oocyte containing solution as a prerequisite of vitrification, although it is just one possibility for increasing cooling rates that may permit decreasing the concentration of cryoprotectants and minimizing their potential toxic and osmotic effects. Many vitrification techniques (for example those earlier forms based on sealed 0.25 ml insemination straws or
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closed cryovials) have been described so far that do not require this direct contact, consequently their use does not invoke more liquid nitrogen-mediated contamination risk than traditional freezing. Accordingly, the most widely emphasized concerns, i.e. toxicity and danger of contamination, are not indispensable elements of the vitrification process, and with the development of future new techniques these negative effects should be minimized or entirely eliminated. Unfortunately, presently available vitrification methods still struggle with these problems. However, the actual harm and danger is most probably much lower than as emphasized endlessly by all critics. Regarding the toxic and osmotic effects, the latest cryoprotectant combinations and concentrations are already not too far from the range of those used for traditional freezing, at least the specific toxic effect of the components cannot be considerably higher. Moreover, in most vitrification methods, the time of exposure to the final cryoprotectant concentrations is very limited at temperatures where this toxic effect may still be significant (i.e. above 20 to 40 8C). On the other hand, as the result of the stepwise concentration increase in slow rate freezing, cells are definitely exposed to similar final cryoprotectant concentration as with vitrification—although at a much lower initial temperature that may minimize the toxic effect [4]. Eventually, survival and in vitro and in vivo developmental statistics provide convincing evidence that the cumulative toxic, osmotic and other harmful effects during the vitrification process are not exceeding but are rather below that caused by slow rate freezing. The danger of liquid nitrogen-mediated contamination is a realistic one, but it should not be overestimated. In spite of the enormous number of samples stored in liquid nitrogen, so far no disease transmission attributable to this mechanism has been documented in domestic animal or human reproductive biology [6]. Based on an earlier idea [11], many recent vitrification techniques separate the cooling and the storage phases; in this way the requirement for direct contact is limited to a relatively small amount of liquid nitrogen that can be filtered, UV sterilized or just purchased as sterile stock. On the other hand, the probability of disease transmission from a factory derived, separately stored liquid nitrogen source – considering that at 196 8C pathogens may survive but cannot proliferate – is most probably lower than to get bacterial or viral infection through the disposable surgical masks commonly used during oocyte retrieval, embryo handling and embryo transfer. It would of course be preferable to eliminate even this negligible danger, and there have been many
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such attempts in the past few years [12–17], but almost all of them resulted in somewhat compromised cooling and warming rates that may also jeopardize the results, especially when sensitive samples are vitrified. 3. Minimum volume vitrification methods Minimizing the volume of the vitrification solution containing oocytes or embryos not only offers the obvious benefit of increasing both cooling and warming rates, but also decreases the chance of ice crystal nucleation/formation in the small sample [9]. Curiously, this approach was and still is disregarded by many scientist, who just use traditional tools including cryovials and standard 0.25 ml insemination straws. Even these approaches may result in survival and development rates comparable to that of traditional freezing, but miss potential benefits of high-rate cooling including not only decreased cryoprotectant concentration but also reduced chilling injury that occurs between +15 8C (in human GV oocytes even +25 8C) [18] and 5 8C, which can be minimized by passing the embryos or oocytes rapidly through this temperature zone. According to the few systems where measurement of cooling rates is possible and was performed, the critical value required to avoid chilling injury and to benefit from the lower cryoprotectant concentration required should be around 20,000 8C/min [19], and from both points of view, the most critical period is the initial cooling [20]. Accordingly, seemingly negligible differences in practical performance of cooling, for example the thickness of the cold nitrogen vapor layer over the surface of the solution and the speed at which the sample passes through this layer, may have decisive consequences for the final outcome. By using liquid nitrogen slush for cooling with a commercially available device (Vitmaster) [21], the cooling rate can be increased between +20 and 10 8C, with considerable decrease of both chilling injury and the required cryoprotectant concentration. Minimum volume vitrification methods may also be helpful to avoid zona pellucida and embryo fracture damage. This type of injury occurs frequently when samples are cryopreserved in standard insemination straws and warmed rapidly afterwards. By using small samples and especially with vitrification in open systems, fracture damage rarely occurs, and it can be entirely eliminated with appropriate adjustment of warming parameters. There is no clear definition of what volume can be defined as ‘‘minimum’’ for vitrification, but according to the common use of this term it should be considerably
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less than 1 ml for direct dropping of samples into liquid nitrogen or the open pulled straw (OPS) method; approaches requiring approximately 4–5 or 1 ml solution are usually not considered to be minimum volume techniques [19,22]. The minimum drop size (MSD) method was developed by Arav [23] and Arav et al. [24,25]. Droplets of 0.1–0.5 ml were prepared on glass coverslip strips and immersed in liquid nitrogen or nitrogen slush. The method has allowed up to a 50% reduction in the concentration of cryoprotectants needed compared to traditional protocols, while preserving safe ice- and fracture-free vitrification of material [20]. A similar approach developed for cryopreservation of Drosophila melanogaster embryos [26] was successfully used later for mammalian oocytes and embryos [27], eventually resulting in human pregnancies and babies born after embryo cryopreservation [28,29]. In this method, the sample in a minimal amount of solution was placed on an electron microscope copper grid, then most of the solution was removed by placing the grid on a filter membrane. The procedure allowed separation of the cooling and storage phase, the latter being done in cryovials that were previously filled with liquid nitrogen [30]. Unfortunately the storage component still seems to be a fragile part of this approach, because all cryovial producers strongly advise not to fill the vials with liquid nitrogen, and not to submerge vials during storage but keep them in the liquid nitrogen vapor. These measures are intended to avoid explosion of the vials due to the extreme pressure changes during evaporation of liquid nitrogen that may enter the vials during the cooling and warming processes. Accordingly, apart from the difficulties related to handling of grids under liquid nitrogen, potential explosion of the vials may hamper widespread application of this technology. Another possibility is to use a solution film formed between files of a nylon loop for holding the sample. This method, the Cryoloop, was first applied for flash freezing of protein solutions for analysis in crystallography [31,32] and was used later also for cryopreservation of mammalian embryos and oocytes [33–36] resulting in births from vitrified human and monkey blastocysts [37– 39]. Due to the absence of solid support and the extremely small volume, the cooling rate upon immersion in liquid nitrogen may be as high as 700,000 8C/min [40] thus also permitting cooling in liquid nitrogen vapor. However, in spite of the unquestionable benefits of this system from the point of potential disease transmission, one may have concerns regarding the safety of storage, as this very sensitive and fragile system may increase the risk of accidental warming over the safe temperature zone,
especially if storage of the container is also performed in the vapor of liquid nitrogen [41]. In parallel with these special approaches, there have been repeated attempts at practical application of the original idea to place a very small drop of solution onto a solid surface and immersing both into liquid nitrogen. Hamawaki et al. [42] placed the embryos in a very small volume onto the inner wall of a 0.25 ml standard insemination straw and immersed the straw into liquid nitrogen after sealing. This manipulation eliminated disease transmission problems but decreased the rate of cooling, and it could be regarded as technically difficult. In the hemi-straw system [43,44], the end of a standard 0.25 ml insemination straw was cut with a scalpel and an approximately 0.3 ml droplet of cryoprotectant solution was pipetted onto the inner face of the straw. The straw was then immersed vertically into liquid nitrogen and eventually inserted and closed into a 0.5 ml straw for storage. This system has been found equally or more efficient than the Cryoloop method for cryopreservation of human zygotes and early stage embryos [45] and resulted in pregnancies after blastocyst vitrification [44,46]. 4. The Cryotop method The Cryotop method is probably the latest minimum volume vitrification approach [47–49]. A special tool consisting of a narrow, thin film strip (0.4 mm wide, 20 mm long 0.1 mm thick) attached to a hard plastic holder, has been developed. To protect the tool from mechanical damage during storage, a 3 cm long plastic tube cap can be attached to cover the film part. The tool and the solutions for vitrification and warming are now commercially produced and available at Kitazato Co., Fujinomiya, Japan. (It should be mentioned that the striking similarity of the Cryotop system to the Medicult Vitrification Freeze–Thaw Kit (sic!) http:// www.medicult.com/B659EFC2-7E60-430D-A8C02F34F7CAC9AC?frames=no&, p. 62–6 cannot be regarded as a pure coincidence.) After a two-step equilibration in a vitrification solution containing ethylene glycol, dimethylsulphoxide and sucrose, are loaded with a narrow glass capillary onto the top of the film strip in a volume of <0.1 ml. After loading, almost all the solution is removed to leave only a thin layer covering the oocytes or embryos, and the sample is quickly immersed into liquid nitrogen. Subsequently, the plastic cap is pulled over the film part of the Cryotop, and the sample is stored under liquid nitrogen. At warming, the protective cap is removed from the Cryotop while it is still submerged in liquid
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nitrogen and the polypropylene strip is immersed directly into a 37 8C medium containing sucrose to counterbalance the osmotic shock caused by the permeable cryoprotectants accumulated intracellularly. The embryos or oocytes are then sequentially incubated in diluent solutions before further in vitro culture or transfer. A variety of cryoprotectant solutions can be used with the Cryotop vitrification method and equilibration and dilution parameters can be varied according to the specific requirements of the species or the developmental stage. According to our experience, an equal proportion of dimethylsulphoxide and ethylene glycol was the most efficient combination, by using a two-step equilibration and supplementation with sucrose in the final concentration. Ethylene glycol as a highly permeable cryoprotectant with moderate toxicity is regarded as a standard component of most successful vitrification solutions [50]. Its combination with DMSO was successfully used first by Ishimori et al. [51–53], and DMSO was reported to increase the permeability of ethylene glycol [54]. According to our experience, the Cryotop vitrification method is easy to learn. Anybody with basic experience in embryology can perform it appropriately after a few hours’ training period. The method is simple and reliable, provides consistent results and variations between operators are minimal. Additionally, exactly the same solutions and equilibration/dilution parameters can be applied to human MII phase oocytes, zygotes and embryos of all phases of preimplantation development, making the application flexible and easy with the use of commercially available, ready-made media. The minimal volume approach of the Cryotop method increases the cooling and especially the warming rates (up to 40,000 8C/min) which may contribute to the improved and consistent survival, and both in vitro and in vivo developmental rates. 5. Results achieved with Cryotop vitrification in human As described in our previous publication [49], vitrification of 5881 human pronuclear stage embryos resulted in 100% morphological survival, 93% cleavage and 52% blastocyst rates; the latter percentage was significantly higher than that achieved by traditional freezing. Vitrification of 6328 human blastocysts resulted in 90% survival, and the 5659 transferred blastocysts resulted in 53% clinical pregnancies and 45% live births [what is this 45%? I assume 45% of the transfers. What % of vitrified embryos survived to
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term?]. These results are by far the highest published so far [see my comment above] for transfer of vitrified human embryos and provide strong evidence of the value of the Cryotop method. Another publication demonstrated a significant improvement achieved by Cryotop vitrification versus slow freezing for cryopreservation of human blastocysts [55]. The morphological survival rate after vitrification of 111 MII phase human oocytes was 95% [define endpoint for survival]. After intracytoplasmic sperm injection, 91% of them normally fertilized and 50% of them developed to the blastocyst stage in vitro. Twenty-nine embryo transfers with a mean number of 2.2 embryos per transfer on days 2 and 5 resulted in 12 initial pregnancies (pregnancy rate 41%) [48] and eventually 11 healthy babies were born from 10 deliveries. Similar survival and pregnancy rates, and the first vitrified oocyte baby in the USA obtained with the Cryotop method, were reported earlier [56]. The same Cryotop technique in Colombia has resulted in 57% pregnancy rates with an average of 4.6 embryos transferred to 23 patients [57]. Including all published and unpublished information, worldwide approximately 50 babies were born after oocyte vitrification with the Cryotop, more than after any other cryopreservation technology used for this purpose. These results allowed us to establish oocyte banks in Japan, providing a possibility for women suffering from cancer to have babies after chemo- and radio-therapy. Our oocyte banking is offered free of charge for cancer patients to cheer them up while enduring the harsh therapy. 6. Cryotop vitrification in animal biotechnology Detailed description of various results achieved with the Cryotop approach in animal biotechnology is beyond the scope of this manuscript; accordingly we only summarize here some of the recent successful applications. Cryotop vitrification has been successfully used for cryopreservation of immature and in vitro matured horse oocytes [58], bovine, ovine and buffalo oocytes [59–62], rabbit zygotes [63], in vitro produced and in vivo derived porcine embryos [64–66], porcine blastocysts produced by parthenogenetic activation or somatic cell nuclear transfer from delipidated in vitro matured oocytes [67], and bovine and buffalo embryos [68], as well as some exotic species such as minke whale oocytes [69]. 7. Biosafety issues Cryotop vitrification is an open method where a direct contact between liquid nitrogen and the solution
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containing oocytes and embryos is required. As discussed earlier, this approach may raise biosafety concerns and may not be acceptable in some countries. A similar but closed method was described recently [49] based on the CryoTip1 device. The CryoTip1 is a narrow capillary that can be sealed after loading with a minimum volume solution; accordingly, there is no direct contact between the biological sample and liquid nitrogen. This method has been successfully tested for cryopreservation of human embryos [49], but results in slightly reduced developmental rates compared to those with Cryotop vitrification when used for human oocytes. Further refinement of the technology may be needed to meet all requirements for safe and efficient cryopreservation. Another possibility is separation of the cooling and storage phases of Cryotop vitrification, i.e. performing the cooling aspect in a small volume of sterile or sterilized liquid nitrogen, then sealing the device into a sterile pre-cooled 1 ml straw for hermetical isolation at storage. A device to perform this manipulation has been developed for OPS vitrification by Minitub Germany, and with slight adjustment it can be used for Cryotop vitrification, as well.
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8. Conclusion Based on the highest number of vitrified-transferred human embryos and healthy babies obtained after cryopreservation of human oocytes, it can be concluded that Cryotop vitrification is an efficient method for both purposes. The method is based now on commercially available tools and ready to use solutions, it is easy to learn and apply, and consistent results are achieved. Although no disease transmission has been registered so far, slight modification of the technique including hermetical wrapping for storage may be necessary to exclude the possibility of cross-contamination and provide a safety level that meets legal requirements worldwide.
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Acknowledgement [18]
The author is very grateful to Dr. Noriko Kagawa for her helpful advise, excellent technical assistance of the experiments and statistical analysis.
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blastocysts after vitrification in a hemi-straw (HS) system. Fertil Steril 2000;74:S215–6. Vanderzwalmen P, Bertin G, Debauche Ch, Standaert V, Bollen N, van Roosendaal E, et al. Vitrification of human blastocysts with the hemi-straw carrier: application of assisted hatching after thawing. Hum Reprod 2003;18:1501–11. 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. Zech NH, Lejeune B, Zech H, Vanderzwalmen P. Nitrification of hatching and hatched human blastocysts: effect of an opening in the zona pellucida before vitrification. Reprod Biomed 2005;11: 355–61 [Online]. Kuwayama M, Kato O. All-round vitrification method for human oocytes and embryos. J Assist Reprod Genet 2000;17:477 [Abstract]. Kuwayama M, Vajta G, Kato O, Leibo S. Highly efficient vitrification method for cryopreservation of human oocytes. Reprod Biomed 2005;11:300–8 [Online]. Kuwayama M, Vajta G, Ieda S, Kato O. Vitrification of human embryos using the CryoTipTM method. Reprod Biomed 2005;11: 608–14 [Online]. Kasai M, Mukaida T. Cryopreservation of animal and human embryos by vitrification. Reprod Biomed 2004;9:164–70 [Online]. Ishimori H, Takahashi Y, Kanagawa H. Viability of vitrified mouse embryos using various cryoprotectant mixtures. Theriogenology 1992;37:481–7. Ishimori H, Takahashi Y, Kanagawa H. Factors affecting survival of mouse blastocysts vitrified by a mixture of ethylene glycol and dimethylsulfoxide. Theriogenology 1992;38:1175–85. Ishimori H, Saeki K, Inai M, Nagao Y, Itasaka J, Miki Y, et al. Vitrification of bovine embryos in a mixture of ethylene glycol and dimethyl sulfoxide. Theriogenology 1993;40:427–33. 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. Stehlik E, Stehlik J, Katajama P, Kuwayama M, Jambor V, Brohammer R, et al. Vitrification demonstrates significant improvement versus slow freezing of human blastocysts. Reprod Biomed 2005;11:53–7 [Online]. Katayama P, 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. Lucena E, Bernal DP, Lucena C, Rojas A, Moran A, Lucena A. Successful ongoing pregnancies after vitrification of oocytes. Fertil Steril 2006;85:108–11. Bogliolo L, Ariu F, Rosati I, Zedda MT, Pau S, Naitana S, et al. Vitrification of immature and in vitro matured horse oocytes. Reprod Fertil Dev 2006;18:149–50 [Abstract]. Chian R-C, Kuwayama M, Tan L, Kato O, Nagai T. High survival rate of bovine oocytes matured in vitro following vitrification. J Reprod Dev 2004;50:685–96. Kelly J, Kleemann D, Kuwayama M, Walker S. Effect of cysteinamine on survival of bovine and ovine oocytes vitrified using the minimum volume cooling (MVC) Cryotop method. Reprod Fertil Dev 2006;18:158 [Abstract]. Succu S, Leoni G, Berlinguer F, Mossa F, Galioto M, Naitana S. Vitrification devices affect developmental competence and biochemical properties of IVM ovine oocytes. Reprod Fertil Dev 2006;18:163 [Abstract].
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[62] Gasparrini B, Attanasio L, De Rosa A, Monaco E, Di Palo R, Campanile G. Cryopreservation of in vitro matured buffalo (Bubalus bubalis) oocytes by minimum volumes vitrification methods. Anim Reprod Sci; in press. [63] Hochi S, Terao T, Kamei M, Kato M, Hirabayashi M, Hirao M. Successful vitrification of pronuclear stage rabbit zygotes by minimum volume cooling procedure. Theriogenology 2004;61: 267–75. [64] Esaki R, Ueda H, Kurome M, Hirakawa K, Tomii R, Yoshioka H, et al. Cryopreservation of porcine embryos derived from in vitromatured oocytes. Biol Reprod 2004;71:432–7. [65] Ushijima H, Yoshioka H, Esaki R, Takahashi K, Kuwayama M, Nakane T, et al. Improved survival of vitrified in vivo-derived porcine embryos. J Reprod Dev 2004;50:481–6. [66] Hiruma K, Ueda H, Saito H, Tanaka C, Maeda N, Kurome M, et al. Successful pregnancies following transfer of vitrified
porcine embryos derived from in vitro matured oocytes. Reprod Fertil Dev 2006;18:157 [Abstract]. [67] Du Y, Kragh PM, Zhang X, Yang H, Vajta G, Bolund H. Successful vitrification of parthenogenetic porcine blastocysts produced from delipated in vitro-matured oocytes. Reprod Fertil Dev 2006;18:153 [Abstract]. [68] Laowtammathron C, Lorthongpanich C, Ketudat-Cairns M, Hochi S, Parnpai R. Factors affecting cryosurvival of nucleartransferred bovine and swamp buffalo blastocysts: effects of hatching stage, linoleic acid-albumin in IVC medium and Ficoll supplementation to vitrification solution. Theriogenology 2005; 64:1185–96. [69] Iwayama H, Hochi S, Kato M, Hirabayashi M, Kuwayama M, Ishikawa H, et al. Effect of cryodevice type and donors’ sexual maturity on vitrification of minke whale (Balaenoptera bonaerensis) oocytes at germinal vesicle stage. Zygote 2004;12:333–8.
Highly efficient vitrification for cryopreservation of human oocytes and embryos: The Cryotop method Masashige Kuwayama * Kato Ladies’ Clinic, 7-20-3 Nishishinjuku, Shinjuku, Tokyo 160-0023, Japan
Abstract Vitrification is frequently referred to as a novel technology of cryopreservation in embryology, although some young embryologists were born after its first successful application. Unfortunately, in spite of the accumulated evidence regarding its enormous potential value, most domestic animal and human laboratories use exclusively the traditional slow-rate freezing with its compromised efficiency and inconsistency. The purpose of this paper is to clarify terms and conditions, to summarize arguments supporting or disapproving the use of vitrification, and to outline its role among assisted reproductive technologies. To provide evidence for the potential significance of vitrification, achievements with the Cryotop technology, an advanced version of the ‘‘minimal volume approaches’’ is analyzed. This technology alone has resulted in more healthy babies after cryopreservation of blastocysts than any other vitrification technique, and more successful human oocyte vitrification resulting in normal births than any other cryopreservation method. The value of this method is also demonstrated by achievements in the field of domestic animal embryology. A modification of the technique using a hermetically sealed container for storage may help to eliminate potential dangers of disease transmission and open the way for widespread application for cryopreservation at all phases of oocyte and preimplantation embryo development in mammals. # 2006 Elsevier Inc. All rights reserved. Keywords: Cryodevice; Blastocyst; Pregnancy
1. Introduction One of the eternal ambitions of humankind is to overcome limits created by dimensions. After initial signs of successes to overcome horizontal barriers with railways almost 200 years ago, a real breakthrough only occurred in the 20th century, accompanied by expansion also into the third dimension, i.e. into the air and also into space. Today, the once demanding 42 km horizontal distance presents a challenge only for a marathon runner; practically all points of the globe can be reached in less than 48 h, while traveling at most
* Tel.: +81 3 3366 3777; fax: +81 3 5332 7373. E-mail address: [email protected]. 0093-691X/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2006.09.014
at 10 km above the surface of the earth. In parallel, the astonishing advancement of information technology has helped us to eliminate virtually all remaining distances by enabling communication between continents exactly as easily as between neighboring offices. Compared to that, almost nothing has happened with the fourth, maybe even more important dimension: time. We cannot travel forwards nor backwards, cannot slow down our limited available time, and cannot probably even use it better than previous generations. Among the very limited achievements in this debate we may list the cooling and later deep-freezing of biological materials, mainly food to slow down post mortem degradation; and hibernation to expand the lifespan of living tissues and organs under temporarily unfavorable conditions.
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However, the second half of the last century has resulted in considerable advances allowing the biological clock of live objects to be stopped for an unlimited (or practically unlimited) period, made possible by cooling them to extremely low temperatures, below 150 8C. This approach was first applied to simple structures including bacteria or single cells, and then advanced to some multicellular organisms and organs. Currently, major limitations are size and complexity: generally the smaller the sample and the simpler its structure, the better the chances for successful cryopreservation. Accordingly, embryology is in a rather privileged situation because of the relatively small size of the biological samples we work with, although even seemingly negligible variations in size may cause considerable differences in survival rates. Since the middle of the last century, in parallel with the development of in vitro reproductive technologies, remarkable successes have been achieved in cryopreservation of spermatozoa, embryos and oocytes, enabling us to stop and restart time both shortly before and after fertilization. Apart from the biological success, this possibility may have profound philosophic and moral consequences, especially when human material is concerned. Among other manipulations, stopping of time seem to be tolerable in the case of gametes, but it has been forbidden for embryos in many countries for ethical and legal reasons. As a curious consequence, to avoid restrictions with serious consequences, developmental stages have been re-defined in some of these countries by replacing the name ‘zygotes’ or ‘one-cell embryos’ with the term ‘pronuclear stage oocytes’, an expression that is rather paradoxical for experimental and domestic animal embryologists, but one that permits more legal freedom in assisted reproduction in humans. Another example is the euphemistic category of ‘‘pre-embryo’’ in human assisted reproduction. In spite of these controversial situations, the need for efficient cryopreservation of oocytes and embryos is enormous both for theoretical and practical reasons. However, in spite of the vast efforts invested, advances are rather slow. The main problems are the lack of consistency [1] as well as differences in survival and developmental rates after warming between species, developmental stages and quality. While the latter is easy to understand, we have only partial and not entirely supported information regarding the reasons for the lack of consistency in results. On the other hand, basic research in this field follows only empirically obtained advances: instead of indicating future directions for improvements, its main role is restricted to explaining retrospectively the achievements (a very recent example
for this situation is the history of the discovery of somatic cell nuclear transfer. In contrast to spermatozoa, where slow-rate freezing is almost exclusively used for cryopreservation and alternative methods are applied only at an experimental level [2], in cryopreservation of mammalian embryos and especially oocytes, vitrification has become a viable and promising alternative to traditional approaches. The convincing evidence that has been accumulated regarding the huge potential importance of vitrification has been reviewed recently [3–6]. Consequently, this paper will focus only on the latest achievements, and will discuss in detail the Cryotop vitrification method that has resulted in the highest number of babies born after vitrification of human embryos and after cryopreservation of human oocytes worldwide, and is now also successfully applied in various areas of animal biotechnology. 2. Vitrification In spite of the fact that mammalian embryo and oocyte vitrification was the subject of more than 500 publications in the past 10 years, and no comparison in any systems has proved its efficiency to be inferior to that of traditional freezing, vitrification is still regarded as experimental [3,4] and its practical use is restricted to a very few human and domestic animal embryology laboratories. Apart from the theoretical and practical problems, many misunderstandings also hamper its large-scale application. There are problems even with the definition. Vitrification is just a vitreous, transparent, ice-free solidification of water-based solutions at subzero temperatures [7]. Accordingly, even the first paper describing successful cryopreservation of mammalian spermatozoa used this term in a context that is not applicable today [4,8]. In some papers, vitrification is described as the result of extremely high cryoprotectant concentrations and extremely high cooling rates. However, vitrification does not necessarily require high cryoprotectant concentrations, because even pure water can be vitrified if the cooling rate is high enough ( 107 8C/s) [9] and, on the other hand, with concentrated cryoprotectant solutions, vitrification can also be achieved with a moderate or even slow cooling rate [10]. Other papers regard direct contact between liquid nitrogen and the embryo-oocyte containing solution as a prerequisite of vitrification, although it is just one possibility for increasing cooling rates that may permit decreasing the concentration of cryoprotectants and minimizing their potential toxic and osmotic effects. Many vitrification techniques (for example those earlier forms based on sealed 0.25 ml insemination straws or
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closed cryovials) have been described so far that do not require this direct contact, consequently their use does not invoke more liquid nitrogen-mediated contamination risk than traditional freezing. Accordingly, the most widely emphasized concerns, i.e. toxicity and danger of contamination, are not indispensable elements of the vitrification process, and with the development of future new techniques these negative effects should be minimized or entirely eliminated. Unfortunately, presently available vitrification methods still struggle with these problems. However, the actual harm and danger is most probably much lower than as emphasized endlessly by all critics. Regarding the toxic and osmotic effects, the latest cryoprotectant combinations and concentrations are already not too far from the range of those used for traditional freezing, at least the specific toxic effect of the components cannot be considerably higher. Moreover, in most vitrification methods, the time of exposure to the final cryoprotectant concentrations is very limited at temperatures where this toxic effect may still be significant (i.e. above 20 to 40 8C). On the other hand, as the result of the stepwise concentration increase in slow rate freezing, cells are definitely exposed to similar final cryoprotectant concentration as with vitrification—although at a much lower initial temperature that may minimize the toxic effect [4]. Eventually, survival and in vitro and in vivo developmental statistics provide convincing evidence that the cumulative toxic, osmotic and other harmful effects during the vitrification process are not exceeding but are rather below that caused by slow rate freezing. The danger of liquid nitrogen-mediated contamination is a realistic one, but it should not be overestimated. In spite of the enormous number of samples stored in liquid nitrogen, so far no disease transmission attributable to this mechanism has been documented in domestic animal or human reproductive biology [6]. Based on an earlier idea [11], many recent vitrification techniques separate the cooling and the storage phases; in this way the requirement for direct contact is limited to a relatively small amount of liquid nitrogen that can be filtered, UV sterilized or just purchased as sterile stock. On the other hand, the probability of disease transmission from a factory derived, separately stored liquid nitrogen source – considering that at 196 8C pathogens may survive but cannot proliferate – is most probably lower than to get bacterial or viral infection through the disposable surgical masks commonly used during oocyte retrieval, embryo handling and embryo transfer. It would of course be preferable to eliminate even this negligible danger, and there have been many
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such attempts in the past few years [12–17], but almost all of them resulted in somewhat compromised cooling and warming rates that may also jeopardize the results, especially when sensitive samples are vitrified. 3. Minimum volume vitrification methods Minimizing the volume of the vitrification solution containing oocytes or embryos not only offers the obvious benefit of increasing both cooling and warming rates, but also decreases the chance of ice crystal nucleation/formation in the small sample [9]. Curiously, this approach was and still is disregarded by many scientist, who just use traditional tools including cryovials and standard 0.25 ml insemination straws. Even these approaches may result in survival and development rates comparable to that of traditional freezing, but miss potential benefits of high-rate cooling including not only decreased cryoprotectant concentration but also reduced chilling injury that occurs between +15 8C (in human GV oocytes even +25 8C) [18] and 5 8C, which can be minimized by passing the embryos or oocytes rapidly through this temperature zone. According to the few systems where measurement of cooling rates is possible and was performed, the critical value required to avoid chilling injury and to benefit from the lower cryoprotectant concentration required should be around 20,000 8C/min [19], and from both points of view, the most critical period is the initial cooling [20]. Accordingly, seemingly negligible differences in practical performance of cooling, for example the thickness of the cold nitrogen vapor layer over the surface of the solution and the speed at which the sample passes through this layer, may have decisive consequences for the final outcome. By using liquid nitrogen slush for cooling with a commercially available device (Vitmaster) [21], the cooling rate can be increased between +20 and 10 8C, with considerable decrease of both chilling injury and the required cryoprotectant concentration. Minimum volume vitrification methods may also be helpful to avoid zona pellucida and embryo fracture damage. This type of injury occurs frequently when samples are cryopreserved in standard insemination straws and warmed rapidly afterwards. By using small samples and especially with vitrification in open systems, fracture damage rarely occurs, and it can be entirely eliminated with appropriate adjustment of warming parameters. There is no clear definition of what volume can be defined as ‘‘minimum’’ for vitrification, but according to the common use of this term it should be considerably
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less than 1 ml for direct dropping of samples into liquid nitrogen or the open pulled straw (OPS) method; approaches requiring approximately 4–5 or 1 ml solution are usually not considered to be minimum volume techniques [19,22]. The minimum drop size (MSD) method was developed by Arav [23] and Arav et al. [24,25]. Droplets of 0.1–0.5 ml were prepared on glass coverslip strips and immersed in liquid nitrogen or nitrogen slush. The method has allowed up to a 50% reduction in the concentration of cryoprotectants needed compared to traditional protocols, while preserving safe ice- and fracture-free vitrification of material [20]. A similar approach developed for cryopreservation of Drosophila melanogaster embryos [26] was successfully used later for mammalian oocytes and embryos [27], eventually resulting in human pregnancies and babies born after embryo cryopreservation [28,29]. In this method, the sample in a minimal amount of solution was placed on an electron microscope copper grid, then most of the solution was removed by placing the grid on a filter membrane. The procedure allowed separation of the cooling and storage phase, the latter being done in cryovials that were previously filled with liquid nitrogen [30]. Unfortunately the storage component still seems to be a fragile part of this approach, because all cryovial producers strongly advise not to fill the vials with liquid nitrogen, and not to submerge vials during storage but keep them in the liquid nitrogen vapor. These measures are intended to avoid explosion of the vials due to the extreme pressure changes during evaporation of liquid nitrogen that may enter the vials during the cooling and warming processes. Accordingly, apart from the difficulties related to handling of grids under liquid nitrogen, potential explosion of the vials may hamper widespread application of this technology. Another possibility is to use a solution film formed between files of a nylon loop for holding the sample. This method, the Cryoloop, was first applied for flash freezing of protein solutions for analysis in crystallography [31,32] and was used later also for cryopreservation of mammalian embryos and oocytes [33–36] resulting in births from vitrified human and monkey blastocysts [37– 39]. Due to the absence of solid support and the extremely small volume, the cooling rate upon immersion in liquid nitrogen may be as high as 700,000 8C/min [40] thus also permitting cooling in liquid nitrogen vapor. However, in spite of the unquestionable benefits of this system from the point of potential disease transmission, one may have concerns regarding the safety of storage, as this very sensitive and fragile system may increase the risk of accidental warming over the safe temperature zone,
especially if storage of the container is also performed in the vapor of liquid nitrogen [41]. In parallel with these special approaches, there have been repeated attempts at practical application of the original idea to place a very small drop of solution onto a solid surface and immersing both into liquid nitrogen. Hamawaki et al. [42] placed the embryos in a very small volume onto the inner wall of a 0.25 ml standard insemination straw and immersed the straw into liquid nitrogen after sealing. This manipulation eliminated disease transmission problems but decreased the rate of cooling, and it could be regarded as technically difficult. In the hemi-straw system [43,44], the end of a standard 0.25 ml insemination straw was cut with a scalpel and an approximately 0.3 ml droplet of cryoprotectant solution was pipetted onto the inner face of the straw. The straw was then immersed vertically into liquid nitrogen and eventually inserted and closed into a 0.5 ml straw for storage. This system has been found equally or more efficient than the Cryoloop method for cryopreservation of human zygotes and early stage embryos [45] and resulted in pregnancies after blastocyst vitrification [44,46]. 4. The Cryotop method The Cryotop method is probably the latest minimum volume vitrification approach [47–49]. A special tool consisting of a narrow, thin film strip (0.4 mm wide, 20 mm long 0.1 mm thick) attached to a hard plastic holder, has been developed. To protect the tool from mechanical damage during storage, a 3 cm long plastic tube cap can be attached to cover the film part. The tool and the solutions for vitrification and warming are now commercially produced and available at Kitazato Co., Fujinomiya, Japan. (It should be mentioned that the striking similarity of the Cryotop system to the Medicult Vitrification Freeze–Thaw Kit (sic!) http:// www.medicult.com/B659EFC2-7E60-430D-A8C02F34F7CAC9AC?frames=no&, p. 62–6 cannot be regarded as a pure coincidence.) After a two-step equilibration in a vitrification solution containing ethylene glycol, dimethylsulphoxide and sucrose, are loaded with a narrow glass capillary onto the top of the film strip in a volume of <0.1 ml. After loading, almost all the solution is removed to leave only a thin layer covering the oocytes or embryos, and the sample is quickly immersed into liquid nitrogen. Subsequently, the plastic cap is pulled over the film part of the Cryotop, and the sample is stored under liquid nitrogen. At warming, the protective cap is removed from the Cryotop while it is still submerged in liquid
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nitrogen and the polypropylene strip is immersed directly into a 37 8C medium containing sucrose to counterbalance the osmotic shock caused by the permeable cryoprotectants accumulated intracellularly. The embryos or oocytes are then sequentially incubated in diluent solutions before further in vitro culture or transfer. A variety of cryoprotectant solutions can be used with the Cryotop vitrification method and equilibration and dilution parameters can be varied according to the specific requirements of the species or the developmental stage. According to our experience, an equal proportion of dimethylsulphoxide and ethylene glycol was the most efficient combination, by using a two-step equilibration and supplementation with sucrose in the final concentration. Ethylene glycol as a highly permeable cryoprotectant with moderate toxicity is regarded as a standard component of most successful vitrification solutions [50]. Its combination with DMSO was successfully used first by Ishimori et al. [51–53], and DMSO was reported to increase the permeability of ethylene glycol [54]. According to our experience, the Cryotop vitrification method is easy to learn. Anybody with basic experience in embryology can perform it appropriately after a few hours’ training period. The method is simple and reliable, provides consistent results and variations between operators are minimal. Additionally, exactly the same solutions and equilibration/dilution parameters can be applied to human MII phase oocytes, zygotes and embryos of all phases of preimplantation development, making the application flexible and easy with the use of commercially available, ready-made media. The minimal volume approach of the Cryotop method increases the cooling and especially the warming rates (up to 40,000 8C/min) which may contribute to the improved and consistent survival, and both in vitro and in vivo developmental rates. 5. Results achieved with Cryotop vitrification in human As described in our previous publication [49], vitrification of 5881 human pronuclear stage embryos resulted in 100% morphological survival, 93% cleavage and 52% blastocyst rates; the latter percentage was significantly higher than that achieved by traditional freezing. Vitrification of 6328 human blastocysts resulted in 90% survival, and the 5659 transferred blastocysts resulted in 53% clinical pregnancies and 45% live births [what is this 45%? I assume 45% of the transfers. What % of vitrified embryos survived to
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term?]. These results are by far the highest published so far [see my comment above] for transfer of vitrified human embryos and provide strong evidence of the value of the Cryotop method. Another publication demonstrated a significant improvement achieved by Cryotop vitrification versus slow freezing for cryopreservation of human blastocysts [55]. The morphological survival rate after vitrification of 111 MII phase human oocytes was 95% [define endpoint for survival]. After intracytoplasmic sperm injection, 91% of them normally fertilized and 50% of them developed to the blastocyst stage in vitro. Twenty-nine embryo transfers with a mean number of 2.2 embryos per transfer on days 2 and 5 resulted in 12 initial pregnancies (pregnancy rate 41%) [48] and eventually 11 healthy babies were born from 10 deliveries. Similar survival and pregnancy rates, and the first vitrified oocyte baby in the USA obtained with the Cryotop method, were reported earlier [56]. The same Cryotop technique in Colombia has resulted in 57% pregnancy rates with an average of 4.6 embryos transferred to 23 patients [57]. Including all published and unpublished information, worldwide approximately 50 babies were born after oocyte vitrification with the Cryotop, more than after any other cryopreservation technology used for this purpose. These results allowed us to establish oocyte banks in Japan, providing a possibility for women suffering from cancer to have babies after chemo- and radio-therapy. Our oocyte banking is offered free of charge for cancer patients to cheer them up while enduring the harsh therapy. 6. Cryotop vitrification in animal biotechnology Detailed description of various results achieved with the Cryotop approach in animal biotechnology is beyond the scope of this manuscript; accordingly we only summarize here some of the recent successful applications. Cryotop vitrification has been successfully used for cryopreservation of immature and in vitro matured horse oocytes [58], bovine, ovine and buffalo oocytes [59–62], rabbit zygotes [63], in vitro produced and in vivo derived porcine embryos [64–66], porcine blastocysts produced by parthenogenetic activation or somatic cell nuclear transfer from delipidated in vitro matured oocytes [67], and bovine and buffalo embryos [68], as well as some exotic species such as minke whale oocytes [69]. 7. Biosafety issues Cryotop vitrification is an open method where a direct contact between liquid nitrogen and the solution
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containing oocytes and embryos is required. As discussed earlier, this approach may raise biosafety concerns and may not be acceptable in some countries. A similar but closed method was described recently [49] based on the CryoTip1 device. The CryoTip1 is a narrow capillary that can be sealed after loading with a minimum volume solution; accordingly, there is no direct contact between the biological sample and liquid nitrogen. This method has been successfully tested for cryopreservation of human embryos [49], but results in slightly reduced developmental rates compared to those with Cryotop vitrification when used for human oocytes. Further refinement of the technology may be needed to meet all requirements for safe and efficient cryopreservation. Another possibility is separation of the cooling and storage phases of Cryotop vitrification, i.e. performing the cooling aspect in a small volume of sterile or sterilized liquid nitrogen, then sealing the device into a sterile pre-cooled 1 ml straw for hermetical isolation at storage. A device to perform this manipulation has been developed for OPS vitrification by Minitub Germany, and with slight adjustment it can be used for Cryotop vitrification, as well.
[3] [4] [5] [6]
[7] [8]
[9] [10]
[11]
[12]
[13]
8. Conclusion Based on the highest number of vitrified-transferred human embryos and healthy babies obtained after cryopreservation of human oocytes, it can be concluded that Cryotop vitrification is an efficient method for both purposes. The method is based now on commercially available tools and ready to use solutions, it is easy to learn and apply, and consistent results are achieved. Although no disease transmission has been registered so far, slight modification of the technique including hermetical wrapping for storage may be necessary to exclude the possibility of cross-contamination and provide a safety level that meets legal requirements worldwide.
[14]
[15]
[16]
[17]
Acknowledgement [18]
The author is very grateful to Dr. Noriko Kagawa for her helpful advise, excellent technical assistance of the experiments and statistical analysis.
[19]
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