Theriogenology 81 (2014) 96102

Contents lists available at ScienceDirect

Theriogenology
journal homepage: www.theriojournal.com

40th Anniversary Special Issue

Cryopreservation of oocytes and embryos

A. Arav*
FertileSafe, Shlomzion Hamalca, Tel Aviv, Israel 62266

a r t i c l e i n f o
Article history: Received 15 July 2013 Received in revised form 11 September 2013 Accepted 11 September 2013 Keywords: Freezing Vitrication Drying Oocytes Embryos Cryopreservation

a b s t r a c t
Two hundred years have passed since the rst description of supercooled water by GeyLussac to the recently high survival rates of embryo and oocytes after vitrication. This review discusses important milestones that have made vitrication the method of choice for oocytes and embryos cryopreservation. We will go through the rst cells ever to survive low temperature exposure in the beginning of the last century, the nding of glycerol in the late 1940s and the rst mouse and bovine embryos freezing in the 1970s. During the 1980s, embryo vitrication began and the time since is a tribute to the development of oocytes vitrication. Standardization and an automatic vitrication procedure are currently under development. The next evolutionary step in oocyte and embryo cryopreservation will be preserving them in the dry state at room temperature, allowing home storage for future use a reality. 2014 Elsevier Inc. All rights reserved.

1. Two hundred years of vitrication Going back to the book Life and Death at Low Temperatures by Basile J. Luyet and Marie Pierre Gehenio, published at 1940 [1], I found out that the small volume vitrication (the minimum drop size technique) that is generally attributed to my work was already thought at the beginning of the 19th century. It was done by the great French chemist and physicist Joseph Louis Gay-Lussac. He is known mostly for his two laws of gases and for his work on alcoholwater mixtures. Gay Lussac found that water can be cooled to 12  C without freezing, nding with this discovery the basis of vitrication [2]. In 1804, Gay-Lussac was ascended in a hot air balloon (Fig. 1) and noticed that the drops in the clouds are not frozen despite the subzero temperatures. He published later the discovery of the effect of small volume of water droplets on supercooling. The size of water drops in clouds is around 8 to 10 mm, which maintains them at a liquid state at a subfreezing temperature of 5  C. Luyet described it in his book: Some of the oldest investigations on subcooling were made by Gay-Lussac
* Corresponding author. Tel.: 972 523 638022. E-mail address: arav@agri.huji.ac.il. 0093-691X/$ see front matter 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2013.09.011

(1836) who observed that water can be subcooled to 12  C when it is enclosed in small tubes [1,3]. Already in 1858, Johann Rudolf Albert Mousson sprayed droplets of water less than 0.5 mm in diameter on a dry surface and observed that the smaller the drops the longer they stayed subcooled [4]. Not only was volume important to achieve supercooling, among other factors that might have an inuence in inducing crystallization, as mentioned by Luyet, are cooling velocity and concentration of the supercooled or supersaturated solutions. Luyet wrote, To avoid freezing, the temperature should drop at a rate of some hundred degrees per second, within the objects themselves, and The only method of vitrifying a substance is to take it in the liquid or gas state and cool it rapidly so as to skip over the zone of crystallization temperatures in less time than is necessary for the material to freeze. Luyet further wrote, It is evident that when crystals grow faster one must traverse the crystallization zone more rapidly if one wants to avoid crystallization [1].

2. Basic principles The velocity of cooling depends on the thermal mass of the sample and on its surface area. To achieve rapid cooling,

A. Arav / Theriogenology 81 (2014) 96102

97

Later attempts at vitrifying pure water have been made by a few investigators; Hawkes [8] published an experiment in which a drop of solid amorphous water was obtained, by chance, during rapid cooling. Burton and Oliver [9] obtained from steam some solid water in which x-ray analysis did not reveal any crystalline structure. As we can see, these achievements were mainly owing to the samples small volume and not the velocity of cooling. A review on supercooling of water can be found also in Cryobiology by Meryman published in1966 [10]. 3. The rst cells survival after vitrication The most important year for cryobiology was 1938; Basile J. Luyet and Eugene L. Hodapp [11] published the rst successful vitrication of sperm. Luyet began his research with colloids (gelatin, milk, or agar) and found that their water content determines the possibility or impossibility of vitrication. In general, with 50% gelatin solutions, they had vitried layers of 0.3 mm thick (by the method of immersion in liquid nitrogen [LN]), whereas with solutions containing 90% water, they could vitrify only smears of few microns thick [3]. They were the rst to demonstrate successful cryopreservation of frog sperm by vitrication using 2 mol/L sucrose and small drops. 4. The beginning of slow freezing In 1949, Christopher Polge, Audrey Smith, and Alan Parkes [12], when trying to duplicate Luyets results, discovered by mistake the cryoprotective property of glycerol and so opened the eld of slow freezing. Currently, there are two methods for gametes cryopreservation: slow freezing and vitrication. Slow freezing has the advantage of using low concentrations of CPs, which are associated with chemical toxicity and osmotic shock. Vitrication is a rapid method, which reduces chilling sensitivity and crystallization damage caused to cells. Sherman and Lin [13] showed that mouse oocytes need 8 to 10 minutes for equilibration in a freezing solution containing 5% glycerol at 37  C. In addition, he demonstrated that mouse oocytes will survive supercooling to 20  C after slow cooling at 0.6  C/min, however, oocytes that were cooled faster or to lower temperatures were damaged owing to intracellular crystallization. In the early 1970s, two groups were competing on achieving the rst success of slow freezing of embryos. One group included Whittingham, Leibo, and Mazur, and the other Wilmut and Polge. Whittingham had partially succeeded in freezing embryos to 79  C for 30 minutes using polyvinylpyrrolidone; however, these experiment could not be duplicated [1416]. Both groups published in 1972 the rst survival of mouse embryos after slow freezing [15,16] and live offspring [15]. The technique included cooling at a slow rate in the presence of 1 mol/L DMSO, which most likely was the ingredient that enabled this. In 1976, sheep embryos were slow frozen by Willadsen using 1.5 mol/L DMSO and a cooling rate of 0.3  C/min. [17]. However, the rst farm animal to be born after transplantation of frozen and thawed embryos was a calf. This was published by Wilmut and Rowson at 1973 [18]. Since

Fig. 1. .Joseph Louis Gay Lussac (17781850) and portrait and an illustration of from 1804 of Gay Lussac and Jean Baptiste Biot in the hot air balloon at 4000 m.

we should use material with the lowest heat mass and maximum surface to volume ratio. Indeed, Gregory M. Fahy and William F. Rall published in 2007 the critical cooling rates needed to vitrify aqueous solutions that contain different concentrations of cryoprotectants (CPs) [5]. It was extrapolated that for pure water over 100 106  C/min are needed to form a glass state without crystallization. Also, it is interesting to note that for 15% (v/v) of most CPs almost 1 106  C/min is needed, which is difcult to achieve. We have shown that 15% (v/v) of CPs can vitrify at relative slow cooling rates when the volume of the drop is 0.07 mL [6]. Therefore, it is much more feasible to achieve vitrication by lowering the volume than by increasing the cooling rate. James H. Walton and Roy C. Judd measured the velocity of ice crystal growth and found that it is in the range of 65 mm/s [7]. This means that if we want to avoid crystallization in a drop placed on a cold metal surface and that has a diameter of 0.1 mm, we need a velocity of 1/6500 mm/s, which is 0.0001 second. If we cool from room temperature to 180  C, this means we need to reduce 200  C at a rate of 0.1 ms or at 78 106  C/min. This is actually the cooling rate that was estimated by Balds and by Bruggeller [1,5]. However, because this cooling rate is impossible to achieve, the ability to reach vitrication of pure water in a small drop can be achieved in relatively slow cooling rates, which indicate that the small volume has an independent effect on the probability of vitrication.

98

A. Arav / Theriogenology 81 (2014) 96102

then, dozens of species have been successfully cryopreserved by slow freezing (for a review, see [19]).

5. The comeback of vitrication For many years, slow freezing and not vitrication was the method of choice for embryo cryopreservation. This was because vitrication was not achieved easily owing to the need of high CPs concentrations and relatively high volume samples. In 1985, the rst successful vitrication of mouse embryos using a relatively large volume sample was done [20]. Rall and Fahy vitried mouse embryos with mixture of DMSO, acetamide, and polyethylene glycol and in a relatively large volume inside a 0.25-mL straw plunged into LN. At that time, I was a veterinary student at the University of Bologna, Italy, and I met Bill Rall, who told me about the exciting work he did on mouse embryos. Two years later, I started to work on cryomicoscopy of oocytes and embryos. As in our laboratory, we used to prepare oocytes for histology evaluation by xing them with a small drop over a microscopic slide. I had the idea of using the same technique for vitrication in a small drop, which I later named as the minimum drop size [6,2124]. As noted, the probability of vitrication increases as the volume of the sample decreases. Pure water is vitried only in very small droplets obtain from aerosols. Vitrication of thin layers (<1 mm) of viruses in water suspension was achieved in rapid cooling for the purposes of electron microscopy [25]. The method we developed in 1989 was based on this concept and called the minimum drop size [21]. The volume we used for the vitrication was in the range of 0.07 mL (70 nL) and the concentration of the vitrication solution (VS) was about 50% lower than of the VS used for large-volume vitrication [22]. It was called minimum drop size because this was the minimal size that maintained oocytes or embryos without damage owing to desiccation. Vitrication of embryos, on the other hand, although initially attempted in the late 1980s, has not been applied clinically until recently. Vitrication is currently producing very satisfactory outcomes by means of methodologies that use a minimal volume [26,27]. Three important factors should be considered. 1. Cooling rate and warming rate: A high cooling rate is achieved with LN or LN slush and a warm water bath for warming. When using LN, the sample is plunged into LN, resulting in cooling rates of hundreds to tens of thousands degrees Celsius per minute, depending on the container, volume, thermal conductivity, solution composition, and so on [28]. To achieve LN slush, the LN needs to be cooled close to its freezing point (210  C), for example, by applying a negative pressure above LN [29]. When LN slush is formed, the cooling rate is dramatically increased. The cooling rate is especially enhanced in the rst stage of cooling when cooling down from room temperature to 0  C. The cooling rate is enhanced two to six times more than plunging to LN (196  C) with 0.25-mL straws or any other device, such as open-pulled straws or electron

microscope grids [23]. It was shown for oocytes and embryos that increasing the cooling rate improves survival rates by up to 37% [19]. Recently, it was found that warming rate is an important variable for successful vitrication of mouse oocytes [30]. 2. Viscosity of the medium in which the embryos are suspended or the glass transition coefeciency of the solution at low temperatures is dened by the concentration and behavior of various CPs and other additives during vitrication. The higher the concentration of CPs, the higher the glass transition temperature (Tg), thus lowering the chance of ice nucleation and crystallization. Different CPs and other additives have different toxicity, penetration rate, and Tg. Viscosity of the medium in which the embryos are suspended is dened by the concentration and behavior of various CPs and other additives during vitrication. The combination of different CPs is often used to increase viscosity, increase Tg, and reduce the level of toxicity. In the cattle industry, to avoid handling of the post warmed embryos and allow direct transfer, ethylene glycol (which was found by Voelkel and Hu [31]) is often used as the permeating CP owing to its high penetration rate [32]. 3. Volume: The smaller the volume, the higher the probability of vitrication [13,21,23,24]. Smaller volumes allow better heat transfer, thus facilitating greater cooling rates. Furthermore, small volume has an independent effect on the probability of nucleation as was discovered 200 years ago by Guy-Lassac. Many techniques have been developed to reduce sample volume with an explosion of methods appearing in the literature during the last decade.

These techniques can generally be divided into two categories: surface techniques and tubing techniques [19]. The surface techniques include electron microscope grid [33], minimum drop size technique [21], Cryotop [34,35], Cryoloop [36,37], Hemi-straw [38], solid surface [39], nylon mesh [40], Cryoleaf [41], direct cover vitrication [42], ber plug [43], vitrication spatula [44], Cryo-E [45], plastic blade [46], and Vitri-Inga [47]. To the tubing techniques belongs the plastic straw [20], open-pulled straw [48,49], closed pulled straw [50], exipet-denuding pipette [51], superne open-pulled straw [52], CryoTip [53], pipette tip [54], high-security vitrication device [55], sealed pulled straw [56], Cryopette [57], Rapid-i [58], and JY Straw (RC Chian, personal communication). Each of these two groups has its specic advantages. In the surface methods, the size of the drop (0.1 mL) can be controlled, a high cooling rate is achieved because these systems are open, and high warming rates are achieved by direct exposure to the warming solution. The tubing systems have the advantage of achieving high cooling rates in closed systems, thus making them safer and easier to handle. Decreasing the vitried volume and increasing the cooling rate allow a moderate decrease in CP concentration so as to minimize its toxic and osmotic hazardous effects [22,56]. Combining these three factors can result in the following general equation for the probability of vitrication:

A. Arav / Theriogenology 81 (2014) 96102

99

Probability of vitrification

cooling and warming rate viscocity volume

6. Oocyte vitrication Oocytes are very different from sperm or embryos with respect to cryopreservation. The volume of the mammalian oocyte is in the range of three to four orders of magnitude larger than that of the spermatozoa, thus substantially decreasing the surface-to-volume ratio. However, this is not the reason why the oocytes are sensitive to low temperatures and slow freezing. Mature oocytes are very sensitive to slow freezing; however, after fertilization the volume of the oocytes remains the same but their sensitivity is reduced to minimum and in fact this is the best stage for freezing human oocytes [59]. The reason for many oocytes susceptibility to low temperatures is owing to their chilling sensitivity which occurs at different cellular levels: the zona pellucida, plasma membrane, meiotic spindles, cytoskeleton, and so on. The plasma membrane of oocytes at the metaphase (M) II stage has a low permeability coefcient, thus making the movement of CPs and water slower [60,61]. In addition, the freezethaw process causes premature cortical granule exocytosis, leading to zona pellucida hardening and making sperm penetration and fertilization impossible [62,63], a process that can be overcome by the use of intracytoplasmic sperm injection or subzonal sperm insertion. Oocytes also have high cytoplasmic lipid content, which increases chilling sensitivity [60]. They have less submembranous actin microtubules [64], making their membrane less robust. Cryopreservation can cause cytoskeleton disorganization, and chromosome and DNA abnormalities [65]. The meiotic spindle, which has been formed by the MII stage, is very sensitive to chilling and may be compromised as well [66]. It does, however, tend to recover to some extent after thawing or warming and IVC, a recovery that is faster after vitrication than after slow freezing [66]. Oocytes are also more susceptible to damaging effects of reactive oxygen species [67]. Many of these parameters change after fertilization, making embryos less sensitive to chilling and easier to cryopreserve [64,67,68].

Vitrication requires the presence of high concentrations of CPs. It is therefore important to minimize the damage caused to cells by the osmotic stress or chemical toxicity. No ideal CP that meets the requirements of all different species and developmental embryonic stages has been found; vitrication studies should therefore be preceded by osmotic and cytotoxic studies. The presence of CP in the VS decreases the probability of intracellular crystallization which is considered to cause most damage when very rapid cooling takes place, but the high concentration of the CP required is toxic and causes osmotic injury to the oocytes even without cooling. Different methods have been used to reduce this solution effect: (i) short time of exposure to CPs [6769], (ii) use of low toxicity CPs [31,70] or mixtures of them [71], (iii) addition of nonpermeating CPs [72], (iv) reduction the CP concentration [70], and (v) exposure at low temperatures [70]. Of these methods, the use of nonpermeating CPs is very useful either because the shrinkage of the oocyte and consequently the amount of water inside the cell that may crystallize during rapid cooling and warming is lower [70] or because of the reduction of the amount of the CP that penetrates the cell thus reducing the possible toxic effect [73]. In addition, the carbohydrates used as a nonpermeating CPs have a stabilizing effect on membranes [74]. In the study reported by us [75], trehalose was less harmful than sucrose. Determination of the Boyle Vant Hoff relationship for both sucrose and trehalose produced the same regression line, so it is possible that this benecial effect could be a consequence of its interaction with the membrane polar lipid groups [74]. Only 10 minutes of exposure is required for equilibration in propylene glycol and DMSO solutions or mixtures of them. The membrane is very permeable to both of them. The results of the vitrication provide evidence that propylene glycol can be used successfully. Indeed, the IVF rate of the oocytes vitried in a solution containing 40% (w/v) propylene glycol was 37% and is not different from the results obtained using a slow freezing protocol [76]. The viability

Fig. 2. Dry oocytes on a Cryotop carrier after vitrication and drying and on the right after rehydration and staining with live/dead uorescent stains (SYTO/PI).

100

A. Arav / Theriogenology 81 (2014) 96102

of vitried mouse embryos was successfully increased by reducing the concentration of the CP [56]. However, concentrated solutions of permeating CP are required for successful cryopreservation of oocytes when rapid cooling and warming rates are used. In earlier reports on immature pig oocytes, we showed that when lower concentrations of CPs are used, despite apparent vitrication, membrane destruction was unavoidable [77]. In 1990, Kasai. et al. [78] were the rst to describe the use of ethylene glycol for mouse embryo vitrication. Today, the most common solutions are based on a mixture of DMSO and ethylene glycol [53]. 7. Small volume is the solution for most problems occur during vitrication There are three major problems associated with vitrication: (1) crystallization (during cooling), (2) devitrication (crystallization during storage or during warming), and (3) fractures of the glassy solution, which can cause devitrication owing to release of energy by the fracture. Surprisingly, at 1 mL, fractures appeared only when the concentration of the VS is high (100% VS 38% ethylene glycol, 0.5 mol/L Trehalose, and 4% bovine serum albumin in TCM medium), but not at lower volumes [24]. This means that the probability of fractures increases with the increasing of Tg or the viscosity of VS. At the low concentration of VS (50% VS), fractures were observed only at very high cooling rate. We suggest here a simple explanation of this phenomenon, based on the following equations:

probability of fracturing also increases. Finally, the results of vitrication of bovine oocytes at the MII or germinal vesicle stage, with a concentration of 75% VS, have been reported [79]. We achieved 72% and 38% cleavage and blastocyst rates formation, respectively, for the vitried MII oocytes and 27% and 14% cleavage and blastocyst rates formation, respectively, for oocytes vitried at the germinal vesicle stage. We conclude that the new vitrication procedure, which features small volumes, direct contact with supercooled LN, and low concentrations of VS, reduces chilling injury and provides a high probability of vitrication in the absence of glass fractures. 8. Drying bovine oocytes after vitrication: A technological breakthrough Storage of cryopreserved oocytes in LN is very demanding in terms of maintenance, storage space, equipment, and costs. We, therefore, sought an alternative method for gamete preservation: vitrication followed by drying. Storage is done in a dry state. We perform a comparative study between freeze-drying and vitrication drying. Three experiments in parallel compared various cooling methods on the recovery and survival after freeze or vitrication drying of in vitro-matured MII bovine oocytes (n 68). Ten oocytes were cryopreserved with slow freezing (using the MTG-1314 device, IMT Israel) at a cooling rate of 4  C/min (group A); 24 oocytes with rapid freezing (using MTG 516 device IMT, Israel), at a cooling rate of 150  C/min (group B); and 34 with vitrication using minimum drop size in IMT-4 solution (trehalose based solution) at a cooling rate of greater than 20,000  C/min.

Probability of fracturing CR and WR m V

Because the probability of vitrification is CR and WR m 1=V

1. Increasing the cooling rate (CR) increases the probability of vitrication; however, it also increases the probability of fractures. 2. Increasing the viscosity (m) increases the probability of vitrication because Tg increases [28]; therefore, it increases the probability of fractions. 3. The only parameter that increases the probability of vitrication and at the same time decrease the probability of fractures is reducing the volume (V) to the value of the minimum drop size.

The reason for the increasing probability of fractures in high concentrations of VS is thought to be related to the glass transition temperature (Tg). We know that fractures can form only at temperatures below that at which the liquid turns into glass (Tg) and above the LN temperature (196  C). We also know that a solution with a higher CPs concentration have a higher Tg. Therefore, if the temperature gradient increases, as in the case of higher Tg, then the

The lyophilization process was carried out with the VirTis wizard for 24 hours (shelf temperature was set to 55  C and vacuum was 10 mTorr). The rehydration process took place at room temperature using equilibrated TCM199 supplemented with 0.5 mol/L trehalose. Oocyte survival was assessed with live/dead uorescent stains (SYTO/PI). For group A, 70% of the oocytes were recovered after rehydration but only one in seven was stained as viable (14%). For group B, 71% were recovered and 10 of 17 were stained as viable (59%). For group C, 88% were recovered and 23 of the 30 stained as viable (77%; P < .05; Fig. 2). In conclusion, lyophilization of oocytes is a groundbreaking innovation for gamete bio-banking; vitrication is conrmed as an essential method not only for preservation in LN, but also in the dry state. References
[1] Luyet BJ, Gehenio PM. Life and death at low temperatures. Normandy, MO: Biodynamica; 1940.

A. Arav / Theriogenology 81 (2014) 96102 [2] von Humboldt A, Gay-Lussac JL. Observations sur lintensit et linclinaison des forces magntiques, faites en France, en Suisse, en Italie et en Allemagne. Mmoires de physique et de chimie de la Socit dArcueil; 1807. Tome: France, 1. p. 122. [3] Luyet BJ. The vitrication of organic colloids and of protoplasm. Biodynamics 1937;1:114. [4] Mousson A. Einige Tatsachen betreffend das Schmelzen und Gefrieren des W a s s e r s. An Pfyaft 1858;105:161174. [5] Fahy GM, Rall WF. Vitrication: an overview. In: Liebermann J, Tucker MJ, editors. Vitrication in assisted reproduction: a users manual and troubleshooting guide. Zug, Switzerland: Informa Healthcare; 2007. [6] Arav A. Vitrication of oocytes and embryos. DVM Thesis. Bologna: Bologna University; 1989. [7] Walton Jr JH, Judd RC. The velocity of crystallization of under cooled water. J Phys Chem 1914;18:7228. [8] Hawkes L. Super-cooled water. Nature 1929;123:244. [9] Burton EF, Oliver WF. The crystal structure of ice at low temperatures. Proc Roy Soc (London) A 1935;153:16672. [10] Meryman HT. Crystallization of ice. In: Maryman HT, editor. Cryobiology. London: Academic Press; 1966. p. 1125. [11] Luyet BJ, Hodapp R. Revival of frog spermatozoa vitried in liquid air. Proc Soc Exp Biol N Y 1938;39:4334. [12] Polge C, Smith AU, Parkes AS. Revival of spermatozoa after vitrication and dehydration at low temperatures. Nature 1949;164:666. [13] Sherman JK, L. Teh Ping. Survival of unfertilized mouse eggs during freezing and thawing. Exp Biol Med (Maywood) 1958;98:902.. [14] Whittingham DG. Survival of mouse embryos after freezing and thawing. Nature 1971;233:1256. [15] Whittingham DG, Leibo SP, Mazur P. Survival of mouse embryos frozen to 196 and 269  C. Science 1972;178:4114. [16] Wilmut I. Effect of cooling rate, warming rate, cryoprotective agent and stage of development on survival of mouse embryos during cooling and thawing. Life Sci 1972;11:10719. [17] Willadsen SM, Polge C, Rowson LEA, Moor RM. Deep freezing of sheep embryos. J Reprod Fertil 1976;46:1514. [18] Wilmut I, Rowson LEA. Experiments on the low-temperature preservation of cow embryos. Vet Rec 1973;92:68690. [19] Saragusty J, Arav A. Current progress in oocyte and embryo cryopreservation by slow freezing and vitrication. Reproduction 2011; 141:119. [20] Rall WF, Fahy GM. Ice-free cryopreservation of mouse embryos at 196  C by vitrication. Nature 1985;313:5735. [21] Arav A, Gianaroli L, Bafaro G, Diotalevi L. A new vitrication technique for 8-cell stage mouse embryos. Presented at: IVF Meeting. Barcelona, 1987. Abs 373, p. 118. [22] Arav A. Vitrication of oocyte and embryos. In: Lauria A, Gandol F, editors. New trends in embryo transfer. Cambridge, UK: Portland Press; 1992. p. 25564. [23] Arav A, Zeron Y. Vitrication of bovine oocytes using modied minimum drop size technique (MDS) is affected by the composition and the concentration of the vitrication solution and by the cooling conditions. Theriogenology 1997;47:341. [24] Arav A, Yavin S, Zeron Y, Natan Y, Dekel I, Gacitua H. New trend in gametes cryopreservation. Mol Cell Endocrinol 2002; 187:7781. [25] Adrian M, Dubochet J, Lepault J, McDowall AW. Cryo-electron microscopy of viruses. Nature 1984;308:326. [26] Kuwayama M, Vajta G, Kato O, Leibo SP. Highly efcient vitrication method for cryopreservation of human oocytes. Reprod BioMed Online 2005;11:3008. [27] Cobo A, Domingo J, Prez S, Crespo J, Remoh J, Pellicer A. Vitrication: an effective new approach to oocyte banking and preserving fertility in cancer patients. Clin Transl Oncol 2008;10:26873. [28] Yavin S, Arav A. Measurement of essential physical properties of vitrication solutions. Theriogenology 2007;67:819. [29] Steponkus PL, Myers SP, Lynch DV, Gardner L, Bronshteyn V, Leibo SP, et al. Cryopreservation of Drosophila melanogaster embryos. Nature 1990;345:1702. [30] Seki S, Mazur P. Ultra-rapid warming yields high survival of mouse oocytes cooled to 196  C in dilutions of a standard vitrication solution. PLoS One 2012;7:e36058. [31] Voelkel SA, Hu YX. Use of ethylene glycol as a cryoprotectant for bovine embryos allowing direct transfer of frozen-thawed embryos to recipient females. Theriogenology 1992;37:68797. [32] Saha S, Otoi T, Takagi M, Boediono A, Sumantri C, Suzuki T. Normal calves obtained after direct transfer of vitried bovine embryos using ethylene glycol, trehalose, and polyvinylpyrrolidone. Cryobiology 1996;32:50510.

101

[33] Martino A, Songsasen N, Leibo SP. Development into blastocysts of bovine oocytes cryopreserved by ultra-rapid cooling. Biol Reprod 1996;54:105969. [34] Hamawaki A, Kuwayama M, Hamano S. Minimum volume cooling method for bovine blastocyst vitrication. Theriogenology 1999;51:165. [35] Kuwayama M, Vajta G, Ieda S, Kato O. Comparison of open and closed methods for vitrication of human embryos and the elimination of potential contamination. Reprod Biomed Online 2005;11: 60814. [36] Lane M, Bavister BD, Lyons EA, Forest KT. Containerless vitrication of mammalian oocytes and embryos. Nat Biotechnol 1999;17: 12346. [37] Lane M, Schoolcraft WB, Gardner DK, Phil D. Vitrication of mouse and human blastocysts using a novel cryoloop container-less technique. Fertil Steril 1999;72:10738. [38] Vanderzwalmen P, Bertin G, Debauche C, Standaart V, Schoysman E. In vitro survival of metaphase II oocytes (MII) and blastocysts after vitrication in a hemi-straw (HS) system. Fertil Steril 2000;74: S2156. [39] Dinnyes A, Dai Y, Jiang S, Yang X. High developmental rates of vitried bovine oocytes following parthenogenetic activation, in vitro fertilization, and somatic cell nuclear transfer. Biol Reprod 2000;63:5138. [40] Matsumoto H, Jiang JY, Tanaka T, Sasada H, Sato E. Vitrication of large quantities of immature bovine oocytes using nylon mesh. Cryobiology 2001;42:13944. [41] Chian RC, Son WY, Huang JY, Cui SJ, Buckett WM, Tan SL. High survival rates and pregnancies of human oocytes following vitrication: preliminary report. Fertil Steril 2005;84:S36. [42] Chen SU, Chien C-L, Wu M-Y, Chen T-H, Lai S-M, Lin C-W, et al. Novel direct cover vitrication for cryopreservation of ovarian tissues increases follicle viability and pregnancy capability in mice. Hum Reprod 2006;21:2794800. [43] Muthukumar K, Mangalaraj AM, Kamath MS, George K. Blastocyst cryopreservation: vitrication or slow freeze. Fertil Steril 2008;90: S4267. [44] Tsang WH, Chow KL. Mouse embryo cryopreservation utilizing a novel high-capacity vitrication spatula. Biotechniques 2009;46: 5502. [45] Petyim S, Makemahar O, Kunathikom S, Choavaratana R, Laokirkkiat P, Penparkkul K. The successful pregnancy and birth of a healthy baby after human blastocyst vitrication using Cryo-E, rst case in Siriraj Hospital. J Med Assoc Thai 2009;92:111621. [46] Sugiyama R, Nakagawa K, Shirai A, Sugiyama R, Nishi Y, Kuribayashi Y, Inoue. M. Clinical outcomes resulting from the transfer of vitried human embryos using a new device for cryopreservation (plastic blade). J Assist Reprod Genet 2010;27:1617. [47] Almodin CG, Minguetti-Camara VC, Paixao CL, Pereira PC. Embryo development and gestation using fresh and vitried oocytes. Hum Reprod 2010;25:11928. [48] Vajta G, Holm P, Greve T, Callesen H. Vitrication of porcine embryos using the open pulled straw (OPS) method. Acta Vet Scand 1997;38:34952. [49] Vajta G, Holm P, Kuwayama M, Booth PJ, Jacobsen H, Greve T, et al. Open pulled straw (OPS) vitrication: a new way to reduce cryoinjuries of bovine ova and embryos. Mol Reprod Dev 1998;51: 538. [50] Chen SU, Lien YR, Cheng YY, Chen HF, Ho HN, Yang YS. Vitrication 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;16:23506. [51] Liebermann J, Tucker M, Graham J, Han T, Davis A, Levy M. Blastocyst development after vitrication of multi pronuclear zygotes using the exipet denuding pipette. Reprod Biomed Online 2002;4: 14650. [52] Isachenko V, Folch J, Isachenko E, Nawroth F, Krivokharchenko A, Vajta G, et al. Double vitrication of rat embryos at different developmental stages using an identical protocol. Theriogenology 2003;60:44552. [53] Kuwayama M. Highly efcient vitrication for cryopreservation of human oocytes and embryos: the Cryotop method. Theriogenology 2007;67:7380. [54] Sun X, Li Z, Yi Y, Chen J, Leno GH, Engelhardt JF. Efcient term development of vitried ferret embryos using a novel pipette chamber technique. Biol Reprod 2008;79:83240. [55] Camus A, Clairaz P, Ersham A, Van Kappel AL, Savic G, Staub C. Principe de la vitrication: cintiques comparatives. The comparison

102

A. Arav / Theriogenology 81 (2014) 96102 of the process of ve different vitrication devices. Gynecol Obstet Fertil 2006;34:73745. Yavin S, Aroyo A, Roth Z, Arav A. Embryo cryopreservation in the presence of low concentration of vitrication solution with sealed pulled straws in liquid nitrogen slush. Hum Reprod 2009;24: 797804. Portmann M, Nagy ZP, Behr B. Evaluation of blastocyst survival following vitrication/warming using two different closed carrier systems. Hum Reprod 2010;25:i261. Larman MG, Gardner DK. Vitrifying mouse oocytes and embryos with super-cooled air. Hum Reprod 2010;25:i265. Testart J, Lassalle B, Belaisch-Allart J. High pregnancy rate after early human embryo freezing. Fertil Steril 1986;46:268. Rufng NA, Steponkus PL, Pitt RE, Parks JE. Osmometric behavior, hydraulic conductivity, and incidence of intracellular ice formation in bovine oocytes at different developmental stages. Cryobiology 1993;30:56280. Van den Abbeel E, Schneider U, Liu J, Agca Y, Critser JK, Van Steirteghem A. Osmotic responses and tolerance limits to changes in external osmolalities, and oolemma permeability characteristics, of human in vitro matured MII oocytes. Hum Reprod 2007;22: 195972. Carroll J, Depypere H, Matthews CD. Freezethaw-induced changes of the zona pellucida explains decreased rates of fertilization in frozenthawed mouse oocytes. J Reprod Fertil 1990;90:54753. Mavrides A, Morroll D. Bypassing the effect of zona pellucida changes on embryo formation following cryopreservation of bovine oocytes. Eur J Obstet Gynecol Reprod Biol 2005;118:6670. Gook DA, Osborn SM, Johnston WIH. Cryopreservation of mouse and human oocytes using 1,2-propanediol and the conguration of the meiotic spindle. Hum Reprod 1993;8:11019. Luvoni GC. Current progress on assisted reproduction in dogs and cats: in vitro embryo production. Reprod Nut Dev 2000;40:50512. Ciotti PM, Porcu E, Notarangelo L, Magrini O, Bazzocchi A, Venturoli S. Meiotic spindle recovery is faster in vitrication of human oocytes compared to slow freezing. Fertil Steril 2009;91: 2399407. [67] Gupta MK, Uhm SJ, Lee HT. Effect of vitrication and betamercaptoethanol on reactive oxygen species activity and in vitro development of oocytes vitried before or after in vitro fertilization. Fertil Steril 2010;93:26027. [68] Ghetler Y, Yavin S, Shalgi R, Arav A. The effect of chilling on membrane lipid phase transition in human oocytes and zygotes. Hum Reprod 2005;20:33859. [69] Fahy GM. The relevance of cryoprotectant toxicity to cryobiology. Cryobiology 1986;23:113. [70] Rall WF, Wood MJ, Kirby C, Whittingham DG. Development of mouse embryos cryopreserved by vitrication. J Reprod Fertil 1987; 80:499504. [71] Massip A, Van der Zwalmen P, Ectors F. Recent progress in cryopreservation of cattle embryos. Theriogenology 1987;27:6979. [72] Fahy GM, MacFarlane DR, Angell CA, Meryman HT. Vitrication as an approach to cryopreservation. Cryobiology 1984;21:40726. [73] Szll A, Shelton JN. Osmotic and cryoprotective effects of glycerolsucrose solutions on day-3 mouse embryos. J Reprod Fertil 1987; 80:30916. [74] Crowe JH, Crowe LM, Mouradian R. Stabilization of biological membranes at low water activities. Cryobiology 1983;20:34656. [75] Arav A, Shehu D, Mattioli M. Osmotic and cytotoxic study of vitrication of immature bovine oocytes. J Reprod Fertil 1993;99:3538. [76] Schellander K, Brackett BG, Fuhrer F, Schleger W. In vitro fertilization of frozen-thawed cattle oocytes. In: Proceedings of the 11th Congress on Animal Reproduction and Articial Insemination; 1988 p. 2630. [77] Rubinsky B, Arav A, DeVries AL. Cryopreservation of oocyte using directional cooling and antifreeze proteins. Cryo Letters 1991;12: 93106. [78] Kasai M, Komi JH, Takakamo A, Tsudera H, Sakurai T, Machida T. A simple method for mouse embryo cryopreservation in a low toxicity vitrication solution, without appreciable loss of viability. J Reprod Fertil 1990;89:907. [79] Arav A, Zeron Y, Ocheretny A. A new device and method for vitrication increases the cooling rate and allows successful cryopreservation of bovine oocytes. Theriogenology 2000;53:2489.

[56]

[57]

[58] [59] [60]

[61]

[62]

[63]

[64]

[65] [66]