MOLECULAR REPRODUCTION AND DEVELOPMENT 29:373-378 (1991)
Vitrification of Mouse Oocytes: Improved Rates of Survival, Fertilization, and Development to Blastocysts P.W. SHAW,l*’B.J. FULLER: A. BERNARD:
AND
R.W. SHAW’
Academic 2Departments of Obstetrics and Gynaecology, and 3Surgery, Royal Free Hospital School of Medicine, London, United Kingdom
ABSTRACT Rall and Fahy’s (1985)vitrification procedure for the cryopreservation of 8-cell embryos was applied to unfertilized mouse oocytes. Unchanged, this method resulted in a mean of 24.1%of vitrified oocytes fertilizing and developing to blastocysts in vitro. Exposure of oocytes to the cryoprotectant media, but without the vitrification, resulted in 30.8%developing to blastocysts. Modifications to the durations of and media used in the dilution and equilibration steps of the procedure produced a final protocol giving a mean of 55.4%of vitrified oocytes and 72.4%of nonvitrified VS1-exposed oocytes developing to blastocysts; 85.7%of control oocytes develop to blastocysts. Osmotically induced damage was found to be the most important cause of loss of viability in these methods. Cooling of oocytes t o 5-8°C during the procedure had no significant effect on their viability. No parthenogenetic activation of oocytes occurred as a result of exposure to the procedure. Key Words: Oocyte cryopreservation, Dilution lysis, Cooling
INTRODUCTION Despite the establishment of routinely successful methods for the cryopreservation of mammalian embryos and semen, a dependable method for use with unfertilized oocytes remains elusive (see Friedler et al., 1988; Siebzehnrubl, 1989). The ability to cryopreserve unfertilized eggs would alleviate a number of ethical and legal problems resulting ,from frozen storage of “spare” embryos in human IVF programmes, assist ovum donation, and confer maximum flexibility upon strategies for human and domestic animal assisted reproduction. Vitrification procedures, developed for the rapid and straightforward cryopreservation of mammalian embroys (e.g., Rall and Fahy, 1985; Massip et al., 19861, have shown some promise for use with oocytes (Trounson 1986; Feichtinger et al., 1987; Kola et al., 1988), but overall rates of survival, fertilization, and subsequent development have been poor compared to those achieved with embryos. The present study set out to apply an established method for embryo vitrification, that of Rall and Fahy
0 1991 WILEY-LISS, INC.
(1985), to mouse oocytes to establish whether the protocol could be tailored to the particular properties of these cells to improve success rates. Previous experiments (Shaw et al., 1990) had demonstrated that when exposed to the above procedure oocytes exhibited a variety of extreme osmotically induced morphological changes during the equilibration and dilution steps, which it was postulated might result in a reduction of cell viability. The present study applied the following sequence of changes to the original protocol to assess the effect on cell viability of attempts to reduce the osmotic problems described in Shaw et al. (1990): 1) adding intermediate steps to the multistep dilution procedure to modulate osmotic pressure changes, 2) replacing the multistep dilution with a single-step sucrose dilution to control osmotically induced volume changes, 3) extending the duration of the dilution step to enhance temperature and osmotic equilibration before the wash steps, and 4) extending the duration of the VS1-equilibration steps to ensure full equilibration before vitrification.
METHODS Oocyte Retrieval Unfertilized mature ovulated oocytes were obtained from virgin C57BL x CBA F1 hybrid female mice as described previously (Bernard and Fuller, 1983). Oocytes were stripped of their surrounding cumulus cells by incubation with hyaluronidase (150 units m1-l Bovine testicular hyaluronidase, Sigma UK) supplemented with 4 mg . ml-’ bovine serum albumin (BSA), and then washed three times in Dulbeccos phosphatebuffered saline (PBS) medium plus 4 mg * ml-’ BSA (PB1).
Culture/Manipulation Conditions Morphologically abnormal oocytes were discarded at this stage, and the remaining oocytes distributed to three experimental groups. The first group was incu-
Received November 29, 1990; accepted March 27, 1991. Address reprint requests to Dr. P.W. Shaw, Academic Department of Obstetrics and Gynaecology, Royal Free Hospital School of Medicine, Pond Street, London NW3 2QG, United Kingdom.
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bated for 2 hours in modified Tyrode’s solution (T& Quinn et al., 1982) at 37°C under 5% C 0 2 + 5% O2 + 90% N2 to serve as a control. The second group was put through the vitrification procedure described below. The third group was put through the same procedure but without the vitrification step (“VS1exposed”).Eggs morphologically intact after processing were inseminated and cultured on for 5 days as described previously (Bernard and Fuller, 1983). Eggs were defined as morphologically intact if the cell was spherical in shape, possessed an intact plasma membrane, the cytoplasm was refractile, there were no cytoplasmic fragments inside the zona pellucida, and the zona pellucida was undamaged.
Vitrification Protocol The vitrification protocol used was that of Rall and Fahy (19851, which utilises a vitrification solution (VS1)containing permeating (dimethylsulphoxide, 1,2propanediol, acetamide) and nonpermeating (polyethylene glycol) agents in buffered saline. Briefly, oocytes were equilibrated in 25% VS1 at room temperature (20-22°C) for 15 minutes, and then dehydrated successively in 50% VS1 and 90% VS1 for 10 minutes each in a cold room (5-8’0, before transfer into a 45 pl drop of 90% VS1 in a plastic insemination straw (L’AIGLE, France) and plunging into liquid nitrogen. Visual examination of the straws confirmed the absence of visible ice in the medium. Straws were thawed in an iced-water bath and the contents expelled into 50%VS1 at 5-8°C for 10 minutes. Further steps of rehydration and elution of cyroprotectant consisted of 10 minutes in each of 25% VS1 at 5-8”C, and 12.5%VS1 and 6% VS1 at room temperature, before a final three washes in PB1. Experimental Groups Initially, a number of test runs were made using 8-cell mouse embryos, produced in vitro, to ensure that the level of survival described by Rall and Fahy (1985) could be reproduced, and therefore that the conditions used with the oocytes would be comparable to their results with embryos. Next, groups of oocytes were processed through the method as described above unchanged. Subsequently, based on observations of morphological and functional survival of processed oocytes at each step, the modifications described briefly in the Introduction above were introduced to the procedure t o attempt to improve survival. These will be described more fully in the results section together with their effects on oocyte viability.
transferred direct to culture without insemination after exposure to the vitrification, VS1-exposed, or control treatments.
Effect of Cooling The protocols used here employed periods of exposure of oocytes to room temperature (20-22°C) and cold room temperature (543°C). Such cooling of metaphase I1 oocytes has been reported to result in reduced rates of subsequent fertilization due to damage caused to the metaphase spindle (Johnson et al., 1988). To assess whether the cooling involved contributes to any reduction in fertilization rates seen in the experimental groups, oocytes were exposed to the temperature changes and durations prescribed in droplets of PB1 before insemination and culture. Further groups were similarly exposed to cooling but were incubated in T6 at 37°C under 5% COz + 5% O2 + 90% N2 for periods of 1 or 2 hours before insemination to see if a recovery period was needed for reassembly of the spindle, as described by Pickering and Johnson (1987). Viability of oocytes from all experimental and control groups was assessed at three levels: number of oocytes morphologically normal immediately post-treatment, number of oocytes fertilizing and cleaving to 2-cells 24 hours post-insemination, and number of oocytes developing to expanded blastocysts. Data Analysis Batches of oocytes from all straws within treatments each day were pooled immediately after dilution and washing, and then cultured on and assessed as one group. Any differences between groups of data of interest were tested by standard chi-square analysis. RESULTS The results of the standard vitrification procedure and all subsequent modifications as applied to mouse unfertilized oocytes, together with their corresponding VS1-exposed groups, are presented in Table 1. Also presented are data for the pooled control groups and the vitrification of 8-cell embryos. All figures represent the pooled results of a number of individual batches (one batch per straw, around 15 oocytes per batch) of oocytes processed through each treatment on at least 3 separate days. Vitrification of 8-Cell Embryos After some practice we managed to attain similar levels of survival and development to blastocysts of vitrified %cell embryos [R and F (Embryos) in Table 11 as described by Rall and Fahy (1985)-75.6% compared to 83.5%in controls.
Parthenogenetic Activation Oocyte Vitrification: Rall and Fahy’s Method Parthenogenetic activation of oocytes after exposure (R and F in Table 1) to a cryoprotectant has been reported by Shaw and Trounson (1989). In order to elucidate whether any When oocytes were vitrified by Rall and Fahy’s such activation took place in oocytes exposed to the (1985) method, 65.7% appeared morphologically norprotocol used in this study, groups of oocytes were mal in ‘culture after treatment, with 79% of these
VITRIFICATION OF MOUSE OOCYTES
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TABLE 1. Survival of Oocytes Vitrified by Methods Modified From Rall and Fahy (1985) and Feichtinger et al. (1987) Percent morphological survival
Percent fertilized Percent (percent 2 - c e l l ~ ) ~ blastocystsa
N Treatment R and F (embryos) Control 97 100 90.7 (91) Vitrified 123 73.2 76.4 (100) Oocytes 543 100 89.7 controls R and F Vitrified 137 65.7 51.8 (79) VS1 exposure 120 76.7 60.0 (78) R and F (+18%) Vitrified 51 45.1 35.3 (78) VS1 exposure 56 96.4 78.6 (81) F “10 10” Vitrified 80 75.0 52.5 (70) VS1 exposure 35 91.4 45.7 (50) F -10 2079 Vitrified 58 60.3 51.7 (86) VSl exposure 47 93.6 91.4 (98) F “15 25” Vitrified 139 79.9 66.2 (83) VS1 exposure 127 97.6 82.7 (85) aFigures in brackets represent percentage of previous step.
+
+ +
fertilizing and cleaving to 2 cells. Only 46% of these %cell embryos developed to expanded blastocysts, giving an overall rate of development to blastocysts of vitrified oocytes of 24.1%. In the corresponding VS1exposed group rates of morphological survival, fertilizationkleavage, and development to blastocysts were very similar to the vitrified group, with an overall rate to blastocysts of 30.8%.
83.5 (92) 75.6 (99) 84.7 (94) 24.1 (46) 30.8 (51) 29.4 (83) 75.0 (95) 33.8 (64) 45.7 (100) 43.1 (83) 72.3 (79) 55.4 (84) 72.4 (88)
logical survival of the process, the overall success rate to blastocysts was only marginally improved over the original protocol (29.4% versus 24.1%, respectively).
One-Step Sucrose Dilution (F“10-t10” in Table 1)
An accepted method for controlling osmotically induced excursions in cells during the elution of permeating cryoprotectants is a l-step dilution with a sucrose solution, as suggested by Feichtinger et al. (1987) for Modified Rall and Fahy Method [R and F (+18%) use with this vitrification method. This modification in Table 11 (F“lO+lO” in Table 1) uses a 10 minute equilibration An obvious point of destruction of VS1-exposed step with 25% VS1, followed by 5 minutes each in 50% oocytes during the R and F protocol occurred by lysis of VS1 and 90% VS1, and a 10 minute l-step dilution with the cells at the first dilution step at room temperature, 0.8 M sucrose. Application of this method resulted in between 25% VS1 and 12.5%VS1. This was probably a much improved morphological survival of vitrified result of over-rapid increase in cell volume, involving oocytes (75.0%,x2 = 12.0P < 0.05) but reduced rates of “blistering” by the plasma membrane as described by subsequent fertilization and development (only 64% 2 Shaw et al. (19901, resulting in rupture of the mem- cells to blastocysts). Similarly, morphological survival brane and lysis. In order to reduce the magnitude of the of VS1-exposed oocytes were high (91.4%)but overall osmotic pressure change at this point the first modifi- rates to blastocysts were lower than with the R&F cation of the protocol was made: introduction of a (+ 18%)method due to reduced fertilization. further dilution step with corresponding scaling of Extended One-Step Dilution steps below it (25% VS1-18% VS1-10% VS1-5% VS1(F“lOt20” in Table 1) wash). The result of this modification [R and F (+ 18%) The R and F (+ 18%)modification demonstrated that in Table 11 was a dramatic increase in both the morphological survival of VS1-exposed oocytes (to modulating the severity of osmotic (and temperature) 96.4%)and, perhaps more interestingly, in the propor- changes during the dilution steps could improve subtion of 2-cell embryos developing to blastocysts (95%). sequent development of fertilized eggs, as well as the This gave an overall rate of VS1-exposed oocytes to immediate benefits to oocyte survival. Therefore, the blastocysts of 75.0% (the proportion of morphologically next modification tried was an extension of the dilution normal eggs fertilizing was unaltered by the modifica- step of the F‘lOt10” method to 20 minutes, to allow tion, x2 = 0.216 P > 0.05). The rate of development of 2 more controlled temperature equilibration (10 minutes cells to blastocysts resulting from vitrified oocytes was in cold room, 10 minutes at room temperature) and thus similarly improved, but due to a reduction in morpho- transmembrane movement of water and permeating
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cyroprotectants. The results (F“10+20” in Table 1) indeed show a small, and mostly nonsignificant, increase in fertilization rate of surviving oocytes (86%in vitrified, x2 = 2.97 P > 0.05; 98% in VS1-exposed, = 24.3 P > 0.05) and subsequent development (83% in vitrified, x2 = 3.16 P > 0.05). This results in a return of overall rates of survival of VS1-exposed oocytes to blastocysts to greater than 70% as seen in R and F (+18%),and an improvement in overall success with vitrified oocytes (to 43.1%) despite a slight nonsi nificant reduction in morphological survival = 3.57 P > 0.05).
x2
(3
Extended CPA Equilibration (F“15+25” in Table 1) It was noted that mouse oocytes did not appear to fully equilibrate with 25% VS1 at room temperature after 10 minutes (i,e., the oocytes did not reexpand to the volume seen in oocytes exposed for 15 minutes, personal observation), so equilibration time was extended to 15 minutes to assess whether this had any advantageous (or detrimental) effects. The result (F“15+25”in Table 1)was an improvement in morphological survival of vitrified oocytes (to 79.9%),without any substantial change to subsequent fertilization and developmental potential, giving a final overall survival of vitrified oocytes to blastocysts of 55.4%. No single modification resulted in a significant improvement over the prior protocol in overall survival to blastocysts of vitrified oocytes (x2 = 0.73 to 3.20, all P > 0.05). However the improvement in overall survival of vitrified oocytes to blastocysts between the initial (R and F) and final (F“15t25”)procedures was very significant (x2 = 27.21, P < 0.001). Parthenogenetic Activation In the tests for parthenogenetic activation of oocytes the F‘15+25” protocol was used for the vitrified and VS1-exposed groups. No signs of activation or cleavage were seen up to 3 days after treatment in any of the vitrified (30 oocytes), VS1-exposed (42 oocytes), or control (20 oocytes) groups. The control inseminated group (27 oocytes) demonstrated normal levels of fertilization (93%) and development to blastocysts (89%).
Effects of Cooling The results of the experiments to assess the effect on fertilization and development of oocytes subjected to temperature changes and durations prescribed by the F“15+25”protocol are displayed in Table 2. None of the experimental groups show significantly different rates of fertilization and development to blastocysts compared to the control group (x2 = 0.05 to 232, all P > 0.05). DISCUSSION Since the first demonstrations of successful cryopreservation of oocytes (Tsunoda et al., 1976; Whittingham, 1977),some considerable achievements have been made in improving morphological survival, fertilization, embryonic development, and even the production of live young from frozen or vitrified oocytes (Critser et al., 1986; Nakagata, 1989; Lewin et al., 1990; Schroeder et al., 1990). The present study shows that significant improvements can be made on existing methods by small modifications of procedure tailoring the method towards the material involved. More important perhaps, this study suggests some links between changes in specific parts of a protocol and effects on different aspects of oocyte survival such as morphological integrity and the potential for fertilization and further development. Such information can be of use in further attempts to improve success rates. One general point to be made from this data is that modulating osmotic stresses to which oocytes are exposed during the dilution phase not only improves morphological survival but also appears to correlate with improved potential of fertilized oocytes to develop further. Improved morphological survival resulting from regulating volume excursions (multistep dilution) or allowing time for temperature equilibration (sucrose dilution) presumably relates to maintaining plasma membrane integrity and avoiding lysis. A number of mechanisms have been proposed to explain membrane damage resulting from osmotic forces, including over expansion of cell membranes after loss of membrane surface area during dehydration (Steponkus and Wiest, 1979) and disruption of membrane integrity by mass movement of water (Muldrew and McGann, 1990).How such processes could affect the ability of resulting embryos to develop is difficult to explain at present.
TABLE 2. Effect of Cooling on Oocyte Fertilization and Development Treatment
N
Percent morphological survival
Percent fertilized (percent 2-cells)
Percent blastocysts
Control Cooled Number of incubation 1 hour incubation 2 hour incubation Pooled
64
100
82.8
79.7
87 108 120 315
100 100 100 100
80.5 81.5 85.0 82.5
75.9 68.5 70.0 71.1
VITRIFICATION OF MOUSE OOCYTES The results presented here also support the hypothesis that osmotically induced damage is much more important than any detrimental effects resulting from biochemical toxicity of the VS1 solution over the durations and temperatures of exposure utilized. Large improvements in survival and viability of oocytes were gained by solving problems of osmotic damage without any concomitant detrimental effects from the increased periods of exposure used. Therefore, for several protocols examined overall rates of development to blastocysts of VS1-exposed oocytes approached those seen in controls, given the small losses in morphological survival and the possible effects of cooling on development (see below). This indicates that concern regarding the biochemical toxicity of some of the cryoprotectants used (Weisburger et al., 1969; Baxter and Lathe, 1971; Rall, 1987; Van der Elst et al., 1988; and see Fahy et al., 1990) may not be relevant to the short exposure protocols used in vitrification, an advantage that would support the application of these methods. The possible effects of vitrification methods in producing chromosome anomalies and defects in young born from vitrified oocytes needs to be investigated further given the inconclusive reports for their occurrence (Kola et al., 1988) or absence (Whittingham, 1977; Nakagata, 1989). Whether oocytes need complete equilibration with a certain dilution of the VS1 solution to protect them from damaging instantaneous osmotic stresses or dehydration effects during dehydration and vitrification is open to question. Extending equilibration time (F‘10+20”versus F“15+25”) improved survival in this study, and most published methods for successful embryo vitrification employ an equilibration period (e.g., Rall and Fahy, 1985; Scheffen et al., 1986; Rall, 1987; Kasai et al., 1990). However, Nakagata (1989) reports a method of vitrification of mouse oocytes which achieves 87.6% morphological suvival using only a 5 to 10 second exposure to the undiluted vitrification solution before plunging into LN2.In our hands only 54%of mouse oocytes vitrified by the Nakagata method survive morphologically (unpublished results), but it still suggests that previtrification equilibration may not be of major importance compared to conditions of vitrification and, especially, dilution. Pre-equilibration of oocytes with cryoprotectants may still be advisable however, particularly in the case of more sensitive human material, if periods of cooling are employed, in order to protect them from cooling damage, as suggested by the work of Van der Elst et al. (1988). The present data also demonstrate that the vitrification and thawing steps definitely have a detrimental effect on oocyte survival, ,but that these are only exhibited in the form of immediate post-vitrification losses and have no effect on the ability of surviving oocytes to fertilize and develop. This is illustrated by a comparison of all pairs of vitrified and corresponding VS1-exposed groups in Table 1. If the percentage development from the previous step is examined in each case it can be seen that for almost all pairs of
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figures there is very little difference between vitrified and VS1-exposed groups except in the figures for morphological survival of processing. In the final protocol used (F‘15+25”) the difference in immediate losses due to physical destruction of oocytes between the VS1-exposed and vitrified groups is 15.2%. What could cause such losses? One obvious source of destruction of oocytes is by “zona cracking”: Rall and Meyer (1989) concluded that splits in the zona of bovine ova were most likely caused by fracturing of the vitrified medium resulting from thermal stresses created during warming. We have previously concluded (Shaw et al., 1990)that the same phenomenon results in the damage seen in vitrified mouse ova. In this study such damage accounts for the destruction of 4% to 5% of oocytes on average. The remaining 10%of losses may be caused by ice damage to the cells as a result of devitrification of the medium during thawing. Although the vitrification medium used (90% VS1 of Rall and Fahy, 1985) successfully vitrifies, we consistently observe a 2-3 second period of devitrification (the medium becomes opaque white) during thawing in an ice bath. Takahashi et al. (1986)reported that ice crystals formed both extra- and intra-cellularly during devitrification of a similar medium and that the presence of intracellular ice corresponded to a significant reduction in survival of the vitrified monocytes. Our finding that the only detrimental effects of vitrification and thawing on oocytes are immediate is in contrast to the results of Kola et al. (1988) who reported that there were also differences in rates of fertilization, cleavage, and development on between vitrified and VS1-exposed oocytes. A number of studies have suggested that cooling of unfertilized oocytes, even to room temperature, may impair their ability to be fertilized and develop normally (Magistrini and Szollosi, 1980; Pickering and Johnson, 1987;Johnson et al., 1988; Van der Elst et al., 1988). This study shows that for mouse oocytes, under the conditions used in these protocols, no significant reduction in fertilization and development occurs. However, mouse oocytes are known to be able to recover from changes induced by cooling (Glenister et al., 1987; Pickering and Johnson, 1987), so caution would still be needed if such methods were transferred to human oocytes. One interesting point in the data was that although there were no significant changes in ability of fertilized cooled oocytes to develop and divide, all three cooled groups exhibited a decrease compared to the control group. If this is a real effect it would explain a similar decrease in viability in the VS1-exposed experimental groups, as mentioned above. Overall, the results presented here suggest that vitrification could be an acceptable method for cryopreservation of unfertilized ova. The final rates of development to blastocysts of vitrified mouse oocytes are comparable with the best results achieved by standard slow-freezing methods (e.g., Schroeder et al., 1990)or ultra-rapid freezing methods (e.g.,Lewin et al., 1990).
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This work is supported by grant number SP2032 from the Cancer Research Campaign.
REFERENCES Baxter SJ, Lathe GH (19711: Biochemical effects on kidney of exposure to high concentrations of dimethyl sulphoxide. Biochem Pharmacol 30:1079-1091. Bernard A, Fuller BJ (1983): Cryopreservation of in vitro fertilized 2-cell mouse embryos using a low glycerol concentration and normothermic cryoprotectant equilibration: A comparison with in vitro fertilized ova. Cryo-Lett 4:171-178. Critser J K , Arneson BW, Aaker DV, Ball GD (1986): Cryopreservation of hamster oocytes: Effects of vitrification or freezing on human sperm penetration of zona-free hamster oocytes. Fertil Steril 46:277-284. Fahy GM, Lilley TH, Linsdell H, St. John Douglas M, Meryman HT (1990) Cryoprotectant toxicity and cryoprotectant toxicity reduction: In search of molecular mechanisms. Cryobiology 27:247-268. Feichtinger W, Benko I, Kemeter P (1987): Freezing human oocytes using rapid techniques. In Feichtinger W, Kemeter P (eds):“Future Aspects in In Vitro Fertilisation.” Berlin: Springer-Verlag, pp 101-110. Friedler S, Giudice LC, Lamb E J (1988):Cyropreservation of embryos and ova. Fertil Steril 49:743-764. Glenister PH, Wood MJ, Kirby C, Whittingham DG (1987):Incidence of chromosome anomalies in first cleavage mouse embryos obtained from frozen-thawed oocytes fertilized in vitro. Gam Res 16:205-216. Johnson MH, Pickering SJ, George MA (1988): The influence of cooling on the properties of the zona pellucida of the mouse oocyte. Hum Reprod 3:383-388. Kasai M, Komi J H , Takakamo A, Tsudera H, Sakurai T, Machida T (1990):A simple method for mouse embryo cryopreservation in a low toxicity vitrification solution, without appreciable loss of viability. J Reprod Fertil 89:91-97. Kola I, Kirby C, Shaw J, Davey A, Trounson A (1988):Vitrification of mouse oocytes results in aneuploid zygotes and malformed fetuses. Teratology 38:467474. Lewin A, Tal Z, Zohav E, Schenker J G (1990):Ultrarapid freezing and thawing of hamster oocytes: Morphologic parameters, Trypan Blue staining and sperm penetration assay for evaluating survival. J Reprod Med 35:136-140. Magistrini M, Szollosi D (1980): Effects of cold and isopropy1-Nphenylcarbonate on the second meioyic spindle of mouse oocytes. Eur J Cell Biol 22:699-707. Massip A, Van der Zwalmen P, Sceffen B, Ectors F (1986):Pregnancies following transfer of cattle embryos preserved by vitrification. Cryo-Lett 7:270-273.
Muldrew K, McGann LE (1990): Mechanisms of intracellular ice formation. Biophys J 57:525-532. Nakagata N (1989): High survival rate of unfertilized mouse oocytes after vitrification. J Reprod Fert 87:479-483. Pickering SJ, Johnson MH (1987): The influence of cooling on the organisation of the meiotic spindle of the mouse oocyte. Hum Reprod 2:207-216. Quinn P, Barros C, Whittingham DG (1982):Preservation of hamster oocytes to assay the fertilizing capacity of human spermatozoa. J Reprod Fertil 66:161-168. Rall WF (1987): Factors affecting the survival of mouse embryos cryopreserved by Vitrification. Cryobiology 24:387402. Rall WF, Fahy GM (1985):Ice-free cryopreservation of mouse embryos a t - 196°C by vitrification. Nature 313573-575. Rall WF, Meyer TK (1989): Zona fracture damage and its avoidance during the cryopreservation of mammalian embryos. TheriogenolOgy 31~683-692. Scheffen B, Van der Zwalmen P, Massip A (1986): A simple and efficient procedure for the preservation of mouse embryos by vitrification. Cryo-Lett 7:260-269. Schroeder AC, Champlin AK, Mobraaten LE, Eppig JJ (1990):Developmental capacity of mouse oocytes cryopreserved before and after maturation in vitro. J Reprod Fertil 89:43-50. Shaw JM, Trounson A 0 (1989): Effect of dimethylsulphoxide and protein concentration on the viability of two-cell mouse embryos frozen with a rapid freezing technique. Cryobiology 26:413421. Shaw PW, Bernard AG, Fuller BJ, Shaw RW (1990): Morphological and functional changes in unfertilized mouse oocytes during a vitrification procedure. Cryo-Lett 11:427432. Siebzehnrubl ER (19891: Cryopreservation of gametes and cleavage stage embryos. Hum Reprod 4 (Supp1):105-110. Steponkus PL, Wiest SC (1979): Freeze-thaw induced lesions in the plasma membrane. In Lyons JM, Graham D, Raison J K (eds):“Low Temperature Stress in Crop Plants.” London: Academic Press, pp 231-254. Takahashi T, Hirsch A, Erbe EF, Bross JB, Steers RL, Williams R J (1986) Vitrification of human monocytes. Cryobiology 23:103-115. Trounson A (1986): Preservation of human eggs and embryos. Fertil Steril 46:l-12. Tsunoda Y, Parkening TA, Chang MC (1976):In vitro fertilization of mouse and hamster eggs after-freezing and thawing. Experientia 32:223-224. Van der Elst J, Van den Abeel F, Jacobs R, Wisse E, Van Steirteghem A (1988): Effect of 1,2-propanediol and dimethylsulphoxide on the meiotic spindle of the mouse oocyte. Hum Reprod 3:960-967. Weisburger JH, Yamamoto RS, Glass RM, Frankel HH (1969): Prevention by arginine glutamate of the carcinogenicity of acetamide in rats. Toxicol Appl Pharmacol 14:163-175. Whittingham DG (1977): Fertilization in vitro and development to term of unfertilized mouse oocytes previously stored a t -196°C. J Reprod Fertil 49:89-94.
Vitrification of Mouse Oocytes: Improved Rates of Survival, Fertilization, and Development to Blastocysts P.W. SHAW,l*’B.J. FULLER: A. BERNARD:
AND
R.W. SHAW’
Academic 2Departments of Obstetrics and Gynaecology, and 3Surgery, Royal Free Hospital School of Medicine, London, United Kingdom
ABSTRACT Rall and Fahy’s (1985)vitrification procedure for the cryopreservation of 8-cell embryos was applied to unfertilized mouse oocytes. Unchanged, this method resulted in a mean of 24.1%of vitrified oocytes fertilizing and developing to blastocysts in vitro. Exposure of oocytes to the cryoprotectant media, but without the vitrification, resulted in 30.8%developing to blastocysts. Modifications to the durations of and media used in the dilution and equilibration steps of the procedure produced a final protocol giving a mean of 55.4%of vitrified oocytes and 72.4%of nonvitrified VS1-exposed oocytes developing to blastocysts; 85.7%of control oocytes develop to blastocysts. Osmotically induced damage was found to be the most important cause of loss of viability in these methods. Cooling of oocytes t o 5-8°C during the procedure had no significant effect on their viability. No parthenogenetic activation of oocytes occurred as a result of exposure to the procedure. Key Words: Oocyte cryopreservation, Dilution lysis, Cooling
INTRODUCTION Despite the establishment of routinely successful methods for the cryopreservation of mammalian embryos and semen, a dependable method for use with unfertilized oocytes remains elusive (see Friedler et al., 1988; Siebzehnrubl, 1989). The ability to cryopreserve unfertilized eggs would alleviate a number of ethical and legal problems resulting ,from frozen storage of “spare” embryos in human IVF programmes, assist ovum donation, and confer maximum flexibility upon strategies for human and domestic animal assisted reproduction. Vitrification procedures, developed for the rapid and straightforward cryopreservation of mammalian embroys (e.g., Rall and Fahy, 1985; Massip et al., 19861, have shown some promise for use with oocytes (Trounson 1986; Feichtinger et al., 1987; Kola et al., 1988), but overall rates of survival, fertilization, and subsequent development have been poor compared to those achieved with embryos. The present study set out to apply an established method for embryo vitrification, that of Rall and Fahy
0 1991 WILEY-LISS, INC.
(1985), to mouse oocytes to establish whether the protocol could be tailored to the particular properties of these cells to improve success rates. Previous experiments (Shaw et al., 1990) had demonstrated that when exposed to the above procedure oocytes exhibited a variety of extreme osmotically induced morphological changes during the equilibration and dilution steps, which it was postulated might result in a reduction of cell viability. The present study applied the following sequence of changes to the original protocol to assess the effect on cell viability of attempts to reduce the osmotic problems described in Shaw et al. (1990): 1) adding intermediate steps to the multistep dilution procedure to modulate osmotic pressure changes, 2) replacing the multistep dilution with a single-step sucrose dilution to control osmotically induced volume changes, 3) extending the duration of the dilution step to enhance temperature and osmotic equilibration before the wash steps, and 4) extending the duration of the VS1-equilibration steps to ensure full equilibration before vitrification.
METHODS Oocyte Retrieval Unfertilized mature ovulated oocytes were obtained from virgin C57BL x CBA F1 hybrid female mice as described previously (Bernard and Fuller, 1983). Oocytes were stripped of their surrounding cumulus cells by incubation with hyaluronidase (150 units m1-l Bovine testicular hyaluronidase, Sigma UK) supplemented with 4 mg . ml-’ bovine serum albumin (BSA), and then washed three times in Dulbeccos phosphatebuffered saline (PBS) medium plus 4 mg * ml-’ BSA (PB1).
Culture/Manipulation Conditions Morphologically abnormal oocytes were discarded at this stage, and the remaining oocytes distributed to three experimental groups. The first group was incu-
Received November 29, 1990; accepted March 27, 1991. Address reprint requests to Dr. P.W. Shaw, Academic Department of Obstetrics and Gynaecology, Royal Free Hospital School of Medicine, Pond Street, London NW3 2QG, United Kingdom.
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bated for 2 hours in modified Tyrode’s solution (T& Quinn et al., 1982) at 37°C under 5% C 0 2 + 5% O2 + 90% N2 to serve as a control. The second group was put through the vitrification procedure described below. The third group was put through the same procedure but without the vitrification step (“VS1exposed”).Eggs morphologically intact after processing were inseminated and cultured on for 5 days as described previously (Bernard and Fuller, 1983). Eggs were defined as morphologically intact if the cell was spherical in shape, possessed an intact plasma membrane, the cytoplasm was refractile, there were no cytoplasmic fragments inside the zona pellucida, and the zona pellucida was undamaged.
Vitrification Protocol The vitrification protocol used was that of Rall and Fahy (19851, which utilises a vitrification solution (VS1)containing permeating (dimethylsulphoxide, 1,2propanediol, acetamide) and nonpermeating (polyethylene glycol) agents in buffered saline. Briefly, oocytes were equilibrated in 25% VS1 at room temperature (20-22°C) for 15 minutes, and then dehydrated successively in 50% VS1 and 90% VS1 for 10 minutes each in a cold room (5-8’0, before transfer into a 45 pl drop of 90% VS1 in a plastic insemination straw (L’AIGLE, France) and plunging into liquid nitrogen. Visual examination of the straws confirmed the absence of visible ice in the medium. Straws were thawed in an iced-water bath and the contents expelled into 50%VS1 at 5-8°C for 10 minutes. Further steps of rehydration and elution of cyroprotectant consisted of 10 minutes in each of 25% VS1 at 5-8”C, and 12.5%VS1 and 6% VS1 at room temperature, before a final three washes in PB1. Experimental Groups Initially, a number of test runs were made using 8-cell mouse embryos, produced in vitro, to ensure that the level of survival described by Rall and Fahy (1985) could be reproduced, and therefore that the conditions used with the oocytes would be comparable to their results with embryos. Next, groups of oocytes were processed through the method as described above unchanged. Subsequently, based on observations of morphological and functional survival of processed oocytes at each step, the modifications described briefly in the Introduction above were introduced to the procedure t o attempt to improve survival. These will be described more fully in the results section together with their effects on oocyte viability.
transferred direct to culture without insemination after exposure to the vitrification, VS1-exposed, or control treatments.
Effect of Cooling The protocols used here employed periods of exposure of oocytes to room temperature (20-22°C) and cold room temperature (543°C). Such cooling of metaphase I1 oocytes has been reported to result in reduced rates of subsequent fertilization due to damage caused to the metaphase spindle (Johnson et al., 1988). To assess whether the cooling involved contributes to any reduction in fertilization rates seen in the experimental groups, oocytes were exposed to the temperature changes and durations prescribed in droplets of PB1 before insemination and culture. Further groups were similarly exposed to cooling but were incubated in T6 at 37°C under 5% COz + 5% O2 + 90% N2 for periods of 1 or 2 hours before insemination to see if a recovery period was needed for reassembly of the spindle, as described by Pickering and Johnson (1987). Viability of oocytes from all experimental and control groups was assessed at three levels: number of oocytes morphologically normal immediately post-treatment, number of oocytes fertilizing and cleaving to 2-cells 24 hours post-insemination, and number of oocytes developing to expanded blastocysts. Data Analysis Batches of oocytes from all straws within treatments each day were pooled immediately after dilution and washing, and then cultured on and assessed as one group. Any differences between groups of data of interest were tested by standard chi-square analysis. RESULTS The results of the standard vitrification procedure and all subsequent modifications as applied to mouse unfertilized oocytes, together with their corresponding VS1-exposed groups, are presented in Table 1. Also presented are data for the pooled control groups and the vitrification of 8-cell embryos. All figures represent the pooled results of a number of individual batches (one batch per straw, around 15 oocytes per batch) of oocytes processed through each treatment on at least 3 separate days. Vitrification of 8-Cell Embryos After some practice we managed to attain similar levels of survival and development to blastocysts of vitrified %cell embryos [R and F (Embryos) in Table 11 as described by Rall and Fahy (1985)-75.6% compared to 83.5%in controls.
Parthenogenetic Activation Oocyte Vitrification: Rall and Fahy’s Method Parthenogenetic activation of oocytes after exposure (R and F in Table 1) to a cryoprotectant has been reported by Shaw and Trounson (1989). In order to elucidate whether any When oocytes were vitrified by Rall and Fahy’s such activation took place in oocytes exposed to the (1985) method, 65.7% appeared morphologically norprotocol used in this study, groups of oocytes were mal in ‘culture after treatment, with 79% of these
VITRIFICATION OF MOUSE OOCYTES
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TABLE 1. Survival of Oocytes Vitrified by Methods Modified From Rall and Fahy (1985) and Feichtinger et al. (1987) Percent morphological survival
Percent fertilized Percent (percent 2 - c e l l ~ ) ~ blastocystsa
N Treatment R and F (embryos) Control 97 100 90.7 (91) Vitrified 123 73.2 76.4 (100) Oocytes 543 100 89.7 controls R and F Vitrified 137 65.7 51.8 (79) VS1 exposure 120 76.7 60.0 (78) R and F (+18%) Vitrified 51 45.1 35.3 (78) VS1 exposure 56 96.4 78.6 (81) F “10 10” Vitrified 80 75.0 52.5 (70) VS1 exposure 35 91.4 45.7 (50) F -10 2079 Vitrified 58 60.3 51.7 (86) VSl exposure 47 93.6 91.4 (98) F “15 25” Vitrified 139 79.9 66.2 (83) VS1 exposure 127 97.6 82.7 (85) aFigures in brackets represent percentage of previous step.
+
+ +
fertilizing and cleaving to 2 cells. Only 46% of these %cell embryos developed to expanded blastocysts, giving an overall rate of development to blastocysts of vitrified oocytes of 24.1%. In the corresponding VS1exposed group rates of morphological survival, fertilizationkleavage, and development to blastocysts were very similar to the vitrified group, with an overall rate to blastocysts of 30.8%.
83.5 (92) 75.6 (99) 84.7 (94) 24.1 (46) 30.8 (51) 29.4 (83) 75.0 (95) 33.8 (64) 45.7 (100) 43.1 (83) 72.3 (79) 55.4 (84) 72.4 (88)
logical survival of the process, the overall success rate to blastocysts was only marginally improved over the original protocol (29.4% versus 24.1%, respectively).
One-Step Sucrose Dilution (F“10-t10” in Table 1)
An accepted method for controlling osmotically induced excursions in cells during the elution of permeating cryoprotectants is a l-step dilution with a sucrose solution, as suggested by Feichtinger et al. (1987) for Modified Rall and Fahy Method [R and F (+18%) use with this vitrification method. This modification in Table 11 (F“lO+lO” in Table 1) uses a 10 minute equilibration An obvious point of destruction of VS1-exposed step with 25% VS1, followed by 5 minutes each in 50% oocytes during the R and F protocol occurred by lysis of VS1 and 90% VS1, and a 10 minute l-step dilution with the cells at the first dilution step at room temperature, 0.8 M sucrose. Application of this method resulted in between 25% VS1 and 12.5%VS1. This was probably a much improved morphological survival of vitrified result of over-rapid increase in cell volume, involving oocytes (75.0%,x2 = 12.0P < 0.05) but reduced rates of “blistering” by the plasma membrane as described by subsequent fertilization and development (only 64% 2 Shaw et al. (19901, resulting in rupture of the mem- cells to blastocysts). Similarly, morphological survival brane and lysis. In order to reduce the magnitude of the of VS1-exposed oocytes were high (91.4%)but overall osmotic pressure change at this point the first modifi- rates to blastocysts were lower than with the R&F cation of the protocol was made: introduction of a (+ 18%)method due to reduced fertilization. further dilution step with corresponding scaling of Extended One-Step Dilution steps below it (25% VS1-18% VS1-10% VS1-5% VS1(F“lOt20” in Table 1) wash). The result of this modification [R and F (+ 18%) The R and F (+ 18%)modification demonstrated that in Table 11 was a dramatic increase in both the morphological survival of VS1-exposed oocytes (to modulating the severity of osmotic (and temperature) 96.4%)and, perhaps more interestingly, in the propor- changes during the dilution steps could improve subtion of 2-cell embryos developing to blastocysts (95%). sequent development of fertilized eggs, as well as the This gave an overall rate of VS1-exposed oocytes to immediate benefits to oocyte survival. Therefore, the blastocysts of 75.0% (the proportion of morphologically next modification tried was an extension of the dilution normal eggs fertilizing was unaltered by the modifica- step of the F‘lOt10” method to 20 minutes, to allow tion, x2 = 0.216 P > 0.05). The rate of development of 2 more controlled temperature equilibration (10 minutes cells to blastocysts resulting from vitrified oocytes was in cold room, 10 minutes at room temperature) and thus similarly improved, but due to a reduction in morpho- transmembrane movement of water and permeating
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cyroprotectants. The results (F“10+20” in Table 1) indeed show a small, and mostly nonsignificant, increase in fertilization rate of surviving oocytes (86%in vitrified, x2 = 2.97 P > 0.05; 98% in VS1-exposed, = 24.3 P > 0.05) and subsequent development (83% in vitrified, x2 = 3.16 P > 0.05). This results in a return of overall rates of survival of VS1-exposed oocytes to blastocysts to greater than 70% as seen in R and F (+18%),and an improvement in overall success with vitrified oocytes (to 43.1%) despite a slight nonsi nificant reduction in morphological survival = 3.57 P > 0.05).
x2
(3
Extended CPA Equilibration (F“15+25” in Table 1) It was noted that mouse oocytes did not appear to fully equilibrate with 25% VS1 at room temperature after 10 minutes (i,e., the oocytes did not reexpand to the volume seen in oocytes exposed for 15 minutes, personal observation), so equilibration time was extended to 15 minutes to assess whether this had any advantageous (or detrimental) effects. The result (F“15+25”in Table 1)was an improvement in morphological survival of vitrified oocytes (to 79.9%),without any substantial change to subsequent fertilization and developmental potential, giving a final overall survival of vitrified oocytes to blastocysts of 55.4%. No single modification resulted in a significant improvement over the prior protocol in overall survival to blastocysts of vitrified oocytes (x2 = 0.73 to 3.20, all P > 0.05). However the improvement in overall survival of vitrified oocytes to blastocysts between the initial (R and F) and final (F“15t25”)procedures was very significant (x2 = 27.21, P < 0.001). Parthenogenetic Activation In the tests for parthenogenetic activation of oocytes the F‘15+25” protocol was used for the vitrified and VS1-exposed groups. No signs of activation or cleavage were seen up to 3 days after treatment in any of the vitrified (30 oocytes), VS1-exposed (42 oocytes), or control (20 oocytes) groups. The control inseminated group (27 oocytes) demonstrated normal levels of fertilization (93%) and development to blastocysts (89%).
Effects of Cooling The results of the experiments to assess the effect on fertilization and development of oocytes subjected to temperature changes and durations prescribed by the F“15+25”protocol are displayed in Table 2. None of the experimental groups show significantly different rates of fertilization and development to blastocysts compared to the control group (x2 = 0.05 to 232, all P > 0.05). DISCUSSION Since the first demonstrations of successful cryopreservation of oocytes (Tsunoda et al., 1976; Whittingham, 1977),some considerable achievements have been made in improving morphological survival, fertilization, embryonic development, and even the production of live young from frozen or vitrified oocytes (Critser et al., 1986; Nakagata, 1989; Lewin et al., 1990; Schroeder et al., 1990). The present study shows that significant improvements can be made on existing methods by small modifications of procedure tailoring the method towards the material involved. More important perhaps, this study suggests some links between changes in specific parts of a protocol and effects on different aspects of oocyte survival such as morphological integrity and the potential for fertilization and further development. Such information can be of use in further attempts to improve success rates. One general point to be made from this data is that modulating osmotic stresses to which oocytes are exposed during the dilution phase not only improves morphological survival but also appears to correlate with improved potential of fertilized oocytes to develop further. Improved morphological survival resulting from regulating volume excursions (multistep dilution) or allowing time for temperature equilibration (sucrose dilution) presumably relates to maintaining plasma membrane integrity and avoiding lysis. A number of mechanisms have been proposed to explain membrane damage resulting from osmotic forces, including over expansion of cell membranes after loss of membrane surface area during dehydration (Steponkus and Wiest, 1979) and disruption of membrane integrity by mass movement of water (Muldrew and McGann, 1990).How such processes could affect the ability of resulting embryos to develop is difficult to explain at present.
TABLE 2. Effect of Cooling on Oocyte Fertilization and Development Treatment
N
Percent morphological survival
Percent fertilized (percent 2-cells)
Percent blastocysts
Control Cooled Number of incubation 1 hour incubation 2 hour incubation Pooled
64
100
82.8
79.7
87 108 120 315
100 100 100 100
80.5 81.5 85.0 82.5
75.9 68.5 70.0 71.1
VITRIFICATION OF MOUSE OOCYTES The results presented here also support the hypothesis that osmotically induced damage is much more important than any detrimental effects resulting from biochemical toxicity of the VS1 solution over the durations and temperatures of exposure utilized. Large improvements in survival and viability of oocytes were gained by solving problems of osmotic damage without any concomitant detrimental effects from the increased periods of exposure used. Therefore, for several protocols examined overall rates of development to blastocysts of VS1-exposed oocytes approached those seen in controls, given the small losses in morphological survival and the possible effects of cooling on development (see below). This indicates that concern regarding the biochemical toxicity of some of the cryoprotectants used (Weisburger et al., 1969; Baxter and Lathe, 1971; Rall, 1987; Van der Elst et al., 1988; and see Fahy et al., 1990) may not be relevant to the short exposure protocols used in vitrification, an advantage that would support the application of these methods. The possible effects of vitrification methods in producing chromosome anomalies and defects in young born from vitrified oocytes needs to be investigated further given the inconclusive reports for their occurrence (Kola et al., 1988) or absence (Whittingham, 1977; Nakagata, 1989). Whether oocytes need complete equilibration with a certain dilution of the VS1 solution to protect them from damaging instantaneous osmotic stresses or dehydration effects during dehydration and vitrification is open to question. Extending equilibration time (F‘10+20”versus F“15+25”) improved survival in this study, and most published methods for successful embryo vitrification employ an equilibration period (e.g., Rall and Fahy, 1985; Scheffen et al., 1986; Rall, 1987; Kasai et al., 1990). However, Nakagata (1989) reports a method of vitrification of mouse oocytes which achieves 87.6% morphological suvival using only a 5 to 10 second exposure to the undiluted vitrification solution before plunging into LN2.In our hands only 54%of mouse oocytes vitrified by the Nakagata method survive morphologically (unpublished results), but it still suggests that previtrification equilibration may not be of major importance compared to conditions of vitrification and, especially, dilution. Pre-equilibration of oocytes with cryoprotectants may still be advisable however, particularly in the case of more sensitive human material, if periods of cooling are employed, in order to protect them from cooling damage, as suggested by the work of Van der Elst et al. (1988). The present data also demonstrate that the vitrification and thawing steps definitely have a detrimental effect on oocyte survival, ,but that these are only exhibited in the form of immediate post-vitrification losses and have no effect on the ability of surviving oocytes to fertilize and develop. This is illustrated by a comparison of all pairs of vitrified and corresponding VS1-exposed groups in Table 1. If the percentage development from the previous step is examined in each case it can be seen that for almost all pairs of
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figures there is very little difference between vitrified and VS1-exposed groups except in the figures for morphological survival of processing. In the final protocol used (F‘15+25”) the difference in immediate losses due to physical destruction of oocytes between the VS1-exposed and vitrified groups is 15.2%. What could cause such losses? One obvious source of destruction of oocytes is by “zona cracking”: Rall and Meyer (1989) concluded that splits in the zona of bovine ova were most likely caused by fracturing of the vitrified medium resulting from thermal stresses created during warming. We have previously concluded (Shaw et al., 1990)that the same phenomenon results in the damage seen in vitrified mouse ova. In this study such damage accounts for the destruction of 4% to 5% of oocytes on average. The remaining 10%of losses may be caused by ice damage to the cells as a result of devitrification of the medium during thawing. Although the vitrification medium used (90% VS1 of Rall and Fahy, 1985) successfully vitrifies, we consistently observe a 2-3 second period of devitrification (the medium becomes opaque white) during thawing in an ice bath. Takahashi et al. (1986)reported that ice crystals formed both extra- and intra-cellularly during devitrification of a similar medium and that the presence of intracellular ice corresponded to a significant reduction in survival of the vitrified monocytes. Our finding that the only detrimental effects of vitrification and thawing on oocytes are immediate is in contrast to the results of Kola et al. (1988) who reported that there were also differences in rates of fertilization, cleavage, and development on between vitrified and VS1-exposed oocytes. A number of studies have suggested that cooling of unfertilized oocytes, even to room temperature, may impair their ability to be fertilized and develop normally (Magistrini and Szollosi, 1980; Pickering and Johnson, 1987;Johnson et al., 1988; Van der Elst et al., 1988). This study shows that for mouse oocytes, under the conditions used in these protocols, no significant reduction in fertilization and development occurs. However, mouse oocytes are known to be able to recover from changes induced by cooling (Glenister et al., 1987; Pickering and Johnson, 1987), so caution would still be needed if such methods were transferred to human oocytes. One interesting point in the data was that although there were no significant changes in ability of fertilized cooled oocytes to develop and divide, all three cooled groups exhibited a decrease compared to the control group. If this is a real effect it would explain a similar decrease in viability in the VS1-exposed experimental groups, as mentioned above. Overall, the results presented here suggest that vitrification could be an acceptable method for cryopreservation of unfertilized ova. The final rates of development to blastocysts of vitrified mouse oocytes are comparable with the best results achieved by standard slow-freezing methods (e.g., Schroeder et al., 1990)or ultra-rapid freezing methods (e.g.,Lewin et al., 1990).
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This work is supported by grant number SP2032 from the Cancer Research Campaign.
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