Cryopreservation of mouse 2-cell embryos and ova by vitrification: methodologic studies

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1 FERTILITY AND STERILITY Copyright c 1987 The American Fertility Society Vol. 48, No.2, August 1987 Printed in U.S.A. Cryopreservation of mouse 2-cell embryos and ova by vitrification: methodologic studies Shevach Friedler, M.D.* Eve Shen, M.Sc. EmmetJ. Lamb, M.D. Department of Gynecology and Obstetrics, Stanford University, Stanford, California Cryopreserv'ation of unfertilized mouse ova and 2-cell embryos by a vitrification technique was examined. Survival was defined by development to the hatching blastocyst stage after in vitro fertilization. With 19 embryos at the 2-cell stage, the authors obtained 1% morphologic survival and 89% development to hatching blastocyst stage. To define the optimal conditions for vitrification of ova, the authors treated a total of 845 unfertilized ova. In experiments done at C, the concentration of vitrification solution (VS1) and the length of exposure of ova to VS1 both had significant (P <.1) effects on survival. The mean survival rate for controls in ten experiments was 52%. VS1 1% or 9% in HEPES buffered saline and 1 minutes' exposure yielded rates that did not differ significantly from controls. Significantly lower survival rates followed the use of 7 and 8% solution and exposure for 5, 15, 2, or 3 minutes. Thus, under these conditions, exposure of unfertilized mouse ova to VS1 and cooling to C did not interfere with in vitro fertilization and development of embryos. However, in five experiments in which a total of 11 ova were plunged into liquid nitrogen after treatment with VS1 under the optimal conditions, none could be fertilized in vitro. Fertil Steril 48:36, 1987 Most cellular damage occurring during freezing and thawing of cells or tissue is related to the formation of intracellular and extracellular ice crystals.1 Recently, successful ice-free preservation of mouse 8-cell embryos at -196 C by a process termed vitrification has been reported by Rall and Fahy. 2 This approach relies on the ability of highly concentrated aqueous solutions of cryoprotectants to supercool to very low temperatures. At sufficiently low temperatures, these solutions become so viscous that they solidify into a glass-like state without the formation of ice crystals. As a first stage in the development of a new approach to cryopreservation of human ova, we decided to examine the cryopreservation of mouse Received February 13, 1987; revised and accepted April 7, *Reprint requests: Emmet J. Lamb, M.D., Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, California Friedler et al. Vitrification of mouse embryos and ova ova by vitrification. Success in freezing and thawing of mouse embryos with development into normal living young after transfer into foster mothers had been obtained in Successful cryopreservation of unfertilized mouse ova using dimethyl sulfoxide (DMSO) as a cryoprotectant was reported in In that report by Whittingham, insemination after thawing resulted in fertilization, development, and birth of normal young. However, the rate of fertilization was significantly lower than that of the freshly inseminated control ova. A great deal of knowledge relating to cryopreservation of embryos of various mammalian species is now available. 5 Embryo preservation is now so successful in the cow that it is of great commercial importance. It has even been proposed that banks of frozen gametes and embryos should be used to preserve the gene line of species threatened with extinction and to aid in many areas of research in zoology. At the time of this writing, more than ten human pregnancies with subsequent normal devel-

2 opment of the infants have been achieved by embryo transfer after cryopreservation of embryos consisting of 1 to 8 cells Since several groups of investigators are studying the techniques of embryo cryopreservation, reports of success should continue to increase rapidly. The problem posed by excess embryos in programs for human in vitro fertilization and embryo transfer (IVF-ET) is a stimulus for research in human embryo cryopreservation. The protocols for ovulation enhancement used in such programs are designed to result in the harvest of ova from many follicles. The number of embryos available for transfer often is greater than that number desired by the couple and/or the program for the ratio of chance-of-anypregnancy to chance-of-multiple pregnancy that they consider optimal. Therefore, only a portion of available embryos is transferred. Moreover, part of the high rate of failure of implantation by transferred embryos may result from the hormonal effects of ovarian hyperstimulation on the endometrium. Delayed transfer of cryopreserved and thawed embryos to a uterus with more normal endometrium in subsequent unstimulated cycles has been proposed as a method of increasing the yield of pregnancies for each harvest procedure. Development of techniques for freezing unfertilized human ova might provide an even better solution to these problems. This approach would bypass some of the ethical and legal problems of embryo cryopreservation in humans, although it would present other such problems. If donation of cryopreserved ova were elected after a successful embryo transfer, insemination with sperm from the recipient's own partner would be possible. In this setting, cryopreservation would also facilitate appropriate synchronization with the menstrual cycle of the recipient. 9 In addition to its application for IVF, ovum cryopreservation might be used to preserve a fertility potential for women for whom there exists a threat of loss of ovarian function because of disease or the therapy of disease. Women may face such a threat before they have met or identified their future partner. Ovum preservation is, therefore, preferable to embryo preservation. When this study was begun, successful vitrification of unfertilized mouse ova had not been accomplished. Russel et al. 1 had reported survival of mouse ova after cryopreservation by vitrification, but the abstract lacked information about the numher of ova or the number of experiments. Since the completion of our research, Feichtinger and Ke- Vol. 48, No. 2, August 1987 meter, 11 Quinn and Kerin, 12 and Trounson8 have each reported their preliminary experience with the vitrification technique for cryopreservation of human eggs and embryos. The results are contradictory and none of the three groups had specific suggestions to change the vitrification protocol of Rall and Fahy 2 to accomodate to the needs of the unfertilized egg. Therefore, the methodologic studies reported in this article should be of interest. The major objective of our research was to define a protocol for vitrification of unfertilized mouse ova that would give optimal survival after freezing to -196 C. For these experiments, survival was defined by formation of blastocysts after in vitro fertilization and incubation. This required the following subsidiary objectives: (1) to adapt a previously described system for IVF of mouse ova that we could use as a functional end point indicating successful cryopreservation; (2) to demonstrate cryopreservation by vitrification of 2-cell mouse embryos by modifying the methods reported by Rall and Fahy2 for 8-cell embryos; and (3) to define the optimal concentration of vitrification solution and the optimal length of exposure at ooc for survival of unfertilized mouse ova. Sources of MATERIALS AND METHODS were obtained from five to 6-week-old Swiss Webster mice superovulated by the technique of Whittingham. 4 Hyperstimulation of the ovaries was obtained by intraperitoneal injection of 5. to 7.5 IU of pregnant mare serum gonadotropin (PMSG) followed 48 hours later by intraperitoneal injection of 5. to 7.5 IU of human chorionic gonadotropin (hcg). The mice were killed 14 to 16 hours after the hcg injection. The ova were released from the oviducts into modified Tyrode's medium (Table 1) enriched with.5% bovine serum albumin (BSA). Cumulus cells were separated by incubation in 1 ml of 1% hyaluronidase in HB1 (Table 1) for 1 minutes followed by three washes of 1 ml HB1 each. Preliminary unpublished studies in our laboratory using ova with attached cumulus had shown failure of morphologic preservation of ova. Experiment 1: Effects of the Concentration of Cryoprotectant and of the Length of Exposure to Cryoprotectant Without Freezing Experiment 1 assessed the fertilizability of mouse ova in relation to different final concentra- Friedler et al. Vitrification of mouse embryos and ova 37

3 Table 1 Composition of Media Used Effect of Four Different Final Concentrations Modified Components Tyrode's HB1 VS1 gmll NaCl KCl.2.2 CaCl2.2.4 MgCl2 6H2.1.5 NaH2P4 H2.5 KH2P4.12 NaHC3 1. Glucose Na Lactate 2.42 Na Pyruvate.4.4 BSA HEPES-Na form Penicillin.6 Phenol red.5% 2. ml DMSO ml Acetamide 155. Propylene glycol 1. Polyethylene glycol (mw 8) 6. NaOH as needed to ph= 8 ph= 7 H2 as needed to 1 ml D-PBS" as needed to 1 ml a = D-PBS, Dulbecco's phosphate-buffered saline (GIBCO Laboratories, Life Technologies, Inc., Chagrin Falls, OH) was prepared in bulk from powder. tions of vitrification solution, VS1 (Table 1), at predefined lengths of exposure without subsequent freezing and thawing. Common Pretreatment Groups of 15 (±9) eggs were permeated with a 25% solution ofvs1 in a Petri dish at room temperature (about 2 C) for 15 minutes. Dilutions were prepared in HB1 at ph 8.. The eggs then were transferred to a cold room (4 C), cooled to C in an ice-water bath, and exposed for 1 minutes to a 5% concentration of VS1 in HBl. Effect of Five Different Exposure Times This experiment was done to determine the length of exposure to VS1 that gave optimal survival rates. Groups of approximately 5 ova each, given the common pretreatment, were exposed to 1% VS1 at ooc for a controlled length of time: 5, 1, 15, 2, or 3 minutes. This experiment was done to determine the concentration ofvs1 for optimal survival. Four groups of approximately 6 eggs each, given the common pretreatment, were exposed to four concentrations (7, 8, 9, or 1%) of VS1 in HB1 for 1 minutes at C. Gradual Dilution and Rapid Warming Groups of ova exposed to each final concentration for each interval were immediately transferred to a 5% VS1 solution at ooc for 15 minutes and then to a 25% VS1 solution for 1 minutes. The eggs were then rapidly warmed to about room ternperature (2 C) by shaking on a heat block prewarmed to 37 C. The cryoprotectant solution was removed from the ova by sequential transfer to gradually decreasing concentrations (12.5, 6.25, and %) of VS1 in HB1 for 5 minutes at each step. Assessment of Survival by In Vitro Fertilization Survival was defined by development of expanded blastocysts after IVF of processed ova. The percentage reaching this stage was compared with that obtained in simultaneous control studies of untreated ova taken from the same mice and inseminated with the same sperm sample. from both treatment and control groups were washed three times in HB1 prior to IVF using the procedures described by Whittingham. 4 Epididymal sperm from Swiss Webster males were used in a dilution of 1 million motile sperm/ml, prepared in modified Tyrode's medium. After a period of 8 to 18 hours' incubation in a Queue cell culture incubator (Queue Systems, Inc, Box 191, Parkersburg, WV) at 37 C with 5% C 2 in air, the ova were washed free of sperm and transferred to Ham's F-1 embryo culture media ( G IBCO laboratories, Life Technologies, Inc., Chagrin Falls, OH) enriched with 2% BSA. Development was assessed every 24 hours for 4 days. The number of embryos that cleaved and developed to the expanded blastocyst stage was recorded and the percentages of the total eggs processed in each group that developed to that stage were calculated. The corrected fertilization and development in vitro rate for each treatment group was expressed as a percentage of the fertilization and development in vitro rate in the corresponding control group. 38 Friedler et al. Vitrification of mouse embryos and ova

4 Experiment 2: Effect of Vitrification and Rapid Cooling to -196 C Experiment 2 was designed to examine the rate of morphologic survival and fertilization of mouse ova that have been frozen in liquid nitrogen and rapidly thawed. Common Pretreatment Groups of 5 to 2 eggs were exposed to a 25% solution of VS1 in HB1 for 15 minutes at room temperature. The eggs were transferred to a cold room, cooled in an ice-water bath to C, and exposed for 1 minutes to 5% VS1 in HB1, followed by 1 minutes in 9 or 1% VSl. The eggs then were pipetted into the center of a drop, approximately 4 ~1, of the final concentration of VS1 solution and transferred to an individual.25-cm diameter plastic insemination straw (Instruments de Medicine Veterinaire, l'aigle, France) which was then heat-sealed. Rapid Cooling in Liquid Nitrogen The straws, each containing groups of about ten eggs in the center 4-~l drop, were directly cooled to -196 C by dropping them into a liquid nitrogen tank for 24 to 72 hours. Rapid Thawing Thawing to C was obtained by immersing the plastic straws in an ice-water bath, a procedure that gives an approximate warming rate of 25 C/min. 2 The content of each straw was expelled into a Petri dish containing a drop of 5% VS1 in HB1 to equilibrate for 15 minutes at C. Gradual dilution and rapid warming were then done as described in experiment 1. Assessment of Survival by In Vitro Fertilization Survival was assessed as described in experiment 1. Statistical Analysis The effects of the concentration of VS1 and of the length of exposure to VS1 on ova survival were examined by analysis of variance. Differences in survival between groups in each experiment also were assessed by the multiple range test. Computer methods were used. 13 Experiment 3: Cryopreservation by Vitrification of 2-Cell Mouse Embryos To test our ability to effectively use vitrification under our experimental conditions, we cryopreserved mouse embryos fertilized in vivo. Although Rall and Fahy 2 had used 8-cell embryos, we used 2-cell embryos for vitrification. Female Swiss Webster mice were treated with PMSG and hcg, as described previously. They were then caged with males from the same strain. Forty-eight hours after the hcg the mice were killed and the oviduct flushed with phosphate-buffered saline (PBS) enriched with 2% BSA to obtain the embryos. We used 1-minute equilibration steps at C to reach a maximal concentration of 1% VS1 before plunging the straws into liquid nitrogen for rapid deep-freezing to -196 C. After 3 days, the embryos were rapidly thawed by shaking the plastic insemination straws in an ice bath and the cryoprotectant was gradually removed by serial dilution, as described previously for ova. The embryos were cultured in Ham's F-1 media (GIBCO Laboratories, Life Technologies, Inc., Chagrin Falls, OH) enriched with heat-inactivated 2% human preovulatory serum in an incubator with 5% C 2 in air at 37 C for 94 hours. RESULTS The Effect of Increasing Length of Exposure Groups of ova were processed as described and exposed to 1% VS1 for various lengths of time between and 3 minutes (Table 2). Each experimental run consisted of several treatment groups and a control group of eggs harvested from the same mice, fertilized in vitro with the same batch of sperm, and placed in the same culture media in the same incubator. A total of 257 ova in 17 treatment groups and 128 ova in 6 control groups were used. The length of exposure to 1% VS1 had a significant effect on survival, as shown by one-way analysis of variance (P <.1; F = 23.1). Exposure for 1 minutes yielded a corrected survival (89.2%) that was not significantly lower than the survival of controls. exposed for 5, 15, 2, or 3 minutes had a significantly lower corrected survival using the least significant difference (LSD) procedure in the multiple range test at P = Vol. 48, No. 2, August 1987 Friedler et al. Vitrification of mouse embryos and ova 39

5 Table2 Effect of Length of Exposure at ooc in 1% VSl Rate(%) of Corrected rate (%) Length of No. of ova fertilization and of fertilization and Group exposure processed development development Mean± SE Control Control Control Control Control Control 1 6. Rate among treated Corrected rate = X 1. Rate among controls b ± ± ± ± ± b Mean (±SE) fertilization and development of controls = 52.7 ± 11%. Effect of the Concentration of Cryoprotectant Groups of ova were processed as described previously to four different final concentrations of vitrification solution (Table 3). Each experimental run consisted of several treatment groups and a control group of eggs harvested from the same mice, fertilized in vitro with the same batch of sperm, and placed in the same culture media in the same incubator. A total of 243 ova in 19 treatment groups and a total of 117 ova in 6 control groups were used. The concentration of VS 1 had significant effect on survival, as shown by one-way analysis of variance (P <.5, F = 3.93). The best corrected survival rates were with 1% VS1 (89.2%) and with 9% VS1 (8.9%). Neither was significantly lower than the control survival, whereas the rate achieved after processing the ova to 7 or 8% VS1 was significantly decreased according to the multiple range test at P =.1. Mouse In Vitro Fertilization The control groups shown in Tables 2 and 3, 189 ova in ten experiments, yielded a fertilization rate of 52.6% (±11.3%). 31 Friedler et al. Vitrification of mouse embryos and ova Effect of Vitrification and Rapid Cooling to -196 C on 2-Cell Embryos and Unfertilized All 19 of the frozen embryos survived morphologically and 17 (89.4%) continued to develop to the hatching blastocyst stage (Table 4). However, in five separate experiments using vitrification and deep-freezing to -196 C of unfertilized ova, a total of 11 eggs were processed as described previously in experiment 2 (Table 4) and no ova survived the procedure. DISCUSSION Rationale of Methods Used The successful cryopreservation experiments of Rall and Fahy 2 were conducted with 8-cell mouse embryos. Unfertilized ova might require an exposure time for complete permeation and dehydration different from that of the embryo. Several transport systems in both invertebrate and mammalian ova are altered simply upon fertilization of the egg even before cell division. For example, membrane potential, the rate of electrolyte and

6 Table 3 Effect of VS1 Concentration Rate(%) of Corrected rate (%) Length of No. of ova fertilization and of fertilization and Group exposure processed development development Mean± SE 1b b b b b b Control Control b Control Control Control Control Rate among treated Corrected rate = X 1. Rate among controls b Groups 1 to 5 and 2 and 22 are identical to groups 4 to 8 and 18 and 2 in Table 2, respectively ± ± ± ± d 1 1Qd In groups 1 and 17 of the experiment, no control group was run because of insufficient number of ova. d Mean (±SE) fertilization and development of controls = 48.1 ± 12%. amino acid transport, and passive permeability to other nonelectrolytes all increase after fertilization of the eggy Jackowski et aly demonstrated a slower rate of glycerol permeation through the cell membrane of unfertilized mouse ova. The same authors have shown that the extent of solute permeation can be controlled by altering the length of time and/or the temperature of incubation of ova or early embryos in glycerol. This is probably true also for other permeating cryoprotectants. Rall and Fahy 2 have demonstrated that exposure to vitrifi- cation solution produces time-dependent injury to multicellular embryos and that this injury can be prevented by use of lower temperatures. We decided that the unfertilized mouse ovum might require a longer exposure time than fertilized ova and that this would carry an increased risk of toxicity. Therefore, we examined the effect of using a lower temperature than that proposed for embryos by Rall and Fahy. 2 We began our studies with ova still within the cumulus mass. Whittingham 4 had reported that the Table 4 Effect of Freezing and Thawing Group Number Status Morphologic survival Fertilization and development cell embryo Vol. 48, No. 2, August 1987 Friedler et al. Vitrification of mouse embryos and ova 311

7 presence of cumulus cells during freezing and thawing did not affect the morphologic survival of mouse ova nor their subsequent fertilization in vitro. Moreover, because of the cellular connections between granulosa cells and the ovum, we looked upon the cumulus mass containing the ovum as functionally similar to a multicellular system. Cryopreservation of multicellular systems by vitrification proved to be efficient for whole-organ preservation as well as for the 8-cell embryo. 2 However, our initial trials, conducted using ova with cumulus cells still attached, failed. Having decided to continue our experiments with denuded ova, we noted several advantages. We could directly observe consecutive morphologic changes during freezing. Moreover, we could count the exact number of ova and calculate a success rate for the different procedures whereas, with an intact cumulus mass, we could not determine the exact number of ova present. Effect of Length of Exposure Vitrification solution was designed to reduce toxicity in several ways. Use of a nonpermeating agent, polyethylene glycol, reduces the required concentration of permeating agents. The use of a combination of acetamide, dextrose, and propylene glycol substantially reduces the required amounts of other more toxic permeating cryopreservants. However, substantial toxicity remains if exposure to this solution is prolonged. The rate of survival of 8-cell mouse embryos exposed to VS1 at 4 C declines after 15 minutes. 2 We found that the corrected survival rate for unfertilized ova was less than 5% when the exposure time exceeded 15 minutes. In this regard, unfertilized ova and embryos reacted in a similar way. At the two extremes of exposure time, however, ova and embryos differ. At one extreme, ova exposed to VS1 for only a short period (5 minutes) showed decreased survival, an effect not seen with embryos. A possible explanation is found in the different permeabilities of unfertilized and fertilized ova demonstrated by Jackowsky et aj.l 4 Five minutes' equilibration may be sufficient, only after the changes that occur with fertilization, for entry by the permeating components of the vitrification mixture. Therefore, the same length of time that suffices for embryos may be insufficient for permeation of unfertilized ova to the extent that the reagents can exert their protective effect. Thus, ova to a greater degree than early embryos may be ex- 312 Friedler et al. Vitrification of mouse embryos and ova posed to damage from sudden changes in osmolarity of the bathing extracellular solution. At the other extreme of exposure time, opposite results were obtained. After 3 minutes' exposure to VS1, ova had a mean corrected survival rate of 36%, whereas 2-cell embryos did not survive at all. Again, differences in the permeability of fertilized and unfertilized ova may explain this finding. Two-cell embryos allow more rapid permeation; thus, their cell contents are actually exposed to the toxic effects for a longer time than are the contents of unfertilized ova. Effect of Cryoprotectant Concentration Our findings of a direct correlation between the concentration of the vitrification solution and survival of ova are in agreement with the results with embryos found by Rail and Fahy. 2 More than 8% of embryos that had undergone equilibration, rapid freezing, and thawing, survived if concentrations of VS1 higher than 85% were used. Conversely, with lower concentrations ofvs1 (75%), only about half of the embryos survived rapid cooling and none survived slow cooling. Embryos permeated with lower concentrations of vitrification mixture probably still have enough intracellular water to form ice crystals during freezing or thawing. Ice formation during thawing is called devitrification. Effect of Deep Freezing In experiments carried out at C, ova survived exposure to high concentrations of VSl. However, in experiments involving exposure to -196 C, no ova survived to form blastocysts. The damage must have occurred during freezing or thawing. Factors that could result in failure of ova to survive the freezing experiments will be discussed subsequently. Lack of a subzero holding temperature may have been a factor. A holding period at temperatures around -3 C may have a beneficial effect on recovery of embryos and tissue culture cell suspensions after cryopreservation The method employed in the present study, lacking this holding period, may fail to provide sufficient time for dehydration and equilibration of the unfertilized oocyte. A second untested possibility is excessive volume of VS1 in the plastic straws. Critser et al. 19 reported significantly better rates of fertilization of hamster ova frozen in straws with 45 Jtl of VS1 versus 9 JLL They speculated that devitrification is more likely

8 to occur in the larger volume because of impairment of rapid heat transfer. The drops we used in the straws containing either the ova or embryos was about 4 p.l, but we did not attempt to accurately measure the volume nor to hold it strictly constant. This might have played some role in our poor results with ova. Developmental Stage Although we obtained survival of 2-cell embryos using the same procedures, equipment, strain of mice, and composition of VSl, we failed to obtain any survival of unfertilized ova. The changes in ovum membrane permeability that occur with fertilization were discussed previously. Additional changes certainly occur during subsequent developmental stages. Therefore, studies of the cryopreservation techniques most appropriate for different developmental stages have been conducted with both mouse 2 and human embryos. 21 DMSO, the main component of VSl, appears to be the optimal cryopreservative for intermediate, 4-cell to 8-cell, stages in the mouse as well as in the human. 4 s.s Conversely, propanediol seems best for the earlier, 2-cell stage, and glycerol for the later, blastocyst, stage Volumetric changes observed in unfertilized mouse ova 14 or human ova 23 indicate a rate of permeation by DMSO that is intermediate between the faster rate of propanediol and the slower rate of glycerol. A vitrification solution with a higher ratio of propanediol to DMSO might prove to be more suitable for use with unfertilized eggs than the one we used. Species Specificity Having observed lower survival of ova from the squirrel monkey than of ova from the hamster after DMSO cryopreservation, De Mayo et al., 24 postulated a difference in oocyte membrane permeability between the two species. In their studies of vitrification of hamster ova, Critser et ap 9 obtained both morphologic and functional survival, penetration by human sperm, after exposure to VSl without freezing. However, as in our studies of mouse ova, greater damage did occur after rapid cooling by direct plunging into liquid nitrogen and thawing. Less than 75% of the hamster ova survived morphologically and these demonstrated a lower frequency of penetration by human sperm. 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