Ultrarapid freezing of early cleavage stage human embryos and eight-cell mouse embryos*

FERTILITY AND STERILITY Copyright 1988 The American Fertility Society Printed in U.S.A. Ultrarapid freezing of early cleavage stage human embryos and eight-cell mouse embryos* Alan Trounson, Ph.D.t:!: Anita Peura, B.Sc. t Lesley Freemann Carol Kirbyt Centre for Early Human Development, Monash University, Monash Medical Centre, and Infertility Medical Centre, Epworth Hospital, Melbourne, Victoria, Australia Early cleavage stage human embryos and 8-cell mouse embryos were snap-frozen after a brief exposure to high concentrations of dimethyl sulfoxide (DMSO; 2 or 3.5 M) and 0.25 M sucrose and thawed in a warm water bath. Eleven of 12 3- to 8-cell human embryos survived freezing and thawing with more than 50% of their original blastomeres intact. However, pregnancy was not initiated when the 11 embryos were transferred to six patients. It was shown that continuation of embryonic development in vitro and in vivo was significantly better when 8-cell mouse embryos were snap-frozen in 3.5 M DMSO than in 2 M DMSO. When frozen in 3.5 M DMSO, 78% of 8-cell embryos survived on thawing, 84% developed to blastocysts in vitro, 63% implanted, and 42% developed to fetuses. Ultrarapid freezing is a quick and inexpensive method for mouse embryo cryobanking, but further studies are required to confirm the viability of frozen human embryos. Fertil Steril 49: 822, 1988 It has been shown that 2-cell mouse embryos may be successfully cryopreserved after a brief exposure to high concentrations of dimethyl sulfoxide (DMSO) at room temperature by plunging them directly into liquid nitrogen and thawing them in a warm water bath. l This ultrarapid freezing method could be used for the routine cryopreservation of mouse strains 2 and of embryos and cells of other species if it could be shown to be successful over a range of species and embryonic cleavage stages. In the present article, we have examined the effects of Received October 19, 1987; revised and accepted January 28, 1988. * Supported by a grant from the National Health and Medical Research Council, Canberra, Australia. t Centre for Early Human Development, Monash University, Monash Medical Centre. :j: Reprint requests: Alan Trounson, Ph.D., Centre for Early Human Development, Monash Medical Centre, 246 Clayton Road, Clayton, Victoria, Australia 3168. Infertility Medical Centre, Epworth Hospital. 822 Trounson et al. Ultrarapid embryo freezing ultrarapid freezing on the survival of early cleavage stage (3- to 8-cell) human embryos and on the survival and development of 8-cell mouse embryos in vitro and their viability when transferred to foster mothers in vivo. MATERIALS AND METHODS Freezing Human Embryos Six patients consented to have 12 of their 3- to 8-cell embryos ultrarapidly frozen, thawed, and replaced in utero. These embryos were in excess of the patients' needs following established procedures of in vitro fertilization (IVF) for the treatment of their infertility.3 In our own IVF program, a maximum of three embryos are normally replaced in utero on the cycle of IVF treatment and any excess embryos are cryopreserved for the patients. 4 The embryos were frozen 48 to 64 hours after insemination using the ultrarapid technique described previously. 1 The embryos were trans- Fertility and Sterility

ferred from culture medium (Whittingham's T6 + 10% maternal serum) into Dulbecco's phosphate-buffered medium (PBl)/ containing 20% fetal calf serum (FCS), 2 M DMSO, and 0.25 M or 0.5 M sucrose, drawn into a 0.25-ml plastic freezing pailette, l the pailette heat-sealed and then plunged into liquid nitrogen. The time of exposure of the embryos to DMSO before plunging into liquid nitrogen was 2 to 4 minutes. Embryos were kept stored in liquid nitrogen for 4 to 8 months and thawed at the appropriate time in the ovulatory cycle of replacement' by transferring the pailette from liquid nitrogen to a water bath at 37 C. The contents of the pailette was gently expelled into PBI, containing 20% FCS and 0.25 M sucrose, for 10 minutes at room temperature. Embryos then were transferred to culture medium (Whittingham's T6 + 10% maternal serum) and incubated for 2 to 4 hours at 37 C before transfer to the patient's uterus. 5 The number of cells immediately after thawing and again just before transfer was noted. Freezing Eight-Cell Mouse Embryos Eight-cell mouse embryos were obtained from superovulated Fl hybrid females (CBA X C57) and handled using previously described protocols. l,6 The embryos were flushed from excised oviducts approximately 68 hours after administration of 5 IV human chorionic gonadotropin (hcg; Chorulon, Intervet, Sydney, Australia) to control the time of ovulation. Only morphologically normal8-cell embryos were used in the experiments. Embryos from up to five females were pooled on each occasion of freezing and the embryos randomly allocated to an untreated control group, a solution control group, and a freezing group. Embryos in the control group were kept in PBl + 20% FCS at room temperature for the duration of the experimental procedures (0.5 to 1 hour) and placed in droplets of culture medium (Whittingham's T6 + 20% FCS) under light-weight paraffin oil at 37 C in 5% CO2 in air, after handling and freezing of embryos in the other groups. The embryos were cultured for 48 hours. Embryos in the solution control group were exposed to the cryoprotective solutions for the same periods of time as those embryos that were frozen. Two different cryoprotective solutions were used. The first solutions consisted of 2 M DMSO and 0.25 M sucrose dissolved in PBl + 20% FCS, and the second contained 3.5 M DMSO instead of 2 M DMSO. Embryos in the solution control group were exposed to 2 or 3.5 M DMSO solutions at room temperature for 3 minutes and then were transferred to PBl + 20% FCS containing 0.25 M sucrose for 5 to 7 minutes at room temperature, washed in PBl + 20% FCS and then T6 + 20% FCS, and placed in droplets of culture medium (T6 + 20% FCS) equilibrated with 5% CO2 under light paraffin oil, and cultured at 37 C in 5% CO2 in air for 48 hours. Embryos in the freezing groups were exposed to either 2 or 3.5 M DMSO for 3 minutes at room temperature (20 C to 22 C). The embryos were pipetted into 0.25 ml pipettes, as previously described/ and the pipette heat-sealed during the 3 minutes of equilibration of embryos in the cryoprotective solutions. The pailettes then were plunged into liquid nitrogen at the end of the 3- minute equilibration period and left in liquid nitrogen storage for 1 hour to 4 weeks. The embryos were thawed in a 37 C water bath and the contents of the pailette expelled into PBl + 20% FCS containing 0.25 M sucrose for 5 to 7 minutes, then washed and cultured in vitro as described for the solution controls. The number of apparently normal expanded and hatching blastocysts was recorded at the completion of 48 hours of culture in vitro. A sample of the blastocysts from each group was fixed and spread on microscope slides and stained with 10% Giemsa 7 (British Drug Houses, BDH, Poole, England). The number of nuclei stained with Giemsa were counted to determine the number of cells in each blastocyst and the mean number of cells in blastocysts for each group determined. A random sample of embryos from each group was transferred to the uteri of pseudopregnant recipient mice that had been mated to vasectomized males. Embryos were transferred after 24 hours of culture in vitro to recipients 1 day earlier in their ovulatory cycles than the donors of the 8-cell embryos. Each recipient had embryos of two different groups transferred to opposite uterine horns. The groups were coded before transfer in order to remove any bias. The number of normal fetuses, abnormal or resorbing fetuses, and implantation sites in recipient mice were recorded 12 days after transfer, and the group codes broken at the completion of the experiments. Statistical Analysis Data on the survival and development of embryos in vitro and in vivo were analyzed by chi- Trounson et ai. Ultrarapid embryo freezing 823

Table 1 Ultrarapid Freezing of Early Cleavage Stage Human Embryos Patient Cleavage No. of cells intact no. stage frozen after thawing 1 8-cell 8 cells 4 cells a 2 0 3-cell 3 cells 4 cells 3 3 cells 4 6-cell 5 cells 5 4 cells 4 cells 7-cell 5 cells 6 7-cell 7 cells 7-cell 5 cells a Cleaved to 8 cells within 3 hours of thawing. square tests and data on blastocyst cell number analyzed by the Student's t-test. RESULTS Survival of Ultrarapidly Frozen Human Embryos The survival of human embryos after ultrarapid freezing is shown in Table 1. Only 1 of the 12 embryos failed to survive with more than 50% of its cells intact after thawing. Seven of the 12 embryos had their full complement of cells intact. One embryo divided from 4 to 8 cells during the brief period (3 hours) of culture after thawing and before transfer to the patient. All surviving embryos appeared to have well-preserved morphologic features as assessed by dissecting microscopy. None of the six patients became pregnant after replacement of the apparently surviving frozen-thawed embryos in utero. Survival and Development of Ultrarapidly Frozen Eight-Cell Mouse Embryos In Vitro There was no significant difference in the proportion of embryos developing to expanded and hatching blastocysts between the untreated control groups and those embryos of the solution control groups exposed to either 2 or 3.5 M DMSO solutions (Table 2). There was no significant difference in the survival of embryos after ultrarapid freezing and thawing in the 2 or 3.5 M DMSO solutions (Table 2). The proportion of embryos developing to blastocysts was significantly reduced from 98% to 84% (X 1 2 = 12.5; P < 0.001) when 8-cell embryos were frozen in 3.5 M DMSO. There was a further significant reduction to 60% blastocysts (x/ = 13.5; P < 0.001) when embryos were frozen in 2 M DMSO. Viability In Vivo of Ultrarapidly Frozen Eight Cell Mouse Embryos Each recipient foster mother had control embryos transferred to one uterine horn and treated embryos to the other, so that failure to become pregnant was likely to be due to a maternal factor. Hence, data on embryonic viability are presented and analyzed for recipients that became pregnant (Table 3) in order to remove maternal effects. There were no significant differences in implantation rates or the proportion of fetuses from embryos in the untreated control group and the embryos exposed to 3.5 M DMSO solutions or frozen in 3.5 M DMSO (Table 3). For some unknown reason, the proportions of implantations and fetuses were significantly higher in embryos exposed to 2 M DMSO than for untreated (control) embryos (Table 3). However, for embryos frozen in 2 M DMSO, there was a significant reduction in the number of implantations and fetuses below that for Table 2 Survival and Development in Culture of Ultrarapidly Frozen Eight-Cell Mouse Embryos No. of embryos Proportion of embryos Treatment Concentration Survived after developed to blastocysts group ofdmso Frozen thawing in culture M (%) (%) Control 97/98 (9W Solution control 2.0 107/109 (98)b Frozen 2.0 283 223 (79)a 56/93 (60)< Control 109/111 (9W Solution control 3.5 132/134 (9W Frozen 3.5 304 236 (78)a 98/117 (84)d a-d Figures in columns with different subscripts are significantly different (P < 0.001). 824 Trounson et al. Ultrarapid embryo freezing Fertility and Sterility

Table 3 Implantation and Development to Fetuses of Ultrarapidly Frozen Eight-Cell Mouse Embryos Transferred to Pseudopregnant Recipients No. of embryos Treatment Concentration transferred to No. of No. of group ofdmso pregnant recipients implantations fetuses M (%) (%) Control 81 46 (57)a 41 (51)d Solution Control 2.0 78 62 (7W 57 (73)e Frozen 2.0 95 40 (42)' 10 (11)1 Solution Control 3.5 77 46 (60)a 43 (56)d Frozen 3.5 93 59 (63)a 39 (42)d a 1 Figures in columns with different subscripts are significantly different (P < 0.01). all other groups (Table 3). More embryos frozen in 3.5 M DMSO than 2 M DMSO implanted (X 1 2 = 7.7; P < 0.01) and developed to fetuses (X12 = 22.5; P < 0.001). In the untreated (control) group, there were four malformed fetuses and one intrauterine death. There were lower numbers of malformations in the other groups (one in the 2-M DMSO solution control group, two in the 2-M DMSO frozen group, zero in the 3.5-M solution control group, and one in the 3.5-M frozen group) and no other intrauterine deaths. Cell Numbers of Blastocysts The mean numbers of cells in blastocysts in the various groups are shown in Table 4. There was a smaller number of cells in blastocysts derived from embryos frozen in 3.5-M DMSO than in the control or solution control groups, but this was not statistically significant. However, there was a significant reduction (t = 2.66; P < 0.02) in cell numbers of blastocysts derived from embryos frozen in 2 M DMSO (Table 4). Table 4 Cell Numbers of Blastocysts Developing from Ultrarapidly Frozen Eight-Cell Mouse Embryos Cultured In Vitro No. of Treatment Concentration blastocysts group ofdmso analyzed Control 33 Solution control 2.0 23 Frozen 2.0 14 Solution control 3.5 18 Frozen 3.5 24 M a Significantly different from other group means. No. of cells (mean ± 8D) 69.3 ± 17.4 67.3 ± 16.2 39.4 ± 13.4a 72.2 ± 18.7 57.4 ± 23.8 DISCUSSION The present experiments confirm our earlier repore that early cleavage stage embryos can be successfully snap-frozen using the simple technique of ultrarapid freezing. Human embryos survived freezing and thawing at exceptionally high rates when compared with other methods of cryopreservation. 4 8 The failure to establish pregnancy after transfer of the ultrarapidly frozen embryos to the six patients may have been due to the small sample size, the retarded cleavage stage of some of the embryos frozen, or the reduced viability of embryos frozen in 2 M DMSO. The latter is suggested by the results obtained for freezing 8-cell mouse embryos. In this case, despite equivalent survival of embryos after freezing and thawing in 2 and 3.5 M DMSO, development of mouse embryos to blastocysts in culture and viability of embryos when transferred to foster mothers was significantly reduced after freezing in 2 M DMSO when compared with 3.5 M DMSO. The marked reduction in fetal development of implanting blastocysts noted in our original report 1 was again evident in 8-cell mouse embryos frozen in 2 M DMSO (only 25% of implantation sites were represented by fetuses) and to a lesser extent in embryos frozen in 3.5 M DMSO (66% of implantation sites represented by fetuses). This may be explained by the reduction in cell numbers of blastocysts (Table 4) in both groups. Ultrarapid freezing probably results in damage to some blastomeres that is not evident at the dissecting microscope level or a decrease in cell division rate of the surviving blastomeres; this would reduce the capacity of embryos to continue normal growth and development beyond the implanting blastocyst stage in vivo. It would be of interest to use more sensitive tests of cell function, such as exposure to trypan Trounson et al. Ultrarapid embryo freezing 825

blue or fluorescein diacetate, or assessment of their metabolic activity by incorporation of radiolabelled precursors, in order to determine whether a difference in the number of viabile blastomeres after freezing and thawing in 2 and 3.5 M DMSO contributes to the difference in cell numbers and embryo viability at the blastocyst stage. However, despite some reduction of cells at the blastocyst stage, the results of ultrarapid freezing in 3.5 M DMSO are at least as good as the more conventional slow-freezing techniques,9 making this method an attractive alternative for the cryopreservation and banking of mouse embryos.2 There have been reports of more involved methods of rapid freezing of 8- to 16-cell lo and morula stage ll mouse embryos in liquid nitrogen vapor. In these experiments, high concentrations of glycerol (3 M) and sucrose (0.25 M)10 or glycerol (2 M) without sucrose ll were found to result in relatively high rates of survival after freezing and development to blastocysts in vitro. However, survival rates, development to blastocysts in vitro, and viability after transfer to foster mothers ll,12 appears to be higher using the ultrarapid freezing technique described in this article and the method is simpler and faster than freezing in liquid nitrogen vapor as described in these reportsy,12 It is of interest to note that our attempts to ultrarapidly freeze mouse and cattle blastocysts have not been successful to date using the method described in this article, but the vapor freezing of early blastocysts in 2-M glycerol appears to be successful. ll The development of simple, low-cost, and rapid embryo freezing techniques will be of major benefit to reproductive technologies in all species. The results obtained for the ultrarapid freezing of human embryos have encouraged us to initiate a major clinical trial of the technique in our IVF programs. This trial will take considerable time because we are required by law (Infertility Medical Procedures Act of the State of Victoria) to transfer all frozen embryos to the patients rather than evaluate them by any damaging procedure. This necessitates that we wait until the patients request the thawing and transfer of their frozen embryos, which varies from 3 months to several years. It is possible that early cleavage stage human embryos could be successfully frozen ultrarapidly in 3.5-M DMSO and 0.25- M sucrose, but it should be noted that we are unable to confirm the viability of human embryos frozen in this manner with the data presently available. REFERENCES 1. Trounson A, Peura A, Kirby C: Ultrarapid freezing: a new low-cost and effective method of embryo cryopreservation. Fertil Steril 48:843, 1987 2. Mobraaten LE: Mouse embryo cryobanking. J In Vitro Fert Embryo Transfer 3:28, 1986 3. Trounson A: In vitro fertilization and embryo transfer. In In Vitro Fertilization and Embryo Transfer, Edited by A Trounson, C Wood. Edinburgh, Churchill Livingstone, 1984, p 111 4. Trounson A: Preservation of human eggs and embryos. Fertil Steril 46:1, 1986 5. Leeton J, Trounson A, Jessup D, Wood C: The technique of human embryo transfer. Fertil Steril 38:156, 1982 6. Caro CM, Trounson A: The effect of protein on preimplantation mouse embryo development in vitro. J In Vitro Fert Embryo Transfer 1:183, 1984 7. Kola I, Folb PI: The effects of cyclophosphamide on alkaline phosphatase activity and on in vitro post-implantation murine blastocyst development. Dev Growth Differ 27:645, 1985 8. Cohen J, Simons RF, Edwards RG, Fehilly CB, Fishel SB: Pregnancies following the frozen storage of expanding human blastocysts. J In Vitro Fert Embryo Transfer 2:59, 1985 9. Whittingham DG: Some factors affecting embryo storage in laboratory animals. In The Freezing of Mammalian Embryos, Edited by K Elliot, J Whelan. Amsterdam, Excerpta Medica, Ciba Foundation, 1977, p 97 10. Szell A, Shelton IN: Sucrose dilution of glycerol from mouse embryos frozen rapidly in liquid nitrogen vapour. J Reprod Fertil 76:401, 1986 11. Miyamoto H, Ishibashi T: Liquid nitrogen vapour freezing of mouse embryos. J Reprod Fertil 78:471, 1986 12. Szell A, Shelton IN: Osmotic and cryoprotective effects of glycerol-sucrose solutions on day-3 mouse embryos. J Reprod Fertil 80:309, 1987 826 Trounson et al. Ultrarapid embryo freezing Fertility and Sterility