Problems in the cryopreservation of unfertilized eggs by slow cooling in dimethyl sulfoxide*

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1 FERTILITY AND STERILITY Vol. 52, No.5, November 1989 Copyright" 1989 The American Fertility Society Printed on acid-free paper in U.S.A. Problems in the cryopreservation of unfertilized eggs by slow cooling in dimethyl sulfoxide* Alan Trounson, Ph.D. t Carol Kirby:j: Centre for Early Human Development, Monash University, Monash Medical Centre, Melbourne, Victoria, Australia The survival, fertilization, development, and viability in vitro and in vivo of~nfertilized mouse eggs frozen by slow cooling to -36oC or -80oC in 1.5M dimethyl sulphoxide (DMSO) was examined in a series of experiments which explored some of the problems in freezing the egg. DMSO was added to the eggs at either room temperature or at ooc. Maximum success rate (42% of frozen eggs developing to-two cells) was obtained when DMSO was added at OOC and the eggs slow cooled to -80 C. Removal of cumulus failed to improve freezing success rates. Addition of DMSO at temperatures above ooc significantly reduced the fertilizing capacity of eggs. Excessive exposure of eggs to temperatures. around l5 C also caused a significant reduction in fertilization rates. The effects of DMSO and cooling on fertilization are likely to be due to zona hardening by cortical granule release and to disorganization of the egg cytoskeleton and plasma membrane. These problems will be difficult to overcome if cryopreservation of the unfertilized human egg is preferred to the fertilized egg or early cleavage stage embryo in clinical in vitro fertilization. Fertil Steril 52:778, 1989 Unfertilized mouse eggs have been successfully cryopreserved by equilibrating in 1.5M dimethyl sulfoxide (DMSO) at o c, slow cooling (0.39.C/ min) to -8o c and slow warming (7.8.C) from -65 to -100CY In the experiments reported by Whittingham, 1 66% to 72% of eggs survived freezing and thawing and 50% to 62% fertilized and developed to two cells. Glenister et al. 2 reported that 36% of frozen eggs fertilized and developed to two cells. In both studies the proportion of fertilized eggs which implanted was the same for frozen and nonfrozen eggs. Interest in the cryopreservation of unfertilized eggs has increased with the clinical application of in vitro fertilization (IVF) for the treatment of hureceived March 6th, 1989; revised and accepted July 7, * Supported by a grant from the National Health and Medical Research Council, Canberra, Australia. t Reprint requests: Alan Trounson Ph.D., Centre for Early Human Development, Monash Medical Centre, 246 Clayton Road, Clayton, Victoria, 3168, Australia. :j: Present address: Surgicentre, Wesley Hospital, Auchenftower, Queensland, Australia. 778 Trounson and Kirby Freezing unfertilized eggs man infertility. It may be considered preferable to cryopreserve human gametes rather than embryos to avoid ethical issues of disposal, donation, or dispute about frozen embryos. Chen3 4 reported that at least 80% of unfertilized human eggs and 75% of mouse eggs survived freezing and thawing when added to 1.5M DMSO at o c, equilibrated for 20 minutes, ice nucleated (seeded) at -7 c, and slow cooled (0.5.C/min) to -36 c to -4o c before rapid cooling to -196 C. Eggs were thawed rapidly in a 45 c waterbath. Al-Hasani et al. 5 were unable to repeat the high success rates claimed by Chen3 4 and reported that only 20 of 144 (14%) human oocytes fertilized after freezing and thawing in DMSO. Furthermore, "28% of the fertilized eggs were polyspermic. Similar problems of low survival rates (5% to 29%) were encountered by the same group when freezing unfertilized rabbit eggs6 and our own group when freezing human eggs. 7 In the present studies we have examined the survival and viability of unfertilized mouse eggs, frozen by slow cooling in 1.5M DMSO using some of the variations reported in the literature. We have

2 also attempted to determine the procedural steps in the slow-freezing procedure which have the major effect on survival, fertilization, and viability of mouse eggs to enable the design of more appropriate freezing procedures to improve the success rate of cryopreserving the unfertilized mammalian egg. MATERIALS AND METHODS Source and Collection of Eggs Cumulus masses containing ovulated eggs were dissected from the ampullae of 3 to 4 week old superovulated F1 (C57BL/6J X CBA/CaHWehi) hybrid mice mated to F1 males. Female mice were superovulated with 5 IU pregnant mare serum gonadotrophin (Folligon, Intervet, Lane Cove, New South Wales, Australia) followed 50 hours later with 5 IU human chorionic gonadotrophin (hcg) (Chorulon, Intervet, Lane Cove, New South Wales, Australia). Eggs were recovered 14 to 15 hours after hcg injection by teasing the cumulus mass out of the ampulla in organically buffered M2 medium. 8 All cryoprotective and wash solutions were made up in M2 medium. Freezing Methods Four primary variations of freezing by slow cooling in 1.5M DMSO were used to cryopreserve eggs. DMSO was added to the eggs at either ooc or at room temperature (20 C to 22 C) and slow cooled to either -36oC or -sooc before rapid cooling to -196 C. Method A was identical to that reported by Whittingham 1 and Glenister et al. 2 Eggs or cumulus masses (4 to 8 masses/vial) were placed in 0.3 ml of M2 medium in glass freezing vials and the vial placed in ice water for 10 minutes. An equal volume (0.3 ml) of ice cold 3M DMSO was added slowly to the vial containing eggs, the vial capped and equilibrated at ooc for 15 minutes. The vials were cooled to -7oC at 0.5 C/minute in a Planar biological freezer (Planar PTC 200, Sunbury on Thames, England), ice was seeded by touching the vial at the fluid meniscus.with precooled (-196oC) forceps and ice permitted to grow by holding the vial at -7oC for 15 minutes. Eggs were cooled slowly (0.5oC/minute) to -sooc and then rapidly to -196oC and the vials stored in liquid nitrogen for 1 to 14 days. Eggs were thawed by warming the vials slowly from -sooc to +4 oc at soc/minute. A total of 2.4 ml of M2 medium was added slowly to Vol. 52, No.5, November 1989 the vial and the diluted contents of the vial allowed to stand for 20 minutes at room temperature. The eggs or cumulus masses were removed from the vial and washed twice in M2 medium before culture in T6 medium 8 and insemination 1 to 2 hours later. Method B was the same as that reported by Chen. 3 4 DMSO was added at ooc, equilibrated for 15 minutes, cooled to -7 C, the solution seeded, held for 15 minutes, and the eggs cooled to -36oC at 0.5 C/min before rapid cooling to -196oC and storage in liquid nitrogen. Eggs were thawed by transferring the vials to a 37oC waterbath and 2.4 mls of M2 added to the 0.6 ml of 1.5M DMSO solution at room temperature. Otherwise the procedure was the same as described for method A. Method C was similar to that reported by Trounson. 7 DMSO (0.3 ml of 3M) was added to 0.3 ml M2 with the eggs or cumulus masses and equilibrated at room temperature (20oC to 22 C) for 5 minutes. The freezing vial was capped and cooled to -7oC at 2oC/minute. Ice was seeded in the vial and held for 15 minutes at -7oC, and the eggs were slow cooled (0.5 C/minute) to -sooc, ~tored in liquid nitrogen and thawed as described for method A. Method D involved the addition of DMSO at room temperature and cooling to -7oC as described for method C. The eggs were slow cooled (0.5 C/ min) to -36 C, stored in liquid nitrogen and thawed as described for method B. The method of freezing was coded so that after thawing the method of cryopreservation was unknown until the final results were obtained. In Vitro Fertilization Sperm were prepared from F1 hybrid males as described by Glenister et al. 2 and the eggs or cumulus masses, transferred 1 to 2 hour later to 50 ~L of sperm suspension in T6 medium containing 15 mg/ ml Fraction V BSA (bovine serum albumin, Sigma, St. Louis, Mo., USA) under light weight paraffin oil (BDH, Kilsyth, Australia). Eggs were incubated in the sperm solution (1 to 2 X 10 6 /ml) for 4 to 5 hours at 37oC in a humidified atmosphere of 5% C0 2 in air. At the termination of insemination, eggs were removed from the sperm solution, washed twice in T6 medium containing 10% fetal calf serum (FCS) (Flow Laboratories, North Ryde, New South Wales, Australia), and cultured overnight (20 hours) in droplets of T6 + 10% FCS under oil in a humidified atmosphere of 5% C02 in air at 37 C. The numbers of degenerate eggs and those with or without pronuclei were noted before culture Trounson and Kirby Freezing unfertilized eggs 779

3 overnight and the number of two cell embryos recorded the next day. Embryo Transfer Two cell embryos were transferred to the oviducts of day 1 pseudopregnant F1 (C57BL X CBA) hybrid recipients mated to vasectomized random bred Swiss male mice. Transfers were carried out in the afternoon after detection of copulation plugs in recipient mice in the morning (day 1). Recipients were anaesthetized with tribromoethyl alcohol in tertiary amyl alcohol and one to six two cell embryos transferred to each oviduct. Two different treatment groups of embryos were transferred separately to each oviduct of a recipient. Recipient mice coded by an ear clipping system were killed on day 14 or 15 of pregnancy and the number of implantation sites and normal fetuses recorded for separate uterine horns. Removal of Cumulus Cells Cumulus cells were removed by transferring cumulus masses immediately after recovery from the oviducts of donor mice, to M2 medium containing 300 JLg/mL hyaluronidase (Type IV-S, Sigma). Cumulus cells were dispersed (within 5 minutes) by gentle swirling in a petrie dish on a 37 C warm plate. The cumulus denuded eggs were washed twice in M2 and transferred to droplets of T6 medium under oil until frozen or inseminated (within 1 to 2 hours). Cumulus denuded eggs were frozen by methods A, C and D, or not frozen. Eggs were inseminated after thawing and the number of two cell embryos recorded as described earlier. Effect of Cooling and DMSO on Unfertilized Eggs Cumulus masses obtained from donor mice were transferred to 0.3 ml M2 medium in a glass-freezing vial and placed in an incubator at 37oC or a Planar biological freezer at 15,4, 0, or -7oC for 15 minutes. The vials were brought to 37oC and the cumulus masses removed, transferred to T6 medium, inseminated, and the number oftwo cell embryos recorded as described earlier. The two cell embryos were transferred to droplets of fresh T6 + 10% FCS under oil and cultured at 37oC in a humidified atmosphere of 5% C0 2 in air for 72 hours. The proportion of expanded and hatching blastocysts were recorded at the end of culture. In a second series of experiments, we examined: (1) the effect of ice seeding after cooling oocytes to -7 C in M2 medium; (2) the effect of addition of DMSO to oocytes at room temperature, cooling to -7 C, and ice seeding; (3) continued cooling (0.3oC/min) to -36oC; (4) rapid cooling to -196oC; and ( 5) the complete freezing procedure with an additional 15 minutes holding period at 0 C. Eggs were warmed rapidly from the final temperature to which they were cooled to (a,b -7 ; c -36 ; d,e -196 C) by immersion of the freezing vials in a 3TC waterbath. DMSO was removed and eggs inseminated and cultured to two cells and to blastocysts as described earlier. The effect of DMSO on survival, fertilization, and capacity of eggs to develop to blastocysts was examined by exposing eggs to 1.5M DMSO at 37, 15, 4, and ooc for 5, 10, 15, and 20 minutes. DMSO was removed, eggs inseminated and the number of two cell embryos and blastocysts recorded after culture as described earlier. Statistical Analysis Data were analysed by x 2 tests. RESULTS Survival, Fertilization, and Viability of Frozen-thawed Eggs As shown in Table 1, the highest survival rate (72%) of eggs after freezing and thawing was obtained when DMSO was added at ooc and the eggs slow cooled to -80oC (method A). The lowest survival rate (15%) occurred when DMSO was added at ooc and the eggs slow cooled to -36oC (method B). When DMSO was added at ooc and eggs were slow cooled to -80"C, 42% fertilized and developed to two cells. Significantly less eggs fertilized and developed to two cells (16%) when DMSO was added at room temperature and very few (3% to 4%) developed to two cells when eggs were cooled to -36oC when DMSO was added at either ooc or room temperature. Even with the best freezing method (method A), the proportion of eggs that survived and developed to two cells was only about 50% of nonfrozen eggs fertilized in vivo or in vitro and grown in vivo or cultured in vitro (Table 1). There was no significant difference between freezing methods in the viability of two cell embryos obtained after insemination of frozenthawed eggs, as determined by the proportion of embryos which developed to fetuses when transferred to recipients (Table 2). However, the two cell 780 Trounson and Kirby Freezing unfertilized eggs

4 Table 1 The Survival and Fertilization of Frozen-Thawed Mouse Eggs No. of eggs No. of eggs intact after freezing and thawing" No. of frozen eggs fertilized and developed to two cellsb Method of fertilization and culture Fertilized in vivo/grown in vivo Fertilized in vivo/cultured in vitro Fertilized in vitro/cultured in vitro Freezing method A. DMSO added o /slow cooled to -so B. DMSO added o /slow cooled to -as C. DMSO added RT /slow cooled to -so D. DMSO added RT /slow cooled to -as S S13 S21 41S 7S2 441 (72) 93 (15) 17S (43) 19S (25) 310 (SS) 213 (79) 4S7 (91) 2SO (42) 17 (3) S5 (1S) 2S (4) a Survival rates for freezing methods were significantly different (P < 0.001) to one another. Values in parentheses are percents. b The number of eggs developing to two cells was significantly (P < 0.001) reduced when fertilized in vivo and cultured in vitro embryos derived from frozen-thawed eggs produced only about 50% of normal fetuses that were obtained with nonfrozen eggs fertilized in vivo or in vitro and grown in vivo or cultured in vitro to the two cell stage (Table 2). This was due to an increase in both the failure of implanting embryos to continue development to fetuses and a reduced implantation rate of embryos derived from frozenthawed eggs (Table 2). Survival and Fertilization of Cumulus Denuded Frozen-thawed Eggs There was a marked reduction in the survival rate of eggs (3% to 5%) in which the cumulus had when compared with the other two methods of fertilization and culture. Development of eggs frozen by method A was significantly (P < 0.001) reduced when compared with nonfrozen culture controls and development of eggs frozen by methods B, C, and D were significantly (P < 0.001) less than by method A. been removed before freezing and thawing, irrespective of the freezing method used (Table 3). Less than 1% of frozen-thawed cumulus denuded eggs fertilized and developed to two cells, compared with 91% of nonfrozen eggs fertilized in vitro and cultured in vitro (Table 3). Effect of Cooling on the Survival, Fertilization, and Development of Eggs There was a small but significant decrease in the proportion of eggs fertilized and developed to two cells when eggs were cooled from 37oC (94%) to 4 oc to -7 C (85% to 86%) but a further reduction was Table 2 Viability of Two Cell Embryos Derived from Frozen-Thawed Eggs when Transferred to Foster Mothers No. of two cell embryos transferred to No. of No. of normal recipients implantations a fetusesb Method of fertilization and culture Fertilized in vivo/grown in vivo Fertilized in vivo/cultured in vitro Fertilized in vitro/cultured in vitro Freezing method A. DMSO added o /slow cooled to -so B. DMSO added o /slow cooled to -as c. DMSO added RT/slovy cooled to -so D. DMSO added RT /slow cooled to -as 10S S (S9) ss (S3) S1 (SS) 50 (54) 117 (77) 97 (S4) S4 (55) 4S (30) 2 (40). 1 (20) 20 (42) 11 (23) 10 (53) 5 (2S) a The number of implantatio~s was significantly (P < 0.01) reduced by all freezing methods when compared with eggs fertilized in vivo and grown in vivo, or eggs fertilized in vitro and cultured in vitro. However, freezing methods A and D were not significantly different to eggs fertilized in vivo and cultured in vitro. There were no significant differences between freezing methods. Values in parentheses are percents. b The numbers of normal fetuses were significantly (P < 0.01) reduced by all freezing methods. Vol. 52, No.5, November 19S9 Trounson and Kirby Freezing unfertilized eggs 781

5 Table 3 The Effect of Removing the Cumulus of Unfertilized Eggs on Survival after Freezing No. of eggs No. of eggs intact after freezing and thawing" No. of frozen eggs fertilized & developed to two cells Method of fertilization and culture Fertilized in vitro/cultured in vitro Freezing method A. DMSO added o /slow cooled to -so C. DMSO added RT /slow cooled to -so D. DMSO added RT /slow cooled to SO 1594 S4 (5) 41 (5) 51 (3) 467 (91) s (1) 0 (0) 1 (0) " Values in parentheses are percents. observed if eggs were cooled and held at 15oC (70%) for 15 minutes (Table 4). It was also of interest to note that cooling of eggs to temperatures of 4 octo -7 C did not reduce the number of two cell embryos developing to blastocysts (Table 4). Effect of Different Steps in the Freezing Procedure on Survival, Fertilization, and Development of Eggs In Vitro As shown in Table 5, the survival rate of eggs was dramatically reduced from 94% to 41% when rapidly cooled below -36 C. Fertilization and development to two cells was reduced by the addition of DMSO at room temperature from 92% to 43%, but was not effected by the cooling procedures used (Table 5). The proportion of two cells developing to blastocysts was not significantly effected by the freezing procedures tested. The additional 15 minutes holding time at ooc significantly reduced the survival rate of frozen eggs and tended to reduce fertilization rate and development to two cells and to blastocysts, although this was not statistically significant (Table 5). Effect of DMSO on Survival, Fertilization, and Development of Eggs When DMSO was added to eggs at ooc for 5 to 20 minutes, there was no effect on the survival, fertilization, or development of eggs to two cells or blastocysts (Table 6). There were no significant effects on the survival of eggs after exposure to DMSO, but there were significant effects ofdmso on the proportion of eggs which fertilized and developed to two cells. When eggs were exposed to DMSO for 5, 10, and 15 minutes, there was a significant reduction (P < 0.01) in fertilization at temperatures above 0 C. The effect ofdmso on fertilization rate was most dramatic at 15 C, particularly when exposed to DMSO for 10 to 15 minutes. While the reduction in fertilization rate was still apparent when eggs were exposed to DMSO for 20 minutes (Table 6), the differences between the various temperatures did not reach statistical significance (x 3 2 = 3.31; NS). There was no significant effect of DMSO on the capacity of fertilized eggs to develop to blastocysts in vitro, although the pro- Table 4 Effect of Cooling on the Survival, Fertilization, and Development of Mouse Eggs Temperature ("C) to Proportion of Proportion of which eggs were No. of normal eggs fertilized two cell embryos cooled & held No. of eggs after and developed to developed to for 15 min eggs cooling" two cells : blastocysts b (96) (96) +4 50S 500 (9S) S9 (99) (94) " The proportion of eggs developing to two cells was significantly (P < 0.05) reduced when cooled to +4, 0 and -7 C and further significantly (P < 0.05) reduced when cooled and held at+ 1s c. Values in parentheses are percents. 767 /S20 (94) 656/767 (S5) 310/442 (70) lsl/215 (SS) 3S7 /445 (S7) 193/202 (96) 36S/432 (S5) 226/254 (90) 422/491 (S6) 273/301 (91) b The proportion of embryos developing to blastocysts was highest when eggs were cooled and held at +4 c, not different at -7 c but significantly (P < 0.05) reduced at 0, + 15 and +37 C. 782 Trounson and Kirby Freezing unfertilized eggs

6 Table 5 Effect of the Addition ofdmso, Cooling and Freezing on Survival, Fertilization, and Development of Unfertilized Mouse Eggs Procedure No. of eggs Proportion of Proportion of eggs fertilized two cell embryos No. of eggs and developed to developed to recovered" two cellsb blastocysts 1. Cooled in culture medium to -7", seeded & held for 15 min DMSO added RT & cooled to -7", seeded & held for 15min As for 2. then cooled to -36" As for 3. then plunged into LN As for 4. with additional15 min holding at o before cooling to -7" 389 The number of eggs recovered was significantly (P < 0.001) reduced by procedure 4 and further significantly (P < 0.001) reduced by procedure 5. Values in parentheses are percents. b The proportion of eggs developing to two cells was significantly (P < 0.001) reduced by procedures 2, 3, and 4 and further 153 (94) 58/63 (92) 51/58 (88) 209 (94) 51/119 (43) 43/51 (84) 231 (94) 63/141 (41) 53/63 (84) 121 (41) 31/71 (44) 24/31 (77) 103 (26) 15/58 (26) 8/15 (53) significantly (P < 0.05) reduced by procedure 5 when compared with procedures 2 and 3. The proportion of embryos developing to blastocysts was significantly (P < 0.05) reduced by procedure 5 when compared with procedures 1, 2, and 3. portions of blastocysts were reduced when eggs were exposed to DMSO at 4oC and 15oC (Table 6). DISCUSSION The results of the present study support the observations of Whittingham 1 and Glenister et al. 2 that unfertilized mouse eggs can be cryopreserved by slow cooling in 1.5 M D MSO to low subzero ternperatures ( -80 C). Survival and fertilization rates in these three studies were comparable when DMSO was added at ooc. Glenister et al. 2 reported 36% of frozen eggs fertilized and developed to two cells compared with 42% in the present study. The survival rates reported by Whittingham 1 varied from 66% to 72% and 50% to 62% of eggs fertilized and developed to two cells compared with 72% survival and 59% fertilization rates, respectively, in the present study. The survival rate of eggs after freezing and thawing was reduced if DMSO was added to eggs at room temperature. Irrespective of the freezing method, the viability of eggs which fertilized and developed to two cells when transferred to foster mothers, was significantly less than two Table6 Effect of Temperature and Time of Exposure to DMSO on Survival, Fertilization, and Development of Unfertilized Mouse Eggs Temperature Proportion of Proportion of at which Equilibration fertilized and two cell embryos DMSOadded time No. of No. of normal developed to develping to ("C) (minutes) eggs eggs recovered two cells blastocysts (97) 83/125 (64) 42/55 (76) (96) 78/138 (57) 45/48 (94) (97) 103/169 (61) 65/73 (89) (91) 88/139 (63) 63/71 (82) (99) 40/66 (61) 28/40 (70) (93) 11/64 (17) 8/11 (73) (100) 17/52 (37) 10/17 (54) (95) 35/56: (63) 20/35 (57) (98) 59/62 (95) 42/59 (71) (92) 32/56 (57) 23/32 (72) (96) 26/52 (50) 20/26 (77) (99) 53/68 (78) 42/53 (79) (98) 169/181 (93) 70/87 (80) (98) 147/155 (95) 99/110 (90) (99) 140/150 (93) 81/85 (95) (98) 106/123 (86) 57/71 (80) Values in parentheses are percents. Vol. 52, No.5, November 1989 Trounson and Kirby Freezing unfertilized eggs 783

7 cell embryos developing from nonfrozen eggs. This reduction in viability was not observed by Whittingham/ but it was of interest to note in his study that only 58% of implanting embryos derived from frozen eggs, developed to normal fetuses compared with 100% of embryos from nonfrozen eggs. We were unable to repeat the observations of Chen3 4 that high survival rates of unfertilized mouse eggs can be obtained by slow cooling in 1.5 M DMSO to -36 C. Using the method described by Chen,3 4 only 15% of eggs survived freezing and thawing and 3% offrozen eggs fertilized and developed to two cells. Chen3.4 did not examine the fertilization or viability of the mouse eggs that were frozen by the slow-cooling method he reported, but Michaelis and Hahn 9 claimed high fertilization and viability rates for mouse eggs slow cooled (0.3 I minute) to -32oC before plunging into liquid nitrogen. Studies reported by others on the freezing of human eggs by slow-cooling methods are at variance with the high survival ( 80%) and fertilization (83%) rates reported by Chen.3 4 In these other studies, survival rates varied from 25% to 36% and fertilization rates varied from 32% to 58%, giving a low overall rate of embryo development of eggs frozen. Polyploidy was not mentioned by Chen3 4 but Al-Has ani et al. 5 reported 28% of fertilized eggs were polyploid and Mandelbaum et al.12 reported 42% were polyploid, presumably a result of damage to the zona which cannot prevent the entry of multiple sperm when inseminated. Fertilized eggs and embryonic cells are not surrounded by a cumulus mass, but the removal of the cumulus did not improve the survival rate of unfertilized eggs. In the present study removal of the cumulus depressed the survival rate of eggs. Whittingham1 found that the removal of cumulus cells had no effect on the survival of frozen mouse eggs and similar results were reported for frozen human eggs by Mandelbaum et al.12 Some of the reasons for the loss of eggs during the freezing process were identified in the present study. Cooling of eggs to the seeding temperature (-7 C) had a very minor effect on their capacity to be fertilized. However, if eggs were kept at 15oC there was a much larger decrease in fertilizing capacity. This may be due to a nonreversible alteration of the egg cell plasma membrane because at this temperature there are critical changes in plasma membrane fluidity allowing rapid diffusion of surface antigens.13 Any undue exposure of eggs to temperatures around 15oC may alter the capacity of the egg cell membrane to bind or to fuse with sperm, causes release of some cortical granules which may harden the zona to sperm penetration, or change eggs in some other way which reduces their fertilizing capacity. Johnson et al.14 reported that cooling of mouse oocytes to 4 oc significantly reduced fertilization rate and hardened the zona to digestion by chymotrypsin and concluded that these effects were consistent with release at cortical granules during cooling. Dimethyl sulfoxide had a dramatic effect on the capacity of oocytes to be fertilized at temperatures above 0 C. Even at 4 oc, more than 5 minutes equilibrations reduced the egg's capacity to be fertilized. These results strongly support the view that DMSO should be added to eggs at OOC or below. Johnson and Pickering15 have shown that DMSO has profound effects on the egg microtubules, pericentriolar material, and chromosomes when added at temperatures between 4 oc and 37 C. Dimethyl sulfoxide causes disassembly of the spindle microtubules and movement of peri -centriolar material to the center for the egg. These effects are not completely reversible. The peri-centriolar material remains around the spindle after removal of DMSO resulting in the abnormal segregation of chromosomes and peri-centriolar material when eggs are parthenogenetically activated. They observed stray chromosomes which were not located on the equatorial plate in oocytes after removal ofdmso, raising the likelihood of aneuploidy if these eggs are fertilized. This prediction was confirmed by Kola et al.16 who found a dramatic increase in aneuploid zygotes after exposure of mouse eggs to vitrifying solutions containing high levels of DMSO and after vitrifying or freezing eggs by slow cooling in 1.5 M DMSO. While Johnson and Pickering 14 did not explore the effects of DMSO at ooc or lower, it is likely that these effects ofdmso would also occur at these lower temperatures and be at least partly responsible for the reduced viability of embryos derived from frozen-thawed eggs. In the present study the reduced embryo viability was expressed as a reduced development to fetuses when compared with nonfrozen eggs. It should, however, be noted that Glenister et al.2 were unable to show increased aneuploidy in zygotes formed from frozen-thawed mouse eggs. The direct effect of DMSO on fertilization rate shown in the present study is supported by the reduced fertilization rates reported by others for frozen-thawed eggs. 1 2 It is likely that the disruption to microtubular organization by DMSO observed by Johnson and Pickering15 may also effect the fer- 784 Trounson and Kirby Freezing unfertilized eggs

8 tilizing capacity of the egg. It is known that microtubule formation is required during fertilization, particularly in association with the 12 to 15 cytoplasmic asters which are assembled in the mouse egg during sperm incorporation and extrusion of the second polar body Inhibition of formation of the microtubules prevents the normal decondensation of the incorporated sperm nucleus and meiotic chromosomes. 17 The nonreversible effect of DMSO on the formation of these microtubules may contribute to the failure of fertilization and development to two cells observed in the present experiments. Dimethyl sulfoxide may also cause the release of cortical granules, inducing the zona reaction and preventing sperm penetration of the zona, resulting in reduced fertilization rates. Dimethyl sulfoxide may be more effective than cooling on cortical granule release than <;:ooling because of its known membrane fusagenic properties. Although the effect of DMSO was temperature dependent, exposure of eggs to this cryoprotectant caused a more dramatic reduction in fertilization rate than cooling. The possibility of using other cryoprotectants to avoid the problems of low survival, reduced fertilization capacity, and disruption to the egg cytosketal organization is remote at the present time. It has been shown that 1,2-propanediol is a potent parthenogenetic activator of unfertilized eggs 19 and problems similar to those encountered with freezing human eggs by slow cooling in DMSO are observed with 1,2-propanediol Glycerol is a more slowly-permeating cryoprotectant for mouse eggs than DMSO and extensive permeation of the cell is required for cryoprotection, 20 but like DMSO, glycerol has stabilizing effects on microtubules 21 and would probably result in the same disruption to microtubular organization as DMSO. In fact, the strqng hydrogen-bonding characteristics of cryoprotectants may equate with their capacity to stabilize microtubules because they reduce effective intracellular water. The dramatic reduction in survival rate of eggs when rapidly cooled from -36oC to -196oC (Table 5) would suggest that there is substantial intracellular water which nucleat~s during rapid cooling below -36oC forming lethal intracellular ice. This is supported by the increased survival rate of eggs when slow cooled to -sooc before rapid cooling to -196 C. Continued slow cooling below -36oC would increase cellular dehydration and reduce the chance of intracellular ice formation. The addition of extracellular solutes which increase dehydration of the cell may improve survival of eggs slow cooled to -36 C. However, an additional15 minutes equilibration time at ooc to increase the permeation of the cell by DMSO did not i~prove survival of the eggs. Human eggs have been cryopreserved by vitrification 7 which avoids the problems of ice crystal formation. However, this involves the use of high concentrations of cryoprotective solutes which exacerbate the problems for chromosomal normality and has been reported to cause fetal abnormalities when used to cryopreserve mouse eggs. 16 While the substance causing these abnormalities was not identified, acetamide which was included in vitrifying solutions to reduce the toxicity of DMSO and has mild carcinogenic properties, has been removed in more recent recipes for vitrifying solutions. 22 With these concerns, it would be unwise to use vitrification as a method of cryopreserving eggs for patients being treated for infertility. The results of the present study highlight some of the major difficulties faced in the cryopreservation of the mature unfertilized egg. The efficiency of freezing human eggs needs to be better than freezing the cleaving embryo because of the reduced pregnancy rate in the cycle of treatment for IVF if freezing eggs is preferred to embryos. 23 It is unlikely that this can be achieved because DMSO and probably other cryoprotectants, reduce the fertilizing capacity of eggs and increase the chance of chromosomal abnormalities. The survival of eggs is usually lower than cleaving embryos after freezing and thawing and in those eggs which do fertilize, the rate of polyploidy is dramatically increased. While it is difficult to know how all these problems can be solved, it is evident from studies on mouse eggs that DMSO should be added to eggs at temperatures below ooc and the eggs slow cooled totemperatures of -sooc or lower before storage in liquid nitrogen. It is also apparent that any undue exposure of eggs to temperatures around 15oC should be avoided and this should be noted by medical and scientific staff responsible for the handling of human eggs in clinical in vitro fertilization. REFERENCES 1. Whittingham DG: Fertilization in vitro and development to term of unfertilized mouse oocytes previously stored at -196 C. J Reprod Fertil 49:89, Glenister PH, Wood MJ, Kirby C, Whittingham DG: The incidence of chromosome anomalies in first-cleavage mouse embryos obtained from frozen-thawed oocytes fertilized in vitro. Gamete Res 16:205, 1987 Vol. 52, No.5, November 1989 Trounson and Kirby Freezing unfertilized eggs 785

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