Preservation of human eggs and embryos

FERTILITY AND STERILITY Copyright 986 The American Fertility Society Vol. 46, No., July 986 Printed in UBA. Preservation of human eggs and embryos Alan Trounson, Ph.D. Centre for Early Human Development, Monash University, Queen Victoria Medical Centre, Melbourne, Australia The introduction of superovulation techniques l into human in vitro fertilization (IVF) raised an immediate dilemma that large numbers of human eggs and embryos would be produced because it has never been possible to control precisely the number of follicles that develop in response to the administration of high doses of ovulationinducing drugs such as clomiphene citrate and human menopausal gonadotropin. The alternatives have been to restrict the number of eggs recovered or eggs inseminated to that required for replacement in utero; to replace all the embryos that develop; to discard eggs or embryos in excess of the number required for immediate replacement in utero; and to use the excess eggs or embryos for research purposes. It is apparent that most IVF groups have chosen one or more of these alternatives because very few groups have successfully integrated egg or embryo cryopreservation into their clinical IVF programs, thereby avoiding the inherent disadvantages of the other alternatives to the infertile patients. For example, the restricted collection of eggs risks that the viable eggs remain in follicles, that nonviable eggs are inseminated, and that unaspiratedfollicles persist as ovarian or luteal cysts. If the number of eggs inseminated is restricted and the excess discarded, the number of embryos available for replacement in utero will be reduced and have the direct effect of reducing the IVF pregnancy success rate. 2 The replacement of large numbers of embryos is an exceedingly risky proposition for maternal health and fetal normality. The sequelae of triplet, quadruplet, and larger multiple pregnancies are well known, and the consequences in IVF are already becoming apparent in perinatal statistics. 3 Vol. 46, No., July 986 The discarding of eggs and embryos is undoubtedly a widespread practice in human IVF. However, in my own view, this procedure is the least desirable or ethical alternative. It would be much better to study excess eggs or embryos so that we might improve IVF procedures, reduce embryonic defects, and increase the application of IVF and related technologies. It is unfortunate that most IVF groups are ill-equipped for embryonic research and that ethical approval of this type of research is becoming difficult to obtain. Given these situations and the present attitude of ma~y communities toward embryonic research, egg and embryo cryopreservation offers an attractive alternative to those mentioned earlier. This consideration was the basis for our own arguments 4 for the development of human embryo cryopreservation. Our own hospital's Ethics Committee supported the proposal in 98, and funding for the work was provided in 982 by our government's National Health and Medical Research Council. DEVELOPMENTS IN EMBRYO FREEZING The first successful reports of mammalian embryo freezing were published in 972. 5,6 Live mice were obtained following slow cooling of 8-cell embryos to temperatures of - 96 C in a dimethyl sulfoxide (DMSO)-based cryoprotectant. Modification of these methods was used successfully to cryopreserve rabbit,7 sheep,8 goat,9 and cow embryos.lo, These animal experiments showed that slow freezing of embryos to low subzero temperatures (- 80 C) requires slow thawing,5 transfer of embryos to liquid nitrogen at relatively high subzero temperatures (- 30 to Trounson Freezing human eggs and embryos

- 40 C) requires rapid thawing,2 embryos of many species have stage-specific requirements for successful cryopreservation,l and glycerol could also be used successfully as a cryoprotectant for late-stage embryos.3 These important observations became the basis for which a substantial commercial industry developed in cattle and sheep embryo banking, which is used extensively in these animal breeding industries. Mouse embryo banks have also been extensively developed for the genetic conservation and breeding of rapidly proliferating mouse strains.4, 5 Analyses of the data generated from embryo banking in the mouse and a wide variety of other species have shown that embryo freezing is a safe and effective method in animal breeding and that there is no evidence to suggest genetic alteration of welldefined, inbred strains of mice, increased birth defects, or any other abnormality, even when embryos are exposed to high levels of ionizing radiation;4 a consideration in the long-term preservation of embryonic material. DEVELOPMENT OF HUMAN EMBRYO FREEZING In 98 we began to apply the principles developed for mouse and cattle embryo freezing to cryopreservation of the human embryo. Our preliminary studies6 showed that embryos of all the developmental stages examined (2-cell to blastocysts) survived freezing and thawing in DMSOand glycerol-based cryoprotectants when frozen to either relatively high or low subzero temperatures. The survival of 4- to 8-cell human embryos is in direct contrast to the failure of embryos of many other species, in particular, domestic animals such as the cow, which survive freezing only at the late morula to expanded blastocyst stages.7 This observation led us to concentrate on the 4- to 8-cell human embryo because nonfrozen embryos of these stages resulted in pregnancies when replaced in utero in routine IVF practice. These observations were reported at a number of conferences in 98,8, 9 and by the end of 982 we were successful in establishing a pregnancy from a frozen-thawed 8-cell human embryo.20 The observations of interest at that time included the success of a DMSO-based cryoprotective medium for freezing, the apparent cellfusagenic properties of glycerol-based cryoprotective medium and the relatively low cell-permeability of glycerol in early cleavage stage 2 Trounson Freezing human eggs and embryos embryos/6 the success of freezing to relatively low subzero temperatures (- 80 C), and the viability of embryos despite freezing damage to some of the original blastomeres.2o We also made detailed observations on the ultrastructure of frozen-thawed human embryos2-23 and thereby confirmed the fusagenic properties of glycerol, which is lethal for embryonic development, and showed that extensive cell damage can result from intracellular ice formation, that abnormal embryo development can occur if damage to more than half the number of original blastomeres results, and that embryos may survive freezing and thawing with no apparent intracellular or membrane damage. The clinical results of pregnancies and births obtained in the initial studies on human embryo cryopreservation using DMSO as the cryoprotectant were published in 985 by Mohr. 24 It was apparent in this report that slow freezing to low subzero temperatures (- 80 C) before storage in liquid nitrogen resulted in significantly higher embryo survival rates than slow cooling to higher subzero temperatures (- 30 to - 40 C). Zeilmaker et al. 25 reported two pregnancies and births from a method of freezing in DMSO to a relatively high subzero temperature (- 40 C). However, no further reports have been published by this group, and high rates of success in the use of this method have not been confirmed by others. Successful freezing and thawing of human blastocysts by use of the freezing methods developed for cryopreservation of cow blastocysts has been reported by Cohen et al.26 They used a glycerolbased cryoprotective medium normally used to preserve cattle embryos and slow-cooled blastocysts to relatively high subzero temperatures ( - 40 C) before storage in liquid nitrogen. The cryopreservation of later stage embryos by use of this method appears to be a good alternative to slow-freezing early cleavage stage embryos to low subzero temperatures. 20 REQUIREMENTS OF HUMAN EMBRYO FREEZING A number of additions to normal IVF facilities are required for incorporating human embryo freezing clinically into infertility practice. A controlled biologic freezer is necessary for cooling accurately to subzero temperatures at rates of 0. to 0.5 C/minute. In our program we use Planar Biological Freezers (Planar Products, Sunbury- Fertility and Sterility

Table. Method for Slow Freezing Early Cleavage Embryos at Monash University Process Embryo culture Addition of cryoprotectant Packaging in glass ampoule Cooling to - 6 C Seeding at - 6 C Cooling to - 32 C Cooling to - 35 C Cooling to - SO C Cooling to - no c Storage in liquid nitrogen Medium Time or freezing rate T6 + 0% MHsa 40-S4 hours PBI + 0% FCS b 0 minutes 0.25 M DMSO 0.5 MDMSO.0 M DMSO 0 minutes 0 minutes 0 minutes 0 minutes 2 minutes 2 C/minute 30 minutes 0.3 C/minute O.PC/minute 0.3 C/minute 0 C/minute < 5 years C awhittingham's T6 + 0% maternal human serum. bphosphate-buffered Dulbecco's medium plus 0% fetal calf serum. cmaximum determined by Victoria's Infertility Medical Procedures Bill, 9S4. on-thames, UK), but a number of other controlled biologic freezers are also suitable. Liquid nitrogen facilities are also needed, including substantial liquid nitrogen storage tanks that must be maintained by automatic liquid nitrogen filling or by close supervision. It is absolutely essential that the embryologists who do the embryo freezing have substantial experience in the cryopreservation of animal embryos. We would normally expect embryologists to achieve a consistent survival rate of more than 60% of 2-cell mouse embryos frozen, developing the blastocyst stage when cultured in vitro after thawing. If survival rates are reduced after thawing or if embryos fail to develop normally to blastocysts, errors have been made in the cryopreservat ion technique; and these must be corrected before progressing to human embryo freezing. Some support of the importance of this experience is that all IVF groups that have been successful in developing and introducing embryo freezing into their IVF programs have experienced cryobiologists. There have been a large number of other groups who have had no success to date, and very few of the latter have had embryologists with any experience in cryobiology. There is also a need for additional clinical responsibilities for counseling patients on the implications of embryo preservation and for the management and tracking of patients for ovula- Vol. 46, No., July 9S6 tion during the cycle of thawing and embryo transfer. METHODS OF EMBRYO FREEZING The methods used for routine embryo freezing and thawing at Monash University are shown in Tables and 2. These methods follow the stepwise evaluation of different cooling and warming rates. The continued cooling from - 80 C to -0 C reduces damage to the zona pellucida, and the step of cooling very slowly between - 32 and - 35 C increases the survival rate of embryos, presumably through achieving greater dehydration of blastomeres. Other methods that have been investigated include the replacement of with 0% glycerol, 20, 26 cooling to higher subzero temperatures ( - 35 to - 40 C) before plunging into liquid nitrogen, 20, 25 reduction of the equilibration time at - 6 C after seeding,27 and the use of different base culture medium for mixture with the cryoprotectant.26,27 The replacement of DMSO with glycerol reduced early cleavage stage embryo viability, 20 and the transfer of embryos to liquid nitrogen at higher subzero temperatures reduced embryo survival. 24 It is not certain whether the other modifications benefit embryo survival and viability. In addition to these methods, our own group and others in Australia and France are examining variations of the procedure described by Renard and Babinet28 for the rapid thawing of mouse embryos frozen in propanediol, the cryoprotectant. Preliminary data indicate that high survival rates may be obtained with very early cleavage stages (I-cell pronuclear and 2-cell embryos), a phenomenon that has been difficult to achieve in the past.6 Table 2. Method for Thawing Early Cleavage Stage Embryos at Monash University Process Thawing between -SOD to +4 C Mixing during dissol ution of ice Removal of cryoprotectant Embryo culture Medium.25 M DMSO.0 M DMSO 0.75 M DMSO 0.5 M DMSO 0.25 M DMSO T6 + 0% FCS Time or thawing rate SOC/minute minute 2-5 minutes 0-2 minutes 0-2 minutes 7.5-0 minutes 7.5-0 minutes 5-7 minutes -4 hours Trounson Freezing human eggs and embryos 3

The freezing of human blastocysts 26 29 involves the use of glycerol instead of DMSO and slow freezing to about - 40 C before plunging into liquid nitrogen. Embryos are always thawed rapidly from - 96 C by agitation of the frozen ampule in a water bath at + 20 to + 30 C, when transferred to liquid nitrogen at high subzero temperatures. The reason for rapid thawing is to prevent the regrowth of minute intracellular ice crystals that may be formed during freezing through incomplete dehydration of the embryos. There are some critical steps in the embryo freezing and thawing technique, irrespective of the methods used. It is important to mix cryoprotective solutions completely because cryoprotectants such as DMSO, glycerol, and propanediol do not immediately mix when added to culture medium. One must adequately expose the embryos stepwise to increasing (before freezing) or decreasing (after thawing) concentrations of cryoprotectant to ensure that equilibration occurs before moving to the next concentration. This step requires careful pipetting of embryos through the various solutions. During dissolution of ice when thawing, it is important to mix completely the cryoprotective solution to avoid exposing embryos to excessive concentrations of cryoprotectant caused by layering when these solutions thaw. When ice crystallization (seeding) is induced, it is essential not to precool the ampule below - looc and to induce the formation of ice crystallization at the liquid meniscus rather than inducing the rapid and complete solidification of the medium at the time of seeding, a procedure that risks rapid precooling and rewarming. One should check the ampule to ensure ice formation after seeding and before beginning to cool to lower temperatures. Embryos may also be frozen successfully in plastic straws. The straws are sealed with a heat sealer, and air spaces are usually used to isolate the embryo from the extremities of the plastic straws. The straws are also seeded at - 6 C with precooled forceps, and the straws are touched toward their extremities and not directly over the embryos. THAWING AND TRANSFER OF FROZEN EMBRYOS Patients requesting the thawing and transfer of their embryos advise the IVF program at the beginning of their menstrual cycle. The method we 4 Trounson Freezing human eggs and embryos use for determining the onset of the luteinizing hormone (LH) surge has been described by Freemann et al.30 The 95% confidence interval for the LH surge is calculated from past menstrual cycle lengths, and blood samples are obtained twice daily, beginning on the first day of this confidence interval, and assayed for estrogen, progesterone, or LH. Less precision is obtained with urine samples, although a self test urinary dipstick assay3 is presently under evaluation for timing of the onset of the LH surge and transfer of frozen embryos. Ovulation is confirmed by assay of plasma progesterone levels on the day before embryo transfer. Embryos are thawed usually 4 to 8 hours before the calculated time of exact synchrony of postinsemination age and the time after ovulation (40 hours after the onset of the plasma LH surge). Embryos are thawed slowly (8 C/minute) from - 80 to + 4 C in a biologic freezer, the cryoprotectant removed (Table 2), and embryos cultured in vitro for to 4 hours before transfer to the patients. Only embryos with 50% or more of their original cell numbers are transferred, and no more than 3 embryos are transferred on any one occasion. RESULTS OF EMBRYO CRYOPRESERVATION The value of embryo cryopreservation may be assessed in many different ways. One could argue that the 20 pregnancies achieved in the Monash IVF Program (Table 3) would never have occurred in the absence of cryopreservation; the eggs or embryos would have been discarded or used in other ways. It could be argued that unless cryopreservation achieves success rates approaching that of normal IVF, then it is not worth the effort. It is almost impossible for pregnancy rates after frozen embryo transfer to be equated Table 3. Summary of Pregnancies from Frozen Thawed Embryos at Monash University 983 to 985 No. of pregnancies No. of chemical pregnancies No. of spontaneous abortions No. of therapeutic abortions No. of ongoing pregnancies No. of births Stillborn femalea Single male Single female Twin females 20 2 3 4 0 3 5 adue to premature membrane rupture and intrauterine infection. 20 Fertility and Sterility

0.4 >- u C tv c 0.3 ~e CII /e GI... Ii. - e -0 e >- 0.2 / /.tl tv.tl 0... Ii. 0. e /e 2 3 4 5 6 No. of Embryos Transferred Figure Calculated probability of pregnancy from the Monash University data with increasing numbers of embryos transferred. The point above the curve is the probability of pregnancy when five embryos are obtained and three embryos are replaced on the cycle ofivf treatment and the other two embryos frozen and thawed transferred in another ovulatory cycle. (Reproduced with permission of the publisher. From Trounson A, Hoppen H-O, Lutjen PJ, Mohr LR, Rogers PAW, Sathananthan AH: In vitro fertilization: the state of the art. In Gamete Quality and Fertility Regulation, Edited by R Rolland, MJ Heineman, SG Hillier, H Verner. Amsterdam, Excerpta Medica, 985, p 325.) with that obtained after routine IVF, because fewer embryos are replaced on average, and the embryos have been subject to a major manipulation (frozen and thawed). If this view predominated, there would clearly be no progress in new IVF techniques and applications, nor would IVF itself have been developed in the first place. The pregnancies established in our own IVF program involving cryopreserved embryos are tabulated in Table 3. There have been ten births, including a set of twins and a stillborn baby following premature membrane rupture and development of a septic infection at 26 weeks' gestation. 20 One therapeutic abortion has been carried out because of a severe fetal limb deformity. This defect was not due to chromosomal abnormality and is of concern, although it is highly unlikely for it to have arisen as a result of cryopreservation. The spontaneous pregnancy loss rate is equivalent to that observed for our own IVF program and lower than that recorded for Australian IVF pregnancies. 3 Vol. 46, No., July 986 There is only one publication 30 with sufficient un selected data to make meaningful predictions concerning cryopreservation success rates. Freemann et al. 30 reported the results for 205 patients who had all of their excess 402 embryos frozen. Of these frozen embryos, 396 were thawed and 229 (58%) were transferred to 44 (70%) of the patients, resulting in 6 (%) pregnancies. All excess embryos were frozen, irrespective oftheir age after insemination and morphologic quality from the 2-cell to the 2-cell stage of development. The mean number of embryos transferred was.6 embryos per patient, and the mean embryo viability was 8% (number of fetuses per number of embryos transferred). The pregnancy rate per patient who had embryos frozen was also 8%. This means in practice that a further 8% may be added to IVF success rates calculated per laparoscopy for patients having four or more embryos available for transfer. When five embryos are available, and only three are transferred and two frozen, then thawed, and replaced during a subsequent ovulatory cycle, there is an advantage for IVF patients as illustrated in Figure. There also appears to be a distinct advantage in terms of pregnancy rate in freezing three or more embryos during the cycle of IVF treatment (Table 4). This concept is consistent with the observation that the pregnancy rate increases with the number of embryos produced and transferred in routine IVF. 2 The calculated success rates in published reports 32 of embryo cryopreservation programs vary from % to 28% pregnancies of patients in whom embryos were transferred and 8% to 4% pregnancies of patients who had embryos frozen. In some reports it is difficult to determine the criteria of selection for the data presented. Cohen et al. 26, 29 reported a relatively high success rate of thawing and transfer of blastocysts: 23 blastocysts of 44 thawed were transferred to 8 patients, resulting in eight pregnancies. However, Table 4. Relationship of the Number of Embryos Frozen and Pregnancy Rate After Freezing and Thawing (Modified from Freemann et al. 30 ) No. of embryos No. of patients No. of patients Pregnancy frozen each IVF with embryos transferred em- rate/transfer cycle frozen bryos % 9 52 0 2 70 5 2 3 23 20 30 4 27 5+ 0 0 0 Trounson Freezing human eggs and embryos 5

F Table 5. Success Rate of Embryo Freezing at Specific Cell Stagesa % Pregnancy rate 4-cell Frozen embryos 5% Transferred embryos 8% 6-cell 4% 9% 7 cell 2% 4% 8-cell afrom Trounson A, Freemann L: The use of embryo cryopreservation in human IVF programs. Clin Obstet Gynaecol 2:825, 985. bp < 0.00. data were excluded on the results of freezing and thawing when blastocysts were thawed and transferred on days 3 and 5 after ovulation. Data from Bourn Hall33 indicate that about 40% of fertilized eggs develop to blastocysts in vitro and that about 25% are arrested from the 8-cell stage onward. The freezing of blastocysts selects only those emb.ryos capable of continued development and excludes embryos that are arrested, fragment, or develop abnormally. This phenomenon would account for some of the differences between the results obtained with early cleavage stages and those with blastocysts. However, it is probably necessary to carry out a large-scale trial within one clinic for the determination of whether a significant advantage occurs in the results of freezing with one of the two different stages of embryonic development. The criteria used for selection of embryos for freezing will also have a major influence on the success rates achieved. For example, if only 8-cell embryos had been chosen for freezing (Table 5), the success rate achieved in our own IVF program would have been substantially higher. FACTORS AFFECTING SUCCESS RATE OF EMBRYO CRYOPRESERV ATION Factors influencing the success rate of cryopreservation have been reviewed recently by Trounson and Freemann.32 The number of embryos frozen (Table 4) and the developmental cell stage (Table 5) both affect the success rate of embryo cryopreservation. Embryo viability appears to increase with increasing stage of development.29. 3 Embryo quality as assessed by the degree of regularity or fragmentation also has a significant effect3o. 32 on embryo viability. Completely regular and unfragmented embryos had the highest viability (5% developed to pregnancies), compared with those with a few fragments (2%), those with a number of irregular cells and fragments (5%), and those that were badly frag- 6 Trounson Freezing human eggs and embryos mented (0%). It has also been noted that survival rates of "irregular blastocysts" were lower than those classified as "regular.,,29 The more rapidly dividing embryos were also more likely to result in pregnancies in the data reported by Cohen et al.,29 but this trend was only apparent at the 8-cell stage in the data reported by Freemann et al. 30 It has been surprising that damage to the blastomeres of embryos after freezing and thawing does not correlate closely with the viability of embryos, except when more than 50% of the original cell number are damaged. 30. 32 However, it was notable that 47% of undamaged 8-cell embryos contributed to all the pregnancies reported by Freemann et al.30 and that both pregnancies obtained by Zeilmaker et al. 25 were undamaged embryos. Additional data may be required for adequate demonstration of the significant effects of freezing damage. Cohen et al. 29 reported that disruption to the zona occurs more frequently in early cleavage stage embryos and is lethal. Removal of the zona pellucida per se is probably not responsible for this effect, and damaged zonae may be associated with even more serious damage to cell function. There is a remarkable association of pregnancy in the cycle ofivf treatment and pregnancy with the same cohort of embryos after freezing and thawing (Table 6). Only 6% of patients who failed to become pregnant on the cycle of IVF treatment became pregnant when transferred frozenthawed embryos, compared with 4% of patients who became pregnant on the cycle of IVF treatment. It is tempting to speculate that this significant (p < 0.00) association is due to the collection of a cohort of viable embryos. It is not possible to exclude a hostile uterine environment in patients who fail to conceive on both the cycle of IVF treatment and the natural ovulatory cycle when frozen-thawed embryos are transferred. Table 6. Association of Pregnancy in the Cycle of IVF Treatment and Pregnancy After Freezing and Thawing the Same Cohort of Embryosa Outcome of transfer of frozen-thawed embryos Not pregnant Aborted (C) Ongoing/delivered (D) C+D Outcome of transfer of fresh embryos on cycle of IVF treatment (no. of patients and pregnancy rate) Not Aborted Delivered A + B pregnant (A) (B) 3 2 5 7 (6%) 2 6 7 (37%) 2 (67%) 3 (9%) 2 (50%) 7 (58%) 9 (4%) acryopreservation data modified from Freemann et al. 30 Fertility and Sterility

Table 7. Pregnancy Rate and Degree of Synchrony Between Embryo Age and Time After Ovulation for Embryo Transfer Factor No. of patients 3 2 transferred embryos No. of patients pregnant 0 0 % Time (hrs.) from calculated exact synchrony +7 to +2 +3 to +6-2 to +2-3 to -6-7to -2-2to -24 67 27 30 8 9 4 3 3% 5% 0% 6% The life-table data reported by Kovacs et al.34 show that it takes six treatment cycles by IVF before there is a significant decline in IVF success rates. This observation would suggest that a hostile uterine environment is most unlikely to be governing the association observed in Table 6 because such defects at present require repeated failure of embryo transfer to be identified. Cohen et a. 26,29 transferred their frozenthawed 5- to lo-cell embryos on day 2 after ovulation, a day earlier than the postinsemination age of the embryos, and reported that blastocysts only implant when transferred on day 4 but not on day 3 or day 5. Quinn and Kerin27 also argued that embryos should be replaced a day earlier than the postinsemination age of the embryos. With the method described by Freemann et al.30 for identifying the onset of the LH surge, the trend to the highest pregnancy rate in our own data is closest to exact synchrony of embryo age after insemination and the time after expected ovulation (Table 7). The time embryos are stored in liquid nitrogen appears to have no effect on the survival or viability of embryos.30, 32 Pregnancies have been obtained from embryos stored up to 2 months in liquid nitrogen,32 and it could be expected that embryos will survive indefinitely in liquid nitrogen, provided they remain fully immersed. PRESERVATION OF MANIPULATED EMBRYOS Our own group has an interest in the micromanipulation of embryos, particularly for the identification of genetic and embryonic developmental abnormalities. We have conducted a series of experiments,35 summarized in Table 8, in which we enzymatically removed the zona pellucida of mouse embryos and then cooled them to - 6 C, froze them to - 80 C, or froze 'and stored them in liquid nitrogen using the procedures described in Table. The embryos were thawed and then cultured in vitro to the expanded blastocyst Vol. 46, No., July 986 stage. The data shown in Table 8 demonstrate a number of interesting principles of cryobiology:. The freezing and thawing of embryos to - 80 C may have a relatively minor effect on embryo viability. 2. The handling of embryos at temperatures below - 80 C can have a major effect on embryo viability, despite the presumption that slow cooling to - 80 C should have completed blastomere dehydration and ice formation. It is frequently presumed that damage to embryos occurs at relatively high subzero temperatures. This presumption may be erroneous. 3. The removal of the zona pellucida reduces embryo viability; and the effects are greater, the earlier the cleavage stage. The role ofthe zona in protecting the embryo against freezing damage is not certain, but the zona may protect the embryos against intracellular ice formation during dehydration. Blastomeres may also lose their intercellular connections during freezing and thawing, and an intact zona pellucida will prevent separation and loss of blastomeres from the embryonic cell mass. It has been reported that in the human embryos disruption of the zona is lethal. 29 Our own observations in women show that the capacity of further embryonic development is severely reduced when the zona is disrupted, although this damage may not be lethal. 2 The effect of Table 8. Survival and Development of Mouse Embryos Frozen With or Without a Zona Pellucida Cell stage Presence of the zona 2-cell 8-cell Morula Yes No Yes No Yes No % Development of blastocysts of embryos frozen Cooled to Frozen to Frozen and - 6"C - 80"C stored at -96"C 96 88 3 65 36 2 95 95 42 72 60 20 85 76 43 6 43 30 Trounson Freezing human eggs and embryos 7

removing the zona may be seen even on cooling embryos to - 6 C (Table 8). 4. The viability of early cleavage stage embryos following freezing and thawing is generally lower than that of later cleavage stages. Similar observations have been made with human embryos.29, 30 This phenomenon may be due to the difference in the size of the component blastomeres, a difference which will affect the degree of dehydration, osmotic damage, and the probability of intracellular ice formation. The intracellular composition of lipid and other temperature-sensitive substances and organelles may vary according to the cell stage, possibly prejudicing the survival of certain cell stages when frozen and thawed. 7 Statistically there is also a greater chance of affecting embryo viability by the damage of a small number of large cells than a small number of small cells. The extreme example of this effect is observed with the oocyte in which, if any part of the cell membrane is damaged, survival cannot occur, whereas loss of up to 50% of cells of an 8-cell embryo will not necessarily mean the loss of embryo viability.30, 32 The implication of these observations for micromanipulation is that biopsied embryos36 should probably be grown to the morula or blastocyst stage before cryopreservation. Studies on the biopsy and preservation of embryos, with a zona partially opened for retrieval of cells, indicate that disruption of the zona has less effect on embryo viability than complete removal. CRYOPRESERVATION OF HUMAN EGGS Our group has studied the cryopreservation of human eggs. Our initial studies37 some years ago showed that immature eggs recovered from ovarian wedge biopsies and matured in culture for 48 hours could be frozen and thawed successfully by the use of the methods of slow freezing employed for mouse eggs38 or human embryos, but survival rates were only about 20% of those frozen. At the time the possibility of increased aneuploidy resulting from exposure of mature eggs to cryoprotectants and to freezing and thawing was of concern. The ovulating oocyte is arrested at the metaphase of the second meiotic division with chromosomes attached to the temperature-sensitive spindle microtubules.39 Depolymerization of the microtubules by cryoprotectants or by cooling may prevent the normal separation of sister chro- 8 Trounson Freezing human eggs and embryos matids at fertilization and thus lead to chromatid nondisjunction and the state of aneuploidy after extrusion of the second polar body. The egg is theoretically more susceptible to aneuploidy because normal separation of sister chromatids requires intact spindle microtubules at the time of sperm penetration for the immediate activation of the egg and resumption of meiosis. Aneuploidy as a result of failure to eject one chromosome would result in trisomy, and the ejection of an additional chromosome would result in monosomy. These chromosomal abnormalities are, for the most part, lethal; but if embryos do survive, infants may be born with major developmental and mental defects, such as Down's syndrome. Whittingham and his colleagues have undertaken a major study of the chromosome complement of embryos derived from frozen-thawed mouse eggs and compared the results with the rate of defects found in eggs fertilized in vivo and in vitro. The results have been summarized by Wood,40 who reported reduced fertilization rates and an increased incidence of chromosomal defects in embryos derived from frozen-thawed eggs; these were due primarily to an increase in polyploidy. Transfer of embryos derived from frozenthawed embryos to recipients resulted in the same proportion of normal fetuses as embryos derived from nonfrozen eggs. Given this information, our own group continued experiments to locate a suitable freezing method for human eggs. We have examined a number of methods for egg cryopreservation; these methods are summarized in Table 9. Since mature human oocytes were not available for screening embryo freezing methods, we used aged human eggs that had failed to fertilize when inseminated from patients enrolled in the Monash IVF program. These aged eggs (24 to 72 hours after insemination) are not viable4 and are normally discarded, but serve as an appropriate model for studying the response of frozenthawed mature human oocytes. Survival of these aged eggs, which have an intact plasma membrane, after thawing was considered a good index for the survival of mature unfertilized eggs. The limitations of mouse eggs and those of other species include the disparity in size and suscepttbility to cooling damage because of differences in membrane and intracellular composition. Slow and rapid cooling of aged human eggs in yielded results (Table 0) similar to those obtained in the original studies with ovarian wedge oocytes. Increased egg survival rates of Fertility and Sterility

Table 9. Summary of Freezing Methods Used for the Cryopreservation of Human Eggs Procedure Slow a Addition of cryoprotectant Temperature 20 Time lo-minute steps Cooling rate 0.3 /min to - 50 Thawing 8 /min -80 to +4 Removal of cryoprotectant Temperature 20 Time lo-minute steps ain DMSO or propanediol cryoprotectant. Rapid Ultrarapid Vitrification 20 20 4 0-minute steps 2-3 minutes 0-minute steps 0.3 /min to + 35 Plunge to Plunge to -96-96 30 37 0 Water bath Water bath Water bath 20 20 4 0-minute steps 0 minutes in 0 minute steps sucrose about 50% were obtained with the use of a new ultrarapid freezing method developed in our own laboratory.42 The use of a modified vitrification technique43 also gave very encouraging survival rates of 75% of those eggs frozen (Table 0). A number of mature human oocytes has been donated by IVF patients for studies of egg freezing. A summary of the results of these studies is included in Table. Even though small numbers of mature oocytes were involved in these experiments, it has been shown that oocytes may survive freezing and thawing and be fertilized after insemination. At least two different methods give reasonable survival rates. Oocytes vitrified with their cumulus intact, fertilized, and developed to at least the 8-cell stage and oocytes slow-frozen with propanediol as the cryoprotectant, resulted in high survival and fertilization rates (Table ). It is likely that other methods will also be devised for egg cryopreservation, and recently a pregnancy following egg freezing has been announced publicly.44 Despite the development of these methods for egg cryopreservation in mid 985, our group has been unable to take the next obvious step and transfer developing embryos to patients. This situation has developed in response to the Victorian State's Infertility Medical Procedures Bill, which prevents embryo experimentation. We have indicated to our own hospital's Ethics Committee that a small number of embryos derived from frozenthawed eggs should be analyzed for their chromosomal and developmental normality before progressing to transfer of such embryos to patients in order to confirm the experiments on mouse eggs.40 We agreed to a self-imposed moritorium on this work while the state government's Standing Review and Advisory Committee on IVF considered our application to study these embryos. No decision has been made on the matter to date. The hospital's Ethics Committee has also been awaiting this decision before agreeing to the transfer of embryos resulting from egg cryopreservation. The ultimate result is that we cannot progress either to examine the embryos or to transfer them to patients. APPLICATIONS OF HUMAN EGG AND EMBRYO CRYOPRESERV ATION Egg and embryo cryopreservation has numerous and diverse applications in human medicine. These applications include the following:. A solution to the collection of excess oocytes and the development of more embryos than is required for transfer in human IVF. 2. An adjunct for procedures where oocytes are transferred directly to the oviducts (gamete intrafallopian transfer or GIFT procedure)45 or to the uterus46 for fertilization and embryo development in vivo. It is usual to transfer a maximum of four eggs in these programs, and excess eggs may be cryopreserved or fertilized in vitro and the embryos cryopreserved for transfer in later ovulatory cycles. Table 0. Survival of Aged Human Eggs After Freezing and Thawing by a Variety of Methods Freezing method No. of eggs frozen No. of eggs surviving after thawing Slow cooling in 7 3 (8%) Rapid cooling in 8 2 (25%) Ultrarapid cooling in 62 32 (52%) in 3.0 M DMSO 33 7 (52%) Vitrification 2 9 (75%) Vol. 46, No., July 986 Trounson Freezing human eggs and embryos 9

Table. Freezing and Thawing Mature Human Eggs Freezing method No. of eggs frozen Survival on thawing Slow cooling 3 0.5 M propanediol 6 4 Ultrarapid cooling 8 9 Vitrification Cumulus removed 6 4 Cumulus intact 6 0 apronuclear eggs not cultured in vitro. 3. A method for overcoming problems of synchronization between donors and recipients in egg or embryo donation programs. In our own donation program we have recently achieved a pregnancy using cryopreserved embryos in a woman who had primary ovarian failure.47 4. A method for preserving eggs or embryos for patients who are at risk oflosing ovarian function through pelvic disease, surgery, or therapy involving radiotherapy or chemotherapy. Semen banking has been available for men at risk of losing reproductive function, and the availability of egg or embryo banking for young women seems entirely reasonable. The creation of frozen egg banks for donation would provide selection of the alternatives that are available in donor semen banks. 5. Use in new methods being developed for the diagnosis of genetic defects in preimplantation embryos. The rapid proliferation of dioxyribonucleic acid probes for the identification of chromosomal and gene defects will enable the identification of genetic abnormalities in embryonic biopsies. Embryo cryopreservation may be needed to enable sufficient time to amplify the dioxyribonucleic acid of biopsied cells and to enable in situ hybridization or the use of other molecular biologic techniques for identifying genetic abberations. In the future it may be possible for couples to defer the formation of families by using semen, egg, or embryo banking facilities. At present there is little medical or community support for this proposal. However, this could be viewed as insurance against the development of sterility and the increased incidence of birth defects that occurs with increasing age. CLINICAL CONSIDERATIONS OF EGG AND EMBRYO CRYOPRESERVATION In our own IVF program the majority of patients choose to have their embryos cryopreserved 0 Trounson Freezing human eggs and embryos Survival after culture Fertilized 4 4 0 Developed to 8 cells or more 4 3 0 0 4 3 in preference to other alternatives presented to them. The other options include inseminating no more than fouroocytes, donation of oocytes or embryos to other infertile couples or to research, and disposal of eggs or embryos.48 This results in about one-third of all IVF patients using cryopreservation facilities. The clinical implications of egg and embryo cryopreservation need to be carefully and fully explained to all couples receiving IVF treatment, particularly those requesting cryopreservation. Counseling on this subject is mandatory in the state of Victoria. The couple advises the IVF program's management in writing of their wishes in the event of disagreement, separation, divorce, or death of one or both partners regarding the destination of any cryopreserved embryos or eggs. More than 75% of couples wish their eggs or embryos to be donated to other couples, 5% wish them to be donated to research, and less than 5% wish them discarded. The importance of these stipulations is exemplified by the controversy surrounding the death of a wealthy American couple in an airplane accident who had two cryopreserved embryos in our own IVF program.48 In this instance, written advice from the couple on the destination of these embryos was lacking, and the attorney general of the state of Victoria decided that the embryos would be donated to another infertile couple and included this procedure in the Infertility Medical Procedures Bill. Under the Status of Children Act in Victoria, couples agreeing to be recipients of donated eggs, sperm, or embryos are considered the parents in law, so that the donating biologic parents are not responsible for the care or financial support of children arising through donation procedures. Therefore, inheritance from the biologic parents is no longer an issue in the state of Victoria. There are limitations for the length of time embryos can remain in cryostorage. The Infertility Medical Procedures Bill limits cryostorage to Fertility and Sterility

5 years in Victoria unless there are particular circumstances requiring notification to the government. Under the Australian National Health and Medical Research Council's guidelines for IVF, cryostorage is limited to 0 years. Unlimited cryostorage, particularly across generation intervals, raises concerns in the community, although in Australia the majority view supports embryo cryopreservation in IVF.49 The cryopreservation of eggs appears to raise few ethical concerns, and at the present time the only regulations relating to eggs are in the area of donation. REFERENCES. Trounson AO, Leeton JR, Wood C, Webb J, Wood J: Successful human pregnancies by in vitro fertilization and embryo transfer in the controlled ovulatory cycle. Science 22:68, 98 2. Wood C, McMaster R, Rennie G, Trounson A, Leeton J: Factors influencing pregnancy rates following in vitro fertilization and embryo transfer. Fertil Steril 43:245, 985 3. Lancaster P: In vitro fertilization pregnancies Australia and New Zealand, 979 to 984. Report of the National Perinatal Statistics Unit, Sydney, University of Sydney, 985, p 4. Trounson AO, Wood C, Leeton J: Freezing of human embryos: an ethical obligation. Med J Aust 2:332, 982 5. Whittingham DG, Leibo SP, Mazur P: Survival of mouse embryos frozen to -96 C and 269 C. Science 78:4, 972 6. Wilmut I: The effect of cooling rate, cryoprotective agent and stage of development on survival of mouse embryos during freezing and thawing. Life Sci :07, 972 7. Whittingham DG, Adams CE: Low temperature preservation of rabbit embryos. J Reprod Fertil 47:269, 976 8. Willadsen SM, Polge C, Rowson LEA, Moor RM: Deep freezing of sheep embryos. J Reprod Fertil 46:5, 976 9. Bilton FJ, Moore NW: In vitro culture, storage and transfer of goat embryos. Aust J BioI Sci 29:25, 976 0. Willadsen SM, Polge C, Trounson AO, Rowson LEA: Transplantation of sheep and cattle embryos after storage at - 96 C. 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In Clinical In Vitro Fertilization, Edited C Wood, A Trounson. Berlin, Springer-Verlag, 984, p 99 23. Mohr LR: Assessment of human embryos. In In Vitro Fertilization and Embryo Transfer, Edited by A Trounson, C Wood. Edinburgh, Churchill Livingstone, 984, p 59 24. Mohr LR, Trounson A, Freemann L: Deep-freezing and transfer of human embryos. J In Vitro Fertil Embryo Trans 2:, 985 25. Zeilmaker GH, Alberda AT, van Gent I, Rifkmans CMPM, Drogendijk AC: Two pregnancies following transfer of intact frozen-thawed embryos. Fertil Steril 42:293, 984 26. Cohen J, Simons RF, Edwards RG, Fehilly CB, Fishel SB: Pregnancies following the frozen storage of expanding human blastocysts. J Vitro Fertil Embryo Trans 2:59, 985 27. Quinn P, Kerin JPF: Experience with the cryopreservation of human embryos using the mouse as a model to establish successful techniques. J In Vitro Fertil Embryo Trans 3:4Q, 986 28. Renard JP, Babinet C: High survival of mouse embryos after rapid freezing and thawing inside plastic straws with -2 propanediol as cryoprotectant. J Exp Zool 230: 443, 984 29. Cohen J, Simons RF, Fehilly CB, Edwards RG: Factors affecting survival and implantation of cryopreserved human embryos. J In Vitro Fertil Embryo Trans 3:46, 986 30. Freemann L, Trounson A, Kirby C: Cryopreservation of human embryos: progress on the clinical use of the technique in human IVF. J In Vitro Fertil Embryo Trans 3:53, 986 3. Martinez F, Trounson A, Besanko M: Detection of the LH surge for AID, AIH and embryo transfer using a twice daily dipstick assay. J Clin Reprod Fertil. In press 32. Trounson A, Freemann L: The use of embryo cryopreservation in human IVF programmes. Clin Obstet Gynecol 2:825, 985 Trounson Freezing human eggs and embryos

33. Fishel S: Embryo culture and replacement. Presented at Jan Pfalfin Symposium, September 985, Antwerp, Belgium 34. Kovacs GT, Rogers P, Leeton JF, Trounson AO, Wood C, Baker G: In vitro fertilization and embryo transferprospects of pregnancy by life table analysis. Med J Aust. In press 35. Jessup D, Trounson A: Unpublished data 36. Trounson A: Recent progress in human in vitro fertilization and embryo transfer. In Manipulation of Mammalian Development, Edited by R Gwatkin. New York, Plenum Press, 985, p 49 37. Whittingham DG, Trounson A: Unpublished data 38. Whittingham DG: Fertilization in vitro and development to term of unfertilized mouse oocytes previously stored at -96 C. J Reprod Fertil 49:89, 977 39. Magistrini M, Szollosi D: Effects of cold and of isopropyl N-phenyl carbamate on the second meiotic spindle of mouse oocytes. Eur J Cell BioI 22:699, 980 40. Wood MJ: Recent progress in animal embryo cryopreservation. In Proceedings ofthe Fourth World Congress on in Vitro Fertilization, Edited by I Johnston. New York, Plenum Press. In press 4. Trounson A, Webb J: Fertilization of human oocytes following reinsemination in vitro. Fertil Steril 4:86, 984 42. Peura A, Trounson A, Freemann L: Ultra-rapid embryo freezing. (Abstr.) Presented at the Proceedings of the Fourth World Congress on In Vitro Fertilization, Melbourne, Australia, November 985, p 244 43. RaIl WF, Fahy GM: Ice-free cryopreservation of mouse embryos at -96 C by vitrification. Nature 33:573, 985 44. Chen C: Unpublished data 45. Asche RH: GIFT (gamete intra fallopian transfer), a new treatment of infertility. (Abstr.) Presented at the Proceedings of the Fourth World Congress on In Vitro Fertilization, Melbourne, Australia, November 985, p 203A 46. Craft IL, Djahanbakheh 0, McLeod F, Bernand A, Green S, Twigg H, Smith W, Edmonds DK, Lindsay KS: Human pregnancies following intrauterine transfer of preovulatory oocytes and sperm. Lancet 2:03, 982 47. Lutjen P, Trounson A, Leeton J, Findlay J, Wood C, Renou P: The establishment and maintenance of pregnancy using in vitro fertilization and embryo donation in a patient with primary ovarian failure. Nature 307:74,984 48. Wood C, Downing B, Trounson A, Rogers P: Clinical implications of developments in in vitro fertilization. Br Med J 289:978, 984 49. Kovacs GT, Wood C, Morgan G, Brumby M: The attitudes of the Australian community to treatment of infertility by in vitro fertilization and associated procedures. J Vitro Fertil Embryo Trans 2:23, 985 This study was supported by the National Health and Medical Council of Australia. Reprint requests: Alan Trounson, Ph.D., Centre for Early Human Development, Monash University and Queen Victoria Medical Centre, Melbourne, Australia, 3000. 2 Trounson Freezing human eggs and embryos Fertility and Sterility