Screening of conditions for rapid freezing of human oocytes: preliminary study toward their cryopreservation

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1 FERTILITY AND STERILITY Copyright~ 1989 The American Fertility Society Vol. 52, No.5, November 1989 Printed on acid-free paper in U.S.A. Screening of conditions for rapid freezing of human oocytes: preliminary study toward their cryopreservation Manuel Pensis, B.Sc. * Ernest Loumaye, M.D. lv Psalti, M.Sc. Physiology of Human Reproduction Research Unit, Department of Obstetric and Gynecology, University of Louvain, Brussels, Belgium One hundred and twenty-one freshly-collected human oocytes and 839 unfertilized human oocytes after insemination were cryopreserved by vitrification. The cryoprotectants used were dimethylsulfoxide (DMSO) and sucrose. Vital staining and morphological criteria were used to assess injuries to cells. Variation of the time exposure to DMSO and sucrose, and cryoprotectants concentrations, followed by extraction-dilution in sucrose without freezing made it possible to study chemical toxicities. Variation of cryoprotectant concentrations followed by immersion in liquid nitrogen, thawing, extraction, and dilution made it possible to choose optimal conditions for vitrification. The sucrose concentration upon extraction after freezing and thawing which was lower than that during soaking enhanced the oocyte survival rate as did the choice of duration and temperature of soaking. No parthenogenetical activation of these unfertilized ovum was observed. This study indicates that with a certain combination of DMSO and sucrose concentrations up to 80% of morphologically intact human oocytes can be recovered after rapid freezing and thawing. Fertil Steril52:787, 1989 Controlled ovarian hyperstimulation (COH) for in vitro fertilization (IVF) often leads to the recovery of more oocytes than are needed to obtain three to four embryos, the optimal number required for transfer. In addition, the embryo implantation rate after IVF and transfer remains quite low1 and postimplantation loss of pregnancy is relatively high. 2 All these facts have led to the development of cryopreservation of surplus embryos. The use of this method has so far led to an increase in the pregnancy rate per ovum pick-up (0PU), 3 and facilitates procedures such as embryo donation. 4 There are, however, ethical reasons for using the supernumerary oocytes through oocyte cryopreservation rather than embryo freezing, as well as med- Received January 11, 1989; revised and accepted July 13, *Reprint requests: Manuel Pensis, B.Sc., Physiology of Human Reproduction Research Unit, University oflouvain 5330, 53 Avenue Em. Mounier, B-1200 Brussels, Belgium. ical reasons such as, for example, oocyte preservation before castrating therapy in young, single women. The freezing and thawing of oocytes has proved difficult from a technical point of view. 5 6 Moreover, some controversy persists concerning the putative risk of the induction of aneuploidy by such procedure.7-11 In addition, the transposition of results obtained with animal cells to human oocytes are disappointing. Nevertheless, a few pregnancies have been obtained in humans after the transfer of embryos produced by the IVF of thawed oocytesp-14 As regards embryo cryopreservation methods, considerable progress has been made toward simplification and reduction in cost.15 However, so far no reliable, simple, and inexpensive method is available for the cryopreservation of human oocytes. The aim of our study was to seek technical conditions which permit ultrarapid freezing and thawing Vol. 52, No.5, November 1989 Pensis et al. Human oocyte cryopreservation 787

2 of human oocytes. This led to the establishing of conditions allowing the recovery of 80% of morphologically intact oocytes. Despite the fact that an intact morphology does not necessarily correspond to oocyte viability, chromosomal and functional integrity, the scarcity of data on human oocyte preservation, and the promising results obtained prompted us to report on this preliminary study. Patients MATERIALS AND METHODS All patients underwent COH with human menopausal gonadotropin (hmg) (Humegon, Organon, Oss, The Netherlands) combined with a gonadotropin-releasing hormone agonist (GnRHa) (Buserelin, Suprefact, Hoechst, Frankfurt, West Germany). This agonist was administered intranasally (3 X 300 ~g/d) from the 1st day of the menstrual cycle or from the 21st day of the preceding cycle up to the human chorionic gonadotropin (hcg) injection.16 Daily injections of 225 Ul of hmg were administered from day 3 of the follicular phase. When plasma levels of estradiol (E 2) reached 200 to 300 pg/ml per follicle larger than 15 mm and when two follicles reached a diameter of 20 mm, 5000 IU of hcg (Pregnyl, Organon, Oss, The Netherlands) were administered. Thirty-five hours later, the OPU was performed by ultrasound guided transvaginal puncture. These data concerned 136 couples admitted for different indications (29% male infertility, 28% male and female infertility, 32% female infertility, 11% unexplained infertility). Oocytes Over a period of 15 months, 165 out of 566 OPU prqvided some unfertilized oocytes or an excess of fresh oocytes suitable for experimental cryopreservation. Nine hundred and sixty human oocytes selected for their morphological integrity were handled. Most of them (839) were unfertilized despite insemination. These aged oocytes were treated 52, 76, or 100 hours after the OPU. During the days between the OPU and the_ experiments they were suspended in our regular IVF Earle's culture medium (Gibco, Paisley, Scotland), supplemented by 2.2 mg NaHC0 3, mg glutamine, 200 IU of penistrepto, 1 mg glucose, mg Na pyruvate/ ml, and 10% (v/v) heat inactivated fetal cord serum. One hundred and twenty-one freshly collected oocytes were subsequently used. The fresh oocytes were cleared of the cumulus cells by brief exposure to hyaluronidase (Sigma, St. Louis, MO) (300 to 600 IU /ml for 5 to 8 minutes at 37oC) or by dissection with needles under an inverted phase microscope (Leitz, Wetzlar, West Germany). Experiments were performed with small groups of 3 to 12 oocytes. Freezing and Thawing Methods The oocytes were vitrified in DMSO (Sigma), sucrose (Merck, Darmstadt, West Germany) and Earle's medium supplemented as described above except that the fetal cord serum was replaced by 5 mg/ml of human albumin and that 15 mm Hepes buffer was added. The cells were soaked at 4 oc or 25oC for between 2 and 30 minutes in precooled mixtures of Earle's medium containing DMSO which ranged from 2.8 to 4.2 M and sucrose from 0.25 to M. At the beginning of this study, the oocytes were transferred into transparent freezing straws of 0.25 ml (IMV, l'aigle, France) with a fine-drawn glass pipette. They are now aspirated directly in the straws before being plunged into liquid nitrogen (LN 2) for about 5 seconds. The straw sealing, initially effected with sucrose 1 M, is now done with the vitrification solution itself, thus preventing the straw breaking on thawing. Straws were thawed at 27oC for 10 seconds and in a water bath at 37oC until the contents of the straws had melted completely. The contents of the melted straws were expelled in 1 ml of Earle's sucrose solutions (O M to 1 M) at 27 to 37 C. Extraction of DMSO was performed in a similar sucrose solution for 9 minutes, followed by an intermediate concentration step of 30 seconds before final dilution in Earle's modified culture medium buffered with Hepes. Cell Assessment To assess the morphological integrity of oocytes after freezing and thawing, the oocytes were photographed before the exposure to cryoprotectants, during extraction after thawing, and 24 hours later. This last examination represents the assessment of the oocytes' integrity taken into account for data analysis. This delayed examination of oocytes made it far easier to distinguish between intact and damaged oocytes than an intermediate examination after thawing. The morphological criteria used to assess cell integrity were: unfractured zona pellucida, clear perivitelline space, unfragmented 788 Pensis et al. Human oocyte cryopreservation Fertility and Sterility

3 A1 ated the role of sucrose concentration during the extraction of the cryoprotectant after freezing and thawing on the oocytes' survival rate. This was repeated with different soaking mixtures. We then studied the effect of time and temperature of soaking. Finally, the protocols which allowed the best recovery of intact human oocytes were applied to mouse oocytes. Vitrification Figure 1 Example of freezing of two fresh human oocytes (AI, Bl) from the same patient. Both were soaked simultaneously in 3.5 M DMSO and 0.5 M sucrose and frozen. After thawing, extraction was performed (A2, B2) with M sucrose. A2 didn't react to osmotic pressure of sucrose although B2 did. Twenty-four hours after thawing A3 had collapsed into an atretic form and B3 was morphologically intact. ooplasm, preserved oocyte shape and diameter, and a nongranular cytoplasm (Fig. 1). In addition, to validate this morphological assessment, 213 aged eggs were also stained with Trypan Blue (Sigma) (0.05% solution for 10 minutes at room temperature 24 hours after thawing). The relationship between different sucrosedmso-earle's mixtures and the quality of vitrification in the straws obtained during freezing and thawing was assessed. Low concentrations of DMSO and sucrose gives the solution a milky appearance when exposed to LN2 and thawed (Fig. 2). This corresponds to crystallization of the solution. Mixtures made with higher concentrations of DMSO looked perfectly transparent after freezing and thawing. This corresponds to real vitrification. It was noted, however, that the association of a high concentration of DMSO and a high c;:oncentration of sucrose led to considerable turbidity of the solutions before any exposure to LN 2 This part is indicated in Figure 2 by the dotted lines. This turbidity makes difficult to pipette the cells suspended in these solutions. The concentrations of DMSO sucrose solutions chosen were those which allowed vitrification on Sucrose RESULTS.5 Our study followed a seven-step experimental design. The first part investigated the relationship between the concentrations of DMSO and sucrose, and the quality of vitrification in straws when the mixtures were plunged directly into LN 2, and thawed. This part of the study was performed without any cells. In the second step of the procedure, we studied the influence of the soaking time upon the integrity of the oocytes. The third step evaluated the chemical toxicity of several cryoprotectant concentrations on the morphology of the oocytes. Both steps were performed without freezing. The fourth step examined the ability of several cryoprotectant concentrations to protect oocytes submitted to freezing and thawing. We then evaluvol. 52, No.5, November !5 (II) milky 2.8 Figure 2 Physical appearance ofdmso-sucrose concentrations in modified Earle's culture medium when plunged into LN2 and thawed. Vitrification is complete when the solution remains transparent during thawing (right to the continuous line on the far right). Between the two continuous lines a transient devitrification is observed upon thawing (T/m/T). With lowest concentrations in cryoprotectants no vitrification is obtained upon freezing (milky). With the highest concentrations of DMSO and sucrose, turbidity is observed before freezing (area delimited by two dotted lines). Pensis et al. Human oocyte cryopreservation 789

4 % of intact oocytes N-7 N-5 N-11 N=9 N=6 N-8 the exposure to the cryoprotectant was detected but no clear relationship between the concentrations and the toxicity was observed (not significant) (NS). Physicochemical Toxicities soaking tjme (minutes). Figure 3 Relationship between DMSO-sucrose soaking time in 3.5 M/0.375 M and the proportion of intact oocytes recovered 24 hours after extraction without freezing. freezing and a milky aspect for 2 or 3 seconds during thawing. These concentrations are indicated by two continuous lines in Figure 2 (Transparent/ milky/transparent: T/m/T). Whether or not this very transient devitrification during thawing is deleterious for the cells has not been established here. Chemical Toxicity To assess time dependence of chemical toxicity of the cryoprotectant solutions, 51 oocytes were exposed to a mixture of 3.5 M DMSO and M sucrose for various periods of time (Fig. 3). Cells were not frozen and cryoprotectants were extracted. Twenty-four hours later, the effect of varying the soaking time was studied. Sixty to eighty percent of exposed oocytes appeared intact. A deleterious chemical effect was thus observed (20% to 40%) but it did not seem to be strictly time dependent (P > 0.05). On the basis of these results, we decided to opt for a soaking time of 15 minutes in further experiments before further testing on the effect of soaking time including the freezing step. The chemical toxicity of several DMSO and sucrose concentrations was.then studied 24 hours after extraction without freezing. Forty-five oocytes were used in this experiment. The extraction was performed at 27 to 37c for 10 minutes with the concentration of sucrose which was used for soaking. Tested concentrations ranged from 2.8 to 4.2 M for DMSO and 0.25 to 0.5 M for sucrose (Fig. 4, S:). Some deleterious effect (0% to 29%) of Ultrarapid freezing followed by thawing was then studied after soaking the oocytes in DMSO concentrations ranging from 2.8 to 4.2 M and sucrose concentrations from 0.25 to M. After thawing, extractions were performed at 2TC to 37c for 10 minutes with the sucrose concentration used for soaking. One hundred and fourteen oocytes were used for these experiments. This freezing and thawing procedure resulted in the destruction of the vast majority.(60% to 100%) of the oocytes. However, when the sucrose soaking concentrations ranged between M to 0.5 M, and DMSO concentrations between 2.8 and 4.2 M a significantly'higher proportion (P < 0.05) of morphologically intact oocytes was recovered (Fig. 4, F:). It was also noted that in experiments that included freshly collected oocytes (n = 15) better results were obtained even with a lower sucrose concentration. A sucrose concentration of 0.5 M and a DMSO concentration of 3.5 M therefore allowed a reasonable oocyte recovery rate despite the fact that only aged oocytes were used for these experiments. We adopted this mixture as a soaking solution, 15 minutes for the soaking time, and 4 c for DMSO(M) Sucrose (M) S: 4/4 S: 4/4 S: 3/4 F: 1/9 F: 0/3 F: 0/7 S: 6/7 S: 3/4 S: 4/6 F: 0/4 F: 1/5 F: 0/4 S: 4/5 S: 5/7 S: 3/4 F: 4/1Q F: 12/34 F: 2/ F: 0/10 F: 0/13 F: 0/5 Figure 4 Relationship between DMSO-Sucrose concentration used for soaking (4C 15 min) and the retrieval of intact oocytes after freezing and thawing (F:), or after soaking and extraction without freezing (8:). The extraction of DMSO was performed in sucrose with the concentration used for soaking. 790 Pensis et al. Human oocyte cryopreservation Fertility and Sterility

5 80 N J!! 60 il' 0 0 N-19 I N 18 >- N-11 4~ ~ 24 c :g Q N34 E 0 N N N Sucrose concentration of the extraction solution Figure 5 Proportion of morphologically intact oocytes 24 hours after freezing and thawing. The soaking solution was DMSO 3.5 M and sucrose 0.5 M. The role of sucrose concentration during extraction has been investigated. The black columns represent experiments using aged oocytes, the white columns represent experiments using freshly collected oocytes. the soaking temperature for the next following experiments. Role of Sucrose Concentration for the Cryoprotectant Extraction Three hundred and fourteen oocytes were exposed to 0.5 M sucrose and 3.5 M DMSO for 15 minutes, and plunged into LN 2 After thawing, the straws contents were expelled to a different sucrose solution to study the role of sucrose concentration during the DMSO extraction. Absence of sucrose or very high concentrations of sucrose (1M) did not allow any recovery of intact oocytes. In these circumstances, evident injuries were observed immediately after extraction (Fig. 5). Between these two extremes, a "bell shape" effect of sucrose concentrations in the extraction mixture was observed. It should be noted that with the concentrations used here for soaking, the oocytes floated over the extraction-solution when it contained more than M sucrose. The most efficient concentrations of sucrose for aged oocytes (n = 232) ranged between and 0.5 M. They allowed a consistent recovery of 32% to 45% of the oocytes. When freshly collected oocytes (n = 82) were tested, an average recovery rate of 54% was obtained with sucrose concentrations between and 0.5 M. In (M) N5 these conditions, freshly collected oocytes supported the cryopreservation procedure significantly better than aged oocytes (P < 0.025). A sucrose concentration of 0.2 M appeared to be particularly efficient, allowing the recovery of 80% of frozen-thawed oocytes. A low concentration (0.06 M) in sucrose showed a detrimental effect on freshly collected oocytes (P < 0.05) as was observed with aged oocytes. The sucrose extraction effect was tested with a lower and a higher DMSO concentration for soaking. Respectively, 118 and 105 aged oocytes were exposed for 15 minutes to 0.5 M sucrose and 2.8 or 4.2 M DMSO, and plunged into LN 2 After thawing, the straws contents were expelled to sucrose concentrations ranging from 0.06 to 0.5 M (Fig. 6). The overall survival rate for these 118 and 105 eggs were respectively, 16% and 52%. The maximum survival rate for the cells submitted to a soaking in 2.8 M DMSQ and a sucrose extraction in 0.06 M sucrose was 29% (NS). These results indicate less cryoprotection with this reduced DMSO concentration compared with the 3.5 M DMSO soaking protocol (P < ). The maximum survival rate for the cells submitted to soaking in 4.2 M DMSO and M sucrose extraction was 73% (P < 0.025). For extraction in sucrose between and 0.5 M sucrose overall, soaking in 4.2 M DMSO gives a better cryoprotection than soaking in 3.5 M DMSO (P < 0.025) N21 20 soaking in N MDMSO = 4.2MDMSO N 26 N26 Sucrose concentration of the extraction solution (M} N27 Figure 6 Proportion of morphologically intact aged oocytes 24 hours after freezing and thawing on the basis of the sucrose concentration during extraction with a soaking solution of 2.8 M or 4.2 Min DMSO and 0.5 Min sucrose. Vol. 52, No.5, November 1989 Pensis et al. Human oocyte cryopreservation 791

6 Role of Temperature and Time of Soaking Two hundred and thirteen aged oocytes were exposed to 0.5 M sucrose and 3.5 M DMSO for 5 minutes at 25oC, 10 minutes at 4 oc and 20 minutes at 4 oc. After freezing and thawing, the straws were expelled to a sucrose solution of 0.25 M to compare with the experiments of Figure 6 where 33 oocytes passed through the same solution with a soaking of 15 minutes at 4 C. A 58% significantly higher percentage of survival was observed (P < 0.05), when the oocytes were soaked during 20 minutes at 4 oc. The soaking at 25 oc didn't seem to be efficient for the time tested. Nevertheless, other time of exposure at this laboratory temperature will be tested. All these oocytes were Trypan Blue stained 24 hours after thawing and morphologically assessed.!!:60 :;; n-72 n-33 n-59 Cell Assessment It should be noted that, in all experiments, in those oocytes which failed to survive the degenerative aspect did not appear to be related to the presence of gas bubbles. In these dying cells we usually observed a faster cell expansion during extraction followed by a ooplasm contraction during culture leading to an atresia aspect: a dark cytoplasm in a 60% of normal diameter. Twenty-four hours after thawing about 5% to 10% of the aged oocytes displayed an ooplasm fragmentation and 5 to 10 other percent presented a normal cleavage similar to that which is observed with aging unfertilized oocytes. It must be stressed that with the freshly collected uninseminated oocytes, no cleavage or fragmentation was observed. No parthenogenesis thus occured in our experiments. A very good relationship was obtained between the delayed morphological assessment and the vital staining which demonstrate that all intact oocytes were indeed alive (Fig. 7). Mice Oocytes Finally, we submitted mouse oocytes to these cryopreservation protocols. Only 1% of the 480 mouse oocytes displayed an intact morphology 24 hours after freezing and thawing. DISCUSSION The cryopreservation of oocytes remains a technical challenge. Slow-cooling techniques have been applied with some success in mice But extrapo- Smin 10mln 25"C 4"C 15mln 4"C Temperature and duration of soaking 20m in 4"C Figure 7 Freezing and thawing of aged oocytes. Role of time and temperature during soaking in 3.5 M DMSO and 0.5 M sucrose. Thawing was followed by extraction in 0.25 M sucrose. lation of these methods to human oocytes yields insufficient results to be applied routinely in the clinical program of medically-assisted reproduction. 14 In addition, the recent evolution in embryo cryopreservation indicates a development from a slowcooling multistep procedure toward a fast-cooling single step procedure. These methods are simpler, less time consuming, and do not require expensive and fragile freezing equipment The aim of our study was to establish conditions of ultrarapid freezing with the advantages of vitrification 19 for the cryopreservation of human oocytes. We plan to determine such conditions for human oocytes directly, to avoid the difficulties which are met when animal methods are transposed to humans. The choice of permeating cryoprotectant was based on the following elements. (1) Despite its low-toxicity, glycerol was not retained owing to its low-efficiency for vitrification, (2) Propylene glycol (PROH) was not retained owing to its ability to induce ploidic abnormalities on unfertilized oocytes,10 and (3) DMSO was selected for its murine embryos vitrification efficiency/ 9 its successful use in ova and embryo rapid freezing techniques, 5 and its presumed ability to protect the meiotic spindle during the cooling period. 20 Sucrose was selected as a nonpermeating cryo- 792 Pensis et al. Human oocyte cryopreservation Fertility and Sterility

7 protectant, as the most positive experience has been gained with this substance.21 The cryoprotective role of sucrose and even of albumin is certainly important, as can be seen from this study, but is not easily distinguishable here from the role of the DMSO. We retain the fact that sucrose exerts its cryoprotective effect at least by causing cellular dehydration increasing intracellular concentration of permeating cryoprotectant, DMSO here, into the cell. The role of albumin is not yet demonstrated in this study. The concentrations used (0.3% to 0.4% w/v) are probably not negligible when one considers the Harrison study22 in which similar concentrations of albumin present cryoprotective properties in slow-cooling techniques. It is interesting to note that the concentrations window of sucrose and DMSO, which allows acceptable oocyte integrity after freezing and thawing, is quite narrow. This emphasizes the value of our study which has systematically investigated a wide range of concentrations for each cryoprotectant using a large number of aged human oocytes to specify the more interesting conditions to test with the freshly-collected ones. The network method enables us to avoid the need to use a great number of oocytes in each experiment to obtain statistical value. The sucrose concentration during the DMSO extraction after thawing also has a significant influence on the outcome of the procedure.23 These data indicate that for the DMSO extraction a sucrose concentration lower than the one used for soaking may contribute toward optimizing the technique. This effect depends on the permeability of the oocytes (aged versus fresh) and thus on the soaking conditions (time, temperature, concentration). Damage to the oocytes during this procedure concerns almost the ooplasm and its cytoplasmic membrane. This is obvious during extra,ction where sucrose has no osmotic effect on damaged oocytes. Fractured zona pellucida are indeed very rare. Destroyed oocytes look like atretic ones. This indicates that the short crystallization state of the cryoprotective mixture during thawing could be sufficient to induce fatal damage. A concentration-dependent chemical toxicity of the cryoprotectants is certainly responsible for some of this damage. This is suggested by the preliminary experiments of cell soaking which are not followed by freezing and thawing even if the number of cells involved is not sufficient to show this significantly. It also appears that some oocytes are more sensitive than others to this toxicity. The membrane maturity may contribute to this difference in sensi- tivity to cryoprotectants.24 It should be stressed that in optical microscopy the cytoplasmic damage we observed is not always apparent during the hours which follow the thawing procedure, but becomes obvious 24 hours later. At this stage, we have a perfect concordance with the cells viability assessment with the trypan blue staining. A significant difference in cryotolerance was also observed between fresh oocytes and aged oocytes. If aging in vitro reduces oocyte membrane permeability, this will result in a decreased penetration of DMSO during the soaking and will lead to increased sensitivity of the cell to physical aggression during freezing and thawing. This is confirmed by the experiment where a longer soaking (20 minutes against 15 or 10) makes it possible to save 60% of the aged oocytes. This suggests that some intrinsic characteristics of the oocyte determine its ability to undergo optimal soaking for vitrification and to support freezing and thawing. This difference indicates the need to study human oocyte cryopreservation with fresh oocytes. With freshly collected oocytes the percentage of morphologically intact cells is higher after soaking with or without freezing despite the strong selection applied on the morphological quality of aged oocytes chosen for the experiments. If no parthenogenesis appear amongst the fresh oocytes treated, the fragmentation and cleavage of aged oocytes inseminated and not fertilized is related to the high percent of andrologic indication (57%) of the OPU selected. In these attempts we observe delayed fragmentation and cleavage without any treatment, incubating the oocytes several days. The encouraging data presented are a first step towards a goal. Essential questions remain unsolved. Chemical toxicity may reduce the survival rate without apparent morphological lesion to the cells. These experiments did not demonstrate a retained capacity of fertilization, cleavage, and the forming of normal embryos. Indeed, some damage in the meiotic spindle may arise from cooling.7 This must be investigated once the entire procedure has been applied to fresh human oocytes. Increased resistance of the zona pellucida must be also investigated as a putative consequence of the procedure which will impair subsequent fertilization attempts In this experimental design, now that apparently satisfactory concentrations of DMSO and sucrose have been established for soaking, and sucrose concentration for extraction, it is necessary to study in greater depth the duration and temperature of the soaking period. A better com- Vol. 52, No.5, November 1989 Pensis et al. Human oocyte cryopreservation 793

8 promise between the toxicity of DMSO at high temperature and the physical aggression of cooling to the spindle may still be found. A visualisation of the meiotic spindle is currently being investigated. This could confirm or inform the results obtained by Kola 11 where two protocols of vitrification were tested on the basis of the Rall and Fahy method. Finally, this human protocol allows the recovery of only 1% of mouse oocytes 24 hours after thawing. Therefore, to progress, we are currently screening for another animal model which will allow the use of this cryoprotection protocol to perform fertilization, cleavage, and fetal tests. In conclusion, this study has defined easy and inexpensive freezing and thawing conditions which lead to the recovery of 80% of morphologically intact human oocytes. We specified the conditions in which aged and fresh human oocytes can be rapidly cryopreserved. Cell losses seem to be due to chemical as well as physical aggression depending on the intrinsic quality of the protoplasm. Further study is required to demonstrate the innocuity of the method before its application as therapy for infertile couples. REFERENCES 1. Edwards RG, Steptoe PC: Current states of in vitro fertilization and implantation of human embryos. Lancet 2: 1265, Lancaster P A: High incidence of preterm birth and early losses in pregnancy after in vitro fertilization. Br Med J 291:1160, Mandelbaum J, Junca AM, Plachot M, Alnot MO, Salat Baroux J, Alvarez S, Tibi C, Cohen J, Debache C, Tesquier L: Cryopreservation of human embryos and oocytes. Hum Reprod 1:117, Devroey P, Braekmans P, Camus M, Khan I, Smitz J, Staessen C, Van den Abbeel E, Van Waesberghe L, Wisanto A, Van Steirteghem AC: Pregnancies after replacement of fresh and frozen-thawed embryos in a donation program. In Future Aspects in Human In-Vitro Fertilization, Edited by W Feichtinger, P Kemeter. Springer Verlag, Berlin, 1987, p Trounson A: Preservation of human eggs and embryos. Fertil Steril46:1, Friedler S, Shen E, Lamb EJ: Cryopreservation of mouse 2- cell embryos and ova by vitrification: methodological studies. Fertil Steril48:306, Magistrini M, Szi:illosi D: Effects of cold and of isopropyl N -phenylcarbonate on the second meiotic spindle of mouse oocytes. Eur J Cell Biol 22:699, Glenister PH, Wood MJ, Kirby C, Wittingham DG: Incidences of chromosome anomalies in first cleavage-mouse embryos obtained from frozen-thawed oocytes fertilised in vitro. Gamete Res 16:205, Pickering SJ, Johnson MH: The influence of cooling on the organization of the meiotic spindle of the mouse oocyte. Hum Reprod 2:207, Vander Elst J, Van den Abbeel E, Jacobs R, Wisse E, Van Steirteghem A: Effect of 1,2-propanediol and dimethylsulfoxide on the meiotic spindle of the mouse oocyte. Hum Reprod 8:960, Kola I, Kirby C, Shaw J, Davey A, Trouson A: Vitrification of mouse Oocytes. Results in Aneuploid Zygotes and Malformed Fetuses. Teratology 38:467, Chen C: Pregnancy after human oocyte cryopreservation. Lancet 1:884, Van Uem JF, Siebzehnrubl ER, Schuh B, Koch R, Trotnow S, Lang N: Birth after cryopreservation of unfertilized oocytes. Lancet 1:752, Al-Hasani S, Diedrich K, V ~n der Ven H, Reinecke A, Hartje M, Krebs D: Cryopreservation of human oocytes. Hum Reprod 2:695, Trounson A, Peura A, Kirby C: Ultrarapid freezing: a new low-cost and effective method of embryo cryopreservation. Fertil Steril 48:843, Loumaye E, Vankrieken L, Depreester S, Psalti I, de Cooman S, Thomas K: Hormonal changes induced by shortterm administration of a gonadotropin-releasing hormone agonist during ovarian hyperstimulation for in vitro fertilization and their consequences for embryos development. Fertil Steril51:105, Whittingham DG: Fertilization in vitro and development to term of unfertilized mouse oocytes previously stored at -196 c. J Reprod Fertil 49:89, Fahy GM, Me Far lane DR, Angell CA, Meryman HT: Vitrification as an approach to cryopreservation. Cryobiology 21:407, Rall WF, Fahy GM: Ice-free cryopreservation of mouse embryos at -196c by vitrification. Nature 313:573, Johnson MH, Pickering SJ: The effect of dimethylsulfoxide on the microtubular system of the mouse oocyte. Development 100:313, Friedler S, Giudice LC, Lamb EJ: Cryopreservation of embryos and ova. Fertil Steril49:743, Harrison KL, Pope AK, Wilson LM, Breen TM, Cummins JM: The optimum concentration of albumin as an embryo cryoprotectant. J In Vitro Fert Embryo Transfer 5:288, Schneider U, Mazur P: Osmotic consequences of cryoprotectant permeability and its relation to the survival of frozen-thawed embryos. Therionology 21:68, Mazur P: Freezing of lhing cells: mechanisms and implications. Am J Physiol247(Cell Physiol16:C125), Johnson MH, Pickering SJ, George MA: The influence of cooling on the properties of the zona pellucida of the mouse oocyte. Hum Reprod 3:383, Pensis et al. Human oocyte cryopreservation Fertility and Sterility