University Hospital of Ghent, Ghent, Belgium

FERTILITY AND STERILITY VOL. 74, NO. 3, SEPTEMBER 2000 Copyright 2000 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Cryopreservation of human germinal vesicle stage and in vitro matured M II oocytes: influence of cryopreservation media on the survival, fertilization, and early cleavage divisions Anuradha Goud, Ph.D., a Pravin Goud, M.D., D.N.B., Ph.D., a Chen Qian, M.D., a Josiane Van der Elst, Ph.D., a Georges Van Maele, Ph.D., b and Marc Dhont, M.D., Ph.D. a University Hospital of Ghent, Ghent, Belgium Received December 3, 1999; revised and accepted March 7, 2000. Presented at the Conjoint Annual Meeting of the American Society for Reproductive Medicine and the Canadian Fertility and Andrology Society, Toronto, Ontario, Canada, September 25 30, 1999. Supported by the University of Ghent, Belgium. Reprint requests: Pravin Goud, M.D., D.N.B., Ph.D., Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, Wayne State University School of Medicine, Hutzel Hospital, 4707, St. Antoine Boulevard, Detroit, Michigan 48201 (FAX: 313-745-7037; E-mail: anupravin@aol.com). a Infertility Center, Department of Obstetrics and Gynecology. b Department of Medical Informatics and Statistics, University Hospital of Ghent. 0015-0282/00/$20.00 PII S0015-0282(00)00672-5 Objective: To study the influence of low-sodium cryopreservation media (CPM) on the survival and development of frozen-thawed germinal vesicle (GV) stage and in vitro matured human oocytes. Design: Prospective experimental study. Setting: Academic hospital-based fertility center. Patient(s): Experimental groups: Oocytes cryopreserved at the GV (group A, n 63 and group B, n 64) or M II stage (group C, n 62) with use of conventional (group A) or low-sodium CPM (groups B and C). Control groups: Sibling GV stage oocytes subjected to in vitro maturation (IVM; control group A, n 64; control group B, n 64). Intervention(s): IVM, intracytoplasmic sperm injection and subsequent culture. Main Outcome Measure(s): Rates of survival, maturation, fertilization, and cleavage. Result(s): The postthaw survival was significantly lower in groups A (57.1%) and B (48.4%) compared to C (84.4%). In group A, maturation and cleavage rates were significantly lower, and fertilization rate was similar to controls (GVBD: 72.2% vs. 90.6%; progression to M II: 33.3% vs. 76.6%; cleavage: 42.9% vs. 88.2%; and fertilization: 58.3% vs. 69.4% in group A vs. control group A, respectively). There was no such difference in group B. In group C, despite a slight but significant lowering of the rate of 2 PN and an increase in that of 3 PN (2 PN: 47.4% vs. 70.2% and 3 PN: 15.8% vs. 3.2% in group C vs. total controls, respectively), embryonic cleavage per GV oocyte was significantly higher (25.8%) compared to group A (4.8%) but not to group B (15.6%). The rate of maturation and cleavage per surviving GV oocyte was significantly higher in group B than group A. Conclusion(s): Low-sodium-based CPM is beneficial for in vitro matured M II stage oocytes and is significantly better than the conventional sodium-based media for the GV stage oocytes. (Fertil Steril 2000; 74:487 94. 2000 by American Society for Reproductive Medicine.) Key Words: Oocyte cryopreservation, in vitro oocyte maturation, cryopreservation media, fertilization, cleavage Oocyte cryopreservation could be an attractive alternative to embryo cryopreservation. However, despite a number of potential advantages, the contemporary techniques of mature oocyte cryopreservation are marred by low rates of survival and further development that consequently lead to staggeringly low rates of viable pregnancies (1). One of the factors leading to this adverse outcome in the mature oocytes is the vulnerability of their microtubular spindle to temperature changes that are inherent to cryopreservation and thawing (2, 3). Therefore, freezing of immature germinal vesicle (GV) stage oocytes has been suggested as an alternative (4, 5). But in vitro maturation (IVM) of GV stage oocytes after freeze-thaw is yet to be perfected. Thus, there is a need to investigate the techniques of both mature and immature oocyte cryopreservation to improve the results. 487

One of the important components of the cryopreservation process is the cryopreservation medium (CPM) that plays a vital role in preventing cellular injury during freeze-thaw. Of the various macromolecular components of the CPM, importance of the cryoprotective agent (CPA) has been stressed previously, and the use of 1,2-propanediol is associated with relatively better results for human oocyte cryopreservation (6). During cryopreservation, optimal results are obtained when the oocytes are cooled at a rate slow enough to prevent the formation of intracellular ice crystals. However, at the same time it should also be fast enough to prevent or minimize the detrimental affects of high solute concentrations as water leaves the intracellular compartment. Recently, attention has been drawn to the adverse influence of this solute effect on the viability of cells undergoing cryopreservation. Accordingly, partial replacement of the major solute in the cryopreservation medium, namely, sodium (Na) with choline was found to be beneficial for cryopreservation of mouse (7, 8) and possibly also human M II oocytes (9). However, it is not known if a similar approach could be beneficial to immature or in vitro matured oocytes undergoing cryopreservation. In the present study, we examined the influence of low- Na-based and conventional (Na-based) cryopreservation media on the survival and development of human GV stage and in vitro matured M II stage oocytes. An improved maturation medium supplemented with gonadotropins and epidermal growth factor (EGF) was used for in vitro maturation (10), and intracytoplasmic sperm injection (ICSI) was used as a method to achieve optimum fertilization (6). Lowering of the concentration of Na in the CPM resulted in high postthaw survival of the frozen in vitro matured M II stage and a significantly improved maturation and embryonic cleavage rate of the frozen-thawed GV stage oocytes. MATERIALS AND METHODS Study Design Institutional ethical review board approval was obtained for the current experimental study. Normal appearing sibling germinal vesicle stage oocytes obtained from consenting patients having at least two spare GV stage oocytes were assigned for IVM (control groups A and B) or cryopreservation using the conventional (group A) or the low-na-based (group B) cryopreservation media. In a third group, normal appearing GV stage oocytes were subjected to IVM and the M II stage, therefore, were cryopreserved with use of the low-na-based protocol (group C). The postthaw survival, maturation, fertilization, and subsequent development up to early cleavage divisions in the sibling and nonsibling oocytes groups were statistically compared. Inclusion Criteria To maintain similarity between the three groups, GV oocytes were collected only from those consenting patients that were 40 years and had at least two GV stage oocytes with similar size and status of cumulus cells in addition to an adequate number of normal appearing M II oocytes and that the total number of GV oocytes did not exceed 50% of the cohort collected at the retrieval. Cryopreservation, Thawing, In Vitro Maturation, and ICSI The ovarian stimulation protocols, oocyte collection and processing, and identification of the GV stage oocytes were performed as described before (10). Before randomization, all the GV stage oocytes were allowed to remain in medium M-199 (Gibco BRL, N.V. Life Technologies, Brussels, Belgium) supplemented with 0.27 mm pyruvate (Sigma Chemical Company, St. Louis, MO), 0.4% human serum albumin (HSA, Albumine 20%; Belgian Red Cross, Brussels, Belgium) and 1 mm dbcamp (Sigma) for preventing spontaneous maturation. The collected GV stage oocytes were thoroughly examined under DIC or Hoffman contrast with use of a 400 magnification, and similar appearing GV stage oocytes having at least one to three layers of cumulus cells were assigned for IVM or cryopreservation. In vitro maturation was performed in a medium supplemented with pyruvate, albumin, estradiol, gonadotropins, and epidermal growth factor (10). The cryopreservation was performed with use of a slow freezing method using 1,2-propanediol (PROH) as the cryoprotective agent (CPA), similar to that described by Lassalle et al. (11). However, the CPM used in the study groups B and C differed from that in group A. Briefly, in group A, the oocytes were cryopreserved in modified Dulbecco s phosphate-buffered saline (PBS) supplemented with 10% fetal bovine serum (FBS; Sigma). Oocytes were equilibrated sequentially in 1.5 M PROH and 1.5 M PROH plus 0.1 M sucrose for 10 and 5 minutes each at room temperature. The oocytes were then loaded into sterile straws and subjected to cryopreservation using an automated freezing unit (Kryo-10, Planer Biomed, Middlesex, United Kingdom) programmed to reach 7 C with the initial rate of freezing at 2 C/minute, held at this point for 5 minutes for manual seeding and cooled subsequently to 33 C at the rate of 0.3 C/min. Straws were then plunged into liquid nitrogen ( 196 C) where the oocytes were stored for at least 1 week before thawing. In groups B and C, oocytes were cryopreserved by the method described by Stachecki et al. (7) with use of the specially prepared low-na based medium (CJ1) where Na was partially replaced with choline (137 mm) and the medium was supplemented with 10% FBS. Briefly, the oocytes were sequentially equilibrated in CJ1 containing 1.5 M 1,2- propanediol (10 minutes) and that containing 1.5 M 1,2- propanediol 0.1 M sucrose (10 minutes), both at room temperature. Within the latter 10-minute interval, they were loaded into sterile freezing straws and were subjected to the 488 Goud et al. Human GV and in vitro matured M II oocyte cryopreservation Vol. 74, No. 3, September 2000

TABLE 1 Patient characteristics and ART cycle profile in different groups. Group A Group B Group C Total (Groups A B) No. of patients 48 43 26 91 Mean ( SD) age (y) 31.1 4.2 31.6 4.8 32.6 4.2 31.3 4.5 Total no. of oocytes (mean SD) 848 (17.7 10.1) 804 (18.7 8.5) 406 (18.1 5.1) 1652 (18.1 9.3) Total no. of GV oocytes (mean SD) 147 (3.1 1.6) 143 (3.3 1.6) 82 (3.1 1.4) 290 (3.2 1.6) GV stage oocytes used for study (mean SD) 127 (2.6 1.0) 128 (2.9 1.2) 62 (2.4 1.1) 255 (2.8 1.1) Note: GV germinal vesicle. All P values were not statistically significant. Goud. Human GV and matured MII oocytes. Fertil Steril 2000. same freezing rate for group A and plunged in liquid nitrogen and stored for at least a week before thawing. Thawing in all the three groups was achieved by exposure first for 30 seconds to RT in air followed by 30 C in water bath for an additional 10 seconds (7). Thawed oocytes were rinsed through serial dilutions of freezing medium in the following order, namely, 1.0 M PROH 0.2 M sucrose, 0.2 M sucrose 0.5 M PROH, 0.2 M sucrose, 0.1 M sucrose, and finally, freezing media alone. After this, the GV stage oocytes were kept for an additional 5 minutes in the M-199 with HEPES at 37 C and then subjected to IVM in a medium supplemented with gonadotropins, estradiol, and epidermal growth factor (10). The M II stage oocytes were transferred to HTF (Irvine Scientific, Irvine, CA) supplemented with 0.4% HSA and preequilibrated at 37 C in 5% CO 2, examined for survival, and subjected to ICSI. Generally, ICSI was performed 2 4 hours after maturation or equilibration as described before (12, 13). Fertilization check was performed 16 20 hours later and the 2 PN zygotes were followed for another 24 36 hours for cleavage. Statistical Methods The data were analyzed with use of SPSS (version 7.5, SPSS Inc., Chicago, IL). Patient parameters such as age, stimulation protocol, number of mature and immature oocytes, and fertilization rates from the three groups were compared with use of the Kruskal-Wallis one-way ANOVA test. Comparisons between different parameters among the sibling and nonsibling oocyte groups were performed with use of the 2 test with Yates correction. RESULTS The patient parameters for age, stimulation protocol, numbers of mature, immature, and total oocytes retrieved were similar in all the three groups (Table 1). In addition, the control groups A and B that were subjected to in vitro maturation had similar rates of maturation, fertilization, and cleavage (Table 2). In groups A and B the oocytes had at least one to three layers of attached cumulus-corona cells before the start of cryopreservation. However, after thawing, most of these oocytes were seen with only loosely attached or partially or completely detached cumulus-corona cells (Fig. 1B). Some oocytes, however, had persistently attached cumulus-corona cells. But in the latter case, the oocytes were found to be damaged as seen from the dark cytoplasm and abnormal appearance (Fig. 1A). The rates of survival in groups A and B were similar but significantly lower than that in group C (Table 2). However, among the oocytes surviving the freeze-thaw, significantly fewer oocytes in group A underwent GVBD (P.04), progression to the M II stage (P.0001), and cleavage to two or more cells (P.03) compared with their sibling controls (Table 2). On the other hand, the surviving GV stage oocytes from group B had similar rates of maturation, fertilization, and cleavage compared with their sibling controls (Table 2). In group C, the rate of 2 PN was significantly lower and that of 3 PN significantly higher (P.03 for both) compared with the total controls (A B). Nevertheless, the efficiency of the cryopreservation procedure, as judged from the rate of cleavage per GV oocyte, was significantly higher in group C compared with group A (P.04), whereas in group B, the per GV oocyte cleavage rate was statistically similar to that of group C. Overall, a significant decrease in the per GV cleavage rates was, however, noted in all the groups (Fig. 2) compared with their sibling (groups A and B, P.005) and nonsibling control oocytes (group C, P.03). Thus, cryopreservation significantly influenced the development of the oocytes to the cleavage stage embryos, irrespective of the use of the conventional Na-based or the new low-na-based CPM. Finally, a direct comparison between the groups A and B indicated that a significantly higher surviving GV stage oocytes from group B underwent maturation to M II (P.05) and cleavage (P.04) compared with group A (Table 2, Fig. 2). Photomicrographs of oocytes from different groups and at different stages are presented in Figure 1. FERTILITY & STERILITY 489

TABLE 2 Survival, maturation, and development after ICSI in GV stage and in vitro matured M II stage oocytes subjected to cryopreservation and thawing in conventional and low Na-based media. Control group A Group A Control group B Group B Group C Total controls (A B) P value No. of oocytes 64 63 64 64 62 128 No. of oocytes that survived after freeze-thaw (%) 36 (57.1) 31 (48.4) 38/45.005 c (84.4).0001 d No. of oocytes that underwent GVBD (%) 58 (90.6) 26 (72.2) 59 (92.2) 28 (90.3) 56 (90.3) 117 (91.4).04 a No. of oocytes that matured to the M II stage (%) 49 (76.6) 12 (33.3) 45 (70.3) 19 (61.3) 45 (72.6) 94 (73.4).0001 a No. of oocytes that developed.05 b 2 PN after ICSI (%) 34 (69.4) 7 (58.3) 32 (71.1) 11 (57.9) 18 (47.4) 66 (70.2).03 e No. of oocytes that developed 1 PN (%) 3 (6.1) 1 (8.3) 3 (6.7) 1 (5.3) 3 (7.9) 6 (6.4) NS No. of oocytes that developed 3 PN (%) 2 (4.1) 0 1 (2.2) 2 (10.5) 6 (15.8) 3 (3.2).03 e No. of cleavage stage embryos f 30 3 26 10 16 56 Note: NS not significant. Control group A vs. group A. Group A vs. group B. Group A vs. group C. Group B vs. group C. Group C vs. total controls (A B). f Refer to Figure 2 for the comparisons between cleavage rates. Goud. Human GV and matured MII oocytes. Fertil Steril 2000. Despite the similarity in the survival rate between groups A and B, group B had significantly better maturation and cleavage rates than group A. Furthermore, the per GV cleavage rates were superior in both groups B and C. Thus, the use of low Na-based CPM was associated with improved results compared with the conventional low-na-based medium. DISCUSSION Oocyte cryopreservation has a tremendous potential in clinical assisted reproduction. This is mainly due to the possibility of it being developed as an alternative technique to embryo cryopreservation and to develop an ovum bank. Cryopreservation of immature oocytes adds another dimension to this issue by raising a possibility of rescuing the fertility of young women undergoing ablative therapies for malignancy. However, both mature and immature oocyte cryopreservation have drawbacks of being less efficacious compared with the contemporary techniques of embryo cryopreservation (1, 14). After the initial successes of human oocyte cryopreservation in the 1980s (14 16), there were no further similar reports during the next decade until recently (17). The dormancy of a decade was due to the low levels of success of oocyte cryopreservation and concerns about the normality of the resultant embryos, whereas the current resurgence of interest is attributed to the encouraging reports of successful pregnancies from oocytes cryopreserved at the mature (17, 18) and immature stage (19). The recent successes could be attributed to the use of PROH as a CPA and the application of ICSI for the fertilization. The latter was shown to be highly successful in developing human embryos to the hatched blastocyst stage (6). Similarly, the technique of immature oocyte cryopreservation has received a boost due to the success in achieving viable pregnancies from in vitro matured oocytes (20, 21). Despite these encouraging reports, concerns about the normality of the embryos derived from cryopreserved oocytes are yet present. This is due to the vulnerability of the oocytes to temperature changes and to the possibility of damage inflicted to the cytoskeleton and the cytoplasmic organelles during cryopreservation and thawing (2, 22 24). Nevertheless, this could possibly be avoided by cryopreserving the oocytes at the GV stage when the chromosomes are within the nuclear membrane and not arranged on the spindle that could be disrupted due to the cryopreservation and thawing process (5). Human immature oocyte cryopreservation was previously performed by Toth et al. (4) using GV oocytes obtained after ovarian stimulation. The results were encouraging in terms of maturation and fertilization postthaw. However, the developmental potential remained unknown, and the results 490 Goud et al. Human GV and in vitro matured M II oocyte cryopreservation Vol. 74, No. 3, September 2000

FIGURE 1 Cryopreservation and thawing at the GV stage frequently resulted in detachment of the cumulus-corona cells from the oocytes, whereas those with the cells attached, often underwent damage. (A), An oocyte that underwent damage with persistently attached cumulus cells (arrow indicates the GV). (B), A surviving oocyte with cumulus cell detachment. (C) and (E), Oocytes that were frozen-thawed either after or before in vitro maturation, respectively, in the low-na medium. (D), A control GV oocyte showing both persistence and growth of the attached cumulus cells after culture. (F) and (G) are zygotes bearing 2 PN obtained after ICSI on M II oocytes that developed from GV oocytes frozen-thawed in conventional and low-na media respectively. (H), (I) and (J) are four- to eight-cell embryos that cleaved from in vitro matured oocytes from groups (B), (A), and (C), respectively. All pictures taken under Hoffman contrast at 200 400 original magnification. Scale bars in D (to represent D), H (to represent H and I) and G (to represent all the rest) correspond to 50 m. Goud. Human GV and matured MII oocytes. Fertil Steril 2000. could not be reproduced for GV stage oocytes retrieved from unstimulated ovaries (25, 26). Similarly, another study reported an increase in the chromosome and spindle abnormalities in oocytes cryopreserved at the GV stage and matured thereafter in vitro (27). These results are, however, somewhat different from those reported earlier (5). Nonetheless, this could have been related to the source of oocytes (unstimulated ovaries) and perhaps to certain patient characteristics, such as age, that were not specified in the former study. In fact, successful birth has recently been reported from oocytes frozen at the GV stage (19). In the present study, we applied low Na-based media for the cryopreservation of immature GV stage and in vitro FERTILITY & STERILITY 491

FIGURE 2 A bar chart representing the rates of cleavage in the experimental and control groups in the context of the zygotes with 2 PN and the total and the surviving GV stage oocytes. Significant differences are indicated as follows: *P.03, P.058 (insignificant after Yates correction), P.0001, P.003, P.002, P.03, and **P.04. Goud. Human GV and matured MII oocytes. Fertil Steril 2000. matured M II stage oocytes. The aim was to optimize the method and to draw conclusions that could help improve the results of both immature and mature oocyte cryopreservation. We previously standardized a technique for the maturation of the spare GV stage oocytes retrieved from patients undergoing ICSI cycles (10). Completion of nuclear and perhaps also cytoplasmic maturation from these oocytes has been confirmed because of their capability of undergoing normal fertilization, cleavage, and also redistribution and increase in the inositol 1,4,5-trisphosphate sensitive receptors that are the vital components of the cellular calcium release machinery (28). Therefore, in the current study, we opted for a similar approach addressing the issue of low Na-based vs. conventional Na-based CPM for immature and in vitro matured oocyte cryopreservation. We used sibling GV stage oocytes for the present study because they are likely to be similar in terms of patient characteristics. Moreover, similarity between the size and cumulus-corona cell status of the GV stage oocytes was confirmed before freezing. This enabled us to have comparable oocytes in the study and the control groups. Even among the three groups, we compared the patient characteristics such as age, stimulation protocol, oocytes retrieved, and found them to be similar. This ultimately enabled us to compare between the sibling and nonsibling oocyte groups. We observed that the GV oocytes that were covered with attached cumulus-corona cells before the start of cryopreservation frequently lost the cumulus attachments, and in those oocytes with intact looking attached cumuls cells frequently underwent damage. These findings are somewhat similar to those reported in the mouse (29) and may have been due to the shrinking and swelling of the oocytes during the different stages of cryopreservation and thawing. The survival after cryopreservation and thawing was similar between groups A and B. These figures are similar to those published previously (4, 26). However, the survival of the in vitro matured M II oocytes in group C was significantly better as found previously in mouse (30) and bovine (31) oocytes. This may have been due to the differences in the permeability characteristics and osmotic responses between the mature vs. immature oocytes (32) and could also be related to the differences in cytoskeletal and organelle distribution between immature and mature oocytes (28, 33, 34). Overall, the survival rate of in vitro matured M II oocytes in our study is relatively superior to that reported before (5) and 492 Goud et al. Human GV and in vitro matured M II oocyte cryopreservation Vol. 74, No. 3, September 2000

further compares favorably with those reported for in vivo matured M II oocytes (6). Despite the lack of significant improvement in the immediate survival after freeze-thaw, the oocytes in group B that survived, underwent GVBD, maturation to the M II stage, fertilization and cleavage at rates that were similar to the unfrozen controls. This was in contrast to the oocytes cryopreserved with use of the conventional, Na-based medium (group A) where significantly lower rates of GVBD, maturation to M II, and cleavage were observed. Furthermore, direct comparison between the groups A and B indicated a significantly higher maturation to M II and cleavage in group B among the surviving GV oocytes. Thus, nuclear (as judged from the GVBD and maturation to MII) and cytoplasmic maturation (as judged from fertilization and cleavage) were improved in the oocytes cryopreserved and thawed in the low-na CPM. In the oocytes from group C, however, there was a small but significant drop in the rate of normal fertilization and a rise in the rate of tripronuclear fertilization. A decrease in the fertilization rates of the cryopreserved mature oocytes has been reported before, and the etiology was considered to be related to the changes in the property of zona pellucida (35). However, in the current study, we bypassed the zona using ICSI. Therefore, the decrease in the normal fertilization rate may be related to some other cause, e.g., the influence of cryopreservation on the oocyte s ability to form the male pronucleus (36, 37). However, the low 2 PN rate in group C was also paralleled by an increase in the 3 PN rate, which may have indirectly contributed to the low 2 PN rate because the total number of oocytes developing 2 or 3 PN was similar to the controls. This was, however, not seen in the GV stage oocytes from groups A and B where the M II oocytes had similar incidence of 2 PN formation as that in their sibling controls. The occurrence of 3 PN after ICSI is mostly due to digyny, which may occur more commonly in cryopreserved and thawed oocytes (37, 38). Although the precise mechanism to explain the digyny in the frozen-thawed M II oocytes is not known, one likely reason may be related to the damage to the microfilament cytoskeleton that may be inflicted during the process of freeze-thaw. Microfilaments are known to play a role in anchoring the spindle in the oocyte cortex and also in ensuring the extrusion of the polar body after oocyte activation (39). The difference in the distribution pattern of the microfilaments between the GV vs. M II oocytes may have led to the lack of increase in 3 PN in the GV stage oocytes (33, 34). Thus, cytoskeleton of the GV stage oocytes may either be less prone to disruption or may even be capable of repairing the damage inflicted during the process of cryopreservation and thawing. Similar differential sensitivity between GV stage and in vitro matured M II stage oocytes for the meiotic spindle has been noted before (5). Therefore, cytoskeleton of the in vitro matured oocytes may be more susceptible to the process of cryopreservation and thawing. Overall, our results indicate that oocytes frozen in the low Na CPM retained their capability to undergo maturation and further development to early embryonic cleavage stages better than those cryopreserved in the conventional Na-based CPM. This confirms the earlier finding of Stachecki et al. (7) in mouse oocytes and adds supportive evidence to Lovelock s hypothesis of solute effect occurring during cryopreservation (40). These results are encouraging for the routine application of oocyte freezing. However, further elucidation is now required to confirm the normality of the resultant embryos in terms of chromosomal complement and developmental potential. References 1. Mandelbaum J, Belaish-Allart J, Junca A, Antoine J, Plachot M, Alvarez S, et al. Cryopreservation in human assisted reproduction is now routine for embryos but remains a research procedure for oocytes. Hum Reprod 1998;13(Suppl 3):161 77. 2. Pickering SJ, Braude PR, Johnson MH, Cant A, Currie J. Transient cooling to room temperature can cause irreversible disruption of the meiotic spindle in the human oocyte. Fertil Steril 1990;54:102 8. 3. Van der Elst J, Van den Abbeel E, Jacobs R, Wisse E, Van Steirteghem A. Effect of 1,2 propanediol and dimethylsulphoxide on the meiotic spindle of the mouse oocyte. Hum Reprod 1988;8:960 7. 4. Toth TL, Baka SG, Veeck LL, Jones HW Jr, Muasher S, Lanzendorf SE. Fertilization and in vitro development of cryopreserved human prophase I oocytes. Fertil Steril 1994;61:891 4. 5. Baka SG, Toth TL, Veeck LL, Jones HW Jr, Muasher SJ, Lanzendorf SE. Evaluation of the spindle apparatus of in-vitro matured human oocytes following cryopreservation. Hum Reprod 1995;10:1816 20. 6. Gook DA, Schiewe MC, Osborn SM, Asch RH, Jansen RPS, Johnston WIH. Intracytoplasmic sperm injection and embryo development of human oocytes cryopreserved using 1,2-propanediol. Hum Reprod 1995;10:2637 41. 7. Stachecki JJ, Cohen J, Willadsen S. Detrimental effects of sodium during mouse oocyte cryopreservation. Biol Reprod 1998;59:395 400. 8. Stachecki JJ, Cohen J, Willadsen SM. Cryopreservation of unfertilized mouse oocytes: the effect of replacing sodium with choline in the freezing medium. Cryobiology 1998;37:346 54. 9. Stachecki JJ, Cohen J, Willadsen S. Detrimental effects of sodium during mouse oocyte cryopreservation. [abstract no. O-001] In: Abstracts of the Scientific Oral and Poster Sessions Program Supplement to Fertil Steril 16th World Congress on Fertility and Sterility and 54th Annual Meeting of the American Society for Reproductive Medicine, October 4 9, 1998. 10. Goud PT, Goud AP, Qian C, Laverge H, Van der Elst J, De Sutter P, et al. In-vitro maturation of human germinal vesicle stage oocytes: role of cumulus cells and epidermal growth factor in the culture medium. Hum Reprod 1998;13:1638 44. 11. Lasalle B, Testart J, Renard J-P. Human embryo features that influence the success of cryopreservation with the use of 1,2 propanediol. Fertil Steril 1985;44:645 51. 12. Dozortsev D, De Sutter P, Rybouchkin R, Dhont M. Methodology of intracytoplasmic sperm injection in the human. Assist Reprod Rev 1996;6:38 44. 13. Goud P, Rybouchkin A, De Sutter P, Dhont M. Fine points of technique: ICSI (Letter). Fertil Steril 1997;67:979 80. 14. Al-Hasani S, Diedrich K, Van de Ven H, Reinecke A, Hartje M, Krebs D. Cryopreservation of human oocyte. Hum Reprod 1987;2:695 700. 15. Chen C. Pregnancy after human oocyte cryopreservation. Lancet 1986; 1:884 6. 16. Van Uem JFHM, Siebzenrübl ER, Schuh B, Koch R, Trotnow S, Lang N. Birth after cryopreservation of unfertilized oocytes. Lancet 1987;1: 752 3. 17. Porcu E, Ciotti PM, Fabbri R, Magrini O, Seracchioli R, Flamigni C. Birth of a healthy female after intracytoplasmic sperm injection of cryopreserved human oocytes. Fertil Steril 1997;68:724 6. 18. Young EY, Kenny A, Puigdomenech E, Van Thillo G, Tiveron M, Piazza A. Triplet pregnancy after intracytoplasmic sperm injection of cryopreserved oocytes: case report. Fertil Steril 1998;70:360 1. 19. Tucker MJ, Wright G, Morton PC, Massey JB. Birth after cryopreser- FERTILITY & STERILITY 493

vation of immature oocytes with subsequent in vitro maturation. Fertil Steril 1998;70:578 9. 20. Cha KY, Koo JJ, Ko JJ, Choi DH, Han SY, Yoon TK. Pregnancy after in vitro fertilization of human follicular oocytes collected from nonstimulated cycles, their culture in vitro and their transfer in a donor oocyte program. Fertil Steril 1991;55:109 13. 21. Russell JB, Knezevich KM, Fabian KF, Dickson JA. Unstimulated immature oocyte retrieval: early versus midfollicular endometrial priming. Fertil Steril 1997;67:616 20. 22. Santhananthan AH, Trounson A, Freeman L, Brady T. The effects of cooling human oocytes. Hum Reprod 1988;3:968 77. 23. Aigner S, Van der Elst J, Siebzehnrubl E, Wildt L, Lang N, Van Steirteghem AC. The influence of slow and ultra-rapid freezing on the organization of meiotic spindle of the mouse oocyte. Hum Reprod 1992;7:857 64. 24. Van Blerkom J, Davis PW. Cytogenetic, cellular, and developmental consequences of cryopreservation of immature and mature mouse and human oocytes. Microsc Res Tech 1994;27:165 93. 25. Toth TL, Hassen WA, Lanzendorf SE, Hansen K, Sandow BA, Hodgen GD, et al. Cryopreservation of human prophase I oocytes collected from unstimulated follicles. Fertil Steril 1994b;61:1077 82. 26. Son WY, Ko JJ, Park SE, Yoon TK, Lee KA, Cha KY, et al. Effects of 1,2-propanediol and freezing-thawing on the in vitro developmental capacity of human immature oocytes. Fertil Steril 1996;66:995 9. 27. Park SE, Lee KA, Son WY, Ko JJ, Lee SH, Cha KY. Chromosome and spindle configurations of human oocytes matured in vitro after cryopreservation at the germinal vesicle stage. Fertil Steril 1997;68:920 6. 28. Goud PT, Goud AP, Van Oostveldt P, Dhont M. Presence and dynamic redistribution of type I inositol 1,4,5-trisphosphate receptors in human oocytes and embryos during in-vitro maturation, fertilization and early cleavage divisions. Mol Hum Reprod 1999;5:441 51. 29. Cooper A, Paynter SJ, Fuller BJ, Shaw RW. Differential effects of cryopreservation on nuclear or cytoplasmic maturation in vitro in immature mouse oocytes from stimulated ovaries. Hum Reprod 1998; 13:971 8. 30. Schroeder AC, Champlin AK, Mobraaten LE, Eppig JJ. Developmental capacity of mouse oocytes cryopreserved before and after maturation in vitro. J Reprod Fertil 1990;89:43 50. 31. Lim JM, Fukui Y, Ono H. Developmental competence of bovine oocytes frozen at various maturation stages followed by in vitro maturation and fertilization. Theriogenology 1992;37:351 61. 32. Agca Y, Liu J, Peter AT, Critser ES, Critser JK. Effect of developmental stage on bovine oocyte plasma membrane water and cryoprotectant permeability characteristics. Mol Reprod Dev 1998;49:408 15. 33. Kim NH, Funahashi H, Prather RS, Schatten G, Day BN. Microtubule and microfilament dynamics in porcine oocytes during oocyte maturation. Mol Reprod Dev 1996;43:248 55. 34. Kim NH, Chung HM, Cha KY, Chung KS. Microtubule and microfilament organization in maturing human oocytes. Hum Reprod 1998;13: 2217 22. 35. Carroll J, Depypere H, Matthews CD. Freeze-thaw-induced changes of the zona pellucida explains decreased rates of fertilization in frozenthawed mouse oocytes. J Reprod Fertil 1990;90:547 53. 36. Quinn P, Barros C, Whittingham DG. Preservation of hamster oocytes to assay the fertilizing capacity of human spermatozoa. J Reprod Fertil 1982;66:161 8. 37. Goud P, Goud A, Van Oostveldt P, Van der Elst J, Dhont M. Fertilization abnormalities and pronucleus size asynchrony after intracytoplasmic sperm injection are related to oocyte postmaturity. Fertil Steril 1999;72:245 52. 38. Carroll J, Warnes GM, Matthews CD. Increase in digyny explains polyploidy after in-vitro fertilization of frozen-thawed mouse oocytes. J Reprod Fertil 1989;85:489 94. 39. Maro B, Johnson MH, Pickering SJ, Flach G. Changes in actin distribution during fertilization of the mouse egg. J Embryol Exp Morphol 1984;81:211 37. 40. Lovelock J. The protective action of neutral solutes against haemolysis by freezing and thawing. Biochem J 1954;56:265. 494 Goud et al. Human GV and in vitro matured M II oocyte cryopreservation Vol. 74, No. 3, September 2000