Effects of sucrose concentration on the developmental potential of human frozen thawed oocytes at different stages of maturity

Human Reproduction Vol.19, No.10 pp. 2345 2349, 2004 Advance Access publication August 6, 2004 DOI: 10.1093/humrep/deh442 Effects of sucrose concentration on the developmental potential of human frozen thawed oocytes at different stages of maturity Z.J.Chen 1,2, M.Li 1, Y.Li 1, L.X.Zhao 1, R.Tang 1, Y.Sheng 1, X.Gao 1, C.H.Chang 2 and H.L.Feng 3,4 1 Center for Reproductive Medicine, Shandong Provincial Hospital, Shandong University, Jinan 250021, China, 2 Department of Medicine, Case Western Reserve University, Cleveland, Ohio 44106 and 3 Center for Human Reproduction, North Shore University Hospital, NYU School of Medicine, Manhasset, NY 11030, USA 4 To whom correspondence should be addressed at: Center for Human Reproduction, North Shore University Hospital, 300 Community Drive, Manhasset, NY 11030, USA. E-mail: hfeng@nshs.edu BACKGROUD: Success of human oocyte cryopreservation depends on multiple cryobiological factors that could influence the developmental potential of the oocytes. The objective of this study was to examine the effects of different sucrose concentrations on the developmental potential of human frozen thawed oocytes at different maturity stages. METHODS: A total of 355 oocytes collected from small follicles were randomly divided into three groups and two groups (B and C) were cryopreserved using slow-freezing method. included 131 oocytes at different maturity stages without freezing. Another 119 oocytes in were cryopreserved with 0.1 M sucrose and 105 oocytes in with 0.2 M sucrose concentration. RESULTS: The post-thaw survival rate of the oocytes and the cleavage rate in were significantly higher than that of (P <0.05). For immature metaphase I (MI) stage oocytes, a significant difference was found in the maturation rate between and (P <0.05). The maturation rate for the GV oocytes in A and C was significantly higher than (P <0.01). CONCLUSIONS: The results suggested that sucrose concentration of 0.2 M in the cryoprotectant solution is more suitable for human oocyte cryopreservation. Key words: cryopreservation/embryo/human fertilization in vitro/ooctyes Introduction In humans, the main application of oocyte cryopreservation technique is fertility restoration in women at risk of premature menopause, which can have several causes: recurrent or severe ovarian diseases such as cysts, benign tumors and endometriomas; ovary removal to treat endometriosis or genital cancer; and chemotherapy or radiotherapy to treat cancer or other systemic diseases (Amorim et al., 2003). For women with certain types of chromosomal anomalies, such as Turner s syndrome (Aubard et al., 2000), which can also cause premature ovarian failure, oocyte cryopreservation might be a good option for fertility treatment. It has been reported (Hreinsson et al., 2002) that follicles may be found in ovaries from adolescent girls with Turner s syndrome. This finding is very important as it indicates that infertility in these patients can be avoided by rescuing ovarian fragments for cryopreservation procedures (Amorim et al., 2003). Oocyte cryopreservation technique could also be used in healthy women who choose to delay childbearing until later in life. It would also facilitate oocyte donation, as the procedure for harvesting the ovarian fragment is very simple and does not require hormonal stimulation, which can cause side effects to the donor (Van den Hurk et al., 2000). In addition, oocyte cryopreservation is a successful alternative for storing the excess of oocytes during the ART therapies, thus avoiding ethical, moral and religious dilemmas. However, in spite of several successes being reported (Porcu et al., 1997; Kuleshova et al., 1999; Porcu et al., 2000; Van der Elst, 2003), there are still technical problems associated with oocyte freezing. Studies have shown that oocyte survival rates after cryopreservation could be affected by morphological and biophysical factors. Morphological characteristics of oocytes such as maturity and size are particularly important. Biophysical factors such as cryoprotectant compositions are also important. For oocyte cryopreservation with slow-freezing method, cryoprotectant solution consists of usually, 1.5 M membrane-permeating cryoprotectant (i.e. propanediol) and 0.1 M sucrose. To limit damage occurring during the cryopreservation procedure, it is necessary to reduce the amount of intracellular water by an increasing dehydration process. It was reported that increasing the sucrose concentrations could benefit Human Reproduction vol. 19 no. 10 q European Society of Human Reproduction and Embryology 2004; all rights reserved 2345

Z.J.Chen et al. the survival of the frozen-thawed human oocytes (Fabbri et al., 2001). In the present study, in order to evaluate the effects of different concentrations of sucrose in the cryoprotectant solution on the developmental potential of human oocytes at different maturity stages, we have frozen different maturity oocytes with a slow-freezing rapid-thawing protocol using 1.5 M 1,2-propanediol with 0.1 M or 0.2 M sucrose. Materials and methods Patients Human oocytes were obtained from counselled polycystic ovarian syndrome (PCOS) patients undergoing an immature follicle puncture treatment, who donated their oocytes punctured from the small follicles. The average age of the patients was 30.23 ^ 3.81 years. All the patients were treated by ovarian stimulation protocol. The PCOS patients were given 150 IU human menopausal gonodotrophen (HMG) by muscle injection from menstrual cycle day 3 for 4 days. A 10 000 IU of HCG was given when the follicles in the two ovaries were about 8 to 10 mm in diameter. Thirty-four hours later, the oocytes were retrieved by transvaginal oocyte aspiration using a specially designed single-lumen aspiration needle (K-OPS-1235- Wood, Cook, Australia). The Human Sperm Bank of Shandong Provincial Hospital provided the human sperm used for intracytoplasmic sperm injection of the matured oocytes. The Shandong University Hospital Institutional Review Board approved the use of patients oocytes and donor sperm for the present study. Cryoprotectant solutions All cryoprotectant solutions were prepared by using Dulbecco s phosphate-buffered solution (DPBS) (Sigma), 1,2-propanediol (PROH) (Sigma) and 5 mg/ml human serum albumin (HSA) (Sigma). We used the following freezing solutions: 1.5 M PROH þ 5 mg/ml HSA in the PBS solution (S1) and 1.5 M PROH þ sucrose (0.1 M or 0.2 M) þ 5 mg/ml HSA (S2). For thawing procedure, the oocytes were thawed in solutions with decreased PROH concentration gradients of 1.0 M, 0.5 M and 0.0 M in the presence of 0.2 M sucrose (Fabbri et al., 2001). Retrieval of oocytes The recovered oocytes from large follicles were used for the patients in vitro maturation (IVM)/IVF procedure. The oocytes from the small follicles (diameter, 1 cm) were included in this study. The participants signed the consent forms. The cells of the cumulus and corona radiata were completely removed by a brief exposure to HEPES-buffered solution containing 80 IU/ml hyaluronidase (Irvine Scientific, USA) and by gentle aspiration in and out of a hand-drawn glass pipette (Sage BioPharma, NJ). The denudated oocytes were then evaluated by assessment of their nuclear maturation stage under the microscope. The assessment included identification of germinal vesicle (GV) stage, metaphase I (MI) stage and metaphase II (MII) stage oocytes. Different maturity oocytes were divided randomly into three experimental groups: was not frozen; was frozen with cryoprotectant solution containing 0.1 M sucrose and 1.5 M PROH; with 0.2 M sucrose and 1.5 M PROH. Both groups (B and C) had the same concentration of 1.5 M PROH. Cryopreservation and thawing The oocytes from the two cryopreservation groups were incubated in human tubal fluid (HTF) plus 10% serum substitute supplement 2346 (SSS) (Irvine scientific, CA) for no more than 3 h before being transferred to culture dishes containing DPBS supplemented with 5 mg/ml human serum albumin (HSA) (Irvine scientific, CA) at room temperature. After pretreatment in S1 solution for 10 min, the oocytes were transferred to S2 solutions. Then the oocytes were loaded into 0.25 ml plastic straws (IMV-Technologies Lalgle, France) and transferred into an automated program freezer (Planer Kryo-10, England). The time of the oocytes in the cryoprotectant was,20 min in all. The initial chamber temperature was 208C in the freezer. The temperature was reduced slowly to 278C at a rate of 28C/min. Ice nucleation was induced manually by ice-forceps. After a hold time of 10 min at 278C, the temperature was cooled slowly to 2308C at a rate of 0.38C/min followed by rapid cooling to 21208C at a rate of 308C/min. The straws were transferred into liquid nitrogen tanks and stored until thawing. All the cryopreserved oocytes were stored in the liquid nitrogen for 6 months. To thaw, the oocytes were air-warmed for 40 s and then immersed in a 308C water bath for 40 s until all traces of ice had disappeared. The cryoprotectant was removed at room temperature by stepwise dilution of PROH in the thawing solutions. The contents of the melted straws were expelled in the 1.0 M PROH and 0.2 M sucrose solution and equilibrated for 5 min and then in 0.5 M PROH and 0.2 M sucrose for another 5 min. After that the oocytes were transferred to 0.2 M sucrose for 5 min, and placed into the final solution of DPBS supplemented with 5 mg/ml HSA. Finally, the immature oocytes were transferred to the M-199 culture medium for in vitro maturation; the mature oocytes were cultured in HTF for further incubation in a 378C, 5% CO 2 incubator. Two hours after thawing, the oocytes were checked for survival according to morphological criteria. The oocytes were classified as high-quality oocytes and low-quality oocytes. High-quality oocytes means that the zona pellucida and plasma membrane were intact, the perivitelline space was clear and normal in size and there was no evidence of cytoplasmic leakage or oocyte shinkage, which could be observed under the inverted microscope. They also had good refractive cytoplasm and smooth plasma membrane. In the but low-quality oocytes, the perivitelline space was enlarged and the cytoplasm was darker, though refraction was just fair under the dissecting microscope. In vitro maturation, ICSI and embryo culture The fresh immature oocytes and frozen-thawed immature oocytes were subjected to (IVM) in M-199 medium supplemented with 20% fetal bovine serum (Gibco, USA), penicillin G 50 IU/ml, streptomycin sulfate 50 mg/ml, pyruvate sodium 25 mm (Sigma, USA), recombinant FSH 0.075 IU/ml, HCG 0.15 IU/ml (Gonal-F; Serono Laboratories) and epidermal growth factor (EGF) 2 ng/ml (Goud et al., 1998). After in vitro culture for 24 48 h, the matured highquality oocytes were subjected to ICSI if the first polar bodies were extruded and the cytoplasm was homogeneous with good refraction. The thawed MII oocytes were cultured in HTF supplemented with 10% SSS for 2 4 h, and then ICSI performed. A fertilization check was performed 16 20 h later and the 2PN zygotes were followed for another 40 48 h in P-1 (Irvine Scientific, USA) for cleavage. According to Puissant s standard for embryo grades, four cell embryos with, 30% fragmentation were specified as high quality embryos (Puissant et al., 1987). Embryos in the non-frozen group were cryopreserved for future replacement into the patient s uterus. Some of the embryos in the frozen MII oocytes group were transferred to patients who needed embryo donation, and obtained data will be submitted for publication elsewhere.

Oocyte cryopreservation in humans Table I. The effect of different sucrose concentration on the developmental potential of frozen thawed MII stage oocytes MII oocytes oocytes oocytes fertilized a oocytes cleaved a high-quality embryos a Non-frozen control 51 N/A 39 (76.5) 35 (68.6)** 29 (56.9)* 0.1 M sucrose 45 22 (48.9) 13 (59.1) 5 (22.7) 3 (13.6) 0.2 M sucrose 69 54 (78.3)** 39 (72.2)* 30 (55. 6)* 22 (40.7)** a The percentage was calculated using the number of surviving oocytes as the denominator. Table II. The effect of different sucrose concentration on the developmental potential of frozen thawed MI stage oocytes MI oocytes oocytes Statistical analysis Differences in the rates of survival, maturation, fertilization, cleavage and embryo quality were analyzed with Fisher s exact test or x 2 test as appropriate. SPSS version 10.0 was used for all statistical analyses. Values were considered significant when P, 0.05. oocytes matured oocytes fertilized a oocytes cleaved a Non-frozen control 58 N/A 42 (72.4)** 25 (59.5) 14 (33.3) 9 (21.4) 0.1 M sucrose 41 32 (78.1) 11 (34.4) 6 (54.6) 3 (27.3) 0 0.2 M sucrose 15 12 (80.0) 9 (75.0)* 6 (66.7) 3 (33.3) 0 a The percentage was counted using the number of the matured oocyte number as the denominator. Table III. The effect of different sucrose concentration on the developmental potential of frozen thawed GV stage oocytes GV oocytes oocytes matured oocytes that oocytes that underwent GVBD oocytes that fertilized a embryos that cleaved a high-quality embryos a high-quality embryos a Non-frozen control 22 N/A 20 (90.9) 15 (68.2)** 10 (66. 7) 4 (26.7) 3 (20.0) 0.1 M sucrose 33 27 (81.8) 19 (70.4) 4 (14.8) 0 0 0 0.2 M sucrose 21 18 (85.7) 18 (100.0)* 13 (72.2)** 9 (69.2) 3 (23.1) 0 GVBD, germinal vesicle breakdown. a The percentage was calculated by taking the matured oocyte number as the denominator. Results A total of 355 oocytes retrieved from 78 PCOS patients undergoing ovary puncture were included in these experiments. A total of 224 oocytes were frozen thawed with slow-freezing rapid-thawing program. The patient parameters for age, stimulation protocol and numbers of the retrieved total oocytes were similar in the three groups. The results of the MII oocytes cryopreservation indicated that the 0.2 M sucrose concentration is beneficial for the survival of frozen thawed human oocytes. The survival rate of the frozen thawed MII oocytes in was significantly higher than that in (P, 0.01). There was also a significant difference in the cleavage rate of the zygotes from B and C (22.73% versus 55.56%, P, 0.05). No such significant difference was found in the fertilization and highquality embryos rates between B and C. However, cleavage rate and high-quality embryo rate in were much less than that in (P, 0.01) (Table I). When it came to the immature MI oocytes, A and C had higher maturation rates than after in vitro maturation (P, 0.01, P, 0.05). No significant difference was found between these groups for other parameters studied, though it seemed that the results in A and C were better than in. No high-quality embryos occurred in the two cryopreservation groups for MI oocytes. High-quality 2347

Z.J.Chen et al. embryos resulted from MI oocytes only in (Table II). There was no significant difference in the survival rates of oocytes at different stages of maturity in both cryopreservation groups. The oocytes that did not survive and became degenerated showed a breakdown of the cytoplasmic membrane and cytoplasmic leakage. According to observation of the oocytes, the surviving oocytes showed a clear cytoplasm and a morphologically healthy appearance in spite of slight roughness in the cytoplasm. As shown in Table III, the GVBD (germinal vesicle broken down) rate of the GV oocytes in was significantly higher than that in (P, 0.05). The matured oocyte rates of the immature GV oocytes after 48 h of in vitro maturation in A and C were also significantly higher than that in (P, 0.01). No matured oocyte was fertilized in. The oocytes in showed a good fertilization rate, but no high-quality embryo developed. It seemed that the developmental potential of the oocytes in was better than that in, but no significant difference was found except that higher-quality embryos developed. Nevertheless, the 0.2 M sucrose concentration gave better results for the cryopreservation of GV oocytes until embryo cleavage (4 8 cells stage). Discussion Human embryo cryopreservation provides an effective technique in the treatment of infertility. However, oocyte cryopreservation offers another method, giving young women at risk of reducing or losing ovarian function because of chemotherapy or professional career development a chance to extend their fertility. Successful pregnancies, achieved through oocyte cryopreservation have been reported at their mature (Porcu et al., 1997; Young et al., 1998; Boldt et al., 2003; Chen et al., 2003; Hreinsson et al., 2003) and immature stage (Tucker et al., 1998). However, both mature and immature oocyte cryopreservation have drawbacks of giving lower survival rates as compared with the contemporary techniques of embryo cryopreservation (Goud et al., 2000). Several plausible explanations have been offered to address the difficulty of storing oocytes (MII) at low temperatures. First of all, there is evidence that the zona pellucida (ZP) hardens and results in premature cortical granule exocytosis during the cryopreservation process (Pickering and Johnson, 1987; Vincent et al., 1991). This change halts the penetration of spermatozoa and inhibits embryonic hatching. This problem can be bypassed using micromanipulation techniques (Gook et al., 1995). Secondly, it is of great concern that there is the possibility of raising the incidence of aneuploidy by depolymerization of the spindle apparatus of the mature oocyte during cellular cooling. The spindle is a dynamic structure composed of microtubules that are being assembled continuously at one end and removed at the other in a treadmill fashion. Although the chromosomes reassemble and align along the spindle equator at cell rewarming, there is a risk of chromosomal loss and of the occurrence of aneuploidy during the first maturation division (Pickering et al., 2348 1998). Thirdly, it has been show that the oocyte cytoskeleton gets damaged by the cryopreservation process, which could lead to significant changes in the organization and trafficking of molecules and organelles (Vincent and Johnson, 1992). Therefore, the quality of frozen oocytes after thawing is a crucial factor to guarantee oocyte fertilization and development potential. The better the quality, the higher the survival rate will be. The high survival rate is particularly affected by freeze thaw protocols. An overview of the literature shows that most studies use a similar freezing and thawing procedure (slow-freezing rapidthawing), similar seeding points and cryoprotectants (1,2- propanediol and sucrose) (Fabbri et al., 2001). The slowfreezing program, as a standard method suitable for large cells, has been successfully used in the cryopreservation of human embryos. In the present study, human oocytes were cryopreserved by using slow-freezing program. The cryoprotectants generally used in oocyte freezing protocols are 1,2- propanediol (membrane-permeating cryoprotectant) and sucrose (membrane non-permeating cryoprotectant). Their protective action is very complex and attributable to a number of properties, the most important of which is the beginning of the dehydration process. In particular, sucrose does not enter the cell, but exerts its beneficial effects by causing cellular dehydration through changes in osmotic pressure (Fabbri et al., 2001). In this study, the effects of different sucrose concentrations on oocyte freezing were investigated. The results indicated that 0.2 M sucrose was more beneficial to the survival of the frozen thawed oocytes than the 0.1 M sucrose (80% vs 68.07%, P, 0.05). A significantly higher developmental potential was found in the second group (0.2 M sucrose) with respect to the first group (0.1 M sucrose). We suggest that the increase in the sucrose concentration generates an osmotic gradient across the cell membrane, which draws water out of the cell, causing the cell to dehydrate sufficiently before and during the freezing procedure. Similar results were obtained in the previous study on the matured oocytes; 0.3 M and 0.2 M sucrose concentrations in the freezing solutions gave higher oocyte survival rates (82% and 60%, respectively) than 0.1 M sucrose (34%) (Fabbri et al., 2001). It was also reported that using Na-depleted media along with other alterations in freezing and thawing procedures in human oocyte cryopreservation can provide excellent survival (74.4%), fertilization (59%) and live birth rates (36.4%) (Boldt et al., 2003). Furthermore, several babies have been born by using oocyte cryopreservation according to a recent report (Fosas et al., 2003). There were also studies suggesting that longer exposure time in cryoprotectants could increase the survival rate pertaining to the dehydration of oocytes, an observation that needs further investigation (Fabbri et al., 2001). Although the survival rates of the frozen thawed immature oocytes appeared to be higher than the frozen thawed mature oocytes, no significant difference in the survival rate was found between oocytes at different maturity stages (P. 0.05). Our results indicated that there was a higher fertilization rate in mature oocytes as compared to immature oocytes, which is similar to previous reports (Fabbri et al., 2001). The cleavage

Oocyte cryopreservation in humans rate was not superior to other studies (91% vs 100%; Gook et al., 1995; Park et al., 2001). At the same time, the GV oocytes could be matured in vitro, but the fertilization and cleavage rates were not as good as in normal mature oocytes, which could be due to freezing injury of the cellular cytoskeleton, resulting in fertilization failure and embryo development arrest (Van der Elst, 2003). The cryopreservation of immature GV oocytes raises a lot of controversial questions. First, no definite conclusion has been drawn about immature oocytes being more suitable for cryopreservation then the mature oocytes. Secondly, the most critical problem is the in vitro maturation of the frozen thawed immature oocytes. A good in vitro maturation system not only means benefit for nucleus maturation, but also benefit for cytopasmic maturation. Although we had a slightly better overall maturation rate of 65.17% with MI and GV oocytes than that reported by Park et al. (61%) (Park et al., 1997), the maturation rate is still lower than the rates obtained with the maturation of fresh oocytes using the same protocol in our center (83%) (Li et al., 2003). The cryopreservation of immature oocytes has been successfully used in animals (Amorim et al., 2003). However, only a few pregnancies and births of normal infants from embryos derived from frozen thawed immature oocytes have been reported in human (Tucker et al., 1998; Goud et al., 2000; Wu et al., 2001). These reports suggest that immature human oocytes could survive the freezing and thawing and process of fertilization in vitro as used for mature oocytes, but only with a very low developmental efficiency. So it seems that the same problems are encountered in human as in mouse immature oocyte maturation with or without freezing. Namely, not the nucleus but the cytoplasm was of concern (Van der Elst, 2003). Therefore, it is necessary in the future to optimize in vitro maturation systems and freeze thawing procedures for immature oocytes. In summary, our study indicates that an increased sucrose concentration (0.2 M) enhanced oocyte survival and quality, which is beneficial for the developmental potential of the embryos. Further studies are required to optimize freezing and thawing conditions in order to improve the oocyte survival rate and increase the potential of oocyte fertilization and embryonic cleavage rates after thawing. Acknowledgements Appreciation is extended to Dr Mathew Cohen for his review and comment. This study was supported by NSFC of China (No. 39300136). References Amorim CA, Goncàlves PB and Figueiredo JR (2003) Cryopreservation of oocytes from pre-antral follicles. Hum Rep Update 9,119 129. Aubard Y, Piver P and Teissier MP (2000) Indications de la cryopreservation du tissu ovarien. La Presse Medicale 29,960 964. 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Submitted on January 2, 2004; resubmitted on May 24, 2004; accepted on July 6, 2004 2349