Comparison of survival and embryonic development in human oocytes cryopreserved by slow-freezing and vitrification

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1 Comparison of survival and embryonic development in human oocytes cryopreserved by slow-freezing and vitrification Yun-Xia Cao, M.D., Ph.D., Qiong Xing, M.D., Li Li, M.D., Lin Cong, M.D., Zhi-Guo Zhang, M.S., Zhao-Lian Wei, M.D., and Ping Zhou, M.D. Center for Reproductive Medicine, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Anhui Medical University, Hefei, People s Republic of China Objective: To compare the survival, fertilization, early embryonic development, and meiotic spindle assembly and chromosome alignment in frozen-thawed human oocytes after slow-freezing and vitrification. Design: A randomized study. Setting: A university-affiliated assisted reproductive center. Patient(s): Donated extra eggs from women undergoing assisted reproduction treatment. Intervention(s): A total of 605 mature oocytes were divided into a slow-freezing group and a vitrification group for cryopreservation. Main Outcome Measure(s): After frozen-thawing, the oocyte survival rate, spindle assembly, and chromosome alignment were compared. The surviving oocytes were inseminated by intracytoplasmic sperm injection, and the rate of fertilization and embryo development were also compared in two groups. Result(s): The oocyte survival rate was statistically significantly lower in the slow-freezing group (75 out of 123, 61.0%) than the vitrification group (268 out of 292, 91.8%). The fertilization rate was the same for both groups, but the cleavage rate of zygotes was statistically significantly different between two groups: (slow-freezing, 25/46 (54.4%) versus vitrification, 142 out of 182 (78.0%). There was a considerable difference in the percentage of high-quality embryos between slow-freezing and vitrification groups: 6 out of 25 (24.0%) versus 60 out of 142 (42.3%), respectively. The percentage of blastocyst development was statistically significantly higher in the vitrification group (47 out of 60, 33.1%) than in the slow-freezing group (3 out of 25, 12.0%). There was a much higher percentage of oocyte abnormalities in terms of spindle assembly and chromosome alignment in the slow-freezing group (25 out of 64, 39.1%) compared with the vitrification group (11 out of 62, 17.7%). Conclusion(s): Vitrification is superior to the slow-freezing method, leading to improved oocyte survival rate, fertilization, and embryonic development in vitro. These results may be related to vitrified human oocytes incurring less damage to spindle integrity and chromosome alignment. (Fertil Steril Ò 2009;92: Ó2009 by American Society for Reproductive Medicine.) Key Words: Slow-freezing, vitrification, human, oocyte, meiotic spindle, chromosome, fertilization, development An effective oocyte cryopreservation program would be a useful treatment option for women who do not have male partners and are at risk of losing their ovarian function due to pelvic diseases, surgery, or radio/chemotherapy. An effective oocyte cryopreservation program would also benefit infertile couples who have moral or religious objections to embryo cryopreservation. In addition, a successful oocyte cryopreservation protocol could facilitate the logistics of coordinating egg donors with recipients. Since the first pregnancy and live birth reported from frozen-thawed human oocytes (1), there have been a limited Received July 11, 2008; revised August 4, 2008; accepted August 6, 2008; published online October 20, Y-X.C. has nothing to disclose. Q.X. has nothing to disclose. L.L. has nothing to disclose. L.C. has nothing to disclose. Z-G.Z. has nothing to disclose. Z-L.W. has nothing to disclose. P.Z. has nothing to disclose. Reprint requests: Yun-Xia Cao, M.D., Ph.D. Center for Reproductive Medicine, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Anhui Medical University, Hefei , People s Republic of China (FAX: ; caoyunxia6@126.com). number of live births from frozen-thawed human oocytes using the conventional slow-freezing method (2 4). The main limitation of frozen oocytes has been the poor survival rate after thawing when using the conventional slow-freezing method. Recently, improved survival rates have been reported with the use of modified slow-freezing protocols such as sodium-free freezing solutions (5 8) and increased sucrose concentrations to 0.2 M or 0.3 M (9 15). Although the oocyte survival rate was improved by the modified slow-freezing methods, no data have directly compared the survival rate and early embryonic development of human oocytes with modified slow-freezing procedures versus vitrification. Recent advances in vitrification methods have led to oocyte survival rates over 85%, resulting in high pregnancy rates and live births (16 24). It has been demonstrated that less damage was detected in the vitrified mouse oocytes compared with slow-frozen oocytes in terms of meiotic spindle integrity and chromosome alignment (25). However, no systematic studies have compared slow-freezing versus 1306 Fertility and Sterility â Vol. 92, No. 4, October /09/$36.00 Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc. doi: /j.fertnstert

2 vitrification in terms of the survival rate of human oocytes and embryonic development as well as meiotic spindle and chromosome configuration. Our study compared the survival rate, fertilization, and early embryonic development of frozen-thawed human oocytes after slow-freezing or vitrification. In addition, the meiotic spindle assembly and chromosome alignment of frozen-thawed human oocytes were evaluated and compared after slow-freezing versus vitrification. MATERIALS AND METHODS Source of Mature Oocytes Mature oocytes were obtained from patients who were undergoing intracytoplasmic sperm injection (ICSI) treatment at our infertility center. The patients were given standard ovarian stimulation using a long protocol. After down-regulation with a gonadotropin-releasing hormone (GnRH) antagonist, the patients were stimulated with human menopausal gonadotropin (HMG; Lizhu Pharmaceutical Ltd., Zhuhai, China). The collected cumulus oocytes complexes (COCs) were denuded from cumulus cells using hyaluronidase (80 IU/mL; Sigma Chemical, St. Louis, MO) and mechanical disruption with fine-bore glass pipettes for assessment of the maturity. Oocyte maturation was examined for the presence of the first polar body. When more than 15 mature oocytes were collected from a patient undergoing ICSI treatment, additional surplus oocytes were used for the present study: a total of 605 mature oocytes were obtained from 111 patients. Patients were offered the following options: oocytes could be cryopreserved either by slow-freezing or vitrification for the patient s own use in the future; or oocytes donated to another couple if the patient became pregnant in the treatment cycle; or oocytes could donated for research purposes without fertilization. The study was approved by the institutional review board of the First Affiliated Hospital of Anhui Medical University. All patients signed the informed consent forms before participating in this study, and there were no conflicts of interest related to any product used in the study. Modified Slow-Freezing and Thawing Procedures The slow-freezing procedure was based on the method from Fabbri et al. (10). Briefly, for 10 minutes the oocytes were placed in human tubal fluid (HTF) medium supplemented with 30% serum substitute supplement (SSS; Irvine Scientific, Santa Ana, CA), which contained 6% total protein and with human serum albumin and human globulins, containing 1.5 M of 1,2-propanediol (PROH). Oocytes were then transferred to a freezing medium (30% SSS) containing 1.5 M PROH and 0.3 M sucrose for 10 minutes. Two to three oocytes were loaded into 0.25-mL French straws (Cryo Bio System, Paris, France). The straws were heated and sealed at both ends and placed in a programmable freezer (Cryobath CL3300; CryLogic, Mulgrave, Australia) set at 25 C. Cooling started at a rate of 2 C/minute. At 7 C, seeding was performed by touching the side of each straw with forceps that had been cooled in liquid nitrogen (LN 2 ). The straws were held at 7 C for 10 minutes and were cooled at a rate of 0.3 C/minute to 33 C before being plunged into LN 2. The oocytes were cryopreserved at 196 C for storage. Thawing was performed by exposing the straws to air at room-temperature for 40 seconds, then immersing them into a 31 C water bath for an additional of 60 seconds. The oocytes were rinsed sequentially through six drops of medium (5 minutes in each drop) to remove the PROH and sucrose: [1] freezing medium (30% SSS) supplemented with 0.3 M sucrose and 1.0 M PROH, [2] 0.3 M sucrose and 0.5 M PROH, [3] 0.3 M sucrose and 0.2 M PROH, [4] 0.1 M sucrose and 0.2 M PROH, [5] 0.1 M sucrose and 0.1 M PROH, and [6] HTF medium (30% SSS) alone. While still in the HTF medium, the oocytes were placed in an incubator at 37 C for 2 to 3 hours of culture before ICSI. Vitrification and Thawing Procedures The oocytes were vitrified with a vitrification kit (MediCult Company, Jyllinge, Denmark). Briefly, the oocytes were suspended in equilibration medium containing 7.5% (v/v) ethylene glycol (EG) þ 7.5% (v/v) (PROH) for 5 minutes at room temperature, and then were transferred to vitrification medium containing 15% (v/v) EG þ 15% (v/v) PROH þ 0.5 M sucrose at room temperature for 45 to 60 seconds. They were loaded on a specially designed vitrification device, the McGill Cryoleaf (MediCult), and were plunged immediately into LN 2 for at least 1 month of storage. For the thawing procedure, we followed by the instructions of the thawing kit (MediCult). Briefly, the McGill Cryoleaf was directly inserted into thawing medium containing 1.00 M sucrose for 1 minute at 37 C. The thawed oocytes were transferred to diluents medium-i containing 0.50 M sucrose and then to diluents medium-ii containing 0.25 M of sucrose for 3 minutes each. Oocytes were washed twice in washing medium for 3 minutes each time. The oocyte survival rate after thawing was evaluated microscopically 2 to 3 hours after culture based on the morphology of the oocyte membrane integrity. The surviving oocytes underwent insemination. Based on the preliminary results, the oocytes from each patient were randomly allocated to slow-freezing or vitrification with a ratio of 1 versus 2 for cryopreservation. For thawing, the oocytes from each patient were thawed on the same day, regardless of slow-freezing or vitrification protocol. Insemination and Embryo Culture The surviving oocytes (n ¼ 343) were inseminated by ICSI. The sperm used for ICSI was from the patient s or recipient s husband. Couples with severe male factor infertility were excluded from the study. Inseminated oocytes were transferred to G-1 medium (G-1 TM ; Vitrolife, Sverige, Sweden) for 3 days of culture Fertility and Sterility â 1307

3 and then were transferred to G-2 medium (G-2 TM ; Vitrolife) for 5 days of incubation in 5% CO 2 at 37 C with high humidity. A fertilization check was performed 17 to 19 hours after ICSI. Embryo quality was evaluated on day 3 after ICSI based on the scoring criteria described by Tomas et al. (26). Ultimately, the embryos were scored as follows: grade 1, equal sized blastomeres with no fragmentation; grade 2, equal sized blastomeres with <20% fragmentation; grade 3, unequal sized blastomere with <20% fragmentation; grade 4, equal and unequal blastomeres with 20% to 50% fragmentation; grade 5, equal and unequal blastomeres with >50% fragmentation. High-quality embryos comprised grades 1 and 2 based on the embryonic morphology with little or without fragmentation. On day 5 after ICSI, the number of blastocyst formations in each group were also recorded. The blastocysts were scored as early blastocyst, expanded blastocyst, or hatched blastocyst, which were all transferable-blastocysts. Some blastocysts without inner-cell mass were not counted as transferable blastocysts. The resulting blastocysts were revitrified for storage without further use. Fixation and Staining for Meiotic Spindle and Chromosome Alignment of the Oocytes Fixation and immunofluorescent staining of tubulin and chromatin were adapted from previously published methods. The oocytes (18 fresh, 64 survived from slow-freezing, and 62 survived from vitrification) were fixed in 4% paraformaldehyde for 5 minutes. After fixation, the oocytes were washed in Dulbecco s phosphate-buffered saline (DPBS) and transferred to 0.01% Triton X-100 in DPBS for 40 minutes at room temperature. Oocytes were then washed extensively and blocked at 4 C in wash medium (DPBS supplemented with 0.02% NaN 3, 0.2% nonfat dry milk, 2% goat serum, 2% bovine serum albumin, and 0.1 mol/l glycine) for 45 minutes at room temperature. After rinsing in DPBS, the oocytes were incubated with b-tubulin monoclonal antibody diluted 1:150 in DPBS at 4 C overnight. After washing twice in DPBS, the microtubulins were stained with fluorescein isothiocyanate (FITC) conjugated antimouse IgG (Molecular Probes/Invitrogen, Eugene, OR) diluted 1:320 in DPBS for 60 minutes in the dark at room temperature. After washing in DPBS, the samples were mounted onto a slide under a coverslip in the Vectashield mounting medium (Vector Laboratories, Burlingame, CA) containing 0.5 mg of 4,6-diamidino-2-phenylindole (DAPI). Laser scanning confocal microscopic observations (SLM- 510; Carl Zeiss Inc., G ottingen, Germany) were performed. The images were recorded and analyzed with three-dimensional (3D) software (LSM, Germany). Morphologically, the normal meiotic spindles and chromosome alignments were barrel-shaped with microtubules traversing between both poles, and metaphase chromosomes aligned in a compact group along the metaphase plate; otherwise, the meiotic spindles and chromosome configuration were considered as abnormal. The meiotic spindle plate and chromosome configurations classified as being normal or abnormal are shown in Figure 1. Statistical Analysis Statistical analysis was performed using SPSS 13.0 (SPSS, Inc., Chicago, IL). The data were analyzed using chi-square. P<.05 was considered statistically significant. RESULTS As shown in Table 1, there was a statistically significant difference (P<.01) in oocyte survival rates of after thawing between the slow-freezing group (75 out of 123, 61.0%) and vitrification group (268 out of 292, 91.8%). Although oocyte fertilization rates did not show major differences between the two groups, the zygote cleavage rate was statistically significantly lower (P<.01) in the slow-freezing group (25 out of 46, 54.4%) than in the vitrification group (142 out of 182, 78.0%). The percentage of high-quality embryos were also statistically significantly different (P<.01) between the slow-freezing and vitrification groups (6 out of 25, 24.0% vs. 60 out of 142, 42.3%, respectively). In addition, the percentage of developed blastocysts was 12.0% (3 out of 25) in the slow-freezing group and 33.1% (47 out of 142) in the vitrification group, a statistically significant difference between the two groups (P<.05). Table 2 shows the comparison of meiotic spindle and chromosome configurations of the oocytes after the slow-freezing and vitrification methods. There were statistically significantly higher percentages (P<.05) of oocyte abnormalities in slow-freezing group (25 out of 64, 39.1%) compared with the control group (3 out of 18, 16.7%) and the vitrification group (11 out of 62, 17.7%). The rates of oocyte abnormalities between the control and vitrification group showed no statistically significant difference. DISCUSSION The results of the present study demonstrate that vitrification is superior to the modified slow-freezing method for cryopreservation of human oocytes, leading to improved oocyte survival and fertilization as well as improved early embryonic development in vitro (see Table 1). Although improved survival and pregnancy rates have been reported using modified slow-freezing methods such as the increasing the sucrose concentration from 0.2 M to 0.3 M in suspending solution (12 15, 27 30), the oocyte survival and pregnancy rates have not been sufficiently satisfactory for clinical application (4). Our results have shown that not only an improved survival rate of oocytes was obtained in the vitrification group but also a statistically significantly higher percentage of high-quality embryo and blastocyst development when compared with the slow-freezing group. It is unclear why slow-frozen oocytes have lower cleavage and embryonic development rates compared with vitrified oocytes. Nottola et al. (31) reported that 1308 Cao et al. Human oocyte cryopreservation Vol. 92, No. 4, October 2009

4 FIGURE 1 Normal and abnormal images of spindle plate and chromosome alignment in the mature human oocytes cryopreserved by slow-freezing or vitrification. Oocytes were stained immunocytochemically with an anti-atubulin monoclonal antibody and fluorescein isothiocyanate to visualize the spindle (green) and counterstained with propidium iodide to visualize chromosomes (red). (a) The oocyte shows with normal spindle plate with chromosome alignment and the second polar body. (b) The same oocyte with (a) with different magnification. (c) The oocyte shows abnormal spindle plate. (d) The oocyte shows dispersed spindle plate and chromosomes. Scale bar indicates 30 mm. Cao. Human oocyte cryopreservation. Fertil Steril the freezing and thawing procedures are associated with ultrastructural alterations in specific human oocyte domains, presumably linked to the reduced developmental potential of mature cryopreserved oocytes. Therefore, a number of analytical methods, including polarized light and confocal microscopy assessment of the meiotic spindle assembly and chromosome alignment, have been used to analyze oocytes for the possible damage caused by freezing procedures (32). It is possible that the reduced embryonic development may be related directly to the ultrastructural organelles of the oocytes. The vitrification procedure may have less damaging effects on the human oocytes in terms of the ultrastructural organelles compared with the slow-freezing procedure. Vitrification, the ice-free solidification of an aqueous solution by extremely rapid cooling, may be a promising, more effective technique than slow-freezing for oocyte cryopreservation (33). Recent reports have indicated that vitrification of human oocytes resulted in relatively high survival and pregnancy rates (16, 18, 19, 21 24). More recently, Chian et al. (20) reported a large series of obstetric and perinatal outcomes in infants conceived from vitrified oocytes in which the mean birth weight and the incidence of congenital anomalies were comparable to spontaneous conceptions in fertile women or infertile women undergoing in vitro fertilization (IVF) treatment. Thus, oocyte vitrification does not appear to be associated with an increased risk of adverse obstetric Fertility and Sterility â 1309

5 TABLE 1 Comparison of survival, fertilization, and embryonic development of human oocytes cryopreserved after slow-freezing or vitrification. Slow-freezing Vitrification P value No. of oocytes frozen-thawed No. of oocytes survived (%) 75 (61.0) 268 (91.8) <.01 No. of oocytes fertilized (%) 46 (61.3) 182 (67.9) NS No. of oocytes cleaved (%) 25 (54.4) 142 (78.0) <.01 No. of high-quality embryos (%) 6 (24.0) 60 (42.3) <.01 No. of blastocysts developed (%) 3 (12.0) 47 (33.1) <.05 Note: NS, not statistically significant. Cao. Human oocyte cryopreservation. Fertil Steril and perinatal outcomes in terms of the oocyte survival rate, pregnancies, and infants conceived. To date, no study has compared the effect of slow-freezing versus vitrification in terms of human oocyte survival rate or embryonic development in vitro. In this study, we systematically compared the survival rate and embryonic development of human oocytes cryopreserved by slow-freezing and vitrification procedures. In an animal model study, Aigner et al. (34) reported no difference in the mouse oocyte spindle morphology between slow-freezing and vitrification, although the morphologic assessment of oocyte survival, spindle integrity, and chromosome alignment after thawing may have been inadequate (35). However, it is common belief that a consequence of freezing thawing is that the spindles of oocytes cannot hold the chromosomes correctly at the metaphase plate before polar body extrusion (36 38), leading to chromosomal dispersion, failure of normal fertilization, increased incidence of aneuploidy or polyploidy, and termination of embryonic development (39 42). Our results indicated less damage detected in the vitrified oocytes compared with the slow-freezing oocytes in terms of meiotic spindle integrity and chromosome alignment (see Table 2). It is interesting that we found the rate of spindle integrity and chromosome alignment abnormalities in the vitrified oocytes to be similar to that of fresh oocytes without cryopreservation. This may provide indirect evidence that the pregnancy rates obtained from vitrified oocytes are comparable to those achieved with the fresh oocytes. A major concern with vitrification has been the use of higher concentrations of cryoprotectant (such as PROH and EG) in the suspending solution. Exposure to higher concentrations of cryoprotectant for longer periods of time may damage the oocytes through toxicity of the cryoprotectant, leading to chromosomal dispersion, failure of normal fertilization, and termination of embryonic development. However, this did not occur in our study, and the spindle organization of the frozen-thawed human oocytes was directly correlated with subsequent embryonic development. Vitrification appears to be superior to slow-freezing methods, leading to improved rates of oocyte survival, fertilization, and embryonic development in vitro. There also was less damage of spindle integrity and chromosome alignment in the vitrified human oocytes compared with the slow-frozen human oocytes. Therefore, the lower rate of embryonic development in the slow-frozen human oocytes may be related TABLE 2 Comparison of meiotic spindle and chromosome configuration of human oocytes after slow-freezing and vitrification. Control Slow-freezing Vitrification P value No. of oocytes frozen-thawed No. of oocytes (62.8) 62 (88.6) <.01 survived and examined (%) No. of oocytes with abnormalities (%) 3 (16.7) 25 (39.1) 11 (17.7) <.05 Note: Slow-freezing group had higher percentage of abnormalities compared with vitrification group. There was no statistically significant difference between control and vitrification group. Cao. Human oocyte cryopreservation. Fertil Steril Cao et al. Human oocyte cryopreservation Vol. 92, No. 4, October 2009

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