Mouse MII oocytes can tolerate a wide range of exposure time to high concentration of cryoprotectants through vitrification procedure

combind vitrification protocol. Statistical analysis using the Kruskal-Wallis, non-parametric dunns post test demonstrated that the vitrified group (n ¼ 42) was significantly different compared with the frozen group (n ¼ 28) (P<0.01). The tissue was implanted beneath the renal capsule or in an orthotopic location. We have detected follicle growth in both conventionally cryopreserved and vitrified ovarian tissues without follicle stimulating. However, because of the difficultly in determining whether a follicle is progressively maturing or decaying using only H & E staining techniques, we decided to use proliferating cell nuclear antigen (PCNA) and apoptosis (caspase-3 and CD-95) analysis to supplement our evaluation. Although there was follicle growth in frozen tissues, we found apoptotic oocytes of the explanted tissues. Follicle degeneration was found in frozen samples, which showed oocyte loss and presence of ‘‘glassy membrane’’. CONCLUSIONS: We demonstrated oocyte loss, follicle degeneration and cell apoptosis in frozen ovarian grafts. Vitrification has no negative effect on follicle formation. The work we have been performing demonstrates ice-free cryopreservation could be an alternative storage method for ovary grafts. Supported by: NIH grant, R43 HD047060. P-749 EFFECTS OF BLASTOMERE LOSS ON DEVELOPMENTAL COMPETENCY OF FROZEN-THAWED HUMAN BLASTOCYSTS. Y. Shu, J. Watt, G. Janice, J. Dasig, J. Jensen, B. Behr. IVF Program Department of OB&GYN, Stanford University Medical Center, Palo Alto, CA. OBJECTIVE: Blastomere loss is a common occurrence in cryopreserved embryos. Several studies have shown negative effects of blastomere loss on developmental potential of frozen-thawed cleavage stage embryos. However, data is still lacking for cryopreserved blastocysts. In this study, the implantation rate and pregnancy outcomes were compared between transfers of frozen-thawed blastocysts with or without blastomere loss. DESIGN: Academic assisted reproductive program. MATERIALS AND METHODS: A total of 404 frozen-thawed blastocysts from 233 non-donor transfer cycles performed from January 2004 to June 2006 were included. Blastomere loss of thawed blastocysts was carefully evaluated under an inverted microscope immediately after being transferred to blastocyst medium for 2–4 h further culture prior to transfer. In general, the thawed blastocysts were categorized as fully intact blastocysts and partially damaged blastocysts according to presence or absence of blastomere loss. Clinical pregnancy and implantation rates were compared between transfer cycles with all blastocysts fully intact (group A) and those with exclusively partially damaged blastocysts (group B). Combined transfers with fully intact and partially damaged blastocysts were excluded. RESULTS: Similar characteristics existed among groups in fresh cycles in terms of mean age at blastocyst freezing, number of oocytes, number of 2PNs, and blastocysts frozen. The outcomes of frozen transfers were shown in the table. The thickness of endometrium and mean number of embryos transferred were comparable between group A and group B. Significantly higher clinical pregnancy and implantation rates were obtained in group A. TABLE. Charactristics of frozen transfer cycles between group A and group B

No. of transfers cycles Thickness of endometrium No. of thawed blastocysts No. of survived blastocyst (%) No. of Tranferred blastocysts Mean No. of transferred blastocysts Positive HCG (%) Clinical pregnancy (%) Implantation (%)

Group A

Group B

P Value

113 9.08  1.46 198 188 (94.9) 190 1.68  0.57 59 (52.2) 43 (38.1) 54 (28.4)

120 9.06  1.43 206 102 (49.5) 200 1.67  0.55 19 (15.8) 14 (11.7) 16 (8)

NA >0.05 NA <0.01 NA >0.05 <0.01 <0.01 <0.01

CONCLUSIONS: Developmental competence was significantly reduced in blastocysts with blastomere loss after freezing and thawing when compared to fully intact ones. Further studies need to be performed to investigate the extent of blastomere loss on the clinical outcomes. Supported by: None. P-750 MOUSE MII OOCYTES CAN TOLERATE A WIDE RANGE OF EXPOSURE TIME TO HIGH CONCENTRATION OF CRYOPROTECTANTS THROUGH VITRIFICATION PROCEDURE. H. Liu, L. C. Krey, J. A. Grifo. NYU Fertility Center, New York University School of Medicine, New York, NY.

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Abstracts

OBJECTIVE: Vitrification uses high cryoprotectant concentrations that raise toxic effect concerns. In general, it is believed that the shorter exposure to high concentration of vitrification solution, the better viability of oocytes following vitrification-thaw. However, a short exposure time to final vitrification solution poses technical challenges. This study examines the impact of extending the exposure time to final vitrification solution on the survival and embryonic potential of vitrified mouse oocytes. DESIGN: An animal experiment. MATERIALS AND METHODS: Metaphase II (MII) oocytes were collected at 14–15 h after stimulation with PMSG þ hCG from 6–8 week old CB6F1 mice, denuded with hyaluronidase and exposed to a DMSO-EG-sucrose based vitrification procedure at room temperature with different times (60, 120, 180 and 240 s) in the final vitrification solution (2.7 M ethylene glycol; 2.1 M DMSO; 0.5 M sucrose). Oocytes were loaded in pulled straws and plunged into liquid nitrogen. After thaw and recovery culture for 1 h, the surviving oocytes were inseminated by capacitated sperm. Embryos were cultured at 37 C and 5% CO2 in Quinn’s Advantage cleavage/blastocyst media. RESULTS: When exposed to the final vitrification solution for 60 s, viability and pre-implantation embryonic development of the vitrified MII oocytes were not different from that of fresh oocytes. Extending the exposure time to final vitrification solution to 120 s, 180 s and even 240 s, did not compromise the viabilities of the vitrified oocytes. CONCLUSIONS: Our findings indicate that exposures to a highly concentrated vitrification solution for up to 240 s are without significant effect on oocyte viability post-thaw and subsequent pre-implantation embryonic potential. These findings disagree with the previous views that extended exposure times to concentrated vitrification solutions increase their toxic effect(s) on oocytes and compromise their viability post-thaw. Whether human oocytes can tolerate a wide range of exposure times to final concentration of vitrification solution similar to mouse oocytes remains to be determined.

Groups

MII

Survival

2 Cell

Blastocyst

R3 BB

Fresh VS60 VS120 VS180 VS240

96 35 40 30 30

100 (100) 34 (97) 39 (98) 30 (100) 30 (100)

77 (80) 29 (83 32 (80) 25 (83) 27 (90)

63 (66) 21 (60) 30 (75) 20 (67) 24 (80)

60 (63) 19 (54) 25 (63) 19 (63) 19 (63)

X2 test: P>0.05; Value in parenthesis indicates percentage. Supported by: None. P-751 EFFECT OF PROCEDURE SLOW-FREEZING AND VITRIFICATION PROTOCOL ON ULTRA MICROSTRUCTURE, ER AND MHC-II ANTIGEN EXPRESSIONS OF RABBIT OVARIAN TISSUES AND THE PROLIFERATIVE ACTIVITY OF THE FOLLICLES. L. Sun, G. Zhu. Department of Obstetrics and Gynecology, Tong Ji, Huazhong University of Science and Technology, Wuhan, Hubei, China; Department of Obstetrics and Gynecology, the Second Hospital Affiliated Zhengzhou University, Zhengzhou University, Zhengzhou, Henan, China. OBJECTIVE: To investigate the effect of PROH procedure slow-freezing protocol and DMSO þ EG vitrification protocol on ultra microstructure, ER and MHC-II antigen expressions of rabbit ovarian tissues and the proliferative activity of the follicles. DESIGN: Experiment research. MATERIALS AND METHODS: Nine healthy female rabbits were randomly divided into fresh control group and two freezing groups which were freezed by PROH procedure slow-freezing and DMSO þ EG vitrification method. After thawing, the effects of two freezing methods on ultra microstructure of ovarian tissues were observed by transmission electron microscope, and the effects on ER and MHC-II antigen expressions of ovarian tissues were observed by immunohistochemistry. Follicles were mechanically dissected from ovarian tissue. Twenty small OGCs (diameter 100–150 mm, wrapped up by 25 layers of granulosa cells) and twenty big OGCs (diameter 200–300 mm, wrapped up by many layers of granulosa cells) with normal appearance from every groups were tested with 3H-TdR uptake method. RESULTS: In PROH group, partial chondriosomes of the oocyte from primordial follicle with vacuole swelled, and some were normal. The nucleus appearance was normal, its membrane was integrity and clear. The nuclear membranes of granular cells were integrity;chondriosomes were normal and gap junctions were intact. In DMSO þ EG groups, the most oocyte

Vol. 88, Suppl 1, September 2007