technique, oocytes frozen by slow freezing protocols have no direct contact with liquid nitrogen, dramatically reducing the possibility of contamination through liquid nitrogen. Supported by: None. in basic oocyte physiology. Further biochemical studies should lead to optimized oocyte cryopreservation protocols. Supported by: None. P-203 P-202 OOCYTE CRYOPRESERVATION: AN ANALYSIS OF CYCLIN B AND MAP KINASE AS AN INDICATOR FOR OOCYTE ACTIVATION FOLLOWING CYROPRESERVATION IN CALCIUM CONTAINING AND CALCUIM FREE MEDIUM. J. R. Fredrickson, D. L. Walker, I. D. Acu, J. L. Salisbury, D. E. Morbeck, C. C. Coddington III. Mayo Clinic Coll. of Medicine, Rochester, MN. OBJECTIVE: Human oocyte cryopreservation is clinically available and although preliminary studies show promising pregnancy rates, implantation rates remain low. Cryopreservation can cause inappropriate oocyte activation by a cryoprotectant-induced increase in intracellular calcium, as evidenced by cortical granule exocytosis, zona hardening, and meiosis resumption. The objective of this study is to assess the effects of different cryopreservation mediums on bovine oocyte activation as monitored through microscopy and cell cycle proteins. DESIGN: Fresh control and chemically activated oocytes were compared to oocytes cryopreserved by controlled-rate freezing and vitrification in calcium containing and calcium free mediums. Assessments by bright field microscopy and Western Blot analysis for Cyclin B1 and mitogen-activated protein kinase (MAPK) proteins were performed in oocytes post-thaw. MATERIALS AND METHODS: Fresh bovine cumulus-oocyte complexes (n
90) were alternately allocated into six groups: 1) Control - not cryopreserved (C), 2) Control - ethanol activated (ACT), 3) Controlled-rate freezing in medium containing calcium (CRCa), 4) Controlled-rate freezing in calcium free medium (CRCa-) 5) Vitrification (ethylene glycol/ trehalose) in medium containing calcium (VCa), and 6) Vitrification in calcium free medium (VCa-). After thawing, cumulus complexes were removed, oocytes were observed by microscopy and Western Blot analysis was performed on groups of five oocytes per treatment for quantitative assessment of cyclin B1 and MAPK as determined by optical densitometry. RESULTS: Oocytes cryopreserved by both CRCa and CRCa- methods had an intact membrane and uniform cytoplasm post-thaw similar to control oocytes. Oocytes vitrified by both VCa and VCa- showed an altered morphology with a high degree of granularity, heterogeneous cytoplasm and a broken membrane post-thaw. Cyclin B1 and MAPK were higher in oocytes cryopreserved by CRCa- medium, with nearly a two-fold increase in MAPK in the CRCa- group compared to CRCa. Interestingly, although not significant (P
0.1241), there was a trend for increased levels of both proteins in the calcium free mediums. (Figure 1) Oocytes vitrified using both mediums showed no detectable levels of Cyclin B1 or MAPK. SINGLE FROZEN EMBRYO TRANSFER AS COMPARED TO MULTIPLE EMBRYO TRANSFER. A. R. Anderson, K. J. Graff, J. Distefano, J. L. Crain. REACH, Charlotte, NC. OBJECTIVE: Recently the request for single embryo transfers with frozen embryo transfer cycle has become common. The objective of this study was to determine if the single embryo transfer would impair pregnancy rates. DESIGN: Prospective cohort study for all blastocyst frozen embryo transfers (SET) where only one embryo was electively transferred in comparison to multiple embryo transfers (MET). MATERIALS AND METHODS: All patients presented for frozen embryo transfer were treated with a hormone replacement cycle with the vivelle estrogen patch. On day 12 of the cycle, and the estradiol was above 250, the patient underwent progesterone injections for 6 days. Embryos were thawed on the sixth day of progesterone using a 7 step thaw protocol. Following an air thaw at ambient temperature embryos were exposed to 6% glycerol/0.4 M Sucrose. Subsequently, rehydration continued through 4%, 3%, 2%, 1% and 0% glycerol/0.2 M Sucrose for 5 minutes in each dilution. Final rehydration into modified HEPES medium supplemented with 20% human sera albumin. Embryos were allow to re-expand for 2 to 4 hours prior to transfer. Transfers took place under ultrasound guidance with either a Wallace catheter or Sage catheter. RESULTS: A total of 240 frozen blastocyst embryo transfers were evaluated in this study. Where 192 transfers were in the MET subgroup and 48 transfers in the SET subgroup. In the MET subgroup there were 99 clinical pregnancies (51%), and implantation rate of 30% and an average of 2.2 embryos transferred per patient. In comparison to the SET subgroup there were 22 clinical pregnancies reported with a 40% implantation rate and only 1 embryo per patient. There was no significant difference in clinical pregnancy rates between the two treatment groups. There was however a 30% multiples rate in the MET subgroup, where 23 resulted in twins, 6 triplets and 1 quadruplet. The average age between the two treatment groups was 34.2 and 31.6 for MET and SET respectively. CONCLUSION: These data however small numbers are an encouraging indicator that a single embryo transfer is a viable option without effecting overall pregnancy rates. The impact on the overall multiples is exceptional and has promise on the road to all SET. It has historically been an acceptable measure of success for frozen embryo transfers to have lower pregnancy rates. However, these data suggest that does not have to be the standard. Supported by: None. P-204 FROZEN DONOR OOCYTES TO RESCUE CYCLES FOR PATIENTS WITH A KNOWN POOR-PROGNOSIS FOR OOCYTE RETRIEVAL. C. M. Briton-Jones, Q. S. Yeung, J. M. Steinberg. The Fertility Institutes, Encino, CA. CONCLUSION: This study suggests that controlled-rate freezing of oocytes in medium without calcium resulted in higher levels of Cyclin B1 and MAPK. This implies that oocyte activation was minimized and calcium free medium could be a superior medium for cryopreservation of oocytes. These data also suggest that the vitrification protocol used is not suitable for bovine oocyte cryopreservation as reflected by undetectable levels of Cyclin B1 and MAPK and microscopic morphology assessment. Since the oocyte confers much of the development potential to a newly formed embryo, it is critical that cryopreservation protocols be designed to minimize alterations FERTILITY & STERILITY威 OBJECTIVE: To examine a role for quarantined frozen-thawed donor oocytes as a ‘backup’ option for patients with a high risk of oocyte collection failure. DESIGN: Case Report. MATERIALS AND METHODS: A 37 year old, (G1,P0) woman presented to our clinic requesting surrogacy treatment as her pregnancy loss two years earlier had been due to complications caused by chronic pregnancy induced hypertension. The patient had a history of successful treatment of Non-Hodgkin’s lymphoma three years before the spontaneous pregnancy. Despite previous chemo and radio therapy the patient showed only a slightly elevated cycle day 3 FSH serum concentration of 9.8 IU. The patient wished to undergo controlled ovarian hyperstimulation (COH) and conventional in vitro fertilization treatment with transfer of the consequent embryos to a gestational surrogate. The potential use of frozen donor oocytes should the COH fail was discussed and consent given by the couple. The patient’s primary physician confirmed that the patient was cleared to undergo COH. Oocyte cryopreservation studies were approved by The Human Subjects Clinical Ethics Committee of Encino Tarzana Hospital S207