Impact of pH buffers on expression of key imprinting genes within the mouse preimplantation embryo

reduce E2 levels. However, AIs alter traditional stimulation markers such as E2 and follicular size and may complicate clinical decision making. This study seeks to determine whether continuous AI use throughout stimulation impacts oocyte maturity and function. DESIGN: Retrospective. MATERIALS AND METHODS: FP patients with breast cancer treated with an AI (letrozole) were compared to FP patients with other malignancies not using AIs. The AI group had oocyte maturation induced when leading follicles reached 20mm as suggested by Oktay et al, compared with the standard of 18mm. Endocrine levels, oocyte maturity (MII/retrieved oocytes), MII yield (MII/follicles R14mm) and fertilization rates were compared. RESULTS: 24 AI patients were compared with 22 controls. The age, AMH, and antral follicle counts were equivalent. In contrast to prior reports, total gonadotropin usage was similar. The AI group had lower peak E2 levels (536 vs 1972 pg/mL; P<0.001), but 58% still reached supraphysiologic levels (>400 pg/mL). The AI group had higher peak P levels (1.1 vs 0.7 ng/mL; P<0.01) and a trend toward a higher risk of premature P rise R1.5 ng/mL (29% vs 5%; P¼0.06). Despite larger follicles, the AI group had a lower maturity rate (68% vs 80%; P<0.001). The MII yield was lower in the AI group (P¼0.04). Among those who underwent ICSI (15 AI, 14 noAI), the fertilization rate was lower in the AI group (76% vs 84%; P¼0.03). CONCLUSION: Despite efforts to prevent supraphysiologic E2 in breast cancer FP cycles, most AI stimulations result in elevated E2 levels. Although the impact of elevated P on oocyte quality in FP cycles is not established, it may reflect an altered intrafollicular endocrine milieu and post-maturity. Caution should be exercised when changing decision making in FP patients receiving AIs. O-385 Wednesday, October 16, 2013 05:30 PM GnRH AGONIST (GnRHa) PRIMING INCREASES THE NUMBER OF IN VITRO MATURED (IVM) OOCYTES AVAILABLE FOR CRYOPRESERVATION IN CANCER PATIENTS SEEKING URGENT FERTILITY PRESERVATION (FP). H. El Hachem,a M. Poulain,b S. Le Parco,a R. Fanchin,a N. Frydman,b M. Grynberg.a aReproductive Medicine, H^ opital Antoine Beclere, Clamart, France; bReproductive Biology, H^ opital Antoine Beclere, Clamart, France. OBJECTIVE: To compare the number of immature and IVM oocytes obtained after GnRHa or hCG priming in cancer patients candidates for urgent FP. DESIGN: Prospective study. MATERIALS AND METHODS: 72 cancer patients candidates for urgent FP using IVM of oocytes were studied. They all had 2 ovaries and no history of chemotherapy. Serum AMH levels and antral follicle count (AFC) were assessed prior to FP, irrespective of cycle day. Patients were sorted into 2 groups according to the priming used 36 hours before egg collection: Group GnRHa, triptorelin 0.2 mg, SC, n¼36 and Group hCG, human chorionic gonadotropin, 10,000 IU, IM, n¼36. Patients were matched according to age, BMI, serum AMH, AFC and the phase of the cycle at which germinal-vesicle stage oocytes were retrieved. Number of immature oocytes recovered, maturation rates, and total number of oocytes cryopreserved were analyzed. RESULTS: By design, in both groups, women were comparable in terms of age (30.60.7 vs 30.60.7 years, respectively), BMI (22.10.5 vs 22.40.6 Kg/m2) and markers of the follicular ovarian status (serum AMH levels (4.00.8 vs 3.80.5 ng/mL, respectively) and AFC (19.41.7 vs 19.41.8 follicles, respectively)). Overall, the number of immature oocytes recovered was significantly higher in the GnRHa-primed group when compared to hCG group (12.51.1 vs 8.00 oocytes, P<0.004, respectively). Although the maturation rates were similar in both groups (60.0%3.7 vs 63.0%4.3, NS), the total number of mature oocytes available for cryopreservation was significantly higher in women primed with GnRHa (7.30.8 vs 5.00.6, P<0.03, respectively). CONCLUSION: The present investigation shows that GnRHa priming increases the number of oocytes retrieved at germinal-vesicle stage and the total number of IVM oocytes available for cryopreservation. These results suggest that GnRHa priming should be prefered to the conventionnal hCG admininistration in IVM cycles performed for urgent FP. O-386 Wednesday, October 16, 2013 05:45 PM CAN STATINS IMPROVE OVARIAN GRAFT RECEPTION? Y. Cohen, H. Dafni, R. Avni, L. Fellus, M. Neeman. Biological Regulation, Weizmann Institute of Science, Rehovot, Israel. OBJECTIVE: Rapid revascularization is essential for reducing hypoxic damage and protecting the primordial follicles reserve following

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ovarian graft transplantation. We have previously shown that endothelial activation of Akt1, a key mediator of angiogenesis, promotes angiogenesis in ovarian grafts. Recent studies suggest that simvastatin activates Akt1 and induces angiogenesis. The aim of this study was to evaluate whether simvastatin administration improves ovarian graft vascularization. DESIGN: A mouse model of heterotopic ovarian graft transplantation. Ovarian graft revascularization was explored by in vivo dynamic contrast enhanced magnetic resonance imaging (DCE-MRI). MATERIALS AND METHODS: Ovaries from 6 weeks old mice were transplanted into the leg muscle of immunocompromised recipient mice. Activated simvastatin (0.1 mg/kg/day) (group A) or saline (group B) were injected intraperitoneally to recipient mice (n¼7,7), starting from one day prior to ovarian grafting and throughout the following 7 days. Vascular properties of the grafts were estimated from the MRI derived parameters vascular density (blood volume fraction, fBV) and permeability (PS). The changes in fBV and PS were examined in each group, on days 2 and 7 after transplantation, and following hormonal stimulation with PMSG/hCG. RESULTS: Mean fBV was increased by 76% in group A on day 7 compared to day 2 (0.0370.009 vs. 0.0210.005, P<0.05). Furthermore, group A showed a tendency towards higher fBV values following PMSG/ hCG (mean fBV: 0.058 0.018 vs. 0.0410.016, post and pre treatment, respectively, P¼0.052). The differences in fBV values over time in group B were not significant. PS was lower on day 7 compared to day 2 in both groups, possibly due to blood vessel maturation at the transplantation site. CONCLUSION: Simvastatin therapy, given for 7 days following ovarian transplantation, during the period of hypoxic ischemia, improves revascularization and vascular support of ovarian grafts. Supported by: ERC advanced grant IMAGO. EMBRYO BIOLOGY II O-387 Wednesday, October 16, 2013 04:00 PM IMPACT OF PH BUFFERS ON EXPRESSION OF KEY IMPRINTING GENES WITHIN THE MOUSE PREIMPLANTATION EMBRYO. N. A. Clark,a J. Ding,a,b G. D. Smith,a,b,c J. E. Swain.a a Obstetrics and Gynecology, University of Michigan, Ann Arbor, MI; bMolecular & Integrative Physiology, University of Michigan, Ann Arbor, MI; c Urology, University of Michigan, Ann Arbor, MI. OBJECTIVE: To determine the impact of commonly used pH buffers on expression of key imprinting genes within the preimplantation mouse embryo. DESIGN: Animal experimental study. MATERIALS AND METHODS: KSOMAA media were formulated with various buffers including 1) 21mM HEPES, 2) 21mM MOPS, 3) 10.5mM of HEPES and MOPS (HM) or 4) phosphate buffered medium (PBS). Controls consisted of bicarbonate-only buffered media. All media contained 25mM bicarbonate and maintained a pH of 7.25-7.35 at 37 C inside the same incubator with 6% CO2. Osmolality was maintained at 280-290mOsm. 1-cell mouse embryos from B6C3F1 mice were flushed and cultured for 18h in respective media until the 2-cell stage and frozen for analysis. 2-cell mouse embryos were flushed to serve as in vivo controls. Primers were developed for igf2, igf-2R, meg-3 and peg-1. RNA was extracted from embryos and rt-qPCR utilized to determine expression levels, using actin as an internal control. Expression levels for in vivo derived embryos were normalized to 1 and treatments expressed as a fold increase or decrease. Means were compared using ANOVA and Tukey. RESULTS: No statistical differences were apparent in expression of igf2. Expression of igf-2R of all treatments was similar (HEPES: 0.310.02, MOPS: 0.350.03, HM: 0.310.03, PBS: 0.370.07, Bicarb:0.360.07), and differed significantly (p<0.05) from in vivo controls (1.0). No significant differences were apparent between any treatments for expression of meg-3 or peg-1. CONCLUSION: This is the first report examining impact of pH buffers on expression of key imprinting genes in the preimplantation embryo. HEPES, MOPS, HM and PBS yield similar expression of imprinted genes compared to bicarbonate buffered media. All buffers yielded similar gene expression compared to in vivo controls, except for ifg-2R, which may be a sensitive imprinted gene. Future studies examining extended embryo culture may yield more information as to the impact of pH buffers on gene expression and imprinting.

Vol. 100, No. 3, Supplement, September 2013