Effects of cyclic adenosine monophosphate modulators on integrity and maturation of vitrified-warmed mouse germinal vesicle oocytes

-The most effective route of administration of G-CSF is the subcutaneous one. - Further study is required. P-76 Tuesday, October 18, 2016 CRYO-INJURY OF HUMAN BLASTOCYSTS IN A CLOSED SYSTEM, RE-VITRIFICATION MODEL: COMPARISON OF UNSTABLE AND METASTABLE SOLUTIONS. M. C. Schiewe, L. A. Gamboa, J. Borba, K. Waggoner, S. Zozula. ART Lab, Ovation Fertility, Newport Beach, CA. OBJECTIVE: Commercial vitrification solutions (VS) possessing 30-32% cryoprotective agents (EG-DMSO or EG-PPG; 4.4 to 4.7M) are relatively unstable solutions, susceptible to ice-induced cryo-injury under insufficient cooling/warming conditions. Alternatively, a more concentrated EG-Glycerol VS (I.C.E.; >7.9M, Schiewe et al., 2015) is metastable under lower cooling/warming rates. We have recently shown that the latter VS mixture is equally non-toxic to blastocysts (BL) for up to a 10 min exposure. The aim of this study was to evaluate the physical stability of an unstable versus metastable VS when challenged to repeated re-vitrification (rVTF). DESIGN: Research consented, discard BL were randomly assigned to a 3x2 factorial design. Three rVTF treatments (1x,3x, 5x) were conducted in a closed VTF model, without intermittent VS elution and isotonic equilibration, using either a non-DMSO VS (I.C.E. BL-VS) or a standard 30% EG-DMSO solution (n¼50 BL/VS). The latter solutions were contrast to a control apriori treatment where standard elution and dilution equilibration of I.C.E. BL-VS treated human BLs (n¼50) was allowed between each rVTF (1-5x). Differences in %survival and %24hr development were statistically compared by Chi-square analysis (p<0.05). MATERIALS AND METHODS: Human BL (AA to BA quality) were repeatedly vitrified directly in flexipettes, sealed securely inside CBS 0.3ml embryo straws and vitrified. Standard rapid warming was performed each time in a 37 C medium bath, tips wiped dry and then re-vitrifed after 1 min. All BLs were eventually eluted in 1.0M sucrose and diluted stepwise (4 steps, 3min each), equilibrated and cultured. Embryo survival assessments were performed at 0 and 24hr post-final warming. RESULTS: A total 6 min exposure to Glycerol/EG following up to 5x-rVTF by the microSecure VTF method had no effect on post-warming survival / development (98%), being similar to the control, equilibrated rVTF group (100%). Meanwhile, the DMSO/EG treatment experienced seemingly unhindered survival (98%), but reduced (p<0.05) development at 3x or 5x rVTF (35% and 20%, respectively) compared to 1x rVTF (90%). CONCLUSIONS: VS induced cryo-sensitivity of DMSO/EG exposed revitrified human BL to cryo-injury, brings into question the true safety of unstable solutions attempting to reduce cytotoxicity. Our rVTF model exerted a repeated physical stress to an unstable VS incapable of inhibiting recrystallization events under the slower cooling and warming of a closed system. Similarly, the intra- and inter-facility variation seen with open VTF systems in clinical use may well be due to unstable VS sensitivity to suboptimal handling events. As aseptic closed VTF systems (e.g., mS-VTF, HSV, VitriSafe) continue to prove their excellence in clinical practice, perhaps it is time we produce and use more metastable solutions capable of maintaining their vitrified ‘‘glasseous’’ state without the need for ultra-rapid cooling and even higher warming rates to optimize sustained embryo viability. References: 1. Schiewe MC, S Zozula, RE Anderson, GM Fahy. Validation of microSecure vitrification (mS-VTF) for the effective cryopreservation of human embryos and oocytes. Cryobiology(2015), 71:264-272. Supported by: Conducted, in part, in our Summer Student Science Training Program using internal funding. P-77 Tuesday, October 18, 2016 EFFECTS OF CYCLIC ADENOSINE MONOPHOSPHATE MODULATORS ON INTEGRITY AND MATURATION OF VITRIFIEDWARMED MOUSE GERMINAL VESICLE OOCYTES. J. Youm,a,b H. Lee,a S. Kim,a,b J. Lee,a,b B. Jee,a,b C. Suh,a,b S. Kim,b S. Kim.c aDepartment of Obstetrics and Gynecology, Seoul National University Bundang Hospital, Seongnam-si, Korea, Republic of; bDepartment of Obstetrics and Gynecology, Seoul National University College of Medicine, Seoul, Korea, Republic of; cCenter for Fertility and Reproduction, American-Sino Women’s and Children’s Hospital, Kansas City, KS.

FERTILITY & STERILITYÒ

OBJECTIVE: To achieve a successful cryopreservation and in-vitro maturation (IVM) of Germinal vesicle (GV) stage oocytes, synchronization of nuclear and cytoplasmic maturation is critical. The aim of this study is to evaluate whether cyclic adenosine monophosphate (cAMP) modulator treatment can improve the outcomes of vitrification and IVM of GV oocytes. DESIGN: Experimental animal study. MATERIALS AND METHODS: GV oocytes were obtained from 4-week-old BDF1 mice and treated with two different cAMP modulators, dibutyl cAMP salt (dbcAMP) or 3-isobutyl-1-methylxantine (IBMX), for 30 min. These reversible GV arresting agents were used to synchronize nuclear and cytoplasmic maturation of the oocytes by postponing meiosis. All GV oocytes in the control and treated groups were vitrified with our new vitrification solution. After warming the chromatin integrity of the nucleus was assessed and meiotic resumption of vitrified-warmed GV was analyzed by MPM-2 and cyclin B1 labeling. In addition, F-actin expression for cytoskeletal integrity was assessed. After IVM the rate and quality of matured oocytes including chromosome and spindle organization were evaluated. RESULTS: The ratio of GV with a normal chromatin pattern was significantly higher with dbcAMP and IBMX treatment (84.9 and 96.0%) compared to that with the control (36.7%). MPM-2 and cyclin B1 expression within the nucleus was not significantly different, but suppressed in the cytoplasm with cAMP modulator treatment. F-actin expression was significantly higher in the dbcAMP group (29.6%) compared to the control (16.3%). The maturation rate was increased in both treatment groups, although the difference was not statistically significant (57.9, 67.8, and 65.6% in control, dbcAMP, and IBMX groups, respectively). The survival rates and chromosome-spindle organization patterns after IVM did not show any statistical difference among groups. CONCLUSIONS: Treatment with cAMP modulators may assist synchronization of meiosis and the integrity of chromatin and cytoskeleton of the GV oocytes when vitrifying GVoocytes, which can lead to better IVM outcomes with vitrified-warmed GV stage oocytes. P-78 Tuesday, October 18, 2016 REDUCED DEVELOPMENTAL EFFICIENCY OF VITRIFIED DONOR OOCYTES (DOS) IS EXPLAINED BY WIDE VARIATION IN FERTILIZATION SUCCESS. S. J. Morin,a T. A. Molinaro,b J. M. Franasiak,a C. R. Juneau,a R. T. Scott.a aReproductive Medicine Associates of New Jersey, Basking Ridge, NJ; bReproductive Medicine Associates of New Jersey, Eatontown, NJ. OBJECTIVE: While the diminished efficiency of vitrified DOs has been largely attributed to failed survival during warming, less is known about how they perform in the laboratory thereafter. Our objective was to compare the efficiency of fresh versus vitrified donor oocytes as each progresses through laboratory milestones to the blastocyst stage. DESIGN: Retrospective cohort. MATERIALS AND METHODS: All anonymous DO cycles between 1/2012 and 4/2016 were included in the analysis. All oocytes originated from donors between 21 and 31 years of age. Patients under study utilizing vitrified DOs were guaranteed at least 6 metaphase II (M2) oocytes following warming by commercial oocyte banks. As a result, efficiency evaluation began at the M2 stage of development. Each M2 was followed to assess whether it achieved the following milestones: normal fertilization (2PN) following ICSI and progression to usable blastocyst (UB) (defined as suitable for vitrification or transfer). The percentage (%) of M2s to achieve each milestone was compared between those that originated as fresh versus vitrified DOs. Chi-squared analyses were performed. RESULTS: A total of 8045 fresh and 1070 vitrified DOs were evaluated. The % of M2s that progressed to UB was significantly higher in the fresh group. The stage that contributed most to this difference was fertilization, as fresh M2s were significantly more likely to demonstrate normal fertilization. Vitrified M2s also exhibited much greater variation in fertilization success than fresh M2s. For example, <50% fertilization was attained in 15.6% of vitrified cycles versus 1.8% of fresh cycles (p<0.001). However, once an M2 achieved fertilization, there was no difference in the proportion that progressed to UB between the 2 groups. CONCLUSIONS: Even after surviving warming, vitrified donor M2s are significantly less likely than fresh M2s to develop into UBs due to a decreased fertilization rate. Vitrified M2s also demonstrated much greater per cycle variability in fertilization potential and blastocyst formation. While vitrified DOs offer many advantages, patients should take the reduced

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