Temperatures in the devitrification process is essential for preserved morphological membrane integrity and sperm function in human spermatozoon

OBJECTIVE: To compare blastocyst (BL) slow freeze to vitrification protocols over a 27 month period. Survival, post-thaw expansion and implantation rates of the two freezing protocols were compared. DESIGN: Retrospective Analysis. MATERIALS AND METHODS: Part 1: Patients undergoing FET with either 1 or 2 embryos were included in the study: n¼170 with 225 BL in the slow-freezing group and n¼137 with 325 BL in the vitrification group. Post-thaw blastocyst survival was assessed immediately after thawing and prior to embryo transfer; implantation rate was defined as number of gestational sacs per number BL transferred. Part 2: Group 1) BL expanded at time of transfer (classified as 100% survived) and Group 2) BL contracted at time of transfer. BL expansion was evaluated retrospectively using embryo transfer pictures. RESULTS: Part 1: BL survival rate was significantly higher in vitrified vs. slow-freeze group (98% vs. 84%, p<0.05). Similarly, implantation rates of vitrified BL were significantly higher than in the slow-freeze group (49% vs. 29%, p<0.05). Part 2: When comparing only expanded BL (100% survived) used for ET, implantation rate in vitrified BL remained significantly higher (51%) than in the slow-freeze group (39.9%, p<0.05). Similarly, implantation rate of contracted vitrified BL (44.1%) was higher than their slowfreeze contracted counterparts (18.9%, p<0.05). In the slow-freezing group, expanded BL exhibited higher implantation rate than contracted BL (39% vs. 18.9%, p<0.05). In contrast, no difference in implantation rates was found between expanded and contracted vitrified BL (51% vs. 44.1%). CONCLUSION: Due to significantly higher post-thaw survival and implantation rates, vitrification has become the freezing method of choice in our program. While post-thaw expansion is a useful predictor of BL viability after slow freeze, it does not appear to be a good predictor of implantation of vitrified BL, indicating better overall embryo viability after vitrification. Supported by: Institutional. P-125 Tuesday, October 15, 2013 ARTIFICIALLY COLLAPSED (AC) BLASTOCYSTS HAVE IMPROVED POST-THAW SURVIVAL WHETHER THE EMBRYO IS EUPLOID OR ANEUPLOID. J. Keltz, B. Hodes-Wertz, K. Goldman, C. McCaffrey, E. Ampeloquio, J. A. Grifo. Obstetrics and Gynecology, NYU Langone Medical Center, New York, NY. OBJECTIVE: To compare post-thaw survival of biopsied blastocysts that underwent AC prior to vitrification to unscreened blastocysts. DESIGN: Retrospective cohort study at an academic institution. MATERIALS AND METHODS: From 2010 to 2012, 646 unscreened day 5 or 6 blastocysts, 259 euploid blastocysts, and 47 aneuploid blastocysts based on array-comparative genomic hybridization (aCGH) were thawed. An additional 24 blastocysts that underwent single gene defect testing (SGD) alone were thawed. In contrast to the unscreened embryos, all biopsied blastocysts (Euploid, Aneuploid, SGD) were AC as a result of the trophectoderm biopsy procedure prior to vitrification (Collapsed). All blastocysts were vitrified and warmed at our institution using established standard procedures. The outcome measure of post-thaw survival defined as R50% of the embryo having survived was judged by the embryologist at time of warming. Survival rates of the three biopsied groups (Euploid, Aneuploid, and Collapsed) were individually compared to the unscreened blastocysts using a c2 analysis. RESULTS: Post thaw survival was better with collapsed blastocysts despite an older age in this group and was independent of ploidy status. Euploidy did not improve thaw survival rates (p value¼0.18).

Unscreened (n¼646) Euploid (n¼251) Aneuploid (n¼47) Collapsed (n¼330)

Age at Cryopreservation

Survival Rate

p value compared to Unscreened

34.7 36.8 39.0 37.1

87% (n¼565) 98% (n¼245) 100% (n¼47) 98% (n¼324)

<0.0001 0.001 <0.0001

FERTILITY & STERILITYÒ

CONCLUSION: Despite an older patient population, AC blastocysts have an improved post-thaw survival compared to non-collapsed blastocysts, irrespective of ploidy status. Euploid blastocysts did not have a post-thaw survival advantage compared to the aneuploid blastocysts which may be due to the small aneuploid sample size. It does corroborate prior publications showing that AC embryos are better able to tolerate the vitrification and warming process than non-AC embryos. Collapsing the blastocysts prior to vitrification creates an overall survival advantage. P-126 Tuesday, October 15, 2013 TEMPERATURES IN THE DEVITRIFICATION PROCESS IS ESSENTIAL FOR PRESERVED MORPHOLOGICAL MEMBRANE INTEGRITY AND SPERM FUNCTION IN HUMAN SPERMATOZOON. R. Sanchez,a J. Fontecilla,b E. Isachenko,c B. Mora,b V. Isachenko,c M. E. Cabrillana.d aDepartment of Preclinical Science, Faculty of Medicine, BIOREN-CEBIOR, Universidad de La Frontera, Temuco, Araucania, Chile; bBIOREN-CEBIOR, Universidad de La Frontera, Temuco, Araucania, Chile; cDepartment of Obstetric and Gynecology, University of Cologne, Cologne, Germany; dInstitute of Histology and Embryology, Faculty of Medical Sciences, National University of Cuyo, Mendoza, Argentina. OBJECTIVE: To determine the optimal warming temperature for vitrified human spermatozoa in order to maintain their fertilization potential. DESIGN: The effect of temperature on vitrification technique was analyzed. MATERIALS AND METHODS: Spermatozoa were cryopreserved by direct plunging into N2L and warmed at different temperatures (38 , 40 and 42 C). The following parameters as: progressive motility (CASA), plasma membrane integrity (SYBR-14/PI; flow cytometry), oxidation of thiol groups (monobromobimane, confocal laser microscopy), lipid peroxidation (TBARS, spectrophotometer) and sperm morphology (electron microscopy) were evaluated. For statistical analysis, the nonparametric ANOVA (Kruskal-Wallis), and the multiple comparison test of Dunn to establish differences between groups were used (p<005). RESULTS: Progressive motility of warmed at 38 , 40 and 42 C spermatozoa was: 26.4%; 56.6% and 65.4% respectively, p< 00.5 compared to 38 / 40 C and 38 /42 C. Temperatures at 40 C (49.8%) and 42 C (45.2%) protected the plasma membrane compare to warming at 38 C (19%) (P<0.05). As well as, the high temperature protect the cytoplasmic membranes against of lipid peroxidation, the lipid absorbance was significantly higher at 40 C (0.018 A) and 42 C (0.022 A) compare to warming at 38 C (0.082 A). It was not established effect of all warming temperature on the protection of cell membrane against of oxidation of thiol groups 38 C (50.6%); 40 C (48.7%); 42 C (50.3%). Sperm morphology showed after warming at 42 C more stable and regular compare to warming at 38 C (P<0.05). CONCLUSION: The warming temperature can generate the morphological and biochemical changes which can affect the sperm function. To check the latent cry-damage of sperm membrane it is necessary with different timeintervals after warming The warming of vitrified spermatozoa at 42 C temperature, is the most optimal for preservation of spermatozoa physiological parameters. Supported by: Grant DI12-2014 by Direccion de Investigacion, Universidad de La Frontera, Temuco, Chile. P-127 Tuesday, October 15, 2013 EFFECTS OF EQUILIBRATION TIME ON CLINICAL OUTCOMES IN EMBRYO VITRIFICATION PROCEDURE. S. Xiong, J. X. Liu, W. Han, G. Huang. Chongqing Maternity and Children Health Care Hospital, Chongqing, China. OBJECTIVE: Vitrification is an efficient way to store human embryos. During this procedure, the duration that embryo suspends in vitrification solution is commonly considered to be critical, which should be strictly controlled within 1 min. However, the duration that embryo suspends in equilibration solution is flexible, ranged from 5 min to 15 min. The purpose of this study is to examine the effects of different equilibration time on the outcomes of frozen-thawed embryo transfer cycles. DESIGN: Retrospective. MATERIALS AND METHODS: Data of all patients undergoing FET in our center from Jan 2012 to Jan 2013 was retrospectively analyzed. The following cycles were excluded: blastocyst vitrification, PGD, only one embryo transferred, slow freezing, and female age older than 38 years. A total of

S183