Transfer of day-3 embryos in the fresh cycle and surplus blastocyst in the thawing cycle: its summary pregnancy chance

MATERIALS AND METHODS: Cycles of IVF conducted between January 2010 and January 2013 at Maria Fertility Hospital in which late cleaved and rescue ICSI embryos were reviewed. The patients were divided in five groups: group A (n¼17), Frozen-thawed late cleaved embryo transfer cycles, group B (n¼5), Frozen-thawed rescue ICSI embryo transfer cycles in failed conventional insemination (CI), group C (n¼9), Frozen-thawed rescue ICSI embryo transfer cycles in additional IVM, group D (n¼ 16), Fresh rescue ICSI embryo transfer cycles in failed CI, group E (n¼66), Fresh rescue ICSI embryo transfer cycles in additional IVM. Cryopreserved embryos were transferred to patients either in natural cycle or in a hormonal cycle. Late cleaved and rescue ICSI embryos were frozen on day 4 using vitrification. Late cleaved and rescue ICSI embryos were thawed on 1 day before ET. Fresh embryo transfer was performed on day 3. RESULTS: There were no differences in patient age, mean number of transferred embryos and endometrial thickness. Otherwise, pregnancy rates were improved in froze-thawed late cleaved embryo transfer (36.8%). In rescue ICSI cycle, the pregnancy rates were significantly higher in group B and C (60.0% and 22.2%) than in group D and E (6% and 3%). CONCLUSION: Low pregnancy potentials of the late cleaved and rescue ICSI embryos can be overcome by frozen-thawed procedure. It seems that the frozen-thawed procedure of the late cleaved and rescue ICSI embryos enabled more appropriate synchronization between embryonic and endometrial development. Therefore the late cleaved and rescue ICSI embryo cryopreservation may be an effective alternative to fresh embryo transfer. Supported by: Self Supported. P-118 Tuesday, October 15, 2013 KRIOBLASTÔ, A SYSTEM FOR KINETIC VITRIFICATION BY HYPERFAST COOLING: APPLICATIONS FOR REPRODUCTIVE & STEM CELLS. I. I. Katkov,a,b V. F. Bolyukh,a,c Y. Liu,b D. Wu,b E. Y. Snyder,b S. Agarwal.d aCELLTRONIX, San Diego, CA; bStem Cell Core, Sanford-Burnham Medical Research Intsitute, La Jolla, CA; cElectrical Engineering, National Technical University ‘‘KhPI’’, Kharkov, Kharkov Region, Ukraine; dReproductive Medicine, UCSD School of Medicine, University of California at San Diego (UCSD), La Jolla, CA. OBJECTIVE: Kinetic (very rapid) vitrification (K-VF) is a promising approach for cryopreservation (CP) of biological materials as it is simple, robust, and can achieve VF for practically any type of cells (Katkov et al, 2012). DESIGN: Several methods of superfast K-VF, particularly for CP of oocytes, embryos, sperm and human embryonic stem cells have been proposed but practically all of them either require very small (in range 0,5-10 mcl) size of the sample or/and cannot avoid the Leidenfrost effect (LFE), which substantially impedes the rate of cooling. MATERIALS AND METHODS: Here, we are reporting an entirely new system for hyper-fast cooling of one-two order of magnitude larger samples developed in CELLTRONIX, which we called ‘‘KrioBlast(TM)’’ that completely eliminates LFE and a need of potentially toxic and osmotically damaging vitrificants such as DMSO or PG used in the current methods of VF. RESULTS: The system allowed us to vitrify up to 4,000 mcl of 15% glycerol solutions (used as a cooling rate marker), which theoretically corresponds to the critical cooling rate up to 600,000 C/min (Warkentin et al, 2008) or even higher. Particular applications for CP of reproductive and stem cells for reproductive and regeneration medicine, husbandry, and cryopreservation of wildlife genetic resources are discussed. Some promising preliminary data of hyper-fast of human sperm and embryonic stem cells are also presented. CONCLUSION: The hyperfast method we proposed is scalable, automated, and robust to the human’s error. This approach can ‘‘unify’’ the equipment, the acesssoirs, and the vitrifcation media in one platform. This KrioBlast(TM) platform can be considered as a step toward the ‘‘Universal Cryopreservation Protocol’’ that will largely benefit Assisted Reproductive Technologies and Regenrative Medicine. Supported by: This work is Supported by NIH 1R43OD012396-01 grant to CELLTRONIX. P-119 Tuesday, October 15, 2013 VITRIFICATION OF OOCYTES IS USEFUL FOR FERTILITY PRESERVATION IN COUPLE OF NON-OBSTRUCTIVE AZOO-

FERTILITY & STERILITYÒ

SPERMIA PATIENTS. E. K. Kim, E. A. Kim, J. H. Kim, H. Kwon, J. E. Shin, D. H. Choi. Fertility Center, CHA Bundang Medical Center, CHA University, Seongnam-si, Gyeonggi-do, Republic of Korea. OBJECTIVE: Oocyte vitrification method has been improved over the past decade. The couples with severe male infertility are insufficient for fertilization of retrieved oocytes, and especially the couples suffered from nonobstructive azoospermia could not possible to fertilize with retrieved oocytes in IVF. In that case, vitrification of oocytes could be an effective method for future reference.The aim of this study is to find the outcomes of vitrified oocytes using donor sperm. DESIGN: Retrospective study. MATERIALS AND METHODS: Between Jan. 2009 and Dec. 2012, frozen oocytes from 6 patients of total 16 patients were thawed. And R 41yrs. and R4 cycles were excluded. Vitrification-thawed methods are followed our previous study, vol. 90 supl. Sep. 2008 S282: F&S. RESULTS: Clinical results are as follows.

Clinical Results.

No.of patients Age(M SD)(yrs.) Infertility duration(M SD) No. of retrieved oocytes(M SD) No. of vitrified oocytes(M SD) No. of survived oocytes(%) No. of 2PN(%) No. of embryos(%) No. of transferred embryos(M SD) Pregnancy rate(%)

6 29.22.8 3.12.0 12.94.8 89(12.75.0) 85/89(95.5) 65/80(81.3) 60/65(92.3) 24(3.41.0) 2/6(33.3)

CONCLUSION: Vitrification has been applied to human oocyte cryopreservation successfully so far. Furthermore this can induce more effective results in severe male factor like non-obstructive azoospermia in IVF. P-120 Tuesday, October 15, 2013 TRANSFER OF DAY-3 EMBRYOS IN THE FRESH CYCLE AND SURPLUS BLASTOCYST IN THE THAWING CYCLE: ITS SUMMARY PREGNANCY CHANCE. F.-T. Kung, Y.-J. Lin, F.-J. Huang, K.-C. Lan, P.-Y. Lin, H.-J. Chiang. OBGYN, Kaohsiung Chang Gung Memorial Hospital, Niao Sung District, Kaohsiung, Taiwan. OBJECTIVE: To examine the pregnancy potential of thawing blastocysts which were originally frozen on day 5 after extended culture of day-3 surplus embryos in fresh cycles and to calculate the subsequent summary pregnancy rate. DESIGN: Prospective observational study. MATERIALS AND METHODS: All of surplus embryos on the day 3 of embryo transfer were kept cultured till day 5. Only the embryos able to grow up to blastocyst stage were frozen by using vitrification method. Those whose original fresh transfers were canceled with any reasons and who did not return for transfer yet were excluded from the study. Endometrium was prepared by clomiphene citrate stimulation or hormone replacement, or naturally. The summary pregnancy rate was defined as the number of pregnancies after transfer of sibling embryos both in fresh and thawing cycles divided by the number of patients. RESULTS: A total of 107 fresh cycles had available blastocysts for freezing. Among them, 46 (43%) cycles in 46 patients had at least one thawing embryo transfer. Originally, the clinical pregnancy and implantation rates were 28.3% and 13.7%, respectively, in those 46 fresh cycles. Subsequently, 102 blastocysts were thawn and 99% of them survived. The mean of storage tome was 9.5  10 months. Thirteen (28.3%) patients succeeded in pregnancies with an implantation rate 14.8%. There were no differences between success and failure in pregnancy groups in age, body mass index, embryo storage time, endometrial thickness and prefreezing blastocyst quality. The summary pregnancy rate up to now was 56.5%.

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CONCLUSION: Combination of day-3 embryo transfer in the fresh cycle and surplus blastocyst transfer in the thawing cycle gave a favorable summary pregnancy chance. P-121 Tuesday, October 15, 2013 EFFECT OF GnRH-ANTAGONIST VERSUS AGONIST ON EMBRYO QUALITY BY COMPARING THE OUTCOMES OF FETs. J. H. Lee, K. H. Lee, S. G. Kim, I. H. Park, H. G. Sun, Y. Y. Kim. Mamapapa&baby OBGY, Ulsan, Republic of Korea. OBJECTIVE: It is still unclear whether detrimental effects of GnRH antagonist are oocyte quality or endometrial maturation. Frozen-thawed embryo transfer cycle provides a model to eliminate any detrimental effect of GnRH antagonist in the oocyte retrieval cycle. To assess whether the GnRH antagonist had a detrimental effect on embryo quality, we compared the frozen-thawed embryo transfer (FET) outcomes of GnRH agonist or GnRH antagonist protocols for COS in the previous oocyte retrieval cycle. DESIGN: Retrospective study. MATERIALS AND METHODS: We analyzed 559 cycles from patients underwent FETs from January 2010 to November 2012. The patients were divided into two groups based on whether they had been treated using a GnRH agonist long protocol (group A, n¼331) or a GnRH antagonist protocol (group B, n¼228) during the previous oocyte retrieval cycle. FETs were performed to patients either in natural cycle or in a hormonally manipulated artificial cycle following preparation of the endometrium with estradiol and progesterone. Embryos were frozen on day 3 using a vitrification protocol and FETs were performed by same physician. We compared survival rates, embryo quality, pregnancy and ongoing rates. RESULTS: There were no differences between both groups in patient age (354.4 in group A vs. 363.9 in group B), endometrial thickness (9.22.1 vs. 9.51.8), fully intact rates (69.2% vs. 66.9%), and survival rates (83.6% vs. 81.3%). And numbers of good quality embryos transferred were similar for the GnRH agonist and GnRH antagonist groups(57.5% vs. 60.5%). Pregnancy and ongoing rates were 33.8% vs. 37.3% and 32.0% vs. 33.8%, respectively, which was not statistically significantly different. CONCLUSION: The use of GnRH agonists or GnRH antagonists in oocyte retrieval cycles did not result in different outcomes for frozenthawed embryo transfer in terms of pregnancy or ongoing rates. Our results suggest that GnRH antagonists do not have a detrimental effect on embryo quality.

P-122 Tuesday, October 15, 2013 OOCYTE VITRIFICATION IS A VIABLE REPRODUCTIVE OPTION FOR WOMEN SEEKING FERTILITY PRESERVATION. T. Schlenker, J. Stevens, R. Smith, M. Rawlins, M. G. Katz-Jaffe, W. B. Schoolcraft. Colorado Center for Reproductive Medicine, Lone Tree, CO. OBJECTIVE: Vitrification has proven to be a highly efficient method for donor oocyte cryopreservation. Fertility preservation is another indication for oocyte cryopreservation allowing female cancer patients and women seeking oocyte banking alternative reproductive choices. This study investigated the clinical efficiency of oocyte vitrification and warming techniques for fertility preservation patients. DESIGN: IRB approved research study. MATERIALS AND METHODS: Female cancer patients and women seeking oocyte banking underwent controlled ovarian stimulation and oocyte vitrification using the Cryotop method (n¼29; age ¼ 34.8 years). At a later date, patients were scheduled for a frozen embryo transfer with oocytes warmed, fertilized and embryos cultured to either day 3 of embryonic development or the blastocyst stage for transfer. Donor oocyte cryopreservation patients were utilized as a control group (n¼97; oocyte age ¼ 28.9 years; recipient age ¼ 39.1 years). Statistical analysis included Fisher’s exact test and unpaired t-test. RESULTS: Following warming, a total of 352 oocytes survived the vitrification process, significantly lower than the control group (83.3% vs. 92.0%; P<0.01). However, 266 warmed oocytes completed normal fertilization which was comparable between groups (75.6% vs. 79.8% control; ns). Nearly half (48.3%) of the fertility preservation patients received a D5 transfer with total blastocyst development only slightly lower than controls (42.1% vs. 48.9%; P<0.05). A trend towards lower clinical pregnancy rate

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ASRM Abstracts

(65.5% vs. 81.4% control; ns) and live birth rates (62.1% vs. 73.2% control; ns) for fertility preservation patients did not achieve statistical significance. CONCLUSION: Fertility preservation using oocyte vitrification techniques for cancer patients and women seeking oocyte banking, results in excellent clinical outcome. As would be expected, excellent outcome was observed for donor oocyte cryopreservation cycles highlighting the exceptional efficiency of oocyte vitrification and warming methods. P-123 Tuesday, October 15, 2013 NO EMBRYOS LEFT BEHIND: EMERGENT VITRIFICATION OF 90 EMBRYOS DURING HURRICANE SANDY (HS). K. N. Goldman, C. McCaffrey, M. Ghosh, A. Adler, A. Chin, J. A. Grifo. Obstetrics and Gynecology, Reproductive Endocrinology and Infertility, New York University Langone Medical Center, New York, NY. OBJECTIVE: To describe our fertility center (FC)’s experience with disaster management during HS, and to report outcomes of emergently vitrified embryos. DESIGN: Descriptive study. MATERIALS AND METHODS: HS struck Manhattan on October 29, 2012 and disrupted power to our hospital and FC. Back-up generators failed after 12 hours, at which time 2 uninterruptible power supply (UPS) units were used to maintain short term embryo culture conditions. Based on prior emergency planning, 2 options were considered for embryos in culture: 1) immediately cryopreserve all embryos, or 2) transport embryos to another lab. Given limited options, the team opted to vitrify all embryos irrespective of developmental stage. RESULTS: 90 embryos ranging from 2pn to blastocyst stage were methodically vitrified using UPS units to power 2 dissecting microscopes. Embryos were transferred to cryostorage tanks and lab operations were suspended until conditions normalized. Since then, 6 patients have undergone frozen embryo transfer (FET). 2 others had no euploid embryos after preimplantation genetic screening (PGS), 1 patient has 2 euploid embryos, and 1 had no ET. All 6 who underwent FET have ongoing clinical pregnancies, and 2 have surplus embryos re-frozen.

HS emergency embryo vitrification/warming outcomes

# Embryos Embryo emergently # Embryos Pregnancy developmental day vitrifieda warmed # ET outcome GS (FH) 1 2 3 5

8 8 16 1 13 13 12 5 11 3

8 8b 9 1 2 13 12b 5b,d 2 2

0 0 1 0 2 2 1 0 2 2

1 (1) 2 (2) 1 (1) 1 (1) 1 (1) 1 (1)

a Represents each patient’s embryo cohort. b Embryos underwent PGS. d No euploid embryos for embryo transfer (ET). GS¼gestational sac. FH¼fetal cardiac activity. CONCLUSION: HS had a profound impact on our FC despite disaster management planning. Urgent decision-making led to timely and successful vitrification of 90 embryos at various stages. No embryos were lost, and there are currently 6 ongoing pregnancies (6/6) from subsequent FET cycles. This experience underscores the need for a comprehensive disaster management strategy. P-124 Tuesday, October 15, 2013 BLASTOCYST VITRIFICATION VERSUS SLOW FREEZE: A RETROSPECTIVE COMPARISON OF BLASTOCYST SURVIVAL, EXPANSION AND IMPLANTATION RATE. R. J. Bodine, Q. Zhan, J. Park, S. Jones, N. Zaninovic, Z. Rosenwaks. Reproductive Medicine CRM, Weill Medical College, NY, NY.

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