Sodium Hyaluronate Supports High Survival and Clinical Pregnancy Rates of Blastocysts Using a MicroSecure, DMSO-free Vitrification System

pregnancy, spontaneous abortion and multiple pregnancy rates. Furthermore, when limiting the analysis to women younger than 35 years, the difference lost its significance. FINANCIAL SUPPORT: None. P-27 Clinical Pregnancy Rates are Similar between Fresh and DMSO-free Vitrified Blastocysts; A Multi-center Study. James J. Stachecki,a Sharon H. Anderson.b aInnovative Cryo Enterprises (ICE) LLC, Linden, NJ 07036; bMain Line Fertility Center, Bryn Mawr, PA. BACKGROUND: Although blastocyst vitrification is commonplace among IVF clinics, the need for a closed, easy-to-learn and use system remains. Using a DMSO-free system will help avoid possible cryoprotectant toxicity. Micro-volume containers and rapid procedure times remain technically challenging for some embryologists. The I.C.E. vitrification system relies on a relaxed protocol, closed device, and vitrification regime that is closer to equilibrium vitrification, and does not depend on ultra fast cooling rates and small volumes. OBJECTIVE: Compare pregnancy rates of fresh and vitrified blastocysts between eight clinics that use the ICE vitrification system. MATERIALS AND METHODS: Retrospective data was collected from eight IVF clinics. Blastocysts were 1) produced and transferred fresh or 2) vitrified, warmed, and transferred in a subsequent cycle. Embryo production (stimulation, culture, etc.) as well as quality of blastocysts varied between clinics. Briefly, expanded, non-collapsed blastocysts (D5 or D6) were vitrified by incubation at 23 C-26 C in I.C.E. vitrification media: V1-V3 media for 5min, 5min, and 1-2min; respectively. Blastocysts were loaded into the device of choice and stored in liquid nitrogen. Embryos were warmed and rehydrated by sequential dilution in thaw media (T1-T5). Clinics used a variety of storage devices ranging from micro-volume (micro-secure and HSV) to large volume (0.25cc straw). RESULTS: TABLE 1. Pregnancy results from fresh and vitrified blastocysts in 8 IVF clinics

Age <35 35-37 38-40 41-42 43+ Donor

Survival 745/819 (91.0%) 287/312 (92.0%) 196/215 (91.2%) 39/45 (86.7%) 12/12 (100%) 112/121 (92.6%)

Transfers Clin. Preg Rate Fresh Preg Rate 501 189 110 23 9 59

246/501 (49.1%) 187/337 (55.5%) 98/189 (51.8%) 76/140 (54.3%) 57/110 (51.8%) 32/78 (41.0%) 6/23 (26.1%) 9/44 (20.4%) 4/9 (44.4%) 1/4 (25%) 35/61 (57.4%) 102/178 (57.3%)

Blastocyst survival was 91.3% for all age groups combined. Clinical pregnancy rates were similar between fresh and vitrified blastocysts, except for patients ages 38-40, where the rate was higher (p<0.05) in the vitrified group. CONCLUSIONS: The collective data demonstrate the effectiveness of vitrification using a simple, closed, system in a variety of clinical settings, despite all of the variations between patient demographics, stimulation protocols, and embryo culture. I.C.E. vitrification represents an alternative approach to traditional DMSO systems and can be successfully utilized with a variety of storage devices. SUPPORT: None. P-28 Sodium Hyaluronate Supports High Survival and Clinical Pregnancy Rates of Blastocysts Using a MicroSecure, DMSO-free Vitrification System. J. J. Stachecki,a R. E. Anderson,b,c M. C. Schiewe.b aInnovative Cryo Enterprises LLC (I.C.E.), Linden, NJ, USA; bSouthern California Institute for Reproductive Sciences (SCIRS), Newport Beach, CA, USA; c Southern California Center for Reproductive Medicine (SCCRM), Newport Beach, CA, USA. BACKGROUND: The success of blastocyst vitrification (BL-VTF) has led to an increase in SET and reduction of multiples. Hyaluronate (HA) is a known extracellular adhesion molecule that stabilizes membranes and promotes growth, proliferation, and embryonic development. HA acts to increase solution viscosity and promote vitrification. In 2012, we prospectively

FERTILITY & STERILITYÒ

vitrified Day 6 PGS-BLs with (+) or without (-) HA and determined there was no difference (PR 0.2) in implantation or clinical pregnancy rates (+HA: 94.1%; -HA: 84.6%). OBJECTIVE: In 2013, we prospectively applied HA to all BL–VTF, and evaluated its effectiveness on cellular integrity and embryonic viability in PGS and non-PGS cycles. MATERIALS AND METHODS: All BLs were vitrified in HA-enriched I.C.E. VTF media. Aseptic microSecure VTF was used (flexipettes sealed inside 0.3ml CBS embryo straws). Warming cycles were conducted 1-3 hr prior to ET, using a gradual 5-step elution. BL were cultured in Life Global medium + 7.5% LGPS and 1% HA under Ovoil in MCO-5M tri-gas incubators. Chi-squared analysis was performed to determine differences (P<0.05) in survival, implantation, and pregnancy outcomes. RESULTS: TABLE 1. VFET Clinical results for BLs vitrified in HA-enriched I.C.E. vitrification media.

Group

Age

<38 38-42 w/PGS <38 38-42 Fresh Donor <38 38-42 NPGS

Survival

Patients

Implantation

Clin. Preg Rate

78/80 (97.5%) 19/20 (95%) 67/67 (100%) 36/36 (100%) (2010-11) N/A N/A N/A

41 10 55 27 48 92 61

54.5%b 30.4%c 79.1%a 85.7%a 76.0%a 88/175 (50.3%)b 25/165 (12.7%)d

73.2%b 60.0%c 85.5%a 88.9%a 93.5%a 64.1%bc 32.8%d

HA supplemented VTF media supported high rates of survival and pregnancy across all groups. Implantation and pregnancy outcomes were significantly higher in the PGS groups compared to non-PGS, but similar to our fresh 2010-11 Donor Egg ET rates, which are among the highest in the country (2011 SART). Furthermore, there has been no difference between the fresh ET and VFET-nonPGS groups for patients <38 y.o. (64.1% vs. 73.2%). However, VFET pregnancy outcomes were higher than the fresh ET group for patients 38-42. VFET-PGS success is independent of age when one or two euploid BLs are transferred. CONCLUSION: The addition of a large macromolecule like HA can offer dual support by increasing both the viscosity of the VTF solution and providing for cellular adhesion/membrane stabilization. Using I.C.E. media and mS-VTF we could optimize pregnancy success, with BLs maintaining full developmental competence. The data show that BLs vitrified in HA enriched media are equally effective, if not improved, in producing pregnancy outcomes comparable to fresh BLs. In fact, older women (38-42 yo) appear to benefit from VFET involving an unstimulated uterus, thus supporting the trend toward VFET only cycles. SUPPORT: None. Reference 1. Schiewe MC. MicroSecure Vitrification for oocytes and embryos: optimum simplicity, security. and cost and effectiveness combining FDA-approved products. Journal of Clinical Embryology 2010;13(2): p. 33–51.

P-29 Sildenafil Rescues First Trimester Trophoblasts from Acute Alcohol Exposure Through Downstream Protein kinase G Signaling. Jay M. Bolnick,a Alan D. Bolnick,a Brian A. Kilburn,a a,b,c a D. Randall Armant. Departments of Obstetrics and Gynecology, Wayne State University, C.S. Mott Center for Human Growth and Development, Detroit, MI; bAnatomy and Cell Biology, Wayne State University, C.S. Mott Center for Human Growth and Development, Detroit, MI; cProgram in Reproductive and Adult Endocrinology, NICHD, NIH, DHHS, Bethesda, MD. BACKGROUND: Ethanol adversely aff5cts human trophoblast cells by inducing programmed cell death. Intracellular Ca2+ becomes elevated in the cells, causing subsequent apoptosis. We previously found that sildenafil (Viagra) can induce integrin switching, increase invasion, and rescue trophoblasts from apoptosis caused by oxidative stress. These effects require both NO and cGMP signaling pathways. Therefore, we hypothesize that protein kinase G (PKG) downstream signaling activated by

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