Purpose: The objective of this stud), was to obtain expanded blastocysts .following intracytoplasmic sperm injection (ICSI) and Vero-cell co-culture, cpyopreserve

Innovative Cryo Enterprises) vitrification solutions. Differences in %survival and %24 hr development were statistically compared by Chisquare analysis (P < 0.05). Materials & methods: Biopsied blastocysts (AA to BB quality) were vitrified using microSecure vitrification (μS-VTF), an aseptic closed system. Embryos approved for research use were randomly assigned a treatment group and discarded within 24 hr. Rapid warming was performed on all embryos, with stepwise sucrose elution of blastocysts and 30 min isotonic equilibration at 37°C in LG medium+7.5% SS in tri-gas, humidified incubation. All BL were then re-exposed to standard VTF dilution protocols with extended ambient air exposure (20-22°C) in the final VTF solution (up to 20 min), before being returned to 1.0M sucrose and a 4-step serial dilution (3 min each), concluding in a 5 min exposure to isotonic H-LG medium before being returned to 24 hr in vitro culture. Embryo survival assessments were performed at 0 and 24 hr (i.e.,continued expansion) post-treatment. Results: 24 hr development was reduced (P < 0.05) by 1-5 min exposures in the EG/DMSO group (83.3%), compared to EG/PPG (93.3%) or Glycerol/EG (96.7%). Interestingly, the developmental integrity of Glycerol/EG treated blastocysts reduced (75%; P < 0.05) by 10 min and was significantly lower at 15/20 min (30-35%) than either EG/ DMSO (55-70%) or EG/PPG (55-60%). Conclusion:Our experiments have dramatically demonstrated the plasticity and cryotolerance of blastocyst to rVTF treatments (Expt.1) and extended exposures to 3 different VTF solutions (Expt.2). Since Glycerol/EG has a higher solute concentration solution, it was more toxic after 10 min exposure. However, it is also more metastable in its vitrified 'glasseous' state without the need for ultra-rapid cooling, as long as sufficiently high warming rates are achieved. The genotoxicity of all treatment solutions to extended exposure remains unknown, especially >5 min. Whereas, up to 3 min exposures and 2× VTF in the control group is regarded as safe, having previously yielded proven healthy live births.