P-107: The Effects of Cryotop Vitrification on Heat Shock Protein 72 Expression in Mouse 2-Cell Embryos by Nested Quantitative PCR

Background: The aim of the study was to compare the effects of two different concentrations of cryoprotectants by Cryotop vitrification on survival and Heat shock protein 72 (Hspa1a) expression of two-cell mouse embryos. Materials and Methods: Different cryoprotectants’ concentrations of the combination of dimethyl sulfoxide (DMSO) and ethylene glycol (EG) were used and compared with each other and the non-vitrified 2-cell mouse embryos. The combinations were 15% (vit1: 7.5% of each EG and DMSO), 30% (vit2: 15% EG + 15% DMSO), and control group with no cryoprotectants. Cryotop was the instrument of choice for vitrification. Vitrified and fresh 2-cell embryos were incubated under mineral oil for 1 hour at 37ºC to obtain survival rate. Embryos were defined “morphologically survived”, if the cells possessed an intact zona pellucida, blastomeres and refractive cytoplasm. Transcript analysis of Hspa1a gene was performed on survived non-vitrified and vitrified embryos via a nested quantitative polymerase chain reaction (PCR) approach subsequent to normalization with Hprt1 as the reference gene. Results: The relative expression of Hspa1a in vit2 (30% v/v) was significantly higher than vit1 (15% v/v). Survival rates were the same for both vitrification treatments and significantly lower than control group (p<0.05). Conclusion: Our data demonstrated that vit1 treatment (7.5% EG and 7.5% DMSO) was more efficient than vit2 (15% EG and 15% DMSO) in mouse 2-cell embryos. The Cryotop vitrification with two concentrations of cryoprotectants caused the relative changes of Hpsa1a transcript level, but the stability of the gene in vit1 was significantly higher than vit2 and closer to the fresh 2-cell embryos.