P-31: Effect of Cryptop Vitrification on DNAMethylation Pattern of Oct4 and Mest Genes inMurine Preimplantation Embryos

Background: Because of the protection of surplus embryos, Cryopreservation is usually used in ART. It is not clear, the vitrified-warmed embryos that have normal morphology, be normal in genetic level, too. DNA methylation of gene regulatory regions can causes inhibition of gene expression. We study effect of vitrification method of cryopreservation on DNA methylation and gene expression level in investigated genes. Materials and Methods: Two-cell embryos of superovulated mouse were collected by oviduct flushing method, then were divided in control and vitrified-warmed groups. In control group, these embryos were cultured to blastocysts and in other group, they were cultured to 4-8 cell embryos, then were vitrified with CryoTop and after 2-6 months, they thawed and cultured to blastocysts. DNA and RNA of morphologically normal embryos were purified. For investigation of DNA methylation status and quantitative expression of these genes were used Bisulfite- Sequencing Technology and Real time RT-PCR, respectively. Results: The results of quantitative PCR analysis showed that expression level of both genes, Oct4 and Mest, in vitrified- warmed group relative to control group has reduced, also investigation of DNA methylation appeared that in vitrified-warmed group, imprinting status of Mest gene has disturbed and in this group, one CpG dinucleotide in promoter of Oct4 gene has been hypermethylated. Conclusion: This study revealed that CryoTop vitrification has a negative effect on expression level of Oct4 and Mest genes and it seems that this vitrified method can change DNA methylation pattern of these two genes. Presumably, reduction of expression level of Oct4 gene is related to hypermethylation of mentioned CpG dinucleotide in this gene