P-94: Mouse Embryo Vitrification Cannot Effect on Global DNA Methylation in Preimplantation Stage

Background: Embryo vitrification was effectively used for assisted reproductive techniques. Despite the undeniable benefits of vitrification, cooling and warming stress, and cytotoxicity of cryoprotectant may affect the DNA methylation that have an important role in gene activation and silencing. In the present study effects of 2-cell embryo vitrification on DNA methylation in hatched blastocyst was evaluated. Materials and Methods: Six-to-eight week-old female mouse was superovulated by 10 IU PMSG, followed 46- 48 hours later with 10 IU of hCG and mated with NMRI male. Female mice were sacrificed in day 1.5 after hCG injection for collection of the 2-cell embryos. These embryos were vitrified by using cryotop as mentioned by Kuwayama et al. After warming, survival rate were evaluated. The recovered embryos were cultured in G1/ G2 medium. Immunofluorescence staining on hatched blastocysts was done by a mouse monoclonal antibody against 5-methylcytosine (5 MC). Intensity of 5 MC was analyzed by Image J software. Results: 359 and 357 embryos randomly inserted to control and vitrified groups, respectively. The survival rate for vitrified group was 97.2%. In the vitrified group blastocyst (81.3%) and hatched blastocyst (65.4%) formation rates were significantly lower than the control group (90.8% and 78.3%, respectively) (p<0.01). This decrease suggests that vitrification may negatively affect the embryo development. Although that the lowest level of fluorescent intensity for 5mC was related to vitrified group, this intensity was not significantly different from control group (p>0.05). This numerically reduction may be related to DMSO which can affect the DNA methyltransfereases. In vitro culture also may make environmental stress and have related to hypermethylation. Thus, it may be reason of have no significant difference between two treatments for 5 MC. Conclusion: Numerical reduction of 5mC in hatched blastocyst of vitrified group may restrict to specific region of DNA such as DMRs or region that related to gene expression.