P-98: Effect of Mouse Embryo Vitrification on Histone Modifications

Background: Vitrification has been usually used as an assisted reproductive technology in animals and humans. This method needs high concentrations of cryoprotectants that can be toxic with high cooling degrees. Then, vitrification could be change histone modifications such as methylation and acetylation can performance as regulatory controls of gene transcription. So, the purpose of the present study was to assess the effects of embryo vitrification and warming in mouse 2-cell embryo on blastocyst histone modifications. Facts found from the present study using this animal embryo model provide a vision into the epigenetic events that happen in human embryos after vitrification. 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 mouse monoclonal anti-H3K9ac, rabbit polyclonal anti-H4K12ac and mouse monoclonal anti-H3K4me3. Intensity of these epigenetic modifications 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 results show that, the vitrification procedure in mouse embryos did not affect the acetylation level of H4K12 and H3K9 biomarks, while this procedure promotes the tri-methylation level of H3K4 significantly (p<0.05). Conclusion: Vitrification procedure can increase H3K4me3 significantly that related to gene expression.