DNA Methylation Patterns of bcl2, bax, bag1 and casp3 Genes in Fragmented and Normal Human Embryos Derived from ART

Piltan A *, Eftekhari-Yazdi P , Toutounchi M , Gourabi H , Valojerdi M.R , Karimian L , Esalaminejad M.B ,


Objective: Fragmentation is common event observed in more than 50% human embryos that grows in vitro culture. The main mechanism of fragmentation is apoptosis. To achieve the correlation between apoptosis and fragmentation, we study the role of DNA methylation in anti-pro apoptotic genes expression inhibitation and its final effect on fragmentation. Materials and Methods: Fragmented and normal human 8-cell embryos were scored according to the degree of fragmentation , into four grades (grade І: normal or least fragmentation embryos, grade ІІ: embryos with lower than 25% fragmentation, grade ІІІ: embryos with more than 25% fragmentation, grade ІV: embryos to induced with a apoptotic inducer Actinomycin D). In this study, TUNEL labeling was used to detect apoptosis, also Bisulfite-Sequencing Technology characterized methylation status of the bag1, bcl2, casp3 and bax enhancer/promoter regions, and then Real-Time PCR. confirmed analysis of gene expression in human embryos. Results: The results of TUNEL labeling showed that embryos with higher fragmentation had a high number of apoptotic bodies. Bisulfite sequencing and quantitative PCR analysis were used respectively to indicate the level of gene expression and DNA methylation profiles of the above genes in four different embryo grades. To determine DNA methylation changes between these embryo grades, we analyzed the CpG islands states of various regulatory regions of bag1, bax, bcl2, casp3 and bax genes. Conclusion: The primery data revealed that bax enhancer/promoter region was hypomethylated through grade I to IV whereas it seems that methylation of Bag1 varied between these embryo grades, the finding should be confirm by their expression level that is ongoing.