Backgrund: Imprinted genes are a unique subset of few genes, which have been differentially methylated region (DMR) in a parental origin-dependent manner during gametogenesis, and these genes are highly protected during pre-implantation epigenetic reprogramming. Several studies heve shown that the particular vulnerability of imprinting genes during suboptimal pre- and peri-conception microenvironments often occur by assisted reproduction techniques (ART). This study investigated the methylation status of H19/IGF2 DMR at high-quality expanding/expanded human blastocysts donated by healthy individuals to evaluate the risks linked to ART. Method: Methylation levels of H19/IGF2 DMR were analyzed by bisulfite conversion and sequencing at 18 CpG sites (CpGs) located in this region. Result: Results showed that the overall percentage of methylated CpGs and the proportion of hyper-methylated clones of H19/IGF2 DMR in analyzed blastocysts were 37.85±4.87% and 43.75±5.1%, respectively. For validation of our technique, the corresponding methylation levels of peripheral human lymphocytes were defined (49.52±1.86% and 50%, respectively). Conclusion: Considering the absence of in vivo produced human embryos, it is not possible to conclude that the methylation found in H19/IGF2 DMR is actually normal or abnormal. Regarding the possible risks associated with ART, the procedures should be optimized in order to at least reduce some of the epigenetic risks.