A Preliminary Study On H19and MEST DNA Methylation Of Imprinted Gene In Sperm Of Infertile Males

Male infertility is affected by genetic, environmental and other multiple factors. Themechanisms of etiology in70%of the male infertile cases are unknown, even in those casesin which the causes of male infertility are clear. Apparently, more studies are needed tounderstand the mechanisms of male infertility. The diagnosis and treatment of male infertilityare complex. There has been evidence that abnormal epigenetic modifications may playimportant roles in male infertility. DNA methylation, a key modification of gene imprinting,is erased in primordial germ cells and specifically reestablished during gametogenesis. Thus,abnormal imprinting DNA methylation is associated with male infertility.In order to investigate the relationship between the abnormal imprinting DNAmethylation and the male infertility, we determined the percentage of methylation of paternalimprinted H19gene and maternal imprinted MEST gene from normozoospermic controls andolig-oastheno-teratozoospermia. Routine semen analysis and morphological observation wereperformed, using the sperm purified by density gradient centrifugation, and DNA wasextracted and treated with bisulfite before PCR. The purified PCR products were cloned intopMD18-T vector and proceed to chemical transformation and restriction enzyme reaction.Positive clones were selected for sequencing analysis and DNA methylation analysis. Theresults showed that all the control individuals had a high H19methylation (100%), while thatof the methylation percentage of the olig-oastheno-teratozoospermia patients wassignificantly lower (93.05%). Significant differences were found in the percentage ofmethylation between the control individuals and olig-oastheno-teratozoospermia (P<0.01).And the methylation percentage of the olig-oastheno-teratozoospermia patients at the CpG1,CpG3and CpG6is significant differences which compares to the control individuals. As toMEST gene, all the control individuals showed a low methylation degree of MEST (1.62%),while the olig-oastheno-teratozoospermia patients had a MEST hypermethylation methylationpercentage of4.03%, although the difference was statistically insignificant (P>0.05), And themethylation percentage of the single CpG between the control individuals andolig-oastheno-teratozoospermia is also statistically insignificant.In conclusion, this study revealed that the male infertility was associated with the aberrantmethylation of H19differentially methylated region (DMR), and it may be potentially abiomarker for human spermatogenesis defect.