Ultra-performance liquid chromatography/tandem mass spectrometry for accurate quantification of global DNA methylation in human sperms.

Aberrant DNA methylation in human sperms has been proposed to be a possible mechanism associated with male infertility. We developed an ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) method for rapid, sensitive, and specific detection of global DNA methylation level in human sperms. Multiple-reaction monitoring (MRM) mode was used in MS/MS detection for accurate quantification of DNA methylation. The intra-day and inter-day precision values of this method were within 1.50-5.70%. By using 2-deoxyguanosine as an internal standard, UPLC-MS/MS method was applied for the detection of global DNA methylation levels in three cultured cell lines. DNA methyltransferases inhibitor 5-aza-2'-deoxycytidine can significantly reduce global DNA methylation levels in treated cell lines, showing the reliability of our method. We further examined global DNA methylation levels in human sperms, and found that global methylation values varied from 3.79% to 4.65%. The average global DNA methylation level of sperm samples washed only by PBS (4.03%) was relatively lower than that of sperm samples in which abnormal and dead sperm cells were removed by density gradient centrifugation (4.25%), indicating the possible aberrant DNA methylation level in abnormal sperm cells. Clinical application of UPLC-MS/MS method in global DNA methylation detection of human sperms will be useful in human sperm quality evaluation and the study of epigenetic mechanisms responsible for male infertility.