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Given the role of the  deleted in azoospermia  gene in male infertility, whether the somatic  deleted in azoospermia  methylation status is associated with idiopathic asthenospermia should be determined. To investigate the methylation levels of the  deleted in azoospermia  promoter in peripheral white blood cells from idiopathic asthenospermia patients relative to those in normozoospermia controls, 61 ethylene diamine tetraacetic acid anticoagulant blood samples were drawn from all participants for DNA isolation. The  deleted in azoospermia  promoter methylation ratio was detected by MassARRAY-based methylation quantification and confirmed by quantitative methylation-specific polymerase chain reaction. A MassARRAY-based methylation analysis showed that the  deleted in azoospermia 3  promoter (0 to − 2 kbp) was significantly hypomethylated in peripheral white blood cells from idiopathic asthenospermia males, specifically one CpG site (− 246 to − 247). Quantitative methylation-specific polymerase chain reaction data further confirmed that the methylation level of the  deleted in azoospermia 3  promoter region in idiopathic asthenospermia patients was significantly lower than that in normozoospermia males. The area under the receiver operating characteristic curve determined by quantitative methylation-specific polymerase chain reaction was 0.737 (95% confidence interval: 0.552 to 0.924), with a sensitivity of 53.9% and a specificity of 88.2% at a cut-off level of 74.7%. Therefore, our results suggested that methylation ratio detection of the  deleted in azoospermia 3  promoter region by real-time polymerase chain reaction assay is a promising and feasible tool for liquid biopsy in the clinical laboratories. The methylation status of other reported infertility-related genes should also be investigated in peripheral white blood cells.

Hypomethylation of the DAZ3 promoter in idiopathic asthenospermia: a screening tool for liquid biopsy