Repeated assessment by high-throughput assay demonstrates that sperm DNA methylation levels are highly reproducible

ObjectiveTo assess reliability of a high-throughput assay of sperm DNA methylation.DesignObservational study comparing DNA methylation of sperm isolated from 3 divided and 12 longitudinally collected semen samples.SettingAcademic medical center.Patient(s)One man undergoing screening semen analysis during evaluation of an infertile couple and 2 healthy fertile male volunteers.Intervention(s)Spermatozoa were separated from seminal plasma and somatic cells using gradient separation. DNA was extracted from spermatozoa, and DNA methylation was assessed at 1,505 DNA sequence–specific sites.Main Outcome Measure(s)Repeatability of sperm DNA methylation measures, estimated by correlation coefficients.Result(s)DNA methylation levels were highly correlated within matched sets of divided samples (all r ≥ 0.97) and longitudinal samples (average r = 0.97).Conclusion(s)The described methodology reliably assessed methylation of sperm DNA at large numbers of sites. Methylation profiles were consistent over time. High-throughput assessment of sperm DNA methylation is a promising tool for studying the role of epigenetic state in male fertility.