A semen analysis is routinely used as a clinical tool to investigate male infertility, though it rarely predicts spermatozoal function. Men with ostensibly normal semen parameters can therefore, be subfertile or infertile due to the presence of an otherwise unknown male genetic factor. Next-generation sequencing of spermatozoal RNAs has emerged as a novel technique, which objectively measures the paternal genetic contribution to idiopathic infertility (1). This is a prospective pilot study to investigate the epigenetic basis of idiopathic infertility using next-generation sequencing of spermatozoal RNAs, thereby identifying candidate genes central to spermatozoal competence. Three control (conceived naturally) and five study (unable to achieve pregnancy via IVF-ICSI) semen samples were included in this study. Female partners of the study patients were known to have patent fallopian tubes. They were also ruled out for endometriosis and ovulatory disorders. RNA was isolated from 25x106 human spermatozoa using a hybrid isolation protocol utilizing TRIzol® Reagent (ThermoFisher Scientific, USA) and a commercially available spin column kit (RNeasy Mini Kit, QIAGEN, Germany). The nucleic acid quality and spermatozoal RNA concentration were assessed by 2100 Agilent Bioanalyzer and by NanoDrop™. Illumina stranded RNA-Seq library preparation with Ribozero purification was used to construct the paired-end libraries. Pilot sequencing expanded to 50-60M reads at 2x76bp utilizing an Illumina platform (NextSeq 500). Data was then processed and analyzed following the Tuxedo Protocol (1). RNA transcripts were classified and associated with spermatogenesis, sperm development, and sperm function. By analyzing fold change in relation to P-values determined by differential analysis, we identified 86 genes with statistically different expression. Within this group of genes, 7 differentially expressed genes (APLF, CYUB5R4, ERCC4, MORC1, PIWIL1, TNFRSF21, and ZFAND6) known to regulate DNA repair mechanisms or protect spermatozoa from reactive oxygen species throughout spermatatogenesis showed significant under expression in the study group when compared to the control study (see table 1). Of note, all under-expressed genes were located on autosomes. Our findings suggest that under-expression of genes involved in DNA repair or protection from apoptosis may be responsible for infertility in the small study sample. While these preliminary results are encouraging, extensive prospective data are required to elucidate the epigenetic mechanisms involved in male infertility. This may influence the choice of fertility treatments, thereby reducing the time to pregnancy.