Investigating the role of sperm-specific RNA to screen men with unexplained infertility

Abstract

To identify the compartmentalization of candidate gene clusters within the human spermatozoon that contribute to spermatozoal fertilization capacity and embryo developmental competence. Sequencing and differential expression of sperm-specific RNAs in men with unexplained infertility. Consenting men screened for infertility donated their ejaculated semen samples during a 15-month period. Donors with proven fertility served as controls. RNA was isolated from 25x106 human spermatozoa using a commercially available spin column kit. Spermatozoal RNA concentration was subsequently assessed. Illumina stranded RNA sequencing (RNA-Seq) library preparation was used to construct paired-end libraries. Pilot sequencing was expanded to 50-60M reads at 2x76bp. RNA expression was calculated in fragments per kilobase of transcript per million mapped reads (FPKM). Differentially expressed RNA transcripts were identified and classified into gene clusters based on sperm-specific components i.e., the acrosome, nucleus, mid-piece, and flagellum. The expression of these transcripts was compared with standard semen parameters, and between the study and control groups. Thirty-one men with a mean age of 39.6±5 years had the following semen parameters: 46.3±19 x 106/mL concentration and 44.8±14% motility. The age of the female partner in the study and control groups was comparable. Differentially expressed RNA transcripts between the study and control groups were grouped into sperm-specific clusters: DPY19L2 and ATP6V1E2 (acrosomal); APLF, CYUB5R4, ERCC4, MORC1, PIWIL1, TNFRSF21, TSSK6 and H1FNT (nucleus); ADCY10, SMCP, PLK4 and AGPAT2 (mid-piece); and, AKAP4 and CATSPER1 (flagellum). There was a strong correlation between the expression of ATP6V1E2 and fertilization rates (R2 0.63; P<0.001) in men who underwent ICSI. A positive correlation between AKAP4 and motility was also noted (R2 0.40; P=0.01). The gene cluster localized to the nucleus was significantly under-expressed in the study group when compared to the control group (P<0.001). Interestingly, the expression of APLF, CYUB5R4, ERCC4, and TNFRSF21 was found to be higher in men <40 years (47.2±15 FPKM) compared to those >40 years (13±9 FPKM; P=0.02). Functional assessment of the gene cluster specific to certain compartments of the human spermatozoon may supplement standard semen analysis and serve as a vital tool in the diagnosis of unexplained infertility. This may further facilitate tailoring of assisted fertility treatments, thereby reducing the time to pregnancy.