DNA methylation at imprinted genes and DNA fragmentation in sperm of men diagnosed with severe oligozoospermia and azoospermia

Abstract

(1) To evaluate DNA methylation (DM) at imprinted genes in men affected by severe oligozoospermia, azoospermia and fertile men undergoing vasectomy reversal (VR). 2) To study the correlation between aberrant DM and DNA fragmentation (DF) levels in each study group. DM and DF were evaluated in sperm from each study group. All samples were collected with patients' consent. Ejaculate sperm from severe oligozoospermic men (SO, N=13) and fertile men with normal semen parameters (N=20) were obtained. Testicular sperm from men with non-obstructive azoospermia (NOA, N=6), VR (N=5) and epididymis sperm from obstructive azoospermic (OA, N=11) men were picked using a micromanipulator. DM status of differentially methylated regions (DMRs) at imprinted genes (H19, GTL2, LIT1, and MEST)was detected in sperm. The one-way ANOVA and T-test were used to compare the means of control as well as study groups. Mean DM at LIT1 and MEST was significantly greater in SO group than fertile control group (9.7% vs 5.2%, P=0.005; and 13.5% vs 7.0%, P=0.009; respectively). NOA (9.82% vs 4.93 % P=0.004), OA (8.26% vs 4.93%, P=0.039) and VR (18.26% vs 4.93%) groups show significantly higher DM levels at LIT1 than fertile men. Mean DM at H19 was significantly lower in VR (77.93% vs 88.48%) and NOA (82.01% vs 88.48%) groups compared to fertile control group. Also, the rate of DF was significantly greater in all study groups compared to the controls (VR 56.24 %, OA 71.43%, SO 60.69%, NOA 66.81% vs 27.4%, P=0.0001). No significant correlations of DM and DF at H19 GTL2 MEST and LIT1 were detected in the groups. LIT1 was susceptible to have higher rate of imprinting abnormalities in sperm of all study groups. Testicular sperm (NOA and VR) had abnormal DM levels at H19 and LIT1. Fertile men with vasectomy showed significantly higher DM and DF levels than fertile control group. DF was highest in epididymis sperm among the study groups.