Sperm chromatin and DNA integrity, methyltransferase mRNA levels, and global DNA methylation in oligoasthenoteratozoospermia.

Abstract

ObjectiveTo investigate sperm chromatin/DNA integrity, global DNA methylation, and DNMT mRNA transcription in men with oligoasthenoteratozoospermia (OAT) compared with normozoospermic men.MethodsSemen samples from 32 OAT patients who comprised the case group and 32 normozoospermic men who comprised the control group were isolated and purified using a standard gradient isolation procedure according to World Health Organization criteria. DNMT1, DNMT3A, and DNMT3B transcripts were then compared between groups using real-time quantitative reverse-transcription polymerase chain reaction. Global DNA methylation in sperm was determined by an enzyme-linked immunosorbent assay. Protamine deficiency and the proportion of apoptotic spermatozoa were evaluated using chromomycin A3 (CMA3), aniline blue (AB), and toluidine blue (TB) staining, as well as the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The p-values <0.05 were considered to indicate statistical significance.ResultsSignificantly higher proportions of AB+, TB+, CMA3+, and TUNEL+ spermatozoa, as well as DNMT3A and DNMT3B transcription, were found in the OAT group. Positive correlations were detected between sperm parameters, DNA/chromatin damage, and DNMT3A and DNMT3B transcripts. Global DNA methylation was significantly higher in the OAT patients and had a significant correlation with abnormal results of all sperm chromatin integrity tests, but was not associated with DNMT1, DNMT3A, or DNMT3B expression.ConclusionOligoasthenoteratozoospermic men showed abnormal sperm parameters, abnormal chromatin/DNA integrity, and a higher global DNA methylation rate, as well as overexpression of DNMT mRNA.