Differential Expression of miR-335-5p, miR-424-5p, miR-181a-5p, and hsa-miR-27a-3p and Their Association with Semen Quality Among Infertile Men

Background: Semen analysis alone may not accurately diagnose male infertility. This study investigated four semen miRNAs, miR-335-5p, miR-424-5p, miR-181a-5p, and hsa-miR-27a-3p-as potential non-invasive biomarkers and examined their associations with infertility and sperm quality. Methods: Semen samples (n=54) were collected from couples who attended an IVF center for male-factor infertility between October 2022 and May 2025. Samples were classified into groups of normozoospermia (Control group; n=8), asthenozoospermia (n=12), oligozoospermia (n=11), teratozoospermia (n=12), and oligoasthenoteratozoospermia (OAT, n=11). Expression levels of selected miRNAs and the genes BCL2, BAX, Caspase-3, PRM1, and PRM2 were assessed using quantitative PCR (qRT-PCR). Sperm quality was evaluated through chromatin integrity, chromatin condensation, viability, acrosome status, and membrane integrity staining assays. One-way ANOVA with Tukey’s post hoc test was performed using Prism 8, with p< 0.05 considered statistically significant. Results: miR-424-5p, miR-335-5p, and miR-181a-5p expression levels were significantly reduced in infertility groups (p<0.01, p=0.0003, and p=0.001, respectively), whereas hsa-miR-27a-3p expression was increased (p=0.0003). All investigated miRNAs showed significant expression changes in the OAT group compared with controls (p<0.05). miR-181a-5p, miR-424-5p, and miR-335-5p were negatively correlated with BAX and Caspase-3 and positively correlated with BCL2, PRM1, and PRM2. The OAT group exhibited the poorest sperm chromatin quality and highest acrosomal damage (p<0.001). Conclusion: These miRNAs are significantly associated with male infertility and sperm quality and may serve as potential non-invasive biomarkers for differential diagnosis of male infertility.