Assessment the effect of freezing on the microRNAs involved in spermatogenesis and their correlation with apoptosis in the sperm of infertile men

Abstract

Infertility is one of the most common problems in the world. Men are responsible for half of all infertility cases. Genetic factors can be known as the main result of male infertility. One of the genetic factors involved in male infertility is microRNAs. Spermatogenesis is also regulated by various microRNAs. MicroRNAs can also be used as molecular biomarkers to diagnose infertility in men Materials and Methods: In this study, Density-gradient centrifugation method was used to prepare sperm and separate it from semen plasma, Diff Quick staining was used to study the sperm morphology. Sperm chromatin dispersion test was conducted to check for DNA fragmentation. Antioxidant enzymes was evaluated by spectrophotometry. Targeted microRNAs and genes are examined by real-time PCR. Graph pad Prism 8 software is used for statistical analysis of the results. Results: The results showed that the morphology and motility of sperm decreased significantly after the freezing process. Freezing increased DNA failure in the MOAT group. In MOAT and SOAT groups, freezing increased oxidative stress and SOD, but GPx showed a decreasing trend. Expression of P53 and CYCLIN D1 decreased in MOAT, SOAT and non-obstructive azoospermia due to cryopreservation, but increased in patients with obstructive azoospermia. Caspase9 also had a significant increase in MOAT, SOAT and obstructive azoospermia freezing groups but, it showed significant decrease in non-obstructive azoospermia. Expression of mir-34c, miR-184, miR-383, miR-15b and miR-122 decreased in MOAT and SOAT groups after freezing. In the non-obstructive azoospermia group, the expression of mir-34c, miR-184, miR-383 and miR-15b increased significantly after cryopreservation. In the obstructive azoospermia group, cryopreservation significantly reduced the expression of miR-15b and miR-122, while the expression of miR-34c was significantly increased.