Sperm motility is essential for oocyte penetration and fertilization. According to the World Health Organization (WHO), asthenozoospermia—defined as total sperm motility below 42%—is a leading cause of male infertility. Melatonin is known for its antioxidant properties in various cell types. This study investigated the in vitro effects of melatonin supplementation on sperm from asthenozoospermic men during the freeze-thaw process. Semen samples from 30 asthenozoospermic men were collected and divided into three groups: control, freeze, and freeze + melatonin (1 mM, 30-min incubation). Sperm motility was assessed based on the (WHO, 2021) guidelines, viability was evaluated using eosin- nigrosin staining, DNA fragmentation was analyzed using the Halosperm assay, and apoptosis-related genes expression (Bcl-2 and Bax) was measured by real-time qPCR. Data were analyzed using repeated measures analysis with Bonferroni post hoc test. Cryopreservation significantly reduced sperm motility, viability, and Bcl-2 expression (p < 0.001). while it increased DNA fragmentation and Bax expression compared to the control group (p < 0.01). However, these adverse effects were significantly reversed in the freeze + melatonin group compared to the freeze group (p < 0.01). Sperm motility, viability, and DNA integrity are key factors in successful fertilization. Melatonin supplementation during sperm cryopreservation improved post-thaw sperm quality and reduced DNA damage and apoptosis. Therefore, melatonin may enhance the success rate of assisted reproductive techniques and help preserve the genetic integrity of future generations.
