Introduction: Sperm cryopreservation is a vital procedure in assisted reproductive technologies; however, the freeze–thaw process inevitably induces oxidative stress, leading to mitochondrial dysfunction and activation of apoptotic pathways in spermatozoa. These changes impair sperm survival and function, particularly in asthenozoospermic men, who already suffer from compromised motility and quality. Melatonin, a powerful antioxidant with anti-apoptotic properties, is capable of crossing cellular and mitochondrial membranes, thereby directly neutralizing free radicals and stabilizing mitochondrial function. Through the regulation of apoptosis-related pathways, melatonin may protect sperm cells from cryo-induced apoptosis, preserving their functional integrity during freezing and thawing. This study aimed to investigate the protective effect of melatonin on apoptosis-related gene expression (Bax, Bcl-2, and Caspase-3) and overall sperm quality in asthenozoospermic men during in vitro cryopreservation.
Methods: Semen samples were collected from 30 asthenozoospermic men after 2–5 days of sexual abstinence. Each sample was divided into three groups: control (fresh), freeze (freezing medium only), and freeze + melatonin (1 mM). Levels of reactive oxygen species (ROS) were measured using flow cytometry. Sperm membrane integrity was assessed with the hypo-osmotic swelling (HOS) test. Expression of apoptosis-related genes (Bax, Bcl-2, and Caspase-3) was analyzed using RT-qPCR. Data were statistically evaluated using repeated measures analysis followed by the Bonferroni post-hoc test.
Results: Compared with the control group, the freeze group showed a significant increase in ROS levels and upregulation of pro-apoptotic genes Bax and Caspase-3, along with a decrease in anti-apoptotic Bcl-2 expression (p < 0.05). Sperm membrane integrity was also significantly reduced in the freeze group (p < 0.01). In contrast, the freeze + melatonin group showed a significant reduction in ROS, downregulation of Bax and Caspase-3, upregulation of Bcl-2, and improvement in sperm membrane integrity compared with the freeze group (p < 0.05).
Conclusion: Melatonin effectively protects sperm from freeze–thaw-induced apoptosis and membrane damage by regulating apoptosis-related genes (Bax, Bcl-2, Caspase-3) and reducing oxidative stress. These findings suggest that melatonin may serve as a promising antioxidant to enhance sperm quality in asthenozoospermic men during cryopreservation.