Cryopreservation increases sperm DNA fragmentation in normozoospermic Libyan men: the role of oxidative stress and the protective effect of melatonin

Cryopreservation of sperm is routinely used in assisted reproduction technology (ART) for male fertility preservation. However, this method has been associated with oxidative stress and DNA fragmentation that may impair sperm quality. Additionally, antioxidant interventions such as melatonin supplementation have not been thoroughly explored in this setting. Although Libya is reported to have one of the highest global prevalence rates of male infertility, Libya-specific data remain limited. This study aimed to determine the effect of a single freeze-thaw cycle on sperm DNA fragmentation and oxidative stress markers, and to evaluate whether melatonin has an impact on post-thaw oxidation profiles. This prospective cohort study was conducted at the Fertility and Reproductive Medicine Center, Beirut Hospital, Benghazi. Semen samples of 104 normozoospermic Libyans were evaluated before and after freezing. DNA fragmentation index (DFI) was measured by sperm chromatin dispersion (SCD) test, and reactive oxygen species (ROS) were quantified by using luminol-enhanced chemiluminescence. In a subset of ejaculates, aliquots were supplemented with 2 mM of melatonin prior to cryopreservation. Cryopreservation was associated with a statistically significant increase in DFI (46.3 ± 18.3% to 60.0 ± 23.0%; <i>p </i>< 0.001) and ROS levels (3.2 × 10³ to 14.7 × 10³ RLU/s; <i>p </i>< 0.001). Smokers presented significantly higher DFI at both pre-freeze and post-thaw evaluations (<i>p</i> < 0.001). We detected a positive correlation between ROS and post-thaw DFI (r = 0.68; <i>p </i>< 0.001). Melatonin-treated samples exhibited moderate but significant differences in ROS (12%, <i>p</i> = 0.045) and DFI (11%, <i>p</i> = 0.004) compared to untreated aliquots. These findings suggested that the freeze-thaw process may contribute to oxidative and genomic stress in spermatozoa, while melatonin supplementation appears to provide limited protection. Larger, multicenter studies incorporating ART endpoints are required to determine the potential translational relevance of these findings.