Background
Male infertility may reflect molecular and biochemical sperm defects that are not captured by conventional semen analysis. Oxidative stress is common in male infertility and can impair motility while promoting lipid peroxidation, protein oxidation and sperm DNA damage. However, the relationships between oxidative stress, conventional semen parameters and complementary molecular biomarkers after sperm cryopreservation remain incompletely defined. This study investigated oxidative stress and sperm DNA damage in relation to post-thaw semen quality in fertility preservation patients.

Methods
Cryopreserved semen samples from fertility preservation patients (n=53) were assessed before and after thawing. Semen parameters were measured according to World Health Organization 2021 guidelines. Static oxidation-reduction potential was quantified using the MiOXSYS system. Sperm DNA damage was assessed using the HaloSperm sperm chromatin dispersion assay and sperm chromatin structure assay. A novel combined Halo-8-hydroxy-2′-deoxyguanosine assay was also applied to detect oxidative DNA lesions in relation to DNA fragmentation. To further explore the relationship between molecular biomarkers and sperm functional status, sperm flagellar movement was assessed using Flagellar Analysis and Sperm Tracking (FAST).

Paired comparisons and association analyses were performed, with statistical significance set at p < 0.05.

Results
Cryopreservation significantly reduced conventional semen parameters and increased sperm DNA fragmentation (p < 0.005). Higher oxidative stress was significantly associated with poorer post-thaw sperm quality (p < 0.005). Patient age, duration of sexual abstinence and cryostorage duration were not significantly associated with post-thaw semen quality or molecular markers of sperm health. No significant differences were detected between the DNA damage assays.

Conclusion
Oxidative stress and sperm DNA damage provide information beyond conventional semen analysis when evaluating cryopreserved sperm. Incorporating these biomarkers may strengthen sperm quality assessment and improve risk stratification in fertility preservation programs.