Sperm cryopreservation has become an essential fertility preservation technique in modern medicine. However, the freez- ing process induces oxidative stress through the generation of reactive oxygen species (ROS), leading to cellular damage. This study investigates the cryoprotective potential of thymoquinone (TQ) as a natural antioxidant to improve semen qual- ity during cryopreservation. In this prospective in vitro study, twenty-five semen samples from asthenozoospermic patients (25–40 years) were divided into: (1) Control (Fresh), (2) Freeze, and (3) Freeze+TQ (5 µg/mL) groups. Evaluated param- eters included: motility (total/progressive), morphology (Papanicolaou stain), viability (Eosin–Nigrosin, WHO standards), membrane integrity (hyposmotic swelling test), mitochondrial membrane potential (MMP), DNA fragmentation (SDFA kit), and protamine deficiency (Chromomycin A3). Oxidative stress markers (MDA, TAC, SOD, catalase) were quantified via ELISA. Results showed cryopreservation significantly impaired all sperm quality parameters (motility, morphology, viability, membrane integrity, MMP, antioxidant enzymes, TAC, and protamine levels), while increasing MDA and DNA fragmentation compared to control. TQ supplementation remarkably attenuated these cryodamage effects, showing significant improvement in all parameters versus freeze group. Thymoquinone demonstrates useful cryoprotective effects by mitigating oxidative stress and preserving sperm function during freeze-thaw cycles. Its antioxidant properties make it a promising candidate for improving cryopreservation outcomes in male fertility preservation.
