Background: Today, sperm cryopreservation is one of the most common approaches in assisted reproductive techniques (ART) that produces irreparable damage and reduces the fertility of human sperm by producing reactive oxygen species (ROS) and oxidative stress. In this regard, researchers are looking for ways to improve freezing conditions. The aim of this study was to evaluate the antioxidant effect of follicular fluid on sperm quality of normospermic men during the freezing process. Materials and Methods: Semen samples from 30 healthy normospermic men were divided into three groups: sham (0 min), control (1 hour), and treatment (1-hour incubation with 50% follicular fluid). After two weeks of cryopreservation, samples were thawed and analyzed for sperm parameters (motility, morphology, viability), plasma membrane integrity, mitochondrial membrane potential, DNA fragmentation, and oxidative stress markers (MDA, TAC, catalase) using ELISA. Data were analyzed using SPSS with repeated measures and Paired-Samples T Test; differences were considered significant at P≤0.05. Results: In the control group, significant declines were observed in sperm motility (total, progressive, and non-progressive), viability, plasma membrane integrity, mitochondrial membrane potential, CAT, and TAC levels compared to the sham group. Conversely, DNA fragmentation and MDA levels significantly increased. No difference was seen in normal sperm morphology. Treatment with follicular fluid significantly improved motility, viability, membrane integrity, and TAC levels, and reduced DNA fragmentation compared to both control and sham groups. However, it showed no significant effect on morphology, mitochondrial membrane potential, CAT, or MDA levels relative to the sham group. Conclusion: The results of this study demonstrated that the use of follicular fluid as an antioxidant in the freezing medium of sperm can improve sperm parameters after the thawing process, which may be due to the increase in total antioxidant capacity and the reduction of oxidative stress.
