Background: Asthenozoospermia is characterized by reduced sperm motility, which can lead to male infertility. Sperm cryo- preservation, commonly used in assisted reproductive tech- niques, often causes considerable damage to sperm quality. The addition of antioxidants to the cryopreservation medium may help mitigate the adverse effects of the freezing process. Theobromine (TB), a plant-derived alkaloid belonging to the methylxanthine family, is widely recognized for its antioxidant properties and potential protective effects. The aim was to in- vestigate the effects of theobromine on the expression of ap- optotic and anti-apoptotic genes in cryopreserved sperm from asthenozoospermic men. Materials and Methods: thirty semen samples were collected from Asthenozoospermic patients. Each sample was divided into three groups: Control (fresh), Freeze (treated with cryo- protectant), and Freeze+ TB (treated with cryoprotectant and 10 mmol/L theobromine). Cryopreservation was performed through the rapid freezing method. In each sample, the expres- sion of the genes Bax, Bcl2 and Caspase 3, were analyzed us- ing Real-time PCR. The results were reported as fold change (ΔΔCt formula). Data were analyzed statistically using Repeat- ed Measure Analysis and Bonferroni post-hoc test. Results: The expression levels of Bax and Caspase 3 genes in the freezing group were significantly increased compared to the control group (P<0.001), while the expression level of Bcl2 gene did not change significantly (P>0.05). Also, the expres- sion levels of and Caspase 3- genes in the freezing + TB group were not significantly changed compared to the freezing group (P<0.001), while the expression level of Bcl2 gene decreased significantly (P<0.001). Conclusion: Our data show that the addition of TB to the sperm freezing medium does not affect the expression of Bax and Cas- pase-3 genes, but significantly reduces the expression of the Bcl2 gene.
