Tehran University of Medical Sciences Journal of Family and Reproductive Health 1735-8949 19 2 2025 08 05 152 160 Mohamadnabi Holakouie Naini Department of Gynecology, Clinical Research Development Unit of Fatemieh Hospital, School of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran Elahe Talebi Ghane Modeling of Non-communicable Diseases Research Center, Hamadan University of Medical Sciences, Hamadan, Iran Shamim Pilehvari Department of Gynecology, Clinical Research Development Unit of Fatemieh Hospital, School of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran AND Fertility and Infertility Research Center, Hamadan University of Medical Sciences, Hamadan, Iran 2025 03 29 2025 06 29 Objective: Unfortunately, the prevalence of infertility in Iran and the world is increasing. There is limited research on the relationship between vitamin D and DNA fragmentation index (DFI). DFI is a measure of DNA damage in sperm cells and is used to evaluate male fertility. Some studies suggest that vitamin D may play a role in sperm health and fertility. Materials and methods: In this cross-sectional study, 789 eligible men referred to the Omid Infertility Clinic in Hamadan-Iran in 2021-2023. Serum levels of vitamin D, DFI, spermogram indexes and demographic characteristics (occupation, age, and body mass index) were collected by face-to-face interview and records review in case files. Data were analyzed with SPSS version 27 software. Results: There is no significant relationship between DFI and vitamin D before and after removing the effect of confounding variables. Clearly, increasing age has been associated with increasing (DFI 95% Cl: 1.06-3.03; p<0.05) odds ratio (OR) = 1.79. The relationship between the increase in DFI and the decrease in normal sperm morphology (95% CI: 1.43-4.20; p<0.01) OR = 2.45 and the decrease in the progressive motility of sperm was seen (95% Cl: 1.66-4.87; p<0.05) OR= 2.85 and also. The relationship between DFI and the decrease in sperm count after removing the effect Confounding variables were observed (95%Cl: 1.06-8.38; p<0.05) OR=2.98. Conclusion: No correlation between DFI and vitamin D serum level was found. However, a clear association was observed between increased DFI and advanced age, reduced progressive motility, abnormal sperm morphology, and decreased sperm count. https://jfrh.tums.ac.ir/index.php/jfrh/article/view/3195 https://jfrh.tums.ac.ir/index.php/jfrh/article/download/3195/727