Tehran University of Medical Sciences Journal of Family and Reproductive Health 1735-8949 19 4 2025 12 31 280 285 Pushpalatha Marisamy Department of Commerce, Sri Krishna Arts and Science, Coimbatore, India Premkumar Jayapal Department of Food Technology, Nehru Institute of Technology, Coimbatore, India Ramesh Mageswaran Department of Aerospace Technology, SNS College of Technology, Coimbatore, India Mohanraj Krishnan Selvaraj Department of Mechanical Engineering, Erode Sengunthar Engineering College, Erode, India Karthikeyan Arthanari Department of Aeronautical Engineering, Excel Engineering College, Namakkal, India 2025 09 20 2025 12 30 Objective: To evaluate the association between serum 25-hydroxyvitamin D [25(OH)D] concentration and sperm DNA fragmentation index (DFI), and to identify clinical predictors of elevated DFI among men referred for infertility. Materials and methods: We conducted a cross-sectional study of 783 men referred to a tertiary infertility clinic. Participants underwent semen analysis according to WHO criteria, measurement of serum 25(OH)D by standard immunoassay, and assessment of sperm DNA fragmentation using a validated laboratory method. Demographic and clinical variables (age, BMI, smoking, duration of infertility) were recorded. Associations between 25(OH)D and DFI were evaluated using correlation analysis and multivariable linear and logistic regression models adjusted for potential confounders. Elevated DFI was predefined based on laboratory cutoffs. Results: Among 783 men, vitamin D deficiency was common and a wide range of DFI values was observed. Serum 25(OH)D concentration showed no significant association with sperm DFI after adjustment for age and conventional semen parameters (adjusted effect estimates not significant). In multivariable analyses, older age, lower progressive motility, abnormal morphology and reduced sperm concentration were independently associated with higher DFI (all p < 0.05). These associations persisted when DFI was analyzed as a dichotomous outcome (elevated vs. normal). Conclusion: In this large clinic-based sample, serum 25(OH)D was not independently associated with sperm DNA fragmentation. Conventional semen quality measures and age were stronger and consistent predictors of elevated DFI. These findings suggest that routine assessment of vitamin D status is unlikely to inform sperm DNA integrity beyond established semen parameters; prospective studies are needed to confirm causality. https://jfrh.tums.ac.ir/index.php/jfrh/article/view/3486 https://jfrh.tums.ac.ir/index.php/jfrh/article/download/3486/741