Background and Objective: Protamine deficiency and DNA fragmentation have a profound effect on embryo gene expression. Incubation of sperm in culture medium leads to morphological changes of the nucleus and DNA release. However, the relationship between protamine deficiency and DNA fragments released from incubated sperm and the presence of the DNMT1 gene is not known. Materials and Methods: Semen analysis was performed according to the WHO criteria. CMA3 staining was used to determine protamine deficiency. Twenty million sperms per sample were incubated in Ham’s F10 medium for 24 hr at 37 °C. After centrifugation, DNA was extracted from the supernatant. DNA concentration was measured by biophotometer. The presence of the DNMT1 gene was confirmed by PCR using specific primers and agarose gel electrophoresis. Results:A significant correlation was observed between the DNA fragments released from sperm in culture media and CMA3 staining (P<0.05). The presence of DNMT1 gene was significantly correlated with DNA fragments released from the sperm (P<0.05). Protamine deficiency and the presence of DNMT1 gene were not significantly correlated. Conclusion:These results demonstrate that incubation of protamines-deficient sperm leads to the release of additional DNA fragments that eventually could have detrimental effectson embryo’s health.