A fluorescent enzyme-assited biosensor for analyzing the sperm DNA breakpoints in the freezing process

Abstract Background: The freezing and thawing process can cause irreversible structural and functional changes in human sperm, especially sperm DNA damage. Choosing a more accurate and sensitive detection method to evaluate sperm DNA integrity will be especially important. Methods: In this study, an innovative terminal deoxynucleotidyltransferase (TDT) and Endonuclease IV assisted fluorescent fluorescent biosensor (TdT/Endo IV Fluo-biosensor) was developed to detect DNA breakpoints in the freezing process. The performance of our biosensor was assessed by comparison with the conventional DNA fragmentation index (DFI) measured by chromatin structure analysis (SCSA). Results: It was found that the sperm DFI was significantly higher in the frozen group than in the fresh group, and there was no significant difference between the antioxidant group and the frozen group. However, using our method, the number of DNA breakpoints in the antioxidant group was significantly lower than that in the frozen group. Conclusion: Our established biosensor is more accurate and effective than conventional SCSA in assessing sperm DNA integrity during freezing. We anticipate that our biosensors can be widely used in reproductive medicine.