BACKGROUND Sperm DNA integrity is crucial for normal fertilization, implantation, and embryo development. Several assays are available to assess sperm DNA fragmentation but are limited by high price, complicated processes and low accuracy. Additionally, the evaluation parameter cannot accurately show the degree of sperm DNA damage. METHODS We developed a secondary amplification detection system based on terminal deoxynucleotidyl transferase (TdT) and endonuclease IV (Endo IV), which could efficiently detect the number of 3'-OH. We used this detection system to detect the amount of 3'-OH in DNA single strand at standard concentration. We then interrupted the double strand of genomic DNA through ultrasound and enzyme digestion, and then used the detection system. Finally, we used this method to detect the number of breakpoints of human sperm DNA and calculated the mean number of breakpoints of sperm DNA. RESULTS We successfully detected the number of 3'-OH in DNA single strand at standard concentration and created the standard curve. The linear range for the increase rate of fluorescence intensity over the concentration of substrate DNA was from 0.1 nM to 15 nM. The detection method was successfully verified on λ DNA and human sperm DNA. CONCLUSION This method, which involves direct detection of actual DNA fragmentation, can measure the specific degree of sperm DNA fragmentation. It also has advantages, such as short time-consumption, simple operation, high analytical sensitivity, and low requirement for instrumentation, which makes it conducive to clinical application.

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