Evaluation of Sperm DNA Integrity by Mean Number of Sperm DNA Breaks Rather Than Sperm DNA Fragmentation Index

Abstract

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. Methods: We developed a secondary amplification detection system based on terminal deoxynucleotidyl transferase and endonuclease IV, which could efficiently measure the number of 3'-OH (equivalent to the number of breakpoints). We applied this detection system in single stranded DNA with standard concentrations to obtain the standard curve. We then broke the double stranded genomic DNA by ultrasound and enzyme digestion and used the detection system to monitor the increase of DNA breakpoints. Finally, we used this method to measure the mean number of sperm DNA breakpoints (MDB) in 80 sperm samples. Results: We successfully measured the number of 3'-OH in single stranded DNA with standard concentration and obtained the standard curve. The linear range for the number of DNA breakpoints was from 0.1 nM to 15 nM. The detection method was successfully validated on λ DNA and 80 human sperm samples. The results of real clinical samples revealed that the mean number of DNA breakpoints (MDB) had a stronger relevance with the sperm motility and clinical pregnancy outcomes than the commonly used parameter of DNA fragmentation index (DFI). Conclusion: We have developed a straight-forward method for direct measurement of the mean number of DNA breakpoints in sperms. The method has advantages of short time-consumption, simple operation, high analytical sensitivity, and low requirement for instrumentation, which makes it conducive to clinical application. The proposed new parameter (MDB) could be a more direct, accurate and clinically significant indicator for evaluating the sperm DNA integrity.