Evaluation of DNA Double-Strand Breaks in Human Sperm Following Selection by Density Gradient Centrifugation, ZyMōt, and Felix Techniques

Purpose: Density gradient centrifugation (DGC) is widely used for sperm preparation, but centrifugation-induced oxidative stress may cause DNA damage. This study compared sperm DNA double-strand breaks (DSBs) among DGC, ZyMōt, and Felix, and evaluated sperm recovery, motility, and processing time. Methods: Fifteen fresh semen samples collected from January to June 2025 were processed in parallel using DGC, ZyMōt, and Felix. Following sperm preparation, γH2AX immunostaining was performed, and at least 200 spermatozoa per sample were analyzed to determine the DSB-positive rate. Sperm recovery, motility, and processing time were also recorded. Statistical analyses were conducted using the Friedman test followed by Wilcoxon signed-rank tests with Bonferroni correction, and data were expressed as medians with interquartile ranges. Results: The DSB-positive rate was significantly lower in the ZyMōt (11.8%) and Felix (10.0%) groups compared with the DGC group (16.0%; p < 0.01). DGC yielded the highest sperm recovery, ZyMōt achieved the highest motility, and Felix required the shortest processing time, indicating that the three methods exhibit distinct performance characteristics. Conclusions: The non-centrifugal systems ZyMōt and Felix significantly reduced DSB-positive sperm compared with DGC while maintaining comparable overall performance, highlighting their potential usefulness as optimized sperm preparation approaches in assisted reproductive technology.