Abstract
Neutral comet assay has been available for two decades to evaluate sperm double-strand breaks (DSBs). However, its clinical usability is limited due to its complex and time-consuming procedure, as well as the lack of a standardized scoring system. The aim of this study was to: develop a rapid diagnostic method for DSBs, Sperm DNA Fragmentation Releasing Assay (SDFR), and explore the association between DSBs and reproductive outcomes. We pioneered the use of polyacrylamide (PA) for embedding sperm chromatin and optimized the porosity of PA to be between 10 and 13%. The refined PA network allowed the trapping of DSBs, which dispersed halo on an immunological slide; in contrast, intact chromatin failed to develop a halo. A strong correlation was showed between reproducible values obtained from SDFR and neutral comet assay. SDFR were responsive to dose-/time-dependent simulated DSBs, indicating high sensitivity and specificity. Furthermore, we conducted a retrospective study of couples with embryonic aneuploidy screening, and recording DSB profiles of the male partners. Our findings revealed that DSB enabled to predict embryonic aneuploidy whereas basic semen parameters did not. In conclusion, SDFR offers a rapid and user-friendly approach for evaluating DSBs, with potential implications for predictive healthcare in reproductive medicine.