TdT/Cas12a-based biosensor for sensitive detection of DNA breakpoints in sperm cryopreservation.

With the advancement of assisted reproductive technology (ART), sperm cryopreservation has become an essential technique in both clinical and research applications. However, maintaining DNA integrity during the freezing and thawing process remains a significant challenge. This study introduces an innovative TdT/Cas12a-based biosensor to enable precise detection and quantification of sperm DNA breakages. Compared to traditional methods such as the sperm chromatin structure assay (SCSA), this biosensor exhibits exceptional sensitivity (capable of detecting DNA breakages at as low as 0.001 nM) and molecular-level resolution, allowing for the precise localization and quantification of DNA breakpoints. By integrating TdT for nucleotide labeling and Cas12a for signal amplification, the system achieves high specificity and reliability. Additionally, the introduction of a standard strand as a quality control significantly enhances reproducibility. Furthermore, the study explores the protective effects of natural antioxidants on sperm DNA integrity during the freeze-thaw process. The results reveal a marked increase in DNA breakpoints caused by cryopreservation, with the TdT/Cas12a system outperforming traditional methods in providing detailed and accurate assessments. Notably, the addition of antioxidants effectively mitigates DNA damage during the freeze-thaw process, safeguarding sperm DNA integrity. This platform not only represents a significant advancement in DNA damage detection and monitoring but also supports the optimization of cryopreservation protocols and contributes to improving the success and safety of ART. It offers valuable insights and innovative tools for the field of reproductive medicine.