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Precise detection of sperm DNA breaks and AP sites based on TDT and strand displacement probes
Bei Yan1, Ziyang Liu2, Heng Fan3
1Human sperm bank, Institute of Medical Sciences, General Hospital of Ningxia Medical University, Key Laboratory of Fertility Preservation and Maintenance of Ministry of Education, Ningxia Medical University, Yinchuan, 750004, China; Institute of Reproductive Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China; Hunan Provincial Key Laboratory of Regional Hereditary Birth Defects Prevention and Control, Changsha Hospital for Maternal & Child Health Care Affiliated to Hunan Normal University, Changsha, 410007, China.
Background:
Sperm DNA damage is an important factor associated with male infertility, miscarriage, and adverse health outcomes in offspring. Current detection methods face challenges such as inaccurate assessments, limited evaluation indicators, and poor reproducibility, largely due to design flaws and constraints in measurable parameters. Therefore, there is an urgent need to develop more reliable detection technologies and to establish more precise and comprehensive evaluation standards to address this critical issue.
Results:
This study developed an innovative biosensor based on terminal deoxynucleotidyl transferase (TdT) and strand displacement (SD) probes, establishing a method for the simultaneous detection of two key biomarkers of sperm DNA damage: DNA breakpoints and apurinic/apyrimidinic (AP) sites. By incorporating standardized reference controls, the assay significantly improved detection accuracy and reproducibility, thereby enhancing clinical assessment efficacy. The method was applied to analyze sperm samples from 40 men with normal semen parameters, 50 asthenozoospermia patients, 48 fertile individuals, and 44 subjects with recurrent pregnancy loss (RPL). Receiver operating characteristic (ROC) curve analysis demonstrated that the combined detection of the mean number of sperm DNA breakpoints (MDB) and AP sites significantly outperformed individual assessments of MDB, DNA fragmentation index (DFI), or AP alone in predicting RPL, exhibiting superior sensitivity and specificity.
Significance:
This advanced technology enables a more comprehensive and precise quantification of sperm DNA damage, overcoming the limitations of traditional methods that have singular assessment indicators, poor effectiveness, and low reproducibility. In addition to demonstrating better clinical relevance in the diagnosis of recurrent pregnancy loss (RPL) and asthenozoospermia, this approach also provides a powerful tool for research in male infertility and holds significant promise for clinical applications.
Insights
A new biosensor accurately detects sperm DNA damage, including DNA breakpoints and AP sites. This method improves diagnosis for male infertility and recurrent pregnancy loss, outperforming older techniques.
Area of Science:
- Reproductive biology and genetics
- Biomedical engineering
- Clinical diagnostics
Background:
- Sperm DNA damage is linked to male infertility, miscarriage, and offspring health issues.
- Existing detection methods lack accuracy, comprehensive indicators, and reproducibility.
- There's a critical need for advanced detection technologies and precise evaluation standards.
Purpose of the Study:
- To develop an innovative biosensor for simultaneous detection of sperm DNA damage biomarkers.
- To improve the accuracy and reproducibility of sperm DNA damage assessment.
- To enhance clinical efficacy in diagnosing male reproductive health issues.
Main Methods:
- Developed a biosensor utilizing terminal deoxynucleotidyl transferase (TdT) and strand displacement (SD) probes.
- Enabled simultaneous detection of DNA breakpoints and apurinic/apyrimidinic (AP) sites.
- Incorporated standardized reference controls to enhance assay accuracy and reproducibility.
Main Results:
- The novel assay accurately quantified sperm DNA breakpoints and AP sites simultaneously.
- Applied to analyze sperm samples from diverse patient groups, including asthenozoospermia and recurrent pregnancy loss (RPL).
- Combined detection of DNA breakpoints and AP sites showed superior sensitivity and specificity in predicting RPL compared to individual markers.
Conclusions:
- The advanced technology provides comprehensive and precise quantification of sperm DNA damage.
- Demonstrated superior clinical relevance for diagnosing RPL and asthenozoospermia.
- Offers a powerful tool for male infertility research and holds significant clinical application promise.
