In a recent study published in Scientific Reports, researchers developed a rapid diagnostic method for double-strand breaks (DSBs), Sperm deoxyribonucleic acid (DNA) Fragmentation (SDF) Releasing Assay (SDFR). Additionally they explored the association between sperm DNA double-stranded breaks (DSBs) and reproductive outcomes.
Study: Modern technology for evaluation of sperm DNA double-strand breaks diagnoses male factor infertility and prevents reproductive failures. Image Credit: Shidlovski/Shutterstock.com
Tens of millions of couples worldwide are affected by infertility, with male aspects accounting for a substantial variety of instances. In rare circumstances, sperm examinations similar to volume, concentration, motility, and morphology may fail to detect abnormalities.
Sperm DNA DSBs are a diagnostic feature in instances of idiopathic infertility and are related to reproductive failures.
The neutral comet test, which correlates with DSBs in clinical settings, has been used to look at DSBs; nonetheless, on account of its technique sensitivity and absence of a consistent grading system, its clinical applicability is restricted. Further research is required to extend its clinical use.
In regards to the study
In the current prospective cohort study, researchers designed a novel technology to guage DSBs, the R11 rapid assay, based on unique molecular features. They validated its specificity, sensitivity, and reliability and used it to evaluate the link between DSBs and reproductive outcomes.
The study included 640 infertile males aged 20 to 60 years who sought assisted reproductive therapy at Lee Women’s Hospital in Taiwan between May 2020 and December 2021. Individuals with retrograde ejaculation or azoospermia were excluded.
The participants masturbated to offer sperm samples, which were examined for sperm concentration, progressive motility, and total motility.
Sperm Chromatin Dispersion experiments utilizing bright-field microscopes were used to find out the overall DNA fragmentation index (DFI). A medium to large halo denoted intact sperm DNA, whereas a small to negligible halo denoted fragmented DNA.
Sperm DNA was described to have DSBs within the neutral comet assay if the DNA tail was 40 m or longer and had ≥20% fraction of the deoxyribonucleic acid tail over the complete signal.
The samples were treated at 37 °C with 5.0 U/ml, 10 U/ml, 20 U/ml, and 40 U/ml of deoxyribonuclease I (DNase I) and 5.0 U/ml, 15 U/ml, 25 U/ml, and 50 U/mL of Alu I endonucleases, that are routinely used to model DSBs.
To provide oxidative stress and cause DNA strand breakage, the sperm samples were treated with 0.030%, 0.150%, and 0.300% hydrogen peroxide (H2O2).
Further, the researchers conducted a retrospective investigation of 140 couples with not less than two embryos under preimplantation genetic testing for aneuploidy (PGT-A). Based on the worldwide genetic testing community consensus, embryonic aneuploidy was determined based on mosaicism levels exceeding 80%; biopsied blastocyst counts ascertained the aneuploidy rates.
The modified PA network enabled the trapping of DSBs, which distributed a halo on an immunological slide; undamaged chromatin, alternatively, didn’t form a halo. A big association was found between reproducible SDFR and the neutral comet assay readings.
SDFR responded to dose/time-dependent simulated DSBs with great specificity and sensitivity. DSB predicted embryonic aneuploidy, although basic semen characteristics didn’t.
The DSB DFI didn’t change significantly between the R11 test (mean, 11) and the comet assay (mean, 10). The Bland-Atman plot evaluation revealed that the 2 tests were in good agreement.
R11 DFI rose when treatment duration and dosages were increased using dose- and time-dependent Alu I and DNase I treatment. Nevertheless, after H2O2 treatment, the R11 DNA fragmentation index remained unaltered, demonstrating that R11 could selectively discover sperm DSBs.
R11 DFI had significant negative relationships with normal morphology and total motility and showed substantial associations with progressive motility. The DFI values for R11 were significantly positively linked with the male’s age.
Among the many sperm characteristics, sperm concentration, normal morphology, total motility, R10 (total DNA fragmentation index), and R11 (double-stranded break DNA fragmentation index), R11 [area under the receiver-operating characteristic curve (AUC), 0.7] was shown to predict aneuploidy prevalence (a 50% or higher aneuploidy rate).
On condition that maternal age (AUC of 0.7) and paternal age (AUC of 0.7) were likewise linked to embryonic aneuploidy, the researchers performed adjusted modeling excluding females above 38 years, accounting for confounding variables.
In comparison with the opposite semen quality indicators, R11 exhibited the very best AUC in aneuploidy prediction (0.7). The R11 cutoff was higher than 8.0%, and the specificity and sensitivity were at their best.
Overall, the study findings highlighted the design of the R11 sperm DSB assay, which employs polyacrylamide to capture DSBs, providing a faster technique in comparison with neutral comet assays.
R11 reduces the subjectivity of DNA fragmentation index interpretation by providing consistent binary halo categorizations for double-stranded breaks and undamaged DNA.
Image-based data could also be combined with a man-made intelligence-integrated automated system, making DSB assessments more efficient, uniform, and controllable within the laboratory.
R11 was significantly linked with morphology and total and progressive motility in 640 males. The researchers established the predictive ability of the R11 assay in detecting embryonic aneuploidy within the preimplantation stage, establishing it as a beneficial tool to evaluate paternal risk determinants and minimize the consequences of DSBs.
More clinical trials with larger sample sizes are required to find out the link between R11 and ART failures.