In a recently published study, Dr Scientific report, researchers developed a rapid diagnostic method for double-strand breaks (DSBs), sperm deoxyribonucleic acid (DNA) fragmentation (SDF) and releasing assay (SDFR). They also explored the link between sperm DNA double-stranded breaks (DSBs) and reproductive outcomes.
Study: Innovative technology to assess sperm DNA double-strand breaks diagnoses male factor infertility and prevents reproductive failure. Image credit: Shidlovski/Shutterstock.com
Background
Millions of couples worldwide are affected by infertility, with male factors responsible for a significant number of instances. In rare circumstances, sperm tests such as volume, concentration, motility and morphology may fail to detect abnormalities.
Sperm DNA DSBs are a diagnostic feature in idiopathic infertility and associated with reproductive failure.
The unbiased comet test, which correlates with DSBs in clinical settings, has been used to test for DSBs; However, due to its technical sensitivity and the absence of a consistent grading system, its clinical applicability is limited. Further research is needed to enhance its clinical use.
About the study
In the current prospective cohort study, researchers designed a novel technology to assess DSB, R11 rapid test based on unique molecular characteristics. They verified its specificity, sensitivity and reliability and used it to assess the link between DSBs and reproductive outcomes.
The study included 640 infertile men aged 20 to 60 who sought fertility therapy at Li Women’s Hospital in Taiwan between May 2020 and December 2021. Subjects with retrograde ejaculation or azoospermia were excluded.
Participants masturbated to provide sperm samples, which were tested for sperm concentration, progressive motility, and total motility.
Sperm chromatin dispersion assays using a bright-field microscope were used to determine the total DNA fragmentation index (DFI). A moderate to large halo indicates intact sperm DNA, while a small to negligible halo indicates fragmented DNA.
Sperm DNA was described as having a DSB in the neutral comet assay if the DNA tail was 40 m or longer and the deoxyribonucleic acid tail fraction was ≥20% above the total signal.
Samples were treated at 37 °C with 5.0 U/ml, 10 U/ml, 20 U/ml, and 40 U/ml deoxyribonuclease I (DNase I) and 5.0 U/ml, 15 U/ml, 25 U/ml. ml. ml, and 50 U/mL of Alu I endonucleases, which are routinely used to model DSBs.
To induce oxidative stress and DNA strand breaks, sperm samples were treated with 0.030%, 0.150% and 0.300% hydrogen peroxide (H2And2)
Further, researchers conducted a retrospective investigation of 140 couples with at least two embryos undergoing preimplantation genetic testing for aneuploidy (PGT-A). Based on the consensus of the global genetic testing community, fetal aneuploidy was defined as mosaicism levels greater than 80%; Biopsied blastocyst counts determine the rate of aneuploidy.
result
The modified PA network enables trapping of DSBs, which distribute a halo on an immunological slide; On the other hand, damaged chromatin did not form a halo. A significant correlation was found between reproducible SDFR and neutral comet assay readings.
SDFR responded to dose/time-dependent simulated DSBs with excellent specificity and sensitivity. DSB predicted fetal aneuploidy, whereas early sperm characteristics did not.
DSB DFI did not vary significantly between the R11 assay (mean, 11) and the Comet assay (mean, 10). Bland-Atman plot analysis revealed that the two tests were in good agreement.
R11 DFI increased when treatment duration and dose were increased using Alu I and DNase I treatment in a dose- and time-dependent manner. However, later H2And2 treatment, the R11 DNA fragmentation index was unchanged, demonstrating that R11 can selectively detect sperm DSBs.
R11 DFI had significant negative correlations with normal morphology and total mobility and showed significant correlation with progressive mobility. DFI values for R11 were significantly positively associated with male age.
Among sperm characteristics, sperm density, 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 the prevalence of aneuploidy (a 50% or higher aneuploidy rate).
Given that maternal age (AUC of 0.7) and paternal age (AUC of 0.7) were similarly associated with fetal aneuploidy, the researchers performed adjusted modeling excluding women older than 38 years, accounting for confounding variables.
Compared to other sperm quality indices, R11 exhibited the highest AUC in predicting aneuploidy (0.7). The R11 cutoff was greater than 8.0% and the specificity and sensitivity were their highest.
Conclusion
Overall, the study results highlighted the design of the R11 sperm DSB assay, which uses polyacrylamide to capture DSBs, providing a faster technique compared to neutral comet assays.
R11 reduces the subjectivity of DNA fragmentation index interpretation by providing a consistent binary halo categorization for double-stranded breaks and intact DNA.
Image-based data can be combined with an artificial intelligence-integrated automated system, making DSB assessment in the laboratory more efficient, uniform and controllable.
R11 was significantly associated with morphology and total and progressive mobility in 640 men. Researchers have established the predictive ability of the R11 assay to detect fetal aneuploidy at the preimplantation stage, establishing it as a valuable tool to assess paternity risk determinants and minimize the effects of DSBs.
Further clinical trials with larger sample sizes are needed to determine the association between R11 and ART failure.