Men with oligozoospermia, or low sperm counts, and high levels of reactive oxygen species, known as ROS, showed extensive differences in sperm DNA methylation in a small human study. The changes were predominantly hypomethylated, meaning the DNA carried less of this chemical marking than the comparison samples. The authors describe the pattern as an association, not proof that ROS caused it. The study also did not test whether the methylation differences affect embryo development or offspring health.
The result came from a narrow sample
The researchers measured ROS in 50 normozoospermic samples and 50 oligozoospermic samples. They then selected eight samples for genome-wide methylation profiling: two low-ROS and two high-ROS samples from each semen group. The analysis compared high-ROS samples with low-ROS samples within each group, as well as the two semen groups overall.
Participants were aged 25 to 45. Inclusion of the patient group required primary infertility lasting more than one year, while normozoospermic men had normal semen parameters under the study's stated 2021 criteria. The eight profiled samples were split evenly between the two semen groups. Mean age was comparable, but sperm count, concentration and motility were lower in the oligozoospermic group.
The test used two ROS bands
ROS was quantified with flow cytometry using H2DCFDA/DCF conversion. Samples with a mean ROS value of 500 or less were classified as low ROS, while those with a mean value of 1,000 or more were classified as high ROS. These cutoffs were specific to the study; there is no universally accepted intracellular ROS threshold established by the analysis.
The team prepared DNA libraries with an enzymatic methyl-sequencing kit, pooled them and sequenced them on a NovaSeq 6,000 instrument using paired-end reads. It then aligned the sequences and searched for methylation differences across the genome. DNA sites called CpGs were excluded when they had fewer than five sequencing reads. The analysis required at least a 15% methylation difference and an FDR-adjusted p-value below 0.05, a threshold used to limit false positives when many sites are tested.
Why the finding remains provisional
The study was observational and cross-sectional, so it measured ROS and methylation at one point rather than changing ROS experimentally and following the samples over time. With only two profiled samples in each ROS and semen subgroup, the findings are vulnerable to chance and need replication in larger, independent groups. The normozoospermic men were described as presumptively fertile, not as a group whose fertility had been formally established.
The molecular results also do not show what the methylation differences mean for reproduction. The study did not measure gene expression, embryo development or long-term offspring health. Its gene-ontology analysis, which groups genes by related biological roles, is not direct evidence of those outcomes. Nor does the study demonstrate clinical benefit from ROS screening, antioxidant treatment or changes to assisted reproduction.
Laboratory checks
The sequencing libraries were processed in one batch. As a quality check, CHH methylation ranged from 0.4% to 0.7%, corresponding to reported conversion efficiency of 99.3% to 99.6%. One sample showed hypermethylation at two of the 14 DLK1 sites examined. These checks support the technical quality of the sequencing, but they do not remove the uncertainty created by the small sample.
What needs testing next
The authors say larger studies should test whether the pattern is reproducible and whether clinically useful ROS thresholds can be established. Future work would also need to determine whether reducing ROS changes sperm methylation and whether either measure is linked to reproductive outcomes. For now, the study offers a molecular association for further investigation, not a clinical recommendation.
Paper data and sources
Original title: Oligozoospermic infertile sperm with high ROS are more prone to epigenetic alterations
Authors: Kumari A, Waseem Andrabi S, Singh R
Journal/Repository: Frontiers in reproductive health
Status: Peer-reviewed
First online: 2026-10-08
DOI: Not available
Original paper · Full text