Advancements in semen sample analysis: evaluating the efficacy and progress in human DNA removal techniques

Microorganism types and quantities in male semen correlate with male semen parameters and male infertility. There has been a growing body of evidence suggesting that identifying microorganisms in male semen through metagenomic sequencing is useful for assessing male fertility and planning appropriate treatment interventions. Nevertheless, non-targeted metagenomic sequencing of semen samples is challenging due to the large number of sperm cells and high amounts of intracellular and extracellular human DNA, which can obscure microbial DNA. In this study, we assessed and refined three methods for DNA extraction from semen. Using low-speed centrifugation, propidium monoazide (PMA) photochemical induction, and the HostZERO Microbial DNA Kit, metagenomic sequencing was performed to detect and evaluate its efficacy. Our findings indicate that low-speed centrifugation effectively reduces sperm and sperm-associated DNA but may inadvertently remove microbial DNA. Increasing sample quantities is a potential strategy for enhancing the microbial DNA ratio in semen samples. PMA and the HostZERO Microbial DNA Kit utilization and optimization show promise in reducing total semen DNA and augmenting microbial DNA representation. Preliminary results suggest that traditional disinfection methods during semen sample collection do not increase microbial contamination significantly. In particular, due to the limited sample size, further investigation with a larger cohort is necessary for conclusive validation of our findings.