L26/P-022 High-resolution analysis of sperm mitochondrial DNA integrity using droplet digital PCR

Abstract

Study question How can sperm mitochondrial DNA integrity be assessed with high sensitivity and quantitative resolution? Summary answer A ddPCR-based tetraplex approach enables sensitive and quantitative assessment of mitochondrial DNA fragmentation and copy number in sperm with high analytical resolution. What is known already Sperm DNA integrity is a key determinant of sperm quality and reproductive potential. While nuclear DNA fragmentation is widely investigated and used in clinical practice, mitochondrial DNA (mtDNA) integrity remains comparatively less explored despite its higher susceptibility to oxidative stress. Previous studies have reported an association between nuclear and mitochondrial DNA damage, suggesting shared mechanisms underlying sperm dysfunction. In parallel, microfluidic sperm selection technologies are increasingly adopted in reproductive laboratories to better reproduce physiological conditions, highlighting the need for sensitive analytical methods to detect differences in sperm functional parameters under different experimental conditions. Study design, size, duration This laboratory-based experimental study was conducted on paired semen samples obtained from 35 men over a one-year period. All samples were analysed within the same experimental workflow to allow intra-sample comparison. Participants/materials, setting, methods Semen samples were processed using swim-up and microfluidic sperm selection techniques. DNA extracted from paired sample fractions was analysed using a tetraplex droplet digital PCR (ddPCR) assay combining three mitochondrial targets to enhance the sensitivity of mitochondrial DNA fragmentation detection and one nuclear target to enable accurate estimation of mitochondrial DNA copy number per sperm cell. Main results and the role of chance The ddPCR tetraplex assay successfully quantified mitochondrial DNA fragmentation and the mean mitochondrial DNA copy number per spermatozoon simultaneously in all analysed samples, demonstrating high analytical sensitivity and reproducibility. Paired analyses revealed significantly higher mitochondrial DNA fragmentation in sperm fractions obtained after swim-up compared with microfluidic sperm selection (p = 1.91 × 10−-6). Conversely, the mean mitochondrial DNA copy number was significantly higher in sperm fractions selected by microfluidics (p = 3.15 × 10−⁵). These differences were consistently observed across paired samples, indicating that the findings are unlikely to be attributable to random variation. Overall, the results demonstrate the ability of ddPCR to simultaneously assess mitochondrial DNA fragmentation and copy number through an integrated multiparametric approach. Limitations, reasons for caution The study is limited by the absence of direct clinical outcome correlations and by the moderate sample size. Further investigations are currently in progress to confirm the translational relevance of mitochondrial DNA integrity assessment and to better characterise sperm functional features through combined evaluation of mt-DNA fragmentation and copy number. Wider implications of the findings High-resolution assessment of sperm mitochondrial DNA integrity by ddPCR may represent a valuable tool for advanced functional sperm evaluation and support the development of improved diagnostic and selection strategies in male fertility research. Trial registration number No