Reduced eccDNA content in idiopathic asthenozoospermia sperm is associated with compromised DNA repair capacity and elevated DNA damage.

BACKGROUND: Despite representing approximately 30% of male infertility cases, idiopathic asthenozoospermia (iAZS) remains etiologically uncharacterized. Extrachromosomal circular DNA (eccDNA) is mobile and circular DNA outside of linear chromosomes. Although eccDNA has been identified in human sperm, its biogenesis and potential role in iAZS pathogenesis requires further study. METHODS: We enrolled 31 patients with idiopathic asthenozoospermia (iAZS) and 31 healthy controls (normozoospermia, NZS), collecting sperm samples with progressive motility (PR) ranging from 0.3% to 90.9%. Sperm eccDNAs were purified and characterized using Circle-seq. Outward PCR, Sanger sequencing, and Nanopore long-read sequencing were employed to investigate eccDNA biogenesis and its potential genomic effects. In addition, spermatozoa RNA sequencing (RNA-seq) and immunofluorescence staining were conducted to identify DNA repair-related candidate genes. A CRISPR/Cas9-mediated APLF knockout model was established to explore DNA repair mechanisms in eccDNA formation in vitro. RESULTS: Comprehensive analysis of eccDNAs derived from the sperm samples revealed a significant positive correlation between sperm motility and eccDNA abundance. Larger eccDNAs (≥ 3 kb) showed inverse associations with meiotic recombination rates and coding gene density, while smaller eccDNAs lacked these trends. Although eccDNA formation broadly aligned with transposable element (TE) densities, larger eccDNAs (≥ 3 kb) were negatively correlated with short interspersed nuclear elements (SINEs) (mainly Alu elements). Microhomology-mediated end joining (MMEJ) likely drove eccDNA biogenesis, as 58% of eccDNAs harbored 3 bp direct repeat (DR) pairs. Nanopore and variant analyses suggested eccDNAs may arise from genomic deletions and later reintegrate. Importantly, sperm motility and eccDNA abundance correlated positively with DNA repair capacity but negatively with DNA damage. Finally, we identified APLF, a downregulated DNA repair protein in low-motility sperm, as a key regulator of eccDNA formation in vitro. CONCLUSIONS: These findings emphasize the potential interplay between genomic elements and sperm eccDNA formation, highlight that impaired DNA repair and elevated DNA damage level may be major causes of reduced sperm motility and pathogenesis of iAZS, and offer new insights into strategies for improving male fertility.