Epigenetic dynamics of human spermatogenesis and their dysregulation in non-obstructive azoospermia

Spermatogenesis is an intricately regulated epigenetic process, yet the exact chromatin dynamics during human meiosis and their systemic failure in non-obstructive azoospermia remain elusive due to the sparsity of single-cell epigenomic data in previous studies. Here, leveraging a high-quality scATAC-seq approach, we mapped over 810,000 cis-regulatory elements across human spermatogenesis. We uncovered a highly synchronized, wave-like activation of master transcription factor networks that precisely orchestrates meiotic progression. Crucially, we traced the dynamic process of opening and closing of chromatin states at meiotic DNA double-strand break hotspot regions at single-cell resolution, defining a transient epigenetic window where thousands of these predefined regions become specifically accessible. In NOA patients, this delicate architecture collapses, leading to severe spermatogenic arrest predominantly at the zygotene stage. We characterize this pathogenesis as the temporal dephasing and decoupling of the activities of master TFs. Furthermore, aberrantly reduced chromatin accessibility at DSB hotspot regions in NOA is potentially associated with defective DSB formation, accompanied by a two- to three-fold increase in sperm aneuploidy. Finally, we establish a functional paradigm for interpreting male infertility by linking non-coding GWAS variants (e.g., the MHC II locus) and rare point mutations to the disruption of cis-regulatory elements. Together, our study provides an unprecedented epigenetic roadmap of human spermatogenesis and redefines NOA pathogenesis from descriptive phenotypes to precise, network-level regulatory failures.