Nanopore long-read sequencing refines STS-defined AZFb deletions and provides spermatogenesis assessment

Deletions in the azoospermia factor b (AZFb) region of the Y chromosome contribute to male infertility. However, the clinical significance of different deletion architectures remains insufficiently resolved by conventional sequence-tagged site (STS) analysis. In this study, four men carrying AZFb deletions were analyzed by multiplex polymerase chain reaction (PCR), Nanopore long-read sequencing, and modified multiplex ligation-dependent probe amplification (MLPA). Nanopore long-read sequencing enabled high-resolution mapping of deletion boundaries and comprehensive characterisation of coding gene loss across the region. Distinct deletion architectures involving RBMY, EIF1AY, HSFY, KDM5D, PRY and RPS4Y2 were identified and showed potentially different associations with spermatogenic phenotypes. One case carried a complete deletion and exhibited non-obstructive azoospermia (NOA). Two cases with partial deletions retained subsets of coding genes and exhibited residual spermatogenesis, enabling successful intracytoplasmic sperm injection (ICSI) outcomes. Notably, one deletion initially classified as proximal partial deletion by STS analysis was shown by Nanopore long-read sequencing to remove all annotated coding genes, and was associated with NOA and failed testicular sperm retrieval. These results demonstrate that Nanopore long-read sequencing refines STS-defined AZFb deletions through delineation of deletion intervals and gene content, may provide an improved assessment of spermatogenesis in male infertility.