Busulfan plus cyclophosphamide induced spermatogenic dysfunction and recovery: A dynamic change perspective.

Busulfan combined with cyclophosphamide (BuCy) is a common conditioning regimen before hematopoietic stem cell transplantation, but it causes severe gonadotoxicity, with nearly half of male patients suffering from irreversible infertility. Existing mouse models mostly use busulfan alone, which does not fully mimic clinical treatment. Here, we applied a BuCy treatment regimen in mice and performed a longitudinal characterization of testicular injury and recovery. BuCy treatment caused marked testicular atrophy in mice, severely disrupted seminiferous tubule architecture, and dramatically reduced sperm count and motility, with partial recovery at later time points. ScRNA-seq revealed a stepwise decline in germ cell populations, with spermatogonia disappearing earlier than spermatogonial stem cells (SSCs). In addition, we observed the highest number of differentially expressed genes (DEGs) at day 28. Functional enrichment highlighted disruptions in spermatogenesis, RNA metabolism, and chromatin regulation. This study systematically characterized the long-term, multi-time-point dynamics of BuCy-induced testicular damage and recovery in mice, with single-cell transcriptomic profiling providing complementary observations at the cellular level.