Study on the Mechanism of Acetylcarnitine in Improving Sperm Quality in Busulfan-Induced Azoospermia Mice

Background Azoospermia is a severe form of male infertility. This study examined acetyl L-carnitine (ALC)'s therapeutic effects and molecular mechanisms in busulfan-induced mouse azoospermia. Methods Random forest analysis of metabolomics data identified key metabolites in azoospermia patients. Azoospermia was induced in male C57BL/6J mice by intraperitoneal injection of busulfan. Mice were then treated with 0.3% ALC for six consecutive weeks. Sperm concentration, motility, and morphology were assessed. Testicular and epididymal histopathology was evaluated by hematoxylin and eosin staining. ALC levels in testicular tissues were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Transcriptome sequencing was performed to identify differentially expressed genes (DEGs), followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Protein expression of laminin subunit beta 2 (LAMB2) was detected by Western blot. Results Azoospermia patients exhibited profound acyl-CoA metabolic disturbances. ALC is the critical shuttle for transporting acyl-CoA into mitochondria. ALC treatment significantly restored sperm concentration and motility, reduced sperm malformation rates, and improved testicular and epididymal histopathology in busulfan-induced azoospermic mice. Endogenous ALC levels in the testis were markedly decreased by busulfan and recovered after ALC supplementation. Transcriptomic analysis revealed 35 upregulated and 190 downregulated DEGs in the Busulfan + ALC group compared to the Busulfan group. DEGs enriched in different pathways (e.g., ECM-receptor interaction). ALC reversed the busulfan-induced upregulation of LAMB2. Conclusion Acyl-CoA metabolism disorder is a critical metabolic feature in azoospermia. ALC improved busulfan-induced azoospermia in mice, potentially by reversing the upregulation of LAMB2.