Low-intensity pulsed ultrasound (LIPUS) has emerged as a non-invasive therapeutic approach with regenerative potential in various tissues. This study explores its effects on testicular steroidogenesis in male rats. Twenty-four 9-month-old rats were allocated into three groups: control, LIPUS, and LIPUS-M. The LIPUS group underwent pulsed ultrasound therapy targeting the testes twice weekly for 4 weeks, whereas the LIPUS-M group continued with maintenance therapy administered once every 2 weeks for 6 months. At the end of the experiment (16 months), blood and testicular tissues were collected for analysis. Our results showed that LIPUS therapy significantly elevated serum testosterone level and increased the number of Leydig cells (LCs), as indicated by steroidogenic acute regulatory protein (STAR) and 3β-hydroxysteroid dehydrogenase (3β-HSD) immunofluorescence. Collagen IV staining showed increased interstitial vascularization, and senescence-associated β-galactosidase (SA-β-Gal) staining suggested reduced cellular senescence. Although the LIPUS-M group also showed increased LC counts and angiogenesis, serum testosterone levels were not elevated. These findings demonstrate that LIPUS can activate LCs and enhance endogenous testosterone production, supporting its potential as a noninvasive regenerative approach for hypogonadism.

Keywords: hypogonadism; Leydig cells; low-intensity pulsed ultrasound; regenerative therapy; testosterone