Effects of Midazolam on the Development of Adult Leydig Cells From Stem Cells In Vitro

Xingyi Zhao, Minpeng Ji, Xin Wen, Dan Chen, Fu Huang, Xiaoju Guan, Jing Tian, Jiajia Xie, Jingjing Shao, Jiexia Wang, Luoqi Huang, Han Lin, Leping Ye, Haolin Chen

Research output: Contribution to journalArticlepeer-review


Background: Midazolam is a neurological drug with diverse functions, including sedation, hypnosis, decreased anxiety, anterograde amnesia, brain-mediated muscle relaxation, and anticonvulsant activity. Since it is frequently used in children and adolescents for extended periods of time, there is a risk that it may affect their pubertal development. Here, we report a potential effect of the drug on the development of Leydig cells (LCs), the testosterone (T)-producing cells in the testis. Methods: Stem LCs (SLCs), isolated from adult rat testes by a magnetic-activated cell sorting technique, were induced to differentiate into LCs in vitro for 3 weeks. Midazolam (0.1–30 μM) was added to the culture medium, and the effects on LC development were assayed. Results: Midazolam has dose-dependent effects on SLC differentiation. At low concentrations (0.1–5 μM), the drug can mildly increase SLC differentiation (increased T production), while at higher concentrations (15–30 μM), it inhibits LC development (decreased T production). T increases at lower levels may be due to upregulations of scavenger receptor class b Member 1 (SCARB1) and cytochrome P450 17A1 (CYP17A1), while T reductions at higher levels of midazolam could be due to changes in multiple steroidogenic proteins. The uneven changes in steroidogenic pathway proteins, especially reductions in CYP17A1 at high midazolam levels, also result in an accumulation of progesterone. In addition to changes in T, increases in progesterone could have additional impacts on male reproduction. The loss in steroidogenic proteins at high midazolam levels may be mediated in part by the inactivation of protein kinase B/cAMP response element-binding protein (AKT/CREB) signaling pathway. Conclusion: Midazolam has the potential to affect adult Leydig cell (ALC) development at concentrations comparable with the blood serum levels in human patients. Further studies are needed to test the effects on human cells.

Original languageEnglish (US)
Article number765251
JournalFrontiers in Endocrinology
StatePublished - Nov 12 2021
Externally publishedYes


  • AKT-CREB signaling
  • midazolam
  • progesterone
  • stem Leydig cell
  • steroidogenesis
  • testosterone

ASJC Scopus subject areas

  • Endocrinology, Diabetes and Metabolism


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