Towards advanced in vitro models of testicular steroidogenesis for endocrine disruption testing

Male reproductive health has declined over recent decades, characterized by rising rates of reproductive birth defects, reduced semen quality, and an increased risk of testicular cancer. This global trend has been linked to exposure to endocrine-disrupting chemicals (EDCs), which can interfere with the endocrine system including disrupting steroidogenesis, the process by which steroid hormones are synthesized. Steroid hormones, not least testosterone, are essential for male sex differentiation, masculinization, and spermatogenesis. During human fetal development, Leydig and Sertoli cells of the testis together produce androgens, whereas in adults, Leydig cells can synthesize testosterone alone. EDCs can disrupt this process through various mechanisms. Advances in in vitro models of the steroidogenic system, particularly three-dimensional (3D) cell cultures, have provided a more accurate representation of the in vivo environment, enabling more realistic responses to external stimuli. These models offer a powerful tool for studying the effects of EDCs on steroidogenesis and will deepen our understanding of the mechanisms underlying endocrine disruption, knowledge that is important in developing new strategies for chemical safety assessment. This review provides a comprehensive analysis of the steroidogenic system, with a focus on key enzymes involved. The current regulatory test strategies to reduce the impact of EDCs on male fertility is discussed. Furthermore, 3D testis models are compared, highlighting their strengths and limitations for testing effects on testis steroidogenesis. Emphasis is placed on the urgent need for innovative, practical, and cost-effective alternatives to traditional in vivo testing to improve chemical risk assessments.