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. Plain language summaryMale reproductive health problems, such as lower sperm quality, birth defects, and testicular cancer, have increased worldwide in recent years. One possible cause is exposure to endocrine-disrupting chemicals (EDCs), which can interfere with hormones like testosterone that are important for male development and fertility. This review explains how the testis produces these hormones and how EDCs may disturb this process. It also discusses current regulatory testing methods used to evaluate EDC effects. Special attention is given to new three-dimensional (3D) testis models currently being developed. These models resemble the human testis better than traditional cell cultures and can improve research on endocrine disruption and chemical safety. Scientifically, these models help researchers better understand how EDCs affect male reproductive health. In line with the 3Rs principle (replacement, reduction, and refinement), they may also reduce the need for animal testing by providing more reliable and cost-effective alternative methods.