The second-to-fourth digit (2D:4D) ratio is thought to be associated with prenatal androgen exposure. However, the relationship between the 2D:4D ratio and hypospadias is poorly understood, and its molecular mechanism is not clear. In this study, by analyzing the hand digit length of 142 boys with hypospadias (23 distal, 68 middle, and 51 proximal) and 196 controls enrolled in Shanghai Children's Hospital (Shanghai, China) from December 2020 to December 2021, we found that the 2D:4D ratio was significantly increased in boys with hypospadias (P < 0.001) and it was positively correlated with the severity of the hypospadias. This was further verified by the comparison of control mice and prenatal low testosterone mice model obtained by knocking out the risk gene (dynein axonemal heavy chain 8 [DNAH8]) associated with hypospadias. Furthermore, the discrepancy was mainly caused by a shift in 4D. Proteomic characterization of a mouse model validated that low testosterone levels during pregnancy can impair the growth and development of 4D. Comprehensive mechanistic explorations revealed that during the androgen-sensitive window, the downregulation of the androgen receptor (AR) caused by low testosterone levels, as well as the suppressed expression of chondrocyte proliferation-related genes such as Wnt family member 5a (Wnt5a), Wnt5b, Smad family member 2 (Smad2), and Smad3; mitochondrial function-related genes in cartilage such as AMP-activated protein kinase (AMPK) and nuclear respiratory factor 1 (Nrf-1); and vascular development-related genes such as myosin light chain (MLC), notch receptor 3 (Notch3), and sphingosine kinase 1 (Sphk1), are responsible for the limitation of 4D growth, which results in a higher 2D:4D ratio in boys with hypospadias via decreased endochondral ossification. This study indicates that the ratio of 2D:4D is a risk marker of hypospadias and provides a potential molecular mechanism.
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