Intermittent Exposure to High Ambient Heat during the Second Half of Gestation in Mice Causes Mild Alterations of Reproductive End Points in Male Embryos

Abstract Periods of elevated ambient temperature challenge the body’s ability to maintain internal homeostasis, and heat stress poses particular risks during pregnancy. Epidemiological studies associate gestational heat exposure with higher rates of congenital anomalies such as hypospadias, yet the direct link between gestational heat exposure and reproductive anomalies remains unknown. In this study, we examined the effects of intermittent heat exposure on reproductive development in male mouse offspring. Pregnant C57BL/6J dams either remained at a constant temperature of 22 °C (control) or were exposed to 38 °C for 2 h daily (experimental) from embryonic day (E)10 to E18, modeling intermittent heat exposure during mid-to-late gestation. A total of 10 experimental and 6 control dams were analyzed, and all embryos were collected at E18.5 for analysis. While heat exposure did not affect pregnancy outcomes, including placental development, litter size, sex ratio, or fetal growth, male embryos exhibited significantly reduced anogenital distance and increased hypospadias scores, both of which are markers of disrupted androgen signaling. Despite these phenotypic changes, expression of genes involved in androgen synthesis in the fetal testis, as well as gene expression in external genitalia, remained unchanged. Instead, transcriptomic analysis revealed significant alterations in testicular pathways related to RNA splicing and mRNA processing. Together, these findings reveal that maternal heat stress disrupts reproductive development of male offspring, with altered gene regulatory processes being a potential driver.