4In human opposite-sex twins, certain phenotypic traits of the female are affected 2 5 negatively by testosterone transfer from the male, while the male may or may not be 2 6 affected by the female in utero. However, no study was carried out to uncover the 2 7 epigenetic basis of these effects. Here, we generated DNA methylation data from 54 2 8 newborn twins and histone modification data from 14 newborn twins, including 2 9 female-female (FF), female-male (FM), and male-male (MM) newborn twins, to 3 0 exclude the effects of postnatal environment and socialization, and investigated the 3 1 epigenetic consequences of prenatal interactions between female and male gonadal 3 2hormones. We found that FM-Fs (female in FM twins) were distinguishable from FF 3 3 twins by their DNA methylome, as were FM-Ms (male in FM twins) from MM twins.3 4The correlation between genome-wide DNA methylation of females and males 3 5 showed that FM-Fs, but not FFs, were closer to males from FM-Ms and MMs.
6Interestingly, the DNA methylomic differences between FM-Fs and FFs, but not those 3 7 between FM-Ms and MMs, were linked to cognition and the nervous system.
8Meanwhile, FM-Ms and MMs, but not FM-Fs and FFs, displayed differential histone 3 9 modification of H3K4me3, which were linked to immune responses. These findings 4 0 provide epigenetic evidence for the twin testosterone transfer hypothesis and explain 4 1 how prenatal hormone exposure is linked to reported and novel traits of childhood and 4 2 adult through the epigenome in opposite-sex twins. 4 3 4 4 3 Author Summary 4 5 Prenatal exposure to testosterone may affect physiological, cognitive, and 4 6 behavioral traits in females with male co-twins, while the males in opposite-sex twins 4 7 present weak and inconsistent influences. In this study, we systematically investigated 4 8 the hormonal interactions between opposite-sex twins in newborns from epigenetics 4 9 7 1 breaking rules[11], having poorer performance in high school and college, being less 7 2 likely to marry, having lower fertility, and earning less[12]. Female OS twins as 7 3 children, but not as adults, also have larger total brain and cerebellum volumes[13]. 7 4 Male OS twins, however, show weak and inconsistent evidence of behavioral and 7 5 biological changes[12, 14, 15]. 7 6Although previous research has reported that a female exposed to prenatal 7 7 testosterone from her brother exhibits specific behavioral and cognitive traits, the 7 8 mechanism is still unclear, especially for epigenetic mechanisms. Animal studies have 7 9shown that testosterone triggers a DNA demethylation event in mouse embryonic 8 0 neural stem cells and can also affect the global acetylation pattern of histone H3[16]. 8 1 Cisternas et al[17] found that inhibiting DNA methylation in neonatal mice disrupted 8 2 the testosterone-dependent masculinization of neurochemical phenotypes. Besides, 8 3 human germline cells undergo global DNA demethylation from 7 to 19 weeks, 8 4 coincident with testosterone production[18].8 5To study the underlyin...