In this large sample population, our findings reinforce the observation that boys have a higher prevalence of NAFLD compared to girls, regardless of age and Tanner stage. The gender difference is apparent in pre-puberty and narrows in late puberty. For girls, estradiol and PRL were inversely associated with the prevalence of NAFLD, while in boys, estradiol was positively related to NAFLD prevalence, whereas LH and testosterone had a negative correlation. Principal component analysis identified three important components representing the potential impact of markers of sex hormones and fat distribution, lipid metabolism, and glucose metabolism. Logistic regression analysis reveals the importance of sex hormones and fat distribution as factors associated with the prevalence of fatty liver in both sexes, and puberty may increase the risk of fatty liver.

Although the susceptibility to pediatric NAFLD in boys, as found in our study cohort, has been demonstrated in previous studies [4, 11, 12], our study still provided some new and interesting findings. As a supplement to the results discovered in a meta-analysis based on Asian children, NAFLD is becoming prevalent among obese children, especially boys, after the age of 10 [4]. Furthermore, our study revealed a rapid increase in NAFLD prevalence in children after the age of 6, coinciding with the emergence of sexual differences. Meanwhile, the Healthy Start Study of 286 children aged 4–8 years, which investigated liver fat composition by MRI-H1 in children before pubertal onset, reported that hepatic steatosis might begin in early childhood. They found a positive association between fasting glucose, triglycerides, and liver fat deposition, but only in boys [13]. Together with our results, these findings provide stronger evidence and clinical significance that young boys are more susceptible to NAFLD.

Notably, our study revealed that the prevalence of fatty liver in obese preschool children reached 21–28%, which is much higher than we expected. Severe obesity might be one reason for this high prevalence. It also underscores the importance of genetic metabolic screening for young children with NAFLD since many rare, inherited metabolic disorders can also cause fatty liver in infants and children [14]. Additionally, the gender difference became apparent at around 6 years of age when the sex hormone axis remains inactive. Interestingly, focal development of the zona reticularis (ZR) of the human adrenal cortex begins at around 5 years of age, coinciding with the onset of increased levels of dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS) [15]. We speculate that, rather than testosterone and estradiol, other hormones like DHEA and DHEAS, which are related to the sex of the subject, may influence the prevalence of NAFLD. Some studies have reported a negative association between DHEAS and the severity of NAFLD in both teenagers and adults [16, 17]. Further studies are needed to investigate the relationship between adrenal androgens and NAFLD in pre-pubertal children. Meanwhile, central obesity, insulin resistance, and weight gain are major risk factors for NAFLD [18]. Additionally, other factors related to sex, such as sex-chromosome complement, genetic and epigenetic factors, circadian rhythm, gut microbiota, individual lifestyle, cultural sex bias in parental feeding practices, and societal ideals of body size, might also play a role in the sexual difference in pre-pubertal NAFLD prevalence [5, 19, 20]. Another interesting finding was the peak in the prevalence rate in girls at around 11 years of age. Insulin resistance increased significantly during the pubertal transition, peaked at Tanner stage 3, and returned to pre-pubertal levels at Tanner stage 5 in girls [12, 21]. Insulin resistance causes NAFLD by increasing de novo lipogenesis and increasing FFA flux to the liver [22], which might explain the fluctuation of NAFLD prevalence during the pubertal transition in girls.

Our results regarding the association between sex hormones and NAFLD prevalence were consistent with previous findings in human and animal models [17, 23, 24]. In contrast to males, estradiol serves as a protective factor against liver disease in female mice. Hepatic steatosis is linked to a slight reduction in circulating testosterone levels in male mice [23]. Testosterone levels were positively correlated with improvements in steatosis and fibrosis in boys but displayed an inverse association in girls. Furthermore, higher estradiol levels were positively linked to the severity of hepatic ballooning in both sexes [17]. Estrogen signaling in both sexes is intertwined with lipid metabolism in the liver [19]. Under physiological conditions, it prevents hepatic fat accumulation in females by inhibiting the expression of genes involved in de novo lipogenesis [19]. Furthermore, androgen signaling also regulates hepatic lipid metabolism. In males, the androgen signaling pathway suppresses the genes involved in DNL and lipid storage by up-regulating PPARα (peroxisome proliferator-activated receptor α) signaling to promote fatty acid oxidation; however, it disrupts hepatic lipid metabolism in female mice [19]. These findings can help explain the divergent associations between different sex hormones and NAFLD. Zhang et al. [25], reported that hepatic lipid accumulation can be improved by PRL/prolactin receptor via the CD36 pathway. Another study by Shao et al. [26] found that hepatic TG accumulation could be reduced in female mice, and liver steatosis could be alleviated in male mice when given PRL in a high-fat diet-induced NAFLD model. Considering that a decrease in PRL has been observed in NAFLD patients of both genders [24, 27], whether there are sexual differences between serum PRL and NAFLD remains unknown.

During this study, we observed a weak correlation between sex hormones and ALT (Supplementary Table 3). There was a strong association between NAFLD and parameters related to body fat distribution and sex hormones. Denzer et al. [11] also arrived at the same conclusion as the one we stated earlier. Waist and hip circumferences serve as indicators of body fat distribution. Gender dimorphism in fat tissue across various body areas is influenced by sex hormones through depot- and time-specific estrogen receptor expression, the active–passive transition of lipid metabolism-related proteins, circulating adipokines, immune response, and more [28, 29]. Males tend to accumulate abdominal and visceral fats, such as Android fat, which are risk factors for NAFLD, while females store white adipose tissues in the subcutaneous and femoral regions, like Gynoid fats [29, 30]. A previous study by our group reported that the Android/Gynoid ratio in pre-pubertal boys, an index for gender-specific abdominal fat distribution, increases before puberty onset [30]. In addition to the effects of sex hormones, differential fat distribution partially explains the early increase in NAFLD prevalence in pre-pubertal boys.

Despite the large sample size of obese children and adolescents with comprehensive clinical examinations of obesity complications, our research has several limitations. Since the study was cross-sectional, it could not dynamically observe the disease progression in different pubertal stages. Furthermore, the sex hormone levels were very low before puberty, with some falling below the detection limits of the assays, which might have introduced certain statistical errors. However, the low sex hormone levels emphasize the intriguing sexual dimorphism of NAFLD before puberty. Additionally, this study population predominantly consisted of Chinese children with moderate to severe obesity who were hospitalized, with the number of boys nearly double that of girls. These factors might lead to selection bias and an under-representation of other racial groups and the general population.

In conclusion, the prevalence of NAFLD is higher in obese children in China, and the sex difference becomes apparent in early childhood, at around 6 years of age, long before sex hormones start to rise. Liver function and liver imaging examinations might be necessary for such young children, especially those who are obese. Sex hormones are related to the severity of NAFLD, and they are influenced by puberty and abdominal fat accumulation. While our study has provided new findings regarding sexual differences in obese children, there are still unanswered questions, such as the sex-specific prevalence of NAFLD in young children, the gender-related effects of NAFLD beyond hormones, and the mechanism of NAFLD sexual dimorphism in kids.