The results showed that the height and weight of children in the ICPP group increased significantly compared with healthy children of the same age, indicating that children in the ICPP group developed earlier. The analysis of GM with ICPP group prompt, the abundances of Roseburia, Prevotella, Lachnospiracea incertae sedis, Ruminococcus, Parabacteroides, Alistipes, Fuscatinibacter, Clostridium sensu stricto, and Clostridium XVIII were significantly higher than that of the healthy group. Combined with literature analysis, we know that Roseburia belongs to actinomycetes, is one of the most abundant bacteria producing butyric acid in the gut, can promote butyric acid production by decomposing a variety of carbohydrates into SCFAs (13, 14). Prevotella can decompose carbohydrates into SCFAs and participate in the production of butyrate, which helps to maintain the homeostasis of the gut microenvironment (14, 15). Both Ruminococcus and Lachnospiracea incertae sedis, are the two most abundant families of Clostridium in the phylum Firmicutes, they can decompose a variety of fiber polysaccharides. Metabolites of SCFAs are beneficial to the gut, can also promote protein synthesis (16). Clostridium scindens can produce testosterone (17). Fusobacterium was positively correlated with FSH (18). The abundance of Alistipes is closely related to the frequency of abdominal pain, and it is speculated that it may lead to gut inflammation (19). The increase of the abundance of Parabacteroides can cause the reduction of the synthesis of tight junction protein (tight junction protein-1 and atresia protein), thus leading to the occurrence of "gut leakage" (20). These results suggest that the change of GM in the ICPP group can lead to the increase of SCFAs production, especially butyrate content, and the increase of some microbiota abundance can lead to chronic gut inflammation. Some studies (21, 22) believe that butyrate can directly stimulate the secretion of glucagon-like peptide-1 (GLP-1) by endocrine cells gpr43 or gpr41 in colon and ileum, and the latter can promote insulin secretion. Also found that butyrate can stimulate the increase of nitric oxide (NO) synthesis when the concentration of butyrate is significantly increased (23).
This study confirmed the existence of intestinal dysbacteriosis in children with ICPP. We further analyzed the function of GM and analyzed the correlation between GM and endocrine hormones with the ICPP group. Hence, we speculate that the changes of GM with ICPP and PCOS were related to endocrine hormone disorders in the two diseases. PCOS patients have high calorie and high-fat diet, insulin resistance, hyperandrogenemia, and so on. Combined with our research results, we analyzed ICPP children from these three aspects.
4.1 Dietary structure
Previous investigation (7) suggested that precocious puberty children preferred high-fat and high-calorie diets. This study also found that children in the ICPP group preferred sweet food (high sugar diet) and high-fat food, we know that the high sugar diet belonged to low molecular carbohydrate. Moreover, we found the abundance of bacteria such as Prevotella that carbohydrate metabolizing in GM was significantly increased, and carbohydrate metabolism in GM was significantly enhanced with the ICPP group, so we speculate that a high sugar diet may be an important factor in precocious puberty.
It has been reported (24) that high fat and high sugar diet can induce metabolic disorder and ovarian changes in female rats. Similar clinical manifestations to PCOS patients have been observed. It has been confirmed that low calorie and low-fat diet is an effective therapy to improve metabolism and ovarian function in obese women with PCOS (25).
The negative feedback of hyperandrogenemia caused the decrease of estradiol and progesterone secretion and the increase of GnRH and LH levels (26). The synthesis of androgen in women is closely related to HPGA. The former mainly comes from the androgen secreted by the ovarian tissue. The testosterone and A2 secreted by the ovary account for two-thirds of the circulating androgens (27). The androgen synthesis in the ovary is regulated by LH. Total serum testosterone is the most common and widely used biochemical test index to evaluate androgen excess (28). However, there are many defects in the detection method of testosterone in clinical practice. At present, androgen level is evaluated by A2 in PCOS patients (29). Wang Quanxian et al. (30) believe that A2 expression is closely related to endocrine and metabolic disorders in PCOS patients. In this project, A2 in the ICPP group was significantly higher than that in healthy group, which may be caused by the following factors: 1) According to the investigation, the children in the ICPP group like to eat sweet food (carbohydrate intake is more), and the correlation analysis between GM and hormone level of children also indicates that the metabolism of carbohydrate in children in the ICPP group is significantly higher than that in the healthy group, and high sugar diet can cause an immune response and oxidative stress to produce inflammatory mediators and stimulate ovaries to produce androgen (31). 2) Compared with healthy children, the bacterial abundance of butyric acid-producing bacteria in the ICPP group was significantly increased, butyric acid-induced insulin secretion through GLP-1 (26, 32), insulin enhanced GnRH gene transcription by enhancing mitogen-activated protein kinase pathway, and increased GnRH secretion in the hypothalamus, resulting in high levels of androgen and LH secretion. On the other hand, insulin can improve the utilization of IGF-1 by inhibiting the expression of IGFBP-1 in the ovary and liver. Insulin can also improve IGF-1 activity in the liver and ovary, reduce the level of SHBG in the liver and ovary, and increase free androgen in vivo (26, 33). 3) In the ICPP group, the abundance of butyric acid-producing bacteria such as Roseburia, Prevotella, Lachnospiracea incertae sedis, and Ruminococcus increased, which may lead to the rich energy of intestinal mucosal cells, the secretion of adrenocorticotropic hormone(ACTH) and other hormones in the gut tract, and also affect the HPGA to promote the synthesis of androgen (33). 4) The abundance of Clostridium sensu stricto and Clostridium XVIII in the ICPP group is increased, the latter can convert glucocorticoid into androgen, studies have confirmed that the bacterium can produce testosterone (34, 35). 60% of testosterone in women is converted from its direct precursor A2.
Liu et al. (36) reported that LPS-producing bacteria such as Bacteroides and Escherichia/Shigella increased in PCOS patients. The correlation analysis showed that the above bacteria were positively correlated with androgen level, suggesting that the changes of GM could lead to hyperandrogenism. The female newborn rats were given a large dose of testosterone once, which not only showed similar phenotypes of PCOS, such as weight gain and insulin resistance but also caused the continuous changes of GM. It suggested that androgen was related to GM disorder, the former could promote the development of PCOS (37).
4.3 Insulin resistance
Insulin resistance is one of the main pathogenesis of precocious puberty, which can enhance the regulation effect of upstream LH on the ovary and strengthen the function of the adrenal gland to synthesize androgen; it can also inhibit the synthesis of SHBG in the liver, reduce the binding of androgen and SHBG in serum, and cause the increase of free testosterone level (38). Analysis of the causes of insulin resistance in the ICPP group: on the one hand, the increase of androgen level can cause gut mucosal damage (39), and the abundance of Alistipes and Parabacteroides increased can lead to gut inflammation (20, 40). As a result, the permeability of gut mucosa is increased. Many inflammatory mediators produced by GM, such as lipopolysaccharide (LPS) and branched-chain amino acid (BCAA), can activate the immune response. Meanwhile, inflammatory mediators can stimulate the production of various inflammatory factors by activating toll-like receptors 4 (TLR4). At the same time, through nuclear factor - κB (NF-κB) and other signaling pathways, it can promote the phosphorylation of insulin receptor substrate-1 (IRS-1) in the insulin signaling pathway, resulting in the decrease of insulin sensitivity. On the other hand, the increase of butyric acid-producing bacteria can also stimulate the increase of GLP-1 content (26, 32), in our study, the levels of IGF-1 and insulin in the ICPP group were significantly higher than those in the same age healthy group, which also supported the above conclusion. In addition, the insulin resistance index of ICPP children was significantly increased in this study, we analyzed the reasons for the existence of insulin resistance: first, LPS produced by GM can induce chronic inflammation in the intestine, and lead to insulin resistance by activating TLR4 (38). Second, our previous study also found that (41) ICPP children have increased NO synthesis in GM, which is positively correlated with FSH and insulin levels, and NO can promote insulin resistance. Last, Prevotella abundance was increased in ICPP children in our study, and Prevotella could increase circulating BCAA level, the latter was positively correlated with free testosterone level and insulin resistance (42, 43).
Liu et al. (36) analysis of GM in PCOS patients showed that the abundance of LPS-producing bacteria such as Bacteroides and Escherichia/Shigella increased, and the abundance of intestinal barrier protection bacteria such as Akkermensia decreased, which led to the increase of LPS production. Compared with the serum LPS binding protein (LBP) in PCOS and healthy people, Zhu et al. (44) found that the level of LBP in PCOS patients was significantly increased, and the combination of LPS and LBP could cause the expression of tumor necrosis factor-α (TNF-α) and IL-6, which is related to insulin resistance (45). Roychoudhury et al. (46) also found that the blood valine and leucine content in PCOS patients increased, 3-hydroxybutyrate (3-HIB), the metabolite of valine, stimulated the intake of fatty acids in muscle tissue, which could cause fat accumulation and insulin resistance (47).
4.4 Summary and thinking
Barrea et al. (48) considered that the interaction between high carbohydrate intake, hyperinsulinemia, hyperandrogenemia, and chronic low-grade inflammation are the four key factors for pathophysiological changes of PCOS. The correlation between hyperandrogenemia, insulin resistance, and chronic low inflammation in patients with ICPP and PCOS was analyzed. In this project, the children in the ICPP group liked to eat sweet food (which may lead to excessive carbohydrate intake), and increased carbohydrate metabolism in GM, so we think that ICPP and PCOS have similar pathogenesis. However, there are also differences between ICPP and PCOS. The latter has a low estrogen level, and the estrogen level of the ICPP group is higher than that of the healthy group. Based on the above research and analysis, we believe that the changes of GM in ICPP children will affect the changes of intestinal short-chain fatty acid composition, thus promoting the secretion of sex hormones such as androstenedione, IGF-1, and insulin. The structural changes of GM will lead to chronic low-grade inflammation, hyperandrogenemia, and insulin resistance, and finally start the HPGA in advance. These factors can also activate the GH-IGF-1 axis, promote growth hormone secretion through IGF-1, and lead to the sudden increase in height and weight, which may be the pathogenesis of ICPP children. The sample size of this study is small, and the fecal samples are only for 16S rRNA sequencing. The next step is to carry out the large sample and multi-center research. It is better to take macro genre sequencing for fecal samples, combined with the analysis of GM metabolites and blood metabolites, which may be more helpful to clarify the pathogenesis of ICPP.