Metabolic dysregulation and gut dysbiosis linked to hyperandrogenism in female mice.

INTRODUCTION: Polycystic ovary syndrome (PCOS) is a common endocrine pathology in women. In addition to infertility, women with PCOS have metabolic dysregulation which predisposes them to Type 2 diabetes, cardiovascular disease and non-alcoholic fatty liver disease. Moreover, women with PCOS have changes in their gut microbial community that may be indicative of dysbiosis. While hyperandrogenism is associated with both the development of metabolic dysfunction and gut dysbiosis in females, the mechanisms involved are not well understood. METHODS: We used dihydrotestosterone (DHT) and ovariectomy (OVX) mouse models coupled with metabolic assessments and 16S rRNA gene sequencing to explore the contributions of hyperandrogenism and oestrogen deficiency to the development of insulin resistance and gut microbial dysbiosis in pubertal female mice. RESULTS: We demonstrated that, while DHT treatment or OVX alone were insufficient to induce insulin resistance during the pubertal-to-adult transition, combining OVX with DHT resulted in insulin resistance similar to that observed in letrozole-treated mice with elevated testosterone and decreased oestrogen levels. In addition, our results showed that OVX and DHT in combination resulted in a distinct shift in the gut microbiome compared to DHT or OVX alone, suggesting that the substantial metabolic dysregulation occurring in the OVX + DHT model was accompanied by unique changes in the abundances of gut bacteria including S24-7, Rikenellaceae and Mucispirillum schaedleri. CONCLUSIONS: While hyperandrogenism plays an important role in the development of metabolic dysregulation in female mice, our results indicate that investigation into additional factors influencing insulin resistance and the gut microbiome during the pubertal-to-adult transition could provide additional insight into the pathophysiology of PCOS.

Genetic hypogonadal mouse model reveals niche-specific influence of reproductive axis and sex on intestinal microbial communities.

BACKGROUND: The gut microbiome has been linked to many diseases with sex bias including autoimmune, metabolic, neurological, and reproductive disorders. While numerous studies report sex differences in fecal microbial communities, the role of the reproductive axis in this differentiation is unclear and it is unknown how sex differentiation affects microbial diversity in specific regions of the small and large intestine. METHODS: We used a genetic hypogonadal mouse model that does not produce sex steroids or go through puberty to investigate how sex and the reproductive axis impact bacterial diversity within the intestine. Using 16S rRNA gene sequencing, we analyzed alpha and beta diversity and taxonomic composition of fecal and intestinal communities from the lumen and mucosa of the duodenum, ileum, and cecum from adult female (n = 20) and male (n = 20) wild-type mice and female (n = 17) and male (n = 20) hypogonadal mice. RESULTS: Both sex and reproductive axis inactivation altered bacterial composition in an intestinal section and niche-specific manner. Hypogonadism was significantly associated with bacteria from the Bacteroidaceae, Eggerthellaceae, Muribaculaceae, and Rikenellaceae families, which have genes for bile acid metabolism and mucin degradation. Microbial balances between males and females and between hypogonadal and wild-type mice were also intestinal section-specific. In addition, we identified 3 bacterial genera (Escherichia Shigella, Lachnoclostridium, and Eggerthellaceae genus) with higher abundance in wild-type female mice throughout the intestinal tract compared to both wild-type male and hypogonadal female mice, indicating that activation of the reproductive axis leads to female-specific differentiation of the gut microbiome. Our results also implicated factors independent of the reproductive axis (i.e., sex chromosomes) in shaping sex differences in intestinal communities. Additionally, our detailed profile of intestinal communities showed that fecal samples do not reflect bacterial diversity in the small intestine. CONCLUSIONS: Our results indicate that sex differences in the gut microbiome are intestinal niche-specific and that sampling feces or the large intestine may miss significant sex effects in the small intestine. These results strongly support the need to consider both sex and reproductive status when studying the gut microbiome and while developing microbial-based therapies.

Microbial communities in the intestinal tract, known as the gut microbiome, regulate many critical aspects of host physiology. Previous studies have shown that the diversity of the gut microbiome differs between the sexes. There are also many diseases with a sex bias linked to the gut microbiome, including autoimmune, metabolic, neurological, and reproductive disorders. The gut microbiome differentiates during puberty, but it is unknown if the reproductive axis, the system responsible for sexual maturation and production of gonadal sex hormones, is critical for this process. Furthermore, since most studies use feces to examine the gut microbiome, it is unknown how sex influences the microbial communities within different segments of the small and large intestine. To address this gap in knowledge, we used DNA-based molecular methods to compare the intestinal-specific microbiomes of a mouse model with a genetically inactivated reproductive axis to that of wild-type mice. We found that both sex and the reproductive axis impacted gut microbial diversity in an intestinal section-specific manner. We also detected significant differences in intestinal microbial diversity between male and female mutant mice, suggesting that sex chromosome factors also affect the gut microbiome. We also showed that fecal samples were dissimilar to small intestine microbial communities, indicating that studies only sampling feces likely miss sex differences specific to the small intestine. Our results strongly support the need to consider both sex and reproductive status when studying the gut microbiome and while developing microbial-based therapies.

Alterations in Gut Microbiota Do Not Play a Causal Role in Diet-independent Weight Gain Caused by Ovariectomy.

Acute estrogen deficiency in women can occur due to many conditions including hyperprolactinemia, chemotherapy, GnRH agonist treatment, and removal of hormone replacement therapy. Ovariectomized (OVX) rodent models, often combined with a high-fat diet (HFD), have been used to investigate the effects of decreased estrogen production on metabolism. Since evidence suggests that gut microbes may facilitate the protective effect of estrogen on metabolic dysregulation in an OVX + HFD model, we investigated whether the gut microbiome plays a role in the diet-independent weight gain that occurs after OVX in adult female mice. 16S rRNA gene sequence analysis demonstrated that OVX was not associated with changes in overall gut bacterial biodiversity but was correlated with a shift in beta diversity. Using differential abundance analysis, we observed a difference in the relative abundance of a few bacterial taxa, such as Turicibacter, 3 to 5 weeks after OVX, which was subsequent to the weight gain that occurred 2 weeks postsurgery. A cohousing study was performed to determine whether exposure to a healthy gut microbiome was protective against the development of the metabolic phenotype associated with OVX. Unlike mouse models of obesity, HFD maternal-induced metabolic dysregulation, or polycystic ovary syndrome, cohousing OVX mice with healthy mice did not improve the metabolic phenotype of OVX mice. Altogether, these results indicate that changes in the gut microbiome are unlikely to play a causal role in diet-independent, OVX-induced weight gain (since they occurred after the weight gain) and cohousing with healthy mice did not have a protective effect.

Letrozole treatment of pubertal female mice results in activational effects on reproduction, metabolism and the gut microbiome.

Polycystic ovary syndrome (PCOS) is a common endocrine disorder in reproductive-aged women that is comprised of two out of the following three features: hyperandrogenism, oligo- or amenorrhea, or polycystic ovaries. In addition to infertility, many women with PCOS have metabolic dysregulation that increases the risk of developing type 2 diabetes, hypertension, and non-alcoholic fatty liver disease. Changes in the gut microbiome are associated with PCOS and gut microbes may be involved in the pathology of this disorder. Since PCOS often manifests in the early reproductive years, puberty is considered to be a critical time period for the development of PCOS. Exposure to sex steroid hormones during development results in permanent, organizational effects, while activational effects are transient and require the continued presence of the hormone. Androgens exert organizational effects during prenatal or early post-natal development, but it is unclear whether androgen excess results in organizational or activational effects during puberty. We recently developed a letrozole-induced PCOS mouse model that recapitulates both reproductive and metabolic phenotypes of PCOS. In this study, we investigated whether letrozole treatment of pubertal female mice exerts organizational or activational effects on host physiology and the gut microbiome. Two months after letrozole removal, we observed recovery of reproductive and metabolic parameters, as well as diversity and composition of the gut microbiome, indicating that letrozole treatment of female mice during puberty resulted in predominantly activational effects. These results suggest that if exposure to excess androgens during puberty leads to the development of PCOS, reduction of androgen levels during this time may improve reproductive and metabolic phenotypes in women with PCOS. These results also imply that continuous letrozole exposure is required to model PCOS in pubertal female mice since letrozole exerts activational rather than organizational effects during puberty.

Exposure to a Healthy Gut Microbiome Protects Against Reproductive and Metabolic Dysregulation in a PCOS Mouse Model.

Polycystic ovary syndrome (PCOS) is a common endocrine disorder affecting ∼10% to 15% of reproductive-aged women worldwide. Diagnosis requires two of the following: hyperandrogenism, oligo-ovulation or anovulation, and polycystic ovaries. In addition to reproductive dysfunction, many women with PCOS display metabolic abnormalities associated with hyperandrogenism. Recent studies have reported that the gut microbiome is altered in women with PCOS and rodent models of the disorder. However, it is unknown whether the gut microbiome plays a causal role in the development and pathology of PCOS. Given its potential role, we hypothesized that exposure to a healthy gut microbiome would protect against development of PCOS. A cohousing study was performed using a letrozole-induced PCOS mouse model that recapitulates many reproductive and metabolic characteristics of PCOS. Because mice are coprophagic, cohousing results in repeated, noninvasive inoculation of gut microbes in cohoused mice via the fecal-oral route. In contrast to letrozole-treated mice housed together, letrozole mice cohoused with placebo mice showed significant improvement in both reproductive and metabolic PCOS phenotypes. Using 16S rRNA gene sequencing, we also observed that the overall composition of the gut microbiome and the relative abundance of Coprobacillus and Lactobacillus differed in letrozole-treated mice cohoused with placebo mice compared with letrozole mice housed together. These results suggest that dysbiosis of the gut microbiome may play a causal role in PCOS and that modulation of the gut microbiome may be a potential treatment option for PCOS.

Letrozole treatment of adult female mice results in a similar reproductive phenotype but distinct changes in metabolism and the gut microbiome compared to pubertal mice.

BACKGROUND: A majority of women with polycystic ovary syndrome (PCOS) have metabolic dysfunction that results in an increased risk of type 2 diabetes. We previously developed a pubertal mouse model using the aromatase inhibitor, letrozole, which recapitulates many of the reproductive and metabolic features of PCOS. To further our understanding of the effects of androgen excess, we compared the effects of letrozole treatment initiated in puberty versus adulthood on reproductive and metabolic phenotypes as well as on the gut microbiome. RESULTS: Letrozole treatment of both pubertal and adult female mice resulted in reproductive hallmarks of PCOS, including hyperandrogenemia, anovulation and polycystic ovaries. However, unlike pubertal mice, treatment of adult female mice resulted in modest weight gain and abdominal adiposity, minimal elevation in fasting blood glucose and insulin levels, and no detectable insulin resistance. In addition, letrozole treatment of adult mice was associated with a distinct shift in gut microbial diversity compared to letrozole treatment of pubertal mice. CONCLUSIONS: Our results indicate that dysregulation of metabolism and the gut microbiome in PCOS may be influenced by the timing of androgen exposure. In addition, the minimal weight gain and lack of insulin resistance in adult female mice after letrozole treatment indicates that this model may be useful for investigating the effects of hyperandrogenemia on the hypothalamic-pituitary-gonadal axis and the periphery without the influence of substantial metabolic dysregulation.