Reproductive and Behavior Dysfunction Induced by Maternal Androgen Exposure and Obesity Is Likely Not Gut Microbiome-Mediated

Abstract

Polycystic ovary syndrome (PCOS) is a common endocrine and metabolic disorder of unclear etiology in women and is characterized by androgen excess, insulin resistance, and mood disorders. The gut microbiome is known to influence conditions closely related with PCOS, and several recent studies have observed changes in the stool microbiome of women with PCOS. The mechanism by which the gut microbiome interacts with PCOS is still unknown. We used a mouse model to investigate if diet-induced maternal obesity and maternal DHT exposure, mimicking the lean and obese PCOS women, cause lasting changes in the gut microbiome of offspring. Fecal microbiome profiles were assessed using Illumina paired-end sequencing of 16S rRNA gene V4 amplicons. We found sex-specific effects of maternal and offspring diet, and maternal DHT exposure on fecal bacterial richness and taxonomic composition. Female offspring exposed to maternal obesity and DHT displayed reproductive dysfunction and anxietylike behavior. Fecal microbiota transplantation from DHT and diet-induced obesity exposed female offspring to wild-type mice did not transfer reproductive dysfunction and did not cause the expected increase in anxietylike behavior in recipients. Maternal obesity and androgen exposure affect the gut microbiome of offspring, but the disrupted estrous cycles and anxietylike behavior are likely not microbiome-mediated.

Keywords: androgens; anxiety; diet-induced obesity; gut microbiota; polycystic ovary syndrome.

Figure 1.

Study design for the maternal DHT-HFHS-exposure mouse model. n = 50 dams at start of experiment and 9 to 12 offspring/group. An additional group of dams was sacrificed at GD 18.5 and fecal material collected for gut microbiome analysis (n = 4 to 6/group). GD, gestational day.

Figure 2.

Study design for the FMT experiment. n = 12/FMT group. ABX, antibiotic.

Figure 3.

Differentially abundant genera in fecal samples from dams and female and male offspring. Log twofold change (FC) of relative abundance for genera, which were found by DESeq2 analysis to be significantly changed in (A) dams, (B–D) female and (E, F) male offspring due to the (A) factors diet, (B, E) maternal diet, (C, F) offspring diet, and (D) maternal DHT exposure. No genera were significantly changed due to DHT exposure in dams or maternal DHT exposure in male offspring. P < 0.05 for all after Benjamini-Hochberg false discovery rate correction. #Firmicutes; †Bacteroidetes; §Proteobacteria; ¶Actinobacteria. Square brackets indicate a Silva suggested taxonomic assignment. n = 9 to 11 animals per tested factor for dams and 34 to 50 animals per tested factor for offspring.

Figure 4.

Alpha diversity of fecal samples from dams and female and male offspring. Number of observed RSVs (left panel) and Shannon Index (right panel) are presented for (A, B) dams, (C, D) female offspring, and (E, F) male offspring. Two-/three-way ANOVA main effect of (a) maternal diet and (b) maternal DHT exposure (P < 0.05). Two-/three-way ANOVA interaction between (1) diet and DHT exposure, (2) maternal DHT exposure and offspring diet, and (3) maternal diet and offspring diet (P < 0.05). Mean and SEM are shown. n = 4 to 6 animals per group for dams and 7 to 10 animals per group for offspring.

Figure 5.

Beta diversity of fecal samples from dams and female and male offspring. MDS plots of Bray-Curtis distances for the (A) factors diet, (B) DHT exposure, and (C) group in dams and (D, H) maternal diet, (E, I) offspring diet, (F, J) maternal DHT exposure, and (G, K) group in offspring. Each dot represents the total bacterial community composition of one sample. The amount of variation explained by each MDS coordinate is indicated in square brackets. Factors were compared using Adonis.

Figure 6.

Estrous cyclicity in FMT recipients. The percent of time spent in (A) proestrus, (B) diestrus, and (C) the number of proestrus to estrus transitions over 10 consecutive d. Mean and SEM are shown. n = 12 mice per group.

Figure 7.

Anxietylike behavior in FMT recipients. Percentage of time spent in the open arms of the (A) EPM and (C) center of the OF and (B, D) the total distance traveled. Two-way ANOVA main effect of (b) donor maternal DHT exposure (P < 0.05). Mean and SEM are shown. n = 12 mice per group.

Figure 8.

Efficiency of the FMT based on the number of genera shared between FMT inoculate and recipient samples and Bray-Curtis dissimilarity. (A) Genera that were shared between FMT inoculate and at least one recipient sample. (B) Genera that were shared between FMT inoculate and all recipient samples. (C) Bray-Curtis dissimilarity. Samples from all groups were pooled together to evaluate dissimilarities of microbiome profiles between groups, within groups, between FMT recipients and No FMT controls, and between FMT recipients and the inoculate. Mean and SEM are shown. n = 12 animals per group.

Figure 9.

Differentially abundant genera in FMT recipient. Log twofold change (FC) of relative abundance for genera that were found by DESeq 2 analysis to be significantly changed due to (A) donor diet and (B) DHT exposure. P < 0.05 for all after Benjamini-Hochberg false discovery rate correction. #Firmicutes; †Bacteroidetes; §Proteobacteria; ||Deferribacteres; ‡Saccharibacteria. n = 24 animals per tested factor.

Figure 10.

Alpha diversity of fecal samples from FMT recipients and inoculates. The number of observed (A) RSVs and (B) Shannon index. Diamonds show the diversity of the respective FMT inoculate. Two-way ANOVA main effect of (a) donor diet and (b) donor maternal DHT exposure (P < 0.05). Two-way ANOVA interaction between (1) donor diet and donor maternal DHT exposure (P < 0.05). P < 0.05 for (d) all groups compared with No FMT. Mean and SEM are shown. n = 12 animals per group.

Figure 11.

Beta diversity of fecal samples from FMT recipients and inoculates. MDS plots of Bray-Curtis distances for the (A) factors donor diet, (B) donor maternal DHT exposure, and (C) donor group. No FMT animals received PBS instead of fecal inoculate. Each dot represents the total bacterial community composition of one sample. The amount of variation explained by each MDS coordinate is indicated in brackets. Groups were compared using Adonis. PBS, phosphate-buffered saline.