Probiotic Bifidobacterium lactis V9 Regulates the Secretion of Sex Hormones in Polycystic Ovary Syndrome Patients through the Gut-Brain Axis

Abstract

Although a few studies have investigated the intestinal microbiota of women with polycystic ovary syndrome (PCOS), the functional and metabolic mechanisms of the microbes associated with PCOS, as well as potential microbial biomarkers, have not yet been identified. To address this gap, we designed a two-phase experiment in which we performed shotgun metagenomic sequencing and monitored the metabolic parameters, gut-brain mediators, and sex hormones of PCOS patients. In the first stage, we identified an imbalance in the intestinal microbiota of the PCOS patients, observing that Faecalibacterium, Bifidobacterium, and Blautia were significantly more abundant in the control group, whereas Parabacteroides and Clostridium were enriched in the PCOS group. In the second stage, we monitored the impact of the probiotic Bifidobacterium lactis V9 on the intestinal microbiome, gut-brain mediators, and sex hormones of 14 PCOS patients. Notably, we observed that the levels of luteinizing hormone (LH) and LH/follicle-stimulating hormone (LH/FSH) decreased significantly in 9 volunteers, whereas the levels of sex hormones and intestinal short-chain fatty acids (SCFAs) increased markedly. In contrast, the changes in the indices mentioned above were indistinct in the remaining 5 volunteers. The results of an analysis of the number of viable Bifidobacterium lactis V9 cells in the two groups were highly consistent with the clinical and SCFA results. Therefore, effective host gut colonization of the probiotic Bifidobacterium lactis V9 was crucial for its ability to function as a probiotic. Finally, we propose a potential mechanism describing how probiotics regulate the levels of sex hormones by manipulating the intestinal microbiome in PCOS patients. IMPORTANCE Polycystic ovary syndrome (PCOS) is a common metabolic disorder among women of reproductive age worldwide. Through a two-phase clinical experiment, we first revealed an imbalance in the intestinal microbiome of PCOS patients. By binning and annotating shotgun metagenomic sequences into metagenomic species (MGS), 61 MGSs were identified as potential PCOS-related microbial biomarkers. In the second stage, we monitored the impact of the probiotic Bifidobacterium lactis V9 on the intestinal microbiota, metabolic parameters, gut-brain mediators, and sex hormones of PCOS patients. Notably, we observed that the PCOS-related clinical indices and the intestinal microbiotas of the participating patients exhibited an inconsistent response to the intake of the B. lactis V9 probiotic. Therefore, effective host gut colonization of the probiotic was crucial for its ability to function as a probiotic. Finally, we propose a potential mechanism by which B. lactis V9 regulates the levels of sex hormones by manipulating the intestinal microbiome in PCOS patients.

Keywords: Bifidobacterium lactis V9; brain-gut axis; intestinal microbiome; polycystic ovary syndrome; probiotics; short-chain fatty acid.

FIG 1

The alterations in the intestinal microbiota of the PCOS patients. (A and B) Principal-component-analysis (PCoA) score plot based on weighted (A) and unweighted (B) UniFrac metrics for all participants. Each color dot (green for the control group and pink for the PCOS group) represents the composition of the intestinal microbiota based on the results of 16S rRNA gene high-throughput sequencing performed for each subject. (C) The differences in intestinal microbes between the PCOS and control groups at the genus level. Significance was calculated by the Wilcoxon rank sum test.

FIG 2

Taxonomic characterization of the intestinal microbiota (metagenomic species [MGS]) in the different groups. Differentially abundant MGS networks that were enriched in the control group (n = 31; panel A, green) and the PCOS individuals (n = 30; panel B, pink). The network was constructed based on Spearman's rank correlation coefficient values determined in comparisons of different MGS. The edge width is proportional to the correlation strength. The node size is proportional to the mean abundance in the respective population. Each node represents an MGS, and the identified MGS is annotated with its species as indicated in the footnote.

FIG 3

The log-transformed reporter Z scores of the metabolic pathways showing significant differences between the PCOS and control groups. The green nodes represent the metabolic pathways (in level 3) enriched in the control group (CO), and the pink nodes represent the metabolic pathways (in level 3) enriched in the PCOS group (PCOS). The node size represents the changed score. The metabolic pathways in level 2 are displayed in different colors. CoA, coenzyme A.

FIG 4

The correlation network constructed using the identified metagenomic species (MGS), metabolic parameters (including TGs, TC, and FPG), SCFAs (including acetic, propionic, butyric, and valeric acid), gut-brain mediators (PYY and ghrelin), and sex hormones (including LH, FSH, LH/FSH, E2, PRL, and T) based on Spearman's rank correlation coefficient (R greater than 0.4 or R less than −0.4). The edge width and color (red, positive; green, negative) are proportional to the strength of the correlation. The node size is proportional to the mean abundance in the respective population. Nodes with the same color are classified as the same type of indicators. Bif, Bifidobacterium.

FIG 5

Dynamic changes in the levels of sex hormones (panel A for LH and panel B for LH/FSH), brain-gut mediators (panel C for ghrelin and panel D for PYY), and SCFAs (panels E, F, G, and H for acetic acid, propionic acid, butyric acid, and valeric acid, respectively) during the Bifidobacterium lactis V9 consumption stage. Each panel is composed of two groups: the response group (group R; the changes in the indices listed above were notable for this group) and the nonresponse group (group N; the changes in the indices listed above were limited for this group). The significance data were calculated using the Kruskal-Wallis test (for comparisons of data from multiple time points) and the Wilcoxon rank sum test (for comparisons of data from two [paired] time points). The variation trends in the indices listed above for each participant are indicated with a solid line.

FIG 6

Quantification of live Bifidobacterium lactis V9 in fecal samples during the intake and washout stages for the response group (group R, n = 9) and the nonresponse group (group N, n = 5). The different experimental stages, including the baseline (point A), probiotic consumption (points B, D, and F), and washout (points C, E, and G) stages, are arranged at the bottom, the top, and the middle of the Sankey diagram. The thicknesses of the curves in different colors represent the average amounts of viable Bifidobacterium lactis V9 in fecal samples. As shown in the figure, the lines in panel A are thick, which indicates that the average amount of viable Bifidobacterium lactis V9 in group R was high, whereas the lines in panel B are thin, which indicates that the average amount of viable Bifidobacterium lactis V9 in group N was low.

FIG 7

The intestinal microbiota exhibited an inconsistent response to the Bifidobacterium lactis V9 probiotic intake. (A) The structure of the intestinal microbiota changed markedly in the response group (group R, n = 9) based on the 16S rRNA gene high-throughput-sequencing weighted UniFrac distance. The dots exhibiting different depths of green color (the green color gradually darkened from 0 to 10 weeks) represent the composition of the intestinal microbiota of the subjects. (B) The influence of the probiotic Bifidobacterium lactis V9 on the gut microbiota in the nonresponse group (group N, n = 5) was limited according to the clusters based on the weighted UniFrac distance. The dots with the same color represent the composition of the intestinal microbiota of the same participant at different time points. (C) A heat map representing the intestinal microbial changes at the genus level in group R at different Bifidobacterium lactis V9 consumption stages. The degrees of depth of the color represents the relative abundances of the related genus. The P value was calculated for each genus by the Kruskal-Wallis test, and the data are annotated on the left side. P values of less than 0.05 are marked in red.

FIG 8

The potential mechanism by which the probiotic Bifidobacterium lactis V9 regulates the levels of sex hormones through the intestinal microbiome in PCOS patients.