Sex is associated with differences in gut microbial composition and function in hepatic encephalopathy

Abstract

Background & aims: Altered microbiota can affect the gut-liver-brain axis in cirrhosis and hepatic encephalopathy (HE), but the impact of sex on these changes is unclear. We aimed to determine differences in fecal microbiota composition/functionality between men and women with cirrhosis and HE on differing treatments.

Methods: Cross-sectional stool microbiome composition (16s rRNA sequencing) and microbial functional analyses were performed in men and women with cirrhosis, and controls. Patients with HE on rifaximin+lactulose (HE-Rif), patients with HE on lactulose only (HE-Lac) and those with cirrhosis without HE (No-HE) were compared to controls using random forest classifier. Men and women were also compared.

Results: A total of 761 individuals were included, 619 with cirrhosis (466 men, 153 women) and 142 controls (92 men, 50 women). Men were older and more frequently used proton pump inhibitors (PPIs), but model for end-stage liver disease score, No-HE (n = 319), HE-lac (n = 130) and HE-Rif (n = 170) proportions were similar. PPI/age-adjusted AUC of differentiation between controls vs. all cirrhosis, and controls vs. No-HE were higher within women than men, but the adjusted AUC for No-HE vs. HE-Rif was higher in men. Control vs. HE-Rif differentiation was similar across sexes. Men vs. women were different in all cirrhosis, No-HE and HE-Lac but not HE-Rif on PERMANOVA and AUC analyses. Autochthonous taxa decreased and pathobionts increased with disease progression regardless of sex. In men, Lactobacillaceae were higher in HE-Lac but decreased in HE-Rif, along with Veillonellaceae. Pathways related to glutamate and aromatic compound degradation were higher in men at all stages. Degradation of androstenedione, an estrogenic precursor, was lower in men vs. women in HE-Rif, likely enhancing feminization.

Conclusions: There are differences in gut microbial function and composition between men and women with cirrhosis, which could be implicated in differential responses to HE therapies. Further studies linking these differences to sex-specific outcomes are needed.

Lay summary: Patients with cirrhosis develop changes in their brain function, and men often develop feminization with disease progression. However, the interaction between sex, microbiota and disease severity is unclear. We found that as disease progressed in men, their microbial composition began to approach that observed in women, with changes in specific microbes that are associated with male hormone metabolism.

Keywords: Androgen; Artificial Intelligence; Cirrhosis; Lactobacillus; Lactulose; Random forest classifier; Rifaximin.

Figure 1:. Composition Changes Between Healthy and Diseased Groups in Cirrhosis within sex

These comparisons are within sex and a heatmap of bacterial families that were high in importance in comparison between all groups, within men and within women. Warmer colors indicate negative relationship while cooler colors indicate positive linkages in the more advanced category of comparison. Blank or white areas indicate that there is no linkage. HE: Hepatic encephalopathy, Rif: rifaximin, Lac: Lactulose, Cir: Cirrhosis, Con: healthy control.

Figure 2:. Microbial Composition Changes between Men and Women

These comparisons are between sex and a heatmap of bacterial families that were high in importance in comparison between men and women are shown. Darker colors indicate greater importance of that family in differentiating between men and women. Blank or white areas indicate that there is no linkage. HE: Hepatic encephalopathy, Rif: rifaximin, Lac: Lactulose

Figure 3:. Microbial taxa composition whose pattern of change over disease stages remains relatively similar between sex

Median and IQR of microbial taxa in men and women across disease states whose relative abundance changed similarly regardless of sex are presented. HE: Hepatic encephalopathy, Rif: rifaximin, Lac: Lactulose

Figure 4:. Microbial taxa composition whose pattern of change after HE development is different between sex

Median and IQR of microbial taxa in men and women across disease states whose relative abundance changed in different patterns with disease progression are presented. HE: Hepatic encephalopathy, Rif: rifaximin, Lac: Lactulose

Figure 5:. Differential enriched pathways volcano plot

Volcano plot showing Pathway fold changes on X-axis and the negative logarithm (base 10) of the Bonferroni-adjusted p-value on Y-axis. Dashed vertical and horizontal lines reflect the filtering criteria (fold change= ±1.0 and Bonferroni-adjusted p-value > −log (0.05)). Blue or Orange dots represent pathways that their log2 foldchange is greater or less than 1.0. The green dots represent common pathways which their absolute value of their foldchange is lower than 1. To be considered as a significant feature; the foldchange value should be greater than 1 or less than −1, and the negative logarithm (base 10) of the Metastats p-value should be above 1.13(-log (0.05)). We labeled top 20 of significant pathways for each group. Fold-change directions of men versus women are presented with log10 p value and log 2-fold change. Orange: pathways expressed more in women, Purple: pathways expressed more in men, Green: pathways expressed similarly in both. Pathways that are found above and to the right of the crossing of the dashed lines are significant. All pathway comparison details are in supplementary tables 1-4. 5A: Comparison between men and women with cirrhosis 5B: Comparison between men and women without hepatic encephalopathy (HE) 5C: Comparison between men and women with HE only on lactulose 5D: Comparison between men and women with HE on rifaximin also