The Effect of Cross-Sex Fecal Microbiota Transplantation on Metabolism and Hormonal Status in Adult Rats

Abstract

Increasing evidence of sexual dimorphism in the pathophysiology of metabolic complications caused by sex steroids is under investigation. The gut microbiota represents a complex microbial ecosystem involved in energy metabolism, immune response, nutrition acquisition, and the health of host organisms. Gender-specific differences in composition are present between females and males. The purpose of this study was to use cross-sex fecal microbiota transplantation (FMT) for the detection of sex-dependent metabolic, hormonal, and gut microbiota changes in female and male recipients. Healthy non-obese female and male Wistar rats were divided into donor, same-sex, and cross-sex recipient groups. After a 30-day period of FMT administration, biochemical markers (glucose and lipid metabolism) and sex hormones were measured, and the gut microbiota was analyzed. The cross-sex male recipients displayed a significantly lower testosterone concentration compared to the males that received same-sex FMT. Sex-dependent changes caused by cross-sex FMT were detected, while several bacterial taxa correlated with plasma testosterone levels. This study represents the first to study the effect of cross-sex changes in the gut microbiome concerning metabolic and hormonal changes/status in adult non-obese Wistar rats. Herein, we present cross-sex FMT as a potential tool to modify sex-specific pathologies.

Keywords: cross-sex; fecal microbiota transplantation; gut microbiota; metabolic disease; microbiome; sex differences; testosterone.

Figure 1

Body weight change in female (A), and male (B) donor and recipient animals and food consumption in female (C) and male (D) donor and recipient animals. Data are presented as mean ± SEM. *—differences between same-sex recipient female rats and donors; +—differences between cross-sex female rats and donors; * p < 0.05; ** p < 0.01; *** p < 0.001; ++ p < 0.01; +++ p < 0.001.

Figure 2

Biochemical analysis of plasma: (A) Body weight; (B) Fasting glycemia; (C) Fasting insulin concentrations; (D) Quantitative index of insulin sensitivity (QUICKI); (E) Total cholesterol concentration; (F) Concentration of triacylglycerols; (G) HDL cholesterol concentrations; (H) LDL cholesterol concentrations; (I) AST concentration; (J) ALT concentration; (K) Uric acid concentration. Data are presented as mean ± SEM. *—sex difference; #—the difference between donor, same-sex, and cross-sex rats in individual sex groups; * p < 0.05; *** p < 0.001; # p < 0.05; HDL—high-density lipoproteins; LDL—low-density lipoproteins; AST—aspartate aminotransferase; ALT—alanine aminotransferase.

Figure 3

Plasma testosterone concentrations and gut microbiota composition analysis: (A) Plasma testosterone concentrations; (B) Principal component analysis of the gut microbiota—all recipients; (C) Redundancy analysis of all female recipients according to different FMT donor sex (same-sex females, cross-sex females); (D) Redundancy analysis of all male recipients according to different FMT donor sex (same-sex males, cross-sex males); (E) Redundancy analysis of all mice receiving FMT from female donors (same-sex females, cross-sex males); (F) Redundancy analysis of all mice receiving FMT from male donors (same-sex males, cross-sex females). Data are presented as mean ± SEM. *—sex difference; #—the difference between donor, same-sex, and cross-sex rats in individual sex groups; ** p < 0.01; *** p < 0.001; ## p < 0.01.

Figure 4

Correlation of testosterone concentration with specific bacterial species: (A) Correlation between plasma testosterone concentration and Bacteroides abundance in recipient males; (B) Correlation between plasma testosterone concentration and Frisingicoccus abundance in recipient males; (C) Correlation between plasma testosterone concentration and Lachnospiraceae abundance in recipient males; (D) Correlation between plasma testosterone concentrations and Bacteria N/A (ASV_314) in recipient males; (E) Correlation between plasma testosterone concentration and Bacteroides abundance in recipient females; (F) Correlation between plasma testosterone concentration and Frisingicoccus abundance in recipient females; (G) Correlation between plasma testosterone concentration and Lachnospiraceae abundance in recipient females; (H) Correlation between plasma testosterone concentrations and Bacteria N/A (ASV_314) in recipient females.

Figure 5

Experimental design: (A) Preparation of fecal microbiota transplantation (FMT) and experimental groups. (B) Timeline of the experiment—(1)—Recipient sample collection; (2)—Blood collection from the tail vein and plasma preparation; (3)—Biochemical analysis of plasma samples; (4)—Isolation of bacterial DNA from stool samples; (5)—DNA analysis and quantity control and amplification of 16S rRNA V4 regions; (6)—Endpoint Ruby Taq electrophoresis; (7)—Sample sequencing using MiSeq instrument; (8)—Data processing. Created with BioRender.com.