Excess vitamin intake during pregnancy leads to obesogenic phenotypes, and folic acid accounts for many of these effects in male, but not in female, offspring. These outcomes may be modulated by another methyl nutrient choline and attributed to the gut microbiota. Pregnant Wistar rats were fed an AIN-93G diet with recommended vitamin (RV), high 10-fold multivitamin (HV), high 10-fold folic acid with recommended choline (HFol) or high 10-fold folic acid without choline (HFol-C) content. Male and female offspring were weaned to a high-fat RV diet for 12 weeks post-weaning. Removing choline from the HFol gestational diet resulted in obesogenic phenotypes that resembled more closely to HV in male and female offspring with higher body weight, food intake, glucose response to a glucose load and body fat percentage with altered activity, concentrations of short-chain fatty acids and gut microbiota composition. Gestational diet and sex of the offspring predicted the gut microbiota differences. Differentially abundant microbes may be important contributors to obesogenic outcomes across diet and sex. In conclusion, a gestational diet high in vitamins or imbalanced folic acid and choline content contributes to the gut microbiota alterations consistent with the obesogenic phenotypes of in male and female offspring.

Objectives Excess gestational folic acid and insufficient choline intakes as observed in the North American populations may increase the risk of obesity in offspring. It is well-established that adverse health outcomes may arise due to shifts in the gut microbial communities, but whether high vitamin intakes or an imbalance between methyl nutrients contributes to gut microbiota alterations is unclear. The objective of this research was to determine the gut microbiota composition of male and female offspring in relation to the vitamin composition of the gestational diet. Methods Pregnant Wistar rats (n = 10/group) were fed the AIN-93G diet with either the recommended vitamin (RV), high multivitamin (HV), high folic acid (HFol) or high folic acid without choline (HFol-C) content. Male and female offspring were weaned to a high-fat control diet for 12 weeks. Fecal samples were collected from the colon upon termination for gut microbiota profiling by 16S rRNA sequencing and data analyses in QIIME2. Results The overall gut microbial communities as assessed by unweighted UniFrac distances differed among the gestational diet groups for male (PERMANOVA P = 0.04) and female (PERMANOVA P = 0.05) offspring. The covariates gestational diet and sex predicted the gut microbiota differences in the offspring (Q2 = 0.07 in Songbird) whereas diet alone resulted in overfitting of the multinomial regression model (Q2 < 0). High ranked features from the natural log-ratios of microbial abundance were Shigella, Clostridiales, Clostridiaceae for HV, and Odoribacter, Akkermansia muciniphila, Blautia for both HFol and HFol-C compared to RV. Low ranked features were Odoribacter for HV, Clostridiaceae and Clostridiales for HFol, and Bifidobacterium, Allobaculum, Lactobacillus vaginalis for HFol-C compared to RV. In male offspring, Lactobacillus vaginalis, Sutterella and Clostridiales were high ranked and Odoribacter was low ranked compared to female offspring. These differentially abundant microbes may be important contributors to obesity across diet and sex. Conclusions Increased vitamin content or an imbalance between folic acid and choline in the gestational diet leads to a shift in the gut microbiota composition in the offspring toward obesity. These effects differed by sex. Funding Sources Utah Agricultural Experiment Station and USU Research Catalyst. UNM supported by USU URCO.