Fig. 4: Prenatal exposure to maternal diet-induced obesity and a nonoptimal vaginal microbiota alter the transcriptional landscape in the placenta and fetal ileum of male offspring.

A Schematic of experimental design to determine whether compounding maternal adversities, such as diet and G. vaginalis vaginal colonization, impact fetal development that may contribute to increased offspring mortality risk. We induced the pregestational phenotype and dams were colonized with G. vaginalis 11E4 on gestational day 13.5 and 15.5. At gestational day 18.5, tissue from one cohort of male offspring was collected for analysis of gene expression patterns in the placenta and ileum. A second cohort of offspring were colonized with the nonoptimal CST IV human vaginal microbiota. Created with BioRender.com. B Venn diagram displaying the number of differentially expressed genes in the embryonic day 18.5 placenta of male offspring exposed to a maternal high-fat low-fiber diet, G. vaginalis 11E4 vaginal colonization, or a combination (linear fit model, FDR < 0.1, log(FC) = 1.5; n = 351 genes). LFt-HFb = low-fat high-fiber; HFt-LFb = high-fat low-fiber; Gv = G. vaginalis 11E4. C Heatmap depicting mean expression of genes in the placenta of embryonic day 18.5 male offspring exposed to a maternal high-fat low-fiber diet, G. vaginalis 11E4 vaginal colonization, or a combination (HFt-LFb+Gv) (linear fit model, FDR < 0.1, log(FC) = 1.5). These comparisons reveal a unique cluster of genes that are differentially expressed in HFt-LFb+Gv male placenta compared with other treatment groups. Z-scores plotted across individuals for each gene. LFt-HFb = low-fat high-fiber; HFt-LFb = high-fat low-fiber; Gv = G. vaginalis 11E4. Color based on row Z-scores for each gene. Six clusters of differentially regulated genes in each treatment group are indicated. N = 3 males per treatment. D Venn diagram displaying the number of differentially expressed genes in the embryonic day 18.5 ileum of male offspring exposed to a maternal high-fat low-fiber diet, G. vaginalis 11E4 vaginal colonization, or a combination (HFt-LFb+Gv) These comparisons reveal a unique cluster of genes that are differentially expressed in the fetal ileum of HFt-LFb+Gv males compared with other treatment groups (linear fit model, FDR < 0.1, log(FC) = 1.5; n = 781 genes). LFt-HFb = low-fat high-fiber; HFt-LFb = high-fat low-fiber; Gv = G. vaginalis 11E4. E Heatmap depicting mean expression of genes in the ileum of embryonic day 18.5 male offspring exposed to a maternal high-fat low-fiber diet, G. vaginalis 11E4 vaginal colonization, or a combination (linear fit model, FDR < 0.1, log(FC) = 1.5). Color based on row Z-scores for each gene. Five clusters of differentially regulated genes in each treatment group are indicated. N = 3 males per treatment. F Survival of offspring from dams that experience a single or multiple compounding adversities in a second validation cohort. All pups were C-section delivered and gavaged with human CST IV inoculant, showing the highest offspring mortality risk in HFt-LFb+Gv male offspring exposed to CST IV at birth. LFt-HFb = low-fat high-fiber; HFt-LFb = high-fat low-fiber; Gv = G. vaginalis 11E4. Kaplan–Meier survival analysis. N = 24–40 offspring per treatment condition.