Fetal Metabolite Effects on Placental Phenotypes

To test the effects of the candidate microbiome-dependent fetal metabolites, the Metab cocktail or vehicle control was administered intraperitoneally once daily from E0.5 to E14.5 to minimize stress to pregnant dams. Metabolite dosages were calculated on the basis of fetal serum metabolomic data and physiologically relevant metabolite concentrations reported in literature, to reflect the daily levels needed to match those observed in SPF fetal serum (table S2). Metabolite concentrations were calculated on the basis of physiological levels in mouse or human blood (table S2), total blood volume of pregnant mouse dams [approximately 58.5 ml/kg (48 (link))], and relative reductions observed in fetal sera of ABX fetuses compared to SPF fetuses (table S2). The metabolite stock solution consisted of: 29.64 μM imidazole propionate, 714.096 μM N,N,N-trimethyl-5-aminovalerate, 13.452 μM 4-hydroxyphenylacetate, 316.008 μM phenol sulfate, 428.868 μM indolepropionate, 360.24 μM indoxyl glucuronide, 323.76 μM N-methylproline, 595.080 μM phenylacetylglycine, 957.6 μM trimethylamine N-oxide, 118.56 μM taurodeoxycholate, 160.8768 μM biotin, 893.76 μM hippurate, 42.864 μM 2-(4-hydroxyphenyl)propionate, 222.072 μM cinnamoylglycine, 273.6 nM equol glucuronide, 433.2 μM 2-aminophenol sulfate, 1.55952 mM 3-indoxyl sulfate, and 268.128 μM p-cresol sulfate in 0.1 M PBS. The stock solution was then diluted 1:100 in sterile 0.1 M PBS, and 200 μl of the working solution was injected intraperitoneally into E0.5 ABX dams once a day daily for 14 days. For SCFA treatment, sodium propionate (25 mM), sodium butyrate (40 mM), and sodium acetate (67.5 mM) were supplemented to drinking water of pregnant ABX dams from E0 to E14.5. These concentrations were determined on the basis of existing studies demonstrating that these concentrations were able to sufficiently penetrate host tissues distal from the gut and restore circulating physiological concentrations (12 (link)). SCFA-supplemented drinking water and sodium-matched control drinking water were sterile filtered and made fresh every 4 days. To assess placental phenotypes, dams treated with metabolites were euthanized on E14.5, and placentas were harvested and processed as described in sections above.