Investigating the Impact of Short-Chain Fatty Acids (SCFAs) from the Maternal Microbiome on Pregnancy and Fetal Health of C57BL/6J Mice: A Research Protocol

Abstract

Short-chain fatty acids (SCFAs), produced by commensal gut bacteria through fiber fermentation, are essential for regulating immune responses during pregnancy, benefiting both maternal and fetal health. Propionate, for example, activates G protein-coupled receptors (GPCRs), promoting colonic T regulatory cell (Treg) differentiation that is critical in maintaining inflammatory homeostasis. These molecules are not confined to the gut and can transverse the placental barrier, directly reaching the fetal compartment, where they may influence placental efficiency, impact pregnancy outcomes, and potentially predispose the child to autoimmunity and allergy. Despite the known association between dysbiosis and adverse pregnancy outcomes, few direct mechanistic studies have investigated the interaction between the maternal microbiome and the developing immune system during pregnancy. 

To develop a robust protocol, we designed a murine model experiment with differential SCFA exposure prior to pregnancy using C57BL/6J mice. Separate groups of female mice will be fed different diets prior to pregnancy: standard diet, high-fiber, or SCFA-supplemented diets. An SCFA-depleted group (via antibiotic treatment) will serve as a comparison.

Maternal fecal samples will be collected during the perinatal stage for SCFA analysis using mass spectrometry and for microbiome profiling via 16S ribosomal RNA gene sequencing. Fecal samples from neonatal mice will be collected shortly after birth for the same analyses. Systemic immune alterations will be examined using maternal and fetal blood samples to quantify key cytokines using a multi-plex ELISA. Flow cytometry will be conducted to compare differences in immune cell composition between different maternal diet groups. Other data including birth weight, litter size and gestation duration will also be compared to assess SCFA influence on pregnancy outcomes. We hypothesize that SCFA supplementation fosters an anti-inflammatory microbiome, elevating Tregs and anti-inflammatory cytokines while downregulating proinflammatory responses. This may lead to fewer pregnancy complications and improved fetal development. This research will help elucidate the role of SCFAs in maternal-fetal immune crosstalk and could inform dietary or therapeutic strategies to reduce immune-related diseases in infants and support long-term health.