(23) Exploring the Role of Microbial Metabolites in Trophoblast Differentiation and Energy Metabolism

Ruschman GL¹; and Day-Walsh PE^1,2; Charnock-Jones DS^1,2

1. Department of Obstetrics and Gynaecology, University of Cambridge, School of Clinical Medicine Cambridge, UK.

2. The Loke Centre for Trophoblast Research (LCTR), Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK

Abstract Text:

Alterations in the maternal gut microbiome are implicated in pregnancy complications which contribute to millions of maternal and neonatal deaths annually. These complications primarily stem from impaired placental development, which hinges on proper trophoblast differentiation from the cytotrophoblast stem cells (CTBs) to the differentiated syncytiotrophoblast (STBs) cells and the migratory extravillous trophoblast. We investigated the effects of short-chain fatty acids (SCFAs), key gut derived metabolites, on human primary trophoblast energy metabolism and CTB differentiation to STBs. We hypothesised that maternal gut-derived SCFAs mediate microbial-host interactions during pregnancy by regulating trophoblast energy metabolism and differentiation.  We used a comprehensive approach to investigate the effects of physiologically relevant doses of SCFAs (acetate, propionate, and butyrate) on human primary trophoblast cells to assess energy metabolism, differentiation, and protein expression through various experimental techniques and bioinformatic analyses. Human primary trophoblast cells isolated from male (n=2) and female (n=3) placentas were treated with a physiologically relevant doses of acetate, propionate, and butyrate. Trophoblast energy metabolism was assessed via Seahorse XF Extracellular Flux Analyzer. Trophoblast differentiation was evaluated by measuring Human chorionic gonadotropin (hCG) release and by ZO-1 immunostaining. Additionally, using online bioinformatic and metagenomic databases (UniProtKB, NCBI, Gmrepo2) we characterized microbes and genes responsible for SCFA production. We determined which SCFA-producing taxa present in maternal/neonatal faecal and maternal vaginal samples were associated with adverse pregnancy outcomes including low birth weight, preterm birth, preeclampsia, and gestational diabetes. ZO-1 staining indicated characteristic trophoblast phenotype with multinucleated cells indicating differentiating trophoblast. No significant differences were observed in trophoblast energy metabolism between SCFA treated and untreated control cells. Trophoblast hCG release showed a trend of increasing as butyrate concentrations decreased. A non-parametric Friedman test revealed a significant difference in hCG release between the control and butyrate-treated groups on day 3 (P = 0.0065, n = 5) and day 4 (p=0.0351, n=5) of treatment. Post-hoc Dunn’s multiple comparisons test indicated that hCG release on day 3 was significantly higher than the control after treatment with 0.195 µM butyrate (P=0.0497) and 0.0975 µM butyrate (P = 0.0077), while other concentrations did not reach statistical significance. Acetate and propionate had no significant effect on trophoblast differentiation. Microbial genes involved in butyrate production included butyrate kinase, butyryl-CoA: acetate CoA-transferase, and phosphotransbutyrylase. These genes were expressed by species including Clostridium butyricum, Peptostreptococcus anaerobius, Butyrivibrio crossotus, Roseburia faecis, and Roseburia inulinivorans among others. In further work we will aim to characterise associations of these species with pregnancy outcomes. Preliminary data demonstrated trophoblast differentiation increased with decreasing concentrations of butyrate. Our findings suggested potential mechanisms through which butyrate and possibly other SCFAs influence placental development and function, offering insights into the role of the microbiome in the pregnancy complications. This research contributes to understanding the complex interactions between microbial metabolites and placental physiology, potentially informing future therapeutic strategies for managing pregnancy-related health challenges.