Molecular effects of maternal smoking on the early human placenta

Abstract

Maternal smoking during pregnancy is the most important avoidable cause of gestational morbidity and mortality, exerting permanent impacts on fetal health. This behavior is causally associated with fetal growth restriction (FGR), fetal malformations, and an increased vulnerability to disease later in life. The placenta is the functional interface between mother and fetus during pregnancy and is essential for healthy fetal development. This transient organ controls nutrient and gas exchange in-utero and secretes pregnancy modulating hormones. Molecular mechanisms that disturb the early development and function of the placenta by smoking remain largely unknown. This thesis uses a multi-omics approach to elucidate the phenotype-specifc molecular effects of maternal smoking on the developmental phase of the early human placenta. To this end, I evaluate tissue gene expression via single nuclei RNA sequencing (n = 5 smokers, n = 6 controls) and tissue relative protein abundances using ultra-sensitive phenotype-specific proteomics (n = 3 smokers, n = 3 controls). Results are validated in an independent human cohort (n = 23 smokers, n = 15 non-smokers), and functionally by in-vitro cigarette smoke component exposure using a primary human trophoblast stem cell model. Smoking status stratification for all cohorts was done via maternal plasma cotinine levels, a stable metabolite of nicotine and clinical gold standard. Results demonstrate that the syncytiotrophoblast (STB), the functional barrier of the placenta in direct contact with maternal blood, is profoundly affected and constitutes the only phenotype with compositional differences between smokers and non-smokers. The molecular profiling of the STB at a high resolution has been seldom performed, as its syncytial multinucleated nature poses challenges for its isolation. Trophoblast differentiation modelling demonstrates a preferential differentiation towards the STB lineage in smokers, in agreement with STB nuclei abundance increases in smokers. There are increases in pro-angiogenic secreted placental growth factor (PGF) gene expression (p < 0.001) with systemic relevance, as PlGF protein is increased in the blood of maternal smokers (p = 0.03). Smoking affected genes and proteins associated with cellular signalling, transmembrane transport, cellular stress and extracellular matrix remodelling. Molecular mechanisms associated with dysregulated markers at the transcriptomic and proteomic levels include increases in oxidative stress, activation of the xenobiotic detoxification aryl-hydrocarbon receptor pathway and disturbed mitochondrial energy metabolism capacity. Significantly higher numbers of mitochondrion are present in the STBs of smokers, as evaluated by immunofluorescence staining. Identified pathways can deregulate biological processes important for placental development and function, consequently impacting fetal growth and development. Beyond smoking, these findings provide a framework for understanding modulators of placental dysfunction that contribute to FGR – a leading cause of neonatal mortality and fetal morbidity – early in pregnancy, before the onset of clinical symptoms. I anticipate this data to be instrumental towards advancing FGR diagnostics and therapies to enhance pregnancy outcomes.