Single-cell RNA-seq of amniotic fluid provides unique insight into fetal development

Background: Human fetal development is difficult to study due to ethical and practical constraints on the collection and use of human fetal tissue. Amniotic fluid (AF) is routinely collected during clinical amniocentesis and contains live fetal cells shed from developing tissues. Thus, we use single-cell RNA-sequencing (scRNA-seq) to build the first cell atlas of amniotic fluid from healthy second-trimester fetuses. Methods: scRNA-seq was performed on AF samples collected from seven euploid, structurally normal pregnancies at 15-25 weeks' gestation, all of which resulted in live births of healthy infants. Mapping to a modified transcriptome was used to help reduce counts sparsity. Supervised cell type annotation, unsupervised clustering and marker gene analyses were used to determine the cell types present in AF. Gestational age-related differences between earlier (<=19 weeks) and later (>= weeks) gestation samples were examined using differential gene expression (DGE) and differential proportion analyses. Results: Fetal skin cells were the most abundant cell type in AF, followed by myeloid and lymphoid cell types. We observed significant gestational age-related shifts in cell type composition, with an increased proportion of lung epithelium cells in later-age samples, coinciding with the canalicular phase of lung development. DGE analysis of fetal skin cells between earlier and later gestation revealed numerous gene expression changes that recapitulated known developmental processes across the second trimester. These were also enriched in an independent AF dataset from pregnancies with diverse fetal pathologies. Our dataset provides an annotated atlas of 11,414 healthy fetal human cells, permitting further study into normal second trimester development. We provide an integrated fetal cell atlas of 45,048 cells combining previous data with our healthy samples. Conclusions: This study applied novel analytical approaches to demonstrate the utility of scRNA-seq in characterising AF and its capacity to capture a diverse range of viable fetal cell types. Our results suggest that changes in AF cellular composition mirror organ development, underscoring the potential of AF scRNA-seq to deepen our understanding of normal and pathological fetal biology.