Single-nuclei RNA-sequencing fails to detect molecular dysregulation in the preeclamptic placenta

Introduction: Single-cell RNA-seq (scRNA-seq) revolutionized our understanding of tissue complexity in health and disease and revealed massive transcriptional dysregulation across placental cell classes in early-onset, but not late-onset preeclampsia (PE). However, the multinucleated syncytium is largely inaccessible to cell dissociation. Nuclei isolation and single-nuclei RNA-seq may be preferable in the placenta; not least considering compatibility with long-term tissue storage. Yet, nuclei contain a subsample of the cells' transcriptional profile. Mature transcripts critical to cellular function and disease may be missed.

Methods: We analyzed placenta from pregnancies using single-cell and single-nuclei RNA-seq. The datasets comprise 45,836 cells and 27,078 nuclei, from 10 to 7 early-onset preeclampsia (EPE) cases and 3 and 2 early idiopathic controls (ECT), respectively. We compared the methods' sensitivities, cell type detection, differential gene expression in PE, and performed histological validations.

Results: Mature syncytiotrophoblast were sampled ∼50x more efficiently after nuclei extraction. Yet, scRNA-seq was more sensitive in detection of genes, molecules and mature transcripts. In snRNA-seq, nuclei of all placental cell classes suffered ambient trophoblast contamination. Transcripts from extravillous trophoblast, stroma, vasculature and immune cells were profiled less comprehensively by single-nuclei RNA-seq (snRNA-seq), restricting cell-type detection. In EPE, we found dysregulation of angiogenic actors FLT1/PGF both in prefused syncytiotrophoblast after cell extraction, and mature syncytiotrophoblast after nuclei isolation. Disease-related stress and inflammation were undetected from nuclei.

Discussion: scRNA-seq has important advantages over snRNA-seq for comprehensive transcriptomics studies of the placenta, especially to understand cell-type resolved dysregulation in pathologies. Yet, to address the dilemma of an underrepresented syncytium, studies benefit from complementary nuclei extraction.