Unlocking placenta accreta spectrum with single-cell gene targets

Gene targets may be effective for tracking the progression of placenta accreta spectrum (PAS) disorders, according to a recent study published in the American Journal of Obstetrics & Gynecology.

One in 272 US births are impacted by PAS disorders, which can potentially be life-threatening pregnancy complications. PAS may lead to cesarean hysterectomy, hemorrhage, and severe maternal and neonatal morbidity. Risk factors include cesarean delivery, dilation and curettage, uterine procedures, and placenta previa.

There is little information about the molecular pathophysiology of PAS, with recent literature questioning the current understanding of PAS and highlighting the potentially significant role of the scarred decidua and uterine scar dehiscence. Therefore, it is necessary to evaluate the environment where placental trophoblasts are located.

Investigators conducted a study to evaluate the cellular heterogeneity and transcriptomic changes in PAS using single-cell and spatial RNA sequencing (RNAseq). Participants included women recruited from April 2021 to August 2022 with and without PAS who underwent cesarean delivery from 34 to 40 weeks’ gestation.

Prior uterine surgery, in vitro fertilization, and placenta previa were reported as risk factors. A PAS adherent (PAS-A) sample and a PAS nonadherent (PAS-NA) sample were provided by almost all participants.

The PAS cohort included patients with ultrasound-suspected PAS selected in the third trimester, with PAS confirmed by pathology after birth. Placenta tissue samples were obtained within 1 hour following birth.

Placenta samples were placed in Dulbecco’s phosphate-buffered saline (PBS; Gibco, Carlsbad, California) and underwent processing in a room temperature laboratory. Biopsies were mechanically disassociated, then broken down into smaller pieces for digestion. Centrifuging was performed at 1200 revolutions per minute for 10 minutes.

Frozen cells were thawed in a 37°C water bath, and thawed cell suspension was performed using the Chromium 10x Genomics 3' Single Cell Gene Platform (10xGenomics). Sequencing was accomplished using the Illumina NovaSeq S2 (Ilumina), with samples merged into a single expression matrix using the cellranger aggr pipeline.

Cell clustering was accomplished using the R package Seurat (version 3.1.2). Cells with under 500 or over 50,000 transcripts, under 100 genes, or over 50% of mitochondrial expression were considered low quality and excluded.

The FindAllMarkers function was used to identify cluster marker genes, while cell-to-cell communication was assessed using CellChat. A gene ontology analysis was performed using Metascape. This allowed biological processes, protein functions, and roles in canonical pathways to be defined.

There were 31,406 individual cells analyzed by single-cell RNAseq, revealing an average 1493 genes, 4293 unique transcripts per cell, and mitochondrial expression below 30%. Twenty-four clusters with distinct transcriptome profiles were found from gene expression, comprised of all 8 samples across PAS-A, PAS-NA, and controls.

Cytotrophoblasts (CTB) cluster 0 expressed 2 markers of nonproliferative interstitial CTBs, while CTBs cluster 12 expressed over 20 genes linked to villous CTBs, as well as an endogenous retroviral (ERV) transcript connected to syncytial cell fusion. Multiple ERVs were also identified in syncytiotrophoblasts clusters.

A combination of decidual cells, endothelial cells, fibroblasts, extravillous trophoblasts, and immune cells was reported in cells of the maternal-fetal interface. Endothelial cells were in the middle of these cluster groups, and classic markers expressed included CD34, PLVAP, PECAM1 and MYH11.

Gene expression for PAS was assessed by gene phototype, revealing different representation of PAS-A cells in clusters previously defined as endothelial cells. Additionally, the top differentially expressed genes (DEGs) for PAS-A were highly expressed within populations of endothelial and decidual cells.

Significant regulation was observed in PAS genes impacting blood vessel development, growth factors, and the cytoskeletal vs control genes. Differences in transcriptome were also observed for PAS-A and PAS-NA cell types vs controls. PAS-A had an increased expression of endothelial cells at least 3-fold vs PAS-NA and controls.

Investigators identified 453 genes differentially expressed in PAS-A when compared to controls, and 861 when comparing PAS-NA to controls. Ninety-nine DEGs were found during differential analysis between PAS-A and PAS-NA.

A PAS-specific increase in collagen deposition was also reported. Potential PAS disorder markers could be determined using single-cell and spatial transcriptomics, with the epidermal growth factor–like domain multiple 6 found to be significantly specific to PAS.

These results indicated success when characterizing PAS disorders at single-cell resolution to improve understanding of disease pathophysiology. Investigators concluded gene targets, “may be used to refine diagnostic assays in early pregnancy, track disease progression over time, and inform therapeutic discoveries.”

Reference

Afshar Y, Yin O, Jeong A, et al. Placenta accreta spectrum disorder at single-cell resolution: a loss of boundary limits in the decidua and endothelium. Am J Obstet Gynecol. 2024;230:443.e1-18. doi:10.1016/j.ajog.2023.10.001