Unlocking the placenta accreta spectrum through single-cell gene targeting

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

PAS disease affects 1 in 272 births in the United States and can be a life-threatening pregnancy complication. PAS may result in caesarean hysterectomy, hemorrhage, and severe maternal and neonatal morbidity. Risk factors include caesarean section, dilatation and curettage, uterine surgery, and placenta previa.

Little information is available on the molecular pathophysiology of PAS, and recent literature has questioned the current understanding of PAS and highlighted the potentially important role of decidual scarring and uterine scar dehiscence. Therefore, it is necessary to evaluate the environment of placental trophoblasts.

We conducted a study to assess cellular heterogeneity and transcriptomic changes in PAS using single-cell and spatial RNA sequencing (RNAseq). Participants included women with or without PAS who underwent caesarean section between 34 and 40 weeks of pregnancy recruited between April 2021 and August 2022.

Previous uterine surgery, in vitro fertilization, and placenta previa have been reported as risk factors. Nearly all participants provided PAS-adhesive (PAS-A) and PAS-non-adhesive (PAS-NA) samples.

The PAS cohort included patients with ultrasound-suspected PAS selected in the third trimester of pregnancy and pathologically confirmed PAS after birth. Placental tissue samples were obtained within 1 hour of birth.

Placental samples were placed in Dulbeccos phosphate-buffered saline (PBS; Gibco, Carlsbad, CA) and processed in a room temperature laboratory. The biopsy tissue is mechanically separated and then broken down into smaller pieces for digestion. Centrifuge at 1200 rpm for 10 minutes.

Thaw frozen cells in a 37°C water bath and thaw cell suspension using the Chromium 10x Genomics 3′ single-cell gene platform (10xGenomics). Sequencing was done using Illumina NovaSeq S2 (Ilumina), and samples were merged into a single expression matrix using the cellranger aggr pipeline.

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

The FindAllMarkers function was used to identify cluster marker genes, and cell-to-cell communication was assessed using CellChat. Gene ontology analysis was performed using Metascape. This allows the definition of biological processes, protein functions and roles in canonical pathways.

31,406 single cells were analyzed through single-cell RNAseq, with an average of 1493 genes and 4293 unique transcripts per cell, and mitochondrial representation was less than 30%. Twenty-four clusters with distinct transcriptome profiles were discovered from gene expression, consisting of all 8 samples of PAS-A, PAS-NA, and control.

Cytotrophoblast (CTB) cluster 0 expresses 2 markers of nonproliferative stromal CTB, whereas CTB cluster 12 expresses more than 20 genes associated with villous CTB, as well as endogenous retroviruses associated with syncytial cell fusion ( ERV) transcripts. Multiple ERVs were also found in syncytiotrophoblast clusters.

A combination of decidual cells, endothelial cells, fibroblasts, extravillous trophoblasts, and immune cells has been reported among the cells at the maternal-fetal interface.Endothelial cells are located in the middle of these clusters and express classic markers including CD34, PLVAP, PECAM1 and MYH11.

Assessment of PAS gene expression by gene photopatterns revealed differential expression of PAS-A cells in clusters previously defined as endothelial cells. Furthermore, the highest differentially expressed genes (DEGs) of PAS-A were highly expressed in endothelial and decidual cell populations.

Significant regulation was observed in PAS genes affecting vascular development, growth factors, and cytoskeleton versus control genes. There were also differences in the transcriptomes of PAS-A and PAS-NA cell types compared to controls. Endothelial cell expression of PAS-A increased at least 3-fold compared with PAS-NA and controls.

The researchers found that 453 genes were differentially expressed in PAS-A compared with controls, and 861 genes were differentially expressed in PAS-NA compared with controls. Difference analysis between PAS-A and PAS-NA found 99 DEGs.

PAS-specific increases in collagen deposition have also been reported. Potential PAS disease markers can be identified using single-cell and spatial transcriptomics, and multiple 6 epidermal growth factor-like domains were found to be significantly specific for PAS.

These results demonstrate success in characterizing PAS disease at single-cell resolution to improve understanding of disease pathophysiology. The genetic targets identified by the researchers could be used to refine diagnostic assays in early pregnancy, track disease progression over time, and inform treatment discovery.

refer to

Afshar Y, Yin O, Jeong A, et al. Invasive placental lineage disorders at single-cell resolution: loss of decidual and endothelial cell boundary constraints. Am J Obstet Gynecol. 2024;230:443.e1-18. doi:10.1016/j.ajog.2023.10.001