Transcriptomic analysis of the interaction of choriocarcinoma spheroids with receptive vs. non-receptive endometrial epithelium cell lines: an in vitro model for human implantation

Abstract

Purpose Several in vitro systems have been reported to model human implantation; however, the molecular dynamics of the trophoblast vs. the epithelial substrate during attachment have not been described. We have established an in vitro model which allowed us to dissect the transcriptional responses of the trophoblast and the receptive vs. non-receptive epithelium after coculture. Methods We established an in vitro system based on co-culture of (a) immortalized cells representing receptive (Ishikawa) or non-receptive (HEC-1-A) endometrial epithelium with (b) spheroids of a trophoblastic cell line (JEG-3) modified to express green fluorescent protein (GFP). After 48 h of co-culture, GFP+ (trophoblast cells) and GFP− cell fractions (receptive or nonreceptive epithelial cells) were isolated by fluorescence-activated flow cytometry (FACS) and subjected to RNA-seq profiling and gene set enrichment analysis (GSEA). Results Compared to HEC-1-A, the trophoblast challenge to Ishikawa cells differentially regulated the expression of 495 genes, which mainly involved cell adhesion and extracellular matrix (ECM) molecules. GSEA revealed enrichment of pathways related to cell division, cell cycle regulation, and metabolism in the Ishikawa substrate. Comparing the gene expression profile of trophoblast spheroids revealed that 1877 and 323 genes were upregulated or downregulated when co-cultured on Ishikawa substrates (compared to HEC-1-A), respectively. Pathways favorable to development, including tissue remodeling, organogenesis, and angiogenesis, were enhanced in the trophoblast compartment after co-culture of spheroids with receptive epithelium. By contrast, the co-culture with less receptive epithelium enriched pathways mainly related to trophoblast cell proliferation and cell cycle regulation. Conclusions Endometrial receptivity requires a transcriptional signature that determines the trophoblast response and drives attachment.

Association of the Cumulative Live Birth Rate with the Factors in Assisted Reproductive Technology: A Retrospective Study of 16,583 Women

“…This could be due to high-quality embryos being preferentially selected for transfer, and women without obtained live birth needed to perform repeated embryo transfers. It is an important reminder for clinicians not to pursue the number of transfers, and prepared endometrium is an important factor affecting the success of ART when top-quality embryos are transferred [ 59 ].…”

Trophoblast attachment to the endometrial epithelium elicits compartment-specific transcriptional waves in an in-vitro model

“…In order to analyse the transcriptional dynamics during the first 24 h of trophoblast-epithelium interaction, an in-vitro system modelling embryo attachment was established as previously described (Vergaro et al, 2019). Briefly, Ishikawa cells were used as the substrate (representing the receptive endometrial epithelium), while GFP+ JEG-3 spheroids represented the embryo trophoblast.…”

“…This group has recently determined that the receptive epithelium mounts a transcriptional response to trophoblast challenge that is severely muted when the epithelium is non-receptive (Vergaro et al, 2019). Combining cell sorting and RNA-seq analysis, the transcriptional responses that the trophoblast (modelled by spheroids of the JEG-3 trophoblastic cell line) and the receptive epithelium (modelled by the Ishikawa endometrial epithelial cell line) undergo during the first 8 and 24 h of attachment in vitro have been characterized.…”

Culture strategy as a modulator of target assessments: Functionality of suspension versus hanging drop‐derived choriocarcinoma spheroids as in vitro model of embryo implantation

“…Coculture of provided spheroids on the top of a monolayer of Ishikawa cell line is a conventional method of attachment assay. 2,19,20 In a time-scheduled manner (10,20,40,60,90, 120 min) determined number of spheroids (100 per each well of 96-well culture plates) seeded on the confluent monolayer Ishikawa cell line. At each time point, the media were removed and the wells were washed with PBS to remove unattached spheroids.…”

“…12,15,17 SC of choriocarcinoma cell lines has been extensively applied as the routine technique to study multiple molecular mechanisms that are integrated into different stages of the implantation process. 2,11,[18][19][20][21] Since functional characteristics of spheroids to motivate endometrium response is a unique challenge in resembling implantation, understanding the functionality of 3D spheroids in endometrium confrontation experiments may be helpful to achieve a consensus about a standard strategy to provide them. Although two previous studies have focused precisely on morphological and functional characteristics of SC choriocarcinoma spheroids, 1,3 no comparative functional studies have been performed to understand spheroids-endometrium cross-talks beyond the cell aggregation protocols.…”