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

  • Paula Vergaro
  • Gustavo Tiscornia
  • Amelia Rodríguez
  • Josep Santaló
  • Rita VassenaEmail author
Reproductive Physiology and Disease



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 co-culture.


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 non-receptive epithelial cells) were isolated by fluorescence-activated flow cytometry (FACS) and subjected to RNA-seq profiling and gene set enrichment analysis (GSEA).


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.


Endometrial receptivity requires a transcriptional signature that determines the trophoblast response and drives attachment.


Implantation Attachment Endometrial receptivity Transcriptomics 



The authors wish to thank all members of the Basic Laboratory from Clínica EUGIN, especially Montserrat Barragán and Anna Ferrer, for critical discussion; José Buratini from Sao Paulo State University (Brasil) for critical revision of the manuscript; Camille Stephan Otto from the Biostatistics/Bioinformatics facility of the Institute for Research in Biomedicine (Barcelona) for bioinformatics analysis; Charles Pineau, Natalie Melaine, and Emmanuelle Com from Proteomics Core Facility Biogenouest (Rennes) for assistance with data analysis; and Prof. Daniel Grinberg from Universitat de Barcelona for technical support.

Author’s contribution

Paula Vergaro: experimental execution, study design, data analysis, and manuscript preparation. Gustavo Tiscornia: study design and supervision, data analysis, manuscript edition, and expert knowledge. Amelia Rodríguez: study supervision. Josep Santaló: study supervision, expert knowledge, and manuscript edition. Rita Vassena: study design and supervision, expert knowledge, and manuscript edition.


This work was supported by intramural funding of Clínica EUGIN and by the Secretary for Universities and Research of the Ministry of Economy and Knowledge of the Government of Catalonia (GENCAT 2015 DI 050).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10815_2019_1442_MOESM1_ESM.pdf (5 kb)
Supplementary Fig. 1 Principal component analysis representing all samples from experimental triplicates according to principal component 1 (PC1) and principal component 2 (PC2): HEC-1-A control (H-c), HEC-1-A substrates co-cultured with JEG-3 spheroids (H-co-S), Ishikawa control (I-c), Ishikawa substrates co-cultured with JEG-3 spheroids (I-co-S), JEG-3 spheroids control (S-c), JEG-3 spheroids co-cultured with HEC-1-A substrates (S-co-H) and JEG-3 spheroids co-cultured with Ishikawa substrates (S-co-I). (PDF 5 kb)
10815_2019_1442_MOESM2_ESM.xlsx (6.4 mb)
Supplementary file S1 List of differentially expressed genes between HEC-1-A control (H-c) vs. Ishikawa control (I-c). Statistical significance was set at absolute log2FC cutoff of 1 and adjusted p-value <0.05. Negative fold changes mean that expression in H-c is lower than that in I-c; positive fold changes mean expression in H-c is higher than that in I-c. (XLSX 6561 kb)
10815_2019_1442_MOESM3_ESM.xlsx (5.6 mb)
Supplementary file S2 List of differentially expressed genes between Ishikawa substrates co-cultured with JEG-3 spheroids (I-co-S) vs. Ishikawa control (I-c). Statistical significance was set at absolute log2FC cutoff of 1 and adjusted p-value <0.05. Negative fold changes mean that expression in I-co-S is lower than that in I-c; positive fold changes mean expression in I-co-S is higher than that in I-c. (XLSX 5739 kb)
10815_2019_1442_MOESM4_ESM.xlsx (2.6 mb)
Supplementary file S3 List of differentially expressed genes between HEC-1-A substrates co-cultured with JEG-3 spheroids (H-co-S) vs. HEC-1-A control (H-c). Statistical significance was set at absolute log2FC cutoff of 1 and adjusted p-value <0.05. Negative fold changes mean that expression in H-co-S is lower than that in H-c; positive fold changes mean expression in H-co-S is higher than that in H-c. (XLSX 2680 kb)
10815_2019_1442_MOESM5_ESM.xlsx (5.7 mb)
Supplementary file S4 List of differential expression patterns between HEC-1-A substrates co-cultured with JEG-3 spheroids (H-co-S) vs. HEC-1-A control (H-c) compared to Ishikawa substrates co-cultured with JEG-3 spheroids (I-co-S) vs. Ishikawa control (I-c). Statistical significance was set at absolute log2FC cutoff of 1 and adjusted p-value <0.05. Negative fold changes mean that difference in gene expression is enriched in H-co-S vs. H-c; positive fold changes mean that difference in gene expression is enriched in I-co-S vs. I-c. (XLSX 5788 kb)
10815_2019_1442_MOESM6_ESM.xlsx (6.1 mb)
Supplementary file S5 List of differentially expressed genes between JEG-3 spheroids co-cultured with Ishikawa substrates (S-co-I) vs. JEG-3 spheroids control (S-c), Statistical significance was set at absolute log2FC cutoff of 1 and adjusted p-value <0.05. Negative fold changes mean that expression in S-co-I is lower than that in S-c; positive fold changes mean expression in S-co-I is higher than that in S-c. (XLSX 6195 kb)
10815_2019_1442_MOESM7_ESM.xlsx (5.7 mb)
Supplementary file S6 List of differentially expressed genes between JEG-3 spheroids co-cultured with HEC-1-A substrates (S-co-H) vs. JEG-3 spheroids control (S-c), Statistical significance was set at absolute log2FC cutoff of 1 and adjusted p-value <0.05. Negative fold changes mean that expression in S-co-H is lower than that in S-c; positive fold changes mean expression in S-co-H is higher than that in S-c. (XLSX 5796 kb)
10815_2019_1442_MOESM8_ESM.xlsx (5.8 mb)
Supplementary file S7 List of differentially expressed genes between JEG-3 spheroids co-cultured with HEC-1-A substrates (S-co-H) vs. JEG-3 spheroids co-cultured with Ishikawa substrates (S-co-I). Statistical significance was set at absolute log2FC cutoff of 1 and adjusted p-value <0.05. Negative fold changes mean that expression in S-co-H is lower than that in S-co-I; positive fold changes mean expression in S-co-H is higher than that in S-co-I. (XLSX 5954 kb)
10815_2019_1442_MOESM9_ESM.xlsx (11 kb)
Supplementary file S8 List of the 208 common genes between JEG-3 spheroids co-cultured with Ishikawa substrates and Ishikawa substrates co-cultured with JEG-3 spheroids (“S-co-H vs. S-co-I” and “I-co-S vs. I-c” comparisons). (XLSX 10 kb)


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Paula Vergaro
    • 1
    • 2
  • Gustavo Tiscornia
    • 1
    • 3
  • Amelia Rodríguez
    • 1
  • Josep Santaló
    • 2
  • Rita Vassena
    • 1
    Email author
  1. 1.Clínica EUGINBarcelonaSpain
  2. 2.Facultat de Biociències, Departament de Biologia Cel·lular, de Fisiologia i d’ImmunologiaUniversitat Autònoma de BarcelonaBarcelonaSpain
  3. 3.Centro de Investigação em Biomedicina (CBMR)Universidade do AlgarveFaroPortugal

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