Transcriptomic analysis of the interaction of choriocarcinoma spheroids with receptive vs. non-receptive endometrial epithelium cell lines: an in vitro model for human implantation
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.
KeywordsImplantation 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.
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.
- 7.Weimar CH, Kavelaars A, Brosens JJ, Gellersen B, de Vreeden-Elbertse JM, Heijnen CJ, et al. Endometrial stromal cells of women with recurrent miscarriage fail to discriminate between high- and low-quality human embryos. PLoS One. 2012;7(7):e41424. https://doi.org/10.1371/journal.pone.0041424.Google Scholar
- 9.Brighton PJ, Maruyama Y, Fishwick K, Vrljicak P, Tewary S, Fujihara R, Muter J, Lucas ES, Yamada T, Woods L, Lucciola R, Hou Lee Y, Takeda S, Ott S, Hemberger M, Quenby S, Brosens JJ Clearance of senescent decidual cells by uterine natural killer cells in cycling human endometrium. eLife. 2017;6. doi: https://doi.org/10.7554/eLife.31274.
- 17.Dominguez F, Avila S, Cervero A, Martin J, Pellicer A, Castrillo JL, et al. A combined approach for gene discovery identifies insulin-like growth factor-binding protein-related protein 1 as a new gene implicated in human endometrial receptivity. J Clin Endocrinol Metab. 2003;88(4):1849–57. https://doi.org/10.1210/jc.2002-020724.Google Scholar
- 21.Tamm-Rosenstein K, Simm J, Suhorutshenko M, Salumets A, Metsis M. Changes in the transcriptome of the human endometrial Ishikawa cancer cell line induced by estrogen, progesterone, tamoxifen, and mifepristone (RU486) as detected by RNA-sequencing. PLoS One. 2013;8(7):e68907. https://doi.org/10.1371/journal.pone.0068907.Google Scholar
- 24.Boggavarapu NR, Berger C, von Grothusen C, Menezes J, Gemzell-Danielsson K, Lalitkumar PG. Effects of low doses of mifepristone on human embryo implantation process in a three-dimensional human endometrial in vitro co-culture system. Contraception. 2016;94(2):143–51. https://doi.org/10.1016/j.contraception.2016.03.009.Google Scholar
- 25.Carver J, Martin K, Spyropoulou I, Barlow D, Sargent I, Mardon H. An in-vitro model for stromal invasion during implantation of the human blastocyst. Hum Reprod. 2003;18(2):283–90.Google Scholar
- 33.Morgan M FSaGR. Morgan M, Falcon S and Gentleman R (2017). GSEABase: Gene set enrichment data structures and methods. R package version 1.40.1. 2017.Google Scholar
- 37.Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002;3(7):RESEARCH0034.Google Scholar
- 38.Borthwick JM, Charnock-Jones DS, Tom BD, Hull ML, Teirney R, Phillips SC, et al. Determination of the transcript profile of human endometrium. Mol Hum Reprod. 2003;9(1):19–33.Google Scholar
- 39.White FJ, Burghardt RC, Hu J, Joyce MM, Spencer TE, Johnson GA. Secreted phosphoprotein 1 (osteopontin) is expressed by stromal macrophages in cyclic and pregnant endometrium of mice, but is induced by estrogen in luminal epithelium during conceptus attachment for implantation. Reproduction. 2006;132(6):919–29. https://doi.org/10.1530/REP-06-0068.Google Scholar
- 40.Quenby S, Anim-Somuah M, Kalumbi C, Farquharson R, Aplin JD. Different types of recurrent miscarriage are associated with varying patterns of adhesion molecule expression in endometrium. Reprod BioMed Online. 2007;14(2):224–34.Google Scholar
- 47.Altmae S, Reimand J, Hovatta O, Zhang P, Kere J, Laisk T, et al. Research resource: interactome of human embryo implantation: identification of gene expression pathways, regulation, and integrated regulatory networks. Mol Endocrinol. 2012;26(1):203–17. https://doi.org/10.1210/me.2011-1196.Google Scholar
- 51.Chan C, Virtanen C, Winegarden NA, Colgan TJ, Brown TJ, Greenblatt EM. Discovery of biomarkers of endometrial receptivity through a minimally invasive approach: a validation study with implications for assisted reproduction. Fertil Steril. 2013;100(3):810–7. https://doi.org/10.1016/j.fertnstert.2013.04.047.Google Scholar
- 61.Chobotova K, Spyropoulou I, Carver J, Manek S, Heath JK, Gullick WJ, et al. Heparin-binding epidermal growth factor and its receptor ErbB4 mediate implantation of the human blastocyst. Mech Dev. 2002;119(2):137–44.Google Scholar
- 64.Modarres P, Tanhaei S, Tavalaee M, Ghaedi K, Deemeh MR, Nasr-Esfahani MH. Assessment of DPY19L2 deletion in familial and non-familial individuals with globozoospermia and DPY19L2 genotyping. Int J Fertil Steril. 2016;10(2):196–207.Google Scholar
- 70.Diaz-Gimeno P, Ruiz-Alonso M, Sebastian-Leon P, Pellicer A, Valbuena D, Simon C. Window of implantation transcriptomic stratification reveals different endometrial subsignatures associated with live birth and biochemical pregnancy. Fertil Steril. 2017;108(4):703–10 e3. https://doi.org/10.1016/j.fertnstert.2017.07.007.Google Scholar
- 71.Huang J, Qin H, Yang Y, Chen X, Zhang J, Laird S, et al. A comparison of transcriptomic profiles in endometrium during window of implantation between women with unexplained recurrent implantation failure and recurrent miscarriage. Reproduction. 2017;153(6):749–58. https://doi.org/10.1530/REP-16-0574.Google Scholar
- 72.Wang H, Pilla F, Anderson S, Martinez-Escribano S, Herrer I, Moreno-Moya JM, et al. A novel model of human implantation: 3D endometrium-like culture system to study attachment of human trophoblast (Jar) cell spheroids. Mol Hum Reprod. 2012;18(1):33–43. https://doi.org/10.1093/molehr/gar064.Google Scholar
- 74.Enciso M, Carrascosa JP, Sarasa J, Martinez-Ortiz PA, Munne S, Horcajadas JA, et al. Development of a new comprehensive and reliable endometrial receptivity map (ER map/ER grade) based on RT-qPCR gene expression analysis. Hum Reprod. 2018;33(2):220–8. https://doi.org/10.1093/humrep/dex370.Google Scholar
- 82.Franasiak JM, Holoch KJ, Yuan L, Schammel DP, Young SL, Lessey BA. Prospective assessment of midsecretory endometrial leukemia inhibitor factor expression versus alphanubeta3 testing in women with unexplained infertility. Fertil Steril. 2014;101(6):1724–31. https://doi.org/10.1016/j.fertnstert.2014.02.027.Google Scholar
- 83.Lessey BA, Castelbaum AJ. Integrins and implantation in the human. Rev Endocr Metab Disord. 2002;3(2):107–17.Google Scholar
- 84.Hirota Y, Osuga Y, Hasegawa A, Kodama A, Tajima T, Hamasaki K, et al. Interleukin (IL)-1beta stimulates migration and survival of first-trimester villous cytotrophoblast cells through endometrial epithelial cell-derived IL-8. Endocrinology. 2009;150(1):350–6. https://doi.org/10.1210/en.2008-0264.Google Scholar
- 86.Kurarmoto H, Hamano M, Imai M. HEC-1 cells. Hum Cell. 2002;15(2):81–95. https://doi.org/10.1111/j.1749-0774.2002.tb00103.x.Google Scholar
- 88.Rahimipour M, Salehnia M, Jafarabadi M. Morphological, ultrastructural, and molecular aspects of in vitro mouse embryo implantation on human endometrial mesenchymal stromal cells in the presence of steroid hormones as an implantation model. Cell J. 2018;20(3):369–76. https://doi.org/10.22074/cellj.2018.5221.Google Scholar