title: Deregulation of the endometrial stromal cell secretome precedes embryo implantation failure.
abstract: Is implantation failure following ART associated with a perturbed decidual response in endometrial stromal cells (EnSCs)?
  
  
  
  Dynamic changes in the secretome of decidualizing EnSCs underpin the transition of a hostile to a supportive endometrial microenvironment for embryo implantation; perturbation in this transitional pathway prior to ART is associated with implantation failure.
  
  
  
  Implantation is the rate-limiting step in ART, although the contribution of an aberrant endometrial microenvironment in IVF failure remains ill defined.
  
  
  
  In vitro characterization of the temporal changes in the decidual response of primary EnSCs isolated prior to a successful or failed ART cycle. An analysis of embryo responses to secreted cues from undifferentiated and decidualizing EnSCs was performed. The primary clinical outcome of the study was a positive urinary pregnancy test 14 days after embryo transfer.
  
  
  
  Primary EnSCs were isolated from endometrial biopsies obtained prior to IVF treatment and cryopreserved. EnSCs from 10 pregnant and 10 non-pregnant patients were then thawed, expanded in culture, subjected to clonogenic assays, and decidualized for either 2 or 8 days. Transcript levels of decidual marker gene [prolactin (PRL), insulin-like growth factor binding protein 1 (IGFBP1) and 11β-hydroxysteroid dehydrogenase (HSD11B1)] were analysed using real-time quantitative PCR and temporal secretome changes of 45 cytokines, chemokines and growth factors were measured by multiplex suspension bead immunoassay. The impact of the EnSC secretome on human blastocyst development was scored morphologically; and embryo secretions in response to EnSC cues analyzed by multiplex suspension bead immunoassay.
  
  
  
  Clonogenicity and induction of decidual marker genes were comparable between EnSC cultures from pregnant and non-pregnant group groups (P > 0.05). Analysis of 23 secreted factors revealed that successful implantation was associated with co-ordinated secretome changes in decidualizing EnSCs, which were most pronounced on Day 2 of differentiation: 17 differentially secreted proteins on Day 2 of decidualization relative to undifferentiated (Day 0) EnSCs (P < 0.05); 11 differentially secreted proteins on Day 8 relative to Day 2 (P < 0.05); and eight differentially secreted proteins on Day 8 relative to Day 0 (P < 0.05). By contrast, failed implantation was associated with a disordered secretome response. Blastocyst development was compromised when cultured for 24 h in medium conditioned by undifferentiated EnSCs when compared to decidualizing EnSCs. Analysis of the embryo microdroplets revealed that human blastocysts mount a secretory cytokine response to soluble decidual factors produced during the early (Day 2) but not late phase (Day 8) of differentiation. The embryo responses to secreted factors from decidualizing EnSCs were comparable between the pregnant and non-pregnant group (P > 0.05).
  
  
  
  Not applicable.
  
  
  
  Although this study uses primary EnSCs and human embryos, caution is warranted when extrapolating the results to the in vivo situation because of the correlative nature of the study and limited sample size.
  
  
  
  Our finding raises the prospect that endometrial analysis prior to ART could minimize the risk of treatment failure.
  
  
  
  This work was supported by funds from the Biomedical Research Unit in Reproductive Health, a joint initiative of the University Hospitals Coventry & Warwickshire NHS Trust and Warwick Medical School, the University of Nottingham and Nurture Fertility, and the National Medical Research Council, Singapore (NMRC/BNIG14NOV023), the "Instituut voor Innovatie door Wetenschap en Technologie" (IWT, Flanders, Belgium), the "Fonds voor Wetenschappelijk Onderzoek" (FWO, Flanders, Belgium) and the "Wetenschappelijk Fonds Willy Gepts" (WFWG, UZ Brussel). The authors have declared that no conflict of interest exists.
authors.affiliation: Division of Biomedical Sciences, Clinical Science Research Laboratories, Warwick Medical School, University of Warwick, Coventry CV2 2DX, UK.
authors.forename: Ruban Rex
authors.initials: RR
authors.lastname: Peter Durairaj
authors.affiliation: Reproductive Immunology and Implantation, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, Brussels, Belgium.
authors.forename: Asma
authors.initials: A
authors.lastname: Aberkane
authors.affiliation: Division of Child Health, Obstetrics & Gynaecology, School of Medicine, University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, UK.|Department of Obstetrics and Gynaecology, Addenbrooke's Hospital, Cambridge CB2 0QQ, UK.
authors.forename: Lukasz
authors.initials: L
authors.lastname: Polanski
authors.affiliation: Division of Biomedical Sciences, Clinical Science Research Laboratories, Warwick Medical School, University of Warwick, Coventry CV2 2DX, UK.
authors.forename: Yojiro
authors.initials: Y
authors.lastname: Maruyama
authors.affiliation: Division of Child Health, Obstetrics & Gynaecology, School of Medicine, University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, UK.|Department of Obstetrics and Gynaecology, Addenbrooke's Hospital, Cambridge CB2 0QQ, UK.
authors.forename: Miriam
authors.initials: M
authors.lastname: Baumgarten
authors.affiliation: Division of Biomedical Sciences, Clinical Science Research Laboratories, Warwick Medical School, University of Warwick, Coventry CV2 2DX, UK.|Department of Reproductive Medicine, KK Women's and Children's Hospital, 100 Bukit Timah Road, Singapore 229899, Singapore.
authors.forename: Emma S
authors.initials: ES
authors.lastname: Lucas
authors.affiliation: Division of Biomedical Sciences, Clinical Science Research Laboratories, Warwick Medical School, University of Warwick, Coventry CV2 2DX, UK.|Department of Reproductive Medicine, KK Women's and Children's Hospital, 100 Bukit Timah Road, Singapore 229899, Singapore.
authors.forename: Siobhan
authors.initials: S
authors.lastname: Quenby
authors.affiliation: KK Research Centre, KK Women's and Children's Hospital, 100 Bukit Timah Road, Singapore 229899, Singapore.|Nurture Fertility, The East Midlands Fertility Centre, Bostocks Lane, Nottingham NG10 5QG, UK.
authors.forename: Jerry K Y
authors.initials: JKY
authors.lastname: Chan
authors.affiliation: Division of Child Health, Obstetrics & Gynaecology, School of Medicine, University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, UK.|Obstetrics & Gynaecology-Academic Clinical Program, Duke-NUS Medical School, 8 College Road, Singapore 169857, Singapore.
authors.forename: Nick
authors.initials: N
authors.lastname: Raine-Fenning
authors.affiliation: Division of Biomedical Sciences, Clinical Science Research Laboratories, Warwick Medical School, University of Warwick, Coventry CV2 2DX, UK.|Department of Reproductive Medicine, KK Women's and Children's Hospital, 100 Bukit Timah Road, Singapore 229899, Singapore.
authors.forename: Jan J
authors.initials: JJ
authors.lastname: Brosens
authors.affiliation: Reproductive Immunology and Implantation, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, Brussels, Belgium.
authors.forename: Hilde
authors.initials: H
authors.lastname: Van de Velde
authors.affiliation: KK Research Centre, KK Women's and Children's Hospital, 100 Bukit Timah Road, Singapore 229899, Singapore.|Department of Obstetrics and Gynaecology, Addenbrooke's Hospital, Cambridge, CB2 0QQ, UK.
authors.forename: Yie Hou
authors.initials: YH
authors.lastname: Lee
mesh_terms: D043205:11-beta-Hydroxysteroid Dehydrogenase Type 1 / Q000235:genetics / Q000378:metabolism; D000328:Adult; D015415:Biomarkers / Q000378:metabolism; D001755:Blastocyst / Q000166:cytology / Q000378:metabolism*; D002454:Cell Differentiation; D016207:Cytokines / Q000235:genetics / Q000378:metabolism; D003656:Decidua / Q000166:cytology / Q000378:metabolism*; D010064:Embryo Implantation; D005260:Female; D005307:Fertilization in Vitro; D005786:Gene Expression Regulation; D006801:Humans; D018970:Insulin-Like Growth Factor Binding Protein 1 / Q000235:genetics / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D016018:Least-Squares Analysis; D008297:Male; D011247:Pregnancy; D011388:Prolactin / Q000235:genetics / Q000378:metabolism; D017154:Stromal Cells / Q000166:cytology / Q000378:metabolism*
chemical_list: D015415:Biomarkers; D016207:Cytokines; C032231:IGFBP1 protein, human; D018970:Insulin-Like Growth Factor Binding Protein 1; D036341:Intercellular Signaling Peptides and Proteins; D011388:Prolactin; D043205:11-beta-Hydroxysteroid Dehydrogenase Type 1; C468386:HSD11B1 protein, human
keywords: decidualization; embryo; endometrium; implantation; secretome; stem cells