Integrin α4-positive human trophoblast progenitors: functional characterization and transcriptional regulation.

STUDY QUESTION: What are the functional characteristics and transcriptional regulators of human trophoblast progenitor cells (TBPCs)? SUMMARY ANSWER: TBPC lines established from the human smooth chorion by cell sorting for integrin α4 expressed markers of stemness and trophoblast (TB) stage-specific antigens, invaded Matrigel substrates and contributed to the cytotrophoblasts (CTBs) layer of smooth chorion explants with high-mobility group protein HMGI-C (HMGA2) and transcription factor GATA-4 (GATA4) controlling their progenitor state and TB identity. WHAT IS KNOWN ALREADY: Previously, we reported the derivation of TBPC lines by trypsinization of colonies that formed in cultures of chorionic mesenchyme cells that were treated with an activin nodal inhibitor. Microarray analyses showed that, among integrins, α4 was most highly expressed, and identified HMGA2 and GATA4 as potential transcriptional regulators. STUDY DESIGN, SIZE, DURATION: The aim of this study was to streamline TBPC derivation across gestation. High-cell surface expression of integrin α4 enabled the use of a fluorescence-activated cell sorter (FACS) approach for TBPC isolation from the human smooth chorion (n = 6 lines). To confirm their TBPC identity, we profiled their expression of stemness and TB markers, and growth factor receptors. At a functional level, we assayed their invasive capacity (n = 3) and tropism for the CTB layer of the smooth chorion (n = 3). At a molecular level, we studied the roles of HMGA2 and GATA4. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Cells were enzymatically disassociated from the human smooth chorion across gestation. FACS was used to isolate the integrin α4-positive population. In total, we established six TBPC lines, two per trimester. Their identity was determined by immunolocalization of a suite of antigens. Function was assessed via Matrigel invasion and co-culture with explants of the human smooth chorion. An siRNA approach was used to down-regulate HMGA2 and GATA4 expression and the results were confirmed by immunoblotting and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses. The endpoints analyzed included proliferation, as determined by 5-bromo-2'-deoxyuridine (BrDU) incorporation, and the expression of stage-specific antigens and hormones, as determined by qRT-PCR and immunostaining approaches. MAIN RESULTS AND THE ROLE OF CHANCE: As with the original cell lines, the progenitors expressed a combination of human embryonic stem cell and TB markers. Upon differentiation, they primarily formed CTBs, which were capable of Matrigel invasion. Co-culture of the cells with smooth chorion explants enabled their migration through the mesenchyme after which they intercalated within the chorionic CTB layer. Down-regulation of HMGA2 showed that this DNA-binding protein governed their self-renewal. Both HMGA2 and GATA4 had pleitropic effects on the cells' progenitor state and TB identity. LIMITATIONS, REASONS FOR CAUTION: This study supported our hypothesis that TBPCs from the chorionic mesenchyme can contribute to the subpopulation of CTBs that reside in the smooth chorion. In the absence of in vivo data, which is difficult to obtain in humans, the results have the limitations common to all in vitro studies. WIDER IMPLICATIONS OF THE FINDINGS: The accepted view is that progenitors reside among the villous CTB subpopulation. Here, we show that TBPCs also reside in the mesenchymal layer of the smooth chorion throughout gestation. We theorize that they can contribute to the CTB layer in this region. This phenomenon may be particularly important in pathological situations when CTBs of the smooth chorion might provide a functional reserve for CTBs of the placenta proper. STUDY FUNDING/COMPETING INTERESTS: Research reported in this publication was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health under award P50HD055764. O.G., N.L., K.O., A.P., T.G.-G., M.K., A.B., M.G. have nothing to disclose. S.J.F. received licensing fees and royalties from SeraCare Life Sciences for trisomic TBPC lines that were derived according to the methods described in this manuscript. TRIAL REGISTRATION NUMBER: N/A.

Proteomics of embryonic implantation.

Implantation is a complex process involving an intricate cascade of molecular interactions between the implanting blastocyst and the receptive endometrium. The molecular basis of endometrial receptivity and the mechanisms by which the blastocyst first adheres to the luminal epithelium and then penetrates into the stroma are only just beginning to be resolved. Advances in "omics" technologies, particularly proteomics and metabolomics, are set to have a major impact on the development of this field. In the wake of this information, novel targets for contraceptive intervention may become apparent.

Proteomic analysis of the human receptive versus non-receptive endometrium using differential in-gel electrophoresis and MALDI-MS unveils stathmin 1 and annexin A2 as differentially regulated.

BACKGROUND: The transcriptome of the endometrium throughout the menstrual cycle has been described in recent years. However, the proteomic of the window of implantation remains unknown. The aim of this study was to compare the proteome of the human endometrium in the pre-receptive phase versus the receptive phase by identifying and quantifying the proteins differentially expressed using differential in-gel electrophoresis (DIGE) and mass spectometry (MS). METHODS: Endometrial biopsies were collected at days 2 (pre-receptive) and 7 (receptive) after the urinary luteal hormone surge in the same menstrual cycle from eight fertile women (corresponding to days 16 and 21 of the menstrual cycle). Proteins were extracted and labeled with CyDye DIGE fluorofores and separated using two-dimensional gel electrophoresis. RESULTS: Image analysis using the DeCyder software followed by protein identification by matrix-assisted laser desorption/ionization-MS and database searching revealed 32 differentially expressed proteins, although only annexin A2 and stathmin 1 were consistently regulated in the two experiments performed. Validation and localization of annexin A2 and stathmin 1 were performed by western blot and immunohistochemistry. Annexin A2 and stathmin 1 were investigated using an endometrial refractoriness model. The results highlight the key potential of these proteins as possible targets for human endometrial receptivity and interception. CONCLUSION: This study shows that the human endometrium has a differential proteomic repertoire during the window of implantation. Consequently, we identified annexin A2 and stathmin 1 as differentially expressed molecules in the receptive endometrium.

How endometrial secretomics can help in predicting implantation.

The receptive endometrium represents a physiologic state of the uterus when embryo implantation is possible. It occurs at a discreet stage of the menstrual cycle referred to as window of implantation, outside of which the uterus is refractory to the initiation of pregnancy. In modern society, assisted reproductive technologies (ART) are an ever-growing demand to counter infertility; however, pregnancy rates remain below expectations, not least because current diagnostic tools fail to provide accurate assessment of endometrial receptivity. In the last decade, widespread arrival of large-scale analytical techniques has brought a stream of studies seeking to identify specific biomarkers of endometrial receptivity by extracting global molecular information from endometrial biopsies. The latter are an undesired requirement for dating the endometrium, which has prompted development of alternative strategies whereby large-scale analyses and non-invasive methods can converge. In this context, secretomics represents an attractive possibility to assess endometrial maturation and receptivity. Endometrial-cell secretions poured into the uterine cavity are suitable for collection and analysis without the need of biopsying, and might provide important additional molecular information reflective of endometrial physiology and day of cycle. If properly validated, the outgoing results would represent a step forward in the development of diagnostic tools to assess endometrial receptivity.