Endometrial exosomes/microvesicles in the uterine microenvironment: a new paradigm for embryo-endometrial cross talk at implantation.

Exosomes are nanoparticles (∼100 nm diameter) released from cells, which can transfer small RNAs and mRNA via the extracellular environment to cells at distant sites. We hypothesised that exosomes or the slightly larger microvesicles (100-300 nm) are released from the endometrial epithelium into the uterine cavity, and that these contain specific micro (mi)RNA that could be transferred to either the trophectodermal cells of the blastocyst or to endometrial epithelial cells, to promote implantation. The aim of this study was to specifically identify and characterise exosomes/microvesicles (mv) released from endometrial epithelial cells and to determine whether exosomes/mv are present in uterine fluid. Immunostaining demonstrated that the tetraspanins, CD9 and CD63 used as cell surface markers of exosomes are present on the apical surfaces of endometrial epithelial cells in tissue sections taken across the menstrual cycle: CD63 showed cyclical regulation. Exosome/mv pellets were prepared from culture medium of endometrial epithelial cell (ECC1 cells) and from uterine fluid and its associated mucus by sequential ultracentifugation. Exosomes/mv were positively identified in all preparations by FACS and immunofluorescence staining following exosome binding to beads. Size particle analysis confirmed the predominance of particles of 50-150 nm in each of these fluids. MiRNA analysis of the ECC1 cells and their exosomes/mv demonstrated sorting of miRNA into exosomes/mv: 13 of the 227 miRNA were specific to exosomes/mv, while a further 5 were not present in these. The most abundant miRNA in exosomes/mv were hsa-miR-200c, hsa-miR-17 and hsa-miR-106a. Bioinformatic analysis showed that the exosome/mv-specific miRNAs have potential targets in biological pathways highly relevant for embryo implantation. Thus exosomes/mv containing specific miRNA are present in the microenvironment in which embryo implantation occurs and may contribute to the endometrial-embryo cross talk essential for this process.

Twist modulates human trophoblastic cell invasion via regulation of N-cadherin.

The invasion of extravillous cytotrophoblasts (EVT) into the underlying maternal tissues and vasculature is a key step in human placentation. The molecular mechanisms involved in the development of the invasive phenotype of EVT include many that were first discovered for their role in cancer cell metastasis. Previous studies have demonstrated that N-cadherin and its regulatory transcription factor Twist play important roles in the onset and progression of cancers, but their roles in human trophoblastic cell invasion is not clear. The goal of the study was to examine the role of Twist and N-cadherin in human trophoblastic cell invasion. Twist and N-cadherin mRNA and protein levels were determined by RT-PCR and Western blotting in human placental tissues, highly invasive EVT, and poorly invasive JEG-3 and BeWo cells. Whether IL-1ß and TGF-ß1 regulate Twist mRNA and protein levels in the EVT was also examined. A small interfering RNA strategy was employed to determine the role of Twist and N-cadherin in HTR-8/SVneo cell invasion. Matrigel assays were used to assess cell invasion. Twist and N-cadherin were highly expressed in EVT but were poorly expressed in JEG-3 and BeWo cells. IL-1ß and TGF-ß1 differentially regulated Twist expression in EVT in a time- and concentration-dependent manner. Small interfering RNA specific for Twist decreased N-cadherin and reduced invasion of HTR-8/SVneo cells. Similarly, a reduction in N-cadherin decreased the invasive capacity of HTR-8/SVneo cells. Twist is an upstream regulator of N-cadherin-mediated invasion of human trophoblastic cells.

Twist regulates cadherin-mediated differentiation and fusion of human trophoblastic cells.

CONTEXT: The formation of the multinucleated syncytiotrophoblast of the human placenta by the terminal differentiation and fusion of mononucleate cytotrophoblasts is a critical step in pregnancy. Previous studies have demonstrated that this cellular event is dependent on a progressive decrease in the levels of the cell-adhesion molecule, E-cadherin. OBJECTIVE: The aim of the study was to examine the role of Twist, a transcription factor identified as a key repressor of E-cadherin expression, in the differentiation of human trophoblastic cells. DESIGN: The expression of Twist or E-cadherin were first examined in first-trimester chorionic villi by immunohistochemistry. Gain- or loss-of-function studies on Twist were then performed in BeWo choriocarcinoma cells. The presence or absence of multinucleated syncytium was confirmed by indirect immunofluorescence using antibodies directed against Twist, E-cadherin, or desmoplakin, a cellular marker of mononucleate cytotrophoblasts. RESULTS: The formation of multinucleated syncytium was associated with increased Twist and a decreased E-cadherin expression. Similarly, exogenous expression of Twist resulted in a continuous and progressive decrease in E-cadherin expression and the subsequent formation of syncytium in BeWo cells maintained under normal culture conditions. In contrast, small interfering RNA specific for Twist inhibited 8-Br-cAMP (8-bromoadenosine 3',5'-cyclic monophosphate)-mediated differentiation and fusion of over time in culture. CONCLUSIONS: Twist is an upstream regulator of the E-cadherin-mediated terminal differentiation and fusion in a human trophoblastic cell line in vitro.

Differential effects of interleukin-1beta and transforming growth factor-beta1 on the expression of the inflammation-associated protein, ADAMTS-1, in human decidual stromal cells in vitro.

BACKGROUND: The pro-inflammatory cytokine, interleukin-1 beta (IL-1beta) promotes the proteolytic degradation of the extracellular matrix (ECM) of maternal decidua, a critical step in pregnancy that is counterbalanced by the expression of the anti-inflammatory cytokine, transforming growth factor-beta 1 (TGF-beta1). Recently, the inflammation-associated protein, ADAMTS-1, a member of the ADAMTS (A Disintegrin And Metalloproteinase with ThromboSpondin repeats) gene family of metalloproteinases has been assigned a central role in the formation and organization of tissues. In view of these observations, we have hypothesized that ADAMTS-1 contributes to the cytokine-mediated remodelling of decidual ECM. METHODS: The spatiotemporal expression of ADAMTS-1 in human endometrium was examined by immunohistochemistry. A quantitative-competitive (QC)-PCR strategy and western blot analysis was then employed to determine whether IL-1beta and TGF-beta1 regulate ADAMTS-1 mRNA and protein expression levels in primary cultures of stromal cells isolated from first trimester decidua. RESULTS: ADAMTS-1 expression is associated with decidualization of the endometrial stroma in vivo. IL-1beta increased whereas TGF-beta1 decreased ADAMTS-1 mRNA and protein levels in decidual stromal cell cultures in a concentration- and time-dependent manner. These regulatory effects were attenuated by function-perturbing antibodies specific for either cytokine. CONCLUSION: IL-1beta and TGF-beta1 differentially regulate ADAMTS-1 expression in human decidual stromal cells.