216. Activin A regulates trophoblast cell adhesion: implications for uterine receptivity and embryo implantation

2008 ◽  
Vol 20 (9) ◽  
pp. 16
Author(s):  
C. J. Stoikos ◽  
A. O.'Connor ◽  
L. A. Salamonsen ◽  
E. Dimitriadis
Keyword(s):  
Embryo Implantation ◽  
Uterine Cavity ◽  
Activin A ◽  
Trophoblast Cell ◽  
Collagen I ◽  
Trophoblast Invasion ◽  
Uterine Receptivity ◽  
Adhesive Properties ◽  
Secretory Phase ◽  
Human Trophoblast

Embryo implantation involves blastocyst attachment to the endometrial luminal epithelium, followed by trophoblast invasion. This process involves a coordinated crosstalk between the implanting blastocyst and the endometrium. Adhesion molecules play an instrumental role during implantation and are regulated by a variety of factors including cytokines and growth factors. Activin A, a TGF-β superfamily member, has been detected in uterine washings,1 and its subunit, β A, is produced by endometrial glands during the secretory phase of the menstrual cycle.2 In endometriosis, a disease that associated with sub-fertility, β A immunostaining is increased in endometrial glands,3 suggesting higher levels of activin A secreted into the uterine lumen could contribute to sub-fertility observed in endometriosis. Therefore we hypothesised that activin A secretion into the uterine cavity affects the adhesive properties of the cells present at the maternal-fetal interface. The aims of the study were to measure and compare activin A secretion in uterine washings from women with and without endometriosis and to demonstrate whether activin A regulates adhesion to extracellular matrix (ECM) components. Uterine washings (5 mL of sterile saline) were collected from women with and without endometriosis during the secretory phase. Activin A was measured by ELISA. HTR8 (human trophoblast cell-line) cells were treated with rhActivin A (50 ng/mL) and assessed for binding to fibronectin, laminin, vitronectin, collagen I and IV. Activin A (>10pg/mL) was detectable in uterine washings from women with and without endometriosis and levels were elevated in endometriosis patients. Untreated HTR8 cells adhered maximally to fibronectin, collagen I and collagen IV with low binding to vitronectin and laminin. Following activin treatment, HTR8 cell binding to fibronectin, collagen I and IV was significantly decreased (n = 3, P < 0.05). These results suggest that activin A regulates the adhesive properties of the blastocyst during implantation. This study also implies that abnormalities in local activin A levels during endometrial receptivity may contribute to sub-fertility in women. (1) Petraglia et., al. (1998) J Clin Endocrinol Metab. (2) Jones et., al. (2000) Mol Hum Reprod. (3) Rombauts et., al. (2006) Aust N Z J Obstet Gynaecol.

155. UNDERSTANDING THE CROSSTALK IN THE HUMAN UTERINE CAVITY: ROLES FOR SOLUBLE MEDIATORS DURING EMBRYO IMPLANTATION

2010 ◽  
Vol 22 (9) ◽  
pp. 73
Author(s):  
N. Hannan ◽  
P. Paiva ◽  
K. L. Meehan ◽  
C. Hincks ◽  
L. J. F. Rombauts ◽  
...  
Keyword(s):  
Menstrual Cycle ◽  
Embryo Implantation ◽  
Uterine Cavity ◽  
Uterine Receptivity ◽  
Adhesive Properties ◽  
Infertile Women ◽  
Functional Studies ◽  
Glandular Secretion ◽  
Uterine Fluid ◽  
Insight Into

Embryo implantation requires synchronized dialogue between a receptive endometrium and an activated blastocyst via locally produced soluble mediators. During the mid-secretory (MS) phase of the menstrual cycle there is increased glandular secretion into the uterine lumen. These secretions contain important mediators that modulate the endometrium and support the conceptus during implantation. Analysis of the composition of uterine fluid across the menstrual cycle and in fertile and infertile women will, therefore, provide new insights into uterine receptivity. We hypothesized that multiplex platform analysis of human uterine lavages would identify soluble mediators important for the establishment of pregnancy in humans. Lavages were collected (by flushing the uterine cavity with 5mL of saline) from fertile and infertile women during the MS phase and from fertile women during the mid-proliferative (MP) phase of the menstrual cycle. Comparison of lavages from the three cohorts was performed using quantitative MilliplexTM Luminex® cytokine/chemokine assays containing 42-analytes. Luminex analysis detected a number of cytokines in uterine fluid, revealing 8 soluble mediators previously unknown in the endometrium and present in human uterine fluid including, PDGF-AA, TNFβ, sIL-2Rα, Flt-3 ligand, sCD40L, IL-7, IFNα2 and GRO. Furthermore comparison of the three cohorts revealed VEGF levels were significantly higher in the fertile (MS) fluid when compared to infertile. Functional studies demonstrated that rhVEGF treatment significantly increased the adhesive properties in cells present at the maternal-fetal interface. These findings suggest VEGF plays a role in regulating embryo implantation. Furthermore identifying the soluble mediators in uterine fluid may provide potential markers of endometrial receptivity, insight into the unique microenvironment essential for pregnancy and a profile of maternal factors that influence the implanting blastocyst.

scholarly journals Activin A Increases Human Trophoblast Invasion by Inducing SNAIL-Mediated MMP2 Up-Regulation Through ALK4

2015 ◽  
Vol 100 (11) ◽  
pp. E1415-E1427 ◽  
Author(s):  
Yan Li ◽  
Christian Klausen ◽  
Hua Zhu ◽  
Peter C. K. Leung
Keyword(s):  
Cell Invasion ◽  
Primary Cultures ◽  
First Trimester ◽  
Activin A ◽  
Trophoblast Cell ◽  
Trophoblast Invasion ◽  
Type I ◽  
Dependent Manner ◽  
Human Trophoblast ◽  
Protein Levels

Context: Activin A increases matrix metalloproteinase (MMP) 2 expression and cell invasion in human trophoblasts, but whether the expression of MMP2 is essential for the proinvasive effect of activin A has yet to be determined. Moreover, the identity of the activin receptor-like kinase (ALK; TGF-β type I receptors) and downstream transcription factors (eg, SNAIL and SLUG) mediating the effects of activin on MMP2 expression and trophoblast cell invasion remains unknown. Objective: To elucidate the role of MMP2 in activin A-induced human trophoblast cell invasion as well as the involvement of ALK4 and SNAIL. Design: HTR8/SVneo immortalized human extravillous cytotrophoblast (EVT) cells and primary cultures of human first-trimester EVT cells were used as study models. Small interfering RNA (siRNA)-mediated knockdown approaches were used to investigate the molecular determinants of activin A-mediated functions. Main Outcome Measures: Levels of mRNA and protein were examined by reverse transcription-quantitative real-time PCR and Western blot, respectively. Cell invasiveness was measured by Matrigel-coated transwell assays. Results: Treatment of HTR8/SVneo cells with activin A increased the production of SNAIL, SLUG, and MMP2 without altering that of MMP9, TIMP1, TIMP2, TWIST, RUNX2, ZEB1, or ZEB2. Similarly, activin A up-regulated the mRNA and protein levels of SNAIL and MMP2 in primary EVT cells. Knockdown of SNAIL attenuated activin A-induced MMP2 up-regulation in HTR8/SVneo and primary EVT cells. In HTR8/SVneo cells, activin A-induced production of SNAIL and MMP2 was abolished by pretreatment with the TGF-β type I receptor (ALK4/5/7) inhibitor SB431542 or siRNA targeting ALK4, SMAD2/3, or common SMAD4. Likewise, knockdown of ALK4 or SMAD4 abolished the stimulatory effects of activin A on SNAIL and MMP2 expression in primary EVT cells. Importantly, activin A-induced HTR8/SVneo and primary EVT cell invasion were attenuated by siRNA-mediated depletion of ALK4 or MMP2. Conclusion: Activin A induces human trophoblast cell invasion by inducing SNAIL-mediated MMP2 expression through ALK4 in a SMAD2/3-SMAD4-dependent manner.

532. SOLUBLE MEDIATORS IN THE HUMAN UTERINE CAVITY: POTENTIAL ROLES IN EMBRYO IMPLANTATION

2009 ◽  
Vol 21 (9) ◽  
pp. 130
Author(s):  
N. Hannan ◽  
K. L. Meehan ◽  
L. J. F. Rombauts ◽  
L. A. Salamonsen
Keyword(s):  
Chemokine Receptors ◽  
Embryo Implantation ◽  
Uterine Cavity ◽  
Cycle Phase ◽  
Human Trophoblast ◽  
Endometrial Epithelial Cell ◽  
Glandular Secretion ◽  
Uterine Fluid ◽  
Human Chemokines ◽  
Insight Into

Embryo implantation requires synchronized dialogue between a receptive endometrium and an activated blastocyst via locally produced soluble mediators. During the mid-secretory (MS) phase of the menstrual cycle there is increased glandular secretion into the uterine lumen. These secretions likely contain important mediators that modulate the endometrium and support the conceptus during implantation. Previously we identified that several chemokines were maximally produced during the MS phase by endometrial glandular epithelium (GE) (1, 2) and the presence of chemokine receptors on GE and human trophoblast (3). Furthermore recombinant human chemokines and endometrial epithelial cell-conditioned media stimulated trophoblast migration; this was attenuated by neutralizing specific chemokines (3). Chemokines also regulate a variety of adhesion and ECM molecules on trophoblast (4). Thus chemokines have important roles during embryo implantation. We hypothesized that chemokines are secreted into the uterine cavity and may act on the implanting blastocyst and the endometrium. This study aimed to identify chemokines in uterine fluid (collected by flushing the uterine cavity with 5mls of saline) from fertile women during the proliferative (non-receptive; n=4) and MS (receptive; n=4) phases of the cycle, and from women with unexplained infertility during the MS phase (n=4). Uterine fluid was analyzed using quantitative MilliplexTM Luminex® 42-plex cytokine/chemokine assays revealing the presence of IL-8, CCL2, CCL4, CCL7, CCL11, CCL22 and CX3CL1 in uterine fluid from all women. Importantly chemokine profiles were altered with both cycle phase and fertility; for example CCL4 and CCL22 levels were lower in the infertile cohort, where as CCL2 levels were higher in uterine fluid collected during the proliferative phase. Identifying the soluble mediators in human uterine fluid may provide potential markers of endometrial receptivity, insight into the unique microenvironment essential for pregnancy and a profile of maternal factors that influence the implanting blastocyst.

scholarly journals The Cytokine Gene CXCL14 Restricts Human Trophoblast Cell Invasion by Suppressing Gelatinase Activity

Endocrinology ◽  
2009 ◽  
Vol 150 (12) ◽  
pp. 5596-5605 ◽  
Author(s):  
HaiBin Kuang ◽  
Qi Chen ◽  
Ying Zhang ◽  
Li Zhang ◽  
HongYing Peng ◽  
...  
Keyword(s):  
Trophoblast Cell ◽  
Trophoblast Invasion ◽  
Trophoblast Cells ◽  
Human Trophoblast ◽  
Gelatinase Activity ◽  
Invasion And Migration ◽  
Human Pregnancy ◽  
Decidual Cells ◽  
And Migration ◽  
Maternal Fetal Interface

Abstract Well-controlled trophoblast invasion into uterine decidua is a critical process for the normal development of placenta, which is tightly regulated by various factors produced within the trophoblast-endometrial microenvironment. CXCL14 is involved in tumor growth and metastasis, and its expression in placenta is temporally regulated during pregnancy. However, the role of CXCL14 in trophoblast function during human pregnancy is not clear. In this study, by using RT-PCR through human pregnancy, we found that CXCL14 was selectively expressed at early but not late pregnancy. Immunostaining revealed that CXCL14 proteins were strongly expressed in villous cytotrophoblasts and moderately in decidualized stromal cells but very weakly in syncytiotrophoblasts and extravillous trophoblasts. The effect of CXCL14 on trophoblast invasion were examined by using human villous explants cultured on Matrigel and further proved by invasion and migration assay of primary trophoblast cells and trophoblast cell line HTR-8/SVneo. Our data showed that CXCL14 significantly inhibited outgrowth of villous explant in vitro; this effect is due to suppression of trophoblast invasion and migration through regulating matrix metalloproteinases activities, whereas the trophoblast proliferation was not affected. Moreover, because a receptor for CXCL14 has not been identified, we performed further cell-specific CXCL14 binding activities with regard to different cell types within the maternal-fetal interface. Our data revealed that CXCL14 could specifically bind to trophoblast cells but not decidual cells from the maternal-fetal interface. These results suggest that CXCL14 plays an important role in regulating trophoblast invasion through an autocrine/paracrine manner during early pregnancy.

scholarly journals Activin A increases human trophoblast invasion by upregulating integrin β1 through ALK4

2020 ◽  
Author(s):  
Shiqin Zhu ◽  
Zeyan Li ◽  
Linlin Cui ◽  
Yanli Ban ◽  
Peter C. K. Leung ◽  
...  
Keyword(s):  
Activin A ◽  
Trophoblast Invasion ◽  
Integrin Β1 ◽  
Human Trophoblast

Activin A, B and AB increase human trophoblast cell invasion by up-regulating N-cadherin

Placenta ◽  
2013 ◽  
Vol 34 (9) ◽  
pp. A49
Author(s):  
Yan Li ◽  
Jung-Chien Cheng ◽  
Christian Klausen ◽  
Peter Leung
Keyword(s):  
Cell Invasion ◽  
Activin A ◽  
Trophoblast Cell ◽  
Human Trophoblast

scholarly journals Bone Morphogenetic Protein 2 Increases Human Trophoblast Invasion by Up-Regulating Integrin Beta3

2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. A747-A748
Author(s):  
Cuiping Hu ◽  
Junhao Yan
Keyword(s):  
Bone Morphogenetic Protein ◽  
Trophoblast Cell ◽  
Bone Morphogenetic Protein 2 ◽  
Extravillous Trophoblast ◽  
Trophoblast Invasion ◽  
Trophoblast Cells ◽  
Human Trophoblast ◽  
Protein Levels ◽  
Integrin Β3 ◽  
Morphogenetic Protein

Abstract The adequate invasion of extravillous trophoblast cells (EVTs) is indispensable for the implantation of embryos and subsequent remodeling of uterine spiral arteries in early human gestation. Bone morphogenetic protein 2 (BMP2), which is abundantly expressed at the maternal-fetal interface, has been shown to promote the human EVT invasion process (1). Integrin switching (i.e., a switch from α6β4 to αvβ3) plays essential roles in cell-extracellular matrix adhesion and has been reported to influence EVT migration and invasion (2). Moreover, integrin β3 has been found to promote the adhesion, invasion, and migration abilities of embryonic trophoblasts (3). However, whether integrin β3 participates in BMP2 signaling and mediates BMP2-increased-human trophoblast invasion remains unknown. The purpose of our study was to explore the effects of BMP2 on integrin αvβ3 expression and the possible mediation role of integrin β3 in BMP2-regulated human trophoblast invasion. We used immortalized human trophoblast cell line (HTR8/SVneo) and primary human extravillous trophoblast cells (EVTs) isolated from first-trimester villi as study models. RT-qPCR and Western blot assay were respectively utilized to detect the messenger RNA and protein levels of intergrin αv and β3. The function of the target protein was studied by siRNA knockdown, and the trophoblast invasion ability was checked by Matrigel-coated transwell invasion assays. Our results demonstrated that the mRNA and protein levels of integrin β3, rather than integrin αv, were up-regulated after BMP2 treatment in HTR8/SVneo and primary EVT cells. Importantly, siRNA-mediated down-regulation of integrin β3 significantly inhibited basal and BMP2-induced HTR8/SVneo cell invasionas measured by transwell invasion assay. In conclusion, we findings support that BMP2 promotes human trophoblast cell invasion by up-regulating integrin β3 expression, benefiting the in-depth understanding of the pro-invasive effect of BMP2 on human trophoblasts during early pregnancy. Reference: (1) Hong-Jin Zhao et al., Cell Death Dis 2018;9:174. (2) Damsky, C.H. et al, Development 1994; 120, 3657-3666. (3) Dong-Mei He et al., Reproduction 2019;157:423-430.

scholarly journals Annexin A1/Formyl Peptide Receptor Pathway Controls Uterine Receptivity to the Blastocyst

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1188 ◽  
Author(s):  
Cristina B. Hebeda ◽  
Silvana Sandri ◽  
Cláudia M. Benis ◽  
Marina de Paula-Silva ◽  
Rodrigo A. Loiola ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Embryo Implantation ◽  
Formyl Peptide Receptor ◽  
Annexin A1 ◽  
Uterine Receptivity ◽  
Mucin 1 ◽  
Human Trophoblast ◽  
Junction Molecules ◽  
Formyl Peptide

Embryo implantation into the uterine wall is a highly modulated, complex process. We previously demonstrated that Annexin A1 (AnxA1), which is a protein secreted by epithelial and inflammatory cells in the uterine microenvironment, controls embryo implantation in vivo. Here, we decipher the effects of recombinant AnxA1 in this phenomenon by using human trophoblast cell (BeWo) spheroids and uterine epithelial cells (Ishikawa; IK). AnxA1-treated IK cells demonstrated greater levels of spheroid adherence and upregulation of the tight junction molecules claudin-1 and zona occludens-1, as well as the glycoprotein mucin-1 (Muc-1). The latter effect of AnxA1 was not mediated through IL-6 secreted from IK cells, a known inducer of Muc-1 expression. Rather, these effects of AnxA1 involved activation of the formyl peptide receptors FPR1 and FPR2, as pharmacological blockade of FPR1 or FPR1/FPR2 abrogated such responses. The downstream actions of AnxA1 were mediated through the ERK1/2 phosphorylation pathway and F-actin polymerization in IK cells, as blockade of ERK1/2 phosphorylation reversed AnxA1-induced Muc-1 and claudin-1 expression. Moreover, FPR2 activation by AnxA1 induced vascular endothelial growth factor (VEGF) secretion by IK cells, and the supernatant of AnxA1-treated IK cells evoked angiogenesis in vitro. In conclusion, these data highlight the role of the AnxA1/FPR1/FPR2 pathway in uterine epithelial control of blastocyst implantation.

scholarly journals Investigation of human trophoblast invasion in vitro

2020 ◽  
Vol 26 (4) ◽  
pp. 501-513 ◽  
Author(s):  
Yassen Abbas ◽  
Margherita Y Turco ◽  
Graham J Burton ◽  
Ashley Moffett
Keyword(s):  
Cell Lines ◽  
First Trimester ◽  
Trophoblast Cell ◽  
Extravillous Trophoblast ◽  
Trophoblast Invasion ◽  
Human Trophoblast ◽  
Uterine Environment ◽  
Microfluidic Assays ◽  
Invasion Assays

Abstract BACKGROUND In humans, inadequate trophoblast invasion into the decidua is associated with the ‘great obstetrical syndromes’ which include pre-eclampsia, foetal growth restriction (FGR) and stillbirth. The mechanisms regulating invasion remain poorly understood, although interactions with the uterine environment are clearly of central importance. Extravillous trophoblast (EVT) cells invade the uterus and transform the spiral arteries. Progress in understanding how they invade has been limited due to the lack of good in vitro models. Firstly, there are no non-malignant cell lines that have an EVT phenotype. Secondly, the invasion assays used are of limited use for the small numbers of primary EVT available from first-trimester placentas. We discuss recent progress in this field with the generation of new EVT lines and invasion assays using microfluidic technology. OBJECTIVE AND RATIONALE Our aim is to describe the established models used to study human trophoblast invasion in vivo and in vitro. The difficulties of obtaining primary cells and cell lines that recapitulate the phenotype of EVT are discussed together with the advantages and pitfalls of the different invasion assays. We compare these traditional end point assays to microfluidic assays where the dynamics of migration can be measured. SEARCH METHODS Relevant studies were identified by PubMed search, last updated on February 2020. A search was conducted to determine the number of journal articles published using the cell lines JEG-3, BeWo, JAR, HTR-8/Svneo, Swan-71 and primary human extravillous trophoblast in the last 5 years. OUTCOMES Deep trophoblast invasion into the maternal decidua is a particular feature of human pregnancy. This invasion needs to be finely regulated to allocate resources between mother and baby. A reliable source of EVT is needed to study in vitro how the uterine environment regulates this process. First, we critically discuss the issues with the trophoblast cell lines currently used; for example, most of them lack expression of the defining marker of EVT, HLA-G. Recently, advances in human stem cell and organoid technology have been applied to extraembryonic tissues to develop trophoblast cell lines that can grow in two (2D) and three dimensions (3D) and differentiate to EVT. This means that the ‘trophoblast’ cell lines currently in use should rapidly become obsolete. Second, we critically discuss the problems with assays to study trophoblast invasion. These lack physiological relevance and have simplified migration dynamics. Microfluidic assays are a powerful tool to study cell invasion because they require only a few cells, which are embedded in 3D in an extracellular matrix. Their major advantage is real-time monitoring of cell movement, enabling detailed analysis of the dynamics of trophoblast migration. WIDER IMPLICATIONS Trophoblast invasion in the first trimester of pregnancy remains poorly understood despite the importance of this process in the pathogenesis of pre-eclampsia, FGR, stillbirth and recurrent miscarriage. The new technologies described here will allow investigation into this critical process.

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