Extravillous Trophoblast Cells Research Articles

MiR-675-5p accelerates the invasive ability of extravillous trophoblast cells by the upregulation of matrix metalloproteinases

MiR-675-5p accelerates the invasive ability of extravillous trophoblast cells by the upregulation of matrix metalloproteinases

Enhanced expression of αVβ3 integrin in villus and extravillous trophoblasts of placenta accreta.

Placenta accreta is one of the most serious complications in obstetrics and gynecology. Villous trophoblasts (VT) and extravillous trophoblasts (EVT) play a central role in normal placentation. Placenta accreta is characterized by abnormal invasion of EVT cells through the uterine layers, due to changes in several parameters, including adhesion proteins. Although αvβ3 integrin is a central adhesion molecule, participating in multiple invasive pathological conditions including cancer, data on placenta accreta are lacking. To study the expression pattern of αvβ3 integrin in placenta accreta in comparison with normal placentas. We collected tissue samples from placentas defined as percreta, the most severe presentation of placenta accreta and from normal control placentas (n = 10 each). The samples underwent protein extractions for analyses of αvβ3 expression by Western blots (WB) and a parallel tissue assessment by immunohistochemistry (IHC). WB results indicated significantly elevated αvβ3 integrin expression in the percreta samples compared to normal placentas. These elevated levels were mainly contributed by EVT cells, as demonstrated by IHC. αvβ3 integrin demonstrated a classical membranal expression in the VT cells, whereas a uniformly distributed expression was documented in the EVT cells. These patterns of the αvβ3 integrin localization were similar in both accreta and normal placental samples. Enhanced αvβ3 integrin expression, mainly in extra villous trophoblasts of placenta percreta, implies for a role of this adhesion molecule in pathological placentation.

Trimethylamine N-Oxide increases soluble fms-like tyrosine Kinase-1 in human placenta via NADPH oxidase dependent ROS accumulation

BackgroundsPreeclampsia (PE) is characterized as placental vascular disturbance and excessive secretion of soluble fms-like tyrosine kinase 1 (sFlt-1) into the maternal circulation. Trimethylamine N-oxide (TMAO, a gut microbe-derived metabolite) is strongly associated with various cardiovascular and cerebrovascular diseases. Recently, we observe that higher maternal circulating TMAO and sFlt-1 in patients with PE. The aims of the present study are to explore the effects of TMAO on placental sFlt-1 production and the underlying mechanism in human placenta. MethodsHuman placental explants, human placental primary trophoblasts and the extravillous trophoblasts (EVT) cell line (HRT-8/SVneo) were exposured to various concentrations of TMAO (100, 150, 300, and 600 μM). The mRNA expression and protein secretion of sFlt-1 in placental explants, primary trophoblasts and HRT-8/SVneo cells were determined with qPCR and ELISA, respectively. The levels of intracellular reactive oxygen species (ROS) production in primary trophoblasts and HRT-8/SVneo cells were measured by peroxide-sensitive fluorescent probe dichlorofluorescein diacetate. ResultsExposure of placental explants, primary trophoblasts and HRT-8/SVneo cells to TMAO significantly enhanced sFlt-1 at both mRNA and protein levels in a dose dependent manner. Moreover, inhibition of NADPH oxidase with apocynin significantly attenuated TMAO-induced ROS production in primary trophoblasts and HRT-8/SVneo, and suppressed sFlt-1 secretion in placental explants, primary trophoblasts and HRT-8/SVneo. ConclusionsOur findings indicated the NADPH oxidase dependent ROS pathway played a critical role in mediating TMAO-induced sFlt-1 generation in human placenta. TMAO may become a potential novel target for pharmacological or dietary interventions to reduce the risk of developing PE.

Trophoblast Stem-Cell-Derived Exosomes Improve Doxorubicin-Induced Dilated Cardiomyopathy by Modulating the let-7i/YAP Pathway

Trophoblast stem cells (TSCs) have been confirmed to play a cardioprotective role in heart failure. However, whether TSC-derived exosomes (TSC-exos) can protect against cardiac injury remains unclear. In the present study, TSC-exos were isolated from the supernatant of TSCs using the ultracentrifugation method and characterized by transmission electron microscopy and western blotting. Utilizing the public Gene Expression Omnibus (GEO) database, we found that let-7i and Yes-associated protein 1 (YAP) could participate in the development of heart failure. In vitro, AC16 cardiomyocytes subjected to doxorubicin (DOX) were treated with TSC-exos or let-7i mimic. Flow cytometry showed that TSC-exos and let-7i both decreased cardiomyocyte apoptosis. In vivo, mice that were intraperitoneally injected into DOX received either PBS, TSC-exos, or AAV9-let7iup for let-7i overexpression. Mice receiving TSC-exos and AAV9-let7iup showed improved cardiac function and decreased inflammatory responses, accompanied by downregulated YAP signaling. Mechanistically, TSC-exos could transfer let-7i to cardiomyocytes and silence the YAP signaling pathway. In conclusion, TSC-exos could alleviate DOX-induced cardiac injury via the let-7i/YAP pathway, which sheds new light on the application of TSC-exos as a potential therapeutic tool for heart failure.

Transient loss of endothelial cells in human spiral artery remodelling during early pregnancy: Challenging the dogma

Spiral artery (SpA) remodelling is essential for a successful pregnancy and is best described by its morphological features; vascular smooth muscle cell separation and loss, vessel dilatation, and invasion by extravillous trophoblast cells (EVT). Current opinion holds that EVT fully replace the endothelial cells (EC) of the SpA and take on EC-like characteristics. Placental bed biopsies (6–20 weeks gestation) were immunostained for EC and EVT, showing transient loss of EC. In vessels containing an endovascular EVT plug (n = 28) 77.6 ± 19.3% of the lumen was covered by EC while in vessels without endovascular EVT (n = 100) it was 100 ± 0%.

Galectin-9 regulates HTR8/SVneo function via JNK signaling.

To obtain a successful pregnancy, trophoblasts must provide a physical barrier, suppress maternal reactivity, produce immunosuppressive hormones locally, and enhance the production of blocking factors that are able to bind to several antigenic sites. Inadequate placental perfusion has been closely associated with several pregnancy-associated diseases. Galectin-9 (Gal-9) has a wide variety of regulatory functions in innate and adaptive immunity during infection, tumor growth, and organ transplantation. We utilized immortalized human first-trimester extravillous trophoblast cells (HTR8/SVneo) for our functional study and examined the effects of Gal-9 on apoptosis, cytokine production and angiogenesis of HTR8/SVneo cells. Gal-9 inhibited the apoptosis and IFN-γ and IL-17A production, promoted IL-4 production, and coordinated the crosstalk between HTR8/SVneo cells and human umbilical vein endothelial cells via its interaction with Tim-3. Blockade of JNK signaling inhibited Gal-9 activities in HTR8/SVneo cells. In addition, we detected a correlation between low levels of Gal-9 and spontaneous abortion. So Gal-9 could inhibit the apoptosis and proinflammatory cytokine expression, and promote the angiogenesis and IL-4 production in HTR8/SVneo cells via Tim-3 in a JNK dependent manner to help the maintenance of normal pregnancy. These findings possibly identify Gal-9 as a key regulator of trophoblast cells and suggest its potential as a biomarker and target for the treatment of recurrent pregnancy loss.

UDP-glucose, a cellular danger signal, and nucleotide receptor P2Y14 enhance the invasion of human extravillous trophoblast cells

IntroductionP2Y14, one of the P2Y purinergic G-protein coupled receptors, is expressed in a variety of cells and tissues. Its ligand, UDP-glucose (UDPG), is released from damaged and stress-stimulated cells and acts as a danger signal via P2Y14. Thus, P2Y14 plays an important role in immunological defense systems. Here, we aimed to elucidate the expression, localization, and role of P2Y14 in human trophoblasts and the placenta. MethodsHuman chorionic villus and placental tissues were subjected to immunostaining for P2Y14 protein and an extravillous trophoblast (EVT) marker, HLA-G. We examined the expression of P2Y14 and the effect of UDPG on cell proliferation and invasion in an EVT cell line, HTR-8/SVneo, using an MTS assay and a Transwell assay, respectively. We tested the effect of UDPG on cell invasion in P2Y14-underexpressing HTR-8/SVneo clones established by the lentiviral introduction of shRNA for P2RY14 mRNA. ResultsImmunostaining revealed that P2Y14 was exclusively expressed by EVTs. P2RY14 mRNA and P2Y14 protein were expressed in HTR-8/SVneo cells. UDPG did not affect cell proliferation but it did enhance invasion. Inhibition of P2Y14 and decreasing the expression of P2Y14 suppressed UDPG-mediated invasive activity. ConclusionsThese results showed that EVT selectively expressed P2Y14 and that P2Y14 was positively involved in UDPG-enhanced EVT invasion. It suggests the possible existence of a danger signal-mediated physiological system at the fetomaternal interface where UDPG released from maternal tissues through destruction by EVT invasion may accelerate EVT invasion, allowing EVTs to undergo successful placentation and vascular remodeling.

Establishment and differentiation of long-term trophoblast organoid cultures from the human placenta.

The human placenta is essential for successful reproduction. There is great variation in the anatomy and development of the placenta in different species, meaning that animal models provide limited information about human placental development and function. Until recently, it has been impossible to isolate trophoblast cells from the human placenta that proliferate in vitro. This has limited our ability to understand pregnancy disorders. Generating an in vitro model that recapitulates the unique features of the human placenta has been challenging. The first in vitro model system of human trophoblast that could be cultured long term and differentiated to syncytiotrophoblast (SCT) and extravillous trophoblast (EVT) was a two-dimensional (2D) culture system of human trophoblast stem cells. Here, we describe a protocol to isolate trophoblast from first-trimester human placentas that can be grown long term in a three-dimensional (3D) organoid culture system. Trophoblast organoids can be established within 2-3 weeks, passaged every 7-10 d, and cultured for over a year. The structural organization of these human trophoblast organoids closely resembles the villous placenta with a layer of cytotrophoblast (VCT) that differentiates into superimposed SCT. Altering the composition of the medium leads to differentiation of the trophoblast organoids into HLA-G+ EVT cells which rapidly migrate and invade through the Matrigel droplet in which they are cultured. Our previous research confirmed that there is similarity between the trophoblast organoids and in vivo placentas in their transcriptomes and ability to produce placental hormones. This organoid culture system provides an experimental model to investigate human placental development and function as well as interactions of trophoblast cells with the local and systemic maternal environment.

Effects of cytomegalovirus infection on extravillous trophoblast cells invasion and immune function of NK cells at the maternal-fetal interface.

Cytomegalovirus (CMV) is one of the most common intrauterine infection virus, which can cause intrauterine transmission through the placenta, resulting in abortion, stillbirth and congenital malformations. In this study, the co‐culture extravillous trophoblast (EVT) HTR8/SVneo cell model of CMV infection was established in vitro. The toxicity of CMV infected EVT was determined, and then, the cell invasion experiment was conducted to evaluate the effect on the invasion ability of EVT cell lines. Western blot and real‐time PCR were used to detect the related cytokines in the PI3K/AKT signalling pathway in cells. Flow cytometry was used to detect the immune function related factors of the supernatant of CMV culture on decidual NK cells. The TCID50 of CMV virus was 10−5.4. The results of immunofluorescence showed that a large number of fluorescent green of CMV pp65 antigen signals appeared in the cytoplasm of CMV infection group. CMV could infect and replicate EVT cells and inhibited cell proliferation. The expression of proteins PDK1, AKT‐S473 and AKT‐S308 was significantly increased in CMV infection group. The levels of IL‐17, IL‐4 and IFN‐γ were 8.7 ± 0.48%, 12.17 ± 0.61% and 6.66 ± 0.25%, respectively, in CMV infection group. The above results indicated that CMV infection inhibited EVT cells proliferation, weakened the invasion ability and inhibited the immune function of NK cells at the maternal‐fetal interface, resulting in the abnormal maternal‐fetal crosstalk.

SARS-CoV-2 Entry Factors: ACE2 and TMPRSS2 Are Expressed in Peri-Implantation Embryos and the Maternal-Fetal Interface.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread throughout the world, leading to large-scale population infection. Angiotensin-converting enzyme 2 (ACE2) is the receptor of both severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2. However, it is still controversial whether vertical transmission exists. In order to investigate the potential risk of SARS-CoV-2 vertical transmission, we explored ACE2 and TMPRSS2 (encoding transmembrane protease serine 2) expression patterns in peri-implantation embryos and the maternal–fetal interface using previously published single-cell transcriptome data. The results showed that day 6 (D6) trophectoderm (TE) cells in peri-implantation embryos, as well as syncytiotrophoblast (STB) at 8 weeks of gestation (STB_8W) and extravillous trophoblast (EVT) cells at 24 weeks of gestation (EVT_24W) in the maternal–fetal interface, strongly co-expressed ACE2 and TMPRSS2, indicating a SARS-CoV-2 infection susceptibility. The ACE2 positive-expressing cells in the three cell types mentioned above were found to share common characteristics, which were involved in autophagy and immune-related processes. ACE2 showed no gender bias in post-implantation embryos but showed a significant gender difference in D6_TE, D6 primitive endoderm (PE) cells, and ACE2 positive-expressing STBs. These findings suggest that there may be different SARS-CoV-2 infection susceptibilities of D6 embryos of different genders and during the gestation of different genders. Our results reveal potential SARS-CoV-2 infection risks during embryo transfer, peri-implantation embryo development, and gestation.

MiR-125b participates in the occurrence of preeclampsia by regulating the migration and invasion of extravillous trophoblastic cells through STAT3 signaling pathway

Preeclampsia (PE) is a major risk factor for maternal and fetal mortality. Studies showed that microRNAs (miRNAs) play important roles in PE, and are closely related to extra-villous trophoblastic proliferation and invasion. The current study determined miR-125b expression in PE patients, and explored the role of miR-125b in the occurrence and development of PE and its possible mechanism, aiming to provide a novel basis for the diagnosis and treatment of PE. The level of miR-125b in serum derived from pregnant women was measured by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation, invasion and migration of HTR-8/SVneo were determined by Cell Counting Kit-8 (CCK-8), Transwell and scratch assay, respectively. The target gene of miR-125b was predicted by Targetscan, and verified by luciferase reporter assay. The expressions of related proteins were determined by Western Blotting. The miR-125b level in the serum of PE patients was up-regulated as compared with normal pregnant women, and high level of miR-125b reduced cell proliferation, inhibited invasion and migration of HTR-8/SVneo as well as the expressions of STAT3, p-STAT3 and SOCS3, while low level of miR-125b produced the opposite results. STAT3 was predicted as the target gene of miR-125b, and the high level of miR-125b inhibited STAT3 signaling pathway. High expression of miR-125b may be involved in the occurrence of PE through inhibiting STAT3 pathway to inhibit the migration and invasion of extra-villous trophoblastic cells.

Stomatin-like protein 2 (SLP2) regulates the proliferation and invasion of trophoblast cells by modulating mitochondrial functions

IntroductionStomatin-like protein 2 (SLP2) is highly expressed in human first trimester trophoblast cells, but its functions in placental morpho-physiology remain unknown. This study aimed to determine the role of SLP2 in the proliferation and invasion of human first trimester trophoblast cells. MethodsImmunofluorescence was used to determine the expression and localization of SLP2 in normal and miscarriage human first trimester placenta. Western blot was used to determine the expression of SLP2, PCNA, Cyclin D3, N-cadherin, Vimentin, PGC1α and PPARα in HTR-8/SVneo cells. SLP2 was knocked down in the HTR-8/SVneo cells by using si-Slp2. Wound healing and migration assays were used to determine the effect of SLP2 knockdown on the migration and invasion in the HTR-8/SVneo cells. Mitochondrial membrane potential, reactive oxygen species (ROS), ATP production and biogenesis were measured to assess the effects of SLP2 knockdown on mitochondrial functions. ResultSLP2 was strongly expressed in the cytotrophoblasts (CTB), syncytiotrophoblast (STB) and extravillous trophoblasts (EVT) of normal pregnancy placenta as compared to miscarriage placenta. SLP2 was highly expressed in the invasive EVT cell lines, HTR-8/SVneo and HPT-8 compared to the CTB cell line JAR. Knockdown of SLP2 significantly inhibited the migration and invasion of HTR-8/SVneo cells and placental villous explants, and repressed mitochondrial biogenesis and functions in HTR-8/SVneo cells. DiscussionSilencing of SLP2 inhibited the proliferation, migration and invasion of HTR-8/SVneo cells via the impairment of mitochondrial functions. This indicates that the downregulation of SLP2 in miscarriage placenta could be part of the pathogenesis and pathophysiology of the disease.

Adenosine A3 Receptor Mediates ERK1/2- and JNK-Dependent TNF-α Production in Toxoplasma gondii-Infected HTR8/SVneo Human Extravillous Trophoblast Cells.

Toxoplasma gondii is an intracellular parasite that causes severe disease when the infection occurs during pregnancy. Adenosine is a purine nucleoside involved in numerous physiological processes; however, the role of adenosine receptors in T. gondii-induced trophoblast cell function has not been investigated until now. The goal of the present study was to evaluate the intracellular signaling pathways regulated by adenosine receptors using a HTR-8/SVneo trophoblast cell model of T. gondii infection. HTR8/SVneo human extravillous trophoblast cells were infected with or without T. gondii and then evaluated for cell morphology, intracellular proliferation of the parasite, adenosine receptor expression, TNF-α production and mitogen-activated protein (MAP) kinase signaling pathways triggered by adenosine A3 receptor (A3AR). HTR8/SVneo cells infected with T. gondii exhibited an altered cytoskeletal changes, an increased infection rate and reduced viability in an infection time-dependent manner. T. gondii significantly promoted increased TNF-α production, A3AR protein levels and p38, ERK1/2 and JNK phosphorylation compared to those observed in uninfected control cells. Moreover, the inhibition of A3AR by A3AR siRNA transfection apparently suppressed the T. gondii infection-mediated upregulation of TNF-α, A3AR production and MAPK activation. In addition, T. gondii-promoted TNF-α secretion was dramatically attenuated by pretreatment with PD098059 or SP600125. These results indicate that A3AR-mediated activation of ERK1/2 and JNK positively regulates TNF-α secretion in T. gondii-infected HTR8/SVneo cells.

Tert-Butyl Hydroperoxide Stimulated Apoptosis Independent of Prostaglandin E2 and IL-6 in the HTR-8/SVneo Human Placental Cell Line.

Significant gaps exist in our knowledge of how cellular redox status, sometimes referred to as oxidative stress, impacts placental trophoblasts. The present study used tert-butyl hydroperoxide (TBHP) as a known generator of reactive oxygen species (ROS) in the extravillous trophoblast cell line HTR-8/SVneo to examine the role of cellular redox disruption of prostaglandin E2 (PGE2) and the cytokine IL-6 in cell death. Cells were exposed to 0, 12.5, 25, or 50μM TBHP for 4, 8, and 24h to ascertain effects on cell viability, caspase 3/7 activity, PGE2 release, PTGS2 mRNA expression, and IL-6 release. Experiments with inhibitors included the cyclooxygenase inhibitor indomethacin, mitogen-activated protein kinase inhibitors (PD169316, U0126, or SP600125), or treatments to counter expected consequences of TBHP-stimulated generation of ROS (deferoxamine [DFO], butylated hydroxyanisole [BHA], and N,N'-diphenyl-1,4-phenylenediamine [DPPD]) using 24-h exposure to 50μM TBHP. Cell viability, measured by ATP content, decreased 24% relative to controls with a 24-h exposure to 50μM TBHP, but not at lower TBHP concentrations nor at earlier time points. Exposure to 50μM TBHP increased caspase 3/7 activity, an indicator of apoptosis, after 8 and 24h. Antioxidant treatment markedly reduced TBHP-stimulated caspase 3/7 activity, PGE2 release, and IL-6 release. TBHP-stimulated IL-6 release was blocked by PD169316 but unaltered by indomethacin. These data suggest that TBHP-stimulated IL-6 release and caspase 3/7 activation were independent of PGE2 yet were interrupted by treatments with known antioxidant properties, providing new insight into relationships between PGE2, IL-6, and apoptosis under conditions of chemically induced cellular oxidation.

1336-P: Trimethylamine N-Oxide Inhibits the Formation of Neutrophil Extracellular Traps to Regulate Placental Development in Gestational Diabetes Mellitus

Objective: Trimethylamine N-oxide (TMAO) is associated with risk of gestational diabetes mellitus (GDM), and TMAO levels in peripheral/cord blood of GDM mothers increased. Diabetes primes neutrophils to produce neutrophil extracellular traps (NETs), but it has not been reported in GDM. We determined the relationship between NETs and GDM, and explored potential role of TMAO. Methods: Maternal peripheral blood was collected during OGTT tests, and cord blood and placenta were collected during delivery. Plasma concentrations of cell-free double-stranded DNA (dsDNA) were compared between GDM and control mothers and association analysis was performed. Expression of myeloperoxidase (MPO) in placental tissues of GDM and control mothers was examined. Neutrophils from the blood of healthy donors were used to observe effects of TMAO on NETs formation. The effect of TMAO on human placental extravillous trophoblast cell line HTR-8/Svneo was examined. Results: Compared with controls, plasma concentrations of dsDNA in peripheral blood and cord blood were significantly increased in GDM mothers. Association analysis found that concentrations of dsDNA in peripheral and cord blood plasma were positively correlated with BMI and blood glucose levels, but not blood pressure. In addition, the placental thickness was positively correlated with dsDNA levels in cord blood plasma. MPO expression in the placental tissues of GDM mothers was also significantly higher than that of control mothers. TMAO-treated neutrophils could resist the formation of NETs stimulated by PMA or LPS, and TMAO promotes the proliferation, migration, invasion and angiogenesis of the cells. Conclusion: The formation of NETs was increased in GDM, and was positively correlated with BMI and blood glucose. TMAO could inhibit NETs formation and promote invasion and angiogenesis of human placental trophoblast cells, which may be involved in the development of the placenta. Disclosure X. Lin: None. X. He: None. X. Xiao: None.

Decorin levels in early- and late-onset preeclampsia

Preeclampsia (PE) is a pregnancy complication caused by typically limited proliferation, apoptosis, migration, and invasion of extra-trophoblast cells. Decorin (DCN) is a decidua-derived transforming growth factor (TGF)-binding proteoglycan which exerts multiple physiological functions such as collagen fibrillogenesis, myogenesis, angiostasis, and restraining placental invasiveness by adversely regulate proliferation, migration, and invasiveness of human extravillous trophoblast cells. Preeclampsia is mainly classified as early- and late-onset PE according to the timing of the disease onset. In the present study, we aimed to investigate the DCN levels in early-onset PE (EOPE, < 34 weeks) and late-onset severe PE (LOPE, ≥ 34 weeks) and uncomplicated pregnancies. In this case-control study, serum samples were obtained from 21 pregnant women with EOPE and 29 pregnant women with LOPE, as well as from 38 healthy controls (n = 12 early-onset controls and n = 26 late-onset controls) with uncomplicated pregnancies. The mean DCN level was statistically significantly higher in the early-onset PE controls than late-onset PE controls (p = 0.040). Although the mean DCN level was higher in the early-onset PE controls than EOPE and LOPE groups, it did not reach statistical significance (p = 0.119 and p = 0.117, respectively). Although DCN has been thought to play a role in the pathophysiology of PE, our study results show that DCN is not a useful predictive marker of EOPE and LOPE. Further large-scale studies are needed to draw a definitive conclusion.

Investigation of human trophoblast invasion in vitro.

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. 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. 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 5years. 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. 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.

Delta-Like 4-Notch signaling regulates trophoblast migration and invasion by targeting EphrinB2

The migration and invasion of trophoblasts during early pregnancy in known to play an important role in placental development, which ensures the oxygen and nutrients to the fetus. Accumulating evidences suggest that Delta-Like 4(DLL4)-Notch signaling may be involved in the process of trophoblast regulation. However, the potential role of DLL4-Notch signaling as well as its molecular mechanism in trophoblast controlling has not been fully studied. This study is designed to investigate the effects of DLL4-Notch signaling on trophoblast functions in human extravillous trophoblast cell line, HTR-8/SVneo. The possible molecular mechanism of DLL4-Notch signaling in trophoblast was also explored. We observed that activation of DLL4-Notch signaling enhanced cell migration and invasion ability while blockage of DLL4-Notch signaling impaired. Control of DLL4-Notch signaling did not affect cell viability. The expression of EphrinB2 was regulated by DLL4-Notch signaling. In addition, up-regulation of EphrinB2 resulted in the similar effects on trophoblast cell functions as DLL4-Notch signaling activation. Moreover, activation of DLL4-Notch signaling reversed the negative impact of EphrinB2 knock-down on trophoblasts migration and invasion. Our study suggested that DLL4-Notch signaling involved in the regulation of trophoblast migration and invasion, which may be induced by direct regulation of EphrinB2 expression.

Optimization Protocol of Fixation Method for Trophoblast Retrieval from the Cervix (TRIC): A Preliminary Study.

Extravillous trophoblast cells (EVTs) secreted by the uterine cavity may help overcome limitations associated with prenatal testing currently in use. EVTs are isolated using a routine safe liquid-based Pap test (called ThinPrep); however, the ThinPrep solution contains alcohol that hinders the isolation of intact EVTs. We compared the trophoblastic cell isolation efficiency of two different methods of fixation: Thinprep (pre-fixation method) and formalin (post-fixation method). We analyzed EVTs from 20 pregnant women (5–20 weeks of gestation) who underwent invasive prenatal testing. The percentages of placental β-human chorionic gonadotropin (β-hCG)-expressing cells were calculated. The presence of XY chromosomes were used to confirm pure trophoblast cells by fluorescence in situ hybridization. The β-hCG-positive cells obtained from pre- and post-fixation were 66.4 ± 13.3% and 83.2 ± 8.1% (p = 0.003), respectively, and fluorescence-positive cells were 11.1 ± 2.1% and 23.8 ± 4.8%, respectively (p = 0.001). Post-fixation was found to be more efficient in isolating non-trophoblast cells than pre-fixation. For the successful clinical application of trophoblast retrieval and isolation from the cervix in prenatal genetic testing, each step should be optimized for consistent and reliable results.

MON-025 Activin a Increases Human Trophoblast Invasion by Up-Regulating Integrinb1 Through ALK4

Activin A Increases Human Trophoblast Invasion by Up-regulating Integrin β1 Through ALK4Following implantation, extravillous trophoblast cells (EVTs) derived from trophectoderm invade into the maternal decidua to a certain extent, which is tightly regulated by a variety of factors. Activin A, a member of the TGF-β superfamily, has been shown to stimulate the invasion of human trophoblasts (1). Integrin β1 has been implicated in cancer cell invasion and is consistently expressed in human preimplantation embryos (2). However, whether integrin β1 is integrated in activin A signaling and mediates activin A increased-human trophoblast invasion remain unknown. The objective of our study was to investigate the possible mediation role of integrin β1 in the pro-invasive effect of activin A on trophoblasts and illustrate the underlying molecular mechanisms. Primary and immortalized (HTR8/SVneo) cultures of human trophoblast cells were employed as study models. Real-time qPCR, Western blot, and small interfering RNA (siRNA)-mediated knockdown approaches were used to investigate the molecular determinants of activin A-mediated functions. The integrin β1 protein levels in poorly invasive BeWo,JAR and JEG-3 human choriocarcinoma cells were lower than that in highly invasive HTR8/SVneo cells and primary human EVTs, suggesting the possible essential role of integrin β1 in mediating human trophoblast invasion. The expression levels of integrin β1 were up-regulated in a time-dependent manner after activin A treatment in HTR8/SVneo cells. Importantly, siRNA-mediated down-regulation of integrin β1 significantly attenuated both basal and activin A-induced cell invasion in HTR8/SVneo cells as measured by transwell invasion assay. Interestingly, the TGF-β type I receptors (ALK4/5/7) inhibitor SB431542 abolished activin A-induced activation of SMAD2/SMAD3 as well as activinA-up-regulated integrin 1 expression. Moreover, siRNA-mediated down-regulation of ALK4 or SMAD4 attenuated activin A-up-regulated integrin β1 in both HTR8/SVneo cells and human primary EVT cells. These results reveal that activin A promotes human trophoblast cell invasion by up-regulating integrin β1 expression through ALK4-activated SMAD2/3-SMAD4 signaling pathway.Reference: (1) Bearfield et al., Eur J Endocrinol 2005;152:909–16. (2) Campbell et al., Hum Reprod 1995;10:1571–8.