Human Extravillous Trophoblast Research Articles

Bisphenol S Impairs Invasion and Proliferation of Extravillous Trophoblasts Cells by Interfering with Epidermal Growth Factor Receptor Signaling.

The placenta supports fetal growth and is vulnerable to exogenous chemical exposures. We have previously demonstrated that exposure to the emerging chemical bisphenol S (BPS) can alter placental endocrine function. Mechanistically, we have demonstrated that BPS interferes with epidermal growth factor receptor (EGFR) signaling, reducing placenta cell fusion. Extravillous trophoblasts (EVTs), a placenta cell type that aids with vascular remodeling, require EGF to invade into the maternal endometrium. We hypothesized that BPS would impair EGF-mediated invasion and proliferation in EVTs. Using human EVTs (HTR-8/SVneo cells), we tested whether BPS could inhibit the EGF response by blocking EGFR activation. We also evaluated functional endpoints of EGFR signaling, including EGF endocytosis, cell invasion and proliferation, and endovascular differentiation. We demonstrated that BPS blocked EGF-induced phosphorylation of EGFR by acting as a competitive antagonist to EGFR. Transwell assay and a three-dimensional microfluidic chip invasion assay revealed that BPS exposure can block EGF-mediated cell invasion. BPS also blocked EGF-mediated proliferation and endovascular differentiation. In conclusion, BPS can prevent EGF-mediated EVT proliferation and invasion through EGFR antagonism. Given the role of EGFR in trophoblast proliferation and differentiation during placental development, our findings suggest that maternal exposure to BPS may contribute to placental dysfunction via EGFR-mediated mechanisms.

Human embryonic development: from peri-implantation to gastrulation.

The basic body plan of the mammalian embryo is established through gastrulation, a pivotal early postimplantation event during which the three major germ layers (endoderm, ectoderm, and mesoderm) are specified with cellular and spatial diversity. Despite its basic and clinical importance, human embryo development from peri-implantation to gastrulation remains shrouded in mystery. Recent advances in the elongated in vitro culture of rodent and non-primate embryos and the construction of embryo-like structures have helped to improve understanding of the mechanisms of human early embryonic development. Here, we review the recent advances and possible future directions in the development of in vitro models to better understand human embryogenesis from peri-implantation to gastrulation.

Interleukin-17 promotes proliferation, migration, and invasion of trophoblasts via regulating PPAR-γ/RXR-α/Wnt signaling

ABSTRACT To investigate the effect of Interleukin 17 (IL-17) on the invasive capacity of trophoblast cells and the underlying mechanism, we collected placental tissues samples from pregnant women with preeclampsia (PE) and healthy pregnant women. The expression levels of IL-17 mRNA and protein in tissue samples were determined using qRT-PCR and Western blot, respectively. Cell viability and cell proliferation was determined using CCK-8 assay, and colony formation assay, respectively. Cell migration and invasion capacity were determined using transwell cell migration assay. Our results showed that the mRNA expression of IL-17 was significantly increased in PE patients and may be used as a sensitive biomarker for PE (P < 0.01). IL-17 overexpression promoted cell viability, migration, and invasion of human extravillous trophoblast cell line, HTR8/SVneo; however, IL-17 knockdown inhibited these effects. Additionally, IL-17 activated PPAR-γ/RXR-α signaling pathway, which promoted proliferation, migration, and invasion of trophoblast cells. Moreover, PPAR-γ/RXR-α heterodimers activated Wnt signaling. In conclusion, our study provides evidence that IL-17 is overexpressed in PE and promotes proliferation, migration and invasion of trophoblast cells via activating PPAR-γ/RXR-α/Wnt signaling.

Hyperosmolarity Impairs Human Extravillous Trophoblast Differentiation by Caveolae Internalization.

We recently reported that an intact caveolar structure is necessary for adequate cell migration and tubulogenesis of the human extravillous trophoblast (EVT) cells. Emerging evidence supports that hyperosmolarity induces the internalization of caveolae into the cytoplasm and accelerates their turnover. Furthermore, signaling pathways associated with the regulation of trophoblast differentiation are localized in caveolae. We hypothesized that hyperosmolarity impairs EVT differentiation and caveolae/caveolin−1 (Cav-1) participates in this process. EVT cells (Swan 71 cell line) were cultured in complete Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 and exposed to hyperosmolar condition (generated by the addition of 100 mM sucrose). Hyperosmolarity altered the EVT cell migration and the formation of tube-like structures. In addition, cell invasion was decreased along with a reduction in the latent and active forms of matrix metalloproteinase-2 (MMP−2) secreted by these cells. With respect to Cav-1 protein abundance, we found that hyperosmolarity enhanced its degradation by the lysosomal pathway. Accordingly, in the hyperosmolar condition, we also observed a significant increase in the number of vacuoles and the internalization of the caveolae into the cytoplasm. Taken together, our findings suggest that hyperosmolarity may induce caveolae internalization and increase their turnover, compromising the normal differentiation of EVT cells.

EG-VEGF Induces Invasion of a Human Trophoblast Cell Line via PROKR2.

Extravillous trophoblast (EVT) invasion is important for embryo implantation, placental development, and successful remodeling of the uterine spiral artery. Endocrine gland derived-vascular endothelial growth factor (EG-VEGF) and matrix metalloproteinases (MMPs) are implicated in EVT invasion; however, the high con-centrations found in pregnancy pathologies have not been investigated in non-tumor trophoblasts. The roles of EG-VEGF, prokineticin receptors (PROKR1/2), MMP-2, and MMP-9 in EVT invasion during spiral artery remodeling were evaluated using human EVT from HTR-8/SVneo cell lines. The expression of MMP-2, MMP-9, and mitogen-activated protein kinase (MAPK), and Akt pathways in HTR-8/SVneo cells treated with recom-binant EG-VEGF alongside anti-PROKR1 and/or anti-PROKR2 antibodies was evaluated using quantitative reverse transcription-PCR and western blotting. Wound-healing and cell invasion assays were performed to assess the migration and invasion of these treated cells. Interestingly, 20 nM EG-VEGF activated ERK1/2 sig-naling and upregulated MMP-2 and MMP-9. This effect was suppressed by anti-PROKR2 antibody via ERK1/2 downregulation. Anti-PROKR2 antibody inhibited the migration and invasion of EG-VEGF-stimulated HTR-8/SVneo cells. Elevated concentrations of EG-VEGF enhance EVT invasion in a human trophoblast cell line by upregulating MMP-2 and MMP-9 via PROKR2. These new insights into the regulation of epithelial cell invasion may help in developing therapeutic interventions for placental-related diseases during pregnancy.

Effects of triclosan exposure on placental extravillous trophoblast motility, relevant IGF2/H19 signaling and DNA methylation-related enzymes of HTR-8/SVneo cell line

Triclosan (TCS) is an antimicrobial agent widely used in personal care products and a potential endocrine disruptor chemical (EDC). TCS can pass through the placental barrier. Any influence of EDCs on epigenetic changes of placenta and embryo may bring profound impact on later health. This study aimed to investigate the effects of TCS exposure on cell proliferation and migration, and the expression of imprinted genes IGF2/H19 and DNA methylation-related enzymes in human placental extravillous trophoblast cell line HTR-8/SVneo. After exposure to TCS levels of 0 (DMSO Control), 10−11, 10−10, 10−9, 10−8, 10−7, 10−6, 10−5, 3 × 10−5, 6 × 10−5, 10−4 M and incubated for up to 36 h, cell proliferation and migration were examined by CCK-8, EdU incorporation assay and wound healing assay; the mRNA levels of IGF2, H19, DNA methyltransferases (Dnmt3a, Dnmt3b and Dnmt1), ten-eleven translocation enzymes (Tet1, Tet2 and Tet3) and IGF2 Receptor (IGF2R) were analyzed by qRT-PCR. The protein levels of IGF2 were measured by Western blot and ELISA. The cell viability turned to decline at TCS treatment of 3 × 10−5 M and above (all p < 0.05, compared to the DMSO Control). The cell migration decreased at TCS 10−5 and 3 × 10−5 M treatment (p < 0.05), but consistently unchanged at low dose of TCS from 10−9 to 10−7 M (p > 0.05). TCS treatments below cytotoxicity doses (< 10−5 M) did not significantly alter the mRNA levels of IGF2, Dnmt1, Dnmt3a, Dnmt3b, Tet1, Tet2 and Tet3, compared to DMSO Control treatment (all p > 0.05). The transcription level of H19 was up-regulated by TCS at 3 × 10−5 M. TCS at 10−7 and 6 × 10−5 M increased the protein level of IGF2 in cell supernatant. Our data suggest that high TCS exposure may suppress HTR-8/SVneo cells viability and migration, increase H19 gene expressions and IGF2 protein secretion. The exact mechanism of TCS action in human trophoblast needs further studies.

MicroRNA-3935 promotes human trophoblast cell epithelial-mesenchymal transition through tumor necrosis factor receptor-associated factor 6/regulator of G protein signaling 2 axis

BackgroundInsufficient migration and invasion during trophoblast epithelial-mesenchymal transition (EMT) results in the occurrence and development of preeclampsia (PE), and our previous study has screened 52 miRNAs, whose expression levels are altered in the placental samples from PE patients, compared with the normal group. Among those, miR-3935 is one of the miRNAs being most significantly down-regulated, indicating its involvement in PE. However, the exact effect and molecular mechanisms remain unknown.MethodsIn the present study, we investigate the roles and underlying mechanisms of miR-3935 in trophoblast EMT by use of the human extra-villous trophoblast cell line HTR-8/SVneo as well as human placental tissues and maternal blood samples obtained from 15 women with normal pregnancies and 15 women with PE. Experimental methods include transfection, quantitative reverse transcription-PCR (qRT-PCR), western blot, immunofluorescence staining, dual-luciferase assays, in vitro invasion and migration assays, RNA-Seq analysis, bisulfite sequencing and immunohistochemistry staining.ResultsMiR-3935 expression is significantly decreased in both placentas and peripheral blood specimens of PE, and functionally, miR-3935 promotes EMT of trophoblast cells. Mechanistically, TRAF6 is identified to be a direct target of miR-3935 and TRAF6 exerts its negative effect on EMT of trophoblast cells by inhibition of RGS2, which down-regulates the methylation status of promoter of CDH1 gene that encodes E-Cadherin protein through induction of ALKBH1, resulting in increase of E-Cadherin and subsequently insufficient trophoblast EMT.ConclusionsTogether these results uncover a hitherto uncharacterized role of miR-3935/TRAF6/RGS2 axis in the function of human trophoblasts, which may pinpoint the molecular pathogenesis of PE and may be a prognostic biomarker and therapeutic target for such obstetrical diseases as PE.

Transcriptomic Drivers of Differentiation, Maturation, and Polyploidy in Human Extravillous Trophoblast.

During pregnancy, conceptus-derived extravillous trophoblast (EVT) invades the endomyometrium, anchors the placenta to the maternal uterus, and remodels the spiral arteries in order to establish maternal blood supply to the fetoplacental unit. Recent reports have described early gestation EVT as polyploid and senescent. Here, we extend these reports by performing comprehensive profiling of both the genomic organization and transcriptome of first trimester and term EVT. We define pathways and gene regulatory networks involved in both initial differentiation and maturation of this important trophoblast lineage at the maternal–fetal interface. Our results suggest that like first trimester EVT, term EVT undergoes senescence and endoreduplication, is primarily tetraploid, and lacks high rates of copy number variations. Additionally, we have highlighted senescence and polyploidy-related genes, pathways, networks, and transcription factors that appeared to be important in normal EVT differentiation and maturation and validated a key role for the unfolded protein response in this context.

IFN-B DISRUPTS INVASIVE EXTRAVILLOUS TROPHOBLASTS FUNCTION ON IMPLANTATION-ON-A-CHIP MODEL

Chronic inflammatory conditions, such as systemic lupus erythematosus, have been linked to implantation and early pregnancy failures. Studies suggest this may be linked to changes in interferon signaling during implantation. Supporting animal data demonstrates that excessive stimulation of the interferon-β (IFN-β) pathway during gestation, via infectious or non-infectious etiologies, causes breakdown of the maternal-fetal barrier and poor pregnancy outcomes (i.e. IUGR, fetal demise). In this study, we aim to examine the role of IFN-β in regulating human extravillous trophoblast (EVT) invasion and spiral artery remodeling using a microfluidic organ-on-a-chip platform, Implantation-On-A-Chip (IOC).

Dynamic Regulation of the Chromatin Landscape during Human Extravillous Trophoblast Cell Lineage Development

Dynamic Regulation of the Chromatin Landscape during Human Extravillous Trophoblast Cell Lineage Development

Circular RNA hsa_circ_0081343 promotes trophoblast cell migration and invasion and inhibits trophoblast apoptosis by regulating miR-210-5p/DLX3 axis

BackgroundVarious circular RNAs (circRNAs) are dysregulated in the placenta of fetal growth restriction (FGR) fetuses, but their roles and regulatory mechanisms have not been fully elucidated. Herein, we aimed to elucidate the role of hsa_circ_0081343 in regulating the migration, invasion, and apoptosis of human extravillous trophoblast HTR-8 cells.MethodsCircRNA and miRNA levels were examined by quantitative reverse transcription PCR (qRT-PCR). Overexpression plasmid constructs and siRNAs were used to overexpress and knockdown hsa_circ_0081343, respectively. Transwell assays and flow cytometry analyses were performed to evaluate the effects of hsa_circ_0081343 or miR-210-5p on migration, invasion, and apoptosis. Protein levels were analyzed by western blotting. Dual luciferase activity and anti-AGO2 RNA immunoprecipitation (RIP) assays were performed to identify the relationship between miR-210-5p and hsa_circ_0081343.ResultsHsa_circ_0081343 expression was significantly downregulated in 37 FGR placental tissues compared to healthy placental control tissues. Hsa_circ_0081343 overexpression may inhibit apoptosis by downregulating the expression of cleaved caspase 3 and caspase 9 and alleviating the migration and invasion of HTR-8 cells by inducing the expression of MMP2 and MMP9. The dual luciferase activity and anti-AGO2 RIP assay results showed that hsa_circ_0081343 binds to miR-210-5p. miR-210-5p overexpression eliminated the effect of hsa_circ_0081343 overexpression in HTR-8 cells. Finally, DLX3 was identified as a direct target of miR-210-5p.Conclusionshsa_circ_0081343 expression levels are significantly downregulated in FGR placental tissues. Hsa_circ_0081343 regulates the migration, invasion, and apoptosis of HTR-8 cells via the hsa-miR-210-5p/DLX3 axis.

P–379 Human platelet lysate improves trophoblast spheroid attachment to primary endometrial epithelial cells from patients with recurrent implantation failure

Abstract Study question Could non-autologous platelet lysate (PL) increase attachment of HTR–8 spheroids in vitro to primary endometrial epithelial cells (EECs) from patients with recurrent implantation failure (RIF)? Summary answer Increased quantity of HTR–8 spheroids attached to primary EECs, isolated from patients with RIF, suggests in vitro treatment with non-autologous PL could improve endometrial receptivity. What is known already Inadequate endometrial receptivity and thickness are major causes for RIF. Recent studies suggest that platelet-rich plasma (PRP) may improve pregnancy outcomes for RIF and/or thin endometrium (TE) patients. Our previous results show that a commercially sourced and non-autologous human PRP/PL (HPL) promotes EC proliferation in vitro, suggesting that HPL may help to standardize future clinical treatments. In addition to EC proliferation, HPL treatment may improve embryo attachment to primary EECs isolated from patients with a history of RIF. In vitro attachment assays with trophoblast spheroids (embryo model) could help elucidate the effect of HPL on endometrial receptivity in RIF patients. Study design, size, duration Endometrial tissue was collected from nine RIF patients at the CReATe Fertility Centre, Toronto, Canada (Veritas REB#16580): five with (RIF+TE) and four without a TE (RIF only). Primary EECs were enzymatically isolated and treated with serum-free culture media (SFM) or 1% HPL in SFM for 48 hours before performing the attachment assay. Trophoblast cells (HTR–8/SVneo) were grown in suspension on a rocker to form 70–100 uM spheroids over 24 hours before use in the assay. Participants/materials, setting, methods Spheroids were fluorescently labelled with calcein-AM for 30 minutes and size-selected to capture spheroids similar in size to a human blastocyst. Spheroids were seeded on top of EEC monolayers and calcein fluorescence was immediately measured by a spectrophotometer. Following the 1-hour incubation, unattached spheroids were aspirated, and fluorescence was measured again. Spheroids were also individually quantified by fluorescent microscopy and ImageJ™ software. The percentage of spheroid attachment was calculated for calcein fluorescence and ImageJ™ quantification. Main results and the role of chance The HTR–8/SVneo cell line, derived from human first-trimester extravillous trophoblast cells (EVT), has been shown to be a suitable cell line to assess adhesion and invasion in vitro. Trophoblast spheroids generated from this cell line visually resembled a blastocyst and maintained expression of the EVT and implantation biomarkers: GATA3, ITGA5, and LIF. Primary EECs, treated for 48 hours with SFM supplemented with 1% commercially sourced and non-autologous HPL, overall exhibited increased attachment to HTR–8 spheroids. The percentage of spheroid attachment, as measured by fluorescence alone, significantly increase from 47.98% to 64.27% (P &amp;lt; 0.01) of seeded spheroids in RIF+TE EEC cultures, and from 48.12% to 85.77% (P &amp;lt; 0.001) of seeded spheroids in RIF only EEC cultures. Quantification by fluorescent microscopy and ImageJ™ software for individual calcein-stained spheroids, revealed a significant increase in spheroid attachment, from 57.52% to 86.5% (P &amp;lt; 0.01) in RIF+TE EEC cultures, and from 42.58% to 68.90% (P &amp;lt; 0.01) in RIF only EEC cultures. Limitations, reasons for caution Although there was a positive correlation between calcein fluorescence and spheroid quantity, quantification by fluorescence alone may be unreliable due to the variable numbers of cells in each spheroid. Our data suggest a more precise increase in attachment is detected when quantified by fluorescent microscopy and ImageJ™ software. Wider implications of the findings: We report a method for functional assessment of endometrial receptivity in vitro. HPL appears to promote implantation in RIF patients in a model of embryo attachment. We predict that the observed increase in attachment is due to increased endometrial receptivity gene expression, which will be our next investigative avenue. Trial registration number N/A

Tim-3/CTLA-4 pathways regulate decidual immune cells-extravillous trophoblasts interaction by IL-4 and IL-10.

To obtain a successful pregnancy, the establishment of maternal-fetal tolerance and successful placentation are required to be established. Disruption of this immune balance and/or inadequate placental perfusion is believed to be associated with a lot of pregnancy-related complications, such as recurrent spontaneous abortion, pre-eclampsia, and fetal intrauterine growth restriction. Extravillous trophoblasts (EVTs) have the unique ability to instruct decidual immune cells (DICs) to develop a regulatory phenotype for fetal tolerance. Utilizing immortalized human first trimester extravillous trophoblast cells and primary EVTs, we found that DICs promote EVT function and placental development. We have previously shown that checkpoints T-cell immunoglobulin mucin-3 (Tim-3) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) are important for DIC function. In the present study, we showed that blockade of Tim-3 and CTLA-4 pathways leaded to the abnormal DICs-EVTs interaction, poor placental development, and increased fetal loss. Treatment with IL-4 and IL-10 could rescue the adverse effects of targeting Tim-3 and CTLA-4 on the pregnancy outcome. Hence, the reproductive safety must be a criterion considered in the assessment of immuno-therapeutic agents. In addition, IL-4 and IL-10 may represent novel therapeutic strategies to prevent pregnancy loss induced by checkpoint inhibition.

MiR-3074-5p/CLN8 pathway regulates decidualization in recurrent miscarriage.

Decidualization is essential for the successful establishment of pregnancy, and the dysregulated decidualization may lead to early pregnancy loss. It was previously reported by us that miR-3074-5p could promote apoptosis but inhibit invasion of human extravillous trophoblast (EVT) cells in vitro, and the expression level of miR-3074-5p in villus tissues of recurrent miscarriage (RM) patients was significantly increased. The aim of this study was to preliminarily explore the role of miR-3074-5p played in the decidualization of human endometrial stromal cells (ESCs). It was found that the decidual expression level of miR-3074-5p in RM patients was remarkably higher than that in the control group. The overexpression of miR-3074-5p in the immortalized human ESC line, T-HESCs, showed suppressive effects not only on the cell proliferation, as well as the intracellular expression levels of cyclin B1 (CCNB1), CCND1 and CCNE1 but also on the in vitro-induced decidualization. CLN8 mRNA, encoding an endoplasmic reticulum (ER)-associated membrane protein, was validated to be directly targeted by miR-3074-5p. And, the expression level of CLN8 was continuously increased along with the decidualization process, whereas down-regulated CLN8 expression could inhibit the decidualization of T-HESCs in vitro. Furthermore, contrary to the increased expression level of miR-3074-5p, a significantly decreased CLN8 expression was observed in decidual tissues of RM patients. Collectively, these data suggested that an increased miR-3074-5p expression in ESCs might cause early pregnancy failure by disturbing decidualization of ESCs via the miR-3074-5p/CLN8 pathway, providing a potential diagnostic and therapeutic target for RM.

BMP6 Mediates BMP2-Increased Human Trophoblast Invasion

TGF-β superfamily proteins play divergent roles in regulating human extravillous trophoblast (EVT) invasion and their coordinated effects are essential for adequate placentation during pregnancy 1. Bone morphogenetic protein 2 (BMP2), which belongs to the BMP subfamily of TGF-β superfamily, has been shown to promote human EVT invasion and the acquisition of endothelial-like phenotype 2,3. It has been reported that BMP2 promotes EVT invasion by up-regulating Activin A, a growth factor which also belongs to TGF-β superfamily. However, whether BMP6 mediates the pro-invasive effect of BMP2 has yet to be determined. Herein, we firstly treated immortalized trophoblast cells (HTR8/SVneo) with recombinant BMP2 protein for 6 and 24 hrs, and our bulk-RNA sequencing results demonstrated significantly increased BMP6 mRNA levels after BMP2 treatment. Furthermore, we confirmed the up-regulatory effects of BMP2 on BMP6 mRNA and protein levels in both HTR8/SVneo and primary EVTs isolated from first-trimester villi. Notably, siRNA-mediated down-regulation of BMP6 significantly attenuated both basal and BMP2-induced cell invasion in HTR8/SVneo cells as measured by Matrigel-coated transwell invasion assay. In summary, our results firstly demonstrated the up-regulatory effect of BMP2 on BMP6 expression in human trophoblasts and identified the mediation role of BMP6 in BMP2-promoted EVT invasion, suggesting the interplay between BMP subfamily members during EVT invasion regulation. Our ongoing research focusing the underlying molecular mechanisms and signaling pathways could further benefit the advancement of diagnostic and therapeutic strategies for EVT invasion dysregulation-related pregnancy disorders, e.g., pre-eclampsia. Reference: (1) Li Yan et al., Trends Endocrinol Metab 2021 18: S1043-2760(20)30266-6. (2) Hong-Jin Zhao et al., FASEB J 2020;34(2):3151-3164. (3) Hong-Jin Zhao et al., Cell Death Dis 2018;9(2):174.

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

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.

Aquaporin 3 promotes human extravillous trophoblast migration and invasion

ProblemDoes aquaporin 3 (AQP3) affect the migration and invasion of human extravillous trophoblast (HTR8/Svneo) cells?Method of studyA lentivirus infection system was used to construct stable cell lines with either AQP3 knockdown or overexpression. RT-PCR and western blotting were used to verify the efficiencies of AQP3 knockdown or overexpression in HTR8/Svneo cells at mRNA and protein levels, respectively. Cell Counting Kit-8 and flow cytometry assays were used to detect the influence of AQP3 knockdown or overexpression on proliferation and apoptosis of HTR8/Svneo cells. In addition, wound healing and Transwell invasion assays were used to detect the effects of AQP3 knockdown or overexpression on migration and invasion capabilities of HTR8/Svneo cells. An Agilent gene chip was used to screen for significant differentially expressed genes after AQP3 knockdown. Finally, mechanisms by which AQP3 influences the migration and invasion of HTR8/Svneo cells were explored using bioinformatic analysis.ResultsCompared with controls, migration and invasion capabilities of HTR8/Svneo cells were significantly reduced after AQP3 knockdown, and significantly increased after AQP3 overexpression. Subsequent bioinformatic analysis of gene chip expression profiles indicated downregulation of genes related to adhesion such as PDGF-B, as well as signaling pathways (such as PIK3/AKT, NF-κB, and TNF) after AQP3 knockdown.ConclusionsAQP3 could significantly promote migration and invasion capabilities of human extravillous trophoblasts, it may mediate embryo invasion and adhesion to endometrium by regulating PDGF-B, PIK3/AKT signaling pathways, although this requires further verification.

Upregulation of miR-29a suppressed the migration and invasion of trophoblasts by directly targeting LOXL2 in preeclampsia.

Preeclampsia is a pregnancy-specific disorder that is a major cause of maternal and foetal morbidity and mortality, with a prevalence of 6-8% of pregnancies. Although the downregulation of lysyl oxidase (LOX) and LOX-like protein 2 (LOXL2), which leads to reduced trophoblast cell migration and invasion through activation of the TGF-β1/Smad3/collagen pathway, is relevant to preeclampsia, the mechanisms regulating differences in the gene expression of LOX and LOXL2 in placentas are not yet understood. This study aimed to investigate the mechanisms regulating differences in the gene expression of LOX and LOXL2 in placentas. The expression of miRNAs, LOX and LOXL2 in preeclamptic placentas and control placentas was analysed by qPCR. Localisation of miR29a and LOXL2 in preeclamptic placentas was performed by RNA-Fluorescence in-situ hybridization assay. The direct regulation of LOXL2 by miR-29a was assessed by dual-luciferase reporter assays in human extravillous trophoblast cells (HTR8/SVneo). Cell migration and invasion were evaluated by Transwell assays in HTR8/SVneo cells. miR-29a expression was upregulated in preeclamptic placentas and negatively correlated with LOXL2 mRNA expression levels. RNA-Fluorescence in-situ hybridization assay revealed a clear overlap between miR-29a and LOXL2 in the placentas of preeclampic women. LOXL2 was a direct target gene of miR-29a, as confirmed by a dual-luciferase reporter assay in HTR8/SVneo trophoblast cells. miR-29a suppressed HTR8/SVneo trophoblast cell migration and invasion. LOXL2 overexpression reversed the inhibitory effects of miR-29a on HTR8/SVneo trophoblast cell migration and invasion. Our results suggest that the upregulation of miR-29a suppresses the migration and invasion of HTR8/SVneo trophoblast cells by directly targeting LOXL2 in preeclampsia.

Delta-9-tetrahydrocannabinol inhibits invasion of HTR8/SVneo human extravillous trophoblast cells and negatively impacts mitochondrial function

Prenatal cannabis use is a significant problem and poses important health risks for the developing fetus. The molecular mechanisms underlying these changes are not fully elucidated but are thought to be attributed to delta-9-tetrahydrocannabinol (THC), the main bioactive constituent of cannabis. It has been reported that THC may target the mitochondria in several tissue types, including placental tissue and trophoblast cell lines, and alter their function. In the present study, in response to 48-h THC treatment of the human extravillous trophoblast cell line HTR8/SVneo, we demonstrate that cell proliferation and invasion are significantly reduced. We further demonstrate THC-treatment elevated levels of cellular reactive oxygen species and markers of lipid damage. This was accompanied by evidence of increased mitochondrial fission. We also observed increased expression of cellular stress markers, HSP70 and HSP60, following exposure to THC. These effects were coincident with reduced mitochondrial respiratory function and a decrease in mitochondrial membrane potential. Taken together, our results suggest that THC can induce mitochondrial dysfunction and reduce trophoblast invasion; outcomes that have been previously linked to poor placentation. We also demonstrate that these changes in HTR8/SVneo biology may be variably mediated by cannabinoid receptors CB1 and CB2.

Cadmium triggers oxidative stress and mitochondrial injury mediated apoptosis in human extravillous trophoblast HTR-8/SVneo cells

Cadmium (Cd) is a bioaccumulative heavy metal element with potential placental toxicity during pregnancy. Up to now, however, the precise toxic effects of Cd on human placentae, particularly as they pertain to trophoblast cells remain obscure. We therefore sought to investigate the cytotoxic effects of Cd on human extravillous trophoblast HTR-8/SVneo cells and the mechanisms involved in the processes. Results in this present study showed that CdCl2 treatment significantly suppressed cell viability and induced noticeable oxidative stress in HTR-8/SVneo cells. Further studies showed that CdCl2 treatment caused distortion of mitochondrial structure, reduction of mitochondrial membrane potential (Δψm), DNA damage and G0/G1 phase arrest. Under the same condition, CdCl2 treatment increased Bax/Bcl-2 ratios by up-regulating Bax expression and down-regulating Bcl-2 expression, and activated apoptotic executive molecule caspase-3, which irreversibly induced HTR-8/SVneo cell apoptosis. N-acetyl-l-cysteine (NAC), ROS scavenger, significantly attenuated CdCl2-caused mitochondrial injury, DNA damage, G0/G1 phase arrest and apoptosis. In addition, in vivo assay suggested that CdCl2 induced trophoblast cells apoptosis but not other cells in mice placental tissue. Taken together, these data suggest that Cd selectively triggers oxidative stress and mitochondrial injury mediated apoptosis in trophoblast cells, which might contribute to placentae impairment and placental-related disorders after Cd exposure. These findings may provide new insights to understand adverse effects of Cd on placentae during pregnancy.