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It was to study trophoblast cell (TC) adhesion molecules regulated by different genes in the placental tissue (PT) of patients with pregnancy-induced hypertension (PIH), and the correlation with the severity of PIH. 42 patients with PIH (13 cases in the mild PIH group, 11 cases in the moderate PIH group, and 18 cases in the severe PIH group) and 40 patients with normal pregnancy (NP group) were included. mRNA and protein levels in matrix metalloproteinase (MMP)-9, MMP-2, tissue inhibitor of metalloproteinases (TIMP)-1, and TIMP-2 of all patients were determined by semi-quantitative polymerase chain reaction (PCR) and Western blotting (WB), respectively. Compared to the NP group, MMP-9 and MMP-2 mRNA levels as well as their proteins in PT significantly decreased in PIH groups (P<0.05). MMP-9 mRNA was greatly lower in the severe PIH group than mild PIH group (P<0.05). MMP-2 mRNA in moderate and severe PIH groups was much lower than NP and mild PIH groups, and that in the severe PIH group was considerably lower than the moderate PIH group (P<0.05). TIMP-1 mRNA and its protein highly increased in PT in PIH groups than NP group (P<0.05). TIMP-2 mRNA was remarkably higher in the severe PIH group than in the NP group (P<0.05). mRNA and proteins of MMP-9 and MMP-2 decreased in PT of PIH patients, while TIMP-1 mRNA and its protein increased, which were correlated with the severity of PIH. MMP-9, MMP-2, and TIMP-1 were involved in the pathogenesis of PIH by regulating the infiltration of TCs.

Mono(2-ethylhexyl) phthalate (MEHP) is a metabolite of DEHP which is one of phthalic acid esters (PAEs) widely used in daily necessities. Moreover, MEHP has been proven to have stronger biological toxicity comparing to DEHP. In particular, several recent population-based studies have reported that intrauterine exposure to MEHP results in adverse pregnancy outcomes. To explore the mechanisms and metabolic biomarkers of MEHP exposure, we examined the metabolic status of HTR-8/Svneo cell lines exposed to different doses of MEHP (0, 1.25, 5.0, 20 μM). Global and dose-response metabolomics tools were used to identify metabolic perturbations and sensitive markers associated with MEHP. Only 22 metabolic features (accounted for <1 %) were significantly changed when exposed to 1.25 μM. However, when the exposure dose was increased to 5 or 20 μM, the number of significantly changed metabolic features exceeded 300 (approximately 10 %). In particular, amino acid metabolism, pyrimidine metabolism and glutathione metabolism were widely affected according to the enrich analysis of those significant altered metabolites, which has and have previously been reported to be closely related to fetal development. Moreover, 5'-UMP and N-acetylputrescine with the lowest effective concentrations (EC -10 = 0.1 μM and EC +10 = 0.11 μM, respectively) were identified as sensitive endogenous biomarkers of MEHP exposure., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Bizarre (atypical/symplastic) cells have been described in various gynecologic normal tissues and benign neoplasms. This type of bizarre cytologic change is usually an incidental finding and is regarded as a benign process. We describe 17 cases of bizarre chorionic-type trophoblast in second-trimester and third-trimester placentas that created concern for an underlying/undersampled or incipient intraplacental trophoblastic neoplasm, predominantly found in intervillous trophoblastic islands (11/17), placental septae (6/17), chorionic plate (1/17), and/or the chorion layer of fetal membranes (2/17). The bizarre trophoblastic cells exhibited sheet-like or nested architecture, had a multifocal/patchy distribution, and/or were present as individual cells within hyaline stroma; they were characterized by large nuclei with smudgy chromatin and occasional intranuclear pseudoinclusions. The degree of atypia was classified as mild (0/17), moderate (3/17), or severe (14/17). Mitotic figures and necrosis were not identified. A dual immunohistochemical stain for trophoblast (hydroxyl-delta-5-steroid dehydrogenase) and a proliferation marker (Ki-67), performed in 15 cases, demonstrated 0% to very low proliferative activity within the bizarre trophoblast (0% to 2% [10/15], 3% to 8% [5/15]). Immunohistochemical stains for fumarate hydratase showed intact/retained expression in the bizarre cells in 7 of 7 cases. Clinical follow-up ranged from 1 to 45 months, and all patients were alive and well without subsequent evidence of a gestational trophoblastic or other neoplasms. We conclude that bizarre chorionic-type trophoblast in second-trimester or third-trimester placentas have the potential to mimic an intraplacental trophoblastic neoplasm but are likely a benign degenerative change. This study expands the spectrum of bizarre cells that occur in the gynecologic tract., Competing Interests: Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Introduction: Excessive activation of maternal systemic inflammation is one of the underlying causes of pathology during the disease course of preeclampsia (PE). The triggering receptor expressed on myeloid cells-1 (TREM-1) participates in the development and persistence of inflammation. We hypothesized that dysregulated TREM-1 may be involved in the pathogenesis of PE by promoting the secretion of trophoblastic pro-inflammatory cytokines that augment inflammation., Methods: The localization of TREM-1 in placenta and the extravillous trophoblast cell line (TEV-1) was determined by immunohistochemical staining. The expression level of TREM-1 and pro-inflammatory cytokines in placentas were compared between normal pregnancies and PE. We used lipopolysaccharide (LPS) to simulate trophoblastic inflammation. TEV-1 cells were transfected with TREM-1 plasmid and si-TREM-1 respectively, and then were incubated with LPS. The expression levels of pro-inflammatory cytokines and key molecules featured in nuclear transcription factor-kappaB (NF-κB) pathway were detected. Transwell assays were used to detect the effects of TREM-1 on cell migration and invasion., Results: TREM-1 was localized on both villous trophoblasts (VTs) and extravillous trophoblasts (EVTs). TREM-1 and pro-inflammatory cytokines were up-regulated in preeclamptic placenta. Overexpression of TREM-1 promoted the activation of NF-κB pathway and the release of pro-inflammatory factors induced by LPS, and enhanced migration and invasion of TEV-1 cells. Inhibition of TREM-1 significantly attenuated LPS-induced effects and suppressed migration and invasion., Discussion: This study suggested that TREM-1 was up-regulated in PE, and may promote the production of downstream inflammatory factors by activating NF-κB pathway in trophoblastic cells, thus exerting pro-inflammatory effects in the pathogenesis of PE., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Preeclampsia (PE) and intrauterine growth retardation (IUGR) are the results of defective placentation associated with the downregulation of different genes in the human trophoblast including the Placental Growth Factor (PGF). TrophEctoderm (TE) biopsy is increasingly performed for Pre-implantation Genetic Testing of Aneuploidies and it involves the traumatical removal of an unpredictable number of mural TE cells from the human blastocyst. We observed strikingly similar obstetrical and neonatal complications in pregnancies where the placenta bears PGF downmodulation or a TE biopsy has been done. In both groups, the risk of PE, IUGR, congenital cardiac ventricular septal defects, caesarean section, sex ratio in favour of males and preterm birth is significantly increased compared to controls. Given the high degree of correlation, the observation may not be a casual one. We postulate herein that the TE biopsy may induce persistent dysregulation of different genes in the placenta including PGF. The mechanism proposed is the disruption of tight junctions caused by the TE biopsy., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Pregnancy-specific glycoproteins (PSGs) are fetal proteins secreted by the placenta during pregnancy. The PSG level in maternal serum is an indicator of risk for pregnancy complications. However, little is known about the molecular mechanisms underlying PSG gene expression. Recently, the importance of epigenetic regulation of placental genes has been emphasized in the study of developmental defects and placental disease. In this study, the role of the CCCTC-binding factor (CTCF) in regulation of PSG expression was investigated to better understand the epigenetic regulatory mechanisms of the PSG genes. Inhibition of CTCF expression disturbed transcription of several PSG genes: PSG1, PSG2, PSG4, PSG5, PSG8, and PSG9 were upregulated and PSG6 and PSG11 were downregulated. These transcriptional changes were correlated with decreased CTCF binding and changes in histone modification at the PSG promoters. Our data demonstrate that CTCF is a potential mediator in the regulation of PSG gene expression., (© 2021 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was demonstrated in the placenta; however, the data on the prevalence of placental infection and associated histopathology are limited. To identify the frequency and features of SARS-CoV-2 involvement, we performed a clinicopathologic analysis of 75 placental cases from women infected at the time of delivery and 75 uninfected controls. Placental samples were studied with anti-SARS-CoV-2 immunohistochemistry and/or in situ hybridization. Positive results were confirmed by electron microscopy and quantitative reverse-transcription polymerase chain reaction (qRT-PCR). During delivery, only one woman had symptoms of coronavirus disease 2019, six women reported previous symptoms, and 68 women were asymptomatic. All neonates tested negative for SARS-CoV-2 as per nasopharyngeal swab PCR results. Obstetric histories were unremarkable in 29 of 75 SARS-CoV-2-positive and 8 of 75 SARS-CoV-2-negative women. Placental examination was normal in 12 of 75 infected and 3 of 75 uninfected subjects, respectively. In the remaining cases, placental pathology correlated with obstetric comorbidities without significant differences between SARS-CoV-2-positive and SARS-CoV-2-negative women. SARS-CoV-2 was identified in one placenta of an infected, but asymptomatic, parturient. Viral staining was predominantly localized to the syncytiotrophoblast (STB) which demonstrated marked damage accompanied by perivillous fibrin deposition and mixed intervillositis. A significant decrease of viral titers was detected in the attached umbilical cord compared with the villous parenchyma as per qRT-PCR. SARS-CoV-2 is seldom identified in placentas of infected women. Placental involvement by the virus is characterized by STB damage disrupting the placental barrier and can be seen in asymptomatic mothers without evidence of vertical transmission., (Copyright © 2020. Published by Elsevier Inc.)

Human trophoblast stem cells (TSCs) have been confirmed to play a cardioprotective role in heart failure. However, whether trophoblast stem cell-derived exosomes (TSC-Exos) can protect cardiomyocytes from doxorubicin (Dox)-induced injury remains unclear. In the present study, TSC-Exos were isolated from the supernatants of human trophoblasts using the ultracentrifugation method and characterized by transmission electron microscopy and western blotting. In vitro, primary cardiomyocytes were subjected to Dox and treated with TSC-Exos, miR-200b mimic or miR-200b inhibitor. Cellular apoptosis was observed by flow cytometry and immunoblotting. In vivo, mice were intraperitoneally injected into Dox to establish a heart failure model. Then, different groups of mice were administered either PBS, adeno-associated virus (AAV)-vector, AAV-miR-200b-inhibitor or TSC-Exos via tail vein injection. Then, the cardiac function, cardiac fibrosis and cardiomyocyte apoptosis in each group were evaluated, and the downstream molecular mechanism was explored. TSC-Exos and miR-200b inhibitor both decreased primary cardiomyocyte apoptosis. Similarly, mice receiving TSC-Exos and AAV-miR-200b inhibitor exhibited improved cardiac function, accompanied by reduced apoptosis and inflammation. The bioinformatic prediction and luciferase reporter results confirmed that Zeb1 was a downstream target of miR-200b and had an antiapoptotic effect. TSC-Exos attenuated doxorubicin-induced cardiac injury by playing antiapoptotic and anti-inflammatory roles. The underlying mechanism could be an increase in Zeb1 expression by the inhibition of miR-200b expression. In summary, this study sheds new light on the application of TSC-Exos as a potential therapeutic tool for heart failure.

Objectives: Complete hydatidiform moles (CHMs) are androgenetic and have a high rate of progression to gestational trophoblastic neoplasia (GTN). CHMs are negative when immunostained for p57KIP2 protein, the product of the maternally expressed gene on chromosome 11p15.5, whereas biparental partial hydatidiform moles and hydropic abortion are positive for p57KIP2. This study presents two cases of p57KIP2-positive androgenetic CHMs and explores the cause of this inconsistency., Methods: Androgenetic CHMs were diagnosed using multiplex short tandem repeat polymorphism analysis. Single-nucleotide polymorphism arrays were performed for molecular karyotyping., Results: Among the consecutive 188 androgenetic CHMs, two cases were positive for p57KIP2. The first case remitted spontaneously, whereas the second case developed into low-risk GTN. The first case was positive for p57KIP2 in all villi. The karyotype was 48,XX,+7,+11, with the additional chromosome 11 confirmed to be of maternal origin. The second case presented a mosaic of both positively and negatively stained villi. The karyotype was 46,XX., Conclusions: The cause of one of the CHMs was trisomy with an additional maternal chromosome 11. Although rare, the confirmation of p57KIP2-positive androgenetic CHM status is necessary to manage GTN risk., (© American Society for Clinical Pathology, 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Introduction: While the COVID-19 pandemic continues to have a significant global health impact, rates of maternal to infant vertical transmission remain low (<5%). Parenchymal changes of placentas from COVID-19 infected mothers have been reported by several groups, but the localization and relative abundance of SARS-CoV-2 viral proteins and cellular entry machinery has not been fully characterized within larger placental tissue cohorts., Methods: An extended placental tissue cohort including samples from 15 COVID-19 positive maternal-fetal dyads (with n = 5 cases with evidence of fetal transmission) in comparison with 10 contemporary COVID-19 negative controls. Using comparative immunofluorescence, we examined the localization and relative tissue abundance of SARS-CoV2 spike glycoprotein (CoV2 SP) along with the co-localization of two SARS-CoV2 viral entry proteins angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2)., Results/conclusions: CoV2 SP was present within the villous placenta in COVID-19 positive pregnancies with and without evidence of fetal transmission. We further identified the predominance of ACE2 expression in comparison with TMPRSS2. Importantly, both CoV2 SP and ACE2 expression consistently localized primarily within the outer syncytiotrophoblast layer placental villi, a key physiologic interface between mother and fetus. Overall this study provides an important basis for the ongoing evaluation of SARS-CoV-2 physiology in pregnancy and highlights the importance of the placenta as a key source of primary human tissue for ongoing diagnostic and therapeutic research efforts to reduce the global burden of COVID-19., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The biological links between cancer and pregnancy are of recent interest due to parallel proliferative, immunosuppressive and invasive mechanisms between tumour and trophoblast development. Therefore, understanding "cancer-like" mechanisms in pregnancy could lead to the development of novel cancer therapeutics, however, little is understood on how tumour and trophoblast cells recapitulate similar molecular mechanisms. Based on our observations from a previous study, it was not only evident that exosomal miRNAs are involved in the pathophysiology of preeclampsia but also contained cancer-specific miRNAs, which suggested that "pseudo-malignant-like" exosomal-mediated mechanisms exist in pregnancy. The presented study therefore aimed to identify exosomal miRNAs (exomiR) in pregnancy which can be repurposed towards preventing tumour metastasis and immunosuppression. It was identified that exomiR-302d-3p, exomiR-223-3p and exomiR-451a, commonly associated with cancer metastasis, were found to be highly expressed in pregnancy. Furthermore, computational merging and meta-analytical pathway analysis (DIANA miRPath) of significantly expressed exomiRs between 38 ± 1.9 vs. 30 ± 1.11 weeks of gestation indicated controlled regulation of biological pathways associated with cancer metastasis and immunosuppression. Therefore, the observations made in this study provide the experimental framework for the repurposing of exosomal miRNA molecular mechanisms in pregnancy towards treating and preventing cancer.

Placenta-derived extracellular vesicles (EVs) are involved in communication between the placenta and maternal immune cells possibly leading to a modulation of maternal T-cell signaling components. The ability to identify EVs in maternal blood may lead to the development of diagnostic and treatment tools for pregnancy complications. The objective of this work was to differentiate EVs from bovine placenta (trophoblast) and peripheral blood mononuclear cells (PBMC) by a label-free, non-invasive Raman spectroscopy technique. Extracellular vesicles were isolated by ultracentrifugation. Dynamic light scattering (DLS) and scanning electron microscopy (SEM) were applied to verify the presence and the size distribution of EVs. Raman peaks at 728 cm-1 (collagen) and 1573 cm-1 (protein) were observed only in PBMC-derived EVs, while the peaks 702 cm-1 (cholesterol) and 1553 cm-1 (amide) appeared only in trophoblast-derived EVs. The discrimination of the Raman spectral fingerprints for both types of EVs from different animals was performed by principal component analysis (PCA) and linear discriminant analysis (LDA). The PCA and LDA results clearly segregated the spectral clusters between the two types of EVs. Moreover, the PBMC-derived EVs from different animals were indistinguishable, while the trophoblast-derived EVs from three placental samples of different gestational ages showed separate clusters. This study reports for the first time the Raman characteristic peaks for identification of PBMC and trophoblast-derived EVs. The development of this method also provides a potential tool for further studies investigating the causes and potential treatments for pregnancy complications., Competing Interests: The authors have declared that no competing interests exist.

In mice, the labyrinth zone of the placenta exchanges nutrients and gases between mother and fetus. This placental zone is complex in structure and defects in its morphogenesis can compromise substrate exchange and thus, fetal growth and viability. Numerous mouse models involving genetic and environmental manipulation show abnormalities in labyrinth zone size. However, further structural analysis, normally undertaken using ultrathin resin sections, can pose practical constraints. Here, we validate the use of stereology on paraffin-embedded sections double-labelled for lectin and cytokeratin as a cheap, fast and robust alternative for analysing the structure of the mouse placental labyrinth., Competing Interests: Declaration of competing interest The authors declare that they do not have any financial competing interests or any other interests that could influence the interpretation of the article., (Crown Copyright © 2020. Published by Elsevier Ltd. All rights reserved.)

Background: It has been reported that during the culture of human placental explants, the syncytiotrophoblast dies between 3 and 24 h and is then replaced within 48 h by a new syncytiotrophoblast layer formed by the fusion of underlying cytotrophoblasts. Most frequently the death of the syncytiotrophoblast is indicated by the uptake of nuclear stains such as propidium iodide (PI). This process is reportedly similar in both early and late gestation placental explants., Methods: We cultured first trimester placental explants for up to 48 h and tested membrane intactness by exposure to PI. Connexin and pannexin mRNAs were quantified by RT-PCR and protein levels determined by immunofluorescence. The syncytiotrophoblast membrane leak was determined by culturing explants in the presence of hemichannel blockers. Extrusion of extracellular vesicles from the syncytiotrophoblast was quantified., Results: Nuclei of the syncytiotrophoblast were stained with PI following approximately 4 h of culture and this was prevented by culturing the explants with pannexin-1 blockers. Expression of pannexin-1 hemichannels increased during explant culture (p = 0.0027). Extracellular vesicles were most abundantly extruded from the explants during the first 3 h of culture and the temporal pattern of extrusion was unaltered by blocking hemichannels., Discussion: We show the mechanism of uptake of nuclear non-viability stains into the syncytiotrophoblast during explant culture is via upregulation of pannexin 1 hemichannels. Contrary to suggestions by some, the production of extracellular vesicles from cultured placental explants is not an in vitro artefact resulting from the apparent death of the syncytiotrophoblast in explant cultures., Competing Interests: Declaration of competing interest None of authors have a conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Small extracellular vesicles (sEVs) released from the extravillous trophoblast (EVT) are known to regulate uterine spiral artery remodeling during early pregnancy. The bioactivity and release of these sEVs differ under differing oxygen tensions and in aberrant pregnancy conditions. Whether the placental cell-derived sEVs released from the hypoxic placenta contribute to the pathophysiology of preeclampsia is not known. We hypothesize that, in response to low oxygen tension, the EVT packages a specific set of proteins in sEVs and that these released sEVs interact with endothelial cells to induce inflammation and increase maternal systemic blood pressure. Using a quantitative MS/MS approach, we identified 507 differentially abundant proteins within sEVs isolated from HTR-8/SVneo cells (a commonly used EVT model) cultured at 1% (hypoxia) compared with 8% (normoxia) oxygen. Among these differentially abundant proteins, 206 were up-regulated and 301 were down-regulated (P < 0.05), and they were mainly implicated in inflammation-related pathways. In vitro incubation of hypoxic sEVs with endothelial cells, significantly increased (P < 0.05) the release of GM-CSF, IL-6, IL-8, and VEGF, when compared with control (i.e. cells without sEVs) and normoxic sEVs. In vivo injection of hypoxic sEVs into pregnant rats significantly increased (P < 0.05) mean arterial pressure with increases in systolic and diastolic blood pressures. We propose that oxygen tension regulates the release and bioactivity of sEVs from EVT and that these sEVs regulate inflammation and maternal systemic blood pressure. This novel oxygen-responsive, sEVs signaling pathway, therefore, may contribute to the physiopathology of preeclampsia., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Background: Human syncytiotrophoblast mitochondria require the activity of the isocitrate dehydrogenase type 2 (IDH2) to obtain reduced coenzymes for progesterone (P4) synthesis. Data from the literature indicate that mitochondrial steroidogenic contact sites transform efficiently cholesterol into P4. In this research, we identified the IDH2 as a member of the steroidogenic contact site and analyzed the steroidogenic role of its activity., Method: Human syncytiotrophoblast mitochondria were isolated by differential centrifugation, and steroidogenic contact sites were obtained by osmotic shock and sucrose gradient ultracentrifugation. In-gel native activity assay, mass spectroscopy, and western blot were used to identify the association of proteins and their activities. P4 was determined by immunofluorescence., Results: The IDH2 was mainly identified in steroidogenic contact sites, and its activity was associated with a complex of proteins with an apparent molecular mass of ~590 kDa. Mass spectroscopy showed many groups of proteins with several metabolic functions, including steroidogenesis and ATP synthesis. The IDH2 activity was coupled to P4 synthesis since in the presence of Ca 2+ or Na 2 SeO 3 , inhibitors of the IDH2, the P4 production decreased., Conclusions: The human syncytiotrophoblast mitochondria build contact sites for steroidogenesis. The IDH2, a non-membrane protein, supplies the NADPH required for the synthesis of P4 in a complex (steroidosome) that associate the proteins required to transform efficiently cholesterol into P4, which is necessary in pregnancy to maintain the relationship between mother and fetus., General Significance: The IDH2 is proposed as a check point in the regulation of placental steroidogenesis., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Peroxisome proliferator-activated receptors (PPARs) are members of a nuclear receptor family of ligand-dependent transcription factors. Three isoforms of PPAR named PPARα, PPARβ/δ, and PPARγ have been described, each encoded by a separate gene: PPARA, PPARD, and PPARG, respectively. In the present study, we examined the profiles of PPAR and retinoid X receptor (RXR; PPAR heterodimer partner) mRNA expression and PPAR DNA binding activity in porcine trophoblast tissue collected on days 15, 20, 25, and 30 of pregnancy and in day-20 embryos. Placenta trophoblast cells isolated on day 25 of pregnancy were used to determine effects of (1) cytokines on PPAR and RXR mRNA expression and (2) PPAR agonists on prostaglandin (PG) E2 synthesis and the expression of genes involved in steroidogenesis, fatty acid binding, and PG transport, as well as on cell proliferation. The mRNA expression of PPARA and RXRB was greater in trophoblast tissue collected on days 25 and 30 of pregnancy compared with day 15 (P < 0.05), while DNA binding activity of PPARα decreased between day 15 and 25 (P < 0.05). Increased concentrations of PPARD and RXRA transcripts were observed in trophoblasts collected on day 20 compared to trophoblasts from days 15 and 30 (P < 0.05). Moreover, concentrations of DNA-bound PPARβ/δ and PPARγ proteins increased in day-30 trophoblasts compared to day 15 (P < 0.01) and day 20 (P < 0.05), respectively. On day 20 of gestation, the mRNA expression of PPARD, PPARG, and RXRA and protein levels of PPARα and PPARγ isoforms were greater in trophoblast than embryonic tissue (P < 0.01). Interleukin 1β and/or interferon γ, but not IL6 and leukemia inhibitory factor, upregulated PPAR and RXR mRNA expression in placenta trophoblast cells in vitro (P < 0.05). Rosiglitazone (a PPARγ agonist) stimulated prostaglandin E synthase mRNA expression in trophoblast cells and PGE2 accumulation in incubation medium (P < 0.05). Moreover, activation of PPAR isoforms differentially affected the expression of genes involved in steroidogenesis, fatty acid binding, and PG transport in studied cells. Finally, PPARα and PPARγ agonists stimulated trophoblast cell proliferation (P < 0.05), and this effect was abolished by the addition of a respective PPAR antagonist (P < 0.05). Overall, these results point to a role of PPAR isoforms in porcine placenta development and function., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Introduction: The placenta produces microRNAs (miRNA) that may traffic to the maternal or fetal compartments and influence the physiology of pregnancy. The trafficking patterns of miRNA expressed from the large human chromosome 19 and chromosome 14 clusters (C19MC and C14MC), remains unclear. We interrogated the cross-sectional landscape of miRNA expression within the human placenta, fetal and maternal plasma to elucidate miRNA trafficking. We hypothesized that C19MC and C14MC miRNAs have similar expression patterns across the maternal-fetal compartments., Methods: Placental biopsies, maternal and fetal venous plasma were collected from 25 pregnancies, and RNA was quantified using next generation sequencing. We identified expression and correlations differences among the compartments, and uncovered distinct miRNA expression patterns using consensus clustering., Results: We found that the placenta exhibits the highest total abundance, average miRNA expression and lowest variance of both C19MC and C14MC miRNAs. The C19MC miRNAs had a comparable expression and variance in fetal and maternal plasma and higher expression in the placenta. In contrast, the C14MC miRNAs had comparable expression between the placenta and fetal plasma, which was higher than the maternal plasma. We also identified 5 distinct groups of trophoblastic miRNAs with different expression patterns in each compartment., Discussion: This is the first comprehensive analysis of C19MC and C14MC miRNA expression patterns in the human placental, maternal and fetal compartments. Our findings suggest that C14MC miRNAs are produced by both the fetus and placenta, but C19MC miRNAs are produced primarily in the placenta and are trafficked to the fetal and maternal compartments., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Trophoblast giant cells (TGCs) are one of the cell types that form the placenta and play multiple essential roles in maintaining pregnancy in rodents. TGCs have large, polyploid nuclei resulting from endoreduplication. While previous studies have shown distinct gene expression profiles of TGCs, their chromatin structure remains largely unknown. An appropriate combination of canonical and non-canonical histones, also known as histone variants, allows each cell to exert its cell type-specific functions. Here, we aimed to reveal the dynamics of histone usage and chromatin structure during the differentiation of trophoblast stem cells (TSCs) into TGCs. Although the expression of most genes encoding canonical histones was downregulated, the expression of a few genes encoding histone variants such as H2AX, H2AZ, and H3.3 was maintained at a relatively high level in TGCs. Both the micrococcal nuclease digestion assay and nucleosome stability assay using a microfluidic device indicated that chromatin became increasingly loose as TSCs differentiated. Combinatorial experiments involving H3.3-knockdown and -overexpression demonstrated that variant H3.3 resulted in the formation of loose nucleosomes in TGCs. In conclusion, our study revealed that TGCs possessed loose nucleosomes owing to alterations in their histone composition during differentiation.

Ex vivo culture of human placental explants has long allowed placentologists to study the milieu of soluble factors secreted by the human placenta throughout gestation while retaining the correct three-dimensional structure of the placental villi. Here, we detail the placental explant culture method employed in our laboratory to collect extracellular vesicles which are known to be released by the human placenta throughout pregnancy from 6 weeks of gestation. Using this method, at least three different populations of placental extracellular vesicles can be simultaneously collected from each placental sample, allowing for comparative analysis of the cargos and downstream effects of the different types of extracellular vesicles produced by the human placenta.

In human placenta, the multinucleated syncytiotrophoblast (ST) allows all the exchanges between the maternal and fetal circulation and is also the site of placental hormonal functions. Absence or disturbances of ST formation are associated with a defect or pathologies of pregnancy such as preeclampsia (PE) and intrauterine growth retardation (IUGR). All along pregnancy, the ST is regenerated by fusion of underlying mononucleated villous cytotrophoblasts (VCT). The protocol described here provides details on how GATA3 or TWIST1 immunostaining and analysis can be used to easily assess the in vitro differentiation of human placental cytotrophoblast.

Background: Observational studies have reported associations between maternal phthalate levels and adverse outcomes at birth and in the health of the child. Effects on placental function have been suggested as a biologic basis for these findings., Objective: We evaluated the effects of phthalates on placental function in vitro by measuring relevant candidate genes and proteins., Materials and Methods: Human trophoblast progenitor cells were isolated at 7-14 wk of pregnancy (two female and three male concepti), and villous cytotrophoblast cells (vCTBs) were isolated at 15-20 wk (three female and four male concepti). Cells were cultured in vitro with four phthalate metabolites and their combination at concentrations based on levels found previously in the urine of pregnant women: mono- n -butyl (MnBP, 200 nM), monobenzyl (MBzP, 3μM), mono-2-ethylhexyl (MEHP, 700 nM), and monoethyl (MEP, 1.5μM) phthalates. mRNA levels of CGA , CGB , PPARG , CYP19A1 , CYP11A1, PTGS2, EREG , and the intracellular β subunit of human chorionic gonadotropin (hCGβ) and peroxisome proliferator activated receptor γ (PPARγ) were measured in the cellular extracts, and protein levels for four forms of secreted hCG were measured in the conditioned media., Results: Previously reported associations between maternal phthalates and placental gene expression were reproduced experimentally: MnBP with CGA , MBzP with CYP11A1 , and MEHP with PTGS2 . CGB and hCGβ were up-regulated by MBzP. In some cases, there were marked, even opposite, differences in response by sex of the cells. There was evidence of agonism in female cells and antagonism in male cells of PPARγ by simultaneous exposure to multiple phthalates., Conclusions: Concentrations of MnBP, MBzP and MEHP similar to those found in the urine of pregnant women consistently altered hCG and PPARγ expression in primary placental cells. These findings provide evidence for the molecular basis by which phthalates may alter placental function, and they provide a preliminary mechanistic hypothesis for opposite responses by sex. https://doi.org/10.1289/EHP1539.

Endogenous hydrogen sulfide (H2S), mainly synthesized by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CTH), has been implicated in regulating placental angiogenesis; however, the underlying mechanisms are unknown. This study was to test a hypothesis that trophoblasts synthesize H2S to promote placental angiogenesis. Human choriocarcinoma-derived BeWo cells expressed both CBS and CTH proteins, while the first trimester villous trophoblast-originated HTR-8/SVneo cells expressed CTH protein only. The H2S producing ability of BeWo cells was significantly inhibited by either inhibitors of CBS (carboxymethyl hydroxylamine hemihydrochloride, CHH) or CTH (β-cyano-L-alanine, BCA) and that in HTR-8/SVneo cells was inhibited by CHH only. H2S donors stimulated cell proliferation, migration, and tube formation in ovine placental artery endothelial cells (oFPAECs) as effectively as vascular endothelial growth factor. Co-culture with BeWo and HTR-8/SVneo cells stimulated oFPAEC migration, which was inhibited by CHH or BCA in BeWo but CHH only in HTR-8/SVneo cells. Primary human villous trophoblasts (HVT) were more potent than trophoblast cell lines in stimulating oFPAEC migration that was inhibited by CHH and CHH/BCA combination in accordance with its H2S synthesizing activity linked to CBS and CTH expression patterns. H2S donors activated endothelial nitric oxide synthase (NOS3), v-AKT murine thymoma viral oncogene homolog 1 (AKT1), and extracellular signal-activated kinase 1/2 (mitogen-activated protein kinase 3/1, MAPK3/1) in oFPAECs. H2S donor-induced NOS3 activation was blocked by AKT1 but not MAPK3/1 inhibition. In keeping with our previous studies showing a crucial role of AKT1, MAPK3/1, and NOS3/NO in placental angiogenesis, these data show that trophoblast-derived endogenous H2S stimulates placental angiogenesis, involving activation of AKT1, NOS3/NO, and MAPK3/1., (© The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In the present study, we aimed to 1) demonstrate the presence of all 10 toll-like receptors (TLRs) in ovine trophoblasts, and 2) investigate the expression profiles of TLR1-10 mRNAs in peripheral blood leukocytes (PBLs) in ewes during early pregnancy. For those purposes, ovine trophoblasts (n = 6) were collected from pregnant ewes on day 13. PBLs were collected from non-pregnant (n = 6) and pregnant ewes (n = 17) on days of mating (d) 0 and 18. TLR mRNAs in ovine trophoblasts were visualized by free-floating in situ hybridization (ISH). To assess the expression profiles of TLR1-10 in PBLs, total RNA was isolated and transcribed to cDNA. TLR1-10 mRNA levels were determined by real-time PCR in triplicate. The Relative Expression Software Tool (REST 2009) was used for statistical analysis. We detected mRNAs for TLR2, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR10 but not for TLR1, TLR3, and TLR9 in trophoblasts. TLR2, TLR5, TLR6, TLR7, TLR8, and TLR10 mRNAs were expressed by all trophoblasts, whereas TLR4 mRNA and protein in trophoblasts were more limited. In PBLs, TLR expression did not differ between day 0 and day 18 in non-pregnant ewes; however, ewes in early pregnancy exhibited significantly upregulated expression of TLR2 (2.3-fold), TLR4 (3.1-fold), TLR6 (1.7-fold), and TLR8 (2.2-fold) on day 18 compared with day 0. In contrast, TLR10 was downregulated (2-fold) on day 18 by pregnancy. Similar results were also obtained for TLR2, TLR4, TLR6, TLR8 and TLR10 from the comparison between day 18 non -pregnant and day 18 pregnant groups. According to these results, the presence of TLRs in early ovine trophoblasts suggests that these cells play an immunological role at the maternal-fetal interface. The results also suggest that tight regulation of some components of TLRs in PBLs due to embryo- and/or pregnancy-related factors is necessary for successful establishment of early pregnancy in ewes., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The aim of this study was to explore the role of viperin in the prevention of intrauterine infection of hepatitis B virus (HBV). Placental samples were collected from seven HBV-positive pregnant women with their infants infected via intrauterine transmission (infected group), 30 HBV-positive women with non-infected infants (non-infected group), and 30 HBV-negative women (controls). The expression of viperin in placenta was analyzed with immunohistochemistry and Western blotting. The expression of viperin of placental trophoblast cell line Swan71 was determined after exposed to HBV. Viperin was localized to syncytiotrophoblast, and there was a significant difference in placental viperin levels among control, non-infected group and infected group (F = 12.824, p < 0.001). The expression of viperin was significantly higher in non-infected group than in control (p = 0.019) and in infected group (p < 0.001), and viperin expression was significantly lower in infected group than in control (p = 0.001). The exposure to HBV significantly increased the expression of viperin in Swan 71 (p < 0.001). Exposure to HBV up-regulates viperin expression in vivo and in vitro in placental trophoblast, and lack of this up-regulation is associated with intrauterine transmission of HBV., (© 2016 APMIS. Published by John Wiley & Sons Ltd.)

We report the case of a 43-year-old woman, who was admitted for intense pelvic pains. Biological investigations showed an increase of β-HCG level. The abdomino-pelvic computed tomography revealed a voluminous mass infiltrating the bladder. A transurethral resection was performed. The histopathological examination evidenced an invasion of the bladder by a massive carcinomatous proliferation composed of clear epithelial cells with in some places syncytiotrophoblastic cells and rarely cytotrophoblastic cells. Immunohistochemical analyses revealed a staining of GATA-3 by the two components, an expression of p63 by the clear cell urothelial component and the immunoreactivity of β-HCG by the trophoblastic component. Ki-67 labeling index was very high. The diagnosis of mixt urothelial carcinoma with clear cells and trophoblastic differentiation was proposed. This is a rare difficult histopathological diagnosis which should be known by pathologists. It is correlated to a poor clinical outcome with a high tendency to lymph node or visceral metastases., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

The aim of this study was to investigate the expression of vascular adhesion molecule (VCAM)-1 in the maternal serum, cord blood, and placental tissue of pregnant women from Xingtai, Hebei, with gestational hypertension (GH) combined with fetal growth restriction (FGR). A total of 108 patients with GH combined with FGR (GH-FGR), 60 patients with GH alone (GH), and 50 healthy pregnant women (control) were recruited to this study. VCAM- 1 expression was detected in the maternal serum and cord blood by enzyme-linked immunosorbent assay, and in the placental tissue by immunohistochemistry. VCAM-1 expression was significantly higher in the maternal serum of patients with GH-FGR (164.38 ± 60.35) and GH alone (103.85 ± 54.47) than in the serum of the control population (46.70 ± 21.79; P < 0.05). On the other hand, VCAM-1 expression in the cord blood of GH-FGR (163.19 ± 69.46), GH (149.82 ± 58.20), and control (128.89 ± 43.59) subjects was not significantly different (P > 0.05). Moreover, the VCAM-1 expression rates were significantly higher and lower in the vascular endothelial and trophoblastic cells of the placenta of patients with GH-FGR (74.71 and 56.1%) and GH (72.98 and 55.36%), respectively, compared to those in the control subjects (46.48 and 95.11%). Therefore, we concluded that VCAM- 1 plays an important role in the development and generation of GH. Additionally, the low VCAM-1 expression in the trophoblastic cell could be correlated to the pathogenesis and progression of GH., Competing Interests: The authors declare no conflict of interest.

Human embryonic stem cells (hESCs) have been used to derive trophoblasts through differentiation in vitro. Intriguingly, mouse ESCs are prevented from differentiation to trophoblasts by certain epigenetic factor proteins such as Dnmt1, thus necessitating the study of epigenetic factor proteins during hESC differentiation to trophoblasts. We used stable isotope labeling by amino acids in cell culture and quantitative proteomics to study changes in the nuclear proteome during hESC differentiation to trophoblasts and identified changes in the expression of 30 epigenetic factor proteins. Importantly, the DNA methyltransferases DNMT1, DNMT3A, and DNMT3B were downregulated. Additionally, we hypothesized that nuclear proteomics of hESC-derived trophoblasts may be used for screening epigenetic factor proteins expressed by primary trophoblasts in human placental tissue. Accordingly, we conducted immunohistochemistry analysis of six epigenetic factor proteins identified from hESC-derived trophoblasts-DNMT1, DNMT3B, BAF155, BAF60A, BAF57, and ING5-in 6-9 week human placentas. Indeed, expression of these proteins was largely, though not fully, consistent with that observed in 6-9 week placental trophoblasts. Our results support the use of hESC-derived trophoblasts as a model for placental trophoblasts, which will enable further investigation of epigenetic factors involved in human trophoblast development.

SALL4 has important functions in embryonic stem cells. The aim of this study was to investigate SALL4 expression in gestational trophoblastic neoplasia. We hypothesized that it could help to distinguish choriocarcinoma, the presumed most primitive form of gestational trophoblastic neoplasia, from placental site trophoblastic tumor and epithelioid trophoblastic tumor, which would be more differentiated variants. This study included 31 gestational trophoblastic neoplasias: 19 choriocarcinomas, 9 placental site trophoblastic tumors, 1 epithelioid trophoblastic tumor, and 2 mixed tumors comprising a placental site trophoblastic tumor and an epithelioid trophoblastic tumor. Unlike usual markers of gestational trophoblastic neoplasia (p63, human chorionic gonadotrophin and human placental lactogen), SALL4 was expressed in 100% of choriocarcinomas and it was not detected in any placental site trophoblastic tumor and epithelioid trophoblastic tumor. However, the proportion of positive cells varied in a wide range, from 10% to 70%, reflecting the fact that SALL4 was specifically present in mononuclear cells consistent with neoplastic cytotrophoblast. So, SALL4 may be helpful in the differential diagnosis of gestational trophoblastic neoplasias., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objective: To investigate the relationship between placental transthyretin (TTR) level and preeclampsia., Materials and Methods: Placental tissues from uncomplicated and preeclamptic pregnancies were analyzed using immunohistochemistry and image analysis. We measured the mean optical density (OD) of immunohistochemical staining of TTR across multiple sections using Image Pro Plus 6.0. To avoid bias, we used placental tissue array, which contained preeclamptic placentas (n=8) and the control placentas (n=6) on the same slide., Results: The mean TTR OD of the syncytiotrophoblast layer of placentas (95% confidence interval) from the first trimester was higher than those from the second/third trimester, and term placentas [0.149 (0.014-0.285) for the 1(st) trimester, 0.037 (0.000-0.073) for the 2(nd)/3(rd) trimester, and 0.011 (0.035-0.056) for term; p<0.01]. Although the OD of the second/third trimester placentas appeared greater than that of term placentas, this was not statistically significant. The mean TTR OD of the syncytiotrophoblast layer of the severe preeclampsia group was lower than that of controls [0.010 (0.005-0.016) vs. 0.027 (0.013-0.041), p<0.05]., Conclusion: The immunohistochemical expression of TTR in the syncytiotrophoblast layer of the placenta decreased significantly after 12 weeks of gestation, paralleling the changing demands of thyroid hormone uptake into the placenta. The reduced TTR expression in the syncytiotrophoblast layer of the preeclamptic placenta might impair thyroid hormone uptake and contribute to the pathophysiology of the disease., (Copyright © 2016. Published by Elsevier B.V.)

Introduction: α-klotho is an anti-aging protein, potentially important in preeclampsia (PE). Produced by kidney, brain and placenta, and by mRNA splicing is both a full-length membrane-bound and a truncated soluble protein in the circulation. The membrane-bound protein is an obligate co-receptor for fibroblast growth factor 23 (FGF23) and its action on receptor (FGFR), but ADAM proteinases also cause its shedding. The aims of this study were to investigate levels of maternal plasma, placental, and fetal membrane α-Klotho and their association with placental accelerated villous maturation (AVM) in PE. In addition, placental and membrane levels of ADAM17 and FGFR were measured in the same patients., Methods: Maternal blood, placenta and fetal membranes from 61 women (31 with PE and 30 controls) between 32 and 40 weeks gestation were collected. Plasma α-klotho was measured by ELISA, and quantitative immunohistochemistry used for α-klotho, ADAM17 and FGFR1 in tissues. Placental AVM was histologically assessed., Results: Maternal plasma levels of α-Klotho were higher in PE compared to controls (p = 0.01) and patients with the highest levels had significantly less AVM (p = 0.03). α-Klotho, ADAM17, and FGFR were all present in syncytiotrophoblast and cytotrophoblast of membranes. Between 32 and 40 weeks gestation, all placental levels decreased in controls respectively (p = 0.04, p = 0.004, p = 0.05), but not in PE. Fetal membrane levels were unchanged., Discussion: Maternal plasma α-Klotho was increased in PE and its levels associated with reduced placental AVM. Changes in placental α-Klotho, ADAM17, and FGFR suggest their involvement in the pathophysiology of PE., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The aim of the present study was to investigate immunohistochemical distribution of heat shock protein 70 (Hsp70) and proliferating cell nuclear antigen (PCNA) in the mouse placenta at different gestational stages. For this purpose a total of 18 Swiss albino female mice at 12-14 weeks of age were used. Females were sacrificed on days 3 (early), 10 (mid-), and 17 (late) of pregnancy and the implantation sites of the pregnant uterus were sampled. The sections were made transversely through the central region of the implantation site and stained with hematoxylin and eosin for histological examination. PCNA and Hsp70 was stained immunohistochemically. Since the definitive placenta was not still formed on day 3 of pregnancy, Hsp70 and PCNA positivity were evaluated in only luminal epithelium and decidual-stromal cells. On days 10 and 17 of pregnancy, Hsp70 and PCNA positivity were evaluated in labyrinth zone, junctional zone and decidual layer of placenta. Hsp70 expression was observed trophoblast cells and decidual cells and was relatively constant throughout the pregnancy. This protein was strongly labeled in the trophoblast cells; while decidual cells were displayed moderate staining. In early pregnant mouse uteri, PCNA was mainly localized in decidual-stromal cells. The trophoblast cells and decidual cells displayed highly proliferative activity at the midgestational period. However there was a significant decrease in the percentage of PCNA positive cells in late gestation., (© 2016 Wiley Periodicals, Inc.)

Objective: To investigate the blastocoelic fluid (BF) for the presence of DNA that could be amplified and analyzed; the extent to which its chromosomal status corresponds to that found in trophectoderm (TE) cells, polar bodies (PBs), or blastomeres; and the identification of segmental abnormalities., Design: Longitudinal cohort study., Setting: In vitro fertilization unit., Patient(s): Fifty-one couples undergoing preimplantation genetic screening or preimplantation genetic diagnosis for translocations by array-comparative genomic hybridization on PBs (n = 21) or blastomeres (n = 30)., Intervention(s): BFs and TE cells were retrieved from 116 blastocysts, whose chromosome status had already been established by PB or blastomere assessment. Separate chromosome analysis was performed in 70 BFs., Main Outcome Measure(s): Presence of DNA in BFs, evaluation of the chromosome condition, and comparison with the diagnosis made in TE cells and at earlier stage biopsies., Result(s): DNA detection was 82%, with a net improvement after refinement of the procedure. In 97.1% of BFs, the ploidy condition corresponded to that found in TE cells, with one false positive and one false negative. The rate of concordance per single chromosome was 98.4%. Ploidy and chromosome concordance with PBs were 94% and 97.9%, respectively; with blastomeres, the concordances were 95% and 97.7%, respectively. Segmental abnormalities, which were detected in PBs or blastomeres of 16 blastocysts, were also identified in the corresponding BFs., Conclusion(s): BF represents to a good extent the blastocyst ploidy condition and chromosome status when compared with TE cells. If the proportion of clinically useful BFs is improved, blastocentesis could become the preferred source of DNA for chromosomal testing., (Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

The bradykinin type 2 receptor (B2R), main effector of the pleiotropic kallikrein-kinin system (KKS), has been localized in the key sites related to placentation in human, rat and guinea pig utero-placental units. The present study was directed to characterize the content, the cellular and subcellular localization of B2R in the villi and basal plate of placentas from normal and preeclamptic pregnancies by means of western blotting, immunohistochemistry and immunoelectron microscopy. The protein content of B2R was demonstrated in both placental zones. The villous placenta of normal and preeclamptic pregnancies expressed B2R in syncytiotrophoblast and fetal endothelium; the basal plate displayed B2R in extravillous trophoblasts and decidual cells. Lastly, immunogold electron microscopy revealed B2R in fetal endothelium, syncytiotrophoblast, extravillous cytotrophoblasts and decidual cells; in all cell types the receptor was mainly located in the cytosol and nucleus. The protein content of placental homogenates and the immunoreactivity in the different cells types did not differ between both study groups; however the abundance of nuclear immunogold B2R positive beads in extravillous trophoblasts was greater in the normal than in the preeclamptic placentas. The purpose of describing nuclear B2R in the utero-placental unit, and its increment in normal extravillous trophoblasts, is to stimulate the study of the functional pathways that may be relevant to understand the local role of the B2R in normal and preeclamptic gestation., (Copyright © 2016 International Society for the Study of Hypertension in Pregnancy. Published by Elsevier B.V. All rights reserved.)

Implantation of the mammalian embryo requires profound endometrial changes for successful pregnancy, including epithelial-mesenchymal transition of the luminal epithelium and stromal-epithelial transition of the stromal cells resulting in decidualization. Claudins (Cldn) determine the variability in tight junction paracellular permeability and may play a role during these epithelial and decidual changes. We here localized Cldn3, Cldn7 and Cldn10 proteins in the different compartments of murine endometrium up to day 8.5 of pregnancy (dpc) as well as in human endometrium and first trimester decidua. In murine estrous endometrium, luminal and glandular epithelium exhibited Cldn3 and Cldn7, whereas Cldn10 was only detectable in glandular epithelium. At 4.5 dpc, Cldn3 protein shifted to an apical localization, whereas Cldn7 vanished in the epithelium of the implantation chamber. At this stage, there was no stromal signal for Cldn3 and Cldn7, but a strong induction of Cldn10 in the primary decidual zone. Cldn3 proteins emerged at 5.5 dpc spreading considerably from 6.5 dpc onward in the endothelial cells of the decidual blood sinusoids and in the decidual cells of the compact antimesometrial region. In addition to Cldn3, Cldn10 was identified in human endometrial epithelia. Both proteins were not detected in human first trimester decidual cells. Cldn3 was shown in murine trophoblast giant cells as well as in human extravillous trophoblast cells and thus may have an impact on trophoblast invasion in both species. We here showed a specific claudin signature during early decidualization pointing to a role in decidual angiogenesis and regulation of trophoblast invasion.

The human placenta releases multiple types and sizes of syncytiotrophoblast (STB) extracellular vesicles (EV) into the maternal circulation that exhibit diverse biological activities. The placental perfusion technique enables isolation of these STBEV, but conventional flow cytometry can only be used to phenotype EV down to ∼300 nm in size. Fluorescence Nanoparticle Tracking Analysis (fl-NTA) has the potential to phenotype EV down to ∼50 nm, thereby improving current characterisation techniques. The aims of this study were to prepare microvesicle and exosome enriched fractions from human placental perfusate (n=8) and improve fl-NTA STBEV detection. Differential centrifugation and filtration effectively removed contaminating red blood cells from fresh placental perfusates and pelleted a STB microvesicle (STBMV) fraction (10,000×g pellet - 10KP; NTA modal size 395±12 nm), enriched for the STB marker placental alkaline phosphatase (PLAP) and a STB exosome (STBEX) fraction (150,000×g pellet - 150KP; NTA modal size 147±6 nm), enriched for PLAP and exosome markers Alix and CD63. The PLAP positivity of 'standard' 10KP and 150KP pools (four samples/pool), determined by immunobead depletion, was used to optimise fl-NTA camera settings. Individual 10KP and 150KP samples (n=8) were 54.5±5.7% (range 17.8-66.9%) and 30.6±5.6% (range 3.3-51.7%) PLAP positive, respectively. We have developed a reliable method for enriching STBMV and STBEX from placental perfusate. We also standardised fl-NTA settings and improved measurement of PLAP positive EV in STBMV. However, fl-NTA is not as sensitive as anti-PLAP Dynabead capture for STBEX detection, possibly due to STBEX having lower surface expression of PLAP. These important developments will facilitate more detailed studies of the role of STBMV and STBEX in normal and pathological pregnancies., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: This study investigated the roles of progesterone and leptin in placenta invasion, which is closely related to pregnancy prognosis. We examined the effects of leptin and progesterone on the invasion of BeWo cells, a human trophoblastic cell line, and the effect of concurrent treatment., Methods: Cells were treated with leptin (0, 5, 50, or 500 ng/mL) or progesterone (0, 2, 20, or 200 µM) and cultured in an invasion assay. Cells treated with 500 ng/mL leptin were also treated with progesterone (0, 2, 20, or 200 µM) in the invasion assay for 48 h. The number of cells that invaded the lower surface was counted in five randomly chosen fields using a light microscope with a 200× objective. The mRNA expression levels of MMP-9, TIMP1, TIMP2, and E-cadherin were detected by semi-quantitative PCR., Results: Invasion of BeWo cells was promoted by leptin and influenced by both leptin concentration and treatment duration. Invasion was most effective at 500 ng/mL leptin and 48 h culture. Leptin-induced invasiveness was suppressed by progesterone in a dose-dependent manner. Leptin significantly decreased the expression levels of TIMP1 and E-cadherin, whereas progesterone significantly decreased expression of MMP-9 and significantly increased levels of TIMP1, TIMP2, and E-cadherin., Conclusions: Leptin promotes invasion of BeWo cells, and progesterone suppresses leptin-induced invasion by regulating the expressions of MMP-9, TIMP1, TIMP2, and E-cadherin. The balance between leptin and progesterone may play an important role in human placenta formation during early pregnancy.

Introduction: Magnesium (Mg(2+)) is essential for cellular growth and the maintenance of normal cellular processes. However, little is known about how maternal hypomagnesemia during pregnancy affects fetal growth and development. This study investigated the effects of maternal hypomagnesemia on the late gestation placenta and fetus, and postnatal outcomes until weaning., Methods: Female CD1 mice consumed a control (0.2% w/w Mg(2+)), moderately Mg(2+) deficient (MMD; 0.02% w/w Mg(2+)) or severely Mg(2+) deficient (SMD; 0.005% w/w Mg(2+)) diet for 4 weeks prior to mating and throughout pregnancy. Dams were killed at E18.5 for embryonic studies or allowed to litter naturally and the offspring studied up to postnatal day 21., Results: At E18.5, both Mg(2+) deficient diets decreased maternal plasma and bone Mg(2+) but only the SMD diet decreased fetal plasma Mg(2+). Maternal hypomagnesemia led to fetal loss and fetal growth restriction. Maternal Mg(2+) deficiency increased placental glycogen cell area and decreased spongiotrophoblast cell area while upregulating mRNA expression of the MagT1 Mg(2+) transporter in spongiotrophoblast cells. The SMD animals also displayed instances of gross placental abnormalities. After birth, pups in the SMD group had increased early postnatal mortality and failed to thrive. Pups in the MMD group underwent catch-up growth but remained shorter than controls at PN21 and were hypomagnesemic and hypoglycemic., Conclusions: These changes suggest that maternal Mg(2+) deficiency during pregnancy impairs placental development and fetal growth, which may have long-term health consequences for offspring. Collectively, these results have important implications for women who are Mg(2+) deficient during pregnancy., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

This study aimed to gain new insights into both systemic and placental leptin and its receptors, with reference to the maternal prepregnancy body mass index (BMI). Thus, 84 women (29 lean, 24 overweight, and 31 obese) were recruited and maternal, cord blood, and placental tissues collected prior to term labor. Plasma levels were measured by enzyme-linked immunosorbent assay and for placenta, immunohistochemistry and messenger RNAs (mRNAs) were quantitated. We confirmed that maternal leptin increased linearly as the soluble receptor decreased with BMI (P = .001). Fetal leptin increased with maternal BMI (P = .02) and birth weight (P = .006) and was higher in female infants (P < .001). Placental mRNA levels of leptin and its receptors showed no change in BMI. However, we show a significant (P = .043) linear increase in leptin in the placental vascular endothelial cells with maternal obesity, while leptin in syncytiotrophoblast showed no statistical change. Leptin receptors localized to syncytiotrophoblast and intravillous macrophages and were unchanged with BMI., (© The Author(s) 2014.)

Human endogenous retroviruses (ERVs) represent 8% of the total human genome. Although the majority of these ancient proviral sequences have only retained non-coding long terminal repeats (LTRs), a number of "endogenized" retroviral genes encode functional proteins. Previous studies have underlined the implication of these ERV-derived proteins in the development and the function of the placenta. In this review, we summarize recent findings showing that two ERV genes, termed Syncytin-1 and Syncytin-2, which encode former envelope (Env) proteins, trigger fusion events between villous cytotrophoblasts and the peripheral multinucleated syncytiotrophoblast layer. Such fusion events maintain the stability of this latter cell structure, which plays an important role in fetal development by the active secretion of various soluble factors, gas exchange and regulation of fetomaternal immunotolerance. We also highlight new studies showing that these ERV proteins, in addition to their localization at the cell surface of cytotrophoblasts, are also incorporated on the surface of various extracellular microvesicles, including exosomes. Such exosome-associated proteins could be involved in the various functions attributed to these vesicles and could provide a form of tropism. Additionally, through their immunosuppressive domains, these ERV proteins could also contribute to fetomaternal immunotolerance in a local and more distal manner. These various aspects of the implication of Syncytin-1 and -2 in placental function are also addressed in the context of the placenta-related disorder, preeclampsia.

Introduction: The interaction between trophoblast cells and maternal uterine endothelium is important for placental vascular modelling. Nitric oxide (NO) is a potent vasorelaxant that regulates systemic blood pressure and is reduced in preeclampsia. NO may affect placental cell interaction., Objectives: This study was to examine whether NO plays a role in regulating TNF-α induced inhibition of trophoblast cell integration into endothelial cellular networks in-vitro., Methods: Red fluorescent-labelled human uterine myometrial microvascular endothelial cells (UtMVECs) were seeded on Matrigel. After endothelial cellular networks formed, green fluorescent-labelled HTR-8/SVneo trophoblast cells were co-cultured with endothelial cells, together with/without TNF-α (0.5 ng/ml) and/or NO donor, sodium nitroprusside dihydrate (SNP) (100 μM). Images were captured after 24 h. The effects on HTR-8/SVneo cell integration were quantified by Image Analysis software. The cells were then recovered from Matrigel to extract mRNA. Quantitative PCR was performed to evaluate the expression of eNOS, VCAM-1 and E-selectin. The concentrations of sVCAM-1 and sE-selectin in the conditioned medium were measured by ELISA., Results: TNF-α inhibited HTR-8/SVneo trophoblast cell integration into endothelial cellular networks, as well as decreased eNOS mRNA expression. Increases in VCAM-1 and E-selectin in cellular mRNA and protein concentrations in the conditioned medium were also seen. The NO donor reversed the inhibitory effect of TNF-α on trophoblast integration and increased eNOS mRNA expression. SNP also reduced sE-selectin and sVCAM-1 expressions which were increased by TNF-α in the conditioned medium., Conclusion: Our data suggest the inhibitory effect of TNF-α on trophoblast integration may be mediated by NO, via reducing endothelial cell activation., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Introduction: Intrauterine growth restriction (IUGR) has been documented to increase placental apoptosis at term. HSP27 has been shown to be involved in the control of apoptosis. Our objective is to determine the expression of phosphorylated HSP27 (p-HSP27) in human IUGR, and to determine the role of HSP27 during gestation in an ovine hyperthermia induced model of IUGR., Methods: Human placenta tissue samples were collected at term to quantify p-HSP27. Pregnant sheep were placed in hyperthermic (HT) conditions to induce IUGR. Placental tissues were collected at 55 (early), 95 (mid-gestation) and 130 (near-term) days gestational age (dGA) to determined phosphorylated and total HSP27 across the development of IUGR., Results: Phosphorylated HSP27 was significantly reduced in human placenta IUGR compared to controls at term. HSP27 was increased throughout gestation during the development of IUGR in the sheep. P-HSP27 was increased in early gestation (55 dGA), and decreased near term (130 dGA). The near term decrease was localized to the trophoblast cells of the placenta., Discussion and Conclusion: We conclude that decreased p-HSP27 at term is present when placental apoptosis is increased during IUGR. This could be a factor leading to the decreased placental weight observed during IUGR., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Immunoelectron microscopy is wrought with technical limitations that complicate its use. However, advances in correlative light and electron microscopy have recently lead to improvements in this field. We report the development of a semi-correlative approach to investigate the ultrastructural location of an antiphospholipid antibody within the syncytiotrophoblast. This method offers several advantages over existing methodologies, since it preserves antigenicity, shows good immunolabel penetrability and does not require specialized equipment. The use of a pre-embedding screen has also allowed us to target individual placental villi and overcome sampling limitations of the electron microscope. This simple, cost-effective method is likely to find widespread application in placental research., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Aim: To investigate whether there is difference in trophoblast apoptosis between infants with asymmetrical idiopathic intrauterine growth retardation (IUGR) and those with symmetrical fetal growth appropriate for gestational age (AGA)., Methods: Data and placentas from 52 singleton term pregnancies with idiopathic IUGR, from which a subgroup of 33 (63.4%) infants with asymmetrical growth and malnutrition was identified using the ponderal index served as a study group. The control group included 60 (86.9%) infants with symmetrical growth, identified by the same criterion among 69 normal singleton pregnancies with AGA. IUGR was defined by birthweight less than the 10th percentile of standard values. Ponderal index value was considered as the measurement of fetal growth proportionality., Results: The proportion of fetal thinness up to ponderal index value was greater in the IUGR group than control (χ(2) = 9.2; P = 0.002). There was no statistically significant difference in the cytotrophoblast proliferation (t = 0.88; P = 0.373), trophoblast expression of the Bcl-2 anti-apoptotic factor (z = 0.66; P = 0.505), total trophoblast apoptotic index (t = 0.45; P = 0.651), as in cytotrophoblast (t = 0.01; P = 0.988) and syncytiotrophoblast apoptotic index (t = 0.34; P = 0.730) between the idiopathic asymmetrical IUGR and control group., Conclusion: Asymmetry of fetal growth is a result of rather long-term placental nutritive insufficiency in which trophoblasts have a central role. Although being crucial for its functioning, the proliferative and apoptotic trophoblast activity remains unaltered in the placentas from pregnancies with idiopathic IUGR and asymmetrical fetal growth. The results obtained in this study indicate that placental nutritive insufficiency may develop without any deviation in the physiological trophoblast regeneration via apoptosis., (© 2013 The Authors. Journal of Obstetrics and Gynaecology Research © 2013 Japan Society of Obstetrics and Gynecology.)

Objective: To investigate the roles of COX-2, TNF-α, IL-6 in the pathogenesis of autoimmune-type recurrent spontaneous abortion (RSA)., Methods: RT-PCR was used to detect the mRNA of COX-2, TNF-α, IL-6 in the trophoblast cells of murine RSA and normal pregnant models. The COX-2, TNF-α, IL-6 protein expressions were determined by using immunohistochemisry staining method. The COX-2, TNF-α, IL-6 protein expressions were determined by ELISA., Results: The embryo loss rates in experiment group was significantly higher than that in normal pregnancy control group, the expression of COX-2, TNF-α, IL-6 in the trophoblast cells of murine RSA and normal pregnant models. The expression of COX-2 in autoimmune-type recurrent spontaneous abortion was significantly lesser than in normal pregnant models. The expression of TNF-α, IL-6 in autoimmune-type recurrent spontaneous abortion was significantly higher than in normal pregnant models. There was a positively correlation between TNF-α and IL-6. There was no relationship between COX-2, TNF-α and IL-6., Conclusions: The abnormal expression of COX-2, TNF-α and IL-6 may result in RSA., (Copyright © 2013 Hainan Medical College. Published by Elsevier B.V. All rights reserved.)

The study was conducted to evaluate the effects of human relaxin on apoptosis in the human trophoblast derived HTR-8/SV neo cell line, which is a possible model of human extravillous trophoblasts (EVTs). HTR-8/SV neo cells, cultured in phenol red free RPMI1640 medium, were treated with different doses of human recombinant (rH2) relaxin in serum-deprived conditions. RT-PCR was used for evaluating relaxin receptor: RXFP1 and RXFP2 expression in HTR-8/SV neo cells. The cell death was examined by TUNEL assay. Furthermore, we investigated caspase-3, cleaved PARP and Bcl-2 expressions by Western blot analysis to recognize the translational effects of anti-apoptotic and pro-apoptotic proteins. RXFP1 and RXFP2 mRNA expression was observed in HTR-8/SV neo cells. Compared with untreated control cultures, treatment with rH2 relaxin, decreased TUNEL-positive rate in HTR-8/SV neo cells was observed. Western blot analysis revealed that treatment with rH2 relaxin decreased the expression of caspase-3 and cleaved PARP, but in contrast increased Bcl-2 expression in those cells. These results suggest that rH2 relaxin has anti-apoptotic effects on HTR8/SV neo cells by decreasing pro-apoptotic caspase-3 and cleaved PARP expression and up-regulating anti-apoptotic Bcl-2 expression.

A disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1) is a secreted protease. Through the regulation of extracellular matrix remodeling or developmental processes or both, ADAMTS1 is involved in several biological functions, including ovulation and embryo receptivity. However, the expression and possible role of ADAMTS1 in bovine endometrium is unknown. In this study, we analyzed ADAMTS1 mRNA expression in bovine endometrium during the estrous cycle, peri-implantation period, and at different stages of gestation by using quantitative real-time RT-PCR (qPCR) and in situ hybridization. The qPCR results indicated that the expression of ADAMTS1 mRNA was not affected by the day of the estrous cycle and was similar to cyclic levels on day 35 of gestation; however, the expression was more abundant in cotyledonary tissues of the placenta during late gestation. The in situ hybridization study showed that ADAMTS1 mRNA was detected mainly in uterine luminal epithelia and stromal cells during the estrous cycle and peri-implantation period. A disintegrin and metalloproteinase with thrombospondin motifs 1 mRNA was also expressed in the peri-implantation conceptus as well as in trophoblast cells, which include binucleate cells, and increased during late gestation. Furthermore, treatment of stromal cell with progesterone (300 nM) stimulated the expression of ADAMTS1 mRNA. This study indicates that ADAMTS1 participates in bovine endometrial remodeling, which is required for implantation and placental development in coordination with ovarian steroids., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Introduction: ATP-sensitive potassium channels (KATP channels) have been identified in a variety of tissues. Nevertheless, the presence and role of such metabolism-sensitive K+ channels still remain to be unraveled in the reproductive system., Methods: The study evaluates the presence of KATP channel subunits in human term placental explants by immunohistochemistry, proximity ligation assay, Western blot and RT-PCR techniques. The potential involvement of KATP channels in human placental lactogen (hPL) and human chorionic gonadotrophin (hCG) release has been assessed radioimmunologically from human term placental explants incubated in the presence of different KATP channel modulators., Results: Immunolocalization of the KATP channel subunits documented both the Kir6.2 and SUR2 subunits in the syncytiotrophoblast of human term placenta. Their colocalization was demonstrated by proximity ligation assay and their presence was further confirmed by immunoblotting and RT-PCR. Kir6.1 subunit was immunolocalized in blood vessels media. SUR1 was not expressed at the mRNA level. Incubation of human term placental explants in the presence of increasing concentrations of modulators of KATP channels such as glibenclamide, tolbutamide, pinacidil or diazoxide did not affect the measured hCG and hPL secretory rates., Discussion: Our study reports, for the first time, the presence of the KATP channel subunits Kir6.2 and SUR2 in the human term syncytiotrophoblast. However, under the present experimental conditions, the activation or inhibition of these putative KATP channels by different pharmacological agents did not affect the hPL and hCG secretory rate of human term placental explants., Conclusion: The present findings suggest that the human term syncytiotrophoblast might be endowed with KATP channels. Further studies should clarify their implication in the syncytiotrophoblast ionic homeostasis and hormone regulation., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Placental site nodules (PSNs) and epithelioid trophoblastic tumors (ETTs) respectively represent non-neoplastic and neoplastic lesions of chorionic-type intermediate trophoblasts (ITs). Many patients with a PSN have a history of a cesarean section (CS) or therapeutic abortion. Recent evidence shows that a PSN may progress to an ETT. Herein, we describe a coexisting ETT and placental site trophoblastic tumor (PSTT) intimately associated with PSNs in the post-cesarean lower uterine segment of a 41-year-old woman. The patient presented with abnormal vaginal bleeding 1 year after a cesarean delivery for her most recent pregnancy. We speculated that the neoplasms had transformed from PSNs, the formation of which was related to faulty expulsion of the placental tissue or abnormal colonization of chorionic-type ITs during the CS. Neoplastic trophoblastic cells derived from PSNs displayed differentiation plasticity toward chorionic-type ITs and implantation site ITs that were respectively constituted of an ETT and PSTT., Virtual Slides: The virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1597949195882123.