Trophoblast cellular adhesion molecule expression regulated by different genes and their correlation with pregnancy-induced hypertension.

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.

Dose-response mapping of MEHP exposure with metabolic changes of trophoblast cell and determination of sensitive markers.

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.

Bizarre Chorionic-type Trophoblast in Second-trimester and Third-trimester Placentas: Clinicopathologic Characterization of a Placental Pseudoneoplastic Lesion.

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.

TREM-1 amplifies trophoblastic inflammation via activating NF-κB pathway during preeclampsia.

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.

Hypothesis: human trophectoderm biopsy downregulates the expression of the placental growth factor gene.

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.

Depletion of CTCF disrupts PSG gene expression in the human trophoblast cell line Swan 71.

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.

Trophoblast damage with acute and chronic intervillositis: disruption of the placental barrier by severe acute respiratory syndrome coronavirus 2.

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.

Human trophoblast-derived exosomes attenuate doxorubicin-induced cardiac injury by regulating miR-200b and downstream Zeb1.

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.

Exosomal MicroRNAs in Pregnancy Provides Insight into a Possible Cure for Cancer.

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.

Consistent localization of SARS-CoV-2 spike glycoprotein and ACE2 over TMPRSS2 predominance in placental villi of 15 COVID-19 positive maternal-fetal dyads.

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.

Androgenetic Complete Hydatidiform Moles With p57KIP2-Positive Immunostaining.

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.

Raman Spectroscopy characterization extracellular vesicles from bovine placenta and peripheral blood mononuclear cells.

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.

Double-label immunohistochemistry to assess labyrinth structure of the mouse placenta with stereology.

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.

Upregulation of pannexin-1 hemichannels explains the apparent death of the syncytiotrophoblast during human placental explant culture.

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.

Hypoxia-induced small extracellular vesicle proteins regulate proinflammatory cytokines and systemic blood pressure in pregnant rats.

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.

Isocitrate dehydrogenase type 2 (IDH2) is part of a multiprotein complex for placental steroidogenesis.

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 Ca2+ or Na2SeO3, 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.

Expression and role of peroxisome proliferator-activated receptors in the porcine early placenta trophoblast.

Peroxisome proliferator-activated receptors (PPARs) are members of a nuclear receptor family of ligand-dependent transcription factors. Three isoforms of PPAR named PPARα, PPARß/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 PPARß/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 1ß 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.

Distinct communication patterns of trophoblastic miRNA among the maternal-placental-fetal compartments.

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.

Nucleosomes of polyploid trophoblast giant cells mostly consist of histone variants and form a loose chromatin structure.

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.

Isolation and Characterization of Extracellular Vesicles from Ex Vivo Cultured Human Placental Explants.

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.