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Perilla leaves are known to be a rich source of polyphenols, which have been shown to exhibit various biological effects. This study aimed to compare the bioefficacies and bioactivities of fresh (PLEf) and dry (PLEd) Thai perilla (Nga-mon) leaf extracts. Phytochemical analysis indicated that both PLEf and PLEd were abundant in rosmarinic acid and bioactive phenolic compounds. PLEd, which had higher levels of rosmarinic acid but lower levels of ferulic acid and luteolin than PLEf, exhibited greater effectiveness in a free radical scavenging assay. Furthermore, both extracts were found to suppress intracellular ROS generation and exhibit antimutagenic activity against food-borne carcinogens in S. typhimurium. They also attenuated lipopolysaccharide-induced inflammation in RAW 264.7 cells by inhibiting the expression of nitric oxide, iNOS, COX-2, TNF-α, IL-1β, and IL-6 through the suppression of NF-κB activation and translocation. However, PLEf exhibited a higher ability to suppress cellular ROS production and higher antimutagenic and anti-inflammatory activities than PLEd, which can be attributed to its combination of phytochemical components. Overall, PLEf and PLEd have the potential to serve as natural bioactive antioxidant, antimutagenic, and anti-inflammatory agents to achieve potential health benefits.

Air pollution is one of the largest global environmental health hazards that threaten premature mortality or morbidity. Particulate matter 10 (PM10) has been demonstrated to contribute to several human diseases via dysregulated miRNA expression. Trophoblast cells play a key role in implantation and placentation for a successful pregnancy. Nonetheless, the PM10 associated trophoblast cell functions during pregnancy and miRNA expression are still unknown. Our study showed that PM10 affected HTR-8/SVneo cell viability and also decreased cell proliferation, migration, and invasion. A high concentration of PM10 caused an increase in HTR-8/SVneo cell apoptosis. Treatment with PM10 induced inflammation through the upregulated IL-1β, IL-6, and TNF-α expression in trophoblast cells. In PM10-treated HTR-8/SVneo cells, miR-125b-5p expression was considerably increased and TXNRD1 was found to be negatively related to miR-125b-5p. Collectively, our findings revealed that PM10 could alter miR-125b-5p expression by targeting TXNRD1 and suppressing trophoblast cell functions. Additional investigations relating to the function of miR-125b-5p and its target on particulate pollution exposure in trophoblast are warranted for future biomarker or effective therapeutic approaches.

Doxorubicin (DOX) is one of the most commonly used drugs for the treatment of childhood cancers, including leukemia and lymphomas. Despite the high survival rate, female leukemia survivors are at higher risk of ovarian failure and infertility later in life. Treatment with chemotherapeutic drugs like DOX is associated with damage in ovarian follicles, but the affectation grade of granulosa cells remains unclear. To assess and avoid the possible side-effects of DOX, early biomarkers of ovarian injury and chemotherapy-induced ovarian toxicity should be identified. MicroRNAs (miRNAs) have emerged in recent years as a promising new class of biomarkers for drug-induced tissue toxicity. In this study, the effects of DOX on cell viability, steroidogenesis, and miRNA expression were studied in primary granulosa cells (GCs) and in two cellular models (COV434 and KGN cells). We report that compared to other chemotherapeutic drugs, DOX treatment is more detrimental to granulosa cells as observed by decrease of cell viability. Treatment with DOX changes the expression of the aromatase gene (CYP19A1) and the secretion of 17β-estradiol (E2) in a cell-specific manner. miR-132-3p is dose-dependently increased by DOX in all cellular models. In absence of DOX, miR-132-3p overexpression in COV434 cells has no effect on E2 secretion or CYP19A1 expression. Altogether, these findings contribute to understanding the hormonal disbalance caused by DOX in human ovarian cells and suggest miR-132 as a putative sensor to predict DOX-induced ovarian toxicity.

Industrially, after the removal of oil from perilla seeds (PS) by screw-type compression, the large quantities of residual perilla seed meal (PSM) becomes non-valuable waste. Therefore, to increase the health value and price of PS and PSM, we focused on the biological effects of perilla seed oil (PSO) and rosmarinic acid-rich fraction (RA-RF) extracted from PSM for their role in preventing oxidative stress and inflammation caused by TNF-α exposure in an A549 lung adenocarcinoma culture model. The A549 cells were pretreated with PSO or RA-RF and followed by TNF-α treatment. We found that PSO and RA-RF were not toxic to TNF-α-induced A549 cells. Both extracts significantly decreased the generation of reactive oxygen species (ROS) in this cell line. The mRNA expression levels of IL-1β, IL-6, IL-8, TNF-α, and COX-2 were significantly decreased by the treatment of PSO and RA-RF. The Western blot indicated that the expression of MnSOD, FOXO1, and NF-κB and phosphorylation of JNK were also significantly diminished by PSO and RA-RF treatment. The results demonstrated that PSO and RA-RF act as antioxidants to scavenge TNF-α induced ROS levels, resulting in decreased the expression of MnSOD, FOXO1, NF-κB and JNK signaling pathway in a human lung cell culture exposed to TNF-α.

Particulate matter from forest fires (PMFF) is an environmental pollutant causing oxidative stress, inflammation, and cancer cell metastasis due to the presence of polycyclic aromatic hydrocarbons (PAHs). Perilla seed meal contains high levels of polyphenols, including rosmarinic acid (RA). The aim of this study is to determine the anti-oxidative stress, anti-inflammation, and anti-metastasis actions of rosmarinic acid rich fraction (RA-RF) from perilla seed meal and its underlying molecular mechanisms in A549 cells exposed to PMFF. PMFF samples were collected via the air sampler at the University of Phayao, Thailand, and their PAH content were analyzed using GC-MS. Fifteen PAH compounds were detected in PMFF. The PMFF significantly induced intracellular reactive oxygen species (ROS) production, the mRNA expression of pro-inflammatory cytokines, MMP-9 activity, invasion, migration, the overexpression of c-Jun and p-65-NF-κB, and Akt phosphorylation. Additionally, the RA-RF significantly reduced ROS production, IL-6, IL-8, TNF-α, and COX-2. RA-RF could also suppress MMP-9 activity, migration, invasion, and the phosphorylation activity of c-Jun, p-65-NF-κB, and Akt. Our findings revealed that RA-RF has antioxidant, anti-inflammatory, and anti-metastasis properties via c-Jun, p-65-NF-κB, and Akt signaling pathways. RA-RF may be further developed as an inhalation agent for the prevention of lung inflammation and cancer metastasis induced by PM exposure.

Members of the placenta-specific miRNA cluster C19MC, including miR-519d, are secreted by fetal trophoblast cells within extracellular vesicles (EVs). Trophoblast-derived EVs can be internalized by the autologous trophoblast and surrounding maternal immune cells, resulting in coordination of cellular responses. The study of functions and targets of placental miRNAs in the donor and recipient cells may contribute to the understanding of the immune tolerance essential in pregnancy. Here, we report that miR-519d-3p levels correlate positively with cell proliferation and negatively with migration in trophoblastic cell lines. Inhibition of miR-519d-3p in JEG-3 cells increases caspase-3 activation and apoptosis. PDCD4 and PTEN are targeted by miR-519d-3p in a cell type-specific manner. Transfection of trophoblastic cell lines with miR-519d mimic results in secretion of EVs containing elevated levels of this miRNA (EVmiR-519d). Autologous cells enhance their proliferation and decrease their migration ability when treated with EVmiR-519d. NK92 cells incorporate EV-delivered miR-519d-3p at higher levels than Jurkat T cells. EVmiR-519d increases the proliferation of Jurkat T cells but decreases that of NK92 cells. Altogether, miR-519d-3p regulates pivotal trophoblast cell functions, can be transferred horizontally via EVs to maternal immune cells and exerts functions therein. Vesicular miRNA transfer from fetal trophoblasts to maternal immune cells may contribute to the immune tolerance in pregnancy.

Proviral insertion in murine (PIM) lymphoma proteins are mainly regulated by the Janus Kinase/Signal Transducer Activator of Transcription (JAK/STAT) signaling pathway, which can be activated by members of the Interleukin-6 (IL-6) family, including Leukemia Inhibitory Factor (LIF). Aim of the study was to compare PIM1, PIM2 and PIM3 expression and potential cellular functions in human first and third trimester trophoblast cells, the immortalized first trimester extravillous trophoblast cell line HTR8/SVneo and the choriocarcinoma cell line JEG-3. Expression was analyzed by qPCR and immunochemical staining. Functions were evaluated by PIM inhibition followed by analysis of kinetics of cell viability as assessed by MTS assay, proliferation by BrdU assay, and apoptosis by Western blotting for BAD, BCL-XL, (cleaved) PARP, CASP3 and c-MYC. Apoptosis and necrosis were tested by flow cytometry (annexin V/propidium iodide staining). All analyzed PIM kinases are expressed in primary trophoblast cells and both cell lines and are regulated upon stimulation with LIF. Inhibition of PIM kinases significantly reduces viability and proliferation and induces apoptosis. Simultaneously, phosphorylation of c-MYC was reduced. These results demonstrate the involvement of PIM kinases in LIF-induced regulation in different trophoblastic cell lines which may indicate similar functions in primary cells.

Functional foods (FF) are commonly consumed by Asians, and this trend has increased in recent years. Despite the reported health benefits of FF, it is necessary scrutiny and updates of the underpinning research are important. The first international conference on functional food innovation in Asia (IFFA 2018) took place on January 22nd- 24th, 2018, at the University of Phayao, Thailand. Domestic and international speakers, researchers, nutritionists, dieticians, research scholars and students shared their knowledge and experience in FF research. Key features were the potential beneficial roles of FF in health and disease, the current situation with FF in Asia and innovative trends. The IFFA 2018 involved 2 keynote speakers, 34 invited speakers and 10 sessions. About 250 people from across Asia participated. Key themes, discussions, innovative opportunities, and future directions to link research in academia with health-directed applications as FF are summarised.

IntroductionLeukemia Inhibitory Factor (LIF) regulates behavior of trophoblast cells and their interaction with immune and endothelial cells.In vitro, trophoblast cell response to LIF may vary depending on the cell model. Reported differences in the miRNA profile of trophoblastic cells may be responsible for these observations. Therefore, miRNA expression was investigated in four trophoblastic cell lines under LIF stimulation followed byin silicoanalysis of altered miRNAs and their associated pathways.MethodsLow density TaqMan miRNA assays were used to quantify levels of 762 mature miRNAs under LIF stimulation in three choriocarcinoma-derived (JEG-3, ACH-3P and AC1-M59) and a trophoblast immortalized (HTR-8/SVneo) cell lines. Expression of selected miRNAs was confirmed in primary trophoblast cells and cell lines by qPCR. Targets and associated pathways of the differentially expressed miRNAs were inferred from the miRTarBase followed by a KEGG Pathway Enrichment Analysis. HTR-8/SVneo and JEG-3 cells were transfected with miR-21-mimics and expression of miR-21 targets was assessed by qPCR.ResultsA similar number of miRNAs changed in each tested cell line upon LIF stimulation, however, low coincidence of individual miRNA species was observed and occurred more often among choriocarcinoma-derived cells (complete data set athttp://www.ncbi.nlm.nih.gov/geo/under GEO accession number GSE130489). Altered miRNAs were categorized into pathways involved in human diseases, cellular processes and signal transduction. Six cascades were identified as significantly enriched, including JAK/STAT and TGFB-SMAD. Upregulation of miR-21-3p was validated in all cell lines and primary cells and STAT3 was confirmed as its target.DiscussionDissimilar miRNA responses may be involved in differences of LIF effects on trophoblastic cell lines.

Perilla frutescens is commonly used as an n-3-fatty acid source for people living in Northern Thailand. However, cultivated areas are limited because it apparently requires specific growth conditions. Our results suggested that perilla seeds grown in higher altitude in Maehongson province contained more ALA contents than seeds collected from Chiangrai and Nan province. Furthermore, mass spectrometry (MS) proteomic and gene expression analysis revealed that the increase of KASII and FAD3 proteins associated with the mRNA expression and the ALA production. Predictive bioinformatics analysis demonstrated two important transcription factor binding sites, AP2 and B3, which responsible for KASII and FAD3 genes, respectively. The AP2 and B3 transcription factor families were known to be responsible for abiotic stress such as drought and temperature changes. In conclusion, high production of ALA by perilla apparently relies on the altitudes. Moreover, plant responses to abiotic stress environments contribute to the increase of KASII and FAD3 gene and protein expression. However, responsive transcription factors will be further studied for a proof of concept and additionally improving the growing methods in order to increase perilla productivity.

Epidermal growth factor (EGF) is expressed by decidual and trophoblast cells and influences manifold cellular functions during embryo implantation. Thus far, signaling of EGF via Signal Transducer and Activator of Transcription 5 (STAT5) has been only partially investigated. STAT5 stimulates proliferation and cell cycle progression in several cell types. Its dysregulation is associated with pregnancy. The aim of this study was to investigate STAT5 activation and function mediated by EGF in 2 trophoblastic cell lines, namely, HTR8/SVneo and JAR. Additionally, expression of STAT5B messenger RNA (mRNA) in trophoblast models has been compared to that of primary cells isolated from term placentas. Our results demonstrate the highest STAT5B mRNA expression in isolated trophoblast cells, lower expression in HTR8/SVneo cells, and the significantly lowest in JAR cells. Moreover, EGF-mediated STAT5 activation increases cell proliferation and viability in both cell lines. The STAT5 knockdown results in significant decrease in cell viability induced by EGF. Only in HTR8/SVneo cells, invasion decreases after STAT5 silencing and this effect cannot be rescued by further addition of EGF. These results demonstrate that STAT5 activated by EGF constitutes an important cascade for the regulation of cell proliferation and invasion in trophoblast cells.

Trophoblast proliferation and invasion are controlled by cytokines and growth factors present at the implantation site. Members of the Interleukin-6 (IL-6) family of cytokines trigger their effects through activation of intracellular cascades including the Janus Kinase/Signal Transducer and Activator of Transcription (JAK-STAT) pathway. Functions of several STAT molecules in trophoblast cells have been described, but the role of STAT1 remained unclear. Here, potential functions of STAT1 and its activation by Oncostatin M (OSM) have been investigated in an in vitro model. STAT1 expression and phosphorylation were analyzed in human term placenta tissue by immunohistochemistry. HTR-8/SVneo cells (immortalized human extravillous trophoblast cells) were stimulated with OSM, IL-6, IL-11, Leukemia Inhibitory Factor (LIF) and Granulocyte Macrophage Colony-Stimulating Factor. Expression and phosphorylation of STAT1 were analyzed by Western blotting and immunocytochemistry. Fludarabine and STAT1 siRNA were employed for STAT1 depletion. STAT1 transcriptional activity was evaluated by DNA-binding capacity assay. Cell viability and invasion were assessed by MTS and Matrigel assays, respectively. STAT1 was expressed in villous and extravillous trophoblast cells. Low phosphorylation was detectable exclusively in extravillous trophoblast cells. Only OSM and LIF induced phosphorylation of STAT1 in the in vitro model. Challenge with OSM increased cell invasion but not proliferation. Inhibition of STAT1 by fludarabine treatment or STAT1 siRNA transfection reduced cell viability and invasiveness in presence and absence of OSM. These results indicate the potential involvement of STAT1 in the regulation of trophoblast behavior. Furthermore, STAT 1 functions are more efficiently inhibited by blocking its expression than its phosphorylation.

Background MicroRNAs (miRNAs) are small single-stranded RNA molecules working as post-transcriptional modulators of gene expression. Trophoblast cells are a heterogenous group of fetal cells forming the feto–maternal interface and displaying a wide spectrum of functions. The regulation of their behavior may partly underly the control through miRNAs. Therefore, we aimed to compare the miRNA profile of primary first and third trimester trophoblast cells with that of different trophoblastic cell lines. Material and methods Total RNA was obtained from isolated cytotrophoblast cells from healthy term and first trimester placentae and the cell lines HTR-8/SVneo (immortalized trophoblast cells), JEG-3 (choriocarcinoma), ACH-3P and AC1-M59, which are choriocarcinoma cells fused with first and third trimester trophoblast cells, respectively. The expression level of 762 different miRNAs was quantitatively analyzed by using a TaqMan Human MicroRNA Array. For testing the reproducibility of the array technique, the expression of 9 selected miRNAs has been re-analyzed by individual qPCR. Results The analyzed cell types share many similar patterns of miRNAs, but are significantly distinct in the expression of three miRNA clusters: chromosome 19 miRNA cluster (C19MC; containing 54 different miRNAs), C14MC (34 miRNAs) and a minor cluster (miRNA-371 to miRNA-373 cluster), also located on chromosome 19. Expression of miRNAs within C19MC increases significantly from first to third trimester trophoblast while that of C14MC members decreases. MiRNAs within the miR-371-3 cluster augment slightly. C19MC and the miR-371-3 cluster are not expressed by HTR-8/SVneo cells whilst C14MC is almost not detectable in the choriocarcinoma-derived cell lines complete array data available at NCBI Gene Expression Omnibus accession number GSE32346 : http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE32346 ). Beside the miRNAs within the mentioned clusters, further 27 miRNAs are differentially expressed (>100 fold) between term and first trimester trophoblast cells. The placenta-specific miRNAs miR-141 and miR-21 as well as let-7g are expressed in all tested cells with the highest expression in primary trophoblast cells. Conclusion Primary first trimester and term trophoblast cells and trophoblastic cell lines display major differences in their miRNA fingerprints which may be involved in their different behavior and characteristics.

Pregnancy is personally special to every woman expecting a child, but is also interesting from the perspective of an immunologist. During a physiological pregnancy, the mother's immune system decides to tolerate and foster an incorporated, non-self, non-dangerous organism. Whether the maternal reaction stems from deciphering the foreigness or safeness of this new individual, it is the general consensus that there is a foeto-maternal, bidirectional "dialogue" occurring and that the "messages" that are "spoken" are relayed through signaling mediators, which are capable of transmitting a functional command to a target cell. Much information dedicated to this theme has been gleaned in the past decade; however, the complex nature of cytokine networks jeopardizes clarity. In this review, we touch upon a list of mediators that are vital for reproduction. These factors are divided according to their receptor family, because this elucidates the characteristic signal transducing pathway, which is expected to mediate their signal within the target cell. The target cells of interest are the trophoblast, upon which we focus for several reasons: 1. the trophoblast represent the foetal compartment while participating in foeto-maternal interplay (e.g. while invading the decidua, trophoblasts are in constant communication with uterine, maternal immunocytes, which check and contain this function), 2. trophoblasts are responsible for foetal well-being (e.g. nutrition, protection from the environment) and 3. dysfunctional trophoblast function results in several pregnancy complications (e.g. preeclampsia, intrauterine growth retardation, miscarriage, preterm delivery). We summarize what is described in the literature on how these mediators are distributed within the reproductive tract, which cells are expressing their respective receptors (especially which trophoblast subsets) and how they modify trophoblast function

The objective of this study was to investigate the in vitro bioactivities associated with the content of phytochemicals in the extracts from perilla seed meal extract (PSME) compared with dietary seed (PSE). PSE had higher total phenolics and flavonoids content than PSME. However, hydrophilic phytochemical contents in PSME were quantitatively equivalent to PSE. Rosmarinic acid was predominantly found in both extracts. Cell viability and anti‐mutagenicity testing demonstrated that PSE and PSME were biosecured and non‐genotoxic. Both extracts strongly scavenged free radicals and significantly reduced reactive oxygen species (ROS) production. The extracts drastically diminished nitric oxide (NO) production of LPS‐treated RAW 264.7 cells via iNOS mRNA expression. The expression of IL‐6 and COX‐2 were evidently inhibited by these extracts. It could be concluded that PSE and PSME clearly showed in vitro anti‐mutagenicity, antioxidant and anti‐inflammatory capacities. In particular, the by‐product perilla seed meal could be considered as a high nutritive functional food. PRACTICAL APPLICATIONS: This study suggests that the seed meals, a by‐product from seed oil industry, can be utilized as a valuable dietary source for humans and animals. The high content of polyphenols and their bioactivities can be developed as functional foods, and excipients and fillers in pharmaceuticals and nutraceuticals production. Moreover, recycling of the by‐product seed meals should also reduce environmental and sanitary pollution.

Preeclampsia (PE) is one of the leading causes of maternal and perinatal morbidity and mortality worldwide. Abnormal expression of microRNAs (miRNAs) occurs in several pregnancy diseases including PE. Placental trophoblast cells express a specific set of miRNAs which changes during pregnancy. These miRNAs can be released within extracellular vesicles (EVs) and mediate intercellular communication. miR-141 is a pregnancy-related miRNA which is expressed by trophoblast cells at increased levels in maternal plasma in the third trimester. We hypothesize that miR-141 is abnormally expressed in PE placentae, controls trophoblast, and immune cell functions and is involved in the intercellular communication between fetal trophoblast and maternal immune cells. Expression of miR-141 was analyzed by quantitative real-time PCR (qPCR) in normal and preeclamptic placentae and in 2 different trophoblastic cell lines, JEG-3 and HTR-8/SVneo. Changes in JEG-3 and HTR-8/SVneo cell proliferation and invasion were investigated after miR-141 inhibition and overexpression by MTS-, BrdU-, and Matrigel assays. EVs from miR-141 transfected cells were isolated from supernatants and characterized by NanoSight analysis and qPCR. Proliferation of Jurkat T cells and invasion of HTR-8/SVneo cells were investigated after treatment with EVs containing different miR-141 levels. miR-141 expression was higher in placentae from PE patients compared with those from normal pregnancies. miR-141 inhibition in trophoblastic cells resulted in decreased cell viability and reduced invasion capability. After transfection with miR-141-mimic, trophoblastic cells secreted EVs with increased miR-141 content. These vesicles did not exert effects on trophoblastic cell invasion but reduced Jurkat T cell proliferation. In conclusion, miR-141 regulates major functions of trophoblastic and immune cells. Trophoblast cells release EVs whose miRNA content can be modified by transfection of origin cells. Furthermore, elevated levels of miR-141 can be transferred from trophoblast to immune cells by release and internalization of EVs suggesting their role in the immune regulation of normal and pathologic pregnancies.

decreases insulin sensitivity and is associated with obesity. Recent data indicates that subclinical endotoxemia is associated with inflammation in obese women in late pregnancy. Objectives: The objective of this study was to quantify circulating endotoxin across pregnancy in lean and obese women, and assess the relationship between endotoxin and markers of inflammation and insulin sensitivity. Methods: Endotoxin was measured in sterile maternal EDTA plasma samples from 24 lean pregnant women (BMI = 22.4 ± 1.9 kg/m) and 45 obese pregnant women (BMI= 32.6 ± 2.1 kg/m), and 6 non-pregnant women. Samples were collected at 10.5 ± 3.1, 21.3 ± 4.6 and 35.2 ± 2.1 weeks gestation. Endotoxin was quantified using the PyroGene Recombinant Factor C endotoxin detection assay from LONZA, inter-assay variability

Introduction Migration and trophoblast invasion are controlled functionally along with the active participation of cytokines and growth factors. Two important intracellular signaling pathways are the Janus kinase/signal transducer and activator of transcription (JAK-STAT) and extracellular regulated kinase1/2 (ERK1/2). These pathways have been associated with the regulation of gene expression, cellular proliferation, differentiation, angiogenesis, embryo development and invasion in tumor and trophoblast cells. Objectives The aim of our study is to characterize and analyze the regulation and crosstalks of STAT1 and ERK1/2 in trophoblast cells and the identification of activating cytokines. Methods The trophoblast derived cell line HTR-8/svneo and a choriocarcinoma cell line (JEG-3) were stimulated with interleukin-6 (IL-6), IL-11, granulocyte-macrophage colony-stimulating factor (GMC-SF), leukemia inhibitory factor (LIF) or oncostatine M (OSM). The the expression and phosphorylation of STAT1(tyr705) and ERK1/2 were analyzed by gel electrophoresis and Western blotting. Expression of STAT1 was inhibited by administration of 50μM fludarabine (2-fluoro-ara-AMP) for 2, 4, 8, 24, 48 or 72h or by using small interfering RNA (siRNA). The full activation of STAT1 was assessed by using an STAT1 DNA-binding assay. Finally, proliferation and invasion assays were performed (Grant Deutscher Akademischer Austausch Dienst A/10172477). Results LIF and OSM induce STAT1 and ERK1/2 phosphorylation in HTR-8 and JEG-3 cells. Fludarabine inhibits the so induced phosphorylation of STAT1 when administered 48 or 72h before stimulation. Simultaneously, ERK phosphorylation increases. In contrast, silencing of STAT1 by application of specific siRNA induces reduction of ERK1/2 phosphorylation. Fludarabine reduces STAT1 DNA-binding capacity. LIF and OSM increase proliferation. Silencing of STAT1 slightly decreases invasiveness of analyzed cells. Conclusion STAT1 in trophoblast cells can be activated by placental cytokines. Suppression of STAT1 by fludarabine or siRNA influences activity of ERK1/2 which indicates a crosstalk between both pathways. Current studies will clarify the reason for the different effects on ERK1/2 in trophoblastic cells.

Trophoblast cells express a singular miRNA expression profile which varies during pregnancy and whose alteration may be associated with pregnancy complications. miR-21, a widely known oncomir, is highly expressed in human placenta but its role in regulating trophoblast cells remains unclear. The aim of this study was to investigate miR-21 functions and targets in HTR-8/SVneo immortalized trophoblast and JEG-3 choriocarcinoma cells, which are trophoblast cell models that differ in their cellular origin. Cells were transfected with miR-21-antagomir, -mimic or their respective controls. Following, cell proliferation (BrdU), migration (Transwell and scratch wound-healing assays), invasion (Matrigel assays) and apoptosis (flow cytometry, TUNEL assay and Western blotting) were assessed. Expression of the potential miR-21 targets phosphatase and tensin homolog (PTEN) and programmed cell death 4 (PDCD4) were analyzed by Western blotting. Inhibition of miR-21 decreased cell proliferation, migration, and invasion in JEG-3 and HTR-8/SVneo cells and additionally, induced apoptosis in JEG-3 cells. Silencing of miR-21 enhanced PDCD4 expression only in JEG-3 cells, and PTEN expression only in HTR-8/SVneo cells. Inhibition of miR-21 significantly increased phosphorylation of AKT in HTR-8/SVneo cells. In conclusion, miR-21 has cell-specific targets depending upon the origin of trophoblastic cells. Furthermore, miR-21 regulates major cellular processes including cell growth, migration, invasion and apoptosis suggesting that its impairment may lead to placental disorders.

Introduction Preeclampsia is associated with complications especially in severe forms. Recognize this condition can direct to adoption of different behaviors. Objectives Evaluate epidemiological characteristics and compare maternal and neonatal outcomes of mild and severe preeclampsia in pregnancies that results in delivery after 37 weeks of gestation. Methods Prospective study with 40 women was developed in Guilherme Alvaro Hospital-Santos/Brazil, during December/2015 and May/2016. Study group: 20 women with preeclampsia (13 mild and 7 severe), according to NHBPEP – National High Blood Pressure Education Program (2000) criteria and gestational age > 37 weeks. Control group: 20 pregnant women with single and live fetus, > 38 weeks, without comorbidities. Exclusion criteria for study group: twin pregnancy, fetal anomalies and gestational age under 37 weeks. Control group exclusion criteria: twin pregnancy, gestational age under 38 weeks, cesarean/forceps/induced vaginal delivery and comorbidities. Maternal variables: age, gestational age at delivery, previous pregnancy history, body mass index (BMI), comorbidities, length of hospital stay and intensive care unit (ICU) admission, previous preeclampsia and delivery route. Neonatal variables: birth weight, adequacy weight for gestational age, Apgar score, meconium fluid at delivery, intensive care unit admission, acute respiratory distress syndrome, cardiopulmonary arrest, jaundice, oxygen therapy and intrahospital mortality. The statistical comparisons were made using Fisher’s exact test and Tukey multiple comparisons method. The significance level was p 0,05. Results Comparison between the three groups in maternal and neonatal outcomes (* = p 0,05): Download : Download high-res image (199KB) Download : Download full-size image Conclusions The present study showed that the mild preeclampsia group has better results than the severe group in some variables. This first group had no need of mother ICU admission, their BMI is higher and was the group with more previous preeclampsia. The severe preeclampsia group has a significant level of arterial hypertension, stayed longer in the hospital and their newborn had presented more general complications. Data shows more favorable maternal and neonatal outcomes in the control group.

Leukaemia inhibitory factor (LIF) and oncostatin M (OSM) are pleiotropic cytokines present at the implantation site that are important for the normal development of human pregnancy. These cytokines share the cell membrane receptor subunit gp130, resulting in similar functions. The aim of this study was to compare the response to LIF and OSM in several trophoblast models with particular regard to intracellular mechanisms and invasion. Four trophoblast cell lines with different characteristics were used: HTR-8/SVneo, JEG-3, ACH-3P and AC1-M59 cells. Cells were incubated with LIF, OSM (both at 10 ng mL–1) and the signal transducer and activator of transcription (STAT) 3 inhibitor S3I-201 (200 µM). Expression and phosphorylation of STAT3 (tyr705) and extracellular regulated kinase (ERK) 1/2 (thr202/204) and the STAT3 DNA-binding capacity were analysed by Western blotting and DNA-binding assays, respectively. Cell viability and invasiveness were assessed by the methylthiazole tetrazolium salt (MTS) and Matrigel assays. Enzymatic activity of matrix metalloproteinase (MMP)-2 and MMP-9 was investigated by zymography. OSM and LIF triggered phosphorylation of STAT3 and ERK1/2, followed by a significant increase in STAT3 DNA-binding activity in all tested cell lines. Stimulation with LIF but not OSM significantly enhanced invasion of ACH-3P and JEG-3 cells, but not HTR-8/SVneo or AC1-M59 cells. Similarly, STAT3 inhibition significantly decreased the invasiveness of only ACH-3P and JEG-3 cells concomitant with decreases in secreted MMP-2 and MMP-9. OSM shares with LIF the capacity to activate ERK1/2 and STAT3 pathways in all cell lines tested, but their resulting effects are dependent on cell type. This suggests that LIF and OSM may partially substitute for each other in case of deficiencies or therapeutic interventions.

Objectives Analyze epidemiological data and the relation with potential markers of lipotoxicity in pregnancies with and without preeclampsia in a reference hospital. Methods A case-control study, 110 blood samples were collected from (August 2011–2013) in the immediate postpartum and biological potential markers of lipotoxicity analyzed which can be related to preeclampsia. Inclusion for the study group followed the NHBPEP – 2000: systolic blood pressure (⩾140 mmHg) and/or diastolic blood pressure (>−90 mmHg) two times, associated to proteinuria/24 h ⩾2 g/dL or tape reagent ⩾1 cross. Inclusion for the control group: healthy women, vaginal birth, newborn term. Exclusion criteria for all postpartum women were: comorbidities such as collagen diseases, diabetes, smoking, gemelarity and fetal malformations. Data analyzed: maternal age, body mass index (BMI) [weight/(height)[2], high urine protein, HDL (high density lipoprotein), VLDL (very low-density lipoprotein), triglycerides, mode of delivery, maternal clinical outcome, gestational age, birth weight, Apgar newborn score at one and five minutes and admission to the neonatal Intensive Care Unity (ICU). For the numerical variables of the research we used the Student t test, the Pearson’s Qui-square test and Fisher’s exact test and categorical variables. Data analysis was performed by calculating odds ratio adoption hypothesis rejection level of 0.05. Results The total of 110 blood samples (55 from the study group and 55 from the control group). Comparing the results analyzed biological potential markers of lipotoxicity (∗p Variable Control Group Study Group Variable Control Group Study Group Maternal age (average/years) 25 30∗ Neonatal ICU admission 4% 9.1%∗ Cesarean section 42.6% 77.3%∗ VLDL (mg/dl) 29 35∗ Comorbidities 19.6% 77.3%∗ Triglyderides (mg/dl) 150 185∗ Gestation age 40 35∗ HDL (mg/dl) 65 57∗ Weight newborn average 3150g 2500g∗ High urine protein (mg) 185 500∗ BMI 27 33∗ Conclusions The present study shows that the potential markers of lipotoxicity are related to preeclampsia, and eventually may indicate likely prognostics associated with clinical expressions. Additional studies can clarify this correlation and these results might be useful to orientate the adoption of practices which are able to decrease maternal/fetal risks. Disclosures B. Zeiger: None. A. Bergamo: None. D. Vidal: None. F. Aires: None. D. Ferreira: None. M. Scarpelini: None. J. Garcia: None. H. Korkes: None. M. Paltronieri: None. S. de Toledo: None. W. Chaiwangyen: None. F. Sousa: None. N. Sass: None.

MicroRNAs (miRNAs) regulate the expression of a large number of genes in plants and animals. Placental miRNAs appeared late in evolution and can be found only in mammals. Nevertheless, these miRNAs are constantly under evolutionary pressure. As a consequence, miRNA sequences and their mRNA targets may differ between species, and some miRNAs can only be found in humans. Their expression can be tissue- or cell-specific and can vary time-dependently. Human placenta tissue exhibits a specific miRNA expression pattern that dynamically changes during pregnancy and is reflected in the maternal plasma. Some placental miRNAs are involved in or associated with major pregnancy disorders, such as preeclampsia, intrauterine growth restriction or preterm delivery and, therefore, have a strong potential for usage as sensitive and specific biomarkers. In this review we summarize current knowledge on the origin of placental miRNAs, their expression in humans with special regard to trophoblast cells, interspecies differences, and their future as biomarkers. It can be concluded that animal models for human reproduction have a different panel of miRNAs and targets, and can only partly reflect or predict the situation in humans.

Introduction Trophoblast cell invasion is one of key steps for successful pregnancy, which is controlled by several factors including cytokines that activate intracellular signaling cascades including the Janus kinase / signal transducer and activator of transcription (JAK-STAT) system. Objectives We aimed to analyze the effect of cytokines on STAT1 expression and trophoblast cell functions. Methods Immortalized HTR-8/SVneo cells were treated with interleukin-6 (IL-6) IL-11, granulocyte macrophage colony-stimulating factor (GM-CSF), leukemia inhibitory factor (LIF) and oncostatin M (OSM). STAT1 phosphorylation (tyr-701) was analyzed by Western blotting and immunofluorescence staining. Simultaneously, cells were incubated with 50 μ M Fludarabine (specific STAT1 inhibitor) or small interfering RNA (siRNA) to inhibit the expression of STAT1 and followed by cytokine stimulation. STAT1 DNA-binding activity, cell viability and invasion were analyzed by DNA-binding, MTS and Matrigel invasion assays. Results Western blot analysis and immunofluorescence staining of STAT1 phosphorylation revealed that OSM induced phosphorylation of STAT-1 stronger than LIF in HTR-8/SVneo. IL-6, IL-11 and GM-CSF had no effect on STAT1 activation. STAT1 phosphorylation was inhibited by STAT1 siRNA and Fludarabine. STAT1 inhibition reduced STAT1 DNA binding activity and OSM could not rescue this effect. OSM stimulated HTR-8/SVneo cell viability and invasion, whereas STAT1 inhibition decreased trophoblast cell invasion. This reduction could not be prevented by OSM. Conclusions These findings demonstrate that the OSM-STAT1 axis plays an important role in trophoblastic cells and STAT1 activation may induce a variety of cell functions

Introduction The onset of preeclampsia is related to nutritional status. Anthropometric measurements can indicate prognosis and underlie different directions. Objectives To assess the anthropometry of preeclampsia patients with and without comorbidities. Methods A transversal study, developed at the Guilherme Alvaro Hospital, located at Santos/Brazil. The anthropometric data from 72 women in the mediate postpartum was collected from January 2015 to May 2016. Inclusion criteria: mothers with PE according to the criteria of NHBPEP (2000). After recruitment and acceptance to participate in the study, the patients signed an informed consent. Data analyzed: body mass index (BMI-kg/m2), waist circumference (WC, cm) – with reference to the distance from the last rib to the iliac crest, divided by two, and comorbidities associated: diabetes mellitus (DM) and systemic arterial hypertension (SAH). The frequency was calculated by Fisher’s exact test and comparisons between groups, by the Mann-Whitney test (significance value of p Results In the table are expressed the mean values of BMI and WC after patients have been separated into groups: PE (total), PE + DM, PE + SAH, PE + DM + SAH and PE without comorbidities (pure). There was a significant difference in mean BMI when the groups were compared (PE + DM vs PE without comorbidities, p = 0.038; PE + SH vs PE without comorbidities, p = 0.027, and PE + DM + SAH vs PE without comorbidities, p = 0.012). Regarding the waist circumference values, it was only significant difference when the PE group + DM + SAH was compared with PE group without comorbidity (p = 0.007). Download : Download high-res image (116KB) Download : Download full-size image Conclusions It can be concluded that the more comorbidities are associated with PE, more changes are observed in anthropometric parameters. It means that a multidisciplinary team monitoring the anthropometric data of these patients is essential to an early observation of comorbidities development that compromises the health status of patients with PE.

Introduction The use of such markers in predicting preeclampsia, such as PlGF, can serve in predicting the syndrome, and precocious identification can anticipate management and decrease the risks. Objective To evaluate among pregnant women, both those who developed pre-eclampsia as those who did not developed hypertensive disorder complications, the role of placental growth factor (PlGF) as predictor of the syndrome in question, pre-eclampsia. Methods From the MEDLINE database, articles were extracted using the following keywords: (placental growth factor OR PLGF) AND pre-eclampsia/diagnosis by selecting, from a total of 193 retrieved in the search, papers published from 2014 to produce a systematic review. Were excluded narrative and systematic reviews, and in addition, the selected study models included cohort and case-control, which analyzed PlGF alone, sFlt-1/PlGF ratio, PlGF associated with other biomarkers of PE, Doppler Velocimetry uterine artery added to the PlGF and other biomarkers, getting a total of 25 articles after three independent reviewers to analyze the search results and confront their findings. Results Although there is no agreement in all literature about the PlGF levels during pregnancy, the studies evaluated showed a standardization of variations in the marker values during gestation, which are reduced in the preeclampsia group compared to controls, and further reduced in early and severe preeclampsia. On average, pregnancies that resulted in the disease did not show values above 208 pg/ml, unlike the pregnancies in the control group who always obtained values above 300 pg/ml, usually in the second quarter. Recalling that, in the latter group, it kept growing with the evolution of pregnancy. The association of PlGF to Doppler velocimetry of the uterine artery, or other biomarkers or both leads to an increase in the accuracy of the tests. But we can see that the evaluation of sFlt-1 ratio/PlGF is the parameter that brings greater sensitivity and specificity in predicting preeclampsia. Conclusion According to gestational age, PlGF levels have a pattern of variability that differs in healthy women compared to that present the syndrome in question. The latter has lower PlGF values than the control group, and the difference is even greater when you have early or severe PE. Disclosure of interest None declared.

MicroRNAs (miRNAs) are expressed in the placenta and can be detected in maternal plasma. An increasing number of studies have been published on the cellular origin, distribution and function of miRNAs in pregnancy. Specific miRNA profiles have been described for the placenta, maternal plasma and several pregnancy disorders. It has been observed that numerous miRNAs, which are predominantly or exclusively expressed during pregnancy, are clustered in chromosomal regions, may be controlled by the same promoters, may have similar seed regions and targets, and work synergistically. The three most eminent clusters are the chromosome 19 miRNA cluster (C19MC), C14MC and miR-371-3 cluster, which is also localized on chromosome 19. MiRNA members of these clusters are not only detected in the placenta, but also in other compartments, e.g. in serum where they have the potential to become novel biomarkers of pregnancy disorders. Additionally, some members are also expressed in a variety of tumors. Antagonism of selected miRNAs or their targets may lead to novel strategies for the development of new drug classes in pregnancy disorders or other diseases. This review summarizes current knowledge on the pregnancy-related miRNA clusters - the C19MC, C14MC and miR-371-3 cluster - in regard to pregnancy and also other, mostly pathological circumstances.

Curcumin (Cur), a component of turmeric ( Curcuma longa ), has been reported to exhibit antimetastatic activities, but the mechanisms remain unclear. Other curcuminoids present in turmeric, demethoxycurcumin (DMC) and bisdemethoxycurcumin (BDMC) have not been investigated whether they exhibit antimetastatic activity to the same extent as curcumin. The regulation of matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA) play important role in cancer cell invasion by cleavage of extracellular matrix (ECM). In this line, we comparatively examined the influence of Cur, DMC and BDMC on the expressions of uPA, MMP-2, MMP-9, membrane Type 1 MMP (MT1-MMP), tissue inhibitor of metalloproteinases (TIMP-2), and in vitro invasiveness of human fibrosarcoma cells. The results indicate that the differential potency for inhibition of cancer cell invasion was BDMC≥DMC>Cur, whereas the cell migration was not affected. Zymography analysis exhibited that curcumin, DMC and BDMC significantly decreased uPA, active-MMP-2 and MMP-9 but not pro-MMP-2 secretion from the cells in a dose-dependent manner, in which BDMC and DMC show higher potency than curcumin. The suppression of active MMP-2 level correlated with inhibition of MT1-MMP and TIMP-2 protein levels involved in pro-MMP-2 activation. Importantly, BDMC and DMC at 10 μM reduced MT1-MMP and TIMP-2 protein expression, but curcumin slightly reduced only MT1-MMP but not TIMP-2. In addition, three forms of curcuminoids significantly inhibited collagenase, MMP-2, and MMP-9 but not uPA activity. In summary, these data demonstrated that DMC and BDMC show higher antimetastasis potency than curcumin by the differentially down-regulation of ECM degradation enzymes.