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The physical connection of mother and offspring during pregnancy allows the bi-directional exchange of a small number of cells through the placenta. These cells, which can persist long-term in the recipient individual are genetically foreign to it and therefore fulfill the principle of microchimerism. Over the last years, pioneer research on microchimeric cells revealed their role in immune adaptation during pregnancy and priming of tolerogenic responses in the progeny. However, the mechanisms involved in cell transfer across the placenta barrier remain poorly investigated. In this review, we summarize the evidence of fetomaternal microchimerism, propose a mechanism for cell trafficking through the placenta and discuss the different models and techniques available for its analysis. Likewise, we aim to generate interest in the use of ex vivo placenta perfusion to investigate microchimerism in physiological and pathological settings.

Introduction Research on human placental development and function lacks a conclusive in vivo model. To investigate the intracellular molecular mechanisms in trophoblast cells, different cell lines have been established during the last decades. So far, none of these accomplishes all features of primary trophoblast, thus their suitability as well as the transferability of the results has been discussed. The aim of this study is to assess molecular markers and features matching different trophoblast subpopulations in trophoblastic cell lines to provide orientation on their suitability and relevance for distinct research questions. Methods The commonly used trophoblastic cell lines, BeWo, JEG-3, HTR-8/SVneo, AC1-M59, AC1-M32, ACH-3P and Swan71 were selected. qPCR and immunoblotting were used to determine expression of characteristic molecular markers. C14MC, C19MC and miR-371-3 miRNA expression were investigated by real time PCR. Proliferation, migration and network stabilization assays were performed. Hormone secretion was determined by chemiluminescent-immunoassays. DNA profiles were obtained by Short Tandem Repeat (STR)-genotyping. Results Immortalized cell lines differ from choriocarcinoma-derived ones in the expression of HLA-G, E-cadherin, N-cadherin, VE-cadherin, cadherin-11, cytokeratin 7, vimentin, ADAM12 and PRG2. Compared to choriocarcinoma-derived cell lines, expression of C19MC and hormone secretion were almost absent in immortalized cell lines. Conversely, they express C14MC and exhibit higher migration and network stabilization. Discussion The data presented will help justify the use of a cell line to evaluate distinct features of trophoblast biology and pathology. In general, characteristics and markers of choriocarcinoma derived cell lines seem to be more similar to in vivo trophoblast than immortalized cell lines and thus might be regarded as more suitable models.

Purpose Several roles are attributed to the myometrium including sperm and embryo transport, menstrual discharge, control of uterine blood flow, and labor. Although being a target of diabetes complications, the influence of high glucose on this compartment has been poorly investigated. Both miRNAs and IGF1R are associated with diabetic complications in different tissues. Herein, we examined the effects of high glucose on the expression of miRNAs and IGF1R signaling pathway in the human myometrium. Methods Human myometrial explants were cultivated for 48 h under either high or low glucose conditions. Thereafter, the conditioned medium was collected for biochemical analyses and the myometrial samples were processed for histological examination as well as miRNA and mRNA expression profiling by qPCR. Results Myometrial structure and morphology were well preserved after 48 h of cultivation in both high and low glucose conditions. Levels of lactate, creatinine, LDH and estrogen in the supernatant were similar between groups. An explorative screening by qPCR arrays revealed that 6 out of 754 investigated miRNAs were differentially expressed in the high glucose group. Data validation by single qPCR assays confirmed diminished expression of miR-215-5p and miR-296-5p, and also revealed reduced miR-497-3p levels. Accordingly, mRNA levels of IGF1R and its downstream mediators FOXO3 and PDCD4, which are potentially targeted by miR-497-3p, were elevated under high glucose conditions. In contrast, mRNA expression of IGF1, PTEN, and GLUT1 was unchanged. Conclusions The human myometrium responds to short-term exposure (48 h) to high glucose concentrations by regulating the expression of miRNAs, IGF1R and its downstream targets.

A complex network composed of at least 1900 microRNA (miRNA) species orchestrates the development and function of the human placenta. These molecules regulate genes and pathways operating major functional processes in trophoblast cells such as proliferation, invasion, differentiation, and metabolism. Nevertheless, the cellular localization and role of most placental miRNAs remain to be determined. The existence of eutherian- (C14MC) and primate-specific miRNA clusters (C19MC), together with human placenta-specific miRNAs, indicate the relevance of these molecules in evolution and diversification of the placenta, including the acquisition of its unique features in humans. They may be related also to diseases that are exclusively present in primates, such as preeclampsia. Changes in the miRNA expression profile have been reported in several placental pathologies. Which miRNAs are involved in the pathomechanism of these diseases or act to maintain placental homeostasis is uncertain. Placenta-derived miRNAs are packed into extracellular vesicles (EVs) and distributed through the maternal circulation to distant organs, where they contribute to adaptations required during pregnancy. Similarly, the placenta also receives molecular information from other tissues to adapt fetoplacental metabolic demands to the maternal energetic supply. These processes can be impaired in pathologic conditions. Therefore, the collection of circulating placental miRNAs constitutes potentially a minimally-invasive approach to assess the fetoplacental status and to diagnose pregnancy diseases. Future therapies may include manipulation of miRNA levels for prevention and treatment of placental complications to protect maternal health and fetal development.

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.

Extracellular vesicles (EVs), including small EVs (sEVs), are involved in neuroinflammation and neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. Yet, increased neuroinflammation can also be detected in the aging brain, and it is associated with increased glial activation. Changes in EV concentration are reported in aging tissues and senescence cells, suggesting a role of EVs in the process of aging. Here, we investigated the effect of peripheral sEVs from aged animals on neuroinflammation, specifically on glial activation. sEVs were isolated from the peripheral blood of young (3 months) and aged (24 months) C57BL/6J wildtype mice and injected into the peripheral blood from young animals via vein tail injections. The localization of EVs and the expression of selected genes involved in glial cell activation, including Gfap, Tgf-β, Cd68, and Iba1, were assessed in brain tissue 30 min, 4 h, and 24 h after injection. We found that sEVs from peripheral blood of aged mice but not from young mice altered gene expression in the brains of young animals. In particular, the expression of the specific astrocyte marker, Gfap, was significantly increased, indicating a strong response of this glial cell type. Our study shows that sEVs from aged mice can pass the blood-brain barrier (BBB) and induce glial cell activation.

Extracellular vesicles (EVs), including small EVs (sEVs), are involved in neuroinflammation and neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Yet, increased neuroinflammation can also be detected in the aging brain, and it is associated with increased glial activation. Changes in EV concentration are reported in aging tissues and senescence cells, suggesting a role of EVs in the process of aging. Here, we investigated the effect of peripheral sEVs from aged animals on neuroinflammation, specifically on glial activation. sEVs were isolated from the peripheral blood of young (3 months) and aged (24 months) C57BL/6J wildtype mice and injected into the peripheral blood from young animals via vein tail injections. The localization of EVs and the expression of selected genes involved in glial cell activation, including

The concept of biological identity has been traditionally a central issue in immunology. The assumption that entities foreign to a specific organism should be rejected by its immune system, while self-entities do not trigger an immune response is challenged by the expanded immunotolerance observed in pregnancy. To explain this "immunological paradox", as it was first called by Sir Peter Medawar, several mechanisms have been described in the last decades. Among them, the intentional transfer and retention of small amounts of cells between a mother and her child have gained back attention. These microchimeric cells contribute to expanding allotolerance in both organisms and enhancing genetic fitness, but they could also provoke aberrant alloimmune activation. Understanding the mechanisms used by microchimeric cells to exert their function in pregnancy has proven to be challenging as per definition they are extremely rare. Profiting from studies in the field of transplantation and cancer research, a synergistic effect of microchimerism and cellular communication based on the secretion of extracellular vesicles (EVs) has begun to be unveiled. EVs are already known to play a pivotal role in feto-maternal tolerance by transferring cargo from fetal to maternal immune cells to reshape their function. A further aspect of EVs is their function in antigen presentation either directly or on the surface of recipient cells. Here, we review the current understanding of microchimerism in the feto-maternal tolerance during human pregnancy and the potential role of EVs in mediating the allorecognition and tropism of microchimeric cells.

Tobacco smoking is an important public health issue recognized by the world health organization as one of the most serious, preventable risk factors for developing a series of pregnancy pathologies. Maternal smoking is positively associated with intrauterine growth restriction (IUGR) and gestational diabetes (GDM), but negatively associated with preeclampsia (PE). In this review, we examine epidemiological, clinical and laboratory studies of smoking effects on immunoregulation during pregnancy, trophoblast function, and placental vasculature development and metabolism. We aim to identify effects of tobacco smoke components on specific placental compartments or cells, which may contribute to the understanding of the influences of maternal smoking on placenta function in normal and pathological pregnancies. Data corroborates that in any trimester, smoking is unsafe for pregnancy and that its detrimental effects outweigh questionable benefits. The effects of maternal smoking on the maternal immune regulation throughout pregnancy and the impact of different tobacco products on fetal growth have not yet been fully understood. Smoking cessation rather than treatment with replacement therapies is recommended for future mothers because also single components of tobacco and its smoke may have detrimental effects on placental function.

Epithelial membrane proteins (EMP1-3) are involved in epithelial differentiation and carcinogenesis. Dysregulated expression of EMP2 was observed in various cancers, but its role in human lung cancer is not yet clarified. In this study, we analyzed the expression of EMP1-3 and investigated the biological function of EMP2 in non-small cell lung cancer (NSCLC). The results showed that lower expression of EMP1 was significantly correlated with tumor size in primary lung tumors (p = 0.004). Overexpression of EMP2 suppressed tumor cell growth, migration, and invasion, resulting in a G1 cell cycle arrest, with knockdown of EMP2 leading to enhanced cell migration, related to MAPK pathway alterations and disruption of cell cycle regulatory genes. Exosomes isolated from transfected cells were taken up by tumor cells, carrying EMP2-downregulated microRNAs (miRNAs) which participated in regulation of the tumor microenvironment. Our data suggest that decreased EMP1 expression is significantly related to increased tumor size in NSCLC. EMP2 suppresses NSCLC cell growth mainly by inhibiting the MAPK pathway. EMP2 might further affect the tumor microenvironment by regulating tumor microenvironment-associated miRNAs.

Extracellular vesicle (EV)-mediated communication has been implicated in the cooperative alliance between trophoblast and immune cells toward maternal tolerance and placentation. Syncytiotrophoblast cells secrete EVs directly into the maternal circulation, which are taken up by immune cells, endothelial cells, and other cell types. Initial evidence also shows that EVs produced by immune cells are, in turn, incorporated by trophoblast cells and modulate placental responses. Non-coding RNAs (ncRNAs), proteins, and lipid mediators transported in EVs are able to influence proliferation, differentiation, cytokine production, and immunological responses of recipient cells. The molecular alphabet and cellular targets involved in this dialogue are being revealed. Nevertheless, several questions regarding the whole content, surface markers, and biological functions of EVs still remain to be investigated in both physiological and pathological conditions. Analysis of circulating EVs in maternal blood has the potential to serve as a minimally invasive approach to monitoring placental functions and immunological features of pregnancy, aiding in the diagnostics of complications. This review addresses the immunomodulatory properties of EVs and their tasks in the communication between placental and immune cells.

Problem Extracellular vesicles (EVs) released by the placenta are packed with biological information and play a major role in fetomaternal communication. Here, we describe a comprehensive set-up for the enrichment and characterization of EVs from human placenta perfusion and their application in further assays. Method of study Human term placentas were used for 3 h ex vivo one-sided perfusions to simulate the intervillous circulation. Thereafter, populations of small (sEVs) and large EV (lEVs) were enriched from placental perfusate via serial ultracentrifugation. Following, EV populations were characterized regarding their size, protein concentration, RNA levels, expression of surface markers as well as their uptake and miRNA transfer to recipient cells. Results The sEV and lEV fractions from an entire perfusate yielded, respectively, 294±32 µg and 525±96 µg of protein equivalents and 2.6±0.5 µg and 3.6±0.9 µg of RNA. The sEV fraction had a mean diameter of 117±47 nm, and the lEV fraction presented 236±54 nm. CD63 was strongly detected by dot blot in sEVs, whereas only traces of this marker were found in lEVs. Both EV fractions were positive for the trophoblast marker PLAP (placental alkaline phosphatase) and annexin A1. EV internalization in immune cells was visualized by confocal microscopy, and the transfer of placental miRNAs was detected by quantitative real-time PCR (qPCR). Conclusions Enriched EV populations showed characteristic features of sEVs and lEVs. EV uptake and transfer of miRNAs to recipient cells demonstrated their functional integrity. Therefore, we advocate the ex vivo one-sided placenta perfusion as a robust approach for the collection of placental EVs.

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.

Dienogest (DNG) administration is a well-established treatment for endometriosis but bleeding irregularities remain its main disadvantage. Changes in diet, mainly to vegetable consumption, are beneficial in the treatment of estrogen-related pathologies but their use for endometriosis has been poorly studied. In this study, addition of the phytochemical 3,3′-diindolylmethane (DIM) to DNG therapy has been investigated in in vitro and ex vivo models for endometriosis and in a small cohort of women with endometriosis. Endometrial Ishikawa cells were treated with DNG or DIM at dosages from 10−10 M to 10−5 M for up to 72 h. Cell proliferation was measured by assessing BrdU incorporation. Endometrial tissue from women with endometriosis and controls was incubated with DNG or a combination of DNG and DIM. Tissue viability was determined using a modified colorimetric MTS assay. 17β-estradiol secretion was quantified by an electro-chemiluminescence immunoassay. Finally, DNG as monotherapy or in combination with DIM was randomly administered to women with endometriosis (n = 8) over 3 months. Bleeding patterns and associated pelvic pain were assessed by Visual Analogue Scale (VAS). DNG and DIM significantly reduced cell proliferation in Ishikawa cells. Ex vivo, DIM reduced viability and estradiol secretion specifically in endometriotic but not in normal endometrial tissue. This effect was enhanced by combination with DNG. Endometriosis associated pelvic pain was significantly reduced in patients taking the DNG-DIM combination therapy compared to those taking DNG alone. Bleeding pattern (number and duration of episodes) was significantly improved by addition of DIM to the DNG treatment. In conclusion, addition of DIM enhances effects of DNG ex vivo and may ameliorate bleeding patterns in endometriosis patients.

Throughout history, pandemics of infectious diseases caused by emerging viruses have spread worldwide. Evidence from previous outbreaks demonstrated that pregnant women are at high risk of contracting the diseases and suffering from adverse outcomes. However, while some viruses can cause major health complications for the mother and her fetus, others do not appear to affect pregnancy. Viral surface proteins bind to specific receptors on the cellular membrane of host cells and begin therewith the infection process. During pregnancy, the molecular features of these proteins may determine specific target cells in the placenta, which may explain the different outcomes. In this review, we display information on Variola, Influenza, Zika and Corona viruses focused on their surface proteins, effects on pregnancy, and possible target placental cells. This will contribute to understanding viral entry during pregnancy, as well as to develop strategies to decrease the incidence of obstetrical problems in current and future infections.

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.

BackgroundExosomes are involved in intracellular communication and contain proteins, mRNAs, miRNAs, and signaling molecules. Exosomes were shown to act as neuroinflammatory mediators involved in neurodegenerative diseases including Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS). Brain aging has been associated to increased neuroinflammation. In addition, a decreased extracellular vesicle concentration was observed in aging tissues. The specific mechanisms how exosomes and aging are connected are not known yet.ResultsHere we have shown that peripheral injection had almost no effect on selected gene expression in the liver. However, exosome injection has led to changes in the specific markers of glial cell activation (CD68, Iba1, GFAP). Interestingly, only injection of exosomes isolated from aged mice induced significant activation of astrocyte cells, as shown by increased GFAP expression.ConclusionTranscription levels of genes GFAP, TGF-β, CD68, Iba1 known to be involved in glial cell function are significantly changing after introduction of peripheral extracellular vesicles. Exosomes were able to pass blood brain barrier and induce glial cell activation. GFAP known to be a specific astrocyte activation marker was significantly higher expressed after injection of old but not young exosomes, indicating a possible role of exosomes in the mechanisms of brain aging.

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.

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.

The development of the follicle and competent oocyte is highly coordinated, requiring interplay among several systems. These implicate endocrine, immune, and metabolic signals, intrafollicular paracrine factors from theca, mural, and cumulus granulosa cells, and the oocyte itself. Granulosa cells play a key role in their interaction. COV434 is one of the few human granulosa cell lines that can be used as an in vitro model for ovarian research. We aimed to evaluate the possible activation of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway by IL-6-type cytokines leukemia inhibitory factor (LIF) and oncostatin M (OSM) in COV434 cells. Expression of GP130 (glycoprotein 130), STAT3 (signal transducer and activators of transcription 3), PIAS3 (protein inhibitor of activated STAT 3), and SOCS3 (suppressor of cytokine signaling 3) genes after stimulation with LIF or OSM was assessed using RT-qPCR (real-time PCR). GP130 transcripts were significantly upregulated after incubation with LIF or OSM for 24h. Expression of the STAT3 gene was stimulated only after incubation with LIF, but not OSM. SOCS3 showed significant upregulation for all time periods and the levels of PIAS3 were initially down- and after 24h upregulated. Furthermore, the major signaling components of the JAK/STAT pathway, GP130 and STAT3, and the kinase activation patterns of STAT3, were examined at protein level. We found constitutive protein expression for GP130, STAT3, pSTAT3(ser727) and upregulation of pSTAT3(tyr705) by LIF and OSM. Our results demonstrate the activation of the JAK/STAT pathway by LIF and OSM in human granulosa cells.

International audience; Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2016 there were twelve themed workshops, four of which are summarized in this report. These workshops addressed challenges, strengths and limitations of techniques and model systems for studying the placenta, as well as future directions for the following areas of placental research: 1) placental imaging; 2) sexual dimorphism; 3) placenta and development of other organs; 4) trophoblast cell lines.

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.

Author(s): Albrecht, Christiane; Baker, Julie C; Blundell, Cassidy; Chavez, Shawn L; Carbone, Lucia; Chamley, Larry; Hannibal, Roberta L; Illsley, Nick; Kurre, Peter; Laurent, Louise C; McKenzie, Charles; Morales-Prieto, Diana; Pantham, Priyadarshini; Paquette, Alison; Powell, Katie; Price, Nathan; Rao, Balaji M; Sadovsky, Yoel; Salomon, Carlos; Tuteja, Geetu; Wilson, Samantha; O'Tierney-Ginn, PF | Abstract: Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2016 there were twelve themed workshops, four of which are summarized in this report. These workshops covered innovative technologies applied to new and traditional areas of placental research: 1) genomic communication; 2) bioinformatics; 3) trophoblast biology and pathology; 4) placental transport systems.

Background During pregnancy, syncytiotrophoblast vesicles contribute to maternal tolerance towards the fetus, but also to pathologies such as pre-eclampsia. The aim of the study was to address whether Plasmodium falciparum and HIV infections in pregnancy affect the secretion, microRNA content and function of trophoblast microparticles. Methods Microparticles were isolated and characterized from 122 peripheral plasmas of Mozambican pregnant women, malaria- and/or HIV-infected and non-infected. Expression of placenta-related microRNAs in microparticles was analysed by qPCR and the effect of circulating microparticles on dendritic cells assessed by phenotype analysis and cytokine/chemokine measurement. Results Concentrations of total and trophoblast microparticles detected by flow cytometry were higher in HIV-positive (P = 0.005 and P = 0.030, respectively) compared to non-infected mothers, as well as in women delivering low birthweight newborns (P = 0.032 and P = 0.021, respectively). miR-517c was overexpressed in mothers with placental malaria (P = 0.034), compared to non-infected. Microparticles from HIV-positive induced a higher expression of MHCII (P = 0.021) and lower production of MCP1 (P = 0.008) than microparticles from non-infected women. Conclusions In summary, alterations in total and trophoblast microparticles associated with malaria and HIV in pregnant women may have an immunopathogenic role. The potential for placental-derived vesicles and microRNAs as biomarkers of adverse outcomes during pregnancy and malaria infection should be confirmed in future studies.

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.

Cytokines are involved in almost all processes during the menstrual cycle, the fertilization period and pregnancy. They are expressed in numerous reproduction-related body fluids and tissues. Disorders of cytokine expression patterns may cause pregnancy pathologies. Therefore, cytokines have the potential as new biomarkers in different body compartments for a variety of such pathologies. Furthermore, cytokines may also serve to treat fertility and pregnancy disorders. The IL-6-like family of cytokines is an intensively investigated group of cytokines with well-accepted functions in fertility and pregnancy. This article summarizes current knowledge on IL-6-like cytokines in regard of their role in reproduction and their potential for new strategies in the treatment of reproductive pathologies.

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

Galectin-1 (gal-1), a member of the mammalian β-galactoside-binding proteins, exerts biological effects by recognition of glycan ligands, including those involved in cell adhesion and growth regulation. In previous studies, we demonstrated that gal-1 induces cell differentiation processes on the membrane of choriocarcinoma cells BeWo, including the receptor tyrosine kinases (RTKs) REarranged during Transfection (RET), Janus Kinase 2 (JAK2) and Vascular endothelial growth factor receptor 3 (VEGFR3). Furthermore, Mitogen-Activated Protein Kinases (MAPK) and serine/threonine kinases were phosphorylated by gal-1. In addition, gal-1 in trophoblast cells in vitro induced syncytium formation especially after concentration dependent stimulation of the cells with this galectin. This is in contrast to MAPK-inhibitor U0126 that reduced syncytium formation of BeWo cells. The aim of this study was to analyze the syncytium formation abilities of BeWo cells that were gal-1 silenced. We found a significantly reduced syncytium formation rate in gal-1 silenced BeWo cells. In addition, these cells show a different miRNA expression profile. In summary, we found that gal-1 is a major trigger for fusion processes in BeWo cells. This function is accompanied by different regulation of miRNA synthesis in the BeWo cell culture model.