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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.

Species differences are among the main reasons for the high failure rate of preclinical studies. A better awareness and understanding of these differences might help to improve the outcome of preclinical research. In reproduction, the placenta is the central organ regulating fetal exposure to a substance circulating in the maternal organism. Exact information about placental transfer can help to better estimate the toxic potential of a substance. From an evolutionary point of view, the chorioallantoic placenta is the organ with the highest anatomical diversity among species. Moreover, frequently used animal models in reproduction belong to rodents and lagomorphs, two groups that are characterized by the generation of an additional type of placenta, which is crucial for fetal development, but absent from humans: the inverted yolk sac placenta. Taken together, the translatability of placental transfer studies from laboratory animals to humans is challenging, which is supported by the fact that numerous species-dependent toxic effects are described in literature. Thus, reliable human-relevant data are frequently lacking and the toxic potential of chemicals and pharmaceuticals for humans can hardly be estimated, often resulting in recommendations that medical treatments or exposure to chemicals should be avoided for safety reasons. Although species differences of placental anatomy have been described frequently and the need for human-relevant research models has been emphasized, analyses of substances with species-dependent placental transfer have been performed only sporadically. Here, we present examples for species-specific placental transfer, including that of nanoparticles and pharmaceuticals, and discuss potential underlying mechanisms. With respect to the COVID 19-pandemic it might be of interest that some antiviral drugs are reported to feature species-specific placental transfer. Further, differences in placental structure and antibody transfer may affect placental transfer of ZIKA virus.

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.

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.

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.

The cynomolgus monkey is increasingly considered in toxicological research as the most appropriate model for humans due to the species' close physiological contiguity, including reproductive physiology. Here, literature on the cynomolgus monkey placenta is reviewed in regards to its similarity to the human placenta and particularly for its immunological role, which is not entirely mirrored in humans. Pertinent original data are included in this article. The cynomolgus monkey placenta is evaluated based on three aspects: first, morphological development; second, the spatial and temporal appearance of maternal and fetal immune cells and certain immune cell products of the innate and adaptive immune systems; and third, the expression of relevant immune tolerance-related molecules including the homologs of anti-human leucocyte antigen, indoleamine 2,3-dioxygenase, FAS/FAS-L, annexin II, and progesterone. Parameters relevant to the immunological role of the placenta are evaluated from the immunologically immature stage of gestational day (GD) 50 until more mature stages close to birth. Selected comparisons are drawn with human and other laboratory animal placentas. In conclusion, the cynomolgus monkey placenta has a high degree of morphological and physiological similarity to the human placenta. However, there are differences in the topographical distribution of cell types and immune tolerance-related molecules. Three basic features are recognized: (1) the immunological capacity of the placenta changes throughout the lifetime of the organ; (2) these immunological changes include multiple parameters such as morphological adaptations, cell type involvement, and changes in immune-relevant molecule expression; and (3) the immune systems of two genetically disparate individuals (mother and child) are functionally intertwined at the maternal-fetal interface.

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.

PurposeInfertility is a debilitating situation that millions of women around the world suffer from, but the causal relationship between infertility and endometriosis is still unclear. We hypothesize that the immune cell populations of uterine natural killer cells (uNK) and plasma cells (PC) which define chronic endometritis could differ in patients with or without endometriosis and therefore be the link to endometriosis-associated infertility.MethodsOur retrospective study includes 173 patients that underwent an endometrial scratching in the secretory phase of the menstrual cycle and subsequently immunohistochemical examination for uNK cells and PC. Sixty-seven patients were diagnosed with endometriosis, 106 served as the control cohort.ResultsThe risk for an elevated number of uNK cells in women with endometriosis is not increased as compared to the control group. Our findings suggest that patients with endometriosis are 1.3 times more likely to have chronic endometritis (CE) as compared to those without and that the treatment with doxycycline might increase pregnancy rates. Endometriosis and an increased number of uNK cells seem to be unrelated.ConclusionsIn contrast to the lately published connection between endometriosis, infertility and increased uNK cells, we could not find any evidence that patients with endometriosis are more prone to elevated uterine uNK cells. Counting of PC in endometrial biopsies might be a new approach in the search of biomarkers for the nonsurgical diagnosis of endometriosis since our findings suggest a connection.

The use of placenta preparations as an individual puerperal remedy can be traced back to historical, traditional practices in Western and Asian medicine. To evaluate the ingestion of processed placenta as a puerperal remedy, the potential risks (trace elements, microorganisms) and possible benefit (hormones in the placental tissue) of such a practice are discussed in this article based on a literature review.Die Einnahme der eigenen Plazenta durch die Mutter als individuelles Heilmittel im Wochenbett ist zurückzuführen auf historische, traditionelle Anwendungen in abendländischer und asiatischer komplementärer Medizin. In dieser Übersichtsarbeit wurden mittels Literaturrecherche sowohl die möglichen Risiken (Spurenelemente, Mikroorganismen) als auch möglicher Nutzen (Hormone im Plazentagewebe) erfasst, um die Einnahme der verarbeiteten Plazenta als Heilmittel im Wochenbett zu bewerten.

Breast cancer is one of the most common malignancies diagnosed in pregnancy. Although the tumor is often detected at an advanced stage, placental metastases are rare. Here, we describe the case of a woman with breast cancer recurrence during pregnancy and subsequent metastases. The focus of this study is the large amount of placenta metastases, which have been analyzed immunohistochemically. Staining with trophoblast markers (placenta alkaline phosphatase, beta human chorionic gonadotropin and human placental lactogen) showed the strict localization of metastases in the intervillous space without invasion into fetal tissue. They have a large spheroidal shape and are free of blood vessels. Staining with Ki-67 revealed an outer proliferative shell and inner necrotic core. At week 28, a healthy newborn was born by elective cesarean section. A few weeks later, after surgery and FEC60 (fluorouracil, epirubicin, cyclophosphamide) cycles, the patient died. Breast cancer metastases in the placenta are rarely described. The special immunological environment in pregnancy may influence phenotype, growth, and behavior of tumor and metastases.

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.

Introduction: Trophoblast homing to maternal spiral arteries is mandatory for successful placentation. Cell-cell adhesion molecules regulate this process and adhesion molecule expression is altered in impaired placentation. We hypothesize that, similar to immune cell recruitment, trophoblast cell adherence and rolling are primarily mediated by adhesion molecules like, cadherins, immunoglobulins, selectins and their partnering ligands. Here, the interdependence of adhesion molecule expression in trophoblastic cell lines of diverse origin was investigated in relation to their interaction with endothelial cell networks on Matrigel® co-cultures and the effect of specific adhesion molecule knockdown analyzed. Methods: Trophoblastic cells were labeled in red and co-cultured with green HUVEC networks on Matrigel®. Association was quantified after collection of fluorescence microscopy pictures using Wimasis® internet platform and software. Expression of adhesion molecules was analyzed by PCR and Western blot, immuno-fluorescence and flow cytometry. The impact of adhesion molecules on trophoblast-endothelial-cell interaction was investigated using siRNA technique. Results: N-cadherin and CD162 were specifically expressed in the trophoblast cell line HTR-8/SVneo, which closely adhere to and actively migrate toward HUVEC networks on Matrigel®. Suppression of N-cadherin led to a significant alteration in trophoblast-endothelial cell interaction. Expression of VE-cadherin in closely interacting trophoblast cells was not confirmed in vitro. Discussion: We identified N-cadherin to mediate specific interaction between HUVEC and the migrating trophoblast cells HTR-8/SVneo in a Matrigel® co-culture model. VE-cadherin contribution could not be confirmed in vitro. Our results support the hypothesis that impaired N-cadherin but not VE-cadherin expression is involved in trophoblast recruitment to the maternal endothelium.

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.

Body fluids contain extracellular vesicles expressing tissue factor on their surface and serve as an additional trigger for coagulation. During the menstrual cycle ovarian tissue restoration is mandatory and it is unknown whether follicular fluid might provide procoagulant substances. Within an observational study, follicular fluid from women undergoing IVF/intracytoplasmic sperm injection (ICSI) was analysed by fluorescence-activated cell sorting (FACS), electron microscopy, resistive pulse sensing (RPS), nanoparticle-tracking analysis (NTA) and fibrin generation tests (FGT). The presence of extracellular vesicles, especially CD9-positive extracellular vesicles in follicular fluid, was proven. However, clotting tests revealed no procoagulant properties of the detected extracellular vesicles.

Background: Breast cancer is one of the most common malignancies during pregnancy. Guidelines for breast cancer treatment during pregnancy demonstrating that breast cancer during pregnancy can be treated similarly to non-pregnancy-associated breast cancer, except for hormonal and anti-HER2 therapies. Nonetheless, a decreased birth weight is often observed in newborns. Therefore, this project aims to analyze the effects of chemotherapy and the impact of cancer progression on the placenta. Methods: Placentas from breast cancer patients (n=63) and non-cancer participants (n=20) enrolled in Breast Cancer in Pregnancy (BCP) study were collected after delivery and embedded in paraffin. Sections were stained with Hematoxylin-Eosin (HE) and IHC (Ki-67, cPARP, p27Kip1); trophoblast morphology was evaluated by an established scoring system and distribution of immunohistochemical markers was assessed. Additionally, epigenetic analyses (cytosine methylation) for the following genes were performed: LINE-1 (general methylation), IGF2-H19 (birth weight), EPO (hypoxia), BDNF (neurobiological development), glucocorticoid receptor, HSD11B2 (inactivation of glucocorticoids), estrogen receptor, P-glycoprotein, CYP-3A4 (drug metabolism). Results: HE staining revealed significant damage of trophoblast nuclei and membranes in placentas from breast cancer patients compared to controls (mean score of damage 1.9/1.8 vs 0.8/0.7, p Citation Format: Karolin Fröhlich, Torsten Plösch, Fenja Seither, Volkmar Müller, Thomas Karn, Elmar Stickeler, Christian Schem, Christine Solbach, Amelie Lupp, Rikst N Verkaik-Schakel, Gitta Turowski, Peter Mallmann, Marion van Mackelenbergh, Bruno Sinn, Valentina Nekljudova, Carsten Denkert, Pamela Schittler, Udo Markert, Sibylle Loibl. Histological and epigenetic analyses of placenta tissue from breast cancer patients and healthy participants [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-04-08.

Objective To investigate whether chronic endometritis (CE) affects the immune status of peripheral blood and endometrium in patients with recurrent reproductive failure (RRF). Design Retrospective study. Setting Private fertility center. Patients(s) A total of 524 RRF patients, including 324 women with recurrent miscarriage (RM) and 200 women with recurrent implantation failure (RIF). Intervention(s) Peripheral blood and endometrium samples were collected in the midluteal phase before in vitro fertilization treatment or pregnancy. The number of peripheral T, natural killer (NK), and B cells, as well as cytotoxicity of NK cells and expression of TH1 cytokines were analyzed with the use of flow cytometry, and uterine immune cells were subjected to immunohistochemistry. Main Outcome Measure(s) Peripheral immune cells, cytokines, NK cytotoxicity, and endometrial immune cells were compared in RRF patients with versus without CE. Result(s) The proportion and function of the analyzed immune cell subsets in peripheral blood as well as the percentages of CD56+ NK cells, CD163+ M2 macrophages, and CD1a+ immature dendritic cells in the endometrium were not significantly altered between non-CE and CE patients, whereas the proportions of uterine CD68+ macrophages, CD83+ mature dendritic cells, CD8+ T cells, and Foxp3+ regulatory T cells were significantly elevated in CE patients. After antibiotic treatment, the percentage of CD68+ macrophages, CD83+ mature dendritic cells, CD8+ T cells, and Foxp3+ regulatory T cells in endometrium were significantly reduced in patients with cured CE. Conclusion(s) CE contributes to elevated endometrial infiltration levels of immune cells. The excessive presence of endometrial immune cells in CE patients may be involved in reduced endometrial receptivity and recurrent pregnancy failures.

Background Breast cancer is one of the most frequently diagnosed malignancies during pregnancy. Tumours often present characteristics of high malignancy and are hormone receptor negative/HER2 positive or triple negative. In general, pregnancy, including the postpartum period, is associated with a transiently increased risk of developing breast cancer but followed by a long-lasting protective period. Placental metastases are very rare and, thus far, breast cancer metastases in the foetal compartment have not been described. To discuss these apparently contradictory observations, this narrative review resumes immunological and hormonal alterations during pregnancy potentially affecting breast cancer risk as well as tumour growth and behaviour. Observations Upregulation of breast cancer–associated genes involved in immunological and reproductive processes has been observed in parous women and is potentially responsible for a transiently increased risk in pregnancy. In contrast, maternal immunisation and immunoglobulin production against antigens expressed on trophoblast cells, such as specific glycosylation patterns of mucin-1 or RCAS1-associated truncated glycans, seem to prevent breast cancer development in later years. Animal and human studies indicate that T cells are involved in these processes. Several placenta-derived factors, especially kisspeptin, have direct anti-tumour effects. The pregnancy-related increase of estrogen, progesterone, and other hormones influence growth and characteristics of breast cancer while the role of further placenta-secreted factors is still controversially discussed. Conclusion Several factors and cells are involved in altered breast cancer risk during and after pregnancy and have potential for developing novel treatment strategies in future.

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.

Introduction Human maternal placentophagy, the behavior of ingesting the own raw or processed placenta postpartum, is a growing trend by women of western societies. This study aims to identify the impact of dehydration and steaming on hormone and trace element concentration as well as microbial contamination of placental tissue. Methods A total of nine placentas have been processed: six were studied for hormone and trace element concentrations; eight were studied for microbial contamination. The concentrations of CRH, hPL, oxytocin and ACTH in samples of raw, steamed dehydrated and raw dehydrated placental tissue were detected using ELISA. A yeast bioassay was performed in order to detect estrogen equivalent (EEQ) and gestagen equivalent (PEQ) active substances. Elements (As, Cd, Fe, Pb, Se, Hg) were analyzed using ICP-MS. Isolated colonies from tissue and placenta swab samples were identified using Vitek MS. Results Following mean hormone concentrations were detected in raw placental tissue: CRH (177.88 ng/g), hPL (17.99 mg/g), oxytocin (85.10 pg/g), ACTH (2.07 ng/g), estrogen equivalent active substances (46.95 ng/g) and gestagen equivalent active substances (2.12 μg/g). All hormones were sensitive to processing with a significant concentration reduction through steaming and dehydration. Microorganisms mainly from the vaginal flora were detected on placenta swab samples and samples from raw, steamed, dehydrated and steamed dehydrated tissue and mostly disappeared after dehydration. According to regulations of the European Union the concentrations of potentially toxic elements (As, Cd, Hg, Pb) were below the toxicity threshold for foodstuffs. Conclusion The commonly used protocols for preparation of placenta for its individual oral ingestion reduce hormone concentrations and bacterial contamination.

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.

The immunological milieu in the placenta may be crucial for priming the developing foetal immune system. Early imbalances may promote the establishment of immune-mediated diseases in later life, including allergies. The initial exposure to allergens seems to occur in utero, but little is known about allergen-induced placental cytokine and chemokine release. The release of several cytokines and chemokines from placenta tissue after exposure to mast cell degranulator compound 48/80 or apple allergen in placentas from allergic and healthy mothers was to be analysed. Four placentas from women with apple allergy and three controls were applied in a placental perfusion model with two separate cotyledons simultaneously perfused with and without apple allergen (Mal d 1). Two control placentas were perfused with compound 48/80. In outflow, histamine was quantified spectrophotofluorometrically, IL-2, IL-4, IL-6, IL-10, TNF and IFN-gamma by a cytometric multiplex bead array and IL-13 and CXCL10, CXCL11, CCL17 and CCL22 with an in-house multiplex Luminex assay. Compound 48/80 induced a rapid release of histamine, CXCL10, CXCL11, CCL17 and CCL22, but not of the other factors. Apple allergen induced a time-dependent release of IL-6 and TNF, but not of histamine, in placentas of women with apple allergy compared with the unstimulated cotyledon. CCL17 levels were slightly increased after allergen stimulation in control placentas. Allergens can induce placental cytokines and chemokines distinctly in allergic and healthy mothers. These mediators may affect the prenatal development of the immune system and modify the risk of diseases related to immune disorders in childhood such as allergies. Funding Agencies|Institut Danone (Haar, Germany); European Network of Excellence within the 6th Framework Programme of the European Union [512040]; Swedish Research Council [K2011-56X-21854-01-06]; Cancer and Allergy Association; Olle Engkvist Foundation

The immune system represents a key defense mechanism against potential pathogens and adverse non-self materials. During pregnancy, the placenta is the point of contact between the maternal organism and non-self proteins of the fetal allograft and hence undoubtedly fulfils immune functions. In the placenta bacteria, foreign (non-self) proteins and proteins that might be introduced in toxicological studies or by medication are barred from reaching the progeny, and the maternal immune system is primed for acceptance of non-maternal fetal protein. Both immunologic protection of the fetus and acceptance of the fetus by the mother require effective mechanisms to prevent an immunologic fetomaternal conflict and to keep both organisms in balance. This is why the placenta requires toxicological consideration in view of its immune organ function. The following articles deal with placenta immune-, control-, and tolerance mechanisms in view of both fetal and maternal aspects. Furthermore, models for experimental access to placental immune function are addressed and the pathological evaluation is elucidated. “The Placenta as an Immune Organ and Its Relevance in Toxicological Studies” was subject of a continuing education course at the 2012 Society of Toxicologic Pathology meeting held in Boston, MA.

This review summarizes the potential and also some limitations of using human placentas, or placental cells and structures for toxicology testing. The placenta contains a wide spectrum of cell types and tissues, such as trophoblast cells, immune cells, fibroblasts, stem cells, endothelial cells, vessels, glands, membranes, and many others. It may be expected that in many cases the relevance of results obtained from human placenta will be higher than those from animal models due to species specificity of metabolism and placental structure. For practical and economical reasons, we propose to apply a battery of sequential experiments for analysis of potential toxicants. This should start with using cell lines, followed by testing placenta tissue explants and isolated placenta cells, and finally by application of single and dual side ex vivo placenta perfusion. With each of these steps, the relative workload increases while the number of feasible repeats decreases. Simultaneously, the predictive power enhances by increasing similarity with in vivo human conditions. Toxic effects may be detected by performing proliferation, vitality and cell death assays, analysis of protein and hormone expression, immunohistochemistry or testing functionality of signaling pathways, gene expression, transport mechanisms, and so on. When toxic effects appear at any step, the subsequent assays may be cancelled. Such a system may be useful to reduce costs and increase specificity in testing questionable toxicants. Nonetheless, it requires further standardization and end point definitions for better comparability of results from different toxicants and to estimate the respective in vivo translatability and predictive value.

Summary Background The upper (UAW) and lower (LAW) airways of patients with cystic fibrosis (CF) have the same ion-channel defects, but little is known about similarities and differences in host immunological responses at the two levels. Aim Identification and comparison of both levels' pathogen colonization and resulting immunological host responses. Methods The UAW and LAW of 40 CF patients were non-invasively assessed by nasal lavage and induced sputum. Pathogen colonization, cytology, and the concentrations of inflammatory mediators (TNF-α, MPO, matrix metalloprotease (MMP)-9, tissue inhibitor of metalloprotease (TIMP)-1, regulated upon activation, normal T-cell expressed and presumably secreted (RANTES), and interleukin (IL)-1β, -5, -6, -8, and -10) were measured. Results Inflammatory responses were more pronounced in the LAW than the UAW. Pseudomonas aeruginosa LAW colonization is accompanied by a significantly enhanced neutrophil (PMN)-dominated response (P = 0.041) and IL-8 concentration (P = 0.01) not observed in P. aeruginosa UAW colonization. In contrast, sinonasal P. aeruginosa colonization resulted in elevated RANTES (P = 0.039) and reduced MMP-9 (P = 0.023) and TIMP-1 (P = 0.035) concentrations. Interestingly, LAW P. aeruginosa colonization was associated with reduced sinonasal concentrations of MMP-9 (P = 0.01) and TIMP-1 (P = 0.02), a finding independent of UAW colonization for MMP-9. Conclusion CF UAW and LAW show distinct inflammatory profiles and differentiated responses upon P. aeruginosa colonization. Assessment of UAW colonization and MMP-9 are predictive of chronic pulmonary colonization with P. aeruginosa. Thus, this linkage between CF UAW and LAW can provide new clinical and scientific implications. Pediatr Pulmonol. 2014; 49:1065–1075. © 2014 Wiley Periodicals, Inc.

Problem Women with antiphospholipid antibodies (aPLs) present a risk of pregnancy morbidity (PM), vascular thrombosis (VT), or both (PM/VT). aPLs affect trophoblast function, and the aim of this study was to determine the modulation of this aPL-induced damage by different drugs. Method of study IgG was obtained from women with PM and PM/VT positive to aPLs. Binding of IgG to trophoblastic cells, proliferation, mitochondrial membrane integrity, and trophoblast invasion were assessed. The effect of enoxaparin, aspirin, and aspirin-triggered lipoxin (ATL) were evaluated as well as signal transducer and activator of transcription 3 (STAT3) phosphorylation. Results IgG from women with aPLs strongly binds to trophoblastic cells. Integrity of mitochondrial membrane was reduced, and proliferation was increased by IgG-PM/VT. Both IgG-PM and IgG-PM/VT decreased trophoblast invasion, which was restored by enoxaparin, aspirin, and ATL. IgG-PM triggered reduction in STAT3 phosphorylation. Conclusion Some drugs used to prevent aPL-induced PM modulated the alteration of trophoblast function.

Preeclampsia (PE) and intrauterine growth retardation (IUGR) are rare but severe pregnancy complications that are associated with placental insufficiency often resulting in premature birth. The clinical pathologies are related to gross placental pathologies and trophoblastic deficiencies that might derive from inflammatory processes and oxidative stress injury. The mesenchymal core of placental villi has been identified as a possible niche for trophoblast progenitor cells that are called upon to replenish the injured syncytiotrophoblast layer. These progenitor cells are known to express trophoblast stem cell (CDX2) and pluripotency (SOX2, NANOG and OCT4A) markers, however only little data is available characterizing the expression of these transcription factors beyond the blastocyst stage. We aimed to describe the expression of these factors in healthy 1st and 3rd trimester placentae as well as PE, IUGR and combined PE+IUGR placentae. We analyzed 8 respective samples derived from 1st trimester (elective abortions), and 3rd trimester (healthy controls, PE, IUGR and combined PE+IUGR). We accomplished immunoperoxidase staining to detect the stem cell markers: CDX2 (trophectoderm), SOX2, NANOG and OCT4A (embryonal). Immunoreative scoring was used for objective analyses of staining patterns. All markers display clearly elevated signals in 1st trimester villous samples as compared to healthy 3rd trimester counterparts. Especially CDX2 and NANOG were specific to the cytotrophoblast layer and the mesenchymal core. Specific and differential expression patterns were visible in the villous/extravillous compartment of each placenta-associated pregnancy complication (PE: pan elevated expression; IUGR elevated SOX2 in basal plate; combined PE+IUGR pan loss of expression). Reduction of stem cell transcription factor expression in term placentae indicates temporal regulation, and probably a specific function which is yet to be elucidated. The differential expression patterns within placentae complicated with placenta-associated pregnancy complications indicate that PE, IUGR and combined PE+IUGR are separate entities. It is unclear whether the alterations are the cause or the effect of the clinical pathology.

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.

Citation Braunschweig A, Poehlmann TG, Busch S, Schleussner E, Markert UR. Signal transducer and activator of transcription 3 (STAT3) and suppressor of cytokine signaling 3 (SOCS3) balance controls cytotoxicity and IL-10 expression in decidual-like natural killer cell line NK-92. Am J Reprod Immunol 2011; 66: 329–335 Objectives In previous studies, we have shown that sHLA-G reduces cytotoxicity of decidual NK cells, which was dependent upon reduction in signal transducer and activator of transcription 3 (STAT3) and perforin. In this study, we aimed to confirm the role of STAT3 for induction of cytotoxicity and to analyze the regulative role of its antagonist suppressor of cytokine signaling 3 (SOCS3). Furthermore, the influence of both factors on cytokine expression should be analyzed. Methods All experiments were performed on NK-92 cells. STAT3 and SOCS3 have been silenced using two different small interfering RNA sequences each. Silencing efficiency and STAT3 tyrosine phosphorylation have been analyzed by Western blotting. Cytotoxicity to K562 target cells has been assessed by flow cytometry. Expression of IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, TNF-α, and TNF-β has been measured using cytometric bead arrays for flow cytometry. Results STAT3 and SOCS3 have been successfully silenced. STAT3 silencing reduced cytotoxicity. SOCS3 silencing induced increase in STAT3 tyrosine phosphorylation and cytotoxicity. STAT3 silencing reduced IL-10 expression significantly, while SOCS3 silencing induced, also significantly, the opposite effect. The other cytokines were expressed at very low concentration or not constantly affected. Conclusion STAT3 and SOCS3 are involved in regulation of NK cell cytotoxicity and IL-10 expression.

Citation Junovich G, Mayer Y, Azpiroz A, Daher S, Iglesias A, Zylverstein C, Gentile T, Pasqualini S, Markert UR, Gutierrez G. Ovarian stimulation affects the levels of regulatory endometrial NK cells and angiogenic cytokine VEGF. Am J Reprod Immunol 2011; 65: 146–153 Problem Endometrial NK cells play a critical role in uterine vascularization producing angiogenic factors. Impact of ovarian stimulation on endometrial expression of NK cells and VEGF in normal fertile oocyte donors and the effect of endometrial injury treatment on these parameters have been investigated. Method of study Endometrial tissue was obtained from oocyte donors during natural and stimulated cycles. NK cell subsets were measured by flow cytometry. VEGF was determined by ELISA and flow cytometry. Endometrial angiogenic parameters were determined by ultrasound Doppler. Local injury was induced by scratching endometrial tissue previous to implantation window. Results Ovarian stimulation decreased endometrial levels of NK cells and vascularization index but increased VEGF levels. Local injury normalized only the CD56+ NK cell count. Conclusion Hormonal therapy for ovarian stimulation may be associated with poor endometrial vascularization. Local injury before the implantation window seems not to influence endometrial angiogenic parameters altered by ovarian stimulation.

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

This review summarizes several aspects especially of regulating factors governing trophoblast invasion. Those include the composition of the extracellular matrix containing a variety of matrix metalloproeinases and their inhibitors, but also intracellular signals. Furthermore, a newly described trophoblast subtype, the endoglandular trophoblast, is presented. Its presence may provide a possible mechanism for opening and connecting uterine glands into the intervillous space. Amongst others, two intracellular signalling pathways are crucial for regulation of trophoblast functions and development: Wnt- and signal transducer and activator of transcription (STAT)3 signalling. Wnt signalling promotes implantation, placentation and trophoblast differentiation. Several Wnt-dependent cascades and regulatory mechanisms display different functions in trophoblast cells. The STAT3 signalling system is fundamental for induction and regulation of invasiveness in physiological trophoblastic cells, but also in tumours. The role of galectins (Gal) in trophoblast regulation and placenta development comes increasingly into focus. The Gal- 1-4, 7-10 and 12-14 have been detected in humans. Detailed information is only available for Gal-1, -2, -3, -4, -9 and -12 in endometrium and decidua. Gal-1, -3 and -13 (-14) have been detected and studied in trophoblast cells.

Problem Homing of lymphocytes and NK cells into the decidua and its regulation has been very controversially discussed. Therefore, we aimed to establish an in vivo simulation method for analysis of homing behavior, which might be also useful for other cells such as stem or tumor cells. Method of study A human term placenta has been perfused with medium to elute blood and then with maternal autologous carboxyfluorescein succinimidyl ester (CFSE)-labeled peripheral blood lymphocytes for 3 hr and rinsed for another 2 hr. Tissue was analysed histologically for detection of labeled cells. Labeled lymphocytes and beads in perfusate have been identified and counted by flow cytometry. Results At the moment of tissue fixation for histology, the perfusate was free of labeled cells. Labeled perfused lymphocytes have been found adhered and integrated in vessel wall structures, in decidual stroma and as colonies in individual villi. Conclusion Placenta perfusion with a lymphocyte suspension is feasible without plugging the tube system. Time is sufficient for cells to adhere and to migrate into the stroma. Also some villi have been infiltrated which might be caused by inflammatory stimuli. The perfusion system might be useful to test substances for their capacity to influence homing of lymphocytes or other cells.

It has recently been reported that the increased prevalence in childhood allergy may be linked to deviations in fetal immune development. One reason may be impaired nutrient supply. Hence, a well-differentiated placenta together with an optimal fetal nutrition via the mother are important prerequisites for the establishment of a functional immune system with normal immune responses. Fatty acids and their derivatives can influence both the early immune development and immune maturation by regulating numerous metabolic processes and the gene expression of important proteins such as enzymes and cytokines. The present review summarises the impact of nutritional fatty acids on the development of the immune system as well as the fetal development. It describes the mechanisms of action of PUFA,transfatty acids and conjugated linoleic acids in programming the fetus with regard to its risk of acquiring atopic diseases in childhood.

Trophoblast cells display a very unique capability: they physiologically invade into the surrounding tissue. This capability is widely associated with tumours, and, indeed, the invasive behaviour of both is rather similar. The imposing difference is that trophoblast cell invasion is temporally and locally controlled in contrast to unlimited tumour invasion. It initiates immediately after embryo implantation into the endometrium. Parallel to tumours, trophoblasts secrete proteases, such as matrix metalloproteinases, which dissolve the extracellular matrix and the surrounding tissue. Thereby, these proteases prepare and allow true invasion of trophoblasts. The invasive capacities of trophoblasts are positively and negatively regulated by numerous cytokines including leukaemia inhibitory factor (LIF), interleukin-6, hepatocyte growth factor, granulocyte macrophage-colony stimulating factor and others. They interact via specific receptors with the trophoblast cells, in which they activate intracellular signalling cascades. These will then induce expression of invasion relevant genes. One of these signalling pathways is the Janus kinase/signal transducers and activators of transcription (STAT) pathway. Especially phosphorylated STAT3 enhances invasiveness of tumours and trophoblast cells, where it is mainly activated by LIF. One of its most efficient physiological antagonists is suppressor of cytokine signalling 3. The balance of these two intracellular molecules seems to be a key regulator of tumour and trophoblast invasion.

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.

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.

Objectives Trophoblast progenitor cells express stem cells markers (SCM) to maintain the proliferative characteristic of stem cells. Beyond blastocyst stage or in preeclampsia (PE) or intrauterine growth restriction (IUGR) little is known about expression of SCMs. We examined the expression of trophoblast and other SCMs in 1st and 3rd trimester placenta and in preeclampsia (PE) and intrauterine growth restriction (IUGR) in order to discriminate if these markers might be involved in progenitor cell functions. Methods 8 samples each of 1st trimester placentae (elective abortions), 3rd trimester IUGR, PE and control (normal term pregnancy placentae) were stained by immunoperoxidase to detect the SCMs: CDX2 (trophectoderm SCM), SOX2, NANOG and OCT4A (embryonic SCMs) and NOTCH1 (endothelial SCM). Results In 1st trimester all SCM were detected, expressed homogenous in syncytio- and cytotrophoblast, and grow increasingly mosaic-like towards the end of 1st trimester. These signals are lost or diminished in 3rd trimester, whereby the syncytiotrophoblast loses these signals first. NOTCH1, however, remains highly expressed in all trophoblast subtypes of both IUGR and PE pregnancies. Conclusion Both embryonic and trophoblast SCMs are expressed in 1st trimester trophoblast and appear most vivid among the villous trophoblast of very early pregnancy. Loss of stem cell transcription factor expression in term placentae indicates temporal regulation, and a so far unknown specific function.

Introduction Endothelial dysfunction is thought to be the cause of preeclampsia (PE) symptoms. Severe forms of PE are correlated to the release of syncytiotrophoblastic microparticles (STBM), which triggers inflammatory processes on the endothelium. The thrombogenic potential of STBMs is not well characterized. We investigated the pro-coagulant activity of STBMs. Methods STBMs were derived from placentae perfused under norm- and hypoxic conditions and quantified with our house-“ELISA”. Surface phospholipid-dependent thrombin formation of microparticles was determined with a commercial ELISA. Rate and absolute platelet aggregation in platelet-rich plasma (PRP), while fibrin production in platelet free plasma, was measured in absence and presence of STBMs using a PAP-4 aggregometer. Results STBM concentration and STBM-induced thrombin formation is stable under normoxic and elevated under hypoxic conditions. STBMs derived from hypoxic placentae increase the rate of fibrinogenesis. Maximum platelet aggregation is stable under normoxic, while highly variable under hypoxic conditions. STBMs of hypoxic placentae cannot alter the rate of platelet aggregation, while normoxic STBMs adjust the rate according to need. Conclusion Hypoxia negatively affects fibrinogenesis and the platelet aggregation-modulating effects of STBMs, which might be due to a phenotype alteration. All observed results may contribute to the impaired microcirculation seen in PE.