BackgroundPregnant women represent a vulnerable group in pharmaceutical research due to limited knowledge about drug metabolism and safety of commonly used corticosteroids like prednisone due to ethical and practical constraints. Current preclinical models, including animal studies, fail to accurately replicate human pregnancy conditions, resulting in gaps in drug safety and pharmacokinetics predictions. To address this issue, we used a three-organ microphysiological system (MPS) combined with a digital twin framework, to predict pharmacokinetics and fetal drug exposure.MethodsThe here shown human MPS integrated gut, liver, and placenta models, interconnected via the corresponding vasculature. Using prednisone as a model compound, we simulate oral drug administration and track its metabolism and transplacental transfer. To translate the generated data from MPS to human physiology, computational modelling techniques were developed.ResultsOur results demonstrate that the system maintains cellular integrity and accurately mimics in vivo drug dynamics, with predictions closely matching clinical data from pregnant women. Digital twinning closely aligned with the generated experimental data. Long-term exposure simulations confirmed the value of this integrated system for predicting the non-toxic metabolization of prednisone.ConclusionThis approach may provide a potential non-animal alternative that could contribute to our understanding of drug behavior during pregnancy and may support early-stage drug safety assessment for vulnerable populations.

Released from trophoblast and other fetal cells, placental extracellular vesicles (EVs) reach the maternal peripheral blood and modulate immune responses. Increased EVs in plasma of preeclampsia (PE) patients indicate their involvement in the etiology of this condition. This study addresses the uptake of plasma EVs by peripheral blood mononuclear cells (PBMCs) and explores the underlying internalization mechanisms. Plasma EVs were isolated from women with normotensive pregnancy (EVNP) and those with PE (EVPE), and characterized by cryo-transmission electron microscopy, nanoparticle tracking analysis, Western blotting, flow cytometry, and micro bicinchoninic acid assay (micro-BCA). To investigate whether the origin of PBMCs affects uptake, samples from males, pregnant women, and non-pregnant women were included. Primary PBMCs and macrophages derived from the human leukemia monocytic cell line THP-1 were incubated with PKH-stained EVs, and uptake was assessed by flow cytometry and confocal microscopy. Key molecules involved in monocyte differentiation and macrophage function were evaluated in EV-treated cells using LEGENDplexTM assay and real-time polymerase chain reaction (RT-PCR). Independent of the PBMC source, EVs were mostly captured by monocytes and in a lower proportion by T lymphocytes. Capture of EVPE was higher than of EVNP in primary T lymphocytes, monocytes, and THP-1-derived macrophages. After inhibition by Wortmannin and Cytochalasin D, EV internalization by THP-1-derived macrophages was significantly inhibited but not completely abolished. No defined polarization profile of treated THP-1-derived macrophages could be identified. These findings provide evidence of EV modifications in PE, which enhance their uptake by monocytes and other immune cells, mainly through phagocytosis and endocytosis.

Extracellular vesicles (EVs) are cell-derived particles that constitute a mechanism of intercellular communication transporting cell-type-specific cargos involved in multiple physiological and pathological processes. The concentration of EVs increases in plasma of normal pregnancy (NP) throughout gestation exhibiting particular molecular profiles and is higher in patients with preeclampsia (PE). PE is characterized by new-onset hypertension and associated with cerebrovascular and neurological impairment. EVs are related with neuroinflammation through activation of microglia and with neurodegenerative conditions. Despite few studies have suggested that EVs are involved in the relationship between PE and central nervous system injury, the underlying mechanisms are unknown. Here we investigated effects of plasma-derived EVs from PE on microfluidically supported and static models of the human blood-brain barrier (BBB) and microglia. EVs were isolated from NP and PE plasma through differential ultracentrifugation, and characterized by nanotracking analysis, cryogenic electron microscopy and Western blotting. Human brain endothelial cells (hCMEC/D3) were cultured with EndoGRO supplemented with exosome-depleted FBS in microfluidically supported biochips -that mimic cerebral blood flow and induce endothelial polarization, and stimulated with NP- and PE-derived EVs for 24 h. A co-culture of hCMEC/D3 cells and human microglial cells (HMC3) in a two-compartment static model of the BBB was also used. PKH67-labeled EVs, endothelial adhesion proteins and microglial activation markers were analyzed by confocal microscopy. Cytokines released in the cell culture supernatant were measured by flow cytometry. Enriched fractions of small (<150 nm) and large (>150 nm) EVs were obtained exhibiting polydisperse spherical morphology. Compared to NP, elevated concentrations of EVs were observed in PE. sEV harbored CD63, ALIX and TSG101, suggesting exosome-enrichment, and HLA-G and PLAP, demonstrating the presence of their placental origin. In assessing the effect of EVs on the integrity of the BBB under flow conditions, PE-EVs were taken up by hCMEC/D3 cells and triggered decrease of endothelial claudin-5 and occludin compared to NP-EVs. Furthermore, PE-EVs crossed the endothelial monolayer in the static co-culture set-up and were taken up by HMC3 microglial cells, inducing increase of the microglial activation markers CD11b, IBA1, and release of TNF-α, IL-1β and IFN-γ. Our results show that placenta derived EVs reach the maternal periphery. PE-EVs disrupt and cross the BBB, are taken up by endothelial and microglial cells and induce microglial activation in vitro. This study suggests that placenta-derived EVs play a role in the mechanism of inter-organ communication in the context of pregnancy disorders, and could be the link between PE and the cerebrovascular and neurological impairment observed in those patients.

Research question: Do microRNAs (miRNAs) play a role in regulating endoplasmic reticulum stress (ERS) and unfolded protein response (UPR) in decidualized cells and endometrium associated with reproductive failures?Design: Endometrial stromal cell line St-T1b was decidualized in vitro with 8-Br-cAMP over 5 days, or treated with the ERS inducer thapsigargin. Expression of ERS sensors, UPR markers and potential miRNA regulators was analysed by quantitative PCR. Endometrial biopsies from patients with recurrent pregnancy loss (RPL) and recurrent implantation failure (RIF) were investigated for the location of miRNA expression.Results: Decidualization of St-T1b cells resulted in increased expression of ERS sensors including ATF6a, PERK and IRE1a, and the UPR marker, CHOP. TXNIP, which serves as a link between the ERS pathway and inflammation, as well as inflammasome NLRP3 and interleukin 1b expression increased in decidualized cells. An in-silico analysis identified miR-17-5p, miR-21-5p and miR-193b-3p as miRNAs potentially involved in regulation of the ERS/UPR pathways and inflammation associated with embryo implantation. Their expression decreased significantly (P <= 0.0391) in non-decidualized cells in the presence of thapsigargin. Finally, expression of the selected miRNAs was localized by in-situ hybridization in stromal and glandular epithelial cells in endometrial samples from patients with RPL and RIF. Expression in stroma cells from patients with RPL was lower in comparison with stroma cells from patients with RIF.Conclusions: Decidualization in St-T1b cells is accompanied by ERS/UPR processes, associated with an inflammatory response that is potentially influenced by miR-17-5p, miR-21-5p and miR-193b-3p. These miRNAs are expressed differentially in stromal cells from patients with RPL and RIF, indicating an alteration in regulation of the ERS/UPR pathways.

Placenta accreta spectrum (PAS) is one of the major causes of maternal morbidity and mortality worldwide with increasing incidence. PAS refers to a group of pathological conditions ranging from the abnormal attachment of the placenta to the uterus wall to its perforation and, in extreme cases, invasion into surrounding organs. Among them, placenta accreta is characterized by a direct adhesion of the villi to the myometrium without invasion and remains the most common diagnosis of PAS. Here, we identify the potential regulatory miRNA and target networks contributing to placenta accreta development. Using small RNA-Seq followed by RT-PCR confirmation, altered miRNA expression, including that of members of placenta-specific miRNA clusters (e.g., C19MC and C14MC), was identified in placenta accreta samples compared to normal placental tissues. In situ hybridization (ISH) revealed expression of altered miRNAs mostly in trophoblast but also in endothelial cells and this profile was similar among all evaluated degrees of PAS. Kyoto encyclopedia of genes and genomes (KEGG) analyses showed enriched pathways dysregulated in PAS associated with cell cycle regulation, inflammation, and invasion. mRNAs of genes associated with cell cycle and inflammation were downregulated in PAS. At the protein level, NF-κB was upregulated while PTEN was downregulated in placenta accreta tissue. The identified miRNAs and their targets are associated with signaling pathways relevant to controlling trophoblast function. Therefore, this study provides miRNA:mRNA associations that could be useful for understanding PAS onset and progression.

During pregnancy, molecules and cells in the maternal circulation can be transferred to the fetal tissues causing changes in fetal tolerance affecting pregnancy outcome. Although studies have been performed to explain the responsible mechanisms for trafficking across the maternal-fetal interface, these involved mostly animal or static models that differ significantly from the in vivo situation. Here, we established an in vitro placenta-on-chip model (PoC). The trophoblastic cell line BeWo was used. BeWo cells were cultured under static and perfusion conditions in a biochip until monolayer formation. The integrity of the PoC was evaluated by ZO-1 and β-catenin staining and FITC-Dextran permeability. The expression of adhesion molecules PSGL-1, ICAM-1, VCAM-1, and JAM-B was analyzed by immunofluorescence/confocal microscopy. Syncytialization was evaluated by BeWo cell-cell fusion ratio. PoC ultrastructure was visualized by scanning electron microscopy. The PoC model based on BeWo cell monolayer exhibits a homogeneous expression of ZO-1 and β-catenin and a constitutive expression of the adhesion molecules PSGL-1, LFA-1, VLA-4, and JAM-B. Compared with the static condition, PoC under flow conditions showed a continuous cell layer with higher signs of spontaneous syncytialization and syncytin expression, resulting in a low permeability rate. SEM showed clean and homogeneous microvilli formation in the BeWo monolayer under perfusion. A microfluidically supported placenta-on-chip model resembling the constitutive expression of adhesion molecules and microvilli formation has been successfully established. This system can potentially study drugs and cell trafficking across the placenta barrier.

Fetomaternal communication plays a decisive role during pregnancy. Soluble factors, such as hormones and neuropeptides but also extracellular vesicles (EV) are transferred from the placenta to the maternal circulation. EVs are membrane-linked vesicles that are released by cells and play an important role in the intercellular communication. They can be classified into various classes including exosomes and microvesicles. EVs contain a multitude of molecules, such as proteins, lipids, nucleic acids and microRNAs (miRNAs), which can be taken up by target cells through a variety of mechanisms. They contribute to the communication between the placenta and maternal organs and cells, including the immune system. Placental miRNAs play an important role in the regulation of cell functions in the placenta but can also be transported into immune cells via EVs. Their uptake can influence the immune response and contribute to the development of fetomaternal immune tolerance. In pregnancy disorders an diseases the amount and composition of placental EVs in maternal blood can be altered. Therefore, they have the potential to be used as diagnostic markers for complications during pregnancy in the future.

Die fetomaternale Kommunikation spielt eine entscheidende Rolle während der Schwangerschaft. Dazu werden lösliche Faktoren wie Hormone und Neuropeptide, aber auch extrazelluläre Vesikel (EV) von der Plazenta in den mütterlichen Kreislauf abgegeben. EV sind membrangebundene Vesikel, die von Zellen freigesetzt werden und eine wichtige Rolle in der interzellulären Kommunikation spielen. Sie können in verschiedene Klassen eingeteilt werden, darunter Exosomen und Mikrovesikel. EV enthalten eine Vielzahl von Molekülen wie Proteine, Lipide, Nukleinsäuren und microRNAs (miRNAs), die durch verschiedene Mechanismen von Zielzellen aufgenommen werden können. EV tragen zur Kommunikation zwischen Plazenta und mütterlichen Organen und Zellen einschließlich des Immunsystems bei. Plazentare miRNAs spielen eine wichtige Rolle bei der Regulation von Zellfunktionen in der Plazenta, gelangen aber auch mittels EV in Immunzellen. Deren Aufnahme kann die Immunantwort beeinflussen und zur Entwicklung der maternofetalen Immuntoleranz beitragen. Bei Schwangerschaftsstörungen und -erkrankungen kann die Menge und Zusammensetzung plazentarer EV verändert sein. Daher haben sie das Potenzial, zukünftig als diagnostische Marker für Schwangerschaftskomplikationen genutzt zu werden.

Hypertension and inflammation are the main characteristics of preeclampsia (PE), a pregnancy-driven disorder. PE serves as a predisposing factor for cardiovascular conditions later in life. The brain is one of the early victims of hypertension-induced organ damage characterized by cerebrovascular malfunction. Therefore, PE patients present anomalies such as brain edema and blood-brain barrier (BBB) failure. We have previously reported that exogenous EVs can reach the BBB, be internalized by endothelial cells, cross through and induce microglia activation. The placenta releases extracellular vesicles (EVs) to the maternal peripheral blood. Placenta-derived EVs are important carriers of bioactive mediators such as proteins, RNAs, lipids, and miRNAs. Furthermore, PE-EVs may trigger inflammatory responses and induce endothelial injury in the maternal vasculature. However, whether PE-EVs can reach the BBB and their possible effects on brain microvasculature are still to be explored. Plasma was obtained from patients with normal pregnancy (NP) and diagnosed with PE. EVs from plasma were enriched by differential ultracentrifugation and characterized by nanotracking analysis (NTA), Western blotting and transmission electron microscopy (TEM). The BBB representing endothelial hCMEC/D3 cell line was cultured in EndoGRO™-MV complete media in static and flow conditions. Cells were treated with unstained or PKH67-stained EVs from NP, PE or PBS as vehicle. The transendothelial electrical resistance (TEER) and permeability (FITC-dextran) were assessed in a trans-well BBB static model. Tight junction integrity was evaluated using a dynamic 3D on-chip BBB model. Localization of tight junction proteins and EV-uptake was imaged by confocal microscopy. In agreement with previous reports, we found elevated concentrations of sEVs and lEVs in plasma from PE patients compared to NP as measured by NTA and visualized by TEM. Enriched EVs contained placenta-specific markers including HLA-G, PLAP and miR-519d-3p, which differed between PE and NP samples. Using a trans-well BBB static model, uptake of EVs by hCMEC/D3 cells was demonstrated. Treatment with PE-EVs resulted in TEER reduction and increased FITC-dextran permeability. EV uptake was also confirmed under flow conditions using the BBB on-chip model. Increased paracellular permeability associated with decreased expression of tight junction proteins ZO-1 and β-catenin was observed upon treatment with PE-EVs. PE diagnosed pregnant women have higher plasma EV concentration in comparison with NP. The presence of placenta-specific markers confirms that plasma contains placenta-derived-EVs. PE-EVs can interact with endothelial cells in the BBB damaging its integrity and increasing paracellular transport. Altogether, our results suggest that PE-derived EVs may be associated with increased permeability of proinflammatory elements from the periphery into the brain increasing the risk for cerebrovascular disease.

A better understanding of how the spatial configuration of cities, understood as urban structure and forms, can achieve sustainable development is needed. This paper presents spatial data and an automated workflow for studying the urban structures (i.e., road and transportation networks) and forms (i.e., building size, position, function and density) of two medium-sized European cities - Bergen, Norway and Zürich, Switzerland. The data focuses on examining correlations between the densification patterns and transport energy usage of these cities de Koning et al., (2020). Spatial and tabular datasets for (i) urban structures, (ii) urban forms, (iii) building density, (iv) road centre lines and (v) transport energy usage are obtained as georeferenced files from OpenStreetMap (OSM) and upon request from collaborating local and national authorities. Transport energy data is derived from traffic data collected from the Norwegian Public Road Authorities or simulated via a traffic model. Open-source data is used wherever possible. Data gaps within proprietary data are supplemented with proxies or open-source data.Hand-drawn axial maps drawn by the authors using the Space Syntax methods and analysed via depthmapX software are a crucial dataset presented here. All analysed data are then returned to a Geographical Information System (GIS) platform and processed via an automated workflow of 19 steps built via the ModelBuilderTM tool in ESRI® ArcGIS. The automated workflow allows for repetitive cross-city comparison and the compilation of diverse spatial data sources for analysis.In combination with the novel workflow, the dataset can be used for future comparative studies in spatial planning, transport planning and management of energy systems to facilitate informed decision-making towards more sustainable developments.

A better understanding of how the spatial configuration of cities, understood as urban structure and forms, can achieve sustainable development is needed. This paper presents spatial data and an automated workflow for studying the urban structures (i.e., road and transportation networks) and forms (i.e., building size, position, function and density) of two medium-sized European cities - Bergen, Norway and Zürich, Switzerland. The data focuses on examining correlations between the densification patterns and transport energy usage of these cities de Koning et al., (2020). Spatial and tabular datasets for (i) urban structures, (ii) urban forms, (iii) building density, (iv) road centre lines and (v) transport energy usage are obtained as georeferenced files from OpenStreetMap (OSM) and upon request from collaborating local and national authorities. Transport energy data is derived from traffic data collected from the Norwegian Public Road Authorities or simulated via a traffic model. Open-source data is used wherever possible. Data gaps within proprietary data are supplemented with proxies or open-source data.Hand-drawn axial maps drawn by the authors using the Space Syntax methods and analysed via depthmapX software are a crucial dataset presented here. All analysed data are then returned to a Geographical Information System (GIS) platform and processed via an automated workflow of 19 steps built via the ModelBuilderTM tool in ESRI® ArcGIS. The automated workflow allows for repetitive cross-city comparison and the compilation of diverse spatial data sources for analysis.In combination with the novel workflow, the dataset can be used for future comparative studies in spatial planning, transport planning and management of energy systems to facilitate informed decision-making towards more sustainable developments.

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.

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.