Extracellular vesicles (EVs) derived from human placenta have a crucial role in regulating maternal immune responses related with successful pregnancy outcome. MicroRNA miR-193b-3p is strongly expressed in the human placenta and dysregulation may be associated with pathologies. We aim to examine its localization in extravillous trophoblast in healthy placenta and placenta accreta spectrum (PAS), and to elucidate its transport via EVs, its effect on Jurkat T cells functions and its target proteins. The localization of miR-193b-3p was investigated in decidua of human term placenta by miRNA in situ hybridization. Trophoblast cells were identified by cytokeratin-7 immunostaining. miR-193b-3p-mimic or -inhibitor transfected JEG-3 cells were subjected to label-free LC-MS/MS proteomics to identify its potential targets. Proteomics data were further validated by qPCR. EVs were collected from miR-193b-3p transfected JEG-3 cells (EVmiR-193b-3p) or from ex vivo perfused human placentas and isolated by ultracentrifugation. They were characterized by Nanoparticle Tracking Analysis (NTA), Western blotting and transmission electron microscopy. The EVs were stained by PKH67 for large EVs (lEVs) and PKH26 for small EVs (sEVs) to be further used for Jurkat T cells uptake assay tested by flow cytometry and confocal microscopy. The degradation kinetics of miR-193b-3p in Jurkat T cells treated with EVmiR-193b-3p was examined by qPCR. The proliferation of Jurkat T cells treated with EV miR-193b-3p or transfected with miR-193b-3p-mimic as positive control was assessed through an xCELLigence real-time cell analyzer and was later confirmed by colorimetric BrdU-incorporation ELISA. In decidual tissue of both healthy and PAS samples, miR-193b-3p was expressed in extravillous and endovascular trophoblast cells. A higher number of miR-193b-3p positive extravillous trophoblast cells were observed in PAS decidua. 48 proteins were significantly changed between miR-193b-3p-mimic transfected and non-transfected cells. qPCR confirmed 3 targets (MCL1, TMPPE and KRAS) in PAS and JEG-3. CD63 and ALIX were enriched in sEV and hardly detected in lEV fractions. GAPDH and CD47 were present in sEV and lEV fractions from both JEG-3 EVs as well as placental perfusate EVs. Flow cytometry and confocal microscopy of Jurkat T cells treated with JEG-3 EVs showed a tendency of cells to take up more lEVs than sEVs when they were treated with both simultaneously. Treatment of Jurkat Tcells with EV miR-193b-3p resulted in increased intracellular levels of miR-193b-3p persisting up to 7 days. The miR-193b-3p concentration was higher in Jurkat cells treated with lEV miR-193b-3p than with sEV miR-193b-3p. The presence of miR-193b-3p decreased Jurkat T cell proliferation. miR-193-3p is expressed in extravillous trophoblast cells and increased in PAS. It regulates expression of several intracellular signalling molecules. miR-193b-3p can be transported via EVs and incorporated by T cells where it reduces proliferation. These capacities might contribute to increased trophoblast invasiveness and immunotolerance.

Recent trends in 3D cell culturing has placed organotypic tissue models at another level. Now, not only is the microenvironment at the cynosure of this research, but rather, microscopic geometrical parameters are also decisive for mimicking a tissue model. Over the years, technologies such as micromachining, 3D printing, and hydrogels are making the foundation of this field. However, mimicking the topography of a particular tissue-relevant substrate can be achieved relatively simply with so-called template or morphology transfer techniques. Over the last 15 years, in one such research venture, we have been investigating a micro thermoforming technique as a facile tool for generating bioinspired topographies. We call them MatriGrid®s. In this research account, we summarize our learning outcome from this technique in terms of the influence of 3D micro morphologies on different cell cultures that we have tested in our laboratory. An integral part of this research is the evolution of unavoidable aspects such as possible label-free sensing and fluidic automatization. The development in the research field is also documented in this account.

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 humanrelevant 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.

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.

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.

BACKGROUND:The use of fetal bovine serum (FBS) as growth supplement for human cell and tissue culture is widely spread in basic research as well as in clinical approaches, although several limitations must be considered, such as unstable composition and availability, biosafety and ethical aspects. Regarding interspecies differences, xenogeneic growth factors may evoke incompatibilities and non-desired interactions with human cells resulting in imprecise outcome of human-relevant data.METHODS:In this study the functionality of human serum (HS) has been investigated in comparison to FBS by assessing proliferation, migration and invasion of the human cervical cancer cell lines SiHa and HeLa. The effects of both sera on spheroid formation were analyzed microscopically.RESULTS:Both, FBS and HS, stimulate cell proliferation and migration similarly, whereas HS significantly enhanced cell invasion. The spheroid formation assay revealed remarkable differences between both sera, especially for SiHa cells. While in FBS supplemented medium cells only formed loose aggregates, HS induced regularly shaped spheroids under all tested conditions.CONCLUSION:We were able to demonstrate that HS and FBS differently influence behavior of cells in culture which may have an impact on experimental results, especially in 3D cultures.

The current Zika virus (ZIKV) outbreak is associated with neurological malformations and disorders in neonates. Areas of increased incidence of malformations may overlap with dengue-hyperendemic areas. ZIKV infection is enhanced by antibodies against dengue virus (DENV) in cell culture and inbred mice. Sufficiently powered clinical studies or primate studies addressing the enhancement of fetal ZIKV infection after previous dengue infection are not available. The human placenta is susceptible to ZIKV in vitro, but it is unknown whether antibody-dependent enhancement of ZIKV infection occurs at the placental barrier. Here we studied ZIKV infection in placental tissue in the presence of DENV-immune sera. Explants from the amniochorionic membrane, the chorionic villi, and the maternal decidua were infected with ZIKV in the presence of DENV type 1-, 2-, or 4-immune sera, or controls. Presence of DENV antibodies of any type enhanced the percentage of successful infections of organ explants between 1.42- and 2.67-fold, and led to a faster replication as well as significantly increased virus production. No enhancement was seen with yellow fever or chikungunya virus control sera. Pre-existing DENV antibodies may pose an increased risk of trans-placental ZIKV transmission.

The proteomic analysis of complex body fluids by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis requires the selection of suitable sample preparation techniques and optimal parameter settings in data analysis software packages to obtain reliable results. Proteomic analysis of follicular fluid, as a representative of a complex body fluid similar to serum or plasma, is difficult as it contains a vast amount of high abundant proteins and a variety of proteins with different concentrations. However, the accessibility of this complex body fluid for LC-MS/MS analysis is an opportunity to gain insights into the status, the composition of fertility-relevant proteins including immunological factors or for the discovery of new diagnostic and prognostic markers for, for example, the treatment of infertility. In this study, we compared different sample preparation methods (FASP, eFASP and in-solution digestion) and three different data analysis software packages (Proteome Discoverer with SEQUEST, Mascot and MaxQuant with Andromeda) combined with semi- and full-tryptic databank search options to obtain a maximum coverage of the follicular fluid proteome. We found that the most comprehensive proteome coverage is achieved by the eFASP sample preparation method using SDS in the initial denaturing step and the SEQUEST-based semi-tryptic data analysis. In conclusion, we have developed a fractionation-free methodical workflow for in depth LC-MS/MS-based analysis for the standardized investigation of human follicle fluid as an important representative of a complex body fluid. Taken together, we were able to identify a total of 1392 proteins in follicular fluid.

Für toxikologische Fragestellungen ist die humane Plazenta ein vielversprechendes Untersuchungsobjekt. Sie enthält eine Vielzahl verschiedener Zelltypen und bleibt nach der Geburt als gesundes Gewebe humanen Ursprungs für einen begrenzten Zeitraum vital. Für Analysen, welche einen längeren Zeitraum umfassen, können Explantate aus villösem Gewebe genutzt werden. Da Krebserkrankungen in der Schwangerschaft zunehmen, war es das Ziel, die Toxizität etablierter Chemotherapeutika auf plazentares Gewebe zu untersuchen.

We studied the role of multidrug resistance proteins in regulating transplacental transmission of corticosteroids and protease inhibitors.We performed quantitative polymerase chain reaction and FACS analyses to study MDR1 (encodes P-glycoprotein) and MRP-1 expression in extravillous (HTR-8/SVneo) and villous (BeWo) trophoblast cells treated with saquinavir, a multidrug resistance protein substrate. We measured H3-dexamethasone and H3-ritonavir transfer across confluent, syncytialized BeWo cells before and after treatment with agents that inhibit multidrug resistance proteins.Compared with baseline expression, messenger RNA and protein levels were increased significantly in trophoblast cells after treatment with saquinavir. H3-dexamethasone and H3-ritonavir levels increased in BeWo cells after treatment with anti-P-glycoprotein antibodies or cyclosporine A. Transfer of H3-labeled drugs from the apical (eg, maternal) to basolateral (eg, fetal) side of the syncytialized BeWo cell monolayer was increased significantly when cells were pretreated with anti-P-glycoprotein antibodies.Multidrug resistance proteins regulate drug levels in trophoblast cells and may mediate transmission of therapeutic agents across the placenta.

Wolbachia are inherited, endocytoplasmic bacteria that infect a wide range of arthropods. Here is the first systematic report on the study of Wolbachia infection in mosquitoes and their predators from both Thailand and China. In Thailand, 632 mosquito specimens (20 spp.) and 424 insect predators (23 spp.) were collected from the rice agroecosystem, mostly from the Central region, followed by the Northeast, the North and the South and were inhabiting rice fields, wetlands and ditches. In China, 928 mosquitoes (15 spp.) and 149 insect predators (16 spp.) were collected from rice fields along the Weishan Lake in Shandong province. Specimens were classified in the orders Diptera, Coleoptera, Odonata and Hemiptera. Using wsp, ftsZ, 16S rRNA and groE gene amplifications, Wolbachia were detected in 12 mosquito spp. and 6 predator spp. from Thailand and 11 mosquito spp. and 5 predator spp. from China. The relative Wolbachia densities of these species were determined using quantitative real-time PCR. The mosquito, Aedes albopictus, and the predator, Agriocnemis femina, had the highest bacterial densities. These results imply that Wolbachia of supergroup B are distributed throughout these insects, probably via horizontal transmission in rice agroecosystems.

Human placental mesenchymal stem/stromal cells (MSC) are an attractive source of MSC with great therapeutic potential. However, primary MSC are difficult to study in vitro due to their limited lifespan and patient-to-patient variation.Fetal and maternal MSC were prepared from cells of the chorionic and basal plates of the placenta, respectively. Fetal and maternal MSC were transduced with the human telomerase reverse transcriptase (hTERT). Conventional stem cell assays assessed the MSC characteristics of the cell lines. Functional assays for cell proliferation, cell migration and ability to form colonies in soft agar were used to assess the whether transduced cells retained properties of primary MSC.Fetal chorionic and maternal MSC were successfully transduced with hTERT to create the cell lines CMSC29 and DMSC23 respectively. The lifespans of CMSC29 and DMSC23 were extended in cell culture. Both cell lines retained important MSC characteristics including cell surface marker expression and multipotent differentiation potential. Neither of the cell lines was tumourigenic in vitro. Gene expression differences were observed between CMSC29 and DMSC23 cells and their corresponding parent, primary MSC. Both cell lines show similar migration potential to their corresponding primary, parent MSC.The data show that transduced MSC retained important functional properties of the primary MSC. There were gene expression and functional differences between cell lines CMSC29 and DMSC23 that reflect their different tissue microenvironments of the parent, primary MSC. CMSC29 and DMSC23 cell lines could be useful tools for optimisation and functional studies of MSC.