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Aerosol deposition (AD) is a room‐temperature film deposition method for the fabrication of scalable ferroelectric ceramic films on different substrates, which is particularly appealing for thick film energy storage applications. However, the electrical and mechanical properties of AD ferroelectric films are not yet satisfactorily understood. Here, we report the dielectric, energy storage, and mechanical properties of aerosol‐deposited BaTiO3 (AD‐BT) thick films with nanosized grains by combining macroscopic electric measurements with indentation tests. We find that thermal annealing is an effective way to improve dielectric permittivity and polarization of the AD‐BT film, as well as to increase the hardness and Young's modulus of the film. However, crack formation in the annealed AD‐BT film is promoted in comparison to the as‐processed sample, suggesting that the interplay between the nanosized grains and release of the internal stress during annealing may have major consequences for the mechanical properties and hence should be taken into consideration in application. [ABSTRACT FROM AUTHOR]

External thermal, electrical, and mechanical fields can induce structural phase transitions in lead‐free Li‐modified Na0.5K0.5NbO3 ferroelectrics, which significantly influence the macroscopic electromechanical response. In particular, the relative stability of the polar monoclinic (or orthorhombic) and tetragonal phases under temperature and stress is critical to realize the ferroelectric and piezoelectric response. In this study, the effect of mechanical and thermal fields on the local structure in the vicinity of the monoclinic‐tetragonal (M‐T) phase boundary was investigated using a novel in situ combined uniaxial compressive stress‐ and temperature‐dependent Raman spectroscopy experimental arrangement. Experiments were performed up to 300°C and −200 MPa, clearly demonstrating stress‐induced M‐T phase transition in Li‐modified Na0.5K0.5NbO3. A stress‐temperature phase diagram has been established based on the change in vibrational modes. It was possible to correlate the relative permittivity singularities previously observed to a given stage of the M‐T phase transition using ratio between characteristic Raman band areas. In addition, the measurement method reported here can be applied to other functional ceramics to investigate the influence of mechanical fields on local structure. [ABSTRACT FROM AUTHOR]

This work demonstrates the successful deposition of bioactive glass (BG) 45S5 coatings on various metallic and ceramic substrates at room temperature under low vacuum condition by using aerosol deposition (AD). This room temperature and particle impact consolidation-based deposition method enabled us to deposit well-adhered and dense BG coatings directly on metallic and ceramic substrates. In vitro tests with human osteoblast-like cells on substrates with a 45S5 BG coating demonstrated high cell activity on the surfaces. All tested materials exhibited high in vitro biocompatibility as no inhibition in cell proliferation could be observed. The utilization of AD process for achieving non-crystalline BG coatings is promising for practical bio-medical applications, e.g. , bioactive coatings on bioinert metallic and ceramic substrates. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Keywords: exosome; extracellular vesicle; microparticle; microvesicle; nanovesicle; pregnancy EN exosome extracellular vesicle microparticle microvesicle nanovesicle pregnancy 1 3 3 02/17/21 20210201 NES 210201 EXTRACELLULAR VESICLES IN REPRODUCTION The literature relating to extracellular vesicles (EVs) has grown exponentially in the past decade, and EVs are now acknowledged to be important in many aspects of biology. The MISEV guidelines recommend referring to these as, 1) small extracellular vesicles (30-150 nm in diameter), which were commonly previously referred to as exosomes (or nanovesicles) and 2) large extracellular vesicles previously commonly referred to as microvesicles or microparticles (150-1000 nm in diameter). Produced by placental cells especially the syncytiotrophoblast during pregnancy, as well as other cells, regulatory RNAs can be packed into EVs and delivered to target cells where they exert regulatory functions on gene expression. Exosome, extracellular vesicle, microparticle, microvesicle, nanovesicle, pregnancy. [Extracted from the article]

In the last few years, it has been shown that extracellular vesicles (EVs), including small (sEVs) and large EVs (lEVs) play a key role in intercellular communication in both healthy and pathological conditions. Gestational diabetes (GDM), a pregnancy disease occurring in approximately 6-13% of all pregnancies, is one of the most common complications during gestation. Although the etiology of GDM remains largely unknown, there have been several studies indicating a potential involvement of EVs in impaired beta cell adaptations in GDM pregnancies. Therefore, in this study we evaluated the influence of maternal blood sEVs from healthy and GDM patients on different cellular responses of 1.1B4 pancreatic beta cell line. Maternal blood samples were collected from both healthy and GDM patients. A protocol for the enrichment of sEV population using ultracentrifugation was employed. The influence of maternal blood sEVs on 1.1B4 pancreatic beta cells proliferation, apoptosis, glucose consumption, and lactate production was evaluated. Maternal blood sEVs from healthy and GDM patients reduced 1.1B4 cell proliferation. This effect was greater with sEVs from GDM patients compared to healthy sEVs. 1.1B4 cells treated with sEVs from healthy patients had a higher glucose consumption and lactate production compared to those treated with GDM sEVs. Both GDM and healthy sEVs did not alter 1.1B4 apoptosis. Our study illustrates that maternal blood sEVs modulate the metabolism and proliferation of a beta cell line and that this effect is altered by sEVs from GDM patients. Further investigations are warranted in order to fully understand the influence of EVs on beta cell functions and the development of GDM [ABSTRACT FROM AUTHOR]

A vinylcarbenoid approach toward N-functionalization of NH-pyrazoles is presented. The rhodium(II)-catalyzed reaction of methyl styryl-diazoacetate (1) or dimethyl 2-diazoglutaconate (3) with 3,5-disubstituted pyrazoles gave products of carbenoid N–H insertion in high combined yields, although regioselectivity issues posed by the pyrazole or the vinylcarbenoid moiety as well as positional and configurational isomerism concerning the C,C double bond of the latter led to product mixtures. The ambident reactivity of the vinylcarbenoid derived from 1 could be steered by the catalyst: while Rh2(OAc)4 yielded products of direct carbenoid insertion preferentially, silver(I) catalysis strongly favored reaction at the vinylogous site of the carbenoid resulting in an N-allylation of the pyrazoles. [ABSTRACT FROM AUTHOR]

Background: The age of technology, where information and knowledge perform important roles in the organisational context, creates an opportunity for local government organisations (such as metropolitan municipalities) in South Africa to support knowledge-sharing. One such technology that supports knowledge-sharing is an intranet. If an intranet is not effectively managed, knowledge-sharing in an organisation shall not be augmented. Objective: To investigate whether or not an intranet augments knowledge-sharing in the selected organisation of eThekwini Municipality. Methods: In this study a quantitative research approach was adopted. Results: The results of this survey suggest that firstly the intranet appears to be at a medium maturity level; secondly, whilst there is information sharing, the intranet does not appear to be effective as a knowledge-sharing structure; and thirdly there appears to be scope for improvement of the content on the intranet. The implication thereof is that eThekwini Municipality's recently formed Municipal Institute of Learning (MILE) may be ideally poised to address the identified shortcomings. Conclusion: Intranet technology plays an important role in an organisation by enabling the effective acquisition, sharing and presentation of knowledge. Because of this an intranet must be effectively managed to readily augment knowledge-sharing in the organisational context of local government organisations (such as metropolitan municipalities) in South Africa. [ABSTRACT FROM AUTHOR]

Abstract: Since the discovery of non-coding RNAs, several families of small regulatory molecules have been described including small nucleolar RNAs, piwi-interacting RNAs and microRNAs (miRNAs). MiRNAs are small single-stranded RNA molecules which play an important role in the regulation of gene expression at the transcriptional level. Recent studies demonstrated that about 30% of human genes are regulated by miRNAs and their deregulation has been associated with malignancies and poor outcome. Therefore, it is not surprising that profiling of miRNAs expression and studies on their regulation became a great field of interest in the last decade. However, miRNA-mediated regulation in pregnancy remains poorly investigated although several independent processes associated with placenta development have been shown to be miRNA-regulated. This review provides a general overview of the current data on profiles and functions of microRNAs in the peri-implantation period, embryonic stem cells, placentation and pregnancy, as well as in several pregnancy-related pathologies. We conclude that miRNAs present in the maternal circulation may provide a new promising diagnostic tool for pregnancy disorders. [Copyright &y& Elsevier]

Abstract: Trirutheniumdodecacarbonyl (Ru3(CO)12) reacts with 2-hydroxy-6-methylpyridine and with 2-hydroxy-5,6,7,8-tetrahydroquinoline in toluene to form centrosymmetric tetranuclear complexes of the type [Ru(η2, μ-L)(CO)2(μ3-L)Ru(CO)2]2, where L is the respective (N,O)-pyridonate ligand (2 and 3). The structures of these complexes, which are almost insoluble in all common solvents, could be determined by single-crystal X-ray diffraction. Reaction of Ru3(CO)12 with 2-hydroxy-4,6-diphenylpyridine in methanol includes ortho-metallation at the phenyl ring, furnishing the dinuclear complex [Ru(κ2 N,C–L)(CO)2(μ-OCH3)2Ru(CO)2 (κ2 N,C–L)] (4), where L=(2-(6-hydroxy-4-phenylpyridin-2-yl)phenyl), according to an X-ray crystal structure determination. [Copyright &y& Elsevier]

The transport of drugs across the placenta is a point of great importance in pharmacotherapy during pregnancy. However, the knowledge of drug transport in pregnancy is mostly based on experimental clinical data, and the underlying biological mechanisms are not fully understood. In this review, we summarize the current knowledge of drug transporters in the human placenta. We only refer to human data since the placenta demonstrates great diversity among species. In addition, we describe the experimental models that have been used in human placental transport studies and discuss their availability. A better understanding of placental drug transporters will be beneficial for the health of pregnant women who need drug treatment and their fetuses. [ABSTRACT FROM AUTHOR]

COVID-19 vaccination seems to be safe in regard to female fertility because of • Low homology of corona spike protein and placental syncytin-1. • Fertility not altered after SARS-CoV-1 and SARS-CoV-2 infections and after antibody therapy against multiple sclerosis associated retrovirus (>80 % homology with syncytin-1). This opinion paper briefly presents arguments that support the unlikelihood of an impact on female fertility from current covid-19 vaccines. [ABSTRACT FROM AUTHOR]

Non-coding RNAs (ncRNAs) play a central and regulatory role in almost all cells, organs, and species, which has been broadly recognized since the human ENCODE project and several other genome projects. Nevertheless, a small fraction of ncRNAs have been identified, and in the placenta they have been investigated very marginally. To date, most examples of ncRNAs which have been identified to be specific for fetal tissues, including placenta, are members of the group of microRNAs (miRNAs). Due to their quantity, it can be expected that the fairly larger group of other ncRNAs exerts far stronger effects than miRNAs. The syncytiotrophoblast of fetal origin forms the interface between fetus and mother, and releases permanently extracellular vesicles (EVs) into the maternal circulation which contain fetal proteins and RNA, including ncRNA, for communication with neighboring and distant maternal cells. Disorders of ncRNA in placental tissue, especially in trophoblast cells, and in EVs seem to be involved in pregnancy disorders, potentially as a cause or consequence. This review summarizes the current knowledge on placental ncRNA, their transport in EVs, and their involvement and pregnancy pathologies, as well as their potential for novel diagnostic tools. [ABSTRACT FROM AUTHOR]

The IL-36 subfamily of cytokines has been recently described as part of the IL-1 superfamily. It comprises three pro-inflammatory agonists (IL-36α, IL-36β, and IL-36γ), their receptor (IL-36R), and one antagonist (IL-36Ra). Although expressed in a variety of cells, the biological relevance of IL-36 cytokines is most evident in the communication between epithelial cells, dendritic cells, and neutrophils, which constitute the common triad responsible for the initiation, maintenance, and expansion of inflammation. The immunological role of IL-36 cytokines was initially described in studies of psoriasis, but novel evidence demonstrates their involvement in further immune and inflammatory processes in physiological and pathological situations. Preliminary studies have reported a dynamic expression of IL-36 cytokines in the female reproductive tract throughout the menstrual cycle, as well as their association with the production of immune mediators and cellular recruitment in the vaginal microenvironment contributing to host defense. In pregnancy, alteration of the placental IL-36 axis has been reported upon infection and pre-eclampsia suggesting its pivotal role in the regulation of maternal immune responses. In this review, we summarize current knowledge regarding the regulatory mechanisms and biological actions of IL-36 cytokines, their participation in different inflammatory conditions, and the emerging data on their potential role in normal and complicated pregnancies. [ABSTRACT FROM AUTHOR]

Aerosol deposition (AD) is a dynamic loading process that can be envisioned as a shock wave loading, necessitating the consideration of the elastic/plastic response of solid materials. Due to the dynamic nature of this process, however, experimental determination of the local pressures during the deposition process is difficult. This work addresses this by investigating the compression and subsequent structure modification of a silicate glass after room‐temperature AD on a silicate glass substrate with Raman spectroscopy. Clear structural changes in the short‐ and middle‐range order of the silicate glass were observed, both as intertetrahedral angle distribution and as ring statistic. Therefore, the AD induced permanent densification of the glass, equivalent, in a hydrostatic approximation, to a minimal pressure of 10.5 ± 1.5 GPa during the film deposition process. Furthermore, the analysis of the Nd3+ photoluminescence of the 4F3/2 − 4I9/2 transition provided complementary information on the glass network modifications occurring during film formation. More than a pure hydrostatic densification, the AD seems to present a very intense shear deformation. This work opens up the perspective of evaluating the mechanical response of film‐substrate and of the particles themselves, and provides critical information on the mechanisms responsible for the AD film formation. [ABSTRACT FROM AUTHOR]

The rhodium-catalyzed reactions of methyl styryl-diazoacetate or dimethyl 2-diazoglutaconate with the title pyrazoles lead to product mixtures as a result of the regioselectivity issues of the pyrazole or the vinylcarbenoid moiety as well as positional and configurational isomerism due to the C=C double bond of the latter. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Serotonin (5-HT) is a biogenic monoamine with diverse functions in multiple human organs and tissues. During pregnancy, tightly regulated levels of 5-HT in the fetoplacental unit are critical for proper placental functions, fetal development, and programming. Despite being a non-neuronal organ, the placenta expresses a suite of homeostatic proteins, membrane transporters and metabolizing enzymes, to regulate monoamine levels. We hypothesized that placental 5-HT clearance is important for maintaining 5-HT levels in the fetoplacental unit. We therefore investigated placental 5-HT uptake from the umbilical circulation at physiological and supraphysiological levels as well as placental metabolism of 5-HT to 5-hydroxyindoleacetic acid (5-HIAA) and 5-HIAA efflux from trophoblast cells. Methods: We employed a systematic approach using advanced organ-, tissue-, and cellular-level models of the human placenta to investigate the transport and metabolism of 5-HT in the fetoplacental unit. Human placentas from uncomplicated term pregnancies were used for perfusion studies, culturing explants, and isolating primary trophoblast cells. Results: Using the dually perfused placenta, we observed a high and concentration-dependent placental extraction of 5-HT from the fetal circulation. Subsequently, within the placenta, 5-HT was metabolized to 5-hydroxyindoleacetic acid (5-HIAA), which was then unidirectionally excreted to the maternal circulation. In the explant cultures and primary trophoblast cells, we show concentration- and inhibitor-dependent 5-HT uptake and metabolism and subsequent 5-HIAA release into the media. Droplet digital PCR revealed that the dominant gene in all models was MAO-A, supporting the crucial role of 5-HT metabolism in placental 5-HT clearance. Conclusions: Taken together, we present transcriptional and functional evidence that the human placenta has an efficient 5-HT clearance system involving (1) removal of 5-HT from the fetal circulation by OCT3, (2) metabolism to 5-HIAA by MAO-A, and (3) selective 5-HIAA excretion to the maternal circulation via the MRP2 transporter. This synchronized mechanism is critical for regulating 5-HT in the fetoplacental unit; however, it can be compromised by external insults such as antidepressant drugs. [ABSTRACT FROM AUTHOR]

This document is a video forum article titled "Robot-assisted laparoscopic descending colon carcinoma resection with D3 lymph node dissection using the triple bipolar technique and intracorporeal delta anastomosis." The article discusses the standardized surgical technique for robot-assisted laparoscopic surgeries (RALS) for descending colon cancer resection. The technique involves a triple bipolar technique and intracorporeal delta anastomosis, which allows for minimal dissection and the possibility of avoiding mobilization of the splenic flexure or rectum. The article provides a video demonstration of the procedure and includes information on the operating time, console time, and blood loss. [Extracted from the article]

AIM: The aim of this study was to analyze human seminal plasma proteins in association with male fertility status using the proteomic mass spectrometry technology Surface-Enhanced Laser Desorption Ionization Time-of-Flight (SELDI-TOF-MS). MATERIALS AND METHODS: Semen analysis was performed using conventional methods. Protein profiles of the seminal plasma were obtained by SELDI-TOF mass spectrometry over a strong anion exchanger, ProteinChip® Q10 array. RESULTS AND CONCLUSION: We found statistically significant differences in motility and sperm count between fertile and infertile men. In addition, we observed ten seminal proteins that are significantly up-regulated in the infertile group. In conclusion, comparison of seminal plasma proteome in fertile and infertile men provides new aspects in the physiology of male fertility and might help in identifying novel markers of male infertility. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The article presents a study on the role of leukemia inhibitory factor (LIF) in inducing the reduction in miR-141 and in regulating trophoblast functions. The study investigated the influence of LIF on miRNA expression in trophoblastic cells. The study concluded that the expression on miRNA in trophoblastic cells is regulated by LIF and is also believed to be responsible for various functional effects which are induced by LIF.

Abstract: In pregnancy, the endometrium represents the hosting surface for invading semi-allogeneic cells. A very fine tuning of cellular interactions is indispensible for the successful symbiosis of mother and fetus. Such interactions include a variety of immune cells, which are present in extremely high concentrations, and trophoblast cells. A wide spectrum of soluble and surface molecules is involved in homing, activation and switching of lymphocyte functions, but also in regulation of embryo implantation and control of trophoblast invasion. This mini-review provides a brief overview of immunological features of the endometrium. [Copyright &y& Elsevier]

Problem: The choriocarcinoma cell line Jeg3 suppresses immunity in vitro by secretion of soluble factors like leukemia inhibitory factor suppressing leukocyte activation. The cells lack expression of classical human leukocyte antigen (HLA)-A and -B alleles but express some HLA-C, and non-classical HLA-G and -E. Upon binding to killing inhibitory receptor on natural killer (NK) cells, HLA-G prevents activation of cytolytic activity. We investigated whether Jeg3 cells are capable of immune stimulation after complementation with classical HLA and T cell costimulatory signal CD80. Method of study: Jeg3 cells were transduced to express HLA-A*0201 and/or CD80. Parental Jeg3 or transfectants Jeg3-A2, Jeg3-CD80 or Jeg3-CD80-A2 were used to stimulate allogeneic resting and activated peripheral blood lymphocytes (PBL). The different cell lines were loaded with a HLA-A2-restricted Epstein-Barr virus (EBV) recall antigen peptide epitope and antigen presenting ability was examined. T cell lines specific for Jeg3 and transfectants were generated from HLA-A2 matched and nonmatched donors and compared for expansion, phenotypes and cytolytic activity. Results: While all Jeg3 cell lines induced only marginal proliferation of resting T cells, phytohemagglutinin (PHA)-activated T cells were stimulated by CD80 or CD80-A2 expressing Jeg3. Only the transfectant Jeg3-CD80-A2 was capable of specific T cell stimulation by EBV recall antigen presentation. T cell lines of HLA-A2 non-matched donors stimulated with the Jeg3 transfectants showed significant expansion only when HLA-A2 and the costimulus CD80 were present. T cells from HLA-A2 positive donors did not expand significantly or differentially. No NK cells grew under any condition. In Jeg3-CD80-A2 stimulated T cells lines CD8+ cells expanded preferentially. These T cells exerted cytolytic activity toward all Jeg3 cell lines. Conclusion: Our data suggest that, in spite of immunosuppressive mechanisms, proliferative and cytolytic T cell responses are induced by Jeg3 cells when classical HLA- and/or costimulatory signals are present on the cells. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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

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. [ABSTRACT FROM AUTHOR]

Wengenmayer T, Poehlmann TG, Markert UR. Inhibition of HLA-G production in JEG-3 choriocarcinoma cells by RNA interference. AJRI 2004; 51:189–191 © Blackwell Munksgaard, 2004 Human leukocyte antigen-G (HLA-G) plays a major role in escape of trophoblast cells from maternal cytotoxicity. Unless its malignant transformation, Jeg-3 choriocarcinoma cell line maintained the capacity of HLA-G production. For the analysis of function and mechanisms of HLA-G-induced immune regulation, a human cellular model with suppressed HLA-G would be very helpful. RNA interference (RNAi) is a very elegant method for this approach, but the design of appropriate oligonucleotide sequences may provide difficulties. We designed oligonucleotides to interfere exclusively with HLA-G mRNA, which were applied at different concentrations to 50% confluent Jeg-3 cells. After 36 hr, the HLA-G content in Jeg-3 cells was analyzed by Western blots. Applying the described RNAi method and oligonucleotides the cellular content of HLA-G was dose-dependently reduced as assessed in several independent Western blots. This method provides a tool for extensive investigation of HLA-G functions in vitro and in vivo. [ABSTRACT FROM AUTHOR]

Purpose: Infertility 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. Methods: Our 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. Results: The 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. Conclusions: In 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. [ABSTRACT FROM AUTHOR]

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

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? 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. Decidualization of St-T1b cells resulted in increased expression of ERS sensors including ATF6α, PERK and IRE1α, and the UPR marker, CHOP. TXNIP, which serves as a link between the ERS pathway and inflammation, as well as inflammasome NLRP3 and interleukin 1β 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. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Molecular communication between a pathogen and its host is crucial for a successful interplay. Extracellular vesicles (EVs) act as mediators for the delivery of molecular signals among pathogens or between pathogens and the host. Toxoplasma gondii (T. gondii), an intracellular parasite with a worldwide presence, produces EVs itself, or induces the secretion of EVs from infected host cells potentially having capacities to modulate the host immune response. T. gondii infection is particularly important during pregnancy. Depending on the gestational age at the time of infection, the parasite can be transmitted through the placenta to the fetus, causing clinical complications such as jaundice, hepatosplenomegaly, chorioretinitis, cranioencephalic abnormalities, or even death. T. gondii infection is related to a pro-inflammatory immune response in both mother and fetus, which may enhance parasite transmission, but the implication of EV signaling in this process remains unclear. In this review, we summarize the current knowledge on EV release from T. gondii and its human host cells in regard to the immunological consequences and the passage through the placenta. • Toxoplasma gondii releases extracellular vesicles (EVs) in the host. • Infected host cells, mainly dentritic cells (DCs), release EVs containing T. gondii components. • EVs from T. gondii or DCs can modulate the immune response. • EVs from T. gondii or DCs can influence the virulence of T. gondii. • Novel diagnostic, treatment and vaccine strategies are based on T. gondii -related EVs. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The placenta is the fetal-maternal interface that supplies the fetus with nutrients, oxygen and immunoprot ective factors but also exposes it to exogenous noxae. Several viral pathogens like cytomegaly, rubella and Zika virus are known to be able to pass the transplacental barrier with far-reaching consequences such as miscarriage, stillbirth, malformations or severe fetal infection. Logically the emergence of the SARS-CoV-2 pandemic was associated with the concern of a possible vertical transmission which seemed to be confirmed in the first case reports. Fortunately over time large cohort studies and meta-analyses could show that vertical transmission with direct fetal infection of the fetal organs is rare complicating only 1-3% of SARS-CoV-2 infections in pregnancy We will present our own data confirming and explaining why vertical transmission of SARS-CoV-2 with direct fetal infection seems to occur only in a minority of cases. Our comprehensive placental analyses were able to identify a paucity of SARS-CoV-2 viral expression ex vivo in term placentae under acute infection and in convalescent pregnant women. Furthermore we could show inefficient SARS-CoV-2 replication in placental tissues in vitro , which provides a rationale for the low ex vivo viral expression. We detected specific SARS-CoV-2 T cell responses in mothers within a few days upon infection, which is undetectable in cord blood. Nevertheless current studies show a significantly elevated risk for perinatal mortality including stillbirth. There is growing evidence that the major cause of perinatal deaths among fetuses and neonates having placentas compromised by SARS-CoV-2 is due to placental insufficiency. SARS-CoV-2 placentitis is defined by increased perivillous fibrin deposition, histiocytic intervillositis and trophoblast necrosis. These changes can cause extensive placental damage leading to placental malperfusion and insufficiency that is incompatible with intrauterine survival. This locally confined changes of the placenta need our further vigilance and evaluation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Problem Uterine natural killer (uNK) cells are major players during implantation and early pregnancy. The aim of our study was to analyze uNK cell concentration in the endometrium of idiopathic recurrent miscarriage (iRM) patients and fertile controls. Method of study Out of n=130 couples with ≥3 consecutive, clinical RM screened according to a standardized diagnostic protocol, n=58 patients with iRM were identified. Endometrial biopsies were investigated in patients and n=17 fertile women (controls) via immunohistochemistry. Results Compared to controls, the concentration of uNK cells was significantly higher in iRM patients (257±212 vs. 148±73 uNK cells/mm², P=.04). IRM patients showed a higher prevalence of >300 uNK cells/mm² than controls (34.5% vs. 5.9%, P=.02). In 88% of controls and 62% of iRM patients, uNK cells were detected within the range of 40-300/mm². Conclusion Idiopathic recurrent miscarriage patients showed higher uNK cell levels than controls supporting the possible impact of uNK cells in the pathophysiology of miscarriage. Our cutoff levels might help to select RM patients which may benefit from immunomodulatory treatment. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Context: Antiphospholipid antibodies (aPL) are related with a high risk of pregnancy morbidity (PM) and also of vascular thrombosis. On the basis of recent studies, we expect that in women with PM associated with antiphospholipid syndrome (APS), further factors may be deregulated and involved in pathophysiology of the disease. Such factors may have the potential to become novel biomarkers for APS and its stages. Settings and Design: Descriptive study from a recurrent pregnancy loss program. Aims: To study the protein expression in sera from women with PM with or without aPL. Materials and Methods: Protein profiles were determined by surface enhanced laser desorption and ionization - time of flight mass spectrometry (SELDITOF MS) in the serum samples from women with PM, 10 of them with aPL and 12 without aPL. On the basis of the mass-to-charge ratio (m/z) of the protein, signals differentially expressed between the two groups were compared with data banks to approach candidate proteins. Statistical Analysis Used: To determine the differential expression of each protein, a no paired t-test was performed using Ciphergen Express Client 3.1 software. Results: SELDI-TOF analysis makes it possible to discriminate between several proteins in women with PM with and without aPL, although it does not allow protein identification. Nine proteins were found in significantly higher levels in aPL-positive women. Conclusion: The results underline that further factors beyond autoantibodies are involved in PM associated with APS and might lead to the development of new biomarkers. [ABSTRACT FROM AUTHOR]