Villous Trophoblast Research Articles

Galectin-8 Contributes to Human Trophoblast Cell Invasion.

Galectins are a class of lectins that are extensively expressed in all organisms. Galectins are involved in a range of functions, including early development, tissue regeneration, cancer and inflammation. It has been shown that galectin-8 is expressed in the villous and extravillous trophoblast (EVT) cells of the human placenta; however, its physiological role in pregnancy establishment has not been elucidated. Taking these factors into account, we investigated the functional role of galectin-8 in HTR-8/SVneo cells-a human EVT cell line-and human primary cytotrophoblast cells isolated from a first-trimester placenta. We analyzed the effects of recombinant human galectin-8 (rh galectin-8) on the adhesion, migration and invasion of HTR-8/SVneo cells. We used qPCR, cell-based ELISA (cELISA) and gelatin zymography to study the effects of galectin-8 on mediators of these processes, such as integrin subunits alpha-1 and beta-1 and matrix metalloproteinases (MMPs)-2 and -9, on the mRNA and protein levels. Further, we studied the effects of galectin-8 on primary cytotrophoblast cells' invasion. Galectin-8 stimulated the adhesion, migration and invasion of HTR-8/SVneo cells, as well as the invasion of primary cytotrophoblasts. In addition, the MMP-2 and -9 levels were increased, while the expression of integrins alpha-1 and beta-1 was not affected. Galectin-8 has the ability to positively affect EVTs' invasion, so it can be considered a significant factor in the trophoblast cell invasion process.

Novel extracellular nanoparticles secreted by human villous trophoblast cells influence peripheral blood mononuclear cells

Novel extracellular nanoparticles secreted by human villous trophoblast cells influence peripheral blood mononuclear cells

Isolation of pure primary term human cytotrophoblasts and their differentiation into syncytiotrophoblast-like cells as an ex vivo model of the human placenta

The placenta plays a fundamental role in fetal growth and maintenance of pregnancy. Its cellular components include a large multinucleated syncytiotrophoblast (STB) and its progenitor, cytotrophoblasts (CTBs), both of which perform vital functions in the human placenta. Primary cytotrophoblasts isolated from term human placentas that spontaneously fuse and differentiate into syncytiotrophoblast-like cells in vitro have been utilized to investigate the functions of the syncytiotrophoblast and placenta with multiple modifications. Although recent advances have enabled the use of trophoblast stem cell-derived organoids as a model for villous trophoblasts, primary CTBs offer several advantages, including spontaneous differentiation, easy access to materials (from term-delivered human placentas), and simple methodology. Here, we present a precise step-by-step process for isolating pure CTBs from term human placenta based on previously reported placenta digestion, density centrifugation, and CTB purification using anti-HLA-A, B, C antibody. Subsequently, we provide a method to improve CTB viability and differentiation into STB-like cells using epidermal growth factor (EGF) and a ROCK inhibitor (Y-27632) that ensures long-term and stable cultures without altering their proliferation. Because these cells can grow on standard tissue culture plates, this model can be easily utilized for various placental investigations, including innate immune responses, drug resistance, and STB metabolism. Employing this approach considerably enhances our understanding of placental functions, which are key to maternal and offspring health.

Oxidative stress induces release of mitochondrial DNA into the extracellular space in human placental villous trophoblast BeWo cells.

Circulating cell-free mitochondrial DNA (ccf-mtDNA) is an indicator of cell death, inflammation, and oxidative stress. ccf-mtDNA in pregnancies with placental dysfunction differs from that in healthy pregnancies, and the direction of this difference depends on gestational age and method of mtDNA quantification. Reactive oxygen species (ROS) trigger release of mtDNA, yet it is unknown whether trophoblast cells release mtDNA in response to oxidative stress, a common feature of pregnancies with placental pathology. We hypothesized that oxidative stress would induce cell death and release of mtDNA from trophoblast cells. BeWo cells were treated with antimycin A (10-320 µM) or rotenone (0.2-50 µM) to induce oxidative stress. A multiplex real-time quantitative PCR (qPCR) assay was used to quantify mtDNA and nuclear DNA in membrane-bound, non-membrane-bound, and vesicle-bound forms in cell culture supernatants and cell lysates. Treatment with antimycin A increased ROS (P < 0.0001), induced cell necrosis (P = 0.0004) but not apoptosis (P = 0.6471), and was positively associated with release of membrane-bound and non-membrane-bound mtDNA (P < 0.0001). Antimycin A increased mtDNA content in exosome-like extracellular vesicles (vesicle-bound form; P = 0.0019) and reduced autophagy marker expression (LC3A/B, P = 0.0002; p62, P < 0.001). Rotenone treatment did not influence mtDNA release or cell death (P > 0.05). Oxidative stress induces release of mtDNA into the extracellular space and causes nonapoptotic cell death and a reduction in autophagy markers in BeWo cells, an established in vitro model of human trophoblast cells. Intersection between autophagy and necrosis may mediate the release of mtDNA from the placenta in pregnancies exposed to oxidative stress.NEW & NOTEWORTHY This is the first study to test whether trophoblast cells release mitochondrial (mt)DNA in response to oxidative stress and to identify mechanisms of release and biological forms of mtDNA from this cellular type. This research identifies potential cellular mechanisms that can be used in future investigations to establish the source and biomarker potential of circulating mtDNA in preclinical experimental models and humans.

USING A MULTI-OMICS LANDSCAPE OF THE MATERNAL-FETAL INTERFACE TO MODEL THE LONGITUDINAL PATHOGENESIS OF EARLY-ONSET PRE-ECLAMPSIA

Objective: Pre-eclampsia (PE) is a syndrome that affects multiple organ systems and is the most severe hypertensive disorder in pregnancy. It frequently leads to preterm delivery, maternal and fetal morbidity and mortality and life-long complications. We currently lack efficient screening tools and early therapies to address PE. Design and method: To identify candidate biomarkers and operative pathways in early onset PE (eoPE, severe PE with onset before week 34), we performed spatio-temporal multi-omics profiling of human eoPE placentae and healthy controls, and validated targets in early gestation in a longitudinal clinical cohort. We used a single-nuclei RNA-sequencing combined with spatial proteo- and transcriptomics and mechanistic in vitro signalling analyses to bridge the gap from late pregnancy disease to early pregnancy pathomechanisms. Results: We discovered a key disruption in villous trophoblast differentiation, which is driven by the increase of transcriptional coactivator p300, that ultimately ends with a senescence-associated secretory phenotype (SASP) of trophoblasts in eoPE. We found a significant increase in the senescence markers in preeclamptic maternal serum in early gestation and late gestation, before the development of clinical symptoms, indicating that the placental syndrome drives systemic maternal syndrome, even before clinical manifestation of eoPE. Conclusions: Our work describes a new disease progression model, starting with dysregulated transition in villous trophoblast differentiation. Our study identifies potential pathophysiology-relevant biomarkers for the early diagnosis of the disease as well as possible targets for interventions, which would be crucial steps toward protecting the mother and child from gestational mortality and morbidity and an increased risk of cardiovascular disease later in life.

Expression of IMP3 and LIN28A RNA-Binding Proteins in Placentas of Patients with Pre-Eclampsia with and without Severe Features.

this study aimed to determine the expression of RNA-binding oncofetal proteins IMP3 and LIN28A in extravillous (EVT) and villous trophoblast (VT) cells of placentas from pre-eclamptic (PE) pregnancies to better understand the pathogenesis of PE. placental tissue of 10 patients with PE with severe features, 10 patients with PE without severe features and 20 age-matched healthy pregnancy controls were analyzed by immunohistochemistry, double immunofluorescence and qPCR. We found a decreased percentage of IMP3-positive EVT cells in PE with and without severe features compared to that of the healthy control (p < 0.001). IMP3 expression was significantly low in VT of PE placentas compared to that of the healthy control (p = 0.002). There was no significant difference in LIN28A expression between groups of PE and the control group. Additionally, we noticed the trend toward downregulation of IMP3 mRNA and LIN28A mRNA in severe PE compared to that of healthy controls. We demonstrated that IMP3 expression is decreased in EVT and VT cells of placentas from pregnancies complicated with both PE with and without severe features. However, additional functional investigations are needed to clarify the role of IMP3 as a potential therapeutic target in the management of PE.

Placental expression of inflammatory Galectin-12 is associated with gestational diabetes

ObjectivesGestational diabetes mellitus (GDM) is a growing health concern. Since members of the galectin-family are identified to play a role in the pathogenesis of GDM, we determined galectin-12 as an essential protein due to its influence in lipolysis and inflammation processes. This study investigates the expression of galectin-12 in the placentas of women with GDM. Study designThe study population includes 40 expectant women suffering from GDM and 40 healthy controls. The expression of galectin-12 in the syncytiotrophoblast (SCT) and the extra villous trophoblast (EVT) of the placenta was analyzed by immunohistological staining and double immunofluorescence. Immunoreactivity Score (IRS) was used for evaluation. ResultsThe results demonstrate a significant overexpression of galectin-12 in the nucleus of the SCT and the EVT of placentas with GDM compared to the healthy control group.Additionally, double immunofluorescence visualizes corresponding results with an overexpression of galectin-12 in the extra villous trophoblast of GDM placentas representing maternal cells. ConclusionThis study identifies galectin-12 to be associated with the process of gestational diabetes mellitus. These findings are in correspondence with the involvement of galectin-12 in inflammatory processes. Maternal BMI and male sex seem to be confounder for the expression of galectin-12 in the nuclear syncytiotrophoblast, but not in other parts of the investigated placental areas. Further investigations are necessary to verify the correlation between gestational diabetes mellitus and the expression of galectin-12 in the placenta and to further elucidate its distinct role.

HLA-G isoforms, HLA-C allotype and their expressions differ between early abortus and placenta in relation to spontaneous abortions

IntroductionSpontaneous abortion (SAB) affects approximately 10% of clinically recognized pregnancies. Fetal trophobalst invasion and remodeling of maternal spiral arteries is reported to be dependent on crosstalk between HLA-C/HLA-G expressed on extra villous trophoblast (EVTs)and Killer cell Immunoglobin like receptors (KIRs) of decidual NK (dNK). Immune dysfunction in decidua contributes to early miscarriage. MethodologyThe study used mother neonate paired cord blood and term placenta samples (n = 46), elective abortus (n = 17,gestational age = 10–12 weeks of pregnancy) and SAB abortus (n = 24, gestational age = 12–15 weeks of pregnancy) for HLA-G, KIR2D and HLA-C. In addition, term placenta was collected from women with history of spontaneous pregnancy loss (n = 24) and women with history of live birth (n = 32). SSP-PCR was used for genotyping, RT-PCR for gene expression, copy number variation (CNVs) and HLA-C allotyping and ELISA for protein expression studies. ResultsMembrane bound HLA-G4 isoform proportion was higher 39.28%, p = 0.02) in term placenta. SAB abortus had higher proportion of HLA-G3 (50%),while elective abortus exhibited higher proportion of soluble isoforms (HLA-G5, = 5.9, HLA-G6 = 5.9%, HLA-G7 = 11.8%).Higher inhibitory KIR2DL1 content and copy numbers with lower HLA-C2 in SAB contrasted with higher copy numbers of KIR2DS1(p = 0.001), KIR2DS1+/2DL1+- HLA-C2 combined genotype in healthy placenta. Elevated KIR2D protein levels (p = 0.001), and concurrently, HLA-C levels were upregulated in healthy placenta. ConclusionOur data supports lower cognate receptor ligand KIR2DS1+/2DL1+ HLA–C2 together with predominance of HLA-G3 isoform in SAB as confounding factors in spontaneous pregnancy loss. HLA-G isoforms and expression differed between first trimester abortus and term placenta suggesting temporal modulation and marks novelty of the study.

Engineering placental trophoblast fusion: A potential role for biomechanics in syncytialization

The process by which placental trophoblasts fuse to form the syncytiotrophoblast around the chorionic villi is not fully understood. Mechanical features of the in vivo and in vitro culture environments have recently emerged as having the potential to influence fusion efficiency, and considering these mechanical cues may ultimately allow predictive control of trophoblast syncytialization. Here, we review recent studies that suggest that biomechanical factors such as shear stress, tissue stiffness, and dimensionally-related stresses affect villous trophoblast fusion efficiency. We then discuss how these stimuli might arise in vivo and how they can be incorporated in cultures to study and enhance villous trophoblast fusion. We believe that this mechanical paradigm will provide novel insight into manipulating the syncytialization process to better engineer improved models, understand disease progression, and ultimately develop novel therapeutic strategies.

Multi-organ dysfunction and outcomes in pregnancy associated COVID-19 infection – descriptive review of pathological findings

Objective(s)Comparative clinical and morphological characterization of lesions of the vascular and nervous system in cases of maternal death associated with COVID-19.Study designThe study included autopsy in 12 cases of maternal death with a positive intravital result for SARS-CoV-2 by reverse transcription polymerase chain reaction. For histopathology, tissue samples were taken from the internal organs of each patient. Pieces of organs were fixed and stained according to the standard protocol. The relative number of microvessels with vasculitis and fibers of the peripheral nervous system with infiltration by immune cells was studied. All morphological changes were classified depending on the severity of the damage.ResultsThe average age of patients with a fatal outcome was 35 ± 4.4 years. Time to death after onset of symptoms averaged 16 ± 4.4 days. Dystrophic lesions (necrosis and apoptosis) of the villous and extravillous trophoblast and decidual tissue were observed in the studied placentas. Histopathological signs of mild and severe lesions of the peripheral nervous system in the organs of the gastrointestinal tract were detected in 2 (16.7%) and 10 (83.3%) cases, respectively, in the myocardium in 4 (33.3%) and 8 (66.7%) cases. Histopathological signs of severe damage to the microvascular bed in the organs of the gastrointestinal tract were registered in 9 (75%) cases.Conclusion(s)The main clinical feature of this cohort was that death occurred in a long-term period, in most cases with a negative PCR. The histopathological pattern was a non-acute injury with an immune component of the microvascular bed and the autonomic nervous system with predominant damage to the myocardium and intestines.What does this study add to the clinical workThis study makes it possible to even better study the immunopathological profile in organs and tissues in pregnant women with a fatal outcome when affected by a viral infection, in particular Covid-19. This knowledge can be used when humanity encounters other viral pandemics in the future.

Cytoplasmic and nuclear DROSHA in human villous trophoblasts

In villous trophoblasts, DROSHA is a key ribonuclease III enzyme that processes pri-microRNAs (pri-miRNAs) into pre-miRNAs at the placenta-specific, chromosome 19 miRNA cluster (C19MC) locus. However, little is known of its other functions. We performed formaldehyde crosslinking, immunoprecipitation, and sequencing (fCLIP-seq) analysis of terminal chorionic villi to identify DROSHA-binding RNAs in villous trophoblasts. In villous trophoblasts, DROSHA predominantly generated placenta-specific C19MC pre-miRNAs, including antiviral C19MC pre-miRNAs. The fCLIP-seq analysis also identified non-miRNA transcripts with hairpin structures potentially capable of binding to DROSHA (e.g., SNORD100 and VTRNA1–1). Moreover, in vivo immunohistochemical analysis revealed DROSHA in the cytoplasm of villous trophoblasts. DROSHA was abundant in the cytoplasm of villous trophoblasts, particularly in the apical region of syncytiotrophoblast, in the full-term placenta. Furthermore, in BeWo trophoblasts infected with Sindbis virus (SINV), DROSHA translocated to the cytoplasm and recognized the genomic RNA of SINV. Therefore, in trophoblasts, DROSHA not only regulates RNA metabolism, including the biogenesis of placenta-specific miRNAs, but also recognizes viral RNAs. After SINV infection, BeWo DROSHA-binding VTRNA1–1 was significantly upregulated, and cellular VTRNA1–1 was significantly downregulated, suggesting that DROSHA soaks up VTRNA1–1 in response to viral infection. These results suggest that the DROSHA-mediated recognition of RNAs defends against viral infection in villous trophoblasts. Our data provide insight into the antiviral functions of DROSHA in villous trophoblasts of the human placenta.

Placental Trophoblast Cell Isolation from the Term Placenta.

Multiple cell lines have been utilized over time in studying placental biology. Still, most of them rely on choriocarcinoma cells or immortalized trophoblast cells that may not be entirely comparable with actual human placental trophoblast cells. Term placentas can be a source of primary villous trophoblasts. However, challenges remain in isolating them and maintaining them in extended culture. This manuscript describes our three-phase protocol utilizing enzymatic/mechanical digestion, modified Percoll gradient density separation, and immunopurification using magnetic beads. The resulting trophoblast culture remains viable for an extended period and highly pure after initial passaging.

Морфофункциональные, молекулярные и иммунологические изменения в плаценте и периферической крови при патологическом прикреплении плаценты

Introduction. Placenta accreta spectrum is an abnormal invasion of villous trophoblast into the myometrium and one of the most severe complications of pregnancy. We aimed to study morphofunctional, molecular, and immunological changes in the placenta and peripheral blood in various types of placenta accreta spectrum.Materials and methods. The study involved 45 pregnant women who underwent ultrasound examination at weeks 35–38. According to the ultrasound data, 15 women had placenta accreta and 15 women developed placenta increta. The comparison group included 15 pregnant women without placenta accreta spectrum with a uterine scar after cesarean section. Histological and immunohistochemical studies were performed on paraffin sections of the placentas. We used mouse antibodies to insulin-like growth factor-binding protein-1, pregnancy-specific β-1 glycoprotein, and human placental lactogen, proteinase 3 and measured them using ELISA. We also studied tumor necrosis factor. Immunophenotyping of peripheral blood lymphocytes was performed using flow cytometry. Results. Рlacenta accreta spectrum is accompanied by increased intensity of IHC staining of placental villi for all the studied markers, in contrast to those in the comparison group. The maximum intensity of insulin-like growth factor-binding protein-1 staining was found in placenta increta. Increased serum concentration of proteinase 3 and placental lactogen (placenta increta) and decreased level of insulin-like growth factor-binding protein-1 were found in placenta accreta spectrum.Conclusion. In placenta accreta spectrum, there is a functional redundancy of placental proteins and an increased pro-inflammatory status. An imbalance between aggressive factors are likely to contribute to pathological villous invasion. Keywords: placenta accreta spectrum, immune status, placental proteins, proteinase 3

Placental pathology in a large (Swedish) cohort of SARS-CoV-2 infected mothers

IntroductionSARS-CoV-2 placentitis is associated with placental destruction and insufficiency and can affect perinatal outcome. The aim of the current study was to contribute with increased knowledge regarding placental histology in maternal SARS-CoV-2 infection during the pregnancy, as well as the correlation to the severity of maternal SARS-CoV-2 infection. Material and methodsThis retrospective observational study included 116 women who had a verified SARS-CoV-2 infection during pregnancy and gave birth between April 2020 and February 2022 in theStockholm region, Sweden. Placental tissue was evaluated regarding several histopathological parameters, amongst them detection of the triad of characteristics of placental SARS-CoV-2 infection: chronic histiocytic intervillositis, fibrin deposition and villous trophoblast necrosis, and immunohistochemistry for ORF-3 protein expression was used for confirmation. Medical records were reviewed for maternal characteristics and neonatal outcome. ResultsSARS-CoV-2 placentitis was present in one-fifth of the examined placentas admitted to the institute due to maternal SARS-CoV-2 infection, out of which 86,4 % were delivered by acute caesarian section (ACS), all on fetal indication, and one pregnancy ended in stillbirth. Half of the cases without placentitis were delivered by ACS, out of which 50 % were on fetal indication. There was a clear tendency of a shorter time gap between confirmed maternal SARS-CoV-2 infection and delivery in the placentitis group. DiscussionThe presence of SARS-CoV-2 placentitis does not seem to correlate with maternal factors or the severity of infection. It does correlate with development of placental dysfunction of acute/subacute onset and is often manifested as reduced fetal movements.

Cytology Techniques Can Provide Insight into Human Placental Structure Including Syncytiotrophoblast Nuclear Spatial Organisation.

The aim of this study was to provide the first systematic description of human placental cytology appearances and to investigate syncytiotrophoblast nuclear organisation patterns using cytology techniques. Term placentas from normal pregnancies were sampled using fine-needle aspiration (FNA) and direct scrapes. Standard histological examination was also performed to exclude pathological changes in the placentas being studied. Both Papanicolaou-stained cytospin preparations and air-dried Giemsa slides from FNA provided high-quality material for cytological assessment with good cellularity. Among the key features of the cytology preparations were villous "microbiopsies" that allowed for the three-dimensional appreciation of villous branching patterns. Cytological appearances, including nuclear characteristics of villous cytotrophoblast and syncytiotrophoblast, were also well demonstrated. In microbiopsies and detached villous trophoblast sheets, complex patterns of syncytiotrophoblast nuclear organisation, not previously described cytologically, were observed, including irregular spacing of nuclei, syncytioplasm windows and linear nuclear arrangements. This study showed that placental cytology (a) provides technically excellent material for cytological evaluation, (b) confirms the presence of complex nuclear organisational patterns in the syncytiotrophoblast by eliminating the possibility of tangential sectioning artefact, (c) provides superior nuclear detail over standard histological sections and (d) may be an untapped research resource for the investigation of normal and pathological processes because of its ability to look at the placenta in a novel way and through its potential for both ex vivo and in vivo placental sampling.

Three-Dimensional In Vitro Human Placental Organoids from Mononuclear Villous Trophoblasts or Trophoblast Stem Cells to Understand Trophoblast Dysfunction in Fetal Growth Restriction.

The placenta plays a critical role in the efficient delivery of nutrients and oxygen from mother to fetus to maintain normal fetal growth. Human placental development and function is a highly orchestrated process, which is spatially and temporally controlled by hormones and growth factors. Specialized epithelial cells called trophoblasts play key roles in placental exchange capacity, and their abnormal function and development contribute to many pregnancy complications, including fetal growth restriction (FGR), a condition in which the fetus does not reach its full growth potential in utero. Great variation in the anatomy and development of the placenta in animal model systems (in vivo) and 2D culture model systems of trophoblasts (in vitro) limits our ability to understand pregnancy disorders. Generating in vitro models that recapitulate the unique features of the human placenta has been challenging. Here, we describe detailed methods to isolate mononuclear villous trophoblasts (containing cytotrophoblasts and trophoblast stem cells) from first trimester placentae, and use both these and trophoblast stem cell populations that can be grown long term in a three-dimensional (3D) placental organoid culture system.

First Evidence for the Daily Oscillation of Circadian Rhythm Gene, BMAL1, in Human Villous Trophoblast Cells

First Evidence for the Daily Oscillation of Circadian Rhythm Gene, BMAL1, in Human Villous Trophoblast Cells

İnsan Trofoblast Hücre Hattında Sialik Asit Bağlayıcı Reseptörlerin (Siglecs) Ekspresyonu

Purpose: Cell surface proteins known as Siglecs exhibit a specific affinity for sialic acid. Primarily located on the surface of immune cells, Siglecs belong to the subset of lectins called I-type lectins. Siglecs have important roles in maternal-fetal immune tolerance. We aimed to analyze the expression of Siglecs on Human Villous Trophoblasts (HVT) cells.&#x0D; Materials and methods: Total RNA was extracted from the HVT cell line, cDNA was synthesized, and real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) was performed the determine the changes in Siglec -3, -5, -6, -7, -8, -9, -10, -11, and -16 mRNA levels. In addition, Siglec levels were assessed by using immunohistochemical staining. Immunoreactivity against Siglec-6 and Siglec-9 was evaluated separately according to the intensity of brown color. &#x0D; Results: Expression levels of Siglec genes by qRT-PCR and melting curve analyses were performed using RNA extracted from the HVT cell line. Siglec -3, -5, -6, -7, -9, -10, -11, and -16 genes were found to be expressed in the HVT cell line. Differently, Siglec-8 results were undetected after cycle 40, which was considered a negative result. The immunocytochemical examination of the HVT cell line revealed that Siglec-6 expression was moderate in the cytoplasm (Score:2). Siglec-9 expression was prominent in the cytoplasm (Score:3). &#x0D; Conclusion: The results showed that Siglec-6 and Siglec-9 were expressed more than other Siglec proteins in the human trophoblast cells. Immunocytochemistry results also support these findings. Our study is the first to show Siglec-9 expression in human trophoblast cells.

Shear stress in the intervillous space promotes syncytial formation of iPS cells-derived trophoblasts†.

The intervillous space of human placenta is filled with maternal blood, and villous trophoblasts are constantly exposed to the shear stress generated by maternal blood pressure and flow throughout the entire gestation period. However, the effects of shear stress on villous trophoblasts and their biological significance remain unknown. Here, using our recently established naïve human pluripotent stem cells-derived cytotrophoblast stem cells (nCTs) and a device that can apply arbitrary shear stress to cells, we investigated the impact of shear stress on early-stage trophoblasts. After 72h of exposure to 10dyn/cm2 shear stress, nCTs became fused and multinuclear, and mRNA expression of the syncytiotrophoblast (ST) markers, such as glial cell missing 1, endogenous retrovirus group W member 1 envelope, chorionic gonadotropin subunit beta 3, syndecan 1, pregnancy specific beta-1-glycoprotein 3, placental growth factor, and solute carrier family 2 member 1 were significantly upregulated compared to static conditions. Immunohistochemistry showed that shear stress increased fusion index, human chorionic gonadotropin secretion, and human placental lactogen secretion. Increased microvilli formation on the surface of nCTs under flow conditions was detected using scanning electron microscopy. Intracellular cyclic adenosine monophosphate significantly increased under flow conditions. Moreover, transcriptome analysis of nCTs subjected to shear stress revealed that shear stress upregulated ST-specific genes and downregulated CT-specific genes. Collectively, these findings indicate that shear stress promotes the differentiation of nCTs into ST.

MRNA and Protein Expression in Human Fetal Membrane Cells: Potential Biomarkers for Preterm Prelabor Rupture of the Fetal Membranes?

Clinically, unique markers in fetal membrane cells may contribute to the search for biomarkers for preterm prelabor rupture of the fetal membranes (pPROM) in maternal blood. pPROM is associated with overwhelming inflammation and premature cellular senescence causing "biological microfractures" of the fetal membranes. We hypothesize that these pathological processes are associated with the shedding of fetal membrane cells into the maternal circulation. The aim of this study was to identify markers expressed exclusively in fetal membrane cells to facilitate their isolation, characterization, and determination of biomarker potential in maternal blood. We have (1), by their transcriptomic profile, identified markers that are upregulated in amnion and chorion tissue compared to maternal white blood cells, and (2), by immunohistochemistry, confirmed the localization of the differentially expressed proteins in fetal membranes, placenta, and the placental bed of the uterus. RNA sequencing revealed 31 transcripts in the amnion and 42 transcripts in the chorion that were upregulated. Among these, 22 proteins were evaluated by immunohistochemistry. All but two transcripts were expressed both on mRNA and protein level in at least one fetal membrane cell type. Among these remaining 20 proteins, 9 proteins were not significantly expressed in the villous and extravillous trophoblasts of the placenta.