Human Chorionic Gonadotropin Secretion Research Articles

Modeling the Progression of Placental Transport from Early- to Late-Stage Pregnancy by Tuning Trophoblast Differentiation and Vascularization.

The early-stage placental barrier is characterized by a lack of fetal circulation and by a thick trophoblastic barrier, whereas the later-stage placenta consists of vascularized chorionic villi encased in a thin, differentiated trophoblast layer, ideal for nutrient transport. In this work, predictive models of early- and late-stage placental transport are created using blastocyst-derived placental stem cells (PSCs) by modulating PSC differentiation and model vascularization. PSC differentiation results in a thinner, fused trophoblast layer, as well as an increase in human chorionic gonadotropin secretion, barrier permeability, and secretion of certain inflammatory cytokines, which are consistent with in vivo findings. Further, gene expression confirms this shift toward a differentiated trophoblast subtype. Vascularization results in a molecule type- and size-dependent change in dextran and insulin permeability. These results demonstrate that trophoblast differentiation and vascularization have critical effects on placental barrier permeability and that this model can be used as a predictive measure to assess fetal toxicity of xenobiotic substances at different stages of pregnancy.

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.

Self-assembled human placental model from trophoblast stem cells in a dynamic organ-on-a-chip system.

The placental barrier plays a key role in protecting the developing fetus from xenobiotics and exchanging substances between the fetus and mother. However, the trophoblast cell lines and animal models are often inadequate to recapitulate the key architecture and functional characteristics of human placental barrier. Here, we described a biomimetic placental barrier model from human trophoblast stem cells (hTSCs) in a perfused organ chip system. The placental barrier was constructed by co-culture of hTSCs and endothelial cells on the opposite sides of a collagen-coated membrane on chip. hTSCs can differentiate into cytotrophoblasts (CT) and syncytiotrophoblast (ST), which self-assembled into bilayered trophoblastic epithelium with placental microvilli-like structure under dynamic cultures. The formed placental barrier displayed dense microvilli, higher level secretion of human chorionic gonadotropin (hCG), enhanced glucose transport activity. Moreover, RNA-seq analysis revealed upregulated ST expression and activation of trophoblast differentiation-related signalling pathways. These results indicated the key role of fluid flow in promoting trophoblast syncytialization and placental early development. After exposure to mono-2-ethylhexyl phthalate, one of the endocrine disrupting chemicals, the model showed inhibited hCG production and disturbed ST formation in trophoblastic epithelium, suggesting impaired placental structure and function elicited by environmental toxicants. Collectively, the hTSCs-derived placental model can recapitulate placenta physiology and pathological response to external stimuli in a biomimetic manner, which is useful for the study of placental biology and associated diseases.

Monosomy X in isogenic human iPSC-derived trophoblast model impacts expression modules preserved in human placenta

Mammalian sex chromosomes encode homologous X/Y gene pairs that were retained on the Y chromosome in males and escape X chromosome inactivation (XCI) in females. Inferred to reflect X/Y pair dosage sensitivity, monosomy X is a leading cause of miscarriage in humans with near full penetrance. This phenotype is shared with many other mammals but not the mouse, which offers sophisticated genetic tools to generate sex chromosomal aneuploidy but also tolerates its developmental impact. To address this critical gap, we generated X-monosomic human induced pluripotent stem cells (hiPSCs) alongside otherwise isogenic euploid controls from male and female mosaic samples. Phased genomic variants in these hiPSC panels enable systematic investigation of X/Y dosage-sensitive features using invitro models of human development. Here, we demonstrate the utility of these validated hiPSC lines to test how X/Y-linked gene dosage impacts a widely used model for human syncytiotrophoblast development. While these isogenic panels trigger a GATA2/3- and TFAP2A/C-driven trophoblast gene circuit irrespective of karyotype, differential expression implicates monosomy X in altered levels of placental genes and in secretion of placental growth factor (PlGF) and human chorionic gonadotropin (hCG). Remarkably, weighted gene coexpression network modules that significantly reflect these changes are also preserved in first-trimester chorionic villi and term placenta. Our results suggest monosomy X may skew trophoblast cell type composition and function, and that the combined haploinsufficiency of the pseudoautosomal region likely plays a key role in these changes.

Placental Nanoparticle Uptake-On-a-Chip: The Impact of Trophoblast Syncytialization and Shear Stress.

The placenta is a dynamic and complex organ that plays an essential role in the health and development of the fetus. Placental disorders can affect the health of both the mother and the fetus. There is currently an unmet clinical need to develop nanoparticle-based therapies to target and treat placental disorders. However, little is known about the interaction of nanoparticles (NPs) with the human placenta under biomimetic conditions. Specifically, the impact of shear stress exerted on the trophoblasts (placental epithelial cells) by the maternal blood flow, the gradual fusion of the trophoblasts along the gestation period (syncytialization), and the impact of microvilli formation on the cell uptake of NPs is not known. To this end, we designed dynamic placenta-on-a-chip models using BeWo cells to recapitulate the micro-physiological environment, and we induced different degrees of syncytialization via chemical induction with forskolin. We characterized the degree of syncytialization quantitatively by measuring beta human chorionic gonadotropin (β-hCG) secretion, as well as qualitatively by immunostaining the tight junction protein, ZO-1, and counter nuclear staining. We also characterized microvilli formation under static and dynamic conditions via F-actin staining. We used these models to measure the cell uptake of chondroitin sulfate a binding protein (CSA) conjugated and control liposomes using confocal microscopy, followed by image analysis. Interestingly, exposure of the cells to a dynamic flow of media intrinsically induced syncytialization and microvilli formation compared to static controls. Under dynamic conditions, BeWo cells produced more β-hCG in conditions that increased the cell exposure time to forskolin (p < 0.005). Our cell uptake results clearly show a combined effect of the exerted shear stress and forskolin treatment on the cell uptake of liposomes as uptake increased in forskolin exposed conditions (p < 0.05). Overall, the difference in the extent of cell uptake of liposomes among the different conditions clearly displays a need for the development of dynamic models of the placenta that consider the changes in the placental cell phenotype along the gestation period, including syncytialization, microvilli formation, and the expression of different transport and uptake receptors. Knowledge generated from this work will inform future research aiming at developing drug delivery systems targeting the placenta.

Maternal physiology and blastocyst morphology are correlated with an inherent difference in peri-implantation human embryo development

To determine what patient and embryo characteristics are correlated with the developmental potential of the peri-implantation embryo. Retrospective study. Research laboratory. Six hundred fifty-one cryopreserved human blastocysts donated for research with informed patient consent. Not applicable. Blastocyst attachment to fibronectin-coated plates, trophectoderm outgrowth area, epiblast cell number, total cell number, human chorionic gonadotropin secretion. Patients' body mass index, age, follicle-stimulating hormone: luteinizing hormone ratio on menstrual cycle day 3, antral follicle count on menstrual cycle day 3, antimüllerian hormone level on menstrual cycle day 3, and blastocyst morphological grade were correlated with peri-implantation development outcomes. After controlling for good-quality morphological grades, blastocysts from patients of advanced maternal age developed fewer epiblast cells than blastocysts from younger patients. Extended embryo culture during the peri-implantation period mirrors several disparities in fertility treatment outcome that we see clinically, including those from patients with advanced maternal age, high body mass index, and low ovarian reserve and from embryos with lower-quality morphological grades. This model system may be useful by providing an alternative or more sensitive endpoint assessment in studying patient, clinical, or laboratory factors that may influence preimplantation embryo developmental potential.

Roles of Tumor Markers in Central Nervous System Germ Cell Tumors Revisited with Histopathology-Proven Cases in a Large International Cohort.

Simple SummaryThe optimal cut-offs for tumor markers in CNS germ cell tumors (GCTs) to specify non-germinomatous GCTs are under active investigation. This study investigated 162 cases with histopathological confirmation and tumor markers, including 77 cases with robust specimens obtained from tumor resection. This study demonstrates that tumor markers are elevated in germinomas and teratomas, especially HCG in germinomas and AFP in immature teratomas, emphasizing the clinical significance of tissue diagnosis. Similarly, some biopsy cases without malignant findings on histopathology demonstrated tumor marker elevations, highlighting the limitations of reliable tissue diagnosis from a diminutive specimen. This study concludes that an integrated diagnosis based on tumor markers and histopathology is the key to precise diagnosis and, therefore, the avoidance of over- or under-treatment. This study also invites novel questions regarding whether marker-positive germinomas or teratomas should be treated as intensively as malignant NGGCTs, as well as the optimal treatment strategy for marker-negative immature teratomas.The central nervous system germ cell tumor (CNS GCT) is a rare and incompletely understood disease. A major outstanding question in the 2015 consensus document for CNS GCT management was the utility and interpretation of the tumor markers human chorionic gonadotropin (HCG) and alpha fetoprotein (AFP) in the diagnosis of malignant non-germinomatous GCTs (hereafter NGGCTs) prior to treatment. In the current study, we assembled two geographically and ethnically different clinical cohorts from the Mayo Clinic (1988–2017) and the intracranial GCT Genome Analysis Consortium (iGCT Consortium) in Japan to address this question. Patients with both histopathological diagnosis and tumor markers available were eligible for inclusion (n = 162). Biopsy and surgical resection were performed in 85 and 77 cases, respectively. Among 77 resections, 35 demonstrated positivity for HCG, AFP, or both (45%). Seventeen of the marker-positive cases had no malignant non-germinomatous component identified on histopathology, but they were composed strictly of germinoma, teratoma, or both (49%). One embryonal carcinoma was the only marker-negative NGGCT in the study sample. Among 85 biopsies, 18 were marker positive (21%). Seven of these patients had no malignant non-germinomatous component on histopathology, suggesting the potential limitations of limited tissue sample volumes. Neither histopathological diagnosis nor tumor markers alone reliably diagnose NGGCTs due to the secretion of HCG and AFP by germinomas and teratomas. Treatment planning should incorporate integrated histopathological and laboratory-based diagnosis to optimize diagnostic and treatment strategies for this unusual and histologically heterogeneous tumor.

The effect of buprenorphine vs. methadone on opioid-mediated responses in human placental explants

Buprenorphine treatment for maternal opioid use disorder (OUD) is associated with less severe neonatal withdrawal compared to methadone, but little is known about the effects of a partial opioid agonist on the placenta. Opioids are known to stimulate in vitro secretion of basal human chorionic gonadotropin (hCG) and acetylcholine (ACh), but chronic exposure has been shown to downregulate these responses. Given its partial agonist activity, we hypothesized that buprenorphine would be associated with less downregulation, and therefore more robust opioid-mediated responses as compared to methadone, a full agonist.

FURIN and placental syncytialisation: a cautionary tale

FURIN is a pro-protein convertase previously shown to be important for placental syncytialisation (Zhou et al. [1]), a process of cell fusion whereby placental cytotrophoblast cells fuse to form a multinucleated syncytium. This finding has been broadly accepted however, we have evidence suggesting the contrary. Spontaneously syncytialising term primary human trophoblast cells and BeWo choriocarcinoma cells were treated with either FURIN siRNA or negative control siRNA or the protease inhibitor, DEC-RVKR-CMK, or vehicle. Cells were then left to either spontaneously syncytialise (primary trophoblasts) or were induced to syncytialise with forskolin (BeWo). Effects on syncytialisation were measured by determining human chorionic gonadotrophin secretion and E-cadherin protein levels. We showed that FURIN is not important for syncytialisation in either cell type. However, in primary trophoblasts another protease also inhibited by DEC-RVKR-CMK, may be involved. Our results directly contrast with those published by Zhou et al. Zhou et al. however, used first trimester villous explants to study syncytialisation, and we used term primary trophoblasts. Therefore, we suggest that FURIN may be involved in syncytialisation of first trimester trophoblasts, but not term trophoblasts. What is more concerning is that our results using BeWo cells do not agree with their results, even though for the most part, we used the same experimental design. It is unclear why these experiments yielded different results, however we wanted to draw attention to simple differences in measuring syncytialisation or flaws in method reporting (including omission of cell line source and passage numbers, siRNA concentration and protein molecular weights) and choice of immunoblot loading controls, that could impact on experimental outcomes. Our study shows that careful reporting of methods by authors and thorough scrutiny by referees are vital. Furthermore, a universal benchmark for measuring syncytialisation is required so that various studies of syncytialisation can be validated.

A Comparative, Randomized Control Trial in Patients of Per Vaginal Bleeding Comparing Efficacy of Oral Dydrogesterone Versus Vaginal Progesterone in Successful Pregnancy Outcome for Patients with Recurrent Pregnancy Loss.

Progesterone is essential for the maintenance of a healthy pregnancy. Any defect in the secretion of human chorionic gonadotropin or progesterone is associated with a significantly increased risk of first-trimester abortion. Progesterone is frequently prescribed to patients presenting with per vaginal (PV) bleeding in early pregnancy and a history of recurrent pregnancy loss. Pregnant women up to 12weeks of gestation with a history of more than two early pregnancy losses and presenting with vaginal bleeding were included in this study. All subjects were randomized to receive either vaginal progesterone 600mg/day or oral dydrogesterone 30mg/day. A detailed history-including menstrual history, previous pregnancies, previous miscarriages, and other risk factors-was obtained. The mean time required for the cessation of PV bleeding and continuation of pregnancy up to 24weeks and till term was compared. A total of 200 patients were randomized to vaginal progesterone 600mg/day (n = 100) or oral dydrogesterone 30mg/day (n = 100). While 74 patients had two miscarriages in the progesterone group, 68 patients had two miscarriages in the dydrogesterone group. The time required for complete cessation of bleeding was significantly lesser among patients who received oral dydrogesterone compared to those who received intravaginal progesterone (53.90 ± 9.09 vs. 94.60 ± 7.29h, p < 0.0001). Numerically higher number of patients receiving oral dydrogesterone had a successful continuation of pregnancy up to 24weeks of gestation, as well as till full term compared to progesterone group (70 vs. 75). Oral dydrogesterone is preferred over vaginal progesterone in patients presenting with vaginal bleeding during early pregnancy and a history of recurrent early pregnancy loss.

Urban PM2.5 Induces Cellular Toxicity, Hormone Dysregulation, Oxidative Damage, Inflammation, and Mitochondrial Interference

Epidemiological studies have found air pollution to be a driver of adverse pregnancy outcomes but biological mechanisms are not clear. A first trimester trophoblast cell line (HTR-8/SVneo) was exposed to various concentrations of PM2.5 in order to elucidate the effect of urban particulate matter (PM) of size &lt;2.5 μm on placental function. PM2.5 were collected at a site representative of urban traffic and dispersed in cell media by indirect and direct sonication. The HTR-8 cells were grown under standard conditions. Cellular uptake was studied after 24 and 48 h of exposure by transmission electron microscopy (TEM). The secretion of human chorionic gonadotropin (hCG), progesterone, and Interleukin-6 (IL-6) was measured by ELISA. Changes in membrane integrity and H2O2 production were analyzed using the CellToxTM Green Cytotoxicity and ROSGloTM assays. Protease activity was evaluated by MitoToxTM assay. Mitochondrial function was assessed through high resolution respirometry in an Oroboros O2k-FluoRespirometer, and mitochondrial content was quantified by citrate synthase activity. Results: TEM analysis depicted PM2.5 cellular uptake and localization of the PM2.5 to the mitochondria after 24 h. The cells showed aggregated cytoskeleton and generalized necrotic appearance, such as chromatin condensation, organelle swelling and signs of lost membrane integrity. The mitochondria displayed vacuolization and disruption of cristae morphology. At 48 h exposure, a significant drop in hCG secretion and a significant increase in progesterone secretion and IL-6 production occurred. At 48 h exposure, a five-fold increase in protease activity and a significant alteration of H2O2 production was observed. The HTR-8 cells exhibited evidence of increased cytotoxicity with increasing exposure time and dose of PM2.5. Conclusion: PM may contribute to the placental dysfunction associated with pregnancy outcomes, such as preeclampsia and intrauterine growth restriction, through their direct and indirect effects on trophoblast protein secretion, hormone regulation, inflammatory response, and mitochondrial interference.

The effects of high-density lipoprotein and oxidized high-density lipoprotein on forskolin-induced syncytialization of BeWo cells

IntroductionThe negative relationship between maternal high-density lipoprotein-cholesterol (HDL-c) level during pregnancy and infant birth weight has been found. Syncytialization (differentiation and fusion) of trophoblast cells is important to fetal development. HDL has an antioxidant effect, and has been proved to protect trophoblast functions including hormone secretion and invasion. However, HDL is susceptible to oxidation, and high concentrations of HDL impair cell growth and oxidized HDL (oxHDL) inhibits cell proliferation and migration. Moreover, the effects of HDL and oxHDL on trophoblast syncytialization have not been characterized. The aim of this study was to investigate the effects of HDL and oxHDL on trophoblast syncytialization. MethodsHuman choriocarcinoma trophoblasts (BeWo cells) were treated with human HDL or oxHDL and then induced to differentiate by forskolin in syncytialization assays. Expression levels of mRNAs and proteins regulating syncytialization were detected by real-time PCR and western blotting, respectively. ResultsTreatments of HDL at high concentrations reduced human chorionic gonadotropin (hCG) secretion, placental alkaline phosphatase activity and fusion rates, and decreased the expressions of GCM1 and ERVW-1 mRNA as well as phospho-MAPK1/3 (p-MAPK1/3) and total MAPK1/3 protein in the forskolin-induced syncytialization of BeWo cells. Furthermore, treatment of oxHDL (20 μg/ml) decreased hCG secretion, but increased the expression of p-MAPK1/3 protein. DiscussionThese data suggested that both HDL at high concentrations and oxHDL inhibited BeWo cells syncytialization, and might be harmful to placental and fetal development.

One of the trends of oxidative protection in in vitro fertilization programs

Here we review published data from experimental and clinical international studies examining pathogenetic effects of melatonin upon using programs of In Vitro Fertilization (IVF); highlighting various viewpoints on its biological action as a regulator of circadian rhythms: on the one hand, the inhibitory effect of melatonin on pulsating secretion of gonadotropin-releasing hormone was considered, thereby achieving a contraceptive effect; on the other hand, its ability to induce the secretion of human chorionic gonadotropin ensuring ovulation process, was discussed. We also review the data on melatonin acting as a highly active antioxidant. While using melatonin as a metabolic supplement in IVF programs, its positive effect on oocyte morphology and quality of fertilization, embryo division was observed. Moreover, we also highlight the results of studies examining melatonin-related effects on quality of fertilization and embryo division after adding it to culture medium. Such effects demonstrated dose-depended pattern. Taking into account the data of the analyzed publications, adding exogenous melatonin to culture medium may represent a new strategy for personalized approach to improve outcome of IVF programs. Its effectiveness should be further investigated and considered for introduction within the framework of pregravid preparation.

Gestational Trophoblastic Disease Spectrum: Case Report and Anaesthetic Implications

Gestational Trophoblastic Disease (GTD) is a spectrum of tumours with abnormal placental trophoblastic proliferation. They can be benign or malignant lesions. When they invade locally or metastasise, they are called Gestational Trophoblastic Neoplasia (GTN). Clinically, women present with history of amenorrhoea, abdominal pain, mild to severe vaginal bleed with or without symptoms due to metastases. Based on the organ of metastasis, symptoms may vary from breathlessness and cough with chest involvement to lethargy, loss of memory or seizures with brain metastasis. This entity ranges from pre-malignant conditions like hydatidiform mole and partial hydatidiform mole to neoplastic invasive mole, choriocarcinoma or rare type of epitheloid trophoblastic tumour. [1,2] Undetected hyperthyroidism can complicate GTD and present with potential significant complications like cardiac failure and arrhythmias. Manifestations of the disease are attributed to excess secretion of human chorionic gonadotropin (HCG) that has thyrotrophic activity due to its structural similarity. [3,4] The incidence reported in Asian population is as high as 1:400, three times higher. In majority of cases, early diagnosis and treatment provide complete cure in GTD. In 20%, even locally invasive disease, with or without metastasis can be life threatening. [5] Surgical removal is the definitive treatment. Peri-operative anaesthetic management of patients with multiple system involvement as a result of extensive disease can be challenging, hence from anaesthetic perspective it is essential to understand the pathophysiology, clinical presentations and potential complications of molar pregnancy. We hereby report successful management of two cases presenting at different areas of the disease spectrum. Keywords: Molar pregnancy; Choriocarcinoma; Tropoblastic disease.

Delta-9-tetrahydrocannabinol disrupts mitochondrial function and attenuates syncytialization in human placental BeWo cells.

The psychoactive component in cannabis, delta‐9‐tetrahydrocannabinol, can restrict fetal growth and development. Delta‐9‐tetrahydrocannabinol has been shown to negatively impact cellular proliferation and target organelles like the mitochondria resulting in reduced cellular respiration. In the placenta, mitochondrial dysfunction leading to oxidative stress prevents proper placental development and function. A key element of placental development is the proliferation and fusion of cytotrophoblasts to form the syncytium that comprises the materno‐fetal interface. The impact of delta‐9‐tetrahydrocannabinol on this process is not well understood. To elucidate the nature of the mitochondrial dysfunction and its consequences on trophoblast fusion, we treated undifferentiated and differentiated BeWo human trophoblast cells, with 20 µM delta‐9‐tetrahydrocannabinol for 48 hr. At this concentration, delta‐9‐tetrahydrocannabinol on BeWo cells reduced the expression of markers involved in syncytialization and mitochondrial dynamics, but had no effect on cell viability. Delta‐9‐tetrahydrocannabinol significantly attenuated the process of syncytialization and induced oxidative stress responses in BeWo cells. Importantly, delta‐9‐tetrahydrocannabinol also caused a reduction in the secretion of human chorionic gonadotropin and the production of human placental lactogen and insulin growth factor 2, three hormones known to be important in facilitating fetal growth. Furthermore, we also demonstrate that delta‐9‐tetrahydrocannabinol attenuated mitochondrial respiration, depleted adenosine triphosphate, and reduced mitochondrial membrane potential. These changes were also associated with an increase in cellular reactive oxygen species, and the expression of stress responsive chaperones, HSP60 and HSP70. These findings have important implications for understanding the role of delta‐9‐tetrahydrocannabinol‐induced mitochondrial injury and the role this might play in compromising human pregnancies.

The (pro)renin receptor (ATP6AP2) does not play a role in syncytialisation of term human primary trophoblast cells

IntroductionIn the placenta, the (pro)renin receptor (ATP6AP2) is localised to the syncytiotrophoblast. ATP6AP2 can activate the placental renin-angiotensin system (RAS), producing Angiotensin II (Ang II) which, acting via the angiotensin II type 1 receptor (AGTR1), is important for placental development and function. ATP6AP2 can also independently stimulate intracellular signalling pathways known to regulate trophoblast syncytialisation. We proposed that ATP6AP2 plays a role in trophoblast syncytialisation. MethodsPrimary trophoblast cells were isolated from human placentae and transfected with an ATP6AP2 siRNA, a negative control siRNA or vehicle and allowed to spontaneously syncytialise. Syncytialisation was determined by secretion of human chorionic gonadotrophin (hCG) and by decreased CDH1 (E-cadherin) levels. Expression of RAS mRNAs and proteins were measured by qPCR and immunoblotting, respectively. ResultsPrimary trophoblast cells spontaneously syncytialised in culture. Syncytialisation did not affect ATP6AP2 mRNA or protein levels. However, the expression of REN, AGT and AGTR1 mRNAs were increased (P = 0.02, P = 0.01 and P = 0.03, respectively). ATP6AP2 siRNA had no effect on syncytialisation. DiscussionIn primary trophoblasts, syncytialisation was associated with increased expression of the RAS. hCG was increased during syncytialisation and is known to stimulate REN and possibly AGT, however further experiments are needed to confirm that this was the mechanism via which the RAS was activated. Therefore, syncytialisation of primary trophoblasts may involve hCG-induced RAS activation and downstream activation of signalling pathways and growth factors, which can be stimulated via the interaction of Ang II with AGTR1. Nevertheless, it appears that the (pro)renin receptor is not involved.

Exposure to wood smoke particles leads to inflammation, disrupted proliferation and damage to cellular structures in a human first trimester trophoblast cell line

The ongoing transition to renewable fuel sources has led to increased use of wood and other biomass fuels. The physiochemical characteristics of biomass combustion derived aerosols depends on appliances, fuel and operation procedures, and particles generated during incomplete combustion are linked to toxicity. Frequent indoor wood burning is related to severe health problems such as negative effects on airways and inflammation, as well as chronic hypoxia and pathological changes in placentas, adverse pregnancy outcome, preterm delivery and increased risk of preeclampsia. The presence of combustion-derived black carbon particles at both the maternal and fetal side of placentas suggests that particles can reach the fetus. Air pollution particles have also been shown to inhibit trophoblast migration and invasion, which are vital functions for the development of the placenta during the first trimester. In this study we exposed a placental first trimester trophoblast cell line to wood smoke particles emitted under Nominal Burn rate (NB) or High Burn rate (HB). The particles were visible inside exposed cells and localized to the mitochondria, causing ultrastructural changes in mitochondria and endoplasmic reticulum. Exposed cells showed decreased secretion of the pregnancy marker human chorionic gonadotropin, increased secretion of IL-6, disrupted membrane integrity, disrupted proliferation and contained specific polycyclic aromatic hydrocarbons (PAHs) from the particles. Taken together, these results suggest that wood smoke particles can enter trophoblasts and have detrimental effects early in pregnancy by disrupting critical trophoblast functions needed for normal placenta development and function. This could contribute to the underlying mechanisms leading to pregnancy complications such as miscarriage, premature birth, preeclampsia and/or fetal growth restriction. This study support the general recommendation that more efficient combustion technologies and burning practices should be adopted to reduce some of the toxicity generated during wood burning.

“Real life” polycyclic aromatic hydrocarbon (PAH) mixtures modulate hCG, hPL and hPLGF levels and disrupt the physiological ratio of MMP-2 to MMP-9 and VEGF expression in human placenta cell lines

Using JEG-3 and BeWo cells, we examined the effect of “real life” mixtures of polycyclic aromatic hydrocarbons (PAHs), at doses reported in maternal blood (Mix I) and in placental tissue (Mix II), on human chorionic gonadotropin (hCG), placental lactogen (hPL) and placental growth factor (hPLGF) secretion, protein expression and immunolocalization. Additionally, the action of PAH mixtures on basal and hormone-stimulated matrix metalloproteinase-2 (MMP-2), MMP-9 and vascular endothelial growth factor (VEGF) protein expression was evaluated. Under basal conditions, the PAH mixtures increased hCG and decreased hPLGF levels in both cell lines, while hPL expression was stimulated in JEG-3 and inhibited in BeWo. There was no effect on the MMP-2/MMP-9 ratio or VEGF expression. In hormone-stimulated cells, PAH mixtures changed the MMP-2/MMP-9 ratio in JEG-3 cells in favor of MMP-9, while in BeWo MMP-2 was favored. The effect on VEGF expression was cell specific and dependent on the mixture. In hCG-treated cells, only Mix II inhibited VEGF expression in JEG-3 cells. Neither PAH mixtures affected this protein in BeWo cells. In hPL-treated cells, Mix I had a stimulatory effect in JEG-3 cells, while Mix II exerted an inhibitory effect in BeWo cells. In hPLGF-treated cells, Mix II decreased in JEG-3 cells, but in BeWo cells, both mixtures increased VEGF expression. Considering that the evaluated protein hormones play crucial roles in angiogenesis and neovascularization in the placenta, “real life” PAH mixtures by disrupting protein hormones levels, the MMP-2/MMP-9 ratio and VEGF expression can lead to insufficiency and many pregnancy-related disorders.

A comparative study of key physiological stem cell parameters between three human trophoblast cell lines

Human trophoblast stem cells (TSCs) play a key role in the placenta. These cells are proliferative, undifferentiated, and can differentiate into mature trophoblast cell types. However, primary human TSCs are difficult to obtain. In our previous study, we established TSCs from human induced pluripotent stem cells (TShiPSC). Here, we aimed to characterize the identity of these TShiPSC cells by comparing them with BeWo choriocarcinoma cells and primary TSCs (CT cells). Compared with BeWo cells, CT and TShiPSC cells showed high secretion of human chorionic gonadotrophin (hCG) and syncytiotrophoblast differentiation ability. Global gene microarray analysis results showed that CT and TShiPSC cells, unlike BeWo cells, could be classified in the same group. Compared with BeWo cells, CT and TShiPSC cells showed high expression levels of TSC-specific genes and low expression of cancer adhesion and invasion genes. Analysis of placental barrier integrity showed that TShiPSC cells could form a good barrier. Prospective studies using TShiPSC cells hold great promise for elucidating the pathogenesis of infertility due to trophoblast defects.

Urban PM2.5 Induces Cellular Toxicity, Hormone Dysregulation, Oxidative Damage, Inflammation, and Mitochondrial Interference in the HRT8 Trophoblast Cell Line.

Objective: Epidemiological studies have found air pollution to be a driver of adverse pregnancy outcomes, including gestational diabetes, low term birth weight and preeclampsia. It is unknown what biological mechanisms are involved in this process. A first trimester trophoblast cell line (HTR-8/SVneo) was exposed to various concentrations of PM2.5 (PM2.5) in order to elucidate the effect of urban particulate matter (PM) of size <2.5 μm on placental function.Methods: PM2.5 were collected at a site representative of urban traffic and dispersed in cell media by indirect and direct sonication. The HTR-8 cells were grown under standard conditions. Cellular uptake was studied after 24 and 48 h of exposure by transmission electron microscopy (TEM). The secretion of human chorionic gonadotropin (hCG), progesterone, and Interleukin-6 (IL-6) was measured by ELISA. Changes in membrane integrity and H2O2 production were analyzed using the CellToxTM Green Cytotoxicity and ROSGloTM assays. Protease activity was evaluated by MitoToxTM assay. Mitochondrial function was assessed through high resolution respirometry in an Oroboros O2k-FluoRespirometer, and mitochondrial content was quantified by citrate synthase activity.Results: TEM analysis depicted PM2.5 cellular uptake and localization of the PM2.5 to the mitochondria after 24 h. The cells showed aggregated cytoskeleton and generalized necrotic appearance, such as chromatin condensation, organelle swelling and signs of lost membrane integrity. The mitochondria displayed vacuolization and disruption of cristae morphology. At 48 h exposure, a significant drop in hCG secretion and a significant increase in progesterone secretion and IL-6 production occurred. At 48 h exposure, a five-fold increase in protease activity and a significant alteration of H2O2 production was observed. The HTR-8 cells exhibited evidence of increased cytotoxicity with increasing exposure time and dose of PM2.5. No significant difference in mitochondrial respiration or mitochondrial mass could be demonstrated.Conclusion: Following exposure to air pollution, intracellular accumulation of PM may contribute to the placental dysfunction associated with pregnancy outcomes, such as preeclampsia and intrauterine growth restriction, through their direct and indirect effects on trophoblast protein secretion, hormone regulation, inflammatory response, and mitochondrial interference.