Human Syncytiotrophoblast Research Articles

Research Progress on the Impact of Human Chorionic Gonadotropin on Reproductive Performance in Sows

Human chorionic gonadotropin is a glycoprotein hormone produced by human or humanoid syncytiotrophoblasts that differentiate during pregnancy. Due to its superior stability and long-lasting effects compared to luteinizing hormone, it is often used to replace luteinizing hormone to regulate reproductive performance in sows. Human chorionic gonadotropin promotes oocyte maturation, follicle development, and luteinization, thereby increasing conception rates and supporting early embryonic development. In sow reproductive management, the application of human chorionic gonadotropin not only enhances ovulation synchrony but also improves the success rate of embryo implantation by regulating endometrial receptivity and immune mechanisms, significantly enhancing overall reproductive performance. This article primarily reviews the application of human chorionic gonadotropin in sow follicle development, luteal maintenance, and embryo implantation, providing theoretical support for its use in improving reproductive performance in sows.

Disturbance of Fetal Growth by Azithromycin Through Induction of ER Stress in the Placenta.

Aim: Azithromycin (AZM) is widely used to treat mycoplasma infection in pregnancy. However, there is no adequate evaluation of its side effect on the placenta. In this study, using human placental syncytiotrophoblasts and a mouse model, we investigated whether AZM use in pregnancy might adversely affect placental function and pregnancy outcome. Results: Transcriptomic analysis of AZM-treated human placental syncytiotrophoblasts showed increased expression of endoplasmic reticulum (ER) stress-related genes and decreased expression of genes for hormone production and growth factor processing. Verification studies showed that AZM increased the abundance of ER stress mediators (phosphorylated eIF2α, activating transcription factor 4 [ATF4], and C/EBP Homologous Protein [CHOP]) and decreased the abundance of enzymes involved in progesterone and estradiol synthesis (STS, CYP11A1, and CYP19A1) and insulin-like growth factor binding protein (IGFBP) cleavage (PAPPA and ADAM12) in human placental syncytiotrophoblasts. Inhibition of ER stress blocked AZM-induced decreases in the expression of CYP19A1, CYP11A1, PAPPA, and ADAM12, suggesting that the inhibition of AZM on those genes' expression was secondary to AZM-induced ER stress. Further mechanism study showed that increased ATF4 in ER stress might repressively interact with C/EBPα to suppress the expression of those genes, including CEBPA itself. Mouse studies showed that AZM administration decreased fetal weights along with increased ER stress mediators and decreased levels of insulin-like growth factor, estrogen, and progesterone in the maternal blood, which could be alleviated by inhibition of ER stress. Innovation and Conclusion: These findings first support the fact that AZM, often used during pregnancy, may affect fetal growth by inhibiting crucial enzymes for estrogen and progesterone synthesis and disrupting crucial proteases for IGFBP cleavage via inducing ER stress in placental syncytiotrophoblasts.

Association of Maternal Serum Vitamin D Level with Hydatidiform Mole-A Systemic Review

Hydatidiform mole is an abnormal type of pregnancy, in which a potentially anomalous ovum is abnormally fertilized resulting in a subsequently non-viable conceptus becoming an enlarged growth in the uterus with dangerous complications. These moles can occur as either complete or partial, each with its own unique features. Like other risk factor, nutritional risk factors are associated with increased likelihood of development of HM. Low dietary intake of carotene, inadequate protein and animal fats are thought to be associated with increased risk of HM. Vitamin D is a steroid hormone with receptors found throughout the body including ovary, uterus, and placenta and has natural immunomodulator with anti-inflammatory properties. At the ovarian level, vitamin D has been shown to enhance ovulation. In the human syncytiotrophoblast, vitamin D and its components act together to enhance the expressions of human chorionic gonadotropin (hCG), human placental lactogen, estrogen and progesterone. Furthermore, vitamin D co-regulates cell proliferation, differentiation and apoptosis in numerous tissues and 1,25(OH)2D3 also inhibits angiogenesis and metastasis. Deficiency of vitamin D may lead to increased ROS and decreased antioxidative defense systems that could lead to oxidative injury in patients with HM. In the human syncytiotrophoblast, vitamin D and its components act together to enhance the expressions of human chorionic gonadotropin (hCG), human placental lactogen, estrogen and progesterone. Furthermore, vitamin D co-regulates cell proliferation, differentiation and apoptosis in numerous tissues and 1,25(OH)2D3 also inhibits angiogenesis and metastasis. Deficiency of vitamin D may lead to increased ROS and decreased antioxidative defense systems that could lead to oxidative injury in patients with HM. Studies showed there is significant association of low vitamin D level and obstetric complications like early pregnancy loss, premature rupture of membrane, fetal growth restriction and also many extra-skeletal diseases like ovary and colon cancer. In developing countries like-Bangladesh, vitamin D deficiency is much more common and starting in early life and persisting across the life span due to limited exposure to sunlight and low consumption of animal sources food. Therefore, this study was intended to determine the association of vitamin D and hydatidiform mole in a Bangladeshi woman, which may help in deciding about vitamin D supplementation as a preventive measure of hydatidiform mole.

The differential regulation of placenta trophoblast bisphosphoglycerate mutase in fetal growth restriction: preclinical study in mice and observational histological study of human placenta.

Fetal growth restriction (FGR) is a pregnancy complication in which a newborn fails to achieve its growth potential, increasing the risk of perinatal morbidity and mortality. Chronic maternal gestational hypoxia, as well as placental insufficiency are associated with increased FGR incidence; however, the molecular mechanisms underlying FGR remain unknown. Pregnant mice were subjected to acute or chronic hypoxia (12.5% O2) resulting in reduced fetal weight. Placenta oxygen transport was assessed by blood oxygenation level dependent (BOLD) contrast magnetic resonance imaging (MRI). The placentae were analyzed via immunohistochemistry and in situ hybridization. Human placentae were selected from FGR and matched controls and analyzed by immunohistochemistry (IHC). Maternal and cord sera were analyzed by mass spectrometry. We show that murine acute and chronic gestational hypoxia recapitulates FGR phenotype and affects placental structure and morphology. Gestational hypoxia decreased labyrinth area, increased the incidence of red blood cells (RBCs) in the labyrinth while expanding the placental spiral arteries (SpA) diameter. Hypoxic placentae exhibited higher hemoglobin-oxygen affinity compared to the control. Placental abundance of Bisphosphoglycerate mutase (BPGM) was upregulated in the syncytiotrophoblast and spiral artery trophoblast cells (SpA TGCs) in the murine gestational hypoxia groups compared to the control. Hif1α levels were higher in the acute hypoxia group compared to the control. In contrast, human FGR placentae exhibited reduced BPGM levels in the syncytiotrophoblast layer compared to placentae from healthy uncomplicated pregnancies. Levels of 2,3 BPG, the product of BPGM, were lower in cord serum of human FGR placentae compared to control. Polar expression of BPGM was found in both human and mouse placentae syncytiotrophoblast, with higher expression facing the maternal circulation. Moreover, in the murine SpA TGCs expression of BPGM was concentrated exclusively in the apical cell side, in direct proximity to the maternal circulation. This study suggests a possible involvement of placental BPGM in maternal-fetal oxygen transfer, and in the pathophysiology of FGR. This work was supported by the Weizmann Krenter Foundation and the Weizmann - Ichilov (Tel Aviv Sourasky Medical Center) Collaborative Grant in Biomedical Research, by the Minerva Foundation, by the ISF KillCorona grant 3777/19.

Transcriptomic Profiling of Placental Cells in Preeclampsia as an Effective Tool for Personalized Medicine

At present transcriptomics is one of the fastest developing fields of molecular biology, which allows to obtain detailed information about the functional activity of the genome both in normal and pathological conditions. We used modern transcriptomic technologies to comprehensively characterize the whole genome gene expression profile of human placental syncytiotrophoblast cells (STB) in physiological pregnancy and preeclampsia (PE). As a result of our analysis, we identified 26 differentially expressed genes (DEGs) in the STB cells between healthy and diseased states. The cluster of DEGs contains not only well-known candidate genes identified earlier in many foreign whole genome studies of the placenta (for example, LEP, INHBA and FLT1), but also new genes (AC098613.1, AC087857.1, FCRLB, TENM4, PTP4A1P7, LINC01225, etc.) that can be considered as new biological markers of PE and are of interest for further study. Functional enrichment annotation indicated that most of the DEGs were implicated in the signaling pathways of regulation of hormonal secretion, MAPK cascade, ERK1 and ERK2 cascade, positive regulation of cell adhesion and proliferation of endothelial cells. These processes may be associated with the development of PE at the STB cells level. Additionally, we revealed that alternative splicing of the FLT1 gene indicate the important role of this RNA processing mechanism in the pathogenetics of PE due to a significant increase in the transcriptional diversity of genes in STB cells. The expression level of the transcript encoding the protein isoform FLT-1 e15a was significantly increased in patients with PE compared to the control group. This study expands understanding of the molecular mechanisms involved in PE and can serve as a basis for developing of preventive, prognostic and therapeutic strategies in the field of personalized obstetrics.

Adrenomedullin has a pivotal role in trophoblast differentiation: A promising nanotechnology-based therapeutic target for early-onset preeclampsia.

Early-onset preeclampsia (EOPE) is a severe pregnancy complication associated with defective trophoblast differentiation and functions at implantation, but manifestation of its phenotypes is in late pregnancy. There is no reliable method for early prediction and treatment of EOPE. Adrenomedullin (ADM) is an abundant placental peptide in early pregnancy. Integrated single-cell sequencing and spatial transcriptomics confirm a high ADM expression in the human villous cytotrophoblast and syncytiotrophoblast. The levels of ADM in chorionic villi and serum were lower in first-trimester pregnant women who later developed EOPE than those with normotensive pregnancy. ADM stimulates differentiation of trophoblast stem cells and trophoblast organoids in vitro. In pregnant mice, placenta-specific ADM suppression led to EOPE-like phenotypes. The EOPE-like phenotypes in a mouse PE model were reduced by a placenta-specific nanoparticle-based forced expression of ADM. Our study reveals the roles of trophoblastic ADM in placental development, EOPE pathogenesis, and its potential clinical uses.

Atypical protein kinase C isoforms promote human syncytiotrophoblast fusion

Atypical protein kinase C isoforms promote human syncytiotrophoblast fusion

Computational Modelling of Paracellular Diffusion and OCT3 Mediated Transport of Metformin in the Perfused Human Placenta

Metformin is an antidiabetic drug, increasingly prescribed in pregnancy and has been shown to cross the human placenta. The mechanisms underlying placental metformin transfer remain unclear. This study investigated the roles of drug transporters and paracellular diffusion in the bidirectional transfer of metformin across the human placental syncytiotrophoblast using placental perfusion experiments and computational modelling. 14C-metformin transfer was observed in the maternal to fetal and fetal to maternal directions and was not competitively inhibited by 5 mM unlabelled metformin. Computational modelling of the data was consistent with overall placental transfer via paracellular diffusion. Interestingly, the model also predicted a transient peak in fetal 14C-metformin release due to trans-stimulation of OCT3 by unlabelled metformin at the basal membrane. To test this hypothesis a second experiment was designed. OCT3 substrates (5 mM metformin, 5 mM verapamil and 10 mM decynium-22) added to the fetal artery trans-stimulated release of 14C-metformin from the placenta into the fetal circulation, while 5 mM corticosterone did not. This study demonstrated activity of OCT3 transporters on the basal membrane of the human syncytiotrophoblast. However, we did not detect a contribution of either OCT3 or apical membrane transporters to overall materno-fetal transfer, which could be represented adequately by paracellular diffusion in our system.

Siglec-6 mediates the uptake of extracellular vesicles through a noncanonical glycolipid binding pocket

Immunomodulatory Siglecs are controlled by their glycoprotein and glycolipid ligands. Siglec-glycolipid interactions are often studied outside the context of a lipid bilayer, missing the complex behaviors of glycolipids in a membrane. Through optimizing a liposomal formulation to dissect Siglec–glycolipid interactions, it is shown that Siglec-6 can recognize glycolipids independent of its canonical binding pocket, suggesting that Siglec-6 possesses a secondary binding pocket tailored for recognizing glycolipids in a bilayer. A panel of synthetic neoglycolipids is used to probe the specificity of this glycolipid binding pocket on Siglec-6, leading to the development of a neoglycolipid with higher avidity for Siglec-6 compared to natural glycolipids. This neoglycolipid facilitates the delivery of liposomes to Siglec-6 on human mast cells, memory B-cells and placental syncytiotrophoblasts. A physiological relevance for glycolipid recognition by Siglec-6 is revealed for the binding and internalization of extracellular vesicles. These results demonstrate a unique and physiologically relevant ability of Siglec-6 to recognize glycolipids in a membrane.

Genome-Wide Analysis of Hypoxia-Inducible Factor Binding Reveals Targets Implicated in Impaired Human Placental Syncytiotrophoblast Formation under Low Oxygen

Preeclampsia (PE) is a common and serious complication of pregnancy with no cure except premature delivery. The root cause of PE is improper development of the placenta-the temporary organ supporting fetal growth and development. Continuous formation of the multinucleated syncytiotrophoblast (STB) layer via differentiation and fusion of cytotrophoblasts (CTBs) is vital for healthy placentation and is impaired in preeclamptic pregnancies. In PE, there is reduced/intermittent placental perfusion, likely resulting in a persistently low O2 environment. Low O2 inhibits differentiation and fusion of CTBs into STB and may thus contribute to PE pathogenesis; however, the underlying mechanisms are unknown. Because low O2 activates a transcription factor complex in cells known as the hypoxia-inducible factor (HIF), the objective of this study was to investigate whether HIF signaling inhibits STB formation by regulating genes required for this process. Culture of primary CTBs, the CTB-like cell line BeWo, and human trophoblast stem cells under low O2 reduced cell fusion and differentiation into STB. Knockdown of aryl hydrocarbon receptor nuclear translocator (a key component of the HIF complex) in BeWo cells restored syncytialization and expression of STB-associated genes under different O2 levels. Chromatin immunoprecipitation sequencing facilitated the identification of global aryl hydrocarbon receptor nuclear translocator/HIF binding sites, including several near genes implicated in STB development, such as ERVH48-1 and BHLHE40, providing new insights into mechanisms underlying pregnancy diseases linked to poor placental O2 supply.

Metformin Impacts Human Syncytiotrophoblast Mitochondrial Function from Pregnancies Complicated by Obesity and Gestational Diabetes Mellitus in a Sexually Dimorphic Manner.

Maternal obesity and gestational diabetes mellitus (GDM) are associated with placental dysfunction, small for gestational age (SGA) offspring, and programming of adult-onset disease. We examine how metformin, commonly used to treat type A2 GDM, affects placental metabolism as well as mitochondrial content and function. Syncytiotrophoblasts (STBs) were prepared from placentas of male and female fetuses collected at term cesarean section from lean (pre-pregnancy BMI < 25), obese (BMI > 30), and obese A2GDM women. Metformin treatment (0.001-10 mM) of STB caused no change in non-mitochondrial respiration but significant concentration-dependent (1 and 10 mM) decreases in basal, maximal, and ATP-linked respiration and spare capacity. Respiration linked to proton leak was significantly increased in STB of male A2GDM placentas at low metformin concentrations. Metformin concentrations ≥1 mM increased glycolysis in STB from placentas from lean women, but only improved glycolytic capacity in female STB. Whereas metformin had little effect on superoxide generation from male STB of any group, it gave a concentration-dependent decrease in superoxide generation from female STB of lean and obese women. Fewer mitochondria were observed in STB from obese women and male STB from lean women with increasing metformin concentration. Metformin affects STB mitochondrial function in a sexually dimorphic manner but at concentrations above those reported in maternal circulation (approximately 0.01 mM) in women treated with metformin for GDM.

Directed Differentiation of Human Pluripotent Stem Cells to Cytotrophoblast and Syncytiotrophoblast.

Human pluripotent stem cells (hPSCs) form an ideal system to study the formation of placental cells, from an undifferentiated human embryonic stem cell state. The conventional human in vitro model systems to study the human placenta cannot be employed for understanding placental dysfunctions or the development of specialized placental cell types. Hence, human PSCs make an ideal model system to study human placental development and disorders. Here, we describe an efficient and validated protocol to reproducibly study the formation of human cytotrophoblasts (CTBs) and syncytiotrophoblast (STBs) from undifferentiated hPSCs. CTBs are the trophoblast stem cells that can differentiate into specialized placental cell types such as STBs. The multinucleated STB plays vital role in the exchange of nutrients and gases across the placenta and secretes several hormones during pregnancy, such as human chorionic gonadotropin β (hCGβ). Here we describe two methods of seeding the hPSCs: chemical (clumps method) and enzymatic methods (single cells) to differentiate them to CTB and STB, activating BMP (B) signaling and inhibiting ACTIVIN/NODAL and FGF signaling pathways (2i), thus naming our protocol as "B2i" (Sudheer et al., Stem Cells Dev 21:2987-3000, 2012). This protocol forms the perfect model system for understanding in vitro placentation, modeling diseases arising from abnormal placentation that cause complications such as miscarriage, preeclampsia or intrauterine growth restriction (IUGR), and drug discovery for placental disorders.

The multifaceted role of GCM1 during trophoblast differentiation in the human placenta

Remodeling of the uterine vasculature by invasive extravillous trophoblasts (EVTs) is a critical aspect of human placentation. Insufficient EVT invasion can lead to severe obstetrical complications like preeclampsia, intrauterine growth restriction, and preterm birth. Glial cells missing-1 (GCM1) is a transcription factor that is crucial for proper placentation in mice, and is highly expressed in human syncytiotrophoblast (ST) and EVTs. GCM1 is classically considered a master regulator of ST formation, but little is known about its contribution to the development and function of EVTs. Therefore, in this study we test the hypothesis that GCM1 is a critical regulator of both EVT and ST development and function. We show that GCM1 is highly expressed in human trophoblast stem (TS) cells differentiated into either ST or EVTs. Knockdown of GCM1 in TS cells hindered differentiation into both ST and EVT pathways. When placed in ST media, GCM1-knockdown cells formed small, unstable clusters; when placed in EVT media, cells had altered morphology and transcript profiles resembling cells trapped in an intermediate state between CT and EVT, and invasive capacity through matrix was reduced. RNA sequencing analysis of GCM1-deficient TS cells revealed downregulation of EVT-associated genes and enrichment in transcripts related to WNT signaling, which was linked to decreased expression of the EVT master regulator ASCL2 and the WNT antagonist NOTUM. Our findings reveal an essential role of GCM1 during ST and EVT development, and suggest that GCM1 regulates differentiation of human TS cells into EVTs by inducing expression of ASCL2 and NOTUM.

O-GlcNAc transferase contributes to sex-specific placental deregulation in gestational diabetes

IntroductionGestational diabetes (GDM) is traditionally thought to emerge from placental endocrine dysregulations, but recent evidence suggests that fetal sex can also impact GDM development. Understanding the molecular mechanisms through which sex modulates placenta physiology can help identify novel molecular targets for future clinical care. Thus, we investigated the nutrient-sensing O-GlcNAc pathway as a potential mediator of sex-specific placenta dysfunction in GDM. MethodsExpression levels of O-GlcNAc enzymes were measured in male and female (n = 9+/gender) human placentas based on the maternal diagnosis of GDM. We then simulated the observed differences in both BeWo cells and human syncytiotrophoblasts primary cells (SCT) from male and female origins (n = 6/gender). RNA sequencing and targeted qPCR were performed to characterize the subsequent changes in the placenta transcriptome related to gestational diabetes. ResultsO-GlcNAc transferase (OGT) expression was significantly reduced only in male placenta collected from mothers with GDM compared to healthy controls. Similar downregulation of OGT in trophoblast-like BeWo male cells demonstrated significant gene expression deregulations that overlapped with known GDM-related genes. Notably, placental growth hormone (GH) production was significantly elevated, while compensatory factors against GH-related insulin resistance were diminished. Inflammatory and immunologic factors with toxic effects on pancreatic β cell mass were also increased, altogether leaning toward a decompensatory diabetic profile. Similar changes in hormone expression were confirmed in male human primary SCTs transfected with siOGT. However, down-regulating OGT in female primary SCTs did not impact hormone production. ConclusionOur study demonstrated the significant deregulation of placental OGT levels in mothers with GDM carrying a male fetus. When simulated in vitro, such deregulation impacted hormonal production in BeWo trophoblast cells and primary SCTs purified from male placentas. Interestingly, female placentas were only modestly impacted by OGT downregulation, suggesting that the sex-specific presentation observed in gestational diabetes could be related to O-GlcNAc-mediated regulation of placental hormone production.

Human early syncytiotrophoblasts are highly susceptible to SARS-CoV-2 infection

Direct invivo investigation of human placenta trophoblast's susceptibility to SARS-CoV-2 is challenging. Here we report that human trophoblast stem cells (hTSCs) and their derivatives are susceptible to SARS-CoV-2 infection, which reveals heterogeneity in hTSC cultures. Early syncytiotrophoblasts (eSTBs) generated from hTSCs have enriched transcriptomic features of peri-implantation trophoblasts, express high levels of angiotensin-converting enzyme 2 (ACE2), and are productively infected by SARS-CoV-2 and its Delta and Omicron variants to produce virions. Antiviral drugs suppress SARS-CoV-2 replication in eSTBs and antagonize the virus-induced blockage of STB maturation. Although less susceptible to SARS-CoV-2 infection, trophoblast organoids originating from hTSCs show detectable viral replication reminiscent of the uncommon placental infection. These findings implicate possible risk of COVID-19 infection in peri-implantation embryos, which may go unnoticed. Stem cell-derived human trophoblasts such as eSTBs can potentially provide unlimited amounts of normal and genome-edited cells and facilitate coronavirus research and antiviral discovery.

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.

How trophoblasts fuse: an in-depth look into placental syncytiotrophoblast formation.

In humans, cell fusion is restricted to only a few cell types under normal conditions. In the placenta, cell fusion is a critical process for generating syncytiotrophoblast: the giant multinucleated trophoblast lineage containing billions of nuclei within an interconnected cytoplasm that forms the primary interface separating maternal blood from fetal tissue. The unique morphology of syncytiotrophoblast ensures that nutrients and gases can be efficiently transferred between maternal and fetal tissue while simultaneously restricting entry of potentially damaging substances and maternal immune cells through intercellular junctions. To maintain integrity of the syncytiotrophoblast layer, underlying cytotrophoblast progenitor cells terminate their capability for self-renewal, upregulate expression of genes needed for differentiation, and then fuse into the overlying syncytium. These processes are disrupted in a variety of obstetric complications, underscoring the importance of proper syncytiotrophoblast formation for pregnancy health. Herein, an overview of key mechanisms underlying human trophoblast fusion and syncytiotrophoblast development is discussed.

Functional characterization of dopamine and norepinephrine transport across the apical and basal plasma membranes of the human placental syncytiotrophoblast

The human placenta represents a unique non-neuronal site of monoamine transporter expression, with pathophysiological relevance during the prenatal period. Monoamines (serotonin, dopamine, norepinephrine) are crucial neuromodulators for proper placenta functions and fetal development, including cell proliferation, differentiation, and neuronal migration. Accumulating evidence suggests that even a transient disruption of monoamine balance during gestation may lead to permanent changes in the fetal brain structures and functions, projecting into adulthood. Nonetheless, little is known about the transfer of dopamine and norepinephrine across the placental syncytiotrophoblast. Employing the method of isolated membranes from the human term placenta, here we delineate the transport mechanisms involved in dopamine and norepinephrine passage across the apical microvillous (MVM) and basal membranes. We show that the placental uptake of dopamine and norepinephrine across the mother-facing MVM is mediated via the high-affinity and low-capacity serotonin (SERT/SLC6A4) and norepinephrine (NET/SLC6A2) transporters. In the fetus-facing basal membrane, however, the placental uptake of both monoamines is controlled by the organic cation transporter 3 (OCT3/SLC22A3). Our findings thus provide insights into physiological aspects of dopamine and norepinephrine transport across both the maternal and fetal sides of the placenta. As monoamine transporters represent targets for several neuroactive drugs such as antidepressants, our findings are pharmacologically relevant to ensure the safety of drug use during pregnancy.

Bilirubin Reduces the Uptake of Estrogen Precursors and the Followed Synthesis of Estradiol in Human Placental Syncytiotrophoblasts via Inhibition and Downregulation of Organic Anion Transporter 4.

Estrogen biosynthesis in human placental trophoblasts requires the human organic anion transporter 4 (hOAT4)-mediated uptake of fetal derived precursors such as dehydroepiandrosterone-3-sulfate (DHEAS) and 16α-hydroxy-DHEA-S (16α-OH-DHEAS). Scant information is available concerning the contribution of fetal metabolites on the impact of placental estrogen precursor transport and the followed estrogen synthesis. This study substantiated the roles of bilirubin as well as bile acids (taurochenodeoxycholic acid, taurocholic acid, glycochenodeoxycholic acid, chenodeoxycholic acid) on the inhibition of hOAT4-mediated uptake of probe substrate 6-carboxylfluorescein and DHEAS in stably transfected hOAT4-Chinese hamster ovary cells, with the IC50 of 1.53 and 0.98 μM on 6-carboxylfluorescein and DHEAS, respectively, for bilirubin, and 90.2, 129, 16.4, and 12.3 μM on 6-CF for taurochenodeoxycholic acid, glycochenodeoxycholic acid, taurocholic acid, and chenodeoxycholic acid. Bilirubin (2.5-10 μM) concentration-dependently inhibited the accumulation of estradiol precursor DHEAS in human choriocarcinoma JEG-3 cells (reduced by 60% at 10 μM) and primary human trophoblast cells (reduced by 80% at 10 μM). Further study confirmed that bilirubin (0.625-2.5 μM) concentration-dependently reduced the synthesis and secretion of estradiol in primary human trophoblast cells, among which 2.5 μM of bilirubin reduced the synthesis of estradiol by 30% and secretion by 35%. In addition, immunostaining and Western blot results revealed a distinct downregulation of hOAT4 protein expression in primary human trophoblast cells pretreated with 2.5 μM of bilirubin. In conclusion, this study demonstrated that bilirubin reduced the uptake of estrogen precursors and the followed synthesis of estradiol in human placenta via inhibition and downregulation of organic anion transporter 4. SIGNIFICANCE STATEMENT: Fetal metabolites, especially bilirubin, were first identified with significant inhibitory effects on the hOAT4-mediated uptake of estrogen precursor DHEAS in hOAT4-CHO, JEG-3 and PHTCs. Bilirubin concentration-dependently suppressed the estradiol synthesis and secretion in PHTCs treated with DHEAS, which was synchronized with the decline of hOAT4 protein expression. Additionally, those identified bile acids exhibited a weaker inhibitory effect on the secretion of estradiol.

Abstract P227: Gq Signaling In The Placental Syncytiotrophoblast Layer During Preeclampsia

Hormones implicated in preeclampsia (PE) such as angiotensin, endothelin, and vasopressin signal via receptors coupled to the Gq cascade, and Regulator of G protein Signaling-2 (RGS2) buffers this signaling. We have published that RGS2 expression is decreased in human PE placenta, and reducing RGS2 in placenta causes development of key features of PE in mice. New in situ hybridization data indicate that in both humans and mice, RGS2 is abundant among many cell types in the placenta, including the syncytiotrophoblast (STB) layer. In addition, RGS2 expression in the human STB layer is reduced during PE. As this layer is strongly implicated in PE, these data lead us to hypothesize a critical Gq-buffering role for RGS2 in STB cells to prevent PE. To explore the effect of excess Gq signaling within the STB layer, we utilized a Cre-Lox approach to cause expression of the Gq-coupled hM3Dq DREADD throughout the fetoplacental unit (dam: hM3Dq+, sire: Actb-Cre+) or only within the STB layer (dam: hM3Dq+, sire: Gcm1-Cre+), and then activated the hM3Dq receptor via clozapine N-oxide (CNO, 0.5 to 2 mg/kg) injection in mid-gestation (GD12.5-14.5) before tissue collection at GD14.5. Gαq activation throughout the fetoplacental unit (Actb-Cre model) severely restricted fetoplacental growth compared to saline-injected controls (n=2 vs 3; placenta: 0.027±0.006 vs 0.115±0.021 g; p&lt;0.05, and fetus: 0.048±0.007 vs 0.268±0.010 g; p&lt;0.05). Similarly, placentas expressing hM3Dq only in STB cells (Gcm1-Cre model) had reduced placental (n=3 0.116±0.022 vs 0.201±0.036 g; p=0.05) and possibly fetal (n=3 0.1112±0.036 vs 0.247±0.028 g; p=0.06) masses after CNO. Vascularization (assessed by CD31 immunostain) was disproportionately reduced in the labyrinth layer of the Actb-Cre model after CNO (n=2 vs 3; 20.189±3.382 vs 35.762±1.976 % area; p&lt;0.05), despite no relative change in layer (ie, decidua/junctional zone/labyrinth) thicknesses. Preliminary results indicate similar findings in the Gcm1-Cre model (n=1 17 vs 25 % area). These data highlight the pathological consequence of excess Gq signaling in the STB layer. Ongoing studies are aimed at characterizing maternal phenotypes in these models and the consequence of STB-specific deletion of RGS2 upon sensitivity to Gq stimulators.