Trophoblast Differentiation Affects Crucial Nutritive Functions of Placental Membrane Transporters.

Cytotrophoblasts are progenitor cells that proliferate and fuse to form the multinucleated syncytiotrophoblast layer, implicated in placental endocrine and transport functions. While membrane transporters play a critical role in the distribution of nutrients, hormones, and xenobiotics at the maternal-fetal interface, their selectivity to the syncytiotrophoblast layer is poorly characterized. We aimed to evaluate the regulation of placental transporters in response to trophoblast differentiation in vitro. Experiments were carried out in isolated primary human trophoblast cells before and after syncytialization. Gene expression of six molecular markers and thirty membrane transporters was investigated by qPCR analysis. Subsequently, functional expression was evaluated for proteins involved in the transplacental transfer of essential nutrients i.e., cholesterol (ABCA1, ABCG1), glucose (SLC2A1), leucine (SLC3A2, SLC7A5), and iron (transferrin receptor, TfR1). We identified that human chorionic gonadotropin, placental lactogen, endoglin, and cadherin-11 serve as optimal gene markers for the syncytialization process. We showed that trophoblast differentiation was associated with differential gene expression (mostly up-regulation) of several nutrient and drug transporters. Further, we revealed enhanced protein expression and activity of ABCG1, SLC3A2, SLC7A5, and TfR1 in syncytialized cells, with ABCA1 and GLUT1 displaying no change. Taken together, these results indicate that the syncytiotrophoblast has a dominant role in transporting essential nutrients cholesterol, leucine, and iron. Nonetheless, we present evidence that the cytotrophoblast cells may also be linked to transport functions that could be critical for the cell fusion processes. Our findings collectively yield new insights into the cellular functions associated with or altered by the trophoblast fusion. Importantly, defective syncytialization could lead to nutrient transfer imbalance, ultimately compromising fetal development and programming

Primary Human Trophoblasts Mimic the Preeclampsia Phenotype after Acute Hypoxia-Reoxygenation Insult.

Preeclampsia (PE) is a pregnancy-specific disorder that affects 3 to 5% of pregnancies worldwide and is one of the leading causes of maternal and fetal morbidity and mortality. Nevertheless, how these events occur remains unclear. We hypothesized that the induction of hypoxic conditions in vitro in primary human trophoblast cells would mimic several characteristics of PE found in vivo. We applied and characterized a model of primary cytotrophoblasts isolated from healthy pregnancies that were placed under different oxygen concentrations: ambient O2 (5% pCO2, 21%pO2, 24 h, termed "normoxia"), low O2 concentration (5% pCO2, 1.5% pO2, 24 h, termed "hypoxia"), or "hypoxia/reoxygenation" (H/R: 6 h intervals of normoxia and hypoxia for 24 h). Various established preeclamptic markers were assessed in this cell model and compared to placental tissues obtained from PE pregnancies. Seventeen PE markers were analyzed by qPCR, and the protein secretion of soluble fms-like tyrosine kinase 1 (sFlT-1) and the placenta growth factor (PlGF) was determined by ELISA. Thirteen of seventeen genes associated with angiogenesis, the renin-angiotensin system, oxidative stress, endoplasmic reticulum stress, and the inflammasome complex were susceptible to H/R and hypoxia, mimicking the expression pattern of PE tissue. In cell culture supernatants, the secretion of sFlT-1 was increased in hypoxia, while PlGF release was significantly reduced in H/R and hypoxia. In the supernatants of our cell models, the sFlT-1/PlGF ratio in hypoxia and H/R was higher than 38, which is a strong indicator for PE in clinical practice. These results suggest that our cellular models reflect important pathological processes occurring in PE and are therefore suitable as PE in vitro models

Effects of aldosterone on the human placenta: Insights from placental perfusion studies.

INTRODUCTION In pregnancy, aldosterone is linked to maternal plasma volume expansion, improved fetal and placental growth/angiogenesis and reduced maternal blood pressure. Aldosterone levels are low in women with pre-eclampsia. Given the placental growth properties of aldosterone in pregnancy, we hypothesised that increased aldosterone improves placental function ex vivo. We applied aldosterone in the dual human placenta perfusion model and analysed specific regulatory markers. METHODS A single cotyledon was perfused using a trimodal perfusion setup consisting of a control phase (CP; basic perfusion medium (BPM) alone) and two consecutive experimental phases (EP1/EP2; BPM supplemented with 1.5 x 10-9M and 1.5 x 10-7M aldosterone, respectively). CP and EP1/EP2 were conducted in closed circuits lasting 2 h each. Quality/time control perfusions using BPM alone were performed for 360 min to distinguish time-dependent effects from aldosterone-related effects. Perfusates were assessed for control parameters (pH/pO2/pCO2/glucose/lactate/creatinine/antipyrine). Maternal perfusates were analysed for placental growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt-1), interleukin-10 (IL-10) and tumour necrosis factor-alpha (TNF-α) using ELISAs. mRNA expression of abovementioned factors was measured by qPCR in post-perfusion tissue. RESULTS Data from quality/time control perfusions indicated that TNF-α and IL-10 release continuously increased over time. Contrary, in the trimodal perfusion setup the application of aldosterone decreased TNF-α secretion (P < 0.05, EP1/EP2 vs CP, 120 min) and increased PlGF release (P < 0.05, EP1 vs CP, 90/120 min) into the maternal perfusates. mRNA expression followed similar trends, but did not reach significance. DISCUSSION Our ex vivo placental perfusion data suggest that increasing aldosterone promotes anti-inflammatory and pro-angiogenic factors, which could positively contribute to healthy pregnancy outcomes

IFPA meeting 2018 workshop report II: Abnormally invasive placenta; inflammation and infection; preeclampsia; gestational trophoblastic disease and drug delivery

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2018 there were nine themed workshops, five of which are summarised in this report. These workshops discussed new perspectives and knowledge in the following areas of research: 1) preeclampsia; 2) abnormally invasive placenta; 3) placental infection; 4) gestational trophoblastic disease; 4) drug delivery to treat placental dysfunction

Materno-fetal cholesterol transport during pregnancy.

Cholesterol is a major nutrient required for fetal growth. It is also a precursor for the synthesis of steroid hormones and essential for the development and maturation of fetal organs. During pregnancy, the placenta controls the transport of cholesterol from the mother to the fetus and vice versa. Cholesterol originating from the maternal circulation has to cross two main membrane barriers to reach the fetal circulation: Firstly, cholesterol is acquired by the apical side of the syncytiotrophoblast (STB) from the maternal circulation as high-density lipoprotein (HDL)-, low-density lipoprotein (LDL)- or very-low-density lipoprotein (VLDL)-cholesterol and secreted at the basal side facing the villous stroma. Secondly, from the villous stroma cholesterol is taken up by the endothelium of the fetal vasculature and transported to the fetal vessels. The proteins involved in the uptake of HDL-, LDL-, VLDL- or unesterified-cholesterol are scavenger receptor type B class 1 (SR-B1), cubulin, megalin, LDL receptor (LDLR) or Niemann-Pick-C1 (NPC1) which are localized at the apical and/or basal side of the STB or at the fetal endothelium. Through interaction with apolipoproteins (e.g. apoA1) cholesterol is effluxed either to the maternal or fetal circulation via the ATP-binding-cassette (ABC)-transporter A1 and ABCG1 localized at the apical/basal side of the STB or the endothelium. In this mini-review, we summarize the transport mechanisms of cholesterol across the human placenta, the expression and localization of proteins involved in the uptake and efflux of cholesterol, and the expression pattern of cholesterol transport proteins in pregnancy pathologies such as pre-eclampsia, gestational diabetes mellitus and intrauterine growth retardation

Placental Expression of Bile Acid Transporters in Intrahepatic Cholestasis of Pregnancy.

Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-related condition characterized by increased maternal circulating bile acids (BAs) having adverse fetal effects. We investigated whether the human placenta expresses specific regulation patterns to prevent fetal exposition to harmful amounts of BAs during ICP. Using real-time quantitative PCR, we screened placentae from healthy pregnancies (n = 12) and corresponding trophoblast cells (n = 3) for the expression of 21 solute carriers and ATP-binding cassette transporter proteins, all acknowledged as BA- and/or cholestasis-related genes. The placental gene expression pattern was compared between healthy women and ICP patients (n = 12 each). Placental SLCO3A1 (OATP3A1) gene expression was significantly altered in ICP compared with controls. The other 20 genes, including SLC10A2 (ASBT) and EPHX1 (EPOX, mEH) reported for the first time in trophoblasts, were comparably abundant in healthy and ICP placentae. ABCG5 was undetectable in all placentae. Placental SLC10A2 (ASBT), SLCO4A1 (OATP4A1), and ABCC2 mRNA levels were positively correlated with BA concentrations in ICP. Placental SLC10A2 (ASBT) mRNA was also correlated with maternal body mass index. We conclude that at the transcriptional level only a limited response of BA transport systems is found under ICP conditions. However, the extent of the transcriptional response may also depend on the severity of the ICP condition and the magnitude by which the maternal BA levels are increased

The polarized localization of lipoprotein receptors and cholesterol transporters in the syncytiotrophoblast of the placenta is reproducible in a monolayer of primary human trophoblasts.

INTRODUCTION The uptake of low- and high-density lipoproteins (LDL and HDL) through the LDL receptor (LDLR) and the scavenger receptor class B type I (SR-BI) mediates maternal to fetal cholesterol transfer in syncytiotrophoblast (STB) cells. STB cells deliver cholesterol via cholesterol efflux through the ATP-binding cassette transporters A1 (ABCA1, to ApoA-I), G1 (ABCG1, to HDL), and SR-BI (to HDL). In the human placenta, these proteins are localized in the apical (LDLR, SR-BI, ABCA1) and basal (SR-BI, ABCA1, ABCG1) membrane of STB cells. However, whether these proteins in polarized primary culture models of STB show a similar localization to those in the human placenta is currently unknown. METHODS Primary human trophoblasts (PHT) were isolated from normal placentas and cultured in Transwells® with Matrigel to obtain a polarized STB monolayer, proteins were determined by immunofluorescence and cholesterol efflux determined to different acceptors. RESULTS At day 5, LDLR and ABCA1 localized mainly in the apical membrane, ABCG1 in the basal membrane, and SR-BI in both. Cholesterol efflux towards the apical compartment was higher to adult and neonatal HDL compared to ApoA-I. When acceptors were added in the basal compartment, cholesterol was retained in the Matrigel. DISCUSSION Polarized STB monolayers express LDLR, SR-BI, ABCA1 and ABCG1, and their apical/basal localization resembles the one described in human placental tissue. This study confirms the high physiological value and suitability of this model for use in functional studies. Our findings also suggest that ABCA1 and SR-BI participate in cholesterol efflux to the maternal side of the cells

Comparative expression patterns of selected membrane transporters in differentiated BeWo and human primary trophoblast cells.

Although placental membrane transporters have an important impact on materno-fetal nutrient transfer, placental cell models are poorly characterized regarding transporter expression. We assessed the mRNA expression of 26 physiologically important solute carriers and ABC transporters in BeWo (b30 clone) and primary human trophoblast cells (PHT) before and after syncytialization. 77% of the transporters showed similar mRNA expression changes between BeWo and PHT after syncytialization. Selected transporters, however, were either lacking in BeWo or showed different trends after syncytialization. In conclusion, BeWo cells generally represent an apt model for transporter studies, but their suitability should be confirmed for each transporter by comparison with PHT

Revisiting Steroidogenic Pathways in the Human Placenta and Primary Human Trophoblast Cells.

Steroid hormones play a crucial role in supporting a successful pregnancy and ensuring proper fetal development. The placenta is one of the principal tissues in steroid production and metabolism, expressing a vast range of steroidogenic enzymes. Nevertheless, a comprehensive characterization of steroidogenic pathways in the human placenta and potential developmental changes occurring during gestation are poorly understood. Furthermore, the specific contribution of trophoblast cells in steroid release is largely unknown. Thus, this study aimed to (i) identify gestational age-dependent changes in the gene expression of key steroidogenic enzymes and (ii) explore the role of trophoblast cells in steroid biosynthesis and metabolism. Quantitative and Droplet Digital PCR analysis of 12 selected enzymes was carried out in the first trimester (n = 13) and term (n = 20) human placentas. Primary trophoblast cells (n = 5) isolated from human term placentas and choriocarcinoma-derived cell lines (BeWo, BeWo b30 clone, and JEG-3) were further screened for gene expression of enzymes involved in placental synthesis/metabolism of steroids. Finally, de novo steroid synthesis by primary human trophoblasts was evaluated, highlighting the functional activity of steroidogenic enzymes in these cells. Collectively, we provide insights into the expression patterns of steroidogenic enzymes as a function of gestational age and delineate the cellular origin of steroidogenesis in the human placenta

Novel Insights into Concepts and Directionality of Maternal–Fetal Cholesterol Transfer across the Human Placenta

Cholesterol is indispensable for cellular membrane composition and function. It is also a precursor for the synthesis of steroid hormones, which promote, among others, the maturation of fetal organs. A role of the ATP-binding-cassette-transporter-A1 (ABCA1) in the transport of maternal cholesterol to the fetus was suggested by transferring cholesterol to apolipoprotein-A-1 (apo-A1), but the directionality of the apoA-1/ABCA1-dependent cholesterol transport remains unclear. We isolated primary trophoblasts from term placentae to test the hypotheses that (1) apoA-1/ABCA1 dispatches cholesterol mainly towards the fetus to support fetal developmental maturation at term, and (2) differentiated syncytiotrophoblasts (STB) exert higher cholesterol transport activity than undifferentiated cytotrophoblasts (CTB). As experimental models, we used (1) trophoblast monolayers grown on Transwell&reg; system consisting of apical (maternal-like) and basal (fetal-like) compartments, and (2) trophoblasts grown on conventional culture plates at CTB and STB stages. Surprisingly, apoA-1-mediated cholesterol efflux operated almost exclusively at the apical-maternal side, where ABCA1 was also localized by immunofluorescence. We found greater cholesterol efflux capacity in STB, which was increased by liver-X-receptor agonist treatment and decreased by ABCA1 inhibition. We conclude that at term the apoA-1/ABCA1 pathway is rather involved in cholesterol transport to the mother than in transfer to the fully developed fetus