Obesogenic diet in pregnancy disrupts placental iron handling and ferroptosis and stress signalling in association with fetal growth alterations.

Obesity and gestational diabetes (GDM) impact fetal growth during pregnancy. Iron is an essential micronutrient needed for energy-intense feto-placental development, but if mis-handled can lead to oxidative stress and ferroptosis (iron-dependent cell death). In a mouse model showing maternal obesity and glucose intolerance, we investigated the association of materno-fetal iron handling and placental ferroptosis, oxidative damage and stress signalling activation with fetal growth. Female mice were fed a standard chow or high fat, high sugar (HFHS) diet during pregnancy and outcomes were measured at day (d)16 or d19 of pregnancy. In HFHS-fed mice, maternal hepcidin was reduced and iron status maintained (tissue iron levels) at both d16 and d19. However, fetal weight, placental iron transfer capacity, iron deposition, TFR1 expression and ERK2-mediated signalling were reduced and oxidative damage-related lipofuscin accumulation in the placenta was increased in HFHS-fed mice. At d19, whilst TFR1 remained decreased, fetal weight was normal and placental weight, iron content and iron transporter genes (Dmt1, Zip14, and Fpn1) were reduced in HFHS-fed mice. Furthermore, there was stress kinase activation (increased phosphorylated p38MAPK, total ERK and JNK) in the placenta from HFHS-fed mice at d19. In summary, a maternal HFHS diet during pregnancy impacts fetal growth trajectory in association with changes in placental iron handling, ferroptosis and stress signalling. Downregulation of placental iron transporters in HFHS mice may protect the fetus from excessive oxidative iron. These findings suggest a role for alterations in placental iron homeostasis in determining perinatal outcomes of pregnancies associated with GDM and/or maternal obesity.

Evidence for hypoxia-induced dysregulated cholesterol homeostasis in preeclampsia: Insights into the mechanisms from human placental cells and tissues.

Preeclampsia (PE) poses a considerable risk to the long-term cardiovascular health of both mothers and their offspring due to a hypoxic environment in the placenta leading to reduced fetal oxygen supply. Cholesterol is vital for fetal development by influencing placental function. Recent findings suggest an association between hypoxia, disturbed cholesterol homeostasis, and PE. This study investigates the influence of hypoxia on placental cholesterol homeostasis. Using primary human trophoblast cells and placentae from women with PE, various aspects of cholesterol homeostasis were examined under hypoxic and hypoxia/reoxygenation (H/R) conditions. Under hypoxia and H/R, intracellular total and non-esterified cholesterol levels were significantly increased. This coincided with an upregulation of HMG-CoA-reductase and HMG-CoA-synthase (key genes regulating cholesterol biosynthesis), and a decrease in acetyl-CoA-acetyltransferase-1 (ACAT1), which mediates cholesterol esterification. Hypoxia and H/R also increased the intracellular levels of reactive oxygen species and elevated the expression of hypoxia-inducible factor (HIF)-2α and sterol-regulatory-element-binding-protein (SREBP) transcription factors. Additionally, exposure of trophoblasts to hypoxia and H/R resulted in enhanced cholesterol efflux to maternal and fetal serum. This was accompanied by an increased expression of proteins involved in cholesterol transport such as the scavenger receptor class B type I (SR-BI) and the ATP-binding cassette transporter G1 (ABCG1). Despite these metabolic alterations, mitogen-activated-protein-kinase (MAPK) signaling, a key regulator of cholesterol homeostasis, was largely unaffected. Our findings indicate dysregulation of cholesterol homeostasis at multiple metabolic points in both the trophoblast hypoxia model and placentae from women with PE. The increased cholesterol efflux and intracellular accumulation of non-esterified cholesterol may have critical implications for both the mother and the fetus during pregnancy, potentially contributing to an elevated cardiovascular risk later in life.

LAT1-dependent placental methionine uptake is a key player in fetal programming of metabolic disease.

The Developmental Origins of Health and Disease hypothesis sustains that exposure to different stressors during prenatal development prepares the offspring for the challenges to be encountered after birth. We studied the gestational period as a particularly vulnerable window where different stressors can have strong implications for fetal programming of the offspring's life-long metabolic status via alterations of specific placentally expressed nutrient transporters. To study this mechanism, we used a murine prenatal stress model, human preeclampsia, early miscarriage, and healthy placental tissue samples, in addition to in vitro models of placental cells. In stressed mice, placental overexpression of L-type amino acid transporter 1 (Lat1) and subsequent global placental DNA hypermethylation was accompanied by fetal and adult hypothalamic dysregulation in global DNA methylation and gene expression as well as long-term metabolic abnormalities exclusively in female offspring. In human preeclampsia, early miscarriage, and under hypoxic conditions, placental LAT1 was significantly upregulated, leading to increased methionine uptake and global DNA hypermethylation. Remarkably, subgroups of healthy term placentas with high expression of stress-related genes presented increased levels of placental LAT1 mRNA and protein, DNA and RNA hypermethylation, increased methionine uptake capacity, one-carbon metabolic pathway disruption, higher methionine concentration in the placenta and transport to the fetus specifically in females. Since LAT1 mediates the intracellular accumulation of methionine, global DNA methylation, and one-carbon metabolism in the placenta, our findings hint at a major sex-specific global response to a variety of prenatal stressors affecting placental function, epigenetic programming, and life-long metabolic disease and provide a much-needed insight into early-life factors predisposing females/women to metabolic disorders.

Revisited role of the placenta in bile acid homeostasis.

To date, the discussion concerning bile acids (BAs) during gestation is almost exclusively linked to pregnancy complications such as intrahepatic cholestasis of pregnancy (ICP) when maternal serum BA levels reach very high concentrations (>100 µM). Generally, the placenta is believed to serve as a protective barrier avoiding exposure of the growing fetus to excessive amounts of maternal BAs that might cause detrimental effects (e.g., intrauterine growth restriction and/or increased vulnerability to metabolic diseases). However, little is known about the precise role of the placenta in BA biosynthesis, transport, and metabolism in healthy pregnancies when serum BAs are at physiological levels (i.e., low maternal and high fetal BA concentrations). It is well known that primary BAs are synthesized from cholesterol in the liver and are later modified to secondary BA species by colonic bacteria. Besides the liver, BA synthesis in extrahepatic sites such as the brain elicits neuroprotective actions through inhibition of apoptosis as well as oxidative and endoplasmic reticulum stress. Even though historically BAs were thought to be only "detergent molecules" required for intestinal absorption of dietary fats, they are nowadays acknowledged as full signaling molecules. They modulate a myriad of signaling pathways with functional consequences on essential processes such as gluconeogenesis -one of the principal energy sources of the fetus- and cellular proliferation. The current manuscript discusses the potential multipotent roles of physiologically circulating BAs on developmental processes during gestation and provides a novel perspective in terms of the importance of the placenta as a previously unknown source of BAs. Since the principle "not too much, not too little" applicable to other signaling molecules may be also true for BAs, the risks associated with fetal exposure to excessive levels of BAs are discussed.

The Placenta-A New Source of Bile Acids during Healthy Pregnancy? First Results of a Gene Expression Study in Humans and Mice.

Bile acids (BAs) are natural ligands for several receptors modulating cell activities. BAs are synthesized via the classic (neutral) and alternative (acidic) pathways. The classic pathway is initiated by CYP7A1/Cyp7a1, converting cholesterol to 7α-hydroxycholesterol, while the alternative pathway starts with hydroxylation of the cholesterol side chain, producing an oxysterol. In addition to originating from the liver, BAs are reported to be synthesized in the brain. We aimed at determining if the placenta potentially represents an extrahepatic source of BAs. Therefore, the mRNAs coding for selected enzymes involved in the hepatic BA synthesis machinery were screened in human term and CD1 mouse late gestation placentas from healthy pregnancies. Additionally, data from murine placenta and brain tissue were compared to determine whether the BA synthetic machinery is comparable in these organs. We found that CYP7A1, CYP46A1, and BAAT mRNAs are lacking in the human placenta, while corresponding homologs were detected in the murine placenta. Conversely, Cyp8b1 and Hsd17b1 mRNAs were undetected in the murine placenta, but these enzymes were found in the human placenta. CYP39A1/Cyp39a1 and cholesterol 25-hydroxylase (CH25H/Ch25h) mRNA expression were detected in the placentas of both species. When comparing murine placentas and brains, Cyp8b1 and Hsd17b1 mRNAs were only detected in the brain. We conclude that BA synthesis-related genes are placentally expressed in a species-specific manner. The potential placentally synthesized BAs could serve as endocrine and autocrine stimuli, which may play a role in fetoplacental growth and adaptation.

Functional reorganization of monoamine transport systems during villous trophoblast differentiation: evidence of distinct differences between primary human trophoblasts and BeWo cells.

BACKGROUND: Three primary monoamines-serotonin, norepinephrine, and dopamine-play major roles in the placenta-fetal brain axis. Analogously to the brain, the placenta has transport mechanisms that actively take up these monoamines into trophoblast cells. These transporters are known to play important roles in the differentiated syncytiotrophoblast layer, but their status and activities in the undifferentiated, progenitor cytotrophoblast cells are not well understood. Thus, we have explored the cellular handling and regulation of monoamine transporters during the phenotypic transitioning of cytotrophoblasts along the villous pathway. METHODS: Experiments were conducted with two cellular models of syncytium development: primary trophoblast cells isolated from the human term placenta (PHT), and the choriocarcinoma-derived BeWo cell line. The gene and protein expression of membrane transporters for serotonin (SERT), norepinephrine (NET), dopamine (DAT), and organic cation transporter 3 (OCT3) was determined by quantitative PCR and Western blot analysis, respectively. Subsequently, the effect of trophoblast differentiation on transporter activity was analyzed by monoamine uptake into cells. RESULTS: We present multiple lines of evidence of changes in the transcriptional and functional regulation of monoamine transporters associated with trophoblast differentiation. These include enhancement of SERT and DAT gene and protein expression in BeWo cells. On the other hand, in PHT cells we report negative modulation of SERT, NET, and OCT3 protein expression. We show that OCT3 is the dominant monoamine transporter in PHT cells, and its main functional impact is on serotonin uptake, while passive transport strongly contributes to norepinephrine and dopamine uptake. Further, we show that a wide range of selective serotonin reuptake inhibitors affect serotonin cellular accumulation, at pharmacologically relevant drug concentrations, via their action on both OCT3 and SERT. Finally, we demonstrate that BeWo cells do not well reflect the molecular mechanisms and properties of healthy human trophoblast cells. CONCLUSIONS: Collectively, our findings provide insights into the regulation of monoamine transport during trophoblast differentiation and present important considerations regarding appropriate in vitro models for studying monoamine regulation in the placenta.

Ex vivo dual perfusion of an isolated human placenta cotyledon: Towards protocol standardization and improved inter-centre comparability.

Since the full development of the ex vivo dual perfusion model of the human placenta cotyledon, the technique has provided essential insight into how nutrients, lipids, gases, immunoglobulins, endocrine agents, pharmaceuticals, chemicals, nanoparticles, micro-organisms and parasites might traverse the maternofetal barrier. Additionally, the model has been instrumental in gaining a better understanding of the regulation of vascular tone, endocrinology and metabolism within this organ. The human placenta is unique amongst species in its anatomy and transfer modalities. This orthologous diversity therefore requires an appropriate consideration of placental transfer rates of compounds, particles and micro-organisms specific to humans. Different research centres have adapted this model with a wide variation in perfusion parameters, including in the establishment of perfusion, perfusate composition, gassing regime, cannulation method, flow rates, perfused tissue mass, and also in the application of quality control measures. The requirement to harmonise and standardise perfusion practice between centres is largely driven by the need to obtain consistency in our understanding of placental function, but also in the qualification of the model for acceptance by regulatory agencies in drug and toxicology testing. A pilot study is proposed, aiming to describe how existing inter-centre variation in perfusion methodology affects placental metabolism, protein synthesis, oxygen consumption, the materno-fetal transfer of key molecular markers, and placental structure.

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.

The Consequences of Assisted Reproduction Technologies on the Offspring Health Throughout Life: A Placental Contribution.

The use of assisted reproductive technologies (ART) worldwide has led to the conception and birth of over eight million babies since being implemented in 1978. ART use is currently on the rise, given growing infertility and the increase in conception age among men and women in industrialized countries. Though obstetric and perinatal outcomes have improved over the years, pregnancies achieved by ART still bear increased risks for the mother and the unborn child. Moreover, given that the first generation of ART offspring is now only reaching their forties, the long-term effects of ART are currently unknown. This is important, as there is a wealth of data showing that life-long health can be predetermined by poor conditions during intrauterine development, including irregularities in the structure and functioning of the placenta. In the current review, we aim to summarize the latest available findings examining the effects of ART on the cardiometabolic, cognitive/neurodevelopmental, and behavioral outcomes in the perinatal period, childhood and adolescence/adulthood; and to examine placental intrinsic factors that may contribute to the developmental outcomes of ART offspring. Altogether, the latest knowledge about life outcomes beyond adolescence for those conceived by ART appears to suggest a better long-term outcome than previously predicted. There are also changes in placenta structure and functional capacity with ART. However, more work in this area is critically required, since the potential consequences of ART may still emerge as the offspring gets older. In addition, knowledge of the placenta may help to foresee and mitigate any adverse outcomes in the offspring.

Materno-fetal iron transfer and the emerging role of ferroptosis pathways.

A successful pregnancy and the birth of a healthy baby depend to a great extent on the controlled supply of essential nutrients via the placenta. Iron is essential for mitochondrial energy supply and oxygen distribution via the blood. However, its high reactivity requires tightly regulated transport processes. Disturbances of maternal-fetal iron transfer during pregnancy can aggravate or lead to severe pathological consequences for the mother and the fetus with lifelong effects. Furthermore, high intracellular iron levels due to disturbed gestational iron homeostasis have recently been associated with the non-apoptotic cell death pathway called ferroptosis. Therefore, the investigation of transplacental iron transport mechanisms, their physiological regulation and potential risks are of high clinical importance. The present review summarizes the current knowledge on principles and regulatory mechanisms underlying materno-fetal iron transport and gives insight into common pregnancy conditions in which iron homeostasis is disturbed. Moreover, the significance of the newly emerging ferroptosis pathway and its impact on the regulation of placental iron homeostasis, oxidative stress and gestational diseases will be discussed.

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.

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.

Glucose Transporter 9 (GLUT9) Plays an Important Role in the Placental Uric Acid Transport System.

BACKGROUND: Hyperuricemia is a common laboratory finding in pregnant women compromised by preeclampsia. A growing body of evidence suggests that uric acid is involved in the pathogenesis of preeclampsia. Glucose transporter 9 (GLUT9) is a high-capacity uric acid transporter. The aim of this study was to investigate the placental uric acid transport system, and to identify the (sub-) cellular localization of GLUT9. METHODS: Specific antibodies against GLUT9a and GLUT9b isoforms were raised, and human villous (placental) tissue was immunohistochemically stained. A systemic GLUT9 knockout (G9KO) mouse model was used to assess the placental uric acid transport capacity by measurements of uric acid serum levels in the fetal and maternal circulation. RESULTS: GLUT9a and GLUT9b co-localized with the villous (apical) membrane, but not with the basal membrane, of the syncytiotrophoblast. Fetal and maternal uric acid serum levels were closely correlated. G9KO fetuses showed substantially higher uric acid serum concentrations than their mothers. CONCLUSIONS: These findings demonstrate that the placenta efficiently maintains uric acid homeostasis, and that GLUT9 plays a key role in the placental uric acid transport system, at least in this murine model. Further studies investigating the role of the placental uric acid transport system in preeclampsia are eagerly needed.

New Insights into the Physiology of Iron Transport: An Interdisciplinary Approach.

The TransCure project entitled 'Iron Transporters DMT1 and FPN1' took an interdisciplinary approach combining structural biology, chemistry and physiology to gain new insights into iron transport. Proteins studied included Divalent Metal Transporter 1 (DMT1, SLC11A2), enabling the import of Fe2+ into the cytoplasm, and the iron efflux transporter Ferroportin (FPN1, SLC40A1). The physiology and pathophysiology, and the mechanisms underlying iron transport in the gut, across the placenta and in bone were investigated. Small molecule high-throughput screening was used to identify improved modulators of DMT1. The characterization of DMT1 inhibitors have provided first detailed insights into the pharmacology of a human iron transport protein. In placental physiology, the identification of the expressional and functional alterations and underlying mechanisms in trophoblast cells clarified the association between placental iron transport by DMT1/FPN1 and gestational diabetes mellitus. In bone, iron metabolism was found to differ between cells of the monocyte/ macrophage lineages, including osteoclasts. Osteoclast development and activity depended on exogenous iron, the expression of high levels of the transferrin receptor (TFR) and low levels of FPN1 suggesting the expression of an "iron storage" phenotype by these cells. The principles and main findings of the TransCure studies on transmembrane iron  transport physiology are summarized in this review.

Structure, Function and Pharmacology of SLC7 Family Members and Homologues.

Amino acids are essential components of all living cells serving as building blocks of proteins, as energy source, and as precursors of metabolites and signaling molecules. Amino acid transporters are membrane proteins that mediate the transfer of amino acids across the plasma membrane, and between compartments in cells, different cells and organs. The absence, overexpression or malfunction of specific amino acid transporters have been associated with human disease. One of the projects within the Swiss National Centre of Competence in Research (NCCR) TransCure was directed at SLC7 family amino acid transporters, with a particular focus on the heteromeric amino acid transporters 4F2hc-LAT1 (SLC3A2-SLC7A5) and 4F2hc-LAT2 (SLC3A2-SLC7A8), and the bacterial homologue AdiC. The project addressed questions of basic research (function and structure), pharmacology (identification of potent inhibitors and activators), and pre-clinical medicine (e.g., physiological role in the placenta) and disease models (e.g., tumor progression) of specific SLC7 family amino acid transporters. This review presents, summarizes and discusses selected main results obtained in this NCCR TransCure project.

Dysregulated Autophagy Leads to Oxidative Stress and Aberrant Expression of ABC Transporters in Women with Early Miscarriage.

Early miscarriage (EMC) is a devastating obstetrical complication. ATP-binding cassette (ABC) transporters mediate cholesterol transfer across the placenta and enhance cell survival by effluxing substrates from target cells in the presence of stressors. Recent evidence reports an intricate interplay between autophagy and ABC transporters. We hypothesized that dysregulated autophagy and oxidative stress (OS) in the placenta leads to abnormal expression of membrane transporters contributing to poor pregnancy survival in EMC. We determined mRNA and protein expression of autophagy genes (Beclin-1/Bcl-2/LC3I/LC3II/p62) and ABC transporters (ABCA1/ABCG1/ABCG2) in placentae from EMC patients (n = 20), term controls (n = 19), first trimester (n = 6), and term controls (n = 5) controls. Oxidative/antioxidant status and biomarkers of oxidative damage were evaluated in maternal serum and placentae from EMC and healthy controls. In EMC, placental expression of LC3II/LC3I as well as of the key autophagy regulatory proteins Beclin-1 and Bcl-2 were reduced, whereas p62 was increased. Both in the serum and placentae of EMC patients, total OS was elevated reflected by increased oxidative damage markers (8-OHdG/malondialdehyde/carbonyl formation) accompanied by diminished levels of total antioxidant status, catalase, and total glutathione. Furthermore, we found reduced ABCG1 and increased ABCG2 expression. These findings suggest that a decreased autophagy status triggers Bcl-2-dependent OS leading to macromolecule damage in EMC placentae. The decreased expression of ABCG1 contributes to reduced cholesterol export to the growing fetus. Increasing ABCG2 expression could represent a protective feedback mechanism under inhibited autophagy conditions. In conclusion, dysregulated autophagy combined with increased oxidative toxicity and aberrant expression of placental ABC transporters affects materno-fetal health in EMC.

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.

Establishment and validation of an approach allowing unequivocal fetal sex determination based on placental sex-specific genes.

The use of human placenta as a matrix for the prediction of the baby's sex has been recently documented, but evaluation methods for placental sex-determining genes allowing reliable sex prediction are still lacking. We compared the accuracy of the retrospective prediction of the baby's sex using placental mRNA expression of RPS4Y1, DDX3Y, and XIST analyzed by an already reported method and a newly developed evaluation approach. Full concordance between the predicted and the actual baby sex was only obtained when analyzing placental RPS4Y1 expression with the newly proposed method, which was found to be robust and reliable.

Counter-directed leucine gradient promotes amino acid transfer across the human placenta.

The developing fetus is highly vulnerable to imbalances in the supply of essential amino acids (AA). Transplacental AA transfer depends on complex interactions between accumulative transporters, exchangers and facilitators, which maintain both intra-extracellular and materno-fetal substrate gradients. We determined physiological AA gradients between maternal and fetal blood and assessed their importance by studying maternal-fetal leucine transfer in human trophoblasts. Maternal-venous and corresponding fetal-arterial/fetal-venous sera were collected from 22 healthy patients at partum. The acquisition of the full AA spectra in serum was performed by ion exchange chromatography. Physiological materno-fetal AA levels were evaluated using paired two-way ANOVA with Tukey's correction. AA concentrations and gradients were tested for associations with anthropometric data by Spearman correlation analysis. Functional effects of a physiological leucine gradient versus equimolar concentrations were tested in BeWo cells using L-[3H]-leucine in conventional and Transwell-based uptake and transfer experiments. The LAT1/SLC7A5-specific inhibitor JPH203 was used to evaluate LAT1-transporter-mediated leucine transport. Maternal AA concentrations correlated with preconceptional and maternal weights at partum. Interestingly, low materno-fetal AA gradients were associated with maternal weight, BMI and gestational weight gain. Leucine uptake was promoted by increased extracellular substrate concentrations. Materno-fetal leucine transfer was significantly increased against a 137µM leucine gradient demonstrating that transplacental leucine transport is stimulated by a counter-directed gradient. Moreover, leucine transfer was inhibited by 10µM JPH203 confirming that Leu transport across the trophoblast monolayer is LAT1-dependent. This study demonstrates a currently underestimated effect of transplacental AA gradients on efficient leucine transfer which could severely affect fetal development.

Ex vivo dual perfusion of an isolated cotyledon of human placenta: History and future challenges.

This is an introductory chapter for the Special Issue: Ex vivo dual perfusion of an isolated cotyledon of the human placenta. It covers the early stages of development of this technique in the laboratory of Professor Maurice Panigel. It was successfully further developed by Henning Schneider during his stay in Paris as a research assistant. Together with Professor Joseph Dancis in New York they managed to overcome initial problems of acceptance. Its value was finally confirmed by studies performed by several groups in different parts of the world and it became a valuable additional tool for research on the human placenta.