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.

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.

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.

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.

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.

Small molecule inhibitors provide insights into the relevance of LAT1 and LAT2 in materno-foetal amino acid transport.

The placenta supplies the foetus with critical nutrients such as essential amino acids (AA, eg leucine) for development and growth. It also represents a cellular barrier which is formed by a polarized, differentiated syncytiotrophoblast (STB) monolayer. Active Na+ -independent leucine transport across the placenta is mainly attributed to the System L transporters LAT1/SLC7A5 and LAT2/SLC7A8. This study explored the influence of trophoblast differentiation on the activity of LAT1/LAT2 and the relevance of LAT1/LAT2 in leucine uptake and transfer in trophoblasts by applying specific small molecule inhibitors (JPH203/JG336/JX009). L-leucine uptake (total dose = 167 µmol/L) was sensitive to LAT1-specific inhibition by JPH203 (EC50  = 2.55 µmol/L). The inhibition efficiency of JPH203 was increased by an additional methoxy group in the JPH203-derivate JG336 (EC50  = 1.99 µmol/L). Interestingly, JX009 showed efficient System L inhibition (EC50  = 2.35 µmol/L) and was the most potent inhibitor of leucine uptake in trophoblasts. The application of JPH203 and JX009 in Transwell® -based leucine transfer revealed LAT1 as the major accumulative transporter at the apical membrane, but other System L transporters such as LAT2 as rate-limiting for leucine efflux across the basal membrane. Therefore, differential specificity of the applied inhibitors allowed for estimation of the contribution of LAT1 and LAT2 in materno-foetal AA transfer and their potential impact in pregnancy diseases associated with impaired foetal growth.

Dynamics of Tryptophan Metabolic Pathways in Human Placenta and Placental-Derived Cells: Effect of Gestation Age and Trophoblast Differentiation.

L-Tryptophan is an essential amino acid and a precursor of several physiologically active metabolites. In the placenta, the serotonin and kynurenine metabolic pathways of tryptophan metabolism have been identified, giving rise to various molecules of neuroactive or immunoprotective properties, such as serotonin, melatonin, kynurenine, kynurenic acid, or quinolinic acid. Current literature suggests that optimal levels of these molecules in the fetoplacental unit are crucial for proper placenta functions, fetal development and programming. Placenta is a unique endocrine organ that, being equipped with a battery of biotransformation enzymes and transporters, precisely orchestrates homeostasis of tryptophan metabolic pathways. However, because pregnancy is a dynamic process and placental/fetal needs are continuously changing throughout gestation, placenta must adapt to these changes and ensure proper communication in the feto-placental unit. Therefore, in this study we investigated alterations of placental tryptophan metabolic pathways throughout gestation. Quantitative polymerase chain reaction (PCR) analysis of 21 selected genes was carried out in first trimester (n = 13) and term (n = 32) placentas. Heatmap analysis with hierarchical clustering revealed differential gene expression of serotonin and kynurenine pathways across gestation. Subsequently, digital droplet PCR, Western blot, and functional analyses of the rate-limiting enzymes suggest preferential serotonin synthesis early in pregnancy with a switch to kynurenine production toward term. Correspondingly, increased function and/or protein expression of serotonin degrading enzyme and transporters at term indicates efficient placental uptake and metabolic degradation of serotonin. Lastly, gene expression analysis in choriocarcinoma-derived cell lines (BeWo, BeWo b30, JEG-3) revealed dissimilar expression patterns and divergent effect of syncytialization compared to primary trophoblast cells isolated from human term placentas; these findings show that the commonly used in vitro placental models are not suitable to study placental handling of tryptophan. Altogether, our data provide the first comprehensive evidence of changes in placental homeostasis of tryptophan and its metabolites as a function of gestational age, which is critical for proper placental function and fetal development.

Gestational diabetes mellitus affects placental iron homeostasis: Mechanism and clinical implications.

Clinical studies suggest that pregnant women with elevated iron levels are more vulnerable to develop gestational diabetes mellitus (GDM), but the causes and underlying mechanisms are unknown. We hypothesized that hyperglycemia induces cellular stress responses leading to dysregulated placental iron homeostasis. Hence, we compared the expression of genes/proteins involved in iron homeostasis in placentae from GDM and healthy pregnancies (n = 11 each). RT-qPCR and LC-MS/MS analyses revealed differential regulation of iron transporters/receptors (DMT1/FPN1/ZIP8/TfR1), iron sensors (IRP1), iron regulators (HEPC), and iron oxidoreductases (HEPH/Zp). To identify the underlying mechanisms, we adapted BeWo trophoblast cells to normoglycemic (N), hyperglycemic (H), and hyperglycemic-hyperlipidemic (HL) conditions and assessed Fe3+ -uptake, expression patterns, and cellular pathways involving oxidative stress (OS), ER-stress, and autophagy. H and HL induced alterations in cellular morphology, differential iron transporter expression, and reduced Fe3+ -uptake confirming the impact of hyperglycemia on iron transport observed in GDM patients. Pathway analysis and rescue experiments indicated that dysregulated OS and disturbed autophagy processes contribute to the reduced placental iron transport under hyperglycemic conditions. These adaptations could represent a protective mechanism preventing the oxidative damage for both fetus and placenta caused by highly oxidative iron. In pregnancies with risk for GDM, antioxidant treatment, and controlled iron supplementation could help to balance placental OS levels protecting mother and fetus from impaired iron homeostasis.

Placental miR-340 mediates vulnerability to activity based anorexia in mice.

Anorexia nervosa (AN) is a devastating eating disorder characterized by self-starvation that mainly affects women. Its etiology is unknown, which impedes successful treatment options leading to a limited chance of full recovery. Here, we show that gestation is a vulnerable window that can influence the predisposition to AN. By screening placental microRNA expression of naive and prenatally stressed (PNS) fetuses and assessing vulnerability to activity-based anorexia (ABA), we identify miR-340 as a sexually dimorphic regulator involved in prenatal programming of ABA. PNS caused gene-body hypermethylation of placental miR-340, which is associated with reduced miR-340 expression and increased protein levels of several target transcripts, GR, Cry2 and H3F3b. MiR-340 is linked to the expression of several nutrient transporters both in mice and human placentas. Using placenta-specific lentiviral transgenes and embryo transfer, we demonstrate the key role miR-340 plays in the mechanism involved in early life programming of ABA.

Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes.

CRISPR-Cas9 enables efficient sequence-specific mutagenesis for creating somatic or germline mutants of model organisms. Key constraints in vivo remain the expression and delivery of active Cas9-sgRNA ribonucleoprotein complexes (RNPs) with minimal toxicity, variable mutagenesis efficiencies depending on targeting sequence, and high mutation mosaicism. Here, we apply in vitro assembled, fluorescent Cas9-sgRNA RNPs in solubilizing salt solution to achieve maximal mutagenesis efficiency in zebrafish embryos. MiSeq-based sequence analysis of targeted loci in individual embryos using CrispRVariants, a customized software tool for mutagenesis quantification and visualization, reveals efficient bi-allelic mutagenesis that reaches saturation at several tested gene loci. Such virtually complete mutagenesis exposes loss-of-function phenotypes for candidate genes in somatic mutant embryos for subsequent generation of stable germline mutants. We further show that targeting of non-coding elements in gene regulatory regions using saturating mutagenesis uncovers functional control elements in transgenic reporters and endogenous genes in injected embryos. Our results establish that optimally solubilized, in vitro assembled fluorescent Cas9-sgRNA RNPs provide a reproducible reagent for direct and scalable loss-of-function studies and applications beyond zebrafish experiments that require maximal DNA cutting efficiency in vivo.