Ultrastructural cilia defects in multi-ciliated uterine glandular epithelial cells from women with reproductive failure.

In brief: The causes of subfertility and recurrent pregnancy loss are often unclear. This study shows that endometrial gland cilia from women with subfertility have ultrastructural defects. Abstract: Endometrial glands secrete products into the endometrium and are necessary for embryo implantation and successful pregnancy. However, structural and functional abnormalities in endometrial gland cilia from women with reproductive failure remain poorly understood. This was a cross-sectional study where endometrial biopsies were collected at days 19-23 of the menstrual cycle from women with unexplained recurrent pregnancy loss (n = 15), unexplained subfertility (n = 11) or from egg donor control participants (n = 10). Endometrial gland cilia ultrastructure was imaged by transmission electron microscopy and cilia defects assessed by an electron-microscopist from a national primary ciliary dyskinesia diagnostic centre. Endometrial glands were isolated, and the cilia beat frequency recorded by high speed video. Subfertile women have proportionately lower ultrastructurally normal cilia (P < 0.05); higher frequency of absent dynamin arms (P < 0.01) or inner arm defects (P < 0.01) and lower cilia beat frequency (P < 0.05). The mechanisms underlying these obversions have yet to be determined. Recent studies have identified cilia related gene expression changes associated with reproductive failure and this study adds to the growing body of literature revealing structural and functional changes. The observation that cilia defects occurred at a higher frequency in endometrial glands of subfertile women raises the question of its mechanistic role in implantation.

Serial block-face scanning electron microscopy reveals novel intercellular connections in human term placental microvasculature.

The placental microvasculature is a conduit for fetal blood allowing solute exchange between the mother and the fetus. Serial block-face scanning electron microscopy (SBF SEM) allows ultrastructure to be viewed in three dimensions and provides a new perspective on placental anatomy. This study used SBF SEM to study endothelial cells within the human placental microvasculature from uncomplicated pregnancies. Term human placental villi were aldehyde-fixed and processed for imaging by SBF SEM. Manual segmentation was carried out on a terminal villous capillary and an intermediate villous arteriole and venule. Twenty-seven SBF SEM stacks from terminal villi were analysed using stereological approaches to determine the volumes of microvascular components and the proportions of pericyte coverage. SBF SEM analysis of capillary endothelial cells revealed the presence of interendothelial protrusions (IEPs) originating from the donor cell at the endothelial junction and forming deep thin projections up to 7 µm into the adjacent endothelial cells. IEP density was estimated to be in the order of 35 million cm-3 placental tissue. Pericytes cover 15% of the fetal capillary surface area in terminal villi. In comparison, the cytotrophoblast covered 24% of the syncytiotrophoblast basal membrane. A trans-endothelial channel was observed in a region of the vasculo-syncytial capillary. Pericyte coverage was extensive in both arteriole and venule. Three-dimensional imaging of the placental microvasculature identified novel ultrastructural features and provided an insight into factors that may influence capillary permeability and placental function. We hypothesise that the IEPs may allow mechanosensing between adjacent endothelial cells to assist in the maintenance of vessel integrity. The numbers of endothelial junctions, the presence of trans-endothelial channels and the extent of pericyte coverage all provide an insight into the factors determining capillary permeability.

Ursodeoxycholic acid inhibits uptake and vasoconstrictor effects of taurocholate in human placenta.

Intrahepatic cholestasis of pregnancy (ICP) causes increased transfer of maternal bile acids to the fetus and an increased incidence of sudden fetal death. Treatment includes ursodeoxycholic acid (UDCA), but it is not clear if UDCA protects the fetus. This study explores the placental transport of the bile acid taurocholate (TC) by the organic anion-transporting polypeptide, (OATP)4A1, its effects on the placental proteome and vascular function, and how these are modified by UDCA. Various methodological approaches including placental villous fragments and Xenopus laevis oocytes were used to investigate UDCA transport. Placental perfusions and myography investigated the effect of TC on vasculature. The effects of acute TC exposure on placental tissue were investigated using quantitative proteomics. UDCA inhibited OATP4A1 activity in placental villous fragments and oocytes. TC induced vasoconstriction in placental and rat vasculature, which was attenuated by UDCA. Quantitative proteomic analysis of villous fragments showed direct effects of TC on multiple placental pathways, including oxidative stress and autophagy. The effects of TC on the placental proteome and vasculature demonstrate how bile acids may cause fetal distress in ICP. UDCA inhibition of OATP4A1 suggests it will protect the mother and fetus against the vascular effects of TC by inhibiting its cellular uptake. UDCA may protect the fetus in ICP by inhibiting OATP4A1-mediated bile acid transfer and TC-induced placental vasoconstriction. Understanding the physiologic mechanisms of UDCA may allow better therapeutic interventions to be designed specifically for the fetus in the future.-Lofthouse, E. M., Torrens, C., Manousopoulou, A., Nahar, M., Cleal, J. K., O'Kelly, I. M., Sengers, B. G., Garbis, S. D., Lewis, R. M. Ursodeoxycholic acid inhibits uptake and vasoconstrictor effects of taurocholate in human placenta.

Maternal Obesity during Pregnancy Alters Daily Activity and Feeding Cycles, and Hypothalamic Clock Gene Expression in Adult Male Mouse Offspring.

An obesogenic diet adversely affects the endogenous mammalian circadian clock, altering daily activity and metabolism, and resulting in obesity. We investigated whether an obese pregnancy can alter the molecular clock in the offspring hypothalamus, resulting in changes to their activity and feeding rhythms. Female mice were fed a control (C, 7% kcal fat) or high fat diet (HF, 45% kcal fat) before mating and throughout pregnancy. Male offspring were fed the C or HF diet postweaning, resulting in four offspring groups: C/C, C/HF, HF/C, and HF/HF. Daily activity and food intake were monitored, and at 15 weeks of age were killed at six time-points over 24 h. The clock genes Clock, Bmal1, Per2, and Cry2 in the suprachiasmatic nucleus (SCN) and appetite genes Npy and Pomc in the arcuate nucleus (ARC) were measured. Daily activity and feeding cycles in the HF/C, C/HF, and HF/HF offspring were altered, with increased feeding bouts and activity during the day and increased food intake but reduced activity at night. Gene expression patterns and levels of Clock, Bmal1, Per2, and Cry2 in the SCN and Npy and Pomc in the ARC were altered in HF diet-exposed offspring. The altered expression of hypothalamic molecular clock components and appetite genes, together with changes in activity and feeding rhythms, could be contributing to offspring obesity.

A systems perspective on placental amino acid transport.

Placental amino acid transfer is a complex process that is essential for fetal development. Impaired amino acid transfer causes fetal growth restriction, which may have lifelong health consequences. Transepithelial transfer of amino acids across the placental syncytiotrophoblast requires accumulative, exchange and facilitated transporters on the apical and basal membranes to work in concert. However, transporters alone do not determine amino acid transfer and factors that affect substrate availability, such as blood flow and metabolism, may also become rate-limiting for transfer. In order to determine the rate-limiting processes, it is necessary to take a systems approach which recognises the interdependence of these processes. New technologies have the potential to deliver targeted interventions to the placenta and help poorly growing fetuses. While many factors are necessary for amino acid transfer, novel therapies need to target the rate-limiting factors if they are going to be effective. This review will outline the factors which determine amino acid transfer and describe how they become interdependent. It will also highlight the role of computational modelling as a tool to understand this process.

Estrone sulphate uptake by the microvillous membrane of placental syncytiotrophoblast is coupled to glutamate efflux.

Organic anion transporters (OATs) and organic anion transporting polypeptides (OATPs) are transport proteins that mediate exchange of metabolites, hormones and waste products. Directional transport by these transporters can occur when exchange is coupled to the gradients of other substrates. This study investigates whether the activity of OATP4A1 and OATP2A1 on the maternal facing microvillus membrane of the placental syncytiotrophoblast is coupled to the glutamate gradient. OAT and OATP transporter proteins were over expressed in Xenopus oocytes to study their transport characteristics. Further transport studies were performed in term human placental villous fragments. Xenopus oocytes expressing OATP4A1 mediated glutamate uptake. No glutamate transport was observed in oocytes expressing OAT1, OAT3, OAT7 or OATP2A1. In oocytes expressing OATP4A1, uptake of estrone sulphate, thyroid hormones T3 and T4 and the bile acid taurocholate stimulated glutamate efflux. In term placental villous fragments addition of estrone sulphate and taurocholate trans-stimulated glutamate efflux. Coupling of OATP4A1 to the glutamate gradient may drive placental uptake of estrone-sulphate and thyroid hormone while also facilitating uptake of potentially harmful bile acids. In contrast, if OATP2A1 is not coupled to a similar gradient, it may function more effectively as an efflux transporter, potentially mediating efflux of prostaglandins to the mother. This study provides further evidence for glutamate as an important counter-ion driving transport into the placenta.

Serial block-face scanning electron microscopy of erythrocytes protruding through the human placental syncytiotrophoblast.

The syncytiotrophoblast forms a continuous barrier between the maternal and fetal circulations. Here we present a serial block-face scanning electron microscopy (SBFSEM) study, based on a single image stack, showing pooling of fetal blood underneath a region of stretched syncytiotrophoblast that has become detached from the basement membrane. Erythrocytes are protruding from discrete holes in the syncytiotrophoblast suggesting that, under specific circumstances, the syncytiotrophoblast may be permeable to fetal cells. This observation represents a pathological process but it poses questions about the physical properties and permeability of the syncytiotrophoblast and may represent an early stage in the formation of fibrin deposits in areas of syncytial denudation. This study also illustrates how the 3D images generated by SBFSEM allow the interpretation of structures that could not be understood from a single histological section.

Glutamate cycling may drive organic anion transport on the basal membrane of human placental syncytiotrophoblast.

The organic anion transporter OAT4 (SLC22A11) and organic anion transporting polypeptide OATP2B1 (SLCO2B1) are expressed in the basal membrane of the placental syncytiotrophoblast. These transporters mediate exchange whereby uptake of one organic anion is coupled to efflux of a counter-ion. In placenta, these exchangers mediate placental uptake of substrates for oestrogen synthesis as well as clearing waste products and xenobiotics from the fetal circulation. However, the identity of the counter-ion driving this transport in the placenta, and in other tissues, is unclear. While glutamate is not a known OAT4 or OATP2B1 substrate, we propose that its high intracellular concentration has the potential to drive accumulation of substrates from the fetal circulation. In the isolated perfused placenta, glutamate exchange was observed between the placenta and the fetal circulation. This exchange could not be explained by known glutamate exchangers. However, glutamate efflux was trans-stimulated by an OAT4 and OATP2B1 substrate (bromosulphothalein). Exchange of glutamate for bromosulphothalein was only observed when glutamate reuptake was inhibited (by addition of aspartate). To determine if OAT4 and/or OATP2B1 mediate glutamate exchange, uptake and efflux of glutamate were investigated in Xenopus laevis oocytes. Our data demonstrate that in Xenopus oocytes expressing either OAT4 or OATP2B1 efflux of intracellular [(14)C]glutamate could be stimulated by conditions including extracellular glutamate (OAT4), estrone-sulphate and bromosulphothalein (both OAT4 and OATP2B1) or pravastatin (OATP2B1). Cycling of glutamate across the placenta involving efflux via OAT4 and OATP2B1 and subsequent reuptake will drive placental uptake of organic anions from the fetal circulation.

Quantitative microCT imaging of a whole equine placenta and its blood vessel network.

Placental structure is linked to function across morphological scales. In the placenta, changes to gross anatomy, such as surface area, volume, or blood vessel arrangement, are associated with suboptimal physiological outcomes. However, quantifying each of these metrics requires different laborious semi-quantitative methods. Here, we demonstrate how, with minimal sample preparation, whole-organ computed microtomography (microCT) can be used to calculate gross morphometry of the equine placenta and a range of additional metrics, including branching morphometry of placental vasculature, non-destructively from a single dataset. Our approach can be applied to quantify the gross structure of any large mammalian placenta.

Gastroschisis associated changes in the placental transcriptome.

The congenital condition gastroschisis is associated with delayed villous development and placental malperfusion, suggesting placental involvement. This study uses RNA sequencing to compare the placental transcriptome in pregnancies with and without gastroschisis. 180 coding genes were differentially expressed, mapping to multiple gene ontology pathways. Altered placental gene expression may represent fetal signalling to the placenta, and these changes could contribute to the pathogenesis of gastroschisis and associated morbidities, including fetal growth restriction.

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.

Regulation of human trophoblast gene expression by endogenous retroviruses.

The placenta is a fast-evolving organ with large morphological and histological differences across eutherians, but the genetic changes driving placental evolution have not been fully elucidated. Transposable elements, through their capacity to quickly generate genetic variation and affect host gene regulation, may have helped to define species-specific trophoblast gene expression programs. Here we assess the contribution of transposable elements to human trophoblast gene expression as enhancers or promoters. Using epigenomic data from primary human trophoblast and trophoblast stem-cell lines, we identified multiple endogenous retrovirus families with regulatory potential that lie close to genes with preferential expression in trophoblast. These largely primate-specific elements are associated with inter-species gene expression differences and are bound by transcription factors with key roles in placental development. Using genetic editing, we demonstrate that several elements act as transcriptional enhancers of important placental genes, such as CSF1R and PSG5. We also identify an LTR10A element that regulates ENG expression, affecting secretion of soluble endoglin, with potential implications for preeclampsia. Our data show that transposons have made important contributions to human trophoblast gene regulation, and suggest that their activity may affect pregnancy outcomes.

The placental exposome, placental epigenetic adaptations and lifelong cardio-metabolic health.

The placental exposome represents the sum of all placental exposures, and through its influence on placental function can affect an individual's susceptibility to cardio-metabolic disease later in life. The placental exposome includes direct exposures during gestation, as well as those prior to gestation that affect the gametes or aspects of maternal physiology that influence placental function. This review will discuss the evidence for placental responses to environmental signals and its involvement in programming offspring health. A wide range of exposures may influence the placenta including maternal metabolic and endocrine status, nutrition, stress and toxins. Epigenetic changes within the placenta induced by these exposures may mediate persistent effects on placental function. Identifying which exposures are most influential in terms of placental function and offspring health is key to focusing future research and developing stratified and personalised interventions.

Endometrial gland specific progestagen-associated endometrial protein and cilia gene splicing changes in recurrent pregnancy loss.

Endometrial glands are essential for fertility, consisting of ciliated and secretory cells that facilitate a suitable uterine environment for embryo implantation. This study sought to determine whether an endometrial gland specific transcriptome and splicing profile are altered in women with recurrent pregnancy loss. Our data provide a comprehensive catalogue of cilia and PAEP gene isoforms and relative exon usage in endometrial glands. We report a previously unannotated endometrial gland cilia transcript GALNT11 and its susceptibility to exon skipping. Key endometrial receptivity gene transcripts are also reported to change in endometrial glands of women with recurrent pregnancy loss. The endometrial gland cilia and PAEP targets identified in this study could be used to identify a perturbed endometrium, isolate causes of recurrent pregnancy loss and develop targeted therapies in personalised medicine.

Microvillous tip vesicles may be an origin of placental extracellular vesicles.

INTRODUCTION: Extracellular vesicles are now believed to be important mediators of placental-maternal communication. However, little is known about the formation of extracellular vesicles by human placenta. This study uses nanoscale three-dimensional imaging to investigate how and where placental extracellular vesicles form. METHODS: Term and first trimester human placental villi were imaged by serial block face scanning electron microscopy. These images were analysed to quantify vesicle surface density. Segmentation was performed to reconstruct three-dimensional images of extracellular vesicles. Live imaging light microscopy of first trimester villous explants was performed. RESULTS: Vesicles were observed on the tips of placental microvilli in term and first trimester placenta. In term placenta these microvillous tip vesicles had a median size of 0.55 µm and their surface area density exceeded 22000 per mm2. Microvillous tip vesicle membranes had a lower electron density than the microvillous plasma membrane. Thirty seven percent of vesicles had a complex membrane structure including double membranes, internal vesicles and vesicle chains. Budding of smaller secondary vesicles from microvillous tip vesicle membranes was observed. Live imaging of a first trimester villus explant observed formation of vesicles which were larger but visually similar to the secondary vesicles observed by electron microscopy. DISCUSSION: These observations suggest that extracellular vesicles are forming on the tips of placental microvilli prior to release into maternal blood. However, it cannot be discounted that there are maternal extracellular vesicles that have bound to microvilli. In either case, the high surface area density of microvillous tip vesicles is consistent with an important role in placental-maternal signalling.

3D visualization of trans-syncytial nanopores provides a pathway for paracellular diffusion across the human placental syncytiotrophoblast.

The placental syncytiotrophoblast, a syncytium without cell-cell junctions, is the primary barrier between the mother and the fetus. Despite no apparent anatomical pathway for paracellular diffusion of solutes across the syncytiotrophoblast, size-dependent paracellular diffusion is observed. Here we report data demonstrating that the syncytiotrophoblast is punctuated by trans-syncytial nanopores (TSNs). These membrane-bound TSNs directly connect the maternal and fetal facing sides of the syncytiotrophoblast, providing a pathway for paracellular diffusion between the mother and fetus. Mathematical modeling of TSN permeability based on their 3D geometry suggests that 10-37 million TSNs per cm3 of placental tissue could explain experimentally observed placental paracellular diffusion. TSNs may mediate physiological hydrostatic and osmotic pressure homeostasis between the maternal and fetal circulations but also expose the fetus to pharmaceuticals, environmental pollutants, and nanoparticles.

Maternal and Fetal Genetic Variation in Vitamin D Metabolism and Umbilical Cord Blood 25-Hydroxyvitamin D.

CONTEXT: Single nucleotide polymorphisms (SNPs) in vitamin D metabolism pathway genes are associated with circulating 25-hydroxyvitamin D (25(OH)D) in adults. Less is known about the relationships between mother and offspring SNPs and umbilical cord blood 25(OH)D. OBJECTIVE: (1) To undertake a meta-analysis of the relationships of maternal and offspring SNPs in the vitamin D metabolism pathway and cord blood 25(OH)D in pregnant women including novel data; and (2) to examine these relationships in women who received antenatal cholecalciferol supplementation in a clinical trial. METHODS: Novel data analysis from an observational mother-offspring cohort study (Southampton Women's Survey) and the MAVIDOS double-blind, randomized, placebo-controlled trial of 1000 IU/day cholecalciferol supplementation in pregnancy, and an electronic literature search of published studies in PubMed up to 31 July 2021. Studies reporting associations between rs12785878 (DHCR7), rs10741657 (CYP2R1), rs6013897 (CYP24A1), or rs2282679 (GC) and cord blood 25(OH)D. One published study was included in addition to the novel data analysis. Associations between both maternal and offspring SNPs at rs2282679 (GC) and rs12785878 (DHCR7), and cord blood 25(OH)D were identified. When maternal genotype was adjusted for offspring genotype, and vice versa, there was persisting evidence for associations with maternal rs12785878 (ß [95% CI] 1.6 nmol/L [0.3, 2.8] per common allele), and offspring rs2282679 (ß 3.1 nmol/L ]2.0, 4.4] per common allele). Maternal and offspring SNPs at rs1074657 and rs613897 were not associated with cord blood 25(OH)D. RESULT: Associations between both maternal and offspring SNPs at rs2282679 (GC) and rs12785878 (DHCR7), and cord blood 25(OH)D were identified. When maternal genotype was adjusted for offspring genotype, and vice versa, there was persisting evidence for associations with maternal rs12785878 (ß [95% CI] 1.6 nmol/L [0.3, 2.8] per common allele), and offspring rs2282679 (ß 3.1 nmol/L ]2.0, 4.4] per common allele). Maternal and offspring SNPs at rs1074657 and rs613897 were not associated with cord blood 25(OH)D. CONCLUSION: Common genetic variation in the vitamin D metabolism pathway is associated with umbilical cord blood 25(OH)D.

Folding of the syncytiotrophoblast basal plasma membrane increases the surface area available for exchange in human placenta.

INTRODUCTION: The placental syncytiotrophoblast is the primary barrier between the mother and the fetus. To cross the placenta, nutrients and wastes must be transported across the apical microvillous and basal plasma membranes. While the syncytiotrophoblast basal plasma membrane is typically represented as relatively smooth, it has been shown to have invaginations that may increase its surface area. This study aimed to quantify how folding of the syncytiotrophoblast basal membrane contributes to its surface area and to visualise three-dimensional structures of the basal membrane and cytotrophoblast cell structures. METHODS: Transmission electron microscope images of human term placenta were analysed using stereological approaches to quantify how folding of the syncytiotrophoblast basal plasma membrane affected surface area. Serial block-face scanning electron microscopy was used to visualise the three-dimensional structure of the syncytiotrophoblast basal membrane and cytotrophoblast cells. RESULTS: Syncytiotrophoblast basal membrane covered 69.1% of the basal lamina, with cytotrophoblast cells covering the remaining 30.9%. In basal lamina adjacent to syncytiotrophoblast, 34% was adjacent to smooth basal membrane and 66% to folded basal membrane. Syncytiotrophoblast basal membrane folds increased the surface area adjacent to basal lamina by 305%. Including regions overlying the cytotrophoblast cells, basal membrane folds increased syncytiotrophoblast basal membrane surface area by 4.4-fold relative to the basal lamina in terminal villi. Terminal and intermediate villi were similar in terms of trophoblast coverage of the basal lamina and basal membrane folding. The three-dimensional structures of the syncytiotrophoblast basal plasma membrane and cytotrophoblast cells were generated from serial block-face scanning electron microscopy image stacks. DISCUSSION: These findings indicate that the surface area of the syncytiotrophoblast basal plasma membrane is far larger than had been appreciated. We suggest that these folds increase the surface area available for transport to and from the fetus. Changes in the extent of basal membrane folding could affect nutrient transfer capacity and underlie pathological fetal growth, including fetal growth restriction and macrosomia.

Placental uptake and metabolism of 25(OH)vitamin D determine its activity within the fetoplacental unit.

Pregnancy 25-hydroxyvitamin D [25(OH)D] concentrations are associated with maternal and fetal health outcomes. Using physiological human placental perfusion and villous explants, we investigate the role of the placenta in regulating the relationships between maternal 25(OH)D and fetal physiology. We demonstrate active placental uptake of 25(OH)D3 by endocytosis, placental metabolism of 25(OH)D3 into 24,25-dihydroxyvitamin D3 and active 1,25-dihydroxyvitamin D [1,25(OH)2D3], with subsequent release of these metabolites into both the maternal and fetal circulations. Active placental transport of 25(OH)D3 and synthesis of 1,25(OH)2D3 demonstrate that fetal supply is dependent on placental function rather than simply the availability of maternal 25(OH)D3. We demonstrate that 25(OH)D3 exposure induces rapid effects on the placental transcriptome and proteome. These map to multiple pathways central to placental function and thereby fetal development, independent of vitamin D transfer. Our data suggest that the underlying epigenetic landscape helps dictate the transcriptional response to vitamin D treatment. This is the first quantitative study demonstrating vitamin D transfer and metabolism by the human placenta, with widespread effects on the placenta itself. These data demonstrate a complex interplay between vitamin D and the placenta and will inform future interventions using vitamin D to support fetal development and maternal adaptations to pregnancy.

N-acetylcysteine, xCT and suppression of Maxi-chloride channel activity in human placenta.

INTRODUCTION: Placental oxidative stress features in pregnancy pathologies but in clinical trials antioxidant supplementation has not improved outcomes. N-acetylcysteine (NAC) stimulates glutathione production and is proposed as a therapeutic agent in pregnancy. However, key elements of N-acetylcysteine biology, including its cellular uptake mechanism, remains unclear. This study explores how the cystine/glutamate transporter xCT may mediate N-acetylcysteine uptake and how N-acetylcysteine alters placental redox status. METHODS: The involvement of xCT in NAC uptake by the human placenta was studied in perfused placenta and Xenopus oocytes. The effect of short-term N-acetylcysteine exposure on the placental villous proteome was determined using LC-MS. The effect of N-acetylcysteine on Maxi-chloride channel activity was investigated in perfused placenta, villous fragments and cell culture. RESULTS: Maternoplacental N-acetylcysteine administration stimulated intracellular glutamate efflux suggesting a role of the exchange transporter xCT, which was localised to the microvillous membrane of the placental syncytiotrophoblast. Placental exposure to a bolus of N-acetylcysteine inhibited subsequent activation of the redox sensitive Maxi-chloride channel independently of glutathione synthesis. Stable isotope quantitative proteomics of placental villi treated with N-acetylcysteine demonstrated changes in pathways associated with oxidative stress, apoptosis and the acute phase response. DISCUSSION: This study suggests that xCT mediates N-acetylcysteine uptake into the placenta and that N-acetylcysteine treatment of placental tissue alters the placental proteome while regulating the redox sensitive Maxi-chloride channel. Interestingly N-acetylcysteine had antioxidant effects independent of the glutathione pathway. Effective placental antioxidant therapy in pregnancy may require maintaining the balance between normalising redox status without inhibiting physiological redox signalling.