Plasma S1P and Sphingosine are not Different Prior to Pre-Eclampsia in Women at High Risk of Developing the Disease.

Sphingolipids like sphingosine-1-phosphate (S1P) have been implicated in the pathophysiology of pre-eclampsia. We hypothesized that plasma S1P would be increased in women at high risk of developing pre-eclampsia who subsequently develop the disease. Low circulating placental growth factor (PlGF) is known to be associated with development of pre-eclampsia; so further, we hypothesized that increased S1P would be associated with concurrently low PlGF. This was a case-control study using stored maternal blood samples from 14 to 24 weeks of pregnancy, collected from 95 women at increased risk of pre-eclampsia. Pregnancy outcome was classified as uncomplicated, preterm pre-eclampsia (<37 weeks), or term pre-eclampsia. Plasma lipids were extracted and analyzed by ultraperformance liquid chromatography coupled to electrospray ionization MS/MS to determine concentrations of S1P and sphingosine. Median plasma S1P was 0.339 nmol/ml, and median sphingosine was 6.77 nmol/l. There were no differences in the plasma concentrations of S1P or sphingosine in women who subsequently developed pre-eclampsia, no effect of gestational age, fetal sex, ethnicity, or the presence of pre-existing hypertension. There was a correlation between S1P and sphingosine plasma concentration (P < 0.0001). There was no relationship between S1P or sphingosine with PlGF. Previous studies have suggested that plasma S1P may be a biomarker of pre-eclampsia. In our larger study, we failed to demonstrate there are women at high risk of developing the disease. We did not show a relationship with known biomarkers of the disease, suggesting that S1P is unlikely to be a useful predictor of the development of pre-eclampsia later in pregnancy.

Watermelon-Derived Extracellular Vesicles Influence Human Ex Vivo Placental Cell Behavior by Altering Intestinal Secretions.

SCOPE: During pregnancy, mother-to-fetus transfer of nutrients is mediated by the placenta; sub-optimal placental development and/or function results in fetal growth restriction (FGR), and the attendant risk of stillbirth, neurodevelopmental delay, and non-communicable diseases in adulthood. A maternal diet high in fruit and vegetables lowers the risk of FGR but the association cannot be explained fully by known macro- and micronutrients. METHODS AND RESULTS: This study investigates if dietary-derived extracellular vesicles (EVs) can regulate placental function. The study characterizes the microRNA and protein cargo of EVs isolated from watermelon, show they are actively internalized by human intestinal epithelial cells in vitro, use mass spectrometry to demonstrate that they alter the intestinal secretome and bioinformatic analyses to predict the likely affected pathways in cells/tissues distal to gut. Application of the watermelon EV-modified intestinal secretome to human placental trophoblast cells and ex vivo tissue explants affects the trophoblast proteome and key aspects of trophoblast behavior, including migration and syncytialization. CONCLUSION: Dietary-derived plant EVs can modify intestinal communication with distal tissues, including the placenta. Harnessing the beneficial properties of dietary-derived plant EVs and/or exploiting their potential as natural delivery agents may provide new ways to improve placental function and reduce rates of FGR.

Trophectoderm differentiation to invasive syncytiotrophoblast is promoted by endometrial epithelial cells during human embryo implantation.

STUDY QUESTION: How does the human embryo breach the endometrial epithelium at implantation? SUMMARY ANSWER: Embryo attachment to the endometrial epithelium promotes the formation of multinuclear syncytiotrophoblast from trophectoderm, which goes on to breach the epithelial layer. WHAT IS KNOWN ALREADY: A significant proportion of natural conceptions and assisted reproduction treatments fail due to unsuccessful implantation. The trophectoderm lineage of the embryo attaches to the endometrial epithelium before breaching this barrier to implant into the endometrium. Trophectoderm-derived syncytiotrophoblast has been observed in recent in vitro cultures of peri-implantation embryos, and historical histology has shown invasive syncytiotrophoblast in embryos that have invaded beyond the epithelium, but the cell type mediating invasion of the epithelial layer at implantation is unknown. STUDY DESIGN, SIZE, DURATION: Fresh and frozen human blastocyst-stage embryos (n = 46) or human trophoblast stem cell (TSC) spheroids were co-cultured with confluent monolayers of the Ishikawa endometrial epithelial cell line to model the epithelial phase of implantation in vitro. Systems biology approaches with published transcriptomic datasets were used to model the epithelial phase of implantation in silico. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human embryos surplus to treatment requirements were consented for research. Day 6 blastocysts were co-cultured with Ishikawa cell layers until Day 8, and human TSC spheroids modelling blastocyst trophectoderm were co-cultured with Ishikawa cell layers for 48 h. Embryo and TSC morphology was assessed by immunofluorescence microscopy, and TSC differentiation by real-time quantitative PCR (RT-qPCR) and ELISA. Single-cell human blastocyst transcriptomes, and bulk transcriptomes of TSC and primary human endometrial epithelium were used to model the trophectoderm-epithelium interaction in silico. Hypernetworks, pathway analysis, random forest machine learning and RNA velocity were employed to identify gene networks associated with implantation. MAIN RESULTS AND THE ROLE OF CHANCE: The majority of embryos co-cultured with Ishikawa cell layers from Day 6 to 8 breached the epithelial layer (37/46), and syncytiotrophoblast was seen in all of these. Syncytiotrophoblast was observed at the embryo-epithelium interface before breaching, and syncytiotrophoblast mediated all pioneering breaching events observed (7/7 events). Multiple independent syncytiotrophoblast regions were seen in 26/46 embryos, suggesting derivation from different regions of trophectoderm. Human TSC spheroids co-cultured with Ishikawa layers also exhibited syncytiotrophoblast formation upon invasion into the epithelium. RT-qPCR comparison of TSC spheroids in isolated culture and co-culture demonstrated epithelium-induced upregulation of syncytiotrophoblast genes CGB (P = 0.03) and SDC1 (P = 0.008), and ELISA revealed the induction of hCGß secretion (P = 0.03). Secretory-phase primary endometrial epithelium surface transcriptomes were used to identify trophectoderm surface binding partners to model the embryo-epithelium interface. Hypernetwork analysis established a group of 25 epithelium-interacting trophectoderm genes that were highly connected to the rest of the trophectoderm transcriptome, and epithelium-coupled gene networks in cells of the polar region of the trophectoderm exhibited greater connectivity (P < 0.001) and more organized connections (P < 0.0001) than those in the mural region. Pathway analysis revealed a striking similarity with syncytiotrophoblast differentiation, as 4/6 most highly activated pathways upon TSC-syncytiotrophoblast differentiation (false discovery rate (FDR < 0.026)) were represented in the most enriched pathways of epithelium-coupled gene networks in both polar and mural trophectoderm (FDR < 0.001). Random forest machine learning also showed that 80% of the endometrial epithelium-interacting trophectoderm genes identified in the hypernetwork could be quantified as classifiers of TSC-syncytiotrophoblast differentiation. This multi-model approach suggests that invasive syncytiotrophoblast formation from both polar and mural trophectoderm is promoted by attachment to the endometrial epithelium to enable embryonic invasion. LARGE SCALE DATA: No omics datasets were generated in this study, and those used from previously published studies are cited. LIMITATIONS, REASONS FOR CAUTION: In vitro and in silico models may not recapitulate the dynamic embryo-endometrial interactions that occur in vivo. The influence of other cellular compartments in the endometrium, including decidual stromal cells and leukocytes, was not represented in these models. WIDER IMPLICATIONS OF THE FINDINGS: Understanding the mechanism of human embryo breaching of the epithelium and the gene networks involved is crucial to improve implantation success rates after assisted reproduction. Moreover, early trophoblast lineages arising at the epithelial phase of implantation form the blueprint for the placenta and thus underpin foetal growth trajectories, pregnancy health and offspring health. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by grants from Wellbeing of Women, Diabetes UK, the NIHR Local Comprehensive Research Network and Manchester Clinical Research Facility, and the Department of Health Scientist Practitioner Training Scheme. None of the authors has any conflict of interest to declare.

Altered protein O-GlcNAcylation in placentas from mothers with diabetes causes aberrant endocytosis in placental trophoblast cells.

Women with pre-existing diabetes have an increased risk of poor pregnancy outcomes, including disordered fetal growth, caused by changes to placental function. Here we investigate the possibility that the hexosamine biosynthetic pathway, which utilises cellular nutrients to regulate protein function via post-translationally modification with O-linked N-acetylglucosamine (GlcNAc), mediates the placental response to the maternal metabolic milieu. Mass spectrometry analysis revealed that the placental O-GlcNAcome is altered in women with type 1 (n = 6) or type 2 (n = 6) diabetes T2D (≥ twofold change in abundance in 162 and 165 GlcNAcylated proteins respectively compared to BMI-matched controls n = 11). Ingenuity pathway analysis indicated changes to clathrin-mediated endocytosis (CME) and CME-associated proteins, clathrin, Transferrin (TF), TF receptor and multiple Rabs, were identified as O-GlcNAcylation targets. Stimulating protein O-GlcNAcylation using glucosamine (2.5 mM) increased the rate of TF endocytosis by human placental cells (p = 0.02) and explants (p = 0.04). Differential GlcNAcylation of CME proteins suggests altered transfer of cargo by placentas of women with pre-gestational diabetes, which may contribute to alterations in fetal growth. The human placental O-GlcNAcome provides a resource to aid further investigation of molecular mechanisms governing placental nutrient sensing.

Pulsatility effects of flow on vascular tone in the fetoplacental circulation.

INTRODUCTION: The regulation of vascular tone in the fetoplacental circulation is governed by endocrine and mechanical forces yielding a relaxed basal state in normal pregnancy. Flow mediated vasodilation, induced by shear stress and endothelial nitric oxide signalling, is key to driving vasorelaxation in this circulation. The pulsatile property of blood flow, as opposed to the flow rate, could provide an additional factor in this regulation, but its effects and signalling have never been explored in the fetoplacental microvasculature. METHODS: Here, we studied the effects of non-pulsatile and pulsatile flow modalities on vascular resistance in the fetoplacental microcirculation of the human placenta using an ex vivo perfusion model; and examined a potential role for nitric oxide. We also explored whether the placental Doppler velocimetry waveform is sustained within subchorial arteries in vivo. RESULTS: Pulsatile flow reduced basal impedance to flow during steady state perfusion compared to non-pulsatile flow, signalled through enhanced nitric oxide production. Doppler velocimetry waveforms were visible within the subchorial arteries in vivo. CONCLUSION: This work suggests that the pulsatile property of flow through the fetoplacental circulation is sensed by the fetoplacental vasculature to mediate a signalling response and provide additional vasodilation of this microcirculation. We speculate that in pregnancy disease, altered amplitude and frequency of the subchorial pulse might impact on vascular function in a compromised high-resistance placental microcirculation.

Protein O-GlcNAcylation Promotes Trophoblast Differentiation at Implantation.

Embryo implantation begins with blastocyst trophectoderm (TE) attachment to the endometrial epithelium, followed by the breaching of this barrier by TE-derived trophoblast. Dynamic protein modification with O-linked ß-N-acetylglucosamine (O-GlcNAcylation) is mediated by O-GlcNAc transferase and O-GlcNAcase (OGA), and couples cellular metabolism to stress adaptation. O-GlcNAcylation is essential for blastocyst formation, but whether there is a role for this system at implantation remains unexplored. Here, we used OGA inhibitor thiamet g (TMG) to induce raised levels of O-GlcNAcylation in mouse blastocysts and human trophoblast cells. In an in vitro embryo implantation model, TMG promoted mouse blastocyst breaching of the endometrial epithelium. TMG reduced expression of TE transcription factors Cdx2, Gata2 and Gata3, suggesting that O-GlcNAcylation stimulated TE differentiation to invasive trophoblast. TMG upregulated transcription factors OVOL1 and GCM1, and cell fusion gene ERVFRD1, in a cell line model of syncytiotrophoblast differentiation from human TE at implantation. Therefore O-GlcNAcylation is a conserved pathway capable of driving trophoblast differentiation. TE and trophoblast are sensitive to physical, chemical and nutritive stress, which can occur as a consequence of maternal pathophysiology or during assisted reproduction, and may lead to adverse neonatal outcomes and associated adult health risks. Further investigation of how O-GlcNAcylation regulates trophoblast populations arising at implantation is required to understand how peri-implantation stress affects reproductive outcomes.

Tracking placental development in health and disease.

Pre-eclampsia and fetal growth restriction arise from disorders of placental development and have some shared mechanistic features. Initiation is often rooted in the maldevelopment of a maternal-placental blood supply capable of providing for the growth requirements of the fetus in later pregnancy, without exerting undue stress on maternal body systems. Here, we review normal development of a placental bed with a safe and adequate blood supply and a villous placenta-blood interface from which nutrients and oxygen can be extracted for the growing fetus. We consider disease mechanisms that are intrinsic to the maternal environment, the placenta or the interaction between the two. Systemic signalling from the endocrine placenta targets the maternal endothelium and multiple organs to adjust metabolism for an optimal pregnancy and later lactation. This signalling capacity is skewed when placental damage occurs and can deliver a dangerous pathogenic stimulus. We discuss the placental secretome including glycoproteins, microRNAs and extracellular vesicles as potential biomarkers of disease. Angiomodulatory mediators, currently the only effective biomarkers, are discussed alongside non-invasive imaging approaches to the prediction of disease risk. Identifying the signs of impending pathology early enough to intervene and ameliorate disease in later pregnancy remains a complex and challenging objective.

The effects of hyaluronate-containing medium on human embryo attachment to endometrial epithelial cells in vitro.

STUDY QUESTION: Does embryo transfer medium containing hyaluronate (HA) promote the attachment phase of human embryo implantation? SUMMARY ANSWER: HA-containing medium does not promote human blastocyst attachment to endometrial epithelial cells in vitro. WHAT IS KNOWN ALREADY: Embryo transfer media containing high concentrations of HA are being used to increase implantation and live birth rates in IVF treatment, although the mechanism of action is unknown. STUDY DESIGN SIZE DURATION: Expression of HA-interacting genes in frozen-thawed oocytes/embryos was assessed by microarray analysis (n = 21). Fresh and frozen human blastocysts (n = 98) were co-cultured with human endometrial epithelial Ishikawa cell layers. Blastocyst attachment and the effects of a widely used HA-containing medium were measured. PARTICIPANTS/MATERIALS SETTING METHODS: Human embryos surplus to treatment requirements were donated with informed consent from several ART centres. Blastocyst-stage embryos were transferred at day 6 to confluent Ishikawa cell layers; some blastocysts were artificially hatched. Blastocyst attachment was monitored from 1 to 48 h, and the effects of blastocyst pre-treatment for 10 min with HA-containing medium were determined. MAIN RESULTS AND THE ROLE OF CHANCE: Human embryos expressed the HA receptor genes CD44 and HMMR, hyaluronan synthase genes HAS1-3, and hyaluronidase genes HYAL1-3, at all stages of preimplantation development. Attachment of partially hatched blastocysts to Ishikawa cells at 24 and 48 h was related to trophectoderm grade (P = 0.0004 and 0.007, respectively, n = 34). Blastocysts of varying clinical grades that had been artificially hatched were all attached within 48 h (n = 21). Treatment of artificially hatched blastocysts with HA-containing medium did not significantly affect attachment at early (1-6 h) or late (24 and 48 h) time points, compared with control blastocysts (n = 43). LIMITATIONS REASONS FOR CAUTION: Using an adenocarcinoma-derived cell line to model embryo-endometrium attachment may not fully recapitulate in vivo interactions. The high levels of blastocyst attachment seen with this in vitro model may limit the sensitivity with which the effects of HA can be observed. WIDER IMPLICATIONS OF THE FINDINGS: Morphological trophectoderm grade can be correlated with blastocyst attachment in vitro. HA-containing medium may increase pregnancy rates by mechanisms other than promoting blastocyst attachment to endometrium. STUDY FUNDING/COMPETING INTERESTS: This work was funded by a grant from the Wellbeing of Women, the NIHR Local Comprehensive Research Network and NIHR Manchester Clinical Research Facility, the Department of Health Scientist Practitioner Training Scheme, and the Ministry of Higher Education, The State of Libya. None of the authors has any conflict of interest to declare.

The effect of high glucose on lipid metabolism in the human placenta.

Diabetes mellitus (DM) during pregnancy can result in fetal overgrowth, likely due to placental dysfunction, which has health consequences for the infant. Here we test our prediction from previous work using a placental cell line that high glucose concentrations affect placental lipid metabolism. Placentas from women with type 1 (n = 13), type 2 (n = 6) or gestational (n = 12) DM, BMI-matched to mothers without DM (n = 18), were analysed for lipase and fatty acid transport proteins and fatty acid and triglyceride content. Explants from uncomplicated pregnancies (n = 6) cultured in physiological or high glucose were similarly analysed. High glucose levels did not alter placental lipase or transporter expression or the profile and abundance of fatty acids, but triglyceride levels were higher (p < 0.05), suggesting reduced ß- oxidation. DM did not affect placental protein expression or fatty acid profile. Triglyceride levels of placentas from mothers with pre-existing DM were similar to controls, but higher in obese women with gestational DM. Maternal hyperglycemia may not affect placental fatty acid uptake and transport. However, placental ß-oxidation is affected by high glucose and reduced in a subset of women with DM. Abnormal placental lipid metabolism could contribute to increased maternal-fetal lipid transfer and excess fetal growth in some DM pregnancies.

The role of insulin-like growth factor 2 receptor-mediated homeobox gene expression in human placental apoptosis, and its implications in idiopathic fetal growth restriction.

Fetal growth restriction (FGR) is caused by poor placental development and function early in gestation. It is well known that placentas from women with FGR exhibit reduced cell growth, elevated levels of apoptosis and perturbed expression of the growth factors, cytokines and the homeobox gene family of transcription factors. Previous studies have reported that insulin-like growth factor-2 (IGF2) interacts with its receptor-2 (IGF2R) to regulate villous trophoblast survival and apoptosis. In this study, we hypothesized that human placental IGF2R-mediated homeobox gene expression is altered in FGR and contributes to abnormal trophoblast function. This study was designed to determine the association between IGF2R, homeobox gene expression and cell survival in pregnancies affected by FGR. Third trimester placentas were collected from FGR-affected pregnancies (n = 29) and gestation matched with control pregnancies (n = 30). Functional analyses were then performed in vitro using term placental explants (n = 4) and BeWo trophoblast cells. mRNA expression was determined by real-time PCR, while protein expression was examined by immunoblotting and immunohistochemistry. siRNA transfection was used to silence IGF2R expression in placental explants and the BeWo cell-line. cDNA arrays were used to screen for downstream targets of IGF2R, specifically homeobox gene transcription factors and apoptosis-related genes. Functional effects of silencing IGF2R were then verified by ß-hCG ELISA, caspase activity assays and a real-time electrical cell-impedance assay for differentiation, apoptosis and cell growth potential, respectively. IGF2R expression was significantly decreased in placentas from pregnancies complicated by idiopathic FGR (P < 0.05 versus control). siRNA-mediated IGF2R knockdown in term placental explants and the trophoblast cell line BeWo resulted in altered expression of homeobox gene transcription factors, including increased expression of distal-less homeobox gene 5 (DLX5), and decreased expression of H2.0-Like Homeobox 1 (HLX) (P < 0.05 versus control). Knockdown of IGF2R transcription increased the expression and activity of caspase-6 and caspase-8 in placental explants, decreased BeWo proliferation and increased BeWo differentiation (all P < 0.05 compared to respective controls). This is the first study linking IGF2R placental expression with changes in the expression of homeobox genes that control cellular signalling pathways responsible for increased trophoblast cell apoptosis, which is a characteristic feature of FGR.

IGF signalling and endocytosis in the human villous placenta in early pregnancy as revealed by comparing quantum dot conjugates with a soluble ligand.

A complex combination of trafficking and signalling occurs at the surface of the placenta. The system delivers maternal nutrients to the fetus and facilitates gaseous exchange, whilst mediating signal transduction to support and stimulate the growth of the placenta itself. IGF-I is acknowledged as a maternally-derived ligand important in the regulation of placental growth. Here we show that quantum dots bearing IGF can stimulate IGF receptor (IGF1R) phosphorylation in the syncytio- (maternal-facing) and cyto- (fetal-facing) trophoblast bilayer that forms the outer boundary of the placenta, in a distribution similar to the one resulting from exposure to a soluble ligand. The conjugates are internalised by a clathrin-dependent pathway and delivered to a syncytioplasmic compartment that differs from conventional late endosomes and lysosomes. Two discrete downstream responses are evident in different cellular compartments: phosphorylation of P70S6K in the non-proliferative syncytiotrophoblast and of AKT in the cytotrophoblast. Co-conjugation of IGF-quantum dots with an RGD-containing ligand permits penetration beyond the syncytium, into the cytoplasm of the underlying cytotrophoblast. These data reveal the existence of a trans-syncytial pathway that allows maternal mitotic signals to penetrate to the inner progenitor cells, which must proliferate to support placental and consequently fetal growth.

Characterisation of Osteopontin in an In Vitro Model of Embryo Implantation.

At the onset of pregnancy, embryo implantation is initiated by interactions between the endometrial epithelium and the outer trophectoderm cells of the blastocyst. Osteopontin (OPN) is expressed in the endometrium and is implicated in attachment and signalling roles at the embryo-epithelium interface. We have characterised OPN in the human endometrial epithelial Ishikawa cell line using three different monoclonal antibodies, revealing at least nine distinct molecular weight forms and a novel secretory pathway localisation in the apical domain induced by cell organisation into a confluent epithelial layer. Mouse blastocysts co-cultured with Ishikawa cell layers served to model embryo apposition, attachment and initial invasion at implantation. Exogenous OPN attenuated initial, weak embryo attachment to Ishikawa cells but did not affect the attainment of stable attachment. Notably, exogenous OPN inhibited embryonic invasion of the underlying cell layer, and this corresponded with altered expression of transcription factors associated with differentiation from trophectoderm (Gata2) to invasive trophoblast giant cells (Hand1). These data demonstrate the complexity of endometrial OPN forms and suggest that OPN regulates embryonic invasion at implantation by signalling to the trophectoderm.

Osmotic stress induces JNK-dependent embryo invasion in a model of implantation.

In vitro culture during assisted reproduction technologies (ART) exposes pre-implantation embryos to environmental stressors, such as non-physiological nutritional, oxidative and osmotic conditions. The effects on subsequent implantation are not well understood but could contribute to poor ART efficiency and outcomes. We have used exposure to hyperosmolarity to investigate the effects of stress on the ability of embryos to interact with endometrial cells in an in vitro model. Culturing mouse blastocysts for 2h in medium with osmolarity raised by 400mOsm induced blastocoel collapse and re-expansion, but did not affect subsequent attachment to, or invasion of, the endometrial epithelial Ishikawa cell line. Inhibition of stress-responsive c-Jun N-terminal kinase (JNK) activity with SP600125 did not affect the intercellular interactions between these embryos and the epithelial cells. Four successive cycles of hyperosmotic stress at E5.5 had no effect on attachment, but promoted embryonic breaching of the epithelial cell layer by trophoblast giant cells in a JNK-dependent manner. These findings suggest that acute stress at the blastocyst stage may promote trophoblast breaching of the endometrial epithelium at implantation, and implicates stress signalling through JNK in the process of trophectoderm differentiation into the invasive trophoblast necessary for the establishment of pregnancy. The data may lead to increased understanding of factors governing ART success rates and safety.

Vitamin D, the placenta and early pregnancy: effects on trophoblast function.

Pregnancy is associated with significant changes in vitamin D metabolism, notably increased maternal serum levels of active vitamin D, 1,25-dihydroxyvitamin (1,25(OH)2D). This appears to be due primarily to increased renal activity of the enzyme 25-hydroxyvitamin D-1α-hydroxylase (CYP27B1) that catalyzes synthesis of 1,25(OH)2D, but CYP27B1 expression is also prominent in both the maternal decidua and fetal trophoblast components of the placenta. The precise function of placental synthesis of 1,25(OH)2D remains unclear, but is likely to involve localized tissue-specific responses with both decidua and trophoblast also expressing the vitamin D receptor (VDR) for 1,25(OH)2D. We have previously described immunomodulatory responses to 1,25(OH)2D by diverse populations of VDR-expressing cells within the decidua. The aim of the current review is to detail the role of vitamin D in pregnancy from a trophoblast perspective, with particular emphasis on the potential role of 1,25(OH)2D as a regulator of trophoblast invasion in early pregnancy. Vitamin D deficiency is common in pregnant women, and a wide range of studies have linked low vitamin D status to adverse events in pregnancy. To date, most of these studies have focused on adverse events later in pregnancy, but the current review will explore the potential impact of vitamin D on early pregnancy, and how this may influence implantation and miscarriage.

The Glycosyltransferase EOGT Regulates Adropin Expression in Decidualizing Human Endometrium.

In pregnancy, resistance of endometrial decidual cells to stress signals is critical for the integrity of the fetomaternal interface and, by extension, survival of the conceptus. O-GlcNAcylation is an essential posttranslational modification that links glucose sensing to cellular stress resistance. Unexpectedly, decidualization of primary endometrial stromal cells (EnSCs) was associated with a 60% reduction in O-linked ß-N-acetylglucosamine (O-GlcNAc)‒modified proteins, reflecting downregulation of the enzyme that adds O-GlcNAc to substrates (O-GlcNAc transferase; OGT) but not the enzyme that removes the modification (O-GlcNAcase). Notably, epidermal growth factor domain-specific O-linked GlcNAc transferase (EOGT), an endoplasmic reticulum-specific OGT that modifies a limited number of secreted and membrane proteins, was markedly induced in differentiating EnSCs. Knockdown of EOGT perturbed a network of decidual genes involved in multiple cellular functions. The most downregulated gene upon EOGT knockdown in decidualizing cells was the energy homeostasis-associated gene (ENHO), which encodes adropin, a metabolic hormone involved in energy homeostasis and glucose and fatty acid metabolism. Analysis of midluteal endometrial biopsies revealed an inverse correlation between endometrial EOGT and ENHO expression and body mass index. Taken together, our findings revealed that obesity impairs the EOGT-adropin axis in decidual cells, which in turn points toward a mechanistic link between metabolic disorders and adverse pregnancy outcome.

Vitamin D attenuates sphingosine-1-phosphate (S1P)-mediated inhibition of extravillous trophoblast migration.

INTRODUCTION: Failure of trophoblast invasion and remodelling of maternal blood vessels leads to the pregnancy complication pre-eclampsia (PE). In other systems, the sphingolipid, sphingosine-1-phosphate (S1P), controls cell migration therefore this study determined its effect on extravillous trophoblast (EVT) function. METHODS: A transwell migration system was used to assess the behaviour of three trophoblast cell lines, Swan-71, SGHPL-4, and JEG3, and primary human trophoblasts in the presence or absence of S1P, S1P pathway inhibitors and 1,25(OH)2D3. QPCR and immunolocalisation were used to demonstrate EVT S1P receptor expression. RESULTS: EVTs express S1P receptors 1, 2 and 3. S1P inhibited EVT migration. This effect was abolished in the presence of the specific S1PR2 inhibitor, JTE-013 (p < 0.05 versus S1P alone) whereas treatment with the S1R1/3 inhibitor, FTY720, had no effect. In other cell types S1PR2 is regulated by vitamin D; here we found that treatment with 1,25(OH)2D3 for 48 or 72 h reduces S1PR2 (4-fold; <0.05), but not R1 and R3, expression. Moreover, S1P did not inhibit the migration of cells exposed to 1,25(OH)2D3 (p < 0.05). DISCUSSION: This study demonstrates that although EVT express three S1P receptor isoforms, S1P predominantly signals through S1PR2/Gα12/13 to activate Rho and thereby acts as potent inhibitor of EVT migration. Importantly, expression of S1PR2, and therefore S1P function, can be down-regulated by vitamin D. Our data suggest that vitamin D deficiency, which is known to be associated with PE, may contribute to the impaired trophoblast migration that underlies this condition.

Apposition to endometrial epithelial cells activates mouse blastocysts for implantation.

STUDY QUESTION: How do interactions between blastocyst-stage embryos and endometrial epithelial cells regulate the early stages of implantation in an in vitro model? SUMMARY ANSWER: Mouse blastocyst apposition with human endometrial epithelial cells initiates trophectoderm differentiation to trophoblast, which goes on to breach the endometrial epithelium. WHAT IS KNOWN ALREADY: In vitro models using mouse blastocysts and human endometrial cell lines have proven invaluable in the molecular characterisation of embryo attachment to endometrial epithelium at the onset of implantation. Genes involved in embryonic breaching of the endometrial epithelium have not been investigated in such in vitro models. STUDY DESIGN, SIZE, DURATION: This study used an established in vitro model of implantation to examine cellular and molecular interactions during blastocyst attachment to endometrial epithelial cells. PARTICIPANTS/MATERIALS, SETTING, METHODS: Mouse blastocysts developed from embryonic day (E) 1.5 in vitro were hatched and co-cultured with confluent human endometrial adenocarcinoma-derived Ishikawa cells in serum-free medium. A scale of attachment stability based on blastocyst oscillation upon agitation was devised. Blastocysts were monitored for 48 h to establish the kinetics of implantation, and optical sectioning using fluorescence microscopy revealed attachment and invasion interfaces. Quantitative PCR was used to determine blastocyst gene expression. Data from a total of 680 mouse blastocysts are reported, with 3-6 experimental replicates. T-test and ANOVA analyses established statistical significance at P < 0.05, P < 0.01 and P < 0.001. MAIN RESULTS AND THE ROLE OF CHANCE: Hatched E4.5 mouse blastocysts exhibited weak attachment to confluent Ishikawa cells over the first 24 h of co-culture, with intermediate and stable attachment occurring from 28 h (E5.5 + 4 h) in a hormone-independent manner. Attached embryos fixed after 48 h (E6.5) frequently exhibited outgrowths, characterised morphologically and with antibody markers as trophoblast giant cells (TGCs), which had breached the Ishikawa cell layer. Beginning co-culture at E5.5 also resulted in intermediate and stable attachment from E5.5 + 4 h; however, these embryos did not go on to breach the Ishikawa cell layer, even when co-culture was extended to E7.5 (P < 0.01). Blastocysts cultured from E4.5 in permeable transwell inserts above Ishikawa cells before transfer to direct co-culture at E5.5 went on to attach but failed to breach the Ishikawa cell layer by E6.5 (P < 0.01). Gene expression analysis at E5.5 demonstrated that direct co-culture with Ishikawa cells from E4.5 resulted in downregulation of trophectoderm transcription factors Cdx2 (P < 0.05) and Gata3 (P < 0.05) and upregulation of the TGC transcription factor Hand1 (P < 0.05). Co-culture with non-endometrial human fibroblasts did not alter the expression of these genes. LARGE SCALE DATA: None. LIMITATIONS, REASONS FOR CAUTION: The in vitro model used here combines human carcinoma-derived endometrial cells with mouse embryos, in which the cellular interactions observed may not fully recapitulate those in vivo. The data gleaned from such models can be regarded as hypothesis-generating, and research is now needed to develop more sophisticated models of human implantation combining multiple primary endometrial cell types with surrogate and real human embryos. WIDER IMPLICATIONS OF THE FINDINGS: This study implicates blastocyst apposition to endometrial epithelial cells as a critical step in trophoblast differentiation required for implantation. Understanding this maternal regulation of the embryonic developmental programme may lead to novel treatments for infertility. STUDY FUNDING AND COMPETING INTEREST(S): This work was supported by funds from the charities Wellbeing of Women (RG1442) and Diabetes UK (15/0005207), and studentship support for SCB from the Anatomical Society. No conflict of interest is declared.

Metabolites involved in glycolysis and amino acid metabolism are altered in short children born small for gestational age.

BACKGROUND: Later life metabolic dysfunction is a well-recognized consequence of being born small for gestational age (SGA). This study has applied metabolomics to identify whether there are changes in these pathways in prepubertal short SGA children and aimed to compare the intracellular and extracellular metabolome in fibroblasts derived from healthy children and SGA children with postnatal growth impairment. METHODS: Skin fibroblast cell lines were established from eight SGA children (age 1.8-10.3 y) with failure of catch-up growth and from three healthy control children. Confluent cells were incubated in serum-free media and the spent growth medium (metabolic footprint), and intracellular metabolome (metabolic fingerprint) were analyzed by gas-chromatography mass spectrometry. RESULTS: Nineteen metabolites were significantly altered between SGA and control cell lines. The greatest fold difference (FD) was seen for alanine (fingerprint FD, SGA: control 0.3, P = 0.01 and footprint FD = 0.19, P = 0.01), aspartic acid (fingerprint FD = 5.21, P = 0.01), and cystine (footprint FD = 1.66, P = 0.02). Network analysis of the differentially expressed metabolites predicted inhibition of insulin as well as growth (ERK) signaling in SGA cells. CONCLUSION: This study indicates that changes in cellular metabolism associated with both growth failure and insulin insensitivity are present in prepubertal short children born SGA.

The impact of a human IGF-II analog ([Leu27]IGF-II) on fetal growth in a mouse model of fetal growth restriction.

Enhancing placental insulin-like growth factor (IGF) availability appears to be an attractive strategy for improving outcomes in fetal growth restriction (FGR). Our approach was the novel use of [Leu(27)]IGF-II, a human IGF-II analog that binds the IGF-II clearance receptor IGF-IIR in fetal growth-restricted (FGR) mice. We hypothesized that the impact of [Leu(27)]IGF-II infusion in C57BL/6J (wild-type) and endothelial nitric oxide synthase knockout (eNOS(-/-); FGR) mice would be to enhance fetal growth and investigated this from mid- to late gestation; 1 mg·kg(-1)·day(-1) [Leu(27)]IGF-II was delivered via a subcutaneous miniosmotic pump from E12.5 to E18.5. Fetal and placental weights recorded at E18.5 were used to generate frequency distribution curves; fetuses <5th centile were deemed growth restricted. Placentas were harvested for immunohistochemical analysis of the IGF system, and maternal serum was collected for measurement of exogenously administered IGF-II. In WT pregnancies, [Leu(27)]IGF-II treatment halved the number of FGR fetuses, reduced fetal(P = 0.028) and placental weight variations (P = 0.0032), and increased the numbers of pups close to the mean fetal weight (131 vs. 112 pups within 1 SD). Mixed-model analysis confirmed litter size to be negatively correlated with fetal and placental weight and showed that [Leu(27)]IGF-II preferentially improved fetal weight in the largest litters, as defined by number. Unidirectional (14C)MeAIB transfer per gram placenta (System A amino acid transporter activity) was inversely correlated with fetal weight in [Leu(27)]IGF-II-treated WT animals (P < 0.01). In eNOS(-/-) mice, [Leu(27)]IGF-II reduced the number of FGR fetuses(1 vs. 5 in the untreated group). The observed reduction in FGR pup numbers in both C57 and eNOS(-/-) litters suggests the use of this analog as a means of standardizing and rescuing fetal growth, preferentially in the smallest offspring.