Glucose influences endometrial receptivity to embryo implantation through O-GlcNAcylation-mediated regulation of the cytoskeleton.

Phenotypic changes to endometrial epithelial cells underpin receptivity to embryo implantation at the onset of pregnancy but the effect of hyperglycemia on these processes remains poorly understood. Here, we show that physiological levels of glucose (5 mM) abolished receptivity in the endometrial epithelial cell line, Ishikawa. However, embryo attachment was supported by 17 mM glucose as a result of glucose flux through the hexosamine biosynthetic pathway (HBP) and modulation of cell function via protein O-GlcNAcylation. Pharmacological inhibition of HBP or protein O-GlcNAcylation reduced embryo attachment in cocultures at 17 mM glucose. Mass spectrometry analysis of the O-GlcNAcylated proteome in Ishikawa cells revealed that myosin phosphatase target subunit 1 (MYPT1) is more highly O-GlcNAcylated in 17 mM glucose, correlating with loss of its target protein, phospho-myosin light chain 2, from apical cell junctions of polarized epithelium. Two-dimensional (2-D) and three-dimensional (3-D) morphologic analysis demonstrated that the higher glucose level attenuates epithelial polarity through O-GlcNAcylation. Inhibition of Rho (ras homologous)A-associated kinase (ROCK) or myosin II led to reduced polarity and enhanced receptivity in cells cultured in 5 mM glucose, consistent with data showing that MYPT1 acts downstream of ROCK signaling. These data implicate regulation of endometrial epithelial polarity through RhoA signaling upstream of actomyosin contractility in the acquisition of endometrial receptivity. Glucose levels impinge on this pathway through O-GlcNAcylation of MYPT1, which may impact endometrial receptivity to an implanting embryo in women with diabetes.NEW & NOTEWORTHY Understanding how glucose regulates endometrial function will support preconception guidance and/or the development of targeted interventions for individuals living with diabetes wishing to embark on pregnancy. We found that glucose can influence endometrial epithelial cell receptivity to embryo implantation by regulating posttranslational modification of proteins involved in the maintenance of cell polarity. Impaired or inappropriate endometrial receptivity could contribute to fertility and/or early pregnancy complications caused by poor glucose control.

Use of 'omics for endometrial timing: the cycle moves on.

For some years, the prospect of precise and personalized timing of the endometrial cycle for optimal embryo replacement has been held out as a potential solution to low implantation rates. It is envisaged that a receptive state can be defined and reached at a predictable time, and embryo replacement performed in synchrony. In the last century, morphological changes characteristic of the mid secretory phase were defined in precisely timed cycles in women of proven fertility, but when deviations from this standardized schedule occur, their significance for implantation has remained uncertain. 'Omics technologies have been widely advocated for staging the endometrial cycle and defining a set of biochemical requirements for implantation, but after two decades of research, improvements to pregnancy rates have not followed, and there is a striking lack of agreement regarding the molecular characterization of the receptive state. Some of the rationale underlying these problems is now emerging with the application of higher-level computational and biological methodology. Here, we consider the challenges of defining an endometrial phenotype that can support implantation and continuing pregnancy. Receptivity may be an emergent trait depending on contributions from multiple proteins that have low pathway connectivity. We recommend that authors choose language which rigorously avoids the implication that protocols for molecular staging of the mid secretory phase inherently identify a state of receptivity to the implanting blastocyst.

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.

Ryanodine receptor calcium release channels in trophoblasts and their role in cell migration.

Trophoblasts are specialized epithelial cells of the placenta that are involved in invasion, communication and the exchange of materials between the mother and fetus. Cytoplasmic Ca2+ ([Ca2+]c) plays critical roles in regulating such processes in other cell types, but relatively little is known about the mechanisms that control this second messenger in trophoblasts. In the current study, the presence of RyRs and their accessory proteins in placental tissues and in the BeWo choriocarcinoma, a model trophoblast cell-line, were examined using immunohistochemistry and Western immunoblotting. Contributions of RyRs to Ca2+ signalling and to random migration in BeWo cells were investigated using fura-2 fluorescent and brightfield videomicroscopy. The effect of RyR inhibition on reorganization of the F-actin cytoskeleton elicited by the hormone angiotensin II, was determined using phalloidin-labelling and confocal microscopy. RyR1 and RyR3 proteins were detected in trophoblasts of human first trimester and term placental villi, along with the accessory proteins triadin and calsequestrin. Similarly, RyR1, RyR3, triadin and calsequestrin were detected in BeWo cells. In this cell-line, activation of RyRs with micromolar ryanodine increased [Ca2+]c, whereas pharmacological inhibition of these channels reduced Ca2+ transients elicited by the peptide hormones angiotensin II, arginine vasopressin and endothelin 1. Angiotensin II increased the velocity, total distance and Euclidean distance of random migration by BeWo cells and these effects were significantly reduced by tetracaine and by inhibitory concentrations of ryanodine. RyRs contribute to reorganization of the F-actin cytoskeleton elicited by angiotensin II, since inhibition of these channels restores the parallelness of these structures to control levels. These findings demonstrate that trophoblasts contain a suite of proteins similar to those in other cell types possessing highly developed Ca2+ signal transduction systems, such as skeletal muscle. They also indicate that these channels regulate the migration of trophoblast cells, a process that plays a key role in development of the placenta.

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 influences of cycle stage and pregnancy upon cell glycosylation in the endometrium of the mare.

From Day 6.5-7 post-conception until its loss around Day 22, the equine embryo is enclosed in a mucinous capsule that prevents direct intercellular interaction between the trophectoderm and uterine epithelium. The embryo is, however, bathed in glycoprotein-rich secretions. In this study, lectin histochemistry was used to characterise the distribution and glycan composition of uterine glycoproteins destined for secretion, and to ascertain the local effect of an embryo on glycosylation in the endometrium. Endometrial biopsies were taken from mares in estrus, on Days 5, 8, 12 and 15 of diestrus, and on Days 12 and 15 of pregnancy and processed for lectin histochemistry. During estrus, lumenal epithelial cells were as truncated pyramids and mainly non-ciliated with glycosylated granules in the cytoplasm. Occasional ciliated cells contained few granules. Five days post-ovulation, non-ciliated cells of the lumenal epithelium were taller, and had accumulated many highly glycosylated apical granules. By Days 12 and 15 post-ovulation these cells were more cuboidal and some showed fewer secretory granules. In marked contrast, by Days 12 and 15 of pregnancy, the ciliated cells were distended, with numerous granules but non-ciliated cells had only a few in the apical cytoplasm. Glycosylation changed dramatically in pregnancy in the luminal and superficial gland epithelium, with fewer fucosylated termini, more N-acetyl galactosamine residues, together with an overall reduction in sialic acid and several other sugar structures. Glycosylation in ciliated cells on Days 12 and 15 of pregnancy showed a striking similarity to that of the blastocyst capsule. The data strongly suggests that glycoprotein production by luminal epithelial cells is influenced by the presence of a conceptus. We speculate that, as well as providing nourishment for the developing embryo, epithelial secretory glycoproteins may contribute components to the capsule, which develops only partially in embryos cultured in vitro.

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.

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.

Vasoactive intestinal peptide shapes first-trimester placenta trophoblast, vascular, and immune cell cooperation.

BACKGROUND AND PURPOSE: Extravillous trophoblast (EVT) cells are responsible for decidual stromal invasion, vascular transformation, and the recruitment and functional modulation of maternal leukocytes in the first-trimester pregnant uterus. An early disruption of EVT function leads to placental insufficiency underlying pregnancy complications such as preeclampsia and fetal growth restriction. Vasoactive intestinal peptide (VIP) is a vasodilating and immune modulatory factor synthesized by trophoblast cells. However, its role in first-trimester placenta has not been explored. Here, we tested the hypothesis that VIP is involved in first-trimester EVT outgrowth, spiral artery remodelling, balancing angiogenesis, and maintenance of immune homeostasis. EXPERIMENTAL APPROACH: First-trimester placental tissue (five to nine weeks of gestation) was collected, and was used for EVT outgrowth experiments, immunofluorescence, isolation of decidual natural killer (dNK) cells and decidual macrophages (dMA), and functional assays. Peripheral blood monocytes were differentiated with GM-CSF and used for angiogenesis assays. KEY RESULTS: In decidua basalis, VIP+ EVT were observed sprouting from cell columns and lining spiral arterioles. EVT migrating from placental explants were also VIP+. VIP increased EVT outgrowth and IL-10 release, whereas it decreased pro-inflammatory cytokine production in EVT, dNK cells, and dMA. VIP disrupted endothelial cell networks, both directly and indirectly via an effect on macrophages. CONCLUSION AND IMPLICATIONS: The results suggest that VIP assists the progress of EVT invasion and vessel remodelling in first-trimester placental bed in an immunologically "silent" milieu. The effects of VIP in the present ex vivo human placental model endorse its potential as a therapeutic candidate for deep placentation disorders.

Decidual leucocytes infiltrating human spiral arterioles are rich source of matrix metalloproteinases and degrade extracellular matrix in vitro and in situ.

PROBLEM: During pregnancy, the decidual spiral arterioles (SpAs) that supply maternal blood to the placenta undergo a series of changes to optimise the transfer of nutrients and oxygen to the developing foetus. Recent studies have shown that initiation of SpA transformation coincides with decidual leucocyte infiltration. Leucocytes are known to be a source of matrix metalloproteinases (MMPs); however, the complete profile of MMPs expressed by decidual NK cells (dNK) and macrophages has not been characterised. We hypothesised that leucocyte-derived MMPs contribute to SpA remodelling. METHODS: Decidual NK cells and macrophages were isolated from first trimester decidua and their MMP repertoire profiled by qRT-PCR (n = 10; 5-11 weeks). Dual immunofluorescence was used to localise MMP expression in situ (n = 3; 5-12 weeks). Gelatin zymography was carried out to assess whether leucocyte-derived MMPs can degrade ECM. In situ zymography and immunofluorescence identified MMP activity in tissue-resident dNK and macrophages. RESULTS: Decidual NK cells cells and macrophages expressed MMP2, -7, -9, -11, -16, -19 and tissue inhibitors of metalloproteinase-1, -2, and -3. Both cell types degraded gelatin using MMP2 and MMP9 and broke down collagen in an in vitro model of the SpA. Extravillous trophoblasts (EVTs) expressed a similar repertoire of MMPs. CONCLUSION: We suggest that matrix remodelling in SpA is initiated by infiltrating leucocytes, while EVTs become involved at later stages.

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.

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.

Selective Targeting of a Novel Vasodilator to the Uterine Vasculature to Treat Impaired Uteroplacental Perfusion in Pregnancy.

Fetal growth restriction (FGR) in pregnancy is commonly caused by impaired uteroplacental blood flow. Vasodilators enhance uteroplacental perfusion and fetal growth in humans and animal models; however, detrimental maternal and fetal side effects have been reported. We hypothesised that targeted uteroplacental delivery of a vasodilator would enhance drug efficacy and reduce the risks associated with drug administration in pregnancy. Phage screening identified novel peptides that selectively accumulated in the uteroplacental vasculature of pregnant mice. Following intravenous injection, the synthetic peptide CNKGLRNK selectively bound to the endothelium of the uterine spiral arteries and placental labyrinth in vivo; CNKGLRNK-decorated liposomes also selectively bound to these regions. The nitric oxide donor 2-[[4-[(nitrooxy)methyl]benzoyl]thio]-benzoic acid methyl ester (SE175) induced significant relaxation of mouse uterine arteries and human placental arteries in vitro; thus, SE175 was encapsulated into these targeted liposomes and administered to healthy pregnant C57BL/6J mice or endothelial nitric oxide synthase knockout (eNOS-/-) mice, which exhibit impaired uteroplacental blood flow and FGR. Liposomes containing SE175 (0.44mg/kg) or PBS were administered on embryonic (E) days 11.5, 13.5, 15.5 and 17.5; fetal and placental weights were recorded at term and compared to mice injected with free PBS or SE175. Targeted uteroplacental delivery of SE175 had no effect on fetal weight in C57BL/6J mice, but significantly increased fetal weight and mean spiral artery diameter, and decreased placental weight, indicative of improved placental efficiency, in eNOS-/- mice; free SE175 had no effect on fetal weight or spiral artery diameter. Targeted, but not free SE175 also significantly reduced placental expression of 4-hydroxynonenal, cyclooxygenase-1 and cyclooxygenase-2, indicating a reduction in placental oxidative stress. These data suggest that exploiting vascular targeting peptides to selectively deliver SE175 to the uteroplacental vasculature may represent a novel treatment for FGR resulting from impaired uteroplacental perfusion.

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.

Extravillous Trophoblast and Endothelial Cell Crosstalk Mediates Leukocyte Infiltration to the Early Remodeling Decidual Spiral Arteriole Wall.

Decidual spiral arteriole (SpA) remodeling is essential to ensure optimal uteroplacental blood flow during human pregnancy, yet very little is known about the regulatory mechanisms. Uterine decidual NK (dNK) cells and macrophages infiltrate the SpAs and are proposed to initiate remodeling before colonization by extravillous trophoblasts (EVTs); however, the trigger for their infiltration is unknown. Using human first trimester placenta, decidua, primary dNK cells, and macrophages, we tested the hypothesis that EVTs activate SpA endothelial cells to secrete chemokines that have the potential to recruit maternal immune cells into SpAs. Gene array, real-time PCR, and ELISA analyses showed that treatment of endothelial cells with EVT conditioned medium significantly increased production of two chemokines, CCL14 and CXCL6. CCL14 induced chemotaxis of both dNK cells and decidual macrophages, whereas CXCL6 also induced dNK cell migration. Analysis of the decidua basalis from early pregnancy demonstrated expression of CCL14 and CXCL6 by endothelial cells in remodeling SpAs, and their cognate receptors are present in both dNK cells and macrophages. Neutralization studies identified IL-6 and CXCL8 as factors secreted by EVTs that induce endothelial cell CCL14 and CXCL6 expression. This study has identified intricate crosstalk between EVTs, SpA cells, and decidual immune cells that governs their recruitment to SpAs in the early stages of remodeling and has identified potential key candidate factors involved. This provides a new understanding of the interactions between maternal and fetal cells during early placentation and highlights novel avenues for research to understand defective SpA remodeling and consequent pregnancy pathology.

Tumor-homing peptides as tools for targeted delivery of payloads to the placenta.

The availability of therapeutics to treat pregnancy complications is severely lacking mainly because of the risk of causing harm to the fetus. As enhancement of placental growth and function can alleviate maternal symptoms and improve fetal growth in animal models, we have developed a method for targeted delivery of payloads to the placenta. We show that the tumor-homing peptide sequences CGKRK and iRGD bind selectively to the placental surface of humans and mice and do not interfere with normal development. Peptide-coated nanoparticles intravenously injected into pregnant mice accumulated within the mouse placenta, whereas control nanoparticles exhibited reduced binding and/or fetal transfer. We used targeted liposomes to efficiently deliver cargoes of carboxyfluorescein and insulin-like growth factor 2 to the mouse placenta; the latter significantly increased mean placental weight when administered to healthy animals and significantly improved fetal weight distribution in a well-characterized model of fetal growth restriction. These data provide proof of principle for targeted delivery of drugs to the placenta and provide a novel platform for the development of placenta-specific therapeutics.

Functional changes in Hofbauer cell glycobiology during human pregnancy.

INTRODUCTION: This study examines the glucose metabolism and glycosylation of villous macrophages (Hofbauer cells) over the course of pregnancy. MATERIALS AND METHODS: Sections of placentae from 6 weeks to term were stained with antibodies to α-amylase, glycogen synthase, glycogen phosphorylase and glucose transporters 1 and 3 (GLUT-1 and GLUT-3) while a panel of 24 lectins was applied to resin sections from 4 weeks onwards. Hofbauer cells were identified by the binding of anti-CD 163 antibody. RESULTS: Little stored glycogen could be demonstrated by Bandeiraea simplicifolia-II agglutinin binding and, by immunocytochemistry, low levels of glycogen synthase were located within the cells, though glycogen phosphorylase expression, an enzyme releasing glucose from glycogen chains, was intense. Glucose transporter-3 but not -1 was present in the cells as has been found in other types of macrophage. Lectin histochemistry showed that many classes of glycan were present in the cells, both N and O-linked, though simple fucose residues could not be demonstrated. Glycan profiles were obtained for Hofbauer cell plasma membranes, cytosol, lysosomes and small granules. With some lectins, the intensity of binding diminished after the second trimester. Morphological changes also occurred over the course of pregnancy. DISCUSSION: Hofbauer cells have properties commensurate with their phagocytic activity with numerous lysosomal vacuoles and heavily glycosylated plasma membranes and granules, most evident in the first half of pregnancy. Their carbohydrate metabolism appears to rely on glucose mobilisation rather than storage as glycogen, reflecting their peripatetic mode of existence.

MiR-145 suppresses embryo-epithelial juxtacrine communication at implantation by modulating maternal IGF1R.

Successful implantation requires the synchronization of viable embryonic development with endometrial receptivity. The mechanisms allowing for the initiation of crosstalk between the embryo and the endometrium remain elusive; however, recent studies have revealed that there are alterations in endometrial microRNAs (miRs) in women suffering repeated implantation failure and that one of the altered miRs is miR-145. We assessed the role of miR-145 and its target IGF1R, in early implantation. miR-145 overexpression and IGF1R knockdown were achieved in Ishikawa endometrial cells. Quantitative PCR, western blotting and 3'UTR luciferase reporter assays confirmed that IGF1R is a direct target of miR-145 in the endometrium. Attachment of mouse embryos or IGF1-coated beads to endometrial epithelial cells was used to study the effects of altered miR-145 and/or IGF1R expression on early implantation events. miR-145 overexpression or specific reduction of IGF1R impaired attachment in both cases. An IGF1R target protector prevented the miR-145-mediated reduction in IGF1R and reversed the effect of miR-145 overexpression on attachment. The data demonstrate that miR-145 influences embryo attachment by reducing the level of IGF1R in endometrium.

Elastin-derived peptides stimulate trophoblast migration and invasion: a positive feedback loop to enhance spiral artery remodelling.

Elastin breakdown in the walls of uterine spiral arteries during early pregnancy facilitates their transformation into dilated, high-flow, low-resistance channels. Elastin-derived peptides (EDP) can influence cell migration, invasion and protease activity, and so we hypothesized that EDP released during elastolysis promote extravillous trophoblast (EVT) invasion and further elastin breakdown. Treatment of the trophoblast cell line SGHPL4 with the elastin-derived matrikine VGVAPG (1 µg/ml) significantly increased total elastase activity, promoted migration in a wound healing assay and increased invasion through Matrigel-coated transwells compared with vehicle control (0.1% DMSO) or the scrambled sequence VVGPGA. Furthermore, treatment of first-trimester placental villous explants with this EDP significantly increased both the area of trophoblast outgrowth and distance of migration away from the villous tips. Primary first-trimester cytotrophoblast exposed to VGVAPG (1 µg/ml) for 30 min showed increased phosphorylation of endothelial nitric oxide synthase and activation of the mitogen activated protein kinase pathway, events also associated with tumour cell migration and invasion. These in vitro observations suggest liberation of bioactive EDP during induction of elastolysis in the uterine spiral arteries may orchestrate a positive feedback loop that promotes EVT invasion and further elastin breakdown, contributing to the process of vascular remodelling.

The role of the osteopontin-integrin αvß3 interaction at implantation: functional analysis using three different in vitro models.

STUDY QUESTION: Does the interaction between integrin and its ligand osteopontin (OPN) mediate embryonic attachment to endometrial epithelium at implantation? SUMMARY ANSWER: OPN of epithelial origin binds the receptor integrin αvß3 at the maternal surface to support adhesion during the early stages of implantation. WHAT IS KNOWN ALREADY: Integrin αvß3 and OPN are both present in the endometrial luminal epithelium in the mid-secretory phase. STUDY DESIGN, SIZE, DURATION: Microscopy of attachment sites of blastocysts (mouse, n = 151, human, n = 8) and OPN- or BSA-coated beads (n = 488) interacting with Ishikawa cell monolayers at 24 and 48 h. Levels of epithelial OPN or integrin αvß3 were altered by siRNA-mediated targeting and the results compared with non-targeting siRNA or mock-transfected controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: In vitro modelling of early implantation with human endometrial cells (Ishikawa) and mouse or human embryos or ligand-coated beads. Immunolocalization of antigen around attached embryos was measured by image analysis with multiple repeats (n > 3), allowing a gradient of relative intensity to be detected. Attachment was quantified using a stability scale and protein expression documented by indirect immunofluorescence. Protein associations were probed by pulldown assays. MAIN RESULTS AND THE ROLE OF CHANCE: Integrin and OPN levels were increased in epithelial cells near to attached embryos. The pulldown assay confirmed OPN-integrin αvß3 binding (n > 3). Decreased attachment stability of mouse embryos observed after siRNA knock-down of integrin αvß3 or OPN itself, or OPN-coated beads after knock-down of integrin αvß3, was tested for significance using Kruskal-Wallis with Dunn's post hoc tests. LIMITATIONS, REASONS FOR CAUTION: In vitro model. Attachment data using human embryos is limited by embryo availability. Mouse embryo attachment to human cells involves a species crossover so must be interpreted with caution. Ligand-coated beads allow specific molecular interactions mediating attachment to be probed, but obviously lack the adhesion and signaling repertoire of a live embryo. WIDER IMPLICATIONS OF THE FINDINGS: Some of the literature identifies reduced integrin αvß3 expression in infertile endometrium; these findings predict that embryo attachment stability will be reduced in vivo if integrin levels are low. We suggest that the robustness of the initial attachment of the embryo affects its ability to progress to the post-epithelial phase of implantation; some poorly attached embryos will be lost. STUDY FUNDING/COMPETING INTEREST(S): No external funds were used for this study, which was supported by funds from the Universities of Manchester and Oxford. None of the authors has any conflict of interest to declare. TRIAL REGISTRATION NUMBER: N/A.