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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.

In view of controversy about the source of placental multinuclear giant cells, we have re-examined the literature which clearly shows they are derived from trophoblastic elements that have populated the decidua. Archival material for electron microscopy from 17 to 18 week placentae demonstrates they can be found connected via desmosomes to the outer extravillous cytotrophoblast cells of anchoring columns, thus identifying a primary source. We suggest their formation is a terminal differentiation step occurring at all stages of invasion from the cell column to the myometrium, progressively reducing the invasive population.

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 Ca 2+ ([Ca 2+] 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 Ca 2+ 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 [Ca 2+] c, whereas pharmacological inhibition of these channels reduced Ca 2+ 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 Ca 2+ 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.

Matters Arising article, arising from H. Bové et al. Nature Communications https://doi.org/10.1038/s41467-019-11654-3 (2019).

Placental dysfunction is the underlying cause of pregnancy complications such as fetal growth restriction (FGR) and pre-eclampsia. No therapies are available to treat a poorly functioning placenta, primarily due to the risks of adverse side effects in both the mother and the fetus resulting from systemic drug delivery. The use of targeted liposomes to selectively deliver payloads to the placenta has the potential to overcome these issues. In this study, we assessed the safety and efficacy of epidermal growth factor (EGF)-loaded, peptide-decorated liposomes to improve different aspects of placental function, using tissue from healthy control pregnancies at term, and pregnancies complicated by FGR. Phage screening identified a peptide sequence, CGPSARAPC (GPS), which selectively homed to mouse placentas in vivo, and bound to the outer syncytiotrophoblast layer of human placental explants ex vivo. GPS-decorated liposomes were prepared containing PBS or EGF (50–100 ng/mL), and placental explants were cultured with liposomes for up to 48 h. Undecorated and GPS-decorated liposomes containing PBS did not affect the basal rate of amino acid transport, human chorionic gonadotropin (hCG) release or cell turnover in placental explants from healthy controls. GPS-decorated liposomes containing EGF significantly increased amino acid transporter activity in healthy control explants, but not in placental explants from women with FGR. hCG secretion and cell turnover were unaffected by EGF delivery, however, differential activation of downstream protein kinases was observed when EGF was delivered via GPS-decorated vs. undecorated liposomes. These data indicate that targeted liposomes represent a safe and useful tool for the development of new therapies for placental dysfunction, recapitulating the effects of free EGF.

Macrophages are innate immune cells that fight infection but also regulate tissue regeneration and remodelling. In the uterus, although tissue remodelling is essential for establishment and maintenance of pregnancy, the specialisation of macrophages is not well characterised compared to other mucosal tissues. Here we show that uterine macrophages are functionally specialised, expressing multiple markers of alternative activation associated with tissue remodelling and repair, and responding more highly to the type 2 cytokine IL-4 than other mucosal tissue macrophages. Uterine macrophages were continuously replenished from circulating bone marrow-derived CCR2+monocytes that fluctuated dramatically in number throughout the reproductive cycle, and had properties distinct from the macrophages that they became, including differential responses to microbial stimulation. Importantly, many of these properties of uterine monocytes and macrophages were conserved between mice and humans. These findings further our understanding of immune regulation of uterine tissue integrity and have important implications for differences in immune responses to infections at different phases of the reproductive cycle.SUMMARYUterine macrophages are specialised, alternatively activated cells that are replenished from circulating bone marrow-derived monocytes. Monocyte and macrophage properties fluctuate markedly throughout the reproductive cycle, with many features conserved between mice and humans, and exhibiting differential responses to microbial stimulation.

Implantation failure is an important impediment to increasing success rates in assisted reproductive technologies. Knowledge of the cascade of morphological and molecular events at implantation remains limited. Cell surface CD44 and hyaluronate (HA) have been reported in the uterus, but a role in intercellular interaction at implantation remains to be evaluated. Mouse embryos were co-cultured with human Ishikawa endometrial epithelial monolayers over 2 days. Attachment was tenuous during the first 24 h, after which it became stable, leading to breaching of the monolayer. The effects of enzymatically reducing the density of HA, or introducing a function-blocking antibody to CD44, were monitored during progression from weak to stable embryonic attachment. Hyaluronidase-mediated removal of surface HA from the epithelial cells enhanced the speed of attachment, while a similar treatment of embryos had no effect. The antibody to CD44 caused retardation of initial attachment. These results suggest that CD44–HA binding could be employed by embryos during initial docking, but the persistence of HA in epithelial cells might be detrimental to later stages of implantation by retarding attainment of stable attachment.

Fetal development may be compromised by adverse events at the placental interface between mother and fetus. However, it is still unclear how the communication between mother and fetus occurs through the placenta. In vitro - models of the human placental barrier, which could help our understanding and which recreate three-dimensional (3D) structures with biological functionalities and vasculatures, have not been reported yet. Here we present a 3D-vascularized human primary placental barrier model which can be constructed in 1 day. We illustrate the similarity of our model to first trimester human placenta, both in its structure and in its ability to respond to altered oxygen and to secrete factors that cause damage cells across the barrier including embryonic cortical neurons. We use this model to highlight the possibility that both the trophoblast and the endothelium within the placenta might play a role in the fetomaternal dialogue.

Invasive trophoblast cells are critical to spiral artery remodeling in hemochorial placentation. Insufficient trophoblast cell invasion and vascular remodeling can lead to pregnancy disorders including preeclampsia, preterm birth, and intrauterine growth restriction. Previous studies in mice identified achaete-scute homolog 2 (ASCL2) as essential to extraembryonic development. We hypothesized that ASCL2 is a critical and conserved regulator of invasive trophoblast cell lineage development. In contrast to the mouse, the rat possesses deep intrauterine trophoblast cell invasion and spiral artery remodeling similar to human placentation. In this study, we investigated invasive/extravillous trophoblast (EVT) cell differentiation using human trophoblast stem (TS) cells and a loss-of-function mutant Ascl2 rat model. ASCL2 transcripts are expressed in the EVT column and junctional zone, which represent tissue sources of invasive trophoblast progenitor cells within human and rat placentation sites, respectively. Differentiation of human TS cells into EVT cells resulted in significant up-regulation of ASCL2 and several other transcripts indicative of EVT cell differentiation. Disruption of ASCL2 impaired EVT cell differentiation, as indicated by cell morphology and transcript profiles. RNA sequencing analysis of ASCL2-deficient trophoblast cells identified both down-regulation of EVT cell-associated transcripts and up-regulation of syncytiotrophoblast-associated transcripts, indicative of dual activating and repressing functions. ASCL2 deficiency in the rat impacted placental morphogenesis, resulting in junctional zone dysgenesis and failed intrauterine trophoblast cell invasion. ASCL2 acts as a critical and conserved regulator of invasive trophoblast cell lineage development and a modulator of the syncytiotrophoblast lineage.

BACKGROUND Villous cytotrophoblast (vCTB) is a precursor cell population that supports the development of syncytiotrophoblast (vSTB), the high surface area barrier epithelium of the placental villus, and the primary interface between maternal and fetal tissue. In light of increasing evidence that the placenta can adapt to changing maternal environments or, under stress, can trigger maternal disease, we consider what properties of these cells empower them to exert a controlling influence on pregnancy progression and outcome. OBJECTIVE AND RATIONALE How are cytotrophoblast proliferation and differentiation regulated in the human placental villus to allow for the increasing demands of the fetal and environmental challenges and stresses that may arise during pregnancy? SEARCH METHODS PubMed was interrogated using relevant keywords and word roots combining trophoblast, villus/villous, syncytio/syncytium, placenta, stem, transcription factor (and the individual genes), signalling, apoptosis, autophagy (and the respective genes) from 1960 to the present. Since removal of trophoblast from its tissue environment is known to fundamentally change cell growth and differentiation kinetics, research that relied exclusively on cell culture has not been the main focus of this review, though it is mentioned where appropriate. Work on non-human placenta is not systematically covered, though mention is made where relevant hypotheses have emerged. OUTCOMES The synthesis of data from the literature has led to a new hypothesis for vCTB dynamics. We propose that a reversible transition can occur from a reserve population in G0 to a mitotically active state. Cells from the in-cycle population can then differentiate irreversibly to intermediate cells that leave the cycle and turn on genes that confer the capacity to fuse with the overlying vSTB as well as other functions associated with syncytial barrier and transport function. We speculate that alterations in the rate of entry to the cell cycle, or return of cells in the mitotic fraction to G0, can occur in response to environmental challenge. We also review evidence on the life cycle of trophoblast from the time that fusion occurs, and point to gaps in knowledge of how large quantities of fetal DNA arrive in maternal circulation. We critique historical methodology and make a case for research to re-address questions about trophoblast lifecycle and dynamics in normal pregnancy and the common diseases of pre-eclampsia and fetal growth restriction, where altered trophoblast kinetics have long been postulated. WIDER IMPLICATIONS The hypothesis requires experimental testing, moving research away from currently accepted methodology towards a new standard that includes representative cell and tissue sampling, assessment of cell cycle and differentiation parameters, and robust classification of cell subpopulations in villous trophoblast, with due attention to gestational age, maternal and fetal phenotype, disease and outcome.

Glycosylation of the reproductive tract of an adult female red-necked ostrich (Struthio camelus camelus) carrying a fully formed calcified egg in her uterus when accidently killed by a blow to the head was examined using lectin histochemistry on samples from the infundibulum, magnum, uterus and vagina. Glycans in the luminal epithelium and underlying glands were described after staining with 23 lectins after neuraminidase pre-treatment in some cases. Ciliated and non-ciliated cells were evident at all levels in the luminal epithelium, the latter full of richly glycosylated secretory granules. The ciliated cells also showed glycosylation and, in the magnum, these cells often stained more intensely than the non-ciliated cells. High mannose and complex N-glycans, α1,6-linked fucosyl and sialic acid residues were present throughout the tract and there was a complete absence of GalNAcα1,3(LFucα1,2)Galß1,3/4GlcNAcß1- and rare terminal GalNAcα1- residues. Fucose in α1,2-linkage as H2 antigen and Ley was also rare in the luminal epithelium and completely absent in glands. Terminal galactose was present in the luminal epithelium apart from in the infundibulum. Gland epithelium showed similar glycosylation to the luminal epithelium except in the magnum where there were significant differences and here the glands were packed full of large secretory granules, unlike the glands in the rest of the tract. Each section of the tract had its own specific pattern of glycosylation which could be related to the stage of egg formation.

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 &beta, 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.

At implantation, trophoblast derived from the trophectoderm of the blastocyst-stage embryo invades the endometrium to establish pregnancy. To understand how embryos breach the endometrial epithelium, we modelled human implantation using blastocysts or trophoblast stem cell spheroids cultured with endometrial epithelial cells (EEC). Blastocyst invasion of the EEC layer was initiated by multinuclear syncytiotrophoblast. Spheroids also invaded the epithelium with syncytiotrophoblast, and EEC induced upregulation of syncytiotrophoblast markers. Modelling implantation in silico using blastocyst and EEC transcriptomes revealed gene networks that exhibited greater connectivity and organisation in trophectoderm of the polar region of the embryonic axis. However, gene ontologies and machine learning suggested that EEC drives syncytiotrophoblast differentiation in polar and mural trophectoderm. This is the first evidence for endometrial epithelium-induced trophectoderm differentiation to invasive syncytiotrophoblast as the cellular mechanism of embryonic breaching of the endometrium in humans, with implications for reproductive medicine and our understanding of human embryonic development.

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. Pre-eclampsia and fetal growth restriction are diseases of pregnancy that arise from disorders of placental development. This Review discusses healthy development of the placenta and considers disease mechanisms, biomarkers and diagnosis of pre-eclampsia and fetal growth restriction.

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 INTEREST(S) 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.

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.

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 β-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

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.

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&ndash, 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.

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. © 2019 The British Pharmacological Society

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 [Leu27]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 [Leu27]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 [Leu27]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 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 [Leu27]IGF-II preferentially improved fetal weight in the largest litters, as defined by number. Unidirectional 14CMeAIB transfer per gram placenta (System A amino acid transporter activity) was inversely correlated with fetal weight in [Leu27]IGF-II-treated WT animals ( P < 0.01). In eNOS−/− mice, [Leu27]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.

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.

STUDY QUESTION: Can vaginal bioelectrical impedance (VZ) electrophysiologically determine alterations of the endometrium in preparation for implantation? SUMMARY ANSWER: VZ can electrophysiologically detect the sulfation and sialylation changes in the uterine glycocalyx in preparation for implantation. WHAT IS KNOWN ALREADY: Uterine receptivity is associated with various glycosylation changes that affect negative charge density at the luminal epithelial cell surface. VZ has been used to monitor the oestrous cycle. STUDY DESIGN, SIZE, DURATION: Pathogen-free Jcl:ICR mice, aged 8-10 weeks, were used in this study. We conducted the following three steps to test our hypothesis that VZ may be used to determine uterine receptivity. First, to investigate whether VZ could determine alteration of sulfation and sialylation in the uterine glycocalyx, VZ was measured in mice with induced artificial sulfation and sialylation changes in the uterine glycocalyx (galactose-3-O-sulfotransferase 2 (GP3ST) + α(1,3/1,4) fucosyltransferase gene (FucT-III)-transferred group (n = 15) and in LacZ (encoding for β-galactosidase)-transferred mice as a control group (n = 12)). Second, to investigate whether VZ could determine alterations of the endometrium in preparation for implantation, we measured VZ during the early stage of pregnancy (n = 12 each). Third, to investigate whether VZ could be used to evaluate uterine receptivity prospectively, VZ was measured in an implantation failure model mice. In 21 mice, local and transient suppression of signal transducer and activator of transcription-3 (Stat3) in the uterus were evaluated 1 day before implantation began, and 23 scramble decoy-transferred mice were used as a control group. PARTICIPANTS/MATERIALS, SETTING, METHODS: The VZ was measured at a frequency of 1 kHz in Jcl:ICR mice. Data were analysed using the Kruskal-Wallis test with Dunn's multiple comparisons, or the Student's t-test or Wilcoxon's rank-sum test with the Shapiro-Wilk normality test. The values of VZ were analysed using receiver operating characteristic (ROC) curve analysis to identify the optimal cut-off point to determine if this parameter predicted non-pregnancy. MAIN RESULTS AND THE ROLE OF CHANCE: Sulfation and sialylation changes induced in the luminal epithelial glycocalyx decreased the value of VZ. VZ showed a significant daily decrease during the early stage of pregnancy (Day 1.5 versus 2.5 p.c.: P < 0.005; Student's t-test, Day 2.5 versus 3.5 p.c.: P < 0.001; Wilcoxon's rank-sum test, Day 3.5 versus 4.5 p.c.: P < 0.005; Student's t-test, Day 4.5 versus 5.5 p.c.: P < 0.05; Student's t-test). One day before implantation began, VZ in the implantation failure model mice was significantly higher than in the control mice (P < 0.001, Wilcoxon's rank-sum test). The ROC curve analysis of VZ as a predictor of non-conception showed areas under the ROC curve of 0.91 (95% CI: 0.83-0.99). LIMITATIONS, REASONS FOR CAUTION: Although it is influenced by surface charge in the uterine epithelium, the mechanism whereby VZ changes during early pregnancy is still unexplained. WIDER IMPLICATIONS OF THE FINDINGS: Local bioelectrical impedance may help to prospectively evaluate uterine receptivity in women. Including the measurement of local bioelectrical impedance as part of a frozen-thawed embryo transfer strategy may improve the efficiency of ART. STUDY FUNDING/COMPETING INTEREST(S): This work was supported in part by the Japan Society for the Promotion of Science JSPS KAKENHI Grant (Nos. 19390429, 21390453, 16K11086 and 16K11087) from the Ministry of Education, Science and Culture of Japan (Tokyo, Japan) and Suzuken Memorial Foundation (Nagoya, Japan). The authors declare that they have no conflict of interest.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2017 there were four themed workshops, all of which are summarized in this report. These workshops discussed new knowledge and technological innovations in the following areas of research: 1) placental bed; 2) 3D structural modeling; 3) clinical placentology; 4) treatment of placental dysfunction.

The placenta is a highly specialized structure, with many functions. Here, we describe the development of the human placental villus from the earliest stages of implantation, tracing the initial formation of villi and the different derivative villus types that are found in placenta up to term. The generative cytotrophoblast cells which develop into villous syncytiotrophoblast—the interface between the mother's circulating blood and the fetus—and the inner core of stromal and vascular cells are described and illustrated as well as their contributions to the functions of the placenta.

Introduction The human placenta is a site of both hematopoiesis and vasculogenesis. There are reports of hemangioblastic foci (HAF) in the first trimester placenta, but little published information about their spatiotemporal incidence. Methods We have used semi-thin sections and whole mount staining techniques on archival early pregnancy hysterectomy material as well as freshly-collected termination tissue. Results We report a description of the distribution of HAF, their gestational profile, and some characteristics of the constituent cells. We show crypt-shaped HAF are present in villi at different levels from 4 to 11 weeks and in the chorionic plate from 4 to 9 weeks. In the villous placenta, the foci often approach closely at one end to the trophoblast basement membrane. Morphologically they show remarkable similarity to those found in the yolk sac at similar stages. In some crypts, all cells are CD34+, but CD34 and nestin progressively segregate into the endothelial lineage. Brachyury is present in less differentiated cells. The erythroid lineage is dominant, as shown by the widespread expression of CD235a/glycophorin and characteristic erythroid morphologies, indicating various degrees of differentiation. However, CD41 is also present in non-endothelial cells. Initially a discontinuous UEA-1/CD31-positive endothelium forms at the periphery of the foci. These cells appear to become integrated into the developing vasculogenic/angiogenic vessel network. We also demonstrate that, independent of HAF, vasculogenesis occurs near the tips of growing villi during the first trimester. Discussion We suggest HAF interface with the developing vascular network, producing communication channels that allow erythrocytes to enter the placental-embryonic circulation. We speculate that the erythroid cells act as oxygen reservoirs during the period before flow of maternal blood through the intervillous space of the placenta, allowing a slow feed of oxygen-rich cells to the developing embryo.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialised topics. At IFPA meeting 2016 there were twelve themed workshops, four of which are summarized in this report. These workshops related to various aspects of placental biology but collectively covered areas of decidual-trophoblast interaction, regulation of trophoblast invasion, immune cells at the maternal-fetal interface, and placental inflammation.

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.

At implantation, with the acquisition of a receptive phenotype in the uterine epithelium, an initial tenuous attachment of embryonic trophectoderm initiates reorganisation of epithelial polarity to enable stable embryo attachment and the differentiation of invasive trophoblasts. In this Cell Science at a Glance article, we describe cellular and molecular events during the epithelial phase of implantation in rodent, drawing on morphological studies both in vivo and in vitro, and genetic models. Evidence is emerging for a repertoire of transcription factors downstream of the master steroidal regulators estrogen and progesterone that coordinate alterations in epithelial polarity, delivery of signals to the stroma and epithelial cell death or displacement. We discuss what is known of the cell interactions that occur during implantation, before considering specific adhesion molecules. We compare the rodent data with our much more limited knowledge of the human system, where direct mechanistic evidence is hard to obtain. In the accompanying poster, we represent the embryo–epithelium interactions in humans and laboratory rodents, highlighting similarities and differences, as well as depict some of the key cell biological events that enable interstitial implantation to occur.

Placental cell growth depends on an adaptable combination of an endogenous developmental program and the exogenous influence of maternal growth factors, both of which may be influenced by microRNA (miR)-dependent effects on gene expression. We have previously shown that global miR suppression in placenta accelerates proliferation and enhances levels of growth factor signaling mediators in cytotrophoblast. This study aimed to identify miRs involved in regulating placental growth. An initial array revealed 58 miR species whose expression differs between first trimester, when cytotrophoblast proliferation is rapid, and term, by which time proliferation has slowed. In silico analysis defined potential growth-regulatory miRs; among these, hsa-miR-145, hsa-miR-377, and hsa-let-7a were predicted to target known placental growth genes and were higher at term than in the first trimester, so they were selected for further analysis. Overexpression of miR-377 and let-7a, but not miR-145, in first trimester placental explants significantly reduced basal cytotrophoblast proliferation and expression of ERK and MYC. PCR arrays, in silico analysis, Western blotting, and 3'-UTR luciferase reporter assays revealed targets of miR-145 within the insulin-like growth factor axis. Analysis of proliferation in placental explants overexpressing miR-145 demonstrated its role as a mediator of insulin-like growth factor-induced trophoblast proliferation. These findings identify miR-377 and let-7a in regulation of endogenous cell growth and miR-145 in the placental response to maternal stimulation and will aid the development of therapeutic strategies for problem pregnancies.

The rapid rise in obesity, metabolic syndrome and type 2 diabetes is one of the major healthcare problems of the Western world. Affected individuals are often treated with statins (3-hydroxy-3-methylglutaryl co-enzyme A [HMG CoA] reductase inhibitors) to reduce circulating cholesterol levels and the risk of developing cardiovascular disease; given the evolving demographic profile of these conditions, such drugs are increasingly prescribed to women of reproductive age. We have previously shown that exposure of placental tissue to statins inhibits the action of insulin-like growth factors (IGF)-I and -II which are key regulators of trophoblast proliferation and placental development. N-linked glycans in the IGF receptor, IGF1R, influence its presentation at the cell surface. This study aimed to determine whether statins, which are known to affect N-glycosylation, modulate IGF1R function in placenta. Treatment of first trimester villous tissue explants with statins (pravastatin or cerivastatin) or inhibitors of N-glycosylation (tunicamycin, deoxymannojirimycin or castanospermine) altered receptor distribution in trophoblast and attenuated proliferation induced by IGF-I or IGF-II (Ki67; P < 0.05, n = 5). Decreased binding of Phaseolus vulgaris lectin and phytohaemagglutinin to IGF1R immunoprecipitated from treated explants demonstrated reduced levels of complex N-linked glycans. Co-incubation of tissue explants with statins and farnesyl pyrophosphate (which increases the supply of dolichol intermediates), prevented statin-mediated disruption of IGF1R localization and reversed the negative effect on IGF-mediated trophoblast proliferation. These data suggest that statins attenuate IGF actions in the placenta by inhibiting N-linked glycosylation and subsequent expression of mature IGF1R at the placental cell surface.

INTRODUCTION: Placental functional impairment in pregnancies with fetal growth restriction (FGR) can arise from fetoplacental vascular abnormalities. We aimed to compare the micro and macrovasculature of placentas from normal pregnancies with those showing late onset FGR. METHODS: Placental arterial casts (n�=�12 normal, 6 FGR) were prepared. Chorionic arterial number and inter-branch length were examined. Microvascular features were quantified in CD34-stained tissue sections obtained by systematic (n�=�12 normal, 12 FGR) and targeted (n�=�6 normal, 6 FGR) sampling from the placental periphery and centre. RESULTS: Adjusted for the weight of the placenta or the surface area of the chorionic plate, the number of chorionic arteries was similar in normal and FGR arterial casts. Inter-branch length per unit placental weight was greater in the first generation of arterial branches in FGR (p�

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 ?v?3 at the maternal surface to support adhesion during the early stages of implantation. WHAT IS KNOWN ALREADY: Integrin ?v?3 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 ?v?3 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 ?v?3 binding (n > 3). Decreased attachment stability of mouse embryos observed after siRNA knock-down of integrin ?v?3 or OPN itself, or OPN-coated beads after knock-down of integrin ?v?3, 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 ?v?3 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.

INTRODUCTION: Blood vessel glycosylation at the fetomaternal interface of four near-term specimens of tammar wallaby, Macropus eugenii, has been examined at days 23-26 of the 26.5 day pregnancy and compared with that of other species. METHODS: A panel of 23 lectins was used to compare vasculature in tammar with non-mammalian (shark, skink) and eutherian species at early and late gestation (camel, horse and alpaca), and term/near-term (cat, lion, dog, mink and elephant). RESULTS: Strikingly low levels of all the glycans tested, apart from sialic acids, were found in capillary endothelium of both the trilaminar omphalopleure and underlying surface endometrium of the tammar, though deeper endometrial vessels showed normally high levels of glycosylation. Only maternal vasculature of the mink placenta showed a comparable lack of expression. DISCUSSION: One reason for a reduced endothelial glycocalyx may be to facilitate diffusion of gases and nutrients as the tammar trophoblast lacks the indentation by overlying vessels that is seen in the other near-term placentae. Early epitheliochorial placentae of other species with equal diffusion distances to the tammar, showed normal vascular glycosylation. However, their pregnancies are much longer. CONCLUSION: The hypoglycosylation of tammar vessels at the fetomaternal interface may allow continued transfer of nutrients and gaseous exchange during the extremely rapid period of organogenesis which occurs during the short 26.5 day pregnancy of this marsupial. Given the short gestation period of the tammar, we suggest that a thinner endothelial glycocalyx has evolved to facilitate diffusion of gases and nutrients between the maternal and fetal compartments.

Introduction Syncytial nuclear aggregates (SNAs) are increased in pregnancy complications; however, little is known about their origin or function. This study aimed to characterise SNAs in more detail than has been reported previously. Methods Immunohistochemistry and morphological examination at the light and ultrastructural level were used to determine the nature and structure of SNAs. Results SNAs comprising bridges and syncytial knots had similar frequency with 974 per mm3 of villous tissue (IQR 717–1193) and 833 per mm3 (IQR 766–1190), respectively while there were approximately four times as many sectioning artefacts than knots and bridges combined. SNAs had increased proportions of condensed nuclei compared to the remaining syncytiotrophoblast (33.3% vs. 8.9%) and decreased proportions of euchromatic nuclei (0.0% vs. 16.2%), as assessed by examination of an electron micrograph archive. SNAs showed little evidence of apoptosis, with weak positivity for the apoptosis markers M30-neoepitope at 16.6% and TUNEL at 10.0%; strong staining was rarely seen for either marker. Immunofluorescence demonstrated rare association of actin (α, β or γ) with SNAs, whereas tubulin was in close proximity to SNAs and cytokeratin was seen within and surrounding SNAs. Discussion M30-positive SNAs traced through serial sections were significantly more likely to be syncytial knots or sectioning artefacts than bridges. Nuclei within SNAs showed signs consistent with degeneration; however, this is unlikely to be an apoptotic process. There are few changes in configuration of cytoskeletal proteins around SNAs. Conclusions These data suggest that the biogenesis and functional significance of SNAs still require resolution.

The use of nanoparticles in medicine is ever increasing, and it is important to understand their targeted and non-targeted effects. We have previously shown that nanoparticles can cause DNA damage to cells cultured below a cellular barrier without crossing this barrier. Here, we show that this indirect DNA damage depends on the thickness of the cellular barrier, and it is mediated by signalling through gap junction proteins following the generation of mitochondrial free radicals. Indirect damage was seen across both trophoblast and corneal barriers. Signalling, including cytokine release, occurred only across bilayer and multilayer barriers, but not across monolayer barriers. Indirect toxicity was also observed in mice and using ex vivo explants of the human placenta. If the importance of barrier thickness in signalling is a general feature for all types of barriers, our results may offer a principle with which to limit the adverse effects of nanoparticle exposure and offer new therapeutic approaches.

Tumor-homing peptides have been exploited to create nanocarriers for targeted delivery of therapeutic agents 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.

A simple method for the demonstration of placental cytotrophoblast cells is described, utilising the affinity of the lectin from Bandeiraea simplicifolia-II (BSA-II) for intracellular amylase-sensitive glycogen and a protocol using biotinylated BSA-II followed by an avidin-peroxidase revealing system. In early pregnancy, cytotrophoblast cells in chorionic and anchoring villi are deeply stained and with ongoing differentiation the staining gradually decreases in intensity, suggesting that this lectin can be a useful marker for these cells.

Insulin-like growth factors (IGFs) influence placental cell (cytotrophoblast) kinetics. We recently reported that the protein tyrosine phosphatase (PTP) SHP-2 positively regulates IGF actions in the placenta. In other systems, the closely related PTP, SHP-1, functions as a negative regulator of signaling events but its role in the placenta is still unknown. We examined the hypothesis that SHP-1 negatively regulates IGF actions in the human placenta. Immunohistochemical (IHC) analysis demonstrated that SHP-1 is abundant in cytotrophoblast. SHP-1 expression was decreased in first-trimester placental explants using siRNA; knockdown did not alter IGF-induced proliferation but it significantly enhanced proliferation in serum-free conditions, revealing that placental growth is endogenously regulated. Candidate regulators were determined by using antibody arrays, Western blotting, and IHC to examine the activation status of multiple receptor tyrosine kinases (RTKs) in SHP-1-depleted explants; amongst the alterations observed was enhanced activation of EGFR, suggesting that SHP-1 may interact with EGFR to inhibit proliferation. The EGFR tyrosine kinase inhibitor PD153035 reversed the elevated proliferation seen in the absence of SHP-1. This study demonstrates a role for SHP-1 in human trophoblast turnover and establishes SHP-1 as a negative regulator of EGFR activation. Targeting placental SHP-1 expression may provide therapeutic benefits in common pregnancy conditions with abnormal trophoblast proliferation.

This study characterises HERV-W (syncytin 1) expression in normal and pathologic placenta and in BeWo cells. HERV-W mRNA levels were higher in the first trimester than at term, and similar patterns were observed with another retrovirally-derived mRNA species, ERV-3. N-glycosylated syncytin 1 precursor (73 kDa) is cleaved to surface-associated (SU) and transmembrane (TM) subunits. Both were evident in villous trophoblast, where perinuclear and punctate cytoplasmic deposits were observed, and linear TM subunit immunoreactivity was seen at the syncytial microvillous membrane. Punctate immunoreactivity was seen in BeWo cells with antibodies to SU and TM, and the two were co-localised. SU immunoreactivity was observed in association with fetal endothelium, and this effect was increased in tissue from pre-eclamptic placentas, which also showed a higher level of total SU protein. Absence of the TM subunit from endothelium suggests it is not a biosynthetic source. We suggest that SU is released from trophoblast into fetal circulation where it may bind vascular endothelium.

Envelope glycoproteins of human endogenous retrovirus (HERV), such as syncytin 1 (HERV-W), are highly expressed in the placenta and some family members have immunomodulatory properties. Placental microvesicles (MV), which are shed into the maternal circulation during pregnancy, have been demonstrated to induce immune cell activation. Therefore, the aim of this study was to investigate the immunological properties of the highly expressed placental HERV-W protein, syncytin 1, and its potential involvement in placental MV modulation of immune cell activity. The MV shed from first trimester, normal term and pre-eclamptic term placentas, and from the BeWo trophoblast cell line, all contain syncytin 1. Recombinant syncytin 1 and syncytin 1-positive BeWo trophoblast MV both induced peripheral blood mononuclear cell (PBMC) activation, indicated through production of cytokines and chemokines. Reducing syncytin 1 content in BeWo MV inhibited PBMC activation. Recombinant syncytin 1 and syncytin-1-positive BeWo MV dampened PBMC responses to lipopolysaccharide challenge. Our findings suggest that syncytin 1 is shed from the placenta into the maternal circulation in association with MV, and modulates immune cell activation and the responses of immune cells to subsequent lipopolysaccharide stimulation. These studies implicate placental MV-associated HERV in fetal regulation of the maternal immune system.

Infertility and recurrent pregnancy loss (RPL) are prevalent but distinct causes of reproductive failure that often remain unexplained despite extensive investigations. Analysis of midsecretory endometrial samples revealed that SGK1, a kinase involved in epithelial ion transport and cell survival, is upregulated in unexplained infertility, most prominently in the luminal epithelium, but downregulated in the endometrium of women suffering from RPL. To determine the functional importance of these observations, we first expressed a constitutively active SGK1 mutant in the luminal epithelium of the mouse uterus. This prevented expression of certain endometrial receptivity genes, perturbed uterine fluid handling and abolished embryo implantation. By contrast, implantation was unhindered in Sgk1-/- mice, but pregnancy was often complicated by bleeding at the decidual-placental interface and fetal growth retardation and subsequent demise. Compared to wild-type mice, Sgk1-/- mice had gross impairment of pregnancy-dependent induction of genes involved in oxidative stress defenses. Relative SGK1 deficiency was also a hallmark of decidualizing stromal cells from human subjects with RPL and sensitized these cells to oxidative cell death. Thus, depending on the cellular compartment, deregulated SGK1 activity in cycling endometrium interferes with embryo implantation, leading to infertility, or predisposes to pregnancy complications by rendering the feto-maternal interface vulnerable to oxidative damage.

Insulin-like growth factor 2 (IGF2) enhances proliferation and survival of human first-trimester cytotrophoblasts (CTB) by signaling through the insulin-like growth factor 1 receptor (IGF1R). However, the role of the IGF2 receptor (IGF2R) in regulating trophoblast kinetics is unclear: It could act as a clearance receptor for trafficking excess ligand to lysosomes for degradation and/or directly mediate IGF2 signaling. We used an IGF2R knockdown strategy in BeWo cells and placental villous explants to investigate trophoblast proliferation and survival in response to stimulation by IGF. Both IGF1 and IGF2 significantly (P < 0.001) increased mitosis and reduced apoptosis in serum-starved BeWo cells. Small interfering RNA (siRNA)-mediated knockdown of IGF2R further enhanced IGF2-stimulated mitosis (P < 0.01), and IGF2-mediated rescue of apoptosis (P < 0.001) in these cells. Leu27IGF2, an IGF2 analogue that binds to IGF2R but not IGF1R, also protected IGF2R-expressing BeWo cells from apoptosis but did not increase mitosis. IGF treatment of term placental villous explants with reduced syncytial expression of IGF2R increased CTB proliferation (P < 0.001) and decreased apoptosis (P < 0.01) compared to untreated controls. Moreover, IGF2-mediated rescue of CTB apoptosis was significantly greater than that in tissue with normal IGF2R expression. Leu27IGF2 promoted mitogenesis and survival only in explants with intact IGF2R expression. Given that altered CTB turnover is observed in pregnancies complicated by fetal growth restriction, the development of strategies to manipulate the IGF2R signaling axis in the syncytiotrophoblast may provide a therapeutic avenue for treating this condition.

Macrophages accumulate within stromal tissue subjacent to the luminal epithelium in the mouse uterus during early pregnancy after seminal fluid exposure at coitus. To investigate their role in regulating epithelial cell expression of fucosylated structures required for embryo attachment and implantation, fucosyltransferase enzymes Fut1, Fut2 (Enzyme Commission number [EC] 2.4.1.69), and Fut4 (EC 2.4.1.214) and Muc1 and Muc4 mRNAs were quantified by quantitative real-time PCR in uterine epithelial cells after laser capture microdissection in situ or after epithelial cell coculture with macrophages or macrophage-secreted factors. When uterine macrophage recruitment was impaired by mating with seminal plasma-deficient males, epithelial cell Fut2 expression on Day 3.5 postcoitus (pc) was reduced compared to intact-mated controls. Epithelial cell Fut2 was upregulated in vitro by coculture with macrophages or macrophage-conditioned medium (MCM). Macrophage-derived cytokines LIF, IL1B, and IL12 replicated the effect of MCM on Fut2 mRNA expression, and MCM-stimulated expression was inhibited by anti-LIF and anti-IL1B neutralizing antibodies. The effects of acute macrophage depletion on fucosylated structures detected with lectins Ulex europaeus 1 (UEA-1) and Lotus tetragonolobus purpureas (LTP), or LewisX immunoreactivity, were quantified in vivo in Cd11b-dtr transgenic mice. Depletion of macrophages caused a 30% reduction in luminal epithelial UEA-1 staining and a 67% reduction in LewisX staining in uterine tissues of mice hormonally treated to mimic early pregnancy. Together, these data demonstrate that uterine epithelial Fut2 mRNA expression and terminal fucosylation of embryo attachment ligands is regulated in preparation for implantation by factors including LIF and IL1B secreted from macrophages recruited during the inflammatory response to insemination.

Angiotensin II (Ang II) is locally generated in the placenta and regulates syncytial transport, vascular contractility and trophoblast invasion. It acts through two receptor subtypes, AGTR1 and AGTR2 (AT1 and AT2), which typically mediate antagonising actions. The objectives of this study are to characterise the cellular distribution of AGTR1 and AGTR2 at the maternal–fetal interface and explore the effects on cytotrophoblast turnover. Low levels ofAGTR2mRNA were detected in first trimester placental homogenates using real-time PCR. Immunohistochemistry using polyclonal antibodies against AGTR1 and AGTR2 detected the receptors in first trimester placenta, decidua basalis and villous tip outgrowths in culture. Serial staining with cytokeratin-7 was used to identify extravillous trophoblasts (EVTs). AGTR1 was found in the syncytiotrophoblast microvillous membrane, in a subpopulation of villous cytotrophoblasts, and in Hofbauer cells. AGTR1 was strongly upregulated in cytotrophoblasts in cell columns and villous tip outgrowths, but was absent in interstitial and endovascular EVTs within the decidua. AGTR2 immunostaining was present in Hofbauer cells and villous cytotrophoblasts, but was absent from syncytiotrophoblast. Faint staining was detected in cell column cytotrophoblasts and villous outgrowths, but not in EVTs within the decidua. Both receptors were detected in placental homogenates by western blotting. Ang II significantly increased proliferation of cytotrophoblasts in both villous explants and villous tip outgrowths, but did not affect apoptosis. Blockade of AGTR1 and AGTR2 together abrogated this effect. This study shows specific expression patterns for AGTR1 and AGTR2 in distinct trophoblast populations at the maternal–fetal interface and suggests that Ang II plays a role in placental development and generation of EVTs.

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.

Lpar3 is upregulated in the preimplantation uterus, and deletion of Lpar3 leads to delayed uterine receptivity in mice. Microarray analysis revealed that there was higher expression of Col3a1 and Col6a3 in the Preimplantation Day 3.5 Lpar3−/− uterus compared to Day 3.5 wild-type (WT) uterus. Since extracellular matrix (ECM) remodeling is indispensable during embryo implantation, and dynamic spatiotemporal alteration of specific collagen types is part of this process, this study aimed to characterize the expression of four main uterine collagen types: fibril-forming collagen (COL) I and COL III, basement membrane COL IV, and microfibrillar COL VI in the peri-implantation WT and Lpar3−/− uterus. An observed delay of COL III and COL VI clearance in the Lpar3−/− uterus may be associated with higher preimplantation expression of Col3a1 and Col6a3. There was also delayed clearance of COL I and delayed deposition of COL IV in the decidual zone in the Lpar3−/− uterus. These changes were different from the effects of 17beta-estradiol and progesterone on uterine collagen expression in ovariectomized WT uterus, indicating that the altered collagen expression in Lpar3−/− uterus is unlikely to be a result of alterations in ovarian hormones. Decreased expression of several genes encoding matrix-degrading metallo- and serine proteinases was observed in the Lpar3−/− uterus. These results demonstrate that pathways downstream of LPA3 are involved in the dynamic remodeling of ECM in the peri-implantation uterus.

Objectives Histiotrophe is now recognized as being an important feature of early human pregnancy, providing nutrients and growth factors to the developing embryo. Our aim was to examine the glycan composition of histiotrophe from first trimester decidua and to compare it with secretions present in endometrial glands from the late secretory phase of the menstrual cycle. Study design Twenty samples of decidua from pregnancies between 8 and 11 weeks were processed into epoxy resin and sections stained with a panel of 22 lectins, together with six late secretory phase endometrial biopsies. Main outcome measures Specimens were analysed using a semi-quantitative ranking system and the density of lectin binding to the glandular secretions and the epithelium assessed. Results With the onset of pregnancy, β-galactose, α-N-acetyl galactosamine and N-Acetyl lactosamine bound by Arachis hypogaea, Glycine max , Helix pomatia and Erythrina crystagalli agglutinins appeared in terminal positions on oligosaccharide chains, suggesting loss of the capping sialic acid residues present in the non-pregnant state. Conclusions Suppression of terminal sialylation is evident during early pregnancy, suggesting that modifications to endometrial glandular activity occur in response to placental signals. The changes may facilitate absorption of histiotrophe by the trophoblast and enhance availability of substrates for degradation.