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This study investigated the hypothesis that different types of recurrent miscarriage history are associated with different markers of endometrial receptivity. A secondary objective was to compare the distribution in endometrial epithelium of a group of cell surface components with roles in cell adhesion. Of 54 women who had an implantation window endometrial biopsy, 17 had idiopathic recurrent fetal loss, 17 had idiopathic recurrent loss of empty gestation sacs, 10 had recurrent implantation failure and 10 had two or more normal pregnancies. Immunohistochemistry and HSCORE was used with frozen sections for integrins (alpha1beta1, alpha4beta1, alpha(v)beta3), and MUC1 (BC2) and paraffin sections for osteopontin and MUC1 (BC3). Epithelial beta1 integrins were located primarily in the basolateral membrane compartment. Consistently greater expression of alpha4beta1, alpha1beta1 and alpha(v)beta3 was seen in the luminal epithelium and greater expression of alpha4beta1 and alpha1beta1 in the glandular epithelium of women with recurrent fetal loss when compared with those with recurrent loss of empty gestation sacs. There were no significant differences in the expression of osteopontin or MUC1 between groups. Different endometrial integrin distribution was found in women suffering different types of recurrent pregnancy loss. It is postulated that impairment of the implantation barrier contributes to recurrent fetal loss.

Hemomonochorial placentation occurs in diverse species. We have examined placental glycosylation in five widely separated mammals with this type of placentation – lesser hedgehog tenrec (Echinops telfairi), spotted hyena (Crocuta crocuta), nine-banded armadillo (Dasypus novemcinctus), human (Homo sapiens) and guinea pig (Cavia porcellus) – in order to assess whether evolutionary convergence to the hemomonochorial state is accompanied by a similar convergence of glycan expression. Placentae from 2 E. telfairi, 3 C. crocuta, 1 D. novemcinctus, 4 womenand 1 C. porcellus were fixed and processed into epoxy resin. Binding of twenty-three lectins was assessed using a semiquantitative ranking system. The trophoblast apical/microvillous membrane of all five species showed marked similarities in glycosylation. In the N-linked series, there were abundant bi/tri-antennary complex chains, while the non-bisected variants were much scarcer. All species had plentiful N-acetyl lactosamine sequences; at chain termini, binding to Galβ1,4GlcNAc and Galβ1,3GalNAc sequences was greatly enhanced after neuraminidase treatment. In all species, terminal NeuNAcα2,3 residues were detected. The tenrec had unusually abundant terminal N-acetyl galactosamine. The basal plasma membrane/basal lamina showed glycosylation patterns distinct from the microvillous membrane in each case, indicating chemical diversity of the two opposite faces of trophoblast. Similar classes of glycan at the hemochorial interface suggest conservation of function. The observed lectin binding patterns suggest broad similarities of glycosylation that may have arisen by convergent evolution.

s / Placenta 36 (2015) A1eA60 A6 lactogen signalling, and their brains processed for microarray analysis and immunohistochemistry. Results: Maternal behaviour was significantly altered. We identified a deficit in the dam’s ability to retrieve pups and build a nest dependent of the genotype of the pups. Importantly, there were also marked differences in anxiety like behaviour across the cohorts. Microarray analysis demonstrated significant alterations in gene expression in the maternal brain, which preceded the aberrant maternal behaviours whilst BrdU Immunohistochemistry revealed variations in cell proliferation across the cohorts. Conclusion: This work directly demonstrates a link between placental signalling and the induction of maternal behaviour during pregnancy regulated by an imprinted gene. Additionally, it may have translational relevance as Phlda2 has been identified as aberrantly expressed in 25% of pregnancies blighted by fetal growth restriction potentially explaining the association between low birth weight and prenatal depression. NI2.6. TARGETED PLACENTAL DELIVERY OF INSULIN-LIKE GROWTH FACTOR-II INCREASES FETAL WEIGHT IN P0 MICE Anna King , John Aplin , Nicola Tirelli , Lynda Harris . Maternal and Fetal Health Research Centre, Institute of Human Development, The University of Manchester, Manchester, UK; Manchester Pharmacy School, The University of Manchester, Manchester, UK Objectives: Insulin-like growth factor-II (IGF-II) enhances placental function and improves fetal viability in animal models of fetal growth restriction (FGR); however, as the IGF-1 receptor is expressed ubiquitously, IGF-II cannot be safely administered to humans as a treatment for FGR. We have previously shown that targeted delivery of IGF-II to the placenta of wild type mice, using liposomes decorated with the placental homing peptide iRGD, significantly increased placental weight. Here we examine the outcome of targeted placental delivery of IGF-II in the placental-specific Igf2 knockout (P0) mouse, a well characterised model of FGR. Methods: Liposomes were prepared by the thin film extrusion method from the natural phospholipid distearoyl-phosphatidylcholine (DSPC; 32.5mM), cholesterol (15mM), polyethylene glycol (PEG; 1.875mM) and PEG-maleimide (0.625mM). The iRGD homing peptide (1.25mmol) was covalently attached to 25% of surface lipids and IGF-II (13mM) was encapsulated. Vehicle control (PBS), IGF-II (1mg/kg) or iRGD liposomes containing IGF-II (0.3mg/kg) were administered intravenously to P0 mice on embryonic (E) day 11.5, 13.5, 15.5 and 17.5 (N1⁄48/group). Animals were sacrificed on E18.5 and fetal and placental weights were collected. Results: Targeted administration of IGF-II significantly increased fetal weight (*P

Two methods are described for the study of human trophoblast invasion. When first-trimester placental villi are explanted on gels of a permissive extracellular matrix (ECM), a population of pure extravillous trophoblast cells grows out during the following several days from villous tips into the adjacent ECM. The outgrowths, which show a polarity and pattern of marker expression that replicates anchoring columns in vivo, may be used as the basis for preparation of small numbers of cells for gene expression studies, or for investigations of cell function and behavior. For quantitative studies, a standard trans-filter trophoblast migration experiment is described that starts from a homogeneous preparation of primary cells released from the tissue.

Two methods are described for the study of human trophoblast invasion. When first-trimester placental villi are explanted on gels of a permissive extracellular matrix (ECM), a population of pure extravillous trophoblast cells grows out during the following several days from villous tips into the adjacent ECM. The outgrowths, which show a polarity and pattern of marker expression that replicates anchoring columns in vivo, may be used as the basis for preparation of small numbers of cells for gene expression studies, or for investigations of cell function and behavior. For quantitative studies, a standard trans-filter trophoblast migration experiment is described that starts from a homogeneous preparation of primary cells released from the tissue.

Extravillous trophoblasts (EVTs) invade and remodel uterine spiral arteries. Regulatory factors may include inherent vessel susceptibility, local oxygen levels and tumour necrosis factor alpha (TNFalpha). We have used an in vitro model to investigate interstitial and endovascular invasion of myometrial spiral arteries from pregnant and non-pregnant uteri and also omental arteries. To model endovascular invasion, fluorescent-labelled EVTs were perfused into the lumen of these dissected vessels. For interstitial invasion, labelled EVTs were layered on top. Cultures were either maintained in 17% or 3% oxygen, or cultured with TNFalpha. The invasion of arteries from pregnant women occurred via both routes at 17% oxygen, with endovascular invasion more efficient than interstitial. In omental arteries and spiral arteries from non-pregnant women, endovascular invasion was limited. Endovascular and interstitial invasion were lower in all arteries at 3% oxygen. Typically, endovascular events were clustered, with an associated disruption in the adjacent endothelium and smooth muscle. A role for TNFalpha in limiting invasion was also supported. In conclusion, priming of uterine arteries may be necessary prior to EVT invasion. Oxygen is a sensitive regulator within this physiological model and increased invasion at higher pO2 may explain the homing of EVT to maternal arteries rather than veins. Adequate vascular transformation may therefore rely on a balance between vascular receptivity, oxygen partial pressure, and exposure to inflammatory mediators.

Experimental studies of human implantation in which embryos hatch and attach to endometrial epithelial cells in monolayer culture have shown that Mucin (MUC1) disappears from a small area of cells that surrounds the attached embryo. The term 'marker' is used advisedly because, while the inclusion criteria include the ability to function as an adhesion molecule, as well as presence at the implantation site, in vivo functional (IVF) evidence is so far mostly lacking. Heparin-binding EGF-like growth factor (HB-EGF) has been suggested to play two different roles: in addition to accelerating embryo development, it has a membrane-bound variant that can mediate intercellular attachment by binding to ErbB4 and heparan sulphate proteoglycan, both of which are present on the blastocyst. Critically, the biochemical dialogue between the embryo and the endometrium that determines endometrial receptivity at the attachment site cannot emerge from an analysis of either one alone.

Villous fibrosis is associated with oxygen deprivation in placental pathology, but the signaling networks and growth factors involved in activating the relevant cellular repair mechanisms are largely unknown. TGF is a powerful enhancer of extracellular matrix (ECM) production and an important immune suppressor that has been linked with fibrosis in several tissues. Here, cell culture methods were used to investigate possible links between hypoxia, elevated TGF beta 1, and altered ECM production in placenta. Term placental fibroblasts were isolated and cultured under hypoxia (3% O(2)) or in the presence of TGF beta 1, and the expression of fibronectin, collagen I, and collagen IV was examined using immunohistochemistry, ELISA of cell monolayers with associated ECM, and real-time RT-PCR. The effect of hypoxia on endogenous production of TGF beta 1-3 was also examined. Both TGF beta 1 and hypoxia increased fibronectin, collagen I, and collagen IV protein and mRNA in placental fibroblasts. However, TGF beta 1-3 production was not increased by culturing the cells under hypoxic conditions for 5 d. Thus, increased ECM expression under hypoxia was not mediated directly by increased TGF beta. We conclude that ECM production can be stimulated independently by hypoxia and TGF beta 1.

IGFs are critical in fetal growth because of their role in placental development and function. In this study, we used adenovirus (Ad-IGF) to deliver sense or antisense IGF-I or IGF-II cDNA to human primary placental fibroblasts (PPF) in vitro to determine whether this could lead to enhanced placental cell function. PPFs virally transfected with Ad-IGF-I or Ad-IGF-II showed 7-fold (P < 0.01) and 3-fold (P < 0.01) increases in [(3)H]thymidine incorporation at 48 h post infection compared with nontransfected controls. In a coculture system designed to assess cell migration, nontransfected PPF cells positioned over a monolayer transfected by Ad-IGF-I or Ad-IGF-II showed a more than 10-fold (P < 0.01) and a 7-fold (P < 0.01) increase in migration compared with cells positioned above a nontransfected monolayer. After 96 h in culture, PPFs transfected with sense Ad-IGF-I or Ad-IGF-II showed 2% apoptosis compared with 16% of nontransfected cells, whereas 37% and 25% of cells transfected with antisense Ad-IGF-I or Ad-IGF-II were apoptotic. This work has established that cells of placental origin are amenable to adenoviral transfection and that IGFs exert autocrine and paracrine effects on proliferation, migration, and survival, suggesting that enhancement of IGF levels in the placenta may augment placental function and increase fetal growth.

Growth in utero depends on adequate development and function of the fetal/maternal interface. During pregnancy, the insulin-like growth factors (IGFs), which are known to be critically involved in placental development, are controlled by a binding protein-IGFBP-1-produced by maternal decidualized endometrium. We have previously found that decidua also produces a protease that cleaves IGFBP-1; because proteolysis of IGFBP-1 may represent a mechanism for increasing IGF bioavailability, the present study aimed to identify the protease and its regulators to understand the control of IGF activity at the maternal/fetal interface. Immunochemical methods were used to show that decidualized endometrial cells from first-trimester pregnancy produced matrix metalloprotease (MMP)-3; incubation of IGFBP-1 with either this enzyme or MMP-9, which is produced by the trophoblast, produced a series of fragments that were unable to bind IGF-I. Western immunoblot analysis and immunocytochemistry demonstrated that decidual cells also produce tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-2, and alpha(2)-macroglobulin, and all three inhibitors attenuated the proteolysis of IGFBP-1 by MMPs. The N-terminal sequence analysis of the fragments revealed that the enzymes cleave IGFBP-1 at (145)Lys/Lys(146), resulting in a small (9-kDa) C-terminal peptide of IGFBP-1. These findings suggest cleavage of IGFBP-1 as a novel mechanism in the control of placental development by matrix metalloproteases.

Objective To characterize endometrial development in unexplained and tubal factor infertility. Design Prospective study of 20 women with unexplained infertility, 22 with tubal factor infertility, and 21 fertile controls in the midproliferative, periovulatory, and midluteal phases of the menstrual cycle. Setting Reproductive Medicine Department of St. Mary's Hospital, Manchester, United Kingdom. Patient(s) Women awaiting assisted conception. Investigation(s) Serum hormone assays, transvaginal ultrasound, Doppler, and midluteal endometrial biopsies. Main outcome measure(s) Serum levels of E2, P, and LH, endometrial ultrasound morphometry, uterine and subendometrial artery Doppler, and endometrial histology and biochemistry. Result(s) Women with unexplained infertility demonstrated significantly reduced uterine artery flow velocity in all phases, significantly elevated uterine and subendometrial artery impedance in the periovulatory and midluteal phases, and significantly reduced endometrial texture in the midproliferative phase. Women with tubal factor infertility demonstrated significantly reduced uterine artery flow velocity, without a concomitant increase in impedance, and significantly greater expression of endometrial glandular and luminal keratan sulphate. Conclusion(s) Unexplained infertility is associated with a profound impairment of endometrial perfusion that might be amenable to treatment by perfusion enhancers. Tubal factor infertility is associated with endometrial developmental defects that might be corrected by salpingectomy. Endometrial ultrasound and Doppler studies are likely to become a vital tool in the investigation of infertility.

Remodelling of the extracellular matrix (ECM) occurs during decidualization of the endometrium in mice. Previously we have documented the appearance of large-diameter collagen fibrils around mature decidual cells between day 5 and day 7 of pregnancy. Proteoglycans are important in the regulation of collagen fibrillogenesis, and the present study analysed four members (decorin, biglycan, lumican and fibromodulin) of the family of small leucine-rich proteoglycans (SLRPs) in the uterus from day 1 to day 7 of pregnancy. Decorin was present together with lesser amounts of lumican in the stroma before the onset of decidualization, whereas biglycan and fibromodulin were almost absent. Biglycan and, less significantly, lumican were expressed in decidualized regions of the endometrium, but decorin was absent. Fibromodulin was weakly expressed in the non-decidualized stroma, but only after implantation. Decorin and lumican were strongly expressed in the undifferentiated interimplantation site stroma, whereas biglycan and fibromodulin were expressed only weakly. These results indicate that the SLRP profile of the uterine ECM alters with differentiation of endometrial stromal cells. The large decidual collagen fibrils are thought to arise by lateral association of smaller diameter fibrils. As decorin has been shown to inhibit lateral association of collagen fibrils, its disappearance between day 2 and day 5 of pregnancy may be a prerequisite for the formation of large fibrils in decidua in mice.

Database mining revealed 102 extracellular matrix (ECM) genes amongst about 10000 mRNA species expressed in human placenta, and these were classified into collagens (23), non-collagenous glycoproteins (59) and proteoglycans (23). A panel of antibodies to selected collagens and glycoproteins was used to examine ECM distribution in the placental villous stroma. Collagens I and IV, fibronectin and fibrillin I were abundant in first trimester and term tissue. Some areas lacked collagen I, while collagen IV was clearly evident in interstitial locations. At term, laminin was present in the stroma as well as in trophoblastic and vascular basement membranes. Thrombospondin I, tenascin C and elastin showed more restricted distributions. Fibrosis has been reported in association with ischaemia, so ECM production by cultured term and first trimester placental fibroblasts was evaluated at three different oxygen concentrations. Fibronectin and collagen IV were more strongly expressed than collagen I, fibrillin I or thrombospondin I, while the production of laminin and elastin was very low. Reducing the oxygen tension led to a selective increase in fibronectin and collagen IV production. Thus both quantitative and qualitative alterations in ECM composition may be expected to accompany prolonged hypoxia.

The cell surface mucin MUC1 is expressed by endometrial epithelial cells with increased abundance in the secretory phase of the menstrual cycle, when it is found both at the apical cell surface and in secretions. This suggests the presence of a maternal cell surface glycoprotein barrier to embryo implantation, arising from the anti-adhesive property of MUC1. In previous work, we demonstrated alternatively spliced MUC1 variant forms in tumour cells. The variant MUC1/SEC lacks the transmembrane and cytoplasmic sequences found in the full-length variant. We now show that MUC1/SEC mRNA is present in endometrial carcinoma cell lines, endometrial tissue and primary cultured endometrial epithelial cells. The protein can be detected using isoform-specific antibodies in uterine flushings, suggesting release from endometrium in vivo. However, on the basis of immunolocalisation studies, MUC1/SEC also remains associated with the apical epithelial surface both in tissue and in cultured cells. Transmembrane MUC1 and MUC1/SEC are both strikingly localised to the apical surface of tubal epithelium. Thus MUC1 may contribute to the anti-adhesive character of the tubal surface, inhibiting ectopic implantation. The mechanism by which this barrier is overcome in endometrium at implantation is the subject of ongoing investigation.

Recent data on recurrent miscarriage (RM) is discussed in the framework of the selection failure hypothesis which states, 'Recurrent miscarriage is the result of failure of the prevention of 'poor quality' embryos implanting, allowing embryos that are destined to fail to implant and present clinically as recurrent miscarriage. Thus, recurrent miscarriage is a failure of nature's quality control.' The assumption that RM results from the maternal rejection of normal fetuses is challenged and evidence reviewed regarding the contribution of abnormal embryos and endometrial receptivity. Further research is needed to understand the mechanisms of maternal tract-embryo interaction and move towards improved management of recurrent pregnancy loss.

s / Placenta 35 (2014) A1eA112 A92 being the catalytic a subunit and the regulatory bII subunit the proteins mainly located in the inner membrane. The experiments with specific inhibitors of cyclases, phosphatases and kinases in JEG-3 cells (choriocarcinoma cells) are in process. 1. Endocrinology, 1997, 138, 2172-83 P2.98-N. THE HEXOSAMINE BIOSYNTHETIC PATHWAY: A ROLE IN NUTRIENT REGULATION OF GROWTH SIGNALLING IN THE HUMAN PLACENTA Victoria Palin, Ian O'Neil, Jim Warwicker, Sterre Paijens, John Aplin, Melissa Westwood The University of Manchester, Manchester, UK Objective: Glucose or glucosamine-stimulated flux through the hexosamine biosynthetic pathway (HBP) leads to the post-translational addition of N-acetyl-glucosamine (GlcNAc) at Ser/Thr residues of intracellular proteins (O-GlcNAcylation), influencing their function by altering location, interactions and phosphorylation. We have previously shown that increased HBP flux inhibits trophoblast proliferation; here we investigate the pathways responsible. Methods: An O-GlcNAcylation site prediction algorithm, developed using information on amino acid sequences flanking published O-GlcNAcylated residues, was used to screen proteins known to influence trophoblast proliferation. BeWo cells were cultured in serum-free DMEM/F12 containing glucose (5-25mM) or glucosamine (0.5-10mM) for 24h, then switched to control medium for a further 48h. Cell number and viability were assessed by counting and trypan-blue exclusion. Global protein GlcNAcylation was confirmed by Western blotting using an anti-O-GlcNAc antibody. The localisation and GlcNAcylation status of tyrosine-protein phosphatase non-receptor type 11 (SHP-2) were assessed by immunocytochemistry and Western blotting using SHP-2 immunoprecipitates with an anti-O-GlcNAc antibody or wheat-germ agglutinin (WGA) respectively. Results: Protein GlcNAcylation increased with increasing glucosamine/ glucose. 24h exposure to 10mM glucosamine led to BeWo cell death; lower concentrations (2.5-5mM) inhibited growth and cells returned to control medium remained in a slower proliferative cycle for at least 48h. 25mM glucose also inhibited proliferation. The prediction algorithm revealed potential O-GlcNAcylation sites in numerous transcription factors and signalling molecules, including Ser/Thr sites in SHP-2 that are close to Tyr residues needed for its activation. Blotting of SHP-2 immunoprecipitates with an anti-O-GlcNAc antibody orWGA demonstrated O-GlcNAcylation of SHP-2. Punctate SHP-2 membrane and cytoplasmic staining was present under all conditions tested suggesting glucose/glucosamine treatment does not affect its expression/localisation. Conclusion: SHP-2, a known regulator of trophoblast proliferation, is posttranslationally modified by O-GlcNAc; regulation of transcription factors / signalling molecules by GlcNAcylation suggests a mechanism by which placental growth can be altered by nutrient availability. P2.99-N. MIR-21 AND ITS ROLE ON TROPHOBLAST CELLS BY TARGETING PHOSPHATASE TENSIN HOMOLOG (PTEN) AND PROGRAMMED CELL DEATH 4 (PDCD4) Wittaya Chaiwangyen, Stephanie Ospina-Prieto, Diana M. MoralesPrieto, Ekkehard Schleussner, Udo R. Markert Placenta-Lab, Department of Obstetrics, University Hospital Jena, Jena, Germany Objectives:MicroRNAs (miRNAs) are a class of small non-coding RNAs (2124 nucleotides) that regulate gene expression by translational inhibition or transcript degradation via targeting the 3’ untranslated region of messenger RNA. MiR-21 is an oncomir and has been associated with various types of cancer. Our group has reported miR-21 as the highest expressed miRNA, out of 762 analyzed, in first trimester isolated trophoblast cells. In this study, we investigated the functions of miR-21 in trophoblastic cell lines and its possible target genes. Methods: The immortalized human trophoblast cell line HTR-8/SVneo and the choriocarcinoma cell line JEG-3 were transfectedwithmiR-21 inhibitor or mimic. Total RNA was extracted and RNA quality and quantity were determined by spectrophotometer. MiRNA-21 expression was quantified by qPCR. Cell growth and invasion were measured after treatment with miR-21 inhibitor or mimic for up to 72 h by using a colorimetric cell proliferation and a Matrigel invasion assay. Migration was assessed by a transwell migration assay and apoptosis by flow cytometry. The expression of the potential targets phosphatase and tensin homolog (PTEN) and programmed cell death 4 (PDCD4) of miR-21 was analyzed by RT-PCR and Western blotting. Results: Constitutive expression of miR-21 was higher in HTR-8/SVneo cells than in JEG-3 cells. MiR-21 inhibition in JEG-3 or HTR-8/SVneo cells significantly decreased proliferation, while overexpression resulted in a significantly increased proliferation. Invasiveness of JEG-3 cells was significantly reduced after silencing of miR-21. Overexpression of miR-21 significantly inducedmigration in HTR-8/SVneo cells but not in JEG-3 cells. Inhibition of miR-21 expression increased apoptosis in JEG-3 cells, but not in HTR-8/SVneo cells. Silencing of miR-21 enhanced expression of PDCD4 in JEG-3, but not in HTR8/SVneo cells, and PTEN expression in HTR-8/ SVneo, but not in JEG-3 cells. Overexpression of miR-21 significantly induced the opposite effects on the respective proteins in both cell lines. Conclusion: Both cell lines express miR-21 but at different levels. MiR-21 is involved in regulating cell growth, migration, invasion and apoptosis in trophoblast cells potentially by controling the intracellular signalling via PTEN or PDCD4. P2.100. RREPROGRAMMING BY GENE INDUCTION: THE FACTORS INVOLVED IN THE ESTABLISHMENT OF CANINE STEM CELLS Natalia Goncalves, Fabiana Bressan, Fl avio Meirelles, Carlos Ambr osio Faculty of Animal Science and Food Engineering, University of Sao Paulo, Pirassununga, Brazil Takahashi and Yamanaka established a technique where transcription factors related to pluripotency were incorporated in the genome of somatic cells in order to reprogram these cells. The expression of these transcription factors enables a somatic cell that presents a differentiated condition to reverse it to an embryonic state, generating induced pluripotent stem cells (iPSC). The production of iPSC from canine fetal fibroblasts was performed with lentiviral integrative systems by policistronic humam and mouse vectors (Stemcca), aiming to obtain cultures of pluripotent stem cells that would develop patterns of gene expression and epigenetic similar to cells derived from the animal model. Canine iPSC lines were produced and these clonal lines were cultivated with bFGF and independently of LIF and presented morphological characteristics that are more close to iPSCs produced in other species such as bovine than to previously described canine iPSC. In the first day after transduction, no differences were observed between murine and human infection. The cells in the reprogramming process presented early colonies at 11 days after transduction, who kept stable and similar to the stem cell morphological patterns. These colonies were picked by hand at the first passage and thereafter Tryple was successfuly used. Such cell lines were alkaline phosphatase positive, had the ability to form embryonic bodies, to spontaneously differentiate and to express pluripotency-related genes (Oct4, Sox2). This work demonstrates it was possible to efficiently reprogram canine somatic cells using lentiviral vectors and proves these cell lines are pluripotent through posive results on the pluripotency tests. Also, these new results combining the use of Tryple enzyme and bFGF supplementation only, unlike what has been done for this specie. P2.101-N. INTERACTIONS OF UMBILICAL MESENCHYMAL STEM CELLS WITH UTERINE AND UMBILICAL ENDOTHELIAL CELLS Neven Ebrahim, Jennifer Sedcole, Olivia Volk, Lopa Leach School of Life Sciences, Nottingham University, Nottingham, UK

s / Placenta 35 (2014) A1eA112 A42 uterine loop arteries were isolated to assess vascular function ex vivo using wire myography. The constriction response to phenylephrine (PE, 10-5M) and the endothelium-dependent relaxation response to acetylcholine (Ach, 10-9 e10-5M) were evaluated. All data are expressed as mean±SEM; statistical analysis was performed using unpaired t test or two-way ANOVA. Result: Data (treated vs untreated) confirmed that melatonin supplementation increased WT fetal weights (1.21±0.01 vs 1.15±0.02; grams, p 20 weeks (FD rate of 1.6/1000). The incidence of pPROM was similar (0.5% vs 0.0%, in LPAPP-A and the normal group respectively, p1⁄4ns).

Fetal alcohol spectrum disorder (FASD) describes developmental issues from high maternal alcohol intake, which commonly results in fetal growth restriction and long term morbidity. We aimed to investigate the effect of alcohol and acetaldehyde, on the first trimester placenta, the period essential for normal fetal organogenesis. Normal invasion and establishment of the placenta during this time are essential for sustaining fetal viability to term. We hypothesise that alcohol (ethanol) and acetaldehyde have detrimental effects on cytotrophoblast invasion, turnover and placental function. Taurine is an important amino acid for neuronal and physiological development, and so, its uptake was assayed in cells and placental explants exposed to alcohol or acetaldehyde. First trimester villous explants and BeWo cells were treated with 0, 10, 20, 40 mM ethanol or 0, 10, 20, 40 µM acetaldehyde. The invasive capacity of SGHPL4, a first trimester extravillous cytotrophoblast cell line, was unaffected by ethanol or acetaldehyde (p>0.05; N = 6). The cells in-cycle were estimated using immunostaining for Ki67. Proliferating trophoblast cells treated with ethanol were decreased in both experiments (explants: 40% at 20 mM and 40 mM, p

Insulin-like growth factors (IGFs) are crucial for many aspects of development, growth, and metabolism yet control of their activity by IGF-binding proteins (IGFBPs) remains controversial. The effect of IGFBP-1 depends on its phosphorylation status; phosphorylated IGFBP-1 inhibits IGF actions whereas the nonphosphorylated isoform is stimulatory. In order to understand this phenomenon, we purified phosphorylated IGFBP-1 from normal human plasma by immunoaffinity chromatography. Unexpectedly, the resulting preparation enhanced IGF-stimulated 3T3-L1 fibroblast proliferation, due to the presence of a co-purified protein of approximately 700 kDa. Matrix-assisted laser desorption ionization-mass spectrometry and Western immunoblotting analysis identified this co-purified protein as alpha(2)-macroglobulin (alpha(2)M). Anti-alpha(2)M antibodies co-immunoprecipitated IGFBP-1 from human plasma and from (125)I-IGFBP-1.alpha(2)M complexes formed in vitro. The (125)I-IGFBP-1/alpha(2)M association could be inhibited with excess unlabeled IGFBP-1. Surface plasmon resonance analysis indicated that alpha(2)M preferentially associates with the phosphorylated isoform of IGFBP-1 and that when complexed to alpha(2)M, IGFBP-1 can still bind IGF-I. These findings have functional significance since alpha(2)M protects IGFBP-1 from proteolysis and abrogates the inhibitory effect of phosphorylated IGFBP-1 on IGF-I stimulated 3T3-L1 cell proliferation. We conclude that alpha(2)M is a binding protein of IGFBP-1 which modifies IGF-I/IGFBP-1 actions resulting in enhanced IGF effects. In line with its role in regulating the clearance and activity of other growth factors, we predict that alpha(2)M has a novel and important role in controlling the transport and biological activity of IGFs.

Epidemiological evidence suggests that size at birth may affect health in later life. The growth of the fetus may be adversely affected by a suboptimal maternal environment. Understanding placental development and function will help unravel the mechanisms controlling fetal growth. This article poses the problem: how does the maternal environment (uterine or systemic) influence placental development? Critical human placental functions include remodelling maternal uterine spiral arteries to increase the flow of blood to the maternofetal interface, and transferring oxygen and nutrients into the fetal vasculature, all processes involving trophoblast. Gene ablations that affect pregnancy outcome in mice lead to some interesting hypotheses.