IFPA Meeting 2009 Workshops Report

Article Outline

• Abstract
• 1. Introduction
• 2. Workshop 1: immune response to pregnancy
• 3. Workshop 2: signaling between fetus and placenta
• 4. Workshop 3: bioactive lipids in placenta
• 5. Workshop 4: placenta in agricultural species
• 6. Workshop 5: epigenetics and placentation
• 7. Workshop 6: trophoblast deportation
• 8. Workshop 7: glucocorticoids and placental function
• 9. Workshop 8: endothelium
• 10. Workshop 9: placental transport: what don't we know?
• 11. Workshop 10: uteroplacental blood flow: from morphology to Doppler waveforms
• 12. Workshop 11: placental stem cells
• 13. Workshop 12: genes and placenta
• Conflict of interest
• Copyright

Abstract 

Workshops are an important part of the annual meeting of the International Federation of Placenta Associations (IFPA). At IFPA Meeting 2009 diverse topics were discussed in twelve themed workshops. Topics covered included: immune response to pregnancy; signaling between fetus and placenta; bioactive lipids in placenta; placenta in agricultural species; epigenetics and placentation; trophoblast deportation; glucocorticoids and placental function; endothelium; placental transport; genes and placenta; uteroplacental blood flow and placental stem cells. This report is a full summary of the various topics covered.

Keywords: Placenta, Trophoblast, Workshops

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1. Introduction 

IFPA Meeting 2009 was held in Adelaide, Australia 6–9 October 2009. One of the major aims of IFPA meetings is to promote discussion and debate on a wide range of topics relating to the placenta. To this end twelve diverse 2 h long workshops were held. The following is a summary of the topics covered and discussions held.

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2. Workshop 1: immune response to pregnancy 

Organizers: Sarah Robertson, University of Adelaide, Australia; Shigeru Saito, University of Toyama, Japan.

Speakers: Judith Cartwright, University of London, UK; Larry Chamley, University of Auckland, New Zealand; Ted Golos, University of Wisconsin–Madison, USA; Leigh Guerin, University of Adelaide, Australia; Lachlan Moldenhauer, University of Adelaide, Australia; Peggy Petroff, Kansas University Medical Centre, USA; Tamara Tilburgs, Harvard University, USA.

Aim: Cells, cytokines and regulatory molecules of the immune system are important determinants of placental development and function, acting through direct effects on trophoblast cell survival, proliferation, differentiation and interactions with the uterine vasculature and decidual tissues. Both the innate and adaptive compartments of the immune response are involved and are essential for constraining adverse responses to placental antigens which are implicated in implantation failure, recurrent miscarriage and pre-eclampsia. The aim of this workshop was to explore the latest information on the identity, regulation and function of key leukocyte subsets that mediate maternal immune tolerance and tissue remodeling in development and maintenance of the placenta, and to examine how their dysregulation might underlie pathologies of pregnancy.

Summary: Lachlan Moldenhauer discussed recent data on how T cell phenotype in early pregnancy may determine pregnancy success. It has been hypothesized that the cytokine environment at the time of initial maternal T cell activation to paternal antigens influences the maternal T cell phenotype and pregnancy success. Data were presented on the effect of cytokine environment on T cell effector function utilizing a transgenic male mouse expressing ovalbumin (OVA) to provide paternal antigen and OVA-reactive CD8+ OT-I T cells. Females that received cytotoxic OT-I T cells activated in vitro with interleukin (IL)-2 had 60% fetal resorption, but only when conceptus tissue expressed OVA. However, high fetal resorption rates were not observed in OVA-expressing pregnancies which received either naïve OT-I T cells activated in vitro with transforming growth factor (TGF)-β1 or IL-10. This demonstrates that the cytokine environment at the outset of the immune response to pregnancy influences the T cell phenotype, impacting upon maternal immune tolerance and pregnancy outcome. In particular, TGF-β1 and IL-10 may be key cytokines in conferring a tolerogenic phenotype to paternal antigen reactive T cells.

Continuing the theme of tolerance to placental antigens Peggy Petroff presented data on the role of CD4+ cells in this process. To examine the mechanisms of maternal tolerance to the semi-allogeneic fetus, the fate of maternal CD8+ and CD4+ T cells specific for a defined fetal antigen, ovalbumin, was investigated. OT-I or OT-II T cells, which express a T cell receptor (TCR) specific for class I or class II-restricted epitopes of ovalbumin, respectively, were examined in females carrying pups expressing paternally-inherited ovalbumin. While both maternal CD4+ and CD8+ T cells clearly encountered fetal ovalbumin, patent differences between the fates of each population were evident. Maternal antigen-specific CD4+ T cells were subjected to deletion, TCR downregulation and upregulation of inhibitory receptors; CD8+ T cells failed to undergo similar changes. CD4+ T cells in pregnant mice also prominently displayed regulatory T cell properties. Finally, heightened fetal loss was observed in OT-I but not OT-II mice. Collectively, these data suggest that distinct mechanisms regulate maternal tolerance of fetal antigen-specific CD4+ and CD8+ T cells.

Leigh Guerin discussed how the abundance of CD4⁺Foxp3⁺ T regulatory cells is influenced by IL-10 together with fetal alloantigens. Several pathways are required to work in concert to achieve maternal immune tolerance of the allogeneic fetus. Two examples of tolerogenic mechanisms employed during gestation are cytokine pathways dominated by type 2 cytokines such as IL-10 and dynamic coordination of regulatory T (Treg) cells. To examine the possible link between these two pathways the kinetics of Treg cell abundance in IL-10 deficient (IL-10−/−) mice was assessed throughout pregnancy. Data were presented showing that in the absence of IL-10 there was an almost 10-fold increase in Treg cell numbers during mid-gestation that were primarily localised to uterine draining para-aortic lymph nodes (PALN). The deficiency of IL-10 did not cause any form of pregnancy pathology or compromise the suppressive capacity of the Treg cells, as assessed in an in vitro suppression assay. Therefore, it was postulated that these changes in Treg cell populations may act to compensate for the loss of IL-10-regulated T cell subsets such as Tr1 cells of Th2 cells.

Tamara Tilburgs presented data on decidual T cell reactivity to placental MHC antigens in humans. It is well established that human extravillous trophoblast cells (EVT) do not express HLA-A, HLA-B, HLA-DR, HLA-DQ and HLA-DP molecules that are the main targets for allo-reactive lymphocytes in transplantation. However, EVT do express HLA-C, HLA-E, HLA-F and HLA-G molecules by which they can avoid decidual NK cell mediated cytotoxicity. The role of decidual T cells and their interaction with HLA molecules on EVT is less well understood. In pregnancies containing an HLA-C mismatched child (compared to the mother) there was an increased percentage of CD4+CD25^dim activated T cells in decidual tissue. In addition, HLA-C mismatched pregnancies exhibited a decidual lymphocyte response to fetal cells and contained functional CD4+CD25^bright regulatory T cells in decidual tissue, whereas HLA-C matched pregnancies did not. These data suggest that decidual T cells specifically recognize fetal HLA-C at the fetal–maternal interface but are prevented by Treg cells from inducing a destructive immune response in uncomplicated pregnancies.

Larry Chamley went on to discuss macrophage activation in response to necrotic trophoblast cells in humans. The human placenta is bathed in maternal blood and like other epithelia the surface layers of trophoblast cells are desquamated as they age and die. This dead trophoblast material is shed into the maternal circulation and deported away from the placental site. A range of material of trophoblast origin is shed from the placenta and collectively this material is referred to as deported trophoblast. It is very difficult to obtain deported trophoblast from maternal blood but this group has recently developed a model employing placental explants that allows the harvest and study of deported trophoblast material. Using this model it has been shown that deported trophoblast material is normally apoptotic and can be phagocytosed by macrophages leading to a tolerogenic response associated with increased IL-10 and indoleamine dioxygenase (IDO) expression. Deported trophoblast material that is induced to become necrotic is also phagocytosed but is associated with increased class II HLA expression and inflammatory cytokine production by macrophages. It has been hypothesized that phagocytosis of apoptotic deported trophoblast material may be part of the mechanism the fetus uses to establish tolerance within the maternal immune system in normal pregnancy but that when deported trophoblast material undergoes necrotic death their phagocytosis may lead to inappropriate maternal immune responses.

Judith Cartwright presented preliminary data supporting the hypothesis that decidual macrophage phenotype is altered in pre-eclampsia. Cytokine secretion by macrophages alters depending on their activation state, potentially impacting on trophoblast migration and invasion. Poor trophoblast invasion has been implicated in the pregnancy disorder pre-eclampsia. Data were presented on CD14+ decidual macrophages isolated from pregnancies at 10–14 weeks gestational age characterized by uterine artery Doppler ultrasound to be at the highest (30%) and lowest (<1%) risk of developing pre-eclampsia if the pregnancy had progressed. They characterized MHC Class I and II expression, cytokine secretion profiles and the effect of macrophage cell culture supernatants on trophoblast invasion. Macrophages from higher risk pregnancies had lower MHC Class II expression, which may represent a less activated phenotype, and an altered cytokine secretion profile as determined by protein array. Of particular note was that the pro-inflammatory cytokines IL-1β and TNF-α were decreased in the higher risk pregnancy group. Macrophage cell culture supernatants from low risk pregnancies promoted a greater level of trophoblast invasion than those from higher risk pregnancies. It was concluded that these data support the hypothesis that macrophages have important roles in regulating trophoblast functions and that this may be altered in pregnancies that go on to be complicated by pre-eclampsia.

Ted Golos described recent data from his laboratory on in vivo perturbation of the placental–maternal immune dialog in a non-human primate model. The cells at the primate maternal–fetal interface that engage in placental-immune dialog have been studied extensively, particularly in the human, where HLA-G and putative targets (NK cells, macrophages, T cells) are readily available for in vitro studies. However, the significance of MHC-leukocyte interactions is exceedingly difficult to interrogate with in vivo studies. They have recently developed two approaches for studying the trophoblast–leukocyte dialog in vivo in the rhesus monkey, where the primary trophoblast MHC class I molecule, Mamu-AG, is considered homologous to HLA-G. Passive immunization studies with anti-Mamu-AG antibodies have demonstrated disruption of placental villous growth, decidual differentiation and spiral arteriole remodeling by endovascular trophoblast, giving in vivo evidence of a physiological role in early pregnancy. Based on this approach, a protocol for immunodepletion of peripheral blood leukocytes in rhesus monkeys has been developed to modulate the decidual leukocyte population. Successful paradigms for depleting peripheral blood NK cells have been established and although studies are in the early stages, a rapid negative impact on pregnancy maintenance has been observed. While complexities of interpretation remain, the preliminary results suggest that non-human primate implantation is extremely sensitive to immune manipulation and could provide a model for early pregnancy loss.

Conclusions: It is clear from this workshop that we are beginning to understand the mechanisms underlying activation of the adaptive immune response in pregnancy. However, it was also recognised that there is likely a fine balance in the regulation of these processes towards immune tolerance. What is not clear is how the adaptive immune response interacts with the innate immune response in early pregnancy development and how these interactions may contribute to adverse pregnancy outcomes.

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3. Workshop 2: signaling between fetus and placenta 

Organizers: Gernot Desoye, Medical University of Graz, Austria; Miguel Constancia, University of Cambridge, UK.

Speakers: Miguel Constancia, University of Cambridge, UK; Rohan Lewis, University of Southampton, UK; Annette Osei-Kumah, University of Adelaide, Australia; Claire Roberts, University of Adelaide, Australia; Richard Saffery, University of Melbourne, Australia.

Aim: The mammalian fetus, although entirely dependent on its mother's nutrients, is not just a passive recipient, but influences its own development and growth. It subverts many of the mother's physiological activities to its own end, to ensure adequate mobilization of nutrients and oxygen through the placenta. Fetal signals also regulate placental growth and development. Understanding signaling systems between the fetus and the placenta is thus a central issue in placental and fetal development. The aim of this workshop was to focus on the complex regulatory mechanisms by which nutritional and oxygen status, endocrine factors and genetic factors work together to regulate signaling networks operating at the fetal–placental interface, with particular emphasis on novel aspects related to (1) genetic basis of supply and demand for maternal nutrients; (2) role of the placenta as the “environmental” sensor of the fetus; (3) potential links between epigenetic modulation of gene expression and adverse pregnancy outcomes related to “environmental” mechanisms; (4) pathways linking genetic drive for growth, placental efficiency and endocrine regulation and (5) the multisystem nature of fetal signals that promote placental adaptations. A key goal of the workshop was to facilitate collaborative interactions among investigators and stimulate the development of this exciting emerging area in reproductive biology. Moreover, it aimed to identify key areas for future research.

Summary: Rohan Lewis hypothesized that placental–fetal cycling of amino acids may provide a mechanism for signaling between the placenta and fetus. Fetal placental cycling of glutamate and glutamine occurs in sheep and may also occur in humans. This cycle involves the placenta taking up fetal glutamate, converting it to glutamine and releasing it back to the fetus where it is converted to glutamate and returned to the placenta. Preliminary evidence suggests that this process occurs in the human; the isolated perfused human placenta takes up fetal glutamate, converts this glutamate to glutamine, and releases it back to the fetus. Placental production of glutamine and its release to the fetus may signal the fetus regarding maternal nutrient availability and placental capacity. In contrast fetal production of glutamate may signal to the placenta regarding fetal metabolic status. It was further hypothesized that this may provide one mechanism by which fetal growth can be modulated to match maternal capacity to support ongoing pregnancy.

Claire Roberts discussed the contribution of both maternal and paternal genetics to complications of pregnancy. The mother and father contribute one copy of each gene to the fetus and placenta while the mother also contributes the environment in which the fetus grows. However, the role of the father has been under appreciated with men conferring a strong genetic influence on pregnancy outcome. Both men and women who were themselves born small for gestational age (SGA) have a 3–5 times increased risk of parenting an SGA infant. A number of genes that are imprinted and expressed from only the paternal allele are known to affect placental invasion and function, and hence fetal growth. A strong candidate is insulin-like growth factor (IGF)-2 that is abundantly expressed both in placental trophoblast cells and in a number of fetal tissues. Evidence was presented that polymorphisms in IGF2 and related genes in the father are associated with adverse pregnancy outcomes including pre-eclampsia and preterm birth. It seems likely that these polymorphisms affect placental differentiation and function.

Miguel Constancia discussed the role of imprinted genes in the regulation of supply and demand for maternal nutrients. Imprinted genes are expressed from only one of the parental chromosomes and control the provision and use of maternal resources during pregnancy and lactation. His group focuses on feto-placental signaling of nutrient demand in mouse models with mismatched placental and fetal growth. Using molecular, morphological and physiological analyses it has been demonstrated that decreasing or increasing fetal demand genetically, significantly impacts on placental supply capacity through a variety of morphological and functional adaptations. This work provides direct evidence that genetic fetal drive for growth is a major determinant of placental nutrient supply. One mechanism by which the placenta responds to fetal demand signals is through regulation of specific transport systems. Altered System A amino acid transporter activity is a common “denominator” in models of mismatched growth, strongly suggesting that System A is an essential component of a dynamic system that orchestrates the cross talk among mother, placenta and fetus. Recent data were discussed that provide strong experimental evidence for a role of imprinting in the regulation of placental endocrine function, with important implications for maternal metabolism and resource allocation to the fetus.

Annette Osei-Kumah discussed sex specific differences in placental microRNA (miR) in normal and pathophysiological pregnancies. MicroRNAs are non-coding small RNAs and act as important post-transcriptional regulators of gene expression by altering the abundance or translational efficiency of mRNA. miR-mediated gene regulation is important for normal physiological and cellular functions. Recent evidence suggests an important role for miRs in pathological conditions. Sex specific differences in placental gene expression are associated with strategies for optimal growth and fetal survival in the presence of maternal asthma. It has been hypothesized that post-transcriptional regulation of genes by miRs may play an important role in conferring sexual dimorphism in placental gene expression during development. Data were presented from a study that examined sex differences in miR expression in the placenta of normal pregnancies and in the presence of maternal asthma. miR expression analysis was determined on male (n = 12) and female (n = 12) placenta from normal pregnancies and pregnancies complicated by asthma using Exiqon arrays. A target prediction algorithm database was used to identify predicted targets for identified miRs while Ingenuity Pathways Analysis software was used to identify functional networks. In the presented study 106 miRs were differentially expressed between male and female placentae in normal pregnancy (P < 0.05). In the presence of maternal asthma 75 miRs were differentially expressed between male and female placentae. This included miR-203 and miR-223 which have been associated with chronic inflammatory disease and cytokine response. The majority of the differentially expressed miRs were clustered on chromosomes 13, 14, 16, 17, 19, 20 and X. Pathway analysis identified networks involved in innate immune activation, cytokine signaling, glucocorticoid receptor signaling, cellular growth and proliferation. It was concluded that there are differentially expressed miRs between male and female placenta in normal pregnancies and pregnancies complicated by asthma, which target genes involved in cytokine expression and other immune pathways in the placenta. This may be related to the differential gene regulatory mechanisms initiated by males and females in utero for growth and survival.

Richard Saffery discussed the role of epigenetics in the regulation of vitamin D homeostasis at the fetomaternal interface. Plasma concentrations of biologically active vitamin D [1α,25β(OH)₂D] are tightly regulated via feedback regulation of renal 1β-hydroxylase (CYP27B1; positive) and 24-hydroxylase (CYP24A1; catabolic) enzymes. In pregnancy, this regulation is uncoupled and maternal 1α,25β(OH)₂D levels are significantly elevated, suggesting a specific role in pregnancy development. The mechanisms underlying this differential regulation remain to be fully elucidated. The potential role of epigenetic modification of genes regulating vitamin D bioavailability and activity at the fetomaternal interface was investigated. Data were presented demonstrating that no methylation of the vitamin D receptor (VDR) and CYP27B1 genes was found in any placental tissues tested. In contrast, the CYP24A1 gene is methylated in human placenta, purified first trimester cytotrophoblast cells and primary and cultured excess chorionic villous sampling tissue. No methylation was detected in any somatic human tissue tested. It was further confirmed that CYP24A1 promoter methylation directly downregulates basal promoter activity and abolishes Vitamin D-mediated feedback transcriptional activation in trophoblast cells. The data presented suggest that pregnancy-specific (placental) epigenetic modification plays an important role in decoupling maternal regulation of vitamin D feedback catabolism during pregnancy, thereby maximising active Vitamin D at the fetomaternal interface.

Conclusions: It was clear from this workshop that the mechanisms of signaling between placenta and fetus continue to be largely unknown. Key areas for future research are the identification of placental signals of maternal nutrient availability to the fetus and fetal demand signals for growth to the placenta. It will be important to characterize environmental induced epigenetic changes in the placenta as these are likely to be important modulators of placental–fetal signaling. The extent to which gender and parental specific actions affect this signaling circuitry also needs to be established.

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4. Workshop 3: bioactive lipids in placenta 

Organizers: Denise Hemmings, University of Alberta, Canada; Jeff Keelan, University of Western Australia, Australia.

Speakers: Louiza Belkacemi, University of California Los Angeles, USA; Daniele Evain-Brion, Université Paris Descartes, France; Denise Hemmings, University of Alberta, Canada; Jeff Keelan, University of Western Australia, Australia; Tomomo Kotani, Nagoya Graduate University School of Medicine, Japan; Stuart Pitson, Institute of Medical and Veterinary Science/Hanson Institute, Australia; Ambika Singh, University of Western Australia, Australia.

Aim: The major theme of this workshop was “The unappreciated importance of bioactive lipids and their metabolites as regulators of placental growth, invasion, differentiation and function”. The effects of growth factors, cytokines and polypeptide hormones on placental development and function have been extensively investigated. However, virtually all aspects of cellular function are also regulated by lipids, which are typically derived enzymatically from abundant substrates in the cellular or extracellular environment. Despite this, the role of bioactive lipids in regulating placental growth, differentiation, survival, migration and function has received relatively little attention, and our understanding of this area remains in its infancy. This may be due, in part, to the complex nature of lipid biochemistry and the complex array of potential mediators and receptors to be studied. In this workshop, recent findings relating to the biological functions of lipids with respect to various aspects of placental function were discussed. The workshop was focused on three groups of lipids known for their widespread and potent biological actions: lysophospholipids, sphingolipids and long-chain fatty acids and their oxidative metabolites. The role and significance of lipid receptors, both nuclear and on the cell surface, in mediating these effects were also explored.

Summary: Jeff Keelan gave a brief review of the major bioactive lipid mediator classes and their receptors. The wide array of mediators derived from fatty acid through cyclooxygenase, lipoxygenase and epoxygenase metabolism were introduced. These include the prostanoids, leukotrienes, lipoxins, HODEs, HETEs, resolvins, protectins, maresins and endocannabinoids. The products of non-enzymatic oxidation, peroxidation and nitrosylation (e.g. isoprostanes, nitro-alkenes) were also introduced. Many of these lipids act via G protein-coupled receptors (GPCR), which segregate phylogenetically into distinct groups, although ligand specificity does not always neatly correlate with GPCR sequence homology. However, some of these lipids are also ligands for nuclear receptors such as the PPARs (e.g. 9-HODE, 15-HETE, 15d-PGJ2, nitro-alkenes). Some of the most widely studied lipid mediators are the phosphorylated lipids such as lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P). These ligands have pleiotropic actions in many cells and tissues, exerted through binding to multiple GPCRs (members of the EDG family and others). Despite an abundance of knowledge concerning the actions of LPA and S1P in many biological systems, their roles in pregnancy remain essentially unexplored. Preliminary evidence was presented that the placenta and extraplacental membranes express the major lysophospholipid generating enzyme, lysophospholipase-D (LPLD). Immunoblotting data suggested that in human placental cells in culture the majority of this protein is secreted and is readily detected in media. RT-PCR data were also presented demonstrating that these same tissues express receptors for LPA and S1P. Receptor expression was altered with trophoblast differentiation in vitro, suggesting roles for these ligands in the differentiated phenotype. A more recently identified class of bioactive lipids, the resolvins, also acts via binding to GPCRs, namely BLT1, GPCR1 and Chemr23. The production and actions of n3-PUFA-derived resolvins in the placenta is currently being investigated to determine their potential role in mediating protection from inflammation associated with dietary n3-PUFA supplementation. Preliminary evidence was presented demonstrating that all three receptors are expressed in gestational tissues; BLT1 and GPCR1 have been localised immunohistochemically to the amniotic epithelium, chorionic trophoblast and placental syncytium. The effects of activation of these receptors by n3-PUFA metabolites are yet to be determined.

Daniele Evain-Brion discussed the potential role of peroxisome proliferator-activated receptor γ (PPARγ) ligands in human trophoblast differentiation. In human placenta PPARγ, a nuclear receptor essential for placental development, is specifically expressed by both extravillous and villous trophoblast. In primary cultures of human EVT, PPARγ agonists inhibit EVT invasion in a concentration-dependent manner. The presence of oxidized-LDLs at the materno-fetal interface suggests that oxidized-LDLs from maternal sera might be a source of potential PPARγ ligands for trophoblast cells. The oxidized LDL receptor (LOX1) is expressed in trophoblast. Oxidized-LDLs decrease trophoblast invasion in vitro and analysis of their content revealed that they contain potent PPARγ agonists such as eicosanoids, but also oxysterols, which are ligands for another nuclear receptor, the liver-X-Receptor (LXR). LXRβ was found to be expressed in trophoblast, and LXR agonists shown to inhibit trophoblast invasion. Data were also discussed on the role of lipids at the materno-fetal interface on human trophoblast differentiation in physiological and pathological clinical conditions.

Louiza Belkacemi presented data on the role of PPARγ in placental apoptosis. Although a relationship between placental function and fetal well-being is well established, the complex regulation of placental growth and development remains poorly understood especially in pregnancies complicated by intrauterine growth restriction (IUGR). PPARγ has emerged in recent years as a pivotal regulator, not only of placental development and trophoblast differentiation, but importantly of apoptosis. Recent studies demonstrate that maternal food restriction (MFR) during rat gestation causes marked reduction in maternal, fetal and placental basal (site of hormone production) and labyrinth zone (site of fetomaternal exchange) weights at near term gestation (E20). Further, the levels of apoptosis in both placental zones are significantly increased at E20. Consistent with this, PPARγ protein expression is significantly downregulated in the MFR placentas. The impact of MFR on placental growth and the potential role of PPARγ activation through its natural or synthetic ligands in MFR-induced placental apoptosis were also discussed.

Tomomo Kotani showed data concerning the role of LPA in regulating trophoblast invasion. LPA is a lysophospholipid mediator of diverse cellular processes including blastocyst implantation in mice and sheep. In humans, serum lysophospholipase-D activity, which produces LPA, increases during pregnancy and LPA is involved in angiogenesis of first trimester decidua. However, the role of LPA in EVT function remains unknown. The expression of LPA receptors and function of LPA in trophoblast was investigated using the human trophoblast-like cell line, HTR-8/SVneo. RT-PCR analysis and immunohistochemistry revealed that mRNA and protein for LPA₁-₃ receptors were expressed in HTR-8/SVneo cells. LPA-induced ERK1 and ERK2 phosphorylation, and significantly stimulated HTR-8/SVneo migration, an effect that was associated with stimulation of phosphorylation of signal transducer and activator of transcription (STAT) 3 protein and AKT. Inhibition of STAT3 by pertussis toxin or addition of MEK/AKT inhibitors attenuated LPA-induced cell migration. A potential role of IL-6 as a mediator of the effects of LPA was discussed. The data presented suggest that LPA may stimulate human EVT migration via phosphorylation of STAT3 and therefore play a role in placentation. However, validatory experiments using a primary source of EVT are required.

Stuart Pitson discussed how stem cell differentiation and survival may be regulated by sphingolipids. Sphingosine kinase (SphK) phosphorylates the lipid sphingosine to generate the bioactive phospholipid sphingosine-1-phosphate (S1P). SphK and S1P have widespread physiological and pathophysiological actions, controlling events within the reproductive, gastrointestinal, vascular, nervous and immune systems, and also has a prominent role in cancer. There is now an additional emerging role for S1P in the regulation of stem and progenitor cell differentiation. SphK and S1P contribute to maintenance of human embryonic stem cells in the undifferentiated state in culture. SphK1 also regulates the rate and direction of endothelial progenitor cell differentiation. Thus, SphK1 appears a critical player in stem and progenitor cell differentiation, at least in human embryonic stem and endothelial progenitor cells, and suggests it could be a target for the specific manipulation of these cells both in vitro and in vivo. The divergent mechanisms for regulating SphK1 that may represent targets for therapeutic control of this enzyme were also discussed.

Ambika Singh discussed the potential role of ceramides and sphingolipids in trophoblast differentiation. Sphingolipids such as S1P and ceramide have been shown to be powerful regulators of differentiation and apoptosis in various cellular systems. Their production, metabolism and roles in differentiation of primary trophoblast were examined using an in vitro model. Changes in endogenous sphingolipids have been documented during differentiation using LC-MS/MS; with altered ratios of sphingolipids accompanied by marked changes in levels of key enzymes of sphingolipid biosynthesis and metabolism. Use of pharmacological or molecular manipulation of enzyme activity/expression altered the rate and progress of trophoblast differentiation as assessed by measurement of hCG production, without overt changes in viability/apoptosis. However, some evidence was presented that manipulation of sphingolipid levels or activity also regulates hCG production directly.

An alternative role for S1P in pregnancy was discussed by Denise Hemmings. S1P has been implicated in regulation of vascular tone in animal models. The role of S1P in human vascular tone regulation, particularly during pregnancy with its unique vascular adaptations, has been examined. S1P stimulates constriction of human chorionic plate and stem villous arteries. S1P-mediated constriction in stem villous arteries was significantly more dependent on activation of Rho-associated kinases (ROK), Ca²⁺ sensitization and nitric oxide modulation than the chorionic plate arteries. Variability of S1P responses and modulation of those responses were also seen in human myometrial and omental arteries. These results were discussed in light of recent data showing increased turnover of S1P in human decidua with increasing gestational age. Identification and characterization of these vasoactive actions of S1P are important for understanding both normal, and potentially abnormal, vascular adaptations in pregnancy.

Conclusions: It is clear from this workshop that lipids have the potential to exert pleiotropic effects on many aspects of placental function, including cell survival, differentiation, migration, angiogenesis and vascular function. Results are beginning to surface which shed some light on a few of these functions, and much can be gleaned from studies of lipid signaling and regulation in other cellular systems. However, as yet there is little known on the importance of lipid mediators in placental pathologies or abnormal pregnancies. This could be fertile ground for future studies.

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5. Workshop 4: placenta in agricultural species 

Organizers: Vibeke Dantzer, University of Copenhagen, Denmark; Christiane Pfarrer, University of Veterinary Medicine Hannover, Germany.

Speakers: Vibeke Dantzer, University of Copenhagen, Denmark; Marc Dilly, University of Veterinary Medicine Hannover, Germany; Jan-Dirk Haeger, University of Veterinary Medicine Hannover, Germany; Greg Johnson, Texas A&M University, USA; Rita Lee; AgResearch, New Zealand; Paula Papa, University of Sao Paulo, Brazil; Christiane Pfarrer, University of Veterinary Medicine Hannover, Germany.

Aim: The workshop was aimed to present and discuss state of the art placenta research in agricultural species (cattle, sheep, pig). The role of various factors involved in control of implantation, placentation and consequently of placental function were discussed. Gene expression, proteomics and their alteration were also highlighted with respect to specific reproductive techniques, thus giving some indications for reasons for the adverse outcome of pregnancies. The methodological approaches included classical in/ex vivo studies and in vitro models. The potential impact on placental function was discussed.

Summary: Greg Johnson discussed gene regulation associated with placental transport in sheep and pigs. S1P modulating enzymes, receptors and regulated genes have been localised to synepitheliochorial sheep placentomes. Therefore it was hypothesized that S1P stimulates angiogenesis within developing sheep placentomes through activation of S1P receptors and that their antagonism in vivo would alter placentome angiogenesis and subsequent uteroplacental nutrient exchange. The administration of FTY720 (S1P receptor antagonist) leads to an alteration of the placentomal histoarchitecture at day 60 (decreased maternal stromal in contrast to increased cotyledonary tissue). FTY720 treatment also affected transport of amino acids across placentae and fetal development as observed by a decrease in methionine levels in allantoic fluid, aspartate and glutamate levels in amniotic fluid, and fetal crown-rump length. These data suggest there is a downstream reduction in DNA synthesis and therefore cell proliferation and angiogenesis. In pig epitheliochorial placenta, gene expression was compared between areolae, where histotroph is absorbed, and the chorioallantoic placenta. The areolar epithelium was found to be unique compared to the rest of the chorion. Both Cathepsin L, a lysosomal cysteine or aspartic proteinase that can induce apoptosis, degrade ECM, catabolize intracellular proteins and process pro-hormones into active forms, and the facilitated glucose transporter SLC2A2 were expressed specifically in the areolar chorionic epithelium, but not in the chorion between the areola. These results support the idea that areolae express specific genes that may be required to facilitate transplacental transport of gases, micronutrients and macromolecules.

Vibeke Dantzer discussed the involvement of Tra-1-60 and Tra-1-81 in L-selectin mediated adhesion of the porcine conceptus to the endometrium. L-selectin expression in the early human embryo is involved in trophoblast adhesion during implantation. The aim of the presented study was to investigate the potential role of the L-selectin adhesion-system in the adhesion and implantation of porcine embryos. Endometrial samples from early pregnant gilts and non-pregnant cycling sows and corresponding embryos were examined by quantitative real time RT-PCR and immunohistochemistry for L-selectin and its ligands PNAd, PEN5 as well as the podocalyxin epitopes Tra-1-60 and Tra-1-81. In pregnant gilts the mRNA expression of L-selectin was significantly up-regulated at Days 15 and 18 compared to Day 10 p.i. and cyclic sows, while L-selectin ligands mRNA for CSPG-2 and podocalyxin were significantly up-regulated in Day 11 blastocysts compared to Day 9 blastocysts. Staining for L-selectin was observed in the endometrial luminal epithelium, while the ligands (PNAd, PEN5, Tra-1-60, Tra-1-81) occurred exclusively in the embryonic trophoblast and/or hypoblast or epiblast. It was concluded that key components of the L-selectin adhesion-system are differentially expressed in the porcine uterus and embryo around the time of initial implantation. Interestingly, those components were expressed in a pattern opposite to that found in humans. In the pig, L-selectin is expressed in the endometrium and the ligands in the trophoblast with the reverse being true in humans.

Rita Lee presented data on the anatomical and functional characteristics of placenta from bovine somatic cell nuclear transfer (SCNT) pregnancies. The most common cause of pregnancy failure and fetal mortality in SCNT pregnancies is the failure of the placenta to form or abnormal placentation. In bovine SCNT, the initiation of cotyledon formation occurs with similar efficiency as in pregnancies generated by artificial insemination (AI). At Day 50 of gestation, vascularization of the SCNT cotyledons appeared more advanced but fewer of the cotyledon/caruncle interactions successfully form placentomes later, resulting in generally fewer placentomes in SCNT pregnancies. Reduced placentome numbers were over-compensated by increased total placental weight in SCNT compared with AI pregnancies. Binucleate cell formation was not apparently impaired in SCNT placentomes. There are indications that the endocrine function of the SCNT placenta may be compromised (no rise in maternal estrone levels around Day 90–100 in failing pregnancies and surrogate dams show little signs of preparation for parturition or readiness for lactation). Data were presented on the steroidogenic potential of placenta from SCNT and AI pregnancies. The expression levels of some key steroidogenic enzymes differed from AI samples at different stages of gestation. Thus, one of the functional abnormalities of the SCNT placenta may be aberrant steroid hormone homeostasis resulting in failure to completely support fetal development to term.

Paula Papa discussed the expression of angiogenic growth factors in relation to steroidogenesis in cloned and non-cloned bovine placenta. Angiogenic growth factors (EG-VEGF, VEGF and bFGF) promote proliferation, survival and chemotaxis of endothelial cells in the placenta. The reason for poor outcome of somatic cell nuclear transfer (SCNT) pregnancies in cattle is still unclear although the placentomes frequently show alterations. Data were presented on the spatial distribution of EG-VEGF, VEGF and bFGF and their receptors in non-cloned and cloned bovine near term placentae, as determined by immunohistochemistry and quantified by Western blot and real time RT-PCR. Placental cells were cultured in the presence of these growth factors and levels of progesterone and estrone sulphate measured in culture medium after 24, 48 and 96 h. The growth factors immunolocalised to the same cell types in normal and cloned bovine placenta. However, relative quantification of protein and mRNA in cloned placentae was different and showed a gender related pattern in most cases. Placental cells derived from cloned placenta did not respond to growth factor stimuli regarding steroid production but were able to produce higher amounts of estrone sulphate but not progesterone than non-cloned placental cells in culture. It was concluded that the altered expression of angiogenic growth factors in bovine placentae derived from cloned gestations may lead to an imbalance of the local regulation of steroid production.

Christiane Pfarrer discussed the various different in vitro culture systems available for bovine placental cells. The frequent appearance of early pregnancy loss and pathological phenomena during bovine gestation have been tackled in a multitude of studies and lead to important findings. However, in vivo studies do not allow study of the action of single ‘factors’, therefore an in vitro model of the bovine synepitheliochorial placenta containing maternal and fetal epithelia lying directly opposed to each other is being developed. To date, bovine placental trophoblast and caruncular endometrial epithelial cell lines have been established and thoroughly characterized and examined for functional properties. These include different two- and three-dimensional applications for which this in vitro model has been used: (1) drug transportation by MDR-1 transporter; (2) growth factor stimulation studies and (3) the development of a 3-dimensional cell culture model for bovine trophoblast cells also allowed invasion assays to be performed. The results of these studies have shown that the combination of classical in/ex vivo studies and in vitro models enhances knowledge of the bovine placenta. These models may allow the development of prophylactic and/or therapeutic agents in the future.

Using the isolated cell types described above Marc Dilly has investigated the epidermal growth factor (EGF) upregulation of matrix metalloproteinase (MMP)-9 and tissue inhibitor of metalloproteinase (TIMP)-1. The regulatory mechanisms of restricted trophoblast invasion observed in bovine placenta are not yet fully understood. Candidates likely involved are MMPs and their counteracting inhibitors (TIMPs). Proteolysis of MMP-9, present in the bovine placenta, is predominantly regulated by TIMP-1. EGF is capable of upregulating MMP-9 activity in a variety of cells types; therefore it was hypothesized that EGF may stimulate MMP-9 and TIMP-1 expression in the bovine placenta. To test this hypothesis, and to dissect the underlying signaling pathways, cultured maternal caruncular epithelial cells (BCEC-1) and trophoblast cells (F3) isolated from bovine placenta were treated with EGF and mitogen-activated protein kinase (MAPK) inhibitor. Densitometric analysis of specific RT-PCR products revealed that EGF increased both MMP-9 and TIMP-1 mRNA expression in BCEC-1 and F3 cells. This effect was abolished by inhibiting MAPK activation. Western blot analysis showed a strong activation of MAPK exclusively in F3 cells. Zymographic analysis revealed that EGF elevated pro-MMP-9 and active MMP-9 in BCEC-1, whereas the active form was expressed predominantly in F3 cells. It was concluded that EGF activates the MAPK pathway in bovine placenta cells, and proposed that this activation is necessary for the upregulation of MMP-9 and TIMP-1 expression. Thus EGF may be involved in the regulation of restricted trophoblast invasion and tissue remodeling during bovine gestation.

Jan-Dirk Haeger went on to describe differential signaling of fibroblast growth factor (FGF) in bovine caruncular epithelial cells and trophoblast cells in vitro. In the bovine placenta FGF1, -2, -7 and FGFR1 are expressed in trophoblast and caruncular epithelial cells throughout gestation. Since FGFs can ‘act’ very differently they hypothesized that FGF1, -2, or -7 stimulate different signaling pathways in trophoblast and caruncular epithelial cells in vitro. To test this hypothesis cell lines were stimulated (FGF1, -2, -7) and analyzed for Ras-, Erk1/2-, p38 MAPK- and Akt activation. FGF1 and -2 activate Ras and Erk1/2 in trophoblast cells but not in caruncular epithelial cells whereas FGF7 did not activate Erk1/2 in either cell type. It was also observed that in FGF-stimulated trophoblast cells Mek1/2-Erk-pathway-inhibition leads to a downregulation of p38 MAPK activation. The docking protein FRS2, which is essential for the initiation of FGF signaling, was expressed in the bovine placenta and in the cell lines used. It was concluded that the FGF system is important for the ‘physiology’ of bovine trophoblast and further work is required to elucidate the FGF-mediated biological processes in bovine trophoblast.

Conclusions: These in/ex vivo studies and in vitro models provide a strong platform for future studies in the complex regulatory mechanisms of placental development and function in different species which may lead to the development of therapeutic agents in the future.

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6. Workshop 5: epigenetics and placentation 

Organizers: Stefan Hiendleder, University of Adelaide, Australia; Richard Saffery, Murdoch Childrens Research Institute, Melbourne, Australia.

Speakers: Miguel Constancia, University of Cambridge, UK; Ying Liu, University of Adelaide, Australia; Marilyn Renfree, University of Melbourne, Australia; Richard Saffery, Murdoch Childrens Research Institute, Australia.

Aim: It is now widely accepted that a major mediator of environmental risk is epigenetic modification of specific genes within the genome. While gene sequence is largely determined at conception, ’epigenetic’ modifications are dynamic and modifiable, raising the possibility of novel preventive and/or therapeutic opportunities. The most widely studied epigenetic mark is DNA methylation of the CG dinucleotide sequence and disruption of methylation profile has been associated with many different adverse health outcomes. The placenta has a unique epigenetic profile amongst tissues. This includes global DNA hypomethylation relative to somatic tissues, a placenta-specific profile of imprinted genes (expressed in a parent of origin specific manner) and the recent description of placenta-specific tumour-suppressor gene methylation. Such modifications are likely to play a fundamental role in trophoblast functioning, placental development, and pregnancy outcome. This workshop examined emerging evidence for a role of epigenetic modification in placental development. Specific presentations included discussion of the unique features of human placental DNA methylation, inter- and intra-species heterogeneity in placental epigenetic profile and the role of imprinted genes in placental development and fetal growth across evolution.

Summary: Ying Liu presented and discussed unpublished data on polymorphic imprinting of IGF2R in bovine placenta. The insulin-like growth factor-2 receptor (IGF2R) facilitates endocytosis and subsequent clearance or activation of a variety of ligands involved in cell growth and motility. The IGF2R gene thus has a major role in placental function and fetal growth control. Murine Igf2r is subject to genomic imprinting and is maternally expressed in peripheral fetal tissues and placenta. However, data on imprinting of IGF2R in humans are still highly controversial with biallelic expression, partial imprinting and monoallelic expression reported for placenta. Similar to human, the bovine is monotocous and outbred; as a model, the bovine embryo and fetus delivers suitable amounts of tissue for the analysis of early developmental stages. Day 153 fetuses (55% to term) of purebred Bos primigenius taurus (Angus) and B. p. indicus (Brahman), and reciprocal crosses, were examined to determine the imprinting status of IGF2R in cotyledon tissue (Placenta fetalis). PCR amplicons from IGF2R exon 48 were sequenced and demonstrated a polymorphic (TG)n microsatellite and single nucleotide polymorphisms. The analysis of 40 concepti identified 15 heterozygous fetuses informative for imprinting analyses. There was a striking variation in imprinting with relative paternal expression levels that ranged from 0 to 70% of maternal expression. This data indicates extensive plasticity in imprinting of IGF2R in placenta that could explain the seemingly contradictory data obtained in human.

Marilyn Renfree discussed how study of the platypus genome has given us insights into eggs, embryos and the evolution of imprinting. Genomic imprinting is an epigenetic phenomenon that results in monoallelic gene expression. It is widespread in eutherian mammals, and also occurs in marsupials. Although there have been many hypotheses to explain why genomic imprinting evolved in mammals, few have examined how it arose. The host defence hypothesis suggests that imprinting evolved from existing mechanisms within the cell that act to silence foreign DNA elements that insert into the genome. However, the changes to the mammalian genome that accompanied the evolution of imprinting have been hard to define due to the absence of large scale genomic resources between all extant classes. The recent release of the platypus genome has provided the first opportunity of comparisons between prototherian (monotreme; which appear to lack imprinting) and therian (marsupial and eutherian; which have imprinting) mammals. The distribution of repeat elements known to attract epigenetic silencing across the genome was compared in monotremes and therian mammals particularly focusing on the orthologous imprinted regions. There was a significant accumulation of certain repeat elements within imprinted regions of therian mammals compared to the platypus. This analysis showed that the platypus has significantly fewer repeats of certain classes in the regions of the genome that have become imprinted in therian mammals. The accumulation of repeats, especially LTRs and DNA elements, in therian imprinted genes and gene clusters therefore appears to be coincident with, and may have been a potential driving force in, the development of mammalian genomic imprinting. These data provide strong support for the host defence hypothesis.

Miguel Constância discussed genomic imprinting as a paradigm of epigenetic gene regulation and the importance of imprinting during mammalian pregnancy. Imprinted genes are expressed from either the maternal or paternal genomes and play key roles in resource transfer from mother to offspring. A major emerging question is how epigenetic mechanisms integrate environmental signals with physiological outcomes. The paternally expressed Igf2P0 placental-specific transcript has been examined to provide a link between epigenetic marking and physiology of growth in the mouse. It was found that DNA methylation in both maternal and paternal alleles silences the P0 promoter in fetal tissues. In placenta, the paternal allele is unmethylated and expressed. Loss of P0 expression results in intrauterine growth restriction caused by nutrient supply insufficiency. Conversely, P0 expression from both parental alleles, associated with loss of repressive methylation on the maternal allele, results in placental overgrowth and increased nutrient supply. Non-coding RNAs, as modulators of gene activity, represent another area of increasing interest. Several miRNAs were recently found to be located within imprinting clusters. Ongoing (unpublished) investigation of the ICR1 region on mouse chromosome 7 was described. These studies are aimed at elucidating the role of these imprinted miRNAs on fetal growth and placental function.

Richard Saffery went on to discuss the wider view of epigenetics and human placentation. Specification of early extraembryonic tissue and subsequent stages in placental differentiation are accompanied by a unique pattern of gene-specific epigenetic modifications. Several research areas of the placental epigenome were described. (1) One of the earliest lineage differentiation events separating extraembryonic and embryonic lineages is the change in global DNA methylation levels, such that extraembryonic (and ultimately placental) tissue is globally hypomethylated in relation to embryonic counterparts. The mechanisms underlying this remain unclear. The potential for ‘self regulation’ of DNA methyltransferase (DNMT) genes as a contributor to this phenomenon was examined. Both full term and first trimester placentas show specific hypermethylation of the maintenance DNA methyltransferase-1 (DNMT1) gene. This has not previously been described in any tissue. Although tracking with global DNA methylation levels in human placenta there was evidence for DNMT1 methylation-independent genomic hypomethylation in non-primate species and an in vitro model of extraembryonic differentiation. The link between DNMT regulation and global hypomethylation appears complex and requires further investigation. (2) The human placenta displays many similarities to human tumours, such as growth in a low oxygen environment, cell migration, invasion of surrounding tissue and escape from immune detection. Interestingly, these similarities have been extended to include hypermethylation of specific tumour-suppressor gene promoter regions, including genes involved in Ras-signaling inhibitors, Wnt-signaling inhibitors, immune modulators and genes regulating Vitamin D homeostasis. These genes are typically only methylated in human cancers. (3) It is clear that many adverse pregnancy outcomes are associated with disruption of early (first trimester) placentation. As a first step to identifying potential epigenetic targets for environmental perturbation in early pregnancy, a genome-wide placenta methylation profile using isolated 8 and 12 week villi and purified 7 week CK7+ cytotrophoblasts was generated. This has revealed specific promoter methylation of genes involved in several cellular pathways, including multiple transcription factors, regulators of apoptosis, oxidative stress response, and cancer development and progression. Locus specific analysis has revealed that many of these individual promoter methylation events track with phylogeny, independent of placental barrier type.

Conclusions: Taken together these data have revealed many novel targets for environmentally-induced epigenetic disruption that are believed to contribute to poor placental development and pregnancy-associated diseases such as pre-eclampsia and IUGR. Objectives for future research include (i) identification of DNA sequences with placenta-specific DNA methylation patterns, (ii) characterization of genetic and environmental factors that contribute to plasticity of imprinting in placenta, and (iii) dissection of interacting epigenetic and genetic factors involved in regulation of gene expression, epigenetic modification, and placental phenotype.

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7. Workshop 6: trophoblast deportation 

Organizers: Larry Chamley, University of Auckland, New Zealand; Ian Crocker, University of Manchester, UK.

Speakers: Graham Burton, University of Cambridge, UK; Qi Chen, University of Auckland, New Zealand; Ian Crocker, University of Manchester, UK; Rebecca Dragovic, University of Oxford, UK; Berthold Huppertz, Medical University of Graz, Austria; Claire Whitehead, University of Edinburgh, UK.

Aim: Deportation of trophoblast material from the placenta into the maternal circulation was first described in human pregnancy in 1893 by a German pathologist, Schmorl, who observed multinucleated fragments of syncytiotrophoblast (syncytial knots) lodged in the lungs of women who had died in eclampsia. Trophoblast deportation was then rapidly confirmed by several authors and shown to be a physiological phenomenon in normal pregnancy which appears to be altered in some way in pre-eclampsia. Despite the process of trophoblast deportation being widely acknowledged, more research is required in this field. It is now known that not only syncytial knots but also mononuclear cytotrophoblasts and subcellular micro- and nano- particles are released from the placenta into the maternal blood. All of this trophoblast material is immunologically foreign to the maternal immune system and has the potential to affect maternal physiology, but does it? What do we really know about trophoblast deportation? What is the quantity of deported material and what is its nature? Does apoptosis really contribute to the shedding and deportation of trophoblastic material? These are some of the questions that were addressed in the workshop.

Summary: Berthold Huppertz discussed the role of oxygen in villous trophoblast turnover. The villous trophoblast of the human placenta is composed of two layers, a layer of mononucleated cells, the villous cytotrophoblast, and a second multinucleated layer, the syncytiotrophoblast. Turnover of the villous trophoblast includes the following steps: (1) cytotrophoblast proliferation, (2) cytotrophoblast differentiation, (3) early apoptosis of a few cytotrophoblast cells, (4) fusion of a few cytotrophoblast cells with the overlying syncytiotrophoblast, (5) further differentiation within the syncytiotrophoblast, (6) signs of late apoptosis and accumulated apoptotic nuclei in specific sites of the syncytiotrophoblast, (7) release of late apoptotic nuclei packed in syncytial knots into the maternal circulation, (8) engulfment of syncytial knots by macrophages in the maternal lungs. It has been proposed that oxygen concentrations not only influence the proliferation rate of cytotrophoblast cells but also the accumulation and release of syncytial nuclei. This becomes obvious in placenta from cases with IUGR where there are also alterations in the flow of maternal blood that may have an impact on the distribution of the syncytial nuclei.

Ian Crocker posed the question – Can trophoblast apoptosis be modulated to regulate deportation? Exaggerated apoptosis (programmed cell death) of placental villous trophoblast is associated with pre-eclampsia and IUGR. Several factors have been recognised to be important in the regulation of trophoblast cell turnover; including (1) uteroplacental growth factors, particularly EGF and IGF in suppressing trophoblast apoptosis, (2) cell cycle disruption in promoting trophoblast differentiation and (3) oxygen in influencing cell turnover of villous trophoblast in vitro and in vivo. Recently, the importance of p53 in controlling these events has been examined with a view to manipulating this transcription factor as a way of augmenting placental syncytiotrophoblast decline. Data were presented from recent studies confirming the importance of p53 in exaggerated villous trophoblast turnover in pre-eclampsia and IUGR. In addition, agent-induced ways of mimicking and alleviating p53-induced cell death were discussed. These studies provide a potentially novel approach to curtailing excessive trophoblast deportation.

Qi Chen discussed the maternal endothelial cell responses to deported trophoblast material. In normal pregnancy 150,000 syncytial knots are estimated to be shed into the maternal blood daily as a result of apoptotic death in the syncytiotrophoblast. In pre-eclampsia it is hypothesized that syncytial knots may be produced by a more necrosis-like cellular death. How syncytial knots are cleared from the maternal lungs is not clear but it has been shown that endothelial cells can phagocytose syncytial knots. Phagocytosis of apoptotic syncytial knots is silent but phagocytosis of necrotic syncytial knots activates the phagocytosing endothelium with the secretion of IL-6 and TGF-β1 that activate bystander endothelial cells thereby spreading endothelial cell activation. In addition, IL-6 and TGF-β1 also induce placental explants to shed more necrotic syncytial material.

Claire Whitehead discussed whether placental RNA in the maternal circulation could be used to transcriptionally profile the placenta. Syncytiotrophoblast microparticles are continually shed into the maternal circulation as part of normal and diseased pregnancies. Free fetal RNA is believed to be encased within these microparticles, providing the RNA with remarkable stability from degradation in the maternal circulation. It may therefore be possible to non-invasively profile dynamic changes in placental mRNA transcripts simply by taking a venous sample of maternal blood. A suite of clinical and animal studies were described that attempt to prove the concept that acute changes in placentally derived mRNA transcripts can be detected in peripheral blood. Acute changes in hypoxia can occur in different clinical settings, therefore maternal whole blood was collected and hypoxic genes measured in clinical situations where there is predicted to be feto-placental hypoxia; fetal acidaemia with labour, severe early-onset growth restriction, and twin-to-twin transfusion pre and post laser surgery.

Rebecca Dragovic discussed characterization of microparticles and exosomes released in three placental culture systems. Three different methods to model the release of placental microparticles and exosomes in normal pregnancy and pre-eclampsia were used. These include the standard ’mechanical’ method, eluates of perfused isolated placental lobes and a placental explant perfusion system. Placental particles have been shown to express a general microparticle marker (maleimide), a syncytiotrophoblast marker, VEGF-R1 and endoglin using four colour flow cytometry. However, flow cytometry can only detect particles down to 300 nm in size and therefore a new technology, Nanosight Tracking Analysis (NTA) which detects particles ranging from 30 nm to 1 μm, was also used. This demonstrated that the predominant population of particles are in the size range of 50–300 nm and therefore are not detectable by flow cytometry. These findings have important implications for the detection of placental particles in the maternal circulation in relation to pre-eclampsia.

Graham Burton discussed the morphological distinctions between syncytial ‘sprouts’ and ‘knots’. The terms ‘syncytial sprouts’ and ‘syncytial knots’ are often used synonymously in the literature, although when originally coined were used to distinguish between accumulations of syncytial nuclei displaying contrasting morphological and developmental properties. Sprouts occur at all stages of gestation, and consist of aggregates of euchromatic syncytial nuclei. Because they are frequently pedunculated, sprouts are easily dislodged and deported as classical ‘tear-drop’ masses into the maternal blood. In contrast, true knots are only seen after approximately 32 weeks of gestation, being predominantly a feature of the post-mature placenta or fetal vascular compromise. The nuclei are closely apposed and display dense heterochromatin, often forming rounded elevations on the villous surface. They must be distinguished from false knots, which are apparent aggregates of euchromatic nuclei created by tangential sectioning through the syncytiotrophoblast. Because of their appearances, it has been proposed that true knots represent the end-stage in a transit of nuclei through the syncytium that culminates in apoptotic nuclei being shed from the villous surface. There can be no doubt that these nuclei are effete, but the evidence that they are apoptotic and deported in a regular fashion was questioned.

Conclusions: This workshop provided lively debate with good interaction between speakers and audience throughout as a number of controversial issues were discussed. It became apparent that the current terminology in this field is not adequate, although no alternative was agreed on at this stage. Two areas in particular stood out. Firstly, it is not yet clear whether structures referred to by some workers as syncytial knots are structurally and/or functionally homologous with structures referred to by others as syncytial sprouts. The latter term is particularly confusing as there is more than one definition of a syncytial sprout in the literature. Furthermore, it was questioned whether syncytial knots, as defined histologically in later gestation, are real structures containing material that is about to be deported from the placenta or are sectioning artefacts. Secondly, the mode of cell death occurring in the syncytiotrophoblast is referred to by many workers as being apoptosis but our discussion highlighted the fact that apoptosis is a process that occurs in mononuclear cells, and was originally defined as a process occurring specifically in lymphocytes. The process of programmed cell death occurring in the multinucleated syncytiotrophoblast may be somewhat analogous to apoptosis but it is not apoptosis per se. Therefore, the term apoptosis is not appropriate to the syncytiotrophoblast. These terminology questions were not resolved. However, clearly identifying these issues is a first step towards the introduction of terms which are widely accepted. We may need to define new terms to clarify these issues so that all workers in the field are using the same terminology to describe structures and processes involved in syncytiotrophoblast death and deportation.

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8. Workshop 7: glucocorticoids and placental function 

Organizers: Vicki Clifton, University of Adelaide, Australia; Brendan Waddell, University of Western Australia, Australia.

Speakers: Vicki Clifton, University of Adelaide, Australia; Peter Mark, University of Western Australia, Australia; Stephen Matthews, University of Toronto, Canada; Michael Stark, University of Adelaide, Australia; Brendan Waddell, University of Western Australia; Euan Wallace, Monash University, Australia.

Aim: Glucocorticoids provide key signals for differentiation of fetal organs yet excess exposure to glucocorticoids limits both placental and fetal growth. Therefore, regulation of glucocorticoid access to the placenta and fetus is recognised as an important determinant of pregnancy outcome and subsequent development of the postnatal phenotype. Glucocorticoid access to the placenta and fetus is determined principally by the ‘placental glucocorticoid barrier’ (PGB), the major component of which is the enzyme 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2). In addition, the multidrug resistant P-glycoprotein (P-gp), a membrane-bound efflux protein, has recently been identified as a potential novel contributor to this barrier. Thus, as maternal glucocorticoid levels rise throughout gestation, their access to the glucocorticoid receptor in both the placenta and in fetal tissues is limited by 11β-HSD2 inactivation or by P-gp-mediated efflux at the maternal interface.

The aims of this workshop were to clarify the key components of the placental glucocorticoid barrier and the consequences to the placenta and fetus of compromised barrier function. A particular focus of the workshop was the impact of inappropriate glucocorticoid exposure (i.e. either an excess or deficiency) on placental structure and function.

Summary: Peter Mark discussed regulation of rat placental P-gp and 11β-HSD2 by steroid hormones. Glucocorticoids potently inhibit fetal and placental growth but are crucial for the maturation of fetal organs prior to parturition. In normal pregnancy, placental and fetal glucocorticoid exposure are determined by the PGB, which serves to limit access of maternal glucocorticoids to the placenta and fetus. Placental expression of 11β-HSD2 is well recognised as the major component of this PGB. Furthermore, syncytiotrophoblast expression of the ATP-driven efflux pump, P-glycoprotein (P-gp/Abcb1) augments the PGB by pumping glucocorticoids within the placenta back into the maternal circulation, thereby reducing fetal and placental glucocorticoid exposure. Consequently, factors affecting the regulation of these PGB components will alter the extent of glucocorticoid exposure in fetal and placental tissues. Data were discussed exploring the roles of two potential regulators of PGB components, namely glucocorticoids themselves and progesterone, during rat pregnancy, since placental exposure to both is highly dynamic across the period of maximal fetal growth.

Michael Stark discussed antenatal betamethasone exposure and sex specific alterations in placental 11β-HSD2 activity. Fetal sex is an acknowledged risk factor for neonatal morbidities and mortality. However, the importance of sexual dimorphisms in the response to pathophysiologic pregnancy remains poorly understood. Placental 11β-HSD2 activity is altered by conditions affecting pregnancy. At term, sexually dimorphic differences in 11β-HSD2 activity occur in response to maternal inflammation, conferring a female advantage. For women at risk of preterm delivery, administration of the synthetic glucocorticoid betamethasone decreases the incidence of major neonatal morbidities. While betamethasone is incompletely metabolised by placental 11β-HSD2, alterations in 11β-HSD2 activity have been demonstrated following maternal exposure. Such responses are sex specific. There are sexually dimorphic differences in preterm placental and fetal responses to antenatal betamethasone exposure, protecting females from excess glucocorticoid exposure and enabling appropriate adrenal responses to physiological stressors. With placental 11β-HSD2 influencing preterm neonatal physiological stability, these findings highlight potential mechanisms underlying excess male morbidity and mortality following preterm birth.

Stephen Matthews discussed fetal vulnerability and the placental gatekeeper. Synthetic and endogenous glucocorticoids have powerful acute and long-term effects within the placenta and fetus. The fetal brain contains high levels of glucocorticoid receptors in late gestation and it has been shown that glucocorticoid exposure leads to rapid upregulation of transcriptional activity in the brain in late gestation. Unlike the situation in the adult, the immature brain is not capable of glucocorticoid receptor autoregulation. In addition, transporter systems that normally exclude a spectrum of drugs from the fetal brain including synthetic glucocorticoids are not developed until late in gestation. As such the fetal brain remains vulnerable to the effects of glucocorticoids that pass across the placenta from mother to fetus and this is exacerbated in situations of placental compromise.

Euan Wallace discussed the relative impact of maternal betamethasone treatment in normal and preterm growth-restricted fetuses. A series of “in parallel” animal and human studies have shown that antenatal glucocorticoids induce opposing systemic vascular changes and altered cardiac output (CO) in normal and IUGR fetuses. Human studies suggest that the extent of these effects may be predictive of clinical outcome, and therefore could guide clinical management. Sheep studies have shown that in the normal fetus the administration of betamethasone increases systemic vascular resistance and reduces cardiac output (CO), whereas in the IUGR fetus it reduces resistance and increases blood flow, most notably to the placenta, heart and brain. These increases were associated with increased oxidative stress and brain injury. Collectively, these studies suggest that glucocorticoids should be used with caution in the IUGR fetus.

Vicki Clifton discussed the complexities of placental glucocorticoid receptor expression in the human placenta. A sex specific growth response of the human fetus in response to increased maternal cortisol exposure has been described. The female fetus reduces growth while the male fetus continues to grow normally in response to high concentrations of cortisol suggesting the male fetus may be glucocorticoid resistant. The placental glucocorticoid receptor (GR), hnRNA, mRNA and protein were examined in male and female fetuses from normal pregnancies and pregnancies complicated by asthma to determine if there are sex differences in either GR expression that may confer a sex specific difference in the response to cortisol. Total GR mRNA was decreased in female placentae of pregnancies complicated by asthma and unchanged in male placentae relative to the control population. GR protein was not altered by sex or the presence of maternal asthma suggesting alterations in protein function rather than expression may confer a differential response to cortisol in male and female placentae.

Brendan Waddell discussed the placental phenotype of the glucocorticoid receptor null mouse that demonstrates evidence for tonic suppression of placental growth by endogenous glucocorticoids. Glucocorticoids act via GR to promote late fetal maturation, but in excess they compromise both placental and fetal growth. However, the role of endogenous glucocorticoids in regulating normal placental differentiation, growth and function remains unclear. Therefore, the placental phenotype of the GR-null (GRKO) mouse was compared with that of the wildtype (WT) by quantitative morphology and measurement of placental expression of several glucocorticoid-responsive genes. Although GRKO fetal weights were similar to those of WTs, GRKO placental weights were 13% higher than WTs. Unbiased stereology showed that placental regions were all slightly larger in the GRKO, but mean harmonic thickness of the placental barrier was reduced (21%). Expression of 11β-HSD2 was increased (46%) in the GRKO placentas whereas that of several other genes including Vegf and Igf2 were similar between genotypes. These data support the hypothesis that endogenous glucocorticoids tonically suppress placental growth.

Conclusions: Excessive exposure of the fetus to glucocorticoids during pregnancy is associated with reduced fetal growth and the subsequent development of diseases in adult life. Even so, the use of betamethasone for fetal lung maturation in women at risk of preterm delivery is currently accepted practice and associated with an increased chance of neonatal survival. The placenta attempts to protect the fetus from excessive glucocorticoid exposure via 11β-HSD2 activity and P-glycoprotein-mediated efflux. Current findings suggest these protective mechanisms are influenced by the sex and size of the fetus, with females adapting placental glucocorticoid metabolism in response to glucocorticoid concentration, and the placenta of the IUGR fetus responding differently to exogenous glucocorticoid treatment. Moreover, similar protective mechanisms operate at the blood brain barrier, effectively providing a second line of defence against excess glucocorticoid exposure. The current workshop highlighted that placental growth and function are highly sensitive to both exogenous glucocorticoids and endogenous glucocorticoids across the full physiological range, with several effects being dependent on fetal sex.

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9. Workshop 8: endothelium 

Organizers: Padma Murthi, University of Melbourne, Australia; Luis Sobrevia, Pontificia Universidad Catolica de Chile, Chile.

Speakers: Gernot Desoye, Medical University of Graz, Austria; Berthold Huppertz, Medical University of Graz; Lopa Leach, University of Nottingham, UK; Donald Morrish, University of Alberta, Edmonton, Canada; Niroshani Pathirage, University of Melbourne, Australia.

Aim: The aim of this workshop was to discuss the role of blood vessels in establishing and maintaining a healthy pregnancy. The discussions focused on the vessels at the feto/maternal interface namely the vessels of chorionic villi and in the maternal spiral arteries. Since endothelium from macrovasculature and microvasculature exhibit phenotypic, genotypic and metabolic differences it is likely that agents that alter endothelial cell function in diseases of pregnancy (such as gestational diabetes, pre-eclampsia and IUGR) could elicit differential action in these two vascular beds. The specific aims of the workshop included characterization of endothelial cells in the two vascular beds and their interaction with other placental cell types. Knowledge of these mechanisms will allow better understanding of the vascular biology of placental insufficiency.

Summary: Gernot Desoye discussed how placental endothelial cells (ECs) may be targets for fetal regulation of placental function. The endothelial cells covering the feto-placental vasculature represent the surface exposing placental tissue to the fetal endocrine and metabolic signals. ECs from umbilical cord and placenta proper differ in many regards because of the different functions of these vascular beds. Methods have become available for isolating and culturing arterial and venous ECs from the placenta allowing not only studies of placental ECs in general, but also into differences between ECs from both types of vasculature. Such differences can be seen in the mitogenic response to vascular endothelial growth factors (VEGFs) paralleling the differential expression of VEGF-Rs. Insulin represents a further endocrine fetal signal acting on the placenta. Activation of insulin receptors on ECs results in glycogen accumulation. Oxysterols are an example of a metabolic signal. They are formed by cytochrome P450-mediated cholesterol oxidation in the fetus and can bind to and activate liver-X-receptors in ECs. As a result cholesterol efflux transporters are up-regulated and more cholesterol is released into the fetal circulation. Thus the fetus can regulate its own supply of cholesterol by signals that act upon ECs.

Lopa Leach discussed the phenotypic plasticity of fetal endothelial cells. Whilst the majority of the vascular world uses human umbilical vein endothelial cells (HUVEC) to investigate endothelial mechanisms, there still appears to be residual doubt on the suitability of this cell type to inform on archetypical endothelial cell behaviour. A similar concern also applies to placental microvascular cells; are they pre-programmed fetal cells that can only behave in a certain way? Endothelial cells line the entire vascular system and heart. EC micro-heterogeneity is likely influenced by the microenvironment. The neighbouring cells and matrix with the circulating growth factors coax these cells to don appropriate clothing and deliver the required function. It has been shown that HUVEC lose their fidelity of origin and alter from continuous to fenestrated endothelium when placed in conditions that mimic the outer retina. Phosphorylation events lead to leaky, angiogenic cells i.e. the diabetic phenotype, when HUVEC, placental microvessels or villous explants are challenged with high glucose, high insulin or VEGF. These can be reversed towards the blood–brain barrier (BBB) phenotype by addition of cAMP and angiopoietin-1. Alterations in the ratio between pro- and anti-angiogenic growth factors allow dynamic remodeling. Endothelial cells from mature vascular beds also show this plasticity: removal of glial cells causes loss of tight and adherens junctions in the BBB; sheer stress alters aortic endothelial cell barriers whilst flow can dictate artery–vein identity. Whether these induced changes in the developing fetus become trans-generational in the face of prolonged chronic insult is a question one now needs to reflect on. Will the diabetic endothelial phenotype seen in offspring of diabetic mothers continue into adulthood? Human placental and cord endothelial cells are fetal/neonatal in origin and this confers them the plasticity one desires when trying to understand how diseases impair function. In the era of stem cells and the importance of extraembryonic stem cells from the placenta and cord why is one surprised that the placenta is one of the most valuable human experimental models for a curious scientist?

Niroshani Pathirage discussed differential homeobox gene expression in arterial and venous endothelial cells. Endothelial cells play important roles in the development and remodeling of vasculature, maintenance of vascular tone, blood fluidity, coagulation, nutrient exchange and organ development. The heterogeneity between macro- and microvascular endothelial cells is well established and given the functional diversity of arterial and venous systems there is now emerging evidence that arterial and venous ECs also have distinct molecular ontogeny. Homeobox genes encode transcription factors which function in body axis patterning in the developing embryo and have recently been implicated in both physiological and pathological vascular remodeling and angiogenesis. Low density DNA microarrays were used to define the homeobox gene expression profile of placental arterial and venous endothelial cells derived from human term placenta. Specification of the arterial and venous molecular identity, whilst enhancing our understanding of vascular development and arteriovenous specification, may also explain the vascular malformations seen in pathologies such as pregnancies complicated by IUGR.

Berthold Huppertz discussed recent work on the potential of placental stem cells to differentiate towards endothelial cells. Recent research in stem cell biology has identified the fetal membranes of the human placenta as a source of cells which show phenotypical similarities to bone marrow-derived mesenchymal progenitor cells in terms of their multipotent differentiation potential. The characterization of amnionic membrane derived human mesenchymal stromal cells (am-hmsc) is mostly limited to flow cytometry. Immunohistochemical studies on cultured cells and term placental cryosections were performed to compare the in situ with the in vitro situation. Furthermore, cells were cultured under angiogenic conditions in order to investigate their endothelial differentiation potential. Am-hmsc were cultured in the presence or absence of VEGF and further characterized by the ability to take up dii-acldl and to form networks in a matrigel assay. Am-hmsc displayed a hybrid epithelial–mesenchymal phenotype, they were concordantly positive for the mesenchymal marker vimentin, though some cells also expressed the epithelial marker cytokeratin. The in situ and in vitro expression of common mesenchymal stem cell markers (cd73, cd90, cd105) and the embryonic stem cell markers oct-4 and ssea-4 indicated a multipotent differentiation potential. In the presence of VEGF the cells altered their morphology towards an endothelial cobblestone-like phenotype and VEGF-R2 was up-regulated. This receptor can also be found on vasculogenic and angiogenic precursor cells. In contrast to undifferentiated control cells, they take up dii-acldl and form distinct vascular networks in the matrigel assay. It was concluded that these results suggest that am-hmsc have the potential to differentiate along the endothelial lineage and might be useful tools for the development of vascular grafts.

Don Morrish reported on recent observations on endothelial progenitor cells. Endothelial progenitor cells are primitive precursors of mature endothelial cells and are thought to arise from bone marrow and vessel wall sources. Previous studies using fibronectin as a substrate showed normal or increased endothelial progenitor cell colony formation in normal pregnancy, normal or decreased colony numbers in pre-eclampsia, and usually decreased colony numbers in diabetes. Data were presented suggesting that endothelial progenitor cell colony numbers, using collagen I as a substrate, do not show a difference between normal and diabetic pregnancies. It was concluded that the role of endothelial progenitor cells in the vascular dysfunction in normal pregnancy, pre-eclampsia or diabetes in pregnancy is still unclear.

Conclusions: This workshop highlighted the importance of heterogeneity and phenotypic plasticity of feto-placental endothelium in placental vasculature. There was also discussion on how the neighbouring cells influence the fate of endothelial cells in feto-placental angiogenesis. It was concluded that strengthening collaboration and communication between researchers in the field of placental endothelium is essential to address some of the important questions about endothelial interaction with neighbouring cells which would provide further insight into the molecular and functional role of feto-placental vasculature.

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10. Workshop 9: placental transport: what don't we know? 

Organizers: Yoshiko Kudo, Hiroshima University, Japan; Rohan Lewis, University of Southampton, UK.

Speakers: Gernot Desoye, Medical University of Graz, Austria; Valaria Dietrich, Universidad de Buenos Aires, Argentina; Rohan Lewis, University of Southampton, UK; Amanda Sferruzzi-Perri, University of Cambridge, UK; Colin Sibley, University of Manchester, UK.

Aim: The aim of this workshop was to promote discussion on the key questions for future research in this area. The workshop focused on a range of placental transport related topics including transfer of specific nutrients and the regulation of placental transport.

Summary: Colin Sibley discussed passive permeability of hydrophilic solutes to the placenta. There is good evidence from a significant number of studies in several species that the rate of transfer across the placenta of hydrophilic molecules, that are not substrates for transport processes, is inversely proportional to their molecular size. The most likely explanation of these data is that the molecules are diffusing across the placenta through an extracellular water filled paracellular route. However it is hard to reconcile these physiological observations with the morphology of the syncytiotrophoblast which is a true syncytium with no apparent lateral intercellular spaces separating its nuclei. Nevertheless better understanding of this paracellular route is important as approximately 90% of the unidirectional flux of small solutes across the human placenta likely takes place through this pathway. The important questions to answer include: (1) What is the paracellular route in the syncytiotrophoblast? There is evidence that both transtrophoblastic channels and areas of denudation of the syncytiotrophoblast are involved. (2) Is it possible to regulate paracellular diffusion? There is now evidence from the mouse that IGF2 might regulate the formation of the paracellular route during placental development. (3) Is the rate of solute transfer via paracellular diffusion altered in pregnancy diseases? This is currently unknown but is being addressed in relation to IUGR.

Amanda Sferruzzi-Perri discussed how IGF2 deficiency modifies placental adaptation to maternal undernutrition during mouse pregnancy. During maternal undernutrition (UN), the placenta adapts to maintain fetal growth, despite its small size by increasing amino acid transport in late gestation. In part, these adaptations may be due to insulin-like growth factor-2 (IGF2), a known regulator of placental nutrient transfer capacity. The adaptation to UN in wildtype mice does not occur in small placentas deficient in the Igf2P0 transcript and likely results from delayed labyrinthine development and perturbed signaling down the growth regulating protein kinase B (AKT) pathway. The placental-specific transcript of the Igf2 gene may be involved in placental adaptation to UN.

Gernot Desoye discussed lipid transport across the placenta. Lipids are a complex class of molecules comprising fatty acids, triglycerides, phospholipids, cholesterol and cholesterol esters and lipid-soluble vitamins. Their bulk circulates in the form of lipoproteins (Lp) of various density classes such as VLDL, LDL, HDL. Microvillous Lp-receptors endocytose these Lps either unmodified (VLDL, LDL), after hydrolysis of triglycerides and phospholipids by endothelial lipase to release free fatty acids and/or after uptake of cholesterol esters (HDL). The free fatty acids are taken up by various microvillous transporter molecules. Once within the syncytiotrophoblast a yet unknown proportion of free fatty acids are released to the fetal circulation, the remainder are re-esterified to triglycerides and/or phospholipids and along with cholesterol esters stored in lipid droplets. Mechanisms and conditions of lipid droplet hydrolysis are unknown as are the transport mechanisms for lipid moieties across the basal syncytiotrophoblast membrane and further across the endothelial cell layer. Cholesterol is taken up by the endothelium, from which it egresses by the coordinated action of two transporters (ABCA1 and ABCG1). This process appears to be regulated by fetal demand.

Rohan Lewis discussed mechanisms of placental transport across the basal membrane of placental syncytiotrophoblast. Recent work has described the mechanisms by which amino acids are transported across the basal membrane of placental syncytiotrophoblast. Net efflux of amino acids to the fetus is mediated by 3 recently described facilitated transporters TAT1, LAT3 and LAT4. However, these transporters can only explain the transport of seven of the 20 amino acids. It has been proposed that the amino acids transported by the facilitated transporters provide substrates for amino acid exchangers. Unlike the facilitated transporters, the amino acid exchangers cannot quantitatively increase fetal amino acid concentrations as they transport one amino acid in and one amino acid out. However, working in conjunction, the facilitated transporters and the exchangers can mediate the transport of most amino acids into the feto-placental circulation. The facilitated transporters quantitatively increase fetal amino acid concentrations and the amino acid exchangers then qualitatively alter this by exchanging substrates of the facilitated transporters for other amino acids required by the fetus.

Valaria Dietrich discussed the molecular expression of NHE-3 in human pre-eclamptic placenta. The exchange barrier between maternal and fetal circulation in the human placenta consists essentially of two cellular layers, the syncytiotrophoblast and the fetal capillary endothelium. Transport activity of the syncytiotrophoblast is essential for a supply of a range of solutes required for fetal growth, as well as homeostasis of the cell itself. The sodium hydrogen exchanger isoform 3 (NHE-3) plays an important role in electrolyte and water homeostasis. These functions are compromised in pregnancies complicated with pre-eclampsia. At present it is not known whether NHE-3 expression is altered during pre-eclampsia. The molecular expression of NHE-3 was examined in syncytiotrophoblast of term placentas obtained from uncomplicated and pre-eclamptic pregnancies. RT-PCR and Western blot assays showed that the expression of NHE-3 was significantly reduced in syncytiotrophoblast from pre-eclamptic placentas. In normal placentas NHE-3 was localised in the apical and basal membranes and in the cytoplasm. However, in pre-eclamptic placentas NHE-3 was almost undetectable. Further studies are needed to determine whether the diminished expression of NHE-3 in pre-eclamptic placentas may compromise placental function and may contribute to the development of this syndrome.

Valaria Dietrich also discussed the regulation of aquaporin 9 in human placenta. In many tissues, aquaporins (AQPs) have been implicated in transmembrane water transport. It has been previously reported that aquaporin 9 (AQP9) is expressed in syncytiotrophoblast of normal human placenta. In pre-eclamptic placentas AQP9 expression is increased but it is functionally decreased. Therefore, studies are focused on determining the mechanisms that may modulate AQP9 expression and functionality. It has been demonstrated that hypoxia–ischemia observed in pre-eclampsia may be responsible for the upregulation in AQP9 protein. In addition, insulin levels are increased in pre-eclampsia and despite the fact that AQP9 gene has a negative insulin-response-element, insulin does not downregulate AQP9 expression. On the other hand, it has been suggested that altered phospholipid composition of the syncytiotrophoblast from pre-eclamptic placentas may contribute to a non-favorable environment for AQP9 and the reduced expression of CFTR failed to regulate AQP9 activity. However, the placental AQP9 physiological function(s) remains unknown.

Conclusions: This workshop provided a stimulating overview of some of the key issues affecting placental transport. Important questions still remain both in regard to the mechanisms of transport and the factors determining fetal growth. Issues raised in the discussion included the role of the endothelium in determining placental nutrient transport and the importance of developing new strategies to identify key placental determinants of fetal growth.

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11. Workshop 10: uteroplacental blood flow: from morphology to Doppler waveforms 

Organizers: Graham Burton, University of Cambridge, UK; Sascha Drewlo, Samuel Lunenfeld Research Institute, Canada.

Speakers: Graham Burton, University of Cambridge, UK; Sascha Drewlo, Samuel Lunenfeld Research Institute, Canada; Lynda Harris, University of Manchester, UK; Rosemary Keogh, University of Melbourne, Australia; Yee Khong, University of Adelaide, Australia; Samantha Smith, University of Manchester, UK; Oleksiy Solovyov, University of Kyiv, Ukraine.

Aim: This workshop aimed to explore the relationship between uterine spiral arterial remodeling, intraplacental maternal blood flow and placental pathology. Remodeling occurs during early pregnancy under the influence of EVT invasion and failure of the process is associated with major complications of pregnancy, such as miscarriage, IUGR and pre-eclampsia. But how are these complications linked to deficient conversion? Is it through placental hypoxia or changes in haemodynamic forces, or is deficient conversion a proxy marker for some more profound disorder of placentation? And how can we monitor the process in vivo? Topics covered ranged from molecular mechanisms underpinning remodeling, through mathematical modelling of blood flow through to placental phenotypes and uterine Doppler waveform analysis.

Summary: Sascha Drewlo introduced the basic mechanisms of spiral artery remodeling and possible downstream effects on the villous trophoblast and their clinical implications. Spiral artery remodeling is a key mechanism for placental formation and represents a hallmark in human placentation. It is a crucial process which results in adaptation of the maternal blood flow to the needs of the growing fetus.

Samantha Smith discussed data from studies into the role of chemokines in recruitment of decidual leukocytes to remodeling spiral arteries. Previous work has identified extensive disruption and loss of vascular smooth muscle cells (VMSC) and endothelium prior to EVT entry into arteries, suggesting EVT-independent remodeling occurs. In addition, CD45+ leukocytes infiltrate the vessel wall during EVT-independent remodeling, suggesting a key role in this process, similar to that described in the mouse. Significantly higher numbers of leukocytes, identified as uterine NK cells and macrophages, infiltrate remodeling spiral arteries in the early stages. Leukocytes do not infiltrate the wall of arteries distant from the implantation site (decidua parietalis) suggesting a local trigger for leukocyte recruitment. Trophoblast-secreted factors may activate spiral arteries to produce chemokines, which chemoattract leukocytes into remodeling vessels. Using a gene array approach, a panel of chemokines were identified as being differentially expressed in both VSMC and decidual endothelial cells in response to EVT conditioned medium. This suggests that factors secreted by EVT promote chemokine expression by vascular cells. In addition, chemoattractants released by vascular cells may recruit macrophages (CCL5, CCL7, CCL3 and CCL4) and uNK cells (CXCL12 and CX3CL1) to spiral arteries, where they participate in the early stages of remodeling.

Rosemary Keogh also discussed the role of chemokines in vascular remodeling in human pregnancy. Trophoblast cells instigate the remodeling of the vessel structure by migrating away from the placenta and invading the maternal spiral arteries of the uterine wall. Not only does the remodeling process occur during a defined time frame, but it also proceeds in a defined manner. Trophoblast cell movement is directed predominantly into the maternal uterine spiral arteries and is limited in extent to only penetrate into the first third of the myometrium before ceasing. Knowledge of the factors that regulate this migration of trophoblast cells and how the vessels themselves may contribute to controlling trophoblast movement is limited. Trophoblast cells are known to show directional movement with greater persistence and speed in the presence of vascular cells. The most probable cause of this directed movement is chemokines, a subgroup of cytokines that can cause chemotaxis (directed movement) of nearby responsive cells. Vascular cells are known to produce chemokines which have a direct role in vascular remodeling and trophoblast cells are known to express receptors for several chemokines, including those made by vascular cells. In addition to acting as chemoattractants, there is now evidence that chemokines have other important functions including regulating cell proliferation, survival and adhesion. Given their ability to regulate migration, proliferation and adhesion, functions that are characteristic of trophoblast cells, vascular-derived chemokines may play critical roles in regulating trophoblast cell behaviour.

Lynda Harris discussed the importance of elastolysis in the spiral arteries as a prerequisite for successful remodeling. Rapid development of the villous placenta in early gestation is necessary to support a successful pregnancy. To ensure that blood is delivered to the intervillous space at an optimal pressure and velocity, EVT colonise and remodel the uterine spiral arteries. The resulting vascular structures lack smooth muscle and elastic fibres and are heavily dilated. Catabolism of the vascular extracellular matrix is central to arterial transformation and breakdown of elastin is vital to achieve permanent vasodilatation and abolish maternal vasomotor control. EVT can be observed migrating through breaks in the internal elastic lamina of colonised arteries in vivo, and can engulf and degrade elastin in vitro. Although they express a wide variety of proteases to facilitate matrix breakdown, only select matrix metalloproteinases (MMP) are capable of degrading elastin. MMP-12 (macrophage metalloelastase) is expressed by EVT and decidual leukocytes in the first trimester and MMP-12 expression is induced in the smooth muscle cells (SMC) of spiral arteries perfused with trophoblast-conditioned medium. Inhibition of MMP-12 activity significantly reduced the elastase activity of both trophoblasts and SMC. It is hypothesized that the coordinated actions of EVT, SMC and decidual leukocytes mediate local elastin catabolism during arterial transformation. Reduced elastolysis may impede spiral artery remodeling, leading to the persistence of narrow bore vessels and perturbed blood flow within the intervillous space.

Graham Burton discussed how mathematical modelling may help our understanding of the consequences of spiral artery conversion on uteroplacental blood flow. Haemochorial placentation presents unique challenges for maternal perfusion of the placenta; a high volumetric flow must be achieved at a low pressure to avoid compressing the fetal capillaries, and at sufficiently low velocity to prevent damage to the villous trees. Dilation of the mouths of the spiral arteries achieves this compromise. Using measurements obtained from serial sections of placenta-in-situ specimens they have calculated that dilation from 0.5 mm diameter in the myometrium to 2.5 mm at the mouth of the artery is associated with a reduction in velocity from 2 to 3 m s⁻¹ to approximately 10 cm s⁻¹. By contrast, conversion is associated with a more modest increase in volumetric flow. These findings correlate with ultrasound observations that maternal blood flow into the placenta in cases of deficient spiral artery conversion is often jet-like and turbulent, and associated with the formation of thrombus-lined intervillous lakes. Conversion of the myometrial segment of the spiral artery is probably related more to the removal of the highly contractile segment that prevents excessive blood loss during menstruation, so reducing the risk of spontaneous vasoconstriction during pregnancy and placental ischemia-reperfusion injury.

Oleksiy Solovyov discussed how a ‘jelly-like’ heterogeneous placenta, as identified through clinical, ultrasound and morphological features, may be a predictor of adverse pregnancy. A study was discussed that investigated the outcome of 13 pregnancies with sonographic findings of thick and heterogeneous placenta, comparing histopathological, clinical and Doppler data. A ‘jelly-like’ placenta was defined as a thick heterogeneous placenta with a layer of decreased echogenecity with visible slow swirling movements of contents, which quivered like jelly when the abdominal wall was tapped. Doppler evaluation of the placenta and uteroplacental circulation was performed. The pregnancy course and neonatal outcome were also reviewed and the placenta morphologically evaluated after delivery. Perinatal death occurred in 5 cases that were all associated with uterine artery Doppler that was abnormal or secondary ‘normalized’ after previously increased resistance for uterine arteries blood flow. In 4 cases neonatal outcome was good, with associated normal uterine artery Doppler. In 4 women with boundary uterine Doppler indices neonatal outcome was satisfactory, but the babies were low-birthweight. Morphologically typical vascular changes were observed, namely infarcts and thrombosis, local blood stasis in vessels of the decidua, villi and chorionic membranes, and arterial obliteration of some secondary and tertiary stem villi. The presence of distally located parts of villi fixed in fibrinoid, pushed aside from the intervillous blood flow, along with narrowing of the intervillous space due to infarcts, thrombosis, and conglomerates of villi fixed by fibrinoid were seen. The sonographic presence of a thick heterogeneous ‘jelly-like’ placenta is strongly associated with an adverse pregnancy outcome, especially in the case of abnormal uterine artery Doppler or secondary ‘normalisation’ after a previously abnormal one.

Yee Khong discussed correlations between the maternal vascular phenotype and placental pathology. Growth and decidualisation are basic features of the response of uterine spiral arteries prior to pregnancy. In pregnancy unique structural changes occur first in endometrial and subsequently in myometrial segments in response to EVT invasion. Ultimately physiological changes are achieved that allow blood flow of some 600 ml/min into the intervillous space. The term ‘placental bed’ was deliberately chosen to emphasize that it includes “not only basal decidua but also underlying myometrium containing the origins of the uteroplacental (spiral) arteries”. Abnormal decidual and myometrial spiral artery remodeling has been described in pre-eclampsia and IUGR. Maternal vascular alterations are also described in placenta accreta. Additionally, other vascular lesions are seen that could explain the placental phenotype. The effect of prior pregnancy on maternal vascular changes has been described but are often not taken into account in experimental studies. Pathological lesions were discussed that are ascribed to perturbations of maternal uteroplacental blood flow but which are poorly defined, using diffuse chorioamniotic haemosiderosis as an example.

Conclusions: This workshop highlighted our greater understanding of the molecular mechanisms underlying spiral artery remodeling and the contribution of both leukocytes and trophoblast cells to this process. There is also a greater understanding of how remodeling impacts on placental blood flow and the pathological changes induced when the process fails. Further work is required however to fully determine the contribution of different cell types to spiral artery remodeling, and the mechanisms by which they act. The challenge remains as to how this greater understanding may be translated to treat adversely affected pregnancies.

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12. Workshop 11: placental stem cells 

Organizers: Bill Kalionis, University of Melbourne, Australia; Ursula Manuelpillai, Monash University, Australia.

Speakers: Chie-Pein Chen, Mackay Memorial Hospital, Taiwan; Yuben Moodley, Monash University, Australia; Sean Murphy, Monash University, Australia; David Natale, University of Calgary, Canada; Rishika Pace, University of Melbourne, Australia; Veronique Tache, University of California, USA.

Aim: Stem cells are readily isolated from the placenta, umbilical cord, fetal membranes and amniotic fluid. The stem cell numbers can be expanded greatly in cell culture, display considerable plasticity in their transdifferentiation potential and may have immunomodulatory characteristics. These properties make gestational tissue derived stem cells attractive candidates for a wide variety of potential therapeutic applications. However, there are major gaps in our knowledge of the basic biology of placental stem cells, their role in the normal function of the placenta and their potential role in placental pathologies. This knowledge is also crucial to understanding the behaviour of these stem cells when they are xenotransplanted in various animal model systems and ultimately into humans. The aim of the workshop was to provide an overview of contemporary research into the biology of placental stem cells and studies to determine their potential for therapeutic applications.

Summary: Veronique Tache discussed the role of PPARγ in mouse trophoblast stem (TS) cells. Trophoblast cells, the epithelial cells of the placenta, can be divided into two main lineages: villous trophoblast, which mediate gas/nutrient exchange, and extravillous trophoblast, which invade the uterus and establish blood supply to the placenta. Little is known about trophoblast lineage specification. Mouse trophoblast stem cells derived from E3.5 blastocysts are invaluable for studying this process in vitro. Using this system it was shown that PPARγ, a ligand-activated transcription factor, directs trophoblast down the villous (labyrinthine) pathway. PPARγ-null TS cells differentiate prematurely, and exclusively, into invasive extravillous trophoblast (trophoblast giant cells). In addition, it was found that PPARγ expression is turned on when TS cells differentiate in normoxia, and is downregulated under hypoxic conditions. When reintroduced into wildtype TS cells differentiating under hypoxia, PPARγ induced differentiation specifically into labyrinthine trophoblast. Finally, PPARγ-null TS cells showed increased expression of sVEGF-R1, which has been associated with development of pre-eclampsia.

David Natale discussed some of the molecular mechanisms underlying differentiation and organization of trophoblast cells in the mouse. The labyrinth layer of the mouse placenta is primarily derived from cells of the chorion. An understanding of the molecular mechanisms controlling the differentiation and organization of the trophoblast cells within this layer will provide tremendous insight into development and pathology of the placenta. It is well understood that Gcm1 is an important regulator of labyrinth development. It has been shown recently that syncytiotrophoblast associated with either the maternal or fetal blood spaces can be identified by the layer-restricted expression of Syncytin a and Gcm1 respectively. In addition, these genes are expressed in a similar spatially related manner in the chorion, suggesting pre-patterning of labyrinth development. Data were presented characterizing the expression of lymphocyte antigen 6, locus E (Ly6e), in trophoblast of the mouse placenta. Ly6E is a membrane-associated protein that is expressed in the hematopoietic lineage and is a marker of trophoblast cell precursors. Using trophoblast stem cell cultures, it was found that Ly6e was expressed in undifferentiated TS cells and increased upon differentiation. Interestingly, in vivo, Ly6e mRNA was first detectable in a subset of cells in the chorion beginning at E8.5. This pattern of expression differed from Gcm1 but was similar to that of Syncytin a, and at later stages of gestation, Ly6e expression was restricted to syncytiotrophoblast cells in the labyrinth. Furthermore, siRNA knockdown of Ly6e in TS cells results in a decrease in expression of Syncytin a, suggesting that Ly6e may be important for syncytiotrophoblast differentiation.

Chie-Pein Chen discussed data showing that human placental multipotent mesenchymal stromal cells (hPMSCs) could repair oxidative injury to the placenta. Reactive oxygen species cause oxidative damage in the placenta. It was postulated that damage to placental endothelial cells caused by reactive oxygen species can be repaired through activation of antioxidant enzymes and by paracrine factors from hPMSCs. In the described study, hPMSC conditioned medium was prepared to test for anti-oxidative and anti-apoptotic effects on HUVECs subjected to oxidative stress with tert-butyl hydroperoxide. hPMSCs produced several cytokines including those of the IL-6 cytokine family, IL-6 and LIF. hPMSC conditioned medium supported EC survival and reduced EC intracellular peroxides and apoptosis. This protective effect of hPMSC conditioned medium did not require cell–cell contact and was mediated by the gp130/STAT3 pathway. The result of gp130/STAT3 activation was upregulation of manganese superoxide dismutase (MnSOD), but not other antioxidant enzymes. MnSOD scavenges oxidative stress-induced cellular reactive oxygen species that may inactivate pro-apoptotic caspase-3. Other mechanisms not assessed by these experiments may also be involved, but at least one pathway by which hPMSCs protect against oxidative damage was described, namely activation of gp130/STAT3, leading to an increase in MnSOD.

Rishika Pace discussed the role of the microenvironment in maintaining mesenchymal stem cells in vitro. Cultured mesenchymal stem cells derived from human term placenta (PMSCs) are intensely studied because of their potential utility in regenerative medicine. Regulation and maintenance of MSC behaviour involves both intrinsic and extrinsic cues. An important extrinsic cue is integrin-mediated cell adhesion to the extracellular matrix. Integrins help define and shape the microenvironment in which stem cells are found i.e. the stem cell niche. Adhesion to the basement membrane is important in the stem cell niche. Antibodies to integrin alpha 1 (ITGA1) are routinely used to enrich for cells with colony forming ability (CFU), which is an important property of stem cells. siRNA was used to reduce expression of ITGA1 in PMSCs and alterations in expression of extracellular matrix proteins examined. ITGA1 mRNA and protein expression were successfully knocked down in PMSCs. ITGA1 knockdown did not affect PMSC proliferation or migration but affected the ability of PMSCs to adhere to specific substrates. PMSC adhesion to the substrates laminin, vitronectin and collagen I was significantly affected following ITGA1 knockdown. Using a real time RT-PCR array it was also determined that the intrinsic production of specific extracellular matrix and adhesion molecules was affected following ITGA1 knockdown. Lack of adhesion is one of the causes of poor cell survival in stem cell transplantation and hence this study may have relevance to the clinical application of PMSCs.

Yuben Moodley discussed how placental derived mesenchymal stem cells and amnion epithelial cells might be used in treatments for lung injury. Chronic lung diseases are characterized by loss of lung tissue, inflammation and fibrosis. Cellular therapies that potentially restore pneumocytes and reduce inflammation would be a major advance in management. The role of term human umbilical cord cells derived from Wharton's jelly with the phenotype consistent with mesenchymal stem cells (uMSCs) and human amnion epithelial cells (hAECs) in treating a Bleomycin-induced model of lung injury was examined. Cells were administered systemically into a mouse model of acute lung injury 24 h after intra-nasally administered Bleomycin. Lungs were analyzed for inflammation, fibrosis and alveolar epithelial differentiation. It was demonstrated that both hAECs and uMSCs reduced inflammation in association with decreased macrophage chemotactic protein-1 (MCP-1), TNF-α, IL-1 and IL-6 and pro-fibrotic TGF-β in mouse lungs. In addition, collagen in the lung was significantly reduced by both uMSCs and hAECs as a possible consequence of increased degradation by matrix metalloproteinase-2 (MMP-2) and downregulation of their endogenous inhibitors, TIMP-1 and TIMP-2. uMSCs did not demonstrate lung differentiation while hAECs developed an alveolar phenotype. Taken together, these findings suggest that both uMSCs and hAECs, have anti-inflammatory properties and reduce fibrosis in lung injury but that hAECs can adopt a lung phenotype.

Sean Murphy introduced animal product-free methods of amnion epithelial cell isolation, cryopreservation and culture in accordance with current guidelines on preparation of cells for clinical use. Human amnion epithelial cells (hAECs) are easily accessible and do not have the limitations of adult and embryonic stem cells for their potential clinical application. They are a heterogeneous population positive for stem cell markers and display multi-lineage differentiation potential, forming cells of the endoderm (liver, lung epithelium), mesoderm (bone, fat), and ectoderm (neural cells). They may have a low immunogenic profile and possess potent immunosuppressive properties. Collectively, these findings indicate that hAECs may elicit minimal immune recognition following transplantation to an allogeneic recipient. The animal product-free isolation method described produces a high yield of viable hAECs with minimal mesenchymal cell contamination. Following cryopreservation using an animal product-free cryopreservation protocol, hAECs maintain high viability and metabolic activity. Cryopreserved hAECs which are thawed and expanded in vitro using animal product-free culture media maintain a normal karyotype, cell cycle distribution and long telomeres over 5 passages. These cells also display multipotent differentiation potential in vitro, differentiating into lineages of the three primary germ layers. Term amnion is an attractive alternative source to cells derived from the embryo or adult for the derivation of pluripotent cells for research and possible clinical application.

Conclusions: The workshop highlighted our increasing fundamental and practical knowledge of placental stem cells, which derives from basic studies in the mouse, investigating the properties of in vitro cultured human placental stem cells, optimizing the preparation of stem cells for human clinical use and from promising clinical studies in animal disease models that accentuate the therapeutic potential of human placental derived stem cells. However, significant gaps in our knowledge were also revealed. The placental stem cell niche, and the factors that control stem cell behaviour in the niche, are poorly understood. The efficient use of human stem cells at the clinical level will require further optimization of preparation and storage methods. Finally, a better understanding of the factors that influence the in vivo homing and engraftment of placental stem cells into damaged or diseased tissues, and their long-term safety, will significantly increase their therapeutic potential.

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13. Workshop 12: genes and placenta 

Organizers: David Natale, University of Calgary, Canada; Amanda Sferruzzi-Perri, University of Cambridge, UK.

Speakers: Edward Chuong, Stanford University, USA; Wee-Ching Kong, University of Adelaide, Australia; Craig Park, McGill University, Canada; Amanda Sferruzzi-Perri, University of Cambridge, UK; Stephen Tong, Monash Institute of Medical Research, Australia; Simon Tunster, Cardiff University, UK.

Aim: The placenta performs an array of functions, which are essential for both fetal growth and the maintenance of maternal health during pregnancy. Thus it is not surprising that a multitude of genes have been implicated in the regulation of placental development and function. Over the last several years, research focused on the genetic regulation of placentation has grown to include not only the identification of genes and their function in the placenta but also to characterize the processes by which these genes are regulated. For instance, epigenetic mechanisms and more recently, non-coding RNAs, have been shown to regulate the placental expression of many genes. In addition, there is a growing appreciation for interactions between the genome and the in utero environment that are thought to influence placental development, phenotype and disease. The aim of this workshop was to explore recent advances in understanding the role of novel genes in the placenta. Focus was on their unique patterns of expression, mechanisms of regulation and potential relevance to placental disease.

Summary: Amanda Sferruzzi-Perri discussed microRNA regulation of the retrotransposon-derived gene Rtl1. Rtl1 is a retrotransposon-derived gene located in a cluster of imprinted genes of ∼1.2 Mb on murine chromosome 12. It is a paternally expressed intron-less imprinted gene expressed by the placenta and fetus. Recently, several microRNAs within the sense and antisense Rtl1 transcripts were identified which suggest there are complex regulatory mechanisms designed to control Rtl1 expression (and potentially have other targets). Mir-127 is the most highly expressed anti-Rtl1 microRNA thought to play a major role in the imprinting of Rtl1 as it is expressed exclusively from the maternal allele and degrades paternal Rtl1 transcript. The biological role of RTL1 in placental morphogenesis was investigated using specifically designed paternally transmitted Rtl1 and maternally-transmitted mir127 knockout mice, which have reduced or increased RTL1 expression, respectively. These studies reveal that RTL1 expression is essential for placental labyrinthine growth via its impacts on fetal capillary formation. Furthermore, knockout of the paternal Rtl1 transcript severely impaired fetal growth and increased mortality, while a 2.5-fold increase in Rtl1 expression in the mir127 knockout does not affect fetal growth despite placentomegaly. Work is currently underway to define the pathway by which RTL1 acts in placental vasculogenesis and determine whether Mir-127 has targets additional to Rtl1.

Craig Park discussed the role of Nodal in the uterus. Nodal, a member of the TGF-β superfamily, is a critical factor in many aspects of embryonic development. In order to investigate the potential role of Nodal in the uterus during pregnancy, a tissue-specific conditional knockout of Nodal was established for the female reproductive tract. Interestingly, uterine Nodal knockout mice exhibit various reproductive abnormalities including IUGR and fetal abortion late into development (d17.5). Strikingly, the maternal basal plate of the placenta was extremely diminished, or in most cases, completely absent. Furthermore, fetal tissue layers that comprise the placenta appeared disorganized. Based on these observations it was hypothesized that maternal Nodal in the uterus plays a critical role in decidualization and developing adequate vascularization within the placenta. Understanding the mechanisms that underlie IUGR and preterm delivery are paramount in the ultimate goal of eliminating complications during pregnancy leading to pre-eclampsia, preterm birth and embryonic loss.

Simon Tunster discussed the role of the imprinted gene Phlda2 in mouse placental development. Imprinted genes, which are expressed from only one parental allele, act in concert to precisely regulate fetal and placental growth. Phlda2 is a rheostat for placental growth, with Phlda2 deficiency resulting in placentomegaly due to a disproportionate expansion of the junctional zone and an increase in stored glycogen. Excess Phlda2 causes the cells of the junctional zone to mislocalise with a relative failure of glycogen cell migration at E16.5 and a loss of the distinct boundary between junctional and labyrinth zones at E18.5. The junctional zone of the mouse placenta comprises two cell types, namely the spongiotrophoblast and glycogen cell populations. Normal expression of glycogen cell-specific markers and enzymes critical for glycogen synthesis suggested that the glycogen storage defect was not intrinsic to this cell type. In contrast, the spongiotrophoblast-specific Prl8a8 gene was significantly downregulated. These data identify an unforeseen role for the spongiotrophoblast cells in regulating the ability of the glycogen cell population to store glycogen. In addition to a major placental defect, increased Phlda2 progressively restricts embryonic growth from E16.5. These data suggest that stores of placental glycogen are essential to support late embryonic growth.

Stephen Tong discussed how forskolin-induced syncytialisation of BeWo cells altered microRNA expression. MicroRNAs are endogenously produced non-coding RNAs possibly regulating a third of all genes. The potential role of microRNAs in regulating syncytialisation was examined. BeWo cells were syncytialised with forskolin (a well known model of syncytialisation), total RNA extracted, and a microRNA microarray performed on two separate occasions. Six microRNAs with significantly changed expression (miR-20a, 20b, 21, 32, 455 and 513) were identified. All six are highly expressed in placenta and widely distributed across the genome. Gene Ontogeny analysis suggested target genes for all six microRNAs are enriched for those involved in nuclear and membrane function. Genes were also identified that are putative targets of a multiple number of these six microRNAs, including ten potentially targeted by four microRNAs, and 65 genes that are putative targets of three microRNAs (20a, 20b, 21 and 32). It was concluded that microRNAs may play an active role in regulating syncytialisation.

Wee-Ching Kong described the characterization of microRNA expression throughout gestation in the mouse placenta. MicroRNA expression in the mouse placenta increases substantially between mid and late gestation and many differentially expressed microRNAs are predicted to target differentiation of various trophoblast cell types and nutrient transport. Some are located in the imprinted region of mouse chromosome 12, including the anti-RTL1 gene, known to be essential in placental development and fetal survival. MicroRNA and mRNA expression patterns in labyrinth and junctional zones of the late gestation (mouse) placenta were also described and regulatory modules identified that are consistent with their regional functional differences. Further work is needed to localise microRNA expression to specific placental cell types and directly test microRNA actions on predicted target mRNAs.

Edward Chuong discussed data from a genomic investigation of a conserved placental gene network. An intrinsic obstacle to investigating placental developmental genetics is the immense diversity of placental morphologies among mammals. Now, the current wealth of mammalian comparative genomic data and high-throughput sequencing technologies enables detailed examination of placental development at a genome-wide level across multiple species. High-throughput sequencing of mRNA (RNA-seq) was performed to generate quantitative transcriptomes over a time course of placental development in both the mouse and the tammar wallaby marsupial; these data help to highlight genes with deeply conserved roles in placental evolution, as well as those involved in eutherian-specific placental adaptations. 1445 genes expressed in the wallaby placenta were identified, where 1362 have a direct mouse ortholog. Of these, 995 are non-housekeeping genes that are also expressed in the mouse trophoblast. Further analysis of expression patterns will help elucidate the conserved placental gene networks that define the ancestral placenta.

Conclusions: In keeping with the title of the workshop, the studies that were presented highlighted the diverse nature of work related to “genes and placenta”. There was good discussion surrounding all of the talks. We were reminded that interactions with the uterus are integral to both placental and embryonic development and we were shown how current genomic investigations are providing insight into genes that are important for placental development across many different species. It is clear that while studies surrounding gene regulation by imprinting as well as emerging fields such as microRNAs will add complexity, ultimately they will also increase our understanding of placental development, function and pathologies.

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Conflict of interest 

The authors do not have any potential or actual personal, political, or financial interest in the material, information, or techniques described in this paper.