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Abstract: The pivotal role of glucocorticoids in the initiation of parturition has been very well documented in several domestic mammalian animal species. However the role of glucocorticoids in human parturition remains controversial mainly because of the absence of effect of synthetic glucocorticoids, given to promote fetal organ maturation in pregnant women with threatened preterm delivery, on the length of gestation. This article will review studies of glucocorticoids in human parturition and provide evidence for an important role of glucocorticoids in human parturition as well but a simultaneous high concentration of estrogen within the intrauterine tissues may be necessary for GCs to initiate parturition. The synthetic GCs dexamethasone and betamethasone pass through the placenta intact resulting in potent negative feedback on the fetal HPA axis and diminished production of DHEA from fetal adrenal glands for estrogen synthesis by the placenta. This may negate the effect of systemic administration of GCs on the induction of labor, especially in cases where the myometrium is not yet fully primed by estrogen. Endogenous glucocorticoids are inactivated by the placental 11β-HSD2 thus limiting the negative feedback of maternal cortisol on the fetal HPA axis and allowing the simultaneous rise of cortisol and estrogen levels towards the end of gestation. Therefore, endogenous glucocorticoids, particularly glucocorticoids produced locally in the intrauterine tissues may play an important role in parturition in humans by enhancing prostaglandin production in the fetal membranes and stimulating estrogen and CRH production in the placenta. [Copyright &y& Elsevier]

Abstract: Biobanks provide an important repository of samples for research purposes. However, for those samples to reflect the in vivo state, and for experimental reliability and reproducibility, careful attention to collection, processing and storage is essential. This is particularly true for the placenta, which is potentially subjected to stressful conditions during delivery, and sample collection may be delayed owing to routine postpartum inspection by clinical staff. In addition, standardisation of the collection procedure enables samples to be shared among research groups, allowing larger datasets to be established. Here, we provide an evidence-based and experts' review of the factors surrounding collection that may influence data obtained from the human placenta. We outline particular requirements for specific techniques, and propose a protocol for optimal sample collection. We recognise that the relevance of these factors, and of the sample types collected to a particular study will depend on the research questions being addressed. We therefore anticipate that researchers will select from the protocol to meet their needs and resources available. Wherever possible, we encourage researchers to extend their collection to include additional samples that can be shared on an international collaborative basis, with appropriate informed consent, to raise the quality, as well as quantity, of placental research. [Copyright &y& Elsevier]

Abstract: Preeclampsia (PE) affects 5–8% of pregnancies and is responsible for 18% of maternal deaths in the US, and for long-term complications in mother and child. PE is an inflammatory state and may influence placental function in a sex-specific manner. We determined if there is a sexual dimorphism in the placental inflammatory and apoptotic responses in preeclamptic pregnancies. Placentas were collected from normotensive and preeclamptic pregnancies with either male or female fetuses (MPE and FPE respectively) after c-section at term with no labor. Expression patterns of markers of inflammation measured by ELISA, as well as hypoxia, apoptosis and angiogenesis markers measured by Western blotting were determined in the placenta. Consistent with previous studies, an increase in inflammation, hypoxia, and apoptotic cell death was observed in PE compared to normotensive pregnancies. Levels of TNFα, IL-6 and IL-8, and HIF-1α were significantly greater, whereas the angiogenic marker VEGF was significantly reduced in MPE vs. FPE. Sexual dimorphism was also observed in the activation of cell death: the number of TUNEL-positive cells, and the expression pro-apoptotic markers PUMA and Bax being higher in MPE vs. FPE. We also found an increase in the levels of protein and DNA-binding activity of NFκB p65 in MPE vs. FPE. In summary, we show here that in preeclamptic pregnancies the placentas of males were associated with significantly higher expression of inflammatory, hypoxia and apoptotic molecules but reduced expression of a pro-angiogenic marker compared to placentas of female fetuses. We propose that the transcription factor NFκB p65 might, at least partially, be involved in sexual dimorphism during PE. [Copyright &y& Elsevier]

Abstract: Preeclampsia (PE) affects 5–8% of all pregnancies and is associated with significant maternal and fetal morbidity and mortality. Placental mitochondrial dysfunction has been reported in PE. MicroRNAs (miRNA) are small non-coding RNAs that regulate gene expression through mRNA degradation and translational repression. MiR-210 has been previously shown to be upregulated in placentas from pregnancies complicated by PE. We hypothesized that placental mitochondrial dysfunction during PE can be mediated by miR-210. Placentas were collected at term from normotensive pregnancies (CTRL) and those complicated by severe PE (n = 6 each) following c-section (no labor). Villous tissue from PE showed significantly increased levels of HIF-1α compared to CTRL with no change in corresponding mRNA expression but with reduced DNA-binding activity. Mitochondrial complex III was significantly decreased in PE along with significantly reduced protein expression in complex I and IV during PE. Among the four miRNAs tested, miR-210 showed significant up regulation in PE and significant downregulation of its target, ISCU mRNA. To understand the role of miR-210 in PE, loss- and gain-of-function studies were performed using primary trophoblasts. Trophoblasts were transfected with miR-210 inhibitor or pre-miR-210 and mitochondrial function was measured using Seahorse Extracellular Flux Analyzer. Cells transfected with pre-miR-210 showed significant reduction in oxygen consumption. In contrast, transfection of trophoblast with AntagomiR-210 was sufficient to prevent the DFO-mediated respiratory deficiency. These data collectively suggest that miR-210 overexpression during PE could be responsible for placental mitochondria dysfunction. [Copyright &y& Elsevier]

Abstract: Hypothesis and study objectives: We hypothesized that maternal obesity is associated with increased placental amino acid transport and hyperleptinemia. Our objectives were to study placental amino acid transport and the effect of leptin on placental amino acid transport in vitro in the setting of maternal obesity. Materials and methods: Seven lean, BMI at entry 22.4, and seven obese, BMI at entry 31.5 (p < 0.001), pregnant women were studied at 39 weeks. We measured baseline and leptin-stimulated placental system A sodium-dependent neutral amino acid transporter (SNAT) activity, placental immunoreactive protein expression of SNAT, leptin and leptin receptor, and maternal and fetal plasma leptin concentrations, with significance set at p ≤ 0.05. The primary outcome measure was placental SNAT activity. Results: The obese group had decreased placental SNAT activity (p = 0.005), maternal hyperleptinemia (p = 0.01) and decreased syncytiotrophoblast expression of leptin receptor (p = 0.01) and SNAT-4 (p < 0.001). Placental amino acid uptake was significantly stimulated by leptin in the lean group as compared to the obese group. Maternal weight gain and offspring birth weights were not different between groups. Conclusion: Maternal obesity was accompanied by decreased placental SNAT activity associated with maternal hyperleptinemia and placental leptin resistance in spite of appropriate maternal weight gain and normally grown neonates. These findings suggest altered placental function that may have clinical implications in obese pregnant women. [ABSTRACT FROM AUTHOR]

Abstract: Maternal obesity is an increasing problem in obstetrics associated with adverse pregnancy outcomes and delivery complications. As an inflammatory state, where elevated levels of pro-inflammatory cytokines are found, obesity can lead to the increased incidence of oxidative and nitrative stress. These stresses may result in protein oxidation and protein nitration respectively, which are post- translational covalent modifications that can modify the structure and subsequently alter the function of a protein. The objective of this study was to examine whether placental oxidative and nitrative stress increase with increasing maternal body mass index. Placental tissue was collected from three groups of patients categorized as lean, overweight and obese. The presence of nitrotyrosine residues, a marker of nitrative stress, and antioxidant enzymes, as markers of oxidative stress, were assessed by immunohistochemistry, Western blot and ELISA. Protein carbonyl formation, a specific measure of protein oxidation, was measured by OxyBlot™ kit. Nitrotyrosine residues were increased in obese compared to lean and overweight groups although localization was unaltered across the three groups. Superoxide dismutase enzyme expression, localization and activity was unaltered between the groups. Protein carbonyl formation was greater in the lean compared to the overweight individuals. This study demonstrates that with increasing maternal body mass index there is an increase in placental nitrative stress. There does not appear to be a corresponding increase in oxidative stress and indeed we demonstrate some evidence of a decrease in oxidative effects in these placenta samples. Potentially the formation of peroxynitrite may be consuming reactive oxygen species and reducing oxidative stress. There may be a shift in the balance between nitrative and oxidative stress, which may be a protective mechanism for the placenta. [Copyright &y& Elsevier]

Abstract: Crucial roles of the placenta are disrupted in early and mid-trimester pregnancy loss, preeclampsia, eclampsia and intrauterine growth restriction. The pathophysiology of these disorders includes a relative hypoxia of the placenta, ischemia/reperfusion injury, an inflammatory response and oxidative stress. Reactive oxygen species including nitric oxide (NO), carbon monoxide and superoxide have been shown to participate in trophoblast invasion, regulation of placental vascular reactivity and other events. Superoxide, which regulates expression of redox sensitive genes, has been implicated in up-regulation of transcription factors, antioxidant production, angiogenesis, proliferation and matrix remodeling. When superoxide and nitric oxide are present in abundance, their interaction yields peroxynitrite a potent pro-oxidant, but also alters levels of nitric oxide, which in turn affect physiological functions. The peroxynitrite anion is extremely unstable thus evidence of its formation in vivo has been indirect via the occurrence of nitrated moieties including nitrated lipids and nitrotyrosine residues in proteins. Formation of 3-nitrotyrosine (protein nitration) is a “molecular fingerprint” of peroxynitrite formation. Protein nitration has been widely reported in a number of pathological states associated with inflammation but is reported to occur in normal physiology and is thought of as a prevalent, functionally relevant post-translational modification of proteins. Nitration of proteins can give either no effect, a gain or a loss of function. Nitration of a range of placental proteins is found in normal pregnancy but increased in pathologic pregnancies. Evidence is presented for nitration of placental signal transduction enzymes and transporters. The targets and extent of nitration of enzymes, receptors, transporters and structural proteins may markedly influence placental cellular function in both physiologic and pathologic settings. [Copyright &y& Elsevier]

Abstract: The aim of the present study was to examine the effect of lipopolysaccharide (LPS) on the secretion of the pro-inflammatory cytokine interleukin-1β (IL-1β) and of its natural inhibitor interleukin-1 receptor antagonist (IL-1Ra), by perfused human term and preterm placental tissue. Eight term and eight preterm placentae were collected immediately after delivery; four term and four preterm placentae were perfused with control medium (without LPS) and the other four term and four preterm placentae were perfused with medium containing LPS. The release of IL-1β into the maternal compartment by term placenta was significantly higher than the release by preterm placenta (p <0.001). However, there were no significant differences between IL-1β levels released into the fetal compartments of term and preterm placentae. No significant differences were observed in the release of IL-1Ra into the maternal and fetal compartments of term placenta, when compared to preterm placenta. Exposure to LPS significantly decreased the capacity of term placenta to release IL-1β into the maternal compartment (p <0.001) and increased the capacity of term placenta to release IL-1Ra into the maternal and fetal compartments (p <0.001 and p =0.017, respectively). However, the capacity of preterm placentae to release IL-1β and IL-Ra into the maternal and fetal compartments was not affected by LPS. IL-1β was expressed by both term and preterm placentae before and after perfusion (+/− LPS), by epithelial cells of the amnion, chorion, by syncytiotrophoblast and stromal cells of villous tissue and by the decidua. IL-1Ra in term and preterm placentae was expressed before perfusion mainly in epithelial cells of the amnion. After perfusion of term placentae (+/− LPS), additional IL-1Ra expression was seen in epithelial cells of the amnion and in syncytiotrophoblast and stromal cells of villous tissue and by the decidua. However, perfusion of preterm placentae (+/− LPS) did not affect IL-1Ra expression. The localization of IL-1β and IL-1Ra in both term and preterm human placental tissue suggests a their physiologic role. The data presented indicates that the IL-1 system in term and preterm placentae seems to be differently affected by LPS. Down-regulation in the release of the pro-inflammatory cytokine IL-1β and the up-regulation of its antagonist (IL-1Ra) may be a part of the inflammatory response to infection in human term, but not preterm, placentae. The IL-1 system in term and preterm placentae seems to be differently affected by LPS. [Copyright &y& Elsevier]

Abstract: P2X4 receptors are activated by extracellular ATP to raise intracellular calcium, thus altering cell signalling. ATP release occurs under pathophysiological, stress and adverse cell conditions; these are all increased in preeclampsia. Although P2X4 is abundantly expressed in normal placenta neither the differences in the amount of protein nor its post-translational modifications have been studied in placentae from pregnancies complicated by preeclampsia. Thus we examined P2X4 protein expression, localization and post-translational modifications in normotensive controls, term and preterm preeclamptic placentae. Densitometric analysis of Western blots showed a significant increase in P2X4 protein expression in both term (p =0.002) and preterm preeclamptic (p =0.0008) placental samples compared to normotensive controls however the tissue localization of this receptor subtype was unaltered across the groups. Our data showed that P2X4 is a nitrated protein in the placenta and this nitration is upregulated in preterm preeclamptic placenta compared to normotensive controls (p =0.03). We also demonstrated that P2X4 is heavily glycosylated in the placenta by deglycosylation with PNGase F which reduced the protein product size by 23kDa. We propose that P2X4 acts within the syncytiotrophoblast to alter intracellular calcium and subsequent signalling pathways thereby restoring placental cell homeostasis following ATP-induced changes during pathophysiological conditions such as preeclampsia. We also propose that the post-translational modifications of nitration and glycosylation are required for the normal functioning of P2X4. [Copyright &y& Elsevier]

OBJECTIVE: The effects of prostaglandin E2 (PGE2) are mediated through G-protein coupled receptors, acting via different second messengers. The aim of this study was to characterize the temporal and tissue specific localization and expression of the PGE2 receptor subtypes (EP1-4) in uteroplacental tissues during human pregnancy. STUDY DESIGN: Placenta and fetal membranes were collected after delivery at preterm or term, each with or without labor. The localization and expression of the PGE2 receptor subtypes were determined by immunohistochemistry and Western blot. RESULTS: All 4 receptors were expressed in the placenta and fetal membranes; only EP3 was present in the syncytiotrophoblast layer. EP1 to EP4 were consistently expressed across gestation in the fetal membranes; however, a different cellular localization with labor was observed in the amnion for EP1, EP2, and EP4. CONCLUSION: The presence of these receptors in the placenta and fetal membranes may indicate autocrine roles for PGE2 in the signaling pathways associated with placental function and parturition. [ABSTRACT FROM AUTHOR]

Abstract: Increased oxidative stress in the placenta has been associated with preeclampsia (PE), a clinical syndrome involving placental pathology. The enzymatic sources of reactive oxygen species in the human placenta are as yet unidentified. We hypothesized that NADPH oxidase is a main source of reactive oxygen species in the placenta and its expression may change in PE. Employing RT-PCR, we have amplified a novel NADPH oxidase isoform Nox1 from human choriocarcinoma BeWo cells. Using polyclonal anti-peptide antiserum recognizing unique Nox1 peptide sequences, we identified by immunohistochemistry and cell fractionation that Nox1 protein localizes in the BeWo cell membrane structures. Immunohistochemistry of normal placental tissues showed that Nox1 was localized in syncytiotrophoblasts, in villous vascular endothelium, and in some stromal cells. At the immunohistochemical level Nox1 expression was significantly increased in syncytiotrophoblast and endothelial cells in placentas from patients with preeclampsia as compared to gestational age-matched controls. Western blot analysis of whole placental homogenate confirmed this increase. Our data suggests that increased Nox1 expression is associated with the increased oxidative stress found in these placentas. [Copyright &y& Elsevier]

Abstract: Studies suggest that preeclampsia (PE) originates in the placenta and is associated with deficient trophoblast invasion of spiral arteries. The direct cause remains unknown, but preeclampsia is often associated with circulating factors that can induce generalized endothelial dysfunction. Antiphospholipid antibodies (APA) in circulation are also associated with vascular diseases. Although the quantification of APA is not currently used as a prognostic of the risk of PE, studies suggest that thrombophilias play a role in PE pathogenesis. In fact, the pathology of placentae from PE and Antiphospholipid syndrome patients is similar; atherosis, thrombosis and infarction, and endothelium activation represent the pathological mechanisms. We identified a new antibody which recognizes non-bilayer phospholipid arrangements (NPA) in membrane models and in cell membranes in vivo, and which triggered an autoimmune-like disease in mice. We evaluated the presence of NPA in the placentae and in sera, and whether NPA induced NPA antibodies in patients with hypertensive disorders of pregnancy (HDP). Results showed increased levels of NPA in the syncytiotrophoblast, extravillous cytotrophoblast, syncytial knots and the amnion epithelial cell membranes of the placenta, as well as increases in NPA and NPA antibodies in sera from HDP patients, when compared with controls. This suggests that NPA derived from placenta could be one of multiple factors associated with pregnancy pathologies. [Copyright &y& Elsevier]

Objective: To investigate the interaction of dexamethasone and interleukin-1β (IL-1β) on the expression of cytosolic phospholipase A2 (cPLA2), the enzyme catalyzing the first reaction in the formation of prostaglandins, in cultured primary human amnion fibroblasts. Design and methods: Human amnion fibroblasts were prepared from fetal amnion collected at term and were treated with dexamethasone with or without interleukin-1β for 24h. Prostaglandin E2 (PGE2) output and cPLA2 expression in cultured amnion fibroblasts were measured with radioimmunoassay, quantitative real-time polymerase chain reaction, Western blotting and cPLA2 promoter-driven luciferase reporter gene activity. Results: Both dexamethasone and IL-1β caused a significant increase in prostaglandin E2 output, cPLA2 mRNA and protein expression in cultured human amnion fibroblasts. Both dexamethasone and IL-1β stimulated cPLA2 promoter-driven luciferase reporter gene activity. There was no obvious antagonistic or synergistic effect of combined treatment of dexamethasone and IL-1β on PGE2 output, cPLA2 expression or cPLA2 promoter-driven luciferase reporter gene activity in cultured human amnion fibroblasts. Conclusion: The above findings suggest that paradoxically dexamethasone is a stimulator for both prostaglandin synthesis and cPLA2 expression in human amnion fibroblasts. The interaction between dexamethasone and IL-1β on prostaglandin synthesis and cPLA2 expression is neither synergistic nor conventionally antagonistic. [Copyright &y& Elsevier]

Objective: The purpose of this study was to identify the isoforms and splicing patterns of prostaglandin H synthase present in pregnant human lower-segment myometrium and determine whether there is differential expression of the isoforms or splice variants with respect to gestational age or parturition.Study Design: Lower-segment myometrium was collected at cesarean section at term (>37 weeks) or preterm (<37 weeks) from patients who were or were not in labor. Total messenger ribonucleic acid was isolated and reverse transcribed. Polymerase chain reaction for prostaglandin H synthase isoforms 1 and 2 and calponin were performed. Primers designed to characterize the splicing patterns of exon 9 of prostaglandin H synthase-1 were used.Results: The predominant polymerase chain reaction product in all samples corresponds to prostaglandin H synthase-1 messenger ribonucleic acid spliced to include exon 9, but a less-abundant polymerase chain reaction product corresponding to prostaglandin H synthase-1 messenger ribonucleic acid spliced at the internal donor site of exon 9 was also detected. Prostaglandin H synthase-2 messenger ribonucleic acid was detected in human myometrium at a lower abundance than prostaglandin H synthase-1, and neither prostaglandin H synthase-1 or prostaglandin H synthase-2 messenger ribonucleic acid expression changed significantly with gestational age or labor.Conclusion: Both prostaglandin H synthase-1 and prostaglandin H synthase-2 isoforms are present in human myometrium. The prostaglandin H synthase-1 messenger ribonucleic acid that includes all of exon 9 encodes the predominant prostaglandin H synthase-1 isoform present in human myometrium. No significant alterations in the expression or splicing patterns for prostaglandin H synthase-1 were detected with respect to gestational age or the onset of labor; but prostaglandin H synthase-1 expression appeared higher at term in anticipation of labor. Although prostaglandin H synthase-2 is present in human myometrium, induction of prostaglandin H synthase-2 does not occur in lower-segment myometrium at parturition. [ABSTRACT FROM AUTHOR]

Objective: Nitric oxide is a potent vasodilator released by endothelial cells that may play an important role in modulating maternal and fetal vascular tone in normal pregnancy. The current study was designed to evaluate whether plasma or urine nitrite and nitrate (the metabolites of nitric oxide) concentrations are elevated in pregnant compared with those of nonpregnant sheep and whether the nitrate concentrations in the fetal circulation were increased in comparison with the maternal circulation.Study Design: Eleven pregnant sheep and seven nonpregnant oophorectomized sheep were instrumented with catheters in the maternal and fetal femoral arteries and veins, uterine and umbilical veins, and amniotic cavity. Blood, urine, and amniotic fluid samples were collected for nitrate determination at least 5 days after surgery. After extraction nitrate was reduced to nitrite and quantitated with the Greiss reagent.Results: Arterial plasma nitrate concentrations in the pregnant sheep were significantly elevated compared with those of nonpregnant sheet (5.0 +/- 0.9 vs 2.5 +/- 0.6 micromol/L, p < 0.05). The urinary nitrate concentrations were also significantly increased in the pregnant sheep compared with those of nonpregnant sheet (89.9 +/- 16.3 vs 23.1 +/- 4.5 nmol/mg creatinine, p < 0.01). Fetal plasma nitrate concentrations were ninefold higher than the maternal nitrate concentrations (43.9 +/- 7 vs 5.0 +/- 0.9 micromol/L, p < 0.01), whereas amniotic fluid concentrations were extremely high (133.8 +/- 13.8 micromol/L, n = 3). No venous-arterial differences were measurable across either the maternal or fetal sides of the placenta.Conclusion: Nitrate concentrations in pregnant sheet and their fetuses are increased. The increased nitrate concentrations in the maternal and fetal circulations may reflect the increased nitric oxide synthesis, which may in part mediate the cardiovascular adaptations to normal pregnancy and the low systemic and umbilical vascular resistance in the fetus. [ABSTRACT FROM AUTHOR]

Abstract: Pregnancy is a state of oxidative stress, which becomes exaggerated under pathological conditions, such as preeclampsia, IUGR, diabetes and obesity, where placental mitochondrial dysfunction is observed. The majority of investigations utilize isolated mitochondria when measuring mitochondrial activity in placenta. However, this does not provide a complete physiological readout of mitochondrial function. This technical note describes a method to measure respiratory function in intact primary syncytiotrophoblast from human term placenta. [Copyright &y& Elsevier]

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2015 there were twelve themed workshops, three of which are summarized in this report. These workshops covered areas of placental regulation and nutrient handling: 1) placental epigenetics; 2) placental mitochondrial function; 3) placental transport systems. [ABSTRACT FROM AUTHOR]

Abstract: The placenta regulates fetal growth and development via transport of nutrients and gases, and synthesis and secretion of steroid and peptide hormones. These functions are determined by vascular development and blood flow and by growth and differentiation of the trophoblast, which contains receptors, transporters and enzymes. The placenta generates reactive oxygen species which may contribute to the oxidative stress seen even in normal pregnancy but this is increased in pregnancies complicated by preeclampsia, IUGR and pregestational diabetes where oxidative and nitrative stress have been clearly documented. Nitrative stress is the covalent modification of proteins and DNA by peroxynitrite formed by the interaction of superoxide and nitric oxide. We have demonstrated nitrative stress by localizing nitrotyrosine residues in these placentas and found increased expression of NADPH oxidase (NOX) enzyme isoforms 1 and 5 as a potential source of superoxide generation. The presence of nitrative stress was associated with diminished vascular reactivity of the fetal placental circulation, a situation that could be reproduced by treatment with peroxynitrite in vitro. We find many nitrated proteins in the placenta, including p38 MAP kinase which has a role in development of the villous vasculature. Nitration of p38 MAPK was increased in the preeclamptic placenta and associated with loss of catalytic activity. We hypothesize that nitration of proteins in the placenta including receptors, transporters, enzymes and structural proteins can alter protein and placental function and this influences fetal growth and development. Increasing nitrative stress but a decrease in oxidative stress, measured as protein carbonylation, is found in the placenta with increasing BMI. Formation of peroxynitrite may then consume superoxide, decreasing nitrative stress. As protein carbonylation is a covalent modification at Lys, Arg, Pro and Thr residues the switch from carbonylation to nitration at tyrosine residues may alter protein function and hence placental function. [Copyright &y& Elsevier]