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PubMed2006

Regional expression of prostaglandin E2 and F2alpha receptors in human myometrium, amnion, and choriodecidua with advancing gestation and labor.

The change from uterine quiescence to enhanced contractile activity may be due to the differential expression of prostaglandin receptors within the myometrium and fetal membranes, in a temporal and topographically distinct manner. To address this question, we determined the localization and expression of the PGE2 receptor subtypes (PTGER1-4) and the PGF2alpha receptor (PTGFR) in paired upper and lower segment myometrium, amnion, and choriodecidual samples throughout human pregnancy, with and without labor. All receptor subtypes were found throughout the muscle layers in both the upper and lower uterine segments, colocalizing with alpha smooth muscle actin. A change in intracellular localization was observed at term labor, where PTGER1 and PTGER4 were predominately associated with the nucleus. Minimal changes in the expression of the PGE2 and PGF2alpha receptor subtypes were observed with gestational age, labor, or between the upper and lower myometrial segments. Receptor expression in maternal and fetal tissues differed between the receptor subtypes; PTGER1 and PTGER4 were predominately expressed in the fetal membranes, PTGER2 was greatest in the myometrium, whereas PTGER3 and PTGFR were similarly expressed in the myometrium and fetal membranes. Myometrial activation through the prostaglandin receptors is perhaps more subtle and may be mediated by a balance between one or several of the prostaglandin receptor subtypes together with other known contraction associated proteins. Lack of coordination in receptor expression between the myometrium and fetal membranes may indicate different regulatory mechanisms between these tissues, or it may suggest a function for these receptors in the amnion and choriodecidua that is independent of that seen in the myometrium.

Amnion↗

PubMed1992

Evidence of a role for phosphatidylinositol synthesis in human amnion cell proliferation.

Phosphatidylinositol (PtdIns) is the key precursor of phosphoinositide-derived intracellular mediators. The effects of changing the rate of PtdIns synthesis on mitogenic activity of human amnion-derived WISH cells were investigated. Incubation of the cells with [3H]inositol caused a time- and dose-dependent PtdIns labeling. Exogenous Ca2+ inhibited [3H]inositol incorporation in a dose-dependent fashion; half-maximal inhibition occurred with 0.3-1.0 mM Ca2+. In contrast, removal of cytosolic Ca2+ by ionophore A23187 and 1 mM EGTA induced enhancement of the PtdIns labeling as a function of A23187 concentration, perhaps through release of inhibitory effects of endogenous Ca2+. The A23187-stimulated PtdIns labeling with [3H]inositol was not abolished by additional unlabeled inositol, suggesting that [3H]inositol labeling of PtdIns occurred mainly through de novo synthesis catalyzed by PtdIns synthase (EC 2.7.8.11). In cells with PtdIns synthase activity decreased by exogenous Ca2+, [3H]thymidine incorporation was also inhibited, while A23187 caused dose-dependent enhancement of thymidine incorporation. The changes in PtdIns synthase activity occurred in parallel with changes in mitogenic activity caused by increasing the dose of exogenous Ca2+ or A23187. A similar lowering of mitogenic activity was observed upon suppression of PtdIns synthase by pemirolast potassium (9-methyl-3-1H-tetrazol-5yl-4H-pyrido[1,2-a]pyridin-4-one potassium) via a Ca(2+)-independent mechanism. These data demonstrate that changes in PtdIns synthase activity by some agents acting via different mechanisms are associated with parallel changes in thymidine incorporation, and suggest that PtdIns production is tightly coupled to cell proliferation in human amnion cells.

Amnion↗

PubMed1995

Effectors of cyclic adenosine 5'-monophosphate up-regulating-oxytocin receptors in rabbit amnion cells: isoproterenol, parathyroid hormone-related protein, and potentiation by cortisol.

Forskolin (FSK; an activator of adenylyl cyclase) and cortisol synergistically increase the concentration of oxytocin receptors (OTRs) in rabbit amnion cells. The aims of this study were to characterize potential physiological regulators of OTR concentrations acting through adenylyl cyclase and to clarify the mechanisms of potentiation by cAMP and cortisol. Both isoproterenol (ISO) and parathyroid hormone-related protein (PTHrP) elevated amnion cell cAMP levels and OTR concentrations. The effects of ISO and PTHrP on OTR were potentiated by cortisol. Cortisol had no effect on the ability of ISO or PTHrP to stimulate adenylyl cyclase activity, and cAMP did not affect the number or affinity of glucocorticoid receptors in whole cells or in cytosol. Adenylyl cyclase activation, however, caused conversion of mifepristone (RU486) from a glucocorticoid antagonist to agonist. Thus, mifepristone elevated OTR receptor concentrations in the presence of FSK. In contrast, a structurally related glucocorticoid antagonist, onapristone (ZK98 299), was unaffected by cAMP. Because glucocorticoid receptors bound to mifepristone are capable of interacting with DNA, whereas onapristone-occupied receptors are not, we conclude that cAMP affects glucocoticoid receptor-DNA interactions, accounting for the synergistic effects of cAMP and cortisol on OTRs.

Adenylyl Cyclases↗

PubMed2000

Corticotropin-releasing hormone increases the expression of the prostaglandin E(2) receptor subtype EP1 in amnion WISH cells.

The purpose of this study was to investigate the effect of corticotropin-releasing hormone (CRH) on the expression of the prostaglandin (PG) E(2) EP1 receptor subtype and PGE(2) production in amnion WISH cells (AWC). AWC cultures were incubated with CRH. Culture fluid was collected for PGE(2) measurement, and the cells were collected and analyzed for EP1 protein and mRNA. Immunohistochemical localization of the EP1 receptor was also performed. Incubation of AWC with CRH resulted in a dose-dependent increase (r = 0.97) in the level of EP1 receptor protein (P < 0.001). Coincubation of AWC with CRH and indomethacin resulted in the decreased production of PGE(2) while having no effect on EP1 receptor expression. A significant but not dose-dependent increase in EP1 mRNA expression was also observed (P < 0.01). Immunohistochemical evaluation verified cell membrane localization of the receptor in both stimulated and unstimulated cells and confirmed the increased expression of EP1 receptor in response to CRH. Incubation of AWC with CRH also resulted in increased culture fluid PGE(2) levels (P < 0.01). These results suggest that the role CRH plays in the initiation of labor may also involve the promotion of elevated PGE(2) levels and increased expression of the EP1 receptor in amnion.

Amnion↗

PubMed1981

Monitoring, banking, and clinical use of amnion as a burn wound dressing.

The preparation of human amniotic membrane obtained from cesarean delivery placentas for use in the treatment of acutely burned pediatric patients is presented. The technique of membrane harvesting and cleansing, bacteriological monitoring during various phases of preparation, and a method of ultracold long-term banking are described. A total of 187 membranes were stripped under aseptic conditions, cultured, treated in 0.025% sodium hypochlorite (NaOCl) for one minute and recultured. Fourteen organisms were recovered prior to NaOC1 treatment, and only 8 organisms after treatment. Amnions that grew any organism after hypochlorite treatment (23%) were deemed unsuitable for clinical use. Clinical experience with amniotic membranes in 36 acutely burned patients suggests that amnion is a cost-effective biological dressing, acceptable as an immediate dressing on superficial second-degree burns. It is also acceptable as an adjunct to meshed autograft over fascia, and as a temporary covering on recently excised wounds prior to autografting.

Adolescent↗

PubMed1987

Effect of relaxin on prostaglandin E production by human amnion: changes in relation to the onset of labour.

The effect of relaxin on prostaglandin E (PGE) production by human amnion in vitro was investigated. When amniotic discs were incubated in the presence of increasing concentrations of relaxin, two distinct effects were observed. Discs prepared from women delivered by caesarean section before the onset of labour showed a significant decrease in PGE output at relaxin concentrations of 0.5-2 micrograms/ml; the effect was abolished at higher relaxin concentrations. Discs obtained from women delivered after labour of spontaneous onset responded to the addition of relaxin (4-8 micrograms/ml) with a significant increase in PGE output, although this increase was only evident in patients in whom labour had started with intact membranes. These results suggest that relaxin, which is present in decidua and chorion laeve at term, may have a paracrine effect on the amnion, inhibiting PGE production during continuing pregnancy but favouring its production during spontaneous labour.

Amnion↗

PubMed1987

Investigation of the expression of amnion antigens by spiral arteries in human utero-placental tissues.

Two monoclonal antibodies raised against human amnion, GB3 and GB5, were used in an indirect immunoperoxidase method to investigate the expression of amnion antigens by spiral arteries in pregnant and nonpregnant uterine tissues. GB3 showed focal reactivity with occasional spiral arteries in the placental bed throughout pregnancy, but no GB3-staining was observed in nonpregnant endometrium. In contrast, GB5 showed bandlike circumferential reactivity with spiral arteries at all gestational ages examined. GB5-positivity showed no relation to the presence of endovascular or perivascular trophoblast. In nonpregnant endometrium, GB5 labeled rare spiral arteries. However, in a premenstrual specimen showing pseudodecidual change, there was circumferential reactivity with GB5 resembling that in pregnancy. The reaction patterns of GB3 or GB5 were not similar to those for two other basement-membrane components, fibronectin and type IV collagen. The results suggest that expression of the GB5 antigen may in part be regulated by hormones.

Amnion↗

PubMed1997

The effect of interleukin-1 beta and interleukin-4 on the expression of prostaglandin receptors EP1 and EP3 in amnion WISH cells.

PROBLEM: Although prostaglandin E2 (PGE2) is believed to modulate biochemical and immunological events leading to parturition, the role of prostaglandin E receptors during labor has not been investigated. METHOD OF STUDY: Amnion WISH cells were incubated in media containing increasing concentrations of either interleukin-1 beta (IL-1 beta) or IL-4. Increased EP1 and EP3 protein expression was determined by Western blot analysis with peptide-specific antibodies. Concomitant measurements of culture media PGE2 were made by an enzyme immunoassay. RESULTS: Incubation of WISH cells with IL-1 beta or IL-4 caused a two- to three-fold increase in EP1 protein levels. IL-1 beta and IL-4 also caused six- and two-fold increases, respectively, in culture fluid PGE2 concentrations. IL-1 beta or IL-4 had no effect on EP3 protein levels. CONCLUSIONS: Based on these results, it is proposed that IL-1 beta and IL-4 may be involved in the initiation and promotion of labor by inducing EP1 levels and PGE2 production in amnion.

Amnion↗

PubMed2005

Effect of bacterial virulence on IL-18 expression in the amnion infected with Escherichia coli.

PROBLEM: The upregulation of inflammatory substances threatens pregnancy. Interleukin-18 (IL-18) is elevated in women who miscarried. The purpose of this study was to develop a pig model of chorioamnionitis to study the effect of bacterial virulence on IL-18 response in experimentally infected amnion. METHOD OF STUDY: A total of 20,000 colony-forming units of Escherichia coli (an enteropathogenic O55 strain, EPEC or O86 non-pathogenic strain) were administered into the amniotic cavity of pig fetuses at 70% of gestation for 10 hr. Fetal amniotic fluid samples were analyzed for IL-18 levels by enzyme-linked immunosorbent assay. The expression of IL-18 was studied also by immunohistochemistry on cryostat sections through amniotic membranes and pathological changes were observed by electron microscopy. RESULTS: Both E. coli strains propagated in amniotic fluids and reached similar counts. Only EPEC, however, caused a significant increase of IL-18 amniotic fluid levels (P < 0.001) and cytokine expression in the amniotic epithelium. CONCLUSIONS: The levels of IL-18 in infected amniotic fluids correlated with bacterial virulence and pathological changes in the amnion.

Amnion↗

PubMed1984

Pulmonary surfactant: hydrophobic nature of the mucosal surface of the human amnion.

The contact angle has been measured for a drop of saline placed upon the rinsed mucosal surface of the amnion in eleven human placental membranes obtained from normal births at full term. The contact angle averaged 70 degrees, indicating a hydrophobic surface comparable with graphite (86 degrees), polyethylene (94 degrees) or oxyntic tissue (85 degrees) which is also exposed to endogenous surface-active phospholipids in vivo. By comparison, four pre-term placentas with an average gestation period of 29.5 weeks gave a mean contact angle of 32 degrees, indicating that hydrophobicity of the placenta increases with maturity (41 weeks) and might well be imparted by adsorbed surfactants present in amniotic fluid and known to render other surfaces hydrophobic. Since the mucosal epithelium of the amnion is exposed to the same surfactants in the same physical state as the fetal alveolar wall, the above results imply that this surface may also be hydrophobic, as indicated in the adult lung by other studies. The concept of surfactant directly adsorbed to the pulmonary tissue surfaces is discussed in connexion with its possible functional advantages in 'de-watering' the lung at birth, maintaining homeostasis by water repellency , releasing airway surfaces and lymph ducts glued by protein and lubricating tissue respiratory movement.

Adsorption↗

PubMed2005

Novel targeting of cyclooxygenase-2 (COX-2) pre-mRNA using antisense morpholino oligonucleotides directed to the 3' acceptor and 5' donor splice sites of exon 4: suppression of COX-2 activity in human amnion-derived WISH and myometrial cells.

Increased expression of cyclooxygenase-2 (COX-2) has been implicated in the onset of both term and preterm labor. In this context, both selective and nonselective COX-2 inhibitors have been used in clinical trials to determine their efficacy in delaying preterm labor. However, recent evidence indicates that these tocolytics may have potentially adverse fetal and maternal side effects. Therefore, the development of more specific and nontoxic agents to inhibit COX-2 needs to be considered. We have evaluated whether antisense morpholino oligonucleotides have therapeutic potential in inhibiting COX-2 by specifically targeting both the 3' and 5' acceptor and donor sites of exon 4 of COX-2's pre-mRNA sequence. Confocal microscopy on "live" cells illustrated high levels of penetrance of antisense morpholino oligonucleotides using the Endo-Porter formula (Gene-Tools, LLC, Philomath, OR), with delivery efficiencies of 82 and 78%, respectively, in amnion-derived WISH and myometrial cells. Substantial inhibition by the morpholino oligonucleotides of COX-2 expression, induced by lipopolysaccharide administration, was observed at both the mRNA and protein levels. Loss of enzymic activity of COX-2 was confirmed using a sensitive COX enzyme activity assay, which reflects the rate of conversion of arachidonic acid to prostaglandin H2. Our results indicate that antisense morpholino oligonucleotides significantly inhibit expression and activity of this enzyme in in vitro cultures of amnion-WISH and myometrial cells. The potential thus exists that a similar approach can be mimicked in vivo to produce a highly specific and nontoxic strategy to inhibit COX-2 activity with its subsequent effects on the better management of preterm labor and other inflammatory conditions.

Amnion↗

PubMed2002

Regulation of cyclooxygenase-2 expression by phospholipase D in human amnion-derived WISH cells.

Prostaglandins (PGs) are known to play a key role in the initiation of labor, but the mechanisms regulating their synthesis in amnion are largely unknown. In this study, the regulatory mechanisms for PGE(2) production during phospholipase D (PLD) and p38-dependent activation of WISH cells were investigated. We found that the stimulation of WISH cells with interleukin (IL)-1 beta elicited dose-dependent synthesis of cyclooxygenase-2 (COX-2) mRNA, protein, and their products, PGE(2). Moreover, the treatment of [(3)H]myristate-labeled cells in the presence of 1-butanol caused the dose-dependent formation of [(3)H]phosphatidylbutanol (PBt), a product specific to PLD activity. Pretreating the cells with 1-butanol and Ro 31-8220 inhibited the IL-1 beta-induced COX-2 expression, but 3-butanol did not affect this response. In addition, evidence that PLD was involved in the stimulation of COX-2 expression was provided by the observations that COX-2 expression was stimulated by the dioctanoyl phosphatidic acid (PA) and that the prevention of PA dephosphorylation by 1-propranolol potentiated COX-2 expression by IL-1 beta. Moreover, IL-1 beta stimulation of the cells caused the phosphorylation of p38 and extracellular signal-regulated kinase (ERK), and IL-1 beta-induced COX-2 expression was inhibited by the pretreatment of WISH cells with a p38 inhibitor, in contrast ERK upstream inhibitor had no effect. Furthermore, Ro 31-8220 inhibited IL-1 beta-induced p38 phosphorylation but not ERK phosphorylation. The results of this study indicate that in human amnion cells, IL-1 beta might activate PLD through an upstream protein kinase C to elicit p38 and finally induce COX-2 expression.

Amnion↗

PubMed1966

Immunochemical study of parainfluenza virus (type 2) in amnion cells.

de Vaux St. Cyr, C. Columbia University, New York, N.Y.),and C. Howe. Immunochemical study of parainfluenza virus (type 2) in amnion cells. J. Bacteriol. 91:1911-1916. 1966.-Immunoelectrophoretic analysis of stable amnion cells in which parainfluenza virus (type 2) was being actively synthesized revealed at least three precipitating antigens not found in normal cells. These "new" antigens differed from viral neuraminidase and hemagglutinin in both specificity and electrophoretic mobility; their identity and function remain to be elucidated.

Amnion↗

PubMed1979

JC human papovavirus replication in human amnion cells.

JC human papovavirus was found to replicate in primary human amnion cells. The virus has undergone eight passages in amnion cells and was identified by serological methods as JC virus. By restriction endonuclease analysis of the viral DNA, the fragments observed were identical to those previously reported for the prototype strain.

Amnion↗

PubMed1985

Amnionicity and chorionicity in twin pregnancies: prediction using ultrasound.

A total of 66 twin pregnancies was studied antenatally with ultrasound, followed up clinically, and the placentae examined pathologically to determine if, solely on the basis of antenatal sonography, an accurate assessment of amnionicity and chorionicity could be made. Sonographic features noted included number of placental sites, whether a membrane separating the fetuses could be visualized, fetal position and amount of amniotic fluid, and in some cases fetal positioning vis-a-vis each other and the uterine wall. Documentation of two placental sites confirms the presence of a dichorionic and, therefore, a diamniotic pregnancy. Documentation of a membrane separating the fetuses confirms the presence of a diamniotic pregnancy; lack of visualization of a separating membrane does not reliably predict, but does suggest, a monoamniotic pregnancy. Other features frequently assist in prediction of amnionicity, chorionicity, and zygosity; the most helpful of these was the "stuck twin" sign, in which one twin moved freely but the other remained in a fixed position adjacent to the sidewall of the uterus despite changes in maternal positioning, indicating the presence of a nonvisualized membrane. Presence of absence of entanglement or intermingling of fetal parts or umbilical cords may also be helpful. In the absence of such sonographic features, antenatal sonographic diagnosis of a monoamniotic pregnancy cannot be made reliably.

Amnion↗

PubMed1989

Hydraulic conductivity of endothelial cell monolayers cultured on human amnion.

Hydraulic conductivity (Lp) of endothelial cell monolayers cultured on a supporting matrix of human amnion was measured. Bovine aortic (Baec), bovine microvascular (Bmec), and human umbilical vein endothelial cell (Huvec) monolayers were mounted in a water-jacketed chamber maintained at 37 degrees C. The lower compartment of the chamber was connected to an electronic sensor-aspiration system that served to volume clamp the lower compartment. The aspirated volume was displayed on a chart recorder, providing continuous measurements of volume flow per unit area (Jv). In 36 monolayers, Jv was linearly related to hydrostatic pressure. The average slope of this relationship (Lp) was 1.14 +/- 0.8 X 10(-6), 1.67 +/- 1.49 X 10(-6), and 2.9 +/- 0.85 X 10(-6) cm.s-1.cmH2O-1 for Baec, Bmec, and Huvec, respectively. The effect of increased luminal oncotic pressure was studied in seven monolayers. The oncotic pressure difference (delta pi) ranged from 3.1 to 7.1 cmH2O (measured with a Wescor oncometer). In all cases, there was an immediate fall in Jv and a displacement to the right of the x-intercept of the line Jv vs. change in pressure (delta P) without a change in the slope (Lp). These results indicate that the Lp of endothelial cell monolayers cultured on amnion is within the range of values previously reported for single capillaries in vivo and that these monolayers appear to sustain delta pi sufficient to generate an osmotically driven water flow.

Amnion↗

PubMed2000

Evidence for de novo cholesterol synthesis by term human fetal amnion and chorion: a comparative study using the reverse-isotope dilution technique.

The cholesterol biosynthetic activity was assessed using [2-(14)C]-acetate as substrate in the homogenates of amnion and chorion obtained from women (n = 6, age 26-39 years) after spontaneous labour at term (37-40 weeks of gestation) having uncomplicated pregnancies. Reverse-isotope dilution analysis gave positive identification of [(14)C]-cholesterol acetate in all incubations of viable tissues. This metabolite was not evident in heat-denatured homogenates which served as controls. The extent of enzymic conversion for amnion at 2.6 x 10(-3) to 0.19% was persistently higher than that of the chorion at 1.7 x 10(-3) to 9.0 x 10(-3)%. The results indicate that human term fetal membranes possess the full complement of enzymes to catalyze the transformation of acetate to cholesterol. This study provides evidence that fetal membranes possess the capacity for de novo cholesterol biosynthesis, the sterol being essential for steroidogenesis as well as in embryo viability during pregnancy.

Acetates↗

PubMed1983

Metabolic pathways in chick amnion muscle. A cytochemical study.

By means of histochemical techniques different reactions were studied in the amnion musculature of the chick during their most active period of contractions between the 5th and the 9th days of incubation. The muscle cells showed strong activity for cholinesterase and glutamate dehydrogenase and low activity for monoaminoxidase. The glycogen, phosphorylase and UDPG-glycogen glycosyltransferase were negative. Whereas, lipids and enzymes involved in fat metabolism were positive. The supposed energy source of the amnion musculature is discussed.

Adenosine Triphosphatases↗