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Introduction: Sphingosine-1-phosphate (S1P), a bioactive lipid, has been reported to regulate inflammation processes. The onset of labor is thought to be related to inflammation. We therefore hypothesized that S1P might be involved in the onset of labor., Methods: The expression of sphingosine kinase (SPHK)-1, which produces S1P, and S1P lyase (SPL)-1, which irreversibly inactivates S1P, were examined in the fetal membranes. The expression levels were compared between amnions from cases of elective Caesarean deliveries (pre-labor) and those from vaginal deliveries (post-labor). In primary cultured human amnion cells, the expression levels of prostaglandin-endoperoxide synthase (PTGS)-2 were examined in the presence or absence of S1P treatment., Results: SPHK-1 and SPL-1 were both expressed in the amnion. The expression of SPHK-1 in the post-labor amnions increased compared with that in the pre-labor amnions. The expression of PTGS-2, a key regulator of labor, also increased in the post-labor amnion. However, the SPL-1 expression in the pre-labor amnion was not significantly different from that in the post-labor amnion. S1P1-3 and 5, which were coupled with Gi protein, were consistently found in the amnion cells. The treatment with S1P increased the expression of PTGS-2, and this was completely suppressed by a Gi inhibitor in the amnion cells., Discussion: We are herein provide the first evidence of increased SPHK-1 expression in post-labor amnions, and that S1P increases the PTGS-2 expression in amnion cells., Conclusions: Our results suggest that S1P might play a role in the onset of labor via the induction of PTGS-2., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Objective: Amnionitis (inflammation of the amnion) is the final stage of extra-placental chorioamniotic inflammation. We propose that patients with "amnionitis", rather than "chorionitis" have a more advanced form of intra-uterine inflammation/infection and, thus, would have a more intense fetal and intra-amniotic inflammatory response than those without "amnionitis"., Study Design: The relationship between the presence of amnionitis, and a fetal and an intra-amniotic inflammatory response was examined in 290 singleton preterm births (

Detailed information regarding the subcellular distribution of proteins within amnion epithelial cells is a goal of numerous placental biologists. In this report, we describe a versatile technique for in situ immunolabeling in amnion that is as technically permissible as traditional immunolabeling of cultured cells and, when coupled with confocal laser scanning microscopy, is similarly capable of providing detailed information regarding subcellular protein distribution. Using antibodies directed against sequential enzymes of the prostaglandin E biosynthesis cascade, we compared this novel method with immunofluorescent labeling using amnion cells in primary culture and cryosections of reflected fetal membrane rolls. By several criteria, we observed morphological variation between the cells cultured in vitro and the tissue specimens. Despite general consistencies in immunostaining patterns between the cryosectioned specimens and those labeled in situ, morphological preservation was superior using the latter technique. Relative to the cryosectioned specimens, in situ immunostaining was advantageous in that it permitted improved sampling efficiency, and allowed regional variations in labeling to be observed in a more global context within the tissue. Our results demonstrate that in situ immunolabeling provides a useful adjunct or alternative to immunolabeling using membrane roll preparations.

Increased matrix metalloproteinase (MMP)-9 proteolytic activity is associated with term birth, preterm birth and premature rupture of membranes. However, most studies show no changes with MMP-2, which binds tightly to cell and matrix proteins. We hypothesized better protein extraction would reveal new MMP patterns. Human amnion and chorion were collected from 25 patients at preterm or term, extracted with 2% SDS (a high concentration), and the MMP protein levels and pro-enzyme activities were determined by Western immunoblotting and zymography. MMP-2 protein and MMP-2 and -9 pro-enzyme activities in the amnion increased significantly (p<0.05) with labor at term, and were higher than at preterm labor (p<0.05), when extracted with high SDS concentration. There were no changes in chorion MMPs under any condition. These associations suggest MMP-2 may be another regulator of membrane rupture and other labor-associated mechanisms at term parturition, and its role(s) should be examined further.

Objective: To investigate the interaction of dexamethasone and interleukin-1beta (IL-1beta) on the expression of cytosolic phospholipase A(2) (cPLA(2)), 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-1beta for 24h. Prostaglandin E(2) (PGE(2)) output and cPLA(2) expression in cultured amnion fibroblasts were measured with radioimmunoassay, quantitative real-time polymerase chain reaction, Western blotting and cPLA(2) promoter-driven luciferase reporter gene activity., Results: Both dexamethasone and IL-1beta caused a significant increase in prostaglandin E(2) output, cPLA(2) mRNA and protein expression in cultured human amnion fibroblasts. Both dexamethasone and IL-1beta stimulated cPLA(2) promoter-driven luciferase reporter gene activity. There was no obvious antagonistic or synergistic effect of combined treatment of dexamethasone and IL-1beta on PGE(2) output, cPLA(2) expression or cPLA(2) 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 cPLA(2) expression in human amnion fibroblasts. The interaction between dexamethasone and IL-1beta on prostaglandin synthesis and cPLA(2) expression is neither synergistic nor conventionally antagonistic.

The cross-linking of the connective tissues in the fetal membranes and placenta is important for their tensile strength and elasticity. We have studied the expression of lysyl oxidase (LOX) because it is the classical enzyme responsible for the cross-linking of collagen and elastin. We have also studied the two recently described, genetically distinct lysyl oxidase-like genes and proteins, lysyl oxidase-like (LOXL) and lysyl oxidase-like 2 (LOXL2), of unknown functions. Specific antisera have been used for immunolocalization in fetal membranes and placentae from early pregnancy terminations and after caesarean section at both preterm and term, prior to labour. In addition, the steady state mRNA levels of the three genes has been quantitated in separated amnion, chorion, decidua and placentae collected at term before labour. The immunocytochemistry shows that the spatial expression of the three lysyl oxidases is similar in early pregnancy in both the fetal membranes and placentae. However, by preterm this pattern had diverged and becomes greatest at term. The expression of the genes found at term was similar to the results of protein expression obtained by immunocytochemistry, with the exception of LOXL which had high placental gene expression, but low levels of immunolocalized protein. Thus by term, LOX was expressed predominantly in the amniotic epithelium, with little expression in the placenta, while LOXL showed highest gene expression in the placenta and lowest expression in the amnion. LOXL2 expression was again different and was expressed predominantly in the chorionic cytotrophoblast of the membranes with low expression in both the amnion and placentae. These results suggest that these three members of the lysyl oxidase family may have similar roles in early pregnancy during the development of the placenta and fetal membranes, but their divergence as pregnancy advances to term, may reflect changes in substrate specificity and connective tissue composition., (Copyright 2001 Harcourt Publishers Ltd.)

Maternal genital tract infections and the associated inflammatory response may contribute to the onset of many cases of preterm labour. Type II phospholipase A(2)(PLA2) hydrolyses glycerophospholipids, releasing free fatty acid for conversion into potent biological mediators, such as prostaglandins, which play a significant role in both the onset and progression of human labour and the activation of inflammatory reactions. The aim of this study was to quantify immunoreactive (ir) Type II PLA2 in placenta, amnion and choriodecidua collected from women delivering prematurely or due to histological chorioamnionitis, and to compare levels to those at term. Tissues were assayed for ir Type II PLA2 by ELISA and expressed as ng/mg tissue protein. Ir Type II PLA2 tissue content was significantly higher in preterm (n=26) amnion and choriodecidua, but not in the placenta when compared to tissues obtained at term (n=42). When the data were stratified with respect to labour status, ir Type II PLA2 content was significantly higher in the preterm not-in-labour group (NIL, n=17) than the preterm in labour group (IL, n=9) in the amnion. When the NIL group was analysed with respect to membrane rupture, women who had spontaneously ruptured membranes (n=6) expressed significantly greater ir Type II PLA2 than those that had intact membranes (n=11) in both the amnion and choriodecidua but not in the placenta. No significant difference was observed between the preterm IL group (n=9) and the group with histological chorioamnionitis (n=14). The data obtained in this study support a role for Type II PLA2 in association with spontaneous rupture of membranes., (Copyright 2001 Harcourt Publishers Ltd.)

Carbon monoxide (CO) is a novel gaseous chemical messenger, formed during heme oxygenase (HO)-catalysed oxidation of heme. CO is proposed to play a key role(s) in cell function in many organ systems, including vasodilator action in the cardiovascular system. Recently, it has been demonstrated that there is expression of HO protein in the human placenta and this appears to have a regulatory role in placental perfusion. The objective of the present study was to determine HO enzymatic activity in vitro in five different regions of term human placenta. HO activity was determined in the microsomal fraction of tissue homogenate by measuring the rate of formation of CO from heme, using a gas-chromatographic method. HO activity, expressed as nmol CO formed/g tissue wet weight/h, was higher (P< 0.05) in the chorionic plate, chorionic villi, basal plate and chorio-decidua compared with the amnion. The finding that HO enzymatic activity is present in different regions of term human placenta supports the concept that the heme-CO (HO) pathway plays a complementary role with the L -arginine-nitric oxide (nitric oxide synthase) pathway in the regulation of placental haemodynamics., (Copyright 2000 Harcourt Publishers Ltd.)

Tumour necrosis factor (TNF)-alpha-stimulated prostaglandin (PG) E(2)biosynthesis by amnion-derived AV3 cells is accompanied by increased prostaglandin H synthase (PGHS)-2 mRNA expression. PGHS-1 mRNA expression is unchanged. PGHS-2 promoter-reporter constructs (-891/+9 and 5' deletions thereof) were prepared. The regions containing concensus nuclear factor kappaB (NF-kappaB) elements (-447/-438 and -222/-213) did not enhance promoter activity. Elements associated with both basal and TNF-alpha-stimulated expression lie between bases -52 and -203. Site-directed mutagenesis of nuclear factor of interleukin-6 (NF-IL6) and cyclic AMP response elements (CREs) in this region reduced both basal and induced transcriptional activity of the -203/+9 construct by over 95 per cent. Electrophoretic mobility-shift assays using oligonucleotides derived from these sites demonstrated formation of specific DNA-protein complexes. Both NF-IL6 and CRE unlabelled oligonucleotides inhibited complex formation with the NF-IL6 oligonucleotide probe. Unlabelled CRE oligonucleotide also effectively inhibited formation of the complex with the CRE probe, but reduced effectiveness was observed when the NF-IL6 oligonucleotide was the competitor. Finally, unlabelled, consensus NF-kappaB oligonucleotide failed to compete for either probe. TNF-alpha treatment did not increase levels of these complexes. Thus NF-kappaB does not enhance basal or TNF-alpha-responsive PGHS-2 transcription in amnion-derived AV-3 cells. A permissive role for NF-IL6/CRE binding proteins in regulating PGHS-2 expression in these cells is indicated, but requires further clarification., (Copyright 2000 Harcourt Publishers Ltd.)

The metabolism of arachidonic acid results in the production of prostaglandins (PGs), which are involved in the initiation of labour at term and preterm. The fetal membranes are a source of pro-inflammatory cytokines which promote increased PG biosynthesis via increased release of arachidonic acid and its conversion to biologically active metabolites such as PGE2 and PGF2alpha. In the amnion, the liberation of arachidonic acid from membrane glycerophospholipid stores can be catalysed by cytosolic phospholipase A2 (cPLA2). In amnion-derived WISH cells, the addition of tumour-necrosis factor alpha (TNF-alpha) (50 ng/ml) provoked a time-dependent increase in the expression of the cPLA2 mRNA which was greatest at 8 and 16 h post-treatment (3.62+/-0.52 and 3.15+/-0.45-fold of control, n=3). The increase in cPLA2 mRNA expression by TNF-alpha was unaffected by the prior addition of interleukin-4 (IL-4) (10 ng/ml), a known inhibitor of prostaglandin endoperoxide H synthase (PGHS)-2 mRNA and protein expression in WISH cells. TNF-alpha also increased the level of immunoreactive cPLA2 protein in a time-dependent manner with the highest levels evident after 8 and 16 h. As with the mRNA, cPLA2 protein levels were unaffected by pre-incubation with IL-4. The inclusion of the cPLA2-specific inhibitor arachidonyl trifluoromethyl ketone (AACOCF3) resulted in a concentration-dependent inhibition of PGE2 biosynthesis in WISH cells treated with TNF-alpha (>95 per cent at 2 microM). We conclude that TNF-alpha increases the abundance of the cPLA2 mRNA and protein in amnion epithelial cells, an effect which plays an important role in amnion PG biosynthesis in the presence of intrauterine infection.

Arachidonic acid mobilization by phospholipase A2 (PLA2) and subsequent prostaglandin synthesis is thought to be a pivotal event in the onset and/or maintenance of human labour. The purpose of this study was to quantify secretory PLA2 (sPLA2) and non-secretory PLA2 enzymatic activity and type II sPLA2 immunoreactivity in human gestational tissues before, during and after labour of spontaneous onset. Placental tissue and fetal membranes were collected from women before, during and after spontaneous-onset labour at term and stored at - 80 degrees C. PLA2 activity in supernatants was quantified by radiolabelled substrate assay (14C-phosphatidylethanolamine) with and without 12.5 mm dithiothreitol (DTT) to separate enzymatic activity contributed by secretory and non-secretory PLA2 components. Immunoreactive type II PLA2 in supernatants was determined by a monoclonal, non-competitive sandwich ELISA. Total PLA2 enzymatic activity in amnion, choriodecidua and placenta was not significantly different before, during and after labour (n=18-20). Likewise, non-secretory enzymatic activity was not significantly different before (n=9), during (n=10) and after labour (n=9) in any of the three types of gestational tissue examined. Although immunoreactive type II PLA2 was significantly higher in the placenta (P<0.01) compared to amnion and choriodecidua, there was no significant difference in immunoreactive type II PLA2 within each tissue group according to labour status (n=18-20). Overall, no change in PLA2 secretory or non-secretory enzymatic activity or immunoreactive type II PLA2 could be detected throughout the peripartum period.

Recently, a model has been proposed for the involvement of secretory phospholipase A2 (sPLA2) in the onset of and/or progression of human labour via the metabolism of cell membrane glycerophospholipids to generate biologically active, phospholipid-derived mediators. The recent molecular cloning and characterization of a cell-surface receptor for sPLA2 raise the possibility that sPLA2 enzymes may also affect cell function in intrauterine tissues via a receptor-mediated pathway. The aim of this study was to determine the expression profile of the PLA2 receptor messenger RNA in human gestational tissues at term. Messenger RNA for the PLA2 receptor was detected in amnion, choriodecidua and placenta by RT-PCR and transcripts of similar size to the 6.5- and 5.4-kb transcripts previously reported in various other human tissues were detected in choriodecidua by Northern blot analysis. However, smaller transcripts of approximately 4, 2.3 and 1 kb were also detected in choriodecidua by Northern blot analysis and the 2.3-kb transcript and the 1-kb transcript were the only major transcripts detected in amnion and placenta, respectively. The presence of PLA2 receptor mRNA in human gestational tissues indicates that an alternative non-catalytic pathway may contribute to the regulatory effects of sPLA2 isozymes in these tissues. While the specificity and affinity of the various transcripts identified in this study have yet to be determined, PLA2 isozymes released from human gestational tissues during pregnancy and at the time of labour may function as paracrine or autocrine mediators to affect cell function.

Ultrastructurally, all cells of human fetal membranes strongly exhibit a large amount of lipid deposits throughout pregnancy. Their origin and function is still unknown. The aim of this study was to investigate the localization of key components of lipid metabolism in this tissue. Using immunohistochemical techniques, the distribution of lipoprotein lipase (LPL), low density lipoprotein receptors (LDL receptors), and apo-lipoprotein B and E was investigated in 20 human fetal membranes at term. In addition, electron microscopy was used to study the intracellular localization of lipoprotein-sized particles. Amnionic epithelium and trophoblast cells reacted strongly for LPL. LDL receptors and apo-lipoproteins were present in amnionic epithelium and fibroblasts of the amnion. In none of the investigated cells were lipoprotein-sized particles identified. Similar results were obtained in all 20 cases. The findings indicate that lipoprotein from the amniotic fluid or from the maternal circulation may serve as substrate for lipids in human fetal membranes.

Human pregnancy is associated with extensive growth and remodelling of the uterus and placenta, and restructuring of these tissues during specific stages of gestation likely involves the degradative activity of various matrix metalloproteinases (MMPs). In this investigation, we used in situ hybridization and immunohistochemistry to identify the sites and cell source of collagenase-1 (MMP-1), stromelysin-1 (MMP-3), matrilysin (MMP-7), and 92 kDa gelatinase (MMP-9), a subgroup of MMPs with the combined ability to degrade essentially all matrix proteins. Human tissues were recovered from uncomplicated pregnancies at various gestational ages and from pregnancies complicated by chorioamnionitis, pre-eclampsia, and placenta accreta. Our results show prominent expression of all four MMPs in specific cells of human placentae involved in trophoblast invasion and placental maturation. Collagenase-1 and stromelysin-1 were detected in cells of the amnion, decidua, and chorionic villi at all stages of pregnancy. Ninety-two kilodalton gelatinase was present in granulocytes whenever present. Matrilysin was seen in cytotrophoblasts and syncytiotrophoblasts during early pregnancy but only in cytotrophoblasts by the third trimester. In addition, we found that matrilysin is over expressed and is produced by more cell types in placentae from pregnancies complicated by pre-eclampsia suggesting that the proteolytic activity of this MMP contributes to the pathology of this condition. We conclude that certain MMPs produced by resident cells of the human placenta, and in particular trophoblasts, participate in the physiological progress human gestation and parturition.

Although Type II phospholipase A2 (PLA2) immunoactivity has been identified in homogenates of human placenta and fetal membranes, there is a paucity of information concerning the sites of synthesis of this secreted PLA2 isozyme. The aim of this study, therefore, was to establish the cellular localization of Type II PLA2 messenger RNA (mRNA) in human term placental and fetal membranes by in situ hybridization. In addition, the co-localization of immunoreactive Type II PLA2 in gestational tissues was determined, and the effect of labour status and pre-eclampsia on immunolabelling intensity were established. Type II PLA2 mRNA was identified in all tissue sections examined and was localized principally in placental villous vasculature and mesenchymal elements of placenta, chorion and amnion. Within the vasculature, Type II PLA2 mRNA was associated with smooth muscle cells. Immunoreactive Type II PLA2 was identified in the fibroblast and spongy layers of the amnion, the fibroblast and reticular layer of the chorion, and in the mesenchymal core and trophoblasts of placental villi. Immunolabelling staining intensity was greater in placenta and chorion than that observed in amnion, however, staining intensity was unaffected by labour status. Pre-eclampsia was associated with increased immunolabelling for Type II PLA2 in placenta but not fetal membranes. The data obtained clearly established that Type II PLA2 is synthesized by multiple cell types within human gestational tissues and co-localization of Type II PLA2 mRNA and immunoreactive protein has been established. The role of Type II PLA2 in gestational tissue phospholipid metabolism and, in particular, in vascular and mesenchymal elements, has yet to be established. Possible roles for this isozyme may include, the provision of substrate for eicosanoid synthesis, maintaining cell membrane phospholipid asymmetry and prevention of clot formation within the placental vascular bed.

Using reverse transcriptase-polymerase chain reaction we have established that mRNAs for prostaglandin H synthases 1 and 2 (PGHS-1 and PGHS-2) are present in amnion, chorion and decidua from women both at term before and after the onset of labour and from women at 28-35 weeks of gestation before the onset of labour. By Western blot analyses we have demonstrated that epidermal growth factor, interleukin 1 beta and phorbol 12-myristate 13-acetate all increase PGHS-2 amounts in amnion cells. The degree of stimulation caused by these substances (218-311 per cent) is less than the increase in prostaglandin production usually generated (five- to 10-fold). Hence we believe that these substances may have multiple sites of action in the pathways of arachidonic acid metabolism.

To determine whether the kinetics of prostaglandin endoperoxide synthase (PGHS, commonly known as cyclooxygenase) in human amnion change with labor onset or between preterm and term labor, a specific enzyme assay was developed and characterized. The assay was linear for time (0-8 min) and protein concentration (5-30 micrograms/250 microliters incubation volume). The optimum pH was 8.0-8.5, and the enzyme reaction reached saturation at 10-20 microM arachidonic acid. Flufenamic acid was more efficacious than ibuprofen in the presence of 1 mM tryptophan in inhibiting enzyme activity. The Km and Vmax of PGHS were determined in 10 amnions obtained at elective caesarean section before labor onset (CS) at 39.3 +/- 0.8 wk gestational age (mean +/- SD, range = 38.5-41 wk) and 9 amnions obtained following spontaneous labor and vaginal delivery (SL) at 39.6 +/- 0.8 wk (range = 38.5-41 wk). The Km values were 1.4 +/- 1.2 mumol/l (CS) and 2.2 +/- 1.5 mumol/l (SL) (not different). However, the Vmax increased significantly (p < 0.05) from 11 +/- 8 (CS) to 19 +/- 4 (SL) pg PGE2/micrograms protein/min. In eight preterm amnions obtained following spontaneous labor and delivery at 32.9 +/- 2.1 wk (range = 29-36 wk), the Km and Vmax were 2.0 +/- 1.2 mumol/l and 17 +/- 9 pg PGE2/micrograms protein/min, respectively. Neither of these values was different from those of CS or SL amnions. None of the preterm pregnancies displayed histological evidence of infection. These results suggest that an increase in the mean amnion PGHS maximum velocity occurs in association with the onset of labor at term. The mean Vmax of PGHS in amnions obtained from idiopathic preterm spontaneous deliveries is between the CS and SL term values, reflecting, perhaps, multiple etiologies for preterm delivery.