Impact of Mothers' Age on Telomere Length and Human Telomerase Reverse Transcriptase Expression in Human Fetal Membrane-Derived Mesenchymal Stem Cells.

Age-related cellular changes and limited replicative capacity of adult mesenchymal stem cells (MSCs) are few of the challenges confronting stem cell research. MSCs from human fetal membranes (hFM-MSCs), including placental, umbilical cord, and amniotic membrane, are considered an alternative to adult MSCs. However, the effect of mothers' age on hFM-MSC cellular properties is still not clearly established. This study aimed to evaluate the effect of mothers' age on hFM-MSC telomere length, telomerase activity, and proliferation ability in three different age groups: GI (20-29 years), GII (30-39 years), and GIII (≥40 years). hFM samples were collected from pregnant women ≤37 weeks after obtaining consent. hFM-MSCs were isolated and cultured to characterize them by flow cytometry and assess proliferation by MTT assay and doubling time. Telomere length and expression levels of human telomerase reverse transcriptase were assessed by quantitative real-time polymerase chain reaction (qRT-RCR). hFM-MSCs in the three age groups were spindle-shaped, plastic-adherent, and exhibited high proliferation rates and strong expression of hMSC markers. GI showed the longest telomere length in hMSCs in various FM regions, whereas GIII showed the highest level of telomerase expression. There was no difference in telomere length between GII and GIII, and both groups showed the same hMSC characteristics. In conclusion, although the hFM-MSCs derived from different fetal membranes maintained the MSC characteristics in all study groups, the hFM-MSCs of older mothers had shorter telomeres and higher telomerase activity and proliferation rate than did those derived from younger mothers. Thus, the hFM-MSCs of older mothers could be unsuitable for expansion in vitro or stem cell therapy. Determination of telomere length and telomerase expression level of hFM might help characterizing and understanding the biological differences of hFM-MSCs in different age groups.

Smoking alters hydroxyprostaglandin dehydrogenase expression in fetal membranes.

INTRODUCTION: The way in which tobacco smoking increases the risk of preterm labor remains uncertain. Altered prostaglandin metabolism is one potential mechanism. METHODS: Proteins in fetal membrane samples (amniochoriodecidua) from 20 women were relatively quantified using Tandem Mass Tagging nano-liquid chromatography mass spectrometry. RESULTS: Prostaglandin synthases and two enzymes involved in prostaglandin degradation, hydroxyprostaglandin dehydrogenase (HPGD) and CBR1, were detected by the mass spectrometer. The expression of HPGD was significantly lower in smokers relative to non-smokers (0.43 fold, p = 0.016). There was no effect of labor, inflammatory status or gestational age on the HPGD levels. DISCUSSION: We describe for the first time an association between maternal smoking and HPGD expression. We propose that reduced expression of HPGD is one mechanism through which smoking may contribute to preterm labor. Lower levels of this enzyme, key to metabolising prostaglandins, may result in higher levels of prostaglandins and therefore precipitate labor prematurely.

In Vitro Screening of Synthetic Fluorogenic Substrates for Detection of Cancer Procoagulant Activity.

Cancer procoagulant (CP), a direct activator of coagulation factor X, is among one of the tumour cell products or activities which may promote fibrin formation and has been suggested to be selectively associated with the malignant phenotype. At present, the most reliable assay for the quantification of CP activity is the three-stage chromogenic assay which utilises the ability of CP to activate factor X. In this assay, the activation of factor X leads to the formation of activated thrombin from prothrombin and the eventual hydrolyses of a thrombin chromogenic substrate which contains a p-nitroaniline leaving group. The complexity of the three-stage chromogenic assay suggests a need for a direct method of assaying CP activity. This study focuses on the design of a fluorogenic substrate that would enable the direct quantification of CP activity. The results of the study show two promising substrates for the determination of CP activity: Boc-PQVR-AMC and PQVR-AMC. Further analysis showed that Boc-PQVR-AMC could be excluded as a potential substrate for CP since it was also cleaved by collagenase.

Screening of lysyl oxidase (LOX) and lysyl oxidase like (LOXL) enzyme expression and activity in preterm prelabor rupture of fetal membranes.

OBJECTIVE: Lysyl oxidase (LOX) and LOX like enzymes (LOXL1-4) physiologically remodel extracellular matrix and pathologically contribute to cellular senescence under oxidative stress (OS). We characterized LOX and LOXL expressions and activity in human fetal membranes. METHODS: Human fetal membranes from women with uncomplicated pregnancies at term, preterm birth with intact membranes (PTB) or preterm prelabor rupture of membranes (pPROM), and in vitro fetal membranes stimulated with water-soluble cigarette smoke extract (CSE), an OS inducer, were analyzed by real-time PCR and immunohistochemistry for LOX and LOXL (1-4) expression and localization. LOX activity was measured by fluorometric assay. RESULTS: LOX gene expression was ∼2.5-fold higher in fetal membranes from pPROM compared to PTB and term (P=0.02). LOX and LOXL1, 2 and 4 were localized to both amniotic and chorionic cells, whereas LOXL3 was limited to chorion. LOX and LOXL isoform expressions were not different between CSE treated and untreated groups, while LOX activity was increased in the presence of an antioxidant (P=0.02). CONCLUSIONS: Increase of LOX expression in pPROM, an OS-related disease, and the apparent inhibition of LOX activity by CSE restored by antioxidant treatment suggest that reactive oxygen species might influence LOX-mediated tissue remodeling in fetal membranes. Balanced antioxidant supplementation during pregnancy may reduce the risk of pPROM by increasing LOX activity.

Differences in extracellular matrix remodeling in the placenta of mares that retain fetal membranes and mares that deliver fetal membranes physiologically.

INTRODUCTION: In mammals, placenta separation at term may involve degradation of the extracellular matrix by matrix metalloproteinases (MMPs). The activity of MMPs is modulated by TIMPs. We hypothesized that the placentas of mares that deliver fetal membranes physiologically and those that retain fetal membranes (FMR) differ in terms of histology; mRNA expression of MMP-2 and MMP-9; protein expression of MMP-2, MMP-9, and TIMP-2; and the potential activity of both MMPs. METHODS: Placenta biopsies were taken from mares (n = 9; 4 FMR, 5 controls) immediately after foal expulsion. Retention was defined as failure to expel all fetal membranes within 3 h of expulsion. All mares were monitored for time of expulsion. The degree of allantochorial/endometrial adhesion was determined in FMR mares, and biopsies from all mares were histologically examined. mRNA expression, protein immunolocalization, protein amount and potential enzyme activity were determined with RT-PCR, immunohistochemistry, Western Blotting and zymography, respectively. RESULTS: FMR mares had strong to extremely strong allantochorial/endometrial adhesion, and significantly more connective tissue in the allantochorial villi than controls. The range of MMP-2 mRNA expression levels was more than 13 times greater in FMR mares than in controls. Protein content of both MMPs and TIMP-2 differed significantly between groups. The range of potential MMP-2 and MMP-9 activity was larger in FMR mares, and MMP-2 potential activity was 1.4 times higher in controls (P = 0.02). DISCUSSION: These results indicate differences in extracellular matrix remodeling in FMR mares and controls, and suggest dysregulation of MMP expression and activation in FMR mares.

Expression of 8-oxoguanine glycosylase in human fetal membranes.

PROBLEM: The most common DNA lesion generated by oxidative stress (OS) is 7, 8-dihydro-8-oxoguanine (8-oxoG) whose excision repair is performed by 8-oxoguanine glycosylase (OGG1). We investigated OGG1 expression changes in fetal membranes from spontaneous preterm birth (PTB) and preterm premature rupture of the membranes (pPROM) and its changes in vitro in normal fetal membranes exposed to OS inducer water-soluble cigarette smoke extract (CSE). METHOD OF STUDY: DNA damage was determined in amnion cells treated with CSE by comet and FLARE assays. OGG1 mRNA expression and localization in fetal membranes from clinical specimens and in normal term membranes exposed to CSE were examined by QRT-PCR and by immunohistochemistry. RESULTS: DNA strand and base damage was seen in amnion cells exposed to CSE. OGG1 expression was 2.5-fold higher in PTB samples compared with pPROM (P = 0.045). No significant difference was seen between term and pPROM or PTB and term. CSE treatment showed a nonsignificant decrease in OGG1. OGG1 was localized to both amnion and chorion with less intense staining in pPROM and CSE-treated membranes. CONCLUSION: Increased OS-induced DNA damage predominated by 8-oxoG is likely to persist in fetal cells due to reduced availability of base excision repair enzyme OGG1. This can likely lead to fetal cell senescence associated with some adverse pregnancy outcome.

Expression profiles of fetal membrane nicotinamide adenine dinucleotide phosphate oxidases (NOX) 2 and 3 differentiates spontaneous preterm birth and pPROM pathophysiologies.

INTRODUCTION: Nicotinamide adenine dinucleotide phosphate oxidases (NOX 1-5) are enzymes that generate cellular reactive oxygen species (ROS) besides mitochondria and might be important ROS sources associated with pregnancy complications, particularly preterm premature rupture of membranes (pPROM), that has been related to ROS. OBJECTIVE: To characterize NOX enzymes expression in human fetal membranes. METHODS: Differential expression and localization of NOX isoforms in human fetal membranes collected from women with uncomplicated pregnancies at term, preterm birth (PTB) or pPROM and in vitro in normal term membranes maintained in an organ explant system stimulated with water-soluble cigarette smoke extract (wsCSE) were documented by real time PCR and immunohistochemistry. RESULTS: Fetal membranes from term deliveries, PTB and pPROM expressed NOX 2, 3 and 4 mRNAs whereas NOX 1 and 5 were not detected. NOX 2 expression was 2.3-fold higher in PTB than pPROM (p = 0.005) whereas NOX 3 was 2.2-fold higher in pPROM compared to PTB (p = 0.04). NOX 2 and 3 expressions at term mimicked pPROM and PTB, respectively. No difference in NOX 4 expression was observed among the studied groups. NOX 2, 3 and 4 were localized to both amniotic and chorionic cells. Expression of NOX 2, 3 and 4 were not significant in wsCSE-stimulated membranes compared to untreated controls. DISCUSSION/CONCLUSIONS: NOX enzymes are present in the fetal membranes and are differentially expressed in PTB and pPROM. Absence of any changes in NOXs expression after wsCSE stimulation suggests ROS generation in the membranes does not always correlate with NOX expression.

Antioxidative signalling pathways regulate the level of reactive oxygen species at the endometrial-extraembryonic membranes interface during early pregnancy.

Conceptus (embryo and associated extraembryonic membranes) implantation and development require a reciprocal biochemical and physical interactions between the extraembryonic membranes and the endometrium. However, the enzymatic antioxidative pathways controlling reactive oxygen species production at the endometrial-extraembryonic membrane interface early in pregnancy are not known. We aimed therefore to determine the content of malondialdehyde, as biomarkers of lipid peroxidation, and the activities of the major antioxidant enzymes, copper-zinc containing and manganese containing superoxide dismutases, catalase and glutathione peroxidase, in sheep extraembryonic membranes, caruncular and intercaruncular endometrium zones sampled at specific stages of pregnancy corresponding to the conceptus implantation (day 16) and the early post-implantation period (day 21). Malondialdehyde content in caruncular, intercaruncular and extraembryonic tissues was not different between stages of the pregnancy. Extraembryonic membranes demonstrated increased manganese containing superoxide dismutase and glutathione peroxidase activities, whereas catalase activity in these tissues decreased from day 16 to day 21. Caruncular tissues demonstrated increased manganese containing superoxide dismutase activity from day 16 to day 21. Intercaruncular tissues demonstrated increased copper-zinc containing superoxide dismutase, manganese containing superoxide dismutase and catalase activities from day 16 to day 21. The ovine extraembryonic membranes exhibit dynamic changes in enzymatic antioxidative pathways different from those of endometrial tissues during the transition from implantation to post-implantation period. This biochemical data provides novel insights into the developmental changes in antioxidative pathways of extraembryonic membranes and endometrium during early conceptus development.

An experimental mixed bacterial infection induced differential secretion of proinflammatory cytokines (IL-1ß, TNFα) and proMMP-9 in human fetal membranes.

Overall, 1-4% of all births in the US are complicated by choriamnionitis. Choriamnionitis is a polymicrobial infection most often due to ascending genital microbes which, in over 65% of positive amniotic fluid cultures, involves two or more organisms. In this study, we determine the cytokines expression (IL-1ß, TNFα) and prometalloproteinase activation (proMMP-2 and proMMP-9) after double o single infection an in vitro model of human fetal membranes. Fetal membranes at term were mounted in the Transwell culture system and after 24 h of infection, choriodecidual, and amnion media was collected. IL-1ß and TNFα were evaluated by ELISA, whereas proMMP-9 and proMMP-2 were determined by substrate gel zymography. The choriodecidual and amnion compartments actively respond to the infectious process, which induced the secretion of IL-1ß, TNFα, and proMMP-9 after either mixed or single infection. The proMMP-2 secretion profile was the same after all experimental conditions. There was no synergy between Streptococcus agalactiae and Escherichia coli for inducing the secretion of inflammatory factors or degradative metalloproteinase. In conclusion, these two bacteria could initiate different pathways to induce chorioamnioitis.

Alpha-lipoic acid inhibits thrombin-induced fetal membrane weakening in vitro.

Cytokine-mediated inflammation and abruption-induced thrombin generation are separately implicated in matrix metalloproteinase (MMP)-mediated weakening of fetal membranes (FM) leading to preterm premature rupture of the fetal membranes (PPROM). At term, FM of both labored vaginal and unlabored Cesarean deliveries exhibit a weak zone overlying the cervix exhibiting ECM remodeling characterized by increased MMP9 protein and activity. We have reproduced these biochemical changes as well as FM weakening in vitro using tumor necrosis factor-alpha (TNF) and interleukin (IL)-1ß, inflammatory cytokines implicated in PPROM. Additionally, we have reported that the antioxidant and NFκB inhibitor alpha-lipoic Acid (LA) blocks these TNF-induced effects. We now present the first direct evidence that thrombin also can induce FM weakening in vitro, and LA treatment inhibits this thrombin-induced-weakening. Full thickness FM fragments from unlabored Cesarean deliveries were incubated with increasing doses of thrombin (0-100 u/ml) for 48 h. Fragments were then strength tested (breaking force and work to rupture) using our published methodology. MMP3 and 9 levels in tissue extracts were determined by Western blot and densitometry. To determine the effect of LA, FM fragments were incubated with control medium or 10 u/ml thrombin, with or without 0.25 mM LA. Strength testing and MMP induction were determined. Thrombin induced a dose-dependent decrease in FM strength (42% baseline rupture force and 45% work to rupture) coupled with a dose-dependent increase in MMP3 and 9 expression (all p < 0.001). Treatment of FM with 0.25 mM LA completely inhibited thrombin-induced FM weakening and MMP expression (all p < 0.001). Thrombin treatment of cultured FM induces mechanical weakening and increased MMP3 and 9. Treatment of FM with LA inhibits these thrombin-induced effects. We speculate LA may prove clinically useful in prevention of PPROM associated with abruption.

The impact of vitamin C supplementation in pregnancy and in vitro upon fetal membrane strength and remodeling.

Generation of reactive oxygen species (ROS) has been suggested as a mechanism of fetal membrane (FM) weakening leading to rupture, particularly with preterm premature rupture of the fetal membranes (PROM). In vitro, FM incubation with tumor necrosis factor (TNF) mimics physiological FM weakening, concomitant with generation of ROS and collagen remodeling. Proinflammatory cytokines are also postulated to have a role in the development of the FM physiological weak zone where rupture normally initiates in-term gestations. We hypothesized that antioxidant treatment may block ROS development and resultant FM weakening. Two studies examining antioxidant effects upon FM strength were conducted, one in vivo and the other in vitro. Fetal membrane of patients enrolled in a multicenter placebo-controlled trial to determine the effect of vitamin C (1 g/day) and vitamin E (400 IU/day) upon complications of pre-eclampsia were examined for FM biomechanical properties and biochemical remodeling at birth. Separately, biomechanics and biochemical markers of remodeling were determined in FM fragments incubated with TNF with or without vitamin C preincubation. Supplemental dietary vitamin C in combination with vitamin E did not modify rupture strength, work to rupture, or matrix metalloproteinase-9 (MMP9; protein or activity) either within or outside the term FM physiological weak zone. In vitro, TNF decreased FM rupture strength by 50% while increasing MMP9 protein. Vitamin C did not inhibit these TNF-induced effects. Vitamin C alone had a weakening effect on FM in vitro. We speculate that vitamin C supplementation during pregnancy will not be useful in the prevention of preterm PROM.

Differential expression of the enzymatic system controlling synthesis, metabolism, and transport of PGF2 alpha in human fetal membranes.

The present study investigated the expression of genes and proteins associated with PGF2alpha biosynthesis, catabolism, and transport in matched amnion and choriodecidua of human term placenta. The concentration of PGF2alpha within fetal membranes depends on the balance between complex enzymatic systems responsible for, respectively, its synthesis-by prostaglandin-endoperoxide synthase 2 (PTGS2) and members of the aldo-keto reductase (AKR) family, AKR1C3 and AKR1B1-and its catabolic inactivation-through hydroxy-prostaglandin-dehydrogenase (HPGD). We observed that AKR1C3 shows equal basal expression (mRNA and protein) in choriodecidua and amnion but that AKR1B1 exhibits preferential expression in the choriodecidua. Expression of HPGD and solute carrier organic anion transporter family member 2A1 (SLCO2A1) was found primarily in the choriodecidua. We also evaluated whether an inflammatory environment induced by the gram-negative bacterial endotoxin lipopolysaccharide (LPS) affects expression of each candidate enzymes. The amnion responded to LPS with a small but significant decrease of AKR1B1 mRNA expression. In contrast, we found a significant increase in PTGS2 and AKR1C3 mRNA expression in choriodecidua after LPS challenge, but such regulation was confirmed only at protein levels for PTGS2 and not for AKR1C3. Our results suggest that the choriodecidua appears to be the main tissue, which expresses maximally all the components (synthesis, degradation, and transport) controlling PGF2alpha levels.

The effect of progesterone on gelatinase expression in the decidua and fetal membranes before and after contractions.

OBJECTIVE: This study was aimed to explore the effect of progesterone on gelatinase expression in the decidua and fetal membranes before and after contractions. STUDY DESIGN: Zymography was conducted for matrix metalloproteinase (MMP) secretion. Semiquantitative reverse transcriptase-polymerase chain reaction was performed to examine MMP2 transcripts, and the effect of progesterone on MMP2 promoter activity was determined with the use of luciferase activity. RESULTS: Progesterone decreased pro-MMP2 secretion, expression, and promoter activity in decidua before contractions began. The effect of progesterone was reversed completely by mifepristone (RU486). Progesterone failed to inhibit MMP2 expression in the amnion and chorion before contractions began. After contractions, progesterone failed to inhibit MMP2 expression in both the decidua and fetal membranes. CONCLUSION: MMP2 expression is inhibited by progesterone only in the decidua and only before contractions begin.

Advanced glycation endproducts mediate pro-inflammatory actions in human gestational tissues via nuclear factor-kappaB and extracellular signal-regulated kinase 1/2.

Processes of human labour include increased oxidative stress, formation of inflammatory mediators (e.g. cytokines) and uterotonic phospholipid metabolites (e.g. prostaglandins). In non-gestational tissues, advanced glycation endproducts (AGE) induce the expression of pro-inflammatory molecules through mitogen-activated protein kinase and nuclear factor kappaB (NF-kappaB)-dependent pathways. Thus, the aim of this study was to investigate the effects of AGE on 8-isoprostane (a marker of oxidative stress), pro-inflammatory cytokine and prostaglandin release in human gestational tissues, and to define the signalling pathways involved. Human placenta and gestational membranes (amnion and choriodecidua combined; n=5) were incubated in the absence or presence of AGE-BSA (0.25, 0.5, 1 and 2 mg/ml) for 18 h. AGE significantly increased in vitro release of tumour necrosis factor-alpha, interleukin (IL)-1beta, IL-6, IL-8, prostaglandin (PG)E(2), PGF(2alpha) and 8-isoprostane from human placenta and gestational membranes. This was associated with a concomitant increase in NF-kappaB p65 activation and ERK 1/2 phosphorylation. AGE-stimulated 8-isoprostane, cytokine and prostaglandin production was significantly suppressed by the ERK 1/2 inhibitor U0126 and the NF-kappaB inhibitor BAY 11-7082. In conclusion, AGE mediates inflammatory actions in human gestational tissues. Protein kinases and the NF-kappaB pathway play an essential role in AGE signalling in human gestational tissues.

The matrix metalloproteinases (MMPS) in the decidua and fetal membranes.

The role of the matrix metalloproteinases (MMPs) in the decidua, fetal membranes and amniotic fluid (AF) has been receiving more and more attention. The MMPs are not only important intermediaries in pathological processes leading to preterm labor but it seems that they also play a crucial role in the activation of labor at term. During normal gestation MMP-1, -2, -3, -7 and -9 are found in the amniotic fluid and fetal membranes. MMP-2 and MMP-3 are expressed constitutively while MMP-9 is barely detectable until labor. At labor, while MMP-9 is the major MMP responsible for gelatinolytic activity in the membranes, MMP-2 is dominant in the decidua. MMP-7 (AF) increases with gestation but does not appear to play a major role in labor. The expression of MMPs is attenuated through the expression of relaxins, integrins and extracellular matrix metalloproteinase inducer (EMMPRIN). Spontaneous preterm delivery (PTD) may be a product of preterm labor (PTL), preterm premature rupture of membranes (P-PROM) or placental abruption. Each of these processes may have differing pathways but the presence of an intrinsic inflammatory response with or without infection seems to involve all etiologies. The inflammatory response is mediated with cytokines such as interleukins -1, -6 and -8 and tumor necrosis factor alpha. MMP-3, MMP-7 and MMP-8 appear to be important in these processes. MMP-9, which is the major MMP involved in normal labor, plays an important role in pathological labor as well. Finally, apoptosis seems to play a role in pathological labor, particularly deliveries involving P-PROM. African-American are at greater risk of PTD than white or Hispanic Americans. Environmental differences may not suffice to explain this phenomenon. Genetic polymorphisms of the MMP genes may help explain the greater risk among this population. Finally, manipulating MMPs may have a role in the prevention of PTD. Agents suggested include indomethacin, N-acetylcysteine, progesterone and specific inhibitors of phosphodiesterase 4.

Tumour necrosis factor-alpha converting enzyme in human gestational tissues from pregnancies complicated by chorioamnionitis.

Chorioamnionitis increases the risk of preterm labour and is associated with adverse neonatal outcomes including cerebral palsy. Tumour necrosis factor-alpha (TNF-alpha) derived from the gestational tissues (placenta, fetal membranes and maternal decidua) is thought to play a pivotal role in the induction of cytokine response in chorioamnionitis. Tumour necrosis factor-alpha converting enzyme (TACE) is essential for the release of TNF-alpha. Our aim was to determine whether the expression of TACE is increased in human gestational tissues from pregnancies complicated by chorioamnionitis, and whether lipopolysaccharide (LPS) causes increased expression of TACE in the human gestational tissues in vitro. The immunostaining of TACE was generally more intense, in particular in the syncytiotrophoblast and stromal cells, in villous samples from pregnancies complicated by chorioamnionitis than those from normal pregnancies. Increased immunoreactivity of TACE was also noted in the amnion and choriodecidua. In parallel, there was an increased infiltration of monocytes/macrophages within the villous stroma and choriodecidua. As a complement to our in vivo findings, LPS significantly increased the levels of mRNA and protein of TACE in a dose-dependent response in villous and fetal membrane explant cultures. Together, our results imply a potential role of TACE in the pathogenesis of chorioamnionitis.

Regulation of phospholipase isozymes by nuclear factor-kappaB in human gestational tissues in vitro.

Phospholipid-derived mediators are implicated in the initiation and progression of human labor and delivery, particularly in relation to infection-induced preterm labor. We previously demonstrated that, in human intrauterine tissues, lipopolysaccharide (LPS)-stimulated nuclear factor-kappaB (NF-kappaB) DNA binding activity, and subsequent cytokine release can be suppressed by sulfasalazine (SASP) concentrations greater than 5 mM. The aim of this study was to elucidate the effect the SASP on secretory type II phospholipase A(2) (PLA(2)), cytosolic PLA(2) (cPLA(2)), cyclooxygenase (COX) isozymes, and subsequent prostaglandin F(2alpha) (PGF(2alpha)) production in human gestational tissues. Human placenta, amnion, and choriodecidua (n = 4-9 separate placentas) were incubated in the presence of SASP (0.1, 1, 5, and/or 10 mM) under either basal or LPS (10 microg/ml) conditions. After 6 h incubation, the tissues were collected and assayed for type II PLA(2) by ELISA and cPLA(2), COX-1, and COX-2 content by Western blotting. The incubation medium was collected and assayed for type II PLA(2) and 13,14-dihydro-15-keto PGF(2alpha) release by ELISA and PGF(2alpha) by RIA. Treatment of placenta, amnion, and choriodecidua with SASP concentrations greater than 5 mM significantly inhibited basal and/or LPS-stimulated type II PLA(2) content and release, 13,14-dihydro-15-keto PGF(2alpha) release, and cPLA(2) protein content (ANOVA, P < 0.05); however, no effect of SASP was observed on basal or LPS-stimulated COX-1 or COX-2 protein. Although no effect of SASP was observed on basal and LPS-stimulated PGF(2alpha) release from placenta and amnion, it significantly increased both basal and LPS-stimulated PGF(2alpha) release from choriodecidua. In addition, SASP concentrations of 5 mM or greater significantly suppressed NF-kappaB DNA binding activity. These data are consistent with the hypothesis that NF-kappaB regulates the expression and release of phospholipase isozymes.

Indoleamine 2,3-dioxygenase: distribution and function in the developing human placenta.

Studies in mice have suggested that the placenta is protected from immune rejection by maternal T cells by means of localised indoleamine 2,3-dioxygenase dependent depletion of tryptophan. To determine whether such mechanisms might operate in the human placenta, we have studied the physiological importance of human placental indoleamine 2,3-dioxygenase immunohistochemically and functionally. Indoleamine 2,3-dioxygenase is detectable immunohistochemically from day 6 human blastocysts and thereafter throughout pregnancy in syncytiotrophoblasts, extravillous cytotrophoblasts and macrophages in the villous stroma and in the fetal membranes. Interferon-gamma added to villous explants markedly stimulates indoleamine 2,3-dioxygenase protein expression in macrophages. Indoleamine 2,3-dioxygenase-mediated tryptophan degradation in the first trimester villous and decidual tissue explants is stimulated by interferon-gamma and inhibited by 1-methyl-tryptophan (an inhibitor of indoleamine 2,3-dioxygenase). Peripheral blood mononuclear cell proliferation is controlled by indoleamine 2,3-dioxygenase-mediated tryptophan degradation. These results suggest the cellular basis of a mechanism present at the human maternal-fetal interface involved in regulating the maternal immune response to conceptus.

Differential expression and regulation of microsomal prostaglandin E(2) synthase in human fetal membranes and placenta with infection and in cultured trophoblast cells.

We have evaluated the effect of chorioamnionitis on the protein expression of microsomal and cytosolic prostaglandin E(2) synthases (mPGES and cPGES) in preterm human placentae (PL) and fetal membranes (FM), by Western blot and immunohistochemistry, as well as the regulatory effect of IL-1beta and TNF-alpha on mPGES, cPGES, and cyclooxygenase (COX)-2 expression in villous trophoblast (VT) and chorion trophoblast (CT) cell cultures. mPGES localized to the syncytiotrophoblast and vascular endothelium in PL and to the amnion epithelium, CT, and decidual cells in FM. cPGES protein was localized only to the syncytiotrophoblast in PL and had the same profile of expression as mPGES in FM. With infection, there was an increase in mPGES expression in PL and a decrease in the expression in FM. cPGES protein did not change in either PL or FM with infection. In VT cells in culture, IL-1beta up-regulated COX-2 protein expression but did not affect mPGES. However, TNF-alpha increased both mPGES and COX-2 protein expression in these cells. In CT cells in culture, IL-1beta and TNF-alpha increased both mPGES and COX-2 protein levels. Neither IL-1beta nor TNF-alpha affected cPGES in either VT or CT cells. We conclude that protein levels of mPGES, as well as COX-2, can be stimulated by cytokines, potentially contributing to the increased prostaglandin production at the time of infection-driven preterm labor. However, multiple mechanisms, which apparently are inductor- and cell-type-specific, exist for the regulation of these enzymes.

Nitric oxide: does it have an etiological role in pre-eclampsia?

AIM: To determine evidence of nitric oxide (NO) in decidual biopsies and fetal membranes of preeclamptic women. BACKGROUND: Nitric oxide, a potent vasodilator, has been postulated to have a role in the etiology of preeclampsia. Investigations in peripheral blood have led to conflicting results. We therefore decided to study whether immunohistochemically detectable nitric oxide is produced in the decidua and within the fetal membranes. METHODS: Forty-two pregnant women at 28 weeks gestation or more were enrolled. Twenty were normotensive and 22 had preeclampsia. Women with chronic hypertension, diabetes and multiple pregnancies were excluded. Maternal blood samples prior to cesarean section (CS), decidual biopsies during CS, and fetal membrane specimens were obtained. The tissue specimens were fixed immediately (after delivery of the baby and placenta) in formalin, washed, and embedded in paraffin. Immunohistochemical staining for nitric oxide synthases (NOS) I, II, III was performed and reviewed in conjunction with routine hematoxylin and eosin sections, using light microscopy. RESULTS: There was no statistically significant difference in the level of immunostaining of nitric oxide synthases in both normotensive and hypertensive patients. DISCUSSION: The severity of blood pressure did not influence expression of nitric oxide synthases.