Human trophoblast cells are known to release a range of arachidonic acid metabolites into culture medium, including cyclo-oxygenase, lipoxygenase and epoxygenase products. In this study we investigated the effects of dibutyryl cyclic AMP (db cAMP) on arachidonic acid metabolism in human first trimester trophoblast cells, and also determined the distribution of metabolites between intracellular and extracellular compartments.

Previous investigations have implicated epoxygenase metabolites of arachidonic acid in the control of steroidogenesis in luteinised granulosa cells. The aim of this study was to assess this hypothesis further. We first determined the responsiveness of the cells in vitro to three different stimuli, namely luteinising hormone (LH), interleukin-1beta (IL-1beta), and dibutyryl cyclic AMP (db. cyclic AMP). Their effects were time-dependent, in that progesterone production from cells incubated for 3 days prior to stimulation responded strongly to db. cyclic AMP, to a lesser extent to LH and not to IL-1beta. After 6 days of preincubation, all three stimuli increased progesterone production, and this preincubation period was used in the remainder of the study.LH and IL-1beta increased the intracellular levels of 5,6-epoxyeicosatrienoic acid (5,6-EpETrE) maximally after 10 min, whereas db. cyclic AMP had a more rapid effect within 2-5 min. There were no changes in levels of 14,15-epoxyeicosatrienoic acid (14,15-EpETrE), indicating that the effect was specific. Levels of dihydroxy derivatives of arachidonic acid were also increased, suggesting rapid metabolism of 5,6-EpETrE to inactive 5,6-DiHETrE. The effects of 5,6-EpETrE on progesterone production were transient, which may be due to the lability of this compound in solution, and limited passage into the granulosa-luteal cell cytoplasm. These results support a role for 5,6-EpETrE in the production of progesterone by human granulosa-luteal cells.

Fetal membranes are a primary source of prostaglandins and pro-inflammatory cytokines implicated in human parturition, so the inhibition of inflammatory pathways may be of benefit in pregnancies complicated by premature labour. We have therefore investigated the effects of a cytokine-suppressant anti-inflammatory drug (CSAID) on the output of prostaglandin E(2) (PGE(2)) and interleukin (IL)-1 beta from human fetal membranes in vitro. Bacterial endotoxin increased the expression of mRNA for IL-1 beta and type-2 cyclo-oxygenase (COX-2), and there were corresponding increases in the output of IL-1 beta protein and PGE(2). The CSAID decreased IL-1 beta protein, COX-2 expression and PGE(2) output, but not mRNA for IL-1 beta, indicating a post-translational effect on the production of IL-1 beta and a transcriptional affect on COX-2, with an overall reduction in PGE(2). These findings are consistent with the effects of CSAIDs in other systems, and indicate that they are of possible use in premature labour.

BACKGROUND The study was designed to investigate the ultrastructural features of the early human feto-maternal interface when generated by in-vitro co-culture, and compare these with findings reported previously from human pregnancies. METHODS Placental villi and decidua parietalis tissues from 8-12 week pregnancies were co-cultured in vitro over a 4-day period. The co-incubations were ended at 24 h intervals and processed for electron microscopical studies, and for immunocytochemistry using anti-cytokeratin antibody (CAM 5.2) for trophoblast. RESULTS Loss of the syncytium at points of contact with the decidual stroma, cytotrophoblast column formation, differentiation and invasion of extravillous trophoblast (EVT) cells into the decidual stroma over the 4-day period of co-culture were observed. Cellular components, such as actin filaments, microtubules, glycogen granules and lamellipodic processes found in EVT cells were consistent with active cellular locomotion. CONCLUSIONS These ultrastructural studies emphasize the usefulness of this model in investigating the formation of the feto-maternal interface of human pregnancy. The recruitment of cytotrophoblast to the syncytium by a process involving fusion of the intervening plasma membranes, and the migration of EVT cells causing little or no damage to the surrounding decidual cells, resemble in-vivo data.

Objective. To compare the efficacy and safety of atosiban and terbutaline for the inhibition of preterm labor.Methods. Two hundred and forty-nine women diagnosed with preterm labor at 23-33 weeks of gestation were enrolled of whom 245 women received treatment, 116 with atosiban and 129 with terbutaline. At randomization, women were stratified by gestational age (less than or equal to 28 weeks and > 28 weeks). Atosiban (iv bolus dose of 6.75 mg, then 300 mug/min for 3 h and 100 mug/ min thereafter) and terbutaline (5-20 mug/min) were administered by iv infusion for 13-18 h. Re-treatment with study drug or an alternative tocolytic agent was allowed. Tocolytic effectiveness was assessed in terms of the number of women undelivered after 48 hours and 7 days and efficacy and tolerability in terms of the number of women remaining undelivered and not requiring alternative tocolytic therapy after 48 hours and 7 days of starting therapy. Safety was assessed in terms of maternal side effects and neonatal morbidity.Results. Tocolytic effectiveness at 48 hours was 86.1% vs 85.3%; p=0.783, and after 7 days it was 76.5% vs 67.4%; p=0.067, in the atosiban and terbutaline groups, respectively. Tocolytic efficacy and tolerability after 48 hours was 72.2% vs 68.2%; p=0.51 and after 7 days was 55.6% vs 43.4%; p=0.08 in the atosiban and terbutaline groups, respectively. Overall, there were fewer clinically important adverse events with atosiban than with terbutaline.Conclusions. The efficacy of atosiban in the inhibition of preterm labor was shown to be comparable to terbutaline. Atosiban had a superior safety profile compared with terbutaline in terms of maternal and fetal adverse events, and comparable infant outcomes.

Prostaglandins are some of the main mediators which control parturition, and their production by intrauterine tissues can be up-regulated by pro-inflammatory cytokines. Anti-inflammatory cytokines may oppose these effects, and in this study we have investigated how two such cytokines affected fetal membrane function. Interleukin-10 (IL-10) inhibited the output of prostaglandin E2 (PGE2) from intact fetal membranes under basal and lipopolysaccharide (LPS)-stimulated conditions, and there was a parallel decrease in the expression of mRNA for COX-2. IL-10 also inhibited the production of interleukin-1beta (IL-1beta) and the expression of mRNA for IL-1beta, indicating that this cytokine has a broad anti-inflammatory effect. Transforming growth factor-beta1 (TGF-beta1), which is generally considered to be anti-inflammatory had opposite effects on PGE2 production, in that it increased the output of PGE2 for up to 8 hr. TGF-beta1 increased levels of type-2 cyclo-oxygenase (COX-2) and cytosolic phospholipase A2 (cPLA2) protein, and also activated the cPLA2 enzyme present; the profile of effects is similar to that of the pro-inflammatory cytokine IL-1beta, and was not expected. Combinations of TGF-beta1 with IL-1beta also increased PGE2 output and caused appropriate changes in prostaglandin pathway enzymes, whereas TGF-beta1 and IL-1alpha had more limited effects. Further studies are needed to establish the physiological significance of these findings, but TGF-beta1 does not seem to act as an inhibitory cytokine in intact fetal membranes at term.

To examine the effect of region and labour upon prostaglandin synthesis in human fetal membranes, intact membranes from three regions, the cervical region, the periplacental region and a region midway between the two, were collected following spontaneous labour and delivery or at elective caesarean section prior to labour. Discs of 2-cm diameter were cut from each of three regions and incubated for 1, 2, 4, 6, 12 or 24 h after which prostaglandin E2 concentration in the supernatant was measured. We found that there was an overall decrease in prostaglandin synthesis in tissues collected after labour, but that this effect could be reversed if exogenous arachidonic acid substrate was supplied. We found no differences in prostaglandin synthesis between tissues collected from each of the three regions. We conclude that prostaglandin synthesis from the fetal membranes during labour leads to depletion of arachidonic acid substrate and that regional changes in prostaglandin dehydrogenase activity do not appear to have a significant effect upon overall prostaglandin synthesis.

Bacterial endotoxin increased the expression of mRNA (maximal after 4 hr) for interleukin‐1β (IL‐1β) and the release of mature protein from intact human fetal membranes. In contrast, the change in expression of mRNA for type 2 cyclo‐oxygenase (COX‐2) was biphasic, with peaks after 0·5–1 hr and after 8 hr of culture. An antibody to IL‐1β was without effect after 4 hr of culture, inhibited endotoxin‐stimulated prostaglandin E2 (PGE2) production after 8 hr of culture, and caused a parallel decrease in the expression of mRNA for COX‐2. We conclude that endotoxin induced the expression of COX‐2 through IL‐1β‐independent and IL‐1β‐dependent mechanisms, and these differences are time dependent. Corticotrophin‐releasing hormone (CRH) or platelet‐activating factor (PAF) also increased the expression of mRNA for IL‐1β and the release of IL‐1β from some, but not all, fetal membranes. The antibody to IL‐1β did not affect CRH‐stimulated or PAF‐stimulated PGE2 production or COX‐2 expression. We conclude that CRH and PAF can induce the expression of IL‐1β, but this is not obligatory for increased PGE2 release, and the effect of these stimuli on COX‐2 expression is a direct, IL‐1β‐independent effect.

Corticotrophin-releasing hormone (CRH) and platelet-activating factor (PAF) are considered to be involved in the physiological processes of human labour. Both may have dual effects, directly regulating myometrial contractility and fetal membrane prostaglandin production. During this study, we investigated the mechanisms through which CRH and PAF exert their latter effect. CRH and PAF increased prostaglandin production from intact fetal membrane discs, with a maximum stimulation after 8 h of culture. Reverse transcription-polymerase chain reaction (RT-PCR) analyses using primers specific for type-2 cyclo-oxygenase (COX-2) showed that CRH and PAF increased the transcription of COX-2 mRNA two-fold after 8 h culture. These data indicate that the increased fetal membrane prostaglandin production in response to CRH or PAF may involve the induction of COX-2.

Lipoxygenase metabolites may be involved in human parturition. 5-lipoxygenase (5-LOX) catalyses the first steps in the synthesis of leukotrienes from arachidonic acid, and its activity is dependent on 5-LOX activating protein (FLAP). The expression of 5-LOX and FLAP were investigated in fetal membranes to determine whether there are changes with gestational age or at term with the onset of labour. No significant differences were found in the expression of 5-LOX or FLAP mRNA in the amnion at different gestational ages or at term. In the chorion-decidua, 5-LOX mRNA expression was significantly higher in the first trimester of pregnancy than in the second and third trimesters. At term, there was a significant increase in both 5-LOX mRNA and protein expression in the chorion-decidua in the time after labour, compared with the time before labour. The expression of FLAP mRNA was also significantly higher in the chorion-decidua in the first trimester of pregnancy compared with the third trimester, and at term in the time after labour compared with the time before labour. Expression of FLAP protein was not studied, as an antibody is not currently available. These results are consistent with a role for 5-LOX and FLAP in the control of parturition at term, and also suggest an involvement earlier in pregnancy.

SummaryWe recruited 111 patients who were considered to be at significantly increased risk of preeclampsia on the basis of previous obstetric history or preexisting medical disorders. All patients were treated with low dose aspirin (75 mg/day) from the first occasion the patient attended the antenatal clinic, regardless of gestational age. If the maternal mean platelet volume (MPV) increased significantly (by >0.8 fl) from the baseline, antiplatelet treatment was increased. Five pregnancies were lost during the second trimester and 106 of the treated patients had live infants. The incidence of neonatal death (3/106 infants) was much lower than in the previous pregnancies in these patients (32/134 infants). Patients who were treated from the first trimester of pregnancy (group A, 89 patients) did substantially better than those treated from the second trimester (group B, 17 patients) as assessed by the incidence of pre-eclampsia or intrauterine growth restriction (IUGR), gestational age and birthweight at delivery. These data suggest that longitudinal monitoring of the MPV may identify the women who could benefit from increased antiplatelet treatment, and that antiplatelet treatment may be more effective when initiated in the first trimester rather than later in pregnancy.

Fetal membranes from term human pregnancies produce prostaglandins, and may respond to bacterial endotoxin or interleukin-1 beta (IL-1 beta) with increased prostaglandin E2 (PGE2) production. The effects of endotoxin persisted for up to 24 h, whereas those of IL-1 beta were maximal 4-8 h after addition. The maximum levels of PGE2 (200-350 pg/ml) were similar in all experiments, and were independent of the stimulus used. Not all tissues responded to these stimuli; those which did not had basal levels of PGE2 production of 200-350 pg/ml, which was not further increased by endotoxin or IL-1 beta. The basal production from these tissues was therefore similar to the maximal production from those tissues which responded to endotoxin or IL-1 beta. The high basal production of PGE2 was attributed to prior in vivo activation of the membranes such that PGE2 synthesis could not be further stimulated in vitro. Overnight pretreatment with aspirin decreased basal PGE2 production from these activated membranes to < 100 pg/ml/4 h during subsequent culture in aspirin-free medium. Both endotoxin and IL-1 beta increased PGE2 production from the activated aspirin-pretreated membranes during this culture time, but this was transient as after 12 h of culture basal PGE2 production rose to over 200 pg/ml despite aspirin pretreatment.

The addition of live or sonicated Escherichia coli, or endotoxin from E. coli increased the release of prostaglandins (PG) on both sides of intact human fetal membranes after 24h of incubation, indicating that live bacteria were not required to activate prostaglandin production. Time-course studies showed that the levels of PGE2 and PGF2α on the fetal side of the membrane were increased 6 h after the addition of endotoxin, whereas levels on the maternal side increased within 1–2 h. These changes were independent of the side to which the endotoxin was added, indicating that a stimulatory factor passes through the fetal membranes. This factor is not endotoxin, which did not cross the membranes, and further studies are required to identify this endogenous stimulus. Prostaglandin metabolite levels were either unaffected or increased by endotoxin, indicating that the main effect is at the level of increased prostaglandin biosynthesis rather than decreased metabolism.

There is strong evidence that prostaglandins E2 and F2α (PGE2 and PGF2α) are involved in the initiation and maintenance of human parturition and that their production can be stimulated by a number of cytokines and in infection-induced preterm labour by bacterial endotoxin. This study used an intact fetal membrane disk model to investigate the regulation of PGE2 and PGF2α metabolism by interleukin-1 beta (IL-1β and bacterial endotoxin [lipopolysaccharide (LPS)]. Fetal membrane explants were incubated with IL-1β (0.1 or 1.0 ng/ml) or LPS (10 ng/ml) for 24 h. A mixture of 3H-prostaglandin (0.1 μCi) and unlabelled prostaglandin (1 μg) was then added at selected times after the addition of inflammatory mediators. The radiolabelled prostaglandins and their metabolites were then extracted from the culture medium and quantified by high-pressure liquid chromatography. Levels of prostaglandin metabolites were generally decreased following incubation with IL-1β or LPS, which is consistent with a decrease in the activity of 15-hydroxyprostaglandin dehydrogenase (PGDH). It is concluded that IL-1β and LPS moderately decrease the metabolism of prostaglandins, which may contribute to increasing the local levels of active prostaglandins induced by these stimuli.

There is strong evidence for the involvement of inflammatory mediators such as interleukin (IL)-1 in the biochemical mechanisms of parturition. Therefore the effects of the IL-1. family (IL-1 alpha (1 ng/ml), IL-1 beta (1 ng/ml) and the IL-1 receptor antagonist (IL-1ra) (10 ng/ml)) on the regulation of prostaglandin synthesis in term human fetal membranes were investigated. It was found that, after 4 h of culture, IL-1 beta increased prostaglandin E-2 (PGE(2)) output approximately twofold. This was associated with both a significant increase in cyclo-oxygenase-2 (COX-2) mRNA levels (approximately fourfold compared with control) and translocation of cytoplasmic phospholipase A(2) (cPLA(2)) from the cytosol to the membrane fraction. IL-1 alpha was less effective than IL-1 beta at stimulating PGE(2) production through similar mechanisms.IL-1ra had no effect on PGE(2) output. However, in combination treatments, IL-1ra did not inhibit IL1 alpha- or IL-1 beta-stimulated PGE(2) output, and increased PGE(2) production further compared with IL-1 beta alone. IL-1ra decreased IL-1 beta-induced COX-2 mRNA expression by about half and significantly increased cPLA(2) protein levels, as detected by immunoblotting, when used alone and together with IL-1 beta. These results suggest that IL-1ra has partial agonist properties when used together with IL-1 alpha and IL-1 beta in fetal membranes by increasing cPLA(2) protein levels, which leads to an increase in the production of prostaglandins.

Basal progesterone production from first trimester placental cells in culture was high during the first 24 h of culture and fell to less than 30% of the initial level after 96 h in vitro. 22(R)-Hydroxycholesterol had a similar effect on progesterone production at all incubation times, indicating that the decline in basal steroidogenesis was not due to a loss of mitochondrial or post-mitochondrial enzymes. Continuous stimulation with dibutyryl (db) cyclic AMP maintained progesterone synthesis at a relatively constant high level despite the fall in basal progesterone production, and the optimum concentration of db cyclic AMP was 1.0 mM. The calcium ionophore A23187 had no effect on progesterone incubation during short-term cultures (<4 h), and inhibited steroidogenesis after 24 h. Repeated addition of A23187 during 96 h of culture also inhibited progesterone production. These findings indicate that progesterone production in human trophoblast is supported by a local factor which maintains a high level of steroid production through a cyclic AMP-dependent mechanism. The inhibitory effects of calcium ionophore in trophoblast differ from the stimulatory effects of this compound in other steroidogenic cells, but the reasons for the difference are not known at present.