The study of progesterone action in human myometrial explants.

STUDY HYPOTHESIS: Myometrial explants represent a superior model compared with cell culture models for the study of human myometrial progesterone (P4) signalling in parturition. STUDY FINDING: Gene expression analysis showed myometrial explants closely resemble the in vivo condition and the anti-inflammatory action of P4 is not lost with labour onset. WHAT IS KNOWN ALREADY: Circulating P4 levels decline before the onset of parturition in most animals, but not in humans. This has led to the suggestion that there is a functional withdrawal of P4 action at the myometrial level prior to labour onset. However, to date, no evidence of a loss of P4 function has been provided, with studies hampered by a lack of a physiologically relevant model. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Myometrial biopsies obtained at Caesarean section were dissected into explants after a portion was immediately snap frozen (t = 0). Microarray analysis was used to compare gene expression of t = 0 with paired (i) explants, (ii) passage 4 myometrial cell cultures or (iii) the hTERT myometrial cell line. Western blotting and chemokine/cytokine assays were used to study P4 signalling in myometrial explants. MAIN RESULTS AND THE ROLE OF CHANCE: Gene expression comparison of t = 0 to the three models demonstrated that explants more closely resemble the in vivo status. At the protein level, explants maintain both P4 receptor (PR) and glucocorticoid receptor (GR) levels versus t = 0 whereas cells only maintain GR levels. Additionally, treatment with 1 µM P4 led to a reduction in interleukin-1 (IL-1) ß-driven cyclooxygenase-2 in explants but not in cells. P4 signalling in explants was PR-mediated and associated with a repression of p65 and c-Jun phosphorylation. Furthermore, the anti-inflammatory action of P4 was maintained after labour onset. LIMITATIONS/REASONS FOR CAUTION: There is evidence of basal inflammation in the myometrial explant model. WIDER IMPLICATIONS OF THE FINDINGS: Myometrial explants constitute a novel model to study P4 signalling in the myometrium and can be used to further elucidate the mechanisms of P4 action in human labour. LARGE SCALE DATA: Data deposited at http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=gvmpggkurbgxfqf&acc=GSE77830. STUDY FUNDING AND COMPETING INTEREST: This work was supported by grants from the Joint Research Committee of the Westminster Medical School Research Trust, Borne (No. 1067412-7; a sub-charity of the Chelsea and Westminster Health Charity) and the Imperial NIHR Biomedical Research Centre. The views expressed are those of the author(s) and not necessarily those of the NHS or the Department of Health. The authors have no conflict of interest.

Progesterone and the Repression of Myometrial Inflammation: The Roles of MKP-1 and the AP-1 System.

Progesterone (P4) maintains uterine quiescence during pregnancy and its functional withdrawal is associated with increased prostaglandin synthesis and the onset of labor. In primary human myometrial cells, the glucocorticoid receptor (GR) rather than the P4 receptor mediates P4 antagonism of IL-1ß-induced cyclooxygenase-2 (COX-2) expression, the rate-limiting enzyme in prostaglandin synthesis. We now report that P4 also acts via GR to induce MAPK phosphatase (MKP)-1 and knockdown of MKP-1 impairs the ability of P4 to repress IL-1ß-dependent COX-2 induction. Microarray analysis revealed that P4 repressed preferentially activator protein-1-responsive genes in response to IL-1ß. Consistent with these observations, we found that the ability of P4 to reduce c-Jun activation was lost upon GR as well as MKP-1 knockdown. Interestingly, c-Jun levels in human myometrial cells declined upon GR and MKP-1 knockdown, which suggests the presence of an activator protein-1 feedback loop. This is supported by our observation that c-Jun levels declined after an initial rise in primary myometrial cells treated with phorbol 12-myrisatate 13-acetate, a potent activator of c-Jun N-terminal kinase. Finally, we show that MKP-1 is an intermediate in P4-mediated repression of some but not all IL-1ß-responsive genes. For example, P4 repression of IL11 and IRAK3 was maintained upon MKP-1 knockdown. Taken together, the data show that P4 acts via GR to drive MKP-1 expression, which in turn inhibits IL-1ß-dependent c-Jun activation and COX-2 expression.

An evaluation of the ability of leucocyte depletion filters to remove components of amniotic fluid.

Haemorrhage remains an important cause of maternal mortality worldwide. Cell salvage carries a theoretical risk of amniotic fluid embolus syndrome and is too expensive for use in many parts of the world. To explore cheaper options, we investigated whether a leucocyte depletion filter alone removes components of pure amniotic fluid. Amniotic fluid was collected from 10 women during elective caesarean section and passed through a LeukoGuard® RS filter. Pre- and post-filtration samples were compared in the laboratory. Lamellar bodies and fetal squames were almost completely removed (filtration efficacy 96.6% and 99.9%, respectively; p<0.0001 and <0.0004), and hair was completely removed (p=0.002). Filtration had no effect on concentrations of α-fetoprotein, tissue factor or endothelin-1, or on the presence of meconium or vernix. Additional work is required to evaluate whether cell salvage using filtration alone may be useful in maternal haemorrhage in the developing world.

Uterine stretch and progesterone action.

CONTEXT: Progesterone administration reduces the risk of preterm labor in high-risk women with singleton pregnancies but has no effect in women with a multiple pregnancy. OBJECTIVE: We investigated whether progesterone is able to inhibit stretch-induced gene expression and/or whether stretch in turn inhibits progesterone action, perhaps providing an explanation for the functional progesterone withdrawal associated with human labor. METHODS AND RESULTS: In a series of in vitro studies using primary cultures of human myometrial cells, we found that preincubation with progesterone did not block stretch-induced ERK1/2 activation and cyclooxygenase-2 mRNA expression. Furthermore, we found that stretch did not alter the ability of progesterone to: 1) modulate progesterone-responsive gene expression; 2) activate a luciferase-linked progesterone response element; or 3) repress IL-1ß-driven cyclooxygenase-2 mRNA expression. We did find that stretch reduced the expression of progesterone receptor mRNA via nuclear factor κB activation but that this did not alter myometrial progesterone response. CONCLUSION: These data show that progesterone does not inhibit stretch-induced MAPK activation or gene expression, possibly explaining why progesterone is ineffective in the prevention of preterm labor in multiple pregnancy. Although stretch did reduce progesterone receptor expression in a nuclear factor κB-dependent manner, this was not sufficient to inhibit progesterone action, suggesting that it is not responsible for the functional progesterone withdrawal observed with the onset of human labor.

Prostaglandin F2-alpha receptor regulation in human uterine myocytes.

Investigations of the modulation of prostaglandin F(2alpha) receptor (FP) expression in primary cultures of human uterine myocytes showed that FP mRNA expression was reduced by progesterone, unaltered by cAMP (8-bromo cAMP or forskolin), but increased by the PKA antagonist H89. Interleukin (IL)-1beta, tumour necrosis factor-alpha and oxytocin increased FP mRNA expression and IL-6 and prostaglandin E(2) reduced FP mRNA expression. The changes in FP protein levels were similar to the mRNA responses. We found that the IL-1beta-induced increase in FP expression was mediated at least in part via protein kinase C (PKC), but was independent of mitogen-activated protein kinase, phospholipase C and PI3 kinase. Since IL-1beta activates NFkappaB, AP-1 and C/EBP, we over-expressed these transcription factors alone and in combination and found that only NFkappaB alone increased FP mRNA expression. Finally, we found that the IL-1beta-induced increase in FP expression was unaffected by progesterone and/or cAMP, but was accentuated by H89. These data suggest that the pregnancy-induced down-regulation in myometrial FP expression is mediated by progesterone and cAMP and that the increase with labour is induced by inflammatory cytokine activation of PKC and NFkappaB.

Stretch and interleukin 1 beta: pro-labour factors with similar mitogen-activated protein kinase effects but differential patterns of transcription factor activation and gene expression.

IL-1beta and stretch increase uterine smooth muscle cell (USMC) prostaglandin H synthase 2 (PGHS-2) and interleukin (IL)-8 mRNA expression in a mitogen-activated protein kinase (MAPK) dependent mechanism. We have tested our hypothesis that stretch and IL-1beta activate different components of the MAPK cascade in USMC and investigated the effects of specific MAPK inhibitors on these components. Further, we have used a Jun N-terminal kinase (JNK) and p38 activator, anisomycin, to compare the effect of differential MAPK activation on the expression of PGHS-2, IL-8 and oxytocin receptor (OTR) mRNA with that seen in response to stretch and IL-1beta. Stretch, IL-1beta and anisomycin activated similar components of the MAPK cascade and specific inhibitors of MAPK altered phosphorylation of MAPK and downstream cascade components as expected. Expression of OTR mRNA was increased by stretch and anisomycin in a MAPK-independent manner. All three stimuli increased PGHS-2 and IL-8 mRNA expression in a MAPK-dependent manner, but while the MAPK inhibitors reduced the IL-1beta-induced activation of activating transcription factor (ATF)-2, liver activating protein (LAP) and c-jun, the stretch-induced increase in LAP was unaffected by MAPK-inhibition and only JNK inhibition appeared to reduce c-jun activation. These observations show that stretch, IL-1beta and anisomycin activate the same components of the MAPK cascade, but differentially activate LAP and liver inhibitory protein (LIP) perhaps accounting for the increase in OTR by stretch and anisomycin but not IL-1beta observed in this study.

Leptin and bone turnover in monochorionic twins complicated by twin-twin transfusion syndrome.

INTRODUCTION: To test the hypothesis that the bone metabolism of a growth-restricted foetus is regulated by genetic, placental and/or foetal factors through leptin, we investigated the foetal bone turnover in monochorionic pregnancies complicated with or without twin-twin transfusion syndrome (TTTS). METHODS: Maternal and cord bloods were collected from gestational-age-matched monochorionic twins with (n=15) and without (n=15) TTTS. The samples were assayed for leptin, cross-linked carboxyl terminal telo-peptide (ICTP, a marker of bone resorption) and pro-peptide (PICP, a marker of bone formation) of type I collagen by radioimmunoassay (RIA). RESULTS: In the growth-restricted donor twin, the plasma concentration of leptin (P < 0.001), PICP (P < 0.001) was lower, while that of ICTP (P < 0.001) was higher than the recipient twin of the TTTS group. In contrast, leptin, PICP and ICTP were comparable in non-TTTS twins. In the recipient twin of TTTS and non-TTTS twins, leptin was positively associated with PICP (r=0.73; n=45, P < 0.001) and negatively with ICTP (r=-0.68; n=45; P < 0.001). No such association was found between leptin and bone marker in the growth-restricted donor twin of the TTTS group. CONCLUSION: Our data suggest that, in AGA twins, leptin maintains bone metabolism by inhibiting resorption and enhancing bone formation. In contrast, growth-restricted donor twins have high bone turnover and this does not seem to be due to leptin deficiency.

Myometrial prostaglandin E2 synthetic enzyme mRNA expression: spatial and temporal variations with pregnancy and labour.

We have investigated the hypothesis that the expression of the enzymes involved in PGE(2) synthesis in the human uterus is co-ordinated. We have studied (i) the mRNA expression of the enzymes involved in PGE(2) synthesis [phospholipases (cPLA(2) and sPLA(2)), prostaglandin H synthase (PGHS)-2 and PG E synthases (PGES-1 and -2)] and their relationship to the expression of inflammatory cytokines in samples of myometrium obtained from pregnant women undergoing caesarean section (LSCS) either before or after the onset of labour at or before term; and (ii) the effect of IL-1beta, IL-6, TNF-alpha, PGE(2) and stretch on PGE(2) enzyme mRNA expression. We found that cPLA(2), sPLA(2) and PGHS-2 mRNA expression were greater in labour samples; cPLA(2), sPLA(2), PGHS-2, PGES-1 and -2 mRNA expression were greater in lower- than upper-segment samples; and there was no effect of gestational age. PGHS-2 mRNA levels correlated with those of PGES-1, cPLA(2), IL-1beta and IL-8; PGES-1 mRNA levels correlated with those of IL-1beta, IL-8 and cPLA(2). In primary cultures of uterine myocytes, cPLA(2) mRNA expression was increased by IL-1beta and IL-6; PGHS-2 mRNA expression was increased by IL-1beta, PGE(2) and stretch; and PGES-1 mRNA expression was increased by IL-1beta only. These data show that labour is associated with increased expression of the enzymes involved in PGE(2) synthesis and their expression is greater in the lower uterine segment. The presence of associations between the levels of PGE(2) enzyme mRNA expression and the effects of IL-1beta suggest that their expression is co-ordinated and that IL-1beta is the responsible factor.

Flow cytometric characterisation of cells of differing densities isolated from human term placentae and enrichment of villous trophoblast cells.

Cells were isolated from human term placentae by trypsinisation of fragments of chorionic villi and fractionation of cells on a Percoll density gradient into six layers. A panel of 10 monoclonal antibodies to antigens on or in trophoblast cells (placental alkaline phosphatase (PLAP), cytokeratin-7, beta-human chorionic gonadotrophin (beta-hCG), human leucocyte antigen-G (HLA-G)), leucocytes (CD45), monocytes, macrophages, dendritic cells, B cells (HLA class II), mesenchyme cells (vimentin), fibroblasts (fibroblast antigen) and nucleated cells excluding villous trophoblast (HLA class I, CD9) was used to characterise the cells by flow cytometry. For staining intracellular antigens (cytokeratin, vimentin, beta-hCG) the cells were first fixed and permeabilised. The upper two layers from the gradient (density 1.013-1.039 g/ml) contained predominantly PLAP-positive cells or fragments, probably derived from the syncytiotrophoblast. Cytokeratin-positive cells accumulated mainly in the layer of density 1.039-1.052 g/ml and comprised the majority of the cell types identified in this fraction. Few or no cells reactive with antibodies to beta-hCG or HLA-G were identified in any layer. Non-trophoblast cells were heavier, being present mainly at densities 1.052-1.079 g/ml (CD45, HLA class I, vimentin) and 1.066-1.092 g/ml (fibroblast). Fewer than 10% of cells in any layer were HLA class II- or CD9-positive. Further purification of trophoblast cells was by negative immunomagnetic separation with removal of CD45-positive cells and HLA class II-positive cells to less than 1%. On culture of the cells from each layer, those of density 1.039-1.066 g/ml exhibited characteristics of cytotrophoblast cells; they secreted high levels of human chorionic gonadotrophin and formed adherent multinucleate cells. This procedure enabled the selection and enrichment of cytotrophoblast cells and/or syncytiotrophoblast fragments that are suitable for cellular and molecular studies.

The mitogen-activated protein kinase dependent expression of prostaglandin H synthase-2 and interleukin-8 messenger ribonucleic acid by myometrial cells: the differential effect of stretch and interleukin-1{beta}.

Infection and uterine stretch are the common causes of preterm labor. IL-1beta plays a key role in infection-induced preterm labor and increases prostaglandin H synthase 2 (PGHS-2) and IL-8 expression. We have shown that mechanical stretch of uterine myocytes in vitro up-regulates the expression of PGHS-2 and IL-8. In this study, we tested the hypotheses that both IL-1beta and mechanical stretch increase the myometrial expression of PGHS-2 and IL-8 via MAPK activation and that their effects are synergistic. MAPK activation was assessed in myocytes obtained from pregnant women undergoing cesarean section before the onset of labor after exposure to IL-1beta and stretch either alone or in combination. Specific inhibitors of ERK, p38, and c-Jun N-terminal kinase were used to define the role of each in the increased expression of PGHS-2 and IL-8 mRNA. We found that both IL-1beta and stretch activated all three MAPK subtypes but that they had no synergistic effect. The inhibitor studies showed that stretch-induced increases in both PGHS-2 and IL-8 mRNA expression were ERK1/2 and p38 dependent and that IL-1beta-induced increases of PGHS-2 mRNA expression were also ERK1/2 and p38 dependent, but those of IL-8 were dependent only on ERK1/2 activation. These data show that exposure of human uterine myocytes to both stretch and IL-1beta activates the MAPK system, which is responsible for the increase in PGHS-2 and IL-8 mRNA expression. We found no evidence of a synergistic effect of IL-1beta and stretch on myometrial expression of PGHS-2 and IL-8 mRNA.

Prostanoid receptors in human uterine myocytes: the effect of reproductive state and stretch.

In the human, prostanoids are known to be important mediators of uterine relaxation and contraction during pregnancy and parturition. We have previously shown that stretch of uterine smooth muscle cells increased prostaglandin H synthase 2 (PGHS-2) mRNA expression, PGHS-2 peptide synthesis and activity, however, the net effect on uterine contractility of this increase in prostaglandin synthesis would be determined by the expression of the different prostanoid receptors. Therefore, the aims of this study were to establish the expression of prostanoid receptor mRNA in uterine myocytes obtained from women in different reproductive states and to test the hypothesis that stretch of uterine myocytes alters prostanoid receptor mRNA expression to promote uterine contractility. Myocytes were isolated from women undergoing hysterectomy (NP) and pregnant women undergoing LSCS either before (NL) or after the onset of labour (L) and were subjected to 11% stretch for 1 h (n = 6 in all cases). Copy numbers of the individual receptors varied widely with reproductive state but followed the pattern: FP > IP = DP = EP-4 > TP = EP-3 = EP-2 > EP-1. FP mRNA expression was significantly lower in the NL group compared to the NP group and EP-3, EP-4 and TP mRNA expression was significantly lower in both NL and L groups compared to NP group levels. The level of mRNA expression of EP-1, EP-2, DP and IP did not differ between NP, NL and L groups. Stretch of cells derived from the NP group resulted in a significant decrease in EP-4 mRNA expression alone and of the NL group a significant decrease in EP-2 mRNA expression alone. Stretch had no effect on cells derived from the L group. These data show that pregnancy is associated with a significant reduction in 3 of 4 pro-contraction associated prostanoid receptor mRNA expression and 1 of 4 pro-relaxant. Stretch elicited no consistent change in prostanoid receptor mRNA expression.

Mechanical stretch of human uterine smooth muscle cells increases IL-8 mRNA expression and peptide synthesis.

Labour is associated with increased synthesis of interleukin-8 (IL-8) by the fetal membranes and myometrium, which leads to an inflammatory infiltrate. Stretch has been shown to increase the expression of contraction-associated proteins in animal models of labour and in human myocytes in vitro. In this study, we tested the hypothesis that mechanical stretch of human myometrial cells increases IL-8 messenger ribonucleic acid (mRNA) expression. We isolated myocytes from non-pregnant women undergoing hysterectomy and pregnant women undergoing Caesarean section before and after the onset of labour. Myocytes in culture were subjected to stretch of varying intensity (6-16%) and duration (1 or 6 h) using the Flexercell system. IL-8 mRNA expression was lowest in myocytes from pregnant women not in labour, intermediate in those from non-pregnant women and greatest in those from pregnant women in labour. Stretch increased IL-8 mRNA expression independent of reproductive state. The stretch-induced increase in IL-8 mRNA expression was associated with higher IL-8 levels in the culture supernatant and enhanced promoter activity. These data suggest that stretch contributes to the increase in myometrial IL-8 synthesis associated with the onset of labour in humans.

Insulin resistance and pre-eclampsia: a role for tumor necrosis factor-alpha?

Insulin resistance occurs in pre-eclampsia, but the cause is unknown. Furthermore, it is uncertain whether women destined to develop pre-eclampsia have a pre-existing insulin resistance or whether it is acquired with the development of the disease. We carried out this study to test the hypotheses that the increase in insulin resistance associated with pre-eclampsia is related to higher levels of tumor necrosis factor (TNF)-alpha, and that the increase in insulin resistance precedes the clinical onset of the disease. Fasting plasma samples were obtained from ten women who subsequently developed pre-eclampsia and ten normal pregnant controls at 16, 20, 24, 28, 32 and 36 weeks' gestation to measure circulating levels of insulin, glucose and TNF-alpha. Fasting insulin resistance index (FIRI) was calculated from insulin and glucose concentrations. In the normal controls, fasting insulin and TNF-alpha levels, and FIRI increased with gestation, and these were significantly greater than baseline values from 24, 28 and 28 weeks, respectively. In the group of women who developed pre-eclampsia, plasma levels of insulin and the FIRI were significantly higher than baseline from 20 and 24 weeks, respectively, but both were significantly higher than in the control group at 32 and 36 weeks. The increase in TNF-alpha in the pre-eclampsia group was significantly greater than in normal pregnant controls (p < 0.001). However, there was no significant association between TNF-alpha levels and FIRI in either normal pregnancy or pregnancies developing pre-eclampsia. These data suggest that insulin resistance in pre-eclampsia precedes the clinical onset of the disease, but that it is not related to elevated levels of TNF-alpha.

Mechanical stretch activates type 2 cyclooxygenase via activator protein-1 transcription factor in human myometrial cells.

The uterus is subject to stretch throughout pregnancy, which, in the presence of progesterone, is a potent stimulus for uterine growth. However, in the absence of progesterone or when stretch is excessive, as in multiple pregnancy, it may provoke the onset of labour. We have investigated the effect of stretch on prostaglandin synthesis in primary human uterine myocytes [non-pregnant (NP), pregnant not in labour (NL) and pregnant in labour (L)]. The cells were grown on flexible bottom culture plates and subjected to 1 or 6 h static stretch. Expression of type 2 cyclooxygenase (COX-2) mRNA was similar in samples obtained from NP and L groups and both were significantly greater than those found in the NL group. Stretch of cells from all groups resulted in increased COX-2 mRNA expression. In further studies carried out on cells taken from the NL group, 6 h of stretch resulted in increased COX-2 protein levels and, in the media, increases in prostaglandin (PG) I(2) metabolite and PGE(2) concentrations and a reduction in the concentration of PGF(2)alpha metabolites. After stretch, EMSA studies showed increased activator protein-1 (AP-1) nuclear protein DNA binding activity but not of nuclear factor kappaB. These data demonstrate that stretch of human myocytes results in increased COX-2 activity and suggest that this may occur through activation of the AP-1 system.

Impaired retrograde transmission of vasodilatory signals via the endothelium in pre-eclampsia: a cause of reduced tissue blood flow?

There is evidence that tissue blood flow is regulated by retrograde transmission of signals initiated at capillary and post-capillary sites, and transmitted via the endothelium to modulate pre-capillary resistance. We have used pre-eclampsia as a model to test the hypothesis that normal endothelium is required to enable adjustment of blood flow to match tissue requirements. Integrity of the endothelial pathway was assessed by measuring calf blood flow at increasing venous pressures, using an established small cumulative-step venous-congestion plethysmography protocol in ten women with pre-eclampsia, 17 normal pregnant controls and ten non-pregnant women. Endothelial cell activation was assessed by measuring plasma levels of the cell adhesion molecules, intercellular cell-adhesion molecule-1 (ICAM-1), vascular cell-adhesion molecule-1 (VCAM-1) and E-selectin. Baseline calf blood flow was significantly lower in pre-eclampsia than in the other two groups (P<0.0001; ANOVA). In the pre-eclampsia group, there was a fall in blood flow as venous congestion pressure was raised (P<0.0001; ANOVA). No such change was observed in the other two groups. A significant inverse correlation was observed between the reduction in blood flow in pre-eclampsia and the levels of E-selectin (r=-0.92, P=0.0002), VCAM-1 (r=-0.93, P=0.0008) and ICAM-1 (r=-0.86, P=0.001). The differences between the pre-eclamptic women and the other two groups support the notion that the failure to sustain blood flow during a cumulative pressure step protocol in the pre-eclamptic group might be influenced by interference with the retrograde transmission of signals via the endothelium in these patients.

Evidence of impaired microvascular function in pre-eclampsia: a non-invasive study.

The clinical presentation of pre-eclampsia suggests that microvascular dysfunction may play a role in the maternal manifestations of the disease. Isovolumetric venous pressure ( P V(i)) is an index of microvascular function, reflecting local plasma colloid osmotic (oncotic) pressure, and is abnormal in clinical conditions with microvascular dysfunction. We hypothesized that, in pre-eclampsia, post-capillary margination of neutrophils would increase post-capillary resistance, and therefore P V(i). A small cumulative step strain-gauge plethysmography protocol was used to compare P V(i) in 18 women with pre-eclampsia, 16 normal pregnant women and 17 non-pregnant controls. Circulating levels of vascular cell-adhesion molecule-1 (VCAM-1), intercellular cell-adhesion molecule-1 (ICAM-1) and E-selectin, and neutrophil elastase, were measured to assess endothelial and neutrophil activation respectively. P V(i) was significantly greater in the pre-eclampsia group, relative to the normal pregnant and non-pregnant controls ( P <0.001, ANOVA, for both comparisons). P V(i) was significantly lower during normal pregnancy compared with the non-pregnant controls ( P =0.001). Plasma levels of neutrophil elastase, VCAM-1, ICAM-1 and E-selectin ( P =0.001) were significantly greater in the pre-eclamptics than the controls. Significant positive correlations were observed between P V(i) and neutrophil elastase ( r =0.71, P =0.001), VCAM-1 ( r =0.52, P =0.03), ICAM-1 ( r =0.67, P =0.002), E-selectin ( r =0.69, P =0.001), uric acid levels ( r =0.54, P =0.02) and haematocrit ( r =0.64, P =0.004) in pre-eclampsia. The relationship with the platelet count was negative ( r =-0.65, P =0.003). No significant correlations were observed between P V(i) and maternal age, gestational age, total protein, albumin, diastolic blood pressures, age, body mass index and infant birth mass in the normal pregnant and non-pregnant controls. These data suggest that microvascular dysfunction occurs in pre-eclampsia, and that it is related to alterations in endothelial cell and neutrophil activation.

The relationship between insulin and insulin-like growth factor binding protein-1 is modified by pre-eclampsia.

Insulin is the main negative regulator of insulin-like growth factor binding protein-1 (IGFBP-1) in the non-pregnant state. Although changes in insulin resistance and circulating level of IGFBP-1 occur in pre-eclampsia, little is known about the relationship between insulin and IGFBP-1 in pregnancies complicated by the disease. In this study, we have investigated whether the relationship between insulin and IGFBP-1 is modified by pre-eclampsia. Maternal levels of insulin and IGFBP-1 were measured, at 4-weekly intervals between 16 and 36 weeks' gestation, in plasma samples obtained from ten normal pregnant controls and ten women who developed pre-eclampsia. The controls were chosen to be similar in maternal age and booking body mass index to the pre-eclampsia group. Insulin levels increased in both the normal controls and the women who developed pre-eclampsia. The levels in pre-eclampsia were significantly greater than those in normal pregnancy at 32 and 36 weeks' gestation (p = 0.02 and 0.005, respectively). IGFBP-1 levels were unchanged in normal pregnancy and rose in pre-eclampsia. In normal pregnancy, insulin levels were inversely related to IGFBP-1 levels throughout. In women developing pre-eclampsia, the relationship between insulin and IGFBP-1 was negative at 16 weeks and positive from 24 weeks. These data suggest that whereas the inverse relationship between insulin and IGFBP-1 is maintained during normal pregnancy, this relationship is reversed in women who develop pre-eclampsia.

Does endothelial cell activation occur with intrauterine growth restriction?

It is possible that in fetal growth restriction without pre-eclampsia endothelial cell activation does not occur. This might be either because there is no release of 'factor X' or because of maternal resistance to its effects. To test this hypothesis, we took blood samples from 26 women with pre-eclampsia (without fetal growth restriction), 13 women with fetal growth restriction (without pre-eclampsia) and 24 normal pregnant controls, and measured the circulating levels of three markers of endothelial cell activation (soluble VCAM, ICAM and E-selectin) and three cytokines [tumour necrosis factor-a (TNF-alpha), interleukin-6 (IL-6) and -8 (IL-8)]. The levels of the markers of endothelial cell activation were raised in both pre-eclampsia and fetal growth restriction pregnancies compared with controls; however, the levels of TNF-alpha, IL-6 and IL-8 were significantly raised in pregnancies complicated by pre-eclampsia, but not in fetal growth restriction, compared with controls. These data show that endothelial cell activation is common to both pre-eclampsia and fetal growth restriction, but that the circulating levels of cytokines are elevated only in pre-eclampsia. Thus, it seems likely that endothelial cell activation is a consequence of a failure of trophoblast invasion and that a further step is required, possibly involving cytokine release, for the expression of the full clinical picture of pre-eclampsia.