The roles of the cyclo-oxygenases types one and two in prostaglandin synthesis in human fetal membranes at term.

UNLABELLED: The aim of this study was to determine the relative contributions of cyclo-oxygenase (COX) types 1 and 2 to prostaglandin synthesis at term. METHODS: Fetal membranes were collected from 6 pregnancies after elective caesarean section at term, prior to labour. The presence of COX-1 and COX-2 protein was determined using Western analysis. The relative contributions of the two isoforms of COX to prostaglandin synthesis were determined by incubation of fetal membrane discs with either a COX-2 selective inhibitor, SC236, or a COX-1 selective inhibitor, SC560, and measurement of prostaglandin release during 24 h using enzyme-linked immuno-sorbent assay (ELISA). RESULTS: Both COX-1 and COX-2 protein were demonstrated in amnion and chorion-decidua. The COX-2 selective inhibitor, SC-236, significantly reduced prostaglandin synthesis, both in its COX-2 specific and higher, non-specific concentration ranges. The COX-1 selective inhibitor, SC-560, had no effect upon prostaglandin synthesis in its COX-1 specific concentration range, but did significantly reduce prostaglandin synthesis at higher, non-selective concentrations. CONCLUSIONS: Fetal membranes contain both COX-1 and COX-2 at term, but only COX-2 contributes towards prostaglandin synthesis. COX-2 selective NSAI drugs will be as effective as non-selective agents in inhibition of fetal membrane prostaglandin synthesis and may represent a new strategy for tocolysis.

Expression of nitric oxide synthase isoforms in pregnant human myometrium.

1. Endogenous nitric oxide has been proposed to play a role in the control of myometrial contractility in pregnancy. In this study, the expression, localisation and regulation of nitric oxide synthase (NOS) isoforms have been examined in human pregnant myometrium and cultured human myometrial smooth muscle cells, by immunoblotting, immunohistochemistry and reverse transcription-polymerase chain reaction. 2. Immunoblotting of extracts from freshly isolated myometrial tissue, affinity-enriched for NOS proteins by precipitation with ADP-sepharose, revealed expression of endothelial NOS (eNOS or NOS3) in tissues from preterm, term non-labour and active labour at term. Inducible NOS (iNOS or NOS2) and neuronal NOS (nNOS or NOS1) proteins were not detected at any stage of pregnancy. 3. Immunohistochemical detection showed that expression of eNOS protein was restricted to the endothelium of the myometrial vasculature, with no staining detected in myometrial smooth muscle cells. 4. Messenger RNA for all three NOS isoforms was detected, although iNOS and nNOS mRNAs were detectable only with high cycle number, implying a low copy number. 5. NOS isoforms were not detectable in human myometrial smooth muscle cells cultured from term non-labour pregnancies. Cytokine stimulation of cultured myometrial cells did not induce iNOS expression or nitrite accumulation in the culture medium, although both iNOS protein and nitrite release were detected in the human pulmonary epithelial cell line A549. 6. Levels of eNOS protein and of NOS mRNA expression were not correlated with gestational stage, suggesting that endogenously produced NO is not likely to be a modulator of myometrial tone during human pregnancy.

Expression of cyclo-oxygenase types-1 and -2 in human myometrium throughout pregnancy.

Human labour is associated with increased prostaglandin synthesis within the uterus. The aim of this study was to examine the expression of the two isoforms of the central prostaglandin synthetic enzyme, cyclo-oxygenase (COX-1 and COX-2) in human myometrium throughout pregnancy and to test the hypothesis that COX in the myometrium may play a role in labour onset. Expression of COX-1 and COX-2 at the mRNA level was analysed using reverse transcriptase-polymerase chain reaction (RT-PCR) and at the protein level using Western blotting. No significant changes of COX-1 RNA or protein expression were observed either with gestational age or labour. COX-2 mRNA and protein expression increased at term with significant up-regulation occurring prior to the onset of labour (P < 0.005). These data would suggest that up-regulation of COX-2, rather than COX-1, mediates increased prostaglandin synthesis in human myometrium at term. The increased COX-2 expression observed preceded labour onset, suggesting that COX-2 has a role in labour onset, rather than its presence merely a consequence of labour.

Region and labour-dependent synthesis of prostaglandin E2 by human fetal membranes.

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.

Myometrial nitric oxide synthase messenger ribonucleic acid expression does not change throughout gestation or with the onset of labor.

OBJECTIVE: The purposes of this study were to examine expression of nitric oxide synthase isoforms in human myometrium and to determine any changes in expression with gestational age and with the onset of labor at term. STUDY DESIGN: Myometrial samples were collected from patients undergoing cesarean delivery at term before and after the onset of labor (n = 17) and throughout gestation (n = 13). Expressions of inducible, calcium-independent nitric oxide synthase and constitutive, calcium-dependent endothelial nitric oxide synthase were determined by semiquantitative reverse transcriptase-polymerase chain reaction. RESULTS: Messenger ribonucleic acid for inducible, calcium-independent nitric oxide synthase and constitutive, calcium-dependent endothelial nitric oxide synthase is expressed in human myometrium at term and throughout the second and third trimesters. Levels of messenger ribonucleic acid for both inducible, calcium-independent nitric oxide synthase and constitutive, calcium-dependent endothelial nitric oxide synthase do not change with either gestational age or the onset of labor. CONCLUSION: Changes in myometrial nitric oxide synthase expression and thus of levels of endogenous nitric oxide are unlikely to be directly involved in myometrial quiescence or the onset of human parturition.

Nitric oxide synthase mRNA expression in human fetal membranes: a possible role in parturition.

Nitric oxide (NO) is a potent endogenous smooth-muscle relaxant. It is synthesised from 1-arginine by isoforms of nitric oxide synthase (NOS). Whilst it is clear that the uterus responds to NO by relaxation, NOS expression has not been investigated in fetal membranes or myometrium in human pregnancy. This study has shown, using semi-quantitative RT-PCR, expression of cNOS mRNA in human amnion, chorion-decidua, and placenta. iNOS mRNA expression was demonstrated in human amnion, chorion-decidua, and placenta. It is possible that NO synthesised in fetal membranes may act either directly to inhibit myometrial contractility or indirectly to interact with other labour-associated genes, such as cyclo-oxygenase, to coordinate the onset of labour.