Expression of cyclo-oxygenase in human endometrium during the implantation period.

Prostaglandins (PG) are known to be involved in the process of human implantation. In several animal species treatment with prostaglandin synthesis inhibitors will prevent implantation. Cyclo-oxygenase (COX) is the rate-limiting enzyme in the synthesis of PGs and exists in two isoforms, COX-1 and COX-2. Defective implantation in COX-2-deficient mice has been demonstrated recently. In the present study, the expression of COX-1 and COX-2 was studied during the implantation period in healthy fertile women before and after treatment with the antiprogesterone, mifepristone. The study consisted of one control cycle and one treatment cycle. The subjects served as their own control. During the treatment cycle the subjects received 200 mg of mifepristone 2 days after the luteinizing hormone (LH) surge. Endometrial biopsies were obtained in the mid-luteal phase (LH + 6 to LH + 8) in both cycles. Using polyclonal antibodies against COX-1 and COX-2, immunostaining for COX-1 was found mainly in the glandular and luminal epithelium and for COX-2 mainly in the luminal epithelium and the perivascular cells. After treatment with mifepristone, expression of COX-1 in glandular epithelium and COX-2 in luminal epithelium significantly decreased whilst the immunostaining for COX-2 in the perivascular cells remained strong. This study shows the expression of both COX-1 and COX-2 during the implantation period and also indicates that treatment with mifepristone in early luteal phase impairs glandular epithelial function and endometrial receptivity.     

Expression of 5-lipoxygenase and 5-lipoxygenase-activating protein in human fetal membranes throughout pregnancy and at term.

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.     

Expression and modulation of Rho kinase in human pregnant myometrium

There is little information outlining the role of Rho kinase, RhoA, and calcium sensitization in regulation of human uterine contractility during pregnancy. The aims of this study were to investigate the expression of RhoA, and the Rho kinases ROCK I and ROCK II in human pregnant myometrium, to evaluate the effects of Rho kinase inhibition on pregnant human myometrial contractility in vitro, and to compare these effects with those of the calcium channel blocker nifedipine. RT-PCR using primers for RhoA, ROCK I and ROCK II was performed on mRNA isolated from human pregnant myometrium. Isometric recording was performed in isolated myometrial strips obtained at Caesarean section. The effects of the Rho kinase inhibitor Y-27632 (1 nmol/l to 10 mmol/l), and nifedipine (1 nmol/l to 10 mmol/l), on oxytocin (0.5 nmol/l) induced contractions were measured and compared. Expression of RhoA, ROCK I and ROCK II mRNA was identified in human pregnant myometrium (n = 3). Y-27632 exerted a potent relaxant effect on myometrial contractility with a pD(2) value (+/- SEM) of 7.63 +/- 0.38 (n = 6). The maximum net relaxant effect (+/- SEM) was 72.3 +/- 6.1% (n = 6). Corresponding values for nifedipine were 7.24 +/- 0.48 (n = 6; P = 0.469) and 93.40 +/- 3.1% (n = 6; P = 0.028). Rho A/Rho kinase-mediated calcium sensitization may play role in the physiology of human parturition, and pharmacological inhibition of this pathway may therefore provide a novel approach to tocolysis for pre-term labour.     

Identification and localization of prostaglandin E2 receptors in upper and lower segment human myometrium during pregnancy

Mol. Hum. Reprod.     

Expression of superoxide dismutase and xanthine oxidase in myometrium, fetal membranes and placenta during normal human pregnancy and parturition

Superoxide, an agent which attenuates the half-life of nitric oxide, is metabolized and synthesized by superoxide dismutase (SOD) and xanthine oxidase, respectively. Over the last few years much work has focused on the role of nitric oxide in human parturition. The aim of this study was to determine whether the onset of human parturition is associated with a change in the expression of copper/zinc superoxide dismutase (Cu/Zn SOD), manganese superoxide dismutase (Mn SOD) or xanthine oxidase within the uterus. Samples of myometrium, placenta, decidua and fetal membranes were obtained from women before and after the onset of labour at term. Immunocytochemistry was used to localize Cu/Zn SOD, Mn SOD and xanthine oxidase and measure SOD enzyme activity. Cu/Zn and Mn SOD-like immunoreactivity was detected in syncytiotrophoblast cells, villous stromal cells and endothelial cells of blood vessels in the placenta. In the myometrium Cu/Zn and Mn SOD were localized to myocytes and endothelial cells and to some vascular smooth muscle cells. In the fetal membranes we observed staining for Cu/Zn SOD and Mn SOD in the amnion, chorion, extravillous trophoblast and decidua. There was no difference in SOD enzyme activity or staining intensity for SOD between different cell types before and during labour. Xanthine oxidase immunoreactivity was identified in each of the tissues examined and again there was no difference in immunostaining in tissues obtained from women delivered before or after the onset of labour. These results show that the pregnant uterus is capable of both synthesizing and degrading superoxide and suggest that superoxide dismutase and xanthine oxidase may play a role in the maintenance of uterine quiescence during pregnancy, but not in the initiation of parturition.      

Identification and localization of prostaglandin E2 receptors in upper and lower segment human myometrium during pregnancy

Prostaglandin E2 (PGE2) plays a key role in the maintenance of human pregnancy and labour onset. PGE2 can elicit diverse actions within the uterus depending on the PGE2 receptors (EP1, EP2, EP3 and EP4) expressed. By signalling through different intracellular pathways the EP receptors may inhibit or promote smooth muscle contractility. Nine different EP3 receptor splice variants have been identified with divergent signalling pathways. RT-PCR and immunohistochemistry were utilized to identify and localize EP receptor isoforms within the upper segment (US) and lower segment (LS) myometrium. EP1 was significantly increased in the LS myometrium with term labour. EP3 (and EP3 splice variants EP3I(1b), EP3II, EP3III and EP3IV) was down-regulated in pregnancy (US and/or LS) with a further decrease at term labour in the LS. Overall, expression of EP2 was significantly higher in the LS while EP3 was significantly higher in the US. No significant EP4 changes were observed. Consistent with the RT-PCR results, immunohistochemistry confirmed the presence and, interestingly, showed nuclear localization of EP receptors in the myometrium with higher EP1 expression and lower expression of EP3. The differential regulation of EP receptors within the myometrium indicates that they may play a role in controlling the onset and maintenance of human labour.     

Neuropeptide Y and noradrenaline in human uterus and myometrium during normal and pre-eclamptic pregnancy

The myometrial levels of noradrenaline (NA) and neuropeptideV (NPY), a recently discovered neuropeptide that coexists withNA in many sympathetic nerves, have been measured by immunohistochemistryand biochemistry in normal and pre-eclamptic pregnancy and comparedwith non-pregnant controls. In the non-pregnant uterus therewere high levels of NA and NPY and a rich presence of NA/NPYnerves in the myometrium and around blood vessels. In pregnancyboth substances were significantly lower as compared with non-pregnantwomen. Five patients with pre-eclampsia had levels of NA/NPYthat were less markedly reduced than in normal pregnant women.The results show that NA and NPY coexist also in human uterinenerves, and that both decrease significantly during pregnancy.Since both substances are vasoactive, the observed reductionmight be of physiological importance for normal pregnancy.     

Expression and regulation of prostaglandin E synthase isoforms in human myometrium with labour

Since the controversies regarding the use of non-steroidal anti-inflammatory drugs (NSAIDs) and selective cyclo-oxygenase (COX)-2 antagonists for the treatment of preterm labour (PTL), more emphasis has been placed on investigating the terminal synthases involved in the production of prostaglandins (PGs) to allow more targeted therapy in PTL. Prostaglandin E(2) (PGE(2)) is synthesized by one of three enzymes, cytosolic prostaglandin E synthase (cPGES), microsomal PGES-1 (mPGES-1) and microsomal PGES-2 (mPGES-2). We have determined (i) the immuno-localization of all three PGES enzymes in lower segment pregnant human myometrium, (ii) the expression of PGES and COX-2 mRNA expression at term and preterm gestation with and without labour and (iii) the effect of interleukin (IL)-1beta on COX-2 and PGES mRNA and protein expression in human myometrial smooth muscle (HMSM) cell cultures. We show mPGES-1 protein located predominantly in myometrial and vascular smooth muscle cells (SMCs), whilst mPGES-2 protein is largely in stromal cells surrounding the SMC and cPGES is diffusely located throughout the myometrium. Expression of mPGES-2 mRNA increased with term labour and PTL and expression of COX-2 and mPGES-1 mRNA with term labour, whereas cPGES expression did not change. IL-1beta stimulated release of PGE(2) by HMSM cells and increased COX-2 and mPGES-1 mRNA and protein expression. Thus, COX-2 expression and mPGES-1 expression are co-ordinately up-regulated in lower segment myometrium with term labour and with IL-1beta treatment in HMSM cells.     

Changes in PGRMC1, a potential progesterone receptor, in human myometrium during pregnancy and labour at term and preterm

This study assesses the role of progesterone receptor membrane component 1 (PGRMC1) in actions of progesterone (P4) on human myometrium during pregnancy and labour. Myometrial tissues were obtained from non-pregnant patients during hysterectomy or pregnant women undergoing C-section at term and preterm, before and during labour. PGRMC1 expression in myometrial tissues and in a human myometrial cell line (HM9) was assessed by western blots and RT-PCR. The subcellular localization of PGRMC1 in HM9 was performed by immunofluorescence staining. Isometric contractions of myometrial tissues were obtained in response to P4 with and without addition of specific antibodies against PGRMC1. Endogenous and over-expressed PGRMC1 proteins are detected by western blots in myometrial tissues, HM9 and 293 cells, respectively. PGRMC1 is localized to the plasma membrane, cytoplasm and nuclear membranes. PGRMC1 is lower in myometrium of women at term either not in labour (P = 0.004) or in labour (P = 0.005) compared with tissues from women in preterm non-labour. PGRMC1 levels are also decreased (P = 0.02) in myometrial tissues from women during preterm labour compared with preterm non-labour. P4 rapidly inhibits contractions of myometrial tissues compared with control (P < 0.05) in vitro. Pretreatment of myometrial strips with PGRMC1 antibody, suppresses the P4-induced relaxation (P < 0.05). PGRMC1 may mediate the non-genomic action of P4 and the relaxation effect on human myometrium during pregnancy. A decrease in PGRMC1 during term or preterm labour might contribute to the 'functional withdrawal' of P4 action and shift the balance to a state of heightened uterine contractility.     

Prostaglandin E2 release in gastric antral mucosa of guinea-pigs: basal PGE2 release by cyclo-oxygenase 2 and ACh-stimulated PGE2 release by cyclo-oxygenase 1

Prostaglandin E(2) (PGE(2)), which is generated by two isoforms of cyclo-oxygenase (COX(1) and COX(2)), is a key mediator in gastric mucosal defense. In the present study, antral mucosa of guinea-pigs was incubated with various agonists or antagonists in a medium, the PGE(2) concentration of which was measured using a PGE(2) EIA kit. Prostaglandin E(2) was released from the antral mucosa spontaneously (basal PGE(2) release) and acetylcholine (ACh, 10 microM) enhanced the PGE(2) release (ACh-stimulated PGE(2) release) was mediated via intracellular Ca(2+) concentration ([Ca(2+)](i)). Arachidonic acid enhanced both forms of PGE(2) release, and a phospholipase A(2) inhibitor (amylcinnamoyl anthranilic acid) and COX inhibitors (acetylsalicylic acid and indomethacin) decreased them. 5-(4-Chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazol (SC560, 100 nm, a COX(1)-selective inhibitor) inhibited ACh-stimulated PGE(2) release without any decrease in basal PGE(2) release. N-(2-Cyclohexyloxy-4-nitrophenyl) methanesulphonamide (NS398, 20 microM, a COX(2)-selective inhibitor) decreased basal PGE(2) release without any reduction of ACh-stimulated PGE(2) release. However, ionomycin (a Ca(2+) ionophore) increased PGE(2) release from antral mucosa in the presence of SC560 or NS398, suggesting that COX(1) and COX(2) are regulated by [Ca(2+)](i). These findings indicate that COX(1)-containing cells have ACh receptors but COX(2)-containing cells do not. Moreover, in isolated antral epithelial cells, SC560 decreased basal and ACh-stimulated PGE(2) release, but NS398 did not. In conclusion, in antral mucosa, basal PGE(2) release is mainly maintained by COX(2) of non-epithelial cells, and ACh-stimulated PGE(2) release is maintained by COX(1) of epithelial cells.     

Expression of CD69 activation marker by endometrial granulated lymphocytes throughout the menstrual cycle and in early pregnancy.

Flow cytometry studies of first-trimester normal human decidua have proposed that a proportion of endometrial granulated lymphocytes (eGL), the predominant leucocyte population in early human pregnancy, co-express CD69. The purpose of this study was to investigate CD69 expression by immunohistochemistry throughout the menstrual cycle and in first-trimester human decidua and to determine whether CD69 expression by eGL is an in vivo characteristic or whether the antigen is acquired in vitro after tissue disaggregation and cell purification. Single immunoenzymatic and double immunofluorescence labelling of tissue sections, supplemented with double immunoenzymatic staining of purified eGL and flow cytometry, indicated that eGL do not co-express CD69 in situ but that they acquire the molecule in vitro after cell purification and culture.