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Study hypothesis Steroid receptor coactivator interacting protein (SIP/KANK2) is involved in regulating the expression of the prostaglandin (PG)-endoperoxide synthase 2 (PTGS2; also known as cyclo-oxygenase 2, COX2) and PG release in human myometrium. Study finding SIP is phosphorylated in myometrial cells in response to epidermal growth factor (EGF)-stimulation and is required for EGF-stimulated increases in COX2 expression, PGE2 and PGF2α release, and expression of interleukins (IL) 6 and IL8. What is known already Human parturition involves inflammatory and non-inflammatory pathways and requires activation of the intrauterine PG cascade. A key mediator of uterine PG production is the highly inducible enzyme COX2. Regulation of COX2 expression is complex, and novel factors involved in its induction may play an important role during labour. The expression and function of SIP in uterine tissues has never been investigated. Study design, samples/materials, methods Mass spectrometry was used to identify SIP from cultured primary myometrial cells, and its expression in fresh placenta, fetal membranes, decidua and myometrium from pregnant and non-pregnant women was determined by western blotting. SIP expression in myometrial cells was reduced using small interfering RNA (siRNA), and COX2 expression was stimulated with EGF. COX2, IL6 and IL8 mRNA and COX2 protein expression were measured using quantitative RT-PCR (RT-qPCR) and western blotting respectively, and release of PGE2 and PGF2α by enzyme immunoassay. The time course and dose response of SIP phosphorylation in response to EGF were determined, and phosphorylation was measured in the presence of the mitogen-activated protein kinase kinase 1(MEK1) inhibitor PD-184352. Fresh myometrial tissue was used to confirm effects of EGF and MEK1 inhibition on SIP phosphorylation and COX2 expression. A profile of transcription factor (TF) activity after SIP knockdown was carried out using a commercially available array. Main results and the role of chance We have demonstrated expression of SIP in human myometrium. siRNA-mediated knockdown of SIP resulted in decreased EGF-stimulated COX2 protein expression (P2 (P2α (PPPIL6 and IL8 mRNA in a SIP-dependent manner (both PPP Limitations, reasons for caution While we describe a new role for myometrial SIP, we are yet to determine whether SIP phosphorylation is required for its effects on regulating COX2 expression and PG release. Our data are from in vitro studies using fresh tissue and cultured myometrial cells at passage 7 therefore may not fully reflect the conditions in vivo. Wider implications of the findings Our group has previously described increases in myometrial COX2 expression with labour at term and preterm. EGF levels rise in the amniotic fluid near term suggesting it may participate in paracrine signalling events, altering gene expression in the myometrium. Our novel data describe a role for SIP in regulating EGF-stimulated expression of myometrial COX2 and PG release. Moreover, our profile of SIP-dependent TF activation provides a platform for further investigations into additional roles for SIP in uterine function. These findings may facilitate the development of new, targeted drugs for the management of labour. Large scale data: Not applicable. Study funding and competing interest(s) This work was supported by an Action Medical Research grant (SP4612). The authors have no competing interests to declare.

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 H 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 mnol/l) induced contractions were measured and compared. Expression of RhoA, ROCK I and ROCK If 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.

The molecular mechanisms regulating human uterine quiescence and parturition are poorly understood. Potassium channels are central to regulation of cell membrane potential and contractility of smooth muscle. The aim of this study was to examine the expression of ATP-sensitive potassium channel (K-ATP channel) subunits in human myometrium, and to investigate for possible differential expression of these subunits in myometrium obtained from three different functional states: (i) non-pregnant (NP); (ii) late pregnant not in labour (PNL); and (iii) late pregnant in labour (PL). RT-PCR detected the presence of mRNA for four subunits of K-ATP channels (Kir6.1, Kir6.2, SUR1 and SUR2B) in the three tissue types. Quantitative analysis of these subunits was achieved with real-time RT-PCR using Lightcycler(TM) technology. This analysis showed that there were significantly higher levels of Kir6.1 and SUR2B transcripts in NP myometrium compared with those measured in myometrium obtained during pregnancy (P < 0.001). Lower levels of Kir6.2 and SUR1 mRNA expression were found, although higher transcript levels in NP myometrium, (P < 0.05) were still observed. Our results indicate that the major K-ATP channel expressed in human myometrium. is composed of Kir6.1 and SUR2B, and that down-regulation of this channel may facilitate myometrial function during late pregnancy.

The in vitro effect of progesterone and oestrogens on the spontaneous and oxytocin-induced activity of the pregnant and non-pregnant human myometrium was studied. Oestrogens as well as progesterone blocked the spontaneous and oxytocininduced contractions 10–20 min after the steroids were added to the bath. The inhibitory effect was dose-dependent and reversible. Oestradiol-17β and diethylstilboestrol were approximately 10 times more active than progesterone. The possible mechanism of action of oestrogens and progesterone are discussed.

The aim of the present investigation was to obtain further knowledge about the concentration and distribution of progesterone and 20α-dihydroprogesterone in the human myometrium during various stages of pregnancy. For the quantitative determination of these steroids in the myometrium a method was used which consisted of the following main steps: (a) addition of radioactive tracers to the plasma or tissue homogenate (b) precipitation of protein using ethanolether (c) extraction with ethyl acetate (d) paper chromatography (e) reduction of progesterone using crystalline 20β-hydroxysteroid dehydrogenase (f) chloroacetylation of the 20β-reduced progesterone and of 20α-dihydroprogesterone (g) quantitative estimation by gas-liquid chromatography using electron capture detection. With this method the overall recovery for progesterone from the uterine tissue was 36.0 ± 7% and 42.6 ± 6% for 20α-dihydroprogesterone. The precision of the entire method varied between 6 and 8% in the quantitative range of 0.1-3.6 μg of progesterone per sample. The specificity of the method was adequate and it was found that the total progesterone content could be extracted from the uterine muscle by the method. Progesterone was identified and quantitated in pooled extracts of 274.6 g of human myometrium obtained during 74 Caesarian sections in the last month of pregnancy. In these extracts the mean progesterone concentration was 0.16 ± 0.06 μg per g wet tissue. The identification of progesterone was established by the following criteria: (a) ultraviolet absorption (b) behaviour in paper- and thin-layer chromatography (c) retention times in gas-liquid chromatography with argon ionisation and electron capture detectors (d) formation of derivatives after enzymatic reduction with 20β-hydroxysteroid dehydrogenase (e) proof of radiochemical purity of the 14C-labelled derivative of progesterone. Results of 84 individual progesterone determinations in the myometrium of 54 pregnant uteri showed that the mean progesterone concentrations remain fairly constant during pregnancy. During the 10th-19th week of pregnancy the mean progesterone concentration was 0.111 ± 0.081 μg per g (26 determinations), during the 33rd-38th week 0.126 ± 0.097 μg per g (23 determinations) and during the 39th-42nd week 0.124 ± 0.073 μg per g of myometrium (35 determinations). The location of the placenta was not considered in these calculations. 20α-dihydroprogesterone was identified tentatively in extracts of 27.2 g of myometrium obtained from 7 uteri at the end of pregnancy. The identification of 20α-dihydroprogesterone was based on the following criteria: (a) behaviour of the free and the chloroacetylated substance in paper- and thin-layer chromatography (b) 20α-oxidation of the compound using placental 20α-hydroxysteroid dehydrogenase (c) 20β-reduction of the 20α-oxidized compound (d) formation of the chloroacetate of the 20β-reduced material (e) retention times of the formed 20α- and 20β-dihydroprogesterone chloroacetates in gas-liquid chromatography. The concentration of 20α-dihydroprogesterone in pooled myometrial extracts of early pregnancy (13th week) was 0.005 μg per g and in pooled extracts of myometrium at term (40th-42nd week) 0.009 μg per g wet tissue. 20β-dihydroprogesterone was identified tentatively in 210 g of tissue from a uterus in the 14th week of gestation. The mean 20β-dihydroprogesterone concentration in this myometrium was 0.00028 μg per g wet tissue. The identification of 20β-dihydroprogesterone was based on the following criteria: (a) behaviour of the free and the chloroacetylated substance in paper- and thin-layer chromatography (b) 20β-oxidation of the substance with 20β-hydroxysteroid dehydrogenase (c) 20β-reduction of the 20β-oxidized substance using 20β-hydroxysteroid dehydrogenase (d) formation of the chloroacetate of the 20β-reduced material (e) retention times of the formed 20β-dihydroprogesterone chloroacetates in gas-liquid chromatography. Comparative determinations of progesterone in the peripheral venous blood in the myometrium of 34 patients showed that the ratio of progesterone concentration in the myometrium versus the concentration in plasma decreases significantly during various stages of pregnancy. The progesterone concentration in early pregnancy (13th-19th week) is about 3 times higher in the myometrium than in the peripheral plasma. During late pregnancy (33rd-42nd week) the progesterone concentrations between the myometrium and peripheral plasma are to some extent adjusted. The concentrations of progesterone in placental and antiplacental sections of the uterus changed during pregnancy. In early pregnancy (10th-16th week) the mean progesterone concentration in placental sections of the uterus was 0.145 ± 0.056 μg per g and in antiplacental sections 0.059 ± 0.019 μg per g of myometrium (11 uteri). In the last month of pregnancy the mean progesterone concentration in the placental parts of the uterus was 0.167 ± 0.035 μg per g and in the antiplacental parts of the uterus 0.121 ± 0.033 μg per g of myometrium (7 uteri). During labour (40th-42nd week) the mean progesterone concentration in the placental sites of the uterus was 0.133 ± 0.068 μg per g and in the antiplacental sites 0.104 ± 0.031 μg per g of myometrium (12 uteri). The statistical analysis of these data showed no significant difference in the myometrial progesterone concentration at the placental site, whereas the progesterone concentration of the myometrium at the antiplacental site increased significantly during pregnancy (P < 0.01). Quantitative determinations of progesterone and 20α-dihydroprogesterone in the different sections of a whole uterus in the 14th week of gestation showed that the concentration of these two steroids in the myometrium is dependent on the location of the placenta. The sections of this uterus overlying the placenta have a 2-3 times higher progesterone- and 20α-dihydroprogesterone concentration than the myometrium sections located opposite to the placenta. Between the concentrations of progesterone and 20α-dihydroprogesterone an equal relationship is adjusted in the various sections of the uterus during the 14th week of gestation. On an average, the ratio of progesterone to 20α-dihydroprogesterone was 16:1. The statistical evaluation showed that the progesterone concentration in the myometrium during the 14th week of gestation significantly decreases with the distance from the centre of the placenta (P < 0.001). These investigations show that there is an asymmetrical distribution of progesterone and 20α-dihydroprogesterone in the uterus during early pregnancy. Determination of the progesterone concentration in the internal and external layer of the uterus during the 14th week of gestation indicated that the internal layer contains more progesterone than the external layer. At the placental site the mean ratio of the progesterone concentration in the internal layer as against the concentration in the external layer of the myometrium was 1.5:1 and at the antiplacental site 1.3:1. This difference was not statistically significant. With regard to the progesterone concentration in the myometrium our results demonstrate that the uterus changes from an asymmetrical system in early pregnancy towards a more symmetrical system with a rather homogenous distribution of progesterone in the myometrium near term.