Expression of gastrin-releasing peptide is increased by prolonged stretch of human myometrium, and antagonists of its receptor inhibit contractility.

Increased uterine stretch appears to increase the risk of preterm labour, but the mechanism is unknown. The aim of this study was to identify factors that mediate the effect of stretch on human myometrium.Myometrial explants, prepared from biopsies obtained at elective caesarean delivery, were either studied acutely, or were maintained in prolonged culture (up to 65 h) under tension with either a 0.6 g or a 2.4 g mass, and compared using in vitro contractility, whole genome array, and qRT-PCR. Tissue held at tonic stretch with the 2.4 g mass for either 24 or 65 h showed increased potassium chloride (KCl)-induced and oxytocin-induced contractility compared with that held with the 0.6 g mass. Gene array identified 62 differentially expressed transcripts after 65 h exposure to increased stretch. Two probes for gastrin-releasing peptide (GRP), a known stimulatory agonist of smooth muscle, were among the top five up-regulated by stretch (3.4-fold and 2.0-fold). Up-regulation of GRP mRNA by stretch was confirmed in a separate series of 10 samples using quantitative RT-PCR (qRT-PCR) (2.8-fold, P =0.01). GRP stimulated contractions acutely when added to freshly obtained myometrial strips in 2 out of 9 cases, but Western blot demonstrated expression of the GRP receptor in 9 out of a further 9 cases. Prolonged incubation of stretched explants in the GRP antagonists PD-176252 or RC-3095 (65 and 24 h, respectively) significantly reduced KCl- and oxytocin-induced contractility.Tonic stretch of human myometrium increases contractility and stimulates the expression of a known smooth muscle stimulatory agonist, GRP. Incubation of myometrium with GRP receptor antagonists attenuates the effect of stretch. GRP may be a target for novel therapies to reduce the risk of preterm birth in multiple pregnancy.

Effects of medroxyprogesterone acetate on gene expression in myometrial explants from pregnant women.

CONTEXT: Progesterone is important physiologically and therapeutically to maintain uterine quiescence during pregnancy, in part through controlling myometrial gene expression. OBJECTIVE: The objective of the study was to use expression microarray and quantitative reverse transcriptase-PCR (qRT-PCR) validation to determine the changes in gene expression induced by prolonged exposure of human myometrium to a synthetic progestogen. DESIGN: Myometrial explants, obtained at elective cesarean section (n=9), were maintained in culture, under 0.6 g tension, for 65 h in the presence of medroxyprogesterone acetate (100 nm) or vehicle. Expression array was performed using Illumina beadchip arrays. Approximately 30% of differentially expressed transcripts were validated in biological replicates (n=10) by qRT-PCR. RESULTS: The 114 significantly regulated transcripts were significantly enriched in inflammatory response (P=0.00001), growth factor activity (P=0.0004), and cytokine activity genes (P=0.008). Thirty-four transcripts were validated using qRT-PCR in explants obtained from 10 further women. There was very close agreement in the fold changes obtained by array and qRT-PCR (r2=0.9, P<0.0001). We confirmed significant down-regulation of a number of genes that have been well characterized as progesterone sensitive (IL-1B, IL-6, PTGS2, and GJA1). However, the top and sixth most down-regulated transcripts encoded two cytokines, IL-11 and IL-24, respectively, not previously implicated in mediating the effects of progesterone in myometrium. Both were validated by qRT-PCR (4.3- and 2.2-fold down-regulated, both P<0.001). CONCLUSIONS: Medroxyprogesterone acetate controls expression of multiple genes in myometrium, including many that have not previously been characterized as progestogen regulated in this tissue, including IL-11 and IL-24. It is plausible that proteins encoded by some of these genes may have important but as yet uncharacterized effects in controlling human parturition.

Pro-labour myometrial gene expression: are preterm labour and term labour the same?

Preterm labour (PTL) is the most important cause of neonatal morbidity and mortality. While some causes have been identified, the mechanisms involved remain elusive. This study investigates whether term labour (TL) is an appropriate model for PTL by examining pro-labour gene expression, using quantitative rtPCR, and protein synthesis, using Western analysis, in preterm and term myometrial samples obtained from the upper and lower uterine segments before and after the onset of labour. In the lower segment, the levels of prostaglandin H synthase type-2 (PGHS-2), interleukin-1beta (IL-1beta), IL-6 and IL-8 mRNA expression were significantly higher in TL compared with PTL samples. Compared with non-labour controls, the expression of IL-1beta and IL-8 mRNA was increased in both PTL and TL samples and the expression of PGHS-2 and IL-6 mRNA was increased in TL samples only. In the upper segment, there were no differences between PTL and TL samples and the mRNA expression of PGHS-2 and IL-1beta was increased in TL compared with term no labour samples. No effect of PTL or TL was seen on either oxytocin receptor or connexin-43 mRNA expression or protein levels. The multiple regression analysis and studies in primary cultures of uterine myocytes suggest that the inflammatory cytokines, IL-1beta and tumour necrosis factor-alpha, are the most important regulators of PGHS-2 and IL-8. Our data show that preterm and term labouring myometrium are significantly different and that the most marked labour-induced changes in gene expression are in the lower segment. These changes may occur in response to the release of inflammatory cytokines by the labour-associated inflammatory infiltration.