In vitro stimulation of Th2 cells by PGD2 requires much higher concentrations to stimulate IL-10 production compared with IL-4, IL-5 and IL-13.[22, 1] We therefore examined the effect of Pyl A on the Th2-type anti-inflammatory cytokines in the myometrium (Fig. 8). Although no changes in levels of IL-4 were detected, an

increase (non-significant) in IL-5 was observed (Fig. 8). Moreover, a non-significant increase in IL-10 mRNA and protein with LPS and Pyl A treatment was detected consistent with improved protection against LPS-induced fetal loss in mice[65] as well as the reduced rate of naturally occurring fetal loss in IL-10-deficient mice.[24] Although Pyl A led to a small increase in the pro-labour transcription factor NF-κB and the pro-inflammatory cytokines, we did not see an increase in COX-2 protein expression. We therefore examined the direct effect of Pyl A on myometrial contractility ex vivo. Contrary to the expected MAPK Inhibitor Library in vitro uterotonic effect, Pyl A administration resulted in complete inhibition of circular muscle contractility (Fig. 9), but had no effect on longitudinal

muscle. There is limited knowledge on the functional role of the individual muscle layers of the mouse uterus, the inner circular and outer longitudinal muscle, in pregnancy and parturition. In the myometrium of other species such as the pig and rat, it has been suggested that the function of the longitudinal muscle is to move luminal contents by contraction[66] and that tonic contraction of the circular muscle may be required for spacing and retention of embryos/fetuses.[67] Circular muscle cells have a higher spontaneous see more electrical activity than longitudinal muscle cells during rat pregnancy,[68] and weak high-frequency

contractions in the circular muscle layer prevent movement of fetuses Etomidate towards the cervix during pregnancy,[69] supporting its potential role in the maintenance of pregnancy. If circular muscle contraction is necessary for retention of uterine contents, this would explain how inhibition of circular muscle contraction by Pyl A leads to preterm expulsion of the fetuses, as seen in this study. Consistent with this, relaxation of uterine tone is also believed to be important during human labour.[70] It is proposed that relaxation of the lower segment of the uterus, in conjunction with contractions of the fundal region, is required for the passage of the fetus through the birth canal. Alternatively, relaxation of circular muscle may not be important in murine labour. Many rodent studies suggest that by term, the function of circular muscle becomes more similar to the longitudinal layer, and that contractility of both the circular and longitudinal muscle is required for labour.[71-74] It is possible that despite the inhibitory effect on contractions seen with Pyl A ex vivo, that the overwhelming in vivo inflammatory effect was enough to overcome the tocolytic effect resulting in preterm labour.