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Increasing evidence indicates that endoplasmic reticulum (ER) stress is involved in various diseases. In nongestational tissues, several markers of the unfolded protein response (UPR) have been shown to regulate the inflammatory response. Thus, the aim of this study was to determine the effect of human labor on markers of ER stress in fetal membranes and myometrium. In addition, the effect of ER stress inhibition on the expression and secretion of proinflammatory and prolabor mediators was also assessed. The markers of ER stress, GRP78, IRE1, and spliced XBP1 (XBP1s), were significantly increased in fetal membranes and myometrium after term and preterm labor compared to nonlaboring samples. Given that inflammation is considered to be one of the leading causes of spontaneous preterm birth, here we used bacterial endotoxin lipopolysaccharide (LPS) as a model for infection-induced preterm birth. In term nonlabored fetal membranes and myometrium, LPS induced UPR activation as evidenced by a significant increase in the expression of GRP78, IRE1, and XBP1s in fetal membranes and myometrium. The use of the chemical chaperones 4-phenylbutyric acid (4-PBA) and tauroursodeoxycholic acid (TUDCA) alleviated ER stress induced by LPS. 4-PBA and TUDCA also ameliorated the increase in LPS-induced prolabor mediators. Our data suggest that the UPR may regulate the inflammatory responses associated with labor or infection in fetal membranes and myometrium of pregnant term and preterm women. Thus, the use of ER stress inhibitors, in particular 4-PBA or TUDCA, may be a potential therapeutic strategy for the prevention of infection-mediated spontaneous preterm birth., (© 2014 by the Society for the Study of Reproduction, Inc.)

Premature delivery remains a serious risk factor in pregnancy, with currently licensed tocolytics unable to offer significant improvement in neonatal outcome. Further understanding of the regulators of uterine contractility is required to enable the development of novel and more effective tocolytic therapies. The transglutaminase family is a class of calcium-dependent, transamidating enzymes, of which tissue transglutaminase 2 is a multifunctional enzyme with roles in cell survival, migration, adhesion, and contractility. The aim of the present study was to investigate the role of this enzyme in regulating the contractility of pregnant human myometrium. Tissue strips from biopsy samples obtained at elective cesarean section were either allowed to contract spontaneously or induced to contract with oxytocin, phenylephrine, or bradykinin. Activity integrals, used to measure contractile activity, were taken following cumulative additions of the reversible, polyamine transglutaminase inhibitors cystamine and mono-dansylcadaverine and the irreversible, site-specific transglutaminase inhibitors N-benzyloxycarbonyl-l-phenylalanyl-6-dimethylsulfonium-5-oxo-L-norleucine and 1,3-dimethyl-2[(oxopropyl)thio]imidazolium. The ability of cystamine and mono-dansylcadaverine to affect oxytocin-mediated calcium mobilization within primary cultured myometrial cells was also measured utilizing a calcium indicator. All inhibitors attenuated myometrial contractions in a concentration-dependent manner independent of the method of contraction stimulus. Similarly cultured myometrial cells preincubated with cystamine and mono-dansylcadaverine displayed an altered calcium response to oxytocin stimulation. Our findings demonstrate a potential role for tissue transglutaminase 2 in regulating uterine contractility in pregnant human myometrium that may be associated with the calcium signaling cascade required for contraction.

11Beta-hydroxysteroid dehydrogenase (HSD11B) enzymes have important roles in regulating cortisol availability in target tissues. We previously demonstrated that HSD11B1 is expressed and active in bovine endometrium and that cortisol suppresses prostaglandin (PG) F2alpha and PGE2 production in cultured bovine endometrial stromal cells. The present study was conducted to examine whether locally synthesized PGF2alpha and/or PGE2 regulates the enzymatic bioactivity and/or the expression of HSD11B1 in bovine endometrium. The conversion rate of cortisone to cortisol in cultured endometrial stromal cells was significantly stimulated by PGF2alpha (1 and 10 microM). In a dose-dependent manner, PGF2alpha but not PGE2 increased the net conversion of cortisone to cortisol in stromal cells after 4 h of treatment. In addition, the bioactivity of HSD11B1 was significantly inhibited by indomethacin (10 microM). The inhibitory effect of indomethacin on HSD11B1 bioactivity was abolished by PGF2alpha (1 microM) but not by PGE2. Although PGF2alpha (1 microM) did not affect the expression of HSD11B1 mRNA in cultured stromal cells, it significantly stimulated the protein expression of HSD11B1. Cycloheximide, a general translational inhibitor, abolished the stimulatory effects of PGF2alpha on HSD11B1 protein expression in endometrial stromal cells, indicating that PGF2alpha increases HSD11B1 protein expression by stimulating a posttranscriptional process rather than a transcriptional mechanism. These results demonstrate that PGF2alpha but not PGE2 increases HSD11B1 bioactivity and protein expression by stimulating a posttranscriptional mechanism in stromal cells and suggest that cortisol has a physiologically relevant role in preventing excessive uterine PG production in nonpregnant bovine endometrium.

Term and preterm labor are associated with increased fetal hypothalamic-pituitary-adrenal (HPA) activation and synthesis of prostaglandins (PGs) generated through the increased expression of prostaglandin H synthase-II (PGHS-II) in the placenta. Inhibition of PGHS-II has been advocated as a means of producing uterine tocolysis, but the effects of such treatment on fetal endocrine functions have not been thoroughly examined. Because PGE(2) is known to activate the fetal HPA axis, we hypothesized that administration of meloxicam, a PGHS-II inhibitor, to sheep in induced labor would suppress fetal HPA function. Chronically catheterized pregnant ewes were treated with RU486, a progesterone receptor antagonist, to produce active labor, and then treated with either high-maintenance-dose meloxicam, graded-maintenance-dose meloxicam, or a saline infusion. Maternal uterine contraction frequency increased 24 h after the RU486 injection and the animals were in active labor by 48 +/- 4 h. RU486 injection led to increased concentrations of PGE(2), ACTH, and cortisol in the fetal circulation, and increased concentrations of 13,14 dihydro 15-ketoprostaglandin F(2 alpha) (PGFM) in the maternal circulation. Uterine activity was inhibited within 12 h of beginning meloxicam infusion at both infusion regimes. During meloxicam infusion there were significant decreases in fetal plasma PGE(2), ACTH, and cortisol concentrations, and PGFM concentrations in maternal plasma. In control animals, frequency of uterine contractions, maternal plasma PGFM, fetal plasma PGE(2), ACTH, and cortisol concentrations increased after RU486 administration, and continued to rise during saline infusion until delivery occurred. We conclude that RU486-provoked labor in sheep is associated with activation of fetal HPA function, and that this is attenuated during meloxicam treatment to a level considered compatible with pregnancy maintenance.

In the present study we have shown that the genetic expression of prostaglandin (PG)F(2alpha) receptor (R) and cyclooxygenase (COX)-2 increases in laboring rat myometrium. This finding was associated with a relatively weak contractile in vitro response (E:(max)) of isolated uterine strips when challenged with PGF(2alpha). Five days postpartum PGF(2alpha)-R mRNA values exceeded those during labor while COX-2 mRNA was reduced to preparturient values. Maximal contractility of isolated strips stimulated with PGF(2alpha) at this time was enhanced and E:C(50) decreased. Oxytocin treatment of estrogen-primed nonpregnant rats down-regulated uterine contractile responsiveness to PGF(2alpha), leaving mRNA values for this receptor unchanged, whereas oxytocin receptor blockade with atosiban (an oxytocin receptor antagonist) left E:(max) unaltered. In contrast, atosiban treatment of pregnant rats resulted in a 2.5-fold increase in E:(max) and a considerably reduced EC(50) during labor when compared to untreated delivering rats. The increased contractile ability was associated with a threefold increase in PGF(2alpha)-R mRNA production, indicating that the regulation by atosiban of the PGF(2alpha)-induced response is exerted at the genetic level. Based on the present data we suggest that 1) PGF(2alpha)-R stimulation may not primarily exert a contracting role in the normally delivering myometrium, and 2) the presence of the PGF(2alpha)-R system in rat myometrium may explain the apparent functional redundancy of the oxytocinergic system during the process of birth in animals lacking oxytocin or where the oxytocin receptor is blocked. In this context PGF(2alpha) receptor stimulation may, in the absence of oxytocin receptor stimulation, exert the contractile forces needed for proper propulsion of the fetus.

The oxytocin antagonist, atosiban (1-deamino-2-D-tyr(OET)-4-thr-8-orn-vasotocin/oxytocin), was infused i.v. to chronically instrumented pregnant baboons in the last third of pregnancy. Atosiban (6 microg/kg per min) inhibited myometrial electromyographic activity associated with spontaneous myometrial contractions that occurred around the onset of darkness between 134 and 162 days gestation (term 180 days gestation). The effect of atosiban on maternal heart rate was minimal. Maternal blood pressure remained unaltered during atosiban infusion. Fetal carotid arterial PO2 was unchanged during a 2-h infusion of atosiban. Transplacental passage of atosiban from mother to fetus was assessed at cesarean section under halothane anesthesia in four baboons and in two chronically instrumented fetuses in the absence of anesthesia. The maternal:fetal concentration gradient ranged from 9.2 to 22.8. Maternal atosiban clearance rates were 9.2-16.9 ml/kg per min. In conclusion, atosiban was very effective at inhibiting spontaneously occurring nocturnal myometrial contractions during the last third of gestation in the pregnant baboon. Although atosiban crosses the placenta relatively freely, there was no effect on fetal oxygenation.

One of the primary methods used to screen the development of oxytocin antagonists (OTAs) is the rat oxytocic bioassay. The purpose of this study was to determine whether the rat oxytocic bioassay is a good predictor of binding affinity to human and rat uterine oxytocin receptors (OTr). The binding affinities of five OTAs to human and rat uterine OTr were determined and correlated with pA2 values derived from the rat uterine oxytocic bioassay. Human uterine myometrial tissue was obtained from patients at the time of cesarean section. Rat uterine tissue for the OTr assay was taken at Day 21 of pregnancy. OTr assays were accomplished by isolating uterine cell membranes and performing saturation analysis with cold OTAs and tritium-labeled oxytocin. The association constants (Ka; 10(+8)/M) were calculated by nonlinear curve-fitting techniques. The Ka for the five OTAs (Mpa1-OT, Antag I, L366948, Antag II, and Antag III), as estimated from the human OTr assay, were 0.55, 0.60, 2.27, 1.91, and 47.20, respectively. Ka estimates obtained through use of rat uterine membranes were 0.51, 1.16, 5.89, 2.03, and 24.40, respectively. Correlation of the log10 of the rat oxytocic bioassay results with those of the rat and human OTr assay was 0.94 and 0.98, respectively (p < 0.01). Antag III was approximately 55, 48, and 90 times more potent than Mpa1-OT as determined by the rat bioassay and rat and human uterine OTr assays, respectively. Mpa1-OT is an OTA that is currently undergoing clinical evaluation.(ABSTRACT TRUNCATED AT 250 WORDS)