Progesterone suppresses uterine contractility acting through its receptors (PRA/B). The mechanism by which human labour is initiated in the presence of elevated circulating progesterone has remained an enigma since Csapo first theorized of a functional withdrawal of progesterone in 1965. Here we report that in vitro progesterone-liganded nuclear PRB forms a complex including JUN/JUN homodimers and P54(nrb)/Sin3A/HDAC to repress transcription of the key labour gene, Cx43. In contrast, unliganded PRA paradoxically activates Cx43 transcription by interacting with FRA2/JUND heterodimers. Furthermore, we find that while nuclear progesterone receptor (PR) is liganded during human pregnancy, it becomes unliganded during both term and preterm labour as a result of increased expression of the progesterone-metabolizing enzyme 20 alpha HSD and reduced nuclear progesterone levels. Our data provide a mechanism by which human labour can occur in the presence of elevated circulating progesterone and suggests non-metabolizable progestogen might represent an alternative new therapeutic approach to preterm birth prevention.
Telocytes ( TC s), a novel type of interstitial cells, were recently described in the interstitial space of tissues (www.telocytes.com). Telocytes TC s have several very long, moniliform extensions, namely telopodes (Tps). However, the functional role(s) of TC s is not yet understood. Successive photomicrographs of ultrathin sections were concatenated to capture the entire length of Tps which usually measure tens to hundreds of micrometres. Besides the podoms (dilations) and podomers (thin segments), ultrastructural features of Tps include the dichotomous branching and establishing homo‐ and heterocellular contacts. Telopodes make a labyrinthine system by 3D convolution and overlapping, their number being roughly estimated at approximately 20 per 1000 μm 2 . Moreover, the presence of extracellular vesicles (shedding vesicles/exosomes) along the Tps suggests an active intercellular signalling (micro‐ and macromolecules), with possible significance in regulating uterine contractility.
Background Although physiologic transition from rhythmic contractions to uterine retraction postpartum remains a poorly understood process, it has been shown that the latter is essential in the prevention of hemorrhage and its negative consequences. Objective To investigate the transition from oscillatory contractions to tonic contracture in human myometrium after delivery, a mechanism purported to facilitate postpartum hemostasis. Protein kinase C (PKC) plays a key regulatory role in human uterine contractions because it can prevent dephosphorylation of regulatory proteins and sensitize the contractile machinery to low Ca2+ . Thus, activation of PKC by phorbol 12,13-dibutyrate (PDBu) may act as a strong uterotonic agent. Study Design Uterine biopsies were obtained from consenting women undergoing elective caesarian delivery at term without labor (N = 19). Isometric tension measurements were performed on uterine strips (n = 114). The amplitudes and area under the curve of phasic contractions and tonic responses were measured and compared. A total of 1 μM PDBu was added to the isolated organ baths, and maximal tension of the uterine contracture was determined in the absence and presence of either 1 μM of staurosporine, 100 nM nifedipine, or 10 μM cyclopiazonic acid to assess the role of PKC and calcium sensitivity on uterine contractility. Results On the addition of PDBu on either basal or oxytocin-induced activity, consistent contractures were obtained concomitant with complete inhibition of phasic contractions. After a 30-minute incubation period, the mean amplitude of the PDBu-induced tone represented 65.3% of the amplitude of spontaneous contraction. Staurosporine, a protein kinase inhibitor, induced a 91.9% inhibition of PDBu contractures, a process not affected by nifedipine or cyclopiazonic acid, thus indicating that this mechanism is largely Ca2+ independent. Conclusion Pharmacologic activation of PKC leads to a significant contracture of the myometrium. Together, these data suggest that the up-regulation of PKC plays a physiologic role in the modulation of uterine contracture after delivery. A switch from phasic to strong tonic contractions potentially may facilitate postpartum hemostasis.
ProblemInflammation is commonly associated with preterm birth. In non-gestational tissues, the cellular inhibitor of apoptosis protein (cIAP) 1 and 2, independent of their role in apoptosis, have emerged as important regulators of inflammation. There is, however, no data on the effect of human labour on the expression of cIAPs in human gestational tissues, or on their role in modulating TNF- induced inflammation. Method of studyMyometrium and fetal membranes and myometrium were obtained from term non-labouring and labouring fetal membranes and myometrium. Fetal membranes were also obtained from preterm non-labouring and labouring fetal membranes with and without histological chorioamnionitis. The effect of cIAP1 and cIAP2 knockdown by siRNA in myometrial primary cells on pro-labour mediators was also determined. ResultscIAP1 and cIAP2 expression was significantly higher in fetal membranes and myometrium after spontaneous labour, in preterm fetal membranes with infection, and with LPS and TNF-. cIAP2 expression was also higher in fetal membranes after spontaneous preterm labour when compared to non-labouring tissues. Knockdown of cIAP1 and cIAP2 in primary myometrial cells significantly decreased TNF- induced expression and secretion of pro-inflammatory cytokines (IL-6 and IL-8); cyclooxygenase (COX)-2 expression and subsequent release of the prostaglandin PGE(2); the expression and secretion of MMP-9; and NF-B transcriptional activity. ConclusioncIAP1 and cIAP2 are increased after labour in fetal membranes and myometrium and are involved in TNF- induced-expression of pro-labour mediators. Thus, cIAP1 and cIAP2 represent novel therapeutic targets for the prevention of spontaneous preterm birth.
A tenet of contemporary obstetrics is that a significant proportion of preterm births involve bacterial infection. Bacterial endotoxin induces pro-inflammatory cytokines, prostaglandins and proteases via the pro-inflammatory pathway nuclear factor-κB (NF-κB), which plays a key role in initiating uterine contractions and rupture of foetal membranes. In non-gestational tissues, the phytophenols curcumin, naringenin and apigenin exert anti-inflammatory properties via inhibition of NF-κB. The aim of this study was to determine whether these treatments regulate pro-inflammatory and pro-labour mediators in human gestational tissues. Placenta, foetal membranes and myometrium were treated with curcumin, naringenin and apigenin in the presence of lipopolysaccharide (LPS) or interleukin (IL)-1β. In placenta and foetal membranes, all treatments significantly reduced LPS-stimulated release and gene expression of pro-inflammatory cytokines IL-6 and IL-8; placenta decreased cyclooxygenase (COX-2) mRNA expression, subsequent release of prostaglandins PGE2 and PGF2α and expression and activity of matrix-degrading enzyme matrix metalloproteinase (MMP)-9. In myometrial cells, all treatments attenuated IL-1β-induced COX-2 expression, release of PGE2 and PGF2α and expression and activity of MMP-9. Although naringenin significantly attenuated IL-1β-induced IL-6 and IL-8 mRNA expression and release, there was no effect of curcumin and apigenin. LPS-stimulated release of 8-isoprostane, a marker of oxidative stress, was attenuated by all treatments. NF-κB p65 DNA-binding activity was also decreased using these treatments. In conclusion, curcumin, naringenin and apigenin exert anti-inflammatory properties in human gestational tissues by inhibiting the transcriptional activity of NF-κB. Further studies should be undertaken to define a possible implication of these natural spices in the management of preterm labour and delivery.
Preterm deliveries remain the leading cause of neonatal morbidity and mortality. Current therapies target only myometrial contractions and are largely ineffective. As labor involves multiple coordinated events across maternal and fetal tissues, identifying fundamental regulatory pathways of normal term labor is vital to understanding successful parturition and consequently labor pathologies. We aimed to identify transcriptomic signatures of human normal term labor of two tissues: in the fetal-facing choriodecidua and the maternalmyometrium. Microarray transcriptomic data from choriodecidua and myometrium following term labor were analyzed for functional hierarchical networks, using Cytoscape 2.8.3. Hierarchically high candidates were analyzed for their regulatory casual relationships using Ingenuity Pathway Analysis. Selectedmaster regulators were then chemically inhibited and effects on downstream targets were assessed using real-time quantitative PCR (RT-qPCR). Unbiased network analysis identified upstream molecular components in choriodecidua including vimentin, TLR4, and TNFSF13B. In the myometrium, candidates included metallothionein 2 (MT2A), TLR2, and RELB. These master regulators had significant differential gene expression during labor, hierarchically high centrality in community cluster networks, interactions amongst the labor gene set, and strong causal relationships with multiple downstream effects. In vitro experiments highlighted MT2A as an effective regulator of labor-associated genes. We have identified unique potential regulators of the term labor transcriptome in uterine tissues using a robust sequence of unbiased mathematical and literature-based in silico analyses. These findings encourage further investigation into the efficacy of predicted master regulators in blocking multiple pathways of labor processes across maternal and fetal tissues, and their potential as therapeutic approaches. Summary Sentence Combined in silico and in vitro analyses have identified novel transcriptomic regulators of term labor in human choriodecidua and myometrium with further therapeutic potential.
Preterm birth continues to be a significant public health problem. Infection (bacterial and or viral) and inflammation, by stimulating proinflammatory cytokines, adhesion molecules, and matrix metalloproteinase 9 (MMP9), play a central role in the rupture of membranes and myometrial contractions. SMAD7 has been implicated in regulating the inflammatory response; however, no studies have been performed with regard to human labor. In this study, we determined the effect of spontaneous human labor and prolabor mediators on SMAD7 expression in myometrium and fetal membranes. Functional studies were employed to investigate the effect of siRNA knockdown of SMAD7 (siSMAD7) in regulating infection and inflammation-induced prolabor mediators. SMAD7 mRNA and protein expression were significantly higher with spontaneous term labor, compared to no labor, in myometrium and fetal membranes. SMAD7 expression was also significantly higher in amnion from women with preterm chorioamnionitis. The proinflammatory cytokines IL1B and TNF, the bacterial product fsl-1, and the viral dsRNA analog poly(I:C) significantly increased SMAD7 in myometrial cells and amnion cells. In myometrial cells, siSMAD7 cells significantly decreased cytokine (IL6) and chemokine (CXCL1, CXCL8, CCL2 are also known as GRO-alpha, interleukin (IL)-8 and monocyte chemotactic protein-1 (MCP-1)) production induced by IL1B, TNF, and fsl-1. There was also a decrease in the expression of adhesion molecules intercellular adhesion molecule 1 (ICAM1) and vascular cell adhesion molecule 1 (VCAM1) in siSMAD7 cells, and MMP9 expression. In amnion, siSMAD7 cells treated with IL1B also decreased cytokine and chemokine production, ICAM1 and MMP9 expression. In conclusion, we report a proinflammatory role for SMAD7 in human gestational tissues, with SMAD7 silencing attenuating the inflammatory response. Summary Sentence SMAD7 expression is increased in laboring myometrium and by mediators of labor; SMAD7 is involved in the regulation of prolabor mediators induced by inflammation.
Pregnancy, spontaneous term labor (TL), and postpartum (PP) involution are associated with changes in the cellular and extracellular matrix composition of the uterus. Both the uterine smooth muscle (myometrium) and the infiltrating peripheral blood leukocytes involved in the activation of labor secrete extracellular matrix-degrading enzymes (matrix metalloproteinases, MMPs) that can modulate cellular behavior and barrier function. MMP expression is induced by mechanical stretch in several tissues. We hypothesized that the expression and activity of myometrial MMPs and their tissue inhibitors (TIMPs) are modulated in preparation for TL and PP involution and are regulated by mechanical stretch of uterine walls imposed by the growing fetus. Myometrial tissues were collected from bilaterally and unilaterally pregnant rats across gestation, TL, and PP. Total RNA and proteins were subjected to real-time PCR and immunoblotting, respectively, and tissue localization and activity was examined by immunohistochemistry and in situ zymography. We found that Mmp7, Mmp11, and Mmp12 mRNA levels were upregulated during TL and PP, while Mmp2, Mmp3, Mmp8, Mmp9, Mmp10, and Mmp13 mRNAs were only upregulated during PP. Timp1-Timp4 were stably expressed throughout gestation with some fluctuations PP. Active MMP2 was induced in the empty uterine horn during gestation and in the gravid PP uterus, suggesting negative regulation by biological mechanical stretch. We conclude that specific subsets of uterine MMPs are differentially regulated in the rat myometrium in preparation for two major events: TL and PP uterine involution.
Obesity during pregnancy and childbirth is associated with labour dystocia leading to instrumental or operative delivery, but the underlying pathophysiological mechanisms remain unclear and insufficient uterine contractility has been suggested. This study examined whether reduced myometrial mitochondrial capacity or quantity could contribute as a pathophysiological mechanism to labour dystocia. Data did not support reduced myometrial mitochondrial capacity or quantity in the myometrium at term in obese women, but a reduced myocyte density with increased triglyceride content was demonstrated, which could lead to poorer uterine contractility. These results add to the understanding of systemic effects of obesity, placing also the myometrium at term as an affected non‐adipose tissue.
ProblemInterleukin-1 (IL-1) is a pro-inflammatory cytokine that is involved in human parturition, especially in the context of infection-induced preterm birth. Caspase-1 is a key component of inflammasomes, which are activated upon infection to trigger the maturation of IL-1. Method of studyTo determine the effect of human labour on caspase-1 activation in human foetal membranes and myometrium. In addition, the mechanisms by which inflammasome activation regulates IL-1 production were also be assessed. ResultsHigher caspase-1 gene and protein expression were detected in foetal membranes myometrium obtained from term labouring women when compared with samples taken from non labouring women. Lipopolysaccharide induced the transcription and secretion of IL-1 from foetal membranes and myometrium; both events were dependent on nuclear factor kappa B (NF-B). However, levels of extracellular IL-1 were greatly increased by subsequent treatment with the potassium-proton ionophore Adenosine triphosphate (ATP) or nigericin; an effect that was dependent on active caspase-1. Additionally, ATP induced IL-1 secretion via the purinergic P2X7 receptor, whereas the pannexin-1 channel was required for nigericin induced IL-1 secretion. ConclusionTaken together, these results demonstrate that caspase-1 activation is increased with human labour in foetal membranes and myometrium, and is required for infection-induced IL-1 secretion.