Inflammatory Aetiology of Human Myometrial Activation Tested Using Directed Graphs

There are three main hypotheses for the activation of the human uterus at labour: functional progesterone withdrawal, inflammatory stimulation, and oxytocin receptor activation. To test these alternatives we have taken information and data from the literature to develop causal pathway models for the activation of human myometrium. The data provided quantitative RT-PCR results on key genes from samples taken before and during labour. Principal component analysis showed that pre-labour samples form a homogenous group compared to those during labour. We therefore modelled the alternative causal pathways in non-labouring samples using directed graphs and statistically compared the likelihood of the different models using structural equations and D-separation approaches. Using the computer program LISREL, inflammatory activation as a primary event was highly consistent with the data (p = 0.925), progesterone withdrawal, as a primary event, is plausible (p = 0.499), yet comparatively unlikely, oxytocin receptor mediated initiation is less compatible with the data (p = 0.091). DGraph, a software program that creates directed graphs, produced similar results (p = 0.684, p = 0.280, and p = 0.04, respectively). This outcome supports an inflammatory aetiology for human labour. Our results demonstrate the value of directed graphs in determining the likelihood of causal relationships in biology in situations where experiments are not possible

Network evaluation from the consistency of the graph structure with the measured data

<p>Abstract</p> <p>Background</p> <p>A knowledge-based network, which is constructed by extracting as many relationships identified by experimental studies as possible and then superimposing them, is one of the promising approaches to investigate the associations between biological molecules. However, the molecular relationships change dynamically, depending on the conditions in a living cell, which suggests implicitly that all of the relationships in the knowledge-based network do not always exist. Here, we propose a novel method to estimate the consistency of a given network with the measured data: i) the network is quantified into a log-likelihood from the measured data, based on the Gaussian network, and ii) the probability of the likelihood corresponding to the measured data, named the graph consistency probability (<it>GCP</it>), is estimated based on the generalized extreme value distribution.</p> <p>Results</p> <p>The plausibility and the performance of the present procedure are illustrated by various graphs with simulated data, and with two types of actual gene regulatory networks in <it>Escherichia coli</it>: the SOS DNA repair system with the corresponding data measured by fluorescence, and a set of 29 networks with data measured under anaerobic conditions by microarray. In the simulation study, the procedure for estimating <it>GCP </it>is illustrated by a simple network, and the robustness of the method is scrutinized in terms of various aspects: dimensions of sampling data, parameters in the simulation study, magnitudes of data noise, and variations of network structures.</p> <p>In the actual networks, the former example revealed that our method operates well for an actual network with a size similar to those of the simulated networks, and the latter example illustrated that our method can select the activated network candidates consistent with the actual data measured under specific conditions, among the many network candidates.</p> <p>Conclusion</p> <p>The present method shows the possibility of bridging between the static network from the literature and the corresponding measurements, and thus will shed light on the network structure variations in terms of the changes in molecular interaction mechanisms that occur in response to the environment in a living cell.</p

Progesterone, the maternal immune system and the onset of parturition in the mouse

The role of progesterone (P4) in the regulation of the local (uterine) and systemic innate immune system, myometrial expression of connexin 43 (Cx-43) and cyclooxygenase 2 (COX-2) and the onset of parturition was examined in: 1) naïve mice delivering at term; 2) E16 mice treated with RU486 (P4-antagonist) to induce preterm parturition; and 3) in mice treated with P4 to prevent term parturition.In naïve mice, myometrial neutrophil and monocyte numbers peaked at E18 and declined with the onset of parturition. In contrast, circulating monocytes did not change and although neutrophils were increased with pregnancy, they did not change across gestation. The myometrial mRNA and protein levels of most chemokines/cytokines, Cx-43 and COX-2 increased with, but not before, parturition.With RU486-induced parturition, myometrial and systemic neutrophil numbers increased before and myometrial monocyte numbers increased with parturition only. Myometrial chemokine/cytokine mRNA abundance increased with parturition, but protein levels peaked earlier at between 4.5 and 9h post RU486. Cx-43, but not COX-2, mRNA expression and protein levels increased prior to the onset of parturition.In mice treated with P4, the gestation-linked increase in myometrial monocyte, but not neutrophil, numbers was prevented and expression of Cx-43 and COX-2 was reduced. On E20 of P4 supplementation, myometrial chemokine/cytokine and leukocyte numbers, but not Cx-43 and COX-2 expression, increased.These data show that during pregnancy P4 controls myometrial monocyte infiltration, cytokine and prolabour factor synthesis via mRNA dependent and independent mechanisms and, with prolonged P4 supplementation, P4 action is repressed resulting in increased myometrial inflammation

Characteristic Changes in Decidual Gene Expression Signature in Spontaneous Term Parturition

Background: The decidua has been implicated in the "terminal pathway" of human term parturition, which is characterized by the activation of pro-inflammatory pathways in gestational tissues. However, the transcriptomic changes in the decidua leading to terminal pathway activation have not been systematically explored. This study aimed to compare the decidual expression of developmental signaling and inflammation-related genes before and after spontaneous term labor in order to reveal their involvement in this process. Materials and Methods: Chorioamniotic membranes were obtained from normal pregnant women who delivered at term with spontaneous labor (TIL, n=14) or without labor (TNL, n=15). Decidual cells were isolated from snap-frozen chorioamniotic membranes with laser microdissection. The expression of 46 genes involved in decidual development, sex steroid and prostaglandin signaling, as well as pro- and anti-inflammatory pathways was analyzed using high-throughput quantitative real-time polymerase chain reaction (qRT-PCR). Chorioamniotic membrane sections were immunostained and then semi-quantified for five proteins, and immunoassays for three chemokines were performed on maternal plasma samples. Results: The genes with the highest expression in the decidua at term gestation included insulin-like growth factor-binding protein 1 (IGFBP1), galectin-1 (LGALS1), and progestogen-associated endometrial protein (PAEP); the expression of estrogen receptor 1 (ESR1), homeobox A11 (HOXA11), interleukin 1beta (IL1B), IL8, progesterone receptor membrane component 2 (PGRMC2), and prostaglandin E synthase (PTGES) was higher in TIL than in TNL cases; the expression of chemokine C-C motif ligand 2 (CCL2), CCL5, LGALS1, LGALS3, and PAEP was lower in TIL than in TNL cases; immunostaining confirmed qRT-PCR data for IL-8, CCL2, galectin-1, galectin-3, and PAEP; and no correlations between the decidual gene expression and the maternal plasma protein concentrations of CCL2, CCL5, and IL-8 were found. Conclusion: Our data suggests that with the initiation of parturition, the decidual expression of anti-inflammatory mediators decreases, while the expression of pro-inflammatory mediators and steroid receptors increases. This shift may affect downstream signaling pathways that can lead to parturition

NEUTROPHIL PRODUCTS CONTROL THE EXPRESSION OF PROGESTERONE RECEPTORS AND MATRIX METALLOPROTEINASE-1 IN THE DECIDUAL AND MYOMETRIUM AND ARE POSSIBLE REGULATORS OF PREMATURE LABOR

Neutrophils infiltrate myometrium and decidual tissue prior to parturition. Activated neutrophils release reactive oxygen species (ROS) and tumor necrosis factor α (TNFα), which might increase expression of pro-labor genes such as matrix metalloproteinase-1 (MMP-1), progesterone receptor (PR) A/B ratio, and cause demethylation of DNA. These changes might cause labor. Decidual tissue was obtained from consented, healthy women at term (37+ weeks of gestation) not in labor (no contractions, without cervical effacement), term labor and preterm labor (under 37 weeks of pregnancy). Decidual and myometrial cells in culture were treated with (1) ROS, (2) TNFα, or (3) 5-aza-2’-deoxycytidine. Total RNA was extracted, converted to cDNA and evaluated by qRT-PCR for MMP-1, PR-A+B and PR-B. TNFα increased MMP-1 by 17 fold in decidual cells and more than 12 fold in myometrial cells. PR-A/B was increased by 5.6 fold in decidua. ROS up-regulated MMP-1 by 6 fold and elevated the PR-A/B ratio by 4.5 fold in decidual tissue. DNA demethylation increased MMP-1 by about 4 and 11 fold in decidual and myometrium, respectively. The PR-A/B ratio was increased by 4 fold in decidua and the PR-B was decreased by 40% in the myometrium due to DNA demethylation. Decidual tissue in preterm labor showed a 7-fold increase in MMP-1 over term laboring and over a 15-fold increase over term not in labor tissue. In conclusion, MMP-1 expression and PR-A/B ratio was increased by neutrophil products possibly through a mechanism of DNA methylation in decidua and myometrium. Preterm decidua showed a dramatic increase in MMP-1 over normal labor tissue. TNFα and ROS increased expression of MMP-1 to possibly initiate parturition. These data might help explain mechanisms responsible for preterm labor unrelated to infection or premature rupture of membranes

Toll-like receptors and mechanism of human parturition

The process of parturition whether physiological at term or pathological in preterm labour is a complex one driven by fetal, placental and maternal signals. There is compelling evidence to suggest that inflammatory mediators, driven by endogenous factors or pathologically by infection, play a crucial role in stimulating the common parturition pathway leading into cervical ripening, uterine contractions and, finally, expulsion of the foetus and placenta. Toll-like receptors (TLRs) are a relatively recently discovered family of pattern recognition molecules which have been shown to play a pivotal role in innate immune responses and inflammation. Products of micro-organisms, e.g. bacteria, viruses or fungi, can be considered to be primary ligands of Toll-like receptors. In addition, they are also activated by other endogenous ligands such as heat shock protein 60 and 70, extra domain A of fibronectin and surfactant protein A (SPA). Therefore, local activation of Toll-like receptors may be responsible for initiation of inflammatory cascade, in presence or absence of infection, leading to initiation of labouring mechanisms within the myometrium. In this study, we hypothesized that Toll-like receptors (TLR 2 & 4) are expressed in labouring myometrium, and are involved in physiologic and pathophysiologic mechanisms of parturition. In order to test this hypothesis, we compared the pattern of expression of Toll-like receptors in myometrial samples obtained from consenting women before and after onset of term and preterm labour. We also aimed to investigate the activation, regulation of and the functional significance of TLR expression with respect to cytokine production in human myometrium at term, and to determine if progestogens could antagonise the effect of TLR ligands such as Lipopolysaccharide (LPS). We identified the synthesis and presence of TLR2 & 4 in human myometrium as shown by expression of their mRNA and protein signals which appear to be increasing by gestation and 11 possibly by labour status. We also demonstrated a possible role for TLR2 as indicated by upregulation of its protein expression in labour compared with non-labouring myometrium. Although there was also a trend to an increase in TLR4 with labour, this did not reach statistical significance. In our short-term tissue culture model, we demonstrated that Medroxyprogesterone acetate (MPA) significantly suppressed baseline and LPS induced production of IL-1β, IL-6 and IL-8 in pregnant human myometrium. Although LPS stimulated IL-10 production, there was no significant inhibitory effect with MPA. In contrast, we failed to demonstrate significant upregulation of either TNF-α or IFN-γ in response to LPS, and there was no effect of MPA. We also showed that the specific inhibitor for double stranded-RNA dependent protein kinase (PKR) 2-Aminopurine significantly inhibited both baseline and LPS stimulated myometrial cytokine production. In conclusion, findings of this work have demonstrated a potential role for Toll-like receptors 2 & 4 in initiation of term and preterm labour and an anti-inflammatory role for the progestogen Medroxyprogestrone acetate in lipopolysaccahride stimulated myometrial tissue culture model in vitro. These findings highlight the need for further studies to examine the role of other progestogens / progesterone, benefits and risks to mothers and to their babies, in prevention of preterm labour

Birth and Neonatal Transition in the Guinea Pig: Experimental Approaches to Prevent Preterm Birth and Protect the Premature Fetus

The guinea pig (Cavia porcellus) displays many features of gestational physiology that makes it the most translationally relevant rodent species. Progesterone production undergoes a luteal to placental shift as in human pregnancy with levels rising during gestation and with labor and delivery occurring without a precipitous decline in maternal progesterone levels. In contrast to other laboratory rodents, labor in guinea pigs is triggered by a functional progesterone withdrawal, which involves the loss of uterine sensitivity to progesterone like in women. In both species the amnion membrane is a major source of labor-inducing prostaglandins, which promote functional progesterone withdrawal by modifying myometrial progesterone receptor expression. These similar features appear to result from convergent evolution rather than closer evolutionally relationship to primates compared to other rodents. Nevertheless, the similarities in the production, metabolism and actions of progesterone and prostaglandins allow information gained in pregnant guinea pigs to be extended to pregnant women with confidence. This includes exploring the effects of pregnancy complications including growth restriction and the mechanisms by which stressful conditions increase the incidence of preterm labor. The relatively long gestation of the guinea pig and the maturity of the pups at birth particularly in brain development means that a greater proportion of brain development happens in utero. This allows adverse intrauterine conditions to make a sustained impact on the developing brain like in compromised human pregnancies. In addition, the brain is exposed to a protective neurosteroid environment in utero, which has been suggested to promote development in the guinea pig and the human. Moreover, in utero stresses that have been shown to adversely affect long term neurobehavioral outcomes in clinical studies, can be modeled successfully in guinea pigs. Overall, these parallels to the human have led to increasing interest in the guinea pig for translational studies of treatments and therapies that potentially improve outcomes following adverse events in pregnancy and after preterm birth

The role of chemokines/chemokine receptors in labour

Human labour is shown to be an inflammatory process, which involves a marked leukocyte infiltrate into myometrium during labour. My study focused on the role of chemokines, key mediators of leukocyte trafficking, in labour. Previous gene array data obtained from human labouring myometrium showed that the mRNA expression of the following chemokines was increased in term labouring myometrium, CCL2, CCL20, CXCL1, CXCL5, CXCL8. I decided to focus on myometrial expression of these chemokines and also to include CCL5, another important chemokine. My data confirmed that the expression of human myometrial chemokines was increased in labour and that their expression was up regulated by cytokines and mechanical stretch via NFKB and MAPK, but decreased by prostaglandins and oxytocin via PLC. I also studied the expression of myometrial chemokine receptors, which may mediate some of the effects of chemokines on myometrial function and/or act as decoys, minimising the effects of locally produced chemokines. I found that the expression of the chemokine receptors decreased with the onset of labour, mainly through the action of prostaglandins and oxytocin. I then used the established model of LPS-­‐induced preterm labour (PTL) in the mouse and found that chemokines and cytokines both increased in the myometrium and placenta. CCL2 is consistently increased with human labour and has been shown to be important in rodent parturition too. I therefore studied the impact of LPS in the CCR2 (the main receptor for CCL2) knockout mouse. There was less inflammation in both the myometrium and placenta and a better pup survival rate in the CCR2-/- mouse. However, the PTL was not delayed, suggesting that CCR2 is not essential for the induction of PTL labour by LPS in the mouse. I then turned my attention to CCL20, which acts only via CCR6. It is known to drive dendritic cell recruitment and I found that its expression was increased with labour, while that of its receptor was reduced. Functionally, I found that CCL20 up-regulated the myometrial expression of chemokines. Next I used the LPS-induced preterm labour model in the mouse and found that CCR6 knockout delayed LPS-induced preterm delivery and improved pup survival. These findings were associated with much lower inflammation in myometrium and plasma. These data suggest that CCR6 could be a therapeutic target in the management of PTL. Chemokines play an important role both in the induction of term labour and in infection induced PTL. Chemokine inhibitors may delay the onset of PTL and improve the fetal outcome

Exploring the potential role of CRTH2 agonists in the prevention of inflammation induced preterm labour

Introduction: Preterm labour occurs in 76,000 women in England and Wales each year and accounts for 75% of neonatal mortality. Infection and inflammation are key triggers of preterm labour. The transcription factor NF-ĸB plays a key regulatory role in the expression of the labour associated genes such as COX-2, prostaglandins and MMPs, as well as pro-inflammatory cytokines such as IL-8, IL-1β, IFN-γ and TNF-α. The anti-inflammatory prostaglandin 15-deoxyΔ-12,14 Prostaglandin J2, a CRTH2 agonist can inhibit NF-ĸB and delay preterm labour in a murine model of inflammation induced preterm labour. It is well recognised that the immunology of pregnancy is biased towards a Th2 rather than Th1 response. CRTH2 agonists induce the production of the anti-inflammatory cytokines IL-4 and IL-10 in T-helper 2 cells in vitro, thus have potential to further alter the Th1:Th2 ratio in favour of the Th2 bias. Aims: This thesis examines the role of the CRTH2 receptor and its agonists in pregnancy and parturition, and its potential as a therapeutic target for the prevention of preterm labour. Methods: PCR, western analysis and flow cytometry were used for CRTH2 expression studies. To examine the effect of CRTH2 agonists on NF-ĸB activation, western analysis of p65 and phospho-p65 was performed. The effect of the agonists on interleukin profiles was demonstrated by flow cytometry. Myometrial contractility response to agonists was measured using a myograph. To determine the in vivo effect of a small molecule CRTH2 agonist on inflammation induced preterm labour, CD1 mice were given an intrauterine injection of LPS and the CRTH2 agonist. Delivery outcome and the effect on labour associated genes and interleukins were determined by PCR, western analysis and ELISA. Results: Amniocytes and myocytes show low CRTH2 mRNA levels however do not express the protein at detectable levels. The small molecule CRTH2 agonist Pyl A does not inhibit NF-ĸB in amniocytes and myocytes, indicating that the inhibition of NF-ĸB by 15dPGJ2 is independent of CRTH2. Although 15dPGJ2 was able to inhibit the production of pro-inflammatory cytokines in peripheral blood mononuclear cells, Pyl A was unable to alter the Th2 cytokine profile. Pyl A inhibits myometrial contractility, although in a mechanism independent of CRTH2. In the murine model of inflammation induced preterm labour fetal viability was significantly increased by Pyl A, however, preterm labour was augmented rather than prevented. Conclusion: 15dPGJ2 inhibits NF-ĸB and inflammation induced preterm labour in the mouse in a mechanism independent of CRTH2. Contrary to anticipated, Pyl A does not modulate the Th1:Th2 bias or delay preterm labour in vivo. However, 15dPGJ2 suppresses the Th1 response in vitro, thus represents a potential anti-inflammatory therapeutic target for both the prevention of preterm labour and inflammation induced brain injury