The endometrial microbiota and early pregnancy loss.

The human endometrium is a dynamic entity that plays a pivotal role in mediating the complex interplay between the mother and developing embryo. Endometrial disruption can lead to pregnancy loss, impacting both maternal physical and psychological health. Recent research suggests that the endometrial microbiota may play a role in this, although the exact mechanisms are still being explored, aided by recent technological advancements and our growing understanding of host immune responses. Suboptimal or dysbiotic vaginal microbiota, characterized by increased microbial diversity and reduced Lactobacillus dominance, has been associated with various adverse reproductive events, including miscarriage. However, the mechanisms linking the lower reproductive tract microbiota with pregnancy loss remain unclear. Recent observational studies implicate a potential microbial continuum between the vaginal and endometrial niche in patients with pregnancy loss; however, transcervical sampling of the low biomass endometrium is highly prone to cross-contamination, which is often not controlled for. In this review, we explore emerging evidence supporting the theory that a dysbiotic endometrial microbiota may modulate key inflammatory pathways required for successful embryo implantation and pregnancy development. We also highlight that a greater understanding of the endometrial microbiota, its relationship with the local endometrial microenvironment, and potential interventions remain a focus for future research.

15-Deoxy-Delta-12,14-prostaglandin J2 modulates pro-labour and pro-inflammatory responses in human myocytes, vaginal and amnion epithelial cells.

Background: Prematurity is the leading cause of childhood death under the age of five. The aetiology of preterm birth is multifactorial; however, inflammation and infection are the most common causal factors, supporting a potential role for immunomodulation as a therapeutic strategy. 15-Deoxy-Delta-12,14-prostaglandin J2 (15dPGJ2) is an anti-inflammatory prostaglandin and has been shown to delay lipopolysaccharide (LPS) induced preterm labour in mice and improve pup survival. This study explores the immunomodulatory effect of 15dPGJ2 on the transcription factors NF-κB and AP-1, pro-inflammatory cytokines, and contraction associated proteins in human cultured myocytes, vaginal epithelial cell line (VECs) and primary amnion epithelial cells (AECs). Methods: Cells were pre-incubated with 32µM of 15dPGJ2 and stimulated with 1ng/mL of IL-1ß as an in vitro model of inflammation. Western immunoblotting was used to detect phosphorylated p-65 and phosphorylated c-Jun as markers of NF-κB and AP-1 activation, respectively. mRNA expression of the pro-inflammatory cytokines IL-6, IL-8, and TNF-α was examined, and protein expression of COX-2 and PGE2 were detected by western immunoblotting and ELISA respectively. Myometrial contractility was examined ex-vivo using a myograph. Results: 15dPGJ2 inhibited IL-1ß-induced activation of NF-κB and AP-1, and expression of IL-6, IL-8, TNF-α, COX-2 and PGE2 in myocytes, with no effect on myometrial contractility or cell viability. Despite inhibiting IL-1ß-induced activation of NF-κB, expression of IL-6, TNF-α, and COX-2, 15dPGJ2 led to activation of AP-1, increased production of PGE2 and increased cell death in VECs and AECs. Conclusion: We conclude that 15dPGJ2 has differential effects on inflammatory modulation depending on cell type and is therefore unlikely to be a useful therapeutic agent for the prevention of preterm birth.

The vaginal microbiota and innate immunity after local excisional treatment for cervical intraepithelial neoplasia.

BACKGROUND: Vaginal microbiota (VMB) composition is altered in women with cervical intra-epithelial neoplasia (CIN) compared to healthy controls and is associated with disease progression. However, the impact of CIN excision on the VMB and innate immunity is not known. This observational study aims to explore the impact of CIN excision on the VMB, antimicrobial peptides (AMP) and proinflammatory cytokines. METHODS: We sampled 103 non-pregnant, premenopausal women at the time of excisional treatment for CIN and at their 6-month follow-up visit. A further 39 untreated controls with normal cytology were also sampled. We used metataxonomics to group vaginal swab samples into community state types (CSTs) and ELISA to quantify cytokine and AMP levels in matched vaginal secretions. Analyses were performed to compare the bacterial composition and immune analyte levels before and after CIN excision and in healthy controls. RESULTS: Women with CIN had significantly higher rates of Lactobacillus species depletion pre-treatment compared to healthy controls (CST IV 21/103, 20% vs 1/39, 3%, p = 0.0081). Excision did not change the VMB composition, with CST IV remaining significantly more prevalent after excision compared to untreated, healthy controls (CST IV 19/103, 20% vs 1/39, 3%, p = 0.0142). Prevotella bivia and Sneathia amnii were significantly higher in samples before treatment compared to untreated controls, and Prevotella bivia remained significantly higher amongst the treated, with less Lactobacillus crispatus compared to untreated controls. IL-1ß and IL-8 remained significantly elevated pre- (p < 0.0001 and p = 0.0014, respectively) and post-treatment (p < 0.0001 and p = 0.0035, respectively) compared to untreated controls. Levels of human beta-defensin-1 and secretory leukocyte protease inhibitor were both significantly reduced following CIN excision (p < 0.0001); however, their levels remained lower than controls post-treatment. CONCLUSIONS: Women with CIN have an increased prevalence of Lactobacillus sp. depletion, high-diversity VMB composition, and higher levels of proinflammatory cytokines and AMPs compared to normal controls. Surgical excision of the disease reduces levels of vaginal AMPs but does not alter VMB composition or cytokine levels. These findings suggest that women with CIN have an inherent predisposition to a high-diversity proinflammatory environment that is not corrected by disease excision. The failure to re-establish a Lactobacillus-enriched CST may explain why women remain at high risk of pre-invasive and invasive disease recurrence.

Proteome-wide prediction of bacterial carbohydrate-binding proteins as a tool for understanding commensal and pathogen colonisation of the vaginal microbiome.

Bacteria use carbohydrate-binding proteins (CBPs), such as lectins and carbohydrate-binding modules (CBMs), to anchor to specific sugars on host surfaces. CBPs in the gut microbiome are well studied, but their roles in the vagina microbiome and involvement in sexually transmitted infections, cervical cancer and preterm birth are largely unknown. We established a classification system for lectins and designed Hidden Markov Model (HMM) profiles for data mining of bacterial genomes, resulting in identification of >100,000 predicted bacterial lectins available at unilectin.eu/bacteria. Genome screening of 90 isolates from 21 vaginal bacterial species shows that those associated with infection and inflammation produce a larger CBPs repertoire, thus enabling them to potentially bind a wider array of glycans in the vagina. Both the number of predicted bacterial CBPs and their specificities correlated with pathogenicity. This study provides new insights into potential mechanisms of colonisation by commensals and potential pathogens of the reproductive tract that underpin health and disease states.

The association between obesity and weight loss after bariatric surgery on the vaginal microbiota.

BACKGROUND: Obesity and vaginal microbiome (VMB) dysbiosis are each risk factors for adverse reproductive and oncological health outcomes in women. Here, we investigated the relationship between obesity, vaginal bacterial composition, local inflammation and bariatric surgery. METHODS: Vaginal bacterial composition assessed by high-throughput sequencing of bacterial 16S rRNA genes and local cytokine levels measured using a multiplexed Magnetic Luminex Screening Assay were compared between 67 obese and 42 non-obese women. We further assessed temporal changes in the microbiota and cytokines in a subset of 27 women who underwent bariatric surgery. RESULTS: The bacterial component of the vaginal microbiota in obese women was characterised by a lower prevalence of a Lactobacillus-dominant VMB and higher prevalence of a high diversity (Lactobacillus spp., and Gardnerella- spp. depleted) VMB, compared with non-obese subjects (p<0.001). Obese women had higher relative abundance of Dialister species (p<0.001), Anaerococcus vaginalis (p=0.021), and Prevotella timonensis (p=0.020) and decreased relative abundance of Lactobacillus crispatus (p=0.014). Local vaginal IL-1ß, IL-4, IL-6, IL-8, IFNγ, MIP-1α and TNFα levels were all higher among obese women, however, only IL-1ß and IL-8 correlated with VMB species diversity. In a subset of obese women undergoing bariatric surgery, there were no significant overall differences in VMB following surgery; however, 75% of these women remained obese at 6 months. Prior to surgery, there was no relationship between body mass index (BMI) and VMB structure; however, post-surgery women with a Lactobacillus-dominant VMB had a significantly lower BMI than those with a high diversity VMB. CONCLUSIONS: Obese women have a significantly different vaginal microbiota composition with increased levels of local inflammation compared to non-obese women. Bariatric surgery does not change the VMB; however, those with the greatest weight loss 6-month post-surgery are most likely to have a Lactobacillus-dominant VMB. Video abstract.

Miscarriage matters: the epidemiological, physical, psychological, and economic costs of early pregnancy loss.

Miscarriage is generally defined as the loss of a pregnancy before viability. An estimated 23 million miscarriages occur every year worldwide, translating to 44 pregnancy losses each minute. The pooled risk of miscarriage is 15·3% (95% CI 12·5-18·7%) of all recognised pregnancies. The population prevalence of women who have had one miscarriage is 10·8% (10·3-11·4%), two miscarriages is 1·9% (1·8-2·1%), and three or more miscarriages is 0·7% (0·5-0·8%). Risk factors for miscarriage include very young or older female age (younger than 20 years and older than 35 years), older male age (older than 40 years), very low or very high body-mass index, Black ethnicity, previous miscarriages, smoking, alcohol, stress, working night shifts, air pollution, and exposure to pesticides. The consequences of miscarriage are both physical, such as bleeding or infection, and psychological. Psychological consequences include increases in the risk of anxiety, depression, post-traumatic stress disorder, and suicide. Miscarriage, and especially recurrent miscarriage, is also a sentinel risk marker for obstetric complications, including preterm birth, fetal growth restriction, placental abruption, and stillbirth in future pregnancies, and a predictor of longer-term health problems, such as cardiovascular disease and venous thromboembolism. The costs of miscarriage affect individuals, health-care systems, and society. The short-term national economic cost of miscarriage is estimated to be £471 million per year in the UK. As recurrent miscarriage is a sentinel marker for various obstetric risks in future pregnancies, women should receive care in preconception and obstetric clinics specialising in patients at high risk. As psychological morbidity is common after pregnancy loss, effective screening instruments and treatment options for mental health consequences of miscarriage need to be available. We recommend that miscarriage data are gathered and reported to facilitate comparison of rates among countries, to accelerate research, and to improve patient care and policy development.

Differential Response of Gestational Tissues to TLR3 Viral Priming Prior to Exposure to Bacterial TLR2 and TLR2/6 Agonists.

Background: Infection/inflammation is an important causal factor in spontaneous preterm birth (sPTB). Most mechanistic studies have concentrated on the role of bacteria, with limited focus on the role of viruses in sPTB. Murine studies support a potential multi-pathogen aetiology in which a double or sequential hit of both viral and bacterial pathogens leads to a higher risk preterm labour. This study aimed to determine the effect of viral priming on bacterial induced inflammation in human in vitro models of ascending and haematogenous infection. Methods: Vaginal epithelial cells, and primary amnion epithelial cells and myocytes were used to represent cell targets of ascending infection while interactions between peripheral blood mononuclear cells (PBMCs) and placental explants were used to model systemic infection. To model the effect of viral priming upon the subsequent response to bacterial stimuli, each cell type was stimulated first with a TLR3 viral agonist, and then with either a TLR2 or TLR2/6 agonist, and responses compared to those of each agonist alone. Immunoblotting was used to detect cellular NF-κB, AP-1, and IRF-3 activation. Cellular TLR3, TLR2, and TLR6 mRNA was quantified by RT-qPCR. Immunoassays were used to measure supernatant cytokine, chemokine and PGE2 concentrations. Results: TLR3 ("viral") priming prior to TLR2/6 agonist ("bacterial") exposure augmented the pro-inflammatory, pro-labour response in VECs, AECs, myocytes and PBMCs when compared to the effects of agonists alone. In contrast, enhanced anti-inflammatory cytokine production (IL-10) was observed in placental explants. Culturing placental explants in conditioned media derived from PBMCs primed with a TLR3 agonist enhanced TLR2/6 agonist stimulated production of IL-6 and IL-8, suggesting a differential response by the placenta to systemic inflammation compared to direct infection as a result of haematogenous spread. TLR3 agonism generally caused increased mRNA expression of TLR3 and TLR2 but not TLR6. Conclusion: This study provides human in vitro evidence that viral infection may increase the susceptibility of women to bacterial-induced sPTB. Improved understanding of interactions between viral and bacterial components of the maternal microbiome and host immune response may offer new therapeutic options, such as antivirals for the prevention of PTB.

Laser-assisted rapid evaporative ionisation mass spectrometry (LA-REIMS) as a metabolomics platform in cervical cancer screening.

BACKGROUND: The introduction of high-risk human papillomavirus (hrHPV) testing as part of primary cervical screening is anticipated to improve sensitivity, but also the number of women who will screen positive. Reflex cytology is the preferred triage test in most settings but has limitations including moderate diagnostic accuracy, lack of automation, inter-observer variability and the need for clinician-collected sample. Novel, objective and cost-effective approaches are needed. METHODS: In this study, we assessed the potential use of an automated metabolomic robotic platform, employing the principle of laser-assisted Rapid Evaporative Ionisation Mass Spectrometry (LA-REIMS) in cervical cancer screening. FINDINGS: In a population of 130 women, LA-REIMS achieved 94% sensitivity and 83% specificity (AUC: 91.6%) in distinguishing women testing positive (n = 65) or negative (n = 65) for hrHPV. We performed further analysis according to disease severity with LA-REIMS achieving sensitivity and specificity of 91% and 73% respectively (AUC: 86.7%) in discriminating normal from high-grade pre-invasive disease. INTERPRETATION: This automated high-throughput technology holds promise as a low-cost and rapid test for cervical cancer screening and triage. The use of platforms like LA-REIMS has the potential to further improve the accuracy and efficiency of the current national screening programme. FUNDING: Work was funded by the MRC Imperial Confidence in Concept Scheme, Imperial College Healthcare Charity, British Society for Colposcopy and Cervical Pathology, National Research Development and Innovation Office of Hungary, Waters corporation and NIHR BRC.

The vaginal microbiota associates with the regression of untreated cervical intraepithelial neoplasia 2 lesions.

Emerging evidence suggests associations between the vaginal microbiota (VMB) composition, human papillomavirus (HPV) infection, and cervical intraepithelial neoplasia (CIN); however, causal inference remains uncertain. Here, we use bacterial DNA sequencing from serially collected vaginal samples from a cohort of 87 adolescent and young women aged 16-26 years with histologically confirmed, untreated CIN2 lesions to determine whether VMB composition affects rates of regression over 24 months. We show that women with a Lactobacillus-dominant microbiome at baseline are more likely to have regressive disease at 12 months. Lactobacillus spp. depletion and presence of specific anaerobic taxa including Megasphaera, Prevotella timonensis and Gardnerella vaginalis are associated with CIN2 persistence and slower regression. These findings suggest that VMB composition may be a future useful biomarker in predicting disease outcome and tailoring surveillance, whilst it may offer rational targets for the development of new prevention and treatment strategies.

CCR2 mediates the adverse effects of LPS in the pregnant mouse.

In our earlier work, we found that intrauterine (i.u.) and intraperitoneal (i.p.) injection of LPS (10-µg serotype 0111:B4) induced preterm labor (PTL) with high pup mortality, marked systemic inflammatory response and hypotension. Here, we used both i.u. and i.p. LPS models in pregnant wild-type (wt) and CCR2 knockout (CCR2-/-) mice on E16 to investigate the role played by the CCL2/CCR2 system in the response to LPS. Basally, lower numbers of monocytes and macrophages and higher numbers of neutrophils were found in the myometrium, placenta, and blood of CCR2-/- vs. wt mice. After i.u. LPS, parturition occurred at 14 h in both groups of mice. At 7 h post-injection, 70% of wt pups were dead vs. 10% of CCR2-/- pups, but at delivery 100% of wt and 90% of CCR2-/- pups were dead. Myometrial and placental monocytes and macrophages were generally lower in CCR2-/- mice, but this was less consistent in the circulation, lung, and liver. At 7 h post-LPS, myometrial ERK activation was greater and JNK and p65 lower and the mRNA levels of chemokines were higher and of inflammatory cytokines lower in CCR2-/- vs. wt mice. Pup brain and placental inflammation were similar. Using the IP LPS model, we found that all measures of arterial pressure increased in CCR2-/- but declined in wt mice. These data suggest that the CCL2/CCR2 system plays a critical role in the cardiovascular response to LPS and contributes to pup death but does not influence the onset of inflammation-induced PTL.

Comparison of vaginal microbiota sampling techniques: cytobrush versus swab.

Evidence suggests the vaginal microbiota (VM) may influence risk of persistent Human Papillomavirus (HPV) infection and cervical carcinogenesis. Established cytology biobanks, typically collected with a cytobrush, constitute a unique resource to study such associations longitudinally. It is plausible that compared to rayon swabs; the most commonly used sampling devices, cytobrushes may disrupt biofilms leading to variation in VM composition. Cervico-vaginal samples were collected with cytobrush and rayon swabs from 30 women with high-grade cervical precancer. Quantitative PCR was used to compare bacterial load and Illumina MiSeq sequencing of the V1-V3 regions of the 16S rRNA gene used to compare VM composition. Cytobrushes collected a higher total bacterial load. Relative abundance of bacterial species was highly comparable between sampling devices (R2 = 0.993). However, in women with a Lactobacillus-depleted, high-diversity VM, significantly less correlation in relative species abundance was observed between devices when compared to those with a Lactobacillus species-dominant VM (p = 0.0049). Cytobrush and swab sampling provide a comparable VM composition. In a small proportion of cases the cytobrush was able to detect underlying high-diversity community structure, not realized with swab sampling. This study highlights the need to consider sampling devices as potential confounders when comparing multiple studies and datasets.

The interaction between vaginal microbiota, cervical length, and vaginal progesterone treatment for preterm birth risk.

BACKGROUND: Preterm birth is the primary cause of infant death worldwide. A short cervix in the second trimester of pregnancy is a risk factor for preterm birth. In specific patient cohorts, vaginal progesterone reduces this risk. Using 16S rRNA gene sequencing, we undertook a prospective study in women at risk of preterm birth (n = 161) to assess (1) the relationship between vaginal microbiota and cervical length in the second trimester and preterm birth risk and (2) the impact of vaginal progesterone on vaginal bacterial communities in women with a short cervix. RESULTS: Lactobacillus iners dominance at 16 weeks of gestation was significantly associated with both a short cervix <25 mm (n = 15, P < 0.05) and preterm birth <34+0 weeks (n = 18; P < 0.01; 69% PPV). In contrast, Lactobacillus crispatus dominance was highly predictive of term birth (n = 127, 98% PPV). Cervical shortening and preterm birth were not associated with vaginal dysbiosis. A longitudinal characterization of vaginal microbiota (<18, 22, 28, and 34 weeks) was then undertaken in women receiving vaginal progesterone (400 mg/OD, n = 25) versus controls (n = 42). Progesterone did not alter vaginal bacterial community structure nor reduce L. iners-associated preterm birth (<34 weeks). CONCLUSIONS: L. iners dominance of the vaginal microbiota at 16 weeks of gestation is a risk factor for preterm birth, whereas L. crispatus dominance is protective against preterm birth. Vaginal progesterone does not appear to impact the pregnancy vaginal microbiota. Patients and clinicians who may be concerned about "infection risk" associated with the use of a vaginal pessary during high-risk pregnancy can be reassured.

The vaginal microbiota, human papillomavirus infection and cervical intraepithelial neoplasia: what do we know and where are we going next?

The vaginal microbiota plays a significant role in health and disease of the female reproductive tract. Next-generation sequencing techniques based upon the analysis of bacterial 16S rRNA genes permit in-depth study of vaginal microbial community structure to a level of detail not possible with standard culture-based microbiological techniques. The human papillomavirus (HPV) causes both cervical intraepithelial neoplasia (CIN) and cervical cancer. Although the virus is highly prevalent, only a small number of women have a persistent HPV infection and subsequently develop clinically significant disease. There is emerging evidence which leads us to conclude that increased diversity of vaginal microbiota combined with reduced relative abundance of Lactobacillus spp. is involved in HPV acquisition and persistence and the development of cervical precancer and cancer. In this review, we summarise the current literature and discuss potential mechanisms for the involvement of vaginal microbiota in the evolution of CIN and cervical cancer. The concept of manipulation of vaginal bacterial communities using pre- and probiotics is also discussed as an exciting prospect for the field of cervical pathology.

Preterm Birth Prevention Post-Conization: A Model of Cervical Length Screening with Targeted Cerclage.

Women with a history of excisional treatment (conization) for cervical intra-epithelial neoplasia (CIN) are at increased risk of preterm birth, perinatal morbidity and mortality in subsequent pregnancy. We aimed to develop a screening model to effectively differentiate pregnancies post-conization into low- and high-risk for preterm birth, and to evaluate the impact of suture material on the efficacy of ultrasound indicated cervical cerclage. We analysed longitudinal cervical length (CL) data from 725 pregnant women post-conization attending preterm surveillance clinics at three London university Hospitals over a ten year period (2004-2014). Rates of preterm birth <37 weeks after targeted cerclage for CL<25mm were compared with local and national background rates and expected rates for this cohort. Rates for cerclage using monofilament or braided suture material were also compared. Of 725 women post-conization 13.5% (98/725) received an ultrasound indicated cerclage and 9.7% (70/725) delivered prematurely, <37weeks; 24.5% (24/98) of these despite insertion of cerclage. The preterm birth rate was lower for those that had monofilament (9/60, 15%) versus braided (15/38, 40%) cerclage (RR 0.7, 95% CI 0.54 to 0.94, P = 0.008). Accuracy parameters of interval reduction in CL between longitudinal second trimester screenings were calculated to identify women at low risk of preterm birth, who could safely discontinue surveillance. A reduction of CL <10% between screening timepoints predicts term birth, >37weeks. Our triage model enables timely discharge of low risk women, eliminating 36% of unnecessary follow-up CL scans. We demonstrate that preterm birth in women post-conization may be reduced by targeted cervical cerclage. Cerclage efficacy is however suture material-dependant: monofilament is preferable to braided suture. The introduction of triage prediction models has the potential to reduce the number of unnecessary CL scan for women at low risk of preterm birth.

Modeling hormonal and inflammatory contributions to preterm and term labor using uterine temporal transcriptomics.

BACKGROUND: Preterm birth is now recognized as the primary cause of infant mortality worldwide. Interplay between hormonal and inflammatory signaling in the uterus modulates the onset of contractions; however, the relative contribution of each remains unclear. In this study we aimed to characterize temporal transcriptome changes in the uterus preceding term labor and preterm labor (PTL) induced by progesterone withdrawal or inflammation in the mouse and compare these findings with human data. METHODS: Myometrium was collected at multiple time points during gestation and labor from three murine models of parturition: (1) term gestation; (2) PTL induced by RU486; and (3) PTL induced by lipopolysaccharide (LPS). RNA was extracted and cDNA libraries were prepared and sequenced using the Illumina HiSeq 2000 system. Resulting RNA-Seq data were analyzed using multivariate modeling approaches as well as pathway and causal network analyses and compared against human myometrial transcriptome data. RESULTS: We identified a core set of temporal myometrial gene changes associated with term labor and PTL in the mouse induced by either inflammation or progesterone withdrawal. Progesterone withdrawal initiated labor without inflammatory gene activation, yet LPS activation of uterine inflammation was sufficient to override the repressive effects of progesterone and induce a laboring phenotype. Comparison of human and mouse uterine transcriptomic datasets revealed that human labor more closely resembles inflammation-induced PTL in the mouse. CONCLUSIONS: Labor in the mouse can be achieved through inflammatory gene activation yet these changes are not a requisite for labor itself. Human labor more closely resembles LPS-induced PTL in the mouse, supporting an essential role for inflammatory mediators in human "functional progesterone withdrawal." This improved understanding of inflammatory and progesterone influence on the uterine transcriptome has important implications for the development of PTL prevention strategies.

Sulfasalazine augments a pro-inflammatory response in interleukin-1ß-stimulated amniocytes and myocytes.

Preterm birth occurs in 10% of pregnancies and is a major cause of neonatal morbidity and mortality. The majority of cases of early preterm labour are associated with infection/inflammation, which places the fetal central nervous system at risk. Targeting immune activation is therefore an appealing therapeutic strategy for the prevention of preterm labour and neonatal brain injury. The expression of many labour-associated and inflammatory-response genes is controlled by the transcription factors nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), which makes them therapeutic targets of interest. Sulfasalazine (SASP) has been shown to inhibit NF-κB and reduce lipopolysaccharide-induced cytokine concentrations in fetal membrane explants and reduce the rate of Escherichia coli-induced preterm labour in mice. Its effects upon AP-1 in the context of pregnancy are unknown. In this study the effect of SASP on interleukin-1ß (IL-1ß) -induced NF-κB and AP-1 activity, cytokine production and cyclo-oxygenase-2 (COX-2) expression was examined in amniocytes and myocytes. A supra-therapeutic concentration (5 mm) was required to inhibit IL-1ß-induced NF-κB (P < 0·0001) in amniocytes and IL-1ß-induced NF-κB (P < 0·01), AP-1 (P < 0·01) and COX-2 (P < 0·05) in myocytes. Despite inhibiting IL-1ß-induced cytokines, a basal increase in IL-6 (P < 0·01), IL-8 (P < 0·0001) and tumour necrosis factor-α (TNF-α) (P < 0·001) was seen with 5 mm SASP in amniocytes, and significant cytotoxic effects were seen in myocytes. The therapeutic concentration of 0·015 mm had no inhibitory effects on pro-inflammatory mediators, but led to an augmented response to IL-1ß-induced IL-6 (P < 0·01), IL-8 (P < 0·05) and TNF-α (P < 0·05) in amniocytes and IL-8 (P < 0·05) in myocytes. SASP is therefore an unlikely therapeutic candidate for the prevention of inflammation-induced preterm labour.

Oxytocin activates NF-κB-mediated inflammatory pathways in human gestational tissues.

Human labour, both at term and preterm, is preceded by NF-κB-mediated inflammatory activation within the uterus, leading to myometrial activation, fetal membrane remodelling and cervical ripening. The stimuli triggering inflammatory activation in normal human parturition are not fully understood. We show that the neurohypophyseal peptide, oxytocin (OT), activates NF-κB and stimulates downstream inflammatory pathways in human gestational tissues. OT stimulation (1 pM-100 nM) specifically via its receptor (OTR) in human myometrial and amnion primary cells led to MAPK and NF-κB activation within 15 min and maximal p65-subunit nuclear translocation within 30 min. Both in human myometrium and amnion, OT-induced activation of the canonical NF-κB pathway upregulated key inflammatory labour-associated genes including IL-8, CCL5, IL-6 and COX-2. IKKß inhibition (TPCA1; 10 µM) suppressed OT-induced NF-κB-p65 phosphorylation, whereas p65-siRNA knockdown reduced basal and OT-induced COX-2 levels in myometrium and amnion. In both gestational tissues, MEK1/2 (U0126; 10 µM) or p38 inhibition (SB203580; 10 µM) suppressed OT-induced COX-2 expression, but OT-induced p65-phosphorylation was only inhibited in amnion, suggesting OT activation of NF-κB in amnion is MAPK-dependent. Our data provide new insight into the OT/OTR system in human parturition and suggest that its therapeutic modulation could be a strategy for regulating both contractile and inflammatory pathways in the clinical context of term/preterm labour.

Activator protein 1 is a key terminal mediator of inflammation-induced preterm labor in mice.

Activation of uterine inflammatory pathways leads to preterm labor (PTL), associated with high rates of neonatal mortality and morbidity. The transcription factors nuclear factor κB (NFκB) and activator protein 1 (AP-1) regulate key proinflammatory and procontractile genes involved in normal labor and PTL. Here we show that NFκB activation normally occurs in the mouse myometrium at gestation day E18, prior to labor, whereas AP-1 and JNK activation occurs at labor onset. Where labor was induced using the progesterone receptor antagonist RU486, NFkB and AP-1/JNK activation both occurred at the time of labor (20 h compared to 60 h in DMSO-treated controls). Using an LPS (Escherichia coli: serotype O111)-induced PTL model that selectively activates AP-1 but not NFkB, we show that myometrial AP-1 activation drives production of cytokines (Il-6, Il-8, and Il-1ß), metalloproteinases (Mmp3 and Mmp10), and procontractile proteins (Cox-2 and Cx43) resulting in PTL after 7 h. Protein levels of CX43 and IL-1ß, and IL-1ß cleavage, were increased following LPS-induced activation of AP-1. Inhibition of JNK by SP600125 (30 mg/kg) delayed PTL by 6 h (7.5 vs. 13.5 h P<0.05). Our data reveal that NFκB activation is not a functional requirement for infection/inflammation-induced preterm labor and that AP-1 activation is sufficient to drive inflammatory pathways that cause PTL.

The CRTH2 agonist Pyl A prevents lipopolysaccharide-induced fetal death but induces preterm labour.

We have previously demonstrated that the anti-inflammatory prostaglandin 15-deoxy-Δ 12,14-prostaglandin J(2) (15dPGJ(2)) delays inflammation-induced preterm labour in the mouse and improves pup survival through the inhibition of nuclear factor-κB (NF-κB) by a mechanism yet to be elucidated. 15dPGJ(2) is an agonist of the second prostaglandin D(2) receptor, chemoattractant receptor homologous to the T helper 2 cell (CRTH2). In human T helper cells CRTH2 agonists induce the production of the anti-inflammatory interleukins IL-10 and IL-4. We hypothesized that CRTH2 is involved in the protective effect of 15dPGJ(2) in inflammation-induced preterm labour in the murine model. We therefore studied the effects of a specific small molecule CRTH2 agonist on preterm labour and pup survival. An intrauterine injection of lipopolysaccharide (LPS) was administered to CD1 mice at embryonic day 16, ± CRTH2 agonist/vehicle controls. Mice were killed at 4.5 hr to assess fetal wellbeing and to harvest myometrium and pup brain for analysis of NF-κB, and T helper type 1/2 interleukins. To examine the effects of the CRTH2 agonist on LPS-induced preterm labour, mice were allowed to labour spontaneously. Direct effects of the CRTH2 agonist on uterine contractility were examined ex vivo on contracting myometrial strips. The CRTH2 agonist increased fetal survival from 20 to 100% in LPS-treated mice, and inhibited circular muscle contractility ex vivo. However, it augmented LPS-induced labour and significantly increased myometrial NF-κB, IL-1ß, KC-GRO, interferon-γ and tumour necrosis factor-α. This suggests that the action of 15dPGJ(2) is not via CRTH2 and therefore small molecule CRTH2 agonists are not likely to be beneficial for the prevention of inflammation-induced preterm labour.

Chemoattractant receptor homologous to the T helper 2 cell (CRTH2) is not expressed in human amniocytes and myocytes.

BACKGROUND: 15-deoxy-Δ 12,14- Prostaglandin J2 (15dPGJ2) inhibits Nuclear factor kappa B (NF-κB) in human myocytes and amniocytes and delays inflammation induced preterm labour in the mouse. 15dPGJ2 is a ligand for the Chemoattractant Receptor Homologous to the T helper 2 cell (CRTH2), a G protein-coupled receptor, present on a subset of T helper 2 (Th2) cells, eosinophils and basophils. It is the second receptor for Prostaglandin D2, whose activation leads to chemotaxis and the production of Th2-type interleukins. The cellular distribution of CRTH2 in non-immune cells has not been extensively researched, and its identification at the protein level has been limited by the lack of specific antibodies. In this study we explored the possibility that CRTH2 plays a role in 15dPGJ2-mediated inhibition of NF-κB and would therefore represent a novel small molecule therapeutic target for the prevention of inflammation induced preterm labour. METHODS: The effect of a small molecule CRTH2 agonist on NF-κB activity in human cultured amniocytes and myocytes was assessed by detection of p65 and phospho-p65 by immunoblot. Endogenous CRTH2 expression in amniocytes, myocytes and peripheral blood mononuclear cells (PBMCs) was examined by PCR, western analysis and flow cytometry, with amniocytes and myocytes transfected with CRTH2 acting as a positive control in flow cytometry studies. RESULTS: The CRTH2 agonist had no effect on NF-κB activity in amniocytes and myocytes. Although CRTH2 mRNA was detected in amniocytes and myocytes, CRTH2 was not detectable at the protein level, as demonstrated by western analysis and flow cytometry. 15dPGJ2 inhibited phospho-65 in PBMC'S, however the CRTH2 antagonist was not able to attenuate this effect. In conclusion, CRTH2 is not expressed on human amniocytes or myocytes and plays no role in the mechanism of 15dPGJ2-mediated inhibition of NF-κB.