Transcriptome changes in maternal peripheral blood during term parturition mimic perturbations preceding spontaneous preterm birth†.

The complex physiologic process of parturition includes the onset of labor, which requires the orchestrated stimulation of a common pathway involving uterine contractility, cervical ripening, and chorioamniotic membrane activation. However, the labor-specific processes taking place in these tissues have limited use as predictive biomarkers unless they can be probed in non-invasive samples, such as the peripheral blood. Herein, we utilized a transcriptomic dataset to assess labor-specific changes in the peripheral blood of women who delivered at term. We identified a set of genes that were differentially expressed with labor and enriched for immunological processes, and these gene expression changes were strongly correlated with results from prior studies, providing in silico validation of our findings. We then identified significant correlations between labor-specific transcriptomic changes in the maternal circulation and those detected in the chorioamniotic membranes, myometrium, and cervix of women at term, demonstrating that tissue-specific labor signatures are partly mirrored in the peripheral blood. Finally, we demonstrated a significant overlap between the peripheral blood transcriptomic changes in term parturition and those observed in asymptomatic women, prior to the diagnosis of preterm prelabor rupture of the membranes, who ultimately delivered preterm. Collectively, we provide evidence that the normal process of labor at term is characterized by a unique immunological expression signature, which may serve as a useful tool for assessing labor status and for potentially identifying women at risk for preterm birth.

A Peripheral Immune Signature of Labor Induction.

Approximately 1 in 4 pregnant women in the United States undergo labor induction. The onset and establishment of labor, particularly induced labor, is a complex and dynamic process influenced by multiple endocrine, inflammatory, and mechanical factors as well as obstetric and pharmacological interventions. The duration from labor induction to the onset of active labor remains unpredictable. Moreover, prolonged labor is associated with severe complications for the mother and her offspring, most importantly chorioamnionitis, uterine atony, and postpartum hemorrhage. While maternal immune system adaptations that are critical for the maintenance of a healthy pregnancy have been previously characterized, the role of the immune system during the establishment of labor is poorly understood. Understanding maternal immune adaptations during labor initiation can have important ramifications for predicting successful labor induction and labor complications in both induced and spontaneous types of labor. The aim of this study was to characterize labor-associated maternal immune system dynamics from labor induction to the start of active labor. Serial blood samples from fifteen participants were collected immediately prior to labor induction (baseline) and during the latent phase until the start of active labor. Using high-dimensional mass cytometry, a total of 1,059 single-cell immune features were extracted from each sample. A multivariate machine-learning method was employed to characterize the dynamic changes of the maternal immune system after labor induction until the establishment of active labor. A cross-validated linear sparse regression model (least absolute shrinkage and selection operator, LASSO) predicted the minutes since induction of labor with high accuracy (R = 0.86, p = 6.7e-15, RMSE = 277 min). Immune features most informative for the model included STAT5 signaling in central memory CD8+ T cells and pro-inflammatory STAT3 signaling responses across multiple adaptive and innate immune cell subsets. Our study reports a peripheral immune signature of labor induction, and provides important insights into biological mechanisms that may ultimately predict labor induction success as well as complications, thereby facilitating clinical decision-making to improve maternal and fetal well-being.

Inhibition of GPR91 Reduces Inflammatory Mediators Involved in Active Labor in Myometrium.

RESULTS: GPR91 mRNA expression was significantly higher in myometrium from women during term spontaneous labor compared to no labor. Likewise, in mice, GPR91 mRNA expression was significantly upregulated in myometrium during inflammation-induced preterm labor compared to preterm no labor. In myometrial cells, IL1B and TNF significantly increased GPR91 mRNA expression. Knockdown of GPR91 by siRNA in myometrial cells significantly suppressed the secretion and/or expression of IL1B- and TNF-induced proinflammatory cytokines (GM-CSF, IL1A, IL1B, and IL6) and chemokines (CXCL8 and CCL2), myometrial contractility (expression of the contraction-associated proteins PTGFR and CX43, secretion of the uterotonic PGF2α , and in situ collagen gel contraction), and the transcription factor NF-κB. CONCLUSION: Our findings demonstrate that GPR91 is involved in the genesis of proinflammatory and prolabor mediators induced by IL1B or TNF and collectively suggest that GPR91 may contribute to augmentation of the labor processes.

Human labour is associated with altered regulatory T cell function and maternal immune activation.

During human pregnancy, regulatory T cell (Treg ) function is enhanced and immune activation is repressed allowing the growth and development of the feto-placental unit. Here, we have investigated whether human labour is associated with a reversal of the pregnancy-induced changes in the maternal immune system. We tested the hypothesis that human labour is associated with a decline in Treg function, specifically their ability to modulate Toll-like receptor (TLR)-induced immune responses. We studied the changes in cell number, activation status and functional behaviour of peripheral blood, myometrial (myoMC) and cord blood mononuclear cells (CBMC) with the onset of labour. We found that Treg function declines and that Treg cellular targets change with labour onset. The changes in Treg function were associated with increased activation of myoMC, assessed by their expression of major histocompatibility complex (MHC) class II molecules and CBMC inflammatory cells. The innate immune system showed increased activation, as shown by altered monocyte and neutrophil cell phenotypes, possibly to be ready to respond to microbial invasion after birth or to contribute to tissue remodelling. Our results highlight changes in the function of the adaptive and innate immune systems that may have important roles in the onset of human labour.

Incidence of maternal peripartum infection: A systematic review and meta-analysis.

BACKGROUND: Infection is an important, preventable cause of maternal morbidity, and pregnancy-related sepsis accounts for 11% of maternal deaths. However, frequency of maternal infection is poorly described, and, to our knowledge, it remains the one major cause of maternal mortality without a systematic review of incidence. Our objective was to estimate the average global incidence of maternal peripartum infection. METHODS AND FINDINGS: We searched Medline, EMBASE, Global Health, and five other databases from January 2005 to June 2016 (PROSPERO: CRD42017074591). Specific outcomes comprised chorioamnionitis in labour, puerperal endometritis, wound infection following cesarean section or perineal trauma, and sepsis occurring from onset of labour until 42 days postpartum. We assessed studies irrespective of language or study design. We excluded conference abstracts, studies of high-risk women, and data collected before 1990. Three reviewers independently selected studies, extracted data, and appraised quality. Quality criteria for incidence/prevalence studies were adapted from the Joanna Briggs Institute. We used random-effects models to obtain weighted pooled estimates of incidence risk for each outcome and metaregression to identify study-level characteristics affecting incidence. From 31,528 potentially relevant articles, we included 111 studies of infection in women in labour or postpartum from 46 countries. Four studies were randomised controlled trials, two were before-after intervention studies, and the remainder were observational cohort or cross-sectional studies. The pooled incidence in high-quality studies was 3.9% (95% Confidence Interval [CI] 1.8%-6.8%) for chorioamnionitis, 1.6% (95% CI 0.9%-2.5%) for endometritis, 1.2% (95% CI 1.0%-1.5%) for wound infection, 0.05% (95% CI 0.03%-0.07%) for sepsis, and 1.1% (95% CI 0.3%-2.4%) for maternal peripartum infection. 19% of studies met all quality criteria. There were few data from developing countries and marked heterogeneity in study designs and infection definitions, limiting the interpretation of these estimates as measures of global infection incidence. A limitation of this review is the inclusion of studies that were facility-based or restricted to low-risk groups of women. CONCLUSIONS: In this study, we observed pooled infection estimates of almost 4% in labour and between 1%-2% of each infection outcome postpartum. This indicates maternal peripartum infection is an important complication of childbirth and that preventive efforts should be increased in light of antimicrobial resistance. Incidence risk appears lower than modelled global estimates, although differences in definitions limit comparability. Better-quality research, using standard definitions, is required to improve comparability between study settings and to demonstrate the influence of risk factors and protective interventions.

Exhausted and Senescent T Cells at the Maternal-Fetal Interface in Preterm and Term Labor.

Successful pregnancy requires a tightly-regulated equilibrium of immune cell interactions at the maternal-fetal interface (i.e., the decidual tissues), which plays a central role in the inflammatory process of labor. Most of the innate immune cells in this compartment have been well characterized; however, adaptive immune cells are still under investigation. Herein, we performed immunophenotyping of the decidua basalis and decidua parietalis to determine whether exhausted and senescent T cells are present at the maternal-fetal interface and whether the presence of pathological (i.e., preterm) or physiological (i.e., term) labor and/or placental inflammation alter such adaptive immune cells. In addition, decidual exhausted T cells were sorted to test their functional status. We found that (1) exhausted and senescent T cells were present at the maternal-fetal interface and predominantly expressed an effector memory phenotype, (2) exhausted CD4+ T cells increased in the decidua parietalis as gestational age progressed, (3) exhausted CD4+ and CD8+ T cells decreased in the decidua basalis of women who underwent labor at term compared to those without labor, (4) exhausted CD4+ T cells declined with the presence of placental inflammation in the decidua basalis of women with preterm labor, (5) exhausted CD8+ T cells decreased with the presence of placental inflammation in the decidua basalis of women who underwent labor at term, (6) both senescent CD4+ and CD8+ T cells declined with the presence of placental inflammation in the decidua basalis of women who underwent preterm labor, and (7) decidual exhausted T cells produced IFNγ and TNFα upon in vitro stimulation. Collectively, these findings indicate that exhausted and senescent T cells are present at the human maternal-fetal interface and undergo alterations in a subset of women either with labor at term or preterm labor and placental inflammation. Importantly, decidual T cell function can be restored upon stimulation.

Novel anti-inflammatory actions of TIPE2 in human primary amnion and myometrial cells.

Inflammation plays a pivotal role in the terminal process of human labor and delivery, including myometrial contractions and membrane rupture. TNF-alpha-induced protein 8-like-2 (TIPE2) is a novel inflammation regulator; however, there are no studies on the role of TIPE2 in human labor. We report that in myometrium, there is decreased TIPE2 mRNA expression during late gestation which was further decreased in labor. In fetal membranes, TIPE2 mRNA expression was decreased with both term and preterm labor compared to no labor samples. Knockdown of TIPE2 by siRNA in primary myometrium and amnion cells was associated with an augmentation of IL1B and TNF-induced expression of pro-inflammatory cytokines and chemokines; expression of contraction-associated proteins and secretion of the uterotonic prostaglandin PGF2α and expression of extracellular matrix degrading enzymes. In TIPE2-deficient myometrial cells treated with inhibitors of NF-κB or ERK1/2, the secretion of pro-labor mediators was reduced back to control levels. In conclusion, these in vitro experiments indicate that loss of TIPE2 exacerbates the inflammatory response.

NOD-like receptor pyrin domain-containing-3 (NLRP3) regulates inflammation-induced pro-labor mediators in human myometrial cells.

PROBLEM: Inflammation plays a major role in preterm birth. Nucleotide-binding oligomerization domain-like receptor pyrin domain-containing-3 (NLRP3) plays a role in inflammatory diseases. The aims of this study were to determine the effect of term labor on the expression of NLRP3 in human myometrium and the effect of NLRP3 silencing on pro-labor mediators in myometrial cells. METHOD OF STUDY: NLRP3 expression was assessed in myometrium from non-laboring and laboring women by qRT-PCR and Western blotting. Human primary myometrial cells were transfected with NLRP3 siRNA (siNLRP3), treated with pro-inflammatory cytokines and toll-like receptor (TLR) ligands, and assayed for pro-inflammatory mediators' expression. RESULTS: NLRP3 expression was higher in myometrium after term spontaneous labor and by TNF, IL1B, fsl-1, and flagellin. In siNLRP3-transfected cells, there was a significant decrease in the expression of pro-inflammatory cytokines (IL1A, IL6), chemokines (CXCL8, CCL2), and adhesion molecules (ICAM1 and VCAM1) stimulated with IL1B, TNF, or TLR ligands; decrease in IL1B-stimulated PTGS2 and PTGFR mRNA expression and PGF2α release; and increase in TNF-stimulated myometrial gel shrinkage as assessed by an in vitro cell contraction assay. CONCLUSION: NLRP3 is increased with labor in myometrial, and knockdown of NLRP3 is associated with an attenuation of inflammation-induced expression of pro-inflammatory and pro-labor mediators in human myometrium.

Innate lymphoid cells at the human maternal-fetal interface in spontaneous preterm labor.

PROBLEM: Pathological inflammation is causally linked to preterm labor and birth, the leading cause of neonatal morbidity and mortality worldwide. Our aims were to investigate whether (i) the newly described family of innate lymphoid cells (ILCs) was present at the human maternal-fetal interface and (ii) ILC inflammatory subsets were associated with the pathological process of preterm labor. METHODS OF STUDY: Decidual leukocytes were isolated from women with preterm or term labor as well as from gestational age-matched non-labor controls. ILCs (CD15- CD14- CD3- CD19- CD56- CD11b- CD127+ cells) and their subsets (ILC1, T-bet+ ILCs; ILC2, GATA3+ ILCs; and ILC3, RORγt+ ILCs) and cytokine expression were identified in the decidual tissues using immunophenotyping. RESULTS: (i) The proportion of total ILCs was increased in the decidua parietalis of women with preterm labor; (ii) ILC1s were a minor subset of decidual ILCs during preterm and term gestations; (iii) ILC2s were the most abundant ILC subset in the decidua during preterm and term gestations; (iv) the proportion of ILC2s was increased in the decidua basalis of women with preterm labor; (v) the proportion of ILC3s was increased in the decidua parietalis of women with preterm labor; and (vi) during preterm labor, ILC3s had higher expression of IL-22, IL-17A, IL-13, and IFN-γ compared to ILC2s in the decidua. CONCLUSION: ILC2s were the most abundant ILC subset at the human maternal-fetal interface during preterm and term gestations. Yet, during preterm labor, an increase in ILC2s and ILC3s was observed in the decidua basalis and decidua parietalis, respectively. These findings provide evidence demonstrating a role for ILCs at the maternal-fetal interface during the pathological process of preterm labor.

Immune cell and transcriptomic analysis of the human decidua in term and preterm parturition.

STUDY QUESTION: Is labour, both at term and preterm, associated with alterations in decidual lymphocyte densities and widespread changes to the decidual transcriptome? SUMMARY ANSWER: The onset of parturition, both at term and preterm, is associated with widespread gene expression changes in the decidua, many of which are related to inflammatory signalling, but is not associated with changes in the number of any of the decidual lymphocyte populations examined. WHAT IS KNOWN ALREADY: Given its location, directly at the maternal-foetal interface, the decidua is likely to play a pivotal role in the onset of parturition, however, the molecular events occurring in the decidua in association with the onset of labour, both at term and preterm, remain relatively poorly defined. Using flow cytometry and microarray analysis, the present study aimed to investigate changes to the immune cell milieu of the decidua in association with the onset of parturition and define the decidual gene signature associated with term and preterm labour (PTL). STUDY DESIGN, SIZE, DURATION: This study used decidual samples collected from 36 women across four clinical groups: term (38-42 weeks of gestation) not in labour, TNL; term in labour, TL; preterm (<35 weeks of gestation)not in labour, PTNL; and preterm in labour, PTL. PARTICIPANTS/MATERIALS, SETTING, METHODS: Decidual lymphocytes were isolated from fresh decidual tissue collected from women in each of our four patient groups and stained with a panel of antibodies (CD45, CD3, CD19, CD56, CD4, CD8 and TCRVα24-Jα18) to investigate lymphocyte populations present in the decidua (TNL, n = 8; TL, n = 7; PTNL, n = 5; PTL, n = 5). RNA was extracted from decidual tissue and subjected to Illumina HT-12v4.0 BeadChip expression microarrays (TNL, n = 11; TL, n = 8; PTNL, n = 7; PTL, n = 10). Quantitative real-time PCR (qRT-PCR) was used to validate the microarray results. MAIN RESULTS AND THE ROLE OF CHANCE: The relative proportions of decidual lymphocytes (T cells, NK cells, B cells and invariant natural killer (iNKT) cells) were unaffected by either gestation or labour status. However, we found elevated expression of the non-classical MHC-protein, CD1D, in PTL decidua samples (P < 0.05), suggesting the potential for increased activation of decidual invariant NKT (iNKT) cells in PTL. Both term and PTL were associated with widespread gene expression changes, particularly related to inflammatory signalling. Up-regulation of candidate genes in TL (IL-6, PTGS2, ATF3, IER3 and TNFAIP3) and PTL (CXCL8, MARCO, LILRA3 and PLAU) were confirmed by qRT-PCR analysis. LARGE SCALE DATA: Microarray data are available at www.ebi.ac.uk/arrayexpress under accession number E-MTAB-5353. LIMITATIONS REASONS FOR CAUTION: Whilst no changes in lymphocyte number were observed across our patient samples, we did not investigate the activation state of any of the immune cell sub-populations examined, therefore, it is possible that the function of these cells may be altered in association with labour onset. Additionally, the results of our transcriptomic analyses are descriptive and at this stage, we cannot prove direct causal link with the up-regulation of any of the genes examined and the onset of either term or PTL. WIDER IMPLICATIONS OF THE FINDINGS: Our findings demonstrate that the onset of parturition is associated with widespread changes to the decidual transcriptome, and there are distinct gene expression changes associated with term and PTL. We confirmed that an inflammatory signature is present within the decidua, and we also report the up-regulation of several genes involved in regulating the inflammatory response. The identification of genes involved in regulating the inflammatory response may provide novel molecular targets for the development of new, more effective therapies for the prevention of preterm birth (PTB). Such targets are urgently required. STUDY FUNDING AND COMPETING INTEREST(S): This work was supported by Medical Research Council (grant number MR/L002657/1) and Tommy's, the baby charity. Jane Norman has had research grants from the charity Tommy's and from the National Institute for Health Research on PTB during the lifetime of this project. Jane Norman also sits on a data monitoring committee for GSK for a study on PTB prevention and her institution receives financial recompense for this. The other authors do not have any conflicts of interest to declare.

TLR2, TLR3 and TLR5 regulation of pro-inflammatory and pro-labour mediators in human primary myometrial cells.

Preterm birth continues to be a significant global health care issue, due to our lack of understanding of the mechanisms that drive human labour and delivery. Toll-like receptors (TLRs) are essential in triggering an inflammatory response in human gestational tissues, leading to the production of pro-inflammatory and pro-labour mediators, and thus preterm birth. The aims of this study were to determine whether the adaptor molecules associated with TLR2, TLR3 and TLR5 signalling are involved in human myometrium. Primary human myometrial cells were transfected with siRNA against TIRAP, IRAK1, IRAK4, TAK1and stimulated with bacterial product fsl-1 (TLR2); TRIF, TRADD, TRAF6, RIP1, TAK1 and stimulated with dsRNA viral analogue poly(I:C) (TLR3); IRAK1, IRAK4, TAK1 and stimulated with bacterial product flagellin (TLR5), and assayed for production of pro-inflammatory and pro-labour mediators. Cells transfected with TIRAP, IRAK1, IRAK4 or TAK1 all showed a decrease in fsl-1-induced expression of cytokines (IL-1α, IL-1ß, IL-6), chemokines (GRO-α, IL-8, MCP-1), adhesion molecule ICAM-1, cyclooxygenase (COX)-2 mRNA and release of PGF2α and MMP-9 expression. Cells transfected with TRIF, TRAF6, RIP1 or TAK1 all decreased production of poly(I:C)-induced IL-1α, IL-1ß, IL-6, GRO-α, IL-8, MCP-1, ICAM-1 and MMP-9 expression. Cells transfected with IRAK1, IRAK4 or TAK1 all showed decreased expression of flagellin-induced cytokine and chemokine expression, ICAM-1 and MMP-9 expression. Lastly, transfection with these siRNAs decreased fsl-1, poly(I:C) and flagellin-induced NF-κB transcriptional activity. Our study signifies that these adaptor molecules are necessary for the proper production of cytokines, chemokines and pro-labour mediators after TLR ligation.

Cell-free DNA, inflammation, and the initiation of spontaneous term labor.

BACKGROUND: Hypomethylated cell-free DNA from senescent placental trophoblasts may be involved in the activation of the inflammatory cascade to initiate labor. OBJECTIVE: To determine the changes in cell-free DNA concentrations, the methylation ratio, and inflammatory markers between women in labor at term vs women without labor. STUDY DESIGN: In this prospective cohort study, eligible participants carried a nonanomalous singleton fetus. Women with major medical comorbidity, preterm labor, progesterone use, aneuploidy, infectious disease, vaginal bleeding, abdominal trauma, or invasive procedures during the pregnancy were excluded. Maternal blood samples were collected at 28 weeks, 36 weeks, and at admission for delivery. Total cell-free DNA concentration, methylation ratio, and interleukin-6 were analyzed. The primary outcome was the difference in methylation ratio in women with labor vs without labor. Secondary outcomes included the longitudinal changes in these biomarkers corresponding to labor status. RESULTS: A total of 55 women were included; 20 presented in labor on admission and 35 presented without labor. Women in labor had significantly greater methylation ratio (P = .001) and interleukin-6 (P < .001) on admission for delivery than women without labor. After we controlled for body mass index and maternal age, methylation ratio (adjusted relative risk, 1.38; 95% confidence interval, 1.13 to 1.68) and interleukin-6 (adjusted relative risk, 1.12, 95% confidence interval, 1.07 to 1.17) remained greater in women presenting in labor. Total cell-free DNA was not significantly different in women with labor compared with women without. Longitudinally, total cell-free DNA (P < .001 in labor, P = .002 without labor) and interleukin-6 (P < .001 in labor, P = .01 without labor) increased significantly across gestation in both groups. The methylation ratio increased significantly in women with labor from 36 weeks to delivery (P = .02). CONCLUSION: Spontaneous labor at term is associated with a greater cell-free DNA methylation ratio and interleukin-6 compared with nonlabored controls. As gestation advances, total cell-free DNA concentrations and interleukin-6 levels increase. A greater methylation ratio reflects a greater maternal contribution (vs placental) in women with labor, likely resulting from greater levels of neutrophils, lymphocytes, and uterine activation proteins at the time of labor. Although not significant, women in labor had a greater total cell-free DNA concentration and thus could theoretically have more hypomethylated DNA available for interaction with the inflammatory cascade. Larger studies are needed to investigate this theory.

Immunophenotyping and activation status of maternal peripheral blood leukocytes during pregnancy and labour, both term and preterm.

The onset of labour in rodents and in humans is associated with physiological inflammation which is manifested by infiltration of activated maternal peripheral leukocytes (mPLs) into uterine tissues. Here, we used flow cytometry to immunophenotype mPLs throughout gestation and labour, both term and preterm. Peripheral blood was collected from non-pregnant women and pregnant women in the 1st, 2nd and 3rd trimesters. Samples were also collected from women in active labour at term (TL) or preterm (PTL) and compared with women term not-in-labour (TNIL) and preterm not-in-labour (PTNIL). Different leukocyte populations were identified by surface markers such as CD45, CD14, CD15, CD3, CD4, CD8, CD19 and CD56. Their activation status was measured by the expression levels of CD11b, CD44, CD55, CD181 and CD192 proteins. Of all circulating CD45+ leukocytes, we detected significant increases in CD15+ granulocytes (i) in pregnant women versus non-pregnant; (ii) in TL women versus TNIL and versus pregnant women in the 1st/2nd/3rd trimester; (iii) in PTL women versus PTNIL. TL was characterized by (iv) increased expressions of CD11b, CD55 and CD192 on granulocytes; (v) increased mean fluorescent intensity (MFI) of CD55 and CD192 on monocytes; (vi) increased CD44 MFI on CD3+ lymphocytes as compared to late gestation. In summary, we have identified sub-populations of mPLs that are specifically activated in association with gestation (granulocytes) or with the onset of labour (granulocytes, monocytes and lymphocytes). Additionally, beta regression analysis created a set of reference values to rank this association between immune markers of pregnancy and to identify activation status with potential prognostic and diagnostic capability.

Optineurin suppression activates the mediators involved in the terminal effector pathways of human labour and delivery.

Spontaneous preterm birth remains the major cause of neonatal death and morbidity. Studies in non-gestational tissues report that optineurin (OPTN) is critical in the termination of NFKB1 activity and control of inflammation, central features of spontaneous preterm birth. The aims of the present study were to determine: (1) OPTN expression in fetal membranes and the myometrium during labour; (2) the effects of IL1B on OPTN expression in primary myometrial cells; and (3) the effects of OPTN short interference (si) RNA on IL1B-stimulated proinflammatory and prolabour mediators. OPTN mRNA and protein expression was significantly decreased with spontaneous term labour in fetal membranes and the myometrium. Although there was no effect of spontaneous preterm labour on OPTN expression in fetal membranes, there was decreased OPTN expression in membranes with chorioamnionitis and myometrial cells treated with 1ng mL-1 IL1B for 1 or 6h. In cells transfected with OPTN siRNA, significant increases were seen in IL1B-stimulated IL6, tumour necrosis factor, CXCL8 and monocyte chemoattractant protein-1 mRNA expression and release, cyclo-oxygenase-2 and prostanoid PTGFR receptor mRNA expression and the release of prostaglandin F2α. There was no change in IL1B-stimulated NFKBIA expression; however, there was increased NFKB1 p65 DNA-binding activity. The results of the present study suggest that OPTN is a negative regulator of inflammation-induced prolabour mediators.

A Role for the Inflammasome in Spontaneous Labor at Term with Acute Histologic Chorioamnionitis.

Inflammasomes are cytosolic signaling platforms that regulate the activation of caspase (CASP)-1, which induces the maturation of interleukin (IL)-1ß and IL-18. Herein, we determined whether the chorioamniotic membranes from women in spontaneous labor at term with acute histologic chorioamnionitis express major inflammasome components and whether these changes are associated with the activation of CASP-1 and CASP-4 and the release of mature IL-1ß and IL-18. When comparing the chorioamniotic membranes from women in spontaneous labor at term with acute histologic chorioamnionitis to those without this placental lesion, we found that (1) the messenger RNA (mRNA) abundance of NLR family pyrin domain containing 3 ( NLRP3), NLR family CARD domain containing 4 ( NLRC4), absent in melanoma 2 ( AIM2), and nucleotide binding oligomerization domain 2 ( NOD2) was higher; (2) the NLRP3 and NLRC4 protein quantities were increased; (3) the mRNA and protein expressions of CASP-1 and its active forms were greater; (4) CASP-4 was increased at the mRNA level only; (5) the mRNA and protein expressions of IL-1ß and its mature form were higher; and (6) a modest increase in the total protein concentration and abundance of the mature form of IL-18 was observed. In vitro incubation of the chorioamniotic membranes with the CASP-1 inhibitor, VX765, decreased the release of endotoxin-induced IL-1ß and IL-18 (2-fold) but not IL-6 or tumor necrosis factor α. In conclusion, spontaneous labor at term with acute histologic chorioamnionitis is characterized by an upregulation of inflammasome components which, in turn, may participate in the activation of CASP-1 and lead to the release of mature IL-1ß by the chorioamniotic membranes. These results support a role for the inflammasome in the mechanisms responsible for spontaneous labor at term with acute histologic chorioamnionitis.

Lactate produced during labor modulates uterine inflammation via GPR81 (HCA1).

BACKGROUND: Uterine inflammatory processes trigger prolabor pathways and orchestrate on-time labor onset. Although essential for successful labor, inflammation needs to be regulated to avoid uncontrolled amplification and resolve postpartum. During labor, myometrial smooth muscle cells generate ATP mainly via anaerobic glycolysis, resulting in accumulation of lactate. Aside from its metabolic function, lactate has been shown to activate a G protein-coupled receptor, GPR81, reported to regulate inflammation. We therefore hypothesize that lactate produced during labor may act via GPR81 in the uterus to exert in a feedback manner antiinflammatory effects, to resolve or mitigate inflammation. OBJECTIVE: We sought to investigate the role of lactate produced during labor and its receptor, GPR81, in regulating inflammation in the uterus. STUDY DESIGN: We investigated the expression of GPR81 in the uterus and the pharmacological role of lactate acting via GPR81 during labor, using shRNA-GPR81 and GPR81-/- mice. RESULTS: (1) Uterine lactate levels increased substantially from 2 to 9 mmol/L during labor. (2) Immunohistological analysis revealed expression of GPR81 in the uterus with high expression in myometrium. (3) GPR81 expression increased during gestation, and peaked near labor. (4) In primary myometrial smooth muscle cell and ex vivo uteri from wild-type mice, lactate decreased interleukin-1ß-induced transcription of key proinflammatory Il1b, Il6, Ccl2, and Pghs2; suppressive effects of lactate were not observed in cells and tissues from GPR81-/- mice. (5) Conversely, proinflammatory gene expression was augmented in the uterus at term in GPR81-/- mice and wild-type mice treated intrauterine with lentiviral-encoded shRNA-GPR81; GPR81 silencing also induced proinflammatory gene transcription in the uterus when labor was induced by endotoxin (lipopolysaccharide). (6) Importantly, administration to pregnant mice of a metabolically stable specific GPR81 agonist, 3,5-dihydroxybenzoic acid, decreased endotoxin-induced uterine inflammation, preterm birth, and associated neonatal mortality. CONCLUSION: Collectively, our data uncover a novel link between the anaerobic glycolysis and the control of uterine inflammation wherein the high levels of lactate produced during labor act on uterine GPR81 to down-regulate key proinflammatory genes. This discovery may represent a novel feedback mechanism to regulate inflammation during labor, and conveys a potential rationale for the use of GPR81 agonists to attenuate inflammation and resulting preterm birth.

Immune Modifications in Fetal Membranes Overlying the Cervix Precede Parturition in Humans.

In humans, parturition is currently viewed as an intrauterine outbreak of inflammation, accompanied by a massive release of proinflammatory cytokines at the maternal-fetal interface that comprises the maternal decidua, placenta, and fetal membranes. At term, fetal membranes overlying the cervix, the future site of rupture, show altered morphology and are termed the zone of altered morphology (ZAM). These alterations occur in normal fetal membranes during late pregnancy, in preparation for labor. In this study, transcriptome, flow cytometry, electron microscopy, and immunohistochemistry analyses collectively highlight a local shift in gene expression and lymphocyte activation in the ZAM. Just before labor, we show that highly polymorphic HLA-A, -B, and -C determinants of fetal origin are selectively exposed in the ZAM to the maternal immune system. A graft rejection-like program occurs in the ZAM, which involves 1) the activation of cytotoxic decidual NK cells, and 2) the decline of decidual immunotolerant M2-like macrophages. Comparison with a prior cohort of fetal membranes shows that acute inflammation only takes place after these first steps of immune modifications. Our results therefore strongly argue in favor of local immune remodeling at the onset of parturition.

Childbirth and consequent atopic disease: emerging evidence on epigenetic effects based on the hygiene and EPIIC hypotheses.

BACKGROUND: In most high and middle income countries across the world, at least 1:4 women give birth by cesarean section. Rates of labour induction and augmentation are rising steeply; and in some countries up to 50% of laboring women and newborns are given antibiotics. Governments and international agencies are increasingly concerned about the clinical, economic and psychosocial effects of these interventions. DISCUSSION: There is emerging evidence that certain intrapartum and early neonatal interventions might affect the neonatal immune response in the longer term, and perhaps trans-generationally. Two theories lead the debate in this area. Those aligned with the hygiene (or 'Old Friends') hypothesis have examined the effect of gut microbiome colonization secondary to mode of birth and intrapartum/neonatal pharmacological interventions on immune response and epigenetic phenomena. Those working with the EPIIC (Epigenetic Impact of Childbirth) hypothesis are concerned with the effects of eustress and dys-stress on the epigenome, secondary to mode of birth and labour interventions. This paper examines the current and emerging findings relating to childbirth and atopic/autoimmune disease from the perspective of both theories, and proposes an alliance of research effort. This is likely to accelerate the discovery of important findings arising from both approaches, and to maximize the timely understanding of the longer-term consequences of childbirth practices.

Exposures influencing total IgA level in colostrum.

Immunoglobulin A (IgA) is a predominant immunoglobulin present in human breast milk and is known to play an important role in infant gut immunity maturation. Breast milk composition varies between populations, but the environmental and maternal factors responsible for these variations are still unclear. We examined the relationship between different exposures and levels of IgA in colostrum. The objective of this study was to examine whether exposures analysed influence levels of IgA in colostrum. The present study used 294 colostrum samples from the MecMilk International cohort, collected from women residing in London, Moscow and Verona. Samples were analysed in automated Abbott Architect Analyser. We found an inverse correlation between time postpartum and colostrum total IgA level (r=-0.49, P<0.001). Adjusting for maternal parity, smoking, fresh fruit and fish consumption and allergen sensitization, multiple regression model showed that IgA levels were influenced by colostrum collection time (P<0.0001) and country of collection (P<0.01). Mode of delivery influence did not appear to be significant in univariate comparisons, once adjusted for the above maternal characteristics it showed a significant influence on total IgA (P=0.01). We conclude that the concentration of IgA in colostrum drops rapidly after birth and future studies should always consider this factor in analysis. IgA concentration varied significantly between countries, with the highest level detected in Moscow and lowest in Verona. Mode of delivery effect should be confirmed on larger cohorts. Further work is needed to determine ways to correct for IgA decline over time in colostrum, and to find the cause of variations in IgA levels between the countries.

Can membrane progesterone receptor α on T regulatory cells explain the ensuing human labour?

Progesterone acts as an immunosteroid by contributing to the establishment of a pregnancy-protective milieu. It seems that it is the responsibility of progesterone to evade the inflammatory events that lead to parturition. T regulatory lymphocytes (Treg cells) could further explain the inhibition of the inflammatory mechanisms that lead to labour through the rapid action of progesterone on this cell subset. We investigated Treg cells and the membrane progesterone receptor α (mPRα) in these immune cells with in relationship to human parturition. This pilot cohort study was conducted in a single-centre tertiary obstetrical unit with 20 normal pregnant women. Variation in the absolute and relative frequency of CD4(+) T cells, Treg cells, and of mPR(α+) Treg cells was calculated by flow cytometry on three occasions (second and third trimesters; delivery day). Our results show that during normal pregnancy there is a generalised increase in Treg cells and mPR(α+) Treg cells, from the second to the third trimesters (23.4% vs. 52.3% and 4.3% vs. 8.3%, respectively). On the contrary, on delivery day, compared with the values in the third trimester, there is a sudden decrease in both Treg cells (52.3% vs. 17.4%) and mPR(α+) Treg cells (8.3% vs. 6.1%). Our findings suggest that human labour may develop as a consequence of a decline in mPR(α+) Treg cells, which reduces progesterone anti-inflammatory action through Treg cells.