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The incidence of preterm birth in developed countries has risen in the past decades. Underlying causes for this enigmatic pregnancy complication are numerous, yet infectious agents that induce dysregulation of immunity at the maternal-fetal interface pose one of the most probable causes of preterm birth. This paper highlights two factors regarding maternal infections that trigger unscheduled inflammatory sequences that are deleterious to the maternal-fetal balance necessary to maintain pregnancy. Firstly, we discuss the role of Toll-like receptors (TLRs) as sentinels of uterine immunity in the context of response to pathogens. We highlight the idea that particular TLR activations lead to differential immune cascades that induce preterm birth. Secondly, two alternative routes of pathogenic entry may prove to be critical for inducing preterm birth via a cytokine storm or a secondary and currently unknown cell-mediated mechanism of uterine inflammation. This paper summarizes pathways that underlie activation of adverse and diverse immune responses to foreign agents that may result in preterm birth.

Chorioamnionitis (CAM) is a major cause of preterm delivery. Inflammatory cytokines and chemokines play important roles in the pathogenesis of preterm delivery. Interleukin (IL)-17 is a key cytokine which induces inflammation and is critical to host defense. In this study, we examined the role of IL-17 in the pathogenesis of preterm delivery. The levels of cytokines including IL-17, IL-8 and tumor necrosis factor (TNF) alpha were measured by ELISA in amniotic fluid from 154 cases of preterm labor. Flow cytometry and immunohistochemical staining were performed to determine the distribution of IL-17-producing cells. IL-8 secretion was evaluated in primary cultured human amniotic mesenchymal (HAM) cells and human amniotic epithelial (HAE) cells stimulated with IL-17, TNFalpha or IL-1beta. We also studied the signaling pathway of IL-17 and TNFalpha in HAM cells. Levels of inflammatory cytokines in amniotic fluid were higher in preterm delivery cases than in term delivery cases. Furthermore, IL-8, IL-17 and TNFalpha levels were significantly higher in the preterm cases with CAM stage II or III than those without CAM. Flow cytometry and immunohistochemical staining revealed that CD3(+)CD4(+) T cells were the main source of IL-17 in the chorioamniotic membrane. Interestingly, TNFalpha-induced IL-8 secretion was enhanced by IL-17 in a dose-dependent manner in HAM cells. The IKK inhibitor BMS-345541 and mitogen-activated protein kinase (MAPK) inhibitors p38, JNK and p42/44 (ERK1/2 pathway) reduced IL-8 secretion by IL-17-stimulated and TNFalpha-stimulated HAM cells. These results indicate that IL-17, produced by T cells, promotes inflammation at the fetomaternal interface in preterm delivery., (2009 Elsevier Ireland Ltd. All rights reserved.)

Objective: Intra-amniotic infection/inflammation (IAI) is one of the most important mechanisms of disease in preterm birth. Triggering receptor expressed on myeloid cells (TREM)- 1 is a transmembrane glycoprotein expressed by neutrophils, macrophages and mature monocytes. TREM-1 is upregulated in biological fluids and tissues infected by Gram (+) and Gram (-) bacteria and fungi, amplifies the production of pro-inflammatory cytokines and chemokines, and its soluble form (sTREM-1) is released in the presence of infection. The aim of this study was to determine the effect of gestational age, parturition (term and preterm) and IAI in the amniotic fluid (AF) concentrations of sTREM-1., Study Design: This cross-sectional study included 434 patients in the following groups: (1) mid-trimester of pregnancy (14-18 weeks, n = 38); (2) normal pregnant women at term with (n = 39) and without (n = 39) labor; (3) patients with spontaneous preterm labor (PTL) and intact membranes classified into: (a) PTL who delivered at term (n = 99); (b) PTL who delivered preterm (<37 weeks gestation) without IAI (n = 80); and (c) PTL with IAI (n = 59); and (4) women with preterm prelabor rupture of membranes (PROM) with (n = 40) and without (n = 40) IAI. The AF concentration of sTREM-1 was determined by enzyme-linked immunoassay. Non-parametric statistics were used for analyses., Results: (1) sTREM-1 was detected in all the AF samples; (2) the median AF sTREM-1 concentration at term was higher than in the mid-trimester (4277.6 pg/ml vs. 1140.4 pg/ml; p < 0.001); (3) among patients with PTL, the median AF sTREM-1 concentration was higher in patients with IAI than in those without IAI (6154.4 pg/ml vs. 3282.8 pg/ml; p < 0.001) and those with PTL who delivered at term (6154.4 pg/ml vs. 2794 pg/ml; p < 0.001); (4) patients with preterm PROM with IAI had a higher median AF sTREM-1 concentration than those without IAI (7893.1 pg/ml vs. 3386.6 pg/ml; p < 0.001); (5) no differences were observed in the median AF sTREM-1 concentration between patients with spontaneous labor at term and those at term not in labor (4712.4 pg/ml vs. 4277.6 pg/ml; respectively p = 0.4); and 6) an AF sTREM-1 concentration >or=6416 pg/ml (derived from a ROC curve) had a sensitivity of 72% and a specificity of 89% for the diagnosis of intra-amniotic infection., Conclusions: sTREM-1 is a physiologic constituent of the AF, and its concentration: (1) is significantly elevated in the presence of IAI; (2) increases with advancing gestation; and (3) does not change in the presence of spontaneous labor at term. We propose that sTREM-1 play a role in the innate immune response against intra-amniotic infection.

Objective: Fragment Bb is an activator of the alternative pathway of the complement system. Recently, increased first trimester maternal plasma concentrations of this fragment were reported in patients destined to have a spontaneous preterm delivery before 34 weeks of gestation. The aim of this study was to determine whether the amniotic fluid (AF) concentrations of fragment Bb change with gestational age, spontaneous labor (term and preterm) and in the presence of intra-amniotic infection/inflammation (IAI)., Study Design: This cross-sectional study included patients in the following groups: (1) mid-trimester (n = 64); (2) term in spontaneous labor (n = 70); (3) term not in labor (n = 43); (4) spontaneous preterm labor (PTL) who delivered at term (n = 76); (5) PTL without IAI who delivered preterm (n = 73); (6) PTL with IAI (n = 76); (7) preterm prelabor rupture of membranes (PROM) without IAI (n = 71); and (8) preterm PROM with IAI (n = 71). Fragment Bb concentration in AF was determined by an enzyme-linked immunoassay. Non-parametric statistics were used for analyses., Results: (1) Fragment Bb was detected in all AF samples (n = 544); (2) The median AF concentration of fragment Bb in patients at term not in labor was significantly higher than that of those in the mid-trimester [2.42 microg/ml, interquartile range (IQR) 1.78-3.22 vs. 1.64 microg/ml, IQR 1.06-3.49; p < 0.001]; (3) Among patients with PTL, those with IAI had a higher median AF fragment Bb concentration than that of woman without IAI, who delivered preterm (4.82 microg/ml, IQR 3.32-6.08 vs. 3.67 microg/ml, IQR 2.35-4.57; p < 0.001) and than that of women with an episode of PTL, who delivered at term (3.21 microg/ml, IQR 2.39-4.16; p < 0.001); (4) Similarly, among patients with preterm PROM, the median AF fragment Bb concentration was higher in individuals with IAI than in those without IAI (4.24 microg/ml, IQR 2.58-5.79 vs. 2.79 microg/ml, IQR 2.09-3.89; p < 0.001). (5) Among patients at term, the median AF fragment Bb concentration did not differ between women with spontaneous labor and those without labor (term in labor: 2.47 microg/ml, IQR 1.86-3.22; p = 0.97)., Conclusions: (1) Fragment Bb, an activator of the alternative complement pathway, is a physiologic constituent of the AF, and its concentration increases with advancing gestational age; (2) AF concentrations of fragment Bb are higher in pregnancies complicated with IAI; and (3) labor at term is not associated with changes in the AF concentrations of fragment Bb. These findings suggest a role for fragment Bb in the host immune response against IAI.

Intra-amniotic infection/inflammation (IAI) is a major cause of preterm birth, but the mechanisms responsible are not well understood. This study investigates the effects of IAI on vascular endothelial growth factor (VEGF) as well as VEGF receptor (Flt1, KDR2) and coreceptor (neuropilin-1 and -2) messenger RNA (mRNA) and protein expression at the maternal-fetal interface, both in vitro and in vivo. Decidual stromal cells (DSCs) were isolated from term placentae, purified, and treated with 10(-8) mol/L estradiol (E(2)), 10( -7) mol/L medroxyprogesterone acetate (MPA), both, or vehicle for 7 days. Vascular endothelial growth factor expression in cultured DSCs increased in response to stimulation with interleukin 1 beta (IL-1 beta; 0.01-10 ng/mL)--but not tumor necrosis factor alpha (TNF-alpha; 1 ng/mL)--in a concentration-dependent fashion irrespective of the hormonal milieu. This effect appears to be mediated at the level of gene transcription because stimulation with IL-1 beta (but not TNF-alpha) increased expression of VEGF mRNA as measured by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR); a similar increase was seen in neuropilin-1/-2 (but not Flt1 and KDR2) mRNA. Immunohistochemical studies confirmed these observations in vivo. Immunostaining for VEGF and neuropilin-1/-2 (but not Flt1 or KDR2) was increased in serial tissue sections of decidua from women with clinical and histological evidence of IAI versus noninfected controls, and in cultured term DSCs exposed to IL-1 beta. The novel observations that IL-1 beta stimulates VEGF and neuropilin-1/-2 mRNA and protein expression in term DSCs in vitro along with confirmatory in vivo data using immunohistochemistry provide a mechanism by which IAI can alter vascular permeability, thereby facilitating leukocyte trafficking and increasing the risk of abruption, both of which are associated with preterm birth.

Chorioamnionitis is the most significant source of prenatal inflammation and preterm delivery. Prematurity and prenatal inflammation are associated with compromised postnatal developmental outcomes, of the intestinal immune defence, gut barrier function and the vascular system. We developed a sheep model to study how the antenatal development of the gut was affected by gestation and/or by endotoxin induced chorioamnionitis.Chorioamnionitis was induced at different gestational ages (GA). Animals were sacrificed at low GA after 2d or 14d exposure to chorioamnionitis. Long term effects of 30d exposure to chorioamnionitis were studied in near term animals after induction of chorioamnionitis. The cellular distribution of tight junction protein ZO-1 was shown to be underdeveloped at low GA whereas endotoxin induced chorioamnionitis prevented the maturation of tight junctions during later gestation. Endotoxin induced chorioamnionitis did not induce an early (2d) inflammatory response in the gut in preterm animals. However, 14d after endotoxin administration preterm animals had increased numbers of T-lymphocytes, myeloperoxidase-positive cells and gammadelta T-cells which lasted till 30d after induction of chorioamnionitis in then near term animals. At early GA, low intestinal TLR-4 and MD-2 mRNA levels were detected which were further down regulated during endotoxin-induced chorioamnionitis. Predisposition to organ injury by ischemia was assessed by the vascular function of third-generation mesenteric arteries. Endotoxin-exposed animals of low GA had increased contractile response to the thromboxane A2 mimetic U46619 and reduced endothelium-dependent relaxation in responses to acetylcholine. The administration of a nitric oxide (NO) donor completely restored endothelial dysfunction suggesting reduced NO bioavailability which was not due to low expression of endothelial nitric oxide synthase.Our results indicate that the distribution of the tight junctional protein ZO-1, the immune defence and vascular function are immature at low GA and are further compromised by endotoxin-induced chorioamnionitis. This study suggests that both prematurity and inflammation in utero disturb fetal gut development, potentially predisposing to postnatal intestinal pathology.

Rationale: Chorioamnionitis frequently associates with preterm delivery and increased amniotic fluid IL-1, and causes fetal lung and systemic inflammation. However, chorioamnionitis is also associated with a paradoxical reduction in the incidence of surfactant deficiency-related respiratory distress syndrome in preterm infants., Objectives: To identify the role of IL-1 signaling in the mediation of pulmonary and systemic inflammation and lung maturation in a fetal sheep model of lipopolysaccharide (LPS) induced chorioamnionitis., Methods: After confirming the efficacy of recombinant human IL-1 receptor antagonist (rhIL-1ra), fetal sheep were exposed to intraamniotic (IA) injections of Escherichia coli LPS with or without prior IA injections of rhIL-1ra. Preterm lambs were delivered at 82% of term gestation., Measurements and Main Results: rhIL-1ra decreased IA LPS-induced lung inflammation assessed by decreased lung neutrophil and monocyte influx, inducible nitric oxide synthase expression, lung IL-6 and IL-1beta mRNA expression, and airway myeloperoxidase concentrations. rhIL-1ra inhibited IA LPS-induced fetal systemic inflammation assessed by decreased plasma IL-8, protein carbonyls, blood neutrophilia, and the expression of serum amyloid A3 mRNA in the liver. rhIL-1ra also partially blocked the lung maturational effects of IA LPS. Therefore blockade of IL-1 signaling in the amniotic compartment inhibited fetal lung and systemic inflammation and lung maturation in response to LPS-induced chorioamnionitis., Conclusions: IL-1 plays a central role in the pathogenesis of chorioamnionitis-induced fetal inflammatory responses.

Objective: The aim of this study was to characterize the pregnant guinea pig cytokine time course following a maternal inflammatory insult., Study Design: Pregnant guinea pigs (n = 34) were injected intraperitoneally with 100 microg/kg lipopolysaccharide (LPS) at 70% gestation and euthanized at 24 hours, 48 hours, or 5 days. Control animals were euthanized at 70% gestation without LPS exposure. Interleukin (IL)-6, IL-1beta, and tumor necrosis factor-alpha (TNF-alpha) were quantified in the maternal serum and amniotic fluid by an enzyme-linked immunosorbent assay., Results: IL-6 and IL-1beta concentrations were elevated in the maternal serum at 24 hours and returned to control levels by 5 days. In the amniotic fluid, IL-6 peaked at 48 hours and IL-1beta at 24 hours. TNF-alpha levels were not significantly increased., Conclusion: Maternal intraperitoneal LPS injection produces transient increases in cytokine concentrations in the maternal serum and amniotic fluid within 5 days, further implicating the cytokines as mediators of fetal white matter damage.

Objective: We sought to determine the relationship between the degree of cervical shortening and intraamniotic inflammation in patients presenting with a midtrimester short cervix., Study Design: Amniocentesis was performed on singleton pregnancies between 16-24 weeks' gestation with a sonographic cervical length (CL) 1500 pg/mL, CL of 5 mm had an 86% sensitivity, 85% specificity, 58% positive predictive value, and 96% negative predictive value to predict elevated MCP-1 levels. After excluding patients with intraamniotic infection or labor, findings were similar., Conclusion: CL

Epidemiological studies suggest that intra-uterine exposure to inflammation may prime postnatal immune responses. In fetal sheep, intra-amniotic injection of lipopolysaccharide (LPS) induced chorioamnionitis, lung inflammation and maturation, matured lung monocytes to macrophages and initiated systemic tolerance of fetal monocytes to subsequent challenge with LPS. We hypothesized that LPS-mediated chorioamnionitis altered the response of lung and blood monocytes to Toll-like receptor (TLR) ligands such as PamCysK4 (TLR2), flagellin (TLR5), and human CpG-DNA (TLR9). Time-mated ewes were given intra-amniotic injections of LPS or saline. Blood and lung monocytes were assessed after 2 days, 7 days and 2 days and 7 days repetitive LPS injections before delivery at 124 days gestational age (term 150 days). Responsiveness of blood and lung monocytes to TLR-ligands in vitro was assessed by interleukin (IL)-6, tumor necrosis factor-alpha (TNF-alpha) and hydrogen peroxide. Monocytes from preterm controls had minimal responses. Lipopolysaccharide-mediated chorioamnionitis increased IL-6, TNF- alpha and hydrogen peroxide to all TLR agonists in blood and lung monocytes. Repetitive exposure to antenatal LPS reduced IL-6, TNF- alpha and hydrogen peroxide to TLR-ligands suggesting tolerance. Tolerance to TLR-ligands reduced IL-1 receptor associated kinase-4 expression. Thus, repeated fetal exposure to LPS induced tolerance to other TLR-ligands. These modulations of fetal innate immunity have implications for host defense and injury responses in preterm infants.

Background: Porphyromonas gingivalis (Pg) is one of the most harmful periodontal pathogens and it has been reported that Pg is associated with preterm birth (PTB), intrauterine growth retardation (IUGR) and pregnancy-induced hypertension (PIH), discovered by animal experiments and clinical research. The relationship between adverse pregnancy outcomes and maternal antibody response to Pg is controversial. On the other hand, the serum C-reactive protein (CRP) has been recognised as a reliable serum marker of periodontal disease., Aims: To determine the significance of antibody responses to Pg affecting pregnancy outcomes in the first trimester., Methods: A case-control study was carried out on women with PTB (n = 58), IUGR (n = 91), PIH (n = 32) and without any complications (control, n = 98). The serum level of the CRP and IgG1 against 40-kDa outer membrane protein of Pg (anti-40-kDa OMP Pg-IgG1) in the first trimester was measured., Results: The IUGR group, and PTB patients whose placentas were diagnosed as chorioamnionitis or whose vaginal flora included Lactobacilli, showed a lower level of anti-40-kDa OMP Pg-IgG1 than the control group. There was no difference in the serum CRP level between each case group and control group., Conclusions: These results suggest that a lack of humoral immunity against Pg in early pregnancy is associated with IUGR and some PTB.

Chorioamnionitis is associated with increased lung and brain injury in premature infants. Ureaplasma is the microorganisms most frequently associated with preterm birth. Whether Ureaplasma-induced antenatal inflammation worsens lung and brain injury is unknown. We developed a mouse model combining antenatal Ureaplasma infection and postnatal oxygen exposure. Intraamniotic Ureaplasma Parvum (UP) increased proinflammatory cytokines in placenta and fetal lungs. Antenatal exposure to UP or broth caused mild postnatal inflammation and worsened oxygen-induced lung injury. Antenatal UP exposure induced central microgliosis and disrupted brain development as detected by decreased number of calbindin-positive and calretinin-positive neurons in the neocortex. Postnatal oxygen decreased calretinin-positive neurons in the neocortex but combined with antenatal UP exposure did not worsen brain injury. Antenatal inflammation exacerbates the deleterious effects of oxygen on lung development, but the broth effects prohibit concluding that UP by itself is a compounding risk factor for bronchopulmonary dysplasia. In contrast, antenatal UP-induced inflammation alone is sufficient to disturb brain development. This model may be helpful in exploring the pathophysiology of perinatal lung and brain injury to develop new protective strategies.

The co-presence of histoincompatible fetal and maternal cells is a characteristic of human placental inflammation. Villitis of unknown etiology (VUE), a destructive inflammatory lesion of villous placenta, is characterized by participation of Hofbauer cells (placental macrophages) and maternal T cells. In contrast to acute chorioamnionitis of infection-related origin, the fundamental immunopathology of VUE is unknown. This study was performed to investigate the placental transcriptome of VUE and to determine whether VUE is associated with systemic maternal and/or fetal inflammatory response(s). Comparison of the transcriptome between term placentas without and with VUE revealed differential expression of 206 genes associated with pathways related to immune response. The mRNA expression of a subset of chemokines and their receptors (CXCL9, CXCL10, CXCL11, CXCL13, CCL4, CCL5, CXCR3, CCR5) was higher in VUE placentas than in normal placentas (p < 0.05). Analysis of blood cell mRNA showed a higher expression of CXCL9 and CXCL13 in the mother, and CXCL11 and CXCL13 in the fetus of VUE cases (p < 0.05). The median concentrations of CXCL9, CXCL10, and CXCL11 in maternal and fetal plasma were higher in VUE (p < 0.05). Comparison of preterm cases without and with acute chorioamnionitis revealed elevated CXCL9, CXCL10, CXCL11, and CXCL13 concentrations in fetal plasma (p < 0.05), but not in maternal plasma with chorioamnionitis. We report for the first time the placental transcriptome of VUE. A systemic derangement of CXC chemokines in maternal and fetal circulation distinguishes VUE from acute chorioamnionitis. We propose that VUE be a unique state combining maternal allograft rejection and maternal antifetal graft-vs-host disease mechanisms.

Objective: The fetal inflammatory response syndrome (FIRS) is present in a fraction of fetuses exposed to intra-amniotic infection and is associated with the impending onset of labor and multisystem organ involvement. Neonates born with funisitis, the histologic counterpart of fetal systemic inflammation, are at increased risk for cerebral palsy and bronchopulmonary dysplasia. The aim of this study was to determine whether fetal and maternal granulocytes and monocytes have the phenotypic and metabolic characteristics of activation in cases with FIRS., Study Design: A case-control study was conducted with umbilical cord and maternal blood samples obtained from patients who delivered preterm with (n=30) and without funisitis (n=15). The phenotypic characteristics of granulocytes and monocytes were examined using flow cytometry and monoclonal antibodies including CD11b, CD14, CD15, CD16, CD18, CD49d, CD62L, CD64, CD66b, and HLA-DR. Intracellular reactive oxygen species (iROS) were measured at the basal state and after stimulation (oxidative burst). A P<0.01 was considered statistically significant., Results: (1) Funisitis was associated with a significant increase in the median mean channel brightness (MCB) of CD14, CD64, and CD66b on granulocytes and the MCB of CD64 on monocytes collected from umbilical cord blood. (2) The basal iROS production and oxidative burst were higher in the umbilical cord monocytes of neonates with funisitis than in those without funisitis. (3) There were no differences in the immunophenotype, basal iROS production, and oxidative burst in maternal granulocytes or monocytes between the study groups., Conclusion: Fetal systemic inflammation is associated with phenotypic and metabolic changes consistent with activation in fetal immune cells but not in maternal blood.

Purpose of Review: There is increasing evidence that both prenatal and perinatal events influence both allergic diseases and early-life respiratory morbidity., Recent Findings: Studies in the last year have suggested that in-utero exposures including tobacco smoke exposure, dietary exposures including vitamin D, and prenatal infection and exposure to microbial products may modulate both atopy and respiratory disease. There have been studies revealing gene x environment interactions between inflammatory pathway genes and in-utero smoke exposure. There have also been studies which have revealed that prenatal exposure to endotoxin may be protective. On the other hand, a recent study also suggested that chorioamnionitis may increase the risk of recurrent wheezing in combination with preterm birth. Finally, two separate large cohort studies evaluated maternal diet in pregnancy and suggested that vitamin D levels may be protective against asthma and wheezing., Summary: There is epidemiological evidence for multiple prenatal factors impacting early-life respiratory morbidity. The mechanisms of these factors need further investigation and may act via various pathways which include effects on lung development, allergic and nonallergic inflammation, and airway remodeling. It remains to be determined if some of these early-life factors which predispose to wheezing will all translate into increased risk of asthma.

Spontaneous preterm labour and preterm births are still the leading cause of perinatal morbidity and mortality in the developed world. Previous efforts to prevent preterm birth have been hampered by a poor understanding of the underlying pathophysiology, inadequate diagnostic tools and generally ineffective therapies. Clinical, epidemiological and experimental studies indicate that genito-urinary tract infections play a critical role in the pathogenesis of preterm birth. Moreover, intrauterine infection increases perinatal mortality and morbidity, such as cerebral palsy and chronic lung disease, significantly. It has recently been suggested that gene-environment interactions play a significant role in determining the risk of preterm birth. Polymorphisms of certain critical genes may be responsible for a harmful inflammatory response in those who possess them. Accordingly, polymorphisms that increase the magnitude or the duration of the inflammatory response were associated with an increased risk of preterm birth. In contrast polymorphisms that decrease the inflammatory response were associated with a lower risk of preterm birth. This article will review the current understanding of pathogenetic pathways in the aetiology of preterm birth.

Some spontaneous preterm deliveries (PTD) are caused by occult infections of the fetal membranes (histologic chorioamnionitis [HCA]). High levels of infection-related markers, including some cytokines, sampled from maternal circulation in mid-pregnancy have been linked to PTD, but whether these specifically identify HCA has not been established. We have tested associations between 13 Th1, Th2 and Th17 cytokines and PTD with and without HCA in a prospective cohort study. The study sample included 926 Pregnancy Outcomes and Community Health Study subcohort women; women with medically indicated PTD or incomplete data excluded. A panel of cytokines was assessed using a multiplex assay in maternal plasma collected at 15-27 weeks of gestation. Severe HCA was scored by a placental pathologist blinded to clinical variables. Multivariable polytomous logistic regression was used to estimate adjusted odds ratios (OR) per 1 standard deviation (S.D.) increase in cytokine levels using a 5 level outcome variable: PTD <35 weeks with HCA, PTD <35 weeks without HCA, PTD 35-36 weeks with HCA, PTD 35-36 weeks without HCA, and term (referent). Interleukin (IL)-1beta, IL-2, IL-12, interferon-gamma, IL-4, IL-6 and transforming growth factor-beta were all significantly associated with PTD <35 weeks with HCA, with ORs of 1.6-2.3 per S.D. increase. None of these were associated with PTD <35 weeks without HCA or PTD 35-36 weeks with HCA. Although the tissues of origin of circulating cytokines are unclear, the observed elevations across many cytokines among women who later delivered <35 weeks with HCA may represent a robust immune response to infection within gestational tissues. These results suggest that women with HCA could be identified using relatively non-invasive means.

Extremely immature preterm infants rarely present with a leukocytosis exceeding 30,000/microL. The pathogenetic sequence leading to leukemoid reactions in non-malignant diseases remains to be elucidated. Potential triggers for leukemoid reactions in premature infants include prenatal corticosteroids, chorioamnionitis and funisitis or systemic infection. In the two-year period from 2006 to 2007 all infants with a gestational age of less than 26 weeks were screened for leukocytosis. Among our cases, one preterm infant presented with a leukocyte count of 229,300/microL at the age of 48 hours, lasting throughout the first three weeks of life. Impairment of microcirculation and resulting organ dysfunction were not observed. Thus, invasive therapeutic procedures, which are routinely initiated in hyperleukocytosis in accompanying malignant diseases, may not have the same significance in extremely immature preterm infants and should be executed in these patients on an individual basis and with extreme caution.

Problem: Galectin-1 can regulate immune responses upon infection and inflammation. We determined galectin-1 expression in the chorioamniotic membranes and its changes during histological chorioamnionitis., Method of Study: Chorioamniotic membranes were obtained from women with normal pregnancy (n = 5) and from patients with pre-term pre-labor rupture of the membranes (PPROM) with (n = 8) and without histological chorioamnionitis (n = 8). Galectin-1 mRNA and protein were localized by in situ hybridization and immunohistochemistry. Galectin-1 mRNA expression was also determined by quantitative reverse transcriptase polymerase chain reaction., Results: Galectin-1 mRNA and protein were detected in the amniotic epithelium, chorioamniotic fibroblasts/myofibroblasts and macrophages, chorionic trophoblasts, and decidual stromal cells. In patients with PPROM, galectin-1 mRNA expression in the fetal membranes was higher (2.07-fold, P = 0.002) in those with chorioamnionitis than in those without. Moreover, chorioamionitis was associated with a strong galectin-1 immunostaining in amniotic epithelium, chorioamniotic mesodermal cells, and apoptotic bodies., Conclusion: Chorioamnionitis is associated with an increased galectin-1 mRNA expression and strong immunoreactivity of the chorioamniotic membranes; thus, galectin-1 may be involved in the regulation of the inflammatory responses to chorioamniotic infection.

Objective: To estimate whether placental pathological lesions from an index preterm birth are associated with an increased risk of recurrent preterm birth and to estimate whether certain pathologic lesions recur in a woman's next delivery., Methods: We performed a retrospective cohort study of all women who delivered at less than 37 weeks and had their next delivery at our institution during a 5-year period. Women were included in the cohort if placental pathology was available from their preterm birth. Placental pathology from their subsequent birth was also collected. Placental pathology was classified into presence or absence of two classes of lesions-inflammatory and thrombotic. Variables considered as possible confounders included race, gestational age of preterm birth, interpregnancy interval, tobacco use, payor status, years of education, and maternal medical problems., Results: Inflammatory lesions (n=173) were associated with recurrent preterm birth overall as well as recurrent spontaneous preterm birth (P<.001). Thrombotic lesions (n=158) were not associated with recurrent preterm birth or any subtypes of preterm birth. The association between inflammatory lesions and recurrent spontaneous preterm birth remained significant when controlling for gestational age of preterm birth, race, and tobacco use, with an adjusted odds ratio of 2.4 (95% confidence interval 1.2-4.7). Inflammatory placental lesions (n=194) were associated with inflammatory lesions in the subsequent delivery P=.001)., Conclusion: Recurrent preterm birth is more likely among women with inflammatory lesions on placental pathology from a prior preterm birth. Additionally, these women are more likely to have placental inflammatory lesions with their next delivery., Level of Evidence: II.

Objective: To assess the relation between sonographic fetal thymus size and the components of fetal inflammatory response syndrome (FIRS) in women with preterm prelabour rupture of membranes (PPROM)., Design: Prospective cohort study., Setting: University hospital from January through October 2006., Population: Fifty-six women with PPROM., Methods: In these women, fetal thymus perimeter was measured sonographically. At birth, cord venous plasma interleukin-6 (IL-6) level estimation and histopathological examination of the placentas and umbilical cords were performed., Main Outcome Measures: Small thymus size (< 5th percentile for gestational age) and its association with FIRS., Results: From the 56 women with PPROM, 54% had chorioamnionitis (CA), 23% had funisitis. IL-6 level was > 11 pg/ml in 52% of women and > 18 pg/ml in 41%. A small thymus was more associated with male fetuses, shorter preterm prelabour rupture of membranes delivery interval, higher IL-6 level, higher frequency of funisitis and CA. When data were regressed for confounding, only IL-6 level and funisitis remained significant independent factors that influence the thymus size. In the subset of women (n = 19) who delivered within 1 week of first measurements, a small thymus had sensitivity and positive predictive value of 93%, specificity and negative predictive value of 75% and accuracy of 89% in the identification of FIRS (IL-6 >18 pg/ml and/or funisitis)., Conclusions: An association exists between fetal thymic involution and components of FIRS in women with PPROM. Small fetal thymus size may be considered a reliable sonographic marker of fetal involvement in the inflammatory response.

Objective: The purpose of this study was to determine the frequency and clinical significance of intraamniotic inflammation in patients with acute cervical insufficiency., Study Design: Amniocentesis was performed in 52 patients with acute cervical insufficiency (cervical dilation, > or =1.5 cm) and intact membranes and without regular uterine contractions (gestational age, 17-29 weeks). Amniotic fluid (AF) was cultured for aerobic and anaerobic bacteria and genital mycoplasmas and assayed for matrix metalloproteinase-8. Intraamniotic inflammation was defined as an elevated AF matrix metalloproteinase-8 concentration (>23 ng/mL). Nonparametric statistics and survival techniques were used for analysis., Results: The prevalence of intraamniotic inflammation was 81% (42/52); the prevalence of a positive AF culture was 8% (4/52). Intraamniotic inflammation was present in all cases with a positive AF culture. Preterm delivery within 7 days occurred in 50% of cases (19/38), and delivery before 34 weeks of gestation occurred in 84% of cases (32/38) with intraamniotic inflammation but without AF infection. Fifty-five percent of newborn infants (21/38) who were born to mothers with intraamniotic inflammation but without AF infection died immediately after birth (<1 day). The amniocentesis-to-delivery interval was shorter in patients with intraamniotic inflammation than in those without inflammation (P < .05). There were no differences in the interval-to-delivery or the rate of adverse outcome between patients with intraamniotic inflammation and a negative culture and patients with proven AF infection., Conclusion: Intraamniotic inflammation, regardless of AF culture result, is present in approximately 80% of patients with acute cervical insufficiency and is a risk factor for impending preterm delivery and adverse outcomes.

Objective: The objective of the study was to test the hypothesis that inflammation modulates fetal erythroblastosis and/or the release of nucleated red blood cells (NRBCs) independent of hypoxia or fetal stress. We sought to determine whether fetal inflammation is associated with an elevation in neonatal NRBC count in the setting of inflammation-associated preterm birth., Study Design: The relationships between peripheral NRBC count, histological chorioamnionitis, umbilical cord interleukin (IL)-6, erythropoietin (EPO), cortisol, and acid-base status were analyzed in 68 preterm singletons, born to mothers who had an amniocentesis to rule out infection. Proteomic profiling of amniotic fluid identified presence of intraamniotic inflammation according to established parameters. NRBC counts were assessed within 1 hour of birth. Early-onset neonatal sepsis (EONS) was established based on hematological and microbiological indices. IL-6, EPO, and cortisol levels were measured by immunoassays. Fetal acid-base status was determined within 10 minutes of delivery. Parametric or nonparametric statistics were used., Results: Fetuses with EONS (n = 19) were delivered at earlier gestational ages (mean +/- SD: 27.1 +/- 2.8 weeks, P = .001) and more often by mothers with intraamniotic inflammation (P = .022) and histological chorioamnionitis (P < .001). Neonates with EONS had higher absolute NRBC counts (P = .011). NRBC counts were directly correlated with cord blood IL-6 levels (P < .001) but not with EPO, cortisol or parameters of acid-base status levels regardless of EONS status. These relationships remained following correction for gestational age, diabetes, intrauterine growth restriction, and steroid exposure., Conclusion: In the setting of inflammation-associated preterm birth and in the absence of hypoxia, elevations in NRBCs in the early neonatal period may be a direct response of exposure to inflammatory mediators in utero.

In a model of human chorioamnionitis, fetal sheep exposed to a single injection, but not repeated injections, of intra-amniotic endotoxin develop lung injury responses. We hypothesized that repeated exposure to intra-amniotic endotoxin induces endotoxin tolerance. Fetal sheep were given intra-amniotic injections of saline (control) or Escherichia coli LPS O55:B5 (10 mg) either 2 days (2-day group, single exposure), 7 days (7-day group, single exposure), or 2 plus 7 days (2- and 7-day repeat exposure) before preterm delivery at 124 days gestation (term=150 days). Endotoxin responses were assessed in vivo in the lung and liver, and in vitro in monocytes from the blood and the lung. Compared with the single 2-day LPS exposure group, the (2 plus 7 days) repeat LPS-exposed lambs had: 1) decreased lung neutrophil and monocyte inducible NO synthase (NOSII) expression, and 2) decreased lung cytokine and liver serum amyloid A3 mRNA expression. In the lung, serum amyloid A3 mRNA expression decreased in the airway epithelial cells but not in the lung inflammatory cells. Unlike the single 7-day LPS exposure group, peripheral blood and lung monocytes from the repeat-LPS group did not increase IL-6 secretion or hydrogen peroxide production in response to in vitro LPS. Compared with controls, TLR4 expression did not change but IL-1R-associated kinase M expression increased in the monocytes from repeat LPS-exposed lambs. These results are consistent with the novel finding of endotoxin tolerance in preterm fetal lungs exposed to intra-amniotic LPS. The findings have implications for preterm infants exposed to chorioamnionitis for both responses to lung injury and postnatal nosocomial infections.

Fetal development and growth occur in a sterile amniotic cavity while first exposure to microorganisms happens at birth. However, at least 25% of all preterm births, the leading cause of perinatal morbidity and mortality worldwide, occur in mothers with microbial invasion of the amniotic cavity. Microbial attack of the fetus takes place in approximately 10% of pregnancies with intra-amniotic infection, and the human fetus is capable of deploying an inflammatory response (cellular and humoral) in the mid-trimester of pregnancy. The onset of premature labor in the context of infection is mediated by pro-inflammatory cytokines, such as interleukin (IL)-1beta and tumor necrosis factor alpha (TNF-alpha), as these cytokines are produced by intrauterine tissues in response to microbial products, can stimulate prostaglandin production, and induce labor in animals. Moreover, knockout experiments suggest that infection is less likely to lead to premature labor when the IL-1 and TNF signaling pathways are disrupted. A fetal inflammatory systemic response occurs in a fraction of fetuses exposed to microorganisms in utero, and is associated with the impending onset of labor as well as multisystem organ involvement. Neonates born with funisitis, the histologic marker of such inflammation, are at increased risk for neurologic handicap and cerebral palsy. Evidence has begun to accumulate that gene-environment interactions determine the likelihood of preterm labor and delivery and, probably, the risk of fetal injury. Fetal inflammation has emerged as a major mechanism of disease responsible for complications in the perinatal period (in utero and in the first 28 days of life), as well as in infancy. Moreover, reprogramming of the fetal immune response may predispose to diseases in adulthood.

Objective: To correlate cervical and amniotic fluid cytokines and macrophage-related chemokines to the development of histological chorioamnionitis (HCA) in patients with preterm labor (PTL) and preterm prelabor rupture of the membranes (PPROM)., Study Design: Cervical and amniotic fluid interleukin (IL)-6, IL-8, IL-18, monocyte chemotactic protein (MCP)-1, MCP-2, and MCP-3 from pregnant women (at

Ureaplasma urealyticum is the bacterial species most often connected with preterm birth, although it often colonises the amniotic fluid without any adverse effects. The induction of preterm labour seems to depend on whether the bacteria produce an inflammatory reaction. In vitro stimulation of choriodecidual tissue with high amounts of U. urealyticum or with lipopolysaccharide induced a qualitatively similar inflammatory response detected by the production of tumour necrosis factor alpha, followed by secretion of anti-inflammatory cytokine interleukin-10 and of prostaglandin E2. Lower quantities of bacteria failed to induce any response.

Objective: The objective of the study was to determine whether placental abruption is associated with an increased incidence of histologic chorioamnionitis among singleton gestations and whether this association is dependent on its severity., Study Design: Data were derived from the New Jersey-Placental Abruption Study, an ongoing, multicenter, case-control study conducted in New Jersey since August 2002. Subjects were women with a clinical diagnosis of abruption, and controls were matched to cases based on parity and maternal race/ethnicity. Two perinatal pathologists, blinded to the case-control status, performed all histologic examination based on standardized protocol. The association between chorioamnionitis and abruption was quantified based on odds ratio (OR) with 95% confidence interval (CI), after adjustment for potential confounders, and all analyses were stratified based on preterm birth (less than 37 weeks) status., Results: At preterm gestations (n = 141), chorioamnionitis was present in 30.8% and 12.5% of abruption cases and controls, respectively (OR 3.6, 95% CI 1.7 to 10.5). At term gestations (n = 205), the corresponding rates were 34.6% and 20.4%, respectively (OR 2.8, 95% CI 1.3 to 6.1). Severe chorioamnionitis was 7.2 (95% CI 1.6 to 20.1) and 18.3 (95% CI 2.2 to 150.4) times more common in abruption patients at preterm and term gestations, respectively., Conclusion: Histologic chorioamnionitis is associated with placental abruption. The association was strongest in the presence of severe chorioamnionitis at term and, to a lesser extent, at preterm gestations. These observations suggest that the histologic findings in abruption are accompanied by severe inflammation, in both preterm and term gestations.

Objective: Intraamniotic inflammation is a risk factor for adverse pregnancy and neonatal outcome, regardless of the presence or absence of a positive amniotic fluid (AF) culture. The purpose of this study was to determine whether the intensity of a fetal inflammatory response (FIR) differs between cases of intraamniotic inflammation with microbiologically proven infection and cases with negative AF cultures., Study Design: The FIR was examined in 89 cases of women with preterm premature rupture of membranes who delivered singleton preterm newborn infants within 48 hours of amniocentesis. AF was cultured for aerobic and anaerobic bacteria and for genital mycoplasmas. AF white blood cell (WBC) count and matrix metalloproteinase-8 (MMP-8) determinations were performed to assess the presence of intraamniotic inflammation. Intraamniotic inflammation was defined as an elevated AF MMP-8 concentration (>23 ng/mL). The intensity of the FIR was determined by the umbilical cord plasma concentrations of C-reactive protein (CRP). Patients were divided into 3 groups according to the presence or absence of intraamniotic inflammation and AF culture results: group 1, without intraamniotic inflammation and with a negative AF culture (n = 28); group 2, with intraamniotic inflammation and with a negative AF culture (n = 26); group 3, with a positive AF culture (n = 35)., Results: Neonates who were born to mothers with intraamniotic inflammation and negative AF cultures had a significantly higher median umbilical cord plasma CRP concentration than did those without intraamniotic inflammation and a negative AF culture (P < .005) but a significantly lower median cord plasma CRP concentration than did those with proven AF infection (P < .05). Patients with intraamniotic inflammation and a negative AF culture had significantly higher median AF MMP-8 concentrations and WBC count than did those without intraamniotic inflammation and a negative AF culture (P < .001). However, there was no significant difference in the median AF MMP-8 and WBC count between patients with intraamniotic inflammation and a negative AF culture and those with proven AF infection (MMP-8, P > .1, and WBC, P = .09)., Conclusion: Intraamniotic inflammation without documented AF infection is a risk factor for a systemic FIR. However, the magnitude of the FIR in those cases was lower than in those with documented AF infection.

The fetal lung normally contains immature monocytes and very few mature macrophages. The chorioamnionitis frequently associated with preterm birth induces monocyte influx into the fetal lung. Previous studies demonstrated that monocytes in the developing lung can mediate lung injury responses that resemble BPD in humans. We hypothesized that chorioamnionitis would induce maturation of immature monocytes in the fetal lung. Groups of three to seven time-mated ewes received saline or 10 mg of endotoxin (Escherichia coli 055:B5) in saline by intra-amniotic injection for intervals from 1 to 14 days before operative delivery at 124 days of gestational age. Monocytic cells from lung tissue were recovered using Percoll gradients. Monocytic cells consistent with macrophages were identified morphologically and by myosin heavy chain class II expression. An increase in macrophages was preceded by induction of granulocyte-macrophage colony-stimulating factor in the lung and subsequent activation of the transcription factor PU.1. The production of IL-6 by monocytes/macrophages in response to endotoxin challenge in vitro increased 7 and 14 days after exposure to intra-amniotic endotoxin. Recombinant TNF-alpha induced IL-6 production by lung monocytic cells exposed to intra-amniotic endotoxin but not in control cells. Monocytic phagocytosis of apoptotic neutrophils also increased 7 and 14 days after exposure to intra-amniotic endotoxin. Intra-amniotic endotoxin induced lung monocytes to develop into functionally mature cells consistent with macrophages. These findings have implications for lung immune responses after exposure to chorioamnionitis.

Objective: Receptor for advanced glycation end products (RAGE) is a multiligand cell-surface receptor part of the immunoglobulin superfamily with crucial roles in inflammation. S100A12/ENRAGE, a biomarker of amniotic fluid (AF) inflammation, is a ligand for RAGE. sRAGE, a competitive soluble RAGE receptor, inhibits RAGE ligands. Here we aimed to investigate the presence and changes in components of the RAGE system in women with intra-amniotic infection and inflammation., Study Design: AF was retrieved by amniocentesis in 113 women stratified as follows: (1) positive AF culture (+AFC; GA = 27 [20-33] wk; n = 27); (2) negative AF culture (-AFC; GA = 30 [20-36] wk; n = 27); (3) second trimester control (2T-CRL; GA = 19 [15-25] wk; n = 31); (4) third trimester control (3T-CRL; GA = 36 [31-38] wk; n = 28). We used mass spectrometry (SELDI) to detect S100A12/ENRAGE in AF. sRAGE levels were measured using specific immunoassays. Placental pathology was interpreted in relationship to the presence or absence of histologic acute inflammation and immunoreactivity of S100A12/ENRAGE and RAGE. mRNA expression of S100A12/ENRAGE, sRAGE, and RAGE in amniochorion and placental villous tissue was investigated using quantitative real-time polymerase chain reaction (PCR)., Results: Presence of the S100A12/ENRAGE biomarker SELDI peak was confirmed in 70% of the +AFC but in only 10% of the -AFC samples (P < .001). The inflammatory biomarker was absent in all control samples. We further determined that the competitive inhibitor sRAGE is temporally regulated during gestation and that its AF levels are not influenced by the presence of either intra-amniotic infection or inflammation. Histologic choriamnionitis associated with intense staining for S100A12/ENRAGE, particularly in inflammatory cells. The immunoreactivity for extracellular domain of RAGE was localized exclusively to amnion epithelial, decidual, and extravillous trophoblast cells. Yet, acute histologic chorioamnionitis was related to increased gene expression of S100A12/ENRAGE in fetal membranes and decreased sRAGE and RAGE in the placenta., Conclusion: The S100A12/ENRAGE system is markedly upregulated in women with intra-amniotic infection and correlates with the degree of inflammation. Further studies remain to elucidate whether the gestational age dependence of the inhibitor molecule sRAGE may explain the higher incidence of infection-related preterm deliveries and especially rupture of the membranes at earlier gestational age.

Objective: To evaluate the impact of clinical and histopathologic correlates related to maternal and fetal inflammatory responses (MIR and FIR) on degree of preterm birth., Methods: Pathology reports and clinical data from 577 singleton preterm births (<37 weeks of gestation) that took place between 1998 and 2004 were analyzed according to decreasing gestational age (>or=33 weeks, 29-32 weeks, and <29 weeks). MIR was defined by presence of subchorionitis, chorioamnionitis, deciduitis, or free membranitis; FIR was defined by presence of funisitis or chorionic plate vasculitis. The associations between MIR alone and MIR with FIR and gestational age subgroups were assessed using logistic regression., Results: The presence of FIR in addition to MIR was more strongly associated with degree of prematurity than the presence of MIR alone, especially for those born at <29 weeks (OR=10.1 (95% CI 4.3-23.7) and OR=5.3 (95% CI 2.3-12.5), respectively). These associations remained significant after adjusting for maternal race, clinical signs of chorioamnionitis, medically indicated birth, and intrapartum corticosteroid, tocolysis and antibiotic use, and after stratification by clinical signs of chorioamnionitis and medically indicated birth., Conclusions: The combined presence of MIR and FIR is associated with a higher risk of extreme preterm birth (<29 weeks) than MIR alone, suggesting a contributory role of FIR in the pathophysiology of preterm birth.

Intrauterine infection is associated with chorioamnionitis (CAM), which can lead to preterm delivery. We previously reported that the levels of IgM and the incidence of CAM were elevated in preterm infants with neonatal pulmonary emphysema. The pathogen and target of this IgM remain unclear. By using Western blot and amino acid sequences, we have determined one of the target proteins: annexin A2. Immunohistochemical analysis showed that annexin A2 was expressed at fetal chorion and amnion membranes. Among very low birth weight (VLBW) infants with hyper-IgM (> or = 30 mg/dL), 58.8% showed a high titer against annexin A2 (more than x 16), which accounted for about 20%-40% of the total IgM. Anti-annexin A2 IgM antibody inhibited plasmin generation. Furthermore, the median of anti-annexin A2 IgM titer from preterm infants who were delivered with high-grade (grade III) CAM was significantly higher than those from preterm infants without CAM (p = 0.011) and with low-grade CAM (grade I and II) (p = 0.010). Here, we indicate the fetal autoimmunoreactivity against the fetomaternal interface in preterm infants.

Objective: Histologic chorioamnionitis has been associated with preterm birth and low birthweight. Our goal was to evaluate the relationship between polymorphonuclear cell and mononuclear cell placental infiltrations and adverse infant outcomes., Study Design: Data from women who were enrolled in a trial of antibiotics for prevention of mother-to-child transmission of HIV-1 and of preterm birth were analyzed. Women who had HIV and women who did not have HIV were assigned randomly to either metronidazole 250 mg and erythromycin 250 mg at 20 to 24 weeks of gestation 3 times daily for 7 days and metronidazole 250 mg and ampicillin 500 mg every 4 hours during labor or identical placebos. Women with HIV were offered nevirapine at delivery for the prevention of mother-to-child transmission. At delivery, various placental sites were evaluated for polymorphonuclear cell and mononuclear cell infiltration., Results: Polymorphonuclear and mononuclear cell infiltrations were common in the decidua (18% and 43%, respectively) and chorioamnion (28% and 34%, respectively). Gestational age and birthweight were lower among women with polymorphonuclear cell infiltrations, with these relationships generally stronger at earlier gestational age and birthweight. Mononuclear infiltrations were not associated with adverse outcomes. Neither polymorphonuclear cell nor mononuclear placental infiltrations were associated with mother-to-child transmission of HIV. Antibiotic use was not associated with reduced polymorphonuclear or mononuclear cell infiltrations., Conclusion: Polymorphonuclear cell infiltrations were associated with preterm birth and decreased birthweight and gestational age. Antibiotic use was not associated with reductions in polymorphonuclear or mononuclear cell infiltrations. In this nevirapine-treated population, neither polymorphonuclear nor mononuclear cell infiltration was associated with the mother-to-child transmission of HIV.

Histopathological examination of the placenta is the gold standard for evaluating antenatal inflammatory processes that might influence fetal development. Histological chorioamnionitis develops through a well-characterised stereotyped progression of maternal and fetal cellular stages that vary from patient to patient and are amenable to quantification. Increases in the intensity of these responses and their gradual transformation into a chronic phase are important variables that can adversely affect fetal physiology. Under recognised placental inflammatory lesions affecting the decidua, placental villi and fetal vessels are also potentially informative factors that should be taken into account in the studies of adverse pregnancy outcomes. This review summarises the relationships between aetiology, intensity, duration, characteristics and site of histological placental inflammation and suggests how these data may help to better understand the antenatal environment.

Chorioamnionitis is associated with intense neutrophil infiltration of the decidua. We therefore determined whether chorioamnionitis enhances decidual interleukin-8 (IL-8) expression and examined cytokine-regulated decidual IL-8 expression. Decidua from chorioamnionitis-complicated pregnancies, but not term controls, displayed marked IL-8 immunohistochemical staining and a dense neutrophil infiltrate. Reverse transcriptase-polymerase chain reaction of microdissected decidual cells identified IL-8 mRNA, confirming decidual synthesis of IL-8. Confluent leukocyte-free term decidual cells were primed with 10(-8) mol/L estradiol (E2) or E2 + 10(-7) mol/L medroxyprogesterone acetate to mimic the steroidal milieu of pregnancy. Compared with cultures maintained in E2 alone, E2 + medroxyprogesterone acetate neither significantly affected IL-8 levels nor altered the response to the cytokines. The addition of 1.0 ng/ml tumor necrosis factor-alpha (TNF-alpha) or interleukin-1 beta (IL-1beta) increased IL-8 secretion levels by 236.6 +/- 51.4- and 1062.6 +/- 254.3-fold, respectively (n = 8, mean +/- SEM, P < 0.05), as measured by enzyme-linked immunosorbent assay. Concentration-response studies revealed that 0.01 ng/ml TNF-alpha and IL-1beta elevated IL-8 output by 10- and 100-fold, respectively. Western blotting confirmed these results, and quantitative reverse transcriptase-polymerase chain reaction demonstrated parallel changes in mRNA levels. In conclusion, IL-8 is strongly expressed in term decidua during chorioamnionitis, and TNF-alpha and IL-1beta enhance IL-8 expression in term decidual cells, suggesting that these cytokines are important regulators of chorioamnionitis-related decidual neutrophil infiltration.

Chorioamnionitis increases the risk of preterm labour and is associated with adverse neonatal outcomes including cerebral palsy. Tumour necrosis factor-alpha (TNF-alpha) derived from the gestational tissues (placenta, fetal membranes and maternal decidua) is thought to play a pivotal role in the induction of cytokine response in chorioamnionitis. Tumour necrosis factor-alpha converting enzyme (TACE) is essential for the release of TNF-alpha. Our aim was to determine whether the expression of TACE is increased in human gestational tissues from pregnancies complicated by chorioamnionitis, and whether lipopolysaccharide (LPS) causes increased expression of TACE in the human gestational tissues in vitro. The immunostaining of TACE was generally more intense, in particular in the syncytiotrophoblast and stromal cells, in villous samples from pregnancies complicated by chorioamnionitis than those from normal pregnancies. Increased immunoreactivity of TACE was also noted in the amnion and choriodecidua. In parallel, there was an increased infiltration of monocytes/macrophages within the villous stroma and choriodecidua. As a complement to our in vivo findings, LPS significantly increased the levels of mRNA and protein of TACE in a dose-dependent response in villous and fetal membrane explant cultures. Together, our results imply a potential role of TACE in the pathogenesis of chorioamnionitis.

We have shown previously that numerous IgE(+) macrophage-like cells are present in the villous stroma of full term placenta and that there was no difference in the amount of IgE(+) cells between allergic and non-allergic mothers. The presence of such an abundant number of IgE(+) cells in the placenta in allergic as well as non-allergic women suggests that the IgE is of some importance for a successful pregnancy outcome. Here we have investigated the IgE-pattern in 59 placentas from second and third trimesters from Sweden with different degrees of chorioamnionitis and 27 full term placentas from Ghana with and without malaria parasites. The immunohistochemical staining pattern for IgE looked similar to our previous study, with the IgE located on Hofbauer-like cells. We could not find any difference in the amount or distribution of IgE(+) cells between malaria-infected and non-infected placentas, nor between different degrees of chorioamnionitis. The IgE score in the placenta did not correlate with the levels of IgE in maternal serum or plasma. However, the IgE score was significantly higher in second- compared to third-trimester placentas (P = 0.03). This might reflect a maturation time-point in the fetus and in the intrauterine environment during the second trimester, or it might be associated with the increased number of intrauterine fetal deaths in the second trimester.

Intrauterine inflammation may relate to neurologic disability among preterm children. We investigated the relationship between chorioamnionitis, cord serum cytokines, and neurologic outcome. Sixty-one consecutively born very preterm extremely low birth weight (ELBW) infants were prospectively enrolled. Histologic inflammation in placenta and umbilical cord and vascular pathology were evaluated. Cord sera were analyzed for five proinflammatory cytokines. Serial brain ultrasound and magnetic resonance imaging were performed for evaluation of intraventricular hemorrhage (IVH grade I-III) and white matter damage (WMD: cystic periventricular leukomalacia or IVH grade IV). Neurologic and neurocognitive outcomes were assessed at the corrected age of 2 y. The incidences of HCA, WMD, and abnormal neurologic outcome were 48%, 13% and 19%, respectively. HCA or high IL-6 in cord serum predicted spontaneous preterm labor with high accuracy. HCA increased the risk of IVH grade II-III. In HCA, without either clinical chorioamnionitis or histologic placental perfusion defect, the children had a low risk of WMD (0%) and a low risk of abnormal neurologic outcome (6%). In HCA, the concentration of IL-6 in cord serum was lower in children with abnormal neurologic outcome than in children with normal neurologic outcome. In HCA and placental perfusion defect (compound defect) the risk of abnormal neurologic outcome was high. Compound placental defect and WMD additively predicted abnormal neurologic outcome. We propose that HCA together with other insults (placental perfusion defect or maternal systemic infection) increases the risk of poor neurologic outcome in very preterm ELBW infants.

Chorioamnionitis (CA) is a severe infection responsible not only for premature birth but also for many severe neonatal diseases. The aim of the present study was to investigate the expression of CD40L and P-selectin on platelets and the plasma levels of their soluble forms in the umbilical cord blood in infants with documented chorioamnionitis. Umbilical cord blood samples were obtained from 10 healthy term newborns, 10 non-infected preterm infants, 10 preterm infants with premature rupture of membranes and 9 preterm infants with clinical and histological CA. The expression of CD40L and P-selectin on platelets was analyzed by flow cytometry. Soluble P-selectin (sCD62P), soluble CD40L (sCD40L) and interleukine-6 (IL-6) were measured in plasma by ELISA assays. Neonates with CA had significantly higher percentages of platelets expressing CD40L in basal conditions (5.3 +/- 2.9% vs. 1.6 +/- 0.7% and in non-infected preterm infants p < 0.05), while the percentages of P-selectin positive platelets were similar among all groups. In contrast, the level of sP-selectin was higher in infants with CA (222 +/- 128 ng/ml vs. 104 +/- 71 ng/ml in non-infected preterm infants, p < 0.05) but no differences were found in the levels of sCD40L. As expected, the levels of IL-6, a pro-inflammatory cytokine were significantly higher in samples obtained from preterm neonates whose mothers had also elevated inflammatory parameters. Our observations suggest that platelets are involved in the complex inflammatory pathogenesis of CA. Neither P-selectin expression on cord blood platelets nor plasma sP-selectin or sCD40L were suitable platelet markers in CA, whereas CD40L was significantly elevated in histologically proven CA.

Bronchus-associated lymphoid tissue consists of lymphoid follicles with or without a germinal center within the bronchial wall. Bronchus-associated lymphoid tissue is part of the integrated mucosal immune system and present in about 50% of healthy infants. We examined a series of 141 fetal and neonatal lungs and detected bronchus-associated lymphoid tissue in 100% of cases with amniotic infection while postpartum perinatal pneumonia did not elicit bronchus-associated lymphoid tissue formation. Only rarely and in low density, bronchus-associated lymphoid tissue was present in non-infected fetuses. The in utero formation of bronchus-associated lymphoid tissue seems to be a reactive phenomenon and - as has been shown in another study - does not portend an adverse prognosis.

Problem: Previous studies with animal models have shown that injection of lipopolysaccharide (LPS) results in fetal loss and increases production of proinflammatory cytokines at the maternal-fetal interface. Most intrauterine infections, however, are associated with Ureaplasma urealyticum, a microorganism that lacks a cell wall and therefore does not contain LPS. Previous work in our laboratory with an animal model for genital infection with a similar organism, Mycoplasma pulmonis, revealed that widespread infection in maternal and fetal tissues can be experimentally induced with minimal manipulation of the animal. For this project, we tested the hypothesis that administration of the organism by a hematogenous route at gestational day (gd) 14 would result in increased tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 production by the placenta., Study Design: Timed-pregnant, Sprague-Dawley rats were anesthetized on gd 14 and 10(7) CFU of M. pulmonis strain X1048 or an equivalent volume of sterile medium was injected into the heart. Rats were necropsied on gd 18 or 21, and ex vivo production of TNF-alpha and IL-6 was evaluated from six randomly selected placentas from each litter. The remaining placentas were harvested and either snap-frozen or placed in formalin. Frozen placentas were processed for real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of TNF-alpha and IL-6 mRNA. Formalin-fixed placentas were sectioned and stained with hematoxylin and eosin for lesion analysis., Results: Concentrations of TNF-alpha but not IL-6 were significantly higher in conditioned medium from placentas harvested from infected dams at gd 21. Levels of mRNA for IL-6 and TNF-alpha, however, were increased by M. pulmonis at gd 18 and 21. Analysis of gd 21 placentas by light microscopy revealed that significant histological chorioamnionitis was present in infected animals with accumulations of neutrophils in the capsular decidua., Conclusion: These data indicate that experimental infection with M. pulmonis causes histological chorioamnionitis, elevated mRNA levels of TNF-alpha and IL-6 in placental tissues, and the secretion of TNF-alpha by the placenta during late gestation.

Chorioamnionitis and funisitis are associated with preterm labor and postnatal morbidity. Activation of endothelium resulting in up-regulation of adhesion molecules seems to be a key mechanism in development of organ damage. We investigated whether chorioamnionitis with or without funisitis in preterm infants induced expression and shedding of adhesion molecules in the umbilical cord and resulted in increased concentrations of E-selectin, intercellular adhesion molecule (ICAM)-1, IL-1beta, IL-6, and IL-8 in the cord blood. Data were obtained by using immunohistochemistry and ELISA. Thirty-two preterm infants were divided into three groups according to histology: chorioamnionitis with funisitis, chorioamnionitis without funisitis, and controls without signs of inflammation. ICAM-1 expression on arterial endothelium was higher with funisitis compared with chorioamnionitis alone or with the control group. Similar results for ICAM-1 expression were found in venous endothelium, vascular walls, Wharton's jelly, and amnion epithelium. Endothelial E-selectin and vascular cell adhesion molecule (VCAM)-1 expression was only induced significantly with funisitis. Serum-concentrations of soluble ICAM-1 were higher with funisitis compared with chorioamnionitis alone or control group. Similarly, concentrations of soluble E-selectin, IL-1beta, IL-6, and IL-8 were increased exclusively with funisitis. In conclusion, only chorioamnionitis with funisitis was associated with systemic inflammation and endothelial activation with up-regulation and shedding of umbilical cord adhesion molecules. We speculate that this activation of endothelium may not be limited to the umbilical cord but may also involve other organs resulting in neonatal morbidity. This underlines the importance of funisitis as a risk factor for adverse outcome.

The preterm fetus is immune naive and has immature innate immune function. Although the preterm fetus is frequently exposed to chorioamnionitis, the effects of exposure of the fetal lung to inflammation on innate immune responses are unknown. Using the fetal sheep model of chorioamnionitis, cord blood monocytes were isolated from preterm lambs 1 to 14 days after intra-amniotic endotoxin injection, cultured for approximately 16 hours, and challenged with endotoxin in vitro. Compared with monocytes from adult sheep, the preterm monocytes produced less H(2)O(2) and interleukin-6, and toll-like receptor 4 expression was decreased. Three days after intra-amniotic endotoxin exposure, preterm monocyte responses to in vitro endotoxin challenge demonstrated decreased H(2)O(2) and interleukin-6 production and decreased CD14 and major histocompatibility complex class II expression. Preterm monocyte responses 7 to 14 days after endotoxin tended to exceed those of adults and preterm control animals indicating augmented function. In contrast, a second intra-amniotic endotoxin injection 7 days after the initial endotoxin exposure suppressed monocyte function at 14 days. The fetal monocytes demonstrated patterns of responses consistent with endotoxin tolerance (immune paralysis) as well as maturation of function. Modulation of fetal innate immune responses by exposure to inflammation may alter subsequent immune adaptation after birth.

Background: The clinical or histologic diagnosis of chorioamnionitis has been associated with an increased risk of neuropathology and adverse neurologic outcomes in premature and term infants. Inflammatory cytokines have been implicated in the pathogenesis of these processes. The objective of this study was to determine whether exposure to chorioamnionitis and fetal inflammatory syndrome is associated with elevated concentrations of inflammatory cytokines (TNF-alpha, IL-6, and IL-8) in the CSF of term and preterm infants., Methods: Eighty-four mother/infant pairs were studied, 54 infants were premature. Twenty-eight showed signs of maternal (n = 14), or fetal (n = 14) inflammation based on placental pathology; mothers of 24 infants showed signs of clinical chorioamnionitis not confirmed by placental pathology and 32 infants were considered as 'controls' since they had only transient difficulty adjusting to extra-uterine life warranting evaluation for sepsis. The cytokine concentrations in the CSF were measured within 24 h of birth., Results: When compared to the control group (IL-8 = 341 +/- 170 pg/ml and IL-6 = 7.4 +/- 1.8 pg/ml) significantly higher concentrations of IL-8 were detected in the CSF of infants exposed to clinical chorioamnionitis (1,854 +/- 878 pg/ml; p = 0.001) and maternal/fetal inflammation (1,754 +/- 787 pg/ml; p = 0.001) and of IL-6 in infants with maternal/fetal inflammation (47.6 +/- 45.1 pg/ml; p = 0.01)., Conclusions: These results indicate that infants exposed to clinical chorioamnionitis, or inflammation in utero, experience at least a transient elevation in inflammatory cytokines in CSF., (Copyright (c) 2005 S. Karger AG, Basel.)

This article will define the concept of fetal/neonatal inflammatory response, and examine the complex interaction between inflammation and neurotoxicity. There appear to be important interactions between infection/inflammation and hypoxia-ischaemia leading to cytokine release and subsequent brain injury. This article will also define adverse outcome and summarize the complexities inherent to neurodevelopmental assessment. Finally, this article will investigate the currently available evidence suggesting a link between inflammatory response and adverse neurodevelopmental outcome, and focus on those variables that need further study: timing and nature of the infectious/inflammatory process; established and new anti-insult strategies; morbidity in organs other than the brain; genetic influences; and environmental factors.