Changes in Relative Counts of Different Leukocyte Subpopulations in Peripheral and Umbilical Cord Blood of Women With Preterm Prelabor Rupture of Membranes With Respect to Intraamniotic Inflammation and Fetal Inflammatory Response Syndrome.

OBJECTIVE: The aim of this study was to evaluate changes in the relative counts of different leukocyte subsets in peripheral and umbilical cord blood in pregnancies complicated by preterm prelabor rupture of membranes (PPROM) with respect to the presence of intraamniotic inflammation (IAI) and fetal inflammatory response syndrome (FIRS). METHODS: Fifty-two women with singleton pregnancies complicated by PPROM were included in this study. From samples of peripheral and umbilical cord blood, relative counts of these leukocyte subpopulations were determined using multicolor flow cytometry: granulocytes, monocytes, lymphocytes, T cells and their subpopulations, B cells and their subpopulations, and NK cells and their subpopulations. IAI was defined as increased concentrations of interleukin 6 in the amniotic fluid. Amniotic fluid samples were obtained by transabdominal amniocentesis. RESULTS: Women with IAI had higher relative counts of monocytes (p = 0.04) in peripheral blood. There was an increased relative number of granulocytes (p = 0.003) and a decreased number of lymphocytes (p = 0.0048), helper CD4+ T cells (p = 0.019), NK cells (p = 0.0001) within leukocytes, NK cells within lymphocytes (p = 0.003) and CD16+ NK cells within NK cells (p = 0.005) in umbilical cord blood samples of women with FIRS. However, after adjusting the results for gestational age at sampling, all differences disappeared. CONCLUSIONS: The presence of IAI or FIRS is not accompanied by significant changes in the relative counts of immune cells in peripheral blood or umbilical cord blood in pregnancies complicated by PPROM.

IL-1 and TNF mediates IL-6 signaling at the maternal-fetal interface during intrauterine inflammation.

Introduction: IL6 signaling plays an important role in triggering labor and IL6 is an established biomarker of intrauterine infection/inflammation (IUI) driven preterm labor (PTL). The biology of IL6 during IUI at the maternal-fetal interface was investigated in samples from human subjects and non-human primates (NHP). Methods: Pregnant women with histologic chorioamnionitis diagnosed by placenta histology were recruited (n=28 term, n=43 for preterm pregnancies from 26-36 completed weeks of gestation). IUI was induced in Rhesus macaque by intraamniotic injection of lipopolysachharide (LPS, n=23). IL1 signaling was blocked using Anakinra (human IL-1 receptor antagonist, n=13), and Tumor necrosis factor (TNF) signaling was blocked by anti TNF-antibody (Adalimumab n=14). The blockers were given before LPS. All animals including controls (intraamniotic injection of saline n=27), were delivered 16h after LPS/saline exposure at about 80% gestation. Results: IUI induced a robust expression of IL6 mRNAs in the fetal membranes (chorion-amnion-decidua tissue) both in humans (term and preterm) and NHP. The major sources of IL6 mRNA expression were the amnion mesenchymal cells (AMC) and decidua stroma cells. Additionally, during IUI in the NHP, ADAM17 (a protease that cleaves membrane bound IL6 receptor (IL6R) to release a soluble form) and IL6R mRNA increased in the fetal membranes, and the ratio of IL6 and soluble forms of IL6R, gp130 increased in the amniotic fluid signifying upregulation of IL6 trans-signaling. Both IL1 and TNF blockade suppressed LPS-induced IL6 mRNAs in the AMC and variably decreased elements of IL6 trans-signaling. Discussion: These data suggest that IL1 and TNF blockers may be useful anti-inflammatory agents via suppression of IL6 signaling at the maternal-fetal interface.

Olfactomedin-4-Positive Neutrophils in Neonates: Link to Systemic Inflammation and Bronchopulmonary Dysplasia.

INTRODUCTION: Little is known about the interplay between neutrophil heterogeneity in neonates in health and disease states. Olfactomedin-4 (OLFM4) marks a subset of neutrophils that have been described in adults and pediatric patients but not neonates, and this subset is thought to play a role in modulating the host inflammatory response. METHODS: This is a prospective cohort of neonates who were born between June 2020 and December 2021 at the University of Cincinnati Medical Center NICU. Olfactomedin-4-positive (OLFM4+) neutrophils were identified in the peripheral blood using flow cytometry. RESULTS: OLFM4+ neutrophil percentage was not correlated with gestational age or developmental age. Neonates with sepsis had a higher percentage than those without the condition, 66.9% (IQR 24.3-76.9%) versus 21.5% (IQR 10.6-34.7%), respectively, p = 0.0003. At birth, a high percentage of OLFM4+ neutrophils was associated with severe chorioamnionitis at 49.1% (IQR 28.2-61.5%) compared to those without it at 13.7% (IQR 7.7-26.3%), p < 0.0001. Among neonates without sepsis, the percentages of OLFM4+ neutrophils were lower in the BPD/early death group compared to those without BPD, 11.8% (IQR 6.3-29.0%) versus 32.5% (IQR 18.5-46.1%), p = 0.003, and this retained significance in a multiple logistic regression model that included gestational age, birthweight, and race. CONCLUSION: This is the first study describing OLFM4+ neutrophils in neonates and it shows that this neutrophil subpopulation is not influenced by gestational age but is elevated in inflammatory conditions such as sepsis and severe chorioamnionitis, and lower percentage at birth is associated with developing bronchopulmonary dysplasia.

Sustained peripheral immune hyper-reactivity (SPIHR): an enduring biomarker of altered inflammatory responses in adult rats after perinatal brain injury.

BACKGROUND: Chorioamnionitis (CHORIO) is a principal risk factor for preterm birth and is the most common pathological abnormality found in the placentae of preterm infants. CHORIO has a multitude of effects on the maternal-placental-fetal axis including profound inflammation. Cumulatively, these changes trigger injury in the developing immune and central nervous systems, thereby increasing susceptibility to chronic sequelae later in life. Despite this and reports of neural-immune changes in children with cerebral palsy, the extent and chronicity of the peripheral immune and neuroinflammatory changes secondary to CHORIO has not been fully characterized. METHODS: We examined the persistence and time course of peripheral immune hyper-reactivity in an established and translational model of perinatal brain injury (PBI) secondary to CHORIO. Pregnant Sprague-Dawley rats underwent laparotomy on embryonic day 18 (E18, preterm equivalent). Uterine arteries were occluded for 60 min, followed by intra-amniotic injection of lipopolysaccharide (LPS). Serum and peripheral blood mononuclear cells (PBMCs) were collected at young adult (postnatal day P60) and middle-aged equivalents (P120). Serum and PBMCs secretome chemokines and cytokines were assayed using multiplex electrochemiluminescent immunoassay. Multiparameter flow cytometry was performed to interrogate immune cell populations. RESULTS: Serum levels of interleukin-1ß (IL-1ß), IL-5, IL-6, C-X-C Motif Chemokine Ligand 1 (CXCL1), tumor necrosis factor-α (TNF-α), and C-C motif chemokine ligand 2/monocyte chemoattractant protein-1 (CCL2/MCP-1) were significantly higher in CHORIO animals compared to sham controls at P60. Notably, CHORIO PBMCs were primed. Specifically, they were hyper-reactive and secreted more inflammatory mediators both at baseline and when stimulated in vitro. While serum levels of cytokines normalized by P120, PBMCs remained primed, and hyper-reactive with a robust pro-inflammatory secretome concomitant with a persistent change in multiple T cell populations in CHORIO animals. CONCLUSIONS: The data indicate that an in utero inflammatory insult leads to neural-immune changes that persist through adulthood, thereby conferring vulnerability to brain and immune system injury throughout the lifespan. This unique molecular and cellular immune signature including sustained peripheral immune hyper-reactivity (SPIHR) and immune cell priming may be a viable biomarker of altered inflammatory responses following in utero insults and advances our understanding of the neuroinflammatory cascade that leads to perinatal brain injury and later neurodevelopmental disorders, including cerebral palsy.

Direct administration of the non-competitive interleukin-1 receptor antagonist rytvela transiently reduced intrauterine inflammation in an extremely preterm sheep model of chorioamnionitis.

BACKGROUND: Intraamniotic inflammation is associated with up to 40% of preterm births, most notably in deliveries occurring prior to 32 weeks' gestation. Despite this, there are few treatment options allowing the prevention of preterm birth and associated fetal injury. Recent studies have shown that the small, non-competitive allosteric interleukin (IL)-1 receptor inhibitor, rytvela, may be of use in resolving inflammation associated with preterm birth (PTB) and fetal injury. We aimed to use an extremely preterm sheep model of chorioamnionitis to investigate the anti-inflammatory efficacy of rytvela in response to established intra-amniotic (IA) lipopolysaccharide (LPS) exposure. We hypothesized that rytvela would reduce LPS-induced IA inflammation in amniotic fluid (AF) and fetal tissues. METHODS: Sheep with a single fetus at 95 days gestation (estimated fetal weight 1.0 kg) had surgery to place fetal jugular and IA catheters. Animals were recovered for 48 hours before being randomized to either: i) IA administration of 2 ml saline 24 hours before 2 ml IA and 2 ml fetal intravenous (IV) administration of saline (Saline Group, n = 7); ii) IA administration of 10 mg LPS in 2 ml saline 24 hours before 2 ml IA and 2 ml fetal IV saline (LPS Group, n = 10); 3) IA administration of 10 mg LPS in 2 ml saline 24 hours before 0.3 mg/fetal kg IA and 1 mg/fetal kg fetal IV rytvela in 2 ml saline, respectively (LPS + rytvela Group, n = 7). Serial AF samples were collected for 120 h. Inflammatory responses were characterized by quantitative polymerase chain reaction (qPCR), histology, fluorescent immunohistochemistry, enzyme-linked inmmunosorbent assay (ELISA), fluorescent western blotting and blood chemistry analysis. RESULTS: LPS-treated animals had endotoxin and AF monocyte chemoattractant protein (MCP)-1 concentrations that were significantly higher at 24 hours (immediately prior to rytvela administration) relative to values from Saline Group animals. Following rytvela administration, the average MCP-1 concentrations in the AF were significantly lower in the LPS + rytvela Group relative to in the LPS Group. In delivery samples, the expression of IL-1ß in fetal skin was significantly lower in the LPS + rytvela Group compared to the LPS Group. CONCLUSION: A single dose of rytvela was associated with partial, modest inhibition in the expression of a panel of cytokines/chemokines in fetal tissues undergoing an active inflammatory response.

Clinical chorioamnionitis at term X: microbiology, clinical signs, placental pathology, and neonatal bacteremia - implications for clinical care.

OBJECTIVES: Clinical chorioamnionitis at term is considered the most common infection-related diagnosis in labor and delivery units worldwide. The syndrome affects 5-12% of all term pregnancies and is a leading cause of maternal morbidity and mortality as well as neonatal death and sepsis. The objectives of this study were to determine the (1) amniotic fluid microbiology using cultivation and molecular microbiologic techniques; (2) diagnostic accuracy of the clinical criteria used to identify patients with intra-amniotic infection; (3) relationship between acute inflammatory lesions of the placenta (maternal and fetal inflammatory responses) and amniotic fluid microbiology and inflammatory markers; and (4) frequency of neonatal bacteremia. METHODS: This retrospective cross-sectional study included 43 women with the diagnosis of clinical chorioamnionitis at term. The presence of microorganisms in the amniotic cavity was determined through the analysis of amniotic fluid samples by cultivation for aerobes, anaerobes, and genital mycoplasmas. A broad-range polymerase chain reaction coupled with electrospray ionization mass spectrometry was also used to detect bacteria, select viruses, and fungi. Intra-amniotic inflammation was defined as an elevated amniotic fluid interleukin-6 (IL-6) concentration ≥2.6 ng/mL. RESULTS: (1) Intra-amniotic infection (defined as the combination of microorganisms detected in amniotic fluid and an elevated IL-6 concentration) was present in 63% (27/43) of cases; (2) the most common microorganisms found in the amniotic fluid samples were Ureaplasma species, followed by Gardnerella vaginalis; (3) sterile intra-amniotic inflammation (elevated IL-6 in amniotic fluid but without detectable microorganisms) was present in 5% (2/43) of cases; (4) 26% of patients with the diagnosis of clinical chorioamnionitis had no evidence of intra-amniotic infection or intra-amniotic inflammation; (5) intra-amniotic infection was more common when the membranes were ruptured than when they were intact (78% [21/27] vs. 38% [6/16]; p=0.01); (6) the traditional criteria for the diagnosis of clinical chorioamnionitis had poor diagnostic performance in identifying proven intra-amniotic infection (overall accuracy, 40-58%); (7) neonatal bacteremia was diagnosed in 4.9% (2/41) of cases; and (8) a fetal inflammatory response defined as the presence of severe acute funisitis was observed in 33% (9/27) of cases. CONCLUSIONS: Clinical chorioamnionitis at term, a syndrome that can result from intra-amniotic infection, was diagnosed in approximately 63% of cases and sterile intra-amniotic inflammation in 5% of cases. However, a substantial number of patients had no evidence of intra-amniotic infection or intra-amniotic inflammation. Evidence of the fetal inflammatory response syndrome was frequently present, but microorganisms were detected in only 4.9% of cases based on cultures of aerobic and anaerobic bacteria in neonatal blood.

Daily monitoring of vaginal interleukin 6 as a predictor of intraamniotic inflammation after preterm premature rupture of membranes - a new method of sampling studied in a prospective multicenter trial.

OBJECTIVES: (A) To introduce a new technique for vaginal fluid sampling (biocompatible synthetic fiber sponge) and (B) evaluate the collected vaginal fluid interleukine-6 (IL-6vag)-concentration as a new diagnostic tool for daily monitoring of intrauterine inflammation after preterm premature rupture of membranes (PPROM). Secondary objectives were to compare the potential to predict an intrauterine inflammation with established inflammation parameters (e.g., maternal white blood cell count). METHODS: This prospective clinical case-control diagnostic accuracy multicenter study was performed with women after PPROM (gestational age 24.0/7 - 34.0/7 weeks). Sampling of vaginal fluid was performed once daily. IL-6vag was determined by electrochemiluminescence-immunoassay-kit. Neonatal outcome and placental histology results were used to retrospectively allocate the cohort into two subgroups: 1) inflammation and 2) no inflammation (controls). RESULTS: A total of 37 cases were included in the final analysis. (A): Measurement of IL-6 was successful in 86% of 172 vaginal fluid samples. (B): Median concentration of IL-6vag in the last vaginal fluid sample before delivery was significantly higher within the inflammation group (17,085 pg/mL) compared to the controls (1,888 pg/mL; p=0.01). By Youden's index an optimal cut-off for prediction an intrauterine inflammation was: 6,417 pg/mL. Two days before delivery, in contrast to all other parameters IL-6vag remained the only parameter with a sufficient AUC of 0.877, p<0.001, 95%CI [0.670-1.000]. CONCLUSIONS: This study established a new technique for vaginal fluid sampling, which permits assessment of IL-6vag concentration noninvasively in clinical daily routine monitoring.

RNA Sequencing Reveals Diverse Functions of Amniotic Fluid Neutrophils and Monocytes/Macrophages in Intra-Amniotic Infection.

Intra-amniotic infection, the invasion of microbes into the amniotic cavity resulting in inflammation, is a clinical condition that can lead to adverse pregnancy outcomes for the mother and fetus as well as severe long-term neonatal morbidities. Despite much research focused on the consequences of intra-amniotic infection, there remains little knowledge about the innate immune cells that respond to invading microbes. We performed RNA-seq of sorted amniotic fluid neutrophils and monocytes/macrophages from women with intra-amniotic infection to determine the transcriptomic differences between these innate immune cells. Further, we sought to identify specific transcriptomic pathways that were significantly altered by the maternal or fetal origin of amniotic fluid neutrophils and monocytes/macrophages, the presence of a severe fetal inflammatory response, and pregnancy outcome (i.e., preterm or term delivery). We show that significant transcriptomic differences exist between amniotic fluid neutrophils and monocytes/macrophages from women with intra-amniotic infection, indicating the distinct roles these cells play. The transcriptome of amniotic fluid immune cells varies based on their maternal or fetal origin, and the significant transcriptomic differences between fetal and maternal monocytes/macrophages imply that those of fetal origin exhibit impaired functions. Notably, transcriptomic changes in amniotic fluid monocytes/macrophages suggest that these immune cells collaborate with neutrophils in the trafficking of fetal leukocytes throughout the umbilical cord (i.e., funisitis). Finally, amniotic fluid neutrophils and monocytes/macrophages from preterm deliveries display enhanced transcriptional activity compared to those from term deliveries, highlighting the protective role of these cells during this vulnerable period. Collectively, these findings demonstrate the underlying complexity of local innate immune responses in women with intra-amniotic infection and provide new insights into the functions of neutrophils and monocytes/macrophages in the amniotic cavity.

The fetal inflammatory response syndrome: the origins of a concept, pathophysiology, diagnosis, and obstetrical implications.

The fetus can deploy a local or systemic inflammatory response when exposed to microorganisms or, alternatively, to non-infection-related stimuli (e.g., danger signals or alarmins). The term "Fetal Inflammatory Response Syndrome" (FIRS) was coined to describe a condition characterized by evidence of a systemic inflammatory response, frequently a result of the activation of the innate limb of the immune response. FIRS can be diagnosed by an increased concentration of umbilical cord plasma or serum acute phase reactants such as C-reactive protein or cytokines (e.g., interleukin-6). Pathologic evidence of a systemic fetal inflammatory response indicates the presence of funisitis or chorionic vasculitis. FIRS was first described in patients at risk for intraamniotic infection who presented preterm labor with intact membranes or preterm prelabor rupture of the membranes. However, FIRS can also be observed in patients with sterile intra-amniotic inflammation, alloimmunization (e.g., Rh disease), and active autoimmune disorders. Neonates born with FIRS have a higher rate of complications, such as early-onset neonatal sepsis, intraventricular hemorrhage, periventricular leukomalacia, and death, than those born without FIRS. Survivors are at risk for long-term sequelae that may include bronchopulmonary dysplasia, neurodevelopmental disorders, such as cerebral palsy, retinopathy of prematurity, and sensorineuronal hearing loss. Experimental FIRS can be induced by intra-amniotic administration of bacteria, microbial products (such as endotoxin), or inflammatory cytokines (such as interleukin-1), and animal models have provided important insights about the mechanisms responsible for multiple organ involvement and dysfunction. A systemic fetal inflammatory response is thought to be adaptive, but, on occasion, may become dysregulated whereby a fetal cytokine storm ensues and can lead to multiple organ dysfunction and even fetal death if delivery does not occur ("rescued by birth"). Thus, the onset of preterm labor in this context can be considered to have survival value. The evidence so far suggests that FIRS may compound the effects of immaturity and neonatal inflammation, thus increasing the risk of neonatal complications and long-term morbidity. Modulation of a dysregulated fetal inflammatory response by the administration of antimicrobial agents, anti-inflammatory agents, or cell-based therapy holds promise to reduce infant morbidity and mortality.

In utero inflammatory challenge induces an early activation of the hepatic innate immune response in late gestation fetal sheep.

Chorioamnionitis is associated with inflammatory end-organ damage in the fetus. Tissues in direct contact with amniotic fluid drive a pro-inflammatory response and contribute to this injury. However, due to a lack of direct contact with the amniotic fluid, the liver contribution to this response has not been fully characterized. Given its role as an immunologic organ, we hypothesized that the fetal liver would demonstrate an early innate immune response to an in utero inflammatory challenge. Fetal sheep (131 ± 1 d gestation) demonstrated metabolic acidosis and high cortisol and norepinephrine values within 5 h of exposure to intra-amniotic LPS. Likewise, expression of pro-inflammatory cytokines increased significantly at 1 and 5 h of exposure. This was associated with NF-κB activation, by inhibitory protein IκBα degradation, and nuclear translocation of NF-κB subunits (p65/p50). Corroborating these findings, LPS exposure significantly increased pro-inflammatory innate immune gene expression in fetal sheep hepatic macrophages in vitro. Thus, an in utero inflammatory challenge induces an early hepatic innate immune response with systemic metabolic and stress responses. Within the fetal liver, hepatic macrophages respond robustly to LPS exposure. Our results demonstrate that the fetal hepatic innate immune response must be considered when developing therapeutic approaches to attenuate end-organ injury associated with in utero inflammation.

Immune Cells in the Placental Villi Contribute to Intra-amniotic Inflammation.

Intra-amniotic (IA) inflammation is associated with significant morbidities for both the mother and the fetus. Prior studies have illustrated many of the effects of IA inflammation on the uterine lining (decidua) and membranous layers of the placenta at the fetal-maternal interface. However, much less is known about the immunological response occurring within the villous placenta. Using a rhesus macaque model of lipopolysaccharide (LPS)-induced IA inflammation, we showed that pregnancy-matched choriodecidua and villi have distinct immunological profiles in rhesus pregnancies. In the choriodecidua, we show that the abundance of neutrophils, multiple populations of antigen-presenting cells, and two populations of natural killer (NK) cells changes with prenatal IA LPS exposure. In contrast, in immune cells within the villous placenta we observed alterations in the abundance of B cells, monocytes, and CD8 T cells. Prior work has illustrated that IA inflammation leads to an increase in tumor necrosis factor alpha (TNFα) at the fetal-maternal interface. In this study, pretreatment with a TNFα blockade partially reversed inflammation in the placental villi. Furthermore, we report that immune cells in the villous placenta sensed LPS during our experimental window, and subsequently activated T cells to produce proinflammatory cytokines. Moreover, this study is the first report of memory T cells in third-trimester non-human primate placental villi and provides evidence that manipulation of immune cells in the villi at the fetal-maternal interface should be considered as a potential therapeutic target for IA inflammation.

De novo Synthesis of SAA1 in the Placenta Participates in Parturition.

Serum amyloid A1 (SAA1) is an acute phase protein produced mainly by the liver to participate in immunomodulation in both sterile and non-sterile inflammation. However, non-hepatic tissues can also synthesize SAA1. It remains to be determined whether SAA1 synthesized locally in the placenta participates in parturition via eliciting inflammatory reactions. In this study, we investigated this issue by using human placenta and a mouse model. We found that SAA1 mRNA and protein were present in human placental villous trophoblasts, which was increased upon syncytialization as well as treatments with lipopolysaccharides (LPS), tumor necrosis factor-α (TNF-α), and cortisol. Moreover, significant increases in SAA1 abundance were observed in the placental tissue or in the maternal blood in spontaneous deliveries without infection at term and in preterm birth with histological chorioamnionitis. Serum amyloid A1 treatment significantly increased parturition-pertinent inflammatory gene expression including interleukin-1ß (IL-1ß), IL-8, TNF-α, and cyclooxygenase-2 (COX-2), along with increased PGF2α production in syncytiotrophoblasts. Mouse study showed that SAA1 was present in the placental junctional zone and yolk sac membrane, which was increased following intraperitoneal administration of LPS. Intraperitoneal injection of SAA1 not only induced preterm birth but also increased the abundance of IL-1ß, TNF-α, and COX-2 in the mouse placenta. Conclusively, SAA1 can be synthesized in the human placenta, which is increased upon trophoblast syncytialization. Parturition is accompanied with increased SAA1 abundance in the placenta. Serum amyloid A1 may participate in parturition in the presence and absence of infection by inducing the expression of inflammatory genes in the placenta.

Maternal azithromycin therapy for Ureaplasma parvum intraamniotic infection improves fetal hemodynamics in a nonhuman primate model.

BACKGROUND: Ureaplasma parvum infection is a prevalent cause of intrauterine infection associated with preterm birth, preterm premature rupture of membranes, fetal inflammatory response syndrome, and adverse postnatal sequelae. Elucidation of diagnostic and treatment strategies for infection-associated preterm labor may improve perinatal and long-term outcomes for these cases. OBJECTIVE: This study assessed the effect of intraamniotic Ureaplasma infection on fetal hemodynamic and cardiac function and the effect of maternal antibiotic treatment on these outcomes. STUDY DESIGN: Chronically catheterized pregnant rhesus monkeys were assigned to control (n=6), intraamniotic inoculation with Ureaplasma parvum (107 colony-forming units/mL, n=15), and intraamniotic infection plus azithromycin treatment (12.5 mg/kg twice a day intravenously, n=8) groups. At approximately 135 days' gestation (term=165 days), pulsed and color Doppler ultrasonography was used to obtain measurements of fetal hemodynamics (pulsatility index of umbilical artery, ductus venosus, descending aorta, ductus arteriosus, aortic isthmus, right pulmonary artery, middle cerebral artery and cerebroplacental ratio, and left and right ventricular cardiac outputs) and cardiac function (ratio of peak early vs late transmitral flow velocity [marker of ventricular function], Tei index [myocardial performance index]). These indices were stratified by amniotic fluid proinflammatory mediator levels and cardiac histology. RESULTS: Umbilical and fetal pulmonary artery vascular impedances were significantly increased in animals from the intraamniotic inoculation with Ureaplasma parvum group (P<.05). Azithromycin treatment restored values to control levels. Amniotic fluid prostaglandin F2 alpha levels were significantly higher in animals with abnormal umbilical artery pulsatility index (>1.1) than in those with normal blood flow (P<.05; Spearman ρ=0.6, P<.05). In the intraamniotic inoculation with Ureaplasma parvum group, left ventricular cardiac output was significantly decreased (P<.001), and more animals had abnormal right-to-left ventricular cardiac output ratios (defined as >1.6, P<.05). Amniotic fluid interleukin-6 concentrations were elevated in cases of abnormal right-to-left ventricular cardiac output ratios compared with those in normal cases (P<.05). CONCLUSION: Fetal hemodynamic alterations were associated with intraamniotic Ureaplasma infection and ameliorated after maternal antibiotic treatment. Doppler ultrasonographic measurements merit continuing investigation as a diagnostic method to identify fetal cardiovascular and hemodynamic compromise associated with intrauterine infection or inflammation and in the evaluation of therapeutic interventions or clinical management of preterm labor.

TNF-Signaling Modulates Neutrophil-Mediated Immunity at the Feto-Maternal Interface During LPS-Induced Intrauterine Inflammation.

Accumulation of activated neutrophils at the feto-maternal interface is a defining hallmark of intrauterine inflammation (IUI) that might trigger an excessive immune response during pregnancy. Mechanisms responsible of this massive neutrophil recruitment are poorly investigated. We have previously showed that intraamniotic injection of LPS in rhesus macaques induced a neutrophil predominant inflammatory response similar to that seen in human IUI. Here, we demonstrate that anti-TNF antibody (Adalimumab) inhibited ~80% of genes induced by LPS involved in inflammatory signaling and innate immunity in chorio-decidua neutrophils. Consistent with the gene expression data, TNF-blockade decreased LPS-induced neutrophil accumulation and activation at the feto-maternal interface. We also observed a reduction in IL-6 and other pro-inflammatory cytokines but not prostaglandins concentrations in the amniotic fluid. Moreover, TNF-blockade decreased mRNA expression of inflammatory cytokines in the chorio-decidua but not in the uterus, suggesting that inhibition of TNF-signaling decreased the inflammation in a tissue-specific manner within the uterine compartment. Taken together, our results demonstrate a predominant role for TNF-signaling in modulating the neutrophilic infiltration at the feto-maternal interface during IUI and suggest that blockade of TNF-signaling could be considered as a therapeutic approach for IUI, the major leading cause of preterm birth.

Cyclic AMP in human preterm infant blood is associated with increased TLR-mediated production of acute-phase and anti-inflammatory cytokines in vitro.

BACKGROUND: Preterm infants are at high risk of infection and have distinct pathogen recognition responses. Suggested mechanisms include soluble mediators that enhance cellular levels of cAMP. The aim of this study was to assess the relationship between blood cAMP concentrations and TLR-mediated cytokine production in infants during the first month of life. METHODS: Cord and serial peripheral blood samples (days of life 1-28) were obtained from a cohort of very preterm (<30 weeks' gestational age) and term human infants. Whole-blood concentrations of cAMP and FSL-1 and LPS in vitro stimulated cytokine concentrations were measured by ELISA and multiplex bead assay. RESULTS: cAMP concentrations were higher in cord than in peripheral blood, higher in cord blood of female preterm infants, and lower at Days 1 and 7 in infants exposed to chorioamnionitis, even after adjusting for leukocyte counts. TLR2 and TLR4-mediated TNF-α, IL-1ß, IL-6, IL-12p70, and IL-10 production in vitro increased over the first month of life in preterm infants and were positively correlated with leukocyte-adjusted cAMP levels and reduced by exposure to chorioamnionitis. CONCLUSIONS: The ontogeny of blood cAMP concentrations and associations with chorioamnionitis and TLR-mediated production of cytokines suggest that this secondary messenger helps shape distinct neonatal pathogen responses in early life.

The origin of amniotic fluid monocytes/macrophages in women with intra-amniotic inflammation or infection.

Background Monocytes, after neutrophils, are the most abundant white blood cells found in the amniotic cavity of women with intra-amniotic inflammation/infection. However, the origin of such cells has not been fully investigated. Herein, we determined (1) the origin of amniotic fluid monocytes/macrophages from women with intra-amniotic inflammation/infection, (2) the relationship between the origin of amniotic fluid monocytes/macrophages and preterm or term delivery and (3) the localization of monocytes/macrophages in the placental tissues. Methods Amniotic fluid samples (n = 16) were collected from women with suspected intra-amniotic inflammation or infection. Amniotic fluid monocytes/macrophages were purified by fluorescence-activated cell sorting, and DNA fingerprinting was performed. Blinded placental histopathological evaluations were conducted. Immunohistochemistry was performed to detect CD14+ monocytes/macrophages in the placental tissues. Results DNA fingerprinting revealed that (1) 56.25% (9/16) of amniotic fluid samples had mostly fetal monocytes/macrophages, (2) 37.5% (6/16) had predominantly maternal monocytes/macrophages and (3) one sample (6.25% [1/16]) had a mixture of fetal and maternal monocytes/macrophages. (4) Most samples with predominantly fetal monocytes/macrophages were from women who delivered early preterm neonates (77.8% [7/9]), whereas all samples with mostly maternal monocytes/macrophages or a mixture of both were from women who delivered term or late preterm neonates (100% [7/7]). (5) Most of the women included in this study presented acute maternal and fetal inflammatory responses in the placenta (85.7% [12/14]). (6) Women who had mostly fetal monocytes/macrophages in amniotic fluid had abundant CD14+ cells in the umbilical cord and chorionic plate, whereas women with mostly maternal amniotic fluid monocytes/macrophages had abundant CD14+ cells in the chorioamniotic membranes. Conclusion Amniotic fluid monocytes/macrophages can be of either fetal or maternal origin, or a mixture of both, in women with intra-amniotic inflammation or infection. These immune cells could be derived from the fetal and maternal vasculature of the placenta.

Gut and immune effects of bioactive milk factors in preterm pigs exposed to prenatal inflammation.

Prenatal inflammation may predispose to preterm birth and postnatal inflammatory disorders such as necrotizing enterocolitis (NEC). Bioactive milk ingredients may help to support gut maturation in such neonates, but mother's milk is often insufficient after preterm birth. We hypothesized that supplementation with bioactive ingredients from bovine milk [osteopontin (OPN), caseinoglycomacropeptide (CGMP), colostrum (COL)] supports gut, immunity, and NEC resistance in neonates born preterm after gram-negative infection before birth. Using preterm pigs as a model for preterm infants, fetal pigs were given intraamniotic injections of lipopolysaccharide (LPS; 1 mg/fetus) and delivered 3 days later (90% gestation). For 5 days, groups of LPS-exposed pigs were fed formula (FOR), bovine colostrum (COL), or formula enriched with OPN or CGMP. LPS induced intraamniotic inflammation and postnatal systemic inflammation but limited effects on postnatal gut parameters and NEC. Relative to FOR, COL feeding to LPS-exposed pigs showed less diarrhea, NEC severity, reduced gut IL-1ß and IL-8 levels, greater gut goblet cell density and digestive enzyme activities, and blood helper T-cell fraction. CGMP improved neonatal arousal and gut lactase activities and reduced LPS-induced IL-8 secretion in intestinal epithelial cells (IECs) in vitro. Finally, OPN tended to reduce diarrhea and stimulated IEC proliferation in vitro. No effects on villus morphology, circulating cytokines, or colonic microbiota were observed among groups. In conclusion, bioactive milk ingredients exerted only modest effects on gut and systemic immune parameters in preterm pigs exposed to prenatal inflammation. Short-term, prenatal exposure to inflammation may render the gut less sensitive to immune-modulatory milk effects. NEW & NOTEWORTHY Prenatal inflammation is a risk factor for preterm birth and postnatal complications including infections. However, from clinical studies, it is difficult to separate the effects of only prenatal inflammation from preterm birth. Using cesarean-delivered preterm pigs with prenatal inflammation, we documented some beneficial gut effects of bioactive milk diets relative to formula, but prenatal inflammation appeared to decrease the sensitivity of enteral feeding. Special treatments and diets may be required for this neonatal population.

Preterm birth: Born too soon for the developing airway epithelium?

Birth prior to term interrupts the normal development of the respiratory system and consequently results in poor respiratory outcomes that persist throughout childhood. The mechanisms underpinning these poor respiratory outcomes are not well understood, but intrinsic abnormalities within the airway epithelium may be a contributing factor. Current evidence suggests that the airway epithelium is both structurally and functionally abnormal after preterm birth, with reports of epithelial thickening and goblet cell hyperplasia in addition to increased inflammation and apoptosis in the neonatal intensive care unit. However, studies focusing on the airway epithelium are limited and many questions remain unanswered; including whether abnormalities are a direct result of interrupted development, a consequence of exposure to inflammatory stimuli in the perinatal period or a combination of the two. In addition, the difficulty of accessing airway tissue has resulted in the majority of evidence being collected in the pre-surfactant era which may not reflect contemporary preterm birth. This review examines the consequences of preterm birth on the airway epithelium and explores the clinical relevance of currently available models whilst highlighting the need to develop a clinically relevant in vitro model to help further our understanding of the airway epithelium in preterm birth.

Are B cells altered in the decidua of women with preterm or term labor?

PROBLEM: The immunophenotype of B cells at the maternal-fetal interface (decidua) in labor at term and preterm labor is poorly understood. METHOD OF STUDY: Decidual tissues were obtained from women with preterm or term labor and from non-labor gestational age-matched controls. Immunophenotyping of decidual B cells was performed using multicolor flow cytometry. RESULTS: (a) In the absence of acute or chronic chorioamnionitis, total B cells were more abundant in the decidua parietalis of women who delivered preterm than in those who delivered at term, regardless of the presence of labor; (b) decidual transitional and naïve B cells were the most abundant B-cell subsets; (c) decidual B1 B cells were increased in women with either labor at term or preterm labor and chronic chorioamnionitis compared to those without this placental lesion; (d) decidual transitional B cells were reduced in women with preterm labor compared to those without labor; (e) naïve, class-switched, and non-class-switched B cells in the decidual tissues underwent mild alterations with the process of preterm labor; (f) decidual plasmablasts seemed to increase in women with either labor at term or preterm labor with chronic chorioamnionitis; and (g) decidual B cells expressed high levels of interleukin (IL)-12, IL-6, and/or IL-35. CONCLUSION: Total B cells are not increased with the presence of preterm or term labor; yet, specific subsets (B1 and plasmablasts) undergo alterations in women with chronic chorioamnionitis. Therefore, B cells are solely implicated in the pathological process of preterm labor in a subset of women with chronic inflammation of the placenta. These findings provide insight into the immunology of the maternal-fetal interface in preterm and term labor.

A Genome-Wide Analysis of Clinical Chorioamnionitis among Preterm Infants.

OBJECTIVE: To identify single nucleotide polymorphisms (SNPs) associated with clinical chorioamnionitis among preterm infants. STUDY DESIGN: We reanalyzed a genome-wide association study (GWAS) from preterm newborns at less than 30 weeks' gestation. Cases and control definitions were determined using administrative records. There were 213 clinical chorioamnionitis cases and 707 clinically uninfected controls. We compared demographic and clinical outcomes of cases and controls. We performed a GWAS and compared the distribution of SNPs from the background genes and from the immunome genes. We used a Wilcoxon's rank-sum test to compare the SNPs normalized odds ratio and used odds ratios and p-values to determine candidate genes. RESULTS: Infants affected by clinical chorioamnionitis were more likely to have periventricular leukomalacia, high-grade retinopathy, and high-grade intraventricular hemorrhage (IVH). Although a GWAS did not identify SNPs associated with clinical chorioamnionitis at the genome-wide significance level, a direct test on the exonic variants in the human immunome revealed their significant increase of risk in clinical chorioamnionitis. CONCLUSION: Among very preterm infants, clinical chorioamnionitis was associated with periventricular leukomalacia, high-grade retinopathy, and IVH. Our analysis of variants in the human immunome indicates an association with clinical chorioamnionitis in very preterm pregnancies.