Bacteria in the amniotic fluid without inflammation: early colonization vs. contamination.

OBJECTIVES: Intra-amniotic infection, defined by the presence of microorganisms in the amniotic cavity, is often accompanied by intra-amniotic inflammation. Occasionally, laboratories report the growth of bacteria or the presence of microbial nucleic acids in amniotic fluid in the absence of intra-amniotic inflammation. This study was conducted to determine the clinical significance of the presence of bacteria in amniotic fluid samples in the absence of intra-amniotic inflammation. METHODS: A retrospective cross-sectional study included 360 patients with preterm labor and intact membranes who underwent transabdominal amniocentesis for evaluation of the microbial state of the amniotic cavity as well as intra-amniotic inflammation. Cultivation techniques were used to isolate microorganisms, and broad-range polymerase chain reaction coupled with electrospray ionization mass spectrometry (PCR/ESI-MS) was utilized to detect the nucleic acids of bacteria, viruses, and fungi. RESULTS: Patients whose amniotic fluid samples evinced microorganisms but did not indicate inflammation had a similar perinatal outcome to those without microorganisms or inflammation [amniocentesis-to-delivery interval (p=0.31), spontaneous preterm birth before 34 weeks (p=0.83), acute placental inflammatory lesions (p=1), and composite neonatal morbidity (p=0.8)]. CONCLUSIONS: The isolation of microorganisms from a sample of amniotic fluid in the absence of intra-amniotic inflammation is indicative of a benign condition, which most likely represents contamination of the specimen during the collection procedure or laboratory processing rather than early colonization or infection.

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.

Interleukin-6 pathobiology in equine placental infection.

PROBLEM: Ascending placentitis is the leading cause of abortion in the horse. Interleukin (IL)-6 is considered predictive of placental infection in other species, but little is understood regarding its role in the pathophysiology of ascending placentitis. METHOD OF STUDY: Sub-acute ascending placentitis was induced via trans-cervical inoculation of S zooepidemicus, and various fluids/serum/tissues collected 8 days later. Concentrations of IL-6 were detected within fetal fluids and serum in inoculated (n = 6) and control (n = 6) mares. RNASeq was performed on the placenta (endometrium and chorioallantois) to assess transcripts relating to IL-6 pathways. IHC was performed for immunolocalization of IL-6 receptor (IL-6R) in the placenta. RESULTS: IL-6 concentrations increased in allantoic fluid following inoculation, with a trend toward an increase in amniotic fluid. Maternal serum IL-6 was increased in inoculated animals, while no changes were noted in fetal serum. mRNA expression of IL-6-related transcripts within the chorioallantois indicates that IL-6 is activating the classical JAK/STAT pathway, thereby acting as anti-inflammatory, anti-apoptotic, and pro-survival. The IL-6R was expressed within the chorioallantois, indicating a paracrine signaling pathway of maternal IL-6 to fetal IL-6R. CONCLUSION: IL-6 plays a crucial role in the placental response to induction of sub-acute equine ascending placentitis, and this could be noted in amniotic fluid, allantoic fluid, and maternal serum. Additionally, IL-6 is acting as anti-inflammatory in this disease, potentially altering disease progression, impeding abortion signals, and assisting with the production of a viable neonate.

Inflammation with the participation of arachidonic (AA) and linoleic acid (LA) derivatives (HETEs and HODEs) is necessary in the course of a normal reproductive cycle and pregnancy.

Data on arachidonic (AA) and linoleic (LA) acid derivatives and their role in the reproductive cycle are limited. In order to systematize these reports, 54 scientific investigations were analyzed, which revealed the important role of AA and LA in the planning and course of pregnancy. Ovulation, menstruation, pregnancy, and childbirth are strongly related to the occurrence of physiological inflammatory reactions. Ovulation and menstruation are cyclic tissue remodeling processes that cause changes in the synthesis of inflammation mediators, such as prostaglandins and leukotrienes. Thus, the cyclooxygenase (COX) and lipoxygenase-5 (5-LOX) pathway for AA transformation is activated. Only the absence of neutrophils during this process differentiates an embryo implantation from a standard inflammatory response. It has been found that in COX-2 deficiency conditions, incorrect embryo implantation and decidual reaction occur; therefore, the mechanism associated with the activation of the nuclear factor (NF)-κB pathway seems to play an important role in the course of embryo implantation. In addition, 12/15-LOX may be key modulators of uterine activity during the implantation process. According to the current state of knowledge, AA derivatives synthesized throughout the cytochrome P450 (CYP) and LOX pathways play a special role in the late pregnancy period. Decreased 5-HETE levels have been related to slowing down the progression of labor, while 11-HETE and 15-HETrE to its acceleration. It has been also proven that renal 20-HETE contents undergo significant changes in the late pregnancy period, which are caused by an increase in their adrenal medulla and vascular synthesis, leading to decrease of blood pressure and an increase of sodium excretion, finally conditioning a normal course of labor.

Erythropoietin prevents LPS-induced preterm birth and increases offspring survival.

PROBLEM: Preterm delivery is the leading cause of neonatal mortality and contributes to delayed physical and cognitive development in children. At present, there is no efficient therapy to prevent preterm labor. A large body of evidence suggests that infections might play a significant and potentially preventable cause of premature birth. This work assessed the effects of erythropoietin (EPO) in a murine model of inflammation-associated preterm delivery, which mimics central features of preterm infections in humans. METHOD OF STUDY: BALB/c mice were injected i.p. with 20 000 IU/kg EPO or normal saline twice on gestational day (GD) 15, with a 3 hours time interval between injections. An hour after the first EPO or normal saline injection, all mice received two injections of 50 µg/kg LPS, also given 3 hours apart. RESULTS: EPO significantly prevented preterm labor and increased offspring survival in an LPS induced preterm delivery model. EPO prevented LPS-induced leukocyte infiltration into the placenta. Moreover, EPO inhibited the expression of pro-inflammatory cytokines, interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and tumour necrosis factor-α (TNF-α) in maternal serum and amniotic fluid. EPO also prevented LPS-induced increase in placental prostaglandin (PG)E2 and uterine inducible nitric oxide synthase (iNOS) production, while decreasing nuclear factor kappa-B (NF-κß) activity in the myometrium. EPO also increased the gene expression of placental programmed cell death ligand 1 (PD-L1) in LPS-treated mice. CONCLUSIONS: Our results suggest that EPO could be a potential novel therapeutic strategy to tackle infection-related preterm labor.

Successful use of an artificial placenta-based life support system to treat extremely preterm ovine fetuses compromised by intrauterine inflammation.

BACKGROUND: Ex vivo uterine environment therapy is an experimental intensive care strategy for extremely preterm infants born between 21 and 24 weeks of gestation. Gas exchange is performed by membranous oxygenators connected by catheters to the umbilical vessels. The fetus is submerged in a bath of synthetic amniotic fluid. The lungs remain fluid filled, and pulmonary respiration does not occur. Intrauterine inflammation is strongly associated with extremely preterm birth and fetal injury. At present, there are no data that we are aware of to show that artificial placenta-based systems can be used to support extremely preterm fetuses compromised by exposure to intrauterine inflammation. OBJECTIVE: To evaluate the ability of our ex vivo uterine environment therapy platform to support extremely preterm ovine fetuses (95-day gestational age; approximately equivalent to 24 weeks of human gestation) exposed to intrauterine inflammation for a period of 120 hours, the following primary endpoints were chosen: (1) maintenance of key physiological variables within normal ranges, (2) absence of infection and inflammation, (3) absence of brain injury, and (4) gross fetal growth and cardiovascular function matching that of age-matched in utero controls. STUDY DESIGN: Ten ewes with singleton pregnancies were each given a single intraamniotic injection of 10-mg Escherichia coli lipopolysaccharides under ultrasound guidance 48 hours before undergoing surgical delivery for adaptation to ex vivo uterine environment therapy at 95-day gestation (term=150 days). Fetuses were adapted to ex vivo uterine environment therapy and maintained for 120 hours with constant monitoring of key vital parameters (ex vivo uterine environment group) before being killed at 100-day equivalent gestational age. Umbilical artery blood samples were regularly collected to assess blood gas data, differential counts, biochemical parameters, inflammatory markers, and microbial load to exclude infection. Ultrasound was conducted at 48 hours after intraamniotic lipopolysaccharides (before surgery) to confirm fetal viability and at the conclusion of the experiments (before euthanasia) to evaluate cardiac function. Brain injury was evaluated by gross anatomic and histopathologic investigations. Eight singleton pregnant control animals were similarly exposed to intraamniotic lipopolysaccharides at 93-day gestation and were killed at 100-day gestation to allow comparative postmortem analyses (control group). Biobanked samples from age-matched saline-treated animals served as an additional comparison group. Successful instillation of lipopolysaccharides into the amniotic fluid exposure was confirmed by amniotic fluid analysis at the time of administration and by analyzing cytokine levels in fetal plasma and amniotic fluid. Data were tested for mean differences using analysis of variance. RESULTS: Six of 8 lipopolysaccharide control group (75%) and 8 of 10 ex vivo uterine environment group fetuses (80%) successfully completed their protocols. Six of 8 ex vivo uterine environment group fetuses required dexamethasone phosphate treatment to manage profound refractory hypotension. Weight and crown-rump length were reduced in ex vivo uterine environment group fetuses at euthanasia than those in lipopolysaccharide control group fetuses (P<.05). There were no biologically significant differences in cardiac ultrasound measurement, differential leukocyte counts (P>.05), plasma tumor necrosis factor α, monocyte chemoattractant protein-1 concentrations (P>.05), or liver function tests between groups. Daily blood cultures were negative for aerobic and anaerobic growth in all ex vivo uterine environment group animals. No cases of intraventricular hemorrhage were observed. White matter injury was identified in 3 of 6 lipopolysaccharide control group fetuses and 3 of 8 vivo uterine environment group fetuses. CONCLUSION: We report the use of an artificial placenta-based system to support extremely preterm lambs compromised by exposure to intrauterine inflammation. Our data highlight key challenges (refractory hypotension, growth restriction, and white matter injury) to be overcome in the development and use of artificial placenta technology for extremely preterm infants. As such challenges seem largely absent from studies based on healthy pregnancies, additional experiments of this nature using clinically relevant model systems are essential for further development of this technology and its eventual clinical application.

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.

Cellular immune responses in amniotic fluid of women with preterm prelabor rupture of membranes.

Background Preterm birth is the leading cause of perinatal morbidity and mortality. Preterm prelabor rupture of membranes (pPROM) occurs in 30% of preterm births; thus, this complication is a major contributor to maternal and neonatal morbidity. However, the cellular immune responses in amniotic fluid of women with pPROM have not been investigated. Methods Amniotic fluid samples were obtained from women with pPROM and a positive (n = 7) or negative (n = 10) microbiological culture. Flow cytometry was performed to evaluate the phenotype and number of amniotic fluid leukocytes. The correlation between amniotic fluid immune cells and an interleukin-6 (IL-6) concentration or a white blood cell (WBC) count in amniotic fluid was calculated. Results Women with pPROM and a positive amniotic fluid culture had (1) a greater number of total leukocytes in amniotic fluid, including neutrophils and monocytes/macrophages and (2) an increased number of total T cells in amniotic fluid, namely CD4+ T cells and CD8+ T cells, but not B cells. The numbers of neutrophils and monocytes/macrophages were positively correlated with IL-6 concentrations and WBC counts in amniotic fluid of women with pPROM. Conclusion Women with pPROM and a positive amniotic fluid culture exhibit a more severe cellular immune response than those with a negative culture, which is associated with well-known markers of intra-amniotic inflammation.

Amniotic fluid antiphospholipid antibodies: potential role in antiphospholipid syndrome-independent aberrant implantation process.

BACKGROUND: The direct role of antiphospholipid antibodies (aPL) at maternal-fetal interface has not been fully investigated, especially whether they are involved in physiological and pathological implantation conditions, in an antiphospholipid syndrome (APS)-independent manner. In fact, trophoblast cells and placental endothelial cells at the implantation site express potential aPL targeted-phospholipid antigens (PL Ags); thus, the local production and presence of their specific antibodies, not related to APS (characterized by aPL presence in the peripheral blood), could be a potential marker of aberrant invasion, implantation and fetal-maternal immune tolerance processes. METHODS: Anti-Beta2glycoprotein I (anti-ß2GPI) and anticardiolipin (aCL Ab) antibodies (the most clinically relevant aPL) were detected by immunoenzymatic assay (ELISA), in the amniotic fluid (AF) of 167 women with physiological and complicated common pregnancy conditions, sharing an aberrant implantation process, such as recurrent pregnancy loss (RPL), autoimmune hypothyroidism (ahT) and smoking. All women included in the study were negative to peripheral blood aPL. RESULTS: aCL and anti-ß2GPI antibodies were detectable in all the AF samples. RPL, ahT and smoking patients had higher level of anti-ß2GPI Abs (IgM) compared to women with physiological pregnancies (p < 0.0001). Since IgM cannot cross the placenta, their local production in response to maternal-fetal interface stimuli, could be hypothesized. CONCLUSIONS: The presence of aPL in the AF (not related to APS) could reveal a potential clinical significance at maternal-fetal interface in selected pregnancy complications, in which an aberrant implantation process, and in turn an impaired fetal-maternal immune tolerance cross-talk, could occur.

Perinatal immuno/inflammatory responses in the presence or absence of bovine fetal infection.

BACKGROUND: It is known that the bovine fetus can mount an immune and inflammatory reaction to infection, but it is not known whether there is a contemporaneous maternal response. Nor is it known whether the response of calves which die perinatally, with or without infection, differs from that of live perinates. Hence, the objective of this study was to determine if acute phase reactant and immunoglobulin concentrations differed between calves (and their dams) in three groups: live calves (CC; n = 21) and dead calves with (PM INF+; n = 22) or without (PM INF-; n = 89) in utero infection. In calf plasma, serum amyloid A, haptoglobin, immunoglobulins M, G1 and G2 and interleukin-6 were measured. In dam serum, SAA and Hp was measured and in amniotic and abomasal fluid, IL-6 was measured. RESULTS: Live calves had higher plasma concentrations of SAA and IL-6 than dead calves with (PM INF+) or without (PM INF-) in utero infection. Calves in the PM INF-, but not PM INF+ group, had higher Hp concentrations than calves in the CC group. Calves in the PM INF+ group had higher IgG1 concentrations than calves in the PM INF- and CC groups. Except for higher IgG1 and IgG2 concentrations, biomarker values did not differ significantly between dead calves with or without in utero infection. Live calves had higher IL-6 concentrations in abomasal fluid compared to PM INF- calves. There were no significant differences in blood biomarker concentrations between dams of the three groups of calves. Amniotic fluid IL-6 concentrations were higher from the dams of control calves than the dams of uninfected calves. CONCLUSIONS: Differences in biomarkers (higher Hp and IgG1; lower SAA and IL-6) between perinatal mortalities and live perinates probably reflect differences between these two groups in age at sampling (SAA and IL-6) and in utero infection (IgG1). Out of the six analytes measured in calves, only IgG1 and IgG2 were biomarkers of (chronic) in utero infection.

CD200-CD200R imbalance correlates with microglia and pro-inflammatory activation in rat spinal cords exposed to amniotic fluid in retinoic acid-induced spina bifida.

Spina bifida aperta is a congenital malformation characterized by the failure of neural tube closure resulting in an unprotected fetal spinal cord. The spinal cord then undergoes progressive damage, likely due to chemical and mechanical factors related to exposure to the intrauterine environment. Astrogliosis in exposed spinal cords has been described in animal models of spina bifida during embryonic life but its relationship with neuroinflammatory processes are completely unknown. Using a retinoic acid-induced rat model of spina bifida we demonstrated that, when exposed to amniotic fluid, fetal spinal cords showed progressive astrogliosis with neuronal loss at mid-gestation (E15) compared to unexposed spinal cords. The number of microglial cells with a reactive phenotype and activation marker expression increased during gestation and exhibited progressive disruption in the inhibitory immune ligand-receptor system. Specifically we demonstrate down-regulation of CD200 expression and up-regulation of CD200R. Exposed spinal cords demonstrated neuroinflammation with increased tissue water content and cytokine production by the end of gestation (E20), which correlated with active Caspase3 expression in the exposed layers. Our findings provide new evidence that microglia activation, including the disruption of the endogenous inhibitory system (CD200-CD200R), may participate in the pathogenesis of spina bifida through late gestation.

Plasma inflammatory and immune proteins as predictors of intra-amniotic infection and spontaneous preterm delivery in women with preterm labor: a retrospective study.

BACKGROUND: We investigated whether various inflammatory and immune proteins in plasma predict intra-amniotic infection and imminent preterm delivery in women with preterm labor and compared their predictive ability with that of amniotic fluid (AF) interleukin (IL)-6 and serum C-reactive protein (CRP). METHODS: This retrospective cohort study included 173 consecutive women with preterm labor who underwent amniocentesis for diagnosis of infection and/or inflammation in the AF. The AF was cultured, and assayed for IL-6. CRP levels and cervical length by transvaginal ultrasound were measured at the time of amniocentesis. The stored maternal plasma was assayed for IL-6, matrix metalloproteinase (MMP)-9, and complements C3a and C5a using ELISA kits. The primary and secondary outcome criteria were positive AF cultures and spontaneous preterm delivery (SPTD) within 48 h, respectively. Univariate, multivariate, and receiver operating characteristic analysis were used for the statistical analysis. RESULTS: In bivariate analyses, elevated plasma IL-6 level was significantly associated with intra-amniotic infection and imminent preterm delivery, whereas elevated plasma levels of MMP-9, C3a, and C5a were not associated with these two outcomes. On multivariate analyses, an elevated plasma IL-6 level was significantly associated with intra-amniotic infection and imminent preterm delivery after adjusting for confounders, including high serum CRP levels and short cervical length. In predicting intra-amniotic infection, the area under the curve (AUC) was significantly lower for plasma IL-6 than for AF IL-6 but was similar to that for serum CRP. Differences in the AUCs between plasma IL-6, AF IL-6, and serum CRP were not statistically significant in predicting imminent preterm delivery. CONCLUSIONS: Maternal plasma IL-6 independently predicts intra-amniotic infection in women with preterm labor; however, it has worse diagnostic performance than that of AF IL-6 and similar performance to that of serum CRP. To predict imminent preterm delivery, plasma IL-6 had an overall diagnostic performance similar to that of AF IL-6 and serum CRP. Plasma MMP-9, C3a, and C5a levels could not predict intra-amniotic infection or imminent preterm delivery.

Long-Term Hematopoietic Engraftment of Congenic Amniotic Fluid Stem Cells After in Utero Intraperitoneal Transplantation to Immune Competent Mice.

Clinical success of in utero transplantation (IUT) using allogeneic hematopoietic stem cells (HSCs) has been limited to fetuses that lack an immune response to allogeneic cells due to severe immunological defects, and where transplanted genetically normal cells have a proliferative or survival advantage. Amniotic fluid (AF) is an autologous source of stem cells with hematopoietic potential that could be used to treat congenital blood disorders. We compared the ability of congenic and allogeneic mouse AF stem cells (AFSC) to engraft the hematopoietic system of time-mated C57BL/6J mice (E13.5). At 4 and 16 weeks of age, multilineage donor engraftment was higher in congenic versus allogeneic animals. In vitro mixed lymphocyte reaction confirmed an immune response in the allogeneic group with higher CD4 and CD8 cell counts and increased proliferation of stimulated lymphocytes. IUT with congenic cells resulted in 100% of donor animals having chimerism of around 8% and successful hematopoietic long-term engraftment in immune-competent mice when compared with IUT with allogeneic cells. AFSCs may be useful for autologous cell/gene therapy approaches in fetuses diagnosed with congenital hematopoietic disorders.

Are amniotic fluid neutrophils in women with intraamniotic infection and/or inflammation of fetal or maternal origin?

BACKGROUND: Neutrophils are the most abundant white blood cells found in the amniotic cavity of women with intraamniotic infection and/or inflammation. The current belief is that these neutrophils are of fetal origin. However, abundant neutrophils have been found in the amniotic fluid of women with a severe acute maternal inflammatory response but without a severe fetal inflammatory response in the placenta, suggesting that these innate immune cells can also be of maternal origin or a mixture of both fetal and maternal neutrophils. OBJECTIVE: We sought to investigate the origin of amniotic fluid neutrophils from women with intraamniotic infection and/or inflammation and to correlate these findings with acute histologic maternal and fetal inflammatory responses in the placenta. STUDY DESIGN: Amniotic fluid was collected from 15 women with suspected intraamniotic infection and/or inflammation (positive microbiological cultures and/or interleukin-6 concentrations ≥2.6 ng/mL). Amniotic fluid neutrophils were purified by fluorescence-activated cell sorting, DNA was extracted, and DNA fingerprinting was performed. DNA fingerprinting was also performed in the umbilical cord and maternal blood DNA. Fluorescence in situ hybridization was assayed in women with male neonates. Blinded placental histopathological evaluations were conducted. RESULTS: First, DNA fingerprinting revealed that 43% (6/14) of women who underwent a single amniocentesis had mostly fetal neutrophils in the amniotic fluid. Second, DNA fingerprinting showed that 36% (5/14) of the women who underwent a single amniocentesis had predominantly maternal neutrophils in the amniotic fluid. Third, DNA fingerprinting indicated that 21% (3/14) of the women who underwent a single amniocentesis had an evident mixture of fetal and maternal neutrophils in the amniotic fluid. Fourth, DNA fingerprinting revealed that a woman who underwent 2 amniocenteses (patient 15) had fetal neutrophils first, and as infection progressed, abundant maternal neutrophils invaded the amniotic cavity. Fifth, fluorescence in situ hybridization confirmed DNA fingerprinting results by showing that both fetal and maternal neutrophils were present in the amniotic fluid. Sixth, most of the women who had predominantly amniotic fluid neutrophils of fetal origin at the time of collection delivered extremely preterm neonates (71% [5/7]). Seventh, all of the women who had predominantly amniotic fluid neutrophils of maternal origin at the time of collection delivered term or late preterm neonates (100% [6/6]). Eighth, 2 of the women who had an evident mixture of fetal and maternal neutrophils in the amniotic fluid at the time of collection delivered extremely preterm neonates (67% [2/3]), and the third woman delivered a term neonate (33% [1/3]). Finally, most of the women included in this study presented acute maternal and fetal inflammatory responses in the placenta (87% [13/15]). CONCLUSION: Amniotic fluid neutrophils can be either predominantly of fetal or maternal origin, or a mixture of both fetal and maternal origin, in women with intraamniotic infection and/or inflammation. The findings herein provide evidence that both fetal and maternal neutrophils can invade the amniotic cavity, suggesting that both the fetus and the mother participate in the host defense mechanisms against intraamniotic infection.

CXCL10 and IL-6: Markers of two different forms of intra-amniotic inflammation in preterm labor.

PROBLEM: To determine whether amniotic fluid (AF) CXCL10 concentration is associated with histologic chronic chorioamnionitis in patients with preterm labor (PTL) and preterm prelabor rupture of the membranes (PROM). METHOD OF STUDY: This study included 168 women who had an episode of PTL or preterm PROM. AF interleukin (IL)-6 and CXCL10 concentrations were determined by immunoassay. RESULTS: (i) Increased AF CXCL10 concentration was associated with chronic (OR: 4.8; 95% CI: 1.7-14), but not acute chorioamnionitis; (ii) increased AF IL-6 concentration was associated with acute (OR: 4.2; 95% CI: 1.3-13.7) but not chronic chorioamnionitis; and (iii) an increase in AF CXCL10 concentration was associated with placental lesions consistent with maternal anti-fetal rejection (OR: 3.7; 95% CI: 1.3-10.4). (iv) All patients with elevated AF CXCL10 and IL-6 delivered preterm. CONCLUSION: Increased AF CXCL10 concentration is associated with chronic chorioamnionitis or maternal anti-fetal rejection, whereas increased AF IL-6 concentration is associated with acute histologic chorioamnionitis.

Comparison of Amniotic Fluid Cytokine Levels in Postterm and Term Pregnancy: a Prospective Study.

BACKGROUND: To evaluate amniotic fluid pro- and anti-inflammatory cytokine levels in women with postterm and term pregnancies in labor and not in labor. METHODS: The study involved three groups: postterm (Group 1, n = 29), term in labor (Group 2, n = 28), and control (Group 3, n = 30). All groups were compared with respect to age, gravidity, parity, obstetric history, gestation week, cervical dilatation and effacement, maternal serum C-reactive protein and white cell count, amniotic interleukin 4, 6, and 10 levels, birthweight, and cord blood pH. RESULTS: The amniotic fluid interleukin 10 level was 24.4 ± 8.8 pg/mL in the postterm group, 13.5 ± 5.1 pg/mL in the term in labor group, and 19.8 ± 5.4 pg/mL in the control group (p < 0.001). The amniotic fluid interleukin 4 level was 86.5 ± 57.7 pg/mL in the postterm group, 38.2 ± 29.2 pg/mL in the term in labor group, and 81.9 ± 68.4 pg/mL in the control group (p = 0.002). The amniotic fluid interleukin 6 level was 329 ± 135.1 pg/mL in the postterm group, 252.8 ± 138.7 pg/mL in the term in labor group, and 227.9 ± 114.4 pg/mL in the control group (p = 0.02). There was a positive correlation between gestational age and IL-10 levels (p < 0.05). CONCLUSIONS: Amniotic fluid IL-10 and IL-4 cytokine levels were increased in postterm pregnancy and they decreased with active labor.

Immune activation in amniotic fluid from Zika virus-associated microcephaly.

Recent advances in the understanding of neuropathogenesis associated with Zika virus (ZIKV) infection has led to descriptions of neonatal microcephaly cases. However, none of these reports have evaluated the humoral response during ZIKV infection. We report here polyfunctional immune activation associated with increased interferon-gamma-inducible protein 10, interleukin (IL)-6, IL-8, vascular endothelial growth factor (VEGF), monocyte chemoattractive protein 1 (MCP-1), and granulocyte colony-stimulating factor (G-CSF) levels in the amniotic fluid of ZIKV-positive pregnant women with neonatal microcephaly. These cytokines have been associated not only with neuronal damage, but also with differentiation and proliferation of neural progenitor cells. Our results suggested that the immune activation caused by ZIKV infection in the uterine environment could also interfere with fetal development. ANN NEUROL 2017;81:152-156.

Neutrophil Extracellular Traps in the Amniotic Cavity of Women with Intra-Amniotic Infection: A New Mechanism of Host Defense.

OBJECTIVE: Neutrophil extracellular traps (NETs) control microbial infections through their antimicrobial activities attributed to DNA, histones, granules, and cytoplasmic proteins (eg, elastase). Intra-amniotic infection is characterized by the influx of neutrophils into the amniotic cavity; therefore, the aim of this study was to determine whether amniotic fluid neutrophils form NETs in this inflammatory process. METHODS: Amniotic fluid samples from women with intra-amniotic infection (n = 15) were stained for bacteria detection using fluorescent dyes. Amniotic fluid neutrophils were purified by filtration. As controls, neutrophils from maternal blood samples (n = 3) were isolated by density gradients. Isolated neutrophils were plated onto glass cover slips for culture with and without 100 nM of phorbol-12-myristate-13-acetate (PMA). NET formation was assessed by 4',6-diamidino-2-phenylindole (DAPI) staining and scanning electron microscopy. Different stages of NET formation were visualized using antibodies against elastase and histone H3, in combination with DAPI staining, by confocal microscopy. Finally, maternal or neonatal neutrophils were added to amniotic fluid samples from women without intra-amniotic infection (n = 4), and NET formation was evaluated by DAPI staining. RESULTS: (1) NETs were present in the amniotic fluid of women with intra-amniotic infection; (2) all of the amniotic fluid samples had detectable live and dead bacteria associated with the presence of NETs; (3) in contrast to neutrophils from the maternal circulation, amniotic fluid neutrophils did not require PMA stimulation to form NETs; (4) different stages of NET formation were observed by co-localizing elastase, histone H3, and DNA in amniotic fluid neutrophils; and (5) neither maternal nor neonatal neutrophils form NETs in the amniotic fluid of women without intra-amniotic infection. CONCLUSION: NETs are detectable in the amniotic fluid of women with intra-amniotic infection.