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.

Clinical chorioamnionitis at term IX: in vivo evidence of intra-amniotic inflammasome activation.

Background The inflammasome has been implicated in the mechanisms that lead to spontaneous labor at term. However, whether the inflammasome is activated in the amniotic cavity of women with clinical chorioamnionitis at term is unknown. Herein, by measuring extracellular ASC [apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (CARD)], we investigated whether there is in vivo inflammasome activation in amniotic fluid of patients with clinical chorioamnionitis at term with sterile intra-amniotic inflammation and in those with intra-amniotic infection. Methods This was a retrospective cross-sectional study that included amniotic fluid samples collected from 76 women who delivered after spontaneous term labor with diagnosed clinical chorioamnionitis. Intra-amniotic inflammation was defined as an elevated amniotic fluid interleukin (IL)-6 concentration ≥2.6 ng/mL, and intra-amniotic infection was diagnosed by the presence of microbial invasion of the amniotic cavity (MIAC) accompanied by intra-amniotic inflammation. Patients were classified into the following groups: (1) women without intra-amniotic inflammation or infection (n=16); (2) women with MIAC but without intra-amniotic inflammation (n=5); (3) women with sterile intra-amniotic inflammation (n=15); and (4) women with intra-amniotic infection (n=40). As a readout of in vivo inflammasome activation, extracellular ASC was measured in amniotic fluid by enzyme-linked immunosorbent assay. Acute inflammatory responses in the amniotic fluid and placenta were also evaluated. Results In clinical chorioamnionitis at term: (1) amniotic fluid concentrations of ASC (extracellular ASC is indicative of in vivo inflammasome activation) and IL-6 were greater in women with intra-amniotic infection than in those without intra-amniotic inflammation, regardless of the presence of MIAC; (2) amniotic fluid concentrations of ASC and IL-6 were also higher in women with sterile intra-amniotic inflammation than in those without intra-amniotic inflammation, regardless of the presence of MIAC; (3) amniotic fluid concentrations of IL-6, but not ASC, were more elevated in women with intra-amniotic infection than in those with sterile intra-amniotic inflammation; (4) a positive and significant correlation was observed between amniotic fluid concentrations of ASC and IL-6; (5) no differences were observed in amniotic fluid ASC and IL-6 concentrations between women with and without MIAC in the absence of intra-amniotic inflammation; (6) women with intra-amniotic infection had elevated white blood cell counts and reduced glucose levels in amniotic fluid compared to the other three study groups; and (7) women with intra-amniotic infection presented higher frequencies of acute maternal and fetal inflammatory responses in the placenta than those with sterile intra-amniotic inflammation. Conclusion The intra-amniotic inflammatory response, either induced by alarmins or microbes, is characterized by the activation of the inflammasome - as evidenced by elevated amniotic fluid concentrations of extracellular ASC - in women with clinical chorioamnionitis at term. These findings provide insight into the intra-amniotic inflammatory response in women with clinical chorioamnionitis at term.

Twenty-four percent of patients with clinical chorioamnionitis in preterm gestations have no evidence of either culture-proven intraamniotic infection or intraamniotic inflammation.

BACKGROUND: Recent studies on clinical chorioamnionitis at term suggest that some patients with this diagnosis have neither intraamniotic infection nor intraamniotic inflammation. A false-positive diagnosis of clinical chorioamnionitis in preterm gestation may lead to unwarranted preterm delivery. OBJECTIVE: We sought to determine the frequency of intraamniotic inflammation and microbiologically proven amniotic fluid infection in patients with preterm clinical chorioamnionitis. STUDY DESIGN: Amniocentesis was performed in singleton pregnant women with preterm clinical chorioamnionitis (<36 weeks of gestation). Amniotic fluid was cultured for aerobic and anaerobic bacteria and genital mycoplasmas and assayed for matrix metalloproteinase-8 concentration. Microbial invasion of the amniotic cavity was defined as a positive amniotic fluid culture; intraamniotic inflammation was defined as an elevated amniotic fluid matrix metalloproteinase-8 concentration of >23 ng/mL. Nonparametric and survival techniques were used for analysis. RESULTS: Among patients with preterm clinical chorioamnionitis, 24% (12/50) had neither microbiologic evidence of intraamniotic infection nor intraamniotic inflammation. Microbial invasion of the amniotic cavity was present in 34% (18/53) and intraamniotic inflammation in 76% (38/50) of patients. The most common microorganisms isolated from the amniotic cavity were the Ureaplasma species. Finally, patients without microbial invasion of the amniotic cavity or intraamniotic inflammation had significantly lower rates of adverse outcomes (including lower gestational age at delivery, a shorter amniocentesis-to-delivery interval, acute histologic chorioamnionitis, acute funisitis, and significant neonatal morbidity) than those with microbial invasion of the amniotic cavity and/or intraamniotic inflammation. CONCLUSION: Among patients with preterm clinical chorioamnionitis, 24% had no evidence of either intraamniotic infection or intraamniotic inflammation, and 66% had negative amniotic fluid cultures, using standard microbiologic techniques. These observations call for a reexamination of the criteria used to diagnose preterm clinical chorioamnionitis.

Clinical chorioamnionitis at term VIII: a rapid MMP-8 test for the identification of intra-amniotic inflammation.

OBJECTIVE: Clinical chorioamnionitis is the most common infection/inflammatory process diagnosed in labor and delivery units worldwide. The condition is a syndrome that can be caused by (1) intra-amniotic infection, (2) intra-amniotic inflammation without demonstrable microorganisms (i.e. sterile intra-amniotic inflammation), and (3) maternal systemic inflammation that is not associated with intra-amniotic inflammation. The presence of intra-amniotic inflammation is a risk factor for adverse maternal and neonatal outcomes in a broad range of obstetrical syndromes that includes clinical chorioamnionitis at term. Although the diagnosis of intra-amniotic infection has relied on culture results, such information is not immediately available for patient management. Therefore, the diagnosis of intra-amniotic inflammation could be helpful as a proxy for intra-amniotic infection, while results of microbiologic studies are pending. A rapid test is now available for the diagnosis of intra-amniotic inflammation, based on the determination of neutrophil collagenase or matrix metalloproteinase-8 (MMP-8). The objectives of this study were (1) to evaluate the diagnostic indices of a rapid MMP-8 test for the identification of intra-amniotic inflammation/infection in patients with the diagnosis of clinical chorioamnionitis at term, and (2) to compare the diagnostic performance of a rapid MMP-8 test to that of a conventional enzyme-linked immunosorbent assay (ELISA) interleukin (IL)-6 test for patients with clinical chorioamnionitis at term. MATERIALS AND METHODS: A retrospective cohort study was conducted. A transabdominal amniocentesis was performed in patients with clinical chorioamnionitis at term (n=44). Amniotic fluid was analyzed using cultivation techniques (for aerobic and anaerobic bacteria as well as genital Mycoplasmas) and broad-range polymerase chain reaction (PCR) coupled with electrospray ionization mass spectrometry (PCR/ESI-MS). Amniotic fluid IL-6 concentrations were determined by ELISA, and rapid MMP-8 results were determined by Yoon's MMP-8 Check®. Intra-amniotic inflammation was defined as an elevated amniotic fluid IL-6 concentration ≥2.6 ng/mL, and intra-amniotic infection was diagnosed by the presence of microorganisms in the amniotic fluid accompanied by intra-amniotic inflammation. The diagnostic indices of Yoon's MMP-8 Check® for the identification of intra-amniotic inflammation were calculated. In order to objectively compare Yoon's MMP-8 Check® with the ELISA IL-6 test for the identification of intra-amniotic inflammation, we used an amniotic fluid white blood cell (WBC) count ≥50 cells/mm3 to define intra-amniotic inflammation. RESULTS: (1) A positive rapid MMP-8 test had a sensitivity of 82.4% (28/34), specificity of 90% (9/10), positive predictive value of 96.6% (28/29), negative predictive value of 60% (9/15), positive likelihood ratio 8.2 (95% CI 1.3-53.2), and negative likelihood ratio 0.2 (95% CI 0.1-0.4) for the identification of intra-amniotic inflammation (prevalence 77.3%); (2) a positive rapid MMP-8 test had a sensitivity of 91.7% (22/24), specificity of 65% (13/20), positive predictive value of 75.9% (22/29), negative predictive value of 86.7% (13/15), positive likelihood ratio of 2.6 (95% CI 1.4-4.8), and negative likelihood ratio of 0.1 (95% CI 0.03-0.5) for the identification of intra-amniotic infection; (3) the rapid MMP-8 test had a significantly higher specificity than the ELISA IL-6 test in the identification of intra-amniotic inflammation as determined by an amniotic fluid WBC count ≥50 cells/mm3. The sensitivity and accuracy of the rapid MMP-8 test were comparable to those of the ELISA IL-6 test; and (4) importantly, the rapid MMP-8 test had 100% sensitivity and 100% negative predictive value in the identification of neonates affected with fetal inflammatory response syndrome (FIRS). CONCLUSION: The rapid diagnosis of intra-amniotic inflammation is possible by analysis of amniotic fluid using a point-of-care test for MMP-8. Patients with a positive test are at risk of delivering a neonate affected with systemic inflammation, a risk factor for adverse neonatal outcome.

Clinical chorioamnionitis at term VII: the amniotic fluid cellular immune response.

OBJECTIVES: 1) To characterize the cellular composition of the amniotic fluid of patients diagnosed with clinical chorioamnionitis at term, as a function of the presence or absence of microorganisms determined by cultivation techniques, and 2) to characterize the cytokine production by white blood cells present in the amniotic fluid using flow cytometry-based techniques. MATERIALS AND METHODS: Amniotic fluid samples from 20 women who had the diagnosis of clinical chorioamnionitis at term were analyzed using cultivation techniques (for aerobic and anaerobic bacteria as well as genital Mycoplasmas). Amniotic fluid IL-6 concentrations were determined by an enzyme-linked immunosorbent assay. Amniotic fluid leukocytes were visualized by using hematoxylin and eosin staining and immunofluorescence. Immunophenotyping of surface markers and cytokines was performed in amniotic fluid leukocytes using flow cytometry. RESULTS: 1) Neutrophils (CD45+CD15+ cells) were the most common leukocyte subset found in the amniotic fluid, followed by monocytes (CD45+CD14+ cells); other white blood cells (such as lymphocytes and natural killer cells) were scarce in the amniotic fluid; 2) the absolute counts of neutrophils and monocytes were significantly higher in patients with microorganisms found in the amniotic fluid than in those without detectable microorganisms, using cultivation techniques; 3) there was a significant correlation between the absolute counts of neutrophils and monocytes determined by flow cytometry (Spearman's correlation=0.97; P<0.001); 4) there was a significant correlation between the absolute white blood cell count determined with a hemocytometer chamber and by flow cytometric analysis (Spearman's correlation=0.88; P<0.001); and 5) the profile of cytokine expression differed between monocytes and neutrophils; while neutrophils predominantly produced TNF-α and MIP-1ß, monocytes expressed higher levels of IL-1ß and IL-1α. CONCLUSION: Flow cytometry analysis of the amniotic fluid of patients with intra-amniotic infection and clinical chorioamnionitis at term demonstrated that neutrophils and monocytes are the most common cells participating in the inflammatory process. We have characterized, for the first time, the differential cytokine expression by these cells in this important complication of pregnancy.

Enlarging amniotic fluid 'sludge' in preterm labor associated with severe course of sepsis and recurrent tension pneumothorax in the infant.

Amniotic fluid 'sludge' (AFS) is defined as the presence of dense aggregates of hyperechogenic material in close proximity to the internal cervical os. The presence of AFS is an independent risk factor for impending preterm delivery, histological chorioamnionitis, and microbial invasion of the amniotic cavity in patients with spontaneous preterm labor with intact membranes, and preterm prelabor rupture of membranes. We describe a case showing enlarging AFS on transvaginal ultrasound in a patient with impending preterm labor, followed by chorioamnionitis and emergency cesarean section at 28 weeks of gestation, resulting in a severe course of sepsis and recurrent tension pneumothorax in the infant. Such a case has not been reported as far as we know. Based on our case, sonographic findings of enlarging AFS may be a predictor of severe neonatal outcomes in a case with preterm labor even though the maternal symptoms of inflammation are not obvious.

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.

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

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