Placental histology micrographs illustrating histologic chorioamnionitis of increasing severity and the corresponding amniotic fluid Mass Restricted scores are presented in Figure 1, while the quantitative aspects of the relationship are depicted in Figure 2. There was a significant correlation between amniotic fluid inflammation as estimated by the Mass Restricted score and the presence (Spearman r=0.475, P<.001) or stage (Spearman r=0.539, P<.001) of histologic chorioamnionitis (Fig. 2A). This indicates that patients with a Mass Restricted score 3–4 are more likely to have marked neutrophil infiltration of the chorionic plate. Multivariable logistic regression analysis revealed that a diagnosis of histologic chorioamnionitis (any stage) occurred independent of the amniocentesis-to-delivery interval or status of the membranes (intact compared with ruptured) and was best predicted by the combination of a Mass Restricted score 3–4 (odds ratio 10.4, 95% confidence interval [CI] 3.3–32.1], P<.001) and an earlier gestational age at delivery (odds ratio 0.8, 95% CI 0.7–0.9, P<.001). The concordance statistic of the model was 0.789 while true-positive fractions and false-positive fractions values were 81.6% and 30.6%, respectively. The amniotic fluid WBC count, mode of delivery (vaginal or cesarean), steroid, antibiotic exposures, demographic characteristics such as maternal age or race, and results of other rapid laboratory tests of amniotic fluid used clinically (glucose concentration, LDH, or Gram stain) did not show significance in the model, suggesting these variables had less value in predicting histologic chorioamnionitis compared with the Mass Restricted score. For comparison, concordance statistic values for an amniotic fluid-positive Gram stain, a glucose concentration of 15 mg/dL or less, or a LDH activity of 419 International Units/L or more ranged from 0.644 (Gram stain), to 0.667 (LDH), to 0.686 (glucose). True-positive fractions values of the same tests were 33.3%, 39.3%, and 51.5%, whereas false-positive fractions values were 3.6%, 5.9%, and 14.3%, respectively.
To exclude the possibility of a prolonged amniocentesis-to-delivery interval affecting or confounding the presence or emergence of histologic chorioamnionitis, we then restricted our analysis to cases delivered within 48 hours from the time of the amniocentesis (n=89).32 Again, a Mass Restricted score 3–4 and gestational age at delivery were the only predictors retained in our model for this subset (concordance statistic 0.778, true-positive fractions 88.9%, false-positive fractions 36.4%; odds ratios Mass Restricted score 10.0 [95% CI 2.5–40.2], P=.001 and gestational age at delivery 0.8 [95% CI 0.7–0.9], P=.005). The direct and significant correlation of the Mass Restricted score with the stages of histologic chorioamnionitis was also maintained for this analysis (Spearman r=0.505, P<.001).
Next, we analyzed the relationship between intraamniotic inflammation and grades of choriodeciduitis and amnionitis. There was a direct relationship between the Mass Restricted score and severity of inflammation in the choriodecidua (Spearman r=0.465, P<.001) (Fig. 2B) and in amnion (Spearman r=0.536, P<.001) (Fig. 2C). Again, these relationships were independent of the amniocentesis-to-delivery interval, mode of delivery, and steroid or antibiotic exposure.
In a prior study we demonstrated that the appearance of the biomarkers of the Mass Restricted score in amniotic fluid is not random but rather sequential.19 The conversion from “mild” (Mass Restricted score 1–2) to “severe” inflammation (Mass Restricted score 3–4) is marked by the appearance in the amniotic fluid of the peak corresponding to calgranulin C (S100A12) protein followed by that of calgranulin A (S100A8). For clinical relevance we were interested in the relationships between the component biomarkers of the Mass Restricted score and severity of histologic chorioamnionitis. We found that the presence of calgranulin C had the strongest relationship with the presence of a stage III chorioamnionitis (concordance statistic: 0.762; true-positive fractions: 71%; false-positive fractions: 19%; odds ratio: 10.2 [95%CI: 4.8–21.6], P<.001), independent of race, amniocentesis-to-delivery interval or gestational age at amniocentesis or delivery.
This study provides evidence that there is a direct correlation between the degree of intraamniotic inflammatory response (as estimated by the Mass Restricted score) and the severity of chorioamnionitis, choriodeciduitis and amnionitis. This relationship is maintained independent of the amniocentesis-to-delivery interval, mode of delivery, and steroid or antibiotic exposure. Lastly, the presence in amniotic fluid of the biomarker corresponding to calgranulin C (S100A12 protein biomarker) is strongly associated with neutrophil infiltration of the chorionic plate. Calgranulin C is a member of the S100 protein family (S100A12) and a potent ligand for the receptor for advanced glycation end-products (RAGE), activity for which it has been designated as ENRAGE (extracellular newly identified RAGE-binding protein).33
This research was motivated by prior studies which suggested that histologic chorioamnionitis is an important risk factor for neonatal sepsis and poor long-term neurodevelopmental outcome.34–36 The finding that newborns delivered by mothers with “severe” intraamniotic inflammation (Mass Restricted score 3–4) and advanced stage of chorioamnionitis more frequently had a sepsis work-up suggestive of early neonatal sepsis, seems to support for this theory, bearing in mind that early neonatal sepsis is a risk factor for neonatal disease, early death and adverse neurologic events.37 The importance of this model is predicated on the idea that inflammation, a highly orchestrated process designed to fight infection or tissue injury, can turn maladaptive and contribute to the fetal neurologic damage by virtue of the effects of proinflammatory cytokines on blood vessels, astrocytes and neurons.38
The prior description of stages and grades of histologic chorioamnionitis, choriodeciduitis and amnionitis represented a significant step forward by development of pathologic criteria which allowed scientists to provide a uniform description of the distribution of acute placental inflammation in women with preterm birth.26 However, description of a relationship between the intensity of histologic inflammation (based on stages and grades) and that of intraamniotic inflammation (based on cytokines and matrix metalloproteinase cut-offs) proved to be difficult.39,40 Some of the explanations for the imperfect correlation between histologic inflammation and cytokines may be inherent in pathology practice which relies on analysis of tissues rather than biologic fluids. Differences in tissue sampling relative to the lower uterine segment (and the initial site of ascending infection), variation in the integrity of the decidual capillary bed, restricting neutrophil exposure to cytokine gradients may account for the significant regionalization of the inflammatory infiltrate. Perhaps most important is that the current pathology practice still follows the recommendations espoused by Kurt Benirschke in 1961,41 assessing neutrophil number in the different tissues that bound, or are contained in, the amniotic fluid space, while the intervening decades have demonstrated the range of inflammatory effects on connective tissue integrity (via matrix metalloproteinases), cell cycle (cell proliferation and apoptosis). Neutrophil number may also not be the most reliable scale to assess intraamniotic cytokines, nor to measure the risks for infection-associated sequelae such as cerebral palsy. Perhaps, along with other variables (fetal genotype, integrity of the blood brain barrier, gestational age at delivery), this may be an explanation for why 76% of the fetuses exposed to elevated amniotic fluid cytokine concentration do not develop cerebral palsy.42
While some researchers have not found a racial/ethnic difference for chorioamnionitis others have found that clinically diagnosed intraamniotic infection occurs more commonly in African-American women than in other groups.43 Recognizing that clinical signs and symptoms of chorioamnionitis are unreliable, Dammann et al found a significant relationship between histologic chorioamnionitis and African-American ancestry.44 In the present study we found that African-American women were more likely to have “severe” intraamniotic inflammation as indicated by the Mass Restricted score, which proved to be a stronger predictor of histologic chorioamnionitis in multivariable analysis than race per se. Moreover, our data also indicate that a significant proportion of women with histologic chorioamnionitis and/or “severe” intraamniotic inflammation lacked clinical signs of inflammation underscoring the limited value of clinical symptoms in predicting histologic inflammation in the placenta. This is especially important in the context where histologic (but not clinical) chorioamnionitis is a significant predictor of neonatal brain injury.45
From a pathophysiologic perspective, the biomarkers comprising the Mass Restricted score (defensins and calgranulins) are anti-microbial/anti-inflammatory proteins released following neutrophil activation.46 Our study provides evidence in support of the view that there is a direct relationship between stages and grades of placental histologic inflammation and the intensity of the intraamniotic inflammation as determined by the Mass Restricted score. The clinical implication is that the severity of histologic chorioamnionitis can be potentially predicted at the time of amniocentesis and before histologic evaluation of the placenta.
The use of subclinical biomarkers for individual-level prediction and classification in the context of personalized medicine has expanded significantly in recent years with cancer markers at the forefront.30 However, the practical utility of such markers remains to be judged entirely in the context where they are to be used. For instance, when screening for prostate cancer in a healthy population, Baker et al47 recommends a false-positive fractions of less than 2% and a true-positive fractions of 50% or more. These values are significantly different for prognostic markers measured in people with disease. A true-positive fractions of 92% and a false-positive fractions of 42% are quoted by one study which evaluated a gene expression profile in patients with breast cancer as a prognostic marker of distant metastases within 5 years.48 The study also emphasized on the importance of a high true-positive fractions for prognostic markers of poor outcome. Although there are no preset numbers for what should be acceptable or not, we appreciate this scenario resembles more closely to our clinical setting. Our predictive model based on a Mass Restricted score 3–4 in amniocentesis samples from symptomatic women had a true-positive fractions of 82%-89% as compared with 33%-51% for the other clinically implemented tests of amniotic fluid. However, whether it would be acceptable for 30% of women with preterm labor or preterm PROM to undergo additional unnecessary therapy remains to be further evaluated.
A major hypothesis to explain the relationship between adverse neonatal outcomes and histologic chorioamnionitis is that in utero, cytokines, activated inflammatory cells, and other mediators of the innate immune response are released by the placenta into the fetal circulation where they can directly affect the integrity of the fetus.49 Members of our research team (CMS) previously described that acute inflammation of the chorionic plate was the most sensitive indicator of infection and hence inflammation.50 Taken together, these data suggest that vascular pathologic features (arthritis and phlebitis) in the chorionic plate and choriodecidua may be important variables to control for the cytokine exchange between the placental and fetal compartment. The observations reported herein suggest that a more advanced degree of inflammation, as reflected by the presence of calgranulin C (S100A12/ENRAGE) in the amniotic fluid, had the strongest relationship with the presence of a stage III chorioamnionitis. We previously determined that histologic chorioamnionitis was associated with increased placental expression of S100A12/ENRAGE paralleled by a decrease in mRNA levels of its antagonist, soluble RAGE.33 Based on recent data suggesting an important role of this biomarker in vascular remodeling,51 we propose that in women with advanced stages of histologic chorioamnionitis, S100A12/ENRAGE may participate directly in vascular inflammation, promoting access of inflammatory mediators into the fetal circulation. This paradigm requires further testing to clarify the role of S100A12/ENRAGE in placental vascular tissue injury.
In summary, our results provide insight into the relationship between severity of histologic chorioamnionitis and amniotic fluid of biomarkers characteristic for intraamniotic inflammation. Additionally, we propose a novel paradigm related to a direct role for one of the biomarkers, S100A12/ENRAGE, in endothelial cellular activation and placental tissue injury secondary to inflammation and infection.
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