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Background Preterm birth (PTB) is a leading cause of neonatal mortality and longer-term morbidity. Acute chorioamnionitis (ACA) is a common cause of PTB, however, there are limited data on the prevalence of ACA and its association with PTB in resource limited settings. Methods Data and samples came from a clinical trial evaluating novel strategies for the prevention of malaria in HIV infected pregnant women in Uganda. Women were enrolled between 12-28 weeks of gestation and followed through delivery. For each placenta delivered, three placental tissue types (membrane roll, umbilical cord and chorionic plate/villous parenchyma) were collected. Slides were assessed for diagnosis of maternal and fetal ACA by microscopic evaluation of neutrophilic infiltration using a standardized grading scale. The primary outcomes were PTB (<37 weeks), low birth weight (LBW, <2500 grams), and small-for-gestational age (SGA, birth weight <10th percentile for age). Univariate and multivariate logistic regression were used to estimate associations between 1) maternal characteristics (age, education, wealth, gravidity, gestational age at enrollment, placental malaria, anti-malarial prophylaxis treatment regimen, HIV disease parameters) and ACA, and 2) associations between ACA and adverse birth outcomes. Findings A total of 193 placentas were included in the analysis. The prevalence of maternal and fetal ACA was 44.5% and 28.0%, respectively. HIV infected women between 28-43 years of age had a higher risk of maternal ACA compared to those between 17-21 years of age (50.9% vs. 19.1%; aOR = 4.00 (1.10-14.5), p = 0.04) and the diagnosis of severe maternal ACA was associated with a significantly higher risk of PTB (28.6% vs. 6.0%; aOR = 6.04 (1.87- 19.5), p = 0.003), LBW (33.3% vs. 9.4%; aOR = 4.86 (1.65-14.3); p = 0.004), and SGA (28.6% vs. 10.1%; aOR = 3.70 (1.20-11.4), p = 0.02). No maternal characteristics were significantly associated with fetal ACA and the diagnosis of fetal ACA was not associated with adverse birth outcomes. Conclusions Histological evidence of severe maternal ACA was associated with an increased risk of PTB, LBW, and SGA in HIV infected, pregnant Ugandan women.
Clinical data and biological samples for this study came from a double-blinded randomized clinical trial (Registration number {"type":"clinical-trial","attrs":{"text":"NCT02282293","term_id":"NCT02282293"}}NCT02282293) evaluating intermittent preventive therapy for malaria in pregnancy (IPTp) among HIV infected women in Tororo, Uganda [20]. Two hundred HIV infected pregnant women at least 16 years of age were enrolled at 12–28 weeks gestational age, between December 2014 to November 2015, and were randomized to daily trimethoprim-sulfamethoxazole (TMP-SMX) plus monthly dihydroartemisinin-piperaquine (DP) or daily TMP-SMX alone [20]. The study physician estimated gestational age using last menstrual period and fetal measurements obtained by a standardized abdominal ultrasound exam. All women were provided with combination antiretroviral therapy (ART) consisting of Efavirenz (EFV)/tenofovir/lamivudine. Women received routine HIV care per the Uganda Ministry of Health guidelines, and HIV-1 RNA monitoring was additionally performed. Participants were prospectively followed until delivery, at which point the placenta was harvested and prepared (as described below) for microscopic examination and diagnosis of ACA. At enrollment, a long-lasting insecticide treated net was provided to every participant and a detailed history including a household questionnaire and physical assessment/examination was obtained. Study participants were closely monitored throughout pregnancy by monthly assessments and collection of blood samples, and all their health care needs were provided by a dedicated study clinic open seven days a week. Participants were encouraged to deliver at the hospital. However, in the event of a home or other health facility delivery, participants were visited by the study staff at the time of delivery or as soon as possible afterwards. Upon delivery, a comprehensive assessment of birth outcomes was made, including neonatal evaluation for congenital anomalies, measurement of birth weight and gestational age. All placental sampling was done within 30 minutes of delivery. Placental specimens were fixed in 10% Neutral Buffered Formalin for 24 hours, and stored at room temperature in 70% ethanol prior to tissue processing. The following placental tissues were collected: placental membranes (“membrane roll”), umbilical cord (two cross-sectional slices, one proximal and one distal to where the cord inserts into the placental disc), chorionic plate with villous parenchyma, and basal plate with villous parenchyma. The basal plate/villous parenchyma section was used for diagnosis of PM in our separately reported studies [16, 20, 21], while the other three sections (membrane roll, umbilical cord, chorionic plate/villous parenchyma) were used as described below for the diagnosis of ACA in the study reported here. For microscopic diagnosis of ACA, three placental specimens were utilized: membrane roll, umbilical cord, and chorionic plate/villous parenchyma. In the Tororo, Uganda laboratory, specimens were dehydrated through a series of ethanol washes, cleared in xylene and embedded in paraffin wax blocks. A 3 μm thick section from each tissue block was obtained using a rotary microtome and sections mounted onto glass slides via a floatation water bath. Slides were baked in a hot air oven at 60°C for 30 minutes, de-paraffinized in xylene, dehydrated through a series of ethanol washes, stained with Hematoxylin and Eosin (H&E), and mounted with organic mounting media. Diagnosis of ACA was made by standard light microscopic examination (Nikon Eclipse Ci-L microscope with DS-Ri2 color camera) of the three placental biopsy slides (membrane roll, umbilical cord, and chorionic plate/villous parenchyma) (Fig 1). Diagnosis of a maternal acute inflammatory response to infection (“maternal ACA”) was indicated by detection of neutrophils within the chorioamnion layers of the membrane roll and/or the chorionic plate. Diagnosis of a fetal acute inflammatory response to infection (“fetal ACA”) was indicated by detection of neutrophils with or without eosinophils within the vascular smooth muscle of the umbilical cord vessels and/or the chorionic plate vasculature [9]. Diagnosis was rendered in blinded fashion by a placental pathologist (G.R.) and noted on a standardized case record form. (A&B) Normal umbilical cord. *indicates lumen of umbilical vessels. (C) Umbilical vein with heavy neutrophilic infiltrate (arrows point to aggregates of neutrophils) and early degeneration of smooth muscle cells diagnostic of severe fetal ACA. (D) Normal chorionic plate/villous parenchyma. Arrow indicates the chorionic plate, and * indicates lumen of a fetal chorionic plate vessel. (E) Chorionic plate with heavy neutrophilic infiltrate in the walls of a fetal chorionic plate vessel (severe fetal ACA) and neutrophils in the subchorionic fibrin layer (maternal ACA). Arrows point to aggregates of neutrophils. (F&G) Normal membrane roll. (H) Membrane roll with neutrophilic microabscess diagnostic of severe maternal ACA (arrows point to neutrophils). In G and H, {-bracket indicates amnion, [-bracket indicates chorion, and arrow indicates decidua parietalis. Scale bars are 1 mm (panels A, D, F) and 200 μM (panels B, C, E, G, H). Grading of maternal ACA and fetal ACA was assigned using the standardized diagnostic framework of the Perinatal Section of the Society for Pediatric Pathologists [9]. Grade was recorded on the case record form as either: no evidence of ACA, mild (scattered mostly single, isolated neutrophils), moderate (small clusters of neutrophils) and severe (presence of neutrophilic microabscesses). Specifically, severe maternal ACA was defined as the presence of microabscesses measuring greater than or equal to 10 x 20 neutrophils in extent, and present within at least three foci, or with neutrophils present as a contiguous band. Severe fetal ACA was defined as the presence of near confluent intramural neutrophils with attenuation/degeneration of vascular smooth muscle cells. The maternal characteristics examined included: maternal age and gestational age at enrollment, education, household wealth index, and gravity. Maternal characteristics pertaining to malaria included: IPTp regimen (either TMP-SMX plus DP or TMP-SMX alone), and evidence of PM defined by histopathology. Additionally, the following maternal characteristic pertaining to HIV infection were assessed: duration since HIV diagnosed, duration since ART was begun, WHO HIV stage at enrollment, CD4 T-cell count at delivery, and viral load at delivery. None of the women enrolled were WHO HIV stage 4 (defined as AIDS). ACA was categorized as maternal ACA (the presence of maternal acute inflammation, defined above) and fetal ACA (the presence of fetal acute inflammation, defined above). Maternal and fetal ACA were each categorized into a grading scale: none/mild, moderate and severe (defined above). The birth outcomes assessed were PTB (delivery at <37 weeks), LBW (birth weight <2500 grams, and SGA (birth weight <10th percentile for age). Data were coded, double entered into a Microsoft access database and analyzed using Stata 14 (Stata Corp, College Station TX). Participants’ baseline maternal characteristics were expressed as means ± SD for continuous variables, while categorical variables were expressed as proportions. Univariate and multivariate logistic regression were used to 1) measure associations between maternal characteristics and maternal and fetal ACA, and 2) measure associations between maternal and fetal ACA and the risk of adverse birth outcomes. P values < 0.05 were considered statistically significant. Measures of association in both univariate and multivariate models were expressed as odds ratios (OR) or adjusted odds ratios (aOR) and respective 95% confidence intervals (CI). Gravidity was not included in the final multivariate model due to correlation with maternal age. All study participants provided informed written consent. Ethical approval was obtained from the Uganda National Council of Science and Technology (UNCST #HS 1708), Makerere University School of Medicine Research and Ethics Committee, the Makerere University School of Biomedical Sciences Research and Ethics Committee (#SBS-REC 159), and the University of California, San Francisco, Committee on Human Research (CHR #14–13900).
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