0031-3998/07/6103-0318 PEDIATRIC RESEARCH Vol. 61, No. 3, 2007 Copyright © 2007 International Pediatric Research Foundation, Inc. Printed in U.S.A. Proteomic Biomarkers of Intra-amniotic Inflammation: Relationship with Funisitis and Early-onset Sepsis in the Premature Neonate CATALIN S. BUHIMSCHI, IRINA A. BUHIMSCHI, SONYA ABDEL-RAZEQ, VICTOR A. ROSENBERG, STEPHEN F. THUNG, GUOMAO ZHAO, ERICA WANG, AND VINEET BHANDARI Department of Obstetrics, Gynecology and Reproductive Sciences [C.S.B., I.A.B., S.A.-R., V.A.R., S.F.T., G.Z., E.W.], and Department of Pediatrics [V.B.], Division of Perinatal Medicine, Yale University School of Medicine, New Haven, CT 06520 ABSTRACT: Our goal was to determine the relationship between 4 vein inflammatory cytokine levels, but not maternal serum val- amniotic fluid (AF) proteomic biomarkers (human neutrophil de- ues, correlate with the presence and severity of the placental fensins 2 and 1, calgranulins C and A) characteristic of intra-amniotic histologic inflammation and umbilical cord vasculitis (7). inflammation, and funisitis and early-onset sepsis in premature neo- Funisitis is characterized by perivascular infiltrates of in- nates. The mass restricted (MR) score was generated from AF flammatory cells and is considered one of the strongest hall- obtained from women in preterm labor (n ϭ 123). The MR score marks of microbial invasion of the amniotic cavity and fetal ranged from 0–4 (none to all biomarkers present). Funisitis was graded histologically and interpreted in relation to the MR scores. inflammatory syndrome (8,9). While there is some debate with Neonates (n ϭ 97) were evaluated for early-onset sepsis. There was regard to the origin of the amniotic fluid (AF) neutrophilic significant correlation between the severity of AF inflammation and response to infection (10), there is consensus that the fetal the presence (53/123) and grades of funisitis (p Ͻ 0.001). Funisitis inflammatory neutrophilic response originates initially from the occurred independently of the amniocentesis-to-delivery interval or umbilical vein and later from the arteries leading to umbilical status of the membranes and was best predicted by an MR score 3–4 cord vasculitis and funisitis (11). Funisitis is a well-recognized and an earlier gestational age (GA) at delivery. Neonates born to risk factor for early-onset sepsis in the premature neonate (12,13). women with an MR score 3–4 had an increased incidence of sus- We have previously shown that proteomic mapping of the pected/confirmed sepsis, even after adjusting for GA at birth. Cal- AF reveals a profile, designated as the Mass Restricted (MR) granulin C had the highest association with clinically significant score, that is highly characteristic of intra-amniotic inflamma- funisitis, while calgranulin A had the strongest association with tion (14). The presence of 4 protein biomarkers (neutrophil early-onset sepsis. To conclude, AF proteomic analysis shows that women with MR scores 3–4 are more likely to have histologic defensins 2 and 1, calgranulins C and A) were highly predic- funisitis, and deliver neonates with early-onset sepsis. (Pediatr Res tive of PTB, whether inflammation was secondary to infection 61: 318–324, 2007) or bleeding (15). We further found that the interpretation of the MR score is not binary (disease present or disease absent), but rather a gradient progressing from the absence of disease, espite extensive research and a variety of interventions, to minimal, and then severe inflammation. Therefore, grading D the rate of preterm birth (PTB) has actually increased the severity of umbilical cord inflammation with increasing over the past 20 y (1). More than 500,000 preterm infants were numerical values of the MR score was based on the number born in the United States in 2004 (1). Interventions to prevent (from 1 to 4) of protein biomarkers being identified in the AF. PTB and its consequences for the fetus should predominantly Thus far, the relationship between the MR score or the indi- be targeted at the prevention, early recognition and treatment vidual 4 proteomic biomarkers, and histologic funisitis has not of several risk factors including intra-amniotic inflammation/ been addressed. In the current study, we sought to determine infection (2). the relationship between presence in the AF of 4 proteomic Robust scientific evidence has confirmed that when intra- biomarkers characteristic of intra-amniotic inflammation, and amniotic inflammation secondary to, or independent of infec- the likelihood of histologic funisitis and early-onset sepsis in tion is superimposed upon prematurity, the consequences for premature neonates. the neonate can prove devastating (3,4). This association can be explained by an inflammatory cytokine cascade that seems METHODS to be, at least partially, of fetal origin (5,6). Indeed, fetal umbilical Patient population and study design. We conducted a prospective study that included 123 women recruited upon admission to the Labor and Birth unit or the antepartum units at Yale New Haven Hospital. For all participants, Received June 30, 2006; accepted October 6, 2006. following admission with symptoms of preterm labor (PTL), preterm prema- Correspondence: Vineet Bhandari, M.D., Yale University School of Medicine, Divi- sion of Perinatal Medicine, Department of Pediatrics, 333 Cedar Street, LCI 401B, New Haven, CT 06520; e-mail: [email protected] Supported by NICHD RO3 HD 50249-01 and K12 HD 047018-01 WRHR (C.S.B.); Abbreviations: AF, amniotic fluid; LDH, lactate dehydrogenase; MR score, NICHD RO1 HD 047321 (I.A.B.); and NHLBI K08 HL 074195 (V.B.). mass restricted score; PPROM, preterm premature rupture of membranes; DOI: 10.1203/01.pdr.0000252439.48564.37 PTB, pretem birth; PTL, preterm labor; WBC, white blood cells 318 AF PROTEOMIC PROFILE AND NEONATAL SEPSIS 319 ture rupture of membranes (PPROM), advanced cervical dilatation (Ն3 cm) Ciphergen Biosystems, Fremont, California). After1hofincubation the and/or uterine contractions refractory to tocolysis, AF was retrieved by arrays were read in the ProteinChip Reader (Model PBS IIC) (Ciphergen ultrasound-guided amniocentesis. Subjects were enrolled (May 2004–May Biosystems) using the ProteinChip Software 3.1.1. Peaks composing the MR 2006) based on the availability of one of the investigators (CSB), and all score were identified by their conspicuous aspect at or in proximity of their enrolled women were included in the final analysis. known respective masses: 3,377.0 and 3,448.1 Da (corresponding to neutro- The Human Investigation Committee of Yale University approved the phil defensins 2 and 1, respectively) and at 10,443.8 and 10,834.5 Da study and written informed consent was obtained from all participants. (corresponding to calgranulins C and A respectively) (14). The MR score Eligible subjects had: a singleton fetus, gestational age (GA) Ͻ37 wk with a provides qualitative information regarding the presence or absence of intra- clinically indicated amniocentesis to rule-out intra-amniotic infection. Exclu- amniotic inflammation (14). The MR score ranges from 0 to 4, depending sion criteria included: anhydramnios, human immunodeficiency virus or upon the presence or absence of each of the four protein biomarkers (14). A hepatitis infections, and the presence of fetal heart rate abnormalities at categorical value of 1 is assigned if a biomarker peak is present and 0 if enrollment (bradycardia, or prolonged variable decelerations). GA was estab- absent. For the purpose of data analysis, we stratified the study population lished based on an ultrasonographic examination before 20 wk in all in- based on the severity of inflammation (MR 0: no inflammation; MR 1–2: stances. PTL was defined as the presence of regular uterine contractions and minimal inflammation; MR 3–4: severe inflammation). One investigator documented cervical effacement and/or dilatation in patients Ͻ37 wk GA (IAB) performed all the protein chip assays and scored all the samples blindly, (16). The diagnosis of PPROM was confirmed by visualization of AF pooling being unaware of either clinical presentation or outcome. through the cervical os, nitrazine, ferning, or amniocentesis-dye positive tests. Histologic evaluation of umbilical cords and diagnosis of funisitis. At Management of the patients was left to the clinical team. In the absence of Yale-New Haven Hospital, placental pathologic study is a routine part of the clinical laboratory results suggestive of infection, or signs/symptoms of evaluation of a pregnancy complicated by PTL or PPROM. In all 123 cases, clinical chorioamnionitis (fever Ͼ100.4°F, abdominal tenderness, fetal tachy- hematoxylin- and eosin-stained sections of the umbilical cord were read by a cardia), and/or abnormalities of fetal heart rate (variable or late decelerations), perinatal pathologist unaware of the results of the proteomic profiling. Each and/or abruption, PPROM was managed expectantly. In PPROM patients, section was examined systematically for the presence or absence of inflam- digital exams were not permitted. Patients received corticosteroids for lung mation and funisitis was diagnosed when neutrophils infiltrated the umbilical maturity if Ͻ32 wk GA and antibiotic therapy (latency phase: ampicillin/ vessels walls or Wharton’s jelly. Funisitis results were expressed using the erythromycin or clindamycin; group B Streptococcus prophylaxis: penicillin, histologic grading system devised by Salafia et al. (22) as: Grade 0, no cefazolin, clindamycin, erythromycin or vancomycin, based on culture results neutrophils observed; Grade 1, neutrophils within the inner third of the and sensitivity). Eighty percent of women had their amniocentesis performed umbilical vein wall (umbilical phlebitis); Grade 2, neutrophils within the inner before initiation of antibiotic or steroid therapy. Women were monitored by third of at least two umbilical vessel walls; Grade 3, neutrophils in the cardiotocography at least twice daily for the presence of fetal heart abnor- perivascular Wharton’s jelly; or Grade 4, panvasculitis and funisitis extending malities and/or uterine contractions. deep into the Wharton’s jelly. Following amniocentesis, each woman was followed prospectively to Evaluation of early-onset neonatal sepsis.