The Journal of Maternal-Fetal and Neonatal Medicine, 2012; Early Online: 1–6 © 2012 Informa UK, Ltd. ISSN 1476-7058 print/ISSN 1476-4954 online DOI: 10.3109/14767058.2012.735997
ORIGINAL ARTICLE
mniotic fluid myeloperoxidase in pregnancies complicated by preterm A prelabor rupture of membranes Marian Kacerovsky1,2,*,Vojtech Tambor1,*, Marie Vajrychová3, Juraj Lenco3, Helena Hornychova4, Ivana Musilova2,5 & Ramkumar Menon6
J Matern Fetal Neonatal Med Downloaded from informahealthcare.com by Texas Tech Hlth Sci Ctr on 11/28/12 For personal use only.
1Biomedical Research Center, University Hospital Hradec Kralove, Czech Republic, 2Department of Obstetrics and Gynecology, Charles
University in Prague, Faculty of Medicine, Hradec Kralove, Czech Republic, 3Institute of Molecular Pathology, Faculty of Military Health Sciences, University of Defence, Hradec Kralove, Czech Republic, 4Fingerland’s Department of Pathology, Charles University in Prague, Faculty of Medicine and University Hospital, Hradec Kralove, Czech Republic, 5Department of Obstetrics and Gynecology, Hospital Pardubice, Czech Republic, and 6Department of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine & Perinatal Research, The University of Texas Medical Branch at Galveston, TX, USA Microbial invasion of the amniotic cavity (MIAC) is found most frequently in pregnancies complicated by pPROM and occurs in approximately 30–40% of cases [4,5]. Some studies indicate that this rate may be even higher, depending on gestational age and the bacterial detection techniques utilized [4–6]. MIAC sets off host innate immune defense, resulting in a cascade of inflammatory processes initially through pattern recognition receptors. Inflammation is characterized by infiltration of neutrophils and other immune cells into the fetal membranes and placenta, a pathologic condition known as histological chorioamnionitis (HCA) [7–10]. Although more than 70% of HCA are associated with MIAC, a variety of non-infectious stimuli (e.g. fetal hypoxia, meconium) may also activate pattern recognition receptors and may lead to induction of HCA [11]. Both MIAC and HCA are associated with the presence of white blood cells in the amniotic fluid, specifically neutrophils [12,13]. Their assessment in amniotic fluid has been traditionally considered, along with Gram stain, amniotic fluid glucose and lactate levels, as a gold standard for the detection of intra-amniotic infection and inflammation. Intra-amniotic infiltration of neutrophils is typified by the production of myeloperoxidase (MPO), a hemecontaining lysosomal protein stored in granules of the neutrophil that belongs to the first line of defense mechanisms against invading microorganisms [14–16]. MPO is involved in generating reactive oxygen species causing oxidative burst of invading pathogens [17,18]. Elevated MPO level in biological fluids is considered as marker of neutrophil activation and degranulation [19]. Although the assessment of white blood cell count in amniotic fluid has been broadly used, there is only limited information regarding MPO in amniotic fluid. Two pieces of pioneering proteomic work described MPO as a profoundly dysregulated amniotic fluid protein in spontaneous preterm labor complicated by the presence of intra-amniotic infection and inflammation [15,16]. In addition, our recent quantitative proteomic studies identified MPO as one of the most altered proteins in the amniotic fluid from pPROM pregnancies complicated by the presence of both MIAC and HCA [20,21]. Nevertheless, these findings have not been confirmed with a complementary approach, for
Objective: To determine amniotic fluid myeloperoxidase concentration in women with preterm prelabor rupture of the membranes with microbial invasion of the amniotic cavity and histological chorioamnionitis. Methods: One hundred eightyone women with singleton pregnancies with a gestational age between 24+0 and 36+6 weeks were included in this study. Amniocenteses were performed, and myeloperoxidase concentration in the amniotic fluid was determined using ELISA. Result: Women with microbial invasion of the amniotic cavity had higher median myeloperoxidase concentration than women without this condition (149.2 ng/mL vs. 54.6 ng/mL; p = 0.0006). Women with the presence of histological chorioamnionitis had higher median myeloperoxidase concentration than women without histological chorioamnionitis (103.7 ng/mL vs. 50.0 ng/ mL; p = 0.0001). The presence of both microbial invasion of the amniotic cavity and histological chorioamnionitis was associated with higher median myeloperoxidase concentration (456.0 ng/ mL vs. 52.9 ng/mL; p < 0.0001). The results remained significant after adjusting for gestational age. Conclusions: Increased amniotic fluid myeloperoxidase in microbial invasion of the amniotic cavity and histological chorioamnionitis confirm a role of myeloperoxidase in preterm prelabor rupture of the membranes pathophysiology. Keywords: Histological chorioamnionitis, lysosomal protein, microbial invasion of the amniotic cavity, neonatal outcome, preterm delivery
Introduction Preterm delivery is an enduring and principal cause of perinatal morbidity and mortality worldwide. A substantial part of preterm deliveries is caused by preterm prelabor rupture of membranes (pPROM) [1]. Besides requiring acute intensive care, the preterm newborns are often threatened by lifelong adverse sequelae (e.g. cerebral palsy). In addition, these conditions have both direct and long-term consequences on subsequent health-care costs [2,3].
*These authors contributed equally on this paper Correspondence: Marian Kacerovsky MD, PhD, Department of Obstetrics and Gynecology, Charles University, University Hospital, Sokolska 581, 500 05 Hradec Kralove, Czech Republic. Mobile: +420-777657991. Tel: +420-495832676. E-mail:
[email protected]
1
2 M. Kacerovsky et al. example, antibody-based method, which is an important step for the translation of proteomic findings closer to the clinics. Therefore, the main aim of this study was to document amniotic fluid MPO concentrations in pPROM pregnancies complicated by both MIAC and HCA. Additionally, we determined the relationship between MPO concentrations and neonatal outcomes from pPROM pregnancies.
J Matern Fetal Neonatal Med Downloaded from informahealthcare.com by Texas Tech Hlth Sci Ctr on 11/28/12 For personal use only.
Material and methods A retrospective cohort study was performed. Pregnant women with gestational age between 240/7 and 366/7 weeks, who were admitted to the Department of Obstetrics and Gynecology, University Hospital Hradec Kralove, between August 2009 and March 2012 with diagnosis of pPROM were involved. pPROM was defined as the leakage of amniotic fluid prior to the onset of regular uterine contractions (by at least 2 hours). This condition was diagnosed visually using a sterile speculum examination to confirm pooling of amniotic fluid in the vagina together with a positive test for the presence of insulin-like growth factor–binding protein (ACTIM PROM test; MedixBiochemica, Kauniainen, Finland) in the vaginal fluid. In total, 209 women with pPROM were recruited, and 181 were included in our study because histopathologic assessment of the placenta and polymerase chain reaction (PCR) analysis for genital mycoplasmas were not available for 21 (10%) and 7 (3%) women, respectively. The gestational age of all included subjects was assessed by first trimester ultrasound evaluation. Women with singleton pregnancies and older than 18 years were included in the study. Exclusion criteria were signs of small size for gestational age (estimated fetal weight by ultrasound below 10th percentiles for gestational age), fetal structural malformations or chromosomal abnormalities, other maternal complications (hypertension, preeclampsia, diabetes mellitus, and thyroid disease), and vaginal bleeding or signs of fetal hypoxia. Sample collection Ultrasound-guided transabdominal amniocentesis was performed upon admission before the administration of corticosteroids, antibiotics, or tocolytics; approximately 5 mL of amniotic fluid was aspirated. The samples were divided into three polypropylene tubes and processed immediately. Two tubes were transported to the laboratory for detection of genital mycoplasmas using PCR analyses and for aerobic and anaerobic cultivation. The third tube was supplemented with protease inhibitors (40 μL per 1 mL of sample; Complete Mini, EDTA-free Protease Inhibitor Cocktail; Roche Diagnostics, Basel, Switzerland), centrifuged for 15 min at 300 x g, filtered using a syringe-driven 0.22-μm filter (TPP, Trasadingen, Switzerland) to remove cells and debris, and aliquoted. Aliquots were stored at –70°C until analysis. Diagnosis of MIAC MIAC was defined as a positive PCR analyses for the genital mycoplasmas (Ureaplasma parvum, Ureaplasma urealyticum, Mycoplasma hominis) and/or Chlamydia trachomatis and/or growth of any bacteria in the amniotic fluid except for coagulasenegative Staphylococcus epidermidis, which was considered to be a skin contaminant [22]. Diagnosis of HCA At delivery, the placenta, the fetal membranes and the umbilical cord were fixed in 10% neutral buffered formalin. Tissue samples were obtained from the placenta and fetal membranes (at least two samples), umbilical cord (usually one sample) and placental
membranes (at least two samples) and routinely processed and embedded in paraffin. Sections of tissue blocks were stained with hematoxylin and eosin. The degree of polymorphonuclear leukocyte infiltration was assessed separately in the free membranes (amnion and chorion-decidua), in the chorionic plate, and in the umbilical cord according to the criteria given by Salafia et al. [8]. Diagnosis of HCA was made based on the presence of inflammatory changes in the chorion-decidua (grades 3-4), chorionic plate (grades 3-4), umbilical cord (grades1-4) and/or amnion (grades 1-4) [8]. Diagnosis of severe neonatal morbidity Maternal and perinatal medical records were reviewed by two investigators (MK and IM). Data regarding morbidity and mortality were reviewed for all newborns. “Severe neonatal morbidity” in this study is defined as a condition composed of respiratory distress syndrome (defined by the presence of two or more of the following criteria: evidence of respiratory compromise, a persistent oxygen requirement for more than 24 hours, administration of exogenous surfactant, radiographic evidence of hyaline membrane disease), intraventricular hemorrhage (the diagnosis of which was made using cranial ultrasound examinations according to the procedure of Papile et al.) [23], necrotizing enterocolitis (defined as radiologic finding of either intramural gas or free intra-abdominal gas), retinopathy of prematurity (identified using retinoscopy), early- (during the first 72 hours of life) and late-onset (between the ages of 4 and 120 days) sepsis (either proven by bacterial culture or clinically highly suspected sepsis), bronchopulmonary dysplasia (defined as infant oxygen requirement at 28 days of age), pneumonia (diagnosed by an abnormal findings on chest X rays), and neonatal death before hospital discharge. Determination of amniotic fluid myeloperoxidase The myeloperoxidase concentration in the amniotic fluid was determined using sandwich enzyme-linked immunosorbent assay (ELISA) for human myeloperoxidase (R&D Systems, Minneapolis, MN, USA) according to manufacturer’s instructions. The limit of detection was 0.1 ng/mL, whereas the upper limit of quantification was 2000 ng/mL. Amniotic fluid samples were diluted 1:10 for the assay, and the resulting absorbance was read at 450 nm using a λ correction at 540 nm (Paradigm Detection Platform, Beckman Coulter, Brea, CA, USA). Statistical analyses The demographic and clinical characteristics were compared using unpaired t-tests for continuous variables (presented as the mean ± SD) or the Mann–Whitney U test for non-parametric variables [presented as the median (range)]. Categorical variables were compared using the Fisher exact test and are presented as number (%). The normality of the data was tested using the D’Agostino and Pearson omnibus normality test and the Shapiro– Wilk test. Because amniotic fluid MPO concentrations were not normally distributed, the non-parametric Mann–Whitney U test was used for analyses, and data are presented as median and interquartile range (IQR). Receiver operator characteristic (ROC) curve was constructed to determine the predictive value of MPO for the presence of both MIAC and HCA. To evaluate MPO as a determinant of neonatal morbidity, mixed linear models were performed to adjust for gestational age. Differences were considered statistically significant at p < 0.05. All p values were obtained from two-sided tests. All statistical analyses were performed using GraphPad Prism 5.03 for Mac OS X (GraphPad Software, La Jolla, CA, USA) and SPSS 19.0 statistical package for Mac OS X (SPSS Inc., Chicago, IL, USA).
The Journal of Maternal-Fetal and Neonatal Medicine
Amniotic fluid myeloperoxidase 3
J Matern Fetal Neonatal Med Downloaded from informahealthcare.com by Texas Tech Hlth Sci Ctr on 11/28/12 For personal use only.
Results Demographical and clinical characteristic Among 181 women, 34% (61/181) and 64% (115/181) had the presence of MIAC and HCA, respectively. The presence of both MIAC and HCA was found in 25% (45/181) of women. Table I shows the demographic and clinical characteristics of the women with of both MIAC and HCA and women in whom at least one of these conditions was ruled out. Women with MIAC and HCA had lower rate of primiparous, gestational age at the sampling and delivery, birth weight, longer latency, and Apgar score 28 weeks 29 (64%) 119 (88%) < 0.0001 Spontaneous delivery 31 (68%) 97 (71%) 0.85 Cesarean section 14 (32%) 39 (29%) 0.85 Apgar score < 7 in 5 min 7 (16%) 5 (4%) 0.01 Apgar score < 7 in 10 min 4 (9%) 2 (1%) 0.04 CRP, C-reactive protein; HCA, histological chorioamnionitis; MIAC, microbial invasion of the amniotic cavity; PPROM, preterm prelabor rupture of membranes; WBC, white blood cells Continuous variables were compared using parametric t-test (presented as mean ± SD) or non-parametric Mann–Whitney U test [presented as median (range)]. Categorical variables were compared using Fisher exact test and presented as number (%). Statistically significant results are marked in bold.
© 2012 Informa UK, Ltd.
J Matern Fetal Neonatal Med Downloaded from informahealthcare.com by Texas Tech Hlth Sci Ctr on 11/28/12 For personal use only.
4 M. Kacerovsky et al.
Figure 1. The amniotic fluid MPO levels in pregnancies complicated by preterm prelabor rupture of the membranes. Women with MIAC had higher median MPO level than women without MIAC. Women with HCA had higher median MPO level that those without HCA. HCA, histological chorioamnionitis; MPO, myeloperoxidase; MIAC, microbial invasion of the amniotic cavity.
Figure 2. The amniotic fluid MPO levels in pregnancies complicated by preterm prelabor rupture of the membranes. Women with the presence of both MIAC and HCA had higher median MPO level than other women (women without both MIAC and HCA, women with HCA alone, and women with MIAC alone). HCA, histological chorioamnionitis; MPO, myeloperoxidase; MIAC, microbial invasion of the amniotic cavity.
Table II. Amniotic fluid MPO according to the presence and absence of MIAC and HCA in a model with and without adjusting for gestational age. Number of Mean p Value Mean 95% CI p Value women (crude) (crude) (adjusted) (adjusted) (adjusted) The presence of MIAC Yes 61 672.3 0.001 606.5 453.0–759.6 0.003 No 120 276.8 311.9 201.4–420.7 The presence of HCA Yes 115 546.8
