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A prediction model of histological chorioamnionitis and funisitis in preterm prelabor rupture of membranes: analyses of multiple proteins in the amniotic fluid

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A prediction model of histological chorioamnionitis and funisitis in preterm prelabor rupture of membranes: analyses of multiple proteins in the amniotic fluid
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  󰀱󰀹󰀹󰀵 The Journal of Maternal-Fetal and Neonatal Medicine , 2012; 25(10): 1995–2001 © 2012 Informa UK, Ltd.ISSN 1476-7058 print/ISSN 1476-4954 onlineDOI: 10.3109/14767058.2012.666592 Objective : To determine the best prediction model of histological chorioamnionitis and funisitis in preterm prelabor rupture of membranes (PPROM) using selected candidate proteins in the amniotic fluid (AF). Material and methods : Prospective cohort study. Twenty-six AF proteins were assayed by a multiple immunoassay from 107 women with membranes rupture from 23+0 to 36+6 weeks. The Czech Republic policy is active management, and the majority of women were delivered within 72 h after the rupture of membranes, except for women with PPROM <28+0 weeks who were managed conservatively. The best predictive models to diagnose histological chorioamnionitis and funisitis were calculated by logistic regression depending on the gestational age (GA) at membrane rupture. Results : Both IL-6 and a combination of IL-10, and migration inhibiting factor (MIF) were the best predictive models of histological chorioamnionitis and funisitis, respectively, with sensitivity, specificity, positive and negative predictive values and positive likelihood ratio (LR+) of 62, 83, 37, 93 and 3.6 and of 63, 91, 53, 94 and 7.0, respectively. Depending on whether GA at membrane rupture was <32 or ≥ 32 weeks, IL-10, alone or in combination with MIF and triggering receptor expressed on myeloid cells-1, was the strongest inflammatory biomarker for funisitis (LR+10.6 and 36.6, respectively). Conclusion : Regardless of the GA at membrane rupture, IL-6 from the AF was the best predictor of histological chorioamnionitis. Amniotic fluid IL-10 was notably accurate in the prediction of funisitis. Keywords: Funisitis, gestational age at membrane rupture, histological chorioamnionitis, intra-amniotic inflammation Introduction Preterm prelabor rupture of membranes (PPROM) occurs in approximately one-third of preterm deliveries and is responsible for a substantial proportion of neonatal mortality and serious morbidity. Neonatal outcome depends on the occurrence of complications in relation to gestational age (GA) [1].󰀀ere is a strong association between preterm delivery and the occurrence of histological chorioamnionitis being detected in up to 60% of all preterm deliveries [2,3]. Histological chorio-amnionitis and funisitis are also associated to neonatal composite morbidity, including chronic pulmonary disease [4] and adverse neurodevelopment outcome [5,6]. 󰀀us, the presence of funisitis represents evidence of a fetal inflammatory response being asso-ciated with higher neonatal mortality and morbidity than when only a maternal inflammatory response is present [7]. Altogether, these findings suggest that predicting the risk of histological chorioamnionitis and the knowledge of fetal involvement in the inflammatory response seem crucial for improving the outcome in pregnancy management in PPROM.Intra-amniotic inflammation has been proposed as the stron-gest predictor of infection in PPROM [8–10]. Intra-amniotic inflammation  per se  has been considered to be an important risk factor of preterm delivery and composite neonatal morbidity. Several studies have previously proposed different inflammatory markers alone or in combination to predict microbial invasion of amniotic cavity [9,10]. However, few studies have evaluated the role of intra-amniotic inflammation in the diagnosis of histo-logical chorioamnionitis and/or funisitis [11–14].Histological chorioamnionitis prevalence in preterm delivery and its association with intra-amniotic inflammation justify the need for a prediction model based on intra-amniotic inflamma-tory markers that could improve pregnancy management and the parental counseling of women at risk.Due to the active management of PPROM in the Czech Republic, there is a short latency from sampling to delivery in women with a PPROM diagnosis. 󰀀is property provides an accurate evalua-tion of the amniotic fluid (AF), placenta and umbilical cord with little time for discrepancies to appear. We hypothesized that we were able to predict placenta pathology prenatally in women with PPROM measuring selected cytokines in AF by amniocentesis. 󰀀e primary outcome of the study was the prediction of histo-logical chorioamnionitis and of funisitis. 󰀀erefore, the aim of the study was to determine the best prediction model of histological A prediction model of histological chorioamnionitis and funisitis in preterm prelabor rupture of membranes: analyses of multiple proteins in the amniotic fluid  Teresa Cobo 1 , Marian Kacerovsky 2 , Montse Palacio 1 , Helena Hornychova 3 , David M. Hougaard 4 , Kristin Skogstrand 4  & Bo Jacobsson 5,6 1 Maternal Fetal Medicine Department, Hospital Clinic, Institut d’ Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Universitat de Barcelona, Barcelona, Spain, 2 Department of Obstetrics and Gynecology, 3 Fingerland’s Department of Pathology, University Hospital Hradec Kralove, Czech Republic, 4 Department of Clinical Biochemistry and Immunology, Statens Serum Institute, Copenhagen, Denmark, 5 Department of Obstetrics and Gynecology, Sahlgrenska University Hospital, Gothenburg, Sweden, and 6 Institute of Public Health, Oslo, Norway Teresa Cobo and Marian Kacerovsky contributed equally to this paper.Correspondence: Dr. Teresa Cobo, Prematurity Unit, Department of Maternal-Fetal Medicine, Hospital Clinic, Sabino de Arana 1, Barcelona 08028, Catalonia, Spain. Tel: +34 93 2275600. Fax: +34 93 2275605. E-mail: tcobo@clinic.ub.es    J   M  a   t  e  r  n   F  e   t  a   l   N  e  o  n  a   t  a   l   M  e   d   D  o  w  n   l  o  a   d  e   d   f  r  o  m   i  n   f  o  r  m  a   h  e  a   l   t   h  c  a  r  e .  c  o  m   b  y   G  o   t  e   b  o  r  g  s   U  n   i  v  e  r  s   i   t  y  o  n   1   1   /   2   1   /   1   2   F  o  r  p  e  r  s  o  n  a   l  u  s  e  o  n   l  y .  1996  T. Cobo et al.   e Journal of Maternal-Fetal and Neonatal Medicine chorioamnionitis and funisitis in PPROM pregnancies using different proteins in the AF. Material and methods A prospective cohort study was performed in pregnant women with a diagnosis of PPROM who were admitted to the Department of Obstetrics and Gynecology, University Hospital Hradec Kralove, Czech Republic between July 2008 and October 2010. GA was established according to the first-trimester ultrasound scan.󰀀e patients enrolled in this study were singleton pregnant women with PPROM from 23+0 to 36+6 weeks. Multiple preg-nancies, structural/chromosomal anomalies and patients with clinical signs of chorioamnionitis or signs of fetal hypoxia or  vaginal bleeding at admission were not considered eligible for this study.PPROM was defined as leakage of AF, which precedes the onset of uterine contractions. 󰀀is condition was diagnosed by a sterile speculum examination confirming the pooling of AF in the vagina in association with a positive test for the presence of insulin-like growth factor-binding protein (IGFBP, ACTIM PROM test; MedixBiochemica, Kauniainen, Finland) in the  vaginal fluid.A complete course of antenatal steroids, β methasone 12-mg intramuscular injection with two doses given 24 h apart, was administered from 24+0 to 34+0 weeks. Tocolysis was considered for 48 h in the absence of clinical chorioamnionitis, abruptio placentae and fetal compromise. Prophylactic parenteral broad-spectrum antibiotics were given at admission. No treatment except for antibiotics was initiated to delay delivery aer 34 weeks. Management of PPROM women in the Czech Republic is active (except for PPROM pregnancies at <28 weeks of gestation, which are handled with expectant care); the induction of labor or elective caesarean section depends on GA (within 24 h in those with GA above 34+0 weeks, within 48 h in those between 32+0 and 33+6 weeks of gestation and within 72 h aer rupture of the membranes in patients with fetuses of GA between 28+0 and 31+6 weeks) [15]. Fetal and maternal status was closely monitored until delivery. Maternal serum C-reactive protein (CRP) and white blood cell concentrations were assayed upon admission and every day until delivery.Ultrasound-guided transabdominal amniocentesis was performed at admission before the administration of corticoster-oids, antibiotics or tocolytics. Cultures for aerobic and anaerobic bacteria, as well as polymerase chain reaction analysis (PCR) for genital mycoplasmas and Chlamydia trachomatis , were assayed immediately aer collection. 󰀀e results were available for clin-ical management. 󰀀e inhibitors of proteases (Complete ™  Mini, EDTA-free Protease Inhibitor Cocktail; Roche Diagnostics, Basel, Switzerland) were added (40 µL per 1 mL of AF) to the sample of remaining AF. 󰀀ese inhibitors were centrifuged for 15 min at 2000  g   to remove cells and debris, filtered (0.22-µm Syringe-driven filter; TPP, Trasadingen, Switzerland), divided into aliquots and stored at −70°C until analysis. Microbial invasion of amniotic cavity was defined as a positive PCR for genital mycoplasmas and/or Chlamydia trachomatis  and/or a positive AF culture.Aer delivery, tissue-block sections from placenta, umbilical cord and placental membranes were placed in paraffin and stained with hematoxylin and eosin for standard histological examination. Histopathological examination was performed by a single pathologist who was blinded to the clinical status of the patients. 󰀀e degree of polymorphonuclear leukocyte infiltration was evaluated separately in the free membranes (amnion and chorion-decidua), in the chorionic plate, and in the umbilical cord according to the criteria proposed by Salafia [16]. 󰀀e diagnosis of histological chorioamnionitis was determined based on grades 3–4 in chorion-decidua and/or 3–4 in chorionic plate and/or 1–4 amnion and/or 1–4 in umbilical cord. Funisitis was diagnosed based on grades of 1–4 in umbilical cord [16].To evaluate the influence of GA on inflammatory response, data were analyzed for the whole study population, but were also stratified by GA at PPROM, < 32+0 and ≥ 32+0 weeks.Written informed consent was obtained from all subjects. 󰀀e Institutional Review Board approved the collection and use of these samples and information for research purposes (March 19, 2008; No. 200804 SO1P). AF analyses AF Interleukin (IL)-1β, IL-6, IL-8, IL-10, IL-12, IL-17, IL-18, soluble IL-6 receptor (sIL-6r), adiponectin, brain-derived neuro-tropic factor (BDNF), CRP, granulocyte macrophage colony stimu-lating factor (GM-CSF), IGFBP-1, IGFBP-3, interferon-γ (IFN-γ), leptin, monocyte chemotactic protein-1 (MCP-1), migration inhibiting factor (MIF), macrophage inflammatory protein-1α (MIP-1α), matrix metalloproteinasis-9 (MMP-9), neurotropin-3 (NT-3), regulated on activation normal T-expressed and secreted (RANTES), tumour necrosis factor (TNF)-α, TNF-β, soluble TNF receptor-1 (sTNF-R1) and triggering receptor expressed on myeloid cells-1 (TREM-1) were analysed at Statens Serum Institute (Department of Clinical Biochemistry and Immunology, Copenhagen, Denmark) using a multiple sandwich immuno-assay based on flowmetric Luminex xMAP technology in the accordance with the workflow previously published [17–20]. 󰀀e samples of AF were measured undiluted in duplicates. Calibration curves were prepared in assay buffer (Phosphate buffered saline containing 5 mL/L Tween 20 and 10 g/L bovine serum albumine). 󰀀e means of intraassay and interassay coefficient of variation was 6 and 12%, respectively. 󰀀e defined working range described by Skogstrand et al .  was used due to impossibility to obtain cytokine-free AF [20]. 󰀀erefore, this approach was considered as a more accurate of defining sensitivity instead of commonly used signal-to-noise ratio (limit of detection). 󰀀us, the detection level was defined as half the lowest levels in the working range in AF (IL-1β 5; IL-6 19.5; IL-8 2.5; IL-10 10; IL-12 4; IL-17 4; IL-18 10; sIL-6r 19.5; Adiponectin 488.5; BDNF 10; CRP 200; GM-CSF 4; IGFBP-1 97.5; IGFBP-397.5; IFN-γ 4; Leptin 97.5; MCP-1 2.5; MIF49; MIP-1 39; MMP-9 244; Neutropin-3 (NT-3) 39; RANTES 2.5; TNF-α 4; TNF-β 4; sTNF-R1 156.5; TREM-1 97.5 pg/mL). 󰀀e analyses were performed by the investigator who was blinded to the clinical status of the women.Eight proteins (IL-1β, IL-12, IL-17, IL-18, IFN-γ, RANTES, TNF-α and TNF-β) had undetectable AF levels in more than 50% of the samples and where subsequently excluded from further analyses. 󰀀erefore, 18 proteins   were analysed by logistic regres-sion to best predict histological chorioamnionitis and funisitis in the entire study population and in two subpopulations: GA at sampling less than 32+0 and GA at sampling ≥ 32+0 weeks. Statistical analysis Statistical analyses were performed using SPSS 19.0 for Windows XP OS (SPSS Inc., Chicago, IL, USA). Demographic and clinical characteristics were compared using the nonparametric Mann–Whitney U test [presented as median (range)]. Categorical vari-ables were compared using Fisher’s exact test and presented as number (%). All continuous variables were entered into a logistic regression with a forward selection. Differences were considered    J   M  a   t  e  r  n   F  e   t  a   l   N  e  o  n  a   t  a   l   M  e   d   D  o  w  n   l  o  a   d  e   d   f  r  o  m   i  n   f  o  r  m  a   h  e  a   l   t   h  c  a  r  e .  c  o  m   b  y   G  o   t  e   b  o  r  g  s   U  n   i  v  e  r  s   i   t  y  o  n   1   1   /   2   1   /   1   2   F  o  r  p  e  r  s  o  n  a   l  u  s  e  o  n   l  y .  Predictor model of histological chorioamnionitis and funisitis  1997 © 󰀲󰀰󰀱󰀲 Informa UK, Ltd. statistically significant at confidence level  p  < 0.05 with two-sided alternative hypotheses. Results Between July 2008 and October 2010, 174 women with a diag-nosis of PPROM between 23+0 and 36+6 weeks of gestation were admitted to the department. In total, 145 women met the inclu-sion criteria. However, only 107 women were finally included in the analysis because AF samples could not be retrieved ( n  = 30), or the placenta was not sent for histopathological assessment ( n  = 8). 󰀀ere were 18 women (17%) with a diagnosis of PPROM < 28+0 weeks; 20 women (19%) with PPROM from 28+0 to 31+6; 25 women (23%) with PPROM from 32+0 to 33+6 and 44 women (41%) with membranes rupture beyond 34+0 weeks. GA (median) at sampling and GA (median) at delivery of the entire study population were 33+1 (range: 23+6 to 36+5) days and 33+2 (range: 24+0 to 36+5) days, respectively. 󰀀e overall rate of histological chorioamnionitis was 57% (61/107) and the rate of funisitis was 18% (19/107).Maternal and neonatal characteristics regarding the presence or absence of histological chorioamnionitis and funisitis are presented in Tables I and II. GA at sampling, GA at delivery, birth weight and the frequency of a 5-min Apgar score < 7 were signifi-cantly different among groups.Data were classified according to the three different groups: the whole study population ( n  = 107), women with GA at PPROM <32+0 weeks ( n  = 41) and women with GA at PPROM ≥32+0 ( n  = 66). 󰀀e rate of histological chorioamnionitis was 78% in patients with PPROM <32+0 and 43.9% in patients with PPROM ≥32+0 weeks. Finally, funisitis was present in 34.1% of women with PPROM < 32+0 and in 7.6% of women with PPROM ≥32+0 weeks.Comparisons of the levels of AF cytokines and neuropeptides to determine the presence or absence of histological chorioam-nionitis and funisitis, respectively, are summarized in Tables III and IV. AF IL-6, IL-8, IL-10, MCP-1, CRP, BDNF, MMP-9, sIL-6r, adiponectin, sTNF-R1 and MIP-1 levels were significantly higher in women with histological chorioamnionitis compared to those without. Levels of MIP-1, IL-10, IL-6, MCP-1, TREM-1, IL-8, sIL-6r, adiponectin, sTNF-R1 and neutropin-3 in AF were signifi-cantly higher in women with funisitis than in women without. Regarding GA at PPROM, the intensity of the intra-amniotic inflammatory response in both histological chorioamnionitis and funisitis was higher in the subgroup of women with PPROM < 32+0.Different predictive models of histological chorioamnionitis and funisitis in PPROM are proposed in Tables V and VI, respec-tively. Regardless of the GA at the time of membrane rupture, the best predictor of histological chorioamnionitis was IL-6. A predictive model based on the assessment of IL-10 alone or in combination with TREM-1 and MIF allowed a notably prediction of funisitis when GA at PPROM was <32+0 (LR+ 10.6) or ≥ 32+0 weeks (LR+36.6). Discussion 󰀀e intra-amniotic inflammatory response mediated by IL-6 is the strongest predictor of histological chorioamnionitis. AF IL-10, alone or in combination with TREM-1 and MIF, is an accu-rate predictor of funisitis in PPROM. One of the main findings of this study is that regardless of the GA at membrane rupture, intra-amniotic inflammation predicts the presence of histological chorioamnionitis and funisitis.Intra-amniotic inflammation has extensively been considered as a predictor of microbial invasion of amniotic cavity, and IL-6 has been proposed as one of the most relevant inflammatory biomarkers, especially at earlier GAs [8,21]. Previous reports described the influence of AF IL-10 in the mechanisms of labor at preterm and term, as well as in the intra-amniotic inflammatory host response in preterm labor with intact membranes [22,23]. 󰀀e ability of our study to evaluate the presence of histological chorioamnionitis and funisitis rapidly aer AF sampling provides a more complete assessment and better knowledge of the inflam-matory response than previously reported. Our data suggest that IL-6 is one of the most important predictors of the inflamma-tory response not only of microbial invasion of amniotic cavity Table I. Maternal and neonatal outcomes according to the presence and absence of histological chorioamnionitis. Presence of histological chorioamnionitis n  = 61Absence of histological chorioamnionitis n  = 46  p Maternal age (years)30.5 (18–44)31 (19–44)0.468Nulliparity30 (52%)29 (67%)0.174Smoking11 (19%)6 (14%)0.597GA at sampling (week+days)32+0 (23+6 to 36+1)34+1 (24+0 to 36+5)0.000CRP level at admission (mg/L)9 (0–82)5 (0–71.3)0.003WBC count at admission (×10 9 /L)12.3 (4–26.8)12.3 (7–24)0.535MIAC31 (53%)16 (37%)0.118GA at delivery (week+days)32+1 (24+1 to 36+2)34+2 (24+0 to 36+5)0.000Birthweight (g)1827.5 (470–3210)2250 (750–3460)0.0005-min Apgar score < 76 (10%)00.036 Continuous variables were compared using a nonparametric Mann–Whitney U   test [presented as median (range)]. Categorical variables were compared using Fisher’s exact test and presented as number (%).AF, amniotic fluid; CRP, C-reactive protein; GA, gestational age; MIAC, microbial invasion of amniotic cavity; WBC, white blood cells. Table II. Maternal and neonatal outcomes according to the presence or absence of funisitis. Presence of funisitis n  = 19Absence of funisitis n  = 88  p Maternal age (years)31 (20–39)31 (18–44)0.570Nulliparity6 (31%)53 (65%)0.040Smoking7 (37%)10 (12%)0.012GA at sampling (week+days)30+3 (23+6 to 35+0)33+5 (24+0 to 36+5)0.000CRP level at admission (mg/L)9 (1–59)6 (0–82)0.593WBC count at admission (×10 9 /L)13 (4–20)12.15 (6.6–26.8)0.360MIAC15 (79%)32 (39%)0.001GA at delivery (week+days)31+0 (24+1 to 35+0)33+8 (24+0 to 36+5)0.000Birthweight (g)1530 (470–2410)2197.5 (495–3460)0.0005-min Apgar score < 74 (21%)2 (2%)0.009 Continuous variables were compared using a nonparametric Mann–Whitney U   test [presented as median (range)]. Categorical variables were compared using Fisher’s exact test and presented as number (%).AF, amniotic fluid; CRP, C-reactive protein; GA, gestational age; MIAC, microbial invasion of amniotic cavity; WBC, white blood cells.    J   M  a   t  e  r  n   F  e   t  a   l   N  e  o  n  a   t  a   l   M  e   d   D  o  w  n   l  o  a   d  e   d   f  r  o  m   i  n   f  o  r  m  a   h  e  a   l   t   h  c  a  r  e .  c  o  m   b  y   G  o   t  e   b  o  r  g  s   U  n   i  v  e  r  s   i   t  y  o  n   1   1   /   2   1   /   1   2   F  o  r  p  e  r  s  o  n  a   l  u  s  e  o  n   l  y .
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