Proteomic Analysis Using Protein Chips to Detect Biomarkers in Cervical and Amniotic Fluid in Women with Intra-Amniotic Inflammation Ulla Ru1 etschi,† A° sa Rose´ n,‡ Go1 sta Karlsson,† Henrik Zetterberg,† Lars Rymo,† Henrik Hagberg,‡ and Bo Jacobsson*,‡,| Department of Clinical Chemistry and Transfusion Medicine, Perinatal Center, Department of Obstetrics and Gynecology, Institute for the Health of Women and Children, Sahlgrenska University Hospital, Go¨teborg, Sweden, North Atlantic Neuro-Epidemiology Alliances (NANEA), Department of Epidemiology and Social Medicine, University of Aarhus, Aarhus, Denmark Received May 12, 2005
Intra-amniotic inflammation (IAI) may cause preterm birth with poor neonatal out-come. To identify novel biomarkers for IAI, we analyzed amniotic and cervical fluid samples from 27 patients with signs of threatening preterm birth with or without IAI by surface-enhanced laser desorption ionization timeof-flight mass spectrometry (SELDI-TOF-MS). Seventeen proteins were significantly overexpressed in amniotic fluid from IAI cases and more often in women with preterm labor than those with rupture of membranes. Five of these were identified as human neutrophil protein 1-3, calgranulin A and B. Keywords: proteomics • surface-enhanced laser desorption ionization time-of-flight mass spectrometry • SELDITOF-MS • preterm birth • intra-amniotic inflammation • preterm labor • preterm prelabor rupture of membranes
Introduction Preterm birth (PTB) is a major problem in modern perinatal medicine and accounts for 75% of the perinatal mortality and 50% of the perinatal morbidity.1 PTB is a common condition and in some parts of the world, e.g., the US, one of every tenth child is born preterm.2 PTB is related to long-term respiratory problems and neurological morbidity.1,3 A major portion of PTB occurs spontaneously either as preterm prelabor or with preterm labor (PTL) rupture of the membranes (PPROM). The etiology and pathophysiology in spontaneous PTB is complex but previous data indicate that infection/inflammation is important, especially in PTB at very low gestational age (2 provided 100% specificity and sensitivity for IAI. We performed a similar calculation using S/N values for calgranulin A, HNP 1 and 2 and the 10440 peak. In our study group 9 out of 14 women with IAI had an MR score >2. For all non-IAI samples an MR score of 0 was calculated. It is especially interesting to see that out of the five IAI patients with an MR score below or equal to 2 there were four PPROM and one PTL. This further emphasizes our notion that there is a true difference in protein expression between the groups. One of the criteria used to select for peaks that should be used by the algorithm, was that the peaks should not be present in patients with no IAI. However, in this study we found low levels of calgranulin A in some of the samples from women without IAI. Due to this we get slightly higher threshold levels in our calculations of the MR score. Another difference between Buhimschi’s and our study is that we used a different definition of IAI. We chose to use a firmly established cut off level of IL-6 as indication of IAI,12,13 which is also related to the short and long-term outcome of the child (e.g., cerebral palsy),9,32 while Buhimschi et al. used white blood cell count and presence of bacteria to define IAI.
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