Identification of interleukin-6 (IL-6) and squamous cell carcinoma (SCC) as amniotic fluid-specific markers

Objective: To determine if an amniotic fluid (AF)-specific marker is present and if its concentration changes with the presence of labor. Study Design: Twenty-six healthy women who gave birth to healthy newborns at term during the period from July 2009 to January 2010 were included in the study. Six candidate markers were assessed by commercially available ELISA kits: interleukin (IL)-6, squamous cell carcinoma (SCC) antigen, insulin-like growth factor (IGFBP)-1, osteopontin (OPN), CA125, and sialyl Tn (STN). Results: The AF/maternal serum (MS) measurement based on IL-6 or SCC has proved to be superior to IGFBP-1, CA125, OPN and STN. Women with spontaneous labor at term had significantly higher IL-6 and IGFBP-1 concentrations in AF compared with those without labor. No significant differences were observed in the AF concentrations of SCC, OPN, CA125 and STN between women with labor and those not in labor. Conclusion: Our observation of IL-6 and SCC in AF may open a new area of research to assess their usefulness as biological markers of obstetrical disorders.

• Amniotic Fluid; Biomarker; Interleukin-6; Squamous Cell Carcinoma Antigen

1. INTRODUCTION

Several investigators have evaluated the diagnostic value of amniotic fluid (AF)-specific polypeptides or metabolites as indicators of obstetrical disorders, including premature rupture of membranes (PROM) [1-4] or amniotic fluid embolism (AFE) [5-10]. We have conducted a document retrieval to identify gene products that are specifically present only in AF but not present in the maternal serum (MS) or proteins/polypeptides that are present in AF at concentrations extremely higher than those in the MS. Proteomic technologies have been predominantly used in the research to discover new AFspecific markers [11-15]. We have previously reported many candidate markers [16]. Enriched protein functions were tumor markers, cell proliferation and embryonic development, metabolism, nervous system, cytokines, immune or complement processes, signaling, cell adhesion and motility, hormones, detoxification system, and metal carrier [16].

In the present study, among these candidate markers, the following six antigens, interleukin (IL)-6, insulin-like growth factor-binding protein (IGFBP)-1, osteopontin (OPN), Squamous cell carcinoma (SCC) antigen, CA125 and sialosyl Tn (STN), were selected, since the specific ELISA has been commercially available. We have examined if these proposed markers are over expressed specifically in AF and if their concentration changes with the presence of labor.

2. MATERIALS AND METHODS

2.1. Study Population and AF Collection

Twenty-six healthy women who gave birth to healthy newborns at term during the period from July 2009 to January 2010 at the Nara Medical University Hospital, Nara, Japan, were included in the study. Pregnant women who presented to our hospital were asked to participate. Two groups of women were recruited: 1) 7 term pregnant women (gestational age, 37 - 41 wk) carrying a single fetus and undergoing elective cesarean section before labor, whose peripheral bloods were taken just prior to operation (term maternal blood); 2) 9 term pregnant women undergoing elective cesarean section before labor, whose amniotic fluids were taken (AF without labor); 3) 10 similar term pregnant women undergoing emergency cesarean section after spontaneous labor (all women were in the active phase of labor of less than 5 minutes interval), whose amniotic fluids were taken (AF with labor). The main indications for urgent and emergency cesareans were prolonged labor and presumed fetal compromise. Subjects with any symptom of infection were excluded from the study. No subject had a comorbid condition such as hypertension, diabetes, asthma, congenital heart disease, kidney disease, connective tissue disorders, and autoimmune disease. All neonates had normal anatomies. This study was approved by the Nara Medical University ethics committee. Informed consent was obtained from all mothers. Clinical data were obtained by two investigators (K.N. and T.N.) and recorded using a standardized data sheet. The women’s ages, gestational ages, and parity did not differ significantly between the three groups.

2.2. Sampling

Maternal blood was obtained from a cannulated vein a few minutes prior to surgery. AF was obtained during surgery by one investigator (K.N.) and immediately filtered through 0.45 μm pore filter (Millex, Millipore Co. Ltd., Billerica, MA) to avoid the debris. After centrifugation at 4˚C, MS and AF were immediately frozen and stored at −80˚C until further analysis.

2.3. Measurements of Six Biomarker Levels in Maternal Serum and Amniotic Fluid

These antigens were assessed by commercially available ELISA kits: IL-6, R&D systems, Inc. (Minneapolis, MN); Squamous cell carcinoma (SCC) antigen, Osteopontin, STN, and CA125, ECLIA kits, BML Co.Ltd. (Tokyo, Japan); and IGFBP-1, Human ELISA kit, Phoenix Pharmaceuticals, Inc. (Burlingame, CA). Every sample was run in duplicate. Differences between the two measurements were minimal (intra-assay precision coefficients of variation equals 5% - 8%). Statistical analysis was performed using the Mann-Whitney U test (SPSS 15.0J, SPSS Japan Inc., Japan). Statistical significance was set at p < 0.05. All values are expressed as the mean ± S.E.M.

3. RESULTS

Figure 1 is a scatter diagram with bar graphs for AF values of IL-6, IGFBP-1, SCC, osteopontin, CA125, and STN. With the use of a commercially available immunoassay kit developed to detect these antigens, elevated concentrations of the markers are frequently found in AF: IL-6 (1031.90 ± 499.14 pg/ml for without labor and 7347.94 ± 1600.22 pg/ml for with labor, p < 0.05),

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