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Glycosylated Fibronectin As a First-Trimester Biomarker for Prediction of Gestational Diabetes

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Glycosylated Fibronectin As a First-Trimester Biomarker for Prediction of Gestational Diabetes Glycosylated Fibronectin as a First-Trimester Biomarker for Prediction of Gestational Diabetes Juha P. Rasanen, MD, PhD, Caryn K. Snyder, MPH, Paturi V. Rao, MD, PhD, Raluca Mihalache, MPHN, Seppo Heinonen, MD, PhD, Michael G. Gravett, MD, Charles T. Roberts Jr, PhD, and Srinivasa R. Nagalla, MD OBJECTIVE: To evaluate the potential clinical utility of and placental lactogen were significantly associated serum biomarkers for first-trimester prediction of gesta- (P,.001) with GDM. After adjustment for maternal fac- tional diabetes mellitus (GDM). tors and other biomarkers, glycosylated fibronectin dem- METHODS: Maternal serum concentrations of glycosy- onstrated an independent association with GDM , lated (Sambucus nigra lectin–reactive) fibronectin, adipo- (P .001). Adiponectin, high-sensitivity CRP, and placental nectin, sex hormone–binding globulin, placental lactogen, lactogen demonstrated modest classification perfor- and high-sensitivity C-reactive protein (CRP) were mea- mance compared with glycosylated fibronectin (respec- sured at 5–13 weeks of gestation in a case-control study of tively: area under the curve [AUC] 0.63; 95% confidence 90 pregnant women with subsequent development of interval [CI] 0.53–0.71; AUC 0.68; 95% CI 0.60–0.76; and GDM and in 92 control group participants. Ability to AUC 0.67, 95% CI 0.59–0.75; compared with AUC 0.91; detect GDM was assessed using logistic regression mod- 95% CI 0.87–0.96). Glycosylated fibronectin levels above eling and receiver operating characteristic (ROC) curves. a threshold of 120 mg/L correctly identified 57 GDM case Classification performance and positive and negative pre- group participants with a positive predictive value of dictive values were reported at specific thresholds. Glyco- 63% (95% CI 53–72%) and a negative predictive value sylated fibronectin variation across trimesters was of 95% (95% CI 94–95%) at a population prevalence of evaluated using a serial-measures analysis of 35 nondia- 12%. There was no association between sex hormone– betic control group participants. binding globulin and GDM. RESULTS: First-trimester serum concentrations of glyco- CONCLUSION: First-trimester glycosylated fibronectin is sylated fibronectin, adiponectin, high-sensitivity CRP, a potential pregnancy-specific biomarker for early identi- fication of women at risk for GDM. (Obstet Gynecol 2013;122:586–94) From the Departments of Obstetrics and Gynecology, Oregon Health and Science DOI: 10.1097/AOG.0b013e3182a0c88b University, Portland, Oregon, and the University of Washington, Seattle, Washington; the Department of Obstetrics and Gynecology, Kuopio University LEVEL OF EVIDENCE: II Hospital, and the University of Eastern Finland, Kuopio, Finland; Nizam’s Insti- tute of Medical Sciences, Hyderabad, India; and DiabetOmics, Beaverton, Oregon. Standard assessments of diabetes and prediabetes, such Presented in part at the 72nd Annual American Diabetes Association Scientific Sessions, June 8–12, 2012, Philadelphia, Pennsylvania. as fasting insulin and glucose and glycated hemoglobin Funded by DiabetOmics, Inc. A1c, are not recommended for gestational diabetes mel- litus (GDM) screening. The standard screening modal- The authors thank John A. Michaels for expert technical assistance. ity in the United States is currently a 50-g, 1-hour oral Corresponding author: Srinivasa R. Nagalla, MD, DiabetOmics, Inc. 20,000 – NW Walker Rd. Beaverton, OR 97006; e-mail: [email protected]. glucose tolerance test (OGTT) performed at 24 28 1 Financial Disclosure weeks of gestation; however, the pooled sensitivity Caryn K. Snyder and Srinivasa R. Nagalla are employees of DiabetOmics. and specificity of this test are only 74% (95% confidence Michael G. Gravett and Charles T. Roberts Jr are consultants for DiabetOmics. interval [CI] 62–87%) and 77% (95% CI 66–89%), Paturi V. Rao, Charles T. Roberts, and Srinivasa R. Nagalla are shareholders in 2 DiabetOmics. The other authors did not report any potential conflicts of interest. respectively. New diagnostic approaches that allow earlier assessment can facilitate a shift from current © 2013 by The American College of Obstetricians and Gynecologists. Published by Lippincott Williams & Wilkins. standard-of-care practices by enabling earlier treatment. ISSN: 0029-7844/13 The desirability of early detection and treatment is 586 VOL. 122, NO. 3, SEPTEMBER 2013 OBSTETRICS & GYNECOLOGY generally accepted on a theoretical basis,3 but the [10.0 nmol/L], and 155 mg/dL [8.6 nmol/L], respec- availability of a robust early screening test will allow tively). Using the same diagnostic criteria, nondiabetic the evaluation of the efficacy of early treatment in control group participants had normal glucose tolerance reducing gestational and postgestational risks for and did not have development of GDM during preg- mother and newborn. nancy. Exclusion criteria were multiple gestations and Proteins modified by intracellular glycosylation as pregnancy complications, including preeclampsia or opposed to nonenzymatic glycation of factors such as preterm labor. There were no ethnic differences glycated hemoglobin A1c or albumin could be better between the groups. indicators of maternal response to metabolic shifts in Sample size was determined from the degree of pregnancy, and altered secretion of glycoproteins has separation between GDM case and control group been described in GDM.4 Based on our previous pro- participants observed in a pilot study (n529; unpub- teomic analysis of maternal serum that demonstrated lished data). Based on the observed performance char- alterations in the glycoproteome in gestational compli- acteristics of the glycosylated fibronectin assay in this cations,5 we evaluated glycosylated (Sambucus nigra study and minimum expected true-positive and false- lectin–reactive) fibronectin to ascertain its potential positive fractions of 0.65 and 0.25, respectively, a sam- utility as an early GDM biomarker, and we found in plesizeof100GDMcasegroupparticipantsand100 preliminary studies that glycosylated fibronectin was control group participants exhibited 84% power using significantly elevated in first-trimester GDM maternal calculations described by Pepe.17 Of the original 200 serum compared with nondiabetic maternal serum. In participants, 15 were not eligible because of insufficient the current study, glycosylated fibronectin levels were volume of serum for analysis (n513) or pre-existing evaluated in a larger study population, and its perfor- diabetes (n52). Two participants were excluded mance compared with other potential GDM biomarkers, because of a diagnosis of GDM within 2 weeks of sam- including adiponectin,6–9 sex hormone–binding ple collection and one participant was excluded because globulin,7,10–12 high-sensitivity C-reactive protein of missing information about the date of sample collec- (CRP),13,14 and placental lactogen.15,16 tion. The remaining 182 participants (90 GDM case group participants and 92 control group participants) MATERIALS AND METHODS comprised the population described in this study. Study participants were selected from the Finnish Maternal blood was spun, aliquoted, and stored maternity serum bank at the National Institute for at 280°C according to maternal serum bank protocol. Health and Welfare. Recruitment occurred between The OGTT plasma glucose was determined using 2004 and 2010 from maternity clinics in the area of a Konelab 60i Clinical Chemistry Analyzer. Levels of Oulu University Hospital (Oulu, Finland) and Kuopio protein analytes were determined by sandwich University Hospital (Kuopio, Finland). Blood samples enzyme-linked immunoassay using specific monoclo- were taken from women participating in a first-trimester nal and polyclonal antibodies. Primary antibodies used screening program for trisomies. All clinical data were in enzyme-linked immunoassays included the follow- obtained from the Birth Register, a computerized ing: sex hormone–binding globulin monoclonal anti- database containing information about baseline mater- body (31401); placental lactogen monoclonal antibody nal characteristics and pregnancy outcome. The study (L1022-03 G); fibronectin monoclonal antibody was approved by the Ethics Committees at Oulu (MAB1918); CRP polyclonal antibody (842676); and University Hospital and Kuopio University Hospital. adiponectin monoclonal antibody (840965). Primary All participants provided informed consent. coating antibodies were resuspended in carbonate The current study used a case-control design in buffer (pH 9.6), with 100 mL added to each well of which GDM case group participants and nondiabetic a 96-well Reactibind plate and incubated at 4°C over- control group participants were retrospectively selected night. Plates were blocked with 3% bovine serum albu- from the described population (Fig. 1). Participants min in phosphate-buffered saline (pH 7.2). After needed a sufficient quantity of first-trimester serum col- sample addition and incubation for 45 minutes at room lected between 5 weeks and 13 weeks of gestation and temperature, plates were washed with phosphate- had to complete a 75-g, 2-hour OGTT for inclusion. buffered saline–Tris buffer using a Biotek plate washer Eligible GDM case group participants included any and then incubated with detection antibodies for 45 mi- woman with development of GDM during pregnancy, nutes at room temperature. Plates were again washed identified by OGTT. A diagnosis of GDM was made if with phosphate-buffered saline–Tris buffer and then fasting, 1-hour, or 2-hour
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