Subjects

The Proteomic Assessment of Preterm Risk (PAPR) study was conducted under a standardized protocol at 11 Institutional Review Board (IRB)-approved sites across the United States ( Clinicaltrials.gov identifier: NCT01371019 ). Subjects were enrolled between 17 ^0/7 and 28 ^6/7 weeks gestational age (GA). Dating was established using a predefined protocol of menstrual dating confirmed by early ultrasound biometry, or ultrasound alone, to provide the best clinically estimated GA. BMI was derived from height and prepregnancy self-reported weight. Pregnancies with multiple gestations or with known or suspected major fetal anomalies were excluded. Pertinent information regarding subject demographic characteristics, past medical and pregnancy history, current pregnancy history, and concurrent medications was collected and entered into an electronic case report form. Following delivery, data were collected for maternal and infant outcomes and complications. All deliveries were classified by the study sites as term (≥37 ^0/7 weeks GA), spontaneous preterm (including preterm premature rupture of membranes [PPROM]), or medically indicated preterm births. Classification of preterm deliveries was subsequently adjudicated by the chief medical officer (D.H.) at Sera Prognostics, Inc, who was blinded to results from laboratory analysis. As indicated, discrepancies were clarified with the principal investigator at the study site. The adjudication occurred prior to locking down the validation database and conducting laboratory and statistical analysis.

Sample collection

Maternal blood was collected and processed as follows: a 10-minute room temperature clotting period, followed by immediate refrigerated centrifugation or placement in an ice-water bath at 4–8 ^o C until centrifugation. Blood was centrifuged within 2.5 hours of collection and 0.5 mL serum aliquots were stored at −80 ^o C until analyzed. Details regarding sample accessioning can be found in Supplementary Materials and Methods .

Predictor development principles

Development of the insulin-like growth factor–binding protein 4 (IBP4)/sex hormone–binding globulin (SHBG) predictor included independent and sequential discovery, verification, and validation steps consistent with Institute of Medicine (IOM) guidelines for best practices in “omics” research. Analytical validation preceded clinical validation sample analysis and included assessment of inter- and intrabatch precision, carry-over, and limit of detection.

The validation nested case-control analysis was performed on specimens from 81 sPTD cases and controls independent of discovery and verification. Validation sPTD cases were the last to be enrolled in PAPR and included samples from 9 sites in total, with 2 sites being unique to validation. Validation cases and controls underwent 100% on-site source document verification with each subject’s medical record prior to mass spectrometry (MS) analysis. This process ensured that all subjects satisfied the inclusion and exclusion criteria, as well as confirmed medical/pregnancy complications and GA at birth assignments for all subjects at time of sample collection and delivery. Detailed analysis protocols, including the validation study design, analysis plan, and a blinding protocol, were preestablished. Personnel were blinded to subject case, control, and GA at birth data assignments, with the exception of the director of clinical operations (DCO) and clinical data manager. The data analysis plan included prespecified validation claims and a protocol for double independent external analyses. Predictor scores, calculated as described below, were determined for all subject samples by a blinded statistician and subsequently confirmed by 2 external blinded statisticians, 1 of whom was university based (E. Mazzola) and the other an industry consultant (P. Kearney). Case, control, and GA data, linked to the predictor scores by the DCO, were then provided to the 2 external statisticians for analysis. Area under the receiver operating characteristic curve (AUROC) and significance testing results were then transferred back to the DCO. Transfer of data incorporated the use of the SUMPRODUCT function to ensure data integrity. To provide an audit trail of data from each subject through to validation results, real-time digital time-stamping was applied to analytical data, plans, and reports.

Validation study design

In the primary analysis, cases were defined as subjects with deliveries due to PPROM or spontaneous onset of labor with delivery <37 ^0/7 weeks GA. Controls were subjects who delivered at ≥37 ^0/7 weeks GA. Prior discovery and verification analyses investigated 44 candidate biomarkers using serum samples collected across broad GA (17 ^0/7 through 25 ^6/7 weeks GA) ( Supplementary Materials and Methods ). Discovery and verification identified an optimal narrow GA at blood draw (GABD) interval (19 ^0/7 through 21 ^6/7 weeks) and 2 proteins, IBP4, up-regulated in sPTD cases, and SHBG, down-regulated in sPTD cases, used in a ratio (IBP4/SHBG) as the best predictor by AUROC for sPTD ( Supplementary Materials and Methods ). In discovery and verification, subjects without extreme BMI values had improved classification performance by IBP4/SHBG ( Supplementary Results , Appendix ). Following discovery and verification analyses, we proceeded to analytical and clinical validation.

Validation sPTD cases totaled 18 subjects collected between 19 ^0/7 and 21 ^6/7 weeks GABD from a total available of 81 subjects between 17 ^0/7 and 28 ^6/7 weeks GA. Sets of controls, comprising 2 controls per sPTD case matched by GABD, were randomly selected using the R statistical program (R 3.0.2) and compared to the term delivery distribution as outlined in the 2012 National Vital Statistics Report using a chi-square test. Randomly created control sets (in groups of 10) were examined for sets yielding a P value approaching 1.0.

Our primary objective was to validate the performance of the IBP4/SHBG ratio as a predictor for sPTD using AUROC. To control the overall multiple testing error rate (α = 0.05), the fixed sequence approach was applied to GABD increments within the optimal interval (19 ^0/7 through 21 ^6/7 weeks GA) identified in discovery and verification with and without the application of a BMI stratification ( Supplementary Materials and Methods ). Significance was assessed by the Wilcoxon-Mann-Whitney statistic that tests equivalence to AUROC = 0.5 (random chance). For determinations of classification performance at GA boundaries other than <37 ^0/7 vs ≥37 ^0/7 weeks GA (eg, <36 ^0/7 vs ≥36 ^0/7 , <35 ^0/7 vs ≥35 ^0/7 ), cases and controls were redefined as all subjects below and equal to/above the specific boundary, respectively.

Laboratory methods

A systems biology approach was employed to generate a highly multiplexed multiple reaction monitoring (MRM) MS assay ( Supplementary Materials and Methods and Supplementary Results ). The validation assay quantified proteotypic peptides specific to predictor proteins IBP4 and SHBG and other controls. Samples were processed in batches of 32, which were composed of clinical subjects (n = 24), pooled serum standards from healthy nonpregnant donors (HGS) (n = 3), pooled serum standards from healthy pregnant donors (pHGS) (n = 3), and phosphate-buffered saline that served as process controls (n = 2). For all analyses, serum samples were first depleted of high-abundance and nondiagnostic proteins using MARS-14 immunodepletion columns (Agilent Technologies, Santa Clara, CA), reduced with dithiothreitol, alkylated with iodoacetamide, and digested with trypsin. Heavy-labeled stable isotope standard (SIS) peptides (New England Peptide, Gardner, MA) were then added to samples, which were subsequently desalted and analyzed by reversed-phase liquid chromatography (LC)/MRM MS. SIS peptides were used for normalization by generating response ratios (RR), where the peak area of a peptide fragment ion (ie, transition) measured in serum was divided by that of the corresponding SIS transition spiked into the same serum sample.

The IBP4/SHBG predictor

The predictor score was defined as the natural log of the ratio of the IBP4 and SHBG peptide transition response ratios: