Longitudinal cohort

The cohort of 500 pregnant women with previous SPTB was projected to have 30 SPTB <34 weeks (6% of the cohort) and 60 SPTB between 34-37 weeks (12% of the cohort; overall SPTB rate 18%) that could be matched with term deliveries from the same cohort between 39-41 weeks for nested case-control analyses. The number of patients in each group was estimated based on other studies of similar nature and valid estimates of sample size.

Women were eligible for enrollment at the 3 GPN-PBR clinical sites (University of Alabama at Birmingham, University of Texas Medical Branch at Galveston, and University of Utah) if they had a history of at least 1 SPTB of a singleton pregnancy at a gestational age between 20 ^0/7 -36 ^6/7 weeks in a previous pregnancy and a current singleton pregnancy <18 ^6/7 weeks’ gestation. Study participants were enrolled during routine prenatal visits <19 weeks of pregnancy. Gestational dating was based upon the first day of the patient’s last menstrual period (LMP) and ultrasound examination performed before enrollment. If the LMP date was uncertain, the ultrasound measurements obtained at the participant’s first ultrasound examination were used to determine the project gestational age. If the date of the LMP was certain and the ultrasound confirmed this gestational age within 7 days, then the LMP-derived gestational age was used. If the ultrasound-determined gestational age did not confirm the LMP-generated gestational age within 7 days, then the ultrasound was used to determine the projected gestational age.

Exclusion criteria were maternal uterine anomalies, planned cervical cerclage, multifetal gestation, fetal aneuploidy or lethal fetal anomalies, polyhydramnios (amniotic fluid index ≥25 cm or deepest vertical pocket ≥12 cm), planned or probable delivery at a nonnetwork site, no availability for prospective specimen/data collection, and serious maternal medical conditions (eg, renal disease, chronic liver disease, organ transplant recipients, spinal cord injuries, severe pulmonary disorders, severe heart disease, malignancy not in remission, antiphospholipid syndrome, genetic thrombophilia, diabetes mellitus class C or greater, hemoglobinopathy, isoimmunization, chronic conditions requiring medication for control [eg, chronic hypertension, lupus, inflammatory bowel disease, and asthma], and human immunodeficiency virus infection).

Following the enrollment visit, participants were seen again at 19 ^0/7 -23 ^6/7 weeks and at 28 ^0/7 -31 ^6/7 weeks. Maternal peripheral blood samples were collected in serum separator tubes at all study visits and at admission for delivery. The blood samples were allowed to coagulate at 4°C for 30 minutes, then the samples were centrifuged at 1200 g for 10 minutes at room temperature. Aliquots of serum were placed in liquid nitrogen and stored at −80°C.

Demographic and outcome data were recorded by trained research coordinators in a World Wide Web–based database developed by the network’s data coordinating center at Yale University. Serum samples were shipped on dry ice to the network’s analytical core at the University of Pennsylvania, where all proteomics assays were performed by laboratory personnel who were blinded to demographic and outcome data. Proteomics results and clinical outcomes were analyzed at the data coordinating center at Yale University. The GPN-PBR scientific protocol was approved by the investigational review boards at all 5 institutions.

Proteomics techniques

We utilized targeted and shotgun proteomics techniques to develop a panel of peptides to measure in serum samples from subjects in our longitudinal study.

Targeted proteomics were performed using SILAP released by 4 biologically relevant transformed cell lines: endocervical (End1) cells, vaginal mucosal (Vk2) cells, endometrial carcinoma (ECC1) cells, and placental choriocarcinoma (BeWo) cells. Transformed cells were obtained (American Type Culture Collection, Manassas, VA). Methods for SILAC-based targeted proteomics were described previously. Briefly, cells were grown in stable isotope-labeled serum-free Dulbecco’s modified eagle medium/F12 media containing [ ¹³ C ₆ ¹⁵ N ₂ ]-lysine and [ ¹³ C ₆ ¹⁵ N ₁ ]-leucine (Cambridge Isotopes, Cambridge, MA). Cells were passaged 7 times and then supernatant was collected every other day, filtered through a 0.22-μm filter, concentrated through a 5-kDa MW cutoff spin-filter (Millipore Corp, Billerica, MA), pooled, and stored at −80°C until analyzed. Supernatants were depleted of 6 high-abundance plasma proteins (albumin, transferrin, haptoglobin, antitrypsin, IgG, and IgA) using a multiple affinity removal system (Hu6) affinity LC column (Agilent Technologies, Palo Alto, CA). Protein concentration was estimated by Coomassie protein assay (Thermo Scientific, Rockford, IL), and supernatants were stored at −80°C. Equal amounts of protein (100 μg/cell line) from the immunodepleted End1, Vk2, ECC1, and BeWo supernatants were mixed together to create the End1-Vk2-ECC1-BeWo SILAC secretome.

To identify proteins in the End1-Vk2-ECC1-BeWo SILAC secretome, proteins were precipitated using a standard methanol/chloroform protocol and digested with trypsin (Promega, Madison, WI). Strong cation exchange (SCX) chromatography was performed on a PolySulfoethyl A column (Nest Group, Southborough, MA) attached to an HP 1100 high-performance LC system (Agilent Technologies). For each sample, 32 2-minute fractions were collected and pooled into 9 fractions as previously described. These 9 fractions were lyophilized and stored at −80°C until further analysis. Individual SCX fractions were analyzed by microflow reversed phase LC-electrospray ionization/MS/MS using a high-resolution LTQ Orbitrap-XL instrument (Thermo Scientific, San Jose, CA) operating at a resolution of 100,000 at mass number/charge number 400.

The MS/MS spectra were searched against an indexed human RefSeq database (version updated November 2007; 33,439 entries) with TurboSEQUEST (version 27.12; Thermo Scientific, Waltham, MA) and Mascot (version 2.2.03; Matrix Science, Boston, MA). Strict trypsin cleavage rules with maximum of 2 missed cleavages, mass accuracy of 1 Da for the precursor and fragment ion, and variable modifications of methionine oxidation, carboxyamidomethlyation on cysteine, [ ¹³ C ₆ ¹⁵ N ₂ ]-lysine and [ ¹³ C ₆ ¹⁵ N ₁ ]-leucine, were applied in the search criteria. The SEQUEST (Thermo Scientific) and Mascot (Matrix Science) output files were integrated into Scaffold (version 2.01; Proteome Software, Portland, OR) for validating MS/MS-based peptide and protein identifications. Assignment of peptide sequences was performed using the PeptideProphet algorithm. PeptideProphet accounts for the distribution of scores over an entire data set to calculate the probability of a correct assignment for every peptide. PeptideProphet calculates false-positive error rates at specific probability score cutoff values for each data set. A minimum PeptideProphet probability score of ≥0.5 was used to remove low probability peptides. At this cutoff, the estimated false-positive error rate was 10.8%. Protein identifications were accepted with a minimum ProteinProphet probability score of ≥0.8 and at least 2 identified unique peptides. For this dataset, a ProteinProphet probability score of ≥0.8 corresponded to a false-positive error rate of 4%. Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony.

The End1-Vk2-ECC1-BeWo secretome was developed so that it could be used as a SILAP standard to quantitate candidate protein levels in human serum samples by LC-MRM/MS using a high-sensitivity Vantage TSQ mass spectrometer (Thermo Scientific). The protocol for targeted proteomics in serum samples is illustrated in Figure 1 , A. Serum samples collected from women during the second and third study visits and at admission for delivery were pooled in 2 comparison groups: 5 women who experienced SPTB <34 weeks and 5 women who experienced uncomplicated term deliveries at 39-41 weeks. Protein (15 mg) from pooled serum samples from each study visit was added to equal amounts of protein (15 mg) from the End1-Vk2-ECC1-BeWo SILAP secretome, and each sample was digested with trypsin and analyzed by LC-MRM/MS. A total of 264 proteins were identified consistently in the pooled serum samples, and the quantity of each protein was determined by computing its light (L) (endogenous peptide amount)/heavy (H) (SILAP peptide amount) ratio. The L/H ratio of 3 proteins was significantly higher at second/third study visits and delivery visits among pooled serum samples from women who delivered preterm, while the L/H ratio of 5 proteins was significantly lower at second/third study visits and delivery visits among pooled serum samples from women who delivered preterm ( P < .10, Mann-Whitney rank sum test) ( Table 1 ). We selected these 8 proteins for inclusion in our overall protein panel for comparison among individual serum samples from SPTB and term deliveries in the longitudinal cohort. We also added fibronectin and thrombospondin proteins, which have potentially important biological relevance and were detected in the End1-Vk2-ECC1-BeWo SILAP secretome and pooled serum samples (but not at different levels between SPTB and term deliveries), to the list of targeted proteins ( Table 1 ).

Methods for targeted and shotgun serum proteomics

Protocols for A , targeted and B , shotgun proteomics comparing pooled maternal serum samples from 5 spontaneous preterm births (PTB) and 5 term deliveries.

LC , liquid chromatography; MRM , multiple reaction monitoring; MS , mass spectrometry; MS/MS , tandem mass spectrometry; SCX , strong cation exchange; SILAP , stable isotope labeled proteome.

Parry. Proteomic analysis of preterm delivery. Am J Obstet Gynecol 2014 .

Shotgun proteomics was performed by comparing pooled serum samples from SPTB with pooled serum samples from term deliveries. This made it possible to expand the list of candidate biomarker proteins for comparisons between individual SPTB cases and term delivery controls ( Figure 1 , B). Serum samples from each study visit were pooled for 5 SPTB cases and for 5 term delivery controls (5 women/group × 3 visits/woman = 15 samples pooled/group). Pooled serum samples underwent IgY-14 immunodepletion LC (IgY-14 column, Seppro protein depletion; Sigma Life Science, St. Louis, MO) to remove the 14 most abundant plasma proteins, trypsin digestion, SCX fractionation into 10 fractions, and LC-MS/MS analysis. A total of 336 proteins were identified in pooled serum samples from SPTB, while 448 proteins were identified in pooled serum samples from term deliveries. A total of 21 proteins were expressed differentially between pooled samples from SPTB and pooled samples from term deliveries ( P < .10, Mann-Whitney rank sum test) ( Table 2 ).

The 21 proteins identified by shotgun proteomics and the 10 proteins identified by targeted proteomics formed the panel of 31 candidate proteins the levels of which were compared by LC-MRM/MS analysis ( Figure 2 ) in individual serum samples from nested cases and controls. Briefly, 10 mL of serum from individual SPTB cases and term delivery controls was diluted in 490 mL of phosphate-buffered saline (approximate protein concentration = 1 mg/mL), and 40 mL of diluted serum was combined with 8 mg End1-Vk2-ECC1-BeWo SILAP secretome. Proteins in each sample were precipitated, reduced, and digested with trypsin. The levels of digested peptides in each sample were measured by LC-MRM/MS (5-mL injections, 2 peptides/protein, 3 MRM/peptide). The shotgun proteomics data have been deposited and are freely available at the Proteomics, Identifications database of the European Bioinformatics Institute, Hinxton, United Kingdom ( http://www.ebi.ac.uk/pride/ ).

Methods to measure protein expression in serum samples

Protocol for comparing levels of 31 candidate proteins by liquid chromatography (LC)-multiple reaction monitoring (MRM)/mass spectrometry (MS) in individual maternal serum samples from nested preterm birth (PTB) cases and controls.

Approx , approximately; conc , concentration; PBS , phosphate-buffered saline; SILAP , stable isotope labeled proteome.

Parry. Proteomic analysis of preterm delivery. Am J Obstet Gynecol 2014 .

Western blotting

Western blots were performed to measure levels of proteins identified in maternal serum proteomics assays in amnion, chorion, and placenta samples collected from SPTB cases and term delivery controls in a separate GPN-PBR observational study. Biospecimens were collected at cesarean delivery in approximately 120 women in 6 clinical groups (n = 20 in each group): (1) preterm delivery without labor (maternal or fetal indications for delivery at 24 ^0/7 -34 ^6/7 weeks); (2) preterm delivery following idiopathic preterm labor; (3) preterm delivery following preterm premature rupture of membranes without labor at 24 ^0/7 -34 ^6/7 weeks; (4) preterm delivery following preterm premature rupture of membranes and labor; (5) term delivery without labor (fetal malpresentation or elective repeat cesarean delivery at 39 ^0/7 -41 ^6/7 weeks); and (6) term delivery following labor. Protein levels were measured in amnion, chorion, and placenta biopsy samples from 5 cases in clinical groups 1, 2, 5, and 6 (preterm delivery ± labor and term delivery ± labor).

Briefly, protein was extracted from amnion, chorion, and placenta samples using radioimmunoprecipitation assay buffer (G-Biosciences, St. Louis, MO) with Complete Mini tablet (Roche, Indianapolis, IN). Protein quantification was performed using Pierce BCA protein assay kits (Thermo Scientific). Western blots were performed using 20 mg of protein for each sample loaded into Mini-Protean TGX precast gels 12% (BioRad Laboratories, Hercules, CA). All blots were transferred to Immobilon PVDF membranes (Millipore Corp), which were washed in deionized water and blocked with Odyssey blocking buffer (LI-COR Biosciences, Lincoln, NE). Blots were then probed for protein expression using primary rabbit polyclonal antibodies (1:5000) and anti-beta-tubulin antibody (loading control, 1:5000, ab6046; Abcam, Cambridge, MA). After incubating blots with antirabbit IgG (goat) IRDye secondary antibody in Odyssey buffer for IR blots, the blots were read using the Odyssey CLx infrared imaging system (LI-COR).

Protein levels in Western blots were calculated by densitometric analysis of infrared fluorescent signals, which were directly proportional to the amount of antigen on Odyssey Western blots. Normalization was performed against the internal control protein (beta-tubulin).

Statistics

Clinical data and protein expression levels were compared between SPTB and term deliveries using χ ² tests for categorical data, Student t tests for normally distributed continuous data, and rank sum tests for nonnormally distributed continuous data. The primary analysis was the comparison of maternal serum protein levels at all 3 study visits between SPTB cases and term delivery controls. Initial analyses indicated that first-visit serum samples did not yield informative protein levels for further analyses, so more extensive analyses were performed using specimens obtained from second and third study visits. Secondary analyses included: (1) correlation between protein levels and days from study visit to delivery; (2) correlation between protein levels and gestational age (days) at delivery; (3) correlation between changes in protein level between second and third study visits and SPTB; and (4) combinations of proteins (ie, biomarker panel) that were associated with SPTB.

An overall difference in protein levels in Western blots was compared among the 4 clinical groups by 1-way analysis of variance, and differences in protein levels between individual clinical groups were compared using the Newman-Keuls multiple comparison test.