Spontaneous preterm birth is a leading cause of neonatal morbidity and mortality. Early identification of at-risk women by reliable screening tests could reduce the spontaneous preterm birth rate, but conventional methods such as obstetrical history and maternal cervical length screening identify only a minority of spontaneous preterm birth cases. Cervicovaginal fluid might prove to be a useful, readily available biological fluid for identifying spontaneous preterm birth biomarkers.

The objective of the study was to identify cervicovaginal fluid biomarkers of early spontaneous preterm birth in a high-risk cohort of pregnant women with a history of spontaneous preterm birth using targeted and shotgun proteomic analyses.

A nested case control study (cases were spontaneous preterm birth <34 weeks in the current pregnancy; controls were spontaneous labor and delivery at 39–41 weeks) was performed using cervicovaginal fluid samples collected at 3 study visits (10^0/7 to 18^6/7 weeks, 19^0/7 to 23^6/7 weeks, and 28^0/7 to 31^6/7 weeks). All participants had a history of at least 1 prior spontaneous preterm birth. Targeted proteomic analysis was performed using a stable isotope–labeled proteome derived from endocervical and vaginal mucosal cells. This served as a standard to quantitate candidate protein levels in individual cervicovaginal fluid samples from the second and third study visits using liquid chromatography–multiple reaction monitoring mass spectrometry. The ratio of endogenous peptide area/stable isotope–labeled proteome–derived peptide area was used to measure levels of 42 peptides in 22 proteins. To maximize biomarker discovery in the cervicovaginal fluid samples, shotgun proteomic analysis also was performed utilizing liquid chromatography and ion trap mass spectrometry. A validation study was performed in second-trimester cervicovaginal fluid samples from an independent study group (12 spontaneous preterm birth cases, 19 term delivery controls) using enzyme-linked immunosorbent assay for 5 proteins expressed at higher levels in spontaneous preterm birth cases compared with controls in targeted or shotgun proteomic analyses.

For targeted proteomics, cervicovaginal fluid samples from 33 cases and 32 controls at 19^0/7 to 23^6/7 weeks and 16 cases and 14 controls at 28^0/7 to 31^6/7 weeks from the same pregnancies were analyzed. When samples were compared between cases and controls, the relative abundance of 5 proteins was greater (P = .02–.05) in cases at both visits, while the relative abundance of 1 protein was lower (P = .03) in cases at both visits. For shotgun proteomics analyses, cervicovaginal fluid samples were pooled for 9 spontaneous preterm birth cases and 9 term delivery controls at each study visit. Shotgun proteomics yielded 28 proteins that were detected at levels >2 times higher and 1 protein that was detected at a level <0.5 times lower in spontaneous preterm birth cases compared with controls at all 3 study visits. Validation enzyme-linked immunosorbent assay for 5 proteins that were detected at higher levels in cervicovaginal fluid samples from spontaneous preterm birth cases compared with term delivery controls in proteomics analyses did not demonstrate statistically significant differences between spontaneous preterm birth cases and controls.

Potential biomarkers of spontaneous preterm birth were identified by targeted and shotgun proteomics analyses in cervicovaginal fluid samples from high-risk, asymptomatic women. Many of the proteins detected at higher levels in cervicovaginal fluid samples from spontaneous preterm birth cases are extracellular matrix proteins and/or regulate cell membrane physiology. These proteins have substantial biological interest, but validation enzyme-linked immunosorbent assay for 5 of these proteins did not yield clinically useful biomarkers for spontaneous preterm birth.