The amniotic fluid proteome predicts imminent preterm delivery in asymptomatic women with a short cervix

Abstract

Preterm birth, the leading cause of perinatal morbidity and mortality, is associated with increased risk of short- and long-term adverse outcomes. For women identified as at risk for preterm birth attributable to a sonographic short cervix, the determination of imminent delivery is crucial for patient management. The current study aimed to identify amniotic fluid (AF) proteins that could predict imminent delivery in asymptomatic patients with a short cervix. This retrospective cohort study included women enrolled between May 2002 and September 2015 who were diagnosed with a sonographic short cervix (< 25 mm) at 16-32 weeks of gestation. Amniocenteses were performed to exclude intra-amniotic infection; none of the women included had clinical signs of infection or labor at the time of amniocentesis. An aptamer-based multiplex platform was used to profile 1310 AF proteins, and the differential protein abundance between women who delivered within two weeks from amniocentesis, and those who did not, was determined. The analysis included adjustment for quantitative cervical length and control of the false-positive rate at 10%. The area under the receiver operating characteristic curve was calculated to determine whether protein abundance in combination with cervical length improved the prediction of imminent preterm delivery as compared to cervical length alone. Of the 1,310 proteins profiled in AF, 17 were differentially abundant in women destined to deliver within two weeks of amniocentesis independently of the cervical length (adjusted p-value < 0.10). The decreased abundance of SNAP25 and the increased abundance of GPI, PTPN11, OLR1, ENO1, GAPDH, CHI3L1, RETN, CSF3, LCN2, CXCL1, CXCL8, PGLYRP1, LDHB, IL6, MMP8, and PRTN3 were associated with an increased risk of imminent delivery (odds ratio > 1.5 for each). The sensitivity at a 10% false-positive rate for the prediction of imminent delivery by a quantitative cervical length alone was 38%, yet it increased to 79% when combined with the abundance of four AF proteins (CXCL8, SNAP25, PTPN11, and MMP8). Neutrophil-mediated immunity, neutrophil activation, granulocyte activation, myeloid leukocyte activation, and myeloid leukocyte-mediated immunity were biological processes impacted by protein dysregulation in women destined to deliver within two weeks of diagnosis. The combination of AF protein abundance and quantitative cervical length improves prediction of the timing of delivery compared to cervical length alone, among women with a sonographic short cervix.

Figure 1

Odds ratio (and 95% confidence intervals) for the association between amniotic fluid proteins and imminent delivery. (A) Odds ratios for delivery within two weeks and (B) within one week. Odds ratios are adjusted for quantitative cervical length. The odd-ratios are calculated for a two-fold change in protein abundance. Alpha-enolase (ENO1), C–C motif chemokine 2 (CCL2), C–C motif chemokine 4-like (CCL4L1), C–C motif chemokine 7 (CCL7), C-X-C motif ligand 8 (CXCL8), Carbohydrate sulfotransferase 15 (CHST15), Catalase (CAT), Chitinase-3-like protein 1 (CHI3L1), Collectin-12 (COLEC12), Glucose-6-phosphate isomerase (GPI), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Granulocyte colony-stimulating factor (CSF3), Gro-beta/gamma (CXCL3/CXCL2), Growth-regulated alpha protein (CXCL1), ICOS ligand (ICOSLG), Interleukin-6 (IL6), L-lactate dehydrogenase B chain (LDHB), Leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2), Matrix Metalloproteinase-8 (MMP8), Mitogen-activated protein kinase 14 (MAPK14), Myeloperoxidase (MPO), Neutrophil gelatinase-associated lipocalin (LCN2), Oxidized low-density lipoprotein receptor 1 (OLR1), Proteinase 3 (PRTN3), Peptidoglycan recognition protein 1 (PGLYRP1), Resistin (RETN), Small ubiquitin-related modifier 3 (SUMO3), SUMO-conjugating enzyme UBC9 (UBE2I), Synaptosomal-associated protein 25 (SNAP25), Tumor necrosis factor receptor superfamily member 1B (TNFRSF1B), Tyrosine-protein phosphatase non-receptor type 11 (PTPN11).

Figure 2

ROC curves for predicting imminent delivery. Each panel compares prediction by cervical length alone and models that combine cervical length with protein abundance. AUC: area under the ROC curve and 95% confidence interval. CL: cervical length.

Figure 3

Combinations of multiple proteins for improving prediction of imminent delivery. AUC: area under the ROC curve and 95% confidence interval. CL: cervical length.

Figure 4

Kaplan–Meier survival curves for patients with a risk score above and those with a risk score below the cut-off value corresponding to a 10% false positive rate. The risk scores are determined by quantitative cervical length and four proteins (CXCL8, SNAP25, PTPN11, MMP8).

Figure 5

Clustered heatmap of protein data. The 17 proteins that were dysregulated in women destined for imminent delivery independent of cervical length were clustered using hierarchical clustering with correlation distance. Higher abundance of amniotic fluid protein levels is represented by a darker red color.

Figure 6

Gene ontology enrichment analysis of amniotic fluid proteins dysregulated with imminent delivery. Top 20 enriched biological processes (A) and a network representation of proteins involved in the top five enriched biological processes (B). Protein Ratio represents the number of proteins significantly dysregulated per the total number of proteins in a particular biological process. Darker red/blue indicate high/low enrichment, respectively. The size of the dots corresponds to the protein ratio represented by each biological process.