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Objective We aimed to evaluate the correlation and agreement of interleukin (IL)-8 and matrix metalloproteinases (MMP-9) levels between cervicovaginal (CVF) and amniotic fluids (AF) in women with preterm labor (PTL) and to determine the clinical values of these proteins in CVF compared with those in AF. Study Design We designed a retrospective cohort study of 85 singleton pregnant women with PTL at 23 to 34 weeks, who underwent amniocentesis. The AF was cultured, and CVF samples were collected at the time of amniocentesis. Paired AF and CVF samples were assayed for IL-8 and MMP-9 by enzyme-linked immunoassay (ELISA) in duplicate on a single plate, using similar dilution ratios. Results A significant but weak correlation was found for IL-8 levels between AF and CVF (r = 0.333), while no correlation was found for MMP-9 levels between AF and CVF (r = −0.039). Intra-class correlation coefficient for the agreement of IL-8 levels between CVF and AF was 0.4335 and −0.279 for MMP-9, indicating a poor-to-fair level of agreement between the two measured values, respectively. IL-8 and MMP-9 levels in CVF were not associated with the risk of either microbial invasion of the amniotic cavity (MIAC) or spontaneous preterm delivery (SPTD) within 7 days, whereas those in AF provided good-to-excellent predictive values for these two outcomes (area under the curve [AUCs]: 0.82–0.95). AUCs for IL-8 and MMP-9 were significantly larger using AF rather than using CVF for the prediction of MIAC and SPTD. Conclusion In women with PTL, IL-8 and MMP-9 levels in CVF do not precisely reflect the levels of the corresponding proteins in AF. IL-8 and MMP-9 levels in CVF had poor predictive values for the risk of MIAC and SPTD and were significantly inferior to those in AF. Key Points IL-8 and MMP-9 levels in CVF do not precisely reflect levels of the corresponding proteins in AF. Diagnostic accuracy of IL-8 and MMP-9 in CVF alone is not sufficient to predict MIAC and SPTD. IL-8 and MMP-9 levels in AF provide good-to-excellent predictive values for these two outcomes. [ABSTRACT FROM AUTHOR]

Objective We sought to determine whether the levels of complement and other inflammatory and angiogenic mediators in cervicovaginal fluid (CVF) are independently associated with intra-amniotic infection and/or inflammation (IAI) and imminent spontaneous preterm birth (SPTB, £48 hours of sampling) in women with preterm premature rupture of membranes (PPROM). Study Design This was a retrospective study consisting of 85 singleton pregnant women with PPROM at 20 0/7 to 33 6/7 weeks. Amniotic fluid (AF) obtained via amniocentesis was cultured and assayed for interleukin-6. CVF samples collected at the time of amniocentesis were assayed for complement C3a, C4a, and C5a, HSP70 (heat shock protein 70), M-CSF (macrophage colony-stimulating factor), M-CSF-R (macrophage colony-stimulating factor-receptor), S100 A8, S100 A9, thrombospondin-2, VEGF (vascular endothelial growth factor-receptor), and VEGFR-1 (vascular endothelial growth factor-receptor 1) by enzyme-linked immunosorbent assay. Results Multivariate logistic regression analyses revealed that elevated CVF concentrations of complement C3a, 4a, and 5a were significantly associated with an increased risk of IAI and imminent SPTB, whereas those of M-CSF were associated with IAI, but not imminent SPTB (p = 0.063), after adjustment for baseline covariates (e.g., gestational age at sampling). However, univariate, and multivariate analyses showed that the CVF concentrations of angiogenic (thrombospondin-2, VEGF, and VEGFR-1) and inflammatory (HSP70, M-CSF-R, S100 A8, and S100 A9) proteins were not associated with either IAI or imminent SPTB. Conclusion In women with PPROM, elevated CVF concentrations of complement C3a, C4a, and C5a are independently related to an increased risk of IAI and imminent SPTB. These findings suggest that complement activation in CVF is significantly involved in mechanisms underlying preterm birth and in the host response to IAI in the context of PPROM. Key Points Elevated CVF levels of C3a, 4a and 5a are associated with IAI and SPTB. CVF C3a, 4a and 5a have better predictability for SPTB, compared to AF WBC. Elevated CVF levels of M-CSF were associated with IAI, but not SPTB. [ABSTRACT FROM AUTHOR]

Objective: We aimed to determine whether various novel inflammatory, angiogenic, and extracellular matrix-related mediators in amniotic fluid (AF) can independently predict emergency cerclage outcomes in women with acute cervical insufficiency (CI). Methods: This was a retrospective cohort study conducted among 50 singleton pregnant women (18–25 weeks) who underwent emergency cerclage for CI and were subjected to amniocentesis. The AF samples were assayed for endoglin, endostatin, haptoglobin, insulin-like growth factor-binding protein (IGFBP)-3, -4, kallistatin, lumican, macrophage colony-stimulating factor (M-CSF), pentraxin 3, p-selectin, receptor for advanced glycation end products (RAGE), resistin, transforming growth factor beta-induced (TGFBI), and vitamin D-binding protein (VDBP) levels. Interleukin (IL)-6 levels in the AF were also measured for comparison with potential biomarkers assessed in this study. The primary endpoint was spontaneous preterm delivery (SPTD) at <34 weeks following emergency cerclage. Results: The AF levels of pentraxin 3, RAGE, and resistin were significantly higher in women who had SPTD at <34 weeks after cerclage placement (pentraxin-3: P = 0.003; RAGE: P = 0.041; and resistin; P = 0.002). In multivariate analysis, elevated AF levels of pentraxin 3 (P = 0.007) and resistin (P = 0.006), but not those of RAGE (P = 0.069), were independently associated with the occurrence of SPTD at <34 weeks after cerclage, following adjustment for baseline clinical variables (e.g., cervical dilation). The area under the curve (AUC) values of AF pentraxin 3, RAGE, and resistin for the prediction of SPTD at <34 weeks were 0.749, 0.669, and 0.770, respectively, which were similar to those of AF IL-6. However, in univariate analyses, no differences in the AF levels of endoglin, endostatin, haptoglobin, IGFBP-3, IGFBP-4, kallistatin, lumican, p-selectin, TGFBI, and VDBP were found to be associated with SPTD at <34 weeks after cerclage placement. Conclusions: In women with acute CI, the AF levels of pentraxin 3, RAGE, and resistin could be useful novel biomarkers for predicting SPTD following emergency cerclage. However, the clinical utility of these new biomarkers should be validated in larger multicenter studies. [ABSTRACT FROM AUTHOR]

Introduction: This study aimed to investigate amniotic fluid (AF) proteins that were differentially expressed between patients with cervical insufficiency (CI) and asymptomatic short cervix (SCX, ≤ 25 mm), and whether these proteins could be predictive of spontaneous preterm birth (SPTB) in these patients. Method: This was a retrospective cohort study of 129 singleton pregnant women with CI (n = 80) or SCX (n = 49) at 17 to 26 weeks who underwent amniocentesis. An antibody microarray was used to perform comparative proteomic profiling of AF from matched CI (n = 20) and SCX (n = 20) pregnancies. In the total cohort, an ELISA validation study was performed for 15 candidate proteins of interest. Subgroup analyses of patients with CI and SCX were conducted to evaluate the association between the 15 proteins and SPTB at < 32 weeks of gestation. Results: Eighty-six proteins showed intergroup differences. ELISA validation confirmed significantly higher levels of AF EN-RAGE, IL-8, lipocalin-2, MMP-9, S100A8/A9, thrombospondin-2, and TNFR2 in patients with CI than in those with SCX. Multivariable analysis showed that increased AF levels of EN-RAGE, S100A8/A9, and uPA were independently associated with SPTB at < 32 weeks in patients with CI; whereas in patients with SCX, high AF levels of APRIL, EN-RAGE, LBP, and TNFR2 were independently associated with SPTB at < 32 weeks. Conclusions: Multiple AF proteins show altered expression in patients with CI compared with SCX controls. Moreover, several novel mediators involved in inflammation were identified as potential biomarkers for predicting SPTB after the diagnosis of CI and SCX. These results provide new insights into target-specific molecules for targeted therapies to prevent SPTB in patients with CI/SCX. [ABSTRACT FROM AUTHOR]

Problem: To identify proteins present in the amniotic fluid (AF) that could be associated with spontaneous preterm birth (SPTB; delivery < 7 days) in women with preterm labor (PTL). Method of study: First, the AF proteome of 20 women with PTL and SPTB was compared with that of 20 matched women with term deliveries using an antibody microarray. Next, nine identified candidate biomarkers of SPTB were further validated in 267 singleton pregnant women with PTL who underwent amniocentesis at 26–33 weeks of gestation using ELISA, and whether the degree of expression of these proteins was associated with the risk severity for subsequent SPTB was retrospectively assessed. Results: Of the 507 proteins evaluated in the microarray analysis, 27 displayed significant intergroup differences. In particular, ELISA quantification confirmed that the expression of EN‐RAGE, IL‐6, IL‐8, IP‐10, lipocalin‐2, MMP‐8, MMP‐9, S100 A8/A9, and TNFR2 were all increased in the AF of women spontaneously delivering within 7 days of sampling compared with those delivering after 7 days. Moreover, the odds of SPTB within 7 days, even upon adjusting for confounders, tended to significantly increase with each increasing quartile of baseline AF levels of each protein (P‐value for trend <.05). Conclusion: Nine AF proteins were found to be independently associated with higher risk of subsequent SPTB in women with PTL, all of which were immune‐, inflammation‐, and extracellular matrix‐related proteins. Moreover, risk severity for this subsequent SPTB is closely related to the degree of expression of each of these proteins. [ABSTRACT FROM AUTHOR]

Problem: We aimed to identify amniotic fluid (AF) proteins associated with the subsequent rupture of membranes (ROM) occurring in the absence of active labor in women with threatened preterm labor (PTL) using an antibody microarray. Method of the study: This retrospective cohort study included 183 singleton pregnant women with PTL (24–33 weeks) who underwent amniocentesis. A nested case‐control study was conducted using AF samples from 20 women with subsequent ROM within 7 days of sampling (case subjects) and 20 gestational age‐matched women with term delivery (TD) without ROM (control subjects), via protein‐antibody microarray analysis. Seven candidate proteins of interest were validated via ELISA in the total cohort. Results: Seventeen proteins displayed significant intergroup differences. ELISA validation confirmed that the levels of EN‐RAGE, Fas, IL‐8, IP‐10, MMP‐8, and MMP‐9 were significantly higher, whereas IGFBP‐3 levels were significantly lower in the AF of women with subsequent ROM within 7 days of sampling than in that of women with TD without ROM. Moreover, the time interval from sampling to membrane rupture was significantly correlated with the expression levels of AF proteins, except for IL‐10. Conclusion: Using an antibody microarray, we identified various inflammatory, angiogenic, matrix‐degrading, and apoptosis‐related proteins in the AF that were associated with subsequent ROM occurring in the absence of active labor in women with threatened PTL. These findings provide novel insights into the molecular mechanisms underlying membrane rupture without active labor in threatened PTL. [ABSTRACT FROM AUTHOR]

Objective: We sought to identify novel biomarkers in the amniotic fluid (AF) related to imminent spontaneous preterm delivery (SPTD) (≤ 14 days after sampling) in women with early preterm premature rupture of membranes (PPROM), using a protein microarray. Method: This was a retrospective cohort study of a total of 88 singleton pregnant women with PPROM (23+0 to 30+6 weeks) who underwent amniocentesis. A nested case-control study for biomarker discovery was conducted using pooled AF samples from controls (non-imminent delivery, n = 15) and cases (imminent SPTD, n = 15), which were analyzed using an antibody microarray. Quantitative validation of four candidate proteins was performed, using ELISA, in the total cohort (n = 88). IL-8, MMP-9, and Fas levels were additionally measured for the comparison and to examine association of SPTD with the etiologic factors of PPROM. Results: Of all the proteins studied in the protein microarray, four showed significant intergroup differences. Analyses of the total cohort by ELISA confirmed the significantly elevated concentrations of AF lipocalin-2, MMP-9, and S100 A8/A9, but not of endostatin and Fas, in women who delivered within 14 days of sampling. For inflammatory proteins showing a significant association, the odds of SPTD within 14 days increased significantly with an increase in baseline AF levels of the proteins (P for trend <0.05 for each) in each quartile, especially in the 3rd and 4th quartile. Conclusions: We identified several potential novel biomarkers (i.e., lipocalin-2, MMP-9, and S100 A8/A9) related to SPTD within 14 days of sampling, all of which are inflammation-related molecules. Furthermore, the SPTD risk increased with increasing quartiles of each of these inflammatory proteins, especially the 3rd and 4th quartile of each protein. The present findings may highlight the importance of inflammatory mechanisms and the degree of activated inflammatory response in developing SPTD in early PPROM. [ABSTRACT FROM AUTHOR]

Purpose: To investigate whether complement and other immune-related proteins in cervicovaginal fluid (CVF) can predict intra-amniotic infection and/or inflammation (IAI) and spontaneous preterm delivery (SPTD, < 34.0 weeks) in women with preterm labor (PTL) and to compare the predictive abilities of these biomarkers with that of amniotic fluid (AF) white blood cells (WBCs).Methods: We designed a retrospective cohort study of 145 women with PTL at 23.0-33.6 weeks who underwent amniocentesis. AF was cultured and assayed for WBC count and interleukin-6 (IL-6). CVF samples were obtained at the time of amniocentesis. CVF was assayed for complement C3a and C5a, IGFBP-1, and MMP-9 by ELISA.Results: In the multivariate analysis, elevated CVF levels of C5a and IGFBP-1 were significantly associated with IAI and SPTD at < 34 weeks, while those of C3a were associated with IAI, but not SPTD, even after adjusting for other baseline confounders. For C3a, C5a, and IGFBP-1 in the CVF, area under the curve (AUC) values were statistically similar to that of AF WBCs for detecting IAI, whereas these CVF biomarkers had similar or higher AUC values than AF WBCs for predicting SPTD at < 34 weeks. However, univariate analysis showed no significant correlation between high CVF MMP-9 and IAI or SPTD at < 34 weeks.Conclusions: In women with PTL, the CVF levels of C3a, C5a, and IGFBP-1 may be useful as novel non-invasive predictors of IAI and SPTD at < 34 weeks. These biomarkers (especially IGFBP-1) have similar or better diagnostic performance compared to AF WBCs. [ABSTRACT FROM AUTHOR]

Objective: To determine whether vitamin D-binding protein (VDBP) in cervicovaginal fluid (CVF) is independently predictive of intra-amniotic infection and imminent spontaneous preterm delivery (SPTD, delivery within 48 hours) in women with preterm labor with intact membranes (PTL) or preterm premature rupture of membranes (PPROM). Method: This was a single-center retrospective cohort study. CVF samples for VDBP assays were obtained along with serum C-reactive protein (CRP) levels immediately after amniocentesis in consecutive women with PTL (n = 148) or PPROM (n = 103) between 23.0 and 34.0 weeks of gestation. VDBP levels in CVF were determined by enzyme-linked immunosorbent assay kits. The primary outcome measures were intra-amniotic infection [defined as positive amniotic fluid (AF) culture] and SPTD within 48 hours after sampling. Results: In the multivariable analysis, elevated VDBP levels in CVF samples of PTL women were significantly associated with intra-amniotic infection and imminent preterm delivery, even after adjusting for potential confounders (e.g., gestational age at sampling, parity, and serum CRP). However, these relationships were not found in women with PPROM. In women with PTL, the areas under receiver operating characteristic curves of CVF VDBP level for predicting intra-amniotic infection and imminent preterm delivery were 0.66 and 0.71, with cut-off values of 1.76 μg/mL (sensitivity of 64.3% and specificity of 78.4%) and 1.37 μg/mL (sensitivity of 65.4% and specificity of 72.6%), respectively. The CVF VDBP levels were significantly higher in women with PPROM than in those with PTL. Conclusions: VDBP in the CVF independently predicts intra-amniotic infection and imminent preterm delivery in women with PTL, whereas in women with PPROM, an elevated VDBP level in CVF is not associated with increased risks of these two outcome variables. [ABSTRACT FROM AUTHOR]

We sought to identify biomarkers in the amniotic fluid (AF) and specific signaling pathways related to spontaneous preterm delivery (SPTD, < 34 weeks) in women with preterm labor (PTL) without intra-uterine infection/inflammation (IUI). This was a retrospective cohort study of a total of 139 PTL women with singleton gestation (24 + 0 to 32 + 6 weeks) who underwent amniocentesis and who displayed no evidence of IUI. A nested case–control was conducted using pooled AF samples (n = 20) analyzed via label-free liquid chromatography-tandem mass spectrometry. In the total cohort, an ELISA validation study was performed for seven candidate proteins of interest. Proteomic analysis identified 77 differentially expressed proteins (DEPs, P < 0.05) in the AF from SPTD cases compared to term delivery controls. ELISA validation confirmed that women who had an SPTD before 34 weeks had significantly independently lower levels of VEGFR-1 and higher levels of lipocalin-2 and the Fc fragment of IgG binding protein in the AF. Five principle pathways associated with the 77 DEPs were identified, including glycolysis, gluconeogenesis, and iron homeostasis. The proteomic analysis data of AFs from women with PTL identified several novel biomarkers and specific protein pathways related to SPTD in the absence of IUI. [ABSTRACT FROM AUTHOR]

Background: We investigated whether various inflammatory and immune proteins in plasma predict intra-amniotic infection and imminent preterm delivery in women with preterm labor and compared their predictive ability with that of amniotic fluid (AF) interleukin (IL)-6 and serum C-reactive protein (CRP).Methods: This retrospective cohort study included 173 consecutive women with preterm labor who underwent amniocentesis for diagnosis of infection and/or inflammation in the AF. The AF was cultured, and assayed for IL-6. CRP levels and cervical length by transvaginal ultrasound were measured at the time of amniocentesis. The stored maternal plasma was assayed for IL-6, matrix metalloproteinase (MMP)-9, and complements C3a and C5a using ELISA kits. The primary and secondary outcome criteria were positive AF cultures and spontaneous preterm delivery (SPTD) within 48 h, respectively. Univariate, multivariate, and receiver operating characteristic analysis were used for the statistical analysis.Results: In bivariate analyses, elevated plasma IL-6 level was significantly associated with intra-amniotic infection and imminent preterm delivery, whereas elevated plasma levels of MMP-9, C3a, and C5a were not associated with these two outcomes. On multivariate analyses, an elevated plasma IL-6 level was significantly associated with intra-amniotic infection and imminent preterm delivery after adjusting for confounders, including high serum CRP levels and short cervical length. In predicting intra-amniotic infection, the area under the curve (AUC) was significantly lower for plasma IL-6 than for AF IL-6 but was similar to that for serum CRP. Differences in the AUCs between plasma IL-6, AF IL-6, and serum CRP were not statistically significant in predicting imminent preterm delivery.Conclusions: Maternal plasma IL-6 independently predicts intra-amniotic infection in women with preterm labor; however, it has worse diagnostic performance than that of AF IL-6 and similar performance to that of serum CRP. To predict imminent preterm delivery, plasma IL-6 had an overall diagnostic performance similar to that of AF IL-6 and serum CRP. Plasma MMP-9, C3a, and C5a levels could not predict intra-amniotic infection or imminent preterm delivery. [ABSTRACT FROM AUTHOR]