Inflammatory biomarkers in the cervicovaginal fluid to identify histologic chorioamnionitis and funisitis in women with preterm labor.

OBJECTIVE: We investigated the association between altered levels of inflammatory proteins in the cervicovaginal fluid (CVF) and acute histologic chorioamnionitis (HCA) and funisitis in women with preterm labor (PTL). METHODS: In this study, a total of 134 consecutive singleton pregnant women with PTL (at 23+0-34+0 weeks) who delivered preterm (at  < 37 weeks) and from whom CVF samples were collected at admission were retrospectively enrolled. The CVF levels of haptoglobin, interleukin-6/8, kallistatin, lipocalin-2, matrix metalloproteinase (MMP)-8, resistin, S100 calcium-binding protein A8, and serpin A1 were determined using enzyme-linked immunosorbent assay. The placentas were histologically analyzed after delivery. RESULTS: Multiple logistic regression analyses showed significant associations between elevated CVF interleukin-8 and resistin levels and acute HCA after adjusting for baseline covariates (e.g., gestational age at sampling). CVF haptoglobin, interleukin-6/8, kallistatin, MMP-8, and resistin levels were significantly higher in women with funisitis than in those without, whereas the baseline covariates were similar between the two groups (P > 0.1). The area under the receiver operating characteristic curves of the aforementioned biomarkers ranged from 0.61 to 0.77 regarding each outcome. Notably, HCA risk significantly increased with increasing CVF levels of interleukin-8 and resistin (P for trend  < 0.05). CONCLUSIONS: Haptoglobin, interleukin-6/8, kallistatin, MMP-8, and resistin were identified as potential inflammatory CVF biomarkers predictive of acute HCA and funisitis in women with PTL. Moreover, the risk severity of acute HCA may be associated with the degree of the inflammatory response in the CVF (particularly based on interleukin-8 levels).

Degree of expression of inflammatory proteins in the amniotic cavity, but not prior obstetric history, is associated with the risk severity for spontaneous preterm birth after rescue cerclage for cervical insufficiency.

PROBLEM: To examine whether the severity of spontaneous preterm birth (SPTB) risk after rescue cerclage for acute cervical insufficiency (CI) is linked to the degree of inflammatory response in the amniotic fluid (AF) based on the concentrations of various inflammatory proteins and prior obstetric history. METHOD OF STUDY: We conducted a retrospective cohort study of 65 singleton pregnant women (17-25 weeks) who underwent rescue cerclage following the diagnosis of acute CI and were subjected to amniocentesis. EN-RAGE, IL-6, IL-8, and IP-10 as inflammatory mediators and kallistatin, MMP-2/8, and uPA as extracellular matrix remodeling-related molecules were assayed in the AF using ELISA. The level of each inflammatory mediator was divided into quartiles. RESULTS: Intra-amniotic inflammation (IAI; AF IL-6 level ≥2.6 ng/mL) was independently associated with SPTB after cerclage placement. The odds of SPTB at < 32 weeks, even after adjusting for confounders, increased significantly with each increasing quartile of baseline AF levels for each inflammatory mediator (p for trend < .05). Kaplan-Meier survival curves showed that the cerclage-to-delivery intervals were significantly shorter as the quartiles of AF EN-RAGE and MMP-8 increased (log-rank test, p < .01 each). Neither previous term birth nor prior PTB was associated with SPTB risk or cerclage-to-delivery interval after rescue cerclage. Multiparous women who experience CI after term birth showed significantly elevated levels of MMP-8 and reduced kallistatin levels in the AF. CONCLUSION: In patients with CI, SPTB risk (especially risk severity) after rescue cerclage is associated with the degree of the inflammatory response in AF as well as the presence of IAI but not with prior obstetric history.

Extracellular matrix-related and serine protease proteins in the amniotic fluid of women with early preterm labor: Association with spontaneous preterm birth, intra-amniotic inflammation, and microbial invasion of the amniotic cavity.

PROBLEM: We aimed to determine whether altered levels of various extracellular matrix (ECM)-related and serine protease proteins in the amniotic fluid (AF) are associated with imminent spontaneous preterm birth (SPTB; ≤7 days) and intra-amniotic inflammation and/or microbial invasion of the amniotic cavity (IAI/MIAC) in women with early preterm labor (PTL). METHOD OF STUDY: This retrospective cohort study included 252 women with singleton pregnancies undergoing transabdominal amniocentesis who demonstrated PTL (24-31 weeks). The AF was cultured for microorganism detection to characterize MIAC. IL-6 concentrations were determined in the AF samples to identify IAI (≥2.6 ng/mL). The following mediators were measured in the AF samples using ELISA: kallistatin, lumican, MMP-2, SPARC, TGFBI, and uPA. RESULTS: Kallistatin, MMP-2, TGFBI, and uPA levels were significantly higher and SPARC and lumican levels were significantly lower in the AF of women who spontaneously delivered within 7 days than in the AF of those who delivered after 7 days; the levels of the first five mediators were independent of baseline clinical variables. In the multivariate analysis, elevated levels of kallistatin, MMP-2, TGFBI, and uPA and low levels of lumican and SPARC in the AF were significantly associated with IAI/MIAC and MIAC, even after adjusting for the gestational age at sampling. The areas under the curves of the aforementioned biomarkers ranged from 0.58 to 0.87 for the diagnoses of each of the corresponding endpoints. CONCLUSION: ECM-related (SPARC, TGFBI, lumican, and MMP-2) and serine protease (kallistatin and uPA) proteins in the AF are involved in preterm parturition and regulation of intra-amniotic inflammatory/infectious responses in PTL.

Expression of inflammatory, angiogenic, and extracellular matrix-related mediators in the cervicovaginal fluid of women with preterm premature rupture of membranes: Relationship with acute histological chorioamnionitis.

PROBLEM: To investigate whether altered expression of various inflammation-, angiogenesis-, and extracellular matrix-related mediators in cervicovaginal fluid (CVF) could be independently associated with acute histological chorioamnionitis (HCA), microbial-associated HCA, and funisitis in women with preterm premature rupture of membranes (PPROM). METHOD OF STUDY: Clinical data of 102 consecutive singleton pregnant women with PPROM at 23+0 to 34+0 weeks were retrospectively analyzed. CVF samples were collected upon admission. Levels of APRIL, DKK-3, IGFBP-1/2, IL-6/8, lipocalin-2, M-CSF, MIP-1α, MMP-8/9, S100A8A9, TGFBI, TIMP-1, TNFR2, uPA, and VDBP were determined by ELISA. Placentas were histologically examined after birth. RESULTS: Multivariate logistic regression analyses showed that: (1) elevated CVF levels of IL-8 and TNFR2 were independently associated with acute HCA; (2) elevated CVF levels of IL-6, IL-8, M-CSF, MMP-8, and TNFR2 were independently associated with microbial-associated HCA; and (3) elevated CVF IL-8 and MMP-8 levels were independently associated with funisitis when adjusted for gestational age. Areas under the curves of the aforementioned CVF biomarkers ranged within 0.61-0.77, thereby demonstrating poor to fair diagnostic capacity for these clinical endpoints. HCA risk significantly increased as the CVF levels of each inflammatory mediator increased (P for trend < 0.05). CONCLUSIONS: Herein, we identified several inflammatory biomarkers (IL-6/8, M-CSF, MMP-8, and TNFR2) in the CVF that are independently associated with acute HCA, microbial-associated HCA, and funisitis in women with PPROM. Furthermore, the degree of inflammatory response in the CVF, based on the levels of these proteins, demonstrated a direct relationship with HCA risk (especially risk severity).

Plasma IGFBP-1, Fas, kallistatin, and P-selectin as predictive biomarkers of histologic chorioamnionitis and associated intra-amniotic infection in women with preterm labor.

PROBLEM: To determine whether altered levels of 13 plasma biomarkers, alone or in combination, could be independently associated with histologic chorioamnionitis (HCA) and microbial-associated HCA (defined as the presence of HCA along with microbial invasion) in women with preterm labor (PTL). METHODS OF STUDY: This was a retrospective cohort study involving 77 singleton pregnant women with PTL (23-34 gestational weeks) who delivered within 96 h of plasma and amniotic fluid (AF) sampling. DKK-3, E-selectin, Fas, haptoglobin, IGFBP-1, kallistatin, MMP-2, MMP-8, pentraxin 3, progranulin, P-selectin, SAA4, and TGFBI levels were assayed in plasma samples by ELISA. AF obtained via amniocentesis was used for microorganism identification. RESULTS: Multiple logistic regression analyses revealed significant associations between low plasma IGFBP-1 levels and acute HCA, and between low plasma Fas and kallistatin levels, and elevated plasma P-selectin levels and microbial-associated HCA (all p < .05), after adjusting for gestational age. Using a stepwise regression procedure, a multi-biomarker panel for microbial-associated HCA was developed, which included plasma MMP-2, kallistatin, and P-selectin levels (area under the curve [AUC], .867). The AUC for this three-marker panel was significantly or borderline significantly greater than that of any single variable included in the panel. However, a predictive model for acute HCA could not be developed because only one variable (MMP-2) was selected. CONCLUSIONS: These findings demonstrate that IGFBP-1, Fas, kallistatin, and P-selectin are associated with acute HCA and microbial-associated HCA in women with PTL. Their combined use can significantly improve the diagnostic ability for the detection of microbial-associated HCA.

Surface display of human lactoferrin using a glycosylphosphatidylinositol-anchored protein of Saccharomyces cerevisiae in Pichia pastoris.

A cell surface display system was developed in Pichia pastoris using the gene TIP1, encoding the glycosylphosphatidylinositol (GPI)-anchored protein of Saccharomyces cerevisiae (ScTIP). Human lactoferrin cDNA (hLf) was fused to a full-length TIP1 DNA (ScTIP ( 630 )) or a short-TIP1 fragment (ScTIP ( 120 )) encoding the 40 C-terminal amino acids of ScTIP. Both hLf-ScTIP fusion genes were expressed in P. pastoris SMD 1168. The fused protein was detected by western blotting after extraction of the lysed recombinant cells with Triton X-100, urea, and Triton X-100 plus urea, suggesting that the hLf is associated with the membrane. The localization of surface-displayed hLf was confirmed by immunofluorescence confocal microscopy and flow cytometric analysis using FITC-labeled anti-hLf antibody, suggesting that hLf was successfully located at the surface of P. pastoris. The intact recombinant cells and cell lysates showed antibacterial activity against target microorganisms, meaning that the expressed hLf was biologically active. The results indicated that the ScTIP anchoring motif is useful for cell surface display of foreign proteins in P. pastoris.