Abstract
BackgroundThe cervical mucus plug (CMP) is a semi-solid structure with antibacterial properties positioned in the cervical canal during pregnancy. The CMP contains high concentrations of matrix metalloproteinase 8 and 9 (MMP-8, MMP-9) and tissue inhibitor of metalloproteinase 1 (TIMP-1). This indicates a potential to degrade extracellular matrix components depending on the balance between free non-complexed inhibitors and active enzymes.MethodsThirty-two CMPs collected during active labor at term were analyzed. Twelve CMPs were separated into a cellular and an extracellular/fluid phase and analyzed by gelatin and reverse zymography to reveal MMP and TIMP location. Twenty samples were homogenized, extracted and studied by the TIMP activity assay based on gelatin zymography. Enzyme-linked immunosorbent assay (ELISA) was used to determine TIMP-1, MMP-8 and MMP-9 protein concentrations, and gelatin and reverse zymography used to identify gelatinases and TIMPs, respectively. The Western blotting technique was applied for semi-quantification of alpha2-macroglobulin. An ELISA activity assay was used to detect MMP-8 and MMP-9 activity.ResultsProMMP-2, proMMP-9, TIMP-1 and TIMP-2 were almost exclusively located in the fluid phase compared to the cellular phase of the CMP. All the extracted samples contained MMP-8, MMP-9, TIMP-1, TIMP-2 and alpha2-macroglobulin. Free non-complexed TIMP was detected in all the samples analyzed by the TIMP activity assay and was associated with TIMP-1 protein (R = 0.71, p < 0.001) and with the TIMP/MMP molar ratio (1.7 (1.1–2.5) (mean (95% confidence interval)) (R = 0.65, p = 0.002). The ELISA activity assay showed no activity from MMP-8 or MMP-9.ConclusionDue to their extracellular location, potential proteolytic activity from neutrophil-derived MMPs in the CMP could exert a biological impact on cervical dilatation and fetal membrane rupture at term. The functional TIMP activity assay, revealing excess non-complexed TIMP, and a molar inhibitor/enzyme ratio above unity, indicate that refined MMP control prevents CMP-originated proteolytic activity in the surrounding tissue.
Highlights
The cervical mucus plug (CMP) is a semi-solid structure with antibacterial properties positioned in the cervical canal during pregnancy
ProMMP-2, proMMP-9, tissue inhibitor of metalloproteinase 1 (TIMP-1) and TIMP-2 were preferably located in the fluid phase (Figure 2A and 2B)
The ratio of proMMP-2 + proMMP-9 between the fluid phase and the cell phase was estimated by scanning of lysis band intensities and found to be 2.6 (1.4–3.8) (mean)
Summary
The cervical mucus plug (CMP) is a semi-solid structure with antibacterial properties positioned in the cervical canal during pregnancy. The CMP contains high concentrations of matrix metalloproteinase 8 and 9 (MMP-8, MMP-9) and tissue inhibitor of metalloproteinase 1 (TIMP-1). This indicates a potential to degrade extracellular matrix components depending on the balance between free non-complexed inhibitors and active enzymes. We know that the CMP has antibacterial properties, and contains high concentrations of the matrix metalloproteinases (MMPs) gelatinase A (MMP-2), gelatinase B (MMP9) and neutrophil collagenase (MMP-8) together with their specific inhibitors, the tissue inhibitors of metalloproteinases, TIMP-1 and TIMP-2 [4]. The functional balance between matrix-degrading enzymes and their inhibitors [10,11] in the CMP and the heterogeneity of cervical matrix biology [12,13], are fundamental to our understanding of complicated multi-faceted processes like term and preterm birth
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