Objective
The purpose of this study was to investigate the temporal changes in immunoreactive cystatin A and the enzymatic activity of cathepsins B, H, L, and S in human cervicovaginal fluid (CVF) in late pregnancy and spontaneous labor.
Study Design
CVF was collected weekly (n = 95 women) from 36 weeks gestation until spontaneous term labor. Cystatin A was quantified using enzyme-linked immunosorbent assay. The enzyme activity of cathepsins B, H, L, and S was measured with fluorometric enzyme assay kits.
Results
Cystatin A significantly decreased towards ( P = .016, 2-way analysis of variance) and during labor ( P < .001, 2-way analysis of variance). Enzymatic activity of cathepsins B, H, and S did not change with labor onset ( P = .452, P = .703, P = .411, respectively, 2-way analysis of variance).
Conclusion
In late gestation, CVF-decreased expression of the cysteine protease inhibitor, cystatin A, is associated with labor. Although the role and contribution of cystatin A to increased extracellular matrix remodeling has yet to be elucidated, the data that were obtained are consistent with this hypothesis.
Cystatin A (also known as acid cysteine proteinase inhibitor or stefin A) is a nonspecific cysteine protease inhibitor that is abundant in the cytosol of leukocytes and epithelial cells that include squamous epithelial cells of the cervix and vagina and in various body fluids. Although the exact physiologic function of cystatin A is unclear, it is believed to play a role in epidermal defense by protecting cells against the proteolytic activities of cathepsins that are released during cell death from proliferating cancer cells or invading microorganisms. The best characterized cathepsins are B, H, L, and S. Cathepsins B, H, and L, are expressed ubiquitously in cell lysosomes ; cathepsin S is expressed highly in the spleen and in antigen-presenting cells. Cathepsin L degrades laminin, fibronectin, collagen types I, IV, and XVIII, and elastin. Cathepsin B degrades insoluble collagen ; cathepsin S is essential for major histocompatibility complex class II antigen presentation. During inflammation, cathepsins B, L, and S secreted extracellularly by macrophages, degrade elastin, and may contribute to extracellular matrix (ECM) remodeling. As extensive ECM remodeling of the cervix and fetal membranes occurs during pregnancy and parturition, it is plausible that cathepsins may play a role.
Using 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) to characterize human cervicovaginal fluid (CVF) protein expression that is associated with term labor, we previously reported that the expression of cystatin A monomer 1-2 days before labor was 56% significantly lower compared with paired CVF samples that were collected at 26-30 days before labor onset ( P = .003). Cystatin A monomer and dimer both were decreased significantly in labor, compared with 26-30 days before labor. The current study built on these previous 2D-PAGE results by validating the cystatin A changes that are associated with labor in a large sample size with the use of enzyme-linked immunosorbent assay (ELISA). Cathepsins B, H, L, and S have been identified previously in the CVF proteome. However, there are no studies that have investigated cathepsin activity in human CVF in association with labor. The enzyme activity of cathepsins B, H, L, and S in human CVF was determined therefore to further elucidate the roles of cysteine proteases in human parturition.
The specific aims of this study were to (1) investigate the quantitative temporal changes of cystatin A in late pregnancy and during spontaneous term labor; (2) investigate the enzymatic activity of cathepsins B, H, L, and S in term pregnant women until spontaneous labor onset; (3) establish whether common vaginal microflora have an influence on cystatin A concentrations in the CVF of term women; and (4) determine whether unprotected sexual intercourse in the preceding 48 hours influences the concentration of cystatin A in the CVF.
Materials and Methods
Patient recruitment and sample collection
This study was approved by the Mercy Health, Research Ethics Committee and all participants gave written informed consent. This was a longitudinal observational study of CVF in pregnancy. Healthy, pregnant women who attended the Antenatal Clinic at the Mercy Hospital for Women (Heidelberg, Victoria, Australia) were recruited prospectively by a research midwife. CVF samples that were used for subsequent analysis were selected retrospectively, based on pregnancy outcome. Our aim was to use CVF samples from women who experienced spontaneous onset of term labor. The highest likelihood of this occurring is in low-risk, parous women with a singleton pregnancy who previously have experienced a successful vaginal delivery. Serial CVF swabs were obtained weekly starting from 36 weeks gestation and, when possible, an in-labor swab was obtained before the rupture of fetal membranes. Labor was defined as regular painful uterine contractions that lead to the effacement and dilation of the cervix (>3 cm).
Inclusion criteria were healthy parous women, singleton pregnancy, and 37-42 weeks’ gestation at spontaneous labor onset.
Exclusion criteria were first pregnancy, multifetal gestation, <37 or >42 weeks’ gestation at delivery, prelabor rupture of fetal membranes, elective cesarean section delivery or induction of labor, bacterial vaginosis, digital vaginal examination or transvaginal ultrasound scans within 6 hours of sampling, and preexisting/current medical conditions (maternal or fetal).
To collect the CVF, a sterile speculum was inserted into the vagina and a Duo-transtube double-tipped rayon swab (Medical Wire & Equipment Co Ltd, Corsham, Wilts, England) was placed into the posterior vaginal fornix for 30 seconds. The swabs were placed into a polystyrene tube with 1 mL of chilled “extraction” buffer that contained 50 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes), 150 mmol/L NaCl, 0.1% sodium dodecylsulfate, 1 mmol/L ethylenediaminetetraacetic acid, 1 mmol/L Pefabloc SC 4-(2-aminoethyl)benzene sulfonyl fluoride (protease inhibitor; Roche Diagnostics GmbH, Mannheim, Germany). The tubes were left on ice until they were transported to the laboratory for processing, which was usually within 2 hours. Swab samples were centrifuged, and the supernatant fluid was collected and stored at −80°C. The total protein of all CVF samples was determined with the Bicinchoninic acid protein assay (Pierce, Rockford, IL), according to the manufacturer’s instructions.
At each CVF sampling, women were questioned as to whether they had had unprotected sexual intercourse within the last 48 hours, had an internal ultrasound scan within the last 6 hours, experienced recent vaginal bleeding, or were on any medication. Samples that were assayed for cystatin A were stratified into 6 groups: in-labor or 0-7 days, 8-14 days, 15-21 days, 22-28 days, and >28 days (range, 29–47 days) before labor. The enzyme activity of cathepsins B, H, L, and S was measured in a subset of 14 women who each provided a minimum of 3 serial samples. These samples were grouped into in-labor or 0-7 days, 8-14 days, and 15-21 days before labor onset.
Microbial assessment
A high vaginal swab was obtained from 69 women at their first sampling (approximately 36 weeks’ gestation) for microbiology assessment. Microbiologic culture was performed according to standard pathology laboratory protocol. The microbiologic results were matched to the cystatin A concentration of the CVF sample that had been collected on the same day. Subjects were allocated into 5 groups based on microbiologic results: no significant pathogens, Group B Streptococcus (GBS) colonization, Candida spp colonization, Ureaplasma spp colonization, and mixed colonization (consisting of ≥2 of these groups).
Cystatin A ELISA
The cystatin A ELISA was established in the laboratory with a pair of cystatin A antibodies (mouse anti-human cystatin A monoclonal immunoglobulin G2A clone 224705 as capture antibody and goat anti-human cystatin A biotinylated polyclonal immunoglobulin G as detection antibody; both were from R&D Systems Inc, Minneapolis, MN). The specificity of the capture antibody was confirmed by Western blot analysis and was specific for the cystatin A monomer (data not shown). The following ELISA protocol was used: (1) 96-well Maxisorp Immuno plate (Nunc A/S, Roskilde, Denmark) was coated with 100 μL of capture antibody mixture (2 μL in phosphate-buffered saline solution) and incubated overnight; (2) recombinant human cystatin A (R&D Systems, Inc) assay standards (2000, 1000, 500, 250, 125, 62.5, and 31.3 pg/mL) were prepared with assay buffer (phosphate-buffered saline solution, 0.5% bovine serum albumin, 0.1% Tween 20); (3) the plate was washed 6 times (dunking method) with wash buffer (1 mmol/L KH 2 PO 4 , 8 mmol/L K 2 HPO 4 , 1 mmol/L ethylenediaminetetraacetic acid, 0.05% Tween 20, pH 7.4) and blocked for 1 hour with 300 μL of assay buffer; (4) 100 μL of standards, control samples, blanks (assay buffer), and test samples were assayed in duplicate and incubated for 90 minutes, and the plate was washed; (5) 100 μL of detection antibody working mixture (0.2 μg/mL in assay buffer) was added and incubated for 90 minutes, and the plate was washed; (6) 100 μL of streptavidin-horseradish peroxidase working solution (1 in 500 dilution; R&D Systems, Inc) was added and incubated for 30 minutes, and the plate was washed; (7) 100 μL of 3,3′,5,5′-tetramethyl-benzidine was added and incubated for 25 minutes; and (8) 100 μL of 1.8 mol/L of H 2 SO 4 was used to stop the reaction. Absorbance was read at 450 nm. A standard curve was plotted with a logistic curve fit to determine the cystatin A concentration of the samples. The sensitivity, interassay, and intraassay coefficients of variation of the in-house cystatin A ELISA were 58.1 pg/mL, 12.0%, and 5.0%, respectively.
Cathepsin activity assays
The activity of cathepsins B and L was determined using InnoZyme kits (Calbiochem, San Diego, CA) and quantified as free amino-4-methyl-coumarin (expressed as micromoles per milligram of protein per minute). Cathepsins H and S were determined with BioVision activity assay kits (BioVision, Mountain View, CA) and quantified as free amino-4-trifluoromethyl coumarin (expressed as micromoles per milligram of protein per minute). All assays were performed adhering to the manufacturers’ protocol.
Statistical analyses
Statistical analyses were performed with the Statistical Package for Social Sciences (version 17.0; SPSS Inc, Chicago, IL). All CVF assay data were corrected for total protein and expressed as concentration per milligram of protein. Data were assessed for homogeneity of variance with the Kolmogorov-Smirnov test. Cystatin A data were not distributed ( P < .001, Kolmogorov-Smirnov test) normally. Data therefore were transformed logarithmically, and the homogeneity of the transformed data was confirmed ( P = .783, Kolmogorov-Smirnov test). Cathepsins B, H, and S activities were normally distributed ( P = .400, P = .392, P = .862, respectively, Kolmogorov-Smirnov test).
Linear regression analysis with 2-way analysis of variance (ANOVA) was used to examine the relationship between cystatin A expression and sampling-to-labor intervals, with days from labor entered as a continuous factor and subjects entered as a random factor to account for multiple sampling. Temporal changes in cystatin A concentration with impending labor were analyzed between groups (in-labor, 0-7 days, 8-14 days, 15-21 days, 22-28 days, and >28 days before labor) with 2-way ANOVA, with subjects entered as a random factor. Tukey honestly significant difference post hoc testing was done where appropriate. Cathepsin activities were analyzed with the groups as a fixed factor (in-labor, 0-7 days, 8-14 days, and 15-21 days before labor) and subjects as a random factor. To determine the influence of unprotected sexual intercourse or microbial status on cystatin A concentrations, the statistical analysis included days from labor as a continuous factor, with subjects entered as a random factor. Fold changes were calculated with median values. Statistical significance was ascribed when the probability value was < .05.
Results
Demographics
Ninety-five women who had spontaneous onset of term labor with intact membranes were suitable for inclusion into the study ( Table ). Each woman contributed 1-6 CVF samples. Samples ranged from in-labor to 47 days before spontaneous labor onset. One woman reported mild bleeding, and 2 women were receiving antibiotics for chest infections. One woman who was allocated into the mixed colonization group had Mycoplasma sp and Candida spp. One woman who was diagnosed with candidiasis was given vaginal clotrimazole cream for treatment. Sexual intercourse data are reported later.
