Patients and investigation of the clinical characteristics
From February 2015 to February 2021, we enrolled 46 female patients diagnosed with videourodynamic-confirmed DV at the department of urology of a single medical center. All patients underwent VUDS, and their diagnostic details were interpreted according to the International Continence Society terminology.1 The following VUDS parameters were recorded: first sensation of bladder filling (FSF), bladder compliance, cystometric bladder capacity (CBC), detrusor voiding pressure (Pdet), maximal urinary flow rate (Qmax), corrected maximal urinary flow rate (cQmax, defined as Qmax / √CBC), voided volume (Vol), post-void residual volume (PVR), voiding efficacy (VE, defined as vol / CBC), and female bladder outlet obstruction index (BOOIf, defined as Pdet - 2.2*Qmax).13 Patients with an open bladder neck but a narrow membranous urethra or PFM (a spinning top appearance) on real-time fluoroscopy, increased EUS electromyography activities, and low Qmax (with or without a high Pdet) during voiding were diagnosed with DV (Figure 1). In addition, only patients without a history of neurological disease were eligible for this study. The exclusion criteria included genital prolapse, active urinary tract infection, interstitial cystitis, and previous surgery for urinary incontinence. In total, 25 women with stress urinary incontinence but without other significant storage or voiding dysfunction on VUDS were included as controls.
All patients were assessed with the International Prostate Symptom Score (IPSS), which included total IPSS, IPSS storage subscore (IPSS-S), and IPSS voiding subscore (IPSS-V). Biofeedback PFM exercise or EUS BoNT-A 100 U injection was recommended to patients with medically refractory DV according to the reported treatment protocols.7,8 The treatment outcome was evaluated using the Global Response Assessment (GRA). Moreover, it was categorized as −3, −2, −1, 0, 1, 2, and 3, which indicated markedly worse to markedly improved status) based on satisfaction 3 months after treatment. A successful outcome was defined as a GRA score of ≥2 (moderately and markedly improved).
Assessment of urine biomarker levels
Urine samples were collected from all patients with DV and controls. Urine was self-voided by patients who had a full bladder sensation. Then, urinalysis was performed simultaneously to confirm an infection-free status before urine samples were stored. In total, 50-mL urine samples were placed on ice immediately and transferred to the laboratory for preparation. The samples were centrifuged at 1800 rpm for 10 min at 4°C. The supernatant was separated into aliquots in 1.5-mL tubes (1 mL per tube) and was preserved in a freezer at −80°C. Before further analyses were performed, the frozen urine samples were centrifuged at 12,000 rpm for 15 min at 4°C, and the supernatants were used for subsequent evaluations.
Quantification of 8-OHdG
The quantification of 8-OHdG in urine samples was performed in accordance with the manufacturer’s instructions (8-OHdG ELISA Kit, BioVision). Briefly, 50 μL of biotin-detection antibody working solution and 50 μL of sample were sequentially added to 96-well plates (panel kits), and the plates were incubated for 45 min at 37°C. The well contents were removed, and the plates were washed three times with 350 μL of wash buffer. Next, 100 mL of horseradish peroxidase-streptavidin conjugate working solution was added to each well, and the plates were incubated for 30 min at 37°C. The solution was discarded, and 350 μL of wash buffer was used to wash the plates five times. Next, 90 mL of TMB substrate was added into each well; incubation was then performed in the dark for 30 min at 37°C. Finally, 50 μL of stop solution was added, and the plates were evaluated on the microplate reader at 450 nm. The median fluorescence intensities of the targets were analyzed to calculate the corresponding concentrations in the samples.
Quantification of 8-isoprostane
The quantification of 8-isoprostane in urine samples was performed in accordance with the manufacturer’s instructions (8 isoprostane ELIZA kit, Enzo). Briefly, 50 μL of 8-iso-PGF2α conjugate solution, 50 μL of 8-iso-PGF2α antibody solution, and 100 μL of sample were sequentially added to 96-well plates (panel kits), and the plates were incubated 2 h at room temperature on a plate shaker at 500 rpm. The well contents were removed, and the plates were washed three times with 400 μL of wash buffer. In total, 200 mL of the pNpp substrate solution was added to each well, and the plates were incubated for 45 min at room temperature without shaking. Finally, 50 μL of stop solution was added, and the plates were read immediately at 405 nm. The median fluorescence intensities of the targets were analyzed to calculate the corresponding concentrations in the samples. The measurement of urine 8-isoprostane levels was standardized using urinary creatinine levels.
Quantification of TAC
The quantification of TAC in the samples was performed in accordance with the manufacturer’s instructions (Total Antioxidant Capacity Assay Kit, abcam). Briefly, 100 μL of Cu2+ working solution and 100 μL of sample were sequentially added to 96-well plates (panel kits), and the plates were incubated for 90 min at room temperature on a shaker protected from light. Finally, the plates were evaluated on the microplate reader at 570 nm. The median fluorescence intensities of the target were analyzed to calculate the corresponding TAC concentrations in the samples.
Evaluation of inflammatory substances
Inflammatory markers including cytokines and neurotrophins in the urine samples were assayed using commercially available microspheres with the Milliplex® human cytokine/chemokine magnetic bead-based panel kit (Millipore, Darmstadt, Germany).
Seven targeted analytes were used for the multiplex kit, and these included the following inflammatory cytokines: catalog number HCYTMAG-60K (interleukin-1 beta [IL-1β], IL-2, IL-6, IL-8, and tumor necrosis factor alpha [TNFα]), catalog number HADK2MAG-61K (nerve growth factor [NGF]), and catalog number HNDG3MAG-36K (brain-derived neurotrophic factor [BDNF]). The following laboratory procedures for the quantification of these targeted analytes were similar to those reported in our previous studies.14,15
This study was approved by the Institutional Review Board and Ethics Committee of Buddhist Tzu Chi General Hospital (no. IRB107-37-A and IRB: 109-095-B). All methods/ study were performed in accordance with the relevant guidelines and regulations. All patients with DV and controls were provided with information about the rationale and procedures of this study, and they provided informed consent
Statistical analysis
Continuous variables were presented as means ± standard deviations and categorical variables as numbers and percentages. Outliers were defined as values outside the range between the means ± three standard deviations for each biomarker in either the study or the control group, and these were excluded from further analysis. The clinical data and urine biomarker levels between the study and control groups and between the different treatment outcome groups were analyzed using analysis of variance. Linear regression analysis with Pearson correlation was performed to determine the association between clinical characteristics and urine biomarker levels. To predict successful treatment outcomes, univariate and multivariate logistic regression model analyses were conducted with the calculation of odds ratio (OR). All calculations were performed using SPSS Statistics software for Windows version 20.0 (IBM Corp., Armonk, NY, the USA). A p value of <0.05 was considered statistically significant.