**5. Materials and Methods**

This retrospective study included IC/BPS patients who had been treated for the first time with 100 U of BoNT-A (BOTOX, Allergan, Irvine, CA, USA) in 10 mL saline injected at 20 sites. All patients had been diagnosed to have IC/BPS based on the characteristic IC symptoms and cystoscopic findings of glomerulations, petechiae, or mucosal fissures on hydrodistention under anesthesia [30]. Among the IC/BPS patients, at least two treatment modalities had been tried, including lifestyle modification, cystoscopic hydrodistention, intravesical hyaluronic acid instillation, or painkiller medication, but the IC symptoms were persistent or had relapsed.

All patients received a thorough investigation at enrollment and were not included if they failed to meet the inclusion criteria of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) [31]. The treatment outcomes were assessed by the global response assessment (GRA) at 6 months after an intravesical BoNT-A injection. In addition, the IC symptoms were assessed by the O'Leary–Sant symptom score (OSS), including the IC symptom index (ICSI) and IC problem index (ICPI) [32]. The bladder pain was also reported by a self-assessed 10-point visual analog scale (VAS).

This study had been approved by the Research Ethics Committee of Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation (IRB 105-25-B, date of approval: 2 June 2018). The written informed consent was waived due to the nature of retrospective analysis.

A videourodynamic study (VUDS) was performed before the BoNT-A injection, using standard procedures. The VUDS involved the infusion of normal saline at a rate of 20 mL/min. All descriptions and terminologies used in this study were according to the recommendations of the International Continence Society [33]. The reported urodynamic parameters included: FSF, urge sensation, cystometric bladder capacity, pressure, maximum flow rate (Qmax) and detrusor pressure at Qmax during voiding, and post-void residual (PVR) volume. According to the characteristic VUDS findings, the urodynamic diagnosis was recorded as increased bladder sensation, detrusor overactivity, dysfunctional voiding, poor pelvic floor muscle relaxation, or intrinsic sphincter deficiency [34]. The potassium chloride (KCl) test (infusion of 0.4 M KCl in normal saline) was performed after evacuation of the PVR urine. The KCl test was considered positive if there was bladder pain or a severe urge to void during the KCl infusion [35]. The VUDS was performed at baseline to confirm the diagnosis of IC/BPS and search for other bladder conditions mimicking IC/BPS. A repeat VUDS was performed at 6 months after the first BoNT-A injection to evaluate the bladder condition after treatment and as a guide for the selection of the next step treatment.

The injected BoNT-A solution constituted a vial of onabotulinumtoxinA (100 U) diluted with 10 mL of normal saline. Each 1.0 mL solution contained 10 U of BoNT-A in saline. A total of 20 injections were performed with this BoNT-A solution, resulting in 5 U of BoNT-A in each injection site. The injection technique was reported in our previous article [18]. In brief, the BoNT-A solution was injected in four rows (each row with 5 injection sites) from the interureteric ridge to the bladder dome to cover the lateral and posterior wall, at a bladder capacity of 100 mL. The trigone was spared, because glomerulations are usually not involved in this region. After the BoNT-A injections, cystoscopic hydrodistention was routinely performed by a slow infusion of normal saline to an 80 cm H2O intravesical pressure for 15 min. The maximal bladder capacity (MBC) at the end of the infusion and the degree of glomerulations developed after bladder drainage were also recorded [18]. The glomerulation grade was classified as 0, 1, 2, and 3 based on the appearance of glomerulations at none, less than half, more than half, and more than half of the bladder wall and severe waterfall bleeding, respectively. Patients were classified as ulcer type IC/BPS if a Hunner's lesion was present with and without glomerulations [4]. After BoNT-A treatment, patients were indwelled with a 14-Fr Foley catheter overnight and discharged the next day. An antibiotic (cephalexin 500 mg every 6 h) was routinely prescribed for seven days and patients visited the outpatient clinic at 2, 4, and 8 weeks after treatment, followed by monthly visits in the outpatient clinic for outcome assessment. The primary end-point was 6 months after the BoNT-A injection.

We classified patients into five subgroups according to different clinical, urodynamic, and cystoscopic characteristics (MBC and glomerulations) and evaluated the treatment outcome (GRA ≥ 2 or GRA ≤ 1), symptom change (with and without bladder pain), urodynamic findings, and comorbidity coexistence. Predictive factors for a satisfactory outcome were also assessed.

The continuous variables reported in this study are expressed as means ± standard deviations (SDs). The Wilcoxon rank-sum test was used for statistical comparisons of continuous variables between groups, and the Wilcoxon sign-rank test was used to evaluate the difference of the variables between baseline and post-treatment. All statistical assessments were performed by two-sided analysis, and significant differences were considered at a *p*-value < 0.05. SPSS version 15.0 statistical software (SPSS Inc., Chicago, IL, USA) was used in all statistical analyses.

**Author Contributions:** Conceptualization, H.-C.K.; methodology, H.-J.W., W.-R.Y. and H.-L.O.; formal analysis, H.-L.O., H.-J.W. and W.-R.Y.; writing manuscript-draft, H.-J.W. and W.-R.Y.; writing—review and editing, H.-C.K.; supervision, H.-C.K.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.
