**4. Conclusions**

In this study, only 40% of IC/BPS patients had symptomatic improvement after intravesical BoNT-A injections. Patients with less inflammatory bladder conditions characterized by a larger bladder capacity, lower symptom severity, and lower urinary inflammatory and oxidative stress biomarker levels may predict satisfactory outcomes. Patients with severe bladder inflammation might require more intravesical BoNT-A injections to achieve a satisfactory outcome.

#### **5. Materials and Methods**

This study involved IC/BPS patients treated with 100 U BoNT-A from February 2000 to December 2021. All patients were diagnosed with IC/BPS in accordance with established IC/BPS characteristic symptoms and cystoscopic findings of glomerulations, petechiae, or mucosal fissures on anesthesia cystoscope hydrodistention [23]. Among the IC/BPS patients, lifestyle and behavioral modification, cystoscopic hydrodistention, intravesical hyaluronic acid instillation, or painkiller medications and treatment modalities were tried in at least two treatment modalities, but the IC symptoms persisted or relapsed. During the study period, some patients were undergoing BoNT-A injection clinical trials. However, we only recorded the outcomes of their first treatments.

All patients were screened thoroughly at the time of enrollment and were not enrolled if they failed the inclusion criteria of the European Society for the Study of Interstitial Cystitis [24]. This is a retrospective analysis of previous clinical trials of BoNT-A injections for patients with IC/BPS. In these clinical trials, the patient inclusion and exclusion criteria were the same (Appendix A).

The treatment outcomes at 6 months after the intravesical BoNT-A injection were evaluated using the GRA scale. Additionally, the IC symptoms were assessed using OSS, including ICSI and ICPI [25]. The ICSI and ICPI are two instruments used to determine the overall level of severity of each symptom and the significance of the problem from the patient's perspective, respectively [26,27]. Both indices included four questions, one each for nocturia, frequency, urgency, and bladder-associated pain. The total ICSI scores range from 0 to 20. Each of the questions in the ICPI has five response options ranging from 0 to 4 with a maximum total ICPI score of 16, with higher scores indicating more severe IC/BPS symptoms and problem severity [26]. Patients were requested to rate their bladder symptoms as compared to that at baseline using a 7-point centered scale, from markedly ( −3), moderately ( −2), and slightly worse ( −1), no change (0), to slightly (+1), moderately (+2), and markedly improved (+3). Patients with moderately and markedly improved results after treatment were considered to have satisfactory treatment outcomes. Otherwise, the treatment outcome was considered unsatisfactory [19].

VUDS was performed before the BoNT-A injection using the multichannel urodynamic system (Life-Tech, Stafford, TX, USA) and a C-arm fluoroscope (Toshiba, Tokyo, Japan). According to International Continence Society recommendations, this study's descriptions and terminologies all follow the compliance criteria [28]. Based on the characteristic VUDS findings, such as the first sensation of filling, first desire to void full bladder sensation, cystometric bladder capacity, detrusor pressure at maximum flow rate, maximum flow rate, voided-volume, and post-void residual (PVR), patients would be diagnosed with hypersensitive bladder, detrusor overactivity, voiding dysfunction, poor pelvic floor muscle relaxation, or intrinsic sphincter deficiency [29]. The KCl test was considered positive if there was bladder pain or an intense urge to void during the KCl infusion after the emptying of the residual urine [30]. was Patients with increased bladder sensation and positive KCl sensitivity tests were encouraged to undergo Cystoscopic hydrodistention. The VUDS was performed to verify the diagnosis of IC/BPS at baseline and recognize other bladder conditions that resemble IC/BPS. A duplicate VUDS was performed 6 months after the primary BoNT-A injection to estimate the bladder condition after treatment and as an action for instigating subsequent treatment.

After cystoscopic hydrodistention, the patients were treated with consecutive bladdertargeting medications for bladder pain, including nonsteroidal-inflammatory drugs, cyclooxygenase-2 inhibitors, antimuscarinics, alpha-blockers, intravesical hyaluronic acid installations, and 4th line of intravesical BoNT-A injections, according to AUA guideline recommendations [20].

BoNT-A medicinal liquid constituted a vial of onabotulinumtoxin A (100 U) diluted with 10 mL 0.9% saline. Twenty injections were performed with this BoNT-A liquid, lead 5-U BoNT-A in each injection site. For the bladder's posterior and lateral walls, an injection needle was inserted approximately 1 mm in the urothelium, sparing the trigone, using a 23-gauge needle and rigid cystoscopic injection instrument (22 Fr, Richard Wolf, and Knittlingen, Germany). After the BoNT-A injections, cystoscopic hydrodistention was performed under slowly dripping 0.9% saline to an intravesical pressure of 80 cm fluid for 15 min. The MBC and glomerulation grade under hydrodistention was also recorded after intravesical pressure release [9]. Based on the appearance of glomerulations for none, less than half, more than half, and more than half and during serious waterfall bleeding of the bladder wall, if patients have Hunner's lesion combined with or without glomerulations were classified as ulcer-type IC/BPS. After that, the glomerulation grade was classified into 0, 1, 2, and 3 [11]. After the BoNT-A treatment, a 14-Fr indwelling urinary catheter was inserted overnight and removed the next day. An antibiotic (cephradine 500 mg every 6 h) was routinely prescribed for a week, and patients visited the outpatient clinic 2 weeks after treatment, followed by monthly visits to the outpatient clinic for outcome assessment. The primary endpoint was 6 months after the BoNT-A injection.

We not only analyzed the patients' subjective and objective characteristics and VUDS parameters but also collected urine specimens to further analyze the urinary biomarkers at baseline. The urinary biomarkers collected included interleukin-8 (IL-8), CXCL10, MCP-1, BDNF, eotaxin, Interleukin 6 (IL-6), RANTES (also known as CCL5), prostaglandin E2, tumor necrosis factor-alpha, 8-hydroxy-2-deoxyguanosine, 8-isoprostane, and total antioxidant capacity [13]. In brief, before cystoscopic hydrodistention, all patients would collect 50 mL urine samples, obtained by self-urination, when patients had a full bladder sensation, and also excluded those with confirmed urinary tract infections. Before transferring to the laboratory, the urine samples were placed on ice. However, HICs would be excluded from further analysis considering the different pathology [13,31].
