**4. Conclusions**

The results of this comparative case series study revealed that intravesical injections of either PRP or BoNT-A were safe and effective for improving IC/BPS symptoms without significantly increasing the PVR, but the bladder pain reduction was limited. Only half of the study cohort had a GRA ≥2 at six months. Repeat treatment of PRP or BoNT-A would be necessary to achieve long-term success. The BoNT-A injection carries a potential risk of UTI after treatment compared with that of the PRP injection.

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

A total of 56 female patients with confirmed IC/BPS who had failed conventional treatments received either a single intravesical BoNT-A 100 U injection (*n* = 26) or four monthly PRP injections (*n* = 30) within the two previous years. The IC/BPS was diagnosed according to the characteristic symptoms and cystoscopic findings after hydrodistention under anesthesia [32]. All patients had previously received at least two types of treatment modalities, including oral medication and intravesical treatment in recent years, with persistent bothersome symptoms and bladder pain. All potential patients received detailed urological examinations and were excluded if the diagnosis failed to meet the criteria of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) [33]. The patients were not randomly allocated to the BoNT-A or PRP group but were treated based on their choice. This study was approved by the Research Committee of Hualien Tzu Chi Hospital (IRB: 111-257-B, dated 15 December 2022). The requirement for informed consent was waived because this study was a retrospective analysis of data.

Patients' preferred treatment was scheduled after a discussion regarding the advantages and potential adverse effects of each treatment type. They were treated with either (1) an intravesical injection of 10 mL PRP (which was extracted from 50 mL of patient's blood) at 20 sites every month for four months, or (2) one intravesical dose of BoNT-A 100 U at 20 injection sites. Both treatments were followed by cystoscopic hydrodistention in the operation room. The primary endpoint was the global response assessment (GRA) recorded at six months after the first treatment day. The GRA scale included 6 items: worsening of symptoms (less than 0%), no change (0%), mild improvement (less than 50%), moderate improvement (50–75%), marked improvement (more than 75%), and completely cured (100%) [34].

All patients were asked to maintain a three-day voiding diary at baseline for recording the functional bladder capacity (FBC), urinary frequency, and nocturia. The IC symptoms were assessed by the O'Leary-Sant symptom score (OSS), including IC symptom index (ICSI) and IC problem index (ICPI) [35]. The bladder painwas scored by a self-assessed 10-point visual analog scale (VAS) system [36]. A videourodynamic study (VUDS) and a potassium chloride sensitivity test were routinely performed to exclude patients with detrusor overactivity or bladder outlet obstruction. All patients were informed of potential complications related to anesthesia and bladder injections, such as gross hematuria, micturition pain, and increased bladder pain after PRP or BoNT-A injection. Patients were also informed that the BoNT-A injection could lead to a greater reduction in bladder pain, and both BoNT-A and PRP injection could reduce the frequency episodes after treatment. However, BoNT-A injection might have adverse events of difficulty in urination, increased PVR, and UTI after injection, which had not been observed in our previous clinical trials of PRP injection.

The VUDS was performed at baseline and time-points after intravesical injections. The VUDS was performed with an infusion rate of 20 mL/min, and the procedures and terminology were according to the International Continence Society recommendations [37]. After the VUDS study, a potassium chloride (KCl) test using 0.4 M KCl solution infused into the bladder was performed. A positive KCl test was considered when a patient perceived

a painful (VAS score of ≥2) or urgency sensation (urgency severity score increased by ≥1) [38].

PRP was prepared according to previously reported standard procedures [17]. In brief, 50 mL of whole blood was withdrawn at the same day of treatment. The blood was sent to the central laboratory and the blood was centrifuged twice with a slow spin (190× *g*, 20 min, <20 ◦C) and fast spin (2000× *g*, 20 min, <20 ◦C) of the supernatant plasma containing platelets. The lower third of the tube comprised PRP, and the upper part was platelet poor plasma (PPP). The platelet pellets were added to the plasma to obtain the desired concentration of PRP. In this study, 6 mL of sterile PRP was obtained. We sent 1 mL of PRP for culture and platelet count. The remaining 5 mL of PRP was used for intravesical injection. A total of 20 suburothelial injections of the PRP solution were performed, with 0.25 mL of PRP at each site, using a 23-gauge needle and a rigid injection instrument (22 Fr, Richard Wolf, and Knittlingen, Germany). The injection was approximately 1 mm in depth into the suburothelium equally distributed at posterior and lateral bladder walls, Cystoscopic hydrodistention was performed immediately after PRP injection to activate the injected platelets and determine the maximal bladder capacity (MBC). The PRP injection procedure was repeated every month for four months. A total of 10 mL of sterile normal saline was used to dissolve the BoNT-A powder to a concentration of 5 U at each site. The BoNT-A solution was gently shaken and then slowly withdrawn and injected at 20 well-distributed sites at posterior and lateral bladder wall followed by cystoscopic hydrodistention as previously reported [17]. After the PRP or BoNT-A injections, a urethral catheter was indwelled overnight, and oral antibiotics were taken for three days.All patients were followed up at one, three, and six months after the first injection day. Urinalysis was routinely checked at each time point and UTI was considered if patients had bladder pain or micturition pain and a white blood cell count >10/high power field in urinalysis.

The three-day voiding diary data (including FBC, daily frequency and nocturia episodes), ICSI, ICPI symptom score, and bladder pain VAS were recorded at baseline (first PRP and BoNT-A injection), and at one, three (fourth PRP injection), and six (three months after fourth PRP, and six months after BoNT-A injection) months. At the one-, three-, and six-month (primary endpoint) follow-up, patients reported any improvement in IC symptoms and adverse events were recorded. An excellent treatment outcome was considered when patients reported a GRA of ≥2 or no bladder pain (VAS = 0). The outcome was considered improved if there was improvement in the GRA by = 1 or the pain VAS score reduced by two or more and there was at least a 25% decrease in urinary frequency and nocturia. Patients with excellent and improved results were considered as having a successful treatment. After the primary endpoint assessment, the patients were continuously followed up with medications, such as anti-inflammatory agents and pain killers only. If the patient's IC symptoms exacerbated and they requested bladder therapy, the duration from the first injection day to the consecutive bladder therapy was recorded as the effective duration.

The data of voiding diary, VUDS parameters, symptom score, and bladder pain VAS score at baseline, one, three, and six months after the first injection day were compared in each group. A successful result was assessed by a self-reported improvement in GRA and pain VAS score. The data were presented as the mean ± standard deviation. Comparisons between PRP and BoNT-A groups were analyzed by the Student's t-test to compare numerical data, and the Chi-square test for categorical data. Cumulative success rate was also calculated using Kaplan–Meier survival curves to compare the success rates from the initial treatment day to receiving additional treatment between groups. All statistical analyses were performed using the statistical package SPSS for Windows (Version 12, SPSS, Chicago, IL, USA). A *p*-value of less than 0.05 was considered statistically significant.

**Author Contributions:** Concept: H.-C.K.; investigation: J.-F.J. and W.-R.Y.; design: H.-C.K.; draft writing: J.-F.J.; manuscript writing and supervision: H.-C.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Buddhist Tzu Chi Medical Foundation gran<sup>t</sup> TCMF-SP-108-01, and gran<sup>t</sup> TCMF-MP-110-03-01.

**Institutional Review Board Statement:** This study was approved by the Research Committee of Hualien Tzu Chi Hospital (IRB: 111-257-B, dated 15 December 2022).

**Informed Consent Statement:** The informed consent form was waived because of the nature of retrospective analysis.

**Data Availability Statement:** Data can be obtained with permission of the corresponding author.

**Conflicts of Interest:** The author declares no conflict of interest.
