Comparison of vascularization and overall perfusion of the bladder wall between women with and without overactive bladder syndrome

The pathophysiology of female overactive bleeder syndrome (OAB) remains undetermined. Our aim is to elucidate the role of vacularization and overall perfusion of the bladder wall in women with OAB. Between 2010 and 2016, women with OAB and the asymptomatic controls were enrolled. Women with OAB were treated with tolterodine. Women with OAB (n = 40) had higher vascularization index (0.40 ± 0.57 versus 0.17 ± 0.22, p = 0.003), vascularization-flow index (0.15 ± 0.28 versus 0.05 ± 0.08, p = 0.003) and thicker trigone (0.56 ± 0.13 cm versus 0.47 ± 0.11 cm, p = 0.004), compared with the controls (n = 34). The following optimum cut-off values to predict OAB were determined: (1) vascularization index (%) ≥ 0.16, (2) vascularization-flow index ≥ 0.032, and (3) trigone bladder wall thickness ≥ 0.47 cm with an area under the curve of 0.71, 0.71 and 0.70, respectively. Correlation analysis showed that a significant correlation between urgency and vascularization index/vascularization-flow index (Spearman’s rho = 0.34 and 0.35, respectively, all p < 0.01). However, after 12 weeks of tolterodine treatment, the vascularization index, flow index and vascularization-flow index did not differ between baseline and after treatment. In conclusion, women with OAB have higher vascularization and overall perfusion of the bladder wall, compared women without OAB. However, vascularization and overall perfusion did not change after antimuscarinic treatment.

Thus, the primary objective of this study was to compare vacularization and blood flow perfusion of the bladder wall between the OAB and the asymptomatic controls. In addition, we were interested in elucidating (1) whether our novel method of estimating BWV is a reliable and good method for assessing female OAB and (2) the impact of tolterodine on vacularization and blood flow of the bladder, BWT, BWV and female sexual function.

Materials
Between September 2010 and March 2016, all women with OAB were invited to participate in this prospective study. Only the data for women who completed 12 weeks of tolterodine ER (4 mg per day) treatment were included in the analysis. All women underwent transvaginal three-dimensional power Doppler ultrasonographic examinations to measure three sites (trigone, anterior wall and dome of the bladder) of the BWT, BWV, vascularization index (VI), flow index (FI) and vascularization-flow index (VFI) of the bladder wall; they also answered Patient Perception of Bladder Condition, Urgency Severity Scale, Overactive Bladder Symptoms Scores (OABSS), Urogenital Distress Inventory-6, Incontinence Impact Questionnaire-7 and Female Sexual Function Index (FSFI) questionnaires 21 before and after 12 weeks of treatment. The study was approved by the Research Ethics Review Committee of Far Eastern Memorial Hospital. Informed consent was obtained from all subjects.
The inclusion criteria were as follows: (1) female patients who were at least 18 years old with at least one month history of OAB symptoms, including urinary urgency with or without urgency incontinence and (2) an average of > 8 micturitions in 24 hours. The exclusion criteria included clinically significant dysuria, severe stress urinary incontinence or mixed urinary incontinence with dominant stress incontinence, regular urethral catheterization or intermittent self-catheterization, urinary retention, urinary tract infection in the previous two weeks, bladder calculus, a history of pelvic radiotherapy or past/existing malignant pelvic tumors, uncontrolled narrow-angle glaucoma, intestinal obstruction and other symptoms that are contraindications for antimuscarinic medication 22 .
All ultrasonographic data were acquired using a Voluson 730 (GE Medical Systems, Zipf, Austria) ultrasound machine equipped with a 5-MHz transvaginal transducer. Similar preset power Doppler ultrasound settings were used for all examinations: pulse repetition frequency, 0.9 kHz; gain, 0.8; wall motion filter, low 1; and quality, normal.
In ultrasonography, the outer hyperechogenic layer of the bladder wall represent the adventitia, and the inner hyperechogenic layer of the bladder wall represent the mucosa/submucosal tissue 8 . The detrusor muscle appears hypoechogenic and is located between the hyperechogenic lines of the adventitia and mucosa. Measurement of all three layers represents BWT 8 , and measurement of the detrusor muscle only represents detrusor wall thickness 8 . Because the fluid instilled into the bladder would cause a decrease of the BWT when the bladder volume exceeded 50 mL 23 . Measurements of BWT were performed after emptying the bladder and perpendicular to the bladder mucosa, from the outer layer of the adventitia to the inner layer of the mucosa 8 . The post-void residual urine volume was checked to ensure that it was less than 50 mL 24 . If the post-void residual urine volume was more than 50 mL, repeated emptying was requested to ensure that it was less than 50 mL, if possible. The BWT was measured three times at each location, and the mean value was used for statistical analysis.
For three-dimensional ultrasound, we scanned transvaginally. Once a satisfactory grayscale image (longitudinal view) of the urethra and bladder had been obtained, the urethra was centralized onscreen, and a three-dimensional power Doppler dataset for the bladder and urethra was acquired, ensuring that the complete volume of the bladder area had been captured. The truncated sector defining the region of interest was adjusted, and the sweep angle was set to 85° to ensure that as complete a bladder wall scan as possible was obtained. Volumes of satisfactory quality were stored on the hard disk for later analysis. The VOCAL (Virtual Organ Computer-aided Analysis, Kretztechnik AG) imaging program was used to calculate BWV (i.e., shell volume in Fig. 1a) and bladder wall power Doppler flow indices (Fig. 1b).
The outer bladder volume was derived from an outward extension of inner bladder volume with a rounding integer BWT (i.e., shell thickness [mm] in Fig. 1a). BWV (i.e., shell volume in Fig. 1a) was derived by subtracting the inner bladder volume from the outer bladder volume 17 . The VI, measuring the ratio of the number of color voxels to the number of all voxels, is thought to represent the blood vessels density and was expressed as a percentage (%) of the bladder wall volume 24 . The FI represents the intensity of flow at the time of the three-dimensional sweep over the bladder wall and is thought to express the average flow intensity 25 . The VFI is a combination of vascularization and blood flow information; it thus represents overall perfusion 25 .
Multichannel urodynamic equipment (Life-Tech, Houston, TX, USA) with computer analysis and Urovision (Urolab Janus System V, Houston, TX, USA) was used. All terminology conformed to the standards recommended by the International Continence Society and Urodynamic Society 1 . The data was interpreted by a single observer to avoid interobserver variability.
A voiding detrusor pressure at maximum flow rate (PdetQmax) of more than 35 cmH 2 O was considered a high PdetQmax, while 10-35 cmH 2 O was considered a normal PdetQmax, and 10 cmH 2 O or less was considered a low PdetQmax 26 . In this study, patients with the volume at strong desire to void >300 mL, a normal PdetQmax or a low PdetQmax but with a maximum flow rate (Qmax) >12 mL/s, and a post-void residual less than 150 mL were considered urodynamically normal 26 . Additionally, women with a high PdetQmax (>35 cmH 2 O) and a low Qmax (<12 mL/s) were suggested to have bladder outlet obstruction in this study 26 .
The FSFI includes six domains: desire, arousal, lubrication, orgasm, satisfaction and pain 21 . Higher FSFI scores represented greater positive effects on sexual function. Women without sexual intercourse in recent one month did not be requested to complete the FSFI questionnaires 21 . Besides, we used the criteria of FSFI total score of> 26.55 as a diagnosis of sexual dysfunction 27 .
All methods in this study were carried out in accordance with relevant guidelines and regulation. STATA software (Version 11.0; Stata Corp, College Station, TX, USA) was used for statistical analyses. The chi-square test and Wilcoxon rank-sum test were used as appropriate. A p value of less than 0.05 was considered statistically significant.
Since one of the objectives of this study was to estimate the difference of bladder wall VI between the asymptomatic controls and the OAB women, a priori study for the first 15 women in each group showed a mean VI of 0.17 ± 0.20 and 0.64 ± 0.79, respectively. To detect a difference in the VI based on the information above, we conducted a test with a significance level of 0.05 and a power of 0.9 and anticipated that groups of equal size would be required. Thus, we concluded that at least 32 subjects in each group were required to test the above hypothesis.

Results
A total of 40 women completed 12 weeks of tolterodine treatment (Fig. 1c). The above 40 women with OAB and another 34 women without OAB were analyzed in this study. Baseline characteristics are tabulated in Table 1.
The following optimum cut-off values to predict OAB were determined using ROC analysis: (1)   Correlation analysis showed that the strongest correlation with VI, FI, VFI, trigone BWT or average BWT was urgency (i.e., the score of the third question of OABSS, Spearman's rho = 0.31 to 0.39, p < 0.01, Table 2). Nonetheless, BWV did not correlated with urgency (rho = 0.05, p = 0.69, Table 2). Besides, trigone and average BWTs did not correlate with VI, FI or VFI (Table 2).
Correlation analyses between the bladder wall blood perfusion/thickness with the baseline urodynamic parameters in women of OAB were shown in Table 3. Pressure transmission ratio was found to be negatively correlated with VI (rho = −0.35) and VFI (rho = −0.34), and positively correlated with average BWT (rho = 0.44) and BWV (rho = 0.46, Table 3). The other correlation analyses did not show any statistical significance.
After 12 weeks of treatment, the scores on the Patient Perception of Bladder Condition, OABSS, Urogenital Distress Inventory-6 and Incontinence Impact Questionnaire-7 questionnaires were improved; however, the score of FSFI questionnaire, VI, FI, and VFI, all sites of BWT and BWV did not differ before and after treatment (Table 4). Similarly, among the subgroup of OAB women with detrusor overactivity (n = 12, Table 4), the scores on the Patient Perception of Bladder Condition, OABSS and Urogenital Distress Inventory-6 questionnaires were improved; however, the score of FSFI questionnaire, VI, FI, and VFI, all sites of BWT and BWV did not differ before and after treatment (Table 4).
Twenty women in the OAB group and 9 women in the control group had baseline FSFI data. Using the criteria of FSFI total score of > 26.55 as a diagnosis of sexual dysfunction 26 , 7 of 20 (35%) women in the OAB group and 3 of 9 (33%) women in the control group were found to have sexual dysfunction (p = 1.00). After tolterodine treatment, two women became having no sexual dysfunction, two women became having sexual dysfunction, and 5 women had persistent sexual dysfunction (McNemar's test, p = 1.00).

Discussion
In this study, we found that women with OAB had higher VI (Fig. 2a) and VFI (Fig. 2b), compared with the asymptomatic control. In addition, VI and VFI were positively correlated with urgency (Spearman's rho = 0.34 and 0.35, p = 0.004 and 0.003, respectively, Table 2). Urgency is the core symptoms of female OAB 28 . That is, woman with OAB has a higher vascularization and overall blood perfusion of the bladder than woman without OAB. It was reported that C reactive protein was elevated in women with OAB-wet 29 , and hinted that inflammation might play a role in the etiology of OAB. Besides, stimulated macrophages and keratinocytes can produce high levels of proangiogenic factors in the inflammatory response; thus, the level of angiogenesis often correlates with inflammation 30 . The above might partly explain the positive correlation between OAB and higher vascularization of the bladder in our study. However, contrarily to our study, chronic bladder ischemia was reported to be associated with OAB 2,3,5-7 . Kojima et al. used ultrasound-estimated bladder weight as a measure of bladder hypertrophy 17 . In this study, the novel method to estimate BWV had a good intraclass correlation of 0.878 (95% CI = 0.832 to 0.924). In addition, the intraclass correlation of BWV is better than that of BWT (intraclass correlation = 0.613 to 0.775). Thus, the finding might mean that our novel method to estimate BWV is a good method, and could be used in the studies of bladder wall thickness or bladder wall weight.
In this study, we found that trigone BWT and average BWT were positively correlated with baseline urgency (Spearman's rho = 0.39 and 0.37, p = 0.001 and 0.002, respectively, Table 2), but not anterior wall BWT, dome BWT and BWV. It has been reported that BWT is associated with OAB 9,11 , detrusor overactivity 8,13,15 , detrusor pressure and lower cystometric capacity 13 . In our study, our data also demonstrated that trigone and average BWTs is positively correlated with urgency severity ( Table 2). Yilmaz et al. also reported a good correlation between BWT and OABSS (r = 0.48, p = 0.002) 12 .
It has been reported that BWT decreases after antimuscarinic treatment 8,11,14 . Nonetheless, in our study, the changes in BWT and BWV did not differ between baseline and after treatment (Table 4). Similarly, Robinson et al. reported that there was no significant reduction in BWT between the solifenacin and the placebo groups 10 .
Pressure transmission ratio at maximum urethral pressure was found to be negatively correlated with VI and VFI in the OAB group (Table 3). We did not find any similar report. Low pressure transmission ratio was associated with urethral hypermobility 31 . Thus, our result might hint that OAB women with urethral hypermobility have high vasularization and overall perfusion of the bladder wall.
Tolterodine was reported to be associated with a decrease in arterial stiffness 32 . However, in our study, tolterodine failed to demonstrate any effect on bladder wall vascularization and blood perfusion owing to the lack of a difference between baseline and post-treatment in VI, FI and VFI (Table 4). Currently main medications for OAB patients included those with inhibition of detrusor contraction (such as antimuscarinics) or activation of detrusor www.nature.com/scientificreports www.nature.com/scientificreports/ relaxation (such as beta 3 agonist). After tolterodine treatment, significant improvements of OAB symptoms were found in our patients, but not overall perfusion and BWT (Table 4); the above discrepancy might hint that tolterodine do not treat underlying etiology of OAB. Thus, it is not a surprise that recurrence of OAB is not uncommon after discontinuation of antimuscarinics 22 .
Ultrasound for monitoring OAB treatment was reported to be not useful 5 . Rachaneni et al. reported that the levels of measurement error are high for a small measurement of BWT, and transvaginal ultrasound measurements have insufficient reliability and reproducibility to be an accurate diagnostic test 33 . Latthe et al. reported that BWT ≥ 5 mm is not a good criterion for detecting detrusor overactivity owing to its low sensitivity (43%) and specificity (62%) 15 . Rachaneni et al. also reported that BWT has no relationship with detrusor overactivity 16 . Together with our finding, although BWT is positively correlated with OAB severity, BWT is not a useful measure for assessing OAB severity and therapeutic efficacy.    Table 4. Comparison of baseline data and changes from baseline after 12 week of tolterodine treatment for women with overactive bladder syndrome (n = 40). Values are expressed as the mean ± standard deviation. The abbreviations are the same as in Tables 1 and 3. † By Wilcoxon sign-rank test.
OAB women frequently have sexual dysfunction 18 . In our study, FSFI total scores did not differ between the OAB and the control group (Table 1). Similarly, Ergenoglu et al. reported that OAB did not significantly affect female sexual function score 19 .
In our study, female sexual function did not improve after tolterodine treatment (Table 4). Similarly, in an Iranian study, tolterodine did not improve female sexual function 34 . However, tolterodine was reported to have a positive effect on female sexual function in some studies 20,35,36 . One study of the United States reported that tolterodine ER was associated with an improvement of the Pelvic Organ Prolapse/Urinary Incontinence Sexual Questionnaire total score, compared with placebo 35 . Similarly, one Iranian study reported that Arizona Sexual Experience Scale total score was improved after 3 months' tolterodine treatment 36 . Recently, a Greek study reported that FSFI total scores were improved after 3 months' tolterodine ER treatment 20 ; however, a lower mean age (43 ± 8.4 years vs. 52.5 ± 10.8 years) and a lower baseline FSFI total score (17.4 ± 1.2 vs. 23.7 ± 4.9) were noted in the Greek study 20 , compared with our current study.
Prevalence of sexual dysfunction varies with race [37][38][39][40] . For example, Chinese middle-aged women reported more pain during intercourse and less sexual desire than the white middle-aged women 39 . Besides, Asian middle-aged and older women tended to report less frequent sexual activity 40 . Thus, the discrepancy about the impact of tolterodione on female sexual function between our and the above studies might be at least partly related to age and racial differences.
Limitations of this study include the limited sample size, a high drop-out rate and the lack of long-term data.
conclusions Women with OAB have higher vascularization and overall perfusion of the bladder wall, and thicker bladder wall thickness, compared women without OAB. However, vascularization and overall perfusion of the bladder wall and bladder wall thickness seems not change after 12 weeks' tolterodine treatment. Further studies might be performed to elucidate the long-term effect of antimuscarinics.

Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.