The surgical effect on overactive bladder symptoms in women with pelvic organ prolapse

This study aimed to explore the effect of pelvic reconstruction surgery on the relation of pelvic organ prolapse (POP) and overactive bladder (OAB) and the impact of preoperative vaginal oestrogen supplement on vaginal tissue. A total of 100 postmenopausal women with symptomatic POP who underwent pelvic reconstruction surgery (laparoscopic sacrocolpopexy or transvaginal mesh) were enrolled in this study. Preoperative vaginal oestrogen was prescribed in 28 cases. The evaluation tools consisted of POP-Q, urodynamic study, Overactive Bladder Symptom Score (OABSS), and urinary NGF. Vaginal maturation index and vaginal specimens for hormone receptors study were investigated during operation to evaluate the effect of topical oestrogen. Follow-up assessments were performed at 1, 3, and 6 months after surgery. Preoperatively, 58 (58%) were POP with OAB. After reconstruction surgery, the OABSS decreased significantly (6.87 ± 0.85 vs 3.77 ± 0.61, p < 0.001) at postoperative 6 months in the group. Remarkable increasing trends of urinary NGF levels are noted till 3 months postoperatively, then decreasing to the baseline level at 6 months postoperative follow-up. Remarkable decrease of mRNA of the androgen receptor and significant higher expression of progesterone receptor (PR) were noted after use of the vaginal oestrogen cream. The severity of OAB in the POP women shows moderate degree according to OABSS. Pelvic reconstruction surgery can significantly improve the OAB symptoms. The surgery induced inflammation effect lasts for about 6 months. Short-term preoperative supplement of topical oestrogen brings alterations of the vaginal epithelium.


Scientific Reports
| (2021) 11:20193 | https://doi.org/10.1038/s41598-021-99537-w www.nature.com/scientificreports/ urinary NGF/Cr levels were observed compared with patients without BOO; in addition, these levels decreased to normal after successful relief of OAB symptoms 13 . However, there is scant data related to female BOO. POP is frequently occurred in the postmenopausal female population. Vaginal tissue is sensitive to steroid hormones, and the lack of sexual hormones in postmenopausal women leads to insufficient blood circulation and insufficient lubrication of the vagina. Atrophic vaginal tissue appears thin, vulnerable, and less resistant to operative dissection in prolapse surgery. Treating postmenopausal women with local oestrogen before prolapse surgery results in better tissue dissection during the operation. Previous studies have shown that oestrogen therapy may effectively alleviate symptoms of an OAB 14,15 . Vaginal oestrogen cream appears to have some impact on the expression of vaginal hormone receptors, but study results have varied 16,17 .
The study aimed to explore the effect of pelvic reconstruction surgery on the relationship between pelvic organ prolapse and overactive bladder. The secondary aims were investigating the change of urine nerve growth factor level after pelvic reconstruction surgery and the impact of preoperative vaginal oestrogen supplement on vaginal tissue.

Methods
This was a prospective study approved by Chang Gung Memorial Hospital Research Ethics Committee (IRB#201601486A3). The study was in accordance with the Helsinki Declaration of 1975, which was revised in 1983. To ensure patients' confidentiality, only data with the encoded identification numbers were released. Postmenopausal women with symptomatic POP who underwent pelvic reconstruction surgery from September 2017 to December 2019 were enrolled in this study. Menopause is defined as no menstruation for more than 12 months. When the patients with POP visited our out-patient department, we discussed with them about the project, pros and cons, and asked them if they are willing to join the research. Thereafter, written informed consent was obtained before survey initiation. Patients were excluded if they had any neurological disease, symptomatic urinary tract infection, un-investigated haematuria, bladder cancer, interstitial cystitis, had previous pelvic reconstruction or anti-incontinence surgery or would receive concomitant anti-incontinence surgery during reconstruction surgery, or had any condition that was a contraindication for oestrogen treatment. The contraindications of using estrogen included undiagnosed abnormal genital bleeding, known or suspected breast cancer, known or suspected estrogen-dependent neoplasia and active or history of thromboembolic disease. POP severity was graded via the Pelvic Organ Prolapse Quantification system (POP-Q) 18,19 . Patients were classified into two groups according to the presence or absence of OAB symptoms, which were defined according to the patient's complaint of urgency and/or urge incontinence as the core symptom with total OABSS score as 3 or more. Preoperatively, patients willing to receive hormone therapy were prescribed Premarin vaginal cream 1 g (0.625 mg conjugated equine oestrogens per gram of cream) for twice per week. Reconstruction surgery would be performed after the patient used Premarin vaginal cream at least two weeks.
The preoperative assessment included baseline characteristics, POP severity, urodynamic study, overactive bladder symptom score (OABSS) and urinary NGF. OABSS is a validated questionnaire including four questions (daytime frequency, night time frequency, urgency and urge incontinence) to assess the severity of overactive bladder symptoms [20][21][22][23] . Pap smears for VMI was collected from the lateral vaginal wall after anesthesia. When performing reconstruction surgery, excessive vaginal wall would be trimmed during colporrhaphy and 0.5 × 0.5 cm in size at least of this specimen would be sent for evaluating hormone receptor expression. Postoperatively, follow-up assessments comprising the POP-Q system, OABSS, and urinary NGF were performed at 1, 3, and 6 months.
Urine sample collection and urinary NGF measurement. A 30 ml urine sample was obtained at the outpatient clinic interview. Urine samples were immediately placed on ice and transferred to the laboratory. Freshly voided urine was centrifuged at 4 °C and at 4000 rpm for 10 min. The supernatant was separated into aliquots and frozen at -80 °C. One of the aliquots was used to measure the urinary creatinine level.
Urinary NGF measurements were performed with an enzyme-linked immunosorbent assay (Abcam ab193760, Cambridge, UK). All reagents, samples, and standards were prepared as instructed. Exactly 50 µl of each standard or sample, and 50 µl of the antibody cocktail, was transferred into each well, and then incubated for 2.5 h at room temperature with shaking. After the plate was washed, 100 μl of a tetramethylbenzidine (TMB) substrate solution was added to the wells for 10 min at room temperature. Stop Solution was added to terminate the reactions, and the optical density was measured with a Wallac Victor 1420 multilabel HTS counter (PerkinElmer Inc., MA, USA) at 450 nm. All of the samples were run in triplicate, and the values were averaged. The total urinary NGF level was further normalized by the concentration of the urinary creatinine (Cr) (NGF/Cr level).

Results
A total of 113 patients were enrolled in this study initially. Figure 1 shows the flowchart of this study selection process. Finally, 100 patients divided into two groups as POP without OAB (n = 42) and POP with OAB (n = 58) were evaluated in this study. Patients' demographic and characteristics are listed in  Table 2). Figure 2 presents the bar graph of the changes of OABSS at baseline and 1 month, 3 months and 6 months postoperative follow-up. Urgency, urgency urinary incontinence (UUI), and OABSS total were significantly improved in POP with OAB patients at 3 and 6 months, postoperatively. Table 2 provides the values of sequential urinary NGF at baseline and postoperative follow-up in total patients, POP stage II-III, and POP stage IV. There are no significant differences in the sequential levels of urine NGF A total of 28 patients used topical vaginal Premarin cream 1 g twice per week for at least 2 weeks before surgery. While seven (7/42, 16.7%) patients were in the group of POP without OAB, 21 (21/58, 36.2%) patients were in the group of POP with OAB. Figure 3 illustrates the relative expression of hormone receptors mRNA in vaginal wall biopsies of patients. The AR mRNA remarkably decreased after vaginal oestrogen cream supplement, whereas ER and PR retained similar expression (Fig. 3). The immunohistochemistry results are shown in the Fig. 4. From the slides of IHC staining (Fig. 4a), thicker epithelium of vaginal biopsies and significantly higher PR expression in the basal epithelium and the stroma are observed in patients who received vaginal oestrogen cream (Fig. 4b).   www.nature.com/scientificreports/ Vaginal oestrogen cream use changed the VMI on the parabasal cells, which significantly decreased; superficial cells were elevated without significance (Fig. 5).

Discussion
BOO and OAB are common lower urinary tract symptoms LUTS) in POP women. A previous multicentric crosssectional study of 521 women seeking care for pelvic floor disorders showed that LUTS are not independently associated to the prolapse severity 24 . Surgical correction of POP can relieve these related LUTS. Our study showed that 58% of POP women had OAB with a OABSS total as 6.87 ± 0.35 (moderate severity of OAB, OABSS 6-11). Urgency, UUI, and OABSS total were significantly improved at 3 and 6 months postoperatively. Among them, urgency showed immediately significant improvement at postoperative 1 month follow-up.
A prospective study, which included 34 patients who received trans-vaginal mesh for cystocele, revealed that the OABSS improved, except in the aspect of nocturia 25 . Another prospective study found 53% of POP patients had OAB before reconstruction surgery, and in 77% of them, the OAB symptoms improved or disappeared in all domains of the OABSS by the 3-month follow-up 26 . One retrospective study divided POP patients according to severity (stage 1-2 vs stage 3-4) and found that 12 months after correcting anterior or apical prolapse, urinary frequency and UUI improved without statistical difference between groups 7 . Table 2. Levels of urinary NGF at baseline and postoperative follow-up between stage II-III and stage IV pelvic organ prolapse patients. Item listed as mean ± SEM. Student's t test was used to test for statistical significance between stage II-III and IV group. One-way analysis of variance (ANOVA) was used to test for statistical significance between baseline, 1, 3 and 6 months postoperatively group. ***Statistically significant difference in comparison with baseline (p < 0.001). *Statistically significant difference in comparison with 3 months (p = 0.026). † Statistically significant difference in comparison with baseline(p = 0.033).  www.nature.com/scientificreports/ Nerve growth factor is one of neurotrophins to promote growth and survival of sympathetic fibres and sensory nerves. In the bladder, urinary NGF is produced by urothelium and smooth muscle cells. Urinary NGF levels may be elevated in numerous conditions of pathological bladder, for instance, overactive bladder, neurogenic bladder, interstitial cystitis/painful bladder syndrome, bladder outlet obstruction, etc. A meta-analysis in 2017 reported that urine NGF/Cr cannot be used as a biomarker for OAB at present, because of lack of specificity 27 . From an evidence research of urinary biomarkers in OAB published in 2019, urinary NGF, BDNF, and ATP are increased in many OAB patients. Theses biomarkers can help identify OAB phenotypes and select the ideal patients for specific target therapies directed to neurotrophic and purinergic pathways 28 . The involvement of NGF in afferent pathway plasticity after BOO such as in benign prostatic hypertrophy has been proposed 29 . Liu and Kuo reported www.nature.com/scientificreports/ that urinary NGF levels increased in male patients with BOO with OAB and reduced after successful medical treatment 13 . The etiology of BOO in women differs from that of men, POP is the most common cause of female BOO. A few articles specifically reporting the relationship between POP and urinary NGF in female patients. Chan et al. ever conducted a small case-control study (n = 10/10) to evaluate urinary NGF levels from women with anatomic BOO resulting from POP and/or previous anti-incontinence surgery. They showed that these levels were significantly higher when compared with age-matched controls. After surgical correction, the urinary NGF/ Cr levels significantly decreased 1 month after intervention 30 . In this study with a larger patient population, we observed that the urinary NGF increased at the 1-and 3-month postoperative follow-ups and decreased to a level similar to baseline at 6-month after surgery. This trend of urinary NGF change after operation is identical regardless of the severity of POP. The increase in urinary NGF might be due to post-operative inflammation triggered by bladder dissection during the reconstruction surgery and the placement of trans-vaginal mesh. As time progressed to 6 months postoperatively, the inflammation subsided and urinary NGF declined. By tracing of medical records, none of the participants were taking steroids or immunomodulatory agent which might influence the study result by altering the inflammation status during the study period.
In our study, ER α and ER β did not significantly change after local oestrogen supplement for 1 month, while AR mRNA decreased, and PR protein increased. Carlo et al. reported that in postmenopausal patients, the levels of ER were similar to those found in premenopausal women, but as regards PR, the majority of the vagina was devoid of PR after menopause 31   www.nature.com/scientificreports/ reported that the women who received 14 days of vaginal estriol, in comparison with the control group, had a significant increase in ER gene expression in the vagina, while enhanced PR gene expression was found in the endometrium 16 . Various animal studies have investigated the modulation of AR expression in physiological conditions and after sex steroid hormone administration in the vagina, whereas human studies are scarce. In this study, we observed the decreasing expression of AR mRNA (Fig. 3) in intervention group while the IHC quantification of AR protein (Fig. 4b) showed only a decreasing trend without significant difference in epithelium and basal epithelium of the vaginal sections. The discrepancy results between mRNA expression and protein level may involve a number of complicated post-transcriptional mechanisms in turning mRNA into proteins that are not sufficiently well defined. The dynamic processes involved in protein synthesis and degradation are another factors to interfere the correlations between mRNA expression and protein level [33][34][35] . Two previous studies have demonstrated a decline in AR associated with age 36,37 . Anne Fuermetz et al. revealed that after completing 6-weeks of nightly vaginal or vulvar oestrogen cream (0.5 mg), ER α was significantly higher in the intervention group in the basal epithelium, stroma, and connective tissue, while ER β was significantly higher in the intervention group in the basal epithelium. The PR score was significantly higher in the intervention group in the superficial epithelium, stroma, and connective tissue. Local vaginal oestrogen therapy leads to an increase in ER α and PR expression of the vaginal wall in postmenopausal women, while ER β expression remains nearly unchanged 17 . As there is little evidence to support the change of hormone receptor expression after oestrogen supplementation, we could not well explain the results we found; more extensive research should be conducted in the future for better interpretation.
Regarding VMI, the predominance of parabasal cells and the absence of superficial cells indicates a low concentration of circulating oestrogens. Previous studies usually gave patients more than 12 weeks of oestrogen supplement (ex. oral synthetic conjugated oestrogens B 0.3 mg per day, transdermal patch releasing 14 μg of E2 per day, or vaginal ring releasing 7.5 μg of E2 per day), and the proportion of parabasal cells significantly decreased 38,39 . In our study, preoperatively applying vaginal Premarin cream 1 g twice per week for 2-4 weeks decreased the proportion of parabasal cells. This demonstrates that short-term low-dose local oestrogen supplement can also improve the VMI.
There are still some limitations in this study. First, all patients were postmenopausal women, there was a lack of a control group for comparison. Second, there were variations in the duration of preoperative supplement of vaginal oestrogen and it was not randomized. Third, missing data of follow-up collection may interfere the power of analysis.
In conclusion, 58% of POP women had OAB which was graded as moderate severity based on OABSS. Urgency, UUI, and OABSS total were significantly improved at 3 and 6 months postoperatively. Surgical correction of anatomical BOO provided an effective relief of OAB in POP women. Urinary NGF could not be utilized as a predictive factor of changes in OAB after reconstruction surgery because the inflammation reaction triggered by the surgery may induce significant increase in urinary NGF at 3 months postoperatively and be back to the baseline 6 months after surgery. Short-term low dose local oestrogen supplement could improve the VMI, decrease the expression of AR mRNA and increase the expression of PR protein in the vaginal tissue.

Data availability
The data used to support the findings of this study are available from the corresponding author upon reasonable request. License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.