A Pre-Post Quasi-Experimental Study to Evaluate the Effects of Vaginal Estrogen on the Urine Microbiome and Overactive Bladder Symptoms in Menopausal Women
Phase: PHASE4
Conditions: Overactive Bladder (OAB), Menopause, Microbiome
Sponsor: Mahidol University

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Clinical Trial Protocol Template for Ramathibodi EC Submission  
Based on SPIRIT 2013 checklist (https://doi.org/10.7326/0003-4819-158-3-201302050-00583)    
(All information can be written in Thai or English. Descriptions in red must be deleted before submission) 
หมายเหตุ: ห้ามตัดหัวข้อออก คงไว้ตามแบบฟอร์ม หากไม่มีข้อมูลที่เกี่ยวข้อง ให้ระบุว่า” ไม่มี” (ตัดเฉพาะคำอธิบายสีแดงออก) และสามารถระบุรายละเอียดเป็นภาษาไทยได้ 
Study Title (English):  
Effect of Vaginal Estrogen on Alterations in the Urine Microbiome of Menopausal Women with Overactive 
Bladder. 
Study Title (Thai):  
ผลของยาเอสโตรเจนชนิดเหน็บช่องคลอดต่อการเปลี่ยนแปลงแบคทีเรียไมโครไบโอมในปัสสาวะของสตรีวัยหมดประจำเดือนที่มี
ภาวะกระเพาะปัสสาวะบีบตัวไวเกิน 
Principal investigator:  
Papapin Pusayapaibul, MD 
Fellow, Female Pelvic Medicine and Reconstructive Surgery Division 
Department of Obstetrics & Gynaecology, Faculty of Medicine Ramathibodi Hospital, Mahidol University 
270 Rama VI Road, Ratchathewi, Bangkok 10400, Thailand 
Telephone: 080-559-8405 
Co-investigator(s):  
Assist. Prof. Rujira Wattanayingcharoenchai, MD, M.Sc. 
Female Pelvic Medicine and Reconstructive Surgery Division 
Department of Obstetrics & Gynaecology, Faculty of Medicine Ramathibodi Hospital, Mahidol University 
270 Rama VI Road, Ratchathewi, Bangkok 10400, Thailand 
Telephone: 02-201-2167 
 
Assist. Prof. Pisut Pongchaikul, MD, PhD 
Chakri Naruebodindra Medical Institute, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bang 
Phli, Samut Prakan 10540, Thailand 
 
REC-MURA.04 

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Prof. Jittima Manonai, MD, MHM 
Female Pelvic Medicine and Reconstructive Surgery Division 
Department of Obstetrics & Gynaecology, Faculty of Medicine Ramathibodi Hospital, Mahidol University 
270 Rama VI Road, Ratchathewi, Bangkok 10400, Thailand 
Telephone: 02-201-2167 
 
Assist. Prof. Komkrit Aimjirakul, MD 
Female Pelvic Medicine and Reconstructive Surgery Division 
Department of Obstetrics & Gynaecology, Faculty of Medicine Ramathibodi Hospital, Mahidol University 
270 Rama VI Road, Ratchathewi, Bangkok 10400, Thailand 
Telephone: 02-201-2167 
 
Assoc. Prof. Orawee Chinthakanan, MD, MPH 
Female Pelvic Medicine and Reconstructive Surgery Division 
Department of Obstetrics & Gynaecology, Faculty of Medicine Ramathibodi Hospital, Mahidol University 
270 Rama VI Road, Ratchathewi, Bangkok 10400, Thailand 
Telephone: 02-201-2167 
Sponsor or planned sponsor, grant, scholarship <if applicable>: 
The budget outlines a plan to request funding from the Faculty of Medicine Ramathibodi Hospital, Mahidol 
University and Menopausal fund. 
Conflict of Interest: 
None 
Study sites (list all as planned):  
• Outpatient department – Female Pelvic Medicine and Reconstructive Surgery clinic (FPMRS clinic), 
Department of Obstetrics & Gynaecology, Faculty of Medicine Ramathibodi Hospital 
Trial registration: 
The trial registration is planning to register on www.clinicaltrials.gov 
 
 

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Background and Significance:  
Overactive bladder (OAB) is a prevalent condition characterized by urinary urgency, often 
associated with increased frequency and nocturia, with or without urgency urinary incontinence (UUI), and 
occurs in the absence of a urinary tract infection (UTI) or other apparent pathology (1). It affects about 12-
20% of adults and increases incidence with age. It significantly impacts the quality of life, leading to 
physical discomfort, psychological stress, and social embarrassment with higher prevalence observed in 
women, particularly those in the menopausal transition or postmenopausal phase (2). 
Menopause marks a significant hormonal shift in a woman's life, primarily characterized by the 
decline in estrogen production by the ovaries. Estrogen plays a crucial role in maintaining the structural 
and functional integrity of the lower urinary tract, vagina, and pelvic floor muscles. Due to these, the 
reduction in estrogen levels following menopause is considered a potential cause of pelvic floor disorders, 
including stress urinary incontinence (SUI), urgency urinary incontinence (UUI), and pelvic organ prolapse 
(POP), including OAB symptoms in menopausal women (3). 
Estrogen therapy, particularly localized treatments like vaginal estrogen, has been utilized to 
alleviate urogenital symptoms associated with menopause (3). Vaginal estrogen has been shown to 
improve vaginal atrophy and increase urethral closure pressure (4). Moreover, it may alleviate OAB 
symptoms by restoring the health of the urogenital mucosa and improving bladder function. For clinical 
use, many studies have shown a significant reduction in urinary urgency symptoms, urinary incontinence, 
and increased maximal cystometric capacity (5, 6). However, the precise mechanisms by which estrogen 
influences bladder function and symptomatology remain not fully elucidated. 
Recent advances in microbiome research have highlighted the significant role of the urinary 
microbiome (or urobiome) in urinary health and disease. Once considered sterile, the urinary tract is now 
known to host a diverse community of microorganisms that may influence urinary tract function and 
susceptibility to disorders like OAB (7). Alterations in the urinary microbiome composition, known as 
dysbiosis, have been associated with various urinary conditions, including overactive bladder, nocturia, 
interstitial cystitis and recurrent urinary tract infection (8). Understanding the impact of vaginal estrogen on 
the urinary microbiome of menopausal women with OAB could provide valuable insights into new 
therapeutic avenues and personalized treatment strategies. It may help clarify whether alterations in the 
urinary microbiome composition mediate the beneficial effects of estrogen on OAB symptoms. 
This study aims to investigate the effect of vaginal estrogen therapy on changes in the urinary 
microbiome and its association with OAB symptom improvement in menopausal women. By exploring this 
relationship, we hope to enhance the understanding of OAB pathophysiology in menopausal women and 
contribute to developing more effective interventions targeting both hormonal and microbial factors. 
 

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Review Literature: 
Vaginal estrogen used in OAB treatment 
Author/Year/ 
Country 
Study 
Aim 
Results 
Cardozo LD et al (5) 
2001 
London, UK 
• Randomized 
controlled trial 
•  N = 110 
• Vaginal 17β Estradiol 
VS Placebo 
• 12 weeks follow up 
Evaluate efficacy of 17β 
Estradiol in treatment of 
postmenopausal 
women with Frequency, 
Urgency or UUI 
Significant reduction in 
symptom of urinary 
urgency for women with 
sensory urgency 
Simunic V. et al (6) 
2003 
Croatia 
• Multicenter RCT 
• N = 1612 
• Vaginal 17β Estradiol 
VS Placebo 
• 12 months follow up 
Determination of 
efficacy and safety of 
vaginal 17β Estradiol in 
patients with urogenital 
symptoms 
In estrogen group 
• Improve urinary 
atrophy symptoms 
• Reduce urinary 
incontinence 
• Increase maximal 
cystometric 
capacity, first desire 
to void, strong 
desire to void 
 
Overactive bladder/Urgency urinary incontinence and microbiome study 
• Pearce MM, et al., 2014 (9)  
o This study used Expand Quantitative Urine Culture (EQUC) techniques and 16s rRNA gene 
sequencing to analyze the trans-catheterized urine samples of patients with urgency 
urinary incontinence compared with the control group.  
o Results:  
▪ UUI microbiome was composed of increased Gardnerella and decreased 
Lactobacillus 
▪ Nine genera were more frequently cultured from the UUI group (Actinobaculum, 
Actinomyces, Aerococcus, Arthrobacter, Corynebacterium, Gardnerella, Oligella, 
Staphylococcus, Streptococcus) 
▪ More Lactobacillus gasseri in UUI group 
▪ More Lactobacillus crispatus in control group 

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o Conclusion: bacterial communities in the urinary tract may be linked to UUI. Data suggest 
that potential important differences exist in urine microbiomes of women with and 
without UUI 
• Curtiss N, et al., 2017 (10) 
o This study used 16s rRNA gene sequencing to analyze the urine samples from clean catch 
of patients with overactive bladder compared with the control group 
o Results: 
▪ Significant differences were found between the microbiomes of OAB patients and 
controls. Lactobacillus was more prevalent in controls (43%) compared to OAB 
patients (20%). Conversely, Proteus was more commonly found in OAB patients 
(23%) than controls (3%). 
o Conclusion: the female bladder has a diverse microbiome with significantly differences 
bacteria species present in overactive bladder patients and controls 
Vaginal estrogen therapy in Overactive bladder and microbiome study 
• Thomas-White K, et al., 2020 (11) 
o The quasi-experimental study aimed to determine the effect of vaginal estrogen 
treatment (0.5 g of conjugated estrogen twice weekly for 12 weeks) on urobiome diversity 
(using Expand Quantitative Urine Culture (EQUC) techniques) in postmenopausal women 
with overactive bladder. 
o Results:  
▪ A significant increase in Lactobacillus was detected in the bladder (10.8 vs. 25.1; 
P= 0.037) but not in the vaginal (P = 0.33), perineum (P = 0.56) or void urine (P = 
0.28) 
▪ The change in Lactobacillus levels in the bladder was associated with modest 
changes in urgency incontinence symptoms (P = 0.02) 
o Conclusion: vaginal estrogen therapy alters the microbiome in different pelvic niches, 
remarkably increasing Lactobacillus in the bladder, which is associated with symptom 
improvement in postmenopausal women with OAB. 
 
Objectives:  
Primary Objective:   
To evaluate the effects of vaginal estrogen on the level of Lactobacillus in the urine of postmenopausal 
women with OAB. 

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Secondary Objectives (if any): 
1. To evaluate the effects of vaginal estrogen on urine microbiome in postmenopausal women with 
OAB. 
2. To identify the association of alterations of urine microbiome after vaginal estrogen treatment with 
overactive bladder symptoms. 
Study design/methodology: 
Study design: Pre-post quasi-experimental study 
Study flow diagram 
 
 Note: The validated Thai version of OAB-q SF (12) 
Study Population  
Postmenopausal women with overactive bladder 
Inclusion Criteria: 
1. Natural or surgical menopause at least 1 year  

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2. Present with overactive bladder symptoms by using validated Thai version of OABSS (13)  
(score in No.3 ≥ 2 and overall score ≥ 3)  
3. Absence of urinary infection 
4. Post-void residual urine less than 100ml 
Exclusion criteria 
1. Currently or prior use of systemic HRT or vaginal estrogen within the past 3 months 
2. Contraindication or allergy to estrogen therapy 
3. Use of antibiotics, prebiotics and probiotics within the past 2 weeks 
4. Currently on antimuscarinic or β3 agonists medication or within the past 3 months 
5. Pelvic organ prolapse greater than stage II 
 
Study drug /Interventions <if applicable>:  
17β-estradiol 10 mcg (Femiest®, HAUPT PHARMA MUNSTER GMBH, Muenster, Germany)  
1 tab daily for 2 weeks vaginally before bed, then 1 tab twice a week vaginally before bed until 12 
weeks (Participants will be asked to bring the blister pack for pill count at the 12-week follow-up 
(checking compliance)  
Comparator/Control <if applicable>:  
None 
Treatment Allocation and concealment: 
None 
Blinding (masking): 
None 
Participant timeline and Procedures:  

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Urine sample collection 
Sterile urine collection procedures will be done by well-trained investigators on catheterization 
urine. The urine will be transferred immediately to the laboratory and placed at 4°C. 
Outcomes/endpoints: 
Primary Outcome 
• Change in Lactobacillus abundance in urine after vaginal estrogen therapy 
Secondary Outcome 
• Change in urine microbiome diversity after vaginal estrogen therapy 
• Association of change in urine microbiome with overactive bladder symptoms 
Discontinuation/withdrawal criteria: 
1. Adverse events or medical reasons 
a. Severe Adverse Reactions: If a participant experiences severe or unmanageable side 
effects related to vaginal estrogen use (for example unexplained vaginal bleeding, severe 
irritation, or allergic reactions), they should be withdrawn from the study. 
b. New Medical Conditions: development of new medical conditions that could interfere 
with the study outcomes for example cardiovascular issues 
2. Non-Adherence to Treatment Protocol: Missed Follow-Up Assessments 
3. Voluntary Withdrawal 
Adverse Event Reporting:  
1. Adverse events (AEs) 
a. Definition: any unintended, unfavorable medical occurrence in a participant during the 
study, whether it is related to the study intervention (vaginal estrogen therapy) or not. 
b. Examples: vaginal irritation, vaginal discomfort, vaginal discharge, bleeding, allergic reaction 
and possible systemic absorption of estrogen could lead to side effects such as breast 
tenderness or headaches. 
c. Participant Responsibilities: Participants are instructed to immediately report any 
discomfort, symptoms, or health changes they experience during the study, regardless of 
severity. 
d. Investigator Responsibilities: AEs and compliance will be inquired by follow-up phone calls 
from the investigator. Mild or moderate AEs will be managed by providing symptomatic 
treatment or observing the participant closely. 

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2. Serious adverse events 
a. Definition: Any event that results in hospitalization, life-threatening conditions, permanent 
disability, or death. 
b. Investigator Responsibilities: If a serious adverse event occurs, the participant will be 
withdrawn from the study, and immediate medical care will be provided. SAEs must be 
reported to the Ethics Committee and relevant regulatory bodies within 24 hours. 
 
Statistical Analysis Plan:  
• Descriptive statistics for baseline demographics and clinical characteristics were presented as mean 
and standard deviation or medians and interquartile ranges (IQRs) for continuous variables and 
frequency and percentage for categorical variables. 
• Comparisons between pre-and post-intervention were tested using pair t-test or Wilcoxon signed-
rank tests for continuous variables and chi-square or the Fisher exact tests for categorical variables. 
• Association between changes in urine microbiome and changes in the OAB symptom were 
calculated using the Spearman rho test. 
Microbiome study 
• Alpha Diversity (within-sample diversity): measures provide insights into the richness (number of 
species) and evenness (distribution of species) within a sample. 
o Observed species, Chao1, Shannon, Simpson using QIIME software 
o Richness was calculated using Chao1 and observed species indices 
o Shannon and Simpson indices combined the interaction between richness and evenness 
o Wilcoxon rank sum test was applied to identify significant differences 
• Beta Diversity (between-sample diversity): evaluates differences in microbial composition between 
samples. 
o Bray-Curtis and Uni-Frac (weighted and unweighted) 
• Linear Discriminant analysis effect size (LEfSe): investigate microbial biomarkers between groups at 
the genus level 
 
P-values <0.05 were considered statistically significant. 
Sample size determination: 
Using 2 dependent means formula for sample size calculation. 
 

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• From Pearce MM’s study (2014), the median (IQR) percentage of lactobacillus in women with OAB 
was 13.6 (0-61.6); we expected the difference in mean percentage of lactobacillus after vaginal 
estrogen treatment was 25.     
• Alpha (α) = 0.05, Beta (β) = 0.2 
• The sample size was calculated = 27. 
• Then, 10% data loss was added, the required sample size was 30. 
 
Recruitment procedure: 
• Menopausal women with a clinical diagnosis of overactive bladder (OAB) who meet the eligibility 
criteria will be recruited from the outpatient department – FPMRS clinic, Ramathibodi hospital. 
• Interested patients will be directed appointed by research nurse to meet with the investigator. 
• The investigator will give a detailed explanation, confirm eligibility through a detailed review of 
medical history and informed consent procedures. 
Informed Consent Process:  
1. Recruitment: Interested patients will be directed to meet with an investigator at the outpatient 
department – FPMRS clinic. 
2. Detailed Explanation: The study’s purpose, procedures, risks, and benefits are explained clearly. 
3. Voluntary Participation: Emphasis is placed on the voluntary nature of participation and the right 
to withdraw at any time. 
4. Signing the Consent Form: Participants sign the form after understanding the study and asking 
questions. 
5. Ongoing Consent: Participants are reminded of their rights and informed of any protocol changes 
during the study. 
 
Privacy and confidentiality (Data Management Plan): 
1. The principal investigator and research collaborators will manage data and maintain patient 
confidentiality. 
2. Assign Participant IDs to de-identify data and samples. The principal investigator will record 
information such as hospital numbers or names in a codebook. 

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3. Physical Storage: Any physical copies of data (e.g., paper forms and consent documents) will be 
securely stored in locked cabinets, with access limited to authorized personnel. 
4. Biological Samples: Use urine samples only for outlined study purposes, store samples securely 
and label them with study ID and destroy samples according to biohazard regulations after use. 
5. Digital Storage: All electronic data will be stored in encrypted databases, and any data transfers 
will occur over secure, encrypted networks. Regular backups will be performed to prevent data 
loss. Data will be stored for at least five years. 
6. Participants can request access to or withdraw their data. 
Ethical consideration: 
- Risks to participants and how to minimize the risks: 
1. Risks Related to Vaginal Estrogen Therapy 
a. Potential risks: vaginal discomfort, allergic reaction, vaginal discharge 
b. Risk minimization: Screen participants carefully to exclude those with contraindications to 
estrogen therapy, monitoring allergic reactions and educating participants 
2. Risks Related to Urine Sample Collection by transurethral catheterization 
a. Potential risks: discomfort, urinary tract infection and urinary tract injury 
b. Risk minimization: Sterile urine collection procedures by the investigator who is well-
trained, monitoring symptoms of urinary tract infection and educating participants 
- Direct Benefits to Participants  
Participants receive vaginal estrogen therapy as part of the study intervention at no cost. Vaginal 
estrogen therapy has been shown to alleviate symptoms associated with overactive bladder, such as 
urinary urgency, frequency, nocturia (nighttime urination), and urge incontinence. 
- Scientific or social value 
Supporting evidence of vaginal estrogen used in overactive bladder.  
Understanding the changes in the urine microbiome caused by vaginal estrogen could allow for 
more personalized treatment strategies. 
- Justification if enrolling potentially vulnerable subjects. 
The study will not be conducted on vulnerable subjects. 

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- Travel compensation and compensation for injury 
Participants will receive 1,000 Thai Baht per visit to compensate for travel expenses incurred 
while attending study appointments. This compensation is provided after each completed visit. 
- Plan of board consent 
None 
 
Study Timeline: 
Budget: 
Expense 
Estimated cost/unit 
Estimated cost 
Participant Compensation 
1000 Baht/visits 
60,000 Baht 
Intervention (Femiest®) 
1197 Baht/box (18 tablets) 
71,820 Baht 
Urine catheterization 
78 Baht/set 
4,680 Baht 
Urine microbiome analysis 
5000 Baht/specimen 
300,000 Baht 
Total budget 
 
436,500 Baht 
  
References: 
1. Haylen BT, de Ridder D, Freeman RM, Swift SE, Berghmans B, Lee J, et al. An International 
Urogynecological Association (IUGA)/International Continence Society (ICS) joint report on the 
terminology for female pelvic floor dysfunction. Int Urogynecol J. 2010;21(1):5-26. 

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2. Cardozo L. Textbook of Female Urology and Urogynecology: Clinical Perspectives. 5 ed. Staskin D, 
editor: CRC Press; 2003. 
3. Weber MA, Kleijn MH, Langendam M, Limpens J, Heineman MJ, Roovers JP. Local Oestrogen for Pelvic 
Floor Disorders: A Systematic Review. PLoS One. 2015;10(9):e0136265. 
4. Krause M, Wheeler TL, 2nd, Snyder TE, Richter HE. Local Effects of Vaginally Administered Estrogen 
Therapy: A Review. J Pelvic Med Surg. 2009;15(3):105-14. 
5. Cardozo LD, Wise BG, Benness CJ. Vaginal oestradiol for the treatment of lower urinary tract 
symptoms in postmenopausal women--a double-blind placebo-controlled study. J Obstet Gynaecol. 
2001;21(4):383-5. 
6. Simunić V, Banović I, Ciglar S, Jeren L, Pavicić Baldani D, Sprem M. Local estrogen treatment in 
patients with urogenital symptoms. Int J Gynaecol Obstet. 2003;82(2):187-97. 
7. Kenneally C, Murphy CP, Sleator RD, Culligan EP. The urinary microbiome and biological therapeutics: 
Novel therapies for urinary tract infections. Microbiol Res. 2022;259:127010. 
8. Gasiorek M, Hsieh MH, Forster CS. Utility of DNA Next-Generation Sequencing and Expanded 
Quantitative Urine Culture in Diagnosis and Management of Chronic or Persistent Lower Urinary Tract 
Symptoms. J Clin Microbiol. 2019;58(1). 
9. Pearce MM, Hilt EE, Rosenfeld AB, Zilliox MJ, Thomas-White K, Fok C, et al. The female urinary 
microbiome: a comparison of women with and without urgency urinary incontinence. mBio. 
2014;5(4):e01283-14. 
10. Curtiss N, Balachandran A, Krska L, Peppiatt-Wildman C, Wildman S, Duckett J. A case controlled 
study examining the bladder microbiome in women with Overactive Bladder (OAB) and healthy 
controls. Eur J Obstet Gynecol Reprod Biol. 2017;214:31-5. 
11. Thomas-White K, Taege S, Limeira R, Brincat C, Joyce C, Hilt EE, et al. Vaginal estrogen therapy is 
associated with increased Lactobacillus in the urine of postmenopausal women with overactive 
bladder symptoms. Am J Obstet Gynecol. 2020;223(5):727.e1-.e11. 
12. Sangnucktham T, Bunyavejchevin S, Ruanphoo P. Validity and Reliability of Thai Version of the 
Overactive Bladder Questionnaire Short form (OAB-q SF) in Women with Overactive Bladder. Thai 
Journal of Obstetrics and Gynaecology. 2024;32(4):306-13. 
13. Bunyavejchevin S. Reliability of Thai-Version Overactive Bladder Symptom Scores (OABSS) 
Questionnaire and the Correlations of OABSS with Voiding Diary, International Prostate Symptom Score 
(IPSS), and Patient Perception of Bladder Condition (PPBC) Questionnaires. J Med Assoc Thai. 
2015;98(11):1064-74. 
 
 
 

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Signature..................................................... Principal Investigator 
      
 
 
 
 
              (Papapin Pusayapaibul, MD) 
          Date...................................................... 
 
                                                     Signature..................................................... Major Advisor 
                                   (Assist. Prof. Rujira Wattanayingcharoenchai, MD, M.Sc.) 
                                                               Date......................................................