Efficacy of Vaginal 17β-Estradiol on the Urinary Storage Symptoms in Postmenopausal Women: A Randomized Double-Blind, Placebo-Controlled Study
Phase: PHASE4
Conditions: Lower Urinary Tract Symptoms, Female Genitourinary Disease, Urinary Stress Incontinence, Urinary Urge Incontinence, Nocturia, Quality of Life, Voiding Disorders, Vagina Atrophy, Urethral Atrophy, Overactive Bladder, Overactive Bladder Syndrome, Bladder, Overactive
Sponsor: Mahidol University

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Clinical Trial Protocol 
Faculty of Medicine Ramathibodi Hospital, Mahidol University (full version) 
 
1. Research name 
Efficacy of Vaginal 17-Estradiol on the Urinary Storage Symptoms in Postmenopausal Women: A 
Randomized Double-blind, Placebo-controlled Study 
 
2. Principal investigator Pornthip Harncharoenkul, MD 
Fellow in Female Pelvic Medicine and Reconstructive Surgery Division 
Contracted workplace: Female Pelvic Medicine and Reconstructive Surgery Division, Department 
of Obstetrics & Gynecology, Faculty of Medicine Ramathibodi Hospital, Mahidol University 
Tel +66 2201 1412 Mobile +668 1862 9009 
 
3. Co-investigator  
1. Assist.Prof.Rujira Wattanayingcharoenchai, MD, MSC 
Contracted workplace: Female Pelvic Medicine and Reconstructive Surgery Division, Department 
of Obstetrics & Gynecology, Faculty of Medicine Ramathibodi Hospital, Mahidol University 
Email address: rujira.wat@mahidol.ac.th  
Tel: +66 2201 1412 
2. Assist.Prof.Dr.Pisut Pongchaikul, MD, Ph.D  
Contracted workplace: Ramathibodi Medical School, Chakri Naruebodindra Medical Institute,  
111 Bang Pla, Bang Phli, Samut Prakan 
Email address : pisut.pon@mahidol.edu 
Tel : +669 0601 2736 
3. Assist.Prof.Komkrit Aimjirakul, MD  
Contracted workplace: Female Pelvic Medicine and Reconstructive Surgery Division, Department 
of Obstetrics & Gynecology, Faculty of Medicine Ramathibodi Hospital, Mahidol University 
Email address: komkrit.aim@mahidol.ac.th 
Tel: +668 1620 7407 
 
 

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4. Assoc.Prof.Orawee Chinthakanan, MD, MPH 
Contracted workplace: Female Pelvic Medicine and Reconstructive Surgery Division, Department 
of Obstetrics & Gynecology, Faculty of Medicine Ramathibodi Hospital, Mahidol University 
Email address: Orawee.chi@mahidol.ac.th 
Tel: +668 1952 2215 
5. Prof.Jittima Manonai, MD, MHM 
Contracted workplace: Female Pelvic Medicine and Reconstructive Surgery Division, Department 
of Obstetrics & Gynecology, Faculty of Medicine Ramathibodi Hospital, Mahidol University 
Email address: jittima.man@mahidol.ac.th 
Tel +66 2201 1412  Mobile +668 1620 7407 
4. Contact person 
1. Pornthip Harncharoenkul, MD 
Contracted workplace: Female Pelvic Medicine and Reconstructive Surgery Division, Department 
of Obstetrics & Gynecology, Faculty of Medicine Ramathibodi Hospital, Mahidol University 
Tel +66 2201 1412 Mobile +668 1862 9009 
 
2. Prof.Jittima Manonai, MD, MHM 
Contracted workplace: Female Pelvic Medicine and Reconstructive Surgery Division, Department 
of Obstetrics & Gynecology, Faculty of Medicine Ramathibodi Hospital, Mahidol University 
Tel +66 2201 1412  Mobile +668 1620 7407 
5. Background and Rationale 
MENOPAUSE AND LOWER URINARY TRACT SYMPTOMS (LUTS) 
The lower urinary tract is responsible for the storage and elimination of urine. The control of 
this phenomenon is attributed to the innervation of the autonomic, somatic, and sensory systems. 
The process of ageing is widely recognized as a significant determinant of the anatomical and 
functional changes that occur in the pelvic floor and lower urinary tract (LUT). The prevalence of 
pelvic floor diseases, such as pelvic organ prolapse, overactive bladder (OAB), urinary incontinence 
(UI), and sexual dysfunction, tends to rise as individuals age. While it is challenging to distinguish 
the specific impacts of reduced estrogen levels during menopause from the broader process of 
ageing, it is worth noting that the pelvic organs and their associated muscular and connective tissue 
support are influenced by estrogen. Epidemiological research suggests that menopause plays a 
significant role in the increased likelihood of developing pelvic floor disorders. Furthermore, the 

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severity of urinary symptoms associated with these disorders tends to escalate following 
menopause.(1) 
Lower urinary tract symptoms (LUTS) encompass a variety of frequently co-occurring 
issues pertaining to storage symptoms. These symptoms include urinary incontinence, heightened 
frequency of urination during the day or night, a sense of urgency, as well as symptoms related to 
voiding and post-micturition. Voiding symptoms may manifest as hesitancy, a slow stream, and 
intermittent flow, difficulty initiating urination, spraying or splitting of the urinary stream, leakage, 
painful urination, and a sensation of incomplete bladder emptying (urine retention). The 
International Continence Society (ICS) has provided a definition for overactive bladder, which is 
considered the most troublesome subset of lower urinary tract symptoms (LUTS). According to the 
ICS, overactive bladder is characterized by a sense of urgency to urinate, with or without 
involuntary leakage, sometimes accompanied by increased frequency of urination and waking up at 
night to urinate (nocturia). Based on the provided definition, urgency is a fundamental element in 
the diagnostic criteria for Overactive Bladder (OAB), serving as the primary symptom that 
influences the manifestation of other OAB symptoms.(1,2,3) The symptoms of Overactive Bladder 
(OAB) have a significant impact on various dimensions of individuals' lives, including social, 
psychological, occupational, household, physical, and sexual aspects. Consequently, these 
symptoms impose a substantial burden on affected individuals, resulting in significant costs.(4) 
ESTROGEN DEPLETION AND STORAGE SYMPTOMS  
The Role of Estrogens and the Continence Mechanism 
Estrogen plays an important role in the genital and lower urinary tract functions, with 
estrogen receptors found in the bladder, urethra, vagina, and pelvic floor musculature. Estrogen 
receptors contribute to the pelvic support mechanism by modulating the production and breakdown 
of collagen. Furthermore, the tissues of the female urinary continence mechanism are estrogen 
sensitive. Estrogens may disrupt continence by increasing the number of periurethral vessels, which 
account for one-third of urethral pressure. Furthermore, estrogens can diminish the frequency and 
amplitude of detrusor contractions, raising the bladder's sensory threshold and promoting detrusor 
muscle relaxation.(3,5,6,7,8,9)  
Epidemiological studies have corroborated the importance of sex hormone insufficiency in 
the etiology of LUTS, with up to 70% of women attributing the onset of urine incontinence to the 
end of their menstrual cycle. 
Pathophysiology of Estrogen Depletion and Storage Symptoms 
Several mechanisms could explain the role of estrogen deprivation in the onset of urinary 
urgency such as increased detrusor contractility through Rho-kinase pathway activation, increased 

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acetylcholine release, changes in urothelial afferent signalling, or increased connexin-43 
expression.(10,11) Previous study has been documented which demonstrates that the removal of 
ovaries in female mice can lead to a reduction in urethral tone in these mice. The administration of 
E2 supplementation in mice that underwent ovariectomy resulted in the restoration of urethral tone. 
The augmentation of urethral tone in ovariectomized mice through E2 mediation is associated with 
the upregulation of surviving motor neuron expression, reduction in proteolysis, facilitation of 
development, neurophysiological processes, and transcription within the urethra. This knowledge 
will provide insights into the pathophysiology of stress urine incontinence following menopause.(11)  
The depletion of estrogen in the body leads to a reduction in blood supply to the pelvic floor 
tissues. Insufficient blood circulation leads to a reduction in collagen synthesis, hence 
compromising the process of tissue repair. These alterations result in a reduction in the capacity of 
the vagina and bladder, leading to symptoms such as dyspareunia and urgency. Animal studies 
suggest that estrogen inhibits bladder contractility, so one can speculate that estrogen loss might 
contribute to OAB symptoms. A loss of periurethral vascularity lowers the urethral closure 
pressure, in theory contributing to the development of stress incontinence.(12)  
Estradiol has been demonstrated to reduce inflammatory cell infiltration and reverse bladder 
shrinkage via vascular endothelial growth factor, supporting estrogen's therapeutic capabilities.(13,14) 
In clinical trials, vaginal 17estradiol tablets (Vagifem) reduced OAB symptoms while improving 
urodynamic markers such as cystometric capacity, strong need to urinate, and unrestrained detrusor 
contractions.(15) 
PREVALENCE OF STORAGE SYMPTOMS  
The overall prevalence of storage symptom in women was about 16% in US and Europe, but 
sex-specific prevalence differed substantially by severity of symptoms. Prevalence of urge 
incontinence increased with age from 2.0% to 19% with a marked increase after 44 years of 
age.(4,16) In Thailand, the prevalence of overactive bladder was 21.3%. Urgency, the main symptom, 
was found to occur in 32.8%.The common associated symptoms were frequency and urge 
incontinence which were found in 23.4% and 11.4% respectively.(17) 
TREATMENT OF STORAGE SYMPTOMS 
Bladder storage symptoms exhibit a clinical scenario and presentation that bears a striking 
resemblance to those observed in cases of an overactive bladder. In our forthcoming clinical trial, 
we intend to establish a treatment regimen by referencing the established standard guidelines for the 
management of overactive bladder as the foundational framework as will be mentioned in the 
following paragraphs.  

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Behavioral therapies, such as bladder training, bladder control strategies, pelvic floor muscle 
training, and fluid management, are commonly employed as first-line treatments. These therapies 
may be administered independently for patients experiencing mild symptoms that cause minimal 
distress. Alternatively, they may be combined with other more invasive treatment modalities for 
patients with severe symptoms. Pharmacologic management constitutes the subsequent therapeutic 
interventions for second-line treatments. Common pharmacological options for the treatment of 
overactive bladder (OAB) encompass the utilization of both anti-muscarinics and β3-adrenoceptor 
agonists.(18) 
Vaginal estrogen therapy may be administered in the form of conjugated equine estrogen, 
estriol, or estradiol, utilizing vaginal pessaries, vaginal rings, or creams. This treatment aims to 
ameliorate symptoms of overactive bladder (OAB), including urgency linked to vaginal atrophy in 
postmenopausal women. The optimal period of treatment is undetermined.(19) 
Estrogen has the potential to provide therapeutic advantages in managing irritative voiding 
symptoms, such as urgency, frequency, and urge urinary incontinence. The precise mechanism by 
which the medication induces the reversal of urogenital atrophy or its direct impact on the lower 
urinary tract remains uncertain. One challenge in comprehending the contradictory data now 
available on the subject pertains to the utilization of multiple estrogen formulations, varying 
dosages, diverse methods of administration, and the inconsistent use of concomitant progesterone.  
There exists substantial evidence supporting the efficacy of low-dose vaginal estrogen treatment in 
reversing urogenital atrophy, encompassing both the symptoms and cytological changes.(20, 21) 
 
 
 
 
 
 
 
 
 

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Review literatures  
• Comparison: Vaginal estradiol 25 mcg versus placebo 
• Weber MA et al. (2015) 
Local Estrogen for Pelvic Floor Disorders: A Systematic Review 
Comparison: Vaginal estrogen versus placebo or no treatment 
The group comprised of a total of eight research, out of which five studies employed a 
placebo-controlled design. Additionally, two studies compared the use of vaginal estrogen to no 
treatment, while one study compared the use of vaginal estrogen (in combination with 
physiotherapy and electrostimulation) to a group that received no treatment. In the present 
investigation, two separate investigations implemented the administration of vaginal estrogen via 
ovules, while an additional two studies employed estrogen vaginal tablets. Furthermore, three 
studies incorporated the utilization of an estrogen vaginal cream as part of their experimental 
protocol. The study lacked clarity regarding the manner of application. 
In general, the subjective, semi-objective, and urodynamic results exhibited a favorable shift 
in the vaginal estrogen groups when compared to the placebo group. The study conducted by 
Simunic et al. observed that the occurrence of treatment adverse effects was evenly divided across 
Research title 
country 
year 
Dosage 
F/U 
Outcome 
Measurement 
Results 
Low-dose 17beta-estradiol 
vaginal tablets in the treatment 
of atrophic vaginitis: a double-
blind placebo-controlled 
study(14) 
Denmark 
Eriksen 
PS 1991 
estradiol 
25 mcg 
vs placebo 
12 
weeks 
urological symptoms 
change 
Vg E -> improve 62.8% 
placebo -> improve 32.4% 
Local estrogen treatment in 
patients with urogenital 
symptoms(22) 
Croatia 
Simunic 
2003 
estradiol 
25 mcg 
vs placebo 
12 
months 
symptoms/signs of 
urinary atrophy 
Dysuria 
n=330->84 
Frequency/nocturia 
n=371->74 (Improve 80%) 
Urinary tract infection 
n=186->49 
Urinary incontinence 
n=245->106 
Urinary atrophy 
n=406->121 
Vaginal estradiol for the 
treatment of lower urinary 
tract  symptoms in 
postmenopausal women--a 
double-blind placebo-
controlled study(23) 
England 
Cardozo 
2001 
estradiol 
25 mcg 
vs placebo 
12 
weeks 
N/A 
A significantly greater 
reduction in the symptom of 
urinary urgency on visual 
analogue score was detected 
for women in stratum A 
(sensory urgency) 

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the vaginal estrogen and placebo groups. There were no instances of severe adverse effects 
reported. Three further investigations found no statistically significant differences in the occurrence 
of adverse events among the various groups. The researchers reached the conclusion that the 
application of topical estrogen appears to reduce the occurrence of overactive bladder (OAB) and 
urinary incontinence (UI) symptoms.(3) 
• J. A. Simon et al. (2013) 
Ultra-low-dose vaginal estrogen tablets for the treatment of postmenopausal vaginal 
atrophy 
A pivotal study demonstrated that an ultra-low-dose 10-microgram E2 vaginal tablet 
effectively induces vaginal epithelial maturation. This change shifts the atrophic epithelium from a 
predominantly parabasal cell population to a superficial cell population, akin to premenopausal 
women. Significant improvements in vaginal cytology, evident after just 14 days of treatment, were 
maintained up to 52 weeks. This improvement in the cellular profile enhances the vaginal 
epithelium's resistance to bleeding and improves vaginal-cervical paracellular permeability, thereby 
increasing lubrication during intercourse. 
Furthermore, the study revealed that local administration of 10 micrograms E2 significantly 
alleviated the most bothersome both urinary and genital symptoms, such as vaginal dryness, 
irritation, itching, dysuria, and dyspareunia. This symptom relief was more consistent and effective 
compared to systemic hormone therapy. While systemic absorption of estrogen does occur with all 
vaginal estrogen therapy products, studies have shown that absorption from low-dose vaginal 
estrogen therapy is minimal. Overall, the results firmly establish the efficacy of the ultra-low-dose 
10-microgram E2 vaginal tablet in treating symptoms of estrogen deficiency-induced urogenital 
atrophy. The treatment effectively normalizes vaginal pH and cytology profiles and provides 
significant relief from the most bothersome urogenital symptoms soon after therapy initiation.(24) 
• Placebo and nocebo 
The administration of placebo has been observed to have a discernible impact on the 
amelioration of signs and symptoms associated with Overactive Bladder (OAB). Furthermore, the 
collective placebo responses observed across several examined outcomes exhibit statistical 
significance, and in certain cases, may also possess potential therapeutic significance. A recent 
systematic review (SR) encompassing 57 studies and involving a total of 12,901 patients revealed 
several noteworthy findings. Specifically, the standardized mean difference (SMD) for various 
urinary parameters were as follows: -0.45 for daily micturition episodes, -0.33 for daily nocturia 
episodes, -0.46 for episodes of urgency urinary incontinence (UUI), -0.50 for daily urgency 
episodes, -0.51 for daily incontinence episodes, and 0.25 for volume voided per micturition.(25,26)  

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The same group published a SR with meta-analysis of data retrieved from 57 RCTs 
comprising 15,446 patients were included in this systematic review. They selected 13 commonly 
reported adverse events for the meta-analysis. Nocebo effect of pharmacotherapy in patients with 
OAB (up to 80% females). They reported dry mouth as the most common reported adverse event 
with mean rate of 4.9%, followed by constipation 2.6%. The authors concluded that HCPs should 
appreciate the possible positive and negative patient expectation regarding pharmacotherapy for 
OAB in order to optimize the individual outcomes.(26,27) 
The placebo response seems to be non-negligible in OAB, supporting the requirement for 
placebo control in RCTs.(25,26) 
Knowledge gap 
        To date, no prior investigations have been conducted employing robust methodological 
approaches to assess the efficacy of 17β-Estradiol at a dosage of 10 mcg in the treatment of Lower 
Urinary Tract Symptoms (LUTS) in postmenopausal women, particularly in comparison to a 
placebo control group. This notable gap in the existing literature underscores the need for a 
comprehensive and rigorously designed study to investigate the potential therapeutic benefits of 
17β-Estradiol in alleviating LUTS among postmenopausal women, while concurrently establishing 
its superiority or non-inferiority relative to a placebo intervention. Such an investigation would not 
only contribute significantly to our understanding of this therapeutic avenue but also provide 
valuable insights into the management of storage symptoms of LUTS in this specific population. 
6. Objective      
Primary objective 
To determine the efficacy of vaginal 17β-estradiol treatment in alleviating storage symptoms of 
LUTS in comparison to placebo. 
Secondary objective 
- To determine the efficacy of vaginal 17β-estradiol treatment on urethral maturation index 
and vaginal pH in comparison to placebo.  
- To assess the impact of vaginal 17β-estradiol treatment on the overall quality of life and the 
patient's global improvement (PGI) perception. 
 
 
 
 

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7. Study Objectives and Study Design Overview 
7.1) Study design  
The clinical study is a two arm, randomized, double-blind, placebo-controlled trial study of 
postmenopausal women who suffer from storage symptom of LUTS.  
7.2) Population 
Postmenopausal women who suffer from storage symptom of LUTS. 
7.3) Outcomes 
 
Primary outcome     
Storage symptom improvement: will be evaluated using “the International Consultation on 
Incontinence Questionnaire-Female Lower Urinary Tract Symptoms: ICIQ-FLUTS” (Validated 
Questionnaire in Thai version)(28)  
It comprises of 5 items, each represent the storage symptoms in the following items; 
- 
Item 2a) nocturia  
- 
Item 3a) urgency  
- 
Item 5a) daytime frequency  
- 
Item 9a) UUI  
- 
Item 11a) SUI 
Each item will have 2 parts of sub-categories questions are severity and bothersome. 
- 
In severity categories: the questionnaire has established ranging from 0, denoting the 
absence of any severity, to 4, indicating the highest level of symptom severity. The 
cumulative point range across all questionnaire items spans from 0 to 20.  
- 
In bothersome categories: the questionnaire employ ranging from 0, signifying the 
absence of any bothersome effect, to 10, representing the utmost degree of symptom 
bother. The cumulative point range across all questionnaire items encompasses 0 to 
50. 
  
The primary outcome interpretation will be predicated on the assessment of storage 
symptom improvement as follows. 
• Improve outcome mean ICIQ-FLUTS post-treatment score less than pre-treatment score.   
• Not improve outcome mean ICIQ-FLUTS post-treatment score more than or equal to pre-
treatment score.   
 

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Secondary outcomes  
 
Subjective assessment 
• Assess the impact of urinary storage symptoms on quality of life with reference to social 
effects by Thai version of International Consultation on Incontinence Questionnaire Urinary 
Incontinence Quality of Life (ICIQ-LUTSqol), which comprises 20 items. The ranges from 
19 to 76, with greater values indicating increased impact on quality of life. ICIQ-LUTSqol is 
a validated Thai adaptation of King's Health Questionnaire (KHQ).(29) 
• Global Improvement score is evaluated by Thai version of Patient Global Impression of 
Improvement (PGI-I).(30) 
 
Objective assessment 
• Urethral maturation index (UMI) and maturation value (MV) 
• Vaginal pH 
 
7.4) Study procedure 
I. First Visit and Recruitment 
Participants will be recruited from the urogynecology clinic, menopause clinic, and urology 
clinic using hospital and public advertisement. An information sheet in Thai language will be 
provided to health care providers in the designated OPD which can ensure the providers convey the 
correct study information to potential participant. Participants will only be able to enroll into this 
study one time. 
Informed Consent 
Individuals with at least one storage symptom of LUTS presenting to clinics in selected 
OPD will undergo the informed consent process by principal investigator prior to initiating 
screening procedures. Consent form in Thai language describing in detail the study procedures will 
be given to the participant privately in a separate room, and extensive discussion of risks and 
possible benefits of participation in this study will be provided. Participation will be informed that 
they may withdraw consent at any time throughout the course of this study.  
Screening  
After documentation of informed consent, principal investigator will interview the 
participant and perform vital signs and physical examination. The urinalysis will be performed by 
urinalysis test strip before the start of the study. Screening procedures are as follows; 

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• Pelvic exam 
• Pelvic floor muscle strength measured as Brink scale 
• POP-Q, Stress test 
• Gynecologic ultrasonography with post-void residual volumes (PVR) 
• Urinalysis by urinalysis test strip 
• Bladder diary: consecutive three-day diary which the time of each micturition and the 
volume voided, fluid intake, incontinence episodes, episodes of urgency and sensation will 
be recorded, as might be the activities performed during or immediately preceding the 
involuntary loss of urine.(31)  
After screening procedure, the appointment will be made for the next visit, which is one or 
two weeks after first visit.  
  
Bladder diary 
 
The study will incorporate the use of essential diagnostic tool, namely the bladder diary, to 
assess and monitor the outcomes. These assessments will be administered under the careful 
supervision of the study investigators. These diagnostic tests play a pivotal role in our research, 
serving as indispensable instruments for evaluating the progression and ultimate success of the 
treatment interventions. 
 
II. Second Visit and Randomization  
At one or two weeks after 1st visit, the following study procedure will be performed;  
• Bladder diary interpretation  
• ICIQ-FLUTS 
• ICIQ-LUTSqol 
• Vaginal pH 
• Urethral maturation index (UMI) 
After evaluating all the data collected during the screening process at the initial visit and 
combining it with the procedures conducted during the second visit, if the woman does not meet all 
study inclusion or if the woman has an exclusion criterion, she will not be enrolled into this study.  
A screening log will be kept of all who were evaluated for participation to document who is and is 
not enrolled and reason for not enrolling in this study.  

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Women who meet all the following criteria will participate the study. 
 
 
Inclusion criteria 
1. Presenting with storage phase symptom score more than/equal to 1 evaluated by the 
ICIQ-FLUTS questionnaire in these items  
- 
Item 2a) nocturia and/or 
- 
Item 3a) urgency and/or 
- 
Item 5a) daytime frequency and/or 
- 
Item 9a) UUI and/or 
- 
Item 11a) SUI  
2. Being natural or surgical menopause for more than 1 year  
3. Absence of urinary tract infection or other identifiable cause  
4. Not using hormone replacement therapy or any route of estrogen within 4 weeks 
5. Never undergone onabotulinumtoxinA therapy, PTNS, or neuromodulation for OAB 
treatment  
6. Willing to adhere to the research protocol and actively participate in the scheduled 
follow-up appointments as delineated within the framework of this study 
Exclusion criteria 
1. Contraindication for estrogen therapy: undiagnosed abnormal vaginal bleeding, previous 
thromboembolic event, breast cancer, gynecologic/genitourinary malignancy, active liver 
disease 
2. Pelvic organ prolapse of anterior compartment stage III and IV  
3. Immunocompromised patient or taking immunosuppressant drug 
4. History of antibiotics drug use within the past 7 days 
5. History of bladder outlet obstruction 
6. History of using anti-muscarinics, β3-adrenoceptor agonists, vaginal energy-
based devices (laser and radiofrequency) or electromagnetic energy-based therapies 
within the past 2 weeks 
7. History of documented positive urine culture in the past 6 weeks 
8. Have an allergic reaction to study’s drug 
9. Any other significant finding that in the opinion of the investigator would increase the 
risk of having an adverse outcome from participating in this study 
 
 
Women who meet inclusion criteria will be randomized with block randomization technique 
and a ratio of treatments of 1:1. The sequence generation will be performed under supervision of a 
senior statistician at the section of Clinical Epidemiology and Biostatistics in Ramathibodi Hospital. 

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The random sequences will be generated using Stata version 18. This is an automated process with 
no interfere from the investigators.  
Upon a subject's successful enrollment in the clinical trial following eligibility assessment, 
principal investigator or research personnel shall provide participants with a comprehensive Thai 
guidebook for behavior modification which is designed to ensure the harmonization of knowledge 
and behaviors among all trial participants. This guidebook was developed and published by IUGA 
and will be distributed by research assistants.     
Trial Treatment: Dosage Form, Regimen, Route of Administration 
• 17-Estradiol 10 mcg (Femiest Haupt Pharma Munster GMBH, Muenster, Germany 
(ESTRADIOL HEMIHYDRATE 0.0103 milligram, pH 6.94) 
 
17-Estradiol will be administered to group A treatment arm intravaginally. The assigned 
dosage will be one vaginal tablet daily for two weeks then one vaginal tablet twice a week for 10 
weeks. Participants will be carefully monitored for any changes in clinical condition in each follow-
up visits using logbook. It is important to clarify that we are not using 17-Estradiol (Vagifem®) 25 
mcg as the study drug. Although 17-Estradiol 25 mcg is frequently mentioned in literature reviews 
and has been historically used for treating vaginal dryness, this drug is currently not available in 
Thailand. Therefore, we are using 17-Estradiol (Femiest) 10 mcg as an alternative for our study 
drug. Notably, 17-Estradiol 10 mcg has been shown to provide a therapeutic dose equivalent to 
that of 17-Estradiol 25 mcg for treatment, as stated by the FDA. This decision ensures that 
participants receive an effective and appropriate dosage while adhering to the availability of 
medications in our region. 
• Placebo  
 
Placebo will be administered to group B treatment arms intravaginally. The assigned dosage 
will be one vaginal tablet daily for two weeks then one vaginal tablet twice a week for 10 weeks. 
Participants will be carefully monitored for any changes in clinical condition in each follow-up 
visits. Placebo is produced by Chulalongkorn University Drug and Health Products Innovation & 
Promotion Center, Faculty of Pharmaceutical Sciences, Chulalongkorn University. Details for 
presentation and packing are as follows; 
Appearance   
 
White, round, biconvex with "E" imprint one side  
Weight    
 
106 - 110 mg  
Hardness  
 
4.1 - 4.8 kP 

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Thickness  
 
3.26 - 3.3 mm 
Diameter  
 
6.02 - 6.03 mm 
Disintegration time   
4 min 58sec - 7min 42sec 
pH 
 
 
7.18  
Placebo contains  
1. Agglomerated Lactose 
 
2. Microcrystalline cellulose PH 102 
 
3. Magnesium stearate 
Both subject groups will be counseled on potential signs/symptoms of adverse events and 
post-marketing experience, such as breast pain, peripheral edema, and postmenopausal bleedings. 
Due to the vaginal administration and minimal systemic absorption, it is unlikely that any clinically 
relevant drug interactions will occur with 17-Estradiol. However, interactions with other locally 
applied vaginal treatments will be considered and advised to the patients. If a dose is forgotten, 
participants will be advised that it should be taken as soon as he/she remembers. A double dose 
should be avoided. 
Random allocation 
Each participant in the study will receive a prescription for the research drug, an adequate 
supply to cover a 12-week treatment duration. This drug will be uniformly packaged within 
identical containers, featuring a unique alphanumeric code and detailed usage instructions on the 
label, thereby avoiding the inclusion of the original medication name. The data analysis will be 
performed by a statistician who will be the sole individual privy to the allocation of drug A or drug 
B to their respective groups. It is essential to note that the statistician will remain unaware of which 
of the two is the active drug and which is the placebo. Additionally, an independent statistician will 
maintain a sealed randomization list as a contingency. Allocation will be as follows; 
Group A 
17-Estradiol 10 mcg (Femiest Haupt Pharma Munster GMBH, Muenster, Germany 
(ESTRADIOL HEMIHYDRATE 0.0103 milligram) 
Group B 
Placebo  
 
 
 
 

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III. Third Visit  
At four weeks after 2nd visit, the following study procedure will be performed;  
• Bladder diary, the participant will be reminded to provide a three-day diary prior to 
schedule visit date. 
• ICIQ-FLUTS 
• ICIQ-LUTSqol 
• PGI-I 
• Vaginal pH 
• Urethral maturation index (UMI) 
Participants will also have clinical evaluation and vital signs measurements evaluated. 
Compensation for participants will be provided.  
 
IV. Fourth and Final Visit  
At eight weeks after 3rd visit, the following study procedure will be performed;  
• Pelvic exam 
• Pelvic floor muscle strength measured as Brink scale 
• Bladder diary, the participant will be reminded to provide a three-day diary prior to 
schedule visit date. 
• ICIQ-FLUTS 
• ICIQ-LUTSqol 
• PGI-I 
• Vaginal pH 
• Urethral maturation index (UMI) 
Participants will also have clinical evaluation and vital signs measurements evaluated. 
Compensation for participants will be provided. Overall study procedures and flow diagram for study 
are outlined in topic No.8 Protocol flow chart.   
 
7.5) Sample Preparation, Processing, Handling, Storage 
 
• Vaginal pH  
 
To assess pH, a piece of litmus paper (MQuant® pH-indicator stripe) will be placed on the 
lateral vaginal wall until moistened. A pH of 4.6 or greater indicates vulvovaginal atrophy 

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(VVA)(32). According to a preliminary double-blinded study in 67 symptomatic postmenopausal 
women confirmed that atrophic vaginitis is associated with an increase in the lateral wall vaginal 
pH and this finding is paralleled by similar changes in pH in the urethra. Also, locally applied 
vaginal conjugated estrogen cream can normalizes the pH in the vagina and urethra Thus, the 
testing of the vaginal pH serves both as a surrogate for evaluating urethral pH and as a monitor of 
compliance with treatment.(33,34) 
• Urethral maturation index  
 
Cytohormonal urethral analysis serves as the definitive diagnostic approach for female 
urogenital atrophy in both clinical and research contexts, primarily owing to the pivotal role of 
estrogen in facilitating the maturation of the urethral epithelium. The urethral maturation index 
(UMI) emerges as a promising indicator of the estrogenic status within the body, derived from the 
ratio assessment of parabasal, intermediate, and superficial cells within a set of 100 cells. The 
interpretation of UMI values is closely associated with distinctive physiological conditions, wherein 
a decline in estrogen levels or the onset of menopause is characterized by elevated proportions of 
parabasal and intermediate cells compared to the reproductive age phase.(34) 
Procedure: We will use wet cotton buds coated with NSS solution to gently swab the distal urethra 
for cytologic preparation. Smear slides will be prepared for cytologic evaluation by immediately 
immersing them in 95% alcohol for 24 hours and staining them with a Papanicolaou stain. The 
urethral epithelial cell will be examined under a microscope and classified into three types: 
parabasal cells, intermediate cells, and superficial cells by an independent cytologist.(35) Two 
hundred cells were counted at each high-power field which was randomly chosen. Six high-power 
fields were evaluated and the mean of the cell numbers was calculated.(36) 
Maturation value (MV)(37) 
MV = (% parabasal cells × 0) + (% intermediate cells × 0.5) + (% superficial cells × 1.0)   
Interpretation: (34,38)  
MV = 0-49 will indicate low estrogen effect 
MV = 50-64 = moderate estrogen effect 
MV = 65-100 = high estrogen effect  
• Urinalysis (dipstick test strip) 
 
The procedure commonly incorporates the utilization of CybowTM 10-urinalysis test strip, 
which are advanced diagnostic tools designed to analyze multiple urine parameters simultaneously. 
Initially, a fresh clean-voided midstream urine sample is collected and visually inspected for its 

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color, clarity, and odor. Subsequently, the Cybow strip is immersed into the urine specimen for a 
specific duration, enabling the strip's chemical pads to react with the components in the urine. These 
pads, strategically embedded with reagents, undergo color changes in response to the presence and 
concentration of specific analytes, including but not limited to pH, protein, glucose, ketones, 
nitrites, leukocytes, bilirubin, and urobilinogen. The strip is then carefully removed and placed on a 
flat surface, allowing for accurate color comparison with standardized color charts to quantify the 
concentration of each parameter. For this research purpose, exclusion of urinary tract infection is 
imperative for eligible criteria.  
Interpretation: In the event of leukocytes displaying a concentration of one plus (1+) or higher, or 
a positive nitrite result, these findings could be indicative of a urinary tract infection (UTI). It is 
imperative to note that the presence of a UTI serves as an exclusion criterion during the evaluation 
process.(39) 
 
7.6) Statistical analysis   
Demographics for enrolled participants will be tabulated and described for each study group 
including age, ethnicity, occupation etc. All clinical and laboratory data related to objectives will be 
summarized and compared for each group. Appropriate statistics will be calculated to summarize 
and compare the range, means, medians, etc., and variability of numerical data. Confidence limits 
(95%) for means, geometric means and proportions will be calculated. Graphical summaries (e.g., 
box plots, histograms) will be used to describe and compare distributions of numeric variables. 
Frequency tables will be used to summarize distributions for discrete data. All clinical and 
laboratory data related to outcomes will be summarized and compared in the two regimens. T-tests 
and chi-square tests will be used to assess the statistical significance of differences in two means 
(possibly log-transformed) or proportions. Descriptive summaries of serious adverse events, subject 
discontinuations due to adverse events, and potentially clinically significant abnormal values 
(clinical laboratory or vital signs) will also be provided. 
 
7.7) Study Subjects 
Collect data and publicizing invitations to participate in research “Efficacy of Vaginal 17-
Estradiol on the Urinary Storage Symptoms in Postmenopausal Women: A Randomized Double-
blind, Placebo-controlled Study” in Outpatient departments, Department of Obstetrics & 
Gynecology, Faculty of Medicine Ramathibodi Hospital 

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7.8) Research site 
Outpatient departments, Department of Obstetrics & Gynecology, Faculty of Medicine 
Ramathibodi Hospital 
 
7.9) Research plan 
 
Activities 
2023 
2024 
2025 
8 
9 
10 
11 
12 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
1 
2 
3 
1. Literature review and 
proprosal preparation 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
2. Proposal submission and 
approval 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
3. Data collection and 
analysis 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
4. Manuscript writing and 
submission 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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8. Protocol Flow Chart 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Eligible Participants 
(n=86) 
Random allocation 
Group A 
Behavioral modifications + vaginal 17-Estradiol 
(n = 43) 
Group B 
Behavioral modifications + Placebo 
(n = 43) 
Followed Up 4 weeks 
Followed Up 4 weeks       
Completed 12 weeks 
Completed 12 weeks       
Analyzed 
Analyzed 
Bladder diary 
ICIQ-FLUTS 
ICIQ-LUTSqol 
Vaginal pH 
UMI  
Assessed for Eligibility 
Exclusion criteria 
Bladder diary 
Urinalysis  
Bladder diary 
ICIQ-FLUTS 
ICIQ-LUTSqol 
Vaginal pH 
UMI 
PGI-I 
1st visit 
Followed Up 1-2 weeks 
2nd visit 
3rd visit 
4th visit 
Bladder diary 
ICIQ-FLUTS 
ICIQ-LUTSqol 
Vaginal pH 
UMI  
PGI-I 

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9. Sample size calculation and inclusion/exclusion criteria 
• Sample size calculation 
The sample size is calculated using two independent population proportions formula. 
 
 
Proportion in group1 (p1) = 0.63   
Proportion in group2 (p2) = 0.32 
alpha() = 0.05 , Beta() = 0.2 
 
 
According to Eriksen PS, Rasmussen H (1991)(14) , after 12 weeks, 62.8% of the women with 
urological symptoms underwent a change for the better in the estradiol (Vagifem®) group compared 
to 32.4% in the placebo group. (P < 0.001). With sample size (n) equal to 39 subjects per group and 
add drop out at rate of 10%, which is equal to 43 subjects per group. The total number of sample 
size will be 86 subjects.  
• Inclusion criteria 
1. Presenting with storage phase symptom score more than/equal to 1 evaluated by the ICIQ-
FLUTS questionnaire in these items  
- 
Item 2a) nocturia and/or 
- 
Item 3a) urgency and/or 
- 
Item 5a) daytime frequency and/or 
- 
Item 9a) UUI and/or 
- 
Item 11a) SUI  
2. Being natural or surgical menopause for more than 1 year  
3. Absence of urinary tract infection or other identifiable cause  
4. Not using hormone replacement therapy or any route of estrogen within 4 weeks 
5. Never undergone onabotulinumtoxinA therapy, PTNS, or neuromodulation for OAB treatment  
6. Willing to adhere to the research protocol and actively participate in the scheduled follow-up 
appointments as delineated within the framework of this study 

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• Exclusion criteria 
1. Contraindication for estrogen therapy: undiagnosed abnormal vaginal bleeding, previous 
thromboembolic event, breast cancer, gynecologic/genitourinary malignancy, active liver disease 
2. Pelvic organ prolapse of anterior compartment stage III and IV  
3. Immunocompromised patient or taking immunosuppressant drug 
4. History of antibiotics drug use within the past 7 days 
5. History of bladder outlet obstruction 
6. History of using anti-muscarinics, β3-adrenoceptor agonists, vaginal energy-based devices (laser 
and radiofrequency) or electromagnetic energy-based therapies within the past 2 weeks 
7. History of documented positive urine culture in the past 6 weeks 
8. Have an allergic reaction to study’s drug 
9. Any other significant finding that in the opinion of the investigator would increase the risk of 
having an adverse outcome from participating in this study 
10. Duration of data collection  
It will take about 12 months for data collection. analyses and manuscript writing. All steps will 
start after the IRB ethical approval. If the research continues beyond the specificized time or Ethics 
committee License expired, the investigators will request for renewal every year. 
11. Risk of the study  
 
There will be no physical, psychological or legal risks related to this study. Estradiol 10 mcg 
has a low dose and is safe. The adverse events observed with a higher frequency in patients treated 
with Estradiol 10 micrograms as compared to placebo and which are possibly related to treatment 
are presented as follows.(40)  
stem organ class 
Common 
≥1/100 to <1/10 
Uncommon 
≥1/1,000 to <1/100 
Rare 
≥1/10,000 to <1/1,000 
Infections and infestation 
 
Vulvovaginal mycotic 
infection 
 
Nervous system disorders 
Headache 
 
 
Gastrointestinal disorders 
Abdominal pain 
Nausea 
 
Reproductive system and 
breast disorders 
Vaginal haemorrhage, 
vaginal discharge or vaginal 
discomfort 
 
 
Skin and subcutaneous 
tissue disorders 
 
Rash 
 
Investigations 
 
Weight increased 
 
Vascular disorders 
 
Hot flush 
Hypertension 
 

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During the course of the research, participants will be required to undergo pelvic 
examinations. This critical procedural step is imperative for the comprehensive evaluation of 
storage symptoms, aiming to assess the severity and progression of symptoms. The eligibility of 
research participants is also contingent upon the results derived from these pelvic assessments, 
ensuring the selection of suitable candidates and the appropriate alignment of the study's objectives 
with the participants' health profiles. Such meticulous monitoring and assessment not only uphold 
the integrity of the research but also prioritize the well-being and safety of the participants, 
reinforcing the ethical and scientific standards governing the study. 
 
 
Distal urethral cell sampling utilizing a cotton bud will be conducted on the study 
participants. This non-invasive procedure is anticipated to last approximately three to five seconds 
and might induce mild discomfort. The sampling process is scheduled for the second, third and 
fourth visits, primarily to facilitate a comprehensive evaluation of the cellular composition in the 
distal urethra. Despite the potential discomfort, this meticulous sampling approach is instrumental 
in gathering crucial data, enabling a deeper understanding of the cellular dynamics and potential 
changes within the urethral microenvironment over the course of the study. 
12. Benefit of the study  
 
This research will be the foundation of knowledge in the future by using objective and 
subjective assessments results to determine the relationship between low dose vaginal estrogen and 
storage symptoms of LUTS in postmenopausal women. This study will also increase efficiency in 
the care of patients with overactive bladder or storage symptoms of LUTS, especially in 
postmenopausal women, by applying the knowledge gained from this research to provide patients 
with the best and most appropriate treatment, supported by research outcome and information from 
this research as well.  
13. Ethic consideration 
1. Respect for person 
 
Participants will receive all information about the research from an information sheet and will 
be asked to consent for the research, giving them time to make their own decisions. Then sign it in 
writing and have the right to refuse to participate in the research at any time. And the information in 
the research will not be personally identifiable. Instead, it will be assigned a code for research to 
maintain patient confidentiality. 
2. Beneficence/Non-maleficence 
 
Treatment of the participants in this study is standard treatment following steps from a safe and 
easy method for the patients first, namely receiving advice on lifestyle modification that causes 

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storage symptoms of LUTS. The intervention in the study has been certified by the Thai FDA, 
which contains a low dosage and fewer side effects than other drugs used to treat storage symptoms 
of LUTS. 
3.  Justice 
 
Research has clear inclusion and exclusion criteria. All patients have an equal chance of 
receiving treatment using a systematic random sampling method. There is no discrimination against 
any individual resulting in equal benefits. 
14.  Compensation to research participants in the event of harm or adverse effects on the 
subject.   
The research participants will receive a compensation of 200 THB during the initial visit, 
which is intended to cover the time and effort invested in the informed consent process and 
preliminary screening procedures. Following this, during the second visit, once the participant 
qualifies for enrollment in the trial, an additional compensation of 300 THB will be provided. 
Subsequently, for both the third and final visits, each participant will be remunerated with 300 THB 
for every scheduled appointment. These monetary reimbursements not only acknowledge the 
participants' valuable contribution to the study but also serve to offset any inconveniences or 
expenses associated with their active involvement in the research. Such compensation strategies are 
designed to uphold ethical considerations and ensure that the participants' dedication and 
engagement in the research are duly recognized and appreciated.  
Medical care in case of research-related injury or serious event on either an emergency or 
routine basis will be provided free of charge according to the local standard of care by qualified 
medical personnel at Ramathibodi Hospital.  
15. Conflict of Interests and Budget 
 
No conflict of interest is anticipated, as this research study does not engage private enterprises. 
The entire budget allocation is projected to be sourced through a funding application submitted to 
the research grant provided by the Faculty of Medicine, Ramathibodi Hospital, Mahidol University. 
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