Clinical trial NCT04301934
Conditions: Urinary Tract Infections
Sponsor: Mayo Clinic
Start date: 2020-06-01; Completion date: 2023-04-30
Enrollment: 12 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
Randomized controlled trial to determine clinical and microbiome difference between fractional CO2 Laser and vaginal estrogen in treating patients with recurrent urinary tract infection (UTI).

Detailed description:
Recurrent urinary tract infection (rUTI) is common in postmenopausal women and can lead to significant bothersome symptoms. This clinical scenario is a difficult and common clinical problem seen by the urologist and urogynecologist with limited options for treatment. Overuse of antibiotics has led to significant resistant microorganisms and can have adverse side effects such as C. Dificile colitis. Current treatments include topical vaginal estrogen and prophylactic antibiotics. Other supplements such as cranberry pills, methenamine and D-mannose have varying levels of evidence and efficacy.

Fractional CO2 LASER vaginal therapy has recently been studied for the treatment of GSM. LASER therapy is currently commercially available and has FDA clearance for use in gynecology. Current evidence shows that with treatment, histologic changes of the vaginal epithelium have shown regeneration to a premenopausal state, along with subjective improvement in GSM symptoms and sexual function. Recent literature has shown improvements in thickening of vaginal epithelium, decreased vaginal pH and improvement in the vaginal microbiome. Based on this, the investigators propose fractional CO2 laser may have a positive impact on women with rUTI.

1. The investigators therefore hypothesize that fractional CO2 LASER vaginal therapy is non-inferior to topical vaginal estrogen therapy for the treatment of rUTI.

   a. Primary Outcome: Improvement in recurrence of culture positive UTI
2. The investigators also hypothesize that LASER therapy will improve the urinary and vaginal microbiome, decreasing the uropathogenic presence and increasing Lactobacillus.

   1. Measurement 1: The number of UTI with UPEC isolated from patients
   2. Measurement 2: The pH of the vagina as a proxy for presence of Lactobacillus sp.
   3. Measurement 3: Categorize vaginal and urinary microbial communities
3. The investigators hypothesize that LASER therapy will improve symptoms of GSM and be associated with regenerative change in vaginal histology.

   1. Measurement 1: MESA and DIVA surveys of symptoms
   2. Measurement 2: Observations of vaginal biopsy between.

Menopausal women will be recruited if subjects have recurrent UTI defined as 3 culture positive UTIs in 1 year or 2 culture positive UTIs in 6 months. Subjects will be randomized to standard of care with topical vaginal estrogen or 3 treatments of fractional CO2 LASER of the vagina. Samples will be collected to show microbiome and histologic changes with treatment.

Samples include:

Vaginal culture Urine culture vaginal biopsy (optional).

Validated questionnaires and clinical recurrence of UTI will also be gathered over 6 months.

Arms: arm 1: Women randomized to vaginal estrogen therapy will be offered vaginal cream conjugated estrogen (Premarin) 0.5 gm per vaginal twice weekly or estradiol (Estrace): 1gm per vaginal twice weekly arm 2: Women randomized to the laser therapy group will undergo 3 treatments, 6 weeks apart.
Interventions: intervention 1: Mona Lisa Touch intervention 2: Conjugated estrogen intervention 3: Estradiol
Intervention descriptions: intervention 1: Fractional CO2 LASER intervention 2: Current standard treatment for recurrent UTIs in postmenopausal women intervention 3: Current standard treatment for recurrent UTIs in postmenopausal women

Protocol documents:
Prot_SAP_000.pdf: 
 
 
Fractional CO2 Vaginal LASER Therapy for 
Recurrent Urinary Tract Infection 
 
NCT04301934 
 
April 1, 2022 


Page | 1 
 
Fractional CO2 Vaginal LASER Therapy for Recurrent Urinary Tract Infection 
PI: Johnny Yi MD 
Co-I:  
 
 
Protocol Version 5.0- April 1, 2022 
 
Background 
Urinary tract infections (UTI) are common in women with over 50% of women 
having a UTI at some time in their lifetime.[1]  As the most common bacterial infection in 
women, UTI’s have a significant financial and social burden on women, with an 
estimated greater than $1 billion spent per year in lost work alone.[2]  Recurrent UTIs 
(rUTI) are defined by 3 UTI’s in 1 year, or 2 UTI’s in 6 months.[1]  Up to 10% of 
menopausal women will be treated for UTI each year, with 5% of them developing a 
recurrence within 1 year.  Recurrent UTI is a common clinical problem seen by the 
Urologist and Urogynecologist, who have limited options in terms of treating and 
preventing further infections in these women.  Recurrence of UTI can be difficult to treat 
due to antibiotic resistance and comorbidities associated with repetitive antibiotic usage.  
 
Most UTIs are caused by uropathogenic bacteria that originate from the 
gastrointestinal tract with Escherichia coli responsible for the vast majority of infections.  
Menopausal women are at higher risk of developing infections as their vaginal flora 
shifts with significant decrease in Lactobacillus and increased pH.  This degenerative 
shift in flora and lack of estrogen to the vagina is also responsible for the symptoms 
associated with genitourinary syndrome of menopause (GSM).  GSM symptoms include 
vaginal dryness, painful intercourse, vaginal burning, irritation and dysuria.  
Approximately 50% of menopausal women are affected by GSM.  Currently, vaginal 
estrogen is an effective treatment for menopausal women with GSM and also for 
prevention of rUTI.  In a randomized controlled trial of vaginal estrogen to placebo for 
women with rUTI, Raz et al showed a significant decrease in UTI from 5.9 episodes per 
patient-year to 0.5 episodes per patient year.  (p<0.001)  Restoring estrogen to the 
postmenopausal vaginal skin may work by regenerating the vaginal microbiome and 
protective environment from uropathogens.  However, its impact on the urinary 
microbiome has not been studied.  Novel sequencing techniques allow us to now 
investigate the urinary microbiome which previously has been thought to be non-
existent.[3] 


Yi 
 
Protocol Version 4.0 
Fractional CO2 Laser Therapy for rUTI 
 
April 6, 2021 
 
Page | 2 
 
 
Fractional CO2 LASER vaginal therapy has recently been studied for the 
treatment of GSM.  LASER therapy is currently commercially available and has FDA 
clearance for use in gynecology.  Current evidence shows that with treatment, histologic 
changes of the vaginal epithelium have shown regeneration to a premenopausal state, 
along with subjective improvement in GSM symptoms and sexual function.  Using 
LASER therapy, Zerbinati et al described response by the vaginal epithelium with 
thickening, superficial shedding and renewing of stratified epithelium, which was 
maintained 2 months after treatment.[4]  Athanasiou et al also described the impact of 
LASER therapy on the vaginal flora, decreasing the vaginal pH to 4.7 (p<0.001) and 
increasing the Lactobacillus colonies from 30% to 79%.[5]  Based on the impact this 
promising new regenerative therapy has on the vaginal epithelium and microbiome, we 
anticipate this may have a positive impact on women with rUTI.   
1. We therefore hypothesize that fractional CO2 LASER vaginal therapy is 
non-inferior to topical vaginal estrogen therapy for the treatment of rUTI.   
a. Primary Outcome: Improvement in recurrence of culture positive UTI  
 
2. We also hypothesize that LASER therapy will improve the urinary and 
vaginal microbiome, decreasing the uropathogenic presence and 
increasing Lactobacillus.   
a. Measurement 1: The number of UTI with UPEC isolated from patients 
b. Measurement 2: The pH of the vagina as a proxy for presence of 
Lactobacillus sp. 
c. Measurement 3: Categorize vaginal and urinary microbial 
communities  
 
Methods 
We propose a randomized controlled trial to evaluate the clinical difference between the 
LASER and topical vaginal estrogen in treating patients with rUTI.  We expect to enroll 
94 patients, to power this study to 80% with type I error of 5%, expecting a recurrence in 
UTI of 16% and non-inferior margin of 20%.  This is accounting for a 10% drop out rate.  
While this clinical margin is wide, this project will provide us with ample data to apply for 
external funding, along with a wealth of microbiome data to evaluate in Aim 2. 
Menopausal women with rUTI will be recruited from the Departments of Urology and 
Gynecology.  If they meet inclusion criteria, they will be randomized to treatment of 
topical vaginal estrogen or LASER therapy.  LASER therapy will be applied 6 weeks 
apart for a total of 3 treatments.  Topical vaginal estrogen will be prescribed per the 
patient’s formulary and preference.  Patients will be monitored for recurrence of culture 
positive UTI.   


Yi 
 
Protocol Version 4.0 
Fractional CO2 Laser Therapy for rUTI 
 
April 6, 2021 
 
Page | 3 
 
Laser Therapy protocol: 
Mona Lisa Touch is a fractional CO2 LASER that has applications for treatment of the 
vulva and vagina.  Patients randomized to this arm will undergo 3 treatments, 6 weeks 
apart.  Patients will be advised to avoid any vaginal creams, lubricants or ointments, 
along with vaginal intercourse 48 hours prior to and 48 hours following LASER 
treatments.   
Speculum examination will be performed and pH obtained.  Any existing vaginal 
discharge will be removed prior to therapy.  2.5% lidocaine and 2.5% prilocaine will be 
applied to the external vulva for 30 minutes to 1 hour prior to procedure.  This cream will 
be removed from the vulva prior to LASER application.   
Internal treatments settings: 
• First treatment:  Power 30W, Dwell time 1000us, Spacing 1000um, Smart 
Stack 1 
• Subsequent treatment: Power 30W, Dwell time 1000us, Spacing 1000um, 
Smart Stack 3 
External treatments settings: 
• Power 26W, Dwell time 800us, Spacing 800um, Smart Stack 1 
Any deviation from these settings will be at the discretion of the provider performing the 
LASER therapy and will be documented in the patient’s medical record.   
 
Vaginal Estrogen Protocol 
Women randomized to vaginal estrogen therapy will be offered vaginal cream.  Two 
FDA approved formulations exist and patients will be offered the choice between the 
two, which may also be dependent on their insurance formulary preference. 
1. Conjugated estrogen (Premarin):  0.5 gm per vagina twice weekly 
 
2. Estradiol (Estrace): 1gm per vaginal twice weekly 
 
Specific Aim 1:  Evaluate the number of culture positive, symptomatic UTI in women 
following randomization to vaginal estrogen and vaginal LASER therapy. 


Yi 
 
Protocol Version 4.0 
Fractional CO2 Laser Therapy for rUTI 
 
April 6, 2021 
 
Page | 4 
 
Methods: Following randomization and treatment initiation, patients will be 
monitored for recurrence of UTI.  Patients will be encouraged to call the treating 
physician for treatment antibiotics according clinical judgment.  Standard urine cultures 
will be obtained along with information about what type of antibiotic treatment was 
administered.  Validated questionnaires will also be collected to monitor symptoms of 
GSM and also of urinary incontinence symptoms (Appendix A and B).  Patients will be 
followed for 6 months with evaluations at baseline, 4 months, and 6 months.   
 
Specific Aim 2:  Characterize the urinary and vaginal microbiome at baseline and at 4 
months of patients in each group. 
 
Methods:  Baseline vaginal and catheterized urine samples will be collected 
along with samples at 4 months.  Standard catheterized urine cultures will be performed 
and if symptomatic, treated appropriately (per Aim 1).  Microbiome of urine and vaginal 
environments will then be analyzed using 16S ribosomal DNA sequencing along with 
shotgun metagenomics.  For this study, we will perform sequencing and metagenomics 
on the first 16 patients in each arm (32 patients total).  The remaining collection will be 
biobanked for future analysis.  These techniques will allow us to measure the 
abundance of different bacteria, but also evaluate the genetic variation and evolutionary 
effect from treatment.  This will also allow us to evaluate for the differences in the 
microbiome between responders and non-responders in each treatment arm, 
considering factors such as UTI, symptoms, pH, etc. 
 
Vaginal Estrogen 
arm 
Baseline 
4 months 
6 months 
(if can be completed within 
the funding period) 
Informed consent 
X 
 
 
Vaginal pH 
X 
X 
X 
Vaginal swab 
collection 
X 
X 
X (banked) 
Urine collection 
X 
X 
X (banked) 
MESA questionnaire 
X 
X 
X 
DIVA questionnaire 
X 
X 
X 
 
 
 
 
 


Yi 
 
Protocol Version 4.0 
Fractional CO2 Laser Therapy for rUTI 
 
April 6, 2021 
 
Page | 5 
 
LASER arm 
Baseline 1st 
treatment 
6 weeks 
(2nd treatment) 
3 months 
(3rd treatment) 
4 
months 
6 months 
(if can be 
completed within 
the funding 
period) 
Informed 
consent 
X 
 
 
 
 
 
Vaginal pH 
X 
X 
X 
X 
X 
X 
Vaginal swab 
collection 
X 
 
 
 
X 
X (banked) 
Urine 
collection 
X 
 
 
 
X 
X (banked) 
MESA 
questionnaire 
X 
 
 
 
X 
X 
DIVA 
questionnaire 
X 
 
 
 
X 
X 
 
Inclusion Criteria: 
• Female patient >18 years old 
• Postmenopausal status, documented by prior bilateral salpingo-
oophorectomy, or absence of menses >12 months 
• Recurrent urinary tract infections as defined by 3 culture positive urine 
cultures in the last 12 months, or 2 positive urine cultures in the last 6 months. 
o Positive urine cultures defined by >100K colony forming units of 1 or 2 
bacterial species on clean catch sample, or >1000 colony forming units 
of 1 or 2 bacterial species on sample via straight catheterization. 
• Patients on vaginal estrogen must undergo a 1 month washout period prior to 
initiation of the trial. 
Exclusion Criteria: 
• Hematuria without appropriate workup 
• Pelvic organ prolapse beyond the hymen 
• Clinically relevant urinary retention 
• Pelvic reconstructive surgery within 6 months 
• Reconstructive pelvic surgery with “mesh kits” 
• Clinically relevant nephrolithiasis 
• History of breast cancer 
• Contraindication to topical estrogen therapy 
• Anticoagulation therapy 
• Prior pelvic or vaginal radiation therapy 
• Prior gynecologic malignancy 
• Undiagnosed genital bleeding 


Yi 
 
Protocol Version 4.0 
Fractional CO2 Laser Therapy for rUTI 
 
April 6, 2021 
 
Page | 6 
 
 
Subject Recruitment 
Patients will be recruited from both the Departments of Gynecology and Urology.  We 
estimate that the two departments evaluate >200 women with rUTI per year.  We 
anticipate realistic recruitment of 1/3 of these patients that meet inclusion criteria.  We 
will recruit 94 patients per power calculation described above and anticipate completion 
of recruitment in 2 years.  For Specific Aim 2, we will perform microbiome analysis on 
1/3 of these patients for a total of 32 patients.  Further collection of samples will be 
frozen for future analysis once external funding is secured. 
Patients may also be recruited from Social Media, either from a Mayo-approved 
Facebook ad or from Twitter.   
 
Subject Randomization 
Patients will be randomized using 6 as a block size to make sure the balance of the 
randomization at any time point. The randomization will be created by 
 and 
stored in REDcap. The coordinator who is going to do the randomization will get access 
to the REDcap randomization schedule and get the treatment assignment from there for 
the eligible patients.  
 
Data Collection 
Demographic information will be collected, which will include age, race, and 
comorbidities.  Urinary cultures will be collected from prior to studies and subsequent to 
enrollment, with details of results documented.  MESA questionnaire will be used to 
assess baseline and subsequent urinary symptoms to evaluate for change following the 
procedure as a secondary outcome.  DIVA questionnaire will assess change to sexual 
function and symptoms of GSM at baseline and following treatment.  All the data will be 
collected and entered in the REDcap database.   
 
Statistical Analysis 
The 4-month rUTI prevalence and 6-month rUTI prevalence will be estimated using 
binomial method and compared between the two groups using Chi-square test or 
Fisher’s exact test when applicable.  The 6 month rUTI recurrence rate will be estimated 
using Poisson method and compared between the two groups using GEE method.   


Yi 
 
Protocol Version 4.0 
Fractional CO2 Laser Therapy for rUTI 
 
April 6, 2021 
 
Page | 7 
 
 
Overdispersion will be evaluated and if presented, negative binomial method will be 
used to estimate the rUTI recurrence rate.  
Time to first recurrent UTI will also be compared between the two groups using log-rank 
test.  
The MESA responses, DIVA responses and vaginal histology changes over time will be 
summarized and compared between the two groups using general linear mixed model 
or generalized linear mixed model when appropriate.  
 
Clinical outcomes 
Microbiome analysis 
Whole population DNA will be extracted directly from vaginal and urine samples for both 
16S and shotgun-metagenomic sequencing. Sequenced reads will be mapped to the 
human genome, in order to remove any host DNA contamination. For both 16S and 
shotgun-metagenomic sequences, reads not aligning to the human genome will then be 
assigned taxonomic grouping using the Kraken software package. Abundance of 
taxa/species will be determined by calculating counts per million reads. Changes in 
microbiome composition will then be determined by comparing the Shannon’s diversity 
index between treatments and sampling timepoints using ANOVA (Yang et al, 2016).  
This type of analysis will allow us to further identify the unique microbiome that exists in 
the urinary bladder.  This would not be possible with standard urinary cultures, as 
without an abundance of 1-2 species, urinary cultures are limited in identifying an 
existing low count microbiome.  This study will add to the microbiome literature in 
several ways.  This type of analysis has not been described using vaginal cultures in 
patients with rUTI.  Furthermore, analysis of change with treatment has not been 
described in this population as well, from both the urinary and vaginal microbiome 
perspective.  Understanding this information has potential to develop new therapies that 
may help in treating menopausal women with rUTI. 
 
Recurrent UTI 
Our goal is to test the hypothesis that LASER treatment is non-inferior to vaginal 
estrogen in rUTI patients.  If LASER is found to be effective, it would offer clinicians a 
modality of treatment for these patients that avoids antibiotics and hormone 


Yi 
 
Protocol Version 4.0 
Fractional CO2 Laser Therapy for rUTI 
 
April 6, 2021 
 
Page | 8 
 
replacement.  It would also offer hope to patients for whom hormones are 
contraindicated. 
 
Strategic Initiatives 
This translational clinical study fulfills multiple strategic initiatives of the Mayo Clinic 
Enterprise.  This novel therapy provides a new service line in regenerative medicine 
treating women with genitourinary syndrome of menopause with LASER therapy.  
Expanding the clinical utility based on level 1 evidence that we would provide would 
fulfill the clinical goals of the Center for Regenerative Medicine.  Given that 50% of 
menopausal women experience symptoms of GSM, this research has potential to 
impact millions of women in the US and throughout the world.   
Incorporating microbiome analysis to this clinical trial opens up further hypotheses of 
how we can affect an aging microbiome to create a more favorable one within the 
human body utilizing regenerative technology.  This metagenomic data will allow us to 
better understand this specific population, menopausal women with rUTI, and we may 
be able to identify avenues to create novel individualized therapies depending on the 
findings of the microbiome analysis.  This fulfills the Center for Individualized Medicine’s 
goals of targeted therapy.