The effect of the probiotic supplement—ASTARTE™—on the reduction of recurrent urinary tract infection in women aged 18-40 years: protocol for a randomized, double-blind, placebo-controlled study

Abstract

Background

Urinary tract infection (UTI) is one of the most common bacterial infections, contributing to increased antibiotic consumption and high healthcare costs. Clinical studies suggest that probiotics can be effective dietary supplements reducing the risk factors for the development of infections in the intestine and vagina. In this study, we investigate the effect of the orally ingested probiotic combination ASTARTE™ on the composition of bacteria in vaginal and fecal samples, as well as on the occurrence of bacteriologically confirmed UTIs in women of reproductive age.

Methods

A randomized, placebo-controlled, double-blind study evaluating the effect of the probiotic combination ASTARTE™ (Chr. Hansen A/S, Part of Novonesis Group, Denmark) on reducing the number of recurrent urinary tract infections (rUTIs) in women aged 18–40 years. Participants are randomized (1:1) to receive one daily probiotic capsule (5 × 10⁹ CFU) containing Lactobacillus crispatus LBV 88™ (DSM22566), Lacticaseibacillus rhamnosus LBV 96™ (DSM22560), Lactobacillus jensenii LBV 116™ (DSM22567), and Lactobacillus gasseri LBV 150N™ (DSM22583), or placebo, for 6 months. Assessments, including symptom questionnaires, urine culturing and microbiome analysis of vaginal and rectal samples by shotgun sequencing, are conducted at baseline, 2, 4, and 6 months. Compliance is tracked by unused capsule counts. Follow-up visits occur at 8, 10, and 12 months. The primary outcome is the incidence of symptomatic bacteriologically confirmed UTIs during the 6 months intervention period. The secondary objective is the effect of ASTARTE™ on changes in vaginal and gut microbiome.

Discussion

This randomized, placebo-controlled, double-blind study investigates the effectiveness of ASTARTE™ in restoring beneficial microbiomes and reducing UTI recurrence. The findings will provide valuable evidence on the effectiveness of a non-antibiotic strategy for the prevention of rUTI as well as insights into the complex interactions between the vaginal and gut microbiome and the risk of rUTI.

Trial registration

ClinicalTrials.gov Identifier: NCT05553652. Registered 21 September 2022.

Introduction

Background and rationale {6a}

UTIs are among the most prevalent infections and are commonly caused by enteric bacteria such as Escherichia coli and Klebsiella pneumoniae [1–4]. In some cases, vaginal bacteria, including Streptococcus agalactiae, may also be implicated [5, 6]. These infections contribute to high antibiotic usage and place a considerable financial burden on healthcare systems [1, 7]. UTIs encompass various conditions, including cystitis, pyelonephritis, prostatitis, urosepsis, and catheter-associated UTIs [8].

Research indicates that UTI prevalence increases with age [2, 8]. Among women over 65 years old, the prevalence is approximately 20%, which is twice as high as in the general female population, where it stands at around 11% [2–4]. It is estimated that 50% to 60% of adult women will experience at least one UTI in their lifetime, with nearly 10% of postmenopausal women reporting a UTI within the past year [2].

A study conducted in the United States suggests that sexually active women aged 18–39 years have a higher UTI risk than their non-sexually active counterparts [2]. After a first episode of a UTI, 27% of women have a confirmed recurrence within the next 6 months [2]. Factors contributing to frequent recurrences include vaginal colonization by enteric bacteria, reduced urinary flow, incomplete bladder emptying, anatomical and functional urological abnormalities, sexual activity, urinary incontinence, and low physical activity [4, 9].

A rUTI is defined as two infections within 6 months or 3 or more infections within 1 year [1, 2]. Repeated courses of antibiotics for rUTIs may disrupt the gut and vaginal microbiota and increase the risk of antibiotic resistance [10–12]. The frequent prescription of antibiotics raises concerns about the emergence of resistant uropathogenic bacteria. Many preventive measures have shown limited success and often suffer from poor patient adherence [12, 13], emphasizing the urgent need for alternative strategies beyond continuous antibiotic use.

The composition of the gut and vaginal microbiota plays a key role in UTI susceptibility [4, 14–16]. Imbalances in the gut microbiota (dysbiosis) can contribute to chronic inflammation, weakened mucosal barrier function, and increased permeability (“leaky gut”), potentially triggering systemic immune responses and elevating UTI risk [14, 17, 18]. Vaginal dysbiosis, marked by an overgrowth of enteric bacteria, raises the risk of bacterial migration to the urethra and bladder, leading to infection [4, 9]. As the vaginal microbiota naturally fluctuates over time, a decline in protective Lactobacillus species can further heighten susceptibility to UTIs [4, 15].

Several randomized clinical trials have examined the effectiveness of probiotics in preventing UTIs, with mixed outcomes influenced by treatment duration, bacterial strains used, and patient demographics [1, 16, 19–23]. It is hypothesized that consistent probiotic supplementation may help maintain a stable microbiota composition, fostering a protective, Lactobacillus-dominant microbiota in the gut, vagina, and urinary tract, thereby potentially reducing the risk of UTIs. These studies have primarily focused on comparing the incidence of symptomatic UTIs in probiotic-treated groups versus placebo groups over both intervention and follow-up periods. Probiotics are believed to help restore microbial balance, which could contribute to reducing the frequency of rUTIs. A lower recurrence rate among individuals receiving probiotics would reinforce their potential role in UTI prevention, while simultaneously aiding in reducing antibiotic use.

Existing research suggests that probiotics such as Lactobacillus and Bifidobacterium support intestinal health by strengthening the gut mucosal barrier, enhancing immune function, and inhibiting colonization of pathogenic bacteria [1, 24–26]. Since probiotics naturally reside in the gut and vaginal microbiota, they are considered safe and may help regulate microbial communities in the urinary, vaginal, and gastrointestinal tracts, potentially reducing the risk of dysbiosis and infection. The ASTARTE™ product in our study consists of four probiotic Lactobacillus strains derived from the vaginal tract of healthy pregnant women. It has been shown to support a balanced vaginal microbiome and alleviate bacterial imbalances [27–29].

This study aims to explore whether specific combinations of Lactobacillus species can contribute to a reduction in rUTI incidence.

Objectives {7}

The primary objective of the study is to investigate the effectiveness of probiotic combination ASTARTE™ on the microbiome composition in the gut and vagina and the contribution to a reduction in rUTI incidence during 6 months of intervention in women aged 18–40 years. This is measured by the incidence of bacteriologically confirmed UTIs that need to be treated with antibiotics.

The secondary objectives of the study are as follows:

• To investigate the effect of ASTARTE™ on the number of bacteriologically confirmed UTIs that need to be treated with antibiotics within 6 and 12 months.

• To investigate the effect of ASTARTE™ on duration (number of days) of UTI episodes.

• To investigate the effect of ASTARTE™ on changes in vaginal microbiome by analyzing bacterial composition, as well as alpha and beta diversity.

• To investigate the effect of ASTARTE™ on changes in gut microbiome by analyzing bacterial composition, as well as alpha and beta diversity.

The exploratory objectives

• To investigate the effect of ASTARTE™ on antibiotic use during the 6 months intervention period by counting the number of issued prescriptions.

• To investigate the effect of ASTARTE™ on antibiotic use within 12 months by counting the number of issued prescriptions.

• To investigate the effect of ASTARTE™ on the eradication of uropathogenic bacteria in urine by analyzing cultured urine samples and quantifying uropathogenic bacteria throughout the intervention period.

• To investigate the effect of ASTARTE™ on Lactobacillus abundance in the gut and vagina.

• To investigate the effect of ASTARTE™ on UTI symptoms by using the Bristol Female Lower Urinary Tract Symptoms questionnaire (BFLUTS) within the intervention and follow-up period.

Trial design {8}

This study is designed as a superiority trial comparing ASTARTE™ to placebo. Women with rUTI will be invited to participate in a 6-month randomized, double-blind, placebo-controlled study with a 6 months follow-up period. Participants are randomly assigned 1:1 to either the probiotic group or the placebo group, and they remain in their assigned group for the duration of the study.

Methods: participants, interventions, and outcomes

Study setting {9}

Participants will be recruited based on urine samples analyzed at the Clinical Microbiology Departments of Copenhagen University Hospitals Hvidovre and Herlev, Denmark (1,145 general practitioner clinics) and Slagelse Hospital in Region Zealand, Denmark (237 general practitioner clinics). Data on inclusion/exclusion criteria, including urine culture results, UTI frequency within 1 year before recruitment, gender, and age, will be collected from laboratory records. Women with rUTIs are invited to an information and screening visit at Copenhagen University Hospital Hvidovre. Eligible participants who provide informed consent will proceed to a baseline visit (Fig. 1).

Fig. 1.

Study design. Schematic representation of the study design showing intervention and follow-up

At the baseline visit, participants will complete the score form BFLUTS [30], and urine samples and vaginal and rectal swabs will be collected. The intervention must be initiated within 7 days of completing antibiotic treatment.

Follow-up visits will occur after 2, 4, and 6 months. At each visit, participants will complete the BFLUTS questionnaire, report any side effects, and provide samples for collection. Compliance will be monitored by counting unused products, with new supplies provided as needed. A minimum of 80% product consumption is required for protocol adherence. Additional visits will be conducted for symptomatic UTIs, during which participants will receive antibiotics and continue the study. At 8, 10, and 12 months, participants will complete the BFLUTS questionnaire on urinary tract symptoms and a remote follow-up at 12 months will collect UTI episode data from hospital records.

Eligibility criteria {10}

Inclusion criteria

• Women 18–40 years of age

• rUTI (2 UTI episodes within 6 months or ≥3 UTI episodes within 1 year).

• The participants must be able to follow the protocol, have provided voluntary consent, and have signed the declaration of consent.

Exclusion criteria

• Pregnancy or planning for pregnancy.

• Breastfeeding.

• Participation in another study with probiotics within the last 30 days.

• Hypersensitivity to any ingredient in the study product.

• Patients primarily admitted for a disorder other than UTI.

• Antibiotic treatment within the last 30 days for any condition other than UTI at the time of recruitment.

Who will take informed consent? {26a}

Informed consent will be obtained by a qualified member of the research team. All participants in the study will provide written informed consent before inclusion.

Additional consent provisions for collection and use of participant data and biological specimens {26b}

No additional consent provisions are needed for the collection and use of participant data and biological specimens in this study.

Interventions

Explanation for the choice of comparators {6b}

Comparator in this study is a placebo capsule that is identical to the probiotic capsule in appearance, taste, and packaging to maintain blinding. A placebo control is used to account for the placebo effect, where participants may experience perceived or real improvements due to their expectations of treatment. This ensures that any observed effect is due to the probiotics and not psychological or other confounding factors.

Intervention description {11a}

Participants are given either probiotic capsules ASTARTE™ or placebo capsules respectively. One capsule is taken daily in the morning with food for 6 months.

Test product

The active product is an ASTARTE™ oral capsule containing a mixture of four probiotic strains with a combined potency of 5 × 10⁹ CFU/capsule, containing the following:

• 2 × 10⁹ CFU/capsule Lactobacillus crispatus LBV 88™ (DSM22566)

• 2 × 10⁹ CFU/capsule Lacticaseibacillus rhamnosus LBV 96 ™ (DSM22560)

• 0.4 × 10⁹ CFU/capsule Lactobacillus jensenii LBV 116 ™ (DSM22567)

• 0.6 × 10⁹ CFU/capsule Lactobacillus gasseri LBV 150 N ™ (DSM22583)

The product is provided by the Danish company Chr. Hansen A/S, part of Novonesis Group in vials of 31 capsules.

The identity of the capsules is unknown to participants, researchers, and primary investigators during the entire study until the database lock. Only the assigned study supply coordinator at Chr. Hansen A/S, part of Novonesis Group, will be unblinded and have access to the randomization list as necessary to perform packaging, labelling, and re-supply of study products.

Criteria for discontinuing or modifying allocated interventions {11b}

If a participant experiences adverse effects or complications, the intervention may be modified or discontinued to prevent harm. If the intervention proves ineffective (e.g., the participant’s condition worsens), it may be stopped or replaced with an alternative treatment. Adjustments may also be necessary if a participant cannot adhere to the protocol requirements, ensuring the study remains safe and ethical. Participants may withdraw or request changes to their intervention if they experience discomfort, unwanted side effects, or do not want to continue.

Strategies to improve adherence to interventions {11c}

Member of the research team provides participants with thorough information about the intervention, including its benefits, risks, and importance. Members of the research team use scheduled regular check-ins, such as phone calls, emails, or clinic visits, to track participants’ adherence and address any issues early. A member of the research team will maintain regular contact with participants throughout the intervention period. Participants will be contacted every 2 months via scheduled clinic visits, email, and telephone calls. In addition, automated reminders via SMS and email will be used to prompt capsule intake and questionnaire completion. This structured follow-up is intended to support adherence and allow early identification of any challenges.

Relevant concomitant care permitted or prohibited during the trial {11d}

It is not allowed to take other probiotics during intervention and 6 months after. All medications are allowed, and the participants’ medication list will be updated continuously.

Provisions for post-trial care {30}

Participation in this study is voluntary, and no compensation or other financial benefits are provided to participants.

Outcomes {12}

This project will provide greater insight into the impact of probiotic use on the composition of the gut and vaginal microbiota, aiming to reduce rUTI incidence. The primary objective of this study is to evaluate the effectiveness of the probiotic combination ASTARTE™ in modulating the gut and vaginal microbiome composition and to determine whether this modulation contributes to a reduction in the incidence of rUTIs over a 6-month intervention period in women aged 18–40 years. The primary clinical outcome is the incidence of symptomatic, bacteriologically confirmed UTIs requiring antibiotic treatment, measured over the 6-month intervention period.

The secondary outcomes of the study are the following:

1. To assess the impact of ASTARTE™ on the number of bacteriologically confirmed UTIs requiring antibiotic treatment, within 6 and 12 months.

2. To evaluate the effect of ASTARTE™ on the duration of UTI episodes (measured in days).

3. To examine changes in the vaginal microbiome following ASTARTE™ treatment, by analyzing bacterial composition, as well as alpha and beta diversity.

4. To investigate changes in the gut microbiome following ASTARTE™ treatment, by analyzing bacterial composition, as well as alpha and beta diversity.

Participant timeline {13}

Participants in this study will follow a structured schedule of assessments over a 12-month period. The timeline includes the following key activities, see Fig. 2. Screening (t1): Participants will be assessed for eligibility before enrolment. Intervention (Month 0): Participants will receive the assigned intervention (probiotic or placebo) at the start of the study. BFLUTS Questionnaire (Months 0, 2, 4, 6, 8, 10, 12): Participants will complete questionnaires at baseline and at each follow-up visit to assess symptoms, adherence, and other study-related factors. Sample Collection (Months 0, 2, 4, 6): Urine samples, rectal and vaginal swabs will be collected at baseline and at specified follow-up visits for analysis. Compliance Monitoring (Months 2, 4, 6): Participant adherence to the intervention will be assessed at these time points. Safety Monitoring (Adverse Events–AE) (Months 2, 4, 6): Any adverse events or safety concerns will be documented during follow-up visits.

Fig. 2.

Schedule of enrolment, interventions, and assessments

The figure illustrates the study timeline, detailing a 6-month intervention period by a 6-month follow-up phase, outlining key activities and assessments conducted during each stage of the trial. This timeline ensures consistent data collection and monitoring throughout the study duration.

Sample size {14}

We aim to include 738 participants based on a power calculation by Danish company SIGNIFIKANS ApS, Vedbæk, Denmark. The test statistic used is the two-sided Fisher’s Exact test, Software PASS version 12.0.10. Assuming a recurrence rate of 20% in the probiotic group versus 30% in the placebo group, with α = 0.05, β (power) = 80%, and an enrollment ratio of 1:1, the required sample size is 626 participants (313 in the placebo group and 313 in the probiotic group). Accounting for an approximate 15% dropout rate, a total of 738 participants will be enrolled (369 in the placebo group and 369 in the probiotic group). Sample size will be adjusted after an interim analysis after inclusion of 138 patients.

Recruitment {15}

Pre-study planning

The inclusion and exclusion criteria are not overly restrictive, as this can limit the eligible pool of participants. Previous studies and realistic recruitment rates were examined to establish a feasible timeline and determine the necessary resources. Laboratory data from the years 2020 to 2021 were analyzed to estimate the number of participants eligible for invitation to this study.

Efforts are made to engage with other Danish hospitals and relevant institutions while leveraging social media and online platforms. Clear and appealing messaging is used, tailored to the target demographic, and emphasizing study benefits, such as health insights or incentives. Recruitment materials will be sent via digital mail (Danish e-Boks). Potential participants are given sufficient time to review study materials and the option to contact the project phone with any questions before scheduling a screening interview. Personalized communication is maintained every 2 months to ensure participant engagement. Flexible scheduling includes phone and online conversations from 8:00 to 18:00 (Monday to Friday), with the option to collect samples from private addresses. A dedicated member of the research team and coordinators have been hired to focus on recruitment. The recruitment process is continuously assessed and refined to align with the study’s objectives.

Assignment of interventions: allocation

Sequence generation {16a}

Prior to the start of the study, the randomization list is produced by SIGNIFIKANS ApS, Vedbæk, Denmark, using the PROC PLAN procedure in SAS version 9.4. To maintain the study blinding, the randomization list is developed and quality controlled by an external statistician. Particularities of the randomization, such as block size, are stated in the randomization list itself and will not be disclosed to the investigator or study personnel. A computer-generated random number is used to ensure unbiased assignments. The participants are randomized to either the probiotic or placebo group in a 1:1 ratio. The investigational capsules and the placebo capsules are similar in smell, taste, and appearance. All study products are packaged in identical vials with identical labelling, except for the randomization number.

Concealment mechanism {16b}

Participants are included via consecutive numbers that are randomized in advance to either the active or placebo group.

Implementation {16c}

Member of the research team enroll participants consecutively by using a computerized IT system REDCap (https://project-redcap.org/) and assign them a participant number, which is also labeled on the capsules provided. The system is also used for data storage, questionnaire, visit records, statistical analyses, etc.

Assignment of interventions: blinding

Who will be blinded {17a}

Research assistants, coordinators, and participants will be blinded. The identity of the capsules is unknown to participants, researchers, and primary investigators.

Procedure for unblinding if needed {17b}

Unblinding will be performed only when necessary for patient safety or in cases where knowledge of the treatment assignment is essential for clinical decision-making. The process will involve notifying the study sponsor and principal investigator, documenting the reason for unblinding, and accessing the randomization key using the emergency unblinding cards. All instances of unblinding will be recorded, including the date, reason, and personnel involved.

Data collection and management

Plans for assessment and collection of outcomes {18a}

The study involves multiple visits over the course of a year to monitor the participants’ health, collect relevant samples, and assess the participants’ adherence to the intervention. Initial visits include informed consent and screening, followed by regular follow-ups at 2, 4, and 6 months to track progress, collect data, and monitor compliance. If participants experience symptoms of a UTI, additional visits for diagnosis and treatment are included. At each visit the participant will receive the BFLUTS questionnaire on urinary tract symptoms, side effects, and UTI episodes will be retrieved from the laboratory IT register. At each visit, midstream urine samples are cultured to identify uropathogenic bacteria, and antimicrobial susceptibility testing is conducted. Rectal and vaginal swabs are analyzed for microbiome composition using shotgun metagenomic sequencing techniques. After 8, 10, and 12 months, participants will complete the BFLUTS questionnaire and report the number of UTI episodes experienced since discontinuing the capsule regimen.

• Information/screening visit (visit 1)

  Interested participants will attend an information and screening visit at Hvidovre Hospital 1 week before inclusion. They will receive verbal and written study details and be reassessed for eligibility. Those who do not meet the criteria will be excluded. Before the visit, participants will be provided with documents via digital mail including an invitation letter, participant information, a consent form, and the brochure Before You Decide—About Being a Research Participant in Health Science Studies. They will have up to 2 days to consider participation. If they sign the consent form, they will proceed to the inclusion visit (visit 2).

• Inclusion visit (visit 2)

  If the participants meet the criteria for participation and have provided written consent, they will be invited to the inclusion visit. At this visit, the participant will fill out a BFLUTS questionnaire, demographics data (place of birth, height, weight, diet etc.) and urine samples and vaginal and rectal swabs will be collected. The study intervention will begin within 7 days after the completion of any antibiotic treatment.

• 2nd, 4th, and 6th-month visit (visit no 3, 4, and 5)

  At the follow-up visits after 2–4 months and at the end of intervention, the participant will complete a BFLUTS questionnaire about urinary tract symptoms, and urine samples and vaginal and rectal swabs will be collected. The participant’s compliance will be assessed by counting unused capsules. The participant must have consumed at least 80% of the provided capsules during the study period to be considered as having completed the intervention according to the protocol.

• Additional visits

  Additional study visits will be scheduled if the participant develops symptoms of a UTI during the study period. If symptoms occur, the participant should contact the study staff and visit Copenhagen University Hospital Hvidovre within 48 h for urine sample, vaginal, and rectal swab collection. Participants with confirmed UTIs will be offered a prescription for antibiotic treatment and will continue in the study.

Plans to promote participant retention and complete follow-up {18b}

Every 2 months, all participants will receive the BFLUTS questionnaire on urinary tract symptoms via email through REDCap, along with an SMS reminder. The study is designed to minimize inconvenience by eliminating the need for hospital visits for questionnaire completion. Automated follow-up reminders will be sent to participants who have not completed the questionnaire within a specified timeframe. Personalized follow-up calls will be made to encourage participation and address any concerns. Members of the research team will be available to answer questions, provide study-related support, and assist participants with any challenges they may encounter. A designated helpline or email contact will be available for participants who require assistance throughout the follow-up period. Feedback from participants will be actively gathered to improve retention efforts and enhance the overall study experience.

Data management {19}

Data collected throughout the study will be securely stored and managed to ensure confidentiality, integrity, and compliance with ethical and regulatory standards. All participant data will be stored in a secure, encrypted REDCap system, which is designed to meet industry standards for data protection. The REDCap platform employs robust encryption protocols, both during data transmission and while at rest, ensuring that sensitive information is always safeguarded. Access to the system is strictly controlled through login authentication mechanisms, ensuring that only authorized personnel can access or modify data. REDCap provides comprehensive audit trails, enabling full traceability of all data entry, modifications, and user actions. This ensures transparency and accountability in the management of study data. Every interaction with the system, including data input, updates, and deletions, is logged with time stamps and user identification, which allows for accurate monitoring of data integrity throughout the study. Access to participant data will be restricted to authorized study team members based on predefined roles and responsibilities. Regular backups of all study data will be conducted to prevent data loss in the event of system failure. A robust data recovery plan will be in place to restore data quickly and effectively in the event of any unforeseen incidents. Data management practices will adhere to applicable data protection regulations, including the General Data Protection Regulation (GDPR) and other relevant ethical standards. Data anonymization and de-identification procedures will be implemented where applicable to ensure participant privacy and confidentiality.

Confidentiality {27}

Essential personal information will be collected via the secure, encrypted REDCap system, adhering to regulatory data protection standards. Participants will be assigned unique study IDs to avoid direct identification. Only authorized personnel will access identifiable information. Shared data will be de-identified and transferred securely. Collaborators must comply with data protection protocols. Personal data will be stored on secure servers with restricted access and encryption. Identifiable details will be stored separately from research data and linked only via study IDs. Data will be retained only as needed and securely destroyed afterward per regulatory requirements. Data collection will begin after consent, with non-enrolled participants’ data de-identified or deleted. Access to identifiable information will be limited to essential personnel. After the study, identifiable data will be destroyed after 01.08.2032, and de-identified data may be retained for future analysis.

Plans for collection, laboratory evaluation and storage of biological specimens for genetic or molecular analysis in this trial/future use {33}

During the first introductory meeting, the study participants are thoroughly introduced to how sample collection will take place, and an additional introduction is offered at each visit to the clinic. The participant is required to hand over the samples to the laboratory on the same day it is collected to ensure the integrity and accuracy of the analysis. This timely delivery of samples minimizes potential degradation or contamination of the sample, thereby optimizing the reliability of subsequent laboratory results. After sample analysis, the remaining sample material will be frozen in 10% glycerol at −80 °C in a research biobank until the project’s completion, after which it will be destroyed if reanalysis is not required.

Mid-stream urine sample

The urine is collected to identify uropathogenic microorganisms and determine whether the participant has a UTI. After washing the genital area with water or wet wipes, midstream urine should be collected in a glass container with boric acid (SARSTEDT, Denmark). The participants collect the sample themselves, preferably as their first-morning urine. Urine culture is performed within 24 h of collection.

Rectal swab

A rectal swab (Fecal Swab, Copan, Italy) for microbiome analysis is collected for the purpose of using shotgun metagenomic sequencing techniques. A sterile swab is inserted approximately 2 cm into the rectum, gently rotated for 20–30 s, and then carefully withdrawn. The participants collect the sample themselves, which is then placed in the provided transport tube or handed over to a member of the research team during study visits. The sample is processed within 48 h and stored at −80 °C for later microbiome composition analysis.

Vaginal swab

A vaginal swab (ESwab®, Copan, Italy) for microbiome analysis is collected for the purpose of using shotgun metagenomic sequencing techniques. The participants collect the sample themselves. The swab is held in one hand, while the other hand is placed on the labia. The swab is then inserted approximately 5 cm into the vagina and rotated for 30–50 s, ensuring contact with the inner vaginal wall. The swab is then removed and placed in a transport tube. The sample is processed within 48 h and stored at −80 °C for later microbiome composition analysis.

Statistical methods

Statistical methods for primary and secondary outcomes {20a}

The primary outcome, the incidence of bacteriologically confirmed UTIs requiring antibiotic treatment, will be analyzed using a time-to-event analysis with Kaplan–Meier survival curves and log-rank tests to compare differences between intervention groups. A Cox proportional hazards model will be used to estimate UTI ratios, adjusting for potential confounders such as age, previous UTI history, and baseline microbiome composition.

For the secondary outcomes, the number of UTIs within 6 and 12 months will be analyzed using Poisson or negative binomial regression models, depending on data distribution. The duration of UTI episodes will be compared between groups using independent t-tests or Mann–Whitney U tests for non-normally distributed data. Changes in vaginal and gut microbiome composition will be assessed using paired and independent t-tests or Wilcoxon rank-sum tests. Alpha diversity indices will be compared using the Shannon and Simpson indices, while beta diversity will be evaluated through principal coordinate analysis (PCoA) based on Bray–Curtis dissimilarity, with PERMANOVA used to test for significant differences between groups.

Interim analyses {21b}

An interim analysis will be conducted to test and potentially adjust the sample size calculation.

An interim statistical analysis plan (interim SAP) will be produced according to study protocol. The interim analysis is planned to be performed when 138 participants have completed the intervention period of 6 months.

An interim analysis (Fig. 3) will be conducted to assess and, if necessary, adjust the sample size calculation. An interim SAP will be developed according to the study protocol.

Fig. 3.

Interim analysis decision tree: The figure illustrates the decision pathways following the interim analysis. Early efficacy indicates that the interim analysis demonstrates a clear beneficial effect of the intervention, supporting early confirmation of effectiveness and termination of the trial with no further recruitment. Futility indicates that the interim analysis suggests a low probability of achieving the primary endpoint, reflecting insufficient treatment effect and supporting early termination of the trial due to lack of efficacy. If neither early efficacy nor futility is demonstrated, the study will continue as planned or be extended according to the updated sample size calculation

The interim analysis outlines the decision tree following the sample size recalculation, highlighting adjustments made based on observed data to ensure sufficient statistical power for detecting significant differences in the primary outcome. At the interim analysis three outcomes are possible (Fig. 3). If early efficacy is demonstrated, indicating a clear beneficial effect of the intervention, the trial may be terminated early, and no further participants will be recruited. If futility is indicated, reflecting a low probability of achieving the primary endpoint and insufficient treatment effect, the trial may be terminated early due to lack of efficacy. If neither early efficacy nor futility is demonstrated, the study will continue as planned or be extended according to the updated sample size calculation. The interim analysis will be performed by an external statistician to maintain study blinding and the unblinded statistician will keep all material with unblinding information confidential until database lock and unblinding. The result will be presented as a new sample size calculation and will be provided in an encrypted file shared with the decision-making part of the study team.

Methods for additional analyses (e.g., subgroup analyses) {20b}

We conduct antibiotic susceptibility testing on pathogenic bacteria from urine and fecal samples to investigate the presence of resistant bacteria, such as Carbapenemase producing organisms (CPO), extended spectrum beta-lactamase (ESBL), vancomycin-resistant Enterococcus (VRE), and methicillin-resistant Staphylococcus aureus (MRSA), and multidrug resistant bacteria (MDR) defined as non-susceptibility to at least one agent in three or more antimicrobial categories [31]. We will use disc-diffusion according to The European Committee on Antimicrobial Susceptibility Testing (EUCAST) and PCR (polymerase chain reaction) test.

Methods in analysis to handle protocol non-adherence and any statistical methods to handle missing data {20c}

We will use Multiple Imputation, which generates several datasets by imputing missing values based on observed data and its variability, and then combines the results. Multiple Imputation assumes that data are missing at random and accounts for uncertainty in the missing values. The analysis population will be defined based on protocol adherence, with compliance set at a minimum threshold of 80%. Participants who complete at least 80% of the prescribed intervention and adhere to study procedures, including sample collection and follow-up visits, will be included in the per-protocol analysis.

The intention-to-treat population will include all randomized participants, regardless of protocol adherence, following the principle of analyzing data based on initial treatment assignment. Sensitivity analyses may be conducted to assess the impact of non-adherence on study outcomes. Participants with less than 80% compliance will be categorized as protocol deviations and analyzed separately to determine their influence on the results.

Plans to give access to the full protocol, participant level-data and statistical code {31c}

The timeline for releasing materials is after publication upon request to the authors.

Oversight and monitoring

Composition of the coordinating centre and trial steering committee {5d}

Data Monitoring Committee is not convened because the study poses minimal risk to participants. Trial Steering Committee and the principal investigator assume monitoring responsibilities.

Composition of the data monitoring committee, its role and reporting structure {21a}

Not applicable, because the Trial Steering Committee and the principal investigator assume monitoring responsibilities. Data monitoring in REDCap takes place continuously in collaboration with the principal investigator and recruitment staff.

Adverse event reporting and harms {22}

ASTARTE™ contains probiotic strains commonly found in the gut and vaginal microbiota. Previous clinical studies have demonstrated that these bacteria contribute to various beneficial effects, particularly in the intestine and vagina, and are considered safe [24, 25, 28, 29, 32]. Participants are monitored for side effects at each study visit, which occurs every 2 months, and the information is documented in a case report form (CRF).

Frequency and plans for auditing trial conduct {23}

Audits, conducted by the sponsor, will occur at predefined intervals, including during initiation, after 138 participants complete the intervention (Interim analysis), midway, and at trial completion, with additional audits as needed. They will assess protocol adherence, informed consent, data integrity, safety reporting, and compliance. Findings will be documented in reports shared with the principal investigator, sponsor, and trial staff, with follow-up audits conducted to address identified issues.

Plans for communicating important protocol amendments to relevant parties (e.g. trial participants, ethical committees) {25}

Significant amendments will be submitted to ethical committees and regulatory authorities for approval before implementation. Investigators, sponsors, and trial staff will be notified through formal communications, and updated protocols will be provided. Participants will be informed of amendments affecting their rights, safety, or participation, and reconsent obtained if necessary. All communications and updates will be documented, and trial registries will be updated to reflect changes.

Dissemination plans {31a}

Trial results will be shared via publications, conferences, and trial registries, platforms. Participants will receive accessible summaries. Publication restrictions will adhere to prior agreements.

Discussion

Most urinary tract infections originate from the gut microbiota, making it logical to explore the link between gut microorganisms and the development of bacteriuria and UTIs [33]. Recent findings have also challenged the traditional diagnostic and therapeutic paradigms for UTIs by highlighting the complex, symbiotic microbiome present in a healthy urogenital tract [3, 34]. These insights reveal that UTIs are not merely caused by external invasion but often arise from intricate interactions between host-associated microbial communities from gut and vagina [14, 15].

In women, vaginal microbiota significantly influence the pathogenesis of UTIs, acting as an intermediary in the journey of bacteria from the gut to the urinary tract. However, the ultimate source of the bacterial strains responsible for conditions such as cystitis and pyelonephritis is frequently the gut microbiota. This interplay between gut and vaginal microbiota demonstrates a dynamic crosstalk and interconnection between these two niches, emphasizing the need for a holistic approach to understanding and management of UTIs [35]. Such insights not only reshape our perception of UTI pathogenesis but also open new avenues for therapeutic strategies aimed at targeting these microbial ecosystems. By analyzing changes in microbiota composition across fecal, vaginal, and urinary samples, we can assess the role of probiotics in restoring or maintaining a beneficial microbiome. Furthermore, potential correlations between microbiota alterations and symptomatic improvement will be investigated to better understand its therapeutic impact.

The relationship between the gut microbiota and UTIs can be attributed to several interconnected factors. One key aspect is gut dysbiosis, which can contribute to a pro-inflammatory state within the intestine [14]. When there is an imbalance in the types and proportions of gut bacteria, it may elicit an inappropriate immune response, resulting in chronic inflammation [14, 36]. Additionally, a disrupted gut microbiota can weaken the intestinal mucosal barrier, leading to increased intestinal permeability, a condition often referred to as “leaky gut” [17, 18]. This compromised barrier allows bacteria, toxins, and other molecules to translocate from the gut into the bloodstream, potentially triggering systemic immune responses or infections in other regions, such as the urinary tract. Understanding these mechanisms highlights the complex interplay between gut health and susceptibility to UTIs. This study will assess microbiome stabilization, demonstrating whether regular intake of probiotics maintains or restores a protective lactobacilli-dominant microbiome in the gut, vaginal, and urinary tracts, which is associated with a reduced risk of UTIs. The impact of probiotics on reducing risk factors for rUTIs is assessed by primary outcomes such as the incidence of symptomatic UTIs during the intervention and follow-up periods. We aim to evaluate whether the probiotic group demonstrates significant health benefits over the placebo group. The comparative efficacy of the intervention will be assessed by comparing outcomes between the placebo group and the probiotic group. If the probiotic group exhibits fewer episodes of UTI recurrence, it will indicate the potential of probiotics as a preventive strategy.

A study by Adu-Oppong B et al. [37] confirms a significant overlap between symptomatic and asymptomatic genitourinary microbiota states. This raises the question of whether establishing a baseline for gut and vaginal microbiota in the placebo group is necessary. Such a baseline would allow us to compare microbiota states between the placebo and probiotic groups, as well as between individuals with symptomatic and asymptomatic bacteriuria, helping to identify any meaningful patterns or differences.

Shotgun sequencing is a powerful tool for microbiome analysis, offering high-resolution taxonomic and functional insights by sequencing entire genomes from a sample. However, the reliability of shotgun sequencing for microbiome analysis is affected by limitations that can impact sensitivity, specificity, and data interpretation [38]. Limitations of shotgun sequencing may include factors such as the type of reference database used, the sequencing method chosen (e.g., 16S amplicon sequencing or whole genome sequencing (WGS)), the amount of human DNA present in the sample, and other technical considerations. These limitations may influence the accuracy of identifying bacterial species and affect the comparison of microbiome composition between placebo and active groups, as well as between symptomatic and asymptomatic bacteriuria.

This study highlights the potential of probiotics as a non-antibiotic intervention to reduce recurrent UTIs, addressing concerns related to antibiotic resistance and overuse [3, 39]. To support this, we will evaluate the prevalence of uropathogenic bacteria both at baseline and after the intervention. Additionally, we will assess whether probiotics have an impact on the eradication of uropathogenic bacteria.

Trial status

This study started enrolling in September 2022 and will continue until January 2028. To date, 326 participants have been enrolled, and 224 have completed the intervention. Protocol name and version: Prob.rUTI 11.03.2025, Version 8.

Abbreviations

BFLUTS

Bristol Female Lower Urinary Tract Symptoms questionnaire

CPO

Carbapenemase producing organisms

CFR

Case form report

ESBL

Extended spectrum beta-lactamase

EUCAST

The European Committee on Antimicrobial Susceptibility Testing

MDR

Multidrug resistant bacteria

MRSA

Methicillin-resistant Staphylococcus aureus

PCR

Polymerase chain reaction

rUTI

Recurrent urinary tract infection

UTI

Urinary tract infection

VRE

Vancomycin-resistant Enterococcus

WGS

Whole genome sequencing

Authors’ contributions {31b}

KSAG, KAK, NFM, SIH, KHH, AH, AKSF, and AMP contributed to the design of the study. All authors have approved the final version of the protocol. KSAG writes the manuscript and coordinates the study. All authors read and approved the final manuscript.

Funding {4}

Open access funding provided by Copenhagen University. The study is funded by Copenhagen University Hospital, Hvidovre, Denmark and Chr. Hansen A/S, Part of Novonesis Group, Denmark.

Data availability {29}

Data will be analyzed after completion of the study, and therefore the availability of data is not applicable for this manuscript.

Declarations

Ethics approval and consent to participate {24}

The study has been approved by the Danish Data Protection Agency (P-2022–356), and permission for human experiments and recruitment of participants has been obtained from the Scientific Ethics Committee for Copenhagen Regional Hospital before initiation and approved by the Danish Data Protection Agency (H-22014666). As probiotics are considered dietary supplements and not pharmaceuticals, no authorization by the Danish Medicines Agency is required. The study is performed in accordance with the requirements of the Revised Declaration of Helsinki. The study is registered at ClinicalTrials.gov (NCT05553652) before inclusion of the first participant. Consent for participation will be obtained from all participants, and participants can discontinue at any time point on their request. Sample materials are temporarily stored in a Research Biobank if there is a need for reanalysis or possibly additional analysis to collect data related to this project. The end date for the research biobank is 01.08.2032. No additional consent provisions are needed for the collection and use of participant data and biological specimens in this study.

Consent for publication {32}

No personal data is published in this protocol article. Consent from participants is obtained/will be obtained to publish the data as anonymous data when the study is completed. The participant information and consent form are available as supplementary material in both Danish and English.

Competing interests {28}

AKSF is an employee of Chr. Hansen A/S, part of Novonesis Group, but will not be involved in the study conduct, data management or statistical analysis. The rest of the authors report no other conflicts of interest in this work.