The role of probiotics in the treatment of endometriosis (ProMetrioS): a randomised double-blinded placebo-controlled cross-over trial

The role of probiotics in the treatment of endometriosis (ProMetrioS): a randomised double-blinded placebo-controlled cross-over trial | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The role of probiotics in the treatment of endometriosis (ProMetrioS): a randomised double-blinded placebo-controlled cross-over trial Sara Kralj, Karlo Zeman, Mislav Mikuš, Andrija Karačić, Ana-Marija Liberati Pršo, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6603155/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Feb, 2026 Read the published version in Trials → Version 1 posted 5 You are reading this latest preprint version Abstract Background ProMetrioS is a randomised blinded placebo-controlled cross-over trial investigating the effect of specific multispecies probiotics in patients with endometriosis. The aim of this clinical trial is to determine whether probiotic treatment can significantly modulate gut microbiome composition and functionality in endometriosis patients, specifically parameters associated with the estrobolome. Methods This is a double-blinded, randomized, cross-over, placebo-controlled trial. Participants are randomly allocated to receive either 3 g of a probiotic formulation daily or placebo for 8 weeks in two study phases, separated by a 8-week washout period between the two phases. The gut microbiome is analysed in four time-points: start-of-study, after phase 1, after the washout phase, end-of-study (after phase 2) via amplicon sequencing. Quality of life related to endometriosis is assessed via validated questionnaires in the same four time-points. Our primary endpoint is a favorable modulation of the gut microbiome, especially in prospect of the estrobolome. Secondary endpoints include changes in quality of life, reported symptomatology and psychophysical condition. Discussion : The findings of this study will provide the first evidence for the use of a combination of probiotics in the treatment of endometriosis. The results are expected at the end of 2025. Trial registration : pending gut microbiome estroblome endometriosis quality of life symptoms pain probiotics gut-microbiome therapeutics Figures Figure 1 Figure 2 Background and rationale Endometriosis is a chronic gynecological hormonal disease that is defined by the appearance of tissue lesions caused by the growth of endometrial-like tissue outside the uterus, most often inside the abdominal cavity, although lesions can also be found in distant organs 1 . Endometriosis affects around 10% of women of reproductive age, and it is estimated that 196 million women around the world suffer from this disease. 2 It is associated with serious, sometimes severe symptoms, including chronic pelvic pain, dysmenorrhea, and dyspareunia. The most serious consequence of endometriosis is certainly infertility, which affects 30% of patients. 3 , 4 Endometriosis is often under-diagnosed and poorly treated due to the limited options for diagnosis and therapy. Its pathophysiology is highly complex and includes intertwined hormonal and inflammatory mechanisms. 5 , 6 Recent studies have identified the gut microbiome to be an additional player in endometriosis. 7 , 8 , 9 Specifically, the estrobolome, bacterial members of the gut microbiome involved in the metabolism of estrogen. 10 , 11 These bacteria possess enzymes which alter the chemical structure of estrogen, reinforcing its reabsorption in the colon. 9 , 12 Since the disbalance of circulatory estrogen levels is vital for the progression of endometriosis these bacteria are therefore indirectly involved in the development and progression of endometriotic lesions and thus may significantly alter the course of disease. 13 Additionally, the gut microbiome impacts the level of chronic inflammation, which may aggravate symptoms of endometriosis such as chronic pain. 13 , 14 , 15 , 16 Dysbiosis, a disbalanced and dysfunctional gut microbiome has been found in many patients on endometiosis patients. 13 The gut microbiome of endometriosis patients is frequently less diverse, with higher abundances of genera such as Blautia, Dorea, Streptococcus , and lower abundances of genera Paraprevotella, Lachnospira and Turicibacter. 15 , 17 The gut microbiome of endometriosis patients could intensively benefit from gut-microbiome directed therapeutics. One could hypothesize that gut-microbiome directed therapeutics, such as probiotics, could even positively affect the pathophysiological processes active in endometriosis. 9 , 13 , 18 Probiotics are the best researched of all gut-microbiome directed therapeutics, be it in in vitro and in vivo, animal and human studies. Different probiotic strains are known to have anti-inflammatory, but also analgesic, antidepressive, anorexigenic and many other effects. 19 , 20 Thus, probiotics are promising agents for the treatment of the gut microbiome in endometriosis patients. Although confronted with a broad choice of potential formulations for the treatment of endometriosis, the decision was laid upon a well-researched multispecies probiotic formulation (OMNi-BiOTiC® Stress). This formulation has proven anti-inflammatory effects, antidepressive, analgesic effect on the central nervous system targeting gut-brain axis. 21 , 22 , 23 , 24 Its administration has been associated with an increase in the relative abundances of biorelevant genera such as Ruminococcus and Coprococcus. 24 The chosen probiotic contains nine human derived probiotic strains: Lactobacillus casei W56, Lactobacillus acidophilus W22, Lactobacillus paracasei W20, Bifidobacterium lactis W51, Bifidobacterium lactis W52, Lactobacillus salivarius W24, Lactococcus lactis W19, Lactobacillus plantarum W62 and Bifidobacterium bifidum W23. 22 That product was chosen due to the analgetic effect, regulating the production of neurotransmitters implicated in pain perception. Specifically, some of the species present in the multispecies probiotic, namely Lactobacillus acidophilus , Lactobacillus plantarum , Lactobacillus fermentum and Lactobacillus gasseri were mentioned to have that effect in the pilot-study from 2019. 18 In the context of endometriosis, that could be really helpful for suppressing constant chronic pain that is present. Via modulation of the gut-brain axis and immunomodulation, probiotics can act antidepressive. The antidepressive effect of bacteria such as genus Lactobacillus and Bifidobacterium could be helpful in endometriosis treatment since more than 50% of patients with painful endometriosis suffer from depression. 24 , 25 The anti-inflammatory properties of this specific formulation were seen in several studies where it was able to reduce the levels of pro-inflammatory cytokines and immune response modulation. The induced chronic inflammation could alleviate sensations of pain and discomfort, as seen in studies with patients suffering from endometriosis. 25 , 26 Additionally, one component of the multispecies formulation, Lactobacillus gasseri OLL2809 has been experimentally associated with an increase of IL-12 secretion which can successfully suppress the development of endometrioc lesions. 18 , 27 Although this probiotic formulation bears many promising effects, its interaction with the gut microbiome in endometriosis patients, especially the estrobolome, has yet to be explored. Since the gut microbiome is a dynamic and complex eco-system with high interindividual variability, randomized placebo-controlled trials are required to track their potential in altering the gut microbiome. Objectives This study aims to investigate the effects of probiotic administration on the gut microbiome and quality of life of endometriosis patients. The primary outcome will be to compare the composition and functionality of gut microbiome before and after probiotic treatment. As secondary outcomes, quality of life, stress level, perception of health and quality of sleep will be evaluated. Our primary hypothesis is that as a result of probiotic intake there will be significant positive changes, on level of the gut microbiome, but also regarding quality of life and other subjective parameters. Trial design This will be a double-blinded randomized placebo-controlled cross-over trial as shown in Fig. 1. Patients will be recruited among the outpatient population of the National Center for Endometriosis, Department of Gynecological Surgery and Urology, Gynecology Department, University Hospital Center Zagreb following the inclusion and exclusion criteria. To participate in the study all patients will be required to sign the Informed Consent form. All patients will undergo a complete anamnesis via a digital form to investigate their lifestyle (occupation, stress, diet, physical activity level). The study period will be 6 months: eight weeks on either option (verum/placebo), with an eight week washout period in between. The purpose of the washout period is to allow the gut microbiome to return to baseline. The washout period was determined based on the fact that the gut microbiome can change rapidly in few days. 28 The outcome measures (gut microbiome, quality of life) will be assessed in four distinct time points in context of the planned intervention. T1 – before the intervention (start-of-study) T2 – after the first intervention cycle/phase 1 (verum/placebo) of two months T3 – after the wash-out phase of two months T4 – after the second intervention cycle/phase 2 (verum/placebo) of two months (end-of-study) Stool sampling will be performed with the aid of specific kits indepently by the patients. Lifestyle and quality of life will be evaluated by an online form designed specifically for the purpose of this study in Microsoft Office Forms (Microsoft, San Francisco, USA), short Online Form. Through the process of randomization, patients will be allocated to either first receive the verum (probiotic) or the placebo (maltodextrin), and second the placebo or verum inversely. Both patients and researchers involved will be blinded (double-blind study), the patients will be informed that they are going to take a probiotic twice a day for two months with a two-month time division. The intervention will consist of treatment with a multispecies probiotic formulation (OMNi BiOTiC® Stress) and the placebo which is identical in all its characteristics and packaging. Patients will be instructed to consume one sachet a day of each (probiotic) for 8 weeks and to report any symptoms or side effects related to its use. Adherence control will be carried out through the patient’s notes on a separate online form provided, and also by checking the number of sachets used. Since this is a cross-over study, data will be compared between time-points T1 and T2 , and time-points T3 and T4 , with the goal of minimizing the potential effect if we find that the effect of the probiotics lasted longer than eight weeks in those randomized into the intervention arm first. Adverse effects Adverse events during the intervention will be monitored via an online form where not only the intake of the sachets will be documented, but also potential gastrointestinal adverse events such as flatulence, bloating, diarrhea, constipation or pain/colics. Space for other extraintestinal adverse events will be left to the participants. In the case of adverse events, they will be immediately reported to the Human Research Ethics Committee of the University Hospital Center Zagreb. Due to the use of probiotics which are popular dietary supplements adverse events are not expected, besides possible transient digestive issues. Probiotics are generally regarded as safe with no to minimal deteriorating health effects. 29 , 30 , 31 Methods Participants, interventions, and outcomes Study setting The study protocol, including the intervention and all measurements, will be carried out by the study staff at Gut Microbiome Center, Zagreb, Croatia. Eligibility Inclusion and exclusion criteria are presented in Table 1 . Table 1 Eligibility criteria Inclusion criteria • adult subjects with stage III or IV endometriosis diagnosis confirmed by biopsy Exclusion criteria • second stage endometriosis • comorbidities in terms of mental disorders • comorbidities in terms of chronic inflammatory bowel diseases • with stomas • neoadjuvant oncological treatment administered • received antibiotic or probiotic therapy six months prior to presentation to the gynecologist • previous appendectomy performed • following a restrictive diet (vegan, vegetarian, gluten-free, ketogenic diet) Recruitment, screening, and enrollment procedures Recruitment Recruitment will be conducted by the gynecologists treating patients at the National Center for Endometriosis, Department of Gynecological Surgery and Urology, Gynecology Department, University Hospital Center Zagreb. The chief researcher with the aid of the staff of the Gut Microbiome Center will oversee the recruitment process. During regular outpatient visits potential participants will be informed about the study. Previous research participants at the Gut Microbiome Center who approved to be contacted for future research will also be contacted via email to be considered for the trial. Profiles on online social media platforms by Meta and Linkedin of the Gut Microbiome Center and the national association of endometriosis patients will also be used to recruit participants. The trial is planned to gain exposure through articles in popular media outlets. Recruitment will start in December 2024 and finish in July 2025. Screening and enrollment Eligible patients will go through assessments at baseline via the Online Form and will be randomly allocated to 2 groups, each consisting of 20 participants. Randomization will be done by an independent researcher in the biostatistics platform at the Gut Microbiome Center. Randomization will be performed using code written in the R statistical programming language (version 3.5.3). Treatments will be assigned with a 1:1 ratio. The randomization schedule will be transferred into sets of opaque sealed envelopes. After a participant’s visit at time-point T1 , the study staff will open a sealed envelope which will contain the participant’s allocation. The order of the interventions will be blinded for both the investigators and the participants. Treatments will be given in sealed sachets; sachet content will be blinded by the staff of the Gut Microbiome Center, given to the study staff labeled as A or B, and dispensed to participants according to their treatment period. Treatments will not be unblinded until the analyses are complete, unless required due to adverse events during the clinical trial. There will also be a midpoint review of the participants’ clinical data by an independent gynecologist who will be unblinded. Measurement All of the 40 patients will be randomized into two groups. One group will take the verum in the first, and the placebo in the second cycle/phase, the other first the placebo and then the verum in the second cycle/phase. The verum will consist of the supplementation with a multi-strain probiotic containing: Lactobacillus casei W56 Lactobacillus acidophilus W22 Lactobacillus paracasei W20 Bifidobacterium lactis W51 Bifidobacterium lactis W52 Lactobacillus salivarius W24 Lactococcus lactis W19 Lactobacillus plantarum W62 Bifidobacterium bifidum W23 The patients will receive the probiotic in a 3g sachet containing 7.5 billion CFU. During the intervention the participants will be instructed to consume one sachet daily with water at room temperature for a period of 8 weeks. If allocated to the placebo phase the participants will receive identical sachet (one 3 g) with an identical appearance (physical and organoleptic) containing polydextrose/maltodextrin as the placebo. Primary outcome The primary outcome is the modulation of the gut microbiome. The fecal microbiome, as a genuine proxy of the gut microbiome, will be analyzed. Specific attention will be brought to taxa and metabolic pathways involved in the estrobolome. Secondary outcome The secondary outcomes concern changes in quality of life and an eventual decrease in symptomatology of endometriosis patients. Two disease-specific questionnaires evaluating will be utilized to assess quality of life: the short form endometriosis health profile questionnaire (EHP-5) and the WHO Quality of Life Scale-Brief. Further study outcomes Through evaluation of lifestyle factors such as diet (Mediterranean diet adherence), stress perception, sleep quantity and physical activity, potential confounding factors influencing the effects of probiotics will be assessed. Mediator Quality of life Self perception of health will be assed with validated questionnaires short form endometriosis health profile questionnaire (EHP-5) and WHO Quality of Life Scale-Brief. Participants will be asked to complete the questionnaires before and after each of the intake periods. The EHP-5 is an 11-item questionnaire which has been previously translated into Croatian and validated for its psychometric properties. 32 The WHO Quality of Life Scale-Brief is a 26-item measure and is used in English, since a Croatian version has not yet been validated. Dietary intake, sleep quality, stress Dietary questionnaires inquiring on adherence to the Mediterranean diet will be used to understand habitual dietary intake among the study participants at the four respective time-points. The results will monitor diet as a potential confounding factor. The participants will complete Visual Analogue Scales (VAS) regarding sleep quality, perceived stress levels and fatigue. 16S rRNA sequencing of stool samples Changes in intestinal micobiome composition will be analyzed in all participating patients. Stool samples will be collected and submitted to the attending clinic at four different time points. Both patients and medical staff will receive detailed training on the correct handling of the stool samples for subsequent microbiome analyses. The collection kits will be provided by the sponsor. The sample containers contain a preservation solution, allowing the stool samples to be stored in a refrigerator at 4°C. Long-term storage is recommended at -20°C. The samples will be stored at the study center until the end of the study and will be collected for analysis. At the end of the study, the samples will be delivered to an analysis laboratory in Germany for DNA extraction, sequencing, and evaluation. The exact methodology for microbiome analysis is described as follows: For the total DNA isolation from stool samples, the TGuide S96 Magnetic Soil/Stool DNA Kit from the company Tiangen will be used. The extracted DNA will be employed in a PCR with the specific primer for the hypervariable region V3-4 of the 16S region. The resulting amplicons will be used to prepare a library, which will then be sequenced on an Illumina platform. The FastQ raw data files will be archived and used for filtering and analysis with standard bioinformatics tools Statistical methods Sample size The study is designed to test the hypothesis that probiotic intake will lead to significant positive changes, not only in the composition and function of the gut microbiome but also in various quality-of-life metrics and other subjective health parameters. By enrolling 40 participants, we aim to detect these changes with confidence using a two-tailed t-test, ensuring that any observed effects on both microbiome composition and participant-reported outcomes can be statistically validated. Randomization and blinding Crossover randomization of study medication is provided by Institut AllergoSan. The allocation of the recruited patients to the respective intervention group is blinded by issuing the study package with a consecutive participant number. The blinding and randomization of the study packages are carried out centrally by Institute AllergoSan. Statistical analysis Statistical analysis of the results of the gut microbiome analyses will be performed. The demultiplexed FASTQ raw data files from the Illumina sequencer will be used for data analysis with DADA2 in QIIME-2 and for archiving. Cleaned forward and reverse sequences will be paired and analyzed using the Amplicon Sequence Variants (ASVs) method. The underlying bacterial taxa will be identified based on the SILVA reference database. Alpha and Beta diversity analyzes will follow, along with visualization using the R package. For determining Alpha diversity, richness, Shannon index, Simpson diversity, evenness, and Chao1 will be calculated. Statistical analyzes will be performed using ANOVA or the Kruskal-Wallis test. For Beta diversity analysis, Bray-Curtis distances, weighted UniFrac, and Bray-Curtis-PcoA (Principal Coordinates Analysis) will be calculated and tested using Anosim or Adonis Safety, oversight and monitoring The patients will receive an online form to mark the intake of the sachet and write down any symptoms that may appear during the intervention period. Every 60 days, patients will be seen and adherence to treatment will be evaluated through repeated completion of the online form and by counting the sachets that were not consumed. The participants will be instructed to inform the research team about the need to use any other non-routine medications and dietary supplements, as some ingredients may alter the gut microbiome, and also to inform the team when they use a product that contains probiotics. Daily checklists for the sachets will be given to the participants via an online form (sent by e-mail) for each day during the two phases. These will be used to monitor their compliance with the study protocol. Additionally, participants will be instructed to return all empty sachets for counting purposes. Compliance for both groups will be defined as an attendance of ≥ 75% of the scheduled visits with the research team and ≥ 80% of the required sachets consumed according to diary entries and returned sachets. Participants will be monitored every two weeks by the research team through phone or video calls to inquire about the intake of antibiotics or other probiotic formulations, significant lifestyle alterations or digestive tract infections, which could lead to exclusion from the study. r. This log will include participant ID, date of dispensing, randomization code, and the returned sachet counts. Concomitant medication will be recorded at the end each treatment phase. Discussion Research into the gut microbiome is evolving at a rapid pace. In recent years associations between the gut microbiome and an ever-increasing number of chronic extraintestinal disorders have been demonstrated, especially of hormonal nature. Modulation of the gut microbiome via gut microbiome-directed therapeutics, such as probiotics, is drawing more and more attention in the treatment of extraintestinal diseases. Especially for diagnoses like endometriosis, where limited diagnostic and therapeutic options are available, the gut microbiome and its therapeutics seem a promising field of research. The findings from this study will provide evidence for the use of gut-microbiome directed therapeutics in the treatment of endometriosis patients. Trial status The trial is submitted on clinicaltrials.gov, but the status of it is still pending. Declarations Acknowledgements The authors want to thank all study participants. Sponsor The sponsor of this study is “Institut AllergoSan“(Institut AllergoSan, Graz, Austria). The sponsors played no role in study design, collection, management, analysis, interpretation of results, writing of the report, or the decision to submit the report for publication. Authors’ contributions All authors (SK, KZ, IR, AMLP AK, MM and MĆ) will be equally involved in the study. AK will oversee gut microbiome analysis and logistics, MM and MĆ will mostly be involved in patient dissemination and coordination, while IR will have biggest contribution in writing the manuscript. Availability of data and materials Once the study is finished and the data published, the datasets used during the current study will be available from the corresponding author on reasonable request. Consent for publication All authors approved the final version of the manuscript and agreed for all aspects of the work to be published. Competing interests Authors AK and IR have held paid presentations for the sponsor of the study. There is no other competing interest among the authors. Author details 1 Gut Microbiome Center, Zagreb, Croatia. 2 Faculty of Food Technology and Biotechnology, Zagreb, Croatia. 3 University Hospital Center (KBC) Zagreb; Gynecology Department, Zagreb, Croatia. 4 University Hospital Sveti Duh, Zagreb, Croatia. 5 University Hospital Center (KBC) Zagreb; Gynecology Department, Gynecological Surgery and Urology, National Center for Endometriosis, Zagreb, Croatia. Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. Conflicts of Interest The authors declare no conflicts of interest. References D’Alterio, M. N., Giuliani, C., Scicchitano, F., Laganà, A. S., Oltolina, N. M., Sorrentino, F., Nappi, L., Orrù, G., & Angioni, S. (2021). 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Probiotics drive gut microbiome triggering emotional brain signatures. Gut Microbes , 9 (6). https://doi.org/10.1080/19490976.2018.1460015 Institut AllergoSan. (2024). OMNi-BiOTiC® STRESS . Https://Omni-Biotic.Hr/Proizvod/Omni-Biotic-Stress/. Leblhuber, F., Steiner, K., Schuetz, B., Fuchs, D., & Gostner, J. M. (2018). Probiotic Supplementation in Patients with Alzheimer’s Dementia - An Explorative Intervention Study. Current Alzheimer Research , 15 (12). https://doi.org/10.2174/1389200219666180813144834 Reininghaus, E.Z, Platzer, M., Kohlhammer-Dohr, A., Hamm, C., Mörkl, S., Bengesser, S. A., Fellendorf, F. T., Lahousen-Luxenberger, T., Leitner-Afschar, B., Schöggl, H., Amberger-Otti, D., Wurm, W., Queissner, R., Birner, A., Falzberger, V. S., Painold, A., Fitz, W., Brunnmayr, M., Rieger, A., Wagner-Skacel, J., Maget, A., Unterweger, R., Schwalsberger, K., Reininghaus, B., Lenger, M., Bastiaanssen, T. F. S., Dalkner, N. (2020). Provit: Supplementary probiotic treatment and vitamin b7 in depression—a randomized controlled trial. Nutrients , 12 (11). https://doi.org/10.3390/nu12113422 Kreuzer K, Reiter A, Birkl-Töglhofer AM, Dalkner N, Mörkl S, Mairinger M, Fleischmann E, Fellendorf F, Platzer M, Lenger M, Färber T, Seidl M, Birner A, Queissner R, Mendel LS, Maget A, Kohlhammer-Dohr A, Häussl A, Wagner-Skacel J, Schöggl H, Amberger-Otti D, Painold A, Lahousen-Luxenberger T, Leitner-Afschar B, Haybaeck J, Habisch H, Madl T, Reininghaus E, Bengesser S. The PROVIT Study-Effects of Multispecies Probiotic Add-on Treatment on Metabolomics in Major Depressive Disorder-A Randomized, Placebo-Controlled Trial. Metabolites. 2022 Aug 21;12(8):770. https://doi.10.3390/metabo12080770 Mehdizadeh Sari F, Mirkalantari S, Nikoo S, Sepahvand F, Allahqoli L, Asadi A, Talebi M. (2022). Potential of Lactobacillus acidophilus to modulate cytokine production by peripheral blood monocytes in patients with endometriosis. Iran J Microbiol. Oct;14(5):698-704. htps://doi.10.18502/ijm.v14i5.10965 Uchida M, Kobayashi O. (2013s). Effects of Lactobacillus gasseri OLL2809 on the induced endometriosis in rats. Biosci Biotechnol Biochem. 77(9):1879-81. https://doi. 10.1271/bbb.130319 Schlomann BH, Parthasarathy R. Timescales of gut microbiome dynamics. Curr Opin Microbiol. 2019 Aug;50:56-63. https://doi.10.1016/j.mib.2019.09.011 Doron, S., & Snydman, D. R. (2015). Risk and safety of probiotics. Clinical Infectious Diseases , 60 . https://doi.org/10.1093/cid/civ085 Roe, A. L., Boyte, M. E., Elkins, C. A., Goldman, V. S., Heimbach, J., Madden, E., Oketch-Rabah, H., Sanders, M. E., Sirois, J., & Smith, A. (2022). Considerations for determining safety of probiotics: A USP perspective. In Regulatory Toxicology and Pharmacology (Vol. 136). https://doi.org/10.1016/j.yrtph.2022.105266 Zhao, L., Zhang, Y., Liu, Y., Zhong, J., & Zhang, D. (2023). Assessing the Safety and Probiotic Characteristics of Lacticaseibacillus rhamnosus X253 via Complete Genome and Phenotype Analysis. Microorganisms , 11 (1). https://doi.org/10.3390/microorganisms11010140 Mikuš M, Matak L, Vujić G, Škegro B, Škegro I, Augustin G, Lagana AS, Ćorić M. (2023). The short form endometriosis health profile questionnaire (EHP-5): psychometric validity assessment of a Croatian version. Arch Gynecol Obstet. Jan;307(1):87-92. https://doi.10.1007/s00404-022-06691-1 Cite Share Download PDF Status: Published Journal Publication published 20 Feb, 2026 Read the published version in Trials → Version 1 posted Reviewers agreed at journal 09 Aug, 2025 Reviewers invited by journal 04 Aug, 2025 Editor assigned by journal 04 Aug, 2025 First submitted to journal 31 Jul, 2025 Editorial decision: Major revision 28 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6603155","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":495393370,"identity":"0fc1bf2a-e5b9-4c7a-a2bd-f9095fe032a1","order_by":0,"name":"Sara Kralj","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAgUlEQVRIiWNgGAWjYLCCDyBCghQdjDNI1sLMQ5IW+fazDz/b1NhFM0j3PiBOi8GZdGPpnGPJuQ0yxw2I1MKQxiCd23Agt0EijViH9T9j/m1JkhaGG2ls0owkaTG48YzNsgfolzYSHJbGfONHjV1uP/EOgwE2UjWMglEwCkbBKMADACTZJQVH7PS1AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0009-0009-5380-6247","institution":"The Gut Microbiome Center (Centar za crijevni mikrobiom)","correspondingAuthor":true,"prefix":"","firstName":"Sara","middleName":"","lastName":"Kralj","suffix":""},{"id":495393371,"identity":"c7ae0a24-d08f-4d6f-bc24-7f9c7cecfbd0","order_by":1,"name":"Karlo Zeman","email":"","orcid":"","institution":"Gut Microbiome Centar","correspondingAuthor":false,"prefix":"","firstName":"Karlo","middleName":"","lastName":"Zeman","suffix":""},{"id":495393372,"identity":"409d7952-f6b1-4e85-8e44-c4cf836b45e4","order_by":2,"name":"Mislav Mikuš","email":"","orcid":"","institution":"KBC Zagreb: Klinicki Bolnicki Centar Zagreb","correspondingAuthor":false,"prefix":"","firstName":"Mislav","middleName":"","lastName":"Mikuš","suffix":""},{"id":495393373,"identity":"466b8fa0-2a4a-4b29-a2fd-2a9902ae7ede","order_by":3,"name":"Andrija Karačić","email":"","orcid":"","institution":"Klinicka bolnica Sveti Duh","correspondingAuthor":false,"prefix":"","firstName":"Andrija","middleName":"","lastName":"Karačić","suffix":""},{"id":495393374,"identity":"defd3aab-4ca6-44ea-9b43-047809153d61","order_by":4,"name":"Ana-Marija Liberati Pršo","email":"","orcid":"","institution":"Klinicka bolnica Sveti Duh","correspondingAuthor":false,"prefix":"","firstName":"Ana-Marija","middleName":"Liberati","lastName":"Pršo","suffix":""},{"id":495393375,"identity":"fc4c2c71-6349-45eb-abf1-60f4b5b52633","order_by":5,"name":"Mario Ćorić","email":"","orcid":"","institution":"Klinicki Bolnicki Centar Zagreb","correspondingAuthor":false,"prefix":"","firstName":"Mario","middleName":"","lastName":"Ćorić","suffix":""},{"id":495393376,"identity":"3a4899e8-8ca7-43c6-a206-2a4565b2e975","order_by":6,"name":"Ira Renko","email":"","orcid":"","institution":"Gut Microbiome Center","correspondingAuthor":false,"prefix":"","firstName":"Ira","middleName":"","lastName":"Renko","suffix":""}],"badges":[],"createdAt":"2025-05-06 12:42:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6603155/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6603155/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13063-025-09405-5","type":"published","date":"2026-02-20T15:57:16+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":88652128,"identity":"caba7120-1ed2-4fe2-ae98-616cf4d3463d","added_by":"auto","created_at":"2025-08-08 17:54:49","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":93789,"visible":true,"origin":"","legend":"\u003cp\u003eDesign of our double-blind, cross-over, randomized trial\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6603155/v1/2a9f323c30c532ac97019dc5.jpg"},{"id":88652921,"identity":"0367fe97-75e0-4284-a1d2-b566642d72d7","added_by":"auto","created_at":"2025-08-08 18:02:49","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":96373,"visible":true,"origin":"","legend":"\u003cp\u003eOverview on study outcome measures\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6603155/v1/a9bcb107362125651baf4a0c.jpg"},{"id":103251033,"identity":"a6f0dccc-31df-469e-840d-8da50543456d","added_by":"auto","created_at":"2026-02-23 16:01:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":942715,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6603155/v1/b84089e3-3d91-47af-97f6-5cd497a31e69.pdf"}],"financialInterests":"","formattedTitle":"The role of probiotics in the treatment of endometriosis (ProMetrioS): a randomised double-blinded placebo-controlled cross-over trial","fulltext":[{"header":"Background and rationale","content":"\u003cp\u003eEndometriosis is a chronic gynecological hormonal disease that is defined by the appearance of tissue lesions caused by the growth of endometrial-like tissue outside the uterus, most often inside the abdominal cavity, although lesions can also be found in distant organs\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Endometriosis affects around 10% of women of reproductive age, and it is estimated that 196\u0026nbsp;million women around the world suffer from this disease.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e It is associated with serious, sometimes severe symptoms, including chronic pelvic pain, dysmenorrhea, and dyspareunia. The most serious consequence of endometriosis is certainly infertility, which affects 30% of patients.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e Endometriosis is often under-diagnosed and poorly treated due to the limited options for diagnosis and therapy.\u003c/p\u003e\u003cp\u003eIts pathophysiology is highly complex and includes intertwined hormonal and inflammatory mechanisms.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e Recent studies have identified the gut microbiome to be an additional player in endometriosis.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e Specifically, the estrobolome, bacterial members of the gut microbiome involved in the metabolism of estrogen.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e These bacteria possess enzymes which alter the chemical structure of estrogen, reinforcing its reabsorption in the colon.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e Since the disbalance of circulatory estrogen levels is vital for the progression of endometriosis these bacteria are therefore indirectly involved in the development and progression of endometriotic lesions and thus may significantly alter the course of disease.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e Additionally, the gut microbiome impacts the level of chronic inflammation, which may aggravate symptoms of endometriosis such as chronic pain.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eDysbiosis, a disbalanced and dysfunctional gut microbiome has been found in many patients on endometiosis patients.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e The gut microbiome of endometriosis patients is frequently less diverse, with higher abundances of genera such as \u003cem\u003eBlautia, Dorea, Streptococcus\u003c/em\u003e, and lower abundances of genera \u003cem\u003eParaprevotella, Lachnospira and Turicibacter.\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e The gut microbiome of endometriosis patients could intensively benefit from gut-microbiome directed therapeutics.\u003c/p\u003e\u003cp\u003eOne could hypothesize that gut-microbiome directed therapeutics, such as probiotics, could even positively affect the pathophysiological processes active in endometriosis.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e Probiotics are the best researched of all gut-microbiome directed therapeutics, be it in in vitro and in vivo, animal and human studies. Different probiotic strains are known to have anti-inflammatory, but also analgesic, antidepressive, anorexigenic and many other effects.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e Thus, probiotics are promising agents for the treatment of the gut microbiome in endometriosis patients.\u003c/p\u003e\u003cp\u003eAlthough confronted with a broad choice of potential formulations for the treatment of endometriosis, the decision was laid upon a well-researched multispecies probiotic formulation (OMNi-BiOTiC® Stress). This formulation has proven anti-inflammatory effects, antidepressive, analgesic effect on the central nervous system targeting gut-brain axis.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e Its administration has been associated with an increase in the relative abundances of biorelevant genera such as \u003cem\u003eRuminococcus\u003c/em\u003e and \u003cem\u003eCoprococcus.\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eThe chosen probiotic contains nine human derived probiotic strains: \u003cem\u003eLactobacillus casei\u003c/em\u003e W56, \u003cem\u003eLactobacillus acidophilus\u003c/em\u003e W22, \u003cem\u003eLactobacillus paracasei\u003c/em\u003e W20, \u003cem\u003eBifidobacterium lactis\u003c/em\u003e W51, \u003cem\u003eBifidobacterium lactis\u003c/em\u003e W52, \u003cem\u003eLactobacillus salivarius\u003c/em\u003e W24, \u003cem\u003eLactococcus lactis\u003c/em\u003e W19, \u003cem\u003eLactobacillus plantarum\u003c/em\u003e W62 and Bifidobacterium bifidum W23.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e That product was chosen due to the analgetic effect, regulating the production of neurotransmitters implicated in pain perception. Specifically, some of the species present in the multispecies probiotic, namely \u003cem\u003eLactobacillus acidophilus\u003c/em\u003e, \u003cem\u003eLactobacillus plantarum\u003c/em\u003e, \u003cem\u003eLactobacillus fermentum\u003c/em\u003e and \u003cem\u003eLactobacillus gasseri\u003c/em\u003e were mentioned to have that effect in the pilot-study from 2019.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e In the context of endometriosis, that could be really helpful for suppressing constant chronic pain that is present.\u003c/p\u003e\u003cp\u003eVia modulation of the gut-brain axis and immunomodulation, probiotics can act antidepressive. The antidepressive effect of bacteria such as genus \u003cem\u003eLactobacillus\u003c/em\u003e and \u003cem\u003eBifidobacterium\u003c/em\u003e could be helpful in endometriosis treatment since more than 50% of patients with painful endometriosis suffer from depression.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eThe anti-inflammatory properties of this specific formulation were seen in several studies where it was able to reduce the levels of pro-inflammatory cytokines and immune response modulation. The induced chronic inflammation could alleviate sensations of pain and discomfort, as seen in studies with patients suffering from endometriosis.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eAdditionally, one component of the multispecies formulation, \u003cem\u003eLactobacillus gasseri\u003c/em\u003e OLL2809 has been experimentally associated with an increase of IL-12 secretion which can successfully suppress the development of endometrioc lesions.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eAlthough this probiotic formulation bears many promising effects, its interaction with the gut microbiome in endometriosis patients, especially the estrobolome, has yet to be explored. Since the gut microbiome is a dynamic and complex eco-system with high interindividual variability, randomized placebo-controlled trials are required to track their potential in altering the gut microbiome.\u003c/p\u003e\u003cp\u003e\u003cb\u003eObjectives\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThis study aims to investigate the effects of probiotic administration on the gut microbiome and quality of life of endometriosis patients. The primary outcome will be to compare the composition and functionality of gut microbiome before and after probiotic treatment. As secondary outcomes, quality of life, stress level, perception of health and quality of sleep will be evaluated. Our primary hypothesis is that as a result of probiotic intake there will be significant positive changes, on level of the gut microbiome, but also regarding quality of life and other subjective parameters.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTrial design\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThis will be a double-blinded randomized placebo-controlled cross-over trial as shown in Fig.\u0026nbsp;1.\u003c/p\u003e\u003cp\u003e Patients will be recruited among the outpatient population of the National Center for Endometriosis, Department of Gynecological Surgery and Urology, Gynecology Department, University Hospital Center Zagreb following the inclusion and exclusion criteria.\u003c/p\u003e\u003cp\u003e To participate in the study all patients will be required to sign the Informed Consent form.\u003c/p\u003e\u003cp\u003eAll patients will undergo a complete anamnesis via a digital form to investigate their lifestyle (occupation, stress, diet, physical activity level).\u003c/p\u003e\u003cp\u003eThe study period will be 6 months: eight weeks on either option (verum/placebo), with an eight week washout period in between. The purpose of the washout period is to allow the gut microbiome to return to baseline. The washout period was determined based on the fact that the gut microbiome can change rapidly in few days.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eThe outcome measures (gut microbiome, quality of life) will be assessed in four distinct time points in context of the planned intervention.\u003c/p\u003e\u003cp\u003e\u003cb\u003eT1 –\u003c/b\u003e before the intervention (start-of-study)\u003c/p\u003e\u003cp\u003e\u003cb\u003eT2 –\u003c/b\u003e after the first intervention cycle/phase 1 (verum/placebo) of two months\u003c/p\u003e\u003cp\u003e\u003cb\u003eT3 –\u003c/b\u003e after the wash-out phase of two months\u003c/p\u003e\u003cp\u003e\u003cb\u003eT4 –\u003c/b\u003e after the second intervention cycle/phase 2 (verum/placebo) of two months (end-of-study)\u003c/p\u003e\u003cp\u003eStool sampling will be performed with the aid of specific kits indepently by the patients. Lifestyle and quality of life will be evaluated by an online form designed specifically for the purpose of this study in Microsoft Office Forms (Microsoft, San Francisco, USA), short Online Form.\u003c/p\u003e\u003cp\u003eThrough the process of randomization, patients will be allocated to either first receive the verum (probiotic) or the placebo (maltodextrin), and second the placebo or verum inversely.\u003c/p\u003e\u003cp\u003eBoth patients and researchers involved will be blinded (double-blind study), the patients will be informed that they are going to take a probiotic twice a day for two months with a two-month time division. The intervention will consist of treatment with a \u003cb\u003emultispecies probiotic formulation (OMNi BiOTiC® Stress)\u003c/b\u003e and \u003cb\u003ethe placebo\u003c/b\u003e which is identical in all its characteristics and packaging. Patients will be instructed to consume one sachet a day of each (probiotic) for 8 weeks and to report any symptoms or side effects related to its use. Adherence control will be carried out through the patient’s notes on a separate online form provided, and also by checking the number of sachets used.\u003c/p\u003e\u003cp\u003eSince this is a cross-over study, data will be compared between time-points \u003cb\u003eT1\u003c/b\u003e and \u003cb\u003eT2\u003c/b\u003e, and time-points \u003cb\u003eT3\u003c/b\u003e and \u003cb\u003eT4\u003c/b\u003e, with the goal of minimizing the potential effect if we find that the effect of the probiotics lasted longer than eight weeks in those randomized into the intervention arm first.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAdverse effects\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAdverse events during the intervention will be monitored via an online form where not only the intake of the sachets will be documented, but also potential gastrointestinal adverse events such as flatulence, bloating, diarrhea, constipation or pain/colics. Space for other extraintestinal adverse events will be left to the participants. In the case of adverse events, they will be immediately reported to the Human Research Ethics Committee of the University Hospital Center Zagreb. Due to the use of probiotics which are popular dietary supplements adverse events are not expected, besides possible transient digestive issues. Probiotics are generally regarded as safe with no to minimal deteriorating health effects. \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cb\u003eParticipants, interventions, and outcomes\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eStudy setting\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe study protocol, including the intervention and all measurements, will be carried out by the study staff at Gut Microbiome Center, Zagreb, Croatia.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEligibility\u003c/b\u003e\u003c/p\u003e\u003cp\u003eInclusion and exclusion criteria are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEligibility criteria\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"1\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInclusion criteria\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e• adult subjects with stage III or IV endometriosis diagnosis confirmed by biopsy\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eExclusion criteria\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e• second stage endometriosis\u003c/p\u003e\u003cp\u003e• comorbidities in terms of mental disorders\u003c/p\u003e\u003cp\u003e• comorbidities in terms of chronic inflammatory bowel diseases\u003c/p\u003e\u003cp\u003e• with stomas\u003c/p\u003e\u003cp\u003e• neoadjuvant oncological treatment administered\u003c/p\u003e\u003cp\u003e• received antibiotic or probiotic therapy six months prior to presentation to the gynecologist\u003c/p\u003e\u003cp\u003e• previous appendectomy performed\u003c/p\u003e\u003cp\u003e• following a restrictive diet (vegan, vegetarian, gluten-free, ketogenic diet)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e\u003cp\u003e\u003cb\u003eRecruitment, screening, and enrollment procedures\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eRecruitment\u003c/b\u003e\u003c/p\u003e\u003cp\u003eRecruitment will be conducted by the gynecologists treating patients at the National Center for Endometriosis, Department of Gynecological Surgery and Urology, Gynecology Department, University Hospital Center Zagreb. The chief researcher with the aid of the staff of the Gut Microbiome Center will oversee the recruitment process. During regular outpatient visits potential participants will be informed about the study. Previous research participants at the Gut Microbiome Center who approved to be contacted for future research will also be contacted via email to be considered for the trial. Profiles on online social media platforms by Meta and Linkedin of the Gut Microbiome Center and the national association of endometriosis patients will also be used to recruit participants. The trial is planned to gain exposure through articles in popular media outlets. Recruitment will start in December 2024 and finish in July 2025.\u003c/p\u003e\u003cp\u003e\u003cb\u003eScreening and enrollment\u003c/b\u003e\u003c/p\u003e\u003cp\u003eEligible patients will go through assessments at baseline via the Online Form and will be randomly allocated to 2 groups, each consisting of 20 participants. Randomization will be done by an independent researcher in the biostatistics platform at the Gut Microbiome Center. Randomization will be performed using code written in the R statistical programming language (version 3.5.3). Treatments will be assigned with a 1:1 ratio. The randomization schedule will be transferred into sets of opaque sealed envelopes. After a participant’s visit at time-point \u003cb\u003eT1\u003c/b\u003e, the study staff will open a sealed envelope which will contain the participant’s allocation. The order of the interventions will be blinded for both the investigators and the participants. Treatments will be given in sealed sachets; sachet content will be blinded by the staff of the Gut Microbiome Center, given to the study staff labeled as A or B, and dispensed to participants according to their treatment period. Treatments will not be unblinded until the analyses are complete, unless required due to adverse events during the clinical trial. There will also be a midpoint review of the participants’ clinical data by an independent gynecologist who will be unblinded.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMeasurement\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAll of the 40 patients will be randomized into two groups. One group will take the verum in the first, and the placebo in the second cycle/phase, the other first the placebo and then the verum in the second cycle/phase. The verum will consist of the supplementation with a multi-strain probiotic containing:\u003c/p\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eLactobacillus casei\u003c/em\u003e W56\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eLactobacillus acidophilus\u003c/em\u003e W22\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eLactobacillus paracasei\u003c/em\u003e W20\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eBifidobacterium lactis\u003c/em\u003e W51\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eBifidobacterium lactis\u003c/em\u003e W52\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eLactobacillus salivarius\u003c/em\u003e W24\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eLactococcus lactis\u003c/em\u003e W19\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eLactobacillus plantarum\u003c/em\u003e W62\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eBifidobacterium bifidum\u003c/em\u003e W23\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003cp\u003eThe patients will receive the probiotic in a 3g sachet containing 7.5\u0026nbsp;billion CFU. During the intervention the participants will be instructed to consume one sachet daily with water at room temperature for a period of 8 weeks. If allocated to the placebo phase the participants will receive identical sachet (one 3 g) with an identical appearance (physical and organoleptic) containing polydextrose/maltodextrin as the placebo.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePrimary outcome\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe primary outcome is the modulation of the gut microbiome. The fecal microbiome, as a genuine proxy of the gut microbiome, will be analyzed. Specific attention will be brought to taxa and metabolic pathways involved in the estrobolome.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSecondary outcome\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe secondary outcomes concern changes in quality of life and an eventual decrease in symptomatology of endometriosis patients. Two disease-specific questionnaires evaluating will be utilized to assess quality of life: the short form endometriosis health profile questionnaire (EHP-5) and the WHO Quality of Life Scale-Brief.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFurther study outcomes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThrough evaluation of lifestyle factors such as diet (Mediterranean diet adherence), stress perception, sleep quantity and physical activity, potential confounding factors influencing the effects of probiotics will be assessed.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMediator\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eQuality of life\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSelf perception of health will be assed with validated questionnaires short form endometriosis health profile questionnaire (EHP-5) and WHO Quality of Life Scale-Brief. Participants will be asked to complete the questionnaires before and after each of the intake periods. The EHP-5 is an 11-item questionnaire which has been previously translated into Croatian and validated for its psychometric properties.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e The WHO Quality of Life Scale-Brief is a 26-item measure and is used in English, since a Croatian version has not yet been validated.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDietary intake, sleep quality, stress\u003c/b\u003e\u003c/p\u003e\u003cp\u003eDietary questionnaires inquiring on adherence to the Mediterranean diet will be used to understand habitual dietary intake among the study participants at the four respective time-points. The results will monitor diet as a potential confounding factor. The participants will complete Visual Analogue Scales (VAS) regarding sleep quality, perceived stress levels and fatigue.\u003c/p\u003e\u003cp\u003e\u003cb\u003e16S rRNA sequencing of stool samples\u003c/b\u003e\u003c/p\u003e\u003cp\u003eChanges in intestinal micobiome composition will be analyzed in all participating patients. Stool samples will be collected and submitted to the attending clinic at four different time points. Both patients and medical staff will receive detailed training on the correct handling of the stool samples for subsequent microbiome analyses. The collection kits will be provided by the sponsor. The sample containers contain a preservation solution, allowing the stool samples to be stored in a refrigerator at 4°C. Long-term storage is recommended at -20°C. The samples will be stored at the study center until the end of the study and will be collected for analysis.\u003c/p\u003e\u003cp\u003eAt the end of the study, the samples will be delivered to an analysis laboratory in Germany for DNA extraction, sequencing, and evaluation.\u003c/p\u003e\u003cp\u003eThe exact methodology for microbiome analysis is described as follows: For the total DNA isolation from stool samples, the TGuide S96 Magnetic Soil/Stool DNA Kit from the company Tiangen will be used. The extracted DNA will be employed in a PCR with the specific primer for the hypervariable region V3-4 of the 16S region. The resulting amplicons will be used to prepare a library, which will then be sequenced on an Illumina platform. The FastQ raw data files will be archived and used for filtering and analysis with standard bioinformatics tools\u003c/p\u003e\u003cp\u003e\u003cb\u003eStatistical methods\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eSample size\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe study is designed to test the hypothesis that probiotic intake will lead to significant positive changes, not only in the composition and function of the gut microbiome but also in various quality-of-life metrics and other subjective health parameters. By enrolling 40 participants, we aim to detect these changes with confidence using a two-tailed t-test, ensuring that any observed effects on both microbiome composition and participant-reported outcomes can be statistically validated.\u003c/p\u003e\u003cp\u003e\u003cb\u003eRandomization and blinding\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCrossover randomization of study medication is provided by Institut AllergoSan. The allocation of the recruited patients to the respective intervention group is blinded by issuing the study package with a consecutive participant number. The blinding and randomization of the study packages are carried out centrally by Institute AllergoSan.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStatistical analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eStatistical analysis of the results of the gut microbiome analyses will be performed. The demultiplexed FASTQ raw data files from the Illumina sequencer will be used for data analysis with DADA2 in QIIME-2 and for archiving. Cleaned forward and reverse sequences will be paired and analyzed using the Amplicon Sequence Variants (ASVs) method. The underlying bacterial taxa will be identified based on the SILVA reference database. Alpha and Beta diversity analyzes will follow, along with visualization using the R package. For determining Alpha diversity, richness, Shannon index, Simpson diversity, evenness, and Chao1 will be calculated. Statistical analyzes will be performed using ANOVA or the Kruskal-Wallis test. For Beta diversity analysis, Bray-Curtis distances, weighted UniFrac, and Bray-Curtis-PcoA (Principal Coordinates Analysis) will be calculated and tested using Anosim or Adonis\u003c/p\u003e\u003cp\u003e\u003cb\u003eSafety, oversight and monitoring\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe patients will receive an online form to mark the intake of the sachet and write down any symptoms that may appear during the intervention period. Every 60 days, patients will be seen and adherence to treatment will be evaluated through repeated completion of the online form and by counting the sachets that were not consumed.\u003c/p\u003e\u003cp\u003eThe participants will be instructed to inform the research team about the need to use any other non-routine medications and dietary supplements, as some ingredients may alter the\u003c/p\u003e\u003cp\u003egut microbiome, and also to inform the team when they use a product that contains probiotics.\u003c/p\u003e\u003cp\u003eDaily checklists for the sachets will be given to the participants via an online form (sent by e-mail) for each day during the two phases. These will be used to monitor their compliance with the study protocol. Additionally, participants will be instructed to return all empty sachets for counting purposes. Compliance for both groups will be defined as an attendance of ≥ 75% of the scheduled visits with the research team and ≥ 80% of the required sachets consumed according to diary entries and returned sachets. Participants will be monitored every two weeks by the research team through phone or video calls to inquire about the intake of antibiotics or other probiotic formulations, significant lifestyle alterations or digestive tract infections, which could lead to exclusion from the study. r. This log will include participant ID, date of dispensing, randomization code, and the returned sachet counts. Concomitant medication will be recorded at the end each treatment phase.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eResearch into the gut microbiome is evolving at a rapid pace. In recent years associations between the gut microbiome and an ever-increasing number of chronic extraintestinal disorders have been demonstrated, especially of hormonal nature. Modulation of the gut microbiome via gut microbiome-directed therapeutics, such as probiotics, is drawing more and more attention in the treatment of extraintestinal diseases. Especially for diagnoses like endometriosis, where limited diagnostic and therapeutic options are available, the gut microbiome and its therapeutics seem a promising field of research. The findings from this study will provide evidence for the use of gut-microbiome directed therapeutics in the treatment of endometriosis patients.\u003c/p\u003e"},{"header":"Trial status","content":"\u003cp\u003eThe trial is submitted on clinicaltrials.gov, but the status of it is still pending.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors want to thank all study participants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSponsor\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sponsor of this study is \u0026ldquo;Institut AllergoSan\u0026ldquo;(Institut AllergoSan, Graz, Austria). \u0026nbsp;The sponsors played no role in study design, collection, management, analysis, interpretation of results, writing of the report, or the decision to submit the report for publication.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;All authors (SK, KZ, IR, AMLP AK, MM and MĆ) will be equally involved in the study. AK will oversee gut microbiome analysis and logistics, MM and MĆ will mostly be involved in patient dissemination and coordination, while IR will have biggest contribution in writing the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOnce the study is finished and the data published, the datasets used during the current study will be available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors approved the final version of the manuscript and agreed for all aspects of the work to be published.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors AK and IR have held paid presentations for the sponsor of the study. There is no other competing interest among the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eGut Microbiome Center, Zagreb, Croatia. \u003csup\u003e2\u003c/sup\u003eFaculty of Food Technology and Biotechnology, Zagreb, Croatia. \u003csup\u003e3\u003c/sup\u003eUniversity Hospital Center (KBC) Zagreb; Gynecology Department, Zagreb, Croatia. \u003csup\u003e4\u003c/sup\u003eUniversity Hospital Sveti Duh, Zagreb, Croatia. \u003csup\u003e5\u003c/sup\u003eUniversity Hospital Center (KBC) Zagreb; Gynecology Department, Gynecological Surgery and Urology, National Center for Endometriosis, Zagreb, Croatia.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eDisclaimer/Publisher\u0026rsquo;s Note:\u0026nbsp;\u003c/strong\u003eThe statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.\u003c/p\u003e\n\u003cp\u003eConflicts of Interest\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eD\u0026rsquo;Alterio, M. N., Giuliani, C., Scicchitano, F., Lagan\u0026agrave;, A. S., Oltolina, N. M., Sorrentino, F., Nappi, L., Orr\u0026ugrave;, G., \u0026amp; Angioni, S. (2021). Possible role of microbiome in the pathogenesis of endometriosis. In \u003cem\u003eMinerva Obstetrics and Gynecology\u003c/em\u003e (Vol. 73, Issue 2). https://doi.org/10.23736/S2724-606X.21.04788-2\u003c/li\u003e\n\u003cli\u003eWHO. (2023). \u003cem\u003eEndometriosis\u003c/em\u003e. Https://Www.Who.Int/News-Room/Fact-Sheets/Detail/Endometriosis.\u003c/li\u003e\n\u003cli\u003eHarada, T. (2013). Dysmenorrhea and endometriosis in young women. In \u003cem\u003eYonago Acta Medica\u003c/em\u003e (Vol. 56, Issue 4).\u003c/li\u003e\n\u003cli\u003eHughes, E. G., Fedorkow, D. M., \u0026amp; Collins, J. A. (1993). A quantitative overview of controlled trials in endometriosis-associated infertility. \u003cem\u003eFertility and Sterility\u003c/em\u003e, \u003cem\u003e59\u003c/em\u003e(5). https://doi.org/10.1016/s0015-0282(16)55911-1\u003c/li\u003e\n\u003cli\u003eBulun, S. E., Monsavais, D., Pavone, M. E., Dyson, M., Xue, Q., Attar, E., Tokunaga, H., \u0026amp; Su, E. J. (2012). Role of estrogen receptor-\u0026beta; in endometriosis. \u003cem\u003eSeminars in Reproductive Medicine\u003c/em\u003e, \u003cem\u003e30\u003c/em\u003e(1). https://doi.org/10.1055/s-0031-1299596\u003c/li\u003e\n\u003cli\u003eGarc\u0026iacute;a-G\u0026oacute;mez, E., V\u0026aacute;zquez-Mart\u0026iacute;nez, E. R., Reyes-Mayoral, C., Cruz-Orozco, O. P., Camacho-Arroyo, I., \u0026amp; Cerb\u0026oacute;n, M. (2020). Regulation of Inflammation Pathways and Inflammasome by Sex Steroid Hormones in Endometriosis. In \u003cem\u003eFrontiers in Endocrinology\u003c/em\u003e (Vol. 10). https://doi.org/10.3389/fendo.2019.00935\u003c/li\u003e\n\u003cli\u003eDang, C., Chen, Z., Chai, Y., Liu, P., Yu, X., Liu, Y., \u0026amp; Liu, J. (2024). Assessing the relationship between gut microbiota and endometriosis: a bidirectional two-sample mendelian randomization analysis. \u003cem\u003eBMC Women\u0026rsquo;s Health\u003c/em\u003e, \u003cem\u003e24\u003c/em\u003e(1). https://doi.org/10.1186/s12905-024-02945-z\u003c/li\u003e\n\u003cli\u003eTalwar, C., Singh, V., \u0026amp; Kommagani, R. (2022). The gut microbiota: a double-edged sword in endometriosis. In \u003cem\u003eBiology of Reproduction\u003c/em\u003e (Vol. 107, Issue 4). https://doi.org/10.1093/biolre/ioac147\u003c/li\u003e\n\u003cli\u003eXholli, A., Cremonini, F., Perugi, I., Londero, A. Pietro, \u0026amp; Cagnacci, A. (2023). 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The role of gut and genital microbiota and the estrobolome in endometriosis, infertility and chronic pelvic pain. \u003cem\u003eHuman Reproduction Update\u003c/em\u003e, \u003cem\u003e28\u003c/em\u003e(1). https://doi.org/10.1093/humupd/dmab035\u003c/li\u003e\n\u003cli\u003eQin, R., Tian, G., Liu, J., \u0026amp; Cao, L. (2022). The gut microbiota and endometriosis: From pathogenesis to diagnosis and treatment. In \u003cem\u003eFrontiers in Cellular and Infection Microbiology\u003c/em\u003e (Vol. 12). https://doi.org/10.3389/fcimb.2022.1069557\u003c/li\u003e\n\u003cli\u003eGuo, C., \u0026amp; Zhang, C. (2024). Role of the gut microbiota in the pathogenesis of endometriosis: a review. In \u003cem\u003eFrontiers in Microbiology\u003c/em\u003e (Vol. 15). https://doi.org/10.3389/fmicb.2024.1363455\u003c/li\u003e\n\u003cli\u003eBailey, M. T., \u0026amp; Coe, C. L. (2002). 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Jan;307(1):87-92. https://doi.10.1007/s00404-022-06691-1\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"trials","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"trls","sideBox":"Learn more about [Trials](http://trialsjournal.biomedcentral.com/)","snPcode":"13063","submissionUrl":"https://www.editorialmanager.com/trls","title":"Trials","twitterHandle":"MedicalEvidence","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"gut microbiome, estroblome, endometriosis, quality of life, symptoms, pain probiotics, gut-microbiome therapeutics","lastPublishedDoi":"10.21203/rs.3.rs-6603155/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6603155/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProMetrioS is a randomised blinded placebo-controlled cross-over trial investigating the effect of specific multispecies probiotics in patients with endometriosis. The aim of this clinical trial is to determine whether probiotic treatment can significantly modulate gut microbiome composition and functionality in endometriosis patients, specifically parameters associated with the estrobolome.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis is a double-blinded, randomized, cross-over, placebo-controlled trial. Participants are\u003c/p\u003e\n\u003cp\u003erandomly allocated to receive either 3 g of a probiotic formulation daily or placebo for 8 weeks in two study phases, separated by a 8-week washout period between the two phases. The gut microbiome is analysed in four time-points: start-of-study, after phase 1, after the washout phase, end-of-study (after phase 2) via amplicon sequencing. Quality of life related to endometriosis is assessed via validated questionnaires in the same four time-points. Our primary endpoint is a favorable modulation of the gut microbiome, especially in prospect of the estrobolome. Secondary endpoints include changes in quality of life, reported symptomatology and psychophysical condition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDiscussion\u003c/strong\u003e: The findings of this study will provide the first evidence for the use of a combination of probiotics in the treatment of endometriosis. 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