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

The trial is submitted to clinicaltrials.gov, but the status of it is still pending.

The study protocol, including the intervention and all measurements, will be carried out by the study staff at the Gut Microbiome Center, Zagreb, Croatia. Inclusion and exclusion criteria are presented in Table 1 . Table 1 Eligibility criteria Inclusion criteria •Adult subjects with stage III or IV endometriosis diagnosed and confirmed by biopsy Exclusion criteria •Stage II endometriosis •Comorbidities in terms of mental disorders •Comorbidities in terms of chronic inflammatory bowel diseases •Patients with stomas •Neoadjuvant oncological treatment administered •Antibiotic or probiotic therapy recieved six months prior to presentation to the gynecologist •Previous appendectomy performed •Following a restrictive diet (vegan, vegetarian, gluten-free, ketogenic diet) Eligibility criteria •Stage II endometriosis •Comorbidities in terms of mental disorders •Comorbidities in terms of chronic inflammatory bowel diseases •Patients with stomas •Neoadjuvant oncological treatment administered •Antibiotic or probiotic therapy recieved six months prior to presentation to the gynecologist •Previous appendectomy performed •Following a restrictive diet (vegan, vegetarian, gluten-free, ketogenic diet) 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 support 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 agreed to be contacted for future research will also be contacted via email to be considered for the trial. Profiles on online social media platforms (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 is expected to in July 2025. Eligible patients will go through assessments at baseline via the Online Form and will be randomly allocated to two groups, each consisting of 20 participants. Randomization will be done by an independent researcher using 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, labeled as A or B, and provided to the study staff, who will dispense them 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. All of the 40 patients will be randomized into two groups. One group will take the verum in the first cycle/phase, and the placebo in the second cycle/phase, while the other group will take the placebo first 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 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 3 g 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 an identical sachet (one 3 g) with an identical appearance (physical and organoleptic) containing polydextrose/maltodextrin as the placebo. A placebo was chosen as the comparator to control for placebo effects and of the carrier matrix itself. 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 given to taxa and metabolic pathways involved in the estrobolome. The secondary outcomes concern changes in quality of life and an eventual decrease in symptomatology of endometriosis patients. Two disease-specific questionnaires 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. 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. Self-perception of health will be assessed with validated questionnaires short form endometriosis health profile questionnaire (EHP-5) and the 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 questionnaires inquiring into 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 serve to monitor diet as a potential confounding factor. The participants will complete Visual Analogue Scales (VAS) regarding sleep quality, perceived stress levels, and fatigue. Changes in intestinal microbiome 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 primers for the hypervariable region V3-V4 of the 16S rRNA 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. 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. 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 Institut AllergoSan. 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 analyses 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 analyses 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. All study data will be entered electronically and exported into a secure, access-restricted database at the Gut Microbiome Center. Each participant will be assigned a unique study identification code, and all data will be stored in de-identified form. Electronic data entry will include built-in range and logic checks to minimize entry errors. Only authorized study personnel will have access to the database, which is stored on password-protected institutional servers with automated backup. Hard-copy materials (e.g., consent forms) will be stored in locked cabinets at the study site. Microbiome sequencing data and associated metadata will be stored on secure servers with restricted access and routinely backed up. Full data management procedures and standard operating protocols are available from the corresponding author upon reasonable request. 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 if/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 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. This log will include participant ID, date of dispensing, randomization code, and the returned sachet counts. Concomitant medications will be recorded at the end each treatment phase. A Data Monitoring Committee will not be established because the intervention is low risk and uses a commercially available probiotic. Safety will be monitored by the study investigators and an independent gynecologist, with any serious adverse events reported to the Human Research Ethics Committee. No interim analyses or formal stopping guidelines are planned. Early termination would only be considered in the event of a serious safety concern, and the decision would be made by the principal investigator in consultation with the Human Research Ethics Committee. No study personnel will have access to unblinded interim data.

The full trial protocol and statistical analysis plan (SAP) are available from the corresponding author upon reasonable request and will be uploaded to the ClinicalTrials.gov registry once registration is completed.

Endometriosis is a chronic gynecological hormonal disease 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 approximately 30% of patients [ 3 , 4 ]. Endometriosis is often underdiagnosed 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 as an additional player in endometriosis [ 7 – 9 ]. Specifically, the estrobolome, the bacterial members of the gut microbiome involved in the metabolism of estrogen, comprises bacteria that possess enzymes which alter the chemical structure of estrogen, reinforcing its reabsorption in the colon [ 9 – 12 ]. Since the imbalance 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 the disease [ 13 ]. Additionally, the gut microbiome impacts the level of chronic inflammation, which may aggravate symptoms of endometriosis such as chronic pain [ 13 – 16 ]. Dysbiosis, an imbalanced and dysfunctional gut microbiome, has been found in many patients withendometiosis [ 13 ]. The gut microbiome of endometriosis patients is frequently less diverse, with higher abundances of genera such as Blautia , Dorea , and Streptococcus , and lower abundances of genera such as Paraprevotella , Lachnospira , and Turicibacter [ 15 , 17 ] . The gut microbiome of endometriosis patients could substantially benefit from gut-microbiome directed therapeutics. One could hypothesize that gut-microbiome directed therapeutics, such as probiotics, could positively affect the pathophysiological processes active in endometriosis [ 9 , 13 , 18 ] . Probiotics are the best researched of all gut-microbiome directed therapeutics, in both in vitro and in vivo, across 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 made in favor of a well-researched multispecies probiotic formulation (OMNi-BiOTiC® Stress). This formulation has demonstrated anti-inflammatory effects, as well as antidepressive, and analgesic effects on the central nervous system via gut-brain axis [ 21 – 24 ]. Its administration has been associated with an increase in the relative abundances of biologically relevant 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 ]. This product was chosen due to the analgesic 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 reported to have that effect in the pilot-study from 2019 [ 18 ]. In the context of endometriosis, this could be particularly 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 those from the 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 demonstrated in several studies where it was able to reduce the levels of pro-inflammatory cytokines and modulate the immune response. The resulting chronic inflammation relief 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 inIL-12 secretion, which can successfully suppress the development of endometriotic 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 ecosystem with high interindividual variability, randomized placebo-controlled trials are required to track itspotential in altering the gut microbiome. 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 the gut microbiome before and after probiotic treatment. As secondary outcomes, quality of life, stress levels, 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 not only at the level of the gut microbiome but also regarding quality of life and other subjective parameters. This will be a double-blinded, randomized, placebo-controlled cross-over trial, as shown in Figs. 1  and 2 . Fig. 1 Design of our double-blind, cross-over, randomized trial Fig. 2 Overview on study outcome measures Design of our double-blind, cross-over, randomized trial Overview on study outcome measures 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 within a few days [ 28 ]. The outcome measures (gut microbiome, quality of life) will be assessed at four distinct time points in the 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) 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 independently 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), hereafter referred to as the Online Form. Through the process of randomization, patients will be allocated to either first receive the verum (probiotic) or the placebo (maltodextrin), and then 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 washout 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 the 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 in case we find that the effect of the probiotics lasted longer than eight weeks in those randomized into the intervention arm first. 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 provided 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 adverse health effects [ 29 – 31 ]. Participants may discontinue the intervention at any time upon request. The intervention will also be discontinued if a participant experiences a serious adverse event, clinically significant worsening of symptoms, requires antibiotic or probiotic therapy during the study period, or any condition arises that, in the judgment of the investigators, makes continued participation unsafe. Participants who withdraw will be encouraged to complete all outcome assessments whenever possible.

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, particularly those of a 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. This is especially relevant for diagnoses like endometriosis, where limited diagnostic and therapeutic options are available, and where 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.

Supplementary Material 1. Supplementary Material 1.