Probiotic supplementation improves quality of life and modulates oestradiol in women with endometriosis: a randomised double-blind pilot trial

Objective Endometriosis is a chronic, oestrogen-dependent inflammatory disorder affecting up to 10% of reproductive-age women, associated with pelvic pain, infertility, and reduced quality of life. Emerging evidence implicates the vaginal microbiome and oestrogen metabolism in its pathogenesis. This study aimed to evaluate the potential therapeutic role of the oral administration of Ligilactobacillus salivarius CECT 30632, a probiotic with demonstrated ability to metabolise oestrogens in vitro, to improve the quality of life in women with endometriosis.

We conducted a pilot, randomised, double-blind, placebo-controlled clinical trial assessing its effects, alongside standard dienogest treatment, in 37 women with endometriosis. Outcomes included changes in the vaginal microbiota composition (16S rRNA sequencing), in the immunological markers, in serum oestradiol levels, and in the quality-of-life of participants assessed with the EHP-30 questionnaire.

Vaginal microbial diversity remained unchanged between groups; however, ∼20% of participants exhibited individualised microbiota shifts, including transitions to Lactobacillus-dominated profiles. In the immunological markers, the IL-10 levels decreased in 65% of probiotic supplemented participants (p = 0.042). Serum oestradiol levels were significantly reduced (∼50%) in the probiotic group (p = 0.013). The probiotic supplementation was associated with significant improvements in pain, emotional well-being, and perceived self-control (p < 0.05).

Daily oral intake of L. salivarius CECT 30632 was associated with changes in circulating oestradiol levels and improvements in the quality of life in women with endometriosis. These preliminary findings support further powered trials to confirm efficacy and clarify underlying mechanisms. SHORT CONDENSATION This randomised double-blind pilot trial shows that a daily oral intake of a probiotic strain reduced oestradiol levels, improved pain, emotional well-being, and overall quality of life in women with endometriosis under dienogest treatment.

Endometriosis is a chronic inflammatory condition characterised by the growth of endometrial-like tissue outside the uterus, affecting 5–10% of reproductive-age women worldwide [Citation1,Citation2]. It is commonly associated with chronic pelvic pain, dysmenorrhoea, dyspareunia, and infertility, but increasing evidence indicates that it is a systemic disease with metabolic, immunological, and neurological consequences [Citation3]. Although the precise aetiology of endometriosis remains unclear, growing evidence suggests that immune dysfunction, chronic inflammation, and hormonal imbalances are key contributors to its pathophysiology. Recent advances, including a consensus Human Endometrial Cell Atlas (HECA) confirm an abnormal inflammatory environment within the endometrium of patients with endometriosis, driven by dysregulated decidualised stromal cells and macrophage populations [Citation4]. Currently, there is not long-term cure for this disease and, consequently, the quality of life of affected women is highly compromised and often aggravated by misdiagnosis, diagnostic delays and lack of effective therapies [Citation3]. Despite the availability of hormonal therapies, pain management strategies, and surgical interventions, recurrence rates remain high, underscoring the need for alternative therapeutic approaches [Citation5]. Emerging research highlights the role of gut and vaginal microbiota in modulating oestrogen metabolism and inflammatory pathways, suggesting novel therapeutic opportunities [Citation6–8]. In particular, Ligilactobacillus salivarius CECT 30632 has shown promise in improving fertility outcomes in women with habitual abortion or infertility of unknown origin and in degrading oestradiol in vitro [Citation9,Citation10], making it a promising candidate, alone or in combination with hormonal therapies, for improving symptoms associated with endometriosis. Here, we report a randomised, double-blind, placebo-controlled trial evaluating the effects of standard hormonal treatment with dienogest (2 mg/day), with or without L. salivarius CECT 30632 supplementation, in women with endometriosis. Outcomes included quality of life scores, serum immunological and oestradiol levels, and vaginal microbiota composition.

Ethics statement and study design This randomised, double-blind, placebo-controlled pilot trial was approved by the Research Ethics Committee for Medicines of the Hospital Universitario La Paz, Madrid (Spain) (Code HULP: 6460; approval date: 11/05/2023) and conducted in accordance with the Declaration of Helsinki (Registration number ISRCTN11711042; registration date: 18/06/2025). Written informed consent was obtained from all participants. Women aged 18–45 years were recruited from the Gynaecology and Obstetrics units of the Hospital Universitario La Paz (Madrid) and the Hospital Quironsalud (Málaga) between May and September 2023. This age range was selected to include adult women within reproductive age, minimising the potential influence of menopausal hormonal changes. Participants were diagnosed with peritoneal endometriosis confirmed by transvaginal ultrasound performed by expert operators at the Gynaecology and Obstetrics units of the aforementioned hospitals. Diagnostic criteria were based on the consensus statements of the International Deep Endometriosis Analysis (IDEA) group [Citation11,Citation12]. Moreover, participants reported a pelvic pain score ≥ 3 on the Numerical Rating Scale (NRS) and were already undergoing daily treatment with 2 mg dienogest. At the time of enrolment, participants had been under dienogest treatment for a mean duration of 46 weeks. Exclusion criteria included BMI > 30 kg/m2, other inflammatory gynaecological conditions other than endometriosis, polycystic ovarian syndrome, ovulatory dysfunction, immunosuppression, severe acute or chronic illnesses, cardiovascular disease, autoimmune disorders, pregnancy, breastfeeding, abnormal vaginal bleeding, consumption of medications, complements or food supplements that could have an impact on the development of the study, such as antibiotics, other pro- and prebiotics, laxatives, etc. during the course of the study or participation in another clinical trial within 30 days before the study. A total of 37 recruited women were randomised using a parallel design without stratification (Statistical Analysis System, SAS). Both groups were under dienogest therapy. Women in group 1 (n = 19) ingested daily a capsule of L. salivarius CECT 30632 (∼9 log10 colony-forming units (CFU)) and women in group 2 (n = 18) ingested a placebo capsule devoid of the bacterial strain that had the same adjuvants and the same organoleptic properties as the investigational product (i.e., HPMC capsule, ascorbic acid, magnesium stearate and potato starch). Allocation was concealed from participants, care providers, and study personnel. The intervention lasted 16 weeks with assessments at baseline (V1) and at the endpoint of the clinical trial (V2). Primary outcomes were changes in vaginal microbiota composition and inflammatory markers; secondary outcomes included 17β-oestradiol levels and quality-of-life measures. Adverse events (AEs) were monitored throughout the study and graded for causality, severity, and outcome (Supplementary Information, SI). Participants could withdraw at any time, and investigators could discontinue participation for safety or protocol non-compliance; those participants taking < 75% of doses were withdrawn without replacement. Quality of life evaluation Quality of life was measured using the EHP-30 questionnaire [Citation13] at baseline and endpoint. This instrument includes 30 items across five domains (pain, control/powerlessness, emotional well-being, social support and self-image), scored on a 0–4 frequency scale. Scores were transformed to a 0–100 scale, with higher values reflecting worse health (SI for calculation details). Sampling and processing Blood samples were collected at both hospitals and processed at the respective clinical analysis services. The samples were centrifuged, and sera were aliquoted and stored at − 80 °C until analysis. Serum levels of interleukin (IL)-8, IL-10, tumoral necrosis factor (TNF)-α, vascular endothelial growth factor A (VEGF-A), and transforming growth factor β2 (TGF-β2) were quantified using a Luminex XMAP™ Technology analyser available at the clinical analysis service of Hospital Universitario La Paz, Madrid (Spain) and the commercial kit (Milliplex Magnetic Bead Kit – TGFBMAG-64K-03, Millipore). All determinations were performed following the manufacturer’s protocols and standard curves were built for each analyte. Serum 17β-oestradiol concentrations were quantified using a chemiluminometric immunoassay on an Atellica® IM analyser (Siemens Healthineers, Germany). As stated, all the participants were receiving dienogest at the time of study inclusion. Baseline 17β -oestradiol levels were therefore measured during ongoing hormonal treatment. As ovulatory cycles are suppressed under continuous progestin therapy and a true follicular phase cannot be defined, blood sampling was scheduled based on each participant’s menstrual history prior to hormonal treatment in order to approximate the timing of the early follicular phase and to standardise sample collection across participants.Vaginal swabs were collected by clinical staff during hospital visits by inserting the swab 1–2 cm into the vagina and rotating against the vaginal wall. Swabs were placed into tubes containing 1 mL of TE50 buffer (10 mM Tris-HCl, 50 mM EDTA, pH 8.0), vortexed for 1 min, and incubated at 37 °C for 30 min. This incubation step improves rehydration of vaginal mucus and release of cellular material, increasing further DNA recovery. Each vaginal suspension (VS) was distributed (500 µL) into two sterile tubes and stored at − 80 °C for metataxonomic analysis. DNA extraction from vaginal samples For mechanical lysis, 500 µl of VS were placed into Fast-Prep tubes with 50 µl of 10% SDS and processed using a Fast-Prep® FP120 device (Thermo) for 3 min at maximum speed. The tubes were centrifugated at 15,000 × g for 1 min at 4 °C, and the resulting supernatants were transferred to 2 ml Eppendorf tubes. Then, a mix of enzymes consisting of 10 µl of lysozyme (100 mg/ml), 30 µl of mutanolysine (10 KU/ml), and 24 µl of lysostaphin (4,000 U/ml) was suspended in 135 µl of TE50 buffer, mixed with the samples and incubated at 37 °C for 90 min. Subsequently, 25 µl of proteinase K (100 μg/ml) and 500 µl of AL Buffer (QIAamp® DNA Mini Kit 250) were added to the samples and incubated at 56 °C for 30 min. Finally, 100 µl of 3 M sodium acetate and 500 µl of 96% (v/v) ethanol were added to each sample and mixed. DNA purification was carried out using the NucleoSpin® Gel and the PCR Clean-up protocol (QIAGEN). DNA was eluted in 25 µl of molecular-grade water (Sigma). Extracted DNA was stored at − 20 °C until metataxonomic analysis was performed. Metataxonomic analysis of vaginal samples The V3–V4 hypervariable region of the 16S rDNA was amplified by PCR using universal primers S-D-Bact-0341-b-S-17 (CCTACGGGNGGCWGCAG) and S-D-Bact-0785-a-A-21 (GACTACHVGGGTATCTAATCC), and sequenced on an Illumina MiSeq platform (Genomic Platform, Scientific Park of Madrid, Spain). Barcodes were added to the 3′ and 5′ ends of PCR amplicons in a second PCR to enable separation of forward and reverse reads. PCR product concentration was assessed using a 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). Amplicons were pooled at approximately equimolar ratios, gel-purified from the expected-size band using the QIAEX II Gel Extraction Kit (Qiagen, Hilden, Germany), and quantified with PicoGreen (BMG Labtech, Jena, Germany). The pooled, purified, barcoded amplicons were then sequenced using the Illumina MiSeq paired-end protocol. Bioinformatic analysis Raw sequence data were demultiplexed and quality filtered using Illumina MiSeq Reporter. Microbiome bioinformatics was performed in QIIME 2 (v2019.1), with denoised by DADA2 and taxonomic assignment of amplicon sequence variants (ASVs) using q2-feature-classifier (classify-sklearn, naïve Bayes) against SILVA v132. Statistical analyses were performed in R (v3.5.1), generating an OTU count table per sample and normalising taxa abundances to the total number of sequences per sample. Differential relative abundances between groups were assessed using LEfSe (Galaxy/Huttenhower). Alpha diversity was evaluated using Shannon and Simpson indices (vegan v2.5.6). Beta diversity was assessed at genus level by principal coordinates analysis (PCoA) based on Bray–Curtis (relative abundance) and binary Jaccard (presence/absence) dissimilarities. Differences in community composition between groups were tested using Adonis/nonparametric MANOVA (PERMANOVA) with 999 permutations (vegan v2.5.6). Statistical analysis Participants with missing outcome data were excluded from the primary analysis (complete-case analysis). Microbial data were expressed as log10 CFU/mL. Statistical analyses and plotting were performed using the R software (×64) version 4.0.3 (desktop). Data normality was assessed using the Shapiro–Wilk test. Depending on distribution, variables were reported as means ± standard deviation (SD)/95% confidence intervals (CI) or medians/interquartile ranges (IQR). Comparisons between groups were conducted using: Kruskal-Wallis and Wilcoxon-Mann-Whitney tests for non-parametric data, with Bonferroni correction for multiple comparisons when indicated; Student’s t-test and ANOVA for parametric data, with Bonferroni adjustment for multiple comparisons; Wilcoxon’s rank-sum tests and PERMANOVA for microbial differences. Statistical significance was set at p-value < 0.05. This study has the characteristics of a proof of concept, so no formal sample size calculation has been performed.

A total of 37 participants diagnosed with peritoneal endometriosis were randomised (19 in Group 1 and 18 in Group 2). Following randomisation, five participants were excluded from the final analysis due to incomplete data. All exclusions resulted from participants missing a key follow-up visit, which precluded the collection of primary outcome data (). At baseline, there were no statistically significant differences between the intervention group and the placebo group across all demographic and clinical variables (p > 0.05 for all comparisons) (). During the study, 27 women experienced at least one AE. A total of 123 AEs were reported, corresponding to an average of 4.7 events per patient. In both groups, most AEs were mild (110/123), and 99.2% (122) were unrelated to the treatment. The most frequently reported AEs categories were ‘reproductive system and breast disorders’ (n = 37; 30.1%), ‘nervous system disorders’ (n = 28; 22.8%), and ‘gastrointestinal disorders’ (n = 21; 17.1%). No significant difference was observed in the frequency of patients experiencing an AE between the two groups (X-square = 2.91, p-value = 0.0879, Pearson’s Chi-square test) (see SI for more information regarding incidence of AEs and reported AEs). Effect of the intervention on the vaginal bacteriome of women with endometriosis 16S rRNA gene sequencing analysis of vaginal exudate samples (n = 64) yielded over 10 million high-quality filtered sequences. The mean sequence values per sample are detailed in supplementary material (Table S1). Sequences were assigned to a total of 21 phyla and 307 genera. Alpha diversity was assessed using Shannon’s and Simpson’s indices (, respectively). This parameter quantifies the diversity and abundance of bacterial genera within the analysed samples, reflecting the richness of bacterial genera in each microbial community. Results revealed no statistically significant differences between baseline and post-intervention samples in either the probiotic (Group 1; n = 17) or placebo (Group 2; n = 15) groups (p > 0.05, Kruskal-Wallis test). Likewise, between-group comparisons showed no significant differences (p = 0.94 and p = 0.96 for Shannon and Simpson indices, respectively). Beta diversity was studied using principal coordinates analysis (PCoA) to visually display patterns of bacterial profiles at the genus level through a distance matrix containing a dissimilarity value for each pairwise sample comparison. The analysis of the beta diversity at the genus level was calculated according to the relative abundance of bacterial genera (Bray–Curtis distance) and the presence/absence of bacterial genera (Binnary Jaccard distance matrix) (). The Bray-Curtis distance matrix indicated similar bacterial community structures across intervention groups, with no significant differences (p > 0.05, Kruskal-Wallis test) (). Similarly, the binary analysis of the Jaccard distance matrix revealed no significant differences between the intervention groups (p > 0.05; Kruskal-Wallis test) (). However, while both groups exhibited consistent clustering, a subset of vaginal samples (∼20%) displayed distinct microbial composition. Sequences corresponding to the 20 most abundant genera were identified in the vaginal exudate samples (n = 64) from the 32 patients included in this study (). Lactobacillus DNA was detected in all the samples and relative abundance analysis identified this genus as the dominant one in 63% of baseline vaginal samples (20 out of 32) (). In G1, the median [IQR] relative abundance of Lactobacillus in the vaginal samples was (97.69% [41.42–98.87]) and (97.89% [42.87–98.52]) at the beginning and at the end of the intervention, respectively. In the samples from G2, the initial and the end median [IQR] values of Lactobacillus abundance were (96.67% [29.86–98.49]) and (97.89% [96.21–98.43]), respectively. No significant shifts in Lactobacillus abundance occurred between timepoints or groups (p = 0.92, Kruskal-Wallis test). In 37% of the baseline vaginal samples (12 out of 32) the relative abundance of Lactobacillus was below 60%, being other genera such as Gardnerella and Atopobium predominant in these cases. Although not statistically significant, Gardnerella showed a decreasing trend in G1, correlating with an increased Lactobacillus abundance (). To further explore into these observations, a patient-specific analysis of the relative abundances of the detected genera was conducted, categorising data based on sample collection time and intervention group (). This analysis revealed that at baseline, 41% of women in G1 (7 out of 17) and 33% in G2 (5 out of 15) exhibited a non-Lactobacillus-dominated (NLD) microbiota. Three NLD participants in G1 (patients 15010-10; 15015-15 and 25006-21) transitioned towards a Lactobacillus-dominated (LD) microbiota following the intervention. Their initial Lactobacillus relative abundances were 0.36%, 41.41% and 23.69%, respectively, and increased to 62.61%, 99.33% and 52.87% post-intervention. All of them showed a concomitant decrease in Gardnerella abundance, from 42.18%, 34.06% and 72.43% at baseline to 13.16% and 0.01% and 48.89%, respectively. Effect of the intervention on immunological parameters The median [IQR] levels of the immunological parameters that were tested in this work (IL-8, IL-10, VEGF-A, TNFα, TGF-β2) are presented in . No significant inter- or intra-group differences were observed (p > 0.05, Kruskal-Wallis; Wilcoxon rank test). However, a trend towards a reduction in the median levels of VEGF-A, from 269.20 [56.16–439.93] pg/mL to 195.10 [87.54–382.03] pg/mL, and TGF-β2, from (3844.22 [1982.68–4068.08] pg/mL to 3143.78 [2213.74–3764.02]) pg/mL, was observed in G1. Individual-level analysis showed that IL-10 levels significantly decreased in 65% of the G1 participants (p = 0.045, repeated-measures Kruskal-Wallis test) (). Effect of the intervention on the quality of life In the L. salivarius CECT 30632 group, results revealed statistically significant differences at the study endpoint in the categories of pain (p = 0.014; Wilcoxon rank test), control & helplessness (p = 0.0014), and emotional well-being (p = 0.037) (, respectively). These differences corresponded to significant reductions (i.e., significant improvement according to EHP-30 scale) in three categories out of the 5 included in the EHP-30 questionnaire. Concretely, in G1, the feeling of pain decreased from 45.32 [32.95–59.09] to 26.74 [9.09–37.50], the lack of control and helplessness decreased from 50.00 [41.67–68.75] to 20.83 [6.25–39.58], and the emotional well-being from 50.00 [37.50–66.67] to 37.50 [18.75–47.92], respectively) (). Overall, total EHP-30 score in this group decreased from 49.02 [41.06–59.81] to 30.87 [16.44–45.40] (∼37%; p = 0.003; Wilcoxon rank test) (). In contrast, non-significant score reductions (p > 0.05, Wilcoxon rank test) were reported in the placebo group. Effect of the intervention on serum oestradiol levels Participants in each group were first stratified by serum oestradiol levels as low (167 pg/mL). No significant differences in the overall distribution of participants across categories were observed between baseline and endpoint in either the intervention or placebo group (Pearson’s Chi-square test, p = 0.1025 and p = 0.8089, respectively; SI, Table S2). Intra- and inter-group changes in mean oestradiol levels were then assessed. The treatment with L. salivarius CECT 30632 led to a significant reduction (∼50%) in the mean oestradiol values, decreasing from 105.9 to 57.6 pg/mL (p = 0.013, Student’s paired t-test). In contrast, no significant differences were observed in the placebo group (p = 0.488) between the initial and final oestradiol levels (). Individual-level analysis confirmed a significant oestradiol reduction in 71% (12/17) of G1 participants (p = 0.033, repeated-measures Kruskal-Wallis test). No significant associations were found when correlation analysis between oestradiol levels and metataxonomic data were performed (p > 0.05, Kruskal-Wallis test) ().

Findings and interpretation This pilot trial assessed whether the oral intake of L. salivarius CECT 30632 in women with endometriosis under dienogest treatment could change the vaginal microbiota composition, the quality of life and the levels of circulating immunological and hormonal markers. Multidimensional analysis revealed that L. salivarius CECT 30632 oral intake did not significantly alter vaginal microbial composition, likely reflecting the intrinsic stability and Lactobacillus dominance of the vaginal ecosystem. Nevertheless, we found significant improvements in pain-related symptoms, psychological well-being and significant reductions in circulating oestradiol and IL-10 levels. To our knowledge, these findings provide preliminary evidence that this probiotic may modulate biological pathways relevant to endometriosis, particularly those related to hormonal regulation and symptom relief.

in the context of what is known The absence of significant changes in vaginal microbiota is consistent with previous studies in healthy women, where probiotic administration resulted in minimal or no measurable changes in vaginal microbiota composition [Citation14–16]. This is not unexpected, as the vaginal microbiome is typically dominated by a single genus (Lactobacillus), making community-level shifts difficult to detect with 16S rRNA gene sequencing, which lacks species-level resolution. In our cohort, approximately 70% of participants exhibited a Lactobacillus-dominated vaginal microbiota at baseline, as expected for healthy reproductive-age women [Citation17–19]. Although vaginal dysbiosis has been proposed to contribute to endometriosis pathogenesis, available evidence remains inconsistent due to heterogeneous study designs, small samples, and patient-level variation [Citation20–25]. Overall, our findings on vaginal microbiota in women with endometriosis, showing predominantly Lactobacillus-dominated profiles, are broadly consistent with previous reports in both healthy and endometriosis cohorts, although some studies have described an enrichment of Gardnerella, Sneathia, and other taxa in severe endometriosis cases [Citation26–28]. In this trial, all participants received a 2 mg daily oral dose of dienogest, a progestin with anti-proliferative, anti-inflammatory, and antiangiogenic effects on endometriotic tissue [Citation29]. While oestrogen-containing contraceptives usually support Lactobacillus-dominance [Citation7], the impact of progestin-only treatments on the vaginal microbiota remains unclear [Citation30]. In our cohort, Gardnerella spp. was relatively abundant at baseline in 37% of women. Although dienogest reduces menstrual blood loss, irregular or breakthrough bleeding may create iron-rich conditions favourable to Gardnerella proliferation [Citation31–37]. The observed decreased in Gardnerella abundance in the intervention group accompanied by an increasing trend of Lactobacillus, yet non statistically significative, may suggests a modest microbiota stabilising effect of L. salivarius CECT 30632, consistent with previous findings in women with infertility of unknown origin [Citation9]. Endometriosis is characterised by a complex inflammatory milieu, yet its immunological profile remains incompletely defined. Key immunological factors analysed in this study are implicated in disease progression [Citation38–40]. IL-10 plays a pivotal role in endometriosis through immunomodulation, angiogenesis and inducing a pro-fibrotic phenotype in endometriotic stromal cells [Citation41,Citation42]. Some studies have correlated elevated IL-10 levels with endometriosis severity and increased pain [Citation40,Citation42–44] while others found significantly lower IL-10 levels in patients with endometriosis [Citation45], reflecting individual heterogeneity. In our cohort, a reduction in IL-10 was observed in participants of the probiotic group, which is consistent with the reported reduction of pain in this group. As occurs in chronic inflammation, scarring and tumours, VEGF-A is overexpressed in ectopic endometriotic tissue stimulating division, migration, and differentiation of endothelial cells while increasing vascular permeability via its vasodilator effect [Citation42,Citation46]. TGF-β2, a member of the transforming growth factor superfamily, is involved in the proliferation, differentiation, and apoptosis of endometrial cells during the menstrual cycle [Citation47]. None of these markers showed significant changes, although modest downward trends were observed. These results broadly reflect the variability reported in previous works [Citation40,Citation48] and emphasise the need for larger, stratified cohorts to clarify whether microbial or immunological shifts have clinical significance in endometriosis. Women receiving the L. salivarius strain reported significant reductions in pain perception, emotional distress, and feelings of helplessness. Endometriosis-associated pain arises from a complex interplay of peripheral and central pathways, involving inflammatory, neurogenic, and hormonal mechanisms, with oestradiol known to amplify nociceptive signalling [Citation3,Citation49,Citation50]. Oestradiol levels are central to modulate pain signalling in endometriosis by upregulating neurotrophic factors and inflammatory cytokines [Citation3,Citation51]. The 54% reduction in circulating oestradiol levels among women receiving L. salivalirus CECT 30632 is consistent with in vitro evidence suggesting that this strain may modulate oestradiol activity through conjugation/deconjugation mechanisms [Citation52]. Hormonal modulation represents the keystone of endometriosis management. Combined hormonal contraceptives and progestin-based therapies are effective in reducing endometriosis-associated pain and improving quality of life, although differences among regimens and their long-term effects remain incompletely defined [Citation53]. Dienogest has been established as an effective long-term treatment option for endometriosis [Citation54]. In our study, all participants had been receiving dienogest for a mean duration of 46 weeks prior to enrolment, thus representing a relatively stable hormonal treatment background. As pre-dienogest oestradiol levels were not available, our findings should not be interpreted as reflecting the primary endocrine effect of this treatment initiation. Rather, the observed reduction of circulating oestradiol levels in the probiotic group appears to occur within an established dienogest-treated population, suggesting that this probiotic strain may complement the already proven efficacy of dienogest [Citation55]. Clinical implications The findings from this pilot trial indicate that L. salivarius CECT 30632 may offer clinically meaningful benefits when used alongside standard therapy for endometriosis. Women receiving a daily oral intake of the probiotic reported notable improvements in both pain-related and psychological symptoms, suggesting an additive effect beyond that achieved with dienogest alone. The observed reductions in inflammatory and hormonal markers provide a plausible biological basis for these improvements. Importantly, this probiotic can be administered orally without the time limitations associated with other hormonal therapies, such as gonadotropin-releasing hormone (GnRH) antagonists, making it a potentially valuable and safe option for long-term symptom management. Although the persistence of microbial and hormonal effects after discontinuation remains unclear, evidence from other chronic gynaecological and gastrointestinal conditions suggests that prolonged probiotic use may enhance clinical outcomes [Citation56,Citation57]. Research implications These preliminary results show that this probiotic is safe and may have a positive impact as adjunctive strategy on the clinical management of endometriosis. However, larger studies, with adequately calculated samples are essential to validate these results. Future studies should integrate multi-omic analyses, including metagenomics and metabolomics, and include faecal sampling to determine whether modulation occurs primarily at the vaginal or at the gastrointestinal level. Strengths and limitations The combination of a double-blind clinical design and a multidimensional evaluation approach (microbiological, immunological, endocrine, and clinical) reinforces the exploratory value of these results. However, the pilot nature of the study, small sample size, and the exclusion of women with obesity (BMI > 30 kg/m2) limit the generalisability of the findings. Modest microbiota or immune effects may have gone undetected, and results should be interpreted with caution. The absence of an intermediate assessment time point limits an evaluation of the longitudinal progression of the observed changes. A further limitation of this study is that information regarding diet, previous surgical treatment for endometriosis and reproductive history was not collected. These factors may influence clinical characteristics and hormonal or immunological parameters. However, all participants were receiving dienogest therapy at the time of inclusion, which provided a relatively homogeneous background. The diagnosis of peritoneal endometriosis was based on clinical and transvaginal ultrasound criteria without laparoscopic confirmation. Although recently published recommendations of the IDEA group have improved the imaging assessment of superficial lesions, transvaginal ultrasound has limited sensitivity for detecting superficial endometriosis. Therefore, despite the high level of expertise of the personnel involved in this study, the precise characterisation of the endometriosis phenotype may have been affected. All these aspects should be considered in future research.

In this double-blind pilot trial, oral supplementation with L. salivarius CECT 30632 was associated with improvements in pain and psychological well-being, and with reductions in circulating oestradiol and IL-10. These preliminary findings suggest that this probiotic may serve as a well-tolerated adjunct to conventional management of endometriosis. Confirmation in larger, clinically powered studies is essential before routine clinical use can be recommended. Supplemental material 9mars_V3_Jara et al_Supplementary (1).docx Download MS Word (1.3 MB)9mars_V3_Jara et al_Supplementary (1).docxDisclosure statement MD PhD P.A. Regidor is Medical Director Exeltis Europe & Global Projects in the company Exeltis Heathcare. Rocío Gutiérrez is OTC Technical and Regulatory Manager in the company Chemo Research. Beatriz Lázcoz is R&D Scientific Project Leader in the company Exeltis Healthcare. These authors are affiliated with companies involved in product development and were involved in the design and supervision of the clinical trial, as well as in the critical review of the final manuscript. To minimise potential bias, these authors had no role in data analysis or interpretation, and all data were coded and analysed in a blinded manner. The other authors declare no competing financial or non-financial interests. Data availability statement The authors declare that the data supporting the findings of this study are available within the paper. Should any raw data files be needed they are available from the corresponding author upon request. De-identified individual participant data, the data dictionary, and the statistical analysis code are not publicly available due to ethical restrictions and confidentiality agreements. However, these materials can be made available from the corresponding author upon reasonable request and subject to approval by the institutional ethics committee. The datasets generated and/or analysed during the current study are available in the ISRCTN [Registration number ISRCTN11711042; registration date 18/06/2025]. Additional information Funding

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