Oral probiotic containing microbial lysates modulates rectal and vaginal microbial environments but does not change the vaginal community state type – a pilot study

Oral probiotic containing microbial lysates modulates rectal and vaginal microbial environments but does not change the vaginal community state type – a pilot study | 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 Article Oral probiotic containing microbial lysates modulates rectal and vaginal microbial environments but does not change the vaginal community state type – a pilot study Jana URBANKOVA RATHOUSKA, Stepanka BUBENIKOVA, Jana MATULOVA, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7488779/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The main aim of this study was to characterize the changes in the rectal and vaginal microbiota, as well as local inflammatory responses following the use of multi-strain oral probiotic containing microbial lysates. Eleven healthy premenopausal women received a one-month regimen of a multi-strain oral probiotic preparation containing Lactobacillus crispatus , Lactobacillus rhamnosus , and Bifidobacterium animalis , microbial lysates and fructooligosaccharides. Rectal and vaginal swab samples were collected at baseline, after one month of probiotic use, and after a one-month washout period. Microbiota composition was assessed using 16S rRNA sequencing. Local inflammatory response was evaluated via interleukin-6 levels in the swab samples. Following one month of probiotic use, a decrease was observed in rectal bacterial richness (median number of taxa 38 vs. 37; p = 0.03) and rectal IL-6 levels (median 1.1 pg/mL vs. 0.6 pg/mL; p = 0.02), as well as a decrease in vaginal bacterial diversity (median inverse Simpson index 1.1 vs. 1.0; p = 0.03) The distribution of vaginal community state types remained unchanged. Among the probiotic strains, L. crispatus and L. rhamnosus were detected in post-intervention only in the vaginal swab samples of women who harbored them at baseline; B. animalis was not detected in any vaginal or rectal samples. To conclude, the use of a multi-strain oral probiotic containing microbial lysates and fructooligosaccharides did not alter the vaginal microbiota community state type in healthy premenopausal women, but it was associated with a modest modulation of rectal and vaginal microbial environments and a reduction in a local rectal inflammatory response. Health sciences/Medical research Biological sciences/Microbiology 16S rRNA gene bacteria vagina community state type cluster cytokine diversity microbiome microorganism richness taxa taxonomy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION The human microbiota plays a crucial role in maintaining host health, with microbial communities occupying key mucosal sites such as the gastrointestinal and genitourinary tracts 1 . The vaginal and rectal microbiotas are particularly dynamic, contributing critically to the prevention of pathogenic colonization, the modulation of immune response, and the maintenance of mucosal homeostasis 2 . In premenopausal women, a vaginal microbiota dominated by Lactobacillus species (particularly Lactobacillus crispatus or Lactobacillus gasseri ) is recognized as a key indicator of reproductive tract health. In contrast, reduced Lactobacillus abundance and increased microbial diversity are linked to a higher risk of bacterial vaginosis, sexually transmitted infections, and adverse reproductive outcomes, including preterm birth 3 , 4 . Recent research has underscored a bidirectional relationship between the gut and vaginal microbiotas, commonly referred to as the “gut-vaginal axis” 5 , 6 . This axis is hypothesized to mediate microbial and immune signaling between the intestinal and genital mucosa, allowing interventions targeting one site to influence the other 6 – 9 . Oral probiotic supplementation, particularly with formulations containing Lactobacillus and Bifidobacterium species, has been proposed as a strategy to restore or maintain vaginal microbial balance characterized by Lactobacillus dominance 10 – 17 . However, the clinical and microbiological outcomes of such interventions have been inconsistent across studies, largely due to variability in probiotic composition, treatment duration, host-related factors, and endpoint measurements 10 – 16 , 18 – 32 . A novel class of probiotic formulations includes microbial lysates (postbiotics), which are non-viable bacterial fragments containing immunogenic components such as lipoteichoic acids, peptidoglycans, and DNA 33 . These lysates can influence mucosal immune responses and may act synergistically with live microbes to enhance colonization resistance and promote immunotolerance 34 . Despite promising data on bacterial lysate in modulating gut inflammation 35 and respiratory tract infections 34 , 36 , their impact on microbiota composition and inflammatory status in the rectal and vaginal microbial niches remains inadequately understood. We hypothesized that oral administration of a probiotic containing microbial lysates would beneficially influence both the composition of microbial communities and local inflammatory responses in the vaginal and rectal niches. Assessing the localized effect of such a formulation requires a comprehensive, multi-level approach that captures both microbial dynamics and mucosal immune responses, as indicated by the levels of inflammatory mediators. Therefore, this pilot study was conducted to evaluate the impact of a multi-strain oral probiotic formulation containing microbial lysates on the rectal and vaginal microbiota and associated local inflammation in healthy premenopausal women. Microbiota composition was analyzed using 16S rRNA gene sequencing before and after a defined supplementation period, as well as following a subsequent washout phase. Local inflammation was assessed by quantifying interleukin (IL)-6 levels in rectal and vaginal swabs. RESULTS A total of 12 healthy women were recruited, of whom 11 were included in the final analysis. One woman was excluded due to antibiotic administration during the probiotic intervention. Demographic and clinical characteristics of the included women are presented in Table 1. Rectal microbial ecosystem i) Microbial composition Sequencing of rectal samples yielded a total of 1,252,339 high-quality reads, with a mean read count of 37,950 per sample. After exclusion of rare operational taxonomic units (OTUs), defined as < 10 reads per sample, a total of 227 taxa were identified in the rectal microbiota. A heatmap depicting the relative abundance of the 50 most abundant taxa is presented in Figure 1A. Before probiotic administration Among the 11 women, the median (IQR) bacterial richness and diversity were 38 (38-52) and 13.1 (10.6-17.5), respectively (Table 2). Relative abundances of the most prevalent bacterial genus are shown in the Figure 2. None of the probiotic strains used in the intervention ( Lactobacillus crispatus , Lactobacillus rhamnosus and Bifidobacterium animalis ) was detected in any participants rectal microbiota prior to probiotic use. The following clusters were observed: cluster 1 in 1 women (9%), cluster 2 in 6 (55%), cluster 3 in 2 (18%), and cluster 4 in 2 (18%); Figure 3A. After 1 month of use of probiotic After the intervention, a decrease in bacterial richness was observed [median 37, IQR (37-51); p = 0.03] while bacterial diversity remained unchanged [median 13.2, IQR (11.3-15.0); p = 0.56; Table 2]. Relative abundances of the most prevalent bacterial genus are shown in the Figure 2. No differences in the prevalence of the clusters were observed [cluster 1 in 1 woman (9%), cluster 2 in 2 women (18%), cluster 3 in 2 women (18%), cluster 4 in 1 woman (9%), cluster 5 in 4 women (36%), and cluster 6 in 1woman (9%); p = 0.17; Figure 3A]. However, there was an increase of the frequency of cluster 5 compared to its baseline prevalence before probiotic use (0%), although this difference reached only borderline statistical significance ( p = 0.09). None of the probiotic strains used in the intervention ( Lactobacillus crispatus , Lactobacillus rhamnosus and Bifidobacterium animalis ) was detected in any participants rectal microbiota after probiotic use. After a 1-month washout After the 1-month washout, no differences in bacterial richness [median 18, IQR (18-50); p = 0.56] and diversity [median 17.6, IQR (11.5-21.7); p = 0.12] were observed. Relative abundances of the most prevalent bacterial genus are shown in the Figure 2. No changes in distribution of clusters were noted ( p = 0.87); cluster 2 was present in 4 women (36%), cluster 3 in 2 women (18%), cluster 4 in 1 woman (9%), cluster 5 in 2 women (18%), and cluster 6 in 2 women (18%); Figure 3A. Of the probiotic strains used in the intervention, Lactobacillus rhamnosus and Bifidobacterium animalis remained undetectable in any participants rectal microbiota. Additionally, low levels of Lactobacillus crispatus (0.3%) were identified in one woman (Table 3). ii) local inflammation IL-6 levels were detectable in all rectal swab samples. No difference in rectal IL-6 levels were found among the clusters (cluster 1: median 2.0 pg/mL, IQR 0.8-3.2, cluster 2: median 1.1, IQR 0.7-1.5, cluster 3: median 0.6, IQR 0.3-1.2, cluster 4: median 1.1, IQR 0.2-1.6, cluster 5: median 0.7, IQR 0.4-1.1, and cluster 6: median 1.3, IQR 0.6-3.5; p = 0.40; Figure 4A). After 1 month of probiotic use, rectal IL-6 levels were reduced compared to baseline (before: median: 1.1 pg/mL, IQR 0.7-1.5 vs. after: median: 0.6 pg/mL, IQR 0.4-0.9; p = 0.02; Figure 5A). After the 1-month washout period, IL-6 increased again, reaching values higher than those observed immediately after probiotic use (washout: median: 1.3 pg/mL, IQR 0.9-1.7, p = 0.02; Figure 5A). Vaginal microbial ecosystem i) microbial composition Sequencing of vaginal samples yielded a total of 1,185,645 high-quality reads, with a mean of 35,928 reads per sample. After removing rare OTUs, a total of 43 taxa were identified in the vaginal microbiota. A heatmap depicting the relative abundance of the taxa is shown in Figure 1B. Before probiotic administration Among the 11 women, the median (IQR) bacterial richness and diversity were 4 (1-6) and 1.1 (1.0-2.5), respectively (Table 2). Relative abundances of the most prevalent bacterial species are shown in the Figure 6. The distribution of bacterial clusters was as the follows: cluster 1 in 2 (18%) women cluster 2 in 1 (9%) women, cluster 3 in 1 (9%) women, cluster 4 in 1 (9%) women, cluster 5 in 4 (36%) and cluster 6 in 2 (18%) women (Figure 3B). The following CSTs were observed: CST I-A in 4 women (36%), CST II in 2 (18%), CST III-A in 2 (18%), CST IV-B in 2 (18%), and CST IV-C3 in 1 (9%); Figure 3C. Of the probiotic strains used in the intervention, Lactobacillus crispatus and Lactobacillus rhamnosus were detected in 4 and 1 women, respectively, all of whom had CST I-A ( Lactobacillus crispatus ) and CST II ( v rhamnosus ). Bifidobacterium animalis was not detected in any participants prior to probiotic use (Table 3). After 1 month of use of probiotic After intervention, a decrease in bacterial diversity was observed [median 1.0, IQR (1.0-2.1); p = 0.03] while bacterial richness remained unchanged [median 3, IQR (2-4); p = 0.34; Table 2]. Relative abundances of the most prevalent bacterial species are shown in the Figure 6. No changes were observed in the overall distribution of the bacterial clusters (Figure 3B) and CSTs (Figure 3C), except in one woman who shifted from CST IV-C3 to CST II. In this woman, the relative abundance of Lactobacillus gasseri increased from 43% to 51% and the relative abundance of Bifidobacterium breve increased from 44% to 48%. Lactobacillus crispatus , Lactobacillus rhamnosus were detected only in women who already harbored these strains prior to the intervention. Bifidobacterium animalis remained undetectable in all women (Table 3). After a 1-month washout An increase in bacterial richness [median 4, IQR (2-9)] and diversity [median 1.4, IQR (1.0-2.4)] were observed after the 1-month washout period compared to the post-intervention values; however, these results reached only borderline statistical significance (both p = 0.06). Relative abundances of the most prevalent bacterial species are shown in the Figure 6. No further changes in distribution of the bacterial clusters (Figure 3B) and CSTs (Figure 3C) were noted compared to the post-intervention bacterial clusters and CSTs. Lactobacillus crispatus and Lactobacillus rhamnosus remained detectable only in participants who harbored them prior to the intervention. Additionally, low levels of Lactobacillus crispatus (0.3%) were identified in one woman with CST IV-B. Bifidobacterium animalis was not detected in any participant (Table 3). ii) local inflammation IL-6 levels were detectable in all vaginal swab samples. IL-6 levels varied significantly among clusters (cluster 1: median 1.6 pg/mL, IQR 1.1-2.4, cluster 2: median 26.1, IQR 13.4-78.5, cluster 3: median 23.4 pg/mL, IQR 17.7-76.8, cluster 4: median 3.1 pg/mL, IQR 2.4-12.1, cluster 5: median 1.1, IQR 0.4-1.6, and cluster 6: median 5.0 pg/mL, IQR 2.5-14.9; p = 0.0004; Figure 4B) and CST groups (CST I-A: median 1.1 pg/mL, IQR 0.4–1.4, CST II: median 1.7 pg/mL, IQR 1.3–14.3, CST III-A: median 5.0 pg/mL, IQR 2.6-14.9, CTS IV-B: median 12.7 pg/mL, IQR 3.0–38.8, and CSTIV-C3: median 78.5, IQR 78.5-78.5; p = 0.001; Figure 4C). No differences in IL-6 levels were observed between samples collected before and after probiotic administration (before: median: 2.4 pg/mL, IQR 1.4-12.1 vs. after: median 1.8 pg/mL, IQR 0.8-10.3; p = 0.15; Figure 5B) and between post-intervention and washout samples (washout: median 2.4 pg/mL, IQR 0.7-13.4; p = 0.52; Figure 5B). DISCUSSION By simultaneously profiling two mucosal compartments and incorporating both microbial and inflammatory endpoints, this study provides pilot insight into how oral probiotic formulations containing microbial lysates may influence distal mucosal environments. Principal findings of this study In healthy premenopausal women one month of oral supplementation with a multi-oral strain probiotic formulation, containing microbial lysates and fructooligosaccharides, was associated with: i) a decrease in bacterial richness in the rectal microbiota, without significant alterations in its community structure (bacterial clusters); ii) a decrease in local rectal local inflammatory response, as reflected by reduced IL-6 levels, iii) a reduction in microbial diversity in the vaginal microbiota also without changes in its community structure; iv) no new detection of L. gasseri , L. rhamnosus , or B. animalis in the rectal and vaginal microbiota by 16S rRNA gene sequencing, except in participants who harbored these taxa at baseline. The results of the study in the context of what is known In this study, a modest reduction in bacterial richness was observed in rectal samples following probiotic intake, while the overall community structure, as defined by cluster analysis, remained stable. These findings are consistent with previous observations showing that probiotic supplementation in healthy adults generally induces only minor changes in gut microbial composition, particularly in the absence of prior dysbiosis 39 , 40 . It is important to note, however, that those studies analyzed stool samples, whereas this study used rectal swabs, which may more accurately reflect the mucosa-associated microbiota 41 . The reduction in richness observed in this study likely reflects a suppression of low-abundance taxa, rather than a disruption of dominant community members. Interestingly, after probiotic supplementation, a borderline significant increase in the prevalence of a cluster characterized by a moderate to high abundance of Finegoldia magna accompanied by moderate abundances of Peptoniphilus spp (from 0–36%, p = 0.09) was noted, as well as a non-significant decrease in the prevalence of the cluster with a moderate to high abundance of Faecalibacterium prausnitzii (from 55–18%, p = 0.18). Although the small sample size and high inter-interindividual variability limit the interpretation of the cluster-level changes, the observed trends may suggest minor ecological reorganization in response to probiotic intake. In the context of an otherwise stable gut ecosystem, such shifts are not necessarily pathological but may represent transient, adaptive changes. This is in line with the concept of colonization resistance, whereby established microbial communities prevents the displacement or overgrowth of new microbial taxa 21 , 40 IL-6 is a pro-inflammatory cytokine involved in mucosal immunity and has been shown to reflect inflammatory responses in both the gastrointestinal and reproductive tract mucosa 22 , 42 , 43 . Its quantification can therefore serve as a useful biomarker for evaluating the intensity of local immune responses following probiotic intake. Despite the fact that none of the microorganisms included in the probiotic formulation were detected in the rectal microbiota, a decrease in rectal IL-6 levels was observed following supplementation, with levels returning to baseline after washout period. This effect occurred in the absence of probiotic strain colonization or major shifts in microbial composition, suggesting that the anti-inflammatory effect was mediated by indirect mechanisms. This observation is consistent with results of Stene et al., who reported that oral supplementation with Lactiplantibacillus plantarum led to reduced IL-6 secretion in rectal biopsies following ex vivo stimulation with tumor necrosis factor (TNF)-α 41 . These results support previous evidence that probiotics can modulate mucosal immunity through transient metabolic or signaling interactions, rather than stable colonization 21 . In this study, the observed anti-inflammatory effect may also be attributed to the presence of microbial lysates in the formulation. These lysates can interact with pattern recognition receptors on epithelial and immune cells, leading to modulation of local cytokine production 44 . Previous in vitro and in vivo studies have shown that microbial lysates can reduce IL-6 and TNF-α levels, promote regulatory immune responses, and strengthen epithelial barrier function 45 – 47 . Their inclusion in the probiotic formulation likely contributed to the observed immunomodulatory effect, even in the absence of detectable changes in the microbiota. These findings highlight the potential utility of microbial lysates in modulating mucosal inflammation under physiologic conditions. However, the pilot design of the study did not allow us to determine whether the reduction in local rectal inflammatory response was driven by the live probiotic component, microbial lysates or a combination of both. In contrast to the gut, reduced vaginal microbial diversity is generally associated with a healthier vaginal ecosystem 3 . In this study, a modest decrease in vaginal microbial diversity was observed following probiotic use, while dominant bacterial clusters and CSTs remained unchanged. Since a borderline significant increase in diversity ( p = 0.06) was detected after the washout period, the initial decrease may represent a transient shift toward a more stable microbiota, possible influenced by oral probiotic. The stability of vaginal bacterial clusters and CST despite supplementation is consistent with previous studies showing that the vaginal microbiota is relatively unresponsive to oral probiotic interventions, particularly in healthy women with established Lactobacillus -dominant communities 10 , 12 , 14 , 17 – 20 , 23 , 24 , 29 , 48 . This resistance to change may by partly explained by the route of administrations, as orally administered probiotics often fail reach or colonize the vaginal tract 10 , 12 , 23 , 24 . Despite their inclusion in the probiotic formulation, Lactobacillus gasseri , Lactobacillus rhamnosus , and B ifidobacterium animalis were not newly detected in rectal or vaginal samples after the intervention, based on 16S rRNA gene sequencing. Lactobacillus gasseri and Lactobacillus rhamnosus were detected only in women who harbored these species at baseline, and Bifidobacterium animalis was not detected in any samples. These findings suggest that no colonization occurred after oral probiotic administration. This observation aligns with a growing body of evidence indicating that successful vaginal colonization by oral probiotic strains in healthy women is rare 10 , 12 , 23 , 24 . Moreover, even in the gut – which is more directly accessible via oral administration – colonization by probiotic strains remains infrequent 39 , 40 , 49 . For example, Suez et al. demonstrated that probiotic colonization of the gut is highly individualized and often prevented by host-specific microbial and immune mechanisms 40 . Similarly, Zmora et al. reported that even high-dose oral probiotics do not reliably colonize the gastrointestinal mucosa 39 . In the vaginal compartment, colonization via oral intake is even less likely due to anatomical separation and the lack of direct microbial transfer. Studies in both healthy and dysbiotic populations have shown that vaginal colonization is more effectively achieved through local (intravaginal) application 50 . It is important to note that microbial detection in this study relied exclusively on 16S rRNA gene sequencing. While this method allows broad taxonomic profiling, it lacks the sensitivity of strain-specific PCRS assays. This limitation may have reduced the ability to detect low-abundance probiotic strains, potentially resulting to false-negative finding regarding their presence in vaginal and rectal microbiotas. The findings of this pilot study suggest that in healthy premenopausal women, oral supplementation with a multi-strain probiotic preparation with microbial lysates may exert an anti-inflammatory effect on local mucosal immunity, particularly within the rectal environment. The observed reduction in local IL-6 levels, in the absence of major microbial community disruption, may be clinically relevant in conditions characterized by low-grade mucosal inflammation. However, the absence of significant colonization or compositional shifts in the microbiota suggests that in healthy population, probiotic effects may occur primarily through immunomodulatory pathways rather than through microbial displacement. For routine use in healthy women, the findings from this study do not support oral probiotic supplementation as an effective strategy for microbiota modification, particularly in the absence of intestinal or vaginal dysbiosis. Strengths and limitations This study has several notable strengths. First, it assessed both rectal and vaginal environments, allowing a more comprehensive evaluation of the potential interaction along the gut-vaginal axis following oral probiotic administration. Second, combined microbial profiling – based on 16S rRNA gene sequencing – with measurement of local inflammatory response via IL-6 levels provide an integrated view of both microbial and immunological outcomes. Third, the self-collection of samples by participants simplified logistics and increased feasibility, which could support wider implementation in larger studies or real-world settings. However, the study also has important limitations. First, the sample size was small, including only 11 healthy women, which limits the statistical power and generalizability of the findings. The results should therefore be interpreted with caution and validated in larger, more diverse cohorts. Second, the absence of 18S rRNA gene sequencing precluded any assessment of the probiotics’ impact on fungal communities, including yeasts. Third, the rectal microbiota was assessed using rectal swab rather than stool samples, which my limit the comparability to studies based on fecal microbial composition and may not fully capture luminal microbial diversity. Lastly, the intensity of local inflammation was assessed using IL-6 alone; additional markers – such us IL-1β, TNF-α, or calprotectin – could have provided a more comprehensive picture of the local immune response. Lastly, the relative short duration of oral probiotic preparation administration does not preclude that a longer supplementation regimen could yield more pronounced effects. CONCLUSION The use of a multi-strain oral probiotic formulation containing microbial lysates and fructooligosaccharides did not alter the vaginal microbiota community state type, but it was associated with a modest modulation of rectal and vaginal microbial environments and a reduction in a local rectal inflammatory response. MATERIAL AND METHODS Study design Healthy (self-reported) women of reproductive age were eligible to participate in this study. Recruitment was conducted among the staff of the Charles University, Faculty of Pharmacy in Hradec Kralove, Czech Republic, and the Department of Obstetrics and Gynecology, Hospital Most, Czech Republic between August and October 2024. The inclusion criteria were as follows: i) age between 18 and 45 years, ii) female sex, iii) white people, iv) no history of vaginal infections within the previous 6 months, v) absence of acute illnesses within the previous 6onths or any chronic diseases, vi) no use any oral or vaginal probiotics within the previous 3 months, vii) willingness to take dietary supplements, and viii) willingness to comply with the study protocol. The exclusion criteria were: i) vaginal or urinary symptoms; ii) current pregnancy or intention to become pregnant, iii) breastfeeding, iv) use of antibiotics in the last 6 months or during the study period, v) use of vulvovaginal medications; vi) menstrual irregularities, vii) any diagnosed ongoing disease rendering the participant unfit for the study, and viii) intolerance to the study product. All women received the multi-strain oral probiotic preparation containing microbial lysates and prebiotic (GYNIMUN ® TRIO VAGICARE, Onapharm, s.r.o, Prague, Czech Republic) and were instructed to maintain their dietary habits and a usual level of physical activity and throughout the study. The preparation contained Lactobacillus crispatus SP28, Lactobacillus rhamnosus CA15 GIOIA, Lactobacillus rhamnosus GC Onapharm, and Bifidibacterium animalis BB Onapharm (6 × 10 9 colony-forming units of each strain per capsula), microbial lysates (5mg) of Candida albicans , Gardnerella vaginalis , Streptococcus agalactiae , and Escherichia coli , as well as fructooligosaccharides (100 mg). Women were instructed to take two capsules orally twice daily (with food, during breakfast and dinner) for 30 days. Women performed vaginal and rectal swabs themselves, after careful instructions, at three time points during the study: i) before initiating probiotic use, approximately 3–5 days after cessation of menstruation, ii) after 1 month of probiotic use, and iii) after a 1-month washout period. Women were asked to refrain from sexual intercourse and use of intravaginal products (lubricant, spermicides) for 24 hours preceding the obtaining the swabs. All women provided written informed consent prior to the collection of the samples and using of probiotics. This study was approved by the Institutional Review Board of University Hospital Hradec Kralove (August 2024, No. 202409 P04). All experiments were performed in accordance with relevant guidelines and regulations. The study was performed in accordance with the Declaration of Helsinki. Vaginal and rectal swab sampling Vaginal and rectal swab samples were self-collected by participants. All women were thoroughly instructed by the study coordinator on how to correctly obtain the samples and how to avoid contamination of the sample. Vaginal swab was obtained first. A sterile Dacron polyester swab was inserted into the cranial part of the vagina. The swab was rotated three times to collect material on all sides of the tip and left in place for 20 s to allow saturation. It was then carefully removed and placed into a polypropylene tube containing 1.5 mL of phosphate-buffered saline. The tube was immediately stored in a refrigerator. Afterward, the rectal swab was collected. A sterile Dacron polyester swab was inserted approximately 2–3 cm in the rectum, rotated the three times to contact the rectal walls and kept in place for 20 s to achieve saturation. The swab was then carefully removed and placed into a polypropylene tube containing 1.5 mL of phosphate-buffered saline. The tube was immediately stored in a refrigerator. Within 24 hours, the tubes were transferred on ice to the laboratory, where they were shaken for 20 min. After swab removal, the tubes were centrifuged at 300 × g for 15 min. The supernatants were divided into aliquots, and both the pellets and aliquots were stored at − 80 ° C until analyses. The pellets and aliquots of supernatant were used to assess the vaginal and rectal microbiotas and to measure IL-6 levels in vaginal and rectal swabs, respectively. DNA extraction from vaginal and rectal swab samples and 16S rRNA gene sequencing DNA was isolated from the vaginal and rectal pellet samples using a QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. The 16S rRNA (27F; 1492R) gene was amplified by PCR, and long-read sequencing was performed using the Oxford Nanopore Technologies Ligation Sequencing Kit V14 (SQK-LSK114) and PromethION Flow Cell R10.4.1 (FLO-PPRO114M) at SEQme s.r.o (Dobris, Czech Republic). Bioinformatic analysis of vaginal and rectal swabs 16S rRNA gene sequencing The raw 16S rRNA sequencing data were automatically processed and basecalled by MinKnow 24.06.16 software interface at default settings. Demultiplexing was done based on barcodes sequences. Demultiplexed reads were filtered based on reads length (1000-2000nt) and trimming 80 low quality nucleotides from both ends of the reads using NanoFilt version 2.8.0. Relative abundance analysis was performed using EMU software version 3.5.0 using the Silva 138.1 database with keep read counts and keep files options and applying three filters: relative abundance analysis was performed on for FASTQ files with more than 5,000 reads, reads assignment to organism was done with at least 50% certainty, identified organisms had to have at least 100 reads. Classification of microbial ecosystem Relative abundances were defined as dominant (> 50%) very high (> 20%), high (> 10%), and moderate (1–10%). i) Rectal microbial ecosystem The microbial ecosystem was classified based hierarchical clustering using the Bray-Curtis dissimilarity metric and Ward linkage in the following bacterial clusters: i) cluster 1 (a very high abundance of Gemmiger formicalis ], ii) cluster 2 (a high or moderate abundance of Faecalibacterium prausnitzii ), iii) cluster 3 (a moderate or high abundance of both Peptostreptococcus bacterium oral and Streptococcus oralis ), iv) cluster 4 (a moderate or high abundance of Dialister propionicifaciens accompanied with a high abundances Peptoniphilus lacrimalis); v) cluster 5 (a moderate or high abundance of Finegoldia magna accompanied with a moderate abundance of Peptoniphilus species), and vi) cluster 6 (a high abundance of Streptococcus periodonticum ). ii) vaginal microbial ecosystem The microbial ecosystem was classified: based hierarchical clustering using the Bray-Curtis dissimilarity metric and Ward linkage in the following bacterial clusters: i) cluster 1 (a dominant abundance of Lactobacillus gasseri ), ii) cluster 2 (a very high abundance of both Gardnerella vaginalis and Fannyhessea vaginae ), iii) cluster 3 (a very high abundance of both Lactobacillus gasseri and Bifidobacterium breve ), iv) cluster 4 (a very high abundance of both Gardnerella vaginalis and Fannyhessea vaginae accompanied with a high abundance of Lactobacillus jensenii ); v) cluster 5 (a dominant abundance of Lactobacillus crispatus ), and vi) cluster 6 (a dominant abundance of Lactobacillus iners ). Based on the VALENCIA classification, a nearest-centroid based algorithm for the classification of human vaginal microbial community 37 , into VALENCIA community state types (CST) based on similarity of cervical microbiota to a set of thirteen reference centroids available at github.com/ravel-lab/VALENCIA. Assessment of IL-6 in rectal and vaginal swabs The levels of IL-6, in the samples that underwent one freezing/thawing cycle, were assessed using enzyme-linked immunosorbent assays (Human IL-6 Quantikine; R&D Systems Inc., Minneapolis, MN, USA). The sensitivity of the test was less than 0.70 pg/mL, and the inter- and intra-assay coefficients were less than 10%. Statistical analysis Demographic and clinical data were compared using the nonparametric Mann–Whitney U test and Fisher’s exact test. Levels of IL-6 between clusters were compared with Kruskal-Wallis H test. Paired comparisons between samples obtained before probiotic use and after 1 month of use, as well as between samples obtained after 1 month of use of probiotic and after a 1-month washout period, were performed using Wilcoxon matched-pairs signed-rank test. Continuous and categorical variables were presented as medians (interquartile ranges [IQR]) and numbers (%), respectively. The heatmaps of the relative abundance of the 50 most abundant taxa in rectal swab and the taxa in the vaginal swab were created in R using the heatmap method from the NMF package 38 . Hierarchical clustering was performed using the Bray–Curtis dissimilarity metric and Ward’s method, minimizing the total within-cluster variation for linkage analysis. Differences were considered statistically significant at P < 0.05. All P -values were obtained using two-tailed tests. All statistical analyses were performed using GraphPad Prism for MacOS (version 9.3.1) and R (version 4.4.2). Declarations AUTHORS CONTRIBUTIONS Conceptualization: Jana Rathouska Urbankova, Marian Kacerovsky. Data curation: Jana Rathouska Urbankova, Stepanka Bubenikova, Antonin Libra, Filip Vrbacky, and Marian Kacerovsky. Funding acquisition: Marian Kacerovsky. Investigation: Jana Rathouska Urbankova, Stepanka Bubenikova, Jana Matulova, Antonin Libra, Filip Vrbacky, Radka Bolehovska, Ondrej Soucek, Tereza Svadlakova, Ivana Musilova, Marek Lubusky, Bo Jacobsson, Marian Kacerovsky. Methodology: Jana Rathouska Urbankova, Stepanka Bubenikova, Jana Matulova, Antonin Libra, Filip Vrbacky, Radka Bolehovska, Ondrej Soucek, Tereza Svadlakova, Ivana Musilova, Marek Lubusky, Bo Jacobsson, Marian Kacerovsky. Project administration: Jana Rathouska Urbankova and Marian Kacerovsky. Resources: Jana Rathouska Urbankova and Marian Kacerovsky. Writing - original draft: Stepanka Bubenikova, Jana Rathouska Urbankova, and Marian Kacerovsky. Writing - review & editing: Jana Rathouska Urbankova, Stepanka Bubenikova, Jana Matulova Antonin Libra, Filip Vrbacky, Radka Bolehovska, Ondrej Soucek, Tereza Svadlakova, Ivana Musilova, Marek Lubusky, Bo Jacobsson, Marian Kacerovsky. DATA AVAILABILITY STATEMENT: Demographic, clinical and metagenomic data that support the finding of this study have been deposited in the OSF repository (Center for open science). The data are available at https://osf.io (DOI 10.17605/OSF.IO/JWQK7). DECLARATION OF FUNDING STATEMENT This study was supported by University Hospital Hradec Kralove (The Biomedical Indicators for Personalized Medicine project (BIPOLE), project ID CZ.02.01.01/00/23_021/0008439, is co-funded by the European Union). The authors are solely responsible for the content and writing of this paper. COMPETING INTERESTS: The authors declare no competing interests. DISCLAIMER: Onapharm s.r.o. provided the GYNIMUN Ò TRIO VAGICARE product for use in this study. The company had no role in the study design; data collection, analysis, or interpretation; manuscript preparation; or the decision to submit the article for publication. References Aggarwal N, Kitano S, Puah GRY, Kittelmann S, Hwang IY, Chang MW. Microbiome and Human Health: Current Understanding, Engineering, and Enabling Technologies. Chem Rev 2023;123:31-72. Zeng J, He Z, Wang G, Ma Y, Zhang F. Interaction Between Microbiota and Immunity: Molecular Mechanisms, Biological Functions, Diseases, and New Therapeutic Opportunities. MedComm (2020) 2025;6:e70265. Ravel J, Gajer P, Abdo Z, et al. Vaginal microbiome of reproductive-age women. Proc Natl Acad Sci U S A 2011;108 Suppl 1:4680-7. Lewis FMT, Bernstein KT, Aral SO. Vaginal Microbiome and Its Relationship to Behavior, Sexual Health, and Sexually Transmitted Diseases. Obstet Gynecol 2017;129:643-54. Elkafas H, Walls M, Al-Hendy A, Ismail N. Gut and genital tract microbiomes: Dysbiosis and link to gynecological disorders. Front Cell Infect Microbiol 2022;12:1059825. Escorcia Mora P, Valbuena D, Diez-Juan A. The Role of the Gut Microbiota in Female Reproductive and Gynecological Health: Insights into Endometrial Signaling Pathways. Life (Basel) 2025;15. Amabebe E, Anumba DOC. Female Gut and Genital Tract Microbiota-Induced Crosstalk and Differential Effects of Short-Chain Fatty Acids on Immune Sequelae. Front Immunol 2020;11:2184. Brantsaeter AL, Myhre R, Haugen M, et al. Intake of probiotic food and risk of preeclampsia in primiparous women: the Norwegian Mother and Child Cohort Study. Am J Epidemiol 2011;174:807-15. Myhre R, Brantsaeter AL, Myking S, et al. Intake of probiotic food and risk of spontaneous preterm delivery. Am J Clin Nutr 2011;93:151-7. Koirala R, Gargari G, Arioli S, et al. Effect of oral consumption of capsules containing Lactobacillus paracasei LPC-S01 on the vaginal microbiota of healthy adult women: a randomized, placebo-controlled, double-blind crossover study. FEMS Microbiol Ecol 2020;96. Gille C, Boer B, Marschal M, et al. Effect of probiotics on vaginal health in pregnancy. EFFPRO, a randomized controlled trial. Am J Obstet Gynecol 2016;215:608 e1-08 e7. Lyra A, Ala-Jaakkola R, Yeung N, et al. A Healthy Vaginal Microbiota Remains Stable during Oral Probiotic Supplementation: A Randomised Controlled Trial. Microorganisms 2023;11. V DEL, Lazzeri E, Governini L, et al. Vaginal colonization of women after oral administration of Lactobacillus crispatus strain NTCVAG04 from the human microbiota. Minerva Obstet Gynecol 2023;75:432-39. Filardo S, Di Pietro M, Mastromarino P, Porpora MG, Sessa R. A Multi-Strain Oral Probiotic Improves the Balance of the Vaginal Microbiota in Women with Asymptomatic Bacterial Vaginosis: Preliminary Evidence. Nutrients 2024;16. Hanson L, VandeVusse L, Forgie M, et al. A randomized controlled trial of an oral probiotic to reduce antepartum group B Streptococcus colonization and gastrointestinal symptoms. Am J Obstet Gynecol MFM 2023;5:100748. Cocomazzi G, Contu V, De Stefani S, et al. Refining Unfavorable Vaginal Microbial Community in Infertile Women Subjected to Precision Probiotic Intervention: An Exploratory Single-Arm, Prospective, Open-Label, Interventional Study. Microorganisms 2025;13. Malloy E, Kates AE, Dixon J, Riley C, Safdar N, Hanson L. Vaginal and Rectal microbiome changes following administration of a multi-species antenatal probiotic: A randomized control trial. Gut Microbes Rep 2024;1:1-10. Hertz FB, Holm JB, Palleja A, et al. Vaginal microbiome following orally administered probiotic. APMIS 2022;130:605-11. Lin LT, Li CJ, Wu CC, Pan LF, Tsui KH. Pilot Study on Next-Generation Sequencing Analysis of Vaginal Microbiota in Clinically Infertile Patients Treated with Probiotics. J Clin Med 2024;13. Liu P, Lu Y, Li R, Chen X. Use of probiotic lactobacilli in the treatment of vaginal infections: In vitro and in vivo investigations. Front Cell Infect Microbiol 2023;13:1153894. Maldonado Galdeano C, Cazorla SI, Lemme Dumit JM, Velez E, Perdigon G. Beneficial Effects of Probiotic Consumption on the Immune System. Ann Nutr Metab 2019;74:115-24. Mitselou A, Grammeniatis V, Varouktsi A, Papadatos SS, Katsanos K, Galani V. Proinflammatory cytokines in irritable bowel syndrome: a comparison with inflammatory bowel disease. Intest Res 2020;18:115-20. Nachum Z, Suleiman A, Colodner R, et al. Oral Probiotics to Prevent Recurrent Vulvovaginal Infections During Pregnancy-Multicenter Double-Blind, Randomized, Placebo-Controlled Trial. Nutrients 2025;17. Yefet E, Suleiman A, Colodner R, et al. Efficacy of Oral Probiotic Supplementation in Preventing Vulvovaginal Infections During Pregnancy: A Randomized and Placebo-Controlled Clinical Trial. Nutrients 2024;16. Kim SY, Seol D, Jeong M, et al. Assessing the Efficacy of Ligilactobacillus salivarius CLS0420 and Lacticaseibacillus paracasei CLPC0603 on Vaginal Well-Being in Healthy Women: A Pilot, Randomized, Double-Blind, Placebo-Controlled Trial. Probiotics Antimicrob Proteins 2025. Rezazadeh MB, Zanganeh M, Jarahi L, Fatehi Z. Comparative efficacy of oral and vaginal probiotics in reducing the recurrence of bacterial vaginosis: a double-blind clinical trial. BMC Womens Health 2024;24:575. Heczko PB, Tomusiak A, Adamski P, et al. Supplementation of standard antibiotic therapy with oral probiotics for bacterial vaginosis and aerobic vaginitis: a randomised, double-blind, placebo-controlled trial. BMC Womens Health 2015;15:115. Yang S, Reid G, Challis JRG, et al. Effect of Oral Probiotic Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 on the Vaginal Microbiota, Cytokines and Chemokines in Pregnant Women. Nutrients 2020;12. Schenk M, Grumet L, Sternat J, Reinschissler N, Weiss G. Effect of probiotics on vaginal Ureaplasma parvum in women suffering from unexplained infertility. Reprod Biomed Online 2021;43:503-14. Martoni CJ, Frederiksen AKS, Damholt A, Leyer G. Effects of a 10-Strain Oral Probiotic on Parameters of Vaginal Health and Microbial Community: A Pilot Clinical Study. Int J Womens Health 2022;14:29-39. Husain S, Allotey J, Drymoussi Z, et al. Effects of oral probiotic supplements on vaginal microbiota during pregnancy: a randomised, double-blind, placebo-controlled trial with microbiome analysis. BJOG 2020;127:275-84. Ho M, Chang YY, Chang WC, et al. Oral Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 to reduce Group B Streptococcus colonization in pregnant women: A randomized controlled trial. Taiwan J Obstet Gynecol 2016;55:515-8. Thorakkattu P, Khanashyam AC, Shah K, et al. Postbiotics: Current Trends in Food and Pharmaceutical Industry. Foods 2022;11. Triantafillou V, Workman AD, Patel NN, et al. Broncho-Vaxom(R) (OM-85 BV) soluble components stimulate sinonasal innate immunity. Int Forum Allergy Rhinol 2019;9:370-77. Lombardi F, Augello FR, Palumbo P, et al. Bacterial Lysate from the Multi-Strain Probiotic SLAB51 Triggers Adaptative Responses to Hypoxia in Human Caco-2 Intestinal Epithelial Cells under Normoxic Conditions and Attenuates LPS-Induced Inflammatory Response. Int J Mol Sci 2023;24. Suarez N, Ferrara F, Rial A, Dee V, Chabalgoity JA. Bacterial Lysates as Immunotherapies for Respiratory Infections: Methods of Preparation. Front Bioeng Biotechnol 2020;8:545. France MT, Ma B, Gajer P, et al. VALENCIA: a nearest centroid classification method for vaginal microbial communities based on composition. Microbiome 2020;8:166. Gaujoux R, Seoighe C. A flexible R package for nonnegative matrix factorization. BMC Bioinformatics 2010;11:367. Zmora N, Zilberman-Schapira G, Suez J, et al. Personalized Gut Mucosal Colonization Resistance to Empiric Probiotics Is Associated with Unique Host and Microbiome Features. Cell 2018;174:1388-405 e21. Suez J, Zmora N, Zilberman-Schapira G, et al. Post-Antibiotic Gut Mucosal Microbiome Reconstitution Is Impaired by Probiotics and Improved by Autologous FMT. Cell 2018;174:1406-23 e16. Stene C, Rome A, Palmquist I, et al. Administration of probiotics to healthy volunteers: effects on reactivity of intestinal mucosa and systemic leukocytes. BMC Gastroenterol 2022;22:100. Andrews C, McLean MH, Durum SK. Cytokine Tuning of Intestinal Epithelial Function. Front Immunol 2018;9:1270. Stranik J, Kacerovsky M, Andrys C, et al. Intra-amniotic infection and sterile intra-amniotic inflammation are associated with elevated concentrations of cervical fluid interleukin-6 in women with spontaneous preterm labor with intact membranes. J Matern Fetal Neonatal Med 2022;35:4861-69. Khameneh HJ, Bolis M, Ventura PMO, et al. The bacterial lysate OM-85 engages Toll-like receptors 2 and 4 triggering an immunomodulatory gene signature in human myeloid cells. Mucosal Immunol 2024;17:346-58. Wegh CAM, Geerlings SY, Knol J, Roeselers G, Belzer C. Postbiotics and Their Potential Applications in Early Life Nutrition and Beyond. Int J Mol Sci 2019;20. Shahini A, Shahini A. Role of interleukin-6-mediated inflammation in the pathogenesis of inflammatory bowel disease: focus on the available therapeutic approaches and gut microbiome. J Cell Commun Signal 2023;17:55-74. Mercer SD, Doherty C, Singh G, et al. Lactobacillus lysates protect oral epithelial cells from pathogen-associated damage, increase secretion of pro-inflammatory cytokines and enhance barrier integrity. Sci Rep 2025;15:5894. Balaghi Z, Azima S, Motamedifar M, Kaviani M, Poordast T, Zare N. The Effect of Lactofem Oral Probiotic Capsule on Lactobacilli Colonization and Some Vaginal Health Parameters. Gynecol Obstet Invest 2020;85:245-51. Han S, Lu Y, Xie J, et al. Probiotic Gastrointestinal Transit and Colonization After Oral Administration: A Long Journey. Front Cell Infect Microbiol 2021;11:609722. Lehtoranta L, Ala-Jaakkola R, Laitila A, Maukonen J. Healthy Vaginal Microbiota and Influence of Probiotics Across the Female Life Span. Front Microbiol 2022;13:819958. Tables Table 1 Demographical and clinical characteristics of the healthy women stratified based on the presence of Lactobacillus -dominated and Lactobacillus -depleted vaginal microbiota. Characteristic Lactobacillus -dominated vaginal microbiota (n=8) Lactobacillus -depleted vaginal microbiota (n=3) Exact p- value Age [years, median (IQR)] 38 (29-39) 39 (28-39) 0.99 Nulliparous [number (%)] 3 (38%) 1 (33%) 1.00 History of cesarean section [number (%)] 1 (13%) 0 (0%) 1.00 Smoking [number (%)] 3 (38%) 0 (0%) 0.49 Body mass index [kg/m 2 , median (IQR)] 22.2 (19.4-24.6) 22.5 (22.4-24.2) 0.68 Oral hormonal contraception [number (%)] 0 (0%) 1 (33%) 0.27 Intrauterine contraception device [number (%)] 2 (25%) 2 (67%) 0.49 Regular menstrual periods [number (%)] 6 (75%) 1 (33%) 0.49 Abbreviations: IQR, interquartile range Continuous variables, presented as median (interquartile range), were compared using a nonparametric Mann-Whitney U test. Categorical variables, presented as number (%), were compared using Fisher’s exact test. Table 2 The bacterial richness and diversity in the vaginal and rectal microbiotas before probiotic use, after 1 month of probiotic use, and after a 1-month washout period. Before probiotic use After 1 month of probiotic use Exact p -value 1 After 1-month washout Exact p -value 2 Rectal microbiota – bacterial richness [median (IQR)] 38 (38-52) 37 (37-51) 0.03 18 (18-50) 0.08 Rectal microbiota – bacterial diversity [median (IQR)] 13.1 (10.6-17.5) 13.2 (11.3-15.0) 0.56 17.6 (11.5-21.7) 0.12 Vaginal microbiota - bacterial richness [median (IQR)] 4 (1-6) 3 (2-4) 0.34 4 (2-9) 0.06 Vaginal microbiota - bacterial diversity [median (IQR)] 1.1 (1.0-2.5) 1.0 (1.0-2.1) 0.03 1.4 (1.0-2.4) 0.06 Abbreviations: IQR, interquartile range Bacterial richness = species observed. Bacterial diversity = inverse Simpson index. p- value 1 – comparison between before probiotic use and after 1 month of probiotic use. p -value 2 – comparison between after 1 month of probiotic use and after 1-month washout. Continuous variables were compared using Wilcoxon matched pairs signed rank test and presented as median (interquartile range). Statistically significant results are marked in bold. Table 3 The relative abundance of the live microorganisms contained in the oral probiotic in the vaginal and rectal microbiotas before probiotic use, and after 1 month of probiotic use, and after 1-month washout period. Before probiotic use After 1 month of probiotic use After 1-month washout Relative abundance of L. crispatus in the vaginal microbiota Women no. 4 96% 99% 99% Women no. 5 0% 0% 0.3% Women no. 7 98% 98% 86% Women no. 9 100% 100% 100% Women no. 11 100% 99% 100% Relative abundance of L rhamnosus in the vaginal microbiota Women no. 6 0.9% 1.5% 2.0% Relative abundance of L. crispatus in the rectal microbiota Women no. 5 0% 0% 0.4% Lactobacillus rhamnosus was not found in the rectal microbiota and Bifidobacterium animalis was found neither in the vaginal nor rectal microbiota. Abbreviations: L . - lactobacillus Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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. 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11:19:06","extension":"jpg","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":579868,"visible":true,"origin":"","legend":"","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7488779/v1/e049992c57ccd941e3f73170.jpg"},{"id":92167585,"identity":"42cfc0fc-8446-46b9-be84-c63f708fc66e","added_by":"auto","created_at":"2025-09-25 11:19:06","extension":"json","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":12305,"visible":true,"origin":"","legend":"","description":"","filename":"dc79395f2db0487dacb79420a9c2bd64.json","url":"https://assets-eu.researchsquare.com/files/rs-7488779/v1/4f60a462384dc8d7c7eb9262.json"},{"id":92167591,"identity":"03c3e009-4c46-49f1-b74b-ad681260e94e","added_by":"auto","created_at":"2025-09-25 11:19:06","extension":"xml","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":145001,"visible":true,"origin":"","legend":"","description":"","filename":"dc79395f2db0487dacb79420a9c2bd641enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7488779/v1/7fef8cfbb47caecb9f509018.xml"},{"id":92168498,"identity":"ac8bc891-8b2c-4d84-9afe-b41d766860ff","added_by":"auto","created_at":"2025-09-25 11:27:06","extension":"jpg","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":364609,"visible":true,"origin":"","legend":"","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7488779/v1/88263b5ff6e95edc5ce65abd.jpg"},{"id":92167593,"identity":"c6e1ea56-7927-4e46-a8c6-71e2c6468181","added_by":"auto","created_at":"2025-09-25 11:19:06","extension":"jpg","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":664035,"visible":true,"origin":"","legend":"","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7488779/v1/9bdde453535d2aff14c1aefa.jpg"},{"id":92167584,"identity":"8bb021c4-3132-4d2e-88b3-1b883be8ae02","added_by":"auto","created_at":"2025-09-25 11:19:06","extension":"jpg","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":426089,"visible":true,"origin":"","legend":"","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7488779/v1/ee118474d7e4bd6b750d785a.jpg"},{"id":92167592,"identity":"1657752b-009d-467f-a8f1-db3a7c0e182c","added_by":"auto","created_at":"2025-09-25 11:19:06","extension":"jpg","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":300540,"visible":true,"origin":"","legend":"","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7488779/v1/4451d237eb6a613d994eca5b.jpg"},{"id":92168990,"identity":"5b6ec033-2402-43aa-bff3-870e7f35b196","added_by":"auto","created_at":"2025-09-25 11:35:06","extension":"jpg","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":476287,"visible":true,"origin":"","legend":"","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7488779/v1/5763c350cd4164577e1c08fa.jpg"},{"id":92167590,"identity":"f9274018-5fd0-4bf7-99f1-d2607eb980e3","added_by":"auto","created_at":"2025-09-25 11:19:06","extension":"jpg","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":579868,"visible":true,"origin":"","legend":"","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7488779/v1/dd9336e2ccf067754cd62f29.jpg"},{"id":92168503,"identity":"309be1a5-d925-4173-860d-4b69a1804a81","added_by":"auto","created_at":"2025-09-25 11:27:06","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":82801,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7488779/v1/20dafffb71d2a8dc9fbf01f8.png"},{"id":92167580,"identity":"ed1c20be-5681-4572-a7b7-463f08d23dde","added_by":"auto","created_at":"2025-09-25 11:19:06","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":118649,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7488779/v1/dabcaa5e4cdee474b177d2c2.png"},{"id":92167595,"identity":"406763de-5696-4b50-9b6f-d21fe6c85663","added_by":"auto","created_at":"2025-09-25 11:19:06","extension":"png","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":85244,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7488779/v1/813ae14d158bea47282e7404.png"},{"id":92167596,"identity":"582b7860-929c-4d70-aa04-31f09e73a04d","added_by":"auto","created_at":"2025-09-25 11:19:07","extension":"png","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":87237,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7488779/v1/956fd58d79810e13ff6f6adc.png"},{"id":92167594,"identity":"f40a980d-325a-4d58-b123-2ea45da73f16","added_by":"auto","created_at":"2025-09-25 11:19:06","extension":"png","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":124581,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7488779/v1/36c7ab60a416c427a822222b.png"},{"id":92167589,"identity":"ba349602-0b1f-4514-bf0b-eff470b27df9","added_by":"auto","created_at":"2025-09-25 11:19:06","extension":"png","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":120432,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7488779/v1/f193c3c1daa438eaee8a03d1.png"},{"id":92168504,"identity":"ae322d0e-1129-4534-9888-0ca742453f9e","added_by":"auto","created_at":"2025-09-25 11:27:07","extension":"xml","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":141914,"visible":true,"origin":"","legend":"","description":"","filename":"dc79395f2db0487dacb79420a9c2bd641structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7488779/v1/89c04bd1d2f4bbb05fd35449.xml"},{"id":92168500,"identity":"bc21958d-7b5e-48bf-85ba-bff6e9a3b139","added_by":"auto","created_at":"2025-09-25 11:27:06","extension":"html","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":160851,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7488779/v1/a26e310604c4ea27635cf091.html"},{"id":92168492,"identity":"d2faa4f6-d6e0-4035-a653-c2bb7595f008","added_by":"auto","created_at":"2025-09-25 11:27:06","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":364609,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmaps of 50 the most abundant bacterial taxa in the rectal microbiota (A) and the most abundant bacterial taxa in the vaginal microbiota (B) in 33 samples of the rectal and vaginal swabs, respectively, obtained from 11 healthy women in reproductive age. \u0026nbsp;Relative abundances were calculated for each taxon and visualized using a color gradient, where yellow indicates low abundance and magenta indicates high abundance (scale: 0.0-1.0). Hierarchical clustering was performed using Ward linkage and Bray–Curtis dissimilarity metric, resulting in the identification of six distinct microbial clusters. The upper annotation bars represent:\u003c/p\u003e\n\u003cp\u003e· Samples obtained before use of probiotics (A), after 1 month of probiotic use (B), and after 1-month washout (C) in the heatmaps of the most abundant bacterial taxa in the vaginal and rectal microbiotas.\u003c/p\u003e\n\u003cp\u003e· VALENCIA classification of vaginal microbiota (color-coded by CST I-A, CST II, CST III-A, CST IV-B, and IV-C3) in the heatmap of the most abundant bacterial taxa in the vaginal microbiota.\u003c/p\u003e\n\u003cp\u003eThis visualization highlights the associations between vaginal and rectal microbial environments and the use of probiotics\u003c/p\u003e\n\u003cp\u003eAbbreviations:\u003c/p\u003e\n\u003cp\u003eCST, community state type\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7488779/v1/874a9489b1469fcf26d987b7.jpg"},{"id":92167566,"identity":"a302dd6f-4df0-4dfb-b401-4fac5184a046","added_by":"auto","created_at":"2025-09-25 11:19:06","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":664035,"visible":true,"origin":"","legend":"\u003cp\u003eRelative abundances of species in rectal microbiota of the women included in the study before the use of probiotics (A), after 1 month use of probiotics (B), and after 1-month washout (C).\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7488779/v1/4bc6383980265200046e3748.jpg"},{"id":92167568,"identity":"3bfb8d3d-1a21-4170-b514-913db0cf3be9","added_by":"auto","created_at":"2025-09-25 11:19:06","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":426089,"visible":true,"origin":"","legend":"\u003cp\u003ePrevalences of bacterial clusters in rectal (A) and vaginal (B) microbiotas, and community state types in the vaginal microbiota (C) before the use of probiotics, after 1 month use of probiotics, and after a 1-month washout.\u003c/p\u003e\n\u003cp\u003eAbbreviations:\u003c/p\u003e\n\u003cp\u003eCST, community state type\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7488779/v1/816f93235f338b157e1c2d81.jpg"},{"id":92167570,"identity":"0134e824-8ee2-43e3-b2bc-17a095df1882","added_by":"auto","created_at":"2025-09-25 11:19:06","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":300540,"visible":true,"origin":"","legend":"\u003cp\u003eInterleukin-6 levels in rectal and vaginal swabs obtained from healthy women, stratified based on bacteria clusters (A - rectal, B - vaginal) and the community state type of the vaginal microbiota (C). The horizontal bars represent median interleukin-6 levels.\u003c/p\u003e\n\u003cp\u003eClusters are color-coded (1 – red, 2 – orange, 3 – purple, 4 – blue, 5 – powder blue, 6 – mustard).\u003c/p\u003e\n\u003cp\u003eCSTs are color-coded (CST I-A – red, CST II – blue, CST III-A – green, and CST IV-B – yellow, CST IV-C3 - mustard).\u003c/p\u003e\n\u003cp\u003eAbbreviations:\u003c/p\u003e\n\u003cp\u003eCST, community state type\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7488779/v1/7ac45ca4047dd37929608ce4.jpg"},{"id":92170263,"identity":"b6aeb2b3-4479-47c4-9583-f9865578ae7c","added_by":"auto","created_at":"2025-09-25 11:43:06","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":476287,"visible":true,"origin":"","legend":"\u003cp\u003eInterleukin-6 levels in rectal (A) and vaginal (B) swabs, obtained before use of probiotics, after 1 month use of probiotics, and after the 1-month washout.\u003c/p\u003e\n\u003cp\u003eClusters are color-coded (1 – red, 2 – orange, 3 – purple, 4 – blue, 5 – powder blue, 6 – mustard).\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7488779/v1/373ccc9af6306f7b2034fcc8.jpg"},{"id":92168987,"identity":"aac64efb-f8d8-45b3-a950-43a30e25f543","added_by":"auto","created_at":"2025-09-25 11:35:06","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":579868,"visible":true,"origin":"","legend":"\u003cp\u003eRelative abundances of genus in vaginal microbiota of the women included in the study before the use of probiotics (A), after 1 month use of probiotics (B), and after 1-month washout (C).\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7488779/v1/c8251e6828c179fdd0d3a351.jpg"},{"id":95224043,"identity":"2fb00405-a5fb-4c68-9d01-b68c1e35d309","added_by":"auto","created_at":"2025-11-05 16:23:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3900217,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7488779/v1/8259eb5c-006f-42cb-958f-1e2f6dfd8bc0.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Oral probiotic containing microbial lysates modulates rectal and vaginal microbial environments but does not change the vaginal community state type – a pilot study","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe human microbiota plays a crucial role in maintaining host health, with microbial communities occupying key mucosal sites such as the gastrointestinal and genitourinary tracts\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. The vaginal and rectal microbiotas are particularly dynamic, contributing critically to the prevention of pathogenic colonization, the modulation of immune response, and the maintenance of mucosal homeostasis\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn premenopausal women, a vaginal microbiota dominated by \u003cem\u003eLactobacillus\u003c/em\u003e species (particularly \u003cem\u003eLactobacillus crispatus\u003c/em\u003e or \u003cem\u003eLactobacillus gasseri\u003c/em\u003e) is recognized as a key indicator of reproductive tract health. In contrast, reduced \u003cem\u003eLactobacillus\u003c/em\u003e abundance and increased microbial diversity are linked to a higher risk of bacterial vaginosis, sexually transmitted infections, and adverse reproductive outcomes, including preterm birth\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Recent research has underscored a bidirectional relationship between the gut and vaginal microbiotas, commonly referred to as the \u0026ldquo;gut-vaginal axis\u0026rdquo;\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. This axis is hypothesized to mediate microbial and immune signaling between the intestinal and genital mucosa, allowing interventions targeting one site to influence the other\u003csup\u003e\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eOral probiotic supplementation, particularly with formulations containing \u003cem\u003eLactobacillus\u003c/em\u003e and \u003cem\u003eBifidobacterium\u003c/em\u003e species, has been proposed as a strategy to restore or maintain vaginal microbial balance characterized by \u003cem\u003eLactobacillus\u003c/em\u003e dominance\u003csup\u003e\u003cspan additionalcitationids=\"CR11 CR12 CR13 CR14 CR15 CR16\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. However, the clinical and microbiological outcomes of such interventions have been inconsistent across studies, largely due to variability in probiotic composition, treatment duration, host-related factors, and endpoint measurements\u003csup\u003e\u003cspan additionalcitationids=\"CR11 CR12 CR13 CR14 CR15\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan additionalcitationids=\"CR19 CR20 CR21 CR22 CR23 CR24 CR25 CR26 CR27 CR28 CR29 CR30 CR31\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eA novel class of probiotic formulations includes microbial lysates (postbiotics), which are non-viable bacterial fragments containing immunogenic components such as lipoteichoic acids, peptidoglycans, and DNA\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. These lysates can influence mucosal immune responses and may act synergistically with live microbes to enhance colonization resistance and promote immunotolerance\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Despite promising data on bacterial lysate in modulating gut inflammation\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e and respiratory tract infections\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, their impact on microbiota composition and inflammatory status in the rectal and vaginal microbial niches remains inadequately understood.\u003c/p\u003e\u003cp\u003e We hypothesized that oral administration of a probiotic containing microbial lysates would beneficially influence both the composition of microbial communities and local inflammatory responses in the vaginal and rectal niches. Assessing the localized effect of such a formulation requires a comprehensive, multi-level approach that captures both microbial dynamics and mucosal immune responses, as indicated by the levels of inflammatory mediators.\u003c/p\u003e\u003cp\u003eTherefore, this pilot study was conducted to evaluate the impact of a multi-strain oral probiotic formulation containing microbial lysates on the rectal and vaginal microbiota and associated local inflammation in healthy premenopausal women. Microbiota composition was analyzed using 16S rRNA gene sequencing before and after a defined supplementation period, as well as following a subsequent washout phase. Local inflammation was assessed by quantifying interleukin (IL)-6 levels in rectal and vaginal swabs.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003eA total of 12 healthy women were recruited, of whom 11 were included in the final analysis. One woman was excluded due to antibiotic administration during the probiotic intervention. Demographic and clinical characteristics of the included women are presented in Table 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eRectal microbial ecosystem\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ei) Microbial composition\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSequencing of rectal samples yielded a total of 1,252,339 high-quality reads, with a mean read count of 37,950 per sample. After exclusion of rare operational taxonomic units (OTUs), defined as \u0026lt; 10 reads per sample, a total of 227 taxa were identified in the rectal microbiota. A heatmap depicting the relative abundance of the 50 most abundant taxa is presented in Figure 1A.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eBefore probiotic administration\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAmong the 11 women, the median (IQR) bacterial richness and diversity were 38 (38-52) and 13.1 (10.6-17.5), respectively (Table 2). Relative abundances of the most prevalent bacterial genus are shown in the Figure 2. None of the probiotic strains used in the intervention (\u003cem\u003eLactobacillus crispatus\u003c/em\u003e, \u003cem\u003eLactobacillus rhamnosus\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Bifidobacterium animalis\u003c/em\u003e)\u003cem\u003e\u0026nbsp;\u003c/em\u003ewas detected in any participants rectal microbiota prior to probiotic use. The following clusters were observed: cluster 1 in 1 women (9%), cluster 2 in 6 (55%), cluster 3 in 2 (18%), and cluster 4 in 2 (18%); Figure 3A.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAfter 1 month of use of probiotic\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAfter the intervention, a decrease in bacterial richness was observed [median 37, IQR (37-51); \u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.03] while bacterial diversity remained unchanged [median 13.2, IQR (11.3-15.0); \u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.56; Table 2]. \u0026nbsp;Relative abundances of the most prevalent bacterial genus are shown in the Figure 2. No differences in the prevalence of the clusters were observed [cluster 1 in 1 woman (9%), cluster 2 in 2 women (18%), cluster 3 in 2 women (18%), cluster 4 in 1 woman (9%), cluster 5 in 4 women (36%), and cluster 6 in 1woman (9%); \u003cem\u003ep =\u0026nbsp;\u003c/em\u003e0.17; Figure 3A]. However, there was an increase of the frequency of cluster 5 compared to its baseline prevalence before probiotic use (0%), although this difference reached only borderline statistical significance (\u003cem\u003ep\u003c/em\u003e \u003cem\u003e=\u0026nbsp;\u003c/em\u003e0.09).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNone of the probiotic strains used in the intervention (\u003cem\u003eLactobacillus crispatus\u003c/em\u003e, \u003cem\u003eLactobacillus rhamnosus\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Bifidobacterium animalis\u003c/em\u003e)\u003cem\u003e\u0026nbsp;\u003c/em\u003ewas detected in any participants rectal microbiota after probiotic use.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAfter a 1-month washout\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAfter the 1-month washout, no differences in bacterial richness [median 18, IQR (18-50); \u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.56] and diversity [median 17.6, IQR (11.5-21.7); \u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.12] were observed. Relative abundances of the most prevalent bacterial genus are shown in the Figure 2. No changes in distribution of clusters were noted (\u003cem\u003ep =\u0026nbsp;\u003c/em\u003e0.87); cluster 2 was present in 4 women (36%), cluster 3 in 2 women (18%), cluster 4 in 1 woman (9%), cluster 5 in 2 women (18%), and cluster 6 in 2 women (18%); Figure 3A.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Of the probiotic strains used in the intervention, \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e and \u003cem\u003eBifidobacterium animalis\u003c/em\u003e remained undetectable in any participants rectal microbiota. Additionally, low levels of \u003cem\u003eLactobacillus crispatus\u0026nbsp;\u003c/em\u003e(0.3%) were identified in one woman (Table 3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eii) local inflammation\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIL-6 levels were detectable in all rectal swab samples. No difference in rectal IL-6 levels were found among the clusters (cluster 1: median 2.0 pg/mL, IQR 0.8-3.2, cluster 2: median 1.1, IQR 0.7-1.5, cluster 3: median 0.6, IQR 0.3-1.2, cluster 4: median 1.1, IQR 0.2-1.6, cluster 5: median 0.7, IQR 0.4-1.1, and cluster 6: median 1.3, IQR 0.6-3.5; \u0026nbsp;\u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.40; Figure 4A). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAfter 1 month of probiotic use, rectal IL-6 levels were reduced compared to baseline (before: median: 1.1 pg/mL, IQR 0.7-1.5 vs. after: median: 0.6 pg/mL, IQR 0.4-0.9; \u003cem\u003ep\u003c/em\u003e = 0.02; Figure 5A). After the 1-month washout period, IL-6 increased again, reaching values higher than those observed immediately after probiotic use (washout: median: 1.3 pg/mL, IQR 0.9-1.7, \u003cem\u003ep\u003c/em\u003e = 0.02; Figure 5A).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eVaginal microbial ecosystem\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ei) microbial composition\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSequencing of vaginal samples yielded a total of 1,185,645 high-quality reads, with a mean of 35,928 reads per sample. After removing rare OTUs, a total of 43 taxa were identified in the vaginal microbiota. A heatmap depicting the relative abundance of the taxa is shown in Figure 1B.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eBefore probiotic administration\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAmong the 11 women, the median (IQR) bacterial richness and diversity were 4 (1-6) and 1.1 (1.0-2.5), respectively (Table 2). \u0026nbsp; Relative abundances of the most prevalent bacterial species are shown in the Figure 6. The distribution of bacterial clusters was as the follows: cluster 1 in 2 (18%) women cluster 2 in 1 (9%) women, cluster 3 in 1 (9%) women, cluster 4 in 1 (9%) women, cluster 5 in 4 (36%) and cluster 6 in 2 (18%) women (Figure 3B). The following CSTs were observed: CST I-A in 4 women (36%), CST II in 2 (18%), CST III-A in 2 (18%), CST IV-B in 2 (18%), and CST IV-C3 in 1 (9%); Figure 3C. Of the probiotic strains used in the intervention, \u003cem\u003eLactobacillus crispatus\u003c/em\u003e and \u003cem\u003eLactobacillus rhamnosus\u0026nbsp;\u003c/em\u003ewere detected in 4 and 1\u003cem\u003e\u0026nbsp;\u003c/em\u003ewomen, respectively, all of whom had CST I-A (\u003cem\u003eLactobacillus crispatus\u003c/em\u003e) and CST II (\u003cem\u003ev rhamnosus\u003c/em\u003e). \u003cem\u003eBifidobacterium animalis\u0026nbsp;\u003c/em\u003ewas not detected in any participants prior to probiotic use (Table 3).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAfter 1 month of use of probiotic\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAfter intervention, a decrease in bacterial diversity was observed [median 1.0, IQR (1.0-2.1); \u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.03] while bacterial richness remained unchanged [median 3, IQR (2-4); \u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.34; Table 2]. Relative abundances of the most prevalent bacterial species are shown in the Figure 6. No changes were observed in the overall distribution of the bacterial clusters (Figure 3B) and CSTs (Figure 3C), except in one woman who shifted from CST IV-C3 to CST II. In this woman, the relative abundance of \u003cem\u003eLactobacillus gasseri\u0026nbsp;\u003c/em\u003eincreased from 43% to 51% and the relative abundance of \u003cem\u003eBifidobacterium breve\u003c/em\u003e increased from 44% to 48%.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eLactobacillus crispatus\u003c/em\u003e, \u003cem\u003eLactobacillus rhamnosus\u0026nbsp;\u003c/em\u003ewere detected only in women who already harbored these strains prior to the intervention. \u003cem\u003eBifidobacterium animalis\u003c/em\u003e remained undetectable in all women (Table 3). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAfter a 1-month washout\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAn increase in bacterial richness [median 4, IQR (2-9)] and diversity [median 1.4, IQR (1.0-2.4)] were observed after the 1-month washout period compared to the post-intervention values; however, these results reached only borderline statistical significance (both \u003cem\u003ep\u003c/em\u003e = 0.06). Relative abundances of the most prevalent bacterial species are shown in the Figure 6. \u0026nbsp;No further changes in distribution of the bacterial clusters (Figure 3B) and CSTs (Figure 3C) were noted compared to the post-intervention bacterial clusters and CSTs.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eLactobacillus crispatus\u003c/em\u003e and \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e remained detectable only in participants who harbored them prior to the intervention. Additionally, low levels of \u003cem\u003eLactobacillus\u003c/em\u003e \u003cem\u003ecrispatus\u0026nbsp;\u003c/em\u003e(0.3%) were identified in one woman with CST IV-B. \u003cem\u003eBifidobacterium animalis\u003c/em\u003e was not detected in any participant (Table 3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp; \u0026nbsp; ii) \u0026nbsp; \u0026nbsp; local inflammation\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIL-6 levels were detectable in all vaginal swab samples. IL-6 levels varied significantly among clusters (cluster 1: median 1.6 pg/mL, IQR 1.1-2.4, cluster 2: median 26.1, IQR 13.4-78.5, cluster 3: median 23.4 pg/mL, IQR 17.7-76.8, cluster 4: median 3.1 pg/mL, IQR 2.4-12.1, cluster 5: median 1.1, IQR 0.4-1.6, and cluster 6: median 5.0 pg/mL, IQR 2.5-14.9; \u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.0004; Figure 4B) and CST groups (CST I-A: median 1.1 pg/mL, IQR 0.4\u0026ndash;1.4, CST II: median 1.7 pg/mL, IQR 1.3\u0026ndash;14.3, CST III-A: median 5.0 pg/mL, IQR 2.6-14.9, CTS IV-B: median 12.7 pg/mL, IQR 3.0\u0026ndash;38.8, and CSTIV-C3: median 78.5, IQR 78.5-78.5; \u003cem\u003ep\u003c/em\u003e = 0.001; Figure 4C).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNo differences in IL-6 levels were observed between samples collected before and after probiotic administration (before: median: 2.4 pg/mL, IQR 1.4-12.1 vs. after: median 1.8 pg/mL, IQR 0.8-10.3; \u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.15; Figure 5B) and between post-intervention and washout samples (washout: median 2.4 pg/mL, IQR 0.7-13.4; \u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.52; Figure 5B).\u0026nbsp;\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eBy simultaneously profiling two mucosal compartments and incorporating both microbial and inflammatory endpoints, this study provides pilot insight into how oral probiotic formulations containing microbial lysates may influence distal mucosal environments.\u003c/p\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003ePrincipal findings of this study\u003c/h2\u003e\u003cp\u003eIn healthy premenopausal women one month of oral supplementation with a multi-oral strain probiotic formulation, containing microbial lysates and fructooligosaccharides, was associated with: i) a decrease in bacterial richness in the rectal microbiota, without significant alterations in its community structure (bacterial clusters); ii) a decrease in local rectal local inflammatory response, as reflected by reduced IL-6 levels, iii) a reduction in microbial diversity in the vaginal microbiota also without changes in its community structure; iv) no new detection of \u003cem\u003eL. gasseri\u003c/em\u003e, \u003cem\u003eL. rhamnosus\u003c/em\u003e, or \u003cem\u003eB. animalis\u003c/em\u003e in the rectal and vaginal microbiota by 16S rRNA gene sequencing, except in participants who harbored these taxa at baseline.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eThe results of the study in the context of what is known\u003c/h2\u003e\u003cp\u003eIn this study, a modest reduction in bacterial richness was observed in rectal samples following probiotic intake, while the overall community structure, as defined by cluster analysis, remained stable. These findings are consistent with previous observations showing that probiotic supplementation in healthy adults generally induces only minor changes in gut microbial composition, particularly in the absence of prior dysbiosis\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. It is important to note, however, that those studies analyzed stool samples, whereas this study used rectal swabs, which may more accurately reflect the mucosa-associated microbiota\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. The reduction in richness observed in this study likely reflects a suppression of low-abundance taxa, rather than a disruption of dominant community members. Interestingly, after probiotic supplementation, a borderline significant increase in the prevalence of a cluster characterized by a moderate to high abundance of \u003cem\u003eFinegoldia magna\u003c/em\u003e accompanied by moderate abundances of \u003cem\u003ePeptoniphilus\u003c/em\u003e spp (from 0\u0026ndash;36%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.09) was noted, as well as a non-significant decrease in the prevalence of the cluster with a moderate to high abundance of \u003cem\u003eFaecalibacterium prausnitzii\u003c/em\u003e (from 55\u0026ndash;18%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.18). Although the small sample size and high inter-interindividual variability limit the interpretation of the cluster-level changes, the observed trends may suggest minor ecological reorganization in response to probiotic intake. In the context of an otherwise stable gut ecosystem, such shifts are not necessarily pathological but may represent transient, adaptive changes. This is in line with the concept of colonization resistance, whereby established microbial communities prevents the displacement or overgrowth of new microbial taxa\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eIL-6 is a pro-inflammatory cytokine involved in mucosal immunity and has been shown to reflect inflammatory responses in both the gastrointestinal and reproductive tract mucosa\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Its quantification can therefore serve as a useful biomarker for evaluating the intensity of local immune responses following probiotic intake. Despite the fact that none of the microorganisms included in the probiotic formulation were detected in the rectal microbiota, a decrease in rectal IL-6 levels was observed following supplementation, with levels returning to baseline after washout period. This effect occurred in the absence of probiotic strain colonization or major shifts in microbial composition, suggesting that the anti-inflammatory effect was mediated by indirect mechanisms. This observation is consistent with results of Stene et al., who reported that oral supplementation with \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e led to reduced IL-6 secretion in rectal biopsies following \u003cem\u003eex vivo\u003c/em\u003e stimulation with tumor necrosis factor (TNF)-α\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. These results support previous evidence that probiotics can modulate mucosal immunity through transient metabolic or signaling interactions, rather than stable colonization\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn this study, the observed anti-inflammatory effect may also be attributed to the presence of microbial lysates in the formulation. These lysates can interact with pattern recognition receptors on epithelial and immune cells, leading to modulation of local cytokine production\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Previous \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e studies have shown that microbial lysates can reduce IL-6 and TNF-α levels, promote regulatory immune responses, and strengthen epithelial barrier function\u003csup\u003e\u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Their inclusion in the probiotic formulation likely contributed to the observed immunomodulatory effect, even in the absence of detectable changes in the microbiota.\u003c/p\u003e\u003cp\u003eThese findings highlight the potential utility of microbial lysates in modulating mucosal inflammation under physiologic conditions. However, the pilot design of the study did not allow us to determine whether the reduction in local rectal inflammatory response was driven by the live probiotic component, microbial lysates or a combination of both.\u003c/p\u003e\u003cp\u003eIn contrast to the gut, reduced vaginal microbial diversity is generally associated with a healthier vaginal ecosystem\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. In this study, a modest decrease in vaginal microbial diversity was observed following probiotic use, while dominant bacterial clusters and CSTs remained unchanged. Since a borderline significant increase in diversity (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.06) was detected after the washout period, the initial decrease may represent a transient shift toward a more stable microbiota, possible influenced by oral probiotic.\u003c/p\u003e\u003cp\u003eThe stability of vaginal bacterial clusters and CST despite supplementation is consistent with previous studies showing that the vaginal microbiota is relatively unresponsive to oral probiotic interventions, particularly in healthy women with established \u003cem\u003eLactobacillus\u003c/em\u003e-dominant communities\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. This resistance to change may by partly explained by the route of administrations, as orally administered probiotics often fail reach or colonize the vaginal tract\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eDespite their inclusion in the probiotic formulation, \u003cem\u003eLactobacillus gasseri\u003c/em\u003e, \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e, and \u003cem\u003eB\u003c/em\u003eifidobacterium \u003cem\u003eanimalis\u003c/em\u003e were not newly detected in rectal or vaginal samples after the intervention, based on 16S rRNA gene sequencing. \u003cem\u003eLactobacillus gasseri\u003c/em\u003e and \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e were detected only in women who harbored these species at baseline, and \u003cem\u003eBifidobacterium animalis\u003c/em\u003e was not detected in any samples. These findings suggest that no colonization occurred after oral probiotic administration.\u003c/p\u003e\u003cp\u003eThis observation aligns with a growing body of evidence indicating that successful vaginal colonization by oral probiotic strains in healthy women is rare\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Moreover, even in the gut \u0026ndash; which is more directly accessible via oral administration \u0026ndash; colonization by probiotic strains remains infrequent\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. For example, Suez et al. demonstrated that probiotic colonization of the gut is highly individualized and often prevented by host-specific microbial and immune mechanisms\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Similarly, Zmora et al. reported that even high-dose oral probiotics do not reliably colonize the gastrointestinal mucosa\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. In the vaginal compartment, colonization via oral intake is even less likely due to anatomical separation and the lack of direct microbial transfer. Studies in both healthy and dysbiotic populations have shown that vaginal colonization is more effectively achieved through local (intravaginal) application\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIt is important to note that microbial detection in this study relied exclusively on 16S rRNA gene sequencing. While this method allows broad taxonomic profiling, it lacks the sensitivity of strain-specific PCRS assays. This limitation may have reduced the ability to detect low-abundance probiotic strains, potentially resulting to false-negative finding regarding their presence in vaginal and rectal microbiotas.\u003c/p\u003e\u003cp\u003eThe findings of this pilot study suggest that in healthy premenopausal women, oral supplementation with a multi-strain probiotic preparation with microbial lysates may exert an anti-inflammatory effect on local mucosal immunity, particularly within the rectal environment. The observed reduction in local IL-6 levels, in the absence of major microbial community disruption, may be clinically relevant in conditions characterized by low-grade mucosal inflammation.\u003c/p\u003e\u003cp\u003eHowever, the absence of significant colonization or compositional shifts in the microbiota suggests that in healthy population, probiotic effects may occur primarily through immunomodulatory pathways rather than through microbial displacement. For routine use in healthy women, the findings from this study do not support oral probiotic supplementation as an effective strategy for microbiota modification, particularly in the absence of intestinal or vaginal dysbiosis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eStrengths and limitations\u003c/h2\u003e\u003cp\u003eThis study has several notable strengths. First, it assessed both rectal and vaginal environments, allowing a more comprehensive evaluation of the potential interaction along the gut-vaginal axis following oral probiotic administration. Second, combined microbial profiling \u0026ndash; based on 16S rRNA gene sequencing \u0026ndash; with measurement of local inflammatory response via IL-6 levels provide an integrated view of both microbial and immunological outcomes. Third, the self-collection of samples by participants simplified logistics and increased feasibility, which could support wider implementation in larger studies or real-world settings.\u003c/p\u003e\u003cp\u003eHowever, the study also has important limitations. First, the sample size was small, including only 11 healthy women, which limits the statistical power and generalizability of the findings. The results should therefore be interpreted with caution and validated in larger, more diverse cohorts. Second, the absence of 18S rRNA gene sequencing precluded any assessment of the probiotics\u0026rsquo; impact on fungal communities, including yeasts. Third, the rectal microbiota was assessed using rectal swab rather than stool samples, which my limit the comparability to studies based on fecal microbial composition and may not fully capture luminal microbial diversity. Lastly, the intensity of local inflammation was assessed using IL-6 alone; additional markers \u0026ndash; such us IL-1β, TNF-α, or calprotectin \u0026ndash; could have provided a more comprehensive picture of the local immune response. Lastly, the relative short duration of oral probiotic preparation administration does not preclude that a longer supplementation regimen could yield more pronounced effects.\u003c/p\u003e\u003c/div\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThe use of a multi-strain oral probiotic formulation containing microbial lysates and fructooligosaccharides did not alter the vaginal microbiota community state type, but it was associated with a modest modulation of rectal and vaginal microbial environments and a reduction in a local rectal inflammatory response.\u003c/p\u003e"},{"header":"MATERIAL AND METHODS","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eStudy design\u003c/h2\u003e\u003cp\u003eHealthy (self-reported) women of reproductive age were eligible to participate in this study. Recruitment was conducted among the staff of the Charles University, Faculty of Pharmacy in Hradec Kralove, Czech Republic, and the Department of Obstetrics and Gynecology, Hospital Most, Czech Republic between August and October 2024. The inclusion criteria were as follows: i) age between 18 and 45 years, ii) female sex, iii) white people, iv) no history of vaginal infections within the previous 6 months, v) absence of acute illnesses within the previous 6onths or any chronic diseases, vi) no use any oral or vaginal probiotics within the previous 3 months, vii) willingness to take dietary supplements, and viii) willingness to comply with the study protocol. The exclusion criteria were: i) vaginal or urinary symptoms; ii) current pregnancy or intention to become pregnant, iii) breastfeeding, iv) use of antibiotics in the last 6 months or during the study period, v) use of vulvovaginal medications; vi) menstrual irregularities, vii) any diagnosed ongoing disease rendering the participant unfit for the study, and viii) intolerance to the study product.\u003c/p\u003e\u003cp\u003eAll women received the multi-strain oral probiotic preparation containing microbial lysates and prebiotic (GYNIMUN\u003csup\u003e\u0026reg;\u003c/sup\u003e TRIO VAGICARE, Onapharm, s.r.o, Prague, Czech Republic) and were instructed to maintain their dietary habits and a usual level of physical activity and throughout the study. The preparation contained \u003cem\u003eLactobacillus crispatus\u003c/em\u003e SP28, \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e CA15 GIOIA, \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e GC Onapharm, and \u003cem\u003eBifidibacterium animalis\u003c/em\u003e BB Onapharm (6\u003cem\u003e\u0026times;\u003c/em\u003e10\u003csup\u003e9\u003c/sup\u003e colony-forming units of each strain per capsula), microbial lysates (5mg) of \u003cem\u003eCandida albicans\u003c/em\u003e, \u003cem\u003eGardnerella vaginalis\u003c/em\u003e, \u003cem\u003eStreptococcus agalactiae\u003c/em\u003e, and \u003cem\u003eEscherichia coli\u003c/em\u003e, as well as fructooligosaccharides (100 mg). Women were instructed to take two capsules orally twice daily (with food, during breakfast and dinner) for 30 days.\u003c/p\u003e\u003cp\u003eWomen performed vaginal and rectal swabs themselves, after careful instructions, at three time points during the study: i) before initiating probiotic use, approximately 3\u0026ndash;5 days after cessation of menstruation, ii) after 1 month of probiotic use, and iii) after a 1-month washout period. Women were asked to refrain from sexual intercourse and use of intravaginal products (lubricant, spermicides) for 24 hours preceding the obtaining the swabs.\u003c/p\u003e\u003cp\u003e All women provided written informed consent prior to the collection of the samples and using of probiotics. This study was approved by the Institutional Review Board of University Hospital Hradec Kralove (August 2024, No. 202409 P04). All experiments were performed in accordance with relevant guidelines and regulations. The study was performed in accordance with the Declaration of Helsinki.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eVaginal and rectal swab sampling\u003c/h2\u003e\u003cp\u003e Vaginal and rectal swab samples were self-collected by participants. All women were thoroughly instructed by the study coordinator on how to correctly obtain the samples and how to avoid contamination of the sample. Vaginal swab was obtained first. A sterile Dacron polyester swab was inserted into the cranial part of the vagina. The swab was rotated three times to collect material on all sides of the tip and left in place for 20 s to allow saturation. It was then carefully removed and placed into a polypropylene tube containing 1.5 mL of phosphate-buffered saline. The tube was immediately stored in a refrigerator. Afterward, the rectal swab was collected. A sterile Dacron polyester swab was inserted approximately 2\u0026ndash;3 cm in the rectum, rotated the three times to contact the rectal walls and kept in place for 20 s to achieve saturation. The swab was then carefully removed and placed into a polypropylene tube containing 1.5 mL of phosphate-buffered saline. The tube was immediately stored in a refrigerator. Within 24 hours, the tubes were transferred on ice to the laboratory, where they were shaken for 20 min. After swab removal, the tubes were centrifuged at 300 \u003cem\u003e\u0026times; g\u003c/em\u003e for 15 min. The supernatants were divided into aliquots, and both the pellets and aliquots were stored at \u0026minus;\u0026thinsp;80\u003cb\u003e\u0026deg;\u003c/b\u003eC until analyses. The pellets and aliquots of supernatant were used to assess the vaginal and rectal microbiotas and to measure IL-6 levels in vaginal and rectal swabs, respectively.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eDNA extraction from vaginal and rectal swab samples and 16S rRNA gene sequencing\u003c/h2\u003e\u003cp\u003eDNA was isolated from the vaginal and rectal pellet samples using a QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer\u0026rsquo;s instructions. The 16S rRNA (27F; 1492R) gene was amplified by PCR, and long-read sequencing was performed using the Oxford Nanopore Technologies Ligation Sequencing Kit V14 (SQK-LSK114) and PromethION Flow Cell R10.4.1 (FLO-PPRO114M) at SEQme s.r.o (Dobris, Czech Republic).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eBioinformatic analysis of vaginal and rectal swabs 16S rRNA gene sequencing\u003c/h2\u003e\u003cp\u003eThe raw 16S rRNA sequencing data were automatically processed and basecalled by MinKnow 24.06.16 software interface at default settings. Demultiplexing was done based on barcodes sequences. Demultiplexed reads were filtered based on reads length (1000-2000nt) and trimming 80 low quality nucleotides from both ends of the reads using NanoFilt version 2.8.0. Relative abundance analysis was performed using EMU software version 3.5.0 using the Silva 138.1 database with keep read counts and keep files options and applying three filters:\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003erelative abundance analysis was performed on for FASTQ files with more than 5,000 reads,\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003ereads assignment to organism was done with at least 50% certainty,\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eidentified organisms had to have at least 100 reads.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eClassification of microbial ecosystem\u003c/h2\u003e\u003cp\u003eRelative abundances were defined as dominant (\u0026gt;\u0026thinsp;50%) very high (\u0026gt;\u0026thinsp;20%), high (\u0026gt;\u0026thinsp;10%), and moderate (1\u0026ndash;10%).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003ei) Rectal microbial ecosystem\u003c/h2\u003e\u003cp\u003eThe microbial ecosystem was classified based hierarchical clustering using the Bray-Curtis dissimilarity metric and Ward linkage in the following bacterial clusters: i) cluster 1 (a very high abundance of \u003cem\u003eGemmiger formicalis\u003c/em\u003e], ii) cluster 2 (a high or moderate abundance of \u003cem\u003eFaecalibacterium prausnitzii\u003c/em\u003e), iii) cluster 3 (a moderate or high abundance of both \u003cem\u003ePeptostreptococcus bacterium oral\u003c/em\u003e and \u003cem\u003eStreptococcus oralis\u003c/em\u003e), iv) cluster 4 (a moderate or high abundance of \u003cem\u003eDialister propionicifaciens\u003c/em\u003e accompanied with a high abundances \u003cem\u003ePeptoniphilus lacrimalis);\u003c/em\u003e v) cluster 5 (a moderate or high abundance of \u003cem\u003eFinegoldia magna\u003c/em\u003e accompanied with a moderate abundance of \u003cem\u003ePeptoniphilus\u003c/em\u003e species), and vi) cluster 6 (a high abundance of \u003cem\u003eStreptococcus periodonticum\u003c/em\u003e).\u003c/p\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003eii) vaginal microbial ecosystem\u003c/h2\u003e\u003cp\u003eThe microbial ecosystem was classified:\u003c/p\u003e\u003cp\u003e\u003col style=\"list-style-type:lower-alpha;\"\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003ebased hierarchical clustering using the Bray-Curtis dissimilarity metric and Ward linkage in the following bacterial clusters: i) cluster 1 (a dominant abundance of \u003cem\u003eLactobacillus gasseri\u003c/em\u003e), ii) cluster 2 (a very high abundance of both \u003cem\u003eGardnerella vaginalis\u003c/em\u003e and \u003cem\u003eFannyhessea vaginae\u003c/em\u003e), iii) cluster 3 (a very high abundance of both \u003cem\u003eLactobacillus gasseri\u003c/em\u003e and \u003cem\u003eBifidobacterium breve\u003c/em\u003e), iv) cluster 4 (a very high abundance of both \u003cem\u003eGardnerella vaginalis\u003c/em\u003e and \u003cem\u003eFannyhessea vaginae\u003c/em\u003e accompanied with a high abundance of \u003cem\u003eLactobacillus jensenii\u003c/em\u003e); v) cluster 5 (a dominant abundance of \u003cem\u003eLactobacillus crispatus\u003c/em\u003e), and vi) cluster 6 (a dominant abundance of \u003cem\u003eLactobacillus iners\u003c/em\u003e).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eBased on the VALENCIA classification, a nearest-centroid based algorithm for the classification of human vaginal microbial community\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, into VALENCIA community state types (CST) based on similarity of cervical microbiota to a set of thirteen reference centroids available at github.com/ravel-lab/VALENCIA.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003ch2\u003eAssessment of IL-6 in rectal and vaginal swabs\u003c/h2\u003e\u003cp\u003eThe levels of IL-6, in the samples that underwent one freezing/thawing cycle, were assessed using enzyme-linked immunosorbent assays (Human IL-6 Quantikine; R\u0026amp;D Systems Inc., Minneapolis, MN, USA). The sensitivity of the test was less than 0.70 pg/mL, and the inter- and intra-assay coefficients were less than 10%.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eDemographic and clinical data were compared using the nonparametric Mann\u0026ndash;Whitney \u003cem\u003eU\u003c/em\u003e test and Fisher\u0026rsquo;s exact test. Levels of IL-6 between clusters were compared with Kruskal-Wallis \u003cem\u003eH\u003c/em\u003e test. Paired comparisons between samples obtained before probiotic use and after 1 month of use, as well as between samples obtained after 1 month of use of probiotic and after a 1-month washout period, were performed using Wilcoxon matched-pairs signed-rank test. Continuous and categorical variables were presented as medians (interquartile ranges [IQR]) and numbers (%), respectively.\u003c/p\u003e\u003cp\u003eThe heatmaps of the relative abundance of the 50 most abundant taxa in rectal swab and the taxa in the vaginal swab were created in R using the heatmap method from the NMF package\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Hierarchical clustering was performed using the Bray\u0026ndash;Curtis dissimilarity metric and Ward\u0026rsquo;s method, minimizing the total within-cluster variation for linkage analysis. Differences were considered statistically significant at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. All \u003cem\u003eP\u003c/em\u003e-values were obtained using two-tailed tests. All statistical analyses were performed using GraphPad Prism for MacOS (version 9.3.1) and R (version 4.4.2).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAUTHORS CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConceptualization:\u0026nbsp;\u003c/strong\u003eJana Rathouska Urbankova, Marian Kacerovsky.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData curation:\u003c/strong\u003e Jana Rathouska Urbankova, Stepanka Bubenikova, Antonin Libra, Filip Vrbacky, and Marian Kacerovsky.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding acquisition:\u0026nbsp;\u003c/strong\u003e Marian Kacerovsky.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInvestigation:\u0026nbsp;\u003c/strong\u003eJana Rathouska Urbankova, Stepanka Bubenikova, Jana Matulova, Antonin Libra, Filip Vrbacky, Radka Bolehovska, Ondrej Soucek, Tereza Svadlakova, Ivana Musilova, Marek Lubusky, \u0026nbsp;Bo Jacobsson, Marian Kacerovsky.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethodology:\u0026nbsp;\u003c/strong\u003eJana Rathouska Urbankova, Stepanka Bubenikova, Jana Matulova, Antonin Libra, Filip Vrbacky, Radka Bolehovska, Ondrej Soucek, Tereza Svadlakova, Ivana Musilova, Marek Lubusky, Bo Jacobsson, Marian Kacerovsky.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProject administration:\u003c/strong\u003e Jana Rathouska Urbankova and Marian Kacerovsky.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResources:\u003c/strong\u003e Jana Rathouska Urbankova and Marian Kacerovsky.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWriting - original draft:\u003c/strong\u003e Stepanka Bubenikova, Jana Rathouska Urbankova, and Marian Kacerovsky.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWriting - review \u0026amp; editing:\u003c/strong\u003e Jana Rathouska Urbankova, Stepanka Bubenikova, Jana Matulova Antonin Libra, Filip Vrbacky, Radka Bolehovska, Ondrej Soucek, Tereza Svadlakova, Ivana Musilova, Marek Lubusky, Bo Jacobsson, Marian Kacerovsky.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY STATEMENT:\u0026nbsp;\u003c/strong\u003eDemographic, clinical and metagenomic data that support the finding of this study have been deposited in the OSF repository (Center for open science). The data are available at https://osf.io (DOI 10.17605/OSF.IO/JWQK7).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDECLARATION OF FUNDING STATEMENT\u003c/strong\u003e This study was supported by University Hospital Hradec Kralove (The Biomedical Indicators for Personalized Medicine project (BIPOLE), project ID CZ.02.01.01/00/23_021/0008439, is co-funded by the European Union). The authors are solely responsible for the content and writing of this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCOMPETING INTERESTS:\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDISCLAIMER:\u0026nbsp;\u003c/strong\u003eOnapharm s.r.o. provided the GYNIMUN\u003csup\u003e\u0026Ograve;\u003c/sup\u003e TRIO VAGICARE product for use in this study. The company had no role in the study design; data collection, analysis, or interpretation; manuscript preparation; or the decision to submit the article for publication.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAggarwal N, Kitano S, Puah GRY, Kittelmann S, Hwang IY, Chang MW. Microbiome and Human Health: Current Understanding, Engineering, and Enabling Technologies. Chem Rev 2023;123:31-72.\u003c/li\u003e\n\u003cli\u003eZeng J, He Z, Wang G, Ma Y, Zhang F. Interaction Between Microbiota and Immunity: Molecular Mechanisms, Biological Functions, Diseases, and New Therapeutic Opportunities. MedComm (2020) 2025;6:e70265.\u003c/li\u003e\n\u003cli\u003eRavel J, Gajer P, Abdo Z, et al. Vaginal microbiome of reproductive-age women. Proc Natl Acad Sci U S A 2011;108 Suppl 1:4680-7.\u003c/li\u003e\n\u003cli\u003eLewis FMT, Bernstein KT, Aral SO. Vaginal Microbiome and Its Relationship to Behavior, Sexual Health, and Sexually Transmitted Diseases. Obstet Gynecol 2017;129:643-54.\u003c/li\u003e\n\u003cli\u003eElkafas H, Walls M, Al-Hendy A, Ismail N. Gut and genital tract microbiomes: Dysbiosis and link to gynecological disorders. Front Cell Infect Microbiol 2022;12:1059825.\u003c/li\u003e\n\u003cli\u003eEscorcia Mora P, Valbuena D, Diez-Juan A. The Role of the Gut Microbiota in Female Reproductive and Gynecological Health: Insights into Endometrial Signaling Pathways. Life (Basel) 2025;15.\u003c/li\u003e\n\u003cli\u003eAmabebe E, Anumba DOC. Female Gut and Genital Tract Microbiota-Induced Crosstalk and Differential Effects of Short-Chain Fatty Acids on Immune Sequelae. Front Immunol 2020;11:2184.\u003c/li\u003e\n\u003cli\u003eBrantsaeter AL, Myhre R, Haugen M, et al. Intake of probiotic food and risk of preeclampsia in primiparous women: the Norwegian Mother and Child Cohort Study. Am J Epidemiol 2011;174:807-15.\u003c/li\u003e\n\u003cli\u003eMyhre R, Brantsaeter AL, Myking S, et al. Intake of probiotic food and risk of spontaneous preterm delivery. Am J Clin Nutr 2011;93:151-7.\u003c/li\u003e\n\u003cli\u003eKoirala R, Gargari G, Arioli S, et al. Effect of oral consumption of capsules containing Lactobacillus paracasei LPC-S01 on the vaginal microbiota of healthy adult women: a randomized, placebo-controlled, double-blind crossover study. FEMS Microbiol Ecol 2020;96.\u003c/li\u003e\n\u003cli\u003eGille C, Boer B, Marschal M, et al. Effect of probiotics on vaginal health in pregnancy. EFFPRO, a randomized controlled trial. Am J Obstet Gynecol 2016;215:608 e1-08 e7.\u003c/li\u003e\n\u003cli\u003eLyra A, Ala-Jaakkola R, Yeung N, et al. A Healthy Vaginal Microbiota Remains Stable during Oral Probiotic Supplementation: A Randomised Controlled Trial. Microorganisms 2023;11.\u003c/li\u003e\n\u003cli\u003eV DEL, Lazzeri E, Governini L, et al. Vaginal colonization of women after oral administration of Lactobacillus crispatus strain NTCVAG04 from the human microbiota. Minerva Obstet Gynecol 2023;75:432-39.\u003c/li\u003e\n\u003cli\u003eFilardo S, Di Pietro M, Mastromarino P, Porpora MG, Sessa R. A Multi-Strain Oral Probiotic Improves the Balance of the Vaginal Microbiota in Women with Asymptomatic Bacterial Vaginosis: Preliminary Evidence. Nutrients 2024;16.\u003c/li\u003e\n\u003cli\u003eHanson L, VandeVusse L, Forgie M, et al. A randomized controlled trial of an oral probiotic to reduce antepartum group B Streptococcus colonization and gastrointestinal symptoms. Am J Obstet Gynecol MFM 2023;5:100748.\u003c/li\u003e\n\u003cli\u003eCocomazzi G, Contu V, De Stefani S, et al. Refining Unfavorable Vaginal Microbial Community in Infertile Women Subjected to Precision Probiotic Intervention: An Exploratory Single-Arm, Prospective, Open-Label, Interventional Study. Microorganisms 2025;13.\u003c/li\u003e\n\u003cli\u003eMalloy E, Kates AE, Dixon J, Riley C, Safdar N, Hanson L. Vaginal and Rectal microbiome changes following administration of a multi-species antenatal probiotic: A randomized control trial. Gut Microbes Rep 2024;1:1-10.\u003c/li\u003e\n\u003cli\u003eHertz FB, Holm JB, Palleja A, et al. Vaginal microbiome following orally administered probiotic. APMIS 2022;130:605-11.\u003c/li\u003e\n\u003cli\u003eLin LT, Li CJ, Wu CC, Pan LF, Tsui KH. Pilot Study on Next-Generation Sequencing Analysis of Vaginal Microbiota in Clinically Infertile Patients Treated with Probiotics. J Clin Med 2024;13.\u003c/li\u003e\n\u003cli\u003eLiu P, Lu Y, Li R, Chen X. Use of probiotic lactobacilli in the treatment of vaginal infections: In vitro and in vivo investigations. Front Cell Infect Microbiol 2023;13:1153894.\u003c/li\u003e\n\u003cli\u003eMaldonado Galdeano C, Cazorla SI, Lemme Dumit JM, Velez E, Perdigon G. Beneficial Effects of Probiotic Consumption on the Immune System. Ann Nutr Metab 2019;74:115-24.\u003c/li\u003e\n\u003cli\u003eMitselou A, Grammeniatis V, Varouktsi A, Papadatos SS, Katsanos K, Galani V. Proinflammatory cytokines in irritable bowel syndrome: a comparison with inflammatory bowel disease. Intest Res 2020;18:115-20.\u003c/li\u003e\n\u003cli\u003eNachum Z, Suleiman A, Colodner R, et al. Oral Probiotics to Prevent Recurrent Vulvovaginal Infections During Pregnancy-Multicenter Double-Blind, Randomized, Placebo-Controlled Trial. Nutrients 2025;17.\u003c/li\u003e\n\u003cli\u003eYefet E, Suleiman A, Colodner R, et al. Efficacy of Oral Probiotic Supplementation in Preventing Vulvovaginal Infections During Pregnancy: A Randomized and Placebo-Controlled Clinical Trial. Nutrients 2024;16.\u003c/li\u003e\n\u003cli\u003eKim SY, Seol D, Jeong M, et al. Assessing the Efficacy of Ligilactobacillus salivarius CLS0420 and Lacticaseibacillus paracasei CLPC0603 on Vaginal Well-Being in Healthy Women: A Pilot, Randomized, Double-Blind, Placebo-Controlled Trial. Probiotics Antimicrob Proteins 2025.\u003c/li\u003e\n\u003cli\u003eRezazadeh MB, Zanganeh M, Jarahi L, Fatehi Z. Comparative efficacy of oral and vaginal probiotics in reducing the recurrence of bacterial vaginosis: a double-blind clinical trial. BMC Womens Health 2024;24:575.\u003c/li\u003e\n\u003cli\u003eHeczko PB, Tomusiak A, Adamski P, et al. Supplementation of standard antibiotic therapy with oral probiotics for bacterial vaginosis and aerobic vaginitis: a randomised, double-blind, placebo-controlled trial. BMC Womens Health 2015;15:115.\u003c/li\u003e\n\u003cli\u003eYang S, Reid G, Challis JRG, et al. Effect of Oral Probiotic Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 on the Vaginal Microbiota, Cytokines and Chemokines in Pregnant Women. Nutrients 2020;12.\u003c/li\u003e\n\u003cli\u003eSchenk M, Grumet L, Sternat J, Reinschissler N, Weiss G. Effect of probiotics on vaginal Ureaplasma parvum in women suffering from unexplained infertility. Reprod Biomed Online 2021;43:503-14.\u003c/li\u003e\n\u003cli\u003eMartoni CJ, Frederiksen AKS, Damholt A, Leyer G. Effects of a 10-Strain Oral Probiotic on Parameters of Vaginal Health and Microbial Community: A Pilot Clinical Study. Int J Womens Health 2022;14:29-39.\u003c/li\u003e\n\u003cli\u003eHusain S, Allotey J, Drymoussi Z, et al. Effects of oral probiotic supplements on vaginal microbiota during pregnancy: a randomised, double-blind, placebo-controlled trial with microbiome analysis. BJOG 2020;127:275-84.\u003c/li\u003e\n\u003cli\u003eHo M, Chang YY, Chang WC, et al. Oral Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 to reduce Group B Streptococcus colonization in pregnant women: A randomized controlled trial. Taiwan J Obstet Gynecol 2016;55:515-8.\u003c/li\u003e\n\u003cli\u003eThorakkattu P, Khanashyam AC, Shah K, et al. Postbiotics: Current Trends in Food and Pharmaceutical Industry. Foods 2022;11.\u003c/li\u003e\n\u003cli\u003eTriantafillou V, Workman AD, Patel NN, et al. Broncho-Vaxom(R) (OM-85 BV) soluble components stimulate sinonasal innate immunity. Int Forum Allergy Rhinol 2019;9:370-77.\u003c/li\u003e\n\u003cli\u003eLombardi F, Augello FR, Palumbo P, et al. Bacterial Lysate from the Multi-Strain Probiotic SLAB51 Triggers Adaptative Responses to Hypoxia in Human Caco-2 Intestinal Epithelial Cells under Normoxic Conditions and Attenuates LPS-Induced Inflammatory Response. Int J Mol Sci 2023;24.\u003c/li\u003e\n\u003cli\u003eSuarez N, Ferrara F, Rial A, Dee V, Chabalgoity JA. Bacterial Lysates as Immunotherapies for Respiratory Infections: Methods of Preparation. Front Bioeng Biotechnol 2020;8:545.\u003c/li\u003e\n\u003cli\u003eFrance MT, Ma B, Gajer P, et al. VALENCIA: a nearest centroid classification method for vaginal microbial communities based on composition. Microbiome 2020;8:166.\u003c/li\u003e\n\u003cli\u003eGaujoux R, Seoighe C. A flexible R package for nonnegative matrix factorization. BMC Bioinformatics 2010;11:367.\u003c/li\u003e\n\u003cli\u003eZmora N, Zilberman-Schapira G, Suez J, et al. Personalized Gut Mucosal Colonization Resistance to Empiric Probiotics Is Associated with Unique Host and Microbiome Features. Cell 2018;174:1388-405 e21.\u003c/li\u003e\n\u003cli\u003eSuez J, Zmora N, Zilberman-Schapira G, et al. Post-Antibiotic Gut Mucosal Microbiome Reconstitution Is Impaired by Probiotics and Improved by Autologous FMT. Cell 2018;174:1406-23 e16.\u003c/li\u003e\n\u003cli\u003eStene C, Rome A, Palmquist I, et al. Administration of probiotics to healthy volunteers: effects on reactivity of intestinal mucosa and systemic leukocytes. BMC Gastroenterol 2022;22:100.\u003c/li\u003e\n\u003cli\u003eAndrews C, McLean MH, Durum SK. Cytokine Tuning of Intestinal Epithelial Function. Front Immunol 2018;9:1270.\u003c/li\u003e\n\u003cli\u003eStranik J, Kacerovsky M, Andrys C, et al. Intra-amniotic infection and sterile intra-amniotic inflammation are associated with elevated concentrations of cervical fluid interleukin-6 in women with spontaneous preterm labor with intact membranes. J Matern Fetal Neonatal Med 2022;35:4861-69.\u003c/li\u003e\n\u003cli\u003eKhameneh HJ, Bolis M, Ventura PMO, et al. The bacterial lysate OM-85 engages Toll-like receptors 2 and 4 triggering an immunomodulatory gene signature in human myeloid cells. Mucosal Immunol 2024;17:346-58.\u003c/li\u003e\n\u003cli\u003eWegh CAM, Geerlings SY, Knol J, Roeselers G, Belzer C. Postbiotics and Their Potential Applications in Early Life Nutrition and Beyond. Int J Mol Sci 2019;20.\u003c/li\u003e\n\u003cli\u003eShahini A, Shahini A. Role of interleukin-6-mediated inflammation in the pathogenesis of inflammatory bowel disease: focus on the available therapeutic approaches and gut microbiome. J Cell Commun Signal 2023;17:55-74.\u003c/li\u003e\n\u003cli\u003eMercer SD, Doherty C, Singh G, et al. Lactobacillus lysates protect oral epithelial cells from pathogen-associated damage, increase secretion of pro-inflammatory cytokines and enhance barrier integrity. Sci Rep 2025;15:5894.\u003c/li\u003e\n\u003cli\u003eBalaghi Z, Azima S, Motamedifar M, Kaviani M, Poordast T, Zare N. The Effect of Lactofem Oral Probiotic Capsule on Lactobacilli Colonization and Some Vaginal Health Parameters. Gynecol Obstet Invest 2020;85:245-51.\u003c/li\u003e\n\u003cli\u003eHan S, Lu Y, Xie J, et al. Probiotic Gastrointestinal Transit and Colonization After Oral Administration: A Long Journey. Front Cell Infect Microbiol 2021;11:609722.\u003c/li\u003e\n\u003cli\u003eLehtoranta L, Ala-Jaakkola R, Laitila A, Maukonen J. Healthy Vaginal Microbiota and Influence of Probiotics Across the Female Life Span. Front Microbiol 2022;13:819958.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e Demographical and clinical characteristics of the healthy women stratified based on the presence of \u003cem\u003eLactobacillus\u003c/em\u003e-dominated and \u003cem\u003eLactobacillus\u003c/em\u003e-depleted vaginal microbiota.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"690\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 293px;\"\u003e\n \u003cp\u003eCharacteristic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e\u003cem\u003eLactobacillus\u003c/em\u003e-dominated\u003c/p\u003e\n \u003cp\u003evaginal microbiota\u003c/p\u003e\n \u003cp\u003e(n=8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 160px;\"\u003e\n \u003cp\u003e\u003cem\u003eLactobacillus\u003c/em\u003e-depleted\u003c/p\u003e\n \u003cp\u003evaginal microbiota\u003c/p\u003e\n \u003cp\u003e(n=3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003eExact\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cem\u003ep-\u003c/em\u003evalue\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 293px;\"\u003e\n \u003cp\u003eAge [years, median (IQR)]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e38 (29-39)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 160px;\"\u003e\n \u003cp\u003e39 (28-39)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e0.99\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 293px;\"\u003e\n \u003cp\u003eNulliparous [number (%)]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e3 (38%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 160px;\"\u003e\n \u003cp\u003e1 (33%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e1.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 293px;\"\u003e\n \u003cp\u003eHistory of cesarean section [number (%)]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e1 (13%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 160px;\"\u003e\n \u003cp\u003e0 (0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e1.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 293px;\"\u003e\n \u003cp\u003eSmoking [number (%)]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e3 (38%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 160px;\"\u003e\n \u003cp\u003e0 (0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e0.49\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 293px;\"\u003e\n \u003cp\u003eBody mass index [kg/m\u003csup\u003e2\u003c/sup\u003e, median (IQR)]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e22.2 (19.4-24.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 160px;\"\u003e\n \u003cp\u003e22.5 (22.4-24.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e0.68\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 293px;\"\u003e\n \u003cp\u003eOral hormonal contraception [number (%)]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e0 (0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 160px;\"\u003e\n \u003cp\u003e1 (33%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e0.27\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 293px;\"\u003e\n \u003cp\u003eIntrauterine contraception device [number (%)]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e2 (25%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 160px;\"\u003e\n \u003cp\u003e2 (67%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e0.49\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 293px;\"\u003e\n \u003cp\u003eRegular menstrual periods [number (%)]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e6 (75%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 160px;\"\u003e\n \u003cp\u003e1 (33%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e0.49\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAbbreviations:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIQR, interquartile range\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eContinuous variables, presented as median (interquartile range), were compared using a nonparametric Mann-Whitney \u003cem\u003eU\u003c/em\u003e test. Categorical variables, presented as number (%), were compared using Fisher\u0026rsquo;s exact test.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e The bacterial richness and diversity in the vaginal and rectal microbiotas before probiotic use, after 1 month of probiotic use, and after a 1-month washout period.\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"924\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 329px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBefore probiotic use\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 137px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAfter 1 month of probiotic use\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eExact\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003ep\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e-value\u003csup\u003e1\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAfter 1-month\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ewashout\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eExact\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003ep\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e-value\u003csup\u003e2\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 329px;\"\u003e\n \u003cp\u003eRectal microbiota \u0026ndash; bacterial richness [median (IQR)]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e38 (38-52)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 137px;\"\u003e\n \u003cp\u003e37 (37-51)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.03\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e18 (18-50)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 329px;\"\u003e\n \u003cp\u003eRectal microbiota \u0026ndash; bacterial diversity [median (IQR)]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e13.1 (10.6-17.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 137px;\"\u003e\n \u003cp\u003e13.2 (11.3-15.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e0.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e17.6 (11.5-21.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 329px;\"\u003e\n \u003cp\u003eVaginal microbiota - bacterial richness [median (IQR)]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e4 (1-6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 137px;\"\u003e\n \u003cp\u003e3 (2-4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e0.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e4 (2-9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 329px;\"\u003e\n \u003cp\u003eVaginal microbiota - bacterial diversity [median (IQR)]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e1.1 (1.0-2.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 137px;\"\u003e\n \u003cp\u003e1.0 (1.0-2.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.03\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e1.4 (1.0-2.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;Abbreviations:\u003c/p\u003e\n\u003cp\u003eIQR, interquartile range\u003c/p\u003e\n\u003cp\u003eBacterial richness = species observed.\u003c/p\u003e\n\u003cp\u003eBacterial diversity = inverse Simpson index.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ep-\u003c/em\u003evalue\u003csup\u003e1\u003c/sup\u003e \u0026ndash; comparison between before probiotic use and after 1 month of probiotic use.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value\u003csup\u003e2\u003c/sup\u003e \u0026ndash; comparison between after 1 month of probiotic use and after 1-month washout.\u003c/p\u003e\n\u003cp\u003eContinuous variables were compared using Wilcoxon matched pairs signed rank test and presented as median (interquartile range).\u003c/p\u003e\n\u003cp\u003eStatistically significant results are marked in bold.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3\u003c/strong\u003e The relative abundance of the live microorganisms contained in the oral probiotic in the vaginal and rectal microbiotas before probiotic use, and after 1 month of probiotic use, and after 1-month washout period.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"600\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBefore probiotic use\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 137px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAfter 1 month of probiotic use\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAfter 1-month\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ewashout\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\" valign=\"top\" style=\"width: 600px;\"\u003e\n \u003cp\u003eRelative abundance of \u003cem\u003eL. crispatus\u003c/em\u003e in the vaginal microbiota\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003eWomen no. 4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e96%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 137px;\"\u003e\n \u003cp\u003e99%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e99%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003eWomen no. 5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 137px;\"\u003e\n \u003cp\u003e0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e0.3%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003eWomen no. 7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e98%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 137px;\"\u003e\n \u003cp\u003e98%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e86%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003eWomen no. 9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 137px;\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003eWomen no. 11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 137px;\"\u003e\n \u003cp\u003e99%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\" valign=\"top\" style=\"width: 600px;\"\u003e\n \u003cp\u003eRelative abundance of \u003cem\u003eL rhamnosus\u003c/em\u003e in the vaginal microbiota\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003eWomen no. 6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e0.9%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 137px;\"\u003e\n \u003cp\u003e1.5%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e2.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\" valign=\"top\" style=\"width: 600px;\"\u003e\n \u003cp\u003eRelative abundance of \u003cem\u003eL. crispatus\u003c/em\u003e in the rectal microbiota\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 170px;\"\u003e\n \u003cp\u003eWomen no. 5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 137px;\"\u003e\n \u003cp\u003e0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e0.4%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cem\u003eLactobacillus rhamnosus\u0026nbsp;\u003c/em\u003ewas not found in the rectal microbiota and \u003cem\u003eBifidobacterium animalis\u0026nbsp;\u003c/em\u003ewas found neither in the vaginal nor rectal microbiota. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAbbreviations:\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eL\u003c/em\u003e. - \u003cem\u003elactobacillus\u003c/em\u003e\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"16S rRNA gene, bacteria, vagina, community state type, cluster, cytokine, diversity, microbiome, microorganism, richness, taxa, taxonomy","lastPublishedDoi":"10.21203/rs.3.rs-7488779/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7488779/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe main aim of this study was to characterize the changes in the rectal and vaginal microbiota, as well as local inflammatory responses following the use of multi-strain oral probiotic containing microbial lysates. Eleven healthy premenopausal women received a one-month regimen of a multi-strain oral probiotic preparation containing \u003cem\u003eLactobacillus crispatus\u003c/em\u003e, \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e, and \u003cem\u003eBifidobacterium animalis\u003c/em\u003e, microbial lysates and fructooligosaccharides. Rectal and vaginal swab samples were collected at baseline, after one month of probiotic use, and after a one-month washout period. Microbiota composition was assessed using 16S rRNA sequencing. Local inflammatory response was evaluated via interleukin-6 levels in the swab samples. Following one month of probiotic use, a decrease was observed in rectal bacterial richness (median number of taxa 38 vs. 37; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.03) and rectal IL-6 levels (median 1.1 pg/mL vs. 0.6 pg/mL; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.02), as well as a decrease in vaginal bacterial diversity (median inverse Simpson index 1.1 vs. 1.0; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.03) The distribution of vaginal community state types remained unchanged. Among the probiotic strains, \u003cem\u003eL. crispatus\u003c/em\u003e and \u003cem\u003eL. rhamnosus\u003c/em\u003e were detected in post-intervention only in the vaginal swab samples of women who harbored them at baseline; \u003cem\u003eB. animalis\u003c/em\u003e was not detected in any vaginal or rectal samples. To conclude, the use of a multi-strain oral probiotic containing microbial lysates and fructooligosaccharides did not alter the vaginal microbiota community state type in healthy premenopausal women, but it was associated with a modest modulation of rectal and vaginal microbial environments and a reduction in a local rectal inflammatory response.\u003c/p\u003e","manuscriptTitle":"Oral probiotic containing microbial lysates modulates rectal and vaginal microbial environments but does not change the vaginal community state type – a pilot study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-25 11:19:01","doi":"10.21203/rs.3.rs-7488779/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bc9ad804-4cdd-435b-ada5-d8bc0e53a037","owner":[],"postedDate":"September 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":55277077,"name":"Health sciences/Medical research"},{"id":55277078,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2025-11-04T06:24:24+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-25 11:19:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7488779","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7488779","identity":"rs-7488779","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}