Postbiotics originated from Lactobacillus crispatus NCU-31 improves vulvar lichen sclerosus: a randomized, double-blind controlled trial

Postbiotics originated from Lactobacillus crispatus NCU-31 improves vulvar lichen sclerosus: a randomized, double-blind controlled trial | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Postbiotics originated from Lactobacillus crispatus NCU-31 improves vulvar lichen sclerosus: a randomized, double-blind controlled trial Hong Liao, Qifa Huang, Xue Wu, Leiping Ding, Ying Jiang, Fen Wei, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7092244/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Jan, 2026 Read the published version in BMC Microbiology → Version 1 posted 11 You are reading this latest preprint version Abstract Background Vulvar lichen sclerosus (VLS) is a chronic inflammatory skin disorder that severely impairs women's physical and psychological well-being. Topical glucocorticoids are the first-line treatment; however, their long-term efficacy is limited due to frequent symptom relapse after discontinuation and incomplete resolution of lesions. Therefore, effective adjunctive strategies are urgently needed to achieve sustained disease control. Methods In this study, we explored the vulvar skin microbiota composition in VLS patients using 16S rRNA gene sequencing and evaluated the therapeutic potential of a postbiotic derived from Lactobacillus crispatus NCU-31. L. crispatus NCU-31 was isolated and its probiotic properties were confirmed in vitro . A postbiotic formulation was then developed and applied in combination with topical glucocorticoids in VLS patients. Clinical efficacy was assessed using the Investigator’s Global Assessment (IGA), Dermatology Life Quality Index (DLQI), and Vulvar Quality of Life Index (VQLI), alongside microbial profiling of the vulvar skin. Results VLS patients exhibited significantly increased microbial richness and diversity, characterized by reduced Lactobacillus abundance and elevated levels of Prevotella , Gardnerella , Dialister , and Streptococcus ( p < 0.05). Combined postbiotic and glucocorticoid treatment led to significant clinical improvement, evidenced by decreased IGA scores and improved DLQI and VQLI ( p < 0.05). Moreover, microbial dysbiosis was partially reversed, with an increase in Lactobacillus and reduction of pathogenic genera. Conclusions This study demonstrates that L. crispatus -derived postbiotics can enhance the efficacy of glucocorticoid therapy, alleviate clinical symptoms, and help restore microbial homeostasis in VLS patients. These findings provide a promising basis for the development of microbiota-targeted adjunctive therapies in the management of chronic vulvar inflammatory disorders. Clinical trial registration: http://www.chictr.org.cn/,identifier ( ChiCTR2400090750), registration time: 12/10/2024. vulvar lichen sclerosus Lactobacillus crispatus postbiotic 16S rRNA gene sequencing Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Vulvar lichen sclerosus (VLS) is a chronic inflammatory, non-neoplastic skin lesion affecting the vulva in women, characterized by the presence of ivory-white patches or plaques with a shiny surface, predominantly located in the anogenital region [1, 2]. Patients with VLS may undergo symptoms such as itch, pain, and urinary incontinence. More seriously, VLS can result in vulvar atrophy, scarring, and potentially the loss of normal vulvar structure and function, imposing a huge psychological burden and economic pressure on patients [3]. Currently, the primary therapeutic approach for VLS is the topical application of glucocorticoids [4]. However, this treatment has limited durability and is susceptible to relapse after discontinuation. Furthermore, prolonged glucocorticoid administration is accompanied by notable adverse effects, ultimately restricting its overall therapeutic efficacy [5]. Therefore, it is imperative to explore innovative adjuvant therapeutic options for VLS that can provide long-term efficacy. As a chronic inflammatory skin disease, the occurrence and progression of VLS have been implicated in autoimmune responses, genetic predisposition, hormonal influences, and local factors [6–8]. With the development of high-throughput sequencing technology, a growing body of research has highlighted the crucial involvement of skin microbiota in the initiation and progression of many diseases, including VLS [9]. For instance, a clinical study led by Liu et al. found that, compared to healthy controls, the relative abundance of lactobacilli was increased, while that of Kelbsiella , Ezakiella , and Escherichia-Shigella decreased remarkably, suggesting that specific microbiota species may facilitate VLS [10]. Additionally, Brunner et al. reported that postmenopausal women diagnosed with VLS showed elevated levels of inducible antimicrobial peptides in their vulvar, which typically constitute a defensive response to microbial invasions and suggest augmented microbial activity or alterations in the composition of microbial species among VLS patients [11]. Consequently, targeting the vulvar microbiota could potentially become a promising therapeutic strategy for alleviating VLS. Postbiotics are defined as a preparation of inanimate microorganisms and/or their components that confer a health benefit on the host [12, 13]. They promote bodily health by balancing microbial populations, suppressing the proliferation and adherence of pathogenic bacteria, and regulating inflammatory responses [14, 15]. For example, Xu et al. demonstrated that postbiotics from Lactobacillus paracasei can inhibit melanogenesis in mouse melanoma cells by activating the PKA/CREB/MITF signaling pathway, thereby contributing to the alleviation of UVB-induced photoaging [16]. Furthermore, Nam et al. indicated that heat-inactivated Lactococcus chungangensis CAU 1447 has the capacity to modulate the expression of cytokines, including interleukin-4 (IL-4), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α), facilitating the healing process of skin wounds in mice [17]. Moreover, research has demonstrated that the lysate of Lactobacillus plantarum K8 strain can enhance the mRNA expression of key moisturizing factors, such as hyaluronic acid synthase 2 and aquaporin 3, thereby being a potential moisturizing product [18]. Given their pivotal role in modulating skin health and mitigating related diseases, postbiotics intervention may be a promising therapeutic approach. In this study, we utilized 16S rRNA sequencing to compare vulvar microbiota between healthy women and VLS patients. Vaginal swab sample were cultured and identified as Lactobacillus crispatus NCU-31. The effects of L. crispatus postbiotics on skin symptoms, signs, Investigator’s Global Assessment (IGA), Dermatology Life Quality Index (DLQL), Vulvar Quality of Life Index (VQLI) and skin microbiota were assessed in VLS patients. This study aimed to develop novel therapeutic strategies for VLS. Materials and methods Study design and ethical approval This randomized, double-blind trial was conducted at Jiangxi Maternal and Child Health Hospital between September 2024 and December 2024. The inclusion criteria: (1) aged between 18 and 70 years old; (2) healthy women or women with a primary pathological diagnosis of VLS; (3) having signed the informed consent form; and (4) having not undergone any other treatment in the past three months. The exclusion criteria: (1) with acute vaginiti or vulvar infections; (2) who had utilized topical glucocorticoids or antibiotics within the preceding month; (3) with a past medical history of malignancy, radiation/chemotherapy treatment, or systemic skin disorders; and (4) Pregnant or lactating women. The Ethics Committee of Jiangxi Maternal and Child Health Hospital granted approval to the study (approval number EC-KY-2024-108), ensuring that all procedures adhered to the principles outlined in the Declaration of Helsinki. Additionally, the study has been registered with the China Clinical Trials Registration Center (ChiCTR), bearing the unique registration number ChiCTR2400090750, with the registration date of October 12, 2024. This study was conducted and reported in accordance with the CONSORT 2025 guidelines for randomized controlled trials. All participants in clinical experiment also provided a signed written informed consent. Randomization and masking In the observational experiment, healthy volunteers comprised the healthy control group (C group), while patients diagnosed with VLS were designated to the VLS group (V group). For the intervention experiment, patients with VLS were randomly assigned in a 1:1 ratio to either the glucocorticoids combined with placebo group (VG group) or the glucocorticoids combined with the postbiotics group (VGP group) using the random number table method. Allocation of the random numbers to postbiotics and placebo was performed by a staff member not involved in the study. Subsequently, dedicated medication personnel administered the glucocorticoids combined with either postbiotics or placebo according to the assigned random number. The glucocorticoids, postbiotics, and placebo were identical in packaging, color, and odor, ensuring a double-blind design for both investigators and participants. Notably, the product supplier had no role in the trial’s design or conduct and held no financial interest in the study. Bacteria culture and identification To study vaginal microbiota in VLS, vaginal swab sample were collected in a liquid medium, and solid media, such as Man Rogosa Sharpe (MRS), brain heart infusion (BHI), and Luria-Bertani (LB), were utilized to screen the vaginal microbiota. A single, smooth, round colony was picked on the solid plate and introduced into 5 ml of liquid medium, incubated in a constant temperature shaker at 37 ℃ and 200 rpm/minute (min) for 24 hours (h) so that the bacteria reached the stable growth stage. Following this, the bacterial liquid was centrifuged at 8,000 rpm for three minutes. After discarding the supernatant, the bacterial DNA was extracted and sent for sequencing to identify the strain. The specific strain information is shown in Table S2. Subsequently, L. crispatus NCU-31 (CGMCC No.31777) was selected for follow-up experiments. To cultivate this strain, MRS medium enriched with 0.5 g L − 1 L-cysteine was utilized. The incubation process occurred in an aerobic atmosphere at 37°C, continuing until the bacterial population reached a concentration of 5×10 9 CFU mL − 1 . The bacterial liquid was centrifuged in a refrigerated high-speed centrifuge at 4°C and 8,000 rpm for 15 min. The bottom sediment was discarded, and the supernatant was obtained; 10–12 ml of the supernatant was filtered through a 0.22 um sterile filter membrane to remove the bacterial body and other impurities, and the cell-free supernatant of L. crispatus (CFS) was collected, and the CFS in subsequent experiments was either dispensed on the spot or stored at -40°C for subsequent clinical intervention testing. Evaluation of probiotic properties Initially, L. crispatus was inoculated into MRS liquid medium at a concentration of 5% and then incubated in an aerobic incubator set at 37°C for 24 hours. Subsequently, the culture was scaled up to 200 ml in a fermentation medium and further incubated under aerobic conditions at 37 C. To establish a growth curve, samples were taken every 2 h, and the OD 600 was measured spectrophotometrically. Additionally, samples were collected every 12 h, and the number of colonies was counted using a spot plate method. For the acid tolerance test, the activated bacteria were centrifuged at 4,500 x g for 10 min at 4°C, and the resulting cell pellet was resuspended in phosphate buffered saline (PBS). The cell suspension was then diluted in PBS at various pH levels (2, 3, 4, and 7) and incubated at 37°C for 4 h. During antioxidant assays, the bacterial cultures were centrifuged at 4,500 x g for 10 min at 4°C. The postbiotics were employed to evaluate their capacity to scavenge 2,2-diphenyl-1-pyridine hydrazine (DPPH), superoxide anions (O 2− ), hydroxyl radicals (-OH), and their ability to chelate Fe 2+ , as well as to determine the reduction potential of the liquid supernatant. For antimicrobial assays, a variety of foodborne pathogenic microorganisms, including Salmonella typhimurium ATCC 13311, Escherichia coli O157, and Staphylococcus aureus Cowan 1 were cultured on LB agar. Oxford cups were placed onto the agar surface, and 250 ul of postbiotics were dispensed into the wells. Subsequently, the diameter of the inhibition zone around the Oxford cups was measured. Intervention procedure and management Observational experiment (Stage 1): Initially, women were enrolled, and those without any vulvar or skin symptoms were considered healthy controls. Concurrently, women who had been diagnostically confirmed with VLS through vulvar skin biopsy were classified as VLS patients. Of these 60 women, 30 healthy women were categorized as the C group, and the other 30 patients with VLS were assigned as the V group. This group's participants were matched in age with those in the comparison group. Routine vaginal discharge wet pap was conducted to rule out bacterial vaginosis. In the outpatient clinic, skin samples were obtained from both the labia majora and negative labia using sterile swabs. Each sample, collected from an area of approximately 3 cm × 3 cm, was stored in a 2 ml sterile Eppendorf tube and preserved at − 80°C for subsequent high-throughput sequencing analysis. Intervention experiment (Stage 2): As depicted in Figure S2, 100 VLS patients were randomized equally into the VG and VGP groups (1:1 ratio). Both groups received an 8-week standardized regimen of topical 0.05% clobetasol propionate ointment, administered daily at a standardized dosage of one fingertip unit. The VGP group additionally daily received 100 microliters of cell-free supernatant from L. crispatus containing 5×10 9 CFU as a postbiotic therapy. The VG group received an equivalent volume of physiological saline as a placebo. Data collection and outcomes Clinicians gathered comprehensive demographic and clinical data, and evaluated VLS patients using the Cattaneo Clinical Score (0–3: Non to Severe), a 6-point scale for symptoms and IGA (0–5: Absent to Severe), DLQI (scale 0–30) and VQLI (scale 0–45). IGA assessed erythema/whitening, infiltration, lichenification, and excoriation, while symptoms encompassed pain, and dyspareunia.The biological outcomes encompassed an investigation into variations in the microbiota of the vulvar skin, with a particular focus on differences in species diversity and taxonomic composition. DNA extraction and 16S rRNA sequencing Genomic DNA was extracted from vulvar skin samples (both observation and intervention groups) using a bacterial DNA extraction kit (M5636–02, Omega, USA). The concentration and purity of the samples were evaluated using spectrophotometry (NanoDrop, Thermo Fisher). The V4 region of 16S rDNA was amplified with primers (5’-AYTGGGYDTAAAGNG and 5’-TACNVGGGTATCTAATCC − 3’, respectively) and sequenced on the Illumina HiSeq 2000 platform (Illumina, California). Overlapping reads were merged using FLASH and analyzed with UPARSE (V7.1), discarding low-quality, ambiguous bases, improper primers, and chimeras. High-quality sequences were assigned to operational taxonomic units (OTUs) based on 97% sequence similarity. Taxonomic classification of bacterial abundance was conducted at various levels (phylum, order, family, genus). The alpha-diversity indices, including Chao1, Shannon, Simpson, Pielou_e, and Faith_pd, were calculated using QIIME2 software (2019.4) and compared by the Wilcoxon test. The beta diversity was evaluated based on the Bray-Curtis distance and subsequently analyzed through principal coordinate analysis (PCoA) as well as non-metric multidimensional scaling (NMDS). QIIME2 software was utilized to visualize the taxonomic composition at the phylum and genus levels, while the Mann–Whitney U test was employed to assess differences in species abundance among groups. Sample size In our observational experiment, our study group undertook a pre-trial evaluation to assess the severity of VLS among patients. The results indicated a symptom score of 2.88 ± 1.95 for healthy volunteers and 5.13 ± 2.39 for those diagnosed with VLS. Based on these findings, a sample size calculation was conducted, revealing that approximately 17 participants per group would be required (power: 80%; α: 5%). Nevertheless, given the typical sample sizes employed in VLS research and their potential influence on the accuracy of high-throughput sequencing outcomes, we decided to augment the initial sample size to 60 participants, with 30 in each group. Since there is no authoritative literature analyzing the efficacy of post-operative L. crispatus probiotics in patients with VLS, a reasonable power analysis could not be performed. Based on the pre-test analysis, assuming a mean post-treatment symptom score of 4.16 in the VG group and a mean post-treatment symptom score of 3.24 in the VGP group, with a standard deviation of 1.84 in both groups, 63 subjects would need to be recruited for the study in each group (power: 80%; α: 5%). Taking into consideration potential attrition, dropout rates, and any unforeseen events, we adjusted the initial sample size upwards by approximately 10%, thereby setting a target enrollment of 70 participants for each group. Statistical analysis Continuous variables were reported using the mean ± standard deviation format, and the Kolmogorov–Smirnov test was utilized to evaluate the normality of the data. For normally distributed data with equal variances, the Student t-test was applied, while for normally distributed data with unequal variances, an adjusted t-test was used. For non-normally distributed data, the Mann–Whitney U test was employed for analysis. Categorical variables, presented as counts and percentages, were analyzed using the Pearson chi-square test, the continuity-corrected chi-square test, or the Fisher exact test, depending on the circumstances. All statistical analyses and data visualizations were conducted using Prism version 9.5.1 software (GraphPad Software, San Diego, CA, USA), QIIME2 software, R version 4.2.3 software, PASS 15.0, and SPSS version 26. The threshold for statistical significance was set at p < 0.05. Results Demographic and baseline features of the participants in the observational study. The baseline characteristics, Cattaneo Clinical Score, IGA, DLQL and VQLI of participants are summarized in Table S1 and Fig. 1 . No statistically significant differences were observed between the C and V groups in age, body weight, or BMI ( p > 0.05; Table S1 ). Notably, the demographic and clinical profiles of the two groups were generally consistent, except for pruritus degree, skin elasticity, skin color, lesion scope, IGA, DLQL and VQLI ( p < 0.05; Table S1 and Figs. 1 B–F). Alterations in the microbial diversity and composition of the vulvar skin among patients with VLS To characterize the microbiota of the vulvar skin in patients with VLS, we gathered vulvar skin samples from 29 healthy volunteers and 30 VLS patients for 16s rRNA sequencing. To determine differences in microbial diversity between the two groups, we analyzed the alpha-diversity. While no significant difference was observed in the Chao1 index ( p = 0.57) between the two groups (Fig. 2 A), the Shannon index ( p = 0.014), which measures species richness and evenness, the Simpson index ( p = 0.0036), and the Pielou_e ( p = 0.0033) were remarkably higher in VLS patients than in healthy individuals (Figs. 2 B–D). According to the results of the Venn diagram, 502 and 517 otu were found in the vulvar skin of the C and V groups, respectively, of which 350 otu were present in the vulvar skin of these two groups (Fig. 2 E). We then assessed beta-diversity to compare the microbial community composition between the two groups of patients, and the findings indicated an independent aggregation of the various groups (Figs. 2 F and 2 G). In addition, LEfSe analysis revealed that distinct levels of vulvar microbiota were characterized by differences. Figures 2 H and 2 I show 30 differential microorganisms of various taxonomic categories and taxa. At the genus level, the C group was mainly enriched with Lactobacillus , Mogibacterium , Megasphaera , Phascolarctobacterium , and Nesterenkonia , the V group was characterized by Prevotella , Streptococcus , Bacillus , and Acinetobacter as characteristic genera. Collectively, these differential taxa may serve as target microorganisms for VLS therapy. At the phylum level, as shown in Fig. 3 A, Firmicutes, Bacteroidetes, and Actinobacteria emerged as the dominant microbiota, which accounted for 96.12% and 91.27% in the C and V groups, respectively. Among them, the C group (C vs. V = 57.01% vs. 60.73%, p < 0.05) exhibited a significantly lower relative abundance of Firmicutes (Fig. 3 B) compared to the V group, whereas the abundance of Bacteroidetes (Figure. 3C) was remarkably higher in C group (C vs. V = 29.27% vs. 11.39%, p < 0.001) than in V group. Furthermore, Actinobacteria (Fig. 3 D) were less abundant in the C group (9.84% vs. 19.15%) than in the V group. We then further analyzed the microbial composition at the genus level (Fig. 3 E) and discovered that the C group had a significantly higher relative abundance of Lactobacillus compared to the V group (C vs. V = 55.97% vs. 18.72%, p < 0.01), suggesting a reduction in the beneficial vulvar skin microbiota among patients with VLS (Fig. 3 F). However, the presence of conditionally pathogenic bacteria, including Prevotella (C vs. V = 10.32% vs. 21.85%, p < 0.01), Gardnerella (C vs. V = 2.84% vs. 10.29%, p < 0.05), Dialister (C vs. V = 2.85% vs. 6.77%, p < 0.05) and Streptococcus (C vs. V = 0.37% vs. 9.22%, p < 0.05), was significantly increased in patients with VLS (Figs. 3 G–J). Notably, Finegoldia , Peptoniphius , and Fusobacterium were not remarkably distinct between the two groups. However, the V group exhibited a slightly higher relative abundance of these microbiota compared to the C group (Figs. 3 K–M). Taken together, the above results revealed that the occurrence of VLS may be associated with a reduction in beneficial vulvar microbiota and a rise in pathogenic microbial populations, suggesting compromised integrity of the vulvar microbial barrier and increased vulnerability of the microenvironment. Isolation of vaginal secretions microbiota and evaluation of the probiotic properties of L. crispatus Previous results have indicated that patients with VLS are susceptible to vulvar microbiota dysbiosis. To further isolate specific VLS-related bacteria, we performed bacterial culture and identification, and a total of 183 bacterial isolates were obtained, including 123 strains of Lactobacillus , 28 strains of Gardnerella , 19 strains of Streptococcus , 6 strains of Staphylococcus , 4 strains of Escherichia coli , 2 strains of Ligilactobacillus salivarius , and 1 strain of Corynebacterium callunae , as shown in Table S2. Lactobacillus dominates the vaginal microbiota of healthy women, and the abundance of Lactobacillus was significantly reduced in the vulvar skin microbiota of VLS patients. Therefore, we hypothesized that the occurrence of VLS might be associated with the decreased abundance of Lactobacillus . Plate coating and Gram staining were conducted to observe the morphology of Lactobacillus , revealing an elongated, curved, and rod-shaped morphology consistent with previous literature reports (Figures S1 A and B). Subsequently, we performed phylogenetic tree construction to clarify the specific branching of Lactobacillus ; more than 50% of bootstrap values for 1000 replicates are shown in Fig. 4 F. The L. crispatus strain of interest was on branch with L. crispatus TMPC3192H (OM760749.1) with 99% sequence similarity and 50.3% confidence. Combined with the previous morphology of L. crispatus and the phylogenetic strain L. crispatus TMPC3192H was determined to be L. crispatus NCU-31 (Figure S1 F). Then, we performed a comprehensive in vitro evaluation to assess the probiotic efficacy of L. crispatus , including growth characteristics, acid tolerance, antioxidant capacity, drug resistance, and inhibition of common pathogenic bacteria. Our results suggested that L. crispatus demonstrated vigorous growth under aerobic conditions at 37°C, entering a rapid growth phase after eight hours and ultimately a stable phase at 24 h with an OD 600 nm of 2.08 (Figure S1 C). In the acid tolerance assay, the viable count of L. crispatus gradually increased with rising pH (2.0, 3.0, 4.0, and 7.0) and was highest at pH 7, reaching 1.7×10 10 CFU mL − 1 (Figure S1 D). Moreover, the results of the antioxidant capacity evaluation of L. crispatus exhibited its ability to scavenge DPPH and hydroxyl radicals (-OH) by 89.86% and 82.84%, respectively. It also inhibited superoxide radicals (O 2− ) by 39.99% and chelate ferrous ions (Fe 2+ ) by 62.36% (Figure S1 E). We also found that L. crispatus was significantly resistant to common antibiotics, with the most pronounced resistance to cotrimoxazole and ciprofloxacin (Figure S1 F). Additionally, we also assessed the bacteriostatic properties of L. crispatus postbiotics. We discovered that they exhibited notable inhibitory activity against E. coli O157, S. typhimurium ATCC 13311, and S. Cowan 1, with inhibition circles size of 11.73 mm, 9.30 mm, and 10.63 mm, respectively (Figure S1 G). Overall, these data underscore the excellent probiotic properties of L. crispatus , encompassing its vigorous growth, acid resistance, antioxidant potential, robust drug tolerance, and significant bacteriostatic activity. Baseline characteristics and the influence of L. crispatus on vulvar skin symptoms and signs To assess the alleviating effect of L. crispatus postbiotics on vulvar skin symptoms in patients with VLS, we enrolled 140 new volunteers in an intervention study (Stage 2). As depicted in Figure S2, following dermatological diagnosis, 100 patients diagnosed with VLS were equally assigned to the VG and VGP groups in a 50:50 ratio for the intervention study. The remaining 40 patients were excluded from the study: 28 declined participation, and 12 fulfilled the exclusion criteria. Table 1 presents baseline demographics, Cattaneo clinical scores, IGA, DLQL and VQLI of intervention experiment. No significant differences in age, weight, BMI, or clinical profiles were observed between the VG and VGP groups ( p > 0.05). After an eight-week intervention, patients in the VGP group exhibited significantly improved Cattaneo clinical scores, IGA, DLQL and VQLI compared to the VG group ( p < 0.05) (Fig. 4 ). These results illustrate that L. crispatus postbiotic can potentially improve vulvar skin symptoms in VLS. Table 1 “Baseline characteristics of participants in the intervention experiment” Characteristics VG group (n = 50) VGP group (n = 50) t/Z/ X2 p -Value Ages (years) 44.38 ± 11.15 40.74 ± 10.92 − 1.708 0.088 Weight (kg) 55.61 ± 4.82 55.12 ± 3.91 − 0.576 0.565 BMI (kg/m2) 20.88 ± 1.42 20.63 ± 1.22 − 0.476 0.634 Vaginal PH 4.01 ± 0.30 4.04 ± 0.32 − 0.236 0.814 HPV infection, n (%) 5(10.00) 3 (6.00) 0.543 0.461 Hypertension, n (%) 5 (10.00) 2 (4.00) 1.382 0.240 Diabetes, n (%) 2 (4.00) 1 (2.00) 0.344 0.558 Currently smoking, n (%) 11 (22.00) 8 (16.00) 0.585 0.444 Consumed alcohol, n (%) 9 (18.00) 5 (10.00) 1.329 0.249 Ever pregnant, n (%) 43 (86.00) 39 (78.00) 1.084 0.298 Skin color 1.06 ± 0.82 0.58 ± 0.61 − 3.046 0.002 Skin elasticity 1.36 ± 0.80 0.76 ± 0.66 − 3.758 0.000 Pruritus degree 1.04 ± 1.03 0.50 ± 0.68 − 2.713 0.007 Lesion scope 1.20 ± 0.73 0.72 ± 0.67 − 3.384 0.001 Dyspareunia 0.04 ± 0.20 0.02 ± 0.14 − 0.583 0.560 IGA 1.38 ± 0.49 0.38 ± 0.49 − 7.194 0.000 DLQI 3.86 ± 1.01 0.16 ± 0.37 − 9.011 0.000 VQLI 6.36 ± 1.14 0.74 ± 0.66 − 8.756 0.000 VG: Vulvar lichen sclerosus patients with glucocorticoid and placebo; VGP: Vulvar lichen sclerosus patients with glucocorticoid and L. crispatus postbiotics; BMI: Body Mass Index; HPV: Human papillomavirus; IGA:Investigator’s Global Assessment; DLQL:Dermatology Life Quality Index; VQLI:Vulvar Quality of Life Index L. crispatus postbiotic enriches and enhances the compromised vulvar skin microbiota in VLS patients To assess the impact of L. crispatus postbiotic on the vulvar skin microbiota in patients with VLS, vulvar skin samples were analyzed by 16SrRNA-sequencing. Figures 5 A–E shows the alterations in alpha-diversity, where the observed species Chao1 ( p = 2.7 e − 13 ), Shannon (p = 4e − 08 ), Simpson ( p = 2.1e − 06 ), Pielou_e ( p = 7.2e − 05 ), and Faith_pd ( p = 3.4e − 05 ) indices were remarkably decreased in the VGP group, suggesting a reduction in the abundance and diversity of the vulvar skin microbiota. Similar results were observed in Venn, where L. crispatus postbiotic intervention alleviated VLS-induced asv(amplicon aequence variant) increase to some extent (Fig. 5 F). In addition, the beta diversity was assessed by PCoA and NMDS, which showed some clustering of taxa, with a separation of taxa in the VG and VGP groups (p < 0.05; Figs. 5 G and 5 H). LEfSe analysis further emphasized the significant difference in the vulvar skin microbiota composition between the VG and VGP groups (Fig. 5 I). This reveals that L. crispatus postbiotic could remodel the disturbed vulvar skin microbiota. Furthermore, we analyzed the community composition of microbiota in the vulvar skin of different groups at the phylum and genus levels. We found that Firmicutes (VG vs. VGP = 55.19% vs. 56.78%), Bacteroidetes (VG vs. VGP = 18.71% vs. 22.01%, p < 0.05), Actinobacteria (VG vs. VGP = 19.38% vs. 11.64%, p 0.05) were the dominant phyla in the vulvar skin of patients with VLS, accounting for 97.17% and 97.61% of the VG and VGP groups, respectively (Fig. 6 A). Following treatment with L. crispatus postbiotic, the VGP group exhibited a significantly higher relative abundance of Bacteroidetes compared to the VG group, indicating a reversal of the decrease in Bacteroidetes previously observed in the observational study among VLS patients ( p < 0.05, Fig. 6 B). Additionally, Actinobacteria and Fusobacteria phyla were significantly down-regulated after the co-administration of glucocorticoid with L. crispatus postbiotic therapy, indicating that the L. crispatus postbiotic intervention restored some of the microecological dysregulation in vulvar skin ( p < 0.05, Figs. 6 C and D). At the genus level, topical application of L. crispatus postbiotic remarkably altered the microbiota composition of vulvar skin compared to the VG group (Fig. 6 E). In addition, after administration of the L. crispatus postbiotic, the VGP group showed a significantly higher relative abundance of Lactobacillus (16.45% in VG vs. 35.92% in VGP, p < 0.05) and Bifidobacterium (0.63% in VG vs. 2.59% in VGP, p 0.05) following the L. crispatus postbiotic supplementation (Fig. 6 G). Moreover, the co-administration of therapy led to a reduction in the relative abundance of pathogenic bacteria, including Streptococcus , Corynebacterium , Finegoldia , Staphylococcaceae_Staphylococcus , Dialister , and Gardnerella ( p < 0.05, Figs. 6 H–M). Furthermore, the rank abundance curve data revealed that topical L. crispatus postbiotic increased the OUT of the VGP group compared to the VG group, and the heat map results were consistent with the previous findings (Figs. 6 O and P). Collectively, these results indicate that an eight-week treatment with L. crispatus postbiotic may ameliorate VLS-induced vulvar microecological dysbiosis, thereby restoring the diversity of the vulvar microbiota. Correlation analysis between vulvar skin microbiota, vulvar skin symptoms, signs, and other clinical indicators in patients with VLS We employed Spearman's rank correlation coefficient for correlation analysis to explore the complex interplay between vulvar skin microbiota, vulvar skin symptoms, and various clinical indicators. The results showed that Streptococcus , Corynebacterium , Finegolida , Dialister and Gardnerella were positively correlated with pruritus degree, lesion scope, IGA, DLQL and VQLI respectively, in patients with VLS, suggesting that these bacteria could potentially serve as target genera for treating VLS. Furthermore, Lactobacillus was significantly negatively correlated with Prevotella , Streptococcus , Corynebacterium , Finegolida , and Dialister , while the latter was remarkably positively correlated with Prevotella and Finegolida ( p < 0.05, Fig. 3 ). Taken together, these findings indicate a strong link between changes in the vulvar skin microbiota due to VLS and alterations in vulvar skin symptoms, potentially exacerbating the symptoms in patients with VLS. Discussion VLS is a chronic inflammatory skin disease that profoundly impacts the physical and mental health of patients [19]. In recent years, numerous studies have increasingly suggested that the vulvar skin microbiota plays a significant role in the initiation and progression of VLS [20, 21]. However, current research has largely focused on identifying and classifying vulvar microorganisms, with relatively few studies exploring the role of microorganisms and their derivatives in treating VLS [22]. Therefore, we first compared the differences in the vulvar microbiota between healthy women and patients with VLS using high-throughput sequencing. The results revealed that VLS patients exhibited higher microbial diversity and richness in their vulvar skin microbiota compared to healthy individuals. The abundance of Lactobacillus decreased, while the relative abundance of Prevotella , Gardnerella , Dialister , and Streptococcus increased. These findings suggest that restoring the balance of the vulvar skin microbiota may be a promising strategy for improving VLS. Previous findings have shown a significant decrease in Lactobacillus abundance in patients with VLS, and we aimed to investigate whether the development of VLS is associated with Lactobacillus . We conducted microbial screening cultures on vaginal swab samples from healthy volunteers and VLS patients and obtained 123 strains of lactobacilli . Based on the results of the Gram staining and phylogenetic tree, we ultimately identified them as L. crispatus . Subsequently, we performed an in vitro probiotic assessment, which showed that L. crispatus possessed favorable probiotic properties, including strong growth capacity, acid resistance, antioxidant capacity, drug resistance, and the capacity to suppress pathogenic bacteria growth. Research has indicated that Lactobacillus spp .-dominated microbiota has been recognized as a hallmark of female reproductive tract health [23–25]. In a longitudinal cohort study, the predominant presence of L. crispatus in the vagina microbiota was linked to a lower risk of HIV infection compared to the bacterial vaginosis microbiota [26]. Women susceptible to L-type crispatus were also less likely to convert to a bacterial vaginosis microbiota than those with L-type dominance, suggesting that L-type crispatus competes to some extent to exclude bacteria associated with inflammatory vaginitis through the production of lactic acid and anti-microbial metabolites [27, 28]. In vitro modeling suggests that L. crispatus is a major producer of lactic acid in the female genital tract, and that lactic acid may directly inhibit the production of pro-inflammatory cytokines [29, 30]. Furthermore, the incorporation of supernatants enriched with either L. crispatus alone or in combination with other components demonstrated a capacity to decrease the production of proinflammatory cytokines by bacteria associated with bacterial vaginitis and protected the epithelial barrier from disruption in response to proinflammatory stimuli [31]. These findings imply that Lactobacillus and its derivatives may exert their probiotic effects by alleviating vaginal inflammation, modulating the body's immune response, and promoting vaginal ecological balance. Given the pivotal role of L. crispatus and its derivatives in maintaining human health, we elected to utilize L. crispatus postbiotics in our intervention trial. Our findings revealed that the co-administration of glucocorticoid with L. crispatus postbiotic therapy markedly enhanced skin symptoms, signs, IGA, DLQL and VQLI among patients with VLS, in line with the beneficial effects of L. crispatus postbiotics observed in other studies [32]. Song et al. discovered that peptidoglycan isolated from vaginal L. crispatus stimulated the expression of CD207 on Langerhans cells and significantly downregulated the expression of HIV entry receptors [33]. Donnarumma et al. demonstrated that L. crispatus L1 exopolysaccharides possessed the ability to robustly augment the production of human defensin-2 protein by vaginal epithelial VK2 cells while reducing Candida albicans adherence through competitive exclusion mechanisms (48%) [34]. Furthermore, the combination therapy of L. crispatus postbiotics with glucocorticoids was found to reduce alpha-diversity and alter the microbial composition of the skin microbiota. In terms of beta-diversity, a slight separation of sample clusters was observed, indicating that the application of glucocorticoids in this study had a slight impact on the patient cohort's skin microbiota diversity. These data indicate that combined therapy may shift the skin microbiota to a healthier state, potentially improving VLS management. We conducted an in-depth analysis of the skin microbiota composition and observed significant alterations in the abundance of taxonomic groups at the phylum and genus levels after combining glucocorticoid treatment with L. crispatus postbiotic therapy. Specifically, the combined therapy resulted in a higher relative abundance of Bacteroidetes at the phylum level and decreased Actinobacteria and Fusobacteria compared to glucocorticoids alone. Actinobacteria phylum includes pathogenic bacteria, such as Salmonella and Vibrio cholerae , which are crucial in the development and progression of several diseases [35–37]. Conversely, Bacteroidetes is a major component of the human vaginal microbiota, which contains several probiotics [38, 39]. It has been discovered that Bacteroidetes, through their metabolism, produce substances that help the vagina get rid of foreign matter and harmful bacteria, thus maintaining a clean and healthy vagina [40]. This indicates that Bacteroidetes could be key in re-establishing the vaginal microecological balance. Furthermore, at the genus level, the combined therapy led to an increase in the relative abundance of Lactobacillus , while the abundance of Streptococcus , Corynebacterium, Finegoldia , Staphylococcaceae_Staphylococcus , Dialister , and Gardnerella was reduced. Lee et al. found that the vaginal microbiota of HPV-infected women exhibited increased Dialister levels and decreased Lactobacillus levels compared to healthy women [41]. Additionally, patients with genital wart infections had dysbiosis of the vaginal microbiota, which featured a reduction in Lactobacillus abundance and an elevation in the proportion of Gardnerella [42]. These results indicated that an L. crispatus postbiotic may alleviate VLS by restoring the balance of vulvar skin microbiota. In this study, we evaluated the efficacy of the co-administration of glucocorticoid with L. crispatus postbiotic therapy in patients with VLS. The findings demonstrated that the co-administration of therapy significantly improved skin symptoms, signs, IGA, DLQL and VQLI in VLS patients without serious adverse effects. Furthermore, L. crispatus postbiotics were found to effectively restore the vulvar skin microbiota. These results indicate that the topical application of L. crispatus postbiotic could potentially serve as an efficacious and targeted therapy for VLS patients. However, this study also has limitations, such as a short observation period; therefore, the long-term therapeutic effect of L. crispatus postbiotic needs to be further assessed. In addition, the difficulty of recruiting a large sample makes the potential heterogeneity of participants difficult to control, thus hindering the current clinical study. Further comprehensive studies are required to validate these findings. Lastly, while this study examined alterations in skin symptoms, signs, and skin microbiota among VLS patients, it did not delve into potential causal links between these changes due to the absence of metabolomic analysis. Declarations Acknowledgements We gratefully acknowledge the approval of this study by the Ethics Committee. We also thank the Jiangxi Province Key Laboratory of Bioengineering Drugs, the General Science and Technology Program of the Jiangxi Provincial Health Commission, and the National Natural Science Foundation of China for their valuable support of this research. Author Contributions Tingtao chen and Qi Chen: Writing – original draft, Supervision, Methodology, Investigation, Formal analysis,Data curation, Resources. Hong Liao and Qifa Huang: Writing–review & editing,Methodology. Ying Jiang and Fen Wei: Writing – review & editing, Validation, Formal analysis. Xiaoyan Ai and Hailian Luo: Writing – review & editing, Project administration. Xue Wu and Leiping Ding: Writing – review & editing, Project administration, Supervision, Conceptualization, Resources. Funding This work was supported by grants from the National Natural Science Foundation of China (82460528 and 82260507 to Q.C,82460297 and 82260389 to T.C), Jiangxi Province Key Laboratory of bioengineering drugs (No.2024SSY07061 to T.C), and Jiangxi Provincial Health Commission's General Science and Technology Plan (No.202310937 to H.L). Data Availability The authors affirm that the entirety of the data instrumental in substantiating the findings of this study are readily accessible and intricately detailed within the confines of the article itself. The raw sequence data have been submitted to the GenBank database and are accessible under the accession identifier PRJNA1188795. The dataset can be accessed via the following link: The raw sequence data have been submitted to the GenBank database and are accessible under the accession identifier PRJNA1188795. The dataset can be accessed via the following link: https://dataview.ncbi.nlm.nih.gov/object/PRJNA1188795. The data will be released to the public on December 31, 2025. Ethical approval The Ethics Committee of Jiangxi Maternal and Child Health Hospital granted approval to the study (approval number EC-KY-2024-108), ensuring that all procedures adhered to the principles outlined in the Declaration of Helsinki. Additionally, the study has been registered with the China Clinical Trials Registration Center (ChiCTR), bearing the unique registration number ChiCTR2400090750, with the registration date of October 12, 2024. This study was conducted and reported in accordance with the CONSORT 2025 guidelines for randomized controlled trials. All participants in clinical experiment also provided a signed written informed consent. Consent for publication Not applicable. Conflicts of Interest The authors declare no conflict of interest. References Fergus KB, Lee AW, Baradaran N, Cohen AJ, Stohr BA, Erickson BA, et al. 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Supplementary Files CONSORT2025editablechecklist.docx Supplementary.docx Cite Share Download PDF Status: Published Journal Publication published 21 Jan, 2026 Read the published version in BMC Microbiology → Version 1 posted Editorial decision: Revision requested 15 Dec, 2025 Reviews received at journal 12 Dec, 2025 Reviewers agreed at journal 02 Dec, 2025 Reviewers agreed at journal 27 Nov, 2025 Reviews received at journal 18 Oct, 2025 Reviewers agreed at journal 23 Sep, 2025 Reviewers agreed at journal 30 Jul, 2025 Reviewers invited by journal 29 Jul, 2025 Editor assigned by journal 25 Jul, 2025 Submission checks completed at journal 24 Jul, 2025 First submitted to journal 24 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7092244","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":493281889,"identity":"632bae0f-87d2-4d95-9d5d-1329930a00d8","order_by":0,"name":"Hong Liao","email":"","orcid":"","institution":"Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Hong","middleName":"","lastName":"Liao","suffix":""},{"id":493281890,"identity":"31c632ba-ab6b-4e5b-ae31-5200ad264300","order_by":1,"name":"Qifa Huang","email":"","orcid":"","institution":"Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Qifa","middleName":"","lastName":"Huang","suffix":""},{"id":493281891,"identity":"13001b9c-0b41-46ab-a71f-b924b0fb40f1","order_by":2,"name":"Xue Wu","email":"","orcid":"","institution":"Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Xue","middleName":"","lastName":"Wu","suffix":""},{"id":493281892,"identity":"f25d3854-467d-414c-9b51-7d965a2d19d1","order_by":3,"name":"Leiping Ding","email":"","orcid":"","institution":"Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Leiping","middleName":"","lastName":"Ding","suffix":""},{"id":493281893,"identity":"cc2b99fc-78b6-4d2d-94e3-9cc3a4860b3a","order_by":4,"name":"Ying Jiang","email":"","orcid":"","institution":"Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Jiang","suffix":""},{"id":493281894,"identity":"6fbb4f3b-e703-451b-bc82-fec3d41b6b03","order_by":5,"name":"Fen Wei","email":"","orcid":"","institution":"Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Fen","middleName":"","lastName":"Wei","suffix":""},{"id":493281895,"identity":"9db41154-837b-484b-860f-67423bd6e863","order_by":6,"name":"Xiaoyan Ai","email":"","orcid":"","institution":"Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoyan","middleName":"","lastName":"Ai","suffix":""},{"id":493281896,"identity":"c7a8188d-0b9d-4e9d-a6be-2fb440dce590","order_by":7,"name":"Hailian Luo","email":"","orcid":"","institution":"Nanchang Medical College","correspondingAuthor":false,"prefix":"","firstName":"Hailian","middleName":"","lastName":"Luo","suffix":""},{"id":493281897,"identity":"b3d0c4b8-54cf-4069-b25b-16473c205c17","order_by":8,"name":"Qi Chen","email":"","orcid":"","institution":"Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Qi","middleName":"","lastName":"Chen","suffix":""},{"id":493281898,"identity":"97c59ecb-c946-443c-9837-9f02bdd66b28","order_by":9,"name":"Tingtao Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBklEQVRIie3RsUoDMRjA8RyF3JLrrQGlfYVPCje2PkrKwd1yRceMSiG3FFwLio/h6hcCN+UBCnYQhJs63CQdBE0tgsvddRTMfwgJ5EdIQojP9xfjhwGOc2wkHdGwxH6CjjBCAr22w8mQWXECId9kYCI1mj/yS+gU8f2yemuut9Pn0KBhNskVJ4Ls5VP7IdsqB4Q6XbFM6AeZLdTZLQYr+9JKgBcJRzApIwXgzlYLdY5iEKgucvV+JPEOMFKfOeUCekhBD2TKeAE6UlT0Er7JEm7BCLaphXtkeqHcI+uuu8TrtObyw8zCu9Q07ivH47LUr3vZTn6a3/xeYe9+1+yUTT6fz/dP+wKRS16S6SfEEAAAAABJRU5ErkJggg==","orcid":"","institution":"Nanchang University","correspondingAuthor":true,"prefix":"","firstName":"Tingtao","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2025-07-10 10:53:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7092244/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7092244/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12866-026-04738-w","type":"published","date":"2026-01-21T15:58:30+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":88193949,"identity":"eb0131fb-306e-43fc-b4c4-75984f6def68","added_by":"auto","created_at":"2025-08-03 15:26:36","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":94262,"visible":true,"origin":"","legend":"\u003cp\u003eThe Clinical Score. (A) The table of Cattaneo Clinical Score. (B) Pruritus degree. (C) Skin elasticity. (D) Skin color. (E) Lesion scope. (F) IGA. (G) VLQI. (H) VQLI. C: health control; V: vulvar lichen sclerosus patients; IGA: Investigator’s Global Assessment; DLQI: Dermatology Life Quality Index; VQLI: Vulvar Quality of Life Index. The data are presented as means ± standard deviation; * \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7092244/v1/e4ab7d7f4a80b5ad2a938f4d.jpg"},{"id":88193373,"identity":"f24e0c79-fb78-4f0b-abeb-6a4baf292f8c","added_by":"auto","created_at":"2025-08-03 15:10:36","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":119383,"visible":true,"origin":"","legend":"\u003cp\u003eThe microbial composition is distinct between the control and VLS groups. (A) The Chao1 index. (B) The Shannon index. (C) The Simpson index. (D) The Pielou_e index. (E) Venn diagram representation of OTUs. (F) PCoA and (G) NMDS of beta-diversity based on the unweighted UniFrac. (H) Taxonomic cladogram from LEfSe analysis. (I) Histogram of LDA score. C: health control; V: vulvar lichen sclerosus patients. * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7092244/v1/f9cc5be6c4b7e04810818f73.jpg"},{"id":88193546,"identity":"6335e85f-1781-4776-a9b9-a9b3930abfe6","added_by":"auto","created_at":"2025-08-03 15:18:36","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":104661,"visible":true,"origin":"","legend":"\u003cp\u003eDisturbed composition of vulvar skin microbiota in patients with VLS. (A) Microbial composition at the phylum level. The relative abundance of (B) Firmicutes, (C) Bacteroidetes, and (D) Actinobacteria. (E) Microbial composition at the genus level. The relative abundance of (F) \u003cem\u003eLactobacillus\u003c/em\u003e, (G) \u003cem\u003ePrevotella\u003c/em\u003e, (H) \u003cem\u003eGardnerella\u003c/em\u003e, (I) \u003cem\u003eDialister\u003c/em\u003e, (J) \u003cem\u003eStreptococcus\u003c/em\u003e, (K) \u003cem\u003eFinegoldia\u003c/em\u003e, (L) \u003cem\u003ePeptoniphius\u003c/em\u003e, and (M) \u003cem\u003eFusobacerium\u003c/em\u003ewere analyzed. C: health control; V: vulvar lichen sclerosus patients. The data are presented as means ± standard deviation; * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7092244/v1/200465d5bccb4df8188d2278.jpg"},{"id":88193547,"identity":"196a8312-d08b-4893-b2bb-ba4820894f37","added_by":"auto","created_at":"2025-08-03 15:18:36","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":68577,"visible":true,"origin":"","legend":"\u003cp\u003eThe Clinical Score in the intervention experiment. (A) Pruritus degree. (B) Skin elasticity. (C) Skin color. (D) Lesion scope. (E) IGA. (F) VLQI. (G) VQLI. VG: Vulvar lichen sclerosus patients received glucocorticoid combined with placebo; VGP: Vulvar lichen sclerosus patients received glucocorticoid combined with \u003cem\u003eL. crispatus\u003c/em\u003e postbiotics; IGA: Investigator’s Global Assessment; DLQI: Dermatology Life Quality Index; VQLI: Vulvar Quality of Life Index. The data are presented as means ± standard deviation; * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01.”\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7092244/v1/c3f658b4c35c386c671cc099.jpg"},{"id":88193392,"identity":"9b52f60c-65d9-4bb2-a1b7-d5c25570ecb2","added_by":"auto","created_at":"2025-08-03 15:10:37","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":103808,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eL. crispatus\u003c/em\u003epostbiotics improve the diversity of the vulvar skin microbiota. The (A) Chao1, (B) Shannon, (C) Simpson, (D) Pielou_e, and (E) Faith_pd index. (F) Venn diagram of vulvar skin microbial species. The weighted UniFrac distance (G) PCoA, and (H) NMDS of vulvar skin microbiota. (I) LDA Effect Size (LEfSe) analysis. VG: Vulvar lichen sclerosus patients received glucocorticoid combined with placebo; VGP: vulvar lichen sclerosus patients with glucocorticoid and \u003cem\u003eL. crispatus\u003c/em\u003e postbiotics. * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7092244/v1/7ed5e3b028bbab1fd6d2f204.jpg"},{"id":88193379,"identity":"57ae10e0-8f9a-41e6-a82d-722a73bfb137","added_by":"auto","created_at":"2025-08-03 15:10:36","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":126772,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of \u003cem\u003eL. crispatus\u003c/em\u003e postbiotics on the composition of vulvar skin microbiota in patients with VLS. (A) The phylum-level abundance of vulvar skin microbiota. The relative abundance of (B) Bacteroidetes, (C) Actinobacteria, and (D) Fusobacteria. (E) The genus-level abundance of vulvar skin microbiota. The relative abundance of (F) \u003cem\u003eLactobacillus\u003c/em\u003e, (G) \u003cem\u003ePrevotella\u003c/em\u003e, (H) \u003cem\u003eStreptococcus\u003c/em\u003e, (I) \u003cem\u003eCorynebacterium\u003c/em\u003e, (J) \u003cem\u003eFinegoldia\u003c/em\u003e, (K) \u003cem\u003eStaphylococcaceae_Staphylococcus\u003c/em\u003e, (L) \u003cem\u003eDialister\u003c/em\u003e, (M) \u003cem\u003eGardnerella\u003c/em\u003e, and (N) \u003cem\u003eBifidobacterium\u003c/em\u003e. (O) Rank-abundance curve. (P) Microbial community heatmap. VG: Vulvar lichen sclerosus patients received glucocorticoid combined with placebo; VGP: vulvar lichen sclerosus patients with glucocorticoid and \u003cem\u003eL. crispatus\u003c/em\u003e postbiotics. The data are presented as means ± standard deviation; * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7092244/v1/e1c0e6c0c311a70c0347b92c.jpg"},{"id":101152124,"identity":"9feb6b6c-fe5c-4a00-8e3f-51745e09adad","added_by":"auto","created_at":"2026-01-26 16:10:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1718284,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7092244/v1/12100fe3-92c4-4233-ad52-71ce85d30fbc.pdf"},{"id":88193374,"identity":"75b7d9d5-bb80-4c8d-8d71-fc015d2d3075","added_by":"auto","created_at":"2025-08-03 15:10:36","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":31023,"visible":true,"origin":"","legend":"","description":"","filename":"CONSORT2025editablechecklist.docx","url":"https://assets-eu.researchsquare.com/files/rs-7092244/v1/877692f80b4bb1a9af1b1ffc.docx"},{"id":88193382,"identity":"db55d5f2-4add-4c70-8235-2908e1161232","added_by":"auto","created_at":"2025-08-03 15:10:36","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":8682695,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-7092244/v1/e330aaceca6a606761e0e713.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Postbiotics originated from Lactobacillus crispatus NCU-31 improves vulvar lichen sclerosus: a randomized, double-blind controlled trial","fulltext":[{"header":"Introduction","content":"\u003cp\u003eVulvar lichen sclerosus (VLS) is a chronic inflammatory, non-neoplastic skin lesion affecting the vulva in women, characterized by the presence of ivory-white patches or plaques with a shiny surface, predominantly located in the anogenital region [1, 2]. Patients with VLS may undergo symptoms such as itch, pain, and urinary incontinence. More seriously, VLS can result in vulvar atrophy, scarring, and potentially the loss of normal vulvar structure and function, imposing a huge psychological burden and economic pressure on patients [3]. Currently, the primary therapeutic approach for VLS is the topical application of glucocorticoids [4]. However, this treatment has limited durability and is susceptible to relapse after discontinuation. Furthermore, prolonged glucocorticoid administration is accompanied by notable adverse effects, ultimately restricting its overall therapeutic efficacy [5]. Therefore, it is imperative to explore innovative adjuvant therapeutic options for VLS that can provide long-term efficacy.\u003c/p\u003e\u003cp\u003eAs a chronic inflammatory skin disease, the occurrence and progression of VLS have been implicated in autoimmune responses, genetic predisposition, hormonal influences, and local factors [6\u0026ndash;8]. With the development of high-throughput sequencing technology, a growing body of research has highlighted the crucial involvement of skin microbiota in the initiation and progression of many diseases, including VLS [9]. For instance, a clinical study led by Liu et al. found that, compared to healthy controls, the relative abundance of \u003cem\u003elactobacilli\u003c/em\u003e was increased, while that of \u003cem\u003eKelbsiella\u003c/em\u003e, \u003cem\u003eEzakiella\u003c/em\u003e, and \u003cem\u003eEscherichia-Shigella\u003c/em\u003e decreased remarkably, suggesting that specific microbiota species may facilitate VLS [10]. Additionally, Brunner et al. reported that postmenopausal women diagnosed with VLS showed elevated levels of inducible antimicrobial peptides in their vulvar, which typically constitute a defensive response to microbial invasions and suggest augmented microbial activity or alterations in the composition of microbial species among VLS patients [11]. Consequently, targeting the vulvar microbiota could potentially become a promising therapeutic strategy for alleviating VLS.\u003c/p\u003e\u003cp\u003ePostbiotics are defined as a preparation of inanimate microorganisms and/or their components that confer a health benefit on the host [12, 13]. They promote bodily health by balancing microbial populations, suppressing the proliferation and adherence of pathogenic bacteria, and regulating inflammatory responses [14, 15]. For example, Xu et al. demonstrated that postbiotics from \u003cem\u003eLactobacillus paracasei\u003c/em\u003e can inhibit melanogenesis in mouse melanoma cells by activating the PKA/CREB/MITF signaling pathway, thereby contributing to the alleviation of UVB-induced photoaging [16]. Furthermore, Nam et al. indicated that heat-inactivated \u003cem\u003eLactococcus chungangensis\u003c/em\u003e CAU 1447 has the capacity to modulate the expression of cytokines, including interleukin-4 (IL-4), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α), facilitating the healing process of skin wounds in mice [17]. Moreover, research has demonstrated that the lysate of \u003cem\u003eLactobacillus plantarum\u003c/em\u003e K8 strain can enhance the mRNA expression of key moisturizing factors, such as hyaluronic acid synthase 2 and aquaporin 3, thereby being a potential moisturizing product [18]. Given their pivotal role in modulating skin health and mitigating related diseases, postbiotics intervention may be a promising therapeutic approach.\u003c/p\u003e\u003cp\u003eIn this study, we utilized 16S rRNA sequencing to compare vulvar microbiota between healthy women and VLS patients. Vaginal swab sample were cultured and identified as \u003cem\u003eLactobacillus crispatus\u003c/em\u003e NCU-31. The effects of \u003cem\u003eL. crispatus\u003c/em\u003e postbiotics on skin symptoms, signs, Investigator\u0026rsquo;s Global Assessment (IGA), Dermatology Life Quality Index (DLQL), Vulvar Quality of Life Index (VQLI) and skin microbiota were assessed in VLS patients. This study aimed to develop novel therapeutic strategies for VLS.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cb\u003eStudy design and ethical approval\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThis randomized, double-blind trial was conducted at Jiangxi Maternal and Child Health Hospital between September 2024 and December 2024. The inclusion criteria: (1) aged between 18 and 70 years old; (2) healthy women or women with a primary pathological diagnosis of VLS; (3) having signed the informed consent form; and (4) having not undergone any other treatment in the past three months. The exclusion criteria: (1) with acute vaginiti or vulvar infections; (2) who had utilized topical glucocorticoids or antibiotics within the preceding month; (3) with a past medical history of malignancy, radiation/chemotherapy treatment, or systemic skin disorders; and (4) Pregnant or lactating women.\u003c/p\u003e\u003cp\u003e The Ethics Committee of Jiangxi Maternal and Child Health Hospital granted approval to the study (approval number EC-KY-2024-108), ensuring that all procedures adhered to the principles outlined in the Declaration of Helsinki. Additionally, the study has been registered with the China Clinical Trials Registration Center (ChiCTR), bearing the unique registration number ChiCTR2400090750, with the registration date of October 12, 2024. This study was conducted and reported in accordance with the CONSORT 2025 guidelines for randomized controlled trials. All participants in clinical experiment also provided a signed written informed consent.\u003c/p\u003e\u003cp\u003e\u003cb\u003eRandomization and masking\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn the observational experiment, healthy volunteers comprised the healthy control group (C group), while patients diagnosed with VLS were designated to the VLS group (V group). For the intervention experiment, patients with VLS were randomly assigned in a 1:1 ratio to either the glucocorticoids combined with placebo group (VG group) or the glucocorticoids combined with the postbiotics group (VGP group) using the random number table method. Allocation of the random numbers to postbiotics and placebo was performed by a staff member not involved in the study. Subsequently, dedicated medication personnel administered the glucocorticoids combined with either postbiotics or placebo according to the assigned random number. The glucocorticoids, postbiotics, and placebo were identical in packaging, color, and odor, ensuring a double-blind design for both investigators and participants. Notably, the product supplier had no role in the trial\u0026rsquo;s design or conduct and held no financial interest in the study.\u003c/p\u003e\u003cp\u003e\u003cb\u003eBacteria culture and identification\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo study vaginal microbiota in VLS, vaginal swab sample were collected in a liquid medium, and solid media, such as Man Rogosa Sharpe (MRS), brain heart infusion (BHI), and Luria-Bertani (LB), were utilized to screen the vaginal microbiota. A single, smooth, round colony was picked on the solid plate and introduced into 5 ml of liquid medium, incubated in a constant temperature shaker at 37 ℃ and 200 rpm/minute (min) for 24 hours (h) so that the bacteria reached the stable growth stage. Following this, the bacterial liquid was centrifuged at 8,000 rpm for three minutes. After discarding the supernatant, the bacterial DNA was extracted and sent for sequencing to identify the strain. The specific strain information is shown in Table S2. Subsequently, \u003cem\u003eL. crispatus\u003c/em\u003e NCU-31 (CGMCC No.31777) was selected for follow-up experiments. To cultivate this strain, MRS medium enriched with 0.5 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e L-cysteine was utilized. The incubation process occurred in an aerobic atmosphere at 37\u0026deg;C, continuing until the bacterial population reached a concentration of 5\u0026times;10\u003csup\u003e9\u003c/sup\u003e CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The bacterial liquid was centrifuged in a refrigerated high-speed centrifuge at 4\u0026deg;C and 8,000 rpm for 15 min. The bottom sediment was discarded, and the supernatant was obtained; 10\u0026ndash;12 ml of the supernatant was filtered through a 0.22 um sterile filter membrane to remove the bacterial body and other impurities, and the cell-free supernatant of \u003cem\u003eL. crispatus\u003c/em\u003e (CFS) was collected, and the CFS in subsequent experiments was either dispensed on the spot or stored at -40\u0026deg;C for subsequent clinical intervention testing.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEvaluation of probiotic properties\u003c/b\u003e\u003c/p\u003e\u003cp\u003eInitially, \u003cem\u003eL. crispatus\u003c/em\u003e was inoculated into MRS liquid medium at a concentration of 5% and then incubated in an aerobic incubator set at 37\u0026deg;C for 24 hours. Subsequently, the culture was scaled up to 200 ml in a fermentation medium and further incubated under aerobic conditions at 37 C. To establish a growth curve, samples were taken every 2 h, and the OD\u003csub\u003e600\u003c/sub\u003e was measured spectrophotometrically. Additionally, samples were collected every 12 h, and the number of colonies was counted using a spot plate method. For the acid tolerance test, the activated bacteria were centrifuged at 4,500 x g for 10 min at 4\u0026deg;C, and the resulting cell pellet was resuspended in phosphate buffered saline (PBS). The cell suspension was then diluted in PBS at various pH levels (2, 3, 4, and 7) and incubated at 37\u0026deg;C for 4 h.\u003c/p\u003e\u003cp\u003eDuring antioxidant assays, the bacterial cultures were centrifuged at 4,500 x g for 10 min at 4\u0026deg;C. The postbiotics were employed to evaluate their capacity to scavenge 2,2-diphenyl-1-pyridine hydrazine (DPPH), superoxide anions (O\u003csup\u003e2\u0026minus;\u003c/sup\u003e), hydroxyl radicals (-OH), and their ability to chelate Fe\u003csup\u003e2+\u003c/sup\u003e, as well as to determine the reduction potential of the liquid supernatant. For antimicrobial assays, a variety of foodborne pathogenic microorganisms, including \u003cem\u003eSalmonella typhimurium\u003c/em\u003e ATCC 13311, \u003cem\u003eEscherichia coli\u003c/em\u003e O157, and \u003cem\u003eStaphylococcus aureus Cowan\u003c/em\u003e 1 were cultured on LB agar. Oxford cups were placed onto the agar surface, and 250 ul of postbiotics were dispensed into the wells. Subsequently, the diameter of the inhibition zone around the Oxford cups was measured.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIntervention procedure and management\u003c/b\u003e\u003c/p\u003e\u003cp\u003eObservational experiment (Stage 1): Initially, women were enrolled, and those without any vulvar or skin symptoms were considered healthy controls. Concurrently, women who had been diagnostically confirmed with VLS through vulvar skin biopsy were classified as VLS patients. Of these 60 women, 30 healthy women were categorized as the C group, and the other 30 patients with VLS were assigned as the V group. This group's participants were matched in age with those in the comparison group. Routine vaginal discharge wet pap was conducted to rule out bacterial vaginosis. In the outpatient clinic, skin samples were obtained from both the labia majora and negative labia using sterile swabs. Each sample, collected from an area of approximately 3 cm \u0026times; 3 cm, was stored in a 2 ml sterile Eppendorf tube and preserved at \u0026minus;\u0026thinsp;80\u0026deg;C for subsequent high-throughput sequencing analysis.\u003c/p\u003e\u003cp\u003eIntervention experiment (Stage 2): As depicted in Figure S2, 100 VLS patients were randomized equally into the VG and VGP groups (1:1 ratio). Both groups received an 8-week standardized regimen of topical 0.05% clobetasol propionate ointment, administered daily at a standardized dosage of one fingertip unit. The VGP group additionally daily received 100 microliters of cell-free supernatant from \u003cem\u003eL. crispatus\u003c/em\u003e containing 5\u0026times;10\u003csup\u003e9\u003c/sup\u003e CFU as a postbiotic therapy. The VG group received an equivalent volume of physiological saline as a placebo.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eData collection and outcomes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eClinicians gathered comprehensive demographic and clinical data, and evaluated VLS patients using the Cattaneo Clinical Score (0\u0026ndash;3: Non to Severe), a 6-point scale for symptoms and IGA (0\u0026ndash;5: Absent to Severe), DLQI (scale 0\u0026ndash;30) and VQLI (scale 0\u0026ndash;45). IGA assessed erythema/whitening, infiltration, lichenification, and excoriation, while symptoms encompassed pain, and dyspareunia.The biological outcomes encompassed an investigation into variations in the microbiota of the vulvar skin, with a particular focus on differences in species diversity and taxonomic composition.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDNA extraction and 16S rRNA sequencing\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGenomic DNA was extracted from vulvar skin samples (both observation and intervention groups) using a bacterial DNA extraction kit (M5636\u0026ndash;02, Omega, USA). The concentration and purity of the samples were evaluated using spectrophotometry (NanoDrop, Thermo Fisher). The V4 region of 16S rDNA was amplified with primers (5\u0026rsquo;-AYTGGGYDTAAAGNG and 5\u0026rsquo;-TACNVGGGTATCTAATCC\u0026thinsp;\u0026minus;\u0026thinsp;3\u0026rsquo;, respectively) and sequenced on the Illumina HiSeq 2000 platform (Illumina, California). Overlapping reads were merged using FLASH and analyzed with UPARSE (V7.1), discarding low-quality, ambiguous bases, improper primers, and chimeras. High-quality sequences were assigned to operational taxonomic units (OTUs) based on 97% sequence similarity. Taxonomic classification of bacterial abundance was conducted at various levels (phylum, order, family, genus). The alpha-diversity indices, including Chao1, Shannon, Simpson, Pielou_e, and Faith_pd, were calculated using QIIME2 software (2019.4) and compared by the Wilcoxon test. The beta diversity was evaluated based on the Bray-Curtis distance and subsequently analyzed through principal coordinate analysis (PCoA) as well as non-metric multidimensional scaling (NMDS). QIIME2 software was utilized to visualize the taxonomic composition at the phylum and genus levels, while the Mann\u0026ndash;Whitney U test was employed to assess differences in species abundance among groups.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSample size\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn our observational experiment, our study group undertook a pre-trial evaluation to assess the severity of VLS among patients. The results indicated a symptom score of 2.88\u0026thinsp;\u0026plusmn;\u0026thinsp;1.95 for healthy volunteers and 5.13\u0026thinsp;\u0026plusmn;\u0026thinsp;2.39 for those diagnosed with VLS. Based on these findings, a sample size calculation was conducted, revealing that approximately 17 participants per group would be required (power: 80%; α: 5%). Nevertheless, given the typical sample sizes employed in VLS research and their potential influence on the accuracy of high-throughput sequencing outcomes, we decided to augment the initial sample size to 60 participants, with 30 in each group.\u003c/p\u003e\u003cp\u003eSince there is no authoritative literature analyzing the efficacy of post-operative \u003cem\u003eL. crispatus\u003c/em\u003e probiotics in patients with VLS, a reasonable power analysis could not be performed. Based on the pre-test analysis, assuming a mean post-treatment symptom score of 4.16 in the VG group and a mean post-treatment symptom score of 3.24 in the VGP group, with a standard deviation of 1.84 in both groups, 63 subjects would need to be recruited for the study in each group (power: 80%; α: 5%). Taking into consideration potential attrition, dropout rates, and any unforeseen events, we adjusted the initial sample size upwards by approximately 10%, thereby setting a target enrollment of 70 participants for each group.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eContinuous variables were reported using the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation format, and the Kolmogorov\u0026ndash;Smirnov test was utilized to evaluate the normality of the data. For normally distributed data with equal variances, the Student t-test was applied, while for normally distributed data with unequal variances, an adjusted t-test was used. For non-normally distributed data, the Mann\u0026ndash;Whitney U test was employed for analysis. Categorical variables, presented as counts and percentages, were analyzed using the Pearson chi-square test, the continuity-corrected chi-square test, or the Fisher exact test, depending on the circumstances. All statistical analyses and data visualizations were conducted using Prism version 9.5.1 software (GraphPad Software, San Diego, CA, USA), QIIME2 software, R version 4.2.3 software, PASS 15.0, and SPSS version 26. The threshold for statistical significance was set at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eDemographic and baseline features of the participants in the observational study.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe baseline characteristics, Cattaneo Clinical Score, IGA, DLQL and VQLI of participants are summarized in Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e and Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. No statistically significant differences were observed between the C and V groups in age, body weight, or BMI (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05; Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e). Notably, the demographic and clinical profiles of the two groups were generally consistent, except for pruritus degree, skin elasticity, skin color, lesion scope, IGA, DLQL and VQLI (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e and Figs. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB\u0026ndash;F).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAlterations in the microbial diversity and composition of the vulvar skin among patients with VLS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo characterize the microbiota of the vulvar skin in patients with VLS, we gathered vulvar skin samples from 29 healthy volunteers and 30 VLS patients for 16s rRNA sequencing. To determine differences in microbial diversity between the two groups, we analyzed the alpha-diversity. While no significant difference was observed in the Chao1 index (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.57) between the two groups (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA), the Shannon index (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.014), which measures species richness and evenness, the Simpson index (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0036), and the Pielou_e (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0033) were remarkably higher in VLS patients than in healthy individuals (Figs. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB\u0026ndash;D). According to the results of the Venn diagram, 502 and 517 otu were found in the vulvar skin of the C and V groups, respectively, of which 350 otu were present in the vulvar skin of these two groups (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE). We then assessed beta-diversity to compare the microbial community composition between the two groups of patients, and the findings indicated an independent aggregation of the various groups (Figs. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eF and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eG). In addition, LEfSe analysis revealed that distinct levels of vulvar microbiota were characterized by differences. Figures \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eH and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eI show 30 differential microorganisms of various taxonomic categories and taxa. At the genus level, the C group was mainly enriched with \u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003eMogibacterium\u003c/em\u003e, \u003cem\u003eMegasphaera\u003c/em\u003e, \u003cem\u003ePhascolarctobacterium\u003c/em\u003e, and \u003cem\u003eNesterenkonia\u003c/em\u003e, the V group was characterized by \u003cem\u003ePrevotella\u003c/em\u003e, \u003cem\u003eStreptococcus\u003c/em\u003e, \u003cem\u003eBacillus\u003c/em\u003e, and \u003cem\u003eAcinetobacter\u003c/em\u003e as characteristic genera. Collectively, these differential taxa may serve as target microorganisms for VLS therapy.\u003c/p\u003e\n\u003cp\u003eAt the phylum level, as shown in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA, Firmicutes, Bacteroidetes, and Actinobacteria emerged as the dominant microbiota, which accounted for 96.12% and 91.27% in the C and V groups, respectively. Among them, the C group (C vs. V\u0026thinsp;=\u0026thinsp;57.01% vs. 60.73%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) exhibited a significantly lower relative abundance of Firmicutes (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB) compared to the V group, whereas the abundance of Bacteroidetes (Figure. 3C) was remarkably higher in C group (C vs. V\u0026thinsp;=\u0026thinsp;29.27% vs. 11.39%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) than in V group. Furthermore, Actinobacteria (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD) were less abundant in the C group (9.84% vs. 19.15%) than in the V group. We then further analyzed the microbial composition at the genus level (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eE) and discovered that the C group had a significantly higher relative abundance of \u003cem\u003eLactobacillus\u003c/em\u003e compared to the V group (C vs. V\u0026thinsp;=\u0026thinsp;55.97% vs. 18.72%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), suggesting a reduction in the beneficial vulvar skin microbiota among patients with VLS (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eF). However, the presence of conditionally pathogenic bacteria, including \u003cem\u003ePrevotella\u003c/em\u003e (C vs. V\u0026thinsp;=\u0026thinsp;10.32% vs. 21.85%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), \u003cem\u003eGardnerella\u003c/em\u003e (C vs. V\u0026thinsp;=\u0026thinsp;2.84% vs. 10.29%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), \u003cem\u003eDialister\u003c/em\u003e (C vs. V\u0026thinsp;=\u0026thinsp;2.85% vs. 6.77%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and \u003cem\u003eStreptococcus\u003c/em\u003e (C vs. V\u0026thinsp;=\u0026thinsp;0.37% vs. 9.22%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), was significantly increased in patients with VLS (Figs. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eG\u0026ndash;J). Notably, \u003cem\u003eFinegoldia\u003c/em\u003e, \u003cem\u003ePeptoniphius\u003c/em\u003e, and \u003cem\u003eFusobacterium\u003c/em\u003e were not remarkably distinct between the two groups. However, the V group exhibited a slightly higher relative abundance of these microbiota compared to the C group (Figs. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eK\u0026ndash;M). Taken together, the above results revealed that the occurrence of VLS may be associated with a reduction in beneficial vulvar microbiota and a rise in pathogenic microbial populations, suggesting compromised integrity of the vulvar microbial barrier and increased vulnerability of the microenvironment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation of vaginal secretions microbiota and evaluation of the probiotic properties of\u003c/strong\u003e \u003cstrong\u003eL. crispatus\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrevious results have indicated that patients with VLS are susceptible to vulvar microbiota dysbiosis. To further isolate specific VLS-related bacteria, we performed bacterial culture and identification, and a total of 183 bacterial isolates were obtained, including 123 strains of \u003cem\u003eLactobacillus\u003c/em\u003e, 28 strains of \u003cem\u003eGardnerella\u003c/em\u003e, 19 strains of \u003cem\u003eStreptococcus\u003c/em\u003e, 6 strains of \u003cem\u003eStaphylococcus\u003c/em\u003e, 4 strains of \u003cem\u003eEscherichia coli\u003c/em\u003e, 2 strains of \u003cem\u003eLigilactobacillus salivarius\u003c/em\u003e, and 1 strain of \u003cem\u003eCorynebacterium callunae\u003c/em\u003e, as shown in Table S2. \u003cem\u003eLactobacillus\u003c/em\u003e dominates the vaginal microbiota of healthy women, and the abundance of \u003cem\u003eLactobacillus\u003c/em\u003e was significantly reduced in the vulvar skin microbiota of VLS patients. Therefore, we hypothesized that the occurrence of VLS might be associated with the decreased abundance of \u003cem\u003eLactobacillus\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003ePlate coating and Gram staining were conducted to observe the morphology of \u003cem\u003eLactobacillus\u003c/em\u003e, revealing an elongated, curved, and rod-shaped morphology consistent with previous literature reports (Figures \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003eA and B). Subsequently, we performed phylogenetic tree construction to clarify the specific branching of \u003cem\u003eLactobacillus\u003c/em\u003e; more than 50% of bootstrap values for 1000 replicates are shown in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eF. The \u003cem\u003eL. crispatus\u003c/em\u003e strain of interest was on branch with \u003cem\u003eL. crispatus\u003c/em\u003e TMPC3192H (OM760749.1) with 99% sequence similarity and 50.3% confidence. Combined with the previous morphology of \u003cem\u003eL. crispatus\u003c/em\u003e and the phylogenetic strain \u003cem\u003eL. crispatus\u003c/em\u003e TMPC3192H was determined to be \u003cem\u003eL. crispatus\u003c/em\u003e NCU-31 (Figure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003eF).\u003c/p\u003e\n\u003cp\u003eThen, we performed a comprehensive \u003cem\u003ein vitro\u003c/em\u003e evaluation to assess the probiotic efficacy of \u003cem\u003eL. crispatus\u003c/em\u003e, including growth characteristics, acid tolerance, antioxidant capacity, drug resistance, and inhibition of common pathogenic bacteria. Our results suggested that \u003cem\u003eL. crispatus\u003c/em\u003e demonstrated vigorous growth under aerobic conditions at 37\u0026deg;C, entering a rapid growth phase after eight hours and ultimately a stable phase at 24 h with an OD\u003csub\u003e600\u003c/sub\u003e nm of 2.08 (Figure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003eC). In the acid tolerance assay, the viable count of \u003cem\u003eL. crispatus\u003c/em\u003e gradually increased with rising pH (2.0, 3.0, 4.0, and 7.0) and was highest at pH 7, reaching 1.7\u0026times;10\u003csup\u003e10\u003c/sup\u003e CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Figure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003eD). Moreover, the results of the antioxidant capacity evaluation of \u003cem\u003eL. crispatus\u003c/em\u003e exhibited its ability to scavenge DPPH and hydroxyl radicals (-OH) by 89.86% and 82.84%, respectively. It also inhibited superoxide radicals (O\u003csup\u003e2\u0026minus;\u003c/sup\u003e) by 39.99% and chelate ferrous ions (Fe\u003csup\u003e2+\u003c/sup\u003e) by 62.36% (Figure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003eE). We also found that \u003cem\u003eL. crispatus\u003c/em\u003e was significantly resistant to common antibiotics, with the most pronounced resistance to cotrimoxazole and ciprofloxacin (Figure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003eF). Additionally, we also assessed the bacteriostatic properties of \u003cem\u003eL. crispatus\u003c/em\u003e postbiotics. We discovered that they exhibited notable inhibitory activity against \u003cem\u003eE. coli\u003c/em\u003e O157, \u003cem\u003eS. typhimurium\u003c/em\u003e ATCC 13311, and \u003cem\u003eS. Cowan\u003c/em\u003e 1, with inhibition circles size of 11.73 mm, 9.30 mm, and 10.63 mm, respectively (Figure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003eG). Overall, these data underscore the excellent probiotic properties of \u003cem\u003eL. crispatus\u003c/em\u003e, encompassing its vigorous growth, acid resistance, antioxidant potential, robust drug tolerance, and significant bacteriostatic activity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBaseline characteristics and the influence of\u003c/strong\u003e \u003cstrong\u003eL. crispatus\u003c/strong\u003e \u003cstrong\u003eon vulvar skin symptoms and signs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the alleviating effect of \u003cem\u003eL. crispatus\u003c/em\u003e postbiotics on vulvar skin symptoms in patients with VLS, we enrolled 140 new volunteers in an intervention study (Stage 2). As depicted in Figure S2, following dermatological diagnosis, 100 patients diagnosed with VLS were equally assigned to the VG and VGP groups in a 50:50 ratio for the intervention study. The remaining 40 patients were excluded from the study: 28 declined participation, and 12 fulfilled the exclusion criteria.\u003c/p\u003e\n\u003cp\u003eTable \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e presents baseline demographics, Cattaneo clinical scores, IGA, DLQL and VQLI of intervention experiment. No significant differences in age, weight, BMI, or clinical profiles were observed between the VG and VGP groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). After an eight-week intervention, patients in the VGP group exhibited significantly improved Cattaneo clinical scores, IGA, DLQL and VQLI compared to the VG group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). These results illustrate that \u003cem\u003eL. crispatus\u003c/em\u003e postbiotic can potentially improve vulvar skin symptoms in VLS.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003e\u0026ldquo;Baseline characteristics of participants in the intervention experiment\u0026rdquo;\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCharacteristics\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eVG group (n\u0026thinsp;=\u0026thinsp;50)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eVGP group (n\u0026thinsp;=\u0026thinsp;50)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003et/Z/ X2\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e-Value\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAges (years)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e44.38\u0026thinsp;\u0026plusmn;\u0026thinsp;11.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40.74\u0026thinsp;\u0026plusmn;\u0026thinsp;10.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026minus; 1.708\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.088\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWeight (kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e55.61\u0026thinsp;\u0026plusmn;\u0026thinsp;4.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e55.12\u0026thinsp;\u0026plusmn;\u0026thinsp;3.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026minus; 0.576\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.565\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBMI (kg/m2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20.88\u0026thinsp;\u0026plusmn;\u0026thinsp;1.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20.63\u0026thinsp;\u0026plusmn;\u0026thinsp;1.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026minus; 0.476\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.634\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVaginal PH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026minus; 0.236\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.814\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHPV infection, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5(10.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3 (6.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.543\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.461\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHypertension, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (10.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2 (4.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.382\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.240\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDiabetes, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2 (4.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 (2.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.344\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.558\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCurrently smoking, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11 (22.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8 (16.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.585\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.444\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eConsumed alcohol, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9 (18.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 (10.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.329\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.249\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEver pregnant, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e43 (86.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e39 (78.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.084\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.298\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSkin color\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026minus; 3.046\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.002\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSkin elasticity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026minus; 3.758\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePruritus degree\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.04\u0026thinsp;\u0026plusmn;\u0026thinsp;1.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026minus; 2.713\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.007\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLesion scope\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026minus; 3.384\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDyspareunia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026minus; 0.583\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.560\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIGA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026minus; 7.194\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDLQI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.86\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026minus; 9.011\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVQLI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.36\u0026thinsp;\u0026plusmn;\u0026thinsp;1.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026minus; 8.756\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003eVG: Vulvar lichen sclerosus patients with glucocorticoid and placebo; VGP: Vulvar lichen sclerosus patients with glucocorticoid and \u003cem\u003eL. crispatus\u003c/em\u003e postbiotics; BMI: Body Mass Index; HPV: Human papillomavirus; IGA:Investigator\u0026rsquo;s Global Assessment; DLQL:Dermatology Life Quality Index; VQLI:Vulvar Quality of Life Index\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eL. crispatus\u003c/strong\u003e \u003cstrong\u003epostbiotic enriches and enhances the compromised vulvar skin microbiota in VLS patients\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the impact of \u003cem\u003eL. crispatus\u003c/em\u003e postbiotic on the vulvar skin microbiota in patients with VLS, vulvar skin samples were analyzed by 16SrRNA-sequencing. Figures \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA\u0026ndash;E shows the alterations in alpha-diversity, where the observed species Chao1 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.7\u003csup\u003ee\u0026thinsp;\u0026minus;\u0026thinsp;13\u003c/sup\u003e), Shannon (p\u0026thinsp;=\u0026thinsp;4e\u003csup\u003e\u0026minus;\u0026thinsp;08\u003c/sup\u003e), Simpson (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.1e\u003csup\u003e\u0026minus;\u0026thinsp;06\u003c/sup\u003e), Pielou_e (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.2e\u003csup\u003e\u0026minus;\u0026thinsp;05\u003c/sup\u003e), and Faith_pd (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.4e\u003csup\u003e\u0026minus;\u0026thinsp;05\u003c/sup\u003e) indices were remarkably decreased in the VGP group, suggesting a reduction in the abundance and diversity of the vulvar skin microbiota. Similar results were observed in Venn, where \u003cem\u003eL. crispatus\u003c/em\u003e postbiotic intervention alleviated VLS-induced asv(amplicon aequence variant) increase to some extent (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eF). In addition, the beta diversity was assessed by PCoA and NMDS, which showed some clustering of taxa, with a separation of taxa in the VG and VGP groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Figs. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eG and \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eH). LEfSe analysis further emphasized the significant difference in the vulvar skin microbiota composition between the VG and VGP groups (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eI). This reveals that \u003cem\u003eL. crispatus\u003c/em\u003e postbiotic could remodel the disturbed vulvar skin microbiota.\u003c/p\u003e\n\u003cp\u003eFurthermore, we analyzed the community composition of microbiota in the vulvar skin of different groups at the phylum and genus levels. We found that Firmicutes (VG vs. VGP\u0026thinsp;=\u0026thinsp;55.19% vs. 56.78%), Bacteroidetes (VG vs. VGP\u0026thinsp;=\u0026thinsp;18.71% vs. 22.01%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), Actinobacteria (VG vs. VGP\u0026thinsp;=\u0026thinsp;19.38% vs. 11.64%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and Proteobacteria (VG vs. VGP\u0026thinsp;=\u0026thinsp;3.89% vs. 7.18%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) were the dominant phyla in the vulvar skin of patients with VLS, accounting for 97.17% and 97.61% of the VG and VGP groups, respectively (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA). Following treatment with \u003cem\u003eL. crispatus\u003c/em\u003e postbiotic, the VGP group exhibited a significantly higher relative abundance of Bacteroidetes compared to the VG group, indicating a reversal of the decrease in Bacteroidetes previously observed in the observational study among VLS patients (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB). Additionally, Actinobacteria and Fusobacteria phyla were significantly down-regulated after the co-administration of glucocorticoid with \u003cem\u003eL. crispatus\u003c/em\u003e postbiotic therapy, indicating that the \u003cem\u003eL. crispatus\u003c/em\u003e postbiotic intervention restored some of the microecological dysregulation in vulvar skin (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Figs. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC and D).\u003c/p\u003e\n\u003cp\u003eAt the genus level, topical application of \u003cem\u003eL. crispatus\u003c/em\u003e postbiotic remarkably altered the microbiota composition of vulvar skin compared to the VG group (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eE). In addition, after administration of the \u003cem\u003eL. crispatus\u003c/em\u003e postbiotic, the VGP group showed a significantly higher relative abundance of \u003cem\u003eLactobacillus\u003c/em\u003e (16.45% in VG vs. 35.92% in VGP, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and \u003cem\u003eBifidobacterium\u003c/em\u003e (0.63% in VG vs. 2.59% in VGP, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) compared to the VG group (Figs. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eF and N). However, no significant difference was observed in the relative abundance of \u003cem\u003ePrevotella\u003c/em\u003e between the two groups (16.13% in VG vs. 18.9% in VGP, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) following the \u003cem\u003eL. crispatus\u003c/em\u003e postbiotic supplementation (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eG). Moreover, the co-administration of therapy led to a reduction in the relative abundance of pathogenic bacteria, including \u003cem\u003eStreptococcus\u003c/em\u003e, \u003cem\u003eCorynebacterium\u003c/em\u003e, \u003cem\u003eFinegoldia\u003c/em\u003e, \u003cem\u003eStaphylococcaceae_Staphylococcus\u003c/em\u003e, \u003cem\u003eDialister\u003c/em\u003e, and \u003cem\u003eGardnerella\u003c/em\u003e (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Figs. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eH\u0026ndash;M). Furthermore, the rank abundance curve data revealed that topical \u003cem\u003eL. crispatus\u003c/em\u003e postbiotic increased the OUT of the VGP group compared to the VG group, and the heat map results were consistent with the previous findings (Figs. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eO and P). Collectively, these results indicate that an eight-week treatment with \u003cem\u003eL. crispatus\u003c/em\u003e postbiotic may ameliorate VLS-induced vulvar microecological dysbiosis, thereby restoring the diversity of the vulvar microbiota.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrelation analysis between vulvar skin microbiota, vulvar skin symptoms, signs, and other clinical indicators in patients with VLS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe employed Spearman\u0026apos;s rank correlation coefficient for correlation analysis to explore the complex interplay between vulvar skin microbiota, vulvar skin symptoms, and various clinical indicators. The results showed that \u003cem\u003eStreptococcus\u003c/em\u003e, \u003cem\u003eCorynebacterium\u003c/em\u003e, \u003cem\u003eFinegolida\u003c/em\u003e, \u003cem\u003eDialister\u003c/em\u003e and \u003cem\u003eGardnerella\u003c/em\u003e were positively correlated with pruritus degree, lesion scope, IGA, DLQL and VQLI respectively, in patients with VLS, suggesting that these bacteria could potentially serve as target genera for treating VLS. Furthermore, \u003cem\u003eLactobacillus\u003c/em\u003e was significantly negatively correlated with \u003cem\u003ePrevotella\u003c/em\u003e, \u003cem\u003eStreptococcus\u003c/em\u003e, \u003cem\u003eCorynebacterium\u003c/em\u003e, \u003cem\u003eFinegolida\u003c/em\u003e, and \u003cem\u003eDialister\u003c/em\u003e, while the latter was remarkably positively correlated with \u003cem\u003ePrevotella\u003c/em\u003e and \u003cem\u003eFinegolida\u003c/em\u003e (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Taken together, these findings indicate a strong link between changes in the vulvar skin microbiota due to VLS and alterations in vulvar skin symptoms, potentially exacerbating the symptoms in patients with VLS.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eVLS is a chronic inflammatory skin disease that profoundly impacts the physical and mental health of patients [19]. In recent years, numerous studies have increasingly suggested that the vulvar skin microbiota plays a significant role in the initiation and progression of VLS [20, 21]. However, current research has largely focused on identifying and classifying vulvar microorganisms, with relatively few studies exploring the role of microorganisms and their derivatives in treating VLS [22].\u003c/p\u003e\u003cp\u003eTherefore, we first compared the differences in the vulvar microbiota between healthy women and patients with VLS using high-throughput sequencing. The results revealed that VLS patients exhibited higher microbial diversity and richness in their vulvar skin microbiota compared to healthy individuals. The abundance of \u003cem\u003eLactobacillus\u003c/em\u003e decreased, while the relative abundance of \u003cem\u003ePrevotella\u003c/em\u003e, \u003cem\u003eGardnerella\u003c/em\u003e, \u003cem\u003eDialister\u003c/em\u003e, and \u003cem\u003eStreptococcus\u003c/em\u003e increased. These findings suggest that restoring the balance of the vulvar skin microbiota may be a promising strategy for improving VLS.\u003c/p\u003e\u003cp\u003ePrevious findings have shown a significant decrease in \u003cem\u003eLactobacillus\u003c/em\u003e abundance in patients with VLS, and we aimed to investigate whether the development of VLS is associated with \u003cem\u003eLactobacillus\u003c/em\u003e. We conducted microbial screening cultures on vaginal swab samples from healthy volunteers and VLS patients and obtained 123 strains of \u003cem\u003elactobacilli\u003c/em\u003e. Based on the results of the Gram staining and phylogenetic tree, we ultimately identified them as \u003cem\u003eL. crispatus\u003c/em\u003e. Subsequently, we performed an \u003cem\u003ein vitro\u003c/em\u003e probiotic assessment, which showed that \u003cem\u003eL. crispatus\u003c/em\u003e possessed favorable probiotic properties, including strong growth capacity, acid resistance, antioxidant capacity, drug resistance, and the capacity to suppress pathogenic bacteria growth. Research has indicated that \u003cem\u003eLactobacillus spp\u003c/em\u003e.-dominated microbiota has been recognized as a hallmark of female reproductive tract health [23\u0026ndash;25]. In a longitudinal cohort study, the predominant presence of \u003cem\u003eL. crispatus\u003c/em\u003e in the vagina microbiota was linked to a lower risk of HIV infection compared to the bacterial vaginosis microbiota [26]. Women susceptible to \u003cem\u003eL-type crispatus\u003c/em\u003e were also less likely to convert to a bacterial vaginosis microbiota than those with L-type dominance, suggesting that \u003cem\u003eL-type crispatus\u003c/em\u003e competes to some extent to exclude bacteria associated with inflammatory vaginitis through the production of lactic acid and anti-microbial metabolites [27, 28]. In vitro modeling suggests that \u003cem\u003eL. crispatus\u003c/em\u003e is a major producer of lactic acid in the female genital tract, and that lactic acid may directly inhibit the production of pro-inflammatory cytokines [29, 30]. Furthermore, the incorporation of supernatants enriched with either \u003cem\u003eL. crispatus\u003c/em\u003e alone or in combination with other components demonstrated a capacity to decrease the production of proinflammatory cytokines by bacteria associated with bacterial vaginitis and protected the epithelial barrier from disruption in response to proinflammatory stimuli [31]. These findings imply that \u003cem\u003eLactobacillus\u003c/em\u003e and its derivatives may exert their probiotic effects by alleviating vaginal inflammation, modulating the body's immune response, and promoting vaginal ecological balance.\u003c/p\u003e\u003cp\u003eGiven the pivotal role of \u003cem\u003eL. crispatus\u003c/em\u003e and its derivatives in maintaining human health, we elected to utilize \u003cem\u003eL. crispatus\u003c/em\u003e postbiotics in our intervention trial. Our findings revealed that the co-administration of glucocorticoid with \u003cem\u003eL. crispatus\u003c/em\u003e postbiotic therapy markedly enhanced skin symptoms, signs, IGA, DLQL and VQLI among patients with VLS, in line with the beneficial effects of \u003cem\u003eL. crispatus\u003c/em\u003e postbiotics observed in other studies [32]. Song et al. discovered that peptidoglycan isolated from vaginal \u003cem\u003eL. crispatus\u003c/em\u003e stimulated the expression of CD207 on Langerhans cells and significantly downregulated the expression of HIV entry receptors [33]. Donnarumma et al. demonstrated that \u003cem\u003eL. crispatus\u003c/em\u003e L1 exopolysaccharides possessed the ability to robustly augment the production of human defensin-2 protein by vaginal epithelial VK2 cells while reducing \u003cem\u003eCandida albicans\u003c/em\u003e adherence through competitive exclusion mechanisms (48%) [34]. Furthermore, the combination therapy of \u003cem\u003eL. crispatus\u003c/em\u003e postbiotics with glucocorticoids was found to reduce alpha-diversity and alter the microbial composition of the skin microbiota. In terms of beta-diversity, a slight separation of sample clusters was observed, indicating that the application of glucocorticoids in this study had a slight impact on the patient cohort's skin microbiota diversity. These data indicate that combined therapy may shift the skin microbiota to a healthier state, potentially improving VLS management.\u003c/p\u003e\u003cp\u003eWe conducted an in-depth analysis of the skin microbiota composition and observed significant alterations in the abundance of taxonomic groups at the phylum and genus levels after combining glucocorticoid treatment with \u003cem\u003eL. crispatus\u003c/em\u003e postbiotic therapy. Specifically, the combined therapy resulted in a higher relative abundance of Bacteroidetes at the phylum level and decreased Actinobacteria and Fusobacteria compared to glucocorticoids alone. Actinobacteria phylum includes pathogenic bacteria, such as \u003cem\u003eSalmonella\u003c/em\u003e and \u003cem\u003eVibrio cholerae\u003c/em\u003e, which are crucial in the development and progression of several diseases [35\u0026ndash;37]. Conversely, Bacteroidetes is a major component of the human vaginal microbiota, which contains several probiotics [38, 39]. It has been discovered that Bacteroidetes, through their metabolism, produce substances that help the vagina get rid of foreign matter and harmful bacteria, thus maintaining a clean and healthy vagina [40]. This indicates that Bacteroidetes could be key in re-establishing the vaginal microecological balance. Furthermore, at the genus level, the combined therapy led to an increase in the relative abundance of \u003cem\u003eLactobacillus\u003c/em\u003e, while the abundance of \u003cem\u003eStreptococcus\u003c/em\u003e, \u003cem\u003eCorynebacterium, Finegoldia\u003c/em\u003e, \u003cem\u003eStaphylococcaceae_Staphylococcus\u003c/em\u003e, \u003cem\u003eDialister\u003c/em\u003e, and \u003cem\u003eGardnerella\u003c/em\u003e was reduced. Lee et al. found that the vaginal microbiota of HPV-infected women exhibited increased \u003cem\u003eDialister\u003c/em\u003e levels and decreased \u003cem\u003eLactobacillus\u003c/em\u003e levels compared to healthy women [41]. Additionally, patients with genital wart infections had dysbiosis of the vaginal microbiota, which featured a reduction in \u003cem\u003eLactobacillus\u003c/em\u003e abundance and an elevation in the proportion of \u003cem\u003eGardnerella\u003c/em\u003e [42]. These results indicated that an \u003cem\u003eL. crispatus\u003c/em\u003e postbiotic may alleviate VLS by restoring the balance of vulvar skin microbiota.\u003c/p\u003e\u003cp\u003eIn this study, we evaluated the efficacy of the co-administration of glucocorticoid with \u003cem\u003eL. crispatus\u003c/em\u003e postbiotic therapy in patients with VLS. The findings demonstrated that the co-administration of therapy significantly improved skin symptoms, signs, IGA, DLQL and VQLI in VLS patients without serious adverse effects. Furthermore, \u003cem\u003eL. crispatus\u003c/em\u003e postbiotics were found to effectively restore the vulvar skin microbiota. These results indicate that the topical application of \u003cem\u003eL. crispatus\u003c/em\u003e postbiotic could potentially serve as an efficacious and targeted therapy for VLS patients. However, this study also has limitations, such as a short observation period; therefore, the long-term therapeutic effect of \u003cem\u003eL. crispatus\u003c/em\u003e postbiotic needs to be further assessed. In addition, the difficulty of recruiting a large sample makes the potential heterogeneity of participants difficult to control, thus hindering the current clinical study. Further comprehensive studies are required to validate these findings. Lastly, while this study examined alterations in skin symptoms, signs, and skin microbiota among VLS patients, it did not delve into potential causal links between these changes due to the absence of metabolomic analysis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe gratefully acknowledge the approval of this study by the Ethics Committee. We also thank the Jiangxi Province Key Laboratory of Bioengineering Drugs, the General Science and Technology Program of the Jiangxi Provincial Health Commission, and the National Natural Science Foundation of China for their valuable support of this research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTingtao chen and Qi Chen: Writing \u0026ndash; original draft, Supervision, Methodology, Investigation, Formal analysis,Data curation, Resources. Hong Liao and Qifa Huang: Writing\u0026ndash;review \u0026amp; editing,Methodology. Ying Jiang and Fen Wei: Writing \u0026ndash; review \u0026amp; editing, Validation, Formal analysis. Xiaoyan Ai and Hailian Luo: Writing \u0026ndash; review \u0026amp; editing, Project administration. Xue Wu and Leiping Ding: Writing \u0026ndash; review \u0026amp; editing, Project administration, Supervision, Conceptualization, Resources.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the National Natural Science Foundation of China (82460528 and 82260507 to Q.C,82460297 and 82260389 to T.C), Jiangxi Province Key Laboratory of bioengineering drugs (No.2024SSY07061 to T.C), and Jiangxi Provincial Health Commission\u0026apos;s General Science and Technology Plan (No.202310937 to H.L).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors affirm that the entirety of the data instrumental in substantiating the findings of this study are readily accessible and intricately detailed within the confines of the article itself. The raw sequence data have been submitted to the GenBank database and are accessible under the accession identifier PRJNA1188795. The dataset can be accessed via the following link: The raw sequence data have been submitted to the GenBank database and are accessible under the accession identifier PRJNA1188795. The dataset can be accessed via the following link: https://dataview.ncbi.nlm.nih.gov/object/PRJNA1188795. The data will be released to the public on December 31, 2025.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Ethics Committee of Jiangxi Maternal and Child Health Hospital granted approval to the study (approval number EC-KY-2024-108), ensuring that all procedures adhered to the principles outlined in the Declaration of Helsinki. Additionally, the study has been registered with the China Clinical Trials Registration Center (ChiCTR), bearing the unique registration number ChiCTR2400090750, with the registration date of October 12, 2024. This study was conducted and reported in accordance with the CONSORT 2025 guidelines for randomized controlled trials. All participants in clinical experiment also provided a signed written informed consent.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFergus KB, Lee AW, Baradaran N, Cohen AJ, Stohr BA, Erickson BA, et al. Pathophysiology, Clinical Manifestations, and Treatment of Lichen Sclerosus: A Systematic Review. 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Beyond bacterial vaginosis: vaginal lactobacilli and HIV risk. Microbiome. 2021;9(1):239; doi: 10.1186/s40168-021-01183-x.\u003c/li\u003e\n\u003cli\u003eWitkin SS, Mendes-Soares H, Linhares IM, Jayaram A, Ledger WJ, Forney LJ. Influence of vaginal bacteria and D- and L-lactic acid isomers on vaginal extracellular matrix metalloproteinase inducer: implications for protection against upper genital tract infections. mBio. 2013;4(4); doi: 10.1128/mBio.00460-13.\u003c/li\u003e\n\u003cli\u003ePendharkar S, Skafte-Holm A, Simsek G, Haahr T. Lactobacilli and Their Probiotic Effects in the Vagina of Reproductive Age Women. Microorganisms. 2023;11(3); doi: 10.3390/microorganisms11030636.\u003c/li\u003e\n\u003cli\u003eTortelli BA, Lewis WG, Allsworth JE, Member-Meneh N, Foster LR, Reno HE, et al. Associations between the vaginal microbiome and Candida colonization in women of reproductive age. 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Bacteroides fragilis alleviates necrotizing enterocolitis through restoring bile acid metabolism balance using bile salt hydrolase and inhibiting FXR-NLRP3 signaling pathway. Gut Microbes. 2024;16(1):2379566; doi: 10.1080/19490976.2024.2379566.\u003c/li\u003e\n\u003cli\u003eZha X, Liu X, Wei M, Huang H, Cao J, Liu S, et al. Microbiota-derived lysophosphatidylcholine alleviates Alzheimer's disease pathology via suppressing ferroptosis. Cell Metab. 2024; doi: 10.1016/j.cmet.2024.10.006.\u003c/li\u003e\n\u003cli\u003eGreenbaum S, Greenbaum G, Moran-Gilad J, Weintraub AY. Ecological dynamics of the vaginal microbiome in relation to health and disease. Am J Obstet Gynecol. 2019;220(4):324 − 35; doi: 10.1016/j.ajog.2018.11.1089.\u003c/li\u003e\n\u003cli\u003eLee JE, Lee S, Lee H, Song YM, Lee K, Han MJ, et al. Association of the vaginal microbiota with human papillomavirus infection in a Korean twin cohort. PLoS One. 2013;8(5):e63514; doi: 10.1371/journal.pone.0063514.\u003c/li\u003e\n\u003cli\u003eZhou Y, Wang L, Pei F, Ji M, Zhang F, Sun Y, et al. Patients With LR-HPV Infection Have a Distinct Vaginal Microbiota in Comparison With Healthy Controls. Front Cell Infect Microbiol. 2019;9:294; doi: 10.3389/fcimb.2019.00294.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"vulvar lichen sclerosus, Lactobacillus crispatus, postbiotic, 16S rRNA gene sequencing","lastPublishedDoi":"10.21203/rs.3.rs-7092244/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7092244/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eVulvar lichen sclerosus (VLS) is a chronic inflammatory skin disorder that severely impairs women's physical and psychological well-being. Topical glucocorticoids are the first-line treatment; however, their long-term efficacy is limited due to frequent symptom relapse after discontinuation and incomplete resolution of lesions. Therefore, effective adjunctive strategies are urgently needed to achieve sustained disease control.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eIn this study, we explored the vulvar skin microbiota composition in VLS patients using 16S rRNA gene sequencing and evaluated the therapeutic potential of a postbiotic derived from \u003cem\u003eLactobacillus crispatus\u003c/em\u003e NCU-31. \u003cem\u003eL. crispatus\u003c/em\u003e NCU-31 was isolated and its probiotic properties were confirmed \u003cem\u003ein vitro\u003c/em\u003e. A postbiotic formulation was then developed and applied in combination with topical glucocorticoids in VLS patients. Clinical efficacy was assessed using the Investigator\u0026rsquo;s Global Assessment (IGA), Dermatology Life Quality Index (DLQI), and Vulvar Quality of Life Index (VQLI), alongside microbial profiling of the vulvar skin.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eVLS patients exhibited significantly increased microbial richness and diversity, characterized by reduced \u003cem\u003eLactobacillus\u003c/em\u003e abundance and elevated levels of \u003cem\u003ePrevotella\u003c/em\u003e, \u003cem\u003eGardnerella\u003c/em\u003e, \u003cem\u003eDialister\u003c/em\u003e, and \u003cem\u003eStreptococcus\u003c/em\u003e (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Combined postbiotic and glucocorticoid treatment led to significant clinical improvement, evidenced by decreased IGA scores and improved DLQI and VQLI (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Moreover, microbial dysbiosis was partially reversed, with an increase in \u003cem\u003eLactobacillus\u003c/em\u003e and reduction of pathogenic genera.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eThis study demonstrates that \u003cem\u003eL. crispatus\u003c/em\u003e-derived postbiotics can enhance the efficacy of glucocorticoid therapy, alleviate clinical symptoms, and help restore microbial homeostasis in VLS patients. These findings provide a promising basis for the development of microbiota-targeted adjunctive therapies in the management of chronic vulvar inflammatory disorders.\u003c/p\u003e\u003ch2\u003eClinical trial registration: http://www.chictr.org.cn/,identifier (\u003c/h2\u003e\u003cp\u003eChiCTR2400090750), registration time: 12/10/2024.\u003c/p\u003e","manuscriptTitle":"Postbiotics originated from Lactobacillus crispatus NCU-31 improves vulvar lichen sclerosus: a randomized, double-blind controlled trial","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-03 15:10:31","doi":"10.21203/rs.3.rs-7092244/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-15T18:00:51+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-12T19:29:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"296226080668048071336867957197503171576","date":"2025-12-02T15:35:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"180587170332839588641944006782516036976","date":"2025-11-27T14:00:34+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-18T15:24:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"288113420527292739003607666903077941324","date":"2025-09-23T08:25:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"50821444384236206222356492374617019912","date":"2025-07-30T15:20:54+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-29T10:15:51+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-25T19:25:52+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-25T02:44:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Microbiology","date":"2025-07-25T02:41:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ba0b02ab-fa18-4cab-a585-06d2d4b47ca3","owner":[],"postedDate":"August 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-26T16:07:12+00:00","versionOfRecord":{"articleIdentity":"rs-7092244","link":"https://doi.org/10.1186/s12866-026-04738-w","journal":{"identity":"bmc-microbiology","isVorOnly":false,"title":"BMC Microbiology"},"publishedOn":"2026-01-21 15:58:30","publishedOnDateReadable":"January 21st, 2026"},"versionCreatedAt":"2025-08-03 15:10:31","video":"","vorDoi":"10.1186/s12866-026-04738-w","vorDoiUrl":"https://doi.org/10.1186/s12866-026-04738-w","workflowStages":[]},"version":"v1","identity":"rs-7092244","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7092244","identity":"rs-7092244","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}