Clinical Efficacy, Safety, and Multi-Omics Insights of Capilliposide Vaginal Gel in CIN with High-Risk HPV: A Prospective, Multicenter Study

Clinical Efficacy, Safety, and Multi-Omics Insights of Capilliposide Vaginal Gel in CIN with High-Risk HPV: A Prospective, Multicenter Study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Clinical Efficacy, Safety, and Multi-Omics Insights of Capilliposide Vaginal Gel in CIN with High-Risk HPV: A Prospective, Multicenter Study Feng Yue, Zhao Wang, Lili Wu, Xiaoli Chen, Qian Xu, Lijun Ye, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8978688/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 12 You are reading this latest preprint version Abstract Background Cervical intraepithelial neoplasia (CIN) is a precursor of cervical cancer. This prospective, multicenter study evaluated the clinical efficacy, safety, and multi-omics characteristics of Capilliposide vaginal gel (CVG) in women with biopsy-confirmed CIN and high-risk human papillomavirus (HR-HPV) infection. Methods This prospective, multicenter, single-arm study enrolled 81 women with CIN 1/2/3 and HR-HPV infection. In Group 1, participants received CVG intravaginally every other day for 4 weeks to assess preliminary safety. Following the absence of significant adverse events, Group 2 was treated with CVG for 12 weeks to evaluate both safety and efficacy. Vaginal lavage and plasma samples were collected at baseline and 3 months post-treatment for microbiota analysis via 16S rRNA gene sequencing and plasma proteomics. Results Among the 67 participants who completed treatment, CVG was well tolerated, with no grade ≥ 2 or systemic toxicity. Cytological regression to negative for intraepithelial lesion or malignancy occurred in 82% of evaluable participants, and histologic regression was observed in 53.8% of paired biopsies. The overall HR-HPV clearance rates were 72.7% for HPV16 and 60.0% for HPV18. Multi-omics analysis showed a shift to a Lactobacillus-dominant vaginal microbiota and modulation of immune and epithelial repair pathways. Conclusions CVG as a safe, non-invasive, fertility-preserving treatment for CIN with HR-HPV infection, accompanied by changes in the vaginal microbiota and immune-related molecular pathways. Trial registration The trial was retrospectively registered on October 8, 2024, with the International Traditional Medicine Clinical Trial Registry (Registration No. ITMCTR2025002151), and the study has been completed. Cervical intraepithelial neoplasia Capillposide Efficacy Safety Microbiomics Proteomics Figures Figure 1 Figure 2 Figure 3 1 Introduction The management of cervical intraepithelial neoplasia still requires attention.Timely and effective intervention for cervical intraepithelial neoplasia (CIN) is a critical determinant of the long-term success of cervical cancer prevention programs. CIN represents a histopathological continuum ranging from low-grade squamous intraepithelial lesions (LSIL; CIN 1) to high-grade squamous intraepithelial lesions (HSIL; CIN 2/3), the latter conferring a substantially increased risk of progression to invasive cervical cancer. Persistent infection with high-risk human papillomavirus (HR-HPV) is well established as the central etiological driver of this malignant transformation cascade [1–3]. Although approximately 60% of LSIL lesions regress spontaneously within two years [4,5], a clinically significant proportion persists (16–30%), and 4–10% progress to higher-grade disease [6]. These figures underscore a critical clinical dilemma: reliance on spontaneous regression alone exposes a subset of patients to prolonged viral persistence and progressive epithelial dysplasia, thereby undermining the preventive intent of screening-based surveillance strategies. Current clinical guidelines recommend active surveillance for LSIL, while ablative or excisional procedures remain the standard of care for HSIL. However, surgical interventions are inherently invasive and are associated with irreversible cervical damage, as well as increased risks of adverse obstetric outcomes, including preterm birth and cervical insufficiency [7,8]. More importantly, these approaches do not directly address the underlying HR-HPV infection, nor do they offer a satisfactory therapeutic option for patients with persistent viral infection who are unwilling or unsuitable for surgery. This therapeutic void is particularly problematic for women of reproductive age and those seeking non-invasive, fertility-preserving interventions, highlighting an urgent unmet need for effective pharmacological alternatives. Lysimachia capillipes Hemsl., a medicinal herb widely distributed in Southeast China, has long been used in traditional medicine for inflammatory disorders. Accumulating pharmacological evidence—including our previous work—has demonstrated that its major active constituents, Capilliposide B (CPS-B) and Capilliposide C (CPS-C), possess robust anti-inflammatory and anti-tumor activities [9]. These compounds have exhibited pronounced cytotoxic effects across multiple cancer models, including prostate [10,11], colorectal [12], and lung cancers [13], suggesting a broad spectrum of biological activity relevant to epithelial dysregulation and immune modulation. Capilliposide vaginal gel (CVG), formulated with CPS-B and CPS-C, has been approved in China for vaginal antibacterial and symptomatic applications; however, its therapeutic potential in CIN and HR-HPV–associated disease has not been systematically evaluated. Beyond viral oncogenesis, mounting evidence indicates that disruption of the vaginal microbiota constitutes a key cofactor in cervical disease progression. A Lactobacillus-depleted microbial ecosystem is associated with impaired mucosal barrier function, chronic inflammation, immune dysregulation, and reduced viral clearance. Nevertheless, existing CIN therapies largely overlook the role of the vaginal microenvironment and its contribution to both local and systemic immune responses. This conceptual gap limits mechanistic understanding and therapeutic innovation in CIN management. Based on these considerations, we hypothesized that CVG may exert therapeutic effects through a dual mechanism: restoration of vaginal microbial homeostasis and modulation of host immune responses. To test this hypothesis, we conducted a prospective, multicentre, single-arm clinical study to evaluate the clinical efficacy, safety, and tolerability of CVG in women with CIN 1/2/3 and HR-HPV infection. Furthermore, by integrating vaginal microbiota profiling with plasma proteomic analysis, we sought to elucidate the local and systemic biological pathways underlying CVG-mediated lesion regression and viral clearance. This multi-omics framework provides mechanistic insight into CVG as a non-invasive, immune-modulating therapeutic strategy for cervical intraepithelial neoplasia. 2 Methods 2.1 Trial Design This study was designed as a prospective, multicentre, single-arm clinical trial to evaluate the clinical efficacy, safety, and tolerability of Capilliposide vaginal antiseptic gel (CVG) in patients with cervical intraepithelial neoplasia (CIN1/2/3) and high-risk human papillomavirus (HR-HPV) infection. The trial was conducted across three clinical sites in China: Zhejiang Cancer Hospital (Site A), the First People's Hospital of Hangzhou Lin'an District (Site B), and Yiwu Maternity and Children Hospital (Site C).The overall trial profile was shown in Fig. 1 . The study was conducted in accordance with the principles of Good Clinical Practice and the Declaration of Helsinki. The study protocol was reviewed and approved by the Medical Ethics Committee of Zhejiang Cancer Hospital (IRB No. 2023 − 140). The trial was registered with the International Traditional Medicine Clinical Trial Registry (ITMCTR2025002151) and has been completed. Written informed consent was obtained from all participants prior to enrollment. 2.2 Patients Between March 2023 and March 2024, a total of 81 patients with histologically confirmed CIN1/2/3 were enrolled across the three participating centers. Eligibility criteria included: (1) biopsy-confirmed CIN1/2/3, (2) confirmed HPV infection, and (3) ability and willingness to comply with the study protocol. Exclusion criteria comprised: (1) known allergy to CVG; (2) unsatisfactory colposcopic examination; (3) history of invasive cervical carcinoma or other invasive malignancies; (4) active skin or mucosal infections, overt pyometra, or acute inflammation of the female reproductive tract; (5) pregnancy or breastfeeding; and (6) serious comorbidities potentially affecting study compliance, including unstable cardiovascular disease, renal disease, chronic hepatitis, poorly controlled diabetes, or severe mental illness. Participants were withdrawn from the study in the event of adverse reactions, poor compliance, voluntary withdrawal, pregnancy, or investigator-determined inability to continue treatment or follow-up. 2.3 Study medication CVG contains Capilliposide B (CPS-B) and Capilliposide C (CPS-C), two bioactive compounds isolated from Lysimachia capillipes Hemsl. The formulation used in this study contained a total of 1% CPS-B and CPS-C and was manufactured by Hangzhou Yirui Medical Technology Co., Ltd. (China). Treatment was initiated on the second day after completion of menstruation. CVG has been approved for clinical use under the Zhejiang Health Disinfection Certificate (License No. 2022-0006). 2.4 Study Procedures Eligible participants were assigned sequentially to two study phases (Fig. 1 ). In the initial safety evaluation phase (Group 1), 20 participants self-administered CVG intravaginally at a dose of 3 g every other night for 4 weeks. Administration was performed before bedtime according to standardized instructions. Following confirmation of acceptable safety outcomes, the study proceeded to the efficacy evaluation phase (Group 2), in which participants received CVG treatment for 12 weeks. Clinical symptoms were monitored via telephone follow-up at two-week intervals. Adverse events were assessed and graded in real time using the Common Terminology Criteria for Adverse Events (CTCAE), version 5.0, and their relationship to the study medication was evaluated. Combined ThinPrep cytology test (TCT) and HPV testing were performed 3–6 months after treatment completion. For patients presenting with abnormal cytology results, colposcopically directed biopsy was strongly recommended but not mandatory, and patient preference was respected. Clinical efficacy, safety, and tolerability were evaluated comprehensively at the end of follow-up. 2.5 Clinical Outcome Measures and Statistical Analysis The primary endpoint was histologic regression, defined as complete resolution of cervical lesions confirmed by post-treatment pathology. Histologic improvement was defined as a reduction in lesion grade, histologic persistence as unchanged pathology, and histologic progression as an increase in lesion severity. For participants who did not undergo post-treatment biopsy, cytologic regression was assessed based on changes in cervical cytology, including transitions from ASC-US, ASC-H, LSIL, or HSIL to negative for intraepithelial lesion or malignancy (NILM)[14]. Secondary endpoints included HR-HPV clearance, safety, and tolerability. HR-HPV clearance was defined as the absence of baseline HR-HPV genotypes at follow-up[15]. Safety was assessed in all participants receiving at least one dose of CVG, focusing on treatment-related adverse events, while tolerability was defined as completion of the prescribed treatment regimen. Statistical analyses were performed using SPSS software (version 25.0; SPSS Inc.). Baseline and post-treatment comparisons were conducted using standard hypothesis testing methods, as appropriate. 2.6 Sample Collection Vaginal lavage and peripheral blood samples were collected prior to CVG initiation and three months after treatment completion. Vaginal douching or rinsing was prohibited for 48 h before sampling. Vaginal lavage was performed using 10 mL of sterile saline, and recovered fluid was immediately transferred into sterile tubes. Following centrifugation, samples were divided into aliquots for microbiome and physicochemical analyses and stored at − 80 ℃ until use. Blood samples were processed and stored according to established protocols[16–19] . 2.7 Vaginal Microbiota Profiling by 16S rRNA Gene Sequencing 2.7.1 Vaginal Microbiota Profiling by 16S rRNA Gene Sequencing Genomic DNA was extracted from vaginal microbial communities in 54 paired pre- and post-treatment vaginal lavage samples using the FastPure Soil DNA Isolation Kit (Magnetic Bead; MJYH, Shanghai, China), following the manufacturer's instructions. DNA integrity was assessed by 1% agarose gel electrophoresis, and concentration and purity were measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). Full-length bacterial 16S rRNA genes were amplified using universal primers 27F (5′-AGRGTTYGATYMTGGCTCAG-3′) and 1492R (5′-RGYTACCTTGTTACGACTT-3′), each tagged with sample-specific PacBio barcodes[20,21]. PCR was performed in 20 µL reactions containing FastPfu buffer, dNTPs, primers, FastPfu DNA polymerase, and 10 ng of template DNA. Cycling conditions included an initial denaturation at 95 ℃ for 3 min, followed by 29 cycles of 95 ℃for 30 s, 60 ℃ for 30 s, and 72 ℃ for 45 s, with a final extension at 72 ℃ for 10 min. All reactions were conducted in triplicate. PCR products were purified using a PCR Clean-Up Kit (YuHua, Shanghai, China) and quantified with a Synergy HTX microplate reader (BioTek, USA). Purified amplicons were pooled in equimolar concentrations, and SMRTbell libraries were constructed using the SMRTbell Prep Kit 3.0 (Pacific Biosciences, USA). Sequencing was performed on the PacBio Revio System (Pacific Biosciences) at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China). 2.7.2 Sequence Processing and Microbiota Analysis Raw sequencing data were processed using SMRT Link software (v11.0) to generate high-quality HiFi reads, requiring a minimum of three full passes and ≥ 99% sequence accuracy. Reads were demultiplexed by barcode and filtered by length, retaining sequences between 1,000 and 1,800 bp. Denoising and amplicon sequence variant (ASV) inference were performed using the DADA2 plugin within the QIIME2 pipeline (v2024), yielding single-nucleotide–resolved ASVs. To normalize sequencing depth, samples were rarefied to 9,757 reads per sample, achieving an average Good's coverage of approximately 99%[22]. Taxonomic assignment was conducted using the classify-consensus-blast classifier in QIIME2 against the NT_16s database (v2024). Functional potential of the microbial communities was predicted using PICRUSt2 following standard protocols[23]. Alpha diversity metrics, including observed ASVs, Chao1 richness, Shannon index, and Good's coverage, were calculated using Mothur (v1.30.2). Beta diversity was assessed based on Bray–Curtis dissimilarity and visualized by principal coordinate analysis (PCoA) using the vegan package in R. Permutational multivariate analysis of variance (PERMANOVA) was applied to evaluate treatment-associated differences in community composition. 2.8 Plasma Proteomic Analysis 2.8.1 Enrichment of Low-Abundance Plasma Proteins Low-abundance plasma proteins from 54 paired pre- and post-treatment samples were enriched using the EasyPep™ Deep Low-Abundance Enrichment Kit (EasyPep, Catalog No. OSFP0002) according to the manufacturer's instructions.Magnetic beads were equilibrated and incubated with 100 µL of plasma at 37 ℃ for 1 h with constant agitation (1000 rpm). After incubation, beads were washed three times to remove high-abundance proteins, and the bead-bound low-abundance protein fraction was collected for downstream processing[24] . 2.8.2 Protein Digestion and Peptide Preparation Enriched proteins were subjected to reduction and alkylation at 95 ℃ for 5 min, followed by enzymatic digestion at 37 ℃ for 2 h with agitation. Digestion was terminated using stop reagent, and peptide-containing supernatants were collected after centrifugation at 20,000 × g for 1 min. Peptides were desalted using EasyPep™ Desalting Cartridges (Catalog No. OSFP0050-W), vacuum-dried, reconstituted in loading buffer, and quantified using a NanoDrop One spectrophotometer (Thermo Scientific). 2.8.3 LC–MS/MS Analysis (DIA Mode) Peptide samples were analyzed on a Vanquish™ Neo UHPLC system coupled to an Orbitrap™ Astral mass spectrometer (Thermo Fisher Scientific) at Majorbio Bio-Pharm Technology Co., Ltd. Chromatographic separation was performed using a uPAC™ High-Throughput column (75 µm × 5.5 cm). Mobile phases consisted of 0.1% formic acid in water with 2% acetonitrile (buffer A) and 0.1% formic acid in water with 80% acetonitrile (buffer B), using an 8-min gradient[25]. Mass spectrometry data were acquired in data-independent acquisition (DIA) mode over an m/z range of 100–1700. Instrument settings included an AGC target of 500%, maximum injection time of 3 ms, isolation window of 2 m/z, HCD collision energy of 25%, and 300 DIA windows. 2.8.4 Protein Identification and Bioinformatic Analysis DIA data were processed using Spectronaut software (v19, Biognosys). Protein quantification was based on the top six peptides per protein and the top three fragment ions per peptide. Identification thresholds were set at a protein and peptide false discovery rate ≤ 1% and peptide confidence ≥ 99%. Shared and modified peptides were excluded. Protein abundance was calculated as the summed peak area of qualified peptides, and only proteins identified with at least one unique peptide were retained. Proteomic data analysis was conducted using the Majorbio Cloud Platform. Proteins with missing values in more than 70% of samples were excluded, and remaining missing values were imputed using the sequential k-nearest neighbors (seqKNN) algorithm. Differentially expressed proteins (DEPs) were identified using a two-sided Student's t-test in R, with thresholds of fold change ≥ 1.5 or ≤ 0.67 and P < 0.05. Functional annotation and pathway enrichment were performed using GO and KEGG databases, and protein–protein interaction networks were constructed using STRING (v11.5). 2.8.5 Molecular Docking Analysis Protein information was retrieved from the UniProt database ( https://www.uniprot.org ), and the corresponding three-dimensional structures were obtained from the RCSB Protein Data Bank ( https://www.rcsb.org ). Protein structures were imported into Discovery Studio 2019 (BIOVIA, San Diego, CA, USA) for structural preparation, including removal of crystallographic water molecules, addition of hydrogen atoms, assignment of partial charges, completion of missing residues, and reconstruction of incomplete side chains. The optimized protein structures were subsequently saved in PDB format. The chemical structure of the small-molecule ligand was obtained from the PubChem database and subjected to energy minimization using Discovery Studio 2019. The optimized ligand structure was exported in PDB format. Preparation of receptor and ligand PDBQT files was performed using AutoDock Tools (AutoDock 4.0). Molecular docking simulations were conducted using AutoDock Vina (version 1.2.6) with default parameters. Docking poses were ranked based on binding affinity scores, and the most favorable binding conformations were selected for further analysis. Protein–ligand interactions were visualized and analyzed using PyMOL (version 3.1) and Discovery Studio 2019. 3 Result 3.1 Clinical Information Between March 2023 and March 2024, 81 participants meeting the eligibility criteria were enrolled across three clinical centers, including 9 patients from Site A, 35 from Site B, and 37 from Site C. Baseline demographic and clinical characteristics are summarized in Table 1 . The median age of the study population was 42 years. An initial safety run-in cohort of 20 participants was assigned to Group 1 for preliminary safety evaluation. Two participants discontinued the study due to treatment-related local adverse reactions in Group 1, such as vaginal bleeding and pruritus. Following confirmation of acceptable safety, 18 enrolled participants proceeded to Group 2 for further assessment of safety and efficacy. During the study period, 14 participants discontinued the trial, resulting in 67 participants included in the final efficacy and safety analyses (Supplementary Table 1). At baseline, histopathological assessment identified 61 participants (91.0%) with CIN 1 and 6 participants (9.0%) with CIN 2/3. All participants were confirmed to have high-risk HPV (HR-HPV) infection. Specifically, HPV16 was detected in 16.4% (11/67) of participants, HPV18 in 7.5% (5/67), and other HR-HPV genotypes in 76.1% (51/67). Table 1 Characteristics of Evaluable Participants on Baseline. Characteristics N % Age, years < 40 28 41.7 ≥ 40 39 58.2 Education Primary school 11 16.4 Unior high school 20 29.8 Senior high school 12 17.9 University and above 24 35.8 Menstrual history Premenopause 47 70.1 Pausimenia 20 29.8 Smoking status No 67 100 Yes 0 0 Drinking History No 62 92.5 Yes 5 7.4 Past History Hypertension 4 5.9 No 63 94 Previous HPV vaccine No 55 82 Yes 12 17.9 TCT NILM,ASCUS or LSIL 62 92.5 ASC-H or HSIL 5 7.5 Histology CIN 1 61 91 CIN 2/3 6 9 HR-HPV 16、Others (+) 11 16.4 18、Others (+) 5 7.5 Other high-risk HPV (+) 51 76.1 Persistent HR-HPV No 27 40.2 Yes 27 40.2 Not detailed 13 19.4 3.2 Treatment Adherence and Loss to Follow-up A dropout rate of 17% was observed in this clinical trial. Among the 81 enrolled women who initiated CVG treatment, a total of 14 participants discontinued the study during the treatment or follow-up period. Two participants withdrew because of local adverse events, including vaginal bleeding or pruritus in Group 1. Specifically, seven participants were lost to follow-up. One participant withdrew from the study to undergo in vitro fertilization–embryo transfer, and one participant discontinued treatment due to pregnancy. Two participants elected to undergo loop electrosurgical excision procedure owing to concerns about potential progression of squamous intraepithelial lesions. One participant discontinued treatment for unspecified personal reasons. 3.3 CIN Regression and HPV Clearance at Follow-up Because colposcopically directed biopsy is an invasive procedure, post-treatment histological evaluation was performed selectively. Among the 67 participants included in the final analysis, 13 underwent both ThinPrep cytologic testing (TCT) and biopsy, while the remaining 45 participants were evaluated by TCT alone. Among the 13 participants with paired histological assessments, histologic regression was observed in 53.8% (7/13) of cases, whereas histologic persistence was observed in 46.2% (6/13). Specifically, regression occurred in 70% (7/10) of patients with CIN 1 and in 33.3% (1/3) of patients with CIN 2/3. Histologic persistence was observed in 40% of CIN 1 cases. Based on cytologic evaluation, 82% (55/67) of participants demonstrated regression to negative for intraepithelial lesion or malignancy (NILM)(Table 2 ). These included 17 cases initially diagnosed as CIN 1, two as CIN 2/3, two as ASC-H, and 15 as ASC-US. Detailed cytologic outcomes are summarized in Table 3 . No cases of progression to invasive cervical cancer were observed during follow-up. Table 2 TCT Results of Baseline Status and After CVG Treatment. Baseline Status After CVG treatment TCT results N TCT result N NILM 22 NILM 19 ASC-H 0 ASC-US 2 LSIL 1 HSIL 0 ASC-US 18 NILM 15 ASC-H 0 ASC-US 1 LSIL 2 HSIL 0 LSIL 22 NILM 17 ASC-H 0 ASC-US 3 LSIL 2 HSIL 0 ASC-H 2 NILM 2 ASC-H 0 ASC-US 0 LSIL 0 HSIL 0 HSIL 3 NILM 2 ASC-H 1 ASC-US 0 LSIL 0 HSIL 0 Among the 67 participants with baseline HR-HPV infection, 23 patients (34.3%) achieved complete HR-HPV clearance, while 23 patients (34.3%) exhibited persistent infection. Three patients (4.4%) showed progression characterized by the detection of additional HPV genotypes (Table 3 ). The clearance rate for HPV16 was 72.7% (8/11), and for HPV18 was 60.0% (3/5). Among participants with concomitant low-risk HPV infection, four achieved complete clearance of all HPV genotypes, seven exhibited persistence of the same HPV genotype, and two demonstrated progression in HPV genotype composition. Notably, no participants showed progression from low-risk to high-risk HPV types during follow-up. Table 3 HPV Status After CVG Treatment. Characteristics % Clearance 23/67 (34.3%) Persistence 23/67 (34.3%) Progression 3/67 (4.4%) Overall response 41/67(61%) Clearance of HPV 16 positive 8/11 (72%) Clearance of HPV 18 positive 3/5 (60%) 3.4 Safety and Acceptability of CVG All 67 participants included in the final analysis were evaluated for safety. During CVG treatment, 30% (20/67) of participants reported at least one adverse event (Table 4 ). No grade 2, 3, or 4 adverse events were observed, and no systemic adverse reactions were reported. In Group 1, one participant experienced vaginal bleeding and one reported vaginal pruritus. In Group 2, all reported adverse events were local and mild in severity, including vaginal pruritus (n = 7), vaginal pain (n = 3), vaginal discharge (n = 2), vaginal bleeding (n = 13), and pelvic pain (n = 1). No adverse events were considered severe enough to interfere with daily activities. Despite the occurrence of local adverse events, treatment acceptability was high. Overall, 91% of participants reported satisfaction with CVG use, and 94% considered the treatment to be safe. All participants expressed confidence in their ability to correctly administer the vaginal gel. Seventeen participants temporarily interrupted treatment for 2–7 days due to vaginal bleeding or pruritus; treatment was resumed after symptom resolution, and all completed the prescribed 12-week treatment course. Table 4 Side Effects of CVG Treatment. Adverse reactions N Systemic symptoms 0 Vaginal pruritus 7 Vaginal pain 3 Vaginal discharge 2 Vaginal bleeding 13 Ulceration of the treatment site 0 Pelvic pain 1 3.5 Vaginal Microbiota Remodeling by CVG Our analysis revealed significant changes in the vaginal microbiota composition following CVG treatment. A total of 2,850,277 high-quality 16S rRNA sequences were obtained, yielding 6,690 ASVs with 99.9% Good's coverage. PCoA based on Bray–Curtis dissimilarity revealed clear separation between pre- and post-treatment samples (PERMANOVA P = 0.001), indicating substantial microbial community remodeling (Fig. 2 A). Species-level community barplot analysis revealed significant alterations in the relative abundance of key taxa, with a notable increase in Lactobacillus crispatus and a marked decrease in Lactobacillus iners and Gardnerella vaginalis following CVG treatment (Fig. 2 B; Figure S1 B). At the genus level, Gardnerella exhibited a marked decrease following treatment. Furthermore, these findings were consistent with the results of the Wilcoxon rank-sum test (Fig. 2 C; Figure S1 A). This shift suggests that CVG promotes a transition from a dysbiotic microbiota to a Lactobacillus-dominant healthy state, which may play a crucial role in restoring the vaginal mucosal barrier and facilitating the clearance of HR-HPV, thereby contributing to the regression of CIN. In addition, both species- and genus-level analyses showed an increase in the Microbiome Health Index (GMHI) and a concomitant decrease in the Microbial Dysbiosis Index (MDI), further confirming a shift toward a healthy, eubiotic microbial composition.(Fig. 2 D-E; Figure S1 C-D). Species-level correlation network analysis revealed that pre-treatment anaerobic taxa (Peptostreptococcus anaerobius, Anaerococcus jeddahensis, Finegoldia magna) formed highly interconnected modules dominated by positive correlations. Post-treatment, multiple negative correlations and antagonistic interactions emerged, indicating that CVG not only altered individual taxa abundances but also restructured microbial interaction networks (Fig. 2 F). 3.6 Plasma Proteomic Profiling and Immune-Related Targets DIA-MS plasma proteomic profiling identified 6,052 proteins across all samples. Correlation and PLS-DA analyses revealed clear separation between pre- and post-treatment groups, with post-treatment samples clustering more tightly, indicating consistent systemic proteomic remodeling following CVG intervention (Fig. 3 A–B). Comparative analysis identified 389 DEPs (305 upregulated, 84 downregulated) (Fig. 3 C). KEGG enrichment highlighted pathways associated with cell adhesion, chromatin organization, and intracellular signaling, including adherens junctions and ATP-dependent chromatin remodeling (Figure SE). The heatmap illustrates the impact of CVG intervention on the protein expression profile, with clustering analysis revealing differentially expressed genes between the two groups (Fig. 3 D).PPI network analysis identified core nodes (ATP5PD, NDUFV1, HIST2H3C, IMMT, UQCRC2, YWHAZ) coordinating mitochondrial function, chromatin dynamics, and signal transduction (Fig. 3 E). Among candidate immune-related proteins, DDX60 emerged as a key mediator of systemic antiviral responses. DDX60 is an interferon-inducible DExD/H-box RNA helicase that enhances RIG-I-like receptor (RLR)-mediated type I interferon (IFN-I) signaling and interferon-stimulated gene (ISG) expression, thereby promoting innate antiviral immunity [26–29]. DDX60 also participates in viral RNA degradation and translational suppression, functioning as a multifunctional antiviral effector. Upregulation of DDX60 and related signaling pathways after CVG treatment coincided with the restoration of Lactobacillus-dominant vaginal microbiota, suggesting that CVG may enhance HR-HPV clearance by synergistically modulating both mucosal and systemic antiviral immunity. Molecular docking demonstrated stable binding of CPS-B to a core protein within the DDX60 network, supporting potential ligand–target interactions (Fig. 3 F). 4 Discussion CIN and persistent HR-HPV infection continue to pose substantial challenges in clinical management. While surgical interventions remain effective for high-grade lesions, they are associated with cervical damage and adverse reproductive outcomes, and no widely accepted non-invasive therapy is currently available for persistent HPV infection or low-grade CIN requiring active intervention. Spontaneous regression of LSIL/CIN1 is well recognized; however, population-based studies show that a substantial proportion of LSIL persists beyond 12 months, with a subset progressing to higher-grade disease [30–32]. In the present study, histologic regression was observed in 53.8% of patients with paired biopsies, while cytologic regression to NILM occurred in 80% of patients assessed by TCT alone, with no cases of progression to invasive cervical cancer during follow-up. The overall HR-HPV clearance rate of 34.3% compares favorably with pharmacological intervention studies, including interferon-based therapies, which typically report clearance rates of 20–25% [33,34]. Clearance of HPV16 and HPV18 was particularly notable (72.7% and 60.0%, respectively), although interpretation is limited by sample size. Consistent with prior reports, regression rates were higher in CIN1 than in CIN2/3, reflecting differences in lesion biology and immune responsiveness [35]. Collectively, these findings support CVG as a potential non-surgical therapeutic option for selected patients with CIN and HR-HPV infection.CVG demonstrated a favorable safety profile, with mild and transient local adverse events and no systemic toxicity. High treatment adherence and patient satisfaction highlight its acceptability as a self-administered topical therapy, making it an attractive option for women seeking fertility-preserving alternatives to surgical intervention or adjunctive strategies to promote HPV clearance during CIN surveillance. Accumulating evidence links persistent HR-HPV infection to vaginal microbiota dysbiosis, characterized by reduced Lactobacillus dominance and enrichment of anaerobic taxa [36–37], which is associated with impaired epithelial barrier function, chronic inflammation, and weakened local antiviral immunity. Lactobacillus crispatus is widely recognized as the most beneficial Lactobacillus species in the vaginal niche, as it efficiently produces lactic acid to maintain a low vaginal pH (≤ 4.5), secretes antimicrobial peptides, and strengthens the vaginal mucosal barrier by promoting tight junction formation [38]. In contrast, Lactobacillus iners , though common in apparently healthy vaginas, has limited acid production and is linked to dysbiosis, while Gardnerella vaginalis promotes inflammation, epithelial barrier disruption, and impaired antiviral immunity, facilitating HR-HPV persistence and CIN progression [39–40]. It strongly suggests that CVG effectively targets dysbiosis-associated taxa while fostering the growth of beneficial lactobacilli, thereby creating a microenvironment unfavorable for HR-HPV survival and CIN progression. By restoring a Lactobacillus-dominant vaginal microbiota that maintains acidity, inhibits HR-HPV and pathogenic bacteria, and enhances local antiviral immunity and mucosal barrier function, CVG plausibly facilitates HR-HPV clearance and CIN regression, consistent with improved clinical outcomes in treated patients. Beyond local microbial effects, plasma proteomic profiling revealed consistent systemic molecular remodeling following CVG treatment. Differentially expressed proteins were enriched in pathways related to epithelial integrity (adherens junctions), chromatin organization, and intracellular signaling. These pathways are central to epithelial repair, immune cell activation, and transcriptional regulation during antiviral responses [41, 42]. Among immune-related candidates, upregulation of antiviral signaling components, including DDX60-associated networks, suggests enhanced innate immune responsiveness following CVG treatment. DDX60 is an interferon-inducible RNA helicase involved in RIG-I–like receptor signaling and amplification of type I interferon responses, thereby contributing to antiviral defense [43]. Although plasma proteomics cannot localize tissue-specific effects, the coordinated modulation of immune-related pathways supports a systemic component of CVG-associated immunomodulation that may complement local mucosal effects. Taken together, the clinical, microbiome, and proteomic data support a working model in which CVG may mediate CIN regression and HR-HPV clearance through complementary local and systemic mechanisms. Locally, CVG appears to restore vaginal microbial homeostasis, promoting Lactobacillus dominance, suppressing dysbiosis-associated anaerobes, and improving epithelial barrier integrity. Systemically, CVG is associated with modulation of antiviral and immune-related molecular pathways, including DDX60-linked interferon signaling, which may enhance innate immune surveillance. These mechanisms are likely to act synergistically rather than independently, providing a plausible explanation for the observed cytologic regression and partial HR-HPV clearance. While causality cannot be established in the absence of a control group, the convergence of multi-omics signals with clinical outcomes supports further investigation of CVG as a non-invasive, fertility-preserving therapeutic strategy. Several limitations should be noted. The single-arm design limits causal inference, and spontaneous regression cannot be excluded. Incomplete histological follow-up and the small CIN2/3 sample size are additional limitations. Future randomized controlled trials with longer follow-up and mechanistic studies are needed to confirm efficacy and clarify the molecular pathways underlying CVG's effects. 5 Conclusions This study provides evidence supporting the clinical efficacy and safety of CVG in the treatment of CIN and HR-HPV infection. By modulating the vaginal microbiome and enhancing immune responses, CVG offers a novel, non-invasive treatment option that could complement current therapeutic strategies. The therapeutic potential of CVG to induce regression of CIN and clear HR-HPV suggests that it may be an effective alternative treatment, especially for patients seeking non-invasive, patient-controlled management options. Further studies are warranted to validate these findings and explore the broader applicability of CVG in clinical practice. Abbreviations HPV Human Papillomavirus HR-HPV High-Risk Human Papillomavirus CIN Cervical Intraepithelial Neoplasia CVG Capilliposide Vaginal Gel DEPs Differentially Expressed Proteins TCT ThinPrep Cytologic Test LSIL Low-Grade Squamous Intraepithelial Lesion HSIL High-Grade Squamous Intraepithelial Lesion NILM Negative for Intraepithelial Lesion or Malignancy ASC-US Atypical Squamous Cells of Undetermined Significance ASC-H Atypical Squamous Cells, Cannot Exclude High-Grade Squamous Intraepithelial Lesion Declarations Ethics approval and consent to participate The study was conducted in accordance with the principles of Good Clinical Practice and the Declaration of Helsinki. The study protocol was reviewed and approved by the Medical Ethics Committee of Zhejiang Cancer Hospital (IRB No. 2023 − 140). The trial was registered with the International Traditional Medicine Clinical Trial Registry (ITMCTR2025002151) and has been completed. Written informed consent was obtained from all participants prior to enrollment. Consent for publication Not applicable. Competing Interests The authors declare that they have no competing interests. Corresponding author Correspondence to [email protected] ; [email protected] ; Acknowledge Not applicable. Funding This work was supported by grants-in-aid for scientific research from the National Natural Science Foundation of China (number 82405091), the Natural Science Foundation of Zhejiang Province (number Q24H290031), the Zhejiang Province Traditional Chinese Medicine Science and Technology Project (number GZY-ZJ-KJ-23050, number GZY-ZJ-KJ-24063). Author Contribution Y F: Conceptualization, Methodology, Formal analysis, Investigation, Writing - original draft, Supervision, Funding acquisition. LL W, XL C, Z W, Q X, LJ Y, YQ L, JM Z: Clinical Information Collection. HM L, W Z: Investigation, Resources, Project administration, Funding acquisition. H Y, JK T:Validation. All authors have read and agreed to the published version of the manuscript. Data Availability The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. References Qin F, Sun X, Tian M, Jin S, Yu J, Song J, Wen F, Xu H, Yu T, Dong Y. Prediction of lymph node metastasis in operable cervical cancer using clinical parameters and deep learning with MRI data: a multicentre study. Insights Imaging . 2024;15(1):56. doi:10.1186/s13244-024-01618-7. Chou HH, Wang PH, Chen CA, et al. Diagnostic performance of magnetic resonance imaging in detecting lymph node metastasis in cervical cancer. J Gynecol Oncol . 2010;21(4):249–255. Tierney JF, Stewart LA, Ghersi D, Burdett S, Sydes MR. Practical methods for incorporating summary time-to‐event data into meta‐analysis. Trials . 2007;8:16. Wu J, Huang YW, Chen YJ, et al. Comparison of CT, MRI, and PET in the detection of pelvic lymph node metastasis in cervical cancer. Radiology . 2020;296(3):574–583. He LY, Zhao SH, Liu M, et al. MRI radiomics for predicting pelvic lymph node metastasis in early cervical cancer: a multicenter study. Eur Radiol . 2021;31(6):4268–4277. Odunsi K, Zhang Y, Lin C, et al. Integrated PET–CT enhances accuracy in lymph node staging of cervical cancer. Clin Nucl Med . 2001;26(7):567–572. Lee H, Kim DW, Park SY, et al. Radiomic CT texture analysis predicts lymph node metastasis in cervical cancer. Acad Radiol . 2023;30(2):250–260. Zhang Y, Smith K, Li J, et al. Deep learning–based MRI improves preoperative lymph node metastasis detection in cervical cancer. Insights Imaging . 2024;15(1):57. doi:10.1186/s13244-024-01619-6. Moher D, Liberati A, Tetzlaff J, Altman DG; PRISMA Group. Preferred Reporting Items for Systematic Reviews and Meta-Analyses: the PRISMA statement. PLoS Med . 2009;6(7):e1000097. DerSimonian R, Laird N. Meta-analysis in clinical trials. Control Clin Trials . 1986;7(3):177–188. Higgins JP, Thompson SG, Deeks JJ, Altman DG. Measuring inconsistency in meta-analyses. Stat Med . 2002;21(11):1539–1558. Egger M, Davey Smith G, Schneider M, Minder C. Bias in meta-analysis detected by a simple, graphical test. BMJ . 1997;315(7109):629–634. Moher D, Liberati A, Tetzlaff J, Altman DG; PRISMA Group. Preferred Reporting Items for Systematic Reviews and Meta-Analyses: The PRISMA Statement. PLoS Medicine . 2009;6(7):e1000097. Tierney JF, Stewart LA, Ghersi D, Burdett S, Sydes MR. Practical methods for incorporating summary time-to-event data into meta-analysis. Trials . 2007;8:16. DerSimonian R, Laird N. Meta-analysis in clinical trials. Controlled Clinical Trials . 1986;7(3):177–188. Higgins JP, Thompson SG. Quantifying heterogeneity in a meta-analysis. Statistics in Medicine . 2002;21(11):1539–1558. Egger M, Davey Smith G, Schneider M, Minder C. Bias in meta-analysis detected by a simple, graphical test. BMJ . 1997;315(7109):629–634. Additional Declarations No competing interests reported. Supplementary Files SupplementaryTable1.xlsx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 11 May, 2026 Reviews received at journal 06 May, 2026 Reviewers agreed at journal 27 Apr, 2026 Reviewers agreed at journal 25 Apr, 2026 Reviews received at journal 26 Mar, 2026 Reviewers agreed at journal 22 Mar, 2026 Reviewers agreed at journal 16 Mar, 2026 Reviewers invited by journal 05 Mar, 2026 Editor invited by journal 27 Feb, 2026 Editor assigned by journal 26 Feb, 2026 Submission checks completed at journal 26 Feb, 2026 First submitted to journal 26 Feb, 2026 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-8978688","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":601571035,"identity":"84b5a0ba-05ae-4be9-bd22-0264ea399e5f","order_by":0,"name":"Feng Yue","email":"","orcid":"","institution":"Zhejiang Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Feng","middleName":"","lastName":"Yue","suffix":""},{"id":601571036,"identity":"978dd1dd-df58-4df5-8c1e-59478f37d982","order_by":1,"name":"Zhao Wang","email":"","orcid":"","institution":"Zhejiang Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Zhao","middleName":"","lastName":"Wang","suffix":""},{"id":601571037,"identity":"53a7bbbe-ea46-45bc-a9d9-9dd4793182be","order_by":2,"name":"Lili Wu","email":"","orcid":"","institution":"Yiwu Maternity and Children Health Hospital,","correspondingAuthor":false,"prefix":"","firstName":"Lili","middleName":"","lastName":"Wu","suffix":""},{"id":601571038,"identity":"75bf9e8a-0234-4ee4-ac1e-d3bfed127684","order_by":3,"name":"Xiaoli Chen","email":"","orcid":"","institution":"The First People's Hospital of Lin'an District","correspondingAuthor":false,"prefix":"","firstName":"Xiaoli","middleName":"","lastName":"Chen","suffix":""},{"id":601571039,"identity":"b0246821-8f8d-4e4d-ae99-640d61c5341e","order_by":4,"name":"Qian Xu","email":"","orcid":"","institution":"Yiwu Maternity and Children Health Hospital,","correspondingAuthor":false,"prefix":"","firstName":"Qian","middleName":"","lastName":"Xu","suffix":""},{"id":601571040,"identity":"48c26b7c-9272-4ad6-96d2-39c510764415","order_by":5,"name":"Lijun Ye","email":"","orcid":"","institution":"The First People's Hospital of Lin'an District","correspondingAuthor":false,"prefix":"","firstName":"Lijun","middleName":"","lastName":"Ye","suffix":""},{"id":601571041,"identity":"5b09b67a-bf8c-4818-99f1-527f08a6246a","order_by":6,"name":"Hui Ye","email":"","orcid":"","institution":"Zhejiang Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Ye","suffix":""},{"id":601571042,"identity":"832253e4-d21c-4e10-8af2-0966b1a7c123","order_by":7,"name":"Yueqi Li","email":"","orcid":"","institution":"Zhejiang Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yueqi","middleName":"","lastName":"Li","suffix":""},{"id":601571045,"identity":"bfe8b573-2286-4cf3-8e3c-7087723d4f90","order_by":8,"name":"Jiamiao Zhu","email":"","orcid":"","institution":"Yiwu Maternity and Children Health Hospital,","correspondingAuthor":false,"prefix":"","firstName":"Jiamiao","middleName":"","lastName":"Zhu","suffix":""},{"id":601571046,"identity":"5071efd8-fc55-4b68-8c7c-4cd72995bd3a","order_by":9,"name":"Jingkui Tian","email":"","orcid":"","institution":"Zhejiang Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jingkui","middleName":"","lastName":"Tian","suffix":""},{"id":601571047,"identity":"1b5ac11e-3337-4747-8228-c4c3df036400","order_by":10,"name":"Hanmei Lou","email":"","orcid":"","institution":"Zhejiang Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hanmei","middleName":"","lastName":"Lou","suffix":""},{"id":601571048,"identity":"fac96279-b4e6-4165-93f2-78a8c1e762b2","order_by":11,"name":"Wei Zhu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsklEQVRIiWNgGAWjYDACCRBhYMNjQKqWNJK1MBxmIF6L/OweM4kPBedlzMUOP2D4UcMgb05IC+OcM2aSMwxu81jOTjNg7DnGYLizgYAWZokcs9s8BiCUw8DA28CQYHCAgBY2kJY/BufAWhj/EqOFB6SFweAAWAszUbZISKSV/+wxSAb75bDMMQnDDYS0yM9I3mzw44+dvbl08sOHb2ps5AnaggIOwKJpFIyCUTAKRgGFAAAo6DgvOkDZiAAAAABJRU5ErkJggg==","orcid":"","institution":"Zhejiang Cancer Hospital","correspondingAuthor":true,"prefix":"","firstName":"Wei","middleName":"","lastName":"Zhu","suffix":""}],"badges":[],"createdAt":"2026-02-26 14:14:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8978688/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8978688/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104413690,"identity":"212e204e-cacf-4f3a-9a50-9d411758cde1","added_by":"auto","created_at":"2026-03-11 13:05:14","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":238909,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverall Study Design.\u003c/strong\u003e (A) Patient Screening and Eligibility Assessment. The trial was conducted at three clinical sites in China: Zhejiang Cancer Hospital (Site A), the First People's Hospital of Hangzhou Lin'an District (Site B), and Yiwu Maternity and Children's Hospital (Site C). (B) Treatment Administration and Clinical Outcome Evaluation. In the initial safety evaluation phase (Group 1), 20 participants self-administered CVG intravaginally at a dose of 3 g every other night for 4 weeks, following standardized instructions and administration. After confirming acceptable safety outcomes, the study advanced to the efficacy evaluation phase (Group 2), where participants received CVG treatment for 12 weeks. (C) Pre-treatment and Post-treatment Clinical Samples Collection. Vaginal lavage and peripheral blood samples were collected before CVG initiation and three months after treatment completion. (D) Microbiota analysis and proteomic analysis were conducted on clinical samples to evaluate the potential effects of CVG therapy on vaginal microbiota composition and systemic protein expression.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8978688/v1/46b6761d94661d66f9cdea07.jpg"},{"id":104411958,"identity":"23393be7-f99a-4fde-a7d6-f9ef95ddc0d2","added_by":"auto","created_at":"2026-03-11 12:58:18","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":256067,"visible":true,"origin":"","legend":"\u003cp\u003eSpecies-level Vaginal microbiota remodeling following CVG treatment.\u003cstrong\u003e (A)\u003c/strong\u003e PCoA score plot on ASV level comparing the pre-treatment and post-treatment groups, illustrating overall differences in microbial community structure.\u003cstrong\u003e (B) \u003c/strong\u003eDifferential abundance analysis between the pre-treatment and post-treatment groups. \u003cstrong\u003e(C)\u003c/strong\u003e Microbial composition displayed as stacked bar plots, showing changes in community structure after CVG intervention.\u003cstrong\u003e (D)\u003c/strong\u003e Vaginal microbiota health index at the species level, reflecting shifts toward a healthier microbial state following treatment. \u003cstrong\u003e(E)\u003c/strong\u003e Vaginal microbiota dysbiosis index, indicating changes in microbial imbalance associated with CVG intervention.\u003cstrong\u003e (F)\u003c/strong\u003e Correlation network analysis illustrating microbial interaction patterns and network restructuring after treatment.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8978688/v1/21a09916352b04a0846814ce.jpg"},{"id":104413825,"identity":"b73e630c-88e0-4268-adb3-8d843e204f5f","added_by":"auto","created_at":"2026-03-11 13:05:29","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":467325,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePlasma proteomic profiling and identification of immune-related targets following CVG treatment.\u003c/strong\u003e(A) PLS-DA score plot comparing the pre-treatment and post-treatment groups.(B) Sample correlation heatmap illustrating global similarity patterns between the pre-treatment and post-treatment plasma proteomic profiles.(C) Volcano plot showing DEMs between the pre- and post-treatment groups.(D) Hierarchical clustering analysis of DEMs, revealing distinct expression signatures associated with CVG intervention.(E) PPI network constructed from common DEPs; node color and size reflect the degree of network connectivity.(F) Molecular docking analysis of CPS-B with DDX60, supporting potential ligand–target interactions within immune-related antiviral pathways.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8978688/v1/4668d55653b312f40603ee7c.jpg"},{"id":104416378,"identity":"a12181ff-f0ef-4754-99ce-1522b4cfda0e","added_by":"auto","created_at":"2026-03-11 13:15:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2069450,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8978688/v1/411f3db7-4a2a-4945-a778-672eaf0839de.pdf"},{"id":104413546,"identity":"237c4870-dc4b-4a72-80b5-deb998ffdc11","added_by":"auto","created_at":"2026-03-11 13:04:46","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":15896,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8978688/v1/ea58a44bcdfbe9eba6b15c7b.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Clinical Efficacy, Safety, and Multi-Omics Insights of Capilliposide Vaginal Gel in CIN with High-Risk HPV: A Prospective, Multicenter Study","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe management of cervical intraepithelial neoplasia still requires attention.Timely and effective intervention for cervical intraepithelial neoplasia (CIN) is a critical determinant of the long-term success of cervical cancer prevention programs. CIN represents a histopathological continuum ranging from low-grade squamous intraepithelial lesions (LSIL; CIN 1) to high-grade squamous intraepithelial lesions (HSIL; CIN 2/3), the latter conferring a substantially increased risk of progression to invasive cervical cancer. Persistent infection with high-risk human papillomavirus (HR-HPV) is well established as the central etiological driver of this malignant transformation cascade [1\u0026ndash;3].\u003c/p\u003e\u003cp\u003eAlthough approximately 60% of LSIL lesions regress spontaneously within two years [4,5], a clinically significant proportion persists (16\u0026ndash;30%), and 4\u0026ndash;10% progress to higher-grade disease [6]. These figures underscore a critical clinical dilemma: reliance on spontaneous regression alone exposes a subset of patients to prolonged viral persistence and progressive epithelial dysplasia, thereby undermining the preventive intent of screening-based surveillance strategies.\u003c/p\u003e\u003cp\u003e Current clinical guidelines recommend active surveillance for LSIL, while ablative or excisional procedures remain the standard of care for HSIL. However, surgical interventions are inherently invasive and are associated with irreversible cervical damage, as well as increased risks of adverse obstetric outcomes, including preterm birth and cervical insufficiency [7,8]. More importantly, these approaches do not directly address the underlying HR-HPV infection, nor do they offer a satisfactory therapeutic option for patients with persistent viral infection who are unwilling or unsuitable for surgery. This therapeutic void is particularly problematic for women of reproductive age and those seeking non-invasive, fertility-preserving interventions, highlighting an urgent unmet need for effective pharmacological alternatives.\u003c/p\u003e\u003cp\u003eLysimachia capillipes Hemsl., a medicinal herb widely distributed in Southeast China, has long been used in traditional medicine for inflammatory disorders. Accumulating pharmacological evidence\u0026mdash;including our previous work\u0026mdash;has demonstrated that its major active constituents, Capilliposide B (CPS-B) and Capilliposide C (CPS-C), possess robust anti-inflammatory and anti-tumor activities [9]. These compounds have exhibited pronounced cytotoxic effects across multiple cancer models, including prostate [10,11], colorectal [12], and lung cancers [13], suggesting a broad spectrum of biological activity relevant to epithelial dysregulation and immune modulation. Capilliposide vaginal gel (CVG), formulated with CPS-B and CPS-C, has been approved in China for vaginal antibacterial and symptomatic applications; however, its therapeutic potential in CIN and HR-HPV\u0026ndash;associated disease has not been systematically evaluated.\u003c/p\u003e\u003cp\u003eBeyond viral oncogenesis, mounting evidence indicates that disruption of the vaginal microbiota constitutes a key cofactor in cervical disease progression. A Lactobacillus-depleted microbial ecosystem is associated with impaired mucosal barrier function, chronic inflammation, immune dysregulation, and reduced viral clearance. Nevertheless, existing CIN therapies largely overlook the role of the vaginal microenvironment and its contribution to both local and systemic immune responses. This conceptual gap limits mechanistic understanding and therapeutic innovation in CIN management.\u003c/p\u003e\u003cp\u003eBased on these considerations, we hypothesized that CVG may exert therapeutic effects through a dual mechanism: restoration of vaginal microbial homeostasis and modulation of host immune responses. To test this hypothesis, we conducted a prospective, multicentre, single-arm clinical study to evaluate the clinical efficacy, safety, and tolerability of CVG in women with CIN 1/2/3 and HR-HPV infection. Furthermore, by integrating vaginal microbiota profiling with plasma proteomic analysis, we sought to elucidate the local and systemic biological pathways underlying CVG-mediated lesion regression and viral clearance. This multi-omics framework provides mechanistic insight into CVG as a non-invasive, immune-modulating therapeutic strategy for cervical intraepithelial neoplasia.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"2 Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Trial Design\u003c/h2\u003e \u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThis study was designed as a prospective, multicentre, single-arm clinical trial to evaluate the clinical efficacy, safety, and tolerability of Capilliposide vaginal antiseptic gel (CVG) in patients with cervical intraepithelial neoplasia (CIN1/2/3) and high-risk human papillomavirus (HR-HPV) infection. The trial was conducted across three clinical sites in China: Zhejiang Cancer Hospital (Site A), the First People's Hospital of Hangzhou Lin'an District (Site B), and Yiwu Maternity and Children Hospital (Site C).The overall trial profile was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The study was conducted in accordance with the principles of Good Clinical Practice and the Declaration of Helsinki. The study protocol was reviewed and approved by the Medical Ethics Committee of Zhejiang Cancer Hospital (IRB No. 2023\u0026thinsp;\u0026minus;\u0026thinsp;140). The trial was registered with the International Traditional Medicine Clinical Trial Registry (ITMCTR2025002151) and has been completed. Written informed consent was obtained from all participants prior to enrollment.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Patients\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eBetween March 2023 and March 2024, a total of 81 patients with histologically confirmed CIN1/2/3 were enrolled across the three participating centers. Eligibility criteria included: (1) biopsy-confirmed CIN1/2/3, (2) confirmed HPV infection, and (3) ability and willingness to comply with the study protocol. Exclusion criteria comprised: (1) known allergy to CVG; (2) unsatisfactory colposcopic examination; (3) history of invasive cervical carcinoma or other invasive malignancies; (4) active skin or mucosal infections, overt pyometra, or acute inflammation of the female reproductive tract; (5) pregnancy or breastfeeding; and (6) serious comorbidities potentially affecting study compliance, including unstable cardiovascular disease, renal disease, chronic hepatitis, poorly controlled diabetes, or severe mental illness. Participants were withdrawn from the study in the event of adverse reactions, poor compliance, voluntary withdrawal, pregnancy, or investigator-determined inability to continue treatment or follow-up.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Study medication\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eCVG contains Capilliposide B (CPS-B) and Capilliposide C (CPS-C), two bioactive compounds isolated from Lysimachia capillipes Hemsl. The formulation used in this study contained a total of 1% CPS-B and CPS-C and was manufactured by Hangzhou Yirui Medical Technology Co., Ltd. (China). Treatment was initiated on the second day after completion of menstruation. CVG has been approved for clinical use under the Zhejiang Health Disinfection Certificate (License No. 2022-0006).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Study Procedures\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eEligible participants were assigned sequentially to two study phases (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In the initial safety evaluation phase (Group 1), 20 participants self-administered CVG intravaginally at a dose of 3 g every other night for 4 weeks. Administration was performed before bedtime according to standardized instructions. Following confirmation of acceptable safety outcomes, the study proceeded to the efficacy evaluation phase (Group 2), in which participants received CVG treatment for 12 weeks. Clinical symptoms were monitored via telephone follow-up at two-week intervals. Adverse events were assessed and graded in real time using the Common Terminology Criteria for Adverse Events (CTCAE), version 5.0, and their relationship to the study medication was evaluated. Combined ThinPrep cytology test (TCT) and HPV testing were performed 3\u0026ndash;6 months after treatment completion. For patients presenting with abnormal cytology results, colposcopically directed biopsy was strongly recommended but not mandatory, and patient preference was respected. Clinical efficacy, safety, and tolerability were evaluated comprehensively at the end of follow-up.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Clinical Outcome Measures and Statistical Analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe primary endpoint was histologic regression, defined as complete resolution of cervical lesions confirmed by post-treatment pathology. Histologic improvement was defined as a reduction in lesion grade, histologic persistence as unchanged pathology, and histologic progression as an increase in lesion severity. For participants who did not undergo post-treatment biopsy, cytologic regression was assessed based on changes in cervical cytology, including transitions from ASC-US, ASC-H, LSIL, or HSIL to negative for intraepithelial lesion or malignancy (NILM)[14]. Secondary endpoints included HR-HPV clearance, safety, and tolerability. HR-HPV clearance was defined as the absence of baseline HR-HPV genotypes at follow-up[15]. Safety was assessed in all participants receiving at least one dose of CVG, focusing on treatment-related adverse events, while tolerability was defined as completion of the prescribed treatment regimen. Statistical analyses were performed using SPSS software (version 25.0; SPSS Inc.). Baseline and post-treatment comparisons were conducted using standard hypothesis testing methods, as appropriate.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Sample Collection\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eVaginal lavage and peripheral blood samples were collected prior to CVG initiation and three months after treatment completion. Vaginal douching or rinsing was prohibited for 48 h before sampling. Vaginal lavage was performed using 10 mL of sterile saline, and recovered fluid was immediately transferred into sterile tubes. Following centrifugation, samples were divided into aliquots for microbiome and physicochemical analyses and stored at \u0026minus;\u0026thinsp;80 ℃ until use. Blood samples were processed and stored according to established protocols[16\u0026ndash;19] .\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Vaginal Microbiota Profiling by 16S rRNA Gene Sequencing\u003c/h2\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.7.1 Vaginal Microbiota Profiling by 16S rRNA Gene Sequencing\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eGenomic DNA was extracted from vaginal microbial communities in 54 paired pre- and post-treatment vaginal lavage samples using the FastPure Soil DNA Isolation Kit (Magnetic Bead; MJYH, Shanghai, China), following the manufacturer's instructions. DNA integrity was assessed by 1% agarose gel electrophoresis, and concentration and purity were measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). Full-length bacterial 16S rRNA genes were amplified using universal primers 27F (5\u0026prime;-AGRGTTYGATYMTGGCTCAG-3\u0026prime;) and 1492R (5\u0026prime;-RGYTACCTTGTTACGACTT-3\u0026prime;), each tagged with sample-specific PacBio barcodes[20,21]. PCR was performed in 20 \u0026micro;L reactions containing FastPfu buffer, dNTPs, primers, FastPfu DNA polymerase, and 10 ng of template DNA. Cycling conditions included an initial denaturation at 95 ℃ for 3 min, followed by 29 cycles of 95 ℃for 30 s, 60 ℃ for 30 s, and 72 ℃ for 45 s, with a final extension at 72 ℃ for 10 min. All reactions were conducted in triplicate. PCR products were purified using a PCR Clean-Up Kit (YuHua, Shanghai, China) and quantified with a Synergy HTX microplate reader (BioTek, USA). Purified amplicons were pooled in equimolar concentrations, and SMRTbell libraries were constructed using the SMRTbell Prep Kit 3.0 (Pacific Biosciences, USA). Sequencing was performed on the PacBio Revio System (Pacific Biosciences) at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.7.2 Sequence Processing and Microbiota Analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eRaw sequencing data were processed using SMRT Link software (v11.0) to generate high-quality HiFi reads, requiring a minimum of three full passes and \u0026ge;\u0026thinsp;99% sequence accuracy. Reads were demultiplexed by barcode and filtered by length, retaining sequences between 1,000 and 1,800 bp. Denoising and amplicon sequence variant (ASV) inference were performed using the DADA2 plugin within the QIIME2 pipeline (v2024), yielding single-nucleotide\u0026ndash;resolved ASVs. To normalize sequencing depth, samples were rarefied to 9,757 reads per sample, achieving an average Good's coverage of approximately 99%[22]. Taxonomic assignment was conducted using the classify-consensus-blast classifier in QIIME2 against the NT_16s database (v2024). Functional potential of the microbial communities was predicted using PICRUSt2 following standard protocols[23]. Alpha diversity metrics, including observed ASVs, Chao1 richness, Shannon index, and Good's coverage, were calculated using Mothur (v1.30.2). Beta diversity was assessed based on Bray\u0026ndash;Curtis dissimilarity and visualized by principal coordinate analysis (PCoA) using the vegan package in R. Permutational multivariate analysis of variance (PERMANOVA) was applied to evaluate treatment-associated differences in community composition.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Plasma Proteomic Analysis\u003c/h2\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.8.1 Enrichment of Low-Abundance Plasma Proteins\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eLow-abundance plasma proteins from 54 paired pre- and post-treatment samples were enriched using the EasyPep\u0026trade; Deep Low-Abundance Enrichment Kit (EasyPep, Catalog No. OSFP0002) according to the manufacturer's instructions.Magnetic beads were equilibrated and incubated with 100 \u0026micro;L of plasma at 37 ℃ for 1 h with constant agitation (1000 rpm). After incubation, beads were washed three times to remove high-abundance proteins, and the bead-bound low-abundance protein fraction was collected for downstream processing[24] .\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.8.2 Protein Digestion and Peptide Preparation\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eEnriched proteins were subjected to reduction and alkylation at 95 ℃ for 5 min, followed by enzymatic digestion at 37 ℃ for 2 h with agitation. Digestion was terminated using stop reagent, and peptide-containing supernatants were collected after centrifugation at 20,000 \u0026times; g for 1 min. Peptides were desalted using EasyPep\u0026trade; Desalting Cartridges (Catalog No. OSFP0050-W), vacuum-dried, reconstituted in loading buffer, and quantified using a NanoDrop One spectrophotometer (Thermo Scientific).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.8.3 LC\u0026ndash;MS/MS Analysis (DIA Mode)\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003ePeptide samples were analyzed on a Vanquish\u0026trade; Neo UHPLC system coupled to an Orbitrap\u0026trade; Astral mass spectrometer (Thermo Fisher Scientific) at Majorbio Bio-Pharm Technology Co., Ltd. Chromatographic separation was performed using a uPAC\u0026trade; High-Throughput column (75 \u0026micro;m \u0026times; 5.5 cm). Mobile phases consisted of 0.1% formic acid in water with 2% acetonitrile (buffer A) and 0.1% formic acid in water with 80% acetonitrile (buffer B), using an 8-min gradient[25]. Mass spectrometry data were acquired in data-independent acquisition (DIA) mode over an m/z range of 100\u0026ndash;1700. Instrument settings included an AGC target of 500%, maximum injection time of 3 ms, isolation window of 2 m/z, HCD collision energy of 25%, and 300 DIA windows.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e2.8.4 Protein Identification and Bioinformatic Analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eDIA data were processed using Spectronaut software (v19, Biognosys). Protein quantification was based on the top six peptides per protein and the top three fragment ions per peptide. Identification thresholds were set at a protein and peptide false discovery rate\u0026thinsp;\u0026le;\u0026thinsp;1% and peptide confidence\u0026thinsp;\u0026ge;\u0026thinsp;99%. Shared and modified peptides were excluded. Protein abundance was calculated as the summed peak area of qualified peptides, and only proteins identified with at least one unique peptide were retained. Proteomic data analysis was conducted using the Majorbio Cloud Platform. Proteins with missing values in more than 70% of samples were excluded, and remaining missing values were imputed using the sequential k-nearest neighbors (seqKNN) algorithm. Differentially expressed proteins (DEPs) were identified using a two-sided Student's t-test in R, with thresholds of fold change\u0026thinsp;\u0026ge;\u0026thinsp;1.5 or \u0026le;\u0026thinsp;0.67 and P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Functional annotation and pathway enrichment were performed using GO and KEGG databases, and protein\u0026ndash;protein interaction networks were constructed using STRING (v11.5).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e2.8.5 Molecular Docking Analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eProtein information was retrieved from the UniProt database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.uniprot.org\u003c/span\u003e\u003cspan address=\"https://www.uniprot.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and the corresponding three-dimensional structures were obtained from the RCSB Protein Data Bank (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.rcsb.org\u003c/span\u003e\u003cspan address=\"https://www.rcsb.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Protein structures were imported into Discovery Studio 2019 (BIOVIA, San Diego, CA, USA) for structural preparation, including removal of crystallographic water molecules, addition of hydrogen atoms, assignment of partial charges, completion of missing residues, and reconstruction of incomplete side chains. The optimized protein structures were subsequently saved in PDB format. The chemical structure of the small-molecule ligand was obtained from the PubChem database and subjected to energy minimization using Discovery Studio 2019. The optimized ligand structure was exported in PDB format. Preparation of receptor and ligand PDBQT files was performed using AutoDock Tools (AutoDock 4.0). Molecular docking simulations were conducted using AutoDock Vina (version 1.2.6) with default parameters. Docking poses were ranked based on binding affinity scores, and the most favorable binding conformations were selected for further analysis. Protein\u0026ndash;ligand interactions were visualized and analyzed using PyMOL (version 3.1) and Discovery Studio 2019.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3 Result","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Clinical Information\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eBetween March 2023 and March 2024, 81 participants meeting the eligibility criteria were enrolled across three clinical centers, including 9 patients from Site A, 35 from Site B, and 37 from Site C. Baseline demographic and clinical characteristics are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The median age of the study population was 42 years. An initial safety run-in cohort of 20 participants was assigned to Group 1 for preliminary safety evaluation. Two participants discontinued the study due to treatment-related local adverse reactions in Group 1, such as vaginal bleeding and pruritus. Following confirmation of acceptable safety, 18 enrolled participants proceeded to Group 2 for further assessment of safety and efficacy. During the study period, 14 participants discontinued the trial, resulting in 67 participants included in the final efficacy and safety analyses (Supplementary Table\u0026nbsp;1). At baseline, histopathological assessment identified 61 participants (91.0%) with CIN 1 and 6 participants (9.0%) with CIN 2/3. All participants were confirmed to have high-risk HPV (HR-HPV) infection. Specifically, HPV16 was detected in 16.4% (11/67) of participants, HPV18 in 7.5% (5/67), and other HR-HPV genotypes in 76.1% (51/67).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCharacteristics of Evaluable Participants on Baseline.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eCharacteristics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e%\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAge, years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e41.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e58.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eEducation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePrimary school\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUnior high school\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e29.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSenior high school\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUniversity and above\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e35.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMenstrual history\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePremenopause\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e70.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePausimenia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e29.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSmoking status\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eDrinking History\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e92.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePast History\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHypertension\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e94\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePrevious HPV vaccine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e82\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTCT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNILM,ASCUS or LSIL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e92.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eASC-H or HSIL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eHistology\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCIN 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e91\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCIN 2/3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eHR-HPV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16、Others (+)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18、Others (+)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOther high-risk HPV (+)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e76.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003ePersistent HR-HPV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e40.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e40.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNot detailed\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e19.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Treatment Adherence and Loss to Follow-up\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eA dropout rate of 17% was observed in this clinical trial. Among the 81 enrolled women who initiated CVG treatment, a total of 14 participants discontinued the study during the treatment or follow-up period. Two participants withdrew because of local adverse events, including vaginal bleeding or pruritus in Group 1. Specifically, seven participants were lost to follow-up. One participant withdrew from the study to undergo in vitro fertilization\u0026ndash;embryo transfer, and one participant discontinued treatment due to pregnancy. Two participants elected to undergo loop electrosurgical excision procedure owing to concerns about potential progression of squamous intraepithelial lesions. One participant discontinued treatment for unspecified personal reasons.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.3 CIN Regression and HPV Clearance at Follow-up\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eBecause colposcopically directed biopsy is an invasive procedure, post-treatment histological evaluation was performed selectively. Among the 67 participants included in the final analysis, 13 underwent both ThinPrep cytologic testing (TCT) and biopsy, while the remaining 45 participants were evaluated by TCT alone. Among the 13 participants with paired histological assessments, histologic regression was observed in 53.8% (7/13) of cases, whereas histologic persistence was observed in 46.2% (6/13). Specifically, regression occurred in 70% (7/10) of patients with CIN 1 and in 33.3% (1/3) of patients with CIN 2/3. Histologic persistence was observed in 40% of CIN 1 cases. Based on cytologic evaluation, 82% (55/67) of participants demonstrated regression to negative for intraepithelial lesion or malignancy (NILM)(Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These included 17 cases initially diagnosed as CIN 1, two as CIN 2/3, two as ASC-H, and 15 as ASC-US. Detailed cytologic outcomes are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. No cases of progression to invasive cervical cancer were observed during follow-up.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTCT Results of Baseline Status and After CVG Treatment.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eBaseline Status\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eAfter CVG treatment\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTCT results\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTCT result\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eNILM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNILM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eASC-H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eASC-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLSIL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHSIL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eASC-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNILM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eASC-H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eASC-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLSIL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHSIL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eLSIL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNILM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eASC-H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eASC-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLSIL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHSIL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eASC-H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNILM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eASC-H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eASC-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLSIL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHSIL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eHSIL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNILM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eASC-H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eASC-US\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLSIL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHSIL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAmong the 67 participants with baseline HR-HPV infection, 23 patients (34.3%) achieved complete HR-HPV clearance, while 23 patients (34.3%) exhibited persistent infection. Three patients (4.4%) showed progression characterized by the detection of additional HPV genotypes (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The clearance rate for HPV16 was 72.7% (8/11), and for HPV18 was 60.0% (3/5). Among participants with concomitant low-risk HPV infection, four achieved complete clearance of all HPV genotypes, seven exhibited persistence of the same HPV genotype, and two demonstrated progression in HPV genotype composition. Notably, no participants showed progression from low-risk to high-risk HPV types during follow-up.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eHPV Status After CVG Treatment.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCharacteristics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e%\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClearance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e23/67 (34.3%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePersistence\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e23/67 (34.3%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProgression\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3/67 (4.4%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOverall response\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e41/67(61%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClearance of HPV 16 positive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8/11 (72%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClearance of HPV 18 positive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3/5 (60%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Safety and Acceptability of CVG\u003c/h2\u003e \u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eAll 67 participants included in the final analysis were evaluated for safety. During CVG treatment, 30% (20/67) of participants reported at least one adverse event (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). No grade 2, 3, or 4 adverse events were observed, and no systemic adverse reactions were reported. In Group 1, one participant experienced vaginal bleeding and one reported vaginal pruritus. In Group 2, all reported adverse events were local and mild in severity, including vaginal pruritus (n\u0026thinsp;=\u0026thinsp;7), vaginal pain (n\u0026thinsp;=\u0026thinsp;3), vaginal discharge (n\u0026thinsp;=\u0026thinsp;2), vaginal bleeding (n\u0026thinsp;=\u0026thinsp;13), and pelvic pain (n\u0026thinsp;=\u0026thinsp;1). No adverse events were considered severe enough to interfere with daily activities.\u003c/p\u003e\u003cp\u003eDespite the occurrence of local adverse events, treatment acceptability was high. Overall, 91% of participants reported satisfaction with CVG use, and 94% considered the treatment to be safe. All participants expressed confidence in their ability to correctly administer the vaginal gel. Seventeen participants temporarily interrupted treatment for 2\u0026ndash;7 days due to vaginal bleeding or pruritus; treatment was resumed after symptom resolution, and all completed the prescribed 12-week treatment course.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSide Effects of CVG Treatment.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAdverse reactions\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSystemic symptoms\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVaginal pruritus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVaginal pain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVaginal discharge\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVaginal bleeding\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUlceration of the treatment site\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePelvic pain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Vaginal Microbiota Remodeling by CVG\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eOur analysis revealed significant changes in the vaginal microbiota composition following CVG treatment. A total of 2,850,277 high-quality 16S rRNA sequences were obtained, yielding 6,690 ASVs with 99.9% Good's coverage. PCoA based on Bray\u0026ndash;Curtis dissimilarity revealed clear separation between pre- and post-treatment samples (PERMANOVA P\u0026thinsp;=\u0026thinsp;0.001), indicating substantial microbial community remodeling (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Species-level community barplot analysis revealed significant alterations in the relative abundance of key taxa, with a notable increase in \u003cem\u003eLactobacillus crispatus\u003c/em\u003e and a marked decrease in \u003cem\u003eLactobacillus iners\u003c/em\u003e and \u003cem\u003eGardnerella vaginalis\u003c/em\u003e following CVG treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB; Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). At the genus level, \u003cem\u003eGardnerella\u003c/em\u003e exhibited a marked decrease following treatment. Furthermore, these findings were consistent with the results of the Wilcoxon rank-sum test (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC; Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). This shift suggests that CVG promotes a transition from a dysbiotic microbiota to a Lactobacillus-dominant healthy state, which may play a crucial role in restoring the vaginal mucosal barrier and facilitating the clearance of HR-HPV, thereby contributing to the regression of CIN.\u003c/p\u003e \u003cp\u003eIn addition, both species- and genus-level analyses showed an increase in the Microbiome Health Index (GMHI) and a concomitant decrease in the Microbial Dysbiosis Index (MDI), further confirming a shift toward a healthy, eubiotic microbial composition.(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-E; Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC-D). Species-level correlation network analysis revealed that pre-treatment anaerobic taxa \u003cem\u003e(Peptostreptococcus anaerobius, Anaerococcus jeddahensis, Finegoldia magna)\u003c/em\u003e formed highly interconnected modules dominated by positive correlations. Post-treatment, multiple negative correlations and antagonistic interactions emerged, indicating that CVG not only altered individual taxa abundances but also restructured microbial interaction networks (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Plasma Proteomic Profiling and Immune-Related Targets\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eDIA-MS plasma proteomic profiling identified 6,052 proteins across all samples. Correlation and PLS-DA analyses revealed clear separation between pre- and post-treatment groups, with post-treatment samples clustering more tightly, indicating consistent systemic proteomic remodeling following CVG intervention (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u0026ndash;B). Comparative analysis identified 389 DEPs (305 upregulated, 84 downregulated) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). KEGG enrichment highlighted pathways associated with cell adhesion, chromatin organization, and intracellular signaling, including adherens junctions and ATP-dependent chromatin remodeling (Figure SE). The heatmap illustrates the impact of CVG intervention on the protein expression profile, with clustering analysis revealing differentially expressed genes between the two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).PPI network analysis identified core nodes (ATP5PD, NDUFV1, HIST2H3C, IMMT, UQCRC2, YWHAZ) coordinating mitochondrial function, chromatin dynamics, and signal transduction (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eAmong candidate immune-related proteins, DDX60 emerged as a key mediator of systemic antiviral responses. DDX60 is an interferon-inducible DExD/H-box RNA helicase that enhances RIG-I-like receptor (RLR)-mediated type I interferon (IFN-I) signaling and interferon-stimulated gene (ISG) expression, thereby promoting innate antiviral immunity [26\u0026ndash;29]. DDX60 also participates in viral RNA degradation and translational suppression, functioning as a multifunctional antiviral effector. Upregulation of DDX60 and related signaling pathways after CVG treatment coincided with the restoration of Lactobacillus-dominant vaginal microbiota, suggesting that CVG may enhance HR-HPV clearance by synergistically modulating both mucosal and systemic antiviral immunity. Molecular docking demonstrated stable binding of CPS-B to a core protein within the DDX60 network, supporting potential ligand\u0026ndash;target interactions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eCIN and persistent HR-HPV infection continue to pose substantial challenges in clinical management. While surgical interventions remain effective for high-grade lesions, they are associated with cervical damage and adverse reproductive outcomes, and no widely accepted non-invasive therapy is currently available for persistent HPV infection or low-grade CIN requiring active intervention.\u003c/p\u003e\u003cp\u003eSpontaneous regression of LSIL/CIN1 is well recognized; however, population-based studies show that a substantial proportion of LSIL persists beyond 12 months, with a subset progressing to higher-grade disease [30\u0026ndash;32]. In the present study, histologic regression was observed in 53.8% of patients with paired biopsies, while cytologic regression to NILM occurred in 80% of patients assessed by TCT alone, with no cases of progression to invasive cervical cancer during follow-up. The overall HR-HPV clearance rate of 34.3% compares favorably with pharmacological intervention studies, including interferon-based therapies, which typically report clearance rates of 20\u0026ndash;25% [33,34]. Clearance of HPV16 and HPV18 was particularly notable (72.7% and 60.0%, respectively), although interpretation is limited by sample size. Consistent with prior reports, regression rates were higher in CIN1 than in CIN2/3, reflecting differences in lesion biology and immune responsiveness [35]. Collectively, these findings support CVG as a potential non-surgical therapeutic option for selected patients with CIN and HR-HPV infection.CVG demonstrated a favorable safety profile, with mild and transient local adverse events and no systemic toxicity. High treatment adherence and patient satisfaction highlight its acceptability as a self-administered topical therapy, making it an attractive option for women seeking fertility-preserving alternatives to surgical intervention or adjunctive strategies to promote HPV clearance during CIN surveillance.\u003c/p\u003e\u003cp\u003eAccumulating evidence links persistent HR-HPV infection to vaginal microbiota dysbiosis, characterized by reduced Lactobacillus dominance and enrichment of anaerobic taxa [36\u0026ndash;37], which is associated with impaired epithelial barrier function, chronic inflammation, and weakened local antiviral immunity. \u003cem\u003eLactobacillus crispatus\u003c/em\u003e is widely recognized as the most beneficial Lactobacillus species in the vaginal niche, as it efficiently produces lactic acid to maintain a low vaginal pH (\u0026le;\u0026thinsp;4.5), secretes antimicrobial peptides, and strengthens the vaginal mucosal barrier by promoting tight junction formation [38]. In contrast, \u003cem\u003eLactobacillus iners\u003c/em\u003e, though common in apparently healthy vaginas, has limited acid production and is linked to dysbiosis, while \u003cem\u003eGardnerella vaginalis\u003c/em\u003e promotes inflammation, epithelial barrier disruption, and impaired antiviral immunity, facilitating HR-HPV persistence and CIN progression [39\u0026ndash;40]. It strongly suggests that CVG effectively targets dysbiosis-associated taxa while fostering the growth of beneficial lactobacilli, thereby creating a microenvironment unfavorable for HR-HPV survival and CIN progression. By restoring a Lactobacillus-dominant vaginal microbiota that maintains acidity, inhibits HR-HPV and pathogenic bacteria, and enhances local antiviral immunity and mucosal barrier function, CVG plausibly facilitates HR-HPV clearance and CIN regression, consistent with improved clinical outcomes in treated patients.\u003c/p\u003e\u003cp\u003e Beyond local microbial effects, plasma proteomic profiling revealed consistent systemic molecular remodeling following CVG treatment. Differentially expressed proteins were enriched in pathways related to epithelial integrity (adherens junctions), chromatin organization, and intracellular signaling. These pathways are central to epithelial repair, immune cell activation, and transcriptional regulation during antiviral responses [41, 42]. Among immune-related candidates, upregulation of antiviral signaling components, including DDX60-associated networks, suggests enhanced innate immune responsiveness following CVG treatment. DDX60 is an interferon-inducible RNA helicase involved in RIG-I\u0026ndash;like receptor signaling and amplification of type I interferon responses, thereby contributing to antiviral defense [43]. Although plasma proteomics cannot localize tissue-specific effects, the coordinated modulation of immune-related pathways supports a systemic component of CVG-associated immunomodulation that may complement local mucosal effects.\u003c/p\u003e\u003cp\u003eTaken together, the clinical, microbiome, and proteomic data support a working model in which CVG may mediate CIN regression and HR-HPV clearance through complementary local and systemic mechanisms. Locally, CVG appears to restore vaginal microbial homeostasis, promoting Lactobacillus dominance, suppressing dysbiosis-associated anaerobes, and improving epithelial barrier integrity. Systemically, CVG is associated with modulation of antiviral and immune-related molecular pathways, including DDX60-linked interferon signaling, which may enhance innate immune surveillance. These mechanisms are likely to act synergistically rather than independently, providing a plausible explanation for the observed cytologic regression and partial HR-HPV clearance. While causality cannot be established in the absence of a control group, the convergence of multi-omics signals with clinical outcomes supports further investigation of CVG as a non-invasive, fertility-preserving therapeutic strategy.\u003c/p\u003e\u003cp\u003eSeveral limitations should be noted. The single-arm design limits causal inference, and spontaneous regression cannot be excluded. Incomplete histological follow-up and the small CIN2/3 sample size are additional limitations. Future randomized controlled trials with longer follow-up and mechanistic studies are needed to confirm efficacy and clarify the molecular pathways underlying CVG's effects.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"5 Conclusions","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThis study provides evidence supporting the clinical efficacy and safety of CVG in the treatment of CIN and HR-HPV infection. By modulating the vaginal microbiome and enhancing immune responses, CVG offers a novel, non-invasive treatment option that could complement current therapeutic strategies. The therapeutic potential of CVG to induce regression of CIN and clear HR-HPV suggests that it may be an effective alternative treatment, especially for patients seeking non-invasive, patient-controlled management options. Further studies are warranted to validate these findings and explore the broader applicability of CVG in clinical practice.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHPV\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHuman Papillomavirus\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHR-HPV\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHigh-Risk Human Papillomavirus\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCIN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCervical Intraepithelial Neoplasia\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCVG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCapilliposide Vaginal Gel\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDEPs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDifferentially Expressed Proteins\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTCT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eThinPrep Cytologic Test\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLSIL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLow-Grade Squamous Intraepithelial Lesion\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHSIL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHigh-Grade Squamous Intraepithelial Lesion\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNILM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNegative for Intraepithelial Lesion or Malignancy\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eASC-US\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAtypical Squamous Cells of Undetermined Significance\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eASC-H\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAtypical Squamous Cells, Cannot Exclude High-Grade Squamous Intraepithelial Lesion\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003eThe study was conducted in accordance with the principles of Good Clinical Practice and the Declaration of Helsinki. The study protocol was reviewed and approved by the Medical Ethics Committee of Zhejiang Cancer Hospital (IRB No. 2023\u0026thinsp;\u0026minus;\u0026thinsp;140). The trial was registered with the International Traditional Medicine Clinical Trial Registry (ITMCTR2025002151) and has been completed. Written informed consent was obtained from all participants prior to enrollment.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting Interests\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCorresponding author\u003c/h2\u003e \u003cp\u003eCorrespondence to [email protected]; [email protected];\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eAcknowledge\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by grants-in-aid for scientific research from the National Natural Science Foundation of China (number 82405091), the Natural Science Foundation of Zhejiang Province (number Q24H290031), the Zhejiang Province Traditional Chinese Medicine Science and Technology Project (number GZY-ZJ-KJ-23050, number GZY-ZJ-KJ-24063).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eY F: Conceptualization, Methodology, Formal analysis, Investigation, Writing - original draft, Supervision, Funding acquisition. LL W, XL C, Z W, Q X, LJ Y, YQ L, JM Z: Clinical Information Collection. HM L, W Z: Investigation, Resources, Project administration, Funding acquisition. H Y, JK T:Validation. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eQin F, Sun X, Tian M, Jin S, Yu J, Song J, Wen F, Xu H, Yu T, Dong Y. Prediction of lymph node metastasis in operable cervical cancer using clinical parameters and deep learning with MRI data: a multicentre study. \u003cem\u003eInsights Imaging\u003c/em\u003e. 2024;15(1):56. doi:10.1186/s13244-024-01618-7.\u003c/li\u003e\n\u003cli\u003eChou HH, Wang PH, Chen CA, et al. Diagnostic performance of magnetic resonance imaging in detecting lymph node metastasis in cervical cancer. \u003cem\u003eJ Gynecol Oncol\u003c/em\u003e. 2010;21(4):249–255.\u003c/li\u003e\n\u003cli\u003eTierney JF, Stewart LA, Ghersi D, Burdett S, Sydes MR. Practical methods for incorporating summary time-to‐event data into meta‐analysis. \u003cem\u003eTrials\u003c/em\u003e. 2007;8:16.\u003c/li\u003e\n\u003cli\u003eWu J, Huang YW, Chen YJ, et al. Comparison of CT, MRI, and PET in the detection of pelvic lymph node metastasis in cervical cancer. \u003cem\u003eRadiology\u003c/em\u003e. 2020;296(3):574–583.\u003c/li\u003e\n\u003cli\u003eHe LY, Zhao SH, Liu M, et al. MRI radiomics for predicting pelvic lymph node metastasis in early cervical cancer: a multicenter study. \u003cem\u003eEur Radiol\u003c/em\u003e. 2021;31(6):4268–4277.\u003c/li\u003e\n\u003cli\u003eOdunsi K, Zhang Y, Lin C, et al. Integrated PET–CT enhances accuracy in lymph node staging of cervical cancer. \u003cem\u003eClin Nucl Med\u003c/em\u003e. 2001;26(7):567–572.\u003c/li\u003e\n\u003cli\u003eLee H, Kim DW, Park SY, et al. Radiomic CT texture analysis predicts lymph node metastasis in cervical cancer. \u003cem\u003eAcad Radiol\u003c/em\u003e. 2023;30(2):250–260.\u003c/li\u003e\n\u003cli\u003eZhang Y, Smith K, Li J, et al. Deep learning–based MRI improves preoperative lymph node metastasis detection in cervical cancer. \u003cem\u003eInsights Imaging\u003c/em\u003e. 2024;15(1):57. doi:10.1186/s13244-024-01619-6.\u003c/li\u003e\n\u003cli\u003eMoher D, Liberati A, Tetzlaff J, Altman DG; PRISMA Group. Preferred Reporting Items for Systematic Reviews and Meta-Analyses: the PRISMA statement. \u003cem\u003ePLoS Med\u003c/em\u003e. 2009;6(7):e1000097.\u003c/li\u003e\n\u003cli\u003eDerSimonian R, Laird N. Meta-analysis in clinical trials. \u003cem\u003eControl Clin Trials\u003c/em\u003e. 1986;7(3):177–188.\u003c/li\u003e\n\u003cli\u003eHiggins JP, Thompson SG, Deeks JJ, Altman DG. Measuring inconsistency in meta-analyses. \u003cem\u003eStat Med\u003c/em\u003e. 2002;21(11):1539–1558.\u003c/li\u003e\n\u003cli\u003eEgger M, Davey Smith G, Schneider M, Minder C. Bias in meta-analysis detected by a simple, graphical test. \u003cem\u003eBMJ\u003c/em\u003e. 1997;315(7109):629–634.\u003c/li\u003e\n\u003cli\u003eMoher D, Liberati A, Tetzlaff J, Altman DG; PRISMA Group. Preferred Reporting Items for Systematic Reviews and Meta-Analyses: The PRISMA Statement. \u003cem\u003ePLoS Medicine\u003c/em\u003e. 2009;6(7):e1000097.\u003c/li\u003e\n\u003cli\u003eTierney JF, Stewart LA, Ghersi D, Burdett S, Sydes MR. Practical methods for incorporating summary time-to-event data into meta-analysis. \u003cem\u003eTrials\u003c/em\u003e. 2007;8:16.\u003c/li\u003e\n\u003cli\u003eDerSimonian R, Laird N. Meta-analysis in clinical trials. \u003cem\u003eControlled Clinical Trials\u003c/em\u003e. 1986;7(3):177–188.\u003c/li\u003e\n\u003cli\u003eHiggins JP, Thompson SG. Quantifying heterogeneity in a meta-analysis. \u003cem\u003eStatistics in Medicine\u003c/em\u003e. 2002;21(11):1539–1558.\u003c/li\u003e\n\u003cli\u003eEgger M, Davey Smith G, Schneider M, Minder C. Bias in meta-analysis detected by a simple, graphical test. \u003cem\u003eBMJ\u003c/em\u003e. 1997;315(7109):629–634.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Cervical intraepithelial neoplasia, Capillposide, Efficacy, Safety, Microbiomics, Proteomics","lastPublishedDoi":"10.21203/rs.3.rs-8978688/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8978688/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eCervical intraepithelial neoplasia (CIN) is a precursor of cervical cancer. This prospective, multicenter study evaluated the clinical efficacy, safety, and multi-omics characteristics of Capilliposide vaginal gel (CVG) in women with biopsy-confirmed CIN and high-risk human papillomavirus (HR-HPV) infection.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThis prospective, multicenter, single-arm study enrolled 81 women with CIN 1/2/3 and HR-HPV infection. In Group 1, participants received CVG intravaginally every other day for 4 weeks to assess preliminary safety. Following the absence of significant adverse events, Group 2 was treated with CVG for 12 weeks to evaluate both safety and efficacy. Vaginal lavage and plasma samples were collected at baseline and 3 months post-treatment for microbiota analysis via 16S rRNA gene sequencing and plasma proteomics.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eAmong the 67 participants who completed treatment, CVG was well tolerated, with no grade\u0026thinsp;\u0026ge;\u0026thinsp;2 or systemic toxicity. Cytological regression to negative for intraepithelial lesion or malignancy occurred in 82% of evaluable participants, and histologic regression was observed in 53.8% of paired biopsies. The overall HR-HPV clearance rates were 72.7% for HPV16 and 60.0% for HPV18. Multi-omics analysis showed a shift to a Lactobacillus-dominant vaginal microbiota and modulation of immune and epithelial repair pathways.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eCVG as a safe, non-invasive, fertility-preserving treatment for CIN with HR-HPV infection, accompanied by changes in the vaginal microbiota and immune-related molecular pathways.\u003c/p\u003e\u003ch2\u003eTrial registration\u003c/h2\u003e \u003cp\u003eThe trial was retrospectively registered on October 8, 2024, with the International Traditional Medicine Clinical Trial Registry (Registration No. ITMCTR2025002151), and the study has been completed.\u003c/p\u003e","manuscriptTitle":"Clinical Efficacy, Safety, and Multi-Omics Insights of Capilliposide Vaginal Gel in CIN with High-Risk HPV: A Prospective, Multicenter Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-11 11:59:18","doi":"10.21203/rs.3.rs-8978688/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-11T05:21:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-06T06:34:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"133080876379965401241140438758469998287","date":"2026-04-27T07:47:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"259421582171185661540491009023849376937","date":"2026-04-25T06:43:32+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-26T18:58:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"42673324272900510254246035830461076286","date":"2026-03-22T14:13:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"309755346973888430985073710778649134415","date":"2026-03-16T07:29:53+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-05T13:21:50+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-02-27T07:05:25+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-27T04:03:17+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-27T04:00:23+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Cancer","date":"2026-02-26T13:56:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b15b0aa5-3aaf-4f6c-b4ae-ec3123084726","owner":[],"postedDate":"March 11th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-11T05:21:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-06T06:34:44+00:00","index":41,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-11T05:25:50+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-11 11:59:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8978688","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8978688","identity":"rs-8978688","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}