Different effects of CO2 laser and estrogen treatment on vaginal mucosa microbiota and function in genitourinary syndrome of menopause patients

To characterize the effects of CO2 laser treatment and estrogen treatment on vaginal microbiota in patients with genitourinary syndrome of menopause (GSM).


INTRODUCTION
5][6] Hormone replacement is the standard treatment for GSM, involving the application of low-doses of estrogen for topical vaginal treatment.However, patient compliance is suboptimal, and some individuals have contraindications to its usage. 3,7O 2 fractional laser therapy is an emerging treatment for GSM in recent years.Research indicates its efficacy in enhancing vaginal function in patients with GSM, resulting in better vaginal health index (VHI) scores and a more favorable vaginal microenvironment. 8Research demonstrates that following CO 2 laser therapy, there's an increase in the proportion of vaginal Lactobacillus and normal flora, accompanied by a decrease in vaginal pH after the therapeutic regimen. 9his suggests that CO 2 laser therapy may enhance vaginal flora in patients with GSM.However, current studies still have certain limitations.Most of these are short-term studies lacking long-term outcomes.Additionally, the classification of vaginal microflora lacks refinement, and there is an absence of a control group for comparison.
Therefore, this study was conducted to investigate the vaginal microbiota of individuals with GSM undergoing various treatment modes, employing 16S rRNA gene sequencing.The investigation included bioinformatics analysis and assessment of VHI and urogenital distress inventory-6 (UDI-6) scores among GSM patients.Aiming to discern variations in vaginal microbial community structure between CO 2 laser and estrogen treatment, as well as to explore the effects of different treatment modes on GSM at 3 and 12 months after treatment.

Case inclusion
In this study, we anticipated a treatment and follow-up failure rate of 20%.Consequently, this study enrolled 25 patients in each group.The study comprised 75 patients seeking treatment for "genitourinary syndrome" at the gynecology outpatient clinic of the Fifth Affiliated Hospital of Guangzhou Medical University from April 2019 to June 2021.All participants provided informed consent form, and the study was approved by the Medical Ethics Committee of the Fifth Affiliated Hospital of Guangzhou Medical University (KY01-2020-09-02).

Inclusive criteria and exclusion criteria
Inclusion criteria

Grouping and treatment
Following gynecological examination, excretion testing, and assessment of genital tract inflammation, 75 cases were randomly divided into three groups: the control group, the estrogen treatment group, and the CO 2 laser treatment group.The CO 2 laser group utilized the HiScan V2LR laser, equipped with a 360 probe scanning system (Hunan Zexin Medical Equipment Co., Ltd., model: JZ-3D) for the treatment of the vaginal wall.Each treatment course comprised 2-3 laser sessions, spaced at intervals of approximately (4 ± 1) weeks.Treatment parameters included a power range of 35-40 W, dwell time of 800-1000 s, spot spacing of 800-1000 μm, with the option for single or double stacking.Treatment was administered on an outpatient basis without the use of analgesia or anesthesia.Sexual intercourse is prohibited for 7 days following laser treatment.The estrogen treatment group received vaginal estriol suppositories daily, with a dosage of 0.5 mg, for a duration of 3 months.After 3 months of estrogen or CO 2 laser treatment, the patients did not receive any further estrogen or CO 2 laser treatment, and their medication history was confirmed during the follow-up.The control group did not undergo any treatment and underwent evaluation 3 months after enrollment.
During the follow-up period, 7 cases were lost, 2 cases were excluded from the group due to an inability to continue hormone therapy, and 2 cases were discontinued treatment after only one laser session due to personal reasons.Consequently, the control group comprised 22 cases, the hormone group consisted of 21, and the CO 2 laser group included 21 cases.There were no significant adverse effects reported after laser treatment, and all patients tolerated the procedure well.The research process is shown in Figure 1.

DNA extraction and 16S rRNA sequencing
Vaginal swab samples were collected from patients in each group after treatment for 3 months.All samples were placed in liquid nitrogen and stored at À80 C immediately prior to treatment.Microbial DNA was extracted from vaginal swab biopsies using a DNA kit (Omega Biotek, Norcross, GA, USA) according to the manufacturer's protocol.Amplicons were extracted from 2% agarose gels and purified with the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) according to the manufacturer's instructions.Amplicons were identified using QuantiFluor-ST (Promega, USA).Purified amplicons were equimolarly pooled and subjected to double-end sequencing (2 Â 250) using Illumina Hiseq 2500 according to standard protocols.The PCR amplification targeted the 16S rDNA V3-V4 region of the eukaryotic RNA gene.The amplification process included an initial denaturation at 95 C for 2 min, followed by 27 cycles denaturation at 98 C for 10 s, annealing at 62 C for 30 s, and extension at 68 C for 30 s (repeated for a total of 27 cycles), with a final extension at 68 C for 10 min.The primers used for amplification were 341F: CCTACGGGNGGCWGCAG and 806R: GGACTACHVGGGTATCTAAT.It is important to note that each sample had a unique eightbase barcode sequence.PCR was performed in triplicate, with 50 μL of a mixture containing 5 μL of 10 Â KOD buffer, 5 μL of 2.5 mM dNTPs, 1.5 μL of each primer (5 μM), 1 μL of KOD polymerase, and 100 ng of template DNA.

Sequencing data analysis
The raw data was filtered to obtain clean reads by eliminating joint contamination and low-quality sequences.Paired end clean read lengths were merged into raw tags using FLASH (Short Read Length Quick Adjustment, v 1.2.11) with a minimum overlap of 10 bp and a mismatch error rate of 2%. 10 The noisy sequences of raw tags were filtered through the QIIME (v1.9.1) pipeline using specific filtering conditions to obtain high-quality clean tags. 11The labels were then clustered into operational taxonomic units (OTUs) using the USEARCH (v 7.0.1090)software script, with a similarity threshold of 97%. 12 Taxonomic classification of representative OTU sequences was performed using the Ribosome Database Project classifier v.2.2, which was trained on the Greengenes database. 13,14Finally, OTU tables and phylogenetic trees were generated for diversity analysis.To estimate the diversity of the microbial communities in the sample, we used the Wilcoxon rank test to calculate the within-sample (alpha) diversity of the two groups.Beta diversity was estimated by calculating weighted Unifrac distances and visualized by principal coordinate analysis (PCoA).Statistical differences in the relative abundance of bacterial phyla and genera between the two groups were assessed using Metastats, the Kruskal-Wallis test for more than two groups.
F I G U R E 1 Flow chart for collecting clinical information from patients.Seventy-five patients with genitourinary syndrome of menopause (GSM) were randomly divided into three groups for different treatment modalities.A total of 22 cases in the control group, 21 cases in the hormone group, and 21 cases in the CO 2 hormone group were included in the follow-up study.

Enrichment function analysis
Genera were categorized into pre-and post-enrichment based on the mean abundance after patients treatment.Pearson correlation tests were conducted based on abundance to build a network correlating differentially enriched genera in the pre-and post-enrichment groups.The correlation network was visualized with Cytoscape (version 3.3.0).Pearson correlation tests were also performed to investigate the microbial disorder index (MDI) and GSMAI, as well as differential genera and predictive pathways.

Function description
The macrogenome of the vaginal microbiome was estimated through 16S rRNA sequences employing PICRUSt, a method that surveys the community phylogenetically by reconstructing the unobserved state.This approach predicts the abundance of gene family by leveraging phylogenetic information, with an estimated accuracy of 0.8.Closed OTU tables were employed as input for macrogenome interpolation, initially thinned to achieve a consistent sequencing depth before PICRUSt analysis.Subsequently, the resulting OTU tables were normalized based on the copy number of 16S rRNA gene.The predicted gene content of each individual was determined.The predicted profiles of functional composition were subsequently integrated into level 3 of the KEGG database pathway.The output files underwent additional analysis through the Statistical Analysis of Macrogenomic Mapping (STAMP) package.

VHI scores
VHI scores were employed to evaluate vaginal function pre-and post-therapy.The evaluation encompassed five primary parameters: overall elasticity, fluid secretions, epithelial mucosa, moisture, and vaginal pH. 15 The total score ranges from 5 to 25, with a lower VHI score indicating a poorer vaginal condition.Score below 15 suggest a state of vaginal atrophy.

UDI-6 Questionnaire
The UDI-6 Questionnaire was used to evaluate urinary function pre-and post-therapy.It consisted of six items that assessed urinary stress incontinence, detrusor overactivity, and nonbladder obstruction.

Immunohistochemical and HE and staining
Vaginal sections were underwent HE staining to measure the thickness of the vaginal epithelium pre-and post-12 month treatment.Type I and III antigen staining aided in assessing the collagen content and evaluating how different therapies impacted vaginal morphology.Results were analyzed using Image Pro Plus image analysis software.

Baseline characteristics
Among the 64 patients, the average age was 51 years.The average BMI was 21.7 kg/m 2 (ranging from 18.7 to 29.0), and they had an average of two pregnancies (ranging from 1 to 4).There were no notable differences in age, pregnancies, and BMI among the three groups.The other parameters are detailed in Table 1.

Effect of estrogen and CO 2 laser therapy on the abundance and diversity of vaginal mucosal flora
To evaluate the influence of disease activity on the microbial composition of the vaginal mucosa in all groups, we initially collected vaginal mucosal swab samples from participants enrolled in the GSM.Some samples were excluded due to contamination or assay failure, resulting in a final collection of 16 vaginal mucosal swab specimens.The results demonstrated differences in vaginal mucosal microorganisms compared to the control group following estrogen or CO 2 treatment.This implies that both treatments influenced the vaginal mucosal microbiota of patients with GSM.Specifically, the CO 2 laser treatment group exhibited a significantly larger distinction compared to the control group, and the distribution between the groups was more closely clustered (Figure 2a).Weighted non-metric multidimensional scaling (NMDS) showed that a more concentrated distribution of vaginal mucosal flora in the CO 2 group compared to the estrogen treatment, indicating increased stability and similarity of the mucosal flora in the CO 2 group (Figure 2b and Supplementary Material 1).Alpha diversity encompasses the diversity within a specific environment or ecosystem, primarily reflecting species richness and diversity.The results showed a significant reduction in the richness (Figure 2c and Supplementary Material 1), Chao1 index (Figure 2d and Supplementary Material 1) and ACE index (Figure 2e and Supplementary Material 1) of the bacterial flora in the estrogen and CO 2 groups compared to the control group.This suggests a decrease in species richness and OTU count in the vaginal mucosal flora following estrogen and CO 2 laser treatments.In addition, the Shannon index (Figure 2f and Supplementary Material 1) and Simpson index (Figure 2g and Supplementary Material 1) decreased in the treatment groups compared to the control group, indicting a reduction in the diversity of the vaginal mucosal flora decreased after estrogen and CO 2 treatments.The good-s_coverage index exceeded 0.97 in the control group, surpassed 0.99 in the estrogen group, and exceeded 0.98 in the CO 2 group (Figure 2h and Supplementary Material 1).This indicates high sequencing coverage, allowing the results to accurately depict the bacterial flora in the samples, ensuring the reliability of the findings.

LEfSe analysis of the effect of estrogen and CO 2 laser therapy on specific species of vaginal mucosal microbiota
To identify specific bacterial taxa differing between treatment and control groups, we employed LEfSe analysis, a high-dimensional biomarker discovery algorithm that utilizes linear discriminant analysis (LDA) to estimate the effect magnitude of each taxon.LEfSe (Figure 3a and Supplementary Material 1) revealed 52 discriminatory features (LDA ≥3) with significantly different relative abundance between treatment and control groups.Bacilli were enriched in the treatment groups at the phylum level, whereas Actinobacteria was enriched in the control group.At the family level, Lactobacillaceae and Aerococcaceae significantly dominated both treatment groups, while Bifidobacteriaceae and Prevotellaceae were significantly dominant in the control group.The hierarchical clustering diagram (Figure 3b and Supplementary Material 1) illustrates the structure of the vaginal mucosal microbiota and identifies the key bacteria in the treatment and control groups.As shown in the Figure 6b, the most significant compositional changes in the flora occurred primarily in the thick-walled phylum, including Bifidobacteriaceae and Actinobacteria.Notably, in the estrogen group, the vaginal mucosal flora exhibited significant changes, with higher levels of Lactobacillus and Streptococcus compared to the control group.Similarly, in the CO 2 group, the composition of the vaginal mucosal microbiota showed significant changes, characterized by increased levels of Lactobacillus and IheB3_7 compared to the control group.Additionally, the CO 2 group displayed significantly higher relative abundance of Trichoderma, Rumex, and Rhodobacter compared to the control group.

Effect and correlation analysis of estrogen and CO 2 laser therapy on the composition of bacterial flora
To examine specific alterations in vaginal mucosa flora in GSM patients, we assessed the relative abundance of vaginal mucosal flora after different treatment modalities.At the family level, the abundance of Streptococcus and Bifidobacteriaceae was lower in both treatment groups compared to the control group, and Firmicutes was significantly reduced in both treatment groups (Figure 4a and Supplementary Material 1).At the genus level, 65 bacterial taxa showed different abundances between the treatment and control groups.A total of 14 bacterial taxa were enriched, and 51 taxa were reduced in the two treatment groups compared to the control group.Aerococcus, Bifidobacterium, Peptostreptococcus, Atopobium, Veillonella, Mageibacillus, Dialister, Anaerococcus Fastidiosipila, Faecalibacterium, Carboxylicivirga, Bacillus, and Candidatus_Saccharimonas exhibited significantly higher levels in the control group compared to the treatment group (Figure 4b and Supplementary Material 1).Finally, we calculated the microbial dysplasia index (MDI) using these taxa.The results showed a positive correlation between MDI and vaginal health scores (Figure 4c and Supplementary Material 1) and negative correlation with species richness (Figure 4d and Supplementary Material 1), suggesting a robust link between vaginal microbiota disruption and disease activity.and moisture score in the estrogen group, approaching the baseline.In contrast, the VHI score in the CO 2 group remained significantly elevated from baseline ( p < 0.001), and both elasticity and moisture scores showed a consistent upward trend (Figure 5 and Supplementary Material 2).

The effect of different treatment methods on patients' urination function
The UDI-6 score was assessed during every study visit.The 3-month follow-up results showed a significant improvement in urinary dysfunction and a notable decrease in UDI-6 scores ( p < 0.05) in both the CO 2 laser and estrogen treatment groups.Additionally, both therapies exhibited a significant reduction in stress scores.Furthermore, after 1 year, the UDI-6 score for patients receiving both treatment modalities were examined.The results showed a proximity to baseline levels for the estrogen group, whereas the UDI-6 score of the CO 2 group continued to exhibit a significant decrease ( p < 0.05) compared to the baseline (Figure 6 and Supplementary Material 2).
Effect of estrogen and CO 2 laser treatment on vaginal mucosal density and collagen content After 12 months of treatment, histopathological examination of vaginal mucosa samples (0.8 mm thickness) revealed an increase in vaginal mucosal density after estrogen or CO 2 laser treatments (Figure 7a).And there was no significant change in type I collagen between the CO 2 and estrogen groups (Figure 7b), while type III collagen was significantly increased after CO 2 laser treatment compared to the estrogen group (Figure 7c).

DISCUSSION
In this study, we examined alterations in the vaginal mucosal flora among patients with GSM after CO 2 laser therapy and conventional estrogen therapy.16S rRNA sequencing was employed to evaluate the differential efficacy between the two treatments for GSM.Additionally, we integrated pathological observations, the VHI Index, and UDI-6 scores to assess vaginal health status and voiding function.
In recent years, CO 2 laser has gained widespread application in gynecological disorders and emerged as a non-hormonal therapy for the genitourinary tract. 16here is increasing evidence suggests disturbances in the vaginal flora of individuals with GSM, and this disturbance being considered a key factor in inflammation. 12,17eported indicate an increase in normal flora, such as Lactobacilli, and a decrease in pH in the short-term after cessation of CO 2 treatment in perimenopausal women. 18his study employed 16S rRNA sequencing to examine alterations in the vaginal mucosal microbiota of patients with GSM following 3 months of CO 2 laser therapy and estrogen therapy.Alpha diversity analysis showed significant differences in the vaginal flora structure after estrogen or CO 2 laser treatment compared to untreated controls, suggesting a modulation effect of both estrogen and CO 2 laser treatments on the vaginal flora of GSM patients.The LEfSe analysis showed that the estrogen group exhibited more significant changes in Lactobacillus and Streptococcus compared to the control group.In contrast, the CO 2 group showed an increase in Lactobacillus and in IheB3_7.These suggested that the efficacy of CO 2 and estrogen in GSM may be attributed to the regulation of Lactobacillus abundance, which consistent with previous studies. 18Furthermore, research has demonstrated a correlation between the lack of short-chain fatty acids (SCFAs)-producing taxa in GSM and the severity of the disease.CO 2 laser treatment was able to restore the level of SCFA-producing taxa in responsive patients. 8,19,20his study found a significantly higher relative abundance of SCFAs-producing bacteria, including Trichoderma, Rumex, and Rhodobacter in the CO 2 group compared to the control group.The results suggest that the therapeutic effect of CO 2 laser on GSM is associated with an increase in the abundance of SCFA-producing flora.However, due to the small sample size, the conclusions mentioned above are limited to the current study.Subsequent prospective experiments should aim to maximize the sample size in order to minimize the statistical bias resulting from a small sample size.
Studies have shown that CO 2 laser therapy improves the female sexual function index score, UDI-6 score, 21 and VHI score, 10 and is effective for treating vaginal atrophy. 17owever, existing studies often feature short-term followup and lack a characterization of dynamic changes.In this study, we observed the vaginal health status after 3 and 12 months following estrogen and CO 2 laser treatment.The result showed a significant increase in the VIH score and a notable decrease in UDI-6 scores after 3 months of CO 2 and estrogen treatment compared to the control group, which is aligning with previous studies. 10,21However, at the 12 month mark, VHI score and UDI-6 score in the estrogen group were close to baseline levels.In contrast, VHI scores in the CO 2 group remained significantly higher than baseline, UDI-6 scores continued to be significantly lower than baseline, and elasticity and moisture scores continued to increase.Pathologic results demonstrated that after 12 months of treatment, both estrogen and CO 2 laser interventions led to an augmentation in vaginal mucosa and an elevation in levels of type I and type III collagen.Nevertheless, the escalation in type III collagen was more significant after CO 2 laser treatment compared to estrogen.These results indicate that in the short-term, both CO 2 and estrogen exhibit comparable efficacy in treating GSM, while in the long-term, CO 2 laser treatment demonstrates a more substantial efficacy.Meanwhile, the sustained synthesis of type III collagen subsequent to CO 2 laser treatment might underlie its enduring impact on GSM.The atrophy of the external genitalia and vagina is a common symptom in patients with GSM and gynecologic tumor patients. 22A systematic review has shown that CO 2 fractional laser can improve clinical symptoms and sexual function in patients with gynecologic tumors.This includes improvements in VHI, leading to a reduction in symptoms of atrophy in the external genitalia and vagina in patients with gynecologic malignant tumors. 23n a prospective observational study, the CO 2 laser was found to improve the VHI in postmenopausal women.It also promotes collagen regeneration, improves vaginal mucosal blood flow and tissue elasticity. 24Thus, it is considered an emerging effective treatment for atrophy of the external genitalia and vagina.The results of this study showed that 12 months after CO 2 laser treatment, there is a significant improvement in VHI scores and UDI-6 scores.Additionally, there is an increase in elasticity and moisture scores, along with an increase in type III collagen.Suggesting that CO 2 laser therapy might improve vulvar and vaginal atrophy by increasing the type III collagen content.However, substantiating this hypothesis necessitates additional prospective studies and animal experiments.
In conclusion, the study reveals that the short-term efficacy of CO 2 laser therapy is comparable to that of estrogen therapy.Both therapies improved vaginal health and voiding function, as well as regulation the balance of vaginal flora in GSM patients.However, the long-term efficacy of CO 2 laser surpassed that of estrogen.

AUTHOR CONTRIBUTIONS
Yingying Qi: Conception and design, data collection or management, drafting of the manuscript; Kexin Mo: Conception and design, data analysis, drafting of the manuscript; Aiping Wang: Conception and design, data collection or management, revising of the manuscript; Yueming He: Conception and design, obtaining funding, supervision.

F
I G U R E 2 Analysis of the abundance diversity of the vaginal flora.(a) Weighted unified principal component analysis.(b) Weighted unified threat analysis.(c) Alpha diversity analysis richness index.(d) Chao1 index.(e) ACE index.(f) Shannon index.(g) Simpson index.(h) Goods_coverage index.

F I G U R E 3
LEfSe analysis of the overall structural flora of the vaginal flora.Linear discriminant analysis (LDA) plot annotations within the three groups (a) and LEfSe analysis in microbial communities (b).

Figure 2
Figure2illustrates that VHI score ( p < 0.05), elasticity score (p < 0.05), and moisture score (p > 0.05) increased in patients after 3 months of CO 2 laser or estrogen treatment compared to the control group.The VHI score were reassessed after 1 year for patients undergoing both treatments, revealing a decrease in VHI score, elasticity score,

F I G U R E 7
Effect of estrogen and CO 2 laser treatment on vaginal mucosal density and collagen content.(a) HE staining of vaginal mucosa.(b) The immunohistochemical of vaginal mucosa type I collagen.(c) The immunohistochemical of vaginal mucosa type III collagen.
Overall condition of patients before treatment.