Alterations in the uterine microbiome drive the progression of endometriosis via neutrophil-mediated AKT-mTOR signaling pathway.

Alterations in the uterine microbiome drive the progression of endometriosis via neutrophil-mediated AKT-mTOR signaling pathway. | 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 Alterations in the uterine microbiome drive the progression of endometriosis via neutrophil-mediated AKT-mTOR signaling pathway. Jingwei Liu, Wanlin Zheng, Wenjie Yu, Pingping Lu, Xiaohuan Lu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8183082/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background The microbiome of the female reproductive system has been extensively studied; however, the correlation between uterine microbial alterations and endometriosis remains unclear. Methods Endometrial and lesion tissues were collected from patients with endometriosis, followed by 16S rRNA sequencing and RNA sequencing. The sequencing data were analyzed via PCA, random forest analysis, PICRUSt2 functional prediction, as well as GO and KEGG enrichment analyses, and further validated using the GEO database. Finally, the presence of the identified mechanism was confirmed in the pathological sections of endometriosis patients. Results we report that alterations in microbiome abundance were observed in both the endometrium and chocolate cysts of endometriosis patients, which promote the migration, proliferation, and pro-angiogenic capacity of endometrial cells. This process relies on the microbiome-immune cell-target cell axis, wherein abnormal microbiota induced neutrophil infiltration and the release of amounts of neutrophil extracellular traps (NETs), inflammatory cytokines, and chemokines. RNA-seq analysis further revealed that excessive inflammation subsequently activated the AKT-mTOR signaling pathway in endometrial cells, leading to enhanced cell adhesion and angiogenic phenotypes. Conclusion our study suggests that uterine microbiome changes are involved in the onset and progression of endometriosis and may offer a new perspective for its treatment. Endometriosis Microbiome Neutrophil AKT mTOR Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1 Background Endometriosis is characterized by the presence of endometrium-like tissue lesions outside the uterus. Approximately 10% of reproductive-aged women worldwide suffer from this condition, and to date, no curative pharmacological or surgical treatments have been available[ 1 , 2 ]. As a chronic inflammatory disease, endometriosis not only exhibits recruitment of immune cells at the lesion sites but also displays a phenotype of elevated inflammatory factors at the local or systemic level[ 3 ]. Neutrophils, as innate immune cells, have been reported in multiple studies as being persistently recruited in cases of endometriosis[ 3 , 4 ]. Activated neutrophils release a large amount of neutrophil extracellular traps (NETs), which disrupt endothelial cell junctions and promote cell migration. NETs have been shown to facilitate the growth and metastasis of various cancers, including breast, liver, and ovarian cancers[ 5 ]. Ayako Nishimoto-Kakiuchi et al. achieved excellent therapeutic effects in both spontaneous and surgically induced endometriosis monkey models by blocking IL-8, a neutrophil chemokine[ 4 ]. In summary, the abnormal recruitment of neutrophils plays a crucial role in the onset and progression of endometriosis. Neutrophils are recognized as the first line of defense against pathogenic microorganisms. However, in previous studies, the academic community has generally held the view that the endometrium is either sterile or minimally colonized[ 6 ]. With the advancement of sequencing technologies, differences in the cervical mucus microbiome of patients with endometriosis have been identified, showing a significant increase in the abundance of Corynebacterium, Enterobacteriaceae, and Streptococcus[ 7 , 8 ]. Similar findings have also been confirmed in the intestinal and peritoneal fluid of patients with endometriosis[ 9 , 10 ]. These microbiome alterations are correlated with inflammation, angiogenesis, and cell migration[ 11 ]. Research on the endometrial microbiome of patients with endometriosis remains limited, despite the confirmation of microbial colonization in the endometrium[ 12 ]. Given the impact of microbiome dysbiosis on neutrophils and the high-level recruitment of neutrophils at the lesion sites in patients with endometriosis, it is essential to study the microbial composition of the endometrium and lesions in these patients[ 11 ]. In this study, we collected endometrial tissues from non-endometriosis patients, as well as endometrial tissues, cyst walls, and cyst fluids of chocolate cysts from endometriosis patients. Subsequently, we conducted 16S rRNA sequencing (16S rRNA-seq) and RNA sequencing (RNA-seq) on these samples. Our results also revealed that there were no significant changes in the microbial composition of the endometrium in endometriosis patients. However, the abundance of various bacterial species closely associated with inflammation was significantly upregulated. Microorganisms were also detected in chocolate cysts. The alterations in the endometrial microbiome contributed to the maintenance of chronic inflammation in endometriosis and induced the enrichment of neutrophils. RNA-seq demonstrated that the endometrium of endometriosis patients exhibited more pronounced NET formation, which subsequently stimulated the AKT -mTOR signaling pathway in endometrial cells. This led to a more prominent pro-angiogenic phenotype and enhanced migratory capacity of ectopic endometrial cells. Notably, we were the first to correlate the significant enrichment of neutrophils with the changes in the microbial composition in the endometrium of endometriosis patients. We also pointed out that neutrophils might promote the onset and progression of endometriosis by inducing the activation of the AKT-mTOR signaling pathway in endometrial cells through NETs. This work provides new insights into the pathogenesis of endometriosis from a novel perspective and suggests potential new therapies based on the microbiome. 2 Materials and Methods 2.1 Antibodies MPO (22225-1-AP) was purchased from Proteintech (China); Ly6G (E6Z1T) was obtained from Cell Signaling Technology (USA); CD31 (ab182981) was acquired from Abcam (USA); p-mTOR (Ser2448) (AF3308) and p-AKT (Ser473) (AF0016) were sourced from Affinity Biosciences (USA). 2.2 Clinical Sample Collection Endometrial tissue from non-endometriosis patients: For patients undergoing myomectomy for uterine fibroids, diagnostic curettage was performed preoperatively to exclude endometrial pathology. Endometrial tissues with normal pathological findings were included in the study. Endometrial tissue from endometriosis patients: Patients hospitalized for long-term infertility were diagnosed with endometriosis following comprehensive examinations. Endometrial biopsy samples, if available, were collected and included in the study. Chocolate cysts from endometriosis patients: For patients meeting surgical criteria for ovarian chocolate cysts, cyst contents and inner wall tissues were collected post-resection and included in the study. All tissue collections adhered to strict aseptic techniques to prevent contamination by cervical or vaginal microbiota. Sequencing samples were stored at -80°C, while remaining tissues were fixed in paraffin for histological staining. All participants were fully informed of the study’s purpose, potential health risks, and safeguards prior to enrollment, and provided written informed consent. Patient-identifiable information was anonymized to protect privacy. The study protocol was approved by the Institutional Review Board of Wuhu Maternal and Child Health Center (Approval Number: 20250001). 2.3 16S rRNA Sequencing We collected endometrial tissues from non-endometriosis patients (n = 3), endometrial tissues from endometriosis patients (n = 3), and intraluminal fluid from chocolate cysts (n = 3) for 16S rRNA sequencing. The 16S rRNA sequencing libraries were prepared and subjected to high-throughput sequencing by Bioprofile (Shanghai, China). Raw sequencing data were denoised using the DADA2 algorithm to generate non-redundant amplicon sequence variants (ASVs) and their corresponding counts across all samples. Low-abundance ASVs—those detected in only one sample or with total counts < 50 across all samples—were filtered out. For bacterial and archaeal 16S rRNA gene analysis, the Greengenes2 database was selected as the reference due to its established reputation as a concise and accurate prokaryotic database. To mitigate false-positive differences in subsequent differential abundance and feature selection analyses caused by varying sequencing depths, ASV counts were rarefied to uniform levels across all samples. 2.4 Shannon Index and Chao1 Index Analysis The Shannon index, grounded in information entropy theory, provides a composite measure of species richness and evenness, while the Chao1 index, a nonparametric estimator, calculates total species richness based on species occurrence frequencies. Both metrics reflect microbial community richness. Rarefied ASV tables were analyzed using the vegan package (v2.6-10) in R to compute these indices. 2.5 Random Forest Analysis Random Forest is a supervised ensemble machine learning algorithm that constructs a "forest" of decision trees using bootstrap aggregating (bagging) and random feature subspace selection, enhancing model generalization and robustness. For this study, rarefied ASV tables were analyzed using the scikit-learn (sklearn, v1.6.1) package in Python to build a Random Forest classifier. Only ASVs with non-zero counts across all samples were included in the analysis. The model identified the top 20 species contributing most to classification, along with their importance scores. 2.6 PICRUSt2 Functional Prediction PICRUSt2 infers the metabolic functional potential of microbial communities from 16S rRNA gene amplicon sequencing data, primarily based on the "phylogenetic conservation" hypothesis. Using PICRUSt2 software (v2.6.0), functional abundance was predicted from unrarefied ASV tables and mapped to the The Kyoto Encyclopedia of Genes and Genomes (KEGG) and MetaCyc databases. 2.7 RNA Sequencing Endometrial tissues from non-endometriosis patients (n = 3), endometrial tissues from endometriosis patients (n = 3), and inner cyst wall tissues from chocolate cysts (n = 3) were collected for RNA sequencing. RNA sequencing libraries were prepared and subjected to high-throughput sequencing by Bioprofile (Shanghai, China). Raw FASTQ data underwent quality filtering, followed by alignment of cleaned reads to the reference genome using HISAT2. HTSeq was employed to quantify read counts per gene as raw expression values. Normalization was performed using FPKM to standardize transcript or gene expression levels. 2.8 GEO Database RNA-seq datasets related to endometrial tissues from endometriosis were retrieved from the GEO database ( https://www.ncbi.nlm.nih.gov/geo ), specifically from projects GSE11691, GSE25628, and GSE168902[ 13 – 15 ]. The combined dataset included 19 endometriosis patients and 18 healthy controls. Data normalization was performed using the limma R package. 2.9 KEGG and GO Analysis The Gene Ontology (GO) database ( https://www.geneontology.org ) categorizes genes and gene products into three domains—cellular component (CC), molecular function (MF), and biological process (BP)—to identify gene sets significantly enriched in specific biological processes. KEGG ( https://www.genome.jp/kegg ) elucidates higher-order functions and utilities of cells, organisms, and ecosystems through molecular-level information. Differentially expressed genes were uploaded to the Database for Annotation, Visualization, and Integrated Discovery (DAVID, https://david.ncifcrf.gov ) to select the top 20 GO terms and KEGG pathways with the lowest p-values. These were visualized using the bioinformatics platform ( https://www.bioinformatics.com.cn ). 2.10 GSEA Analysis Gene Set Enrichment Analysis (GSEA) evaluates the distribution trend of predefined gene sets within a phenotype-ranked gene list to assess their contribution to phenotypic traits. GSEA was performed using the local version of the GSEA software ( http://www.broadinstitute.org/gsea/index.jsp ). 2.11 CIBERSORT Immune Infiltration Analysis CIBERSORT deconvolutes RNA-seq data using a signature gene set of 22 immune cell types to predict immune cell infiltration levels in tissues. The merged GEO dataset was analyzed for immune infiltration using the R package CIBERSORT. 2.12 Immunofluorescence and Immunohistochemical Staining Immunofluorescence staining detects protein expression levels and spatial distribution in tissues or cells. Paraformaldehyde-fixed paraffin-embedded tissue sections were deparaffinized using xylene and ethanol with gradient concentration, followed by antigen retrieval with citrate buffer and permeabilization with 0.1% Triton X-100 (Aladdin, China). After blocking with 3% BSA (Biosharp, China), sections were incubated overnight at 4°C with primary antibodies, followed by 1-hour incubation with fluorescent secondary antibodies at room temperature. Immunohistochemical staining follows similar steps, with additional treatment using 3% hydrogen peroxide post-antigen retrieval. Sections were incubated with primary antibodies overnight at 4°C and secondary antibodies for 30 minutes at room temperature, followed by staining with 3,3'-diaminobenzidine (DAB) and hematoxylin. Images were captured using a fluorescence inverted microscope (Olympus, Japan). 2.13 Statistical Analysis One-way analysis of variance (ANOVA) was used for comparisons among multiple groups, while Student’s t-test was applied for pairwise comparisons. GraphPad Prism 8.0.2 was utilized for graphical and statistical analyses. Statistical significance was set at P < 0.05. 3 Results 3.1 Microbiome Shifts in the Endometrium with Endometriosis Traditionally, it was widely believed that the female uterus is a sterile or minimally colonized environment with trace amounts of bacteria. However, with the advancement of detection techniques, the critical role of the microbiome in the female reproductive organs in urogenital system diseases has been highlighted[ 6 ]. In this study, we collected in-situ endometrial tissues either from patients undergoing total hysterectomy for fibroids or from patients with endometriosis who underwent uterine lavage. And the ectopic endometrial tissues were collected from patients undergoing chocolate cyst resection surgery. Subsequently, 16S rRNA-seq was performed to explore the alterations in the uterine microbiome. Consistent with previous reports, the microbiome in the endometrium of patients with endometriosis (EC) and that of control patients (NC) was predominantly dominated by Bacillota A 368345 and Bacteroidota, with no significant differences observed across various phyla (Figs. 1 a-b, Supplementary Figs. 1–2 ). Additionally, there were no significant differences in the Shannon index and Chao1 index between them, although the Chao1 index was slightly elevated in the EC group (Figs. 1 c-d). Random forest analysis identified p__Bacillota_A_368345, p__Fusobacteriota, and p__Actinomycetota as characteristic phyla (Fig. 1 e). Specifically, at the species level, Limosilactobacillus mucosae, Lachnospira eligens, Megasphaera_A_38685 indica, Eubacterium_G_180878 ventriosum, and Ruminiclostridium_E siraeum within the p__Bacillota phylum showed significant increases in the EC group (Fig. 1 f). Lachnospira eligens primarily produces short-chain fatty acids, including acetate and butyrate, and alterations in its gastrointestinal abundance have been associated with chronic nephritis[ 16 ]. Megasphaera_A_38685 indica belongs to the same genus as Megasphaera elsdenii, which has been linked to bacterial vaginosis and can induce chronic endometritis or pelvic inflammatory disease[ 17 ]. Eubacterium_G_180878 ventriosum has been shown to have a positive correlation between its respiratory abundance and asthma airway inflammation levels[ 18 ]. Interestingly, Prevotella muris_A within the p__Bacteroidota phylum also exhibited a significant increase in the EC group. Prevotella muris_A belongs to the Prevotella genus, which has been demonstrated to induce the onset and progression of inflammatory diseases in the gut and respiratory tract[ 5 , 19 , 20 ]. In summary, although there were no significant differences in the endometrial microbiome between the EC and NC groups, several microbes potentially associated with inflammation showed significant upregulation, emphasizing the potential association between microbiome-induced inflammation and the onset and development of endometriosis. Subsequently, the PICRUSt algorithm was employed to predict the functions of differentially abundant bacterial genera between the EC and NC groups. Mapping to the KEGG dataset revealed a significant upregulation of the mTOR signaling pathway in the EC group, which has been shown to promote angiogenesis, maintain cellular stemness, and facilitate cell migration[ 21 ] (Fig. 1 g). Enzymatic reaction enrichment analysis indicated a significant increase in phosphatidylinositol-4,5-bisphosphate 4-phosphatase (EC:3.1.3.78) in the EC microbiome, which can catalyze the hydrolysis of PIP2 to PIP and subsequently regulate the host cell's PI3K-AKT-mTOR signaling pathway[ 22 ] (Fig. 1 h). Metacyc enrichment analysis suggested that the microbiome changes in the EC group might lead to a decreased degradation capacity of L-tryptophan (PWY-5655, TRYPTOPHAN-DEGRADATION-1) (Fig. 1 i). L-tryptophan is primarily degraded into kynurenine and other metabolites via IDO/TDO, and effective inhibition of IDO promotes the immune surveillance function of CD8 T cells[ 23 ]. Reduced degradation of L-tryptophan may facilitate immune evasion for the ectopic implantation of endometrial cells[ 24 , 25 ]. Although the pathogenesis of endometriosis has not yet been fully elucidated, there is evidence indicating that ectopic endometrial cells originate from the uterus, as evidenced by identical somatic mutations[ 26 ]. Given the correlation between ectopic tissues and the endometrium, we further examined the microbiome of the endometrial cyst fluid (ECF) in chocolate cysts and conducted an analysis with data from the EC group. The heatmap revealed significant differences in microbiome distribution between the ECF and EC groups (Fig. 2 a, Supplementary Figs. 3–4 ). The abundance of Bacillota I in the ECF group increased significantly by 12.05-fold, while the abundances of Actinomycetota, Bacillota A 368345, Bacteroidota, and Pseudomonadota phyla decreased markedly (Fig. 2 b). Both the Shannon index and the Chao1 index indicated a significant reduction in microbial species richness in the ECF group (Figs. 2 c-d). Random forest analysis identified p_Bacillota_C as a characteristic phylum, with Lactobacillus iners being the predominant bacterial species in ECF (Figs. 2 e-f). Unlike Lactobacillus crispatus, the dominant species in a healthy vagina, the predominance of Lactobacillus iners is often associated with an increased risk of vaginal inflammation[ 27 , 28 ]. KEGG enrichment analysis revealed significant enrichment of the PI3K-Akt signaling pathway, which is closely related to the mTOR signaling pathway (Fig. 2 g). In the endometrium, activation of the PI3K-AKT signaling pathway has been shown to promote angiogenesis and enhance the proliferation, migration, and invasiveness of endometrial cells[ 29 , 30 ]. Enzymatic reaction enrichment analysis showed a significant increase in P-type K + transporter (EC:3.6.3.12), dinitrogen oxidoreductase (EC:1.18.6.1), 6,7-dihydropteridine reductase (EC:1.5.1.34), and thymidylate synthase (EC:2.1.1.148), enzymes directly involved in ATP metabolism, suggesting a higher level of energy metabolism in the ECF microbiome (Fig. 2 h). Metacyc enrichment analysis indicated enhanced anaerobic energy metabolism pathways (PWY-6478) in this group (Fig. 2 i). Considering the hypoxic environment within chocolate cysts, the enhanced anaerobic metabolism pathways and related metabolic enzymes in the ECF microbiome are possibly fundamental characteristics[ 31 ]. In summary, our analysis of NC, EC, and ECF further confirmed alterations in the uterine microbiome of endometriosis patients. The microbiome changes between EC and NC were correlated with local inflammation in endometriosis patients. Notably, microorganisms were also present in theoretically sterile chocolate cysts. Given the anatomical structure of the female reproductive system, these microorganisms likely originated from the patient's endometrium. The significant differences between the two suggest that these microorganisms had already reached the lesion site before cyst formation and underwent compositional changes in the anaerobic environment after cyst formation. 3.2 Neutrophil Infiltration in the Endometrium of Patients with Endometriosis To analyze the molecular changes in the endometrium of patients with endometriosis, we conducted RNA-seq on the tissue samples from the aforementioned patients. PCA revealed that, similar to the results of 16S rRNA-seq, there was no significant change in the RNA composition of the endometrium between the EC and NC groups (Fig. 3 a). The volcano plot showed that 726 genes were upregulated and 189 genes were downregulated in the EC group (Fig. 3 b). Enrichment analysis of the differentially expressed genes (DEGs) was performed. KEGG analysis indicated a significant enrichment of the neutrophil extracellular trap formation pathway. Additionally, the enrichment of the NF-kappa B signaling pathway and the chemokine signaling pathway suggested a higher level of inflammation in EC tissues (Fig. 3 c). Gene Ontology (GO) Biological Process (BP) analysis revealed enrichment of leukocyte activation and immune system process, further validating this finding (Fig. 3 d). GO Cellular Component (CC) and Molecular Function (MF) analyses similarly revealed enrichment in signaling pathways related to cell membrane vesicles and cytokine receptors ( Supplementary Fig. 5 ). Moreover, the enrichment of response to other organism and response to external biotic stimulus suggested a correlation between the occurrence and development of inflammation and the aforementioned microbiome alterations. Reactome analysis showed enrichment of the neutrophil degranulation and transcriptional regulation of granulopoiesis pathways, further emphasizing the importance of neutrophils in the inflammation of EC tissues (Fig. 3 e). Gene Set Enrichment Analysis (GSEA) demonstrated that the Neutrophil activation pathway was activated in the EC group, although with an False Discovery Rate (FDR) of 0.3856 ( Fig. 3 f). We further validated these findings by merging and analyzing three datasets (GSE11691, GSE25628, and GSE168902) from the Gene Expression Omnibus (GEO) database. In the GEO datasets, compared to the NC group, 438 genes were upregulated and 130 genes were downregulated in the EC group (Fig. 3 g). Analysis of DEGs showed similar results, with KEGG indicating enrichment of the neutrophil extracellular trap formation pathway, and GO BP showing enrichment of the neutrophil activation involved in immune response and neutrophil degranulation pathways (Figs. 3 h-i). GO CC and MF analyses equally highlighted the significance of cytokine and immune receptor activity ( Supplementary Fig. 6 ). GSEA analysis also revealed activation of the neutrophil activation pathway (FDR = 0.0000) (Fig. 3 j). The heatmap similarly showed significant upregulation of genes related to the neutrophil activation pathway in the EC group (Fig. 3 k). CIBERSORT analysis for immune cell infiltration deconvolution demonstrated significant upregulation of activated mast cells and neutrophils in the EC group (Fig. 3 l). In conclusion, through transcriptomic analysis, we found a significant upregulation of events related to neutrophil activation in the EC group compared to the NC group. As reported in the literature, additional infiltration of neutrophils contributes to angiogenesis and the release of inflammatory factors, which further recruits macrophages and NK cells and promotes disease progression[ 32 , 33 ]. These findings highlight the role of inflammation in the occurrence of endometriosis and further support the conclusion that microbiome changes in the endometrium of patients with endometriosis may induce the occurrence and development of inflammation. 3.3 Activation of the PI3K-AKT-mTOR signaling pathway and angiogenesis in ectopic endometrial tissue. PCA revealed significant differences in the RNA profiles between the EE and EC groups (Fig. 4 a). In the EE group, 1,621 genes were upregulated and 1,531 genes were downregulated (Fig. 4 b). GO BP analysis of the differentially expressed genes showed enrichment in pathways such as cell adhesion, extracellular matrix organization, and cell-cell adhesion in the EE group (Fig. 4 c). This validated the enhanced invasive and adhesive capabilities of ectopic endometrial tissue compared to eutopic endometrium, explaining its ectopic implantation pathology[ 34 ]. The enrichment of the response to xenobiotic stimulus pathway further underscored the significance of microbiome dysregulation in the development of endometriosis. Both GO BP and GSEA revealed significant upregulation of the acute inflammatory response (FDR = 0.0000) and angiogenesis (FDR = 0.0000) pathways (Figs. 4 c-e). However, unlike the differences between the EC and NC groups, the EE group did not exhibit significant neutrophil activation compared to the EC group (Fig. 4 f). GO CC and MF analyses indicated enrichment in extracellular region and receptor ligand activity. ( Supplementary Fig. 7 ). KEGG enrichment analysis suggested that the PI3K-AKT signaling pathway played a crucial role in the EE group (Fig. 4 g). Combined with the mTOR signaling pathway indicated by 16sRNA sequencing, which is closely related to AKT signaling, GSEA analysis showed significant upregulation of the PI3K-AKT-mTOR pathway in the EE group (Fig. 4 h). Analyzing the differences between the EE and NC groups, PCA also revealed significant differences in their RNA profiles (Fig. 4 i). The volcano plot showed that 2,227 genes were upregulated and 1,769 genes were downregulated in the EE group compared to the NC group (Fig. 4 j). GO BP analysis similarly showed enrichment in pathways such as cell adhesion, cell-cell adhesion, extracellular matrix organization, positive regulation of cell migration, response to lipopolysaccharide, and angiogenesis (Fig. 4 k). The analysis results of GO CC and MF were similar to those of EE/EC ( Supplementary Fig. 8 ). KEGG analysis indicated enrichment in pathways such as ECM-receptor interaction, cell adhesion molecules, and leukocyte transendothelial migration, further emphasizing the importance of the PI3K-Akt signaling pathway in the EE group (Fig. 4 l). GSEA analysis revealed upregulation of the angiogenesis, acute inflammatory response, and AKT-mTOR signaling pathways, albeit without significant differences ( Supplementary Fig. 9 ). These results demonstrated that ectopic endometrium exhibited more pronounced implantation and angiogenesis capabilities than the EC group, with significant activation of the PI3K-AKT-mTOR signaling pathway. Previous literature has shown that the formation of neutrophil extracellular traps (NETs) can effectively promote angiogenesis and facilitate cancer cell metastasis[ 35 ]. Endometrium from patients with endometriosis exhibited higher levels of neutrophil infiltration, accompanied by elevated levels of NETs, which may induce some endometrial cells to acquire a pro-angiogenic phenotype. High levels of NETs have also been shown to promote the phosphorylation of AKT and mTOR in human umbilical vein endothelial cells (HUVECs), which has been proven to promote tumor proliferation and invasiveness[ 36 , 37 ]. Endometrial tissue with a pro-angiogenic phenotype and activated AKT-mTOR signaling has enhanced growth, migration, and implantation capabilities, forming the basis for the occurrence of ectopic tissue. 3.4 High levels of neutrophils accompanied by enhanced angiogenesis and AKT-mTOR phosphorylation in the endometrium. Based on the aforementioned bioinformatics analysis, we further validated the conclusions through immunostaining. Immunofluorescence staining revealed that the proportion of Ly6G + MPO + cells in both the EC group and the EE group was significantly higher than that in the NC group, but there was no significant difference between the EC and EE groups (Figs. 5 a-b, Supplementary Fig. 10 ). This was consistent with the results of the GSEA analysis. Immunohistochemical staining showed that the CD31 + area in the EE group was 4.01 times that in the NC group and 1.46 times that in the EC group. The CD31 + area in the EC group was also significantly higher than that in the NC group, which validated the phenotype of more active angiogenesis in the endometrium and ectopic tissues of patients with endometriosis (Figs. 5 c-d, Supplementary Fig. 11 ). The expression level of vimentin is significantly correlated with the invasiveness of endometrial cancer[ 38 ]. The results indicated that the vimentin expression levels in both the EC and EE groups were elevated compared to the NC group, but only the EE group showed a significant increase compared to the NC group (Figs. 5 e-f, Supplementary Fig. 12 ). Finally, we detected the expression levels of p-AKT and p-mTOR in the patient tissues. The p-AKT expression level in the EC group was 13.8-fold that in the NC group, and in the EE group, it was further increased to 3.26-fold that in the EC group (Figs. 5 g-h, Supplementary Fig. 13 ). As a downstream molecule of the AKT signaling pathway, the phosphorylation level of mTOR was synchronized with that of AKT. The p-mTOR expression level in the EE group was 16.19-fold that in the NC group and 4.6-fold that in the EC group (Figs. 5 i-j, Supplementary Fig. 14 ). The above results were consistent with the bioinformatics analysis, highlighting the potential mechanism by which neutrophils promote invasion and ectopic colonization by activating the pro-angiogenic phenotype of the endometrium and AKT-mTOR phosphorylation. 4 Discussion Nearly 190 million women globally have been affected by Endometriosis, which is strongly associated with chronic pain and infertility[ 39 ]. Unfortunately, the pathogenic mechanism of this disease remains unclear. The prevailing hypothesis is retrograde menstruation, which posits that during menstruation, fragments of endometrial tissue flow backward through the fallopian tubes into the peritoneal cavity, where they implant and form lesions[ 39 , 40 ]. Our study also supported this hypothesis, as we identified a microbiome dominated by Lactobacillus iners in chocolate cysts. This bacterium primarily resides in the vagina under normal physiological conditions, and its dominance as the predominant strain is often linked to the onset of gynecological diseases. This suggests that microbes in patients with endometriosis may enter deep tissue environments via retrograde menstruation and assist in the implantation of ectopic endometrial tissue. The microbial composition in chocolate cysts differed from that in the endometrial microbiome of endometriosis patients, likely due to secondary selection caused by the hypoxic environment within the cysts. The occurrence of endometriosis is inextricably linked to the enhanced migration and implantation capabilities of endometrial cells, given that retrograde menstruation is not a rare event[ 41 ]. As a chronic inflammatory disease, the correlation between inflammatory responses and the onset and progression of endometriosis has been extensively studied[ 42 , 43 ]. Abnormally activated immune cells secrete excessive inflammatory cytokines and chemokines, affecting not only the uterus but also the entire systemic system of patients. An excessive inflammatory environment drives angiogenesis, endometrial cell proliferation, and steroid responses[ 44 – 46 ]. In our study, we highlighted the significant enrichment of neutrophils in the endometrium of endometriosis patients. This differs from previous research that solely focused on ectopic lesions, as the chronic inflammation in the endometrium demonstrated that this inflammation was not merely secondary but deeply involved in lesion formation. RNA-seq analysis highlighted significant activation of the AKT-mTOR signaling pathway in the endometrium of endometriosis patients. Similarly, PICRUSt predictions from 16s rRNA-seq data also underscored the critical role of the mTOR signaling pathway. Yang et al. demonstrated that NET formation can effectively promote angiogenesis in gastric cancer and enhance the phosphorylation of AKT and mTOR in endothelial cells[ 35 ]. This finding further aligned with the observed increase in neutrophils in the uterus of endometriosis patients. The AKT-mTOR signaling pathway has been confirmed in various diseases to promote cell migration, proliferation, implantation, and angiogenesis[ 21 , 47 , 48 ]. GSEA analysis revealed that both cell adhesion and angiogenesis pathways are significantly activated in ectopic endometrium compared to eutopic endometrium in endometriosis patients. These processes collectively contribute to the onset and progression of endometriosis, making AKT-mTOR a promising molecular target for further investigation in the treatment of endometriosis[ 48 , 49 ]. It must be admitted that our research is still at a preliminary stage. Our study has highlighted the microbiome-immune cell-target cell axis, yet it has not delved into the impact of microbial metabolites. For instance, 16S rRNA sequencing indicated the possiblity of tryptophan metabolism disorders in the endometrium of endometriosis, as well as the occurrence of excessive anaerobic glycolysis in chocolate cysts. The effects of these abnormal metabolic patterns on endometrial cells remain to be further explored. Secondly, although we collected tissues from endometriosis patients, we did not further classify the patients according to their phenotypes and disease stages. For example, only endometriosis patients with chocolate cysts was selected, which may mean that our conclusions require further validation before they can be applied to peritoneal endometriosis. Similarly, it is difficult to correlate these conclusions with whether patients experience dysmenorrhea, menstrual disorders, or infertility. Therefore, further work needs to be carried out in the later stage, including building a database to conduct more detailed research. 5. Conclusion In conclusion, we were the first to analyze the endometrial microbiome in endometriosis and have proposed the finding that changes in microbiome abundance induce neutrophil infiltration in the endometrium. The infiltrated neutrophils then release neutrophil extracellular traps (NETs), inflammatory cytokines, and chemokines, which activate the phosphorylation of AKT and mTOR in endometrial cells, thereby promoting the occurrence and development of endometriosis. Declarations Ethics approval and consent to participate All participants were fully informed of the study’s purpose, potential health risks, and safeguards prior to enrollment, and provided written informed consent. Patient-identifiable information was anonymized to protect privacy. The study protocol was approved by the Institutional Review Board of Wuhu Maternal and Child Health Center (Approval Number: 20250001). All human-related research is in strict compliance with the Declaration of Helsinki. Consent for publication Not applicable. Availability of data and materials The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive (Genomics, Proteomics & Bioinformatics 2025) in National Genomics Data Center (Nucleic Acids Res 2025), China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences (GSA-Human: HRA015202 and HRA015176 ) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa-human. Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Funding This work was supported by Anhui Provincial Health Research Projects (AHWJ2024Ab0238) and Scientific Research Projects of Wuhu Municipal Health Commission (WHWJ2023z030). Author Contributions HT: Supervision, Writing – review & editing, Project administration, Funding acquisition. XL: Supervision, Writing – review & editing, Resources, Project administration. JL: Writing – original draft, Writing – review & editing, Resources, Methodology, Investigation, Formal analysis, Data curation, Validation, Conceptualization. WZ: Writing – original draft, Investigation, Validation, Formal analysis. WY: Writing – original draft, Investigation, Conceptualization. PL: Writing – original draft, Methodology. Acknowledgements Not applicable. References As-Sanie S, Mackenzie SC, Morrison L, Schrepf A, Zondervan KT, Horne AW, Missmer SA. Endometriosis: A Review. JAMA. 2025;334(1):64–78. Bonavina G, Taylor HS. Endometriosis-associated infertility: From pathophysiology to tailored treatment. Front Endocrinol (Lausanne). 2022;13:1020827. Symons LK, Miller JE, Tyryshkin K, Monsanto SP, Marks RM, Lingegowda H, Vanderbeck K, Childs T, Young SL, Lessey BA, et al. Neutrophil recruitment and function in endometriosis patients and a syngeneic murine model. FASEB J. 2020;34(1):1558–75. 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Zhang M, Xu T, Tong D, Li S, Yu X, Liu B, Jiang L, Liu K. Research advances in endometriosis-related signaling pathways: A review. Biomed Pharmacother. 2023;164:114909. Dudley AC, Griffioen AW. Pathological angiogenesis: mechanisms and therapeutic strategies. Angiogenesis. 2023;26(3):313–47. Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterial1.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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of the expression levels of species of different bacteria. g-i) KEGG (g), Enzymatic reaction (h), and MetaCyc (i) enrichment analysis of the EC relative to the NC group.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8183082/v1/af0678b1260392c1702fa1c1.png"},{"id":98425598,"identity":"74eb9a75-80af-476f-a129-05e2ac900667","added_by":"auto","created_at":"2025-12-17 16:34:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":612131,"visible":true,"origin":"","legend":"","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8183082/v1/f8edbb1b560dd9df6af96b70.png"},{"id":97990473,"identity":"aebf36f1-e6c9-4ae5-b150-0362c34c47e4","added_by":"auto","created_at":"2025-12-11 14:29:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":729137,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of RNA-seq of the endometrium from the endometriosis and control patients.\u003c/strong\u003e a) PCA analysis of the EC and NC groups. b) Volcano plot expressing differentially expressed genes between EC and NC groups. c-f) KEGG (c), GO BP (d), Reactome (e) and GSEA (f) enrichment analysis of the EC relative to the NC group. g) Volcano plot expressing differentially expressed genes between EC and NC groups in the GEO database. h-j) KEGG (h), GO BP (i) and GSEA (j) enrichment analysis of the EC relative to the NC group. k) Heatmaps showing mRNA expression profiles of the endometrium from endometriosis and control patients. l) CIBERSORT analysis of immune cell composition in the endometrium from endometriosis and control patients.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8183082/v1/606a6f4a16e8bc9a0687b880.png"},{"id":97990470,"identity":"1983a7e8-f502-4b37-b593-6f494129ee54","added_by":"auto","created_at":"2025-12-11 14:29:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":574049,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of RNA-seq of endometrial cyst fluid in chocolate cysts from patients with endometriosis.\u003c/strong\u003e a) PCA analysis of the EE and EC groups. b) Volcano plot expressing differentially expressed genes between EE and EC groups. c) GO BP enrichment analysis of the EE relative to the EC group. d-f) GSEA enrichment analysis of acute inflammatory response (d), angiogenesis (e), and neutrophil activation (f) between the EE and EC groups. g) KEGG enrichment analysis of the EE relative to the EC group. h) GSEA enrichment analysis of PI3K-AKT-mTOR signaling pathway between the EC and NC groups. i) PCA analysis of the EE and NC groups. j) Volcano plot expressing differentially expressed genes between EE and NC groups. k-l) GO BP (k) and KEGG (l) enrichment analysis of the EE relative to the NC group.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8183082/v1/bca6c380ab55fbf0c99d638f.png"},{"id":98424971,"identity":"6ccfa889-85e2-4551-adc5-35217937ea48","added_by":"auto","created_at":"2025-12-17 16:34:08","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":956597,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIn vivo validation of neutrophil enhancement, endometrial angiogenesis, and AKT-mTOR phosphorylation.\u003c/strong\u003e a) Representative fluorescence images of the endometrium with MPO (violet), Ly6G (green), and DAPI (blue) (bar = 50 μm). b) Quantification of MPO\u003csup\u003e+\u003c/sup\u003eLy6G\u003csup\u003e+\u003c/sup\u003e cells in the indicated groups in (a). Data were presented with mean ± S.D., ANOVA. c) Representative white-light images of the endometrium with CD31 (bar = 50 μm). d) Quantification of CD31\u003csup\u003e+\u003c/sup\u003e area in the indicated groups in (c). Data were presented with mean ± S.D., ANOVA. e) Representative white-light images of the endometrium with vimentin (bar = 50 μm). f) Quantification of average intensity of vimentin in the indicated groups in (e). Data were presented with mean ± S.D., ANOVA. g) Representative white-light images of the endometrium with p-AKT (bar = 50 μm). h) Quantification of average intensity of p-AKT in the indicated groups in (g). Data were presented with mean ± S.D., ANOVA. i) Representative white-light images of the endometrium with p-mTOR (bar = 50 μm). j) Quantification of average intensity of p-mTOR in the indicated groups in (i). Data were presented with mean ± S.D., ANOVA. n.s. p ≥ 0.05, *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8183082/v1/8dac094ac031f3bc9784f241.png"},{"id":98444028,"identity":"5205716f-8263-45df-ba93-962e54a324bf","added_by":"auto","created_at":"2025-12-17 17:14:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4608668,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8183082/v1/09daa125-8ba3-4061-812f-6b2459853992.pdf"},{"id":97990474,"identity":"a37412c1-ee5b-406c-9b8b-55d53c7aa3c8","added_by":"auto","created_at":"2025-12-11 14:29:31","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":5575300,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8183082/v1/bc6da4d1dca9beeb5b873064.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Alterations in the uterine microbiome drive the progression of endometriosis via neutrophil-mediated AKT-mTOR signaling pathway.","fulltext":[{"header":"1 Background","content":"\u003cp\u003eEndometriosis is characterized by the presence of endometrium-like tissue lesions outside the uterus. Approximately 10% of reproductive-aged women worldwide suffer from this condition, and to date, no curative pharmacological or surgical treatments have been available[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. As a chronic inflammatory disease, endometriosis not only exhibits recruitment of immune cells at the lesion sites but also displays a phenotype of elevated inflammatory factors at the local or systemic level[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Neutrophils, as innate immune cells, have been reported in multiple studies as being persistently recruited in cases of endometriosis[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Activated neutrophils release a large amount of neutrophil extracellular traps (NETs), which disrupt endothelial cell junctions and promote cell migration. NETs have been shown to facilitate the growth and metastasis of various cancers, including breast, liver, and ovarian cancers[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Ayako Nishimoto-Kakiuchi et al. achieved excellent therapeutic effects in both spontaneous and surgically induced endometriosis monkey models by blocking IL-8, a neutrophil chemokine[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In summary, the abnormal recruitment of neutrophils plays a crucial role in the onset and progression of endometriosis.\u003c/p\u003e\u003cp\u003eNeutrophils are recognized as the first line of defense against pathogenic microorganisms. However, in previous studies, the academic community has generally held the view that the endometrium is either sterile or minimally colonized[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. With the advancement of sequencing technologies, differences in the cervical mucus microbiome of patients with endometriosis have been identified, showing a significant increase in the abundance of Corynebacterium, Enterobacteriaceae, and Streptococcus[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Similar findings have also been confirmed in the intestinal and peritoneal fluid of patients with endometriosis[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. These microbiome alterations are correlated with inflammation, angiogenesis, and cell migration[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Research on the endometrial microbiome of patients with endometriosis remains limited, despite the confirmation of microbial colonization in the endometrium[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Given the impact of microbiome dysbiosis on neutrophils and the high-level recruitment of neutrophils at the lesion sites in patients with endometriosis, it is essential to study the microbial composition of the endometrium and lesions in these patients[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn this study, we collected endometrial tissues from non-endometriosis patients, as well as endometrial tissues, cyst walls, and cyst fluids of chocolate cysts from endometriosis patients. Subsequently, we conducted 16S rRNA sequencing (16S rRNA-seq) and RNA sequencing (RNA-seq) on these samples. Our results also revealed that there were no significant changes in the microbial composition of the endometrium in endometriosis patients. However, the abundance of various bacterial species closely associated with inflammation was significantly upregulated. Microorganisms were also detected in chocolate cysts. The alterations in the endometrial microbiome contributed to the maintenance of chronic inflammation in endometriosis and induced the enrichment of neutrophils. RNA-seq demonstrated that the endometrium of endometriosis patients exhibited more pronounced NET formation, which subsequently stimulated the AKT -mTOR signaling pathway in endometrial cells. This led to a more prominent pro-angiogenic phenotype and enhanced migratory capacity of ectopic endometrial cells. Notably, we were the first to correlate the significant enrichment of neutrophils with the changes in the microbial composition in the endometrium of endometriosis patients. We also pointed out that neutrophils might promote the onset and progression of endometriosis by inducing the activation of the AKT-mTOR signaling pathway in endometrial cells through NETs. This work provides new insights into the pathogenesis of endometriosis from a novel perspective and suggests potential new therapies based on the microbiome.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Antibodies\u003c/h2\u003e\u003cp\u003eMPO (22225-1-AP) was purchased from Proteintech (China); Ly6G (E6Z1T) was obtained from Cell Signaling Technology (USA); CD31 (ab182981) was acquired from Abcam (USA); p-mTOR (Ser2448) (AF3308) and p-AKT (Ser473) (AF0016) were sourced from Affinity Biosciences (USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Clinical Sample Collection\u003c/h2\u003e\u003cp\u003eEndometrial tissue from non-endometriosis patients: For patients undergoing myomectomy for uterine fibroids, diagnostic curettage was performed preoperatively to exclude endometrial pathology. Endometrial tissues with normal pathological findings were included in the study. Endometrial tissue from endometriosis patients: Patients hospitalized for long-term infertility were diagnosed with endometriosis following comprehensive examinations. Endometrial biopsy samples, if available, were collected and included in the study. Chocolate cysts from endometriosis patients: For patients meeting surgical criteria for ovarian chocolate cysts, cyst contents and inner wall tissues were collected post-resection and included in the study. All tissue collections adhered to strict aseptic techniques to prevent contamination by cervical or vaginal microbiota. Sequencing samples were stored at -80\u0026deg;C, while remaining tissues were fixed in paraffin for histological staining.\u003c/p\u003e\u003cp\u003e All participants were fully informed of the study\u0026rsquo;s purpose, potential health risks, and safeguards prior to enrollment, and provided written informed consent. Patient-identifiable information was anonymized to protect privacy. The study protocol was approved by the Institutional Review Board of Wuhu Maternal and Child Health Center (Approval Number: 20250001).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 16S rRNA Sequencing\u003c/h2\u003e\u003cp\u003eWe collected endometrial tissues from non-endometriosis patients (n\u0026thinsp;=\u0026thinsp;3), endometrial tissues from endometriosis patients (n\u0026thinsp;=\u0026thinsp;3), and intraluminal fluid from chocolate cysts (n\u0026thinsp;=\u0026thinsp;3) for 16S rRNA sequencing.\u003c/p\u003e\u003cp\u003eThe 16S rRNA sequencing libraries were prepared and subjected to high-throughput sequencing by Bioprofile (Shanghai, China). Raw sequencing data were denoised using the DADA2 algorithm to generate non-redundant amplicon sequence variants (ASVs) and their corresponding counts across all samples. Low-abundance ASVs\u0026mdash;those detected in only one sample or with total counts\u0026thinsp;\u0026lt;\u0026thinsp;50 across all samples\u0026mdash;were filtered out. For bacterial and archaeal 16S rRNA gene analysis, the Greengenes2 database was selected as the reference due to its established reputation as a concise and accurate prokaryotic database. To mitigate false-positive differences in subsequent differential abundance and feature selection analyses caused by varying sequencing depths, ASV counts were rarefied to uniform levels across all samples.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Shannon Index and Chao1 Index Analysis\u003c/h2\u003e\u003cp\u003eThe Shannon index, grounded in information entropy theory, provides a composite measure of species richness and evenness, while the Chao1 index, a nonparametric estimator, calculates total species richness based on species occurrence frequencies. Both metrics reflect microbial community richness. Rarefied ASV tables were analyzed using the vegan package (v2.6-10) in R to compute these indices.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Random Forest Analysis\u003c/h2\u003e\u003cp\u003eRandom Forest is a supervised ensemble machine learning algorithm that constructs a \"forest\" of decision trees using bootstrap aggregating (bagging) and random feature subspace selection, enhancing model generalization and robustness. For this study, rarefied ASV tables were analyzed using the scikit-learn (sklearn, v1.6.1) package in Python to build a Random Forest classifier. Only ASVs with non-zero counts across all samples were included in the analysis. The model identified the top 20 species contributing most to classification, along with their importance scores.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 PICRUSt2 Functional Prediction\u003c/h2\u003e\u003cp\u003ePICRUSt2 infers the metabolic functional potential of microbial communities from 16S rRNA gene amplicon sequencing data, primarily based on the \"phylogenetic conservation\" hypothesis. Using PICRUSt2 software (v2.6.0), functional abundance was predicted from unrarefied ASV tables and mapped to the The Kyoto Encyclopedia of Genes and Genomes (KEGG) and MetaCyc databases.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 RNA Sequencing\u003c/h2\u003e\u003cp\u003eEndometrial tissues from non-endometriosis patients (n\u0026thinsp;=\u0026thinsp;3), endometrial tissues from endometriosis patients (n\u0026thinsp;=\u0026thinsp;3), and inner cyst wall tissues from chocolate cysts (n\u0026thinsp;=\u0026thinsp;3) were collected for RNA sequencing.\u003c/p\u003e\u003cp\u003eRNA sequencing libraries were prepared and subjected to high-throughput sequencing by Bioprofile (Shanghai, China). Raw FASTQ data underwent quality filtering, followed by alignment of cleaned reads to the reference genome using HISAT2. HTSeq was employed to quantify read counts per gene as raw expression values. Normalization was performed using FPKM to standardize transcript or gene expression levels.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 GEO Database\u003c/h2\u003e\u003cp\u003eRNA-seq datasets related to endometrial tissues from endometriosis were retrieved from the GEO database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/geo\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/geo\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), specifically from projects GSE11691, GSE25628, and GSE168902[\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The combined dataset included 19 endometriosis patients and 18 healthy controls. Data normalization was performed using the limma R package.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 KEGG and GO Analysis\u003c/h2\u003e\u003cp\u003eThe Gene Ontology (GO) database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.geneontology.org\u003c/span\u003e\u003cspan address=\"https://www.geneontology.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) categorizes genes and gene products into three domains\u0026mdash;cellular component (CC), molecular function (MF), and biological process (BP)\u0026mdash;to identify gene sets significantly enriched in specific biological processes. KEGG (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genome.jp/kegg\u003c/span\u003e\u003cspan address=\"https://www.genome.jp/kegg\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) elucidates higher-order functions and utilities of cells, organisms, and ecosystems through molecular-level information. Differentially expressed genes were uploaded to the Database for Annotation, Visualization, and Integrated Discovery (DAVID, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://david.ncifcrf.gov\u003c/span\u003e\u003cspan address=\"https://david.ncifcrf.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to select the top 20 GO terms and KEGG pathways with the lowest p-values. These were visualized using the bioinformatics platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.bioinformatics.com.cn\u003c/span\u003e\u003cspan address=\"https://www.bioinformatics.com.cn\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10 GSEA Analysis\u003c/h2\u003e\u003cp\u003eGene Set Enrichment Analysis (GSEA) evaluates the distribution trend of predefined gene sets within a phenotype-ranked gene list to assess their contribution to phenotypic traits. GSEA was performed using the local version of the GSEA software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.broadinstitute.org/gsea/index.jsp\u003c/span\u003e\u003cspan address=\"http://www.broadinstitute.org/gsea/index.jsp\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.11 CIBERSORT Immune Infiltration Analysis\u003c/h2\u003e\u003cp\u003eCIBERSORT deconvolutes RNA-seq data using a signature gene set of 22 immune cell types to predict immune cell infiltration levels in tissues. The merged GEO dataset was analyzed for immune infiltration using the R package CIBERSORT.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.12 Immunofluorescence and Immunohistochemical Staining\u003c/h2\u003e\u003cp\u003eImmunofluorescence staining detects protein expression levels and spatial distribution in tissues or cells. Paraformaldehyde-fixed paraffin-embedded tissue sections were deparaffinized using xylene and ethanol with gradient concentration, followed by antigen retrieval with citrate buffer and permeabilization with 0.1% Triton X-100 (Aladdin, China). After blocking with 3% BSA (Biosharp, China), sections were incubated overnight at 4\u0026deg;C with primary antibodies, followed by 1-hour incubation with fluorescent secondary antibodies at room temperature.\u003c/p\u003e\u003cp\u003eImmunohistochemical staining follows similar steps, with additional treatment using 3% hydrogen peroxide post-antigen retrieval. Sections were incubated with primary antibodies overnight at 4\u0026deg;C and secondary antibodies for 30 minutes at room temperature, followed by staining with 3,3'-diaminobenzidine (DAB) and hematoxylin. Images were captured using a fluorescence inverted microscope (Olympus, Japan).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e2.13 Statistical Analysis\u003c/h2\u003e\u003cp\u003eOne-way analysis of variance (ANOVA) was used for comparisons among multiple groups, while Student\u0026rsquo;s t-test was applied for pairwise comparisons. GraphPad Prism 8.0.2 was utilized for graphical and statistical analyses. Statistical significance was set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Microbiome Shifts in the Endometrium with Endometriosis\u003c/h2\u003e\u003cp\u003eTraditionally, it was widely believed that the female uterus is a sterile or minimally colonized environment with trace amounts of bacteria. However, with the advancement of detection techniques, the critical role of the microbiome in the female reproductive organs in urogenital system diseases has been highlighted[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In this study, we collected in-situ endometrial tissues either from patients undergoing total hysterectomy for fibroids or from patients with endometriosis who underwent uterine lavage. And the ectopic endometrial tissues were collected from patients undergoing chocolate cyst resection surgery. Subsequently, 16S rRNA-seq was performed to explore the alterations in the uterine microbiome.\u003c/p\u003e\u003cp\u003eConsistent with previous reports, the microbiome in the endometrium of patients with endometriosis (EC) and that of control patients (NC) was predominantly dominated by Bacillota A 368345 and Bacteroidota, with no significant differences observed across various phyla (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-b, \u003cb\u003eSupplementary Figs.\u0026nbsp;1\u0026ndash;2\u003c/b\u003e). Additionally, there were no significant differences in the Shannon index and Chao1 index between them, although the Chao1 index was slightly elevated in the EC group (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec-d). Random forest analysis identified p__Bacillota_A_368345, p__Fusobacteriota, and p__Actinomycetota as characteristic phyla (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). Specifically, at the species level, Limosilactobacillus mucosae, Lachnospira eligens, Megasphaera_A_38685 indica, Eubacterium_G_180878 ventriosum, and Ruminiclostridium_E siraeum within the p__Bacillota phylum showed significant increases in the EC group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). Lachnospira eligens primarily produces short-chain fatty acids, including acetate and butyrate, and alterations in its gastrointestinal abundance have been associated with chronic nephritis[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Megasphaera_A_38685 indica belongs to the same genus as Megasphaera elsdenii, which has been linked to bacterial vaginosis and can induce chronic endometritis or pelvic inflammatory disease[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Eubacterium_G_180878 ventriosum has been shown to have a positive correlation between its respiratory abundance and asthma airway inflammation levels[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Interestingly, Prevotella muris_A within the p__Bacteroidota phylum also exhibited a significant increase in the EC group. Prevotella muris_A belongs to the Prevotella genus, which has been demonstrated to induce the onset and progression of inflammatory diseases in the gut and respiratory tract[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In summary, although there were no significant differences in the endometrial microbiome between the EC and NC groups, several microbes potentially associated with inflammation showed significant upregulation, emphasizing the potential association between microbiome-induced inflammation and the onset and development of endometriosis.\u003c/p\u003e\u003cp\u003eSubsequently, the PICRUSt algorithm was employed to predict the functions of differentially abundant bacterial genera between the EC and NC groups. Mapping to the KEGG dataset revealed a significant upregulation of the mTOR signaling pathway in the EC group, which has been shown to promote angiogenesis, maintain cellular stemness, and facilitate cell migration[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg). Enzymatic reaction enrichment analysis indicated a significant increase in phosphatidylinositol-4,5-bisphosphate 4-phosphatase (EC:3.1.3.78) in the EC microbiome, which can catalyze the hydrolysis of PIP2 to PIP and subsequently regulate the host cell's PI3K-AKT-mTOR signaling pathway[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh). Metacyc enrichment analysis suggested that the microbiome changes in the EC group might lead to a decreased degradation capacity of L-tryptophan (PWY-5655, TRYPTOPHAN-DEGRADATION-1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei). L-tryptophan is primarily degraded into kynurenine and other metabolites via IDO/TDO, and effective inhibition of IDO promotes the immune surveillance function of CD8 T cells[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Reduced degradation of L-tryptophan may facilitate immune evasion for the ectopic implantation of endometrial cells[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAlthough the pathogenesis of endometriosis has not yet been fully elucidated, there is evidence indicating that ectopic endometrial cells originate from the uterus, as evidenced by identical somatic mutations[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Given the correlation between ectopic tissues and the endometrium, we further examined the microbiome of the endometrial cyst fluid (ECF) in chocolate cysts and conducted an analysis with data from the EC group. The heatmap revealed significant differences in microbiome distribution between the ECF and EC groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, \u003cb\u003eSupplementary Figs.\u0026nbsp;3\u0026ndash;4\u003c/b\u003e). The abundance of Bacillota I in the ECF group increased significantly by 12.05-fold, while the abundances of Actinomycetota, Bacillota A 368345, Bacteroidota, and Pseudomonadota phyla decreased markedly (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Both the Shannon index and the Chao1 index indicated a significant reduction in microbial species richness in the ECF group (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec-d). Random forest analysis identified p_Bacillota_C as a characteristic phylum, with Lactobacillus iners being the predominant bacterial species in ECF (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee-f). Unlike Lactobacillus crispatus, the dominant species in a healthy vagina, the predominance of Lactobacillus iners is often associated with an increased risk of vaginal inflammation[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eKEGG enrichment analysis revealed significant enrichment of the PI3K-Akt signaling pathway, which is closely related to the mTOR signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg). In the endometrium, activation of the PI3K-AKT signaling pathway has been shown to promote angiogenesis and enhance the proliferation, migration, and invasiveness of endometrial cells[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Enzymatic reaction enrichment analysis showed a significant increase in P-type K\u0026thinsp;+\u0026thinsp;transporter (EC:3.6.3.12), dinitrogen oxidoreductase (EC:1.18.6.1), 6,7-dihydropteridine reductase (EC:1.5.1.34), and thymidylate synthase (EC:2.1.1.148), enzymes directly involved in ATP metabolism, suggesting a higher level of energy metabolism in the ECF microbiome (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh). Metacyc enrichment analysis indicated enhanced anaerobic energy metabolism pathways (PWY-6478) in this group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei). Considering the hypoxic environment within chocolate cysts, the enhanced anaerobic metabolism pathways and related metabolic enzymes in the ECF microbiome are possibly fundamental characteristics[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn summary, our analysis of NC, EC, and ECF further confirmed alterations in the uterine microbiome of endometriosis patients. The microbiome changes between EC and NC were correlated with local inflammation in endometriosis patients. Notably, microorganisms were also present in theoretically sterile chocolate cysts. Given the anatomical structure of the female reproductive system, these microorganisms likely originated from the patient's endometrium. The significant differences between the two suggest that these microorganisms had already reached the lesion site before cyst formation and underwent compositional changes in the anaerobic environment after cyst formation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Neutrophil Infiltration in the Endometrium of Patients with Endometriosis\u003c/h2\u003e\u003cp\u003eTo analyze the molecular changes in the endometrium of patients with endometriosis, we conducted RNA-seq on the tissue samples from the aforementioned patients. PCA revealed that, similar to the results of 16S rRNA-seq, there was no significant change in the RNA composition of the endometrium between the EC and NC groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). The volcano plot showed that 726 genes were upregulated and 189 genes were downregulated in the EC group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003eEnrichment analysis of the differentially expressed genes (DEGs) was performed. KEGG analysis indicated a significant enrichment of the neutrophil extracellular trap formation pathway. Additionally, the enrichment of the NF-kappa B signaling pathway and the chemokine signaling pathway suggested a higher level of inflammation in EC tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Gene Ontology (GO) Biological Process (BP) analysis revealed enrichment of leukocyte activation and immune system process, further validating this finding (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). GO Cellular Component (CC) and Molecular Function (MF) analyses similarly revealed enrichment in signaling pathways related to cell membrane vesicles and cytokine receptors (\u003cb\u003eSupplementary Fig.\u0026nbsp;5\u003c/b\u003e). Moreover, the enrichment of response to other organism and response to external biotic stimulus suggested a correlation between the occurrence and development of inflammation and the aforementioned microbiome alterations. Reactome analysis showed enrichment of the neutrophil degranulation and transcriptional regulation of granulopoiesis pathways, further emphasizing the importance of neutrophils in the inflammation of EC tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). Gene Set Enrichment Analysis (GSEA) demonstrated that the Neutrophil activation pathway was activated in the EC group, although with an False Discovery Rate (FDR) of 0.3856 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef).\u003c/p\u003e\u003cp\u003eWe further validated these findings by merging and analyzing three datasets (GSE11691, GSE25628, and GSE168902) from the Gene Expression Omnibus (GEO) database. In the GEO datasets, compared to the NC group, 438 genes were upregulated and 130 genes were downregulated in the EC group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg). Analysis of DEGs showed similar results, with KEGG indicating enrichment of the neutrophil extracellular trap formation pathway, and GO BP showing enrichment of the neutrophil activation involved in immune response and neutrophil degranulation pathways (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh-i). GO CC and MF analyses equally highlighted the significance of cytokine and immune receptor activity (\u003cb\u003eSupplementary Fig.\u0026nbsp;6\u003c/b\u003e). GSEA analysis also revealed activation of the neutrophil activation pathway (FDR\u0026thinsp;=\u0026thinsp;0.0000) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ej). The heatmap similarly showed significant upregulation of genes related to the neutrophil activation pathway in the EC group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ek). CIBERSORT analysis for immune cell infiltration deconvolution demonstrated significant upregulation of activated mast cells and neutrophils in the EC group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003el).\u003c/p\u003e\u003cp\u003eIn conclusion, through transcriptomic analysis, we found a significant upregulation of events related to neutrophil activation in the EC group compared to the NC group. As reported in the literature, additional infiltration of neutrophils contributes to angiogenesis and the release of inflammatory factors, which further recruits macrophages and NK cells and promotes disease progression[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. These findings highlight the role of inflammation in the occurrence of endometriosis and further support the conclusion that microbiome changes in the endometrium of patients with endometriosis may induce the occurrence and development of inflammation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Activation of the PI3K-AKT-mTOR signaling pathway and angiogenesis in ectopic endometrial tissue.\u003c/h2\u003e\u003cp\u003ePCA revealed significant differences in the RNA profiles between the EE and EC groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). In the EE group, 1,621 genes were upregulated and 1,531 genes were downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). GO BP analysis of the differentially expressed genes showed enrichment in pathways such as cell adhesion, extracellular matrix organization, and cell-cell adhesion in the EE group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). This validated the enhanced invasive and adhesive capabilities of ectopic endometrial tissue compared to eutopic endometrium, explaining its ectopic implantation pathology[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The enrichment of the response to xenobiotic stimulus pathway further underscored the significance of microbiome dysregulation in the development of endometriosis. Both GO BP and GSEA revealed significant upregulation of the acute inflammatory response (FDR\u0026thinsp;=\u0026thinsp;0.0000) and angiogenesis (FDR\u0026thinsp;=\u0026thinsp;0.0000) pathways (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec-e). However, unlike the differences between the EC and NC groups, the EE group did not exhibit significant neutrophil activation compared to the EC group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). GO CC and MF analyses indicated enrichment in extracellular region and receptor ligand activity. (\u003cb\u003eSupplementary Fig.\u0026nbsp;7\u003c/b\u003e). KEGG enrichment analysis suggested that the PI3K-AKT signaling pathway played a crucial role in the EE group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg). Combined with the mTOR signaling pathway indicated by 16sRNA sequencing, which is closely related to AKT signaling, GSEA analysis showed significant upregulation of the PI3K-AKT-mTOR pathway in the EE group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh).\u003c/p\u003e\u003cp\u003eAnalyzing the differences between the EE and NC groups, PCA also revealed significant differences in their RNA profiles (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei). The volcano plot showed that 2,227 genes were upregulated and 1,769 genes were downregulated in the EE group compared to the NC group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ej). GO BP analysis similarly showed enrichment in pathways such as cell adhesion, cell-cell adhesion, extracellular matrix organization, positive regulation of cell migration, response to lipopolysaccharide, and angiogenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ek). The analysis results of GO CC and MF were similar to those of EE/EC (\u003cb\u003eSupplementary Fig.\u0026nbsp;8\u003c/b\u003e). KEGG analysis indicated enrichment in pathways such as ECM-receptor interaction, cell adhesion molecules, and leukocyte transendothelial migration, further emphasizing the importance of the PI3K-Akt signaling pathway in the EE group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003el). GSEA analysis revealed upregulation of the angiogenesis, acute inflammatory response, and AKT-mTOR signaling pathways, albeit without significant differences (\u003cb\u003eSupplementary Fig.\u0026nbsp;9\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eThese results demonstrated that ectopic endometrium exhibited more pronounced implantation and angiogenesis capabilities than the EC group, with significant activation of the PI3K-AKT-mTOR signaling pathway. Previous literature has shown that the formation of neutrophil extracellular traps (NETs) can effectively promote angiogenesis and facilitate cancer cell metastasis[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Endometrium from patients with endometriosis exhibited higher levels of neutrophil infiltration, accompanied by elevated levels of NETs, which may induce some endometrial cells to acquire a pro-angiogenic phenotype. High levels of NETs have also been shown to promote the phosphorylation of AKT and mTOR in human umbilical vein endothelial cells (HUVECs), which has been proven to promote tumor proliferation and invasiveness[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Endometrial tissue with a pro-angiogenic phenotype and activated AKT-mTOR signaling has enhanced growth, migration, and implantation capabilities, forming the basis for the occurrence of ectopic tissue.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.4 High levels of neutrophils accompanied by enhanced angiogenesis and AKT-mTOR phosphorylation in the endometrium.\u003c/h2\u003e\u003cp\u003eBased on the aforementioned bioinformatics analysis, we further validated the conclusions through immunostaining. Immunofluorescence staining revealed that the proportion of Ly6G\u003csup\u003e+\u003c/sup\u003eMPO\u003csup\u003e+\u003c/sup\u003e cells in both the EC group and the EE group was significantly higher than that in the NC group, but there was no significant difference between the EC and EE groups (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-b, \u003cb\u003eSupplementary Fig.\u0026nbsp;10\u003c/b\u003e). This was consistent with the results of the GSEA analysis. Immunohistochemical staining showed that the CD31\u003csup\u003e+\u003c/sup\u003e area in the EE group was 4.01 times that in the NC group and 1.46 times that in the EC group. The CD31\u003csup\u003e+\u003c/sup\u003e area in the EC group was also significantly higher than that in the NC group, which validated the phenotype of more active angiogenesis in the endometrium and ectopic tissues of patients with endometriosis (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec-d, \u003cb\u003eSupplementary Fig.\u0026nbsp;11\u003c/b\u003e). The expression level of vimentin is significantly correlated with the invasiveness of endometrial cancer[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The results indicated that the vimentin expression levels in both the EC and EE groups were elevated compared to the NC group, but only the EE group showed a significant increase compared to the NC group (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee-f, \u003cb\u003eSupplementary Fig.\u0026nbsp;12\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eFinally, we detected the expression levels of p-AKT and p-mTOR in the patient tissues. The p-AKT expression level in the EC group was 13.8-fold that in the NC group, and in the EE group, it was further increased to 3.26-fold that in the EC group (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg-h, \u003cb\u003eSupplementary Fig.\u0026nbsp;13\u003c/b\u003e). As a downstream molecule of the AKT signaling pathway, the phosphorylation level of mTOR was synchronized with that of AKT. The p-mTOR expression level in the EE group was 16.19-fold that in the NC group and 4.6-fold that in the EC group (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ei-j, \u003cb\u003eSupplementary Fig.\u0026nbsp;14\u003c/b\u003e). The above results were consistent with the bioinformatics analysis, highlighting the potential mechanism by which neutrophils promote invasion and ectopic colonization by activating the pro-angiogenic phenotype of the endometrium and AKT-mTOR phosphorylation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eNearly 190\u0026nbsp;million women globally have been affected by Endometriosis, which is strongly associated with chronic pain and infertility[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Unfortunately, the pathogenic mechanism of this disease remains unclear. The prevailing hypothesis is retrograde menstruation, which posits that during menstruation, fragments of endometrial tissue flow backward through the fallopian tubes into the peritoneal cavity, where they implant and form lesions[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Our study also supported this hypothesis, as we identified a microbiome dominated by Lactobacillus iners in chocolate cysts. This bacterium primarily resides in the vagina under normal physiological conditions, and its dominance as the predominant strain is often linked to the onset of gynecological diseases. This suggests that microbes in patients with endometriosis may enter deep tissue environments via retrograde menstruation and assist in the implantation of ectopic endometrial tissue. The microbial composition in chocolate cysts differed from that in the endometrial microbiome of endometriosis patients, likely due to secondary selection caused by the hypoxic environment within the cysts.\u003c/p\u003e\u003cp\u003eThe occurrence of endometriosis is inextricably linked to the enhanced migration and implantation capabilities of endometrial cells, given that retrograde menstruation is not a rare event[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. As a chronic inflammatory disease, the correlation between inflammatory responses and the onset and progression of endometriosis has been extensively studied[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Abnormally activated immune cells secrete excessive inflammatory cytokines and chemokines, affecting not only the uterus but also the entire systemic system of patients. An excessive inflammatory environment drives angiogenesis, endometrial cell proliferation, and steroid responses[\u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. In our study, we highlighted the significant enrichment of neutrophils in the endometrium of endometriosis patients. This differs from previous research that solely focused on ectopic lesions, as the chronic inflammation in the endometrium demonstrated that this inflammation was not merely secondary but deeply involved in lesion formation.\u003c/p\u003e\u003cp\u003eRNA-seq analysis highlighted significant activation of the AKT-mTOR signaling pathway in the endometrium of endometriosis patients. Similarly, PICRUSt predictions from 16s rRNA-seq data also underscored the critical role of the mTOR signaling pathway. Yang et al. demonstrated that NET formation can effectively promote angiogenesis in gastric cancer and enhance the phosphorylation of AKT and mTOR in endothelial cells[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. This finding further aligned with the observed increase in neutrophils in the uterus of endometriosis patients. The AKT-mTOR signaling pathway has been confirmed in various diseases to promote cell migration, proliferation, implantation, and angiogenesis[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. GSEA analysis revealed that both cell adhesion and angiogenesis pathways are significantly activated in ectopic endometrium compared to eutopic endometrium in endometriosis patients. These processes collectively contribute to the onset and progression of endometriosis, making AKT-mTOR a promising molecular target for further investigation in the treatment of endometriosis[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIt must be admitted that our research is still at a preliminary stage. Our study has highlighted the microbiome-immune cell-target cell axis, yet it has not delved into the impact of microbial metabolites. For instance, 16S rRNA sequencing indicated the possiblity of tryptophan metabolism disorders in the endometrium of endometriosis, as well as the occurrence of excessive anaerobic glycolysis in chocolate cysts. The effects of these abnormal metabolic patterns on endometrial cells remain to be further explored. Secondly, although we collected tissues from endometriosis patients, we did not further classify the patients according to their phenotypes and disease stages. For example, only endometriosis patients with chocolate cysts was selected, which may mean that our conclusions require further validation before they can be applied to peritoneal endometriosis. Similarly, it is difficult to correlate these conclusions with whether patients experience dysmenorrhea, menstrual disorders, or infertility. Therefore, further work needs to be carried out in the later stage, including building a database to conduct more detailed research.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn conclusion, we were the first to analyze the endometrial microbiome in endometriosis and have proposed the finding that changes in microbiome abundance induce neutrophil infiltration in the endometrium. The infiltrated neutrophils then release neutrophil extracellular traps (NETs), inflammatory cytokines, and chemokines, which activate the phosphorylation of AKT and mTOR in endometrial cells, thereby promoting the occurrence and development of endometriosis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll participants were fully informed of the study\u0026rsquo;s purpose, potential health risks, and safeguards prior to enrollment, and provided written informed consent. Patient-identifiable information was anonymized to protect privacy. The study protocol was approved by the Institutional Review Board of Wuhu Maternal and Child Health Center (Approval Number: 20250001). All human-related research is in strict compliance with the Declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw sequence data reported in this paper have been deposited in the Genome Sequence Archive (Genomics, Proteomics \u0026amp; Bioinformatics 2025) in National Genomics Data Center (Nucleic Acids Res 2025), China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences (GSA-Human: \u003cstrong\u003eHRA015202\u003c/strong\u003e and \u003cstrong\u003eHRA015176\u003c/strong\u003e) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa-human.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Anhui Provincial Health Research Projects (AHWJ2024Ab0238) and Scientific Research Projects of Wuhu Municipal Health Commission (WHWJ2023z030).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHT: Supervision, Writing \u0026ndash; review \u0026amp; editing, Project administration, Funding acquisition. XL: Supervision, Writing \u0026ndash; review \u0026amp; editing, Resources, Project administration. JL: Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing, Resources, Methodology, Investigation, Formal analysis, Data curation, Validation, Conceptualization. WZ: Writing \u0026ndash; original draft, Investigation, Validation, Formal analysis. WY: Writing \u0026ndash; original draft, Investigation, Conceptualization. PL: Writing \u0026ndash; original draft, Methodology.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAs-Sanie S, Mackenzie SC, Morrison L, Schrepf A, Zondervan KT, Horne AW, Missmer SA. Endometriosis: A Review. JAMA. 2025;334(1):64\u0026ndash;78.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBonavina G, Taylor HS. 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Angiogenesis. 2023;26(3):313\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Endometriosis, Microbiome, Neutrophil, AKT, mTOR","lastPublishedDoi":"10.21203/rs.3.rs-8183082/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8183082/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eThe microbiome of the female reproductive system has been extensively studied; however, the correlation between uterine microbial alterations and endometriosis remains unclear.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eEndometrial and lesion tissues were collected from patients with endometriosis, followed by 16S rRNA sequencing and RNA sequencing. The sequencing data were analyzed via PCA, random forest analysis, PICRUSt2 functional prediction, as well as GO and KEGG enrichment analyses, and further validated using the GEO database. Finally, the presence of the identified mechanism was confirmed in the pathological sections of endometriosis patients.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003ewe report that alterations in microbiome abundance were observed in both the endometrium and chocolate cysts of endometriosis patients, which promote the migration, proliferation, and pro-angiogenic capacity of endometrial cells. This process relies on the microbiome-immune cell-target cell axis, wherein abnormal microbiota induced neutrophil infiltration and the release of amounts of neutrophil extracellular traps (NETs), inflammatory cytokines, and chemokines. RNA-seq analysis further revealed that excessive inflammation subsequently activated the AKT-mTOR signaling pathway in endometrial cells, leading to enhanced cell adhesion and angiogenic phenotypes.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eour study suggests that uterine microbiome changes are involved in the onset and progression of endometriosis and may offer a new perspective for its treatment.\u003c/p\u003e","manuscriptTitle":"Alterations in the uterine microbiome drive the progression of endometriosis via neutrophil-mediated AKT-mTOR signaling pathway.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-11 14:29:22","doi":"10.21203/rs.3.rs-8183082/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"92bd8c3d-05e5-450f-8c81-346d673628a2","owner":[],"postedDate":"December 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-17T05:38:54+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-11 14:29:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8183082","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8183082","identity":"rs-8183082","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}