Rotenone targeting the oxidative phosphorylation pathway improves invasive lesions in ovarian endometriosis

Endometriosis (EM) is a prevalent gynecological disorder characterized by diagnostic difficulty and high recur - rence rates. In this study, single-cell RNA sequencing (scRNA-seq) was performed to characterize endome - trial stromal cell (ESC) subpopulations in EM, investigate the involvement of the oxidative phosphoryla - tion (OXPHOS) pathway in EM pathogenesis and fibrosis, and assess the therapeutic effects of Rotenone, a mitochondrial electron transport chain complex I inhibitor. Twelve endometrial single-cell samples from the GSE179640 dataset were analyzed to compare cellular composition between EM and control groups, and to identify EM-associated pathways using Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. The role of OXPHOS was further validated using clinical samples, in vitro assays, and in vivo animal models, with evaluation of histopathology, fibrosis, and mitochondrial function. scRNA-seq analysis demonstrated increased proportions of stromal, lymphoid, and myeloid cells in EM. Subcluster analysis of ESC subclusters revealed enrichment of the eStromal_cycling population, with differentially expressed genes significantly associated with the OXPHOS pathway. Ectopic EM lesions exhibited disrupted tissue architecture, increased collagen deposition, elevated mitochondrial complex I activity and adenosinetriphosphate (ATP) levels. OXPHOS- related proteins were co-localized with Vimentin in ectopic tissues. In vitro, Rotenone suppressed proliferation, migration, and invasion of ectopic endometrial stromal cells (ecESCs), and reduced complex I activity, ATP production, and OXPHOS protein expression. In vivo, Rotenone treatment reduced ovarian cystic lesions and adhesions, decreased fibrosis area, restored estradiol and progesterone levels, and reduced the co-localization of OXPHOS and stromal markers. These findings indicate that activation of the OXPHOS pathway in stromal cells is associated with disease progression and fibrosis. Inhibition of mitochondrial complex I attenuated lesion development and fibrosis in experimental models, suggesting that metabolic modulation may represent a potential therapeutic approach for EM.

Ovarian endometriosis · Single-cell analysis · Oxidative phosphorylation · ESC · Rotenone Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-70076-6

Endometriosis (EM) is an estrogen-dependent chronic inflammatory disorder characterized by the presence of ectopic endometrial-like tissue. It affects approximately 10–15% of women of reproductive age and is associated with 30–50% of infertility cases 1–3. The widely accepted retrograde menstruation theory proposes that refluxed endometrial stromal cells (ESC) adhere to ectopic sites, invade surrounding tissues, and promote angiogen - esis, thereby contributing to lesion formation and fibrosis4. Several pathogenic mechanisms have been proposed, including retrograde menstruation, coelomic metaplasia, and immune dysfunction, reflecting the multifacto - rial nature of EM5–9. Increasing evidence indicates that EM pathogenesis involves complex interactions among genetic, hormonal, immune, inflammatory, angiogenic, and metabolic factors. A better understanding of these mechanisms may facilitate the identification of diagnostic biomarkers and therapeutic targets. Recent advances in single-cell transcriptomics (scRNA-seq) have enabled the dissection of cellular heteroge - neity and functional states within EM lesions at single-cell resolution. ESC are not a homogeneous population; rather, they comprise quiescent, activated, proliferative, and secretory subpopulations, each contributing differ - ently to disease progression10. However, largely large-scale scRNA-seq studies of EM have primarily focused on the immune microenvironment or epithelial-mesenchymal transition11, with limited systematic exploration of the metabolic characteristics of stromal subpopulations and their direct contribution to fibrosis. While metabolic reprogramming in endometriosis has attracted increasing attention, most existing studies have relied on bulk tissue metabolomics or have described generalized mitochondrial dysfunction without resolving cell-type specificity or establishing functional causality. For example, Hawkins et al., identified reduced levels of malic acid and flavin adenine dinucleotide (FAD) in endometriotic tissue from a nonhuman primate model using bulk metabolomics; however, these alterations could not be attributed to specific cell populations 12. Similarly, Chen et al., observed swollen mitochondria with disrupted cristae in adenomyotic stromal cells by transmission electron microscopy (TEM), but did not determine whether these structural abnormalities contributed to invasive behavior or were secondary to inflammatory processes 13. Consequently, it remains unclear whether OXPHOS hyperactivation in a specific cell type functionally drives EM progression and whether this pathway represents a viable therapeutic target. Oxidative phosphorylation (OXPHOS) coordinates energy supply with biological processes processes such as collagen deposition and cell migration through the regulation of oxygen species (ROS) production and metabolic intermediates14,15. In EM, ESC exhibit tumor-like invasive and migratory properties, suggesting that OXPHOS remodeling may drive aberrant ESC proliferation, migration, and fibrosis activity. Rotenone, a reversible inhibi- tor of mitochondrial complex I, suppresses OXPHOS and reduces ATP and ROS production, and has been widely used in studies of neurodegenerative disorders and tumor metabolism 16,17. Therefore, If ESC within EM lesion depend on enhanced OXPHOS activity to maintain their invasive phenotype, targeting mitochondrial complex I may disrupt the metabolic support required for lesion establishment and fibrogenesis, potentially complementing hormonal therapy, surgical resection, and anti-inflammatory strategies to prevent recurrence. Here, we integrated publicly available scRNA-seq data (GSE179640) with clinical samples to characterize stromal subpopulations in EM and to identifiy associations between OXPHOS activation and disease phenotypes. We further employed primary ESC isolates from eutopic and ectopic tissues to evaluate the effects of Rotenone on cell proliferation, migration, and mitochondrial function. In addition, we established a mouse ovarian EM model to assess the impact of Rotenone impact on lesion burden, fibrosis, and circulating sex hormone levels. The work aimed to elucidate the role of OXPHOS in EM progression and to explore the potential of metabolism- targeted therapeutic strategies. Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-70076-6

Date mining scRNA-seq data were retrieved from the Gene Expression Omnibus (GEO) database ( h t t p s : / / w w w . n c b i . n l m . n i h . g o v / g e o ) . The dataset GSE179640, generated using the GPL24676 platform and submitted by Tan et al., was selected for analysis. GSE179640 contains 59 samples, including 31 single-cell sequencing samples, 24 bulk-seq samples, and 4 organoid-type samples 10. Bulk-seq and organoid samples were excluded to maintain single-cell resolution and primary tissue relevance. For single-cell samples, those with 20% mitochondrial gene expression, or > 10% doublet rate were excluded. After applying exclusion criteria, 12 samples remained for analysis: 3 controls and 9 endometriosis samples (Table S1). Data preprocessing and principal component analysis Single-cell transcriptomic analysis was performed using Seurat (v5.0.0), and batch effects were corrected using Harmony (v1.2.4). The workflow included quality control filtering, data normalization, and feature selection. Graph-based clustering was conducted to identify distinct cell populations, followed by cell type annotation and evaluation of cellular composition. Principal component analysis (PCA) was used for initial dimensionality reduction, and visualization was performed using t-distributed stochastic neighbor embedding (t-SNE) and uni - form manifold approximation and projection (UMAP). Identification of differentially expressed genes Differential gene expression analysis was conducted on key subtypes using the R package limma. The criterion for differential expression was |log 2FoldChange| > 1. The Benjamini-Hochberg method was applied to adjust P-values (FDR), with a threshold of adjusted P < 0.05 to define differentially expressed genes (DEGs). KEGG pathway analysis Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs was performed using the clusterProfiler (v4.16.0) in R. Statistical significance was defined as an adjusted P value < 0.05. Enrichment

were visualized using bar plots generated with the ggplot2 package (v4.0.0). Clinical sample collection Paired eutopic and ectopic endometrial tissue samples were collected from Fujian Maternal and Child Health Hospital, including 14 eutopic EM tissues (controls) and 14 ectopic EM tissues. All samples were obtained dur- ing the proliferative phase of the menstrual cycle. The patients were aged 25–40 years, with an average age of 35.1 ± 10.4 years. All procedures involving human participants were conducted in accordance with the Declara - tion of Helsinki and relevant institutional guidelines. All patients procedures approved by the Ethics Committee of Fujian Maternal and Child Health Hospital (approval number: 2025KY253). All participants provided written informed consent prior to participation. Cell isolation and culture Postoperative eutopic and ectopic endometrial tissues were processed to obtain a single-cell suspension. Tis - sues were washed with PBS, transferred on ice, and digested with a pre-chilled solution containing Collagenase Type IV (1 mg/mL; Solarbio, Cat# C8160) and DNase I (0.2 mg/mL) in RPMI-1640 (digestion solution; Procell, Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-70076-6 Cat# PM150110) for 60 min at 37 °C with gentle shaking (220 rpm). The digest was passed through a 70-µm cell strainer (Bkmam, Cat# 110426002), and the filtrate washed with PBS. The suspension was subjected to red blood cell lysis in RBC lysis buffer (Solarbio, Cat# R1011), then centrifuged at 400 ×g for 5 min at 4 °C. The cell pellet was resuspended in PBS, counted, and plated in DMEM complete medium (10 mL per dish, Procell, Cat# PM150210) and incubated at 37 °C with 5% CO 2; medium was changed after 24 h. Stromal cells were identi - fied by immunofluorescence for Vimentin (Proteintech, Cat# 10366-1-AP) and Cytokeratin 7 (Proteintech, Cat# 17513-1-AP). For passaging, cells were trypsinized with 0.25% Trypsin-EDTA (0.25% Trypsin, Beyotime, Cat# C0201) for 1–3 min, and subculture ratios were 1:1 or 1:2 for the first passage and 1:3 for the second. Experi - ments used cells from the second passage onward. Cell treatments Cells in the logarithmic growth phase were harvested, prepared as single-cell suspensions and counted. For the CCK-8 assay, 2 × 10^3 cells per well were seeded into 96-well plates with three technical replicates per condition. After overnight attachment, cells were treated with increasing concentrations of Rotenone (0–10 µM; MCE, Cat# HY-B1756) for 48 h. Following treatment, remove medium and add 100 µL of CCK-8 solution per well; incu - bated at 37 °C for 3 h. Absorbance was measured at 450 nm using a microplate reader. Dose-response curves were generated to determine the half-maximal inhibitory concentration (IC50). For subsequent experiments, cells were divided into four groups: inESC (Intact endometrial stromal cells), inESC + Rotenone, ecESC (Ectopic endome- trial stromal cells), ecESC + Rotenone. After 48 h, collect cells for downstream analyses. CCK-8 assay For the four treatment groups, 2 × 10^3 cells per well were seeded into 96-well plate with three replicates per group. Cell viability was assessed using a CCK-8 assay kit (Beyotime, Cat# C0038) according to the manufac - turer’s instructions. Specifically, after 48 h of treatment, the culture medium was removed, add 10 µL of CCK-8 solution per well, incubated at 37 °C for 3 h, and absorbance was measured at 450 nm using a microplate reader. EdU cell proliferation assay Four groups (n = 3 per group) were seeded at a density of 3 × 10^5 cells per well in a 6-well plate. After 48 h of treatment, proliferation was evaluated using the BeyoClick EdU Cell Proliferation Kit (Beyotime, Cat# C0071S) according to the manufacturer’s instructions. Briefly, EdU was added to a final concentration of 10 µM, and incubated 2 h at 37 °C. Cells were then fixed, permeabilized, and subjected to click chemistry labeling, nuclear counterstaining, and observed and imaged under a fluorescence microscope. Transwell assay After 48 h of Rotenone treatment, cells were detached using 1 mL Trypsin-EDTA solution for 1–2 min, collected and counted. The cell suspension was adjust to 2.5 × 10^5 cells/mL. For the Transwell assay, 600 µL of culture medium containing 10% FBS was added to the lower chamber of a 24-well plate. Transwell inserts (with or without Matrigel; Mogengel, Cat# 0827245) were placed into the wells, and added 200 µL cell suspension to the upper chamber. Incubate at 37 °C, 5% CO2 for 24 h. Removed inserts, wiped the interior to remove cells from the upper surface. Cells on the lower surface were fixed with 600 µL formaldehyde for 20 min. Stained with 0.1% crystal violet (Beyotime, Cat# C0121) for 15 min. The inserts were rinsed three times with PBS and air-dried. Migrated or invaded cells were observed under a light microscope, and images were captured. Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-70076-6 MitoTracker Red CMXRos assay Four groups (n = 3 per group) were seeded at a density of 3 × 10^5 cells per well in a 6-well plate. MitoTracker Red CMXRos (200 nM, Beyotime, Cat# C1035) was added and incubated at 37 °C for 15–30 min. After stain- ing, the dye was removed and replaced with fresh pre-warmed medium. Cells were observed and imaged under a fluorescence microscopy. Subsequently, cells were fixed with PBS containing 3.7% formaldehyde for 15–30 min, washed three times with PBS containing 3% BSA, permeabilized with PBS containing 0.3% Triton X-100 for 15 min. Nuclei were counterstained with Hoechst 33,342 (1×) for 10–15 min. Fluorescence images were then acquired using a fluorescence microscope. Protein extraction and western blotting Cells from Four groups ( n = 3 per group) were digested with 1 mL Trypsin-EDTA Solution (Beyotime, Cat# C0201) for 1–2 min, collected, and lysed in RIPA buffer (Beyotime, Cat# P0013B) to extract total protein. Protein extracts were mixed with 5 × SDS loading buffer (Sangon, Cat# C516031) and denatured at 100 °C for 10 min. Samples were separated by SDS-PAGE and transferred onto NC membrane (Merck, Cat# HATF00010). Membranes were blocked with 5% BSA (Solarbio, Cat# SW3015) for 2 h, then incubated overnight at 4 °C with primary antibodies against PPA1 (Proteintech, Cat# 14985-1-AP), ATP5PO (Proteintech, Cat# 10994-1-AP), NDUFB5 (Proteintech, Cat# 23855-1-AP), COX6C (Proteintech, Cat# 11429-2-AP), and GAPDH (1:5000; Pro- teintech, Cat# 10494-1-AP). After washing, membranes were incubated with HRP-conjugated secondary anti - bodies (1:10,000; Proteintech, Cat# SA00001-1, SA00001-2) for 2 h at 37 °C. Protein bands were visualized using enhanced chemiluminescence (ECL) and detected with a chemiluminescence imaging system (Service - bio, Cat# SCG-W2000). Densitometric analysis of target proteins normalized to GAPDH was performed using ImageJ software. Animal experiments Thirty-two female SPF C57BL/6J mice, 5–6 weeks old, were purchased from SPEF (Suzhou) Biotechnology Co., Ltd. All animals were housed under specific pathogen-free (SPF) conditions (25 ± 1 °C; 40–60% relative humidity; 12 h light/dark cycle) with free access to standard chow and water. Mice were acclimated for 1 week. All animal experiments were conducted in accordance with relevant guidelines and regulations. The proce - dures were approved by the Ethics Committee of Fujian Maternal and Child Health Hospital (approval number: AECSFY2025080). Eight mice were used as donors, and the remaining 24 mice were randomly assigned to four groups (n = 8 per group): Sham group, ovarian endometriosis group (OE group), and OE with Rotenone group (OE + Rotenone group). The OE model was established by allogeneic uterine tissue transplantation as previously described 18. Specifically, the donor-to-recipient ratio was 1:2. Prior to modeling, donor and recipient mice were gavaged with 1 mg/kg estradiol valerate for two consecutive days and fasted for 12 h. Under deep anesthesia induced with 3% isoflurane (Ruiwode Life Science Technology Co., Ltd., Cat# 26675-46-7), donor mice were euthanized by cer- vical dislocation. Uteri were excised, trimmed of surrounding fat and connective tissue, rinsed in PBS, minced into approximately ~ 0.5 mm fragments, and digested in Collagenase Type IV (1 mg/mL) at 37 °C for 30 min. The digestion mixture was centrifuged at 8000 ×g for 5 min; the pellet was resuspended in 100 µL PBS and centrifuged again to remove residual collagenase. Tissue fragments were used immediately for transplantation. For OE modeling, recipient mice were anesthetized with 3% isoflurane, a 2–3 cm ventral midline laparotomy was performed to expose the ovaries. The bilateral ovarian bursa were gently distended with 100 µL PBS to facilitate identification of the membranes, and the ovarian capsule were opened. Half of a donor uterus tissue fragment was placed onto each ovary in a uniform manner. The ovaries were then returned to the peritoneal cavity, and the abdominal wall was closed in layers using 3 − 0 Vicryl sutures. The incision site was disinfected Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-70076-6 with povidone-iodine. Mice were allowed to recover on a heating pad and returned to their cages after regain - ing consciousness. Beginning on the day of surgery (day 0), OE mice were gavaged daily with estradiol valerate (1 mg/kg) for 5 consecutive days to promote ectopic lesion growth; On postoperative day 3, mice received an intraperitoneal injection of penicillin (8 IU in 0.2 mL) to prevent infection. The Sham group underwent identical anesthesia and laparotomy procedures, but no uterine tissue was transplanted; instead, PBS was injected into the peritoneal cavity. Starting on postoperative day 2, mice in the OE + Rotenone group were administered 0.5 mg/ kg Rotenone by oral gavaged once daily for 6 weeks19. Blood and ovarian tissue collection After 6 weeks of treatment, mice were deeply anesthetized with 3% isoflurane. Blood was collected from the abdominal aorta and kept on ice for 1 h to allow clotting, followed by centrifuged at 4000 ×g for 20 min at 4 °C. Serum was separated and stored at -80 °C until further analysis. Mice were subsequently euthanized by cervical dislocation under deep anesthesia. The abdominal cavity was opened, and the reproductive organs were carefully exposed. Ovaries were examined for cyst formation and adhesions, then harvested. One ovary from each mouse was fixed in 4% paraformaldehyde for histological analysis, while the contralateral ovary was stored at -80 °C for subsequent experiments. H&E staining Tissues (clinical endometrium tissues and animal ovaries) were processed for paraffin embedding, sectioned, and deparaffinized through graded xylene and graded ethanol. Sections were stained with hematoxylin and eosin (H&E) for 3–5 min, washed in running water, differentiated, blue, and washed again. Slides were counterstained with eosin for 5 min, then dehydrated through graded ethanol and xylene, cleared, and mounted with neutral gum. Images were acquired and analyzed. Masson’s trichrome staining Masson’s trichrome stain kit (Servicebio, Cat# G1006) was used to analyze collagen fiber deposition in clinical endometrial tissues and mouse ovarian tissues according to the manufacturer’s instructions. In brief, paraffin- embedded sections were deparaffinized and rehydrated to distilled water, then immersed in Masson A solution overnight and rinsed with running water. Subsequently, sections were immersed in a mixed solution of Masson B and Masson C (1:1 ratio) for 1 min, rinsed with running water, differentiated in 1% hydrochloric acid in etha- nol and rinsed again. Sections were then stained with Masson D solution for 6 min, rinsed, and counterstained with Masson E solution for 1 min. After brief blotting, sections were directly stained with Masson F solution for 2–30 s. Sections were differentiated in 1% acetic acid, dehydrated in ethanol, cleared in xylene, and mounted with neutral resin. Images were captured under a light microscope for subsequent analysis. Immunofluorescence Paraffin-embedded tissues from different groups (clinical endometrium and animal ovaries) were sectioned at 4–6 μm and mounted on poly-L-lysine-coated slides. After deparaffinization in xylene and rehydration through a graded ethanol series, antigen retrieval was performed in 10 mM sodium citrate buffer (pH 6.0) at 95–100 °C for 10 min. After cooling, sections were washed three times with PBS and blocked with 1% BSA for 30 min. Sections were then incubated overnight at 4 °C with primary antibodies against PPA1 (1:200; Proteintech, Cat# 14985- 1-AP), ATP5PO (1:200; Proteintech, Cat# 10994-1-AP), NDUFB5 (1:200; Proteintech, Cat# 23855-1-AP), COX6C (1:200; Proteintech, Cat# 11429-2-AP ), and Vimentin (1:200; Proteintech, Cat# 10366-1-AP). After washing with PBS, sections were incubated with HRP-conjugated secondary antibodies (1:10000; Proteintech, Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-70076-6 Cat# SA00001-1, SA00001-2) for 1 h at 37 °C. Nuclei were counterstained with 1 µg/mL DAPI (Beyotime, Cat# C1006) for 5–10 min. Sections were mounted and imaged for analysis. Biochemical and ELISA assay Mitochondrial respiratory chain complex I activity and ATP content were measured in clinical samples, four groups of treated cells, and serum samples from three animal groups using the Mitochondrial Complex I/NADH- CoQ Reductase Activity Assay Kit (Solarbio, Cat# BC0515) and the ATP Content Assay Kit (Jonlnbio, Cat# JL-T0633), respectively, according to the manufacturer’s instructions. Serum concentrations of follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), and progesterone (PROG) were measured using commercial ELISA kits: Mouse FSH ELISA Kit (Jonlnbio, Cat# JL10239), Mouse LHR ELISA Kit (Jonlnbio, Cat# JL51835), Mouse Estradiol (E2) ELISA Kit (Jonlnbio, Cat# JL11790), and Mouse Progesterone (PROG) ELISA Kit (Jonlnbio, Cat# JL20678), following the manufacturer’s instructions. Statistical analyses All data are presented as mean ± standard deviation (SD). Normality of data distribution was assessed using the Shapiro-Wilk test. Differences between two groups were analyzed by t-tests, and multiple-group comparisons were conducted using one-way ANOV A followed by Tukey’s multiple comparisons test (GraphPad, La Jolla, CA, USA). Statistical significance was set at P < 0.05. Denotations: * P < 0.05, ** P < 0.01, *** P < 0.001.

Single-cell transcriptomic analysis of endometrial cell subpopulations To focus on stromal cell subpopulations, we rigorously filtered 31 samples from the GSE179640 dataset. Quality control filtering retained high-quality cells with > 500 detected genes, 1,000-100,000 UMIs per cell, and mito - chondrial gene content < 25% (Figure S1A and S1B). The top 20 principal components (PCs) were selected for downstream analyses based on the elbow plot (Figure S1C). Seurat clustering identified 21 distinct cell clusters (Figure S1D). Cell type annotation was performed using canonical markers as previously described10: epithelial cells (EPCAM, CDH1), stromal cells (COL1A1, PDGFRA), endothelial cells (VWF, PECAM1), myeloid cells (PTPRC, CD68, CD14), and lymphoid cells (PTPRC, CD2, CD3G). Violin plots illustrating marker expression across Seurat clusters, identifying 9 epithelial, 5 stromal, 3 endothelial, 5 myeloid, and 1 lymphoid (Fig. 1A and B, Table S2). To eliminate potential doublets, DoubletFinder was applied (PCs = 1:10, pN = 0.25, pK = 0.09), yielding 5 major cell groups (Fig. 1C and F, Table S3). The distribution of subpopulations in control versus EM groups shows stromal, lymphoid, myeloid, endothelial and epithelial cells comprising 76.49%, 76.75%, 74.01%, 50.88% and 54.04% in EM, respectively. Notably, stromal cells, lymphoid cells, and myeloid cells are signifi - cantly enriched in EM compared with controls (Fig. 1E and F; Table S3). Single-cell analysis reveals activation of oxidative phosphorylation in stromal cells of EM patients ESC are recognized for their roles in immune modulation, tissue repair, and anti-inflammatory properties, making them attractive candidates for cell therapy in endometrial diseases (e.g., Asherman syndrome)20. Therefore, ESC were selected as the primary focus of this study. Following Marečková et al.,21, we performed ESC subpopulation analyses of ESC in control and EM groups, identified three subpopulations: quiescent ESC specific to the pro - liferative phase (eStromal), activated eStromal with matrix metalloproteinases (eStromal_MMPs), and cycling Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-70076-6 Fig. 1 Identification of marker genes and cluster analysis. (A) Expression of marker genes across Seurat clusters; (B) Expres- sion of markers in annotated clusters; (C) Doublet prediction in the dataset; (D) Clustering after removal of doublets; (E) Cluster plots comparing control versus EM; (F) Relative abundances of cells in control versus EM. Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-70076-6 eStromal (eStromal_cycling) exhibiting G2/M and S phase markers (Fig. 2A and B). Marker genes expression across Seurat clusters was examined to annotate ESC subpopulations. High expression of MMP1, AR, ESR1, and PGR was observed in specific Seurat clusters. Within the annotated ESC subtypes, eStromal cells exhibited elevated expression of MMP11, MMP10, INHBA, COL8A1, MMP3, MMP1, AR, ESR1, and PGR, whereas eStromal_MMPs were characterized by high expression of MMP11, AR, ESR1, and PGR. The eStromal_cycling subpopulation displayed increased expression of proliferation-associated genes, including MKI67, PCNA and TGFB1 (Fig. 2C and D). Comparison of subpopulation proportions between control and EM groups showed that all three subpopula - tions increased significantly in EM (Fig. 2E). The eStromal_cycling population is regarded as the differentiation “starting point” for stromal cells. Thus, KEGG analysis of DEGs in this subpopulation demonstrated significant enrichment of OXPHOS (Fig. 2F). The OXPHOS signature comprises 50 DEGs. To represent key components of the mitochondrial electron transport chain and ATP production, we selected four representative genes: NDUFB5 (Complex I, NADH dehydrogenase), COX6C (Complex IV , cytochrome c oxidase), ATP5PO (Complex V , ATP synthase), and PPA1 (a mitochondrial enzyme involved in energy metabolism). These genes collectively reflect electron transport and ATP synthesis processes central to OXPHOS function. Their expression patterns across ESC subtypes are shown in Fig. 2G. In addition, KEGG enrichment analysis identified significant enrichment of extracellular matrix-receptor interaction pathway including COL1A1, COL1A2, FN1, COL4A1, and COL4A2 (P < 0.05; data not shown, ranked 47th among enriched terms). Collectively, single-cell transcriptomic analysis indicates enhanced OXPHOS activity in stromal cells from EM patients. Pathology and activation of the oxidative phosphorylation pathway in stromal cells from EM patients We collected 14 eutopic endometrium tissue samples (control) and 14 ectopic endometrial tissue samples from EM patients. H&E and Masson’s trichrome staining (MTS) were performed on serial sections from the same tissue samples for histological evaluation. In control samples, the endometrium was intact, with glands lined by a single layer of epithelial cells, orderly glandular arrangement, and no obvious hemorrhage or inflammatory infiltration. In contrast, ectopic endometrial tissue from EM patients exhibited disrupted or partially lost glands, irregular glandular organization, the presence of foamy cells, and marked hemorrhage and inflammatory infiltra- tion (Fig. 3A). MTS revealed minimal collagen deposition in control endometrium, whereas EM tissue displayed conspicuous blue staining indicative of fibrosis (Fig. 3B). Quantitative analysis of MTS staining further revealed that the collagen volume fraction (CVF) was significantly higher in EM tissue compared with controls (Fig. 3B). Immunofluorescence analysis confirmed the colocalization of PPA1, ATP5PO, NDUFB5, COX6C with Vimen- tin in both eutopic and EM endometrial tissues. Notably, the expression levels of PPA1, ATP5PO, NDUFB5, and COX6C were significantly elevated in EM tissue relative to eutopic endometrium, whereas Vimentin levels remained comparable between groups (Fig. 3C). Consistently, biochemical assays showed that mitochondrial complex I activity and ATP content were significantly increased in EM endometrium compared with controls (Fig. 3D and E). These findings indicate that EM endometrium exhibits pronounced histopathological alterations and fibrotic deposition, accompanied by enhanced OXPHOS activity. Rotenone inhibits proliferation and migration of endometrial stromal cells Endometrial stromal cells in situ (inESC) and ectopic endometrial stromal cells (ecESC) were isolated by mechanical shear and collagenase digestion. Immunofluorescence showed high expression of the stromal marker Vimentin in both inESC and ecESC, whereas the epithelial marker cytokeratin 7 was negative, indicating high cellular purity suitable for downstream experiments (Figure S2A). Dose-response analysis of Rotenone in ecESC yielded an IC50 of 0.9289 µM, which was used for subsequent treatments (Figure S2B). Further analyses assessed the effects of Rotenone on proliferation, migration, and invasion of inESC and ecESC. The EdU assay revealed a significantly more EdU positive cells in ecESC than in inESC, whereas Rotenone treatment reduced EdU Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-70076-6 Fig. 2 Analysis of stromal cell subpopulations. (A) Stromal subpopulation Seurat clusters; (B) Annotated stromal subpopula- tions; (C) Marker gene expression profiles across stromal Seurat clusters (dot plot); (D) Marker gene expression profiles for annotated stromal subpopulations (dot plot); (E) Proportions of stromal subpopulations across treatments; (F) KEGG enrich- ment for cycling stromal cells (bar graph); (G) Expression of oxidative phosphorylation marker genes (dot plot). NDUFB5 (Complex I), PPA1 (a mitochondrial enzyme involved in energy metabolism), COX6C (Complex IV), and ATP5PO (Com- plex V) were selected to span major electron transport chain complexes. Bubble size, percent expressed; color, average expression. Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-70076-6 Fig. 3 Analysis of clinical samples. (A) H&E staining to compare general morphology between control and EM (4×, bar = 500 μm; 10×, bar = 200 μm). (B) Masson’s trichrome staining to compare fibrosis between control and EM (4×, bar = 500 μm; 10×, bar = 200 μm). Quantitative analysis of collagen volume fraction (%), determined as the percentage of blue- stained area per total tissue area using ImageJ. (C) Colocalization analysis of OXPHOS-related pritein (PPA1, ATP5PO, NDUFB5, COX6C) with ESC marker. (D) Mitochondrial complex I activity in control versus EM. (E) ATP content in control versus EM. (H&E, Masson: n = 8; IF: n = 5; Mitochondrial complex I activity, ATP: n = 14); data are presented as mean ± SD. Normality of data distribution was assessed using the Shapiro-Wilk test. Comparisons between the two groups were per - formed using Student’s t-test. Significance is indicated by asterisks, *** P < 0.001. Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-70076-6 incorporation in both cell types, with a greater reduction in Rotenone-treated ecESC than in untreated ecESC (Fig. 4A and B). CCK-8 assay indicated higher viability of ecESC compared with inESC, and Rotenone mark - edly reduced viability in both groups (Fig. 4C). Transwell assays demonstrated increased migration and invasion capacities in ecESC relative to inESC, these abilities were markedly suppressed following Rotenone treatment in both cell types (Fig. 4D and E). Collectively, these results indicate that Rotenone suppressed proliferation, migra- tion, and invasion of ESC. Additionally, we examined the impact of Rotenone on mitochondrial function in inESC and ecESC. Mitochon- drial respiratory chain complex I activity and ATP content were significantly higher in ecESC than in inESC; Rotenone treatment reduced these parameters in both cell types, the magnitude of suppression was greater in ecESC (Fig. 4F and G). MitoTracker Red CMXRos staining showed stronger mitochondrial fluorescence in ecESC compared with inESC. Rotenone markedly diminished mitochondrial fluorescence in both groups, with the lowest intensity observed in ecESC + Rotenone group (Fig. 4H). Western blotting revealed that the OXPHOS- related proteins PPA1, ATP5PO, NDUFB5 and COX6C were upregulated in ecESC relative to inESC, and were significantly downregulated following Rotenone treatment in both groups (Fig. 4I). Rotenone ameliorates lesions and fibrosis in a uterine endometriosis model by suppressing the oxidative phosphorylation pathway At 6 weeks post-surgery, mice in the sham, OE, and OE + Rotenone groups were euthanized, and ovarian tissues were collected to assess surface cyst formation and surrounding adhesions. Ovaries from sham mice appeared morphologically normal. In contrast, OE mice developed evident cystic lesions on one or both ovaries, whereas OE + Rotenone mice exhibited markedly reduced cystic lesions (Fig. 5A). H&E staining revealed intact ovar - ian architecture and smooth surfaces without adhesions in sham mice. OE mice showed cystic dilation of the ovary accompanied by partial parenchymal atrophy, adhesions between the uterus and ovary, chronic inflamma- tory infiltration, and hemosiderin deposition at adhesion sites and within ovarian endometriosis. In comparison, OE + Rotenone mice demonstrated largely preserved ovarian and uterine structure, without ovarian-uterine adhe- sions, although focal ovarian endometriosis remained (Fig. 5B). Fibrosis is a hallmark of EM. MTS showed minimal fibrotic blue staining in sham mice, whereas OE mice exhibited prominent collagen deposition within the cyst wall. The fibrotic blue staining was markedly attenuated in OE + Rotenone mice compared with OE mice, and quantitative analysis further demonstrated that the CVF was markedly elevated in OE mice (44.15 ± 18.60%) compared with the sham group (3.60 ± 2.07%). Notably, CVF in OE + Rotenone mice (5.19 ± 1.43%) was sig - nificantly lower than in OE mice (Fig. 5C). Together, these results indicate that Rotenone can improve disease progression in an OE model. Biochemical analyses were subsequently performed to assess mitochondrial function and sex hormone-related parameters. Compared with sham mice, OE mice showed significantly increased mitochondrial respiratory chain complex I activity and ATP content (Fig. 6A and B), along with elevated serum LH and FSH levels (Fig. 6C and D), while serum E2 and PROG levels were significantly reduced (Fig. 6E and F). In OE + Rotenone mice, com- plex I activity and ATP content were 402.85 ± 29.77 nmol/min/g and 657.77 ± 41.52 µmol/g, respectively. Serum E2, PROG, LH, and FSH levels were 1001.70 ± 16.59 pg/mL, 3037.50 ± 403.92 pg/mL, 88.43 ± 15.77 ng/mL, and 298.13 ± 88.58 ng/mL, respectively, showing significant restoration compared with the OE group (Fig. 6A-F). Furthermore, immunofluorescence analysis of ovarian tissues demonstrated colocalization of OXPHOS-related proteins PPA1, ATP5PO, NDUFB5, COX6C with the stromal cell marker vimentin; OE ovaries showed mark - edly increased colocalization of these markers compared with sham mice, while OE+Rotenone mice showed reduced colocalization relative to OE mice (Fig. 7). Overall, these results suggest that Rotenone alleviated lesions and fibrosis in ovarian endometriosis, potentially through inhibition of OXPHOS. Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-70076-6 Fig. 4 Rotenone inhibits proliferation and migration of ESC. (A) EdU assay showing proliferation of Rotenone-treated inESC and ecESC; bar = 50 μm. (B) EdU/DAPI cell numbers ratio. (C) CCK-8 assay for cell viability after Rotenone treat- ment. (D) Transwell migration after Rotenone treatment. (E) Transwell invasion after Rotenone treatment. (F) Biochemical measurement of mitochondrial complex I activity after Rotenone treatment in inESC and ecESC. (G) ATP content after Rotenone treatment. (H) Mito-Tracker Red CMXRos staining indicating changes in mitochondrial membrane potential after Rotenone treatment; bar = 50 μm. (I) Western blot analysis of oxidative phosphorylation markers PPA1, ATP5PO, NDUFB5, COX6C after Rotenone treatment. Original blots are presented in Supplementary info file. n = 3; data are pre - sented as mean ± SD. Normality of data distribution was assessed using the Shapiro-Wilk test. Comparisons among multiple groups were performed using one-way ANOV A followed by Tukey’s multiple comparisons test. differences versus inESC are marked with *, ** P < 0.01, *** P < 0.001; differences versus ecESC are marked with #, ### P < 0.001. Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-70076-6 Fig. 5 Rotenone improves pathology and fibrosis in the ovarian endometriosis model. (A) At 6 weeks post-surgery, mice were euthanized and ovarian lesions photographed to assess disease status. In the OE group, cystic lesions were clearly observable, whereas in the OE+Rotenone group the lesions were improved. (B) HE staining confirms the severity of ovar - ian endometriosis; upper panels show 4× magnification (bar = 500 μm) and lower panels show the corresponding magnified region (4×, bar = 100 μm). (C) Masson staining confirms the extent of fibrosis in ovarian endometriosis; upper panels show 4× magnification (bar = 500 μm) and lower panels show the magnified region (4×, bar = 100 μm). Quantitative analysis of collagen volume fraction (%), determined as the percentage of blue-stained area per total tissue area using ImageJ. n = 3; data are presented as mean ± SD. Normality of data distribution was assessed using the Shapiro-Wilk test. Comparisons among multiple groups were performed using one-way ANOV A followed by Tukey’s multiple comparisons test. Differences versus sham are marked with *, ** P < 0.01; differences versus OE are marked with #, # P < 0.05. Sham, sham operation; OE, ovar- ian endometriosis. Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-70076-6

In this study, we employed single-cell transcriptomics to systematically characterize the heterogeneity and molecular features of stromal cells in EM and, for the first time, identified a pivotal role of OXPHOS in disease initiation and progression. Through in vitro experiments and animal model, we further validated the therapeutic potential of targeting mitochondrial electron transport chain complex I (Mitochondrial ETC Complex I) using Rotenone, thereby providing a new rationale and target for precision treatment of EM. In the initial phase, we analyzed the GSE179640 dataset to define cellular composition and subtypes in the endometrium. Five major cell types and 21 subtypes were identified, consistent with previously reports10. Nota- bly, the proportions of ESC, lymphocytes, and myeloid cells were increased in EM samples, reaching 76.49%, 76.75%, and 74.01%, respectively. The expansion of ESC likely reflects enhanced extracellular matrix remodel- ing and fibrosis within ectopic tissues. ESCs play essential roles in normal endometrial physiology, inflamma - tion, angiogenesis, and tissue repair 22–24. The increased proportions of lymphocytes and myeloid cells supports the presence of a chronic inflammatory microenvironment in EM, in which infiltrating immune cells may promote survival, proliferation, and invasion of ectopic endometrium tissue 25,26. Subsequent subclustering of ESCs revealed three functionally distinct subpopulations: quiescent eStromal, activated eStromal_MMPs, and Fig. 6 Rotenone treatment alters mitochondrial function and sex hormone levels in the mouse ovarian endometriosis model. At 6 weeks post-surgery, ovarian tissues were collected after euthanasia for biochemical analyses of different treatment groups. (A) Mitochondrial respiratory chain complex I activity in the ovary; (B) ovary ATP levels; and serum concentrations of (C) E2, (D) PROG, (E) LH, (F) FSH in mice from different treatment groups. n = 8; data are shown as mean ± SD. Normal- ity of data distribution was assessed using the Shapiro-Wilk test. Comparisons among multiple groups were performed using one-way ANOV A followed by Tukey’s multiple comparisons test. Differences versus sham are marked with *, *** P < 0.001; differences versus OE are marked with #, ### P < 0.001. Sham, Sham operation; OE, ovarian endometriosis; E2, Estradiol; PG, Progesterone; LH, Luteinizing Hormone; FSH, Follicle-Stimulating Hormone. Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-70076-6 proliferative eStromal_cycling. This classification is broadly consistent with Marečková et al., 21, although we observed distinct expression patterns specific to EM. The eStromal_MMPs subgroup exhibited high expression of matrix metalloproteinases (MMPs, including MMP1, MMP3, MMP10, and MMP11), which are involved in extracellular matrix degradation, angiogenesis, and cell migration 27. In EM, MMP overexpression may facili - tate lesion invasiveness and neovascularization. This subgroup also shows high expression of ESR1, PGR, and AR, suggesting a prominent role for hormone signaling in stromal activation. The eStromal_cycling subset, characterized by high expression of cell cycle–related genes (MKI67, PCNA) and TGFB1, may represent a proliferative progenitor-like population. TGF-β signaling plays a central regulatory role in fibrosis, and may be a key mechanism underlying EM-associated fibrosis28,29. Importantly, this subset showed significant enrichment Fig. 7 Immunofluorescence analysis of colocalization between oxidative phosphor- ylation markers and stromal cell marker in ovaries after Rotenone treatment. Immuno- fluorescence detection of PPA1, ATP5PO, NDUFB5, COX6C with Vimentin in ovaries from the different groups, illustrating the degree of colocalization. Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-70076-6 of OXPHOS-related pathways, suggesting disease-associated metabolic reprogramming in proliferative stromal cells. Metabolic reprogramming is a hallmark of malignant cells, typically involving enhanced glycolysis and altered mitochondrial respiration to meet bioenergetic and biosynthetic demands. Although EM is a benign condition, ectopic endometrial tissue exhibits invasive and migratory properties reminiscent of malignancy. Recent studies report increased glycolytic activity in ectopic EM cells, likely reflecting elevated energy requirements 30. Gly- colysis provides pyruvate for mitochondrial oxidation, fueling the TCA cycle and ATP generation via OXPHOS. Elevated ATP levels may also contribute to EM-associated pain and infertility, as ATP acts as a purinergic signal- ing molecule via receptors such as P2 × 3, P2 × 4, P2 × 7, and P2Y 31. Our in vitro findings demonstrated that OXPHOS was markedly upregulated in ESC derived from EM patients, accompanied by increased expression of PPA1, ATP5PO, NDUFB5 and COX6C. Enhanced mitochondrial respiratory chain complex I activity and ATP production were observed in ecESCs, together with augmented proliferative, migratory, and invasive capacities. These data suggest that OXPHOS activation may provide the energetic support required for ectopic lesion growth and progression. Mitochondrial dysfunction is closely linked to inflammatory responses. Increased OXPHOS activity may elevate ROS production, leading to oxidative stress and inflammatory signaling. In EM, ectopic lesions are frequently associated with chronic inflammatory infiltration, which may be exacerbated by mitochon- drial ROS overproduction32,33. In turn, inflammation may further impair mitochondrial homeostasis, creating a self-perpetuating pathological cycle. Furthermore, Rotenone effectively inhibits proliferation, migration, and invasion of ESC, with more pro - nounced effects in ecESC. In vivo, Rotenone treatment significantly improved pathological features in the OE model, including reducted cystic lesions, fewer adhesions and ameliorated fibrosis. Importantly, serum sex hor - mone levels were also significantly restored in the OE + Rotenone group, suggesting that Rotenone not only mitigates local pathology but also helps restore ovarian endocrine function. Nevertheless, several limitations should be acknowledged. First, the single-cell transcriptomic data were derived from publicly available databases with limited clinical annotation, which may restrict interpretability. Second, the surgically induced murine model does not fully recapitulate human endometriosis. Mice neither menstruate nor spontaneously develop EM; therefore, this model may not entirely reflect the chronic inflamma - tion, hormonal cyclicity, and progressive fibrosis observed in patients 34,35. These interspecies differences limit clinical translation. Third, the human validation cohort was relatively small and requires confirmation in larger populations. Fourth, although Rotenone effectively inhibited complex I activity in our study, its documented neu- rotoxicity precludes clinical application36,37. Here, Rotenone was used solely as a pharmacological tool to estab- lish the functional relevance of OXPHOS/complex I in EM. For clinical translation, Metformin, Mdivi-1, SS-31, and mitochondrial-targeted antioxidants as potential therapeutics for clinical translation 38–40. Finally, while our pharmacological findings implicate OXPHOS/complex I in EM pathogenesis, definitive causal inference will require genetic manipulation combined with rescue experiments, which remain to be addressed in future studies.

Our analysis identifies aberrant activation of the OXPHOS in ESC, which is a key driver of disease progression and fibrosis. Targeted inhibition of mitochondrial complex I markedly suppresses ESC proliferation, migration, and invasion, improves lesion pathology and fibrosis in the OE model, and partially restores ovarian endocrine function. These findings provide a novel metabolism targeted strategy for EM intervention. Supplementary Information The online version contains supplementary material available at https://doi.org/10.1038/ s41598-026-70076-6. Author contributions Xiulan Weng and Shunhe Lin conducted all the experiments in this article, and wrote the manuscript. Zhenna Wang and Chaobin Liu visualized the data and combined the results. Guan Lin provided methodological support. Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-70076-6 Pengming Sun and Jingsong Yi conceived and designed the manuscript, and revised the manuscript. All the authors reviewed the manuscript and approved it for publication. Funding We are very grateful for the funding support from Joint Funds for the innovation of science and Technology, Fujian province (Grant number: 2025Y9611), and Fujian Provincial Traditional Chinese Medicine Science and Technology Program (Category B) (Grant number: 2025YBB014). Data availability The authors confirm that the data supporting the findings of this study are available within the article and/ or its supplementary materials, and from the corresponding authors upon reasonable request. Declarations Competing interests The authors declare no competing interests. Ethical statement The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Ethical approval All patients procedures were approved by the Ethics Committee of Fujian Maternal and Child Health Hospital (approval number: 2025KY253). All animal procedures were approved by the Ethics Committee of Fujian Maternal and Child Health Hospital (approval number: AECSFY2025080). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Authors and Affiliations Xiulan Weng1,2 · Shunhe Lin1 · Zhenna Wang1 · Chaobin Liu1 · Guan Lin1 · Pengming Sun1,2 · Jingsong Yi2,3 Pengming Sun [email protected] Jingsong Yi [email protected] 1 Department of Gynecology, Fujian Maternity and Child Health Hospital (Fujian Obstetrics and Gynecology Hospital), Fuzhou 350001, Fujian, China 2 College of Clinical Medicine for Obstetrics & Gynecology and Pediatrics, Fujian Medical University, Fuzhou 350001, Fujian, China 3 Fujian Maternity and Child Health Hospital, No. 18 Daoshan Road, Gulou District, Fuzhou, Fujian 350001, PR China