A preliminary single-cell map of endometriosis suggests roles for fibroblast metabolism and macrophage polarization in disease progression

Single-cell RNA sequencing (scRNA-seq) serves as a crucial tool for rapidly and accurately quantifying tissue heterogeneity and its associated biological significance within highly organized life systems. In this study, we utilized scRNA-seq to compare and analyze the characteristics of cell populations in eutopic endometrium (EUE) and its paired ectopic endometrium (ECE). Also, through multiple analytical approaches, we revealed the distinct contributions of fibroblasts and immune cells in endometriosis (EMs), with a particular focus on the features of fibroblasts and macrophages during pseudo-time differentiation. Notably, we not only identified Aquaporin 9-positive monocyte/macrophages (AQP9+/CD68+ cells) by anchoring the target protein AQP9 from the macrophage marker gene, but further quantitative analysis confirmed that AQP9 expression abundance was positively correlated with the disease stage of EMs (rASRM stage), i.e., the higher the disease stage, the higher the expression level of AQP9 in ectopic lesions. This suggests that AQP9 may be involved in the pathological progression of EMs, and its expression shows a preliminary association with disease severity. In summary, our results provide a high-resolution, preliminary cellular map of EMs, proposing novel hypotheses regarding disease progression mechanisms, and informing potential research priorities for future investigations. Similar content being viewed by others Data availability The datasets presented in this article are not readily available because of Regulations on the management of human genetic resources in China. Requests to access the datasets should be directed to the corresponding author.

Artemova D, Vishnyakova P, Khashchenko E, Elchaninov A, Sukhikh G, Fatkhudinov T (2021a) Endometriosis and cancer: exploring the role of macrophages. Int J Mol Sci 22(10). https://doi.org/10.3390/ijms22105196 Artemova D, Vishnyakova P, Khashchenko E, Elchaninov A, Sukhikh G, Fatkhudinov T (2021b) Endometriosis and cancer: exploring the role of macrophages. Int J Mol Sci 22(10). https://doi.org/10.3390/ijms22105196 Barcena De Arellano ML, Gericke J, Reichelt U et al (2011) Immunohistochemical characterization of endometriosis-associated smooth muscle cells in human peritoneal endometriotic lesions. Hum Reprod 26(10):2721–2730. https://doi.org/10.1093/humrep/der253 Bettelli E, Carrier Y, Gao W et al (2006a) Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory t cells. Nature 441(7090):235–238. https://doi.org/10.1038/nature04753 Bettelli E, Carrier Y, Gao W et al (2006b) Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory t cells. Nature 441(7090):235–238. https://doi.org/10.1038/nature04753 Blanco LP, Salmeri N, Temkin SM, Shanmugam VK, Stratton P (2025) Endometriosis and autoimmunity. Autoimmun Rev 24(4):103752. https://doi.org/10.1016/j.autrev.2025.103752 Choi YS, Park JH, Yoon J et al (2019) Potential roles of aquaporin 9 in the pathogenesis of endometriosis. Mol Hum Reprod 25(7):373–384. https://doi.org/10.1093/molehr/gaz025 de Araujo-Souza PS, Hanschke SCH, Viola JPB (2015) Epigenetic control of interferon-gamma expression in CD8 t cells. J Immunol Res 2015:849573. https://doi.org/10.1155/2015/849573 Dong Y, Zhang M, OuYang J, Zhou W, Yu B (2023) Tumor-derived endothelial cell: important etiological factors in endometriosis. Arch Med Res 54(7):102891. https://doi.org/10.1016/j.arcmed.2023.102891 Dos Santos Hidalgo G, Meola J, Rosa E, Silva JC, Paro De Paz CC, Ferriani RA (2011) TAGLN expression is deregulated in endometriosis and may be involved in cell invasion, migration, and differentiation. Fertil Steril 96(3):700–703. https://doi.org/10.1016/j.fertnstert.2011.06.052 Godbole G, Suman P, Malik A et al (2017) Decrease in expression of HOXA10 in the decidua after embryo implantation promotes trophoblast invasion. Endocrinology 158(8):2618–2633. https://doi.org/10.1210/en.2017-00032 Guerin L, Wu V, Houser B et al (2015) CD1 antigen presentation and autoreactivity in the pregnant human uterus. Am J Reprod Immunol 74(2):126–135. https://doi.org/10.1111/aji.12375 He L, Jhong J, Chen Q et al (2021) Global characterization of macrophage polarization mechanisms and identification of m2-type polarization inhibitors. Cell Rep 37(5):109955. https://doi.org/10.1016/j.celrep.2021.109955 Hogg C, Horne AW, Greaves E (2020) Endometriosis-associated macrophages: origin, phenotype, and function. Front Endocrinol (Lausanne) 11:7. https://doi.org/10.3389/fendo.2020.00007 Jiang D, Xiao R, Bai J et al (2025) FOLR2(+) macrophages in cancer: allies or enemies. Cell Commun Signal 23(1):261. https://doi.org/10.1186/s12964-025-02257-1 Jin FY, Nathan C, Radzioch D, Ding A (1997) Secretory leukocyte protease inhibitor: a macrophage product induced by and antagonistic to bacterial lipopolysaccharide. Cell 88(3):417–426. https://doi.org/10.1016/s0092-8674(00)81880-2 Jing J, Sun J, Wu Y et al (2021) AQP9 is a prognostic factor for kidney cancer and a promising indicator for m2 TAM polarization and CD8 + t-cell recruitment. Front Oncol 11:770565. https://doi.org/10.3389/fonc.2021.770565 Kang Y, Cho HJ, Lee Y et al (2023) IL-17a and th17 cells contribute to endometrial cell survival by inhibiting apoptosis and NK cell mediated cytotoxicity of endometrial cells via ERK1/2 pathway. Immune Netw 23(2):e14. https://doi.org/10.4110/in.2023.23.e14 Kasvandik S, Samuel K, Peters M et al (2016) Deep quantitative proteomics reveals extensive metabolic reprogramming and cancer-like changes of ectopic endometriotic stromal cells. J Proteome Res 15(2):572–584. https://doi.org/10.1021/acs.jproteome.5b00965 Khan KN, Yamamoto K, Fujishita A et al (2019) Differential levels of regulatory t cells and t-helper-17 cells in women with early and advanced endometriosis. J Clin Endocrinol Metab 104(10):4715–4729. https://doi.org/10.1210/jc.2019-00350 Kim T, Lee SU, Yun S et al (2011) Human microRNA-27a* targets prf1 and GzmB expression to regulate NK-cell cytotoxicity. Blood 118(20):5476–5486. https://doi.org/10.1182/blood-2011-04-347526 Lagana AS, Garzon S, Gotte M et al (2019) The pathogenesis of endometriosis: molecular and cell biology insights. Int J Mol Sci 20(22). https://doi.org/10.3390/ijms20225615 Lagana AS, Salmeri FM, Ban Frangez H, Ghezzi F, Vrtacnik-Bokal E, Granese R (2020) Evaluation of m1 and m2 macrophages in ovarian endometriomas from women affected by endometriosis at different stages of the disease. Gynecol Endocrinol 36(5):441–444. https://doi.org/10.1080/09513590.2019.1683821 Lawson D, Harrison M, Shapland C (1997) Fibroblast transgelin and smooth muscle SM22alpha are the same protein, the expression of which is down-regulated in many cell lines. Cell Motil Cytoskeleton 38(3):250–257. Li W, Wu M, Tsai S (2021) HYPOXIA and REPRODUCTIVE HEALTH: the role of hypoxia in the development and progression of endometriosis. Reproduction 161(1):F19–F31. https://doi.org/10.1530/REP-20-0267 Lin X, Lei Y, Pan M et al (2024) Augmentation of scleral glycolysis promotes myopia through histone lactylation. Cell Metab 36(3):511–525. https://doi.org/10.1016/j.cmet.2023.12.023 Liu X, Xu Q, Li Z, Xiong B (2020) Integrated analysis identifies AQP9 correlates with immune infiltration and acts as a prognosticator in multiple cancers. Sci Rep 10(1):20795. https://doi.org/10.1038/s41598-020-77657-z Ma J, Zhang L, Zhan H et al (2021) Single-cell transcriptomic analysis of endometriosis provides insights into fibroblast fates and immune cell heterogeneity. Cell Biosci 11(1):125. https://doi.org/10.1186/s13578-021-00637-x Mabbott NA, Baillie JK, Brown H, Freeman TC, Hume DA (2013) An expression atlas of human primary cells: inference of gene function from coexpression networks. BMC Genomics 14:632. https://doi.org/10.1186/1471-2164-14-632 Mareckova M, Garcia-Alonso L, Moullet M et al (2024) An integrated single-cell reference atlas of the human endometrium. Nat Genet 56(9):1925–1937. https://doi.org/10.1038/s41588-024-01873-w Mei J, Chang K, Sun H (2017) Immunosuppressive macrophages induced by IDO1 promote the growth of endometrial stromal cells in endometriosis. Mol Med Rep 15(4):2255–2260. https://doi.org/10.3892/mmr.2017.6242 Meyer SU, Krebs S, Thirion C, Blum H, Krause S, Pfaffl MW (2015) Tumor necrosis factor alpha and insulin-like growth factor 1 induced modifications of the gene expression kinetics of differentiating skeletal muscle cells. PLoS ONE 10(10):e0139520. https://doi.org/10.1371/journal.pone.0139520 Palma A, Jarrah AS, Tieri P, Cesareni G, Castiglione F (2018) Gene regulatory network modeling of macrophage differentiation corroborates the continuum hypothesis of polarization states. Front Physiol 9:1659. https://doi.org/10.3389/fphys.2018.01659 Rutihinda C, Haroun R, Saidi NE et al (2023) Inhibition of the CCR6-CCL20 axis prevents regulatory t cell recruitment and sensitizes head and neck squamous cell carcinoma to radiation therapy. Cancer Immunol Immunother 72(5):1089–1102. https://doi.org/10.1007/s00262-022-03313-2 Sarsenova M, Lawarde A, Pathare ADS et al (2024) Endometriotic lesions exhibit distinct metabolic signature compared to paired eutopic endometrium at the single-cell level. Commun Biol 7(1):1026. https://doi.org/10.1038/s42003-024-06713-5 Shi Y, Yasui M, Hara-Chikuma M (2022) AQP9 transports lactate in tumor-associated macrophages to stimulate an m2-like polarization that promotes colon cancer progression. Biochem Biophys Rep 31:101317. https://doi.org/10.1016/j.bbrep.2022.101317 Shinde AV, Humeres C, Frangogiannis NG (2017) The role of alpha-smooth muscle actin in fibroblast-mediated matrix contraction and remodeling. Biochim Biophys Acta Mol Basis Dis 1863(1):298–309. https://doi.org/10.1016/j.bbadis.2016.11.006 Tan Y, Flynn WF, Sivajothi S et al (2022) Single-cell analysis of endometriosis reveals a coordinated transcriptional programme driving immunotolerance and angiogenesis across eutopic and ectopic tissues. Nat Cell Biol 24(8):1306–1318. https://doi.org/10.1038/s41556-022-00961-5 Tello D, Balsa E, Acosta-Iborra B et al (2011) Induction of the mitochondrial NDUFA4l2 protein by HIF-1alpha decreases oxygen consumption by inhibiting complex i activity. Cell Metab 14(6):768–779. https://doi.org/10.1016/j.cmet.2011.10.008 Tufa DM, Chatterjee D, Low HZ, Schmidt RE, Jacobs R (2014) TNFR2 and IL-12 coactivation enables slanDCs to support NK-cell function via membrane-bound TNF-alpha. Eur J Immunol 44(12):3717–3728. https://doi.org/10.1002/eji.201444676 Verkman AS, Hara-Chikuma M, Papadopoulos MC (2008) Aquaporins–new players in cancer biology. J Mol Med (Berl) 86(5):523–529. https://doi.org/10.1007/s00109-008-0303-9 Vinogradov AD, Grivennikova VG (2016) Oxidation of NADH and ROS production by respiratory complex i. Biochim Biophys Acta 1857(7):863–871. https://doi.org/10.1016/j.bbabio.2015.11.004 Vissers G, Giacomozzi M, Verdurmen W, Peek R, Nap A (2024) The role of fibrosis in endometriosis: a systematic review. Hum Reprod Update 30(6):706–750. https://doi.org/10.1093/humupd/dmae023 Wang Z, Chen L, Huang Y et al (2021) Pharmaceutical targeting of succinate dehydrogenase in fibroblasts controls bleomycin-induced lung fibrosis. Redox Biol 46:102082. https://doi.org/10.1016/j.redox.2021.102082 Wang J, Yang P, Yu T et al (2022) Lactylation of PKM2 suppresses inflammatory metabolic adaptation in pro-inflammatory macrophages. Int J Biol Sci 18(16):6210–6225. https://doi.org/10.7150/ijbs.75434 Wang Y, Huang S, Kong W et al (2023) Corilagin alleviates liver fibrosis in zebrafish and mice by repressing IDO1-mediated m2 macrophage repolarization. Phytomedicine 119:155016. https://doi.org/10.1016/j.phymed.2023.155016 Younesi FS, Miller AE, Barker TH, Rossi FMV, Hinz B (2024) Fibroblast and myofibroblast activation in normal tissue repair and fibrosis. Nat Rev Mol Cell Biol 25(8):617–638. https://doi.org/10.1038/s41580-024-00716-0 Zhang D, Wang Y, Shi Z et al (2015) Metabolic reprogramming of cancer-associated fibroblasts by IDH3alpha downregulation. Cell Rep 10(8):1335–1348. https://doi.org/10.1016/j.celrep.2015.02.006 Zhang Q, Duan J, Liu X, Guo S (2016) Platelets drive smooth muscle metaplasia and fibrogenesis in endometriosis through epithelial-mesenchymal transition and fibroblast-to-myofibroblast transdifferentiation. Mol Cell Endocrinol 428:1–16. https://doi.org/10.1016/j.mce.2016.03.015 Zhang Z, Xiong Y, Jiang H et al (2024) Vaginal extracellular vesicles impair fertility in endometriosis by favoring th17/treg imbalance and inhibiting sperm activity. J Cell Physiol 239(4):e31188. https://doi.org/10.1002/jcp.31188

We thank Xiaohua Yao and Junhao Yu from OE Biotech Co., Ltd (Shanghai, China)to help us in sequencing analysis. We also thank all of the project participants for their contributions. Funding This work was supported by Project for Pioneering Clinical Technologies through Open Challenge by Changzhou Municipal Health Commission (WJJBGS202505); Key Medical Research Project of Jiangsu Province Health Commission (K2023058); Study on the Mechanism of AQP9-Mediated Lactate Transport in Macrophages Involved in Endometriosis (YJ202408); and The Application Foundation Project of Changzhou Science and Technology Bureau (CJ20245041). Author information Authors and Affiliations Contributions PZ, PX, and WZ contributed to the study conception and design, and provided supervision and project administration. XC and HT performed the experiments, conducted data analysis, and wrote the original manuscript. XF and YW were responsible for patient recruitment and sample collection. JY assisted with data management and validation. All authors critically reviewed, edited the manuscript, and approved the final version for submission. Corresponding authors Ethics declarations Ethical approval This study was conducted in accordance with the Declaration of the Helsinki. This study involving human participants was reviewed and approved by the Ethics Committee of Changzhou Maternal and Child Health Care Hospital, Affiliated with Nanjing Medical University (No. 202276). Informed consent was obtained from all individual participants included in the study. Competing interests The authors declare no competing interests. Clinical trial number Not applicable. Additional information Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Xiurong Cao and Hui Tao are co-frst author. Supplementary Information Below is the link to the electronic supplementary material. Rights and permissions Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. About this article Cite this article Cao, X., Tao, H., Fan, X. et al. A preliminary single-cell map of endometriosis suggests roles for fibroblast metabolism and macrophage polarization in disease progression. Funct Integr Genomics 26, 78 (2026). https://doi.org/10.1007/s10142-026-01860-w Received: Revised: Accepted: Published: Version of record: DOI: https://doi.org/10.1007/s10142-026-01860-w