{"paper_id":"1aa30b0d-b784-4007-ba26-c05bd687a071","body_text":"Abstract\nIt is generally believed that ovarian hormones regulate uterine functions and their altered levels result in various uteropathies like non-receptive uterus, endometrial hyperplasia, adenomyosis, endometriosis, leiomyomas and cancer. Uterus harbors two populations of stem cells including pluripotent, very small embryonic-like stem cells (VSELs) and tissue-specific progenitors (endometrial stem cells, EnSCs). Unlike endometrial mesenchymal stem/ stromal cells, VSELs/EnSCs express ERα, ERβ and PR which makes them directly vulnerable to perinatal endocrine insults. Present study was undertaken to evaluate whether uteropathies occur due to altered hormones and/or intrinsic changes in stem/progenitor cells. Mice pups, exposed to estradiol (20 µg/pup/day) on postnatal days 3–7 or vehicle, were subjected to bilateral ovariectomy on day 30 and later exposed sequentially to estradiol and progesterone resulting in receptive uterus in control mice. Despite similar hormonal exposure, endocrine disruption resulted in non-receptive uterus with noticeable endometrial and myometrial hyperplasia and up-regulation of stem cell markers (Oct-4A, Oct-4, Sox2, Nanog). Glands were poorly formed and ‘defective’ epithelial progenitors were found disseminated into myometrium and blood vessels revealing how adenomyosis and endometriosis possibly initiate. Progesterone resistance and estradiol dominance due to downregulation of Erα & Pr and upregulation of Erβ transcripts was observed in both intact uterus and stem cells enriched from uterus. Transcripts specific for DNA mismatch repair axis (Pcna, NP95 and Dnmt1), repair enzymes (Brca-1, Rad51 and Mlh1) were dysregulated whereas Ki67 was ten-folds increased suggestive of genomic instability. Study reveals role of stem cells in initiating uteropathies during adult life independent of circulatory ovarian hormones.\nGraphical Abstract\nEndocrine disruption affects tissue resident stem/progenitor cells (VSELs/EnSCs) in both endometrium and myometrium, result in epithelial cells hyperplasia, non-receptive endometrium, adenomyosis and defective stem cells and epithelial progenitors were detected in the perimetrium from where they can mobilize to ectopic sites to initiate endometriosis. Study shows stem cell basis for various uteropathies. VSEL: Very small embryonic like stem cell; EnSC: Endometrial stem cell; E + P: Estradiol + Progesterone; E: Endometrium; P: Perimetrium; M: Myometrium; ACD: Asymmetrical cell division; SCD: Symmetrical cell division; CE: Clonal expansion; G: Gland; S: Stromal cell; US: Undifferentiated stromal cell; LE: Luminal epithelium; GE: Glandular epithelium; EP: Epithelial progenitors; SMC: Spindle-shaped myometrial cell; OMC: Oval-shaped myometrial cell\nSimilar content being viewed by others\nData availability\nSupplementary section provides additional data.\nCode availability\nNot applicable.\nReferences\nMarquardt, R. M., Kim, T. H., Shin, J.-H., & Jeong, J. W. (2019). Progesterone and estrogen signaling in the endometrium: What goes wrong in endometriosis? International Journal of Molecular Sciences, 20(15), 3822. https://doi.org/10.3390/ijms20153822\nMontgomery, G. W., Mortlock, S., & Giudice, L. C. (2020). Should genetics now be considered the pre-eminent etiologic factor in endometriosis? Journal of Minimally Invasive Gynecology, 27(2), 280–286. https://doi.org/10.1016/j.jmig.2019.10.020\nAndaloussi, A. E., Al-Hendy, A., Ismail, N., Boyer, T. G., & Halder, S. K. (2020). Introduction of somatic mutation in MED12 induces Wnt4/β-catenin and disrupts autophagy in human uterine myometrial cell. Reproductive Sciences, 27(3), 823–832. https://doi.org/10.1007/s43032-019-00084-7\nNewbold, R. R., Bullock, B. C., & McLachlan, J. A. (1990). Uterine adenocarcinoma in mice following developmental treatment with estrogens: A model for hormonal carcinogenesis. Cancer Research, 50, 7677–7681.\nNewbold, R. R., Jefferson, W. N., & Padilla-Banks, E. (2009). Prenatal exposure to bisphenol a at environmentally relevant doses adversely affects the murine female reproductive tract later in life. Environmental Health Perspectives, 117(6), 879–885. https://doi.org/10.1289/ehp.0800045\nSuen, A. A., Jefferson, W. N., Williams, C. J., & Wood, C. E. (2018). Differentiation patterns of uterine carcinomas and precursor lesions induced by neonatal estrogen exposure in mice. Toxicologic Pathology, 46(5), 574–596. https://doi.org/10.1177/0192623318779326\nFernung, L. E. P., Yang, Q., Sakamuro, D., Kumari, A., Mas, A., & Al-Hendy, A. (2018). Endocrine disruptor exposure during development increases incidence of uterine fibroids by altering DNA repair in myometrial stem cells†. Biology of Reproduction. https://doi.org/10.1093/biolre/ioy097\nSignorile, P. G., Spugnini, E. P., Mita, L., Mellone, P., D’Avino, A., Bianco, M., … Baldi, A. (2010). Pre-natal exposure of mice to bisphenol A elicits an endometriosis-like phenotype in female offspring. General and Comparative Endocrinology, 168(3), 318–325. https://doi.org/10.1016/j.ygcen.2010.03.030\nFilby, C. E., Rombauts, L., Montgomery, G. W., Giudice, L. C., & Gargett, C. E. (2020). Cellular origins of endometriosis: Towards novel diagnostics and therapeutics. Seminars in Reproductive Medicine, 38(02/03), 201–215. https://doi.org/10.1055/s-0040-1713429\nBulun, S. E., Yilmaz, B. D., Sison, C., Miyazaki, K., Bernardi, L., Liu, S., et al. (2019). Endometriosis. Endocrine Reviews, 40(4), 1048–1079. https://doi.org/10.1210/er.2018-00242\nSimoni, M., & Taylor, H. S. (2018). Therapeutic strategies involving uterine stem cells in reproductive medicine. Current Opinion in Obstetrics & Gynecology, 30(3), 209–216. https://doi.org/10.1097/gco.000000000000045\nBulun, S. E. (2013). Uterine fibroids. New England Journal of Medicine, 369(14), 1344–1355. https://doi.org/10.1056/nejmra1209993\nKim, J. J., Kurita, T., & Bulun, S. E. (2013). Progesterone action in endometrial cancer, endometriosis, uterine fibroids, and breast cancer. Endocrine Reviews, 34(1), 130–162. https://doi.org/10.1210/er.2012-1043\nCousins, F. L., Pandoy, R., Jin, S., & Gargett, C. E. (2021). The elusive endometrial epithelial stem/progenitor cells.Frontiers in Cell and Developmental Biology, 9. https://doi.org/10.3389/fcell.2021.640319\nSantamaria, X., Mas, A., Cervelló, I., Taylor, H., & Simon, C. (2018). Uterine stem cells: From basic research to advanced cell therapies. Human Reproduction Update, 24(6), 673–693. https://doi.org/10.1093/humupd/dmy028\nCousins, F. L., O, D. F., & Gargett, C. E. (2018). Endometrial stem/progenitor cells and their role in the pathogenesis of endometriosis. Best Practice & Research Clinical Obstetrics & Gynaecology, 50, 27–38. https://doi.org/10.1016/j.bpobgyn.2018.01.011\nChan, R. W. S., Ng, E. H. Y., & Yeung, W. S. B. (2011). Identification of cells with colony-forming activity, self-renewal capacity, and multipotency in ovarian endometriosis. The American Journal of Pathology, 178(6), 2832–2844. https://doi.org/10.1016/j.ajpath.2011.02.025\nLi, J., Dai, Y., Zhu, H., Jiang, Y., & Zhang, S. (2016). Endometriotic mesenchymal stem cells significantly promote fibrogenesis in ovarian endometrioma through the Wnt/β-catenin pathway by paracrine production of TGF-β1 and Wnt1. Human Reproduction, 31(6), 1224–1235. https://doi.org/10.1093/humrep/dew058\nSzukiewicz, D., Stangret, A., Ruiz-Ruiz, C., Olivares, E. G., Soriţău, O., Suşman, S., & Szewczyk, G. (2021). Estrogen- and progesterone (P4)-mediated epigenetic modifications of endometrial stromal cells (EnSCs) and/or mesenchymal stem/stromal cells (MSCs) in the etiopathogenesis of endometriosis. Stem Cell Reviews and Reports, 17(4), 1174–1193. https://doi.org/10.1007/s12015-020-10115-5\nMas, A., Cervelló, I., Gil-Sanchis, C., Faus, A., Ferro, J., Pellicer, A., & Simón, C. (2012). Identification and characterization of the human leiomyoma side population as putative tumor-initiating cells. Fertility and Sterility, 98(3). https://doi.org/10.1016/j.fertnstert.2012.04.044\nOno, M., Qiang, W., Serna, V. A., Yin, P., Coon, J. S., Navarro, A., et al. (2012). Role of stem cells in human uterine leiomyoma growth. PLoS ONE, 7(5). https://doi.org/10.1371/journal.pone.0036935\nBarragan, F., Irwin, J. C., Balayan, S., Erikson, D. W., Chen, J. C., Houshdaran, S., et al. C. (2016). Human endometrial fibroblasts derived from mesenchymal progenitors inherit progesterone resistance and acquire an inflammatory phenotype in the endometrial niche in endometriosis. Biology of Reproduction, 94(5). https://doi.org/10.1095/biolreprod.115.136010\nCaplan, A. I. (2017). Mesenchymal stem cells: Time to change the name! STEM CELLS Translational Medicine, 6(6), 1445–1451. https://doi.org/10.1002/sctm.17-0051\nBhartiya, D. (2016). An update on endometrial stem cells and progenitors. Human Reproduction Update, 22(4), 529–530. https://doi.org/10.1093/humupd/dmw010\nSyed, S. M., Kumar, M., Ghosh, A., Tomasetig, F., Ali, A., Whan, R. M., … Tanwar, P. S. (2020). Endometrial Axin2 cells drive epithelial homeostasis, regeneration, and cancer following oncogenic transformation. Cell Stem Cell, 26(1). https://doi.org/10.1016/j.stem.2019.11.012\nSyed, S. M., & Tanwar, P. S. (2020). Axin2 endometrial stem cells: The source of endometrial regeneration and cancer. Molecular & Cellular Oncology, 7(3), 1729681. https://doi.org/10.1080/23723556.2020.1729681\nGhosh, A., Syed, S. M., Kumar, M., Carpenter, T. J., Teixeira, J. M., Houairia, N., et al. (2020). In vivo cell fate tracing provides no evidence for mesenchymal to epithelial transition in adult fallopian tube and uterus. Cell Reports, 31(6), 107631. https://doi.org/10.1016/j.celrep.2020.107631\nSingh, P., & Bhartiya, D. (2020). Pluripotent stem (VSELs) and progenitor (EnSCs) cells exist in adult mouse uterus and show cyclic changes across estrus cycle. Reproductive Sciences, 28(1), 278–290. https://doi.org/10.1007/s43032-020-00250-2\nJames, K., Bhartiya, D., Ganguly, R., Kaushik, A., Gala, K., Singh, P., Metkari, S. M. (2018). Gonadotropin and steroid hormones regulate pluripotent very small embryonic-like stem cells in adult mouse uterine endometrium. Journal of Ovarian Research, 11(1). https://doi.org/10.1186/s13048-018-0454-4\nBhartiya, D., & James, K. (2017). Very small embryonic-like stem cells (VSELs) in adult mouse uterine perimetrium and myometrium. Journal of Ovarian Research, 10(1). https://doi.org/10.1186/s13048-017-0324-5\nGunjal, P., Bhartiya, D., Metkari, S., Manjramkar, D., & Patel, H. (2015). Very small embryonic-like stem cells are the elusive mouse endometrial stem cells- a pilot study. Journal of Ovarian Research, 8(1). https://doi.org/10.1186/s13048-015-0138-2\nRatajczak, M. Z., Ratajczak, J., & Kucia, M. (2019). Very small embryonic-like stem cells (VSELs) An update and future directions. Circulation Research, 124(2), 208–210. https://doi.org/10.1161/circresaha.118.314287\nBhartiya, D., Shaikh, A., Anand, S., Patel, H., Kapoor, S., Sriraman, K., et al. (2016). Endogenous, very small embryonic-like stem cells: Critical review, therapeutic potential and a look ahead. Human Reproduction Update, 23(1), 41–76. https://doi.org/10.1093/humupd/dmw030\nShaikh, A., Nagvenkar, P., Pethe, P., Hinduja, I., & Bhartiya, D. (2015). Molecular and phenotypic characterization of CD133 and SSEA4 enriched very small embryonic-like stem cells in human cord blood. Leukemia, 29(9), 1909–1917. https://doi.org/10.1038/leu.2015.100\nBhartiya, D., Patel, H., Ganguly, R., Shaikh, A., Shukla, Y., Sharma, D., & Singh, P. (2018). Novel insights into adult and cancer stem cell biology. Stem Cells and Development, 27(22), 1527–1539. https://doi.org/10.1089/scd.2018.0118\nKaushik, A., Anand, S., & Bhartiya, D. (2020). Altered biology of testicular VSELs and SSCs by neonatal endocrine disruption results in defective spermatogenesis, reduced fertility and tumor initiation in adult mice. Stem Cell Reviews and Reports, 16(5), 893–908. https://doi.org/10.1007/s12015-020-09996-3\nChapman, J. C., Min, S. H., Freeh, S. M., & Michael, S. D. (2009). The estrogen-injected female mouse: new insight into the etiology of PCOS. Reproductive Biology and Endocrinology, 7(1). https://doi.org/10.1186/1477-7827-7-47\nUimari, O., Järvelä, I., & Ryynänen, M. (2011). Do symptomatic endometriosis and uterine fibroids appear together? Journal of Human Reproductive Sciences, 4(1), 34. https://doi.org/10.4103/0974-1208.82358\nGebril, M., Hirota, Y., Aikawa, S., Fukui, Y., Kaku, T., Matsuo, M., et al. (2020). Uterine epithelial progesterone receptor governs uterine receptivity through epithelial cell differentiation. Endocrinology, 161(12). https://doi.org/10.1210/endocr/bqaa195\nFukui, Y., Hirota, Y., Matsuo, M., Gebril, M., Akaeda, S., Hiraoka, T., & Osuga, Y. (2019). Uterine receptivity, embryo attachment, and embryo invasion: Multistep processes in embryo implantation. Reproductive Medicine and Biology, 18(3), 234–240. https://doi.org/10.1002/rmb2.12280\nHirota, Y. (2019). Progesterone governs endometrial proliferation-differentiation switching and blastocyst implantation. Endocrine Journal, 66(3), 199–206. https://doi.org/10.1507/endocrj.ej18-0431\nJohnatty, S. E., Stewart, C. J. R., Smith, D., Nguyen, A., Dwyer, J. O., O’Mara, T. A., et al. (2020). Co-existence of leiomyomas, adenomyosis and endometriosis in women with endometrial cancer. Scientific Reports, 10(1). https://doi.org/10.1038/s41598-020-59916-1\nWang, Y., Nicholes, K., & Shih, I.-M. (2020). The origin and pathogenesis of endometriosis. Annual Review of Pathology: Mechanisms of Disease, 15(1), 71–95. https://doi.org/10.1146/annurev-pathmechdis-012419-032654\nChantalat, E., Valera, M.-C., Vaysse, C., Noirrit, E., Rusidze, M., Weyl, A., et al. (2020). Estrogen receptors and endometriosis. International Journal of Molecular Sciences, 21(8), 2815. https://doi.org/10.3390/ijms21082815\nGenc, M., Genc, B., & Cengiz, H. (2014). Adenomyosis and accompanying gynecological pathologies. Archives of Gynecology and Obstetrics, 291(4), 877–881. https://doi.org/10.1007/s00404-014-3498-8\nKok, V. C., Tsai, H.-J., Su, C.-F., & Lee, C.-K. (2015). The risks for ovarian, endometrial, breast, colorectal, and other cancers in women with newly diagnosed endometriosis or adenomyosis: A population-based study. International Journal of Gynecologic Cancer, 25(6), 968–976. https://doi.org/10.1097/igc.0000000000000454\nVerit, F. F., & Yucel, O. (2013). Endometriosis, leiomyoma and adenomyosis: The risk of gynecologic malignancy. Asian Pacific Journal of Cancer Prevention, 14(10), 5589–5597. https://doi.org/10.7314/apjcp.2013.14.10.5589\nMaruyama, S., Imanaka, S., Nagayasu, M., Kimura, M., & Kobayashi, H. (2020). Relationship between adenomyosis and endometriosis; Different phenotypes of a single disease? European Journal of Obstetrics & Gynecology and Reproductive Biology, 253, 191–197. https://doi.org/10.1016/j.ejogrb.2020.08.019\nYilmaz, B. D., & Bulun, S. E. (2019). Endometriosis and nuclear receptors. Human Reproduction Update, 25(4), 473–485. https://doi.org/10.1093/humupd/dmz005\nYovich, J. L., Rowlands, P. K., Lingham, S., Sillender, M., & Srinivasan, S. (2020). Pathogenesis of endometriosis: Look no further than John Sampson. Reproductive Bio Medicine Online, 40(1), 7–11. https://doi.org/10.1016/j.rbmo.2019.10.007\nGuo, Y., Schaik, T. V., Jhamat, N., Niazi, A., Chanrot, M., Charpigny, G., et al. (2019). Differential gene expression in bovine endometrial epithelial cells after challenge with LPS; specific implications for genes involved in embryo maternal interactions. Plos One, 14(9). https://doi.org/10.1371/journal.pone.0222081\nIkhena, D. E., & Bulun, S. E. (2017). Literature review on the role of uterine fibroids in endometrial function. Reproductive Sciences, 25(5), 635–643. https://doi.org/10.1177/1933719117725827\nMas, A., Stone, L., O’connor, P. M., Yang, Q., Kleven, D., Simon, C., et al. (2016). Developmental exposure to endocrine disruptors expands murine myometrial stem cell compartment as a prerequisite to leiomyoma tumorigenesis. Stem Cells, 35(3), 666–678. https://doi.org/10.1002/stem.2519\nŁupicka, M., Socha, B., Szczepańska, A., & Korzekwa, A. (2015). Expression of pluripotency markers in the bovine uterus with adenomyosis. Reproductive Biology and Endocrinology, 13(1). https://doi.org/10.1186/s12958-015-0106-0\nCardoso, J. V., Perini, J. A., Machado, D. E., Pinto, R., & Medeiros, R. (2020). Systematic review of genome-wide association studies on susceptibility to endometriosis. European Journal of Obstetrics & Gynecology and Reproductive Biology, 255, 74–82. https://doi.org/10.1016/j.ejogrb.2020.10.017\nMoore, L., Leongamornlert, D., Coorens, T. H. H., Sanders, M. A., Ellis, P., Dentro, S. C., et al. (2020). The mutational landscape of normal human endometrial epithelium. Nature, 580(7805), 640–646. https://doi.org/10.1038/s41586-020-2214-z\nBae-Jump, V. (2020). Unraveling the mystery of clear cell endometrial cancer. Gynecologic Oncology, 158(1), 1–2. https://doi.org/10.1016/j.ygyno.2020.06.159\nKyo, S., Sato, S., & Nakayama, K. (2020). Cancer-associated mutations in normal human endometrium: Surprise or expected? Cancer Science, 111(10), 3458–3467. https://doi.org/10.1111/cas.14571\nJin, S. (2019). Bipotent stem cells support the cyclical regeneration of endometrial epithelium of the murine uterus. Proceedings of the National Academy of Sciences, 116(14), 6848–6857. https://doi.org/10.1073/pnas.1814597116\nOwusu-Akyaw, A., Krishnamoorthy, K., Goldsmith, L. T., & Morelli, S. S. (2018). The role of mesenchymal–epithelial transition in endometrial function. Human Reproduction Update, 25(1), 114–133. https://doi.org/10.1093/humupd/dmy035\nAcknowledgements\nHelp from Praveen, Swati and Shobha from central facilities at NIRRH is acknowledged.\nFunding\nFunding support for the study was provided to DB by Indian Council of Medical Research, Government of India, New Delhi. PS acknowledges DST-INSPIRE fellowship (IF170144).\nAuthor information\nAuthors and Affiliations\nContributions\nDB designed the study, data interpretation, manuscript preparation. PS performed all experiments, data interpretation and manuscript preparation. SMM helped with surgeries. All authors read and approved final draft of manuscript.\nCorresponding author\nEthics declarations\nEthics approval\nThe study was approved by Institute Animal Ethics Committee (IAEC).\nConsent to participate\nNot applicable.\nConsent for publication\nInstitute accession number is RA/1045/03–2021.\nConflicts of interest/Competing interests\nAuthors declare no conflict of interest whatsoever.\nAdditional information\nPublisher's Note\nSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.\nThis article belongs to the Topical Collection: Special Issue on Tissue-resident Stem/Progenitor Cells Endowed with Broader Germ Layer Specification Potential in Normal and Cancerous Tissues\nGuest Editor: Deepa Bhartiya\nSupplementary Information\nBelow is the link to the electronic supplementary material.\nRights and permissions\nAbout this article\nCite this article\nSingh, P., Metkari, S.M. & Bhartiya, D. Mice Uterine Stem Cells are Affected by Neonatal Endocrine Disruption & Initiate Uteropathies in Adult Life Independent of Circulatory Ovarian Hormones. Stem Cell Rev and Rep 18, 1686–1701 (2022). https://doi.org/10.1007/s12015-021-10279-8\nAccepted:\nPublished:\nVersion of record:\nIssue date:\nDOI: https://doi.org/10.1007/s12015-021-10279-8","source_license":"CC0","license_restricted":false}