Regulated Expression of Interleukin-17 System at the Endometrial-Myometrial Interface: Implications for Adenomyosis

Regulated Expression of Interleukin-17 System at the Endometrial-Myometrial Interface: Implications for Adenomyosis | 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 Regulated Expression of Interleukin-17 System at the Endometrial-Myometrial Interface: Implications for Adenomyosis Le-Tien Hsu, Pei-Chen Lu, Yi-Wen Wang, Hsien-Ming Wu, I-Ju Chen, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4317248/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 Adenomyosis involves the infiltration of endometrial glands and stroma deep into the uterine tissue, causing disruption to the interface between the endometrium and myometrium. The role of interleukin-17 (IL-17), a cytokine associated with immune responses, has been extensively studied in endometriosis, but its involvement in adenomyosis remains unclear. This study aimed to investigate the expression of IL-17 in ectopic and eutopic endometrium of individuals with adenomyosis, comparing its levels between these two types of endometrium. Methods Paired tissues of eutopic endometrium and adenomyoma were collected from 16 premenopausal women undergoing hysterectomy due to clinical symptoms related to adenomyosis. IL-17 system was demonstrated in paired tissue samples by the immunochemistry study. Gene expression levels of IL-17A and IL-17 receptor (IL-17R) were assessed through quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). Comparative gene transcript amounts were calculated using the delta-delta Ct method. Results By immunohistochemical staining, CD4, IL-17R, and IL-17A proteins were detected in both endometrium and adenomyosis, with higher expression in epithelial cells. DAB staining revealed greater IL-17A expression in adenomyosis compared to endometrium. Quantitative RT-PCR showed significantly higher fold change levels of IL-17A and IL-17R in adenomyosis (IL-17A: p = 0.047, IL-17R: p = 0.027) versus endometrium. Conclusions We found significantly higher IL-17 levels in adenomyosis compared to endometrium, suggesting immune system involvement in adenomyosis pathogenesis. Interleukin-17 Adenomyosis Endometrial-myometrial interface Cytokine Figures Figure 1 Figure 2 Figure 3 1 Introduction Endometriosis is a common, benign, estrogen-dependent, chronic gynecological disorder associated with pelvic pain and infertility. It is characterized by the presence of uterine endometrial tissue outside of the normal location—mainly on the pelvic peritoneum, but also on the ovaries and in the recto-vaginal septum, and more rarely in the pericardium, pleura, and even the brain. The prevalence of pelvic endometriosis approaches 6–10% in the general female population; in women with pain, infertility, or both, the frequency is 35–50% ( 1 – 3 ). Although multiple theories exist regarding the etiology of the disease, it is most commonly held that retrograde menstruation is believed to allow endometrial tissue to seed the peritoneal cavity ( 4 ). These endometrial implants appear to activate host immune responses, leading to humoral- and cell-mediated inflammation. Activated macrophages and lymphocytes are found in greater numbers in the peritoneal cavity of women with endometriosis ( 5 ). Adenomyosis is a pathological condition of the uterus closely associated with endometriosis ( 6 ). Similar to endometriosis, adenomyosis is reliant on estrogen and involves the ectopic presence of endometrial cells and stromal fibroblasts within the myometrium, either as extensions of the normally located endometrium or as islands surrounded by hypertrophic smooth muscle cells ( 7 – 9 ). Adenomyosis shares similar symptoms with endometriosis, including heavy menstrual bleeding, dysmenorrhea, chronic pelvic pain, and infertility ( 9 , 10 ). Several theories have been proposed to explain the pathogenesis of adenomyosis ( 11 ). The prevailing understanding implicates the profound invasion of endometrial tissue into the inner myometrium, causing disruption to the endometrial-myometrial interface (EMI) ( 12 , 13 ). Multiple lines of evidence suggest that inflammation and immune responses play a pivotal role in the pathogenesis of endometriosis ( 14 , 15 ). In the same way, growing evidence indicates alterations in human and cellular immunity in adenomyosis ( 16 , 17 ). Moreover, various immune-reacted serum proteins have been suggested to contribute to the development of adenomyosis ( 18 ). Hirata et al. first showed the presence of Th17 cells in the peritoneal fluid (PF) of endometriotic women by flow cytometry and the presence of IL-17A-positive cells in endometriotic tissues by immunohistochemistry ( 19 ). IL-17 is secreted by the CD4 + T helper 17 (Th17) cell and is recognized as the hallmark cytokine of the unique cell population. In 1999, IL-17 was first identified through the use of T-cell clones derived from the joints of patients suffering from rheumatoid arthritis ( 20 – 23 ). Currently, the IL-17 family comprises six structurally associated cytokines (IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F), all sharing genetic homology, along with five matching receptors (IL-17RA, IL-17RB, IL-17RC, IL-17RD, and IL-17RE) found on cell surfaces. IL-17A stands as the inaugural member of this family and holds the distinction of being the most extensively studied, previously thought to be predominantly generated by Th17 cells ( 24 – 26 ). IL-17F and IL-17A demonstrate elevated genetic homology and have the capability to create both homodimers and heterodimers for signaling purposes. Moreover, they engage with the same receptor complex, thereby predominantly sharing biological functions, with IL-17A exhibiting greater potency compared to IL-17F. The four other IL-17 isoforms exist exclusively as homodimers ( 22 , 27 – 29 ). So far, scant information exists concerning the role of the IL-17 system in the pathophysiological mechanisms of adenomyosis, and there is no report regarding the presence of IL-17 in women with adenomyosis. The current study aimed to investigate the expression of IL-17 in both ectopic and eutopic endometrium, and to compare its expression between these two types of endometrium in adenomyotic individuals. 2 Materials and Methods 2.1 Ethical disclosure and tissue sampling methodology Approval for our research was granted by the Institutional Review Board of Chang Gung Memorial Hospital (IRB#201700139B0). Prior to enrollment, all participants submitted written informed consent. Paired tissues of their matched endometrium and adenomyoma were obtained from 16 premenopausal women undergoing hysterectomy. Hysterectomy was indicated due to clinical symptoms related to adenomyosis, including hypermenorrhea and dysmenorrhea. The exclusion criteria were: ( 1 ) subjects with past history of pelvic inflammatory disease, ( 2 ) subjects with history of genital tract infection, and ( 3 ) subjects who had undergone any hormone therapies within 6 months prior to surgery. Biopsies were conducted by a skilled gynecologist at Linkou Chang Gung Memorial Hospital. Histological documentation was conducted on the excised tissue samples to confirm the presence of adenomyosis foci, characterized by active endometrial glands and stroma surrounded by hypertrophic myometrium. Section of EMI was collected for immunohistochemistry. 2.2 Immunohistochemistry study of IL-17 system in paired-tissue of EMI. 12 serial sections (each 6 µm thick) of frozen eutopic endometrium with attached myometrium were excised for immunohistochemistry staining. The first and last slides underwent staining using hematoxylin and eosin (H&E) for pathologic confirmation, while the remaining slides were stained for the IL-17 system and analyzed using a Nikon microphot-FXA microscope (Nikon Instruments, Garden City, New York, NY, USA). Tissues were fixed with methanol: acetone (1:1) (Mallinckrodt, USA) and resuspended in phosphate buffered saline (PBS; Sigma Chemical Co, St. Louis, MO, USA) until processed for immunohistochemistry. After washing with PBS, endogenous peroxidases were blocked with 1% H 2 O 2 in 96% methanol, and non-specific binding was blocked with 5% non-fat milk in PBS at room temperature. The EnVision kit facilitated immunohistochemical identification of the IL-17 system with horseradish peroxidase (HRP) (Dako, Glostrup, Denmark) system as the linker and liquid 3, 3-Diaminobenzidine (DAB) as the chromagen. Positive antibody reactions were indicated by the presence of brown granules. All sections underwent counterstaining with Mayer hematoxylin, dehydration, and mounting. 2.3 Quantitative study for IL-17 mRNA expression using real-time reverse transcription polymerase chain reaction (RT-PCR) DNA amplification and data collection were carried out using the LighCycler System. All reactions were performed with the QuantiTect SYBR Green PCR Kit (Qiagen). The reaction components comprised 1X QuantiTect SYBR Green PCR Master Mix, with a final concentration of 0.5µM for each primer. The reactions were cycled with an initial activation step of 15min at 95°C followed by 40 cycles of 15 seconds at 94°C, 30 seconds at 60°C and 20 seconds at 72°C. The raw data were analyzed with the Light Cycler software version 4.0. The quantitative amount of IL-17A and IL-17R was determined. 2.4 IL-17 mRNA expression determined by quantitative PCR (Q PCR) Gene expression levels of IL-17A and IL-17R in paired uterine samples were assessed through quantitative real-time RT-PCR, utilizing the TaqMan methodology with the Applied Biosystems 7900 HT Real-time PCR System. PCR reactions were conducted according to the manufacturer's guidelines, reaching a final volume of 25 µL, which comprised 12.5 µL of 2X TaqMan Universal PCR Master Mix (Life Technologies®, Foster City, CA, USA), 1.25 µL TaqMan assay (20×), 1µL of sample cDNA, and 10.25 µL of RNAse-free water. The levels of IL-17A and IL-17R mRNA in adenomyosis were contrasted with those in the endometrium from the same patient. Comparative gene transcript amounts were calculated using the delta-delta Ct method ( 30 ). 2.5 Data interpretation The data are presented as the mean ± standard deviation. Data analysis was conducted using SPSS 22.0 (SPSS Inc., Chicago, IL, USA), with statistical significance set at a p-value of < 0.05. 3 Results The tissues from both eutopic endometrium and adenomyosis, fixed in formalin, underwent preparation for immunohistochemical staining to reveal the existence of IL-17 system and CD4 proteins at the EMI level. CD4, IL-17R, and IL-17A immunoreactive proteins were detected within both the eutopic endometrium and adenomyosis. CD4, IL-17R, and IL-17A were noted to be expressed in both the glands and stroma of the endometrium and adenomyosis. In both eutopic and ectopic endometrium samples, epithelial cells exhibited greater staining intensity compared to stromal cells. We used DAB as the chromagen for immunohistochemical detection of IL-17 system. Figure 2 shows an example of paired samples from one patient and provides qualitative data on IL-17A expressions within the tissue sample. After the tissues were stained by immunohistochemistry, 5 areas are randomly circled at the endometrium position, 10 areas are randomly circled at the position of adenomyosis. Scattergram–histogram combinations are well suited to visualize the relation between two tissues. The result shows that the mean intensity of DAB stain is higher in adenomyosis (0.54%) than in endometrium (0.30%), demonstrating there are more IL-17A expressions in adenomyosis than in endometrium. In Fig. 3 , we demonstrated the comparison of the fold change levels of IL-17A and IL-17R between the endometrium and corresponding adenomyosis group. Quantitative real-time RT-PCR in paired samples was performed. The fold change of IL-17A is significantly higher in the adenomyosis group compared to the endometrium group (p = 0.047, Fig. 3 (A)). Similarly, the fold change of IL-17R is significantly higher in the adenomyosis group in comparison to the endometrium group (p = 0.027, Fig. 3 (B)). 4 Discussion The endometrium showcases a sophisticated interplay among a network of cells, manifesting as a meticulously orchestrated phase encompassing proliferation, differentiation, and menstrual shedding ( 31 , 32 ). The formation of cytokines, the expression of cytokine receptors, and the modulation of endometrial functions by these factors underscore the pivotal role of cytokines in operating at endocrine, paracrine and autocrine levels within the endometrium. Increasingly, there is compelling evidence pointing to the participation of immune cells and inflammatory mediators in the development of endometriosis ( 33 ). In our study, IL-17A and IL-17R were identified in endometrium and adenomyosis, and the fold change levels of IL-17A and IL-17R were both significantly higher in adenomyosis than in endometrium, implicating that IL-17 system functions as a local immunomodulatory factor in adenomyosis. In endometriosis patients, various active substances, including cytokines, growth factors, hormones, and oxidative stress parameters, have been identified at different stages of the disease ( 34 ). An analysis revealed that the primary contributors to inflammation in endometriotic women were cytokines produced by macrophages ( 35 ). IL-17A has been noted to function as a chemotactic agent for macrophages via its receptor, IL-17RA, and can also trigger M2 macrophage polarization in endometriotic patients ( 36 ). Additionally, the researchers suggest that IL-17A plays a role in recruiting macrophages ( 37 ), and M2 macrophages have indeed been demonstrated to facilitate extracellular matrix remodeling and neovascularization, closely linked to the development of endometriosis ( 38 – 40 ). The heightened production of tumor necrosis factor, triggered by activated macrophages, along with the effect of IL-17 on endometrial cells, hastens the formation of endometriosis, often leading to unexplained pelvic pain and infertility. Moreover, recent research indicated that increased IL-17 levels in PF from patients with endometriosis-associated infertility might stimulate peritoneal macrophages to produce nitric oxide synthase 2 (NOS2) and nitric oxide (NO), which can have detrimental effects on both men’s and women’s reproductive systems ( 41 ). Therefore, elevated levels of IL-17 are associated with infertility in endometriosis. As the research delves deeper into cytokines and endometriosis, there is increased reporting and wider confirmation of elevated IL-17 levels in endometriosis, particularly during the early stages of the disease ( 37 , 42 ). Zhang and colleagues were the first to demonstrate elevated IL-17 levels in the PF of endometriotic individuals. Furthermore, they found a relationship between the IL-17 concentration in PF and the advancement of the illness. Specifically, the IL-17 concentrations in PF were notably elevated in individuals with minimal/mild endometriosis compared to those without endometriosis and individuals with moderate/severe endometriosis. Additionally, their research indicated that the IL-17 concentration in PF was linked to infertility associated with endometriosis ( 43 ). Salmeri et al. proposed that this observation could be explained by the notion that the inflammation appears to diminish in more advanced stages of endometriosis ( 44 ). Furthermore, other studies have shown heightened levels of IL-17A in both the PF and plasma of endometriotic patients compared to controls, along with the production of IL-17A by lesions of endometriosis ( 37 , 42 , 45 ). One research reported that they observed IL-17A levels increased comparably in both follicular fluid and serum in cases where infertility and endometriosis coexisted ( 46 ). Another study showed that cells positive for IL-17A were localized around the vasculature and in the stroma in both endometrium and endometriotic tissues from endometriotic patients ( 47 ). Additionally, one research found a higher percentage of Th17 cells in PF compared to the blood in endometriotic women. The result of this research also revealed in the PF of severe endometriosis, the Th17 cell percentage was higher compared to early-stage (I/II) endometriosis ( 48 ). Considering the facts mentioned above, it's apparent that in women with endometriosis, the IL-17 level commonly rises, often observed in both blood and PF samples. Histologically, adenomyosis is characterized by ectopic endometrial tissue within the myometrium. Despite extensive research, its underlying causes remain unclear, with many aspects of its development still unknown. One identified mechanism contributing to adenomyosis is the heightened ability of certain endometrial cells to invade the myometrial tissue. Additionally, both local and systemic immune responses have been linked to the onset and maintenance of the condition. Immune system involvement coincides with hormonal irregularities and activation of the epithelial to mesenchymal transition pathway, facilitating the migration of endometrial cells ( 5 ). Exploring the IL-17 system may offer novel perspectives on the pathogenesis of adenomyosis. In a prior study, they concluded that regulated by cytokines and estrogen, IL-17 secretion increases, either stemming from Th17 differentiation or stromal cells of endometrium. IL-17 then enhances the development, proliferation, and ectopic invasion, promoting immune evasion and endometriotic development by inducing and recruiting the M2 macrophage differentiation ( 36 ). In conjunction with our results, the IL-17 system might participate in the formation of ectopic endometriotic lesions. Nevertheless, this study also has several limitations. Methodologically, this study is disadvantaged by an inability to completely control for selection and confounding biases. Despite being the first published study reporting on expression of IL-17 system in adenomyosis, the number of patients with adenomyosis was small. Therefore, this population was not generally representative. Furthermore, the study design limited our ability to infer causality between IL-17 system and adenomyosis. 5 Conclusion In conclusion, to our knowledge, this is the first study to compare the expression of IL-17 system between eutopic endometrium and adenomyosis in women with adenomyosis. We found that expression of IL-17 system is significantly higher in adenomyosis than in corresponding endometrium. Our results may provide clues to the pathogenesis of adenomyosis involving the immune system. However, further investigation is needed to assess precisely how the IL-17 system participates in the development of adenomyosis. Declarations Ethics approval and consent to participate The study was approved by the institutional review board of the Human Investigation and Ethical Committee of the Chang Gung Medical Foundation (CGMHIRB# 201700139B0). Informed consent was obtained from all subjects involved in the study. Consent for publication Not applicable Availability of data and materials The datasets used and analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This research work was supported in part under Chang Gung Memorial Hospital Research Grant (CMRPG3E0771, CMRPG3M0791/792). Authors' contributions H.-Y.H. participated in conceptulization and methodology. P.-C.L. and Y.-W.W. helped in the lab work and analysis of data. L.-T.H., H.-M.W., I.-J.C., H.-Y.H. helped in writing the manuscript. All authors reviewed the manuscript. Acknowledgements Not applicable References Houston DE. Evidence for the risk of pelvic endometriosis by age, race and socioeconomic status. Epidemiol Rev. 1984;6:167–91. Kistner RW. Endometriosis and infertility. Clin Obstet Gynecol. 1979;22(1):101–19. Sensky TE, Liu DT. Endometriosis: associations with menorrhagia, infertility and oral contraceptives. Int J Gynaecol Obstet. 1980;17(6):573–6. Chapron C, Marcellin L, Borghese B, Santulli P. Rethinking mechanisms, diagnosis and management of endometriosis. Nat reviews Endocrinol. 2019;15(11):666–82. Bourdon M, Santulli P, Jeljeli M, Vannuccini S, Marcellin L, Doridot L, et al. Immunological changes associated with adenomyosis: a systematic review. Hum Reprod Update. 2021;27(1):108–29. Bulun SE, Yildiz S, Adli M, Wei JJ. Adenomyosis pathogenesis: insights from next-generation sequencing. Hum Reprod Update. 2021;27(6):1086–97. García-Solares J, Donnez J, Donnez O, Dolmans MM. Pathogenesis of uterine adenomyosis: invagination or metaplasia? Fertil Steril. 2018;109(3):371–9. Bird CC, McElin TW, Manalo-Estrella P. The elusive adenomyosis of the uterus–revisited. Am J Obstet Gynecol. 1972;112(5):583–93. Zhai J, Vannuccini S, Petraglia F, Giudice LC, Adenomyosis. Mechanisms and Pathogenesis. Semin Reprod Med. 2020;38(2–03):129–43. Struble J, Reid S, Bedaiwy MA. Adenomyosis: A Clinical Review of a Challenging Gynecologic Condition. J Minim Invasive Gynecol. 2016;23(2):164–85. Benagiano G, Brosens I. The endometrium in adenomyosis. Women's health (London England). 2012;8(3):301–12. Marcus CC. Relationship of adenomyosis uteri to endometrial hyperplasia and endometrial carcinoma. Am J Obstet Gynecol. 1961;82:408–16. Benagiano G, Habiba M, Brosens I. The pathophysiology of uterine adenomyosis: an update. Fertil Steril. 2012;98(3):572–9. Harada T, Iwabe T, Terakawa N. Role of cytokines in endometriosis. Fertil Steril. 2001;76(1):1–10. Lebovic DI, Mueller MD, Taylor RN. Immunobiology of endometriosis. Fertil Steril. 2001;75(1):1–10. Bourdon M, Santulli P, Marcellin L, Maignien C, Maitrot-Mantelet L, Bordonne C, et al. Adenomyosis: An update regarding its diagnosis and clinical features. J Gynecol Obstet Hum Reprod. 2021;50(10):102228. Wang F, Li H, Yang Z, Du X, Cui M, Wen Z. Expression of interleukin-10 in patients with adenomyosis. Fertil Steril. 2009;91(5):1681–5. Xiaoyu L, Weiyuan Z, Ping J, Anxia W, Liane Z. Comparative serum proteomic analysis of adenomyosis using the isobaric tags for relative and absolute quantitation technique. Fertil Steril. 2013;100(2):505–10. Hirata T, Osuga Y, Hamasaki K, Yoshino O, Ito M, Hasegawa A, et al. Interleukin (IL)-17A stimulates IL-8 secretion, cyclooxygensase-2 expression, and cell proliferation of endometriotic stromal cells. Endocrinology. 2008;149(3):1260–7. Aarvak T, Chabaud M, Miossec P, Natvig JB. IL-17 is produced by some proinflammatory Th1/Th0 cells but not by Th2 cells. Journal of immunology (Baltimore, Md: 1950). 1999;162(3):1246-51. Albanesi C, Cavani A, Girolomoni G. IL-17 is produced by nickel-specific T lymphocytes and regulates ICAM-1 expression and chemokine production in human keratinocytes: synergistic or antagonist effects with IFN-gamma and TNF-alpha. Journal of immunology (Baltimore, Md: 1950). 1999;162(1):494–502. Beringer A, Noack M, Miossec P. IL-17 in Chronic Inflammation: From Discovery to Targeting. Trends Mol Med. 2016;22(3):230–41. Harrington LE, Hatton RD, Mangan PR, Turner H, Murphy TL, Murphy KM, et al. Interleukin 17-producing CD4 + effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nat Immunol. 2005;6(11):1123–32. Zou W, Restifo NP. T(H)17 cells in tumour immunity and immunotherapy. Nat Rev Immunol. 2010;10(4):248–56. Fossiez F, Djossou O, Chomarat P, Flores-Romo L, Ait-Yahia S, Maat C, et al. T cell interleukin-17 induces stromal cells to produce proinflammatory and hematopoietic cytokines. J Exp Med. 1996;183(6):2593–603. Rouvier E, Luciani MF, Mattéi MG, Denizot F, Golstein P. CTLA-8, cloned from an activated T cell, bearing AU-rich messenger RNA instability sequences, and homologous to a herpesvirus saimiri gene. Journal of immunology (Baltimore, Md: 1950). 1993;150(12):5445-56. Patel DD, Kuchroo VK. Th17 Cell Pathway in Human Immunity: Lessons from Genetics and Therapeutic Interventions. Immunity. 2015;43(6):1040–51. Miossec P, Kolls JK. Targeting IL-17 and TH17 cells in chronic inflammation. Nat Rev Drug Discovery. 2012;11(10):763–76. Toy D, Kugler D, Wolfson M, Vanden Bos T, Gurgel J, Derry J et al. Cutting edge: interleukin 17 signals through a heteromeric receptor complex. Journal of immunology (Baltimore, Md: 1950). 2006;177(1):36 – 9. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods (San Diego, Calif). 2001;25(4):402-8. Simón C, Piquette GN, Frances A, Polan ML. Localization of interleukin-1 type I receptor and interleukin-1 beta in human endometrium throughout the menstrual cycle. J Clin Endocrinol Metab. 1993;77(2):549–55. Simón C, Frances A, Lee BY, Mercader A, Huynh T, Remohi J, et al. Immunohistochemical localization, identification and regulation of the interleukin-1 receptor antagonist in the human endometrium. Hum Reprod (Oxford England). 1995;10(9):2472–7. Gazvani R, Templeton A. Peritoneal environment, cytokines and angiogenesis in the pathophysiology of endometriosis. Reprod (Cambridge England). 2002;123(2):217–26. Bedaiwy MA, Falcone T. Peritoneal fluid environment in endometriosis. Clinicopathological implications. Minerva Ginecol. 2003;55(4):333–45. Beste MT, Pfäffle-Doyle N, Prentice EA, Morris SN, Lauffenburger DA, Isaacson KB, et al. Molecular network analysis of endometriosis reveals a role for c-Jun-regulated macrophage activation. Sci Transl Med. 2014;6(222):222ra16. Shi JL, Zheng ZM, Chen M, Shen HH, Li MQ, Shao J. IL-17: an important pathogenic factor in endometriosis. Int J Med Sci. 2022;19(4):769–78. Miller JE, Ahn SH, Marks RM, Monsanto SP, Fazleabas AT, Koti M, et al. IL-17A Modulates Peritoneal Macrophage Recruitment and M2 Polarization in Endometriosis. Front Immunol. 2020;11:108. Okizaki S, Ito Y, Hosono K, Oba K, Ohkubo H, Amano H et al. Suppressed recruitment of alternatively activated macrophages reduces TGF-β1 and impairs wound healing in streptozotocin-induced diabetic mice. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie. 2015;70:317 – 25. Weber C, Telerman SB, Reimer AS, Sequeira I, Liakath-Ali K, Arwert EN, et al. Macrophage Infiltration and Alternative Activation during Wound Healing Promote MEK1-Induced Skin Carcinogenesis. Cancer Res. 2016;76(4):805–17. Wynn TA, Vannella KM. Macrophages in Tissue Repair, Regeneration, and Fibrosis. Immunity. 2016;44(3):450–62. Miljkovic D, Trajkovic V. Inducible nitric oxide synthase activation by interleukin-17. Cytokine Growth Factor Rev. 2004;15(1):21–32. Sikora J, Smycz-Kubańska M, Mielczarek-Palacz A, Bednarek I, Kondera-Anasz Z. The involvement of multifunctional TGF-β and related cytokines in pathogenesis of endometriosis. Immunol Lett. 2018;201:31–7. Zhang X, Xu H, Lin J, Qian Y, Deng L. Peritoneal fluid concentrations of interleukin-17 correlate with the severity of endometriosis and infertility of this disorder. BJOG: Int J Obstet Gynecol. 2005;112(8):1153–5. Salmeri FM, Laganà AS, Sofo V, Triolo O, Sturlese E, Retto G et al. Behavior of tumor necrosis factor-α and tumor necrosis factor receptor 1/tumor necrosis factor receptor 2 system in mononuclear cells recovered from peritoneal fluid of women with endometriosis at different stages. Reproductive sciences (Thousand Oaks, Calif). 2015;22(2):165 – 72. Rafi U, Ahmad S, Bokhari SS, Iqbal MA, Zia A, Khan MA, et al. Association of Inflammatory Markers/Cytokines with Cardiovascular Risk Manifestation in Patients with Endometriosis. Mediat Inflamm. 2021;2021:3425560. Sabbaghi M, Aram R, Roustaei H, Fadavi Islam M, Daneshvar M, Castaño AR, et al. IL-17A concentration of seminal plasma and follicular fluid in infertile men and women with various clinical diagnoses. Immunol Investig. 2014;43(7):617–26. Ahn SH, Edwards AK, Singh SS, Young SL, Lessey BA, Tayade C. IL-17A Contributes to the Pathogenesis of Endometriosis by Triggering Proinflammatory Cytokines and Angiogenic Growth Factors. Journal of immunology (Baltimore, Md: 1950). 2015;195(6):2591 – 600. Gogacz M, Winkler I, Bojarska-Junak A, Tabarkiewicz J, Semczuk A, Rechberger T, et al. Increased percentage of Th17 cells in peritoneal fluid is associated with severity of endometriosis. J Reprod Immunol. 2016;117:39–44. Additional Declarations No competing interests reported. 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4317248","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":297618154,"identity":"9053fea2-c83a-4dd4-b34f-602055523bfc","order_by":0,"name":"Le-Tien Hsu","email":"","orcid":"","institution":"Department of Obstetrics and Gynecology, Linkou Medical Center, Chang Gung Memorial Hospital, Chang Gung University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Le-Tien","middleName":"","lastName":"Hsu","suffix":""},{"id":297618158,"identity":"e0c95d0c-1c0f-4563-bb4c-0e22e23c4f55","order_by":1,"name":"Pei-Chen Lu","email":"","orcid":"","institution":"Department of Obstetrics and Gynecology, Linkou Medical Center, Chang Gung Memorial Hospital, Chang Gung University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Pei-Chen","middleName":"","lastName":"Lu","suffix":""},{"id":297618163,"identity":"8481fcee-a1fc-4bc0-afdf-1676ab812d11","order_by":2,"name":"Yi-Wen Wang","email":"","orcid":"","institution":"Department of Obstetrics and Gynecology, Linkou Medical Center, Chang Gung Memorial Hospital, Chang Gung University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yi-Wen","middleName":"","lastName":"Wang","suffix":""},{"id":297618167,"identity":"67cd280c-b24b-45d4-b91c-976f2c6e5402","order_by":3,"name":"Hsien-Ming Wu","email":"","orcid":"","institution":"Department of Obstetrics and Gynecology, Linkou Medical Center, Chang Gung Memorial Hospital, Chang Gung University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Hsien-Ming","middleName":"","lastName":"Wu","suffix":""},{"id":297618170,"identity":"85ca585d-4ec4-4cf0-8c4d-4b3e08fe8f65","order_by":4,"name":"I-Ju Chen","email":"","orcid":"","institution":"Department of Family Medicine, Linkou Medical Center, Chang Gung Memorial Hospital","correspondingAuthor":false,"prefix":"","firstName":"I-Ju","middleName":"","lastName":"Chen","suffix":""},{"id":297618173,"identity":"f0e9706e-3b0a-470c-a4c2-c5b2a34e5a14","order_by":5,"name":"Hong-Yuan Huang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIiWNgGAWjYBACAxiDH4iZSdMi2QDXQkAnXIvBAWK1mLP3Hn7N22aXZ3z8+NXNBQw2+fIO/Mck8Gmx7DmXZs3bllxsdian7PYMhjTLjQeY2fBqMbiRY2bM28acuO0GT9ptHobDBoYNzGw3iNBSn7h5BglajB/zth1O3CDBfgysRZ6BkJYzZ8wY55w7njjjTA7bbR6DNAMDZmbzH3i1HO8x/vCmrDqxv/34s9s8FTYG8u2Njw3waQECNikeMM1jAI4mg8ME1AMB80eIO9gfgCn5BsJaRsEoGAWjYGQBAA9wSFBXWWWFAAAAAElFTkSuQmCC","orcid":"","institution":"Department of Obstetrics and Gynecology, Linkou Medical Center, Chang Gung Memorial Hospital, Chang Gung University College of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Hong-Yuan","middleName":"","lastName":"Huang","suffix":""}],"badges":[],"createdAt":"2024-04-24 09:54:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4317248/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4317248/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":55765426,"identity":"a0857c01-7daf-4df0-be59-c9481e3604e5","added_by":"auto","created_at":"2024-05-02 20:07:13","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":610842,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemistry of IL-17 system in patients with adenomyosis. A black stain (arrow) indicated a diffuse CD4 and IL-17 system positive reaction in luminal epithelium and stromal cells of endometrium \u003cstrong\u003e(E,G,I)\u003c/strong\u003e and adenomyosis \u003cstrong\u003e(F,H,J)\u003c/strong\u003e. \u003cstrong\u003e(A,B)\u003c/strong\u003eshowed both eutopic and ectopic samples stained by H\u0026amp;E, and \u003cstrong\u003e(C,D)\u003c/strong\u003eshowed both samples without staining. \u003cstrong\u003e(A-J)\u003c/strong\u003e scale bar = 100μm. H\u0026amp;E: hematoxylin and eosin.\u003c/p\u003e","description":"","filename":"Figure.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4317248/v1/412a81a8fd1d73c0084f0a2f.jpg"},{"id":55765427,"identity":"8c33fbce-22d9-4625-9f4a-f61b9087c304","added_by":"auto","created_at":"2024-05-02 20:07:13","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":269026,"visible":true,"origin":"","legend":"\u003cp\u003eScattergram–histogram combinations. After the paired tissues are stained by DAB, 5 areas are randomly circled at the endometrium position, and 10 areas are randomly circled at the position of adenomyoma. In both paired tissues, scattergram depicts the mean intensity of H\u0026amp;E stain on the x-axis and the mean intensity of DAB stain on the y-axis. The corresponding histograms add the density information for both parameters separately. DAB: 3-Diaminobenzidine; H\u0026amp;E: hematoxylin and eosin.\u003c/p\u003e","description":"","filename":"Figure.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4317248/v1/c2dd0124326c06e7207dbdf7.jpg"},{"id":55765428,"identity":"8c992a27-c41e-4526-847a-1c56ba6992c8","added_by":"auto","created_at":"2024-05-02 20:07:14","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":178057,"visible":true,"origin":"","legend":"\u003cp\u003eBar graph presentation of the fold change of IL-17A \u003cstrong\u003e(A)\u003c/strong\u003e and IL-17R \u003cstrong\u003e(B)\u003c/strong\u003e transcript quantified by real-time RT-PCR. Data presented are mean ± standard deviation. \u003cstrong\u003e(A)\u003c/strong\u003eIL-17A levels are significantly elevated in the adenomyosis group compared to the endometrium group (p= 0.047). \u003cstrong\u003e(B)\u003c/strong\u003e IL-17R levels are significantly higher in the adenomyosis group compared to the endometrium group (p= 0.027). * p \u0026lt; 0.05. RT-PCR: reverse transcription polymerase chain reaction.\u003c/p\u003e","description":"","filename":"Figure.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4317248/v1/cdbda9c3da2adb301070968c.jpg"},{"id":57549399,"identity":"14770246-aa18-434f-b6d3-9c04be9e15b9","added_by":"auto","created_at":"2024-06-01 16:03:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1502194,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4317248/v1/caa19638-d55e-4356-a196-e09a95efee9c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Regulated Expression of Interleukin-17 System at the Endometrial-Myometrial Interface: Implications for Adenomyosis","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eEndometriosis is a common, benign, estrogen-dependent, chronic gynecological disorder associated with pelvic pain and infertility. It is characterized by the presence of uterine endometrial tissue outside of the normal location\u0026mdash;mainly on the pelvic peritoneum, but also on the ovaries and in the recto-vaginal septum, and more rarely in the pericardium, pleura, and even the brain. The prevalence of pelvic endometriosis approaches 6\u0026ndash;10% in the general female population; in women with pain, infertility, or both, the frequency is 35\u0026ndash;50% (\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Although multiple theories exist regarding the etiology of the disease, it is most commonly held that retrograde menstruation is believed to allow endometrial tissue to seed the peritoneal cavity (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). These endometrial implants appear to activate host immune responses, leading to humoral- and cell-mediated inflammation. Activated macrophages and lymphocytes are found in greater numbers in the peritoneal cavity of women with endometriosis (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAdenomyosis is a pathological condition of the uterus closely associated with endometriosis (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Similar to endometriosis, adenomyosis is reliant on estrogen and involves the ectopic presence of endometrial cells and stromal fibroblasts within the myometrium, either as extensions of the normally located endometrium or as islands surrounded by hypertrophic smooth muscle cells (\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Adenomyosis shares similar symptoms with endometriosis, including heavy menstrual bleeding, dysmenorrhea, chronic pelvic pain, and infertility (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Several theories have been proposed to explain the pathogenesis of adenomyosis (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). The prevailing understanding implicates the profound invasion of endometrial tissue into the inner myometrium, causing disruption to the endometrial-myometrial interface (EMI) (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMultiple lines of evidence suggest that inflammation and immune responses play a pivotal role in the pathogenesis of endometriosis (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). In the same way, growing evidence indicates alterations in human and cellular immunity in adenomyosis (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Moreover, various immune-reacted serum proteins have been suggested to contribute to the development of adenomyosis (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Hirata et al. first showed the presence of Th17 cells in the peritoneal fluid (PF) of endometriotic women by flow cytometry and the presence of IL-17A-positive cells in endometriotic tissues by immunohistochemistry (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIL-17 is secreted by the CD4\u0026thinsp;+\u0026thinsp;T helper 17 (Th17) cell and is recognized as the hallmark cytokine of the unique cell population. In 1999, IL-17 was first identified through the use of T-cell clones derived from the joints of patients suffering from rheumatoid arthritis (\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Currently, the IL-17 family comprises six structurally associated cytokines (IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F), all sharing genetic homology, along with five matching receptors (IL-17RA, IL-17RB, IL-17RC, IL-17RD, and IL-17RE) found on cell surfaces. IL-17A stands as the inaugural member of this family and holds the distinction of being the most extensively studied, previously thought to be predominantly generated by Th17 cells (\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). IL-17F and IL-17A demonstrate elevated genetic homology and have the capability to create both homodimers and heterodimers for signaling purposes. Moreover, they engage with the same receptor complex, thereby predominantly sharing biological functions, with IL-17A exhibiting greater potency compared to IL-17F. The four other IL-17 isoforms exist exclusively as homodimers (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSo far, scant information exists concerning the role of the IL-17 system in the pathophysiological mechanisms of adenomyosis, and there is no report regarding the presence of IL-17 in women with adenomyosis. The current study aimed to investigate the expression of IL-17 in both ectopic and eutopic endometrium, and to compare its expression between these two types of endometrium in adenomyotic individuals.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Ethical disclosure and tissue sampling methodology\u003c/h2\u003e \u003cp\u003e Approval for our research was granted by the Institutional Review Board of Chang Gung Memorial Hospital (IRB#201700139B0). Prior to enrollment, all participants submitted written informed consent. Paired tissues of their matched endometrium and adenomyoma were obtained from 16 premenopausal women undergoing hysterectomy. Hysterectomy was indicated due to clinical symptoms related to adenomyosis, including hypermenorrhea and dysmenorrhea. The exclusion criteria were: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) subjects with past history of pelvic inflammatory disease, (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) subjects with history of genital tract infection, and (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) subjects who had undergone any hormone therapies within 6 months prior to surgery. Biopsies were conducted by a skilled gynecologist at Linkou Chang Gung Memorial Hospital. Histological documentation was conducted on the excised tissue samples to confirm the presence of adenomyosis foci, characterized by active endometrial glands and stroma surrounded by hypertrophic myometrium. Section of EMI was collected for immunohistochemistry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Immunohistochemistry study of IL-17 system in paired-tissue of EMI.\u003c/h2\u003e \u003cp\u003e12 serial sections (each 6 \u0026micro;m thick) of frozen eutopic endometrium with attached myometrium were excised for immunohistochemistry staining. The first and last slides underwent staining using hematoxylin and eosin (H\u0026amp;E) for pathologic confirmation, while the remaining slides were stained for the IL-17 system and analyzed using a Nikon microphot-FXA microscope (Nikon Instruments, Garden City, New York, NY, USA). Tissues were fixed with methanol: acetone (1:1) (Mallinckrodt, USA) and resuspended in phosphate buffered saline (PBS; Sigma Chemical Co, St. Louis, MO, USA) until processed for immunohistochemistry. After washing with PBS, endogenous peroxidases were blocked with 1% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in 96% methanol, and non-specific binding was blocked with 5% non-fat milk in PBS at room temperature. The EnVision kit facilitated immunohistochemical identification of the IL-17 system with horseradish peroxidase (HRP) (Dako, Glostrup, Denmark) system as the linker and liquid 3, 3-Diaminobenzidine (DAB) as the chromagen. Positive antibody reactions were indicated by the presence of brown granules. All sections underwent counterstaining with Mayer hematoxylin, dehydration, and mounting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Quantitative study for IL-17 mRNA expression using real-time reverse transcription polymerase chain reaction (RT-PCR)\u003c/h2\u003e \u003cp\u003eDNA amplification and data collection were carried out using the LighCycler System. All reactions were performed with the QuantiTect SYBR Green PCR Kit (Qiagen). The reaction components comprised 1X QuantiTect SYBR Green PCR Master Mix, with a final concentration of 0.5\u0026micro;M for each primer. The reactions were cycled with an initial activation step of 15min at 95\u0026deg;C followed by 40 cycles of 15 seconds at 94\u0026deg;C, 30 seconds at 60\u0026deg;C and 20 seconds at 72\u0026deg;C. The raw data were analyzed with the Light Cycler software version 4.0. The quantitative amount of IL-17A and IL-17R was determined.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 IL-17 mRNA expression determined by quantitative PCR (Q PCR)\u003c/h2\u003e \u003cp\u003eGene expression levels of IL-17A and IL-17R in paired uterine samples were assessed through quantitative real-time RT-PCR, utilizing the TaqMan methodology with the Applied Biosystems 7900 HT Real-time PCR System. PCR reactions were conducted according to the manufacturer's guidelines, reaching a final volume of 25 \u0026micro;L, which comprised 12.5 \u0026micro;L of 2X TaqMan Universal PCR Master Mix (Life Technologies\u0026reg;, Foster City, CA, USA), 1.25 \u0026micro;L TaqMan assay (20\u0026times;), 1\u0026micro;L of sample cDNA, and 10.25 \u0026micro;L of RNAse-free water. The levels of IL-17A and IL-17R mRNA in adenomyosis were contrasted with those in the endometrium from the same patient. Comparative gene transcript amounts were calculated using the delta-delta Ct method (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Data interpretation\u003c/h2\u003e \u003cp\u003eThe data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Data analysis was conducted using SPSS 22.0 (SPSS Inc., Chicago, IL, USA), with statistical significance set at a p-value of \u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cp\u003eThe tissues from both eutopic endometrium and adenomyosis, fixed in formalin, underwent preparation for immunohistochemical staining to reveal the existence of IL-17 system and CD4 proteins at the EMI level. CD4, IL-17R, and IL-17A immunoreactive proteins were detected within both the eutopic endometrium and adenomyosis. CD4, IL-17R, and IL-17A were noted to be expressed in both the glands and stroma of the endometrium and adenomyosis. In both eutopic and ectopic endometrium samples, epithelial cells exhibited greater staining intensity compared to stromal cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe used DAB as the chromagen for immunohistochemical detection of IL-17 system. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows an example of paired samples from one patient and provides qualitative data on IL-17A expressions within the tissue sample. After the tissues were stained by immunohistochemistry, 5 areas are randomly circled at the endometrium position, 10 areas are randomly circled at the position of adenomyosis. Scattergram\u0026ndash;histogram combinations are well suited to visualize the relation between two tissues. The result shows that the mean intensity of DAB stain is higher in adenomyosis (0.54%) than in endometrium (0.30%), demonstrating there are more IL-17A expressions in adenomyosis than in endometrium.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, we demonstrated the comparison of the fold change levels of IL-17A and IL-17R between the endometrium and corresponding adenomyosis group. Quantitative real-time RT-PCR in paired samples was performed. The fold change of IL-17A is significantly higher in the adenomyosis group compared to the endometrium group (p\u0026thinsp;=\u0026thinsp;0.047, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(A)). Similarly, the fold change of IL-17R is significantly higher in the adenomyosis group in comparison to the endometrium group (p\u0026thinsp;=\u0026thinsp;0.027, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(B)).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eThe endometrium showcases a sophisticated interplay among a network of cells, manifesting as a meticulously orchestrated phase encompassing proliferation, differentiation, and menstrual shedding (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). The formation of cytokines, the expression of cytokine receptors, and the modulation of endometrial functions by these factors underscore the pivotal role of cytokines in operating at endocrine, paracrine and autocrine levels within the endometrium. Increasingly, there is compelling evidence pointing to the participation of immune cells and inflammatory mediators in the development of endometriosis (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). In our study, IL-17A and IL-17R were identified in endometrium and adenomyosis, and the fold change levels of IL-17A and IL-17R were both significantly higher in adenomyosis than in endometrium, implicating that IL-17 system functions as a local immunomodulatory factor in adenomyosis.\u003c/p\u003e \u003cp\u003eIn endometriosis patients, various active substances, including cytokines, growth factors, hormones, and oxidative stress parameters, have been identified at different stages of the disease (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). An analysis revealed that the primary contributors to inflammation in endometriotic women were cytokines produced by macrophages (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). IL-17A has been noted to function as a chemotactic agent for macrophages via its receptor, IL-17RA, and can also trigger M2 macrophage polarization in endometriotic patients (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). Additionally, the researchers suggest that IL-17A plays a role in recruiting macrophages (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e), and M2 macrophages have indeed been demonstrated to facilitate extracellular matrix remodeling and neovascularization, closely linked to the development of endometriosis (\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). The heightened production of tumor necrosis factor, triggered by activated macrophages, along with the effect of IL-17 on endometrial cells, hastens the formation of endometriosis, often leading to unexplained pelvic pain and infertility. Moreover, recent research indicated that increased IL-17 levels in PF from patients with endometriosis-associated infertility might stimulate peritoneal macrophages to produce nitric oxide synthase 2 (NOS2) and nitric oxide (NO), which can have detrimental effects on both men\u0026rsquo;s and women\u0026rsquo;s reproductive systems (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Therefore, elevated levels of IL-17 are associated with infertility in endometriosis.\u003c/p\u003e \u003cp\u003eAs the research delves deeper into cytokines and endometriosis, there is increased reporting and wider confirmation of elevated IL-17 levels in endometriosis, particularly during the early stages of the disease (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). Zhang and colleagues were the first to demonstrate elevated IL-17 levels in the PF of endometriotic individuals. Furthermore, they found a relationship between the IL-17 concentration in PF and the advancement of the illness. Specifically, the IL-17 concentrations in PF were notably elevated in individuals with minimal/mild endometriosis compared to those without endometriosis and individuals with moderate/severe endometriosis. Additionally, their research indicated that the IL-17 concentration in PF was linked to infertility associated with endometriosis (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Salmeri et al. proposed that this observation could be explained by the notion that the inflammation appears to diminish in more advanced stages of endometriosis (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). Furthermore, other studies have shown heightened levels of IL-17A in both the PF and plasma of endometriotic patients compared to controls, along with the production of IL-17A by lesions of endometriosis (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). One research reported that they observed IL-17A levels increased comparably in both follicular fluid and serum in cases where infertility and endometriosis coexisted (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). Another study showed that cells positive for IL-17A were localized around the vasculature and in the stroma in both endometrium and endometriotic tissues from endometriotic patients (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). Additionally, one research found a higher percentage of Th17 cells in PF compared to the blood in endometriotic women. The result of this research also revealed in the PF of severe endometriosis, the Th17 cell percentage was higher compared to early-stage (I/II) endometriosis (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). Considering the facts mentioned above, it's apparent that in women with endometriosis, the IL-17 level commonly rises, often observed in both blood and PF samples.\u003c/p\u003e \u003cp\u003eHistologically, adenomyosis is characterized by ectopic endometrial tissue within the myometrium. Despite extensive research, its underlying causes remain unclear, with many aspects of its development still unknown. One identified mechanism contributing to adenomyosis is the heightened ability of certain endometrial cells to invade the myometrial tissue. Additionally, both local and systemic immune responses have been linked to the onset and maintenance of the condition. Immune system involvement coincides with hormonal irregularities and activation of the epithelial to mesenchymal transition pathway, facilitating the migration of endometrial cells (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Exploring the IL-17 system may offer novel perspectives on the pathogenesis of adenomyosis. In a prior study, they concluded that regulated by cytokines and estrogen, IL-17 secretion increases, either stemming from Th17 differentiation or stromal cells of endometrium. IL-17 then enhances the development, proliferation, and ectopic invasion, promoting immune evasion and endometriotic development by inducing and recruiting the M2 macrophage differentiation (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). In conjunction with our results, the IL-17 system might participate in the formation of ectopic endometriotic lesions.\u003c/p\u003e \u003cp\u003eNevertheless, this study also has several limitations. Methodologically, this study is disadvantaged by an inability to completely control for selection and confounding biases. Despite being the first published study reporting on expression of IL-17 system in adenomyosis, the number of patients with adenomyosis was small. Therefore, this population was not generally representative. Furthermore, the study design limited our ability to infer causality between IL-17 system and adenomyosis.\u003c/p\u003e"},{"header":"5 Conclusion","content":"\u003cp\u003eIn conclusion, to our knowledge, this is the first study to compare the expression of IL-17 system between eutopic endometrium and adenomyosis in women with adenomyosis. We found that expression of IL-17 system is significantly higher in adenomyosis than in corresponding endometrium. Our results may provide clues to the pathogenesis of adenomyosis involving the immune system. However, further investigation is needed to assess precisely how the IL-17 system participates in the development of adenomyosis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by the institutional review board of the Human Investigation and Ethical Committee of the Chang Gung Medical Foundation (CGMHIRB#\u0026nbsp;201700139B0). Informed consent was obtained from all subjects involved in the study.\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 datasets used and analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research work was supported in part under Chang Gung Memorial Hospital Research Grant (CMRPG3E0771, CMRPG3M0791/792).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH.-Y.H. participated in conceptulization and methodology. P.-C.L. and Y.-W.W. helped in the lab work and analysis of data. L.-T.H., H.-M.W., I.-J.C., H.-Y.H. helped in writing the manuscript. All authors reviewed the manuscript.\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\u003eHouston DE. Evidence for the risk of pelvic endometriosis by age, race and socioeconomic status. Epidemiol Rev. 1984;6:167\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKistner RW. Endometriosis and infertility. Clin Obstet Gynecol. 1979;22(1):101\u0026ndash;19.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSensky TE, Liu DT. Endometriosis: associations with menorrhagia, infertility and oral contraceptives. Int J Gynaecol Obstet. 1980;17(6):573\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChapron C, Marcellin L, Borghese B, Santulli P. Rethinking mechanisms, diagnosis and management of endometriosis. Nat reviews Endocrinol. 2019;15(11):666\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBourdon M, Santulli P, Jeljeli M, Vannuccini S, Marcellin L, Doridot L, et al. Immunological changes associated with adenomyosis: a systematic review. Hum Reprod Update. 2021;27(1):108\u0026ndash;29.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBulun SE, Yildiz S, Adli M, Wei JJ. Adenomyosis pathogenesis: insights from next-generation sequencing. Hum Reprod Update. 2021;27(6):1086\u0026ndash;97.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGarc\u0026iacute;a-Solares J, Donnez J, Donnez O, Dolmans MM. Pathogenesis of uterine adenomyosis: invagination or metaplasia? Fertil Steril. 2018;109(3):371\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBird CC, McElin TW, Manalo-Estrella P. The elusive adenomyosis of the uterus\u0026ndash;revisited. Am J Obstet Gynecol. 1972;112(5):583\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhai J, Vannuccini S, Petraglia F, Giudice LC, Adenomyosis. Mechanisms and Pathogenesis. Semin Reprod Med. 2020;38(2\u0026ndash;03):129\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStruble J, Reid S, Bedaiwy MA. Adenomyosis: A Clinical Review of a Challenging Gynecologic Condition. J Minim Invasive Gynecol. 2016;23(2):164\u0026ndash;85.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBenagiano G, Brosens I. The endometrium in adenomyosis. Women's health (London England). 2012;8(3):301\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarcus CC. Relationship of adenomyosis uteri to endometrial hyperplasia and endometrial carcinoma. Am J Obstet Gynecol. 1961;82:408\u0026ndash;16.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBenagiano G, Habiba M, Brosens I. The pathophysiology of uterine adenomyosis: an update. Fertil Steril. 2012;98(3):572\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarada T, Iwabe T, Terakawa N. Role of cytokines in endometriosis. Fertil Steril. 2001;76(1):1\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLebovic DI, Mueller MD, Taylor RN. Immunobiology of endometriosis. Fertil Steril. 2001;75(1):1\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBourdon M, Santulli P, Marcellin L, Maignien C, Maitrot-Mantelet L, Bordonne C, et al. Adenomyosis: An update regarding its diagnosis and clinical features. J Gynecol Obstet Hum Reprod. 2021;50(10):102228.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang F, Li H, Yang Z, Du X, Cui M, Wen Z. Expression of interleukin-10 in patients with adenomyosis. Fertil Steril. 2009;91(5):1681\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXiaoyu L, Weiyuan Z, Ping J, Anxia W, Liane Z. Comparative serum proteomic analysis of adenomyosis using the isobaric tags for relative and absolute quantitation technique. Fertil Steril. 2013;100(2):505\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHirata T, Osuga Y, Hamasaki K, Yoshino O, Ito M, Hasegawa A, et al. Interleukin (IL)-17A stimulates IL-8 secretion, cyclooxygensase-2 expression, and cell proliferation of endometriotic stromal cells. Endocrinology. 2008;149(3):1260\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAarvak T, Chabaud M, Miossec P, Natvig JB. IL-17 is produced by some proinflammatory Th1/Th0 cells but not by Th2 cells. Journal of immunology (Baltimore, Md: 1950). 1999;162(3):1246-51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlbanesi C, Cavani A, Girolomoni G. IL-17 is produced by nickel-specific T lymphocytes and regulates ICAM-1 expression and chemokine production in human keratinocytes: synergistic or antagonist effects with IFN-gamma and TNF-alpha. Journal of immunology (Baltimore, Md: 1950). 1999;162(1):494\u0026ndash;502.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBeringer A, Noack M, Miossec P. IL-17 in Chronic Inflammation: From Discovery to Targeting. Trends Mol Med. 2016;22(3):230\u0026ndash;41.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarrington LE, Hatton RD, Mangan PR, Turner H, Murphy TL, Murphy KM, et al. Interleukin 17-producing CD4\u0026thinsp;+\u0026thinsp;effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nat Immunol. 2005;6(11):1123\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZou W, Restifo NP. T(H)17 cells in tumour immunity and immunotherapy. Nat Rev Immunol. 2010;10(4):248\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFossiez F, Djossou O, Chomarat P, Flores-Romo L, Ait-Yahia S, Maat C, et al. T cell interleukin-17 induces stromal cells to produce proinflammatory and hematopoietic cytokines. J Exp Med. 1996;183(6):2593\u0026ndash;603.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRouvier E, Luciani MF, Matt\u0026eacute;i MG, Denizot F, Golstein P. CTLA-8, cloned from an activated T cell, bearing AU-rich messenger RNA instability sequences, and homologous to a herpesvirus saimiri gene. Journal of immunology (Baltimore, Md: 1950). 1993;150(12):5445-56.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePatel DD, Kuchroo VK. Th17 Cell Pathway in Human Immunity: Lessons from Genetics and Therapeutic Interventions. Immunity. 2015;43(6):1040\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiossec P, Kolls JK. Targeting IL-17 and TH17 cells in chronic inflammation. Nat Rev Drug Discovery. 2012;11(10):763\u0026ndash;76.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eToy D, Kugler D, Wolfson M, Vanden Bos T, Gurgel J, Derry J et al. Cutting edge: interleukin 17 signals through a heteromeric receptor complex. Journal of immunology (Baltimore, Md: 1950). 2006;177(1):36\u0026thinsp;\u0026ndash;\u0026thinsp;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLivak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods (San Diego, Calif). 2001;25(4):402-8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSim\u0026oacute;n C, Piquette GN, Frances A, Polan ML. Localization of interleukin-1 type I receptor and interleukin-1 beta in human endometrium throughout the menstrual cycle. J Clin Endocrinol Metab. 1993;77(2):549\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSim\u0026oacute;n C, Frances A, Lee BY, Mercader A, Huynh T, Remohi J, et al. Immunohistochemical localization, identification and regulation of the interleukin-1 receptor antagonist in the human endometrium. Hum Reprod (Oxford England). 1995;10(9):2472\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGazvani R, Templeton A. Peritoneal environment, cytokines and angiogenesis in the pathophysiology of endometriosis. Reprod (Cambridge England). 2002;123(2):217\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBedaiwy MA, Falcone T. Peritoneal fluid environment in endometriosis. Clinicopathological implications. Minerva Ginecol. 2003;55(4):333\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBeste MT, Pf\u0026auml;ffle-Doyle N, Prentice EA, Morris SN, Lauffenburger DA, Isaacson KB, et al. Molecular network analysis of endometriosis reveals a role for c-Jun-regulated macrophage activation. Sci Transl Med. 2014;6(222):222ra16.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShi JL, Zheng ZM, Chen M, Shen HH, Li MQ, Shao J. IL-17: an important pathogenic factor in endometriosis. Int J Med Sci. 2022;19(4):769\u0026ndash;78.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiller JE, Ahn SH, Marks RM, Monsanto SP, Fazleabas AT, Koti M, et al. IL-17A Modulates Peritoneal Macrophage Recruitment and M2 Polarization in Endometriosis. Front Immunol. 2020;11:108.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOkizaki S, Ito Y, Hosono K, Oba K, Ohkubo H, Amano H et al. Suppressed recruitment of alternatively activated macrophages reduces TGF-β1 and impairs wound healing in streptozotocin-induced diabetic mice. Biomedicine \u0026amp; pharmacotherapy\u0026thinsp;=\u0026thinsp;Biomedecine \u0026amp; pharmacotherapie. 2015;70:317\u0026thinsp;\u0026ndash;\u0026thinsp;25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeber C, Telerman SB, Reimer AS, Sequeira I, Liakath-Ali K, Arwert EN, et al. Macrophage Infiltration and Alternative Activation during Wound Healing Promote MEK1-Induced Skin Carcinogenesis. Cancer Res. 2016;76(4):805\u0026ndash;17.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWynn TA, Vannella KM. Macrophages in Tissue Repair, Regeneration, and Fibrosis. Immunity. 2016;44(3):450\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiljkovic D, Trajkovic V. Inducible nitric oxide synthase activation by interleukin-17. Cytokine Growth Factor Rev. 2004;15(1):21\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSikora J, Smycz-Kubańska M, Mielczarek-Palacz A, Bednarek I, Kondera-Anasz Z. The involvement of multifunctional TGF-β and related cytokines in pathogenesis of endometriosis. Immunol Lett. 2018;201:31\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang X, Xu H, Lin J, Qian Y, Deng L. Peritoneal fluid concentrations of interleukin-17 correlate with the severity of endometriosis and infertility of this disorder. BJOG: Int J Obstet Gynecol. 2005;112(8):1153\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSalmeri FM, Lagan\u0026agrave; AS, Sofo V, Triolo O, Sturlese E, Retto G et al. Behavior of tumor necrosis factor-α and tumor necrosis factor receptor 1/tumor necrosis factor receptor 2 system in mononuclear cells recovered from peritoneal fluid of women with endometriosis at different stages. Reproductive sciences (Thousand Oaks, Calif). 2015;22(2):165\u0026thinsp;\u0026ndash;\u0026thinsp;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRafi U, Ahmad S, Bokhari SS, Iqbal MA, Zia A, Khan MA, et al. Association of Inflammatory Markers/Cytokines with Cardiovascular Risk Manifestation in Patients with Endometriosis. Mediat Inflamm. 2021;2021:3425560.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSabbaghi M, Aram R, Roustaei H, Fadavi Islam M, Daneshvar M, Casta\u0026ntilde;o AR, et al. IL-17A concentration of seminal plasma and follicular fluid in infertile men and women with various clinical diagnoses. Immunol Investig. 2014;43(7):617\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhn SH, Edwards AK, Singh SS, Young SL, Lessey BA, Tayade C. IL-17A Contributes to the Pathogenesis of Endometriosis by Triggering Proinflammatory Cytokines and Angiogenic Growth Factors. Journal of immunology (Baltimore, Md: 1950). 2015;195(6):2591\u0026thinsp;\u0026ndash;\u0026thinsp;600.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGogacz M, Winkler I, Bojarska-Junak A, Tabarkiewicz J, Semczuk A, Rechberger T, et al. Increased percentage of Th17 cells in peritoneal fluid is associated with severity of endometriosis. J Reprod Immunol. 2016;117:39\u0026ndash;44.\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":"Interleukin-17, Adenomyosis, Endometrial-myometrial interface, Cytokine","lastPublishedDoi":"10.21203/rs.3.rs-4317248/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4317248/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eAdenomyosis involves the infiltration of endometrial glands and stroma deep into the uterine tissue, causing disruption to the interface between the endometrium and myometrium. The role of interleukin-17 (IL-17), a cytokine associated with immune responses, has been extensively studied in endometriosis, but its involvement in adenomyosis remains unclear. This study aimed to investigate the expression of IL-17 in ectopic and eutopic endometrium of individuals with adenomyosis, comparing its levels between these two types of endometrium.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003ePaired tissues of eutopic endometrium and adenomyoma were collected from 16 premenopausal women undergoing hysterectomy due to clinical symptoms related to adenomyosis. IL-17 system was demonstrated in paired tissue samples by the immunochemistry study. Gene expression levels of IL-17A and IL-17 receptor (IL-17R) were assessed through quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). Comparative gene transcript amounts were calculated using the delta-delta Ct method.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eBy immunohistochemical staining, CD4, IL-17R, and IL-17A proteins were detected in both endometrium and adenomyosis, with higher expression in epithelial cells. DAB staining revealed greater IL-17A expression in adenomyosis compared to endometrium. Quantitative RT-PCR showed significantly higher fold change levels of IL-17A and IL-17R in adenomyosis (IL-17A: p\u0026thinsp;=\u0026thinsp;0.047, IL-17R: p\u0026thinsp;=\u0026thinsp;0.027) versus endometrium.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eWe found significantly higher IL-17 levels in adenomyosis compared to endometrium, suggesting immune system involvement in adenomyosis pathogenesis.\u003c/p\u003e","manuscriptTitle":"Regulated Expression of Interleukin-17 System at the Endometrial-Myometrial Interface: Implications for Adenomyosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-02 20:07:09","doi":"10.21203/rs.3.rs-4317248/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":"f332e757-9533-4811-9dd7-7c931ea0b016","owner":[],"postedDate":"May 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-06-01T15:54:55+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-02 20:07:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4317248","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4317248","identity":"rs-4317248","version":["v1"]},"buildId":"WvIrzKhiLBfengagbw6Ux","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}