{"paper_id":"2d3bfd1b-c8c8-41b1-b168-f8ffaefdabe5","body_text":"112\nThis is an Open Access article distributed under the terms of the Creative Commons Attribu-\ntion Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits \nunrestricted non-commercial use, distribution, and reproduction in any medium, provided the \noriginal work is properly cited.\n© 2024 THE KOREAN SOCIETY FOR REPRODUCTIVE MEDICINEwww.eCERM.org\nPatterns of proliferation and fibrosis in a rat model \nof endometriosis following administration of Allium \ncepa \nHakan Kula\n1\n, Orkun Ilgen\n1,*\n, Sefa Kurt\n1\n, Filiz Yılmaz\n2\n \n1\nDepartment of Obstetrics and Gynecology, Dokuz Eylul University, Izmir; \n2\nDepartment of Histology and Embryology, Hitit University, Corum, Turkey\nORIGINAL ARTICLE\nhttps://doi.org/10.5653/cerm.2023.06261\npISSN 2233-8233 · eISSN 2233-8241\nClin Exp Reprod Med 2024;51(2):112-119\nReceived: June 8, 2023 ∙ Revised: November 11, 2023 ∙ Accepted: November 14, 2023 \nCorresponding author: Hakan Kula \nDepartment of Obstetrics and Gynecology, Dokuz Eylul University, Izmir 35330, \nTurkey \nTel: +90-5358230685 Fax: +90-2324123149 E-mail: hkula95@gmail.com \n*Current affiliation: Department of Obstetrics and Gynecology, Erzurum Regional \nEducation and Research Hospital, Erzurum, Turkey  \nObjective: Endometriosis is a common gynecological disease among reproductive-age women. Numerous hypotheses exist regarding the \npathogenesis of endometriosis. In Turkey, the consumption of Allium cepa (commonly known as the “onion cure”) is a popular treatment \nemployed to alleviate a variety of gynecological disorders. \nMethods: In this study, our objective was to assess the therapeutic mechanisms of the onion bulb A. cepa using an autologous endometrio-\nsis model in Sprague-Dawley rats. Previous research has shown that A. cepa possesses anti-inflammatory, antioxidant, and antiapoptotic \nproperties. We evaluated the pathological condition of endometriotic implants by employing hematoxylin-eosin staining and Ki67 immuno-\nhistochemistry analysis. Transforming growth factor-beta 1 (TGF-β1) and alpha-smooth muscle actin (α-SMA) have been identified as profi-\nbrotic markers that are highly overexpressed in endometriotic tissues relative to eutopic endometrial tissue. Furthermore, TGF-β1 influences \nthe differentiation and progression of endometriosis. To quantify profibrotic activity, we measured TGF-β1 and α-SMA using the immunosor-\nbent assay method. \nResults: Lower histologic evaluation scores for endometriotic implants were observed in the group receiving high-dose A. cepa relative to \nthe other groups. Ki67 expression was reduced following the high-dose A. cepa regimen, which consisted of 30% A. cepa and 70% normal \nfeed. However, no statistically significant differences in TGF-β1 or α-SMA levels were observed among the groups (p=0.7 and p=0.778, re-\nspectively). \nConclusion: The findings suggest that A. cepa could serve as a therapeutic agent in endometriosis treatment, as evidenced by the reduction \nin proliferative potential. Nevertheless, A. cepa was not associated with significantly lower levels of endometriosis-associated TGF-β1 or \nα-SMA. \nKeywords: Alpha-smooth muscle actin; Fibrosis; Ki67; Proliferation; Transforming growth factor beta1  \nIntroduction \nEndometriosis is a common inflammatory gynecological patholo-\ngy characterized by the ectopic implantation of endometrial tissues \n[1]. Endometriosis can damage the tissue through inflammatory, \nproliferative, and fibrogenic processes. This condition is estimated to \naffect nearly 10% of the general female population and between \n25% and 40% of women who are infertile [2]. Furthermore, endome-\ntriosis is present in approximately 70% of women with chronic pelvic \npain [3]. The primary clinical manifestations of endometriosis include \nchronic pelvic pain, dysmenorrhea, dyspareunia, and infertility. \nThe pathogenesis of endometriosis is multifactorial and continues \nto provoke debate concerning its pathophysiology and treatment \nstrategies. A variety of theories contribute to our understanding of \nendometriosis, including the roles of inflammatory mediators, infec-\ntious agents, endocrine factors, and proangiogenic elements. Recent \nstudies have focused on the inflammatory and immune mechanisms \n\n\nunderlying endometriosis. Increased oxidative damage, as evidenced \nby elevated levels of malondialdehyde, superoxide anions, and hy-\ndrogen peroxide, has also been implicated in the disease process [4]. \nRecommended treatments encompass medical therapies, surgical \ninterventions, herbal remedies, and specific anti-inflammatory and \nantioxidant agents. \nFibrosis refers to a loss of tissue function due to repetitive damage. \nThis condition is marked by increased activity of myofibroblasts and \nheightened collagen production, causing the primary tissue to lose \nits functionality [5]. Histologically, fibrosis in endometriosis is evi-\ndenced by the envelopment of endometrial glands and stroma by \ndense fibrous tissue [6]. Furthermore, fibrosis contributes to the degra-\ndation of both the vasculature and tissue functions, potentially lead-\ning to resistance against medical treatments and hormonal suppres-\nsive therapies [7]. Transforming growth factor-beta 1 (TGF-β1) and al-\npha-smooth muscle actin (α-SMA) have been identified as key profi-\nbrogenic biomarkers that can be used to assess myofibroblast activity \nand the extent of fibrosis in endometriotic tissue [8]. Ki67, a nuclear \nprotein, is intimately associated with cell division, as it is present \nduring all active phases of the cell cycle (G1, S, G2, and mitosis) but is \nconspicuously absent in the resting phase, G0 [9]. Ki67 is a crucial \nmarker for determining a cell’s proliferative capacity. Therefore, due to \nits role in signaling proliferative activity in neoplastic diseases, an in-\ncreased level of Ki67 is considered a key factor for predicting the prog-\nnosis and the risk of recurrence in endometriosis cases [10]. \nAllium cepa, commonly known as the onion bulb, is a member of \nthe Amaryllidaceae family [11]. Prior research has explored its anti-\ncancer and antioxidant properties [12]. Studies have shown that A. \ncepa possesses anti-inflammatory effects, as evidenced by reduced \nlevels of prostaglandin E2, nuclear factor kappa-light-chain-enhancer \nof activated B cells, and thromboxanes, as well as the inhibition of \nchemotaxis [13]. Furthermore, A. cepa has been reported to amelio-\nrate cellular degeneration and mitigate damage caused by ischemia \nand reperfusion in ovarian tissue [14]. Quercetin, flavonoids, and or-\nganosulfur compounds have been identified as the primary active \ncomponents in A. cepa that contribute to its anti-inflammatory ef-\nfects. The literature also describes the inhibition of endometriotic \nproliferation through the regulation of the cyclin pathway by querce-\ntin molecules [15]. The objective of the present study is to investigate \nwhether A. cepa can improve the histological parameters of endo-\nmetriotic lesion proliferation and the levels of profibrotic mediators. \nMethods \nThis study received approval from the Dokuz Eylül University Lab-\noratory Animals Local Ethics Committee (Approval No: 41-2020). \nTwenty-one adult Sprague-Dawley rats, each weighing between 220 \nand 250 g, were acquired from the Dokuz Eylül University Experi-\nmental Animal Laboratory. The trial was conducted in accordance \nwith the Animal Research: Reporting of In Vivo Experiments (ARRIVE) \nguidelines. The rats were housed in standard cages with sawdust \nbedding, maintained at a room temperature of 22±2 °C, and kept \nunder a 12/12-hour dark/light cycle throughout the trial. To deter-\nmine the estrus phase of the rats, vaginal smears were collected four \ntimes daily. Each procedure was documented using a digital camera. \nThe 21 rats were randomly divided into three groups of seven ani-\nmals each. Surgical procedures were carried out under sterile condi-\ntions. Anesthesia was induced with an intraperitoneal injection of \nketamine hydrochloride (50 mg/kg, Ketalar; Pfizer Inc.) and xylazine \nhydrochloride (7 mg/kg, Alfazyne; Alfasan International BV). All labo-\nratory procedures are detailed in Figure 1. \n1. Surgical procedure (step 1: implantation of endometriotic \ntissue) \nBefore the incision was made, the abdominal wall was shaved and \ncleansed with a 10% povidone-iodine solution. With the rat under \nanesthesia, a sterile incision measuring 2 cm was made along the \nmidline of the lower abdomen. The uterine horns, adnexa, and ova-\n• Implantation of right uterine horn into the peritoneum\nImplanting \nendometriotic \ntissues \nValidation of \nendometriotic \nimplants\nA. cepa \nregimen\nEvaluation\n• Relaparotomy is performed at day 14 to establish the model\n• Low-dose Allium cepa regimen: 10% A. cepa+90% normal feed\n• High-dose A. cepa regimen: 30% A. cepa+70% normal feed\n• A. cepa is given by oral gavage per day for 21 days\n• Histological examination\n• TGF-β1 and α-SMA concentrations measured by ELISA\n• Immunohistochemistry of Ki67 expression\nFigure 1. Explanation of the endometriosis model. TGF-β1, transforming \ngrowth factor-beta 1; α-SMA, alpha-smooth muscle actin; ELISA, \nenzyme-linked immunosorbent assay.\nwww.eCERM.org 113\nH Kula et al. A. cepa administration in an endometriosis model\n\nries were identified in each animal. Autologous uterine tissue (1 cm \nlong) was harvested from the distal end of the right uterine horn. \nThis tissue was then immersed in a phosphate-buffered saline solu-\ntion for 2 minutes. Subsequently, the endometrium was isolated, \nand endometrial fragments measuring 5×5 mm were implanted \ninto the abdominal wall (Figure 2). \nThe abdominal wall and skin were closed using 3.0 polyglactin su-\ntures. Following sterilization of the abdominal wall with 10% povi-\ndone-iodine, heating pads were applied until the animal regained \nconsciousness. To induce the formation of endometriotic implants in \nautologous tissues, 50 μg/kg of estrogen was administered subcuta-\nneously to the rats twice weekly [16]. \n2. Validation of endometriotic implants \nRelaparotomy was scheduled for day 14 to validate the presence \nof endometriotic implants on the inner abdominal wall, coinciding \nwith the estrus phase in rats. The anesthetic and surgical protocols \nfrom step 1 were repeated, from the initial incision to the closure of \nthe abdominal cavity. To determine the estrus phase, daily vaginal \nsmears were conducted. Three rats were excluded from the remain-\nder of the study due to the inadequate development of endometri-\notic lesions. \n3. Preparation of A. cepa \nFeed fortified with A. cepa was incorporated into the rats’ regular \ndiet. The A. cepa onion bulbs were peeled, sliced, and air-dried for 1 \nweek before being ground into a fine powder. Group A (n=6), desig-\nnated as the Sham group, was administered only a 0.9% sodium chlo-\nride solution via oral gavage along with their standard diet. Group B \n(n=6), the low-dose group, received a mixture of 10% A. cepa and \n90% standard feed, while group C (n=6), the high-dose group, was \ngiven a blend of 30% A. cepa and 70% standard feed through oral ga-\nvage daily for 21 days. The A. cepa-supplemented diet was initiated \nafter the stabilization period following relaparotomy. \n4. Effect of A. cepa on endometriotic implants \nUnder anesthesia, the rats were euthanized, and their endometri-\notic implants were harvested for both histopathological and bio -\nchemical assessments. Concurrently, blood samples were obtained \nfor biochemical analysis. The collected endometriotic implants were \nsubjected to histological and biochemical examinations. For histo-\nlogical evaluation, the tissues were preserved in 10% formalin. \n5. Histological examination \nEndometriotic implants were embedded in paraffin blocks follow-\ning formalin fixation. Tissue sections, 5 mm thick, were prepared, \nthen stained with hematoxylin-eosin and examined under light mi-\ncroscopy (CX-41; Olympus). Blinded histological assessment was \nconducted by a histologist with experience in a prior study of rat en-\ndometriosis, and the findings were documented photographically \n[17,18]. \nThe epithelial lining of the endometrial implants underwent \nsemi-quantitative evaluation based on a method previously outlined \nin the literature [17,18]. The grading was as follows: grade 0 indicated \nan absence of epithelium, with a corresponding score of 0; grade 1 \nrepresented a poorly preserved epithelium, characterized by the \npresence of only occasional epithelial cells, and was assigned a score \nof 1; grade 2 denoted a moderately preserved epithelium accompa-\nnied by leukocyte infiltration, with a score of 2; and grade 3 de -\nscribed a well-preserved epithelial lining, which received a score of 3. \n6. Biochemical examination \nWhole blood was drawn into blood collection tubes without anti-\ncoagulant and then allowed to clot naturally at 25 °C for 30 minutes. \nFollowing coagulation, the samples were centrifuged at 2,000 ×g \nand 4 °C for 15 minutes. The upper layer of yellow serum was then \ncarefully collected and stored at −80 °C. Upon thawing, the serum \nconcentrations of TGF-β1 and α-SMA were measured using an en-\nzyme-linked immunosorbent assay kit (BTLAB, catalog numbers \nE1688Ra and E2330Ra). All procedures were performed in strict ac-\ncordance with the instructions provided with the kit. \nFigure 2. Macroscopic visualization of endometriotic implants.\nhttps://doi.org/10.5653/cerm.2023.06261114\nClin Exp Reprod Med 2024;51(2):112-119\n\n7. Immunohistochemical analysis \nSequential 5-μm-thick sections were cut from the paraffin blocks. \nFor antigen retrieval, these sections were boiled for 9 minutes in 0.01 \nmol/L sodium citrate buffer at a pH of 6. To suppress endogenous \nperoxidase activity, the sections were then incubated in a 3% hydro-\ngen peroxide solution for 10 minutes. Immunohistochemical stain-\ning was carried out using the avidin-biotin immunoperoxidase \nmethod, employing the Ki67 rabbit polyclonal antibody (catalog no. \nNB500-170SS; Novus Biologicals) as the primary antibody. \n8. Statistical analysis \nStatistical analysis of the data gathered during the study was con-\nducted using SPSS ver. 26.0 (IBM Corp.). Means and standard devia-\ntions were calculated for the evaluation, with biochemical and para-\nmetric data presented as mean±standard deviation. Differences \namong groups were assessed using the Kruskal-Wallis test, and the \nsource of any differences was further investigated with the Mann- \nWhitney U test. A p-value of less than 0.05 was considered indicative \nof statistical significance. \nResults \nThroughout the trial, we observed no adverse effects—such as \nhair loss, fatigue, or loss of appetite—in rats fed A. cepa. All the ani-\nmals survived until the conclusion of the study, and none exhibited \nsigns of wound infection. \nFigure 3 displays representative histopathological images of endo-\nmetriotic implants. Table 1 presents the mean scores from the histo-\npathological evaluation of the implants following the treatment pe-\nriod. A significant reduction in the mean histopathological evalua-\ntion score was observed for group C (1.14±1) relative to groups A \nand B (2.71±0.7 vs. 2.28±1.2, respectively; p<0.05). \nThe levels of tissue biomarkers (TGF-β1 and α-SMA) were assessed \nacross groups. The data for TGF-β1 and α-SMA levels are presented in \nTable 2. Notably, we observed no statistically significant difference in \nTGF-β1 levels among groups A, B, and C (633.21±89.49 vs. 697.66±76.53 \nvs. 636.08±156.11, p=0.7). Similarly, α-SMA levels exhibited no signifi-\ncant changes following treatment with A. cepa in groups A, B, and C \n(36.26±6.81 vs. 32.84±5.75 vs. 33.98±8.90; p=0.778). Immunohisto-\nchemical analysis revealed that Ki67, a marker of cell proliferation, \nwas highly expressed in all areas of the endometriotic implantation \ntissues in group A and in the low-dose A. cepa group (group B). \nHowever, Ki67 expression was diminished in group C, which received \na high-dose of A. cepa (30% A. cepa+70% normal feed), as depicted \nin Figure 4. \nDiscussion \nIn this study, we demonstrated that high-dose A. cepa intake sig-\nnificantly reduced histological evaluation scores and decreased Ki67 \nexpression in a surgically induced rat model. However, the levels of \nthe profibrotic mediators TGF-β1 and α-SMA did not change signifi-\ncantly following A. cepa administration. \nEndometriosis is a common gynecological disorder characterized \nby chronic pelvic pain, infertility, pelvic organ dysfunction, and im-\nFigure 3. Histopathologic images of the ovaries under hematoxylin and eosin staining (×10 magnification). (A) Endometriosis only. (B) \nEndometriosis+low-dose Allium cepa. (C) Endometriosis+high-dose A. cepa.\nTable 1. Mean histopathological evaluation scores \nGroup Histopathological evaluation score\nEndometriosis only 2.71 ± 0.7\nEndometriosis+low-dose Allium cepa 2.28 ± 1.2\nEndometriosis+high-dose A. cepa 1.14 ± 1.0\na)\nValues are presented as mean±standard deviation.\na)\np<0.05.\nAA BB CC\nwww.eCERM.org 115\nH Kula et al. A. cepa administration in an endometriosis model\n\npairment in daily activities and overall quality of life. The clinical \nmanifestations of endometriosis are driven by chronic inflammation, \nwhich is evidenced by elevated local and systemic levels of inflam-\nmatory chemokines and cytokines [19]. These mediators play key \nroles in histopathological cell damage and the clinical symptoms ob-\nserved. Despite this, the underlying mechanisms of endometriosis \nremain incompletely understood, and definitive treatment protocols \nare a subject of ongoing research. Inflammatory mediators such as \ntumor necrosis factor alpha, interleukin 1β (IL-1β), IL-6, IL-12, prosta-\nglandin E2, thromboxane, and others are also involved in the patho-\nphysiology of endometriosis [20]. \nThe progression of this condition is similarly impacted by inflam-\nmatory mediators, cytokines, and angiogenic and fibrogenic factors. \nThe regulation of these mediators may reduce the severity of endo-\nmetriosis and alleviate gynecological symptoms. Strategies that tar-\nget antiproliferative, proapoptotic, autophagic, anti-cell migration \nand invasion, antifibrotic, and anti-angiogenic mechanisms have \nbeen explored to inhibit the progression of endometriosis [21]. Fur-\nthermore, modulators of the immune system and inhibitors of an-\ngiogenesis have been studied as alternatives to hormonal therapy \nand nonsteroidal anti-inflammatory drugs (NSAIDs) for the improve-\nment of endometriosis-related clinical outcomes [22]. \nDeep endometriosis is the primary predisposing factor for poor \noutcomes. It is characterized by a rigid and compromised pelvic \nstructure resulting from extensive fibrosis and the activation of myo-\nfibroblasts [23]. The recurrent tissue injury and repair (ReTIAR) theory \nis among the most prevalent explanations for deep endometriosis \nand the associated fibrosis. The key mechanisms of ReTIAR include \noverexpression of TGF-β1, THY-1, and peroxisome proliferator-acti-\nvated receptor gamma; the transformation of fibroblasts into myofi-\nbroblasts; and recurrent tissue injury [24]. \nIn the present study, hematoxylin-eosin staining revealed a mor-\nphological enhancement in group C (which received a high-dose A. \ncepa regimen) relative to the other groups. Observations of epitheli-\nal integrity, inflammation, glandular structures, and vasculature in \nendometriosis tissue were less frequent in group C. These findings \ncorroborate a significant decrease in histological features associated \nwith endometriosis following the intake of A. cepa. \nA. cepa contains a variety of chemical constituents, including fla-\nvonoids (such as quercetin), lipophilic antioxidants, and isoliquiriti-\ngenin (ISL) [25]. These components are associated with a range of \ntherapeutic benefits, including analgesic, antitumor, antihyperlipid-\nemic, and antithrombotic effects [17]. Two studies have been con-\nducted on the impact of A. cepa on endometriosis [15,26]. The find-\nings from these studies indicate that quercetin and ISL not only im-\nprove endometriosis-associated clinical symptoms and lesions but \nalso may influence antiproliferative and anti-inflammatory agents \nwithin endometriotic tissue. Additionally, ISL exhibits a proapoptotic \nTable 2. Levels of TGF-β1 and α-SMA by group \nVariable Endometriosis only Endometriosis+low-dose Allium cepa Endometriosis+high-dose A. cepa p-value\nTGF-β1 633.21 ± 89.49 697.66 ± 76.53 636.08 ± 156.11 0.705\nα-SMA 36.26 ± 6.81 32.84 ± 5.75 33.98 ± 8.90 0.778\nValues are presented as mean±standard deviation.\nTGF-β1, transforming growth factor-beta 1; α-SMA, alpha-smooth muscle actin.\nFigure 4. Immunohistochemical analysis of Ki67 (×10 magnification). (A) Endometriosis only. (B) Endometriosis+low-dose Allium cepa. (C) \nEndometriosis+high-dose A. cepa.\nAA BB CC\nhttps://doi.org/10.5653/cerm.2023.06261116\nClin Exp Reprod Med 2024;51(2):112-119\n\neffect on endometriotic cells. These results align with prior research \nthat has documented histological improvements in endometriotic \ntissue. \nThe presence of fibrosis within endometriotic tissue can lead to \nanatomical distortion in the pelvis and chronic pelvic pain. Further-\nmore, TGF-β not only plays a critical role in fibrosis but also influences \napoptosis, disease progression, and cellular differentiation in endo-\nmetriosis [27]. Myofibroblast-like cells that are positive for α-SMA \nhave been observed within fibrotic areas in ovarian, deep, and su-\nperficial endometriotic implants. One study noted markedly elevat-\ned levels of fibrotic mediators, such as α-SMA and fibronectin, in en-\ndometriotic lesions relative to the eutopic endometrium [28]. Conse-\nquently, TGF-β and α-SMA, along with associated profibrotic signal-\ning pathways, may serve as potential therapeutic targets for the \ntreatment of endometriosis. \nIn the literature, two studies have investigated the levels of TGF-β1, \nα-SMA, and fibrosis in rats with endometriosis treated with various \nsubstances [23,29]. These studies suggest that targeting the antifi-\nbrotic mechanism could be considered as an alternative treatment \napproach for endometriosis. However, we did not observe significant \nchanges in the levels of the profibrotic mediators TGF-β and α-SMA \nin our surgically induced endometriosis rat model. \nPrevious research has indicated that the activation of the fibrino-\ngen pathway by fibrotic mediators in endometriosis leads to reduced \neffectiveness of cyclooxygenase (COX) inhibitors. This reduction in \ndrug efficacy is attributed to lower levels of prostaglandin E2 and di-\nminished expression of COX-2 [30]. Another study demonstrated \nthat A. cepa exhibits anti- inflammatory and analgesic effects rela-\ntive to indomethacin [31]. That research indicated that A. cepa is as-\nsociated with a significantly shorter pain reaction time on pain stim-\nulation tests compared to indomethacin. The findings suggest that \nthe diminished analgesic efficacy of NSAIDs in the presence of A. \ncepa within endometriotic lesions could be a consequence of fi -\nbrosis. \nKi67, which can be detected through immunohistochemistry, \nserves as a marker of cellular proliferation. This enables the assess-\nment of activity levels within endometriotic lesions, providing valu-\nable insights. An increase in Ki67 expression suggests that cells are \nbecoming autonomous and may exert an influence on adjacent tis-\nsues [32]. Such an increase may be linked to the aggressiveness of \nendometriosis. Previous research has suggested that an increase in \ncell number may indicate uncontrolled cell division, and a correlation \nhas been found between the severity of endometriosis and Ki67 ex-\npression levels [33]. In our study, a high-dose A. cepa regimen (com-\nprising 30% A. cepa and 70% standard feed) resulted in a reduction \nof Ki67 expression compared to that observed in the other groups. \nMoreover, elevated Ki67 expression has been more commonly ob-\nserved in infertile women, with a strong correlation between Ki67 \nlevels and postoperative pregnancy. Notably, 66.7% of patients with \nhigh Ki67 expression have been found to achieve pregnancy within \n1 year after endometriosis surgery, underscoring the importance of \nendometrial resection in the treatment of infertility [34]. Our findings \nsuggest that treatment with A. cepa may reduce Ki67 expression in \nlesions, which could be advantageous for infertile patients and avoid \nthe need for surgical intervention during the follow-up period. \nIn conclusion, A. cepa may enhance histological outcomes as re-\nflected by evaluation scores and reduce Ki67 expression when ad-\nministered in high doses. However, no statistically significant change \nwas detected in the levels of profibrotic mediators. A. cepa appears \nto exert a more pronounced effect on the proliferation aspect of en-\ndometriosis rather than on antifibrotic characteristics. \nConflict of interest \nNo potential conflict of interest relevant to this article was report-\ned. \nORCID \nHakan Kula https://orcid.org/0000-0003-1443-5796 \nOrkun Ilgen https://orcid.org/0000-0002-0296-8504  \nSefa Kurt https://orcid.org/0000-0002-5144-0634 \nFiliz Yılmaz https://orcid.org/0000-0003-0505-3905 \nAuthor contributions \nData curation: HK, OI. 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