{"paper_id":"7e1df115-a732-4c4b-8d7d-51432fb14f7f","body_text":"Vol:.(1234567890)\nBratislava Medical Journal (2026) 127:1132–1141\nhttps://doi.org/10.1007/s44411-026-00510-8\nRESEARCH\nGonadotropin‑Induced Molecular Alterations in Experimental \nEndometriosis: Downregulation of Tumor Suppressor Genes \nand Inflammatory Activation\nErol Karakaş1  · Mehmet Dolanbay2 · Mustafa Ermiş3 · Hülya Akgün4 · Arzu Hanım Yay5 · Eda Köseoğlu 5 · \nEnes Karaman2\nReceived: 8 October 2025 / Revised: 20 January 2026 / Accepted: 22 January 2026 / Published online: 2 February 2026 \n© The Author(s) 2026\nAbstract\nBackground Endometriosis is a hormonally responsive inflammatory disease with a recognized association with certain \novarian cancer subtypes. Gonadotropins are widely used in assisted reproductive technologies; however, their direct molecular \neffects on endometriotic tissue remain insufficiently characterized.\nObjective This study aimed to investigate the effects of gonadotropin treatment on molecular markers potentially associated \nwith early carcinogenesis-related processes in endometriotic lesions, using a surgically induced rat model.\nMethods Twenty-two female Wistar Albino rats underwent surgical induction of endometriosis via autologous peritoneal \nimplantation. The animals were randomly assigned to two groups: a control group receiving saline and a treatment group \nreceiving gonadotropin (2 IU/kg/day) for 14 days. Following treatment, endometriotic lesions were excised and analyzed \nhistologically and immunohistochemically for phosphatase and tensin homolog (PTEN), tumor protein 53 (TP53), and \ntumor necrosis factor-alpha (TNF-α) expression. Statistical analyses were performed using the Mann–Whitney U test based \non non-parametric data distribution.\nResults Histopathological evaluation revealed no significant morphological differences between the groups. However, quan-\ntitative immunohistochemical analysis demonstrated that gonadotropin-treated rats exhibited markedly decreased PTEN \n(p < 0.001) and TP53 (p = 0.001) expression, alongside a significant increase in TNF-α expression (p = 0.035) compared with \ncontrols (Table 1,  Figs.  5, 6). These alterations reflect early molecular changes involving tumor suppressor gene downregula-\ntion and pro-inflammatory cytokine upregulation.\nConclusion These findings suggest that gonadotropin exposure may induce early molecular alterations in endometriotic tis-\nsue. Further translational and clinical studies are warranted to validate these findings in human populations.\nKeywords Endometriosis · Gonadotropins · PTEN · TP53 · TNF-α\n1 Introduction\nEndometriosis is a chronic, estrogen-dependent inflamma-\ntory disorder characterized by the presence of endometrial-\nlike tissue outside the uterine cavity [1 –3]. Affecting up to \n15% of women of reproductive age, it represents a major \ncause of chronic pelvic pain, dysmenorrhea, dyspareu -\nnia, and infertility [4 , 5]. Although histologically benign, \nendometriosis exhibits several malignant-like characteris-\ntics—including local invasion, hormone responsiveness, \nand recurrence—and is associated with an increased risk of \ncertain ovarian cancers, particularly the endometrioid and \nclear cell subtypes [6, 7].\n * Erol Karakaş \n erolkarakas@erciyes.edu.tr\n1 Department of Obstetrics and Gynecology, Op. Dr. Erol \nKarakaş Gynecology And Obstetrics Clinic, Alpaslan Mah. \nAşık Veysel Boulevard Road Number: 21/A Melikgazi, \n38140 Kayseri, Turkey\n2 Department of Obstetrics and Gynecology, Faculty \nof Medicine, Erciyes, University, Kayseri, Turkey\n3 Department of Biochemistry, Faculty of Veterinary \nMedicine, Erciyes University, Kayseri, Turkey\n4 Department of Pathology, Faculty of Medicine, Erciyes, \nUniversity, Kayseri, Turkey\n5 Department of Histology and Embryology, Faculty \nof Medicine, Erciyes, University, Kayseri, Turkey\n\n1133Bratislava Medical Journal (2026) 127:1132–1141 \nApproximately one-third of women with endometriosis \nexperience infertility [8 ]. Assisted reproductive technolo-\ngies (ART), such as ovulation induction with gonadotropins, \nare commonly employed to address endometriosis-related \nsubfertility [9 ]. However, growing concern has emerged \nregarding the potential tumorigenic effects of gonadotro-\npins on hormonally sensitive tissues [10–13]. Gonadotropins \nstimulate ovarian follicular growth and elevate circulating \nestrogen levels, potentially exacerbating proliferative and \ninflammatory activity within ectopic endometrial implants \n[14–16]. While epidemiological findings remain inconclu-\nsive, mechanistic evidence elucidating the direct molecular \neffects of gonadotropin exposure on endometriotic tissues \nis notably scarce.\nThis study was designed to determine whether gonado-\ntropin administration induces molecular alterations relevant \nto pathways implicated in malignant transformation in sur -\ngically induced endometriotic lesions in rats. Specifically, \nwe examined the expression patterns of the tumor suppres-\nsor genes phosphatase and tensin homolog (PTEN) and \ntumor protein 53 (TP53), along with the pro-inflammatory \ncytokine tumor necrosis factor-alpha (TNF-α), which are \nkey regulators implicated in tumorigenesis [17– 19]. The \nfindings of this study may provide mechanistic insights into \nthe molecular effects of gonadotropin exposure in endome-\ntriotic tissue and contribute to ongoing discussions regard-\ning treatment safety [20– 22]. These markers were selected \ndue to their established involvement in endometriosis-asso-\nciated carcinogenesis and inflammation-driven malignant \ntransformation.\n2  Materıals and Methods\n2.1  Study Design and Animals\nThis experimental study was conducted at the Experimental \nResearch Center of Erciyes University following approval \nby the Institutional Animal Ethics Committee (Protocol No: \n17/085). Twenty-two female Wistar Albino rats (8 weeks \nold, 180–260 g) were used. The animals were housed indi-\nvidually under standard laboratory conditions (12-h light/\ndark cycle, 22 ± 2 °C, and 55–60% humidity) with ad libitum \naccess to standard chow and water.\n2.2  Induction of Endometriosis\nA surgical endometriosis model was established according \nto the method described by Vernon and Wilson [ 1] and is \nillustrated in Fig. 1. Under general anesthesia, 1 cm segment \nof the left uterine horn was excised, opened longitudinally, \nand sutured onto the peritoneal surface with the endome-\ntrial side facing inward [1 ]. Cefazolin sodium (50 mg/kg) \nwas administered preoperatively and for three consecutive \nFig. 1  Rat Endometriosis \nModel Setup. A schematic \nrepresentation of the surgically \ninduced endometriosis model \nin Wistar-Albino rats. Endo-\nmetrial tissue was excised from \nthe uterine horn and sutured \nonto the peritoneal surface \nunder anesthesia. The model \nsuccessfully simulates ectopic \nendometrial implantation sites \nafter four weeks of recovery\n\n\n1134 Bratislava Medical Journal (2026) 127:1132–1141\ndays postoperatively to prevent infection. After four weeks, \nendometriotic implants were confirmed in all animals. The \nperitoneal autologous transplantation model was selected \nbecause it allows controlled evaluation of early molecular \nchanges in ectopic endometrial tissue without confound -\ning cystic degeneration, making it suitable for mechanistic \nassessment.\n2.3  Experimental Groups\nRats were randomly allocated into two groups (n = 11 per \ngroup):\n• Control group Received subcutaneous physiological \nsaline at an injection volume equivalent to that of the \ntreatment group for 14 days.\n• Gonadotropin group Received subcutaneous recombinant \nfollicle-stimulating hormone (rFSH; Gonal-F®, Merck \nSerono) at a dose of 2 IU/kg/day for 14 days. This for -\nmulation provides FSH activity only. [2].\nThe gonadotropin dose of 2 IU/kg/day was derived from \nbody surface area (BSA) conversion of standard human \nclinical doses (150–225 IU/day) used for controlled ovarian \nstimulation. Based on the Food and Drug Administration \n(FDA)-recommended formula and the method of Reagan-\nShaw et al., the human equivalent dose (HED) was translated \nto the rat model using Km factors (human: 37; rat: 6). This \nconversion yielded an approximate dose of 2 IU/kg/day in \nrats, representing a moderate ovarian stimulation regimen \ncomparable to clinical practice [23].\nCharacteristic endometriotic lesions observed after four \nweeks are shown in Fig.  2. All 22 rats completed the study \nprotocol without mortality or exclusion during the modeling \nor treatment phases. All animals were included in the final \nhistological and immunohistochemical analyses.\n2.4  Tissue Collection and Histopathology\nAt the end of the treatment period, rats were euthanized, and \nendometriotic implants were excised. Tissues were fixed in \n10% neutral buffered formalin, embedded in paraffin, and \nsectioned for hematoxylin and eosin (H&E) and Masson’s \ntrichrome staining. The epithelial integrity of endometrial \nlesions was assessed and scored using standard histopatho-\nlogical criteria.\n2.5  Immunohistochemistry\nImmunohistochemical staining for PTEN, TP53, and TNF-α \nwas performed using the standard avidin–biotin–peroxidase \ncomplex method. The primary antibodies were as follows: \nanti-PTEN (rabbit polyclonal, Abcam, ab32199, 1:200), \nanti-TP53 (mouse monoclonal, Santa Cruz Biotechnol-\nogy, sc-47698, 1:100), and anti-TNF-α (rabbit polyclonal, \nAbcam, ab6671, 1:100). Rat liver served as the positive con-\ntrol for PTEN and TP53, and rat spleen for TNF-α. Negative \ncontrols were prepared by omitting the primary antibody. \nAntigen retrieval, blocking, and antibody incubation steps \nwere followed by diaminobenzidine (DAB) chromogen visu-\nalization and hematoxylin counterstaining. Staining inten-\nsity was quantified using ImageJ software in ten randomly \nselected high-power fields (HPFs) per sample. Thresholds \nFig. 2  Gross view of induced \nendometriotic lesions. Rep-\nresentative gross images of \nectopic endometriotic lesions \nobserved on the peritoneal \nsurface four weeks after surgi-\ncal induction. Images were \nproportionally cropped to main-\ntain consistent magnification \nacross panels. Endometriotic \nlesions appear as well-defined, \nvascularized nodules and are \nindicated by arrows and circles. \nA scale bar is included to dem-\nonstrate lesion size. Comparable \ngross lesion morphology is \nobserved between the control \nand gonadotropin-treated \ngroups\n\n\n1135Bratislava Medical Journal (2026) 127:1132–1141 \nfor positive staining were standardized across all images. \nAll scorers were blinded to treatment allocation. Although \ninter-observer variability was not statistically analyzed, all \nslides were independently evaluated by two histopathologists \nand finalized by consensus.\n2.6  Statistical Analysis\nAll statistical analyses were performed using SPSS ver -\nsion 22. Data normality was assessed using the Shap-\niro–Wilk test. Group comparisons were performed using \nthe Mann–Whitney U test based on non-parametric data \ndistribution. A p -value < 0.05 was considered statistically \nsignificant (Figs.  3, 4, 5, 6, 7, 8, 9).\n3  Results\n3.1  Histological Evaluation\nHistological scoring based on epithelial integrity showed no \nstatistically significant difference between the control and \ngonadotropin groups (p  = 0.127), indicating comparable \nlesion formation across both groups [24]. The distribution \nof histological epithelial integrity scores is shown in Fig.  8. \nAlthough no statistically significant difference was observed, \ngraphical presentation allows clearer visualization of score \nvariability between groups.\nH&E staining demonstrated inflammatory infiltration, \nedema, and capillary dilation in both groups (Fig.  3), while \nMasson’s trichrome staining revealed collagen deposition \nin both the control and gonadotropin-treated groups. To \nobjectively evaluate fibrosis, we quantified the percentage \nof collagen-positive area using *ImageJ* software across \n10 randomly selected high-power fields per lesion, for all \nanimals (n = 11 per group). The median fibrosis area was \n**21.3% (IQR: 17.0–25.7%)** in the control group and \n**18.6% (IQR: 14.2–22.4%)** in the gonadotropin-treated \ngroup, with **no statistically significant difference** \n(p = 0.43, Mann–Whitney U test). These results support the \ninitial histological impression that gonadotropin treatment \ndid not significantly affect fibrotic remodeling of ectopic \nlesions (Fig.  4). Approximate gross lesion size comparison \nis presented in Fig. 9. Lesion dimensions were obtained dur-\ning macroscopic excision and are provided as supportive, \nsemi-quantitative data.\nLesion volume was not quantitatively measured; however, \ngross inspection did not reveal apparent differences between \ngroups.\n3.2  Immunohistochemical Findings\nImmunohistochemical analysis revealed significant differ -\nences in the expression levels of PTEN, TP53, and TNF-α \nbetween the control and gonadotropin-treated groups \n(Table  1). Representative immunohistochemical staining \nControl\nH&E\n*\n*\nx20 x40\nGonadotropin\nx20 x40\n**\n*\n*\n*\nEpithelium\n* Mononuclear\ncell infiltration\nS\nS\nS Stroma\nFig. 3  Histological Features of Ectopic Lesions (H&E Staining). \nRepresentative hematoxylin–eosin (H&E) stained sections of ectopic \nendometrial lesions from the control and gonadotropin-treated \ngroups. Both groups show glandular structures surrounded by stroma, \nwith visible inflammation, stromal edema, and increased capillary \ndensity. Magnification: × 400. Scale bars (100  μm and 200  μm) are \nshown in the images\n\n1136 Bratislava Medical Journal (2026) 127:1132–1141\npatterns of PTEN and TP53 in both groups are shown in \nFig.  6. Quantitative ImageJ-based analysis demonstrated \nthat PTEN and TP53 expression was significantly reduced \nin the gonadotropin-treated group, whereas TNF-α expres-\nsion was significantly increased compared with controls. \nThese findings indicate that gonadotropin administration is \nassociated with downregulation of tumor suppressor proteins \n(PTEN and TP53) and upregulation of the pro-inflammatory \ncytokine TNF-α, suggesting enhanced inflammatory and \nproliferative activity within ectopic endometriotic tissue. \nAll 22 rats completed the experimental protocol without \nmortality or exclusion, and molecular analyses included all \nsamples (n = 11 per group).\n4  Discussion\nInfertility affects approximately one in seven couples world-\nwide, and the increasing reliance on assisted reproductive \ntechnologies (ART) has raised concerns regarding their \nlong-term safety [25, 26]. Among these interventions, gon-\nadotropins are widely used for ovulation induction [27]. \nHowever, accumulating evidence suggests a potential onco-\ngenic risk, particularly in hormonally responsive conditions \nsuch as endometriosis [28–30].\nIn this study, gonadotropin administration in a rat model \nof surgically induced endometriosis resulted in significant \ndownregulation of the tumor suppressor proteins PTEN and \nFig. 4  Quantitative assessment \nof fibrosis in ectopic endome-\ntriotic lesions. Representative \nMasson’s trichrome–stained \nsections of ectopic endometri-\notic lesions from the control and \ngonadotropin-treated groups. \nCollagen fibers are stained blue, \nwhile cytoplasm is stained red. \nImages are shown at × 400 mag-\nnification; scale bar = 100 μm. \nQuantitative analysis of fibrosis \nexpressed as collagen-positive \narea percentage, measured \nusing ImageJ software. Fibrosis \nwas analyzed in 10 randomly \nselected high-power fields \nper animal (n = 11 per group). \nData are presented as box-\nand-whisker plots showing \nmedian and interquartile range. \nNo statistically significant \ndifference in fibrosis area was \nobserved between the control \nand gonadotropin-treated \ngroups (Mann–Whitney U test, \np = 0.43)\n\n\n1137Bratislava Medical Journal (2026) 127:1132–1141 \nTP53, accompanied by increased expression of the pro-\ninflammatory cytokine TNF-α [18– 20]. These molecular \nalterations are consistent with pathways implicated in early \nneoplastic transformation, which have been described in \nassociation with endometriosis-related malignancies.\nPTEN and TP53 play pivotal roles in maintaining \ngenomic stability and regulating apoptosis. Their suppres-\nsion may facilitate uncontrolled cellular proliferation and \nimpair DNA repair, both hallmarks of carcinogenesis. Con-\ncurrently, elevated TNF-α levels indicate an inflammatory \nmicroenvironment that may favor tumor initiation and pro-\ngression. TNF-α is known to promote angiogenesis, immune \nevasion, and epithelial-to-mesenchymal transition (EMT), \nall of which are central to malignant transformation [21–24, \n31–34].\nThese results align with previous reports describing \nsimilar molecular alterations in atypical endometriosis and \nendometriosis-associated ovarian cancer. The present study \nprovides experimental evidence suggesting that exogenous \nhormonal stimulation via gonadotropins may influence \nmolecular pathways that have been implicated in endome-\ntriosis-related carcinogenesis [14, 35, 36].\nA methodological consideration is the use of saline as a \ncontrol, while the gonadotropin formulation may contain \nstabilizing excipients. Future experiments should include a \nvehicle control prepared with the identical solvent composi-\ntion to exclude potential non-specific effects.\nFurthermore, the present study focused exclusively on \nTNF-α as a representative inflammatory cytokine. Expand-\ning future analyses to include IL-6, IL-8, and IL-1β would \nprovide a more comprehensive understanding of the cytokine \nTNF-α\nControlGonadotropin\nFig. 5  TNF-α Immunohistochemical Expression. Immunohistochemi-\ncal staining for TNF-α in ectopic lesions. Gonadotropin-treated rats \nexhibit more intense cytoplasmic TNF-α expression in both epithelial \nand stromal compartments compared to controls. Increased staining \ncorrelates with enhanced inflammatory activity. Magnification: × 400. \nScale bars (100 μm) are shown in the images\nFig. 6  Expression of PTEN \nand TP53 Tumor Suppressor \nProteins. Immunohistochemi-\ncal staining of PTEN and TP53 \nin endometriotic lesions. Both \nmarkers show reduced nuclear \nexpression in the gonadotro-\npin-treated group relative to \ncontrols, indicating suppression \nof tumor suppressor pathways. \nMagnification: × 400. Scale \nbars (100 μm) are shown in the \nimages\nP53\nControlGonadotropin\nPTEN\n\n1138 Bratislava Medical Journal (2026) 127:1132–1141\nnetwork implicated in endometriosis-associated inflamma-\ntion and malignancy.\nAs illustrated schematically in Fig.  7, gonadotropin \nstimulation may act through receptor-mediated pathways, \npotentially involving activation of the PI3K/AKT and NF-κB \nsignaling cascades, which may contribute to downregulation \nof PTEN and TP53 and upregulation of TNF-α in endometri-\notic tissue. Although the underlying signaling mechanisms \nwere not directly examined, it is biologically plausible that \ngonadotropin stimulation acts through receptor-mediated \npathways. Follicle-stimulating hormone (FSH) and luteiniz-\ning hormone (LH) bind to their respective G protein–coupled \nreceptors (FSHR and LHR), activating downstream cascades \nsuch as the cyclic AMP/protein kinase A (cAMP–PKA) and \nphosphoinositide 3-kinase/protein kinase B (PI3K–AKT) \npathways [37–40]. Activation of PI3K–AKT signaling has \nbeen shown to suppress PTEN activity, which may explain \nthe reduced PTEN expression observed in the present study \n[41]. Additionally, TP53 function may be indirectly modu-\nlated through these pathways, thereby contributing to a pro-\nproliferative cellular milieu [42].\nGonadotropin-induced inflammation may also involve \nthe nuclear factor kappa B (NF-κB) pathway, a key regu-\nlator of inflammatory gene expression including TNF-α \n[43]. NF-κB activation in response to hormonal stimula-\ntion has been demonstrated in endometrial and ovarian \ntissues [44], with downstream effects on cytokine secre-\ntion, angiogenesis, immune modulation, and EMT—all \nhallmarks of early oncogenesis [45, 46]. These mecha-\nnistic hypotheses are consistent with the molecular pro-\nfile observed in our gonadotropin-treated group—spe-\ncifically, concurrent suppression of PTEN and TP53 and \nFig. 7  Hypothetical schematic representation of potential molecular \npathways affected by gonadotropin treatment in endometriotic tissue. \nThis schematic illustrates a hypothetical, literature-based framework \nsummarizing potential signaling pathways that may be involved in \ngonadotropin-associated molecular alterations in endometriotic tis-\nsue. Gonadotropin stimulation may activate the follicle-stimulating \nhormone receptor (FSHR), potentially leading to activation of the \nPI3K/AKT signaling pathway and reduced expression of the tumor \nsuppressor proteins PTEN and TP53. In parallel, gonadotropin signal-\ning may contribute to activation of the NF-κB pathway, resulting in \nincreased expression of the pro-inflammatory cytokine TNF-α. This \ndiagram is intended for conceptual purposes only and does not repre-\nsent a mechanistic pathway directly demonstrated by the experimental \ndata presented in this study\nFig. 8  Distribution of histological epithelial integrity scores in con-\ntrol and gonadotropin-treated groups presented as box-and-whisker \nplots (median and interquartile range)\nFig. 9  Approximate gross lesion size comparison between groups \nbased on macroscopic measurements obtained during tissue excision. \nMeasurements are presented as semi-quantitative values due to tech-\nnical limitations\n\n1139Bratislava Medical Journal (2026) 127:1132–1141 \nupregulation of TNF-α. Future studies incorporating \ngene expression profiling, pathway-specific inhibitors, \nand longer observation periods are warranted to further \nelucidate these signaling relationships.\nFrom a translational perspective, these findings under -\nscore the need for further investigation into the molecu-\nlar effects of gonadotropin exposure before any defini-\ntive clinical conclusions can be drawn. Considering the \nestablished association between PTEN and TP53 altera-\ntions and malignant transformation, the potential clinical \nimplications of these molecular findings warrant further \ninvestigation in well-designed human studies before any \npreventive strategies can be recommended [36, 47]. Addi-\ntional clinical studies are required to determine whether \nthese molecular alterations translate into increased cancer \nincidence among patients undergoing ovulation induction \n[48–51]. Until such data become available, individualized \ntreatment planning and careful risk–benefit assessment \nare essential, particularly for women with severe, recur -\nrent, or long-standing endometriosis [12, 18].\nAlthough fibrosis was initially evaluated qualitatively, \nsubsequent quantitative analysis using *ImageJ* con-\nfirmed that gonadotropin exposure did not significantly \nalter collagen deposition in ectopic endometriotic lesions. \nThis suggests that the molecular alterations induced \nby gonadotropin treatment (e.g., TNF-α upregulation, \nPTEN and TP53 downregulation) occur independently \nof changes in fibrotic tissue remodeling within the short \nexperimental window. The absence of significant histo-\npathological changes despite marked molecular altera-\ntions may be attributable to the relatively short exposure \nperiod. Alternatively, gonadotropin-induced stress signal -\ning could provoke transient inflammatory or pro-neoplas-\ntic molecular events preceding detectable structural alter -\nations. These possibilities warrant further investigation.\n4.1  Limitations\nThis study has several limitations. The relatively short \ntreatment and observation period (14 days) may explain the \nabsence of overt morphological changes despite significant \nmolecular alterations. The sample size was relatively small, \nwhich may limit the statistical power and generalizability of \nthe findings. Functional assays such as Ki-67 or Caspase-3 \nimmunostaining were not performed, and molecular analy -\nsis was limited to three markers: PTEN, TP53, and TNF-α. \nBroader cytokine panels (e.g., IL-6, IL-8, IL-1β), transcrip-\ntomic analyses, and extended follow-up durations should be \nconsidered in future studies. Additionally, the eutopic endo-\nmetrium was not evaluated; thus, systemic effects of gon-\nadotropins cannot be excluded. The findings are restricted \nto ectopic lesions. The absence of a positive control group \nand a vehicle control group using the same excipients as \nthe gonadotropin formulation may also limit the ability to \ndistinguish specific hormonal effects from potential solvent-\nrelated influences.\n5  Conclusion\nThis study provides experimental evidence that gonadotro-\npin administration is associated with early molecular alter -\nations in surgically induced endometriotic tissue. Specifi-\ncally, decreased expression of PTEN and TP53, along with \nincreased expression of the pro-inflammatory cytokine TNF-\nα, was observed in ectopic lesions in rats. These molecular \nchanges may reflect a shift toward a pro-inflammatory and \npro-proliferative microenvironment in hormonally respon-\nsive tissues.\nWhile these findings offer mechanistic insights into \nthe potential effects of gonadotropins on endometriotic \nTable 1  Semi-quantitative \nhistological scores and \nImageJ-based quantitative \nimmunohistochemical \nmeasurements of endometriotic \ntissues\nData are presented as median (interquartile range, IQR). Histological epithelial integrity was evaluated \nusing a semi-quantitative scoring system on a 0–3 scale. PTEN, TP53, and TNF-α values represent ImageJ-\nderived quantitative measurements of staining intensity and/or positive staining area obtained from digital \nimage analysis. For each marker, multiple microscopic high-power fields were analyzed per lesion. n indi-\ncates the total number of microscopic fields evaluated. Statistical comparisons were performed using the \nMann–Whitney U test. p < 0.05 was considered statistically significant. Superscript letters (a, b) indicate \nstatistically significant differences between groups\nControl Gonadotropin p\nSCORE (Histological epithelial \nintegrity score)\n(n = 30)\n1.00 (0.00–2.00)a 2.00 (1.00–2.25)a 0.127\nPTEN (n = 60) 77.69 (70.30–88.38)a 64.89 (60.60–73.16)b 0.001*\nTP53 (n = 60) 63.20 (59.01–69.80)a 57.13 (55.48–60.58)b 0.001*\nTNF-α (n = 60) 87.79 (76.67–103.54)a 95.13 (85.79–101.31)b 0.035*\n\n1140 Bratislava Medical Journal (2026) 127:1132–1141\nlesions, they are based on a short-term experimental model \nand should be interpreted with caution. No morphological \nevidence of malignant transformation was observed, and \nthe current results do not establish a causal link to cancer \ndevelopment.\nTherefore, the results of this study should be considered \npreliminary and hypothesis-generating. Further experimen-\ntal studies with longer observation periods, broader molecu-\nlar profiling, and eventual validation in human models are \nwarranted to better understand the long-term implications \nof gonadotropin exposure in the context of endometriosis.\n5.1  Translational Perspective\nThis experimental study suggests that gonadotropin expo-\nsure may be associated with early molecular alterations \nin ectopic endometriotic tissue, including reduced PTEN \nand TP53 expression and increased TNF-α. These findings \noffer mechanistic insights into potential pathways relevant \nto endometriosis-associated carcinogenesis. However, as \nthey are derived from a short-term animal model, they do \nnot provide direct evidence of cancer risk in humans.\nFurther research is needed to explore the molecular \neffects of gonadotropins in endometriosis using long-term \nexperimental designs and human studies. Until such data \nbecome available, these results should be interpreted as \npreliminary and hypothesis-generating.\nAcknowledgements This study was supported by the Scientific \nResearch Projects Coordination Unit of Erciyes University (Project No: \nTTU-2021-11050). The funding period has concluded, and no active \nfinancial support is currently available.\nAuthors Contribution E.K. (Erol Karakaş): Supervision, Methodology, \nData Curation, Writing—Original Draft, Writing—Review & Editing. \nM.D.: Data Curation, Funding Acquisition, Formal Analysis, Writ-\ning—Original Draft Preparation. M.E.: Investigation, Data Curation. \nH.A.: Writing—Original Draft Preparation, Formal Analysis. E.K. \n(Enes Karaman): Investigation, Data Curation, Writing. A.H.Y. and \nE.K. (Eda Köseoğlu): Funding Acquisition, Formal Analysis, Supervi-\nsion, Validation.\nFunding Open access funding provided by the Scientific and Techno-\nlogical Research Council of Türkiye (TÜBİTAK). This research was \nsupported by the Scientific Research Projects Coordination Unit of \nErciyes University (Project No: TTU-2021-11050). The funding period \nhas concluded, and no active financial support is currently available.\nData Availability The datasets generated and/or analyzed during the \ncurrent study are available from the corresponding author upon rea-\nsonable request.\nDeclarations \nEthics Approval and Consent to Participate All experimental proce-\ndures were approved by the Ethics Committee of Erciyes University \n(Approval No: 17/085). All methods were conducted in accordance \nwith relevant institutional and international guidelines and regulations.\nPatient Consent for Publication  Not applicable. This study did not \ninvolve human participants.\nCompeting interests The authors declare that they have no competing \ninterests.\nOpen Access This article is licensed under a Creative Commons Attri-\nbution 4.0 International License, which permits use, sharing, adapta-\ntion, distribution and reproduction in any medium or format, as long \nas you give appropriate credit to the original author(s) and the source, \nprovide a link to the Creative Commons licence, and indicate if changes \nwere made. The images or other third party material in this article are \nincluded in the article’s Creative Commons licence, unless indicated \notherwise in a credit line to the material. If material is not included in \nthe article’s Creative Commons licence and your intended use is not \npermitted by statutory regulation or exceeds the permitted use, you will \nneed to obtain permission directly from the copyright holder. 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