{"paper_id":"ec98f17b-1a3b-41cb-9e5a-08cf2452a7ab","body_text":"C57BL/6J mice best recapitulate \nfibrosis and inflammatory \npathophysiology in syngeneic \nmouse model of endometriosis\nMegha Anchan1,8, Atharvaraj Hande1,8, Samruddhi Deshpande1, Richa Patel1, \nGuruprasad Kalthur1, Jahnavy Madhukar Joshi2, Ratul Datta3, Swar Shah4, Kriti Sharma5, \nHiral Pandya6 & Rahul Dutta1,7\nEndometriosis (ENDO), a chronic inflammatory disease affecting approximately 190 million women \nglobally, is characterized by fibrosis, a feature often challenging to replicate in murine models. To \nidentify an optimal syngeneic model exhibiting robust fibrosis and inflammation, we evaluated three \ninbred mouse strains: C57BL/6J (n = 27), BALB/c (n = 24), and Swiss albino (n = 27). Uterine fragments \nfrom donor mice were intraperitoneally transplanted into recipient mice (1 donor: 2 recipients) \nusing an established protocol with minor modifications. All ENDO-induced mice displayed reduced \nburrowing and exploratory behaviors, alongside increased mechanical hyperalgesia, indicative of \nENDO-associated discomfort. Peritoneal fluid analysis revealed a pro-inflammatory environment with \na tendency towards an M2 macrophage-dominant profile across all strains. Histological examination \nconfirmed endometriotic lesions with proliferating epithelium (Ki-67+), neovascularization (CD31+), \nand macrophage infiltration (F4/80+). Notably, C57BL/6J mice exhibited the highest ENDO incidence \nand a significantly pronounced fibrotic response, evidenced by increased stromal collagen deposition \nand elevated Col1A1, cytokeratin, α-Smooth Muscle Actin (α-SMA), and Nestin expression. Molecular \nanalysis in C57BL/6J mice further supported epithelial-mesenchymal transition (EMT)-driven fibrosis, \nwith decreased E-cadherin and increased N-cadherin and S100A4 mRNA levels, corroborated by \ncorresponding protein changes (cytokeratin, vimentin, snail). Our findings establish the C57BL/6J \nstrain as the most suitable syngeneic model for ENDO, consistently recapitulating the inflammatory \nand fibrotic pathophysiology observed in human disease, particularly its fibrotic component.\nKeywords Endometriosis, Animal model, Syngeneic, C57BL/6J, Fibrosis, EMT\nEndometriosis (ENDO) is a prevalent, chronic, estrogen-dependent gynaecological disorder affecting \napproximately 6–10% (~ 190 million) of reproductive-aged women globally 1. It is marked by the presence and \nproliferation of viable ectopic endometrial-like tissue outside the uterus, commonly on the pelvic peritoneum or \novaries2. Often simplified as solely dysmenorrhea3. ENDO is an etiologically complex, multifaceted inflammatory \ncondition. It is characterized by dysregulated cell proliferation, impaired hormonal signaling, chronic \ninflammation, immunological dysregulation, angiogenesis, neurogenic inflammation, and notably, epithelial-\nto-mesenchymal transition (EMT)-induced tissue remodeling and fibrosis 4,5. ENDO is a major contributor \nto debilitating chronic pelvic pain (CPP) and infertility. The often underestimated distress of ENDO extends \nbeyond pain, significantly impacting well-being and productivity 6. Laparoscopically, lesions are classified by \ndistribution, showing significant heterogeneity 7. Histological diagnosis requires at least two of: endometrial \nepithelium, glands, stroma, nerve fibers/blood vessels, hemosiderin-laden macrophages, or fibrosis7. Despite its \n1Division of Reproductive Biology, Department of Reproductive Science, Kasturba Medical College, Manipal \nAcademy of Higher Education, Manipal, Karnataka 576104, India. 2Manipal Centre for Biotherapeutics Research, \nManipal Academy of Higher Education, Manipal, Karnataka 576104, India. 3Nova IVF Fertility, Pantaloons Building, \nSix Mile, Guwahati, Assam 781036, India. 4Krishna Nursing Home, 4-D, near Delux Bus Stand, Tashkand Society, \nNizampura, Vadodara, Gujarat 390002, India. 5Harsh Hospital & Maternity Home, Surat, Gujarat 395017, India. \n6Sevashram Hospital, Bharuch, Gujarat 392001, India. 7Center for Animal Research, Ethics & Training, Manipal \nAcademy of Higher Education, Manipal, Karnataka, India. 8Megha Anchan and Atharvaraj Hande contributed \nequally to this work. email: rahul.dutta@manipal.edu\nOPEN\nScientific Reports |        (2025) 15:29024 1| https://doi.org/10.1038/s41598-025-13900-9\nwww.nature.com/scientificreports\n\n\ncommonality, ENDO’s pathogenesis remains unclear, and therapeutic options are limited, emphasizing the need \nfor research8.\nJohn Sampson’s 1927 “retrograde menstruation” theory is the most cited explanation for ectopic tissue \nimplantation9,10. However, only a subset of women experiencing retrograde menstruation develop ENDO. This \nsuggests additional mechanisms, including immune evasion, implantation, proliferation, peritoneal invasion, and \nneovascularization11,12. The inflammatory microenvironment, rich in alternatively activated macrophages and \nneutrophils, cytokines, chemokines, and growth factors, is increasingly recognized as a key driver of ENDO 13. \nNotably, fibrosis, once viewed as secondary, is now considered a defining characteristic of ENDO 14–17with \ngrowing support for its inclusion in diagnostic criteria18,19. The inflammatory milieu in ENDO is closely linked \nto fibrosis, as indicated by altered immune cell profiles and cytokine levels in peritoneal fluid20. Thus, ENDO can \nbe considered both an inflammatory and a fibrotic disease14. However, the interplay between inflammation and \nthe development of fibrotic endometriotic implants remains a critical research gap21.\nDeveloping effective ENDO therapies necessitates evaluation in animal models that accurately mimic the \nhuman disease. However, spontaneous ENDO is limited to menstruating species, with practical limitations 22. \nMurine ENDO models are widely used due to their advantages 23  but rodents do not menstruate, making \nretrograde menstruation an unlikely mechanism 24. Consequently, models involve surgical transplantation of \nuterine tissue or human endometriotic tissue into mice 25–28  often with estrogen supplementation26,29. While \nhuman tissue transplantation has benefits, it faces immune rejection issues 30. Surgical models create lesions \nresembling clinical ENDO but can disrupt peritoneal immunity26. Intraperitoneal injection is also used but may \nnot fully replicate human lesions31,32.\nDespite advancements in developing rodent models of ENDO, their ability to accurately replicate the \nprogressive fibrosis characteristic of chronic human ENDO remains a significant limitation. Existing models \noften emphasize the initial formation of lesions and the accompanying immunological responses, largely \nneglecting the gradual and ongoing progression of fibrosis. This lack of a reliable model that recapitulates the \nfibrotic progression hinders the investigation of the underlying mechanisms of fibrogenesis and the preclinical \nevaluation of potential anti-fibrotic therapies. A key challenge lies in the insufficient representation of the \nprogressive fibrotic process observed in human ENDO lesions. Furthermore, current models often provide an \ninadequate evaluation of key myofibroblast markers such as α-SMA, Collagen I, and Nestin, which are crucial \nfor understanding fibrosis. Comprehensive data on ECM remodeling, with collagen deposition and tissue \nstiffness in rodents mirroring human fibrotic alterations, are absent. Finally, an over-reliance on in vitro model \nsystems may not fully capture the complex cellular interactions and microenvironmental cues driving fibrosis \nin vivo. These shortcomings underscore the pressing need for an optimized mouse model that comprehensively \nreplicates the progressive fibrotic aspects of human ENDO.\nTherefore, our study aims to establish and validate an experimental fibrotic syngeneic mouse model of \nENDO by comparing C57BL/6J, BALB/c, and Swiss albino strains. We seek to identify the strain that most \naccurately replicates the inflammatory and fibrotic pathophysiology of ENDO by thoroughly characterizing \nthe morphological, histological, and functional features of the generated model. This research provides a \ncomprehensive framework for assessing inflammation and fibrosis in ENDO. We aim to select the optimal \nmouse strain for accurate disease modeling, addressing significant discrepancies between experimental models \nand clinical pathology, particularly concerning fibrosis.\n Methodology\nAll methods were carried out in accordance with relevant guidelines and regulations. All procedures were \nperformed as described below.\nEthical approvals\nHuman sample experiments\nThis study involving human samples was approved by the Institutional Ethics Committee (IEC1: 94/2022), \nKasturba Medical College and Kasturba Hospital, Manipal, adhering to the Helsinki Declaration of 1964 and \nits later amendments. Written informed consent was obtained from all patients. Lesions were collected through \nlaparoscopic excision. While patient hormone status was not a primary focus of this study, standard clinical \nprotocols were followed for all sample collection.\nAnimal experiments\nThe Institutional Animal Ethics Committee at Kasturba Medical College, Manipal, approved the use of animals \n(Approval Number: C57BL/6J- IAEC/KMC/88/2024, BALB/c- IAEC/KMC/45/2022, Swiss albino- IAEC/\nKMC/56/2022). Institutional guidelines and the guidelines of the Committee for the Purpose of Control and \nSupervision of Experiments on Animals (CPCSEA)were strictly followed for animal handling, and the reporting \nof animal experiments follows ARRIVE (Animal Research: Reporting of In vivo Experiments) guidelines. The \nstudy utilized adult inbred female mice (8–10 weeks, 23 ± 2  g) of three distinct strains: C57BL/6J, BALB/c, \nand Swiss albino. All the animals were procured from the Central Animal Research Facility, Manipal Academy \nof Higher Education. Mice were housed (6 per cage) in an environment-controlled setting (21 ± 2  °C, 50–\n55% humidity, 12–12 h light-dark cycle) at the Central Animal Research Facility, Manipal Academy of Higher \nEducation, with ad libitum access to water and food.\nPreparation of donor and recipient mice\nA total of C57BL/6J (n = 27), BALB/c (n = 24), and Swiss albino (n = 27) mice were used for ENDO induction. \nFor each strain, recipient mice were assigned as follows: C57BL/6J (n = 14), BALB/c (n = 12), and Swiss albino (n \n= 14). Six control mice were included for each strain. The experimental protocol was adapted from a previously \nScientific Reports |        (2025) 15:29024 2| https://doi.org/10.1038/s41598-025-13900-9\nwww.nature.com/scientificreports/\n\npublished method33 with minor modifications. Syngeneic female donor and recipient mice of the three strains \nwere allowed to acclimate for 5 days before the experimental procedures. During this acclimation period, the \nestrous cycle stages of each animal were monitored using the vaginal lavage method.\nDevelopment of a syngeneic mouse model of endometriosis\nFollowing confirmation of consistent estrous cycling, donor mice were primed with subcutaneous injections of \nestradiol benzoate (EB) (TCI chemicals, #E0329) (3 µg/mouse) for seven consecutive days to synchronize their \nestrous cycles and promote endometrial development. After priming, donor mice were euthanized, and their \neutopic uterine horns were harvested. Excess fat and debris were carefully removed, and the uterine horns were \nrinsed with cold, sterile 1x Phosphate-Buffered Saline (PBS) containing penicillin (100 U/mL) and streptomycin \n(100  mg/mL) (Pen/strep) (ThermoFisher Scientific #15140122). The uterine tissue was then meticulously \nminced into small cell aggregation suspensions of uterine fragments (UFs < 0.1 mm) containing both eutopic \nendometrium and uterine muscle. These UF suspensions were divided into two equal portions, resuspended in \n0.5 mL of PBS in a 1 mL syringe (Dispovan), and randomly injected I/P into recipient mice using an 18-gauge \nneedle (0.5 mL per recipient). Thus, each recipient mouse received endometrial tissue from half of a donor \nuterus. Control mice received subcutaneous injections of estradiol benzoate and I/P injections of 0.5 mL of \nsterile 1x PBS(without pen/strep). No signs of distress or unusual pain behaviors were observed post-injection. \nRecipient animals received a single dose of EB before UF injection to synchronize their estrous cycles. Both the \nrecipients and control animals were subsequently administered EB every two days until sacrifice to maintain \nuniform circulating estrogen levels (as depicted in Fig. 1).\nBehavioral assessment of ENDO mice\nBehavioral assays were conducted to assess pain-like behavior and anxiety levels in ENDO-induced mice. \nAll measurements were performed by the same investigator in a blinded manner to ensure consistency and \nminimize bias. After each test, mice were returned to their home cages.\nBurrowing assay (assessment of spontaneous behavior)\nThe burrowing assay, standardized by Deacon with minor modifications 34 was performed between 16:00 and \n18:00 h (3 h before the dark cycle). Control and recipient mice were individually housed in cages equipped with \na burrow setup. Mice were initially acclimatized to the burrow tube (20 cm × 6 cm) filled with 200 g of chow \ndiet for 30 min without food in the cage hopper. Water was provided ad libitum. The test was initiated after \nacclimatization to the laboratory setting with the burrow setup for at least one hour. Each mouse was placed in \nits cage-burrow setup for two hours. Subsequently, the amount of feed burrowed was measured by weighing the \ndisplaced amount in the morning.\nFig. 1. Experimental design and timeline of ENDO induction and behavioral assessments. UH from a single \ndonor mouse were processed into small fragments (~ 1 mm) and equally distributed via I/P injection into \ntwo recipient mice to induce endometriosis. Behavioral assessments for pain (burrowing, von Frey) and \nexploratory behavior (OFT) were conducted between days 16–18 post-induction. Subsequent tissue collection \nfor ELISA, flow cytometry (FC), histology (H&E), Masson’s trichrome staining (MTS), immunohistochemistry \n(IHC), qRT-PCR, and Western blot (WB) was performed. (Created in part with BioRender.com).\n \nScientific Reports |        (2025) 15:29024 3| https://doi.org/10.1038/s41598-025-13900-9\nwww.nature.com/scientificreports/\n\nElectronic von Frey reflex (EvF) (assessment of mechanical hyperalgesia)\nMechanical hyperalgesia was assessed using an electronic von Frey filament approach (Dynamic Plantar \nAesthesiometer, Ugo Basile) based on methodologies described by Barrot and Gregory 35,36. Mice were \nindividually placed in Plexiglas chambers on a wire mesh floor and allowed to acclimate for 1 h until exploratory \nbehavior ceased. A calibrated von Frey filament touch probe (Ugo Basile) applied a gradually increasing force (up \nto 10 g) to the abdomen. A definite withdrawal response (abdominal retraction, licking, or flinching), along with \nthe corresponding filament force (grams) and duration (seconds), was recorded as the withdrawal threshold. An \ninterval of at least 30 min was maintained between applications.\nOpen field test (OFT) (assessment of exploratory behavior and anxiety)\nExploratory behavior and anxiety levels were assessed 9 days post-UF injection using the OFT 37. Control and \nENDO mice were individually placed in the center of a transparent Plexiglas box (50 cm × 50 cm × 40 cm) \nwith a clean floor. The box was virtually divided into central and peripheral zones. Each mouse was placed in a \ncorner and allowed to explore for 15 min. The number of entries into the central and peripheral zones, as well as \nthe time spent in them, were recorded using a video-tracking system (Logitech HD C930e webcam). Increased \nanxiety is indicated by more time spent in the peripheral zones and fewer entries into the central zone. The data \nwere analyzed via ANY-maze 64-bit version 7.48 software.\nCollection and processing of biological samples\nOn day 12 post-induction, all recipient mice were euthanized by cervical dislocation, and blood, peritoneal fluid \n(PF), and suspected ENDO-like lesions were collected.\nBlood and Serum: Blood was collected via cardiac puncture under Ketamine + Xylazine cocktail (0.1mL/20 g \nmouse wt. IP) anesthesia. Serum was separated by centrifugation at 3000 × g for 10 min at 4 °C and stored at \n−80 °C for ELISA-based cytokine analysis (IL-6, TNF-α, TGF-β, and Estrogen).\nPeritoneal fluid (PF)\nThe PF was collected from the control and the recipient mice in individual tubes collected by peritoneal lavage \nwith 2 mL of sterile 1x PBS. The fluid was treated with RBC lysis buffer(eBioscience™ 1X RBC Lysis Buffer \nCatalog number 00-4333-57) for 15 min and centrifuged to eliminate erythrocytes. The cells were subsequently \nresuspended in sterile 1x PBS for flow cytometry analysis of M1 and M2 macrophage populations.\nEctopic lesions\nEctopic lesions were photographed for documentation using smart phone camera. Selected lesions were either \nfixed in Bouin’s solution for 24 h, then transferred to 70% ethanol and embedded in paraffin for histological \nand immunohistochemical (IHC) analyses, or flash-frozen in liquid nitrogen and stored at −80  °C for RNA \n(E-cadherin, N-cadherin, and S100A4) and protein (Cytokeratin, Snail, and Vimentin) analyses. Recipient mice \nwithout visible lesions were excluded from further investigation.\nValidation of the syngeneic mouse model of ENDO\nModel success rate\nThe overall success rate for each strain was calculated as the percentage of recipients with confirmed ectopic \nlesions. This was done by evaluating recipient mice for ENDO-like lesions based on gross morphology, \nhistological analysis (H&E staining), and the presence of characteristic IHC markers such as Ki67 (proliferation), \nCD31 (neovascularization), and F4/80 (macrophages).\nEstrogen ELISA\nSerum estradiol (E2) levels were quantified using a commercially available ELISA kit (ELK Biotechnology CO., \nLTD, #ELK8407) following the manufacturer’s instructions. Estradiol concentrations (pg/mL) were determined \nby comparing sample absorbance (450 nm) to a standard curve generated with serially diluted estradiol standards, \nanalyzed in duplicate using a microplate reader (MultiSkan FC Microplate Photometer with SkanIt software).\nFlow cytometry analysis of peritoneal fluid (PF)\nPF cells were collected and treated with RBC lysis buffer as described earlier. After washing, 1 × 10 6 cells were \nincubated with fluorophore-conjugated antibodies: anti-MO-CD11b-Alexa Fluor 488, anti-MO-CD86-APC, \nand anti-MO-CD206-PE (all from eBioscience). After incubation, cells were washed and resuspended in PBS. \nFlow cytometry analysis was performed using a BD Accuri™ C6 Plus flow cytometer, and data were analyzed \nusing FlowJo software to determine the percentage of positive cells for each marker.\nCharacterization of ectopic lesions\nHematoxylin and eosin (H&E) staining\nEctopic ENDO lesions and control eutopic endometrium were fixed, embedded in paraffin, and sectioned \n(4  μm). Sections were deparaffinized, rehydrated, stained with hematoxylin (Sigma-Aldrich, #HX03021349) \nand eosin (Sigma-Aldrich, #1.15935), and mounted with DPX mountant (Sisco Research Laboratories Pvt Ltd, \n# 88147). Slides were examined using a bright-field microscope (Nikon Eclipse Ei 4  W), and representative \nimages were captured. Histological assessment confirmed the presence of epithelial glands and stromal cells as \npreviously described38. Samples not exhibiting endometrial morphology were excluded.\nScientific Reports |        (2025) 15:29024 4| https://doi.org/10.1038/s41598-025-13900-9\nwww.nature.com/scientificreports/\n\nImmunohistochemistry (IHC)\nParaffin-embedded ectopic lesions from all three mouse strains were immunostained with primary antibodies \nagainst Ki67 (proliferation), CD31 (blood vessels), and F4/80 (macrophages) (all from ThermoFisher Scientific). \nBriefly, sections were deparaffinized, rehydrated, and antigens were retrieved using sodium citrate buffer \n(Sigma-Aldrich, Missouri, USA #C7254). Sections were permeabilized with bovine serum albumin (BSA) \n(HiMedia, India #MB083) in Triton X-100 (Sisco Research Laboratories Pvt Ltd #2024271), and blocked with \n5% goat serum (Genei, #163018010A) before overnight incubation with primary antibodies at 4 °C, followed \nby incubation with appropriate HRP-conjugated secondary antibodies Immunoreactive signals were visualized \nusing 3,3′-Diaminobenzidine (DAB) (Sigmafast, Sigma-Aldrich, #D4293) (Sigma-Aldrich), and sections were \ncounterstained with hematoxylin (Sigma-Aldrich, #HX03021349). Slides were mounted with DPX mountant, \nand representative images were captured using a Nikon microscope (Nikon Eclipse Ei 4 W , Nikon, Tokyo, Japan) \nand analyzed using ImageJ software. Antibody details are provided in Table 1.\nEvaluation of fibrotic phenotype\nQuantification of collagen deposition by Masson-Trichrome staining (MTS)\nCollagen deposition in ectopic lesions was assessed using MTS39. Sections were fixed, embedded, deparaffinized, \nand rehydrated. They were then sequentially stained with hematoxylin (Sigma-Aldrich #HX03021349), Biebrich \nscarlet-acid fuchsin solution(Loba chemie Pvt Ltd, #3855D), phosphomolybdic-phosphotungstic acid(Loba \nchemie Pvt Ltd, #05265)for 10–15  min, and, and aniline blue (Sisco Research Laboratories Pvt. Ltd.). After \ndehydration and mounting, slides were examined under a light microscope. The area of collagen deposition \n(blue staining) was quantified as a proportion of the total ectopic lesion area using ImageJ software.\nPrimary antibodies\nTarget Primary antibody Species raised in\nDilution used\n(IHC/WB) Manufacturer & catalog number RRID\n Proliferating cells (Ki67) Anti-Ki67 (Monoclonal) Rat 1:100\nIHC ThermoFisher Scientific, #14-5698-82 AB_10854564\n Blood vessels (CD31/PECAM) Anti-CD31 (Monoclonal) Rabbit 1:100\nIHC ThermoFisher Scientific, #14-0311-81 AB_467201\n Macrophages (F4/80) Anti-F4/80 (Monoclonal) Rat 1:50\nIHC ThermoFisher Scientific, #14-4801-82 AB_467558\n Pan-keratin (C11) mouse mAb Pan-keratin (Monoclonal) Mouse\n1:500\nIHC\n1:1000\nWB\nCell Signaling\nTECHNOLOGIES #4545 -\n αSMA Anti- αSMA (Monoclonal) Mouse 1:200\nIHC ThermoFisher Scientific, #14-976080 AB_2572996\n Nestin Anti- αSMA\n(Monoclonal) Mouse 1:200\nIHC ThermoFisher Scientific, #14-584380 AB_1907436\n Vimentin rabbit pAb Vimentin (Polyclonal) Rabbit 1:1000\nWB\nABclonal\n#A11952 AB_2861643\n Snail rabbit pAb Snail (Polyclonal) Rabbit 1:1000\nWB\nABclonal\n#A5243 AB_2766076\nSecondary antibodies for DAB IHC\nTarget Secondary antibody Species raised in Dilution used Manufacturer & catalog number RRID\nRat IgG (H + L) Goat anti-rat IgG (H + L) \n(HRP-conjugated) Goat 1:1000\nIHC ThermoFisher Scientific, #A18865 AB_2535642\nMouse\nIgG Fc\nGoat anti-Mouse IgG Fc \nSecondary Antibody, HRP Goat\n1:1000\nIHC\n1:10000\nWB\nThermoFisher Scientific, #A16084 AB_2534758\nRabbit IgG Fc Goat anti-Rabbit IgG Fc \nSecondary Antibody, HRP Goat\n1:1000\nIHC\n1:10000\nWB\nThermoFisher Scientific, #A16116 AB_2534789\nAntibodies used for flow cytometry\nTarget Primary antibody Species raised in Dilution used Manufacturer & catalog number RRID\nCD 11b\nMouse\nAnti-Mo-CD 11b-Alexa \nflour 488\nMonoclonal\nRat 0.5 µg/test eBioscience\n# 53-0112-80 AB_469901\nCD 86\nMouse\nCD86 (B7-2) monoclonal \nantibody (GL1), APC-\neFluor™ 780\nRat 0.06 µg/test eBioscience\n# 17-0862-81 AB_469418\nCD 206\nMouse\nAnti-Mo-Cd206 (MMR) \nMonoclonal Antibody \n(MR6F3), PE\nRat 0.125 µg/test eBioscience\n# 12-2061-80 AB_2637422\nCD 68\nMouse\nAnti-Mo-CD68 (FA-11), \nPE Rat 0.25 μg/test eBioscience\n#12-0681-80 AB_2572569\nTable 1. Antibodies used in the study primary antibodies.\n \nScientific Reports |        (2025) 15:29024 5| https://doi.org/10.1038/s41598-025-13900-9\nwww.nature.com/scientificreports/\n\nCOL1A1 ELISA\nThe levels of Collagen Type I (COL1A1) in ectopic tissue lysates were quantified using a commercially available \nELISA kit (Krishgen Biosystems, USA, #111111111). Lesions were homogenized in 1x PBS, centrifuged at 12,000 \n× g for 20 min at 4 °C, and the supernatant was collected. ELISA was performed according to the manufacturer’s \ninstructions using 50 µL of lysate per sample, analyzed in duplicate. Optical density was measured at 450 nm, \nand COL1A1 concentrations were calculated using a standard curve.\nQuantification of iron deposition by prussian blue staining\nIron deposits in ectopic lesions were identified using Prussian blue staining based on Perls’ reaction40. Sections \nwere treated with a freshly prepared mixture of 5% potassium ferrocyanide (Sigma Aldrich #244023) and \nhydrochloric acid (v/v), counterstained with nuclear fast red41 dehydrated, and mounted. Images were captured \nusing a Nikon microscope, and the area of iron deposition (blue staining) was measured using ImageJ software \n(https://imagej.nih.gov/ij/download.html, RRID: SCR_003070).\nImmunostaining for EMT markers\nEpithelial-to-mesenchymal transition (EMT) status in ENDO lesions was assessed by immunostaining \nfor Cytokeratin (epithelial marker), α-Smooth Muscle Actin (α-SMA, mesenchymal/fibrotic marker), and \nNestin (marker of cellular plasticity/intermediate mesenchymal state) (all from Cell Signaling Technologies \nor ThermoFisher Scientific; details in Table  1). The immunostaining procedure was performed as described \npreviously.\nMeasurement of pro-inflammatory cytokines\nSnap-frozen serum samples were thawed, and cytokine concentrations (IL-6 (ABclonal, #RK00008), TNF-α \n(ABclonal, #RK00027), and TGF-β (ABclonal, #RP01458), were measured using mouse-specific ELISA kits \naccording to the manufacturer’s instructions. Serum from control mice was used to establish baseline cytokine \nlevels. Standard curves were generated for each cytokine, and samples were analyzed ( n = 6 per group). The \ndetection limits for IL-6, TNF-α, and TGF-β were 7.2, 6.5, and 3.9 pg/mL, respectively.\nRNA extraction and quantitative real-time PCR (qRT-PCR)\nTotal RNA was extracted from frozen ENDO lesions ( n = 3 biological replicates) using a Qiagen RNeasy Mini \nKit (Qiagen, #74104). Complementary DNA (cDNA) was synthesized using the PrimeScript RT Reagent Kit \n(TaKaRa Bio Inc., #RR037A). qRT-PCR was performed using Sybr ® Premix Ex Taq™ II (Tli RNase H Plus, \nTakaraBio, #RR820A) on a StepOne real-time PCR system (Applied Biosystems) to assess the gene expression of \nfibrotic markers: E-cadherin, N-cadherin, and S100A4, with GAPDH as an internal control. Primer efficiencies \nand specificities were confirmed to be between 90% and 110%. Reactions were performed in duplicate, and \namplification included initial denaturation (98 °C, 2 min) followed by 40 cycles of denaturation (98 °C, 30 s), \nannealing (optimal temperature, 30 s), and extension (72 °C, 45 s). All samples were examined in triplicate. \nPrimer sequences were custom-synthesized by Bioserve Biotechnologies (India) Pvt Ltd and are provided in \nTable 2.\nWestern blot analysis\nProtein profiling for fibrosis-associated markers was performed on ectopic lesions by western blotting. Snap-\nfrozen ectopic lesions and corresponding eutopic endometrium were homogenized in RIPA lysis buffer with \nprotease inhibitor cocktail (TCI chemicals, #P2976). Equivalent amounts of protein (30  µg) were separated \nby SDS-PAGE (10–12%) and blotted onto PVDF membranes. Membranes were blocked with 5% non-fat dry \nmilk and incubated overnight at 4 °C with primary antibodies against Cytokeratin (ABclonal, #A5243), Snail \n(ABclonal, #A5243), and Vimentin (ABclonal, #A11952). After washing with TBST, membranes were incubated \nwith appropriate secondary antibodies (1:10000) at RT for 1 h. Immunoreactive bands were visualized using \nenhanced chemiluminescence and a gel documentation system (GE healthCare Systems, Amersham Imager \n600).\nStatistical analysis\nStatistical analysis was performed using GraphPad Prism (version 10.2). Data from two groups were analyzed \nusing Student’s t-test (unpaired, two-tailed, 95% CI, significance defined as p < 0.05). Ordinary one-way ANOV A \nwas used for comparisons of three or more groups. Data are presented as mean ± standard error of the mean \nGene Oligonucleotide sequence (5′ to 3′) Product size (bp)\nGAPDH F-  A T G G G A C G A T G C T G G T A C T G A\nR-  T G C T G A C A A C C T T G A G T G A A A T 117\nE-cadherin F-  A A C C C A A G C A C G T A T C A G G G\nR-  A C T G C T G G T C A G G A T C G T T G 142\nN-cadherin F-  C A C T G C C A T T G A T G C G G A T G\nR-  T G C C A C A G T G A T G A T G T C C C 136\nS100A4 F-  T T G T G G T T G A G C T G T G G G A G\nR-  G G T A A C C G T T G A G A C C C C T C 122\nTable 2. Primer sequences used in the experiments.\n \nScientific Reports |        (2025) 15:29024 6| https://doi.org/10.1038/s41598-025-13900-9\nwww.nature.com/scientificreports/\n\n(SEM) of triplicate measurements. Significant differences are indicated by asterisks in figures (* p < 0.05, ** p < \n0.01, *** p < 0.001, **** p < 0.0001).\nResults\nENDO mice exhibit behavioral alterations suggestive of pain and anxiety\nThe burrowing assay, a measure of well-being and potential pain-related behavior, revealed a significant \nreduction in the amount of chow displaced by ENDO mice compared to their respective controls across all \nstrains. Specifically, C57BL/6J ENDO mice displaced an average of 5.79 ± 7.30 g after 2 h and 19.02 ± 19.43 g \novernight, significantly less than the control group (2 h: 12.9 ± 1.66 g; overnight: 153.38 ± 28.30 g) (Fig.  2A). \nSimilarly, BALB/c ENDO mice showed a substantial decrease in burrowing, with an average displacement of \n31.94 ± 26.13 g at 2 h and 43.61 ± 32.93 g overnight, compared to controls (2 h: 103.99 ± 33.32 g; overnight: \n150.63 ± 34.90  g)(Fig.  2B). Swiss albino ENDO mice also exhibited reduced burrowing, displacing 12.94 ± \n12.02 g at 2 h and 63.52 ± 51.92 g overnight, compared to controls (2 h: 50.13 ± 35.22 g; overnight: 157.67 ± \n63.52 g) (Fig. 2C).\nMechanical hypersensitivity, assessed using the electronic von Frey test, was significantly heightened in \nENDO mice of all strains compared to their respective control groups (Fig.  2D, E, F). This was evidenced by a \nmarkedly lower mechanical threshold (force in grams required to elicit a withdrawal response in ENDO mice, \nindicating an increased sensitivity to tactile abdominal stimulation.\nIn the OFT, used to evaluate exploratory behavior and anxiety levels, ENDO mice across all strains exhibited \nreduced exploratory activity. The trajectory plots (Fig.  2G, H, I) visually confirmed the diminished overall \nmovement in ENDO mice. This is indicated by a statistically significant decrease in the number of entries into the \ncentral and peripheral zones, as well as a shorter duration of time spent in the central, more exposed area of the \narena (Fig. 2J, K, L). Swiss albino mice showed similar trends but without statistical significance for central zone \nentries or time spent. The increased time spent in the peripheral zones further indicates heightened anxiety-like \nbehavior in the ENDO groups, though this was not statistically significant in C57BL/6J mice.\nFig. 2. ( A) C57BL/6J ENDO mice showed significantly reduced burrowing activity, heightened mechanical \nsensitivity (Von Frey), and altered Open Field Test (OFT) parameters (decreased central zone duration/\nentries, increased peripheral zone time) compared to controls, indicative of pain-related behavior. (B) BALB/c \nENDO mice also exhibited significant impairment in burrowing, increased Von Frey sensitivity, and reduced \ncentral zone activity in OFT, suggesting persistent pain or anxiety-like behavior. (C) Swiss albino ENDO mice \ndisplayed diminished burrowing, heightened mechanical sensitivity, and decreased central zone activity in \nOFT, consistent with pain-related behavioral modifications observed in other strains. While no significant \nstrain-specific differences were observed, BALB/c and C57BL/6J mice showed a tendency towards a more \npronounced burrowing deficit. (n = 6 per control group, n = 14 per ENDO group for C57BL/6J and Swiss \nalbino, n = 12 per ENDO group for BALB/c; ***P < 0.001, ****P < 0.0001). Trajectory plots for C57BL/6J \n(G), BALB/c (H), and Swiss albino (I) ENDO and control mice in the Open Field Test, visually confirming \ndiminished overall movement in ENDO mice.(D, E, F) Significantly heightened mechanical hypersensitivity in \nENDO mice of all strains compared to their respective control groups. (J, K, L) Statistically significant decrease \nin the number of entries into the central and peripheral zones, a shorter duration of time spent in the central \narea, and increased time spent in the peripheral zones in the ENDO groups compared to the controls.\n \nScientific Reports |        (2025) 15:29024 7| https://doi.org/10.1038/s41598-025-13900-9\nwww.nature.com/scientificreports/\n\nSuccessful establishment of a syngeneic mouse model of ENDO with high incidence in \nC57BL/6J mice\nI/P injection of minced donor uterine horn tissue fragments into the recipient animals was well-tolerated, with \nno significant mortality or adverse effects on the overall health and body weight of the recipient mice (Fig. 3A). \nThis resulted in the successful development of ectopic lesions across all three inbred strains after 12 days \n(Fig. 3B). Macroscopic examination at necropsy revealed the presence of multiple ectopic lesions in all ENDO-\ninduced mice, varying in size (small to moderate) and morphology (cystic or solid) with diverse coloration \n(white, brown, red, or black) (Fig.  3C). The lesions were predominantly on peritoneal surfaces, adipose tissue, \nand pelvic organs.\nThe prevalence of endometriosis, defined as the percentage of recipient mice exhibiting visible ectopic \nlesions, demonstrated a significant strain-dependent variation (Fig.  3D). The C57BL/6J strain showed the \nhighest incidence at 92.85% (13 out of 14 mice), followed by BALB/c at 83.33% (10 out of 12 mice), and Swiss \nalbino at 64.28% (9 out of 14 mice). Circulating serum estrogen levels, measured by ELISA, were significantly (p \n< 0.01) elevated in ENDO mice compared to their respective sham controls across all three strains, confirming \nthe estrogen-dependent nature of the induced lesions (Fig. 3E). To account for the impact of estrogen priming, \nthe control animal animals were injected with EB every alternate day until sacrifice.\nHistopathological analysis confirms typical endometriotic lesion morphology\nHistological evaluation of the ectopic lesions using H&E staining confirmed the presence of characteristic \nendometriotic features in all three strains (Fig. 4B–D). These included the presence of endometrial glands with \nsurrounding stroma, evidence of previous microhemorrhages indicated by hemosiderin-laden macrophages \nFig. 3. General health, lesion distribution, incidence, and estrogen levels in the endometriosis mouse model. \n(A) Body Weight Monitoring: Cumulative mean ± SEM body weight of control and ENDO mice post-\ninjection. No significant differences (P > 0.05) were observed between groups throughout the experimental \nperiod, indicating that ENDO induction did not compromise the general well-being of recipient mice despite \na transient minor decrease in the ENDO group after day 7. (B) Ectopic Lesion Distribution: Representative \nillustration showing the common anatomical locations of ectopic lesions identified 12 days post-ENDO \ninduction across all mouse strains, including adipose tissue, peritoneal layer, uterine horn, ovaries (adhesions), \nand internal organs (intestine). (C) Macroscopic Appearance of Ectopic Lesions: Representative in situ \nphotographs of ectopic lesions observed across the three mouse strains. Lesions typically presented as \nsuperficial, white cystic or red nodules/lesions exhibiting varying degrees of inflammation. Control mice \ninjected with 1× PBS did not develop any lesions. (D) Incidence of Endometriosis Across Strains: The \nincidence of ENDO varied significantly among strains, with C57BL/6J exhibiting the highest success rate \n(92.85%, 13/14), followed by BALB/c (83.33%, 10/12) and Swiss albino (64.28%, 9/14). (E) Circulating \nEstrogen Levels: ELISA analysis revealed significantly increased circulating estrogen levels in ENDO mice \ncompared to their respective controls across all three strains (p < 0.01, unpaired t-test), indicating systemic \nestrogen dependence and hormonal dysregulation associated with the model. Data are presented as mean ± \nSEM.\n \nScientific Reports |        (2025) 15:29024 8| https://doi.org/10.1038/s41598-025-13900-9\nwww.nature.com/scientificreports/\n\nwithin the stroma, and the formation of new blood vessels. These histological features were consistent with the \nexpected morphology of endometriotic lesions and resembled the structure of the control UH obtained from \nmice in the estrous phase (Fig. 4A).\nIHC analysis of the lesions further characterized their cellular composition. Compared to the uterus (Fig. 4E), \nthere was a significant increase in the number of Ki67-positive cells in the ectopic lesions(Fig.  4H), indicating \nenhanced cell proliferation. Similarly, compared to the control (Fig. 4F), the density of CD31-positive endothelial \ncells, marking neovascularization, was significantly higher in the lesions (Fig.  4I). Furthermore, relative to the \ncontrol (Fig. 4G) a substantial infiltration of F4/80-positive macrophages was evident in the stromal regions of \nthe ectopic lesions across all strains (Fig.  4J). Across all ENDO lesions analyzed by quantitative IHC (Fig.  4K), \nwe observed a high level of cell proliferation (65.18% Ki67+), significant vascularization (15.68% CD31+), and a \nnotable presence of macrophages (41.67% F4/80+). While all strains exhibited these markers, visual inspection \nsuggested potentially higher levels in C57BL/6J lesions, consistent with its heightened fibrotic and inflammatory \nprofile.\nStrain-dependent dysregulation of inflammation and macrophage polarization\nAnalysis of circulating pro-inflammatory cytokines in serum samples revealed a distinct strain-specific \ninflammatory response to ENDO induction (Fig.  5A–C). C57BL/6J ENDO mice exhibited a marked and \nstatistically significant elevation in IL-6 levels compared to controls, with modest but non-significant increases \nin TNF-α and TGF-β. In BALB/c ENDO mice, TNF-α levels were significantly increased compared to controls, \nwith slight, non-significant increases in IL-6 and TGF-β. In contrast, Swiss albino ENDO mice did not show any \nstatistically significant alterations in the circulating levels of these three cytokines.\nFlow cytometric analysis of peritoneal fluid macrophages revealed strain-specific patterns of M1 \n(CD86⁺CD11⁺) and M2 (CD206 ⁺CD11⁺) macrophage populations (Fig.  5D–F). In the C57BL/6J strain, M1 \nand M2 macrophages were present at 26.5% and 27.2% of the CD11b + population, respectively, indicating \na balanced and robust immune cell involvement. BALB/c mice showed M1 and M2 populations at 24.9% and \nFig. 4. Histological confirmation and cellular characterization of ectopic endometriotic lesions. (A) Control \nuterine horn (UH) from healthy mice in the estrous stage displayed normal morphology (H&E, n = 6). \n(B–D) Ectopic lesions harvested at day 12 post-induction from C57BL/6J, BALB/c, and Swiss albino mice \n(H&E, n = 6 per group) exhibited characteristic endometriotic features, including singular or multi-layered \nepithelium, dense immune cell infiltration in the stroma, and the presence of endometrial glands. (E–G) \nImmunohistochemical analysis of control uterus sections (E–G, Control) revealed (H) proliferating epithelial \ncells (Ki67+), (I) established vasculature (CD31/PECAM1+), and (J) a significant presence of macrophages \n(F4/80+). Representative images for (H, I, J) are shown from C57BL/6J ectopic lesions, and similar staining \npatterns were observed across all three strains. (K) Quantitative analysis of IHC staining across all ENDO \nlesions from all strains (n = 3 per group) indicated a high proliferative index (65.18% Ki67+), substantial \nvascularization (15.68% CD31+), and marked macrophage infiltration (41.67% F4/80+). (Magnification and \nscale bars to be consistently applied throughout the figure. Example: Magnification = 4x, scale bar = 40 μm; \nMagnification = 40x, Scale bar = 10 μm).\n \nScientific Reports |        (2025) 15:29024 9| https://doi.org/10.1038/s41598-025-13900-9\nwww.nature.com/scientificreports/\n\n26.9%, respectively, suggesting a similarly active but slightly more M2-skewed profile. Swiss albino mice exhibited \nM1 and M2 populations at 20.7% and 25.3%, respectively, indicating a comparatively lower overall macrophage \nactivation in the peritoneal fluid. These percentages represent average values from flow cytometry analysis across \nmultiple mice for each strain ( n = 3 for C57BL/6J, Swiss albino, and BALB/c). While M2 populations were \nnumerically higher than M1 populations across all strains, suggesting a pro-fibrotic environment, statistical \nsignificance for this difference was not performed in this study.\nEctopic lesions display increased fibrosis markers and iron deposition\nMasson’s trichrome staining, used to visualize collagen deposition, demonstrated a substantial presence of \ncollagen-rich fibrotic areas (blue staining) in the stromal regions of ectopic lesions across all three strains \n(Fig. 6A). Quantitative analysis revealed a significant increase in the collagen-positive area in C57BL/6J ENDO \nmice compared to controls and the other strains, indicating more pronounced fibrotic remodeling. While the \nnumerical difference in percentage between C57BL/6J (69.95%) and BALB/c (69.05%) was small, C57BL/6J \nexhibited a statistically significant difference in collagen deposition when compared to Swiss albino (57.47%) \nand controls, indicating more pronounced fibrotic remodeling in C57BL/6J. Prussian blue staining revealed a \nconsiderable accumulation of iron deposits (blue staining) within the stroma of the ectopic lesions in all strains \n(Fig.  6C) compared to the control horn(Fig.  6B), suggesting chronic microhemorrhaging and inflammation \nwithin the lesions. ELISA of ectopic lesion lysates confirmed significantly elevated protein levels of Col1A1 in \nthe lesions compared to control uterine tissues across all strains. The highest levels of this key component of the \nfibrotic extracellular matrix were observed in C57BL/6J mice (Fig. 6D).\nC57BL/6J ENDO lesions exhibit pronounced EMT and fibrotic marker expression resembling \nhuman ENDO \nImmunohistochemical analysis of fibrotic and EMT-related markers (α-SMA, Nestin, Cytokeratin) revealed \nminimal expression in control uterine tissues (Fig. 7A). In contrast, C57BL/6J lesions showed strong expression \nof these markers (Fig.  7B). BALB/c and Swiss albino lesions showed moderate and lower expression levels, \nrespectively (Fig. 7C, D). Notably, the staining patterns observed in our mouse model, particularly in C57BL/6J \nmice, qualitatively resembled those seen in human ENDO tissue (Fig.  7E), suggesting potential translational \nFig. 5. Strain-dependent systemic inflammation and peritoneal macrophage polarization in endometriosis. \n(A) Serum cytokine analysis in C57BL/6J ENDO mice revealed a significant increase in IL-6, with modest \nelevations in TNF-α and TGF-β. (B) BALB/c ENDO mice showed a prominent rise in TNF-α, accompanied \nby minor increases in IL-6 and TGF-β. (C) In contrast, Swiss albino ENDO mice exhibited no significant \nalterations in circulating levels of these cytokines. (D–F) Peritoneal fluid flow cytometry demonstrated the \npercentage of M1 (CD11b + CD86+) and M2 (CD11b + CD206+) macrophages in ENDO mice of each strain: \n(D) C57BL/6J showed a balanced M1/M2 population (26.5% vs. 27.2%), (E) BALB/c displayed similar M1 \n(24.9%) and M2 (26.9%) percentages, and (F) Swiss albino exhibited M1 (20.7%) and M2 (25.3%) populations.\n \nScientific Reports |        (2025) 15:29024 10| https://doi.org/10.1038/s41598-025-13900-9\nwww.nature.com/scientificreports/\n\nrelevance, though this comparison was not quantitatively analyzed for statistical significance. C57BL/6J lesions \nexhibited the highest percentage of positively stained cells for all three markers (α-SMA: 17.41%, Nestin: 8.28%, \nCytokeratin: 10.08%), indicative of a significant fibrotic and EMT signature. Lesions used for immunostaining \nwere collected at day 12 post-induction and represent various morphologies (cystic or solid) observed across \nthe strains. Figure  7F represents the average percentage of positively stained cells across the tissue field from \nmultiple mice for each strain (n = 3 per group for IHC analysis).\nGene expression analysis using qRT-PCR revealed a classic EMT profile in C57BL/6J lesions, with a consistent \ndownregulation of E-cadherin (0.52-fold change) and significant upregulation of mesenchymal markers \nN-cadherin (1.59-fold change) and S100A4 (42.66-fold change) (Fig. 8A).BALB/c lesions also showed evidence \nof partial EMT, with a significant upregulation of S100A4, alongside reduced E-cadherin and low N-cadherin. \nSwiss albino lesions exhibited inconsistent E-cadherin levels and minimal mesenchymal marker expression. To \nvalidate mRNA expression patterns of C57BL/6j, protein levels of cytokeratin, snail, and vimentin were assessed \nthrough densitometric analysis of immunoreactive bands in ENDO lesions and corresponding control tissues \nfrom C57BL/6J mice ( n = 3 per group). Snail expression was numerically higher in ENDO lesions (mean = \n1.43 µg/mL) than in controls (mean = 0.99 µg/mL), suggesting the transcriptional activation of EMT pathways. \nCytokeratin expression was also numerically elevated in lesions (mean = 1.35 µg/mL), indicating retention of \nepithelial characteristics. Vimentin levels were numerically diminished in ENDO lesions (mean = 0.82 µg/mL) \nrelative to controls, potentially suggesting a potential divergence from the traditional EMT state (Fig. 8B). These \nobservations, while indicative of trends, did not reach statistical significance in this analysis.\nDiscussion\nOur study comprehensively characterizes a syngeneic mouse model of endometriosis across three commonly \nused inbred strains: C57BL/6J, BALB/c, and Swiss albino. We meticulously compared lesion development, \nbehavioral alterations indicative of pain and anxiety, the inflammatory milieu, macrophage polarization, and the \nprogression of fibrosis. The findings suggest the C57BL/6J strain as the most promising model for recapitulating \nthe inflammatory and fibrotic pathophysiology of human ENDO, particularly its fibrotic component. This \nmodel was established through a straightforward, cost-effective, and short intraperitoneal injection protocol. \nThe model effectively mimics key aspects of the human disease, like collagen deposition, pain-related behavioral \nchanges, and the upregulation of crucial EMT biomarkers.\nThe behavioral assays provided compelling evidence for the development of pain and anxiety-like behaviors \nin the ENDO mice. Significant reduction in burrowing activity was observed across all strains. This aligns with \nfindings in other animal models and symptomatic women with ENDO, where discomfort and pain often lead \nto decreased engagement 42. The heightened mechanical hypersensitivity observed in the ENDO mice further \nFig. 6. Assessment of fibrosis and iron accumulation in ectopic lesions across strains. (A) Masson’s trichrome \nstaining (MTS) revealed varying degrees of collagen deposition (blue) in ectopic lesions, with C57BL/6J \nexhibiting the most pronounced fibrosis, followed by substantial fibrosis in BALB/c (quantified at 69.95%), \nand the least in Swiss albino (57.47%). (B, C) Prussian blue staining showed strain-dependent iron deposition \n(blue precipitates, arrowheads) in the lesion stroma, with significant accumulation in C57BL/6J (score: 2.348), \nmoderate in BALB/c (score: 1.208), and minimal in Swiss albino (score: 0.356, OD: 0.084). (D) Collagen Type \nI alpha 1 (Col1A1) ELISA indicated significant stromal fibrosis in C57BL/6J (*p < 0.05), moderate fibrosis in \nBALB/c (lower than C57BL/6J but higher than Swiss albino), and mild fibrosis in Swiss albino. Data are mean \n± SEM, *p < 0.05 (unpaired t-test).\n \nScientific Reports |        (2025) 15:29024 11| https://doi.org/10.1038/s41598-025-13900-9\nwww.nature.com/scientificreports/\n\nsupports the presence of endometriosis-associated hyperalgesia, a hallmark of the human condition 43. The \ndiminished exploratory behavior and increased time spent in the peripheral zones of the OFT suggest elevated \nanxiety levels, which are frequently comorbid with chronic pelvic pain in women with ENDO23. The C57BL/6J \nstrain consistently exhibited the most pronounced behavioral alterations, making it suitable for investigating the \nmechanisms underlying ENDO-associated pain.\nThe successful induction of ectopic lesions in all ENDO-challenged mice, coupled with the absence of \nsignificant morbidity, underscores the feasibility and safety of our syngeneic model 31. The elevated circulating \nestrogen levels were observed in all ENDO groups. This corroborates the well-established estrogen-dependent \nnature of endometriosis29 and validates the hormonal milieu supporting lesion development in our model. The \nsignificantly higher prevalence of lesion formation in the C57BL/6J strain points towards inherent differences in \nimmune responses or hormonal sensitivity.\nHistopathological analysis confirmed that the induced lesions in all strains displayed typical glandular \nand stromal components as observed in human ENDO 44,45. There was increased cell proliferation, \nneovascularization, and macrophage infiltration within the lesions. This is characteristic of human endometriotic \nimplants, highlighting the active and dynamic nature of these ectopic tissues 46,47. The heightened angiogenic \nand macrophage infiltration observed in C57BL/6J lesions suggests a more active inflammatory and fibrotic \nenvironment in this strain.\nThe strain-dependent differences in systemic inflammation and macrophage polarization are noteworthy. \nThe marked elevation of IL-6 in C57BL/6J mice and TNF-α in BALB/c mice suggests distinct inflammatory \npathways are activated in response to ENDO in these strains 48,49. The increased M2 macrophage population in \nthe peritoneal fluid of all ENDO strains aligns with previous findings, indicating the pro-fibrotic environment of \nendometriosis50. The most dramatic alterations in pro-fibrotic immune subsets observed in C57BL/6J mice are \nconsistent with its heightened fibrotic and inflammatory profile51.\nA key finding of our study is the significant increase in collagen deposition and Col1A1 protein levels in the \nectopic lesions, particularly in the C57BL/6J strain. This robust fibrotic response in C57BL/6J mice mimics the \nexcessive collagen deposition that defines fibrotic ENDO in humans 17,18. The substantial iron deposits in the \nlesions further indicate chronic microhemorrhage and inflammation. Iron overload is known to contribute to \nfibrosis in various tissues52,53.\nThe immunohistochemical and molecular analyses of EMT and fibrotic markers provide strong evidence for \nthe involvement of EMT in the fibrotic remodeling. The lesions in C57BL/6J showed high expression of α-SMA, \nFig. 7. Strain-dependent expression of fibrotic markers in ectopic lesions and translational relevance to \nhuman endometriosis. (A, E) Control tissues exhibited negligible immunohistochemical staining for α-SMA, \nNestin, and Cytokeratin (quantified as low percentages). (B, E) C57BL/6J ectopic lesions displayed the most \npronounced fibrotic profile, characterized by the highest percentages of α-SMA, Nestin, and Cytokeratin \npositive cells. (C, E) BALB/c lesions showed intermediate expression levels of these markers, while (D, E) Swiss \nalbino lesions exhibited the lowest expression. Data are presented as the percentage of positively stained cells \nacross the tissue field. (E) Comparative IHC analysis of Cytokeratin, Vimentin, and Nestin in ectopic lesions \nfrom the mouse model and human endometriosis samples revealed similar patterns of marker localization \nand staining intensity, supporting the model’s translational significance. (DAB brown, Hematoxylin blue \ncounterstain; Magnification: 40x, 100x, 1000x). (F) Percentage of positively stained cells for all three markers \n(α-SMA: 17.41%, Nestin: 8.28%, Cytokeratin: 10.08%.\n \nScientific Reports |        (2025) 15:29024 12| https://doi.org/10.1038/s41598-025-13900-9\nwww.nature.com/scientificreports/\n\nNestin, and Cytokeratin, resembling the patterns observed in human ENDO tissue. This underscores the \nactivation of myofibroblasts and the ongoing tissue remodeling54. The gene expression data, showing a classical \nEMT signature in C57BL/6J mice (downregulated E-cadherin, upregulated N-cadherin, and S100A4), further \nsupports this notion. The Western blot findings in C57BL/6J lesions showed elevated Snail and Cytokeratin \nand decreased Vimentin. This suggests a dynamic and potentially complex EMT process, warranting further \ninvestigation to fully understand its role in the fibrotic progression of endometriosis55.\nThis study provides a comprehensive characterization of syngeneic mouse models for endometriosis, but \nwe acknowledge several limitations. Firstly, murine models do not naturally menstruate, making the widely \naccepted theory of retrograde menstruation not replicable in mice. The current model relies on the injection \nof uterine fragments, which may not fully mimic the complex clinical processes leading to human lesion \nestablishment. Secondly, we have compared mouse lesions to human tissue samples to provide qualitative visual \nvalidation. This aspect was not quantitatively analyzed for statistical significance, therefore, direct translational \ncomparability may be missing. Future studies using RNA sequencing or advanced proteomics for in-depth \ncomparative analysis between mouse strains and human samples would provide further insights into the \nmolecular similarities and differences. Additionally, while behavioral assays like burrowing used for the study \nare not specific to endometriosis-associated pain alone. Estrogen and inflammation are the two most prominent \ncharacteristics in endometriosis. In this work, we did not study the interplay of estrogen and inflammatory \npathways. Future research could explore the local estrogen metabolism within the lesions.\nIn conclusion, our comprehensive comparison of three inbred mouse strains demonstrates that the C57BL/6J \nsyngeneic model most accurately recapitulates the key pathological features of human endometriosis. The model \nshowed behavioral alterations indicative of pain and anxiety, a pro-inflammatory and M2 macrophage-skewed \nFig. 8. Differential EMT Marker expression at the transcriptional and translational levels in endometriotic \nlesions. (A) mRNA expression profiles of E-cadherin, N-cadherin, and S100A4 in ectopic lesions across \nthree mouse strains. C57BL/6J lesions displayed a robust EMT signature with E-cadherin downregulation \nand significant upregulation of N-cadherin and S100A4 (**P < 0.01, ****P < 0.0001). BALB/c lesions showed \nevidence of partial EMT with reduced E-cadherin, low N-cadherin, and moderate S100A4. Swiss albino \nlesions exhibited inconsistent E-cadherin levels and minimal mesenchymal marker expression. (B) Western \nblot analysis in C57BL/6J mice (n = 3 per group) revealed increased protein levels of Snail (ENDO: 1.43 µg/\nmL, Control: 0.99 µg/mL) and Cytokeratin (ENDO: 1.35, Control: 0.98) in ENDO lesions, while Vimentin \nexpression was reduced (ENDO: 0.82 µg/mL, Control: 1.11 µg/mL).\n \nScientific Reports |        (2025) 15:29024 13| https://doi.org/10.1038/s41598-025-13900-9\nwww.nature.com/scientificreports/\n\nimmune environment, and a robust fibrotic response driven by EMT-related mechanisms. This model offers a \nsignificant ability to mimic the progressive fibrosis, a critical aspect of chronic ENDO. The C57BL/6J model is \npromising for future research. aimed at understanding molecular pathways of fibrosis in endometriosis and for \nthe preclinical evaluation of novel anti-fibrotic strategies56. Using a traffic light-based classification57 C57BL/6J \nreceives the maximum green lights, indicating confirmation of the clinical phenotype (Fig.  9). BALB/c comes \npromising second in this classification. However, the Swiss albino strain showed inconsistent results across \nvarious parameters, making it unsuitable for ENDO modelling. Further investigations into strain-dependent \nvariations observed in our study are warranted to enhance our understanding of endometriosis pathogenesis \nand to facilitate the development of more effective treatments57,58.\nData availability\nThe original contributions presented in the study are included in the article; further inquiries can be directed to \nthe corresponding author.\nReceived: 30 April 2025; Accepted: 28 July 2025\nReferences\n 1. Leone, R. et al. Epidemiology of infertility in women with endometriosis. Best Practi. Res. Clin. Obstet. Gynaecol.  92, 102454 \n(2024).\n 2. Sherwani, S. et al. 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Nandakumar and Prof. Jayesh Mudgal from the Department of \nPharmacology, Manipal College of Pharmaceutical Sciences, Manipal, in conducting the behavioral assays (OFT, \nhot plate testing, von Frey, Randall’s-Selitto). The authors also acknowledge the contribution of Prof. Raviraja N \nS from Manipal Centre for Biotherapeutics Research for providing access to the flow cytometry facility for our \nwork. Authors extend heartfelt gratitude to Dr. Dinesh Updhyaya of Centre for Molecular Neurosciences for the \nsupport in providing facility access for Western blot and ELISA reading.\nAuthor contributions\nM.A., A.H., S.D., and R.Du. designed the research. M.A., A.H., S.D., R.P ., G.K., J.J., S.S., K.S., H.P ., and R.Da. \ncollected the data. M.A. and R.Du. wrote the original draft. G.K., R.Da., S.S., K.S., H.P ., and R.Du. help optimize \nthe research and proofread the paper. R.Da. and S.S. supplements relevant information and provide guidance. \nAll authors critically reviewed this draft. All authors approved the final draft for submission.\nFunding\nOpen access funding provided by Manipal Academy of Higher Education, Manipal\nDeclarations\nCompeting interests\nThe authors declare no competing interests.\nAdditional information\nSupplementary Information The online version contains supplementary material available at  h t t p s : / / d o i . o r g / 1 \n0 . 1 0 3 8 / s 4 1 5 9 8 - 0 2 5 - 1 3 9 0 0 - 9     .  \nCorrespondence and requests for materials should be addressed to R.D.\nReprints and permissions information is available at www.nature.com/reprints.\nPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and \ninstitutional affiliations.\nOpen Access  This article is licensed under a Creative Commons Attribution 4.0 International License, which \npermits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give \nappropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and \nindicate if changes were made. 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