{"paper_id":"608f4c68-5efe-4aa3-af57-d811d6babc2d","body_text":"ARTICLE IN PRESS\nArticle in Press\nDental pulp–derived mesenchymal stem cells \nreduce lesion progression in a rat model of \nendometriosis\nScientific Reports\nReceived: 28 December 2025\nAccepted: 5 May 2026\nCite this article as: Hortu I., Kandemir \nDemirci G., Şimşek B. et al. Dental pulp–\nderived mesenchymal stem cells reduce \nlesion progression in a rat model of \nendometriosis. Sci Rep (2026). https://\ndoi.org/10.1038/s41598-026-52416-8\nIsmet Hortu, Gözde Kandemir Demirci, Birant Şimşek, Ebru Eroğlu, Özgün Selim \nGermiyan, Aylin Gökhan, Dilek Taşkiran, Mehmet Emin Kaval, Uğur Tekin, Pelin \nGüneri, Timur Köse & Yiğit Uyanikgil\nWe are providing an unedited version of this manuscript to give early access to its \nfindings. Before final publication, the manuscript will undergo further editing. Please \nnote there may be errors present which affect the content, and all legal disclaimers \napply.\nIf this paper is publishing under a Transparent Peer Review model then Peer \nReview reports will publish with the final article.\nhttps://doi.org/10.1038/s41598-026-52416-8\n© The Author(s) 2026. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International \nLicense, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit \nto the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do \nnot have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this \narticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the \narticle’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain \npermission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.\n\n \n \nDental Pulp–Derived Mesenchymal Stem Cells Reduce Lesion Progression \nin a Rat Model of Endometriosis \nIsmet HORTU 1,2 M.D., Ph.D, Assoc. Prof. , Gözde KANDEMİR DEMİRCİ 2,3 DDS, Ph.D., Assoc. \nProf. , Birant ŞİMŞEK2,4, DDS.,Ph.D, Ebru EROĞLU 5 Ph.D-c., Özgün Selim GERMİYAN2 Ph.D-c, \nAylin GÖKHAN5 M.D., Dilek TAŞKIRAN6 M.D., Ph.D, Professor, Mehmet Emin KAVAL3, DDS, \nPh.D Professor, Uğur TEKİN4 DDS, Ph.D, Professor, Pelin GÜNERİ7 DDS, Ph.D, Professor, Timur \nKÖSE8 Ph.D, Professor, Yiğit UYANIKGİL2,5 Ph.D, Professor \n1Department of Obstetrics and Gynecology, Faculty of Medicine, Ege University, 35100, İzmir, Türkiye \n2Department of Stem Cell, Institute of Health Sciences, Ege University, 35100, İzmir, Türkiye \n3Department of Endodontology, Faculty of Dentistry, Ege University, 35100, İzmir, Türkiye. \n4Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Ege University, 35100, İzmir, Türkiye \n5Department of Histology and Embryology, Faculty of Medicine, Ege University, 35100, İzmir, Türkiye \n6Department of Physiology, Faculty of Medicine, Ege University, 35100, İzmir, Türkiye  \n7Department of Oral and Maxillofacial Radiology, Faculty of Dentistry Ege University, 35100, İzmir, Türkiye \n8Department of Biostatistics and Medical Informatics, Faculty of Medicine, Ege University, 35100, İzmir, \nTürkiye \n \n \n \n \n \nCorresponding author:  \nAssoc. Prof. Dr. Gözde KANDEMİR DEMİRCİ \nDepartment of Endodontology,  \nFaculty of Dentistry, Ege University, 35100, İzmir, Türkiye \nTel: +905056822552 \ne-mail:gozde.kandemir.demirci@ege.edu.tr \n           dt. gozdekandemir@hotmail.com \n  \nACCEPTED MANUSCRIPT\nARTICLE IN PRESSARTICLE IN PRESS\n\n \n \nAbstract \nEndometriosis is a chronic, estrogen -dependent inflammatory disease sustained by aberrant \nangiogenesis and progressive fibrosis. We evaluated the therapeutic efficacy of human dental pulp –\nderived mesenchymal stem cells (DP-MSCs) in a surgically induced rat endometriosis model. Twenty-\neight adult female  Wistar rats were randomized (n = 7/group) to sham group (G1), untreated \nendometriosis-group  (G2), single-dose DP-MSCs-group (G3)  (2×106 cells intraperitoneally on day 28) \nand double-dose DP-MSCs-group (G4)  (2×106 cells on days 28 and 35). Endometriosis was induced by \nautologous uterine tissue implantation onto the peritoneal wall and allowed to establish for 28 days; \ntreatment effects were assessed 7 days after the final DP-MSCs dose. Serum and peritoneal TNF-α, IL-\n6, VEGF, and CA-125 were quantified; lesions were evaluated by semi-quantitative histopathology and \nfibrosis grading and by immunohistochemistry for CA-125, VEGF, type I collagen (Col1), and TNF-α. \nUntreated endometriosis showed increased systemic TNF -α (p = 0.0207) and IL -6 (p = 0.0003) and \nmarked peritoneal elevations versus sham (all p < 0.0001). DP -MSCs treatment significantly reduced \nperitoneal TNF-α and IL-6 in both regimens (each p < 0.0001 vs untreated) and decreased peritoneal \nVEGF, with greater suppression after double dosing (p = 0.0100 between regimens). Double dosing \nproduced stronger systemic TNF-α suppression (p = 0.0027 vs untreated). Histopathology and fibrosis \nimproved, most prominently with double dosing (both p < 0.0001), accompanied by reduced CA -125, \nVEGF, Col1 and TNF-α immunoreactivity (CA-125 and TNF-α, p < 0.0001).  DP-MSCs effectively \nresolve the hallmark pathological features of endometriosis in a dose -dependent manner. By \nsynergistically targeting inflammatory, angiogenic, and fibrotic pathways, this cell -based \nstrategy offers a potent, disease-modifying approach for clinical management. \nKeywords: Biomarkers; Cytokines; Dental Pulp; Endometriosis; Histopathology; Mesenchymal Stem \nCells. \n \n \n  \nACCEPTED MANUSCRIPT\nARTICLE IN PRESSARTICLE IN PRESS\n\n \n \nIntroduction \nEndometriosis is a chronic, estrogen-dependent inflammatory disorder defined by ectopic endometrial-\nlike glands and stroma, most commonly on the pelvic peritoneum, and it remains a major cause of pelvic \npain and infertility. Disease persistence is linked to a permissive peritoneal milieu with immune \ndysregulation, sustained inflammation, angiogenic activation, and progressive e xtracellular matrix \nremodeling that supports lesion survival, invasion, and adhesion formation [1]. High symptom burden, \nrecurrence, and the limitations of current medical and surgical options highlight the need for fertility -\nsparing, mechanism-directed approaches that target inflammatory, angiog enic, and fibrotic pathways \n[2]. \nMesenchymal stem cells (MSCs) are increasingly studied as mechanism -oriented therapeutics, with \neffects largely attributed to paracrine signaling, immunomodulation, and regulation of tissue repair. \nSeveral international consensus statements have proposed minimal criteria and surface -marker panels \nto standardize MSC definition and improve comparability across studies [3]. Among available sources, \ndental tissue–derived MSCs are attractive due to practical procurement and relevance to inflammatory \nand regenerative indications. Dental pulp–derived MSCs (DP-MSCs) show strong expansion capacity, \nrelatively low immunogenicity, and translational feasibility, including tissue -engineering applications \n[4]. \nEndometriosis progression depends on angiogenesis and stromal remodeling, domains that DP-MSC \nmay influence through microenvironmental modulation. In addition, emerging work indicates that DP-\nMSC function and heterogeneity are shaped by epigenetic programs, including DNA methylation, which \ncan affect lineage potential and immunobiology and may influence therapeutic consistency and potency. \nDespite growing knowledge of dental stem cell  biology, direct preclinical testing of DP-MSCs in \nendometriosis remains limited, supporting the need for well-controlled in vivo studies that address dose \nregimen and timing [5]. \nIn this context, the present study evaluates the therapeutic potential of human DP-MSCs administration \nin a surgically induced rat model of endometriosis. Key disease axes—local and systemic inflammation, \nangiogenesis, fibrotic remodeling, and lesion activity —were evaluated using complementary \nbiochemical (TNF -α, IL -6, VEGF, CA -125), histochemical (H&E and Masson’s trichrome), and \nimmunohistochemical (CA-125, Col1, VEGF, TNF-α) readouts. Single and double-dose intraperitoneal \nDP-MSCs regimens were compared to assess regimen dependence and to align delivery with peritoneal \ntargeting approaches used to modulate abdominal inflammatory milieus in experimental m odels [6], \nthereby strengthening the translational rationale for peritoneal adm inistration in endometriosis and \nsupporting the development of fertility-sparing treatment strategies.  \nACCEPTED MANUSCRIPT\nARTICLE IN PRESSARTICLE IN PRESS\n\n \n \nMaterials and Methods \nEthical Approval and Experimental Design \nThe human DP-MSCs used in this study were isolated from third molars extracted for clinical reasons \nat the Ege University Faculty of Dentistry. All experimental protocols and the collection of human \nsamples were approved by the Ege University Medical Research Ethics Committee under the approval \nnumber 23-1.1T/37 (Initial approval) and updated/confirmed with the decision 23-9.1T/51. All methods \nwere performed in accordance with the relevant guidelines and regulations (Declaration of Helsinki). \nInformed consent was obtained from all participants and/or their legal guardians prior to the col lection \nof dental tissues.  \nAll experimental procedures were conducted at the Ege University Experimental Animals Application \nand Research Center following approval from the Ege University Animal Experiments Local Ethics \nCommittee (decision date: March 27, 2024; Approval number: 2023-072). All procedures complied with \ninstitutional and national guidelines for laboratory animal care and the ARRIVE guidelines.  Twenty-\neight female Wistar albino rats aged 10 - 12 weeks and weighing 180 to 260 g were included. Female \nrats were housed in single cages in a room with a temperature of 24 ± 1 °C and a 12-h light–dark cycle \n(lights on at 07.00 a.m.) and were fed a standard laboratory diet and water and food were provided ad \nlibitum. All efforts were made to minimize animal suffering and to reduce the number of animals used. \nAt the end of the study, all rats were euthanized to ensure minimum pain and distress. Euthanasia was \nperformed by an intraperitoneal overdose of a combination of 80 mg/kg ketamine (VetaKetam, Vet -\nAgro, Lublin, Poland) and 10 mg/kg xylazine (VetaXyl, Vet-Agro, Lublin, Poland).  \nDP-MSC Isolation, Culture, and Characterization \nHuman DP-MSCs were obtained from impacted third molars using commonly applied dental pulp stem \ncell isolati on procedures [7] . Teeth were transported in sterile Falcon tubes and processed under a \nbiosafety cabinet. Dental pulp tissue was removed under aseptic conditions and processed separately for \neach donor. The pulp was digested with 3 mg/mL type I collagenase at 37 °C for 45 minutes. Enzymatic \ndigestion was stopped by adding complete culture medium to a final volume of 10 mL (α -MEM \nsupplemented with 10% fetal bovine serum, 1% gentamicin, 1% penicillin/streptomycin, and 0.8% \namphotericin B). The suspension was centrifuged at 1 600 rpm for 5 minutes at 4 °C. The supernatant \nwas discarded, the pellet was resuspended in fresh medium (final volume 5 mL), and cells were seeded \ninto T-25 cm² flasks. Cultures were monitored on days 3 and 5 for attachment and early growth. After \nday 5, the medium was replaced every three days. At ≥80% confluency, cells were detached with trypsin-\nEDTA, counted using a hemocytometer, and expanded as required. Cells were cryopreserved in freezing \nmedium containing 90% fetal bovine serum and 10% dimethyl sul foxide, following standard MSC \nACCEPTED MANUSCRIPT\nARTICLE IN PRESSARTICLE IN PRESS\n\n \n \nhandling procedures [8]. After one month, cells were thawed for experiments and recounted, yielding a \npost-thaw concentration of approximately 9 ×105 cells/mL per vial. Representative images of cultured \ncells are shown in Figure 1. \nFlow cytometric immunophenotyping of DP-MSCs \nDP-MSCs were characterized by flow cytometry (FACS)  at the STEMBIO A.Ş. laboratory (TÜBİTAK \nMarmara Technopark R&D and Innovation Center, Gebze, Kocaeli, Türkiye). Cells were expanded to \npassage 1 to lim it passage -related changes in MSC propert ies [9]. At ~80% confluency, cells were \ndetached, washed, and resuspended in Dulbecco’s phosphate -buffered saline (DPBS) at ≥5 × 10^6 \ncells/mL. Immunostaining was performed using the BD Stemflow™ Human MSC Analysis Kit (BD \nBiosciences, San Jose, CA, USA). The panel included MSC -positive markers (CD73, CD90, CD105) \nand negative lineage markers (CD45, CD34, CD19, CD11b, HLA-DR), consistent with commonly used \nMSC phenotyping approaches for DP- MSCs [10]. After staining, cells were washed, resuspended in \nFACSflow solution, and analyzed on a Navios EX flow cytometer using Navios EX tetra software. DP-\nMSCs showed high expression of CD90 (99.81%), CD73 (99.7%), CD105 (98.3%), and CD44 (95.6%); \nCD44 is often reported as an MSC -associated marker [11]. Cells showed low expression of \nhematopoietic/immune markers (combined panel positivity 1.56%). Overall, 98.35% of analyzed cells \nmatched the expected DP-MSCs immunophenotype (Figure 2). \n \nACCEPTED MANUSCRIPT\nARTICLE IN PRESSARTICLE IN PRESS\n\n \n \n \nFigure 1. Morphological and immunophenotypic characterization of DP -MSCs. Representative \ninverted phase-contrast micrographs illustrate the adherent, spindle-shaped, fibroblast-like morphology \nof DP-MSCs. The upper images display the cells during the early expansion phase (Initial isolation, P1-\nP2); the middle images represent the confluent culture prior to cryopreservation; and the lower images \nshow the cells after thawing and subsequent re-culture. The consistent morphology observed across all \nstages indicates that the cryoprese rvation and thawing processes do not adversely affect the typical \nMSC-like appearance or the plastic-adherence capacity of the cells. \n \nACCEPTED MANUSCRIPT\nARTICLE IN PRESSARTICLE IN PRESS\n\n \n \n \nACCEPTED MANUSCRIPT\nARTICLE IN PRESSARTICLE IN PRESS\n\n \n \nFigure 2. Immunophenotypic characterization of DP -MSCs. Representative flow cytometry dot \nplots showing the gating strategy based on forward (FSC) and side scatter (SSC) ( Gate A, 93.83% ). \nAnalysis of MSC markers: DP-MSCs show high expression levels for CD90 (99.91%), CD44 (95.64%), \nCD105 (98.32%) , and CD73 (99.74%) . Exclusion of hematopoietic lineages: cells show negat ive \nexpression for CD34 and CD45 (Gate G, 1.56%), confirming their non-hematopoietic origin. Data tables \nprovide event counts, percentage of gated cells (%Gated), and mean fluorescence intensity (MFI) for \neach marker. \nAnimal Model of Experimental Endometriosis and Study Design \nTwenty-eight adult female Wistar albino rats (10 –12 weeks old; 180–260 g) were randomly allocated \nto four experimental groups (n = 7/group). Experimental endometriosis was induced using a validated \nautologous uterine tissue implantati on method, as previously described [12]. Briefly, under aseptic \nconditions, a full -thickness uterine fragment containing endometrium (approximately 0.5 × 0.5 × 0.1 \ncm) was excised from the right uterine horn and sutured to the peritoneal wall with 5 -0 Vicryl. All \nsurgical procedures were performed by the same operator to reduce inter-operator variability. A 28-day \npost-induction interval was defined as the lesion establishment (baseline) time point, consistent with the \noriginal description of this model  and with the commonly used ~4 -week period required for stable, \nhistologically confirmable ectopic implants [12]. To standardize treatment evaluation across regimens, \nanimals were euthanized seven days after the final DP-MSCs administration in each treatment arm. This \ninterval was selected to capture early MSCs-mediated immunomodulatory and paracrine effects within \nthe peritoneal milieu, consistent with the concept that MSC s efficacy is primarily driven by secreted \nmediators rather than long -term engraftmen t [13]. Intraperitoneal delivery was chosen because it is \nwidely used to target abdominal/peritoneal inflammation and has been shown to ameliorate \nexperimental inflammatory disease by modulating immune cell activation in the peritoneal compartment \n[6,14].  \nGroup Allocation and Interventions \nGroup allocation and interventions were defined a priori based on established endometriosis induction \nmethodology and prior intraperitoneal MSCs administration studies [6, 12,14]. Group 1 (Sham control; \nday 42 endpoint) underwent laparotomy on day 0, followed by peritoneal irrigation with sterile 0.9% \nNaCl and closure without tissue implantation; animals were euthanized on day 42 to provide a time -\nmatched control for the longest study duration. Group 2 (Endometriosis base line; day 28 endpoint) \nunderwent endometriosis induction on day 0 without further intervention and was euthanized on day 28 \nto verify lesion establishment and to define baseline model status [12]. Group 3 (Single-dose DP-MSC; \nday 35 endpoint) underwent endometriosis induction on day 0 and received a single intraperitoneal dose \nof 2×106 DP-MSCs on day 28; animals were euthanized on day 35 (i.e., 7 days after dosing). Group 4 \nACCEPTED MANUSCRIPT\nARTICLE IN PRESSARTICLE IN PRESS\n\n \n \n(Double-dose DP-MSCs; day 42 endpoint) underwent endometriosis induction on day 0 and received \nintraperitoneal DP-MSCs (2×106 cells) on days 28 and 35; animals were euthanized on day 42 (i.e., 7 \ndays after the second dose) (Figure 3). \n \nFigure 3. Macroscopic evaluation and quantitative measurement of endometriotic lesions across \nexperimental groups. Top row (G2, G3, G4) shows the representative in situ macroscopic appearance \nof the lesions during laparotomy. Bottom row  (G2-1, G3 -1, G4 -1) displays the harvested lesions \nmeasured with a digital caliper to calculate the volumes. G2: Untreated endometriotic lesion group \nexhibiting well-vascularized, large cystic structures. G3: Single-dose DP-MSCs therapy group showing \na moderate reduction in lesion size. G4: Double-dose DP-MSCs therapy group showing the most \nsignificant regression in  lesion volume and vascularization. Caliper measurements correspond to the \ndata distribution presented in the volumetric analysis. \nBiochemical Analyses \nSerum and peritoneal lavage fluid were collected at euthanasia. All protein concentrations (TNF-α, IL-\n6, VEGF, and CA -125) were determined using commercially available ELISA kits according to the \nmanufacturer’s instructions. To ensure methodological rigor,  all samples were analyzed in duplicate. \nThe sensitivity of the assays was 0.86 pg/mL for TNF -α, 7.5 pg/mL for IL -6, and 18.75 pg/mL for \nVEGF. The intra -assay and inter -assay coefficients of variation (CV) were confirmed to be less than \n10% and 12%, respec tively, ensuring high reproducibility and minimal experimental variability. \nACCEPTED MANUSCRIPT\nARTICLE IN PRESSARTICLE IN PRESS\n\n \n \nAbsorbance was read at 450 nm using a microplate reader (Thermo Scientific Multiskan Go). All assays \nwere performed under the same conditions to allow valid comparisons between groups [15]. \nHistochemical and Immunohistochemical Analyses \nExcised endometriotic implants were fixed in 4% paraformaldehyde, paraffin-embedded, and sectioned \nat 5 µm [16]. For histochemical assessment, hematoxylin and eosin staining was performed to evaluate \nglandular architecture, stromal organization, and inflammatory cell infiltration, and a histopathological \nscore was calculated [17]. Fibrosis was evaluated using Masson’s trichrome staining with aniline blue \n(BioOptica Milano S.p.A., Milan, Italy , 04 -010802), and a fibrosis score was calculated based on \ncollagen deposition [18]. For immunohistochemistry, sections were incubated with primary antibodies \nagainst CA-125 (Raybiotech, 144-61671-100), collagen I (Col 1; Bioss Antibodies, bs-10423R), VEGF \n(Bioss Antibodies, bs-0279R) and TNF -α ( Elabscience, E-AB-40015) at 1:100 dilution; Col 1  \nimmunostaining is commonly used as a fibrosis-associated marker in endometriosis tissue studies [19], \nand TNF -α/VEGF are widely used section -level inflammatory/an giogenic readouts  [20] \nImmunoreactivity was visualized using DAB chromogen and counterstained with Mayer’s hematoxylin. \nTwo histologists, blinded to group allocation, scored staining using a semi-quantitative scale (0, absent; \n1+, weak; 2+, moderate; 3+, s trong) [21]. Two investigators independently scored all histological \noutcomes, and any discrepancies were resolved by joint re -evaluation to reach a consensus score for \neach section [21]. \nStatistical Analysis \nData analyses were performed using IBM SPSS Statistics for Windows, Version 25.0 (IBM Corp., \nArmonk, NY, USA). The normality of the data distribution was assessed using the Shapiro -Wilk test. \nDescriptive statistics are presented as mean ± standard deviation (SD) for normally distributed variables \nand as median (interquartile range [IQR]) for non -normally distributed variables.  For intergroup \ncomparisons of normally distributed continuous variables, one-way analysis of variance (ANOVA) was \nemployed, followed by the Bonferroni post -hoc test for multipl e comparisons to maintain the family -\nwise error rate. For variables that did not conform to a normal distribution (e.g., serum VEGF and lesion \nvolume), the non -parametric Kruskal -Wallis test was used, with Dunn’s post -hoc test (including \nBonferroni correction) applied for pairwise comparisons. Homogeneity of variances was verified using \nLevene’s test. A p-value of < 0.05 was considered statistically significant for all tests. \n \n \n \nACCEPTED MANUSCRIPT\nARTICLE IN PRESSARTICLE IN PRESS\n\n \n \nResults \nA. Comparative evaluation of cytokines and the tumor marker in serum and peritoneal fluid \nAnalysis of systemic (serum) and local (peritoneal fluid) samples demonstrated that the untreated \nendometriosis group exhibited elevated levels of proinflammatory cytokines (TNF -α, IL -6) and the \nangiogenic factor VEGF. DP-MSCs administration shifted these readouts toward a lower-activity state, \nwith a generally stronger effect observed under the repeated -dose regimen across multiple endpoints.  \nSerum TNF-α levels were significantly increased in the endometriosis group compared with controls (p \n= 0.0207). Single-dose DP-MSCs treatment did not yield a statistically significant reduction in serum \nTNF-α. In contrast, the two -dose regimen produced a significant decrease relative to untreated \nendometriosis (p = 0.0027) and relative to the single-dose group (p = 0.0097), indicating that repeated \nadministration conferred a more robust systemic anti -inflammatory effect. In peritoneal fluid, TNF -α \ndiffered substantially across groups: levels were markedly elevated in endometriosis compared with \ncontrols (p < 0.0001) and were significantly reduced by both DP -MSCs regimens relative to untreated \nendometriosis (p < 0.0001 for both comparisons). However, peritoneal TNF -α remained significantly \nhigher in both treated groups than in controls (p < 0 .0001 for control versus endometriosis plus single \ndose, and for control versus endometriosis plus two doses), supporting partial rather than complete \nnormalization of local inflammatory activity within the observation period (Figure 4). A similar pattern \nwas observed for IL -6. Serum IL -6 was significantly higher in endometriosis than in controls (p = \n0.0003) and decreased in both DP -MSCs-treated groups compared with untreated endometriosis \n(endometriosis versus endometriosis plus single dose, p = 0.0016; e ndometriosis versus endometriosis \nplus two doses, p = 0.0008), consistent with attenuation of systemic inflammatory signaling. In \nperitoneal fluid, IL-6 showed a strong separation between controls and endometriosis (p < 0.0001) and \nwas significantly reduced by both DP-MSCs regimens relative to untreated endometriosis (p < 0.0001 \nfor both). Nevertheless, peritoneal IL-6 remained significantly elevated in treated groups compared with \ncontrols (control versus endometriosis plus single dose, p = 0.0002; control  versus endometriosis plus \ndouble dose , p = 0.0044), again indicating incomplete resolution of local inflammation during the \nfollow-up interval (Figure 4). VEGF levels demonstrated a coherent disease -associated increase and \ntreatment-associated decrease, w ith an overall pattern compatible with dose dependence. In serum, \nVEGF was higher in endometriosis than in controls (p = 0.0278) and was reduced by the double-dose \nDP-MSCs regimen (p = 0.0345). In peritoneal fluid, VEGF was markedly increased in endometrio sis \ncompared with controls (p < 0.0001) and decreased after DP -MSCs treatment (endometriosis versus \nendometriosis plus single dose, p = 0.0002; endometriosis versus endometriosis plus double dose, p = \n0.0073). In addition, the significant difference betwee n treatment regimens (endometriosis plus single \ndose versus endometriosis plus double dose, p = 0.0100) supports a stronger local anti-angiogenic effect \nwith repeated dosing (Figure 4).  \nACCEPTED MANUSCRIPT\nARTICLE IN PRESSARTICLE IN PRESS\n\n \n \nFor CA-125, serum measurements showed a significant reduction in the double- dose group compared \nwith the single -dose group (p = 0.0496), consistent with diminished lesion -related biological activity \nunder repeated DP-MSCs exposure. In peritoneal fluid, CA-125 did not exhibit statistically significant \nseparation in post hoc  analyses, suggesting either limited sensitivity of local CA -125 to short -term \ntreatment-related changes relative to inflammatory and angiogenic mediators, or the need for greater \nstatistical power or longer follow-up to resolve smaller effect sizes (Figure 4). \n  \nFigure 4. Impact of DP-MSCs on Serum and Peritoneal Biomarker Profiles.  Comparative ELISA \nquantification of TNF-α (ng/L), IL-6 (ng/L), and CA-125 (U/mL) levels in serum and peritoneal lavage \nfluid (PF) across experimental groups: Intact control (G1), Endometriosis (G2), Single-dose DP-MSCs \n(G3) and Double-dose DP-MSCs (G4). The untreated endometriosis group (G2) exhibited significantly \nelevated inflammatory and disease-associated markers compared to the sham control. In contrast, DP -\nMSCs treatment led to a dose -dependent reduction in these markers, with the double-dose group (G4) \ndemonstrating the most robust suppression. Data are presented as mean ± standard deviation (SD). \nIndividual p-values for intergroup comparisons are indicated above the brackets. Statistical significance \nwas defined as p<0.05. \n \n \n \nACCEPTED MANUSCRIPT\nARTICLE IN PRESSARTICLE IN PRESS\n\n \n \nB. Dose-Dependent Effects of Stem Cell Therapy on Lesion Volume \nThe analysis of lesion volumes across experimental groups revealed a clear downward trend \nassociated with stem cell administration. Compared to the untreated endometriosis group (G2), \na reduction in lesion volume was observed in the single -dose group (G3) and further \npronounced in the double-dose group (G4). Although the difference between G2 and G4 \napproached statistical significance (p= 0.071), it did not cross the conventional threshold of \np<.05. Furthermore, no statistically significant difference was found between the single -dose \n(G3) and double-dose (G4) treatments (p> 0.999). Notably, the G4 group exhibited the lowest \nvariance among all groups, suggesting that double -dose stem cell application may provide a \nmore consistent therapeutic response in reducing lesion size (Figure 5). \n \nFigure 5. Comparison of Lesion Volumes (mm3) Among Experimental Groups. The distribution of \nlesion sizes following stem cell applications. G2 (Untreated endometriosis group), G3 (Single-dose i.p \n2×106 DP-MSCs group) G4 (Double-dose i.p 2×106 DP-MSCs group). Data are presented as box-and-\nwhisker plots indicating median values and interquartile ranges. While a downward trend in lesion \nvolume is observed with increasing doses, the di fference between G2 and G4 did not reach statistical \nsignificance ( p=0.071). No significant difference was observed between the single and double -dose \ngroups (p>0.999).  \n \nACCEPTED MANUSCRIPT\nARTICLE IN PRESSARTICLE IN PRESS\n\n \n \nC. Histopathological assessment by histochemical stainings  \nSemi-quantitative grading (0 absent to 3 severe) indicated that untreated endometriosis displayed the \nhighest overall histopathology and fibrosis scores, consistent with active lesion biology accompanied \nby stromal remodeling. Both DP-MSCs regimens significantly reduced histological severity relative to \nuntreated endometriosis, with improvement observed in the single-dose group (p = 0.0370) and a more \npronounced reduction in the double-dose group (p < 0.0001). Fibrosis scores followed a comparable \ntrajectory, with DP-MSCs treatment decreasing fibrotic burden compared with untreated endometriosis \n(single dose, p = 0.0370; double doses, p < 0.0001). The parallel improvement across overall \nhistopathology and fibrosis supports the interpretation that DP -MSCs therapy mitigated inflammatory \ntissue injury and collagenous remodeling within endometriotic foci, with greater efficacy after repeated \nadministration (Figure 6). \n \nACCEPTED MANUSCRIPT\nARTICLE IN PRESSARTICLE IN PRESS\n\n \n \nFigure 6. Histochemical evaluation of endometriotic lesions and fibrosis.  Representative \nhematoxylin and eosin (H&E) –stained sectio ns (A) show glandular –stromal architecture and \ninflammatory cell infiltration in untreated endometriotic lesions, with reduced histological activity after \nDP-MSCs treatment, most prominently in the double-dose grou p. Masson’s trichrome staining with \naniline blue (B) highlights collagen deposition, demonstrating marked stromal fibrosis in untreated \nlesions and reduced collagen accumulation following DP-MSCs therapy. G2 (Untreated endometriosis \ngroup), G3 (Single- dose i.p 2×10 6 DP-MSCs group) G4 ( Double- dose i.p 2×10 6 DP-MSCs group).  \nScale bar: 100 µm (10×), 50 µm (20×), 20 µm (40×). \nD. Histopathological assessment by immunohistochemical stainings \nImmunohistochemical scoring demonstrated strong positivity for CA-125, VEGF, Col 1 and TNF-α in \nuntreated endometriosis, consistent with active epithelial or glandular features, heightened angiogenic \nsignaling, extracellular matrix accumulation, and robust  inflammatory pathway activation within \nlesions. DP-MSCs therapy was associated with reductions across these markers, most consistently and \nprominently in the double-dose group. CA -125 immunoreactivity was markedly decreased in the \ndouble-dose group relati ve to untreated endometriosis (p < 0.0001), supporting regression of lesion -\nassociated biological activity. Col 1 staining was significantly reduced in both DP-MSCs-treated groups \ncompared with untreated endometriosis (single dose, p = 0.0242; double dose, p = 0.0002), indicating \nan anti -fibrotic effect at the tissue level that was strengthened by repeated dosing. VEGF \nimmunoreactivity was significantly lower in the double-dose group than in untreated endometriosis (p \n= 0.0002), corroborating the biochemica l evidence for suppression of angiogenic signaling. TNF -α \nstaining was also substantially reduced in the double-dose group compared with untreated endometriosis \n(p< 0.0001), consistent with inhibition of proinflammatory pathways within the local lesion \nmicroenvironment (Figure 7). \n \nACCEPTED MANUSCRIPT\nARTICLE IN PRESSARTICLE IN PRESS\n\n \n \n \nFigure 7. Immunohistochemical assessment of lesion activity, inflammation, angiogenesis, and \nfibrosis. Representative immunohistochemical staining for CA-125, Col 1, VEGF and TNF-α in ectopic \nendometriotic lesions. Untreated endometriosi s shows strong immunoreactivity  while DP -MSCs \ntreatment reduces staining intensity and extent, with the most pronounced decrease in the double-dose \ngroup, consistent with reduced lesion act ivity, inflammatory signaling, angiogenic drive, and \nextracellular matrix remodeling. Scale bar: 20 µm (40×). \nDiscussion \nThe present study provides integrated biochemical and histopathological evidence indicating that dental \npulp–derived mesenchymal stem c ells (DP -MSCs) can modulate several key biological processes \ninvolved in experimental endometriosis. Untreated animals displayed pronounced systemic and \nperitoneal inflammatory activation, reflected by elevated TNF -α and IL -6 levels, increased VEGF \nsignaling, and higher histopathological and fibrosis scores. Administration of DP-MSCs was associated \nwith marked reductions in inflammatory cytokines and angiogenic mediators, accompanied by \nimprovements in lesion histopathology and extracellular matrix remodeli ng. Notably, several of these \neffects were more pronounced following repeated dosing, as evidenced by reductions in serum TNF -α \n(p = 0.0027), serum VEGF (p = 0.0345), and peritoneal VEGF (p = 0.0073). Collectively, these findings \nsuggest that DP-MSC therapy may exert multi-axis regulatory effects on the inflammatory, angiogenic, \nand fibrotic components of the endometriotic microenvironment [22]. \nInflammation is widely recognized as a central driver of endometriosis progression and lesion \npersistence. In line with this concept, untreated animals in the present study exhibited elevated serum \nTNF-α levels compared with controls (p = 0.0207). While a single administration of DP-MSCs did not \nACCEPTED MANUSCRIPT\nARTICLE IN PRESSARTICLE IN PRESS\n\n \n \nsignificantly modify systemic TNF-α levels, repeated dosing resulted in a significant reduction relative \nto untreated endometriosis (p = 0.0027) and relative to the single -dose group (p = 0.0097). These \nfindings suggest that sustained exposure to DP -MSC-derived regulatory signals may be necessary to \nachieve measurable systemic modulation of inflammatory cytokines. \nA similar pattern was observed for IL -6, a cytokine strongly implicated in the inflammatory cascade \nassociated with endometriosis. Serum IL -6 levels were significantly increased in untreated disease \ncompared with controls (p = 0.0003) but were markedly reduced following DP-MSC treatment in both \nintervention groups (single dose, p = 0.0016; double dose, p = 0.0008). Together, these observations \nsupport the concept that DP -MSCs attenuate systemic inflammatory signalin g in experimental \nendometriosis, consistent with the well -established immunomodulatory properties of mesenchymal \nstromal cells [23]. \nInflammatory modulation was even more evident within the peritoneal cavity, which represents the \nprimary inflammatory niche  for ectopic lesions. Both peritoneal TNF -α and IL -6 were markedly \nelevated in untreated disease compared with controls (both p < 0.0001). DP-MSC therapy significantly \nreduced these cytokines relative to untreated animals (all p < 0.0001), although levels remained higher \nthan those observed in controls (TNF-α: both p < 0.0001; IL-6: p = 0.0002 for single dose and p = 0.0044 \nfor double dose). This pattern suggests that DP -MSC treatment substantially attenuates local \ninflammatory activity but may not fully no rmalize the peritoneal inflammatory milieu within the \nrelatively short observation period of the present study [24]. \nAngiogenesis represents another critical process supporting the establishment and survival of ectopic \nendometrial tissue. Consistent with t his paradigm, serum VEGF levels were elevated in untreated \nendometriosis compared with controls (p = 0.0278). A significant reduction was observed following \nrepeated DP-MSC administration (p = 0.0345), whereas the single -dose regimen did not produce a \nstatistically significant change. Within the peritoneal cavity, VEGF concentrations were markedly \nincreased in untreated disease relative to controls (p < 0.0001) and were significantly reduced following \nDP-MSC treatment (single dose, p = 0.0002; double dose, p = 0.0073). The significant difference \nbetween treatment regimens (p = 0.0100) suggests that repeated administration may produce a stronger \nsuppression of angiogenic signaling within the lesion microenvironment. Histological findings further \nsupported these biochemical results, demonstrating reduced VEGF immunoreactivity, particularly in the \nrepeated-dose group (p = 0.0002), indicating concordant molecular and tissue-level responses [25]. \nHistopathological evaluation further supports the possibility that DP-MSC therapy may influence lesion \nseverity and stromal remodeling. Untreated animals exhibited the highest histopathological and fibrosis \nscores, whereas both treatment regimens significantly reduced these parameters (single dose, p = 0.0370; \nACCEPTED MANUSCRIPT\nARTICLE IN PRESSARTICLE IN PRESS\n\n \n \ndouble dose p < 0.0001). The more pronounced improvement observed following repeated dosing \nsuggests that treatment frequency may influence the magnitude of tissue remodeling and repair. \nConsistent immunohistochemical patterns were observed across several lesion-associated markers. CA-\n125 immunoreactivity was markedly reduced in the repeated-dose group (p < 0.0001), which may reflect \ndecreased epithelial or glandular activity within ectopic lesions. Similarly, collagen type I (C ol 1) \nstaining was significantly reduced following DP-MSC therapy (single dose, p = 0.0242; double dose, p \n= 0.0002), indicating attenuation of fibrotic remodeling. Reduced TNF -α immunoreactivity in the \nrepeated-dose group (p < 0.0001) further supports the interpretation that DP-MSC treatment modulates \ninflammatory activity directly within the lesion microenvironment [19]. \nInterestingly, CA-125 demonstrated compartment -dependent dynamics in the present model. Serum \nCA-125 levels differed between treatment regimens (double dose vs single dose, p = 0.0496), suggesting \nreduced systemic biomarker activity following repeated DP-MSC exposure. In contrast, peritoneal CA-\n125 did not show significant post hoc differences between groups. This observation aligns with previous \nstudies indicating th at CA -125 may exhibit limited sensitivity in experimental rodent models of \nendometriosis, particularly over relatively short observation intervals [26]. \nThe multi -axis modulation of inflammation, angiogenesis, and fibrosis observed in this study is \nconsistent with the broader biological profile of mesenchymal stromal cells. Increasing evidence \nindicates that many MSC -mediated therapeutic effects are primarily driven by paracrine and \nimmunomodulatory signaling rather than long -term cellular engraftment [27].  Several xenogeneic \nstudies have demonstrated measurable biological activity despite transient MSC survival [6,23,28]. \nWithin this framework, the therapeutic effects observed here may reflect transient reprogramming of \nthe lesion microenvironment mediated by DP-MSC-derived secretory factors. \nConsistent with this interpretation, lesion volume analysis demonstrated a trend toward dose-dependent \nreduction following DP -MSC administration, with the lowest mean lesion volume observed in the \nrepeated-dose group (G4). Previous experimental studies have reported that MSC -based therapies can \nsuppress the progression of endometriotic lesions through anti -inflammatory and immunomodulatory \nmechanisms [29,30]. Similarly, Sun et al. [29] and Abbas et al. [31] reported that increasing MSC dosage \nmay enhance cytokine regulation and angiogenesis suppression. In the present study, the difference \nbetween the untreated endometriosis group (G2) and the repeated-dose group (G4) approached statistical \nsignificance (p = 0.071). Although this did not reach the conventional p < 0.05 threshold—likely due to \nthe limited sample size—the observed reduction may still be biologically meaningful. The absence of a \nsignificant difference between the single- and repeated-dose groups (G3 vs G4, p > 0.999) may indicate \nthat therapeutic responses plateau beyond a certain exposure threshold or that dosing intervals require \nfurther optimization. \nACCEPTED MANUSCRIPT\nARTICLE IN PRESSARTICLE IN PRESS\n\n \n \nSeveral limitations of the present study should be acknowledged. First, the surgically induced rat model \nmay not fully replicate the complex heterogeneity and hormonal cyclicity characteristic of human \nendometriosis. Second, the study employed a fixed sacrifice time point, preventing evaluation of long-\nterm therapeutic durability, disease recurrence, or fertilit y outcomes. Third, lesion burden was not \nassessed using detailed volumetric or stereological approaches. In addition, the biodistribution, \npersistence, and homing behavior of DP -MSCs were not directly tracked. Mechanistic investigations, \nincluding immune c ell phenotyping, extracellular vesicle analysis, or transcriptomic profiling of the \nlesion microenvironment, were also beyond the scope of the present study. \nAn additional methodological consideration relates to the xenogeneic transplantation of human-derived \ncells into immunocompetent hosts. Host immune recognition typically limits long -term cellular \nengraftment; however, current evidence suggests that MSC therapeutic effects are largely mediated \nthrough transient paracrine and immunomodulatory signaling r ather than permanent cellular \nreplacement [27]. This “hit -and-run” mechanism has been supported in multiple xenogeneic models \ndemonstrating significant biological effects despite limited MSC survival [6,23,28]. \nFinally, the absence of significant alteratio ns in peritoneal CA -125 levels requires cautious \ninterpretation. Although CA-125 is widely used as a clinical biomarker, its translational value in rodent \nmodels of endometriosis remains limited because correlations with lesion burden or therapeutic response \nare inconsistent [32 –34]. Model-specific variability may arise from immune responses and surgical \ninduction techniques [1,35]. Therefore, stable CA -125 levels should not necessarily be interpreted as \nevidence of therapeutic inefficacy but rather highlight the importance of evaluating treatment responses \nusing a multi-parameter framework that integrates inflammatory cytokines, angiogenic mediators, and \nhistopathological outcomes. \nIn conclusion, DP-MSC administration significantly modulated the pathologica l microenvironment of \nexperimental endometriosis by attenuating inflammatory cytokines, suppressing angiogenic signaling, \nand reducing fibrotic remodeling within lesions. Repeated dosing produced the most consistent \nimprovements across both biochemical and histological parameters. These findings support the potential \nof DP -MSCs as a promising cell -based therapeutic strategy for modulating the endometriotic \nmicroenvironment while underscoring the need for further mechanistic and translational studies to \noptimize treatment protocols and clarify the underlying biological mechanisms [36]. \n \n \n \nACCEPTED MANUSCRIPT\nARTICLE IN PRESSARTICLE IN PRESS\n\n \n \nConclusion \nIn summary, human dental pulp -derived MSCs (DP -MSCs) effectively mitigated the hallmark \npathological features of experimental endometriosis, including pro-inflammatory activation, angiogenic \nsignaling, and fibrotic remodeling. Systematic administration significantly reduced systemic and local \nconcentrations of TNF-α, IL-6, and VEGF , with the most pronounced therapeutic efficacy observed \nunder a repeated dosing regimen . These biochemical improvements were corroborated by \nhistopathological recovery, characterized by reduced fibrosis scores and diminished lesional \nimmunoreactivity for Col1, VEGF, and TNF-α. \nWhile peritoneal inflammatory mediators did not ret urn to baseline control levels within the study \nperiod, the concordant improvements across biochemical, histochemical, and immunohistochemical \nparameters underscore the potential of DP -MSCs as a potent disease-modifying, cell-based strategy. \nOur findings highlight a clear regimen-dependent therapeutic profile, suggesting that optimized dosing \nis critical for clinical efficacy. Future investigations incorporating extended follow -up, quantitative \nlesion burden analysis, and functional endpoints are warranted to further define the durability of these \neffects and facilitate the clinical translation of DP-MSCs therapy for endometriosis. \nAckowledgement \nThe authors express their gratitude to the Scientific and Technological Research Council of Türkiye \n(TÜBİTAK), which played a major role in conducting the research. \nFunding Information \nThis study (Project number: SBAG 224S142) received both scientific and financial support from The \nScientific and Technological Research Council of Turkey (TÜBİTAK). \nData Availability Statement \nThe datasets generated during and/or analysed during the current study are available from the \ncorresponding author on reasonable request. \nConflict of Interest \nThe authors have no conflicts of interest to declare that are relevant to the content of this article. \n \n \n \nACCEPTED MANUSCRIPT\nARTICLE IN PRESSARTICLE IN PRESS\n\n \n \nReferences \n1. 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Dental pulp stem cells: from discovery to clinical application. J. Endod. 46, S46–\nS55 (2020). \n8. Tonyalı, G., et al. Simple isolation of human bone marrow adipose tissue-derived mesenchymal \nstem/stromal cells. Curr. Protoc. 5, (2025). \n9. Carmona-Luque, M., et al. The effect of cell culture passage on the efficacy of mesenchymal \nstromal cells as a cell therapy treatment. J. Clin. Med. 13, 2480 (2024). \n10. Łabędź-Masłowska, A., et al. Multilineage differentiation potential of human dental pulp s tem \ncells-Impact of 3D and hypoxic environment on osteogenesis in vitro. Int. J. Mol. Sci. 21, 6172 \n(2020). \n11. Veréb, Z., et al. Vessel wall -derived mesenchymal stromal cells share similar differentiation \npotential and immunomodulatory properties with bone ma rrow-derived stromal cells. Stem \nCells Int. 2020, 1–16 (2020). \n12. Güney, M., Oral, B., Karahan, N., Mungan, T. Regression of endometrial explants in a rat model \nof endometriosis treated with melatonin. Fertil. Steril. 89, 934–942 (2008). \n13. Fu, X., et al. Mesenchymal stem cell migration and tissue repair. Cells 8, 784 (2019). \n14. Nikolic, A., et al. Intraperitoneal administration of mesenchymal stem cells ameliorates acute \ndextran sulfate sodium-induced colitis by suppressing dendritic cells. Biomed. Pharmacother. \n100, 426–432 (2018). \n15. Lequin, R. M. Enzyme immunoassay (EIA)/Enzyme -linked immunosorbent assay (ELISA). \nClin. Chem. 51, 2415–2418 (2005). \n16. Wang, X., et al. Evaluation of prevention and treatment effects of fibroblast growth factor-21 in \nBLM-induced pulmonary f ibrosis. Naunyn-Schmiedebergs Arch Pharmacol . 396, 3299–3313 \n(2023). \nACCEPTED MANUSCRIPT\nARTICLE IN PRESSARTICLE IN PRESS\n\n \n \n17. Stasi, A., et al. PMMA-based continuous hemofiltration modulated complement activation and \nrenal dysfunction in LPS-induced acute kidney injury. Front. Immunol. 12, (2021). \n18. Han, S., et al. 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Harnessing the mesenchymal stem cell secretome for regenerative medicine. \nNature Reviews Urology 16, 363–375 (2019 \n30. Li, X., et al. Mesenchymal stem cells in the treatment of endometriosis: mechanisms and \ntherapeutic potential. Stem Cell Research & Therapy 12, 1–12 (2021) \n31. Abbas, M., et al. Mesenchymal stem cell therapy in endometriosis: immunomodulatory and \nanti-angiogenic mechanisms. Reproductive Sciences 27, 1100–1110 (2020). \n32. Mol, B. W. J., et al. The performance of CA-125 measurement in the detection of endometriosis: \na meta-analysis. Fertility and Sterility 70, 1101–1108 (1998) \n33. May, K. E., et al. Peripheral biomarkers of endometriosis: a systematic review. Human \nReproduction Update 17, 651–674 (2011). \nACCEPTED MANUSCRIPT\nARTICLE IN PRESSARTICLE IN PRESS\n\n \n \n34. Hirsch, M., et al. Diagno stic accuracy of cancer antigen 125 for endometriosis: a systematic \nreview and meta-analysis. BJOG 123, 1761–1768 (2016). \n35. Güney, M., et al. Evaluation of serum and peritoneal CA-125 levels in an experimental rat model \nof endometriosis. European Journal of Obstetrics & Gynecology and Reproductive Biology 137, \n95–99 (2008) \n36. Meligy, F., et al. Adipose tissue-derived mesenchymal stem cells reduce endometriosis cellular \nproliferation through their anti -inflammatory effects. Clin. Exp. Reprod. Med.  48, 322–336 \n(2021).   \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \nACCEPTED MANUSCRIPT\nARTICLE IN PRESSARTICLE IN PRESS\n\n \n \nFigure Legends  \nFigure 1. Morphological and immunophenotypic characterization of DP -MSCs. Representative \ninverted phase-contrast micrographs illustrate the adherent, spindle-shaped, fibroblast-like morphology \nof DP-MSCs. The upper images display the cells during the early expansion phase (Initial isolation, \nP1-P2); the middle images represent the confluent culture prior to cryopreservation; and the lower \nimages show the cells after thawing and subsequent re -culture. The consistent morphology observed \nacross all stages indicates that the cryopreservation and thawing processes do not adversely affect the \ntypical MSC-like appearance or the plastic-adherence capacity of the cells. \nFigure 2. Immunophenotypic characterizati on of DP -MSCs. Representative flow cytometry dot \nplots showing the gating strategy based on forward (FSC) and side scatter (SSC) ( Gate A, 93.83% ). \nAnalysis of MSC markers: DP-MSCs show high expression levels for CD90 (99.91%), CD44 (95.64%), \nCD105 (98.32%) , and CD73 (99.74%) . Exclusion of hematopoietic lineages: cells show negative \nexpression for CD34 and CD45 (Gate G, 1.56%), confirming their non-hematopoietic origin. Data tables \nprovide event counts, percentage of gated cells (%Gated), and mean fluorescen ce intensity (MFI) for \neach marker. \nFigure 3. Macroscopic evaluation and quantitative measurement of endometriotic lesions across \nexperimental groups. Top row (G2, G3, G4) shows the representative in situ macroscopic appearance \nof the lesions during laparo tomy. Bottom row  (G2-1, G3 -1, G4 -1) displays the harvested lesions \nmeasured with a digital caliper to calculate the volumes. G2: Untreated endometriotic lesion group \nexhibiting well-vascularized, large cystic structures. G3: Single-dose DP-MSCs therapy group showing \na moderate reduction in lesion size. G4: Double-dose DP -MSCs therapy group showing the most \nsignificant regression in lesion volume and vascularization. Caliper measurements correspond to the \ndata distribution presented in the volumetric analysis. \nFigure 4. Impact of DP-MSCs on Serum and Peritoneal Biomarker Profiles . Comparative ELISA \nquantification of TNF-α (ng/L), IL-6 (ng/L), and CA-125 (U/mL) levels in serum and peritoneal lavage \nfluid (PF) across experimental groups: Intact control (G1), Endometriosis (G2), Single-dose DP-MSCs \n(G3) and Double-dose DP-MSCs (G4). The untreated endometriosis group (G2) exhibited significantly \nelevated inflammatory and disease-associated markers compared to the sham control. In contrast, DP -\nMSCs treatment led to a dose-dependent reduction in these markers, with the double -dose group (G4) \ndemonstrating the most robust suppression. Data are presented as mean ± standard deviation (SD). \nIndividual p-values for intergroup comparisons are indicated above the brackets. Statistical significance \nwas defined as p<0.05. \nACCEPTED MANUSCRIPT\nARTICLE IN PRESSARTICLE IN PRESS\n\n \n \nFigure 5. Comparison of Lesion Volumes (mm3) Among Experimental Groups. The distribution of \nlesion sizes following stem cell applications. G2 (Untreated endometriosis group), G3 (Single-dose i.p \n2×106 DP-MSCs group) G4 (Double-dose i.p 2×106 DP-MSCs group). Data are presented as box-and-\nwhisker plots indicating median values and interquartile ranges. While a downward trend in lesion \nvolume is observed with increasing doses, the difference between G2 an d G4 did not reach statistical \nsignificance ( p=0.071). No significant difference was observed between the single and double -dose \ngroups (p>0.999).  \nFigure 6. Histochemical evaluation of endometriotic lesions and fibrosis. Representative \nhematoxylin and eos in (H&E) –stained sections ( A) show glandular –stromal architecture and \ninflammatory cell infiltration in untreated endometriotic lesions, with reduced histological activity after \nDP-MSCs treatment, most prominently in the double -dose group. Masson’s trichro me staining with \naniline blue ( B) highlights collagen deposition, demonstrating marked stromal fibrosis in untreated \nlesions and reduced collagen accumulation following DP-MSCs therapy. G2 (Untreated endometriosis \ngroup), G3 (Single - dose i.p 2×10 6 DP-MSCs group) G4 (Double - dose i.p 2×10 6 DP-MSCs group).  \nScale bar: 100 µm (10×), 50 µm (20×), 20 µm (40×). \nFigure 7. Immunohistochemical assessment of lesion activity, inflammation, angiogenesis, and \nfibrosis. Representative immunohistochemical staining for CA-125, Col 1, VEGF and TNF-α in ectopic \nendometriotic lesions. Untreated endometriosis shows strong immunoreactivity while DP -MSCs \ntreatment reduces staining intensity and extent, with the most pronounced decrease in the double -dose \ngroup, consisten t with reduced lesion activity, inflammatory signaling, angiogenic drive, and \nextracellular matrix remodeling. Scale bar: 20 µm (40×). \n \nACCEPTED MANUSCRIPT\nARTICLE IN PRESSARTICLE IN PRESS","source_license":"CC0","license_restricted":false}