{"paper_id":"2b56c5c4-d88a-4d68-9370-1f3eb2fa0a52","body_text":"Citation: Mielke Cabello, L.A.;\nMeresman, G.; Darici, D.; Carnovale,\nN.; Heitkötter, B.; Schulte, M.;\nEspinoza-Sánchez, N.A.; Le, Q.-K.;\nKiesel, L.; Schäfer, S.D.; et al.\nAssessment of the Ferroptosis\nRegulators: Glutathione Peroxidase 4,\nAcyl-Coenzyme A Synthetase\nLong-Chain Family Member 4, and\nTransferrin Receptor 1 in\nPatient-Derived Endometriosis Tissue.\nBiomolecules 2024, 14, 876. https://\ndoi.org/10.3390/biom14070876\nAcademic Editor: Mingqing Li\nReceived: 14 May 2024\nRevised: 14 July 2024\nAccepted: 19 July 2024\nPublished: 21 July 2024\nCopyright: © 2024 by the authors.\nLicensee MDPI, Basel, Switzerland.\nThis article is an open access article\ndistributed under the terms and\nconditions of the Creative Commons\nAttribution (CC BY) license (https://\ncreativecommons.org/licenses/by/\n4.0/).\nbiomolecules\nArticle\nAssessment\nof the Ferroptosis Regulators: Glutathione\nPeroxidase 4, Acyl-Coenzyme A Synthetase Long-Chain Family\nMember 4, and Transferrin Receptor 1 in Patient-Derived\nEndometriosis Tissue\nLidia A. Mielke Cabello 1,*, Gabriela Meresman 2\n ,\nDogus Darici 3, Noelia Carnovale 2, Birthe Heitkötter 4,\nMiriam Schulte 4, Nancy A. Espinoza-Sánchez 1\n ,\nQuang-Khoi Le 1\n ,\nLudwig Kiesel 1\n ,\nSebastian D. Schäfer 1,†\nand Martin Götte 1,*,†\n1 Department\nof Gynecology and Obstetrics, University Hospital of Muenster, 48149 Muenster, Germany;\nludwig.kiesel@ukmuenster.de (L.K.); seb.schaefer@alexianer.de (S.D.S.)\n2 Institute of Biology and Experimental Medicine IBYME-CONICET, Buenos Aires C1428, Argentina;\ngabriela.meresman@gmail.com (G.M.)\n3 Institute of Anatomy and Molecular Neurobiology, University Hospital of Muenster,\n48149 Muenster, Germany\n4 Gerhard-Domagk-Institute of Pathology, University Hospital of Muenster, 48149 Muenster, Germany\n* Correspondence: lidiaandrea.mielke@ukmuenster.de (L.A.M.C.); mgotte@uni-muenster.de (M.G.)\n† These authors contributed equally to this work.\nAbstract: Ferroptosis, an iron-dependent form of non-apoptotic cell death, plays a pivotal role in\nvarious diseases and is gaining considerable attention in the realm of endometriosis. Considering the\nclassical pathomechanism theories, we hypothesized that ferroptosis, potentially driven by increased\niron content at ectopic sites, may contribute to the progression of endometriosis. This retrospective\ncase–control study provides a comprehensive immunohistochemical assessment of the expression\nand tissue distribution of established ferroptosis markers: GPX4, ACSL4, and TfR1 in endometriosis\npatients. The case group consisted of 38 women with laparoscopically and histologically confirmed\nendometriosis and the control group consisted of 18 women with other gynecological conditions.\nOur study revealed a significant downregulation of GPX4 in stromal cells of endometriosis pa-\ntients (M = 59.7% ± 42.4 versus 90.0% ± 17.5 in the control group, t (54) = −2.90, p = 0.005). This\nfinding aligned with slightly, but not significantly, higher iron levels detected in the blood of en-\ndometriosis patients, using hemoglobin as an indirect predictor (Hb 12.8 (12.2–13.5) g/dL versus\n12.5 (12.2–13.4) g/dL in the control group; t (54) = −0.897, p = 0.374). Interestingly, there was no\nconcurrent upregulation of TfR1 (M = 0.7 ± 1.2 versus 0.2 ± 0.4 for EM, t (54) = 2.552, p = 0.014),\nresponsible for iron uptake into cells. Our empirical findings provide support for the involvement of\nferroptosis in the context of endometriosis. However, variances in expression patterns within stromal\nand epithelial cellular subsets call for further in-depth investigations.\nKeywords: endometriosis; ferroptosis; biomarkers; GPX4; ACSL4; TfR1; iron\n1.\nIntroduction\nEndometriosis (EM) is a prevalent, reproductive, estrogen-dependent, inflammatory\ndisorder characterized by the presence of endometrial-like tissue growing abnormally\noutside the uterus [1]. Despite being first described almost a century ago, the precise causes,\nmechanisms, and pathways contributing to the development and progression of various\nEM subtypes remain unclear. With a global prevalence affecting around 10% of women,\nEM represents a significant health concern. Notably, 30 to 50% of symptomatic patients\nexperience chronic pelvic pain and/or infertility, the two hallmark symptoms of EM [2,3].\nBiomolecules 2024, 14,\n876. https://doi.org/10.3390/biom14070876 https://www.mdpi.com/journal/biomolecules\n\nBiomolecules 2024, 14, 876 2 of 13\nEM can be categorized into three main subtypes based on its histopathology and\nanatomical locations: superficial endometriosis (SUP), deep infiltrating endometriosis\n(DIE), and ovarian endometriosis (OMA) [3,4]. Additionally, adenomyosis, found within\nthe uterus, is characterized by endometrial tissue surrounded by smooth muscle cells\nwithin the myometrium. Interestingly, these various forms of EM often coexist, hinting at\nshared developmental pathways among the different types [3,5–7].\nVarious theories, including the widely accepted Sampson’s theory of retrograde men-\nstruation, suggest that endometrial tissue fragments, cells, and protein-rich fluid can reflux\nthrough the fallopian tubes, ultimately reaching the peritoneal cavity, particularly within\nthe pelvis [2,3,8]. These cells utilize a molecular strategy to adhere to the serosal surface and\nendure initial ischemic conditions. Consequently, the immune system perceives the mis-\nplaced endometrium as foreign, triggering an inflammatory response, aided by the high iron\ncontent in menstrual blood [3,6,9]. Individuals with a dysregulated inflammatory response\nmay activate cancer-associated pathways like nuclear factor kappa-light-chain-enhancer of\nactivated B cells (NF-κB), potentially due to genetic and epigenetic alterations [3]. This in-\ntriguingly mirrors the cancer-like characteristics exhibited by EM, despite its benign nature.\nElevated levels of inflammatory mediators, hormones, and immune cells are observed in\nthe tissue microenvironment and peritoneal fluid of patients with EM. These components\ncontribute to survival, implantation, invasion, angiogenesis, and immunosurveillance\nevasion in endometriotic lesions [2,10].\nThe emergence and progression of DIE, which infiltrates deep organ tissue layers\nand structures, remain unclear. Although the notion that it is an extension of SUP is\ninsufficient, the angiolymphatic dissemination and/or stem cell theory appear more fitting,\ndespite its limitations [ 3,5,6]. The tissue injury and repair theory (TIAR) suggests that\nthe elevated estrogen and progesterone resistance in adenomyosis triggers the oxytocin\nreceptors, inducing hyperperistalsis and leading to changes in the endometrial–myometrial\njunctional barrier, myometrial injury, fibrosis, smooth muscle metaplasia, and microlesions,\nallowing cells to migrate like in cancer metastasis [5,7,11,12].\nWhile bone-marrow-derived stem cells may play a role in rare cases, as in individuals\nwith Mayer–Rokitansky–Küster–Hauser syndrome and in men, chronic inflammation and\ndisease progression are more likely to occur due to immune responses and activation of\ncertain pathways in an iron-rich environment [2,6,13].\nOriginally described as “oxytosis” in 1989, the term “ferroptosis”, coined by Dixon et al.\nin 2012, describes a distinct type of iron-dependent cell death. This emerging concept is gain-\ning significance and attention for its implications in various diseases, including EM [9,14,15].\nFerroptosis is morphologically, biochemically, and genetically divergent from apoptosis,\nnecrosis, and autophagy [16,17]. The influx of iron into cells via transferrin receptor 1 (TfR1)\nor through the divalent metal transporter 1 (DMT1) leads to an overproduction of reactive\noxygen species (ROS) due to a redox imbalance, causing substantial lipid peroxidation in\ncell membranes and ultimately resulting in cell death [9,17].\nCellular iron is intricately regulated, with balance mechanisms controlling the stability\nand translation of specific iron-related messenger ribonucleic acids (mRNAs) coding for\nferritin, transferrin, TfR1, DMT1, among others [18].\nPhysiologically, ingested iron is absorbed in duodenal enterocytes, a process regulated\nby hepcidin. Once in the bloodstream, iron can enter cells through TfR1, bound to transfer-\nrin in its ferric form (Fe3+), or through DMT1 in its divalent ferrous form (Fe2+) [18–21]. The\nmetalloreductase six-transmembrane epithelial antigen of prostase 3 (STEAP3) converts\nthe insoluble ferric form of iron into its soluble ferrous form, contributing to the cellular\niron pool required for ferroptosis. Conversely, ferroportin (FPN) facilitates the efflux of\niron from the cell [17,20,22]. In a state of inflammation, this delicate balance is disrupted.\nFerroptosis is tightly regulated at various levels, impacting both systemic and local\niron homeostasis. This process can spread to adjacent cells rapidly, creating a propagating\nwave [15,17].\n\nBiomolecules 2024, 14, 876 3 of 13\nSeveral proteins and genes participate in inducing ferroptosis through diverse path-\nways. Identified antibodies, such as anti-TfR1, have proven effective in detecting cells\nundergoing ferroptosis in various contexts. A study by Feng et al. demonstrated the\nreliability of anti-TfR1 in indicating ferroptosis, applicable in both tissue sections and cell\ncultures [23].\nCentral to ferroptosis, glutathione peroxidase 4 (GPX4) and acyl-coenzyme A syn-\nthetase long-chain family member 4 (ACSL4) play pivotal roles as positive and negative\nregulators, respectively [24].\nGPX4 inhibits the formation of lipid peroxides. It converts glutathione (GSH) into\noxidized glutathione (GSSG) and reduces toxic lipid peroxides (L-OOH) to alcohols (L-OH).\nInhibition or downregulation of GPX4 results in the accumulation of lipid peroxides and\nincreases sensitivity to ferroptosis [9,15,19,25,26].\nACSL4 is crucial for fatty acid metabolism and lipid peroxidation [ 23,26]. Its role\ninvolves enhancing the content of polyunsaturated fatty acids (PUFAs) in phospholipids.\nFree PUFAs undergo esterification into membrane phospholipids and oxidation to transmit\nthe ferroptosis signal. While ACSL4 is generally associated with ferroptosis, it may not be\nrequired in all cases, suggesting that ACSL4-depleted cells can undergo ferroptosis under\nspecific circumstances. Inhibiting the expression of ACSL4 has been shown to reduce lipid\nperoxide accumulation and diminish ferroptosis [15,17,26].\nConsidering the classical pathomechanism theories of EM, we hypothesized that\nferroptosis, potentially driven by increased iron content at ectopic sites, may contribute\nto the progression of the disease. We, therefore, studied the expression of established\nferroptosis biomarkers—GPX4, ACSL4, and TfR1—by immunohistochemical analysis on\nEM tissue and on the eutopic endometrium of the controls.\n2. Materials and Methods\n2.1. Study Population\nThis retrospective case–control study included women aged 19 to 39. Inclusion criteria\nfor the case group were laparoscopic and histological confirmation of EM in women who\nunderwent surgery between 2021 and 2022 by the same surgeon at the Department of\nGynecology and Obstetrics at the University Hospital in Muenster, Germany. Exclusion\ncriteria were ages under 18 or over 50 years and concurrent diagnosis of malignancies. The\nseverity of the disease was classified according to the rASRM score (Revised Classification\nof the American Society for Reproductive Medicine), ranging from stage I (minimal) to\nstage IV (severe). For a precise description, including the anatomical location, the size of\nthe endometriotic lesions, and the involvement of the genital tract and adjacent organs,\nthe ENZIAN and #ENZIAN classifications were utilized [27,28]. In the control group, we\nincluded women with other gynecological conditions such as abnormal uterine bleeding,\npolyps, or infertility who underwent endometrial scraping between 2017 and 2022. Patients\nwith malignant disorders or histologically confirmed endometriosis were excluded from the\ncontrol group. Anthropometric and clinical data were obtained from the patients’ records.\nThe hemoglobin value was obtained from the standard pre-surgical blood analysis.\n2.2. Ethical Approval\nThe study was designed under consideration of the principles of the Helsinki Conven-\ntion and was approved by the ethics committee of Westphalia-Lippe (1 IX Greb, from 19\nSeptember 2001, updated 2012).\n2.3. Immunohistochemistry\nThe surgically obtained tissue samples were immediately fixed in 10% neutral-buffered\nformalin and embedded in paraffin according to the standard procedures. Serial sections of\n5 µm were transferred to poly-L-lysine-coated slides and stained with hematoxylin and\neosin for fine tissue examination. EM samples from different locations were selected for this\nstudy. Samples of eutopic endometrium from women without EM were used as controls.\n\nBiomolecules 2024, 14, 876 4 of 13\nDried slides were deparaffinized, rehydrated, and treated with target retrieval solution\n(pH 6.0) for 10 min in a steamer, followed by a wash in phosphate-buffered saline (PBS). Sec-\ntions were blocked with peroxidase (Dako, Denmark A/S, Glostrup, Denmark) for 10 min,\nfollowed by a second block with 4% BSA (bovine serum albumin, Dako, Denmark A/S,\nGlostrup, Denmark) for 60 min. Sections were then incubated overnight at 4 ◦C with the\nfollowing primary antibodies: mouse anti-TfR1 monoclonal antibody (1:20 = 0.05 mg/mL;\nRRID: AB_10966364; Cat# MA5-11441, ThermoFisher, Waltham, MA, USA), mouse anti-\nACSL4 monoclonal antibody (1:100 = 0.029 mg/mL; RRID: AB_2787171; Cat# MA5-31543,\nThermoFisher, Waltham, MA, USA), and rabbit anti-GPX4 recombinant monoclonal anti-\nbody (1:100 = 0.01 mg/mL; RRID: AB_2810103; Cat# MA5-32827, ThermoFisher, Waltham,\nMA, USA). Subsequently, sections were treated for 60 min with the corresponding sec-\nondary biotinylated antibody (goat anti-rabbit IgG, 1:200, B7389; Sigma-Aldrich, Burlington,\nMA, USA; or goat anti-mouse IgG, 1:200, BA-9200-1.5; Vector-Laboratories, Newark, CA,\nUSA), followed by incubation with streptavidin–peroxidase (Roche Diagnostics GmbH,\nMannheim, Germany) for 30 min. The binding was visualized by incubating sections with\ndiaminobenzidine (DAB; Roche Diagnostics GmbH, Mannheim, Germany) and lightly\ncounterstaining with hematoxylin before permanent mounting. A non-relevant antibody\nof the same species (mouse, rabbit) and the same immunoglobulin isotype (IgG1) was used\nas a negative control. We conducted positive controls with each antibody following the\nproduct information guidelines and referring to previously conducted studies: mouse testis\ntissue for GPX4, mouse epididymis for ACSL4, and mouse liver for TfR1 [19,23,29]. The\ntissue sections for the positive controls were sourced from the Institute of Biology and\nExperimental Medicine (IBYME-CONICET) in Buenos Aires, Argentina.\n2.4. Microscopic Evaluation\nA random subset of the slides (around 10%) was evaluated blindly by three inde-\npendent investigators, two of whom are expert pathologists at the University Hospital in\nMuenster, Germany. The full dataset was evaluated by one pathologist.\nBiopsy tissue sections were analyzed at ×40, ×100, and ×400 magnification using an\nOlympus BX51 Fluorescence microscope with an integrated imaging system from Hologic\nand photographed using a Sony E 18–55 mm f/3.5–5.6 OSS digital pathology scanner (Sony\nCorporation, Minato, Tokyo, Japan).\nFor the immunohistochemical analysis, staining intensity of ACSL4, GPX4, and TfR1\nwas evaluated at ×400 magnification for each cell type. It was assessed according to a score\nof 0: light staining, 1: medium staining, and 2: intense staining.\nEach of the used antibodies showed cytoplasmic staining of epithelial, stromal, or both\ncell types. For the quantitative analysis, a percentage ranging between 0% and 100% was\nassigned separately for each cell type.\n2.5. Statistical Analysis\nStatistical analysis was conducted using SPSS v. 29.0 for Mac (SPSS, Chicago, IL,\nUSA). The graphs were generated using GraphPad Prism v. 9 for Mac (GraphPad Software,\nBoston, MA, USA). For descriptive analysis, mean and standard deviations were calculated\nfor normally distributed continuous variables, while median and quartiles were used for\nnon-normally distributed variables. Additionally, categorical variables were presented\nwith absolute and relative frequencies.\nTwo-sided t-tests for independent samples with Bonferroni correction were applied\nto compare the protein expression between groups. Additionally, non-parametric Mann–\nWhitney U-tests were conducted for non-normally distributed outcomes. Pearson’s correla-\ntion was used to analyze the correlation between the variables. The clinical characteristics\nwere analyzed as categorical variables by the Chi-squared test. All statistical analyses were\nperformed with a significance value of alpha = 0.05, where a p-value < 0.05 was considered\nstatistically significant. A power analysis was conducted with an alpha = 0.05, power = 0.80,\nand effect size d = 0.80.\n\nBiomolecules 2024, 14, 876 5 of 13\n3. Results\n3.1. Patient Characteristics\nThe anthropometric and clinical characteristics of the women included in this study\nare presented in Tables1 and 2. Out of a total of 56 women, 38 had laparoscopically and\nhistologically confirmed EM. The collected samples were derived from different anatomical\nlocations: pelvic sidewall (9), sacrouterine ligament (5), bladder peritoneum (5), recto-\nvaginal space (5), rectum (3), endometrioma (3), vaginal wall (2), pararectal space (2),\ndiaphragm (1), abdominal wall (1), appendix vermiformis (1), and ureter (1). The samples\nwere analyzed altogether (i.e., Table 3). The interrater reliability (one-way random model)\nbetween all three raters was CCC = 0.942, 95% CI: 0.916; 0.960, p < 0.001, showing high\nreliability according to Landis and Koch [30]. In the control group of 18 women, the absence\nof EM was histologically confirmed. No significant correlations between the location and\nstage of the disease were found.\nWe observed slightly, but not significantly higher, hemoglobin levels in patients with\nEM, serving as an indirect predictor of iron levels (Hb 12.8 (12.2–13.5) g/dL versus 12.5\n(12.2–13.4) g/dL in the control group; t (54) = −0.897, p = 0.374). The complete clinical data\nare available in the Supplementary Materials (i.e., Supplementary Table S1).\nThe power analysis (alpha = 0.05, power = 0.80, and effect size d = 0.80) indicated a mini-\nmum sample size of 26 per group. Therefore, the current study was slightly underpowered.\nTable 1. Anthropometric and clinical characteristics of cases and controls.\nAll Women\nN = 56\nMedian (25th–75th\nPercentile)\nEndometriosis\nn = 38\nMedian (25th–75th\nPercentile)\nControls\nn = 18\nMedian (25th–75th\nPercentile)\np-Value\nAge 30.5 (26.0–35.0) 29.8 (25.0–37.0) 32.1 (30.5–36.3) n.s.\nBMI (kg/m2) 24.9 (21.0–26.8) 24.7 (21.0–31.9) 25.4 (21.0–29.5) n.s.\nHb (g/dL) 12.7 (12.2–13.5) 12.8 (12.2–13.5) 12.5 (12.2–13.4) n.s.\nBMI = body mass index; Hb = hemoglobin; p < 0.05 was considered statistically significant. n.s. = not statisti-\ncally significant.\nTable 2. Clinical characteristics of cases and controls.\nAll Women\nN = 56\nEndometriosis\nn = 38\nControls\nn = 18 p-Value\nPast hormone therapy 39 30 9 0.028\nCurrent hormone therapy 17 16 1 0.005\nDysmenorrhea 35 35 0 -\nInfertility 17 5 12 0.000\nIrregular cycle 22 20 2 0.003\nBleeding disorder 25 16 9 n.s.\nAnalgesics 26 26 0 -\nPast hormone therapy = gestagene pill, combined oral contraceptive pill or hormonal intrauterine device\n>3 months; current hormone therapy = gestagene pill, combined oral contraceptive pill or hormonal intrauter-\nine device; infertility = failure to achieve a pregnancy after 12 months or more of regular unprotected sexual\nintercourse (WHO, 2023); irregular cycle = shortest to longest cycle variation > 10 days; bleeding disorder = menor-\nrhagia, metrorrhagia, menometrorrhagia, polymenorrhea, hypermenorrhea, oligomenorrhea, and intermenstrual\nbleeding; analgesics: NSAR (non-steroidal anti-inflammatory drug), opioids. p < 0.05 was considered statistically\nsignificant. n.s. = not statistically significant.\n\nBiomolecules 2024, 14, 876 6 of 13\nTable 3. Endometriosis stage and classification.\nrASRM I\nn = 10\nrASRM II\nn = 9\nrASRM III\nn = 9\nrASRM IV\nn = 10\nSUP 10 9 8 9\nDIE 2 5 9 10\nP 8 9 9 10\nO 0 3 6 5\nT 0 2 5 8\nA 1 1 5 9\nB 0 4 7 10\nC 1 1 4 9\nFA 9 8 9 10\nFB 0 1 2 1\nFI 0 0 1 2\nFU 0 0 0 1\nF 1 1 1 0\nSUP = superficial endometriosis; DIE = deep infiltrating endometriosis; P = peritoneum; O = ovary; T = tube;\nA = rectovaginal space, vagina, retrocervical Area; B = acrouterine ligaments, cardinal ligaments, pelvic sidewall;\nC = rectum; FA = adenomyosis; FB = blader; FI = intestinum; FU = ureter; F = diaphragm, lung, nerve. The\nENZIAN classification describes the deep infiltrating endometriosis lesions with A, B, C, FA, and F. The #ENZIAN\nclassification is an extension of the ENZIAN classification, that also describes superficial lesions with P , O, T, A, B,\nC, FA, FB, FI, FU, F (. . .).\n3.2. Staining Pattern of ACSL4, GPX4, and TfR1\nACSL4, GPX4, and TfR1 exhibit cytoplasmic staining in cells. GPX4 displayed the\nstrongest expression in terms of both the percentage and staining intensity of cells (i.e.,\nFigure 1). Staining was prominently observed in the cytoplasm of stromal cells and in the\nepithelial cells of the glands. In the case of the endometriotic lesion samples, staining was\nobserved throughout the gland, even extending to some nuclei (i.e., Figure 1A).\nBiomolecules 2024, 14, x FOR PEER REVIEW 7 of 15 \n \n \nFigure 1. Expression of GPX4. (A) Ectopic endometrium (vaginal wall) of endometriosis patient. (B) \nEutopic endometrium of control. (C) Mouse testis as positive control. (D) Eutopic endometrium of \ncontrol treated with an unrelated monoclonal antibody of the same isotype as negative control. ×400 \nmagnification. Scale bar = 200 µm. \n \nFigure 2. Expression of ACSL4. (A) Ectopic endometrium (ureter) of endometriosis patient. (B) Eu-\ntopic endometrium of control. (C) Mouse epididymis as positive control. (D) Ectopic endometrium \n(sacrouterine ligament) treated with an unrelated monoclonal antibody of the same isotype as neg-\native control. ×400 magnification. Scale bar = 200 µm. \n \nFigure 3. Expression of TfR1. (A) Ectopic endometrium (abdominal wall) of endometriosis patient. \n(B) Eutopic endometrium of control. (C) Mouse liver as positive control. (D) Eutopic endometrium \nof control treated with an unrelated monoclonal an tibody of the same isotype as negative control. \n×400 magnification. Scale bar = 200 µm. \n3.3. GPX4, ACSL4, and TfR1 Expression Levels \nThe strongest expression was observed in stromal cells with the GPX4 antibody. The \npercentage of stained stromal cells was higher in the control group with M = 90.0% ± 17.5 \nversus 59.7% ± 42.4 in the EM group, t (54) = −2.90, p = 0.005 (i.e., Figure 4, Supplementary \nTable S2). The intensity of the staining of stromal cells was stronger in the EM group with \nM = 1.0 ± 0.7 versus 1.4 ± 0.6 in the control group, t (54) = 2.22, p = 0.031 (i.e., Table 4). \nComparing the expression of the di fferent antibodies between EM patients and the \ncontrol group, we observed a similar staini ng for GPX4 and ASCL4. Both antibodies \nFigure 1. Expression of GPX4. (A) Ectopic endometrium (vaginal wall) of endometriosis patient.\n(B) Eutopic endometrium of control. (C) Mouse testis as positive control. (D) Eutopic endometrium\nof control treated with an unrelated monoclonal antibody of the same isotype as negative control.\n×400 magnification. Scale bar = 200 µm.\nHowever, in the control samples, staining was predominantly restricted to the luminal\nepithelium of the glands (i.e., Figure 1B).\nACSL4 exhibited a similar staining pattern in terms of both cell count and intensity\n(i.e., Figure 2A,B). In the case of TfR1, very mild to almost imperceptible staining was\nobserved in the cytoplasm of both stromal and epithelial cells (i.e., Figure 3A,B).\nThe positive controls displayed a cytoplasmatic staining of the cells of mouse testis (i.e.,\nFigure 1C), mouse epididymis (i.e., Figure 2C), and mouse liver (i.e., Figure 3C). Negative\ncontrols showed no staining (i.e., Figures 1D, 2D and 3D).\n\nBiomolecules 2024, 14, 876 7 of 13\nBiomolecules 2024, 14, x FOR PEER REVIEW 7 of 15 \n \n \nFigure 1. Expression of GPX4. (A) Ectopic endometrium (vaginal wall) of endometriosis patient. (B) \nEutopic endometrium of control. (C) Mouse testis as positive control. (D) Eutopic endometrium of \ncontrol treated with an unrelated monoclonal antibody of the same isotype as negative control. ×400 \nmagnification. Scale bar = 200 µm. \n \nFigure 2. Expression of ACSL4. (A) Ectopic endometrium (ureter) of endometriosis patient. (B) Eu-\ntopic endometrium of control. (C) Mouse epididymis as positive control. (D) Ectopic endometrium \n(sacrouterine ligament) treated with an unrelated monoclonal antibody of the same isotype as neg-\native control. ×400 magnification. Scale bar = 200 µm. \n \nFigure 3. Expression of TfR1. (A) Ectopic endometrium (abdominal wall) of endometriosis patient. \n(B) Eutopic endometrium of control. (C) Mouse liver as positive control. (D) Eutopic endometrium \nof control treated with an unrelated monoclonal an tibody of the same isotype as negative control. \n×400 magnification. Scale bar = 200 µm. \n3.3. GPX4, ACSL4, and TfR1 Expression Levels \nThe strongest expression was observed in stromal cells with the GPX4 antibody. The \npercentage of stained stromal cells was higher in the control group with M = 90.0% ± 17.5 \nversus 59.7% ± 42.4 in the EM group, t (54) = −2.90, p = 0.005 (i.e., Figure 4, Supplementary \nTable S2). The intensity of the staining of stromal cells was stronger in the EM group with \nM = 1.0 ± 0.7 versus 1.4 ± 0.6 in the control group, t (54) = 2.22, p = 0.031 (i.e., Table 4). \nComparing the expression of the di fferent antibodies between EM patients and the \ncontrol group, we observed a similar staini ng for GPX4 and ASCL4. Both antibodies \nFigure 2. Expression of ACSL4. (A) Ectopic endometrium (ureter) of endometriosis patient. (B) Eu-\ntopic endometrium of control. (C) Mouse epididymis as positive control. (D) Ectopic endometrium\n(sacrouterine ligament) treated with an unrelated monoclonal antibody of the same isotype as negative\ncontrol. ×400 magnification. Scale bar = 200 µm.\nBiomolecules 2024, 14, x FOR PEER REVIEW 7 of 15 \n \n \nFigure 1. Expression of GPX4. (A) Ectopic endometrium (vaginal wall) of endometriosis patient. (B) \nEutopic endometrium of control. (C) Mouse testis as positive control. (D) Eutopic endometrium of \ncontrol treated with an unrelated monoclonal antibody of the same isotype as negative control. ×400 \nmagnification. Scale bar = 200 µm. \n \nFigure 2. Expression of ACSL4. (A) Ectopic endometrium (ureter) of endometriosis patient. (B) Eu-\ntopic endometrium of control. (C) Mouse epididymis as positive control. (D) Ectopic endometrium \n(sacrouterine ligament) treated with an unrelated monoclonal antibody of the same isotype as neg-\native control. ×400 magnification. Scale bar = 200 µm. \n \nFigure 3. Expression of TfR1. (A) Ectopic endometrium (abdominal wall) of endometriosis patient. \n(B) Eutopic endometrium of control. (C) Mouse liver as positive control. (D) Eutopic endometrium \nof control treated with an unrelated monoclonal an tibody of the same isotype as negative control. \n×400 magnification. Scale bar = 200 µm. \n3.3. GPX4, ACSL4, and TfR1 Expression Levels \nThe strongest expression was observed in stromal cells with the GPX4 antibody. The \npercentage of stained stromal cells was higher in the control group with M = 90.0% ± 17.5 \nversus 59.7% ± 42.4 in the EM group, t (54) = −2.90, p = 0.005 (i.e., Figure 4, Supplementary \nTable S2). The intensity of the staining of stromal cells was stronger in the EM group with \nM = 1.0 ± 0.7 versus 1.4 ± 0.6 in the control group, t (54) = 2.22, p = 0.031 (i.e., Table 4). \nComparing the expression of the di fferent antibodies between EM patients and the \ncontrol group, we observed a similar staini ng for GPX4 and ASCL4. Both antibodies \nFigure 3. Expression of TfR1. (A) Ectopic endometrium (abdominal wall) of endometriosis patient.\n(B) Eutopic endometrium of control. (C) Mouse liver as positive control. (D) Eutopic endometrium\nof control treated with an unrelated monoclonal antibody of the same isotype as negative control.\n×400 magnification. Scale bar = 200 µm.\n3.3. GPX4, ACSL4, and TfR1 Expression Levels\nThe strongest expression was observed in stromal cells with the GPX4 antibody. The\npercentage of stained stromal cells was higher in the control group with M = 90.0% ± 17.5\nversus 59.7% ± 42.4 in the EM group, t (54) = −2.90, p = 0.005 (i.e., Figure 4, Supplementary\nTable S2). The intensity of the staining of stromal cells was stronger in the EM group with\nM = 1.0 ± 0.7 versus 1.4 ± 0.6 in the control group, t (54) = 2.22, p = 0.031 (i.e., Table 4).\nComparing the expression of the different antibodies between EM patients and the\ncontrol group, we observed a similar staining for GPX4 and ASCL4. Both antibodies denote\na strong staining intensity of stromal cells M = 1.0 ± 0.7 for EM and 1.4 ± 0.6 for controls,\nt (54) = −2.22, p = 0.031 with GPX4 and M = 1.1 ± 0.8 for EM and 1.2 ± 0.4 for controls,\nt (54) = −0.809, p = 0.422 with ACSL4. In the case of epithelial cells, M = 1.2 ± 1.1 for EM\nand 1.3 ± 0.7 for controls, t (54) = −1.235, p = 0.815 with GPX4 and M = 1.1 ± 0.9 for EM\nand 1.1 ± 0.6 for controls, t (54) = 0.085, p = 0.932 with ACSL4.\nTfR1 showed a very low expression level. The staining intensity was stronger in the\nstromal cells of the control group with M = 0.7 ± 1.2 versus 0.2 ± 0.4 for EM, t (54) = 2.552,\np = 0.014 (i.e., Table 4).\nWe further analyzed the association between the different antibodies using correlation-\nbased analyses. In all patients, the strongest correlations were found between GPX4 and\nACSL4, where r = 0.758, p < 0.001 for the percentage of stained stromal cells and r = 0.672,\np < 0.001 for the intensity of the staining of stromal cells, and r = 0.694, p < 0.001 for the\npercentage of stained epithelial cells and r = 0.714, p < 0.001 for the intensity of the staining\nof epithelial cells.\n\nBiomolecules 2024, 14, 876 8 of 13\nBiomolecules 2024, 14, x FOR PEER REVIEW 9 of 15 \n \nTable 4. Staining intensity of GPX4, ACSL4, and TfR1. \n Endometriosis  \nn = 38 \nControls  \nn = 18  p-Value \nGPX4 stromal cells 1.0 ± 0.7 1.4 ± 0.6 0.031 \nGPX4 epithelial cells 1.2 ± 1.1 1.3 ± 0.7 n.s \nACSL4 stromal cells 1.1 ± 0.8 1.2 ± 0.4 n.s \nACSL4 epithelial cells 1.1 ± 0.9 1.1 ± 0.6 n.s \nTfR1 stromal cells 0.2 ± 0.4 0.7 ± 1.2 0.014 \nTfR1 epithelial cells 0.1 ± 0.3 0.3 ± 0.5 n.s \n0 = light staining; 1 = medium staining; 2 = intense staining; p < 0.05 was considered statistically \nsignificant. n.s. = not statistically significant. \n \nFigure 4. Violin plots of the percentage (%) of stained cells. (A) Expression of GPX4 in stromal and \nepithelial cells of patients with endometriosis (EM) and controls (Ctrl). ( B) Expression of ACSL4 in \nstromal and epithelial cells of endometriosis patients and controls. (C) Expression of TfR1 in stromal \nand epithelial cells of endometriosis patients and controls. ** p < 0.01; n.s. = not statistically signifi -\ncant. \n4. Discussion \nThe pathomechanism of EM remains unclear despite years of intensive research. Var-\nious theories have been proposed to explain its origin and development; however, numer-\nous uncertainties persist.  \nConsidering the classical pathomechanism theories of EM, we hypothesized that fer-\nroptosis, potentially driven by increased iron content at ectopic sites, may contribute to \nthe progression of the disease. Our findings showed a significant downregulation of GPX4 \nin the stromal cells of EM patients, linked to a slightly higher hemoglobin value as an \nindirect predictor of the iron level, as the amount of iron in hemoglobin accounts for about \ntwo-thirds of the mass of iron in the human body [18]. \nGPX4 and hemoglobin play crucial roles in managing oxidative stress. Hemoglobin \ncan generate reactive oxygen species (ROS), which trigger ferroptosis, while GPX4 helps \nmitigate the damage caused by ROS, partic ularly lipid peroxides that can damage cell \nmembranes and lead to cell death. In the context of EM, higher levels of hemoglobin and \niron may trigger ferroptosis, potentiated by a downregulation of GPX4 [31]. \nBoth systemic and local iron homeostasis in fluence cell sensitivity to ferroptosis, \nwhich can propagate rapidly to adjacent cells [15,17,32]. Ferroptosis operates through two \nmajor pathways: the extrinsic or transporter-dependent pathway and the intrinsic or \nFigure 4. Violin plots of the percentage (%) of stained cells. (A) Expression of GPX4 in stromal and\nepithelial cells of patients with endometriosis (EM) and controls (Ctrl). (B) Expression of ACSL4 in\nstromal and epithelial cells of endometriosis patients and controls. (C) Expression of TfR1 in stromal\nand epithelial cells of endometriosis patients and controls. **p < 0.01; n.s. = not statistically significant.\nTable 4. Staining intensity of GPX4, ACSL4, and TfR1.\nEndometriosis\nn = 38\nControls\nn = 18 p-Value\nGPX4 stromal cells 1.0 ± 0.7 1.4 ± 0.6 0.031\nGPX4 epithelial cells 1.2 ± 1.1 1.3 ± 0.7 n.s\nACSL4 stromal cells 1.1 ± 0.8 1.2 ± 0.4 n.s\nACSL4 epithelial cells 1.1 ± 0.9 1.1 ± 0.6 n.s\nTfR1 stromal cells 0.2 ± 0.4 0.7 ± 1.2 0.014\nTfR1 epithelial cells 0.1 ± 0.3 0.3 ± 0.5 n.s\n0 = light staining; 1 = medium staining; 2 = intense staining; p < 0.05 was considered statistically significant.\nn.s. = not statistically significant.\n4. Discussion\nThe pathomechanism of EM remains unclear despite years of intensive research.\nVarious theories have been proposed to explain its origin and development; however,\nnumerous uncertainties persist.\nConsidering the classical pathomechanism theories of EM, we hypothesized that\nferroptosis, potentially driven by increased iron content at ectopic sites, may contribute\nto the progression of the disease. Our findings showed a significant downregulation of\nGPX4 in the stromal cells of EM patients, linked to a slightly higher hemoglobin value as\nan indirect predictor of the iron level, as the amount of iron in hemoglobin accounts for\nabout two-thirds of the mass of iron in the human body [18].\nGPX4 and hemoglobin play crucial roles in managing oxidative stress. Hemoglobin\ncan generate reactive oxygen species (ROS), which trigger ferroptosis, while GPX4 helps\nmitigate the damage caused by ROS, particularly lipid peroxides that can damage cell\nmembranes and lead to cell death. In the context of EM, higher levels of hemoglobin and\niron may trigger ferroptosis, potentiated by a downregulation of GPX4 [31].\nBoth systemic and local iron homeostasis influence cell sensitivity to ferroptosis, which\ncan propagate rapidly to adjacent cells [15,17,32]. Ferroptosis operates through two major\npathways: the extrinsic or transporter-dependent pathway and the intrinsic or enzyme-\nregulated pathway [16,17,26]. As mentioned above, cellular iron is intricately regulated;\nthis delicate balance is disrupted in a state of inflammation [18].\nPrevious research has demonstrated iron accumulation in both endometriotic lesions\nand the peritoneal fluid of patients with EM [ 2,33–35]. Consistent with other studies,\n\nBiomolecules 2024, 14, 876 9 of 13\nwhich either detected higher iron levels in endometriosis patients or found no significant\ndifference, our findings suggest that patients with EM exhibit slightly, but not significantly,\nhigher systemic iron levels compared to the control group [13,36,37]. This finding can be\nattributed to the fact that most patients with EM are on hormonal therapy that inhibits\nmenstruation, potentially explaining the slightly higher hemoglobin levels. As the day of\nthe cycle was not documented, it presents a potential source of bias, given that hemoglobin\nlevels are known to decrease in women during menstruation.\nParadoxically, our results showed a downregulation of TfR1 in stromal cells of EM\npatients compared to the controls. This outcome could be attributed to the previously\nidentified aberrant iron transport discussed in several other studies [2,37]. The significant\npresence of iron within endometriotic lesions may trigger a downregulation of TfR1 as\na protective mechanism for cells against excessive iron influx. However, it is crucial to\nacknowledge that TfR1 is not the sole player in iron transport. Other studies conducted\nin patients with EM have shown that iron overload induces the increased expression of\ntwo subtypes of DMT1, which is also responsible for iron influx into cells [38,39]. Akashi\net al. found a downregulation in TfR1 and FPN and an upregulation of DMT1 in samples\nof ovarian endometriosis (OMA) and clear cell carcinoma [40].\nThe influx of high levels of iron into the cells induces a lethal accumulation of ROS\nthrough the Fenton reaction, resulting in a redox imbalance between oxidants and antioxi-\ndants and leading to the extensive lipid peroxidation of cell membranes and, ultimately,\ncell death [9,17,20,21,26].\nGPX4 inhibits the formation of lipid peroxides. Therefore, the inhibition or downreg-\nulation of GPX4 results in the accumulation of lipid peroxides and increases sensitivity\nto ferroptosis [\n9,15,19,25,26]. Our study identified a significant downregulation of GPX4\nin stromal cells of EM patients, suggesting a potential link between ferroptosis and the\npathomechanism of EM. Recent findings also propose the involvement of different genetic\nvariants of GPX4 in the pathomechanism of EM [41,42]. The observation within stromal\ncells adds a layer of complexity, highlighting the distinct involvement of both cell types:\nepithelial and stromal, and deepening the intricacy of understanding the disease.\nQueckbörner et al., utilizing single-cell RNA sequencing, identified ten distinct stromal\ncell subpopulations, establishing cell cluster diversity and highlighting the complexity of\nthe endometrial stromal compartment [ 43,44]. Zhang et al. also described endometrial\nstromal cell subpopulations and observed that iron overload in ectopic endometrial stro-\nmal tissue, followed by ferroptosis, promotes fibrosis and adhesion [ 45]. Furthermore,\nAkashi et al. described a highly proliferative endometrial epithelium infiltrating the stroma\nwith elevated Ki-67 expression in patients with EM, emphasizing the proliferative nature\nof the disorder [40].\nIn the current comprehension of EM pathophysiology, these observations indicate\nthat distinct subpopulations of stromal cells react to ferroptosis triggered by elevated\niron levels. Consequently, a microenvironment characterized by inflammation, hypoxia,\nand angiogenesis emerges, fostering the proliferation and infiltration of epithelial cells.\nThis dynamic contributes to the formation and expansion of endometriotic lesions. The\nintricate processes at play, mediated through diverse inflammatory pathways, that require\nfurther and deeper investigation, may induce ferroptosis-triggered fibrosis, typical of deep\ninfiltrating lesions.\nSupporting our hypothesis, Alvarado-Díaz et al. demonstrated that exposing isolated\nendometrial stromal cells to iron excess stimulates the pro-inflammatory NF-κB pathway,\nenhancing the migration ability of endometriotic cells by promoting the expression of\nmatrix metalloproteinases (MMPs) and exacerbating inflammation, angiogenesis, and cell\nadhesion [3,46,47]. Li et al. demonstrated that inducing ferroptosis in endometriotic stromal\ncells increases the expression of pro-inflammatory and angiogenic cytokines, such as inter-\nleukin 8 (IL-8) and vascular endothelial growth factor A (VEGFA) [10]. Other studies have\nhighlighted the contribution of elevated levels of inflammatory mediators, hormones, and\nimmune cells as cyclooxygenase-2, interleukin-β, interleukin-6, IL-8, tumor necrosis factor\n\nBiomolecules 2024, 14, 876 10 of 13\nalpha, prostaglandin E2, and estradiol in the tissue microenvironment and the peritoneal\nfluid of patients with EM in the survival, implantation, invasion, growth, angiogenesis,\nimmunosurveillance evasion, and establishment of endometrioticlesions [10,45,48–51]. Fur-\nthermore, Akashi et al. observed M2 macrophages engulfed with iron in the stroma of OMA,\nlinking excessive erythrophagocytosis, iron overload, and ferroptosis in macrophages to\nthe chronic inflammatory mechanism of EM [40,52,53].\nRegarding ACSL4, which is generally associated with promoting ferroptosis, our\nstudy revealed a strong correlation between ACSL4 and GPX4 in both the stromal and\nepithelial cells of patients with EM and in the controls. This finding highlights the delicate\nequilibrium between induction and inhibition, as ACSL4 and GPX4 are well established as\npositive and negative regulators of ferroptosis, respectively [24].\nAs a limitation of our pilot study, we analyzed a small group of patients, comparing\nthe eutopic endometrium of non-endometriosis control women with ectopic lesions of en-\ndometriosis patients. While we consider this an appropriate control for comparing healthy\nwith diseased tissue, the inclusion of eutopic endometrium from endometriosis patients\ncould have provided further insights into potential pathogenetic mechanisms. Moreover,\nendometriosis is a hormone-dependent disease characterized by chronic inflammation [1,2].\nAlthough subgroup analysis according to hormonal therapy or analgesics did not reveal\nstatistically significant differences in the expression of ferroptosis markers, the use of these\ndrugs could have acted as a possible confounder. Larger sample sizes are needed to validate\nthese results. Additionally, the hormonal status of the patients was unknown, as it is not\nroutinely tested.\nWhile we identified GPX4 as dysregulated in EM, alternative pathways of ferroptosis\nsurveillance independent of GPX4 have been described in oncological settings. Among\nthese, the phospholipid-modifying enzymes MBOAT1 and MBOAT2 suppress ferropto-\nsis [54] and are expressed in an estrogen- and androgen-dependent manner, marking them\nas worthwhile candidates for future investigations of the steroid-related regulation of\nferroptosis in EM.\nOur observation of decreased GPX4 expression in endometriotic tissue compared to\ncontrol tissue suggests a possible involvement of ferroptosis in EM. While this contrasts\nwith previous suggestions of potential ferroptosis resistance in the disease [ 13], it also\nopens up the possibility of the pharmacological inhibition of ferroptosis in EM. Indeed,\ninhibitors of ferroptosis such as Baicalin, Selenium, Dexmedetomidine, Dexpramipexole,\nand several natural compounds have been proposed as emerging treatments targeting key\nregulators of ferroptosis in the context of neurodegenerative diseases and strokes [55–57]\nand may be worth evaluating in preclinical models of EM.\n5. Conclusions\nIn summary, our investigation into the progression of EM reveals a complex interplay\ninvolving ferroptosis, disrupted iron metabolism, and intricate inflammatory responses.\nThe downregulation of GPX4 in stromal cells, along with elevated systemic iron levels and\nthe complex regulation of iron transporters in patients with EM, suggests a potential link\nbetween ferroptosis and the disease’s pathomechanism.\nThe influx of high iron levels triggers ferroptosis, leading to a cascade of events, includ-\ning inflammation, hypoxia, angiogenesis, and eventual cell death. This microenvironment\nsupports the proliferation and infiltration of epithelial cells, contributing to the establish-\nment and growth of endometriotic lesions. Paradoxically, TfR1 downregulation, potentially\nas a protective mechanism, adds another layer of complexity to iron regulation in EM.\nUnderstanding the involvement of ferroptosis and iron dysregulation in EM opens\nup avenues for further research and potential therapeutic interventions targeting these\npathways. It underscores the necessity for a comprehensive exploration of the molecular\nmechanisms of EM, underlying iron transport, ferroptosis, cell differentiation, and genetic\nvariants of implicated proteins to unravel the fairly unknown pathophysiology of EM.\n\nBiomolecules 2024, 14, 876 11 of 13\nSupplementary Materials: The following supporting information can be downloaded at:https://\nwww.mdpi.com/article/10.3390/biom14070876/s1. Supplementary Table S1—Data; Supplementary\nTable S2—Expression levels of GPX4, ACSL4, and TfR1 in (%).\nAuthor Contributions: L.A.M.C., M.G., L.K. and S.D.S. designed the study. L.A.M.C. conducted\ndatabase searches and data extraction. N.A.E.-S. prepared the tissue samples. L.A.M.C., G.M. and\nN.C. performed the immunohistochemical staining. D.D., L.A.M.C. and M.G. conducted data analysis.\nB.H., M.S. and L.A.M.C. performed microscopic evaluation and immunohistochemical analysis. D.D.\nwas responsible for the statistical analysis and figure creation. L.A.M.C. created the tables, wrote the\nfirst draft of the manuscript, and contributed to critical discussions. Q.-K.L. critically reviewed the\nfindings and contributed significantly to their interpretation. All authors provided critical review and\nfeedback on the first draft with substantial input into the analysis and interpretation of the findings.\nAll authors have read and agreed to the published version of the manuscript.\nFunding: This study was funded by EU H2020-RISE project TRENDO, grant # 101008193 (to MG and\nGM), and the Open Access Publishing Fund of the University of Muenster.\nInstitutional Review Board Statement: This study was designed under consideration of the princi-\nples of the Helsinki Convention and was approved by the ethics committee of Westphalia-Lippe (1 IX\nGreb, from 19 September 2001, updated 2012).\nInformed Consent Statement: Informed consent was obtained from all subjects involved in the study.\nData Availability Statement: All data generated for the manuscript are included in the study and are\navailable upon request.\nAcknowledgments: We would like to thank Birgit Pers, Dorothea Godulla, Magdalena Marciniak,\nand Sofia Dávalos for their expert technical assistance.\nConflicts of Interest: The authors declare no conflicts of interest. 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