{"paper_id":"fa4c60da-eca6-4262-bf56-86dff6c5d056","body_text":"ORIGINAL ARTICLE\nHormones (2024) 23:509–515\nhttps://doi.org/10.1007/s42000-024-00559-6\ninflammatory disease that affects up to 5 to 10% of repro -\nductive-aged women and is associated with pain and/or \ninfertility among 30 to 50% of these women [1]. Retrograde \nmenstruation and peritoneal adhesion of endometrial tis -\nsue are basic elements in the pathogenesis of endometriosis \naccording to Sampson’s classical implantation theory [ 2]. \nHowever, what causes attachment and outgrowth of endo -\nmetrial cells after their appearance in the peritoneal cavity \nremains unclear.\nSimilarly to tumors and metastases, endometriotic lesions \nrequire development of new blood vessels for continuous \noxygen and nutrient supply [ 3]. Endometriotic lesions pro -\nduce cytokines and growth factors that promote their pro -\nliferation and vascularization [ 4]. Interleukin-1β (IL-1β), \nIL-6, and IL-8 are cytokines that have a stimulatory role \nin angiogenesis and neovascularization in endometriotic \nIntroduction\nEndometriosis is a chronic benign gynecological disease \nthat is characterized by the presence of endometriotic tissue \noutside the uterine cavity [ 1]. It is an estrogen-dependent \n \r Apostolos Kaponis\nakaponis@upatras.gr; kaponisapostolos@hotmail.com\n1 Dept. of Obstetrics & Gynecology, Patras University School \nof Medicine, Patras, Greece\n2 Laboratory of Forensic Medicine and Toxicology, School of \nMedicine, Aristotle University of Thessaloniki,  \nThessaloniki 54124, Greece\n3 Dept. of Obstetrics & Gynecology, Tottori University Faculty \nof Medicine, Yonago, Japan\nAbstract\nPurpose Neoangiogenesis is necessary for adhesion and invasiveness of endometriotic lesions in women affected by endo-\nmetriosis. Vascular endothelial growth factor (VEGF) is one of the main components of angiogenesis and is part of the major \npathway tissue factor (TF)-protease activated receptor-2 (PAR-2)-VEGF that leads to neoangiogenesis. Specificity protein \n1 (SP1) is a transcriptional factor that has recently been studied for its crucial role in angiogenesis via a specific pathway. \nWe hypothesize that by blocking angiogenetic pathways we can suppress endometriotic lesions. Gonadotrophin-releasing \nhormone-agonists (GnRH-a) are routinely used, especially preoperatively, in endometriosis. It would be of great interest to \nclarify which angiogenetic pathways are affected and, thereby, pave the way for further research into antiangiogenetic effects \non endometriosis.\nMethods We used quantitative real-time polymerase chain reaction (qRT-PCR) to study mRNA expression levels of TF, \nPAR-2, VEGF, and SP1 in endometriotic tissues of women who underwent surgery for endometriosis and received GnRH-a \n(leuprolide acetate) preoperatively.\nResults VEGF, TF, and PAR-2 expression is significantly lower in patients who received treatment (p < 0,001) compared to \nthose who did not, whereas SP1 expression is not altered (p = 0.779).\nConclusions GnRH-a administration does affect some pathways of angiogenesis in endometriotic lesions, but not all of \nthem. Therefore, supplementary treatments that affect the SP1 pathway of angiogenesis should be developed to enhance the \nantiangiogenetic effect of GnRH-a in patients with endometriosis.\nTrial registration Clinicaltrial.gov ID: NCT06106932.\nKeywords Endometriosis · Angiogenesis · GnRH-a · VEGF · SP1\nReceived: 20 November 2023 / Accepted: 3 April 2024 / Published online: 19 April 2024\n© The Author(s) 2024\nThe effect of GnRH-a on the angiogenesis of endometriosis\nTheodoros Filindris1 · Efthymia Papakonstantinou1 · Maria Keramida1 · Eleftherios Panteris2 · \nSotiris Kalogeropoulos1 · Neoklis Georgopoulos1 · Fuminori Taniguchi3 · George Adonakis1 · Tasuku Harada3 · \nApostolos Kaponis1\n1 3\n\nHormones (2024) 23:509–515\nlesions [5–7]. Many angiogenic growth factors have been \nshown to be overexpressed in endometriotic lesions and in \nthe peritoneal fluid of women with endometriosis [ 8, 9]. \nΤhere is increasing evidence that the vascular endothelial \ngrowth factor (VEGF) family is involved in the etiology and \nmaintenance of peritoneal endometriosis [ 9]. 17β-estradiol \n(E2) up regulates VEGF expression in human endometrial \nstromal cells [10, 11].\nTissue factor (TF) is also known to be involved in angio-\ngenesis via intracellular signaling that utilizes protease acti-\nvated receptor-2 (PAR-2), as indicated in multiple studies \n[12, 13]. Specificity protein 1 (SP1) is thought to regulate \nVEGF expression in several carcinomas, such as pancreatic \nadenocarcinoma and ovarian cancer [14, 15].\nGonadotropin-releasing hormone agonists (GnRH-a) \nhave long been used for the management of endometrio -\nsis. The administration of long-lasting GnRH agonists has \na central effect, causing pituitary down-regulation and a \nreduction in gonadotropin release, thus exerting an impact \non endometriotic lesions [ 16]. According to the latest \nESHRE guideline, it is strongly recommended that GnRH-a \nbe prescribed in patients to reduce endometriosis-associated \npain [17]. Considering the significance of angiogenesis in \nendometriotic lesions, it has been hypothesized that GnRH-\na might influence angiogenic mechanisms in endometrial \ncell growth [ 16]. According to a published study, GnRH-a \n(leuprolide acetate, LA) has a direct effect on endometriotic \ntissue, partly by interfering in inhibiting angiogenesis [ 18]. \nThere is, hence, great interest in utilizing this knowledge \nsince it may pave the way to further investigation regarding \nthe effect of GnRH analogs on angiogenesis in endometri -\notic lesions.\nIn the current study, we explore the effect of the long-\nterm administration of a GnRH-a on angiogenesis factors \nwhich promote neoangiogenesis via two different and inde-\npendent pathways, namely, the TF-PAR-2-VEGF pathway \nand the SP-1-VEGF pathway.\nMaterials & methods\nThe subjects in this study were women of reproductive age. \nFrom January 2016 to December 2022, 60 women with \nknown endometriosis (stages 2 and 3) were recruited. The \nstaging of endometriosis was based on the rASRM classi -\nfication system [19]. Stage 2 includes women with ovarian \nendometrioma and superficial ovarian endometriosis, peri -\ntoneal filmy adhesions, or deep peritoneal endometriosis. \nStage 3 includes women with ovarian endometrioma, deep \nperitoneal endometriosis with dense adhesions, and partial \nobliteration of the cul de sac. Their mean age was 38 years. \nThey were nulliparous and had a mean body mass index \n(BMI) of 27 kg/m2. The ovarian endometrioma present in \nall the participants was diagnosed using ultrasonography \nand/or magnetic resonance imaging. Women with peri -\nmenopausal symptoms such as hot flashes, night sweats, \nand/or irregular menstrual period were excluded from the \ncurrent study. The inclusion and exclusion criteria are pre -\nsented in Table 1.\nThis was a randomized follow-up study with analysis \nof ovarian samples derived from GnRH-a-treated and non-\nGnRH-a-treated women before surgery. The randomiza -\ntion was performed by accessing a central internet-based \nrandomization program MinimRan [ 20]. The random allo -\ncation sequence and the assignment of the participants to \ninterventions were made by two of the authors (A.K. and \nS.K).\nAfter enrollment, the women were randomized into \ntwo groups (Table 2). Group A (GnRHa+) consisted of 30 \nwomen with a mean age of 35.5 years and a mean BMI of \n27 kg/m2. Seventeen of them had stage 2 and 13 had stage \n3 endometriosis. They received GnRH-a (LA) for a period \nof 3 months prior to surgery and had not received any hor -\nmonal treatment within the 12 months before the surgical \nprocedure. Group B (GnRHa-) consisted of 30 women with \na mean age of 38 years and a mean BMI of 27 kg/m2. Six-\nteen of them had stage 2 and 14 had stage 3 endometrio -\nsis. They did not receive GnRH-a treatment before surgery. \nIn addition, no treatment with oral contraceptives or other \nhormonal therapy had been administered within 12 months \nprior to surgery.\nTable 1 Inclusion and exclusion criteria of women\nInclusion criteria Exclusion criteria\nReproductive age Perimenopausal symptoms\n(hot flashes, night sweats, \nirregular menstrual bleeding)\nPersonal history of infertility FSH > 12 IU/dl*\nEndometriosis stage II or greater Obesity**\nBMI < 35 kg/m2 Hormonal treatment 1 year \nbefore the evaluation\nEndometriomas > 5 cm\n*Measured on 2nd day of menstrual period. **BMI >35 kg/m2.\nTable 2 Demographic parameters of the study population and controls. \nStage of endometriosis\nNo. of participants 30 30 -\nGroup A Group B -\nAge [median, 95% Cis] 35.5\n [29–40]\n38.00\n[34–46]\n0.388\nBMI [median, 95% Cis] 27.00 27.00 0.910\nDuration of Treatment \n[months]\n3 - -\nStage 2 endometriosis 17 16 0.924\nStage 3 endometriosis 13 14 0.956\nMenstrual cycle phase Amenorrhea Proliferative -\n1 3\n510\n\nHormones (2024) 23:509–515\nDuring laparoscopy, biopsy specimens of the ovarian \nendometrioma were collected. In group B, surgery was \nperformed during the proliferative phase of the menstrual \ncycle. All biopsy specimens were collected in accordance \nwith the guidelines of the Declaration of Helsinki and with \nthe approval of the ethical committee of the General Uni -\nversity Hospital of Patras. Informed consent was obtained \nfrom all women.\nqRT-PCR\nQuantitative real-time polymerase chain reaction (qRT-\nPCR) is used to study the expression of genes in various \ntissues. This method is one of the most common tools, \nenabling relative quantification of target gene expression by \ncomparison with the expression of a “reference” or “house-\nkeeping” gene. A “housekeeping” gene is defined as being \nconstitutively expressed in the tissue under study [ 21]. The \nreference gene should have stable expression under all \nexperimental conditions (i.e., patients and controls) and \nbe expressed appropriately in the tissue studied, otherwise \nresults may be biased.\nFor primer design\nThe gene sequences of the exons and introns used for \nthe design of the specific primers were obtained from the \nensembl database (EMBL-EBI) (Online Resource 1). Primer \ndesign, purchased from Thermo Fisher Scientific, using the \ngene sequences was performed with the NCBI tool “Primer-\nBlast” according to the instructions of the manufacturer. \nThe following criteria were considered in the development \nof the primers [22]:\n1. Length of 18–24 bases\n2. 40–60% G/C content\n3. Starts and ends with 1–2 G/C pairs\n4. Melting temperature (TM) of 50–65 °C\n5. The two primers of a primer pair should have closely \nmatched melting temperatures for maximizing PCR \nproduct yield\n6. Primer pairs should not have complementary regions\n7. The amplicon length is dictated by the experimental \ngoals. For qPCR, the target length is closer to 100 bp \nand for standard PCR it is near 500 bp (Online Resource \n2; Online Resource 3)\nFresh tissue samples were cut < 0.5 cm and immersed in \n5–10 volumes of RNAlater Stabilization Solution (Invi -\ntrogen, Cat. No. AM7020), stored at 4 °C overnight, and \nthen moved to − 80 °C until RNA extraction for long-term \nstorage.\nTissue lysis and RNA extraction\nPrior to RNA isolation, the samples were lysed and homog-\nenized. The frozen tissue was placed on ice and 0.5 mL of \nTRIzol Reagent (TRIzol Reagent, Cat. No: 15,596,026, \nInvitrogene) was added at optimal sample size (50–70 mg) \nand homogenized at 25 Hz for 3 min. 0.1 mL of chloro -\nform was added to 0.5 mL of Trizol reagent, shaken vigor -\nously by hand for 15 s, and incubated at room temperature \nfor 3 min. The samples were centrifuged at 11.600 x g for \n15 min at 4 °C. An equal volume of ice-cold 75% ethanol \nwas added to the upper phase and transferred to a High Pure \nFilter Tube of the High Pure RNA Isolation Kit (Cat. No. 11 \n828 665 001, Roche) [ 23]. RNA isolation was performed \naccording to the isolation kit protocol [ 24]. The concentra-\ntion and purity of RNA was determined by measuring the \nabsorbance at 260 nm and 280 nm in a spectrophotometer. \nThe yield of total RNA was 0.5–0.8 µg/mg.\nDNA (cDNA) synthesis was performed with a mixture \nof anchored-oligo (dT) primers and 1 µg of total RNA, \naccording to the manufacturer’s instructions (Transcriptor \nFirst Strand cDNA Synthesis Kit, Cat. No. 04897030001; \nRoche Applied Science). Real-time PCR was carried out in \nthe LightCycler 2 Instrument (Roche) using the FastStart \nUniversal SYBR Green Master (Roche Hellas).\nFour independent experiments were analyzed in dupli -\ncates for all data shown. GAPDH was used as a reference \ngene for normalization. To analyze qPCR data, REST-MCS \nbeta software version 2 was used.\nStatistical methods\nPower analysis was performed using GPower 3.1.9.6 for \nthe comparison of patients with and without GnRH-a for a \npower level of = 0.8 with effect size = 0.7. Effect size was \ndeemed to be large (0.7), as a large difference is expected \nbetween RNA expression of the main regulators of angio -\ngenesis between patients with and without GnRH-a [ 25]. A \nsample size of 28 per group was indicated for the specified \npower level; thus, 60 patients were recruited to allow for \ndropouts/analysis issues.\nThe data were analyzed using nonparametric methods \nvia SPSS (Statistical Package for the Social Sciences) v. 26 \n(SPSS, Inc. Chicago, IL, USA) to generate graphs and anal-\nyses. As parameters do not follow the normal distribution as \nper the Shapiro-Wilk normality test, the Mann-Whitney U \ntest was used for multiple variables with a significance level \nof 0.05. Median and 95% confidence intervals (95% CIs) \nwere recorded for all continues variables.\n1 3\n511\n\nHormones (2024) 23:509–515\nDiscussion\nIn normal endometrial stromal cells, VEGF is highly \nexpressed, its levels depending on the effect of estrogen and \nprogesterone [26–28]. It is widely accepted that in women \nwith endometriosis, VEGF is highly expressed in perito -\nneal fluid as well as in ectopic endometrial tissue [9, 26, 29, \n30]. Estrogen is a proangiogenic hormone whose effects on \nneovascularization and angiogenesis in the uterus and endo-\nmetrium through proliferation and migration of endothelial \ncells are widely studied [26]. Previous studies have reached \nthe conclusion that GnRH-a (LA) administration in women \nwith endometriosis or uterine fibroids downregulated VEGF \nexpression and affected the vascular pattern via decrease of \nmicrovessel density in the endometria studied [31–33]. The \nabove observations enable us to formulate the hypothesis \nthat reduction in the size of endometriomas after treatment \nResults\nA sample of 60 women, 30 treated with GnRH-a in the \namenorrhea phase and 30 controls without GnRH-a treat -\nment in the proliferative menstrual cycle phase participated \nin this study. The stage of endometriosis was similar in both \ngroups (p = 0.956). There were no demographic differences \namong the patients as age and BMI were similar in the two \ngroups, with a median age of 38 years old (34–46, 95%CIs) \nfor the control group, who did not receive GnRH-a antago -\nnists, and a median age of 35.5 years old (30–41, 95%CIs) \n(p = 0.388) for the experimental group, who did. The BMI \nwas also identical between the two groups ( p = 0.910). \nTable 2 displays the demographics.\nMedian expressions of the relevant mRNAs were sta -\ntistically differentiated between the two groups. In detail, \nTF mRNA median expression was 3.2 (3.1–3.6, 95%CIs) \nin control vs. 0.7 (0.7–0.9, 95%CIs) in the experimental \ngroup. Similarly, P AR-2 mRNA median expression was 7.65 \n(7.5–8.2, 95%CIs) vs. 2.1 (1.9–2.7, 95%CIs) in the control \nversus the experimental group and VEGF mRNA median \nexpression was also significantly differentiated with 1.52 \n(1.4–1.8 95%CIs) vs. 0.3 (0.3–0.4, 95%CIs) in the control \nversus the experimental group, respectively. In contrast, \nSP1 mRNA did not show any differentiation between the \ntwo groups, with SP1 median expression being 1.57 (1.43–\n1.69, 95%CIs) vs. 1.51 (1.36–1.69, 95%CIs) in the control \nversus the experimental group, respectively. Table 3; Fig. 1 \npresents the latter specifics in detail.\nTable 3 Gene expression between women with endometriosis who \nreceived a GnRH-a (Group A) and those who did not receive GnRH-a \n(Group B)\nGroup A Group B Mann \n-Whit-\nney U \ntest\nMedian 95.0% CΙs Median 95.0% CΙs P value\nTF 0.7 0.70–0.90 3.2 3.10–3.60 < 0.001\nPAR-2 2.1 1.90–2.70 7.65 7.50–8.20 < 0.001\nVEGF 0.3 0.30–0.40 1.52 1.40–1.80 < 0.001\nSP1 1.51 1.36–1.69 1.57 1.43–1.69 0.779\nFig. 1 Gene expression of PAR-2, TF, VEGF, and SP-1 in endometriotic tissues of women with and without GnRH-a administration\n \n1 3\n512\n\nHormones (2024) 23:509–515\nHormonal treatments, such as GnRH-a, are not suitable \nfor women desiring to preserve their fertility and act only \nfor symptomatic relief and not for actual improvement of \nfertility [ 42]. Bevacizumab, an anti-VEGF, non-hormonal \nfactor has been studied for possible treatment of endome -\ntriosis; however, it carries serious, not easily tolerated side \neffects (i.e., gastrointestinal perforation, thrombosis, severe \nbleeding, impaired kidney function, and wound healing) \n[42]. In addition, statins work in a dose-dependent way, \neither promoting angiogenesis (at low doses) or blocking \nangiogenesis (at higher doses). However, their long-term \nside effects, such as myopathy and rhabdomyolysis, as well \nas their controversial effectiveness remain a deterrent factor \nto their widespread use [42]. Cabergoline, a dopamine ago-\nnist, has also been studied for the treatment of endometrio -\nsis and, in some studies, it has been found to downregulate \nVEGF receptors in endometriotic implants [ 42, 43]. Future \nresearch is essential to highlight the role of other treatments, \nhormonal or non-hormonal, in downregulating both the TF-\nPAR2-VEGF and SP1 pathways of angiogenesis, resulting \nin ultimately diminishing endometrioma size and not only \nrelieving the symptoms.\nRecent studies on the treatment of endometriosis have \nfocused on the development of antiangiogenic drugs, such \nas anti-VEGF antibodies, VEGFR tyrosine kinase, COX-2 \ninhibitors, and dopamine agonists [ 44]. This is why the \npresent study is of high originality, given the fact that it is \nthe first time, to our knowledge, that a study has explored \nthe impact of GnRH-a treatment preoperatively on angio -\ngenetic pathways in women with endometriosis. One dis -\nadvantage of the present study is that we studied only the \nmRNA expression of TF, PAR2, VEGF, and SP1 and not \ntheir protein expression using Western blot or enzyme-\nlinked immunosorbent assay (ELISA). While GnRH-a can \ninhibit neoangiogenesis in endometriotic lesions, it cannot \ncompletely block all the angiogenetic pathways, since no \nalterations in expression of the SP1 pathway of angiogen -\nesis have been found. Further research should be conducted \nto discover new, more efficient treatments of endometrio -\nsis. Since endometriosis concerns many reproductive-aged \nwomen, discovering ways to affect its angiogenesis is very \npromising to moderate the role of angiogenesis in its patho-\ngenesis and will give hope and new perspectives to patients \nwith endometriosis.\nAbbreviations\nBMI  Body mass index\nELISA  Enzyme-linked immunosorbent assay\nE2  17β-estradiol\nGnRH-a  Gonadotropin-releasing hormone agonist\nIL-1β  Interleukin 1β\nMVD  Microvessel density\nwith GnRH-a might be caused by reduction of angiogenesis \nin the pathologic lesions.\nMany other components apart from VEGF are involved \nin neoangiogenesis and thus play an equally significant \nrole in this pathway. Angiogenesis has been widely stud -\nied in neoplastic tissues given that angiogenesis is of great \nimportance for tumor viability and progression [34, 35]. TF \nis a cell membrane-bound glycoprotein that binds to circu -\nlating factor VIIa to mediate the activation of both factors \nIX and X and, thus, has a crucial role in hemostasis [ 35]. \nAccording to a number of studies, TF-PAR2 signaling con-\ntributes to angiogenesis. When TF is exposed to the blood -\nstream, FVIIa binds to it on the cell surface, an event that \npromotes hemostasis. Furthermore, the binding of FVIIa to \nTF cleaves PAR-2, a cleavage that results in phosphoryla -\ntion of the TF cytoplasmic domain and inhibits the negative \neffect of PAR-2-mediated signaling, promoting angiogen -\nesis [35]. Several mitogen-activated protein kinase (MAPK) \npathways are then activated, which leads to expression of \nseveral genes, one of them being the VEGF gene. High \nexpression of VEGF has been reported after exposure of \nTF-expressing cells to FVIIa (via PAR-2 activation). Our \nobservation that mRNA of TF and PAR-2 is downregulated \nin women receiving GnRH-a is significant as regards insight \ninto the role of GnRH-a in angiogenesis, since it blocks one \nof the most important pathways, thereby causing endome -\ntriosis regression.\nTranscription factor SP1 promotes tumor angiogenesis \nand invasion by activating VEGF expression in several \ntumors, such as ovarian and pancreatic cancer, follow -\ning a different and independent pathway from that of TF-\nPAR2-VEGF [14, 35]. According to a recent study, SP1 \ncan activate the transforming growth factor-β1/Sma and \nMad proteins 2 (TGF-β1/SMAD2) pathway and promote \nVEGF secretion through TGF-β1, promoting angiogenesis \nin preosteoblasts [ 37]. In gastric cancer as well, transcrip -\ntion factor SP1 is an independent prognostic factor since it \nhas been observed that the higher the expression of SP1, the \nhigher the microvessel density (MVD) of the tumor [38]. In \na recent study, SP1 mRNA and protein levels were found to \nbe increased in ectopic and eutopic endometrium of women \nwith stage III/IV endometriosis [39]. We therefore included \nSP1 transcription factor in our study and found that GnRH-a \ndoes not affect its expression in endometriomas. The non-\ninvolvement of SP1 could be due to its known post-transla-\ntional modification capacity, which regulates its expression, \nwhich action could override potential disruptors. SP1 is a \nunique transcription factor as it both initiates transcription \nand can regulate the activation and repression processes: it \nis thus a key component that must not be affected by exter -\nnal disruptors [40, 41].\n1 3\n513\n\nHormones (2024) 23:509–515\n4. Groothuis PG, Endometriosis (2012) Sci Pract 2013:190–199. \nhttps://doi.org/10.1002/9781444398519.ch19\n5. 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Bustin S, Huggett J (2017) qPCR primer design revisited. \nBiomol Detect Quantif 14:19–28. https://doi.org/10.1016/j.\nbdq.2017.11.001\nMAPK  Mitogen activated protein kinase\nPAR-2  Protease activated receptor 2\nqRT-PCR  Quantitative real-time polymerase chain \nreaction\nSP1  Specificity protein 1\nSMAD2  Sma and Mad proteins from Caenorhabditis \nelegans and Drosophilla, respectively\nTF  Tissue factor\nTGF-β1  Transforming growth factor β1\nVEGF  Vascular endothelial growth factor\nSupplementary Information  The online version contains \nsupplementary material available at https://doi.org/10.1007/s42000-\n024-00559-6.\nAcknowledgements None\nFunding Open access funding provided by HEAL-Link Greece.\nDeclarations\nDisclosure The authors declare no conflict of interest for this article.\nHuman rights statement and informed consent  All procedures fol -\nlowed were in accordance with the ethical standards of the responsible \ncommittee on human experimentation (institutional and national) and \nwith the 1964 Declaration of Helsinki and its later amendments. The \nstudy conformed to the Greek Federal Policy for the Protection of Hu-\nman Subjects. The appropriate ethical review committee approval was \nreceived on 11-05-2015/83. Informed consent was obtained from all \npatients for inclusion in the study.\nOpen Access   This article is licensed under a Creative Commons \nAttribution 4.0 International License, which permits use, sharing, \nadaptation, distribution and reproduction in any medium or format, \nas long as you give appropriate credit to the original author(s) and the \nsource, provide a link to the Creative Commons licence, and indicate \nif changes were made. The images or other third party material in this \narticle are included in the article’s Creative Commons licence, unless \nindicated otherwise in a credit line to the material. 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