{"paper_id":"53d53c98-4734-41a4-b2c9-f0216f96bec5","body_text":"1916\n© Aluna Publishing\nWiadomości Lekarskie Medical Advances, VOLUME LXXVII, ISSUE 10, OCTOBER 2024    \nINTRODUCTION \nHMGB1 is a nuclear protein that is present in almost \nall cell types. It can also be released extracellularly, \nwhere it mediates the activation of innate immune \nresponses, including chemotaxis and cytokine release. \nHMGB1 was originally described as a protein that binds \nnuclear DNA [1,2]. It is evolutionarily highly conserved \nand functions as a nuclear cofactor in transcriptional \nregulation. HMGB1, like many other cofactors, was later \nfound to play another role as an intercellular molecule \nthat is released from various cells into the extracellular \nenvironment to act on specific cell surface receptors. In \nthis latter role, HMGB1 is a proinflammatory cytokine \nthat can contribute to many inflammatory diseases, \nincluding sepsis [3-5]. Through interaction with immune \ncell cell surface receptors, HMGB1 activates intracellular \ncascades that regulate immune cell functions, including \nchemotaxis and immune modulation. HMGB1 is a criti-\ncal mediator of lethality in sterile and infectious inflam-\nmation. Similar inflammatory responses are initiated by \nstrokes caused by sterile trauma or infection. During \ninfection, innate immunity is activated by foreign \nmolecular products called PAMPs, which include, for \nexample, lipopolysaccharides [6]. During sterile injury \nor ischemia, the cells themselves are activated under \nthe influence of endogenous DAMPs, which include \nmolecules such as heat shock proteins, uric acid, an-\nnexin, and IL1β. HMGB1, released by activated immune \ncells and damaged or necrotic cells, plays an important \nrole in host responses to both types of threats; thus, it \nis a critical mediator in the final common pathway to \nmorbidity and mortality during infection and sterile \ntrauma [7-10].\nIn a recent study (Expression of high-mobility group \nbox-1 in eutopic/ectopic endometrium and correlations \nwith inflammation-related factors in adenomyosis Xiu-\nNi Liu and Zhong-Ping Cheng) it was described.\nPossible pathogenesis of adenomyosis: the \nstrong HMGB1/TLR4 system may be involved in the \ninflammatory pathological process of adenomyosis \ndevelopment. It generates a local inflammatory reaction \nand activates the body’s specific immune system, builds \nMolecular-genetic characteristics of HMGB1 mRNA expression in  \nblood of women with endometriosis associated with infertility\nOksana V. Bakun, Natalya Ya. Muzyka, Svitlana B. Semenenko, Tetiana P . Savchuk, Alla I. Peryzhniak\nBUKOVINIAN STATE MEDICAL UNIVERSITY , CHERNIVTSI, UKRAINE\nABSTRACT\nAim: To examine HMGB1 expression in the blood of women with endometriosis-associated infertility and to establish its role in disease progression. \nMaterials and Methods: We analyzed HMGB1 gene expression levels in two groups: 20 women with endometriosis-associated infertility (main group) \nand 10 healthy women (control group). The study was conducted at Bukovinian State Medical University and the Centre of Reproductive Medicine. Primary \ninfertility was significantly higher in the main group. We used real-time reverse transcription polymerase chain reaction (RT-PCR) to determine HMGB1 mRNA \nlevels in mononuclear cells isolated from whole blood. Statistical significance was determined using the Student’s t-test, with p < 0.05 considered significant.\nResults: Results indicated a significant increase in HMGB1 mRNA expression in the main group compared to the control group (p < 0.001). The relative \nnormalized expression ranged from 1.8924 to 33.426 (median = 8.01). Expression levels were categorized into three groups: borderline with the control, \nmoderate increase, and significant increase. Only one sample (5%) had values equal to the control. Fifteen percent of samples had values slightly above control \n(1.89–3.45), 30% had moderate increases (3.45–8.01), and 50% had significant increases (>8.01). In 95% of the main group, HMGB1 mRNA expression was \nelevated, predominantly at high values (p < 0.001).\nConclusions: Women with endometriosis-associated infertility show significantly increased HMGB1 mRNA expression, particularly in moderate and severe \ncases compared to mild cases, indicating HMGB1’s role in disease progression (p < 0.001).\n  KEY WORDS: endometriosis, infertility, assisted reproductive technologies, HMGB1\nWiad Lek. 2024;77(9):1916-1921. doi: 10.36740/WLek/195142 DOI\nORIGINAL ARTICLE CONTENTS\n\nMolecular-genetic characteristics of HMGB1 mRNA expression in  blood of women with endometriosis...\n1917\nand maintains a stable inflammatory inflammatory \nmicroenvironment in the local lesion, and forms a local \ninflammatory pathology of adenomyopathy, which may \ninvolve the pathogenesis of angiomyopathy.\nTherefore, HMGB1 may also play an important role in \nthe pathophysiology of endometriosis, a gynecological \ndisease associated with a chronic immunoinflammatory \nprocess.\nAIM\nThe aim of our study was to examine the expression \nHMGB1  in the blood of women with endometriosis-as-\nsociated and to establish the role of HMGB1 in the \nprogression of endometriosis by analyzing the level of \ngene expression in the blood of women with infertility-\nassociated infertility, depending on the severity of \nendometriosis.\nMATERIALS AND METHODS\nWe determined the level of HMGB1 mRNA gene expres-\nsion in two groups. The first group (main) consisted of \n20 women with endometriosis associated with infertil-\nity. The control group consists of 10 practically healthy \nwomen. The absence of signs of acute inflammatory \nprocesses in the reproductive sphere and the negative \nresults of microbiological and virological studies were \nevidence of a long-overdue inflammatory process that \ncaused tubal obstruction.\nThis study was conducted at the Bukovyna State \nMedical University and the clinic “Yuzko medical center” .\nTo analyze the expression of the HMGB1 gene and \nto determine the relative normalized expression of \nHMGB1 mRNA, the polymerase chain reaction with \nreverse transcription in real time (RT-PCR) was used. \nThe object for molecular genetic studies by the RT-PCR \nmethod was the fraction of mononuclear cells isolated \nfrom the whole blood of patients with endometriosis.\nSpecific pairs of primers for the analysis of the studied \nand reference genes were selected using the Primer-\nΒLAST software (www.ncβi.nlm.nih.gov/tools/primer-\nβlast) and manufactured by Thermo Scientific (USA). \nThe actin, βeta (Actβ) gene was used as a reference gene \nto determine the relative value of the change in the \nexpression level of the studied genes. The comparative \nCt method (ΔΔCt method) was used to express the \nrelative level of gene expression. Calculations were \nmade according to the formulas: ΔCt (target gene) = Ct  \n(target gene) – Ct (calibrator gene / ACT1);  \nΔΔCt = ΔCt (target gene) – ΔCt (base gene); The relative \nexpression level was expressed as 2–ΔΔCt. Statistical \nanalysis of PCR data was performed using CFX Manager ™  \nsoftware (Βio-Rad, USA). Optimal RT-PCR conditions \nwere selected to achieve a linear relationship between \nthe number of cycles and the number of PCR products. \nNegative controls were included in the experiment: \nwithout addition of cDNA matrix in the PCR reaction, \nwithout addition of mRNA matrix in cDNA synthesis, \nwithout addition of enzyme in cDNA synthesis. All \namplification reactions were performed on individual \nsamples in triplicate. Quantitative variables were \nevaluated using the Shapiro-Wilk test (if the number of \nsubjects was less than 50) or the Kolmogorov-Smirnov \ntest (when the number of subjects was more than 50).\nComparison of two groups for a quantitative variable \naccording to a normal distribution, under the condi-\ntion of equality of variances, was performed using the \nStudent’s t-test.\nStatistical processing of the results of this section was \ncarried out using non-parametric methods, namely the \nWilcoxon-Mann-Whitney test. Differences between \ngroups were considered probable at the significance \nlevel of p<0.05. \nRESUL TS\nIn the analysis, depending on the degree of severity of \nendometriosis, stage I and II were combined into so-\ncalled «small» forms of endometriosis, or mild stage, \nstage III corresponded to the average severity of en-\ndometriosis, and stage IV corresponded to the severe \nstage of the disease.\nThe results of the study of the relative normalized \nexpression of HMGB1 mRNA in the whole blood of \nwomen with endometriosis associated with infertility \nare shown in Fig. 1.\nBased on our research, it was established that the ex-\npression of HMGB1 mRNA in the blood of women with \nendometriosis associated with infertility is significantly \nincreased compared to the indicators of the control \ngroup (р< 0.001).\nThe range of all obtained values of the relative \nnormalized amount of mRNA of the HMGB1 gene \nwas 1,8924 – 33,426 (median – 8,01). For a broader \ncharacterization of the features of the level of \nHMGB1 mRNA expression in the blood of women \nwith endometriosis, associated with infertility, and \nnot only the limitation of the data of the average \nvalue, conditionally, the ranges of fluctuations of the \nindicator were separated into the following groups: \nborderline with the control group, lower than the \nmedian (moderate increase HMGB1 expression level) \nand higher than the median (significant increase in \nHMGB1 expression level). As can be seen, the lower \nrange of values of the HMGB1 gene is equal to the \n\nOksana V. Bakun et al. \n1918\ncontrol, however, this indicator was measured only in 1 \nout of 20 studied blood samples, which was 5% of cases \n(p<0,001). There were only three results that slightly \ndiffered from the control (>1,89-<3,45), which was 15% \n(p<0,001). The number of blood samples in which low \nvalues (>3,45-<8,01) of relative normalized expression \nwere found was 6 cases (30%). Relative normalized \nexpression above the median (>8,01) was found in 10 \nblood samples, which accounted for half of the cases - \n50%. Thus, in the blood samples of 95% of the women \nin the study group, increased expression of the HMGB1 \nmRNA gene was detected relative to the control with a \npreference for high values (p<0,001) (Table 1).\nThus, the obtained results indicate an increase in the \nrelative normalized amount of mRNA of the HMGB1 \ngene in the blood of women with endometriosis asso-\nciated with infertility. The results of the work indicated \nin the previous subsection demonstrate a pronounced \nprobable increase in the expression of the HMGB1 \ngene in the blood of women with endometriosis \nassociated with infertility, which coincides with the \ntrends of scientific works of recent years. Free HMGB1IL \nparticipates in all phases of inflammation, from damage \nto repair. It is able to activate endothelial cells and their \nprecursors, which determines a certain contribution \nof HMGB1 to angiogenesis in pathological processes.\nTo implement the tasks of this work unit, patients are \ndivided into three groups depending on the severity \nof endometriosis. Stages of the disease were deter -\nmined according to the recommendations of AFS-85, \nhowever, in order to simplify the analysis, stages I and \nII were combined into the so-called “small” forms of \nendometriosis, or mild stage, stage III corresponded \nto the average severity of endometriosis, and stage IV \ncorresponded to the severe stage of the disease. The \nfirst group included 10 women with mild endometriosis, \nthe second group included 3 patients with moderate \nendometriosis, and the third group included 7 women \nwith severe endometriosis.\nTable 2 shows the results of the HMGB1 mRNA gene \nexpression study in the blood of women depending on \nthe severity of endometriosis.\nAs can be seen from the table 2, the levels of relative \nnormalized expression of the HMGB1 gene increased \nin direct proportion to the increasing stage of endome-\ntriosis. This was reflected in the statistically significant \ndifference found between the indicators of the first \nand third, and second and third studied groups, i.e., \ncompared between the groups of women with mild \nand severe endometriosis. It is worth noting that the \nrange of relative normalized values of HMGB1 gene \nmRNA in mild cases was 1,89 – 7,32 (median – 4,76). No \nspecific features of the distribution, concentration of \nlow values or predominance of lower values of HMGB1 \nexpression in the group of patients with “small” forms \nof endometriosis were found - all values of the relative \nnormalized mRNA expression of the studied genes are \n“scattered” among the entire range of the sample as a \nwhole. In the group of patients with an average degree \nof endometriosis, the range of values of the relative \nFig. 1. Relative normalized HMGB1 mRNA gene expression\nNormalization by the ΔΔCt method with a reference gene Actin, βeta\n\nMolecular-genetic characteristics of HMGB1 mRNA expression in  blood of women with endometriosis...\n1919\nnormalized expression of the HMGB1 gene ranged from \n8,28 to 12,87 (median – 9,94), without certain trends.\nIn the group of women with severe endometriosis, the \nrange of relative normalized expression of the HMGB1 gene \nwas 13,94 – 33,42 (median – 22,97). It should also be noted \nthat (100,00%) of the observations in this group belonged \nto significant deviations from the control and the median \n(p<0,001). Accordingly, the expressed values of HMGB1 \ngene expression were concentrated even in groups of \nwomen suffering from mild endometriosis (I stage).\nThus, statistical differences in the relative normalized \nexpression of HMGB1 were found between groups of \nwomen with mild, moderate, and severe endometrio-\nsis, trends indicate an increase in this indicator directly \nproportional to the increase in the degree of endome-\ntrioid lesions. This is consistent with the assumption of \nsome authors that endometrioid growth is supported \nby the activation of a number of innate immune mol-\necules with subsequent production of factors such as \ncytokines and vascular growth factors [1, 16, 20, 21].\nDISCUSSION\nThe resulting increase in IL1β expression can be ex -\nplained from the standpoint that HMGB1 is part of a sys-\ntem that binds to immunogenic nucleotides to activate \nthe innate immune response during tissue damage or \nmicrobial infection. The biological activity of extracel-\nlular HMGB1 is largely related to the interaction with \ntarget cells involved in inflammatory and immune re -\nsponses, which emphasizes the importance of HMGB1 \nblood levels as a valuable biomarker of inflammation \nor inflammatory diseases [11-15].  HMGB1 provokes \ncell proliferation, invasion and cellular inflammation in \nthe ectopic endometrial environment through Toll-like \nreceptors [16-20]. By itself, HMGB1 has little or no pro-in-\nflammatory activity, but forms complexes with pro-in-\nflammatory factors such as IL 1β or lipopolysaccharide \nand thus enhances their biological activity [21]. Thus, \nHMGB1 complexes stimulate the synthesis of pro-in-\nflammatory cytokines. During infection, innate immu-\nnity is activated by foreign molecular products called \nPAMPs (pathogen-associated molecular patterns), \nwhich include, lipopolysaccharides. During damage or \nischemia, the cells themselves are activated  under the \ninfluence of endogenous DAMPs (damage associated \nmolecular patterns), which include such molecules as \nheat shock proteins, annexin, IL1β.  HMGB1, released \nby activated immune cells and damaged or necrotic \ncells, plays an important role in host responses to both \nTable 1.  Comparative characteristics of relative normalized expression HMGB1 gene mRNA in the blood of women with endometriosis is associated \nwith infertility\nGroups HMGB1 mRNA p\nResearched\nGroup 11,9198 ±0,01 p<0.05\nControl\nGroup 1,00±0,01 *\nNote: Relative normalized amount of HMGB1 gene mRNA. Normalization by the ΔΔCt method with the reference gene Actin βeta p<0,05 reflects a \nprobable difference.* - normalization by relative amount.\nTable 2. Expression of the HMGB1 mRNA gene level in the blood of women, of patients with endometriosis associated with infertility, depending on \nthe severity of endometriosis (M±m)\nNo\n/p Groups of patients\nIndexes\nHMGB1\n1 1 group\n(mild endometriosis (“small” forms) (n=10)) 4,76±0,22\n2 2nd group\n(moderate endometriosis (n=3)) 9,94±0,73\n3 3 group\n(severe endometriosis (n=7)) 22,97 ± 1,23\np\np1 > 0,05\np2 <0,001\np3 < 0,001\nNotes:p reflects the statistical probability of the difference between the indicators of the studied and control groups; p < 0.05, p < 0.01 - probable dif-\nference, p > 0.05 - no probable difference. p1 -   the probability of the difference between the indicators of the 1st and 2nd groups, p2 - the probability of \nthe difference between the indicators of the 2nd and 3rd groups, p3 - the probability of the difference between the indicators of the 1st and 3rd groups.\n\nOksana V. Bakun et al. \n1920\nmation of multiple tumor inflammatory pathological \nmicroenvironments and participates in inflammatory \ntumor proliferation, invasion and metastasis and other \npathological mechanisms [18].\nCONCLUSIONS\nIn the blood of women with endometriosis associated \nwith infertility, there is a significant increase in the level \nof mRNA expression of the HMGB1 gene. In the blood \nof women with endometriosis associated with infer -\ntility, a probable increase in the expression of HMGB1 \n(p<0,001) in women with moderate and severe endo -\nmetriosis compared to the mild course of the disease \nand HMGB1 (p<0.05) in the groups of women with mild \nand severe endometriosis, which indicates the role of \nthese factors in the progression of the disease.\ntypes of threats; thus, it is a critical mediator in the \nfinal common pathway to morbidity during infection \nand trauma. A possible pathogenesis of adenomyosis \na strong HMGB1/TLR4 system may be involved in the \ninflammatory pathological process of adenomyosis de-\nvelopment. It generates a local inflammatory reaction \nand activates the body’s specific immune system, builds \nand maintains a persistant inflammatory microenviron-\nment in the local lesion, and forms a local inflammatory \npathology of adenomyopathy, which may involve the \npathogenesis of angiomyopathy, include IL1β. Etracel-\nlular matrix acts an endogenous  ligand for molecules \nsuch as TLR-4 (tumor like receptor) to stimulate the \nactivation of TLR-4-mediated cellular inflammatory \nsignaling and participate in the inflammatory response \n[17]. It has been proven that HMGB1, TLR-4-mediated \ninflammatory signaling system is involved in the for -\nREFERENCES\n 1.  Broi MG Da, Ferriani RA, Navarro PA. Ethiopathogenic mechanisms of endometriosis-related infertility. 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High expression of high-mobility group box 1 in menstrual blood: implications for endometriosis. \nReprod Sci. 2017;24:1532–7. doi: 10.1177/1933719117692042. DOI\n 20.  Tang D, Kang R, Livesey KM et al.. Endogenous HMGB1 regulates autophagy. J Cell Biol. 2010;190:881–92. doi: 10.1083/jcb.200911078.\n 21.  Li Q, Yu B, Yang P . Hypoxia-induced HMGB1 in would tissues promotes the osteoblast cell proliferation via activating ERK/JNK signaling. \nInt J Clin Exp Med. 2015;8:15087. \nCONFLICT OF INTEREST\nThe Authors declare no conflict of interest\nCORRESPONDING AUTHOR\nOksana V. Bakun\nBukovinian State Medical University\n129 Golovna St, 58000 Chernivtsi, Ukraine\ne-mail: kupchanko06@gmail.com \nORCID AND CONTRIBUTIONSHIP\nOksana V. Bakun: 0000-0002-4742-2265  \nNatalya Ya. Muzyka: 0000-0002-3381-1163  \nSvitlana B. Semenenko : 0000-0002-6124-1938  \nTetiana P . Savchuk: 0000-0002-1782-1812  \nAlla I. Peryzhniak: 0000-0003-0139-0058  \n – Work concept and design,  – Data collection and analysis,  – Responsibility for statistical analysis,  – Writing the article,  – Critical review,  – Final approval of the article\nRECEIVED: 04.06.2024\nACCEPTED: 26.09.2024\n CREATIVE COMMONS 4.0","source_license":"public-domain-us","license_restricted":false}