Molecular-genetic characteristics of hmgb1 mrna expression in blood of women with endometriosis associated with infertility

HMGB1 is a nuclear protein that is present in almost all cell types. It can also be released extracellularly, where it mediates the activation of innate immune responses, including chemotaxis and cytokine release. HMGB1 was originally described as a protein that binds nuclear DNA [1,2]. It is evolutionarily highly conserved and functions as a nuclear cofactor in transcriptional regulation. HMGB1, like many other cofactors, was later found to play another role as an intercellular molecule that is released from various cells into the extracellular environment to act on specific cell surface receptors. In this latter role, HMGB1 is a proinflammatory cytokine that can contribute to many inflammatory diseases, including sepsis [3-5]. Through interaction with immune cell cell surface receptors, HMGB1 activates intracellular cascades that regulate immune cell functions, including chemotaxis and immune modulation. HMGB1 is a criti- cal mediator of lethality in sterile and infectious inflam- mation. Similar inflammatory responses are initiated by strokes caused by sterile trauma or infection. During infection, innate immunity is activated by foreign molecular products called PAMPs, which include, for example, lipopolysaccharides [6]. During sterile injury or ischemia, the cells themselves are activated under the influence of endogenous DAMPs, which include molecules such as heat shock proteins, uric acid, an- nexin, and IL1β. HMGB1, released by activated immune cells and damaged or necrotic cells, plays an important role in host responses to both types of threats; thus, it is a critical mediator in the final common pathway to morbidity and mortality during infection and sterile trauma [7-10]. In a recent study (Expression of high-mobility group box-1 in eutopic/ectopic endometrium and correlations with inflammation-related factors in adenomyosis Xiu- Ni Liu and Zhong-Ping Cheng) it was described. Possible pathogenesis of adenomyosis: the strong HMGB1/TLR4 system may be involved in the inflammatory pathological process of adenomyosis development. It generates a local inflammatory reaction and activates the body’s specific immune system, builds Molecular-genetic characteristics of HMGB1 mRNA expression in blood of women with endometriosis associated with infertility Oksana V. Bakun, Natalya Ya. Muzyka, Svitlana B. Semenenko, Tetiana P . Savchuk, Alla I. Peryzhniak BUKOVINIAN STATE MEDICAL UNIVERSITY , CHERNIVTSI, UKRAINE

Aim: To examine HMGB1 expression in the blood of women with endometriosis-associated infertility and to establish its role in disease progression.

We analyzed HMGB1 gene expression levels in two groups: 20 women with endometriosis-associated infertility (main group) and 10 healthy women (control group). The study was conducted at Bukovinian State Medical University and the Centre of Reproductive Medicine. Primary infertility was significantly higher in the main group. We used real-time reverse transcription polymerase chain reaction (RT-PCR) to determine HMGB1 mRNA levels in mononuclear cells isolated from whole blood. Statistical significance was determined using the Student’s t-test, with p < 0.05 considered significant.

Results indicated a significant increase in HMGB1 mRNA expression in the main group compared to the control group (p < 0.001). The relative normalized expression ranged from 1.8924 to 33.426 (median = 8.01). Expression levels were categorized into three groups: borderline with the control, moderate increase, and significant increase. Only one sample (5%) had values equal to the control. Fifteen percent of samples had values slightly above control (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 elevated, predominantly at high values (p < 0.001).

Women with endometriosis-associated infertility show significantly increased HMGB1 mRNA expression, particularly in moderate and severe cases compared to mild cases, indicating HMGB1’s role in disease progression (p < 0.001). KEY WORDS: endometriosis, infertility, assisted reproductive technologies, HMGB1 Wiad Lek. 2024;77(9):1916-1921. doi: 10.36740/WLek/195142 DOI ORIGINAL ARTICLE CONTENTS Molecular-genetic characteristics of HMGB1 mRNA expression in blood of women with endometriosis... 1917 and maintains a stable inflammatory inflammatory microenvironment in the local lesion, and forms a local inflammatory pathology of adenomyopathy, which may involve the pathogenesis of angiomyopathy. Therefore, HMGB1 may also play an important role in the pathophysiology of endometriosis, a gynecological disease associated with a chronic immunoinflammatory process. AIM The aim of our study was to examine the expression HMGB1 in the blood of women with endometriosis-as- sociated and to establish the role of HMGB1 in the progression of endometriosis by analyzing the level of gene expression in the blood of women with infertility- associated infertility, depending on the severity of endometriosis.

We determined the level of HMGB1 mRNA gene expres- sion in two groups. The first group (main) consisted of 20 women with endometriosis associated with infertil- ity. The control group consists of 10 practically healthy women. The absence of signs of acute inflammatory processes in the reproductive sphere and the negative

of microbiological and virological studies were evidence of a long-overdue inflammatory process that caused tubal obstruction. This study was conducted at the Bukovyna State Medical University and the clinic “Yuzko medical center” . To analyze the expression of the HMGB1 gene and to determine the relative normalized expression of HMGB1 mRNA, the polymerase chain reaction with reverse transcription in real time (RT-PCR) was used. The object for molecular genetic studies by the RT-PCR

was the fraction of mononuclear cells isolated from the whole blood of patients with endometriosis. Specific pairs of primers for the analysis of the studied and reference genes were selected using the Primer- ΒLAST software (www.ncβi.nlm.nih.gov/tools/primer- βlast) and manufactured by Thermo Scientific (USA). The actin, βeta (Actβ) gene was used as a reference gene to determine the relative value of the change in the expression level of the studied genes. The comparative Ct method (ΔΔCt method) was used to express the relative level of gene expression. Calculations were made according to the formulas: ΔCt (target gene) = Ct (target gene) – Ct (calibrator gene / ACT1); ΔΔCt = ΔCt (target gene) – ΔCt (base gene); The relative expression level was expressed as 2–ΔΔCt. Statistical analysis of PCR data was performed using CFX Manager ™ software (Βio-Rad, USA). Optimal RT-PCR conditions were selected to achieve a linear relationship between the number of cycles and the number of PCR products. Negative controls were included in the experiment: without addition of cDNA matrix in the PCR reaction, without addition of mRNA matrix in cDNA synthesis, without addition of enzyme in cDNA synthesis. All amplification reactions were performed on individual samples in triplicate. Quantitative variables were evaluated using the Shapiro-Wilk test (if the number of subjects was less than 50) or the Kolmogorov-Smirnov test (when the number of subjects was more than 50). Comparison of two groups for a quantitative variable according to a normal distribution, under the condi- tion of equality of variances, was performed using the Student’s t-test. Statistical processing of the results of this section was carried out using non-parametric methods, namely the Wilcoxon-Mann-Whitney test. Differences between groups were considered probable at the significance level of p<0.05. RESUL TS In the analysis, depending on the degree of severity of endometriosis, stage I and II were combined into so- called «small» forms of endometriosis, or mild stage, stage III corresponded to the average severity of en- dometriosis, and stage IV corresponded to the severe stage of the disease. The results of the study of the relative normalized expression of HMGB1 mRNA in the whole blood of women with endometriosis associated with infertility are shown in Fig. 1. Based on our research, it was established that the ex- pression of HMGB1 mRNA in the blood of women with endometriosis associated with infertility is significantly increased compared to the indicators of the control group (р< 0.001). The range of all obtained values of the relative normalized amount of mRNA of the HMGB1 gene was 1,8924 – 33,426 (median – 8,01). For a broader characterization of the features of the level of HMGB1 mRNA expression in the blood of women with endometriosis, associated with infertility, and not only the limitation of the data of the average value, conditionally, the ranges of fluctuations of the indicator were separated into the following groups: borderline with the control group, lower than the median (moderate increase HMGB1 expression level) and higher than the median (significant increase in HMGB1 expression level). As can be seen, the lower range of values of the HMGB1 gene is equal to the Oksana V. Bakun et al. 1918 control, however, this indicator was measured only in 1 out of 20 studied blood samples, which was 5% of cases (p1,89-<3,45), which was 15% (p3,45-8,01) was found in 10 blood samples, which accounted for half of the cases - 50%. Thus, in the blood samples of 95% of the women in the study group, increased expression of the HMGB1 mRNA gene was detected relative to the control with a preference for high values (p<0,001) (Table 1). Thus, the obtained results indicate an increase in the relative normalized amount of mRNA of the HMGB1 gene in the blood of women with endometriosis asso- ciated with infertility. The results of the work indicated in the previous subsection demonstrate a pronounced probable increase in the expression of the HMGB1 gene in the blood of women with endometriosis associated with infertility, which coincides with the trends of scientific works of recent years. Free HMGB1IL participates in all phases of inflammation, from damage to repair. It is able to activate endothelial cells and their precursors, which determines a certain contribution of HMGB1 to angiogenesis in pathological processes. To implement the tasks of this work unit, patients are divided into three groups depending on the severity of endometriosis. Stages of the disease were deter - mined according to the recommendations of AFS-85, however, in order to simplify the analysis, stages I and II were combined into the so-called “small” forms of endometriosis, or mild stage, stage III corresponded to the average severity of endometriosis, and stage IV corresponded to the severe stage of the disease. The first group included 10 women with mild endometriosis, the second group included 3 patients with moderate endometriosis, and the third group included 7 women with severe endometriosis. Table 2 shows the results of the HMGB1 mRNA gene expression study in the blood of women depending on the severity of endometriosis. As can be seen from the table 2, the levels of relative normalized expression of the HMGB1 gene increased in direct proportion to the increasing stage of endome- triosis. This was reflected in the statistically significant difference found between the indicators of the first and third, and second and third studied groups, i.e., compared between the groups of women with mild and severe endometriosis. It is worth noting that the range of relative normalized values of HMGB1 gene mRNA in mild cases was 1,89 – 7,32 (median – 4,76). No specific features of the distribution, concentration of low values or predominance of lower values of HMGB1 expression in the group of patients with “small” forms of endometriosis were found - all values of the relative normalized mRNA expression of the studied genes are “scattered” among the entire range of the sample as a whole. In the group of patients with an average degree of endometriosis, the range of values of the relative Fig. 1. Relative normalized HMGB1 mRNA gene expression Normalization by the ΔΔCt method with a reference gene Actin, βeta Molecular-genetic characteristics of HMGB1 mRNA expression in blood of women with endometriosis... 1919 normalized expression of the HMGB1 gene ranged from 8,28 to 12,87 (median – 9,94), without certain trends. In the group of women with severe endometriosis, the range of relative normalized expression of the HMGB1 gene was 13,94 – 33,42 (median – 22,97). It should also be noted that (100,00%) of the observations in this group belonged to significant deviations from the control and the median (p<0,001). Accordingly, the expressed values of HMGB1 gene expression were concentrated even in groups of women suffering from mild endometriosis (I stage). Thus, statistical differences in the relative normalized expression of HMGB1 were found between groups of women with mild, moderate, and severe endometrio- sis, trends indicate an increase in this indicator directly proportional to the increase in the degree of endome- trioid lesions. This is consistent with the assumption of some authors that endometrioid growth is supported by the activation of a number of innate immune mol- ecules with subsequent production of factors such as cytokines and vascular growth factors [1, 16, 20, 21].

The resulting increase in IL1β expression can be ex - plained from the standpoint that HMGB1 is part of a sys- tem that binds to immunogenic nucleotides to activate the innate immune response during tissue damage or microbial infection. The biological activity of extracel- lular HMGB1 is largely related to the interaction with target cells involved in inflammatory and immune re - sponses, which emphasizes the importance of HMGB1 blood levels as a valuable biomarker of inflammation or inflammatory diseases [11-15]. HMGB1 provokes cell proliferation, invasion and cellular inflammation in the ectopic endometrial environment through Toll-like receptors [16-20]. By itself, HMGB1 has little or no pro-in- flammatory activity, but forms complexes with pro-in- flammatory factors such as IL 1β or lipopolysaccharide and thus enhances their biological activity [21]. Thus, HMGB1 complexes stimulate the synthesis of pro-in- flammatory cytokines. During infection, innate immu- nity is activated by foreign molecular products called PAMPs (pathogen-associated molecular patterns), which include, lipopolysaccharides. During damage or ischemia, the cells themselves are activated under the influence of endogenous DAMPs (damage associated molecular patterns), which include such molecules as heat shock proteins, annexin, IL1β. HMGB1, released by activated immune cells and damaged or necrotic cells, plays an important role in host responses to both Table 1. Comparative characteristics of relative normalized expression HMGB1 gene mRNA in the blood of women with endometriosis is associated with infertility Groups HMGB1 mRNA p Researched Group 11,9198 ±0,01 p<0.05 Control Group 1,00±0,01 * Note: Relative normalized amount of HMGB1 gene mRNA. Normalization by the ΔΔCt method with the reference gene Actin βeta p<0,05 reflects a probable difference.* - normalization by relative amount. Table 2. Expression of the HMGB1 mRNA gene level in the blood of women, of patients with endometriosis associated with infertility, depending on the severity of endometriosis (M±m) No /p Groups of patients Indexes HMGB1 1 1 group (mild endometriosis (“small” forms) (n=10)) 4,76±0,22 2 2nd group (moderate endometriosis (n=3)) 9,94±0,73 3 3 group (severe endometriosis (n=7)) 22,97 ± 1,23 p p1 > 0,05 p2 <0,001 p3 < 0,001 Notes:p reflects the statistical probability of the difference between the indicators of the studied and control groups; p < 0.05, 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 the 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. Oksana V. Bakun et al. 1920 mation of multiple tumor inflammatory pathological microenvironments and participates in inflammatory tumor proliferation, invasion and metastasis and other pathological mechanisms [18].

In the blood of women with endometriosis associated with infertility, there is a significant increase in the level of mRNA expression of the HMGB1 gene. In the blood of women with endometriosis associated with infer - tility, a probable increase in the expression of HMGB1 (p<0,001) in women with moderate and severe endo - metriosis compared to the mild course of the disease and HMGB1 (p<0.05) in the groups of women with mild and severe endometriosis, which indicates the role of these factors in the progression of the disease. types of threats; thus, it is a critical mediator in the final common pathway to morbidity during infection and trauma. A possible pathogenesis of adenomyosis a strong HMGB1/TLR4 system may be involved in the inflammatory pathological process of adenomyosis de- velopment. It generates a local inflammatory reaction and activates the body’s specific immune system, builds and maintains a persistant inflammatory microenviron- ment in the local lesion, and forms a local inflammatory pathology of adenomyopathy, which may involve the pathogenesis of angiomyopathy, include IL1β. Etracel- lular matrix acts an endogenous ligand for molecules such as TLR-4 (tumor like receptor) to stimulate the activation of TLR-4-mediated cellular inflammatory signaling and participate in the inflammatory response [17]. It has been proven that HMGB1, TLR-4-mediated inflammatory signaling system is involved in the for -

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