Serum Chemokines and Quality of Life among Patients with Endometriomas and Teratomas

Background : Endometriosis is a chronic inflammatory condition characterized by the presence of endometrial tissue outside the uterus, which can cause pelvic pain, infertility, and other symptoms. The disease may manifest as superficial peritoneal or deep-infiltrating endometriosis or as ovarian endometriomas. Although the mechanisms associated with the regulation and production of inflammatory mediators in endometriosis have been widely investigated, the precise mechanism responsible for inflammation-induced pain remains unclear


Introduction
Endometriosis is a chronic inflammatory condition characterized by the presence of endometrial tissue outside the uterus, which can cause pelvic pain, infertility, and other symptoms [1].The disease may manifest as superficial peritoneal or deep-infiltrating endometriosis or as ovarian endometriomas [2].Endometriosis provokes a neurovascular response mediated by hormones.The growth of ectopic endometrial tissue stimulates an estrogen-dependent chronic inflammatory reaction, causing severe pain, which can be attributed to an increased prostaglandin production accompanied by compression and/or infiltration of approximal nerves [3].Increased expression of nerve growth factors, high nerve fiber density, angiogenesis, and changes in the pattern of uterus innervation may also play a role in the manifestation of endometriosis [3].Chemokines (chemo-tactic cytokines), a small subgroup of cytokines, lead to the chemotaxis of monocytes, neutrophils, eosinophils, lymphocytes, and fibroblasts.One of the main functions of chemokines is to induce leukocytes to migrate to the site of inflammation [4].Cytokines belonging to the CXC chemokine family, as well as their receptors, have been shown to be possibly involved in the proliferation and invasion of endometrial cells [4].Although the mechanisms associated with the regulation and production of inflammatory mediators in endometriosis have been widely investigated, the precise mechanism responsible for inflammationinduced pain remains unclear, and the findings related to the cytokine expression profile and the location of cytokines in cells are contradictory [3,4].
Based on the location, depth, and size of lesions, endometriosis can be categorized into four stages.However, this classification is ineffective in predicting the clinical outcomes, such as disease-related symptoms and associated discomfort [5].The intensity of pain experienced by endometriosis patients is not proportional to the degree and severity of their disease.Pain has a significant impact on women suffering from endometriosis.Patients with chronic pain have a lower quality of life and a worse mental health condition, and are more likely to be depressed than patients living with a chronic disease without pain [6,7].Psychological intervention is recommended for patients suffering from chronic pain [8].Eighty percent of endometriosis patients suffer from chronic pelvic pain [9].Unfortunately, there is currently no one tool available for assessing the level of pain experienced by endometriosis patients [8].Two of the most frequently used pain scales, not just in the case of endometriosis, are the visual analog scale and the numerical pain rating scale [10].In addition, the Nottingham Health Profile (NHP) questionnaire, which is a less commonly used tool, allows assessing health problems and their impact on the daily functioning of patients [11][12][13][14][15].The results of this questionnaire were found to be consistent and strongly correlated with the findings of other tools, indicating that the measures are repeatable and reliable [11][12][13][14][15].
The current study aimed to assess the serum levels of MCP-1/Monocyte Chemoattractant Protein 1, MCP-2/Monocyte Chemoattractant Protein 2, MCP-3/Monocyte Chemoattractant Protein 3, Eotaxin-1/Eosinophil Chemotactic Protein, CCL13/Chemokine CC motif with ligand 13, RANTES/Regulated on Activation, Normal T cell Expressed and Secreted, CXCL9/Chemokine CXC motif with ligand 9, CXCL10/Chemokine CXC motif with ligand 10, CXCL11/Chemokine CXC motif with ligand 11 and determine their relationship with the quality of life or health problems among patients with endometriomas in comparison to patients with teratomas.

Materials
The study sample included patients with endometriomas and teratomas who were treated laparoscopically at the Gynecological and Obstetrics Clinical Hospital of the Medical University of Poznan, during 2019-2020, before the SARS-CoV-2 pandemic.
In the first stage of the study, the patients were subjected to a vaginal ultrasound examination.The initial diagnosis endometriomas (an ovarian tumor filled with hyperechoic fluid) or teratomas (hyper or hypoechoic tumor of the ovary) was made based on the ultrasound images.The final diagnosis was made based on histopathological examination of the tumors removed during surgery.The following inclusion criteria were applied for qualifying patients for surgery: presence of endometriomas vs teratomas, negative pregnancy test result, no prior obstetric and infertility treatment, and good health condition with no dis-eases or coagulation disorders.All the patients underwent laparoscopy during the first phase of the cycle (i.e., after the end of bleeding).
The control group included healthy patients who were undergoing preventive examination in the Gynecological Outpatient Clinic.Inclusion criteria for the study were: no ultrasound ovarian changes, negative pregnancy test result, no prior obstetric and infertility treatment, and good health condition with no diseases or coagulation disorders.After qualifying for the study, the patients came to the hospital the next morning (only to collect blood in a designated place and complete two questionnaires).
After surgery and histopathological examination, the patients were divided into three groups.Group E (endometriomas) included women who had been histologically diagnosed with endometriomas without macroscopic peritoneal endometriosis (n = 24).Group T (teratomas) included women with histologically diagnosed teratomas (n = 14).Group C (control) included healthy women who were undergoing routine, preventive gynecological examination (n = 22).

Methods
Blood samples were collected from all patients on the day of admission to the hospital, in the morning under fasting condition (i.e., 1 day before surgery).The levels of CA125 and HE4 were determined immediately after sample collection.The remaining blood samples collected for chemokine analysis were centrifuged and frozen at -20 °C.
The serum levels of chemokines were determined by ELISA (enzyme-linked immunosorbent assay).All the experiments were performed in duplicate to calculate measurement error.The concentrations of the analyzed parameters were determined in pg/mL by plotting a standard graph.
A total of 60 female patients aged 21-50 years participated in the study.Two questionnaires were used in the study: the questionnaire developed by the authors and the NHP questionnaire.The first one contained 10 questions concerning age, education, marital status, place of residence, financial situation, number of miscarriages and childbirths, and health condition [16,17].There are different scientific tools to asses quality of life among patients, everyone emphasizes some aspects.The NHP questionnaire is a validated research tool in the Polish language.It is a simple questionnaire that allows measuring the perceived physical, social, and emotional health status of an individual.The NHP questionnaire is designed to assess the influence of social and personal factors on illness [15].Worth noticing is also the fact, that NHP was more sensitive to physical aspect of the disease, whereas SF-36 was more coherent in evaluating social functioning [16].In contrary to widely used QoL questionnaires there are also developing tools of narrow use, like the Endometriosis Health Profile (EHP-5) or World Endometriosis Research Foundation tool (WERF) [16].It consists of two parts.The first fundamental part focuses on six dimensions, namely: "VITAL ENERGY," "PAIN", "EMOTIONS", "SLEEP DISORDERS", "SO-CIAL ISOLATION", "PHYSICAL FITNESS", and "TO-TAL".This part assesses the patients' current problems that have an impact on their health status, which includes their physical, psychological, and social functioning.The second part focuses on "INCOMPATIBILITIES IN EVERY-DAY LIFE" and contains questions regarding the impact of the disease on the quality of life.All patients filled in the questionnaires on their own (1 day before the surgery and after their routine annual medical visit to the clinic).

Statistical Analysis
The results of the quantitative data analysis were presented as mean and standard deviation, and the median (Me) values were also calculated.Significant differences in the rank values obtained for parameters with a nonnormal distribution or parameters showing heterogeneous variance were evaluated by nonparametric Kruskal-Wallis test.The results of the statistical analysis of qualitative data obtained from the questionnaire, which concern the characteristics of the studied patients, were presented as the number of individuals in a particular category (n).The differences in qualitative characteristics between the groups were analyzed using the χ 2 independence test.The correlation between particular variables and the strength of this associa-tion was determined by calculating Spearman's rank correlation coefficients.The limit of significance was set at p = 0.05 in all the statistical tests.

Results
Group E included female patients with histopathologically confirmed endometriomas (n = 24), and their mean age was 33.50 ± 7.14 years.Group T included female patients with teratoma cysts (n = 14) who had undergone surgical treatment for benign ovarian lesions, and their mean age was 34.86 ± 8.03 years.Group C (control) comprised healthy women (n = 22) who were undergoing routine, annual gynecological examination, and their mean age was 23.86 ± 5.28 years (Table 1).

General Characteristics of the Studied Groups
All the studied women had good general health condition without coexisting diseases.The general characteristics, the size of ovarian tumors, and the levels of CA125 and HE4 markers determined in each of the analyzed groups are presented in Table 1.

Serum Chemokine Levels
The median serum levels of MCP-1 and MCP-3 were statistically higher in the endometriomas group compared to the other two groups (Table 2).

NHP Questionnaire
The comparison of the subjective health dimensions in individual groups showed that the patients in the endometriomas group experienced a significantly higher intensity of "PAIN" compared to other groups (Table 3).No statistically significant differences were observed between the groups in terms of the seven dimensions of life.

Correlation Analysis between NHP Dimensions and Serum Chemokine Levels
Spearman's rank correlation analysis indicated a statistically significant relationship between the "VITAL EN-ERGY" dimension and the level of MCP-2 (r = -0.295;p = 0.022), MCP-3 (r = 0.254; p = 0.050), and RANTES (r = -0.353;p = 0.006); between the "EMOTIONS" dimension and the level of MCP-3 (r = 0.262; p = 0.043); and between the "INCONVENIENCE IN DAILY LIFE" dimension and the level of Eotaxin-1 (r = -0.283;p = 0.028) and CCL13 (r = -0.287;p = 0.026).No statistically significant associ-ation between the NHP dimensions and the serum levels of chemokines was shown by the analysis for the control and teratomas group of women (Table 4).

Discussion
The predominant complaint reported by endometriosis patients is pain, which often persists even after the disease is treated.Endometriosis-related pain can be caused by several factors, including nociception, inflammation, and alterations in pain processing functions in the nervous systems.As observed in other chronic diseases, pain due to endometriosis often leads to psychological restlessness and fatigue, which can further worsen pain and reduce the quality of life of patients [18,19].Compared to patients with asymptomatic endometriosis, endometriosis patients with pelvic pain have a poorer quality of life and mental health [8].The issue of pain in endometriosis patients has already been studied [16,17].Previous works, as well as the present study, showed that patients with endometriomas ex- perienced a significantly higher intensity of "PAIN" compared to other groups.However, Bień et al. [20] highlighted that women with endometriosis rated the overall quality of life higher than the general state of health, which can be attributed to the patients' acceptance of their disease [20].The degree of disease progression does not correlate with the subjective symptoms; therefore, the quality of life can be measured and compared using validated questionnaires [21].Unfortunately, women with deep-infiltrating endometriosis have issues in various domains of quality of life, regardless of the questionnaire used for the assessment [22].It seems that the NHP questionnaire could be routinely used for endometriosis patients and could be considered as a screening test by family doctors.Patients suffering from severe pain due to endometriosis can be assessed using the NHP questionnaire, and regardless of the COVID-19 pandemic, should be treated immediately.
In continuation of the previous research [23][24][25], the present study investigated the levels of selected chemokines and their relationship with the quality of life of patients with endometriomas.Endometriosis is a well-known chronic inflammatory condition in which endometrial tissue grows outside the uterus, mainly into the peritoneum [26].The main source of endometriosis-related pain appears to be lesions and adhesions.However, removal of the lesions does not help with pain in all patients [9,27].It has been observed that the peritoneal fluid undergoes several biological changes in endometriosis patients.The dynamic interactions between cytokines may contribute to developing an appropriate microenvironment for the implantation of endometrial cells as well as disease progression [26].Chen et al. [28] observed that in patients with endometriosis progression peritoneal fluid chemokines (MCP-1, MCP-3, CXCL1, CXCL2) are produced at a significantly higher level [28].CXCL12 can affect the proliferation, migration, and invasion of endometriotic cells [29].The ligand-receptor complexes such as CXCR4-CXCL12 and CXCL12-CXCR7 are also activated in endometriosis [30].Pizzo et al. [26] reported that the serum levels of MCP-1 and IL-8 decreased with increasing severity of endometriosis, while the concentration of peritoneal fluid significantly increased in severe stages [26].Similarly, Hornung et al. [31] highlighted that the level of eotaxin-1 in the peritoneal fluid was higher in patients with moderate-to-severe endometriosis [31], suggesting that this protein interacts with other cytokines and immune cells, contributing to inflam- mation [31].Măluțan et al. [32] found increased levels of MCP-1 and IL-8 and lower levels of Eotaxin-1 in the serum of endometriosis patients, which may indicate that the immune activity is imbalanced in this disease [32].These findings are in line with the results of the present study which demonstrated that the median values of MCP-1 and MCP-3 cytokines were statistically significantly higher in the endometriomas group.Indeed, it seems that the effects induced by pelvic endometriosis, including the breakdown of peritoneal homeostasis and the production of pro-inflammatory and proangiogenic cytokines, are responsible for the altered innervation and modulation of pain pathways [33,34] and perhaps the higher intensity of pain.In this study, the correlation analysis did not show any statistically significant relationship between "PAIN" and the level of serum chemokines in the studied endometriomas patients.However, a statistically significant relationship was found between the "VITAL ENERGY" dimension and the serum levels of MCP-2, MCP-3, and RANTES; between the "EMOTIONS" dimension and the serum level of MCP-3; and between the "INCONVENIENCE IN DAILY LIFE" dimension and the serum levels of eotaxin-1 and CCL13.According to Roch et al. [35], cytokines and chemokines are not reliable markers for predicting the presence of endometriosis in women who show the symptoms of this disease [35].
Patients suffering from endometriosis, regardless of pathogenesis [36], experience pain every day [8][9][10][18][19][20] and often require immediate consultation, planned surgeries, or even emergency surgical interventions.SARS-CoV-2 pandemic has significant negative consequences for endometriosis patients worldwide, including postponed surgeries [37][38][39], reduced quality of life [40], worsened access to care [41], difficulties in repeating painkiller or hormone prescriptions, and missed appointments [42].Clinicians should remember that patients suffering from endometriosis or its symptoms, especially pain, need longterm treatment and consultation with doctors.In case of contraindications to a face-to-face appointment, patients can use telemedicine techniques to have regular contact with clinicians [43,44].In particular, patients who have had multiple surgeries or who experience sudden, substantial bleeding should be aware of the symptoms that prompt a visit to the gynecological emergency unit.

Conclusions
The chemokines serum levels (i.e., MCP-1 and MCP-3) and intensity of "PAIN" were statistically higher in the endometriomas compared to the teratomas group of women.
Therefore, understanding their role in endometriosis-related pain could help in the development of novel, multidisciplinary treatments.

Table 1 . General characteristics of the examined groups.
* Means difference is significant at 0.05.

Table 2 . Serum chemokine levels.
* Means difference is significant at 0.05.

Table 3 . Nottingham Heath Profile questionnaire.
* Means difference is significant at 0.05.