Increased expression of IL-37 correlates with TNF-α levels and disease stage in endometriosis patients

Background: IL-37 is an anti-inflammatory cytokine that increases in several inflammatory diseases with the main inducing signal for its production being pro-inflammatory cytokines like TNF-α. We aimed to assess the correlation between peritoneal fluid levels of IL-37 and TNF-α in endometriosis patients and investigate their association with disease stage. Levels of IL-37 and TNF-α were assessed in peritoneal fluid of 50 patients with endometriosis and 23 endometriosis-free females using enzyme-linked immunosorbent assay. We also assessed serum levels of IL-37 using enzyme-linked immunosorbent assay and expression of IL-37 mRNA in peritoneal fluid cells using polymerase chain reaction.

Peritoneal fluid levels of IL-37 and TNF-α were higher in endometriosis patients than in control females. Also, levels were higher in patients with late endometriosis than patients with early endometriosis. In addition, serum levels of IL-37, as well as IL-37 mRNA expression in peritoneal fluid cells, were higher in patients than controls. In endome- triosis patients, peritoneal fluid levels of IL-37 positively correlated with levels of TNF-α.

Collectively, our results show increased levels of the anti-inflammatory cytokine IL-37 in endome- triosis patients that correlate with levels of the pro-inflammatory cytokine TNF-α, one of the main signals for IL-37 production.

IL-37, TNF-α, Peritoneal fluid, Endometriosis © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.

Endometriosis is a disease characterized by the pres - ence of functioning endometrial tissue, both glandular epithelium, and stroma, outside of the uterus [1]. It is a common disease that affects around 2–10% of females [2]. Patients typically complain of abdominal pain, dys - menorrhea, dyspareunia, and infertility, however, some patients may be asymptomatic and are accidentally dis - covered during laparoscopy for any other reason [3]. Endometriosis can occur in a variety of locations, the most common of which are the ovary, rectovaginal septum, Douglas pouch, and uterosacral ligaments. It can sometimes occur in the vagina, rectum, fallopian tubes, uterine cervix, or the urinary tract [4]. Symptoms associ - ated with endometriosis have an impact on the patient’s physical, psychological, and social well-being [5]. The exact etiology of endometriosis is not fully eluci - dated, however multiple theories have been proposed, among which are environmental factors, hormones, dysregulated immunity, inflammatory factors as well as genetic and epigenetic factors [6]. One widely accepted theory is retrograde menstruation followed by implan - tation of endometrial fragments. A growing body of lit - erature indicates that the survival of endometrial tissue is enhanced by a concurrently altered immune surveillance that allows the survival of the implanted endometrial tis - sue [7]. Open Access Egyptian Journal of Medical Human Genetics *Correspondence: [email protected] 1 Clinical Pathology, Faculty of Medicine, Ain Shams University, Cairo, Egypt Full list of author information is available at the end of the article Page 2 of 7Ragab et al. Egyptian Journal of Medical Human Genetics (2022) 23:72 Interleukin-37 (IL-37) is an anti-inflammatory cytokine and one of the members of the IL-1 family. IL-37 is mainly expressed in circulating monocytes, tissue macrophages, dendritic cells, tonsil B cells, and plasma cells [8]. IL-37’s anti-inflammatory function is primarily achieved by sup - pressing innate immunity [9]. Expression and production of IL-37 are increased in response to exposure to various toll-like receptor ligands, as well as, pro-inflammatory cytokines including IL-1, TNF-α, IL-18, and IFN-γ [8]. Furthermore, data from multiple studies show that IL-37 plays a role in controlling the inflammatory process in a variety of inflammatory diseases [10–17]. As an anti-inflammatory cytokine, IL-37 is suggested to play a role in endometriosis. Women with endome - triosis had higher IL-37 mRNA expression in ectopic and eutopic endometrium [18] as well as higher serum and intraperitoneal fluid levels [19]. In mice, IL-37 over - expression inhibited endometrial stromal cells prolif - eration and adhesion [20]. The relationship between IL-37 and proinflammatory cytokines in endometriosis patients has not been investigated before. Because pro- inflammatory cytokines, such as TNF-α, are considered to be the primary stimulus for increased IL-37 expres - sion and production [21], herein, we sought to examine the relationship between levels of IL-37 and levels of TNF-α in peritoneal fluid of endometriosis patients. We also assessed serum IL-37 levels as well as the expression of IL-37 mRNA in peritoneal fluid cells of endometriosis patients.

Study participants The study included 73 females recruited from the Gyne - cology and Obstetrics Department at the Faculty of Medicine, Ain Shams University. All study participants had regular menstrual cycles and were in the menstrual cycle’s proliferative phase (according to the last men - strual period date). Females who had received hormonal treatment in the previous 6 months or who had any con - comitant autoimmune disease, endometrial hyperplasia, endometritis, endometrial cancer, or adenomyosis were excluded. The patients’ group included 50 endometriosis patients who were diagnosed through histopathological examination following laparoscopy. The revised Ameri - can Fertility Society (rAFS) classification of endometrio - sis was used to assess endometriosis staging and scoring. The rAFS score was used to categorize patients into four stages: Stage I (minimal; rAFS score 1–5), Stage II (mild; rAFS score 6–15), Stage III (moderate; rAFS score 16–40) and Stage IV (severe; rAFS score > 40). Endometriosis patients in stages I and II were grouped as “early endo - metriosis group” while those in stage III and stage IV were grouped as “late endometriosis group” . As a control group, 23 age-matched females, who underwent laparos - copy for diagnostic reasons, tubal disconnection, ovarian cystectomy, ovarian drilling, and who were confirmed to not have endometriosis during laparoscopy ± histologi- cal examination if needed, were recruited to the study. Before taking part in the study, each participant provided written informed consent. Samples Two milliliters (2  mL) of venous blood were collected into vacutainer tubes (gel type), and the serum was cen - trifuged at 3000  rpm for 15  min before being stored at − 80  °C until it was used later to measure serum IL-37 levels. Peritoneal fluid (2 mL) was collected at the begin - ning of the laparoscopy from the pouch of Douglas in a sterile centrifuge tube. After centrifuging the sample at 3000  rpm for 15  min, the supernatant was stored at − 80  °C until used to assess peritoneal fluid IL-37 and TNF-α levels, while cell pellets were stored in RNA later (Qiagen, Germany) at − 80  °C till used later in mRNA extraction. Assessment of IL‑37 and TNF‑α levels Levels of IL-37 in both serum and peritoneal fluid were measured using an enzyme-linked immunosorbent assay (ELISA) kit provided by Elabscience, USA, while levels of TNF-α in the peritoneal fluid were measured using an ELISA kit provided by R&D Systems, UK. Assessment of IL‑37 mRNA expression mRNA extraction RNeasy Mini Kit (Qiagen, Germany) was used to extract RNA from peritoneal fluid cells. Reverse-transcription of RNA into complementary DNA was done using Quanti - Tect Reverse Transcription kit (Qiagen, Germany) by incubating a total volume of 14 uL containing 1ug RNA, 2  uL gDNA wipe-out buffer, and RNase free water for 2  min at 42  °C. Then 1  uL reverse transcriptase, 4  uL RT buffer, and 1uL RT primer mix were added to reach a total volume of 20 uL. The mixture was incubated for 15 min at 42 °C followed by 3 min at 95 °C. Real‑time PCR TaqMan Gene Expression Master Mix (Applied Biosys - tems, USA) and TaqMan Gene Expression Assay sets (Applied Biosystems, USA) were used for gene expres - sion PCR analysis of IL-37 and GAPDH as the normal - izing house-keeping gene. The reaction volume (total 20 uL) included 10 uL Master Mix, 1 uL Taqman assay, 7  uL nuclease-free water, and 2  uL cDNA. Rotorgene QPCR System was used for amplification according to the following protocol: initial activation for 10  min at 95  °C, followed by 40 cycles of denaturation for 15  s at Page 3 of 7 Ragab et al. Egyptian Journal of Medical Human Genetics (2022) 23:72 95  °C followed by annealing and extension for 1  min at 60  °C. The comparative CT method (also known as the 2−ΔCT method) was used to calculate IL-37 mRNA rela - tive expression by normalizing to GAPDH. Statistical analysis For statistical analysis, Graph Pad Prism 5 (San Diego, CA, USA) was used. The median and interquartile range were used to present quantitative data, while number and percentage were used to present categorical data. To compare groups, the Mann–Whitney test was used. Spearman’s correlation was used to determine the degree of correlation between parameters. A p value of < 0.05 was deemed statistically significant.

Clinical characteristics of study participants The study included 50 patients with endometriosis, with a mean age of 29.7 ± 6.2  years. Patients were classified according to their rAFS stage: seven had stage I endome - triosis, nineteen had stage II endometriosis, nineteen had stage III endometriosis, and five had stage IV endome - triosis. Control samples were collected from 23 women who did not have endometriosis and had mean age of 30.9 ± 5.1 years (Table 1). Elevated IL‑37 levels in endometriosis patients We first wanted to look into IL-37 levels and expression in Egyptian endometriosis patients because they had never been investigated before. And, before assessing the correlation of IL-37 with TNF-α, we wanted to find out if IL-37 levels were higher in endometriosis patients compared to control females. Endometriosis patients showed significantly higher serum IL-37 levels compared to controls (p < 0.0001) (Fig.  1A). Furthermore, perito - neal fluid samples of endometriosis patients showed sig - nificantly higher IL-37 levels compared to the peritoneal fluid of controls (p < 0.0001) (Fig. 1B). Higher IL‑37 levels in late endometriosis stages To evaluate if higher IL-37 levels were related to endome- triosis stage, patients were divided into two groups based on their r-AFS stage: early endometriosis (stages I and II) and late endometriosis (stages III and IV), and IL-37 levels were compared between both groups. Patients with late stages of endometriosis (n = 24) had signifi - cantly higher serum IL-37 levels compared to patients with early stages of endometriosis (n = 26) (p = 0.0018) (Fig.  2A). Furthermore, IL-37 levels in peritoneal fluid were significantly higher in patients with late stage endo - metriosis compared to patients with early stage endome - triosis (p = 0.0001) (Fig. 2B). Increased IL‑37 mRNA expression in endometriosis patients Real-time PCR was used to look at the expression of IL-37 mRNA in peritoneal fluid cells. IL-37 mRNA rela - tive expression was higher in peritoneal fluid cells of endometriosis patients compared to cells from controls (p < 0.0001) (Fig.  3A). In addition, late endometriosis patients had significantly higher IL-37 mRNA relative expression than early endometriosis patients (p = 0.0017) (Fig. 3B). Table 1 Demographical and clinical characteristics of endometriosis patients and controls IQR interquartile range, NA not applicable, rAFS revised American Fertility Society Endometriosis patients (n = 50) Controls (n = 23) Age in years, mean ± SD 29.7 ± 6.2 30.9 ± 5.1 Parity, n (%) P0 38 (76%) 15 (65.2%) P1 6 (12%) 5 (21.7%) P2 6 (12%) 3 (13.1%) rAFS score for endometriosis, median (IQR) 14 (10–26) NA rAFS stage for endometriosis, n (%) Stage I 7 (14%) NA Stage II 19 (38%) Stage III 19 (38%) Stage IV 5 (10%) Serum IL-37 (pg/mL), median (IQR) 128.1 (93.9–198.7) 64.6 (52.7–94.3) Peritoneal fluid IL-37 (pg/mL), median (IQR) 146.2 (106.1–194.4) 75.5 (61.8–108.2) IL-37 mRNA expression, (2−ΔCT ) 0.0239 (0.01393–0.03218) 0.0029 (0.0017–0.0052) Peritoneal fluid TNF-α (pg/mL), median (IQR) 55.55 (39.7–63.5) 15.6 (12.8–19.3) Page 4 of 7Ragab et al. Egyptian Journal of Medical Human Genetics (2022) 23:72 High peritoneal fluid TNF‑α levels in endometriosis patients Endometriosis patients showed significantly higher TNF-α levels in their peritoneal fluid compared to con - trols (p < 0.0001) (Fig. 4A). Moreover, patients with late stages of endometriosis showed significantly higher peritoneal fluid TNF-α levels compared to patients with early stages of endometriosis (p = 0.0006) (Fig. 4 B). IL‑37 levels correlate with TNF‑α levels TNF-α, among other pro-inflammatory cytokines, effectively induces the expression of IL-37 [20]. The Fig. 1 Elevated serum (A) and peritoneal fluid (B) levels of IL-37 in endometriosis patients compared to control females Fig. 2 Higher levels of serum (A) and peritoneal fluid (B) IL-37 in late stages of endometriosis Fig. 3 Higher relative expression of IL-37 mRNA in endometriosis patients compared to control females (A) and in late stages of endometriosis compared to early stages (B) Page 5 of 7 Ragab et al. Egyptian Journal of Medical Human Genetics (2022) 23:72 relationship between IL-37 and TNF-α in endometriosis patients has not been previously investigated. To address this point, we examined the correlation between levels of IL-37 and levels of TNF-α in the peritoneal fluid of endo - metriosis patients. IL-37 levels positively correlated with TNF-α levels in peritoneal fluid (r s = 0.7221, p < 0.0001) (Fig. 5).

According to emerging evidence, immune cells and cytokines play a profound role in the immuno-patho - genesis of endometriosis. Endometriosis is, in fact, thought to be an estrogen-dependent chronic inflamma - tory condition [7, 22]. Supporting this concept are stud - ies focusing on macrophages and natural killer cells in both ectopic endometrial tissue and the peritoneal fluid, suggesting that ectopic endometrial tissue contains an increased number of immune cells that produce a vari - ety of products, including several cytokines and growth factors that enhance ectopic tissue implantation and con- tribute to the inflammation and fibrosis that result from endometriosis [23, 24]. Cytokines are considered among the key players that can participate in modulating the implantation milieu and participating in the inflamma - tory process that is associated with endometriosis [25]. In the present study, we report higher serum and peri - toneal fluid IL-37 levels in endometriosis patients with higher levels in later endometriosis stages compared to early stages. Similar findings were reported by Kaabachi et  al. [19] who studied levels of IL-37 in 30 Tunisian endometriosis patients and found higher IL-37 levels in both serum as well as peritoneal fluid samples. Also, Jiang et  al. [26] found comparable results in a group of 40 Chinese females with endometriosis compared to 32 controls. Meanwhile, in a study involving 27 Chi - nese endometriosis patients and 36 controls, Fan et  al. [27] reported higher serum IL-37 in endometriosis patients but no difference in peritoneal fluid IL-37 lev - els. IL-37 tends to increase in inflammatory conditions in an attempt to dampen the inflammatory process [28]. Administration of recombinant IL-37 in endometriosis mice resulted in a significant decrease in endometriotic lesion size and weight and also lowered pro-inflamma - tory cytokine expression as well as vascular endothelial growth factor and soluble adhesion molecules, in peri - toneal fluid of endometriosis mice [20]. Furthermore, in- vitro transfection of endometrial stromal cells with IL-37 RNA was reported to suppress endometrial stromal cell proliferation, adhesion, migration, and invasion [20]. Consequently, it is possible to postulate that IL-37 might play a potential role in the immune response that occurs in endometriosis. Evidence is accumulating that IL-37 plays a role in limiting excessive inflammation. IL-37 levels have been found to rise in a variety of inflammatory conditions including osteoarthritis [10], rheumatoid arthritis [11], systemic lupus erythematosus [12], atopic dermatitis [13], and inflammatory bowel diseases [16]. Furthermore, the potential use of IL-37 as an anti-inflammatory agent in chronic inflammatory conditions has been investigated Fig. 4 Higher levels of TNF-α in peritoneal fluid of endometriosis patients compared to control females (A) and in late stages of endometriosis compared to early stages (B) Fig. 5 Levels of IL-37 in peritoneal fluid of endometriosis patients positively correlated with TNF-α levels Page 6 of 7Ragab et al. Egyptian Journal of Medical Human Genetics (2022) 23:72 in animals models, where treatment with IL-37 in allergic rhinitis murine models alleviated  allergic inflammation through repressing STAT6 and STAT3  signalling path - ways [ 29]. Treatment with recombinant IL-37 also sup - pressed joint inflammation in mouse models of arthritis [30]. IL-37b gene transfer enhanced the therapeutic efficacy of mesenchymal stem cells in dextran sulfate sodium-induced colitis mice through inducing regulatory T cells and myeloid-derived suppressor cells and regulat - ing cytokine production [31]. Our results show higher expression of IL-37 mRNA in endometriosis patients compared to controls, with higher levels in later endometriosis stages compared to early stages. IL-37 expression increases in response to the pro-inflammatory cytokines e.g., IL-1, IL-18, TNF- α, and IFN-γ [21]. However, the relationship between these cytokines and IL-37 in endometriosis patients has not been studied before. Our results show higher levels of TNF-α in peritoneal fluid of endometriosis patients with higher levels in late endometriosis compared to early endometriosis explaining the high IL-37 levels and expression observed in endometriosis patients in our study. Furthermore, in our study, IL-37 levels in perito - neal fluid were found to positively correlate with TNF- levels. Several studies have reported increased TNF-α in the peritoneal fluid of endometriosis patients [32–34]. Meanwhile, increased IL-37 mRNA expression in perito - neal fluid cells was also reported by Kaabachi et al. [19]. Nevertheless, Jiang et  al. [18] reported higher mRNA expression in ectopic endometrial biopsies as well as eutopic endometrial biopsies in endometriosis patients compared to eutopic endometrium from females without endometriosis. To the best of our knowledge, this is the first study to look at the relationship between TNF-α and IL-37 levels in endometriosis patients. This is also the first study to assess IL-37 levels and expression in Egyptian endome - triosis patients. One limitation of the present study is the relatively small number of patients included. Further research on a larger scale of patients as well as further follow-up studies are needed to assess the levels of IL-37 during endometriosis progression and the possible cor - relation of IL-37 levels with levels of TNF-α and other cytokines throughout the course of the disease.

In conclusion, we report higher serum and perito - neal fluid IL-37 levels as well as higher peritoneal fluid TNF-α levels in endometriosis patients with a positive correlation between TNF-α and IL-37 levels. Our data in addition to previous reports highlight the poten - tial importance of IL-37 in the immune process during endometriosis. Abbreviations IL-37: Interleukin 37; rAFS: Revised American Fertility Society; TNF-α: Tumor necrosis factor α.

Not applicable. Authors’ contributions DR, AA designed the study. AA collected the samples, collected patients’ clinical data and staged the patients. DR, RS carried out the laboratory work, analyzed the data and carried out statistical analysis. DR, RS drafted the paper. AA revised the paper. All authors read and approved the final manuscript. Funding None. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Declarations Ethics approval and consent to participate The study protocol was approved by the ethical committee of the Faculty of Medicine, Ain Shams University. Ethical approval reference number (FWA 000017585). Written informed consent was taken from all subjects before participation in this study. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Author details 1 Clinical Pathology, Faculty of Medicine, Ain Shams University, Cairo, Egypt. 2 Obstetrics and Gynecology, Faculty of Medicine, Ain Shams University, Cairo, Egypt. Received: 13 February 2022 Accepted: 11 March 2022

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