Correlation between inflammatory biomarkers in biological fluids in patients with ovarian endometriosis

Yana A.  Mangileva; Мангилева Яна Андреевна; Yana A. Mangileva; Elena V. Kudryavtseva; Кудрявцева Елена Владимировна; Elena V. Kudryavtseva; Larisa G. Polushina; Полушина Лариса Георгиевна; Larisa G. Polushina; Elvira I.  Shakiryanova; Шакирьянова Эльвира Ильсеновна; Elvira I.  Shakiryanova; Nikolay N. Potapov; Потапов Николай Николаевич; Nikolay N. Potapov; Vladislav V. Kovalev; Ковалев Владислав Викторович; Vladislav V. Kovalev

doi:10.17816/aog630395

Correlation between inflammatory biomarkers in biological fluids in patients with ovarian endometriosis

Authors: Mangileva Y.A.¹, Kudryavtseva E.V.², Polushina L.G.², Shakiryanova E.I.³, Potapov N.N.⁴, Kovalev V.V.⁵
Affiliations:
1. Medical Center « UMMC-Health»
2. Ural State Medical University
3. City Clinical Hospital No. 14
4. City Clinical Hospital No. 40
5. Ural Institute of Health Management n.a. A.B. Blokhin
Issue: Vol 11, No 4 (2024)
Pages: 460-469
Section: Original study articles
Submitted: 16.04.2024
Accepted: 29.09.2024
Published: 22.12.2024
URL: https://archivog.com/2313-8726/article/view/630395
DOI: https://doi.org/10.17816/aog630395
ID: 630395

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Abstract

BACKGROUND: To date, there are no available screening techniques allowing to distinguish groups of women with the risk of endometriosis. Therefore, many researchers are searching for meaningful biomarkers for non-invasive diagnosis. The peritoneal fluid is subject to multidirectional changes in patients with external genital endometriosis, although its sampling requires invasive procedure. Testing of the inflammatory markers in saliva is a simple and safe method of particular interest, given that its sampling is non-invasive.

AIM: To assess the correlations between the level of inflammatory biomarkers in the peritoneal fluid and saliva in patients with ovarian endometriosis.

MATERIAL AND METHODS: A prospective cohort comparative study of 46 women with ovarian endometriosis was carried out. Inclusion criteria: confirmed diagnosis of ovarian endometriosis; age between 18 and 40 years; written informed consent for surgical intervention and for participation in the study; no prior hormonal treatment. Non-inclusion criteria: patient's refusal to participate in the study; age below 18 and above 40 years; contraindications for surgical treatment; oral inflammatory diseases. Patients were excluded if the diagnosis could not be visually confirmed during surgery or if there were histological discrepancies. All patients underwent laparoscopic cystectomy. Peritoneal fluid samples were collected during surgery. Mixed unstimulated saliva was collected before surgery in the morning, on an empty stomach. The levels of interleukins and of vascular endothelial growth factor were assessed in biological fluids.

RESULTS: The mean age of the study subjects was 32,4±6,1 years. Correlation analysis showed a direct statistically significant moderate relationship between the levels of IL-6 (r=0.548; p=0.001) and IL-8 (r=0.360; p=0.026) in the peritoneal fluid and saliva, respectively.

CONCLUSION: These data suggest that the onset and progression of endometriosis are associated with the increase of inflammatory cytokine levels both in the peritoneal fluid and in saliva. This may serve a potential tool for diagnosis and assessment of the severity of endometriosis. Evaluation of IL-6 and IL-8 levels in saliva may be useful in clinical practice in patients with external genital endometriosis.

Keywords

endometriosis, endometrioma, interleukins, saliva, biomarker

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BACKGROUND

Endometriosis is one of the most common gynecological diseases in women of childbearing age. Endometriosis is estimated to affect 176 million women worldwide (approximately 10% of women of childbearing age) [1, 2]. This condition can cause severe dysmenorrhea, severe dyspareunia, chronic pelvic pain, infertility, bowel and bladder symptoms, and chronic fatigue [1, 3, 4]. In addition, the symptom severity often does not correspond to the disease severity. Symptoms of endometriosis have detrimental effects on women’s functional status and physical, mental, social, and sexual well-being [4, 5].

Endometriosis significantly reduces fertility in women of childbearing age, alters abdominal parameters due to inflammatory reactions, and affects the pelvic anatomy. Up to half of people with endometriosis struggle to get pregnant [4, 6]. Many theories attempt to explain the development of endometriosis, but none is considered definitive. Endometrial-like cells can implant outside the uterus and respond to ovarian estrogen stimulation, causing inflammatory changes at the site of invasion, followed by scarring of the surrounding tissue and adhesion [7]. Ectopic endometrioid aggregates secrete chemokines into the surrounding tissue and recruit immune cells, which in turn secrete cytokines and growth factors such as TNF-α (tumor necrosis factor α), IL-1β (interleukin 1β), IL-6 (interleukin 6), IL-8 (interleukin 8), IL-17 (interleukin 17), FGF (fibroblast growth factor), and VEGF (vascular endothelial growth factor), creating a specific local microenvironment that contributes to the reciprocal interaction with the peritoneal fluid (PF) [8–10]. Some publications have addressed the issue of diagnosing endometriosis, but it is still poorly understood. The lack of simple, accessible, and affordable screening techniques impedes the identification of at-risk women who need in-depth diagnosis. However, early recognition and diagnosis are key to timely treatment and prevention of the adverse effects of the advanced endometriosis [11, 12].

Of all the biological fluids, PF is the most susceptible to multidirectional changes in external genital endometriosis, since it is in direct contact with its lesions and both influences endometrioid heterotopias and changes under their influence. Previous studies have demonstrated the clinical diagnostic value of PF levels of various interleukins and VEGF [13]. However, PF sampling is an invasive and expensive procedure associated with certain risks that preclude its use as a screening technique and significantly limit its diagnostic value. Of course, it would be better to develop a reliable, ideally inexpensive and non-invasive test for diagnosing endometriosis with high sensitivity and specificity [11].

In this regard, specific attention should be paid to testing easily accessible biological fluids, especially saliva, for inflammatory markers [14, 15]. Saliva contains a high number of proteins, peptides, enzymes, electrolytes, hormones, antibodies, and cytokines from systemic and local sources and therefore can be used for diagnosis of various diseases and evaluation of their progression. Saliva collection is a simple and safe non-invasive procedure. We believe that the research of the saliva inflammatory profile is one of the most promising areas in the diagnosis and screening of endometriosis.

Study Aim. The aim of the study was to evaluate correlations between PF and salivary levels of inflammatory markers in patients with ovarian endometriosis.

MATERIALS AND METHODS

Study design

A prospective, observational, comparative cohort study enrolled 46 women of childbearing age with confirmed cystic ovarian endometriosis. Figure 1 shows the study design.

Fig. 1. Study design: PF — peritoneal fluid.

Eligibility criteria

Inclusion criteria were as follows: 1) diagnosis of ovarian endometriosis; 2) childbearing age (18–40 years); 3) patient consent for surgery and study participation; 4) no hormonal treatment prior to surgery.

Non-inclusion criteria were as follows: 1) patient refusal to participate in the study; 2) age under 18 years or over 40 years; 3) contraindications to surgical treatment; 4) inflammatory diseases of the oral cavity.

Exclusion criteria were the lack of diagnosis confirmation by intraoperative visual assessment and/or by histology.

Study setting

The study enrolled women who were residents of Yekaterinburg (Russia) and who underwent surgery in 2022–2023 in the Gynecology departments of the Sverdlovsk Regional State Autonomous Healthcare Institutions “City Clinical Hospital No. 40” and “City Clinical Hospital No. 14.”

Study duration

The planned duration of the enrollment period was 12 months. The study did not include post-operative follow-up.

Intervention description

Clinical (medical history, gynecologic examination), laboratory tests, ultrasound, endoscopy, and histology were used to achieve the aim of the study. Saliva was obtained the day before surgery. The PF was obtained intraoperatively.

After insertion of a videoendoscope into the abdominal cavity, the pelvic organs (peritoneum, fallopian tubes, uterus, ovaries, uterine ligaments, rectouterine pouch) were examined. Endometrioid heterotopias were visualized and their location and color were identified, as well as the severity of external genital endometriosis using the revised American Fertility Society (r-AFS) classification (1985; renamed later American Society for Reproductive Medicine’s classification, ASRM classification). Small trocars were then inserted into the abdomen. One was used to insert a surgical aspiration tube into the rectouterine pouch prior to any manipulation. A sterile disposable syringe was attached to the end of the tube to collect PF from the rectouterine and other peritoneal pouches. The syringe PF was then transferred to a 5 mL Eppendorf test tube.

Mixed unstimulated saliva was collected after rinsing the mouth with water in the morning on the day before surgery in the fasting and resting state. Patients were asked to keep their head down in a seated position, refrain from swallowing any saliva and moving their tongue or lips during the entire saliva collection process. Saliva was allowed to accumulate in the oral cavity for two minutes and then transferred to a SalivaCapsSet tube by spitting out the entire contents. All samples obtained were labeled no later than 4 hours after the collection, placed in a cold chamber and stored at -40°C in a biobank of the Central Research Laboratory of the Ural State Medical University. Biological material was thawed and centrifuged in a TsLMN-R10-01-Elekon laboratory centrifuge at 1,500 rpm for 10 minutes prior to testing. Immunochemistry of saliva and PF included determination of IL-1β, IL-6, IL-8, IL-10, TNF, VEGF by heterogeneous enzyme-linked immunosorbent assay using Vector BEST test systems (Russia). The analysis was performed using a system consisting of a Thermo Scientific Multiskan GO (Japan) plate enzyme immunoassay analyzer, a Thermo Scientific Wellwash (Japan) washer, and an Elmi shaker-thermostat (ST-3L, Latvia).

Main study outcome

The main study outcome was to assess the strength of the correlation between PF and salivary levels of cytokines.

Group analysis

The study group included 46 patients of childbearing age with histologically confirmed cystic ovarian endometriosis.

Methods for registration of outcomes

To report the main study outcome, correlation analysis for nonparametric quantitative data was performed using the Spearman’s correlation coefficient, with the strength of association assessed using a Chaddock scale.

Ethical review

The study was approved by the local ethics committee of the Ural State Medical University (Protocol No. 11 dated 24 December 2021).

Statistical analysis

Statistical data were processed using the Jamovi licensed package (The Jamovi project, version 2.3 https://www.jamovi.org). For quantitative parameters, descriptive statistics such as median (Me) and interquartile range (Q1–Q3) were calculated as the distribution was not normal. The relationship between parameters for nonparametric quantitative data was assessed using the Spearman’s correlation coefficient, with the strength of association assessed using the Chaddock scale. The association was considered insignificant at <0.1, weak at 0.1–0.3, moderate at 0.3–0.5, relatively strong at 0.5–0.7, strong at 0.7–0.9, and very strong at 0.9–0.99. The results were considered statistically significant at p<0.05.

RESULTS

Participant characteristics

Forty-six patients with cystic ovarian endometriosis were examined. The mean age of patients enrolled in the study was 32.4±6.1 years. All study participants were Caucasian and residents of the Sverdlovsk region.

Primary findings

Table 1 shows the PF and salivary levels of IL and VEGF.

Table 1. IL and VEGF levels (pg/ml) in peritoneal fluid and saliva

Parameter	Peritoneal fluid		Saliva
Parameter	Ме	Q1–Q3	Ме	Q1–Q3
VEGF	239.5	184.2–313.6	963.6	242.6–1890.0
IL-1β	0.164	0.164–0.164	88.5	28.8–235.8
IL-6	29.4	6.3–128.4	0.726	0.726–0.726
IL-8	21.9	8.8–153.6	127.9	79.1–208.7
IL-10	16.9	8.4–27.6	0.42	0.20–0.63
TNF-α	3.7	3.7–3.7	0.01	0.01–0.01

Salivary levels of TNF-α were in most cases below the sensitivity threshold of the test system (<0.1 pg/mL). Therefore, a correlation between salivary and PF levels of TNF-α could not be assessed. Table 2 shows the analysis of the correlation between other parameters.

Table 2. Correlation between the levels of study parameters in peritoneal fluid and saliva

Parameter	r	p
VEGF	-0.130	0.436
IL-1β	0.100	0.546
IL-6	0.548	0.002*
IL-8	0.360	0.026*
IL-10	0.234	0.158

*Тhe differences are statistically significant (p <0,05).

Correlation analysis showed a direct statistically significant association (moderate strength) between salivary and PF levels of IL-6 (r=0.548; p=0.001) and IL-8 (r=0.360; p=0.026). Figure 2 graphically shows a statistically significant correlation.

Fig. 2. Correlations of IL-6 (А) and IL-8 (В) levels in the peritoneal fluid and saliva

DISCUSSION

The cytokine profile of various biological fluids, especially PF, is altered in ovarian endometriosis. We demonstrated a significant correlation between PF and salivary levels of IL-6 and IL-8. In the future, salivary cytokine levels may be used as a non-invasive diagnostic test for endometriosis.

Chronic inflammation plays a key role in the development and progression of endometriosis; both pro- and anti-inflammatory cytokines are involved in the disease pathogenesis [9]. Analysis of the cytokine profile in patients with cystic endometriosis demonstrated a weak positive direct correlation between PF and salivary levels of IL-1β (although the correlation coefficient in our study did not achieve statistically significant levels). IL-1β is a potent pro-inflammatory cytokine produced primarily by monocytes and macrophages. There are some physiological and pathological effects attributed to IL-1β that may be associated with endometriosis. IL-1β induces prostaglandin synthesis and stimulates fibroblast proliferation, collagen deposition and fibrinogen formation, which could contribute to the fibrosis and adhesion formation associated with endometriosis [16]. In addition, IL-1β stimulates B-cell proliferation and antibody production, which could be related to the autoantibodies associated with the disease IL-1β also stimulates IL-2 secretion by T cells and NK cells, which in turn can induce NK proliferation and T-cell growth [17].

IL-6 is the most investigated interleukin concerning endometriosis [18]. It is secreted in response to injury by various immune cells and participates in several immunological mechanisms. Many studies have demonstrated higher IL-6 levels in serum, PF and follicular fluid in patients with endometriosis compared to healthy women. IL-6 is a macrophage activator that enhances angiogenesis, promotes endometrial cell proliferation, and may regulate its immune environment. Therefore, elevated IL-6 levels are thought to contribute to endometriosis-associated infertility. IL-6 family proteins, especially leukemia inhibitory factor, also play an important role in the early stages of implantation, and one hypothesis is that their decreased expression may cause endometriosis-associated infertility [18–20]. A direct correlation between salivary and PF levels of IL-6 was demonstrated.

IL-8 also plays an important role in the development and progression of endometriosis [11]. Our study demonstrated a statistically significant positive correlation between PF and salivary levels of IL-8. IL-8 is a potent angiogenic, pro-inflammatory and cell proliferative cytokine found in the PF of patients with endometriosis. IL-8, which promotes endometrial cell adhesion, is secreted by leukocytes, macrophages, and endothelial cells stimulated by IL-1β or TNF-α. Studies evaluating IL-8 levels in the PF found higher IL-8 levels in patients with endometriosis associated with infertility [21].

TNF-α levels are also being actively investigated in patients with endometriosis [13]. However, our study showed low salivary levels of TNF-α. In most cases, it was below the sensitivity threshold of the test system. Therefore, currently, it does not seem promising to measure salivary levels of TNF-α.

IL-10 is an anti-inflammatory interleukin produced by type-2 T helpers [13]. We found a positive direct correlation between PF and salivary levels of IL-10, but it was not statistically significant. In addition, in many cases, salivary levels of IL-10 were below the sensitivity threshold of the test system.

VEGF plays an important role in the neovascularization of new endometriotic implants [22]. A negative relationship was found between PF and salivary levels of VEGF, but it was not statistically significant.

Our findings for inflammatory markers are largely consistent with those of other investigators [8, 13]. Detectable salivary levels of inflammatory markers are relatively high, sometimes higher than blood levels, indicating the unique role of saliva in the assessment of inflammatory markers [23, 24]. However, we did not find any medical research showing an association between salivary levels of inflammatory markers and endometriosis. The significant correlation between PF and salivary levels of inflammatory cytokines that we found in women with cystic ovarian endometriosis suggests the use of these markers as a non-invasive diagnostic test for external genital endometriosis. Salivary levels of IL-6 and IL-8 are the most promising tests in this regard.

Study limitations

Based on our data, the use of a larger sample may yield statistically significant results for other parameters. A limitation of our study is the lack of a control group, since it is not ethical to refer healthy women for laparoscopy to obtain PF.

CONCLUSION

Our findings suggest that with the development and progression of endometriosis, levels of inflammatory cytokines increase in both PF and saliva and may be used to diagnose and assess the severity of endometriosis. Salivary levels of IL-6 and IL-8 can be used in clinical practice in patients with external genital endometriosis.

ADDITIONAL INFO

Authors’ contributions. Ya.A. Mangileva reviewed literature data, collected and analyzed literature sources, wrote and edited the manuscript; E.V. Kudryavtseva reviewed literature data, collected and analyzed literature sources, wrote and edited the manuscript; L.G. Polushina collected and analyzed literature sources, prepared and wrote the manuscript; E.I. Shakiryanova performed surgical treatment of patients, reviewed the literature, collected and analyzed literature sources, prepared and wrote the manuscript; N.N. Potapov performed surgical treatment of patients, reviewed the literature, collected and analyzed literature sources, prepared and wrote the manuscript; V.V. Kovalev performed surgical treatment of patients, reviewed the literature, collected and analyzed literature sources, prepared and edited the manuscript. All authors confirm that their authorship meets the international ICMJE criteria (all authors have made a significant contribution to the development of the concept, research and preparation of the article, read and approved the final version before publication).

Funding source. This study was not supported by any external sources of funding.

Competing interests. The authors declares that there are no obvious and potential conflicts of interest associated with the publication of this article.

Patients’ consent. Written consent was obtained from all the study participants before the study screening in according to the study protocol approved by the local ethic committee.

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