Urinary IL-1β and plasma IFN-γ levels reveal complementary immune mechanisms in endometriosis

Objectives: Endometriosis is a multifactorial inflammatory disorder characterized by the coexistence of localized inflammatory ac- tivation and systemic immune dysregulation. Single biomarkers are unlikely to capture this biological complexity, underscoring the need for integrated immune profiling across different biological compartments. This pilot study evaluated interleukin-1β (IL-1β) and interferon-γ (IFN-γ) in paired plasma and urine samples from women with clinically confirmed endometriosis and healthy controls to explore compartment-specific cytokine patterns.

IL-1β and IFN-γ were quantified in plasma and urine samples from patients with endometriosis and healthy control women using the automated microfluidic ELISA system (Ella, ProteinSimple/Bio-Techne, USA). Marker detectability and expression patterns were evaluated individually. The expression levels were compared using the Mann–Whitney U test.

IL-1β was undetectable in plasma samples in both groups but was detected in urine samples from patients with endometri- osis but not in controls (p = 0.0303). In contrast, IFN-γ was detectable in plasma but not in urine. Plasma IFN-γ levels were lower in patients compared with controls; however, this difference was not statistically significant (p > 0.99) and should be interpreted only as a descriptive trend.

The findings of this study suggest compartment-specific immune differences in endometriosis, with urinary IL-1β reflecting localized inflammatory activity and plasma IFN-γ showing a trend toward reduced systemic T helper 1 (Th1)-associated immunity. Larger studies are required to validate these preliminary observations. This complementary biomarker profile underscores the importance of integrated interpretation across biological systems and supports a minimally invasive immune signature for im - proved characterization of endometriosis.

Biomarkers, endometriosis, noninvasive Copyright Dimova-Mihaylova E et al. This is an open access article distributed under the terms of the Creative Commons Attri- bution License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Pharmacia 73: e189520 DOI 10.3897/pharmacia.73.e189520 Research Article Dimova-Mihaylova E, et al.: Potential cytokine biomarkers of endometriosis 2

Endometriosis is a chronic, estrogen-dependent in - flammatory disease that affects an estimated 10–15% of women of reproductive age worldwide and represents a major cause of pelvic pain, dysmenorrhea, and infertility (Garmendia et al. 2025). Despite its significant impact on quality of life, diagnosis remains challenging and is often delayed for several years, largely due to the absence of re- liable noninvasive biomarkers (Agic et al. 2006; Garmen- dia et al. 2025). This diagnostic gap reflects the biological complexity of the disease, which arises from the interplay between hormonal regulation and immune dysfunction rather than a single dominant pathogenic pathway. One of the prevailing theories for the occurrence of endometriosis is the underlying process of immune dys - regulation, which contributes both to lesion establishment and persistence. Alterations have been described across multiple components of the immune system, including enhanced macrophage activation, impaired natural killer (NK) cell cytotoxicity, imbalances in T helper cell subsets, and aberrant B-cell responses (Abramiuk et al. 2022; Vall- vé-Juanico et al. 2022). Together, these changes promote an inflammatory microenvironment that favors ectopic tissue survival while limiting effective immune clearance. Within this context, cytokines play a pivotal role as me - diators of immune communication, tissue remodeling, angiogenesis, and pain signaling (Bergqvist et al. 2001; Uçeyler et al. 2007; Turner et al. 2014). Numerous studies have reported elevated concentra - tions of pro-inflammatory cytokines—including inter - leukin-1 beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α)—in endometriotic lesions and peritoneal fluid, with less consistent findings in peripheral blood (Bergqvist et al. 2001; Krygere et al. 2024). However, translation into clinically useful biomarkers has been lim- ited due to variability across menstrual cycle phase, lesion localization, disease stage, and biological compartment. Cytokine measurements are also influenced by systemic inflammatory conditions and lack disease specificity, lim- iting the utility of single-marker approaches (Agic et al. 2006). As a result, single cytokine measurements often fail to reflect the underlying complexity of the disease. Most immunological studies in endometriosis focus on serum and peritoneal fluid, where numerous cytokines, such as IL-6, IL-8, TNF-α, and MCP-1, are elevated com- pared with controls and correlate with disease activity or severity (Wu and Ho 2003). There are very few direct studies measuring classical cytokines, such as IL-6, IL-8, MCP-1, and TNF-α, in urine from patients with endome- triosis as diagnostic biomarkers (Liu et al. 2015). Some studies have measured solitary urinary proteins related to inflammation, such as cytokeratin-19 fragment (CYFRA 21-1) (Saputra et al. 2025), urinary vitamin D-binding protein (Cho et al. 2012), urocortin, and other non-cy - tokine markers (Toma et al. 2025). Urine has long been shown to be a reliable source of diagnostic material; how- ever, classical cytokines are often present at very low con- centrations or below detection limits. Evidence about the levels of classical cytokines in urine is so far limited and inconclusive. Among the immune mediators of endometriosis, IL-1β and IFN-γ represent distinct and complementary aspects of immune function. IL-1β is a key driver of innate inflam- matory responses and is produced primarily by activated macrophages. It has been repeatedly shown to be upregu- lated in endometriosis tissue and peritoneal fluid, where it promotes leukocyte recruitment, angiogenesis, and local inflammation (Sikora et al. 2016). In contrast, IFN-γ is a hallmark cytokine of T helper 1 (Th1)-mediated immuni- ty and plays a critical role in cellular immune surveillance and macrophage activation (Castro et al. 2018). In this study, IL-1β and IFN-γ levels were measured in paired urine and plasma samples obtained from individuals with endometriosis and from controls without the disease. By evaluating those two protein markers, this study aims to identify a complementary immune signature that reflects both local and systemic aspects of endometriosis. This ap- proach explores the potential of cytokine patterns as a mini- mally invasive strategy for improving disease identification.

Study participants A total of 16 women were included in this study: 10 pa - tients with endometriosis (mean age 34 years) and six healthy control women of the same ethnicity (mean age 22 years). All participants provided written informed consent for participation in the study, and the study protocol was approved by the Ethical Committee of the Scientific Coun- cil of the Medical University—Sofia, Bulgaria (Approval No. 2826/24.06.2025, Protocol No.13/05.06.2025). All women were diagnosed with confirmed, non-surgically treated en- dometriosis and were selected at the University Hospital “Lozenets” or Medical Complex “D-r Shterev” (Table 1). Sample collection and storage Peripheral venous blood was collected in EDTA tubes and centrifuged at 2,000× g for 10 mins to separate the plasma. Plasma samples were aliquoted and stored at –80 °C until analysis. Morning midstream urine samples were collect- ed, aliquoted, and similarly stored at –80 °C. Cytokine quantification using the Ella® system Plasma and urine concentrations of IL-1β and IFN-γ (pg/ mL) were measured using the Ella automated immunoas- say system (ProteinSimple/Bio-Techne, MN, USA). The Ella system is a fully automated, microfluidic-based platform that performs multiplexed sandwich ELISA assays in nano- liter-scale volumes. The system reports mean RFU (relative fluorescence units), RFU %CV , mean concentration, and concentration %CV , enabling direct quantification of cyto- kine concentrations with built-in quality control. Each sam- ple was assayed at a 1:2 dilution for both plasma and urine. Pharmacia 73: e189520 3 Data presentation and statistical anal - ysis IL-1β and IFN-γ levels were quantified in plasma and urine samples from 10 women with endometriosis and six healthy controls and are presented as mean ± standard de- viation (SD). Due to the limited sample size and non-nor- mal distribution of the data, between-group comparisons of protein concentrations (pg/mL) were performed using the non-parametric Mann–Whitney U test. Statistical analyses were conducted using GraphPad Prism version 10.6.1 (GraphPad Software, Boston, MA, USA). A p-value < 0.05 was considered statistically significant.

below the lower limit of quantification (LLOQ) were replaced with LLOQ/2. Results with a coefficient of variation > 25% were excluded.

IL-1β and IFN-γ in plasma and urine IL-1β was undetectable in plasma samples from both pa - tients and controls. Urinary IL-1β levels differed between groups (p = 0.0303), with higher levels observed in women with endometriosis (mean = 4.99 pg/mL) compared with controls (mean = 0.75 pg/mL). The distribution of urinary IL-1β concentrations measured using the Ella® system is shown in Fig. 1. IFN-γ was not detectable in urine samples from ei - ther group. In plasma, IFN-γ was detectable in both groups. Mean levels were lower in patients with endometriosis compared with controls (0.70 pg/mL vs 1.54 pg/mL). However, this difference was not statistically significant (p > 0.99) and is therefore reported only as a descriptive observation. The plasma IFN-γ concentrations are shown in Fig. 2.

In this study, the protein expression of IL-1β and IFN-γ was investigated in paired plasma and urine samples from women with endometriosis and healthy controls. The find- ings suggest differential cytokine detectability depending on the studied biological fluid, as well as on disease status. IL-1β appeared undetectable in plasma samples from both groups, supporting the concept that endometriosis is primarily characterized by localized rather than overt systemic inflammation (Giudice and Kao 2004; Bulun et al. 2009). In contrast, IL-1β was vaguely detectable in urine samples from women with endometriosis, whereas it re - mained largely undetectable in controls. Despite the small Table 1. Clinical and demographic characteristics of the study groups. Number Sample code Age Diagnosis Patients 1 P1-PR 37 endometriosis 2 P2-PR 28 endometriosis 3 P3-PR 27 endometriosis 4 P4-PR 32 endometriosis 5 P9-PR 43 endometriosis 6 PR-P10 37 endometriosis 7 PR-P15 34 endometriosis 8 PR-P16 34 endometriosis 9 PR-P17 36 endometriosis 10 PR-P20 32 endometriosis Controls 1 PR-C2 27 control group 2 PR-C10 19 control group 3 PR-C11 21 control group 4 PR-C12 20 control group 5 PR-C13 21 control group 6 PR-C17 22 control group Figure 1. The distribution of urinary IL-1β concentrations measured by the automated ELISA system Ella® (ProteinSimple/ Bio-Techne, USA) in pg/mL in patients with endometriosis and control individuals. * indicates statistical significance between the analyzed groups (p = 0.0303). Figure 2. The distribution of plasma IFN-γ concentrations measured by the automated ELISA system Ella® (Bio-Techne, Boston, USA) in pg/mL in patients with endometriosis and con- trol individuals. “ns” indicates a lack of statistical significance between the analyzed groups (p > 0.99). Dimova-Mihaylova E, et al.: Potential cytokine biomarkers of endometriosis 4 number of samples, this observation demonstrated a clear trend toward higher urinary IL-1β levels in patients with endometriosis. This is consistent with the participation of IL-1β in inflammasome activation, angiogenesis, and in - flammatory amplification within the pelvic microenviron- ment (Lebovic et al. 2001; Dinarello et al. 2011). IL-1β has been consistently reported as elevated in the peritoneal fluid of women with endometriosis, where it contributes to immune cell recruitment (Lebovic et al. 2001; Sikora et al. 2018). The detection of IL-1β in urine may be caused by localized pelvic inflammation, potentially offering a non- invasive way to monitor the inflammatory activity associ- ated with the disease. In contrast, IFN-γ was undetectable in urine in both groups, suggesting limited urinary excretion or absent lo- cal production in this compartment. Plasma IFN-γ levels were lower in patients with endometriosis compared with controls; however, this difference was not statistically sig- nificant and should be interpreted cautiously as a possible rather than a confirmed biological effect. IFN-γ is predom- inantly produced by Th1 CD4+ T cells, cytotoxic CD8+ T cells, and natural killer (NK) cells and directs macrophage activation and enhanced antigen presentation, which are essential for the effective clearance of aberrant cells (Cas- tro et al. 2018; Alspach et al. 2019). Relatively lower sys - temic IFN-γ levels in patients, if confirmed, could reflect a shift away from Th1 immunity, which is mainly implicated in endometriosis pathogenesis, particularly through NK cell dysfunction and reduced cytotoxicity against ectopic endometrial tissue (Reis et al. 2022; Chen et al. 2023). IFN-γ cannot currently be considered a candidate bio- marker in this dataset, and the trend toward lower plasma IFN-γ in patients warrants further investigation, partic - ularly using larger cohorts or in combination with func - tional assays to assess Th1 and NK cell activity. This may be of interest for future hypothesis-driven studies examin- ing systemic Th1 activity in endometriosis.

of the study Importantly, several limitations must be acknowledged. The study comprises a relatively small cohort (10 patients and six controls), which limits the statistical power and increases the likelihood of type II error. The observed sta- tistical significance for urinary IL-1β should be interpret- ed with caution. Even if the observed trend is biologically consistent with the current understanding of localized in- flammatory activation in endometriosis, it should be vali- dated in a larger number of samples. Several clinically relevant variables that may influence cytokine levels were not controlled in this study, including menstrual cycle phase, hormonal therapy status, BMI, dis- ease stage, and lesion localization. These factors are known to significantly affect immune signaling and cytokine ex - pression in endometriosis. Future studies should incorpo- rate stratification for these variables. Strengths of the study Despite these limitations, the present study has several notable strengths. The parallel assessment of cytokines in two biological compartments (plasma and urine) pro - vides a compartment-specific perspective that is rarely explored in endometriosis research. Most previous stud - ies have focused on serum or peritoneal fluid; therefore, the inclusion of urine expands the perspectives toward the evaluation of a noninvasive sampling matrix. The findings are biologically coherent with the current un - derstanding of endometriosis as a localized inflammato - ry disorder. The selective detection of IL-1β in urine from patients aligns with established evidence of IL-1–medi - ated pelvic inflammation and inflammasome activation (Lebovic et al. 2001; Dinarello et al. 2011).

In summary, this pilot study suggests a distinct compart - ment-specific immune pattern in endometriosis, charac - terized by detectable urinary IL-1β and a nonsignificant reduction in plasma IFN-γ. This combination reflects the dual nature of the disease, involving localized pelvic in - flammation alongside altered systemic Th1-mediated im- mune responses. Larger, controlled studies are required to validate these observations before any possible clinical utility consideration and application. Additional information Conflict of interest The authors have declared that no competing interests exist. Ethical statements The authors declared that no clinical trials were used in the pres- ent study. All participants provided written informed consent for par - ticipation in the study, and the study was approved by the Eth - ics Committee of the Medical University of Sofia (Approval No. 2826/24.06.2025, Protocol No.13/05.06.2025) The authors declared that no experiments on animals were performed in the present study. The authors declared that no commercially available immor- talized human or animal cell lines were used in the present study. Artificial Intelligence (AI) use The authors accept full responsibility for the content of the man- uscript, including the disclosure of any use of AI. No AI tools were used in the preparation of this manuscript. Funding This study is financed by project № D-156/04.06.2025 of the Coun- cil of Medical Sciences, Medical University–Sofia, Sofia, Bulgaria. Pharmacia 73: e189520 5 Author contributions E.Dimova-Mihaylova has collected part of the samples at “Lozenetz” hospital, has performed the experiments, has made the statistical analysis; I.Dimova has participated in the statistical analysis of the data and has suggested some interpretations; P .An- dreeva has collected the blood/urine samples at the Medical com- plex “D-r Shterev” and has selected the patients; D.Nikolova has participated in the experiments and analysis, suggested improve- ments, interpretations and critically revised the manuscript. Author ORCIDs Ivanka Dimova https://orcid.org/0000-0003-4091-9604 Petia Andreeva https://orcid.org/0000-0002-6277-8550 Dragomira Nikolova https://orcid.org/0000-0002-8929-8522 Data availability All of the data that support the findings of this study are available in the main text.

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