The significance of immune microenvironment in patients with endometriosis

Abstract

Endometriosis represents an estrogen-dependent disease of the female reproductive system and intra- and extraperitoneal regions, with chronic feature. Currently, immune cells, such as macrophages and lymphocytes, are considered to play a pivotal role in angiogenesis and invasion of endometriotic cells through matrix remodeling. Additionally, various studies have revealed the role of E-cadherin, β-catenin, along with steroid hormone receptors in endometriosis development. In this context, our study aimed to analyze the relationship between the cellular immune profile and E-cadherin, β-catenin, estrogen receptor alpha (ERα), and progesterone receptor (PR) immunoexpression in endometriosis tissues, along with an analysis of the possible association between serological parameters and immunohistochemical (IHC) markers. The study included 53 patients diagnosed with ovarian or cutaneous abdominal wall endometriosis, which have been investigated by routine histology, immunohistochemistry, and serum analysis. The IHC exam showed an increased density of cluster of differentiation (CD)4+ T-cells, CD8+ T-cells, and CD68+ macrophages, along with variable increased expressions of E-cadherin, β-catenin, ERα, and PR. Statistical analysis revealed an intense positive correlation between CD68 and PR expression (p<0.05), without any other statistically significant correlations between IHC markers or between IHC and serological markers. Our study supports that endometriosis is an immune-dependent disease characterized by an abnormal morphological profile of T-cells and macrophages in endometriotic implants. Our study provides additional data useful in the understanding the immune milieu of endometriosis in the context of its complex pathogenic molecular mechanism. Further research is needed to develop new immunological therapeutic approaches, like immune checkpoint inhibitors administration or T-cell-targeted immunotherapy in these patients.

Introduction

Endometriosis may be defined as a chronic hormonal-dependent disease, characterized by the development of endometrial tissue, containing glands and stroma, in extrauterine sites. Endometriosis is associated with debilitating chronic pelvic pain, being now considered a neuroinflammatory condition [1].

The pathogenesis of endometriosis has been intensely investigated, but still remains elusive. Even though the retrograde menstruation or the stem cell theory can explain the presence of endometrial tissue in the peritoneal cavity, the development of the implants needs to be further investigated, regarding the escape from the immune clearance, their mechanism of attachment to the peritoneal surface, the invasion of the epithelium, the development of vascular support, along with their continuous growth and survival. Recent molecular studies addressed the genetic predisposition, estrogen dependence, progesterone resistance, and inflammation processes [1, 2], in addition to E-cadherin, β-catenin, estrogen receptors (ERs), and progesterone receptors (PRs) abnormal expression registered in endometriotic tissues.

There is a current consensus that endometriosis is associated with a chronic inflammatory process induced and/or supported by the peritoneal microenvironment [3, 4]. A key role played by the immune system has been hypothesized in both the initiation and progression of endometriosis [5].

The inflammatory cellular spectrum associated with endometriosis includes cluster of differentiation (CD)4+ T-cells and CD8+ T-cells [6]. However, their involvement in endometriosis is partially deciphered [6]. Additionally, macrophages are now considered to have critical roles in the growth, development, vascularization, and innervation of endometriotic lesions, as well as in generation and perception of pain [7, 8, 9].

Several studies have demonstrated the role of adhesion molecules, such as cadherins, galectins, heparan sulphate, integrins, selectins, or trophinin–tastin–bystin complex in the molecular mechanism associated with endometriotic cells implantation [4, 10, 11]. Moreover, an imbalance in the activity of sex steroid hormones has been also demonstrated in patients diagnosed with endometriosis, leading to a local proinflammatory status, which supports endometrial cells adhesion, proliferation, along with angiogenesis, in endometriotic foci [12].

Aim

The composition of the intralesional inflammatory infiltrate in different histological types of endometriosis could make a significant contribution to the knowledge about the sequence of events and the complexity of the immune response in endometriosis. In this context, our study aimed to analyze the inflammatory infiltrate profile using a semiquantitative assessment of CD4+ T-cells, CD8+ T-cells, and CD68+ macrophages in ovarian or abdominal wall cutaneous endometriosis. Supplementary, we aimed to identify any possible association with serological parameters, such as serum inflammatory markers [C-reactive protein (CRP), plasma fibrinogen, and erythrocyte sedimentation rate (ESR)], along with blood cell count and the immunohistochemical (IHC) expression of adhesion molecules (E-cadherin and β-catenin), hormone receptors (ERα and PR), based on our experience in previous studies.

Patients, Materials and Methods

Our study was conducted at the Elena Doamna Clinical Hospital and at the Grigore T. Popa University of Medicine and Pharmacy, Iaşi, Romania. The study included a retrospective analysis of information provided by clinical files, along with histopathological investigations of the surgical specimens (nodule excision, cystectomy, oophorectomy, and hysterectomy with adnexectomy) obtained from 53 patients diagnosed with endometriosis between 2018 and 2023.

Hematoxylin–Eosin (HE)-stained slides were reviewed by two independent pathologists to confirm the diagnosis and assess the histological type of endometriosis (ovarian or abdominal wall cutaneous endometriosis). Following the histopathological diagnosis, an IHC exam using CD4 (mouse, monoclonal, clone 4B12, 1:100 dilution, Novocastra, UK), CD8 (mouse monoclonal, clone 1A5, 1:50 dilution, Novocastra, UK), CD68 (mouse monoclonal, clone KP1, 1:400 dilution, Novocastra, UK), E-cadherin (mouse monoclonal, clone 36B5, 1:50 dilution, Novocastra, UK), β-catenin (mouse monoclonal, clone β-catenin-1, 1:200 dilution, Dako, UK), ERα [mouse monoclonal, clone 6711, ready-to-use (RTU) dilution, Novocastra, UK], and PR (mouse monoclonal, clone 312, RTU dilution, Novocastra, UK) has been performed.

3–4 μm thickness sections from the paraffin blocks were deparaffinized in xylene and then gradually rehydrated in ethanol to distilled water. The endogenous peroxidase was blocked using a 3% hydrogen peroxide solution, at room temperature (RT), for 10 minutes. The incubation with the primary antibody tissue was performed in a moist chamber, at RT, for one hour. The samples were washed out in Tris-buffered saline (TBS) and incubations with the secondary antibody (Novocastra Post Primary) and tertiary (Novolink™ Polymer) antibodies were performed. The samples were washed out in TBS following each incubation. Finally, the tissue samples were contrasted by Hematoxylin.

Following the IHC method, a semiquantitative assessment of the inflammatory infiltrate immunoreactivity, along with epithelial and stromal adhesion molecules and hormone receptors expression, using their corresponding scores available in literature [13], as illustrated in Table 1.

Table 1.

Quantification of CD4, CD8, and CD68 immunohistochemical expressions

Percent of immunoreactive cells	Score
˂5%	0 (without expression)
5–20%	1 (weak expression)
20–50%	2 (mild expression)
>50%	3 (strong expression)

CD: Cluster of differentiation.

Additionally, E-cadherin expression was quantified using a scoring system based on staining intensity index (II) multiplied by the percentage of positive cells or index of positivity (IP) [14]. To establish the staining index, the semiquantitative scoring used the quantification values for staining intensity and for positive cells as described in Table 2.

Table 2.

E-cadherin staining intensity quantification and positive cells percentage quantification values for determination of the staining index value

Staining intensity (II)	Staining index
Negative (without expression)	0
Weak (low expression)	1
Moderate	2
Strong	3
Percentage of positive cells (IP)	Staining index
≤10%	1
11–50%	2
≥51%	3

II: Intensity index; IP: Index of positivity

A scoring system based on staining intensity (II) multiplied by the percentage of positive cells or IP has been used for β-catenin immunoexpression evaluation [15]. To establish the staining index, the staining intensity and the percentage of positive cells values (Table 3) have been evaluated according to literature data [15].

Table 3.

β-catenin staining intensity quantification and positive cells percentage quantification values for determination of the staining index value

Staining intensity (II)	Staining index
Negative (without expression)	0
Weak (low expression)	1
Moderate	2
Strong	3
Percentage of positive cells (IP)	Staining index
0	0
≤10%	1
11–50%	2
≥51%	3

II: Intensity index; IP: Index of positivity

ERα and PR expression analysis used a score based on the IP added to the staining II [16, 17], IP (% positive cells) being considered: 0: none; 1: <1/100; 2: 1/100–1/10; 3: >1/10–1/3; 4: >1/3–2/3; 5: >2/3 and II being considered: 0 – absent; 1 – weak; 2 – moderate; 3 – strong. When adding the values, the total score has ranged from 0–8 and was considered positive if >2. The cut-off value for both ERα and PR was considered as 4 (≤4 low and >4 high).

In addition, information from the clinical files was also assessed, including age, menopausal status, along with serological parameters, such as the blood cell count and serum inflammatory markers (CRP, plasma fibrinogen, and ESR).

Statistical analysis

The statistical data has been processed using IBM Statistical Package for the Social Sciences (SPSS) version 23.0 software. Statistical significance was examined using the Spearman’s rank-order correlation coefficient (Spearman’s correlation). The level of significance was p<0.05 in all statistical analyses used.

Ethical considerations

The patients’ informed consent has been obtained in all cases, following the recommendations to ensure that participants’ decision-making is meaningful and that their interests are protected [18]. The present study has been approved by the Research Ethics Commission of Grigore T. Popa University of Medicine and Pharmacy (Approval No. 285/26.03.2023), consistent with the Declaration of Helsinki – Ethical Principles for Medical Research Involving Human Subjects.

Results

The study group included 53 cases with ovarian endometriosis (n=43; 81.13%) (Figure 1A and 1B ) and abdominal wall cutaneous endometriosis (n=19; 1.86%) (Figures 2 and 3 ). The ages of patients of the study group ranged between 21 and 52 years old, with 36.7 years median age and a mean of 39 years. Six cases have been included in the menopausal status at the moment of diagnosis.

Figure 1.

(A and B) Ovarian endometriotic cyst (HE staining, ×100). HE: Hematoxylin–Eosin

Figure 2.

Abdominal wall cutaneous endometriosis (HE staining, ×100)

Figure 3.

Abdominal wall cutaneous endometriosis, with evident hemorrhagic areas (HE staining, ×200)

The IHC exam revealed an increased CD4+ and CD8+ T-cells densities in endometriotic areas in all cases. Most of the samples (encompassing less than half of the cases, 41.5%), showed a moderate expression of CD4 (score 2), while 35.8% displayed a strong CD8 expression (score 3) (Figures 4 , 5 , 6 , 7 ). Additionally, a strong expression (score 3) of CD68 has been registered in most cases (77.4%) in our study group, while a weak CD68-positive expression has been noticed in the endometriotic foci, in 3.8% of cases (Figures 8 and 9 ; Table 4). The graphic representations of the frequency distribution of CD4, CD8, and CD68 are presented in corresponding histograms (Figures 10 , 11 , 12 ).

Figure 4.

CD4 expression in ovarian endometriotic cyst (score 3) (Anti-CD4 antibody immunomarking, ×200). CD4: Cluster of differentiation 4

Figure 5.

CD4 expression in abdominal wall cutaneous endometriosis (score 2) (Anti-CD4 antibody immunomarking, ×200).

Figure 6.

CD8 expression in ovarian endometriotic cyst (score 3) (Anti-CD8 antibody immunomarking, ×400). CD8: Cluster of differentiation 8

Figure 7.

CD8 expression in abdominal wall cutaneous endometriosis (score 3) (Anti-CD8 antibody immunomarking, ×200)

Figure 8.

CD68 expression in ovarian endometriotic cyst (score 3) (Anti-CD68 antibody immunomarking, ×100). CD68: Cluster of differentiation 68

Figure 9.

CD68 expression in abdominal wall cutaneous endometriosis (score 3) (Anti-CD68 antibody immunomarking, ×200).

Table 4.

CD4, CD8, and CD68 scores frequency distribution in our study group

Score	Frequency	Percent	Valid percent	Cumulative percent
1	12	22.6	22.6	22.6
2	19	35.8	35.8	58.5
3	22	41.5	41.5	100
Total	53
1	6	11.3	11.3	11.3
2	28	52.8	52.8	64.2
3	19	35.8	35.8	100
Total	53
1	2	3.8	3.8	3.8
2	10	18.9	18.9	22.6
3	41	77.4	77.4	100
Total	53

CD: Cluster of differentiation

Figure 10.

CD4 percent of immunoexpression frequency distribution

Figure 11.

CD8 percent of immunoexpression frequency distribution.

Figure 12.

Figure 12 – CD68 percent of immunoexpression frequency distribution.

A diffusely ERα and PR nuclear homogeneous expression through all endometriotic areas has been noted in all cases. ERα expression in foci of endometriosis displayed negative scores in five (9.4%) cases and variable positive scores in 48 (90.6%) cases (Figures 13 and 14 ; Table 5), while PR had a negative score in 14 (26.4%) cases and a positive score in 39 (73.6%) cases (Figures 15 and 16 ; Table 5).

Figure 13.

ERα moderate epithelial and stromal expression in ovarian endometriosis (Anti-ERα antibody immunomarking, ×100). ERα: Estrogen receptor alpha

Figure 14.

ERα expression in abdominal wall cutaneous endometriosis (Anti-ERα antibody immunomarking, ×200)

Table 5.

ERα, PR, E-cadherin, and β-catenin scores frequency distribution in the study group

	Score	Frequency	Percent	Cumulative percent
ERα	0	5	9.4	9.4
3	5	9.4	18.9
4	5	9.4	28.3
5	4	7.5	35.8
6	3	5.7	41.5
7	13	24.5	66.0
8	18	34.0	100
Total	53	100
PR	0	14	26.4	26.4
3	1	1.9	28.3
4	6	11.3	39.6
5	8	15.1	54.7
6	6	11.3	66.0
7	7	13.2	79.2
8	11	20.8	100
Total	53	100
E-cadherin	6	46	86.8	86.8
9	7	13.2	100
Total	53	100
β-catenin	6	17	32.1	32.1
8	15	28.3	60.4
9	16	30.2	90.6
12	5	9.4	100
Total	53	100

ERα: Estrogen receptor alpha; PR: Progesterone receptor

Figure 15.

PR strong stromal expression in ovarian endometriotic cyst (Anti-PR antibody immunomarking, ×100). PR: Progesterone receptor.

Figure 16.

PR strong epithelial expression in abdominal wall cutaneous endometriosis (Anti-PR antibody immunomarking, ×200).

A homogeneous, membrane distribution of E-cadherin expression has been registered in glandular epithelium and in few stromal cells in areas of endometriosis, in our group. The evaluation of E-cadherin expression showed a positive reaction in all cases (Figure 17 ), having a staining index score of 6 in most (86.8%) cases.

Figure 17.

E-cadherin epithelial and stromal expression in ovarian endometriosis (Anti-E-cadherin antibody immunomarking, ×100)

The glandular epithelium from endometriotic areas also displayed a homogeneous, membrane and cytoplasmic β-catenin immunoexpression (Figure 18 ). The percentage of positive cells ranged between six to 12, with 30.2% of them having a score of 9 and only five (9.4%) cases having a score of 5 (Table 5).

Figure 18.

β-catenin strong epithelial expression in abdominal wall cutaneous endometriosis (Anti-β-catenin antibody immunomarking, ×400).

Spearman’s non-parametric test revealed a significant strong correlation between CD68 and PR expression (p<0.05) (Table 6). Additionally, an average association in intensity and a proportional correlation type have been observed. Moreover, no correlation was found between CD68 and ERα expression, or between CD4, CD8, and hormone receptors (ERα and PR) in the analyzed samples (Table 6).

Table 6.

Spearman’s correlation test between the expression of inflammatory markers (CD4, CD8, and CD68) and of hormone receptors (ERα and PR)

Spearman’s rho	CD4 percent	Correlation data Coefficient value	1.000	0.438**	0.283*	0.054	0.163
Sig. (2-tailed)		0.001	0.040	0.699	0.243
N	53	53	53	53	53
CD8 percent	Correlation data Coefficient value	0.438**	1.00	0.105	0.125	-0.195
Sig. (2-tailed)	0.001		0.455	0.373	0.163
N	53	53	53	53	53
CD68 percent	Correlation data Coefficient value	0.283*	0.105	1.00	0.133	0.402**
Sig. (2-tailed)	0.040	0.455		0.343	0.003
N	53	53	53	53	53
ERα score	Correlation data Coefficient value	0.054	0.125	0.133	1.00	0.231
Sig. (2-tailed)	0.699	0.373	0.343		0.096
N	53	53	53	53	53
PR score	Correlation data Coefficient value	0.163	-0.195	0.402**	0.231	1.00
Sig. (2-tailed)	0.243	0.163	0.003	0.096
N	53	53	53	53	53

CD: Cluster of differentiation; ERα: Estrogen receptor alpha; N: No. of cases; PR: Progesterone receptor; *Correlation is significant at the 0.05 level (2-tailed); **Correlation is significant at the 0.01 level (2-tailed)

The non-parametric test (Mann–Whitney and Spearman’s tests) revealed no significant correlation between the expression of inflammatory markers (CD4, CD8, and CD68) and of adhesion molecules (E-cadherin and β-catenin) (Tables 7 and 8).

Table 7.

Mann–Whitney test correlation results between the inflammatory markers (CD4, CD8, and CD68) and of E-cadherin expression

	E-cadherin staining index	No. of cases	Mean rank	Sum of ranks
CD4 percent	6	46	27.04	1244.00
9	7	26.71	187.00
Total	53
CD8 percent	6	46	26.38	1213.50
9	7	31.07	217.50
Total	53
CD68 percent	6	46	27.96	1286.00
9	7	20.71	145.00
Total	53

CD: Cluster of differentiation; ERα: Estrogen receptor alpha; PR: Progesterone receptor; *Correlation is significant at the 0.05 level (2-tailed); **Correlation is significant at the 0.01 level (2-tailed)

Table 8.

Spearman’s correlation test between the expression of inflammatory markers (CD4, CD8, and CD68) and of β-catenin expression

	CD4 percent	CD8 percent	CD68 percent	β-catenin staining index
Spearman’s rho	CD4 percent	Correlation data Coefficient value	1.000	0.438**	0.283*	-0.034
Sig. (2-tailed)		0.001	0.040	0.810
N	53	53	53	53
CD8 percent	Correlation data Coefficient value	0.438**	1.00	0.105	0.068
Sig. (2-tailed)	0.001		0.455	0.626
N	53	53	53	53
CD68 percent	Correlation data Coefficient value	0.283*	0.105	1.00	0.061
Sig. (2-tailed)	0.040	0.455		0.665
N	53	53	53	53
β-catenin staining index	Correlation data Coefficient value	-0.034	0.068	0.061	1.00
Sig. (2-tailed)	0.810	0.626	0.665
N	53	53	53	53

CD: Cluster of differentiation; N: No. of cases; *Significant correlation at 0.05 level (2-tailed); **Significant correlation at 0.01 level (2-tailed)

Regarding the serological parameters, the assessment of blood cell count and serum inflammatory markers (CRP, plasma fibrinogen, and ESR) identified a threshold of 6 mg/L for CRP to identify the presence of unspecific inflammatory stimuli. CRP value has been significantly elevated in 64.2% of cases (Table 9).

Table 9.

CRP values frequency distribution

CRP value [mg/L]	Frequency	Percent	Cumulative percent
<6	34	64.2	64.2
≥6	19	35.8	100
Total	53	100

CRP: C-reactive protein

The multivariable analysis did not identify any statistically significant correlations between the serological parameters and tissular IHC markers.

Discussions

Endometriosis is considered a “disease of theories” but none of these theories is generally accepted, as these cannot fully explain the clinical and pathological phenomena [19]. The retrograde menstruation and implantation theory was the first theory that has been proposed [20] and this is still considered the primary mechanism involved in endometriosis pathogenesis [21, 22]. The coelomic metaplasia theory suggests that endometriosis arises from metaplastic changes of the mesothelial cells derived from the coelomic epithelium, located in the peritoneum and pleura [23, 24]. The induction theory holds that coelomic metaplasia is triggered by endogenous factors (hormonal and immunological) [23, 24]. The Müllerian remnants theory implies that residual cells from the embryological Müllerian migration can develop into endometriotic tissue, under hormonal influences (associated with the onset of estrogen secretion at puberty) [24]. More recent theories assign key pathogenic roles to stem cells, either of endometrial origin, arriving via Fallopian tubes and inducing the lesions [25], either of bone marrow origin, contributing to the further development of the already-established lesions [26, 27]. Finally, some authors consider that lymphatic or vascular dissemination could also be related to the ectopic sites of endometrial tissue development [28].

There are ongoing studies and inconsistent reports regarding the inflammatory cells’ spectrum associated with the female reproductive tract [3, 5, 29]. The conflicting results are probably consequences of the differences in the menstrual cycle and the complexity of continuously changing morphology of the epithelial layer and stroma during different menstrual phases. Nevertheless, T-cells are the predominant immune cells followed by dendritic cells, macrophages, neutrophils, mast cells, and natural killer (NK) cells, while B-cells are scarce in the female reproductive system [30].

The inflammatory cellular spectrum associated with endometriosis includes CD4+ T-cells and CD8+ T-cells [6]. CD4+ T-cells or T-helper (Th)-cells play a significant role in organization of the immune response through the stimulation of other immune cells, such as cytotoxic T-cells and B-cells, as a response to an aggression [ 6, 31 ]. CD4 interacts with major histocompatibility complex class II (MHCII) of antigen-presenting cells (APCs), aiding CD4+ cells to recognize and respond to a specific antigen [ 32 ]. Additionally, endometriosis is characterized by an abnormal immune response related to CD8+ cytotoxic T-cells [ 6, 33 ].

The literature reports variable types of involvement of CD4+ or CD8+ T-cells in the pathophysiology of endometriosis [6 ], while hypotheses regarding the possibility that their alterations may be possible causes or consequences of endometriosis are still debated. Additionally, the relationship between the hormonal status, inflammation, and disease progression has been widely studied.

Halme et al. suggested an immune component of endometriosis since 1987, when they identified a higher number of activated macrophages, as part of innate immune system cellular component, in intact endometrial tissues (eutopic endometrium) and in the peritoneal fluid (PF) of patients diagnosed with endometriosis compared to that obtained from patients without endometriosis [34]. Knowing that macrophages are phagocytic cells with a large tissular distribution, which play a key role in various inflammatory and tumoral diseases [35], they are now considered to have critical roles in the growth, development, vascularization, and innervation of endometriotic lesions, as well as in the generation and perception of pain [7, 8, 9].

Studies that have focused on the microenvironment of numerous malignancies and cancer-associated diseases, including endometriosis, have concluded that macrophages represent an important cell population of the inflammatory infiltrate [7, 8, 9, 36, 37]. In response to their abundance in the microenvironment, a maladaptive reaction takes place, which consequently promotes tumor growth by initiating the process of angiogenesis, along with tissue remodeling, and by establishing an immunosuppressive environment [13]. In breast cancer, for example, up to 50% of the tumor mass is represented by tumor-associated macrophages (TAMs) [38] and their number is positively correlated with the angiogenic process and, ultimately, with the reduction of the relapse-free and overall survival [39]. In an analogous manner, there are studies that correlate their presence with the development and progression of endometriotic implants, as another hormone-dependent process [2].

Macrophages are also involved in the pathogenesis and development of endometriosis through various mechanisms. They are able to promote the secretion of inflammatory cytokines and other molecules that can induce inflammation (further leading to pain and tissue growth) and angiogenesis, by secreting basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), interleukin (IL)-6, IL-1β, monocyte chemoattractant protein-1 (MCP-1), and tumor necrosis factor-alpha (TNF-α) [37], and enhancement of cell proliferation and adhesion in endometriosis [3]. Nevertheless, macrophages can exhibit impaired function, such as a reduced ability to clear endometriotic cells, potentially contributing to the survival and growth of ectopic endometrial tissue. Moreover, macrophages can interact with sex hormones, like estrogens and progesterone, which are crucial in endometriosis: they contribute to local estrogens production by expressing aromatase, the enzyme that converts androgens to estrogens [5].

Macrophages may be classified into proinflammatory M1 cells (classically activated macrophages) (CD40+/CD80+/CD86+/HLA-DR+), which secrete chemokines and proinflammatory cytokines that initiate the response against infections and a reparative and anti-inflammatory M2 phenotype (CD163+/CD206+/CD204+) (alternatively activated macrophages) [9, 37, 40]. However, macrophages display a bidirectional polarization in endometriosis, with a tendency of M1 polarization, associated to an increased production of a classical neuronal guidance cue, netrin-1, which is involved in the proliferation, tube formation, along with migration and invasion of vascular endothelial cells, supporting the vascular infiltration in endometriosis [37].

Moreover, macrophages are considered key cells in endometriosis pathogenesis, acting as APCs for T-cells activation, being also involved in tissue repairing processes [41]. A theory is considering that the high amount of immune cells, such as B- and T-lymphocytes and activated macrophages, as well as the inflammatory cytokines characteristic for endometriosis [42], are supporting an immunological dysregulation involved in its pathogenesis [40].

Traditionally, CD4, CD8, and CD68 are considered reliably surface markers to separate macrophages from lymphocytes [43]. There are several classical and novel IHC markers currently used to identify macrophages, such as arginase-1 (Arg-1), C–C motif chemokine receptor (CCR)2, CCR5, CD9, CD14, CD16, CD64, CD68, CD163, CD169, CD206, C–X3–C motif chemokine receptor 1 (CX3CR1), F4/80, galectin-3 (Gal-3), inducible nitric oxide synthase (iNOS), lymphatic vessel endothelial receptor 1 (Lyve1), MHCII, and triggering receptor expressed on myeloid cells 2 (TREM2) [31]. Among them, CD68, a glycosylated transmembrane protein, is largely used as a pan-macrophage marker in different tissues, being able to identify the phagocytic population involved in endometriosis pathogenesis [31]. Recent studies have confirmed that CD68+ cells, most of them being macrophages, have the capability to co-express numerous other markers, including those of lymphocytes, CD4, CD3, or CD8 (cytotoxic T-cells) [43]. Additionally, a high immunoexpression in CD14high macrophages [40] and CD68 expression [43] was reported in peritoneal endometriosis [40]. These data correlate with our results, revealing a moderate to strong expression of CD4+ and CD8+ cells, along with a strong expression of CD68+ cells, suggesting the common involvement of macrophages in all endometriosis sequences, along with different degrees of involvement of T-lymphocytes in various stages of the disease.

However, the significance of macrophages infiltration of endometriotic lesions is more complex and difficult to assess, considering the possible co-expression of CD68, with CD4 and/or CD8, added to their involvement in phagocytosis and degradation of the menstrual blood. However, the expression identified for CD68, CD4, and CD8 markers in the studied cases underlines the most probable initial role of macrophages in promoting the immune response, followed by the contribution of other cell types, including lymphocytes, in maintaining the inflammatory status characteristic for endometriosis.

Local and systemic changes are hypothesized to support the relationship between inflammation and carcinogenesis, including increased circulating levels of adipose tissue-derived estrogens, adipokines, and inflammatory mediators [44]. In this context, some studies have been suggesting that macrophages can regulate proliferation and invasion of ER-positive breast tumors, without a general consensus [45]. The existence of a high expression of both ERα and ERβ isoforms in peritoneal macrophages of patients with endometriosis has been identified, with ERα inducing the inflammatory response, which accompanies endometriosis, and ERβ producing the proinflammatory cytokines [40, 46].

Furthermore, the endometriotic implants express enzymes, such as aromatase and 17β-hydroxysteroid dehydrogenase type 1, that are involved in estradiol production, and are deficient in 17β-hydroxysteroid dehydrogenase type 2, which normally inactivates estrogen, thus creating a hyperestrogenic milieu [47]. In endometriotic implants, a cellular resistance to progesterone may further enhance the estradiol effects [48].

Although estrogens have an important role in normal tissues homeostasis, the regulatory mechanisms work together to produce an extracellular acidic microenvironment, a necessary condition for counteracting stressors in the neuroimmune–endocrine axis disruption characteristic for endometriosis [49].

Although the relationship between inflammation, ER, and PR is complex, our study could not identify any statistically significant correlation between CD68 and ERα expression, nor between CD4, CD8, and hormone receptors in the samples which were analyzed. The only significant statistical value has been registered between CD68 expression and PR score, correlated with literature data, considering that ERα is reported as a promoter of the inflammatory response characteristic for endometriosis [40, 46].

Considering the literature data report alterations of the expressions of ER and PR receptors in endometriosis, our study used ERα, without any discriminatory use of a certain PR isoform. ERα has been chosen as it is generally considered to promote the growth of endometrial cells, while ERβ may have an opposite effect, inhibiting the proliferation and the inflammatory response [50]. The levels of PR, particularly the PR-B isoform, are markedly decreased in endometriotic stromal cells, resulting in a loss of paracrine signaling [51]. PR deficiency is probably involved in the development of progesterone resistance in patients with endometriosis [48, 52]. Samples from our study were collected from patients that underwent surgical treatment, but they were not further divided into different groups according to the presurgical treatment. Considering that some of the patients of the study group underwent medical treatment before surgery, including hormonal treatment, such as combined oral contraceptive pill, progesterone only pill or Levonorgestrel intrauterine device could have contributed to a distortion of the hormone receptor expression, with a change of the ratio between different isoforms or to a regression of the local inflammatory process.

Our study reveals, in most cases, a strong immunoexpression for both ERα and PR, which leads to the conclusion that ERα stimulates PR expression, thus contributing to the complex pathogenic association between hormonal stimuli and the immune processes that accompany endometriosis. Moreover, recent studies have addressed the expression of adhesion molecules (E-cadherin and β-catenin) in different inflammatory processes and in endometriosis [53, 54, 55, 56]. E-cadherin is a transmembrane epithelial adhesion molecule linked to the cytoskeleton of actin filaments, via β- or, alternatively, γ-catenin [57, 58]. Additionally, it intervenes in the Wnt signaling pathway, with an essential role in normal morphogenesis processes and also in pathological processes, such as carcinogenesis [57, 58]. A component of the complex of cadherin adhesion proteins has been originally identified as β-catenin [59]. Cytosolic accumulation of β-catenin is followed by the formation of molecular complexes with various transcription factors, such as lymphoid-enhancer factor-1/T-cell factor (Lef-1/Tcf) family [57, 60]. Alterations in E-cadherin expression leads to reduced intercellular adhesion, including in endometriosis, tumor dedifferentiation, increased invasion capacity, and metastatic potential [57, 61]. Similar to alterations in E-cadherin expression, mutations of the gene encoding β-catenin (CTNNB1) lead to alterations in cell adhesion functions [57, 61].

Regarding the adhesion molecules, we found that all samples showed a high staining index, the strongest expression being noticed only in 13.2% and 9.4% of cases, for E-cadherin and β-catenin, respectively, most cases showing lower scores for both markers, in the present study. Our results confirm the literature data, which reported high scores for both E-cadherin and β-catenin in ovarian endometriosis, values that were visibly lower compared to those in the normal endometrium [62]. In addition, Fujimoto et al. demonstrated that, while the values of adhesion molecules are cyclically variable in normal endometrium, being much higher in the secretory phase, the values of these molecules did not show any cyclic variations in endometriosis [62]. Another study also showed strong immunoexpression for both E-cadherin and β-catenin in gastrointestinal endometriosis [63]. In this sense, a possible correlation of the variations in E-cadherin and β-catenin expression in the studied endometriotic foci with the value of hormone receptors, as well as with the phases of the menstrual cycle or with the menopausal status could explain a potential influence of the endocrine milieu in endometriotic implants, in a similar manner to that encountered in the normal endometrium [62], but comparatively attenuated. This hypothesis is also strengthened by the finding that peritoneal endometriotic lesion express ER and undergo cyclical menstrual events, as in normal endometrium [64].

Nevertheless, a stronger expression of adhesion IHC markers suggested that some of the cases might express a more aggressive phenotype. It is possible that alteration in the expression of these adhesion molecules might intervene in more advanced cases, reflecting different stages of endometriosis. Their alterations could suggest that the cadherin switch occurs later in the development of endometriosis, as the disease is progressing. Similarly, as no significant correlation was found between the expression of inflammatory markers (CD4, CD8, and CD68) and of adhesion molecules, we could suspect that the inflammatory infiltrate could be more prominent in later or different stages of the disease, when more similarities with the carcinogenesis might occur.

Considering that serological parameters are useful in assessing the inflammatory status of endometriosis, the assessment of serum inflammatory markers (CRP, plasma fibrinogen, and ESR) has been performed in our study group. CRP evaluation has considered a threshold of 6 mg/L for CRP to identify the presence of unspecific inflammatory stimuli, considering that either a low increase or even a normal value may be found in genetic polymorphisms, diabetes, obesity, sedentary lifestyle, depression, common cold, and cigarette smoking. However, the multivariable analysis performed in our study could not identify a significant association between the level of serological markers and the analyzed IHC markers from endometriotic foci. In this context, further studies are needed to identify novel markers or a panel of associated serological markers that could be used in early diagnosis and in follow-up of the disease.

Conclusions

The study of the chronic inflammatory infiltrate in endometriosis may provide important details regarding the initiation and development of the pathological mechanisms of the disease, along with possible pathways of interaction between several types of inflammatory cells, probably initiated by inflammatory macrophages, in the context of unbalanced hormonal milieu, and changes of intercellular adhesion molecules.

Further studies that consider previous medical treatment administered to patients are needed to evaluate the exact correlation between the hormone receptors and the inflammatory markers. Understanding these hormone receptors and their roles in endometriosis is an active area of research. Future treatments for endometriosis may target these receptors for a better management of the disease. However, further studies are needed to fully understand these mechanisms, aiming to discover new therapeutic approaches, like immune checkpoint inhibitors administration or T-cell-targeted immunotherapy in endometriosis patients.

The complex involvement of CD4+ cells, CD8+ cells, and macrophages in endometriosis, added to their consistently more prevalent proportion in the inflammatory infiltrate of women with endometriosis, along with the intricate immunological responses related to endometriosis are requiring supplementary studies. A thorough pathophysiological characterization may lead to the development of non-invasive diagnostic biomarkers and to an effective medication repurposing in endometriosis.

Conflict of interests

The authors declare that they have no conflict of interests.